Research article

Vitamin B12 production in soybean fermentation for tempeh

  • Received: 13 January 2020 Accepted: 04 June 2020 Published: 15 June 2020
  • Most studies have found that vitamin B12 in tempeh is produced by contaminating bacteria specifically, Klebsiella sp. and Citrobacter freundii, during fungal fermentation. This study is to evaluate the effect of starter culture on the vitamin B12 and isoflavone aglicone content of soybean fermentation for tempeh production. In this study, soybeans were washed, soaked in water overnight, dehulled and sterilized by boiling at 100 ℃ for 30 min. Three starter cultures (103 CFU g-1) namely Rhizopus oligosporus, Klebsiella sp. and Saccharomyces cerevisiae were then inoculated as follows, soybeans + R. oligosporus + Klebsiella sp. (SRK), soybeans + R. oligosporus + S. cerevisiae (SRSc), soybeans + R. oligosporus + S. cerevisiae + Klebsiella sp. (SRScK), and Soy + R. oligosporus (SR) and soybeans + Klebsiella sp (SK). Inoculated soybeans were then incubated at 30 ±2 ℃ for 40 hours (tempeh-style). The growth of Klebsiella sp., S. cerevisiae, R. oligosporus and the production of vitamin B12 as well as isoflavone aglicones were observed. The results showed the highest vitamin B12 3.15 mg 100 g-1 was found in tempeh SRSc, followed by 2.88 mg 100 g-1 and 1.64 mg 100 g-1 in tempeh SR and SRScK, respectively. In addition, vitamin B12 in tempeh SRK was the lowest (0.81 mg 100 g-1). All the starter cultures were able to hydrolyze daidzin and genistin, but the amount of daidzein and genistein was tripled and doubled, respectively when Klebsiella sp was inoculated to the soybean fermentation. The study suggested that S. cerevisiae contributes to the production of vitamin B12, while Klebsiella sp contributes to production of daidzein and genistein in soybean fermentation for tempeh production.

    Citation: Maria E. Kustyawati, Subeki, Murhadi, Samsul Rizal, Penipuji Astuti. Vitamin B12 production in soybean fermentation for tempeh[J]. AIMS Agriculture and Food, 2020, 5(2): 262-271. doi: 10.3934/agrfood.2020.2.262

    Related Papers:

    [1] Diana Lo, Andreas Romulo, Jia-Ying Lin, Yuh-Tai Wang, Christofora Hanny Wijaya, Ming-Chang Wu . Effect of different fermentation conditions on antioxidant capacity and isoflavones content of soy tempeh. AIMS Agriculture and Food, 2022, 7(3): 567-579. doi: 10.3934/agrfood.2022035
    [2] Birabrata Nayak, Bibhu Prasad Panda . Modelling and optimization of texture profile of fermented soybean using response surface methodology. AIMS Agriculture and Food, 2016, 1(4): 409-418. doi: 10.3934/agrfood.2016.4.409
    [3] Budi Suarti, Sukarno, Ardiansyah, Slamet Budijanto . Bio-active compounds, their antioxidant activities, and the physicochemical and pasting properties of both pigmented and non-pigmented fermented de-husked rice flour. AIMS Agriculture and Food, 2021, 6(1): 49-64. doi: 10.3934/agrfood.2021004
    [4] Rita Khathir, Murna Muzaifa, Yunita, Marai Rahmawati . Study on the physicochemical and sensory profile of pliek-u: A traditional dried fermented coconut endosperm from Aceh, Indonesia. AIMS Agriculture and Food, 2023, 8(2): 534-549. doi: 10.3934/agrfood.2023028
    [5] Samsul Rizal, Maria Erna Kustyawati, Murhadi, Udin Hasanudin, Subeki . The effect of inoculum types on microbial growth, β-glucan formation and antioxidant activity during tempe fermentation. AIMS Agriculture and Food, 2022, 7(2): 370-386. doi: 10.3934/agrfood.2022024
    [6] Nicholas Mawira Gitonga, Gilbert Koskey, Ezekiel Mugendi Njeru, John M. Maingi, Richard Cheruiyot . Dual inoculation of soybean with Rhizophagus irregularis and commercial Bradyrhizobium japonicum increases nitrogen fixation and growth in organic and conventional soils. AIMS Agriculture and Food, 2021, 6(2): 478-495. doi: 10.3934/agrfood.2021028
    [7] Rasyid Sukifto, Rosimah Nulit, Yap Chee Kong, Noorhazira Sidek, Siti Nuratiqah Mahadi, Nurfatiha Mustafa, Roslinda A. Razak . Enhancing germination and early seedling growth of Malaysian indica rice (Oryza sativa L.) using hormonal priming with gibberellic acid (GA3). AIMS Agriculture and Food, 2020, 5(4): 649-665. doi: 10.3934/agrfood.2020.4.649
    [8] Yeon Ju Lee, Jong Hyuk Kim, Ju Hyeon Ha, Ha Yeon Nam, Il Rae Rho . Effects of nitrogen topdressing fertilization on yield and quality in soybeans. AIMS Agriculture and Food, 2024, 9(4): 1004-1026. doi: 10.3934/agrfood.2024054
    [9] Nelson Mpumi, Revocatus L. Machunda, Kelvin M. Mtei, Patrick A. Ndakidemi . Insecticidal Efficacy of Syzygium aromaticum, Tephrosia vogelii and Croton dichogamus Extracts against Plutella xylostella and Trichoplusia ni on Brassica oleracea crop in Northern Tanzania. AIMS Agriculture and Food, 2021, 6(1): 185-202. doi: 10.3934/agrfood.2021012
    [10] Nirawan Gunun, Chatchai Kaewpila, Waroon Khota, Pongsatorn Gunun . Investigation of the effect of different additives on the qualities, in vitro degradation, and rumen fermentation profile of indigo waste silage. AIMS Agriculture and Food, 2024, 9(1): 169-182. doi: 10.3934/agrfood.2024010
  • Most studies have found that vitamin B12 in tempeh is produced by contaminating bacteria specifically, Klebsiella sp. and Citrobacter freundii, during fungal fermentation. This study is to evaluate the effect of starter culture on the vitamin B12 and isoflavone aglicone content of soybean fermentation for tempeh production. In this study, soybeans were washed, soaked in water overnight, dehulled and sterilized by boiling at 100 ℃ for 30 min. Three starter cultures (103 CFU g-1) namely Rhizopus oligosporus, Klebsiella sp. and Saccharomyces cerevisiae were then inoculated as follows, soybeans + R. oligosporus + Klebsiella sp. (SRK), soybeans + R. oligosporus + S. cerevisiae (SRSc), soybeans + R. oligosporus + S. cerevisiae + Klebsiella sp. (SRScK), and Soy + R. oligosporus (SR) and soybeans + Klebsiella sp (SK). Inoculated soybeans were then incubated at 30 ±2 ℃ for 40 hours (tempeh-style). The growth of Klebsiella sp., S. cerevisiae, R. oligosporus and the production of vitamin B12 as well as isoflavone aglicones were observed. The results showed the highest vitamin B12 3.15 mg 100 g-1 was found in tempeh SRSc, followed by 2.88 mg 100 g-1 and 1.64 mg 100 g-1 in tempeh SR and SRScK, respectively. In addition, vitamin B12 in tempeh SRK was the lowest (0.81 mg 100 g-1). All the starter cultures were able to hydrolyze daidzin and genistin, but the amount of daidzein and genistein was tripled and doubled, respectively when Klebsiella sp was inoculated to the soybean fermentation. The study suggested that S. cerevisiae contributes to the production of vitamin B12, while Klebsiella sp contributes to production of daidzein and genistein in soybean fermentation for tempeh production.


