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Identification of lactic acid bacteria from Meliponine honey and their antimicrobial activity against pathogenic bacteria.

INTRODUCTION

Honey is naturally produced by honeybees which are consists of a very concentrated solution of a complex mixture of sugars mainly fructose and glucose (Aljadi and Kamaruddin, 2004). Honey is one of the oldest medicines known which used to treat wounds, ulcers, sunburn, and infections of the eyes, throat and gut. Honey has been increasingly recognized as alternative medicine. The composition and physicochemical properties of honey are variable depending on its floral source (Chen et al., 2000). Honey is often named according to the geographical location where it produced, either floral source or the trees on which the hives are found (Lusby et al, 2002).

There is a growing interest to study lactic acid bacteria (LAB) from honey as it is generally recognized as safe (GRAS) and play an important role in food fermentation and preservation (Yang et al., 2012). LAB belongs to a group of Gram-positive bacteria that produce lactic acid as their main fermentation product into the culture medium. LABs are important for preservation of the nutritional qualities of the raw materials through extended shelf life, enhance flavoring foods and the inhibition of spoilage and pathogenic bacteria on food products (Suhartatik et al., 2014).

Recently, several strains of LAB mainly from Lactobacillus sp. isolated from honey and demonstrated antimicrobial activity against both Gram negative and positive pathogenic and spoilage bacteria (Aween et al., 2010; Ibarguren et al., 2010; Lee et al., 2008, Klaenhammer, 1993). Most of the antimicrobial activity of honey is due to metabolites produced by LAB, such as organics acid and bacteriocins (Aween et al., 2012). However, there was limited study on antimicrobial activity of Meliponine honey, especially in Malaysia.

2. Objectives:

Therefore, the purpose of this study is to isolate LAB from Meliponine honey and identify the isolates using phenotypic and molecular identification methods. Besides, the antimicrobial activity of these isolates was determined against various pathogenic bacteria using agar-well diffusion method.

3. Methods:

3.1. Honey samples:

Meliponine honey from Heterotrigona itama were collected from various local honey beekeeper in Kelantan and Terengganu, located at the East Coast of Peninsular Malaysia. The samples were kept in the sterile container at room temperature (28[degrees]C) before analysis.

3.2. Isolation of lactic acid bacteria from honey:

The isolation of LAB from Meliponine honey followed the methodology described by Aween et al. (2012). Approximately 10 g of honey samples were added to 90 ml of de Man, Rogosa and Sharpe (MRS) broth (Merck, Germany) and incubated at 30[degrees]C for 24 h to 48 hin anaerobic condition using C[O.sub.2] incubator. After incubation, the LAB Count in the sample was determined using appropriate serial dilutions with 0.85% saline. Then, 0.1 ml of the dilution was spread plated on several selective media namely, de Man, Rogosa and Sharpe (MRS) agar, MRS agar with 0.8% CaC[O.sub.3], MRS agar + 1% glucose and M17 agar (Merck, Germany). The dilution factor varied among the samples in a range of [10.sup.-2] to [10.sup.-6] dilutions. All plates were incubated under anaerobic condition using C[O.sub.2] incubator for 48 hours. The presumptive of LAB colonies were tested for Gram staining, catalase test and oxidase test. Cultures with Gram positive bacteria and negative for catalase were maintained in slanted agar and 20% glycerol stock at 4[degrees]C and -20[degrees]C

3.3. Gram stain test:

Morphology of the pure cultures of LAB isolates were discerned using gram staining method. Single colony of LAB from agar plates was mounted and then fixed onto the glass slides with one drop of distilled water. After drying, the slides were stained by flooding in crystal violet before leaving at room temperature for 60 s. The slides were washed with distilled water and flooded again by gram iodine for 60 s before the second distilled water washing. Ethanol and distilled water were used to decolorize slides. The final step involved using anti-stain, safranin for 45 s. Before microscopic examination, the slides were washed with distilled water and dried at room temperature. The prepared slides were monitored by light microscope under 1000X magnification using oil immersion. Blue-purple colour represents Gram-positive temperament of Gram-positive LABs.

