PHD THESIS ABSTRACT - Doctorate...

PHD THESIS ABSTRACT

NEAMȚU BOGDAN 1 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU

PHD THESIS ABSTRACT RESEARCH ON ISOLATION AND INDUSTRIAL

DEVELOPMENT OF PROBIOTIC STRAINS FROM

HUMAN BREAST MILK AND THE PRODUCTION OF

NUTRACEUTICAL PRODUCTS INTENDED FOR

INFANTS FEEDING

SIBIU, 2014

Project co-financed by European Social Fund through Sectoral Operational Programme Human

Resources Development 2013

Priority axis no. 1: "Education and training in support for growth and development of the knowledge

society"

Major Area of Intervention 1.5 : "Doctoral and post-doctoral programmes in support of research "

Project Title: Integration of Romanian research in the context of European research-doctoral

fellowships.

Contract : POSDRU/ 88 / 1.5 / S / 60370

The scientific leader

Professor Phd.Eng. Ovidiu Tița Phd Student

Bogdan Neamțu

PHD THESIS ABSTRACT


Table of Contents

2.1 The main objective of the study ........................................................................... 7

2.2 Secondary objectives ............................................................................................ 7

2.2.1. Analysis of the milk samples collected from nursing mothers ....................... 7

2.2.2. Identification of isolated strains from human breast milk ............................... 7

2.2.3. Bioreactor growth of isolated strains from human milk ................................ 7

2.2.4. The evaluation of strain resistance in different growing environments .......... 7

2.2.5. Microencapsulation ........................................................................................ 7

3.1. Overview ............................................................................................................. 8

3.2 Historical considerations ...................................................................................... 8

3.3 The taxonomy of probiotic microorganisms ........................................................ 9

3.4 The Genus Lactobacillus ...................................................................................... 9

3.5 The Genus Bifidobacterium ................................................................................. 9

4.1 Introduction ........................................................................................................ 10

4.2 The effects of probiotics in enterocolitis ........................................................... 10

4.3 The effects of probiotics in Helicobacter pylori infection ................................. 10

4.4 The effects of probiotics in respiratory infections ............................................. 11

4.5 The effects of probiotics in cancer ..................................................................... 11

4.6 The effects of probiotics in allergies .................................................................. 11

4.7 Effects of probiotics on the immune system ...................................................... 11

4.8 The effects of probiotics in urinary infections ................................................... 11

5.1 Introduction ........................................................................................................ 12

5.2 Isolation of lactic acid bacteria .......................................................................... 12

Chapter 1 .................................................................................................................................... 6 Introduction ................................................................................................................................ 6 Chapter 2 .................................................................................................................................... 7

Objectives of the research .......................................................................................................... 7

The Documentary Part ............................................................................................................... 8 Chapter 3 .................................................................................................................................... 8

The probiotic taxonomy ............................................................................................................. 8

Chapter 4 .................................................................................................................................. 10

Probiotics and health ................................................................................................................ 10

Chapter 5 .................................................................................................................................. 12 Methods for isolation and cultivation of probiotic strains ....................................................... 12

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5.2.1. Isolation from yogurt and other dairy products ............................................. 12

5.2.2 Isolation of lactic bacteria from human milk ................................................ 12

5.3 The phenotypic identification ............................................................................. 13

5.4 In vitro evaluation of potential probiotic bacterial strains ................................. 13

5.4.1 Resistance to gastric acid ............................................................................... 13

5.4.2. Resistance to bile acids .................................................................................. 13

5.4.3. Bacterial concentration .................................................................................. 13

5.4.4 Incubation time ............................................................................................... 13

5.5. Isolation and selection ....................................................................................... 14

5.5.1 Strains preservation. ....................................................................................... 14

5.5.2 Materials used in probiotic growth. ................................................................ 14

5.6 Conditions of cultivation and production of probiotics ..................................... 14

5.6.1 Inoculum culture. ............................................................................................ 14

5.6.2 Micropilot-level biosynthesis. ........................................................................ 15

6.1 Introduction ........................................................................................................ 15

6.2 Nephelometry versus turbidimetry ..................................................................... 15

6.2.1 Nephelometry ................................................................................................. 15

6.2.2 Turbidimetry ................................................................................................... 16

6.2.3 Nephelometry used in milk analysis .............................................................. 16

6.3 Milk bioactive factors ......................................................................................... 16

6.3.1 Proteins .......................................................................................................... 16

6.3.2 Carbohydrates ................................................................................................. 16

6.3.3 Lipids and vitamins ........................................................................................ 17

6.3.4 Nucleotides, nucleic acids and nucleosides .................................................... 17

7.1 Encapsulating materials ...................................................................................... 18

7.1.1 Alginate .......................................................................................................... 18

7.1.2 Chitosan .......................................................................................................... 18

7.1.3 Xanthan gum .................................................................................................. 18

7.1.4 Gellan gum: .................................................................................................... 18

7. 1.5 Carrageenan: .................................................................................................. 19

7.1.6 Cellulose acetate phthalate (CAP) .................................................................. 19

Chapter 6 .................................................................................................................................. 15 Bioactive factors in breast milk ............................................................................................... 15

Chapter 7 .................................................................................................................................. 18 Principles of encapsulation ...................................................................................................... 18

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7.1.7 Starch .............................................................................................................. 19

7. 1.8 Gelatin ........................................................................................................... 19

7.1.9 Milk proteins .................................................................................................. 19

7.1.10 Pectins .......................................................................................................... 20

7.2. Microencapsulation ........................................................................................... 20

7. 2.1. Introduction, definitions, overview .............................................................. 20

7.2.2. Classification of microcapsules ..................................................................... 20

7.2.3 Membrane ....................................................................................................... 20

7.2.4. The Nucleus ................................................................................................... 20

7.2.5. The diameter of the microcapsule correlated with the method ..................... 21

7.3. Microcapsules analysis methods ....................................................................... 21

7.3.1. Determining the form and size of the microcapsule ...................................... 21

7.3.2. Determining the microcapsule concentration ................................................ 21

8.1. Harvesting and analysis of milk samples .......................................................... 22

8.2. Identification of Lactobacillus spp bacterial strains. ......................................... 22

8.3 Bioreactor growth of the strains isolated from breast milk ................................ 22

8.4 In vitro evaluation of probiotic potential of bacterial strains ............................. 22

8.5 Microencapsulation of Lactobacillus paracasei ssp Paracasei (L18) strain ....... 23

9.1 Introduction ........................................................................................................ 24

9.2 Objectives ........................................................................................................... 24

9.3 Materials and methods ....................................................................................... 24

9.4. Results ............................................................................................................... 24

10.1 Introduction ...................................................................................................... 26

10.2 Objectives: ........................................................................................................ 26

10.3. Materials and methods: ................................................................................... 26

10.3.1 Principle of the method: ................................................................................ 26

10.4. Results ............................................................................................................. 27

11.1 Introduction ...................................................................................................... 28

The Experimental Part ............................................................................................................. 22

Chapter 8 .................................................................................................................................. 22 Materials and methods in the proposed research ..................................................................... 22

Chapter 9 .................................................................................................................................. 24 Evaluation of bioactive immune factors in breast milk and milk powder ............................... 24

Chapter 10 ................................................................................................................................ 26 Identification of Lactobacillus ssp strains................................................................................ 26

Chapter 11 ................................................................................................................................ 28 Bioreactor growth of the strains isolated from breast milk...................................................... 28

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11.2 Objectives ......................................................................................................... 28

11.3 Materials and methods ..................................................................................... 28

11.4. Results ............................................................................................................. 28

12.1 Introduction ...................................................................................................... 30

12.2 Objectives ......................................................................................................... 30

12.3 Materials and methods ..................................................................................... 30

12.4 Results .............................................................................................................. 30

13.1 Introduction ...................................................................................................... 32

13.2 Objectives ......................................................................................................... 32

13.3 Materials amd methods ................................................................................... 32

13.4 Results .............................................................................................................. 33

14.1 General conclusions ......................................................................................... 34

14.2 Recommendations ............................................................................................ 35

14.3 Own contributions and future development trends of research ........................ 35

Chapter 12 ................................................................................................................................ 30 In vitro evaluation of probiotic potential of bacterial strains ................................................... 30

Chapter 13 ................................................................................................................................ 32 Microencapsulation of Lactobacillus paracasei ssp Paracasei (L18) strain ............................. 32

Chapter 14 ................................................................................................................................ 34 Conclusions .............................................................................................................................. 34

Bibliography ............................................................................................................................ 37

Publications: ............................................................................................................................. 47

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Chapter 1

Introduction

Probiotics are defined as "live microorganisms which in adequate amounts bring

benefits for the host" (FAO/WHO, 2002) and are extremely important as functional foods

(representing 65% of the world market of food) [4,27]. Probiotics are considered food

supplements with health beneficial role. Positive effects on health correlates with a specific

type of strain [27, 36]. Probiotics are represented by a wide range of micro-organisms in the

genus Lactobacillus prokariote bacteria, (about 116 species in 2008) and the genus

Bifidobacterium (about 30 species), but also in the field of fungi (some yeasts) [27].

The most important benefits of probiotics refer to the maintenance of normal intestinal

microflora, defensive roles in urinary infections, in enteropatogenic bacterial infections, in

Helicobacter Pylori infections, modulation of allergic response (atopic dermatitis, asthma,

recurrent wheezing), anticarcinogenic and antimutagenic activities, and decreasing cholesterol

levels [27,36].

Probiotic bacteria are enumerated and isolated from yoghurt and probiotic dairy

products available on the market. The material used for isolation of probiotics could be human

milk obtained from healthy volunteer mothers. Samples are collected in sterile harvesters and

stored on ice until delivery to the laboratory. Once delivered to the laboratory the procedure

for isolation is initiated [121].

Isolation and industrial development of probiotics from human breast milk is a highly

actual subject and a real challenge for dairy microbiology and biotechnology centers worldwide

(Malek et al 2010). Roles of breast milk (in addition to the nutritional and anti-infective

protection) are the initiation and the development of the infants intestinal flora immediately

after birth, mainly due to natural microbiota of breast milk (staphylococci, streptococci,

Micrococcus, lactobacilli and enterococci) [121].