    Vitamin B12, also known as cobalamin, plays an important role in the functioning of the brain and nervous system, and in the formation of red blood cells. Vitamin B12, occurring naturally in food, derives from bacterial synthesis and therefore occurs only in foods of animal origin. Small amounts can be found in plant products due to the presence of bacteria or due to microbial contamination. Plant food such as blue-green algae contains of pseudo vitamin B12 which is in active in humans. In foods, cobalamins are bound to proteins and glycoproteins and must be released by enzymatic and acid hydrolysis in the gut. Dairy foods, milk and milk products, eggs, meat and seafoods are the main natural sources in the diet. Tempeh, solid state boiled-dehulled soybean fermentation using starter culture of Rhizopus oligosporus, is rich of antioxidants and contains vitamin B12 [1,2]. Few scientific literatures mentioned the vitamin B12 values of tempeh. Areekul et al. [3] found between 0.18 and 4.1 Mcg vitamin B12 analogues per 100 g tempeh from tempeh sold in market in Jakarta, and tempeh sold in Toronto, Canada contained of 148 ng of vitamin B12 per g [4]. During the soybean fermentation, enzymatic activities of R. oligosporus leads to a significant increase in water-soluble nutrients, enhancing the biosynthesis of B vitamins and transformation of soy-isoflavones into antioxidant compounds. Soybean itself contains low or undetectable of vitamin B12, yet when soybeans are fermented to produce tempeh, a considerable amount of vitamin B12 was found, about 0.7 to 0.8 µg/100 g [5]. It is known that vitamin B12 contained in tempeh is synthesized by Klebsiella pneumoniae and Citrobacter freundii [6]. These bacteria were thought as the contaminating bacteria during processing of tempeh making. With these values of vitamin B12, tempeh may be as an additional diet of vitamin B12 from plant-derived food sources. The RDI of vitamin B12 for adults is set at 2.4 µg/day in Indonesia [7], and as well in The US and Japan (RDA) [8]. Recently, industrial production of vitamin B12 has been carried out through microbial fermentation, mostly use Pseudomonas denitrificans, Propionibacterium shermanii [9]; however, this process has some drawbacks such as a long fermentation process and expensive media requirements

    Consumption of soy based foods may have health benefits due to their functional ingredients, especially isoflavones. In soybeans, isoflavones are in the form of isoflavone glycosides. The two most widely studied of isoflavones from soybeans are daidzin and genistin, which are not easily absorbed by the intestinal absorptive cells because of their large hydrophilic structures [10,11]. However, hydrolytic enzyme-producing microorganisms can hydrolyze isoflavone glucosides by the enzyme β-glucosidase to form of aglicones, daidzein and genistein which have antioxidant properties. Hydrolysis of isoflavone glycoside, and variation of individual isoflavone aglycones are influenced by microbial types [12,13,14] reported that bacteria are more effective in converting daidzin and genistin into their aglycone forms than molds. Aspergillus and Streptomyces isolated from soybean fermented foods are known as best producer of ortho-dihydroxyisoflavone (ODI), hydroxylated isoflavones which is produced during fermentation of soybeans. This study conducted to analyze the vitamin B12 and the isoflavone aglycone contents produced during soybean fermentation using co-inoculated of R. oligosporus, S. cerevisiae, and Klebsiella sp as starter cultures.

