RS20150852A1 - NEW PROBIOTIC STARTER CULTURE FOR HUMAN AND ANIMAL APPLICATION - Google Patents

Abstract

The present invention relates to a new probiotic starter culture consisting of three thermophilic natural lactic acid bacteria (BMK) isolates: Streptococcus thermophilus LMG P-28585 (BGVLJl-44), Lactobacillusfermentum LMG P-28583 (BGHI14) and Lactobacillus helvetGillvetMillvetMillvetMillvetMillvetMillvetMillvetMillvetGillvetGM 24226 (BGRA43), in specific relationships. The starter culture of the present invention exhibits proteolytic and antimicrobial activity, as well as an immunomodulatory effect.

Description

NEW PROBIOTIC STARTER CULTURE

FOR HUMAN AND ANIMAL USE

Technical field

The subject invention belongs to the field of biotechnology and relates to a new probiotic starter culture with a unique combination of three bacterial strains within the species Streptococcus thermophilus, Lactobacillus fermentumiLactobacillus helveticus. Its use in the prevention and/or treatment of intestinal infections in mammals, i.e. humans and animals, and its use as an immunomodulator is also described.

Technical problem

The technical problem solved by the present invention can be defined as the provision of a new improved probiotic culture as an alternative to the use of antibiotics for the prevention and/or treatment of intestinal infections in humans and animals, thereby improving the individual's health condition.

It is known that the probiotic property comes from the property of the strain, and the species. Accordingly, the probiotic starter culture according to the present invention is based on the optimal ratio of three clearly defined bacterial strains: Streptococcus thermophilusLMG P-28585 (BGVLJ1-44), Lactobacillus fermentumLMG P-28583 (BGHI14) and Lactobacillus helveticusLMG P-24226 (BGRA43), characterized to the strain level by 16S rDNA sequencing and the method "pulse-field gel eleetrophoresis" (PFGE), and which has been shown to exhibit proteolytic and antimicrobial activity, as well as immunomodulatory effects.

Furthermore, the use of this starter culture for obtaining probiotics and compositions intended for the prophylaxis and/or treatment of disorders associated with the colonization of the gastrointestinal tract of mammals by pathogenic microorganisms is described, as well as for obtaining probiotics and compositions for the colonization of the gastrointestinal tract of mammals after antibiotic therapy.

The subject invention also relates to a probiotic and a composition based on this probiotic starter culture and their use in the prevention and/or treatment of intestinal infections in humans and animals and their use as an immunomodulator in maintaining the natural balance of the immune system and regulating the immune response of humans and animals, including infants and young animals.

Furthermore, the present invention also relates to a probiotic combination based on this probiotic starter culture in the prevention and/or treatment of inflammatory bowel diseases in humans and animals.

In more detail, the subject invention related to the new probiotic starter culture consists of three thermophilic natural isolates of lactic acid bacteria (LMG): Streptococcus thermophilusLMG P-28585 (BGVLJ1-44), Lactobacillus fermentumLMG P-28583 (BGHI14) and Lactobacillus helveticusLMG P-24226 (BGRA43), in specific relationships, its use in prevention and/or treatment intestinal infections in mammals and use as an immunomodulator.

State of the art

Probiotic preparations based on the use of various strains of lactic acid bacteria, bacilli and/or yeasts are well known in the art. Probiotics are known in the art as mono- or combined cultures of living microorganisms which, when used, have a beneficial effect on the mammalian host (humans or animals), improving the properties of the existing microflora.

Probiotic bacteria have a number of therapeutic effects, among others, in the prevention and treatment of diarrhea, they have a favorable effect on immunity (probiotic microorganisms form an intestinal ecosystem that inhibits the growth of pathogenic and opportunistic bacteria (E. coli, Salmonella tvphimurium, Clostridium

perfringens, Clostridium difficile, Pseudonomas sp., Listeria sp., Staphylococcus aureus...), u

prevention of candidiasis, etc.

Currently available preparations for the treatment of intestinal infections in domestic animals are intended for feeding newborn animals (Method for correction of immunobiological indices with piglets in period of weaning - RU2471359 ( Cl)— 2013-01-10), prepared in the form of dry fodder with the addition of various active substances such as e.g. zeolite (Complexprobioticpreparation for beef strains - RU2011153643 ( A) — 2013-07-10), medicinal plants (Chinese herbal medicine

composite probiotic feed additive for pigs and preparation method of additive - CN103549141 ( A)

- 2014-02-05), prebiotics { Feed additive for veterinary drug solid fermentation and preparation method of feed additive - CN103039 700 ( A) — 2013- 04- 17), vitamins ( Pig feed capable of

preventing piglets from diarrhea and traditional Chinese medicine composition - CN102835609 ( A) — 2012- 12- 26) and others.