Isolation of probiotic cultures from human breast milk shows the following advantages:

they are of human origin, they are adapted to nutritional substrates in dairy products and

represent a safe intake in children. Selection of probiotic strains to their encapsulation and the

production of nutraceuticals using milk as a source of isolation from mothers to infants

respiratory infections could be a goal and an interesting research approach. Meta-analysis on

studies that have evaluated the rates of hospitalization in infants with lower respiratory tract

infections (breastfed infants versus formula fed infants) have shown unequivocally a

hospitalization rate 3 times higher in those fed with formula [44,45].

The research infrastructure of Pediatrics Clinical Hospital from Sibiu (bacteriology

department with the possibility of isolation, microbiological analysis, chemical and metabolical

analysis of the strains), SAIAPM Faculty research infrastructure from Lucian Blaga University

of Sibiu (microbiology and biotechnology compartment for micropilot studies), and last but

not least, the research infrastructure in pharmaceutical technology and biotechnology in the

Faculty of Medicine V.Papilian from Sibiu, all of these made possible to carry out a research

of this magnitude.

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Chapter 2

Objectives of the research

2.1 The main objective of the study

The main objective of this study was to conduct a research on isolation and industrial

development of probiotic strains from human breast milk and to obtain a nutraceutical product

intended for infants intake.

2.2 Secondary objectives

Alternatively we aimed specific issues related to: 1.analysis of milk samples, 2

identifying the types of strains with their specific phenotypes, 3 presenting patterns of growth

in the bioreactor and in vitro testing of resistant strains , 4 presenting the differences between

encapsulation methods in terms of microcapsules parameters (diameter, thickness).

2.2.1. Analysis of the milk samples collected from nursing mothers

1.Study of immune protection of breastfed infants compared with those fed with

formula ; 2 Identification of bioactive factors in human breast milk and formula, factors

involved in immune protection; 3 Human breast milk and formula study of IgA levels influence

on immunoglobulins (IgA and IgG) in infants serum; 4 Study of milk lactose levels influence

on infants serum immunoglobulins (IgA and IgG).

2.2.2. Identification of isolated strains from human breast milk

1. Identifying the lactic acid bacteria types of strains isolated from milk; 2. Presenting

identification accuracy for each strain by API CH50 method in order to assess the

appropriateness of using this method as a first step identification mean for probiotic

characterisation; 3. Presenting the sensitivity of sugars fermentation method in identifying

human breast milk probiotic strains

2.2.3. Bioreactor growth of isolated strains from human milk

1.Study of performance differences in DO maximal values between different strains

isolated from maternal milk samples; 2.Study of the optimal combination of the parameters

pH, oxygen concentration, temperature, related to the maximum optical density value for each

strain.

2.2.4. The evaluation of strain resistance in different growing environments

Study of each strain resistance to pepsin, bile and HCl environments simulating in vitro

gastrointestinal conditions.

2.2.5. Microencapsulation

1.Analysis of the microcapsules mean diameters obtained by emulsion (method 1)

versus the microcapsules mean diameters obtained by extrusion (method 2); 2.Analysis of the

sodium alginate concentrations on the microcapsules diameters obtained by the extrusion

method (methods 3 and 4) correlating with their shape; 3) Analysis of the microcapsules wall

thickness obtained by all four methods; 4) Analysis of the sodium alginate concentration on

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the thickness of the microcapsules obtained by methods 3 and 4 (using different combinations

of solutions and substances)

The Documentary Part

Chapter 3

The probiotic taxonomy

3.1. Overview

Probiotics are very important in industrial applications, the concept of probiotic is open

to many different applications in a wide variety of areas relevant to human and animal health.

Probiotic products consist of various enzymes, vitamins, capsules or tablets and some

fermented foods containing microorganisms with beneficial effects on health. They can also

contain one or more species of probiotic bacteria. The majority of the products for human

consumption are fermented milk products or they are administered in the form of tablets or

powders. Capsules or tablets are used for health support. Oral intake of probiotic

microorganisms produces a protective effect on the intestinal flora. There are many studies

showing beneficial effects on intestinal microbial imbalances, but it is really difficult to show

clinical effects of these products. The probiotic strains protect patients from different intestinal

diseases, such as: travelers' diarrhea, antibiotic associated diarrhea, acute diarrheal disease,

lactose intolerance, colon cancer, Helicobacter pylori infection, and other pathological

conditions, such as hypertension, chronic inflammatory diseases, allergic diseases,

osteoporosis, urogenital infections and autoimmune diseases [27,36].

3.2 Historical considerations

Probiotics term is derived from the Greek "pro bios" meaning life, in contrast with

"antibiotic" which means "against life". It is known that since ancient times the Greeks and

Romans ate fermented foods and beverages. In 1908 Elie Metchnikoff, Nobel laureate

suggested beneficial effects of probiotic microorganisms on human health. The term

"probiotic" was used by Lilly and Stillwell 1965 to describe substances that stimulate the

growth of other microorganisms. After 1965 the term was used in accordance with the

mechanism by which probiotic acts and influences health. In 1974 Parker has defined the

probiotics as organisms and substances which contribute to intestinal microbial balance. In

1989 Fuller defined probiotics as living microbial supplement that positively influence health

by improving the intestinal microbial balance. In the following years many researchers have

studied probiotics and completed definitions: 1.Living organisms which ingested in certain

amounts would determine health benefits in addition to the basic nutrition; 2.Adjuvant

beneficial microbial diet affecting host physiology by modulating mucosal and systemic

immunity, and improving the microbial balance in the intestinal tract and nutrition (Naidu et al

1999); 3 A live microbial ingredient beneficial to health (Salminen et al 1998); 4. A preparation

product containing viable micro-organisms in sufficient numbers to alter the microflora (by

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implantation and colonization) in the host and exerting beneficial health effects on the host

Schrezenmeir and Vresse 2001; 5.Live micro-organisms which, when administered in adequate

amounts, confer a health benefit on the host (report in October 2001)[186].

3.3 The taxonomy of probiotic microorganisms

Taxonomy term, meaning in Greek categorizing can be viewed as an attempt to put

order in nature (e.g. classification and systematisation of real domains). Taxonomic hierarchy

is based on the unit named species. The species are then grouped into a Genus, genera in a

Family, families in an Order, the orders in a Class, and classes in a Domain. Three areas of life

have been described including all living organisms, both for prokaryotes the Archaea and

Bacteria, and one for eukaryotes. In the Eukarya, there are four subdomains also recognized,

for example, Protista, Fungi, Animalia, Plantae.

The probiotic strains are defined as subculture of billions of cells almost identical,

ideally derived from the same parent cell. Probiotic strains described so far are divided into

two different groups of microorganisms, namely bacteria and fungi. Taxonomic orders for

microorganisms are species, genus, family, order, class, phylum, and domain[36,186].

3.4 The Genus Lactobacillus

The genus Lactobacillus belongs to LAB (lactic acid bacteria), a definition which

groups species of Gram-positive, non-spore forming, catalase-negative bacteria that produce

lactic acid as a result of carbohydrates fermentation. Catalase is an enzyme that is found in

almost all living organisms that are exposed to oxygen and decompose hydrogen peroxide into

water and oxygen. Regarding the base composition of the genomic DNA, Genus Lactobacillus

has a content of GC (guanine and cytosine) between 32 and 51 %[36]. Guanine-cytosine

content is the percentage of these nitrogenous bases in the DNA molecule, if we refer to the

known four different bases including adenine and thymine. Based on prokaryotes taxonomic

scheme genus Lactobacillus belongs to the phylum Firmicutes, class Bacilli, order

Lactobacillales, family Lactobacillaceae with its closest relatives being grouped in the same

family, represented by Paralactobacillus genres and Pediococcus. In 2014, the genus

Lactobacillus comprised 180 species.

3.5 The Genus Bifidobacterium

Bifidobacteria are Gram-positive branched polymorphic forms rods, occurring in

chains or groups. They have various shapes, including short, curved, bifurcated rods. Their

name derives from the observation that they often exist in the form of a Y (bifidoshaped). Non-

spore forming bacteria, bifidobacteria are nonmotile, and non filamentous. They are anaerobic

having a fermentative type of metabolism, producing acid but not gas from a variety of

carbohydrate. Bifidobacteria are catalase-negative, with some exceptions, such as

Bifidobacterium indicum and Bifidobacterium asteroides when growing in the presence of air.

Genomic GC content ranges from 42 to 67%. Bifidobacteria degrade hexoses using a single

metabolic pathway referred to as fructose-6-phosphate pathway, also known as bifidum shunt.

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Bifidobacterium genus belongs to Actinobacteria phylum, class Actinobacteria,

Actinobacteridae subclass, order Bifidobacteriales, family Bifidobacteriaceae. The following

genera don't belong to this family but are considered probiotics and include Aeriscardovia,

Falcivibrio, Gardnerella, Parascardovia and Scardovia. Currently, more than 30 species of

Bifidobacterium were isolated, validated, and identified [36,186].

Chapter 4

Probiotics and health

4.1 Introduction

Probiotics are considered to be health promoters, however, the underlying mechanisms

have not yet been explained. There are studies on how probiotics work: 1.producing inhibitory

substances (organic acids, hydrogen peroxide and bacteriocins inhibitory to both gram positive

and gram negative, 2.blocking adhesion sites (probiotics and pathogenic bacteria compete,

probiotics adhere to the surface of epithelial cells occupying the areas of adhesion, 3.Nutrients

competition (although there are not many in vivo studies, probiotics inhibit pathogens by

consuming nutrients necessary for pathogens) 4.immunity stimulation (specific and

nonspecific) by some specified wall cell components that can determine a humoral immune

response, 5.toxin receptor degradation on intestinal mucosa, for example Streptomyces

Boulardi protects the host against intestinal infection with Clostridium difficile; other

mechanisms involve suppression of toxin production, reducing intestinal pH, virulence

attenuation , 6. suppression of carcinogenic substances by binding, blocking or removing,7.

modifying pH resulting in altering intestinal microflora activity and solubility of bile [36,186],

8. Lowering serum cholesterol levels [18,164].

4.2 The effects of probiotics in enterocolitis

Există multe studii şi date referitoare la efectele benefice ale probioticelor pe câteva

tipuri de diaree. La copiii cu diaree provocata de rotavirus, Lactobacillusul rhamnosus GG,

Acidophilus, LB1, Bifidobacterium Lactis şi Lactobacillus Reuteri, sunt raportate ca fiind într-

adevăr eficace (scad durata infecției cu rotavirus). Probioticele prezintă efecte benefice şi în

prevenirea unor forme de boală diareica acută (Lactobacillus rhamnosus GG, Acidophilus,

Bulgaricus, Streptococcus thermophilus, Bifidobacterium bifidum). [36,186].

