molecular characteristics of indigenous … · shirota strain (yakult), as from human intestinal,...

International Journal of Probiotics and Prebiotics Vol. 10, No. 4, pp. 109-116, 2015ISSN 1555-1431 print, Copyright © 2015 by New Century Health Publishers, LLC

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MOLECULAR CHARACTERISTICS OF INDIGENOUS PROBIOTIC STRAINS FROM INDONESIA

1Endang S. Rahayu, 2Agung Yogeswara, 1Mariyatun, 1Linda Windiarti, 1Tyas Utami, and 3Koichi Watanabe

1Faculty of Agricultural Technology, Universitas Gadjah Mada, Flora Street No 1 Bulaksumur, Yogyakarta 55281, Indonesia; 2Nutritional Science Department, Universitas Dhyana Pura, Jalan Raya Padang Luwih Tegaljaya Dalung Kuta Utara, Bali 80361, Indonesia; and 3Department of Animal Science and Technology, National Taiwan University, No. 50,

Lane 155, Sec. 3., Keelung Rd., Taipei, Taiwan R.O.C.

[Received February 16, 2015; Accepted September 10, 2015]

[Communicated by Prof. Francesco Marotta]

ABSTRACT: The aims of this study were to screen indigenous probiotic strains of the Indonesian habitat and to identify them based on molecular characteristics. Screening for probiotic candidates were mainly based on the resistance of simulated gastric juice, bile salt, and antimicrobial activities against pathogens. Molecular techniques used for identification in this study were sequence of 16S rRNA, RAPD genetic profile using multiplex primer, RecA gene profile for Lactobacillus plantarum-pentosus group and pepR gene profile for Lactobacillus casei group. Results from this study shown that RAPD genetic profile could be used to distinguish among strain-level belong to Lactobacillus plantarum. RecA gene profile was able to differentiate between Lactobacillus plantarum and Lactobacillus pentosus used in this study, while pepR gene profile could be used to differentiate subspesies belong to L. casei group. Based on the molecular characteristics in this study, four strains isolated from fermented foods (Dad-13, T-3, Mut-7, and Mut-13) were clearly identified as Lactobacillus plantarum, while SNP-2 isolated from fecal of a healthy infant was identified as Lactobacillus paracasei. Based on the screening criteria, all indigenous strains from Indonesia in this study were potential as probiotic candidates.

KEYWORDS: Indigenous; Indonesia; Molecular characteristics; Probiotic

Corresponding Author: Endang S. Rahayu, Faculty of Agricultural Technology, Universitas Gadjah Mada, Flora Street No 1, Bulaksumur, Yogyakarta 55281, Indonesia; Fax. (+6274) 549650; Email: [email protected]

Abbreviations Used: PCR, polymerized chain reaction; RAPD, Random Amplified Polymorphic DNA.

INTRODUCTIONProbiotics are defined as living microorganisms when

administered in adequate amount as part of food confer a health benefit on the host (FAO/WHO, 2001; FAO/WHO, 2002). The health benefits are primarily come from the ability of probiotics in supporting the balance of microbiota in intestine. O’Sullivan et al., (2001) states that in order to effectively used as probiotic, the strains should be of human origin, since these strain are considered to have ability to adapt the complex conditions of gut intestinal tract. Some Bifidobacterium and Lactobacillus species is the natural inhabitant of human digestive tract. However, probiotic strains also frequently isolated from fermented foods, so they can be easily used as starter cultures. The selection of probiotic strain from fermented food that has been consumed for many years in hereditary also expected to be safe as probiotics.

There are several commercial probiotics with health benefits documented from fermented food, such as Lactobacillus casei Shirota strain (Yakult), as from human intestinal, such as Lactobacillus acidophilus NCFM (Danisco), Lactobacillus casei CRL431 (Chr. Hansen), Lactobacillus paracasei F19 (Cultura Aria Food), Lactobacillus rhamnosus ATCC 53013 (LGG) (Valio). Probiotic candidates should meet various criteria, and the main criterion is resistance to bile salt and gastric juice so that the strain is able to survive and grow in the intestine.

