CN117645964B - Staphylococcus succinogenes and application thereof - Google Patents
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Abstract
The invention relates to staphylococcus succinogenes and application thereof, and belongs to the technical field of microbial fermentation. The staphylococcus amber SG052 provided by the invention is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.27529, the preservation date of 2023 and 06 month 02, the preservation address of Beijing, and the classification name of the staphylococcus amber Staphylococcus succinus. The staphylococcus succinogenes SG052 provided by the invention can improve the pH value of a culture medium, can be used for fermenting bean paste and soy sauce, can obviously reduce the total acid content in the bean paste and the soy sauce, can inhibit the growth of mixed bacteria, and can improve the taste and flavor of the bean paste and the soy sauce.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to staphylococcus succinogenes and application thereof.
Background
The soybean paste and soy sauce fermentation belong to a multi-micro co-fermentation system, and on the basis of strict process management, the natural brewing microorganisms are inevitably accessed. The multi-strain co-fermentation process forms rich aroma and taste of the bean paste, but the instability of flavor quality of the fermented semi-finished product is caused by the different microorganism compositions and proportions in the multi-strain co-fermentation process due to the uncertainty of natural inoculation. If in the fermentation of soybean paste and soy sauce, the proliferation of acidogenic microorganisms can increase the variety and content of organic acids after fermentation, enrich the taste of the product, but excessive proliferation can lead to higher total acids after fermentation, which adversely affects the taste of soybean paste and soy sauce.
Coagulase-negative staphylococci (CNS) are generally described as benign bacteria, such as staphylococcus xylosus, staphylococcus sarcoidosis, all belonging to the species useful in food. The results of the flora analysis of traditional fermented foods such as soybean paste, soy sauce also show that staphylococci are the main bacterial group.
Studies have shown that total acid in soybean paste can be reduced by screening Pediococcus acidilactici and inhibiting acid-producing bacillus (Zhou Jiyang, hou Jie. A strain which can promote screening, identification and application of soybean paste flavor lactobacillus [ J ]. Chinese brewing, 2019, 38 (6)). However, in actual production, acid-producing microorganisms in a soybean paste fermentation system have a large amount of lactic acid bacteria besides bacillus, and the method can inhibit the acid production of bacillus, but cannot solve the acid production problem of other types of microorganisms.
Disclosure of Invention
The invention aims to provide a staphylococcus succinogenes which is used for fermenting bean paste and soy sauce and can remarkably reduce the total acid content in the bean paste and the soy sauce.
In order to achieve the above purpose, the present invention provides the following technical solutions: the staphylococcus succinogenes is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), the preservation address is Beijing, the preservation number is CGMCC No. 27529, the preservation date is 2023 and 06 month 02, and the staphylococcus succinogenes is classified and named as staphylococcus succinogenes Staphylococcus succinus.
Strain isolation source: fermenting for 1-2 months to obtain thick broad-bean sauce mash;
primary screening of the culture medium: TSA medium with 10g/L glucose, 100g/L NaCl, 0.02g/L bromocresol purple was added;
Re-screening the culture medium: 5.0 g/L peptone, 10.0 g/L yeast extract powder, 0.5 g/L Tween 80, 0.02g/L, naCl g/L bromocresol purple and 2.0g/L agar;
culture conditions for staphylococcal SG 052: the glycerol tube deposited strain was streaked onto TSA medium containing 100g/L NaCl and incubated at 36℃for 3 days.
The invention also provides a soy sauce mash containing the staphylococcus succinogenes.
Preferably, the inoculum size of the staphylococcus succinogenes is 10 5-107 CFU/g per gram of moromi.
The invention also provides a product containing the staphylococcus succinogenes or the moromi.
Preferably, the product comprises a microbial agent and a fermentation preparation.
The invention also provides application of the staphylococcus succinogenes, or the moromi, or the product in preparing soybean paste and soy sauce.
The invention also provides a preparation method of the soybean paste, which comprises the following steps: the staphylococcus succinogenes is applied during the preparation process.
