Characterization, Phylogenetic Affiliation and Probiotic Properties of High Cell Density Lactobacillus Strains Recovered From Silage

Mariadhas Valan Arasu a, Min-Woong Junga, Soundharrajan Ilavenila, Da Hye Kimb, Hyung Su Parka, Jung Won Parkc, Naif Abdullah Al-Dhabid, Ki Choon Choi a,*

a

Grassland and forage division, National Institute of Animal Science, RDA, Seonghwan-Eup,

Cheonan-Si, Chungnam, 330-801, Korea b

The United Graduate School of Agricultural Sciences, Tottori University, Tottori-Shi, 680-

8553, Japan, c

Animal and Plant Quarantine Agency, Anyang-Si, Gyeonggi, 430-824, Korea

d

Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies,

College of Science, King Saud University, Riyadh 11451, Saudi Arabia

*

Corresponding author: Ki Choon Choi,

Grassland and forage division, National Institute of Animal Science, RDA, Seonghwan-Eup, Cheonan-Si, Chungnam, 330-801, Republic of Korea. Tel: +82-41-580-6752, Fax: +82-41-580-6779 E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6573

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ABSTRACT

BACK GROUND: The aim of the present study was to isolate high cell density Lactobacillus (LAB) from different forages and select the best strains for production of silage with improved the lactic acid production. RESULTS: Twenty hetero-fermentative LAB strains were selected and their probiotic properties were analyzed by evaluating their tolerance to low pH, bile salts, biogenic amine production, enzyme activity, antibiotic susceptibility pattern and antifungal activity. The 16S rRNA gene based phylogenetic affiliation indicated that sixteen strains are Lactobacillus plantarum and others are L. bobalius, L. zymae, L. crustorum and L. diolivorans. Shake flask cultivation of these strains under aerobic conditions showed comparatively higher growth and organic acid production than that achieved using the well-studied LAB strains. In addition, all the strains were highly sensitive towards the Oxgall (0.3 %), but it grows well in the presence of sodium taurocholate (0.3 %). Antimicrobial susceptibility pattern is an intrinsic feature of these LAB strains thus; the consumption does not represent a health risk to the humans. L. plantarum strains exhibited considerable antifungal activity against food pathogens. CONCLUSION: The present finding raises the possibility that high cell density LAB strains with potential probiotic properties could be used to prepare the quality silages for animals.

Keywords: Lactic acid bacteria; Silage; Probiotic property; Antifungal activity INTRODUCTION Lactobacillus bacteria are short-rod and coccus in cell types with wide physiological, biochemical characteristics and low G+C content have been frequently reported from a variety of environments, including milk products, fermented foods and plant additives. However, studies on the isolation of Lactobacillus (LAB) from soil remain scarce, even This article is protected by copyright. All rights reserved

though it is well known that spore-forming LAB exists in soil.1 LAB have played a long and important role in food technology because of their ability to produce desirable changes in taste, flavor and texture as well as suppress the growth of pathogenic and spoilage microorganisms. The importance of LAB is associated mainly with their physiological features such as utilization of various substrates, metabolic capabilities and probiotic properties, etc. The abundant existence of LAB in foods coupled with their long historical uses contributes to their acceptance as GRAS (Generally Recognized as Safe) for consumption.2 Previous research has been shown that LAB is considered as technologically important in various fermentation products, mainly for the preparation of silage for animal feed.3 Among LAB, L. plantarum, L. buchneri and other lactobacilli, Enterococcus faecium are commonly used as the inoculants for silage additives. Homo fermentative LAB exclusively produces lactic acid; whereas, hetero fermentative species produce a mixture of lactic, acetic acid and other by-products like succinic acid and carbon dioxide. The main action of silage production include the high cell growth with rapid production of lactic acid and improving the stability of silage by aerobic or micro aerobic conditions because of the production of acetic acid, detoxification, inhibition of pathogenic microorganisms and good probiotic action4. LAB are generally more active at an initial day of cultivation and it was reduced after a few days because of the production of various organic acids, hydrogen peroxide, diacetyl, antifungal compounds such as fatty acids or phenyl lactic acids thereby pH declines.5 There are clear evidences that the growth characteristics and by-product formation nature of LAB used in silage inoculants were positively affected the quality of silage, but there is little information on the growth and survival of inoculants. The present investigation is to isolate high cell density LAB from different forages and select the best strains for the production of silage with improved lactic acid production.

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MATERIALS AND METHODS

Chemicals and reagents Antibiotic discs and culture media were purchased from Himedia, India. API 50CHB and API-ZYM test kits were acquired from Bio-Merieux SA, Marcy I’Etoile, France. Glucose Tyrosine, ornithine, lysine, histidine and other chemicals were obtained from Sigma-Aldrich.

Sample collection Silage of Italian rye grass, Crimson clover, Soyabean, Barley and Rice were collected from different locations at Cheonan, South Korea. The powdered and sediment samples were collected in sterile poly propylene bags and screw cap bottles, respectively. The collected samples were brought to the laboratory for isolation of high cell density LAB strains and the locations, nature of sample were recorded.

Isolation of Lactobacillus strains Lactobacillus strains were isolated from different forage.6

Biochemical and physiological tests for identification of Lactobacillus strains Biochemical and physiological properties of the isolate was analyzed using routine methods.7

Scanning electron microscope (SEM) The cells were suspended in a primary fixative solution containing glutaraldehyde (2.5 %) and PBS (0.1 M). The fixed cells were washed using PBS. Subsequently, the cells were fixed using osmium tetroxide (1.0 %) and PBS (0.1 mol L-1). Both primary and secondary fixation

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procedures were carried out for 2 h at 4°C. After dehydration through a graded series of ethanol (50, 60, 70, 80, 90, and 95 %), the fixed cells were dried twice with hexamethyldisilizane. Finally the dehydrated cells were sputter coated with gold at 25 mA for 250s and observed by scanning electron microscopy (SEM, JEOL. JSM-6460 LV) at 12 kV.

Amplification of 16S rRNA gene and sequencing The 16S ribosomal RNA gene sequencing was done at Sol-Gent Company, South Korea. Briefly, genomic DNA of LAB strains were isolated and PCR reaction was carried out with Taq DNA polymerase using the primers, 27 forward primer (5' AGA GTT TGA TCG TGG CTC AG 3') and 1492 reverse primer (3' GGT TAC CTT GTT ACG ACT T 5'). The conditions for thermal cycling were as follows: initial denaturation of the target DNA at 95oC for 10 min followed by 30 cycles of amplification, denaturation at 95 ºC for 2 min, primer annealing at 58 ºC for 1 min and primer extension at 72 ºC for 2 min. At the end of the cycle, the reaction mixture was held at 72

oC

for 10 min and cooled to 4 ºC. Amplified DNA was

visualized at 100 V and 400 mA for 25 min on agarose gel (1% (w/v) in TAE buffer 1X, 0.1 µL Ethidium bromide solution). Concentration of DNA was determined by spectrophotometer. The amplified PCR products were purified by QIA quick® PCR purification Kit (Qiagen Ltd., Crawley, UK). The PCR product was ligated into the pGEM-T cloning vector by following the instructions given by the manufacture (Promega, Madison, WI, USA). Plasmids were transformed into Escherichia coli DH5α competent cells. Recombinant transformants were selected by blue/white colony screening. Individual white colonies were grown at 37 ºC over night with rotary shaking in 25 mL of LB medium containing ampicillin. After plasmid preparation, 2 µL (out of 50 µL) of each sample was amplified by PCR (Bio-Rad I cycler) using M13-F and M13-R primers to check for the presence of insert DNA.

