CN111329034B - Method for improving soy sauce quality - Google Patents
Method for improving soy sauce quality Download PDFInfo
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- CN111329034B CN111329034B CN202010211491.XA CN202010211491A CN111329034B CN 111329034 B CN111329034 B CN 111329034B CN 202010211491 A CN202010211491 A CN 202010211491A CN 111329034 B CN111329034 B CN 111329034B
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- soy sauce
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- zygosaccharomyces rouxii
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- tetragenococcus halophilus
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- A—HUMAN NECESSITIES
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- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
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Abstract
The invention discloses a method for improving the quality of soy sauce, belonging to the field of microbial food. The method is characterized in that the high-salt liquid soy sauce is produced by double-strain synergistic fermentation, and zygosaccharomyces rouxii and tetragenococcus halophilus are added in the process of soy sauce mash fermentation. The method for producing the high-salt dilute soy sauce by the synergistic fermentation of the two strains has the advantages that the produced soy sauce has strong sauce fragrance, mellow ester fragrance and sufficient delicate flavor, the content of an aroma component, namely furanone (HEMF), in the soy sauce is 7.4 times of that of a control without adding a strain, the total content of volatile flavor substances is increased by 2.4 times of that of the control, and the method has important significance for producing the high-quality soy sauce in a large scale.
Description
Technical Field
The invention relates to a method for improving the quality of soy sauce, belonging to the field of microbial food.
Background
Soy sauce, a long-standing fermented condiment, has been used for over 3000 years, and consumers are now worldwide. At present, the situation that various soy sauce brewing processes coexist exists in China, but the production of high-quality soy sauce mainly depends on a high-salt dilute state brewing process, and the soy sauce brewed by the process has the advantages of strong sauce fragrance, bright reddish brown color, delicious and pure taste and the like. However, compared with the advanced countries such as Japan and the like, the traditional high-salt dilute brewing process in China still has the defects of deviation of aroma and taste, unstable product flavor and the like.
The soy sauce fermentation is a mixed fermentation process in which various microorganisms participate together. The composition, succession and functional metabolism of microbial communities in the soy sauce brewing process have important influence on the quality of soy sauce. The microorganisms involved in soy sauce brewing are various, and mainly include aspergillus, yeast, lactic acid bacteria and other bacteria. Zygosaccharomyces rouxii is used in early stage of soy sauce fermentation, and can convert glucose into ethanol and produce various flavor substances such as esters and furanone. Tetragenococcus halophilus (Tetragenococcus halophilus) is a salt-tolerant lactic acid bacteria participating in soy sauce fermentation, and some strains have intracellular aminopeptidase activity and can be used for increasing the content of glutamic acid in fish paste. In the process of mixed fermentation of soy sauce, the adding period and the adding amount of yeast and lactobacillus have obvious influence on the fermentation of soy sauce mash, and the influence is different along with the change of strains. Although some researches are made on adding the halophilic tetragenococcus and the yeast in the soy sauce fermentation process at present, the interaction relationship of the halophilic tetragenococcus and the yeast in the soy sauce fermentation process and the influence of the combined action of the halophilic tetragenococcus and the yeast on the metabolism of substances in a soy sauce fermentation system are not clear. Therefore, the research aims at the fermentation process of the high-salt dilute soy sauce, and the influence on the growth of microorganisms in a system and the fermentation of the soy sauce is expected to provide a theoretical basis for the promotion of the modern production technology of the soy sauce through the influence of different addition modes of the halophilic tetragenococcus and the zygosaccharomyces rouxii.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a production method of high-salt dilute soy sauce capable of effectively improving the quality of soy sauce.
The invention provides a Tetragenococcus halophilus R44 for improving the quality of soy sauce, which is preserved in China center for type culture Collection in 1 month and 7 days of 2020, wherein the preservation address is Wuhan university in China, and the preservation number is CCTCC NO: M2020022.
The present invention provides a strain of zygosaccharomyces rouxii ZQ02, which has been disclosed in the article (Zhang Zaran, fang, cheng Jiang, et al. Zygosaccharomyces rouxii's metabolism of ethyl carbamate precursors in soy sauce [ J ]. Microbiol. Report 2016,56 (06): 956-963).
