INVESTIGATION OF THE POTENTIAL UTILIZATIONS OF HUMAN MILK ORIGINATED Lactobacillus gasseri STRAINS IN AQUACULTURE AND SEAFOOD PRODUCTION

In the current study, five Lactobacillus gasseri MA strains from human breast milk were investigated for their usage potentials in aquaculture and seafood products as probiotic and bio-protective. The strains were tested for their susceptibilities to various clinical antibiotics used in fish cultivation, antimicrobial activities against seven fish pathogens, alpha-amylase enzyme activities, and susceptibilities to sodium benzoate which used as a food preservative. The strains were susceptible to Ampicillin, Amoxicillin and Erythromycin antibiotics. Antimicrobial activity test results indicated that the inhibition zone diameters were ranged between 1.66 mm and 11.22 mm against various pathogens originated fish. The highest antimicrobial activity was recorded against the Aeromonas hydrophila ATCC 19570 (11.22 mm) for the MA-2 strain, while the lowest antimicrobial activity was determined against Vibrio anguillarum A4 (1.66 mm) for the MA-6 strain. L. gasseri MA-5, MA-3 and MA-6 showed alpha-amylase activity. The spectrophotometric and live-cell count data showed that all the strains exhibited resistance to sodium benzoate. The results suggest that L. gasseri MA strains could be good sources of probiotic for aquaculture and bio-protectives in seafood products.


Bacterial strains and culture conditions
Five L. gasseri MA strains from human milk were genetically identified by PCRbased molecular identification method (NCBI strain number:ATCC 33323 = JCM 1131). L. gasseri MA strains were cultured in MRS liquid/solid media and stored at 4°C in glycerol stocks for further use.

Determination of antibiotic sensitivity of MA strains
Antibiotic resistance of the strains was determined by a previously reported method by Sharma et al. (2016). The resistance of L. gasseri MA strains to commercial antibiotics used in the treatment of fish diseases i.e., Erythromycin (E, 15 μg), Gentamycin (CN, 10 μg), Kanamycin (K, 30 μg), Ampicillin (AMP, 10 μg), Amoxicillin (AMC, 30 μg) was determined by disc diffusion method. Briefly, the cell densities of active L. gasseri MA strains were adjusted to Mc Farland 0.5 in a physiological saline solution (PSS: 0.865 g NaCl). L. gasseri strains were then inoculated on MRS agar medium (100 μL) and spread with the sterile drigalski spatula. Each antibiotic disc was placed on the MRS agar with three replicates and then left to incubate for 24 h at 37°C. At the end of the incubation period, the inhibition zone diameters around the antibiotic discs were measured with Vernier calliper and the results were evaluated according to CLSI (Clinical and Laboratory Standards Institute) 2012 standards.

Determination of antimicrobial activity
The antimicrobial activity of the strains against various fish pathogens was determined according to the method described by Drago et al. (1997).

Preparation of L. gasseri MA supernatant
For the determination of antimicrobial activity, L. gasseri MA strains were centrifuged at 5000 rpm using a Universal 320 R centrifuge for 15 minutes and the supernatants were sterilized with 0.2 μm pore size microfilters. These sterilized supernatants were stored at 4 °C.

Agar well diffusion assay
In well diffusion assay, 100 μL of pathogen test microorganisms were inoculated onto the specific soft agar mediums and wells (7 mm) were made in triplicates. 100 μL of L. gasseri MA supernatants were loaded into the wells and then allowed to incubate for 24 h at appropriate temperatures for growth of each pathogenic microorganism. At the end of the incubation period, the inhibition zone values around the wells were measured with Vernier calliper and the results are given as averages of triplicates.

Determination of alpha-amylase enzyme activity
The alpha-amylase enzyme activity of the MA strains was determined using the method reported by Gupta et al. (2003) with some slight modifications. Alphaamylase enzyme activity was determined using supernatants of L. gasseri MA strains on nutrient agar medium containing 0.5% soluble starch (w/v) with spot culture method. For this purpose, the twice-activated cultures were centrifuged at 9000 rpm for 15 minutes. The supernatants were dropped on a nutrient agar medium at 20 and 50 μL in two different concentrations and allowed to incubate at 37 °C for 30 min. At the end of the incubation period, the media was stained with iodine and clear zones were appeared around positive colonies.

Sodium benzoate resistance
The sodium benzoate resistance of L. gasseri MA strains was determined using the method reported by Gunyakti and Asan-Ozusaglam (2019). The MA strains cultures adjusted to Mc Farland 0.5 standard were inoculated as 1% to experimental and control groups with ten different concentrations (0.015-1%) of sodium benzoate. The tubes were then allowed to incubate at 37 °C for 24 h. At the end of the incubation period, optical densities (OD) were measured spectrophotometrically at 600 nm. Besides, acid production abilities of strains were determined on sodium benzoate containing (at ten different concentrations) media. MRS containing sodium benzoate at a concentration of 0.1% was used to determine live cell count. After dilution, the culture was inoculated on the MRS-agar medium in two replicates and then incubated at 37° C for 24 h under anaerobic conditions.