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Nucleotide sequence accession numbers The 16S rRNA gene sequences were deposited in Gen Bank database, under the accession numbers: KC422316-KC422325 for KCC-1 to KCC-9, KC430920 (KCC-11), KC571196 (KCC-12), KC571197- KC571202 for KCC-15-KCC-20, respectively.

Analysis of 16S rRNA gene sequence The obtained sequences were subjected to BLAST for comparing the sequence homologies in NCBI

database(http://www.ncbi.nlm.nih.gov/BLAST/)

and

manually

aligned

using

DNAMAN software (version 4.0).The restriction fragment length polymorphism (RFLP) of 16S rRNA sequences were analyzed in-silico by using NEB cutter V2.0 database (http://tools.neb.com/ NEBcutter2/). NEB cutter V2.0 is an on-line DNA sequence tool for analyzing the restriction pattern of large, non-overlapping, open-reading frames. It provides the users to check the different restriction enzyme sites for the nucleotide sequences. Briefly, after submitting the desired sequence by clicking the “Custom digest” command, different restriction enzymes can be selected for analysis. In this report we have analyzed the restriction pattern of “AluI”, and“XmaI”. Shake-flask cultivation of strains and HPLC analysis Strains were grown in 250 mL Erlenmeyer flask containing 50 mL of MRS broth at 30 ºC in an orbital incubator shaker under the aerobic and microaerobic conditions 6. After 24 h incubation, the samples were withdrawn and analyzed for the cell density (OD

600

nm).

Concentrations of fermentation metabolites (Lactic acid, acetic acid and succinic acid) were determined by HPLC (HP1100 Agilent Co. USA). The supernatants, obtained by centrifugation of the culture samples at 16000 g for 10 min, were filtered through Acrodiscsyringe filter (PALL Life Sciences; USA) and eluted through a 300 × 7.8 mm Aminex HPX87H (Bio-Rad; Hercules, CA, USA) column at 60 ºC using 5.0 mmol H2SO4. The HPLC

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analysis was carried out with a flow rate of 0.5 mL min-1 at a column wavelength of 220 nm. The injection sling was 10 μL. Quantification of the different organic acids was based on peak areas and calculated as equivalents of standard compounds.

Tolerance to low pH Tolerance to low pH was determined using the plate count method. Briefly, the active strains grown in MRS broth were inoculated (1 %) in 10 mL of fresh MRS broth adjusted to pH 2.5 with hydrochloric acid (1.0 N) and incubated at 30 ºC for 3 h. Samples were withdrawn at 0 h and at the end of 3 h of incubation to measure the initial bacterial population and residual cell population by plating suitable dilutions on MRS agar plates. The plates were incubated at 30 º

C for 48 h and the number of colonies grown was counted. The experiment was performed in

triplicate.

Bile tolerance The ability of isolated LAB strains to grow in presence of two different bile salts was studied8.

Biogenic amine production Production of biogenic amines was assessed by the method described9 by Bover-Cid, (1999).

Enzymatic activities Enzymatic activities were assayed using kit method according to the manufacturer’s instructions (BioMerieux, France).

Determination of antibiotic sensitivity and resistance pattern Antibiotic sensitivity and resistance of LAB strains were assayed by disc diffusion method1.

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Antifungal activity The inhibition spectrum was assayed by the agar diffusion method11.

Microorganisms Fungi: Aspergillus clavatus (KACC 40071), A. fumigates (KACC 40080), A.niger (KACC 40280), A.oryzae var. brunneus (KACC 44823), Candida albicans (KACC 30003), Curvularia lunata (KACC 40392), Fusarium oxysporum (KACC 40051) Gibberella moniliformis (KACC 44022), Humicolagrisea (KACC 40860), Penicillium chrysogenum (KACC 40399) and Penicillium roqueforti (KACC 41354) and bacteria: Lactobacillus strains used for the experiment were obtained from KACC culture collection.

Statistical analysis The investigated results were expressed as standard error of mean (mean ± SEM). The Statistical analysis was performed by SPSS/16 software, hypothesis testing methods that included the analysis of variance (one ANOVA) followed by least significance difference test (p 0.05 level).

Results Isolation and identification of Lactobacillus strains The present investigation involved in the isolation of LAB strains from five forage samples obtained from Cheonan, Chungnam, South Korea. 245 LAB strains were isolated and purified based on their capability to grow on MRS agar medium and BCP medium. All the 245 strains showed broad growth range either cultivated at aerobic or microaerobic conditions. Twenty high cell density LAB strains have ability to reduce the pH of MRS-broth to 3.8 - 4.3 after 24 h at 30 ºC, were isolated from Italian rye grass; crimson clover, soyabean, This article is protected by copyright. All rights reserved

barley and rice forages were selected for further studies. These strains were identified by biochemical, physiological characteristics, 16S rRNA amplification and sequencing. Further these bacteria were cultivated in shake flask scale and growth characteristics and metabolite production profile have been recorded.

Biochemical and physiological characterization of Lactobacillus strains All new bacterium designated and numbered as Lactobacillus sp. KCC-1 to KCC-22 was recovered from forage samples, all strains are showing good growth as compared with other identified strains. Culture characteristics of these strains were derived on the basis of observations made after 24 and 48 h of incubation on MRS and BCP agar medium. According to the culture characteristics, all the strains grew well on BCP and nutrient agar medium, but the colonies were spreading when grown on MRS. They displayed creamy in color; the surfaces were light yellow in appearance. The colonies of LAB strains were ovoid and lightly elevated and viscous in nature on solid medium. They were observed as Grampositive, aerobic or micro aerobic, rod-shaped (0.6-0.9 µm width and 2.1-3.5 µm length). SEM picture revealed that it did not contain spores on the cell wall and all the strains were motile in nature (Fig. 1). These micro morphological characters shape and motile properties strongly suggested that all the strains are belong to the genus Lactobacillus. Biochemical identifications of isolated strains were performed by the API 50E micro tests (Table-1). Biochemical tests revealed that the LAB strains were mesophilic and unable to reduce citrate or produce hydrogen sulphide. The optimal pH and temperature for growth of the strains were 5.5.0-7.5.0 and 28-37 ºC, respectively. All the strains exhibited good growth on medium amended with sodium chloride up to 2 % and could not liquate gelatin. They displayed positive results for amylase and protease enzyme production. Moreover, based on the biochemical and physiological characteristics, they were divided into five groups. Group

