WO2023125330A1 - 一种低刺激性的γ-氨基丁酸产品及其制备方法和降低刺激性的应用 - Google Patents

一种低刺激性的γ-氨基丁酸产品及其制备方法和降低刺激性的应用 Download PDF

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WO2023125330A1
WO2023125330A1 PCT/CN2022/141654 CN2022141654W WO2023125330A1 WO 2023125330 A1 WO2023125330 A1 WO 2023125330A1 CN 2022141654 W CN2022141654 W CN 2022141654W WO 2023125330 A1 WO2023125330 A1 WO 2023125330A1
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aminobutyric acid
preparation
fermentation
irritation
yeast
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French (fr)
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魏玉洁
陆震
陈清平
薛文萍
郭学平
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华熙生物科技股份有限公司
华熙生物科技(天津)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a low-irritation gamma-aminobutyric acid composition and a preparation method thereof.
  • GABA ⁇ -Aminobutyric acid
  • GABA is a naturally occurring non-protein amino acid, which is widely distributed in animals and plants, but its content is relatively low.
  • GABA exists only in nervous tissue, and is an important inhibitory neurotransmitter in the mammalian central nervous system, participating in various metabolic and physiological activities in the human body.
  • emotions such as anxiety, restlessness, fatigue, and worry will occur.
  • GABA is widely used in food and health products, and can also be used as a drug for treating diseases. It is also an internationally recognized cosmetic raw material and has been included in the international cosmetic raw material catalogue. GABA has a small molecular weight and can quickly penetrate the skin and relax tense muscles, thereby significantly reducing fine lines and wrinkles, but it will bring immediate discomfort when used, especially tingling, which limits its use in cosmetics .
  • compositions containing gamma-aminobutyric acid and its application which is characterized in that through gamma -Aminobutyric acid and N-acetylglucosamine are synergistically compounded to relieve the tingling sensation caused by the use of ⁇ -aminobutyric acid.
  • Chinese patent CN105362091B discloses "a composition containing ⁇ -aminobutyric acid and hyaluronic acid and its application in cosmetics", which is characterized in that the active ingredients of the composition include ⁇ -aminobutyric acid and hyaluronic acid or its Salt can significantly improve and reduce the adverse reaction of ⁇ -aminobutyric acid to the skin, and overcome the skin irritation caused by using ⁇ -aminobutyric acid alone.
  • Chinese patent CN112089637B discloses "skin care composition with synergistic anti-aging effect and its preparation method and application", which is characterized in that the skin care composition is composed of gamma-aminobutyric acid, flower palm tree bark extract and tomato/yeast fermentation product Composed of filtrate, the combined use of the three shows a stronger effect in reducing the number, depth, length, and area of wrinkles and improving skin elasticity, and has a significant synergistic effect, and the combination of the three overcomes the 5% gamma-aminobutyric acid There is a bigger irritation problem when used alone.
  • Existing research is alleviating the tingling sensation of GABA by compounding substances with a soothing effect with GABA.
  • the present invention provides a strain of Saccharomyces cerevisiae WJ-37 with excellent fermentation performance, and its preservation number is CCTCC NO: M 20211621.
  • the invention also provides a preparation method for reducing the irritation of gamma-aminobutyric acid.
  • the present invention relates to the following aspects:
  • a preparation method of the gamma-aminobutyric acid product of low irritation is characterized in that, described preparation method comprises the following steps:
  • the bacterial cells in the fermentation liquid are crushed and filtered to obtain a filtrate containing ⁇ -aminobutyric acid.
  • the preparation method according to item 1 characterized in that the preparation method also includes the step of adding auxiliary materials to the filtrate and spray drying to obtain a low-irritation ⁇ -aminobutyric acid composition.
  • the ⁇ -aminobutyric acid in the solution containing ⁇ -aminobutyric acid is obtained by enzymatic catalysis, preferably, the solution containing ⁇ -aminobutyric acid
  • the mass content of ⁇ -aminobutyric acid in the medium is 10-100g/L
  • the bacterium breaking method includes at least one of stirring, homogenization, ultrasonication, heating, and more preferably, the fermentation condition At 22-27°C, ferment until there is no glucose in the fermentation broth.
  • the solution containing ⁇ -aminobutyric acid also includes 1-30g/L peptone, 1-20g/L yeast powder and 1- 20g/L glucose.
  • auxiliary material is selected from the group consisting of hydroxypropyl- ⁇ -cyclodextrin, hyaluronic acid or its salt, trehalose, maltodextrin, polyglutamic acid, micro One or more of crystalline cellulose, mannitol, lactose, and fucose, preferably a mixture of hydroxypropyl- ⁇ -cyclodextrin and hyaluronic acid or its salts, preferably, the adjuvant and the The mass ratio of the filtrate is 1:2.5-50.
  • a low-irritation ⁇ -aminobutyric acid product prepared by the preparation method described in any one of items 1-5.
  • ⁇ -aminobutyric acid product according to item 6 wherein the ⁇ -aminobutyric acid is the ⁇ -aminobutyric acid composition prepared by the preparation method described in item 2, and in the combination In the product, the mass content of the gamma-aminobutyric acid is 10-50%, and the mass content of the auxiliary materials is 40-80%.
  • a brewer's yeast Sacharomyces cerevisiae
  • CCTCC NO: M 20211621 CCTCC NO: M 20211621.
  • Saccharomyces cerevisiae with deposit number CCTCC NO: M 20211621 or Saccharomyces cerevisiae strain with CGMCC NO: 2.3854 in reducing the irritation of ⁇ -aminobutyric acid products.
