A kind of brewing yeast and its fermentation product and application

Abstract

The present invention relates to a brewer's yeast and its fermentation product and application, belonging to the technical field of fermentation engineering. The brewer's yeast or its fermentation product can be used in a method for preparing anthranilic acid by biodegrading L-tryptophan, the method has the advantages of fast reaction speed, safe process, green environmental protection, few by-products, convenient downstream extraction and separation, etc., and the obtained anthranilic acid has high yield and high purity.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to brewer's yeast and its fermentation products and applications. Background Art

[0002] Methyl anthranilate has fruity, floral, powdery, moldy and grape aromas. It exists naturally in grapes, strawberries, red wine and white wine. It is an important raw material for preparing grape, citrus loganberry, strawberry and watermelon flavors. It is also an essential raw material for preparing artificial orange oil and has a large market demand.

[0003] At present, methyl anthranilate on the market mainly comes from chemical synthesis. With the progress and development of society, human beings are more and more willing to return to nature, which has led to the wider application of natural flavors and fragrances in the food industry. Compared with chemically synthesized products, natural flavors and fragrances obtained through biotransformation using natural substrates as raw materials have higher market acceptance and added value.

[0004] In the process of realizing the present invention, the inventors found that there are at least the following problems in the prior art: In the prior art, the production of natural anthranilic acid methyl ester mainly depends on the oxidative demethylation reaction of N-methyl anthranilic acid methyl ester, and N-methyl anthranilic acid methyl ester can be obtained from Egyptian petitgrain leaf oil. However, the reaction depends on chemical oxidants such as MnO2, which not only causes problems in the natural certification of the produced anthranilic acid methyl ester, but also produces a large amount of waste residue that is difficult to handle, causing environmental pressure, and generates a series of by-products, which is not conducive to subsequent separation and extraction operations. Relying on microbial fermentation to prepare anthranilic acid is another possible way to prepare natural anthranilic acid methyl ester, but the current space-time productivity and product concentration are low, and it has no value for industrial application. For example, Víctor E Balderas-Hernández et al. reported that genetically modified Escherichia coli can produce anthranilic acid using glucose as raw material, and fermentation for 34 hours can only produce 14g / L anthranilic acid. Jannis Kuepper et al. reported that using molecularly modified Pseudomonas putida with glucose as raw material, only 1.54 g / L of anthranilic acid could be obtained within 70 hours.

[0005] Therefore, there is still an urgent need for a method for preparing anthranilic acid and methyl anthranilate that is safe, green and environmentally friendly, has few by-products, is easy to extract and separate, has a high yield and high purity. Summary of the invention SUMMARY OF THE INVENTION

[0007] The purpose of this application document is to provide a brewer's yeast and its fermentation product and application. In the first aspect, a brewer's yeast is provided; in the second aspect, a brewer's yeast fermentation product is provided; in the third aspect, a method for preparing the aforementioned fermentation product is provided; in the fourth aspect, a method for preparing anthranilic acid is provided; in the fifth aspect, a method for preparing methyl anthranilate is provided; in the sixth aspect, an application of the brewer's yeast described in the first aspect or the brewer's yeast fermentation product described in the second aspect is provided. The brewer's yeast provided has good catalytic activity and can be used to degrade L-tryptophan to prepare anthranilic acid. The obtained anthranilic acid has high purity, high yield, fast reaction, high safety, green process, few by-products, convenient downstream extraction and separation, and little solvent residue. DETAILED DESCRIPTION OF THE INVENTION

[0009] This application document provides the following technical solutions.

[0010] In a first aspect, a brewer's yeast is provided.

[0011] A brewer's yeast, the brewer's yeast having the deposit number of: CCTCC M 20211292 (deposited in China Center for Type Culture Collection on October 18, 2021).

[0012] The brewer's yeast was named Saccharomyces cerevisiae OMK-77.

[0013] In a second aspect, a fermentation product of brewer's yeast is provided.

[0014] A fermentation product of the Saccharomyces cerevisiae according to the first aspect.