The invention provides a method for strengthening halophilic tetragenococcus R44 and zygosaccharomyces rouxii ZQ02 in soy sauce, which is characterized in that the halophilic tetragenococcus R44 is added in the 20 th to 40 th days of soy sauce fermented from soy sauce mash, and the halophilic tetragenococcus R44 and the zygosaccharomyces rouxii ZQ02 are added in the addition amount of which the final concentration ratio is 1-1000.
In one embodiment of the present invention, the amounts of said Tetragenococcus halophilus R44 and ZQ02 zygosaccharomyces rouxii are 1.0X 10 5 ～1.0×10 8 CFU·g -1 Soy sauce mash and 1.0X 10 6 ～1.0×10 9 CFU·g -1 Sauce mash.
In one embodiment of the present invention, the addition amounts of said Tetragenococcus halophilus R44 and ZQ02 of ZQ.rouxii are 1.0X 10 6 ～1.0×10 7 CFU·g -1 Fermented soy sauce and 1.0X 10 6 ～1.0×10 8 CFU·g -1 Sauce mash.
The invention provides a method for improving soy sauce flavor substances, which comprises the following steps: preparing yeast from soybean meal, aspergillus oryzae spore powder and wheat particles; mixing the finished koji with saline water to prepare soy sauce mash; fermenting the soy sauce mash, adding zygosaccharomyces rouxii ZQ02 when the soy sauce mash is fermented to 10-20 days, and adding tetragenococcus halophilus R44 when the soy sauce mash is fermented to 30-40 days; fermenting for 65-80 days to obtain the soy sauce.
In one embodiment of the present invention, the zygosaccharomyces rouxii ZQ02 is added in an amount of 1.0X 10 6 ～1.0×10 7 CFU·g -1 Sauce mash.
In one embodiment of the invention, the addition amount of the Tetragenococcus halophilus R44 is 1.0X 10 6 ～1.0×10 7 CFU·g -1 Sauce mash.
In one embodiment of the present invention, the concentration of the brine is 10% to 30% (w/v), and the fermentation temperature is 25 to 40 ℃.
The invention provides a food leavening agent, which contains tetragenococcus halophilus R44 and zygosaccharomyces rouxii ZQ02.
The invention provides a preparation method of the leavening agent, which comprises the following steps: respectively inoculating 200-600 mu L of tetragenococcus halophilus R44 and zygosaccharomyces rouxii ZQ02 into 10-30 mL of MRS liquid culture medium, activating for 2-3 generations at 30-40 ℃, and allowing the tetragenococcus halophilus R44 and the zygosaccharomyces rouxii ZQ02 to reach 1.0 multiplied by 10 6 When the number of viable bacteria is more than CFU/mL, centrifuging for 15-25 min at 2000-6000 rpm, removing supernatant, sequentially adding buffer solution and cryoprotectant in a sterile environment until the cell concentration is 1.0 × 10 5 ～1.0×10 10 And (3) performing vacuum freeze drying treatment when the concentration is CFU/mL, and mixing the obtained freeze-dried powder of the tetragenococcus halophilus R44 and the freeze-dried powder of the zygosaccharomyces rouxii ZQ02 according to the mass ratio of 1-100.
In one embodiment of the invention, the lyophilized powders of tetragenococcus halophilus R44 and zygosaccharomyces rouxii ZQ02 are mixed in a mass ratio of 1.
In one embodiment of the present invention, the buffer is 0.1-0.3M phosphate buffer with pH 6.5-7.5, and the cryoprotectant is 12-18% (v/v) glycerol.
In one embodiment of the invention, the buffer is 0.2M phosphate buffer at ph7.2 and the cryoprotectant is 15% glycerol.
The invention provides a method for preparing soy sauce by using a leaven, which comprises the steps of adding the leaven into soy sauce mash by the adding amount of 1-20% by volume, and fermenting for 65-80 days at 30-40 ℃ to obtain the soy sauce.
The invention provides a method for preparing soy sauce by using a leaven, which comprises the steps of adding the leaven into soy sauce mash by an adding amount of 5-15% by volume, and fermenting for 68-75 days at 35-40 ℃ to obtain the soy sauce.
The invention provides an application of tetragenococcus halophilus R44 and zygosaccharomyces rouxii ZQ02 in preparation of soybean meal fermented food, wherein the soybean meal fermented food comprises soybean paste and soybean milk.
The invention provides an application of a leavening agent in preparing soybean meal fermented food, wherein the soybean meal fermented food comprises soybean paste and soybean milk.
