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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
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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].
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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
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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
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(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].
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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].
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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.
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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
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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.
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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
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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.
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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
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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)
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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.
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NEAMȚU BOGDAN 27 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
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.
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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.
11.3 Materials and methods
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.
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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)
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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
12.3 Materials and methods
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;
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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 ;
3. L.paracasei ssp paracasei strain (p6-L16) shows an increase of 81.53% in the pepsin
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 ';
6. L.paracasei ssp paracasei strain (p9-L22) shows an increase of 75.35% in the pepsin
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
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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.
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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 .
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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 .
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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.
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NEAMȚU BOGDAN 36 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
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)
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Bibliography
1. Abd-Elhamid A.M., Production of Functional Kariesh Cheese by Microencapsulation of Bifidobacterium
adolescentis ATCC 115704, Advance Journal of Food Science and Technology 4(2) : 112-117, 2012, ISSN :
2042-4876;
2. Abd Malek Roslinda, Production of Lactobacillus salivarius, a new probiotic strain isolated from human
breast milk, in semi-industrial scale and studies on its functional characterization; Current Research,
Technology and Education Topics in Applied Microbiology and Microbial Biotechnology,A.Mendez-Vilas
(Ed),pp 1196-1204,ISBN978-84-614-6195-0 FORMATEX 2010;
3. Adhilkari K., Mustapha A., Grun I.U., L.Fernando, Viability of microencapsulated Bifidobacteria in set
yogurt during refrigerated storage, Journal of Dairy Science.83 (2000) 1946-1951; ISSN:0022-0302;
4. Agrawal Renu, Probiotic: an emerging food supplement with health benefits. Food Biotechnology, pp 227-
246,vol 19,number 2, 2005; ISSN: 0890-5436 print DOI: 10.1080/08905430500316474;
5. Akhiar Mohammed,. Enhancement of probiotics survival by microencapsulation with alginate and prebiotics.
MMG 445 Electronic Journal of Biotechnology(6):13-18. 1; 2010
6. Albertini Beatrice, Passerini Nadia, Di Sabatino Marcello, Vitali Beatrice, Brigidi Patrizia , Rodriguez
Lorenzo, Polymerlipid based mucoadhesive microspheres prepared by spray-congealing for the vaginal
delivery of econazole nitrate, European Journal of Pharmaceutical Sciences, Dec 25,pp 591-601, (2008)
ISSN 0928-0987 ;
7. Ali El-Enshasy et al., Optimization of cell mass production of the probiotic strain lactococcus lactis in batch
and fed-bach culture in pilot scale levels; Current Research, Technology and Education Topics in Applied
Microbiology and Microbial Biotechnology. FORMATEX, Spain, pp. 873-879. ISBN 978-84-614-6195-0,
2010;
8. Allan-Wojtas P., Hansen Truelstrup L. and Paulson A.T., Microstructural studies of probiotic bacteria-loaded
alginate microcapsules using standard electron microscopy techniques and anhydrous fixation, LWT Food
Science and Technology Journal. 41, 101-108 (2008) ISSN: 0023-6438.;
9. Ambule A.H., Timande S.P and Soni S.B., Study on Probiotic Potential and Laboratory Scale Production of
Lactic Acid Bacteria; International Journal of Applied Biology and Pharmaceutical Technology; volume 3,
Issue 3,pp 295-300, July–Sept 2012, ISSN:0976-4550; doi:10.2298/CICEQ0703169P;
;http://www.ijab.pt.com/;
10. Anal Anil Kumar and Singh Harjinder. Recent advances in microencapsulation of probiotic for industrial
applications and targeted delivery. Trends in Food Science & Technology. 240-251, 18, 2007
DOI:10.1016/j.tifs.2007.01.004;
11. Anin Tawheed, Thakur Manika, Jain S.C. - Microencapsulation - The future of Probiotic Cultures, Journal of
Microbiology, Biotehnology and FoodSciences 2013: 3(1), pp 35-43 ISSN; ISSN: 1338-5178;
12. Annan NT, Borza AD, Hansen L.-Truelstrup. Encapsulation in alginate-coated gelatin microspheres improves
survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal
conditions. Food Research International, 2008, 41:184-193 http://dx.doi.org/10.1016/j.foodres.2007.11.001 ;
ISSN: 0963-9969;
13. Arsh Chanana, Mahesh Kumar Kateria, Manish Sharma, Ajay Bilandi, Seth C.L. Bihani S.D,
Microencapsulation : Advancements in Applications, International Research Journal of Pharmacy IRJP, 4(2),
ISSN: 2230-8407; www.irjponline.com, 2013;
14. Arup Nag, Development of microencapsulation technique for probiotic bacteria Lactobacillus casei 431 usig