American patent US 5,656,268 describes the process of obtaining a biological product in liquid form or in the form of cream, which consists of pure milk or a milk ingredient and agents for fermentation or the following bacterial strains Lactobacillus fermentum, Lactobacillus bulgaricus, Lactobacillus

acidophilus, Lactobacillus helveticus, Lactobacillus casei and Streptococcus thermophilus. This one

the invention can be considered the closest state of the art, since the composition of the biological product includes milk and at least two bacterial strains from the above. The described process yields a product of defined acidity that can contain oil (olive, vegetable and aromatic oil) as well as fruit, vegetables, plants or honey, and which is based on milk or whey and is used as a supplement to improve the general health of the user, especially when it is in a dry sublimated form. Furthermore, the product is used as a cosmetic cream, creams and liquids for body and massage, as a gum care cream and as an addition to baths.

The probiotic starter culture of the subject invention is significantly different from the mentioned patent, among other things, in that it is based on optimal ratios of only three clearly defined bacterial strains within the species Streptococcus thermophilus, Lactobacillus fermentum, and Lactobacillus helveticus mentioned in the mentioned patent, namely: Streptococcus thermophilusLMG P-28585 (BGVLJ1-44), Lactobacillus fermentumLMG P-28583 (BGHI14) and Lactobacillus helveticusLMG P-24226 (BGRA43), which depends on its use in humans and animals. In the state-of-the-art document, the ratio of the components that make up the biological product is not defined, but only the procedure for obtaining the product, which is significantly different from the procedure for obtaining the probiotic starter culture of the subject invention.

Furthermore, as described in the following text, the probiotic starter culture exhibits proteolytic and antimicrobial activity and immunomodulatory action, unlike the document from the state of the art, where the activities have not been proven in any way. The probiotic starter culture of the present invention does not contain oils or other ingredients listed in the given document, and it also differs according to the forms for use. It is known that the probiotic property is a property of the strain, not the species. In this sense, the combination of strains of the subject invention manifests its probiotic properties, which is supported by the text of the description and as such finds application, unlike the product described in the state of the art, which refers to a type that includes more (all strains), but is biologically completely unusable.

European patent EP1359816 refers to a combination of probiotics containing different combinations of lactobacilli, propionic acid bacteria and bifidobacteria. This probiotic can be combined with a prebiotic to give a synbiotic. The combination of the invention may be consumed as such or with appropriate food products such as dairy products, beverages, juices, soups or baby food or with pharmaceutical products and is useful in immune system stimulation therapy and general health improvement. The given combination is prepared in a unit dosage form, more precisely in the form of a capsule.

European patent EP1541673 describes lactic acid bacteria, more specifically natural isolates of the genera Lactobacillus, Bifidobacterium and Streptococcus (Enterococcus) and their use to obtain a composition for feeding pets (primarily dogs and cats). The resulting compositions are intended for the regulation of the immune response of animals primarily by colonizing the gastrointestinal tract of the animal and exerting a beneficial probiotic effect on the health of the animal.

Furthermore, European patent EP2219658 relates to a composition based on probiotic bacteria and substances with prebiotic properties and their use in the treatment of respiratory pathologies and/or infections while simultaneously improving intestinal function. The symbiotic composition contains a combination of the following bacterial strains: Lactobacillus plantarum (LMG P-21020), Lactobacillus plantarum (LMG P-21021), Lactobacillus rhamnosus (DSM 16605), Lactobacillus rhamnosus (DSM 19739), Bifidobacterium lactis (LMG P-21384) and prebiotic components, i.e. fructo-oligosaccharides or galactosaccharides.

European patent EP2101796 discloses the strain of the microorganism Bacillus smithiiTBMI12 MSCL P737 and compositions containing this strain, compositions for use as a probiotic for antibacterial purposes, for the colonization of the gastrointestinal tract and for the stimulation of the immune system, as well as the use of this strain as a food supplement and as a component in a therapeutic and non-therapeutic probiotic composition. European patent EP0856259 describes a composition for use containing lyophilized live active lactic acid bacteria in combination with oligosaccharides, for use as a food supplement, with the aim of balancing the intestinal flora. The specified lyophilisate contains strains of the genera Bifidobacterium, Lactobacillus, Streptococcus. Furthermore, the invention relates to a kit consisting of two containers, one of which contains food that can be liquid, creamy or dough-like, and the other contains a composition that is added to the food immediately before consumption. The food in this case can be milk, yogurt, another type of fermented milk, a milk dessert, a milk drink or a drink based on milk serum or milk that is enriched with fruit, fruit juice, tomato juice, carrot juice, tea or a drink based on plant extracts.

Therefore, a large number of different probiotic starter cultures and probiotics are known, both as monocultures and as a combination of different bacterial strains. As can be seen, among other things, from these isolated documents from the patent and non-patent documentation given in the text of the description, combinations of different strains of microorganisms are known which can be applied in different forms, and which have a wide range of different effects on both humans and animals. However, to our knowledge, no probiotic product for mammals containing the proposed combination of bacterial strains in a given ratio and with such probiotic potential has been described so far.

Brief description of the invention

Intake of microorganisms through food has a positive effect on human and animal health. While the use of probiotics for humans is widespread, the use of probiotics in livestock is negligible. The latest research is intensively concerned with the development of probiotic preparations for animals, based on the use of lactic acid bacteria (lactic acid bacteria), bacteria of the genus Bacillus or yeasts. Increased interest in the use of probiotics in animal husbandry has arisen due to increasingly rigorous European regulations on the use of antibiotics in animal husbandry. Therefore, probiotics are considered an adequate alternative when it comes to intestinal infections in animals, especially in puppies. Establishing good microflora by colonizing BMK in the intestines of newborn animals immediately after birth results in healthier animals.