4.3 The effects of probiotics in Helicobacter pylori infection

Helicobacter pylori is a gram negative organism which colonizes the stomach lining

and causing gastritis, chronic active ulcer disease and gastric cancer. The species of lactobacilli

and or bifidobacterium inhibit the activity of H. pylori by several proposed mechanisms: 1.the

synthesis of antibacterial compounds (amicoumacin A - Bacillus subtilis); 2 occupation of

surface receptor expressed on epithelial cells ; 3 stimulation of heat shock protein associated

with the cell surface (GroEL) expressed by lactic acid producing microorganisms, which are

involved in aggregation of Helicobacter pylori, preventing it from adhering to the epithelial

cells; 4. Proteas-sensitives substances (Bifidobacterium); 5. Inhibition of interleukin-8

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chemotactic response of neutrophils in inflammation area in the proximity of the infected

gastric epithelial cell.

4.4 The effects of probiotics in respiratory infections

Recently it has been suggested that lactobacilli might induce a positive

immunomodulating effect on other than digestive mucosa (ie respiratory tract). Moreover the

therapeutic potential of probiotics in the upper respiratory tract infections may be derived from

their ability to modulate the immune system. In fact lactobacilli can stimulate B cells from

GALT, causing their migration in lymphoid tissues of upper respiratory tract and a much better

IgA secretion. Several authors have shown that Lactobacillus GG may reduce the incidence

and severity of respiratory infections in children[36,186] .

4.5 The effects of probiotics in cancer

Epidemiological studies have shown an increased incidence of colon cancer by

consuming saturated fat (in Western countries). It is believed that probiotics reduced the risk

of cancer by the decrease of the bacterial enzymes activity, suppression of carcinogens by

binding, removal or suppression of favorable bacterial growth of procarcinogens to the

carcinogens, adjusting the intestinal pH, altering the colonic transit time and more efficient

removal of fecal mutagens, stimulating immune responses [36,186].

4.6 The effects of probiotics in allergies

At birth, infants present increased levels of Th2 cytokines from mother. The balance of

Th1 / Th2 is reequilibrated as they colonize the gut after birth. In infants, allergic disease is

based on IgE-mediated food allergy and an exaggerated response from Th2. Some authors have

shown that allergic infants have a smaller number of Bifidobacterium in the faeces. Other

authors have shown that administration of raffinose and oligosaccharides based on alginate led

to a reduction of Th2 response, probably by stimulation of the Th1 response , rebalance of the

immune response with increasing anti-inflammatory cytokines (IL-10 and TGF-β). [36,186].

4.7 Effects of probiotics on the immune system

The mechanisms by which probiotic bacteria may have positive effects on the immune

system are not yet fully understood. Probiotics affect the immune system in different ways such

as: increasing cytokine production, stimulating macrophages, increasing concentrations of the

secretory IgA. Some of these effects are related to the ability of the probiotics adhesion, while

others are not related to this mechanism. There are studies showing both increased serum IgA,

stimulation of macrophages and production of γ-interferon. [36,186].

4.8 The effects of probiotics in urinary infections

Meta-analysis conducted in 2008 on this subject have shown 14 clinical trials related to

the administration of probiotics for improving urogenital health. From these clinical trials, 6

have shown that there are strains which could normalize urogenital tract flora and improve

antibiotic treatment efficiency of urogenital infections. Lactobacilli probably have an

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immunomodulation capacity on urogenital tract mucosa. The mechanisms by which IL-1β

interleukin levels are reduced in the mucosal vagina, IL-6 levels in the bladder, and the

Th1/Th2 ratio is modulated, are incompletely known, although there are several studies in

vivo/in vitro to elucidate the mechanisms [36,186].

Chapter 5

Methods for isolation and cultivation of probiotic strains

5.1 Introduction

Various beneficial microorganisms and most important lactic acid bacteria have

evolved and have adapted to live in symbiotic association between them and their host at most

sites, including skin and gastrointestinal tract (GIT). Some of the best probiotic bacteria

include Lactobacillus and Bifidobacterium genera members. Lactic bacteria are widely used

in the production of fermented foods and are regarded as safe (GRAS), being considered as

microrganisms which could be used safely in medical and veterinary functions [14,36,111].

In vitro selection criteria of probiotic bacteria for food preparations, which enable them to

adhere the intestinal tract mucosa include bile tolerance and resistance to gastric juice,

surviving and growing capabilities to exert specific roles in gastro intestinal tract (GIT).

Although the tolerance limit required for maximum growth GIT is not known, it makes sense

that in any study of different probiotic strains the most resistant species are to be selected

[36,113].

5.2 Isolation of lactic acid bacteria

5.2.1. Isolation from yogurt and other dairy products

Probiotic bacteria are isolated from commercially available yoghurt and probiotic dairy

products. Lactobacillus delbrueckii ssp. Bulgaricus is characterised and isolated using MRS

agar, incubated anaerobically at 37⁰C for 72 hours. Growth medium M17 agar is used for the

characterisation and isolation of Streptococcus thermophilus. MRS agar and modified MRS

agar (MRS + L-cysteine + LiCl + Na propionate) are used for the isolation and enumeration of

probiotic bacteria [36].

5.2.2 Isolation of lactic bacteria from human milk

The material used for isolation is human milk obtained from healthy volunteer mothers.

Samples are collected in sterile harvesters and stored on ice until delivery to the laboratory.

Once delivered to the lab, the procedure for isolation is initiated. Plate technique is used to

isolate organisms. After incubation, individual colonies are selected and transferred to sterile

broth medium. The next step is purification of selected colony using "Streak" plate technique

[98,99,102,113,117]. Isolates are examined then by colony morphology, catalase reaction and

Gram reaction. Colonies of cocci and Gram-positive and catalase-negative bacilli are treated

with glycerol [121, 142,165,184,188,189,190].

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5.3 The phenotypic identification

Morphological characterisation is carried out by examining the growth of the colony,

cell morphology, and Gram reaction. It also performs catalase test [14,15,20,48,85,88].

Isolates proved to be Gram positive and catalase-negative are subjected to biochemical

characterization using the API 50 CHL kit (bioMérieux) [142,165,184,188,189,190].

5.4 In vitro evaluation of potential probiotic bacterial strains

Assessment of potential probiotic bacteria implies evaluating resistance to gastric

acidity and bile toxicity, adherence to the intestinal epithelial tissue, the ability to colonize the

gastrointestinal tract, the production of antimicrobial substances, and the ability to modulate

immune responses [94, 98, 99,102,113,117,121].

5.4.1 Resistance to gastric acid

Probiotic bacteria must survive the transit through the stomach. Gastric acid secretion

is the first defense mechanism against the most ingested microorganisms. The survival of the

bacterial strain is first measured by the addition of 1x109 CFU Lactobacillus strains and 1x1011

CFU , Bifidobacterium strains respectively, to a modified MRS medium with hydrochloric

acid to pH values between 2.0 and 3.4. Bifidobacteria have been shown to be more acid

resistant compared to the lactobacilli, especially when exposed to human gastric [36,184].

5.4.2. Resistance to bile acids

To assess the potential use of probiotic lactic acid bacteria as effective probiotics, it is

generally considered necessary the assessment of their ability to withstand the effects of bile

acids. Solid medium can be supplemented with bovine bile (eg. Sigma Chemical, Poole, UK),

porcine bile (ex.Sigma) and human bile(obtained by laparoscopic cholecystectomy) with final

concentrations between 0.3% and 7.5 %. These plates can be incubated at 37 ⁰ C, under

anaerobic conditions, and growth being recorded after 24-48 hours [36,184].

5.4.3. Bacterial concentration

Different substrates used to assess the adhesion of probiotics have different levels of

potential binding loci. Considering the gastrointestinal secretions, it should be used

concentrations of probiotic physiologically relevant. Consumption of 108 colony forming units

(CFU)/ml could result in concentrations of 105-106 CFU/ml in the small intestine, assuming

that no growth takes place during passage through the upper gastrointestinal tract. The bacterial

adhesion tests are typically used at concentrations of 107-109 CFU/ml [94,184,186].

5.4.4 Incubation time

The effect of incubation time on the adhesion of probiotics has not been fully

investigated. In most of the assays, the probiotics were incubated for 12 h with the substrate.

Some researchers have reported little or no effect of incubation time on the level of adhesion

of propionic bacteria and bifidobacteria. However, the incubation time could have a major

influence on the adhesion observed. This phenomenon could be explained by sedimentation of

microbes [94,184,186]. Regarding the optimal choice of a probiotic microorganism it should

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be considered the followings: 1. microbial strain should resist to the lytic enzymes from saliva

and digestive tract; 2 probiotic strain has to resist the low pH of the stomach (1.5-3.0) more

than a few hours; 3 the probiotic has to be resistant to bile salts (normally indigenous microflora

has this capability); 4 it should produce a low pH to prevent the growth of pathogens and

reduce production of toxins and unwanted substances; 5 the strain has to be resistant to the

antibiotics added in food; 6. Probiotic microbial strain must present the capacity of adherence

to the cells of the intestinal wall; 7 Probiotic bacteria must be alive to proliferate in vivo and

in vitro; 8 it is important the probiotic microorganism resistance in the process of obtaining

final preparation [36, 94,184,186].

5.5. Isolation and selection

The microorganisms from different habitats are isolated and cultivated on solid or

liquid media, in order to study some of their physiological characteristics. In the first phase is

obtained pure culture using established techniques of microbiology (streaking, successive

dilutions). In the second phase microorganisms are sorted by cultivation on petri dishes and

on agar solidified medium [94].

5.5.1 Strains preservation.

After isolation and selection stages, strains are characterized using morphological,

biochemical, physiological, immunological and toxicological methods. The characterized

strain is recorded in a collection of microorganisms using an identification number. Both the

original strains and mutant strains are preserved and maintained by the special methods at low

temperatures for example by lyophilization or storage in freezer below -80°C or in liquid

nitrogen. The stock culture is used in the biosynthesis process and refrigerated on nutrient agar

in inclined tube. The stock culture is the source for the inoculum culture source [94].

5.5.2 Materials used in probiotic growth.

Milk is a supportive environment for the maintenance and cultivation of lactobacilli.

