1Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign, US2 National Institute of Food Technology Entrepreneurship and management, Sonepat, India

3 Mansinhbhai Institute of Dairy and Food Technology, Mehsana, India4 Division of Nutritional Sciences, University of Illinois, Urbana-Champaign, US

Introduction: Micronutrient undernutrition is a persistent problem in India mainly due to low dietary diversity and poor nutrient density of staple foods. Although dairy

products have been used as fortification vehicles in the Western world, no study exists on the fortification of traditional fermented dairy products in India. Chhash is an

inexpensive fermented milk drink, widely consumed for its hydration and digestive-enhancing properties. The objective was to evaluate the effect of micronutrient

addition on Chhash’s physicochemical and sensory characteristics under different storage conditions.

Method: The yogurt prepared with 2% milk, 20g/L fermentation culture (CHN-11) and 12 h incubation (42°C) was diluted with water (35:100 water:milk) with or without

fortification premix, 32 mg fortificant per 100 mL Chhash. The fortification premix (vitamin A, D, folic acid, Fe, Zn, and I) was designed to provide 25-30% RDA for adults,

per 100 mL serving. A three factorial design consisting of fortification (control vs. fortification), temperature (5°C, 25°C and 40°C) and storage time (1, 2, 3 and 6 d) was

used to evaluate the product’s physicochemical properties (titrable acidity, pH, color, and viscosity). A triangle test was performed with college students (n=58) and

women (n=25), living in Mehsana, India to evaluate differences in sensory discrimination between fortified and unfortified samples (5°C).

Results: Except for temperature, time and their interactions, fortification did not modify most of the physicochemical properties of Chhash. Titrable acidity of samples

increased (p<0.05) whereas pH decreased (p<0.05) with longer storage times and higher temperatures. Fortified samples had a higher b value (yellowness; p<0.05)

compared to the control samples across all time points and temperatures. Temperature increased viscosity and torque, especially among samples stored at 40°C.

Women and students were not able to differentiate between fortified products based on color, aroma, and taste (p>0.05). In conclusion, fortification of Chhash is

feasible, and the sensory and storage effects found are similar between fortified and unfortified products.

Significance: This study provides evidence on the technical feasibility of Chhash fortification to serve as a viable vehicle for micronutrient delivery in India.

[1] Vijayaraghavan K. 2002. Control of Micronutrient Deficiencies in India: Obstacles and Strategies, Nutr. Rev. 60: S73–

S76.

[2] Kotecha PV. 2008. Micronutrient Malnutrition in India: Let Us Say “No” to it Now, Indian J. Community Med. 33: 9–10.

[3] Allen L, Benoist B de, Dary O, Hurrell R. 2006. Guidelines on food fortification with micronutrients; Geneva: World

Health Organization, Food and Agricultural Organization.

[4] FSSAI. 2017. Fortification of Foods, Food Safety Standards Authority of India, India. Retrieved from

https://gain.fas.usda.gov/Recent GAIN Publications/India Implements Food Fortification Regulation_New Delhi_India_5-

25-2017.pdf retrieved on June 21, 2017

[5] Amul Pouch Butter Milk. 2016. AMUL, India Retrieved from http://www.amul.com/products/amul-pouchbuttermilk-

info.php on June 21, 2017

[6] Standard Operating Procedure for Chhash production. 2016. Retrieved from Quality Control Department at

Dudhsagar Dairy, Mehsana, Gujarat, India on August 12, 2016.

[7] AOAC.1990. AOAC Official Methods of Analysis. 15th Edition, Association of Official Analytical Chemists, Arlington.

[8] Isleten M, Karagul-Yuceer Y. 2006. Effects of Dried Dairy Ingredients on Physical and Sensory Properties of Nonfat

Yogurt, J. Dairy Sci. 89:2865–2872.

[9] Meilgaard M, Civille GV, Carr BT. 2007. Sensory evaluation techniques, Taylor & Francis.

[10] DSM, India and Dudhsagar Dairy, India. 2016. Prices retrieved on April 18, 2016.

International Division

064 Abstract

Physicochemical Characterization

• Fortification did not have an effect on most of the physico-chemical

properties of Chhash

• Titrable acidity of samples increased (p<0.05) whereas pH

decreased (p<0.05) with increasing storage time and temperature.

• Viscosity and torque had no fortification effect but changed with

temperature and were higher for samples stored at 40°C compared

with those at lower temperatures.

