Method for increasing yield of tetramethylpyrazine

Abstract

The invention discloses a method for increasing the yield of tetramethylpyrazine, and belongs to the technical field of tetramethylpyrazine preparation. The method for increasing the yield of tetramethylpyrazine comprises the following steps: preparing a fermentation material, adding bacillus for solid-state fermentation, sampling in the solid-state fermentation process, measuring the pH value of the fermentation material, and calculating delta pH; when the delta pH is more than 1, ending the fermentation to obtain a fermentation material containing tetramethylpyrazine; wherein the calculation method of the delta pH is as follows: the pH value of the fermented material after being dried subtracts the pH value of the fermented material before being dried. According to the method for increasing the yield of the tetramethylpyrazine, allogenic materials such as diammonium hydrogen phosphate do not need to be added, the high conversion rate can be achieved at the normal fermentation temperature, the conversion rate from acetoin to the tetramethylpyrazine can be greatly increased through specific heating reaction treatment, the conversion rate is increased to 90% or above, and the yield of the tetramethylpyrazine is increased. The problem of low yield of tetramethylpyrazine in fermentation is effectively solved.

Description

Technical Field

[0001] This application belongs to the technical field of tetramethylpyrazine preparation, and specifically relates to a method for improving the yield of tetramethylpyrazine. Background Art

[0002] Tetramethylpyrazine (2,3,5,6 - tetramethylpyrazine), also known as ligustrazine, is the main active alkaloid component of the traditional Chinese medicine Ligusticum wallichii. It has pharmacological effects such as anti - platelet aggregation, reducing blood circulation, improving microcirculation, dilating blood vessels, and increasing coronary and cerebral blood flow, and is widely used in the treatment of cardiovascular and cerebrovascular diseases. Currently, there are natural extraction methods and chemical synthesis methods for producing tetramethylpyrazine: Tetramethylpyrazine is directly extracted from Ligusticum wallichii. Since the content of tetramethylpyrazine in Ligusticum wallichii is very low and the raw material supply is limited, it is difficult to achieve large - scale production; chemically synthesized products do not fall within the scope of "natural" or "biosynthetic", and safety and quality issues are constantly questioned. Compared with natural extraction methods and chemical synthesis methods, the microbial fermentation method is an important way to achieve large - scale production of tetramethylpyrazine in a green and safe manner.

[0003] The strains for producing tetramethylpyrazine by microbial fermentation are mainly Bacillus spp. (Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, etc.), recombinant Escherichia coli, Lactobacillus, etc. The common technical route is that microorganisms ferment and accumulate the tetramethylpyrazine precursor 3-hydroxy-2-butanone (acetoin) using sugar as a nutrient, and then further carry out a thermal reaction between acetoin and the exogenous substrate diammonium hydrogen phosphate to generate tetramethylpyrazine. Therefore, scholars mainly focus on improving the fermentation level of the precursor acetoin by screening high-yield acetoin-producing strains (CN101445786B, CN115806908A, CN114854617A) or means of genetically recombinant strains (CN116024150A, CN115678819A, CN108728389B.B) to improve the level of tetramethylpyrazine. The fermentation level of acetoin has currently reached a relatively high level, up to 80 - 100 g / L at most, but the conversion rate of acetoin to tetramethylpyrazine is low. Usually, the utilization degree of the added precursor by microorganisms is low, and even when a relatively high amount of pyrazine is produced, the utilization rate of acetoin is less than 1% (CN 113583902 B). Therefore, many efforts have been made in existing research to improve the conversion rate of tetramethylpyrazine. For example, CN101445786B has a conversion rate of 11.6%, CN115386525B adopted adding ammonium dihydrogen phosphate at the stage of thermal reaction to generate tetramethylpyrazine, with a conversion temperature of 60 °C and ensuring air replacement during the conversion process, and the conversion rate reached 22.8%. CN101955980B achieved a conversion rate of 40.3% through the accumulation of endogenous acetoin and the improvement of the in-situ fermentation environment, which is the highest conversion rate for the disclosed data. Therefore, the conversion efficiency of acetoin has become the bottleneck for improving the synthesis efficiency of tetramethylpyrazine. In addition, due to the introduction of fermentation raw materials that cannot be used in food in the current method, it cannot be applied to the actual production of vinegar. Currently, the accumulation of tetramethylpyrazine in vinegar still mainly depends on a large amount of aging time, and it is impossible to efficiently produce vinegar with a high content of tetramethylpyrazine.

[0004] Application Content

[0005] The purpose of this application is to provide a method for improving the yield of tetramethylpyrazine, which can effectively improve the conversion of tetramethylpyrazine in fermentation, thereby increasing the yield.

[0006] To achieve the above application purpose, the following technical solutions are adopted.

[0007] A method for improving the yield of tetramethylpyrazine, comprising the following steps:

[0008] Prepare a seed solution;

[0009] Prepare the fermentation materials and carry out solid-state fermentation. During the solid-state fermentation process, monitor the pH value of the fermentation materials and calculate ΔpH;

[0010] When ΔpH is 1 or above, the fermentation ends to obtain a fermentation material containing tetramethylpyrazine.

[0011] Among them, the calculation method of ΔpH is: the pH value of the dried fermentation material minus the pH value of the fermentation material before drying.

[0012] In some aspects of the present application, the seed liquid is prepared from Bacillus.

[0013] In some aspects of the present application, the Bacillus includes at least one of Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis.

[0014] In some aspects of the present application, the seed liquid is prepared from Bacillus subtilis CGMCC 23362.

