CN105543118A - 一种基于海藻酸钠寡糖的抗逆保护剂及其制备方法和应用 - Google Patents
一种基于海藻酸钠寡糖的抗逆保护剂及其制备方法和应用 Download PDFInfo
- Publication number
- CN105543118A CN105543118A CN201610130770.7A CN201610130770A CN105543118A CN 105543118 A CN105543118 A CN 105543118A CN 201610130770 A CN201610130770 A CN 201610130770A CN 105543118 A CN105543118 A CN 105543118A
- Authority
- CN
- China
- Prior art keywords
- degeneration
- resistant protective
- protective material
- resistant
- stress
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- -1 alginate oligosaccharides Chemical class 0.000 title claims abstract description 29
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 239000003223 protective agent Substances 0.000 title abstract 7
- 229940072056 alginate Drugs 0.000 title abstract 5
- 229920000615 alginic acid Polymers 0.000 title abstract 5
- 235000010443 alginic acid Nutrition 0.000 title abstract 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 43
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
- 239000002904 solvent Substances 0.000 claims abstract description 6
- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 claims abstract description 5
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 230000007850 degeneration Effects 0.000 claims description 62
- 239000000463 material Substances 0.000 claims description 46
- 230000001681 protective effect Effects 0.000 claims description 43
- 239000000661 sodium alginate Substances 0.000 claims description 32
- 235000010413 sodium alginate Nutrition 0.000 claims description 32
- 229940005550 sodium alginate Drugs 0.000 claims description 32
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 11
- 238000013016 damping Methods 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 230000015556 catabolic process Effects 0.000 claims description 7
- 238000006731 degradation reaction Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 4
- 230000002180 anti-stress Effects 0.000 claims description 3
- 230000009977 dual effect Effects 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 8
- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 abstract description 3
- 150000004676 glycans Chemical class 0.000 abstract description 3
- 150000002482 oligosaccharides Chemical class 0.000 abstract description 3
- 229920001282 polysaccharide Polymers 0.000 abstract description 3
- 239000005017 polysaccharide Substances 0.000 abstract description 3
- 239000008103 glucose Substances 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 abstract 1