Other growth media are considered for lactic acid bacteria growth: whey, corn starch, potato

mash and molasses [94]. Lactic bacteria ferment the following sugars: glucose, fructose,

galactose, mannose, rhamnose, arabinose, xylose, [94].

5.6 Conditions of cultivation and production of probiotics

The steps for obtaining probiotics are: 1. isolation of probiotics from source,

2.preparation of culture stock, 3.preparation of inoculum 4. fermentation in dedicated vials, 5.

micropilot level of biosynthesis, 6. processing of the culture medium, Jurcoane et al 2004 [94].

Culture for study is obtained by maintaining stock cultures on nutrient agar medium in inclined

tubes. Maintenance medium is tested for each microorganism [79,81,94,120,125,150,175].

5.6.1 Inoculum culture.

The culture study is multiplied by ensuring an optimum nutrient substrate for

developing which involves an optimal combination of the following parameters: pH,

temperature, agitation, and oxygen concentration.The multiplied culture can be seeded from

the synthetic environment in bioreactor under aseptic conditions Jurcoane et al 2004 [94].

PHD THESIS ABSTRACT


5.6.2 Micropilot-level biosynthesis.

Many biosynthesis media with different nutritional substrates are tested, finally

selecting the optimum one. It also envisages assessing the optimal cultivation parameters in the

bioreactor (temperature, pH, dissolved O2 concentration) [2,7,9,24,43,68,70]. It monitors the

increase of the total number of bacteria/ml (TNB/ml) by measuring the optical density hourly.

For probiotics is estimated TNB/ml greater than or equal to 107 as indicative threshold for

completion of the biosynthesis, Jurcoane et al 2004 [94].

Chapter 6

Bioactive factors in breast milk

6.1 Introduction

Breast milk meets all the nutritional requirements of the neonate, protecting the

newborn against infectious diseases, by antimicrobial compounds, immunoglobulins,

immunity cells, prebiotic substances [140,141,179]. The composition of breast milk is the

following: 1.Fat: AG, PUFA-AG, 2.proteins: casein, α-lactalbumin, albumin, β-lactoglobulin,

lactoferrin, IgA, IgG, lysozyme, 3. carbohydrates: oligosaccharides, lactose, 4. minerals: Ca,

phosphorus, sodium, potassium, chloride, 5. bioactive factors [86,126,140,179].

In spite of its complex nature human milk can be easily fractionated by centrifugation

into three major components, namely: 1 whey, 2.casein micelles, and 3. milk fat globules

(MFGs floating) [17,44,78,86,121 ]. Breast milk contains various ingredients needed for

infant growth and development. Among these components a special role is considered for the

specific proteins from milk serum proteins, such as β-casein (b-CN), kappa-casein (k-CN),

α-lactalbumin (a-LA), serum albumin (SA), lactoferrin (LF), lysozyme (LZ),

immunoglobulin A (IgA), C3, C4 (complement fractions) that have important nutritional and

immunological functions [44, 64, 86, 140, 179].

6.2 Nephelometry versus turbidimetry

Numerous methods have been reported as assays used for the analysis of human milk

proteins such as 1. different types of immunoassays, 2. measurement of enzyme activity, 3

polyacrylamide gel electrophoresis, 4. proteins liquid chromatography, 5.ion exchange

chromatography. On the basis of nephelometric and turbidimetric analysis methods is the

phenomenon of diffusion and absorption of light by particles that are solid, or colloid in a

solution [126].

6.2.1 Nephelometry

Nephelometry is the method based on measuring the intensity of scattered light flux

by solid particles that are in a solution, according to the equation of Rayleigh. Nephelometric

method applies to colloidal solution (fine particles), and the measurement depends on the

volume of the particles in suspension. According to Rayleigh's equation, the amount of

scattered light increases according to the increase in particle size at the same total amount of

PHD THESIS ABSTRACT


suspended matter. Suspension should be stable over time.To enhance the stability of the

suspension, protective colloids are often used.

6.2.2 Turbidimetry

Turbidity measurement approach is based on the measurement of the weakening

intensity of luminous flux that has passed through a solution containing solid particles due to

absorption and diffusion lumen. F turbulence coefficient is proportional to the concentration of

particulate matter, therefore, the analogue turbidimetry equation is the fundamental equation

of Lambert-Bouguer-Beer.

6.2.3 Nephelometry used in milk analysis

It represents the immunological analysis of human milk proteins (b-casein, k-casein, α-

lactalbumin, serum albumin, lactoferrin, and lysozyme). Imunonephelometry is based on the

conventional nephelometric quantification of the scattered light of antigen-antibody complexes

formed during the immunoprecipitation reaction liquid phase, and is usually used for the

determination of human serum proteins including IgA, and fractions of the complement C3,

C4. This technique allows measurement of IgA, complement fractions in mature human milk

C3, C4 with precision and accuracy [136].

6.3 Milk bioactive factors

6.3.1 Proteins

Proteins as a major nutrient groups contain a number of bioactive factors, including

immunoglobulins, lactoferrin, lysozyme, lactalbumin, and casein. Specific immunoglobulins

in breast milk (predominantly IGAS, rather than IgM, IgG), work by binding directly to specific

microbial antigens, blocking binding and adhesion, increasing phagocytosis, modulating the

local immune function and thus contributing to the development of baby's immune system.

Lactoferrin iron chelation (limits siderophilic bacterial growth) blocking bacterial adhesion and

the adsorption/penetration of viruses, contributing to the development of intestinal cells and

restoring them (by maintaining a barrier effect) and decreasing production of IL-1,-2,- 6 and

TNF-α from monocytes (immune system modulation) [17,44,45,57]. Lysozyme causes lysis of

the bacterial cell wall, binds endotoxin (limiting effect), increases the production of IgA, and

contributes to the activation of macrophages (immunomodulatory effects). Lactalbumin

transporting calcium is an essential part of the enzyme complex that synthesizes lactose, and

support the growth of bifidobacteria. After being changed in the gut, a lactalbumin called

"human lactalbumin that kill tumor cells," appears to have a role in apoptosis of malignant cells

(immune modulation and immune protection). Casein inhibits adhesion of different bacteria in

different epithelial loci and promotes growth of Bifidobacteria [64, 86,114,140,179].

6.3.2 Carbohydrates

Carbohydrates in milk include lactose and oligosaccharides as major components and

glycoconjugate compounds. They function as the main nutrients in energy production.

Oligosaccharides act as prebiotics stimulating the growth of Lactobacillus and Bifidobacterium

and binding to microbial antigens. Glycoconjugate compounds bind specific bacterial ligands

PHD THESIS ABSTRACT


(V. cholerae) and viral ligands (rotavirus). The oligosaccharides in milk are mainly derived

from lactose. Almost all of them transport the lactose until final reduction; several

glycosyltransferases add more monosaccharides to this basic structure to synthesize complex

oligosaccharides. Human milk is unique in its complex oligosaccharide content (12 to 14 g /

L), including both neutral form (90%) and sialyl form(10%).

6.3.3 Lipids and vitamins

Lipids, the third major nutrient and energy source in breast milk, include triglycerides,

long-chain polyunsaturated fatty acids (LC-PUFA). They have a lytic effect on a variety of

viruses, protozoa, especially against Giardia. Vitamins A, C, E, in addition to their nutritional

effects, have anti-inflammatory effects due to the removal of oxygen radicals. Different

enzymes in human milk have dual functions: catalase has inflammatory effects due to

degradation of H2O2, and glutathione peroxidase reduces inflammation by preventing lipid

peroxidation [17,140].

6.3.4 Nucleotides, nucleic acids and nucleosides

Nucleotides, nucleosides, nucleic acids and related products make up about 15 to 20%

of non-protein nitrogen content in the human milk. In vivo and in vitro experiments suggest a

variety of roles for nucleotides intake: increased iron absorption; the growth of

Bifidobacterium, improving growth and development of the gastrointestinal mucosa recovery,

increasing the NK cell activity and production of IL-2. Several clinical studies on children who

received formula feed, supplemented with nucleotides, have shown small benefits with fewer

episodes of diarrhea and higher plasma levels of IgM and IgA in the group that received the

supplementation. There are a number of factors in human breast milk, which are considered

immune modulating factors. Most of these factors are cytokines, but also soluble receptors of

these cytokines. The list includes IL-1, -3, - 4, -5, -6, -8, -10, -12, IFN-α, TNF-α and TGF-β,

TGF-α, IL-10 and TNF-α recptors which are associated with anti-inflammatory effects. Real

physiological effects and the functions of each of these factors in children have not been

completely elucidated [17,140].

PHD THESIS ABSTRACT


Chapter 7

Principles of encapsulation

7.1 Encapsulating materials

7.1.1 Alginate

Alginate is the most common biomaterial used for the encapsulation of probiotics.

Natural polysaccharide, it is extracted from various types of algae (particularly brown algae),

made up of α-L-guluronic (G) and β-D-mannuronic blocks (M). M/G ratio determines the

functionality of alginate mix. Alginate gel strength is high when the proportion of (G) is high

[4,5,10,35,40]. The microcapsules of alginate can be obtained by extrusion or emulsion.

Alginate gel is susceptible to precipitation in the presence of divalent calcium ions Ca2 + in

excess or in the presence of chelating agents. The alginate solution may be mixed with the

liquid medium containing probiotic (MRS) in a solution of CaCl2 and then added dropwise to

solidify. Maximum load cell in the microcapsules is limited to 25% of the volume [46, 55, 56,

60, 62, 66].

7.1.2 Chitosan

Chitosan is a positively charged polysaccharide and it is formed by deacetylation of

chitin. It is more frequently used as a coating and not as a capsule and in combination with

alginate. Chitosan capsule provides the best protection in bile salt solution (there is an ion

exchange absorption of bile salt). Chitosan appears to have inhibitory effects on lactic acid

bacteria and the other bacteria, viruses, fungi. The lack of solubility in water is a disadvantage

in preventing the complete release of the biomaterial in the intestine having a pH greater than

5.4 so that its applicability is limited basically to nutraceutical products [156,166,187].

7.1.3 Xanthan gum

It is a polysaccharide synthesized by aerobic fermentation by Xanthomonas

campestris. It consists of D-glucose residues β 1.4 linked with a trisaccharide side-chain

attached to the 0-3 D-glucosyl residues in succession. Side chains present in α positions D-

mannopyranose, in β positions D-mannopyranose (4-1), β-D-glucuronic acid (2-1), which

determines the properties of the anionic hydrocolloid. It is used in combination with gellan for

probiotics encapsulation [27,132].

