CN109762742B - Aspergillus oryzae ZA184 and application thereof - Google Patents
Aspergillus oryzae ZA184 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of food processing and microbial fermentation. In particular, the invention relates to aspergillus oryzae ZA184 and application thereof in soy sauce brewing. The aspergillus oryzae ZA184 disclosed by the invention can improve the pH value of finished koji, enhance the activity of finished koji enzymes (such as neutral protease, alkaline protease, leucine aminopeptidase and glutaminase), and improve the contents of amino nitrogen, total nitrogen and glutamic acid in soy sauce, so that the flavor quality of the soy sauce is improved, the fermentation period can be obviously shortened, and the production efficiency is improved.
Description
Technical Field
The invention relates to the technical field of food processing and microbial fermentation. Specifically, the invention relates to aspergillus oryzae and application thereof in soy sauce brewing.
Background
The soy sauce belongs to traditional fermented seasoning food, the strain is the basis of soy sauce fermentation, and the performance of the strain is very important to the influence of the yield and the quality of the soy sauce. Researches show that various enzyme systems secreted by aspergillus oryzae in the soy sauce koji making stage have direct relations on the fermentation maturity speed of soy sauce mash, the utilization rate of raw materials, the shade of finished products and the delicious degree of taste. The acquisition of excellent aspergillus sojae strains is one of the key factors for determining the raw material cost and market competitiveness of soy sauce enterprises, and is a long-standing struggle target of the soy sauce enterprises.
The brewing process of soy sauce includes inoculating Aspergillus oryzae on soybean (or defatted soybean), wheat, bran, etc. to prepare yeast, mixing with certain amount of salt water, and loading into fermenting container to produce various enzymes to decompose complex organic matters (protein, starch, etc.) into simple taste matters. The enzymes that play a major role in the entire fermentation process are hydrolases and synthetases. The hydrolase hydrolyzes the high molecular substance in the raw material into low molecular substance, and then the color, fragrance, taste and body of the soy sauce are formed by the synthesis enzyme and other chemical actions. It can be said that the hydrolase is the "leading force" for brewing soy sauce. The hydrolase is mainly produced by aspergillus, the more the variety of the strain containing the hydrolase is, the stronger the activity is, the more thoroughly the raw material is decomposed, and the higher the protein utilization rate of the raw material is. The technology for improving the raw material proteolysis rate mainly comprises the improvement of a raw material treatment method and the improvement of fermentation strains. Since most of soy sauce raw materials are decomposed by enzymes produced by aspergillus, the quality of aspergillus is greatly related to the yield and quality of soy sauce products.
The process of soy sauce fermentation is a process of various enzymatic reactions. The influence of the pH on the enzymatic reaction includes two aspects: (1) influence the stability of the enzyme; (2) affecting the binding of the enzyme to the substrate and the enzymatic conversion of the substrate to the product. Under certain conditions, each enzyme has its specific pH optimum. Deviating from this value, the activity of the enzyme is reduced and even the enzyme protein is denatured and thus inactivated.
The pH of the koji is an important control point in koji making because it has a great influence on the action of enzymes and other microorganisms in the fermented moromi. The pH value of the finished koji made by different strains is greatly changed. The pH value of the koji is high, the enzyme amount of the average bacteria is large, the generation amount of protease, leucine aminopeptidase, lipase and the like is high, the speed of protein hydrolysis is accelerated, and the protein decomposition rate is improved.
At present, the production strain commonly used in the soy sauce industry is Shanghai brewing 3.042 series strain (Zhongke As3.951), and the series strain has the characteristics of vigorous growth, strong sporulation capacity, good genetic stability, complete enzyme system and pure flavor of fermented soy sauce, and is a very good strain for production, so the strain is developed in 1958 and is used by various soy sauce production enterprises all the time. However, the pH value of the finished koji obtained by the series of strains is generally lower than 6.5, the optimum pH values of main enzymes for decomposing protein raw materials, such as alkaline protease, neutral protease, aminopeptidase, glutaminase, lipase and the like, are all higher than 7.0, the activity of the enzymes in fermented soy sauce mash is only less than 30%, the decomposition speed of the raw materials is greatly reduced, and the utilization rate of the protein raw materials is low. In view of this, the growth and propagation of lactic acid bacteria are controlled by adopting a lower temperature of about 15 ℃ in the early fermentation stage of soy sauce brewing in Japan, so that the pH value of soy sauce mash is not reduced too fast, and the decomposition of the protein in the raw materials by neutral protease and alkaline protease is facilitated. The low-salt solid fermentation process adopted in soy sauce brewing in China has high fermentation temperature (generally controlled at 40-50 ℃) and low pH value of soy sauce mash, so the quality of the fermented soy sauce is not high. The quality of soy sauce is often improved by adding enzyme preparations or prolonging the fermentation period, so that the production cost of enterprises is increased, and the addition of exogenous enzyme preparations objectively has uncontrollable risks.
