WO2020082419A1 - 一种发酵蛋白纯化多肽及其应用 - Google Patents

一种发酵蛋白纯化多肽及其应用 Download PDF

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WO2020082419A1
WO2020082419A1 PCT/CN2018/114228 CN2018114228W WO2020082419A1 WO 2020082419 A1 WO2020082419 A1 WO 2020082419A1 CN 2018114228 W CN2018114228 W CN 2018114228W WO 2020082419 A1 WO2020082419 A1 WO 2020082419A1
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fermented
fermentation
protein
water
peptides
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PCT/CN2018/114228
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English (en)
French (fr)
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白洪响
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白洪响
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Priority claimed from CN201811245099.6A external-priority patent/CN109266712A/zh
Priority claimed from CN201811245123.6A external-priority patent/CN109295143A/zh
Priority claimed from CN201811245116.6A external-priority patent/CN109295141A/zh
Priority claimed from CN201811245117.0A external-priority patent/CN109295142A/zh
Priority claimed from CN201811247425.7A external-priority patent/CN109295144A/zh
Application filed by 白洪响 filed Critical 白洪响
Publication of WO2020082419A1 publication Critical patent/WO2020082419A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

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  • the invention specifically relates to a polypeptide purified by fractionation from fermented protein and its use as a feed additive for pigs.
  • the purpose of the present invention is to provide a polypeptide purified from fermented protein fractionally and its use as a feed additive for pigs.
  • a fermentation enzymolysis peptide is prepared by the following steps:
  • Step 2 Fermentation and enzymatic hydrolysis of carnation protein (papain + yeast)
  • Step 3 Hierarchical purification of fermentation enzymatic peptides
  • Dissolve the fermented peptides with distilled water add 1mL of fermented peptides with 30mL of distilled water, and then purify the obtained fermented peptides with ultrafiltration membranes with molecular weight cutoffs of 3000u and 1000u, and freeze the components obtained by ultrafiltration Papain 1000u ⁇ 3000u fermentation enzymolysis peptide can be obtained after drying.
  • step 1 a 40 ° C water bath is stirred for 6 hours.
  • step 1 rinse gently with distilled water 3 times.
  • step 2 is inactivated in an oven at 105 ° C for 5 minutes.
  • step 2 centrifugation is performed at 12000 rpm for 25 minutes.
  • a fermentation enzymolysis peptide is prepared by the following steps:
  • Step 2 Fermentation and enzymatic hydrolysis of carnation protein (papain + yeast)
  • Step 3 Hierarchical purification of fermentation enzymatic peptides
  • Dissolve the fermentation enzyme peptide with distilled water add 1mL of fermentation enzyme peptide to 30mL of distilled water, and then use the ultrafiltration membrane with molecular weight cut-off of 5000u and 3000u to fractionally purify the obtained fermentation enzyme peptide, and freeze the components obtained by ultrafiltration Papain 3000u ⁇ 5000u fermentation enzymolysis peptide can be obtained after drying.
  • step 1 a 40 ° C water bath is stirred for 6 hours.
  • step 1 rinse gently with distilled water 3 times.
  • step 2 is inactivated in an oven at 105 ° C for 5 minutes.
  • step 2 centrifugation is performed at 12000 rpm for 25 minutes.
  • a fermentation enzymolysis peptide is prepared by the following steps:
  • Step 2 Fermentation and enzymatic hydrolysis of carnation protein (papain + yeast)
  • Step 3 Hierarchical purification of fermentation enzymatic peptides
  • Dissolve the fermented peptides with distilled water add 1mL of fermented peptides with 30mL of distilled water, and then purify the obtained fermented peptides with ultrafiltration membranes with molecular weight cutoffs of 10000u and 7000u, and freeze the components obtained by ultrafiltration Papain 7000u ⁇ 10000u fermentation enzyme digestion peptide can be obtained after drying.
  • step 1 a 40 ° C water bath is stirred for 6 hours.
  • step 1 rinse gently with distilled water 3 times.
  • step 2 is inactivated in an oven at 105 ° C for 5 minutes.
