A method for extracting malic acid for cosmetics

Abstract

The present invention belongs to the field of microbial fermentation technology, and specifically relates to a method for extracting malic acid for cosmetics. The method comprises the following steps: activation, cultivation and fermentation of Aspergillus oryzae; solid-liquid separation of fermentation liquid; hydrolysis of fermentation filtrate; malic acid chitosan composite salt; separation, purification and crystallization of malic acid. The present invention utilizes bagasse and cellulose hydrolase to improve the fermentation of malic acid by Aspergillus oryzae, and also adds chitosan with a molecular weight of 10KDa-30KDa during the fermentation process to form a malic acid chitosan composite salt, and then obtains malic acid by hydrolysis, separation and purification, thereby improving the fermentation yield of malic acid and the purity of malic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for extracting malic acid for cosmetics. Background Art

[0002] L-Malic acid, also known as 2-hydroxysuccinic acid, is a four-carbon dicarboxylic acid that can enhance liver function and relieve metabolic acidosis. At the same time, malic acid has a taste similar to that of apples. It is a safe, non-toxic, drinkable organic acid that is widely present in plant, animal and microbial cells. As a natural moisturizing ingredient, L-malic acid can easily dissolve the "sticky matter" between dry, scaly dead cells, thereby removing wrinkles on the skin surface and making the skin white, smooth and elastic. Therefore, it is highly favored in cosmetics. In addition, L-malic acid can be used to prepare a variety of flavors and fragrances, and is widely used in a variety of daily chemical products, such as toothpaste, shampoo, etc.

[0003] At present, L-malic acid can be synthesized by direct extraction, chemical synthesis, enzyme conversion, cell conversion and microbial fermentation. Microbial fermentation is a method of synthesizing L-malic acid by utilizing the tricarboxylic acid cycle of microorganisms. L-malic acid is one of the important metabolic intermediates and can accumulate in large quantities under certain conditions to obtain the product L-malic acid. In recent years, as people pay more and more attention to environmental protection, green and pure natural products, fermentation methods have become increasingly popular.

[0004] The current research on malic acid fermentation production can be divided into: one-step fermentation method, that is, the sugar raw material is directly converted into malic acid through the complex multi-enzyme reaction of mold or yeast itself; mixed fermentation method, that is, the raw material is first converted into fumaric acid, and then bacteria and the like further convert it into malic acid. The production of L-malic acid by microbial fermentation has the advantages of a wide source of substrates and green environmental protection. However, during the microbial fermentation process, as the L-malic acid product accumulates, the pH of the fermentation liquid will continue to decrease (usually below 3.0). A large amount of neutralizing agents (such as calcium carbonate, magnesium carbonate, sodium hydroxide or ammonia water, etc.) are added during the production process. This operation not only increases the difficulty of the downstream separation and purification process, increases the production cost, but also causes serious environmental pollution. The extraction method process includes multiple steps such as multi-step acid hydrolysis, filtration, refining, crystallization, etc., which has the problems of complex process, low extraction purity and low yield. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for extracting malic acid for cosmetics. In the method, chitosan is added during the fermentation process of malic acid, and malic acid and chitosan are reacted to generate a malic acid chitosan composite salt, thereby avoiding the accumulation of malic acid during the fermentation process, thereby improving the fermentation yield of malic acid; and high-purity malic acid can be obtained through hydrolysis, separation and purification.

[0006] Specifically, the present invention provides a method for extracting malic acid, comprising the following steps:

[0007] S1 Activation of Aspergillus oryzae: Transfer Aspergillus oryzae to Aspergillus oryzae seed culture medium for activation and culture at 32-36°C for 24-36 hours;

[0008] S2 Aspergillus oryzae liquid culture: inoculating the activated Aspergillus oryzae into an Aspergillus oryzae liquid culture medium, and culturing at 32-36° C. for 24-36 hours to obtain an Aspergillus oryzae seed liquid;

[0009] S3 Aspergillus oryzae fermentation: transfer the seed solution to the fermentation tank culture medium in a volume ratio of 1:8-10, culture at a temperature of 32-36°C, culture for 36-48h, then add chitosan, control the temperature to 34-38°C, continue fermentation for 36-48h, keep the temperature and pressure for fermentation for 12-16h, stop fermentation, and obtain malic acid chitosan composite salt fermentation liquid;

