J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
Nuryshev et al., J Microb Biochem Technol 2016, 8:4
Microbial & Biochemical Technology
New Probiotic Culture of
Opportunities and Prospects
Murat Zh Nuryshev
, Lidia G Stoyanova
and Alexander I Netrusov
LN Gumilyov Eurasian National University, Satpaeva 2, Astana, Kazakhstan
Department of Microbiology, Biological Faculty, MV Lomonosov Moscow State University, Lenin’s Hills, Moscow, Russia
*Corresponding author: Alexander I Netrusov, Department of Microbiology,
Biological Faculty, MV Lomonosov Moscow State University, Lenin’s Hills, Moscow,
Russia, Tel: +7 495 939-10-00; E-mail:
of Lactococcus lactis ssp. lactis: Effective Opportunities and Prospects. J Microb
Biochem Technol 8: 290-295. doi:
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
cin; Sensitivity to antibiotics; Feed additive; Mouse model CBRB-Rb
(8.17) 1Iem; Probiotic
Probiotics are live microorganisms, which refer to a normal
inhabitant of the intestines of healthy animals and human . A
common property of probiotic bacteria is a formation of natural
antibiotic-like substances. Factors associated with the possible effect
of bacteria on the microbiocenosis of intestine, may include the
high antagonistic potential, especially against pathogens, synthesis
of antibiotics, protease, amylase, xylanase, lipase, endoglucanases.
But to define probiotic properties, microbes should be considered
with the ability to provide immunomodulatory impact and
allocate metabolites which stimulate the development of normal
microbiocenosis [2,3]. Generally, probiotics may define as a safe and
“natural” approach that helps to curb the population of bacteria that
cause microbial infections. The largest group of probiotic bacteria in
the intestine is lactic acid bacteria (LAB). LAB widely used in various
food fermentations and has a long history. The genus Lactococcus has
“GRAS” - status (absolutely harmless for human health and animals)
accordingly to the European Commission , while the member of
genera Streptococcus and Enterococcus contain some opportunistic
pathogens. The LAB isolated from the natural sour milk products
draw a special interest among the probiotic correctors of normal
intestinal microbiota and play an important role in human ecology.
Many of them are known to synthesize biologically active peptides
or protein complexes, known as bacteriocins. Bacteriocins differ
from each other by chemical structure and antagonistic activity.
Lactococcus lactis produces bacteriocins as lacticins 48, 3147, several
forms of nisin and lactococcins [1,5]. Nisin is a unique, nontoxic
antibiotic. Nisin is the best studied compound in the latter group.
It is the only antibiotic substance given the status “GRAS”, which is
allowed for application as food preservative under the code E234.
Nisin is the low-molecular mass protein so it is easy to apart it into
amino acids during digestion and does not influence microbiota of
gastrointestinal tract [6,7]. One of the very important properties
of nisin is the activity against Gram-positive bacteria and bacterial
spores of clostridia and bacilli, other non-spore forming bacteria,
as many species of pathogenic Streptococci, Staphylococci, Listeria,
We have isolated several new strains of lactococci from raw milk of Buryatia region of Russia near Lake Baikal
with wide variety of climatic and ecological niches. Physiological and biochemical features of new strains were
studied and compared to the nisin-producing strain Lactococcus lactis ssp. lactis MSU. According to morphological,
cultural, physiological, biochemical properties and gene sequence of 16S rRNA a novel most effective strain 194 was
identified as Lactococcus lactis ssp. lactis (GenBank database DQ 255954), which has status “GRAS” (absolutely
harmless for human health and animals). The strain 194 had inhibitory activity against Gram-positive, Gram-negative
pathogenic bacteria and also on fungi of genera Aspergillus, Fusarium and Candida. This is unique biological property
for natural strains of Lactococcus lactis specie. We also studied the probiotic properties of strain as resistance to HCl
and bile acids, sensitivity to antibiotics and show the therapeutic effect of strain as a food additive on model mice
CBRB-Rb (8,17) 1Iem chronic dermatitis.