[0015] In some aspects of the present application, the seed liquid is prepared from Bacillus amyloliquefaciens CICC 20229.

[0016] In some aspects of the present application, the seed liquid is prepared from Bacillus licheniformis CICC 10084.

[0017] In some aspects of the present application, the formula of the fermentation material includes pea flour, pea protein, and sugar.

[0018] In some aspects of the present application, in the formula of the fermentation material, the pea flour is 25 - 90 parts by mass; the pea protein is 25 - 75 parts by mass.

[0019] In some aspects of the present application, the above sugar is sucrose and / or glucose.

[0020] In some aspects of the present application, the method for increasing the yield of tetramethylpyrazine further includes heating the fermentation material.

[0021] In some aspects of the present application, the fermentation material to be heated is the fermentation material after the fermentation ends.

[0022] In some aspects of the present application, the heating method in the step of heating the fermentation material includes open heating and / or closed heating.

[0023] In some aspects of the present application, the heating method in the step of heating the fermentation material is to first perform closed heating and then perform open heating.

[0024] In some aspects of the present application, the temperature for heating the fermentation material is 70 - 95 °C; the time for heating the fermentation material is 2 - 7 h.

[0025] In some aspects of the present application, the method further includes sealing and placing the fermentation material.

[0026] In some aspects of the present application, the sealing time is 12 - 36 h.

[0027] The present application also discloses a fermented material containing tetramethylpyrazine, which is prepared by the method for increasing the yield of tetramethylpyrazine described above.

[0028] The present application also discloses the use of the above fermented material in the preparation of foods containing tetramethylpyrazine.

[0029] In some aspects of the present application, the above food is vinegar.

[0030] In some aspects of the present application, the vinegar has a roasted flavor.

[0031] In some aspects of the present application, the above vinegar contains 90 mg / mL or more of tetramethylpyrazine.

[0032] Another object of the present application is to provide a preparation method for a fermented material containing tetramethylpyrazine, which is characterized by including the following steps:

[0033] Prepare the fermented material, add Bacillus for solid-state fermentation, sample and measure the pH value of the fermented material during the solid-state fermentation process, and calculate ΔpH;

[0034] When ΔpH is 1 or more, end the fermentation to obtain a fermented material containing tetramethylpyrazine;

[0035] Among them, the method for sampling and measuring the pH value of the fermented material is: measure the pH values of the fermented material and the dried fermented material;

[0036] The calculation method of the above ΔpH is: the pH value of the dried fermented material minus the pH value of the fermented material when not dried.

[0037] Another object of the present application is to provide a food, which includes the fermented material or is prepared from the fermented material;

[0038] Among them, the fermented material contains 3600 mg / kg or more of tetramethylpyrazine; and / or

[0039] The conversion rate of acetoin to tetramethylpyrazine during the preparation of the fermented material reaches 20% or more;

[0040] In some aspects of the present application, the above food includes vinegar and soy sauce.

[0041] Another object of the present application is to provide a preparation method for a food, including the following steps:

[0042] 1) The step of preparing a fermented material containing tetramethylpyrazine, and

[0043] 2) The step of using the fermented material obtained in step 1) to prepare the above food.

[0044] The present application has the following beneficial effects:

[0045] The present application proposes a new fermentation formula and process control method. Using the solid-state fermentation culture method, without adding exogenous substances such as diammonium hydrogen phosphate, a high conversion rate can be achieved at normal fermentation temperatures, and the highest conversion rate can reach 54%. After fermentation, further open and / or closed heating treatment can greatly improve the conversion rate of tetramethylpyrazine, and the conversion rate is increased to more than 90%, effectively solving the problem of low conversion rate of acetoin to tetramethylpyrazine during fermentation, thereby increasing the yield of tetramethylpyrazine. At the same time, the fermented product containing tetramethylpyrazine obtained in the present application can be used for the efficient production of vinegar with a high content of tetramethylpyrazine, and the production method is green and safe. The produced vinegar has a high content of tetramethylpyrazine and a harmonious flavor, with a roasted flavor. Detailed implementation manners

[0046] To enable those skilled in the art to understand the characteristics and effects of the present application, the following provides a general description and definition of the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein shall have the ordinary meaning understood by those skilled in the art regarding the present application. In case of conflict, the definition in this specification shall prevail.

[0047] The theories or mechanisms described and disclosed herein, whether correct or incorrect, shall not limit the scope of the present application in any way, that is, the content of the present application can be implemented without being limited by any specific theory or mechanism.

[0048] In this article, all features defined in the form of numerical ranges or percentage ranges, such as quantity, content, and concentration, are only for the sake of simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be regarded as having covered and specifically disclosed all possible sub-ranges and individual values within the range (including integers and fractions). In this article, unless otherwise specified, the percentage is the mass percentage.

[0049] In this article, for the sake of concise description, all possible combinations of all technical features in each embodiment or example are not described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered as the scope described in this specification.

[0050] The following further elaborates on the present application in the form of examples, but the scope of the present application is not limited by the content of the following examples. The protection scope of the present application is only defined by the claims, and any omission, substitution, or modification made by those skilled in the art based on the disclosed implementation manners of the present application will fall within the protection scope of the present application.

[0051] Conventional instruments and equipment in the art are used in the following examples. For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Various raw materials are used in the following examples, and unless otherwise specified, commercially available products are used. In the specification of this application and the following examples, unless otherwise specified, "%" represents weight percentage, "parts" represents weight parts, and the ratio represents weight ratio.