[0015] In some embodiments, the fermentation product can be a fermentation broth, a fermentation supernatant, a cell suspension and / or intracellular material.

[0016] In a third aspect, a method for preparing the aforementioned fermentation product is provided.

[0017] A method for preparing the fermentation product of the second aspect comprises: activating the brewer's yeast of the first aspect, subjecting the yeast to at least one stage of expansion culture, and collecting the fermentation product.

[0018] In some embodiments, the expanded culture includes primary expanded culture, secondary expanded culture, tertiary expanded culture, or quaternary expanded culture.

[0019] In a fourth aspect, a method for preparing anthranilic acid is provided.

[0020] A method for preparing anthranilic acid comprises the following steps:

[0021]

[0022] L-tryptophan is fermented with a microorganism or a fermentation product of a microorganism to produce anthranilic acid.

[0023] The microorganism comprises at least one selected from the group consisting of Corynebacterium sp., Bacillus sp., Lactobacillus sp., Lactococcus sp., Pseudomonas sp., Clostridium sp., Rhodococcus sp., Rhodobacter sp., Streptococcus sp., Saccharomyces sp., Aspergillus sp. and Streptomyces sp.

[0024] In some embodiments, the microorganism is selected from the genus Saccharomyces; preferably, the microorganism is the Saccharomyces cerevisiae described in the first aspect.

[0025] In some embodiments, the fermentation product of the microorganism is the fermentation product described in the second aspect or the fermentation product obtained by the preparation method described in the third aspect.

[0026] In some embodiments, a method for preparing anthranilic acid comprises:

[0027] (1) Preparation of seed culture fluid: The microorganism strain is subjected to at least one-stage expansion culture, or cultured in a solid slant culture medium and a seed culture medium in sequence to obtain a seed culture fluid;

[0028] (2) inoculating the seed culture solution into a fermentation medium, culturing for 6 h to 12 h, adding L-tryptophan in an amount of 10 g / L to 50 g / L, and continuing to culture until all the L-tryptophan is converted into anthranilic acid to obtain a bioconversion mixture;

[0029] (3) The biotransformation mixture is subjected to post-treatment 1 to obtain anthranilic acid.

[0030] The culturing time of continuing to culture until all L-tryptophan is converted into anthranilic acid can be 40h-60h. In some embodiments, the culturing time of continuing to culture until all L-tryptophan is converted into anthranilic acid is 45h-55h.

[0031] In some embodiments, a method for preparing anthranilic acid comprises:

[0032] (1) Preparation of seed culture fluid: The microorganism strain is subjected to at least one-stage expansion culture, or cultured in a solid slant culture medium and a seed culture medium in sequence to obtain a seed culture fluid;

[0033] (2) inoculating the seed culture solution into a fermentation medium, culturing for 6 h to 12 h, and then adding the first portion of L-tryptophan to induce the expression of enzymes related to the L-tryptophan degradation pathway; then adding L-tryptophan in batches every 8 to 10 h and continuing to culture, and continuing to culture for 8 h to 30 h after the last addition of L-tryptophan to obtain a bioconversion mixture;

[0034] (3) The biotransformation mixture is subjected to post-treatment 1 to obtain anthranilic acid.

[0035] The culture temperature of the bacterial strain in the solid slant medium can be about 25° C. to about 30° C. In some embodiments, the culture temperature of the bacterial strain in the solid slant medium is about 28° C. to 30° C.

[0036] The culture time of the strain in the solid slant medium can be about 40 hours to about 60 hours. In some embodiments, the culture time of the strain in the solid slant medium is 45 hours to 55 hours.

[0037] The culture temperature of the bacterial strain in the seed culture medium can be about 28° C. to about 30° C. In some embodiments, the culture temperature of the bacterial strain in the seed culture medium is 28° C.

[0038] The culture of the strain in the seed culture medium can be primary culture or secondary culture. The primary culture means that the microorganism is cultured only once in the seed culture medium; the secondary culture means that the microorganism is cultured once in the seed culture medium to obtain a primary seed liquid, and then the primary seed liquid is inoculated into a fresh seed culture medium to continue culturing once.