Nakayama et al., (2015) reported that fecal bacterial composition among school-age children in 10 cities of 5 countries in Asia has been studied to address the diversity of their gut microbiota. The microbiota profiled for the 303 subjects were classified into enterotype-like cluster, each driven by Prevotella (P-type) or Bifidobacterium/bacteriodes (BB-type). Majority, in Indonesian and Thailand (Khon Kaen) subjects harbored P-type, while in China, Japan and Taiwan harbored BB-type. The differences in fecal microbiota diversity among

110 Indigenous Probiotic Strains from Indonesia

young Asian is strongly supported by their dietary habits. Indonesian school-age children had P-type microbiota and less population of Bifodobacterium compared to those China, Japan, and Taiwan, which reflecting their high intake of diets rich in resistant starch. Therefore, we suggest that development probiotic based on indigenous strains is very challenging to match the gut microbiota of each region.

Lactic bacteria have been isolated from Indonesian fermented foods (Rahayu, 2003) and fecal material of healthy infant (Purwandhani and Rahayu, 2003; Rahayu and Purwandhani, 2004), and some of them have been screened for their potential for probiotic agent. Screening for probiotic agents were mainly based on resistancy to simulated gastric juice and bile salts. Since probiotics should passed the stomach which gastric acid is the great barrier for the bacteria, and if they survived then they reached to the duodenum where they encounter bile acids. The other important criteria for screening is their antagonisms activity against pathogen. However in the previous studies, identification were mainly based on phenotypic characteristics, which authors could not clearly distinguish between L. plantarum and L. pentosus. According to FAO/WHO (2002) speciation of the probiotic bacteria must be established using the most current, valid methodology and it is recommended using combination of phenotypic and genotypic tests. To meet the requirement for characterization of probiotic candidates, identification based on current method is a must. The aims of this study were to confirm our indigenous probiotic strains isolated from the Indonesian habitat (fermented foods and fecal material of healthy infant) and to identify these strains based on molecular characteristics.

MATERIALS AND METHODS

Bacterial strains usedFive strains from our collection and previously were

identified as Lactobacillus plantarum-pentosus Mut-7 & Mut-13 (isolated from gatot, fermented dried cassava), Lactobacillus plantarum T-3 (growol, fermented raw cassava), Lactobacillus plantarum Dad-13 (dadih, fermented milk), and Lactobacillus acidophilus SNP-2 (fecal material of healthy infant) were used in this study. Pediococcus acidilactici F-11 (bacteriocin produces strain, also from our collection) and commercial Lacto B was used as controls for antibacterial test against pathogens. Pathogenic strains used in this study were Aeromonas hydrophilla dkt-5, Salmonella typhi dky-3 (901), Shigella dysenteriae dky-4, E. coli OK111, and E. coli ST. All strains used in this study were preserved in ampoules and deposited at Food and Nutrition Culture Collection (FNCC), Center for Food and Nutrition Studies, Universitas Gadjah Mada, Yogyakarta.

Preparation of bacterial culturesAll lactic acid bacteria strains were grown in MRS (de

Man, Rogosa and Sharpe) media and pathogenic strains in

NA (Nutrient Agar) media at 30°C for 24 h before used.Simulated gastric juice resistance

Determination of resistance to simulated gastric juice of the strains was developed using method reported by Lian et al., (2003) and Shahidi et al., (2008). Solutions of simulated gastric juice used in this study contained 5% saline, 3 g/l pepsine at increasing pH values (1.5, 2.0 and 3.0 adjusted with HCl 5M). A total volume of 1 ml of 24 h culture of lactic acid bacteria grown in MRS broth was inoculated into 10 ml simulated gastric juice (SGJ), then incubated at 37oC for 0, 4, and 6 hours, which corresponds to the time of food transit in the stomach which about 2‒6 hours. Viable cells were enumerated using dilution and plating method in MRS agar media.

