INTRODUCTION:

Probiotic cultures have been associated historically with cultured of milks and dairy products, from which there is substantial evidence for positive effects on human health and general well-being [36][57]. The transity lactic acid bacteria in the gastrointestinal tract are capable of delivering enzymes and other substances into the intestine which possibly help to control intestinal flora [16]. Also, the antioxidative activity of lactic acid bacteria is reported [66]. Because of inhibitory effect, selected probiotic lactobacilli may be used as biological preservative, so, the aim of this study was to present some data on isolation, growth, and antimicrobial activity, effect of pH, heat, biosurfactant, immunomodulatory and sensitivity to proteolytic enzymes of lactobacillus as probiotic bacteria.

ANTIBACTERIAL ACTIVITY:

Against human multidrug resistant pathogens: The word ‘probiotic’ was coined by Lilly and Stillwell, which means beneficial to human [45]. According to World Health Organization (WHO), probiotic is defined as a live microorganism when administrated in adequate amount into host provides health benefits [77] Most commonly used probiotics are Lactobacilli which are known as helpful intestinal microflora [63]. Probiotics have been declared to promote health by suppression of viral induced diarrhoea [5], antibiotic induced diarrhoea [29], reduce irritable bowel symptoms [51], anti-cancerous activities [56], alleviate lactose intolerance [39] and modulate immune system[65].It was concluded that different strains of probiotics have shown antibacterial property against pathogenic and MDR (Multiple Drug Resistant) strains of S. aureus, E. faecalis, K. pneumonia, P. aeruginosa,

Escherichia coli, S. typhii and other Salmonella spp. [55, 49] by several studies. Lactobacilli of human milk from healthy volunteer mothers shown antibacterial activity against B. cereus, S. entericaserovar Typhi, P. aeruginosa and S. flexneri [62].Hence probiotics and their metabolites can arguably be promoted to have potential value in the treatment and prevention of infectious diseases. Thus, the present study was carried out to isolate, characterize and investigate in-vitro antibacterial activity of Lactobacilli isolated from dairy products against MDR bacteria isolated from human samples[68].The bacterial colonies isolated were identified on the basis of standard microbiological methods[72] and Kirby-Bauer disc diffusion method [76] was used for the susceptibility tests for each isolates. Sample specific lactobacilli shown antibacterial activity against MDR pathogenic bacteria.The result of this study indicates that dairy products contain beneficial probiotics which exhibited antibacterial activity against the selected clinical MDR isolates. More intensive in-vitro and in-vivo model studies need to be carried out revealing the phenotypic and genotypic characteristics of probiotics, in order to elucidate their role in human health.[11]

Activity against poultry pathogens:

Lactobacilli are the major type of lactic acid bacteria, which have been shown to act as a preservative as well as a probiotic agent [43].Probiotics are products used as dietary supplements to enhance the growth and health of humans and animals. They have been shown to be essential in disease control, as digestion aids, immune booster and in supplementing or replacing the use of antimicrobial compounds in the field of health [15]The present study was focused on scrutinize the antibacterial activity of Lactobacillus species isolated from poultry droppings in the treatment of poultry pathogens. Well diffusion method was used for the preparation of cell free supernatant[20] .For the antibacterial activity, agar diffusion method[22] was employed against the test organisms ( Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 25923, Salmonella typhi ATCC 6539, Shigella flexneri ATCC 12022. ) and the activity was evaluated by measuring the zone of inhibition and recorded. All the metabolites showed good inhibitory potency against selected pathogens. The zone of inhibition with the highest potency observed on L.delbruckii against E. coli while the the least zone of inhibition was observed against Staphylococcus aureus, Shigella flexneri and Salmonella typhi by L.caucasicus and L. buchneri. Lactobacillus isolates exhibiting antibacterial activity to demonstrate the probiotic function that can improve the health as well as increase performance parameters in poultry production.[35]

ANTIFUNGAL ACTIVITY:

