Characterization of Lactococcus lactis 11/19-B1 Isolated from Kiwi Fruit as a Potential Probiotic and Paraprobiotic

Probiotics are live bacteria used as food additives that are beneficial to human health. Lactococcus lactis 11/19-B1 strain isolated from kiwi fruit stimulates innate immunity in silkworms. Intake of yogurt containing the living 11/19-B1 strain significantly decreases the level of low-density lipoproteins (LDLs) in high-LDL volunteers and improves atopic dermatitis in humans. In this study, the probiotic properties of the 11/19-B1 strain, such as sensitivity to antimicrobial compounds, biogenic amine production, some virulence genes for human health, antimicrobial activity, tolerance to gastric acid and bile acids, and ability to adhere to the intestinal mucosa, were evaluated. The 11/19-B1 strain did not show resistance to the tested antimicrobial compounds except cefoxitin and fosfomycin. In addition, no production of amines that can harm humans, the antimicrobial activity required for probiotics, and the absence of adhesion to Caco-2 cells suggest that it is unlikely to attach to the intestinal epithelium. The 11/19-B1 strain grew in 0.3% but not in 1% bile salt. In the presence of 2% skim milk, the survival rate of the 11/19-B1 strain under simulated gastrointestinal tract conditions was 67% even after 4 h. These results indicate that the 11/19-B1 strain may function as a probiotic or paraprobiotic to be utilized in the food industry.


Introduction
Lactic acid bacteria (LABs) are defined as bacteria that produce lactic acid by fermenting sugars, such as lactose and glucose, and have been found in many environments, such as animals and plants, fermented foods, and feces [1][2][3].Live bacteria that are beneficial to the human body are called probiotics, and these have attracted particular attention from the viewpoint of health consciousness and preventive medicine in recent years [4].In terms of human safety, only LABs isolated from the human intestinal tract are recommended for human use as probiotics.However, in recent years, it has been reported that more beneficial probiotics have been isolated from the environment and fermented foods.Probiotics need to reach the intestines alive, while a new group, termed paraprobiotics, defined as inactivated (non-viable) microbial cells with a beneficial effect on the human body even in killed bacteria, has been proposed [5].
Lactococcus lactis is a Gram-positive cocci LAB that is a known probiotic and used in the production of many fermented foods such as yogurts and cheese, and some L. lactis strains produce various bacteriocins such as nisin, lactococcin, and diacetin [6][7][8].Many in vitro and in vivo studies on the properties of probiotics and/or paraprobiotics of L. lactis isolated from the environment and food have been reported, and it has been revealed that L. lactis modulates host immunity [2].Therefore, L. lactis is effective in strengthening the host's immunity and improving immune-related symptoms.On the other hand, certain strains of L. lactis produce nisin, which has an antimicrobial effect.Due to its antimicrobial activity, nisin is used as a preservative food additive in more than 50 countries.In this way, it is important to note that although L. lactis is effective as a probiotic and/or paraprobiotic, it is strain-specific and not common to all L. lactis.Therefore, it is important to examine the characteristics of each strain in detail.
In silkworms, the living L. lactis 11/19-B1 strain isolated from kiwi fruit markedly stimulates innate immunity and shows certain differences in sugar utilization and enzymatic characteristics in comparison with other L. lactis strains [9].Furthermore, we reported that the intake of yogurt containing the living 11/19-B1 strain significantly decreased the level of low-density lipoproteins (LDLs) in high-LDL volunteers compared to the control yogurt [10].Moreover, the living 11/19-B1 strain improved atopic dermatitis (AD) in humans, and the killed 11/19-B1 strain improved AD-related dorsal skin and ear lesions in a 1-fluoro-2,4-dinitrobenzen-induced AD mouse model [11].
Probiotics must be generally regarded as safe (GRAS) for application in animal and human systems.The suitable properties of probiotics are: (1) antimicrobial activity to suppress the growth of other bacteria; (2) no virulence factors against the host; (3) no ability to produce amines; (4) sensitivity to antimicrobials; (5) tolerance to gastric acid and bile acids; and (6) ability to adhere to the intestinal mucosa.In this study, we evaluated the probiotic properties of the L. lactis 11/19-B1 strain.

