CN111909923A - Strain immobilization method and application of immobilized strain - Google Patents
Strain immobilization method and application of immobilized strain Download PDFInfo
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- CN111909923A CN111909923A CN202010865196.6A CN202010865196A CN111909923A CN 111909923 A CN111909923 A CN 111909923A CN 202010865196 A CN202010865196 A CN 202010865196A CN 111909923 A CN111909923 A CN 111909923A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/024—Preparation of other alcoholic beverages by fermentation of fruits other than botanical genus Vitis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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Abstract
The invention belongs to the technical field of strain immobilization, and particularly relates to a strain immobilization method and application of immobilized strains, wherein the strain immobilization method comprises the following steps: inoculating; swelling; solidifying into beads; and (5) air drying and preserving. According to the invention, an immobilization technology is introduced, immobilized strain cells are utilized, the survival capability and fermentation characteristic of strains are improved, and a theoretical basis is provided for developing immobilized fermentation and continuously producing fruit wine; the immobilized strain obtained by the invention has similar fermentation characteristics with the free strain, has no influence on fermentation, can be repeatedly used for fermentation, does not reduce the activity of each fermentation, does not change the alcohol production capacity too much, and greatly reduces the production cost.
Description
Technical Field
The invention belongs to the technical field of strain immobilization, and particularly relates to a strain immobilization method and application of immobilized strains.
Background
The cherry belongs to the Rosaceae, Prunus, and the mature fruit of the cherry belongs to the Prunus subgenus, and is one of the earliest fruits on the market in spring, and the cherry is called as the first branch of the spring fruit. The cherry has rich nutrition, and the fruit is rich in sugar, protein, vitamins, calcium, ferrum, phosphorus, potassium and other elements. The iron content of the cherries is rich, and each hundred grams of cherries contain 59 mg of iron and are positioned at the head of the fruits. The vitamin A content in cherry is 4-5 times more than that in grape, apple and orange. However, at present, cherries are mainly sold in the form of fresh fruits, are greatly influenced by seasons, are single in product, and are easy to rot, over-mature and brown after being picked. The cherry fruit wine prepared by fusing the cherry and the fruit wine is convenient for long-term storage, can retain the nutritive value of the cherry and the fragrance of the fruit wine, and has various effects of delaying senescence, preventing cardiovascular and cerebrovascular diseases, effectively reducing cholesterol, preventing arteriosclerosis, beautifying and protecting skin, restoring consciousness and refreshing, reducing fat and helping digestion and the like.
The fermented fruit wine in the existing market is prepared by fermenting free strains basically, and has the defects of poor later-stage survival capability of the strains, incapability of being recycled and high cost. The application of immobilized cell technology and bioreactor to realize rapid fermentation is an effective method for solving the problem, so that the application of biotechnology to realize semi-continuous or continuous fermentation to produce fruit wine is the development direction of the current fruit wine industry.
Disclosure of Invention
Aiming at the problems that most fermented fruit wine in the market at present adopts free strains for fermentation and the strains cannot be recycled, so that the cost is high, the invention provides a strain immobilization method and application of immobilized strains, so as to solve the problems.
A strain immobilization method comprises the following steps: inoculating; swelling; solidifying into beads; and (5) air drying and preserving.
Further, the inoculation step: inoculating the strain into the liquid to be fermented, and performing shake culture at 28 deg.C and 160r/min for 12 h.
Furthermore, the to-be-fermented liquid is cherry juice which is subjected to enzymolysis and added with 200ppm of sodium metabisulfite.
Further, the strain is yeast composition of special yeast for Angel grape wine fruit wine and active dry yeast for Angel grape wine.
Further, the swelling step: adding 1.5-2.5% sodium alginate solution into the inoculated fermentation solution to swell fully, stirring and mixing uniformly.
Further, the step of solidifying into beads comprises the following steps: injecting the swelled mixed bacteria liquid into a sterilizing injector, selecting needles with different sizes, dripping the needles into a calcium chloride solution with the concentration of 0.5-1.5% to solidify the needles into rubber beads, soaking the rubber beads in the calcium chloride solution for 4min, taking out the rubber beads, washing the rubber beads with sterile water, and washing off the calcium chloride solution on the surfaces of the rubber beads.
Further, the air-drying preservation step comprises: drying the surface moisture of the rubber beads at 35 ℃, and preserving at low temperature of 0-4 ℃.
Furthermore, the diameter of the rubber beads is 2-6.5 mm.
