ASSESSMENT OF THE BACTERIOCINOGENICITY OF INDIGEN LACTOBACILLUS ONTO CATTLE CARCASSES

Cattle carcasses of ritual slaughter (Halal) from the lateral and medial side were investigated in order to identify the indigenous strains of lactic acid microorganisms capable of producing bacteriocins. Only 14 strains, from 36 strains of microorganisms isolated from washings taken from carcasses, were used for further research and identified as lactabacilus. In the study of their bacteriocinogenicity by the method of two-layer agars of modified composition, high inhibitory efficiency was proved with respect to the growth of the pathogen Salmonella typhimurium, only 6 isolated strains of lactabacilus. The use of this method of evaluation will not only reveal the presence of the ability of the lactabacilus to produce bacteriocins, but also to study their antagonistic activity against many of the studied microorganisms, which are indicators of compliance with various modes of technological processes. The presence of such microorganisms in the meat will increase the shelf life of meat, due to the suppression of the growth of closely related lactabacilus, some of which are spoilage microorganisms, as well as the growth of opportunistic and pathogenic microflora. In addition, the use of lactabacilus with bacteriocinogenicity in the production technology of raw sausages will reduce the risk of production of unsafe products. Thus, the presence of indigenous lactabacilus in meat is an additional factor that ensures the flow of desired biochemical processes and the safety of raw smoked sausages. УДК /UDC: 579.6:636.2.033 Для цитирования: Батаева Д.С., Соколова о.в., Зайко Е.в., Пашкова в.в. оценка бактериоциногенности индигенных молочнокислых бактерий, полученных с туш крупного рогатого скота. Теория и практика переработки мяса. 2018;3(2):22–32.DOI 10.21323/2414– 438X‐2018–3–2–22–32 FOr cItatIOn: Bataeva D. S., Sokolova O. V., Zaiko E. V., Pashkova V.V. assessment of the bacteriocinogenicity of indigen lactobacillus onto cattle carcasses. Theory and practice of meat processing. 2018;3(2): 22–32. (In russ.) DOI 10.21323/2414–438X‐2018–3–2–22–32 Батаева Д.С.,* Соколова О.В., Зайко Е.В., Пашкова В.В. Федеральный научный центр пищевых систем им. В.М. Горбатова РАН, Москва, Россия Dagmara S. Bataeva*, Olga V. Sokolova, Elena V. Zajko, Victoria V. Pashkova V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Moscow, Russia DOI 10.21323/2414–438X-2018–3–2–22–32


Introduction
Currently, the world pays great attention to food safety.A special place is the protection of food from the effects of xenobiotics, which include microbial contaminants, especially pathogenic bacteria.Ensuring microbiological safety of products is a priority direction of the food industry.
In conjunction with the neurotoxic effect resulting from environmental exposure, food contaminated with pathogens is a great danger to human health.
Despite the introduction of various programs to prevent microbial contamination and control of pathogens -the problem does not lose its relevance.The situation is aggravated by the increasing resistance of a number of pathogens to chemicals, including antibiotics [1].
In connection with the detection of antibiotic resistance of pathogenic microorganisms found in food products, different approaches to deter their reproduction are proposed.Especially rapid development of biological methods.Bacteriophages are actively used abroad.Moreover, they are positioned as alternative to chemical substances means of controlling pathogens and as agents that prolong the shelf life of products [2,3,4].
Among other biological measures to combat pathogens is the use of bacteriocins.Bacteriocins-antibiotic-like substances with bactericidal or bacteriostatic effect, were discovered in the mid of 1960s, at the same time began to study their properties.Despite the rich history of bacteriocin research, their potential has not been fully disclosed yet.The current high level of diagnostic capabilities allows a more in-depth and comprehensive study of the properties of bacteriocins.Bacteriocins are heterogeneous peptides secreted by various microorganisms, including lactabacilus [5].
The mechanism of action of bacteriocins against pathogenic microorganisms is associated with a violation of the permeability of the cytoplasmic membrane of the latter.Bacteriocins are divided into two classes -lantibioticspeptides containing unusual amino acids (for example, lanthionin) and short heat-resistant polypeptides that do not contain such amino acids.A number of authors are inclined to believe that these peptides have the ability to form pores in the cytoplasmic membrane, resulting in cell lysis of the pathogen [6,7,8,9,10].
The most well -known bacteriocin is nisin, which is synthesized by the coccal strain Streptococcus lactis.Currently, nisin is used as a preservative in the production of some foods [11].
For the meat industry, as producers of bacteriocin, the most interesting are the microorganisms that make up the non-pathogenic pool of the indigenous microflora.
Thus, the aim of this study was to identify and study the indigenous lactabacilus of the carcass microbiome of cattle of ritual slaughter (Halal) to assess their bacteriocinogenicity.