Mycobacterium tuberculosis, but it is not effective against Gram-
negative enterobacteria and fungi [5,8].
Fungal spoilage of food is a common and global phenomenon. The
potential production of toxins by fungi is of particular health concern.
As mentioned above, Lactococcus spp. strains generally inhibit only
Gram-positive bacteria and they are not effective against fungi. But
recently it was revealed that some lactococci strains have an ability to
produce antifungal substances which were determined as peptide and
low-molecular phosphoglycolipid . For this reason lactococci can
be considered as potential producers of different antimicrobials with
wider activity spectrum than nisin. In recent years, the concept of
probiotic bacteria has also stimulated work on bacteriocins. In the light
of the increased antibiotic resistance among pathogens, bacteriocins
have attracted attention as an alternative means to prevent infection
by pathogens. In fact, two lantibiotics, nisin and lacticin 3147, have
been found useful in preventing dyskinesia, mastitis, dermatitis, type
impetigo, ecthyma, scalded skin syndrome forms of chronic dermatitis
in humans [6,10-12].
At present, the ways of targeted synthesis of antimicrobial
substances by lactococci are studied in order to obtain new ones
with more valuable properties for application them as probiotics. The
interest in the use of bacteriocin producer cultures as probiotics has
increased tremendously. Screening of effective strains from raw milk
products from Buryatia - North Asiatic region of Lake Baikal, with wide
variety of climatic and ecological niches, which creates conditions for
a variety of LAB and their unique properties are of great scientific and
Prospects. J Microb Biochem Technol 8: 290-295. doi:
Volume 8(4): 290-295 (2016) - 291
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
The aim of this investigation was to isolate and identify the new
lactococci strains from Buryatia with bactericidal and fungicidal
activity and study of their probiotic properties.
Materials and Methods
Media and conditions for isolaton of natural lactococci strains
Raw cow’s milk obtained from the milk farm of Buryatia (Russia)
was used in the work. The tubes with investigated milk were left for
spontaneous self-fermentation at 28°C in steady-state conditions for
17 h. Then bacteria from the tubes, where a dense milk clot formed,
were subjected upon several passages in skimmed milk over 10–17
h. These conditions are allowed to propagate the homo-fermentative
lactococci, which then were inoculated into the agar milk hydrolysate
with bromocresol purple indicator. The individual acid-producing
colonies from the surface of the agar medium were transferred onto
the lawn with the test culture for nisin determination activity - Bacillus
coagulans 429, a thermophilic spore-forming, acid-resistant bacterium,
using a sterile replicator and, in parallel, to the same solid medium
without test culture. The clones forming the largest growth inhibition
zones of test organism were selected as active nisin-producing strains.
To obtain the lawn of the test culture, a 24 h-old culture of B. coagulans,
grown at 55°C on the agar organic medium (g/l): glucose: 10.0, peptone:
5.0, NaCl: 5.0, agar: 20.0 with an addition of Hottinger’s broth (at a
concentration of 28 mg% according to ammonium nitrogen) at pH 7.0
was plated in Petri dishes.
Cultural properties of the isolated strains were examined by
classical microbiological methods of identification and determination
of the culture [13,14].
The identification of the isolated strains
The phylogenetic analysis using the sequences of the 16S rRNA
genes was performed using the programs Vector NTI: ContigExpress
and AlignX. A comparative analysis and search for homologous
sequences were performed using the NCBI database (http://www.ncbi.
