Effect of Storage Conditions on Aspergillus Growth and Aflatoxin

Production in Peanuts: A Study in Ghana

Clara Bernice Darko

Dissertation submitted to the faculty of the Virginia Polytechnic Institute and

State University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

in

Biological Systems Engineering

P. Kumar Mallikarjunan

John V. Perumpral

Maria Balota

Emmanuel A. Frimpong

Komla A. Dzisi

December, 2nd, 2016

Blacksburg, Virginia

Keywords: Aflatoxin production; Aspergillus flavus growth; Hermetic storage

system; Polypropylene woven sacks, Enterprise budget; Partial Budget

Abstract (Academic)

Effect of Storage Conditions on Aspergillus Growth and Aflatoxin Production in

Peanuts: A Study in Ghana

Clara Bernice Darko

Peanuts (Arachis-hypogaea) are one of the staples in Ghana, Sub-Saharan Africa, and

other developing countries. This leguminous crop is frequently contaminated with aflatoxins,

which are secondary metabolites of some Aspergillus fungi, mostly A. flavus and A.

parasiticus. Aflatoxins in foods are known to cause liver cancer, stunted growth in children,

immune system disorders and economic losses. Aflatoxin contamination of peanuts during

storage is worse in the tropics because climatic storage conditions there are almost the same

as the optimum conditions for Aspergillus growth: temperature conditions of about 26-43 °C

and relative humidity of 62-99%. This study investigated the growth of Aspergillus and the

production of aflatoxin in shelled peanuts under varying treatment and packaging conditions.

In addition, the appropriate pre-storage treatments and packaging needed to reduce aflatoxin

production and to maintain quality of shelled and in-shell peanuts in storage under tropical

environments were studied. Another aim was to determine the impact of the switch to

hermetic storage on peanut farming and marketing profitability in Ghana.

Different peanut treatments, with and without Aspergillus flavus fungi, were packaged

in different systems; specifically, polypropylene woven sacks and hermetic packaging.

Peanuts were analyzed for fungi growth, aflatoxin production and lipid oxidation (peroxide

value and p-Anisidine value).

Partial roasting and blanching of peanuts eliminated aflatoxigenic fungi and halted

aflatoxin production in stored peanuts, increased the effectiveness of peanut sorting and,

hence, helped reduce or eliminate aflatoxin levels along the peanut value chain. Additionally,

the results of this study demonstrated that hermetic storage, by suppressing aflatoxin

production, has the potential for maintaining peanut quality vis a vis polypropylene woven

packaging. Profitability analysis conducted as part of this study revealed that the use of the

hermetic storage system would not only improve farmer and trader profits, but also reduce

the incidence of various ailments attributed to aflatoxins.

Abstract (Public)

Effect of Storage Conditions on Aspergillus Growth and Aflatoxin Production in

Peanuts: A Study in Ghana

Clara Bernice Darko

Peanuts are one of the staple crops in Ghana, Sub-Saharan Africa, and other

developing countries. This crop is frequently contaminated with a special type of molds

which produce a poisonous substance called aflatoxin. In foods, it is known to cause liver

cancer, stunted growth in children, immune system disorders and economic losses. Aflatoxin

contamination during storage in the tropical regions is worst because peanuts are mostly

packaged in polypropylene woven sacks and stored under environmental conditions.

Unfortunately, climatic conditions promote mold growth and aflatoxin production. In view of

this problem, this study was aimed at finding appropriate, affordable and adoptable storage

solutions to control fungi growth and aflatoxin production. A portion of the shelled peanuts

was partially roasted to kill the molds and to stop aflatoxin production, and then peeled to

facilitate the sorting of discolored peanuts which are believed to contain aflatoxins. The

partially roasted peanuts and raw ones were packaged in conventional polypropylene woven

sacks and hermetic packaging system and stored. It was demonstrated that partial roasting

and peeling of peanuts can kill molds and halt aflatoxin production in stored peanuts. Partial

roasting increased the effectiveness of peanut sorting and hence, aided in reducing aflatoxin

levels along the peanut value chain. Additionally, the results of this study have shown that,

compared to polypropylene woven sacks, hermetic storage suppresses mold growth because it

eliminates oxygen from the package and results in lower aflatoxin levels and reduces the rate

of quality deterioration. Profitability analysis conducted as part of this study revealed that the

use of the hermetic storage system would not only improve farmer and trader profitability,

but also help reduce the incidence of various ailments that have been attributed to aflatoxins.

With the high potential of making additional profits when the hermetic packaging

system is adopted, I recommend that local production and marketing of a hermetic storage

system be encouraged, along with the active creation of awareness of their benefits in

reducing the incidence of aflatoxins.

v

Dedication

To my children, Jason and Priscilla Osei-Bonsu, and my parents, Cecilia Duah and Nana

Osei-Assibey II

vi

Attribution

The experiments and informal interviews for chapters 3, 4, and 5 were performed by

Clara Darko; and the statistical analysis was guided by Dr. Emmanuel A. Frimpong. Dr. Yaw

B. Ansah and Anthony Aliebe contributed to the data analysis for the budgeting in chapter 5.

The main idea for each chapter was developed by Clara Darko and Prof. Kumar. The

manuscript of each chapter was written by Clara Darko and reviewed by Prof. Kumar.

The following chapters are to be submitted to the following journals for publication:

Chapter 3: Journal of Food Science and Technology, Chapters 4: Journal of Food Science

and Technology, and Chapter 5: Journal of Agricultural Sciences.

vii

Acknowledgements

First of all, glory be to God.

I am very grateful to the following two agencies which funded all my doctoral work:

1) the United States Agency for International Development, as part of the Feed the Future

initiative, under the CGIAR Fund, award number BFS-G-11-00002, and the predecessor fund

the Food Security and Crisis Mitigation II grant, award number EEM-G-00-04-00013; and 2)

the Peanuts Mycotoxin and Innovation Lab.

I would like to thank my advisor, Prof. P. Mallikarjunan Kumar for his support of my

development. This doctoral work would not have been possible without determination and his

guidance. Special thanks to my doctoral committee members, both individually and as a

group, for helping me with advice over the years. Dr. Emmanuel A. Frimpong was especially

helpful with the experimental design of my work as well as with data analysis and the extra

push in helping me finish my program successfully. Prof. although he is currently retired, he

did not hesitate to join my committee. I deeply appreciate his detailed revision of my drafts.

Dr. Maria Balota and Prof. Dzisi helped me refine my objectives. Dr. Balota also supplied the

peanuts for my work in the US laboratory and took me to peanut research fields and handling

and processing factories.

Ing. Joseph Boamah, Chief Director of the Ministry of Food and Agriculture, Ghana,

was extremely helpful with my field research work in Ghana not forgetting his advice and

words of encouragements. Dr. Joeseph Asamoah of Ghana Technology University college,

George Brantuo (Director, Agric Engineering Directory) and Patrick Aboagye, were also

helpful. Drs. Hande Ceiliker-Kaya, Manjeet Chinan, Hillary Mehl and Anton Bouldin were

very helpful with my US laboratory research. Amanda McGoug and Kyle Webb of the

Laboratory for Interdisciplinary Statistical Analysis (LISA-Virginia Tech) helped with some

portion of my statistical analysis. My laboratory mates Fatma and Shivranti also helped me in

various ways.

Emmanuel Quaye, William Appaw, Frank Osei-Boakye, Kofi Dekyi and Redeemer

Agblelegbe were very helpful with my research in Ghana. Anthony Aliebe helped me with

my profitability analysis in chapter 5.

I would also like to acknowledge my parents (Nana Osei-Assibey II and Cecilia

Duah), my siblings and my special friend Dr. Yaw Ansah for their support and encouragement

throughout my academic pursuits. Special thanks to my elder sister, Helena, without whom I

would not have gotten this far. My last year at Virginia Tech has been fruitful, and this

viii

success would not have been possible without the help of Dr. Panford. I am grateful to him

for hosting me and my family, for his patience and the rides to and from the airport.

ix

Table of Contents

Dedication ................................................................................................................................. v

Acknowledgements ................................................................................................................ vii

List of Tables ..........................................................................................................................xiii

List of Figures ......................................................................................................................... xv

Chapter 1: Introduction .......................................................................................................... 1

Chapter 2: Literature Review ................................................................................................. 5

Peanuts and their Importance in Ghana ............................................................................... 5

Aflatoxins and food safety regulations ................................................................................ 5

Factors for Aspergillus spp. growth and aflatoxin production (Pre- and Post-harvest) ....... 6

Pre- and Post-harvest Losses of Grains due to Aflatoxin Contamination ............................ 8

Harvesting ............................................................................................................................ 9

Drying .................................................................................................................................. 9

Shelling and Packaging ...................................................................................................... 10

Storage Conditions ............................................................................................................. 11

Storage Solutions ............................................................................................................... 12

Chemical control ................................................................................................................ 12

Bio-Control ........................................................................................................................ 12

Physical (Roasting) ............................................................................................................ 12

Gas control ......................................................................................................................... 13

Controlled Atmosphere (CA) ............................................................................................. 13

Modified Atmospheres Storage .......................................................................................... 14

Hermetic Storage ............................................................................................................... 14

Active Packaging ............................................................................................................... 15

Lipid Oxidation .................................................................................................................. 15

Conclusion ......................................................................................................................... 16

References ............................................................................................................................... 18

Chapter 3: Effects of Packaging and Pre-storage treatments on Aflatoxin Production in

Peanut Storage under Controlled Conditions. .................................................................... 29

Abstract .............................................................................................................................. 29

Introduction ........................................................................................................................ 30

Materials and Methods ....................................................................................................... 32

Experimental Design .......................................................................................................... 32

Aspergillus spp. Spore Suspension Preparation ................................................................. 32

x

Peanuts Rehydration and Inoculation ................................................................................ 32

Preparation of peanut treatments and packaging ............................................................... 33

Fungi Growth Measurement .............................................................................................. 33

Aflatoxin testing (ELISA test) ........................................................................................... 33

Oil Extraction ..................................................................................................................... 34

Peroxide Value (PV) .......................................................................................................... 34

P-Anisidine Value (p-AV) .................................................................................................. 35

Model Selection Procedure ................................................................................................ 35

Statistical Analysis ............................................................................................................. 36

Results and Discussions ..................................................................................................... 36

Fungi Growth ..................................................................................................................... 36

Aflatoxin Production .......................................................................................................... 40

Peroxide Value ................................................................................................................... 43

P- Anisidine Value .............................................................................................................. 47

Model selection .................................................................................................................. 49

Conclusions and Recommendations .................................................................................. 50

References ............................................................................................................................... 51

Chapter 4: Effects of Packaging and Pre-storage treatments on Aflatoxin Production

and Quality in Storage of Shelled and In-shell Peanuts in Ghana. ................................... 56

Abstract ................................................................................................................................... 56

Introduction ........................................................................................................................ 56

Materials and Methodology ............................................................................................... 58

Study Area .......................................................................................................................... 58

Experimental Design .......................................................................................................... 59

Aspergillus spp. Spore Suspension Preparation ................................................................. 59

Peanut Inoculation ............................................................................................................. 59

Sorting ................................................................................................................................ 59

Sample Preparation and Packaging .................................................................................... 60

Sampling (Quartering Method) .......................................................................................... 60

Moisture Content Analysis ................................................................................................. 61

Aflatoxin Extraction........................................................................................................... 61

Aflatoxin Calculation ......................................................................................................... 61

Oil Extraction ..................................................................................................................... 62

Peroxide Value ................................................................................................................... 62

xi

P-Anisidine Value .............................................................................................................. 62

Statistical Analysis ............................................................................................................. 63

Results and Discussion ...................................................................................................... 63

Sorting ................................................................................................................................ 63

Aflatoxin ............................................................................................................................ 64

Peroxide Value ................................................................................................................... 69

P-Anisidine Value .............................................................................................................. 72

b.) In-shell .......................................................................................................................... 75

Aflatoxin Production .......................................................................................................... 75

Peroxide Value ................................................................................................................... 78

P-Anisidine ........................................................................................................................ 80

Conclusion and Recommendation ..................................................................................... 81

References ........................................................................................................................ 82

Chapter 5: Switching to Hermetic Storage of Peanuts to reduce Aflatoxin in Ghana:

Impact on Farming and Marketing Profitability. ............................................................... 87

Abstract: ................................................................................................................................. 87

Introduction ........................................................................................................................ 87

Materials and Methods ....................................................................................................... 90

Study Area .......................................................................................................................... 90

Budget Analysis ................................................................................................................. 91

Results and Discussions ..................................................................................................... 92

Analysis of Peanut Farmer Profitability ............................................................................ 92

Profitability Analysis of Peanut Traders ............................................................................ 96

Evaluating the Shift from Polypropylene woven Sacks to Hermetic Packs ...................... 96

Conclusions and Recommendations ................................................................................... 100

References ............................................................................................................................. 101

Chapter 6: Summary and Conclusions .............................................................................. 105

Recommended Future Study ............................................................................................... 108

General References .............................................................................................................. 109

Appendices ....................................................................................................................... 113

Appendix A: Experimental design for a.) Shelled and b.) In-shell peanuts for field study

in Ghana ........................................................................................................................... 113

Appendix B: Effects of aflatoxin Least square means and standard errors ..................... 114

xii

Appendix C: Moisture levels for (a). –Partially -roasted-blanched (PR-B), partially

roasted not blanched (PR-NB), raw clean (Raw-Cl), and raw inoculated (Raw-Inf), and

(b) moisture levels in samples in Kumasi (Ashanti Region) and Tamale (Northern region)

over 26 weeks of storage.................................................................................................. 115

Appendix D: Moisture levels for a.) In-shell peanuts in polypropylene woven sacks (PS)

and hermetic bags with oxygen absorber (HPO); and (b) moisture levels of samples in

Ejura (Ashanti region) and Diare (Northern region) over 26 weeks of storage ............... 116

Appendix F: Farm equipment purchased for a 2-acre peanut farm in Northern Ghana .. 117

Appendix G: Depreciation and interest of farm equipment for a 2-acre peanut farm in

Northern Ghana ................................................................................................................ 117

xiii

List of Tables

Table 3.1 Mean fungi growth, and aflatoxin of raw clean, raw inoculated, Partial-roast blanch

and Partial-roast- not-blanch samples in the Polypropylene woven sacks- PS, hermetic pack-

HP, hermetic pack +O2 -HPO, hermetic pack vacuumed –HPV packaging............................ 39

Table 3.2. Percentage reduction of fungi growth, aflatoxin, peroxide value and p- Anisidine

values for the four peanut processing methods (raw clean - Raw-Cl, raw inoculated - Raw-

Inf, Partial-roast-blanched- PR-B and Partial-roast- not-blanched – PR-NB) in three

packaging systems (Polypropylene woven sacks- PS, hermetic pack- HP, hermetic pack +O2

-HPO, hermetic-pack-vacuumed –HPV packaging) with respect to the raw- inoculated

samples in polypropylene woven sacks. .................................................................................. 42

Table 3.3 Mean Peroxide and p-Anisidine values of raw clean, raw inoculated, Partial-roast

blanch and Partial-roast- not-blanch samples in the Polypropylene woven sacks- PS, hermetic

pack- HP, hermetic pack +O2 -HPO, hermetic pack vacuumed –HPV ................................... 46

Peroxide Value/meq/Kg ........................................................................................................... 46

Table 3.4. Statistical Models to predict aflatoxin production and quality deterioration for raw

inoculated peanuts. ................................................................................................................... 49

Table 4.1 Results of aflatoxin production levels of peanuts after sorting for shelled and in-

shell. ......................................................................................................................................... 64

Table 4.2 Mean aflatoxin, peroxide and p-Anisidine values of Raw-Clean, raw-inoculated,

Partial-roast-blanch and Partial-roast- not-blanch samples in the Polypropylene woven sacks-

PS, and hermetic pack +02 -HPO, packaging systems. ........................................................... 67

Table 4.3. Relative humidity and temperatures in Ashanti and Northern regions of Ghana

during the storage period. Minimum-maximum (Average) ..................................................... 68

Table 4.4. Percentage reduction of aflatoxin, peroxide value and P-Anisidine values of the

four pre-treatment (raw clean, raw inoculated, Partial-roast blanch and Partial-roast-not-

blanch) in Polypropylene woven sacks- PS, hermetic pack +O2 -HPO packaging system with

respect to the raw- inoculated samples in polypropylene woven sacks. .................................. 68

Table 4.5. Mean aflatoxin, peroxide and P-Anisidine values of Raw-Clean, and raw-

inoculated samples in the Polypropylene woven sacks-PS, and hermetic pack + 02-HPO

packaging systems. .................................................................................................................. 77

Table 4.6. Percentage reduction of aflatoxin, peroxide value and P- Anisidine values of the

two peanuts conditions (raw clean, raw inoculated in Polypropylene woven sacks-PS,

hermetic pack+02-HPO with respect to the raw-inoculated samples in polypropylene woven

sacks. ........................................................................................................................................ 77

xiv

Table 5.1. Enterprise budget for a 2-acre peanut farm, using polypropylene woven bag

storage, and operating for one year. ......................................................................................... 94

Table 5.2. Partial budget analysis for a peanut farmer switching from polypropylene woven

sack to hermetic storage ........................................................................................................... 95

Table 5.3. Enterprise budget for a peanut trader operating for one year, with sales output of

100 bags. .................................................................................................................................. 98

Table 5.4. Partial budget analysis for a peanut trader switching from polypropylene woven

storage to hermetic packaging system ..................................................................................... 99

xv

List of Figures

Figure 1.1. Map of study area showing the approximate locations of the two political regions

of Ghana (Adapted from Wikimedia Foundation, Inc., 2015) ................................................... 4

Fig. 2.1. Chemical structures of aflatoxins AFB1, AFB2, AFG1, and AFG2 (William et al

2004.) ......................................................................................................................................... 6

Fig. 3.1. Fungi growth of the four peanut treatments (raw clean(a), raw inoculated(b),

partially roasted blanched (c) and partially roasted not blanched (d)) in the four packaging

systems (Polypropylene woven sacks-PS, Hermetic pack-HP, Hermetic pack+02-HPO,

Hermetic pack vacuumed-HPV ............................................................................................... 37

Fig. 3.2. Aflatoxin production results of the raw clean(a), raw inoculated(b), partially roasted

blanched (c) and partially roasted not blanched (d) peanut treatments in the four packaging

(PS, HP, HPO, HPV) system. ................................................................................................... 41

Fig. 3.3 Peroxide value results for (a) raw-clean (b) raw inoculated,(c) partially roasted

blanched and (d) partially roasted not blanched peanut samples in the four packaging (PS, HP,

HPO, HPV) systems................................................................................................................. 44

Fig. 3.4. Effects pretreatments (raw clean (a), raw inoculated (b), partially roasted blanched

(c) and partially roasted not blanched (d)) and packaging (PS, HP, HPO, HPV) of P-

Anisidine results....................................................................................................................... 48

Fig. 4.1. Aflatoxin production results of the raw clean(a), raw inoculated(b), partially roasted

not blanched (c) and partially-roasted-blanched (d) peanut treatments in the two packaging

systems (PS, and HPO) over 26 weeks of storage. .................................................................. 65

Fig. 4.2. Peroxide Value results of the raw clean(a), raw inoculated(b), partially roasted not

blanched (c) and partially roasted blanched (d) peanut treatments in the two packaging (PS,

and HPO) systems ................................................................................................................... 71

Fig. 4.3. Correlation matrix of aflatoxin production and quality (Peroxide and p-Anisidine

value) for shelled raw infested peanuts. ................................................................................... 72

Fig. 4.4 (a, b, c, & d)- P-Anisidine Value results of the raw clean(a), raw inoculated(b),

partially roasted not blanched (c) and partially-roasted-blanched (d) peanut treatments in the

two packaging systems (PS, and HPO)................................................................................... 74

Fig. 4.5 (a&b) Effects of aflatoxin production for raw clean (Raw- Cl) and raw inoculated

(Raw-Inf) in-shell peanuts in polypropylene woven sacks (PS) and hermetic bags with

oxygen absorber (HPO) ........................................................................................................... 76

xvi

Fig 4.6 (a & b) Effects of peroxide value for raw clean (Raw- Cl) and raw inoculated (Raw-

Inf) in-shell peanuts in polypropylene woven sacks (PS) and hermetic bags with oxygen

absorber (HPO) ........................................................................................................................ 79

Fig 4.7 Effects of P-Anisidine value for raw clean (Raw- Cl) and raw inoculated (Raw- Inf)

in-shell peanuts in polypropylene woven sacks (PS) and hermetic bags with oxygen absorber

(HPO). ...................................................................................................................................... 79

Fig. 4.8. Correlation matrix of aflatoxin production and quality (Peroxide and p-Anisidine

value) of peanuts for in-shell raw infested peanuts. ................................................................ 80

Fig. 5.1 Comparism of the percentage profit returns of using polypropylene woven sacks and

hermetic bags for peanut farmer and trader in Ghana .............................................................. 99

1

Chapter 1: Introduction

Staple foods in Sub-Saharan Africa and other parts of the developing world are

frequently contaminated with aflatoxins, metabolites of Aspergillus species, mostly

Aspergillus flavus and Aspergillus parasiticus. The main crops affected include maize (corn)

and peanuts, which are the main dietary staples in many parts of Sub-Saharan Africa (Turner

et al., 2005). According to the Food & Drug Administration (FDA, 1980), in USA, the

maximum tolerable aflatoxin level for humans is 20 parts per billion (ppb), and 100 ppb for

dairy animals. The European Union (EU) permitted a maximum level of 4 ppb for total

aflatoxins in peanuts (EU Legislation on Aflatoxins, 2010).

The aflatoxin contamination of peanuts and peanut-based products has been of great

concern globally due to their carcinogenic effect on humans and livestock (Amaditor, 2010).

The primary disease associated with aflatoxin intake is hepatocellular carcinoma (HCC, or

liver cancer) which is the third-leading cause of cancer death globally, according to the World

Health Organization (2008). In addition to liver cancer, aflatoxins have also been linked to

stunted growth in children and immune system disorders (Jolly et al., 2008). This is of great

concern in Ghana because peanuts are extensively used in preparing all kinds of dishes, and

are mixed with baby food as a source of protein, and also used as bread spread. The

individual level of aflatoxin contamination also creates a trade barrier and results in great

economic losses to exporters and the country at large. A recent World Bank study indicated

that the EU regulation on aflatoxins costs Africa $750 million annually in the export of

cereals, dried fruits and nuts (Agyei, 2013). Aflatoxin contamination of peanuts in African

markets is quite high. William, et al. (2011) reported from a survey conducted in local

African markets that 40% of the commodities found there exceeded permissible aflatoxin

contents (in excess of the international standard of 10-20 ppb).

