CN110846346B - 富含Sn-2位DHA的微生物油脂及其制备方法和应用 - Google Patents
富含Sn-2位DHA的微生物油脂及其制备方法和应用 Download PDFInfo
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- CN110846346B CN110846346B CN201911175787.4A CN201911175787A CN110846346B CN 110846346 B CN110846346 B CN 110846346B CN 201911175787 A CN201911175787 A CN 201911175787A CN 110846346 B CN110846346 B CN 110846346B
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- microbial oil
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- 241000233671 Schizochytrium Species 0.000 claims abstract description 25
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- 125000005457 triglyceride group Chemical group 0.000 claims abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 10
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 claims description 3
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- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 3
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
- A23L29/04—Fatty acids or derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
-
- 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
- C12N1/12—Unicellular algae; Culture media therefor
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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
- C12N1/12—Unicellular algae; Culture media therefor
- C12N1/125—Unicellular algae isolates
-
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Abstract
本发明涉及微生物技术领域,公开了一种富含Sn‑2位DHA的微生物油脂及其制备方法和应用。所述微生物油脂中甘油三酯Sn‑2位上DHA的比例不低于23%,所述微生物油脂的制备方法包括将裂殖壶菌菌株接种到发酵培养基中发酵制得,所述裂殖壶菌菌株为保藏编号为GDMCC No.60733的裂殖壶菌(Schizochytrium)。该微生物油脂中甘油三酯Sn‑2位上DHA的比例显著高于甘油三酯Sn‑1、Sn‑3位上DHA的比例,从而使该微生物油脂中DHA的人体吸收率明显高于常规菌株生产的微生物油脂,促进人体对功能性脂肪酸DHA的吸收与利用。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种富含Sn-2位DHA的微生物油脂及其制备方法和应用
背景技术
