International Food Research Journal 25(2): 566-572 (April 2018) Journal homepage: http://www.ifrj.upm.edu.my Isolation, characterization and screening of folate-producing bacteria from traditional fermented food (dadih) 1Purwandhani, S. N., 2Utami, T., 2Milati, R. and 2*Rahayu, E. S. 1Faculty of Agricultural Technology, Widya Mataram Yogyakarta University, nDalem Mangkubumen KT III/237, Yogyakarta 55123, Indonesia 2Faculty of Agricultural Technology, Gadjah Mada University, Jl. Flora No.1, Bulaksumur, Yogyakarta 55281, Indonesia Article history Abstract Received: 6 January 2017 Folate represents an important B vitamin participating in one-carbon transfer reaction required Received in revised form: in many metabolic pathways, especially purine and pyrimidine biosynthesis which indicates the 25 January 2017 importance of folate in human metabolism. Fermented milk products using lactic acid bacteria Accepted: 2 February 2017 (LAB) are good sources of such vitamins. In order to find suitable strains capable producing high folate, isolation, and characterization of LAB from traditional fermented milk (dadih) were carried out. The isolated bacteria were characterized biochemically, phenotypically, genetically and were screened for their ability to produce folate during fermentation in skim milk. Keywords Phenotypic characterization was performed using API 50 CHL; genotypic characterization was Lactic acid bacteria conducted based on the sequence of 16S rRNA genes, while the determination of folate level Dadih was done using Vita fast folate kit. From this study, 17 isolates from dadih were obtained and Folate based on phenotypic and genotypic, 16 of them were identified as Lactobacillus plantarum. Folate production of the 17 selected isolates was between 12.43 ± 3.13 to 27.84 ± 5.80 μg/L, and the folate production of Lactobacillus plantarum Dad-13 as the control was 29.27 ± 3.91 μg/L. © All Rights Reserved Introduction whereas its concentration in yogurts may be increased depending on the starter cultures used and the storage The lactic acid bacteria (LAB) are a large and conditions, ranging from 20 μg/L to a maximum of heterogeneous group of Gram-positive bacteria 150 μg/L (Sybesma et al., 2003; van Hylckama and characterized by a strictly fermentative metabolism Hugenholtz, 2007). with lactic acid as the major end product during sugar Folate is produced by various kinds of fermentation (Stamer, 1979). Besides producing the microorganisms and green plants. Dairy products lactic acid, LAB contributes to the flavour, texture are the main sources of folate for humans. Among and nutritional value of the fermented foods through these, fermented milk products, especially yoghurt, the production of aroma components, production or can contain even larger amounts of folate (Crittenden modification of exopolysaccharides and proteins, et al., 2003; Verwei et al., 2003; Iyer and Tomar, and the production of nutritional components such 2009). Folate is an essential component of the human as vitamins (Wood and Holzapfel, 1995; Hugenholtz, diet. It is involved, as a cofactor, in many metabolic 2008). Some LAB, such as Lactobacillus and pathways, mainly in carbon transfer reactions such Streptococcus strain are extensively used in food as purine and pyrimidine biosynthesis and amino and pharmaceutical industries due to their healthful acid interconversion, including biosynthesis of the properties (Leroy and De Vuyst, 2004; van Hylckama building blocks of DNA and RNA (Crittenden et al., and Hugenholtz, 2007; Hugenholtz, 2008) 2003; Iyer and Tomar, 2009; Rossi et al., 2011). Many Some strains of LAB have the ability to synthesize plants, fungi, and bacteria are able to synthesise folate; some B vitamins such as riboflavin and niacin however, humans and other animals are incapable of including folic acid in dairy products (Michaelidoua synthesising folic acid and consequently it becomes and Steijnsb, 2006). LAB starter for the production an essential nutrient (Shane, 2010). of cheese actively synthesizes vitamin B12 and folic The Nordic Nutrition suggests a daily folate acid, so that the produced cheese contains quite a intake of 300 μg for men, 400 μg for women and high vitamin B (Shahani, 1983). The amount of folic 500 for pregnant and lactating women (Becker et acid found in cow’s milk ranges from 20 to 50 μg/L, al., 2004). Folate deficiency has been implicated *Corresponding author. Email: [email protected] 567 Purwandhani et al./IFRJ 25(2): 566-572 in a wide variety of disorders from Alzheimer’s to West Sumatra (dadih), and to examine their ability to coronary heart diseases, osteoporosis, increased produce folate during the fermentation of skim milk. risk of breast and colorectal cancer, poor cognitive performance, hearing loss and NTDs (Le-Blanc et Materials and Methods al., 2007; Le-Blanc et al., 2011). Folate quantity and composition in milk and Materials dairy products are affected by several factors such as The household dadih of buffalo milk from Padang the processing technology of milk and the time and West Sumatra