a protein polysaccharide complex" A Thesis Presented in Partial Fulfilment of the Requirements for the
Degree of Masters of Technology in Food Technology at Massey University, Palmerston North, New
Zealand; 2011 , http://hdl.handle.net/10179/2355;
15. Ashraf M. et al, In Vitro Screening of Locally Isolated Lactobacillus Species for Probiotic Properties;
Pakistan Veterinary Journal. 2009, 29(4):186-190; 2009;
16. Avallone Bueno Luciano, Fonseca Maria de Fatima, Marques Djalma, Shinagawa Fernanda Branco, Quintino
Amanda, Locatelli Gabriel, Qudros Cedemir Peseira Evaluation of probiotic containing microcapsules
stability in different media www.icef11.og/main.php/fall paper, 2011, full paper poceedings, ISBN:978-960-
89789-3-5;
Page 38
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 38 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
17. Ballard Olivia et. Al, Human Milk Composition Nutrients and Bioactive Factors, Pediatric Clinics of North
America 60 (2013) 49–74;DOI: 10.1016/j.pcl.2012.10.002 ; www.pediatric.theclinics.com ;
18. Bhathena Jasmine, Tamaro-Duchesnean Catherine, Martoni Cristopher, Malhotva Meenakshi, Kulamarva
Arun, Urbanska Malgarzata Aleksandra, Arghya Paul and Prakash Satya, Effect of Orally Administered
Microencapsulated FA - Producing L. fermentum on Markers of Metabolic Syndrome: An In Vivo Analysis;
Journal of Diabetes &Metabolism 2012, S6 , ISSN: 2155-6156, pp 1-9;
19. Bickerstaff Gordon F., Immobilization of Enzymes and Cells, Methods in Biotechnology, 1st ed. Bickerstaff
GF Eds., Humana Press, Totowa, USA, 1997, vol 1, ISBN 0-896-03386-4;
20. Blazenka Kos et al, Characterization of the Three Selected Probiotic Strains for the Application in Food
Industry, World Journal of Microbiology and Biotechnology (2008) 24(5):699-707, DOI 10.1007/s11274-
007-9528-y, 2008;
21. Bogner Agnes, Jouneau PH, Thollet G, Basset D, Gauthier C. A history of scanning electron microscopy
developments: Towards "wet-STEM" imaging. Micron Journal 2007; 38:390-401; ISSN 09684328;
22. Bos GW; Verrijk R; Franssen O; Bezemer JM; Hennink WE; Crommelin DJ. Hydrogels for the Controlled
Release of Pharmaceutical Proteins, Pharmaceutical Technology North America, 2001, 25(10), 110-120;
ISSN:1534-2131
23. Both Emese, Gyenge Laszlo, Bodar Zsolt, Gyorgy Eva, Lanyi Szabdcs, Abraham Beata, Intensification of
probiotic microorganisms viability by microencapsulation using ultrasonic atomizer U.P.B. Scientific
Bulletin, series B, vol 74, Issue 1, 2012, ISSN 1454-2331;
24. Bouguettoucha, A. ; Balannec, B. ; Nacef, S. ; Amrane, A.; Unstructured Models for Batch Cultures of
Lactobacillus Helveticus; Associazione Italiana di Ingegneria Chimică, CHEMICAL ENGINEERING
TRANSACTIONS ; VOL 14 2008; 14; 121-128, International conference on industrial biotechnology;
IBIC2008,, www.aidic.it/IBIC2008/webpapers;
25. Bryony James, Fonseca Celia. Textures studies and compression behavior of apple flesh. International Journal
of Modern Physics. 2006; 20:3993-3998; ISSN: 0217-9792;
26. Burgain J. , Gaiani C., Francius G., Rend-Juelles A.M., Cailliez-Grimal C., Lebeer S., Tytgat H.L.P.,
Vanderleyden J., In vitro interactions between probiotic bacteria and milk proteins probed by atomic force
microscopy, Colloids and Surfaces B: Biointerfaces Journal (2013) pp 153-162, ISSN: 09227-7765
27. Burgain J., Gaiani C., Linder M., Scher J., Encapsulation of probiotic living cells : From laboratory scale to
industrial applications, Journal of Food Engineering 104 , Issue 4, June (2011) 467-483 ; DOI : 10.
1016/j.foodeng.2010.12.031; ISSN:0260-8774; 2011;
28. Capela P, Hay TKC, Shah NP. Effect of cryoprotectants, prebiotics and microencapsulation on survival of
probiotic organisms in yoghurt and freeze-dried yoghurt, Food Research International 39, issue 2, (2006)
203-211- " "; ISSN 0963-9969; DOI: http://dx.doi.org/10.1016/j.foodres.2005.07.007;
29. Carvalho AS, Silva J, Ho P, Teixeira P, Malcata FX, Gibbs P. Survival of freeze-dried Lactobacillus
plantarum and Lactobacillus rhamnosus during storage in the presence of protectans. Biotechnology Letters
2002; 24:1587-1591; ISSN: 1573-6776
30. Champagne CP, Starter cultures biotechnology: the production of concentrated lactic cultures in alginate
beads and their applications in the nutraceutical and food industries. Chemical Industry & Chemical
Engineering Quarterly 2006; 12:11-17. [doi: 10.2298/CICEQ0601011C]; ISSN 1451-9372;
31. Champagne, CP. Fustier, Microencapsulation for the improved delivery of bioactive compounds into foods.
Current Opinion in Biotechnology 2007; 18:184-190. [doi: 10.1016/j.copbio.2007.03.001] [pmid:
17368017]; ISSN 09581669;
32. Chan LW, Jin Y, Heng PWS. Cross-linking mechanisms of calcium and zinc in production of alginate
microspheres. International Journal of Pharmaceutics, 2002, 242:255-258; ISSN:0378-5173;
33. Chan LW, Lee HY, Heng PWS. Mechanisms of external and internal gelation and their impact on the
functions of alginate as a coat and delivery system. Carbohydrate Polymers, 2006, 63:176-187; ISSN, 0144-
8617 ;
34. Chan LW, Lee HY, Heng PWS. Production of alginate microspheres by internal gelation using an
emulsification method. International Journal of Pharmaceutics, 2002, 242:259-262; ISSN: 0378-5173;
Page 39
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 39 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
35. Chandramouli, V.; Kailsapathy, K.; Peiris, P.; Jones, M. An improved method of microencapsulation and its
evaluation to protect Lactobacillus spp. in simulated gastric conditions. Journal of Microbiological Methods
2004, 56, 27-35; ISSN 0167-7012;
36. Charalampopoulos Dimitris, Rastall Robert A. (Eds) Prebiotics and Probiotics Science and Technology,
Springer Science and Bussiness, LLC 2009, ISBN:978-0-387-79057-2;
37. Chavarri Maria, Izaskun Maranon and Maria Carmen Villaran, Encapsulation Technology to Protect
Probiotic Bacteria, ISBN 978-953-51-0776-7, Published: October 3, 2012 ; DOI: 10.5772/50046;
38. Chavarri Maria, Maranen Izaskun, Ares Raquel, Ibanez Francisco C, Marzo Florencia, Villaran Maria del
Carmen Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves survival in
simulated gastro-intestinal conditions. International Journal of Food Microbiology 142(2010) pp 185-189,
ISSN: 0168-1605, doi: 10.1016/j.ijfood.micro 2010.06.022;
39. Chen G, Yao SJ, Guan Yx, Lin DQ. Preparation and characterization of NaCS-CMC/PDMDAAC capsules.
Colloids Surfaces B: Biointerfaces 2005; 45:136-43; ISSN: 0927-7765;
40. Chen, K.N., Chen M.J., Liu, J.-R., Lin, C.W. and Chiu, H.Y. (2005), Optimization of Incorporated Prebiotics
as Coating Materials for Probiotic Microencapsulation. Journal of Food Science, 70: M260–M266.