Objects and methods
The objects of the study were flushes selected from the lateral and medial sides of the anterior quarter of cattle carcasses immediately after.To exclude contamination of carcasses by third-party microorganisms that may be deposited by dry and/or wet processing, the collection of swabs was carried out from the surface of carcasses Halal slaughter.When Halal slaughter is not used washing operations carcasses, so there is no change in the original microbiome.Washings were taken from two zones of thoracic and rib cuts of each half-carcass both from the inner and outer surface.Sampling of washings was carried out with sterile sponges moistened with 10 cm 3 of saline solution from each zone with an area of not less than 100 cm 2 .Then the sponges were placed in a sterile package, a buffered peptone water with a volume of 90 cm 3 was added, and a number of tenfold dilutions were used to sow in nutrient media.Since the aim of our work was to study the bacteriocinogenicity of lactobacillus, the primary objective of the study was to identify the lactobacillus from the object of study.
For selection of lactic acid microorganisms from the received washouts, a number of tenfold dilutions were prepared and their sowing in the dense nutrient medium of MRS was carried out by deep method.The crops were incubated for 3 days at a temperature of (30±2) °C at the end of incubation, Petri dishes were examined to identify colonies with a typical growth of lactic acid microorganisms (having a lens shape or a star shape) in order to select them for further study.Typical colonies were passaged in liquid medium MRS-broth, where in optimum conditions at temperature (30±2)°C conducted with the accumulation of biomass of cells.The presence of microbial growth was determined by the turbidity of the broth in the lower part of the tube.The turbidity of the broth in the bottom space of the tube is a characteristic feature of the development of microorganisms with microaerophilic and anaerobic type of respiration, which include lactabacilus.The duration of cultivation ranged 1 to 5 days.In the absence of growth within 5 days or growth different from the above -crops were not further investigated.
To assess the belonging of isolated bacteria to lactic acid microorganisms, according to GOST 10444.11-2013«Microbiology of food and animal feed.Methods of detection and calculation of mesophilic lactic acid microorganisms», the following comprehensive assessment of each isolated strain, presumably related to lactic acid bacteria, was carried out: growth on the MRS medium at 30 °C; catalase formation; evaluation of the microscopic preparation: by gram and by spore formation.Lactic acid microorganisms are characterized by the ability to grow on dense and liquid nutrient media MRS at 30 °C, they do not form catalase and gram stained positively, while in the microscopic preparation can be both cocci and sticks, the latter of which do not form a dispute.Culture, identified as lactic acid -passaged in a semi-liquid environment of the MRS for the purpose of long storage and further study.Other microorganisms were culled.
According to the method proposed by these researchers, it was necessary to introduce bacteriocin into the wells of the infected pathogenic microorganisms, agar.Bacteriocin diffused to agar, resulting in a visualized zone of inhibition of growth of the pathogen.Due to the small area of visualization of the results, we have modified the technique.Two specialized nutrient media were used for this purpose.The first medium-modified MRS-agar (MRS-mod), the following composition: non-selective liquid medium MRS; glucose-0,2 %; agar-agar-0,3 %; the second medium-soft agar (MA) having in its composition: pepton -15 g, yeast extract -3 g, NaCl -6 g, glucose -1 g, agar-agar -7 g.
To conduct the study, daily culture of lactabacilus was prepared on the MRS broth, which was applied by touching the surface of the MRS-mod at a certain point using a bacteriological loop.The cups were then incubated with the lids upwards at a temperature of (30±2) °C for 3 days, for maximum accumulation and diffusion of bacteriocin into the culture medium.At the end of incubation, a thin layer of soft agar, inoculated with a test strain was poured over the MRS-mod.As a test strain was chosen culture Salmonella typhimurium ATSS14028, which is a common causative agent of toxicoinfections.Soft agar was infected with a test strain of Salmonella typhimurium ATSS14028 with a titer of 10 6 KOE / cm 3 , and the thermostat was again placed for incubation with lids up.The samples were incubated at a temperature (37±1)°C for 24-48 h.
According to the working hypothesis, during the incubation period in the MRS-mod environment, lactic acid microorganisms must multiply, producing bacteriocin, which diffuses into the agar, and then when applying the second layer of agar with the test strain Salmonella typhimurium, bacteriocin should prevent the growth of the pathogen.

