Research Paper on Comparative Genomics

Humankind has been utilizing the antimicrobial effect of Lactococcus SSP and various lactic acid bacteria in its diverse forms for a long time. The aim assumed the extension of the shelf life for the food products due to the formation of lactic acid with a concomitant decrease in pH, and also biologically active substances that have a bactericidal effect on microorganisms, including the pathogenic forms. In the currently available literature, researchers assign a leading place in explaining the phenomenon of antagonism of lactic acid bacteria to the bacteriocins. In the future, interest in the use of bacteriocins will only increase. One of the foremost aspects of this interest presumes the increased consumer demand for food quality and health safety since the widely used chemical preservatives and antibiotics, which increase the shelf life of food, cause critical concerns. Synthesis of bacteriocins designates a hereditary feature of microorganisms manifested in the fact that each strain is capable of forming some antibiotic substances strictly specific for it. In most populations of lactococci, bacteriocin synthesis could be subjected for inducing by genetic engineering methods in terms of comparative genomics.

The representatives of the scientific world actively discuss the theme of comparative genomics regarding the lactococcus SSP and diverse lactic acid bacteria. Following the material of Tulini et al. (2016), the demand for bactericidal and fungicidal preparations to apply in the food industry, medicine, and agriculture is annually increasing, while the chemical preparations and antibiotics used at the moment remain toxic to humans and animals. They are accumulating in soil and water, while numerous multi-resistant forms of pathogenic and conditionally-pathogenic microbes are spreading. As might proceed from the article by Pessione and Cirrincione (2016), the search for new natural antimicrobial substances synthesized by non-pathogenic microorganisms designates a critical task. Lactic acid bacteria (LAB) are widely distributed in nature: they can be found in the soil, decaying the animal and plant residues, in the intestines of vertebrates, in milk and dairy products. Together with plants and food, they enter the gastrointestinal tracts of humans and animals, making up its microbiota.

As admit Tumbarski, Lante, and Krastanov (2018), the main property of LAB, due to which the bacteria are combined into a separate extensive group of microorganisms, is the ability to form lactic acid as the main product of fermentation. Concerning the lactic fermentation process, it is carried out by bacterial organisms that are heterogeneous in its morphology. Abosereh, El Ghani, Gomaa, and Fouad (2016) mention that they could be rod-shaped and spherical (spherical or elliptical cocci); one also could belong to the Lactococcus, Tetragenococcus, Bifidobacterium, and related items genera. For centuries, humans have used LAB in the preparation of fermented products as a way of preserving milk and food raw materials. Hassan, Zhou and Bullerman (2016) note that LAB are capable of preventing the growth of pathogenic microorganisms through the antimicrobial metabolites (lactic and acetic acids, peroxides, and diacetyl) synthesis. However, the leading place in explaining the phenomenon of antagonism has to be given to specific antibiotic substances of protein nature. Proceeding from Stoyanova, Napalkova, and Netrusov (2016), it is bacteriocins. The last represents mainly heterogeneous antibacterial peptides, varying in level of activity, spectrum, mechanism of action, molecular weight, and physicochemical properties.

Nisin embodies the most studied bacteriocin so far. As note Hager, Rawles, Xiong, Newman, and Webster (2019), it is an exclusive antibiotic because it is granted GRAS (Generally Recognized As Safe) label and is approved for use as a dietary supplement (E234). Nisin represents the basis of the drug Nisaplin, produced by the British firm Aplin & Barrett Ltd. As mentioned by Sadek, Refaat, El-Shakour, Mehanna, and Hassan (2017), recently the Danish-based Chr. Hansen company has also begun to market nisin analog. The preparations of both companies have very similar characteristics. However, Sangcharoen, Klaypradit, and Wilaipun (2017) note that nisin loses activity at neutral and alkaline pH values.