Sentitivity to Antibiotics
The sensitivity to antibiotics of each LAB used in this study was basically assessed with CLSI-M45 standard with some modifications [14].Frozen-stocked bacteria, except B. lactis, were reactivated on De Man, Rogosa, and Sharpe (MRS) medium (Becton, Dickinson, and Company, Franklin Lakes, New Jersey, USA) containing agar with a final concentration of 1.5% at 35 • C under 5% CO 2 for 24 h.B. lactis was reactivated on a MRS agar plate at 35 • C under anaerobic conditions for 24 h.Reactivated bacteria were subcultured once under the same conditions.Tested bacteria were selected and suspended in sterilized saline to the 0.5 McFarland turbidity standard equivalent.To prepare the samples for the antibiotic sensitivity test using a DP32 or DP43 dry plate (Eiken chemical Co., Tokyo, Japan), 25 µL of 11/19-B1 and JCM5805 suspension at 0.5 McFarland were added to 12 mL of MHBII (Becton, Dickinson, and Company) containing lysed horse blood (Nippon BIO-TEST laboratories, Asaka, Saitama, Japan) at a final concentration of 2.5%.On the other hand, 25 µL of L. bulgaricus suspension was added to 12 mL of MHBII containing lysed horse blood at a final concentration of 5%.Furthermore, 100 µL of prepared samples was added into wells of the DP32 or DP43 dry plate, and the plates were incubated for 24 h at 35 • C under aerobic conditions for the 11/19-B1 and JCM5805 strains or under 5% CO 2 for the L. bulgaricus LB-12 strain.

Antibacterial Activity
To detect the antibacterial activity against indicator bacteria (S. aureus, P. aeruginosa, K. pneumoniae, and E. coli), a spot-on-the-lawn assay was carried out with some modification [17].Briefly, each indicator bacterium was incubated in 5 mL brain heart infusion (BHI) broth (Nissui Pharmaceutical Co., Tokyo, Japan) at 35 • C overnight.Moreover, 50 µL of the culture was added to 5 mL of BHI soft agar (0.7% agar), and this mixture was poured onto BHI agar.The tested LAB strains were grown in BHI broth for 48 h, and the supernatants were collected by centrifugation at 8000× g at 4 • C for 10 min.The collected supernatants were adjusted to pH 6.0 with NaOH, treated with or without heating at 80 • C for 10 min, filtrated using a 0.2-µm filter (KURABO industries, Osaka, Japan), and stocked at −30 • C until used.Moreover, 10 µL of the samples were then spotted onto BHI soft-agar plates containing the indicator bacterium.The plates were analyzed for bacterial growth inhibition after incubation at 35 • C for 18 h.The L. lactis NBRC12007 strain, a nisin-producing strain, was used as a positive control for the antibacterial activity test.

Suvival under Simulated Gastrointestinal Tract Conditions
The analysis of bacterial survival under simulated gastrointestinal tract conditions was performed according to the previous report [18].The simulated gastric juice was prepared as follows: 0.06 g of FaSSGF (Boirelevant Com., London, United Kingdom) was dissolved in 1000 mL of 0.2% saline (adjusted to pH 1.6 with HCl), and then pepsin (Sigma-Aldrich Co., Saint Louis, MO, USA) was added at a final concentration of 0.1 mg/mL.This juice was filtrated before use.The tested bacteria, the 11/19-B1 and B. lactis Bb-12 strains, were incubated in 10 mL of MRS broth at 35 • C for 24 h under aerobic or anaerobic conditions, respectively.After centrifugation at 6800× g for 10 min at 4 • C, the resultant pellet from each culture was washed twice with phosphate-buffered saline (PBS) and then resuspended in 0.5% saline.A 200 µL aliquot of the cell suspension was mixed with 300 µL of saline or 10% skim milk and 1 mL of simulated gastric juice.After incubation at 37 • C for 0, 60, 120, 180, and 240 min, the number of viable cells was counted by culturing on MRS agar at 35 • C after 72 h to evaluate tolerance against simulated gastric juice.

Susceptibility to Bile Salts
The susceptibility of the LABs used in this study to NaCl, pH, and bile salts was analyzed according to the method of Drosinos et al. [19], with some modifications.Tested LABs were grown on MRS agar at 35 • C under 5% CO 2 for 24 h and subsequently subcultured in MRS broth at 35 • C under 5% CO 2 for 24 h.Moreover, 50 µL of the cultures was added to 5 mL of modified MRS broth, with the addition of 4.5% or 6% of NaCl and 0.3% or 1% (v/v) of bovine bile, adjusted to pH 2.0 with HCl, and incubated at 35 • C for 3 days.The growth of bacteria strains in the medium was demonstrated by visual turbidity, and the replication ability of the bacteria was examined under different conditions.