二十二碳六烯酸(DHA)是大脑和眼部中的一种主要结构性脂肪酸,占大脑中n-3脂肪酸的97%,在眼部中的比例达93%。研究表明与随机结构的甘油三酯相比,DHA在Sn2位的特殊结构甘油三酯更利于人体吸收。另有研究指出进食特殊结构油脂会使DHA在大脑中含量最高,而进食随机结构的油脂,DHA则在肝脏中含量最高,这表明进食不同结构的甘油三酯会导致脂肪酸代谢的差异,即脂肪酸在Sn-2位比在Sn-1、Sn-3位的吸收利用更有效。胰腺脂肪酶这一主要的脂肪消化酶,通过吸附在油水界面来发挥作用,同时它具有专一的酯键分解作用,即只专注水解Sn-1、Sn-3的酯键,因此,甘油三酯结构的油脂经胰腺脂肪酶分解后,Sn-1、Sn-3位脂肪酸形成游离的脂肪酸、Sn2位脂肪酸形成单甘油酯,而呈游离态的脂肪酸难以渗入胆盐微胶粒被人体吸收,从而容易与肠道中钙、镁离子结合形成不溶性的皂盐损失掉,而Sn2位脂肪酸形成单甘油酯易渗入胆盐微胶粒被人体吸收,所以Sn2位脂肪酸比Sn-1、Sn-3位脂肪酸具有更高的人体吸收率。
微生物油脂作为DHA的主要来源之一,随着人们保健意识的加强,已被广泛应用于婴幼儿食品、保健食品中,其营养功效愈来愈得到公众的认识、认可,而微生物油脂中DHA的吸收率也愈来愈为公众所关注。微生物油脂中90%以上的脂肪酸是甘油三酯结构形式,而目前微生物来源的DHA油脂采用吾肯氏菌属、裂殖壶菌属、破囊壶菌目、隐甲藻属、酵母等微生物发酵所获取,得到的甘油三酯型DHA的甘油骨架上的分布比例是Sn2位的含量远远低于Sn-1、Sn-3位的含量,大量Sn-1、Sn-3位DHA在人体消化过程中形成皂盐而损失,微生物油脂的保健功效受到了限制。
发明内容
本发明的目的是为了克服现有技术存在的微生物油脂中甘油三酯Sn-1、Sn-3位脂肪酸的比例高导致人体对脂肪酸的吸收率低的问题,提供一种富含Sn-2位DHA的微生物油脂及其制备方法和应用,该微生物油脂通过裂殖壶菌发酵生产所得,富含DHA且油脂中甘油三酯Sn2位上DHA的比例不低于23%,有效提高DHA的人体吸收率。
为了实现上述目的,本发明提供了一种富含Sn-2位DHA的微生物油脂,所述微生物油脂中甘油三酯Sn-2位上DHA的比例不低于23%。
本发明进一步提供了上述微生物油脂的制备方法,该方法包括将裂殖壶菌菌株接种到发酵培养基中发酵制得,所述裂殖壶菌菌株为保藏编号为GDMCC No.60733的裂殖壶菌(Schizochytrium)。
优选地,所述发酵培养基包括碳源、氮源、无机盐、微量元素和维生素,发酵的条件包括pH为6-7.5、温度为27-31℃、时间为4-8天、通气比为0.45-1.1VVM、谷氨酸浓度为5-15g/L、碳源浓度为5-70g/L。
优选地,所述裂殖壶菌菌株经活化后制作成摇瓶种子液接种到种子培养基中培养,然后再转接入发酵培养基中发酵培养。
优选地,所述活化的过程包括将所述裂殖壶菌菌株的冷冻甘油管菌种解冻、接种到种子培养基中进行扩大培养。
优选地,所述菌株活化培养的过程包括:将所述裂殖壶菌菌株的冷冻甘油管菌种解冻,用无菌吸管将所述甘油管菌种接种到已灭菌的种子培养基中,在温度为27.5-28.5℃、转速为150-200r/min的条件下培养48-72h,所述种子培养基中碳源为30-50g/L、氮源为25-45g/L、无机盐为25-40g/L、微量元素为0.015-0.025g/L、维生素为0.01-0.02g/L。
优选地,所述碳源为葡萄糖或/和蔗糖,所述氮源为谷氨酸钠、酵母粉、酵母浸膏中的一种或多种,所述无机盐为钙盐、磷酸盐、钾盐、钠盐、镁盐、铵盐中的一种或多种,所述微量元素为镍、铜、钼、钴、锌、铁、锰中的一种或多种,所述维生素为维生素B1、维生素B12、维生素B6、泛酸钙、生物素中的一种或多种。
优选地,该方法还包括对所述发酵的产物进行破壁、萃取得到所述发酵的产物中的油脂产物。
本发明进一步提供了由上述的方法制备得到的微生物油脂,所述微生物油脂中甘油三酯Sn-2位上DHA的比例不低于23%,且甘油三酯型DHA含量不低于38%。
本发明进一步提供了由上述的方法制备得到的微生物油脂在食品中的应用,所述食品优选为婴幼儿配方食品、保健食品以及健康食品。
通过上述技术方案,本发明提供的利用裂殖壶菌菌株发酵生产的微生物油脂,不仅富含DHA,而且甘油三酯Sn2位上DHA的比例显著高于甘油三酯Sn-1、Sn-3位上DHA的比例,提高人体对该微生物油脂中DHA的吸收率,从而有效促进人体对功能保健因子DHA的吸收与利用。