[0010] S4 centrifuge the fermentation broth: centrifuge the malic acid chitosan complex salt fermentation broth to remove the precipitate to obtain a fermentation crude filtrate;

[0011] S5 Fermentation crude filtrate filtration: Fermentation filtrate is filtered through a ceramic membrane to obtain fermentation refined filtrate;

[0012] S6 hydrolysis: the fermentation filtrate is filtered through an ultrafiltration membrane with a molecular weight cut-off of no more than 1000Da, and the retentate is hydrolyzed by adding acid to obtain a malic acid chitosan composite salt hydrolyzate;

[0013] S7 ultrafiltration: The hydrolyzed malic acid-chitosan composite salt solution is filtered through an ultrafiltration membrane with a molecular weight cut-off of no more than 1000 Da, and the filtrate is the L-malic acid solution;

[0014] S8 vacuum distillation: The L-malic acid solution is subjected to vacuum distillation and vacuum drying to obtain L-malic acid.

[0015] The above method, the Aspergillus oryzae seed culture medium described in S1 comprises, by mass percentage: 0.3% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, and the balance is water, pH 6.2;

[0016] Preferably, the S1 culture temperature is 34° C. and the culture time is 24 h.

[0017] The above method, the Aspergillus oryzae liquid culture medium described in S2 comprises, by mass percentage: 0.3% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, and the balance is water, pH 6.2;

[0018] Preferably, the S2 culture temperature is 34°C and the culture time is 24h.

[0019] In the above method, the Aspergillus oryzae fermentation tank culture medium described in S3 comprises, by mass percentage: 0.3% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% chitosan, 1-2% bagasse, 0.01-0.02% cellulose hydrolase, and the balance is water, pH 6.2;

[0020] Preferably, the volume ratio of seed liquid to fermentation tank culture medium is 1:8;

[0021] Preferably, the fermentation temperature of Aspergillus oryzae is 34°C, the culture time is 48h, the continued fermentation time is 36h, and the heat preservation and pressure fermentation time is 12h;

[0022] In the above method, the molecular weight of the chitosan described in S3 is 1KDa-10KDa;

[0023] The preferred molecular weight of chitosan is 8000Da;

[0024] In the above method, the final amount of chitosan added to the Aspergillus oryzae fermentation tank culture medium is 80-100 g / L;

[0025] Preferably, the final amount of chitosan added to the Aspergillus oryzae fermentation tank culture medium is 100 g / L.

[0026] In the above method, the filtration pore size of the ceramic membrane described in S5 is 1000-1500nm.

[0027] Preferably, the filtration pore size of the ceramic membrane described in S5 is 1000 nm.

[0028] In the above method, the acid described in S6 is hydrochloric acid, the hydrolysis pH value is 1.0-4.5, and the hydrolysis time is 1-2h.

[0029] In the above method, the hydrolysis temperature in S6 is 50-80°C;

[0030] Preferably, the hydrolysis pH in S6 is 2, the temperature is 70° C., and the hydrolysis time is 1 h;

[0031] In the above method, the vacuum distillation temperature described in S8 is 60-80°C.

[0032] Preferably, the reduced pressure distillation temperature in S8 is 60°C.

[0033] The present invention has the following beneficial effects:

[0034] 1. The present invention improves the yield of malic acid fermented by Aspergillus oryzae by adding bagasse and cellulose hydrolase during the fermentation process of Aspergillus oryzae.

[0035] 2. The present invention forms a malic acid-chitosan composite salt by adding chitosan to the malic acid fermentation broth of Aspergillus oryzae, thereby avoiding the accumulation of malic acid during the fermentation process and the influence on the activity of Aspergillus oryzae and the yield of malic acid.

[0036] 3. The present invention avoids the use of a large amount of chemical neutralizers by adding chitosan to the Aspergillus oryzae malic acid fermentation broth, is environmentally friendly, and provides a new idea for extracting and fermenting malic acid.