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1 consisted of KCC-1 to KCC-11 and KCC-15-21, and were identified as L. plantarum on the bases of morphological and physiological characteristics, because they could ferment arabinose, galactose, glucose, mannitol but could not ferment glycerol, erythritol, xylose, mannose, sorbose, lactose, melibiose, raffinose, glycogen, fucose, arabitol and potassium gluconate 2-ketogluconate. Group 2 including KCC-22 could grow on adonitol, methyl-D-xylopyraniside, galactose, glucose, fructose, mannitol and salicin. However, it could not grow on xylose, ribose, glycerol, arabinose, amygdalin, rhamnose, sorbose, glycogen and xylitol. Utilization of various carbon sources by LAB strain KCC-22. So, this strain was considered as L. diolivorans. Lactobacillus strain KCC-14 categorized in Group 3 was identified as L. zymae. This strain was able ferment galactose, glucose, fructose, dulcitol, mannitol, sorbitol, cellobiose, maltose, saccharose, gentiobiose and turanose. Strain KCC-12 and KCC-13 were included in group 4 and 5 respectively.

Comparative 16S rRNA analysis The 16S rRNA gene of the strains was completely sequenced and analyzed for the similarities. The NCBI BLAST search program showed that the sequence data of KCC-1 to KCC-11 and KCC-15 to KCC-21 had high identity (99 %) to L. plantarum with E value of 0, whereas KCC-22 shared 100 % similarities towards L. diolivorans NM194-1. Strain KCC-13 and KCC-14 identified as L. bobalius and L. zymae respectively. Based on sequence similarity strain KCC-12 was identified as L. crustorum. A homology tree was constructed using DNAMAN software (Fig. 2a). The percentage of replicate trees showed that the associated taxa clustered together in the boot strap test (1000 replicates). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the

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phylogenetic tree. Restriction digestion analysis revealed that the strains belonged to same species showed a comparatively similar restriction band pattern with AluI and XmaI (Fig. 2b).

Growth characteristics and metabolite production profile All twenty LAB strains showed considerable growth on MRS supplemented with glucose under aerobic and micro-aerobic conditions, as compared to other standard LAB strains which showed less, equivalent, or higher growth (Table 2a & 2b and S1). Lactic acid, acetic acid, and succinic acid, production abilities were determined. Lactobacilli species have been reported to be a determining factor for acid production and flavour development in the presence of sugars. Growth kinetics, fermentation end products profiles are given in Table 2. Results revealed that after 24 h of cultivation pH of the fermented broth was declined to 4.0 in majority of the strains. This clearly indicated that all the LAB strains were able to secrete organic acids. All the strains consumed entire glucose present in the culture medium within 24 h showed similar glucose consumption profiles. L. plantarum strain KCC-12 produced the highest amount of lactic acid (555.43 mmol L-1) after 24 h of incubation under aerobic condition. The acetic acid production ranged from 21.63 to 316.18 mmol L-1. The OD600 recorded with the KCC 22 after 24 h of cultivation under aerobic condition was 3.53. All the LAB strains showed better growth at micro aerobic condition.

Probiotic properties of LAB strains The low pH tolerance level of LAB strains was evaluated by culturing at pH 2.5. All the strains showed different performance to low pH. L. plantarum strains remained unaffected in acidic condition while strain KCC-15 showed a comparatively higher viable count from 7.44 to 7.68 log cfu mL-1 after 3 h of incubation (Table 3). Whereas, L. bobalius, L. zymae and L. crustorum showed decrease in cell viability count 7.61, 7.37 and 7.40 log cfu mL-1, This article is protected by copyright. All rights reserved

respectively. At low pH, L. diolivorans did not exhibit any change in the bacterial viable count (7.66 to 7.59 log cfu mL-1). The physiological function of intestine is very important in animals. Therefore, presence of the LAB in the intestine is depending on the tolerance of bile salts because it plays a major role in maintaining the physiological level. The twenty LAB strains were further grown at the presence of oxgall (0.3 %) and sodium taurocholate (0.3 %). Results revealed that all the strains were highly sensitive towards Oxgall but in the presence of sodium taurocholate they showed slight growth. L. plantarum strain KCC-1, 5, 6, 9, 11 and 16-20 showed positive results for decarboxylase activity against tyrosine, whereas L. bobalius, L. zymae and L. crustorum strains revealed negative results. All the strains were negative for decarboxylase activity against ornithine and histidine. Enzymatic activities measured using kit method showed a different profile (Table 4). All the strains showed weak to moderate level activity of leucine arylamidase and β-Glucosidase whereas, none of the strains exhibited positive response in the activities of alkaline phosphatase, α-fucosidase and α-mannosidase. Average β-glucuronidase activity (10 nmol L-1 of substrate hydrolyzed) was detected in L. plantarum strains. The L.crustorum and L.bobalius showed moderate βglucosidase activities (5–10 nmol of substrate hydrolyzed), and L. plantarum strains exhibited marginal β-glucosidase activities (10 nmol L-1 of substrate hydrolyzed).

Antibiotic sensitivity Antibiotic sensitivity pattern of the LAB strains was conducted against 22 antimicrobial agents including the antibiotics highlighted by EFSA (2008) by disc diffusion method. All strains exhibited comparatively different sensitivity patterns towards various antibiotics which interrupt either protein or cell wall biosynthesis in the bacteria (Table 5).

Antifungal activity

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L. plantarum strains exhibited moderate antifungal activity against fungal pathogens (Table 6). A. niger, F. oxysporum, G. moniliformis and P. chrysogenum were the most sensitive fungi, followed by A. clavatus, A. fumigates and Humicola grisea. P. roqueforti, C. lunata and A. oryzae growth were not inhibited by any LAB strains. Among the LAB strains L. bobalius, L. zymae, L. crustorum and L. diolivorans did not exhibit activity against the tested fungal pathogens.