  • step 2 centrifugation is performed at 12000 rpm for 25 minutes.
  • a fermentation enzymolysis peptide is prepared by the following steps:
  • Step 2 Fermentation and enzymolysis of carnation protein (neutral protease + yeast)
  • the carrageenan protein was added to the neutral protease at 350U / g protein, and Saccharomyces cerevisiae was inoculated at the inoculation amount of 2 ⁇ 108CFU / g, and then 45 ° C warm water was added according to the feed water ratio of 1: 5, and the enzyme was hydrolyzed at room temperature for 24h. After the fermentation is completed, the mixture is stirred evenly, and the sample is immediately put into an oven at 105 ° C for inactivation and cooled to room temperature. After cooling, the sample is centrifuged, the supernatant is taken, and freeze-dried to obtain a fermented peptide.
  • Step 3 Hierarchical purification of fermentation enzymatic peptides
  • Dissolve the fermentation enzyme peptide with distilled water add 1mL of fermentation enzyme peptide to 30mL of distilled water, and then use the ultrafiltration membrane with molecular weight cut-off of 5000u and 3000u to fractionally purify the obtained fermentation enzyme peptide, and freeze the components obtained by ultrafiltration After drying, the neutral protease 3000u-5000u fermentation enzymolysis peptide can be obtained.
  • step 1 a 40 ° C water bath is stirred for 6 hours.
  • step 1 rinse gently with distilled water 3 times.
  • step 2 is inactivated in an oven at 105 ° C for 5 minutes.
  • step 2 centrifugation is performed at 12000 rpm for 25 minutes.
  • a fermentation enzymolysis peptide is prepared by the following steps:
  • Step 2 Fermentation and enzymolysis of carnation protein (neutral protease + yeast)
  • the carrageenan protein was added to the neutral protease at 350U / g protein, and Saccharomyces cerevisiae was inoculated at the inoculation amount of 2 ⁇ 108CFU / g, and then 45 ° C warm water was added according to the feed water ratio of 1: 5, and the enzyme was hydrolyzed at room temperature for 24h. After the fermentation is completed, the mixture is stirred evenly, and the sample is immediately put into an oven at 105 ° C for inactivation and cooled to room temperature. After cooling, the sample is centrifuged, the supernatant is taken, and freeze-dried to obtain a fermented peptide.
  • Step 3 Hierarchical purification of fermentation enzymatic peptides
  • Dissolve the fermented peptides in distilled water add 1mL of fermented peptides to 30mL of distilled water, and then use the ultrafiltration membranes with molecular weight cutoff of 7000u and 5000u to carry out fractional purification of the obtained fermented peptides, and freeze the components obtained by ultrafiltration After drying, the neutral protease 5000u ⁇ 7000u fermentation enzymolysis peptide can be obtained.
  • step 1 a 40 ° C water bath is stirred for 6 hours.
  • step 1 rinse gently with distilled water 3 times.
  • step 2 is inactivated in an oven at 105 ° C for 5 minutes.
  • step 2 centrifugation is performed at 12000 rpm for 25 minutes.
  • the present invention provides papain 1000u ⁇ 3000u fermentation enzymolysis peptide, papain 3000u ⁇ 5000u fermentation enzymolysis peptide, papain 7000u ⁇ 10000u fermentation enzymolysis peptide, neutral protease 3000u ⁇ 5000u fermentation enzymolysis peptide and neutral protease 5000u ⁇ 7000u fermented enzymolysis peptide can significantly reduce the feed-weight ratio of fattening pigs, significantly improve the diarrhea of fattening pigs, and can be used as a feed additive for pigs to improve the growth performance and diarrhea of fattening pigs.
  • Figure 1 shows the growth performance (feed weight ratio) of fattening pigs in each group
  • Figure 2 shows the diarrhea rate (%) of fattening pigs in each group.
  • the Pteris viridis was picked from Suzhou City, Jiangsu Province, and identified as Pteris Pteris multifida Poir, washed and dried for use.
  • Papain was purchased from Shanghai Yuanye Biotechnology Co., Ltd .; neutral protease was purchased from Beijing Aoboxing Biotechnology Co., Ltd.