Aflatoxin producing fungi are found in the soil in many parts of the world. Since the

edible part of the peanut plant grows underground, it is more prone to aflatoxin

contamination. The optimum temperature for Aspergillus growth and aflatoxin development

is 26.7-43.3°C, with a relative humidity of 62-99% and moisture content of 13-20% (Sumner

& Lee, 2012). Since Ghana and most African countries have an average temperature and

humidity of about the same as the optimum range for Aspergillus growth and Aflatoxin

production, this problem is compounded in this region. In addition to the excessive heat and

high humidity, the lack of aeration in stores, and insect and rodent damages also result in the

proliferation and spread of fungal spores. Kaaya & Kyamuhangire (2006) reported that most

food contamination occurs during the postharvest storage, because aflatoxin-producing fungi

2

grow exponentially in conventional multi-month storage as a result of a combination of these

optimum environmental conditions.

In developed countries, such as the USA, Aspergillus growth and aflatoxin production

in stored peanuts are not a problem, because peanuts are maintained in cold storage and good

structures at appropriate moisture content. Unfortunately, in Ghana cold storage is not a

viable option because of the cost of power and frequent power outages. Therefore, peanuts

are packaged in jute or polypropylene woven sacks or stored in bulk on farms, in thatch or

mud houses with thatch roofs or cement buildings. In the market places, it is mostly shelled

peanuts that are packaged in jute or polypropylene woven sacks and stored in cement

buildings, wooden or aluminum sheet structures. Most traders store or package their peanuts

in materials other than polypropylene woven sacks. These packaging materials were also

found to influence (increase) aflatoxin levels in stored peanuts (Mutegi et al., 2013). The

above-mentioned packaging materials are not airtight, and there is evidence that these storage

methods facilitate fungal contamination and aflatoxin production (Hell et al., 2000; Udoh et

al., 2000).

Numerous studies have been conducted to control aflatoxin production in stored

agricultural products. Both biological control (Dorner & Cole, 2002; Dorner, 2004; Dorner

2004; Völkl et al., 2004; Yin et al., 2008) and chemical controls (Omidbeygi et al., 2009;

Kumar et al., 2010; Nogueira et al., 2010; Medeiros et al., 2011; Passone et al., 2013; Kedia

et al., 2014) have been reported. Some of these control methods produced satisfactory results;

however, many of them are not suited for food products because the residues left behind may

be harmful for human consumption or might change the taste of the food product.

The hermetic storage system is one of the technologies that might not significantly

change the composition of the food product or leave chemical residues; and this is an airtight

packaging system which stops oxygen and water movement between the outside atmosphere

and the stored grain. Since it is sealed or airtight, it enables insects and other aerobic

organisms in the stored commodity to generate the modified atmosphere by reducing oxygen

and increasing carbon dioxide concentrations through respiratory metabolism. This modified

atmosphere packaging (MAP) is one of the popular food technologies for extending the shelf-

life of perishable and semi-perishable food products by altering the atmospheric gases that

surround the produce. This technology can also help reduce quality deterioration of foods like

peanuts since it is airtight. High quality peanuts with a low FFA content resulted when peanut

environments were modified with CO2 in a hermetic package, thereby reducing lipase

activity which can cause lipid oxidation (Navarro et al., 2012).

3

Modified atmosphere packaging studies (Ellis et al., 1994; Vamava, 2002; White &

Jayas, 2003; Ramos et al., 2012; Navarro et al., 2012; Chong et al., 2013, etc.) done so far on

peanuts and other foods do not seem adoptable and affordable by most peanut farmers and

traders in Ghana and other sub-Saharan countries because of the high capital cost of gas

packaging machinery associated with its use. There is another type of packaging system

which is hermetic but with different substances added, and the additive can either absorb or

release a specific gas, and control the internal atmosphere within the package. This concept,

called active packaging, was defined by Gómez-Estaca et al. (2014) as a packaging system

that deliberately incorporates components that release or absorb substances into or from the

packaged food or the environment surrounding the food to extend the shelf-life or to maintain

or improve the condition of the packaged food. Thus this system can limit the growth of

microorganisms and reduce other quality deterioration processes. The active component,

which can work as either an absorbing or releasing system, can be an integral part of the

packaging system or a separate component placed inside the package (Yahia, 2009). With the

overall goal of finding appropriate easily accessible, affordable, and adaptable storage

conditions for peanuts in Ghana, and reducing aflatoxin production, the latter packaging

system mentioned will be friendlier to the Ghanaian environment and other Sub-Saharan

African countries.

The potential usefulness of hermetic storage for storing agricultural products has been

widely demonstrated. However, to date, scientific studies evaluating its effectiveness for

controlling aflatoxin growth in agricultural products stored over long periods have been

minimal. Hence, the goal of developing solutions that will reduce or control Aspergillus

growth, aflatoxin production, and maintain quality during the storage of peanuts.

The specific objectives of my research were to:

1. investigate the growth of Aspergillus flavus and the production of aflatoxin in shelled

peanuts under varying treatment and packaging conditions to reduce aflatoxin

production and maintain quality under controlled conditions;

2. determine appropriate pre-storage treatments and packaging to reduce aflatoxin

production and maintain the quality of shelled and in-shell peanuts during storage

under the tropical environment;

3. determine the impact of the switch to hermetic storage on peanut farming and

marketing profitability in Ghana.

This research will be reported as follows. The next chapter, which is chapter 2, is literature

review on the main topic. Chapter 3 answers objective 1 of this study, reporting on aflatoxin

4

levels, quality parameters (Peroxide and p-Anisidine value) for four different pre-treated

peanut samples packaged into four different packaging systems for the 14-week study on the

storage of shelled peanuts done in the laboratory, under controlled conditions. The next

chapter (4) also reports on similar work done in chapter 3, but the difference is that the

samples were stored under the normal ambient conditions in Ghana for twenty-six weeks and

shelled and in-shelled peanuts were stored. Chapter 5 also reports on the impact of the

proposed hermetic packaging (vis a vis the existing one) on farming and marketing

profitability if it is adopted in Ghana to help reduce aflatoxins and quality deterioration. The

last chapter summarizes all the research findings and the general conclusions.

The laboratory studies were conducted in the USA (Virginia Tech) and the field

studies in Ghana, specifically in the Northern and Ashanti regions (Figure 1). Ghana has ten

political regions, but the studies were conducted in only the two regions mentioned above.

The Northern region is one of the leading producers of peanuts in Ghana. The Ashanti region,

which is located in the middle part of Ghana, also produces a sizeable amount of the

commodity. Notably, it has a big market for peanuts.

The two selected regions lie between latitudes 9°25'N and 6°43'N, and longitudes

0°53'W and 1°36'W, and have altitudes of 183m (600 ft.) and 287 m (942 ft.). Tamale, in the

Northern region, and Kumasi, in the Ashanti region have average temperatures of 20° C -37°

C and 20° C -33° C, respectively (Climatemps.com, 2012-13). Also, the average relative

humidity for Tamale ranges from 19% in January to 69% in August; and 75% in January to

87% in August for Kumasi, but it can go as high as 98% (climatemps.com, 2012-2013).

Figure 1.1. Map of study area showing the approximate locations of the two political regions

of Ghana (Adapted from Wikimedia Foundation, Inc., 2015)

5

Chapter 2: Literature Review

Peanuts and their Importance in Ghana

Peanuts are a leguminous crop that belongs to the family of Fabaceae, genus Arachis,

and botanically named as Arachis hypogaea. Peanuts are rich in calories and contain many

nutrients, vitamins, antioxidants and minerals that are essential for optimum health (Settaluri

et al., 2012). They also contain high amounts of fats and proteins and, can be used in curbing

protein energy malnutrition (Eshun et al., 2013). They contain also the essential amino acids

needed for normal body growth and metabolism (Pelto & Armar-Klemesu, 2011). Moreno et

al. (2013) found that peanuts had a positive effect (reduce) on cholesterol levels and nutrient

intake.

Peanuts can be consumed raw, boiled, or roasted; or as peanut oil, peanut butter,

peanut meal in the form of snack foods, energy bars and candies. In Ghana, they are normally

used in soups, stews, baby foods, as the main ingredient for khebab powder, and snacks such

as kulikuli, dakwa, nkati cake, etc. Thus, peanuts are used to prepare several indigenous

foods and are one of the major staple crops in most parts of Ghana (Tsigbey et al., 2003).

Peanuts are cultivated in all ten regions of Ghana. Nonetheless, almost half of the

production of peanuts is concentrated in the Northern region of the country, made up of three

separate administrative regions (Northern, Upper East and Upper West) which account for

94% of the peanut production in the country (FAO, 2013). Although the other regions do not

produce a lot of peanuts, they consume and market them. Aflatoxin contamination of peanuts

in African markets, in general, is quite high. William et al. (2011) reported, from a survey

conducted in local African markets, that 40% of the commodities found there exceeded

permissible aflatoxin levels (i.e., in excess of the international standard of 10–20 ppb), and

that an estimated 4.5 billion people in developing countries are at risk of uncontrolled or

poorly controlled exposure to aflatoxins.

Aflatoxins and food safety regulations

Aflatoxins are secondary metabolites of some Aspergillus fungi, mostly A. flavus and

A. parasiticus. There are four major types of aflatoxins: AFB1, AFB2, AFG1, and AFG2 (see

Fig.2.1) with the letters B and G referring to blue and green colors in the fluorescence under

ultraviolet light, and the numbers indicating relative migration distances on thin-layer

chromatography. There are many other aflatoxins, but AFB1 is the most potent naturally

occurring carcinogen (Klich, 2007). The primary disease associated with this type is

hepatocellular carcinoma (HCC, or liver cancer) and this is the third-leading cause of cancer

6

death globally, according to the WHO (2008). Aflatoxins have also been linked to stunted

growth in children and immune system disorders (Jolly et al., 2008). The individual level of

aflatoxin contamination also creates a trade barrier and results in great economic losses for

exporters and countries at large. A 2012 World Bank study indicated that the EU regulation

on aflatoxins costs Africa $750,000,000 annually in the exportation of cereals, dried fruits,

and nuts (Agyei, 2013).

Regulations on aflatoxins have been established in almost all countries, to protect

humans and animals from their harmful effects. About 98% of all countries have had

regulations for mycotoxin for food and/or feed since 2002 (van-Egmond & Jonker, 2004).

The US began regulating the concentration of aflatoxins in food and feed in 1968; and

according to USFDA, the maximum tolerable aflatoxin level for food is 20 parts per billion

(ppb), and 300 ppb for feed (USFDA, 2009). The EU permits a maximum level of 4 ppb for

total aflatoxins in peanuts (EU Legislation on Aflatoxins, Nov. 2010). Regulatory guidelines

for aflatoxins and other mycotoxins differ from country to country. The tolerable level in

Ghana is 15ppb; 20ppb in Kenya, and 10ppb in Uganda. These levels are normally high when

the environmental conditions of grains or peanuts are favorable for aflatoxigenic fungi

growth. Kaaya et al. (2006) reported that although the limits are set, they are poorly enforced

by some of the developing African countries, and this may be due to the lack of access to

research, materials, and equipment for testing aflatoxin levels.

Fig. 2.1. Chemical structures of aflatoxins AFB1, AFB2, AFG1, and AFG2 (William et al

2004.)

Factors for Aspergillus spp. growth and aflatoxin production (Pre- and Post-harvest)

Aflatoxin-producing fungi need favorable conditions to grow and produce toxins.

Some of these conditions are temperature, oxygen, moisture content or water activity, and the

humidity of the surrounding environment of the commodity. These and other factors can lead

to mold growth and mycotoxin production, and can occur in both pre- and post-harvest

7

stages. Other factors that can facilitate mycotoxin or aflatoxin production are mechanical

damage, insect and bird damage, drought, stress, and excessive rainfall (Miller, 1991).

The incidence and levels of fungal infection and aflatoxin contamination reported

vary from one geographical area to another (Kaaya et al., 2006). In most instances, however,

aflatoxins are formed after harvest, especially when grains are not dried well, and also due to

the improper storage of dried agricultural commodities. The factors that have been singled

out as those that mainly encourage mold growth and aflatoxin production in grains and

kernels are temperature and moisture.

Temperature and Relative humidity: The effect of temperature is difficult to separate from

that of humidity. Under favorable environmental conditions with an optimum temperature of

approximately 33°C and 99% RH, as reported by Milani (2013), Aspergillus fungi grow very

well and produce mycotoxins. Aflatoxigenic fungi grow on certain foodstuffs—most

commonly, grains like cereals and nuts. These fungi can grow under very dry post-harvest

conditions of 0·65–0·75 water activity-aw (water in food which is not bound to food

molecules can support the growth of bacteria, yeasts and molds-fungi).where there is less

competition from the majority of mesophilic fungi (Magan & Eldred, 2008). The production

of aflatoxins is most acute and widespread in warm, humid climates. Tropical conditions, as

in Ghana, are more susceptible to the growth of Aspergillus species. Aspergillus flavus grows

best between 10oC and 45

oC at a relative humidity of 75% or more; conditions often not met

in Ghana‘s environment.

Grain Moisture content: The amount of moisture in peanut grains affects their quality and

storability. The two most important factors that affect the life cycle of all microorganisms,

including mycotoxigenic molds, are water availability and storage temperature (Magan,

2007). These two interacting factors influence germination, growth, sporulation and

mycotoxin production (Sanchis & Magan, 2004). The optimum grain moisture for Aspergillus

growth and aflatoxin development ranges from 13-20%, with a relative humidity of 62–99%

of the surrounding air, (Sumner & Lee, 2012). Soil moisture stress has also been reported to

worsen the pre-harvest aflatoxin contamination of produce. Peanuts exposed to drought stress

in the field have been reported to have more A. flavus-infected kernels than those in irrigated

plots. Irrigation helps increase water activity, and there is evidence (Dorner, et al., 1989) that

seeds with high water activity during germination produce stilbenes (phenolic compounds

with antifungal properties) which inhibit growth and aflatoxin production by fungi.

8

Pre- and Post-harvest Losses of Grains due to Aflatoxin Contamination

Post-harvest losses occur during, pre-harvest, harvesting, handling, processing,

storage and transportation to the final consumer (Mrema & Rolle, 2002). It was once thought

that aflatoxin formations only occur during post-harvest, that is, during storage; but it is now

well documented that aflatoxin production also occurs in the field prior to harvest (Okello,

2013). Extensive heat and drought stress can cause aflatoxigenic fungi infestation, especially

when peanuts are deprived of water for 30-50 days during pod maturation and soil

temperatures are as high as 29-31°C (Cole, et al., 1995). Infection by Aspergillus spp. at pre-

harvest and the environmental factors that lead to colonization, infection of the seeds, plants,

and Aflatoxin accumulation have been reviewed in detail (Payne, 1998). Initial inoculum of

aflatoxigenic fungi in peanuts most likely originates in the soil when the peanut plant is on

the field. Evidence suggests that the use of low technology (e.g., irrigation, applying calcium

to soil, sorting) at the farmer level in Sub-Saharan Africa could substantially reduce the

Aflatoxin contamination in peanuts at both pre- and post-harvest levels (Mkoka, 2007).

Aflatoxin contamination occurs more during post-harvest than during pre-harvest

conditions (Wild & Hall, 2000). Improper management practices and adverse climatic

conditions at and after harvest are predisposing factors for post-harvest aflatoxin

contamination. Significant grain deterioration caused by molds also occurs during storage

because of prevailing ambient conditions. The World Bank (2011) reported that post-harvest

grain losses in Sub-Saharan Africa amount to US$4 billion. The APHILS (2013) also

estimated the loss of grains in 2010 to be 15, 000 tons, which, according to the World Bank

(2011), could meet the minimum annual food requirements of 48 million people. Developing

countries have high losses at the post-harvest and processing stages due to spoilage. In Ghana

and other Sub-Saharan African countries, post-harvest losses of major staple crops (maize

and peanuts) are mostly due to mold or fungi infestation.

Post-harvest activities have a major effect on losses and aflatoxin production.

Globally, about one third of the food produced is lost or wasted (FAO, 2010; World Bank,

2010 & Prusky, 2011). A report by the World Bank (2011) revealed that significant amounts

of food are lost after harvest in Sub-Saharan Africa each year. The Food and Agricultural

Organization of the United Nations (FAO) has estimated that about 25% of the world's food

crops are significantly contaminated with mycotoxins (Adams & Motarjemi, 1999). Losses in

peanuts in Ghana and other Sub-Saharan countries are due to handling and post-harvest

activities.

9

Postharvest Peanut Activities Conducive for Aflatoxin Development

Normal post-harvest losses of many crops, especially peanuts, depend on how farmers

harvest, dry, package, and store these after harvest. Most peanuts take about three to four

months to mature before they are harvested, depending on the variety. They are then dried

and stored. Below are some of the post-harvest activities that are most influential in aflatoxin

production.

Harvesting

Harvesting usually consists of a series of operations comprising digging, lifting,

stacking, and threshing. In harvesting peanuts, the soil is first loosened either by working

with a hand hoe, a plough, or a blade harrow before the pods are lifted from the soil. After

digging and lifting, the soil around the pods are then shaken off and the pods with vine are

then stacked on the field. The hand pulling of peanuts can cause high pod loss and pod

cracking, exposing the pods to infestation (Tsigbey, 2003). The pods are normally left on the

farm land for about 4 days for them to be partially cured from harvesting moisture content of

about 50-20% (Cundiff, et al., 1991). There may be a considerable invasion of seeds by

aflatoxigenic fungi when drying is slow, because the seeds remain very susceptible to

aflatoxin production when the moisture content ranges from 13-20% (Sumner & Lee, 2012)

for extended periods. It is even worse when rain falls on the windrows or stacks, thus

providing fungi with needed moisture to multiply. After curing, the next process is threshing,

which involves quite distinct operations, including separating the pods from the vine, sorting

the pods from the haulms and winnowing the chaff from the pods. Threshing can be done

manually with a wooden comb-type structure with a long handle or by beating the pod-end of

the plants against a rough stone or a thick iron rod or beating with flails. The best results are

gained when the moisture content is between 20-25 % (Young, et al., 1982), because the pods

can then be separated easily and completely. If the moisture content is lower, then the pods

and seeds will be more susceptible to damage. Physical damage to kernels makes them much

more susceptible to invasion by storage molds, including Aspergillus flavus. Cracks and

breaking of kernels normally happen during harvesting and shelling, although insect and

rodent feeding may also be responsible for breaks in the pericarp (Sauer & Tuite, 1987).

Drying

Traditional drying of peanuts in developing countries like Ghana involves the field or

bare ground, which is a major source of fungal contamination. The process is slow, time

consuming, and labor intensive, and it may be delayed by rains that normally persist at

harvesting and drying times. It is difficult to achieve the recommended moisture level for safe

10

storage. It is recommended that harvested commodities be dried as quickly as possible to safe

moisture levels of 10–13% for grains (Hell & Mutegi, 2011). The common practice for

peanuts after threshing is drying the pods in the sun. Achieving this through simple sun-

drying under the high humidity conditions of many parts of Africa is difficult. When drying is

done in the dry season, the safe moisture content of 10% and below is not achieved before

loading the grains into stores, as observed by Mestre, et al. (2004), and the product can be

easily contaminated with aflatoxin. After uprooting, the pods are often left in the field for up

to four weeks to partially dry before being dried at home. In addition, the crop is persistently

exposed to soil contamination, which is the source of fungi (Okello et al., 2010). After drying,

peanuts are shelled if there is ready market for them.

Shelling and Packaging

Peanuts can be marketed either in-shell or shelled, but mostly shelled because of

customers‘ preference. Shelling can be done either manually or with a sheller. To avoid

breaking the kernels during shelling, the pods have to be re-wetted. However, this helps

increase the moisture content and, in turn, contributes to aflatoxin production.

Apart from the condition of the commodity to be stored, other important things to

consider are the packaging and the storage structure. From a survey study conducted, it was

observed that peanuts were packaged in polypropylene woven sacks; and a few were

packaged in jute sacks and stored in bulk on farms, in thatch houses, mud houses with thatch

roofing, or cement buildings. In the market places, shelled peanuts are mostly packaged in

jute or polypropylene woven sacks and stored in cement buildings, or wooden or aluminum

sheet structures (Chapter 1). These packages normally used are found to influence aflatoxin

levels in the stored peanuts (Mutegi et al., 2013). Since these packages can absorb moisture

from the atmosphere, they affect the moisture levels of the grains during storage. Farmers,

especially in the Northern region of Ghana, store their packaged peanuts in an enclosed store

room, or on a veranda. Some of the farmers also do bulk storage in enclosed store rooms.

Poor post-harvest storage management can lead to significant dry matter losses and the

accumulation of post-harvest mycotoxins (Magan et al., 2010). New storage practices, such

as the use of metal or cements bins; offer an improvement over traditional storage methods.

However, high costs and access to improved materials remain major constraints for their

adoption by small-scale farmers (Hell & Mutegi, 2011). As mentioned earlier, polypropylene

woven sacks are commonly used but, because they are not airtight, peanuts are still

susceptible to fungal and aflatoxin contamination (Hell et al., 2000; Udoh et al., 2000). Grain

11

moisture content, mold growth, aflatoxins, and free fatty acid content were significantly

higher in pods stored in jute bags than in those stored in polyethylene-doubled jute bags

(Bulaong, 2002). The use of hermetic triple-layer bags (PICS, Purdue Improved Crop

Storage) for the storage of grains is gaining favor because of their advantages over traditional

storage devices (Hell et al., 2010; Murdock et al., 2003). These triple-layer bags are now

being marketed in Africa (Ben et al., 2009). Hermetic packaging could protect peanuts from

molds and aflatoxin contamination (Paramawati et al., 2006). Studies have indicated that the

efficacy of the storage of peanuts in hermetic bags resulted in minimizing Aspergillus flavus

fungi growth and aflatoxin production and maintaining peanut quality.