  • the present invention finds that the tingling sensation of gamma-aminobutyric acid may partly be caused by impurity components in gamma-aminobutyric acid products, and through the production process of the present invention, part of the impurity-causing impurities can be consumed, and at the same time Produces soothing ingredients to reduce the discomfort caused by gamma-aminobutyric acid.
  • the Saccharomyces cerevisiae WJ-37 of the present invention has been preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20211621, the preservation date is December 13, 2021, and the preservation address is Wuchang, Wuhan City, Hubei province Luojiashan Wuhan University, zip code is 430072.
  • Figure 1 is a photo of the colony morphological characteristics of Saccharomyces cerevisiae WJ-37 CCTCC NO: M 20211621.
  • Figure 2 is a photomicrograph of Saccharomyces cerevisiae WJ-37 CCTCC NO: M 20211621 cells.
  • the invention provides a strain of Saccharomyces cerevisiae with excellent fermentation performance, which is obtained through self-screening from Bohai sea mud.
  • the Saccharomyces cerevisiae is named Saccharomyces cerevisiae WJ-37, and it has been preserved in the China Center for Type Culture Collection in 2021, with the preservation number CCTCC NO: M 20211621, and the preservation address: Wuchang, Wuhan City, Hubei province Luojiashan Wuhan University, Zip Code: 430072.
  • Saccharomyces cerevisiae (Saccharomyces cerevisiae) WJ-37 can be screened by the following method:
  • Bohai sea mud as raw material, disperse the Bohai sea mud with 100 times the mass of sterile water, and use a spreader to evenly spread each solution on a yeast solid medium plate containing 0.1mg/L ampicillin sodium, and in 25 Cultivate statically in an incubator at °C for 1 to 2 days, select a single colony with round and smooth colony shape, inoculate it on the inclined surface of a test tube of yeast solid medium, and culture it in an incubator at 25°C for 1 to 2 days. After the bacteria grow well, Store in refrigerator at 4°C.
  • the yeast solid medium is: peptone 20g/L, yeast powder 10g/L, glucose 20g/L, agar 20g/L, and the rest is water.
  • the present invention provides a method for preparing a low-irritation gamma-aminobutyric acid product, the preparation method comprising: The following steps:
  • Step 1 Inoculate the yeast into the solution containing ⁇ -aminobutyric acid for fermentation
  • Step 2 After the fermentation, the cells in the fermentation liquid are crushed and filtered to obtain a filtrate containing ⁇ -aminobutyric acid.
  • preparation method may include step three:
  • Step 3 adding the filtrate to auxiliary materials for spray drying to obtain a low-irritation ⁇ -aminobutyric acid composition.
  • the gamma-aminobutyric acid product described in the present invention can cover various forms of products containing gamma-aminobutyric acid, for example, it can be a filtrate containing gamma-aminobutyric acid, or it can be dried gamma-aminobutyric acid acid composition.
  • step one the step of inserting the yeast into the solution containing ⁇ -aminobutyric acid for fermentation can directly insert the yeast into the solution containing ⁇ -aminobutyric acid, or activate the yeast and then Let it ferment.
  • said inserting the yeast into the solution containing gamma-aminobutyric acid for fermentation comprises the following steps:
  • the seed solution is inoculated into a solution containing ⁇ -aminobutyric acid for fermentation.
  • the inoculum amount of the seed solution is 1-10%, that is, the volume ratio of the seed solution to the solution containing ⁇ -aminobutyric acid is 1-10%.
  • the yeast used in the preparation method of the present invention may be various yeasts known in the art.
  • the yeast is the above-mentioned Saccharomyces cerevisiae, that is, Saccharomyces cerevisiae WJ-37 with a preservation number of CCTCC NO: M 20211621.
  • the yeast is Saccharomyces cerevisiae numbered 2.3854, which can be purchased from China General Microorganism Culture Collection Center (CGMCC).
  • CGMCC General Microorganism Culture Collection Center
  • the ⁇ -aminobutyric acid in the solution containing ⁇ -aminobutyric acid can be from various sources, such as the ⁇ -aminobutyric acid powder product obtained after fermentation, conversion, and purification, or the decarboxylation of glutamic acid
  • the conversion liquid containing ⁇ -aminobutyric acid obtained after enzyme catalysis can be obtained through fermentation of lactic acid bacteria or Escherichia coli.
  • the conversion solution containing ⁇ -aminobutyric acid can be directly used to prepare a solution containing ⁇ -aminobutyric acid.
  • the mass content of ⁇ -aminobutyric acid in the solution containing ⁇ -aminobutyric acid is 10-100 g/L.
  • the solution containing ⁇ -aminobutyric acid may further include other nutrients for yeast fermentation.
  • the solution containing ⁇ -aminobutyric acid further includes 1-30 g/L peptone, 1-20 g/L yeast powder and 1-20 g/L glucose.
  • the yeast is fermented under the fermentation condition of 22-27°C until no glucose is contained in the fermentation broth.
  • the fermentation method may be static fermentation.
  • step 2 the method of crushing the bacteria in the fermentation broth may be one of stirring, homogenizing, ultrasonic and heating.
  • the auxiliary material can be selected from hydroxypropyl- ⁇ -cyclodextrin, hyaluronic acid, trehalose, maltodextrin, polyglutamic acid, microcrystalline cellulose, mannitol, lactose, fucose one or more of them.
  • the excipient is a mixture of hydroxypropyl- ⁇ -cyclodextrin and hyaluronic acid, wherein hydroxypropyl- ⁇ -cyclodextrin and hyaluronic acid can be mixed in any proportion, such as hydroxypropyl- ⁇ -cyclodextrin
  • the mass ratio of propyl- ⁇ -cyclodextrin and hyaluronic acid can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2 :1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc.
  • the excipients are hydroxypropyl- ⁇ -cyclodextrin and 10KDa sodium hyaluronate, and the mass ratio of hydroxypropyl- ⁇ -cyclodextrin and sodium hyaluronate is 9:1.
  • Spray drying can be carried out using equipment known in the prior art, for example a centrifugal spray dryer can be used.
  • the present invention also provides a low-irritation ⁇ -aminobutyric acid product prepared by the above preparation method.
  • the ⁇ -aminobutyric acid is a low-irritation ⁇ -aminobutyric acid composition prepared by the above preparation method, and the composition includes ⁇ -aminobutyric acid and auxiliary materials.
  • the mass content of the gamma-aminobutyric acid is 10-50%, and the mass content of the auxiliary materials is 40-80%.
  • the auxiliary material can be selected from one of hydroxypropyl- ⁇ -cyclodextrin, hyaluronic acid, trehalose, maltodextrin, polyglutamic acid, microcrystalline cellulose, mannitol, lactose, and fucose. species or two or more.
  • the excipient is a mixture of hydroxypropyl- ⁇ -cyclodextrin and hyaluronic acid.
  • Hyaluronic acid herein may encompass hyaluronic acid or its salts, such as sodium hyaluronate, calcium hyaluronate, zinc hyaluronate, and the like. Among them, the molecular weight of hyaluronic acid is 3KDa-30KDa.
  • composition is prepared by the above-mentioned preparation method.
  • the present invention also provides the application of Saccharomyces cerevisiae strain with preservation number CCTCC NO: M 20211621 or CGMCC NO: 2.3854 in reducing the irritation of ⁇ -aminobutyric acid products.
  • yeast is inoculated into a solution containing ⁇ -aminobutyric acid for fermentation, and the composition obtained by adding auxiliary materials for drying can reduce ⁇ -aminobutyric acid Acid irritation.
  • Saccharomyces cerevisiae with a preservation number of CCTCC NO: M 20211621 of the present invention, as well as auxiliary materials hydroxypropyl- ⁇ -cyclodextrin and hyaluronic acid, can obtain the best irritation-reducing effect.
  • Bohai sea mud as raw material, disperse the Bohai sea mud with 100 times the mass of sterile water, and use a spreader to evenly spread each solution on a yeast solid medium plate containing 0.1mg/L ampicillin sodium, and in 25 Cultivate statically in an incubator at °C for 1 to 2 days, select a single colony with round and smooth colony shape, inoculate it on the inclined surface of a test tube of yeast solid medium, and culture it in an incubator at 25°C for 1 to 2 days. After the bacteria grow well, Store in refrigerator at 4°C. Among them, yeast solid medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L, agar 20g/L, and the rest is water.
  • the screened strain was named WJ-37.
  • the screened strains were sent to Shanghai Sangon Bioengineering (Shanghai) Co., Ltd. for genome sequencing identification, and the results showed that the strains were Saccharomyces cerevisiae.
  • the 26s rDNA gene sequence determination result of the strain is shown in SEQ ID NO: 1:
  • Saccharomyces cerevisiae Saccharomyces cerevisiae (Saccharomyces cerevisiae) WJ-37 on the yeast solid medium plate, the surface of the colony is smooth, moist, with neat edges, uniform texture and uniform color, as shown in Figure 1; the bacterium is single-celled and oval , the shape is simple, budding reproduction, as shown in Figure 2.
  • Seed medium peptone 20g/L, yeast powder 10g/L, glucose 20g/L, and the rest is water.
  • Fermentation medium peptone 10g/L, yeast powder 5g/L, glucose 5g/L, ⁇ -aminobutyric acid (purchased from Bloomage Biotechnology Co., Ltd., product number 1020010002) 60g/L, and the rest is water.
  • Seed medium and fermentation medium are the same as in Example 3.
  • the inoculum amount is 5% of the fermentation medium, and cultured statically at 24°C until there is no residual sugar, the fermentation broth is heated at 85°C for 1 hour, and filtered through a 0.22 ⁇ m polyethersulfone filter element to obtain a filtrate.
  • the above filtrate was added with 9% hydroxypropyl- ⁇ -cyclodextrin and 1% 10KDa sodium hyaluronate, mixed evenly, and sprayed with a centrifugal spray dryer.
  • the air inlet temperature used in the spray drying was 180° C., and the outlet air temperature was 90° C. to obtain GABA-containing powder S2.
  • Seed medium and fermentation medium are the same as in Example 3.
  • the above-mentioned filtrate is added with a mass ratio of 10% 10KDa sodium hyaluronate, mixed evenly, and sprayed with a centrifugal spray dryer.
  • the air inlet temperature used in the spray drying was 180° C., and the outlet air temperature was 90° C. to obtain GABA-containing powder S3.
  • Seed medium and fermentation medium are the same as in Example 3.
  • Seed medium and fermentation medium are the same as in Example 3.
  • the above filtrate was added into trehalose with a mass ratio of 10%, mixed evenly, and sprayed with a centrifugal spray dryer.
  • the air inlet temperature used in the spray drying was 180° C., and the outlet air temperature was 90° C. to obtain GABA-containing powder S5.
  • Seed medium and fermentation medium are the same as in Example 3.
  • the aqueous solution of 60g/LGABA was heated at 85°C for 1h, and filtered through a 0.22 ⁇ m polyethersulfone filter element to obtain a filtrate.
  • the above-mentioned filtrate was added with a mass ratio of 9% hydroxypropyl- ⁇ -cyclodextrin and 1% 10kDa sodium hyaluronate, mixed evenly, and sprayed with a centrifugal spray dryer.
  • the air inlet temperature used in the spray drying was 180° C., and the outlet air temperature was 90° C. to obtain GABA-containing powder C1.