[0037] 4. The present invention adopts multi-level separation technology, utilizes chitosan of specific molecular weight to modify malic acid, then adopts ultrafiltration separation method to intercept malic acid chitosan composite salt, and finally obtains malic acid by hydrolysis, thereby greatly improving the purity of malic acid. The obtained malic acid can be applied to the daily cosmetics industry. DETAILED DESCRIPTION

[0038] The technical scheme of the present invention is further described below in conjunction with specific examples. The Aspergillus oryzae strain used in the following examples is (Aspergillus oryzae FMME 338), with a deposit number of CCTCC NO: M2016401; other raw materials, if not otherwise specified, can be obtained from conventional commercial channels; the processes used, if not otherwise specified, all adopt conventional processes in the art.

[0039] Example 1

[0040] A method for extracting malic acid comprises the following steps:

[0041] S1 Activation of Aspergillus oryzae: The Aspergillus oryzae strain was transferred to an Aspergillus oryzae seed culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, the balance being water, pH 6.2) for activation and cultured at 34° C. for 24 hours;

[0042] S2 Aspergillus oryzae liquid culture: activated Aspergillus oryzae was inoculated with Aspergillus oryzae liquid culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% chitosan, the balance being water, pH 6.2), and cultured at 34°C for 24 hours to obtain Aspergillus oryzae seed liquid;

[0043] S3 Aspergillus oryzae fermentation: according to seed liquid: fermentation tank medium (according to mass percentage, including: 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% 8000Da molecular weight chitosan, 1% bagasse, 0.01 cellulose hydrolase, the balance is water, pH6.2) be transferred into a fermentation tank and cultured at a temperature of 34°C for 48h, and then chitosan is continuously supplemented during the fermentation process until the total addition amount of chitosan in the medium is 100g / L, the temperature is controlled to be 34-38°C and the fermentation is continued for 36h, and the fermentation is kept warm and under pressure for 12h, and the fermentation is stopped to obtain malic acid chitosan composite salt fermentation liquid;

[0044] S4 centrifuge the fermentation broth: centrifuge the malic acid chitosan complex salt fermentation broth to remove the precipitate to obtain a fermentation crude filtrate;

[0045] S5 Fermentation crude filtrate filtration: Fermentation filtrate is filtered through a ceramic membrane to obtain fermentation refined filtrate;

[0046] S6 hydrolysis: the fermentation filtrate was filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, hydrochloric acid was added to the retentate to a pH of 2, and hydrolysis was performed at 70°C for 1h to obtain a hydrolyzate of malic acid chitosan composite salt;

[0047] S7 ultrafiltration: The hydrolyzate of malic acid-chitosan composite salt was filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, and the filtrate was the L-malic acid solution;

[0048] S8 vacuum distillation: The L-malic acid solution is subjected to vacuum distillation at 60° C. and vacuum dried to obtain L-malic acid.

[0049] Example 2

[0050] A method for extracting malic acid comprises the following steps:

[0051] S1 Activation of Aspergillus oryzae: The Aspergillus oryzae strain was transferred to an Aspergillus oryzae seed culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, the balance being water, pH 6.2) for activation and cultured at 34° C. for 24 hours;

[0052] S2 Aspergillus oryzae liquid culture: activated Aspergillus oryzae was inoculated with Aspergillus oryzae liquid culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% chitosan, the balance being water, pH 6.2), and cultured at 34°C for 24 hours to obtain Aspergillus oryzae seed liquid;

[0053] S3 Aspergillus oryzae fermentation: according to seed liquid: fermentation tank medium (according to mass percentage, including: 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% 1000Da molecular weight chitosan, 1.5% bagasse, 0.015% cellulose hydrolase, the balance is water, pH6.2) be transferred into a fermentation tank and cultured at a volume ratio of 1:8, the temperature is 34°C, and the culture is carried out for 48h, and chitosan is continuously supplemented in the fermentation process until the total addition amount of chitosan in the medium is 80g / L, and the temperature is controlled to be 34-38°C and the fermentation is continued for 36h, and the fermentation is kept warm and under pressure for 12h, and the fermentation is stopped to obtain malic acid chitosan composite salt fermentation liquid;

[0054] S4 centrifuge the fermentation broth: centrifuge the malic acid chitosan complex salt fermentation broth to remove the precipitate to obtain a fermentation crude filtrate;

