CN115708547A - Application of functional fermented feed beneficial to improving micropterus salmoides glycolipid metabolism - Google Patents
Application of functional fermented feed beneficial to improving micropterus salmoides glycolipid metabolism Download PDFInfo
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- CN115708547A CN115708547A CN202211297013.0A CN202211297013A CN115708547A CN 115708547 A CN115708547 A CN 115708547A CN 202211297013 A CN202211297013 A CN 202211297013A CN 115708547 A CN115708547 A CN 115708547A
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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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
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Abstract
Description
技术领域:Technical field:
本发明属于动物饲料领域,具体涉及一种有利于改善加州鲈糖脂代谢功能性发酵饲料的应用。The invention belongs to the field of animal feed, and in particular relates to the application of a functional fermented feed that is beneficial to improving the glucose and lipid metabolism of California perch.
背景技术:Background technique:
加州鲈(Micropterus salmoides)隶属鲈形目太阳鱼科(Ceutrarchidae),又名大口黑鲈,为肉食性温水鱼类,我国重要的淡水养殖名特优品种,肉质鲜美,营养价值高。根据《2022中国渔业统计年鉴》显示,我国加州鲈养殖产量已经达到了70.21万吨,目前加州鲈已基本解决了从冰鲜鱼到配合饲料的养殖问题,但由于加州鲈本身对糖脂利用率差,摄食人工饲料易于导致加州鲈糖脂代谢紊乱,腹腔脂肪蓄积、肝脏肿大,严重的影响了加州鲈肉质品质及商品价值。肠道菌群是肠道屏障中的重要成员,影响着宿主的营养吸收、能量代谢以及免疫应答。机体营养代谢障碍和肠道菌群失调有着密切的联系,调节肠道微生物是调控机体营养代谢障碍重要方式。Micropterus salmoides belongs to the family Ceutrarchidae of the order Perciformes, also known as largemouth bass. It is a warm-water carnivorous fish. According to the "2022 China Fishery Statistical Yearbook", the aquaculture output of California perch in my country has reached 702,100 tons. At present, California perch has basically solved the problem of farming from chilled fish to compound feed. Artificial feed can easily lead to glucose and lipid metabolism disorder, abdominal fat accumulation, and hepatomegaly, which seriously affect the meat quality and commodity value of California sea bass. Gut flora is an important member of the intestinal barrier, affecting the host's nutrient absorption, energy metabolism and immune response. There is a close relationship between the body's nutrient metabolism disorder and the intestinal flora imbalance, and the regulation of intestinal microbes is an important way to regulate the body's nutrient metabolism disorder.
饲料经过功能性微生物发酵饲喂养殖动物,通过功能性微生物及次生代谢产物靶向调节肠道菌群及代谢产物是预防或治疗机体营养代谢障碍疾病的非常有效的方法。The feed is fermented by functional microorganisms to feed farmed animals, and the targeted regulation of intestinal flora and metabolites through functional microorganisms and secondary metabolites is a very effective method for preventing or treating nutritional metabolism disorders in the body.
目前未见有关促进加州鲈糖脂代谢功能性发酵饲料制备方法相关专利。At present, there is no relevant patent related to the preparation method of functional fermented feed that promotes glucose and lipid metabolism in California sea bass.
发明内容:Invention content:
本发明的目的是提供发酵饲料在制备改善加州鲈糖脂代谢功能性发酵饲料中的应用。The purpose of the invention is to provide the application of fermented feed in the preparation of functional fermented feed for improving the glucose and lipid metabolism of California perch.