Storage Conditions

Storage is an important link in the post-harvest value chain of almost all food

commodities and this is the stage where most of post-harvest losses occur. These are losses

from insects, rodents or fungi damage. The deterioration of peanuts by fungi is partly due to

conditions that favor mold formation after windrowing and during storage. Aflatoxigenic

fungi grow exponentially in conventional multi-month storage as a result of a combination of

heat and high humidity (Hell et al., 2010; Guchi, 2015). High moisture and temperatures

determine the rate of deterioration of kernels in storage. When grains or peanuts are stored

dry, their storability is increased, and this prevents the formation of storage fungi. Thus, the

best conditions for the normal storage of unshelled peanuts are about 7.5% kernel moisture

content (w.b.) at 10°C and 65% relative humidity (Davidson et al, 1982) and 7-8 % moisture

content, with relative humidity of less than 55% for shelled peanuts (Sablani & Mujumdar,

2006). The conditions stated are almost impossible to achieve in Ghana. Aflatoxin production

by Aspergillus spp. can increase tenfold in a three-day period, if harvested grains are stored

with a high moisture content (Hell et al., 2008). Apart from the condition of the commodity to

be stored, another important thing to consider is the package and the storage structure. From a

recent survey (Clara Darko, unpublished), unshelled peanuts are currently packaged in

polypropylene woven sacks and a few are packaged in jute sacks. Some of the farmers also

do bulk storage in either cement buildings or thatch houses, or mud houses with thatch

roofing. Peanut traders also store their products (shelled peanuts) in the market place mostly

in polypropylene woven sacks kept in wooden or aluminum sheet structures in the Northern

region and in cement structures in the Ashanti region. Poor post-harvest storage management

can lead to significant dry matter loss and the accumulation of post-harvest mycotoxins

(Magan et al., 2010). There has been a lot of work done towards combating aflatoxin

production during storage (Fonseca et al., 1994; Reddy et al., 2009; Hell et al., 2000, 2010;

12

Mutegi et al., 2013; Villers, 2014; Guchi, 2015, etc.). One major intervention for reducing or

eliminating aflatoxin production during storage is the pretreatment of the grains.

Storage Solutions

Chemical control

Chemical control methods using essential oils have shown great promise. Most of

these oils are produced by medicinal plants and that have been tested for their ability to

control aflatoxin contamination in culture medium conditions (Omidbeygi et al., 2009;

Kumar et al., 2010; Nogueira et al., 2010; Medeiros et al., 2011).

There have also been studies on antifungal and anti-aflatoxigenic activity by essential

oils on peanuts, wheat, and chickpeas (Passone et al., 2013; Kedia et al., 2014). Eugenol oil

was used for the inhibition of AFB1 production in stored sorghum grains and that

significantly reduced contamination (Komala et al, 2012). Plant extracts or plant-based

preservatives have been effectively targeted as a viable strategy to enhance shelf life of food

items against microbial deterioration and mycotoxin problems and aflatoxins (Sánchez et al.,

2005; Selvi et al., 2003).

Bio-Control

The effects of bio-control agents on A. flavus and AFB1 Biological control have been

used to reduce aflatoxin contamination in various crops (Dorner, 2004) such as wheat (Völkl

et al., 2004), maize (Dorner, et al., 1999), peanuts (Dorner, Cole, & Blankenship, 1998), and

cotton (Cotty, 1994). In India, farmers had knowledge on seed treatment with bio-control

agents like Trichoderma viride and Trichoderma harzianum, and 17% of the farmers adopted

them (Kumar & Popat, 2007). Additionally, Kimura and Hirano (1988) reported that using the

isolates of Bacillu subtilis and Bacillus pumilus suppressed fungal growth and aflatoxin

production by A. flavus in corn and by A. parasiticus in peanuts. Percentages of inhibition of

aflatoxin production ranged between 98.4% and 99.9% (Munimbazi & Bullerman, 1998).

The culture filtrate of Rhodococcus erythropolis completely inhibited the growth of A. flavus

and AFB1 production (Reddy et al., 2009).

Physical (Roasting)

Some of the methods discussed above showed satisfactory results. However, many of

them might not be suitable to be used for foods, and some of them would leave chemical

residues on the products, which might not be edible by humans or might change the taste of

the final product. Various methods of food processing, e.g., frying, roasting and microwaving

can destroy the aflatoxin contamination to varying degrees (Scott, 1984; Tobata et al., 1992;

Farag, 1996). One of the safer, more economical and practical methods for combating

13

aflatoxin production were roasting. Some studies have shown that roasting causes reduction

in the levels of aflatoxins (Lee et al., 1968, 1969; Waltking, 1971; Escher et al., 1973;

Hamada & Megalla, 1982; Ogunsanwo, et al., 2004) and is one of the effective physical

methods, which can reduce or remove aflatoxin contents in food (Yazdanpanah et al., 2005).

The extent of the reduction normally depends on the initial level of contamination,

temperature, heating time and type of food (Rustom, 1997; Yazdanpanah, et al., 2005).

Arzande and Jinap (2010) reported an 80.2% reduction of AFB1 and 69.7% of AFB2

in naturally contaminated samples with initial aflatoxin concentrations of 174 and 25 ng/g

(ppb) at 150°C and 130°C, respectively. Mobeen et al. (2011) also reported a 60% reduction

of aflatoxin contamination levels in peanuts and locally available peanut products with the

effect of microwave processing.

Gas control

Another method, which does not leave chemical residues or does not change the

physiology of the peanuts is reducing or removing oxygen from the grain or peanut

environment. This can be done hermetically, by controlling or modifying the atmosphere

around the food produce. In the related literature, the terms ―modified atmosphere‖ (MA) and

―controlled atmosphere‖ (CA) differ based on the degree of control exerted over the

atmospheric composition. In MA storage, the gas composition is modified initially and it

changes dynamically depending on the respiration rate of the food product and permeability

of film or storage structure surrounding the food product. In CA storage, the gas atmosphere

is continuously controlled throughout the storage period (Jayas & Jeyamkondan, 2002).

Controlled Atmosphere (CA)

A CA package, as mentioned above, is a modified gas composition, usually produced

artificially and maintained by additionally generating the desired gases (CO2 or N2) or by

further purging the storage with these gases, supplied from pressurized cylinders (Navarro,

2012). Normally, the gas is topped up when the concentration in the sealed container falls

below the required level. This type of system may either be closed or open. In closed

systems, the storage vessel is sealed and the gas phase composition is not actively controlled.

The composition is determined solely by the initial gas composition and any changes that

occur due to respiration (Chong et al, 2013). ). The open system modifies the atmosphere by

purging the vessel with an inert gas stream; typically N2. A CA system can be designed to

reduce oxygen concentration by purging with N2 and injecting CO2 or allowing carbon

accumulation by perishable respiration (Rama & Narasimham, 2003) A controlled

atmosphere is more appropriate and practical for large-scale perishable transport (Fonseca, et

14

al., 2002). As reported by Chong, et al., 2013, this system was used to maintain an

environment that prolonged the lifespan of fruits and vegetables during transportation and

storage.

Modified Atmospheres Storage

Modified atmospheres have shown promise in controlling Aspergillus flavus and the

subsequent aflatoxin production in stored peanuts. Sanders et al. (1968) reported that little

aflatoxin was found on inoculated peanuts maintained in an atmosphere of 60% CO2, 20%

O2, and 20% N2, as compared with 206 pg of aflatoxin per g of peanuts stored in normal air at

25°C and 90% relative humidity. The CO2-enriched atmosphere was similar to that

recommended by Jay (1971) for the control of insects in stored products. Landers et al.

(1967) reported little aflatoxin production on peanuts with a 28.9% moisture content stored in

an atmosphere of 99% N2 and 1% O2. Navarro et al. (2012) stored peanuts under hermetic

conditions and a control. The formation for the control samples was greater than1.3 *104

CFU/G but the controlled atmosphere of 99% CO2 was able to suppress fungi growth and

recorded less than or equal to 9.70 CFU/G.

Ellis et al. (1993) also reported that aflatoxin production by A. flavus was reduced to

safe and acceptable levels (<20ng/g) by reducing oxygen levels to below 1%, elevated levels

of CO2 to about 70% and N2 to about 30% in combination with other environmental factors in

synthetic media, particularly at temperature abuse conditions. Ozonation is an oxidation

method that was developed for the detoxification of aflatoxins in foods (Samarajeewa et al.,

1990). Ozone, or triatomic oxygen (O3), is a powerful disinfectant and oxidizing agent

(Mckenzie et al., 1998). El-Desouky et al. (2012) reported that AFB1 was degraded by about

77% with ozone treatments.

Hermetic Storage

Hermetic storage is a type of modified atmospheric system that can be applied to the

protection of grain, and it is termed "sealed storage" or "air-tight storage". Since this system

is sufficiently sealed or airtight, it enables insects and other aerobic organisms in the

commodity itself to generate the modified atmosphere by reducing oxygen and increasing

carbon dioxide concentrations through respiratory metabolism. The respiration of the living

organism (eg., grain, insects, and fungi) in storage consumes the oxygen, reducing it from

near 21% in air to 1-2% while the production of carbon dioxide (CO2) rises from an ambient

0.035% to near 20% (White & Jayas, 2003). Insect control success due to the hermetic

storage treatments is comparable to that of conventional fumigants (over 99.9% killed), and

losses due to insect activity are minimal (0.15% loss in weight for a storage period of 15

15

months) (Vamava, 2002). This system kills insect and mite pests, and prevents aerobic fungi

from growing (Weinberg et al., 2008). There is evidence that this storage method killed

aflatoxin-producing fungi and maintained aflatoxin levels in corn storage by using cocoons

(Arnold and Navarro, 2008).

Active Packaging

Active packaging is a concept that implies the interaction between the package and

the product or the headspace between the package and the food system to limit the growth of

microorganisms and to reduce other quality deterioration processes. Gómez-Estaca et al.

(2014) defined active packaging as a package system that deliberately incorporates

components that release or absorb substances into or from the packaged food or the

environment surrounding the food to extend the shelf life or to maintain or improve the

condition of the packaged food. Applications of active packaging include the use of oxygen

and carbon dioxide absorbers to manipulate headspace gas concentrations and the addition of

bioactive agents, such as plant extracts, to the packaging system. Polyolefin-based films are

usually used for the development of active packaging systems by combining the good general

properties of polymer (mechanical, barrier, optical, and thermal) and the antimicrobial or

antioxidant efficiency given by the additives. Ramos et al. (2012) analyzed the influence of

the substrate of several packaging materials containing cinnamon (Cinnamomun zeylanicum)

as an active agent on the antifungal activity against A. flavus and this featured excellent long-

term effectiveness and stability.

Lipid Oxidation

Lipid oxidation in foods constitutes a complex chain of reactions that firstly yields to

primary products (peroxides that have neither taste nor flavor) which, once exposed to

extended oxidation conditions, gives rise to secondary oxidation products, including

aldehydes, ketones, epoxides, hydroxy compounds, oligomers and polymers. Most of these

secondary oxidation products produce undesirable sensorial and biological effects (Marquez-

Ruiz et al., 2007; Kanner, 2007) and are related to rancidity in sensorial tests.

Lipid oxidation is a major cause of food quality deterioration and the generation of

odors and bad flavors, a decrease in shelf life, an alteration of texture and color, and a

decrease in the nutritional value of food (Alamed et al., 2009). The aldehydes interact with

sulphydryl and amine groups in proteins, and this may alter the functionality of proteins

(McClements & Decker, 2007). Polyunsaturated fatty acids (PUFA), which are specifically

abundant in vegetable oils, are major substrates of oil or lipid oxidation, and their oxidative

degradation products deteriorate the chemical, sensory, and nutritional properties of the lipids

16

(Velasco and Dobarganes, 2002; Kowalski et al., 2004; Kamal-Eldin; 2006; Merrill et al.,

2008). When PUFAs are exposed to external factors such as light, elevated temperatures, and

oxygen (Kamal-Eldin, 2006; Merrill et al., 2008) and intrinsic factors such as antioxidants,

prooxidants and water in the oils, they (i.e., PUFAs) might simultaneously operate in a

complementary or opposite way to affect fatty acids (Velasco and Dobarganes, 2002;

Kowalski et al., 2004; Kamal-Eldin; 2006; Merrill et al., 2008). It has; therefore, been

difficult to single out a strong predictor that could assume future oil behavior before the oil

oxidation actually proceeds.

As cited by Martínez et al. (2011), beyond their chemical composition, the

susceptibility of vegetable oils to oxidation also depends on processing, packing and storage

conditions. The enzyme lipoxygenase can also initiate oxidation (Shahidi & Naczk, 2004;

Shahidi, 2015).

To reduce lipid oxidation, several strategies have been applied, such as the direct

addition of antioxidants to foods or the design of a suitable packaging technology. Vacuum or

modified-atmosphere packaging combined with high-barrier packaging materials can limit

the presence of oxygen, although it is not always completely and effectively eliminated

because of a residual presence at the time of packing or because it permeates in from the

exterior through the package wall (Lopez-de-Dicastillo et al., 2010). Antioxidants act at

different levels in the oxidative sequence involving lipid molecules. They may decrease

oxygen concentration, intercept singlet oxygen, prevent first-chain initiation by scavenging

initial radicals such as hydroxyl radicals, bind metal ion catalysts, decompose primary

products of oxidation to non-radical species and break chain reaction in order to prevent

continued hydrogen abstraction from substrates (Shahidi, 2015; Shahidi & Naczk, 2004).

Conclusion

All the storage solutions listed above can be used to combat aflatoxin production in

peanuts during storage, but some of them can pose challenges under Ghanaian conditions. As

examples, chemical and bio-control can leave residues on the peanuts during and after

storage. Getting carbon dioxide, nitrogen, and other inert gases to displace oxygen under

modified and controlled atmosphere can also be challenging. However, we can meet our goal

of finding an appropriate, affordable, and adaptable storage system to help reduce or control

Aspergillus growth, aflatoxin production, and maintain the quality of peanuts. From the

extensive literature on finding solutions to reducing aflatoxin, physical treatment (partial

roasting) can also be used to kill most of the fungi and, therefore, reduce or eliminate

aflatoxin production. In addition, the packages which will be friendlier to the Ghanaian

17

environment are hermetic storage and active packaging. The type of packaging used for

storage can also reduce the rate of lipid oxidation or quality deterioration. Based on the

information from the literature, this study was designed to look at the effect of partial roasting

and hermetic packaging to control Aspergillus growth and to reduce aflatoxin production.

18

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Chapter 3: Effects of Packaging and Pre-storage treatments on Aflatoxin Production in

Peanut Storage under Controlled Conditions.

Abstract

Studies have shown that under favorable environmental conditions (27- 33°C and 62-

99% relative humidity), aflatoxigenic fungi growth and aflatoxin production escalate during

storage. Therefore, there is the need to find storage systems to reduce or eliminate aflatoxin

during this period. This study was set to investigate the growth of Aspergillus flavus and the

production of aflatoxin in shelled peanuts under varying treatment and packaging conditions

to reduce aflatoxin production and maintain quality under controlled conditions.

This study reports on aflatoxin production and peanut (Bailey‘s variety) quality, for

four peanut pre-storage treatments (Raw-clean [Raw-Cl], Raw-inoculated-with-A. flavus

[Raw-Inf], Inoculated-partially-roasted-not-blanched [PRN-blanch], and Inoculated-partially-

roasted-blanched-with-discolored-nuts-sorted-out [PR-blanched]) methods. These treated

samples were packaged in four packaging systems (polypropylene woven sacks–PS, hermetic

packs–HP, hermetic packs with oxygen absorbers–HPO, and vacuumed hermetic packs–

HPV) and stored under controlled conditions at a temperature of 30±10C and water activity

of 0.85±0.02, for 14 weeks.

Raw inoculated samples in polypropylene woven sacks had a higher fungi growth of a

mean value of 2.59 *105 CFU/g as compared to the mean values of samples in hermetic

packs-8298.67 CFU/g for HP, 6018.00 CFU/g for HPO, and 6904.33 CFU/g for HPV.

Similarly, the hermetic bags were able to reduce aflatoxin production of the raw inoculated

samples by 71.85% (HP), 82.02% (HPV), and 83.77% (HPO). Partial roasting and blanching

also reduced aflatoxin production by 89.44%. Quality was best for HPO-10.16 meq/kg and

3.95 meq/kg as compared to 19.25 meq/kg and of 6.48 meq/kg for samples in polypropylene

woven sacks, for peroxide value and p-Anisidine, respectively. There was a strong correlation

(r=0.93, r2

=0.87 P-value <0.0001; r=0.96, r2=0.93 P-value <0.0001 RMSE=0.55) between

aflatoxin production and quality (peroxide value and p-Anisidine value, respectively) for

inoculated samples. These results indicate that using zero-oxygen hermetic packaging,

instead of the conventional polypropylene woven sacks, helped suppress aflatoxin production

and quality deterioration. Also, partially-roasted-blanched-sorted peanuts showed a potential

for reducing or eliminating aflatoxin levels during storage.

Keywords: Aflatoxin, Lipid Oxidation, Aspergillus flavus, Peanuts, Hermetic Storage.

Polypropylene woven sacks.

30

Introduction

Aflatoxin contamination of peanuts and peanut-based products is of great concern

globally due to its carcinogenic effect on humans and livestock (Amaditor, 2010). The

primary disease associated with aflatoxin intake is hepatocellular carcinoma (HCC, or liver

cancer) and this disease is the third leading cause of cancer death globally, according to the

World Health Organization (2008). In addition to liver cancer, aflatoxins have been linked to

stunted growth in children and immune system disorders (Jolly et al., 2008). This is a great

concern in Sub-Saharan Africa, because peanuts are extensively used in preparing all kinds of

dishes, mixed with baby food as a protein source, and used as bread spread. The level of

aflatoxin contamination also creates a trade barrier and results in great economic losses to

exporters and the region at large. A recent World Bank study indicated that the European

Union regulation on aflatoxins costs Africa $750 million annually in the export of cereals,

dried fruits and nuts (Agyei, 2013).

The optimum temperature for Aspergillus growth and aflatoxin development ranges

from 26.7-43.3°C, with a relative humidity of 62–99%, and moisture content of 13-20%

(Sumner & Lee, 2012). Since Ghana and most African countries have average temperatures

and humidity around the same optimum range for Aspergillus growth and aflatoxin

production, the problem is especially severe in these regions. In addition to the excessive heat

and high humidity, the lack of aeration in stores, and insect and rodent damage result in the

proliferation and spread of fungal spores. Kaaya and Kyamuhangire (2006) reported that

most food contamination occurs during post-harvest storage and aflatoxin contamination of

foods has been shown to increase with storage period. Aflatoxin-producing molds grow

exponentially in conventional multi-month storage as a result of a combination of heat and

high humidity. In market places, mostly shelled peanuts are packaged in jute or

polypropylene woven sacks, and stored in cement buildings, or wooden or aluminum sheet

structures. Most traders store or package their peanuts in materials other than sisal bags. The

packaging material used was also found to influence aflatoxin levels in the stored peanuts

(Mutegi et al., 2013). The above-mentioned packaging materials are not airtight, and there is

evidence that these methods of storage facilitate fungal contamination and aflatoxin

development (Hell et al., 2000; Udoh et al., 2000).

Physical, chemical and biological control measures are available for controlling

aflatoxin (Jalili et al., 2010). These control methods have shown positive results (e.g.,

Basaran, 2009; Srivasta et al., 2009; Kumar et al., 2010). Unfortunately, these methods are

not suitable for application to foods, and make the resultant products unwholesome for

31

human consumption (Proctor et al, 2004; Akbas & Ozdemir, 2006). Consequently, safer, more

economical and practical methods need to be found. Peanut is mostly consumed in the

roasted form in most parts of the world (Ogunsanwo et al, 2004). Some studies have shown

that roasting causes reduction in the levels of aflatoxins (Ogunsanwo et al., 2004;

Yazdanapanah et al., 2005). The level of the reduction through roasting is dependent on the

initial level of contamination, temperature, heating time and the type of food (Yazdanapanah

et al., 2005).

One of the most effective techniques for managing aflatoxin is sorting. In commercial

settings, electronic color sorting (ECS) is used for sorting a high percentage of the

contaminated, discolored kernels because peanuts that have been colonized by aflatoxigenic

fungi are often discolored. Cole et al. (1995) reported that the combined use of blanching and

ECS produced a 91% reduction in the mean aflatoxin concentration in shelled peanuts. The

major challenge is that, there is the weight and financial loss when peanuts are blanched, and

also electronically sorted (Dorner & Lamb, 2006).

Another loss on the peanut value chain is quality loss during storage, due to lipid

oxidation. The high level of about 81% unsaturated fatty acid (USDA Nutrient Database

2003) in peanuts can affect the quality and flavor of edible peanuts and peanut products (Ul-

Hassan and Ahmed, 2012). Lipid oxidation does not only cause food quality deterioration but

also a decrease in shelf life, alteration of texture and color, and a decrease in the nutritional

value of food (Alamed, Chaiyasit, McClements, & Decker, 2009). Some of the external

factors that affect lipid oxidation are exposition to external factors such as light, elevated

temperatures, and oxygen (Kamal-Eldin, 2006; Merrill et al., 2008). Since oxygen aids in

aflatoxin production and quality deterioration, there is the need to find a storage system

which blocks the in and out flow of oxygen.

Several studies have reported on Aspergillus growth and aflatoxin production on

peanuts under controlled conditions with hermetic packaging (Ellis et al., 1994; Vaamonde et

al., 2006; Garcia et al., 2011; Navarro et al., 2012). Very little was found in the literature on

the use of hermetic storage for preventing aflatoxin, and in longer-term storage. There was

the need to find appropriate conditions for storing peanuts to reduce aflatoxin production and

reduce quality deterioration. Therefore an experimental study was conducted to investigate

the growth of Aspergillus and the production of aflatoxin in shelled peanuts under different

pre-storage treatments and packaging conditions to reduce aflatoxin production and maintain

quality under controlled conditions. It was also to find a statistical model that best predicted

the behavior of fungi growth, aflatoxin production and quality deterioration.

32

Materials and Methods

Experimental Design

Four by four factorial design was used, i.e., four peanut processing methods and four

types of packaging systems. The four processing methods considered included Raw-clean

(Raw-Cl), Raw-inoculated-with-A. Flavus (Raw-Inf), Inoculated-partially-roasted-not-

blanched (PRN-blanch), and Inoculated-partially-roasted-and-blanched-with-discolored-nuts-

sorted-out (PR-blanched). The four packaging systems were polypropylene woven sacks–PS,

hermetic packs–HP, hermetic packs with oxygen absorbers–HPO, and vacuumed hermetic

packs–HPV. Experiments were replicated three times, and samples were drawn randomly at

0, 2, 6, 10, and 14 weeks, providing a sample size (n) of two hundred and forty (240).