In the first days after birth, animals are infected with bacteria from their environment, which is extremely important for the development of their immunity. However, it often happens that newborn animals during this period are also infected with pathogenic intestinal bacteria {Enterobacteriaceae), which leads to

intestinal infections accompanied by diarrhea, weight loss, and not infrequently the death of animals, which represents a significant economic loss for the livestock industry. Since in this case antibiotics are resorted to in the treatment of intestinal infections, this represents an additional problem in the livestock and food industry.

Fermented milk products with probiotic bacteria are products obtained by fermenting milk with the action of probiotic starter cultures or by combining starter cultures with other lactic acid bacteria.

Detailed description of the invention

The invention specifically discloses a new probiotic starter culture for human and animal use, a technological procedure for its preparation as well as the use of this probiotic culture in the prevention and/or treatment of intestinal infections of various etiologies in humans and animals and use as an immunomodulator.

The basic unit of the product is a probiotic culture, which represents a unique combination

bacteria with specific probiotic potential for the prevention and/or treatment of intestinal infections and inflammatory bowel diseases in humans and animals. Probiotic culture is extremely effective in the treatment of intestinal infections of various etiologies and reduces the number of pathogenic bacteria with a unique spectrum of action on pathogenic bacteria.

Probiotic culture has an extremely high probiotic potential, has the ability of immunomodulation, as well as a specific antimicrobial effect on a large number of bacterial species. The new probiotic culture represents an alternative to the use of antibiotics in the treatment of intestinal infections, based on a wide spectrum of antimicrobial activity, especially in the case of hard-to-treat infections, which improves the health of people and animals.

In one embodiment, the subject invention relates to a new probiotic starter culture consisting of three thermophilic natural isolates of BMK: Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 in a specific ratio that produces the desired results, its use for obtaining probiotics and compositions intended for the prevention and/or treatment of colonization-related disorders of the gastrointestinal tract of mammals with pathogenic microorganisms and which also have an immunomodulatory effect.

In the next embodiment, the probiotic starter culture of the subject invention consists of three thermophilic natural isolates of BMK: Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 in an optimal ratio. Their specific relationship is an important factor in the effect of this starter culture in the prevention and/or therapy of intestinal infections in mammals, both humans and animals, and will be described in detail below.

In three experiments, different combinations of strains were tested: Streptococcus thermophilusLMG P-28585 in the amount of 10% to 40%, Lactobacillus fermentumLMG P-28583 in the amount of 10% to 50% and Lactobacillus helveticusLMG P-24226 in the amount of 40% to 80%. The results showed that in all tested combinations, they exhibit probiotic potential.

It has been shown that in the technological sense for the preparation of a fermented milk probiotic product, the optimal ratio of Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 strains in the probiotic starter culture is 30%:30%:40% (3:3:4), as well as the ratio 30%:20%:50 (3:2:5). These ratios ensure a sufficient number of all probiotic strains in the culture, while not lowering the pH of the fermented product below 4.2-4.3 (threshold of tolerance of sour taste).

Further, in one embodiment, the preferred strain ratio is Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 10%:10%:80%

(1:1:8) or 10%:20%:70% (1:2:7). This relationship ensures a greater number of bacteria of the strain Lactobacillus helveticusLMG P-24226, which has been shown to have a broad spectrum of antimicrobial activity against hard-to-treat pathogenic bacteria, including Staphylococcus aureusATCC25923, Staphylococcussp.

ATCC MetR (clinical isolate) collection Torlak,Yer sinia enterocolitica03 (clinical isolate) collection Torlak,Shigella sonnei(Hamburg 105) collection Torlak,Shigella flexneri(London 9950) collection Torlak,Shigella dysenteriae(serotype 5 and 7) collection Torlak,Streptococcus pneumoniaeATCC276,Streptococcus pneumoniaeATCC496,Pseudomonassp.,Escherichia coliC600, Escherichia coli

ATCC25922, Clostridium sporogeneskolekcija Torlak and Clostridium difficile (clinical isolate).

The ratio, Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 40%:20%:40% (4:2:4) has potential application in immunocompromised people, considering that this combination contains the least represented strain Lactobacillus fermentumLMG P-28583, which has been shown to exhibit mild pro-inflammatory effect.

In the next embodiment, the probiotic starter culture of the present invention consists of Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 in a ratio of 30%:40%:30% (3:4:3). In this relationship, the Lactobacillus fermentumLMG P-28583 strain is the most represented, and its properties increase the immune response.

As described in the text of the description, depending on the stated ratios of the given bacterial isolates in the probiotic starter culture, it will exhibit its protcolytic (acidification) and antimicrobial activity, as well as its immunomodulatory effect.