[0055] S5 Fermentation crude filtrate filtration: Fermentation filtrate is filtered through a ceramic membrane to obtain fermentation refined filtrate;

[0056] S6 hydrolysis: the fermentation filtrate was filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, hydrochloric acid was added to the retentate to a pH of 3, and hydrolyzed at 50°C for 2h to obtain a malic acid chitosan composite salt hydrolyzate;

[0057] S7 ultrafiltration: The hydrolyzate of malic acid-chitosan composite salt was filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, and the filtrate was the L-malic acid solution;

[0058] S8 vacuum distillation: The L-malic acid solution is subjected to vacuum distillation at 60° C. and vacuum dried to obtain L-malic acid.

[0059] Example 3

[0060] A method for extracting malic acid comprises the following steps:

[0061] S1 Activation of Aspergillus oryzae: The Aspergillus oryzae strain was transferred to an Aspergillus oryzae seed culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, the balance being water, pH 6.2) for activation and cultured at 34° C. for 24 hours;

[0062] S2 Aspergillus oryzae liquid culture: activated Aspergillus oryzae was inoculated with Aspergillus oryzae liquid culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% chitosan, the balance being water, pH 6.2), and cultured at 34°C for 24 hours to obtain Aspergillus oryzae seed liquid;

[0063] S3 Aspergillus oryzae fermentation: according to seed liquid: fermentation tank medium (according to mass percentage, including: 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% 10KDa molecular weight chitosan, 2% bagasse, 0.02% cellulose hydrolase, the balance is water, pH6.2) be transferred into the fermentation tank and culture, temperature 34 ℃, culture 48h, then continue to supplement chitosan in the fermentation process until the total addition amount of chitosan in the medium is 100g / L, control temperature is 34-38 ℃ and continue fermentation for 36h, keep warm and keep pressure and ferment for 12h, stop fermentation, obtain malic acid chitosan composite salt fermentation liquid;

[0064] S4 centrifuge the fermentation broth: centrifuge the malic acid chitosan complex salt fermentation broth to remove the precipitate to obtain a fermentation crude filtrate;

[0065] S5 Fermentation crude filtrate filtration: Fermentation filtrate is filtered through a ceramic membrane to obtain fermentation refined filtrate;

[0066] S6 hydrolysis: the fermentation filtrate was filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, hydrochloric acid was added to the retentate to a pH of 1, and hydrolysis was performed at 80°C for 2h to obtain a hydrolyzate of malic acid chitosan composite salt;

[0067] S7 ultrafiltration: The hydrolyzate of malic acid-chitosan composite salt was filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, and the filtrate was the L-malic acid solution;

[0068] S8 vacuum distillation: The L-malic acid solution is subjected to vacuum distillation at 60° C. and vacuum dried to obtain L-malic acid.

[0069] Comparative Example 1

[0070] A method for extracting malic acid comprises the following steps:

[0071] S1 Activation of Aspergillus oryzae: The Aspergillus oryzae strain was transferred to an Aspergillus oryzae seed culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, the balance being water, pH 6.2) for activation and cultured at 34° C. for 24 hours;

[0072] S2 Aspergillus oryzae liquid culture: activated Aspergillus oryzae was inoculated with Aspergillus oryzae liquid culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% chitosan, the balance being water, pH 6.2), and cultured at 34°C for 24 hours to obtain Aspergillus oryzae seed liquid;

[0073] S3 Aspergillus oryzae fermentation: according to seed liquid: fermentation tank medium (according to mass percentage, comprising: 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, the balance is water, pH 6.2) be transferred into a fermentation tank and cultured at a temperature of 34°C for 48h, supplemented with pH neutralizer calcium carbonate, it is 6.2 to maintain the pH of the fermentation tank medium, the temperature is controlled to 34-38°C and the fermentation is continued for 36h, the heat-insulating and pressure-maintaining fermentation is fermented for 12h, and the fermentation is stopped to obtain malic acid fermentation liquid;

[0074] S4 centrifugation of fermentation broth: centrifuging the malic acid fermentation broth to remove the precipitate to obtain a malic acid fermentation crude filtrate;

[0075] S5 Fermentation crude filtrate filtration: ceramic membrane filtration of the fermentation filtrate to obtain malic acid fermentation refined filtrate;

[0076] S6 ultrafiltration: The fermentation filtrate is filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, and the filtrate is the L-malic acid solution;

[0077] S7 vacuum distillation: The L-malic acid solution is subjected to vacuum distillation at 60° C. and vacuum dried to obtain L-malic acid.