doi: 10.1111/j.1365-2621.2005.tb09981.x; ISSN0022-1147;
41. Chen M.; Mustapha, A. Survival of freeze-dried microcapsules of α-galactosidase producing probiotics in a
soy bar matrix. Food Microbiology, 2012; 30:68-73; ISSN 07400020;
42. 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
43. ChinFa Hwang et all, Biomass Production of Lactobacillus Plantarum LP02 Isolated from Infant Feces with
Potential Cholesterol-Lowering Ability; African Journal of Biotechnology vol 10(36), pp7010-7020, 18 July
2011; DOI : 10.5897/AJB11.507, ISSN 1684-5315, 2011;
44. Chirico G., Gasparoni A., Immunologic components of human milk, Haematologica Reports 2006; 2 (issue
10):27-30; September 2006; ISSN: 1824-9337
45. Chirico Gaetano, Marzollo Roberto, Cortinovis Sheila, Fonte Chiara, Gasparoni Antonella, Antiinfective
properties of Human Milk, The Journal of Nutrition, 138: 1801S-1806S, 2008, ISSN : 0022-3166/08;
46. Cinquin C., Le Blay G., Fliss I., Lacroix C., Immobilization of Infant Fecal Microbiota and Utilization in
an in vitro Colonic Fermentation Mode, Microbial Ecology 48 (2004) 128-138. ISSN 0095-3628;
47. Corcoran B.M., Ross R.P., Fitzgerald G.F. and Stanton C., Comparative survival of probiotic lactobacilli
spray-dried in the presence of prebiotic substances, Journal of Applied Microbiology 96, 1024-1039 (2004);
ISSN, 1364-5072;
48. Çakmakçi S. et al, Probiotic Properties, Sensory Qualities and Storage Stability of Probiotic Banana Yogurts;
Turkish Journal of Veterinary & Animal Sciences 2012; 36(3):231-237;DOI:10.3906/vet-1007-2; 2012;
49. Darrabie Marcus D, Kendall William FJ, Opara Emmanuel C. Characteristics of poly-L-ornithine-coated
alginate microcapsules. Biomaterials 2005; 26:6846-52; ISSN: 0142-9612;
50. De Castro M, Orive G, Hernandez RM, Gascon AR, Pedraz JL. Comparative study of microcapsules
elaborated with three polycations (PLL, PDL, PLO) for cell immobilization. Journal of Microencapsulation
2005; 22:303-15; ISSN: 0265-2048;
51. Desai K.G.H., Park H.J., Preparation of cross-linked chitosan microspheres by spray drying: Effect of cross-
linking agent on the properties of spray dried microspheres, Journal of Microencapsulation: 22(4), June, p
377-395 (19), 2005 ISSN 1464-5246;
52. Desmond C, Santon C, Fitzgerald GF, Collins K, Ross RP. Environmental adaptation of probiotic lactobacilli
towards improvement of performance during spray drying. International Dairy Journal 2001; 11:801-808;
ISSN 0958-6946;
53. Devi Nirmala, Kakati Dilip Kumar - Smart porous microcapsles based on gelatin/sodium alginate
polyelectrolyte complex, Journal of Food Engineering, 2013 117: pp 193-204, ISSN: 0260-8774;
54. Doleyress Y.; Fliss I.; Lacroix C. Quantitative determination of the spatial distribution of pure and mixed-
strain immobilized cells in gels beads by immunofluorescence. Applied Microbiology Biotechnology 2002,
59, 297-302; ISSN 1432-0614;
Page 40
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 40 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
55. Dong Z., Wang Q, Du Y - Alginate/gelatin blend films and their properties for drug controlled release. Journal
of Applied Membrane Science, 2006, 280:37-44; ISSN 0376-7388;
56. Draget, K.I.; Steinsvag, K.; Onsoyen, E.; Smidsrod, O.Na+ and K+ - Alginate effect on Ca2+ gelation.
Carbohydrate Polymers 1998, 35, 1-6; ISSN 01266039;
57. Ekah Elijah Ella et al. , Studies on the Interaction between IgA, lactoferrin and lysozyme in the breastmilk of
lactating women with sick and healthy babies; Journal of Infectious Diseases and Immunity, vol 3(2), pp 24-
29, 2011 ISSN 2141-2375;
58. Eun Y. Ann, Younghoon Kim, Sejong Oh, Jee-Young Imm , Dong-Jun Park , Kyoung S. Han, Sae H. Kim,
Microencapsulation of Lactobacillus acidophilus ATCC 43121 with prebiotic substrates using a hybridisation
system, International Journal Food Science Technology 2007;vol 42, issue4, pp:411-419.
[doi:10.1111/j.1365-2621.2007.01236.x] ISSN 0950-5423;
59. Eun Young Lee, Effect of Atomization on viability of microencapsulated probiotics, Master Thesis, pp 1-73,
2012, https://www.ideals.illinois.edu ;
60. Fahimdnesh Maryam et al, " Effect of microencapsulation plus resistant starch on survival of Lactobacillus
casei and Bifidobacterium bifidum in mayonnaise sauce " African Journal of Microbiology Research vol
6(40), pp 6853-6858, oct 2012; ISSN 1996-08008;
61. Fatemeh Atyabi Saeed; Manoochehri Shadi, Moghadam Rassoul, Dinarvand. Cross-linked starch
microspheres: Effect of cross-linking condition on the microsphere characteristics Archives of Pharmacal
Research, 2006, 29(12), 1179-1786;
62. Farzanch Lotfipour, Shahla Mirzaeei, Maryam Maghsoodi, Evaluation of the effects of CaCl2 and alginate
concentrations and hardening time on the characteristics of Lactobacillus acidophilus loaded alginate beads
using response surface analysis, Advances Pharmaceutical Bulletin, 2012, 2(1), 71-78 doi:
105681/apb.2012.010; ISSN 2228-5881;
63. Fazaeli M., Tahmaebi M. and Emam Z., Characterization of food texture: application of Microscopic
technology, Current Microscopy Contributions to Advances in Science and Technology, ISBN(13): 978-84-
939843-6-6 (pp 855-871), 2012;
64. Garofalo Roberto,Cytokines in Human Milk, The Journal of Pediatrics. www.jpeds.com, supplement, 2010,
156:S36-40;ISSN 00223476;
65. Galia Salem, Zaiton Hassan and Maryam Abubako , Adhesion of Probiotic Bacteria to Resistant Rice Search,
American Journal of Applied Sciences 10(4): pp 313-321, ISSN: 1546-9239;
66. Gbassi K Gildos., Vandamme Thierry, Probiotic Encapsulation Technology: From Microencapsulation to
Release into the Gut, 4, 149-163; 2012;
67. Gbassi, G K..; Vandamme, T.; Ennahar, S.; Marchioni, E. Microencapsulation of Lactobacillus plantarum
spp in an alginate matrix coated with whey proteins. International Journal of Food Microbiology. 2009, 129,
103-105; ISSN 0168-1605 ;
68. Georgieva R., Koleva P. , Nikolova D., Yankov D. and Danova S., Growth parameters of Probiotic Strain
Lactobacillus Plantarum Isolated from Traditional White Cheese ; Biotechnology&Biotechnology Equipment
23/2009/SE; ISSN 1310-2818 ; 2009;
69. Gharsalaoui A., Roudaut G., Chambin O., Voilley A., Saurel R., Application of spray-drying in
microencapsulation of food ingredients: an overview. Food Reseasrch International Journal, 2007, 40:1107-
1121; doi:10.1016/j.foodres.2007.07.004;
70. Ghosh M. ; Ghosh U. ; Comparative Batch Growth Studies of Pure Lactobacillus Strains and Their Co-
culture in Synthetic Medium with Different Neutralizing Agents;Associazione Italiana di Ingegneria
Chimica; CHEMICAL ENGINEERING TRANSACTIONS ; VOL 14 2008; 14; 221-228 International
conference on industrial biotechnology; IBIC2008; www.aidic.it/IBIC2008/webpapers;
71. Goodwin J.T., Somerville G.R., Microencapsulation by physical methods; Chemtech 4: 623-626,(1974);
72. Gorecka Elzbiela, Jostuzebska Magdalena; Imobilization techniques and biopolymer carriers, Biotechnology
and Food Science 2011(1), 65-86, ISSN: 2084-0136;
73. Gouin Sebastien. Microencapsulation: Industrial appraisal of existing technologies and trends. Trends in Food
Science & Technology. 2004, 15, 330-347; ISSN 0924-2244;
Page 41
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 41 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
74. Govind Babu, Shantanu Rath, V.Nithyalakshmi, Probiotic Viability of Freeze Dried Synbiotic
Microcapsuless in Skim Milk Powder at Ambient Storage Condition 2011, Internet Journal of Food Safety;
vol 13.2011, pp 62-68;
75. Grasdalen H., Larsen B., Smidsrod O. A P.M.R. study of the composition and sequence of uronate residues
in alginates. Carbohydrate Research 1979: 23-31; DOI: 10.1016/S0008-6215(00)84051-3;