The mentioned combinations give exceptional results in the treatment of intestinal infections in mammals, especially animals. More precisely, this starter culture gives excellent results in the treatment of infections caused by bacteria Clostridium perfringensi Clostridium difficile, as well as bacteria of the Enterobacteriaceae family, when the number of living bacteria is in the range Streptococcus thermophilusLMG P-28585 from 6xl0<7> to l,25xlO<u>, Lactobacillus fermentumLMG P-28583 from l,lxl0<8>to 1,25x10" and Lactobacillus helveticusLMG P-24226 4xl0<6>to 2.5xl0<9>.

It is known that the introduction of certain microorganisms through food has a positive effect on the health of humans and animals. Namely, in the first days of life, cubs are infected with bacteria from the environment in which they are, which is extremely important for the development of immunity. However, it often happens that during this period the young are also infected with pathogenic intestinal bacteria {Enterobacteriaceae), which leads to intestinal infections accompanied by diarrhea, weight loss, and not infrequently death. Since in this case antibiotics are resorted to in the treatment of intestinal infections, this represents an additional problem in the livestock and food industry. Therefore, this probiotic culture, in addition to solving the problem of intestinal infections in domestic animals, contributes to reducing the use of antibiotics. Furthermore, most of the probiotics described in the state of the art are intended for feeding cubs, while this probiotic culture (due to the safe status of the bacteria included in its composition) is in one embodiment intended for pregnant animals, where the cubs receive passive immunity from the mother and are immunized already in the first days of life. Also, during the last 15 days of pregnancy, there is a drop in immunity and the number of enterobacteria in the mother's intestines increases, which after birth are transferred to the cubs, who become infected that way. Treatment of pregnant animals with the new probiotic culture does not increase the number of enteropathogens in the mother's intestines and does not infect newborn animals.

In another embodiment, the probiotic starter culture consisting of Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 when the number of live bacteria is in the range Streptococcus thermophilusLMG P-28585 from 4xl09 to 8xl0<10>, Lactobacillus fermentumLMG P-28583 from 3xl0<9>to lxl0<10>and Lactobacillus helveticusLMG P-24226 4xl0<6>to 9xl0<8>CFU/ml.

The new probiotic culture of the subject invention proved to be extremely effective in in vivo experiments in farm conditions in the treatment of intestinal infections of various etiologies, and was specifically shown to successfully reduce (below the detection level) the number of bacteria of the species Clostridiumperfringens, as well as bacteria of the Enterobacteriaceae family.

All the mentioned combinations can be prepared in the form of milk fermented probiotic product, suspension, lyophilizate, premix, spray-dry form, in the form of tablets, capsules and all other forms known to the average expert in this field of technology and as such find human and animal application. All these forms containing the probiotic starter culture of the present invention are obtained according to generally known procedures.

The procedure for preparing a probiotic starter culture includes: 1) Cultivation of natural isolates that are part of the probiotic starter culture in optimal conditions for the growth of these bacteria; 2) The combination of natural isolates that are part of the probiotic starter culture in an optimal ratio.

Thanks to the presence of the Streptococcus thermophilusLMG P-28585 strain in the probiotic culture, this culture can also be used to prepare a fermented milk product that can be used to prevent intestinal infections in non-ruminant mammals including humans and animals (pigs, dogs, cats, etc.).

The procedure for preparing a fermented probiotic product for humans and animals (non-ruminants) involves the fermentation of milk with defined starters and probiotic bacteria, which takes place in the following stages: 1) Pre-cultivation of natural isolates that are part of the probiotic starter culture in optimal conditions for the growth of these bacteria; 2) Inoculation of milk with natural isolates prepared in phase 1 in the optimal ratio; 3) Incubation of milk inoculated in phase 2 in optimal conditions for fermentation.

The fermented product prepared in this way is stable, the number of live probiotic bacteria is reproducible, and the production technology is easily applicable on an industrial scale. In addition, this product has an improved probiotic effect, probably due to the presence of specific bioactive peptides created by the degradation of milk proteins by proteolytic enzymes of bacteria in the composition of the probiotic culture.

Application of probiotic culture in other mammals (ruminants) as well as other animals implies the use of probiotic culture prepared without milk.

Short description of the pictures

Figure 1. Genotyping of strains from the composition of the probiotic culture using the "pulse-field gel electrophoresis" (PFGE) method. • The results showed that the bacteria included in the probiotic culture represent unique strains within the mentioned species.

Figure 2. Antimicrobial activity of individual strains from the composition of probiotic culture and probiotic combination (synergism) on Clostridium difficile.1. ControlC, difficile+ MRS; 2.C. difficile+ LMG P-28585; 3.C. difficile+ LMG P-28583; 4.C. difficile+ LMG P-24226; 5.C. difficile + probiotic combination.

• By testing individual cultures and probiotic combinations, it is clearly observed

that the combination (synergism) successfully suppresses growthC. difficile - none

release of gas or rupture of the nutrient medium, while in the case of individual cultures growthC. difficile was only partially suppressed - a small release of gas and a partial rupture of the nutrient medium. Control - growthC. difficile causes gas production and cracking of the nutrient medium.

Figure 3. Survival of strains A) LMG P-28585, B) LMG P-28583 and C) LMG P-24226 in simulated conditions of the gastrointestinal tract in NaCl solution and in milk matrix. • All tested strains successfully survive the conditions of the simulated gastrointestinal tract.

