Identification of hydrogen peroxide-generating lactobacilli
The screening of Lactobacillus progressed in a three-step process. First, 500 strains with excellent development of hydrogen peroxide were isolated from saliva and feces. Second, Lactobacillus sp. were collected using LBS agar plates to sort through the 500 isolated strains. A total of 50 isolates were identified. Finally, the 50 isolates were tested for their strength against oral microorganisms, and L. gasseri HHuMIN D was the most effective. In fact, L. gasseri HHuMIN D severely inhibited the growth of oral anaerobia, which are known to cause halitosis (data not shown).
L. gasseri HHuMIN D was shown to produce hydrogen peroxide and suppress oral microorganisms. 16S rRNA gene sequencing of L. gasseri HHuMIN D showed that it was a Lactobacillus sp. Multiple alignment of 16S rRNA base sequences of isolated bacteria with the Lactobacillus sp. 16S rRNA sequence was obtained through the similarity matrix listed on GenBank. This genealogy research showed a 99% 16S rRNA homology; L. gasseri HHuMIN D shares 99% homology with L. gasseri.
Lactobacillus species isolated from the oral cavity vary widely and representative oral lactic acid bacteria include L. acidophilus, L. casei, L. fermentum, L. gasseri, L. johnsonii, L. rhamnosus, L. reuteri, L. salivarius and L. vaginalis [43-46]. These probiotic strains are normally used in dairy and health/functional food products. These results indicate that candidate probiotic Lactobacillus strains are present in the oral cavity. The current research confirms the probiotic capabilities of L. gasseri HHuMIN D. The antimicrobial effect of 5% of HHuMIN D supernatant was clearly demonstrated in the control of anaerobic oral bacteria causing halitosis. P. catoniae, P. intermedia, F. nucleatum, and P. gingivalis were inhibited by 90%, 89 ± 1%, 88 ± 1%, and 88 ± 1%, respectively. S. mutans, a dental caries-inducing bacterium, was inhibited by 60 ± 2%. S. mitis and S. gordonii, which cause periodontitis, were inhibited by 22 ± 2% and 19 ± 5%, respectively. Antimicrobial activity was lower against oral facultative anaerobic bacteria (S. sobrinus, S. sanguinis, S. parasanguinis, and S. oralis). On the other hand, the antimicrobial effect of 5% of W. cibaria culture supernatant showed relatively weaker antimicrobial activity than L. gasseri HHuMIN D (Table 2).
Lactobacillus culture medium neutralization and disinfection is an experimental process used to monitor the antibacterial activity of bacteria metabolites, such as bacteriocin. In another study, sterile neutral S. mutans ATCC 25175 supernatant showed >80% inhibition of L. paracasei strains . Another study confirmed that Lactobacillusgasseri, which produces gassericin A, has inhibitory properties against a wide range of oral pathogens, including carcinogenic and periodontal pathogens . In this study, the strong inhibitory effect of the culture supernatant of L. gasseri HHuMIN D against oral anaerobic pathogens was confirmed, and L. gasseri HHuMIN D’s significant antibacterial activity was confirmed.
Inhibition effect of L. gasseri HHuMIN D on oral microorganisms
Oral lactic acid bacteria are known to inhibit harmful oral bacteria by the production of various antibiotics . Lactobacillus, Bifidobacterium, Streptococcus and Streptomyces spp. are the majority of the oral probiotics studied to date. L. gasseri has also been studied as an oral probiotic and there are results similar to the findings of this research that L. gasseri is effective against harmful oral bacteria [50,51].
The interactions between L. gasseri HHuMIN D and oral anaerobic (F. nucleatum, P. gingivalis, P. intermedia, and P. catoniae) and facultative anaerobic bacteria (S. sobrinus KCOM1157, S. mitis KCOM 1356, S. oralis KCOM 1493, S. gordonii KCOM 1788, S. sanguinis KCOM 2167, S. parasanguinis KCOM 2522, and S. mutans KCTC3065) are shown in Fig. 1. All harmful oral bacteria were strongly inhibited by L. gasseri HHuMIN D, but interaction with oral microorganisms also impaired the proliferation of L. gasseri HHuMIN D. Compared to the control, L. gasseri HHuMIN D showed poor growth in most co-cultures. Among them, the lowest viable cell counts occurred when L. gasseri HHuMIN D was co-cultured with P. intermedia.
