CN110964706A - 一种纤维素酶、木聚糖酶和果胶酶的制备方法 - Google Patents
一种纤维素酶、木聚糖酶和果胶酶的制备方法 Download PDFInfo
- Publication number
- CN110964706A CN110964706A CN201911389431.0A CN201911389431A CN110964706A CN 110964706 A CN110964706 A CN 110964706A CN 201911389431 A CN201911389431 A CN 201911389431A CN 110964706 A CN110964706 A CN 110964706A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- cellulase
- xylanase
- pectinase
- aspergillus niger
- 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.)
- Pending
Links
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 26
- 229940106157 cellulase Drugs 0.000 title claims abstract description 26
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title claims abstract description 23
- 108010059820 Polygalacturonase Proteins 0.000 title claims abstract description 23
- 108010093305 exopolygalacturonase Proteins 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 241001122767 Theaceae Species 0.000 claims abstract description 31
- 238000000855 fermentation Methods 0.000 claims abstract description 25
- 230000004151 fermentation Effects 0.000 claims abstract description 25
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000011081 inoculation Methods 0.000 claims abstract description 8
- 238000002791 soaking Methods 0.000 claims abstract description 7
- 235000013616 tea Nutrition 0.000 claims description 29
- 241000628997 Flos Species 0.000 claims description 15
- 235000015092 herbal tea Nutrition 0.000 claims description 14
- 239000000725 suspension Substances 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 8
- 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 claims description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 7
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 230000003213 activating effect Effects 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 238000010563 solid-state fermentation Methods 0.000 abstract description 8
- 108090000790 Enzymes Proteins 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 16
- 230000000694 effects Effects 0.000 description 16
- 229940088598 enzyme Drugs 0.000 description 16
- 239000000243 solution Substances 0.000 description 9
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 101000765308 Aspergillus niger N-(5'-phosphoribosyl)anthranilate isomerase Proteins 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 108091005508 Acid proteases Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241001061264 Astragalus Species 0.000 description 1
- 102000003712 Complement factor B Human genes 0.000 description 1