3.4. Catalase test:

Pure culture of each LAB was place on a clean microscope slide. One drop of 3% Hydrogen peroxide ([H.sub.2][O.sub.2]) was put into LAB colony. The gas production (bubbling) indicated positive catalase test. However, LAB was known as catalase negative.

3.5. Oxidase test:

By using sterile stick of cotton swab, the LAB colonies on the MRS plate agar were scraped and were rubbed on the filter paper. Two to three drops of oxidase reagent were placed on a filter paper. The Gram negative bacteria changed to purple colour while no changes in colour for Gram positive bacteria

3.6. Phenotypic characterization of LAB by API 50 CHL system:

Overnight cultures of the LAB isolates were grown on MRS plates (Merck) at 37[degrees]C for 24 h anaerobically. The pure colonies were suspended in API 50 CH medium (API system, BioMerieux, France). The suspension was transferred into each of the 50 wells of the API 50 CH strips. All wells were overlaid with sterile mineral oil to make it anaerobic. Strips were incubated at 37[degrees]C as recommended by the manufacturer. Changes in color of wells were noticed after 24 and 48 h. The results were analyzed with API Web (BioMerieux).

3.7. Genomic DNA extraction:

Genomic DNA was extracted according to the manufacturer recommended protocol with some modifications as described by Ward et al. (1994). The purity of DNA was determined by using a spectrophotometer and the ratio of the readings at 260 and 280 nm (A260/A280). Amplification of a 518bp fragment of 16S rRNA gene was carried out by using PCR with primers based on the conserved region of 16S rRNA gene27F (5'-AGAGTTTGATCCTGGCTCAG-3') and a reverse primer, 1492R (5' GGTTACCTTGTTACGACTT-3') (Lane, 1991). Polymerase chain reactions (PCR) was performed in 20[micro]l reaction volumes, containing 10 [micro]L1 x Taq Master Mix, 0.20 [micro]L forward primer, 0.20[micro]L reverse primer, 3.5 [micro]L of genomic DNA and 6.1 [micro]L sterile distilled water. The PCR protocols was as follows: denaturation at 95[degrees]C for 2 min, followed by 35 cycles of denaturation at 95[degrees]C for 30 s, annealing at 59[degrees]C for 55 s, elongation at 72[degrees]C for 1 min, and a final extension at 72[degrees]C for 5min. Amplification products were analyzed by electrophoresis (75V, 40 min) on a 1.0% agarose gel containing GelRed dye (Biothium, USA) and visualized by UV fluorescence. Amplified products were purified using the QIAquick PCR purification kit according to the manufacturer's protocol (Qiagen, Germany). Nucleotide sequence determination was done using the ABI 3730XL capillary DNA Analyzer machine and the ABI prism Bigdye terminator cycle sequencing Ready reaction kit ver. 3.1 sequencing program (Applied Biosystems, USA) provided by 1st Base Sdn. Bhd. The DNA sequencing results from each primer were aligned using the BioEdit Sequence Aligment Editor software (Hall, 1999) and the corresponding primers were located using the same programme. The aligned nucleotide sequence was then subjected to the Nucleotide-nucleotide Basic Local Alignment Search Tool (BLASTN) software version 2.2.14 for comparison with other isolates available in the GenBank Database of the National Center for Biotechnology Information (NCBI) for confirmation.

3.8. Antimicrobial activity by agar-well diffusion method:

The antimicrobial activities of identified LAB were determined by using agar well diffusion assay (Liasi, 2009) with Mueller Hinton Agar (Merck, Germany) as medium, all pathogenic bacteria with concentration of 0.5 McFarland (>3.0 x [10.sup.8] CFU/ml) were swabbed on agar. The pathogenic bacteria tested were Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa and Listeria monocytogenes. Then, 6mm of wells were made by using sterile cork borer. 80 pl of LAB broth were seeded into the well and the plates were incubated at 37[degrees]C for 24 h. After 24 h, diameters of the growth inhibition zones were measured (Modified from Liasi, 2009).

RESULT AND DISCUSSIONS

4.1. Isolation and biochemical test of lactic acid bacteria:

A total of 29 isolates were obtained from the Meliponine honey samples. For the pre-identification test, the isolates were subjected to catalase test, oxidase test and Gram staining as shown in Table 1. From the result obtained, only five isolates were catalase negative and oxidase negative. The isolates were Gram positive rod organisms. As the probability to obtain LAB was small (17% probability), it is due to the nature of honey that contains high sugar concentration that limits the detection of LAB (Aween et al., 2012). Some successful of LAB isolates in raw honey were reported by Bahiru et al., 2006; Forsgren et al., 2009; and Hosny et al., 2009. Additionally, many studies reported the isolation of LAB from stomach of bee, flowers and plants (Lubsy, 2012) suggesting that LAB which present in honey may come from plant sources and the bees.

4.2. Phenotypic identification of LAB isolates using API 50CHL Kit:

Five isolated LAB were identified using API 50 CHL as summarized in Table 2. LAB001-B, LAB001-C, LAB001-D and LAB001-E showed high percentage of similarity (97.2%) to Lactobacillus brevis 3, whereas LAB005-F showed very high percentage of similarity (99.9%) to Leuconostoc mesenteroides ssp. These results will be validated with 16S rRNA sequences.

4.3. Identification based on 16S rRNA gene sequences:

This study confirmed that five isolates from Meliponine honey were belongs to Lactobacillus sp. as shown in Table 3. The results showed very high similarity percentage from 94% to 97% based on the database sequence in NBCI. The presence of viable Lactobacilli in the most popular Malaysian stingless honey bee, Heterotrigona itama has showed that lactic acid bacteria (LAB) plays important role as beneficial microorganisms present in Meliponine honey. In previous study by Aween et al, 2012, Lactobacillus acidophilus 1 was the major microorganism isolated from honey. The differences of the result is due to the different types of honey studied, where Aween et al., 2012 studied honey from sting-bee whereas this study used the stingless bee honey.

Lactobacillus classified as an important category in LAB which prevalently found as commensal bacteria and is utilized as a prebiotic for humans and animals (Tajabadi et al., 2013). Ramos et al. (2013) reported that Lactobacillus brevis exhibited potential probiotics properties which mainly found in food. Ho et al. (2009) has reported that the combination of Lactobacillus brevis and Lactobacillus pentosaceus as starter culture conferred the best sensory quality to the fermented products. Further study will lead to the potential of Meliponine honey to be used as probiotic activities and natural food preservatives for food industry.

On the other hand, the result of identification using API systems and molecular identification showed some contradictory where LAB005-F in API 50 CHL was identified as Leuconostoc mesenteroides ssp while in NCBI database, LAB005-F was classified as Lactobacillus brevis. According to Suhartatik et al. (2014) and Riebel and Washington (1990), molecular identification was more valid than phenotypic test.

4.3. Antibacterial activity of isolated LAB against pathogenic bacteria by well diffusion method:

The identified strains were further tested for their antimicrobial activity against pathogenic bacteria as summarized in Table 4. All these pathogenic bacteria were reference culture from ATCC and clinical isolates. All strains exhibited a good inhibitory activity against S. epidermidis, P. aeruginosa and L. monocytogenes as shown in Figure 1. It is believed that the antimicrobial activity produced by metabolites of LAB which contained variety inhibitory substances such as bacteriocins and mainly due to organic acid activity (Lawalata et al., 2010). From the result obtained, LAB005-F exhibit greater inhibition against S. epidermidis, B. subtilis, P. aeruginosa and L. monocytogenes compared to other isolates. Among the pathogens, all LAB isolates show the highest inhibition zone value against S. epidermidis was range from 25 to 32 mm in diameter

5. Conclusion:

The result of this study exhibited that Lactobacilli in the Kelulut honey were dominated with the strain of Lactobacillus brevis. The results obtained in antibacterial activity revealed L. brevis exhibited a good antibacterial activity against S. epidermidis, P. aeruginosa and L. monocytogenes. To our knowledge, this is the first reports on the isolation of LAB in Meliponine honey of Heterotrigona itama. This study highlights the presence of dominant Lactobacilli bacteria in the Meliponine honey as major contributor to its antibacterial component against pathogenic bacteria. Further study will explore the potential probiotics of the identified strains for the application in food industry.