Therefore, it is necessary to find new fermentation tubes to improve the quality of soy sauce and shorten the fermentation period.
Disclosure of Invention
In order to achieve the above object, the present invention provides an aspergillus oryzae by which a koji having a high pH value and a high protease activity can be obtained; the strain is used for brewing seasonings such as soy sauce, and the like, so that the contents of amino nitrogen, total nitrogen and glutamic acid can be improved, the flavor of the soy sauce is improved, the fermentation period can be obviously shortened, and the economic benefit is obvious.
In the fermentation process of seasonings such as soy sauce, it is known in the art that a protein material is decomposed into substances such as polypeptide by protease, and then further decomposed into components such as amino acid by peptidase, thereby forming the color, flavor, taste and body of soy sauce. Thus, it can be seen that proteases play an important role in soy sauce brewing. The protease is mainly produced by aspergillus and comprises alkaline protease, neutral protease and acid protease, and the optimal pH values of the three different proteases are respectively 9.5, 7.2 and 3.5. Peptidases include leucine aminopeptidase, which has an optimum pH of 8. Neutral protease and alkaline protease have significant effect on the primary decomposition of protein, and peptidase can further decompose polypeptide into amino acid. Therefore, the alkaline protease, the neutral protease and the leucine aminopeptidase are closely related to the utilization rate of the raw material protein, and the higher the enzyme activity is, the higher the utilization rate is, and the better the flavor of the soy sauce is.
In addition, the pH of soy sauce koji has a great influence on the action of enzymes, microorganisms in the moromi mash. For example, since the optimum pH of alkaline protease, neutral protease, leucine aminopeptidase and the like is 7.0 or more, it is found that the pH for koji preparation is high, the amount of enzymes produced per unit cell amount such as alkaline protease, leucine aminopeptidase, glutaminase and the like is high, the enzyme activity is also high, and the high pH of the moromi mash at the early stage of fermentation is advantageous for the growth of lactic acid bacteria and for the fermentation of soy sauce having good flavor.
Based on this, the inventors of the present application tried to obtain an Aspergillus oryzae strain capable of increasing koji-forming pH and enhancing protease activity by mutagenesis breeding using a currently commonly used production strain as an initial strain. Specifically, the mutagenesis selection method adopted by the invention is as follows.
1. The aspergillus oryzae As3.951 is used as an original strain, and a known mutagenesis method is adopted to mutate the original strain to obtain a mutant colony. Screening the mutant colonies by alternately using a pH indicator color-changing plate culture medium and a soybean juice plate culture medium, namely, selecting strains which do not change from red to yellow at the periphery of the colonies on the pH indicator color-changing plate to obtain new strains with high finished koji pH value; and selecting strains with vigorous hypha growth, long period, high mycelium content and slightly less sporulation of colonies on the bean juice flat plate. Selecting new strains meeting the double-plate screening condition at the same time, transferring the new strains to a soybean juice inclined plane, culturing the strains to be mature, performing conventional preservation and performing primary screening. Wherein, the formula of the pH indicator plate culture medium is as follows: 1000ml of rice koji juice (9-10 ℃ Be'), 150mM citric acid, 2.5ml of Triton X-100, 1g of phenol red, pH7.0 adjusted, 20g of agar powder, and sterilization at 121 ℃ under 0.1MPa for 20 min. The bean juice plate culture medium adopts a bean juice inclined plane production formula.
2. The primary screening method is to transfer the preserved strain activated by the bean juice slant into a triangular flask for culture and observe the growth condition of aspergillus oryzae. Selecting mutant strains with vigorous hypha growth and normal spore colonization, detecting the germination rate and spore number of the finished koji by adopting a conventional method, and preferably, re-screening the strains with high germination rate.