[0052] This application provides a method for improving the yield of tetramethylpyrazine, which includes the following steps:

[0053] Prepare a seed solution;

[0054] Prepare the fermentation material, carry out solid-state fermentation, monitor the pH value of the fermentation material during the solid-state fermentation process, and calculate ΔpH;

[0055] When ΔpH is above 1, end the fermentation to obtain the fermentation material containing tetramethylpyrazine;

[0056] Among them, the calculation method of the ΔpH is: the pH value of the dried fermentation material minus the pH value of the fermentation material before drying.

[0057] In some embodiments of this application, the seed solution is prepared from Bacillus;

[0058] In some embodiments of this application, the Bacillus is selected from at least one of Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis.

[0059] In some embodiments of this application, the Bacillus subtilis is Bacillus subtilis CGMCC23362.

[0060] In some embodiments of this application, the seed medium used for strain preparation includes one or more of the following components:

[0061] Beef extract, yeast extract, tryptone, sodium chloride, potassium dihydrogen phosphate, glucose.

[0062] In some embodiments of this application, the preparation of the seed solution includes the following specific steps:

[0063] Strain activation: Coat the strain on an LB plate and place it in a constant temperature incubator for cultivation;

[0064] Seed cultivation: Inoculate the activated strain into the seed liquid medium and place it in a shaker for cultivation. The seed solution is obtained after the cultivation ends.

[0065] In some embodiments of this application, the formula of the fermentation material includes pea flour, pea protein, and sugar.

[0066] In some embodiments of the present application, the formula of the fermentation material includes 25-90 parts by mass of pea flour; 25-75 parts by mass of pea protein.

[0067] In some embodiments of the present application, the above sugar is sucrose and / or glucose.

[0068] In some embodiments of the present application, the solid-state fermentation includes the following specific steps:

[0069] Take the cultured seed liquid, add the fermentation material, stir evenly and place it in a container for constant-temperature cultivation; turn the material during the fermentation process to ensure the normal metabolism of the bacteria; sample and monitor the pH of the material during the fermentation process, and end the fermentation cultivation when ΔpH is greater than 1.

[0070] In some embodiments of the present application, the method for increasing the yield of tetramethylpyrazine further includes heating the fermentation material.

[0071] In some embodiments of the present application, the fermented material to be heated is the fermented material after fermentation.

[0072] In some embodiments of the present application, the heating method for the step of heating the fermentation material includes open heating and / or closed heating.

[0073] In some embodiments of the present application, the method of open heating is to spread the fermentation material flat in an open container for heating; the method of closed heating is to seal the fermentation material and then use a forced-air oven for heating.

[0074] In some embodiments of the present application, the heating method for the step of heating the fermentation material is to first perform closed heating and then open heating.

[0075] In some embodiments of the present application, the temperature for heating the fermentation material is 70-95°C; the time for heating the fermentation material is 2-7 h.

[0076] In some embodiments of the present application, the temperature for heating the fermentation material is 90-95°C; the time for heating the fermentation material is 2-5 h.

[0077] In some embodiments of the present application, the solid-state fermentation includes the following specific steps:

[0078] Take the cultured seed liquid, add the fermentation material, stir evenly and place it in a container for constant-temperature cultivation; turn the material during the fermentation process to ensure the normal metabolism of the bacteria; sample and monitor the pH of the material during the fermentation process, and end the fermentation cultivation when ΔpH is greater than 1; after the fermentation is completed, heat the material.

[0079] In some embodiments of the present application, the method further includes sealing and placing the fermentation material.

[0080] In some embodiments of the present application, the sealing time is 12 - 36 h.

[0081] In some embodiments of the present application, the sealing time is 12 - 24 h.

[0082] In some embodiments of the present application, the sealing time is 24 - 36 h.

[0083] In some embodiments of the present application, the solid-state fermentation includes the following specific steps:

[0084] Take the cultured seed liquid, add the fermentation materials, stir evenly and place in a container for constant-temperature cultivation; turn the materials during the fermentation process to ensure the normal metabolism of the bacteria; sample and monitor the pH of the materials during the fermentation process, and when ΔpH is greater than 1, end the fermentation cultivation after sealing and placing.

[0085] In some embodiments of the present application, the solid-state fermentation includes the following specific steps:

[0086] Take the cultured seed liquid, add the fermentation materials, stir evenly and place in a container for constant-temperature cultivation; turn the materials during the fermentation process to ensure the normal metabolism of the bacteria; sample and monitor the pH of the materials during the fermentation process, and when ΔpH is greater than 1, end the fermentation cultivation after sealing and placing; after the fermentation ends, heat the materials.

[0087] The present application also discloses a fermentation material, which is prepared by the method for increasing the yield of tetramethylpyrazine described above.

[0088] The present application also discloses the use of the above fermentation material in the preparation of food.

[0089] In some embodiments of the present application, the above food is vinegar.

[0090] In some embodiments of the present application, the vinegar has a roasted fragrance and a harmonious aroma.

[0091] In some embodiments of the present application, the above food is soy sauce.

[0092] In some embodiments of the present application, the soy sauce has a roasted fragrance and a harmonious aroma.

[0093] In some embodiments of the present application, the above vinegar contains tetramethylpyrazine of 26 mg / mL or more.

[0094] In some embodiments of the present application, using the above fermentation material to prepare food includes the following specific steps:

[0095] Add the above fermentation material containing tetramethylpyrazine to the food, stir evenly and let it stand, and filter and clarify to obtain the food containing tetramethylpyrazine.