- 108090000056 Complement factor B Proteins 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 241000222383 Polyporales Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241001460073 Trichoderma asperellum Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000006533 astragalus Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229960004106 citric acid Drugs 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000019784 crude fat Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229940074391 gallic acid Drugs 0.000 description 1
- 235000004515 gallic acid Nutrition 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 229950006191 gluconic acid Drugs 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000015067 sauces Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 210000004233 talus Anatomy 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 235000014101 wine Nutrition 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01011—Pectinesterase (3.1.1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01015—Polygalacturonase (3.2.1.15)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02002—Pectate lyase (4.2.2.2)
Abstract
本发明公开了一种纤维素酶、木聚糖酶和果胶酶的制备方法，本发明通过设置菌种浓度、接种量、含水量以及适当的浸泡液pH，发酵时长，发酵温度，得到纤维素酶、木聚糖酶和果胶酶的最佳生产工艺。本发明涉及一种利用黑曲霉固态发酵凉茶渣生产的纤维素酶、木聚糖酶和果胶酶的工艺，首次公开了利用黑曲霉固态发酵凉茶渣生产的纤维素酶、木聚糖酶和果胶酶的高效廉价方法。
Description
技术领域
本发明属于微生物领域，特别涉及一种纤维素酶、木聚糖酶和果胶酶的制备方法。
背景技术
为了尽可能地保留凉茶中风味成分，凉茶多采用低温提取，使凉茶渣与原药材具有相似的营养与活性成分。谢月琴等报道凉茶渣干物质含量为20.55％，其中粗纤维28.83％、粗蛋白9.78％、粗脂肪3.52％、粗灰分6.68％。凉茶渣具有较好的营养和药用价值，是一类未被充分利用的生物资源。近年来，随着凉茶产业的快速发展，凉茶渣的排放也在逐年增加，凉茶渣日产量达到680t。传统的凉茶渣多采用直接堆放、填埋等方式处理，造成了严重资源浪费和环境污染。由于场地限制而不能及时处理，凉茶渣甚至对企业生产也产生较大影响。对凉茶渣进行资源化、高值化利用已经成为企业和社会必须面临的重大问题。
黑曲霉(Aspergillus niger)属于真核生物子囊菌亚门丝孢目丛梗孢科，是一种常见的安全无毒性的曲霉属真菌，是制酱、酿酒、制醋、糖化饲料的主要工业菌种，广泛分布于世界各地的粮食、植物性产品和土壤中。黑曲霉可用于生产淀粉酶、酸性蛋白酶、纤维素酶、果胶酶、葡萄糖氧化酶、柠檬酸、葡糖酸和没食子酸等。
纤维素酶、木聚糖酶和果胶酶是降解植物细胞壁的主要酶类，是提高饲料和食品利用率的重要酶类。利用黑曲霉等固态发酵生产的纤维素酶等酶类，经过干燥后依然具有较强的酶活性。
焦巧芳等以筛选的棘孢木霉为菌种，利用黄芪渣、麸皮和淀粉等组成的培养基同时生产纤维素酶、木糖酶和果胶酶，三种酶活力分别约为1200U/mL、1000U/mL、1000U/mL。本发明与该文献相比，不用添加麸皮和淀粉，且加入较少的水进行固态发酵，使工艺更简单，成本更低。
根据现有问题，本发明致力于研究以黑曲霉为菌种，利用凉茶渣固态发酵生产这三种酶的廉价高效的工艺，具有重要的社会意义和经济价值。
发明内容
本发明的首要目的在于提供一种纤维素酶、木聚糖酶和果胶酶的制备方法。
为达到上述目的，本发明所采取的技术方案是：
本发明的第一方面，提供了一种生产纤维素酶、木聚糖酶和果胶酶的方法，包含以下步骤：将黑曲霉菌种制备成菌悬液，接种到凉茶渣培养基上发酵；所述凉茶渣包括鸡蛋花、金银花、菊花、凉粉草、夏枯草、甘草和布渣叶。
根据本发明的实施例，还包括预先将黑曲霉菌活化、传代，所述活化将黑曲霉菌接种到PDA固体培养基培养。
根据本发明的实施例，培养条件为50％～90％湿度，28℃～37℃，培养24～240h。
根据本发明的实施例，所述传代为1～3次。
根据本发明的实施例，所述菌悬液浓度为105～109cfu/mL，优选2×107cfu/mL的黑曲霉菌悬液。
根据本发明的实施例，接种量为5％～25％，发酵温度为28℃～37℃，发酵时间为3～7天。
根据本发明的实施例，发酵时的含水量为65％～85％。
根据本发明的实施例，所述凉茶渣培养基包括：凉茶渣粉末1～5份，加入硫酸铵0.04～0.20份，葡萄糖0～0.1份，磷酸二氢钾0.005～0.02份，磷酸氢二钾0.002～0.020份，水5.5～8.5份。
根据本发明的实施例，凉茶渣粉末2份，加入硫酸铵0.08份，葡萄糖0.04份，磷酸二氢钾0.01份，磷酸氢二钾0.008份，水7份。
根据本发明的实施例，所述凉茶渣粉末制备方法：将鸡蛋花、金银花、菊花、凉粉草、夏枯草、甘草和布渣叶混合，煎制后得到的凉茶渣，再干燥粉碎。
本发明使用黑曲霉为单一菌种，在计算各种酶活力时，扣除酶液中原有的还原糖的影响，且使用酶的原液进行计算。
本发明的有益效果是：
