Isolation and characterization of a native isolate of Leuconostoc for functional attributes

During the past several years, the focus of nutritional sciences has shifted
from deficiency disease prevention to optimizing health and prevention of
chronic diseases. Accordingly the research has encompassed the health effects of
bioactive food components. In this regard, probiotic therapy is being used
increasingly in humans and veterinary medicine due to their apparent high index
of safety and public perception about natural or alternative therapies (Gherty
1995; Sander 1998; FAO Report 2002).
Probiotics are a category of ‘nutraceuticals’ i.e., viable cultures added to
food with the intension of maintaining or improving the nutritional health of
consumers. These bacteria favorably alter the intestinal microflora, inhibit
growth of harmful bacteria, promote good digestion, boost immune function and
increase resistance to infection (Malin et al 1996; Haudault et al 1997).
Lactic acid bacteria (LAB) are in the focus of extensive research because
of their probiotic nature. One important way in which they affect health of the
host is by providing enzymatic activities that improve the utilization of nutrients
within the intestine.
Today, probiotic market promises the disease prevention and better health
for all as a natural alternative therapy. One of the important challenges to the
present day probiotic industry is the eradication of lactose intolerance problem
which is quite common all over the world. It is estimated that 70-90% of adults
are lactose intolerant (Swegerty 2002), suffering from intestinal discomfort with
the symptoms like nausea, cramps and gas (Stiles and Holzapfel 1997). In the
present scenario, with increase desire of consumer for natural food products as
source of providing nutrition and other desirable benefits, research work towards
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the selection of strain with functional properties has become very important
especially for treating lactose intolerance.
Considering all these aspects, in the present work a strain of LAB was
isolated from milk and milk products, characterized and studied for its probiotic
functional attributes to be used in food formulation.
Objectives
1) Isolation and characterization of Leuconostoc from milk products
2) Properties of the isolated bacterium in relation to functional significance
3) Colonization of LAB in relation to homoserine lactone
Chapter 1
Isolation and screening of lactic acid bacteria from milk and milk product
The problem for choosing a culture to be used in health promoting
probiotic ingredients in food and pharmaceutical preparations was apparent even
in the original work of Metchnikoff (1906). Reid (1999) and Sobel (1999)
specify certain properties like ability to adhere intestinal cell wall, exclude/
reduce pathogenic microorganisms, produce antimicrobial compound, resist
microbicides, non-carcinogenic and non-pathogenic character to be present in
the selected strain for their use as probiotic culture.
Now a days, a consensus is emerging for selection criteria of LAB to
achieve positive probiotic effect (Collins et al 1998). Therefore, in the present
work a screening procedure is performed to select a potential probiotic culture
with an ultimate aim of using the culture in functional food for beneficial effect.
In the present work, 45 isolates were screened for basic LAB
characteristics from milk and milk products. As the most critical characteristics
of probiotic strain are tolerance gastrointestinal condition (Ammor and Mayo
2007), the isolated strains were initially screened for their resistance in this harsh
environment and further adapted to grow at low pH and high bile salt
concentration. The strains were further analyzed for their survival under
simulated gastrointestinal condition. The strains that were able to survive under
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such environment were characterized and identified through biochemical assays
and molecular techniques. The identified cultures Leuconostoc mesenteroides
(Lsr-1(W)) and Lactobacillus plantarum (Lsr-12(Cu)) were further studied for their
resistance to digestive enzymes pepsin and trypsin. Leuconostoc mesenteroides
(Lsr-1(W)) that was better resistant to these enzymes was selected for further
studies and was coded as PLsr-12(Cu).
Chapter 2
Probiotic functional properties of culture isolate
Probiotics have health promoting effects including inhibition of
pathogens, antimutagenic, anticarcinogenic activity, prevention of diarrhea,
stimulation of immune response and ability to reduce serum cholesterol levels
(Tannock 1999). The development of new applications such as life vaccines and
probiotic foods reinforces the need for these characteristics.
In this regard, the selected culture of Leuconostoc mesenteroides
(PLsr-1(W)) was evaluated for its functional properties. According to the data
obtained, the culture PLsr-1(W) shows antimicrobial activity against 7 toxic food
pathogens such as E. coli, S. typhi, S. dysenteriae, P. aeroginosa, V. cholerae,
Y. enterocolitica and S. aureus. The isolate was found resistant to 3 antibiotics
tested. The inhibitory activity of intracellular cell free extract of culture to
ascorbate autooxidation, ferrous ion chelating ability and scavenging ability of
oxygen radical represent the antioxidative property of culture isolate. The culture
was also able to assimilate 28 μg/ml of cholesterol from media which shows the
anticholesterol activity of the strain. Analysis of β-galactosidase in culture
indicate the ability of the culture to hydrolyze lactose into simple sugar for easy
absorption. From the results of cell surface hydrophobicity and intestinal
adherence test, the adhesion ability of the culture was confirmed. Analysis of
S-layer adhesion protein by SDS-PAGE showed a prominent protein band of
60 KD. Volatile compounds analyzed by GC and GCMS of culture extract
confirmed the presence of therapeutically important compounds. Stress response
of PLsr-1(W) to low pH and high bile salt indicate the increased content of stress
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proteins and membrane fatty acids (saturated and unsaturated). All these
properties make the present isolate a potent probiotic and will stand out as a
natural cure to many diseases.