  • the yeast is Saccharomyces cerevisiae, the Latin scientific name Saccharomyces cerevisiae, purchased from CGMCC, and the deposited strain number is CGMCC 2.3886.
  • the raw material composition includes corn, soybean meal, fish meal, dicalcium phosphate, stone powder, sodium chloride, lysine, vitamin A, vitamin E, vitamin D3, vitamin B6, copper sulfate , Ferrous sulfate, zinc sulfate, manganese sulfate, sodium selenite, calcium propionate, etc.
  • Dissolve the fermented peptides with distilled water add 1mL of fermented peptides with 30mL of distilled water, and then use the ultrafiltration membranes with molecular weight cut-offs of 10000u, 7000u, 5000u, 3000u, and 1000u to fractionally purify the obtained fermented peptides.
  • the components obtained by ultrafiltration are freeze-dried to obtain peptides of different molecular weights, specifically:
  • Papain 1000u ⁇ 3000u fermentation enzymolysis peptide referred to as wood 1000u ⁇ 3000u;
  • Neutral protease 5000u ⁇ 7000u fermentation enzymolysis peptide (referred to as 5000u ⁇ 7000u);
  • Neutral protease 7000u ⁇ 10000u fermentation enzymatic hydrolysis peptide (abbreviated as 7000u ⁇ 10000u).
  • control group commercial fattening pigs aged 80 to 90 days and weighing about 30 kg were selected and randomly divided into a control group and groups 1 to 8. There are 2 repetitions in each group, a total of 18 pens with 15 pigs per pen.
  • the control group was fed the basic diet, and the experimental groups 1-8 were fed the basic diet supplemented with 10% of the above-mentioned fermentation peptides of different molecular weights.
  • test period is 28 days, free to eat and drink water every day, immunization, disinfection, etc. are carried out according to the immunization program.
  • the fattening pigs were weighed on an empty stomach at the beginning (1d) and the end (28d) of the experiment, and the consumption amount was settled in units of pens. Calculate the average daily gain, weigh each fattening pig at the beginning (1d) and end (28d) of the experiment, record the feed intake of each group in detail, calculate the average daily gain, average daily feed intake and feed-to-weight ratio .
  • Feed-weight ratio average daily feed intake / average daily gain
  • Diarrhea rate (%) total number of diarrhea days of fattening pigs in each group / total observation days of fattening pigs in each group ⁇ 100%
  • the growth performance and diarrhea rate of fattening pigs in each group are shown in Table 1 and Figures 1-2.
  • the results show that papain 1000u ⁇ 3000u fermentation enzymolysis peptide, papain 3000u ⁇ 5000u fermentation enzymolysis peptide, papain 7000u ⁇ 10000u fermentation enzymolysis peptide, neutral protease 3000u ⁇ 5000u fermentation enzymolysis peptide and neutral protease 5000u ⁇ 7000u Fermented enzymatically hydrolyzed peptides can significantly reduce the feed-to-weight ratio of fattening pigs, and significantly improve the diarrhea of fattening pigs, while other fermented enzymatically hydrolyzed peptides have no obvious improvement effect.
  • the present invention provides papain 1000u-3000u fermentation enzymolysis peptide, papain 3000u-5000u fermentation enzymolysis peptide, papain 7000u-10000u fermentation enzymolysis peptide, neutral protease 3000u-5000u fermentation enzymolysis peptide and medium Fermented protease 5000u ⁇ 7000u fermented peptide can significantly reduce the feed-weight ratio of fattening pigs, significantly improve the diarrhea of fattening pigs, and can be used as a feed additive for pigs to improve the growth performance and diarrhea of fattening pigs.