3. The re-screening method is to carry out small-scale starter propagation according to the conventional soy sauce production method, detect the number of starter propagation finished yeasts, the consumption rate of raw materials, neutral protease, alkaline protease, leucine aminopeptidase and glutaminase activity, and preferably select strains with high comprehensive enzyme activity for production trial.
4. The production trial is to carry out soy sauce production according to a conventional soy sauce production method, and detect the starter propagation finished starter index and the quality of fermented soy sauce.
By the above method, the mutagenized strain of the present invention, Aspergillus oryzae (Aspergillus oryzae) ZA 184:
1. the aspergillus oryzae ZA184 is preserved in the Guangdong province microorganism strain collection center in 2018, 8 months and 13 days, and the preservation number is GDMCC No: 60429.
2. the colony characteristics of aspergillus oryzae ZA184 of the present invention are as follows: culturing for 72h with bean juice plate culture medium, wherein the diameter of bacterial colony is about 52mm, the bacterial colony is bright green, the thickness of spore is normal, the surface of bacterial colony grows smoothly, the edge hypha is slightly long, and the thickness of hypha layer is general; the pH indicator plate culture medium is cultured for 96 hours, the diameter of a bacterial colony is about 20mm, the color of the culture medium around the bacterial colony is red and does not change color, hyphae at the edge of the bacterial colony is thin and not obvious, and mesospores are dense and green in color.
3. The aspergillus oryzae ZA184 of the invention is innovative at least in that:
3.1 koji making by Aspergillus oryzae ZA184 of the present invention can obtain a koji having a high pH value and a large amount of enzyme in the average cell amount; the activity of the prepared koji neutral protease, the activity of the alkaline protease, the activity of leucine aminopeptidase and the activity of glutaminase are high; the fermented crude oil has low total acid, high amino nitrogen and high total nitrogen.
3.2 the aspergillus oryzae ZA184 of the invention has fast hydrolysis speed of starter propagation protein and can obviously shorten the fermentation period. The pH value of the finished koji is high, so that the enzyme quantities such as alkaline protease, leucine aminopeptidase and glutaminase generated by unit bacteria quantity are high; and the initial pH value in the fermented soy sauce mash after adding the saline water is high, the effective enzyme activity of various enzymes is high, the hydrolysis is strong and quick, and the fermentation period can be shortened.
Thus, in one aspect, the present invention provides an Aspergillus oryzae (Aspergillus oryzae) ZA184 deposited at 13.8.2018 with the accession number gdmcc. no: 60429, the preservation address is No. 59 building 5 of the No. 100 Dazhong Jie-Lu in Guangzhou city, China.
In another aspect, there is provided the use of aspergillus oryzae ZA184 of the present invention in koji making. In certain embodiments, the koji obtained by said koji making is used for preparing soy sauce.
In another aspect, there is provided the use of aspergillus oryzae ZA184 of the present invention in the preparation of soy sauce.
In another aspect, there is provided the use of aspergillus oryzae ZA184 of the present invention for increasing koji pH, enhancing koji enzyme viability. In certain embodiments, the koji-forming enzyme is selected from the group consisting of a neutral protease, an alkaline protease, a leucine aminopeptidase, a glutaminase, and any combination thereof.
In another aspect, the present invention provides a koji prepared by the invention of aspergillus oryzae ZA 184.
In another aspect, the present invention provides a soy sauce, which is prepared from the koji of the present invention.
The soy sauce of the invention can be prepared according to the traditional soy sauce brewing process. In certain embodiments, the soy sauce of the present invention may be obtained by:
1) bean selection: the selected soybeans are uniform in particle, dry, free of wrinkled skin, high in relative density, free of mildew, rot and deterioration and free of damage;
2) soaking the beans: removing impurities such as mud and sand from soybeans respectively, and soaking in warm water until the hardness is proper and the soybean heart is white;
3) boiling the beans: then, the bean raw materials are respectively cooked in a pressure cooker until the hardness is proper, and the bean raw materials are easy to be utilized by aspergillus oryzae;
4) mixing materials: mixing the cooked soybean with flour, and mixing;
5) preparing yeast: inoculating Aspergillus oryzae ZA184 of the invention, mixing, culturing in a koji pool until mycelium turns white, and collecting koji after koji formation is finished;
6) fermentation: soaking in saline water, fermenting, squeezing, and filtering to obtain soy sauce.