- 238000006116 polymerization reaction Methods 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 41
- 230000035882 stress Effects 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 10
- 229960004756 ethanol Drugs 0.000 description 10
- 238000000034 method Methods 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 238000010257 thawing Methods 0.000 description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 235000013305 food Nutrition 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000001509 sodium citrate Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 231100000225 lethality Toxicity 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000006353 environmental stress Effects 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000199919 Phaeophyceae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- LUVOJBWJNHWVNG-UHFFFAOYSA-N [Na].[Na].[Na].OC(=O)CC(O)(C(O)=O)CC(O)=O Chemical compound [Na].[Na].[Na].OC(=O)CC(O)(C(O)=O)CC(O)=O LUVOJBWJNHWVNG-UHFFFAOYSA-N 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- 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
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
-
- 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
- C12N1/04—Preserving or maintaining viable microorganisms
-
- 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
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
-
- 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
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Botany (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及一种酿酒酵母抗逆保护剂，具体为一种基于海藻酸钠寡糖的抗逆保护剂及其制备方法和应用，本发明的抗逆保护剂主要包括海藻酸钠寡糖和溶剂，所述海藻酸钠寡糖的质量浓度1μg/mL-10mg/mL。所述的海藻酸钠寡糖由甘露糖酸醛、古罗糖醛酸或二者杂合组成，平均聚合度为5-8，平均分子量在2000-3000之间。将本发明提供的抗逆保护剂添加到酿酒酵母的培养基中，扩展了海洋来源多糖和寡糖的应用领域，缩短了高糖发酵的发酵时间，提高了发酵强度，提高了酿酒酵母应对多种胁迫环境的存活率和细胞活性。
Description
技术领域
本发明涉及工业微生物胁迫耐受性领域和海洋多糖/寡糖生物利用领域，具体涉及一种酿酒酵母抗逆保护剂。
背景技术
酿酒酵母因其卓越的产酒性能和良好的生物安全性，在生物能源和食品医药领域(酿酒、发面食品、生物医药等)获得了广泛的应用。特别是近年来，燃料乙醇作为可再生的清洁能源，其生产和使用受到普遍关注，其中以非粮纤维素替代粮食为原料生产燃料乙醇可以有效的解决或缓解粮食危机，缓解石油资源短缺的矛盾，同时也有利于对环境的改善降低空气污染。
但是，在不同工艺(批次、单级/多级连续、重复批次等)的发酵过程中均存在包括产物抑制和底物抑制在内的环境胁迫，使细胞活性受到不同程度的抑制，从而降低乙醇得率和发酵效率。在利用淀粉质原料超高浓度(VeryHighGravity,VHG)乙醇发酵过程中，随着终产物乙醇浓度的提高，乙醇在一众环境胁迫因子中成为最主要的胁迫因素，酵母细胞活性被高浓度乙醇严重抑制甚至出现生长停滞和死亡的现象；同步糖化发酵技术的实现需要酵母耐受高温；以纤维素为原料的乙醇发酵与淀粉或糖质乙醇发酵不同，利用现有技术发酵过程中很难达到对微生物(酵母或细菌)产生抑制作用的乙醇浓度，但木质纤维素预处理环节却成为抑制物主要来源，如弱酸类物质(乙酸)、呋喃醛类等，同时酵母菌会出现氧化胁迫特征。因此，提高酿酒酵母的抗逆性，进而提高细胞活性，获得高乙醇得率和发酵效率具有重要意义。
海藻酸钠(SodiumAlginate)是海带等褐藻类植物细胞壁的主要组成成分，来源广泛，由古洛糖醛酸(G)和甘露糖醛酸(M)相邻或交替排列，被广泛应用于纺织、食品、印染等行业，还被用作固定化载体、增稠剂、稳定剂等。而其降解产物海藻酸钠寡糖(AlginateOligosaccharides,AOS)具有相对于海藻酸钠本身分子量低、易吸收等优点。
