Evaluation of Bacillus Spp. As Dough Starters for Adhirasam-A Traditional
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Brazilian Journal of Microbiology 46, 4, 1183-1191 (2015) Copyright © 2015, Sociedade Brasileira de Microbiologia ISSN 1678-4405 www.sbmicrobiologia.org.br DOI: http://dx.doi.org/10.1590/S1517-838246420140409 Research Paper Evaluation of Bacillus spp. as dough starters for Adhirasam - A traditional rice based fermented food of Southern India Anvar Hussain Noorul Anisha1, Rangasamy Anandham2, Soon Woo Kwon3, Pandiyan Indira Gandhi4, Nellaiappan Olaganathan Gopal2 1Department of Food Science, Tamil Nadu Agricultural University, Tamil Nadu, India. 2Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Tamil Nadu, India. 3Korean Agricultural Culture Collection, National Academy of Agricultural Science, Republic of Korea. 4Regional Research Station, Tamil Nadu Agricultural University, Tamil Nadu, India. Submitted: May 14, 2014; Approved: February 16, 2015. Abstract Adhirasam is a cereal based, doughnut shaped, deep fried dessert consumed in the southern regions of India. The dough used to prepare adhirasam is fermented and contains rice flour and jaggery. The aim of the present study was to characterize the cultivable bacteria associated with this fermented dough and to identify a suitable starter culture for the production of quality adhirasam. In total, one hundred and seventy bacterial isolates were recovered from de Man Rogosa Sharp (MRS) agar, nutri- ent agar, lysogeny agar and tryptic soy agar media. Out of the 170 bacterial isolates, sixteen isolates were selected based on their ability to tolerate glucose and sucrose. All the bacterial isolates tolerated 15% glucose and 30% sucrose. Analyses of 16S rDNA gene sequences of the bacterial isolates showed that the dominant cultivable bacteria were members of the genus Bacillus. These strains were further used as starters and tested for their ability to ferment rice flour with jaggery to produce adhirasam dough. Organoleptic evaluation was carried out to choose the best starter strain. Adhirasam prepared from Bacillus subtilis isolates S4-P11, S2-G2-A1 and S1-G15, Bacillus tequilensis isolates S2-H16, S3-P9, S3-G10 and Bacillus siamensis isolate S2-G13 were highly ac- ceptable to consumers. Adhirasam prepared using these starter cultures had superior product charac- teristics such as softness in texture, flavor and enhanced aroma and sweet taste. Key words: Adhirasam, fermented cereal product, Bacillus, South Indian dessert. mesenteroides) and yeast species (Saccharomyces Introduction cerevisiae, Debaryomyces hansenii, Pichia anomala and Cereals are staple foods for billions of people across Trichosporon pullulans) has been documented during the the globe, especially in the Indian sub-continent, Asia and fermentation of idli batter (Aidoo et al., 2006). Park et al. Africa. Cereals are important substrates in fermented foods (2010) have reported that Bacillus amyloliquefaciens, B. and it is reported that fermented foods comprise one third of subtilis, and Bacillus vallismortis were dominant in the tra- the diets in the world (Marshall and Mejia, 2011). Several ditionally fermented Korean soybean paste, eoyukjang. fermented cereal products have been documented including Hong et al. (2012) have documented an increase in the sourdough from America, Australia, and Europe (Brandt, aroma characteristics of the fermented Korean soybean 2007), Idli and dosa from India (Soni et al., 1985; 1991; paste, Doenjang, upon inoculation with B. Agaliya and Jeevaratnam, 2013), and Puto from South East amyloliquefaciens. Previously, Lee et al. (2013) have iso- Asia (Kelly et al., 1995). The presence of obligate hetero- lated the probiotic, B. subtilis KU201 from kimchi, which fermentative lactic acid bacteria (Leuconostoc has antifungal and antimicrobial properties. Choi et al. Send correspondence to R. Anandham. Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai 625 104, Tamil Nadu, India. E-mail: [email protected]. 1184 Anisha et al. (2010) have developed a fermented soymilk product with Benson (2002). Molecular characterization was performed Bacillus subtilis as the starter culture and have found that according to Kim et al. (2011). Briefly, the gene encoding this fermented soymilk exhibited high antioxidant activity. bacterial 16S rRNA was amplified through Polymerase Cheonggukjang, produced with the co-inoculation of the Chain Reaction (PCR) with forward primer 27F: probiotics B. subtilis W42 and B. amyloliquefaciens MJ1-4, 5’-AGAGTTTGATCCTGGCTCAG-3’ and reverse exhibited high antioxidant and fibrinolytic activities (Cho primer 1492R: 5’-GGTTACCTTGTTACGACTT-3’. The et al., 2014). 