DISCUSSION Isolation and characterization of new microorganisms are never ending process to meet the everlasting demand for probiotic properties in order to use as a food for humans and animals. Therefore, it is more important to characterize LAB strains, because they are the important sources of probiotic properties with less pathogenicity. Food additives, distilleries sites, silages, dairy industries and municipal wastes containing environment are the biggest reservoir of biological diversity with respect to LAB species. Among these, animal feed prepared using silages contained higher amount of LAB species. The quality and nutritional value of the silage is deepening on the amount of LAB and its secretary products. Therefore, research focus on isolation and characterization from silages has been gaining importance in recent years. However, still it has not been fully explored and there is tremendous potential to identify high cell density and higher lactic acid production capability LAB with various probiotic properties. Although fermented foods are considered excellent sources for the isolation of LAB strains with diverse potential.12 several LAB strains have been isolated from silages.13 The isolation and phylogenetic affiliation of LAB from silages was well documented. In this study the entire identified LAB grew well on MRSA and BCP agar medium. We isolated 245 LAB strains from different silages, among that twenty LAB strains were selected for further study because of their higher growth characteristics and lactic acid

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producing nature. Results revealed that all the strains contained smooth surface and creamy in appearance. They utilized most of the sugars that were provided, indicating a wide pattern of carbon assimilation. Among the strains, L. plantarum was the most frequently presented microorganism in this microbial group were dominant (16 out of 20). The L. plantarum was commonly found in silages. In contrast L. buchneri was reported as forage preservation to improve aerobic stability of barley silage to improve the quality of cheese. 4 The abundance of LAB strains in silages depended on several factors, including the preparation conditions, the time of harvesting and the duration of silage preparations.14 The strains showed different biochemical characteristics, relative to each specific species. On the basis of biochemical properties, L. plantarum strains could be divided into two groups, especially nine L. plantarum strains grew in D-arabinose, while the nine strains did not grow in the presence of D-arabinose. In contrast none of the L. plantarum grew on L-arabinose. These results were in close agreement with the findings; they characterized the functional properties of LAB strains isolated from kimch.15 The whole-cell protein analysis, cell wall composition analysis, morphological, physiological, and biochemical analyses are important criteria for the identification of LAB strains. 16 Species level was confirmed by 16s rRNA sequencing and BLAST search showed 99 % similarity towards respective LAB 16S rRNA gene sequences. The morphological and biochemical characteristics also reflected those of LAB genera. Recently, molecular biological tools played a major role in the identification of LAB strains.17 The growth characteristics and organic acid production profile of the isolated strains were evaluated by growing them in Erlenmeyer flasks under aerobic and micro-aerobic conditions. Standard LAB type cultures growth pattern and organic acid production nature also measured by cultivating under aerobic condition in shake flask levels. Results indicated that 20 LAB strains growth profile were comparatively better than type culture L. plantarum (KACC

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15357). After 24 h of incubation pH of the fermented broth were declined to 4.0. The decrease in pH is a good indication of the better acid producing strains. These variations in the pH are mainly due to the secretion of various organic acids especially lactic acid and acetic acids. Lactic acid is considered as a stronger acid with higher pKa (3.86) than the acetic acid pKa (4.73). Secretion of large amounts of organic acids inhibits the cell growth have been reported. Organic acids such as lactic acids and acetic acids affect the microbial cells by causing a decrease in the cytoplasmic pH, which effects on the function of cellular proteins and enzymes.18 Our results indicated that lactic acid and acetic acid were identified as the major metabolites. The other metabolites also produced such as acetic acid and succinic acid. LAB strains fermented the glucose through two major pathways: the glycolysis is used by the homo-fermentative LAB, were one molecule of glucose is completely converted into two molecules of lactic acid and in 6-phosphogluconate/phosphoketolase pathway, one molecule of glucose is converted into one molecule of acetic acid and lactic acid each.19 In our results all the strains produced lactic acids and acetic acids as comparable amount thereby behaves hetero fermentation pathway. The presence of acetic acid in the fermentation medium also may derive from citrate metabolism or lactic acid degradation.20 The accumulation of succinic acid in the fermentation medium suggests that oxaloacetate decarboxylase was inactive in LAB strains.21 The lactic acid production abilities of these strains was more important in the fermentation, because it is easy for us to select the best strain for further research, especially in the production of silages for animal feed preparations. LAB strains were mainly considered as probiotic. Because, probiotic strains are beneficially helping the host animal by improving its intestinal microbial balance.22 Normal pH of human gastric juice is 0.9-1.5 in an empty stomach, and rise to pH 3.0 after consumption of foods. Food stayed in stomach about 2–4 h generally.23 The organisms survived at acidic pH for 2 h were considered to as good probiotic.24

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Whereas, the

concentration of bile salts in the intestine was 0.03–0.3 %.25 Therefore, the probiotic nature of the LAB strains was evaluated under low pH and bile salts for certain conditions. Our results indicated that all the identified strains were able to survive under low pH and highly sensitive against oxgall (0.3 %) but they also showed positive growth in the presence of sodium taurocholate (0.3%) exhibited their novelty in using as probiotic. The nature of probiotic LAB microorganisms vary considerably in their levels of bile tolerance.26 The growth of bacteria was depending on the concentrations of bile acids27. In our results strains belonging to the same species (eg. L. plantarum) showed different resistance towards the bile acids. All the strains exhibited negative results for decarboxylase activity against ornithine, and histidine, whereas L. plantarum and L. bobalius, L. zymae strains were found to possess decarboxylase activity on lysine. Most of the identified L. plantarum strains showed decarboxylase activity on tyrosine. Tyrosine decarboxylase activity found in the LAB species isolated from traditionally fermented sausages, which indicated that the LAB strains had more added values28. All the strains grew well under acidic condition which may be related to the positive influence of the amine level. Therefore, it is suspected that biogenic amine production could be a used to help the probiotic microorganisms under acidic environmental conditions. Even though amine production capability of LAB strains have little health disturbance but have great advantages in surviving under low pH in the intestine for a long time. 9 L. plantarum KCC-7 and KCC-12 exhibited good β-galactosidase activity compared to other L. plantarum strains. Β-galactosidase mainly involved in the degradation of lactose into to glucose and galactose thereby helps to relief from lactose mal-digestion symptoms and other disorders associated with lactose intolerance.29 These results closely support the biochemical study where we examined the growth of the strains using lactose. Results revealed that all the strains could able to grow in the presence of lactose where only KCC-11

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and 12 showed positive results for β-galactosidase.30 The LAB strains isolated from kimchi did not show any activity towards β-galactosidase31. The identified LAB strains have been screened for their susceptibility towards 22 antimicrobial agents by disc diffusion method. All the 20 strains were found to sensitive towards the common antibiotics regardless of their source of origin, suggesting their suitability as probiotics. The LAB strains were susceptible to various antimicrobial agents32. In our results all the strains were highly sensitive to aminoglycoside antibiotics. Lactic acid bacteria were sensitive towards the antimicrobial agents33. Therefore, it is worthy to conclude that consumption of the LAB strains studied in the present study does not represent a health risk to humans and animals due to the emergence of antibiotic resistance and pathogenicity. It is also appeared that the antifungal activities of the LAB strains are typically more pronounced on filamentous fungi. These results are consistent with previous studies of probiotic LAB strains, which showed strong activity against filamentous fungi A. fumigates34 and poor activity against non-filamentous fungi Candida albicans.

35

The reason for the

discrepancy in sensitivity is based on the morphological differences between pathogenic fungi. The L. pentosus exhibited activity against C. albicans, but did not show activity against filamentous fungi36. But, the present study high cell density L. plantarum strains are having inhibitory effects against fungal pathogens which commonly affect the foods. The antifungal properties of these strains are not only to organic acid production, but also may due to the presence of hydrogen peroxide, diacetyl, fatty acids or phenyl lactic acid and bacteriocin formation.15

CONCLUSION From the present study, a total of twenty LAB from five different silages were confirmed by biochemical and molecular biological techniques. It is clear that novel high cell density LAB

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strains with probiotic properties are important for animal feed preparations. Few strains, L. plantarum KCC-11, KCC-18, and KCC-20, showed higher cell growth and considerable lactic acid production. The probiotic property such as considerable tolerance to low pH and bile salts along with different enzyme activities attracts their features. The extracellular products were effective against food pathogenic fungi such as Aspergillus niger, A. fumigates, Fusarium oxysporum, Penicillium chrysogenum, Gibberella moniliformis and Curvularia lunata. Therefore, these strains could be effectively used as the silage production for livestock animals without allowing the fugal attack.

ACKNOWLEDGMENTS This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ008502)"Rural Development Administration, Republic of Korea.

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Table 1 Biochemical characteristics of Lactobacillus strains recovered from different silages.

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No.s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Tests Control Glycerol Erythritol D-Arabinose L-Arabinose D-Ribose D-Xylose L-Xylose D-Adonitol Methyl-D-Xylopyraniside D-Galactose D-Glucose D-Fructose D-Mannose L-Sorbose L-Rhamnose Dulcitol Inositol D-Mannitol D-Sorbitol Methyl-D-Mannopyranoside Methyl-D-Glucopyranoside N-AcetylGlucosamine Amygdalin Arbutin Esculin ferric citrate Salicin D-Celiobiose D-Maltose D-Lactose D-Melibiose D-Saccharose D-Trehalose Inulin D-MeleZitose D-Raffinose Amidon Glycogen Xylitol Gentiobiose D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol potassium Gluconate potassium Gluconate 2-KetoGluconate potassium Gluconate 5-KetoGluconate

Lactobacillus strains KCC-1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 20 21 22

+ + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + -

+: Positive (more than 90 %). −: Negative (more than 90 %).

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+ + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - - + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - + + + + + + + + + + + + + - - - - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + + + + + + + - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - + + + + - - - - - - + + + + + + + + + + + + - - - - - - - - - - - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + - - - - - - + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - + + + + + + + + + + + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + - - - - - - + - - - - - - - - - - - - - - - - - - - - -

Table 2a Aerobic Growth kinetics and fermentation end products profiles of different Lactobacillus strains from silage.

Strains

pH

KCC-1 KCC-2 KCC-3 KCC-4 KCC-5 KCC-6 KCC-7 KCC-8 KCC-9 KCC-11 KCC-12 KCC-13 KCC-14 KCC-15 KCC-16 KCC-17 KCC-18 KCC-20 KCC-21 KCC-22

1.00 4.15 3.95 4.00 4.00 4.50 4.00 4.00 3.78 4.50 4.32 4.15 4.50 4.12 4.00 4.00 3.90 4.10 4.10 3.70

Cell growth

Lactic acid (nM)

Acetic acid (nM)

succinic acid (nM)

3.82 3.68 1.61 1.54 3.78 3.91 2.95 3.27 3.87 4.2 3.97 1.68 2.33 3.27 4.43 3.78 4.27 4.32 4.21 3.53

548.80 345.86 233.52 230.20 256.35 368.69 251.20 310.50 348.43 92.08 555.43 455.62 287.66 356.17 460.41 370.90 285.08 299.82 333.33 219.15

255.19 273.36 279.41 272.49 256.06 248.70 203.29 217.56 204.58 21.63 323.96 292.82 270.33 196.80 294.55 243.08 160.03 177.77 171.71 269.90

30.65 38.76 11.17 10.07 19.05 19.49 22.33 40.07 31.75 0.00 24.96 30.87 40.73 0.00 27.81 35.47 21.90 4.38 19.27 29.12

Strains were grown in 250 mL Erlenmeyer flasks containing 50 mL of MRS broth at 30 ºC in an orbital incubator shaker under the aerobic conditions. After 24 h incubation, the samples were withdrawn for analyzing cell density and metabolites.

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Table 2b Microaerobic Growth kinetics and fermentation end products profiles of different Lactobacillus strains from silage.

pH

Cell growth

Lactic acid (nM)

Acetic acid (nM)

Succinic acid (nm)

KCC-1

4.30

3.97

352.12

169.55

37.66

KCC-2

4.20

3.78

366.11

196.37

33.94

KCC-3

4.00

1.68

219.15

182.96

37.44

KCC-4

4.00

1.65

233.89

192.47

36.57

KCC-5

4.21

3.80

243.83

204.58

40.95

KCC-6

4.30

3.45

432.04

141.44

40.29

KCC-7

4.00

2.74

241.25

179.50

38.76

KCC-8

3.90

3.30

251.57

188.15

39.41

KCC-9

4.10

3.78

355.43

154.41

14.67

KCC-11

4.21

4.00

349.54

160.90

37.22

KCC-12

3.95

3.87

322.65

168.25

43.14

KCC-13

4.20

1.87

319.71

168.25

39.85

KCC-14

4.30

2.17

326.70

190.74

33.50

KCC-15

4.21

3.61

363.54

137.54

36.57

KCC-16

4.25

3.40

313.81

129.76

35.69

KCC-17

3.78

3.64

353.96

143.60

31.97

KCC-18

4.00

3.29

356.54

136.68

42.04

KCC-20

4.00

3.55

336.65

142.73

31.31

KCC-21

4.54

3.45

347.70

155.28

24.74

KCC-22

3.90

3.14

183.06

31.57

31.09

Strains

Strains were grown in 250 mL Erlenmeyer flask containing 50 mL of MRS broth at 30 ºC in an orbital incubator shaker under the microaerobic conditions. After 24 h incubation, the samples were withdrawn for analyzing cell density and metabolites

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Table 3 Probiotic properties of Lactobacillus strains. Strains KCC-1 KCC-2 KCC-3 KCC-4 KCC-5 KCC-6 KCC-7 KCC-8 KCC-9 KCC-11 KCC-12 KCC-13 KCC-14 KCC-15 KCC-16 KCC-17 KCC-18 KCC-20 KCC-21 KCC-22

Viable Lactobacillus counts (log CFU/ml) Tolerance to low pH Bile tolerance Control 7.94±0.05 7.86±0.07 8.00±0.10 7.74±0.21 7.92±0.02 7.98±0.13 7.96±0.02 7.78±0.29 7.93±0.04 7.83±0.05 7.88±0.17 7.61±0.06 7.71±0.25 7.92±0.01 7.94±0.04 7.87±0.07 7.90±0.07 7.89±0.03 7.75±0.4 7.93±0.06

0h 7.44±0.04 7.54±0.08 7.63±0.05 7.55±0.09 7.46±0.11 7.59±0.02 7.62±0.01 7.60±0.07 7.50±0.23 7.59±0.23 7.70±0.22 7.51±0.23 7.73±0.22 7.44±0.15 7.39±0.03 7.38±0.11 7.54±0.07 7.51±0.01 7.49±0.06 7.55±0.05

3h 7.47±0.07 7.56±0.13 7.58±0.11 7.54±0.07 7.44±0.10 7.56±0.18 7.59±0.10 7.58±0.03 7.42±0.16 7.54±0.16 7.61±0.06 7.37±0.08 7.40±0.16 7.68±0.14 7.40±0.05 7.33±0.06 7.59±0.10 7.56±0.04 7.57±0.06 7.59±0.02

Control 7.84±0.06 7.85±0.14 7.89±0.27 7.81±0.20 7.92±0.02 7.97±0.05 7.72±0.22 7.87±0.08 7.85±0.15 7.86±0.04 08.0±0.03 7.96±0.05 7.86±0.04 08.0±0.01 7.89±0.10 7.78±0.17 7.92±0.06 7.76±0.26 7.68±0.30 7.96±0.08

MRS+Na.T 8.70±0.17 8.49±0.17 8.78±0.21 8.55±0.12 8.65±0.22 8.84±0.13 8.14±0.17 8.54±0.06 8.44±0.17 8.72±0.21 8.61±0.15 8.65±0.15 8.32±0.11 8.46±0.11 8.72±0.12 8.32±0. 21 8.49±0.15 8.84±0.14 8.55±0.12 8.60±0.11

Decarboxylase activity MRS+Na.T+Oxgall 7.35±0.22 7.39±0.23 7.48±0.21 7.20±0.08 6.93±0.13 7.12±0.02 6.79±0.21 6.25±0.20 6.17±0.08 6.17±0.08 6.96±0.04 7.17±0.07 7.21±0.08 7.27±0.18 7.25±0.05 7.03±0.08 7.11±0.01 7.12±0.11 7.03±0.07 6.96±0.45

Lysine + + + + + + + +

Tyrosine + + + + + + + + + -

Ornithine Histidin -

Presented values are means of triplicate determinations; ±, indicates standard deviation from the mean; (+), positive activity; (-), negative activity.

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Table 4 Enzyme activities of Lactobacillus strains. Nos 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Intra cellular and extracellular enzymes Alkaline phosphatase Esterase (C4) Esterase lipase (C8) Lipase (C14) Leucine arylamidase Valine arylamidase Cystine arylamidase Trypsin α-Chymotrypsin Acid phosphatase Naphthol-AS-Biphophohydrolase α-Galactosidase β-Galactosidase β-Glucuronidase α-Glucosidase β-Glucosidase N-Acetyl-β-glucosaminidase α-Mannosidase α-Fucosidase

KCC-1 0* 10 5 0 30 5 5 0 0 0 5 5 0 10 0 10 10 0 0

2 0 5 5 0 20 5 5 5 0 0 5 10 0 20 0 10 10 0 0

3 0 0 5 5 10 20 0 0 0 0 5 10 0 20 10 10 5 0 0

4 0 0 10 10 10 0 0 0 0 0 5 5 0 20 5 5 5 0 0

5 0 5 10 10 10 10 0 0 0 0 5 5 0 20 10 10 5 0 0

6 0 0 5 5 5 0 0 0 5 10 0 30 0 10 20 10 10 0 0

7 0 10 5 0 30 10 0 5 0 5 5 5 30 0 10 5 20 0 0

8 0 0 5 5 10 0 0 0 0 0 5 5 0 20 5 5 5 0 0

Lactobacillus strains 9 11 12 13 0 0 0 0 5 0 0 0 10 5 0 5 5 5 0 0 10 20 10 10 10 20 0 5 0 0 0 0 0 0 0 5 0 0 0 0 0 0 5 5 5 5 10 10 5 10 0 5 0 20 20 30 20 40 0 0 10 10 0 0 10 10 5 10 5 5 0 0 0 0 0 0 0 0 0 0

14 0 0 0 0 10 0 0 0 0 5 10 0 0 0 10 0 0 0 0

15 0 0 0 0 10 5 5 5 0 0 5 5 0 20 0 10 10 0 0

16 0 5 5 0 20 5 5 0 0 0 5 5 0 10 0 10 10 0 0

17 0 0 0 0 30 5 5 0 0 0 5 5 0 10 0 10 10 0 0

18 0 0 5 5 10 20 0 0 0 0 5 10 0 20 10 10 5 0 0

20 0 0 0 5 10 20 0 0 0 0 10 10 0 30 10 10 5 0 0

21 0 0 0 5 10 20 0 0 0 0 10 10 0 30 10 10 5 0 0

The enzyme activities of the strains were assayed using API ZYM kit method as approximate nmol L-1 of substrate hydrolyzed during 4 h of incubation at 30 ºC. Strains without showing enzyme activity were represented as 0*; Enzyme activities were represented as 5, 10, 20, 30 and 40 nmol L-1of hydrolyzed substrates.

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22 0 5 0 0 10 5 5 0 0 0 5 10 0 20 0 10 5 0 0

Table 5

Antibiotic sensitivity pattern of Lactobacillus strains towards various antibiotics.

Antibiotic groups

Antibioti c agent

Disc potenc y

Aminogly coside

Amikacin

30

Gentamic in Kanamyc in Streptom ycin Tobramy cin Carbenici llin Ampicilli n Augment in Imipene m Ticarcilli n Ciproflox acin Gatifloxa cin Levoflox acin Moxiflox acin Nalidixic acid Norfloxa cin Ofloxaci n Sparfloxa cin Cefpodox ime Cetriaxon e Colistin

10

CoTrimoxaz ole

25

Corboxyp enicillin Lactomas e

Fluroquin olone

Cephalos porin

Polymixin Sulphona mide

30 10 10 50 50 30 10 75 5 5 5 5 30 5 10 5 10 30 10

KC C-1

KC C-2

KC C-3

KC C-4

KC C-5

KC C-6

KC C-7

KC C-8

KC C-9

KC C11

22.6 ±0.5 16.6 ±0.5 16.0 ±1.0 11.3 ±0.5 24.0 ±1.0 26.3 ±1.1 30.0 ±1.0 25.8 ±1.0 30.6 ±1.1 30.6 ±0.5 17.3 ±0.5 15.5 ±0.7 20.6 ±1.1 20.6 ±0.5 21.3 ±0.5 25.3 ±1.1 26.6 ±1.1 27.6 ±0.5 21.0 ±1.4 25.0 ±1.0 20.3 ±1.5 19.0 ±1.0