A previous study on the co-culture of oral anaerobia (F. nucleatum, P. gingivalis) and beneficial oral bacteria Weissella cibaria showed that F. nucleatum reduced the number of viable cells up to 1.5 Í 108 CFU/mL, and P. gingivalis reduced the number of viable cells to <105 CFU/mL, compared to controls . In this experiment L. gasseri HHuMIN D inhibited F. nucleatum more effectively. Additionally, L. gasseri HHuMIN D inhibited P. intermedia, P. catoniae and seven facultative anaerobic bacteria (S. sobrinus KCOM1157, S. mitis KCOM 1356, S. oralis KCOM 1493, S. gordonii KCOM 1788, S. sanguinis KCOM 2167, S. parasanguinis KCOM 2522, and S. mutans KCTC3065) quite effectively.
In another study, Lactobacillus salivarius, Lactobacillus fermentum, and the fermentation broths of these bacteria showed a definite inhibitory effect against harmful periodontal bacteria. Lactobacillus spp. showed greater direct antibacterial effects against harmful oral bacteria than their microbial supernatants. As the number of lactic acid bacteria and the concentration of the fermentation broth increased, the antibacterial effect also increased . The current research results are similar to those of Chen et al.,  : L. gasseri HHuMIN D's antibacterial effect was greater than L. gasseri HHuMIN D's supernatant antibacterial effect. This indicates the direct inhibition of harmful bacteria by L. gasseri HHuMIN D and indirect inhibition by the metabolites it produces [45, 53]. L. gasseri HHuMIN D strongly inhibited all harmful oral bacteria, but the supernatant of this bacteria inhibited only anaerobic oral bacteria and had different effects. The combination of these two actions efficiently kills pathogenic microorganisms in the oral environment. The results of this study indicate that L. gasseri HHuMIN D can affect various oral diseases caused by periodontal bacteria. Consuming probiotic or lactic acid bacteria-containing health/functional foods can prevent or treat periodontal disease .
Accumulation of hydrogen peroxide by L. gasseri HHuMIN D
Oral Lactobacillus spp. produces bacteriocins, lactic acid and hydrogen peroxide, which act as a defense against pathogens [54,55]. The generation of hydrogen peroxide is a typical function of Lactobacillus spp. (L. bulgaricus, L. lactis, and L. plantarum) isolated from the oral cavity. Several studies have shown that these beneficial bacteria inhibit the growth of pathogenic micro-organisms such as Staphylococcus aureus , Pseudomonas spp. , and psychrotrophic bacteria [58,59]. In the process of separating Lactobacilli from saliva and feces, colony-colored strains were visually chosen using selective agar plates for Lactobacillus with outstanding production of hydrogen peroxide. A Pierce Quantitative Peroxide Assay Kit (aqueous-compatible formulation, Thermo Fisher Scientific, Waltham, MA, USA) was used to test quantitatively using the colorimetric process (Fig. 2). The hydrogen peroxide produced by L. gasseri HHuMIN D increased continuously from 0 μmol/L to 802 μmol/L after 3 hours. After 12 hours, the concentration of hydrogen peroxide decreased steadily to 24 hours. L. johnsonii and L. gasseri have been shown to produce hydrogen peroxide at rates between 400 and 1400 μmol/L . Other research found that S. sanguis released hydrogen peroxide up to 30 μmol/L, S. oralis released hydrogen peroxide up to 640 μmol/L and both inhibited plaque formation by inhibiting S. mutans proliferation. The application of lactoperoxidase and thiocyanate to oxygen-supplied cultures revealed that S. mutans was inhibited, but did not affect the growth of, S. sanguis and S. oralis . Additional research contends that H2O2 produced by beneficial bacteria can decrease halitosis by inhibiting the growth of F. nucleatum and reducing the VSC produced by oral anaerobic bacteria .
L. gasseri HHuMIN D exhibits fairly good hydrogen peroxide production ability and is predicted to suppress harmful anaerobic bacteria that are vulnerable to hydrogen peroxide by continuous production. Further experimentation will be necessary to specifically investigate the effect of L. gasseri HHuMIN D's H2O2 on oral anaerobic bacteria.