The invention has the beneficial effects that:
the invention produces the high-salt dilute soy sauce by the synergistic fermentation of the two strains, and properly adds the zygosaccharomyces rouxii and the halophilic tetragenococcus in the soy sauce fermentation process, thereby effectively improving the utilization rate of raw material protein and the product quality. The content of amino acid nitrogen in the soy sauce mash reaches 7.56g/L. In addition, the content of volatile flavor substances in the soy sauce is increased by 2.4 times, wherein the contents of alcohols, ketones and esters are respectively increased by 282.7%, 475.1% and 895.4%. In addition, furanone HEMF is the main fragrant component in fermented soy sauce, has strong sauce fragrance, and can play a role in softening salty taste of soy sauce. The soy sauce is not easy to volatilize, so that the soy sauce can be stored for a long time or the fragrance is not dispersed after being heated; strong oxidant, strong bactericidal effect, and high antimildew effect. The content of furanone (HEMF) as main aroma component in soy sauce is 7.4 times that of control without addition of strain, and 1.4 times that of Zygosaccharomyces rouxii alone.
Biological material preservation
Tetragenococcus halophilus (Tetragenococcus halophilus) is preserved in China center for type culture Collection in 1 month and 7 days of 2020, with the preservation address of Wuhan university in Wuhan, china and the preservation number of CCTCC NO: M2020022.
Drawings
FIG. 1 is a comparison of the main physicochemical indexes of soy sauce.
FIG. 2 is a comparison of the main volatile substances in soy sauce; control: blank; t: adding Tetragenococcus halophilus R44 on days 10-20; z: adding ZQ02 of ZQ Ludwith on 10-20 days; z + T: adding Z.rouxiiZQ02 and Tetragenococcus halophilus R44 on days 10-20; z + T (35): zygosaccharomyces rouxii ZQ02 was added on days 10-20, and tetragenococcus halophilus R44 was added on day 35.
Detailed Description
MRS culture medium: peptone 10.0 g.L -1 Beef powder 5.0 g.L -1 Yeast powder 4.0 g.L -1 20.0 g.L of glucose -1 Dipotassium hydrogen phosphate 2.0 g.L -1 Sodium acetate 5.0 g. L -1 Triammonium citrate 2.0 g.L -1 Magnesium sulfate 0.2 g. L -1 Manganese sulfate 0.1 g. L -1 Tween-80.0 g. L -1 . When in use, 100g and L are added -1 NaCl, pH 7.0.
Zygosaccharomyces rouxii ZQ02: see Zhang Zaran, fang, chen Jiang, et al, metabolism of ethyl carbamate precursors in soy sauce by Zhang J. Microbiological report 2016,56 (06): 956-963.
Example 1: soy sauce preparation using Tetragenococcus halophilus R44 and ZQ02
(1) Koji making
Soaking bean cake in 1.2 times of water for 2 hr, sterilizing at 121 deg.C for 15min, and cooling to room temperature. Inoculating 1.5 per mill (V: V) of Aspergillus oryzae spore powder according to the weight of the raw materials (the total weight of the mixed soybean meal and wheat grains), uniformly mixing the spore powder and a proper amount of fried wheat grains, and then sufficiently and uniformly mixing the mixture with the soybean meal and the wheat grains (the mass ratio of the soybean meal to the wheat grains is 6); culturing at 30 deg.C in biochemical incubator, controlling humidity at about 90% during starter propagation, turning over starter propagation at appropriate time for 60 hr, and collecting yeast material with yellow-green mycelium on surface. Quality requirement of finished koji: developed hyphae, dense spores, no sticky feeling and no yeast burning phenomenon.
(2) Fermentation of
The koji was mixed with 20% (W: V) brine in a volume ratio of 1.8. And (3) putting the mixed sauce mash into 2L jars, and fermenting for 75d at 30 ℃. Stirring once a day in 1 week of fermentation, once every two days in 2-6 weeks, and twice a week in the later period. Adding Zygosaccharomyces rouxii (to be added to) when fermenting the moromi mash for 15dThe pH of the system is reduced to about 5.2, the zygosaccharomyces rouxii is suitable for growing under the acidic condition), and the pH value is 1.0 multiplied by 10 7 CFU·g -1 Adding into soy sauce mash. The addition time of Tetragenococcus halophilus is 35 days, and the addition amount is 1.0 × 10 6 ～1.0×10 7 CFU·g -1 Sauce mash.
Three control runs were set up simultaneously, the embodiment being as above, except that the substitutions were made with the addition of zygosaccharomyces rouxii at 15d and the addition of tetragenococcus halophilus at 35 d:
control 1: tetragenococcus halophilus alone was added on day 15;
control 2: zygosaccharomyces rouxii was added alone on day 15;
control 3: zygosaccharomyces rouxii and tetragenococcus halophilus were added on day 15.
Example 2: determination of the amount of Tetragenococcus halophilus R44 and ZQ02 zygosaccharomyces rouxii
To confirm that the effect of tetragenococcus halophilus R44 and ZQ02 zygosaccharomyces rouxii can be enhanced by addition of the strain during soy sauce fermentation, the number of tetragenococcus halophilus R44 and ZQ02 zygosaccharomyces rouxii was measured by plate counting. Weighing 1g of soy sauce mash, pouring into a conical flask containing sterilized glass beads and 99mL of physiological saline, mixing uniformly, and diluting in a gradient manner (10 dilution respectively) -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 ) Diluted and applied to MRS (containing 100 mg. L) -1 Natamycin, 100 g.L - 1 NaCl) plates and Bengal agar (sodium propionate 5 g. L) -1 ，100g·L -1 NaCl) plate, incubated at 30 ℃ for 4-6d and counted.
The result shows that the addition of the tetragenococcus halophilus can strengthen the tetragenococcus halophilus in the soy sauce fermentation process. The quantity of the halophilic tetragenococcus can be increased by 1.9 to 3.0 orders of magnitude by independently adding the halophilic tetragenococcus; the addition of Tetragenococcus halophilus under the conditions of enhanced Zygosaccharomyces rouxii increased the number by 2.2 orders of magnitude. The addition of ZQ02 can increase the number of the bacteria in the sauce mash by 1.5-3.1 orders of magnitude compared with the control without ZQ02, and the maximum value reaches 1.6 multiplied by 10 7 CFU·g -1 Gradually decreases in the later stage and stabilizes at 6.0 × 10 4 CFU·g -1 . Therefore, the addition of tetragenococcus halophilus R44 and zygosaccharomyces rouxii ZQ02 can improve the concentration of the strain in the soy sauce fermentation process and strengthen the strain.
Example 3: intensification of influence of Tetragenococcus halophilus R44 and ZQ02 on physicochemical indexes of soy sauce
The moromi of example 1 was sampled on days 15, 20, 25, 30, 35, 40, 45, 60 and 75. 100g of sauce mash is respectively taken and put in a centrifuge tube for 12000 r.min -1 Centrifuging for 30min, collecting supernatant, and filtering with filter membrane with pore diameter of 0.22 μm for physicochemical index determination. The content of amino acid nitrogen in the soy sauce mash is measured by adopting a formaldehyde titration method (the specific steps refer to GB 5009.235-2016).
In this example, it was determined that fortification of tetragenococcus halophilus R44 and zygosaccharomyces rouxii ZQ02 significantly increased the amino acid nitrogen content of the moromi mash. The ammonia nitrogen content in the moromi mash with the addition of zygosaccharomyces rouxii and the addition of tetragenococcus halophilus on day 35 was 7.56g/L, which is 6.8% higher than the control without the addition of the strain.
Example 4: effect of enhanced halophilic Tetragenococcus R44 and zygosaccharomyces rouxii ZQ02 on free amino acid content of soy sauce
The free amino acid has important contribution to the formation of unique taste and aroma of fermented foods, particularly soybean fermented foods taking protein as a main fermentation substrate, and is one of main physicochemical indexes for measuring the quality of soy sauce. The determination of free amino acids is performed by high performance liquid chromatography (the specific steps are Shen Shi Wang, microbial community analysis and function research in soy sauce fermentation process [ D ]. Master academic thesis, 2017, university of south Jiangnan). 1mL of soy sauce sample was diluted 20 times with 5g/L of trichloroacetic acid, left to stand at room temperature in the dark for 30min, and the filtrate was filtered through a water-based filter membrane having a pore size of 0.22. Mu.m, and the measurement was carried out.