[0096] In some embodiments of the present application, in the above steps for preparing vinegar, the standing time is 8 hours or more.

[0097] In some embodiments of the present application, in the above steps for preparing vinegar, the standing time is 24 hours or more.

[0098] The present application also discloses a method for preparing a fermentation material containing tetramethylpyrazine, comprising the following steps:

[0099] Prepare a fermentation material, add Bacillus to carry out solid-state fermentation, sample and measure the pH value of the fermentation material during the solid-state fermentation process, and calculate ΔpH;

[0100] When ΔpH is 1 or more, end the fermentation to obtain a fermentation material containing tetramethylpyrazine;

[0101] Among them, the method for sampling and measuring the pH value of the fermentation material is: measure the pH values of the fermentation material and the dried fermentation material;

[0102] The calculation method of the ΔpH is: the pH value of the dried fermentation material minus the pH value of the fermentation material before drying.

[0103] The present application also discloses a food, which includes the fermentation material or is prepared from the fermentation material;

[0104] Among them, the fermentation material contains 3600 mg / kg or more of tetramethylpyrazine; and / or

[0105] The conversion rate of acetoin to tetramethylpyrazine during the preparation of the fermentation material reaches 20% or more.

[0106] In some embodiments of the present application, the fermentation material contains 4800 mg / kg or more of tetramethylpyrazine.

[0107] In some embodiments of the present application, the fermentation material contains 5800 mg / kg or more of tetramethylpyrazine.

[0108] In some embodiments of the present application, the fermentation material contains 6569 mg / kg or more of tetramethylpyrazine.

[0109] In some embodiments of the present application, the fermentation material contains 8000 mg / kg or more of tetramethylpyrazine.

[0110] In some embodiments of the present application, the conversion rate of acetoin to tetramethylpyrazine during the preparation of the fermentation material reaches 22.8% or more.

[0111] In some embodiments of the present application, the conversion rate of acetoin to tetramethylpyrazine during the preparation of the fermentation material reaches 41% or more.

[0112] In some embodiments of the present application, the conversion rate of acetoin to tetramethylpyrazine during the preparation of the fermentation material reaches more than 51%.

[0113] In some embodiments of the present application, the conversion rate of acetoin to tetramethylpyrazine during the preparation of the fermentation material reaches more than 60%.

[0114] In some embodiments of the present application, the conversion rate of acetoin to tetramethylpyrazine during the preparation of the fermentation material reaches more than 90%.

[0115] In some embodiments of the present application, the food includes vinegar and soy sauce.

[0116] The present application also discloses a method for preparing a food, comprising the following steps:

[0117] 1) The step of preparing a fermentation material containing tetramethylpyrazine, and

[0118] 2) The step of using the fermentation material obtained in step 1) to prepare the food.

[0119] In some embodiments of the present application, the method for preparing a food comprises the following steps:

[0120] 1) The step of preparing a fermentation material containing tetramethylpyrazine, and

[0121] 2) Adding the fermentation material obtained in step 1) to the food, stirring evenly, standing, and filtering to obtain a clarified food containing tetramethylpyrazine.

[0122] Other materials and methods used in the examples are, unless otherwise specified, common materials and methods in the art, and these materials can be obtained through commercial channels.

[0123] The detection and calculation methods in the following examples and comparative examples are as follows:

[0124] Determination method for acetoin content: Voges Proskauer (VP) method;

[0125] Add the diluted sample into a colorimetric tube so that the acetoin concentration range is 2.5 - 12.5 mg / L. Then add 1 ml of 10% NaOH, 0.5% creatine, and 5% α-naphthol into the colorimetric tube respectively, shake well, heat in a water bath at 30°C for 60 min, measure the absorbance at 520 nm, and calculate the acetoin content according to the standard curve;

[0126] Determination method for tetramethylpyrazine content: GBT19777 - 2013;

[0127] Conversion rate of tetramethylpyrazine: Conversion rate = (concentration of tetramethylpyrazine / concentration of acetoin) × 100%.

[0128] Method for measuring pH value: Take 1 g of culture medium, add 5 g of deionized water, mix well by shaking, and then detect with a pH meter;

[0129] In the following examples and comparative examples, "normal temperature" refers to the conventional fermentation temperature of 37 °C.

[0130] In the following examples and comparative examples, the formula of the seed liquid culture medium is as follows:

[0131] Formula 1: Beef extract 3 g / L, Tryptone 30 g / L, Sodium chloride 10 g / L, Potassium dihydrogen phosphate 1 g / L, Glucose 20 g / L, natural pH, sterilize at 121 °C for 15 min before use;

[0132] Formula 2: Yeast extract 10 g / L, Tryptone 20 g / L, Glucose 10 g / L, natural pH, sterilize at 121 °C for 15 min before use.

[0133] In the following examples and comparative examples, the formula of the fermentation material is as follows:

[0134] Formula of fermentation material 1: 75 g of pea powder (peas are pulverized), 25 g of pea protein (from Shandong Shuangta), 15 g of sucrose, 60 g of water;

[0135] Formula of fermentation material 2: 75 g of pea powder (peas are pulverized), 25 g of pea protein (from Shandong Shuangta), 15 g of glucose, 60 g of water;

[0136] Formula of fermentation material 3: 90 g of pea powder (peas are pulverized), 10 g of pea protein (from Shandong Shuangta), 15 g of glucose, 60 g of water;

[0137] Formula of fermentation material 4: 25 g of pea powder (peas are pulverized), 75 g of pea protein (from Shandong Shuangta), 15 g of glucose, 60 g of water.