[0039] The primary culture may be cultured for 4 hours to 10 hours. In some embodiments, the strain is cultured in the seed culture medium for 4 hours to 6 hours.

[0040] The culturing time of each culturing of the secondary culturing may be 8h-14h. In some embodiments, the culturing time of the strain in the seed culture medium is 10h-12h.

[0041] The stirring speed of the bacterial strain during cultivation in the seed culture medium may be 200 rpm-250 rpm.

[0042] When the seed culture solution is inoculated into the fermentation medium, the volume ratio of the seed culture solution to the fermentation medium may be 6:100-12:100.

[0043] The pH of the fermentation medium may be 5.0-6.5.

[0044] The pH of the seed culture solution may be 5.0-6.5.

[0045] The culture temperature in the fermentation medium in step (2) may be 28°C-30°C.

[0046] The stirring speed of the culture in the fermentation medium in step (2) may be 100 rpm-600 rpm. In some embodiments, the stirring speed of the culture in the fermentation medium in step (2) may be 200 rpm-500 rpm. In some embodiments, the stirring speed of the culture in the fermentation medium in step (2) may be 250 rpm-400 rpm.

[0047] The ventilation ratio of the seed culture solution in step (2) when inoculated into the fermentation medium for culturing can be 1:0.2-1:1. In some embodiments, the ventilation ratio of the seed culture solution in step (2) when inoculated into the fermentation medium for culturing is 1:0.4-1:0.6. In some embodiments, the ventilation ratio of the seed culture solution in step (2) when inoculated into the fermentation medium for culturing is 1:0.5.

[0048] In some embodiments, a method for preparing anthranilic acid comprises:

[0049] (1) Preparing a seed culture solution: Spreading the strain of the microorganism on a solid slant culture medium, culturing at 28° C. for 48 h to obtain an activated strain, inoculating the activated strain into a seed culture medium, and shaking culturing at 28° C. to 30° C. and 200 rpm to 250 rpm until the early logarithmic phase, thereby obtaining a seed culture solution;

[0050] (2) inoculating the seed culture liquid obtained in step (1) into the fermentation medium at a volume ratio of 6:100-12:100 of the seed culture liquid to the fermentation medium, culturing for 6-12 hours under the conditions of pH 5.0-pH6.5, 28°C-30°C, stirring speed 100rpm-600rpm, and ventilation ratio 1:0.2-1:1, adding the first portion of L-tryptophan to induce the expression of enzymes related to the L-tryptophan degradation pathway, and continuing to culture for 8-10 hours, adding the second portion of L-tryptophan and continuing to culture; then adding L-tryptophan in batches and continuing to culture, and continuing to culture for 8h-30h after the last addition of L-tryptophan, to obtain a bioconversion mixture;

[0051] (3) The biotransformation mixture is subjected to post-treatment 1 to obtain anthranilic acid.

[0052] In some embodiments, the ventilation ratio is 1:0.4-1:0.6. In some embodiments, the ventilation ratio is 1:0.5.

[0053] The first amount of L-tryptophan added may be 1 g / L-5 g / L. In some embodiments, the first amount of L-tryptophan added is 2 g / L-4 g / L or 2 g / L-3 g / L.

[0054] The second portion of L-tryptophan or the amount of L-tryptophan added in batches each time can be 5g / L-20g / L. In some embodiments, the second portion of L-tryptophan or the amount of L-tryptophan added in batches each time can be 10g / L-15g / L.

[0055] The batch-wise addition of L-tryptophan may be performed by adding L-tryptophan once in an amount of 5g / L-20g / L every 8h-15h after the second portion of L-tryptophan is added. In certain embodiments, the batch-wise addition of L-tryptophan may be performed by adding L-tryptophan once in an amount of 5g / L-20g / L every 10h-15h after the second portion of L-tryptophan is added. In certain embodiments, the batch-wise addition of L-tryptophan may be performed by adding L-tryptophan once in an amount of 5g / L-20g / L every 12h-15h after the second portion of L-tryptophan is added.