  • Seed medium and fermentation medium are the same as in Example 3.
  • Example 3 Insert a single colony of yeast WJ-37 into the seed medium, and culture it statically at 24°C until the concentration of the yeast at the end of the fermentation in Example 3 is the same, mix the yeast fermentation filtrate with the fermentation medium, heat at 85°C for 1 hour, and pass through 0.22 ⁇ m Filter through a polyethersulfone filter to obtain a filtrate.
  • Seed culture medium and fermentation medium with embodiment 3.
  • a single colony of Bacillus subtilis NT-11 (Bacillus subtilis) (preservation number: CCTCC NO: M 2019383, (disclosed in the patent "A strain of Bacillus subtilis and its application in the production of ⁇ -polyglutamic acid") Insert it into the seed culture medium, culture it statically at 24°C until the bacterial concentration is the same as that at the end of the fermentation in Example 3, mix the NT-11 fermentation filtrate with the fermentation medium, heat at 85°C for 1 hour, and filter through a 0.22 ⁇ m polyethersulfone filter element. A filtrate was obtained.
  • the 60g/LGABA aqueous solution 85 was dissolved and heated for 1 hour, and filtered through a 0.22LG polyethersulfone filter element to obtain the filtrate C4.
  • the sample S6 of embodiment 8 and the sample C4 of comparative example 4, and the sample S1-5 of embodiment 3-7 and the sample C1-3 in the comparative example 1-3 are respectively made into the aqueous solution that GABA content is 3%, Put 1 drop on the outer corner of the eye, and after applying it evenly, observe whether there is a tingling sensation.
  • the scoring criteria are shown in Table 2, and the number of people tested is 30.
  • yeast for secondary fermentation is better than simply adding yeast lysates to GABA fermentation broth, which has a better effect on alleviating the irritation caused by GABA, and it may not only be the yeast lysate There are some soothing and irritating substances in the cell product, and the yeast can also consume some irritating impurities during the growth process.

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Abstract

本发明提供一种酿酒酵母(Saccharomyces cerevisiae)，其保藏编号为CCTCC NO:M 20211621。本发明还提供一种低刺激性的γ-氨基丁酸产品的制备方法，所述制备方法包括以下步骤：将酵母菌接种到含有γ-氨基丁酸的溶液中进行发酵，发酵结束后将发酵液中的菌体进行破碎、过滤，得到含有γ-氨基丁酸的滤液，进一步的可将所述滤液加入辅料进行喷雾干燥，得到低刺激性的γ-氨基丁酸组合物。本发明发现γ-氨基丁酸的刺痛感可能部分是由γ-氨基丁酸产品中杂质成分引起的，而通过本发明的生产工艺，既可以消耗掉部分引起刺激性的杂质，同时又能产生舒敏成分，从而降低γ-氨基丁酸带来的不适感。

Description

一种低刺激性的γ-氨基丁酸产品及其制备方法和降低刺激性的应用 技术领域
本发明属于生物医药领域，具体涉及一种低刺激性的γ-氨基丁酸组合物及其制备方法。
背景技术
γ-氨基丁酸，简称GABA，是一种天然存在的非蛋白质氨基酸，广泛分布于动、植物体内，但含量都较低。在动物体内，GABA仅存在于神经组织中，是哺乳动物中枢神经系统中重要的抑制性神经传达物质，参与人体内的多种代谢和生理活动。当人体内GABA缺乏时，会产生焦虑、不安、疲倦、忧虑等情绪。
GABA在食品保健品中应用较多，也可作为治疗疾病的药物，同时也是一种国际公认的化妆品原料，被列入国际化妆品原料目录中。GABA分子量小，能迅速穿透皮肤，放松紧张的肌肉，从而显著减少细纹、淡化皱纹，但在使用时会带来即时的不适感，尤其是刺痛感，从而限制了在化妆品中的使用。
近年来关于如何解决GABA在使用时带来的刺痛感的方法的报道也比较多，如中国专利文献CN113143795A公开了“含γ-氨基丁酸的组合物及其应用”，其特征在于通过γ-氨基丁酸与N-乙酰壳糖胺协同复配，来减缓γ-氨基丁酸使用时带来的刺痛感。中国专利CN105362091B公开了“一种含γ-氨基丁酸和透明质酸的组合物及其在化妆品中的应用”，其特征在于该组合物有效成分包括γ-氨基丁酸和透明质酸或其盐，能显著改善、降低γ-氨基丁酸对皮肤的不良反应，克服了单独使用γ-氨基丁酸对皮肤的刺激。中国专利CN112089637B公开了“具有协同抗衰作用的护肤组合物及其制备方法和应用”，其特征在于护肤组合物由γ-氨基丁酸、花榈木树皮提取物和番茄/酵母菌发酵产物滤液组成，三者联合使用在降低皱纹数量、深度、长度、面积及提高皮肤弹性方面均表现出更强的作用，具有显著的协同作用，而且三者联合后克服了5％γ-氨基丁酸单独使用存在的刺激性较大的问题。现有研究都是通过与GABA复配具有舒缓作用的物质来减轻GABA的刺痛感。
发明内容
针对现有技术存在的上述问题，本发明提供一株发酵性能优异的酿酒酵母(Saccharomyces cerevisiae)WJ-37，其保藏编号为CCTCC NO:M 20211621。
本发明还提供一种降低γ-氨基丁酸刺激性的制备方法。
具体来说，本发明涉及如下方面：
1.一种低刺激性的γ-氨基丁酸产品的制备方法，其特征在于，所述制备方法包括以下步骤：
将酵母菌接种到含有γ-氨基丁酸的溶液中进行发酵；
发酵结束后将发酵液中的菌体进行破碎、过滤，得到含有γ-氨基丁酸的滤液。
2.根据项1所述的制备方法，其特征在于，所述制备方法还包括以下步骤，将所述滤液加入辅料进行喷雾干燥，得到低刺激性的γ激氨基丁酸组合物。
3.根据项1所述的制备方法，其特征在于，所述酵母菌选自保藏编号为CCTCC NO:M 20211621的酿酒酵母或CGMCC NO:2.3854的酿酒酵母。
4.根据项1所述的制备方法，其特征在于，所述含有γ-氨基丁酸的溶液中的γ-氨基丁酸通过酶催化得到，优选的，所述含有γ-氨基丁酸的溶液中γ-氨基丁酸的质量含量为10-100g/L，进一步优选地，所述菌体破碎方式包括搅拌、均质、超声、加热中的至少一种，更进一步优选地，所述发酵条件为22-27℃下，发酵至发酵液中不含葡萄糖，更进一步优选地，所述含有γ-氨基丁酸的溶液还包括1～30g/L蛋白胨、1～20g/L酵母粉和1～20g/L葡萄糖。
5.根据项2所述的制备方法，其特征在于，所述辅料选自羟丙基-β-环糊精、透明质酸或其盐、海藻糖、麦芽糊精、聚谷氨酸、微晶纤维素、甘露醇、乳糖、岩藻糖中的一种或两种以上，优选为羟丙基-β-环糊精和透明质酸或其盐的混合物，优选地，所述辅料与所述滤液的质量比为1:2.5-50。
6.一种低刺激性的γ-氨基丁酸产品，通过项1-5中任一项所述的制备方法制备得到。
7.根据项6所述的γ-氨基丁酸产品，其特征在于，所述γ-氨基丁酸为通过项2所述的制备方法制备得到的γ述氨基丁酸组合物，在所述组合物中，所述γ-氨基丁酸的质量含量为10-50％，所述辅料的质量含量为40-80％。
8.一种酿酒酵母(Saccharomyces cerevisiae)，其保藏编号为CCTCC NO:M 20211621。
9.根据项8所述的酿酒酵母，其26s DNA基因序列如SEQ ID NO：1所示。
10、保藏编号为CCTCC NO:M 20211621的酿酒酵母或CGMCC NO:2.3854的酿酒酵母菌株在降低γ-氨基丁酸产品刺激性中的应用。
本发明发现γ-氨基丁酸的刺痛感可能部分是由γ-氨基丁酸产品中杂质成分引起的，而通过本发明的生产工艺，既可以消耗掉部分引起刺激性的杂质，同时又能产生舒敏成分，从而降低γ-氨基丁酸带来的不适感。
保藏信息
本发明所述酿酒酵母(Saccharomyces cerevisiae)WJ-37已保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 20211621，保藏日期为2021年12月13日，保藏地址为湖北省武汉市武昌珞珈山武汉大学，邮编为430072。
附图说明
图1是酿酒酵母(Saccharomyces cerevisiae)WJ-37 CCTCC NO：M 20211621的菌落形态特征的照片。
图2是酿酒酵母(Saccharomyces cerevisiae)WJ-37 CCTCC NO：M 20211621菌体的显微照片。
具体实施方式
下面结合实施例进一步说明本发明，应当理解，实施例仅用于进一步说明和阐释本发明，并非用于限制本发明。
除非另外定义，本说明书中有关技术的和科学的术语与本领域内的技术人员所通常理解的意思相同。虽然在实验或实际应用中可以应用与此间所述相似或相同的方法和材料，本文还是在下文中对材料和方法做了描述。在相冲突的情况下，以本说明书包括其中定义为准，另外，材料、方法和例子仅供说明，而不具限制性。以下结合具体实施例对本发明作进一步的说明，但不用来限制本发明的范围。