76. Grasdalen H. High-field, H-NMR spectroscopy of alginate: sequential study and linkage conformation.
Carbohydrate Research 1983:225-60;
77. Grasdalen H., Larsen B., Smidsrod O. 13C-NMR studies of monomeric composition and sequence in alginate.
Carbohydr Res 2008:179-91; http://dx.doi.org/10.1016/S0008-6215(00)85243-X ;
78. Guilbert T.W et al, Effect of Breastfeeding on Lung Function in Childhood and Modulation by Maternal
Asthma and Atopy; American Journal of Respiratory and Critical Care Medicine, Vol 176, pp 843-848, 2007,
DOI:10.1164/rccm.200610-1507OC;
79. Gurjot Deepika et al, Influence of Fermentation Conditions on the Surface Properties and Adhesion of
Lactobacillus Rhamnosus GG;. Microbial Cell Factories; 2012, 11:116; DOI : 10.1186/1475-2859-11-116;
80. Hamayoumi Aziz, Ehsami Mohammed Reza, Ayizi Aslan, Yarmand Mohammad Laeid, Razani Hadi S.,
Effect of Lecithin and Calcium Chloride Solution on the Microencapsulation Process Yield of Calcium
Alginate Beads, Iranian Polymer Journal 16(9), 2007, pp 597-606, ISSN: 1735-5265;
81. Homayouni Aziz et al, Spectrophotometricaly Evaluation of Probiotic Growth in Liquid Media;Asian Journal
of Chemestry,vol 20, No 3 (2008) pp 2414-2421; www.asianjournalofchemistry.co ; 2008;
82. Harnsilawat,T.; Pongsawatmanit, R.; McClements, D.J. Characterization of β-lactoglobulin-sodium
alginate interactions in aqueous solutions: A calorimetry, light scattering, electrophoretic mobility and solubility
study. Food Hydrocolloids 2006, 20, 577-585; ISSN 0268005X;
83. Harker FR, White A, Gunson FA, Hallett IC, De-Silva HN. Instrumental measurement of apple texture: a
comparison of the single-edge notched bend test and the penetrometer. Post-harvest Biology and Technology.
2006; 39:185-192; ISSN, 0925-5214;
84. Heinzen Christoph, Marison I., Berger A. and U. von Stockar. Use of vibration technology for jet break-up
for encapsulation of cells and liquids in monodisperse microcapsules. Fundamentals of Cell Immobilisation
Biotechnology(Focus on Biotechnology) Volume 8A, 2004, pp 257-275; ISSN: 1569-268X;DOI
:10.1007/978-94-017-1638-3_14;
85. Herbel Stefan R et al , Timely Approaches to Identify Probiotic Species of the Genus Lactobacillus, Gut
Pathogens 2013; 5:27; DOI:10.1186/1757-4749-5-27; 2013-Review;
86. Honorio-França Adenilda Cristina and Luzia-França Eduardo, Human Milk: An Ecologically Functional
Food; Environmental Sciences “Relevant Perspectives in Global Environmental Change” chapter 4, ISBN
978-953-307-709-3 DOI:10.5772/26520, 2011 December;
87. Huguet, M.L., Neufeld R.J., Dellacherie E. - Calcium-alginate beads coated with polycationic polymers:
Comparison of chitosan and DEAE-Dextran.Process Biochemistry. 1996, 31, 347-353 ISSN:. 0032-9592;
88. Hyun-jue Kim et al, Characterization of Lactic Bacterial Strains Isolated from Raw Milk; Asian-Australasian
Journal of Animal Sciences 2006, vol 19, No1; 131-136; 2006;
89. Illum L.; Fisher AN; Jabbal-Gill I.; Davis SS, Bioadhesive starch microspheres and absorption enhancing
agents act synergistically to enhance the nasal absorption of polypeptides, International Journal of
Pharmaceutics, 2001, 222(1), 109-119;; ISSN: 0378-5173;
90. Jackson, LS. and K. Lee, Microencapsulation and the food industry , Lebensmittel-Wissenschaft Technologie
1991, vol. 24, no4, pp. 289-297 ; ISSN 0023-6438 ;
91. Jafari SM; Assdpoor E; Bhandari B.; ; Nano-particle encapsulation of fish oil by spray drying ,Y. He. Food
Research International, 2008, 41(2), 172-183;2008, ISSN, 0963-9969;
92. Jayalalitha V , Balasundaram B and R, Palanidakai, In vitro Assessment of microencapsulated probiotic
beads, International Journal of Agriculture : Research and Review vol 2(1), 1-6, 2012, ISSN: 2228-7973;
93. Junzhang Lin, Weiting Yu, Xindong Liu, Hanggno Xie, Wei Wang and Xxaajun Ma - In Vitro and in Vivo
characterization of alginate-chitosan-alginate artificial microcapsules for therapeutic oral delivery of live
bacterial cells; Journal of Bioscience and Bioengineering 2008 Jun;105(6):660-5. doi: 10.1263/jbb.105.660;
Page 42
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 42 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
94. Jurcoane Stefana, Sasarman Elena, Lupescu Irina, Rosu Ana, Banu Alexandra; Berehoiu Tamba Radiana,
Radoi Florentina, Tratat de Biotehnologie vol I si II , Editura Tehnica Bucuresti, ISBN: 973-31-2236-X, 973-
31-2235-1, 2004;
95. Jyothi Sri.S et al , Microencapsulation, A Review, International Journal of Pharma & Bio Sciences;Jan-
Mar2012, Vol. 3 Issue 1, pP.509, ISSN 0975-6299 (www.ijpbs.net);
96. Kailasapathy K.,Microencapsulation of probiotic Bacteria: Technology and Potential Applications"; Current
Issues in Intestinal Microbiology. (2002) 3: 39-48; 2002, Horizon Scientific Press; PMID: 12400637;