• Fortified samples stored at 40°C were different in terms of human

eye perception per the delta E-76 value for color.

Discriminatory Sensory Studies

• Sensory tests with students and women revealed no differences

(p>0.05) in color, aroma and taste between fortified and control

Chhash.

Conclusion

• Fortification of Chaash is feasible in terms of the fortification

criteria; nutritional quality, availability, affordability,

acceptability, storability. Chhash has the potential to serve as

a viable vehicle of micronutrient delivery.

Summary

Effect of Micronutrient Fortification on Physicochemical and Sensory Properties of Chhash(A Traditional Indian Yogurt-based Fermented Dairy Drink)

The authors thank Pankaj Nemade, DSM, India for providing the

micronutrient mix and Chr. Hansen, USA for providing the CHN-11 yogurt

culture.

This work was funded by the Office of International Programs, University

of Illinois, Urbana-Champaign.

Acknowledgements

Shashank Gaur1, Anna W. Waller1, Ankur Ojha2, Dharmendra Shukla3, Juan E. Andrade1,4

Results: Storability

• Micronutrient malnutrition is a persistent problem in India, with

unacceptably high morbidity and mortality in children and

pregnant women.[1]

• More than 70% of Indian population consumes less than 50%

RDA of micronutrients, typically due to inadequate food intake,

poor nutrient bioavailability (inhibitors, preparation method,

interactions), and/or presence of infections. [2]

• The fortification of dairy products with vitamins and minerals is a

well-established and successful practice in developed countries

to contribute to good nutrition. [3]

• In India, fortified dairy products are limited in markets.

Nevertheless, in response to a newly implemented “Food

Fortification Regulation,” dairy products, such as Vitamin A and D

fortified milk, are now emerging in the Indian market.[4]

• Dairy products such as flavored milk, yogurt and ice cream are

examples of market-driven fortification vehicles, however no

study exists on the fortification of Chaash, a traditional, low-cost

Indian fermented dairy product.

• Chhash, also known as sour buttermilk, has 1% fat, 5.5% solids-

not-fat and maximum acidity of 0.8%. [5]

Introduction

Figure 7. Change in the viscosity of Chhash

after a 6 day period

Figure 10. Triangle test results show no

significant differences (p<0.05) between fortified

and control chhash. n critical = 13.

Figure 5. Change in the pH of Chhash over 6 day

period

• Systematically analyze the storability and acceptability of

micronutrient fortified Chhash in terms of physicochemical and

sensory characteristics.

Fortification Criteria

Results: Acceptability

Figure 8. Color difference between fortified and

control samples at different temperature at day 6

Figure 2. Success criteria for fortification of Chhash

ReferencesFigure 9. Triangle test results show no

significant differences (p<0.05) between fortified

and control chhash. n critical = 25.3.

pH and Titrable Acidity - Method number 981.12 and Method 947.05 of AOAC, 1990 [7]

All figures show mean ± standard deviation. a>b>c indicate significant differences between treatments at each

timepoint at p<0.05. * indicates significant difference from baseline (time point 0), within a treatment group.

C-5 - control sample at 5°C, F-5 - fortified sample at 5°C, C-25 - control sample at 25°C,

F-25 - fortified sample at 25°C, C-40 - control sample at 40°C, F-40 - fortified sample at 5°C

Discriminatory tests - All figures show a Z critical

line such that if correct answers exceed the line, a

significant difference is detectable in the assigned

sensory attribute.

Figure 3. Flowchart for preparation of fortified Chhash [6]

1 million liters Chhash

reaches 3 million

consumers per day

Objective

Methodology

Figure 1. Status of micronutrient malnutrition in India [2]

∆𝐸 − 76 = (𝐿𝑓 − 𝐿𝑐)2+(𝐴𝑓−𝐴𝑐)

2 + (𝐵𝑓−𝐵𝑐)2

where Lf, Af and Bf are the L, A, and B values of fortified samples

and Lc, Ac and Bc are L, A, and B values of the control sample

𝑍𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙 =𝑘 −

13𝑛

29𝑛

Statistical Analysis – All experiments conducted in triplicates and results reported as mean ±

SD. Differences in outcomes analyzed using ANOVA and post hoc tests (SAS v.9.4). Triangle

test critical values calculation equation:

Discriminatory Sensory Analysis (Triangle test) - IRB, UIUC. Location

- MIDFT, India. Participants - 58 students and 25 women with young

children at home (1-6 y).Written consent, training in Gujarati. Coded

samples (~2 ± 0.5 g) in clear disposable sensory cups. Water (~25 ± 2°C)

and crackers provided for rinsing between the samples. Time - 11:00 AM -

1:00 PM. [9]

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

0 1 2 3 4 5 6 7

pH

Days

C-5 F-5 C-25 F-25 C-40 F-40

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 1 2 3 4 5 6 7

Titr

atab

le A

cid

ity

(% L

acti

c ac

id)

Days

C-5 F-5 C-25 F-25 C-40 F-40

b*b*

0

50

100

150

200

250

300

10 20 30

Vis

cosi

ty (

cP)

Time (s)

C-5 F-5 C-25 F-25 C-40 F-40 Day 0

• In vitro and in vivo assessment of bioavailability and

bioaccessability of nutrients from Chhash samples.

• Preclinical and clinical trials to assess the effectiveness of

Chhash intervention among at-risk populations.

Future Studies

Experimental Design - Three factorial - 2 treatments (control and fortified), 3 temperatures

(5°C, 25°C and 40°C) and 4 time points (1, 2, 3 and 6 days)

Culture taxonomy - Lactococcus lactis subsp. cremoris, Leuconostoc, Lactococcus lactis

subsp. Lactis, Lactococcus lactis subsp. Lactis biovar diacetylactis.

Micronutrient premix - 500 IU Vit A acetate, 120 IU Vit D2, 28 ug Folic acid, 22.5 ug Iodine

(KI), 4.2 mg Iron (Fe Pyrophosphate), 2.33 mg Zinc (ZnO) to provide 25-30% RDA per serving.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5°C 25°C 40°C

ΔE-

76

Val

ue

Temperature

Not perceptible

by human eyes

Perceptible via

close observation

Perceptible

at a glance

0

10

20

30

40

50

60

Color smell taste

Nu

mb

er

of stu

de

nts

Characteristic

Student Participants

Yes No Z Critical(p<0.05)

0

5

10

15

20

25

Color smell taste

Nu

mb

er

of w

om

en

Charasteristic

Women Participants

Yes No Z Critical (p<0.05)

a a a a

b*

b*

b*

b* b*

c*

c*d* c* a

a*

a* a*

b*

c cc

a a a

b b b

Nutritional Quality

Affordability

Commodity Price [10]

Chhash (250 mL) ₹ 6 ~ 10¢

Micronutrient mix (80 mg in 250 mL) ₹ 0.06 ~ 0.09¢

where z critical is 1.64 for α=0.05, k is minimum number of

correct responses to reject the assumption of “no difference”, n

is total number of participants.

Color - HunterLab, LabScan XE, to measure L, A and B values. Measure of change in color

perception calculated as ΔE-76 value: [7]

Viscosity - Brookfield viscometer (Model DV-II Pro, Brookfield Engineering

Laboratories, USA) with a spindle RV2. 130 mL Chhash sample in 250 mL

beakers, analysed at 5 ± 1°C. Shear rate - 60 RPM, 10 s interval. [8]

Figure 4. Student and

women participants during

sensory analysis

Fortification

premix330,000 child deaths per year

Vitamin

A 22,000 pregnant women death per year from severe anemia

Iron Anemia 6.6 M

children born mentally impaired every year

Iodine200,000 babies born with neural tube defects per year

Folic acid

Availability

Affordability

AcceptabilityStorability

Nutritional quality

Packaging and Storage

Form-fill-seal packaging and storage at 5±2°C

Mixing and Homogenization

Two stage homogenization - 1st stage 100 Kg/cm2, 2nd stage 20-30 Kg/cm2

Dilution and fortification

Dilution with fortification premix stock solution in water at the rate of 35% to yield 32 mg forficant per 100 mL of final Chhash

Yogurt Preparation

Heating (42 °C), addition of culture (CHN-11 @ 20g/L), agitation (10 min) and setting (12 h at 42°C) to acidity 0.7-1.0%

Pasteurization (high temperature short time)

80°C for 15 s

Milk Silo Storage

Chilled double toned milk (Fat 1.5% and solids-not-fat 9%) at 4°C

Figure 6. Change in the titrable acidity of Chhash

over 6 day period