Chapter 3
Leuconostoc as a source for β-galactosidase enzyme
Lactose is a non-reducing disaccharide which provides almost half the
total energy required by infants. In presence of lactase/ β-galactosidase, lactose
gets hydrolyzed into galactose and glucose for easy absorption. But in persons
deficient in lactase show symptoms of intestinal discomfort known as lactose
intolerance (Sieber et al 1997). Hence studies were carried out to enhance the
enzyme activity in the present culture isolate.
Leuconostoc mesenteroides PLsr-1(W) that had the ability to survive the
harsh conditions of GIT was studied for its ability to increase lactose tolerance.
Primarily strain was improved by UV irradiation and chemical mutagenesis for
enhanced enzyme activity. UV mutant strain showed 2 folds higher activity than
the parent strain and hence was selected for all further studies (mutant strain
was coded as M7-PLsr-1(W)). Cell permeabilization method was optimized for
maximum release of enzyme. Response surface methodology (RSM) studies
were undertaken for optimization of chemical and physical parameter. The
enzyme produced under optimum condition of pH 7.5 with 1.25 % lactose was
partially purified and studied for kinetics. The results indicate a 25 fold increase
in the activity of partially purified enzyme as compared to the crude extract.
Chapter 4
Enhancement of culture shelf life on storage
LAB in food biotechnology or for any probiotic formulations are strictly
concerned for the preservation techniques employed to ensure stable culture in
terms of viability and bacterial metabolism. It is very important to use suitable
technology or process to enhance and maintain the viability of the culture on
storage for a beneficial effect.
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As the present isolate M7-PLsr-1(W) is very important culture due to its
characteristic multifunctional probiotic properties, it becomes necessary to keep
the culture viable for a longer time. Hence the aim of the present work was to
preserve the culture for longer shelf life. In this regard, the culture was subjected
to different preservation techniques i.e., oven, spray, vacuum and freeze drying.
The viability and resistance of culture to these methods was tested and it was
found that the culture showed maximum survival to freeze drying condition. The
viability under freeze drying condition was further enhanced with
supplementation of different cryoprotective agents (PEG, lactose and sucrose).
Enhanced membrane fatty acid composition and cellular protein confirmed the
adoptive nature of the culture to freeze drying.
In the present work, we also report the viability of the freeze dried culture
for 6 months. It showed resistance to low pH (2.0) and high bile salt (4%). Even
after 6 months of freeze drying, culture showed antimicrobial activity against 6
toxic food pathogens. Data determines that the culture retains the β-galactosidase
enzyme even after 6 months of storage.
The above study determines the capability of culture to remain viable
even after 6 months of storage. It also shows the importance of cryoprotectants
in enhancing the viability and beneficial attributes of culture during storage. This
determines the capability of the culture to be utilized in the form of capsules or
any functional food.
Chapter 5
Functional food with Leuconostoc: a native isolate
The demand for probiotic foods is increasing all over the world reflecting
the heightened awareness among the public relationship between diet and health.
Fermented dairy products are the most widely used food vehicles for these
probiotic bacteria because of their healthy image. During the past twenty years
there has been a tremendous increase in the world wide sales of cultured
products containing probiotic bacteria because of their health effects (Ostlie
2005; Maltila-Sandholm 1999).
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In this regard, the culture M7-PLsr-1(W) that has the potent probiotic
properties was used as starter culture in the preparation of fermented milk
beverage. The product was analyzed to be rich in protein, fat, sugar and minerals
like iron, zinc and magnesium. Hence the viability of culture in the product was
further enhanced with supplementation of different adjuvants (tryptone, casein
hydrolysate, cysteine hydrochloride and ascorbic acid). The results conclude that
the culture maintained maximum viability on supplementation of tryptone
(100 mg/L) after 5 days of storage at 4°C. Fatty acid composition of the product
also confirmed the nutritional property of the product.
Chapter 6
Preservation of fermented milk over shelf storage
Spoilage causes a significant loss to dairy industries and also disease
outbreaks. In this concern, research work was carried out to investigate and
preserve fermented milk beverage prepared with M7-PLsr-1(W).