7.1.4 Gellan gum:

It is an anionic liniar heteropolysaccharide having a repetitive tetrasaccharide unit,

composed of rhamnose, D-glucose and D-glucuronic acid in a 1:2:1 ratio. It has the potential

of partially or totally replacement of gelling agents [27,46,132].

PHD THESIS ABSTRACT


7. 1.5 Carrageenan:

Carrageenan can be obtained from red seaweed and it is a linear anionic sulfated

polysaccharide composed of D-galactopyranoza residues alternating linked α-(1/3) and β -

(1/4). Carrageenan presents three types k, l, λ after modifying enzyme substrates (fraction - μ

fraction - δ fraction - σ) which differ in their disaccharidic structure. K-carrageenan matrix

with immobilised lactic acid bacteria can be emulsified in a stable vegetable oil, in a

thermostated reactor [146,169,180].

7.1.6 Cellulose acetate phthalate (CAP)

Cellulose is a highly hydrophilic polymer in the composition of plants and bacteria; In

pharmaceutical industry it is used to control the release of drugs in the gut [27, 46, 54, 66]. The

advantage of the compound is the insolubility in acidic environment (pH <5), but its solubility

at pH> 6, which gives excellent protection properties in gastric conditions. CAP disadvantage

is that it can't form gel particles by ionotropic gelation. Capsules were developed only by

emulsifying and interfacial polymerisation. CAP is widely used as a coating agent

microcapsules Rein et al 2012 [97, 105, 133, 146].

7.1.7 Starch

The starch is a hydrocolloid biopolymer produced from plants in the form of

hydrophilic granules of various sizes . The sources of starch include: potato, corn, rice, wheat.

Resistant starch is resistant to the action of pancreatic amylase in the small intestine and reaches

the colon where it can be fermented. This specificity enables a superior releasing capacity the

large intestine. Starch has prebiotic functionality for encapsulated bacteria. Starch-based

microcapsules were mainly obtained by means of the cross-linked emulsifying

method[112,115,132].

7. 1.8 Gelatin

It is a protein produced by hydrolyzing the collagen from bones and skin. Gelatin

forms a thermosensitive gel and it was used for the encapsulation of probiotics alone or in

combination with other compounds. Due to its amphoteric nature, is an excellent candidate

for combinations with anionic polysaccharides (e.g. alginate and gellan gum). It does not

form particles but can be considered as material for microencapsulation or as a coating

material. Gelatin and K-carrageenan are widely used polymers for coating alginate and

chitosan microcapsules, since it doesn't present any satisfactory encapsulation properties

[156,168,181].

7.1.9 Milk proteins

Represents natural vehicles for probiotics due to their structural and physicochemical

properties. They have excellent gelling properties and this specificity was recently studied by

Heidelbach et al 2009, Livney et al 2010 [27] for the encapsulation of probiotics. Their physico-

chemical properties (low viscosity, indefinite flavor, the ability to form gel) makes them ideal

as encapsulating matrices.

PHD THESIS ABSTRACT


7.1.10 Pectins

Their source is represented by the the residues resulting from the extraction of sugar -

pectin is an anionic biopolymer water soluble and represents a polysaccharide. They have

applicability as colloidal stabilizers, gelling agents and emulsifiers and can be chemically

modified to modulate their ester content for specific applications [132,162].

7.2. Microencapsulation

7. 2.1. Introduction, definitions, overview

Microcapsules represent particles with a diameter in the range of 1-1000μm. Particles

with diameters below 1 micron are nanoparticles, and the particles with diameters more than

1000 μm are called macroparticles [150]. The probiotic microencapsulation process presents

three stages: The first stage consists in incorporating the bioactive component (e.g. probiotic

growth medium) in a solid or liquid matrix. If the matrix is liquid the incorporation process

implies a dispersion or dissolution in the matrix, while in the case of a solid core it involves

adsorption or agglomeration. In the next stage liquid matrix is dispersed, and finally in the 3rd

process chemical stabilization (polymerization), physico-chemical (gelling) or physical

(evaporation, solidification) can be noticed - Poncelet and Dreffier 2007 [27].

7.2.2. Classification of microcapsules

The microcapsules can be described following several characteristics: 1. according to

structure (homogeneous or heterogeneous microspheres) uninucleate single or double coated

microcapsules, multinucleate single or double coated spherical or irregular shaped [6, 51, 71,

95 , 108, 158], 2. depending on the aggregation state of microencapsulated substance (liquid

core microcapsules and solid core respectively); 3. depending on the nature of the polymer

used in microencapsulation (microcapsule based non-biodegradable synthetic polymers and

natural based polymers); 4. depending on how the release of the active substance

(microcapsules immediate release and modified release/target controlled or extended).

Microcapsules can form clusters in the environment they have been obtained [108,150,158].

7.2.3 Membrane

The properties of the membrane refer to the surface charge and the hydrophobicity or

hydrophilicity of the microcapsules, and the degree of porosity and wall thickness. Surface

electric charge is assessed by determining the zeta potential using laser Doppler anemometer

[5, 8,10,47, 58, 96] . Porosity degree is increased when using sodium alginate, reducing

efficiency protection of lactic bacteria (Gouin 2004) [13,27]. The defect may be solved by

using two types of polymers such as sodium alginate and starch (Truelstrup-Hansen et al 2002,

Krasaekoopt et al 2003) [27].

7.2.4. The Nucleus

The microcapsules may be uninucleated or multinucleated, single or double layer

coated spherical or irregular. The core may be liquid or solid [150]. Lactic bacteria (0.5 - 4μm

diameter) are retained well in alginate gel matrix that is estimated to have a pore size smaller

than 7 μm (Klien et al 1983) [103]. Maximum load in microcapsules is limited to 25% of

PHD THESIS ABSTRACT


volume because of poor mechanical strength (Ducholz Luttmann, Zakrezwski and Schugerl,

1980).

7.2.5. The diameter of the microcapsule correlated with the method

Several technologies can be applied to the encapsulation of probiotics and each of them

causes the microcapsules with different characteristics in terms of particle size range and

capsule type. In the emulsion the largest range of values is between 0.2 and 5000 μm, while

in extrusion method the particles sizes are above 300 μm, up to 5000 μm. Spray-coating

method values range between 5 and 5000μm [14, 23, 38, 60-64] while in coextrusion values

range between 20 and 8000μm (Mc Master et al 2005)[27]. Spray-drying method generates

microcapsules with diameters between 20 and 300μm. It should be noted that in the emulsion

case there is a range of 0.2-1μm which is below the lactic acid bacteria (1-5 μm) [27,74,95-

100,101,112,118].To avoid the negative sensorial impact it is desirable to obtain microcapsules

with less than 100 μm (Truelstrup - Hansen et al 2002) [27,62,104,169] (Lahail A et al 2010)

[127,166].

7.3. Microcapsules analysis methods

7.3.1. Determining the form and size of the microcapsule

Different methods have been described [27,150]: 1.Optical microscopy (1-50 μm),

Scanning electron microscopy (0.05-500 μm), Transmission electron microscopy (0.001-500

μm) [127]; 2.Coulter analysis for the determination of particle size in the range (0.1-1000 μm);

3. Photon correlation spectroscopy applied to microcapsules having sizes of less than 1μm; 4

Laser Doppler Anemometry to assess surface charge by zeta potential determination; 5

Confocal microscopy using FITC-labeled chitosan to penetrate polymer network sodium

alginate; 6. Electron microscopy image using a scanning beam of electrons with the specimen.

7.3.2. Determining the microcapsule concentration

1.Turbidimetry is a method for measuring the concentration of a component in the

solution. Turbidity refers to the opacity of the liquid caused by the particles in suspension

reflecting light; 2 Spectroscopy analysis is an experimental method that measures infrared

electromagnetic radiation interaction with matter [53,131]; 3 Thermal analysis of the matrix

making up the microcapsules involves a group of techniques that measure a physical property

of a substance according to the temperature. For the analysis of probiotic encapsulation

biopolymer matrix in microcapsules there are described several methods: TGA

thermogravimetric analysis, differential thermal analysis TDA, DSC differential scanning

calorimetry; 4 X-ray diffractometry, a technique used to analyze the powders. X-rays are

scattered in all directions. It measures the angles of diffraction in which the peaks occur ; 5

Atomic force microscopy [26,42,93] is a technique at room temperature, using an atomic

force microscope with a cantilever applying a nano-sized tip force of pN (piconewton) orders

on microcapsule surface or cell surface.

PHD THESIS ABSTRACT


The Experimental Part

Chapter 8

Materials and methods in the proposed research

8.1. Harvesting and analysis of milk samples

The study was conducted in Sibiu Pediatric Clinical Hospital between 2011-2012.

There have been collected 100 samples of milk as follows: 52 samples of breast milk and 48

samples of powdered milk for the purpose of biochemical analysis. There have been collected

also blood samples from the 100 infants (breastfed and formula fed) for biochemical analysis

purposes. Analysis of IgA and PCR was performed by Hitachi 912 immunoturbidimetry device

and Genio device was used for protein electrophoresis. Samples from each milk probe were

seeded in the specific medium for the growth of Lactobacilli. Isolates from MRS agar were

transferred to MRS broth, broth (same ingredients without the agar) and preserved with

glycerol in cultures at -80 ° C in a freezer.

8.2. Identification of Lactobacillus spp bacterial strains.

Identification of the isolated and glycerol culture preserved strains in the freezer (-70

° C) was achieved by restoring cultures in MRS broth, MRS agar medium cultivation,

anaerobic incubation for 48 h (10% CO2, temperature 37⁰C). After obtaining isolated colonies,

the strains were examined to test their belonging to the group of lactic acid bacteria: Gram (+)

on Gram stained smears, negative catalase test and oxidase test). Identification of bacterial

strains of breast milk isolated Lactobacillus ssp has been performed using API 50 CH galleries

in Pediatrics Clinical Hospital Sibiu.