20.0 ±1.0 14.6 ±0.5 17.6 ±2.0 10.3 ±1.5 24.6 ±2.5 16.6 ±1.5 31.6 ±1.5 29.0 ±1.0 19.0 ±1.7 17.0 ±1.0 12.6 ±2.0 19.3 ±1.5 25.3 ±0.5 18.3 ±1.5 14.6 ±0.5 26.0 ±1.7 26.0 ±1.7 21.0 ±2.6 21.3 ±1.1 23.6 ±1.5 22.3 ±2.5 16.6 ±1.5

20.3 ±1.5 11.0 ±1.0 13.6 ±3.2 13.6 ±1.5 25.0 ±3.0 19.0 ±1.0 24.6 ±0.5 24.6 ±0.5 21.0 ±1.0 18.6 ±1.1 15.0 ±1.0 18.3 ±1.5 21.3 ±0.5 20.3 ±0.5 18.0 ± 2.6 16.3 ±1.5 20.3 ±1.5 20.6 ±0.5 21.0 ±0.0 21.3 ±0.5 20.0 ±1.0 18.3 ±0.5

21.3 ±1.5 20.0 ±1.0 16.0 ±2.6 12.6 ±2.0 20.0 ±1.0 10.3 ±0.5 14.0 ±0.7 12.6 ±2.5 22.0 ±2.6 27.0 ±1.7 20.0 ±1.0 16.3 ±1.5 20.0 ±1.0 21.6 ±0.5 21.6 ±0.5 24.0 ±1.7 24.3 ±2.0 26.3 ±1.1 19.3 ±0.5 15.6 ±5.0 20.0 ±1.0 21.0 ±1.0

17.3 ±0.5 16.0 ±1.7 20.6 ±0.5 20.3 ±0.5 25.3 ±0.5 29.0 ±3.4 27.3 ±1.1 29.0 ±1.0 23.3 ±2.8 13.3 ±2.8 12.6 ±0.5 19.3 ±1.1 23.3 ±1.1 21.0 ±1.7 20.3 ±0.5 28.6 ±0.5 29.0 ±1.7 24.0 ±1.7 19.3 ±1.1 20.0 ±1.7 22.6 ±2.3 22.0 ±1.7

20.6 ±0.5 17.6 ±0.5 20.6 ±0.5 14.3 ±6.6 22.3 ±2.3 16.3 ±2.3 31.3 ±1.1 27.6 ±5.7 25.3 ±0.5 21.0 ±1.0 19.6 ±7.2 22.6 ±1.1 25.3 ±0.5 19.0 ±1.0 16.0 ±1.0 25.6 ±0.5 27.3 ±0.5 25.3 ±0.5 21.3 ±0.5 21.0 ±1.0 25.3 ±0.5 21.0 ±1.0

22.3 ±1.1 16.0 ±1.7 16.6 ±0.5 11.0 ±1.0 23.0 ±1.0 26.0 ±1.0 30.6 ±0.5 25.8 ±1.0 27.3 ±6.4 30.6 ±0.5 17.3 ±0.5 16.6 ±1.1 21.3 ±1.1 20.3 ±1.1 20.0 ±1.7 25.0 ±1.0 26.3 ±1.5 26.3 ±1.1 19.6 ±0.5 24.0 ±2.0 20.6 ±1.1 20.0 ±1.0

21.0 ±1.1 18.6 ±0.5 21.3 ±1.1 10.3 ±0.5 20.3 ±0.5 13.0 ±1.7 26.6 ±2.8 27.0 ±3.4 24.6 ±0.5 21.6 ±0.5 17.0 ±1.7 24.0 ±1.7 27.3 ±1.1 21.0 ±1.7 19.0 ±1.7 27.6 ±2.3 22.3 ±4.0 25.3 ±0.5 21.6 ±0.5 23.6 ±2.3 25.3 ±0.5 23.0 ±1.7

19.3 ±1.1 13.3 ±1.5 20.0 ±1.7 18.6 ±1.1 27.6 ±1.5 27.0 ±1.7 27.6 ±0.5 29.6 ±0.5 27.0 ±1.7 17.0 ±1.7 14.3 ±1.1 21.3 ±1.1 22.6 ±1.1 20.6 ±0.5 20.6 ±0.5 27.6 ±1.1 30.6 ±0.5 25.6 ±0.5 21.3 ±1.1 19.6 ±0.5 22.6 ±1.1 22.3 ±0.5

21.0 ±1.0 21.3 ±1.1 24.6 ±0.5 12.0 ±1.7 13.6 ±1.1 25.6 ±0.5 24.3 ±1.1 29.6 ±0.5 29.0 ±2.6 31.3 ±1.1 24.3 ±2.0 26.0 ±1.0 26.0 ±1.0 24.0 ±1.7 21.6 ±0.5 22.3 ±1.1 24.0 ±1.7 23.0 ±2.6 17.3 ±2.0 20.0 ±1.0 22.6 ±0.5 24.0 ±1.7

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Table 5

(to be continued)

Antibiotic groups

Antibioti c agent

Disc potenc y

Aminogly coside

Amikacin

30

Gentamic in Kanamyc in Streptom ycin Tobramy cin Carbenici llin Ampicilli n Augment in Imipene m Ticarcilli n Ciproflox acin Gatifloxa cin Levoflox acin Moxiflox acin Nalidixic acid Norfloxa cin Ofloxaci n Sparfloxa cin Cefpodox ime Cetriaxon e Colistin

10

Corboxyp enicillin Lactomas e

Fluroquin olone

Cephalos porin

Polymixin Sulphona mide

CoTrimoxaz ole

30 10 10 50 50 30 10 75 5 5 5 5 30 5 10 5 10 30 10 25

KC C-12

KC C-13

KC C-14

KC C-15

KC C-16

KC C-17

KC C-18

KC C-20

KC C-21

KC C-22

25.7 ±0.5 23.0 ±1.7 31.3 ±2.0 34.7 ±0.5 21.3 ±1.1 35.0 ±0.0 19.0 ±1.0 20.7 ±1.1 29.3 ±1.1 24.3 ±0.5 31.0 ±1.0 32.7 ±0.5 31.7 ±0.5 30.7 ±0.5 30.0 ±1.0 35.7 ±1.1 35.0 ±1.0 39.0 ±1.0 33.0 ±1.7 31.0 ±1.0 33.0 ±1.7 33.0 ±1.0