Susceptibility of the bacteriocin produced by L. gasserie HHuMIN D to hydrolytic enzymes
Several types of hydrolytic enzymes were added to the partially purified bacteriocin solution at a concentration of 10%, reacted, and the residual activity of the antibacterial substance was measured. Antibacterial activity was completely lost by proteinase K, trypsin, and α-chymotrypsin treatment, indicating that the antibacterial active substance is proteinaceous, and possibly bacteriocin (Table 3). The fact that the activity was not lost by pepsin is possibly an enzyme concentration issue or indicative of the lack of a specific cleavage site for pepsin to react at. Since α-amylase did not affect the antibacterial activity, the carbohydrate moiety was either not present in the antibacterial molecule or not related to the antibacterial activity. Bacteriocins in which antibacterial activity is lost only with proteolytic enzyme treatment have previously been reported. For example, the same results were found in the case of Bacillus licheniformis .
The greatest advantage of bacteriocin is that it is composed of proteins or peptides. Bacteriocin is decomposed by proteolytic enzymes in the digestive tract of the human body, so it is considered to be non-toxic and non-persistent [64-67].
Coaggregation of L. gasserie HHuMIN D and oral microorganisms
In this research we evaluated the coaggregation of L. gasseri HHuMIN D with various oral microorganisms and found that L. gasseri HHuMIN D showed the highest cohesion with P. catoniae (70%) and P. intermedia (28 ± 5%). There was no cohesion with P. gingivalis or F. nucleatum. Amongst aerobic oral bacteria, L. gasseri HHuMIN D coaggregated with S. sanguinis best (74 ± 2%), followed by S. gordonii (62 ± 1%), S. mutans (49 ± 4%), S. mitis (46 ± 3%), S.sobrinus (37 ± 2%), S. oralis (30 ± 2%), and S. parasanguinis (6 ± 3%) However, the coaggregation of W. cibaria was relatively weaker than that of L. gasseri HHuMIN D (Table 4). L. gasseri HHuMIN D generally has higher coaggregation with oral facultative anaerobic bacteria than oral anaerobic bacteria with the exception of P. catoniae, which indicates that L. gasseri HHuMIN D possesses effective inhibitory capacity against oral facultative anaerobic bacteria. In another study evaluating the accumulation of 4 strains of human streptococci and 6 strains of lactobacillus used in consumer products, all probiotic strains showed the ability to aggregate with oral pathogens, but the degree of aggregation was different for each strain and dependent on time . Therefore, it is safe to state that only some strains of Lactobacillus can co-aggregate and not all Lactobacillus spp. co-aggregate with harmful bacteria.
There are more than 500 bacterial species in the oral cavity, of which 15-20 bacterial species are known to produce toxic materials and are directly implicated in the evolution of various forms of periodontal diseases . Many of these harmful bacteria are also highly capable of coagulating with other microorganisms, live in the oral cavity and create toxic substances constantly, thus harming oral health . Although the mechanism of aggregation has not been identified, the ability of probiotics to coagulate with harmful bacteria is important as a secondary function to support the main function of probiotics. Oral probiotics, which adhere well to oral mucosa and dental tissues, effectively inhibit oral disease and the growth of harmful oral bacteria [71,72].
L. gasseri HHuMIN D's ability to coagulate with harmful bacteria is essential for creating and sustaining a stable oral cavity. Beneficial bacteria with high coaggregation are not readily eliminated by saliva and physical removal from the oral cavity, since they can be rapidly attached to oral periodontal bacteria in the oral cavity. Additionally, harmful bacteria are exposed directly to antimicrobials such as bacteriocins, lactic acid, and hydrogen peroxide through direct coaggregation with beneficial bacteria .
Effect of L. gasseri HHuMIN Don the formation of artificial dental plaque by S. mutans
S. mutans, a facultative anaerobic coccus in the oral cavity, produces lactic acid as an end-product of glycolysis using sucrose as a substrate, and secretes glucosyl transferase (GTF; EC 18.104.22.168) to make insoluble glucan, a glucose polymer . These glucans are insoluble mucous substances which cling to the tooth surface to help bind bacteria to the tooth surface and cause enamel and dentin degradation through retention of the organic acid produced. S. mutans binds to the tooth surface, creates high amounts of acid by digestion of carbohydrates and erodes the enamel of the tooth, inducing dental caries .
To determine the inhibition by L. gasseri HHuMIN D of the formation of dental plaque by S. mutans, a beaker wire test was performed (Fig. 3). Artificial dental plaques formed on calibration wire suspended in S. mutans inoculated broths, but such plaques were not developed in L. gasseri HHuMIN D and W. cibaria inoculated broths. No artificial dental plaque was formed on calibration wire in broth co-cultured with S. mutans and L. gasseri HHuMIN D, indicating 100% inhibition (Table 5).