Figure 4. Adhesion to Caco-2 intestinal epithelial cells. A) Adhesion of strains LMG P-28585, LMG P-28583 and LMG P-24226. B) AdhesionE. coliATCC25922 in the presence and absence of strains LMG P-28585, LMG P-28583 and LMG P-24226. C) Adhesion of Salmonella 654/7E (veterinary isolate) in the presence and absence of strains LMG P-28585, LMG P-28583 and LMG P-24226. The values in the graph represent the mean values of 3 measurements expressed in percentages. Statistically significant values compared to the control are marked with asterisks (<*>p<0.5;<**>p<0.1). • Figure 4A: adhesion percentage shows the binding success of individual strains to epithelial intestinal cells. • Figure 4B: percentage of adhesionE. coliATCC25922 in the presence of individual strains and the probiotic combination shows that the probiotic combination (synergism) best prevents the binding of E. coliATCC25922 for epithelial intestinal cells. • Figure 4C: percent adhesion of Salmonella 654/7E in the presence of individual strains and the probiotic combination shows that strain LMG P-28585 best inhibits the binding of Salmonella 654/7E to epithelial intestinal cells.

Figure 5. Proliferation of rat GALT cells in the presence of UV-killed strains LMG P-28585, LMG P-28583 and LMG P-24226, live strains LMG P-28585, LMG P-28583 and LMG P-24226 in milk and probiotic cultures prepared in milk. The values in the graph represent the mean values of 4 measurements expressed in percentages. Statistically significant values compared to the control are marked with asterisks (<*>p<0.5;<**>).

P<<>0.1). • The results show that the live strains significantly increase the proliferation of GALT lymphocytes compared to the control.

Figure 6. Reduction of the number of pathogenic bacteria after treatment of animals in farm conditions.

A) Reduction of the number of Enterobacteriaceae bacteria in newborn piglets after treatment of supramammary sows with a milk probiotic fermented product; B) Reduction of the number of Clostridium perfringens bacteria in goats after treatment with a probiotic culture prepared without milk. • Figure 6A: The results show that the number of bacteria of the family Enterobacteriaceae significantly decreases after the treatment of animals with a probiotic, while the number of the same bacteria in untreated animals increases. • Figure 6B: The results show that the number of Clostridium perfringens bacteria decreases below the detection threshold after treatment of the animals with the probiotic.

Figure 7. Body mass of experimental animals.

• The data show that individual strains partially affect the increase in the appetite of experimental animals, which affects the increase in their body weight.

Natural isolates that are part of the probiotic culture

The composition of the probiotic starter culture includes 3 thermophilic natural isolates BMK: Streptococcus thermophilus BGVLJ1-44 (natural isolate from autochthonous, traditionally produced yogurt from uncooked cow's milk, strain from the laboratory collection of the Laboratory for Molecular Microbiology. Institute of Molecular Genetics and Genetic Engineering. University of Belgrade LMM-IMGGI), Lactobacillus fermentumBGHI14 (natural isolate from newborn feces, strain from the laboratory collection LMM-IMGGI) and Lactobacillus helveticusBGRA43 (a natural isolate from the human gastrointestinal tract, strain from the laboratory collection LMM-IMGGI).

The strains were isolated using classic microbiological methods described in the work of Terzic-Vidojcvic et al.

(2007). For the cultivation of strain BGVLJ1-44, liquid and solid Ml7 medium (Merck, GmbH, Darmstadt, Germany) with the addition of 0.5% glucose (GM17) is used, and for the cultivation of strains BGHI14 and BGRA43, liquid and solid MRS medium (Merck, GmbH) is used. Cultures are stored at -80°C in freezers in the appropriate liquid GM17 or MRS medium with the addition of 15% glycerol.

Data on the deposition of biological material

All strains were deposited in the bacterial collection of the Belgian Coordinated Collection of

Microorganisms (BCCM) Laboratory for Microbiology, University of' Gent, Belgium, K. L.

Ledegancstraat 35, Ghent, Belgium.

The strain Lactobacillus helveticusBGRA43 was deposited on 08.05.2007. year; The strain has the deposit number LMG P-24226. Deponent Ljubiša Topisirović, head of the Laboratory for Molecular Genetics of Industrial Microorganisms (previous name Laboratory for Molecular Microbiology), IMGGI, Belgrade, Serbia.

Strain Streptococcus thermophilusBGVLJ1-44 was deposited under the number LMG P-28585 on 28.10.2014. year. Deponent Dr. Milan Kojić, head of the Laboratory for Molecular Microbiology, IMGGI, Belgrade, Serbia.

Lactobacillus fermentumBGHI14 strain was deposited under the number LMG P-28583 on 28.10.2014. Depositor Dr. Milan Kojić, head of the Laboratory for Molecular Microbiology, IMGGI, Belgrade. Serbia.

Characterization and identification of LMG P-28585, LMG P-28583 and LMG P-24226 strains

Natural isolates Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 were characterized in detail biochemically and molecularly genetically.

Phenotypic characteristics of natural isolates LMG P-28585, LMG P-28583 and LMG P-24226 are shown in Table 1.

Identification of LMG P-28585, LMG P-28583 and LMG P-24226 strains to species level was done by 16S rDNA sequencing (Jovcic et al., 2009).

Additional identification up to the strain level was done by the PFGE ("pulse-field gel electrophoresis") fingerprint method (Figure 1). Total DNA of Streptococcus thermophilusLMG P-28585, Lactobacillus helveticusLMG P-24226 and Lactobacillus fermentumLMG P-28583 strains were cleaved with non-restriction enzyme. The obtained restriction profiles represent unique fingerprint profiles of the analyzed strains.

Proteolytic activity

Given that proteolytic activity is important for the process of preparing a dairy fermented product, the proteolytic activity of strains Streptococcus thermophilusLMG P-28585, Lactobacillus helveticusLMG P-24226 and Lactobacillus fermentumLMG P-28583 was tested according to the method described by Kojic et al. (1991). The ability to degrade α-, β- and K-casein, milk protein, was tested.

Strains LMG P-28585 and LMG P-24226 completely cleave a-, B- and K-casein, while strain Lactobacillus fermentumLMG P-28583 does not show proteolytic activity towards any type of casein (Table 1).

Antimicrobial activity

Antimicrobial activity was tested by the delayed antagonism method using overnight cultures of strains Streptococcus thermophilusLMG P-28585, Lactobacillus helveticusLMG P-24226 and Lactobacillus fermentumLMG P-28583 and different indicator strains. Briefly, soft agar of the appropriate medium (0.7% v/v) for the growth of the indicator strains, containing the indicator strains was poured over GM17 or MRS petri dishes with wells to which 50 µl of the analyzed strain was added. Petri dishes were incubated overnight at 37°C. Zones of inhibition of the growth of the indicator strain around the well represent a positive signal for antimicrobial activity.

In the case of anaerobic bacteria (Clostridium sp.), the antimicrobial activity was tested as follows: An overnight culture of Clostridium sporogenes or Clostridium difficile (1 ml) was poured into a test tube, and then soft agar for the growth of C. difficile, and over it soft GM17 with 200 μl of overnight culture Streptococcus thermophilusLMG P-28585, or MRS with 200 μl of overnight culture Lactobacillus helveticusLMG P-24226 or Lactobacillus fermentumLMG P-28583 or 200\x\mixed probiotic cultures. Gas production by strains of Clostridium sporogenes or Clostridium difficile, which manifests itself in the form of bursting of agar, represents the growth of strains of Clostridium sporogenes or Clostridium difficile, that is. absence of antimicrobial activity. The antimicrobial activity of probiotic strains on Clostridium sporogenesiliClostridium difficile is manifested by the absence of gas production, i.e. the agar remains intact.L. helveticusLMG P-24226 exhibits antimicrobial activity against various strains of Lactococcus sp. and Lactobacillus sp., on Bacillus subtilisATCC8, Bacillus cereusATCC11778, Bacillus mycoides, as well as on pathogenic bacteriaStaphylococcus aureus ATCC25923, Staphylococcussp. ATCC MetR (clinical isolate) Torlak collection, Yersinia enterocolitica03 (clinical isolate) Torlak collection, Shigella sonnei (Hamburg 105) Torlak collection, Shigella flexneri (London 9950) Torlak collection, Shigella dysenteriae (serotype 5 and 7) Torlak collection, Streptococcus pneumoniae ATCC276, Streptococcus pneumoniae ATCC496, Pseudomonas sp., Escherichia coli coliC600, Escherichia coliATCC25922, Clostridium sporogeneskolekcija Torlak and Clostridium difficile (clinical isolate).

Strains Streptococcus thermophilusLMG P-28585 and Lactobacillus fermentumLMG P-28583 exhibit weaker antimicrobial activity on Clostridium difficile, while the probiotic combination shows a synergistic effect and exhibits the strongest antimicrobial effect on Clostridium sporogenes and Clostridium difficile (Figure 2).

Antibiotic resistance

Antibiotic resistance was tested using methods prescribed by European Food Safety

Authority (EFSA, 2012).

Antibiotic resistance testing revealed that the strains LMG P-24226, LMG P-28583 and LMG P-28585 are sensitive to the recommended concentrations of antibiotics selected based on the recommendation of EFSA (2012).

Survival of natural isolates in simulated conditions of the gastrointestinal tract (GIT).

The survival of probiotics in the gastrointestinal tract is of crucial importance for its action. Survival of natural isolates in simulated GIT conditions was performed according to the method previously described by Uroić et al. (Uroić et al, 2014). Simulated GIT conditions involve bacterial growth in artificial gastric juice (NaCl [7.31 g/l], KC1 [0.52 g/l], NaHCO3 [3.78 g/l], pH 2 using concentrated HCl; filtered pepsin [3 g/l] was added after sterilization) and intestinal juices A (simulation of duodenal conditions: 0.6% (w/w) bile salts, pH 8.0 using ION NaOH) and B (simulation of conditions in the ileum: 0.3% (w/w) bile salts and pancreatin [1 g/l]). Samples were taken in the period from: t=0 min - CFU/ml overnight culture; t=90 min - CFU/ml of culture incubated for 90 min in gastric juice; t=100 min - CFU/ml of culture incubated for 10 min in intestinal juice A, t=160 min and t=220 - CFU/ml of culture incubated for 60 and 120 min in intestinal juice B.

The results showed that strain LMG P-28585 successfully survives in simulated conditions of the gastrointestinal tract in NaCl solution, while strains LMG P-28583 and LMG P-24226 successfully survive simulated conditions of the GIT when applied in a milk matrix (Figure 3).

Adhesion of strains in probiotic culture and pathogens to Caco-2 intestinal cell lines

Adhesion to epithelial intestinal cell (EIC) lines is an essential prerequisite for the probiotic strain to exert positive effects on the health of the host over a longer period of time. On the one hand, a higher percentage of adhesion ensures better antagonistic activity against enteropathogens by competing for the same binding sites for EIC (Coconnier et al., 1993). On the other hand, the strain that exhibits a higher degree of adhesion to EIĆ has the ability to colonize the gastrointestinal tract and survive longer in the gastrointestinal tract and makes better contact with EIĆ through which it stimulates the host's immune system (Schiffrin et al., 1995).

The adhesion experiment for EIC Caco-2 was performed according to the method previously described by Nikolić et al. (2012).

The test showed that strain LMG P-24226 exhibited the highest degree of adhesion, while strains LMG P-28583 and LMG P-28585 exhibited a slightly lower degree of adhesion to Caco-2 cells (Figure 4A).

In addition, the adhesion of Escherichia coli ATCC25922 (Figure 4B) and Salmonella 654/7E (veterinary isolate, collection of the Scientific Institute of Veterinary Medicine, Novi Sad) (Figure 4C) to Caco-2 cells in the presence of bacteria included in the probiotic culture and in the presence of the milk fermented probiotic product was also tested.

The results showed that the LMG P-24226 strain significantly reduces the number of attached E. coli ATCC25922 bacteria (p<0.5), while the probiotic combination in milk exhibits a synergistic effect and even more significantly (p<<>0.1) reduces the number of attached bacteriaE. coli ATCC25922. Also, the strains LMG P-28585 (p<0.1) and LMG P-24226 (p<0.5) significantly reduce the number of Salmonella654/7E bound bacteria.

Proliferation of rat GALT ("gut associated lymphoid tissue") cells in the presence of individual

strains and probiotic cultures

Monitoring the probiotic effect of strains LMG P-28585, LMG P-28583 and LMG P-24226 killed by UV rays, living strains LMG P-28585, LMG P-28583 and LMG P-24226 in milk and probiotic cultures prepared in milk on the primary culture of GALT cells of rats in order to determine the immune parameters activated by the action of probiotic bacteria and combination. Isolation and proliferation of rat GALT cells was performed according to the method described by Hidalgo-Cantabrana et al. (2014).

The results showed that the live strains LMG P-28585, LMG P-28583 and LMG P-24226 grown in milk and the probiotic culture prepared in milk (synergism) have a stimulatory effect on the proliferation of lymphocytes, indicating that the strains and the probiotic combination have an immunomodulatory effect (Figure 5).

Testing probiotic combinations in vivo under farm conditions

The effect of the probiotic combination on the improvement of animal health was monitored in vivo under farm conditions. 50 farrowing sows were monitored, 25 of which were treated with a milk fermented probiotic product for 7-15 days before farrowing (during the period when immunity naturally declines). High-bred sows received the probiotic every day during the first morning feeding. Twenty-five control sows were not treated with probiotics. Before and after the treatment with probiotics, the general health condition of the sows was monitored, as well as the total number of bacteria (BMK and Enterobacteriaceae) in the feces of the pigs according to the previously described procedures (Terzic-

Vidojevic et al., 2007; Anonymous, 1996). After dusting, the general state of health was monitored and

the immune status of piglets, as well as the total number of bacteria (BMK and Enterobacteriaceae) in piglet feces. As a control, the general health and immune status of sows that were not treated with probiotics and their piglets were simultaneously monitored, as well as the total number of bacteria (BMK and Enterobacteriaceae) in the feces of the control group of sows and piglets (Figure 5a).

The results showed that after probiotic treatment, the number of enteropathogenic bacteria in piglet feces was significantly lower compared to the number of the same bacteria in sow feces before the start of treatment, while the number of enteropathogenic bacteria in the control sample was higher in piglet feces than in the feces of suprafatty sows before farrowing (Figure 6a). Also, it was observed that piglets whose mothers were treated with the probiotic combination did not have diarrhea, while in the control group diarrhea occurred sporadically in a larger number of piglets.

In parallel, testing the effect of probiotic culture on goats infected with Clostridium perfringens. Ten goats spontaneously infected with the bacterium Clostridium perfringens were treated with probiotic culture 10 days before lambing (during the period when immunity naturally declines).

The results showed that the probiotic culture successfully reduced the number of Clostridium perfringens bacteria below the detection level (Figure 6b). In addition, it was observed that the number of other sporogenic bacteria decreased after treatment with probiotic culture, and the general health condition of the goats improved.

