JFS: Microbiology and Safety

Natto Characteristics as Affected by Time, Bacillus Strain, and Time Q. WEI, C. WOLF-HALL, AND K.C. CHANG

ABSTRACT: Natto was made in a laboratory scale from using 2 steaming times, 9 Bacillus natto strains, and 6 fermentation times. Natto characteristics including color, firmness, viscosity, ammonia content, and population were determined. The highest viscosity value of the final products was the natto inoculated with B. natto “Itobiki strain”; the 2nd was natto with B. natto NRRL B-3383 strain. A higher steaming time reduced fermentation time and ammonia content in the final products. A combination of soaking at the room temperature for 20 h (weight increase ratio: 2.1 to 2.3), steaming at 121 °C for 40 min, inoculated with “Itobiki strain” or NRRL-3383 strain, and fermented at 40 to 42 °C for 18 h was able to produce good natto products in a laboratory scale. Key words: natto quality, fermentation, Bacillus subtilis (natto), soybean, processing

Introduction 102 to 104 cells/g of the starter. From the standpoint of the ap- ATTO IS A TRADITIONAL FERMENTED SOYBEAN FOOD, ORIGI- pearance, color, and hardness, 18 to 20 fermentation hours Nnating in the northern parts of Japan about 1000 yeas were sufficient. Matsumoto and others (1995) investigated the ago (Fukushima 1986). Similar products popular in the effect of soybean steaming conditions during manufacturing are “shi” in China, “thua-nao” in , on the quality of natto products. From sensory evaluation of “chung-kook-jong” in , and “tao-si” in Philippines the final natto products, they concluded that the optimum (Ohta 1986). There are 3 types of natto in Japan: itohiki-natto, steaming time for was 30 to 40 min at 1.5 kg/cm2 yukiwai-natto, and hama-natto. Itohiki-natto is very popular pressure. There is a controversy on fermentation time from and produced in large amounts. The word “natto “ usually the different aspects of consideration. Sakurai (1960) believed refers to itohiki-natto (Fukushima 1986). 6 h (40 to 42 °C) of fermentation to be favorable, judging from Natto is a low-cost, high-nutrition, and long shelf-life food. the palatability, digestibility, and yield of finished product. Good quality natto products should be covered with a white- Ohta (1986) demonstrated 18 to 20 h (40 to 42 °C, 80 to 90% of colored mucous substance, have characteristic flavor, palat- RH) to be necessary for complete fermentation. Maruo and ably soft texture, light yellow color and is able to generate silky Yoshkawa (1989) suggested that natto product was considered and sticky mass when mixed/stirred with a pair of chopsticks. to be of good quality after 16 to 18 h of fermentation (42 °C, 85 During the manufacturing process, nutritional value is im- to 90% of RH). After studying the changes in molecular weight proved through heat denaturation of soy proteins, trypsin in- of proteins during fermentation, Ikeda and Tsuno (1985) im- hibitors, and bacteria- hydrolysis of the proteins into plied that natto formation appears to occur at 14 h of fermen- easily digestible peptides. After sufficient fermentation, the in- tation of soybean. tact soybeans are covered with a white-colored viscous sub- Complex biochemical reactions occur during natto fer- stance and have a slimy appearance, softer texture, and mentation (Hayashi 1959; Ikeda and Tsuno 1984, 1985; Kanno unique flavor (Steinkraus 1983). After fermentation the natto and Takamatsu 1987; Kanno and others 1982, 1985; Kusano products may be eaten without heating, and are stored in a 1969, 1971; Maruo and Yoshkawa 1989; Ohno and others 1990; cooler or freezer in the supermarket for purchasing. In Japa- Ohta 1986; Taira and others 1983; Taguchi and others 1986). nese homes, traditional natto is eaten with a mixture of thinly The degree and speed of the reactions, and substances pro- chopped green onion, seaweed, , and a small amount duced depend on the conditions of soaking, steaming, fer- of soy and served as a side dish along with steamed mentation, and bacteria strains as well. Natto has different (Ohta 1986). Natto also can be used as a flavoring agent for aroma, texture, flavor, viscosity, and amount of mucus under preparing meat, seafood, and vegetable dishes and as an in- various combinations of processing. In the literature, there are Food Microbiology and Safety gredient for sauce production (Lin 1991). no small-scale laboratory methods available for evaluating the Natto contains about 59.5% moisture, 16.5% protein, and suitability of soybean cultivar for natto processing. The objec- 10.0% lipid (Ohta 1986). Natto manufacture generally includes tive of this study was to determine natto product characteris- steps of soaking, steaming/cooking, inoculating, and incubat- tics as affected by various treatments of steaming times, bac- ing (Steinkraus 1983; Ohta 1986; Maruo and Yoshkawa 1989). teria strain, and fermentation times. It is imperative that a lab- The quality of natto product varies greatly with the conditions processing scheme could be identified for future investigation of soaking, steaming, fermentation, and cultivars of soybean of the suitability of soybeans for natto making. (Sakurai 1960; Maruo and Yoshkawa 1989). Matsumoto and others (1993) studied the effect of temperature and inoculum size on natto’s appearance, number of B. natto, temperature, Materials and Methods hardness, and color during fermentation. The results indicat- ed that natto’s fermentation preceded in good condition at Soybean 35 °C, with an inoculation of 104 to 106 cells/g, or at 40 °C, with Soybean samples SSND91-2330 (Danatto) were obtained