The invention also provides a preparation method of the soy sauce, which comprises the following steps: the staphylococcus succinogenes is applied during the preparation process.
The invention also provides a method for reducing the total acid content in the product, which comprises the steps of inoculating staphylococcus succinogenes into raw materials of the product and fermenting.
Compared with the prior art, the invention has the beneficial effects that: the invention separates a staphylococcus succinogenes SG052 from the soy sauce mash, the staphylococcus succinogenes can improve the pH value of a culture medium, and the staphylococcus succinogenes can be used for fermenting the soy sauce and the soy sauce, so that the total acid content in the soy sauce and the soy sauce can be obviously reduced, the growth of mixed bacteria can be inhibited, and the taste and flavor of the soy sauce and the soy sauce can be improved.
Drawings
FIG. 1 is a graphical representation of inoculation of Staphylococcus succinogenes SG052 in a rescreened medium in an example of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made more apparent and fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the following examples, starter propagation fermentation of soybean paste and soy sauce is achieved by the following steps:
Bean paste
1) The starter propagation process comprises the following steps:
the pretreatment of soybeans comprises: after cleaning the soybeans, controlling the temperature to 98-120 ℃ and steaming for 8-10 minutes; the pretreatment of wheat flour comprises: the temperature is controlled to 98-120 ℃, the mixture is steamed for 20-30 s, and the water content is controlled to 22% -24%;
mixing the pretreated soybeans and wheat flour according to a ratio of 1:10-1:1, inoculating aspergillus oryzae, and then making starter at 28-35 ℃ for 38-45 hours to obtain the fermented soy sauce starter.
2) Fermentation:
Mixing the fermented soy sauce yeast with brine to obtain soy sauce mash, wherein the concentration of the brine is 20.5% -23.5%; the adding amount of the salt water is 0.9-1.5 times of the weight of the fermented soy sauce. The fermentation temperature is maintained at 25-35 ℃, and the fermentation time is 70-100 days; detecting the content change of tyrosine, tyramine and amino acid nitrogen after fermentation, and organizing sensory evaluation.
Soy sauce
1) The starter propagation process comprises the following steps:
The pretreatment of defatted soybeans comprises: after the defatted soybeans are cleaned, controlling the temperature to be 115-120 ℃ and the pressure to be 0.05-0.15 MPa, and steaming for 9 minutes; pretreatment of the roasted wheat comprises the following steps: the temperature is controlled at 300-400 ℃, the pressure is controlled at 0.01-0.05 MPa, the moisture is controlled at 0.8% after wheat frying, and finally the wheat is crushed, wherein the particle size is 20-100 meshes after crushing. Mixing the pretreated defatted soybean and fried wheat according to a ratio of 1:1.5, inoculating Aspergillus oryzae, and making starter at 28-40 ℃ for 42h to obtain soy sauce starter.
2) Fermentation:
Mixing soy sauce yeast and brine to prepare soy sauce mash, wherein the concentration of the brine is 16-20%, and mixing the soy sauce yeast and the brine according to the weight ratio of 1:1-1:1.5. The primary fermentation temperature is 8-10 ℃, and the fermentation is carried out for 1-1.5 months; the medium-term fermentation temperature is 20-25 ℃, and the fermentation time is 2.5-3 months; the later fermentation temperature is 30-32 ℃, the time is 1-2 months, and the total of the earlier stage, the middle stage and the later stage is hermetically fermented for 6 months.
In the following examples, the strain isolation and culture conditions are as follows:
strain isolation source: fermenting for 1-2 months.
Primary screening of the culture medium: 10g/L glucose, 100g/L NaCl, 0.02g/L bromocresol purple in TSA medium were added.
Re-screening the culture medium: 5.0 g/L peptone, 10.0 g/L yeast extract powder, 0.5 g/L Tween 80, 0.02g/L, naCl g/L bromocresol purple and 2.0g/L agar.
