Food Flavor and Safety - American Chemical Society


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Chapter 16

Rapid Fermentation of Soy Sauce Application to Preparation of Soy Sauce Low in Sodium Chloride 1

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Shunsuke Muramatsu , Yoshihito Sano , and Yasuyuki Uzuka 1

Takeda Shokuryo Company, Ltd., 9-30 Saiwai-cho, Kofu, Yamanashi 400, Japan Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University, Kofu, Yamanashi 400, Japan

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Because of the importance of soy sauce as a food flavorant, a new method for its production was developed. The importance of this novel processing procedure is that it significantly shortens the fermentation processing time by half or to about three months. Attempts at further modification of the process to prepare low-sodium-chloride-containing soy sauce are also described. Soy sauce is a uniquely Japanese seasoning prepared from soy beans and wheat. Although soy sauce is made from soy beans, it has a highly desirable flavor without the beany aroma. Soy sauce is currently used not only in the preparation of many Japanese cuisines but also in the preparation of many popular western dishes such as beef steaks, poultry, and stews. Japan has more than 2,300 soy sauce producing firms. Soy sauce production in Japan is l,200,000kL per year at a cost of about 239.4 billion yen ($1,840 million U.S.). The per capita consumption of soy sauce in Japan is 2,947 yen ($22.70 U.S.) per year. By comparison, the per capita yearly consumption of sugar in Japan is 2,318 yen ($17.80 U.S.). This indicates the relative impact of soy sauce as a food adjuvant for the Japanese consumer. Conventional production of Soy Sauce Kofi making: The conventional process of soy sauce production is summarized in Figure 1. Soy beans are boiled for 10 minutes at 120°C or higher. Wheat grains, the second extremely important component in soy sauce production, are roasted and crushed. The pulverized wheat and the boiled soy beans are mixed with the spores of a food grade strain of Aspergillus oryzae (AO). The mixture is placed in a well-ventilated room with high humidity. The mold, AO, utilizes the starch of the wheat and the humidity of the storage area as a source of substrate for its growth. At the same time the mold is growing it is hydrolytically converting die starch rich wheat to glucose by amylase. The glucose will serve as substrate for lactic acid bacteria and yeast described below. The mold and its associated mycelia at this point are called "to/i." The koji is a rich source of other hydrolytic enzymes such as proteases, peptidases, and amylases (2,2). Protein rich soy beans and wheat grains are proteolytically converted to peptides and amino acids by enzymes from the koji. The distinctive urnarni taste of soy 0O97-6156/93/0528-O200$06.00/0 © 1993 American Chemical Society

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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sauce comes from the degradation of glutamic acidrichproteins from the wheat and soy bean.

SOY BEANS (DEFATTED SOY BEANS)

I WHEAT!

SOAK|NGJ

I ROASTING!

I STEAMING I

I CRASHING! -I KOJI SEED\ MIXING

SATURATED SODIUM CHLORIDE IMOROMII

I

I PRESSING

1

1 RAW SOY SAUCE | I PASTEURIZATION |

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PRODUCT (SOY SAUCE) Fig. 1. Conventional Method of Soy Sauce Production

Moromi making: When koji mixed with saturated sodium chloride solution and allowed to stand, the mixture is called "moromi" The mixing of the koji with the salt solution makes the internal region of the moromi mix anaerobic, a condition that kills Aspergillus oryzae. On the other hand the high salt, anaerobic conditions are quite

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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suitable for the growth of other microorganisms. Halotolerant microorganisms such as Pediococcus halophilus are the first to increase in population densities. Pediococcus halophilus, a lactic bacteria, produces lactic acid to sour the soy sauce and makes the saltiness milder. As the lactic acid concentration in the moromi increases, the pH decreases, leading to the cellular densities of Pediococcus halophilus to reach maximal levels. At the sametime,as the pH approaches 5.0, halophilic yeast species such as Zygosaccharomyces rouxii begin to grow. By 60 days these yeast have produced large amounts of ethanol (Figure 2 & 3) (3). The switch from lactic acid fermentation to alcohol fermentation in the moromi is mainly due to the difference in growth characteristics of the bacteria and yeast based on the pH. The hydrolysis of starch, lactic acid fermentation, and alcohol fermentation, proceed very slowly in the moromi vessel.

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Fig. 2. Changes of P. halophilus, Z. rouxii and C. versatilis in Moromi Mash (Reproduced with permission from ref. 3. Copyright 1988 Brewing Society of Japan)

Once the lactic acid and alcohol fermentation is complete, the moromi is aged until its moldy odor has disappeared. During this portion of the aging period other flavor molecules, particularly 4-ethylguaiacol and 4-ethylphenol are added to the moromi by Candida versatilis (4). These compounds are the major components contributing to the flavor of soy sauce. The entire moromi process is completed in about six months.

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Fig. 3. Changes of pH, Lactic Acid and Ethanol in Moromi Mash (Reproduced with permission from ref. 3. Copyright 1988 Brewing Society of Japan)

Final soy sauce product: When the moromi aging process is complete, the material is pressed yielding a raw soy sauce. The raw pressed material is pasteurized and filtered prior to sending it to the market. Rapid Fermentation of Soy Sauce Analysis of each process in the conventional method of soy sauce production indicated that moromi required three processes: hydrolysis, fermentation, and aging. It was hypothesized that the time required for the moromi process could be reduced into two process, i.e. autolysis and fermentation. These are discussed below. Effect of Salt on Autolysis of koji. In addition to causing the interior of the koji mixture to become anaerobic the high salt used in the conventional method of soy sauce production uses high salt to avoid putrefaction of the autolysate (5). Since high dietary levels of sodium chloride can exacerbate pre-existing medical conditions such as hypertension, it would be desirable to prepare soy sauce preparations low in sodium chloride. Since high temperatures are used in the beer brewing industry to assist in the saccharification of malt, it seemed to be a likely choice to apply to the autolysis of koji. Figure 4 shows the autolysis of koji in the absence of sodium chloride at several different temperatures over a three day period (6\ 7). Maximal levels of total soluble nitrogen were obtained at temperatures of 60°C. The lower the temperature the less total soluble nitrogen was observed with similar amounts of total nitrogen being found at 45°, 50° , and 55°C. On the other hand, the maximum levels of glutamic acid were

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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obtained at temperatures of 45°C anc 50°C. Higher temperatures produced lower yields of glutamic acid. A similar temperature response was observed for formol nitrogen.. The pH of the koji was significantly affected by the temperature at which the autolysis was carried out. At 45° and 50° the pH dropped below 4.5 within one day of autolysis suggesting that the lower pH might be due to lactic acid or other acidifying materials produced by unexpected microorganisms. Since the acidified autolysis is not suitable as the starting material for the next fermentation step, it was determined that autolysis should be carried out at 55°C.

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Fig: 4. Effect of Temperature on Autodigestion of Soy Sauce Koji Materials in the Absence of Sodium Chloride (Reproduced with permissionfromret & Copyright 1991 Brewing Society of Japan)

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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The effect of sodium chloride (NaCl) on autolysis of koji was also examined. NaCl concentrations were varied while the temperature of autolysis was maintained at 5S°C. Maximum total nitrogen was obtained when NaCl was absent. The yield of total nitrogen decreased with increasing concentrations of NaCl. Similar responses were observed for glutamic acid and formol nitrogen. As opposed to the traditional means of soy sauce production the require high levels of NaCl, these data suggested that maximal levels of total nitrogen, glutamic acid and formol nitrogen could be obtained in the absence this salt (8,9). Having determined the effect of temperature and NaCl on koji autolysis, the next step in finding an optimal protocol for the rapid production of soy sauce with high flavor quality was to examine the effect of independent and combined effect of lactic acid fermentation and alcohol on koji autolysis.

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Effect of Lactic Acid Fermentation on Koji Autolysis. The effect of NaCl on lactic acid fermentation (70) is seen in Figure 5. Four autolysates were prepared

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PERIOD (day)

Fig. 5. Effect of Sodium Chloride Concentration on Lactic Acid Fermentation by P. halophilus (Reproduced with permissionfromret 10. Copyright 1992 Brewing Society of Japan)

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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with 7, 8,9, and 10% NaCl. The autolysates were inoculated with 105 cells/mL of P. halophilus. The mixture was incubated at 30°C with the initial pH adjusted to 5.8. P. halophilus was observed to have a more rapid growth rate at lower concentrations of NaCl. As the fermentation proceeded, the pH of the autolysate would drop to 4.5 regardless of the NaCl concentration. TTie yield of lactic acid in this system was more than 15g/L, which is about twice that of regular soy sauce.

PERIOD (day)

Fig. & Effect of Initial pH on the Growth of Ζ rouxii (Reproduced with permissionfromret 10. Copyright 1992 Brewing Society of Japan)

Effect of Alcohol Fermentation on Koji Autolysis. Z. rouxii was used as an inoculum in the study of alcohol fermentation. Reports have suggested that the initial pH of the inoculum is very important to the proliferation of yeast such as Z. rouxii when grown under high concentrations of NaCl. Five autolysates were prepared

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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containing 13.8% NaCl with the pH of the inoculum adjusted in 0.5 pH increments from 4.0 to 6.0 (Figure 6). Z. rouxii was inoculated at 10* cells/mL and the inoculum incubated at 30°C. The maximal density of cells was reached by 10 days when pH levels were below 5.0. On the other hand, maximum cell levels were reached at 13 days if the initial pH was 5.5 or greater (70). Ethanol concentrations at the end of incubation was more the 20g/L, which is enough for soy sauce. Scale-up of Soy Sauce Preparation. A potentially useful method for the large scale production of soy sauce was developed based on a combination of the data and optimal protocols determined in the experiments described above. The pH change, reducing sugar, ethanol and lactic acid concentration of a 1 ton scale incubation in which lactic acid and alcohol fermentation are set to proceed sequentially is shown in Figure 7. P. halophilus inoculations of 10 cells/mL were used for lactic acid fermentation with the mixture incubated at 30° in the presence of 10% NaCl. Lactic acid levels reached lOg/L by 14 days of incubation in the 1 ton vessel. After 14 days the autolysate was mixed with an equal amount of newly prepared autolyzate and was inoculated with Z. rouxii and C. versatillis and the inoculum incubated. Alcohol fermentation proceeded with ethanol levels reaching their maximum plateau level of 23g/L. The pH in this 1 ton vessel slowly dropped during the first 23 days and reached a low of 4.8 (77).

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Fig. 7. Fermentation of Moromi i n One Ton Vessel (Reproduced with permission from ref. 11. Copyright 1992 Brewing Society of Japan)

Further scaled-up of the soy sauce production to a 40 ton reaction vessel yielded result very similar to that obtained for the 1 ton vessel and comparable to the of the conventional method. Data on the composition of these raw soy sauces are seen in

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Table I. The soy sauce produced by the scaled-up methods yielded a soy sauce with lower NaCl concentrations than the soy sauce produced by conventional means. Glutamic acid concentrations, while somewhat lower than that of the soy sauce produced by conventional method, were still quite high. While the lengthy moromi aging process of the conventional method produces pyroglutamic acid, a tasteless product of glutamic acid, the scale-up procedure reduces the pyroglutamic acid conversion to half that found in the conventional method. Consumer evaluating soy sauce produced by the scale-up method were unable to distinguish taste differences between the new (scale-up) soy sauce and soy sauce produced by the conventional method. This led to die opening of a new production plant that is currently producing the new soy sauce for marketing.

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Table I. Comparison of Autolyzate Compositions* of Raw Soy Sauce

Contents

Raw Soy Sauce Autolyzate Obtained from (Unfermented) 1 Ton Vessel

NaCl Total Nitrogen Glutamic Acid Pyroglutamic Acid Reducing Sugars Alcohol Lactic Acid pH

0 1.99 1.98 0.11 6.68 0 0 5.92

14.5 1.90 1.63 0.16 1.20 2.20 1.00 4.80

Raw Soy Sauce Raw Soy Sauce Obtained by Obtained from Conventional Method 40 Ton Vessel 14.5 1.83 1.75 0.15 2.05 1.97 1.02 4.77

16.9 1.80 1.49 0.29 3.13 1.83 1.20 4.77

*A11 values, exsept pH, are given as g/dL Preparation of Low-Sodium-Chloride-Containing Soy Sauce. While soy sauce is a highly flavorful seasoning its continuous use by consumers suffers from a major drawback, i.e., high NaCl level are potentially deleterious to the individuals with hypertension, hypertensive tendencies, and renal problems, to name just a few. Thus, it would be advantageous to a soy sauce producing company to develop a method that would produce a low-sodium-chloride-containig soy sauce that still maintains the high flavor quality of the sauce. Conventional methods have attempted to use potassium chloride (KC1) as a replacement for NaCl. Unfortunately this yields a soy sauce with an unsatisfactory taste for most consumers. Since the new scaled-up method described above yields a soy sauce with lower than conventional NaCl levels attempts were made to redesign the protocol with even lower levels of NaCl. Soy sauce containing a final concentration of 4.6% NaCl were prepared by the scale-up method described above. Since traditional (conventional) soy sauce contains approximately 16.2% NaCl, experiments were designed to use the addition of several salt substitutes to make up the difference in salt concentration between the scale-up soy sauce (4.6%) and the traditional soy sauce (16.2%). Salt substitutes used included KC1, glycine ethyl ester hydrochloride, lysine hydrochloride, taurine and glutamic acid. According to Tamura et al, (72), glycine ethyl ester hydrochloride, lysine hydrochloride and glutamic acid have the effect of enhancing the saltiness of NaCl. Taurine was expected to have an affect on the final salty taste since the presence of a salty peptide, ornithyltaurine, had been previously reported (72). Sensory evaluation of the various

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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samples by five panelists included a dilution of the samples with distilled water such that the final concentration of the additives were adjusted to the following percentages (%): 2.0, 1.0, 0.5, 0.125, and 0.063%. Threshold values were determined for each salt substitute. The results of the sensory evaluation of the salt substitute investigation are shown on Table II. The threshold concentration for the perception of the "salty" taste remained the same for all groups. This suggested that the perception mechanism of each of the compounds was similar, if not identical, i.e., they enhanced the salty taste perception. The samples containing taurine yielded desirable flavor result, but the mechanism of how taurine effected this response remains unclear at this time. While glutamic acid and glycine ethyl ester hydrochloride enhanced the saltiness of the soy sauce, they also imparted a sour note. On the other hand, the panelists did not find the sourness unfavorable and reported that the net sensory response of both glutamic acid and glycine ethyl ester hydrochloride was to give a better flavor to the soy sauce. Lysine hydrochloride gave the soy sauce an unfavorable aftertaste which disappeared when diluted samples were tasted. Acceptable flavors and saltiness were unattainable even in diluted samples when using KC1 as the salt replacement suggesting that KC1 could not serve as a commercially marketable NaCl substitute in soy sauce. Table II. Results of Sensory Analysis Original Concentration Threshold Value of NaCl (%) (%) of Additives Comments

Samples Conventional Soy Sauce

16.2

0.25

Tastes like soy sauce.

Soy Sauce Prepared by Our Method

4.6

0.25

Contains a little roasty flavor.

Soy Sauce Prepared by a New Method + 11.6% KC1

4.6

0.25

Unfavorable taste of KC1 was detected even at lower concentration where saltiness could not sensed.

Soy Sauce Prepared by a 4.6 New Method + 11.6 % Glycine Ethyl Ester Hydrochloride

0.25

Contains a sourness.

Soy Sauce Prepared by a New Method + 11.6% Lysine Hydrochloride

4.6

0.5

Unfavorable taste of LysHCl was detected at the higher concentration.

Soy Sauce Prepared by a New Method + 11.6% Taurine

4.6

0.5

Delicious.

Soy Sauce Prepared by a New Method + 11.6% Glutamic Acid

4.6

0.25

Contains a sourness.

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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A n interesting outcome o f this investigation is the birth o f a potentially new seasoning. B y dividing the moromi process one can produce a koji autolysate with the same chemical composition and flavors as soy sauce expect for die salt and saltiness, i.e. the koji autolysate contains the same amount o f total nitrogen and glutamic acid as raw soy sauce but no salt. The new type o f N a d - f r e e seasoning has the potential o f being applied to new food materials.

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Conclusion A new method for the preparation of soy sauce has been developed. The new scaledup method divides the moromi process into two processes: autolysis and fermentation. Because o f the utilization of high temperatures, the new process permits the production of a N a C l free autolyzate from koji. D i v i s i o n o f the fermentation process into two separated processes permit better control o f lactic acid fermentation and alcohol fermentation processed which used to require great skill. The new scale-up procedure for soy sauce production yields a product i n half the time required by the traditional (conventional) method and still produces a soy sauce with high levels o f the desirable flavor component, glutamic acid. Utilization of this protocol by the soy sauce producing industry should have significant economic impact to both producers and consumers.

Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

Yokosuka, Y; Iwasa, T; Fuji, S. J. Jpn. Soy Sauce Res. Inst, 1987, 13, 18. Terada, M; Hayashi, K; Mizunuma, T. J. Jpn. Soy Sauce Res. Inst., 1981, 7, 158. Kadowaki, K. in Shoyu no Kagaku to Gijyutsu,Tochikura, T., Ed; Nippon Jozo Kyokai; Tokyo; 1988. Uchida, D. In Shoyu no Kagaku to Gijyutsu; Tochikura, T., Ed.; Nihon Jyozou Kyokai: Tokyo (1988). Terada, M.; Hayashi, K.; Mizunuma, T.; Mogi, K. Seasoning Sci., 1973, 2, 23. Muramatsu, S; Sano, Y; Takeda, T; Uzuka, Y. J. Brew. Soc. Jpn., 1991, 86, 610. Muramatsu, S; Sano, Y; Uzuka, Y. J. Brew. Soc. Jpn., 1992, 87, 219. Muramatsu, S; Sano, Y; Uzuka, Y. J. Brew. Soc. Jpn., 1992, 87, 150. Muramatsu, S; Sano, Y; Uzuka, Y. J. Brew. Soc. Jpn., 1992, 87, 295. Muramatsu, S; Ito, N; Sano, Y; Uzuka, Y. J. Brew. Soc. Jpn., 1992, 87, 378. Muramatsu, S; Ito, N; Sano, Y; Uzuka, Y. J. Brew. Soc. Jpn., 1992, 87, 538. Tamura, M.; Seki, T.; Kawasaki, Y.; Tada, M.; Kikuchi, E.; Okai, H. Agric, Biol. Chem., 1989, 53, 1625. Tada, M.; Shinoda, I.; Okai, H. J. Agric. Food Chem., 1984, 32, 992.

Received October 28, 1992

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.