nlm.nih.gov/blast). Multiple alignments of the sequences were obtained
by the program ClustalX; the alignments were constructed and the
genetic distances between the strains compared were computed by the
The strains were compared according to their cultural, physiological,
and biochemical properties. The physiological and biochemical
properties of strains displaying the most pronounced antibiotic activity
were assessed according to the fermentation of carbohydrates, the
demand for growth factors, the level of inhibitory activity, and the
range of antimicrobial action. We used the series of carbohydrates:
D-arabinose, D-xylose, D-ribose, D-glucose, L-rhamnose, D-maltose,
D-sucrose, D-mannose, D-lactose, D-galactose, raffinose, D-fructose,
D-sorbitol, dulcitol, mannitol, dextrin, glycerol and starch, which were
added to the base fermentation medium in amounts of 1.0% each. For
growth factors DL-valine, DL-threonine, DL-leucine, L-serine, L-lysine,
DL-cystine, L-glutamate, L-aspartate, L-isoleucine, L-methionine, and
L-cysteine were used. Each amino acid was introduced into the base
fermentation medium in the amount of 0.01%.
To obtain an inoculum, the culture from skimmed milk was re-
inoculated into the inoculation medium, which contained 1% glucose,
yeast autolysate (35 mg% of ammonium nitrogen), and tap water (pH
6.8-7.0). The culture was grown under steady-state condition at 28
of 5 vol.% into the base fermentation medium, which contained (g/l):
: 20.0; glucose: 10.0; NaCl: 1.0; MgSO
: 0.2 and yeast autolysate
grow at 28
the diffusion into agar by measuring of the growth inhibition zone of
test cultures in mm .
The spectrum of inhibitory effects of the strains was studied by
culturing strains under steady-state conditions in the fermentation
medium of the above composition. The microorganisms used in these
experiments were from the Collection of Microbes of the Department
of Microbiology of Moscow State University: 6 strains of Gram-positive
bacteria (Micrococcus luteus 128, Bacillus mycoides 32, B. subtilis 2,
B. coagulans 429, B. cereus 112 and Staphylococcus aureus 144); 6
strains of Gram-negative bacteria (Alcaligenes faecalis 82, Escherichia
coli 52, Proteus vulgaris 206, Pseudomonas aeruginosa 54, Salmonella
gallinarum); 3 strains of fungi (Fusarium oxysporum 61, Penicillium
chrysogenum 37, Aspergillus niger 369); and two yeast strains (Candida
guilliermondii 17 and Rhodotorula aurantiaca 226).
The bacilli were grown on an organic medium containing (g/l):
glucose: 10.0, peptone: 5.0, NaCl: 5.0 and agar: 25.0; the medium was
supplemented by Hottinger's broth (25 mg% amine nitrogen) and
had a pH of 7.0. The yeasts were grown in a wort (6-8
similar (wort 3-4
B with 2.0% agar; pH 6.0).
the case of bacilli, staphylococci, and micrococci was 37°C; E. coli was
grown at 42°C; B. coagulans at 55°C, and yeasts and fungi, at 28°C. Petri
dishes were inoculated with 1-day cultures of test microorganisms (0.1
ml cell suspension in physiological saline per dish; the concentrations
were adjusted to 10
cells per 1 ml using a bacterial turbidity standard).
production judged by the suppression of growth of the indicator
culture B. coagulans, which s was introduced into the agar medium as
a suspension with a density of 10
cell/ml. Antibiotic substances were
acetone, acetic acid, and water (55°C, 1.5 h).
Quantitative determination of the antimicrobial activity was
performed by measuring the zones of growth suppression with
subsequent calculation involving a calibration plot for standard nisin
solutions. Solutions of the preparations served as the standards:
“Nisaplin” (activity 1 000 000 IU/g, "Aplin & Barrett, Ltd" Co., UK)
– for Gram-positives; chloramphenicol (HiMedia Laboratories Ltd,
Mumbai) - for Gram-negative bacteria; nystatin (4670 U/mg, Sigma)
– for fungi. The fungicidal activity was assessed with indicator culture
Aspergillus niger 369 .
Physiological and biochemical features of new strains were studied
and compared to the nisin-producing strain Lactococcus lactis ssp.
lactis MSU. This strain was a reference strain which produces nisin
A identical to that contained in the commercial preparation Nisaplin
(Aplin & Barrett Ltd., UK).The strain MSU was stored as lyophilized in a
household refrigerator at 4
C. The lyophilized culture was reconstituted
The dynamics of growth and bacteriocin accumulation in the
culture liquid of strains were followed for 24 h. The amount of the
biomass was determined by optical density (540 nm).