Culture conditions for staphylococcal SG 052: the glycerol tube deposited strain was streaked onto TSA medium containing 100g/L NaCl and incubated at 36℃for 3 days.
EXAMPLE 1 isolation and screening of Staphylococcus succinogenes
Adding 25 g fermented soybean paste mash for 1 month into 225 mL sterile physiological saline, beating with a homogenizer for 2min to obtain 10 -1 dilution, and gradient diluting to obtain 10 -2、10-3、10-4、10-5、10-6 dilution. 100 mu L of the diluted solution is respectively coated on a primary screening culture medium containing 10% NaCl, and the culture is carried out for 3 days at 36 ℃, single bacterial colony which has better growth vigor and purple culture medium around the bacterial colony is selected, and the TSA containing 10% NaCl is used for separation and purification, thus obtaining a pure culture.
And selecting single bacterial colony, culturing the bacterial colony by using a re-screening culture medium containing 10% NaCl in a streaking way, and screening out bacterial strains with the culture medium surrounding the bacterial colony changed from yellow green to purple after culturing for 3 days at 36 ℃, namely, the bacterial strain capable of raising the pH value of the culture medium. After rescreening, strain SG052 grew fastest on the salt-containing plates and rescreened plates changed from yellow-green to purple. As shown in FIG. 1, the areas of SG052 where colonies are less aggregated are still yellowish green in the initial culture medium, while the areas where colonies are more aggregated are purple. Since bromocresol purple has a pH discoloration range of 5.2 (yellow) to 6.8 (purple), SG052 can turn the medium purple, indicating that SG052 can raise the pH of the medium and weaken the acidity of the medium.
Molecular identification: the strain SG052 was subjected to 16S sequencing, and the sequencing results were compared and analyzed in NCBI database, and the result shows that the strain SG052 is staphylococcus succinogenes Staphylococcus succinus. The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No. 27529.
EXAMPLE 2 safety evaluation of Strain SG052
Plasma clotting enzyme assay: and (3) carrying out streak culture on the strain SG052 until a single colony exists, picking the single colony by using an inoculating needle, transferring the single colony into a sterile liquid culture medium test tube, and culturing for 24 hours at 36 ℃ after shaking uniformly. Taking freeze-dried rabbit plasma which is preserved at low temperature, adding 500 mu L of sterile normal saline until the freeze-dried powder is completely dissolved, adding 250 mu L of bacterial liquid which is uniformly vibrated, uniformly shaking, placing in a 36 ℃ incubator for observation every half hour, and observing for 6 hours; if clotting (i.e., clotting when the tube is tilted or inverted) or clotting volume is greater than half of the original volume, a positive result is determined. A broth culture of staphylococcus aureus positive and staphylococcus epidermidis negative for the plasma coagulase test was also used as a control.
Hemolysis experiment: strains SG052 were streaked onto blood plates and incubated at 36℃for 24h, while positive strains Staphylococcus aureus and negative strains Staphylococcus epidermidis were streaked as experimental controls. After the staphylococcus aureus of the positive strain is cultured on a blood agar plate at 36 ℃ for 18-24 hours, a colony is large, round, smooth and convex, moist and golden (sometimes white) is formed, and a completely transparent hemolytic ring is visible around the colony. The staphylococcus epidermidis of the negative strain is cultured on a blood agar plate at 36 ℃ for 18-24 hours, and then is a colony with medium size, bulges, neat edges, smooth surface, wetness, white or lemon color, and has no hemolysis transparent ring. After the strain SG052 is cultured, white colonies are observed, no plasma coagulation phenomenon exists, and no hemolytic ring appears on a blood plate, namely, SG052 has no coagulability and hemolysis, and is safe and available.