In one study, S. mutans created artificial dental plaque was suppressed by 53% by E. faecium T7 co-culture . In another experiment, Lactobacillus lactis 1370 suppressed plaque formation by 95% . Compared to other organisms tested, L. gasseri HHuMIN D strongly inhibits dental plaque formation by S. mutans. L. gasseri HHuMIN D whole genome sequencing revealed the presence of the glucosyltransferase gene (gtfD) that generates GTF-S (P49331), a strongly branched water-soluble beta-glucan synthetic enzyme (alpha 1,6-glucose). L. gasseri HHuMIN D only produces water-soluble glucan and prevents plaque production by inhibiting the growth of harmful bacteria in the oral cavity at the same time. S. mutans plaque formation in the oral cavity is affected by many factors, one of which is the glucan binding domain (GBD) in the S. mutans GTF enzymes (GTF-I, GTF-S, GTF-SI) that generate glucan, a major plaque element. Based on the type, the glucan produced has various effects on the GBD, such that the form and volume of glucan eventually released are different . The water-soluble glucan produced by L. gasseri HHuMIN D mediated the GBD of water-insoluble glucan produced by S. mutans in the co-culture of L. gasseri HHuMIN D and S. mutans while inhibiting the overall development of water-insoluble glucan. Several researchers have isolated and analyzed other species of Lactobacillus that produce water-soluble glucans such as those produced by L. gasseri HHuMIN D [78,79].
Adhesion assay of L. gasseri HHuMIN D
Harmful oral bacteria may bind to epithelial cells in the oral cavity, which can endanger oral health if the number of bacteria is high. We assessed L. gasseri HHuMIN D’s ability to prevent the adhesion of harmful oral bacteria to KB cells, a typical oral epithelial cell, using inhibition, competition, and displacement assays. Oral epithelial cell monolayer testing is one of the methods used to identify beneficial bacteria and has also been used as a guide to test the attachment of beneficial bacteria to the oral epithelium. According to an earlier study, if the number of attached bacteria per KB cell is 1.5 or more, the attachment capacity is considered to be very strong (+ + + +) and if the number of attached bacteria is 1.5 to 1, the adhesion capacity is considered to be strong (+ + +). If the number of bacteria attached is 1 to 0.5, the ability to adhere is moderate (+ +) and if the number of the bacteria attached is less than 0.5, the ability to adhere is considered weak (+) . The KB cell adhesion ability of L. gasseri HHuMIN D was determined to be 4.41 ± 1.4 cells per cell-- very strong. F. nucleatum showed a very high adhesion capacity of 18.35 ± 4.0, and S. mutans showed a strong adhesion capacity of 2.57 ± 0.1 (Table 6). Strains with strong adhesion are likely to adhere to oral epithelial cells, form colonies, and inhibit the attachment of pathogenic bacteria to epithelial cells. In a previous analysis, the adhesion abilities of harmful bacteria were measured for P. intermedia from 4.1Í104 ± 2.7Í104 to 152 ± 57 per 105 KB cells, for P. gingivalis at 9.6Í105 ± 1.0Í105, and for E. coli at 278 ± 133 . In another study, F. nucleatum’s KB cell adhesion at the initial dose was from 19.2 ± 0.3% to 1.5 ± 0.4, suggesting that F. nucleatum has very strong adhesion capabilities .
Competition between L. gasseri HHuMIN Dand oral microorganisms for cell adhesion
The ability of L. gasseri HHuMIN D to inhibit cell adhesion by harmful bacteria was assessed by conducting protection and displacement assays. In the protection assays, L. gasseri HHuMIN D was allowed to bind to KB cells before introducing harmful oral bacteria. L. gasseri HHuMIN D decreased cell adhesion by F. nucleatum and S. mutans by 63% and 71%, respectively, and that of F. nucleatum and S. mutans by 100% and 90%, respectively. L. gasseri HHuMIN D strongly inhibited F. nucleatum adhesion and it seems that the cell adhesion of L. gasseri HHuMIN D might also have been lowered by competition with the harmful bacteria (Table 7).