In this regard, the functional fermented milk beverage prepared by
M7-PLsr-1(W) was studied for the predominant bacterial cultures responsible for
spoilage of the product. Pseudomonas sp was identified as dominant spoilage
bacteria and through TLC, GC and GCMS, the signal molecule for spoilage was
investigated as hexanoyl homoserine lactone (HHSL).
This culture releases HHSL and forms a quorum when high cell density is
reached. Inhibition of bacterial growth in this biofilm by using biocides,
antibiotic and bacteriophages has many obstacles such as cell permeability,
specificity and efficacy in mode of delivery. Hence the natural furanones which
are non-toxic are used in present study because of its small size and ease of
delivery. Because of its structural similarity they specifically interfere with
signal molecule without any adverse effect on the beneficial bacterial consortia.
Results show that 2(5H)-furanone tested was having better inhibitory activity
against Pseudomonas than bromofuranone. This was also in concurrence with
reduction in rhamnolipid content, reduced motility and exoprotease enzyme
activity. Using 2(5H)-furanone about 5-6 log of Pseudomonas culture was
reduced in the fermented beverage.
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Chapter 7
In-vivo studies using Leuconostoc for functional attributes
The health benefits described for probiotic lactic acid bacteria make them
a good agent for preparation of functional food and hence a number of bacterial
strains are being identified and incorporation into these foods. As more probiotic
organisms are discovered, it is important to carefully document the efficacy of
the strain for its potential application and safety.
Looking into the probiotic characteristic of the present isolate
M7-PLsr-1(W) of being resistant to GIT condition, adherence ability,
antimicrobial, β-galactosidase activity and anticholesterol activity through
in-vitro assays the present study was aimed to assess its safety and functions in
in-vivo model using albino rats.
According to the result there was no deleterious effect on probiotic
feeding for 3 months. No bacterial translocation was observed and hence it is
likely to be safe for human consumption. Increase in the general body weight
and serum urea concentration provides a potential proof for the health promoting
effect of the culture. High LAB count in feces and ceacum shows the ability of
the culture to resist the GIT condition and adhere to exert beneficial effect.
Results of cholesterol assay confirmed the anticholesterol effect of the culture.
The ability of the isolate to protect against pathogens was determined by
decrease in E. coli count.
Lactose intolerance, a clinical problem associated with unpleasant
abdominal discomfort is due to undigested lactose. According to a survey, about
70-80% of the world population are lactose intolerant (Swagerty 2002).
Innovative approaches have been tried as alternative to antibiotics in treating
lactose intolerance because of the growing antibiotic resistance problem.
Alternative methods such as exogenous β-galactosidase administration in
functional foods or in pharmaceutical preparation is advised but the draw back is
the inactivation of the enzyme in gastrointestinal transit. The other approach is
gene therapy or exclusion of milk and dairy products with lactose from diet
which may cause nutritional disadvantages.
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Considering this, the present culture M7-PLsr-1(W) was tested for its
ability to reduce lactose intolerance problem by in-vivo experiments. Single dose
study and long term (3 months) experiments were carried out with albino Wister
rats. From the study, the effective dose of culture was determined to be
108 cfu/ml. Disappearance of diarrhea in lactose intolerant induced rats after
culture feeding confirmed the positive impact of culture in treating lactose
intolerance.
From the report presented here, the culture M7-PLsr-1(W) was found to
have probiotic characters in terms of resistance to GIT, antimicrobial activity,
anticholesterol activity and adherence ability. The culture was also able to
reduce the symptoms of lactose intolerance and hence can be used as an
alternative source to treat the problem.
Achievement of the work
The culture is native, isolated in laboratory and shows potential probiotic
characteristics along with high β-galactosidase activity. The in-vivo experiments
conducted with albino Wister rats conclude the potential probiotic functional
properties and its importance in reducing lactose intolerance problem. The
culture preserved by freeze drying shows viability even after 6 months of
storage. The fermented milk beverage prepared with the present isolate is having
a high nutritive value so that it can be used by all ages for its beneficial effects.
This fermented milk beverage was preserved over a storage time by interrupting
the signal molecules produced by of spoilage bacteria using 2(5H)-furanone
which is a natural compound produced by an algae and known to be safe.
Social and scientific relevance
With the rise in the consumer’s awareness of individual health, nutrition
and well being, the interest and demand for value added foods and beverages has
expanded. Although some companies have marketed the probiotic products, they
are either expensive or are specified for one specific cure. Most of these products
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are not indigenous in our country and the viable count of the culture is
questionable or not to the mark as labeled.
Today WHO and Indian five year plans both have a common priority to
replace antibiotics with natural means of cure. In this regard, the present isolate
having probiotic functional properties promises to be as a source of natural
alternative cure. The fermented beverage prepared by Leuconostoc
mesenteroides (M7-PLsr-1(W)) has many functional attributes.