© 2001 Institute of Food Technologists Vol. 66, No. 1, 2001—JOURNAL OF FOOD SCIENCE 167 1.03, Ϫ = L A portion of 1.8-mL double distilled water was added to a A portion of 1.8-mL double distilled water cooled to room After steaming, the steamed beans were Fifteen grams of the natto product was weighed and shak- The ammonia content of the natto product was deter- After soaking to achieve weight increase ratio of 2.1 to 2.3 increase ratio of to achieve weight After soaking beans was weighed out after A 50-g portion of steamed was measured The color of the steamed beans and natto = +1.14). L mashed sample of 3 g of natto. The pH value was measured mashed sample of 3 g of natto. The pH 61, Orion Research, with a digital pH/millivolt meter (Model Boston, Mass., U.S.A.). prod- Natto testing firmness property. before temperature inhibit fermentation, ucts were stored in a refrigerator to before testing and were equilibrated to room temperature (Model Machine Testing Universal firmness with an Instron A Kramer sheer 1011, Instron Corp., Canton, Mass., U.S.A.). used. The speed of the cell and a weight beam of 500 kg were set at 20 mm/min. crosshead and the recording chart was (kg) to sheer through The results were expressed as the force 100 g of the steamed beans or natto. en vigorously in 15 mL of pH 7.4, 0.2 M sodium phosphate buffer for 2 min. The mixture was allowed to settle for at least a minute, so that the foam rose to the top. An aliquot of 500 mL of the extract was pipetted into the plate cup of a Mass., viscometer (Stoughton, Cone/Plate Wells-Brookfield U.S.A.) and measured at 3.0 rpm at 30C. mined using a modified colorimetric method (AOAC Method 973.25 1995). The modified bromine solution was prepared with 10 mL of NaOH solution (50% of NaOH in carbon diox- ide-free water) and 90 mL of double distilled water by adding 1.0 mL of concentrated bromine reagent. After mixing a por- tion of sodium hydroxide, fresh thymol, and bromine solu- tion, the color intensity of the mixture in a 2.5% phospho- tungstic acid solution (180 mL for 20-g bean sample) was re- lated to the ammonia content. Separation of the clear color solution and the turbid portion was conducted by using n- butanol. The absorption of the color solution was measured and steaming (121and steaming 25, 30, 35, or a period of time (20, °C) for then the was recorded, weight of the sample 40 min), the weighed. of the sample and were picked out broken beans beans of the weight of broken of the percentage The ration bean ratio as the broken beans were recorded to the intact of the steamed beans. room temperature on an aluminum cooling to the ambient a steamed bean was mashed by using film. A portion of the The moisture content was determined mortar and a pestle. after drying at 105 Sci- °C for 16 h in a hot air oven (Precision Ill., U.S.A.), and expressed as the per- entific Inc., Chicago, of the sample. centage of the weight Gardner CL-23 Lab Colorimeter, with a colorimeter (Model U.S.A.) using a stan- Laboratory Instrument, Bethesda, Md., dard white tile as reference (L = 91.94, a pH value of natto products products Firmness of steamed beans and natto Viscosity of natto Ammonia content of natto Broken bean ratio of steamed beans steamed ratio of bean Broken of steamed beans Moisture content beans and natto products Color of steamed b B. 660 was added ) for 20, 25, 30, 35 B. natto used in this study. used in this 2 values were measured —Vol. 66, No. 1, 2001 66, No. —Vol. 660 Bacillus natto cfu/mL. The cell pellet from 200 mL boxes (90 × 90 × 35 mm). The inocu- 8 ® to 10 7 JOURNAL OF FOOD SCIENCE population in NB increased dramatically after 2 to 14 population in NB increased dramatically 20To- of Corporation Food natto (Takahashi °C. Itobiki Five milliliter inoculum preparation of After removing impurities, the soybean samples were After removing impurities, the soybean Several colonies from nutrient agar or 10 g of a com- Several colonies from nutrient agar There were 9 strains of 9 strains were There Ϫ and 40 min in a retort (Reid Boiler Works, Inc., Bellingham, Inc., Works, Boiler (Reid and 40 min in a retort was built up in 15 min in the retort U.S.A.). Pressure Wash., and released gradually in 5 min after the steaming time was ended. Steamed beans were then covered immediately with an aluminum foil and weight was measured. The beans (30 to 40 C) were inoculated quickly with the bacteria inoculum. to 120 g of steamed beans. Steamed beans were placed into sandwich Styrofoam washed, soaked in water at the room temperature (21 to washed, soaked in water at the room 23 increase ratio °C) for approximately 16 h until the weight (WIR) to the original dry of 2.1 to 2.3, in the range weight was and then drained. Soaked beans were placed in a steaming shelf and steamed at 121 °C (1.2 kg/cm of NB centrifuged at 12,000 rpm (22100 × g) (BECKMAN J2- of NB centrifuged at 12,000 rpm (22100 Alto, Calif., U.S.A.) for HS, Beckman Instruments, Inc., Palo phosphate 25 min was diluted in 20 mL of Butterfield’s was used for inocu- buffer to prepare a suspension, which lating the steamed soybeans. mercial natto product were cultured in a sterilized nutrient mercial natto product were cultured in flask) at 40broth (NB) (200 ml per 250-ml Erlenmeyer °C shaker (200 rpm). for approximately 16 h on a rotary natto h of growth at 40 to 42 °C. The OD 168 lum and beans were thoroughly mixed together, and covered mixed together, thoroughly lum and beans were with a piece of perforated plastic film on the top of packaged beans. Inoculated beans were fermented in a bacteriological Convection, Gravity Type, Dry incubator (Stabil-Therm BLUEM, Blue Island, Ill., U.S.A.) at 40 to 42 °C for 10, 12, 14, to 90% of RH. under 85 18 and 20 h, respectively, Inoculation and incubation Preparation of soybean as fermentation medium Preparation of soybean as fermentation value of the NB inoculated with the 10-gram commercial value of the NB inoculated with the populations were in bean was about 1.5, and the bacteria the range of 10 with a spectrophotometer (Model 20D, Milton Roy Compa- Roy Milton 20D, (Model with a spectrophotometer The cells produced from an in- U.S.A.). N.Y., Rochester, ny, population were cubation period of 16 h with a high viable 16 h of growth, OD harvested from 200 mL of NB. After Preparation of inoculum Bacteria strains Bacteria from the Agronomy Seed Farm (Casselton, N.D., U.S.A.). The U.S.A.). N.D., (Casselton, Farm Seed the Agronomy from (21 to room temperatures stored at the ambient beans were 23 used. °C) until Five of them were obtained from the National Center of Ag- the National Center were obtained from Five of them Research (Agricultural Research Service ricultural Utilization B- Peoria, Ill., U.S.A.). They were NRRL Midwest Area, USDA, B-3384 (IAM 1109), NRRL B-3385 3383 (IAM 1071), NRRL B-3386 (IAM 1207), and NRRL B-14202. (IAM 1143), NRRL came from the 15245) (patent strain) (ATCC strain One U.S.A.). Md., (Rockville, Collection Culture Type American from commercial natto products The other 3 were cultured (Yamada Yamada and Mito). Yamada, (brand name: Itobiki, natto Japan) (Kumamoto, and Mito Akita, Japan) , from Japan and stored in a freezer products were obtained at kyo, Japan) was purchased, frozen, from a local grocery kyo, Japan) was purchased, store. Natto Quality as Affected by Processing . . . Processing by as Affected Quality Natto