本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。
生物保藏
本发明的菌株如下:
裂殖壶菌(Schizochytrium),于2019年8月8日被保藏在广东省微生物菌种保藏中心(地址:广州市先烈中路100号大院59号楼5楼,广东省微生物研究所,邮政编码:510070)(保藏单位的缩写为GDMCC),保藏编号为GDMCC No.60733。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
第一方面,本发明提供了一种富含Sn-2位DHA的微生物油脂,所述微生物油脂中甘油三酯Sn-2位上DHA的比例不低于23%。
第二方面,本发明提供了上述微生物油脂的制备方法,该方法包括将裂殖壶菌菌株接种到发酵培养基中发酵制得,所述裂殖壶菌菌株为保藏编号为GDMCC No.60733裂殖壶菌(Schizochytrium)。
本发明提供的裂殖壶菌经过发酵产生富含DHA的微生物油脂,所述发酵的方法没有特别的要求,只要是能使所述裂殖壶菌增殖即可。
所述裂殖壶菌发酵的pH、温度、时间、通气比、碳源和氮源没有特别的要求,可以是获得裂殖壶菌发酵产物常用的选择,为了获得较多的发酵产物,优选地,所述发酵培养基包括碳源、氮源、无机盐、微量元素和维生素,发酵的条件包括pH为6-7.5、温度为27-31℃、时间为4-8天、通气比为0.45-1.1VVM、谷氨酸浓度为5-15g/L、碳源浓度为5-70g/L。
其中,所述裂殖壶菌接种到发酵培养基中发酵的过程中,在发酵培养基中投加部分碳源、部分氮源,在发酵过程中根据碳氮比的调控工艺不断流加碳源、氮源,在接近发酵终点时不再补充碳源直至培养基中残糖含量为0,以此来调控碳氮比形成营养缺陷型营养条件,以确保裂殖壶菌发酵产油量的水平。
为了获得较多的发酵产物,所述裂殖壶菌菌株经活化后制作成摇瓶种子液接种到所述种子培养基中培养,然后再转接入发酵培养基中发酵培养。
更优选地,所述活化的过程包括将所述裂殖壶菌菌株的冷冻甘油管菌种解冻、接种到种子培养基中进行扩大培养。
进一步优选地,所述菌株活化培养的过程包括:将所述裂殖壶菌菌株的冷冻甘油管菌种解冻,用无菌吸管将所述甘油管菌种接种到已灭菌的种子培养基中,,在温度为27.5-28.5℃、转速为150-200r/min的条件下培养48-72h,所述种子培养基中碳源为30-50g/L、氮源为25-45g/L、无机盐为25-40g/L、微量元素为0.015-0.025g/L、维生素为0.01-0.02g/L。具体地,所述甘油管菌种的接种量按一支甘油管转接4-6个三角瓶的方式,三角瓶的规格为500mL,每个三角瓶中的种子培养基体积为200-300mL。
本发明中所述碳源可以为葡萄糖、蔗糖或其他任何可以提供碳源的物质,氮源可以为谷氨酸钠、酵母粉、酵母浸膏或其他任何可以提供氮源的物质。优选地,所述碳源为葡萄糖或/和蔗糖,所述氮源为谷氨酸钠、酵母粉、酵母浸膏中的一种或多种,所述无机盐为钙盐、磷酸盐、钾盐、钠盐、镁盐、铵盐中的一种或多种,所述微量元素为镍、铜、钼、钴、锌、铁、锰中的一种或多种,所述维生素为维生素B1、维生素B12、维生素B6、泛酸钙、生物素中的一种或多种。
本发明可以进一步对上述发酵的产物进行处理,以获得微生物油脂,所述处理的方法没有特别的限制,只要是能从所述发酵的产物中将微生物油脂分离即可,为了获得较高的微生物油脂收率,所述处理包括对所述发酵的产物进行破壁、萃取得到所述发酵的产物中的油脂产物。
第三方面,本发明提供了由上述的方法制备得到的微生物油脂,该微生物油脂中甘油三酯Sn-2位上DHA的比例不低于23%,且甘油三酯型DHA含量不低于38%。
第四方面,本发明提供了由上述方法制备的微生物油脂在食品中的应用,所述食品优选为婴幼儿配方食品、保健食品或健康食品。
以下将通过实施例对本发明进行详细描述。
以下实施例中,微生物油脂中DHA的含量及脂肪酸组分测定通过GB26401-2011方法测得;甘油三酯Sn-2位上DHA的比例测定采用“GB/T24984-2010/ISO6800:1997动植物油脂甘三酯分子2-位脂肪酸组分的测定”的方法测定;;甘油三酯Sn-1、Sn-3位上DHA的比例是在全样脂肪酸、Sn-2位脂肪酸分析测定的基础上采用1,3-随机-2-随机分布学说计算而成;人体对DHA的吸收率测定方法为“人体功效试验的方法”:征集男女志愿者食用本专利方法生产的DHA油脂和对比DHA油脂,然后抽取血液测定Sn2位DHA脂肪酸含量来计算吸收率。裂殖壶菌常规菌株来自于中国工业微生物菌种保藏管理中心,编号为CICC 11091s;葡萄糖、蔗糖、酵母粉、谷氨酸钠、酵母浸膏、氯化钠、硫酸镁、氯化钙、磷酸二氢钾、硫酸镍、硫酸铜、钼酸钠、氯化钴、硫酸锌、硫酸亚铁、氯化锰、维生素B1、维生素B12、维生素B6、泛酸钙、生物素、碳酸氢钠、硫酸钠、硫酸铵、氯化钾均为市售品。
本发明的实施例中,微量元素溶液的配方为:硫酸镍2g/L,硫酸铜1.9g/L,钼酸钠0.04g/L,氯化锰2.8g/L,氯化钴0.04g/L,硫酸锌3.2g/L,硫酸亚铁9g/L;维生素溶液的配方为:维生素B1 10.3g/L、维生素B12 0.16g/L、泛酸钙3.2g/L、生物素0.008g/L。
实施例1、裂殖壶菌的30L发酵生产
本实施例中种子培养基配方为:葡萄糖40g/L,谷氨酸钠31g/L,氯化钠19g/L,酵母浸膏5.8g/L,硫酸镁8g/L,磷酸二氢钾5.7g/L,微量元素1g/L,维生素1g/L;
种子罐培养基配方为:葡萄糖30g/L,谷氨酸钠6.3g/L,氯化钠1.45g/L,酵母浸膏8.3g/L,酵母粉8.3g/L,硫酸镁5.18g/L,磷酸二氢钾1.66g/L,氯化钙0.25g/L,碳酸氢钠0.25g/L,硫酸钠9.34g/L,硫酸铵1.04g/L,氯化钾0.83g/L,微量元素1g/L,维生素1g/L,消泡剂0.3g/L;
发酵培养基配方为:葡萄糖50g/L,谷氨酸钠15g/L,氯化钠2.6g/L,酵母浸膏10.9g/L,硫酸镁5.8g/L,磷酸二氢钾2.4g/L,氯化钙0.25g/L,碳酸氢钠0.22g/L,硫酸钠3.62g/L,硫酸铵1.13g/L,氯化钾0.94g/L,微量元素1.1g/L,维生素1.1g/L,消泡剂0.19g/L。
本实施例具体步骤如下:
(1)分别将裂殖壶菌常规菌株和本发明所使用的裂殖壶菌菌株(保藏编号为GDMCCNo.60733)进行活化,接种量为1支解冻后的甘油管菌种转接到4个装有200mL种子培养基的摇瓶中,在转速为180r/min、温度为28℃的摇床上培养48h;
(2)将步骤(1)所得的种子活化液接种到放有200mL种子培养基的摇瓶中进行扩大培养,接种量为3%,在转速为180r/min、温度为28℃的摇床上培养72h;
(3)种子罐中装有3L种子罐培养基,将200mL步骤(2)所得的种子液接入种子罐中,以温度为28℃、搅拌速度为180r/min、通气量为0.6vvm、罐压为0.03Mpa的条件培养50h;
(4)发酵罐中装有14L发酵培养基,将步骤(3)所得的种子罐培养液全部转接入发酵罐中,以温度为29℃、搅拌速度为140r/min、通气量为0.95vvm、罐压为0.03Mpa、pH为6.8的条件培养5-6天,发酵过程中流加葡萄糖浓度为250g/L的无菌液和谷氨酸钠浓度为250g/L的无菌液,以保持谷氨酸浓度为5-8g/L、碳源浓度为10-23g/L,96h后停止补充碳源和谷氨酸,最终获得发酵液;
(5)将5L步骤(4)所得的发酵液经酶解破壁后,用实验室高速离心机将水相、渣相、油相分离,得到微生物油酯;
(6)将步骤(5)所得的微生物油脂进行DHA含量、脂肪酸组分和油脂中甘油三酯Sn2位DHA的比例分析测定,结果见表1。
表1实施例1中微生物油脂中各参数的数据
实施例2、裂殖壶菌的100L发酵生产
本实施例中种子培养基配方为:葡萄糖30g/L,谷氨酸钠20g/L,氯化钠15g/L,酵母浸膏5g/L,硫酸镁6g/L,磷酸二氢钾4g/L,微量元素0.8g/L,维生素0.75g/L;
种子罐培养基配方为:葡萄糖25g/L,谷氨酸钠5.5g/L,氯化钠1.1g/L,酵母浸膏7g/L,酵母粉7g/L,硫酸镁4g/L,磷酸二氢钾1.2g/L,氯化钙0.15g/L,碳酸氢钠0.15g/L,硫酸钠7g/L,硫酸铵0.8g/L,氯化钾0.6g/L,微量元素0.8g/L,维生素0.75g/L,消泡剂0.2g/L;
发酵培养基配方为:葡萄糖30g/L、蔗糖10g/L,谷氨酸钠12g/L,氯化钠2g/L,酵母浸膏9g/L,硫酸镁4.5g/L,磷酸二氢钾2g/L,氯化钙0.2g/L,碳酸氢钠0.2g/L,硫酸钠2.8g/L,硫酸铵1g/L,氯化钾0.8g/L,微量元素0.9g/L,维生素0.9g/L,消泡剂0.15g/L。