本发明的发酵饲料,即提高加州鲈糖脂代谢功能性发酵饲料,其制备方法是:Fermented feed of the present invention, promptly improves the functional fermented feed of California perch glucose and lipid metabolism, and its preparation method is:
将枯草芽孢杆菌、酿酒酵母、产罗伊氏菌素的罗伊氏乳杆菌菌液混合,得到复合菌剂,将复合菌剂接种到固体培养基中进行厌氧发酵,厌氧发酵后再加入加州鲈预混合饲料和魔芋精粉,制粒获得发酵饲料;Mix Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus reuteri producing reuterin to obtain a composite bacterial agent, inoculate the composite bacterial agent into a solid medium for anaerobic fermentation, and then add it after anaerobic fermentation California perch premixed feed and konjac powder, pelleted to obtain fermented feed;
所述的固体培养基是:按质量分数计,包括玉米粉10%-20%,豆粕35-55%,鱼粉25-45%,麦麸5-10%;混合后加水至水分含量为25-40%,混合均匀得到固体培养基。The solid culture medium is: in terms of mass fraction, it includes 10%-20% of corn flour, 35-55% of soybean meal, 25-45% of fish meal, and 5-10% of wheat bran; after mixing, add water until the water content is 25- 40%, mixed evenly to obtain a solid medium.
优选,是将枯草芽孢杆菌、酿酒酵母、产罗伊氏菌素的罗伊氏乳杆菌菌液按照体积比1-3:1-3:4-8混合,得到复合菌剂,将复合菌剂按照固体培养基重量10-20%接种到固体培养基中,28-35℃恒温厌氧发酵,发酵时间为72-96h,厌氧发酵结束后,在厌氧发酵料中添加厌氧发酵料重量0.5%-1%加州鲈预混合饲料和0.1-0.5%的魔芋精粉,按不同生长阶段加州鲈饲料粒径要求,常温制软颗粒饲料。Preferably, Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus reuteri bacteria liquid producing reuterin are mixed according to the volume ratio of 1-3:1-3:4-8 to obtain a composite bacterial agent, and the composite bacterial agent Inoculate 10-20% of the weight of the solid medium into the solid medium, perform anaerobic fermentation at a constant temperature of 28-35°C, and the fermentation time is 72-96h. After the anaerobic fermentation is completed, add the weight of the anaerobic fermentation material to the anaerobic fermentation material 0.5%-1% California perch premixed feed and 0.1-0.5% konjac fine powder, according to the particle size requirements of California perch feed at different growth stages, soft pellet feed is made at room temperature.
优选,枯草芽孢杆菌、酿酒酵母、产罗伊氏菌素的罗伊氏乳杆菌菌液,其活菌数都在1.0×109cfu/mL以上。Preferably, the number of live bacteria of Bacillus subtilis, Saccharomyces cerevisiae, and reuterin-producing Lactobacillus reuteri are all above 1.0×10 9 cfu/mL.
优选,所述的枯草芽孢杆菌菌液的制备方法是:挑取枯草芽孢杆菌接种到LB培养基中,37±1℃摇床活化16-24h;然后将活化好的的菌液按体积比3-6%接种量接种到种子液体培养基中,37±1℃培养16-24h得到枯草芽孢杆菌种子液;Preferably, the preparation method of the Bacillus subtilis bacterial liquid is as follows: pick the Bacillus subtilis and inoculate it into LB medium, activate it on a shaker at 37±1°C for 16-24h; then mix the activated bacterial liquid with a volume ratio of 3 Inoculate -6% of the inoculation amount into the seed liquid medium, and cultivate at 37±1°C for 16-24 hours to obtain the Bacillus subtilis seed liquid;
所述的种子液体培养基为:按质量分数计,包括葡萄糖0.5%,可溶性淀粉1.5%,蛋白胨2%,酵母粉0.5%,硫酸镁0.2%,磷酸氢二钾0.2%,硫酸锰,0.03%,碳酸钙0.24%,pH7.0±0.2。The seed liquid culture medium is: by mass fraction, including glucose 0.5%, soluble starch 1.5%,
优选,所述的酿酒酵母菌液是挑取酿酒酵母斜面菌种接种到PDA液体培养基中,28±1℃摇床活化20-24h;然后将活化好的菌液按体积比2-5%接种量接种到种子液体培养基中,28±1℃培养18-24h得到酿酒酵母种子液;Preferably, the Saccharomyces cerevisiae liquid is inoculated into PDA liquid culture medium by picking Saccharomyces cerevisiae slant strains and activating it on a shaker at 28±1°C for 20-24h; then the activated bacterium liquid is 2-5% by volume The inoculation amount was inoculated into the seed liquid medium, and cultivated at 28±1°C for 18-24 hours to obtain the Saccharomyces cerevisiae seed liquid;
所述的种子液体培养基为:按质量分数计,包括葡萄糖2%,蔗糖2%,大豆蛋白胨3%,酵母粉1%,磷酸氢二钾0.5%,尿素0.5%,pH6.0±0.2。The seed liquid culture medium is: by mass fraction, including 2% of glucose, 2% of sucrose, 3% of soybean peptone, 1% of yeast powder, 0.5% of dipotassium hydrogen phosphate, 0.5% of urea, pH 6.0±0.2.