97. Kailasapathy, K., Encapsulation technologies for functional food and nutraceutical product development.
CAB Reviews perspectives in agriculture veterinary science nutrition and natural resources 2009 Vol. 4 No.
033 pp. 1-19; ISSN 1749-8848;
98. Karna B.K.L. et al, Lactic Acid and Probiotic Bacteria from Fermented and Probiotic Dairy Products ; Science
Diliman (July – December 2007 ) 19:2, 23-24, ISSN 0115-7809; 2007;
99. Khalil Rowaida et al., Evaluation of the Probiotic Potential of Lactic Acid Bacteria Isolated from Faeces of
Breast – Fed Infants in Egypt; African Journal of Biotechnology vol 6 (7), pp 939-949 April 2007, ISSN:
1684-5315; 2007;
100. Khasravi Zenjani Mohammad Ali et al Microencapsulation of Lactobacillus casei with calcium alginate-
resistant starch and evaluation of survival and sensory properties in cromfilled cake, African Journal of
Microbiology Research, vol 6(26) pp 5511-5517, ISSN 1996-0808;
101. Ki.Yong Lec et al, 2000; Survival of Bifidobacterium longum immobilized in calcium alginate beads in
simulated gastric juices and bile salt solution; Applied and Environmental Microbiology, Feb 2000, p 869-
873, ISSN: 0099-2240;
102. Kizerwetter-Swida Magdalena and Binek Marian; Selection of Potentially Probiotic Lactobacillus Strains
Towards their Inhibitory Activity against Poultry Enteropathogenic Bacteria Polish Journal of Microbiology
2005, vol 54, No4, 287-294; ISSN 1733-1331;
103. Klien J., Stock J., Vorlop K.D.. Pore size and properties of spherical calcium alginate biocatalysts, European
Journal Applied Microbiology Biotechnology, 18, 86-91; 1983, ISSN: 1432-0614;
104. Kotikalapudi Lakshmi B. - Characterization and encapsultion of probiotic bacteria using a Pea - protein
Alginate matrix, A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment
of the Requirements for the Degree of Master of Sciencein the Department of Food and Bioproduct Sciences
University of Saskatchewan Saskatoon, Saskatchewan, Canada , July 2009; http://hdl.handle.net/10388/etd-
08282009-170854 ;
105. Krasaekoopt W., Bhandari B., Deeth H. - Evaluation of encapsulation techniques of probiotics for yoghurt,
International Dairy Journal, 13 (2003) 3-13. ISSN 0958-6946;
106. Krasaekoopt, W.; Bhandari, B.; Deeeth, H. The influence of coating on some properties of alginate beads and
survivability of microencapsulated probiotic bacteria. International Dairy Journal 2004, 14, 737-743;ISSN
0958-6946;
107. Krishnan; R. Bhosale; RS Singhal. Microencapsulation of cardamom oleoresin: Evaluation of blends of gum
arabic, maltodextrin and a modified starch as wall materials; Carbohydrate Polymers, 2005, 61(1), 95-102,
ISSN, 0144-8617;
108. Lachman Leon, Lieberman Herbert A., Kanig Joseph L.,The Theory and Practice of Industrial Pharmacy, 3rd
edition, pp.420;
109. Lee J.S., Cha D.S., Park H.J., Survival of freeze-dried Lactobacillus bulgaricus KFRI 673 in chitosan-coated
calcium alginate microcapsles, Journal of Agricultural and Food Chemistry. 52(2004) 7300-7305; ISSN
0021-8561;
110. Lee MJ, Scog EJ, Lee JH. Physicochemical properties of Chaga (Inonotus obliquus) mushroom powder as
influenceed by drying methods. Food Science and Nutrition, 2007; 12:40-45; ISSN, 1226-332X;
111. Leuschner R. , Kneife W., Vernoux J.-P. , Stanton C. and Aldamiz P. Methods for the official control of
probiotics used as food additives, volume 2, European Comission Community Research 2002, ISBN 92-894-
6251-5;
112. Lisova Ivana, Horackova Sarka, Kovacova Renata, Kaeda Voitech and Plackova Milada Emulsion
Encapsulation of Bifidobacterium animalis subsp. lactis BB12 with the addition of Lecithin - Czech Journal
of Food Sciences, vol 31, 2013, No 3: pp 270-274, ISSN 1805-9317;
Page 43
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 43 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
113. Lukacova Drahomira et al, In Vitro Testing of Selected Probiotic Characteristics of Lactobacillus Plantarum
and Bifidobacterium Longum , Journal of Food and Nutrition Research, vol 45, 2006, No 2, pp 77-83,
ISSN:1336-8672 2006;
114. M. Lawrence Robert, A. Pane Camille, Human Breast Milk: Current Concepts of Immunology and Infectious
Diseases, Current Problems in Pediatric and Adolescent Health Care, January 2007, 37:7-36, DOI:
10.1016/j.cppeds.2006.10.002;
115. Malafaya PB, Stappers F; Reis RL. Starch-based microspheres produced by emulsion crosslinking with a
potential media dependent responsive behavior to be used as drug delivery carriers, Journal of Materials
Science. Materials in Medicine, 2006, 17(4), 371-377;
116. Mangione MR, Giacomazza D., Bulone D, Martorana V., Cavallaro G., San Biagio PL. K+ and Na+ effects
on the gelation properties of k-carrageenan. Biophysical Chemistry journal, 2005, 113:129-135.