Volume 8(4): 290-295 (2016) - 292
The sensitivity experiments to the antibiotics: ampicillin,
erythromycin, tetracycline, lincomycin, kanamycin, streptomycin,
rifampicin, neomycin and oleandomycin was performed by disk
In our experiments the isolated lactococci were studied as probiotic
cultures. It was conducted in model experiments of the effects of
adverse conditions of the gastrointestinal tract by the action of high
concentrations of bile acids (0-50%) and hydrochloric acid (0 to 1.0%),
added to fermentative medium, on the viability of strains during the
incubation for 1–3 h.
We checked the effect of culture of novel strain 194 on the
symptoms of dermatoses on male mice CBRB-Rb (8.17) 1Iem at the
age of 23,0 ± 1,3 weeks, as an adequate model of spontaneous chronic
skin dermatitis, type impetigo, ecthyma and scalded skin syndrome.
Animals were fed by complete pelleted feed. As a standard feed used
cereal grains (5 g per mouse per day) with the addition of sunflower
oil and vitamins A, E, D, F; water was unrestricted. Males mice of the
experimental group (n=17) were added daily for two weeks with the
culture of Lactococcus in feed by 194 to 300 mkl per mouse per day.
Assessment of symptoms of dermatitis conducted a double-blind study
once a week in all mice individually. The following information was
recorded: 1) the degree of ulceration or manifestation of alopecia of the
skin on their backs by the 7-point scale; 2) the area of the affected area
of the back, in mm
; 3) weight gain. The experiment comprised three
Screening for the effective bacteriocins-synthesizing strains of
Lactococcus lactis was performed. Lactococci were isolated from raw
milk from Ulan-Ude city area (Buryatia, Russia).
The lactococcal colonies were selected in the media with the
indicator bromocresol purple, which changed the color of the medium
from violet to yellow indicating the acidification degree. It is known
that the major product of homolactic acid fermentation is lactic
acid. Overall, 18 colonies of mesophilic acid-producing bacteria
were selected from which 5 isolated colonies inhibited the growth of
test-microorganism B. coagulans, 2 of them displayed the highest
activity. The best strain named 194 was chosen for further study. The
morphology and cultural properties of it were compared to the nisin-
producing strain Lactococcus lactis ssp. lactis MSU.
The morphology of the isolated strains demonstrated that the
cultures were represented by cocci assembled in pairs or short chains of
various lengths: two, four or 12 cells. in compared to strain L. lactis ssp.
lactis MSU (Figure 1).
The bacteria were non-motile and Gram-positive. They did not
differ in their cultural features from the bacteria belonging to the genus
Lactococcus (Table 1).
Growth was absent in the meat–peptone broth containing 6.5%
NaCl and at pH 9.6. The pH from 6.6 to 7.2 was optimal for the growth
and development of these strains. The optimal incubation temperature
was 28°C; at 10°C the growth was minimal and was completely absent
at 45°C. Uniform growth of bacteria along the entire inoculum’s length
in a stab of an agar medium is characteristic of facultative anaerobes
Ability to consume of various carbohydrates, including sugars,
alcohols and organic acids is the basis of the distinguishing features
in the identification of lactic acid bacteria. Studies have found that all
strains utilized glucose, sucrose, lactose, galactose, maltose, mannose,
fructose, and did not utilize - rhamnose, the trisaccharide raffinose and
did not hydrolyze starch
Strain 194 utilize not only arabinose and xylose, but also the
alcohols: dulcitol, mannitol and sorbitol.
The hallmark of lactic acid bacteria is a great need for complex
nutrients: purines, pyrimidines, amino acids and vitamins, especially
of group B. Amino acids are essential for the construction of the cell
and for the bacteriocin formation. It is known that threonine, serine,
cysteine, lysine, and aspartic acid are precursors of lanthionine and
methyl-lanthionine and part of the nisin molecules .