[FIGURE 1 OMITTED]

ACKNOWLEDGMENT

The work is supported by The Ministry of Education, Malaysia, through the FRGS research project Vot No. 59329..

REFERENCES

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(1) Nor Hazwani Mohd Hasali, (1) Amir Izzwan Zamri, (1) Mohd Nizam Lani, (1) Aidilla Mubarak and (2,3) Zarizal Suhaili

(1) School of Food Science and Technology, University Malaysia Terengganu, 21030, Kuala Terengganu

(2) Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Tembila Campus, 22200 Besut, Terengganu

(3) East coast Environmental Research Institute, Universiti Sultan Zainal Abidin, Gong Badak Campus, 21030 Kuala Terengganu.

Received 25 July 2015; accepted 1 September 2015; Available online 19 September 2015

Address For Correspondence:

Nor Hazwani Mohd Hasali, School of Food Science and Technology, University Malaysia Terengganu, 21030, Kuala Terengganu.

E-mail: ian.wanihasali@gmail.com
Table 1: Result of biochemical test for each LAB colony.

Samples    Catalase Test   Oxidase Test   Gram staining

LAB001-B     Negative        Negative     Gram positive
LAB002-C     Negative        Negative     Gram positive
LAB003-D     Negative        Negative     Gram positive
LAB004-E     Negative        Negative     Gram positive
LAB005-F     Negative        Negative     Gram positive

Table 2: Phenotypic identification of isolates using
API 50 CHL Kits and API web.

Samples    Similarity (%)         Identification ID

LAB001-B       97.2%            Lactobacillus brevis 3
LAB002-C       97.2%            Lactobacillus brevis 3
LAB003-D       97.2%            Lactobacillus brevis 3
LAB004-E       97.2%            Lactobacillus brevis 3
LAB005-F       99.9%        Leuconostoc mesenteroides ssp

Table 3: Nucleotides sequence alignment analysis of 16S rRNA
gene of isolates.

Samples     Most closely related type     Similarity%    Accession
                     strain              of similarity    numbers

LAB001-B   Lactobacillus brevis strain       (97%)       HM130535.1
                     lbr-42
LAB002-C   Lactobacillus brevis strain       (94%)       KC755092.1
                      37901
LAB003-D     Lactobacillus sp safa1          (96%)       KM819111.1
LAB004-E   Lactobacillus brevis strain       (97%)       KJ914900.1
                      NJ42
LAB005-F   Lactobacillus brevis strain       (96%)       EU620612.1
                      KLDS

Table 4: Antibacterial activity of isolated LAB against
pathogenic bacteria by well diffusion method *.

Pathogens                     LAB     LAB     LAB     LAB     LAB
                             001-B   002-C   003-D   004-E   005-F

Staphylococcus epidermidis    25      26      28      32      32
Bacillus subtilis              5       5       5      12      19
Pseudomonas aeruginosa        13      14      15      16      15
Escherichia coli               0       0       0       5       5
Salmonella Typhimurium         5       5       5       5       5
Staphylococcus aureus          0       0       0       5       5
Listeria monocytogenes        14      14      16      18      24

* Diameter of inhibition zone around the well (mm).
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Author:Hasali, Nor Hazwani Mohd; Zamri, Amir Izzwan; Lani, Mohd Nizam; Mubarak, Aidilla; Suhaili, Zarizal
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Date:Sep 19, 2015
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