doi:10.1016/j.bpc.2004.08.005; ISSN:0301-4622;
117. Mahdhi Abdelkarim et al, Preliminary Characterization of the Probiotic Properties of Candida famata and
Geobacillus thermoleovorans; Iranian Journal of Microbiology, 129-134, volume 3, No 3, September
2011,ISSN 2008-3289 ; 2011;
118. Mandal S., Puniya A.K., Singh K., Effect of alginate concentrations on suvival of microencapsulated
Lactobacillus casei NCDC-298, International Dairy Journal, 16, 1190-1195, 2006ISSN, 0958-6946;
119. Marsich E, Borgogna M, Donati I, Mozetic P, Strand BL, Salvador SG et al. Alginate /lactose-modified
chitosan hydrogels: a bioactive biomaterial for chondrocyte encapsulation. Journal of Biomedical Materials
Research 2008; 84:364-76; ISSN, 1549-3296;
120. Matlock Brian C., Beringer Richard W. et al; Analyzing Differences in Bacterial Optical Density
Measurements between Spectrophotometers; Abstract 1730, 2011 Thermo Fisher Scientific Inc.
www.thermoscientific.com ;
121. Mehanna Nayra S.H., Tawfik Nabil F., Salem Moussa M.E., Effat Baher A.M. and Gad El-Rab D.A.,
Assessment of Potential Probiotic Bacteria Isolated from Breast Milk Middle-East Journal of Scientific
Research 14 (3): 354-360, ISSN 1990-9233,IDOSI Publications, 2013, DOI:
10.5829/idosi.mejsr.2013.14.3.2102;
122. Mladenovska K., Cruaud O., Richomme P., Belamie E., Raicki R.S., M. C. Venier-Juliene, PopovskE. i,
Benoit J.P. and Goracinova K.. 5-ASA loaded chitosan-Ca-alginate microcapsles: Preparation and
physicochemical chracterization. International Journal of Pharmaceutics. 345, 59-69 (2007b); ISSN: 0378-
5173;
123. Mladenovska K., Raicki R.S., Janevik E.I., Ristoski T., Pavlova M.J., Kavrakovski Z., Dodov M.G. and
Goracinova K., Colon-specific delivery of 5-aminosalicylic acid from chitosan-Ca-alginate microcapsles.
International Journal of Pharmaceutics. 342.124-136 (2007a); ISSN:0378-5173;
124. Mokarram R.R., Mortazavi S.A., Habibi Najafi M.B., Shahioti,F. The influence of multi stage alginate
coating on survivability of potential probiotic bacteria in simulated gastric and intestinal juice, Food Research
International 42(2009) ; pp 1040-1045, ISSN: 0963-9969;
125. Monteiro SM et al; A Procedure for High-Yield Spore Production by Bacillus Subtilis; Pubmed (PMID-
16080679) ; DOI:10.1021; ISSN:8756-7938; Biotechnology
Progress 21,4, 1026-1031, 5 sept 2008;
126. Morais Inês P. A., Tóth Ildikó V., Rangel António O. S. S., Turbidimetric and Nephelometric Flow Analysis:
Concepts and Applications; Spectroscopy Letters Journal vol. 39, no. 6, pp. 547-579, 2006
DOI: 10.1080/00387010600824629;
127. Mortazavian A, Razavi SH, Ehsani MR, Sohrabvandi S. Principles and methods of microencapsulation of
probiotic microorganisms. Iranian Journal of Biotechnology (IJB) 2007; 5(1):1-18, ISSN : 1728-3043;
128. Morgan CA, Herman N, White PA, Vesey G. Preservation of microorganisms by drying: an review. Journal
of Microbiological Methods, 2006, 66:183-193 ISSN 01677012;
129. Motohiro Shima, Yasunobu Morita., Matsatsugu Yamashita.; Shuji Adachi . Protection of Lactobacillus
acidophilus from the low pH of a model gastric juice by incorporation in a W/O/W emulsion. Food
Hydrocolloids 2006, 20, 1164-1169; ISSN 0268-005X;
130. Muhr AH, Blanshard JMV. Diffusion in gels. Polymer 1982; 23:1012-26 http://dx.doi.org/10.1016/0032-
3861(82)90402-5;
Page 44
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 44 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
131. Murtaza Ghulam, Ahamal Mahmood, Achtar Naved, Rasad Fatima, A comparative study of various
Microencapsulation techniques effect on polymer viscosity on microcapsule characteristics, Pakisan Journal
of Pharmaceutical Sciences, vol 22, No 3, July 2009, pp 291-300;
132. Naiaretti Daniela , Tita Ovidiu, Biopolymers used in Food Packaging –A review, Acta Universitatis
Cibiniensis Series E: FOOD TECHNOLOGY Vol. XVI (2012), no.2, ISSN 2344 – 150X;
133. Nedovic V.A., Obradovic B., Poncelet D., Goosen M.F.A., Leskosek Cukalovic I., Bugarski B. - Cell
immobilization by electrostatic droplet generation, Landbauforschung Volkenrode SH 241(2002) 11-17;
ISSN1569-268X;
134. Nedovic V.A., Obradovic B., Leskosek-Cukalovic I., Trifunovic O., Pesic R., Bugarski B., Electrostatic of
alginate microbeads loaded with brewing yeast, Process Biochemistry Journal. 37 (2001) 17-
22; DOI:10.1016/S0032-9592(01)00172-8; ISSN 00329592;
135. 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
136. 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
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
138. Neamțu Bogdan, Grupul Roman de Experti in Nutritie Pediatrică, Recomandări nutriționale în practica
pediatrică, Capitol Nutriția în Terapia Intensivă, Editura Universitară Carol Davila București, 2013, ISBN :
978-973-708-697-6;
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
Childhood 2012 Oct: 97(Suppl 2): 1-58 ISSN 14682044;
140. Oddy H Wendy, Why Breast Milk Has Health Benefits for Infants and Children : A Review, Pakistan Journal
of Nutrition 1(3), 106 – 118, 2002, ISSN 1680 – 5194,
141. Oddy H Wendy., A Review of the Effects of Breastfeeding on Respiratory Infections, Atopy and Childhood
Asthma; Journal of Asthma vol 41, No.6, pp. 605-621, 2004, Review Article, DOI :10.1081/JAS-200026402;
142. Olfat S. Barakat et al, Identification and Probiotic Characteristics of Lactobacillus Strains Isolated from
Traditional Domiati Cheese; International Journal of Microbiology Research, vol 3, issue 1, 2011, pp 59-66,
ISSN: 0975-5276 & E-ISSN : 0975 – 9174, 2011;
143. Orive G, Hernandez RM, Gascon AR, Igartua M, Pedraz JL. Development and optimisation of alginate-
PMCG-alginate microcapsules for cell immobilisation. International Journal of Pharmaceutics 2003; 259:57-
68; ISSN: 0378-5173
144. Ouled-Haddar Houria, Idoni Tayeb, Sifour Mohamed, Guezira Messaouda, Bouthabet Messaouda, Isolation,
Characterization and Microencapsulation of Probiotic Lactobacillus curvatus 67 from Chicken Crop, The
Online Journal of Science and Technology - 2012, vol 2, ISSUE-1, ISSN:2146-7390 ;
145. Paul de Vos, Bucko Marck, Gemeiner Peter, Navratil Marian, Snitel Juraj, Faas Maurijke, Stiand Lakensgard
Berif, Skjak-Brack Gudmund, March Yrr A., Vikartovska,Alica Lacik Igor, Kallarikova Gabriela, Onive
Gorka, Poncelet Dennis, Pedraz Jase Luis, Ansorge-Schummacher Marian B., Multiscale requirements for
bioencapsulation in medicine and biotechonology, Biomaterials 2009, ISSN: 0142-9612, pp: 2559-2570
146. Petrovic Tanja et al ,“Protection of probiotic microorganisms by microencapsulation”, Chemical Industry and
Chemical Engineering Quarterly 13 (3) 2007, 169-174; DOI :10.2298/CICEQ 0703169P
147. Petreska Tania Ivanovska et al, “Microencapsulation of Lactobacillus Casei in Chitosan Microencapsulation
of Lactobacillus Casei in Chitosan-Ca- Alginate Microparticles using Spray-Drying Method” Macedonian
Journal of Chemistry and Chemical Engineering; 2012, ISSN :1857-5552, vol 31, No1, pp 115-123
148. Picot Arnaud and Lacroix Christophe, Encapsulation of bifidobacteria in whey protein-based microcapsules
and survival in simulated gastrointestinal conditions and in yoghurt, International Dairy Journal, 505-515,
14, (2004); ISSN 09586946;
Page 45
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 45 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