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Abstract

一种发酵蛋白纯化多肽及其应用，木瓜蛋白酶1000u～3000u发酵酶解肽、木瓜蛋白酶3000u～5000u发酵酶解肽、木瓜蛋白酶7000u～10000u发酵酶解肽、中性蛋白酶3000u～5000u发酵酶解肽和中性蛋白酶5000u～7000u发酵酶解肽可以显著降低育肥猪的料重比，显著改善育肥猪的腹泻情况，可以用作猪饲料添加剂改善育肥猪的生长性能和腹泻情况。

Description

一种发酵蛋白纯化多肽及其应用 技术领域
本发明具体涉及一种从发酵蛋白中分级纯化的多肽及用作猪饲料添加剂的用途。
背景技术
随着我国经济快速发展和进步，人们的物质生活水平得到了显著提高，对动物产品的需求量也在不断提高。猪规模化养殖技术不断完善，养殖户在规模和养殖的数量方面得到了显著的提高，但是随着规模化养殖的不断开展，由于养殖户养殖技术存在欠缺，导致了猪的养殖效益不高，有时还会对养殖户造成严重的经济损失。
如何通过改善饲料提高猪的生产性能，降低料肉比，对收益影响很大。
发明内容
本发明的目的在于提供一种从发酵蛋白中分级纯化的多肽及用作猪饲料添加剂的用途。
本发明上述目的通过如下技术方案实现：
一种发酵酶解肽，通过如下步骤制备得到：
步骤1、凤尾草蛋白的提取
将干燥的凤尾草粉碎成50～60目粉末，加入水，用氢氧化钠溶液调节其pH值至8.5，40℃水浴搅拌，过滤，收集滤液；滤液用盐酸调节pH值至等电点4.5，室温静置过夜，离心，弃上清得到乳白色蛋白质沉淀，用蒸馏水轻轻漂洗，备用。
步骤2、凤尾草蛋白的发酵酶解(木瓜蛋白酶+酵母菌)
将凤尾草蛋白按300U/g蛋白加入木瓜蛋白酶，按2×108CFU/g接种量接种酿酒酵母，之后按料水比1:5加入45℃温水，置于室温条件下发酵酶解24h。发酵完成后搅拌均匀，样品立刻放入105℃烘箱中灭活，冷却至常温；冷却后，离心，取上清，冷冻干燥得发酵酶解肽。
步骤3、发酵酶解肽的分级纯化
将发酵酶解肽用蒸馏水溶解，1g发酵酶解肽加30mL蒸馏水，然后依次用截留分子量为3000u、1000u的超滤膜对所得到的发酵酶解肽进行分级纯化，将超滤所得组分冷冻干燥即得木瓜蛋白酶1000u～3000u发酵酶解肽。
优选地，步骤1中1kg粉末加入5L水。
优选地，步骤1中40℃水浴搅拌6h。
优选地，步骤1中用蒸馏水轻轻漂洗3次。
优选地，步骤2中105℃烘箱中灭活5min。
优选地，步骤2中12000rpm离心25分钟。
上述发酵酶解肽用作猪饲料添加剂的用途。
一种发酵酶解肽，通过如下步骤制备得到：
步骤1、凤尾草蛋白的提取
将干燥的凤尾草粉碎成50～60目粉末，加入水，用氢氧化钠溶液调节其pH值至8.5，40℃水浴搅拌，过滤，收集滤液；滤液用盐酸调节pH值至等电点4.5，室温静置过夜，离心，弃上清得到乳白色蛋白质沉淀，用蒸馏水轻轻漂洗，备用。
步骤2、凤尾草蛋白的发酵酶解(木瓜蛋白酶+酵母菌)
将凤尾草蛋白按300U/g蛋白加入木瓜蛋白酶，按2×108CFU/g接种量接种酿酒酵母，之后按料水比1:5加入45℃温水，置于室温条件下发酵酶解24h。发酵完成后搅拌均匀，样品立刻放入105℃烘箱中灭活，冷却至常温；冷却后，离心，取上清，冷冻干燥得发酵酶解肽。
步骤3、发酵酶解肽的分级纯化