The immobilized strain obtained by the strain immobilization method is applied to fermentation preparation of cherry fruit wine, and comprises the following steps: adding immobilized strains with a volume of 3% of the cherry juice into the cherry juice, and fermenting at 28 deg.C under stirring for 12 hr; then taking out the immobilized strains, cooling to 23 ℃, and continuing to ferment for 120 h.
The invention has the beneficial effects that:
the strain immobilization method provided by the invention introduces an immobilization technology, utilizes immobilized strain cells to improve the survival capability and fermentation characteristic of strains, and provides a theoretical basis for developing immobilized fermentation and continuously producing fruit wine. In the immobilization process, the concentration of the strain directly influences the bacterium content of the glue beads; the concentration of sodium alginate determines the molding and reaction performance of the gel beads, if the concentration is too low, beading cannot be realized, if the concentration is too high, beading is difficult to extrude, and if the concentration is too high, the sensitivity of strains and external fermentation is reduced due to the fact that the gel beads are too compact; the concentration of calcium chloride determines the hardness of the gel beads and the reaction rate. The optimal concentrations of sodium alginate and sodium chloride are determined through experiments, so that the beading performance, the hardness of the rubber beads, the surface strength of the rubber beads and other performances are optimal.
The immobilized strain obtained by the invention has similar fermentation characteristics with the free strain, has no influence on fermentation, can be repeatedly used for fermentation, does not reduce the activity of each fermentation, does not change the alcohol production capacity too much, and greatly reduces the production cost.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A strain immobilization method comprises the following steps: inoculating; swelling; solidifying into beads; and (5) air drying and preserving.
Inoculation: taking 30ppm of strain, the strain is yeast composition of Angel wine fruit wine special yeast and Angel wine active dry yeast with weight ratio of 1:2, inoculating the yeast composition into cherry juice which is subjected to enzymolysis and added with 200ppm of sodium metabisulfite, and performing shake cultivation at 28 ℃ and 160r/min for 12 h.
Swelling: adding sodium alginate solution with concentration of 1.5%, 2.0%, and 2.5% into three inoculated fermentation solutions respectively to swell, stirring, and mixing.
And (3) solidifying into beads: injecting the three swollen mixed bacteria solutions into a sterilization syringe, selecting needles with the same size, respectively dripping the needles into a calcium chloride solution with the concentration of 1.0% to solidify the mixed bacteria solutions into rubber beads, soaking the rubber beads in the calcium chloride solution for 4min, taking out the rubber beads, washing the rubber beads with sterile water, and washing off the calcium chloride solution on the surfaces of the rubber beads.
Air drying and preserving: drying the surface moisture of the rubber beads at 35 ℃, and preserving at low temperature of 0-4 ℃.
According to the percentage, the beading performance (40 min), the hardness (40 min) of the rubber beads and the surface strength (20 min) of the rubber beads are taken as indexes, 10 persons are scored and averaged, and the beading performance under different sodium alginate concentrations is compared, as shown in table 1:
TABLE 1 beading Performance at different sodium alginate concentrations
| Sodium alginate concentration (%) | 1.5 | 2.0 | 2.5 |
| Average score | 75 | 82.1 | 81 |
As can be seen from Table 1, 2.0% sodium alginate has the highest bead forming performance score, proper strength and elasticity, and no breaking phenomenon of the rubber beads occurs in the shaking table and stirring fermentation experiments, so 2.0% is selected as the optimal concentration of the sodium alginate solution.
Example 2
A strain immobilization method comprises the following steps: inoculating; swelling; solidifying into beads; and (5) air drying and preserving.
Inoculation: taking 30ppm of strain, the strain is yeast composition of Angel wine fruit wine special yeast and Angel wine active dry yeast with weight ratio of 1:2, inoculating the yeast composition into cherry juice which is subjected to enzymolysis and added with 200ppm of sodium metabisulfite, and performing shake cultivation at 28 ℃ and 160r/min for 12 h.
Swelling: adding sodium alginate solution with concentration of 2.0% into the inoculated fermentation solution to swell fully, stirring and mixing well.
And (3) solidifying into beads: and dividing the swelled mixed bacteria liquid into three parts, respectively injecting the three parts into a sterilization syringe, selecting needles with the same size, respectively dripping the three parts into calcium chloride solutions with the concentrations of 0.5%, 1.0% and 1.5% to solidify the calcium chloride solutions into rubber beads, soaking the rubber beads in the calcium chloride solution for 4min, then taking out the rubber beads, washing the rubber beads with sterile water, and washing off the calcium chloride solution on the surfaces of the rubber beads.