149. Pongjanyakul, T.Alginate-magnesium aluminum silicate films: Importance of alginate block structures.
International Journal of Pharmaceutics. 2009, 365, 100-108; ISSN: 0378-5173;
150. Popovici Iuliana, Lupuleasa Dumitru, Tehnologie Farmaceutica voulumul 3 , Editura Polirom, 2008,
ISBN:978-683-635-5
151. Preetz C, Hauser A, Hause G, Kramer A, Mader K. Application of atomic force microscopy and ultrasonic
resonator technology on nanoscale: distinction of nanocapsules European Journal of Pharmaceutical Science.
2010; 39:141-151 ISSN 09280987;
152. Pruesse U., Vorlop K.D. - The jetcutter technology, Int. Fundamentals of cell imobilization biotechnology,
V. Nedovic, R. Willaert (Eds.), Kluwer Academic Publishers, Fundamentals of Cell Immobilisation
Biotechnology, Focus on Biotechnology Volume 8A, 2004, pp 295-309 (2004) pp. 295-309; ISSN1569-268X
153. Prüsse U., Dalluhn J., Breford J., Vorlop K.-D., Production of spherical beads by jetcutting, Chemical
Engineering and Technology, 23 , issue
12,(2000)11051110;DOI: 10.1002/15214125(200012)23:12<1105::AIDCEAT1105>3.0.CO;2-V
154. Reis CP, Neufeld RJ, Vilela S, Ribeiro AJ, Veiga F. Review and current status of emulsion/dispersion
technology using an internal gelation process for the design of alginate particles. Journal of
Microencapsulation 2006, 23:245-257; ISSN 1464-5246;
155. Robinson Siân and Fall Caroline; Infant Nutrition and Later Health: A Review of Current Evidence, Nutrients
2012, 4(8), 859-874; doi:10.3390/nu4080859, ISSN 2072 – 6643, www.mdpi.com/journal/nutrients;
156. Rokka, S., Rantamaki, P. - Protecting probiotic bacteria by microencapsulation: Challenges for industrial
applications. European Food Research and Technology 2010, 231-1-12; ISSN 1438-2385;
157. Shitara K.; Ikami I.; Munakata M; Muto O.; Sakata Y.. Hepatic Arterial Infusion of Mitomycin C with
Degradable Starch Microspheres for Unresectable Intrahepatic Cholangiocarcinoma, Clinical Oncology,
2008, 20(3), 241-246; a, DOI: 10.1016/j.clon.2007.12.007
158. Sparks R.E., Norbert M., Method for coating particles or liquid droplets; US Patent 4675140, June 6, (1987)
159. Sandoval-Castilla O., Lobato-Calleros C., Garcia-Galindo H.S, Alvarez-Ramirez J., Vernon-Carter EJ.
Textural properties of alginate-pectin beads and survivability of entrapped L.casei in simulated
gastrointestinal conditions and in yoghurt. Food Research International 2010; 43:111-117. doi:
10.1016/j.foodres.2009.09.010] ISSN, 0963-9969.
160. Sasimar-Wbraharn et al 2010, African Journal of Microbiology Research vol 4(20); pp 2086-2093, 18 oct
2010. ISSN 1996-0808 - "Survival enhancement of probiotic Lactobacillus plantarum CMU-FP002 by
granulatio and encapsulation techniques"
161. Schmidt JJ, Rowley J, Kong HJ. Hydrogels used for cell-based drug delivery. Journal of Biomedical
Materials Research 2008; 87:1113-32; ISSN 0976 – 3333
162. Sefton M V, May M H, Lahooti S & Babensee J E (2000) Making microencapsulation work, conformal
coating, immobilization gels and in vivo performance, Journal of Controlled Release 65, 173-186; ISSN:
0168-3659.
163. Se-Jin Kim, Seung Yong Cho, Sae Hun Kim, Ok-Ja Song, Il-Shik Shin, Dong Su Cha, Hyun Jin Park, Effect
of microencapsulation on viability and other characteristics in Lactobacillus acidophillus ATCC 43121 LWT-
Food Science and Tehnology 41(2008) pp 493-500 ISSN: 0023-6438,
164. Simon Benita. Microencapsulation: Methods and Industrial Applications. 2nd ed. vol 158, Drugs and The
Pharmaceutial Siences; Informa Healthcare, New York, USA, 2006; ISBN -13 : 978-0-8247-2317-0
165. Siriwan Nawong et al, Isolation and Selection of Probiotic Lactic Acid Bacteria from Cassava Pulp for
Cholesterol Lowering Property, 13th ASEAN Food Conference, 9-11 September 2013, Singapore, Meeting
Future food Demands : Security & Sustainability, 2013; www.cvent.com;
166. Sohail A et al - Survivability of probiotics encapsulated in alginate gel microbeads using a novel impinging
aerosols method, International Journal of Food Microbiology 2011 Ian 31 145(1) : 162-8,
DOI:10.1016/j.ijfoodmicro.2010.12.007. Epub 2010, dec 28;; ISSN:0168-1605;