Specific results are shown in table 1: the addition of zygosaccharomyces rouxii and the addition of tetragenococcus halophilus on day 35 increased the total amino acid content in soy sauce by 23.9% over the control without the added strain and by 9.27% and 6.41% over the addition of tetragenococcus halophilus R44 or zygosaccharomyces rouxii ZQ02 alone, respectively. The increase of glutamic acid, threonine, valine, leucine and lysine is obviously improved, and is respectively improved by 18.76%, 26.77%, 22.18% and 21.92% compared with a control group without adding the strain, wherein the glutamic acid is an important delicate flavor source in the soy sauce; threonine is an important sweet amino acid in soy sauce.
TABLE 1 content of free amino acids in soy sauce
Note: control: blank; t: day 15 addition of Tetragenococcus halophilus R44; z: zygosaccharomyces rouxii ZQ02 was added on day 15; z + T: zygosaccharomyces rouxii ZQ02 and tetragenococcus halophilus R44 were added on day 15; z + T (35): zygosaccharomyces rouxii ZQ02 was added on day 15, and tetragenococcus halophilus R44 was added on day 35.
Example 5: effect of enhanced Tetragenococcus halophilus R44 and ZQ02 zygosaccharomyces rouxii on volatile substances in Soy sauce
The influence of enhanced halophilic tetragenococcus R44 and zygosaccharomyces rouxii ZQ02 on soy sauce flavor was studied by SPME-GC-MS analysis (specific steps refer to Wavenwang. Soy sauce fermentation Process microbial community analysis and functional study [ D ]. A Master academic thesis, 2017.).
The addition of ZQ02 and Tetragenococcus halophilus R44 at day 35 increased the volatile flavor content of soy sauce by 2.4 times, wherein the alcohol, ketone, ester and phenol contents were increased by 282.7%, 475.1%, 895.4% and 9.1%, respectively. In addition, the content of furanone (HEMF) as an aroma component in soy sauce was 7.4 times that of the control without the addition of the strain and 1.4 times that of Zygosaccharomyces rouxii alone.
TABLE 2 content of various volatile substances (g/kg) in different soy sauce samples
Note: control: blank; t: tetragenococcus halophilus R44 was added on day 15; z: zygosaccharomyces rouxii ZQ02 was added on day 15; z + T: zygosaccharomyces rouxii ZQ02 and tetragenococcus halophilus R44 were added on day 15; z + T (35): zygosaccharomyces rouxii ZQ02 was added on day 15, and tetragenococcus halophilus R44 was added on day 35.
TABLE 3 content of major volatile substances (g/kg) in different soy sauce samples
Note: control: blank; t: tetragenococcus halophilus R44 was added on day 15; z: zygosaccharomyces rouxii ZQ02 was added on day 15; z + T: zygosaccharomyces rouxii ZQ02 and tetragenococcus halophilus R44 were added on day 15; z + T (35): zygosaccharomyces rouxii ZQ02 was added on day 15 and tetragenococcus halophilus R44 was added on day 35.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A method for improving flavor substances of soy sauce is characterized in that zygosaccharomyces rouxii ZQ02 is added into soy sauce mash on the 15 th day of fermentation, and Tetragenococcus halophilus R44 which is the Tetragenococcus halophilus R44 and is stored in China center for type culture collection (CCTCC NO: M2020022) on the 1 st and 7 th days of 2020 is added to the soy sauce mash for fermentation on the 35 th day.
2. The method of claim 1, wherein the moromi is made by mixing koji with brine; the finished koji is prepared by using bean pulp, aspergillus oryzae spore powder and wheat particles; the fermentation lasts for 65-80 days.
3. The method according to claim 1 or 2, wherein the Tetragenococcus halophilus is added in an amount of 1.0 x 10 5 ～1.0×10 9 CFU·g -1 The addition amount of Zygosaccharomyces rouxii in soy sauce mash is 1.0 × 10 5 ～1.0×10 9 CFU·g -1 Sauce mash.
4. The method as claimed in claim 3, wherein the ZQ02 is added in an amount of 1.0X 10 6 ～1.0×10 7 CFU·g -1 Sauce mash.
5. The method of claim 3, wherein the amount of Tetragenococcus halophilus R44 added is 1.0 x 10 6 ～1.0×10 7 CFU·g -1 Sauce mash.
6. The method according to claim 5, wherein the brine has a concentration of 10% to 30% (w/v).
7. The method according to claim 6, wherein the temperature of the fermentation is 25 to 40 ℃.
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