The results of an experiment to determine the needs of strains in
amino acids and other growth components revealed some features of the
isolates (Table 2). Strain 194 showed good growth in media, indicating
the specific needs in glutamine, asparagine, uracil, aspartic acid and
arginine. Strain 122 does not require adenine and phenylalanine, as
strain 105 did not require methionine, threonine, and diaminopimelic
acid. Strain 205 did not need the following growth components:
Figure 1: Morphology of perspective strains Lactococcus lactis subsp. lactis: A – strain MSU, B - strain 194.
Figure 1: Morphology of perspective strains Lactococcus lactis subsp. lactis: A – strain MSU, B - strain 194.
Volume 8(4): 290-295 (2016) - 293
The preferential location of the most
chains (to 4-7 cocci)
chains to 7 cocci
Growth at 10°C
Growth in the presence of 6.5%
Sensitivity to oxygen
All are facultative anaerobes
thymine, valine, proline, glycine. Rapid growth of the strain MSU
was detected in the media, which included histidine, glutamine,
pyridoxine, isoleucine, uracil, biotin, valine, arginine, alanine. In
medium containing glutamine, asparagine, uracil, aspartic acid,
arginine, and which is a blend of vitamins to the amino acid alanine,
grew all strains, indicating their need for uracil, arginine and vitamins.
Studied lactococci grew equally well in media containing a mixture of
amino acids in combination with vitamin B
and biotin, as well as with
environment lactococci did not grow, confirming that they belong to
auxotrophic microorganisms. Strains 194 and MSU need the presence
of glycine in the medium. The absence of a serine or glycine had no
effect on the growth of strains, except strain 194. Strain 105 has a very
limited need for growth components, but proline and uracil stimulated
its growth. All strains were in need of arginine.
Taxonomic description of isolates
To confirm the taxonomic status were conducted molecular
genetic studies. Computer processing of the results of 16S rRNA gene
sequencing and comparative analysis of our data with the sequences of
the type strains revealed high similarity between them. Based on the
phylogenetic distance, reflecting the differences between strains in the
number of nucleotide substitutions per 100 base pairs (bp), 16S rRNA
homology genes were calculated. All strains show a high degree of DNA
homology 98.9-100%) relative to the reference strain of L. lactis subsp.
lactis. The level of genetic similarity (in %%) of all the strains studied
in relation to closely related strains L. lactis subsp. cremoris was 95.4-
96.6% (Figure 2).
The nucleotide sequences of the 16S rRNA genes of novel strains
were deposited to the GenBank database under following accession
numbers: DQ255952 - strain MGU and DQ255954 - strain 194.
Antimicrobial activity testing
Only four of the selected strains expressed a broad spectrum of
activity against pathogens: Listeria monocytogenes, Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris,
Salmonella and fungi of Aspergillus, Fusarium, Penicillium genera, as well
as against yeasts Rhodotorula aurantiaca and Candida guilliermondii.
The results of our study of the spectra of antibiotic activity of
culture liquids of the strains of L. lactis subsp. lactis, grown in the
fermentation medium, are summarized in Table 3. The strain MSU
suppressed the growth of Gram-positive bacilli and micrococci
in a manner similar to that of nisin. The strain 194 exhibited
Volume 8(4): 290-295 (2016) - 294
the highest antibiotic activity and exerted broad-spectrum of
antibacterial and fungicidal effects, which has not been reported
so far for this specie. The study of the antimicrobial spectrum of
action showed that the strains suppressed the growth of Gram-
positive bacteria, including Bacillus coagulans, Staphylococcus
aureus; Gram-negative, such as: Alcaligenes faecalis, Escherichia
coli, Pseudomonas aeruginosa, Proteus vulgaris and also possessed
fungicidal action - suppressed the growth of microscopic fungi:
Fusarium oxysporum, Penicillium chryzogenum, Aspergillus niger,
what is rare biological property for the natural strains of lactococci.