优选,所述的产罗伊氏菌素的罗伊氏乳杆菌菌液是挑选产罗伊氏菌素的罗伊氏乳杆菌种接种到MRS液体培养基中,37±1℃静置培养18-26h,然后将活化的液体菌种以体积比3-6%的接种量转接到液体种子培养基的发酵罐中,发酵罐装液量为60-80%,发酵温度37±1℃,密闭静置培养,发酵时间为18-26h,制成种子液;Preferably, the reuterin-producing Lactobacillus reuteri bacteria liquid is selected to inoculate the reuterin-producing Lactobacillus reuteri species into the MRS liquid medium, and cultured statically at 37±1°C for 18 -26h, then transfer the activated liquid strain to the fermenter of the liquid seed culture medium with an inoculation amount of 3-6% by volume, the liquid volume of the fermenter is 60-80%, and the fermentation temperature is 37±1°C. Sealed static culture, fermentation time is 18-26h, made into seed liquid;
将种子液以体积比3-6%的接种量转接到含有产甘油脱水酶发酵培养基中进行发酵,发酵罐装液量为50-65%,发酵温度28-32℃,微氧培养,转速80-100rpm,控制整个发酵过程pH5.5-7.0,发酵时间18-25h后,以变速流加的方式流加已灭菌冷却的甘油到菌液中,甘油浓度150-450mmol/L,甘油溶液和菌液体积比:1-5∶99-95,酶转化温度:28-32℃,时间1-5h,由此制备得到含罗伊氏菌素的罗伊氏乳杆菌菌液;The seed liquid is transferred to the fermentation medium containing glycerol dehydratase with a volume ratio of 3-6% for fermentation, the liquid volume of the fermentation tank is 50-65%, the fermentation temperature is 28-32 °C, micro-aerobic culture, The rotation speed is 80-100rpm, and the pH of the whole fermentation process is controlled to be 5.5-7.0. After the fermentation time is 18-25h, the sterilized and cooled glycerin is fed into the bacterial liquid by feeding at a variable speed. The glycerol concentration is 150-450mmol/L Volume ratio of solution and bacterial liquid: 1-5:99-95, enzyme conversion temperature: 28-32°C, time 1-5h, thus preparing Lactobacillus reuteri bacterial liquid containing reuterin;
所述的MRS液体培养基,液体种子培养基,其每升制备方法均为:将蛋白胨10g、酵母粉5g、牛肉膏10g、乙酸钠5g、柠檬酸氢二胺2g、磷酸氢二钾2g、硫酸镁0.2g、硫酸锰0.05g、吐温80 1mL、碳酸钙10g加水至1000mL,pH6.8;Described MRS liquid culture medium, liquid seed culture medium, its preparation method per liter is: peptone 10g, yeast powder 5g, beef extract 10g, sodium acetate 5g, diamine hydrogen citrate 2g, dipotassium hydrogen phosphate 2g, Magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, calcium carbonate 10g, add water to 1000mL, pH6.8;
所述的产甘油脱水酶发酵培养基,每升制备方法为:将糖蜜25g、牛肉膏4g、酵母粉4g加入到水中,加水至1000ml,pH 6.0。The preparation method per liter of the fermentation medium for producing glycerol dehydratase is as follows: add 25g of molasses, 4g of beef extract, and 4g of yeast powder into water, add water to 1000ml, and pH 6.0.
经罗伊氏菌素含量和罗伊氏乳杆菌活菌数检测,罗伊氏菌素含量120-200mmol/L,罗伊氏乳杆菌活菌数1.0×109cfu/mL~5.0×109cfu/mL。The content of reuterin and the number of live bacteria of Lactobacillus reuteri were detected, the content of reuterin was 120-200mmol/L, and the number of live bacteria of Lactobacillus reuteri was 1.0×10 9 cfu/mL~5.0×10 9 cfu/mL.
优选,所述的改善加州鲈糖脂代谢是提高加州鲈对饲料中糖脂的利用和营养的消化吸收,提高动物的饲料转化率。Preferably, the improvement of glycolipid metabolism in California perch is to improve the utilization of glycolipids in feed and the digestion and absorption of nutrients by California perch, and improve the feed conversion rate of animals.
本发明能够调控肠道微生物菌群结构,改善肠道菌群分布,拮抗有害菌定植,同时能靶向调节加州鲈肠道菌群和代谢物,促进加州鲈对饲料中糖脂的利用率和营养的消化吸收,提高动物的饲料转化率,且可有效提高加州鲈对对饲料糖脂代谢的利用,减少腹腔脂肪蓄积,降低脂肪肝的发生,增强机体免疫能力,减少疾病发生,提高商品鱼经济和市场价值。The invention can regulate the structure of intestinal microbial flora, improve the distribution of intestinal flora, antagonize the colonization of harmful bacteria, and at the same time, it can target and regulate the intestinal flora and metabolites of California perch, and promote the utilization rate of glycolipids in feed and nutrition of California perch Digestion and absorption, improve the feed conversion rate of animals, and can effectively improve the utilization of California perch on the metabolism of feed glucose and lipids, reduce the accumulation of abdominal fat, reduce the occurrence of fatty liver, enhance the body's immunity, reduce the occurrence of diseases, and improve the economy and market of commercial fish value.
附图说明:Description of drawings:
图1是实施例4的实验结果,每组柱形图从左至右分别为Ctr0、试验组I和II;Fig. 1 is the experimental result of embodiment 4, and each group of histograms is respectively Ctr0, test group I and II from left to right;
图2是肠道代谢物生物胺的含量;Figure 2 is the content of intestinal metabolite biogenic amine;
图3是肠道代谢物锻炼脂肪酸含量,每组柱形图从左至右分别为Ctr0、试验组I;Figure 3 shows the fatty acid content of intestinal metabolite exercise, and the bar graphs of each group are Ctr0 and test group I from left to right;
图4是脂代谢含量,每组柱形图从左至右分别为Ctr0、试验组I和II;Fig. 4 is lipid metabolism content, and each group of histograms is respectively Ctr0, test group I and II from left to right;
图5是酶活图,每组柱形图从左至右分别为Ctr0、试验组I和II。Fig. 5 is a graph of enzyme activity, and each group of histograms is respectively Ctr0, test groups I and II from left to right.
具体实施方式:Detailed ways:
以下实施例是对本发明的进一步说明,而不是对本发明的限制。The following examples are to further illustrate the present invention, rather than limit the present invention.
以下使用的加州鲈预混合饲料,每1000kg由以下质量配比的原料配制而成:维生素A 0.8kg,维生素D3 0.24kg,维生素K3 2.16kg,dl-α-生育酚乙酸酯8.40kg,维生素B10.86kg,维生素B2 1.20kg,维生素B6 0.73kg,维生素B12 0.30kg,烟酰胺6.19kg,D-泛酸钙2.45kg,叶酸0.49kg,D-生物素0.60kg,L-抗坏血酸-2-磷酸酯20.57kg,肌醇3.67kg,乙氧基喹啉2.40kg,一水硫酸镁20.63kg,一水硫酸亚铁12.83kg,一水硫酸锌5.74kg,五水硫酸铜3.52kg,一水硫酸锰3.46kg,1%硫酸钴13.20kg,1%碘酸钙9.9kg,1%亚硒酸钠3.3kg,其余为沸石粉和稻壳粉质量各半,将上述原料混合均匀,即得加州鲈预混合饲料。其中,所述的1%硫酸钴、1%碘酸钙和1%亚硒酸钠均从广州志专饲料有限公司购买。The premixed feed for California perch used below is prepared from the following raw materials in mass ratio per 1000kg: vitamin A 0.8kg, vitamin D3 0.24kg, vitamin K3 2.16kg, dl-α-tocopheryl acetate 8.40kg, vitamin B10 .86kg, vitamin B2 1.20kg, vitamin B6 0.73kg, vitamin B12 0.30kg, niacinamide 6.19kg, D-calcium pantothenate 2.45kg, folic acid 0.49kg, D-biotin 0.60kg, L-ascorbic acid-2-phosphate 20.57 kg, inositol 3.67kg, ethoxyquinoline 2.40kg, magnesium sulfate monohydrate 20.63kg, ferrous sulfate monohydrate 12.83kg, zinc sulfate monohydrate 5.74kg, copper sulfate pentahydrate 3.52kg, manganese sulfate monohydrate 3.46kg , 1% cobalt sulfate 13.20kg, 1% calcium iodate 9.9kg, 1% sodium selenite 3.3kg, all the other are zeolite powder and rice husk powder quality half and half, above-mentioned raw material is mixed, obtains California perch premixed feed. Wherein, the 1% cobalt sulfate, 1% calcium iodate and 1% sodium selenite were purchased from Guangzhou Zhizhuan Feed Co., Ltd.
