New Probiotic Culture of Lactococcus lactis ssp lactis: Effective Opportunities and Prospects



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Volume 8(4): 290-295 (2016) - 290

J Microb Biochem Technol 

ISSN: 1948-5948 JMBT, an open access journal

Nuryshev et al., J Microb Biochem Technol 2016, 8:4

http://dx.doi.org/10.4172/1948-5948.1000299

Research Article

Open Access

Journal of

Microbial & Biochemical Technology

Jo

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f M

icr

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 Biochem

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l T

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olo

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ISSN: 1948-5948

New Probiotic Culture of 

Lactococcus lactis

 ssp. 


lactis

: Effective 

Opportunities and Prospects

Murat Zh Nuryshev

1

, Lidia G Stoyanova

2

 and Alexander I Netrusov

2

*

1

LN Gumilyov Eurasian National University, Satpaeva 2, Astana, Kazakhstan

2

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: 

anetrusov@mail.ru



Received May 13, 2016; Accepted June 29, 2016; Published July 09, 2016

Citation: Nuryshev MZ, Stoyanova LG, Netrusov AI (2016) New Probiotic Culture 

of Lactococcus lactis ssp. lactis: Effective Opportunities and Prospects. J Microb 

Biochem Technol 8: 290-295. doi: 

10.4172/1948-5948.1000299



Copyright: © 2016 Nuryshev MZ, et al. This is an open-access article distributed 

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.



Keywords:

 Lactococcus lactis subsp. Lactis; Identification; Bacterio-

cin; Sensitivity to antibiotics; Feed additive; Mouse model CBRB-Rb 

(8.17) 1Iem; Probiotic

Introduction

Probiotics are live microorganisms, which refer to a normal 

inhabitant of the intestines of healthy animals and human [1]. 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 [4], 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, 



Abstract

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 [9]. 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 

practical interest.




Citation: Nuryshev MZ, Stoyanova LG, Netrusov AI (2016) New Probiotic Culture of Lactococcus lactis ssp. lactis: Effective Opportunities and 

Prospects. J Microb Biochem Technol 8: 290-295. doi: 

10.4172/1948-5948.1000299

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 

program Mega2.

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

o

C. 



Thereafter, the inoculum (OD

540 


0.14-0.19) was introduced in an amount 

of 5 vol.% into the base fermentation medium, which contained (g/l): 

KH

2

PO



4

: 20.0; glucose: 10.0; NaCl: 1.0; MgSO

4

: 0.2 and yeast autolysate 



(35 mg% ammonium nitrogen), pH 6.8-7.0 and culture was allow to 

grow at 28

o

C.

Antimicrobial testing



The antimicrobial activity of lactic acid bacteria was determined by 

the diffusion into agar by measuring of the growth inhibition zone of 

test cultures in mm [14].

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

o

B) medium 



supplemented with 2.5% agar (pH 6.8). The medium for the fungi was 

similar (wort 3-4

o

B with 2.0% agar; pH 6.0).



The bacteria were cultured at 28-:-55°C: the temperature used in 

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

9

 cells per 1 ml using a bacterial turbidity standard).



The bacteriocin-synthesizing activity was assessed as nisin 

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

9

 cell/ml. Antibiotic substances were 



extracted from the cells and culture liquid using a 4:1:5 mixture of 

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 [14].

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

o

C. The lyophilized culture was reconstituted 



by sterile non-fat (skimmed) milk.

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). 




Citation: Nuryshev MZ, Stoyanova LG, Netrusov AI (2016) New Probiotic Culture of Lactococcus lactis ssp. lactis: Effective Opportunities and 

Prospects. J Microb Biochem Technol 8: 290-295. doi: 

10.4172/1948-5948.1000299

Volume 8(4): 290-295 (2016) - 292

J Microb Biochem Technol 

ISSN: 1948-5948 JMBT, an open access journal

The sensitivity experiments to the antibiotics: ampicillin, 

erythromycin, tetracycline, lincomycin, kanamycin, streptomycin, 

rifampicin, neomycin and oleandomycin was performed by disk 

diffusion method. 



Testing the Lactococcus lactis ssp. lactis strain 194 as probiotic 

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

2

; 3) weight gain. The experiment comprised three 



series of triplicate measurements each and the results were statistically 

processed. 



Results and Discussion

Screening for the strains and their isolation

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 

[11].


Biochemical testing of isolates

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 [5].

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.