Results and discussion
Lactobacillus has potential for use due to their wide range of potential antagonistic activity.The use of bacteriocins produced by lactabacilus is positioned as agents capable of replacing antibiotics and increasing the safety of meat [13,14,15].
The most promising lactabacilus from the point of view of their potential as inhibitors of pathogens, consider the indigenous lactabacilus, that is, isolated from the natural environment.As a rule, bacteria that have the potential of bacteriocinogenic isolated from food products [16,17,18,19].
It is logical to assume that the meat processing industry is most appropriate to use bacteriocins isolated from the indigenous microflora of meat.Since microorganisms from meat can be most adapted to it and be most competitive than those isolated from other sources, a study of it was conducted.
As a rule, lactabacilus are isolated from fermented meat products [20,21], that is, from ready-to-use.In our work, the premise of the study of the indigenous microflora of carcasses was the assumption that the lactic microflora enters the meat products at the initial stages of processing, that is, at the stage of slaughter, nutration and boning of carcasses.
As a result of monitoring the lateral and medial sides of cattle carcasses for the presence of lactic acid bacteria, with a dense nutrient medium, MRS36 microbial colonies were selected for further research.Only those forms of colonies of which, on selective agar, were typical for lactic acid microorganisms: in the form of a three-beam star or a lens (Figure 1).At this stage, 15 samples were excluded from the study.Of the remaining 21 strains, all were positively gramstained, but 2 were catalozopositive, presumably Brochothrix thermosphacta, and did not have a typical lactic acid micro-organisms microscopic picture.According to studies by canadian scientists, the bacteria Brochotrix thermosphacta can develop together with lactabacilus, reaching high titers [22].
Further studies are 19 conducted by cultivating the strains in MRS broth.When assessing the growth pattern of the selected colonies in this liquid nutrient medium, not all microorganisms showed growth typical of microaerophilic and facultative anaerobic microorganisms, which include the required prokaryotes.For Figure 2 the illustration from the reference book with the scheme of typical growth of microaerophilic microorganisms (a) and the photo of growth on the broth of the experimental sample (b) is presented.
Thus, after studying the cultural and morphological properties of 36 cultures of microorganisms isolated from the washings from cattle carcasses, to study the ability to form bacteriocin were selected only 14 strains.For Figure 3 a photograph of a control sample of CS infected with a test strain that has not been infested with lactobacillus is presented.As a result of the growth of the test strain, a continuous dense turbidity of the agar is observed., which confirms the ability of this agar to ensure the growth of Salmonella typhimurium.
For Figure 4,5,6 photos of experimental samples after cultivation of Salmonella typhimurium on top of growth of lactobacillus on Petri dishes are presented.Since the MRS-mod environment is a semi-liquid agar, lactobacillus grow on it closer to the bottom of the Petri dish with the formation of vague colonies of bizarre shapes, they are shown by arrows and marked with the number «1».According to the research methodology, CS infected with Salmonella typhimurium was applied to the surface of MRS-mod with pre-grown lactobacillus.
For Figure 4 it can be seen that the area of agar, where it was supposed to be bacteriocin, marked with the number «2», lost transparency due to the growth of the test strain.At the same time in the zone of lactobacillus growth the environment remained completely transparent.Figure 3 and Figure 4 shows a transparent zone, which is the result of inhibiting the growth of Salmonella typhimurium lactobacillus.This fact may be the result of the absence of exogenously produced bacteriocin by this type of lactobacillus or antagonistic activity of only the native culture.For Figure 5 the boundary between the growth and suppression zones of the test strain of the second of the studied lactobacillus was determined.In contrast to the growth shown in Pic.4, the growth pattern of Salmonella typhimurium in pic.5 it is characterized by the absence of growth in the zone of visible growth of lactobacillus, and the zone of its absence.This is proof that this strain lactobacillus has bacteriocinogenic.
For Figure 6 the results are similar to those shown in Figure 6.5.The third strain of lactobacillus produces bacteriocin, but differs in the cultural and morphological nature of growth.
The presented figures show that the growth suppression zone of the test strain Salmonella typhimurium in the pres-ence of two of the three studied lactobacillus reached 3/4 of the Petri dish area, which clearly demonstrates the high antagonistic activity of the studied lactobacillus and their bacteriocins.As a result of the screening of lactobacillus for bacteriocinogenicity, only 6 strains of lactobacillus were selected for further study as the most promising.
The use of this method of evaluation will not only reveal the presence of the ability of lactobacillus to produce bacteriocins, but also to study the antagonistic activity of both the native culture and their bacteriocins in relation to many studied microorganisms, which many researchers position as indicators of the correct flow of technological processes [23].The presence of such indigenous microorganisms in meat may allow to increase the shelf life of meat due to minimal barrier technologies, such as vacuum packaging or MGA [24].In particular, due to the suppression of the growth of closely related lactobacilli, some of which are spoilage microorganisms [25], as well as the growth of opportunistic and pathogenic microflora [26].In addition, the use of lactic acid bacteria with bacteriocinogenicity in the production technology of raw sausages will reduce the risk of production of unsafe products.Thus, the presence of indigenous lactobacillus in meat is an additional factor that ensures the flow of desired biochemical processes and the safety of raw smoked sausages [20,27].