Aspergillus spp. Spore Suspension Preparation

Aspergillus flavus NRRL 3357 (Aflatoxigenic producing fungi) from stock cultures

was inoculated onto potato dextrose agar and incubated at room temperature for 5 days to

enable significant sporulation to take place. After incubation, 10-15 ml of sterile distilled

water was added to each plate. A sterile plastic inoculation loop was used to loosen the

conidia from the PDA plates. The suspension was then filtered through sterile cheese cloth

into a sterile 50 ml Falcon tube. Spores were enumerated using a hemocytometer (Bright-

line™, Sigma-Aldrich, St. Louis, MO, USA) and a microscope (Leica DME, Meyer

Instruments, Inc., Houston, TX, USA), and appropriate dilutions were made from the spore

suspension.

Peanuts Rehydration and Inoculation

Virginia type peanut variety (Bailey‘s) was obtained from Tidewater Agricultural

Research & Extension Center. Shelled peanuts were visually inspected and defective peanuts

were discarded. The remaining peanuts were sterilized under UV light in a biosafety cabinet

for 30 minutes and then it was adjusted to the desired water activity level (0.85) by adding

sterile distilled water to the substrate in sterile bottles. The bottles were cooled down to 4 ºC

for 48 hours with periodic shaking. The amount of water necessary to reach the 10% moisture

content was determined as described by Garcia et al. (2011). Moisture content was confirmed

with an infrared moisture meter (Ohaus MB 200, H & C Weighing Systems, and Columbia,

MD, USA). For inoculation, each kernel was infected by pipetting 10 μl spore suspensions

(105-10

6 CFU/ml) of A. flavus NRRL 3357 on the surface (Dufault, 2008). Infected peanuts

were then incubated at a temperature of 30 ±1 0C and water activity of 0.85± 0.03 for 48

hours before various treatments were applied and the peanuts were packaged. Twenty micro

33

liters (20 µl) of spore suspension was applied to the peanuts‘ surface and left in the incubator

for two days.

Preparation of peanut treatments and packaging

After manually removing discolored, moldy and defective peanuts, those used were

divided into four. The first batch did not receive any treatments and was labelled ‗Raw-clean‘

(Raw-Cl). This batch was then packed into the four different packaging systems and sealed.

The second batch, labelled ‗Raw-inoculated-with-A. Flavus‘ (Raw-Inf), was prepared by

adding 2g of inoculated peanuts to 98g of clean peanuts. The third and the fourth batches

were partially roasted. These samples were roasted in sub batches of 1 kg for 10 minutes at

145 ºC. Half of this roasted lot was blanched (peeling off the testa), and the remaining not

blanched. The inoculated samples were partially roasted separately and 2g added to each

package. The peanuts from the different processing methods were packaged in the four

different packaging systems, each pack weighed 100g and stored in an incubator with 30 ±1

°C temperature, with about 85% humidity (regulated by saturated potassium chloride salt

solution). The hermetic bags used were a special type of polyethylene storage bag (Super

Grain-bag III, Grain Pro. Inc., Concord, MA, USA) with an oxygen transmission rate of 4.28

cc/m2/day and water vapor transmission rate of 2.14 g/cm

2/day. The hermetic packs and

polypropylene woven sacks (Sandbag Depot, Perth, Australia) used for the experiment had

the same size of 7‖ x 8‖. Oxygen absorbers (Oxyfree ®, Marietta, GA, and U.S.A) were used

in one group of the packaging systems that were designated as ‗HPO‘. The various treatment

combinations were analyzed for fungi growth, aflatoxin level, peroxide value, and p-

Anisidine value.

Fungi Growth Measurement

Measurement of fungi growth was based on the colony plate count method described

by Dorner (2002). Peanut water slurry was prepared by grinding shelled peanuts with an

equal weight of distilled water for 7 minutes in a food processor. For quantification of A.

flavus NRRL 3357, 50 g of sub-sample of the slurry was transferred to an autoclaved,

stainless steel blender jar and 50 ml of distilled water added. The diluted slurry was blended

for 1 min at low speed. After that, serial dilutions on modified Dichloran-Rose Bengal

medium (DRBA) were done. The media was incubated for 3 days at 37°C and then colonies

were counted manually.

Aflatoxin testing (ELISA test)

Post-storage peanut samples were analyzed for aflatoxins as described by Dorner

(2002). For quantification of aflatoxins, a separate 10g subsample of original slurry, as

34

prepared for the mold count above, was transferred to a clean jar, 17.5 ml methanol was

added, and was blended at high speed for 1 min. After that, aflatoxin content characterization

was performed by the ELISA test (Agra Quant Total Aflatoxin Test Kit, Romer Labs).

Briefly, 20g of the first slurry of each sample was mixed with 100 ml of 70% methanol in a

vial, and then shaken for 2 min. The mixture was filtered and the filtrate was directly tested

with an ELISA kit according to the manufacturer‘s instructions.

Oil Extraction

As described by Lee et al. (2010), total lipids were extracted by mixing chloroform–

methanol (1:1v/v) with all the samples in a proportion of 1:1, using a slightly modified

version of Bligh and Der method (1959). Twenty four grams (24g) of the original peanut

slurry which was prepared for the fungi growth above was used for the oil extraction. The

slurry was transferred to Erlenmeyer flask and 15ml of chloroform and 30 ml of methanol

were added. The mixture was vortexed for 1-2 minutes. Then another 15ml of chloroform

was added and vortexed. Afterwards, 15 ml of distilled water was added and vortexed. The

mixture was centrifuged at 1000 rpm for 5 minutes and the bottom phase of the solution

collected. The chloroform in the bottom phase was evaporated with a vacuum evaporator at

40°C for 15 min. A vacuum pump (Model R-3000; Buchi, rotavapor, Newcastle, USA) was

used to ensure complete solvent removal. The lipid was transferred to centrifuge tubes,

flushed with nitrogen, and stored at -20°C until it was analyzed.

Peroxide Value (PV)

The peroxide values of the oil extracted from the different samples were determined

using the American Oil Chemists‘ Society (1998) official method Cd 8-53. Briefly, about 3g

of oil was put in a 125ml glass Erlenmeyer flask, 30 ml acetic acid-chloroform solution (3:2,

v/v) was added and shaken. Then 0.5 ml saturated potassium iodide solution was also added,

and swirled gently for exactly one minute, the flask was stoppered and shaken vigorously to

liberate the iodine from the organic layer. Starch indicator (1 ml) was added into the mixture

and then titrated with 0.1 N sodium thiosulfate until the blue grey color disappeared. The

volume of the titrant was recorded to the nearest 0.01 ml.

The peroxide value (milliequivalents peroxide/ 1,000g) was computed using the

following equation:

m

NBS=PV

1000--------- (1)

where B is the titration of blank, mL, mL N is the normality of the sodium thiosulfate

solution and m is the weight of the sample.

35

P-Anisidine Value (p-AV)

Oil was extracted from the peanut sample after storage, and the amount of non-

volatile aldehydes (principally 2-alkenals) in the oil was measured using p-Anisidine value by

following AOCS official method Cd 18-90 (The American Oil Chemists‘ Society, 1998).

About 1g of the oil sample was transferred into a volumetric flask, and mixed with 24 ml iso-

octane. The absorbance of this solution (Ab) was measured at 350 nm with a

spectrophotometer, using iso-octane as the blank. Then 5ml of the mixture was measured and

the blank was transferred separately into a new test tube. 1 ml of p-Anisidine was added to

each test tube and incubated for 10 mins. The absorbance (As) was recorded and the p-

Anisidine value were calculated using the formula below:

mAbAs=AVp /1.225 ------------- (2)

where As is the absorbance of the fat solution after reaction with the p–Anisidine reagent, Ab

is the absorbance of the solution of the fat, and m is the mass, in grams, of the test portion.

Model Selection Procedure

Data of fungi growth, aflatoxin production, peroxide value, p-Anisidine values of raw

inoculated samples in polypropylene woven sacks was fitted to quadratic regression and log

transformed models. The best model was determined for each data set based on Akaike

Information Criterion (AIC) and root mean Square error. The Statistical Analysis software

used was JMP Pro. 11 (SAS Institute Inc., Cary, NC, USA). The Akaike information criterion

(AIC): AIC measures the relative quality of statistical models for a given set of data. It

provides a means for comparison among models, with the model with lowest AIC is

considered the best (Tsay 2005). The AIC is defined as:

AIC= 2K -2* ln L( )------------(3)

where: K is the number of model parameters; L is the minimum value of the likelihood

function for the statistical model.

Root mean square error (RMSE): May be used to determine the differences between

predicted values by a model and the values actually observed. This calculates the real error

between predicted and observed values. The RMSE is more appropriate for assessing model

performance (Chai and Draxler, 2014):

n

XXRMSE

n

i idelmoiobs

1

2

,, )(----------(4)

36

where Xobs is observed values and Xmodel is modelled or predicted values, and n is the sample

size.

Statistical Analysis

Data was subjected to analysis of variance (ANOVA) using JMPRO version 11 (SAS

Institute Inc., Cary, N.C., U.S.A.). Means of all the treatment (pre-storage treatments and

packaging system) combinations were separated using Turkey‘s Kramer Test with α=0.05 and

Student‘s t-test when a significant F-value was obtained. Data was transformed to log10 for

fitting model equations.

Results and Discussions

Fungi Growth

The effects of storage temperature and humidity on raw clean (Raw- Cl), raw inoculated

(Raw-inf, partially roasted blanch (PR-blanch), and partially roasted not blanched (PRN-

blanch) peanut samples over a fourteen-week period are shown in figure 3.1 (a, b, c and d)

below. Apart from the raw inoculated samples, regardless of the packing system, the other

three pre-treatment methods (raw clean, partially roasted blanch and partially roasted not

blanched) had little or no fungi growth. Although the peanuts were first sorted (infested,

damaged, and discolored were removed) and sterilized under ultra violet light, it was

observed that there was very little fungi growth (mean of 109.5 CFU/g) on the raw clean

samples. Although U.V light is known to be good for killing micro-organisms, it is best for

disinfecting surfaces (Meechan & Wilson, 2006). It is likely that it was unable to kill possible

fungi inside the peanuts because some of the raw clean peanut samples had little quantities of

fungi despite the U.V sterilization. Also, exposing peanuts to 145oC for 10 minutes for the

partially roasted sample may have killed the fungi on these samples. There were no

significant differences in fungi growth on the PR-blanch, PRN-blanch, or raw clean peanuts

packaged in all the four packaging systems. Similarly, the fungi growth on raw-inoculated

samples in the three hermetic systems did not increase significantly over the fourteen weeks

of storage. Neither were there any significant differences among all the hermetic packaging

systems. Although the samples had Aspergillus flavus introduced and were stored under

favorable environmental conditions, there was no growth because this hermetic system is

known to kill insects, mite pests, and to prevent aerobic fungi growth (Weinberg et al., 2008).

These results are comparable to the findings of Navarro et al (2012) who reported that

peanuts stored under controlled atmospheric conditions with 99% CO2 concentration were..

37

Fig. 3.1. Fungi growth of the four peanut treatments (raw clean(a), raw inoculated(b), partially roasted blanched (c) and partially roasted not

blanched (d)) in the four packaging systems (Polypropylene woven sacks-PS, Hermetic pack-HP, Hermetic pack+02-HPO, Hermetic pack

vacuumed-HPV

38

able to suppress fungi growth to approximately 97CFU/g. Conversely, fungi growth in the

peanut samples in polypropylene woven sacks displayed dormancy for about 6 weeks. During

the next 8 weeks, the growth increased exponentially and recorded an average of 7.7*

105CFU/g, although the surrounding conditions (31 ± 1°C temperature and 85±2 % relative

humidity) were ideal for Aspergillus growth and aflatoxin development. The lag phase for

fungi growth was longer (6 weeks), compared to less than 7 days for peanuts with water

activity of 0.92 and more, and could be attributed to the relatively low moisture content or

water activity (Ellis et al, 1994).

Regardless of the type of packaging used, there was no significant difference in the

fungi growth (at 95% confidence level) in raw-clean, partially-roasted-blanch, and partially-

roasted-not-blanch peanut samples. The mean value of 2.59*10

5 CFU/g recorded by the raw-

inoculated samples stored in the polypropylene woven sacks was significantly different from

that of the other three packages (104 CFU/g for hermetic-pack-vacuumed, 88.67 CFU/g for

hermetic pack +O2, and 134 CFU/g for hermetic pack vacuumed) as shown in Table 3.1.

These hermetic bags were able to reduce the fungi growth of the raw inoculated samples to

around 97% and partial roasting was also able to almost completely eliminate the growth of

fungi by 99.9 % (Table 3).

39

Table 3.1 Mean fungi growth, and aflatoxin of raw clean, raw inoculated, Partial-roast blanch and Partial-roast- not-blanch samples in the

Polypropylene woven sacks- PS, hermetic pack- HP, hermetic pack +O2 -HPO, hermetic pack vacuumed –HPV packaging

Fungi growth/ CFU/g

Treatment/

Package

HP HPO HPV PS Mean

Raw-Cl 166.67±7673.6

b 34.00±0.11

b 22.67 ± 7673.6

b 214.67 ± 7673.6

b 109.50 ± 3836.8

b

Raw-Inf 8298.67 ± 7673.6

b 6018.00± 7673.6

b 6904.33± 7673.6

b 259283.0 ± 7673.6

a 70126.00 ± 3836.8

a

PR-B 2.67 ± 7673.6

b 0.67± 7673.6

b 9.33 ± 7673.6

b 400.67 ± 7673.6

b 103.33 ± 3836.8

b

PR-NB 30.00 ± 7673.6

b 0.67± 7673.6

b 8.67 ± 7673.6

b 308.67 ± 7673.6

b 86.85 ± 3836.8

b

Overall-Mean 2124.5 ± 3836.8

b 1513.18 ± 3836.8

b 1736.25 ± 3836.8

b 65051.75 ± 3836.8

a

Aflatoxin production Levels/PPb

Raw-Cl 7.03± 1.45

b 6.99± 1.45

b 6.46 ± 1.45

b 7.48± 1.45

b 6.99 ± 0.72

b

Raw-Inf 1 0.77 ± 1.45

b 6.21± 1.45

b 6.88 ± 1.45

b 38.26 ± 1.45

a 15.53 ± 0.72

a

PR-B 5.26 ± 1.45

b 4.04 ± 1.45

b 5.19 ± 1.45

b 4.74

± 1.45 b 4.81 ± 0.72

b

PR-NB 5.67 ± 1.45

b 6.01 ± 1.45

b 6.10 ± 1.45

b 6.37 ± 1.45

b 6.04 ± 0.72

b

Overall-mean 7.19 ± 0.72

b 5.81 ± 0.72

b 6.16± 0.72

b 14.21 ± 0.72

a

Levels not connected by same letter are significantly different. P < 0.05

40

Aflatoxin Production

The aflatoxin production results from the combined effects of raw clean (Raw Cl),

raw inoculated (Raw-inf), partially roasted blanch (PR-blanch), and partially roasted not

blanch (PRN-blanch) peanut samples packaged in the Polypropylene woven sacks-PS,

Hermetic pack-HP, Hermetic pack+02-HPO, Hermetic pack vacuumed–HPV across the

fourteen (14) weeks of storage are shown in figure 3.2. The raw Cl, PR-blanch, and PRN-

blanch peanut samples had relatively lower aflatoxin mean values than raw-inf samples

(Table 3.1). This could be because, as explained earlier, these aflatoxin-producing fungi may

have been destroyed due to U.V radiation and roasting. Early detection and elimination of

aflatoxin-producing fungi are critical to preventing this mycotoxin from entering the food

chain. Aflatoxin concentration can be correlated with the density levels of aflatoxigenic

species detected on naturally contaminated samples (Shapira et al., 1996).

Statistically, there were no significant differences in aflatoxin production in all the

four packaging systems for the entire storage period for the three processing methods (raw

Cl, PR-blanch and PRN-blanch) at P>0.05. Although the aflatoxin levels did not increase

over the fourteen weeks of storage (Figure 3.2) it is obvious that there were little doses of

aflatoxin in the peanut samples which were not visible enough to be sorted out. The aflatoxin

production for the raw inoculated samples in the hermetic bags was relatively lower

(HP=10.77 ppb, HPO=6.21 ppb and HPV=6.88 ppb) than for the same samples in

polypropylene woven sacks (38.26ppb). This could be attributed to the fact that hermetic

bags help remove or reduce oxygen levels. Since oxygen is essential for aflatoxin production

in aflatoxigenic fungi, its absence in the packages (hermetic packaging) will minimize fungi

growth (Paramawati et al., 2006). Ellis, et al. (1993) also reported that aflatoxin production

by A. flavus was reduced to safe and acceptable levels (<20ng/g) by reducing oxygen levels.

Overall, inoculated peanut samples in polypropylene woven sacks recorded the highest

aflatoxin levels. Although all treatment combinations were stored under aflatoxin producing

favorable environmental conditions (30± 1°C, water activity 0.85 ± 0.02, and humidity 85%),

the presence of oxygen may have accounted for the difference. This can be compared to

Vaamonde et al (2005) who recorded the highest aflatoxin value of 4,450 µg/kg in peanuts

stored with 0.86 water activity at 30°C for 28 days. It was observed that all the hermetic bags

were able to reduce aflatoxin production of the raw inoculated samples by 71.85% (HP),

82.02% (HPV) and 83.77% (HPO) and partial roasting and blanching was also able to reduce

aflatoxin production by 89.44% (Table 3.2).

41

Fig. 3.2. Aflatoxin production results of the raw clean(a), raw inoculated(b), partially roasted blanched (c) and partially roasted not blanched

(d) peanut treatments in the four packaging (PS, HP, HPO, HPV) system.

`

42

Table 3.2. Percentage reduction of fungi growth, aflatoxin, peroxide value and p- Anisidine

values for the four peanut processing methods (raw clean - Raw-Cl, raw inoculated - Raw-

Inf, Partial-roast-blanched- PR-B and Partial-roast- not-blanched – PR-NB) in three

packaging systems (Polypropylene woven sacks- PS, hermetic pack- HP, hermetic pack +O2

-HPO, hermetic-pack-vacuumed –HPV packaging) with respect to the raw- inoculated

samples in polypropylene woven sacks.

Pre-storage treatments &

Packaging

Fungi

Growth %

Aflatoxin

Production /%

Peroxide-

Value /%

P-Anisidine

Value/%

Raw-Cl/HP 100.0 81.6 65.2 67.1

Raw-Cl/HPO 100.0 81.7 71.7 69.8

Raw-Cl/HPV 100.0 83.1 69.5 68.5

Raw-Cl/PS 99.9 80.5 69.3 64.5

Raw-Inf/HP 96.8 71.9 62.5 49.5

Raw-Inf/HPO 97.7 83.8 60.6 57.5

Raw-Inf/HPV 97.3 82.0 0.0 56.13

Raw-Inf/PS 0.0 0.0 0.0 0.0

PR-B/HP 100.0 86.3 62.3 59.0

PR-B/HPO 100.0 89.4 67.3 66.0

PR-B/HPV 100.0 86.4 64.8 60.1

PR-B/PS 99.8 87.6 38.1 48.5

PR-NB/HP 99.9 85.2 65.7 58.1

PR-NB/HPO 100.0 84.3 66.3 62.0

PR-NB/HPV 100.0 84.1 63.3 59.6

PR-NB/PS 99.8 83.4 42.2 40.7

43

Peroxide Value

Peroxide value, as a measure of lipid oxidation (quality), showed a slight increase in

oil extracted from all the treatment combinations stored over the fourteen-week period, (fig.

3.3). During lipid oxidation, there is a web of complex oxidation processes involved in the

generation of oxidative degradation products from their precursor fatty acids. There are

intrinsic factors, such as antioxidants, prooxidants, and water in the oils, that might

simultaneously operate in a complementary or opposite way to affect the fatty acids (Velasco

& Dobarganes, 2002; Kowalski et al, 2004; Kamal-Eldin; 2006; Merrill et al, 2008) and, with

a relatively high moisture content of 10± 1% in the samples, this could have been a

contributing factor to this increasing trend of the peroxide value during the storage.

The increase in peroxide values for raw inoculated peanut samples packaged in

polypropylene woven sacks was the highest among all the treatments and had a mean value

of 30.74 Meq/kg compared to the least mean value of 8.71 meq/kg for raw clean samples in

hermetic bags with oxygen absorber (Table 3.3). This could be explained by the free oxygen

passage in the package as the presence of oxygen in the sample can increase lipid oxidation

and this affects the quality because of the fatty acid composition of the lipids (Ul-Hassan and

Ahmed 2012). External factors, such as light, elevated temperatures, and oxygen contribute to

the generation of oxidative degradation products from their precursor fatty acid (Kamal-

Eldin, 2006; Merrill et al, 2008). Furthermore, fungi growth resulting in aflatoxin production

can increase oxidation. This could be a result of the release of enzymes (lipase) by the

aflatoxin-producing fungi which hydrolyze the fatty acids in peanuts which, in turn, initiate

lipid oxidation which leads to rancidity, as reported by Lam and Protor (2003). Thus,

microbial growth is correlated with free fatty acid (FFA) formation. There was a very good

correlation (r = 0.93, r2

= 0.87, RMSE- 7.26, P-value <0.0001) between aflatoxin production

and quality (peroxide value) for inoculated samples. Pattee and Sessoms (1967) also reported

that increase in fat acidity.

44

Fig. 3.3 Peroxide value results for (a) raw-clean (b) raw inoculated,(c) partially roasted blanched and (d) partially roasted not blanched peanut

samples in the four packaging (PS, HP, HPO, HPV) systems

45

highly correlated with visible A. flavus growth. Studies have shown simultaneous increases in

FFA levels and microbial growth (Leob & Mayne, 1952; Lam & Protor, 2003).