(1)分别将裂殖壶菌常规菌株和本发明所使用的裂殖壶菌菌株(保藏编号为GDMCCNo.60733)进行活化,接种量为1支解冻后的甘油管菌种接种到5个装有200mL种子培养基的摇瓶中,在转速为150r/min、温度为28.5℃的摇床上培养72h;
(2)将步骤(1)所得的种子活化液接种到5个装有200mL种子培养基的摇瓶中进行扩大培养,接种量为:将步骤(1)中得到的种子活化液2瓶接种到5瓶种子培养基中,在转速为150r/min、温度为28℃的摇床上培养48h;
(3)种子罐中装有6L种子罐培养基,将400mL步骤(2)所得的种子液接入种子罐中,以温度为28℃、搅拌速度为150r/min、通气量为0.5vvm、罐压为0.03Mpa的条件培养48h;
(4)发酵罐中装有45L发酵培养基,将步骤(3)所得的种子罐培养液全部转接入发酵罐中,以温度为28-29℃、搅拌速度为90--120r/min、通气量为0.5-0.8vvm、罐压为0.03Mpa的条件培养5-6天,发酵过程中流加葡萄糖浓度为250g/L的无菌液和谷氨酸钠浓度为250g/L的无菌液,以保持谷氨酸浓度为8-12g/L、碳源浓度为40-65g/L,96h停止补充碳源和谷氨酸,最终获得发酵液;
(5)将10L步骤(5)所得的发酵液经酶解后,用实验室离心机分离得到微生物油酯;
(6)将步骤(5)所得的微生物油脂进行DHA含量、脂肪酸组分和油脂中甘油三酯Sn2位DHA的比例分析测定,结果见表2。
表2实施例2中微生物油脂中各参数的数据
实施例3、裂殖壶菌的45M3工业发酵生产
本实施例中种子培养基配方为:葡萄糖50g/L,谷氨酸钠35g/L,氯化钠22g/L,酵母浸膏10g/L,硫酸镁10g/L,磷酸二氢钾8g/L,微量元素1.4g/L,维生素1.5g/L;
一级种子罐培养基配方为:葡萄糖35g/L,谷氨酸钠8g/L,氯化钠1.9g/L,酵母浸膏10g/L,酵母粉10g/L,硫酸镁6g/L,磷酸二氢钾2.1g/L,氯化钙0.4g/L,碳酸氢钠0.4g/L,硫酸钠12g/L,硫酸铵1.3g/L,氯化钾1.1g/L,微量元素1.2g/L,维生素1.2g/L,消泡剂0.4g/L;
二级种子罐培养基配方为:葡萄糖50g/L,谷氨酸钠5.65g/L,氯化钠1.3g/L,酵母浸膏5.65g/L,硫酸镁4.65g/L,磷酸二氢钾1.49g/L,氯化钙0.22g/L,碳酸氢钠0.22g/L,硫酸钠8.39g/L,硫酸铵0.93g/L,氯化钾0.74g/L,微量元素0.93g/L,维生素0.93g/L,消泡剂0.3g/L;
发酵培养基配方为:葡萄糖40g/L、蔗糖20g/L,谷氨酸钠20g/L,氯化钠3.2g/L,酵母浸膏12g/L,硫酸镁7g/L,磷酸二氢钾3g/L,氯化钙0.4g/L,碳酸氢钠0.3g/L,硫酸钠4.5g/L,硫酸铵1.5g/L,氯化钾1.2g/L,微量元素1.6g/L,维生素1.6g/L。
本实施例具体步骤如下:
(1)分别将裂殖壶菌常规菌株和本发明所使用的裂殖壶菌菌株(保藏编号为GDMCCNo.60733)进行活化,接种量为1支解冻后的甘油管菌种接种到6个装有200mL种子培养基的摇瓶中,在转速为180r/min、温度为28℃的摇床上培养60h;
(2)将步骤(1)所得的2瓶活化摇瓶种子液接种到5个装有200mL种子培养基的三角瓶中进行扩大培养,在转速为180r/min、温度为28±0.5℃的摇床上培养60h;
(3)一级种子罐中装有500L一级种子罐培养基,灭菌冷却至40℃以下,然后将1L步骤(2)所得的种子液转接入一级种子罐中,以温度为28±0.5℃、搅拌速度为150r/min、pH为6.8的条件培养55h;
(4)二级种子罐中装有6M3二级种子罐培养基,灭菌冷却至40℃以下,然后将步骤(3)所得的一级种子液全部在无菌条件下转接到二级种子罐中,以温度28±0.5℃、搅拌150r/min、pH6.8的条件培养18h;
(5)主发酵罐中装有22M3发酵培养基,灭菌冷却至40℃以下,将步骤(4)所得的二级种子液全部在无菌条件下转接到主发酵罐中,以温度28±0.5℃、搅拌100r/min、通气比为1.0VVM、pH7.5的条件培养5天,发酵过程中流加葡萄糖浓度为250g/L的无菌液和谷氨酸钠浓度为250g/L的无菌液,以保持谷氨酸浓度为12-15g/L、碳源浓度为55-70g/L,96h停止补充碳源和谷氨酸,最终获得发酵液;