[0056] The total number of times the L-tryptophan is added may be 2-8 times. In some embodiments, the total number of times the L-tryptophan is added may be 2, 3, 4, 5, 6, 7 or 8 times.

[0057] In some embodiments, the culture is continued for 8 h to 30 h after the last addition of L-tryptophan. In some embodiments, the culture is continued for 10 h to 30 h after the last addition of L-tryptophan. In some embodiments, the culture is continued for 10 h to 25 h after the last addition of L-tryptophan. In some embodiments, the culture is continued for 15 h to 25 h after the last addition of L-tryptophan. In some embodiments, the culture is continued for 18 h to 25 h after the last addition of L-tryptophan.

[0058] The post-treatment 1 may include: filtering to remove the bacterial cells, adjusting the pH with sulfuric acid to precipitate anthranilic acid, filtering to collect the filter residue to obtain a crude anthranilic acid product, and recrystallizing the obtained crude anthranilic acid product using water as a solvent.

[0059] The adjusting the pH with sulfuric acid may include adjusting the pH to 2-3 with sulfuric acid.

[0060] Calculated by the total mass of the seed culture medium, the seed culture medium may include or consist of the following components: 1.5wt%-5wt% peptone, 0.5wt%-3wt% yeast extract, 2wt%-5wt% glucose, and a solvent, wherein the solvent is water. In some embodiments, calculated by the total mass of the seed culture medium, the seed culture medium includes: 2.0wt% peptone, 1.0wt% yeast extract, 2.0wt% glucose, and the balance is water.

[0061] Calculated by the total mass of the solid slant culture medium, the solid slant culture medium may include or consist of the following components: 1.5wt%-5wt% peptone, 0.5wt%-3wt% yeast extract, 2wt%-5wt% glucose, 1wt%-5wt% agar, and a solvent, wherein the solvent is water. In some embodiments, calculated by the total mass of the solid slant culture medium, the solid slant culture medium includes: 2.0wt% peptone, 1.0wt% yeast extract, 2.0wt% glucose, 2.0wt% agar, and the balance is water.

[0062] Calculated by the total mass of the fermentation medium, the fermentation medium may include or consist of the following components: 3.0wt%-5.0wt% glucose, 0.5wt%-1.0wt% diammonium phosphate, 0.2wt%-0.5wt% potassium dihydrogen phosphate, 0.1wt%-0.2wt% magnesium sulfate, 0.05wt%-0.1wt% calcium sulfate, 0.2wt%-1.0wt% yeast extract, and a solvent; the solvent is water. In some embodiments, calculated by the total mass of the fermentation medium, the fermentation medium includes: 4wt% glucose, 1wt% diammonium phosphate, 0.5wt% potassium dihydrogen phosphate, 0.2wt% magnesium sulfate, 0.1wt% calcium sulfate, 0.5wt% yeast extract, and the balance is water.

[0063] In a fifth aspect, a method for preparing methyl anthranilate is provided.

[0064] A method for preparing methyl anthranilate, comprising esterifying the anthranilic acid obtained by the method of the second aspect to obtain methyl anthranilate.

[0065] The esterification may include any method mentioned in the prior art.

[0066] In some embodiments, a method for preparing methyl anthranilate comprises dissolving the anthranilic acid obtained by the method of the second aspect in methyl tert-butyl ether, adding natural methanol and Novozyme 435, reacting at 30°C-50°C for 16h-20h, and performing post-treatment 2 to obtain methyl anthranilate.

[0067] The post-treatment 2 may include: filtering and recovering Novozyme 435, distilling the filtrate to recover methyl tert-butyl ether to obtain pot bottom; and rectifying the obtained pot bottom to obtain methyl anthranilate.

[0068] In the sixth aspect, a use of the brewer's yeast described in the first aspect or the fermentation product described in the second aspect is provided.

[0069] A use of the brewer's yeast of the first aspect or the fermentation product of the second aspect in biodegrading L-tryptophan to prepare anthranilic acid.

[0070] Beneficial Effects

[0071] Compared with the prior art, one of the above technical solutions has one of the following beneficial technical effects:

[0072] (1) Using the above technical solution, brewer's yeast Saccharomyces cerevisiae OMK-77 ferments and degrades L-tryptophan to prepare anthranilic acid. The space-time yield of anthranilic acid can reach 0.72 g / (L*h), and the product concentration can reach 30.2 g / L, which is much higher than the existing production process. The production cost is greatly reduced, and the subsequent production of anthranilic acid methyl ester can be achieved through a simple enzyme-catalyzed esterification reaction, which has good industrial application prospects.

[0073] (2) The brewer's yeast Saccharomyces cerevisiae OMK-77 using the above technical solution ferments L-tryptophan to prepare anthranilic acid, and the purity of the obtained anthranilic acid can reach >99% and the yield is as high as 85%. The purity of the anthranilic acid methyl ester further prepared can be >99% and the yield is as high as 78%.

[0074] (3) The preparation method of the above technical solution is used to prepare anthranilic acid and methyl anthranilate, without using organic solvents or greatly reducing the use of organic solvents, and without the need for chemical oxidants such as MnO2. The preparation method has the advantages of fast reaction, high safety, green process, few by-products, convenient downstream extraction and separation, and little solvent residue.

[0075] Definition of terms:

[0076] Unless otherwise specifically defined, all technical and scientific terms used in this application document have the same meaning as commonly understood by ordinary technicians in the relevant fields. The term "ventilation ratio" means the ratio of the ventilation volume per minute to the actual liquid volume of the tank. For example, "ventilation ratio 1:04" means that the ratio of the ventilation volume per minute to the actual liquid volume of the tank is 1:0.4.

[0077] The term "weight percent" or "percent by weight" or "wt %" is defined as the weight of a single component in a composition divided by the total weight of all components of the composition multiplied by 100.

[0078] The term "g / L" refers to the ratio of the weight of the substance being described to the volume of the mixture containing the substance.

[0079] The term "rpm" means rotational speed in revolutions per minute.

[0080] The term "room temperature" refers to the indoor ambient temperature, which may be 15°C to 30°C, 20°C to 30°C, or 25°C to 30°C, or about 25°C.

[0081] In this article, all the numbers disclosed herein are approximate values, regardless of whether the words "about" or "approximately" are used. Based on the disclosed numbers, the value of each number may vary by less than ±10% or reasonable differences considered by those skilled in the art, such as ±1%, ±2%, ±3%, ±4% or ±5%.

[0082] The term "natural methanol" refers to methanol obtained by fermentation, for example, methanol obtained by fermentation using sucrose as a raw material.

[0083] The term "space-time yield" refers to the mass of anthranilic acid that can be obtained per unit volume of culture medium (including seed culture medium and fermentation medium) per unit time under given reaction conditions.

[0084] The description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application document. In the present application document, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 This is the HPLC chart of the production of natural anthranilic acid by fermentation and degradation of L-tryptophan by Saccharomyces cerevisiae OMK-77 in Example 2. DETAILED DESCRIPTION

[0086] In order to enable those skilled in the art to better understand the technical solution of the present application, some non-limiting embodiments are further disclosed below to further explain the present application in detail.

[0087] The reagents used in this application document can be purchased from the market or prepared by the method described in this application document. Example 1: Production of anthranilic acid (triangular shake flask fermentation)

[0088] (1) Obtaining strains

[0089] A strain that can degrade L-tryptophan to anthranilic acid was isolated from the surface of apple peel and identified as Saccharomyces cerevisiae OMK-77 by 18S rDNA and ITS sequencing. The strain was deposited in the China Center for Type Culture Collection (Wuhan University, Wuhan, China) on October 18, 2021, with the deposit number: CCTCCM 20211292.