[0138] Example 1:

[0139] Preparation of seed liquid:

[0140] The strains used are: Bacillus subtilis CGMCC23362, Bacillus amyloliquefaciens CICC20229, Bacillus licheniformis CICC10084.

[0141] 1.1 Activation of Bacillus subtilis strain: Spread the strain on an LB plate and place it in a constant temperature incubator at 37 °C for 24 h;

[0142] Seed culture of Bacillus subtilis: Scrape one loop of the activated strain and inoculate it into the seed liquid culture medium of Formula 1, place it in a shaker at 37 °C and 220 rpm for 18 - 20 h to obtain seed liquid a1;

[0143] Bacillus subtilis seed culture: Scrape a loopful of the activated strain and inoculate it into the seed liquid medium formula 2, place it in a shaker, and culture it at 37°C and 220 rpm for 18 - 20 h to obtain seed liquid a2;

[0144] 1.2 Bacillus amyloliquefaciens strain activation: Spread the strain on an LB plate and place it in a constant temperature incubator, culture it at 37°C for 24 h;

[0145] Bacillus amyloliquefaciens seed culture: Scrape a loopful of the activated strain and inoculate it into the seed liquid medium formula 1, place it in a shaker, and culture it at 37°C and 220 rpm for 18 - 20 h to obtain the seed liquid;

[0146] 1.3 Bacillus licheniformis strain activation: Spread the strain on an LB plate and place it in a constant temperature incubator, culture it at 37°C for 24 h;

[0147] Bacillus licheniformis seed culture: Scrape a loopful of the activated strain and inoculate it into the seed liquid medium formula 1, place it in a shaker, and culture it at 37°C and 220 rpm for 18 - 20 h to obtain the seed liquid.

[0148] (2) Solid - state fermentation culture:

[0149] 2.1 Take 5 ml of the cultured Bacillus subtilis seed liquid (seed liquid a1), add it to fermentation material 1, stir evenly and place it in a 500 - ml beaker, and culture it at 37°C. During the fermentation process, turn the material every 24 h to ensure the normal metabolism of the bacteria. During the fermentation process, sample to monitor the pH of the material, measure the pH of the dried material and the pH of the material before drying respectively, and calculate ΔpH (ΔpH = pH after drying - pH before drying); When ΔpH is 1.04, end the fermentation. At this time, the fermentation time is 54 h, the normal - temperature conversion rate during the fermentation process is 33.5%, and the material at the end of fermentation is A.

[0150] 2.2 Take 5 ml of the cultured Bacillus subtilis seed liquid (seed liquid a2), add it to fermentation material 1, stir evenly and place it in a 500 - ml beaker, and culture it at 37°C. During the fermentation process, turn the material every 24 h to ensure the normal metabolism of the bacteria. During the fermentation process, sample to monitor the pH of the material, measure the pH of the dried material and the pH of the material before drying respectively, and calculate ΔpH (ΔpH = pH after drying - pH before drying); When ΔpH is 1.08, end the fermentation. At this time, the fermentation time is 50 h, the normal - temperature conversion rate during the fermentation process is 34.5%, and the material at the end of fermentation is A1.

[0151] 2.3 Take 5 ml of the cultured Bacillus subtilis seed liquid (seed liquid a1), add it to Fermentation Material 2, stir evenly, place it in a 500-ml beaker, and conduct cultivation at 37 °C. During the fermentation process, turn the material every 24 h to ensure the normal metabolism of the bacteria. During the fermentation process, sample and monitor the pH of the material, measure the pH of the dried material and the pH of the material before drying respectively, and calculate ΔpH (ΔpH = pH after drying - pH before drying); when ΔpH is 1.07, end the fermentation. At this time, the fermentation time is 54 h, the normal-temperature conversion rate during the fermentation process is 34.2%, and the material at the end of fermentation is B.

[0152] 2.4 Take 5 ml of the cultured Bacillus subtilis seed liquid (seed liquid a1), add it to Fermentation Material 3, stir evenly, place it in a 500-ml beaker, and conduct cultivation at 37 °C. During the fermentation process, turn the material every 24 h to ensure the normal metabolism of the bacteria. During the fermentation process, sample and monitor the pH of the material, measure the pH of the dried material and the pH of the material before drying respectively, and calculate ΔpH (ΔpH = pH after drying - pH before drying); when ΔpH is 1.02, end the fermentation. At this time, the fermentation time is 44 h, the normal-temperature conversion rate during the fermentation process is 34.0%, and the material at the end of fermentation is B1.

[0153] 2.5 Take 5 ml of the cultured Bacillus amyloliquefaciens seed liquid, add it to Fermentation Material 4, stir evenly, place it in a 500-ml beaker, and conduct cultivation at 37 °C. During the fermentation process, turn the material every 24 h to ensure the normal metabolism of the bacteria. During the fermentation process, sample and monitor the pH of the material, measure the pH of the dried material and the pH of the material before drying respectively, and calculate ΔpH (ΔpH = pH after drying - pH before drying); when ΔpH is 1.09, end the fermentation. At this time, the fermentation time is 61 h, the normal-temperature conversion rate during the fermentation process is 35.6%, and the material at the end of fermentation is B2.