本发明提供一株发酵性能优异的酿酒酵母(Saccharomyces cerevisiae)，从 渤海海泥中经过自行筛选得到。该酿酒酵母(Saccharomyces cerevisiae)命名为酿酒酵母(Saccharomyces cerevisiae)WJ-37，已于2021年在中国典型培养物保藏中心进行保藏，保藏编号为CCTCC NO:M 20211621，保藏地址：湖北省武汉市武昌珞珈山武汉大学，邮编：430072。
在本发明的一个具体的实施方式中，酿酒酵母(Saccharomyces cerevisiae)WJ-37可以通过以下方法筛选得到：
以渤海海泥为原料，将渤海海泥用100质量倍无菌水分散，用涂布器将各溶液均匀涂布在含0.1mg/L氨苄青霉素钠的酵母菌固体培养基平板上，于25℃培养箱中静置培养1～2d，挑选菌落形态圆润光滑的单菌落，接种于酵母菌固体培养基试管斜面上，于25℃培养箱中静置培养1～2d，菌体长好后，于4℃冰箱保藏。
其中，酵母菌固体培养基为：蛋白胨20g/L，酵母粉10g/L，葡萄糖20g/L，琼脂20g/L，其余为水。
该酿酒酵母(Saccharomyces cerevisiae)WJ-37的26s DNA基因序列如SEQ ID NO：1所示：
Figure PCTCN2022141654-appb-000001
针对现有技术中γ-氨基丁酸产品所带来的不适感，尤其是刺痛感的问题，本发明提供一种低刺激性的γ-氨基丁酸产品的制备方法，所述制备方法包括以下步骤：
步骤一：将酵母菌接种到含有γ-氨基丁酸的溶液中进行发酵，
步骤二：发酵结束后将发酵液中的菌体进行破碎、过滤，得到含有γ-氨基丁酸的滤液。
进一步地，所述制备方法可以包括步骤三：
步骤三：将所述滤液加入辅料进行喷雾干燥，得到低刺激性的γ-氨基丁酸组合物。
即本发明所述的γ-氨基丁酸产品可以是涵盖含有γ-氨基丁酸的各种形式的产品，例如可以为含有γ-氨基丁酸的滤液，也可以是干燥后的γ基氨基丁酸组合物。
在步骤一中，所述将酵母菌接入含有γ-氨基丁酸的溶液中进行发酵的步骤可以直接将酵母菌接入含有γ-氨基丁酸的溶液中，也可以将酵母菌活化后再进行发酵。
在一个具体的实施方式中，所述将酵母菌接入含有γ-氨基丁酸的溶液中进行发酵包括以下步骤：
将酵母菌接入种子培养基，培养得到种子液，
将所述种子液接种到含有γ-氨基丁酸的溶液中进行发酵。
进一步地，所述种子液的接种量为1-10％，即所述种子液与所述含有γ-氨基丁酸的溶液中的体积比为1-10％。
本发明制备方法中所用的酵母菌可以是本领域已知的各种酵母菌。在一个具体的实施方式中，所述酵母菌为上述酿酒酵母，即保藏编号为CCTCC NO:M 20211621的酿酒酵母(Saccharomyces cerevisiae)WJ-37。
在一个具体的实施方式中，所述酵母菌为编号为2.3854的酿酒酵母，该酿酒酵母可以从中国普通微生物菌种保藏管理中心(CGMCC)购买得到。
含有γ-氨基丁酸的溶液中的γ-氨基丁酸可以是各种来源的，比如可以是经过发酵、转化、纯化后得到的γ-氨基丁酸粉末产品，也可以是经过谷氨酸脱羧酶催化后得到的含有γ-氨基丁酸转化液。其中，谷氨酸脱羧酶可以是经过乳酸菌或者大肠杆菌发酵得到的。例如，当γ-氨基丁酸的来源是含有γ-氨基丁酸的转化液时，可以直接将含有γ-氨基丁酸的转化液用于配制含有γ-氨基丁酸的溶液。
其中，所述含有γ-氨基丁酸的溶液中γ-氨基丁酸的质量含量为10-100g/L。
所述含有γ-氨基丁酸的溶液还可以进一步包括用于酵母菌发酵的其他营养成分。在一个具体的实施方式中，所述含有γ-氨基丁酸的溶液还包括1～30g/L蛋白胨、1～20g/L酵母粉和1～20g/L葡萄糖。
在一个具体的实施方式中中，酵母菌在发酵条件为22-27℃的下，发酵至发酵液中不含葡萄糖。具体地，发酵方式可以为静置发酵。
在步骤二中，将发酵液中的菌体进行破碎的方式可以为搅拌、均质、超声、加热中的一种。
在步骤三中，所述辅料可以选自羟丙基-β-环糊精、透明质酸、海藻糖、麦芽糊精、聚谷氨酸、微晶纤维素、甘露醇、乳糖、岩藻糖中的一种或两种以上。在一个具体的实施方式中，所述辅料为羟丙基-β-环糊精和透明质酸的混合物，其中羟丙基-β-环糊精和透明质酸可以以任意比例混合，例如羟丙基-β-环糊精和透明质酸的质量比可以为10:1、9:1、8:1、7:1、6:1、5:1、4:1、3:1、2:1、1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10等。
在一个具体的实施方式中，辅料为羟丙基-β-环糊精和10KDa的透明质酸钠，羟丙基-β-环糊精和透明质酸钠的质量比为9:1。
喷雾干燥可以采用现有技术中已知的设备的进行，例如可以使用离心式喷雾干燥机。
本发明还提供一种由上述制备方法制备得到的低刺激性的γ-氨基丁酸产品。在一个具体的实施方式中，所述γ-氨基丁酸为上述制备方法制备得到的低刺激性的γ-氨基丁酸组合物，所述组合物包括γ-氨基丁酸和辅料。
进一步地，所述组合物中，所述γ-氨基丁酸的质量含量为10-50％，所述辅料的质量含量为40-80％。
其中，所述辅料可以选自羟丙基-β-环糊精、透明质酸、海藻糖、麦芽糊精、聚谷氨酸、微晶纤维素、甘露醇、乳糖、岩藻糖中的一种或两种以上。在一个具体的实施方式中，所述辅料为羟丙基-β-环糊精和透明质酸的混合物。
本文中的透明质酸可以涵盖透明质酸或其盐，例如透明质酸钠、透明质酸钙、透明质酸锌等。其中，透明质酸的分子量3KDa-30KDa。
进一步地，所述组合物通过上述的制备方法制备得到。
本发明还提供保藏编号为CCTCC NO:M 20211621的酿酒酵母或CGMCC NO:2.3854的酿酒酵母菌株在降低γ-氨基丁酸产品刺激性中的应用。