Bile salts resistance Determination of resistance to bile salts of the strains was

developed using method reported by Lian et al., (2003) and Shahidi et al., (2008). Concentrations of bile salt used to screen the probiotic candidate in this study were 0.5, 2 and 3% due to its equivalent with physiological concentrations of bile salt in duodenum. A total volume of 1 ml of 24 h culture of lactic acid bacteria grown in MRS broth was inoculated into 10 ml of sterile distilled water with 0.5, 2.0 and 3.0% of bile salt respectively, then incubated at 37oC for 0, 4 and 6 hours. Viable cells were enumerated using dilution and plating method in MRS agar media.

Antibacterial activity using in vitro testAntibacterial activity was test based on the ability to inhibit

the growth of pathogens using well diffusion methods as described by Papamanoli et al., (2003). Lactic acid bacteria culture to be tested was grown 24 hours in MRS agar, as for pathogens in nutrient broth (24 hours).

Pathogen culture (0.1 ml) was inoculated on Petridish then pour plate with melting NA media (warm temperature). This Petridish were put in cool room for 1‒1.5 hours, and then plug the media for making well (7 wells for each Petridish). For antibacterial activity test, 50 µl of 24 hours lactic acid bacterial cultures were dropped in every well and put Petridish in cool room for 3‒4 hours, for the diffusion of metabolits of cultures. Petridish was then incubated at 37oC for 24 hours. The ability to inhibit the growth of pathogens showed by clear zone surrounding the well. In this study, the antibacterial activities of LAB strains were determined by diameter of clear zones minus the diameter of well in cm.

Molecular analysis based on PCR and 16S rRNA gene sequence

RAPD (Random Amplified Polymorphic DNA) analysis used to differentiate strain level on bacteria through genetic profiles produced by randomly amplification. In this study, 3 primers were used to show the genomic profiles between strains (Table 1).

For genus-species confirmation, general molecular

Indigenous Probiotic Strains from Indonesia 111

identification based on 16S rRNA gene was done using method described by Watanabe et al. (2008). The process included DNA isolation, amplification with 3 primer pairs, and sequencing the amplicon obtained. Sequences obtained then compared with available sequences in NCBI (National Center for Biotechnology Information) via online using BLAST (The Basic Local Alignment Search Tool).

Multiplex primers were used to distinguish between L. plantarum group and L. casei group. RecA gene used to differentiate L. plantarum and L. pentosus according to Torriani et al., (2001). Primers used for amplification of recA regions of the strains were pRev – reverse primer and three forward primers, i.e., paraF, pentF, and planF. Meanwhile to differentiate species belong to Lactobacillus casei group pepR

gene was used according to Watanabe et al., (2008), with the primers used pepR LbC, pepR LbR and pepR g-LbC. Amplicons were passed through electrophoresis gel, and DNA bands were stained and photographed.

RESULTS

Simulated gastric juice and bile salts resistanceResult of resistancees to gastric juice and bile salt of strains

used in this study are shown in Figure 1 respectively. From simulated gastric juice resistance test using variation pH (Figure 1A) shows that all strains are not resistant in pH 1.5, but in pH 2 all strains are still survived within 6 hours incubation, with mortality around 3‒4 log cycles. While for bile salts resistance test (Figure 1B) appears that five

TABLE 1. Information about the primers used in this study.

FIGURE 1. (A) Resistance of lactic acid bacteria strain to simulated gastric juice ; (B) Resistance of lactic acid bacteria strain to bile salt


strains used in this study are resistant to bile salts up to 3% of concentration and incubation up to 6 hours. The average mortality in this condition is about 2‒3 log cycles. Resistance to low pH and bile salts are an important key requirement as a candidate probiotic strains. With the resistance up to pH 2 and 3% of bile salts, the five strains used in this study are suggested to be a potential as a candidate probiotic.