    Starter cultures used in this study: Rhizopus oligoporus was isolated from tempeh, Saccharomyces cerevisiae isolated from commercial Fermipan dried yeast product, and Klebsiella sp non-pathogenic bacteria were isolated from the tempeh [15]. Except for Klebsiella sp which was cultured on nutrient agar and its stock culture was maintained at −20 ℃ in 20% glycerol, all the starter cultures were freshly prepared. For inoculums preparation, the bacteria and yeast were grown in nutrient broth at 37 and 32 ℃ respectively for 24 h. The cells were harvested and resuspended in sterile distilled water and properly adjusted to obtain a concentration of 105 and 105 CFU mL−1 respectively. For mold preparation, the mycelium was streaked on potatoes dextrose agar and grown at 27 ℃ for 120 h. The spores were harvested, resuspended in sterile distilled water and properly adjusted to obtain a concentration of 105 cell mL−1.

    The data obtained was analysed by the use of Excel Microsoft program 2016.

    Soybeans in this research were purchased in the Primkopti Bandar Lampung. Tempeh was produced in a microbiology laboratory as follows; 300 g of soybeans were soaked in clean water overnight at room temperature, and then dehulled manually. Dehulled soybeans were boiled in clean water with a ratio of 1:3 (soybeans: water) for 30 minutes, drained, air dried at room temperature, and then 100g aseptically inoculated with the cultures at defined number of cells which was 105, 105, and 105 CFU 100g−1 for R.oligosporus, S. cerevisiae, and Klebsiella sp, respectively. Inoculated soybeans were packed in perforated plastic packaging and incubated at 32 ± 2 ℃ for 40 hours. Five types of fermented soybeans with the addition of different inoculated cultures were produced in this study, namely (1) soybeans + R. oligosporus + Klebsiella sp (SRK), soybeans + R. oligosporus + S. cerevisiae (SRSc), soybeans + R. oligosporus + S. cerevisiae + Klebsiella sp. (SRScK), and Soybean + R. oligosporus (SR) and soybeans + Klebsiella sp. (SK). Separate soybeans were also fermented as a control without inoculation. The experiments were made in three replication.

    The measurement of pH was carried out with a pH meter model 501 (Crison, Barcelona, Spain). All samples were homogenized prior to pH measurements. Measurements were made three times for testing the accuracy then the mean was taken.

    Each of fermented soybeans was analyzed for its total number of bacteria, yeast and molds at the starting and the end of fermentation. A total of 15 g tempeh was taken, mixed with 135 mL of 0.1% peptone water, homogenized with a stomacher for 30 seconds, and a series of dilutions from 10−1 to 10−8 was made in duplicate. Then one mililiter is taken from certain dilutions for plating microorganisms done by spread plate method on EMB agar (BBL Microbiology Systems. Cockeysville, Md.), MEA (Difco), and PDA (Oxoid) for Klebsiella sp., S. cerevisiae, and R. oligosporus, respectively. Incubation of the plates was at 37, 27, 32 ℃ for Klebsiella sp., S. cerevisiae, and R. oligosporus respectively, for 24 to 48 hours.

    The method of vitamin B12 analysis was performed according to the procedure run by Lawrence [16] with small modification on the application of UAE. Sample preparation method applies water ultrasonic assisted extraction (UAE), in which ultrasonic water extraction was used for extraction. A total of 0.5 grams of ground tempeh was weighed and place into 100 mL Erlenmeyer containing of 20 mL of milli-Q water. Sample was extracted by using ultrasonic equipped with heater for 30 minutes. Then, sample volume was set for 25 mL by adding milli-Q-water, and centrifuged at 3000 rpm for 10 min. Supernatant was pipetted using syringe equipped with filter holder so that 2 mL of supernatant was obtained, and then filtered using paper filter of 13 mm in diameter and 0.2 µm pore size. The filtrate was then placed in the vial bottle. The sample was ready to be injected into the HPLC (Shimadzu, CBM-20A controller, LC 20AD solvent delivering unit, CTO 10A column oven, SPD M20-A photo diode array detector). HPLC running condition was using Agilent C-18 5 um (125 × 4.6 mm) column, column temperature of 35 ℃, mobile phase (water: acetonitrile: buffer phosphate 10 nM = 80:10:10), isocratic mobile phase method with flow rate 1 mL min−1, injection volume of 20 µL, wavelength detector 360 nm, and running time of 20 min.

    Standard solution was prepared as following, weigh 5 mg of standard cyanocobalamin, dissolve it in 100 mL of milli-Q-water so that a concentration of 50 µg.mL−1 is obtained. Then the standard was diluted into a working solution with a concentration of 0.5 µg.mL−1, 1 µg.mL−1, 2 µg.mL−1, and 4 µg.mL−1.

    For quantification of daidzein and genistein, fermented soybean samples were freeze-dried and stored at −20 ℃ until used. The extraction of isoflavones from fermented soybeans and unfermented soybeans, and their quantification by HPLC were assayed followed the procedures worked by El-Shazy et al. [17] with small modification and was assayed at the Centre Technology and Innovation Lab. Univ. of Lampung. HPLC-MWD instrument (Agilent 1200 series), Zorbax 5µm (4.6 mm × 150 mm) column, and UV detector (λmax = 254 nm). The mobile phase consisted of 100% methanol and 10 mmol L-1 of ammonium acetate buffer (60:40) containing 1 mL of trifluoro-acetic acid per liter of solvent mixture. This was set at a flow rate of 0.6 mL min−1. Standards of daidzein and genistein were obtained from Sigma. 1gram of the freeze-dried sample was added to 10mL of 80% (v v−1) aqueous methanol and extracted under agitation for 24 hours at room temperature. The homogenates were centrifugated at 15000rpm for 30min and the methanol obtained were used to determine daidzein and genistein concentrations. Injection volumes of isoflavone standards and of the samples were set at 100 µL throughout the run time of 30min. Single standards were also prepared for peak identification. Calculation of the isoflavone concentration was dry basis (µg g−1 fermented soybean).