本发明的低刺激性的γ-氨基丁酸组合物的制备方法，将酵母菌接种到含有γ-氨基丁酸的溶液中进行发酵，并加入辅料进行干燥得到的组合物可以降低γ-氨基丁酸的刺激性。尤其是使用本发明的保藏编号为CCTCC NO:M 20211621的酿酒酵母，以及辅料羟丙基-β-环糊精和透明质酸，可以获得最佳的降低刺激性的效果。
实施例1酿酒酵母(Saccharomyces cerevisiae)WJ-37筛选及鉴定过程
以渤海海泥为原料，将渤海海泥用100质量倍无菌水分散，用涂布器将各溶液均匀涂布在含0.1mg/L氨苄青霉素钠的酵母菌固体培养基平板上，于25℃培养箱中静置培养1～2d，挑选菌落形态圆润光滑的单菌落，接种于酵母菌固体培养基试管斜面上，于25℃培养箱中静置培养1～2d，菌体长好后，于4℃冰箱保藏。其中，酵母菌固体培养基：蛋白胨20g/L，酵母粉10g/L，葡萄糖20g/L，琼脂20g/L，其余为水。
筛选得到的菌种命名为WJ-37。
将筛选得到的菌种送至上海生工生物工程(上海)股份有限公司进行基因组测序鉴定，结果显示菌种为酿酒酵母(Saccharomyces cerevisiae)。该菌株的26s rDNA基因序列测定结果如SEQ ID NO：1所示：
Figure PCTCN2022141654-appb-000002
实施例2酿酒酵母(Saccharomyces cerevisiae)WJ-37的形态学观察
酿酒酵母(Saccharomyces cerevisiae)WJ-37在酵母菌固体培养基平板上，菌落表面光滑、湿润、边缘整齐，质地均匀，颜色均一，如图1所示；该菌体为单细胞，成卵圆形，形态简单，出芽生殖，如图2所示。
实施例3
种子培养基：蛋白胨20g/L，酵母粉10g/L，葡萄糖20g/L，其余为水。
发酵培养基：蛋白胨10g/L，酵母粉5g/L，葡萄糖5g/L，γ-氨基丁酸(购买自华熙生物科技股份有限公司，产品编号为1020010002)60g/L，其余为水。
将酵母菌WJ-37单菌落接入种子培养基中，24℃静置培养24h，转接入发酵培养基中，接种量为发酵培养基的5％，24℃静置培养至无残糖，将发酵液85℃加热1h，经过0.22μm聚醚砜滤芯过滤，得到滤液。
将上述滤液中加入质量比为9％的羟丙基-β-环糊精和质量比为1％的10KDa透明质酸钠，混匀，使用离心式喷雾干燥机喷雾。喷雾干燥中采用的进风温度为180℃，出风温度为90℃，得到含GABA的粉末S1。
实施例4
种子培养基和发酵培养基，同实施例3。
将购自中国普通微生物菌种保藏管理中心(CGMCC)编号为2.3854(CGMCC NO:2.3854)的酵母菌单菌落接入种子培养基中，24℃静置培养24h，转接入发酵培养基中，接种量为发酵培养基的5％，24℃静置培养至无残糖，将发酵液85℃加热1h，经过0.22μm聚醚砜滤芯过滤，得到滤液。
将上述滤液加入质量比为9％的羟丙基-β-环糊精和质量比为1％的10KDa透明质酸钠，混匀，使用离心式喷雾干燥机喷雾。喷雾干燥中采用的进风温度为180℃，出风温度为90℃，得到含GABA的粉末S2。
实施例5
种子培养基和发酵培养基，同实施例3。
将酵母菌WJ-37单菌落接入种子培养基中，24℃静置培养24h，转接入发酵培养基中，接种量为发酵培养基的5％，24℃静置培养至无残糖，将发酵液85℃加热1h，经过0.22μm聚醚砜滤芯过滤，得到滤液。
将上述滤液加入质量比为10％的10KDa透明质酸钠，混匀，使用离心式 喷雾干燥机喷雾。喷雾干燥中采用的进风温度为180℃，出风温度为90℃，得到含GABA的粉末S3。
实施例6
种子培养基和发酵培养基，同实施例3。
将酵母菌WJ-37单菌落接入种子培养基中，24℃静置培养24h，转接入发酵培养基中，接种量为发酵培养基的5％，24℃静置培养至无残糖，将发酵液85℃加热1h，经过0.22μm聚醚砜滤芯过滤，得到滤液。
将上述滤液加入质量比为10％的羟丙基-β-环糊精，混匀，使用离心式喷雾干燥机喷雾。喷雾干燥中采用的进风温度为180℃，出风温度为90℃，得到含GABA的粉末S4。
实施例7
种子培养基和发酵培养基，同实施例3。
将酵母菌WJ-37单菌落接入种子培养基中，24℃静置培养24h，转接入发酵培养基中，接种量为发酵培养基的5％，24℃静置培养至无残糖，将发酵液85℃加热1h，经过0.22μm聚醚砜滤芯过滤，得到滤液。
将上述滤液加入质量比为10％的海藻糖，混匀，使用离心式喷雾干燥机喷雾。喷雾干燥中采用的进风温度为180℃，出风温度为90℃，得到含GABA的粉末S5。
实施例8
种子培养基和发酵培养基，同实施例3。
将酵母菌WJ-37单菌落接入种子培养基中，24℃静置培养24h，转接入发酵培养基中，接种量为发酵培养基的5％，24℃静置培养至无残糖，将发酵液85℃加热1h，经过0.22μm聚醚砜滤芯过滤，得到滤液S6。
对比例1
将60g/LGABA的水溶液85℃加热1h，经过0.22μm聚醚砜滤芯过滤，得到滤液。
将上述滤液加入质量比为的9％羟丙基-β-环糊精和1％的10kDa透明质酸钠，混匀，使用离心式喷雾干燥机喷雾。喷雾干燥中采用的进风温度为180℃，出风温度为90℃，得到含GABA的粉末C1。
对比例2
种子培养基和发酵培养基，同实施例3。
将酵母菌WJ-37单菌落接入种子培养基中，24℃静置培养至与实施例3发酵结束时菌浓相同，将酵母发酵滤液与发酵培养基混合后85℃加热1h，经过0.22μm聚醚砜滤芯过滤，得到滤液。
将上述溶液加入9％羟丙基-β-环糊精和1％10kDa透明质酸钠，混匀，使用离心式喷雾干燥机喷雾。喷雾干燥中采用的进风温度为180℃，出风温度为90℃，得到含GABA的粉末C2。
对比例3
种子培养基和发酵培养基，同实施例3.