Antibacterial activity against pathogensResults of the antibacterial activity against pathogens of

the strains used in this study are shown in Table 2. The result shows that all strains used in this study were able to inhibit the three pathogenic bacteria, i.e., S. dysenteriae dky-4, E. coli OK111, and E. coli ST, as shown in Table 2. All strains could not inhibit the growth of Aeromonas hydrophilla, while only Dad-13 strain is able to inhibit S. typhi. Antimicrobial activity of Dad-13 is the biggest among to the other strains. Photos of antibacterial activity indicated by clear zone of the strains against the growth of the two pathogens, E. coli and S. dysenteriae is shown in Figure 2.

RAPD genetic profileResult of RAPD genetic profile are shown in Figure 3, it

shows that 6 lactic acid bacterial strains used in this study have different RAPD genetic profiles one to another. Four strains of Lactobacillus plantarum group isolated from fermented foods (Dad-13, T-3, Mut-7, and Mut-13) showed

FIGURE 2. Growth inhibition of E. coli OK 111 and Shigella dysenteriae dky-4 by LAB strains, showing by the clear zones

FIGURE 3. RAPD genetic profiles of lactic acid bacteria strains using 3 primers

TABLE 2. Antibacterial activities (clear zone, cm) of lactic acid bacteria strains to the growth of some pathogens


different profile, which means that each strain is different. The other two strains, F-11 (Pediococcus acidilactici) and SNP-2 (from fecal material of healthy infant) have a very

different RAPD genetic profiles with the others.

16S rRNA sequence based identificationIdentification results based on similarity of 16S rRNA

sequence are shown in Table 3. The four isolates from fermented foods remains undetermined species, based on 16s rRNA, this isolates was still identified as L. plantarum group. The used of other characters are needed to distinguish between L. plantarum and L. pentosus. As well as identification of SNP-2, that previously was stated as L. acidophilus (Purwandhani and Rahayu, 2003).

RecA gene profile and PepR gene profileResult of recA gene profile is shown in Figure 4A,

according the profile, Dad-13, T-3, Mut-7, and Mut-13 showed a similar profile with L. plantarum subsp. plantarum. Result of pepR gene profile is shown in Figure 4B, and according to the profile SNP-2 was identified as L. paracasei.

TABLE 3. Identification of lactic acid bacteria strain based on 16S rRNA gene andmultiplex primers

FIGURE. 4 (A) RecA gene profile to distinguish species on L. plantarum group and (B) pepR gene profile to distinguish species on L. casei group


DISCUSSION

According to FAO/WHO, (2001) for food application, probiotics microorganisms should not only be capable or surviving passage through the digestive tract but also have the capability to proliferate the gut. It means they must be resistant to gastric juices and be able to grow in the presence of bile under conditions in the intestines, or be consumed in a food vehicle that allows them to survive passage through the stomach and exposure to bile. Therefor the main criteria for screening of probiotic candidate are resistance to bile salt and low pH, as well as antimicrobial activity against pathogens. Lactic acid bacteria are able to produce several antibacterial compounds during their growth, such as, organic acids (lactic and acetic acid), hydrogen peroxide, CO2, and bacteriocins. Result from this study indicate that strains used in this study are resistant to gastric juice and low pH and have different abilities to inhibit pathogenic bacteria.

As a probiotic agents, lactic acid bacteria strains must be identified based on morphology, physiology, and molecular characteristics (FAO/WHO, 2001; FAO/WHO, 2002). One of the requirements as a probiotic agent is the clear identity of the strain, with accurate genus-species identification.

Ben-Amor et al., (2007) stated that DNA-based molecular analysis can provide proper identification results as required in the legal process and good manufacturing practice in the field of industry, this technique can also be used to track the presence of probiotic strains in production, in food and in the digestive tract after consumption. Ben-Amor et al., (2007) also stated that generally 16s rRNA-based identification is stronger and superior and can be use to construct phylogeny tree, compared than phenotype identification that sometimes are not able to be used to distinguish between species. Mori et al., (1997) stated that on his studies, 16s rRNA can be used to distinguish L. casei or other related taxa.