16S rRNA nucleotide sequences were obtained by PCR di- Adhirasam is an ethnic fermented cereal-based food rect sequencing using the fluorescent dye terminator of South India, especially Tamil Nadu state. It is a dough- method (ABI PrismTM BigdyeTM Terminator cycle se- nut-shaped, spongy, deep fried food that is consumed quencing ready reaction kit v.3.1) and the products were during festivals and special occasions. People may also oc- purified using the Millipore-Montage dye removal kit. casionally consume the adhirasam dough without deep fry- Finally, the products were run in an ABI3730XL capillary ing. To the best of our knowledge, there is no literature DNA sequencer (50 cm capillary). Nearly complete 16S describing the microbiology of the fermented adhirasam rRNA gene sequences from the automatic sequencer were dough. Therefore, this study was formulated to investigate aligned and bacterial identities were deduced from the the cultivable bacteria involved in the fermentation of EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/) to as- adhirasam dough and to identify a promising starter cul- certain their closest relatives. The sequences obtained from ture. this study were submitted to NCBI with accession numbers KC851825 to KC851840. Materials and Methods Determination of antimicrobial activity Sample collection and isolation of microorganisms To examine the antimicrobial activity, 14 Bacillus Thirty samples of 3-day fermented adhirasam dough spp. isolated from adhirasam dough were used. Bacillus were collected from adhirasam producers in Madurai dis- spp. isolates were grown in 100 mL of nutrient broth trict, Tamil Nadu state, India. The samples were packed in (Himedia, India) at 30 °C, and 120 rpm for 24 h. The polyethylene pouches and stored in the refrigerator at 4 °C antimicrobial activity was investigated against human for subsequent analysis. Ten grams of adhirasam dough pathogens viz., Escherichia coli MTCC 2622, Staphylococ- from each sample was aseptically transferred into a 250 mL cus aureus MTCC 1144, Listeria monocytogens MTCC flask containing 90 mL sterile saline solution (0.85 g/L 1143, Saccharomyces cerevisiae MTCC 36 and Bacillus NaCl) and the contents were mixed thoroughly for 30 min cereus MTCC 1272 as described previously (Zheng et al., at 150 rpm. Serial dilutions (10-1 to 10-8) were made for 2013). These pathogenic bacteria were obtained from the each sample, and 100 mL of the each dilution was spread Microbial Type Culture Collection (MTCC), Chandigarh, onto each de Man Rogosa Sharpe agar (MRSA), Nutrient India. agar (NA), Lysogeny agar (LA), trypticase soy agar media Preparation of adhirasam dough, inoculation with (TSA) (Himedia, India). After two days of incubation at starter cultures and estimation of population 30 °C, bacterial colonies were isolated and purified. Fungi dynamics (yeasts and molds) were enumerated on Rose Bengal cho- ramphenicol agar (Himedia, India) as described previously Bacillus spp. isolates were grown in 25 mL of nutrient (Zheng et al., 2013). broth, in an incubator shaker at 120 rpm for 2 days at 30 °C. The bacterial cells were harvested by centrifugation at Identification of bacterial isolates 8,000 g at 4 °C for 15 min. The bacterial cells were washed Cell morphology, Gram staining, catalase and oxi- 2-3 times with 0.85% NaCl and resuspended in physiologi- dase activity, curdling, spore formation, sugar fermentation cal saline. (glucose, sucrose and lactose) and glucose and sucrose tol- Rice (Oryza sativa L.), local variety ‘IR20’, and erance tests were performed to characterize the isolates jaggery were purchased from the local market. Adhirasam (Gerhardt et al., 1994). Glucose tolerance was tested in a dough was prepared in the laboratory following the tradi- nutrient broth with 5%, 10%, 15% or 20% glucose after tional method. One kilogram of rice was sorted, washed 2 days of fermentation at 30 °C. Sucrose tolerance of bacte- and soaked at ambient temperature (28-30 °C) for 1 h. Wa- ria was tested in nutrient broth (devoid of glucose) having ter was decanted, and the soaked rice was pounded and sucrose concentrations ranging from 10% to 80% with sieved to get rice flour. Jaggery syrup was prepared sepa- 10 unit increments, by incubation for 2 days at 30 °C. Cur- rately by mixing 750 g of jaggery in 300 mL distilled water dling activity was tested in toned milk medium prepared and heating to 80 °C to get desired consistency. Immedi- from bacteria free toned milk (Terzic-Vidojevic et al., ately, one kilogram of rice flour was mixed with this 2009). The hemolytic property of Bacillus spp. isolated 300 mL of hot jaggery syrup and kneaded into soft dough from adhirasam dough was determined according to under aseptic conditions. After the dough reached room Starter culture for Adhirasam 1185 temperature, equal quantities (100 g) of dough were distrib-