The experimental results are shown in table 1:
TABLE 1 SG052 plasma coagulase assay and hemolysis assay results
EXAMPLE 3 Small test of Strain SG052 for fermentation of Bean paste
The brewing sauce is prepared into yeast and brine with the concentration of 22 percent according to the weight ratio of 1:1, mixing to form soy sauce mash, inoculating the strain SG052 after activating and culturing to the soy sauce mash fermented for 0 day, wherein the inoculum size is 10 6 CFU/g (soy sauce mash), and the group is used as an experimental group; fermenting for 0 days without inoculating strain SG052 to obtain sauce mash as control group; the experimental group and the control group are controlled according to the normal fermentation process of the bean paste (the fermentation temperature is maintained at 25-30 ℃ and the fermentation time is 90 days). After fermentation, the soybean paste is pasteurized to obtain the original paste, and total acid and amino acid nitrogen are detected, wherein the total acid content detection refers to the determination of total acid in national food safety Standard of food in GB 12456-2021, and the amino acid nitrogen content detection refers to the determination of amino acid nitrogen in national food safety Standard of food in GB 5009.235-2016. Compared with the control group, the total acid content of the experimental group is obviously reduced, the pH value is obviously increased, and the strain SG052 has the effect of reducing the total acid content of the fermented sauce. The specific detection results are shown in Table 2.
TABLE 2 detection of sauce mash index at maturity
Example 4 pilot plant application of Strain SG052 in fermentation of Bean paste
Mixing the fermented soy sauce with 21% saline solution at a ratio of 1:1.5 to obtain soy sauce mash.
Experimental group: inoculating strain SG052 into soy sauce mash fermented for 0 day, wherein the inoculation amount is 10 5 CFU/g (soy sauce mash);
Control group: fermenting the soy sauce mash for 0 day without inoculating strain SG052;
The experimental group and the control group are controlled according to the same fermentation process of the soybean paste, the temperature is controlled to be 25-30 ℃, the pH value is controlled to be 4-6, the fermentation time is 90 days, and the soybean paste raw paste is obtained, and the total acid and amino acid nitrogen are detected, and the detection method is the same as that of example 3. After obtaining the finished product, 15 professional panelists were selected to evaluate and score from the group consisting of body, color, aroma, sour, sweet, bitter, salty, umami, soy sauce and comprehensive mouthfeel, wherein the score was 0-5 points, and the higher the score indicated that the index was better.
As can be seen from Table 3, compared with the control group, the total acid content of the experimental group is obviously reduced, and the pH value is increased, which indicates that the strain SG052 has the effect of reducing the total acid content of the fermented sauce; in addition, the content of amino acid nitrogen in the experimental group is increased compared with that in the control group, which shows that the strain SG052 inhibits the growth of partial mixed bacteria and reduces the consumption of amino acid nitrogen. As shown in Table 4, the finished product obtained by fermentation in the experimental group is obviously improved in sour taste, and the comprehensive taste is better than that of the control group, which shows that the strain SG052 has a certain effect of improving the flavor of the soybean paste.
TABLE 3 detection of sauce mash index at maturity
TABLE 4 sensory evaluation results of fermented Soy products
Example 5 pilot plant application of Strain SG052 in soy sauce koji making fermentation
Mixing soy sauce yeast with 18% saline solution at a ratio of 1:1.5 to obtain soy sauce mash.
Experimental group: inoculating strain SG052 into soy sauce mash fermented for 0 day, wherein the inoculation amount is 10 7 CFU/g (soy sauce mash);
Control group: fermenting the soy sauce mash for 0 day without inoculating strain SG052;
The experimental group and the control group are controlled according to the same soy sauce fermentation process, the initial fermentation temperature is 8-10 ℃, and the fermentation is carried out for 1.5 months; the medium-term fermentation temperature is 23-25 ℃, and the fermentation time is 2.5 months; the later fermentation temperature is 30-32 ℃, the time is 2 months, and the total of the earlier stage, the middle stage and the later stage is sealed and fermented for 6 months. Squeezing after fermentation to obtain soy sauce crude oil, and detecting total acid and amino acid nitrogen, wherein the detection method is the same as in example 3. After obtaining the finished product, 15 professional panelists were selected to evaluate and score from the group consisting of body, color, aroma, sour, sweet, bitter, salty, umami, soy sauce and comprehensive mouthfeel, wherein the score was 0-5 points, and the higher the score indicated that the index was better.