Statistical analysis
Statistical analysis was performed with the Mann-Whitney U-test to identify significant differences in antimicrobial activity and sodium benzoate resistance assay results. The differences were considered significant at a p-value of <0.05. The statistical analyses were conducted using SPSS version 22 (SPSS Inc, Chicago, IL, USA).

Determination of antibiotic sensitivity of MA strains
As previously mentioned, resistance characteristics of MA strains to various commercial antibiotics were determined according to the disk diffusion method. Lactic acid bacteria with inhibition zone diameter as greater than 20 mm, 14-19 mm and below 14 mm were considered as sensitive, moderately resistant and fully resistant, respectively according to CLSI 2012 standards (Table 1). All the L. gasseri MA strains were observed resistant to Gentamycin, Kanamycin and sensitive against Ampicillin, Amoxicillin, Erythromycin antibiotics. Among the strains, the highest sensitivity was observed for MA-3 strains against erythromycin antibiotics (30.60 mm). The highest resistance was observed for all the tested lactic acid bacteria against Kanamycin (no inhibition) and for MA-3 and MA-6 strains against Gentamicin (no inhibition) antibiotics.  In a previous study, the antibiotic resistance properties of two L. gasseri strains from human breast milk were observed as resistant to Gentamycin and Kanamycin while susceptible to Ampicillin and Erythromycin antibiotics (Martín et al., 2005). In another study, L. gasseri NLRI-312 strain isolated from feces of newborn infants was determined as resistant to Kanamycin while susceptible to Erythromycin antibiotics (Kim et al., 2006). Some sources have been reported trouble that lactic acid bacteria used as probiotic or starter cultures can transfer antibiotic resistance genes to other lactic acid bacteria or pathogenic microorganisms. The results of the present study are similar to the above reports which cleared that L. gasseri MA strains exhibit resistance profiles to some clinically tested antibiotics. However, it has been also reported that probiotic microorganisms contain antibiotic resistance genes in genomic structures, structural resistance (chromosomal resistance) do not constitute a safety concern on their own as long as these genes are not transferred by horizontal transfer (Darsanaki et al., 2013).

Determination antimicrobial activity
Lactic acid bacteria inhibit the development of many enteric pathogens and play an important role in the treatment of gastrointestinal disorders in humans and animals (Fernández et al., 2003). Therefore, the inhibition of pathogenic microorganisms is expected from a probiotic strain by secreting various antimicrobial substances. In this study, antimicrobial activities of L. gasseri MA strains against fish pathogens were determined by well diffusion method ( Table 2). The inhibition zone diameters for MA strains against the seven fish pathogens varied from 1.66 mm to 11.22 mm. The highest antimicrobial activity was determined for the MA-2 strain against A. hydrophila ATCC 19570 while the lowest activity was determined against the V. anguillarum A4 for MA-6 strain. Among the tested strains, only MA-1 strain did not show any inhibitory activity against the only Y. ruckeri. There are average differences between the obtained data of antimicrobial activity among L. gasseri MA strains. However, the statistical analysis indicated that there are no significant difference between these results (p>0.05). The probable cause is that all the bacteria tested are different strains but the same bacteria (L. gasseri). For this reason, the use of L. gasseri MA strains in aquaculture may reduce or prevent epidemic diseases caused by pathogenic microorganisms. L. gasseri MA strains with antimicrobial activity may be recommended as a bio-preservative agent in aquaculture, seafood and canned fish products. The in-vitro results of the current study will also provide a basis for in-vivo studies.

Determination of alpha-amylase enzyme activity
The alpha-amylase is one of the most important industrial enzymes that cleave the alpha-1,4-glucosidic linkages of starch and produces different products such as glucose, maltose and maltotriose units (Gupta et al., 2003). Amylases and many other exogenous enzymes have been reported to be widely used as fish feed supplements. In the present study, alpha-amylase activity was observed for MA-5 (with 20 μL of L. gasseri supernatant) and MA-3, MA-5, MA-6 (with 50 μL of L. gasseri supernatant) (Figure 1). However, MA-1 and MA-2 strains showed no activity at both concentrations. Carter et al. (1992) observed that the alphaamylase enzyme used as dietary supplement affects positively the growth and development of fish. Amylases from bacteria promote starch digestion in the intestine of Cyprinus carpio (common carp) (Fusheng et al., 1994). The diets contain additional exogenous α-amylase enzyme might raise the enzyme activity of the fish and improve feed digestibility (Ji et al., 2012). However, the purification of enzymes from the microbial sources could be expensive. Therefore, the usage of bacterial strains producing enzymes in feeding is a more economic way. Although most lactic acid bacteria do not have amylolytic enzymes, a limited number of lactic acid bacteria can produce alpha-amylase (Asoodeh et al., 2010) (Oh et al., 2015). This study will give a biotechnological perspective to the MA strains. Because of this property, the MA strains that produce amylase enzyme can be a new source for the feed industry and enzyme technology.