The strain 194 was more effective against Rhodotorula aurantiaca
and Candida guilliermondii.
It has been revealed, that the strain 194 produced antibiotic complex
which differed from nizin.
The strain 194 had higher antimicrobial productivity, up 3600
IU/ml (with test-culture B. coagulans) as compared with strain MSU
(2000 IU/ml). The inhibition activity against Gram-negative bacteria
consisted of 370 U/ml (was detected with chloramphenicol), and its
antifungal activity (with nistatin as a standard and test-culture .A.
niger) was 1700 u/ml. The strain MSU did not inhibited the growth of
Gram-negative bacteria and fungi.
Sensitivity to antibiotics and other probiotic properties
Accordingly to the results of the study of the sensitivity of isolated
strains to antibiotics, they were sensitive to: ampicillin, erythromycin,
tetracycline, lincomycin, and to a lesser degree to aminoglucoside
antibiotics - kanamycin, streptomycin, to the macrolactam antibiotic -
rifampicin, but they are resistant to neomycin and oleandomycin.
In our experiments isolated lactococci were studied for its probiotic
properties. The food entering the stomach is exposed to the gastric
juice. Pure gastric juice has hydrochloric acid concentration of about
0.3-0.5%, this corresponds to a pH of 1 to 3 in the healthy stomach. It
was revealed that addition of 0.2% of hydrochloric acid to the cultural
medium the growth rates were reduced by 30% to 49% after 3 hours
of incubation. But the strain is resistant to high concentrations of
hydrochloric acid (0.3%-0.5%). The survival rate of strain 194 was 96%
after 1 h of exposure, 94% - after 2 h and after 3 h - 92%.
In the investigations on the influence of culture broth (lived
lactococci with metabolites) of strain 194 on symptoms of chronic
dermatitis on model mice CBRB-Rb (8,17) 1Iem during two weeks of
feeding (used daily as food additive and twice application on the skin of
the back) showed the degree of ulceration and the square of damaged
skin of the back of treated mice decreased by 21% after one week of
culture application and the weight of the animals increased (by 4.5%) to
the end of experiment. Thus, we showed that use of 194 strain as food
additive clearly reduced the severity of dermatitis symptoms.
Screening for the effective strains from raw milk of Buryatia in
accordance with its geographical and natural characteristics of this
Figure 2: Relationships of new isolated lactococci strains on the base of gene sequence of 16S rRNA.
L. diacetilactis AY920469
L. diacetilactis AY920468
L. cremoris AB100792
Volume 8(4): 290-295 (2016) - 295
North Asiatic region of lake Baikal, revealed the isolation of new
lactococci cultures. This region includes a wide variety of climatic
and ecological niches, thus creating conditions for a variety of new
LAB with their unique properties. Thus, our studies are of great
scientific and practical interest for the operation. On the basis of
morphological, cultural, physiological, biochemical properties
and accordingly to 16S rRNA gene sequence of isolates they were
identified as Lactococcus lactis subsp. lactis. The most promising
strain was 194 (accession number in GenBank is DQ255954),
which synthesized antibiotic complex with a wide spectrum of
antimicrobial activity, effective against pathogens. This strain have
“GRAS” status. Thus, the properties of these strains with the wide
spectrum of bactericidal and fungicidal action to the pathogens, the
absence of toxicity and medical effect in a mouse model of chronic
dermatitis enable to recommend the strain L. lactis ssp. lactis 194 for
using as probiotic culture or as potential perspective biopreservative
for preventing fungal spoilage of foodstuffs and edible raw materials
with probiotic effect.
Lidia G. Stoyanova and Alexander I. Netrusov
acknowledge a funding from
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Culture of Lactococcus lactis ssp. lactis: Effective Opportunities and Prospects.
J Microb Biochem Technol 8: 290-295. doi: