CN116459203A - 一种双歧杆菌/糙米发酵产物溶胞物的制备方法及其应用 - Google Patents
一种双歧杆菌/糙米发酵产物溶胞物的制备方法及其应用 Download PDFInfo
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- CN116459203A CN116459203A CN202310598475.4A CN202310598475A CN116459203A CN 116459203 A CN116459203 A CN 116459203A CN 202310598475 A CN202310598475 A CN 202310598475A CN 116459203 A CN116459203 A CN 116459203A
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- brown rice
- bifidobacterium
- lysate
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9794—Liliopsida [monocotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- 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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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- 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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
本发明属于微生物化妆品原料生产技术领域,本发明提供了一种双歧杆菌/糙米发酵产物溶胞物的制备方法及其应用,本发明以糙米水解液作为双歧杆菌的糙米发酵培养基进行厌氧发酵。双歧杆菌/糙米发酵液经过破碎、除菌处理后得到溶胞物原液。本发明制备的双歧杆菌/糙米发酵产物溶胞物首次在化妆品原料领域将双歧杆菌溶胞物与糙米精华相结合,以较为温和的方式对溶胞物除菌,原料中富含γ‑氨基丁酸等多种活性成分,不仅具有显著的DPPH自由基清除效果以及酪氨酸酶活抑制效果,而且还规避了传统酵母发酵糙米水的方式所带来的皮肤刺激性酒精残留问题。
Description
技术领域
本发明属于微生物化妆品原料生产技术领域,具体涉及一种微生物发酵产物溶胞物的制备方法。
背景技术
双歧杆菌是肠道益生菌之一,具有较强的肠道定植能力,其代谢过程可产生共轭亚油酸(conjugated linoleic acid,CLA)、胞外多糖(exopolysaccharides,EPS)、短链脂肪酸(indole-3-lactic acid,ILA)以及吲哚-3-乳酸(indole-3-lactic acid,ILA)等多种活性物质,因此双歧杆菌的应用价值极高。双歧杆菌也被广泛用于化妆品领域,二裂酵母发酵产物溶胞物就是由双歧杆菌经培养、灭活及破壁得到的代谢产物、细胞质片段、细胞壁组分及多糖复合体。二裂酵母发酵产物溶胞物作为一种天然活性化妆品原料,具有较强的促进DNA修复、预防皮肤光老化、加强角质层代谢能力。然而,双歧杆菌的培养基活性成分较少且种类单一,只能起到支持菌体生长的作用,选择可被双歧杆菌利用且活性成分丰富的培养基成分可期盼成为双歧杆菌发酵类化妆品原料的优化方向。
糙米是常用发酵营养物,用于化妆品发酵类功效原料的制备,其含有大量的生物活性化合物,如γ-氨基丁酸、维生素、植物甾醇、酚酸、α-生育酚等,这些成分均高度集中在糙米的麸皮层中。γ-氨基丁酸具有抗氧化、清除自由基、抗肿瘤、增强免疫力、延缓衰老、降胆固醇、降血脂、降血糖等功效,是糙米中被广为关注的活性成分。糙米在发芽的过程中利用自身酶的转化能产生更多的γ-氨基丁酸等活性成分,因此糙米通常需经过浸泡发芽来富集更多的γ-氨基丁酸。然而,直接从糙米中提取γ-氨基丁酸不仅产量低,且工艺复杂,还容易造成糙米中其余活性成分的浪费。如何使用温和手段使糙米中活性成分释放成为能否成功利用糙米的关键,微生物发酵法正好符合了这一需求。
目前国内米类原料主要应用场景主要在食品行业,其在化妆品领域的应用主要有水解大米提取物、酵母/大米发酵产物滤液、酵母/糙米发酵产物滤液、糙米水等。由此可见微生物发酵已成为制备米类化妆品原料的主流趋势。目前最多的是酵母类的发酵产品。然而,酵母类的发酵糙米水的缺陷是使用酵母作为糙米的发酵菌种,会造成发酵产物酒精含量过高的问题,从而对使用者的皮肤产生刺激性。
发明内容
为了解决现有米类化妆品原料使以酵母为发酵菌种发酵糙米或精米导致的酒精残留问题,本发明提供了以发芽糙米精华为发酵基底的双歧杆菌发酵产物溶胞物的制备方法。发芽糙米经过α-淀粉酶和葡糖淀粉酶水解后形成大量单糖可为双歧杆菌提供生长所需的碳源,双歧杆菌生长代谢可促进糙米中的γ-氨基丁酸等活性物的释放,且无酒精代谢物的产生,制备出的双歧杆菌/糙米发酵产物溶胞物从根源上解决了酵母发酵的酒精残留问题,双歧杆菌发酵产物溶胞物作为一种被广泛认可的功能性化妆品原料,将其与糙米精华相结合,其营养密度和生理活性将大幅度提高,功效远远超过了糙米本身且该原料富含γ-氨基丁酸,具有突出的DPPH自由基清除效果和酪氨酸酶活抑制效果。其具体技术方案如下:
一种双歧杆菌/糙米发酵产物溶胞物的制备方法,包括如下步骤:
(1)将糙米清洗浸泡在2-3倍质量的水中催芽24-72h,60℃-95℃烘干5-10h,粉碎过100-200目筛网;
(2)糙米粉加水混为匀浆,其中糙米粉质量分数为1-3%,加入1:1质量分数α-淀粉酶和葡糖淀粉酶酶解;95℃灭酶活5min;
(3)将冷却的糙米匀浆液离心,保留上清液得到澄清的糙米水解液;
(4)将所述糙米水解液补充纯水至还原糖含量为20g/L后,再向其中额外添加氮源、缓冲盐与微量元素,并高压蒸汽灭菌;制得糙米发酵培养基;
(5)将糙米发酵培养基接种2%-10%的对数期双歧杆菌,厌氧发酵24-72h,发酵终点为双歧杆菌的生长密度达到最高值且不再变化;
(6)将发酵液低温高压破碎,冷水机控温2℃-8℃,反复破碎3次,收集破碎液;将破碎液保温60℃/30min后,离心收集上清液,上清液过滤除菌后得到最终产物双歧杆菌/糙米发酵产物溶胞物。
优选地,所述双歧杆菌株包括长双歧杆菌、青春双歧杆菌、婴儿双歧杆菌和短双歧杆菌中的一种或几种。
优选地,步骤(2)中加入0.2-1%质量分数的α-淀粉酶和0.2-1%质量分数的葡糖淀粉酶。
优选地,步骤(2)中酶解时间为3-12h,酶解温度为50-65℃,酶解pH为5.0-7.0,以还原糖含量不再变化的酶解时间作为酶解终点。
优选地,步骤(4)中向1L糙米水解液添加20g酵母提取物、2.0gK2HPO4、5.0g醋酸钠、2.0g柠檬酸三铵、0.25g MgSO4·7H2O、0.05g/MnSO4·H2O、1.0g吐温80、1g半胱氨酸盐酸盐。
优选地,步骤(5)中厌氧发酵温度为35~40℃。
优选地,步骤(6)中具体压力为1500Mpa-1900Mpa。
优选地,步骤(6)中上清液采用0.22μm聚醚砜滤芯过滤除菌。
一种所述制备方法制备的双歧杆菌/糙米发酵产物溶胞物的应用,将其作为化妆品原料。
有益效果
一、本发明首次将双歧杆菌与糙米精华相结合,直接将菌体破碎后的胞内产物释放到糙米发酵液中,将双歧杆菌溶胞物与发酵后的糙米精华相融合,促进了糙米中γ-氨基丁酸活性物的释放。
二、本发明所制备的双歧杆菌/糙米发酵产溶胞物具有较好的DPPH自由基清除效果。
三、本发明所制备的双歧杆菌/糙米发酵产溶胞物具有较好的酪氨酸酶活抑制效果。
四、本发明采用温和灭菌方式,保温60℃/30min后进行过滤除菌,既确保了双歧杆菌/糙米发酵产物溶胞物的稳定性,又达到了除菌的目的。
具体实施方式
下面结合实施例对本发明作进一步的说明。
实施例1
本实施例提供一种长双歧杆菌/糙米发酵产物溶胞物的制备方法,包括如下步骤:
(1)取600g糙米浸泡在1.2L超纯水中,25℃催芽40h,65℃烘干5h,使用高速研磨机将烘干后的糙米粉碎后过200目筛网;
(2)在500g糙米粉中加入16.5L纯水混为匀浆,加入0.3%的α-淀粉酶和0.3%的葡糖淀粉酶,55℃水浴搅拌,6h后每隔1h使用还原糖含量检测试剂盒进行还原糖含量的检测,当还原糖含量不再发生变化时停止酶解,最终还原糖含量为29g/L。将糙米酶解液升温至95℃,保温5min灭酶活;
(3)将冷却的糙米酶解液9000rpm/min离心10min,保留上清液得到16L澄清的糙米水解液,加入7.2L纯水并混匀得到还原糖含量为20g/L的糙米水解液;
(4)向1L糙米水解液中添加20g酵母提取物、2.0g K2HPO4、5.0g醋酸钠、2.0g柠檬酸三铵、0.25g MgSO4·7H2O、0.05g MnSO4·H2O、1.0g吐温80、1g半胱氨酸盐酸盐,并于115℃高压蒸汽灭菌20min;得到双歧杆菌的糙米发酵培养基;
(5)接种质量分数为3%的对数期长双歧杆菌菌液于所述糙米发酵培养基中,于37℃的厌氧工作站中厌氧发酵,当长双歧杆菌发酵液的OD600达到2.2时终止发酵,共发酵36h;
(6)收集15L发酵液,在1550Mpa高压、3℃条件下反复破碎3次,收集破碎液;保温60℃/30min后,8000rpm/min离心10min收集上清液14.5L,上清液采用0.22μm聚醚砜滤芯过滤除菌后得到双歧杆菌/糙米发酵产物溶胞物S1。
实施例2
本实施例提供一种青春双歧杆菌/糙米发酵产物溶胞物的制备方法,包括如下步骤:
(1)取300g糙米浸泡在0.7L超纯水中,25℃催芽48h,95℃烘干10h,使用高速研磨机将烘干后的糙米粉碎后过100目筛网;
(2)在150g糙米粉中加入8.25L纯水混为匀浆,加入1%的α-淀粉酶和1%的葡糖淀粉酶,65℃水浴搅拌,9h后每隔1h使用还原糖含量检测试剂盒进行还原糖含量的检测,当还原糖含量不再发生变化时停止酶解,最终还原糖含量为27g/L。将糙米酶解液升温至95℃,保温5min灭酶活;
(3)将冷却的糙米酶解液9000rpm/min离心10min,保留上清液得到8L澄清的糙米水解液,加入3.6L纯水并混匀得到还原糖含量为20g/L的糙米水解液;
(4)向1L糙米水解液中添加20g酵母提取物、2.0gK2HPO4、5.0g醋酸钠、2.0g柠檬酸三铵、0.25g MgSO4·7H2O、0.05g MnSO4·H2O、1.0g吐温80、1g半胱氨酸盐酸盐,并于115℃高压蒸汽灭菌20min;得到双歧杆菌的糙米发酵培养基;
(5)接种质量分数为9%的对数期青春双歧杆菌菌液于糙米发酵培养基中,于37℃的厌氧工作站中厌氧发酵,当青春双歧杆菌发酵液的OD600达到2.2时终止发酵,共发酵72h;
(6)收集15L发酵液,在1900Mpa高压、7℃条件下反复破碎3次,收集破碎液。保温60℃/30min后,5000rpm/min离心10min收集上清液14.5L,上清液采用0.22μm聚醚砜滤芯过滤除菌后得到双歧杆菌/糙米发酵产物溶胞物S2。
对比例1
本对比例以相同工艺制备了市面上常见的酵母/糙米发酵产物滤液,具体制备方法步骤如下:
(1)糙米水解液的制备步骤如同实施例1;
(2)向糙米水解液中添加10g/L酵母提取物、20g/L蛋白胨,并于115℃高压蒸汽灭菌15min,得到酵母的糙米发酵培养基;
(3)接种质量分数为5%的对数期酿酒酵母(C1)、扣囊复膜孢酵母(C2)、解脂复膜孢酵母(C3)、产朊假丝酵母(C4)于糙米发酵培养基中,于30℃,220rpm培养至OD600稳定不变;
(4)各收集1L发酵液,在1800Mpa高压、4℃条件下反复破碎3次,收集破碎液。保温60℃/30min后,8000rpm/min离心10min收集上清液950ml,上清液采用0.22μm聚醚砜滤芯过滤除菌后得到酵母/糙米发酵产物溶胞物C1、C2、C3、C4,常温或4℃保藏。
对比例2
本对比例在制备工艺相同的基础上,使用传统双歧杆菌培养基MRS发酵培养制备了双歧杆菌发酵产物溶胞物。发酵菌种为长双歧杆菌CICC6190,MRS培养基配方:20g/L葡萄糖、20g/L酵母提取物、2.0g/L K2HPO4、5.0g/L醋酸钠、2.0g/L柠檬酸三铵、0.25g/L MgSO4·7H2O、0.05g/L MnSO4·H2O、1.0g/L吐温80、1g/L半胱氨酸盐酸盐,并于115℃高压蒸汽灭菌20min。其余工艺同实施例1,得到双歧杆菌发酵产物溶胞物C5。
对比例3
本对比例将发酵菌种改为青春双歧杆菌,培养基改为MRS培养基,其余工艺同实施例1不变,得到双歧杆菌发酵产物溶胞物C6。
试验例
1、γ-氨基丁酸含量的测定
本实验采用γ-氨基丁酸(GABA)比色法试剂盒进行样品中γ-氨基丁酸含量的测定,具体结果如表1所示。
γ-氨基丁酸含量(g/L) | |
S1 | 1.71 |
S2 | 2.25 |
C1 | 0.84 |
C2 | 1.19 |
C3 | 1.15 |
C4 | 0.76 |
表1不同样品γ-氨基丁酸含量的测定
由表1可得,S1、S2的γ-氨基丁酸含量显著高于C1、C2、C3、C4,说明双歧杆菌发酵糙米释放的γ-氨基丁酸含量显著高于酵母发酵糙米所释放的γ-氨基丁酸,因此由双歧杆菌进行糙米的发酵是更好的促进γ-氨基丁酸释放的方法。
2、DPPH自由基清除效果(针对对比例1)
配置0.02mM DPPH溶液:准确称量DPPH 40mg,将其加入到500ml无水乙醇中定容,避光4℃低温保存。
样品准备:使用纯化水分别将S1、S2、C1、C2、C3、C4稀释为体积浓度为10%、2%的溶液。
编号 | DPPH(μl) | 待测样品(μl) | 无水乙醇(μl) |
A1 | 500 | 500 | 0 |
A2(样品背景) | 0 | 500 | 500 |
A3(空白对照) | 500 | 0 | 500 |
表2清除DPPH自由基反应体系
反应体系:按表2添加各组反应液,室温避光放置30min,取各组样品与517nm处测定吸光值,DPPH自由基清除率计算公式如下:
结果如表3所示,从表中可以看出实例S1、S2的自由基清除效果是显著优于对比例C1、C2、C3、C4的。该结果说明了双歧杆菌的糙米发酵代谢产物的抗氧化能力优于酵母菌的糙米发酵产物溶胞物。
2%原液 | 10%原液 | |
S1 | 45.56% | 95.68% |
S2 | 43.16% | 94.51% |
C1 | 8.09% | 26.05% |
C2 | 8.25% | 38.16% |
C3 | 7.28% | 38.17% |
C4 | 9.15% | 32.34% |
表3各样品的DPPH自由基清除效果。
3、酪氨酸酶活抑制效果(针对对比例1)
PBS(pH=6.8)缓冲液的配置:分别配置0.2M的磷酸二氢钠和0.2M的磷酸氢二钠,取51ml的磷酸二氢钠和49ml的磷酸氢二钠混合即得到100ml pH=6.8的PBS(0.2M)溶液。
0.03%多巴溶液的配置:取0.03g多巴粉末溶于100ml纯化水中即得0.03%多巴溶液。
酪氨酸酶溶液:用PBS溶液配置120U/ml的酪氨酸酶溶液。
1%熊果苷:本实验以1%熊果苷作为阳性对照,取0.1g熊果苷粉末溶于10ml纯化水中即可得到1%熊果苷溶液。
表4酪氨酸酶活抑制反应体系
反应体系:按照表4进行混合反应后,于475nm处测定吸光度,酪氨酸酶活抑制计算公式:D1(待测组)=B1-B0、D2(阴性对照)=A1-A0、D3(阳性对照)=C1-C0
结果如表5所示,从表中可以看出实例S1、S2的酪氨酸酶活抑制效果是显著优于对比例C1、C2、C3、C4的,其中S2的酪氨酸酶活抑制效果尤其突出。
这可能是由于S1、S2中富集了较多的γ-氨基丁酸,从而起到了较好的美白效果。
10%原液 | |
S1 | 84.32% |
S2 | 90.96% |
C1 | 72.87% |
C2 | -43.82% |
C3 | -71.56% |
C4 | 40.89% |
表5各样品的酪氨酸酶活抑制率。
4、DPPH自由基清除效果(针对对比例2、对比例3)
试验方法同试验例1,比较糙米发酵培养基与MRS发酵培养基发酵双歧杆菌所制备出的原液的自由基清除效果,结果如表6所示。
2%原液体积浓度 | |
S1 | 46.56% |
S2 | 43.16% |
C5 | 30.79% |
C6 | 36.14% |
表6不同培养基制备的发酵产物溶胞物DPPH自由基清除效果
如表6所示,可知S1、S2的自由基清除效果是高于对比例C5、C6的。该结果说明了使用糙米精华作为发酵培养基确实有提高双歧杆菌发酵产物溶胞物抗氧化能力的效果。
5、酪氨酸酶活抑制效果(针对对比例2、对比例3)
试验方法同试验例2,比较糙米发酵培养基与MRS发酵培养基发酵双歧杆菌所制备出的原液的酪氨酸酶活抑制效果,结果如表7所示。
10%原液体积浓度 | |
S1 | 90.96% |
S2 | 84.32% |
C5 | 33.82% |
C6 | 44.93% |
表7不同培养基制备的发酵产物溶胞物酪氨酸酶活抑制效果。
如表7所示,可知S1、S2的酪氨酸酶活抑制效果显著高于对比例C5、C6。该结果说明了以糙米精华作为双歧杆菌的发酵培养基是S1、S2酪氨酸酶活抑制效果较高的主要原因。
6、稳定性实验
将S1、S2密封于5ml西林瓶中,分别放置于-20℃、4℃、45℃的稳定性培养箱中,每日进行气味、颜色、杂质析出、分层等性状的记录,观察期为40天,观察结果如表8、表9。
表8S1的外观形状稳定性测试
表9S2的外观形状稳定性测试
由表8、表9可知,45℃的条件下:不加防腐剂的S1,超过一个月容易发生颜色变化;不加防腐剂的S2,在第30天会有固形物析出以及颜色变深的变化。而在其他温度条件下,S1、S2未发生任何性状的变化。由此得知该原液可以室温保存一定的时间,低温可延长其稳定保存时间。
实施例3
如前述长双歧杆菌/糙米发酵产物溶胞物的制备方法制备的双歧杆菌/糙米发酵产物溶胞物S1或青春双歧杆菌/糙米发酵产物溶胞物的制备方法制备的双歧杆菌/糙米发酵产物溶胞物S2,作为化妆品原料使用。
Claims (9)
1.一种双歧杆菌/糙米发酵产物溶胞物的制备方法,包括如下步骤:
(1)将糙米清洗浸泡在2-3倍质量的水中催芽24-72 h,60℃-95℃烘干5-10 h,粉碎过100-200目筛网;
(2)糙米粉加水混为匀浆,其中糙米粉质量份数为1-3%,加入1:1质量份数α-淀粉酶和葡糖淀粉酶酶解;95℃灭酶活5min;
(3)将冷却的糙米匀浆液离心,保留上清液得到澄清的糙米水解液;
(4)将所述糙米水解液补充纯水至还原糖含量为20g/L后,再向其中额外添加氮源、缓冲盐与微量元素,并高压蒸汽灭菌;制得糙米发酵培养基;
(5)将糙米发酵培养基接种2%-10%的对数期双歧杆菌,厌氧发酵24-72h,发酵终点为双歧杆菌的生长密度达到最高值且不再变化;
(6)将发酵液低温高压破碎,冷水机控温2℃-8℃,反复破碎3次,收集破碎液;将破碎液保温60℃/30min后,离心收集上清液,上清液过滤除菌后得到最终产物双歧杆菌/糙米发酵产物溶胞物。
2.根据权利要求1所述的双歧杆菌/糙米发酵产物溶胞物的制备方法,其特征在于:
所述双歧杆菌株包括长双歧杆菌、青春双歧杆菌、婴儿双歧杆菌和短双歧杆菌中的一种或几种。
3.根据权利要求1所述的双歧杆菌/糙米发酵产物溶胞物的制备方法,其特征在于:步骤(2)中加入0.2-1%质量分数的α-淀粉酶和0.2-1%质量分数的葡糖淀粉酶。
4.根据权利要求1所述的双歧杆菌/糙米发酵产物溶胞物的制备方法,其特征在于:步骤(2)中酶解时间为3-12h,酶解温度为50-65℃,酶解pH为5.0-7.0,以还原糖含量不再变化的酶解时间作为酶解终点。
5.根据权利要求1所述的双歧杆菌/糙米发酵产物溶胞物的制备方法,其特征在于:步骤(4)中向1L糙米水解液添加20g酵母提取物、2.0gK2HPO4、5.0g醋酸钠、2.0g柠檬酸三铵、0.25g MgSO4·7H2O、0.05g MnSO4·H2O、1.0g吐温80、1g 半胱氨酸盐酸盐。
6.根据权利要求1所述的双歧杆菌/糙米发酵产物溶胞物的制备方法,其特征在于:步骤(5)中厌氧发酵温度为35~40℃。
7.根据权利要求1所述的双歧杆菌/糙米发酵产物溶胞物的制备方法,其特征在于:步骤(6)中具体压力为1500Mpa-1900Mpa。
8.根据权利要求1所述的双歧杆菌/糙米发酵产物溶胞物的制备方法,其特征在于:步骤(6)中上清液采用0.22μm聚醚砜滤芯过滤除菌。
9.一种根据权利要求1-8之一所述制备方法制备的双歧杆菌/糙米发酵产物溶胞物的应用,其特征在于:将其作为化妆品原料。
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