Citation: Nuryshev MZ, Stoyanova LG, Netrusov AI (2016) New Probiotic Culture of Lactococcus lactis ssp. lactis: Effective Opportunities and 

Prospects. J Microb Biochem Technol 8: 290-295. doi: 

10.4172/1948-5948.1000299

Volume 8(4): 290-295 (2016) - 293

J Microb Biochem Technol 

ISSN: 1948-5948 JMBT, an open access journal



Properties

Llactis subsp. 

lactis*

Strain MSU  

Strain 194

Strain 105

Strain 122

Strain 205

The preferential location of the most 

typical cells

short chains

chains (to 4-7 cocci) 

diplo-cocci and 

long chains 

chains to 7 cocci 

diplo-cocci

chains to 7 cocci 

Mobility 

-

-



-

-

-



-

Growth at 10°C

+

+

+



+

+

+



Growth at 45°C

-

-



-

-

-



-

pH 9.6


-

-

-



-

-

-



Growth in the presence of 4% NaCl

+

+



+

+

+



+

Growth in the presence of 6.5% 

NaCl

-

-



-

-

-



-

Sensitivity to oxygen

All are facultative anaerobes

The ability



 to consume carbohydrates

Xylose


-

-

+_



-

-

-



Fructose

+

+



+

+

+



+

Lactose 


+

+

+



+

+

+



Sucrose

+

+



+

+

+



+

Maltose


+

+

+



+

+

+



Raffinose

-

-



-

-

-



-

Starch 


-

-

-



-

-

-



Mannitol

-

-



+

-

-



+

Note: 


(negative); (positive)

Table 1: Differentiating features of isolated lactococcal strains.

Growth components of strain

Strain  194

Strain 105

Strain 122

Strain 205

Strain MSU

Alanine 


+

-

+



+

-

Arginine



+

+

+



+

+

Asparagine



+

+

-



+

-

Aspartic acid



+

+

+



+

+

Glycine



+

-

-



-

+

Glutamine



+

-

-



+

+

Glutamine



-

+

-



-

+

Thymine



+

-

+



+

-

Uracil 



+

+

+



+

+

Note: 



(negative); ( positive )

Table 2: The need for growth components of isolated strains lactococci.

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

1

 and biotin, as well as with 



uracil. In control experiments in the absence of growth factors in the 

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 



Citation: Nuryshev MZ, Stoyanova LG, Netrusov AI (2016) New Probiotic Culture of Lactococcus lactis ssp. lactis: Effective Opportunities and 

Prospects. J Microb Biochem Technol 8: 290-295. doi: 

10.4172/1948-5948.1000299

Volume 8(4): 290-295 (2016) - 294

J Microb Biochem Technol 

ISSN: 1948-5948 JMBT, an open access journal

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.



Conclusion

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.

 

 A10



 

 A4


 

 L. lactis AB100798

 

 A6



 

 L .lactis AJ419572

 

 A14



 

 A5


 

 A9


 

 A3


 

 A1


 

 L. lactis AB118034

 

 A7



 

 L. diacetilactis AY920469

 

 L. diacetilactis AY920468

 

 A2



 

 A8


 

 A13


 

 A11


 

 Lcremoris AB100802

 

 L. cremoris AB100792

 

0.005 


 

Isolated strains А: 1 - 109; 2 – 129; 3 - 116; 4 – 105; 5 – strain MSU; 6 - 95; 7 - 116; 8 – 122; 9 – 220; 10 – 112; 11 – 122; 13 – 194; 14 – 805.

Reference strains: L. lactis ssp. lactis: AB100798, AJ419572, AB118034; 

L. lactis bv. diacetylactis: AY920469, AY9204684; L. cremoris: AB100802 and AB100792.


Citation: Nuryshev MZ, Stoyanova LG, Netrusov AI (2016) New Probiotic Culture of Lactococcus lactis ssp. lactis: Effective Opportunities and 

Prospects. J Microb Biochem Technol 8: 290-295. doi: 

10.4172/1948-5948.1000299

Volume 8(4): 290-295 (2016) - 295

J Microb Biochem Technol 

ISSN: 1948-5948 JMBT, an open access journal

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. 



Acknowledgement

Lidia G. Stoyanova and Alexander I. Netrusov

 

acknowledge a funding from 



Russian Scientific Fund (Grant #14–50-00029).

References

1.  Fuller R, Gibson GR (1998) Probiotics and prebiotics: microflora management 

for improved gut health. Clin Microbiol Infect 4: 477–480.

2.  Holzapfel WH, Haberer P, Geisen R (2001) Taxonomy and important features 

of probiotic microorganisms in food and nutrition. Am J Clin Nutr 73: 365-373.

3.  Madden  JA,  Hunter  JO  (2002) A  review  of  the  role  of  the  gut  microflora  in 

irritable bowel syndrome and the effects of probiotics. Br J Nutr 88 Suppl 1: 

S67-72.


4.  European Parliament and Council. Regulation (EC) 248/97 of the European 

Parliament and of the Council of 27 Jan 1997 concerning novel foods and novel 

food ingredients.

5.  Stoianova LG, Ustiugova EA, Netrusov AI (2012) Antibacterial metabolites of 

lactic acid bacteria: their diversity and properties. Prikl Biokhim Mikrobiol 48: 

259-275.