[0078] Comparative Example 2

[0079] A method for extracting malic acid comprises the following steps:

[0080] S1 Activation of Aspergillus oryzae: The Aspergillus oryzae strain was transferred to an Aspergillus oryzae seed culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, the balance being water, pH 6.2) for activation and cultured at 34° C. for 24 hours;

[0081] S2 Aspergillus oryzae liquid culture: The activated Aspergillus oryzae was inoculated with Aspergillus oryzae liquid culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% 8000Da chitosan, the balance being water, pH 6.2), and cultured at 34°C for 24 hours to obtain Aspergillus oryzae seed liquid;

[0082] S3 Aspergillus oryzae fermentation: according to seed liquid: fermentation tank medium (comprising according to mass percentage: 0.3% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 2% 8000Da molecular weight chitosan, the remainder is water, pH6.2) be transferred into fermentation tank and cultivate at a volume ratio of 1:8, temperature 34 ℃, cultivate 48h, then continue to supplement chitosan in the fermentation process until the total addition amount of chitosan in the medium is 100g / L, control temperature is 34-38 ℃ and continue fermentation for 36h, keep warm and keep pressure and ferment for 12h, stop fermentation, obtain malic acid chitosan composite salt fermentation liquid;

[0083] S4 centrifuge the fermentation broth: centrifuge the malic acid chitosan complex salt fermentation broth to remove the precipitate to obtain a fermentation crude filtrate;

[0084] S5 Fermentation crude filtrate filtration: Fermentation filtrate is filtered through a ceramic membrane to obtain fermentation refined filtrate;

[0085] S6 hydrolysis: the fermentation filtrate was filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, hydrochloric acid was added to the retentate to a pH of 2, and hydrolysis was performed at 70°C for 1h to obtain a hydrolyzate of malic acid chitosan composite salt;

[0086] S7 ultrafiltration: The hydrolyzate of malic acid-chitosan composite salt was filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, and the filtrate was the L-malic acid solution;

[0087] S8 vacuum distillation: The L-malic acid solution is subjected to vacuum distillation at 60° C. and vacuum dried to obtain L-malic acid.

[0088] Comparative Example 3

[0089] A method for extracting malic acid comprises the following steps:

[0090] S1 Activation of Aspergillus oryzae: The Aspergillus oryzae strain was transferred to an Aspergillus oryzae seed culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, the balance being water, pH 6.2) for activation and cultured at 34° C. for 24 hours;

[0091] S2 Aspergillus oryzae liquid culture: activated Aspergillus oryzae was inoculated with Aspergillus oryzae liquid culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, the balance being water, pH 6.2), and cultured at 34°C for 24 hours to obtain Aspergillus oryzae seed liquid;

[0092] S3 Aspergillus oryzae fermentation: according to the volume ratio of 1:8 of seed liquid: fermentation tank culture medium (comprising, by mass percentage, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate heptahydrate, 3% sucrose, 2% agar, 1% bagasse, 0.01 cellulose hydrolase, the balance being water, pH 6.2), cultured at 34°C for 48h, supplemented with pH neutralizer calcium carbonate, maintaining the pH of the fermentation tank culture medium at 6.2, controlling the temperature to continue fermentation at 34-38°C for 36h, fermenting under heat and pressure for 12h, stopping the fermentation, and obtaining malic acid fermentation liquid;

[0093] S4 centrifugation of fermentation broth: centrifuging the malic acid fermentation broth to remove the precipitate to obtain a malic acid fermentation crude filtrate;

[0094] S5 Fermentation crude filtrate filtration: ceramic membrane filtration of the fermentation filtrate to obtain malic acid fermentation refined filtrate;

[0095] S6 ultrafiltration: The fermentation filtrate is filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000Da, and the filtrate is the L-malic acid solution;

[0096] S7 vacuum distillation: The L-malic acid solution is subjected to vacuum distillation at 60° C. and vacuum dried to obtain L-malic acid.