Air drying and preserving: drying the surface moisture of the rubber beads at 35 ℃, and preserving at low temperature of 0-4 ℃.
According to the percentage, the beading performance (40 minutes), the hardness (40 minutes) of the rubber beads and the surface strength (20 minutes) of the rubber beads are taken as indexes, 10 persons are scored and averaged, and the beading performance under different calcium chloride concentrations is compared, as shown in table 2:
TABLE 2 beading Performance at different calcium chloride concentrations
| Calcium chloride concentration (%) | 0.5 | 1.0 | 1.5 |
| Average score | 78.7 | 82.1 | 79 |
As can be seen from Table 2, the beading performance of 1.0% calcium chloride is the highest, and it is found in the experiment that the calcification time required for 0.5% calcium chloride solution is longer, and the surface of the rubber beads is too hard to spread the strains although the calcification time is short in 1.5% calcium chloride solution, so that 1.0% is selected as the optimum concentration of calcium chloride solution.
Example 3
A strain immobilization method comprises the following steps: inoculating; swelling; solidifying into beads; and (5) air drying and preserving.
Inoculation: taking 30ppm of strain, the strain is yeast composition of Angel wine fruit wine special yeast and Angel wine active dry yeast with weight ratio of 1:2, inoculating the yeast composition into cherry juice which is subjected to enzymolysis and added with 200ppm of sodium metabisulfite, and performing shake cultivation at 28 ℃ and 160r/min for 12 h.
Swelling: adding sodium alginate solution with concentration of 2.0% into the inoculated fermentation solution to swell fully, stirring and mixing well.
And (3) solidifying into beads: injecting the swelled mixed bacteria liquid into a sterilization injector, selecting three needles with different sizes, respectively dripping the needles into a calcium chloride solution with the concentration of 1.0% to respectively solidify the needles into rubber beads with the diameters of 2-2.5 mm, 4-4.5 mm and 6-6.5 mm, soaking the rubber beads in the calcium chloride solution for 4min, taking out the rubber beads, washing the rubber beads with sterile water, and washing away the calcium chloride solution on the surfaces of the rubber beads.
Air drying and preserving: drying the surface moisture of the rubber beads at 35 ℃, and preserving at low temperature of 0-4 ℃.
The three kinds of glue beads (immobilized strains) with different diameters obtained by the strain immobilization method are respectively used for cherry juice fermentation, and the method comprises the following steps: taking 6g of the immobilized strains with different diameters, respectively adding the immobilized strains into 200ml of cherry juice, and stirring and fermenting at 28 ℃ for 12 h; then taking out the immobilized strains, and cooling to 23 ℃ for continuous fermentation. Wherein, the change of the alcoholic strength is measured every 24h, which is specifically shown in table 3:
TABLE 3 Effect of glue bead size on alcohol content
As can be seen from Table 3, the alcohol production speed of the small size glue beads (diameter 2-2.5 mm) and the medium size glue beads (diameter 4-4.5 mm) is significantly faster than that of the large size glue beads (diameter 6-6.5 mm). The small-size rubber beads and the medium-size rubber beads have similar alcohol production trend along with the change of time, and the rubber beads have too small diameter and are not easy to recover in actual production, so that the medium-size rubber beads (with the diameter of 4-4.5 mm) are more suitable for industrial production.
Comparative example 1
The free strain is used for fermenting cherry juice, and comprises the following steps: taking 30ppm of strain, wherein the strain is a yeast composition of Angel wine fruit wine special yeast and Angel wine active dry yeast with a weight ratio of 1:2, inoculating the yeast composition into cherry juice which is subjected to enzymolysis and added with 200ppm of sodium metabisulfite, and performing shake cultivation for 12h at 28 ℃ and 160r/min to obtain seed solution; inoculating 6ml seed solution into 200ml cherry juice to be fermented, stirring and fermenting at 28 deg.C for 12 hr, and cooling to 23 deg.C for further fermentation. Wherein, the change of the alcoholic strength is measured every 24h, and the comparison of the alcoholic strength of the free strain of the comparative example and the alcoholic strength of the medium size glue bead (immobilized strain with the diameter of 4-4.5 mm) in the fermentation process of the example 3 is shown in the table 4:
TABLE 4 comparison of alcohol content during fermentation of free and immobilized bacteria
As can be seen from Table 4, the alcohol produced by the immobilized strain and the free strain is basically similar with the change of time in the fermentation process, so that the fermentation characteristics of the immobilized strain and the free strain are similar and have no influence on the fermentation.