8.3 Bioreactor growth of the strains isolated from breast milk

The experiments were conducted in the Laboratory of Microbiology and

Biotechnology, in Faculty of Agricultural Sciences, Food Industry and Environmental

Protection. For the initial propagation of lactic acid bacteria one cryogenic vial stored at -80 °

C containing 1 ml for each strain has been used. Bioreactor growth was carried out in order to

obtain biomass using an IKA fermenter, Economy 10, 2l capacity, with thermostat interface,

pH sensor, temperature, oxygen. The substrate used medium had a specific composition g/l

(glucose-25, yeast extract 25, Peptone-25). The optical density was recorded hourly using a

spectrophotometer at 600 nm following a proper dilution, between 0-24 hours(Malek et al

2010).

8.4 In vitro evaluation of probiotic potential of bacterial strains

Experiments were conducted in the Laboratory of Microbiology and Biotechnology

from the Faculty of Agricultural Sciences, Food Industry and Environmental Protection.

Strains of the preserved lactic acid bacteria were passed in MRS agar medium. After incubation

Lactobacillus strains were transferred to three different culture media simulating the digestive

PHD THESIS ABSTRACT


conditions (the first growth medium with pepsin, the second growth medium with bile, the third

growth medium with HCl). We have analyzed and compared statistically (Annova test) for

each growth medium the average values of optical densities at each point in time. For each

strain there has been expressed as percentages the increases and the decreases of the values

compared with the initial starting values.

8.5 Microencapsulation of Lactobacillus paracasei ssp Paracasei (L18) strain

Encapsulation of Lactobacillus paracasei ssp Paracasei (L18) strain isolated from

breast milk and kept in culture with glycerol at -70 ° C in the freezer was performed in the

biochemistry and pharmaceutical technology laboratories from the Faculty of Medicine

V.Papilian of Sibiu, by restoring MRS broth culture (bacterial suspension) which was added in

the encapsulating agent sodium alginate 2%. We have used four methods: Method 1

emulsification / gelation ionotropic (MRS broth, 2% sodium alginate, solution buffers, calcium

carbonate suspension 500 mM Ca2+, distilled water, CaCl2 0.05mmol / l, sunflower oil or

other vegetable oil, TWEEN 80, 80% acetic acid; Methods 2,3,4 - extrusion(MRS broth, 1%

or 2% sodium alginate, ¼ strength Ringer's solution +/- distilled water, buffer solution, CaCl2

0.05mmol / l, syringe, filter paper, magnetic stirrer, filtration instalation with vacuum pump).

After harvesting the filtered material from the very low porosity filter, the samples were spread

on a slide and stained with Lugol and/or methylene blue (laboratory techniques used in the

laboratory department of Pediatrics Clinical Hospital Sibiu). The obtained microcapsules were

analyzed under a microscope.

PHD THESIS ABSTRACT


Chapter 9

Evaluation of bioactive immune factors in breast milk and milk powder

9.1 Introduction

Some studies have shown that the same chemokine (CCL28) is responsible for both the

accumulation of secretory IgA-producing cells in the mammary glands to stimulate secretion

of IgAs in breast milk but also for the passive transfer control of IgAs in breast fed infants.

Other studies indicate that there are two mechanisms that regulate the ontogeny of circulating

IgA in the newborn and infant: 1.The first mechanism is represented by intestinal absorption

of IgA in breast milk (especially colostrum where the concentration of IgA which is greater);

2. The second mechanism is the endogenous production of IgA (eg intestinal) [44,57].

9.2 Objectives

1. Study of immune protection of breast fed infants compared with those fed with milk

powder formula

2. Identification of bioactive factors involved in immune protection, in breast milk and

formula milk powder

3.Impact study of IgA levels in breast milk and milk powder on serum

immunoglobulins (IgA and IgG) in infants

4. Study of milk lactose levels impact on immunoglobulins (IgA and IgG) in serum of

breast fed infants

9.3 Materials and methods

The study was conducted in Sibiu Pediatric Clinical Hospital between 2011-2012.

There have been collected 100 samples of milk as follows: 52 samples of breast milk and 48

samples of powdered milk for the purpose of biochemical analysis. There have been collected

also blood samples from the 100 infants (breastfed and formula fed) for biochemical analysis

purposes. Analysis of IgA and PCR was performed by Hitachi 912 immunoturbidimetry device

and Genio device was used for protein electrophoresis. Samples from each milk probe were

seeded in the specific medium for the growth of Lactobacilli. Isolates from MRS agar were

transferred to MRS broth, broth (same ingredients without the agar) and preserved with

glycerol in cultures at -80 ° C in a freezer. Assessment of human milk physical and

biochemical parameters was performed using ultrasonic analyzer. Pearson correlations were

studied according to the objectives of the study

9.4. Results

1. The maternal milk protein content was 3.45 g/dl (mean) with a minimum of 3.24 g/dl

and a maximum of 3.5 g/dl using the ultrasound Ekomilk total. The same samples were dosed

on electrophoresis apparatus Genio S with the following values (mean 1.021 g/dl with a

PHD THESIS ABSTRACT


minimum of 0.268 and a maximum value of 1.48 g/dl). There is a discrepancy between the

measured values provided by the ultrasonic analyzer and the protein electrophoresis device.

2 Fat content of the milk samples had a mean of 3.85 g/dl with a minimum of 0.79 g/dl

and a maximum value of 7.64 g / dl. Dosage of fat content was made only ultrasonic analyzer

and is appropriate.

3. Lactose content of milk samples had a mean of 5.021 g/dl with a minimum of 4.76

g/dL and a maximum value of 5.25 g/dl. The results obtained using ultrasonic analyzer are

comparable to those reported in the composition of cow's milk (4.5 g/dl up to 5 g/dl).

4 Mean Ph value was 7.042 with a minimum of 6.8 and a maximum of 7.16. It should

be noted that low levels of pH together with lactose promote lactic acid bacteria growth on

intestinal mucosa.

5 IgA levels from breast milk samples have shown positive correlations with β-

globulins (p <0.05) and did not show any correlation with lactose (p = 0.35> 0.05), with α1, α2

globulins or with the values of serum CRP, IgA, IgG in breastfed infants .

6 IgA levels in samples centrifuged from formula showed negative correlations with

infants serum CRP levels and didn’t show any correlation with α2, β, γ globulins from the

formula. Average concentration of IgA in breast milk samples was significantly higher (83.71

mg/dl) than the average of IgA concentration in milk powder samples (9.45 mg/dl)

7 β-globulin levels in breast milk were statistically significant in negative correlation

with the levels of γ-globulin and α1-globulin, suggesting a major role in family of globulines;

γ-globulinele positively correlated with infants levels of serum of IgA and IgG .

8 Lactose correlated negatively, statistically significant, with IgA serum titers in

infants. In milk powder formula we couldn’t fiind any statistically significant correlations

between the various globulin fractions α2, β, γ, or between globulin fractions and IgA . There

are, however, positive correlations between α2 titers in formula and PCR serum levels in

infants suggesting an antiinfective protection role.

Fig. 9.1- IgA LM vs LP (personal archive) Fig. 9.2- globulins LM vs LP(personal archive)

PHD THESIS ABSTRACT


Chapter 10

Identification of Lactobacillus ssp strains

10.1 Introduction

Isolation of lactic acid bacteria from various sources is performed on a special medium

MRS (Man Rogosa Sharpe) using the plate technique. Isolates are examined by colony

morphology, catalase reaction and Gram reaction. Lactic bacteria are stained gram positive and

catalase-negative [14,98,102,117]. Phenotypic identification, besides morphological

characterization (optical microscopy, Gram stain), catalase test (negative) implies also the

fermentation of sugars (hexoses) [14,102,113,117,142]. Fermentation of sugars can be

highlighted by API-20CH and API-50CH tests for lactobacilli [20,48,117,188,189,190].

10.2 Objectives:

1 Identifying lactic acid bacteria types of strains isolated from breast milk

2 Specifying the accuracy of identification for each strain using API CH50 method

3 Evaluating the opportunity to use metabolic fermentation testing as the first step

approach in identifying the probiotics isolated from breast milk

10.3. Materials and methods:

Identification of Lactobacillus ssp isolated from human milk was carried out by means

of API 50 CH gallery. This is a standardized system that combines 50 biochemical tests,

allowing the study of microorganisms carbohydrate metabolic profiles. API 50 CH galleries

are used in conjunction with API 50 CHL medium, to identify genus and species of lactobacilli.

10.3.1 Principle of the method:

API CH 50 galleries are composed of 50 microtubes and each microtube contains a

material belonging to the family of carbohydrates and their derivatives: heterosides,

polyalcohols, uronic acids. A suspension of microorganisms in the API 50 CHL is prepared to

inoculate each tube of the gallery, rehydrating the substrate and incubating it in anaerobic

atmosphere. Meanwhile carbohydrate fermentation will result in changing the color in the tube,

due to the production of acid and highlighted by the environmental pH indicator. Oxidative and

fermentative pathways determine the color change due to pH change. Initially the color is

purple, and it turns to yellow because of acid formation during anaerobic conditions

[20,113,189]. A color between green and yellow is considered unsatisfactory [15,20,165,184].

The results represent the biochemical profiles of isolated bacterial strains and enable their

identification using a dedicated software.

PHD THESIS ABSTRACT


Fig 10.1- Synopsis of the samples worked on API kits

(personal archive)

10.4. Results

In our study using sugar fermentation method, we have been identified 10 strains of

probiotic lactic acid bacteria as follows: 7 strains of Lactobacillus paracasei ssp paracasei, 1

strain of Lactobacillus fermentum, 1 strain of Lactobacillus acidophilus ssp acidophilus and

1 strain of Lactococcus lactis ssp lactis.

Identification of 7 heterofermentative strains of Lactobacillus paracasei ssp paracasei

was performed with a percentage of 99.8% and a T index over 0.88, more than the percentages

described in the literature.

Identifying the strain of Lactobacillus fermentum was made with 99.8% identification

rate and a 0.96 T index, more than the values described in the literature (63%). Identifying

Lactobacillus acidophilus ssp acidophilus strain was performed with a percentage of 89.6%

and a T index of 0.51, representing a low taxonomic significance, below the values quoted in

the literature (over 93%).

Identifying Lactoccocus lactis ssp lactis strain in the study was carried out with a rate

of 50% considered an unsatisfactory identification as to the values described in the literature

(84.2%). Of the 10 strains isolated in this study only two strains (Lactobacillus acidophilus ssp

acidophilus and Lactoccocus lactis ssp lactis) were identified below the percentages described

in the literature.