将枯草芽孢杆菌NT-11(Bacillus subtilis)(保藏编号为：CCTCC NO:M 2019383，(在“一株枯草芽孢杆菌及其在生产γ-聚谷氨酸中的应用”专利中公开)单菌落接入种子培养基中，24℃静置培养至与实施例3发酵结束时菌浓相同，将NT-11发酵滤液与发酵培养基混合后85℃加热1h，经过0.22μm聚醚砜滤芯过滤，得到滤液。
将上述滤液加入9％羟丙基-β-环糊精和1％10kDa透明质酸钠，混匀，使用离心式喷雾干燥机喷雾。喷雾干燥中采用的进风温度为180℃，出风温度为90℃，得到含GABA的粉末C3。
对比例4
将60g/LGABA的水溶液85溶加热1h，经过0.22LG聚醚砜滤芯过滤，得到滤液C4。
上述各实施例和对比例的具体条件如表1所示。
表1
Figure PCTCN2022141654-appb-000003
Figure PCTCN2022141654-appb-000004
试验例1
将实施例8的样品S6和对比例4的样品C4，并将实施例3-7的样品S1-5与对比例1-3中的样品C1-3分别配成GABA含量为3％的水溶液，在人外眼角处滴1滴，涂抹均匀后，观测是否有刺痛感，评分标准如表2所示，试验人数为30人。
表2皮肤刺激反应标准
麻痒刺痛感  
无任何不适感 0
轻微麻痒感 1
严重麻痒感 2
轻微刺痛感 3
严重刺痛感 4
严重刺痛灼烧感 5
结果如表3所示，通过S1、S6和C1、C4的结果可以看出，使用酵母菌发酵可以有效降低GABA的刺激性，而通过加入辅料喷雾干燥后可进一步降低GABA的刺激性。从S1、S3、S4、S5，同时可以看出，不同的辅料对降低GABA刺激性的作用也不相同，其中羟丙基-β-环糊精和HA作用较好，海藻糖作用稍小一些。从样品S1、S2和C3可以看出，通过不同菌种发酵，作用效果不同，其中酵母菌WJ-37发酵后效果最好。从样品S1和C2的结果来看，使用酵母菌进行二次发酵，比单纯的将酵母菌溶胞产物加入GABA发酵液中，对于缓解GABA带来的刺激性效果要好，可能是不光酵母菌溶胞产物中有一些舒缓刺激性的物质，酵母菌在生长过程中还能消耗掉一些带来刺激性的杂质。
表3
  皮肤刺激性评分
S1 1.42±0.37
S2 2.01±0.65
S3 1.59±0.68
S4 1.48±0.32
S5 1.95±0.76
S6 1.98±0.76
C1 2.65±0.54
C2 2.09±0.39
C3 2.59±0.78
C4 3.01±0.25

Claims (9)

  1. 一种低刺激性的γ-氨基丁酸产品的制备方法，其特征在于，所述制备方法包括以下步骤：
    将酵母菌接种到含有γ-氨基丁酸的溶液中进行发酵；
    发酵结束后将发酵液中的菌体进行破碎、过滤，得到含有γ-氨基丁酸的滤液。
  2. 根据权利要求1所述的制备方法，其特征在于，所述制备方法还包括以下步骤，将所述滤液加入辅料进行喷雾干燥，得到低刺激性的γ-氨基丁酸组合物。
  3. 根据权利要求1所述的制备方法，其特征在于，所述酵母菌选自保藏编号为CCTCC NO:M 20211621的酿酒酵母或CGMCC NO:2.3854的酿酒酵母。
  4. 根据权利要求1所述的制备方法，其特征在于，所述含有γ-氨基丁酸的溶液中的γ-氨基丁酸通过酶催化得到，进一步优选地，所述菌体破碎方式包括搅拌、均质、超声、加热中的至少一种，更进一步优选地，所述发酵条件为22-27℃下，发酵至发酵液中不含葡萄糖，更进一步优选地，所述含有γ-氨基丁酸的溶液还包括1～30g/L蛋白胨、1～20g/L酵母粉和1～20g/L葡萄糖。
  5. 根据权利要求2所述的制备方法，其特征在于，所述辅料选自羟丙基-β-环糊精、透明质酸或其盐、海藻糖、麦芽糊精、聚谷氨酸、微晶纤维素、甘露醇、乳糖、岩藻糖中的一种或两种以上，优选为羟丙基-β-环糊精和透明质酸或其盐的混合物，优选地，所述辅料与所述滤液的质量比为1:2.5-50。
  6. 一种低刺激性的γ-氨基丁酸产品，通过权利要求1-5中任一项所述的制备方法制备得到。
  7. 根据权利要求6所述的γ-氨基丁酸产品，其特征在于，所述γ-氨基丁酸为通过权利要求2所述的制备方法制备得到的γ述氨基丁酸组合物，在所述组合物中，所述γ-氨基丁酸的质量含量为10-50％，所述辅料的质量含量为40-80％。
  8. 一种酿酒酵母(Saccharomyces cerevisiae)，其保藏编号为CCTCC NO:M 20211621。
  9. 保藏编号为CCTCC NO:M 20211621的酿酒酵母或CGMCC NO:2.3854的酿酒酵母菌株在降低γ-氨基丁酸产品刺激性中的应用。
PCT/CN2022/141654 2021-12-28 2022-12-23 一种低刺激性的γ-氨基丁酸产品及其制备方法和降低刺激性的应用 WO2023125330A1 (zh)

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