The attachment of L. gasseri HHuMIN D after the attachment of harmful bacteria was measured in the displacement assays. With F. nucleatum and S. mutans, the cell adhesion of L. gasseri HHuMIN D decreased to 70% and 73%, respectively. However, the attachment of harmful bacteria decreased by 89% and 90%, respectively. Taken together, these two results showed a stronger ability to inhibit the binding of harmful bacteria when first bound to L. gasseri HHuMIN D. This suggests that L. gasseri HHuMIN D is more effective in preventing than inhibiting the attachment of oral harmful bacteria. Consequently, continuous intake is likely necessary to enable adhesion of L. gasseri HHuMIN D to highly concentrated oral cavity cells. Other researchers have theorized that there is competition for common adhesion receptors between harmful bacteria and beneficial oral bacteria  and that antibacterial or antiadhesive factors produced by beneficial bacteria inhibit oral harmful bacteria from adhering after the beneficial bacteria aggregate .
Safety evaluations of L. gasseri HHuMIN D
As a potential novel probiotic bacterium, L. gasseri HHuMIN D was evaluated for its potential as a clinical pathogen on the basis of its phenotypic properties and genomics.
The evaluation of ammonia production confirmed the safety of L. gasseri HHuMIN D; no ammonia was produced. On the other hand, Enterococcus faecium ATCC19433, a positive control, produced 109 ± 7 μg/mL of ammonia. L. gasseri HHuMIN D's ammonia production is below the level of concern in South Korea's Ministry of Food and Drug Safety's milk product quality . Microorganisms may create various poisonous substances by nitrogen derivatives through decomposing the proteins, peptides and amino acids in saliva or food . When a microorganism enters the large intestine, it is able to generate poisonous substances such as phenol, ammonia and indole by decomposition of proteins . Ammonia formed by microorganisms is known to migrate to the liver and cause cell damage cofactors and chronic hepatic damage. The production of ammonia from microorganisms is closely related to human health and must be assessed to demonstrate the safety of commercial probiotics. According to Vince and Burridge , Clostridia, Enterobacter, Bacillus spp., and Gram-negative anaerobes create large amounts of ammonia. Furthermore, certain Streptococci, Micrococci, and Gram-positive non-spore forming anaerobes release small quantities of ammonia, and Gram-positive aerobic rods generate trace amounts of ammonia. Certain strains of Lactobacillus can produce small amounts of ammonia during growth.
Evaluation of biogenic amine production
L. gasseri HHuMIN D did not produce cadaverine, histamine, putrescine, or tyramine. Since ammonia and/or BAs are used as a quality indicator for fermented foods, L. gasseri HHuMIN D's absence of ammonia and BA activity suggests that L. gasseri HHuMIN D is suitable for use in the manufacture of fermented and non-fermented foods. Biogenic amines (BAs) derived from amino acids are common anti-nutritional compounds in animals and humans. Fresh meat, potatoes, and cheese commonly contain these compounds. Ingestion of massive amounts of BAs may cause symptoms similar to significant allergic reactions. BAs have been identified as causative agents in many cases of food poisoning and are critical from a hygienic point of view because they can induce a variety of pharmacological reactions . BAs are involved in numerous mammalian metabolic and intracellular processes, such as synaptic transmission, modulation of blood pressure, allergic reactions, and management of cellular growth. Probiotic bacteria, commonly used in the food industry, produce BAs through microbial metabolic activities such as decarboxylation and protein molecule transamination .
Hemolytic property test
L. ivanovii developed β-hemolysis colorless zones around colonies in BL agar added 5% sheep blood but L. gasseri HHuMIN D cultivated in the same medium did not reveal colorless zones around the colonies (Fig. 4). Therefore, L. gasseri HHuMIN D does not cause hemolysis. The Guidelines for the Evaluation of Probiotics in Food, produced by FAO and WHO joint research, states, “If the strain under evaluation belongs to a species with known hemolytic potential, determination of hemolytic activity is required” [28, 89]. The hemolytic characteristics of microorganisms are an important measurement criterion for the safety of bacteria since they may liquefy/degrade red blood cells and ultimately cause anemia and edema. Among probiotics, Lactobacillus spp. are graded as α-hemolytic microorganisms . According to research , several Lactobacillus spp. (L. sakei MBSa1 bac+, L. curvatus MBSa3 bac+ and L. lactis 368 bac–) demonstrate strong β-hemolysis.