In another aspect, the present invention provides a method for preparing koji comprising inoculating the aspergillus oryzae ZA184 of the present invention into a soy sauce material and culturing.
In certain embodiments, the soy sauce material is a mixture of a legume material and a starchy adjunct.
In another aspect, the present invention provides a method for preparing soy sauce, comprising the steps of koji making and fermentation, wherein the koji making comprises inoculating and culturing aspergillus oryzae ZA184 of the present invention into soy sauce raw material.
In certain embodiments, the soy sauce material is a mixture of a legume material and a starchy adjunct.
In certain embodiments, the fermentation is performed using a low salt solid state fermentation process.
In certain embodiments, the method comprises the steps of: preparing yeast; mixing finished starter obtained by starter propagation with saline water to prepare soy sauce mash; and fermenting the soy sauce mash.
In certain embodiments, the soy sauce mash is fermented using a low salt solid state fermentation process.
In certain embodiments, the method comprises one or more of the following steps:
pretreating bean raw materials;
pretreating starchy auxiliary materials;
mixing the pretreated bean material and starchiness adjuvant, adding Aspergillus oryzae ZA184, culturing, and making into koji;
mixing the finished koji with saline water to prepare soy sauce mash;
fermenting soy sauce mash;
after the fermentation is finished, separating fermented sauce from the fermentation product;
and blending, sterilizing and filling the fermented sauce or any combination thereof to obtain the sauce.
Advantageous effects of the invention
The invention has at least the following technical advantages and positive effects:
1. the screening method of ZA184 strain of the invention has universality and can be widely applied to the transformation of filamentous fungi.
2. The Aspergillus oryzae ZA184 provided by the invention has high pH value in koji making, high yield of neutral protease, alkaline protease, leucine aminopeptidase and glutaminase with high enzyme activity, can be used for brewing seasonings such as soy sauce by using the strain, can obviously shorten the fermentation period, is particularly suitable for brewing soy sauce in a low-salt solid state, and has obvious economic benefit.
The soy sauce enterprises can improve the existing strains by utilizing the method provided by the invention to try to screen out a good aspergillus oryzae strain with the characteristics; the strain can shorten the fermentation period of soy sauce production and realize energy conservation and efficiency improvement.
Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.
Drawings
FIG. 1 is a flow chart of the acquisition of Aspergillus oryzae ZA 184.
FIG. 2 is a colony morphology of Aspergillus oryzae ZA184 on soy bean juice medium.
Description of biological Material preservation
The invention relates to the following biological materials which are preserved in Guangdong province microbial strain preservation center (No. 59 building 5 of the No. 100 institute of the Middleway, Guangzhou, China):
aspergillus oryzae (Aspergillus oryzae) ZA184 having accession number gdmcc.no: 60429, and the preservation date is 2018, 8 and 13.
Detailed Description
The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The media and assay conditions used in the examples of the present invention are those conventional in the art unless otherwise specified. The reagents used in the examples of the present invention were all commercially available unless otherwise specified.
Example 1 obtaining Aspergillus oryzae ZA184
FIG. 1 shows a flow chart for obtaining Aspergillus oryzae ZA184 of the present invention.
1. Strain mutagenesis
Mature slant spores of As3.951 strain of Aspergillus oryzae of family As3 are taken to prepare spore suspension, and ARTP mutagenesis is carried out on the spore suspension. Gradually diluting the mutagenized spore suspension to 10% with 0.85% sterile physiological saline-1、10-2、10-3、10-4、10-5、10-6And (5) concentration, and preparing a spore diluent.
0.2mL of the spore diluent was spread on a pH indicator plate medium (1000mL of rice koji (9-10 ℃ Be'), 150mM citric acid, 2.5mL of Triton X-100, 1g of phenol red, pH adjusted to 7.0, 20g of agar powder, sterilized at 0.1MPa and 121 ℃ for 20min), and cultured at 31 ℃ in the dark for 96 h. And observing and recording the color change of the culture medium around the colony, the growth of hyphae, the sporulation and the like in the culture process. And selecting the mutant strain which has no change in the color of the culture medium around the colony and has normal growth.