Example 4
After the medium size glue beads (immobilized strains with the diameter of 4-4.5 mm) in example 3 are fermented and used, the medium size glue beads are taken out, washed clean by normal saline, added into 200mL of cherry pulp and continuously fermented, and the steps are repeated for a plurality of times. In each fermentation process, the change of the alcoholic strength in the fermentation process is measured after 96 hours of fermentation, and then the change of the alcoholic strength in the fermentation process is measured every 24 hours, wherein the measured change of the alcoholic strength in the fermentation process is shown in the table 5:
TABLE 5 results of repeated fermentation test of immobilized yeast beads
As can be seen from Table 5, in the recycling test, the alcoholic strength changes of the first four fermentations are relatively close, and the alcoholic strength of the fifth fermentation does not reach the value of the first four fermentations, which indicates that the activity of the gelatin beads is not reduced and the alcohol production capacity is not changed too much in the test of the four fermentations. Therefore, by utilizing the characteristic of recycling immobilized strains, the strains can be recycled for fermentation for many times during production.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A strain immobilization method is characterized by comprising the following steps: inoculating; swelling; solidifying into beads; and (5) air drying and preserving.
2. The method for immobilizing bacteria according to claim 1, wherein the inoculation step comprises: inoculating the strain into the liquid to be fermented, and performing shake culture at 28 deg.C and 160r/min for 12 h.
3. The method of claim 2, wherein the fermentation broth is cherry juice that has been enzymatically hydrolyzed and added with 200ppm sodium metabisulfite.
4. The strain immobilization method according to claim 2, wherein the strain is a yeast composition of Angel wine dedicated yeast and Angel wine active dry yeast.
5. The method for immobilizing bacteria according to claim 1, wherein the swelling step: adding 1.5-2.5% sodium alginate solution into the inoculated fermentation solution to swell fully, stirring and mixing uniformly.
6. The method of claim 1, wherein the step of solidifying into beads comprises: injecting the swelled mixed bacteria liquid into a sterilizing injector, selecting needles with different sizes, dripping the needles into a calcium chloride solution with the concentration of 0.5-1.5% to solidify the needles into rubber beads, soaking the rubber beads in the calcium chloride solution for 4min, taking out the rubber beads, washing the rubber beads with sterile water, and washing off the calcium chloride solution on the surfaces of the rubber beads.
7. The strain immobilization method according to claim 1, wherein the air-drying preservation step comprises: drying the surface moisture of the rubber beads at 35 ℃, and preserving at low temperature of 0-4 ℃.
8. A method according to claim 6 or 7, wherein the diameter of the gel beads is 2 to 6.5 mm.
9. The application of the immobilized strain in the fermentation preparation of the cherry fruit wine is characterized in that the immobilized strain is prepared by the method of any one of claims 1 to 8.
10. The use of the immobilized strain of claim 9 for the fermentative preparation of cherry fruit wine, wherein the fermentative preparation of cherry fruit wine comprises the steps of: adding immobilized strains with a volume of 3% of the cherry juice into the cherry juice, and fermenting at 28 deg.C under stirring for 12 hr; then taking out the immobilized strains, cooling to 23 ℃, and continuing to ferment for 120 h.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170138801A (en) * | 2016-06-08 | 2017-12-18 | 경북대학교 산학협력단 | Development of Saccharomyces cerevisiae starter by using Ca-alginate bead after air-blast drying |
| CN111040923A (en) * | 2019-06-03 | 2020-04-21 | 烟台工程职业技术学院(烟台市技师学院) | Preparation method of agar-agar strawberry health-care vinegar |
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- 2020-08-25 CN CN202010865196.6A patent/CN111909923A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170138801A (en) * | 2016-06-08 | 2017-12-18 | 경북대학교 산학협력단 | Development of Saccharomyces cerevisiae starter by using Ca-alginate bead after air-blast drying |
| CN111040923A (en) * | 2019-06-03 | 2020-04-21 | 烟台工程职业技术学院(烟台市技师学院) | Preparation method of agar-agar strawberry health-care vinegar |
Non-Patent Citations (3)
| Title |
|---|
| 杜双奎 等: "酿酒酵母细胞固定化研究", 《西北农业学报》, vol. 15, no. 3, pages 208 - 211 * |
| 邬全喜: "固定化酵母生产樱桃果酒工艺研究", 《食品工业》, vol. 34, no. 6, pages 41 - 43 * |
| 黄达明 等: "海藻酸钠固定化啤酒酵母及应用的研究", 《食品科技》, vol. 38, no. 03, pages 31 - 34 * |
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