Food Microbiology and Safety Natto Quality as Affected by Processing . . . with a spectrophotometer (Model 20D, Milton Roy Co., Table 1—Effect of steaming times on selected physical Rochester, N.Y., U.S.A.) at 580 nm. The ammonia content properties of steamed beans1 (mg/100g natto product) was calculated by using a standard Steaming times (min) curve of ammonia. Properties 20 25 30 35 40 Population of B. natto Moisture content 60.09a 59.55a 59.48a 60.72a 60.05a The viable populations of cells in the inoculum and fin- (%) (0.51) (1.20) (0.40) (1.77) (0.85) ished natto products were determined by the aerobic plate Broken bean ratio 1.49a 2.29a 2.10a 2.07a 2.53a count procedure using the spread plate method (FDA, Bac- (%) (0.72) (1.45) (0.81) (0.58) (1.90) teriological Analytical Manual 1995). Color (L value) 42.13a 42.35a 40.50b 40.42b 39.41c (0.84) (0.40) (0.27) (0.75) (1.03) Statistical analysis Firmness (kg) 45.8a 45.5a 46.5a 45.2a 46.1a Two completely randomized single-factor designs with (1.79) (2.45) (1.73) (1.92) (0.74) five-level intervals of steaming time (20, 25, 30, 35, and 40 a-cMeans (SD) of at least 3 replicates. Means within the same row with different letters differed significantly (p < 0.05). min) and 9-level types of bacterial species (NRRL B-3383, 1Soybean sample soaked in water at the room temperature for 16 h to and steamed at 121 °C. NRRL B-3384, NRRL B-3385, NRRL B-3386, NRRL B-14202, ATCC 15245, Yamada natto strain, Mito natto strain, and Ito- Table 2—Effect of steaming times on selected physical biki natto strain) were applied for determining the effect of and chemical properties of natto1 the conditions of steaming time and bacteria strain. One ran- domized complete block design with factorial arrangement Steaming times (min) was used to determine the effect of fermentation time (10, Properties 20 25 30 35 40 12, 14, 16, 18, and 20 h) of natto processing in which there Color 35.74a 36.10a 34.79a 35.61a 35.98a were 4 observations per treatment of incubating time within (L value) (0.84) (0.57) (1.94) (0.80) (0.47) each block of steaming time. The other factor to affect fer- pH Value 266.78a 240.50a 218.90a 212.43a 220.85a mentation time was bacteria strains with two levels (two (0.18) (0.17) (0.19) (0.54) (0.29) types of B. natto strains) of effect. Analyses of variance Firmness (kg) 51.8a 44.4b 38.3c 37.4c 35.0c (ANOVA) and Duncan’s Multiple Range Test were conducted (2.75) (3.09) (2.06) (1.80) (1.87) using the statistical analysis system (SAS Institute, Inc. 1989) Viscosity (cps) 21.5a 30.2a 31.9a 42.2a 21.5a for color, pH, firmness, viscosity, ammonia content, and bac- (6.10) (17.33) (19.19) (4.57) (2.23) teria population of steamed bean and natto product analy- Ammonia content 266.78a 240.50a 218.90a 212.43a 220.85a ses, respectively. (mg/100g)2 (25.28) (13.70) (21.36) (11.13) (17.77) Bacteria 5.5a 2.8b 2.9b 4.6ab 6.6a population (1.70) (0.65) (0.90) (1.88) (2.43) Results and Discussion ×109 (cfu/g)3 a-cMeans (SD) of at least 3 replicates. Means within the same row with Effect of steaming time different letters differed significantly. (p < 0.05). 1Soybean sample soaked in water at the room temperature for 16 h, steamed Statistical analysis showed that different steaming times at 121 °C inoculated with a commercial Itobiki natto culture (Takahashi Food did not significantly (p > 0.05) affect moisture content, bro- Corporation of Tokyo, Japan), and fermented at 40–42 °C and 85–90% of RH for 16 h. ken bean ratio, and firmness of the steamed beans (Table 1). 2Data based on a dry matter basis. Moisture content of the natto samples in Steaming time longer than 25 min made the steamed beans the range of 59.0–61.0%. 3Data based on a wet matter basis. darker gradually; however, steaming time did not affect the color of the final fermented product (Table 2). In a study of suitability of soybean varieties grown in Ibaraki prefecture (Japan) for natto production, Taira and others (1983) report- changing the steaming time (Table 1); however, the firmness ed that the average of moisture content and broken bean ra- of the final fermented natto product was affected (Table 2). tio after steaming were 59.5% (58.1 to 60.6%) and 5.8% (1.5 to When steamed for 20 min, the natto product had the highest 15.0%), respectively. In their study, WIR of the samples of Instron reading (kg) compared with that of the beans various soybean cultivars after soaking was 2.48 (2.55 to steamed for 25, 30, 35 and 40 min. There was no significant 2.41). The small seed size cultivars were steamed for 23 min difference (p > 0.05) in the firmness of natto products be- at 2.0 kg/cm2, the middle and large seed size cultivars were tween steaming times of 30, 35 and 40 min (Table 2). In the steamed for 35 min at 1.9 kg/cm2. In the other study of suit- studies of Taira and others (1983 and 1987), the firmness de-

ability of small seed cultivars for natto processing, Taira and termination method differed from the method in this study. Food Microbiology and Safety others (1987) reported that the moisture content and broken Although different steaming periods did not significantly (p > bean ratio after steaming were in the range of 58.2 to 61.6% 0.05) influence the firmness of steamed beans, however, the and 0 to 18%, respectively. In their research, WIR of the culti- longer steaming times might have made the microstructure vars of the small seed type after soaking was 2.19 to 2.49 and of the beans more penetrable to bacteria, thereby, affecting the samples were steamed for 20 to 30 min under the pres- the texture of the final products. sure of 1.7 to 2.0 kg/cm2 (Taira and others 1987). The broken The viscosity (21.5 to 42.2 cps) and ammonia-nitrogen bean ratios in this study were lower (1.49 to 2.53%) than that content (approximately 85 to 107 mg/100g), important crite- reported by (Taira and others 1987) and the moisture con- ria for the quality of natto products, were statistically the tents were similar to their values. In our study, the WIR of same (p > 0.05) in the different steaming periods (Table 2). Danatto soybean after soaking was in the range of 2.1 to 2.3 Taira and others (1983 and 1987) did not report viscosity val- and the steam pressure used to cook Danatto was 1.2 kg/cm2 ues of the natto products, but ammonia-nitrogen contents (121 °C). (215 to 360 mg/100g on a dry matter basis) were generally The firmness of steamed beans was not affected by higher than that observed in our study. In Table 2, the firm-

Vol. 66, No. 1, 2001—JOURNAL OF FOOD SCIENCE 169 cfu/g), the firm texture (62.6 kg) of its natto firm texture (62.6 cfu/g), the 9 There seems to be a disagreement in the literature about There seems to be a disagreement in the 6.25. Fermentation The pH value of the steamed bean was optimum duration of fermentation time for industrial natto optimum duration of fermentation time 1960) to 20 h making, which ranges from 6 h (Sakurai the fermenta- (Beuchat 1983). Most of the reports preferred Yoshkawa Maruo and tion time of 16 to 18 h (Ohta 1986; 1989). 1). Using “Itobiki lead to an increase in pH value (Figure natto products strain” and under 40 min steaming condition, during 10 h of fer- showed the quick increase in pH value mentation (pH = 7.21) (Figure 1). There were no significant changes (p > 0.05) in pH from the 10 to 20 h of fermentation (pH = of natto beans cooked for 35 min 7.58). The pH value not had a significant was 6.59 in 10 h of fermentation and did (pHchange (p > 0.05) until 20 h of fermentation = 7.87). In- steamed for 40 min, oculated with NRRL B-3383 strain and 14 h of fermenta- natto products had the pH value 7.08 until Figure 2—Firmness of the natto products made with dif- ferent fermentation times at 40 to 42 °C Effect of fermentation time products, and the low viscosity (5.9 cps). The pH of the (5.9 cps). The and the low viscosity products, and that acidic (pH 6.2 to 6.8) beans was slightly steamed 7.6) (Maruo alkaline (pH 7.2 to was slightly natto products of the insufficient fermentation The 1989). Yoshkawa and natto pH with a lower strain was associated NRRL B-3384 made with the strain from the com- value (6.79). The natto had a soft texutre (48.2 kg), high viscosi- mercial Itobiki natto content (0.121% on a wet matter ty (23.0 cps), and ammonia is 301.55 mg/100 g on a dry basis), which basis equivalent to (0.1 to 0.15% or 100 to 150 mg/100g in the acceptable range consumers as described by Maruo and on a wet basis) of strain” with “Itobiki The natto produced (1989). Yoshkawa aroma of natto and a dense mucous possessed a very strong of natto made from NRRL B-3383, appearance. The quality the (43.9 kg), and 9.9 cps viscosity was with a softer texture strain”. “Itobiki from next best to that made ly, a poor one for natto processing, because of its low popu- of its low because natto processing, one for a poor ly, × 10 lation (0.4 6 cfu/g to 10 7 4 to 10 6 —Vol. 66, No. 1, 2001 66, No. —Vol. cells/g of the starter, judging cells/g of the starter, 4 grew well on the PCA plates when grew well on the PCA cfu/g bean. 9 to 10 2 cultures, such as the NRRL B-3386, NRRL B. natto and 10 8 natto

B. cfu/g) after 16 h growth on the Danatto soybean. cfu/g) after 16 h growth on the Danatto 9 JOURNAL OF FOOD SCIENCE The 9 strains of natto fermentation proceeded According to the literature, The strains from the commercial Yamada, Mito natto, Yamada, from the commercial The strains A high-quality natto product is supposed to have a high is supposed to have natto product A high-quality from the appearance, color, and hardness for 18 to 20 fer- for 18 to 20 and hardness color, appearance, the from 1993). From a pre- mentation hours (Matsumoto and others initial population in liminary study conducted in our lab, the of 10 the inoculated beans was in the range we checked for the viability of the cultures upon receipt. we checked for the inoculated onto steamed (35 min) Dan- Therefore, they were at 40 to 42atto soybean and fermented 16 h, and the °C for content of the final prod- and ammonia viscosity, firmness, ucts were determined. 35in good condition at °C, with an inoculation of 10 170 Figure 1—pH value of the natto products made with differ- 1—pH value of the natto products Figure ent fermentation times at 40 to 42 °C B-3385, and the strain from the commercial Mito natto, B-3385, and the strain from the commercial to 280.50 mg/100g tended to produce more ammonia (262.65 B-3384 was, obvious- bean on a dry matter basis). The NRRL However, their natto products had much lower viscosity (4.1 had much lower natto products their However, the strain from the to 5.2 cps) than the natto inoculated with incubation condi- commercial Itobiki natto under the same acid (PGA) and polysaccharide 3). Poly-glutamic tions (Table (Fujii and others are responsible for the viscosity of natto 1975). The cells/g or at 40 °C, 10 Effect of bacteria strains Effect of bacteria had high populations (2.9 to and some NRRL strains ATCC, 5.0 × 10 Natto Quality as Affected by Processing . . . Processing by as Affected Quality Natto 20 to 30 from increased time as the steaming decreased ness dif- did not show significant ammonia content min, but the ferences (p > 0.05). tex- a palatably soft and flavor, ammonia-like low viscosity, a wet matter bean (on of 100 to 150 mg/100g ture. A range con- is acceptable by Japanese basis) of ammonia-nitrogen 1989). Yoshkawa (Maruo and sumers in Japan beans, and the population of bacteria in the final products beans, and the population of bacteria was between 10

Food Microbiology and Safety Natto Quality as Affected by Processing . . .

Table 3—Effect of Bacillus natto strains on selected physical and chemical properties of natto1 B. natto Strains NRRL NRRL NRRL NRRL NRRL ATCC Yamada Mito Itobiki Properties B-3383 B-3384 B-3385 B-3386 B-14202 15245 Natto Natto Natto Color (L value) 36.87b 37.34b 34.16de 34.09de 33.15e 34.24de 35.26cd 36.33bc 35.04cd (1.70) (1.06) (2.21) (2.00) (1.44) (1.48) (1.45) (1.86) (1.36) pH Value 7.94a 6.79b 8.02a 7.90a 8.06a 8.01a 7.74ab 8.16a 8.10a (0.62) (1.34) (0.03) (0.22) (0.21) (0.83) (0.33) (0.27) (0.45) Firmness (kg) 43.9d 62.6a 53.4bc 51.2cd 61.2ab 57.3abc 62.8a 53.7abc 48.2cd (3.47) (10.76) (3.09) (3.38) (4.89) (12.94) (8.36) (11.09) (9.07) Viscosity (cps) 9.9b 5.9c 4.5c 5.5c 4.1c 5.2c 4.3c 4.7c 23.0a (6.27) (4.84) (0.89) (0.92) (1.00) (1.48) (1.07) (0.99) (13.30) Ammonia 259.50ab 113.30d 279.53a 280.50a 230.98b 168.33c 124.50d 262.65ab 301.55a Content (35.51) (29.16) (3.94) (10.39) (21.98) (25.45) (13.91) (13.32) (8.90) (mg/100g)2 Bacteria 4.8a 0.4b 2.2ab 3.2ab 2.9ab 3.3ab 5.0a 4.4a 2.6ab Population (1.49) (0.31) (0.73) (2.38) (2.36) (2.13) (2.02) (4.15) (2.54) ×109 (cfu/g)3 a-eMeans (SD) of at least 3 replicates. Means within same row with different letters are significantly different (p < 0.05). 1Soybean sample was soaked in water at the room temperature for 16 h (Weight increase ratio: 2.1–2.3), steamed at 121 °C for 35 min, and fermented at 40–42 °C and 85-90% of RH for 16 h. 2Data based on a dry matter basis. Moisture content of the natto samples in the range of 59.0–61.0%. 3Data based on a wet matter basis.

tion and reached to 7.10 at 20 h of fermentation. This indi- 3 conditions. It is not understood why steaming for 40 min cates that the species of B. natto and steaming time affect would facilitate the continued growth of the NRRL B-3383 the fermentation speed. between 18 and 20 h of fermentation. The NRRL B-3383 The relationships between firmness and fermentation strain lagged behind “Itobiki strain” in population of growth time differed for all 4 conditions of bacteria strain-steaming in the initial fermentation stages of 10 h (Figure 3), and time treatments (Figure 2). Statistical results showed no sig- seemed to catch up with the growth of the Itobiki strain after nificant difference in the firmness of the natto products 14 h of fermentation. Comparing the shapes of the curves in which were produced by steaming soaked beans for 40 min Figures 2 and 3, it can observed that the firmness of the nat- and followed by inoculating with the “Itobiki strain” for all of to finished products were not associated with the population the fermentation times (Figure 2). However, the firmness of of the bacteria in the products. natto products made by steaming soaked beans for 40 min In Japan, when natto is served, it is stirred vigorously with and inoculating with NRRL B-3383 strain had a decreasing trend as fermentation time increased (Figure 2). There were no significant differences (p > 0.05) between the firmness values of natto inoculated with the both strains in 16 and 18 h of fermentation, and 16, 18, and 20 h of fermentation, re- spectively, if beans were steamed for 35 min. The firmness of natto inoculated with NRRL B-3383 strain had an irregular pattern, which was similar to “Itobiki strain” when beans were steamed for 35 min. It is not clear why the natto steamed for 35 min increased firmness when fermentation time increased from 18 to 20 h. Comparing 35 min- and 40- min steaming time, 40-min steaming yielded natto products with substantially lower firmness, which was observed in products made by both strains (Figure 2). Longer steaming might have made nutrients more accessible to bacteria. Bac-

teria colonies and mucilage produced by the Food Microbiology and Safety grown on soybean substrate covered the surface of the beans of 40 min steaming earlier during fermentation than that did on the beans of 35 min steaming. The mucilage might have prevented moisture loss during the later stage of fermentation. The strain of B. natto was a factor that affected the ade- quacy of fermentation time when the other experimental conditions were kept the same. In Figure 3, viable bacterial counts indicated the growth of B. natto followed a similar pattern for 3 of the 4 processing conditions. The bacteria continued to increase from 18 to 20 h of incubation for beans steamed for 40 min with NRRL B-3383 strain, whereas Figure 3—Bacterial population of the natto products made the bacteria seemed to decrease from 18 to 20 h in the other with different fermentation times at 40 to 42 °C

Vol. 66, No. 1, 2001—JOURNAL OF FOOD SCIENCE 171 - Myco- G Reed G FERMENTA

16th ed. Assoc THE

Biotechnology (1989) B Maruo, H Yosh- H (1989) B Maruo, strains. The “Itobiki SHORTEN /Food and Drug Admin, 8th ed.

B. natto COULD

Topics in Secondary Metabolism I Bacillus in Secondary Metabolism Topics Conclusion References TIME

cfu/g (on a wet bean matter basis), and 7 to 10 6 STEAMING

Nr 11. St Paul, MN. APS Press. p 121–149. Official Methods of Analysis of AOAC International Bacteriological Analytical Manual LONGER tion time needed to obtain a good quality natto product. tion time needed to obtain a good quality

The ammonia content, like the viscosity, increased as the increased The ammonia content, like the viscosity, Gaithersburg MD. AOAC International. AOAC MD. Gaithersburg Kaishi 53: 424 [In Hakkokogaku Biology Molecular Application and Industrial subtilis: kawa, Ed. Ch 8. p 152. logia Memoir of Off Anal Chem. Washington DC. Washington Off Anal Chem. of (Ed.) p 479–482. Verlag and Chemie. Weinheim West Germany. West Weinheim and Chemie. Verlag p 479–482. (Ed.) Japanese). Fujii H, Shiraishi A, Kaba H, Shibagaki M, Takahashi S, Honda A. 1975. S, Honda Takahashi M, H, Shibagaki A, Kaba H, Shiraishi Fujii Ch 8 in sauce and other fermented foods of Japan. 1986. Soy D. Fukushima the fermentation time of 18 h were suitable for natto making. Therefore, these combinations of the processing conditions may be used as a laboratory method for evaluating soybean cultivar for natto making. Beuchat LR. 1983. Indigenous fermented foods. Vol 5 in Vol LR. 1983. IndigenousBeuchat fermented foods. AOAC. 1995. FDA. 1995. A 28: 568–572 (In Health. J Nation’s Japanese natto. U. 1959. Japanese Hayashi In the late stage (18 to 20 h) of fermentation, the natto In the late stage (18 to 20 h) of fermentation, lower contents steamed for a longer time (40 min) contained steamed for 35 min. of ammonia as compared to the natto strain” the “Itobiki experimental conditions, the same Under texture natto from the produced higher viscosity and softer strain. Firmer tex- Danatto soybean than the NRRL B-3383 ture, higher concentration of ammonia, and lower quantity of mucilage could result from a prolonged fermentation (20 h). Under our laboratory conditions, a steaming time of 40 an inocula- strain” or NRRL B-3383 strain, min, the “Itobiki tion size of 10 the natto inoculated with the NRRL B-3383 strain, steamed B-3383 the NRRL with inoculated the natto the vis- 18 to 20 h. However, and fermented from for 35 min, natto inocu- (p < 0.05) for the significantly cosity decreased 40 min, and fer- steamed for strain”, the “Itobiki lated with the bacteria h, during which 18 and 20 mented between some of the to break down secreted might have B-3383, with the NRRL For the natto inoculated mucilage. 40 occurred at 18 h of fermentation for the highest viscosity at 20 h of fermentation for 35 min steam- min steaming, and a significantly higher (p < produced strain” The “Itobiki ing. 40 did the NRRL B-3383 strain at 35 and 0.05) viscosity than during 12, 14, 16, 18, and 20 h of fermen- min steaming times fer- increasing trend in viscosity with tation (Figure 4). The that a longer time might be required mentation time implies B-3383 to produce a natto product when using the NRRL of viscosity as that produced by the “Ito- with the same level a rela- 4). However, biki strain” fermentation at 18-h (Figure fer- time may be used to shorten the tively longer steaming a viscous natto. mentation time to obtain were significant fermentation proceeded (Figure 5). There content of the natto differences (p < 0.05) in the ammonia of fermentation for products during every 2-hour interval both steaming times and the 2 ammonia than the NRRL B- tended to produce more strain” times, respec- 3383 did under both 35 and 40 min steaming the and steaming time affected bacteria strain Both tively. Based on the re- ammonia content of the natto products. acceptable to produce may be used strain” the “Itobiki sults, and incubated for natto product when steamed for 40 min upon the de- various periods of times (12 to 20 h) depending of ammonia content and viscosity. range sired —Vol. 66, No. 1, 2001 66, No. —Vol. JOURNAL OF FOOD SCIENCE 172 Figure 5—Ammonia content of the natto products made with different fermentation times at 40 to 42 °C Figure 4—Viscosity of the water extract of the mucus from Figure 4—Viscosity of the water extract fermentation times the natto products made with different at 40 to 42 °C Natto Quality as Affected by Processing . . . Processing by as Affected Quality Natto to the silkiness/stickiness generate to of chopsticks a pair to a high stickiness feeling due of beans. Silky form a mass criterion of a most important the mucilage is the viscosity of objective study we found that In a preliminary good natto. with sensory was correlated viscosity analysis instrumental steamed In general, the natto data). viscosity (unpublished steamed for viscosity than that produced higher for 40 min was a significant increase (p < 0.05) 35 min (Figure 4). There 18 the fermentation times from 10 to of natto viscosity along for The viscosity continued to increase h for all 4 conditions.

Food Microbiology and Safety Natto Quality as Affected by Processing . . .

Ikeda H, Tsuno S. 1984. The component changes during the manufacturing pro- Kogyo Gakkaishi). 40(1): 75–82. (In Japanese). cess of natto. Part I. On the amino nitrogen ammonia nitrogen carbohydrates Ohno Y, Kogawa K, Fujii K. 1990. Lipids of soybean products and soaked and

and vitamin B2 . Shokumotsu Gakkaishi (Kyoto Joshi Daigaku). 39: 19–24 (In boiled Soybeans. Sanyo Gakuen Tanki Daigaku Kenkyu Ronshu 21: 29–38. (In Japanese). Japanese). Ikeda H, Tsuno S. 1985. The component changes during the manufacturing pro- Ohta T. 1986. Natto. Ch 5 in Legume-based Fermented Foods NR Reddy MD Pier- cess of natto. Part II. On the water-soluble protein Shokumotsu Gukkaishi (Ky- son. D Merle, DK Salunkhe, Ed. Boca Raton, FLA: CRC Press. p 85-93 . oto Joshi Daigaku). 40: 27–37. (In Japanese). Sakurai Y. 1960. Report of the research on the production of high protein food Kanno A, Takamatsu H. 1987. Studies on natto. Part IV. Changes in the volatile from fermented soybean products. Food Res Inst Ministry of Agric and Forest- components of natto during manufacturing and storage. J Japanese Soc of ry Fukagawn P O Tokyo Japan. (In Japanese). Food Sci and Tech (Nippon Shokuhin Kogyo Gakkaishi). 34: 258–265. (In Jap- SAS User’s Guide: Statistics Version 6 Edition. 1989. SAS Institute Inc. Cary, NC. anese). Steinkraus KH. Ed. 1983. Indigenous fermented amino acid/peptide and Kanno A, Takamatsu H, Takano N, Akimoto T. 1982. Studies on Natto. Part I. pastes with meatlike flavors: Chinese Japanese shoyu Japanese Change of saccharides in soybeans during manufacturing of natto. J Japanese southeast Asian fish sauces and pastes and related foods Section 5 in Hand- Soc of Food Sci and Tech (Nippon Shokuhin Kogyo Gakkaishi) 29(2): 105-110. bood of Indigenous Fermented Foods Microbiology series. Vol 9 New York: Marcel (In Japanese). Dekker Inc. p 530–547. Kanno A, Takamatsu H, Tsuchihashi N, Watanable T, Takai Y. 1985. Studies on Taguchi K, Kawabata M, Ohtsuki K, Tanaka Y. 1986. Changes in dietary fiber of Natto. Part III. Change in tocopherol contents during manufacture and storage natto and during fermentation. J Japanese Soc of Nutri and Food Sci of natto and hikiwari-natto. J Japanese Soc of Food Sci and Tech. (Nippon Shoku- (Nihon Eiyo Shokuryo Gakkaishi). 39(3): 203–208. (In Japanese). hin Kogyo Gakkaishi). 32(10): 754–758. (In Japanese). Taira H, Takahashi H, Okano H, Nagashima S. 1983. Quality of soybean seeds Kusano A. 1969. Changes of protein in manufacturing natto. Part I. Total N - grown in Japan. Part IV. Suitability of soybean cultivars grown in Ibaraki pre- ter-soluble N TCA soluble N amino N and peptide N Eiyo To Shokuryo 22(9): fecture for natto production. Rept Natl Food Res Inst. (Shokuryo Kenkyusho 615–619. (In Japanese). Kenkyu Hokoku). 43: 62–71. (In Japanese). Kusano A. 1971. Changes of protein in manufacturing natto. Part II. TCA (tri- Taira H, Suzuki N, Tsukamoto C, Kainuma Y, Tanaka H, Saito M. 1987. Quality of chloroacetic acid) soluble total and free amino acids Eiyo To Shokuryo 24(1): 8– soybean seeds grown in Japan. Part CV. Suitability for natto processing of 12. (In Japanese). small seed cultivars and quality of the natto. Rep Natl Food Res Inst. (Shokuryo Lin S. 1991. Fermented soya foods. Ch 5 in Developments in Food Proteins De- Kenkyusho Kenkyu Hokoku). 51: 48-58. (In Japanese). velopment series. p 167–194. London; Applied Science. Englewood NJ. MS 20000411 Maruo B, Yoshkawa H. Ed. 1989. Industrial application of B subtilis. Ch 8 in Topics in Secondary Metabolism I Bacillus subtilis: Molecular Biology and Industrial A major portion of the article was presented at the 1998 IFT annual meeting in Atlanta, Ga. Application B Maruo and H Yoshkawa Ed. New York: Elsevier Science Publishing This research was supported by USDA-CSREES Alternate Crops Research Program and by North Dakota Soybean Council. We thank Dr. L. K. Nakamura, microbiologist in the Na- Co. Inc. p 143–161. tional Center for Agricultural Product Utilization (USDA Agriculture Research Service in Matsumoto I, Akimoto T, Imai S. 1995. Effect of soybean cooking condition on the Peoria, IL), for giving us 6 Bacillus natto strains. quality of natto. J Japanese Soc of Food Sci and Tech (Nippon Shokuhin Kogyo Gakkaishi). 42(5): 338–343. (In Japanese). All authors are with Dept. of Science, North Dakota State Univ., Fargo, Matsumoto I, Akimoto T, Imai S. 1993. Effects of temperature and inoculum size ND 58105. Direct inquiries to author Chang, IACC Rm 322, NDSU, Fargo, on natto’s appearance number of B natto temperature hardness and color tone ND 58105. (E-mail: [email protected]) during fermentation. J Japanese Soc of Food Sci and Tech. (Nippon Shokuhin Food Microbiology and Safety

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