    The number of R.oligosporus, S. cerevisiae, and Klebsiella sp inoculated into soybeans for tempeh production were 103, 105 and 103 cells g−1, respectively. Table 1 shows that the addition of S. cerevisiae and Klebsiella sp. either together or separately did not affect the growth of R. oligosporus. R oligosporus in tempeh is not a contaminant microorganism and instead it has to be deliberately added into the soybeans. It was possible that R. oligosporus played a role in supporting the growth of other microorganism in the soybeans fermentation, because during fermentation R. oligosporus produces several enzymes including lipase, protease, and phytase which hydrolyzes carbohydrate, lipid, and protein from the soybeans and produces fatty acids and amino acids which are then utilized by bacteria and yeast [18]. In addition, they also function as growth control of microorganisms in tempeh, preventing the tempeh consumer from diarrhea and flatulence. R.oligosporus produces antibiotics which actively kill certain bacteria in tempeh i.e. Staphylococcus aureus, Bacillus subtilis, and Clostridium sp which is known to produce gas in the intestines [19]. S. cerevisiae was able to grow together either with R. oligosporus or Klebsiella sp. during fermentation of tempeh making, it is possible to obtain carbon and nitrogen sources from soybeans as well as fatty acid and amino acids produced by R. oligosporus. Meanwhile, the wide pH tolerant and that Klebsiella is a bacterial contaminant in tempeh which may be the reason for the high number of Klebsiella found in tempeh. The pH of tempeh in this experiment was found between 6.60 to 7.40 (Tabel 2).

    Table 1.  The total number of microorganism in soy fermentation inoculated with various cultures.
    Type of isolates in soy fermentation Microbial number (CFU g−1) at
    0 h of fermentation 40 h of fermentation
    Ro Sc. K Ro Sc. K
    SR 2.6 × 103 ND ND 3.4 × 106 ND 2.0 × 108
    SRSc 2.8 × 103 3.6 × 103 ND 4.5 × 106 1.2 × 106 1.1 × 108
    SRScK 4.4 × 103 6.3 × 103 1.0 × 103 5.6 × 105 3.7 × 105 2.5 × 108
    SRK 5.0 × 103 ND 2.7 × 103 3.0 × 105 ND 1.9 × 108
    SK ND ND 2.1 × 103 ND ND 2.7 × 108
    Note: The data in the table were average value of three replications. ND not detected. SR is soy + R. oligosporus, SRSc is soy + R. oligosporus + S. cerevisiae, SRScK is soy + R. oligopsorus + S. cerevisiae + Klebsiella sp, SRK is soy + R.oligosporus + Klebsiella sp, SK is soy + Klebsiella sp. Ro is R. oligosporus, Sc is S. cerevisiae, K is Klebiella sp.

     | Show Table
    DownLoad: CSV
    Table 2.  Effect of isolated cultures in producing vitamin B12, daidzein, and genistein during tempeh fermentation.
    Isolated culture Initial pH Final pH Water content (%) Vit B12 (Mcg100 g−1) Daidzein (Mcg g−1) Genistein (Mcg g−1)
    SR 6.2 6.40 65.1 ± 0.19 2.88 ± 0.50 276.17 ± 0.20 492.96 ± 1.0
    SRSc 6.2 6.10 65.23 ± 0.80 3.15 ± 0.70 224.37 ± 0.20 465.12 ± 0.90
    SRScK 6.2 6.60 65.14 ± 0.10 1.64 ± 0.9 753.96 ± 0.15 1072.87 ± 0.90
    SRK 6.2 6.60 65.24 ± 0.20 0.81 ± 2.00 781.76 ± 0.15 1071.85 ± 1.0
    SK 6.2 7.40 65.91 ± 0.50 0.39 ± 1.9 782.17 ± 0.30 1072.51 ± 0.70
    Soybean only 6.2 6.20 65.0 ± 0.50 0.36 ± 0.42 0.69 ± 0.20 0.25 ± 0.60
    Note: The data in the table were mean of three replications with standard deviation. SR is soy + R. oligosporus, SRSc is soy + R. oligosporus + S. cerevisiae, SRScK is soy + R. oligopsorus + S. cerevisiae + Klebsiella sp, SRK is soy + R.oligosporus + Klebsiella sp, SK is soy + Klebsiella sp.

     | Show Table
    DownLoad: CSV

    Biosynthesis of vitamin B12 is limited to some bacteria and archaea, and its production depends on type of microorganisms involved during fermentation. Soybeans only used as control of the tempeh production in this experiment contained very little vitamin B12 that it would not be of nutritional significance. This finding (0.36 mg100g−1) was close with data reported by Liem et al. [4] and Areekul et al. [3], in which the amount was found to be 0.39 ng/g and 0.15 ng g−1, respectively. R. oligosporus was an essential microorganism in the production of tempeh, but when Klebsiella sp was used as culture to soybean fermentation, tempeh did not produce (Figure 1). When R. oligosporus was as an inoculum in soybean fermentation it yielded vitamin B12 of about 2.88 mg 100g−1, R. oligosporus co-inoculated with Klebsiella sp yielded lower vitamin B12 (0.81 mg 100g−1). The first data (2.88 Mcg 100 g−1) was close with the data reported by Liem et al. [4] which was 66.0 ng g−1. The presence of S. cerevisiae during tempeh fermentation was beneficial from the nutritional point of view. When S. cerevisiae co-inoculated with R. oligosporus, a high vitamin B12 was produced (3.15 Mcg100 g−1) in tempeh. S. cerevisiae is rich in cobalt and a vitamin B12 producer [20,21], which may contribute to an increase in vitamin B12 of the tempeh. In addition, S. cerevisiae co-inoculated with R. oligosporus in soybean fermentation produced tempeh that contain β-glucan [22] and a new aroma of yeasty which is preferred by some tempeh consumers [23]. Yeasty aroma of tempeh produced by alcohol, ester, and aromatic group such as styrene, caryophyllene, phenol, and maltol during fermentation.