6.  Bernbom N, Licht TR, Brogren CH, Jelle B, Johansen AH, et al. (2006) Effects 

of Lactococcus lactis on composition of intestinal microbiota: Role of nisin. Appl 

Environ Microbiol 72: 239-244.

7.  Nes IF, Diep DB, Holo H (2007) Bacteriocin diversity in Streptococcus and 

Enterococcus. J Bacteriol 189: 1189-1198.

8.  Yildirim Z, Yildirim M, Johnson MG (2004) Mode of action of lactococcin R 

produced by Lactococcus lactis R. Nahrung 48: 145-148.

9.  Lowe D, Arendt E (2004) Lactic acid bacteria in malting and brewing with their 

relationships to antifungal activity. Micotoxins and gushing: A review. J Inst 

Brew 110: 163-180.

10. Ryan MP, Meaney WJ, Ross RP, Hill C (1998) Evaluation of lacticin 3147 and 

a teat seal containing this bacteriocin for inhibition of mastitis pathogens. Appl 

Environ Microbiol 64: 2287-2290.

11. Hayashi A, Kimura M, Nakamura Y, Yasui HJ (2009) Anti-atopic dermatitis 

effects and the mechanism of lactic acid bacteria isolated from Mongolian 

fermented milk. Dairy Res 76: 158-164.

12. Iannitti T, Palmieri B (2010) Therapeutical use of probiotic formulations in 

clinical practice. Clin Nutr 29: 701-725.

13. Ludwig W, Schleifer KH, Whitman WB Revised road map to the phylum 

Firmicutes. Bergey’s Manual of Systematic Bacteriology, The Firmicutes. New 

York, Springer-Verlag.

14. Stoianova LG, Egorov NS, Fedorova GB, Katrukha GS, Netrusov AI (2007) 

A comparison of the properties of bacteriocins formed by Lactococcus lactis 

subsp. lactis strains of diverse origin. Prikl Biokhim Mikrobiol 43: 677-684.



 Test

Strains

119

222

229

116

205

115

194

МГУ

Nisaplin 

3000 IU/ml

Diameter of inhibition zone, mm

Bacillus mycoides 

20.0


10.0

16.0


19.5

16.0


11.0

24.0

15.0


17.0

Bacillus subtilis

12.0


10.0

15.0


22.0

20.0


11.0

24.0

16.0


18.0

Bacillus coagulans

17.0


8.0

15.0


18.0

18.0


20.0

23.0

18.0


21.0

Bacillus cereus

18.5


10.0

14.0


21.0

19.0


15.0

12.0

16.0


18.0

Micrococcus luteus

21.5


13.0

20.0


22.5

16.5


21.5

19.0

19.0


25.0

Staphylococcus aureus

16.0


0

16.0


20.0

17.0


17.0

16.0

12.0


15.0

Alcaligenes faecalis

0

9.0



10.0

12.5


12.5

15.0


12.0

0

0



Escherichia coli

0

11.0



0

15.0


14.0

19.0


12.0

0

0



Proteus vulgaris

0

9.0



0

16.5


16.0

14.0


16.0

0

0



Pseudomonas aeruginosa

0

0



0

16.5


15.5

12.0


12.0

0

0



Fusarium oxysporum

0

0



0

16.5


10.0

12.0


10.0

0

0



Penicillium chryzogenum

0

0



0

17.5


10.5

12.0


16.0

0

0



Aspergillus niger

0

0



0

21.0


10.0

14.0


15.0

0

0



Rhodotorula aurantiaca

0

0



0

19.5


10.0

13.0


14.0

0

0



Candida guellermondii

0

0



0

11.0


12.0

10.0


16.0

0

0



Таble 3: Antimicrobial action of isolated strains of Lactococcus lactis ssp. lactis in comparison with Nisaplin.

Citation:  Nuryshev  MZ, Stoyanova LG, Netrusov AI  (2016) New Probiotic 

Culture of Lactococcus lactis ssp. lactis: Effective Opportunities and Prospects. 

J Microb Biochem Technol 8: 290-295. doi: 

10.4172/1948-5948.1000299



Document Outline

  • Title
  • Corresponding author
  • Abstract
  • Keywords
  • Introduction
  • Materials and Methods 
    • Media and conditions for isolaton of natural lactococci strains
    • The identification of the isolated strains
    • Antimicrobial testing
    • Testing the Lactococcus lactis ssp. lactis strain 194 as probiotic 
  • Results and Discussion
    • Screening for the strains and their isolation
    • Biochemical testing of isolates
    • Taxonomic description of isolates
    • Antimicrobial activity testing
    • Sensitivity to antibiotics and other probiotic properties
  • Conclusion
  • Acknowledgement
  • Figure 1
  • Figure 2
  • Table 1
  • Table 2
  • References



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