Food Sci. Technol. Res., 9 (3), 237–241, 2003
Preparation of Low Salt Miso-Like Fermented Seasonings Using Soy-Oncom and Okara-Oncom (Fermented Soybeans and Okara with Neurospora intermedia) and Their Antioxidant Activity and Antimutagenicity
Masako MATSUO and Tokuo TAKEUCHI
Faculty of Home Science, Gifu Women’s University, 80 Taromaru, Gifu 501-2592, Japan
Received July 29, 2002; Accepted April 9, 2003
To develop a new type of Miso, low salt miso-like fermented seasonings were prepared with soybean Oncom(S- Oncom) and Okara Oncom (O-Oncom): those were fermented soybeans and fermented Okara with Neurospora inter- media, respectively. Their antioxidant activity and antimutagenicity were analyzed and both Oncom had higher -glu- cosidase activity than Koji (cooked rice molded with Aspergillus oryzae) with or without the presence of 4% salt and 2% ethanol. The strongest 1,1-diphenyl-2-picryl-hydrazyl radical scavenging, superoxide anion scavenging and anti- mutagenic activities were detected in the extracts prepared from the seasonings with S-Oncom, O-Oncom and Koji with both 70% ethanol and with water. These activities of O-seasoning are attributable to the isoflavone aglycones present and to water soluble substances. Based on these results, O-seasoning is deemed to be healthier than other low salt miso-like fermented seasoning prepared with steamed soybeans and Koji. Furthermore, the dishes prepared using O-seasoning were evaluated as acceptable.
Keywords: low salt miso-like seasoning, antimutagenicity, superoxide scavenging, Oncom, Neurospora intermedia
Miso has been found to be useful in suppressing hypertension pared by the following method described by Matsuo (1997a): (Kanda et al., 1999), colonic aberrant crypt foci (Ohara et al., Okara containing 60% moisture was autoclaved for 20 min under 2002), cerebrovascular diseases (Kanazawa et al., 1995) and ele- steam pressure at 0.18 Mpa, and pressed at 1.1 g/cm2 to a thick- vation of plasma cholesterol (Horii et al., 1990), and has been ness of 2.5 cm. Oncom starter containing spores of Neurospora used for several centuries in Japan, where it is known to contrib- intermedia FGSC 2559 (Fungal Genetics Stock Center, Kansas ute to good health. However, the consumption of Miso in Japan City, KS) was then inoculated to the Okara surface to give a final is currently decreasing due to the popularization of western food concentration of 0.3%. The inoculated Okara was incubated at and also because of its high salt content. In Indonesia, many 30˚C for 18 h tightly covered with plastic sheeting. After the kinds of Oncom, a traditional unsalted food fermented from spores had germinated, the Okara was fermented uncovered at pressed peanut cake or Okara (the solid waste of soybean milk 27˚C for another 19 h under 65% relative humidity. production) by Neurospora intermedia, have been developed for Preparation of S-Oncom S-Oncom was prepared by the consumption (Sastraatmadja et al., 2002). Neurospora belongs to method described above with the exception that dehulled four- a different subdivision (mycotina) than Aspergillus, the fungus cut soybeans (Fukuyutaka, Gifu, 2000) were used instead of used in fermentation of Miso; specifically, Neurospora belongs Okara. to the subdivision Ascomycotina while Aspergillus belongs to Preparation of miso-like seasonings Koji (cooked rice Deuteromycotina. If these fungi were used together in ferment- molded with Aspergillus oryzae) was provided by Marusanai ing Miso, its physiological functions might be improved via a Co., Ltd. (Aichi, Japan). Control seasoning (C-seasoning) con- collaborative effect of these fungi. The purpose of this study was sisting of 1 kg steamed soybeans and 0.5 kg Koji were mixed to develop new directions for Miso by fermenting three kinds of well, and salt and ethanol were added to the mixture at the final low salt miso-like seasoning using soybeans, soybean Oncom (S- concentration of 4% and 2%, respectively. Then the mixture was Oncom) and Okara Oncom (O-Oncom). These three seasonings fermented at 30˚C. S-Oncom seasoning (S-seasoning) and O- were then judged for their sensory effects as seasoning, and their Oncom seasoning (O-seasoning) were prepared following essen- functions as antioxidants and antimutagens were examined. tially