And continuously coating the selected target colony on the pH indicator plate on a bean juice plate culture medium, and culturing at 31 ℃ for 72 h. And observing and recording the hypha growth, sporulation and the like of the colonies during the culture process. And selecting the bacterial colony with vigorous hypha growth, long period, high mycelium content and slightly less sporulation of the bacterial colony.
Selecting colonies which have red color, no discoloration, good colony growth, strong hyphae, vigorous growth and little spore production amount and are arranged around the colonies on a double-plate culture medium (a bean juice plate culture medium and a pH indicator plate culture medium) as target colonies, and transferring the target colonies to bean juice slant culture, wherein the colonies are respectively numbered as HC180, HC181, HC182, HC183, HC184 and HC 185.
2. Screening of mutagenized strains
(1) Preliminary screening of mutagenized strains
Activating and transferring the 6 strains obtained by mutagenesis to a test tube soybean juice slant culture medium, and culturing for four days. Inoculating the mature slant to triangular flask culture medium prepared from testa Tritici, semen glycines powder, flour and water, respectively, and performing propagation. Mutant strains HC180, HC181, HC182, HC183, HC184 and HC185 with vigorous hypha growth and normal spore colonization are selected, and the number of spores and germination rate are detected as koji indexes, and the original strain (As3.951) is used as a control strain, and the detection results are shown in Table 1. Preferably, the strains HC180, HC181, HC182, HC183 and HC184 which grow normally and have high germination rate are rescreened.
Table 1: quality analysis results of koji obtained by different strains
Remarking: the indexes of the control strain As3.951 are 1.00, and other mutant strains are converted into corresponding ratios.
(2) Rescreening of mutagenized strains
Sequentially transferring 5 strains of bacteria HC180, HC181, HC182, HC183, HC184 and the like which are primarily screened to slant activation, performing amplification culture in a triangular flask culture medium, and performing starter propagation and fermentation in a starter propagation culture medium prepared from raw materials such as soybean, wheat and the like. The results of measuring the quality indicators of koji making (pH of finished koji, consumption rate of raw materials, activity of neutral protease, alkaline protease, leucine aminopeptidase and glutaminase) are shown in Table 2. The result shows that the pH of the finished koji prepared by HC184 is obviously improved, and the activities of the neutral protease, the alkaline protease, the leucine aminopeptidase and the glutaminase are obviously superior to those of a control strain and other mutant strains.
Table 2: quality analysis results of koji obtained by different strains
Remarking: the indexes of the control strain As3.951 are 1.00, and other mutant strains are converted into corresponding ratios.
The quality of the fermented crude oil (total acid, amino nitrogen, total nitrogen yield, glutamic acid) was measured for 10 days, 20 days and 30 days of fermentation, and the measurement results are shown in tables 3, 4 and 5, respectively. The results show that the amino nitrogen, total nitrogen and glutamic acid contents of the crude oil obtained after fermentation of the finished koji prepared by HC184 are obviously improved compared with the control strain and other mutagenic strains, and the higher amino nitrogen, total nitrogen and glutamic acid contents can be reached after fermentation for 10 days.
Table 3: fermentation crude oil quality analysis results (fermentation 10 days)
Remarking: the indexes of the control strain As3.951 are 1.00, and other mutant strains are converted into corresponding ratios.
Table 4: fermentation crude oil quality analysis results (fermentation 20 days)
Remarking: the indexes of the control strain As3.951 are 1.00, and other mutant strains are converted into corresponding ratios.
Table 5: fermentation crude oil quality analysis results (fermentation 30 days)
Remarking: the indexes of the control strain As3.951 are 1.00, and other mutant strains are converted into corresponding ratios.
The results show that HC184 is not only beneficial to improving the delicate flavor of the fermented crude oil and enriching the mouthfeel, but also can obviously shorten the fermentation period and improve the production efficiency. Therefore, the HC184 strain obtained by breeding has obvious advantages.
The HC184 obtained by the breeding of this example has been deposited in the collection of microorganisms of Guangdong province, and is named as Aspergillus oryzae (Aspergillus oryzae) ZA184 with the deposition number gdmcc. no: 60429. thus, in the present application, HC184 and ZA184 refer to the same strain.