本发明公开了使用黑曲霉固态发酵凉茶渣生产的纤维素酶、木聚糖酶和果胶酶的工艺，当硫酸铵用量为4％，葡萄糖用量为2％，菌种浓度为2×107cfu/mL，接种量为10％时，含水量为70％，浸泡液pH为9，发酵温度为31℃，发酵7天，是纤维素酶、木聚糖酶和果胶酶的最佳生产工艺。本发明涉及一种利用黑曲霉固态发酵凉茶渣生产的纤维素酶、木聚糖酶和果胶酶的工艺，首次公开了利用黑曲霉固态发酵凉茶渣生产的纤维素酶、木聚糖酶和果胶酶的高效廉价方法。
具体实施方式
下面结合实施例对本发明中的技术方案进行清楚、完整的说明，但并不局限于此。
预先准备
1.菌种活化：
(1)菌种活化在PDA固体培养基上，于电热恒温恒湿培养箱80％的湿度，30℃培养120h；
(2)将黑曲霉菌种连续传代两次后，用生理盐水制成2×107cfu/mL的菌悬液；
PDA固体培养基：马铃薯200g去皮，切成块，煮沸30min，纱布过滤，再加蔗糖20g，溶化后补水至1000mL。加入琼脂20g，加热溶解，然后121℃湿热灭菌20min。
2.凉茶渣培养基的制备：
取6～8份水，加入硫酸铵0.08份，葡萄糖0.04份，磷酸二氢钾0.01份，磷酸氢二钾0.008份，用氢氧化钾将pH调节至5.0～9.0，加入2份凉茶渣，121℃，湿热灭菌20分钟；
凉茶渣：鸡蛋花、金银花、菊花、凉粉草、夏枯草、甘草和布渣叶共7种植物药物混合，煎制后的凉茶渣。
实施例1～8的设置：
一种纤维素酶、木聚糖酶和果胶酶活力的制备方法
按照10％的接种量接种(即1g干凉茶渣接种0.1mL的黑曲霉孢子悬液)将黑曲霉菌接种在凉茶渣培养基上，具体发酵条件如表1所示。
发酵后纤维素酶、木聚酶、果胶酶的测量：
1)发酵结束后，在发酵物中加入15mL的生理盐水，摇匀后过夜静置；
2)15000g高速离心，取上层，检测上清液中纤维素酶、木聚糖酶和果胶酶活力。
表1实施例1～8的设置和酶活力测试结果
注：因素A为含水量，含水量为当体系仅含有凉茶渣和水时，水的百分含量；
因素B为浸泡液pH值，浸泡液是指，一定体积水按照比例要求加入磷酸二氢钾和磷酸氢二钾后，再用3摩尔每升的氢氧化钾调节至固定pH值的溶液；
因素C为发酵温度；
因素D为发酵时间，单位为天。
实施例9
一种纤维素酶、木聚糖酶和果胶酶活力的制备方法
按照10％的接种量接种(即1g干凉茶渣接种0.1mL的黑曲霉孢子悬液)将黑曲霉菌接种在凉茶渣培养基上，在含水量为70％，浸泡液pH为9.0，发酵温度为31℃，发酵7天。
结果：纤维素酶活力为(1.00±0.04)×103U/g，木聚糖酶活力为(5.78±0.37)×103U/g，果胶酶活力为(5.71±0.13)×103U/g，三种酶活力的平均值为(4.16±0.17)×103U/g。
实施例10
一种纤维素酶、木聚糖酶和果胶酶活力的制备方法
按照10％的接种量接种(即1g干凉茶渣接种0.1mL的黑曲霉孢子悬液)将黑曲霉菌接种在凉茶渣培养基上，在含水量为70％，浸泡液pH为9.0，发酵温度为31℃的设置下发酵5天。
结果：纤维素酶活力为(1.05±0.06)×103U/g，木聚糖酶活力为(6.21±0.1)×103U/g，果胶酶活力为(4.11±0.0.02)×103U/g，三种酶活力的平均值为(3.79±0.04)×103U/g。
根据上述实施例，发现实施例9和实施例10的效果较好，能够有效生产三种酶(纤维素酶、木聚糖酶和果胶酶)。当硫酸铵用量为4％，葡萄糖用量为2％，菌种浓度为2×107cfu/mL，接种量为10％时，含水量为70％，浸泡液pH为9，发酵温度为31℃，发酵7天，是三种饲用酶的最佳生产工艺。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (10)
1.一种制备纤维素酶、木聚糖酶和果胶酶的方法，其特征在于，包含以下步骤：将黑曲霉菌悬液，接种到凉茶渣培养基上发酵；所述凉茶渣包括鸡蛋花、金银花、菊花、凉粉草、夏枯草、甘草和布渣叶。
2.根据权利要求1所述的方法，其特征在于，还包括预先将黑曲霉菌活化、传代，所述活化将黑曲霉菌接种到PDA固体培养基培养。
3.根据权利要求2所述的方法，其特征在于，培养条件为50％～90％湿度、28℃～37℃培养24～240h。
4.根据权利要求1所述的方法，其特征在于，所述菌悬液浓度为105～109cfu/mL，优选2×107cfu/mL。
5.根据权利要求1所述的方法，其特征在于，接种量为5％～25％。
6.根据权利要求1所述的方法，其特征在于，发酵温度为28℃～37℃，发酵时间为3～7天，发酵时的含水量为65％～85％，浸泡液pH为5～10。
7.根据权利要求1所述的方法，其特征在于，发酵温度为31℃，发酵时间为7天，发酵时的含水量为70％，浸泡液pH为9。
8.根据权利要求1所述的方法，其特征在于，所述凉茶渣培养基包括：凉茶渣粉末1～5份，硫酸铵0.04～0.20份，葡萄糖0～0.1份，磷酸二氢钾0.005～0.02份，磷酸氢二钾0.002～0.020份，水5.5～8.5份。
9.根据权利要求8所述的方法，其特征在于，所述凉茶渣培养基包括：凉茶渣粉末2份，硫酸铵0.08份，葡萄糖0.04份，磷酸二氢钾0.01份，磷酸氢二钾0.008份，水8份。
10.根据权利要求8或9所述的方法，其特征在于，所述凉茶渣粉末为：将鸡蛋花、金银花、菊花、凉粉草、夏枯草、甘草和布渣叶混合，煎制后得到的凉茶渣。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911389431.0A CN110964706A (zh) | 2019-12-30 | 2019-12-30 | 一种纤维素酶、木聚糖酶和果胶酶的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911389431.0A CN110964706A (zh) | 2019-12-30 | 2019-12-30 | 一种纤维素酶、木聚糖酶和果胶酶的制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110964706A true CN110964706A (zh) | 2020-04-07 |
Family
ID=70037182