26.0 ±1.7 21.3 ±1.1 21.3 ±1.1 19.3 ±1.1 26.3 ±1.1 20.3 ±1.1 31.3 ±1.1 34.0 ±1.7 35.7 ±0.5 27.0 ±1.7 21.3 ±1.1 24.7 ±0.5 19.7 ±0.5 23.0 ±1.7 23.0 ±2.6 24.0 ±1.7 31.3 ±1.1 34.0 ±1.7 34.0 ±1.7 33.7 ±1.5 29.3 ±1.1 27.0 ±1.7

29.6 ±1.5 19.6 ±1.5 23.0 ±1.7 22.3 ±2.5 16.0 ±1.7 21.3 ±1.1 14.0 ±1.7 17.0 ±1.7 26.0 ±1.7 30.6 ±1.1 30.3 ±0.5 31.3 ±1.1 34.0 ±1.7 37.0 ±1.7 27.6 ±2.3 25.6 ±0.5 27.3 ±1.1 20.0 ±1.0 24.0 ±1.7 23.0 ±1.7 24.0 ±1.7 27.0 ±1.7

23.3 ±1.5 20.6 ±1.5 24.3 ±2.8 11.3 ±2.3 22.3 ±2.5 17.3 ±6.8 21.6 ±2.8 22.0 ±2.6 23.6 ±1.5 21.3 ±1.1 19.3 ±1.1 25.3 ±0.5 26.0 ±1.7 21.0 ±1.0 21.3 ±1.1 26.3 ±2.3 23.6 ±3.2 22.3 ±2.5 20.6 ±1.1 21.6 ±2.8 21.6 ±2.8 21.6 ±2.0

24.3 ±1.1 17.6 ±0.5 16.6 ±1.5 13.3 ±2.0 21.0 ±1.7 23.0 ±1.7 33.6 ±2.3 29.0 ±1.7 30.6 ±1.1 30.3 ±0.5 19.3 ±1.1 18.6 ±2.3 21.3 ±1.1 21.6 ±0.5 23.6 ±2.3 24.6 ±0.5 28.6 ±1.1 28.6 ±1.1 27.0 ±1.7 25.3 ±1.5 20.6 ±1.1 21.3 ±1.1

22.3 ±1.1 19.0 ±1.7 21.6 ±0.5 13.3 ±2.8 23.0 ±1.7 17.0 ±1.7 34.0 ±1.7 33.6 ±2.3 27.6 ±2.3 30.0 ±1.0 18.6 ±2.3 20.6 ±1.1 28.3 ±2.8 19.3 ±1.1 17.3 ±1.1 28.0 ±1.7 29.0 ±1.7 27.6 ±2.3 23.6 ±2.3 21.3 ±1.1 25.6 ±0.5 21.3 ±1.1

21.0 ±1.0 14.3 ±1.2 23.3 ±2.1 19.3 ±1.2 29.3 ±1.2 29.3 ±1.2 27.6 ±0.6 31.3 ±1.2 29.3 ±1.2 20.0 ±2.0 16.3 ±1.5 21.0 ±1.0 21.3 ±1.2 22.3 ±1.5 23.6 ±2.3 27.6 ±0.6 33.0 ±1.7 28.6 ±2.3 21.3 ±1.2 21.3 ±1.2 23.3 ±1.5 24.0 ±1.7

19.0 ±1.0 15.3 ±0.5 20.3 ±0.5 10.0 ±1.0 21.3 ±1.1 18.3 ±1.5 34.0 ±1.0 31.0 ±1.0 21.6 ±0.5 19.3 ±1.1 15.6 ±0.5 20.3 ±0.5 25.3 ±0.5 17.6 ±0.5 17.0 ±1.7 24.3 ±1.1 29.3 ±1.1 24.6 ±0.5 24.0 ±1.7 24.0 ±1.7 26.3 ±1.1 19.0 ±1.7

21.6 ±1.5 11.3 ±1.5 16.3 ±1.5 15.3 ±1.1 20.6 ±1.1 19.3 ±1.1 24.6 ±0.5 24.6 ±0.5 22.3 ±2.5 19.3 ±1.1 14.6 ±0.5 16.3 ±1.5 21.6 ±0.5 21.3 ±1.1 20.6 ±1.1 17.3 ±1.1 20.6 ±1.1 21.3 ±0.5 21.0 ±1.0 21.6 ±0.5 19.6 ±0.5 20.0 ±2.0

27.3 ±1.1 27.0 ±1.7 34.0 ±1.7 36.0 ±1.7 24.0 ±1.7 33.0 ±1.7 21.0 ±0.7 23.3 ±2.8 33.3 ±2.8 24.6 ±0.5 31.6 ±0.5 33.0 ±0.0 34.0 ±1.7 33.6 ±2.3 33.3 ±2.8 35.0 ±0.0 36.0 ±0.0 40.6 ±1.5 34.0 ±1.7 33.6 ±2.3 34.0 ±1.7 34.0 ±1.7

Zone of inhibition was expressed as Mean ± SEM of three replicates (p< 0.05 level in mm) after incubating the strains at 30 ºC for 48 h in MRS Agar medium (MRSA).

Table 6 Antifungal activity of Lactobacillus strains against food pathogens.

Lactobacillus strains Fungal Strains Aspergillus clavatus Aspergillus fumigates Aspergillus niger Aspergillus oryzae Curvularia lunata Fusarium oxysporum Gibberella moniliformis Humicola grisea Penicillium chrysogenum Penicillium roqueforti Candida albicans

KCC-1 2

3

4

5

6

7

8

++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + +++ +++ +++ +++ +++ +++ +++ +++ − − − − − − − − − − − − − − − − +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + +++ +++ +++ +++ +++ +++ +++ +++ − − − − − − − − ++ ++ ++ ++ ++ ++ ++ ++

9

11

12

13

14

15

16

17

18

20

21

22

++ + +++ − − +++ ++ + +++ − ++

++ + +++ − − +++ +++ + +++ − ++

− − − − − − − − − − −

− − − − − − − − − − −

− − − − − − − − − − −

− − − − − − − − − − −

− + +++ − − − − + − − +

− − − + + + +++ +++ +++ − − − − − − − − − − − − + + + ++ ++ ++ − − − + + +

++ + +++ − − − − + ++ − ++

− − − − − − − − − − −

−, no visible inhibition; +, weak suppression around the streaks (0.1 to 3.0 % of the Petri dish); ++, good suppression; with detectable clear zones around the streaks (>3.0 to 8.0 % of the Petri dish); +++, strong suppression with large clear zones around the streaks (>8.0 % of the Petri dish).

KCC-9; J-KCC-11; K-KCC-12; L-KCC-13; M-KCC-14; N-CC-15; O-CC-16; P-CC-17; QCC-18; and R- KCC-20.

Fig. 1 .

Fig 2a.

CC-17; Q-CC-18; and R, KCC-20.

Fig 2b.