Several research teams have studied the mucous dissolving capacities of human pathogenic bacteria since 1980, and mucus dissolving capacity is now considered a measure of microbial virulence and microbial toxicity [92-95]. The intestinal mucus gel coating is a membrane made of glycoproteins and is an essential part of the intestine. Even as it acts as a biological shield from microorganisms, the intestinal mucus of infants and immunocompromised hosts are capable of bacterial translocation. This bacterial translocation is regarded one of the most critical probiotic safety tests due to the risk for septicemia and bacteremia endocarditis . Although most microorganisms do not exhibit mucolytic activity, several studies have reported that certain microorganisms do and the genes that induce mucin degradation enzymes have been identified . Various intestinal pathogens are known to hydrolyze glycoprotein-based mucus gel layers and possess the ability to metabolize mucus-derived monosaccharides . The 'Guidelines for the Evaluation of Probiotics' of the FAO/WHO does not offer guidelines on the assessment of the mucin-decomposing function of probiotics , but the Steering Committee of the Norwegian Scientific Committee for Food Safety declared that decreased bowel mucin development or increased mucin degradation should be subject to examination because it may have harmful effects for patients . While there is no need for a safety assessment for mucin deterioration in the oral cavity, this study was performed to take into consideration the potential for ingestion and entry to the intestine by L. gasseri HHuMIN D. L. gasseri HHuMIN D was also assessed for the potential of translocation by in vitro mucolytic assays.
Cell growth levels were evaluated after incubation by measuring the absorption of quinary-modified MRS media (basal medium (glucose-free MRS basal medium with 0.5% mucin, 1.0% mucin, 0.5% glucose, and 1.0% glucose) at a wavelength of 550 nm. Basic sugars (glucose, fructose, maltose, and sucrose) were added to inhibit the development of mucinase through catabolic repression. However, this can lead to negative effects since mucinolytic enzymes can still be produced. Glucose, which is widely used as a source of carbon in MRS media, was therefore omitted from all broths to prevent this flaw.
If L. gasseri HHuMIN D had the ability to produce mucinases, through mucin degradation it would be able to survive and grow aggressively in the presence of mucin and no other carbohydrate sources. As illustrated in Fig. 5, L. gasseri HHuMIN D growth was actively induced by the addition of glucose as the carbon source. But growth was not observed when mucin was added instead of glucose. These results show that L. gasseri HHuMIN D does not use mucin as a source of carbon for growth.
As shown in Table 8, L. gasseri HHuMIN D was sensitive to ampicillin, carbenicllin, cephalothin, chloramphenicol, clindamycin, dicloxacillin sodium salt hydrate, erythromycin, lincomycin, methicillin, penicillin G, tetracycline, and vancomycin (MICs ranged from 0.01 to 4 µg/mL). There was general resistance to bacitracin, gentamicin, katamycin, metronidazole, neomycin, polymyxin B, phosphomycin, streptomycin, and trimethoprim-Sulfamethoxazole (all MICs were greater than 32 µg/mL).
To distinguish antibiotic tolerance from antibiotic-sensitivity, microbiological cut-off values for the antibiotic tolerance of microorganisms used as food were defined by the European Food Safety Authority . Except for gentamicin, streptomycin, and kanamycin, the MIC values of L. gasseri HHuMIN D were less than or equal to the cut-off values proposed by the EFSA. In a study of Lactobacillus spp.'s susceptibility to 23 antibiotics, some lactobacilli showed resistance to kanamycin, vancomycin, and chloramphenicol. The authors hypothesized that strains with these genes did not necessarily indicate cause for concern about the transition of antibiotic resistance and could be used in food and medicinal formulations as natural biopreservatives . Ampicillin, erythromycin, vancomycin, chloramphenicol, and clindamycin were low compared to the cut-off values proposed by EFSA. Antibiotic resistance can be transmitted via plasmids, the assessment of antibiotic resistance is a significant criterion for assessing Lactobacillus sp. safety . Therefore, we used WGS to genetically identify the antibiotic resistance gene of L. gasseri HHuMIN D and ascertain the possibility of transmission to other bacteria through the presence or absence of a plasmid.