(6)发酵液经酶解后,将发酵液预热到85-90℃,用三相离心机分离得到微生物油酯;
(7)将步骤(6)所得的微生物油脂进行DHA含量、脂肪酸组分和油脂中甘油三酯Sn2位DHA的比例分析测定,结果见表3。
表3实施例3中微生物油脂中各参数的数据
以上详细描述了本发明的优选实施方式,但是,本发明并不限于此。在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,包括各个技术特征以任何其它的合适方式进行组合,这些简单变型和组合同样应当视为本发明所公开的内容,均属于本发明的保护范围。
Claims (11)
1.一种Sn-2位DHA含量不低于23%的微生物油脂,其特征在于,所述微生物油脂由裂殖壶菌菌株发酵制得,所述裂殖壶菌菌株为保藏编号为GDMCC No.60733的裂殖壶菌(Schizochytrium)。
2.一种Sn-2位DHA含量不低于23%的微生物油脂的制备方法,其特征在于,该方法包括将裂殖壶菌菌株接种到发酵培养基中发酵制得,所述裂殖壶菌菌株为保藏编号为GDMCCNo.60733的裂殖壶菌(Schizochytrium)。
3.根据权利要求2所述的方法,其特征在于,所述发酵培养基包括碳源、氮源、无机盐、微量元素和维生素,发酵的条件包括pH为6-7.5、温度为27-31℃、时间为4-8天、通气比为0.45-1.1VVM、谷氨酸浓度为5-15g/L、碳源浓度为5-70g/L。
4.根据权利要求2或3所述的方法,其特征在于,该方法包括将所述裂殖壶菌菌株经活化后制作成摇瓶种子液接种到种子培养基中培养,然后再转接入所述发酵培养基中发酵培养。
5.根据权利要求4所述的方法,其特征在于,所述活化的过程包括:将所述裂殖壶菌菌株的冷冻甘油管菌种解冻、接种到种子培养基中进行扩大培养。
6.根据权利要求5所述的方法,其特征在于,所述活化的过程包括:将所述裂殖壶菌菌株的冷冻甘油管菌种解冻后,用无菌吸管将所述甘油管菌种接种到已灭菌的种子培养基中,在温度为27.5-28.5℃、转速为150-200r/min的条件下培养48-72h,所述种子培养基中碳源为30-50g/L、氮源为25-45g/L、无机盐为25-40g/L、微量元素为0.015-0.025g/L、维生素为0.01-0.02g/L。
7.根据权利要求3或6所述的方法,其特征在于,所述碳源为葡萄糖或/和蔗糖,所述氮源为谷氨酸钠、酵母粉、酵母浸膏中的一种或多种,所述无机盐为钙盐、磷酸盐、钾盐、钠盐、镁盐、铵盐中的一种或多种,所述微量元素为镍、铜、钼、钴、锌、铁、锰中的一种或多种,所述维生素为维生素B1、维生素B12、维生素B6、泛酸钙、生物素中的一种或多种。
8.根据权利要求2所述的方法,其特征在于,该方法还包括对所述发酵的产物进行破壁、萃取得到所述发酵的产物中的油脂产物。
9.由权利要求2-8中任意一项所述的方法制得的微生物油脂,所述微生物油脂中甘油三酯Sn-2位上DHA的比例不低于23%,且甘油三酯型DHA含量不低于38%。
10.权利要求9所述的微生物油脂在食品中的应用。
11.根据权利要求10所述的应用,其特征在于,所述食品为婴幼儿配方食品、保健食品以及健康食品。
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IE20200267U1 (en) | 2022-08-03 |
US20210340583A1 (en) | 2021-11-04 |
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EP3896167A4 (en) | 2022-09-14 |
CN110846346A (zh) | 2020-02-28 |
IES87347Y1 (en) | 2022-12-21 |
KR20220088912A (ko) | 2022-06-28 |
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CA3149969A1 (en) | 2021-06-03 |
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