At α =0.05, the peroxide values of all the processing methods were significantly

different from one another apart from the PR-blanch and PRN-blanch which were not

significantly different at P-value of 0.77. Similarly, when the peroxide values of the four

packaging systems were compared with one other, they were individually distinct except that

HP and HPV were not significantly different at P-value = 0.20; nor did HPO differ much

from HPV at p-value= 0.30. Although it is difficult to point out a single factor that can affect

lipid oxidation, with all the peanut samples stored under the same environmental conditions,

it was expected that all the partially roasted samples would have a lower oil quality (i.e.,

higher peroxide value) because they were exposed to elevated temperature during roasting.

With the hermetic packaging, it was possible to reduce quality deterioration in terms of

peroxide value in the range of 62.3% (PR- blanch in HP) to 67.27 % (PR- blanch in HPO) for

all the partially roasted (blanch and not-blanch) samples, compared to raw inoculated samples

in polypropylene woven sacks, which had the worst peroxide value recorded (see table 3.2).

46

Table 3.3 Mean Peroxide and p-Anisidine values of raw clean, raw inoculated, Partial-roast blanch and Partial-roast- not-blanch samples in the

Polypropylene woven sacks- PS, hermetic pack- HP, hermetic pack +O2 -HPO, hermetic pack vacuumed –HPV

Peroxide Value/meq/Kg

Treatment/ Package HP HPO HPV PS Mean

Raw-Cl 10.70 ± 0.56de

8.71 ± 0.56 e 9.37± 0.56

de 9.45 ± 0.56

de 9.55 ± 0.28

c

Raw-Inf 13.90 ± 0.56 c 11.53 ± 0.56

cd 12.12 ± 0.56

cd 30.74 ± 0.56

a 17.07 ± 0.28

a

PR-B 11.59 ± 0.56cd

10.06 ± 0.56 de

10.83 ± 0.56de

19.04 ± 0.56 b 12.88 ± 0.28

b

PR-NB 10.56± 0.56 de

10.35 ± 0.56 de

11.29 ± 0.56cde

17.77 ± 0.56 b 12.49 ± 0.28

b

Overall-Mean 11.69 ± 0.28b 10.16 ± 0.28

c 10.90 ± 0.28

bc 19.25 ± 0.28

a

P-Anisidine Value/meq/Kg

Raw-Cl 3.46 ± 0.11 gh

3.18 ± 0.11 h 3.32± 0.11

gh 3.74± 0.11

efgh 3.43 ± 0.05

d

Raw-Inf 5.32 ± 0.11 c 4.48± 0.11

cd 4.62± 0.11

c 10.53 ± 0.11

a 6.24 ± 0.05

a

PR-B 4.32± 0.11 de

3.58 ± 0.11 fgh

4.20 ± 0.11 def

5.42 ± 0.11 c 4.38 ± 0.05

c

PR-NB 4.41 ± 0.11 de

4.0 ± 0.11 defg

4.25 ± 0.11 de

6.24± 0.11 a 4.72± 0.05

b

Overall-mean 4.38 ± 0.05b 3.81± 0.05

d 4.10± 0.05

c 6.48 ± 0.05

a

Levels not connected by same letter are significantly different. P < 0.05

47

P- Anisidine Value

P-Anisidine value assay measures non-volatile secondary oxidation products in oils.

During secondary oxidation, unstable molecules decompose readily to form a myriad of

products such as aldehydes, ketones, alcohols and hydrocarbons, etc. (Shahidi, 1998). These

impart unpleasant flavors and odors to fats, oils and lipid in foods. As in the peroxide values,

the p-Anisidine values from the three samples from hermetic packages also showed a slight

increase over the 14-week storage period for the raw clean, PR-blanch and PRN-blanch

samples (Figure 3.5). Although hermetic packages are airtight, other factors such as light and

temperature (Merrill et al, 2008) were present, and that explains the slight increase in the p-

Anisidine values. There are many procedures for controlling the rate and extent of lipid

oxidation in foods, but the addition of antioxidants has proven to be the most effective

(Barriuso et al 2013). This may be the reason why samples in hermetic bags with oxygen

absorbers recorded the lowest mean value of 3.85 meq and polypropylene woven sacks

recorded 5.30 meq/kg (Table 3.2). It was observed that, the raw inoculated samples in

polypropylene woven sacks sampled from week 10 onwards had a bad odor. This can be

attributed to mold growth because microbial growth is correlated with free fatty acid

formation. The products from the secondary oxidation have the potential to produce

undesirable sensorial and biological effects (Marquez-Ruiz et al, 2007, Kanner, 2007). The

results also showed a strong correlation (r = 0.96, r2 = 0.93 P-value <0.0001 RMSE= 0.55)

between aflatoxin production and quality (p-Anisidine value) for inoculated samples. The p-

Anisidine value for all four pre-storage treatments were significantly different from one

another at p–values <0.05. The overall mean values were 3.43 meq/kg for raw clean, 6.24

meq/kg for raw-inf, 4.38 meq/kg for PR- blanch, and 4.72 meq/kg for PRN-blanch (table 3.3)

48

Fig. 3.4. Effects pretreatments (raw clean (a), raw inoculated (b), partially roasted blanched (c) and partially roasted not blanched (d)) and

packaging (PS, HP, HPO, HPV) of P- Anisidine results.

49

Overall, the raw inoculated samples in polypropylene woven sacks recorded the

highest p-Anisidine value. Comparing the p-Anisidine values of raw inoculated samples to all

the other treatment combinations, the raw clean samples in the three hermetic bags were able

to reduce the quality deterioration by 67.14% (HP), 68.47% (HPV) and 69.80% (HPO),

followed by partially roasted blanched samples in hermetic bags with oxygen absorbers

recording 66% (Table 3.2).

Table 3.4. Statistical Models to predict aflatoxin production and quality deterioration for raw

inoculated peanuts.

Model Equation RMSE AIC

Fungi growth/CFU/g Log- transform =e(9.2991794+0.3272557*Interval/weeks)

0.70 37.95

Quadratic

Regression

= 25111.889-14715

time/weeks+4886.19

(time/weeks)2

55781.7 379.10

Aflatoxin

Production/ppb

Log- transform = e(1.523348 + 0.217011*time/weeks)

0.51 28.40

Quadratic

Regression

7.2-3.21*time(weeks)+0.77

(time/weeks)2

15.49 133.42

Peroxide- Value/meq/kg Log- transform = e (2.426907 + 0.1243391*time/weeks)

0.14 -10.59

Quadratic

Regression

= 10.94 +1.56*time/weeks +

0.15 (time/weeks)2

4.29 94.93

P-Anisidine

Value/meq/kg

Log- transform = e(1.3615382+0.0589321*Interval/weeks)

0.08 -26.77

Quadratic

Regression

= 3.99+ 0.16 *time/weeks

+0.05 (time/weeks)2

0.55 33.43

Model selection

Data on fungi growth, aflatoxin production, peroxide value and p-Anisidine value for

only raw inoculated samples in polypropylene woven sacks was fitted to quadratic regression

and log transformation to find which of the models could best predict the above-mentioned

responses. The root-mean-square error (RMSE) which describes differences between

predicted values of a model and the actual values observed was better for the log transformed

models describing fungi growth than for the quadratic regression models. The akaike

information criterion (AIC) which is used to estimate the quality of models was also better

50

for the log transformed models describing fungi growth than for the quadratic regression

models. Also, using RMSE and AIC for log transformed models and quadratic regression

models for aflatoxin production and the quality parameters (Peroxide values and p- Anisidine

values), the log transformed models were better in predicting the responses than the quadratic

regression models. Again, the RMSE and AIC were better for the log transformed models;

which means that the differences between values predicted by the model and the actual values

observed were better for the log transformed models for all the responses than for the

quadratic regression models.

Overall, it can be inferred that the log transformed model is the best one for

describing and predicting fungi growth, aflatoxin production, and lipid oxidation (peroxide

value and p-Anisidine value) in Table 3.4.

Conclusions and Recommendations

This study has shown that it is best to clean peanuts either by sorting or disinfecting

the peanuts (finding ways to kill the fungi on the surface) before storage. When peanuts were

cleaned, no significant fungi growth, aflatoxin production and lipid oxidation were observed

in raw clean, partially-roasted-blanch, and partially-roasted-not-blanch peanuts over the

storage period when compared to the infested samples. Overall, although partially-roasted-

blanched peanuts produced lower level of aflatoxin production over the storage period, raw-

clean samples were best in terms of quality maintenance, followed by partially-roasted-

blanched peanut samples. To achieve lower aflatoxin values during storage, it will be best to

partially roast the peanut samples, blanch them and then sort the bad ones out before storage

or processing.

The log transformed model is the best model for describing and predicting fungi

growth, aflatoxin production, and lipid oxidation (peroxide value and p-Anisidine value).

Hermetic packages were robust in controlling fungi growth and aflatoxin as well as

maintaining quality, regardless of the type of pre-treatment. Of all the four packaging

systems, the hermetic bags with oxygen absorbers were the best. Therefore, it is

recommended that peanuts be stored hermetically with zero-oxygen in the package. It is

recommended that a study be conducted to find the effect of storage time on fungi growth and

aflatoxin production, with varying water activity levels under controlled optimum

environmental conditions (for temperature and humidity levels) with specific emphasis on the

lag phase of the fungi growth.

51

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56

Chapter 4: Effects of Packaging and Pre-storage treatments on Aflatoxin Production

and Quality in Storage of Shelled and In-shell Peanuts in Ghana.

Abstract

Peanuts are used in most developing countries, such as Ghana, to prevent malnutrition, due to

their high protein content. In most developing countries peanuts are infested with fungi,

especially Aspergillus spp. (mostly Aspergillus. flavus, and Aspergillus. parasiticus) that

produce aflatoxin. This study was designed to determine the effect of pre-storage treatments

and packaging to control aflatoxin production and quality degradation during storage of

shelled and in-shell peanuts, under tropical conditions. Manually-sorted, shelled peanuts were

divided into batches and exposed to the following four treatments: raw-clean (Raw-Cl), raw-

inoculated-with-A. flavus (Raw-Inf), inoculated-partially-roasted-not-blanch (PRN-blanch),

and inoculated-partially-roasted-blanch-with-discolored-nuts-sorted-out (PR-blanch). Two

pre-storage treatments for in-shell (raw-cl and raw-inf) samples were packaged in

polypropylene woven sacks–PS, and in hermetic packs with oxygen absorbers-GPO and

stored for 26 weeks under ambient environmental conditions in Ghana (i.e., Northern and

Ashanti region). These samples were analyzed approximately every 4 weeks for aflatoxin

levels and quality (peroxide and p- Anisidine values).

The results show that sorting reduced aflatoxin levels by 75% for raw-shelled, 97 %

for blanched and 68% for in-shell peanuts. At the end of the storage period, aflatoxin levels

were 4.13 ppb for PR-blanch, 18.8 ppb for PRN-blanch, 21.25ppb for Raw-Cl, and 39.91 ppb

for Raw-Inf. Reduction in quality as determined by p-Anisidine values was 49% for raw-

clean, 29.88% for partially-roasted-blanch and 17.6% for partially-roasted-not-blanch

samples in hermetic bags.

Partially roasting and blanching peanuts can increase the effectiveness of sorting, and hence

aid in reducing aflatoxin along the peanut value-chain. Also, hermetically storing peanuts can

suppress the growth of aflatoxigenic fungi and the production of aflatoxin under tropical

ambient conditions.

Keywords: Aflatoxin, Lipid Oxidation, Peanuts, Hermetic Storage, Polypropylene woven

sacks, Ghana

Introduction

Peanut is a leguminous crop that belongs to the family of Fabaceae, genus Arachis,

and botanically named Arachis hypogaea. Peanuts are rich in calories and contain many

nutrients, vitamins, antioxidants and minerals that are essential for maintaining optimum

health (Settaluri, et al, 2012). Peanuts also contain a high amount of fats and proteins.

57

According to Eshun et al., (2013), post-harvest losses of major staple crops (maize and

peanuts) in Ghana and other countries in Sub-Saharan Africa are mostly due to fungi

infestation; particularly by Aspergillus fungi mostly Aspergillus flavus and Aspergillus

parasiticus which metabolize to produce aflatoxins.

Aflatoxin contamination of peanuts is a worldwide problem and, in addition to its

effect on food safety, the economic losses are significant (Dorner, 2008). Kaaya and

Kyamuhangire (2006) reported that most food contamination occurs during post-harvest

storage of agricultural products. Aflatoxin contamination of foods has been found to increase

in storage. Aflatoxigenic fungi grow exponentially in conventional storage for a longer

duration as a result of prevalent heat and high humidity (Hell et al., 2010; Guchi, 2015),

ranging from 80 to 110°F (26.7-43.3°C), with a relative humidity of 62–99%, and a moisture

content (seed) of 13-20% (Sumner & Lee, 2012). Ideal conditions for storing in-shell peanuts,

is at kernel moisture content (w.b.) of 7.5% and temperature and relative humidity at 10°C

and 65%, respectively (Davidson et al, 1982). For shelled peanuts the recommended moisture

content, temperature and relative humidity conditions are 7-8 % (w.b.), 10 °C and 55%,

respectively (Sablani & Mujumdar, 2006).

Shelled and in-shell peanuts in Ghana and most parts of Sub-Saharan African

countries are generally stored in jute sacks or polyethylene or polypropylene woven sacks.

These sacks are not airtight, and there is evidence that these methods of storage facilitate

fungal contamination and aflatoxin development (Hell et al., 2000; Udoh et al., 2000). Mutegi

et al. (2013) reported that peanuts stored in polypropylene woven and polyethylene bags were

5.6% and 13.4% more contaminated with total aflatoxin than samples stored in jute bags,

respectively. Jute bags easily absorb moisture but allow good airflow while polypropylene

woven and polyethylene are non-absorptive but trap heat (Kennedy & Devereau 1994).

Results from chapter 3 showed that hermetic packaging controls aflatoxigenic fungi and

hence aflatoxin production.

Aflatoxigenic fungi can grow and produce aflatoxins if the oxygen in the headspace is

not removed. As reported by Navarro et al. (2012), aflatoxigenic fungi may take as long as 30

days to reach a 3% oxygen level in ultra-hermetic storage (Navarro et al., 2012) and this is

too long a period to prevent aflatoxin production under optimum conditions for storage. This

means it is best to remove oxygen from peanut surroundings before storage. Oxygen

scavengers are capable of removing or reducing the oxygen concentration in a package (Miltz

& Perry, 2005) in storage.

58

Oxygen and other factors, e.g., heat and high humidity, do not only aid in fungi

growth and aflatoxin production but also help in lipid oxidation (Nkagawa & Rosolem,

2011). Lipid oxidation is considered as a major cause of food quality deterioration and

generation of undesirable odors and flavors. In addition to altering texture and color, lipid

oxidation may also decrease the nutritional value of food (Alamed et al., 2009).

Polyunsaturated fatty acids (PUFA), which are abundant in vegetable oils, are major

substrates of oil or lipid oxidation, and their oxidative degradation products deteriorate the

chemical, sensory, and nutritional properties of the lipids (Velasco and Dobarganes, 2002;

Kowalski et al, 2994; Kamal-Eldin; 2006; Merrill et al, 2008). When PUFAs are exposed to

external factors such as light, elevated temperatures, and oxygen (Kamal-Eldin, 2006; Merrill

et al, 2008), and intrinsic factors such as antioxidants, pro-oxidants and water in the oils

might simultaneously operate in a complementary or opposite way to affect fatty acids

(Velasco & Dobarganes, 2002; Kowalski et al, 2004; Kamal-Eldin; 2006; Merrill et al, 2008).

Thus, it not easy to single out a strong predictor that identifies future oil behavior before the

oil oxidation actually takes place. Also, Pattee and Sessoms (1967) found that an increase in

fat acidity was highly correlated with visible A. flavus growth and aflatoxin production.

There has been considerable research on aflatoxin production in peanuts during

storage (Dorner & Cole 2002; Navarro 2012; Mutegi et al 2013). However, studies dealing

with the control of aflatoxin production levels in hermetically stored peanuts have been

limited. There is a need to fill in this gap and also maintain quality of peanuts in Ghana and

other Sub-Saharan African countries as a whole. The objective of this study was to find

appropriate pre-storage treatments and packaging to reduce aflatoxin production and maintain

quality during the storage of shelled and in- shell peanuts under tropical conditions.

Materials and Methodology

Study Area

The study was conducted in Ashanti and Northern regions of Ghana. Ghana has ten

political regions, but studies were conducted in only these two mentioned regions. The

Northern region is one of the leading producers of peanuts and the Ashanti region also

produces a sizeable amount of the commodity and has a big market for peanuts. In the

Ashanti region, shelled peanuts were stored in Kumasi Central Market and Asuoyeboah Seed

Company in Kumasi. The Northern region samples were stored in Aboabo Market and Agric-

Ridge Seed Company in Tamale. The in-shell peanuts were also stored in the small towns of

Ejura and Diare, in the Northern and Ashanti regions respectively, noted for peanut

production.

59

Experimental Design

Four by two factorial design was used for shelled peanut samples and two by two for

in-shell samples. For the shelled samples, there were four pre-treatment methods (raw-clean

[Raw-Cl], raw-inoculated-with-A. Flavus [Raw-Inf], inoculated-partially-roasted-not-

blanched [PRN-blanch], inoculated-partially-roasted-and-blanched-with-discolored-nuts-

sorted-out [PR-blanched]. All the pre-storage treatments were packed in two packaging

systems (i.e., polypropylene woven sacks–PS, and ultra-hermetic pack with oxygen absorber–

HPO). Similarly, there were two pre-storage treatments (raw-clean [In-Raw-Cl], and in-shell

raw-inoculated-with-A. Flavus [In-Raw-Inf]) packaged in two packaging systems (i.e.,

polypropylene woven sacks–PS, and ultra-hermetic pack with oxygen absorber– HPO). It

was a nested design: two samples in the two regions (Ashanti and Northern region) for either

shelled or in-shell samples. One of each treatment combination (both shelled and in-shell

peanuts) was randomly sampled from each region for analysis in 0, 2, 6, 10, 14, 18, 22 & 26

weeks.

Aspergillus spp. Spore Suspension Preparation

A. flavus (Aflatoxingenic-producing fungi) was applied to potato dextrose agar and incubated

at room temperature for 5 days to enable significant sporulation to take place. After

incubation, 10-15ml of sterile distilled water was added to each plate. A sterile plastic

inoculation loop was used to loosen the conidia from the potato dextrose agar plates. The

suspension created was then filtered through a sterile cheese cloth into a sterile 50 ml

capacity Falcon tube. Spores were enumerated using a hemocytometer (Neubauer, Hausser

Scientific, Horsham, USA), and appropriate dilutions were made from the stock spore

solution.

Peanut Inoculation

Peanuts types (Chinese) were obtained from Tamale market in the Northern region of

Ghana. Shelled and in-shell peanuts were visually inspected for defects, e.g., discoloration

and mold growth, and defective peanuts were removed from the batch. The amounts of

peanuts needed to be inoculated were infected by spraying 105-10

6 CFU/ml of A. flavus on

the surface. Infected peanuts were then incubated at a temperature of 30 ±1 0C and water

activity of 0.85± 0.01 for 7 days before being applied to the samples and packaged.

Sorting

Thirty-three (33) sacks of raw shelled peanuts, each weighing about 80kg, and 25

bags of in-shell peanuts, also weighing about 45kg, were sorted manually (removing

discolored, moldy, and defective peanuts by hand). Every sack was weighed before sorting,

60

and the aflatoxin infested peanuts were also weighed. To aid in further sorting, 17 sacks of

the sorted peanuts were partially roasted in batches (50 kg at 90 ºC for about 50 minutes).

Aflatoxin levels of the unsorted, sorted, partially roasted and partially roasted blanch and

sorted peanuts were tested. Aflatoxin reduction percentages of the sorted and blanched

peanuts were tested.

Sample Preparation and Packaging

As already explained, thirty-three (33) sacks of raw shelled peanuts, each weighing

about 80kg, and 25 bags of in-shell peanuts, also weighing about 45kg, were sorted manually

(removing discolored, moldy, and defective peanuts by hand). After sorting, mixtures with a

ratio of 100 g of inoculated peanuts to 50 kg of clean peanuts were then partially roasted in

batches in a pot over fire at an average pot temperature of 190 ºC for about 50 minutes in

batches for 17 bags. Half of the partially-roasted peanuts were blanched (removing the testa

manually) and the other half not blanched after cooling. The blanched portion was then hand-

sorted to remove burnt, defective and discolored grains.

Half of the remaining 17 bags of raw shelled peanuts did not receive any further pre-

storage treatment, and half of this was regarded as Raw-clean (Raw-Cl). The other half was

processed for ―raw-inoculated-with-A. flavus‖ - (Raw-Inf) samples; with each pack

containing 10kg of clean peanuts and 20g of inoculated peanuts. For in-shell peanuts, half of

the 25 bags did not receive any further pre-storage treatments either, and was regarded as

Raw-clean (In-Raw-Cl). The other half was also processed or treated for ―Raw-inoculated-

with-A. flavus‖ -(In- Raw-Inf) samples; with each pack containing 10kg of clean peanuts and

20g of inoculated peanuts.

All peanut treatments were packaged in the hermetic bags (special polyethylene

storage bags Super Grain-bag III, Grain Pro. Inc., Concord, MA, USA) with oxygen absorber

(Oxyfree®, Marietta, GA, and U.S.A) and polypropylene woven sacks (from the local

market). Each pack contained 10 kg peanuts. The four shelled treatment samples were stored

in the market places and seed companies in Ashanti and Northern regions of Ghana under

normal environmental conditions for 26 weeks. The two in-shell treatments were also stored

with two farmers in Ejura in the Ashanti region and two farmers in Diare in the Northern

region of Ghana. The various treatment combinations were analyzed for aflatoxin, peroxide

value, and p-Anisidine value.

Sampling (Quartering Method)

Each bag was poured onto a clean, non-absorbent surface and mixed thoroughly and

gathered together into a cone-shaped pile. The top surface was flattened out and then the pile

61

was divided into quarters, A, B, C, and D. Incremental samples of 125g were picked from

each quarter and the procedure was repeated to achieve a laboratory sample of 2 kg.

Moisture Content Analysis

About twenty grams of peanuts sample was placed in moisture (Adam Moisture Meter

Pmb 53). Then the moisture content was recorded after each sampling event with a digital

moisture meter.