近年来，海藻酸钠寡糖的生物活性广泛受到国内外学者的关注，研究表明海藻酸钠寡糖具有多种生物活性，如促进植物生长和诱导抗逆性的作用、抗氧化活性、抑菌活性、诱导动物细胞因子的产生等，但其应用于酿酒酵母发酵与逆境环境保护还没有报道。
发明内容
为解决上述技术问题，本发明的目的在于提供一种基于海藻酸钠的抗逆保护剂及其制备方法和应用。
本发明涉及一种基于海藻酸钠寡糖的抗逆保护剂，包括海藻酸钠寡糖和溶剂，所述海藻酸钠寡糖的质量浓度为1μg/mL-10mg/mL。所述的海藻酸钠寡糖由甘露糖酸醛、古罗糖醛酸或二者杂合组成，平均聚合度为5-8，平均分子量在2000-3000之间。
进一步的，所述溶剂为本领域的任意常规的溶剂，如水或缓冲液，缓冲液比如柠檬酸盐缓冲液、醋酸-醋酸钠缓冲液，水优选去离子水。
所述的抗逆保护剂的制备方法如下：(1)将海藻酸钠溶解于溶剂中作为海藻酸钠底物，海藻酸钠浓度为1-2％(w/v)，向其中加入浓度为0.3％(v/v)的盐酸，在降解温度为90℃的条件下，降解时间24-36小时；(2)降解完成后离心保留上清液并中和至中性。
为避免盐分影响抗逆保护剂发挥作用，比如盐分存在会增加酵母的高渗/高盐胁迫条件，故还可先将盐分除去，再将除去盐分的上清液冷冻干燥或喷雾干燥获得海藻酸钠寡糖粉末，得到抗逆保护剂。优选的，海藻酸钠的浓度为1.5％(w/v)，降解时间24小时。
发明人将抗逆保护剂应用在酿酒酵母抗逆保护中，起到了良好的技术效果，所述的抗逆保护应用包括但不限于在酿酒酵母保藏、发酵及抗胁迫条件下的应用，抗胁迫条件包括高浓度乙醇、高渗、高盐、高温、氧化胁迫、冻融胁迫等所有抑制细胞活性的不利条件。
进一步的，所述抗逆保护中的应用，其应用方式可以为将抗逆保护剂添加至含酿酒酵母的培养基或缓冲液中，再离心除去抗逆保护剂。更好的，将抗逆保护剂添加至含酿酒酵母的培养基或缓冲液中与酿酒酵母共培养0.5-3小时后，再离心除去抗逆保护剂。
进一步的，所述抗逆保护中的应用，其应用方式还可以为将所述的抗逆保护剂添加至含酿酒酵母的培养基中，直至发酵终点。
所述的抗逆保护剂的使用量可根据不同实际需要进行调整，比较适宜的浓度范围为1μg/mL-10mg/mL。
本发明的有益效果如下：
(1)本发明提供的抗逆保护剂提高了酿酒酵母应对多种胁迫环境的存活率和细胞活性；
(2)本发明提供的抗逆保护剂缩短了高糖发酵的发酵时间，提高了发酵强度；
(3)本发明提供的抗逆保护剂扩展了海洋来源多糖和寡糖的应用领域。
附图说明
图1为残糖含量变化示意图；
图2为乙醇含量变化示意图；
图3为保护剂促进酿酒酵母乙醇胁迫下致死率的示意图；
图4保护剂促进酿酒酵母高温胁迫下致死率的示意图；
图5保护剂促进酿酒酵母乙酸胁迫下细胞活性的示意图；
图6保护剂促进酿酒酵母过氧化氢胁迫下细胞活性的示意图；
图7保护剂促进酿酒酵母冻融的示意图。
其中：Control为对照组；AOS为抗逆保护剂组。
具体实施方式
下面结合具体实施例对本发明的技术方案作进一步的说明，但本发明不以任何形式受限于实施例内容。实施例中所述实验方法如无特殊说明，均为常规方法；如无特殊说明，所述试剂和生物材料，均可从商业途径获得。
酿酒酵母菌在发酵过程中普遍存在产物抑制和底物抑制，使细胞活性受到不同程度的抑制，从而降低乙醇得率和发酵效率的现象，本发明的抗逆保护剂对任意酿酒酵母存在的上述现象均有抗逆保护作用，影响抗逆保护效果的因素主要是抗逆保护剂的使用量、添加时机等因素，本发明实施例使用大连大学实验室酿酒酵母菌Sc4126，对抗逆保护剂的的保护效果进行试验。
实施例1
抗逆保护剂的制备
(1)将海藻酸钠溶解于缓冲液中作为反应底物，海藻酸钠浓度为1.5％(w/v)，向其中加入浓度为0.3％(v/v)的盐酸，降解温度为90℃，降解时间24小时；
(2)降解完成后离心保留上清液并中和至中性，使用截留分子量为100D的透析袋将上清液中的盐分除去，再将除盐后的上清液冷冻干燥获得海藻酸钠寡糖粉末；
使用常规方法测定所得海藻酸钠寡糖粉末中还原糖含量与总糖含量，计算得出平均相对分子质量约为2497，海藻酸钠寡糖的平均聚合度约为6。
(3)将海藻酸钠寡糖粉末溶于去离子水中，使其在溶液中的浓度分别为1ug/mL，10ug/mL，10mg/mL，得到不同质量浓度的抗逆保护剂。
实施例2
抗逆保护剂在酿酒酵母发酵中的应用
取对数生长期的酿酒酵母Sc4126，以1％(v/v)接种于高糖发酵培养基(高糖发酵培养基的配方：265g/L葡萄糖，8g/L蛋白胨，6g/L酵母粉)，然后向此培养基中分别添加浓度10ug/mL、10mg/mL的抗逆保护剂，搅拌均匀，于30℃、150rpm发酵48h，每隔6h测定残糖、乙醇；未加抗逆保护剂的组为阴性对照组。
试验结果如图1-2所示，添加抗逆保护剂组较阴性对照组的发酵终点时间缩短2-6小时，且终点乙醇得率各组持平，均在0.46左右。乙醇得率的计算公式如下：
乙醇得率＝终点乙醇质量/初始糖浓度。
实施例3
抗逆保护剂在酿酒酵母胁迫条件下的应用
取处于对数生长期后期到稳定期前期的酿酒酵母菌Sc4126的过夜培养物，3000r/min离心5min，弃上清液，用0.1mol/L的pH5.0的柠檬酸三钠缓冲液洗涤沉淀两次，最后加柠檬酸三钠缓冲液重新溶解沉淀，随时测定其OD600值，直至使其OD600值达到2.0左右，再加入实施例1制备的浓度为1ug/mL、10ug/mL或10mg/mL抗逆保护剂，置于30℃共培养2h，然后离心去掉抗逆保护剂，用柠檬酸三钠缓冲液重悬酿酒酵母菌Sc4126细胞；使酿酒酵母菌Sc4126细胞分别暴露于不同抗逆胁迫条件，抗逆胁迫条件包括：
终浓度为20％(v/v)的无水乙醇处理细胞2h；
55℃热击1.5h；
3g/L乙酸处理2-8h；
7.5mM过氧化氢处理2-8h；
-80℃冻融一次；