Whole genome sequencing (WGS) of L. gasseri HHuMIN D
The size of the entire gene sequence of L. gasseri HHuMIN D was 2,066,663 bp and the GC composition ratio was 34.9%. The average GC content of Lactobacillus spp. is 46.61%, L. gasseri HHuMIN D is lower than the average GC content of Lactobacillus spp. The number of rRNA genes and tRNA genes were 7 and 63, respectively. The number of coding sequences (CDSs) was 2,015, and the average of the coding sequence length was 923.9 bp. Figs. 6 and 7 show a genetic map of L. gasseri HHuMIN D and a functional classification based on COG.16S rRNA analysis can be used to analyze the microbial population composition by analyzing short nucleotide sequences but cannot be used to explain the microbial genome's functional and physiological details . Whole genome sequencing (WGS) is a technique which studies the functional aspects of a microorganism by sequencing a microorganism's entire genome and comparing it to a gene previously identified .
Bacteriocin gene analysis through the WGS
Bacteriocin is a proteinaceous bacterial product with bactericidal activity . Any Gram-positive bacteria that produce bacteriocin are considered to be active against Gram-negative bacteria . This broad spectrum of compounds is important in preventing the growth of harmful bacteria and alleviating disease. They are produced by specific bacteria of the lactic acid bacteria, including lactococci, lactobacilli, and pediococci .
The whole genome sequence of L. gasseri HHuMIN D was verified through CLgenomicsTM and EZBioCloud applications to see if this strain qualifies for GRAS status. All results are summarized in Table 9. We found that the antimicrobial activity against oral harmful bacteria may be the result of one or more of three bacteriocin genes in which the antimicrobial activity of L. gasseri HHuMIN D was found: hlv, lafA, lafX. TheWGS cannot determine which of these genes are responsible for inhibitory activity against the pathogens tested and there is no data for reference because there are insufficient screening studies on bacteriocins produced by L. gasseri HHuMIN D against harmful oral bacteria. Further studies on bacteriocins produced by L. gasseri HHuMIN D and their effects on harmful oral bacteria are needed.
Since 1980, the L. acidophilus group has been classified by DNA-DNA homology into six homologous species: L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri and L. johnsonii [108,109]. Out of these six species L. gasseri is thought to be the primary member inhabiting the human intestine, and studies on the bacteriocins produced by this bacterium have been conducted [110-112]. Lactacin F is the best-studied bacterocin in the LAB class Ⅱb. Gassericin T is a 2-component bacteriocin consisting of GatA and GatX and of the lacticin-F family; it is known as the primary bacteriocin developed by L. gasseri strains . Gassericin T is heat-stable (121°C, 10 min), pH-tolerant (pH 2–11) and bactericidal against several food poisoning gram-positive bacteria such as Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus . Helveticin J was first studied in L. helveticus. Helveticin J can be used as an indicator of closely related species, is active at neutral pH under aerobic or anaerobic conditions and is sensitive to proteases and heat (30 min at 100 °C) . These 2 bacteroicins are good candidate biopreservatives.
Biogenic amines production gene analysis through the WGS
Biogenic amines are substances that have one or more amine groups that are used in eukaryotic cells as precursors to hormones, alkaloids, nucleic acids, and proteins. BAs are naturally produced by animals, plants, and microorganisms, but foods containing high amounts of BAs induce excessive gastric acid production in the body, improve cardiac efficiency, and can contribute to migraines and hypertension. BAs are an essential component in both qualitative and quantitative aspects of foods and beverages. Typical BAs include histamine, tyramine, putrescine, cadaverine, and β-phenyl ethylamine. These compounds are formed by histidine, tyrosine, ornithine, lysine, and β-phenylalanine decarboxylase and decarbonation reactions, so they can exist in acidic conditions. Traditionally, BAs have been used as indicators of microbial activity in food, and high levels of BAs in food implies deterioration in the quality of food.
The whole genome sequence of L. gasseri HHuMIN D was scanned via the CLgenomicsTM and EZBioCloud applications for genes that generate enzymes engaged in the metabolic pathways that create biogenic amines. Fig. 8 demonstrates the biosynthetic mechanism of different biogenic amines.
No genes were identified that produce tyrosine decarboxylase (which generates tyramine from tyrosine), L-tryptophan decarboxylase (which generates tyramine from tyrosine, histamine from histidine, tryptamine from tryptophan, and phenylethylamine from phenylalanine), lysine decarboxylase (which generates cadaverine from lysine), histidine decarboxylase (which generates histamine from histidine), carbamoyl-phosphate synthase (which generates ornithine from NH3), ornithine carbamoyl transferase (which generates citruline from ornithine), argininosuccinate synthase (which generates L-arginosuccinate from citruline), argininosuccinate lyase (which generates arginine from L-arginosuccinate), arginase (which generates ornithine from arginine), polyamine oxidase (which generates putrescine from spermidine), spermine synthase (which generates spermine from spermidine), spermidine synthase (which generates methylthioadenosine from S-adenosylmethionine) (Figs. 8), and genes were identified that produce glutaminase (which generates NH3 from glutamine), ornithine decarboxylase (which generates putrescine from ornithine), S-adenosylmethionine synthetase (which generates S-adenosylmethionine from methionine) (Figs. 8).