    Figure 1.  The profile of tempeh produced by the use of different cultures. SR is soy + R. oligosporus), SRSc is soy + R. oligosporus + S. cerevisiae, SRScK is soy + R. oligosporus + S. cerevisiae + Klebsiella sp, SRK is soy + R.oligosporus + Klebsiella sp, SK is soy + Klebsiella sp. All of the tempeh except SK showed the white mycelium binds the beans into a compact, cake-like texture.

    The recommended dietary allowance of vitamin B12 is 2.4 µg/day for adults. When the concentration of vitamin B12 in tempeh was 3.15 Mcg100g−1, it becomes visible for the consumer to get his or her daily requirement of vitamin B12 by consumption of approximately 76.2 g of tempeh. The bioavailability of vitamin B12 in tempeh in this study was not investigated. Mo et al. [24] found the bioavailability of tempeh was 0.016–0.072 Mcg 100g−1 which is very low. This could be because they made tempeh from soybean fermentation with Rhizopus microsporus var. microsporus LU573 and did not use the vitamin B12 producing bacteria. However, Bor et al. [25] mentioned that a daily vitamin B12 intake of 6 Mcg is sufficient to maintain a steady-state concentration of plasma vitamin B12. Bioavailability of dietary vitamin B12, daily body loss of vitamin B12, and human healthiness of normal gastrointestinal function are important to consider for consumption of tempeh as a source of daily vitamin B12 intake. When compared to cooked lean meat which contains 33 µg 100g−1 of vitamin B12 and the bioavailability of vitamin B12 which is 0.9 µg from 100 g of mutton ground patties, tempeh can be a potential source of vitamin B12 from non-meat origin. Provided that the potential use of bacteria producing vitamin B12 or yeast in soy fermentation is attempted to increase the quantity of vitamin B12 to make tempeh a reliable source of vegetable vitamin B12.

    Recently Propionibacterium freundenreichii, the food grade producer of vitamin B12, has been used in co-culture with R. oryzae to enrich vitamin B12 in lupin tempeh [26]. An increase of vitamin B12 content (up to 0.97 μg100 g−1) was achieved in lupin tempeh and its texture, taste and overall acceptance were not affected by the bacterial co-inoculation [27]. Nevertheless, lupin tempeh have distinct flavour and aroma that may be unacceptable to consumers who are familiar with the taste of soy tempeh. Propionibacterium freundenreichii was usually used in the preparation of cheese making with its contribution to fat compounds, an important flavour in cheese [28]. Regardless of whether tempeh can be fermented with substantial amount of vitamin B12 producing bacteria, its overall digestibility, rich in protein content and antioxidant. This can make it potential meat alternative and functional food.

    In all, the tempeh that was analyzed showed that genistein was larger amount than daidzein (Table 2). Most of the inoculated cultures, R.oligosporus, S. cerevisiae and Klebsiella sp may effectively hydrolyze daidzin and genistin during the soy fermentation. In tempeh fermentation co-inoculated with Klebsiella sp, it was noticed that the amount of daidzein was three-fold, and genistein was doubled compared to that of without Klebsiella sp. Differences in the biotransformation of both daidzin and genistin were found when the addition of Klebsiella sp to the soy fermentation produced high amount of both daidzein and genistein. In addition, inoculation of the mold R. oligosporus into the soy fermentation produced low amount of either daidzein or genistein. Even though the activity of β-glucosidase was not analyzed in this study, the production of daidzein and genistein indicated that there was biotransformation of isoflavones catalysed by the β-glycosidase. Our results are not in agreement with the work done by El-Shazly et al. [17] where the amount of daidzein was larger than genistein in fermented soy flour with Bacillus licheniformis. The type of soybean fermentation applied to measure isoflavones, and the selective action of the β-glucosidase, might explain the different findings in this study. Solid state fermentation as a process in which microorganisms are grown in solid material without the presence of free liquid was applied in this study while submerge fermentation was applied in another study. According to Fang et al. [9], hydrolysis of soy isoflavones by microbial β-glucosidase depend on substrate specificity. Beside hydrolyzed from daidzin and genistin, daidzein and genistein may also be derived from formononetin and biochanin-A [29]. In addition, the bacterial-synthesized β-glucosidase can be bound to the cell walls or secreted into the periplasmic space [30]. Borges et al. [31] found that soaking, cooking and fermentation times in the tempeh making influenced the production of isoflavone, and the highest yield of genistein and daidzein was fermentation with R. oligosporus for18 hours, but the beans and the culture used were no cause for concern.

    R. oligosporus is the main isolated culture in tempeh production. Inoculation of Klebsiella to cooked dehulled soybean fermentation did not produce tempeh. S. cerevisiae is the most potential contributor of vitamin B12 in tempeh. Klebsiella sp. co-inoculation with either R. oligosporus or S. cerevisiae inhibit the production of vitamin B12 in tempeh, although it does not affect their growth. Klebsiella sp contributes to the production of daidzein and genistein in tempeh.

    This research was partly funded by the University of Lampung through the BLU-Fundamental Research, University of Lampung, 2019.

    All authors declare no conflict of interest.