PHD THESIS ABSTRACT


Chapter 11

Bioreactor growth of the strains isolated from breast milk

11.1 Introduction

The multiplication of lactic acid bacteria depends on the quality and quantity of the

inoculum, substrate concentrations, pH, temperature, dissolved oxygen, and includes several

stages: 1. lag phase ; 2 exponential growth phase (logarithmic multiplication); 3. maximum

stationary phase; 4 declining phase. For each strain the increase of optical density (OD) during

the cycle is plotted against time. Modeling growth of lactic acid bacteria in bioreactors [70,

79,175] is performed using a well known model - the model of Monod.

11.2 Objectives

The research on bioreactor growth of probiotic strains isolated from human breast milk

had the following objectives: 1. studying the differences in performances (maximum OD)

between all isolated strains; 2.studying the optimal combination regarding the following

parameters pH, oxygen concentration, temperature in correlation with the maximum optical

density.


The experiments were conducted in the Laboratory of Microbiology and

Biotechnology, in Faculty of Agricultural Sciences, Food Industry and Environmental

Protection. For the initial propagation of lactic acid bacteria one cryogenic vial stored at -80 °

C containing 1 ml for each strain has been used. Bioreactor growth was carried out in order to

obtain biomass using an IKA fermenter, Economy 10, 2l capacity, 7 ports, with thermostat

interface, pH sensor, temperature, oxygen. The substrate used medium had a specific

composition g/l (glucose-25, yeast extract 25, Peptone-25). The optical density (OD) was

recorded hourly, using a spectrophotometer at 600 nm following a proper dilution, between 0-

24 hours(Malek et al 2010).

11.4. Results

The highest values of the optical density were recorded in Lactobacillus paracasei ssp

paracasei. Basically for L16 strain, OD had the highest value (OD = 1.994), for L21 OD was

1.992, for L18 OD value was 1.987, and for L22 OD recorded value was 1.972.

Interestingly, for the remaining three Lactobacillus paracasei ssp paracasei strains the

maximum OD difference is noticeable, 1.897 for L14, 1.895 for L15 at constant temperature

and variable pH and oxygen, and 1.887 for L6, but for L6 the conditions were different in the

sense that the parameters pH and temperature were variable and oxygen concentration was

constant.

PHD THESIS ABSTRACT


Lactoccocus lactis strain reached a maximum of 1.897 OD under constant temperature

conditions with varying pH and dissolved oxygen values, a performance comparable with the

strains of Lactobacillus paracasei ssp paracasei.

L.acidophilus ssp acidophilus (L12) strain reached a maximum OD of 1.878 but all

three parameters (pH, temperature, oxygen) were variable.

In Lactobacillus fementum strain (L1), maximum OD had the lowest value compared

with the other strains, 1.779, while all three parameters (pH, temperature, oxygen) were

variable. Our results on the strains of Lactobacillus paracasei ssp paracasei and Lactobacillus

acidophilus ssp acidophilus (regarding environmental growth conditions) are in accordance

with literature data. These strains grow at the optimal concentration of the glucose/fructose

from 10 to 25% [7,120], pH values between 5.5-6.5 and a temperature of 37 ° C [43,68,175].

Fig 11.1– Synopsis of optical density (for all strains) (personal archive)

PHD THESIS ABSTRACT


Chapter 12

In vitro evaluation of probiotic potential of bacterial strains

12.1 Introduction

In vitro evaluation involves assessing probiotic potential, basically its resistance to

gastric acidity and bile toxicity, adhesion to intestinal epithelial tissue, ability to colonize the

gastrointestinal tract, production of antimicrobial substances, and the ability to modulate

immune responses [10,35,55,56]. Probiotics must survive the transit through the stomach.

Survival of strains is first evaluated by adding 1x109 CFU lactobacillus strains and 1x1011

CFU bifidobacteria strains to the modified MRS medium with hydrochloric acid to obtain a

pH between 1.5 and 3.4. The assessment of probiotic resistance ability by exposure to bile

acids, is performed on liquid or solid medium supplemented with bovine, porcine or human

bile (obtained by laparoscopic cholecystectomy) supplementing the medium so that the final

concentrations would range from 0.3% to 8% .

12.2 Objectives

Research studies on resistance of isolated strains from breast milk were aimed at

studying the resistance of each strain in the pepsin, bile and HCl environments in vitro

conditions that simulate gastrointestinal environment


Preserved Lactobacillus strains are passed on MRS agar medium, incubated for 48

hours at 37 ° C in 10% CO2 atmosphere. After incubation, the lactobacillus strains are

transferred to the three culture media simulating the digestive juices (Medium 1, 2, 3). Culture

media (Medium 1, Medium 2, Medium 3) had the following composition: Medium 1 with

pepsin g/l (1.glucose-3.5, 2.NaCl-2.05, 3.KH2PO4-0,6, 4.CaCl2-0.11, 5.KCl-0.37 ; 6.Pepsine-

13.3); Medium 2 with bile g/l (1.glucoză-3.5; 2.NaCl-2.05; 3.KH2PO4-0,6; 4.CaCl2-0,11;

5.KCl-0.37, 6. pepsin - 13.3; 7.bile-0.05); Medium 3 with HCl g/l (1.glucoză-3.5, , 2.NaCl-

2.05, 3.KH2PO4-0,6, 4.CaCl2-0,11, 5.KCl-0.37, 6. pepsin-13.3; 7.HCL -1 M); Medium 4 was

used to obtain bacterial colonies cultured in fresh MRS agar.

We have analyzed and compared statistically (Annova test) for each growth medium

the average values of optical densities at each point in time. For each strain there has been

expressed as percentages, the increases and the decreases of the optical density values

compared with the initial starting values.

12.4 Results

1. L.paracasei ssp paracasei (p2-L6) shows a 100% increase in the pepsin growth

medium, a 74.22% increase at 30 ', followed by a decrease of 10.3% at 120' in the bile medium

and a decrease of 6.6% in HCl medium;

PHD THESIS ABSTRACT


2. L.paracasei ssp paracasei strain (p5-L15) shows an increase of 78.6% in the pepsin

growth medium at 120 ', a decrease of 20.9% in the bile medium at 120' and a decrease of

2.98% in the HCl medium ;


medium, a decrease of 14.48% in the bile medium and a decrease of 12% in the HCl medium

at 120 ';

4. L.paracasei ssp paracasei strain (p7-L18) shows an increase of 124% in the pepsin

medium, a decrease of 20.5% in the bile medium and an increase of 1.35% in the HCl medium

at 120 ';

5. L.paracasei ssp paracasei strain (p8-L21) shows an increase of 89% in the pepsin

medium, a decrease of 13.2% in the bile medium and a decrease of 8.6% in the HCl medium

at 120 ';


medium, a decrease of 23.8% in the bile medium and an increase of 18.3% in the HCl medium

at 120 ';

7 L.acidophilus ssp acidophilus strain (p3-L12) shows an increase of 49.15% in the

pepsin medium, a decrease of 12.9% in the bile medium with a drop of 33.67% in the HCl

medium at 120 ';

8 L.fermentum strain (p1-L7) shows an increase of 78.8% in the pepsin medium, a

decrease of 2.75% in the bile medium with a drop of 14.1% in the HCl medium at 120 ';

9 Lactoccocus lactis ssp lactis strain (p4-L14) shows an increase of 35.57% in medium

with pepsin, a decrease of 3.7% in the bile medium with a drop of 3.7% in the HCl medium at

120 '.

10 . L.paracasei ssp paracasei strain (p11-L8) shows a 70% increase in the pepsin

medium, a decrease of 29.28% in the bile medium and a decrease of 7.17% in the HCl medium

at 120 ';

11 Survivability capacity is specific for each strain.

Fig. 12.1 – Evolution of the optical density in each medium for each strain

PHD THESIS ABSTRACT


Chapter 13

Microencapsulation of Lactobacillus paracasei ssp Paracasei (L18) strain

13.1 Introduction

Several technologies can be applied to the encapsulation of probiotics and each of them

causes the microcapsules with different characteristics in terms of particle size range and

capsule type. With emulsion method it can be obtained the largest interval with values between

0.2 and 5000μm, while extrusion method causes particles above 300μm to 5000μm.

13.2 Objectives

1 Analysis of the mean diameter of the microcapsules obtained by emulsion (method

1) versus the mean diameter of the microcapsules obtained by extrusion (method 2).

2 Analysis of the sodium alginate concentration influence on the microcapsules

diameters obtained by the extrusion method (3 and 4) also applying a correlation with the shape.

3 Analysis of the wall thickness of the microcapsules obtained by all 4 methods.

4 Analysis of the sodium alginate concentration influence over the thickness of the

microcapsules obtained by the methods 3 and 4.

13.3 Materials amd methods

Encapsulation of Lactobacillus paracasei ssp Paracasei (L18) strain isolated from

breast milk and kept in culture with glycerol at -70 ° C in the freezer was performed in the

biochemistry and pharmaceutical technology laboratories from the Faculty of Medicine

V.Papilian of Sibiu, by restoring MRS broth culture (bacterial suspension) which was added in

the encapsulating agent sodium alginate 2%. We have used four methods:

Method 1 emulsification / gelation ionotropic (MRS broth, 2% sodium alginate,

solution buffers, calcium carbonate suspension 500 mM Ca2+, distilled water, CaCl2

0.05mmol / l, sunflower oil or other vegetable oil, TWEEN 80, 80% acetic acid;

Methods 2,3,4 - extrusion(MRS broth, 1% or 2% sodium alginate, ¼ strength Ringer's

solution +/- distilled water, buffer solution, CaCl2 0.05mmol / l, syringe, filter paper, magnetic

stirrer, filtration instalation with vacuum pump).

After harvesting the filtered material from the very low porosity filter, the samples

were spread on a slide and stained with Lugol and/or methylene blue (laboratory techniques

used in the laboratory department of Pediatrics Clinical Hospital Sibiu). The obtained

microcapsules were analyzed under an inversed microscope Zeiss Axiovert 40 using

AxiovisionLE-Axiovert 4.8 software.

PHD THESIS ABSTRACT


13.4 Results

We have obtained the following results presented in accordance with the objectives of

the microencapsulation study :

1. Mean diameters obtained by emulsion are smaller than the extrusion mean diameters,

only for ranges between 0-99 μm and above 300 μm but in the ranges of 100-199 μm

and 200-299 μm we have obtained higher diameters using emulsion method

Fig 13.1- emulsion vs extrusion Fig 13.2- wall thickness depending on the method

(personal archive) (personal archive)

2. Increasing sodium alginate concentration in the extrusion method we have obtained

increased diameters regardless of the form of microcapsules.