Adhesion Assay to the Human Colon Adenocarcinoma Cell Line Caco2
Adhesion assays were performed according to the method described by Guglielmotti et al. [20], with some modifications.The human colon adenocarcinoma cell line Caco-2 (Riken BioResource Research Center RCB0988) was used in this study.The cells were grown in Dulbecco's Modified Eagle Essential Medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 20% fetal calf serum and L-glutamine.The frozen, stocked 11/19-B1 cells were plated onto MRS agar and cultivated aerobically at 35 • C for 24 h.A few colonies of bacteria were cultured in 10 mL of MRS broth at 37 • C for 22 h under aerobic conditions.Moreover, 100 µL of 10 mL culture were inoculated into fresh MRS broth and cultured at 37 • C for 22 h.The cultures were centrifuged at 4000 rpm.After removing the supernatant, the pellets were resuspended in 2 mL of PBS.The 2 × 10 5 Caco-2 cells were seeded per well of 24-well microplates and cultured in the fresh media mentioned above until 90% confluent.Three wells of Caco-2 monolayers were inoculated with approximately 7.2 × 10 7 of the 11/19-B1 cells/well, and the plates were incubated at 35 • C under microaerophilic conditions.After 1 h of incubation, the monolayers were washed carefully three times with PBS, and the Caco-2 cells were lysed with 1 mL of ice-cooled 0.2% (v/v) Triton-X 100 solution (Sigma-Aldrich Co., Saint Louis, MO, USA) and vigorous pipetting.The Caco-2 cell lysates and the 11/19-B1 strain were collected and serially diluted in saline, and then the 11/19-B1 strain was plated on MRS agar and incubated at 35 • C for 72 h.All viable colonies were counted, and the CFU (colony-forming unit)/mL was calculated.Adhesion values (%) were calculated as follows: % Adhesion = (V1/V0) × 100, where V0 is the viable count of bacteria added initially and V1 is the viable bacteria count that adhered to Caco-2 cells at the end of the experiment.Two independent assays were carried out.

Statistical Analysis
The survival of each LAB under simulated gastrointestinal tract conditions was determined in three independent trials.The average of CFU/mL and the standard deviation of each LAB were calculated.

Sentitivity to Antibacterial Compounds
Probiotics included in foods should not confer antimicrobial-resistant genes on other gut microbiota [21].Minimum inhibitory concentrations (MICs) of antibacterial compounds against three LABs, the L. lactis 11/19-B1 and JCM5805, and L. bulgaricus LB-12 strains, were determined.The test was repeated two or three times, and the highest MIC values are shown in Table 1.The JCM5805 strain was used for comparison with 11/19-B1.The FEEDAP Panel (Table 2 in the 2018 EFSA [21]) establishes the microbiological cut-off values between susceptible and resistant for various LABs.The 11/19-B1, JCM5805, and L. bulgaricus LB-12 strains were inhibited at lower concentrations than the established cut-off values, at least for ampicillin, clindamycin, gentamicin, vancomycin, and erythromycin, with all strains showing resistance to cefoxitin and fosfomycin.The 11/19-B1 strain showed an equal to slightly higher MIC compared to the JCM5805 strain for all tested antibiotics.These results demonstrated that 11/19-B1 did not confer antimicrobial resistance genes on other microbiota and may be used as a food additive.Probiotics need to be safe for humans, but there have been cases of infection in association with some LABs, and there is a debate about safety and risk [22,23].L. lactis does not adhere to the intestinal tract and, except for some cases, is considered safe, although it has been reported to cause bacteremia and endocarditis [24].As shown in Table 1, the 11/19-B1 strain was inhibited at lower concentrations than the established cutoff values, at least for ampicillin, clindamycin, gentamicin, vancomycin, and erythromycin.This indicates that even if an infection occurs due to the 11/19-B1 strain, it can be treated with antibiotics.