将发酵酶解肽用蒸馏水溶解，1g发酵酶解肽加30mL蒸馏水，然后依次用截留分子量为5000u、3000u的超滤膜对所得到的发酵酶解肽进行分级纯化，将超滤所得组分冷冻干燥即得木瓜蛋白酶3000u～5000u发酵酶解肽。
优选地，步骤1中1kg粉末加入5L水。
优选地，步骤1中40℃水浴搅拌6h。
优选地，步骤1中用蒸馏水轻轻漂洗3次。
优选地，步骤2中105℃烘箱中灭活5min。
优选地，步骤2中12000rpm离心25分钟。
上述发酵酶解肽用作猪饲料添加剂的用途。
一种发酵酶解肽，通过如下步骤制备得到：
步骤1、凤尾草蛋白的提取
将干燥的凤尾草粉碎成50～60目粉末，加入水，用氢氧化钠溶液调节其pH值至8.5，40℃水浴搅拌，过滤，收集滤液；滤液用盐酸调节pH值至等电点4.5，室温静置过夜，离心，弃上清得到乳白色蛋白质沉淀，用蒸馏水轻轻漂洗，备用。
步骤2、凤尾草蛋白的发酵酶解(木瓜蛋白酶+酵母菌)
将凤尾草蛋白按300U/g蛋白加入木瓜蛋白酶，按2×108CFU/g接种量接种酿酒酵母，之后按料水比1:5加入45℃温水，置于室温条件下发酵酶解24h。发酵完成后搅拌均匀，样品立刻放入105℃烘箱中灭活，冷却至常温；冷却后，离心，取上清，冷冻干燥得发酵酶解肽。
步骤3、发酵酶解肽的分级纯化
将发酵酶解肽用蒸馏水溶解，1g发酵酶解肽加30mL蒸馏水，然后依次用截留分子量为 10000u、7000u的超滤膜对所得到的发酵酶解肽进行分级纯化，将超滤所得组分冷冻干燥即得木瓜蛋白酶7000u～10000u发酵酶解肽。
优选地，步骤1中1kg粉末加入5L水。
优选地，步骤1中40℃水浴搅拌6h。
优选地，步骤1中用蒸馏水轻轻漂洗3次。
优选地，步骤2中105℃烘箱中灭活5min。
优选地，步骤2中12000rpm离心25分钟。
上述发酵酶解肽用作猪饲料添加剂的用途。
一种发酵酶解肽，通过如下步骤制备得到：
步骤1、凤尾草蛋白的提取
将干燥的凤尾草粉碎成50～60目粉末，加入水，用氢氧化钠溶液调节其pH值至8.5，40℃水浴搅拌，过滤，收集滤液；滤液用盐酸调节pH值至等电点4.5，室温静置过夜，离心，弃上清得到乳白色蛋白质沉淀，用蒸馏水轻轻漂洗，备用。
步骤2、凤尾草蛋白的发酵酶解(中性蛋白酶+酵母菌)
将凤尾草蛋白按350U/g蛋白加入中性蛋白酶，按2×108CFU/g接种量接种酿酒酵母，之后按料水比1:5加入45℃温水，置于室温条件下发酵酶解24h。发酵完成后搅拌均匀，样品立刻放入105℃烘箱中灭活，冷却至常温；冷却后，离心，取上清，冷冻干燥得发酵酶解肽。
步骤3、发酵酶解肽的分级纯化
将发酵酶解肽用蒸馏水溶解，1g发酵酶解肽加30mL蒸馏水，然后依次用截留分子量为5000u、3000u的超滤膜对所得到的发酵酶解肽进行分级纯化，将超滤所得组分冷冻干燥即得中性蛋白酶3000u～5000u发酵酶解肽。
优选地，步骤1中1kg粉末加入5L水。
优选地，步骤1中40℃水浴搅拌6h。
优选地，步骤1中用蒸馏水轻轻漂洗3次。
优选地，步骤2中105℃烘箱中灭活5min。
优选地，步骤2中12000rpm离心25分钟。
上述发酵酶解肽用作猪饲料添加剂的用途。
一种发酵酶解肽，通过如下步骤制备得到：
步骤1、凤尾草蛋白的提取
将干燥的凤尾草粉碎成50～60目粉末，加入水，用氢氧化钠溶液调节其pH值至8.5，40℃水浴搅拌，过滤，收集滤液；滤液用盐酸调节pH值至等电点4.5，室温静置过夜，离心，弃 上清得到乳白色蛋白质沉淀，用蒸馏水轻轻漂洗，备用。
步骤2、凤尾草蛋白的发酵酶解(中性蛋白酶+酵母菌)
将凤尾草蛋白按350U/g蛋白加入中性蛋白酶，按2×108CFU/g接种量接种酿酒酵母，之后按料水比1:5加入45℃温水，置于室温条件下发酵酶解24h。发酵完成后搅拌均匀，样品立刻放入105℃烘箱中灭活，冷却至常温；冷却后，离心，取上清，冷冻干燥得发酵酶解肽。
步骤3、发酵酶解肽的分级纯化
将发酵酶解肽用蒸馏水溶解，1g发酵酶解肽加30mL蒸馏水，然后依次用截留分子量为7000u、5000u的超滤膜对所得到的发酵酶解肽进行分级纯化，将超滤所得组分冷冻干燥即得中性蛋白酶5000u～7000u发酵酶解肽。
优选地，步骤1中1kg粉末加入5L水。
优选地，步骤1中40℃水浴搅拌6h。
优选地，步骤1中用蒸馏水轻轻漂洗3次。
优选地，步骤2中105℃烘箱中灭活5min。
优选地，步骤2中12000rpm离心25分钟。
上述发酵酶解肽用作猪饲料添加剂的用途。
有益效果：
本发明提供的木瓜蛋白酶1000u～3000u发酵酶解肽、木瓜蛋白酶3000u～5000u发酵酶解肽、木瓜蛋白酶7000u～10000u发酵酶解肽、中性蛋白酶3000u～5000u发酵酶解肽和中性蛋白酶5000u～7000u发酵酶解肽可以显著降低育肥猪的料重比，显著改善育肥猪的腹泻情况，可以用作猪饲料添加剂改善育肥猪的生长性能和腹泻情况。
附图说明
图1为各组育肥猪生长性能(料重比)；
图2为各组育肥猪腹泻率(％)。
具体实施方式
下面结合附图和实施例具体介绍本发明实质性内容，但并不以此限定本发明的保护范围。
一、实验材料
凤尾草采摘自江苏省苏州市，鉴定为凤尾草Pteris multifida Poir，洗净后晒干备用。
木瓜蛋白酶购自上海源叶生物科技有限责任公司；中性蛋白酶购自北京奥博星生物技术有限责任公司。酵母菌为酿酒酵母，拉丁学名Saccharomyces cerevisiae，购自CGMCC，保藏菌株编号CGMCC 2.3886。
商品育肥猪购自广西奇昌种猪养殖有限公司。基础日粮购自武汉新农翔饲料有限公司， 原料组成包括玉米、豆粕、鱼粉、磷酸氢钙、石粉、氯化钠、赖氨酸、维生素A、维生素E、维生素D3、维生素B6、硫酸铜、硫酸亚铁、硫酸锌、硫酸锰、亚硒酸钠、丙酸钙等。
二、实验方法
1、凤尾草蛋白的提取
将干燥的凤尾草粉碎成50～60目粉末，1kg粉末加入5L水，用氢氧化钠溶液调节其pH值至8.5，40℃水浴搅拌6h，过滤，收集滤液；滤液用盐酸调节pH值至等电点4.5，室温静置过夜，离心，弃上清得到乳白色蛋白质沉淀，用蒸馏水轻轻漂洗3次，备用。
2、凤尾草蛋白的发酵酶解(木瓜蛋白酶+酵母菌)
将凤尾草蛋白按300U/g蛋白加入木瓜蛋白酶，按2×10 8CFU/g接种量接种酿酒酵母，之后按照料水比1:5(质量比)加入45℃温水，置于室温条件下发酵酶解24h。发酵完成后搅拌均匀，样品立刻放入105℃烘箱中灭活5min，冷却至常温；冷却后，12000rpm离心25分钟，取上清，冷冻干燥得到发酵酶解肽。
3、凤尾草蛋白的发酵酶解(中性蛋白酶+酵母菌)
将凤尾草蛋白按350U/g蛋白加入中性蛋白酶，按2×10 8CFU/g接种量接种酿酒酵母，之后按照料水比1:5(质量比)加入45℃温水，置于室温条件下发酵酶解24h。发酵完成后搅拌均匀，样品立刻放入105℃烘箱中灭活5min，冷却至常温；冷却后，12000rpm离心25分钟，取上清，冷冻干燥得到发酵酶解肽。