    [1] Nout MJR, Sarkar PK, Beuchat LR (2007) Indigenous fermented foods, In: Doyle M, Beuchat L, Food Microbiology: Fundamentals and Frontiers, Third Eds., Washington, DC: ASM Press, 817-835.
    [2] Keuth S, Bisping B (1994) Vitamin B12 Production by Citrobacter freundii or Klebsiella pneumonia during Tempeh Fermentation and Proof of Enterotoxin Absence by PCR. Appl Environ Microbiol 60: 1495-1499. doi: 10.1128/AEM.60.5.1495-1499.1994
    [3] Areekul S1, Pattanamatum S, Cheeramakara C, et al. (1990) The source and content of vitamin B12 in the tempehs. J Med Assoc Thai 73: 152-156.
    [4] Liem IT, Steinkraus KH, Cronk TC (1977) Production of vitamin B12 in tempeh, a fermented soybean food. Appl Environ Microbiol 34: 773-776. doi: 10.1128/AEM.34.6.773-776.1977
    [5] Nout MJR, Rombouts FM (1990) Recent developments in tempeh research. J Appl Bacteriol 69: 609-633. doi: 10.1111/j.1365-2672.1990.tb01555.x
    [6] Denter J, Bisping B (1994) Formation of B-vitamins by bacteria during the soaking process of soybeans for tempe fermentation. Intern J Food Microbiol 22: 23-31. doi: 10.1016/0168-1605(94)90004-3
    [7] Kartono D, Hardinsyah, Jahari AB, et al. (2012) Summary of recommended daily intake for Indonesian 2012, (Ringkasan Angka Kecukupan gizi (AKG) yang dianjurkan bagi orang Indonesia 2012). From Conference Paper, 2012. Available from: http://www.researchgate.net/publication/316095861.
    [8] Watanabe F (2007) Vitamin B12 sources and bioavailability. Exp Biol Med 232: 1266-1274. doi: 10.3181/0703-MR-67
    [9] Fang H, Kang J, Zhang D (2017) Microbial production of vitamin B12: A review and future perspectives. Microb Cell Fact 16: 1-15. doi: 10.1186/s12934-016-0616-2
    [10] Rafli F (2015) The role of colonic bacteria in the metabolism of the natural isoflavone daidzin to equol. Metabolites 5: 56-73. doi: 10.3390/metabo5010056
    [11] Lee PG, Lee UK, Song H, et al. (2018) Recent advances in the microbial hydroxylation and reduction of soy isoflavones. FEMS Microbiology Letters 365: fny 195.
    [12] Xiao Y, Zhang S, Tong H, et al. (2018) Comprehensive evaluation of the role of soy and isoflavone supplementation in humans and animals over the past two decades. Photother Res 32: 384-394. doi: 10.1002/ptr.5966
    [13] Sirilun S, Sivamaruthi BS, Kesika P, et al. (2017) Lactic acid bacteria mediated fermented soybean as a potent nutraceutical candidate. Asian Pac J Trop Biomed 7: 930-936. doi: 10.1016/j.apjtb.2017.09.007
    [14] Deza MAC, de Olmos AR, Garro MS (2019) Solid fermentation to obtain vegetable products bio-enriched with isoflavones aglicones using lacyic cultures. Rev Argent Microbiol 51: 201-207.
    [15] Ayu E, Suwanto A, Barus T (2014) Klebsiella pneumonia from Indonesian tempeh were genetically different from that of pathogenic isolates. Microbiology 8: 9-15.
    [16] Lawrance P (2011) An evaluation of procedures for the determination of vitamin B12 in foods, supplements and premixes using HPLC and UPLC after selective extraction with immunoaffinity cartridges. LGC Limited, 1-12.
    [17] El-Shazly AI, El-Deen AMN, Ibrahim NA, et al. (2017) Assessment of genistein and daidzein production by some local fungal and bacterial isolates. Egypt J Microbiol 52: 49-61.
    [18] Handoyo T, Morita N (2006) Structural and functional properties of fermented soybean (tempeh) by using R. oligosporus. Int J Food Prop 9: 347-355. doi: 10.1080/10942910500224746
    [19] Shurtleff W, Aoyagi A (2007) History of tempeh, a special report on the history of traditional fermented soyfoods, In: Shurtleff W, Aoyagi A, History of Soybeans and Soyfoods: 1100 B.C. to the 1980s, Lafayette, California: Soyinfo Center, 1-85.
    [20] Klis FM, Boorsma A, de Groot PWJ (2006) Cell wall construction in Saccharomyces cerevisiae. Yeast 23: 185-202. doi: 10.1002/yea.1349
    [21] Fakruddin MD, NurHossain Md, Ahmed MM (2017) Antimicrobial and antioxidant activities of Saccharomyces cerevisiae IFST062013, a potential probiotic. BMC Complemet Altern Med 17: 1-11. doi: 10.1186/s12906-016-1505-2
    [22] Rizal S, Murhadi, Kustyawati ME, et al. (2020) Growth optimization of S. cerevisiae and R. oligosporus during fermentation to produce tempeh with high β-glucan content. Biodiversitas 21: 2667-2673.
    [23] Kustyawati ME, Nawansih O, Nurdjanah S (2017) Profile aroma compounds and acceptability of modified tempeh. Int Food Res J 24: 734-740.
    [24] Mo H, Kariluoto S, Piironen V, et al. (2013) Effect of soybean processing on content and bio accessibility of folate, vitamin B12 and isoflavones in tofu and tempeh. Food Chem 141: 2418-2425. doi: 10.1016/j.foodchem.2013.05.017
    [25] Bor MV, von Castel-Roberts KM, Kauwell GPA, et al. (2010) Daily intake of 4 to 7 µg dietary vitamin B-12 is associated with steady concentrations of vitamin B12-related biomarkers in a healthy young population1-4. Am J Clin Nutr 91: 571-577. doi: 10.3945/ajcn.2009.28082
    [26] Wolkers-Rooijackers JCM, Endika MF, Smid EJ (2018) Enhancing vitamin B12 in lupin tempeh by in situ fortification. Food Sci Technol 96: 513-518.
    [27] Kaczmarska KT, Chandra-Hioe MV, Frank D, et al. (2018) Aroma characteristics of lupin and soybean after germination and effect of fermentation on lupin aroma. LWT-Food Sci Technol 87: 225-233. doi: 10.1016/j.lwt.2017.08.080
    [28] Mukdsi MCA, Falentin H, Maillard MB, et al. (2013) The secreted esterase of Propionibacterium freundenreichii has a major role in cheese lipolysis. Appl Environ Microbiol 80: 751-756.
    [29] Ogbuewu IP, Uchegbu MC, Emenalom OO, et al. (2010) Overview of the chemistry of soy isoflavones, potential threats and potential therapeutic benefits. Electric J Environ Agric Food Chem 9: 682-695.
    [30] Pyo YH, Lee TC, Lee YC (2005) Enrichment of bioactive isoflavones in soymilk fermented with β -glucosidase-producing lactic acid bacteria. Food Res Int 38: 551-559. doi: 10.1016/j.foodres.2004.11.008
    [31] Borges CWC, Carrao-Panizzi MC, Mandarino JMG, et al. (2016) Contents and bioconversion of β-glycoside isoflavones to aglycones in the processing conditions of soybean tempeh. Pesq agropec bras, Brasília 51: 271-279.
  • This article has been cited by:

    1. Samsul Rizal, Maria Erna Kustyawati, Udin Hasanudin, Dimitrios Tsaltas, The Growth of Yeast and Fungi, the Formation of β-Glucan, and the Antibacterial Activities during Soybean Fermentation in Producing Tempeh, 2021, 2021, 2314-5765, 1, 10.1155/2021/6676042
    2. Jyoti P Tamang, Anu Anupma, Headstar Nakibapher Jones Shangpliang, Ethno-microbiology of Tempe, an Indonesian fungal-fermented soybean food and Koji, a Japanese fungal starter culture, 2022, 48, 22147993, 100912, 10.1016/j.cofs.2022.100912
    3. Sulaiman Akbar Mahdi, Made Astawan, Nur Wulandari, Tjahja Muhandri, Tutik Wresdiyati, Andi Early Febrinda, Formula Optimization and Physicochemical Characterization of Tempe Drink Powder, 2022, 10, 23220007, 1178, 10.12944/CRNFSJ.10.3.31
    4. Yali Qiao, Kenan Zhang, Zongcai Zhang, Chao Zhang, Yan Sun, Zhen Feng, Fermented soybean foods: A review of their functional components, mechanism of action and factors influencing their health benefits, 2022, 158, 09639969, 111575, 10.1016/j.foodres.2022.111575
    5. Samsul Rizal, Maria Erna Kustyawati, Udin Hasanudin, , The effect of inoculum types on microbial growth, β-glucan formation and antioxidant activity during tempe fermentation, 2022, 7, 2471-2086, 370, 10.3934/agrfood.2022024
    6. H Adhianata, A Pramana, N Rochmawati, Y Ditya, Development of Non-Soybean Tempeh from Cowpea Bean and Koro Bean, 2022, 1059, 1755-1307, 012062, 10.1088/1755-1315/1059/1/012062
    7. Samsul Rizal, Maria Erna Kustyawati, Theresia Santika Kusuma Putri, Teguh Endaryanto, Effect of substrate type and incubation time on the microbial viability of instant starter for premium tempeh, 2023, 8, 2471-2086, 461, 10.3934/agrfood.2023024
    8. Afolake Olanbiwoninu, Anna Greppi, Theresa Awotundun, Elijah Adegoke Adebayo, Giuseppe Spano, Diego Mora, Pasquale Russo, Microbial-based biofortification to mitigate African micronutrients deficiency: A focus on plant-based fermentation as source of B-group vitamins, 2023, 55, 22124292, 102996, 10.1016/j.fbio.2023.102996
    9. Sze Qi Teoh, Nyuk Ling Chin, Chun Wie Chong, Adiratna Mat Ripen, Syahmeer How, Joyce Jen Li Lim, A review on health benefits and processing of tempeh with outlines on its functional microbes, 2024, 9, 26668335, 100330, 10.1016/j.fufo.2024.100330
    10. Brajeshwar Singh, Shruti Sharma, Vitamin B12 Production by Lactobacillus Species Isolated from Milk Products, 2022, 1, 2583-4053, 48, 10.55544/jrasb.1.2.6
    11. Liza Nurohmah, Catur Sriherwanto, Imam Suja’i, Etyn Yunita, Aquafeed Biofloatation through Mycelial Hydrophobic Coating, 2023, 12, 2528-0864, 155, 10.20473/jafh.v12i2.38647
    12. Rania M. M. Abdel-Baki, Galal M. Khalafalla, Olfat S. Barakat, Marwa N. Ahmed, Production of vitamin B12 via microbial strains isolated from marine and food sources in Egypt, 2024, 23, 1687-4315, 309, 10.4103/epj.epj_267_23
    13. Vira Putri Yarlina, Mohammad Djali, Robi Andoyo, Siti Nurmilah, Mohd Nizam Lani, Metagenomic Insights into Enhancing Protein Content and Digestibility in Jack Bean (Canavalia ensiformis) Tempeh: Unraveling Microbial Dynamics During Fermentation, 2024, 27725022, 100588, 10.1016/j.afres.2024.100588
    14. Marcelo Gomes Soares, Gabriel Cicalese Bevilaqua, Érika Maria Marcondes Tassi, Vivian Consuelo Reolon Schmidt, Fermented foods and beverages: a potential in situ vitamin B12 biofortification – a literature review, 2023, 74, 0963-7486, 655, 10.1080/09637486.2023.2248422
    15. Vira Putri Yarlina, Dea Indriani Astuti, Mohammad Djali, Robi Andoyo, Mohd Nizam Lani, Effects of Combined Pure Cultures of Rhizopus sp. (Rhizopus oryzae, Rhizopus oligosporus, and Rhizopus stolonifer) on Tempeh Extract Yogurt as a Functional Food, 2023, 19, 15734013, 307, 10.2174/1573401318666220328101155
    16. Yixiao Zhou, Aien He, Baojun Xu, Natural resources, quantification, microbial bioconversion, and bioactivities of vitamin B12 for vegetarian diet, 2025, 463, 03088146, 140849, 10.1016/j.foodchem.2024.140849