Production of Amines and the Presence of Virulence Genes
Dairy products are made by fermenting milk with LABs, known as starter strains.LABs are known to produce biogenic amines (BAs), which are derived from various amino acids.It was reported that some BAs induce toxic effects such as headaches, heart palpitations, vomiting, diarrhea, and hypertensive crises [25].It has been reported that dairy products contain large amounts of BAs, such as tyramine, histamine, putrescine, 2-phenylethylamine, etc., which are associated with these symptoms [26,27].Therefore, BAs produced by the 11/19-B1 strain were investigated.
E. coli and two strains of the genus Pediococcus (P1 and P2) produced cadaverine and histamine, respectively, while the 11/19-B1 strain, like the control strain E. faecalis JCM5803, did not produce histamine, putrescine, tyramine, or cadaverine (Figure 1).Some Lactococcus species produce histamine, putrescine, or 2-phenyl-ethylamine [25].The production of 2-phenyl-ethylamine by the 11/19-B1 strain is unknown, and the 11/19-B1 strain, unlike other L. lactis strains, does not produce tyramine or putrescine.Histamine and tyramine are known as particularly dangerous BAs as they may cause more severe symptoms [28,29].Upon consumption of large amounts of cheese, the tyramine contained in the cheese can cause symptoms such as diet-induced migraines, increased cardiac output, nausea, vomiting, respiratory disorders, and elevated blood glucose [28,29].High-level histamine intake can also result in harmful physiological effects due to its psychoactive and vasoactive properties, resulting in potentially life-threatening effects [30][31][32][33].Indole, a metabolite of tryptophane, is synthesized by the tryptophanase of some intestinal bacteria, such as Escherichia coli.Once indole is absorbed from the intestinal tract, it accumulates in the liver through the portal vein, where it is converted to indoxyl by oxidative metabolism.Thereafter, indoxyl becomes indoxyl sulfate through the action of sulfotransferase 1A1 (SULT1A1).Indoxyl sulfate acts as a cardiotoxin and uremic toxin, and although it is excreted from the body with urine when renal function is functioning normally, it accumulates in the blood due to a decrease in urine volume due to a decrease in renal function.Indole and indoxyl sulfate are considered to be factors in the progression of renal failure [34].The genome sequence information of the 11/19-B1 strain (Genbank accession No. BLYG01000001.1 to BLYG01000006.1)indicates that the present strain does not possess tryptophanase and estimates that the present strain does not produce indole.It is thought that foods fermented with the 11/19B1 strain do not contain indole, and patients with chronic kidney disease can consume these foods.The selection of specific starter strains that lack the accumulation pathway of these harmful BAs is essential for obtaining high-quality foods with a reduced BA content, and the 11/19-B1 strain could be suitable for use as a starter strain.

Antibacterial Activity
It has been reported that several LABs produce bacteriocin to prevent the growth of pathogenic microbes such as Listeria monocytogenes, Bacillus cereus, or Staphylococcus aureus [36][37][38][39][40]. Nisin A, produced by certain strains of L. lactis, is a bacteriocin consisting of 34 amino acids that shows an antimicrobial effect against Gram-positive bacilli such as the genera Bacillus and Clostridium [41].This bacteriocin is also thought to have very little effect on the intestinal flora in the lower intestinal tract, as it is inactivated by pancreatin, an Indole, a metabolite of tryptophane, is synthesized by the tryptophanase of some intestinal bacteria, such as Escherichia coli.Once indole is absorbed from the intestinal tract, it accumulates in the liver through the portal vein, where it is converted to indoxyl by oxidative metabolism.Thereafter, indoxyl becomes indoxyl sulfate through the action of sulfotransferase 1A1 (SULT1A1).Indoxyl sulfate acts as a cardiotoxin and uremic toxin, and although it is excreted from the body with urine when renal function is functioning normally, it accumulates in the blood due to a decrease in urine volume due to a decrease in renal function.Indole and indoxyl sulfate are considered to be factors in the progression of renal failure [34].The genome sequence information of the 11/19-B1 strain (Genbank accession No. BLYG01000001.1 to BLYG01000006.1)indicates that the present strain does not possess tryptophanase and estimates that the present strain does not produce indole.It is thought that foods fermented with the 11/19B1 strain do not contain indole, and patients with chronic kidney disease can consume these foods.The selection of specific starter strains that lack the accumulation pathway of these harmful BAs is essential for obtaining high-quality foods with a reduced BA content, and the 11/19-B1 strain could be suitable for use as a starter strain.