Additionally, the unsaturated amino acids in its composition easily interact with the phosphate groups of amino acids enzymes contained in the raw materials and products. It leads to a decrease or loss of the drug activity. The group of authors Woraprayote et al. (2016) indicates that nisin is inactivated by proteolytic enzymes present in food raw materials. Besides, the most important thing is that nisin is effective only against gram-positive bacteria, so using it as a preservative does not solve the entire problem of spoiling food and raw materials, the leading cause of microbial spoilage of which under conditions of storage is microbes related to gram-negative bacteria and microscopic fungi.

Synthesis of bacteriocins marks a hereditary feature of microorganisms manifested in the fact that each strain is capable of forming one or more specific antibiotic substances that are strictly specific to it. Nisin products comprise L. lactis SSP lactis (streptococcus serological group N). By systematic position, it is isolated from the group of microorganisms of the Streptococcus genus, including pathogenic forms, and is classified as GRAS under the new name Lactococcus as not causing infectious diseases in humans and animals. Besides, one of the first and crucial stages in the search and selection of a strain that is promising for use in the food industry is the definition of its taxonomic identity. Proper identification of the strain at the species level allows the researcher to get an idea about the safety, origin, habitat and physiological characteristics of the selected microorganism in advance.

Lactic acid bacteria constitute the bacterial basis and (or) ferment of various food products. They are permanent inhabitants of the gastrointestinal tract with the capability to successfully compete with putrefactive bacteria in the intestines, often resistant to antibiotics. Lactic acid bacteria and their bacteriocins can serve as biopreservatives and probiotics for the use in medicine and the food industry. Recently, growing attention has been given to the exploration of new strains with antimicrobial potential deprived of the preservatives and classical antibiotics disadvantages. The purpose of this cross (comparative) genomics study is to screen and identify natural L. lactis SSP lactis strains with antimicrobial activity that are promising for the creation of bio-preservatives.

Some fermented milk products (sour cream, sour milk, cheeses, kefir), plants, and others could serve as the sources for the isolation of lactococci. Besides, according to Linares et al. (2017), L. lactis SSP lactis, being potential producers of nisin bacteriocin, are widely distributed under natural conditions. Many LAB are initially present in milk and cause its spontaneous ripening as a result of lactic fermentation. The main product of this fermentation is lactic acid, which removes calcium from casein in milk. In this case, the protein is converted into paracasein and precipitates, which causes coagulation of milk and the formation of a milk clot. Primary lactic fermentation is carried out by such mesophilic homofermentative lactococci as L. lactis SSP lactis and L. lactis SSP cremoris, and then other lactic acid bacteria join the process. It could be, for example, one of the Lactobacillus genus or microorganisms of another taxonomic group.

There are known several national fermented milk beverages, obtained as a result of mixed lactic acid and alcoholic fermentation. For instance, Zhao et al. (2019) mention koumiss, made from mare’s milk and close in composition to the human female, used to be widely known as a beverage for the treatment of tuberculosis, diseases of the gastrointestinal tract, and the cardiovascular system. Articulating about the benefits of fermented milk products, it should be noted that even with modern technology and the technology of their production it is impossible to completely exclude the ingestion of foreign microorganisms (among which there are pathogens), which cause various defects and reduce the quality of the finished product. Often, the starter microbiota, cooked without adding pure cultures, is clogged with foreign microorganisms. Therefore, as Sacchini, Migliorati, Di Giannatale, Polmilo and Rossi (2017) agree, the search for biological methods to combat undesirable microbiota, based on the use of antibiotically active starters and cultures, is gaining an increasing value. In connection with the intended purpose of screening bacteriocin-forming strains of mesophilic lactococci, one can use national fermented milk drinks, such as, for example, the aforementioned koumiss (from Kyrgyzstan, Asia; fermented mare’s milky drink) and Doogh (from Iran; yogurt mixed with mint and soda), which have inhibitory activity on different groups of microorganisms.