167. Sultana K., Godward G, Reynolds N, Arumugaswamy R, Peiris P, Kailasapaty K, Encapsulation of probiotic
bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions andin yoghurt,
International Journal of Food Microbiology, 62, 47-55, ISSN : 0168-1605
168. Tapia C, Escobar Z, Costa E, Sapag-Hagar J, Valenzuela F, Basualto C, Gai MN, Yazdani-Pedram M.
Comparative studies on polyelectrolyte complexes and mixtures of chitosan-alginate and chitosan-
Page 46
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 46 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
carrageenan as prolonged diltiazem clorhydrate release system. European Journal of Pharmaceutics and
Biopharmaceutics2004, 57:65-75; ISSN: 0939-6411
169. Tița Ovidiu- Aspecte privind utilizarea celulelor de drojdie imobilizate"; Buletinul AGIR nr 3/2003 iulie -
septembrie, - ISSN 2247-3548;
170. Tilxeira P., Castro H., Mohacsi-Farkas C., Kirby R., Identification of sites of injury in Lactobacillus
bulgaricus during heat stress, Journal of Applied Microbiology 83 (1997) 219-226 ; DOI: 10.1046/j.1365-
2672.1997.00221.x; ISSN:1364-5072
171. Umer Hammad, Nigam Hemlata, Tamboli Asif M., Sundara M., Nainar Moorthi, Microencapsulation:
Process, Techniques and Application, Internnational Journal of Research in Pharmaceutical and Biomedical
Sciences, ISSN: 2229-3701, vol 2(2), Aps-Jm 2011, www.ijrpbsonline.com, pp 474-481;
172. Upoahyay Avand Joshi Amborish, "Fabrication of starch based microcapsles by an emulsification
crosslinking method; Journal of Chemical and Pharmaceutical Research 2011, 3(6): 839-845, ISSN 0260-
8774
173. Vasile Aida, Miron Tudor Lucian, Paraschiv Daniela, Bahrim Gabriela, Dima Stefan, "The enhancement of
growth ability and the viability of some probiotic bacteria in media with origanum vulgare L.
extract",Romanian Biotechnological Setters, vol 16, No 6, 2011, pp 6847-6853; ISSN 12245984;
174. Vandamme T F, Lenourry A.Charrueau C & Chaumeil J (2002) The use of polysaccharides to target drugs to
the colon. Carbohydrate Polymers 48, 219-231; ISSN: 0008-6215;
175. Vamanu E; Biotechnologies Used to Obtain Probiotic Biomass in Batch-System; Archiva Zootechnica ; vol
13; No4, 64-71, 2010; ISSN : 1016-4855;
176. Vodnar Dan C., Socaciu Carmen, Green tea increases the survival yield of bifidobacteria in simulated
gastrointestinal environment and during refrigerated conditions"; Chemistry Central Journal 2012, 6:61 doi:
10.1186/1752-153x-6-61" ISSN 1752-153X ;
177. Vidhyalakshmi R. et al, "Encapsulation The Future of Probiotics" - A Review 2009, Advances in Biological
Research 3(3-4): 96-103; ISSN: 1992-0067
178. Wei Ouyang, Hangwei Cheu, Mitchell L. Jones, Tasima Haque, Cristopher Martni, Fatane Afkhami and Satya
Prakash, Novel multilayer APPPA microcapsules for oral delivery: preparation condition, stability and
permeability.; Indian Journal of Biochemistry Biophysics, 2009, vol 46, pp 491-497; ISSN: 0301-1208;
179. Work Group on Breastfeeding, Breastfedding and the Use of Human Milk , SECTION ON
BREASTFEEDING , Pediatrics : Official Journal of the American Academy of Pediatrics. Vol 129 , Number
3 , 2012 , DOI : 10.1542/peds2011-3552, ISSN:0031-4005;
180. Wnek GE, Bowlin GL. Encyclopedia of biomaterials and biomedical engineering, 2nd ed. Wnek GE, Eds.;
Informa Healthcare, New York, USA, 2008, vol 1-4; ISBN 9781420078022
181. Xiao Yan Li, Xi Guang Chen, Doug Su Cha, Mynn Jin Park, Cheng Steng Lin, “Microencapsulation of a
probiotic bacteria with alginate-gelatin and its properties"; Journal of Microencapsulation, 2009, 26(4), pp
315-324 ISSN 1464-5246
182. Y Fang; L Wang; D Li; B Bhandari; X D Chen; Z Mao. Carbohydrate Polymers, 2008,
10.1016/j.carbpol.2008.03.005
183. Yin C, Chia SM, Quek CH, Yu H, Zhuo RX, Leong KW, et al. Microincapsulation with improved mechanical
stability for hepatocyte culture, Biomaterials 2003; 24:1771-80 ISSN: 0142-9612;
184. Yavuzdurmaza Hatice, Harsa Sebnem, Selection of potential probiotic lactobacillus strains from human milk;
International Congress on Engineering and Food, May 2011;
185. Yu, J., Liu, J., Effects of suspension crosslinking reacting conditions on the size of starch microspheres.
Starch/Stäke, Journal of Food Engineering46 (7), 252–255 doi:10.1016/j.jfoodeng.2008.08.011
186. Yuan Kun Lee, Seppo Salminen; Handbook of Probiotics and Prebiotics, 2nd Edition, Wiley, ISBN:978-0-
470-13544-0, 2009
187. Yuguchi, Y.; Thuy, T.T.T.; Urakawa, H.; Kajiwara, K. Structural characteristics carrageenan gels:
Temperature and concentration dependence. Food Hydrocolloids 2002, 16, 515-522;ISSN 0268-005X;
188. Zavisic Gordana et al, Probiotic Features of Two Oral Lactobacillus Isolates ; Brazilian Journal of
Microbiology (2012): 418-428; ISSN:1517-8382 ; 2012;
189. Zinedinde A. and Faid M ; Isolation and Characterization of Strains of Bifidobacteria with Probiotic
Properties In vitro; World Journal of Dairy & Food Sciences 2 (1) : 28-34, 2007, ISSN:1817-308X;
Page 47
PHD THESIS ABSTRACT
NEAMȚU BOGDAN 47 UNIVERSITATEA LUCIAN BLAGA DIN SIBIU
190. Zheng Y. et al, Probiotic Properties of Lactobacillus Strains Isolated from Tibetan Kefir Grains; PLoS ONE
8(7) : e69868 ; DOI : 10.371/journal.pone.0069868, 2013;
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