对于冻融胁迫，直接将抗逆保护剂加入到柠檬酸钠缓冲液中，用此柠檬酸钠缓冲液重悬细胞，置于-80℃冰箱中冷冻24h后，取出放置于室温融化，加20％(v/v)甘油组为阳性对照；其他抗逆胁迫条件的试验以未加抗逆保护剂的组作为阴性对照。
乙醇、高温短时冲击和冻融胁迫试验组采用美蓝染色后显微镜下拍照计数，蓝色细胞为死亡细胞，从而计数确定细胞致死率；乙酸与过氧化氢冲击组采用间隔2h测定细胞OD600的吸光度，即细胞生长抑制程度来评价细胞所受胁迫程度。
试验结果如图3-7所示，结果表明不同浓度的海藻酸钠寡糖对酿酒酵母Sc4126均有一定程度的抗逆保护作用，发挥作用浓度在ug/mL到mg/mL范围内。
实施例4
抗逆保护剂在酿酒酵母保藏条件下的应用
处于保藏状态的酿酒酵母经过了低温冷冻过程，与冻融胁迫条件下，酿酒酵母所处的环境几乎完全相同，因此，本发明的抗逆保护剂在酿酒酵母保藏中的应用的效果可依据实施例3中冻融胁迫条件下的保护效果得到。
Claims (9)
1.一种基于海藻酸钠寡糖的抗逆保护剂，其特征在于，包括海藻酸钠寡糖和溶剂，所述海藻酸钠寡糖的质量浓度为1μg/mL-10mg/mL。
2.如权利要求1所述的抗逆保护剂，其特征在于，所述的海藻酸钠寡糖由甘露糖酸醛、古罗糖醛酸或二者杂合组成，平均聚合度为5-8，平均分子量在2000-3000之间。
3.一种如权利要求1所述的抗逆保护剂的制备方法，其特征在于，包括如下步骤：向浓度为1-2％(w/v)的海藻酸钠底物中加入浓度为0.3％(v/v)的盐酸，降解温度为90℃，降解时间24-36小时；离心保留上清液并中和至中性，将上清液干燥获得海藻酸钠寡糖粉末，得抗逆保护剂。
4.如权利要求3所述的制备方法，其特征在于，还包括如下步骤：将上清液中和至中性后，再除去盐分，将除去盐分后的上清液干燥获得海藻酸钠寡糖粉末，得抗逆保护剂。
5.一种基于海藻酸钠寡糖的抗逆保护剂的新用途，其特征在于，将权利要求1所述的抗逆保护剂应用在酿酒酵母抗逆保护中。
6.如权利要求5所述的抗逆保护剂的新用途，其特征在于，将权利要求1所述的抗逆保护剂应用在酿酒酵母保藏、发酵、抗胁迫条件中。
7.如权利要求5所述的抗逆保护剂的新用途，其特征在于，将权利要求1所述的抗逆保护剂添加至含酿酒酵母的培养基或缓冲液中，再离心除去抗逆保护剂。
8.如权利要求7所述的抗逆保护剂的新用途，其特征在于，将所述的抗逆保护剂添加至含酿酒酵母的培养基或缓冲液中，共培养0.5-3小时后，再离心除去抗逆保护剂。
9.如权利要求3所述的抗逆保护剂的新用途，其特征在于，将权利要求1所述的抗逆保护剂添加至含酿酒酵母的培养基中，直至发酵终点。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610130770.7A CN105543118B (zh) | 2016-03-08 | 2016-03-08 | 一种基于海藻酸钠寡糖的抗逆保护剂及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610130770.7A CN105543118B (zh) | 2016-03-08 | 2016-03-08 | 一种基于海藻酸钠寡糖的抗逆保护剂及其制备方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105543118A true CN105543118A (zh) | 2016-05-04 |
| CN105543118B CN105543118B (zh) | 2020-02-14 |
Family
ID=55822677
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610130770.7A Active CN105543118B (zh) | 2016-03-08 | 2016-03-08 | 一种基于海藻酸钠寡糖的抗逆保护剂及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105543118B (zh) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520844A (zh) * | 2016-10-28 | 2017-03-22 | 大连大学 | 利用微生物疫苗提高酿酒酵母溶剂胁迫耐受性和发酵性能的方法 |
| CN108285474A (zh) * | 2018-04-19 | 2018-07-17 | 成都欧甘拓展有限公司 | 海藻酸钠或海藻酸钾寡糖的制备方法 |
| CN109892330A (zh) * | 2017-12-08 | 2019-06-18 | 中国科学院大连化学物理研究所 | 一种农作物日灼病防治剂及其应用 |
| CN114272260A (zh) * | 2021-12-24 | 2022-04-05 | 大连大学 | 一种海洋天然产物解酒保肝口服液、制备方法以及应用 |
-
2016
- 2016-03-08 CN CN201610130770.7A patent/CN105543118B/zh active Active
Non-Patent Citations (7)
| Title |
|---|
| COSTA V ET AL: "Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases", 《MOL ASPECTS MED》 * |
| 吴海歌 等: "海藻酸钠寡糖抗氧化活性的研究", 《大连大学学报》 * |
| 张小华 等: "酿酒酵母SOD1、SOD2基因缺失对胁迫耐受性的影响", 《中国酿造》 * |
| 张玉娟 等: "海藻酸钠寡糖生物活性的研究进展", 《中国酿造》 * |
| 方青 等: "过表达谷氧还蛋白基因GRX5提高酿酒酵母乙酸耐性", 《化工学报》 * |
| 曹文涛 等: "乙醇对酿酒酵母Saccharomyces cerevisiae应激性的研究进展", 《酿酒科技》 * |