[0097] Determination of L-malic acid

[0098] The 2,7-naphthalene diol colorimetric method was used to take 1.0 mL of the malic acid-chitosan composite salt hydrolyzate in each embodiment, the malic acid fermentation crude filtrate in Comparative Example 1, and the diluted solution of malic acid after drying, respectively, and 6.0 mL of analytically pure concentrated sulfuric acid was added, and then 0.1 mL of 2,7-naphthalene diol solution was added, followed by heating in a 100° C. water bath for 20 min, taking out and cooling to room temperature, and then measuring the absorbance at 385 nm. A standard curve was first made using a standard L-malic acid sample, with the malic acid content as the abscissa and the absorption value at 385 nm as the ordinate, to obtain the content of L-malic acid in the malic acid-chitosan composite salt hydrolyzate in each embodiment, the content of L-malic acid in the malic acid fermentation crude filtrate in Comparative Example 1, and the purity of the malic acid after drying. The results are shown in Table 1.

[0099]

[0100]

[0101] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for extracting malic acid for cosmetics, characterized in that: The steps include: S1 Activation of Aspergillus oryzae strain: Transfer the Aspergillus oryzae strain to Aspergillus oryzae seed culture medium for activation and culture at 32-36°C for 24-36 hours; S2 Liquid culture of Aspergillus oryzae strain: inoculate the activated Aspergillus oryzae strain into Aspergillus oryzae liquid culture medium, and culture at 32-36° C. for 24-36 hours to obtain Aspergillus oryzae seed liquid; S3 Aspergillus oryzae fermentation: transfer the seed solution to the fermentation tank at a volume ratio of 1:8-10, culture at a temperature of 32-36°C, culture for 36-48 hours, then add chitosan, control the temperature at 34-38°C, continue fermentation for 36-48 hours, keep the temperature and pressure for fermentation for 12-16 hours, stop fermentation, and obtain malic acid chitosan composite salt fermentation liquid; S4 centrifugation of fermentation broth: centrifuging the malic acid chitosan complex salt fermentation broth to remove the precipitate and obtain a fermentation crude filtrate; S5 Fermentation crude filtrate filtration: The fermentation filtrate is filtered through a ceramic membrane to obtain a fermentation refined filtrate; S6 Hydrolysis: The fermentation filtrate is filtered through an ultrafiltration membrane with a molecular weight cut-off of no more than 1000 Da, and the retentate is hydrolyzed by adding acid to obtain a malic acid chitosan composite salt hydrolyzate; S7 ultrafiltration: the malic acid chitosan composite salt hydrolyzate is filtered through an ultrafiltration membrane with a molecular weight cut-off of no more than 1000 Da to obtain an L-malic acid solution; S8 vacuum distillation: the permeate is subjected to vacuum distillation and vacuum drying to obtain L-malic acid; The molecular weight of chitosan described in S3 is 1KDa-10KDa; The final addition amount of chitosan described in S3 in the Aspergillus oryzae fermentation tank culture medium is 80-100 g / L, and the fermentation tank culture medium contains 10-20 g / L of bagasse and 0.1-0.2 g / L of cellulose hydrolase; The Aspergillus oryzae strain is Aspergillus oryzae FMME 338, the deposit number is CCTCC NO:M2016401.

2. The method for extracting malic acid according to claim 1, characterized in that: The filtration pore size of the ceramic membrane described in S5 is 1000-1500nm.

3. The method for extracting malic acid according to claim 1, characterized in that: The acid described in S6 is hydrochloric acid.

4. The method for extracting malic acid according to claim 1, characterized in that: The hydrolysis pH value of S6 is 1.0-4.

5.

5. The method for extracting malic acid according to claim 1, characterized in that: The hydrolysis time described in S6 is 1-2h.

6. The method for extracting malic acid according to claim 1, characterized in that: The hydrolysis temperature described in S6 is 50-80°C.

7. The method for extracting malic acid according to claim 1, characterized in that: The reduced pressure distillation temperature in S8 is 60-80°C.

8. The method for extracting cosmetic malic acid according to any one of claims 1 to 7, characterized in that: The purity of the malic acid obtained by the extraction method is higher than 90%.

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