The colony characteristics of Aspergillus oryzae ZA184 were as follows: after the culture is carried out for 72 hours on a bean juice plate culture medium, the diameter of a bacterial colony is about 52mm, the bacterial colony is bright green, the thickness of spores is normal, the surface of the bacterial colony grows more smoothly, the edge hyphae are slightly longer, and the thickness of a hypha layer is common, and the bacterial colony morphology of the Aspergillus oryzae ZA184 strain on the bean juice culture medium is shown in figure 2. Culturing for 96h in a pH indicator plate culture medium, wherein the diameter of a bacterial colony is about 20mm, the color of the culture medium around the bacterial colony is red and does not change, hyphae at the edge of the bacterial colony is thin and not obvious, and mesospores are dense and green in color.
Example 2 application of Aspergillus oryzae ZA184 in Soy sauce brewing
Aspergillus oryzae ZA184 was synchronized with the starting strain As3.951 for soy sauce production. Specifically, soybeans are washed, soaked at normal temperature and cooked in a steam pot, the cooked soybeans are cooled and then mixed with wheat flour, corresponding aspergillus oryzae is inoculated for starter propagation, and low-salt solid state fermentation is carried out after starter propagation is finished.
After the completion of koji preparation, pH values and enzyme activities were sampled and measured, and the results are shown in Table 6. Results show that compared with a control strain, the pH value of the finished koji obtained by the ZA184 of the invention is improved by more than 15%, the activity of neutral protease is improved by more than 23%, the activity of alkaline protease is improved by more than 22%, the activity of leucine aminopeptidase is improved by more than 29%, and the activity of glutaminase is improved by more than 12%.
Table 6: quality analysis of finished koji
The crude oil quality indexes were measured for 10 days, 20 days, 25 days and 30 days of fermentation, and the results are shown in tables 7, 8, 9 and 10, respectively. The results show that the amino nitrogen, total nitrogen and glutamic acid content of the crude oil obtained by the ZA184 of the invention are obviously improved compared with the control strain. In particular, the quality index of the crude oil obtained with ZA184 according to the invention reaches high values at 25 days of fermentation, whereas the control strain takes 30 days of fermentation. This result indicates that under the same starter propagation fermentation process conditions, the fermentation period of the strain ZA184 of the present invention is shortened by more than 5 days compared with the control strain AS 3.951.
Table 7: fermentation crude oil quality analysis (fermentation 10 days)
Table 8: fermentation crude oil quality analysis (fermentation 20 days)
Table 9: fermentation crude oil quality analysis (fermentation 25 days)
Table 10: fermentation crude oil quality analysis (30 days of fermentation)
The results show that the strain ZA184 can obviously improve the pH value of finished koji, the activity of neutral protease, alkaline protease, leucine aminopeptidase and glutaminase, and improve the content of amino nitrogen, total nitrogen and glutamic acid in soy sauce, thereby improving the flavor quality of the soy sauce, obviously shortening the fermentation period and improving the production efficiency. Therefore, the strain ZA184 of the present invention is particularly suitable for the production of soy sauce.
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.
Claims (8)
1. Aspergillus oryzae (Aspergillus oryzae) ZA184 deposited at 13.8.2018 with the accession number gdmcc. no: 60429, the preservation address is No. 59 building 5 of the No. 100 Dazhong Jie-Lu in Guangzhou city, China.
2. Use of aspergillus oryzae ZA184 according to claim 1 in koji making, wherein koji-making is obtained for the preparation of soy sauce.
3. Use of aspergillus oryzae ZA184 according to claim 1 for the preparation of soy sauce.
4. A koji prepared by the aspergillus oryzae ZA184 of claim 1.
5. A soy sauce prepared from the koji claimed in claim 4.
6. A method for producing koji comprising inoculating the Aspergillus oryzae ZA184 of claim 1 into a soy sauce material and culturing.
7. A method for producing soy sauce, comprising the steps of koji-making and fermentation, wherein the koji-making comprises inoculating the aspergillus oryzae ZA184 of claim 1 into soy sauce raw material and culturing.
8. The method of claim 7, wherein the fermentation is performed using a low salt solid state fermentation process.
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