Aflatoxin Extraction

Aflatoxin was extracted using methods described by Sirhan et al. (2014), with a few

modifications. Using an IKA homogenizer, a slurry of peanut and water (1:1 ratio) was

prepared, 2g of slurry weighed into a 15ml centrifuge tube and 3ml of 60:40 (v/v) methanol

acetronitrile solution was added. The resultant mixture was vortexed using a Genie Vortex

machine for 3mins. 1.32g of anhydrous MgSO4 and 0.2g of NaCl were added to the mixture

and then vortexed for 1min. The tube was centrifuged for 5min at 4000rmp and the upper

organic layer filtered through a 0.45m nylon syringe prior to injection. A volume of 100 µl

of the filtered extract was injected into the HPLC.

A Cecil-Adept Binary Pump HPLC coupled with Shimadzu 10AxL fluorescence detector

(Ex: 360nm, Em: 440nm) with Phenomenex HyperClone BDS C18 Column (150 x 4.60mm,

5µm) was used for the aflatoxin analysis. The mobile phase used was methanol (HPLC

grade): dionized water solution (40:60, v/v) at a flow rate of 1ml/min with column

temperature maintained at 40°C. To 1 liter of mobile phase were added 119 mg of potassium

bromide and 350ul of 4M nitric acid (required for post column electrochemical derivatization

with Kobra Cell, R-Biopharm Rhone). Aflatoxin Mix (G1, G2, B1, B2) standards (ng/g) were

prepared from Supelco®

aflatoxin standard of 2.6ng/μL in methanol. The concentrations of

B1 and G1 were 0.5, 1, 2, 8, 16 ppb per 100 µl injection. The concentrations of B2 and G2

were 0.15, 0.3, 0.6, 2.4, 4.8 ppb per 100 µl injection. Limit of Detection and Limit of

Quantification of total aflatoxin were established at 0.5ng/g (ppb) and 1ng/g (ppb).

Aflatoxin Calculation

Aflatoxin level was calculated by:

WI

TA=ppb

g

ng 1------------- (1)

62

Where A= Nano gram of aflatoxin as eluate injected, T=final test solution eluate volume (ul),

I=volume eluate injected into LC (ul), W=mass (g) of commodity represented by final

extract.

Oil Extraction

Oil was extracted from 600g of peanuts sampled by using a hand cranked oil press

(Piteba, brand). The peanuts were put in the hob of the oil press and the crank turned to get

the lipids out.

Peroxide Value

Peroxide value (PV) is one of the most widely used chemical tests for the

determination of the quality of fat and oil. The peroxide values of the oil extracted from the

different samples were determined using the American Oil Chemists‘ Society (AOCS) official

method Cd 8-53 (1998). Briefly, about 5g oil was put in a 125ml glass Erlenmeyer flask,

30ml acetic acid-chloroform solution (3:2, v/v) was added and shaken. Then 0.5ml saturated

potassium iodide solution was added, swirled gently for exactly one minute, and the flask

stoppered and shaken vigorously to liberate the iodine from the organic layer. Starch indicator

(1 ml) was added into the mixture and then titrated with 0.1 N sodium thiosulfate until the

blue grey color disappeared. The volume of the titrant was recorded to the nearest 0.01 ml.

The peroxide value (milliequivalents peroxide/ 1,000g) sample) was determined using the

formula:

m

NBS=PV

1000------------- (2)

where B is the titration of blank (ml), S is the titration of sample (ml), N is the normality of

the sodium thiosulfate solution and m is the weight of the sample

P-Anisidine Value

Oil was extracted from the treatment combinations of peanuts after storage and p-

Anisidine value was determined by following AOCS official method Cd 18-90 (1998). About

3g of the oil sample was weighed into a volumetric flask and mixed with 22ml iso-octane.

The absorbance of this solution (Ab) was measured at 350nm with a spectrophotometer,

using iso-octane as the blank. Then 5ml of the mixture was measured and the blank was

transferred separately into a new test tube. 1ml of p-Anisidine was added to each test tube

and incubated for 10 minutes. The absorbance (As) was recorded and the p-Anisidine value

(p-AV) were given by the formula:

mAbAs=AVp /1.225 ----------- (3)

63

Where As is the absorbance of the fat solution after reaction with the p–Anisidine reagent, Ab

is the absorbance of the solution of the fat and m is the mass, in grams, of the test portion.

Statistical Analysis

The data was subjected to analysis of variance (ANOVA) using JMP Pro version 11

(SAS Institute Inc., Cary, N.C., U.S.A.). Means and interactions of all treatments (pre-

treatments and packaging system) were separated using Turkey‘s Kramer Test with α=0.05

and Student‘s t-test when a significant F-value was obtained.

Results and Discussion

Sorting

Sorting peanuts bought from farmers and traders in the Northern region of Ghana had

a significant effect on the reduction of aflatoxin levels (Table 4.1). Sorting helped reduce

aflatoxin levels from 137.99ppb to 33.54ppb which was about 75.7 %. Manual sorting of

peanuts with high aflatoxin levels can result in very low aflatoxin contents of less than 15 ppb

(Galvez et al, 2003). Also, sorting partially roasted, blanched samples drastically reduced

aflatoxin levels from 137.99 ppb (raw unsorted samples) to 3.7 ppb, which is a 97%

reduction.

Blanching can be a very efficient way of removing aflatoxin-contaminated kernels,

because it is known that peanuts that have been colonized by aflatoxigenic fungi are often

discolored (Dorner, 2008). Therefore, they are easier to sort out than when peanuts are not-

blanch. Electronic color sorting (ECS) is very effective for managing aflatoxin

contamination. Cole et al. (1995) reported that blanching and sorting with an electronic color

sorter reduced aflatoxin levels by 91%, which is similar to the findings in this study.

Aflatoxin levels in partially roasted and not blanched (17.2 ppb) samples were lower than

those of raw clean (33.54 ppb) peanuts. As shown in previous studies (Ogunsanwo et al.,

2004; Yazdanapanah et al., 2005), roasting also helps reduce aflatoxin levels. These

reductions in aflatoxin levels depend on factors such as initial contamination levels, crop or

food type, temperature, and heating time (Yazdanapanah et al., 2005). The in-shell peanuts

used for this studies had relatively high aflatoxin levels (average 93.79ppb), and this can be

explained as having been contaminated before harvesting. Drought can cause peanuts to be

contaminated with aflatoxin during germination. This is supported by Cole and coworkers

(1984), as cited in Diao et al (2015), ―that drought stress and soil temperature of 29 ºC for 85-

100 days yielded the greatest number of colonized edible-grade peanuts and high aflatoxin

levels‖. There was a 68% aflatoxin reduction after sorting in-shell peanuts (Table 4.1).

64

Table 4.1 Results of aflatoxin production levels of peanuts after sorting for shelled and in-

shell.

Treatment Mean-wt/kg %-Infested

after sorting

Wk-0,

Afla/ppb

Percentage

Reduction/(compared

to un-sorted samples)

Shelled Sorted 81.24 2.20 33.54 75.7

Shelled (Un-Sorted) 81.35 Not sorted 137.99

Blanch shell 81.25 1.05 3.7 97.32

Not-Blanch 81.55 Not sorted 28.18 87.54

In-shell (Sorted) 46.55 2.75 29.5 68.55

In-Shell (Un-Sorted) 47.17 Not sorted 93.79

A) Shelled Peanuts

Aflatoxin

The production of aflatoxin of the four shelled peanut treatments in the conventional

polypropylene woven sacks and hermetic bags with oxygen absorbers in Tamale in the

Northern region and Kumasi in the Ashanti region of Ghana is as shown in figures 4.1 a, b, c,

& d. As observed during the laboratory experiments described in chapter 3, the partially-

roasted-blanched samples had relatively low mean aflatoxin levels of 4.13 ppb, followed by

18.8 ppb for partially roasted not blanch samples, 21.25 ppb for raw clean samples, and

39.91ppb for raw inoculated samples (Table 4.2). Roasting can reduce aflatoxin levels

(Yazdanapanah et al., 2005) and blanching and sorting can further reduce them (Cole et al.,

1995). The A. flavus fungi introduced into the raw inoculated samples did not grow and

produce aflatoxin as expected, because the moisture content peanuts used in the experiments

in Ghana was relatively low, in the range of 5-11%.

65

Fig. 4.1. Aflatoxin production results of the raw clean(a), raw inoculated(b), partially roasted not blanched (c) and partially-roasted-blanched

(d) peanut treatments in the two packaging systems (PS, and HPO) over 26 weeks of storage.

66

Moisture level is also critical for fungi growth, sporulation and mycotoxin production

(Sanchis & Magan, 2004; Magan 2007). There were slight increases in aflatoxin levels for the

raw clean samples in polypropylene woven sacks. This shows that although peanuts were

sorted there was evidence of small doses of aflatoxin-producing fungi which produced small

amounts of aflatoxin during the twenty-six (26) weeks of storage, just as in the raw

inoculated samples. The aflatoxin production of the samples in the hermetic bags with

oxygen absorbers was quite stable for the whole storage period, because of the deprivation of

oxygen in the package. Reduced oxygen levels were able to reduce aflatoxin production of A.

flavus to safe and acceptable levels (<20 ng/g (ppb) (Ellis, et al., 1993).

The fungi were dormant from week zero (0), which was in December, to week ten (10) in

February which was the harmattan (Dry-Season) season in Ghana, when relatively low

humidity ranging from 16.1-76.2% for Ashanti region and 11.0-55.0% for Northern region

(Table 4.3) was recorded. There was an increase in aflatoxin level from week 18 onwards

when the weather was relatively humid; but before the 18th week, the aflatoxin levels did not

increase. Fungi can survive under adverse conditions when they produce microscopic spores

(Stajich et al, 2009). Also, Coley-Smith (1971) reported that fungi such as aflatoxigenic ones

can produce dense aggregations of fungal tissue called scelerotia, and these structures help

them survive challenging conditions such as freezing temperatures and long term absence of

a host. These Aspergillus flavus fungi can survive until favorable growth conditions are

available (Sumner & Lee, 2012).

Regardless of the package type, there was no increase of aflatoxin levels the partially-

roasted-blanched and non-blanched samples. It was known that A. flavus cannot survive

temperatures above 80 °C (H. Mehl, personal communication, May 22 2015). Therefore,

partially roasting peanuts at a temperature of 190°C for 50 minutes must have killed all fungi

on the inoculated peanuts. This observation probably explains why there was no aflatoxin

production during the storage period.

Blanching the peanuts and sorting out infested and discolored ones was very effective

in reducing the aflatoxin levels by about 90 %, compared to raw-inoculated samples in

polypropylene woven sacks. Aflatoxin levels in Kumasi and Tamale were significantly

different from each other at p<0.05, (23.17 ppb) at Kumasi where the environment was more

humid than Tamale which recorded 18.89 ppb during the 26 weeks of storage. Overall, at

95% confidence level, there was a significant difference in aflatoxin levels for all the four

pre-storage treatments as well as a significant difference between the two packaging systems.

67

Table 4.2 Mean aflatoxin, peroxide and p-Anisidine values of Raw-Clean, raw-inoculated, Partial-roast-blanch and Partial-roast- not-blanch

samples in the Polypropylene woven sacks- PS, and hermetic pack +02 -HPO, packaging systems.

Aflatoxin/ppb

Treatment/ Package HPO PS Overall Mean

Raw-Clean 19.71 ± 1.13c 22.80 ± 1.13c 21.25 ± 0.80 b

Raw- inoculated 35.12 ± 1.13 b 44.70 ± 1.13a 39.91 ± 0.80 a

Partial-roast-blanch 4.13 ± 1.13d 4.14 ± 1.13d 4.13± 0.80 d

Partial-roast-not-blanch 18.53 ± 1.13 c 19.08 ± 1.13c 18.8 ± 0.80 c

Overall Mean 19.37 ± 0.56 b 22.68± 0.56 a

Peroxide Value/meq/kg

Raw-Clean 8.87 ± 0.19 e 10.54 ± 0.19d 7.71 ± 0.13c

Raw- inoculated 11.77 ± 0.19 c 15.29 ± 0.19 a 13.53± 0.13 a

Partial-roast-blanch 10.69± 0.19 d 13.72 ± 0.19b 12.21± 0.13 b

Partial-roast- not-blanch 10.99 ± 0.19 cd 15.24± 0.19 a 13.12± 0.13 a

Overall Mean 10.58 ± 0.10 b 13.70 ± 0.10a

P-Anisidine Value /meq/Kg

Raw-Clean 5.12 ± 0.18e 6.18± 0.18 d 5.65 ± 0.13 c

Raw- inoculated 8.49 ± 0.18c 10.04± 0.18 b 9.26 ± 0.13 b

Partial-roast-blanch 7.04± 0.18 c 11.26± 0.18 a 9.65 ± 0.13 ab

Partial-roast- not-blanch 8.27± 0.18 c 11.68 ± 0.18a 9.98 ± 0.13 a

Overall Mean 7.49± 0.09 b 9.79± 0.09 a

Levels not connected by the same letter are significantly different

68

Table 4.3. Relative humidity and temperatures in Ashanti and Northern regions of Ghana

during the storage period. Minimum-maximum (Average)

Table 4.4. Percentage reduction of aflatoxin, peroxide value and P-Anisidine values of the

four pre-treatment (raw clean, raw inoculated, Partial-roast blanch and Partial-roast-not-

blanch) in Polypropylene woven sacks- PS, hermetic pack +O2 -HPO packaging system with

respect to the raw- inoculated samples in polypropylene woven sacks.

Treatment/

Package

Aflatoxin/ ppb Peroxide Value/

(Meq/kg)

P- Anisidine Value/

(Meq/kg)

Raw-Cl/HPO 55.9 44.0 49.0

Raw-Cl/PS 49.00 31.1 38.5

Raw-Inf/HPO 21.4 23.0 15.4

Raw-Inf/PS 0.0 0.0 0.0

PR-B/HPO 90.8 30.1 29.9

PR-B/PS 90.7 10.3 -12.2

PR-NB/HPO 58.6 28.1 17.6

PR-NB/PS 57.3 0.3 -16.3

Interval/

weeks

(Shelled and

In- Shell)

Relative

Humidity/%

Temperature/°C

Ashanti Region

Range (Average)

Northern Region

Range (Average)

Ashanti Region

Range (Average)

Northern Region

Range (Average)

Week 0 30-46(39.6) 14-38(21.5) 24.2-31.9(27.6) 17.6-42 (30)

Week 2 30.3-47.1(37.2) 12-47(20.2) 24.2-31.9(28.6) 18-42.5 ( 31.9)

Week 6 16.2-76.2(39.3) 13-55(21.3)28.5 24.8-36.1(30.0) 20- 41.8 (29.5)

Week 10 16.1-71.9 (34.7) 11-49(22.5)24.3 27.1-39(31.9) 17.9 – 42 (31.0)

Week 14 36.0-82.6(62.5) 14-83(48.8) 54.3 25.6- 37.9(32.2) 22- 42.5 ( 33)

Week 18 47-86.5 (70.4) 36-88(59.5) 25.4-35.2(30.9) 22- 39.8 (32.3)

Week 22 52- 94.0 (75.8) 33-87.1 (65.3) 24.6 – 36.5(30.9) 21.5 – 40.5 ( 30.9)

Week 26 62.5 – 90.7 (79.2) 44.0-96.0 (71.25) 24.7 – 34.6 (29.0) 21.50 – 36.50 (29.3)

69

Peroxide Value

Figures 4.2 (a, b, c, &d) present the trend of peroxide values (primary oxidation

parameter) for the peanuts stored under Ghanaian ambient conditions. All the four treatments

experienced only minor increases in peroxide levels during storage. This may be due to the

fact that external factors such as light, elevated temperatures and oxygen may have

contributed to the generation of oxidative degradation products from their precursor fatty

acids (Kamal-Eldin, 2006; Merrill et al, 2008). Additionally, peroxide values from samples in

the polypropylene woven sacks plateaued after week 18. As reported by Hill (1994), the use

of peroxide value as flavor quality indicator is only reliable during the initial stages of lipid

oxidation because the peroxide value increases to a maximum and then decreases as storage

time increases. All four treatment samples stored in the polypropylene woven sacks exhibited

the highest values of peroxide although they were stored under the same environmental

conditions. One possible reason for higher peroxide reading is that the sacks had oxygen and

this facilitated lipid oxidation as compared to hermetic packs.

Statistical analysis (Table 4.2) of quality (Peroxide value) of the peanut samples for

the two storage systems HPO and PS were significantly different from each other at P<

0.0001, indicating that the storage system utilized may have influenced the peroxide levels in

the samples. There were no significant differences in peroxide values between samples stored

in Kumasi and Tamale (P-value=0.56), although it was expected that samples from the

Northern region would be higher because of higher temperatures, compared to Kumasi (Table

4.3). Kumasi samples had relatively higher aflatoxin levels; and it is known that aflatoxin is

highly correlated with peroxide value (Chapter 3), hence high peroxide values for the Kumasi

samples.

Furthermore, there was a very good correlation (r= 0.84, r2

=0.70, RMSE-=1.34, P-

value <0.0001) between aflatoxin production and quality (peroxide value) for inoculated

samples in polypropylene woven sacks (fig. 4.3). As reported by Pattee and Sessoms (1967),

increase in fat acidity was highly correlated with visible A. flavus growth and aflatoxin

production. Overall, there was no significant difference in peroxide values of both raw

inoculated samples and partially roasted non-blanched peanut samples (P-value = 0.13). This

similarity in peroxide values may be due to the fact fungi growth aids in quality.

Deterioration and elevated heat also helps in lipid oxidation. This is confirmed by Nakagawa

and Rosolem (2011), as cited in Santo et al (2016) that high temperatures and relative

humidity can also contribute to the deterioration of seeds because of lipid peroxidation. The

rest of the pre-treatment samples were significantly different from one another at P <0.0001.

70

Comparing peroxide values with respect to the raw-inoculated samples in polypropylene

woven sacks, the raw clean treatment samples in both polypropylene woven sacks and

hermetic bags were best in reducing oxidation by 31% and 42%, respectively. This was

followed by partially-roasted-blanched samples with 30%, and 28.1% for partially roasted

non-blanched samples in hermetic packages.

71

Fig. 4.2. Peroxide Value results of the raw clean(a), raw inoculated(b), partially roasted not blanched (c) and partially roasted blanched (d)

peanut treatments in the two packaging (PS, and HPO) systems

72

Fig. 4.3. Correlation matrix of aflatoxin production and quality (Peroxide and p-Anisidine

value) for shelled raw infested peanuts.

P-Anisidine Value

P-Anisidine trends for the four pre-storage treatments (raw-clean, raw-inoculated,

partially-roasted-not blanch, and partially roasted blanch) in the two packing systems

(Polypropylene woven sacks and hermetic bags with oxygen absorbers) are presented in

figures 4.3 (a, b, c, and d). The amount of p-Anisidine value also increased with storage time,

and the values were higher in the samples stored in the polypropylene woven sacks (PS) as

compared to the hermetic bags with oxygen absorber (HPO). It is known that this hermetic

system suppresses oxidation because of lack of oxygen in the sack. The slight increase of p-

Anisidine values noted during the storage period may be due to increased lipid oxidation

caused by factors such as light and elevated temperatures (Kamal-Eldin, 2006; Merrill et al,

2008).

73

The mean of p-Anisidine values of peanut samples indicated that there were

significant differences in the values between the two packaging systems at p- value < 0.0001

(Table 4.2). Thus the p-Anisidine value in peanuts could have been affected by the packing

system. For the four pre-treatments, the partially-roasted-blanched peanut samples were

statistically not different from the partially roasted not blanched and raw inoculated samples

at p values = 0.27 & 0.14, respectively. The partially roasted not blanched peanuts in the

polyethylene sacks recorded the worst quality degradation of 11.68 meq/kg, as compared to

raw clean samples in hermetic bags recording 5.12 meq/kg. Hence, hermitic bags were the

best in maintaining quality. P-Anisidine values for the partially roasted blanch samples in

polypropylene woven sacks were expected to be higher than those for the partially roasted

non-blanched samples because of the testa (seed coat) as an anti-oxidant (Constanza et al,

2012) which is supposed to protect peanuts from oxidation. However, these not blanched

samples had higher aflatoxin levels, and these in turn can be a factor for higher oxidation (p-

Anisidine) levels

.

74

Fig. 4.4 (a, b, c, & d)- P-Anisidine Value results of the raw clean(a), raw inoculated(b), partially roasted not blanched (c) and partially-roasted-

blanched (d) peanut treatments in the two packaging systems (PS, and HPO).

75

Aflatoxin levels also had a strong correlation with peroxide and p-Anisidine values

(Chapter 3). There was no significant difference in quality (p-Anisidine value) between

peanut samples stored in Ashanti and Northern regions of Ghana at P>0.05, but as explained

in the peroxide value section, values for samples stored in the Northern region were expected

to be higher than for samples from Ashanti region because of higher storage temperatures,

ranging from 17.6°C–42.5 °C in Northern region and 24.2-37.9°C in Ashanti region (Table

4.3), and also because of higher aflatoxin levels in Ashanti region. The p-Anisidine values of

raw clean samples in polypropylene woven sacks (6.19 meq/kg) and hermetic bags (5.12

meq/kg) best maintained quality. This was followed by partially-roasted-blanched peanut

samples in hermetic bags (7.04 meq/kg) and then partially roasted not blanched samples in

hermetic packs followed (8.27 meq/kg), with raw-inoculated recording 8.48 meq/kg

recording the least (Table 4.2).

There was a strong correlation (r= 0.88, r2=0.78, RMSE-= 1.08, p-value <0.0001)

between the aflatoxin production and the quality index (p-Anisidine) (Figures 4.3). The trend

line indicates that the higher the aflatoxin produced, the higher the quality deterioration.

Comparing p-Anisidine values of the raw-inoculated samples in polypropylene woven sacks

to those of the rest of the treatment combinations, raw clean treatment samples in both

polypropylene woven sacks and hermetic bags were best in reducing oxidation by 38% and

49 %, respectively; followed by partially-roasted-blanched in hermetic bags samples

recording 29.88% and 17.6% for partially roasted not blanched samples in hermetic bags

(Table 4.4)

b.) In-shell

Aflatoxin Production

The production of aflatoxin in in-shell peanut samples increased with increased

storage time for infested peanuts in polypropylene woven sacks (Figures 4.5 a & b). The

hermetic bags with oxygen absorbers likely suppressed the fungi growth and aflatoxin

production because of the lack of oxygen in the packs. Oxygen absorbers are capable of

removing or reducing the oxygen concentration in a package (Miltz & Perry, 2005) and this

might have helped to limit the growth of aflatoxigenic fungi.