    17. Reggie Surya, Nurlinah Amalia, William Ben Gunawan, Nurpudji Astuti Taslim, Marwan Ghafoor, Nelly Mayulu, Hardinsyah Hardinsyah, Rony Abdi Syahputra, Felicia Kartawidjajaputra, Gianluca Rizzo, Raymond Rubianto Tjandrawinata, Dionysius Subali, Rudy Kurniawan, Fahrul Nurkolis, Tempe as superior functional antioxidant food: From biomechanism to future development of soybean-based functional food, 2024, 71, 2603-557X, 1, 10.3897/pharmacia.71.e116748
    18. Samsul Rizal, Maria Erna Kustyawati, Defina Zulfa, Firda Rosida, Fairuzsita Naura Amalia Syifani, Ayu Dian Pratiwi Permatahati, Chemical properties, sensory characteristics, and antibacterial activity to Staphylococcus aureus of tempeh gembus fermented with Mosaccha inocula, 2024, 95, 14668564, 103742, 10.1016/j.ifset.2024.103742
    19. Muspirah Djalal, Nurul Fathanah, Serli Hatul Hidayat, Andi Fadiah Ainani, Dewi Sisilia Yolanda, A Nur Farahdiba Suriadi, Utilization of shrimp shell as a substitute ingredient in mineral and protein enrichment of tempeh steak product, 2023, 1230, 1755-1307, 012178, 10.1088/1755-1315/1230/1/012178
    20. Lavinia Florina Călinoiu, Răzvan Odochean, Gheorghe-Adrian Martău, Laura Mitrea, Silvia Amalia Nemes, Bianca-Eugenia Ștefănescu, Dan Cristian Vodnar, In situ fortification of cereal by-products with vitamin B12: An eco-sustainable approach for food fortification, 2024, 460, 03088146, 140766, 10.1016/j.foodchem.2024.140766
    21. Muzaffar Hasan, S.R. Arpitha, Chandrika Das, Rosalin Laishram, Minnu Sasi, Sandeep Kumar, Chirag Maheshwari, Veda Krishnan, Sweta Kumari, Jose M. Lorenzo, Manoj Kumar, Archana Sachdev, Anil Dahuja, Research trends and approaches for the nutritional and bio-functionality enhancement of fermented soymilk, 2023, 107, 17564646, 105698, 10.1016/j.jff.2023.105698
    22. Bożena Stodolak, Anna Starzyńska-Janiszewska, Dagmara Poniewska, Oncom from Surplus Bread Enriched in Vitamin B12 via In Situ Production by Propionibacterium freudenreichii, 2024, 14, 2076-3417, 4879, 10.3390/app14114879
    23. Siti Maryam, I Dewa Sastrawidana, I Ketut Sudiana, I Nyoman Sukarta, Nutritional Profile Analysis of Red Bean Tempeh Fermented Using <i>Rhizopus Oligosporus </i>at Different Time , 2024, 13, 2327-2716, 199, 10.11648/j.ijnfs.20241305.15
    24. Loveggie Bella Maitresya, Reggie Surya, Development of tempeh made from soybeans, black-eyed beans, and winged beans, 2023, 1200, 1755-1307, 012008, 10.1088/1755-1315/1200/1/012008
    25. Jyoti Prakash Tamang, Souvik Das, Sonam Lama, Gemiilang Lara Utama, Ratu Safitri, Roostita Lobo Balia, Namrata Thapa, Analysis of meta-transcriptomics and identification of genes linked to bioactive peptides and vitamins in Indonesian tempe, 2025, 202, 09639969, 115757, 10.1016/j.foodres.2025.115757
    26. Maria Erna Kustyawati, Tegar Suryawan, Samsul Rizal, Esa Ghanim Fadhallah, Khairun Nisa Berawi, In vivo Evaluation of Saccharomyces-Modified Tempeh as Potential Prebiotic and Probiotic Food using Mus musculus as an Animal Model, 2025, 5, 2774-3047, 218, 10.47352/jmans.2774-3047.242
    27. Youla Annatje ASSA, Nelly MAYULU, Reggie SURYA, Nurpudji Astuti TASLIM, William Ben GUNAWAN, Mrinal SAMTIYA, Felicia KARTAWIDJAJA, Aurielle Annalicia SETIAWAN, Alfredo WIJAYA, Fahrul Nurkolis, Soy and Algae Combination Using Tempe Fermentation Method: A Proposed Opinion for the Development of Functional Food , 2023, 43, 1989-208X, 10.12873/433assa
    28. Reggie Surya, Kanta Petsong, Andreas Romulo, David Nugroho, Felicia Tedjakusuma, Olifia Rombot, Microbial profile of agar-supplemented tempeh: a strategy towards enhancing the values of tempeh as a functional food, 2025, 1445, 1755-1307, 012005, 10.1088/1755-1315/1445/1/012005
  • Reader Comments
  • © 2020 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Metrics

Article views(7665) PDF downloads(641) Cited by(28)

Figures and Tables

Figures(1)  /  Tables(2)

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return

Catalog