| 赵素娟 等: "活性氧在酿酒酵母乙醇胁迫中的作用", 《中国优秀硕士学位论文全文数据库 工程科技I辑 B024-360 》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520844A (zh) * | 2016-10-28 | 2017-03-22 | 大连大学 | 利用微生物疫苗提高酿酒酵母溶剂胁迫耐受性和发酵性能的方法 |
| CN109892330A (zh) * | 2017-12-08 | 2019-06-18 | 中国科学院大连化学物理研究所 | 一种农作物日灼病防治剂及其应用 |
| CN109892330B (zh) * | 2017-12-08 | 2021-04-06 | 中国科学院大连化学物理研究所 | 一种农作物日灼病防治剂及其应用 |
| CN108285474A (zh) * | 2018-04-19 | 2018-07-17 | 成都欧甘拓展有限公司 | 海藻酸钠或海藻酸钾寡糖的制备方法 |
| CN114272260A (zh) * | 2021-12-24 | 2022-04-05 | 大连大学 | 一种海洋天然产物解酒保肝口服液、制备方法以及应用 |
| CN114272260B (zh) * | 2021-12-24 | 2024-02-09 | 大连大学 | 一种海洋天然产物解酒保肝口服液、制备方法以及应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105543118B (zh) | 2020-02-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105543118A (zh) | 一种基于海藻酸钠寡糖的抗逆保护剂及其制备方法和应用 | |
| Mountfort et al. | Fermentation of cellulose to methane and carbon dioxide by a rumen anaerobic fungus in a triculture with Methanobrevibacter sp. strain RA1 and Methanosarcina barkeri | |
| Wu et al. | Features of sweet sorghum juice and their performance in ethanol fermentation | |
| CN101880636B (zh) | 酿酒酵母的耐受多种抑制剂的菌株 | |
| CN104371937A (zh) | 一株能以多碳源共发酵的酿酒酵母及其应用 | |
| EP3222732A1 (en) | Lignocellulosic simultaneous saccharification and fermentation method improved by utilizing surfactant | |
| CN103849576B (zh) | 一株具有胁迫耐受性的重组酿酒酵母菌株 | |
| CN105152356A (zh) | 一种用于净化水体的微胶囊复合菌剂及其制备方法 | |
| CN102206689A (zh) | 一种在发酵过程中改性细菌纤维素的方法 | |
| Salim | The Effect of pH on simultaneous saccharification and fermentation process of water hyacinth (Eichhornia crassipes (Mart.) Solms.) using Trichoderma harzianum an | |
| CN105695387A (zh) | 一种基于卡拉胶寡糖的抗逆保护剂及其制备方法和应用 | |
| CN104336574B (zh) | 一种生物-低温冷冻干燥法制备酵母微胶囊的方法 | |
| CN101671698A (zh) | 用木聚糖酶提高淀粉质原料酒精发酵速度和出酒率的方法 | |
| US20160032272A1 (en) | Preparation method of yeast cell immobilization medium and application thereof | |
| CN103981109B (zh) | 一株对啤酒发酵环境具有多种耐受性的啤酒酵母及其应用 | |
| CN102424797A (zh) | 多介质爆破木质纤维素原料制备酒精的装置及其制备酒精的方法 | |
| Widmer et al. | Simultaneous saccharification and fermentation of orange processing waste to ethanol using Kluyveromyces marxianus | |
| Altuntaş et al. | Ethanol production from starch by co-immobilized amyloglucosidase—Zymomonas mobilis cells in a continuously-stirred bioreactor | |
| Bajpai et al. | Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice | |
| CN103146763B (zh) | 一种制备乙醇的方法 | |
| CN103911302A (zh) | 一种酵母菌的培养方法和生产酒精的方法 | |
| Pornpukdeewattana et al. | Optimization of Fermentation Temperature for Very High Gravity Ethanol Production using Industrial Strain of Saccharomyces cerevisiae SC90 | |