[0154] 2.6 Take 5 ml of the cultured Bacillus amyloliquefaciens seed liquid, add it to Fermentation Material 1, stir evenly, place it in a 500-ml beaker, and conduct cultivation at 37 °C. During the fermentation process, turn the material every 24 h to ensure the normal metabolism of the bacteria. During the fermentation process, sample and monitor the pH of the material, measure the pH of the dried material and the pH of the material before drying respectively, and calculate ΔpH (ΔpH = pH after drying - pH before drying); when ΔpH is 1.08, end the fermentation. At this time, the fermentation time is 50 h, the normal-temperature conversion rate during the fermentation process is 29.7%, and the material at the end of fermentation is C.

[0155] 2.7 Take 5 ml of the cultured Bacillus licheniformis seed liquid, add it to Fermentation Material 1, stir evenly, place it in a 500 ml beaker, and incubate at 37°C. During the fermentation process, turn the material every 24 hours to ensure the normal metabolism of the bacteria. During the fermentation process, sample and monitor the pH of the material, and measure the pH of the dried material and the pH of the material before drying respectively (ΔpH = pH of dry material - pH of wet material); when ΔpH is 1.01, end the fermentation. At this time, the fermentation time is 60 hours, the normal-temperature conversion rate during the fermentation process is 22.8%, and the material at the end of fermentation is D.

[0156] (3) Heating of the material:

[0157] After Material A is subjected to open heating treatment at 70°C for 5 hours (spread the fermentation material flat in a glassware, with a height of about 5 cm and an open mouth), the total conversion rate of tetramethylpyrazine is 60%, and the content of tetramethylpyrazine reaches 5000 mg / kg.

[0158] After Material A1 is subjected to open heating treatment at 70°C for 5 hours (spread the fermentation material flat in a glassware, with a height of about 5 cm and an open mouth), the total conversion rate of tetramethylpyrazine is 62%, and the content of tetramethylpyrazine reaches 4900 mg / kg.

[0159] After Material B is subjected to open heating treatment at 70°C for 5 hours, the total conversion rate of tetramethylpyrazine is 61%, and the content of tetramethylpyrazine reaches 5800 mg / kg.

[0160] After Material B is further sealed for 20 hours, ΔpH is 1.11. After Material B is subjected to open heating treatment at 70°C for 5 hours, the total conversion rate of tetramethylpyrazine is 72%, and the content of tetramethylpyrazine reaches 6800 mg / kg.

[0161] After Material B1 is subjected to open heating treatment at 70°C for 5 hours, the total conversion rate of tetramethylpyrazine is 59%, and the content of tetramethylpyrazine reaches 5160 mg / kg.

[0162] After Material B2 is subjected to open heating treatment at 70°C for 5 hours, the total conversion rate of tetramethylpyrazine is 63%, and the content of tetramethylpyrazine reaches 5720 mg / kg.

[0163] After Material C is subjected to open heating treatment at 70°C for 5 hours, the total conversion rate of tetramethylpyrazine is 55%, and the content of tetramethylpyrazine reaches 4800 mg / kg.

[0164] After Material D is subjected to open heating treatment at 70°C for 5 hours, the total conversion rate of tetramethylpyrazine is 49%, and the content of tetramethylpyrazine reaches 3600 mg / kg;

[0165] After the material C was heated in a closed system at 70°C for 5 h and then subjected to open heating for 2 h, the total conversion rate of tetramethylpyrazine was 63%, and the content of tetramethylpyrazine reached 5500 mg / kg;

[0166] After the material D was heated in a closed system at 70°C for 5 h and then subjected to open heating for 2 h, the total conversion rate of tetramethylpyrazine was 55%, and the content of tetramethylpyrazine reached 4040 mg / kg.

[0167] Example 2:

[0168] (1) Preparation of the seed liquid:

[0169] The preparation method was the same as that of the Bacillus subtilis seed liquid a1 in Example 1.

[0170] (2) Solid-state fermentation culture:

[0171] Take 5 ml of the cultured Bacillus subtilis seed liquid and add it to Fermentation Material 1. After stirring evenly, place it in a 500 ml beaker and culture at 37°C. During the fermentation process, turn the material every 24 h to ensure the normal metabolism of the bacteria. During the fermentation process, sample and monitor the pH of the material, measure the pH of the dried material and the pH of the material before drying respectively, and calculate ΔpH (ΔpH = pH after drying - pH before drying);

[0172] When ΔpH was 1.15, the fermentation was terminated. At this time, the fermentation time was 58 h, and the normal-temperature conversion rate during the fermentation process was 41%;

[0173] (3) Material heating:

[0174] After open heating at 70°C for 5 h, the total conversion rate of tetramethylpyrazine was 65%, and the content of tetramethylpyrazine reached 6100 mg / kg;

[0175] After open heating at 95°C for 5 h, the total conversion rate of tetramethylpyrazine was 70%, and the content of tetramethylpyrazine reached 6569 mg / kg.

[0176] Example 3:

[0177] (1) Preparation of the seed liquid:

[0178] The preparation method was the same as that of the Bacillus subtilis seed liquid a1 in Example 1.

[0179] (2) Solid-state fermentation culture:

[0180] Take 5 ml of the cultured Bacillus subtilis seed liquid, add it to Fermentation Material 1, stir well, place it in a 500-ml beaker, and incubate at 37°C. During the fermentation process, turn the material every 24 h to ensure the normal metabolism of the bacteria. During the fermentation process, sample and monitor the pH of the material, measure the pH of the dried material and the pH of the material before drying respectively, and calculate ΔpH (ΔpH = pH after drying - pH before drying).