There was a significant difference in aflatoxin production between the raw clean

peanut samples stored in both HPO and PS packs at 95% significance level. Samples in the

polypropylene woven sacks had higher aflatoxin levels (Table 4.5). This may mean that the

samples already had fungi infection and produced aflatoxin during storage. The same trend

76

was observed in raw inoculated samples packed in HPO and PS, with means of 58.97 ppb for

samples in hermetic pack with oxygen absorbers and 74.12 ppb for samples in polypropylene

woven sacks. If proper post-harvest practices are followed, in-shell peanuts may be stored for

a period of at least six months without fungi or insect treatments if the pods are not damaged

(Santos et al, 2016). A study by De Camargo et al (2012) found no fungi in in-shell samples

kept in storage for months in polyethylene plastic bags.

Hermetic bags with oxygen absorbers were able to prevent aflatoxin production by

suppressing the growth of the A. flavus fungi (Navarro, et al, 2012) in raw inoculated by

about 20%, compared to the raw inoculated samples in polypropylene woven sacks (Table

4.6). It is also known that this type of storage kills insect and mite pests, and prevents aerobic

fungi from growing (Weinberg et al., 2008). The aflatoxin concentration of (54.32 ppb) found

in in-shell peanut samples stored in farmers‘ storage in Ejura (Ashanti region) was slightly

higher than the concentration obtained at Daire (Northern region, 47.32 ppb). This could be

attributed to the difference in the environmental conditions of the two locations. In the

Northern region temperatures and relative humidity ranged from 17.6°C–42.5°C and l 11-

88%, respectively. In the Ashanti region the temperatures ranged from 24.2°C–37.9°C and

relative humidity, 16- 94% (Table 4.3).

Fig. 4.5 (a&b) Effects of aflatoxin production for raw clean (Raw- Cl) and raw inoculated

(Raw-Inf) in-shell peanuts in polypropylene woven sacks (PS) and hermetic bags with oxygen

absorber (HPO)

77

Table 4.5. Mean aflatoxin, peroxide and P-Anisidine values of Raw-Clean, and raw-

inoculated samples in the Polypropylene woven sacks-PS, and hermetic pack + 02-HPO

packaging systems.

Aflatoxin

Treatment/ Package HPO PS Overall mean

Raw Clean 30.52 ± 1.80d 39.70 ± 1.80

c 35.11 ± 1.27

b

Raw Inf 58.97 ± 1.80b 74.12± 1.80

a 66.54± 1.27

a

Overall mean 44.74 ± 1.27 b 56.91 ± 1.27

a

Peroxide Value

Raw Clean 8.05 ± 0.19 d 9.47 ± 0.19

b 8.76 ± 0.13

b

Raw Inf 8.58 ± 0.19 c 10.43± 0.19

a 9.51± 0.13

a

Overall mean 8.31 ± 0.13 b 9.95 ± 0.13

a

P-Anisidine Value

Raw Clean 4.04± 0.11 c 4.68 ± 0.11

b 4.36 ± 0.07

b

Raw Inf 4.88 ± 0.11 b 5.70± 0.11

a 5.29 ± 0.07

a

Overall mean 4.46 ± 0.07 b 5.19± 0.07

a

Levels not connected by the same letter are significantly different

Table 4.6. Percentage reduction of aflatoxin, peroxide value and P- Anisidine values of the

two peanuts conditions (raw clean, raw inoculated in Polypropylene woven sacks-PS,

hermetic pack+02-HPO with respect to the raw-inoculated samples in polypropylene woven

sacks.

Treatment/ Package Aflatoxin/ ppb Peroxide Value/

(meq/kg)

P- Anisidine

Value/(meq/kg)

Raw-Cl/HPO 58.8 22.8 29.1

Raw-Cl/PS 46.4 9.2 17.9

Raw-Inf/HPO 20.4 17.7 14.4

Raw-Inf/PS 0.0 0.0 0.0

78

Peroxide Value

The production of peroxide in in-shell peanuts was highest in the raw inoculated

treatment; its production was however cumulative with the passage of storage time (Figures

4.6 a & b). Peroxide values from samples in polypropylene woven sacks were higher than the

same from samples stored in hermetic bags with oxygen absorbers. There were significant

differences, based on the pre-storage treatments and types of package used for storage at 95%

confidence interval (Table 4.5). Thus the treatments and packaging could have had an

absolute bearing on quality in terms of production of peroxide in in-shell peanuts stored

under the prevailing Ghanaian conditions.

The raw inoculated samples in the polypropylene woven sacks recorded the highest

values because microbial growth rate is responsible for food quality degradation (Peltzer et

al., 2010; Suppakul et al., 2011) and also because of the presence of oxygen. The hermetic

packs likely reduced quality deterioration of these aflatoxigenic inoculated samples by

17.7%, meaning that hermetic bags are a good package for storing peanuts, controlling

aflatoxin production and quality degradation.

With in-shell peanuts too, there was a good correlation (r=0.86, r2=0.74, RMSE-

=0.64, P-value=0.0007) between aflatoxin levels and peroxide value (Figure 4.8). There was

no significant difference in overall quality deterioration (peroxide values) for samples stored

in Ashanti and Northern regions of Ghana at p-value=0.28.

79

Fig 4.6 (a & b) Effects of peroxide value for raw clean (Raw- Cl) and raw inoculated (Raw-Inf) in-shell peanuts in polypropylene woven sacks

(PS) and hermetic bags with oxygen absorber (HPO)

Fig 4.7 Effects of P-Anisidine value for raw clean (Raw- Cl) and raw inoculated (Raw- Inf) in-shell peanuts in polypropylene woven sacks (PS)

and hermetic bags with oxygen absorber (HPO).

80

Fig. 4.8. Correlation matrix of aflatoxin production and quality (Peroxide and p-Anisidine

value) of peanuts for in-shell raw infested peanuts.

P-Anisidine

Overall, the rate of quality deterioration for the raw-clean and raw-inoculated samples

was low, possibly because the shells or pods protected the kernels and helped in reducing the

rate of oxidation as compared to shelled peanuts. Although lipid oxidation was minimal, the

hermetic packaging system was able to protect the samples from quality deterioration for

both of the treatment conditions, by 14.4 % for raw inoculated and 29.1% for raw clean

samples (Table 4.5). There were significant differences in both treatments (Raw Cl and Raw

Inf) and packaging systems (HPO and PS) at P- value < 0.05 (Table 4.4), indicating that the

effect of p-Anisidine on the storage of in-shell peanuts was dependent on the treatment type

and packaging used for storage.

Also, there were significant differences in overall quality deterioration (p-Anisidine

values) for samples stored in Ashanti and Northern regions of Ghana at p-value=0.04. As

with the treatment conditions reported earlier, there was a strong correlation (r=0.78, r2=0.62,

81

RMSE-=0.83, P-value <0.0001) between the aflatoxin production and the quality index (P-

Anisidine) as presented in Figure 4.8.

Conclusion and Recommendation

Sorting could be a very good method for reducing aflatoxin levels before and during

storage. Also, partially roasting, and blanching peanuts, can kill the aflatoxigenic fungi and

halt aflatoxin production during storage, and also increase the effectiveness of peanut sorting;

thus aiding in reducing or eliminating aflatoxin levels along the peanut value chain. However,

hand-sorting consumes time and adds additional preparation cost.

From the study, it was concluded that hermetic storage of peanuts can suppress

aflatoxigenic fungi growth and aflatoxin production under ambient conditions. Therefore, to

reduce and also prevent aflatoxin production in storage under favorable environmental

conditions, it would be best to store peanuts hermetically, since sorted peanuts can still

produce aflatoxin if there is even the least trace of fungi on them.

Raw clean peanuts can best maintain quality during storage but might still have high

aflatoxin levels. To have low levels of aflatoxin before, during, and after storage, as well as to

maintain peanut quality, it would be best to partially roast peanuts, blanch them and sort out

the infested and discolored ones, and hermetically store them.

The perfect window for the field research was missed due to timely access to money,

and so the time for the study (December to June) did not favor aflatoxin production very

much. Therefore, I recommend that research work be done for a whole year under Ghanaian

environments starting from July-August, because that is when actual peanut storage time

starts and also when the temperatures and humidity are conducive to aflatoxin production.

82

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Chapter 5: Switching to Hermetic Storage of Peanuts to reduce Aflatoxin in Ghana:

Impact on Farming and Marketing Profitability.

Abstract:

In Ghana and in other countries Sub-Saharan Africa, postharvest loss in peanuts, is

primarily due to aflatoxin fungi infestation. Polypropylene woven sacks have been use for

storing peanuts. Peanuts stored in polypropylene woven sacks are susceptible to fungal and

aflatoxin contamination because these are not airtight. Present studies have shown that

hermetic packs can be used effectively to suppress fungi growth, aflatoxin production and

quality deterioration in stored peanuts. The objective of this study aimed to determine if the

new hermetic storage technology is more profitable than existing storage methods, before

recommending it for peanut farmers and traders.

An enterprise budget was created to determine the profitability of the current practice

of storing peanuts in polypropylene woven sacks. This was followed by a partial budget

analysis of how switching to storage in hermetic bags affected production costs and revenues,

when all production factors were kept constant.

Switching to the new technology improved profitability in a 10% and 5% for farmers

and traders, respectively. Although production cost increased by about 7% for farmers and

3% for traders. With the switch to hermetic storage technology, the higher quality peanuts

which could result can be sold at a higher price. While the new storage technology improves

the farmer and trader profitability, it has the potential reduce the incidence of various

ailments that have been attributed to aflatoxins. Hence, the local production and marketing of

a hermetic storage system should be encouraged, along with the active creation of awareness

of their benefits in reducing the incidence of aflatoxins.

Keywords: Aflatoxin, Peanuts, Hermetic Storage, Polypropylene woven sacks, Partial

budget, Enterprise budget, Ghana.

Introduction

Approximately one-third of food produced for human consumption is lost globally,

and this amounts to about 1.3 billion tons per year (Gustafson et al., 2011). Postharvest losses

for grains are about 18-25 % of the total amount of grains produced annually (African

Postharvest Losses Information System, 2013). In Sub-Saharan Africa, postharvest grain

losses account for US$4 billion (World Bank, 2011). Based on the latest estimates from FAO

(2015), about 795 million people remain undernourished globally. Among the

undernourished, about 780 million people, are in developing regions and about 239 million

88

are in Sub-Saharan Africa (FAO, 2010). These losses could adequately meet the minimum

annual food requirements of 48 million people (World Bank, 2011). Also according to

African Postharvest Losses Information System-APHILS (2013), the estimated loss of grains

in 2010 was about 15 million metric tons. These losses could adequately meet the minimum

annual food requirements of 48 million people (World Bank, 2011). Maize and peanuts

(groundnuts) are dietary staples for the majority of the rural poor in Sub-Saharan Africa

(SSA), and normally have high postharvest due to mold contamination (Wagacha &

Muthomi, 2008).

Peanuts contain a high amount of fats and proteins, and can be used in curbing protein

and energy malnutrition because they contain all the essential amino acids needed for normal

body growth and metabolism (Pelto & Armar- Klemesu, 2011; Eshun et al., 2013). Peanuts

are dietary staples for the majority of the rural poor in Sub-Saharan Africa (SSA). In Ghana

and other countries in Sub-Saharan Africa postharvest losses of major staple crops (maize and

peanuts) are mostly due to mold or fungus infestation. Chief among these infesting fungi are

members of the Aspergillus genera, mostly A. flavus and A. parasiticus, whose metabolites

produce aflatoxin.

Aflatoxin poisoning is common in this region (Wagacha & Muthomi, 2008). The

primary disease associated with this type is hepatocellular carcinoma (HCC, or liver cancer)

and is the third-leading cause of cancer death globally (World Health Organization, 2008).

Aflatoxins have also been linked to stunted growth in children and immune system disorders

(Jolly et al., 2008). It also creates a trade barrier, which results in great economic losses to

exporters and countries at large. Agyei (2013) reported that, the European Union regulation

on aflatoxins alone costs Africa $750 million annually in the exportation of cereals, dried

fruits, and nuts. Aflatoxin contamination of peanuts in African markets, in general, is quite

high. William, et al. (2011) reported, from a survey conducted on local African markets, that

40% of the commodities found there exceeded permissible aflatoxin levels (i.e., far in excess

of the international standard of 10–20 ppb), and that an estimated 4.5 billion people in

developing countries are at risk of uncontrolled or poorly controlled exposure to aflatoxins.

These high levels are caused by improper postharvest practices, storage and adverse

climatic conditions are predisposing factors for post-harvest aflatoxin contamination.

Unfortunately conditions of temperatures ranging from 26.7-43.3°C with a relative humidity

of 62–99%, (Sumner & Lee, 2012) coincides with the environmental conditions of most Sub

Saharan African countries of which Ghana is no exception. This makes aflatoxin

89

contamination in storage in these regions compounded as a result of this combination of heat

and high humidity (Hell et al., 2010; Guchi, 2015).

Apart from the condition of the commodity to be stored, other important factors to

consider are the package material and the storage structure. In Ghana, peanuts have been

observed to be packaged in polypropylene woven sacks and jute sacks and stored under

different storage structures. In market places, shelled peanuts are mostly packaged in jute

sacks or polypropylene woven sacks and stored in cement buildings, or wooden or aluminum

sheet structures. Ghanaian farmers store in-shell peanuts in bulk in thatch houses, mud houses

with thatch roofing, or cement buildings. Some also package them in these polypropylene

woven sacks in store on verandas, wooden or cement structures (Darko, unpublished data).

New storage practices, such as the use of metal or cement bins are an improvement

over traditional storage methods. However, high costs and access to improved materials

remain major constraints against their adoption by small-scale farmers (Hell & Mutegi,

2011). Polypropylene woven sacks are commonly used, but because these are not airtight,

peanuts are still susceptible to fungal and aflatoxin contamination (Hell et al., 2000; Udoh et

al., 2000). Oxygen is one major factor for aflatoxigenic fungi growth and aflatoxin

production, and by cutting the oxygen source, it will help kill the fungi and control aflatoxin

production. Hermetic packaging is the type of storage that is airtight which stops oxygen and

water movement between the outside atmosphere and grain stored inside it. This packaging

type can protect peanuts from molds and aflatoxin contamination (Paramawati et al., 2006).

Studies have indicated that the storage of peanuts in hermetic bags resulted in minimized A.

flavus fungus growth and aflatoxin production, and maintained the quality of peanuts

(Chapter 3 and Chapter 4).

A laboratory study on the storage of peanuts revealed that hermetic bags with oxygen

absorbers were able to reduce aflatoxin production in artificially inoculated shelled peanuts

by 83.77%, and reduced quality deterioration by about 60% compared to peanuts in

polypropylene woven sacks (Chapter 3). A similar study in Ghana also recorded similar

results, with the hermetic bags plus oxygen absorbers reducing aflatoxin levels of shelled and

in-shell peanuts by about 21% and 20%; quality deterioration by 23.02% (see Chapter 4). The

findings for aflatoxin production and quality deterioration under the Ghanaian environment

was lower than expected, and was attributed to lower moisture content (5-11%). The relative

humidity was also very low, with an overall average of 49% and a range of 11-94% from

90

December-June of the storage period in the two study areas in Ghana. The hermetic bags with

oxygen absorbers were able to reduce both aflatoxin production and quality deterioration.

This technology, therefore, has the potential to reduce aflatoxin levels and help

maintain quality, but is expected to increase the total costs of production and marketing, since

it is more expensive than the current storage technologies. There have been other new storage

technologies, such as the use of cement or metal bins as mentioned earlier, which offer an

improvement over traditional storage methods to combat aflatoxin. However, because of high

costs and accessibility, the adoption of these improved technologies by farmers is still limited

(Hell & Mutegi, 2011). Reduction in post-harvest losses is one of the keys to improving profit

(Waliyar et al, 2015). Moreover, post-harvest management, especially storage, is vital for

increasing food availability without the need for additional production (Kimatu et al., 2012).

The cost effectiveness, sustainability, and technical feasibility of hermetic storage and

hermetic bags need to be evaluated with regard to local contexts and practices as part of

devising strategies for halting post-harvest aflatoxin contamination. It can be deduced from

the above reasons that there is the need to determine if the hermetic storage technology is

more profitable than existing storage methods, before recommending the to peanut farmers

and traders.

Materials and Methods

Study Area

The study was conducted in the Northern and Ashanti region in Ghana. They were

selected for data collection because they are noted for high peanut production and marketing.

The three separate administrative sub-regions in Ghana‘s Northern zone (Northern, Upper

West and Upper East) produce about 92% of the country‘s total production (MoFA, 2012).

This region falls in the Guinea Savannah agro-ecological zone, which has a mono-modal

rainy season that starts in April/May and ends in September/October. This area is recognized

among as a major peanut producer in West Africa Angelucci and Bazzucchi (2013).

Again, according to the Angelucci and Bazzucchi (2013), most of the peanuts produced in

northern Ghana are consumed in urban areas, especially in the Ashanti and Greater-Accra

Regions. The Ashanti Region is one of the key trading areas for peanuts in Ghana.

91

Budget Analysis

In the present study, enterprise and partial budgets were used to evaluate the

profitability of using hermetic storage for farmers and traders in Ghana. An enterprise budget

predetermines the profitability of the indicated venture, in a representative year or specified

period (Engle & Neira, 2005). The budget compares revenues to costs for the stipulated

period, using typical or average values and prices. A partial budget considers a particular

scenario in which costs and revenues alter the outcome of an enterprise budget (Engle &

Neira, 2005). Essentially, a partial budget is used to determine if an additional proposed

output, activity, or technology will either increase or decrease the profitability determined in

an enterprise budget.

For the analyses of budgets, peanut farmers and traders in the Northern and Ashanti

regions of Ghana were identified with the help of the Ministry of Food and Agriculture

(MoFA). Informal meetings with farmers, middlemen and women, and traders from both

regions were used to gain insights into, among others: farmers‘ responses for average size and

cost of farmland; seed quantity and cost; quantity and cost of fertilizers and insecticides;

materials and equipment; labor cost, including the owners‘; polypropylene woven sacks and

storage; quantity of peanuts harvested per year; peanut selling price, on one hand; and on the

other, trader responses for quantity and cost of peanuts purchased from farmers; labor cost,

including traders‘ labor; polypropylene woven sacks and storage; quantity and (cost and

selling) price of peanuts. The data collected was then summarized in Microsoft Excel 2013.

An enterprise budget was done to determine the profitability of the current practice of

storing peanuts in polypropylene woven sacks. This was followed by a partial budget analysis

of how switching to storage in hermetic bags would affect production costs and revenues,

when all production factors were kept constant. Enterprise and partial budgets based on the

survey data were developed for the average-size/typical peanut farmer (with 2 acres of

farmland) and trader (1.3 tons of peanuts), for a one-year operation.

Straight-line depreciation and interest were applied to farm equipment, in terms of

useful life and the salvage value (Table 1). Labor costs included land preparation, planting,

harvest, drying, sorting, storage, bagging, transportation costs and owner‘s labor. The survey

did not include any equipment used by peanut traders. However, unforeseen expenses of 5%

of operating costs were added for any expenditure that might not have been captured by the

survey for both farmers and traders.

Secondary data on variables such as Bank of Ghana‘s Interest Rate and US Dollar

($)–Ghana Cedi (¢) exchange rate, were obtained from secondary sources (Bank of Ghana

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2016; Trading Economics 2016). All reported rates are for August 2016. The data were

analyzed with Graph-pad Prism statistical software version 5.

Results and Discussions

Analysis of Peanut Farmer Profitability

Two different enterprise budgets (and partial budgets) are presented in Tables 5.1–5.4

for peanut farmers and traders in Ghana. Tables 5.1 and 5.2 show enterprise and partial

budgets, respectively, developed for the average peanut farmer who sells shelled peanuts to

middlemen (or women).The size of cultivated peanut farmland used for the analyses was two

(2) acres because this is the size of the farm in the area . A farm of this size harvest an

average of approximately 1.3 tons of in-shell peanuts annually. The harvested in-shell peanuts

are then stored and sold only when money is needed. However, it was noted that the farmers

can increase the value (i.e. revenue) of peanuts by storing longer. The typical storage period

ranged from 2-8 months. Out of the total 1.3 tons of in-shell peanuts harvested, about 70% (1

ton) is normally recovered after shelling by farmers. This confirms with literature, where it is

reported that the composition of peanuts is 21-29% external hull or shell and 71-79% kernels

or seeds (Van Doosselaere, 2013). In Ghana, peanuts are normally sold as approximately 80-

100 kg packages of in-shell grains in polypropylene woven sacks; but farmers shell their

peanuts only when they are ready to sell them.

Normally, a bag of average quality peanuts (in terms of visible molds, discolored

grains, or mechanical damage) sells for GH¢ 350; and very good quality peanuts (with no

visible molds, discolored grains, or mechanical damage) sell for about GH¢ 400.00 per bag.

Hence, it was clear that Aflatoxin infestation of peanuts reduces both revenue and

profitability of both peanut farming and trading. This result is also corroborated by Hell et al.

(2003), Kpodo and Bankole (2008), and N‘dede (2009), who concluded that the type of

storage structure and length of storage significantly affect the incidence of aflatoxin

infestation in harvested peanuts, which, in turn, leads to losses.

The average cost of production totaled about GH¢ 2,000.00, with labor cost

(including owner‘s opportunity cost) constituting 60% of the sum (Table 5.1). Gross revenue

from the sale of shelled peanuts amounted to about GH¢3,000.00, resulting in annual profits

of approximately GH¢ 1,000; which represents about 44% profit from the cost of production

(Table 5.1).

For partial budgeting, the variables whose values changed with hermetic bags were:

peanut selling price, storage cost, interest on operating capital, equipment depreciation, and

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interest (Table 5.2). A shift from polypropylene woven to hermetic sacks appreciated the

selling price to approximately GH¢ 350-400, because this type of packaging gives higher

quality peanuts. Hermetic packaging suppresses mold growth, aflatoxin production, and

quality deterioration during storage; thus, yielding relatively lower aflatoxin levels and higher

quality peanuts than peanuts in polypropylene woven sacks (Navarro et al 2012; Chapter 3).