In previous studies, Rahayu (2003) have identified more than hundred strains of lactic acid bacteria based on morphological and physiological characteristics. Based on this phenotipic characteristics there were several strains, which very difficult to be distinguished between Lactobacillus plantarum and L. pentosus, so they were named as Lactobacillus plantarum-pentosus, although some strains have done the DNA hybridization.

Lactic acid bacteria strains used in this study were Mut-7, Mut-13 (isolated from gatot, a fermented dried cassava), T-3 (from growol, a fermented raw cassava), which identified as Lactobacillus-pentosus group (Rahayu, 2003), Dad-13 isolated from dadih (traditional fermented milk), and Lactobacillus acidophilus SNP-2 from fecal material of a healthy infant (Purwandhani and Rahayu, 2003).

In this study, based on gene sequences, which encode 16S rRNA, both species still could not be separated. For example on Table 3, Mut-7 has 16S rRNA gene similarity over 99.8% with either strains of L. plantarum or L. pentosus. Similar case, with other strains, such as Mut-13, T-3, and Dad-13, all these

strains have 16S rRNA gene similarity either with L. plantarum and L. pentosus more than 99%.

Torriani et al., (2001) in their publication had succeeded to distinguish Lactobacillus plantarum and L. pentosus using recA gene. Based on the same sequence in each group, specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The size of amplicons were 318 bp for L. plantarum and 218 bp for L. pentosus. To distinguish L. plantarum and L. pentosus, this study also used recA gene profile according to Torriani et al. publication (2001). The results of this study are shown in Figure 4A. Based on four recA gene profiles of the indogenous strains (lane 5-8 in Fig 4A), Dad-13, T-3, Mut-7, Mut-13, are identified as Lactobacillus plantarum as their amplicon molecular weights are exactly the same with L. plantarum subsp. plantarum YIT 0102T as reference (lane 1 Fig 4A).

Random amplification of genetic material is reported as a quick, sensitive, and cheap methods for typing genetic level strain from lactic acid bacteria and bifidobacteria (Ben-Amor, 2007). The principle of this technique is to use several primers to amplify genome on unknown locations, which are complementary to primers used followed by gel electrophoresis to detect and confirm amplification. The genetic profiles can be used to distinguish strains from Lactobacillus acidophilus or even other Lactobacillus strains (Tynkkynen et al., 1999; Roy et al., 2000; and Gancheva et al., 1999).

Rahayu (2003), isolated several kinds of lactic acid bacteria from fermented food, from RAPD analysis, shows that even if Mut-7 and Mut-13 are from the same source (gatot, fermented food from cassava) the strain are different. RAPD analysis can also be used to show the diversity level of lactic acid bacteria strain on fermented food.

In previous studies, Purwandhani and Rahayu (2003) isolated lactic acid bacteria from a healthy infant’s feces then identified as Lactobacillus acidophilus SNP-2. But from 16S rRNA sequence, as shown in Table 3, SNP-2 has similarity over 99%, either with L. casei or L. paracasei. PepR gene profile then used to distinguish this two species (Watanabe et al., 2008), as shown in Figure 4B, it is found that SNP-2 identified as Lactobacillus paracasei.

CONCLUSIONS

Four strains isolated from fermented foods (Dad-13, T-3, Mut-7, and Mut-13) were clearly identified as Lactobacillus plantarum, while SNP-2 isolated from fecal of a healthy infant was identified as Lactobacillus paracasei, all these indigenous strains from Indonesia were potential as probiotic candidates.

ACKNOWLEDGEMENTThe authors are greatly thankful to Kementerian Riset

Teknologi dan Pendidikan Tinggi Republik Indonesia (Ministry of Research, Technology and Higher Education Republic of Indonesia) for their financial assistance.


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molecular characteristics of indigenous … · shirota strain (yakult), as from human intestinal,...

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