In the context of comparative genomics, the initial identification includes a complex of phenotypic traits based on the study of morphological, physiological, and biochemical properties of lactococci. During profound incubation, lactococci compose boat-shaped colonies. According Liu, Chan, Chen, Solem and Jensen (2019), differentiation of L. lactis SSP lactis from L. lactis SSP cremoris and from L. lactis SSP lactis diacetylactis requires taking into account the growth pattern on dense media with milk hydrolyzate. L. lactis SSP cremoris form dark round colonies on the surface of the medium, while L. lactis SSP lactis diacetylactis create deep colonies of irregular shape in the form of pieces of cotton wool.

In turn, as Guzel-Seydim, Dibekci, Cagdas, and Seydim (2016) cite, lactic acid bacteria are positively Gram-stained. They are also immobile, do not form a spore, do not form a pigment, do not liquefy gelatin, do not restore nitrates to nitrites, and catalase and oxidase-negative. The fact that bacteria do not possess oxidase activity corresponds to the generic characteristic in the Burgi bacteria determinant. Next, bacteria of the Lactococcus genus have round or slightly oval cells located singly, in pairs, or different lengths in chains. A typical representative of this genus L. lactis SSP lactis is considered by some authors, particularly by Anbi, Razavilar, Naghadehi, and Osalou (2018), to be rod-shaped, since the cells are longer than wide. However, it should be noted that lactococci are polymorphic. They can stretch out and resemble rod-shaped forms and form streptococcus-like chains of different lengths, which is associated with a decrease in the activity of autolytic enzymes in the process of divergence of cells formed during cell division.

Molecular oxygen in homofermentative lactic acid bacteria is not included in the process of lactic fermentation, but they can grow in its presence. Following König and Fröhlich (2017), homofermentative lactic acid bacteria are facultative anaerobes, but some microaerophiles might be present among them when low concentrations of molecular oxygen in the medium promote bacterial growth. Flavin enzymes dwell in the cells of these bacteria, help ing the oxygen to gradually reduce to the hydrogen peroxide. The accumulation of peroxide slows down the oxidation of glucose and inhibits growth, since these bacteria lack hemoproteins, such as cytochromes and catalase, which decomposes hydrogen peroxide. Particular studies have shown that all the strains that might be isolated are facultative anaerobes that might possess a funnel-shaped completion in the agar column.

Thakur, Tomar, and De (2016) consider the idea that a distinctive feature of lactic acid bacteria is a high need for complex nutrients, purines, pyrimidines, amino acids, and vitamins: thiamine, riboflavin, biotin, nicotinic, and pantothenic acids. To a large extent, it explains the growth effect from the addition of various plant extracts to the media (yeast, potatoes, carrots, or corn). In fact, homofermentative bacteria are exclusive as they are the only ones to be found among the representatives of the Lactococcus genus. Following the ideas of Buron-Moles, Chailyan, Dolejs, Forster, and Mikš (2019), they form almost one hundred percent of lactic acid in the process of fermentation as one of the primary end metabolites. Isolation and rapid accumulation of lactate lead to sharp acidification of the medium, caused by a decrease in pH, which inhibits the growth of bacteria that cause food spoilage. It should be remarked that different microorganisms react to the environment acidity in diverse ways.

For a prolonged period, most researchers have associated the antagonistic effect of lactic acid bacteria addressed by Özogul and Hamed (2018) with their ability to produce lactic acid. In a while, the factual data range about the nature of this phenomenon has been significantly enlarged. Along with lactic acid, the role of other metabolites inhibiting the development of bacteria has also been established. The physiological features of many L. lactis SSP lactis lactococcal homofermentative strains include the synthesis of bacteriocins as the low molecular weight inhibitory substances of protein nature. Meanwhile, a genotypic method based on the analysis of similarity of nucleotide sequences in genes confirms the taxonomic position of the isolated cultures received via the classical microbiological methods for the identification of bacteriocin-forming strains of lactococci. Nucleotide sequences serve as a valuable tool for identifying lactococcal intraspecific links. However, lactic acid is not the principal antimicrobial agent, but it has a high level of inhibitory activity.