4、发酵酶解肽的分级纯化
将发酵酶解肽用蒸馏水溶解，1g发酵酶解肽加30mL蒸馏水，然后依次用截留分子量为10000u、7000u、5000u、3000u以及1000u的超滤膜对所得到的发酵酶解肽进行分级纯化，将超滤所得组分冷冻干燥即得不同分子量大小的多肽，具体为：
(1)木瓜蛋白酶1000u～3000u发酵酶解肽(简称木1000u～3000u)；
(2)木瓜蛋白酶3000u～5000u发酵酶解肽(简称木3000u～5000u)；
(3)木瓜蛋白酶5000u～7000u发酵酶解肽(简称木5000u～7000u)；
(4)木瓜蛋白酶7000u～10000u发酵酶解肽(简称木7000u～10000u)；
(5)中性蛋白酶1000u～3000u发酵酶解肽(简称中1000u～3000u)；
(6)中性蛋白酶3000u～5000u发酵酶解肽(简称中3000u～5000u)；
(7)中性蛋白酶5000u～7000u发酵酶解肽(简称中5000u～7000u)；
(8)中性蛋白酶7000u～10000u发酵酶解肽(简称中7000u～10000u)。
5、育肥猪分组及饲喂
试验选用80～90日龄、体重为30kg左右的商品育肥猪，随机分为对照组、试验1～8组。 每组设2个重复，共18栏，每栏15头猪。对照组饲喂基础日粮，试验1～8组分别饲喂添加10％上述不同分子量发酵酶解肽的基础日粮。
6、育肥猪饲养管理
试验周期为28d，每天自由采食和饮水，免疫、消毒等均按免疫程序进行。
7、生长性能检测
分别在试验开始(1d)和结束(28d)对每头育肥猪空腹称重，以栏为单位结算耗料量。计算平均日增重，分别在试验开始(1d)和结束(28d)对每头育肥猪空腹称重，详细记录各组采食量，计算平均日增重，平均日采食量和料重比。
料重比＝平均日采食量/平均日增重
8、腹泻率检测
在整个试验期间，为了记录育肥猪腹泻的严重程度，每天多次观察育肥猪排便情况并记录。最后计算腹泻率。
腹泻率(％)＝各组育肥猪腹泻天数总和/各组育肥猪总观察天数×100％
9、数据处理
采用SPSS17.0软件处理数据。One-Way ANOVA分析用于比较不同处理组细胞之间的差异。以P＜0.05为差异具有统计学意义。
三、实验结果
各组育肥猪生长性能和腹泻率结果见表1和图1～2。结果可见，木瓜蛋白酶1000u～3000u发酵酶解肽、木瓜蛋白酶3000u～5000u发酵酶解肽、木瓜蛋白酶7000u～10000u发酵酶解肽、中性蛋白酶3000u～5000u发酵酶解肽和中性蛋白酶5000u～7000u发酵酶解肽可以显著降低育肥猪的料重比，显著改善育肥猪的腹泻情况，而其他几种发酵酶解肽改善作用不明显。
表1各组育肥猪生长性能和腹泻率
组别 料重比 腹泻率(％)
对照 2.81 22.5
木1000u～3000u 2.45 6.4
木3000u～5000u 2.44 6.7
木5000u～7000u 2.79 20.6
木7000u～10000u 2.47 6.5
中1000u～3000u 2.80 19.8
中3000u～5000u 2.42 6.3
中5000u～7000u 2.43 6.5
中7000u～10000u 2.77 20.2