the same method except that the steamed soybeans were replaced with S-Oncom or the mixture of 90% S-Oncom and Materials and Methods 10% O-Oncom, respectively. During fermenting, pH, titratable Preparation of O-Oncom Okara powder (30 mesh) was acidity, reducing sugars and formol nitrogen of each seasoning provided by Misuzu Co. (Nagano, Japan). O-Oncom was pre- were periodically monitored. The fermenting period until these values reached a constant level was 6 weeks for C-seasoning and E-mail: [email protected] 5 weeks for S-seasoning and O-seasoning. Abbreviations: S-Oncom, soybean-Oncom; O-Oncom, Okara-Oncom; C-sea- Component analysis of the seasonings Titratable acidity, soning, Control seasoning; S-seasoning, seasoning used S-Oncom; O-sea- formol nitrogen, soluble nitrogen and reducing sugars were de- soning, seasoning used O-Oncom; O2 , superoxide anion; DPPH, 1,1-di- phenyl-2-picryl-hydrazyl; IC50, fifty percent inhibition. termined by the standard analytical methods for Miso as de- 238 M. MATSUO & T. TAKEUCHI scribed by the Ministry of Agriculture and Forestry (1977). Pro- egg and beaten well. The butter and flour mixtures were beaten tein solubility and protein hydrolysis were calculated individual- together until smooth, poured into fluted aluminum baking cups, ly from the percentage of soluble nitrogen and formol nitrogen to and baked in a moderate oven at 135˚C for 33 min. the total nitrogen, respectively. Organic acids were analyzed Recipe for sesame miso sauce cooked with miso-like sea- using a Finepak SIL-5 column (4.5 250 nm, Jasco Co., Tokyo) soning Sixty grams miso-like seasoning, 25 g ground sesame and 0.5% phosphoric acid as a mobile phase with monitoring by seeds and 30 g sugar were beaten together until smooth, and then absorbance at 210 nm (Tachi et al., 1993). Ethanol, acetaldehyde mixed well with 22 ml water. The mixture was brought to a boil and ethyl acetate were analyzed by a PEG 6000 glass column to melt the sugar. (25% 545U, 60–80 mesh, 3 mm 2 m, GLC Science, Tokyo) at Recipe for oden miso sauce cooked with miso-like season- 80˚C and detected by FID (GC-15A PFsc, Shimadzu, Tokyo) ing One hundred grams miso-like seasoning, 30 ml sake, 20 g (Honma et al., 1973). Color was defined according to the Hunter honey, 20 g sugar and 5 ml water were beaten together until color scale using a color difference meter (ND-1000 DP, Nippon smooth, and the mixture was boiled for 1 min. Densyoku, Tokyo), and hardness was measured by a creep meter Sensory evaluation In the sensory analysis of the un- (RE-3305, Yamaden Co., Ltd., Tokyo) at compressibility 30% heated miso-like seasoning, 24 female students (21–22 years and speed 5 mm/s. old) were asked to judge specified items. For the test of the Enzyme activities The supernatants of 20% homogenate dishes cooked with miso-like seasoning, 28 female students (21– of Koji, S-Oncom and O-Oncom were dialyzed against water for 22 years old) were asked to evaluate organoleptically using a 5- use as the respective enzyme solution. The activities of protease point rating scale: good ( 2), somewhat tasty ( 1), neither good (E.C. 3.4.21.14), leucine aminopeptidase (E.C. 3.4. 11.1) and nor poor (0), rather poor ( 1), poor ( 2). glutaminase (E.C. 3.5.1.35) were assayed by the methods for soy The data from the sensory test on the unheated miso-like sea- source (Nakadai, 1985), and lipase (E.C. 3.1.1.3) activity was soning were analyzed by the paired preference test (Roessler et assayed by following the procedure described by the Worthing- al., 1978) and the data from the taste tests of the dishes were ana- ton enzyme manual (Lilian, 1977). For -glucosidase (EC lyzed by the Tukey’s multiple range test using the statistical anal- 3.2.1.21), we followed the method described by Iwashita et al. ysis system SPSS. Other data were expressed as the means (1998). Specifically, the mixture of 0.1 M acetate buffer (pH 4.5) standard error. The differences between the values were analyzed 0.25 ml, 8 mM p-nitrophenyl -glucoside 0.25 ml and enzyme by Tukey’s multiple range test solution 0.25 ml was incubated at 40˚C for 30 min, and then the Antioxidant activity and 1,1-diphenyl-2-picryl-hydrazyl reaction was stopped by addition of 0.1 M Na2CO3 2.5 ml. (DPPH) radical scavenging activity The