[0181] During the solid-state fermentation process, when ΔpH exceeds 1, carry out a sealing treatment (cover the material with a plastic film), maintain it at 37°C for 24 h and then end the fermentation. The final ΔpH is 1.20, and the normal-temperature conversion rate during the fermentation process is 52%. The fermented material is H1;

[0182] During the solid-state fermentation process, when ΔpH exceeds 1, carry out a sealing treatment, maintain it at 37°C for 12 h and then end the fermentation. The final ΔpH is 1.18, and the normal-temperature conversion rate during the fermentation process is 51%. The fermented material is H2;

[0183] During the solid-state fermentation process, when ΔpH exceeds 1, carry out a sealing treatment, maintain it at 37°C for 36 h and then end the fermentation. The final ΔpH is 1.24, and the normal-temperature conversion rate during the fermentation process is 54%. The fermented material is H3;

[0184] (3) Heating of the material:

[0185] The fermented material H1 is subjected to a 5-h airtight treatment at 90°C and heated using a forced-air oven, and then subjected to a 2-h open heating treatment. The total conversion rate of tetramethylpyrazine is 90%, and the content of tetramethylpyrazine reaches 8000 mg / kg.

[0186] The fermented material H2 is subjected to a 5-h airtight treatment at 90°C and heated using a forced-air oven, and then subjected to a 2-h open heating treatment. The total conversion rate of tetramethylpyrazine is 91%, and the content of tetramethylpyrazine reaches 8088 mg / kg.

[0187] The fermented material H3 is subjected to a 5-h airtight treatment at 90°C and heated using a forced-air oven, and then subjected to a 2-h open heating treatment. The total conversion rate of tetramethylpyrazine is 92%, and the content of tetramethylpyrazine reaches 8170 mg / kg.

[0188] Example 4:

[0189] Use the fermented material A containing tetramethylpyrazine obtained in Example 1 to prepare food. After preparation, ask sensory evaluators to smell it and evaluate the flavor and aroma of the vinegar:

[0190] The fermented materials containing tetramethylpyrazine were added to vinegar (JINLONGYU Liangfen Shanxi mature vinegar 4.5°, purchased from JD.com) at the addition ratios of 1%, 2%, and 5% respectively. After stirring evenly, they were left standing for 24 h, filtered and clarified to obtain high-concentration tetramethylpyrazine vinegar, which had a good flavor and a roasted flavor.

[0191]

[0192] The fermented materials containing tetramethylpyrazine were added to vinegar (Ta mature vinegar 4.5°, purchased from JD.com) at the addition ratio of 1%. After stirring evenly, they were left standing for 24 h, filtered and clarified to obtain vinegar with a harmonious aroma and a slightly roasted aroma. The content of tetramethylpyrazine in this vinegar reached 50 mg / L.

[0193] The fermented materials containing tetramethylpyrazine were added to soy sauce (JINLONGYU light soy sauce, purchased from JD.com) at the addition ratio of 0.5%. After stirring evenly, they were left standing for 24 h, filtered and clarified to obtain soy sauce with a relatively harmonious aroma. The content of tetramethylpyrazine in this soy sauce reached 26 mg / L.

[0194] Comparative Example 1:

[0195] (1) Preparation of seed liquid:

[0196] Same as Example 2.

[0197] (2) Solid-state fermentation culture:

[0198] Fermentation was carried out using Fermentation Material 1;

[0199] 5 ml of the cultured Bacillus subtilis seed liquid was taken and added to Fermentation Material 1. After stirring evenly, it was placed in a 500 ml beaker and cultured at 37 °C. During the fermentation process, the materials were turned over every 24 h to ensure the normal metabolism of the bacteria. During the fermentation process, samples were taken to monitor the pH of the materials, and the pH of the materials after drying and before drying during the solid-state fermentation were measured respectively, and ΔpH (ΔpH = pH after drying - pH before drying) was calculated; when ΔpH was -0.10, the fermentation was ended. At this time, the fermentation time was 36 h, and tetramethylpyrazine could not be detected in the fermented materials, and the normal-temperature conversion rate during the fermentation process was 0;

[0200] (3) Heating of materials:

[0201] After the fermentation of the fermented materials ended, they were subjected to open heating treatment at 70 °C for 5 h, and the total conversion rate of tetramethylpyrazine was 0.74%.

[0202] Comparative Example 2:

[0203] (1) Preparation of seed liquid:

[0204] Same as Example 2.

[0205] (2) Solid state fermentation culture:

[0206] Differences from Comparative Example 1 are as follows:

[0207] During the solid state fermentation process, when ΔpH is 0.35, the fermentation ends. At this time, the fermentation time is 62 h, and the normal temperature conversion rate during the fermentation process is 8%;

[0208] (3) Material heating:

[0209] After the fermented material after fermentation is subjected to open heating treatment at 70 °C for 5 h, the total conversion rate of tetramethylpyrazine is 25%.

[0210] Comparative Example 3:

[0211] (1) Preparation of seed liquid:

[0212] Same as Example 2.

[0213] (2) Solid state fermentation culture:

[0214] Differences from Comparative Example 1 are as follows:

[0215] During the solid state fermentation process, when ΔpH is 0.45, the fermentation ends. At this time, the fermentation time is 48 h, and the normal temperature conversion rate during the fermentation process is 10%;

[0216] (3) Material heating:

[0217] After the fermented material after fermentation is subjected to open heating treatment at 70 °C for 5 h, the total conversion rate of tetramethylpyrazine is 32%.

[0218] Comparative Example 4:

[0219] (1) Preparation of seed liquid:

[0220] Same as Example 2.