Antifungal activities were depending on microbial community composition as well as environment and growth conditions [69]. In the present study, from animal manure 30 strains were isolated. For further investigation, four lactic acid bacteria were selected based on the cell growth and antifungal efficiency. Arasu et al., 2013 [47], reported that against fungi G.moniliformis and A.fumigatus, the Lactobacillus plantarum K-46 showed potent antifungal activity and often it caused spoilage in bakery products [33].Antifungal activity of these strains may be due to its fermentative metabolites. Okkers et al., 1999 [18], reported the supernatant of LAB having protein-like compounds which inhibit fungal growth. However, in our study, we confirmed the main reasons for these inhibitions are due to fermentative metabolites containing organic acids such as lactic acid, acetic acid, and succinic acid. In addition, all these strains have susceptibility against most of the antibiotics and did not exhibit the hemolytic activity on human blood, so these strains are nontoxic to host and can be considered safe. Overall results suggested that these strains could be used as potent probiotic strains.[64]

ANTIVIRAL ACTIVITY:

The development of resistance against antiviral agents has increased a threat to public health sector. As a result, there is an enlarging demand for the growth of unconventional antiviral agents that could efficiently rearrange the alive in-use drugs. In health care armamentarium, the probiotic strains of lactic acid bacteria (LAB) have received the efficient, economical and safe “nutraceutical” status. Against H1N1 virus, total of 2272 LAB strains were screened. The isolate YML009 exhibit a prominent antiviral activity. Sequencing and biochemical assays identified the isolate as Lactoballius plantarum which, indicate resistance to the damage caused by the acidic conditions such as gastric juice and 5% bile salt. By hemagglutination assay, the anti-H1N1 activity of the strain was confirmed and was found to show increased efficacy in comparison to the commercially available antiviral drug. This is a initial report on anti-influenza activity of a bacterium L.plantarum YML009.[32]

ANTI OXIDANT ACTIVITY:

Nutritional and medical studies point out the benefits of intensified consumption of fruit and vegetables on account of their low caloric value and beneficial effects of their constituents on human organisms. Most important merits of fruit and vegetables is their antioxidative properties. Lactic acid bacteria (LAB) are widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. L. plantarum is a lactic acid bacteria species that is frequently encountered in the fermentation of plant materials where phenolic compounds are abundant. The aims of this work were to study the antioxidant activity and oxygen competition of L. plantarum strain LAB 1 isolated from traditional fermented olives and to apply its antioxidant potential to produce functional foods with high-added-value compounds, such as antioxidants, from the conversion of olive phenolic compounds. Eight lactic acid bacteria strains were used in this study. The DPPH radical-scavenging capacity of lactic acid bacteria strains was determined according to the method described by Kao and Chen. The total antioxidative activity (TAA) was assessed by using the linolenic acid test (LA-test) [25]. Among the 8 strains tested, L. plantarum LAB 1 had the highest radical-scavenging activity with an inhibition rate of 57.07 ± 0.57 % at 8.2 109 CFU/ml. The measurement of antioxidant activity of L. plantarum LAB 1, showed that the antioxidant activity increased with the cell concentrations [37].

ANTAGONISTIC ACTIVITY OF LACTOBACILLUS:

Typhoid fever continues to be a public health problem in developing countries where infections are endemic, since it has been an important cause of illness and death, and it has been exacerbated by the emergence of antibiotic resistance. Although chloramphenicol has been the “gold standard” of therapy, a widespread plasmid-mediated resistance emerged in S. typhi, with outbreaks in 1970 [44]. However, Salmonella typhi and paratyphi A acquired resistance to fluoroquinolones and other antimicrobial agents, causing a major setback in the management of typhoid [13]. The potential use of Lactobacillus to control typhoid fever represents a promising approach, as it may exert protective actions through various mechanisms. Lactobacilli have a long history of safe use, especially in the dairy industry [61]. They resemble a major part of the commensal human mucosal flora [60, 2, 3, 73, 27]. Different Lactobacillus strains can function as microbial barriers against gastrointestinal pathogens through competitive exclusion of pathogen binding, modulation of the host’s immune system, and production of inhibitory compounds, such as organic acid (e.g., lactic acid and acetic acid), oxygen catabolites (e.g., hydrogen peroxide), proteinaceous compounds (e.g., bacteriocins), fat and amino acid metabolites, and other compounds [30, 34, 4, 23]. A total of 32 Lactobacillus isolates, recovered and selected as probiotic candidates in a previous study [1], were cultured in MRS broth and incubated at 37∘ C under anaerobic conditions. Determination of the Antagonistic Effect of Tested Lactobacillus Isolates against a Selected Salmonella typhi Isolate was done by Adherence and invasion assay and trypan blue assay followed by the microdilution technique described by Henry [17]. Lactobacillus isolates were examined firstly to test if they have any cytotoxic potential. The results showed that the tested isolates had not any cytotoxic potential. The cytoprotective effect on Vero cells pretreated with tested Lactobacillus isolates followed by infection with Salmonella typhi was assessed. Lactobacillus isolates nearly caused complete inhibition of Salmonella typhi cytotoxicity on Vero cells. The results of the present study revealed that twelve Lactobacillus plantarum isolates could protect against Salmonella typhi infection through interference with both its growth and its virulence determinants such as adherence, invasion, and cytotoxicity. These probiotic candidates are oxygen tolerant and as a consequence can retain viability during processing and storage. Therefore, they could be novel therapeutic agents for prevention and treatment of typhoid fever after being subjected to in vivo and application studies.Lactobacillus isolates not only showed interference with Salmonella typhi growth, but also showed strong killing activity.

BIOSURFACTANT IN LACTOBACILLIUS:

Microbial surfactants (Bio surfactants) are amphiphilic compounds produced in living spaces or excreted extracellular hydrophobic and hydrophilic moieties that confer on the organism the ability to accumulate between fluid phases thus reducing surface and interfacial tension [7]. Biosurfactants produced by Lactobacilli, in fact, have been shown to reduce adhesion of pathogenic micro-organisms to glass, silicone rubber, surgical implants and voice prostheses. Lactobacillus was isolated into MRS (Man, Rogosa and Sharpe) agar to determine the best medium for the growth of Lactobacillius. Bio surfactant production from isolated bacteria was detected by using oil spreading technique and emulsification stability test. Biosurfactants were identified by TLC. The Lactobacillus strain L2 also produced maximum biosurfactant [8].

CHOLESTEROL-LOWERING PROPERTIES OF LACTOBACILLIUS:

Cholesterol-lowering activity is one of the most promising properties of lactic acid bacteria with probiotic characteristics. In the present study, 58 potentially probiotic lactic acid bacteria were tested for their ability to survive in vitro digestion and reduce cholesterol in a medium containing cholesterol and bile acids. The LAB strains previously possess cholesterol reduction[42] Lactobacillus plantarum, Lactobacillus paracasei [10], Lactococcus lactis [41] The aim of the present work was to assay 58 LAB strains for their ability to remove cholesterol during growth using in vitro assessment to further test for the best-performing strains for their ability to survive gastrointestinal conditions, to survive in cheese during the ripening time at adequate levels to provide beneficial effects for consumers, and to reduce cholesterol to a great extent in cheese. The lactobacilli strains were maintained in de Man, Rogosa and Sharpe (MRS) broth (Biolife, Milan, Italy), whereas lactococci and enterococci were cultured in M17 broth (Biolife). In the experimental trials the bacterial counts were evaluated using MRS agar under anaerobic conditions (AnaerocultA, Merck, Darmstad, Germany) at 37°C for 72 h for lactobacilli, whereas M17 agar and kanamycin aesculin azide agar (Scharlau Science, Barcelona, Spain) at 37°C for 48 h were used for lactococci and enterococci, respectively. Several determination tests were performed like Culture Conditions for Determination of Cholesterol Removal from Media In Vitro [70][54], Survival in Simulated In Vitro Digestion[71], Culture Conditions for Determination of Cholesterol Removal in Cheese, followed by DNA Extraction and Strain Typing and Traceability in Cheese by Randomly Amplified Polymorphic DNA Profile. The experimental results revealed different ability levels in removing cholesterol from the growth medium. Bile tolerance and the ability to assimilate cholesterol were considered primary characteristics to select potential probiotic strains with a cholesterollowering effect. With the exception of Lb. casei BT147, all the LAB strains consumed cholesterol from the medium during static growth in MRS or M17 after 24 h of incubation in the presence of bile salts, but there was a wide variation in the cholesterol-reducing ability among and within the species. The lowest reduction in cholesterol was brought about by Lc. lactis ssp. lactis, whereas Lb. plantarum and Lb. paracasei ssp. paracasei gave the highest decrease. The 7 Lb. casei strains lowered the cholesterol content by an average of 22.8 ± 16.6%, but, interestingly, one (VC199) reduced it with a mean percentage of approximately 54%. Our results demonstrate that 7 potential probiotic LAB strains possess desirable properties in terms of cholesterol-lowering activity [12]