Glutamine can be converted to NH3 through glsA or GLS and converted NH3 can be converted to ornithine through CPS1. As a result of confirming the mechanism, L. gasseri HHuMIN D can be converted from glutamine to NH3 via glsA or GLS but cannot be converted to ornithine since there is no CPS1 thereafter. If ornithine exists, it can be converted via the urea cycle into citriuline, L-arginosuccinate, and arginine; the associated genes have not been found in the L. gasseri HHuMIN D genome. Polyamines such as putrescine, spermidine and spermine are produced using ornithine as the starting substrates through different pathways (Fig. 8). Except for methionine adenosyltransferase (MAT) and putrescine biosynthetic genes (ODC1, speC, speF), no genes encoding polyamine biosynthetic enzymes were found in the L. gasseri HHuMIN D genome. Contrary to the genetic results, the production amount seems to be very small, as bioamines are not measured in BA production capacity evaluation.
It has been reported that platelet aggregation induced by some lactic acid bacteria occurs in endocarditis, so it is a significant part of microbiological safety assessment. Phosphatidylserine plays a crucial role in the development of blood clotting and thrombus, so the genes involved in the metabolic pathways that create this compound were evaluated. L. gasseri HHuMIN D does not have any genes that produce phosphatidylserine in the metabolic pathway (Fig. 9)
Virulence genes lend pathogenicity to microorganisms. L. gasseri HHuMIN D was assumed to be non-pathogenic using VirulenceFinder 2.0, a program that enables pathogenic and non-pathogenic bacteria to be differentiated using data from the whole genome sequence in order to classify potential virulence genes within the genome. The L. gasseri HHuMIN D genome sequence was compared with the genomic sequences of four noted pathogens (Escherichia coli, Enterococcus, Listeria, and Staphylococcus aureus). Virulence factors evaluated included Escherichia coli shiga toxin gene and Staphylococcus aureus exoenzyme genes, host immune alteration or evasion genes and toxin genes. The L. gasseri HHuMIN D genomic sequencing revealed no virulence factors or toxic or pathogenic genes.
Antibiotic resistance and associated genes
L. gasseri HHuMIN D genes related to antibiotic resistance were discovered using the ideal and strict algorithms of CARD (Table 10). The genome of L. gasseri HHuMIN D was found to contain 16 putative genes associated with resistance to beta-lactams (7), bacitracin (1), aminoglycoside (1), aminocoumarin (1), lincomycin (1), polymyxin (1), macrolide (2) and multi antibiotic (2). Antibiotic resistance assays showed that L. gasseri HHuMIN D was resistant to gentamicin, streptomycin, and kanamycin, but genes associated with antibiotic resistance were not detected. Although penicillin, bacitracin, and lincomycin genes linked to resistance have been identified, the organism itself has demonstrated low resistance in antibiotic resistance assays. It must be noted that the L. gasseri HHuMIN D genome and the phenotype for antibiotic resistance do not match completely. It should be considered that the L. fermentum OK genome and the phenotypes of antibiotic resistance may not exactly fit. For DNA replication, GyrA is necessary. Multidrug efflux transporters are involved in several detoxifying activities in cells and are widely distributed across many forms of Lactobacillus spp. . There are also safety factors regarding the use of antibiotic-resistant strains, due to the possibility of transferring antibiotic-resistance genes to intestinal pathogens . Consequently, whole genome sequencing was used to determine whether the antibiotic-resistant genes of L. gasseri HHuMIN D could be transmitted through plasmids to other bacteria. No gene capable of delivering antibiotic resistance in the whole genome of L. gasseri HHuMIN D was found (data not shown). Since genes cannot be spread, antibiotic resistance to L. gasseri HHuMIN D is known to be inherent or normal. Several studies have documented that tolerance to aminoglycoside groups such as gentamicin, streptomycin, kanamycin and neomycin is suspected to be intrinsic to Lactobacillus spp. and is due to the absence of cytochrome-mediated electron transport that mediates drug absorption [117,118].