Determination of sodium benzoate resistance
Sodium benzoate is an artificial food additive used as a preservative in food and beverage industries against bacteria, fungi, and yeast (Chen et al., 2009). SB can be used in some seafood products or ice used in fish conservation (Zhang and Ma, 2013). Probiotic microorganisms naturally present in or added later to food products should be tolerant to SB. For this reason, this study can be crucial in determining the effects of SB during the applications of MA strains as a natural food additive. The development of MA strains in the sodium benzoate medium (OD) was generally reduced compared to the control group (except for 0.075% and 0.1 mg/mL SB concentrations), but the strains never lost their viability (Table 3). There are mean differences between the observational data of spectrophotometric results of L. gasseri MA strains. The statistical analysis also indicated that there are significant differences between L. gasseri MA strains (p|<0.05). Even, the average differences between MA 1 and MA-4, MA-1 and MA 5, MA 2 and MA-5 and MA-5 and MA-6 at various SB concentrations are statistically different at the significance level of 0.05 (p|<0.05). The tested MA strains originated from human milk are the same bacteria (L. gasseri) but different strains. Therefore, the strains showed different resistance activities against various SB concentrations.
The pH values of the MA strains after culturing in media contained different concentrations of SB (Table 4) showed decreases compared to the initial pH values. This indicates that the strains survive throughout the incubation period and that initial pH values decreased with the production of various organic acids. However, MA-2 and MA-3 strains were found to have a value close to their initial pH values in a sodium benzoate medium with a concentration of 1 mg/mL. As the application rate of sodium benzoate as a food preservative in various food industries is generally 0.1%, live-cell counts of strains have been evaluated at this concentration. The number of viable cells of MA strains was found to be in the range of 10.78-6.85 Log CFU/mL ( Table 5). The viable cell counts of four L. gasseri strains (MA-1, MA-2, MA-3, MA-6) showed bit decreases when compared to the control group. However, it was observed that MA-5 showedincreased growth and this result was following the spectrophotometric data. Up to now, no study has been reported on the resistance properties of L. gasseri strains to sodium benzoate. Considering the results obtained in this study, it was determined that all MA strains were resistant to 0.1% mg/mL SB, which is generally used concentration in food products. Sodium benzoate is used as preservatives in the food industry in a pH range below 4.5. The initial pH after the inoculation of the MA strains in the growth medium was nearly 5.6 (growth medium pH). After incubation, the pH of the growth medium was generally decreased under pH 4.5 which is below the pH limit at which sodium benzoate is active (Table 4). The spectrophotometric data showed that the strains did not lose their viability after incubation (below pH 4.5). Besides, the viable cell results slightly decreased as compared with the control group at the application concentration of sodium benzoate (0.1%) in the food industry, suggesting that these strains survived under pH 4.5 at which sodium benzoate was active (Table 5). As a result, when SB as a food preservative and MA strains as probiotic are used together in the food industry, these strains have a potential to be used as natural bio-preservatives against pathogen microorganisms as well as probiotic effects for the food industry (including marinated fish, fish sauces, salad dressings, etc.) since they do not lose their viability.

CONCLUSION
In this study, the usage potential of human milk origin L. gasseri MA strains in aquaculture and various seafood products was investigated. The results of this study demonstrate that L. gasseri MA strains can be good candidates for use as fish probiotics since these strains have a susceptibility to various clinical antibiotics in addition to their capacity to produce antimicrobial compounds against seven fish pathogens responsible for high morbidity and mortality in aquaculture. The results of this study also revealed that L. gasseri MA strains can be suggested as an alternative bio-preservative for chemical preservatives to limit the microbialinduced diseases in fish farming. It is also suggested that strains having positive alpha-amylase enzyme activity may be used as feed supplementation to enhance nutritional value in the fish feed industry and to increase fish feed utilization. Additionally, all the strains were found resistant to sodium benzoate applied at different concentrations. Since strains are resistant to sodium benzoate, which is used as a preservative for canned fish or fish products, it may be advisable to use L. gasseri MA strains as alternative preservatives alone or in combination with sodium benzoate in such foods materials.