[0221] (2) Solid state fermentation culture:

[0222] Differences from Comparative Example 1 are as follows:

[0223] During the solid state fermentation process, when ΔpH is 0.7, the fermentation ends. At this time, the fermentation time is 40 h, and the normal temperature conversion rate during the fermentation process is 25%. The fermented material after fermentation is sealed and placed at 37 °C for 24 h, and the measured ΔpH is 0.72.

[0224] (3) Material heating:

[0225] After the fermented material after fermentation is subjected to open heating treatment at 70 °C for 5 h, the total conversion rate of tetramethylpyrazine is 41%.

[0226] After the fermented material at the end of fermentation is treated by open heating at 95 °C for 5 h, the total conversion rate of tetramethylpyrazine is 40%.

[0227] After the fermented material at the end of fermentation is treated by being kept sealed for 24 h and then treated by open heating at 70 °C for 5 h, the total conversion rate of tetramethylpyrazine is 43%.

[0228] As can be seen from the above examples and comparative examples, in the present application, by controlling the ΔpH of the fermented material to be greater than 1, the conversion rate of acetoin to tetramethylpyrazine can be effectively increased, and the highest can reach 54%; further heating of the fermented material can increase the conversion rate again. Especially after being treated by closed treatment and open heating, the conversion rate is significantly increased, and the conversion rate can reach more than 90%. It can be seen from the comparative examples that when the ΔpH of the fermented material is less than 1, the promoting effect of open heating on the increase of the conversion rate of acetoin to tetramethylpyrazine in the fermented material is limited; after being treated by sealed placement and then open heating, it has a certain promoting effect on the increase of the conversion rate of acetoin to tetramethylpyrazine.

Claims

1. A method for improving the yield of tetramethylpyrazine, comprising the following steps: Prepare a fermentation material, add Bacillus and carry out solid-state fermentation, and sample and measure the pH value of the fermentation material during the solid-state fermentation process to calculate ΔpH; When ΔpH is above 1, end the fermentation to obtain a fermentation material containing tetramethylpyrazine; Among them, The method for sampling and measuring the pH value of the fermentation material is: measure the pH value of the fermentation material and the pH value of the dried fermentation material; The calculation method of ΔpH is: the pH value of the dried fermentation material minus the pH value of the fermentation material when it is not dried.

2. The method for increasing the yield of tetramethylpyrazine according to claim 1, wherein The Bacillus is selected from at least one of Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis; preferably, the Bacillus subtilis is Bacillus subtilis CGMCC23362; preferably, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens CICC20229; preferably, the Bacillus licheniformis is Bacillus licheniformis CICC10084.

3. The method for increasing the yield of tetramethylpyrazine according to claim 1, characterized in that, The formula of the fermentation material includes pea flour, pea protein, and sugar; preferably, the sugar is sucrose and / or glucose; preferably, the pea flour is 25-90 parts by mass; preferably, the pea protein is 25-75 parts by mass.

4. The method for increasing the yield of tetramethylpyrazine according to any one of claims 1-3, characterized in that, The method further includes a step of heating the fermentation material; preferably, heat the fermentation material after fermentation ends.

5. The method for increasing the yield of tetramethylpyrazine according to claim 4, wherein The heating method for heating the fermentation material includes open heating and / or closed heating; preferably, first carry out closed heating, and then carry out open heating.

6. The method for increasing the yield of tetramethylpyrazine according to claim 5, characterized in that, The temperature for heating the fermentation material is 70-95 °C; the time for heating the fermentation material is 2-7 h.

7. The method for increasing the yield of tetramethylpyrazine according to claims 1-6, characterized in that, The method further includes sealing and placing the fermentation material; preferably, the time for sealing and placing is 12-36 h.

8. A fermentation material containing tetramethylpyrazine, characterized in that, The fermentation material containing tetramethylpyrazine is prepared by the method for improving the yield of tetramethylpyrazine according to any one of claims 1-7.

9. Use of the fermentation material according to claim 8 in the preparation of a food containing tetramethylpyrazine and / or increasing the content of tetramethylpyrazine in a food; preferably, the food is vinegar and / or soy sauce.

10. The use according to claim 9, characterized in that, The food has a roasted flavor; preferably, the food contains 26 mg / mL or more of tetramethylpyrazine.

11. A preparation method of a fermentation material containing tetramethylpyrazine, characterized in that, Comprising the following steps: Prepare a fermentation material, add Bacillus and carry out solid-state fermentation, sample and measure the pH value of the fermentation material during the solid-state fermentation process to calculate ΔpH; When ΔpH is above 1, end the fermentation to obtain a fermentation material containing tetramethylpyrazine; Among them, the method for sampling and measuring the pH value of the fermentation material is: measure the pH value of the fermentation material and the pH value of the dried fermentation material; The calculation method of ΔpH is: the pH value of the dried fermentation material minus the pH value of the fermentation material when it is not dried.

12. A food, characterized in that, The food includes the fermentation material or is prepared from the fermentation material; Among them, the fermentation material contains 3600 mg / kg or more of tetramethylpyrazine; and / or The conversion rate of acetoin to tetramethylpyrazine during the preparation process of the fermentation material reaches 20% or more; Preferably, the food includes vinegar and soy sauce.

13. A method for preparing a food, characterized in that, Comprising the following steps: 1) A step of preparing a fermentation material containing tetramethylpyrazine, and 2) A step of using the fermentation material obtained in step 1) to prepare the food; Among them, the usage amount of the fermentation material in step 2) is 0.5 wt% or more.

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