IMMUNOMODULATORYACTIVITY OF LACTOBACILLIUS PLANTRUM:

Probiotics have complex nutritional requirements and are found in a variety of habitats, such ashuman and animal mucosal membranes, material of plantorigin, sewage, and fermented dairy products and spoiledfood [19]. Lactobacilli are able to promote immunity in mice, and this effect is dose and strain reliant [28][53]. Lactobacillus plantarum has immunoregulatory function: activation of Th1 immune responses[38], promotion of IgA secretion and prevention of influenza virus infection[40], enhancement of the cytokine profile against mite allergy[59], and improvement of natural killer (NK) cell activity[30], for instance. L. plantarum KLDS1.0318, a late probiotic, Its effects on the activity of immune cells in vitro were previously investigated and it is considered to be possessed of a higher immunomodulatory activity. Pathogen-free BALB/c mice were used and all mice were randomly divided into six groups which was dispensed with L. plantarum KLDS1.0318 groups with different doses, equivalent volume of sterile phosphate-buffered saline (PBS). Analysis of body weight and Analysis of immune organ index were used for monitoring the result. The activity was determined on different cell activity. The results indicated that L. plantarum KLDS1.0318 could stimulate a T-lymphocyte–specific proliferative response. The impression of L. plantarum KLDS1.0318 on the thymus and spleen indices was determined first when thymus and spleen are such important immune organs in the body and the places of growth and proliferation of immunological cells. Consequently, these results suggest that L. plantarum KLDS1.0318 is an effective immunomodulating agent and may be effectively used to improve the immune function in humans [80][81].

LACTOBACILLUS FROM YAK YOGURT AND ITS PROBIOTICS:

Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” [24]. During the last decade, the use of probiotics for human has received increasing attention as scientific evidence continues to accumulate on the properties, functionality, and beneficial effects of probiotic bacteria on humans. In this study, a lactic acid bacterium was isolated and identified, and its probiotic properties were determined. After cultured in MRS medium, a bacterium was identified as lactic acid bacteria by staining and microscopic observation. And the lactic acid bacteria were further identified as Lactobacillus plantarum by physiological and biochemical tests. Probiotics are good for the parasitifer as microbial colonization in the human intestinal tract and reproductive system, which can produce the exact health effects so as to improve microecological balance of parasitifer, and play a useful role in the activity [31]. Probiotics have been now widely used in bioengineering, industrial and agricultural, food safety and life and health fields. With the rapid development of fermented dairy products, the development of probiotic products with better physiological functions has become an important direction for the development of dairy industry [78]. The natural fermented Yak Yogurt was adopted and was isolated and purified using MRS (De Man, Rogosa and Sharpe agar) medium. Identification and the DNA extraction of the lactic acid bacteria was performed followed by the activity study of acid tolerance, bile salt tolerance and hydrophobicity. The primary identification confirmed to be lactobacillus. By the study of the activity, when probiotics enter the intestinal tract, the bile salts in the small intestine inhibit the probiotics. hence the tolerance of the strains to bile salts is often one of the important indicators for screening probiotics [52]. Generally, the concentration of bile salts in human body fluctuates within the range of 0.03% to 0.3%. Studies have shown that the survival rate of lactic acid bacteria more than 70% in the artificial gastric juice can grow in different concentrations of bile salt, and the growth efficiency decreases with the increase of bile salt concentration. The reason is that high concentration bile salt can make the membrane permeability change, membrane protein dissociation, eventually lead to intracellular material flow, some cell death [9]. In the screening of probiotics in vitro, the surface hydrophobicity of bacteria reflects the ability of bacteria colonization in vivo, as with strong hydrophobic ability, lactic acid bacteria have good adhesion and strong colonization ability. [74][6][79]