Undoubtedly, the increasing number of literature on the theme regularly aggravates the interest to the practical future potential of the empirical-theoretical investigations and tests of Lactococcus SSP and distinct lactic acids. As cited by Panda, Mishra, Kayitesi, and Ray (2016), the central areas of work in optimizing of the of lactic acid production comprise the study of the biological properties of lactic acid fermentation producers, selection of active homofermentative lactic acid bacteria, and optimization of parameters for controlling the process of biosynthesis. The study and application of optimal growth conditions for selected cultures of microorganisms and the effectiveness of the acidification process are of the same value. Following Salar-García et al. (2017), nitrogen and carbon sources, pH, temperature, and culture method make it possible to impact the titer of the lactic acid producer and its productivity. Also, it is accessible to increase the yield of lactic acid by using stress factors, which are titled as critical conditions. It could be the elevated temperature, high or low pH values, high concentrations of initial reagents, high cell concentrations, the introduction of various chemical components not involved in metabolic pathways of lactic fermentation (such as ethanol), and the introduction into the system of different bacteriostatic elements. As follows from the results of comparing the calculated and experimental data, a model that takes into account the stress factors of the biosynthesis process referred by Filannino, Di Cagno and Gobbetti (2018) is recommended to simulate and optimize the process of lactic acid biosynthesis in a membrane fermenter. This model can be elongated to continuous multistage processes. In the future, to reproduce the operation of multistage fermentation plants, it is necessary to introduce into the model the relationships that characterize the effects of the bio-membrane element in the reactor design.

Consequently, the possibilities of comparative genomics allow conducting the screening of bacteriocin-forming strains of mesophilic lactococcal and other lactic acid bacteria from milk, dairy products, and fermented milk drinks. The scientists, as mentioned by Frantzen et al. (2017), continue to elaborate and enhance the method for isolating and differentiating mesophilic lactococcal strains with antimicrobial activity from these products. An essential criterion for assessing the effectiveness of strains encompasses the spectrum of its antimicrobial action, which is the activity against different groups of microorganisms. In this context, the data obtained can be viewed not only as an example of the response of a biological system to adverse environmental factors but also as the systems of relationships between yeast and lactococcal in natural ecosystems. As cited by Lim (2016), using the particular method of microbial selection based on the natural screening of active producers, it is possible to isolate some new original bacteriocin-forming L. lactis SSP lactis strains, to conduct a comparative genomics assessment of producers, and select the most promising of them. The diversity of strains with the different geographical origin and similar functionalities implies a consequence of the specific conditions that are formed in the ecological environment of one’s habitat.

Overall, the results obtained and shown in the literature might significantly contribute to the understanding of the mechanisms of lactococci adaptation to stressful environmental conditions and their respective effects on the human organism. It should be accentuated that these LAB strains have replenished the collection of valuable crops and are of scientific and practical interest in terms of expanding biological diversity, their possible use as biopreservatives, and for the preparation of bacterial starter cultures applied in the manufacture of lactic acid products. At present, scientists in multiple laboratories around the world are studying ways to biologically direct the synthesis of bacteriocins to create different modifications of already known bacteriocins, but with more valuable properties; also, they are trying to obtain new natural balanced bacteriocinogenic complexes that are safe for use as biopreservatives. The possibility of designing new antimicrobial analogs (bacteriocins) in the future might be the paramount method of combating antibiotic-resistant pathogenic bacteria. The lactic streptococci of the serological group N are of particular scientific interest; they have been recently isolated from a group of microorganisms of the Streptococcus genus (including its pathogenic forms) due to the systematic position, and assigned to the GRAS category under the new name Lactococcus. Therefore, lactococci, like their bacteriocins, could be utilized as natural biological preservatives in the further perspective comparative genomics investigations and experiments aimed to improve the life-defining spheres, particularly the food one.

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