Antibacterial Activity
It has been reported that several LABs produce bacteriocin to prevent the growth of pathogenic microbes such as Listeria monocytogenes, Bacillus cereus, or Staphylococcus aureus [36][37][38][39][40]. Nisin A, produced by certain strains of L. lactis, is a bacteriocin consisting of 34 amino acids that shows an antimicrobial effect against Gram-positive bacilli such as the genera Bacillus and Clostridium [41].This bacteriocin is also thought to have very little effect on the intestinal flora in the lower intestinal tract, as it is inactivated by pancreatin, an intestinal enzyme [42].Furthermore, it was recognized as GRAS (Generally Recognized As Safe) by the FDA (Food and Drug Administration) in 1988 as a preservative food additive in more than 50 countries, including Japan.The control strain NBRC12007, a nisin Aproducing strain, showed antimicrobial activity against only S. aureus among the strains tested in this study.The 11/19-B1 strain was shown to possess lactococcin as a bacteriocin based on the genome sequence; however, this strain did not show antibacterial activity against Gram-positive cocci such as S. aureus and Gram-negative bacilli such as E. coli, K. pneumoniae, and P. aeruginosa in this study.Such antibacterial properties of the 11/19-B1 strain may be advantageous for foods produced by the fermentation of multiple lactic acid bacteria, such as yogurt.Furthermore, even if the 11/19-B1 strain reaches the intestines alive, its bacteriocin products, such as lactococcin, are thought to have little effect on the intestinal flora.

Suvival under Simulated Gastrointestinal Tract Conditions
To examine whether the 11/19-B1 strain reaches the intestine alive, we examined its survival under various conditions (simulated gastric tract conditions, low pH, NaCl, and bile salts).In the fasted state, the pH in the stomach of a healthy person is about 1.7 (1-2) [43].In addition, bacteria must be able to withstand pH 2.5 to 3.5 to survive in the stomach [44].As shown in Table 2, none of the LABs in this experiment showed growth at pH 2.
The digestion of foods is known to take 2-4 h, so the viability of each LAB was examined in simulated gastric juice up to 4 h after exposure.Under simulated gastric tract conditions, the viable bacteria numbers of 11/19-B1 and B. lactis decreased from 2.3 × 10 8 to 0 and from 3.2 × 10 7 to 0 within 2 h, respectively.In addition, JCM5805 was reduced from 8.4 × 10 7 to 0 within 1 h.As shown in Figure 2, the survival rate of these bacteria was improved by the presence of skim milk at a final concentration of 2%, with the survival rate of 11/19-B1 reaching 67% even after 4 h (Figure 2).The pH in the stomach rises to 4.5 after eating, but the 11/19-B1 strain was viable in the presence of skim milk, even in simulated gastric juice with pH 1.These findings suggest that the 11/19-B1 strain in yogurt can survive, reach the intestine, and work as a probiotic, even when ingested on an empty stomach.
Along with pH tolerance, bile acid tolerance is an important factor for probiotics that reach the intestine alive.Furthermore, salt tolerance for survival in foods with high salt concentrations is also an important factor.As shown in Table 2, the 11/19-B1 strain showed the same salt tolerance as the other LABs tested in this study.B. lactis showed tolerance to 1% bile salt, as previously reported.Other LABs, including 11/19-B1, grew in 0.3% but not in 1% bile salt.