LACTOBACILLUS ON BACTERIAL VAGINAL PATHOGENS:

The vaginal microbiota is a dynamic ecosystem that in healthy individuals is usually colonized by the Lactobacillus genus but it can rapidly lead to microbiota dysbiosis where a range of microorganisms become predominant and cause polymicrobial bacterial vaginosis (BV) and aerobic vaginitis (AV), respectively.[48][75] Incompetent diagnosis and antibiotic resistance, together with the eradication of some helpful bacteria [21]are the main causes of the undesirable results of conventional antimicrobic treatments of BV and AV. of Lactobacillus species in BV and AV has been decreased and it has given growth to the concept of their substitution to reinstate the natural vaginal flora by utilizing probiotic strains. According to the World Health Organization, probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host.”[26] Even though the use of probiotics to increase the vagina and prohibit or treat infection has been considered for some time, only at a recent time, their effectiveness has been confirmed and, different from that observed for antibiotics, no harmful effects have been reported.[50] Lactobacillus strains were inoculated to MRS (De Man Rugosa Sharpe) broth8 incubated at 37°C under non-agitated aerobic conditions. Here we studied that against four different pathogens the antimicrobial activity of two commercially probiotic strains, L.rhamnosus HN001 and L.acidophilus GLA-14, single or in union is in charge of both BV (G. vaginalis and A. vaginae) and AV (S. aureus and E. coli). The tested probiotic bacteria proclaimed that they possess inhibitory activity towards BV and, mainly, AV pathogenic bacteria having the highest antagonistic effect against anaerobic strains. Such an effect could be due to several mechanisms including the production of toxic compounds such as lactic acid, hydrogen peroxide, and bacteriocins that are enhanced in L. acidophilus rather than L. rhamnosus. [67] Our results reveal that the Lactobacilli combination was symbiosis against E. coli, demonstrating that the combination of two probiotic strains can be valuable to manage bacterial vaginal infections. One hopeful lead towards the treatment of BV and AV is also the vagina colonization by Lactobacilli which forms an obstruction against infection.[14][46].

CONCLUSION:

Given the results of this study, antagonistic effects of produced substances by the bacteria on a wide range of microorganisms have an important role in food preservation and human health. These bacteria can be raised for the production of various kinds of food and pharmaceutical products. They can also be used for the production of new functional foods. Therefore, increasing use of dairy products containing probiotics, identification and production of foods containing highest and most effective lactobacilli are recommended in daily diet. Lactobacillus strains showed strong antagonistic activities against a wide range of pathogens to humans, they could be considered as good potential probiotic candidates for treatment and prevention of infections. They should be studied further as biotherapeutic agents for treatments of specific disease conditions. The strains should also be investigated further for other probiotic bioactivities that have human health benefits.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 22.09.2020 Accepted on 10.07.2021

Int. J. Tech. 2021; 11(2):70-77.

DOI: 10.52711/2231-3915.2021.00010