[0090] (2) Activation of bacterial strains: Streak Saccharomyces cerevisiae OMK-77 from a -80°C glycerol tube onto a solid slant culture medium and incubate at 28°C for 48 h to obtain activated bacterial strains.

[0091] The formula of the solid slant culture medium is as follows in terms of mass percentage: peptone 2.0wt%, yeast extract 1.0wt%, glucose 2.0wt%, agar 2.0wt%, and the balance is water.

[0092] (3) Preparation of seed culture medium: The activated bacterial strain obtained was inoculated into a 500 mL Erlenmeyer shake flask containing 30 mL of seed culture medium, and cultured at 28° C. and 250 rpm for 4-6 h to obtain the seed culture medium.

[0093] The formula of the seed culture medium is as follows by mass percentage: peptone 2.0wt%, yeast extract 1.0wt%, glucose 2.0wt%, the balance is water, initial pH 6.0, and sterilization at 115°C for 30min.

[0094] (4) Fermentation and transformation: 5 mL of seed culture solution was inoculated into a 500 mL Erlenmeyer shake flask containing 50 mL of fermentation medium. After fermentation at 28°C and 250 rpm for 10 h, L-tryptophan was added at a rate of 20 g / L and fermentation was continued at 28°C and 250 rpm for 48 h.

[0095] (5) Precipitation and recrystallization: After fermentation, heat to 60°C to inactivate the cells, and centrifuge to remove the bacteria. Adjust the pH of the fermentation supernatant to 2.0 with sulfuric acid, and collect the precipitate by centrifugation. Dissolve the precipitate in an appropriate amount of 90°C hot water, then cool the system to room temperature to precipitate anthranilic acid crystals. After drying and removing water from the crystals, 0.628 g of pure anthranilic acid was obtained, with a purity of >99% and a yield of 85%.

[0096] The formula of the fermentation medium is as follows by mass percentage: glucose 4wt%, ammonium dihydrogen phosphate 1wt%, potassium dihydrogen phosphate 0.5wt%, magnesium sulfate 0.2wt%, calcium sulfate 0.1wt%, yeast extract powder 0.5wt%, the balance is water, initial pH 6.0, sterilized at 115°C for 30min.

[0097] Example 2: Production of methyl anthranilate (fermentation in a fermenter)

[0098] Steps (1) to (2) are the same as in Example 1;

[0099] (3) Preparing seed culture solution: inoculate the obtained activated strain into a 500 mL triangular shake flask containing 50 mL of seed culture medium, and culture at 28° C. and 250 rpm for 10-12 h to obtain a first-level seed solution; inoculate all 50 mL of the first-level seed solution into a 10 L seed tank containing 6 L of seed culture medium, and culture at 28° C., a stirring speed of 250 rpm, and a ventilation ratio of 1:0.5 for 10-12 h to obtain a second-level seed solution;

[0100] The formula of seed culture medium is the same as that in Example 1.

[0101] (4) Fermentation and transformation: 15.0 L of fermentation medium was placed in a 30 L fermenter and sterilized at 115°C for 30 min. The cultured secondary seed solution was inoculated into the 30 L fermenter at an inoculation rate of 10% for cultivation. The culture conditions were: temperature 28°C, stirring speed 400 rpm, ventilation ratio 1:0.5. After 8 h of fermentation, L-tryptophan was added at an amount of 2 g / L to continue fermentation. Subsequently, L-tryptophan was added once at an amount of 15 g / L at 16 h, 28 h, and 40 h of fermentation (the fermentation time was calculated from the time the secondary seed solution was inoculated into the 30 L fermenter). The total fermentation time was 58 h. (The reaction progress in 58 h is shown in Figure 2.) Figure 1 As shown, L-tryptophan has been almost completely converted into anthranilic acid, with a space-time yield of 0.72 g / (L*h) and a product concentration of 30.2 g / L).