Ability to Adhere to Caco-2 Cells, Human Colon Adenocarcinoma Cells
LABs can influence the adhesion of specific pathogenic microbes by competing for binding sites on intestinal epithelial cells, thereby preventing the development of infections [45,46].Therefore, the adhesion of LAB to the gastrointestinal epithelium is one of the factors that determines whether it functions as a probiotic.To investigate whether the 11/19-B1 strain can adhere to and settle in the colon, we undertook an adhesion assay using the human colon adenocarcinoma cell line, Caco-2 cells.The 11/19-B1 strain did not adhere alone.It was reported that the adhesion of LABs to intestinal epithelial cells is associated with the presence of mucin [47].Goblet cells exist in the gastrointestinal epithelium of mammals, and a large amount of mucus (mucin) is produced from the goblet cells and coats the intestinal epithelium.Therefore, mucin is considered to be the main place of settlement for LABs.Caco2 cells are known to be incapable of mucin production, so it remains possible that the 11/19-B1 strain adheres to the intestinal epithelium along with other bacteria after binding with mucin.and from 3.2 × 10 to 0 within 2 h, respectively.In addition, JCM5805 was reduced from 8.4 × 10 7 to 0 within 1 h.As shown in Figure 2, the survival rate of these bacteria was improved by the presence of skim milk at a final concentration of 2%, with the survival rate of 11/19-B1 reaching 67% even after 4 h (Figure 2).The pH in the stomach rises to 4.5 after eating, but the 11/19-B1 strain was viable in the presence of skim milk, even in simulated gastric juice with pH 1.These findings suggest that the 11/19-B1 strain in yogurt can survive, reach the intestine, and work as a probiotic, even when ingested on an empty stomach.3 shows the in vivo probiotic and paraprobiotic effects of the 11/19-B1 strain and the mechanisms thought to be involved mechanisms, and Table 4 shows the in vitro probiotic properties of this study.Consumption of yogurt containing live 11/19-B1 strains significantly reduced LDL levels, lowered SCORAD scores in human AD patients, and activated innate immunity in silkworms [9][10][11].In addition, in a mouse AD model using DNFB, it was reported that ingestion of the heat-killed 11/19-B1 strain improved AD-related dorsal skin and ear lesions [11].
It is not clear whether the hypocholesterolemic effect is related to changes in the gut microbiota or whether it is a direct function of the strain.In this study, the 11/19-B1 strain did not show anti-microbial activity or adhesion to Caco-2 cells.These results suggest that the hypocholesterolemic effect is a direct function of the strain rather than a result of alterations in the gut microbiota.On the other hand, L. lactis has also been reported to modify the gut microbiota, even when heat-killed bacteria were used [48,49].It is possible that compounds produced by the 11/19-B1 strain and/or bacterial components contained in the yogurt altered the intestinal microbiota as a prebiotic and/or paraprobiotic, resulting in a hypocholesterolemic effect.The mechanism for lowering the SCORAD score of human AD patients by ingestion of 11/19-B1 yogurt remains unclear, but the heat-killed 11/19-B1 strain causes the activation of innate immunity in silkworms and the improvement in AD-related dorsal skin and ear lesions in mouse AD models.These phenomena are thought to be due to the paraprobiotic effects of the 11/19-B1 strain.Previous studies have shown that LABs, whether alive or dead, have an immunoregulatory effect on hosts [50,51].As with the 11/19-B1 strain, another L. lactis strain, JCM20101, also improved AD-related dorsal skin and ear lesions in the mouse AD model [11].This effect may be a common feature of L. lactis strains.The reason for activation of immunity with the 11/19-B1 strain may be due to genomic DNA or cell wall components.Therefore, the 11/19-B1 strain can be used as a paraprobiotic through its application as an additive not only in yogurt but also as an additive in various foods without the risk of infection.

Conclusions
In this study, it was suggested that strain 11/19-B1 may reach the intestines alive when ingested as a food additive in products such as yogurt.In addition, since it has been shown to be sensitive to many antibiotics, the risk of it bestowing drug resistance on other intestinal bacteria upon reaching the intestine alive is low.As no production of bacteriocin was observed, even when used as a food additive, this strain does not prevent the growth of bacteria beneficial to humans, and the risk of dysbiosis in the intestine is also considered to be low.In addition, as the ingestion of sterilized 11/19-B1 improved atopic dermatitis in a 1-fluoro-2, 4-dinitrobenzen-induced atopic dermatitis mouse model, as described above, 11/19-B1 may function as a paraprobiotic.It has been reported that certain killed L. lactis bacteria change the intestinal flora and suppress aging.We have been investigating whether the intake of strain 11/19-B1 affects fecal indole, blood indoxyl sulfate, the improvement of medical conditions, and changes in the gut microbiota in patients undergoing dialysis due to renal failure.Since this study targeted dialysis patients, it would be possible to investigate the risks of ingesting the 11/19-B1 strain.Furthermore, using a mouse model of age-related hearing loss, we are investigating whether ingestion of killed bacteria from the 11/19-B1 strain will inhibit the progression of age-related hearing loss.Yoghurts containing live bacteria are already on the market, but it would be possible to use this killed bacterium as a paraprobiotic in a variety of foods.

Figure 2 .
Figure 2. Survival curves of the L. lactis 11/19-B1 strain and other LABs for comparison under simulated gastrointestinal tract conditions.Bars indicate the standard deviation.Circles: L. lactis 11/19-

Table 1 .
Minimum inhibitory concentrations (MICs; µg/mL) of antibacterial compounds against each LAB strain in this study.

Table 2 .
Tolerance to pH, NaCl, and bile salt of L. lactis 11/19-B1 and other tested LABs.
+: presence of growth; −: absence of growth Testing was carried out in duplicate at one time.* indicates that one of two trials was grown.** indicates lower growth than that in 0.3% bile salt.