As shown in Table 5, compared with the control group, the total acid content of the experimental group is obviously reduced, the pH value is increased, and the amino acid nitrogen content is increased compared with the control group, which proves that the strain SG052 can reduce the total acid and inhibit the growth of partial mixed bacteria, thereby reducing the consumption of amino acid nitrogen. As shown in Table 6, the finished product obtained by fermentation in the experimental group is obviously improved in sour taste, and the comprehensive taste is better than that of the control group, which shows that the strain SG052 has a certain effect of improving the flavor of soy sauce.
TABLE 5 crude oil index detection for soy sauce
TABLE 6 sensory evaluation results of Soy sauce finished products
In summary, the staphylococcus succinogenes SG052 provided by the invention is used for fermenting soybean paste or soy sauce, can improve the pH of a culture medium, reduce the total acid content in the soybean paste or soy sauce, improve the problem of partial acid taste, inhibit the growth of mixed bacteria, and generally improve the taste and flavor of the soybean paste or soy sauce.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the above-described embodiments, and that the above-described embodiments and descriptions are only preferred embodiments of the present invention, and are not intended to limit the invention, and that various changes and modifications may be made therein without departing from the spirit and scope of the invention, and that the changes and modifications fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A staphylococcus succinogenes strain, which is characterized in that,
The staphylococcus succinogenes is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.27529, a preservation date of 2023, 06 and 02 days, a preservation address of Beijing, and a classification of Staphylococcus succinus.
2. A moromi comprising the staphylococcus succinogenes of claim 1.
3. The moromi of claim 2, wherein the staphylococcus amber is inoculated at 10 5-107 CFU/g per gram of moromi.
4. Use of staphylococcus succinogenes according to claim 1, or moromi according to claim 2, for the preparation of soy sauce, soy sauce.
5. A method for preparing soybean paste, comprising: the staphylococcus succinogenes of claim 1 applied during the preparation process.
6. A method for preparing soy sauce, comprising: the staphylococcus succinogenes of claim 1 applied during the preparation process.
7. A method for reducing the total acid content of a product, wherein the product is a soybean paste or sauce, and the staphylococcus succinogenes of claim 1 is inoculated into a raw material of the product and fermented.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101919756B1 (en) * | 2017-05-22 | 2018-11-19 | 경기대학교 산학협력단 | Novel microorganism, starter composition including the same and fermented product by the same |
| KR102186420B1 (en) * | 2019-10-17 | 2020-12-03 | 고려대학교 산학협력단 | Composition for preventing, improving or treating of allergic diseases comprising Staphylococcus succinus 14BME20 as an active ingredient |
| CN114058551A (en) * | 2021-12-03 | 2022-02-18 | 楚雄云泉酱园有限责任公司 | Staphylococcus succinogenes for fermentation of broad bean paste |
| CN116536185A (en) * | 2023-03-13 | 2023-08-04 | 江南大学 | Pediococcus acidilactici for improving color and flavor of low-salt solid soy sauce |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101919756B1 (en) * | 2017-05-22 | 2018-11-19 | 경기대학교 산학협력단 | Novel microorganism, starter composition including the same and fermented product by the same |
| KR102186420B1 (en) * | 2019-10-17 | 2020-12-03 | 고려대학교 산학협력단 | Composition for preventing, improving or treating of allergic diseases comprising Staphylococcus succinus 14BME20 as an active ingredient |
| CN114058551A (en) * | 2021-12-03 | 2022-02-18 | 楚雄云泉酱园有限责任公司 | Staphylococcus succinogenes for fermentation of broad bean paste |
| CN116536185A (en) * | 2023-03-13 | 2023-08-04 | 江南大学 | Pediococcus acidilactici for improving color and flavor of low-salt solid soy sauce |
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