Toxicological analysis

In order to safely use the probiotic combination on humans and animals, acute and subacute toxicological analyzes of individual strains and probiotic combinations were performed. Acute and subacute toxicological analysis were performed on 12 mice of the NMRI HAN strain. The analysis was performed in accordance with the Good Laboratory Practice Guidelines (Official Gazette No. 28/2008) with respect to the general requirements for performing the test specified in the OECD GUIDELINES FOR TESTING OF

CHEMICALS NO. 423.

The results showed that no strain, as well as the probiotic combination, did not exhibit a toxic effect (Table 2-6, Figure 7).

Preparation process of milk fermented probiotic product

An overnight culture of individually grown natural isolates in milk is added to the pasteurized milk in a total amount of 3%, preferably in the ratio LMG P-28585 : LMG P-28583 : LMG P-24226 = 1 : 0.5 : 1, optimally 1 : 1 : 1.3. The inoculated milk is incubated in optimal conditions for bacterial growth until the total number of bacteria is 1<08->10<10>CFU/ml, after which the obtained fermented product can be stored in a refrigerator at 4-6°C for up to 28 days without losing activity and the total number of live bacteria remains the same as immediately after preparation.

Industrial application

The probiotic starter culture of the subject invention will exhibit its proteolytic activity, antimicrobial and/or immunomodulatory activity according to the mutual relationship of the bacterial isolates that are part of the probiotic culture. So, according to the needs and purposes, this probiotic starter culture finds human and animal application in the prevention and/or treatment of intestinal pathologies of various etiologies and as an immunomodulator.

This probiotic starter culture can be prepared as such and in forms suitable for the target group of mammals, i.e. human or animal. The special purpose of the probiotic starter culture is as a food supplement for humans and animals.

For veterinary use, the probiotic starter culture can be prepared in the form of standard pharmaceutical forms used in veterinary medicine, but not exclusively, in the form of a fermented probiotic product, lyophilisate, premix, i.e. granules, spray-dry form (sprinkled on food), powders that are mixed with water and food, suspension, tablets, capsules and all other forms known to the average expert in this technical field. Depending on the case, the probiotic starter culture can be applied especially as a supplement to animal feed, and a particularly desirable spray dry form (sprinkled on the feed).

For human use, the probiotic starter culture/probiotic/composition of the subject invention containing the three thermophilic natural isolates of bacteria of the subject invention, as previously stated, can be obtained in the form of a dairy fermented probiotic product, lyophilized, powder form, oral suspension, lozenges and chewable tablets, tablets, capsules and other forms known to the average person skilled in the art.

Claims (11)

1. Probiotic starter culture, characterized in that it consists of the following thermophilic natural isolates of lactic acid bacteria (LMG): Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226. 2. Probiotic starter culture according to claim 1, characterized in that it contains Streptococcus thermophilusLMG P-28585 in the amount of 10% to 40%, Lactobacillus fermentumLMG P-28583 in the amount of 10% to 50% and Lactobacillus helveticusLMG P-24226 in the amount of 40% to 80%. 3. Probiotic starter culture according to claim 1, characterized in that the ratio between Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 is 30%:30%:40%. 4. Probiotic starter culture according to claim 1, characterized in that the ratio between Streptococcus thermophilusLMG P-28585, Lactobacillus fermentumLMG P-28583 and Lactobacillus helveticusLMG P-24226 is 10%:10%:80%, i.e. 10%:20%:70%, better 30%:20%:50%, and better 30%:40%:30% and even better 40%:20%:40%. 5. Probiotic starter culture according to claim 1, characterized by the number of live bacteria: Streptococcus thermophilusLMG P-28585 from 6x10<7> to 1.25xl0<n>, Lactobacillus fermentumLMG P-28583 from 1.1xl08 to 1.25x10<11> and Lactobacillus helveticusLMG P-24226 from 4xl06 2.5xIO9. 6. Probiotic starter culture according to one of the previous claims as a substitute for the use of antibiotics, for the prevention and/or treatment of bacterial infections of the gastrointestinal tract, disorders associated with the colonization of the gastrointestinal tract, for the mitigation and/or elimination of side effects caused by the use of antibiotics and for use as an immunomodulator in humans and animals. 7. Probiotic starter culture according to claim 6, characterized by the fact that the infections are caused by bacteria of the species Clostridium perfringens, Clostridium sporogenes, Clostridium difficile, bacteria of the family Enterobacteriaceae, bacteria of the species Escherichia coli, Salmonella sp., Staphylococcus aureus, Staphylococcus sp. Met<R>, Yersinia enterocolitica, Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Streptococcus pneumoniaePseudomonassp., in humans and animals. 8. Probiotic starter culture according to one of the previous claims, indicated by the fact that it is in spray-dry form (sprayed on food), in the form of lyophilisates, suspensions and milk fermented probiotic products. 9. Probiotic starter culture according to claims 1-8 for human and animal use as a probiotic and as a food supplement for humans and animals. 10. Composition, characterized in that it contains a probiotic starter culture according to one of the previous claims and a pharmaceutically acceptable carrier and is in the form of capsules, tablets, lyophilisates, spray-dry forms, suspensions, milk fermented probiotic products. 11. Composition according to claim 10 for human and animal use.