seasoning extract (0.3 Enzyme activity was calculated from the increase of absorption ml) was added to a mixture of 0.1 M acetate buffer (pH 5.5, 0.3 which developed by p-nitrophenol at 400 nm. One unit (U) of - ml) and 0.2 mM DPPH in ethanol, and the decrease in absor- glucosidase activity was defined as the amount of -glucosidase bance at 517 nm for 1 min was measured at room temperature. which produced 1 mol of p-nitrophenol per min in the reaction Fifty percent inhibitory concentration (IC50) was assayed as the mixture. weight of the seasoning to scavenge 50% DPPH radical. Preparation of extract of a seasoning For unheated Superoxide anion (O2 ) scavenging activity Following the extract, the lyophilized seasoning was shaken with 5 volumes of method described by Oyanagi (1984), a mixture of 65 mM phos- n-hexane for one night at room temperature. The residue was phate-borate buffer (pH 8.2, 0.1 ml), 0.5 mM xanthine (0.1 ml), shaken with 5 volumes of 70% ethanol, and the residue of 70% 10 mM hydroxylamine (0.05 ml), the seasoning extract, and xan- ethanol extract was subsequently shaken with 5 volumes of water thine oxidase (EC 1.2.3.2, Wako Pure Chemical Industries, Ltd., at room temperature. The seventy percent ethanol extract and the Osaka) diluted 100-fold with 50 l water was incubated for 30 water extract were individually concentrated. For heated extract, min at 37˚C. Naphthylethylenediamine reagent (1 ml) was added the lyophilized seasoning was shaken with the same kinds of sol- to the mixture, and the absorbance at 550 nm was measured. vents as the unheated extract for 12 h at 60˚C, and each extract Antimutagenicity Antimutagenicity against N-nitrosodim- was individually concentrated. ethylamine (NDMA) was assayed by a modified Ames test using
Pretreatment of miso-like seasoning before cooking The Salmonella typhimurium TA 100 in the presence of S9 pretreated seasonings were heated for 10 min at 80˚C to inactivate their with acetone and fasting (Ebata & Furukawa, 1997). The His enzymes produced by A. oryzae and N. intermedia, followed by revertant colonies were counted after incubation for 48 h at 37˚C, homogenization by a grinder mill (Millser 300DG, Iwatani Inter- and the suppression ratio was calculated as follows: 1 (rever- national Corp., Osaka). tants/test plate spontaneous mutants/test plate)/(revertants/con- Recipe for custard cream cooked with miso-like season- trol plate spontaneous mutants/control plate) 100. The data ing Twenty grams egg yolk and 10 g miso-like seasoning were presented in Table 7 show the means standard error of three blended together, and 50 g sugar was added. The mixture was measurements. beaten until smooth, and 13 g sifted soft flour was added, 180 ml Isoflavone analysis The extract from the seasoning with milk was added gradually, and the mixture was boiled gently for 70% methanol was analyzed by HPLC with Crestpack C18S (Jas- 1 min. co Co. Tokyo) using a linear gradient from 10% to 60% metha- Recipe for cupcakes cooked with miso-like seasoning One nol containing 4% acetate as a mobile phase and detected at 262 gram of baking powder was added to 50 g sifted soft flour. Fifty nm (Matsuo, 1997b). grams unsalted butter and 20 g miso-like seasoning were creamed until soft. The butter mixture was mixed with 40 g sugar Results and Discussion until it was light and fluffy, and then blended with 50 g whole Chemical components of steamed soybeans, S-Oncom and Preparation of Low Salt Miso-Like Fermented Seasonings 239
O-Oncom The chemical components of steamed soybeans, S- soft, and the high activities of xylanase and cellulase of N. inter- Oncom and O-Oncom were compared and the results of this media (Matsuo, 1997a) are assumed to have contributed to the comparison are shown in Table 1. The protein content of S- hydrolysis of the soy cell wall. The chemical components of S- Oncom was higher than that of the steamed soybeans. The pro- seasoning were intermediate between O-seasoning and those of tein content of O-seasoning, in which 10% O-Oncom was substi- C-seasoning. tuted for S-Oncom was higher than that of C-seasoning. Enzyme activities of Koji, S-Oncom and O-Oncom Four percent salt and 2% ethanol were the minimum amounts essen- tial for fermentation of Miso (Okada & Takeuchi, 1977). Prior