Fig 13.3- thickness and diameters Fig 13.4- wall thickness depending on the method

depending on the method (personal archive)

(personal archive)

3 The wall thickness mean of the microcapsules obtained by emulsion method is lower

than in extrusion method where it depends upon the concentration of alginate and the presence

of the strengthing substance .

PHD THESIS ABSTRACT


Chapter 14

Conclusions

14.1 General conclusions

Data analysis suggests the following conclusions presented in accordance with the

objectives of the study (analysis of milk samples, identification of probiotic strains isolated

from breast milk, their growth in the bioreactor, analysis of resistance to conditions simulating

the gastrointestinal environment, the benefits of different kinds of encapsulation methods of

isolated strains in accordance to the type of method and characteristics of the microcapsules

diameter and wall thickness):

1. Breastfed infants with respiratory infections have a higher immune protection

compared to those fed with formula primarily through IgA titers and β globulins levels and

secondary by γ-globulin titer.

2 The titer of IgA from human breast milk samples does not affect IgA and IgG titers

in the serum of breastfed infants and the concentration of lactose correlates negatively with

serum IgA titer of these infants.

3 Identification of the 10 strains of lactic acid bacteria by fermentation of sugars

method (API 50 CH test) was performed with a succesful rate of over 99% and the T index of

more than 0.88 for eight strains (7 strains of Lactobacillus Paracasei ssp paracasei and 1 strain

of Lactobacillus fermentum).

4 Identification of lactic acid bacteria by sugar fermentation method (API 50 CH test)

was performed for the remaining 2 strains (L. acidophilus ssp acidophilus, Lactoccocus lactis

ssp lactis) with a percentage below 90% which implies a lower taxonomic significance of

these subspecies;

5 The method of sugar fermentation tests (API 50 CH) can be used as the first

identification step.

6. Growth in bioreactor has shown the highest values for optical density in

Lactobacillus paracasei ssp paracasei strains L16, L18, L14 (1.994, 1.992 and 1.987

respectively) and Lactoccocus lactis ssp lactis L14 (1.897) under the same environmental

conditions (constant temperature, variable pH, variable oxygen), while the minimum optical

density was recorded for Lactobacillus fementum L1 (1.779) in terms of variable pH,

temperature and oxygen.

7. All the strains have shown an increase in optical density values of over 35% after 2

hours of exposure to pepsin environment, a decreasing optical density values of more than

2.75% after 2 hours of exposure to the bile environment.

8.Most strains present a decrease in the optical density values of over 2.98% in HCl

acid medium excepting L.paracasei ssp paracasei strains (L21, L22) showing a slight increase.

9 Mean diameters obtained by emulsion are smaller than the extrusion mean diameters,

only for ranges between 0-99 μm and above 300 μm but in the ranges of 100-199 μm and 200-

299 μm we have obtained higher diameters using emulsion method .

PHD THESIS ABSTRACT


10. Increasing the concentration of sodium alginate with the extrusion technique has

increased the diameters of the generated microcapsules regardless of the shape and has

decreased the thickness of the microcapsule wall.

11. The wall thickness of the generated microcapsules is thinner using the emulsion

compared with the extrusion technique.

14.2 Recommendations

Similar research projects for probiotics isolated from human breast milk, involving milk

analysis, isolation, identification, in vitro resistance testing, growth in bioreactors, it should be

taken into account the following aspects:

1. In order to validate human milk IgA and β-globulins dynamics versus γ-globulin,

further studies on breastfed infants cohorts infants with respiratory infections are needed with

the analysis of human milk .

2 It is desirable that identification of lactobacilli and bifidobacteria strains should

involve also genomic analysis(PCR RFLP, genetic sequencing) because many data from

literature have indicated confusion regarding the taxonomic characterization of strains.

3 Fermentation of carbohydrates using API test method is recommended as the first

step to identify the genus.

4 Lactic acid bacteria isolated from human milk belonging to Lactobacillus paracasei

species require optimal growth conditions in the bioreactor with constant temperature values

and variable values for pH and oxygen.

5 The design of further studies regarding probiotic strains isolated from human breast

milk should include both research on individual environments (eg only pepsin, only bile or

only hydrochloric acid) but also a medium that combines all three components (pepsin, bile ,

and hydrochloric acid).

6 Further studies should target the testing of nonencapsulated lactic acid bacteria versus

encapsulated lactic acid bacteria in the presented growth media

14.3 Own contributions and future development trends of research

The research conducted has revealed many original elements that define practical

personal contributions:

1. With regard to the evaluation of bioactive immune factors in breast milk and milk

powder we have to mention the successful use of immunoturbidimetry for serum analysis after

removal of the supernatant samples from breast milk and milk powder samples. In the Pediatric

Hospital of Sibiu this method was used only for analysis of serum obtained from the blood of

patients but it proved feasible in serum analysis of milk samples.

2. It should be noted that the analysis of the physicochemical properties of human

breast milk was successful for the determination of fat and pH on the ultrasonic analyzer in

food safety testing laboratory from the Faculty of Agricultural Sciences, Food Industry and

Environmental Protection.

PHD THESIS ABSTRACT


3. Interesting results have emerged regarding the immune protection of infants

achieved mainly by of IgA and β-globulin titers and secondary by γ-globulin titers without any

influence on serum IgA, IgG titers in infants.

4. Identification with higher percentages compared with those described in the literature

for 7 strains in Lactobacillus paracasei ssp paracasei species and 1 strain of Lactobacillus

fermentum using the sugar fermentation method to these two subspecies.

5. Presentation of performance differences related to the bioreactor growth for isolates

belonging to Lactobacillus paracasei ssp paracasei in the same environmental conditions (

constant temperature, variable pH, oxygen).

6. Identification of at least two strains of Lactobacillus paracasei ssp paracasei with

increased resistance after 120 minutes of exposure in the HCl medium and increased resistance

in the Lactobacillus fermentum species and Lactoccocus lactis ssp lactis in bile medium.

7. Performing 1299 measures on microcope slides samples (519 measurements on

microcapsules obtained by emulsion versus 780 on extruded microcapsules) almost 13 times

more than the minimum of 100 random measurements recommended in the literature.

8. Documentation of sodium alginate concentration influence and the presence of the

strengthening substance on the results regarding microcapsule wall thickness.

9. Using filters of less than 40 μm we have shown that the distribution of diameters

for the majority of generated microcapsules by emulsion and extrusion is in the range of 0-99

μm.

10. We have obtain a nutraceutical product, namely a Lactobacillus paracasei ssp

paracasei strain of human origin encapsulated in 1-2% sodium alginate microcapsules.

Future directions for the development of this research outlines the followings:

1.The posibility to achieve on an industrial scale the process of encapsulating this strain

2.The posibility of milk fermentation or addition of encapsulated strains derived from

milk products (yogurt, cheese)

PHD THESIS ABSTRACT


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utile în analiza compoziției probelor de lapte matern și lapte praf, Acta Medica Transilvanica nr. 3 septembrie

2014 , ISSN-L 1453-1968

137. Neamțu Bogdan, Tița Ovidiu, Felicia Gligor, Neamțu Mihai, Tița Mihaela, Sbârcea Claudia, Hila Mirela,

Maniu Ionela, A comparative study of emulsion and extrusion as encapsulation methods for probiotic growth

media, International Journal of Science and Advanced Technology, 2014, ISSN: 2221-8386

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139. Neamțu Bogdan, Ketney O, Tița AM, Tița O., Hila M, Melaniea M, Neamtu LM , Neamtu C , Maternal and

Endogenous IgA Protection in Infants with Respiratory Tract Infections Archives of Disease in

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Publications:

1. Neamțu Bogdan, Tița Ovidiu, Felicia Gligor, Neamțu Mihai, Tița Mihaela, Sbârcea Claudia,

Hila Mirela, Maniu Ionela, A comparative study of emulsion and extrusion as encapsulation

methods for probiotic growth media, International Journal of Science and Advanced

Technology, Volume 4, Issue 8, August 2014 ISSNs: 2221-8386

2. Neamțu Bogdan, Tița Ovidiu, Neamțu Mihai, Tița Mihaela, Hila Mirela, Maniu Ionela,

Identification of probiotic strains from human milk in breastfed infants with respiratory

infections, Acta Universitatis Cibiniensis Series E: FOOD TECHNOLOGY Vol. XVIII (2014),

no. 2 , ISSN:2344-150X

3. Neamțu Bogdan, Tița Ovidiu, Neamțu Mihai, Tița Mihaela, Hila Mirela, Maniu Ionela,

Metode de laborator utile în analiza compoziției probelor de lapte matern și lapte praf, Acta

Medica Transilvanica nr. 3 septembrie 2014 , ISSN-L 1453-1968

4. Chicea D, Neamțu B , Chicea R., Chicea L. M. - The Application of AFM for Biological

Samples Imaging , Digest Journal of Nanomaterials and Biostructures, Vol. 5, No 3, July -

September 2010, p. 1033 - 1040;ISSN 1842 - 3582

5. Neamțu B , Ketney O.Tița, M.Tița, Hila M, Melaniea M, Neamțu LM , Neamțu C , Maternal

and Endogenous IgA Protection in Infants with Respiratory Tract Infections Archives of

Disease in Childhood 2012 Oct: 97(Suppl 2): 1-58 ISSN 14682044;

6. Neamțu Bogdan, Grupul Roman de Experți in Nutriție Pediatrică, Recomandări nutriționale

în practica pediatrică, Capitol Nutriția în Terapia Intensivă, Editura Universitară Carol Davila

București, 2013, ISBN : 978-973-708-697-6;

7. S.I. Iurian, S. Iurian, M.L. Neamțu, B.M.Neamțu, V. Bunescu. Statistic Evaluation of

Streptococus Resistance to antibiotics in children. Pediatric Research 68, 591-591

doi:10.1203/00006450-201011001-01195 ISSN: 0031-3998, November 2010

8. S.I. Iurian, S. Iurian, M.L. Neamtu, B.M. Neamțu, B.I. Mehedintu. Epidemiological Aspects of

Salmonella and Shigella infections in children. Statistics of Pediatric Clinic , Pediatric Research

68, 591-591 doi:10.1203/00006450-201011001-011962010 ISSN: 0031-3998 November 2010

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