Indonesia was chosen to find new incubation temperature (Forsse’n et al., 2000; Lin strains with a higher yield of folate. L. plantarum and Young, 2000; Critendent et al., 2003). Numerous Dad-13 isolated from dadih (Rahayu et al., 2015) investigations regarding traditional dairy products was used as a control. have shown that they have a unique microflora which depends on the process conditions as well as on the Sampling, enumeration, and isolation of LAB from ecological localities where they have been produced traditional fermented milk (Zamfir et al., 2006; Mathara et al., 2008; Dana et LAB was isolated from eight samples of dadih. al., 2010). The average pH of dadih was 4.67 with pH values Isolation of indigenous LAB from various ranging from 4.44 to 4.89. Thus, the acidity of dadih fermented foods and other sources has been done by as an Indonesian fermented buffalo milk product is Indonesian researchers (Pramono et al., 2008; Antara comparable to that of most dairy products. et al., 2009; Lawalata et al., 2011; Suhartatik et al., For the enumeration and isolation of LAB, 10 2014) and has been screened for their health benefits millilitres curd of dadih in a bamboo was suspended such as probiotics (Purwandhani and Rahayu, in 90-mL of sterile physiologic water and serial 2003; Rahayu et al., 2015) β-glucosidase producers dilutions (10-1-10-8) were made for each sample; 1 (Djaafar, Cahyanto, Santoso et al., 2013; Suhartatik mL of the appropriate dilution was plated onto the et al., 2014); ACE inhibitor producer (Wikandari Petri dish and covered with de Man Rogosa and et al., 2012); isoflavone and antioxidant properties Sharpe (MRS) agar (Oxoid) supplemented with 1% o (Djaafar, Santoso, Cahyanto et al., 2013). In this calcium carbonate (CaCO3), and incubated at 37 C study, indigenous lactic acid has been isolated again for 48 h. The colonies which formed clear zones from dadih and screened for its ability in folate around by dissolving CaCO3 were enumerated. synthesis during fermentation of skim milk. Isolation was done by picking randomly five of the Dadih, traditional fermented milk of West white colonies with a clear zone around it up from Sumatra, Indonesia, is made by pouring fresh raw MRS agar plate then purified by streak plating using unheated buffalo milk into a bamboo tube capped the same medium (Stamer, 1979). with a banana leaf or plastic wrap, and allowing it to The preliminary characterization of the isolates ferment spontaneously by LAB at room temperature was done by morphological characteristics, Gram for two days. As a naturally fermented milk product, staining, and catalase reaction. The catalase test was dadih is white in colour and the curd texture is like performed by placing a drop of 3% hydrogen peroxide tofu, tastes like yoghurt, and it is generally served as (H2O2) to single colonies of the culture taken in a a complementing meal in some traditional occasions glass slide. The Gram positive and negative catalase as well as a delicacy from Padang West Sumatera. reaction bacteria were then selected and stored as Dadih is a highly nutritive product. Its protein and fat frozen stock at – 40oC in (1:1) 20% glycerol with contents are higher than those of yoghurt. It is rich in 10% skim milk for further analysis. amino acids and bacteria such as Lactobacillus sp. and low in cholesterol (Surono, 2016). Biochemical characterization It has been reported that LAB, such as Basic biochemical characterizations such as gas Streptococcus sp. and Lactobacillus sp. can produce production, the ability to grow at several different folate during milk fermentation (Lin and Young, temperatures, pH and salt tolerant were conducted. 2000; Dana et al., 2010). Therefore, isolation and Gas production was observed for 48 h incubation selection of naturally occurring LAB from traditional by examining the formation of gas from glucose fermented milk which can produce folate are highly in MRS broth using inverted Durham’s tubes. The significant. ability of the isolated bacteria to grow at several The main objective of this research is to isolate different temperatures (10, 15 and 45°C) was and characterize biochemical, phenotypically and evaluated visually after 24, 48 and 72 h of incubation. genetically LAB in traditional fermented milk from Furthermore, growth at pH 4.4, 5.5 and 9.6 was Purwandhani et al./IFRJ 25(2): 566-572 568 determined in MRS agar with pH adjusted by using Primer design and polymerase chain reaction (PCR) hydrogen chloride (HCl) and sodium hydroxide Molecular analysis and construction of (NaOH), respectively. Salt tolerance was determined phylogenetic tree based on the published using MRS broth containing 6.5% and 18% (w/v) data (Suhartatik et al., 2014), primer pairs, sodium chloride (NaCl) with incubation for 72 h at [(1510R (5’GGCTACCTTGTTACGA) and (9F 37oC (Sneath et al., 1986). (5’-GAGTTTGATCCTGGCTCAG)] were used for verifying the putative Lactobacillus strains at The ability of isolates to produce well-coagulated genus level for amplification of the desired fragment fermented milk (1,500 bp).