to Table 3. Physical and chemical characteristics of the miso-like seasonings. the preparation of the miso-like seasoning, the enzyme activities Miso-like seasoning Item of Koji, S-Oncom, O-Oncom, as well as their tolerance to 4% C-Seasoning S-Seasoning O-Seasoning salt and 2% ethanol were compared (Table 2). The lipase and - Water (%) 52.9 52.0 51.8 glucosidase activities of O-Oncom and S-Oncom were higher Salt (%) 3.8 3.7 3.8 than those of Koji, and this ranking did not change under the pH 5.2 5.8 5.7 Titratable acidity I (ml) 11.3 9.1 8.9 presence of 4% salt and 2% ethanol. Soy isoflavone glycosides Titratable acidity II (ml) 11.0 13.2 12.8 are hydrolyzed to aglycones by -glucosidase, and the aglycones Total sugar (%) 16.9 17.5 18.0 Reducing sugar (%) 12.1 13.4 14.5 show higher antioxidant activity than that of glycosides (Hsieh & Sugar hydrolysis (%) 71.9 76.3 81.0 Graham., 2001). If S-seasoning is fermented with the substitution Total nitrogen (%) 1.9 2.1 2.0 of S-Oncom for soybeans, the antioxidant activity of the season- Soluble nitrogen (%) 1.1 1.1 1.2 Formol nitrogen (%) 0.5 0.6 0.7 ing is higher than that of C-seasoning. The -glucosidase activity Protein solubility (%) 56.7 53.0 56.4 of O-Oncom was the greatest among the three kinds of fer- Protein hydrolysis (%) 22.6 27.5 32.5 mented products. The antioxidant activity of O-seasoning, which Ethanol (%) 2.38 1.93 1.57 Acetaldehyde (mg%) 10 19 20 is fermented with a partial substitution of O-Oncom for S- Ethyl acetate (mg%) 7 9 9 Oncom, is stronger than that of S-seasoning . Citric acid (mg%) 107 142 135 Components of seasoning Lactic acid (mg%) 112 93 91 Three kinds of low salt miso- Acetic acid (mg%) 223 230 219 like seasoning were prepared, and their components were com- Pyroglutamic acid (mg%) 29 36 34 pared (Table 3). The titratable acidity of O-seasoning was low Color L 35.2 24.3 26.3 a 9.5 10.9 10.8 and the amount of lactate production was little. The hydrolysis b 16.5 12.6 14.0 ratio of both protein and saccharide was high, and the presence Hardness ( 103 N/m2) 12.2 4.3 4.0 of enzymes of N. intermedia can be attributed to this hydrolysis during both the preparation of O-Oncom and the fermentation of O-seasoning. During the fermentation, the ethanol level de- creased, but the levels of acetaldehyde and ethylacetate in- creased. The color was slightly reddish and dark, indicating that a Table 4. Sensory analysis of the unheated miso-like seasonings. Responder number Maillard reaction with reducing sugars and formol nitrogen Question Items might occur during the fermentation. Hardness was extremely S-Seasoning O-Seasoning Texture 5 19* Color 16 8 Which do you like? Gloss 5 19* Table 1. Chemical components of steamed soybean, S-Oncom and O- Flavor 16 8 Oncom. Taste 19* 5 Total evaluation 18* 6 Components Steamed soybean S-Oncom O-Oncom Sweet 15 9 Moisture 63.5 59.8 50.9 Salty 10 14 Crude protein 13.8 17.4 13.1 Which do you feel strongly? Sour 8 16 Crude fat 7.0 9.0 4.5 Bitter 7 17* Carbohydrate 13.9 11.8 29.7 Umami 19* 5 Ash 1.8 2.0 1.8 Twenty-four female students (21–22 years old) were asked to judge speci- Total 100.0 100.0 100.0 fied items. *p 0.05.
Table 2. Effect of 4% salt and 2% ethanol on the enzyme activities of Koji, S-Oncom and O-Oncom. Salt Ethanol Enzyme activity (U/g) Enzyme activity (%) Enzymes (%) (%) Koji S-Oncom O-Oncom Koji S-Oncom O-Oncom Protease 0 0 65.2 19.7 35.7 100 30 55 4245.4 15.5 9.5 70 24 15 Leucine aminopeptidase 0 0 4.2 2.1 3.0 100 50 71 422.31.40.0 54 33 0 Glutaminase 0 0 0.15 0.15 0.30 100 100 200 220.05 0.17 0.23 30 110 150 Lipase 0 0 0.087 0.131 0.357 100 151 410 420.011 0.025 0.032 12 29 37 -Glucosidase 0 0 113 3000 7750 100 266 688 4287 2650 7420 77 235 658 240 M. MATSUO & T. TAKEUCHI
Table 5. Sensory evaluation scores of the dishes prepared with the miso-like seasonings. Western style dishes Japanese style dishes Evaluation scores Custard cream Cupcake Sesame-Miso sauce Oden-Miso sauce C-Seasoning O-Seasoning C-Seasoning O-Seasoning C-Seasoning O-Seasoning C-Seasoning O-Seasoning Total 33 26433050455048 Mean 1.2 0.9 1.5 1.1 1.8 1.6 1.8 1.7 Twenty-eight female students were asked to evaluate organoleptically using a 5-point rating scale: good( 2); somewhat tasty( 1), neither good nor poor(0); rather poor( 1), poor( 2).