| CN106520846A (zh) | 一种改良的木质纤维素类生物质水热预处理方法 | |
| KR100967694B1 (ko) | 초임계유체를 이용한 녹조류, 홍조류 및 갈조류로부터 에탄올 생산방법 | |
| Kregiel | Amylolytic activity of Kluyver-positive Debaryomyces occidentalis cells immobilized in foamed alginate gel |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Li Qian Inventor after: Wu Haige Inventor after: Yao Ziang Inventor after: Cui Hongli Inventor after: Qin Liyuan Inventor after: Gao Chenhui Inventor after: Wu Chen Inventor before: Wu Haige Inventor before: Li Qian Inventor before: Yao Ziang Inventor before: Cui Hongli Inventor before: Qin Liyuan Inventor before: Gao Chenhui Inventor before: Wu Chen |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20160504 Assignee: Dalian Zhicheng Biotechnology Co.,Ltd. Assignor: DALIAN University Contract record no.: X2023210000188 Denomination of invention: A stress resistant protective agent based on sodium alginate oligosaccharides and its preparation method and application Granted publication date: 20200214 License type: Common License Record date: 20231123 |
|
| EE01 | Entry into force of recordation of patent licensing contract | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20160504 Assignee: Dalian Sanlida Food Co.,Ltd. Assignor: DALIAN University Contract record no.: X2023210000228 Denomination of invention: A stress resistant protective agent based on sodium alginate oligosaccharides and its preparation method and application Granted publication date: 20200214 License type: Common License Record date: 20231129 Application publication date: 20160504 Assignee: Haisenge yaojianguo (Dalian) Food Co.,Ltd. Assignor: DALIAN University Contract record no.: X2023210000227 Denomination of invention: A stress resistant protective agent based on sodium alginate oligosaccharides and its preparation method and application Granted publication date: 20200214 License type: Common License Record date: 20231129 Application publication date: 20160504 Assignee: Dalian Yuqian Industrial Co.,Ltd. Assignor: DALIAN University Contract record no.: X2023210000206 Denomination of invention: A stress resistant protective agent based on sodium alginate oligosaccharides and its preparation method and application Granted publication date: 20200214 License type: Common License Record date: 20231127 |
|
| EE01 | Entry into force of recordation of patent licensing contract | ||
| OL01 | Intention to license declared | ||
| OL01 | Intention to license declared |