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911389431.0A Pending CN110964706A (zh) | 2019-12-30 | 2019-12-30 | 一种纤维素酶、木聚糖酶和果胶酶的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110964706A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114058454A (zh) * | 2021-11-30 | 2022-02-18 | 汉源县昊业科技有限公司 | 一种啤酒澄清剂、制备方法及其应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106085881A (zh) * | 2016-07-12 | 2016-11-09 | 西北农林科技大学 | 一种黑曲霉菌株及其应用 |
| CN107373086A (zh) * | 2017-08-22 | 2017-11-24 | 吉林省农业科学院 | 一种利用中药渣生产的保健饲料添加剂及制备方法 |
-
2019
- 2019-12-30 CN CN201911389431.0A patent/CN110964706A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106085881A (zh) * | 2016-07-12 | 2016-11-09 | 西北农林科技大学 | 一种黑曲霉菌株及其应用 |
| CN107373086A (zh) * | 2017-08-22 | 2017-11-24 | 吉林省农业科学院 | 一种利用中药渣生产的保健饲料添加剂及制备方法 |
Non-Patent Citations (3)
| Title |
|---|
| 张熙等: "黑曲霉发酵产酶研究进展", 《化学与生物工程》 * |
| 王松林等: "黑曲霉菌种的筛选及培养条件的优化", 《造纸科学与技术》 * |
| 谢月琴等: "凉茶渣对育肥猪生长性能、胴体性状和肉品质的影响", 《动物营养学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114058454A (zh) * | 2021-11-30 | 2022-02-18 | 汉源县昊业科技有限公司 | 一种啤酒澄清剂、制备方法及其应用 |
| CN114058454B (zh) * | 2021-11-30 | 2024-02-23 | 汉源县昊业科技有限公司 | 一种啤酒澄清剂、制备方法及其应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100448979C (zh) | 一种酶法生产木糖的方法 | |
| CA3121566C (en) | Aspergillus oryzae blcy-006 strain and application thereof in preparation of galactooligosaccharides | |
| CN106993807A (zh) | 一种生姜酵素的制备方法 | |
| CN116064685B (zh) | 一种冠突散囊菌发酵食用中药的制备工艺及其应用 | |
| Darah et al. | Pomelo Citrus grandis (L.) Osbeck peel as an economical alternative substrate for fungal pectinase production | |
| CN103421851B (zh) | 一种用甘薯废弃物制备糖和乙醇的方法 | |
| CN105255745A (zh) | 一株产β-葡萄糖苷酶根霉及其发酵产酶方法 | |
| CN101020911A (zh) | 一种生物学提取方法及其应用 | |
| CN106974268A (zh) | 一种黑果枸杞酵素的制备方法 | |
| CN104762229B (zh) | 一种枯草芽孢杆菌菌株及其应用 | |
| CN102511650B (zh) | 一种利用菊芋糟渣发酵生产蛋白饲料的方法 | |
| CN110964706A (zh) | 一种纤维素酶、木聚糖酶和果胶酶的制备方法 | |
| CN102517188B (zh) | 一种在酿造过程中产生异麦芽低聚糖的黄酒小曲的制备方法 | |
| CN104789491A (zh) | 地衣芽孢杆菌菌株及其应用 | |
| CN110093281B (zh) | 桑黄液体深层发酵培养工艺 | |
| Shweta | Solid state fermentation for cellulase production | |
| Buenrostro-Figueroa et al. | Juice extraction from mango pulp using an enzymatic complex of Trichoderma sp. produced by solid-state fermentation | |
| CN106755179A (zh) | 一种适于细菌纤维素发酵的培养基 | |
| Aziz et al. | Bioconversion of acid-and gamma-ray-treated sweet potato residue to microbial protein by mixed cultures | |
| CN110938552A (zh) | 一种凉茶渣的降解并制备黑曲霉的方法 | |
| CN105087280B (zh) | 一种鸭血糯银杏低度黄酒的现代化大罐酿造方法 | |
| CN110791436B (zh) | 一株高产果胶酶的黑曲霉菌株及其应用 | |
| CN111019839A (zh) | 一种由黑曲霉和酵母菌联合发酵降解凉茶渣并生产益生菌的方法 | |
| CN114946996A (zh) | 一种柠檬酸菌丝渣液肽饲料及其制备方法 | |
| CN110885760B (zh) | 一种生产果胶酶的液体发酵方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200407 |