Summary
As a result of monitoring of cattle carcass were selected indigenous lactobacillus has bacteriocinogenic against pathogenic test strain Salmonella typhimurium.The working hypothesis about the accumulation of bacteriocin in the nutrient medium was confirmed.Experimentally proved, that the chosen for the study of indigenous lactobacillus of meat have bacteriocinogenic and can be applied to reduce the growth of pathogenic microorganisms.

Figure 1 .
Figure 1.Typical growth of µb in dense MRS nutrient medium

Figure 2 .Figure 3 .
Figure 2. Typical growth of lactobacillus in MRS broth for obligate anaerobic microorganisms: (a) -scheme; (b) -photograph of the test tube with the growth of one of the studied samples

Figure 4 .Figure 5 .
Figure 4. Photograph of Petri dishes with growth of Salmonella typhimurium on top of the growth of lactobacillus: 1 -of lactobacillus colonies in the lower layer of agar; 2 -zone of continuous growth of Salmonella typhimurium in the upper layer of agar; 3 -zone of absence of growth of Salmonella typhimurium in the upper layer of agar

Figure 6 .
Figure 6.Photograph of Petri dishes with growth of Salmonella typhimurium on top of the growth of lactobacillus: 1 -Growth of lactobacillus colonies in the lower layer of agar; 2 -growth zone of Salmonella typhimurium in the upper layer of agar