The new storage technology (hermetic packaging system) increased total costs of

production by 7% (Table 5.2). However, there was a 14% increase in revenue and, therefore,

an overall profit of 54% (Tables 5.1 and 5.2). Lower quality peanuts attract lower revenue,

which is similar to the findings of N‘dede (2009), who reported that lower peanut quality (in

terms of visible mold, lower aflatoxins, and physical damage) in many Sub-Saharan

countries, such as Ghana, commands lower pricing the market, and vice versa.

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Table 5.1. Enterprise budget for a 2-acre peanut farm, using polypropylene woven bag

storage, and operating for one year.

Category Unit Quantity Unit Price

(GHC)

Total

(GHC)

GROSS RECEIPTS

Peanut (bag; 100kg) bags 9.00 350.00 3,150.00

Total Receipts 3,150.00

VARIABLE COSTS

Seeds bags 1.00 200.00 2,00.00

Insecticides # 2.00 25.00 50.00

Casual labor ( land preparation) GH¢ 2.00 80.00 160.00

Casual labor (harvesting and -

transportation)

GH¢ 2.00 235.00 470.00

Owner's labor (opportunity cost) month 12.00 50.00 600.00

Bags # 27.00 2.50 67.50

Packaging charges (bag sewing) # 27.00 1.00 27.00

De-shelling GH¢ 27.00 10.00 270.00

Storage month 6.00 5.00 30.00

Interest on operating capital GH¢ 487.37 0.26 126.72

Unforeseen expenses (5% of TVC) GH¢ 1.00 100.06 100.06

A. Total Variable Costs GH¢ 2,101.28

FIXED COST

Equipment depreciation GH¢ 8.80

Equipment interest GH¢ 12.48

Interest on land (opportunity cost) GH¢ 61.67

B. Total Fixed Cost GH¢ 82.95

C. TOTAL COSTS GH¢ 2,184.22

Above Variable costs GH¢ 1,048.72

Above Variable costs Per Acre GH¢/acre 524.36

Net GH¢ 965.78

Per Acre GH¢ 482.89

Breakeven Yield

Above variable costs bags/acre 3.00

Above total costs bags/acre 3.12

Breakeven Price

Above variable costs GHC/kg 233.48

Above total costs GHC/kg 242.69

*Opportunity cost of investment in Land=Investment in Land*18.5% interest rate

*GH¢3.94 equals USD $1.00 (Trading Economics, 2016).

*Ghana interest rate is 26% (Bank of Ghana, 2016)

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Table 5.2. Partial budget analysis for a peanut farmer switching from polypropylene woven sack to hermetic storage

VALUE OF PARAMETERS THAT CHANGE

Polypropylene woven Sack (GH¢) Hermetic Sack (GH¢) Change in Cost / Revenue (GH¢)

I. Peanut Sales (bag; 100kg) 3,150.00 (350*9bags) 3,600.00 (400* 9bags) 450.00

II. Oxygen absorber 0 27.55 27.55

III. Packaging 67.50 119.68 52.18

IV. Interest on operating capital 126.72 111.99 14.72

V. Equipment depreciation (crib) 8.80 32.00 23.20

VI. Equipment interest ( crib) 12.48 62.40 49.92

A. Additional Costs (II, III, V and VI) 152.85

B. Additional Revenue (I) 450.00

Net Change in Profit (B-A) 297.15

* Therefore, using hermetic bags increase profits by GHC 297.15, when all the other factors, such as size of operation, are kept constant.

*GH¢3.94 equals USD $1.00 (Trading Economics, 2016).

*Ghana interest rate is 26% (Bank of Ghana, 2016).

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Profitability Analysis of Peanut Traders

Middle men normally buy peanuts from farmers and sell them to traders. As

mentioned earlier, they buy average quality peanuts for approximately GH¢350.00. They then

sell them to the traders at a cost of approximately GH¢ 380.00. The traders, in turn, sell these

average quality peanut for approximately GH¢ 450.00, making an average net return (profit)

of GH¢15 per bag (Table 5.3). The major costs incurred by the traders in their business

include: purchasing of shelled peanut, shop rental, and owner‘s labor. On average, these

operational costs summed up to about GH¢ 44,000.00, which represents about 97% of the

gross revenue received (GH¢ 45,000.00); yielding a profit of approximately 3%. Under this

scenario, it is observed that a peanut trader breaks even at around GH¢435/bag for selling 100

bags of peanuts per annum.

With the introduction of hermetic packs and storage cribs for the prevention of rodent

damage, partial budgeting with hermetic packs indicated additional operating costs of about

GH¢1,330.00. The traders interviewed mentioned that they get a premium price for clean (no-

mold) peanuts after storage, and that they prefer storing their products and selling them in the

lean season for higher prices. However, they usually had to sell quickly to avoid mold

infestation. Polypropylene woven sacks are susceptible to fungal and aflatoxin contamination

(Hell et al., 2000; Udoh et al., 2000) and hermetic packaging could protect peanuts from the

aforementioned molds and aflatoxin contamination (Paramawati et al., 2006).

A bag of clean peanuts (free from molds and not discolored) sold for about

GH¢490.00 instead of GH¢450.00, and this increased revenue by about 9%; yielding an

overall profit of about 3.4-8.4% (Tables 5.3 and 5.4). The lower profit for peanuts stored in

polypropylene woven sacks could be attributed to the fact that, higher aflatoxin levels reduce

peanut quality. They can cause economic and trade difficulties at almost all stages of peanut

marketing; presenting an inverse relationship between aflatoxin levels and profit (Latha et al.,

2007). In a nutshell, there was an annual return of 9% (approx. GH¢ 4,000.00) on every

GH¢45,000.00 invested in the peanut trading business using hermetic bags and cribs. Clearly,

this new storage technology can reduce post-harvest losses and make peanut storage more

economically viable.

Evaluating the Shift from Polypropylene woven Sacks to Hermetic Packs

Comparing the profitability of the peanut business for farmers and traders in Ghana, it

was realized that there are positive net returns for both groups (Tables 5.1 and 5.3). With the

use of the traditional storage method (polypropylene woven packaging), the average peanut

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farmer makes a profit of about GHC 1,000.00 (44%), whilst the trader makes about GHC

1,500.00 (about 3%); signifying that the average peanut farmer makes higher returns on

investment than the trader in Ghana (fig. 1). Middlemen also drain some of the profits of the

traders if peanuts are not sold directly to them by the farmers.

With the introduction of the new technology (hermetic storage), the two groups both

had increases in profits. The trader had a 5% increase in profit whilst the farmer had a 10%

increment (fig. 1). A shift to the hermetic storage technology increased the farmer‘s profit to

about GH¢1,200.00 (i.e., 54% returns on cash investment) whilst the trader had an increase in

profits of approximately GH¢ 4,000.00, which translates to about 9% profit returns on cash

investment (fig.1).

The major difference between the polypropylene woven sacks and the hermetic packs,

in terms of storage time, is that as storage time increases, peanut quality deteriorates faster in

polypropylene woven sacks; hence, the decrease in returns. This is in agreement with

findings from N‘dede (2009), who reported that there is an inverse relationship between

peanut selling price and length of storage period. Consumers normally offer low prices for

peanuts of low quality, and this can account for be the reason why revenues increased with

the use of hermetic storage which maintains quality with storage time.

One methodology used by these farmers and traders when consumers are demanding

mold- and aflatoxin-free peanuts is sorting. Sorting has, however, been found to result in

increased labor costs, reduced quantities and, ultimately, reduced profits as confirmed by

Awuah et al., (2000) who reported that peanut farmers and traders throw away up to 15% of

their produce after sorting, which reduces their final revenue. In this study, the new storage

approach to solving the high levels of fungus and aflatoxin produced during storage was

found to inhibit the growth of the fungi and to prevent insect and mice infestation. This

approach proved to be effective and also economically motivating. Though the adoption of

the new storage technology increased cost, it was found to be more profitable since the higher

prices outweighed the higher cost. Therefore, using polypropylene woven sacks could be

more profitable than sorting.

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Table 5.3. Enterprise budget for a peanut trader operating for one year, with sales output of

100 bags.

Category Unit Quantity Unit Price Total (GHC)

GROSS RECEIPTS

Peanut (bag; 100kg) bags 100.00 450.00 45,000.00

Total Receipts GH¢ 45,000.00

VARIABLE COSTS

Peanut (bag; 100kg) bags 100.00 380.00 38,000.00

Shop Rental GH¢ 1.00 216.67 216.67

Owner's labor (opportunity cost) month 12.00 50.00 600.00

Interest on operating capital GH¢ 10,205.00 0.26 2,653.30

Unforeseen expenses (5% of TVC) GH¢ 1.00 2,095.17 2,095.17

Total Variable Costs GH¢ 43,512.71

TOTAL COSTS GH¢ 43,512.71

Returns Above Variable costs GH¢ 1,487.29

Returns

Net GH¢ 1,487.29

Per Bag GH¢ 14.87

Breakeven yield

Above variable costs bags 96.69

Above total costs bags 96.69

Breakeven price

Above variable costs GHC/bag 435.13

Above total costs GHC/bag 435.13

*Ghana interest rate is 26% (Bank of Ghana, 2016).

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Table 5.4. Partial budget analysis for a peanut trader switching from polypropylene woven

storage to hermetic packaging system

VALUE OF PARAMETERS THAT CHANGE

Polypropylene

woven Sack

(GH¢)

Hermetic Sack

(GH¢)

Change in

Cost / Revenue

(GH¢)

I. Peanut Sales (bag; 100kg) 45,000.00 49,000.00 4,000.00

II. Packaging (sack and sewing) 0 936.50 936.50

III. Interest on operating capital* 2,624.01 2,687.31 63.31

IV. Equipment depreciation 0 112.00 112.00

V. Equipment interest 0 218.40 218.00

A. Additional Costs (II, III, IV and V) 1,330.21

B. Additional Revenue (I) 4,000.00

Net Change in Profit (B-A) 2,669.79

* Therefore, using hermetic bags will increase profits by GHC 2,669.79, when all the other

factors, such as size of operation, are kept constant.

Fig. 5.1 Comparism of the percentage profit returns of using polypropylene woven sacks and

hermetic bags for peanut farmer and trader in Ghana

100

Conclusions and Recommendations

This study revealed that peanut business in Ghana is generally profitable. Besides,

farmers and traders stand to make additional revenue and profits from switching from

traditional packaging (mostly in polypropylene woven sacks) to hermetic storage. Although

the latter system incurs additional investment costs (storage cribs, hermetic packs and oxygen

absorbers). Nonetheless, the increase in revenue from sales of higher quality peanuts far

outweighed the additional costs incurred.

The local production and marketing of a hermetic storage system should be

encouraged, along with an active creation of awareness of their benefits in reducing the

incidence of aflatoxins and post-harvest losses. A switch to the new storage technology will

not only have positive implications for farmer and trader profitability, it will also reduce the

incidence of various ailments that have been attributed to aflatoxins. In considering the

significant national economic impacts of aflatoxins, peanut farmers and traders could be

assisted through various financing schemes to acquire the new technology.

101

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Chapter 6: Summary and Conclusions

Peanuts in Ghana and other developing countries are frequently contaminated with

aflatoxins, metabolites of Aspergillus species—mostly the Aspergillus flavus and Aspergillus

parasiticus. Aflatoxin contamination of peanuts and peanut-based products has been of great

concern globally due to their carcinogenic effect on humans and livestock. Some of the

ailments associated with aflatoxin intake is hepatocellular carcinoma (HCC, or liver cancer),

linked to stunted growth in children and immune system disorders .This is of great concern in

Ghana, because peanuts are extensively used in preparing all kinds of dishes, and are mixed

with baby food as a source of protein. The level of aflatoxin contamination also creates a

trade barrier and results in great economic losses to exporters and the country at large. Poor

postharvest handling causes high aflatoxin levels and it is worse during storage. Aflatoxin

contamination is a problem in Ghana because the storage system and environmental

conditions (temperature and humidity) supports aflatoxin production. In view of this, there

was the need to find a storage system that would be able control aflatoxin production in

storage, which would be easily affordable and adaptable. This study was set to develop

solutions for peanut storage that would reduce or control Aspergillus growth and aflatoxin

production and also to maintain quality. Specifically: to investigate the growth of Aspergillus

flavus and the production of aflatoxin in shelled peanuts under varying pre-storage treatment

and packaging conditions to reduce aflatoxin production and maintain quality under

controlled conditions; determine appropriate pre-storage treatments and packaging to reduce

aflatoxin production and maintaining quality in storage of shelled and in-shell peanuts under

the tropical environment; and also determine the impact on peanut farming and marketing

profitability of switching to hermetic storage of peanuts in Ghana.

Storage studies on shelled and in-shell peanuts were conducted under laboratory

settings in the United States of America (Virginia Tech) and the other part on the field in

Ghana; specifically, the Northern and Ashanti regions. Under laboratory settings shelled

peanuts were subjected to four different pre-storage treatments (raw-clean, raw-inoculated-

with-A. flavus, inoculated-partially-roasted , inoculated-partially-roasted-and-blanched-with-

discolored-nuts-sorted-out) and were packed into four packaging systems (polypropylene

woven sacks-PS, hermetic packs- HP, hermetic packs + oxygen absorbers -HPO and

hermetic packs vacuumed- HPV) and stored for the 14-weeks. Storage under Ghanaian

environment lasted for 26 weeks with four pre-storage treatments as the laboratory samples

given to shelled peanuts. Two pre- storage treatments (raw-clean and raw-inoculated-with-A.

flavus) were also given to in-shell peanuts and packaged in two packaging (polypropylene

106

woven sacks-PS and hermetic packs + oxygen absorbers -HPO) systems. All peanuts samples

were first were sorted out (removing all discolored and infested seeds). before all pre-storage

treatments, peanuts were applied. Mostly after every four weeks of storage, peanuts were

sampled and aflatoxin and quality degradation (peroxide and p- Anisidine values) levels were

analyzed. Additionally profitability analyzes was done on the impact on farming and

marketing of the proposed package over the existing package. To access it benefits for peanut

farmers and traders in Ghana and the country as a whole.

This study made a number of key findings which have implications for reducing

aflatoxins on the peanut value chain, for agricultural innovation extension, storage

management, and postharvest policy development in this region.

Firstly it was realized that sorting was a very good tool for reducing aflatoxin levels

before and during storage, and was able to reduce aflatoxin levels of shelled peanuts by 75 %.

Blanching and again sorting peanuts was also able to reduce aflatoxin levels drastically to

about 95%. With possibility of drought, in-shell peanuts could have high levels of aflatoxin,

and sorting as a pre-storage treatment was able to reduce the levels by 65%.

Partially roasting peanuts (1kg) at temperature at 145 °C for 10 minutes was able to

almost reduce or eliminate the fungi growth on raw inoculated samples. Partial roasting was

also able to almost completely eliminate the growth of fungi by 99.9 % fungi growth

reduction and reduce aflatoxin levels by 95% as mentioned earlier. Aflatoxin levels for

partially roasted samples were minimal compared to raw of the same samples. Regardless of

the package used, there was no increase in trend in aflatoxin levels of both the partially

roasted blanch and not blanch samples. It can be deduced that, infested shelled peanut

samples can be partially roasted, and blanched; this process can kill the aflatoxigenic fungi

and halt the aflatoxin production in storage. This pre-storage treatments can also increase the

effectiveness of peanut sorting; thus aiding in reducing or eliminating aflatoxin levels along

the peanut value chain, although it consumes time and adds additional preparation cost.

In terms quality, apart from the raw clean peanuts, partially roasted blanched sorted

peanut samples stored in hermetic packages were second in maintaining quality deterioration.

This can be attributed to sorting out almost all the aflatoxin infected peanuts out, absorbing

oxygen with the oxygen absorbers and blocking oxygen exchange in the package with the

hermetic packs The hermetic package with oxygen absorbers was the best in maintaining

quality among the three hermetic (Hermetic package- HP, Hermetic package plus oxygen

absorbers- HPO and Hermetic package vacuumed- HPV) packages used for the study. This

package type was able to reduce aflatoxin production of shelled raw inoculated peanuts by

107

about 80% and reduce quality deterioration of partially roasted blanch peanuts by more than

60 %. Although these blanched peanuts were supposed to have the worst quality deterioration

parameters, since it was exposed to elevated temperatures during roasting and the testa which

is an anti- oxidant was also peeled off. Additionally, the rate of quality deterioration for the

clean peanuts was low compared to infested peanuts for both shelled and in-shell peanuts

Normally, its advised to store peanuts in-shell because it can help reduce aflatoxin

production and also reduce the rate of oxidation (quality deterioration); but the hermetic

packaging system was able to extra protect against aflatoxin production by about 20% and

quality deterioration about 17 % for the infested peanuts. Overall, hermetic packaging system

was robust in controlling fungi growth, aflatoxin levels as well as maintaining quality

regardless of the type of processing method. This is because of the limitation of oxygen in the

package. Of all the four packaging systems, the hermetic bags with oxygen absorber were the

best in suppressing fungi growth, controlling aflatoxin production and reducing quality

deterioration in storage.

The appropriate model that best describe fungi growth, aflatoxin production, and lipid

oxidation (peroxide value and p-Anisidine value) was log transformed models. With all the

infested samples, there were a strong correlation between aflatoxin production and quality for

all the infested samples in the polypropylene woven sacks.

Enterprise budget, followed by partial budget analysis helped revealed that peanut

business in Ghana is generally profitable. Besides, farmers and traders stand to make

additional revenue and profits from switching from traditional packaging (mostly in

polypropylene woven sacks) to hermetic storage. Although the latter system incurs additional

investment costs of about 7% for farmers and 3% for traders due to cost storage cribs,

hermetic packs and oxygen absorbers. Nonetheless, the increase in revenue from sales of

higher quality peanuts far outweighed the additional costs incurred. Switching to the new

technology resulted in a 10% and 5% increment in profits for farmers and for traders,

respectively.

From these findings, it can be concluded that sorting raw peanuts and storing it

hermetically can best maintain quality and suppress aflatoxin production compared to sorted

peanuts in polypropylene woven sacks which can still aid in producing aflatoxin if there are

aflatoxigenic fungi present. Overall, the best treatment combination that can control or reduce

aflatoxin levels, stop aflatoxin production during storage and also maintain quality, is to

partially roast, blanch and sort out the infested and discolored peanuts, and hermetically store

them.

108

With high potential of making additional profits when hermetic packaging system is

adopted, I recommend that local production and marketing of hermetic storage system should

be encouraged, along with the active creation of awareness of their benefits in reducing the

incidence of aflatoxins along the peanut value chain. Switching to the new storage technology

will not only have positive implications for farmer and trader profitability, but also reduce the

incidence of various ailments that have been attributed to aflatoxins .Therefore to ensure the

successful adoption of this hermetic packaging system, regular well planned workshops

(incorporating effective demonstrations) should be organized to both create awareness and to

create an atmosphere conducive to target peanut policy makers, extension officers, farmers,

middlemen, and traders at multiple stages of the innovation decision process.

Recommended Future Study

Aspergillus flavus was dormant for weeks, but grew rapidly and produce aflatoxin at a

water activity level of 0.85 ± 0.02 and temperature of 30 ± 1 °C. This could mean there are

different lag phases for aflatoxigenic fungi growth and aflatoxin production for different

water activity levels. It is recommended that a study be done to find the effect of time of

fungi growth and aflatoxin production, with varying water activity levels under controlled

optimum environmental conditions (for temperature and humidity levels).

The perfect window was missed due to timely access to money, and so time for the

study (December to June) did not favor aflatoxin production very much, therefore, I

recommend a research work to be done for a whole year under Ghana environment starting

from July-August because that is when actual peanuts storage time starts and also when the

temperatures and humidity is conducive for aflatoxin production.

109

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113

Appendices

Appendix A: Experimental design for a.) Shelled and b.) In-shell peanuts for field study in

Ghana

a.)

b.)

114

Appendix B: Effects of aflatoxin Least square means and standard errors

Shelled Peanuts

Ashanti Region Northern Region

Aflatoxin (ppb) 23.32 ± 0.56a 18.87 ± 0.09b

Peroxide Value (meq/kg) 12.10 ± 0.56c 12.18 ± 0.09c

p- Anisidine (meq/kg) 8.67 ± 0.09d 8.60 ± 0.09d

In - Shell

Aflatoxin (ppb) 54.32 ± 1.27 e 47.32 ± 1.27f

Peroxide Value 9.38 ± 0.13 g 9.80 ± 0.13 g

p-Anisidine Value 4.66 ± 0.07i 4.99 ± 0.07i

Levels not connected by the same letter are significantly different

115

Appendix C: Moisture levels for (a). –Partially -roasted-blanched (PR-B), partially roasted

not blanched (PR-NB), raw clean (Raw-Cl), and raw inoculated (Raw-Inf), and (b) moisture

levels in samples in Kumasi (Ashanti Region) and Tamale (Northern region) over 26 weeks

of storage

a.)

b.)

116

Appendix D: Moisture levels for a.) In-shell peanuts in polypropylene woven sacks (PS) and

hermetic bags with oxygen absorber (HPO); and (b) moisture levels of samples in Ejura

(Ashanti region) and Diare (Northern region) over 26 weeks of storage

a.)

b.)

117

Appendix F: Farm equipment purchased for a 2-acre peanut farm in Northern Ghana

Quantity

(#)

Unit Price

(GH¢)

Total Price

(GH¢)

Useful life

(#)

Salvage Value

(GH¢)

Cutlass 2 20.00 40.00 5 20.00

Hoe 2 15.00 30.00 5 6.00

Storage Crib 1 400.00 400.00 10 80.00

Source: Survey data

***Salvage value is the estimate resale value of an asset at the end of its useful life. (Salvage

percentages are 50%, 20%, 20% for cutlass, hoe, and crib respectively.

*** GH¢3.94 equals USD $1.00 (Trading Economics 2016).

Appendix G: Depreciation and interest of farm equipment for a 2-acre peanut farm in

Northern Ghana

Total Price

(GH¢) Average Annual depreciation (%)

(cost-salvage value) / useful life

Equipment Interest

(cost + salvage

value) /2 x (interest

rate)

Cutlass 40.00 4.00 7.80

Hoes 30.00 4.80 4.68

Storage Crib 400.00 32.00 62.40

Source: Survey Data

*** GH¢3.94 equals USD $1.00 (Trading Economics, 2016).