综上可见，本发明提供的木瓜蛋白酶1000u～3000u发酵酶解肽、木瓜蛋白酶3000u～5000u发酵酶解肽、木瓜蛋白酶7000u～10000u发酵酶解肽、中性蛋白酶3000u～5000u发酵酶解肽和中性蛋白酶5000u～7000u发酵酶解肽可以显著降低育肥猪的料重比，显著改善育肥猪的腹泻情况，可以用作猪饲料添加剂改善育肥猪的生长性能和腹泻情况。
上述实施例的作用在于具体介绍本发明的实质性内容，但本领域技术人员应当知道，不应将本发明的保护范围局限于该具体实施例。

Claims (7)

  1. 一种发酵酶解肽，其特征在于：
    通过如下步骤制备得到：
    步骤1、凤尾草蛋白的提取
    将干燥的凤尾草粉碎成50～60目粉末，加入水，用氢氧化钠溶液调节其pH值至8.5，40℃水浴搅拌，过滤，收集滤液；滤液用盐酸调节pH值至等电点4.5，室温静置过夜，离心，弃上清得到乳白色蛋白质沉淀，用蒸馏水轻轻漂洗，备用。
    步骤2、凤尾草蛋白的发酵酶解(木瓜蛋白酶+酵母菌)
    将凤尾草蛋白按300U/g蛋白加入木瓜蛋白酶，按2×10 8CFU/g接种量接种酿酒酵母，之后按料水比1:5加入45℃温水，置于室温条件下发酵酶解24h。发酵完成后搅拌均匀，样品立刻放入105℃烘箱中灭活，冷却至常温；冷却后，离心，取上清，冷冻干燥得发酵酶解肽。
    步骤3、发酵酶解肽的分级纯化
    将发酵酶解肽用蒸馏水溶解，1g发酵酶解肽加30mL蒸馏水，然后依次用截留分子量为10000u、7000u、5000u、3000u以及1000u的超滤膜对所得到的发酵酶解肽进行分级纯化，将超滤所得组分冷冻干燥即得分子量1000u～3000u、3000u～5000u或7000u～10000u的多肽；
    或通过如下步骤制备得到：
    步骤1、凤尾草蛋白的提取
    将干燥的凤尾草粉碎成50～60目粉末，加入水，用氢氧化钠溶液调节其pH值至8.5，40℃水浴搅拌，过滤，收集滤液；滤液用盐酸调节pH值至等电点4.5，室温静置过夜，离心，弃上清得到乳白色蛋白质沉淀，用蒸馏水轻轻漂洗，备用。
    步骤2、凤尾草蛋白的发酵酶解(中性蛋白酶+酵母菌)
    将凤尾草蛋白按350U/g蛋白加入中性蛋白酶，按2×10 8CFU/g接种量接种酿酒酵母，之后按料水比1:5加入45℃温水，置于室温条件下发酵酶解24h。发酵完成后搅拌均匀，样品立刻放入105℃烘箱中灭活，冷却至常温；冷却后，离心，取上清，冷冻干燥得发酵酶解肽。
    步骤3、发酵酶解肽的分级纯化
    将发酵酶解肽用蒸馏水溶解，1g发酵酶解肽加30mL蒸馏水，然后依次用截留分子量为10000u、7000u、5000u、3000u以及1000u的超滤膜对所得到的发酵酶解肽进行分级纯化，将超滤所得组分冷冻干燥即得分子量3000u～5000u、5000u～7000u的多肽。
  2. 根据权利要求1所述的发酵酶解肽，其特征在于：步骤1中1kg粉末加入5L水。
  3. 根据权利要求1所述的发酵酶解肽，其特征在于：步骤1中40℃水浴搅拌6h。
  4. 根据权利要求1所述的发酵酶解肽，其特征在于：步骤1中用蒸馏水轻轻漂洗3次。
  5. 根据权利要求1所述的发酵酶解肽，其特征在于：步骤2中105℃烘箱中灭活5min。
  6. 根据权利要求1所述的发酵酶解肽，其特征在于：步骤2中12000rpm离心25分钟。
  7. 权利要求1～6任一所述发酵酶解肽用作猪饲料添加剂的用途。
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