[0102] (5) Precipitation and recrystallization: After fermentation, heat to 60°C to inactivate the bacteria, and filter to remove the bacteria. Adjust the pH of the fermentation supernatant to 2.0 with sulfuric acid, and filter to collect the precipitate. Dissolve the precipitate in as little 90°C hot water as possible, then cool the system to room temperature to precipitate anthranilic acid crystals. After drying and removing water from the crystals, 458.3 g of pure anthranilic acid was obtained, with a purity of >99% and a yield of 88%.

[0103] (6) Esterification: 458.3 g of the anthranilic acid crystals obtained in step (5) were dissolved in 7 L of methyl tert-butyl ether, 350 g of Novozyme 435 and 1 kg of activated molecular sieve 3A were added, stirring was started, the reaction temperature was controlled at 45° C. 172 ml of natural methanol was added at 0 h, 4 h, 9 h, and 15 h of the reaction, respectively. The reaction was continued for a total of 22 h to obtain a bioconversion mixture.

[0104] (7) Recovery and distillation of the catalyst and solvent: The bioconversion mixture obtained in step (6) was filtered to recover Novozyme 435 and molecular sieve 3A, and the clear liquid was transferred to a distillation flask to recover methyl tert-butyl ether under normal pressure to obtain a residue; the obtained residue was transferred to a distillation tower for distillation, the distillation temperature was controlled, and the fractions corresponding to the temperature were collected to obtain 447.8 g of methyl anthranilate with a purity of >99% and a total yield of 78%.

[0105] The formula of the fermentation medium is the same as that of the fermentation medium in Example 1.

[0106] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A brewer's yeast, the deposit number of which is CCTCC M 20211292, and the name of which is Saccharomyces cerevisiae OMK-77.

2. A method for obtaining a saccharomyces cerevisiae fermentation product, comprising: The brewer's yeast of claim 1 is activated, cultured for at least one stage, and the fermentation product is collected.

3. A method for preparing anthranilic acid, characterized in that: The method comprises the following steps: fermenting L-tryptophan with microorganisms to generate anthranilic acid, It is characterized in that The name of the brewer's yeast is Saccharomyces cerevisiae OMK-77, and the deposit number is CCTCC M20211292. The fermentation method is: S1: Preparation of seed culture solution: subjecting the strain of Saccharomyces cerevisiae to at least one-stage expansion culture, or to sequential culture on a solid slant culture medium and a seed culture medium, to obtain a seed culture solution; S2: Add the seed culture solution into the fermentation medium at an inoculum rate of 8-12%, and the fermentation temperature is 26-30℃; S3: After 6-10 h of fermentation, L-tryptophan was added at a rate of 1.5-2.5 g / L to continue fermentation. Subsequently, L-tryptophan was added once at a rate of 13-17 g / L at 14-18 h, 26-30 h, and 38-42 h after the start of fermentation. The total fermentation time was 56-60 h.

4. The method according to claim 3, wherein the seed culture medium comprises, calculated based on the total mass of the seed culture medium: 1.5wt%-5wt% of peptone, 0.5wt%-3wt% of yeast extract, 2wt%-5wt% of glucose, and a solvent, the solvent being water; and / or, calculated based on the total mass of the solid slant culture medium, the solid slant culture medium comprises: 1.5wt%-5wt% of peptone, 0.5wt%-3wt% of yeast extract, 2wt%-5wt% of glucose, 1wt%-5wt% of agar, and a solvent, the solvent being water; and / or, calculated based on the total mass of the fermentation culture medium, the fermentation culture medium comprises: 3.0wt%-5.0wt% of glucose, 0.5wt%-1.0wt% of diammonium dihydrogen phosphate, 0.2wt%-0.5wt% of dipotassium dihydrogen phosphate, 0.1wt%-0.2wt% of magnesium sulfate, 0.05wt%-0.1wt% of calcium sulfate, 0.2wt%-1.0wt% of yeast extract, and a solvent, the solvent being water.

5. Use of the brewer's yeast according to claim 1 in the biodegradation of L-tryptophan to prepare anthranilic acid.

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