魔芋精粉购自湖北强森魔芋科技有限公司Konjac fine powder was purchased from Hubei Qiangsen Konjac Technology Co., Ltd.
以下使用的罗伊氏乳杆菌为罗伊氏乳杆菌GDMCC 1.614,其保藏于广东省微生物菌种保藏中心(GDMCC),菌株编号:GDMCC 1.614。Lactobacillus reuteri used below is Lactobacillus reuteri GDMCC 1.614, which is preserved in Guangdong Microbial Culture Collection Center (GDMCC), strain number: GDMCC 1.614.
以下使用的枯草芽孢杆菌为枯草芽孢杆菌GDMCC 1.372,其保藏于广东省微生物菌种保藏中心(GDMCC),菌株编号:GDMCC 1.372。The Bacillus subtilis used below is Bacillus subtilis GDMCC 1.372, which is preserved in Guangdong Microbial Culture Collection Center (GDMCC), strain number: GDMCC 1.372.
以下使用的酿酒酵母为酿酒酵母GDMCC 2.167,其保藏于广东省微生物菌种保藏中心(GDMCC),菌株编号:GDMCC 2.167。The Saccharomyces cerevisiae used below is Saccharomyces cerevisiae GDMCC 2.167, which is preserved in Guangdong Microbial Culture Collection Center (GDMCC), strain number: GDMCC 2.167.
实施例1Example 1
1制备发酵菌种:制备枯草芽孢杆菌、酿酒酵母种子液、产罗伊氏菌素的罗伊氏乳杆菌菌液,其活菌数在1.0×109cfu/mL以上;1 Preparation of fermentation strains: preparation of Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid, the number of viable bacteria of which is above 1.0×10 9 cfu/mL;
1.1枯草芽孢杆菌种子液的制备1.1 Preparation of Bacillus subtilis seed solution
挑取枯草芽孢杆菌接种到LB培养基中,37±1℃摇床活化16h;然后将活化好的菌液按体积比3%接种量接种到种子液体培养基中,37±1℃培养16h得到枯草芽孢杆菌种子液。Pick Bacillus subtilis and inoculate it into LB medium, and activate it on a shaking table at 37±1°C for 16 hours; then inoculate the activated bacterial liquid into the seed liquid medium according to the volume ratio of 3% inoculum, and culture it at 37±1°C for 16 hours to obtain Bacillus subtilis seed liquid.
LB培养基(Luria-Bertani培养基)配方为:将胰蛋白胨10g、酵母粉5g、氯化钠10g加入到1L蒸馏水中,调pH7.0,灭菌备用。The formula of LB medium (Luria-Bertani medium) is: add 10 g of tryptone, 5 g of yeast powder, and 10 g of sodium chloride into 1 L of distilled water, adjust the pH to 7.0, and sterilize for later use.
所述的种子液体培养基为:按质量分数计,包括葡萄糖0.5%,可溶性淀粉1.5%,蛋白胨2%,酵母粉0.5%,硫酸镁0.2%,磷酸氢二钾0.2%,硫酸锰,0.03%,碳酸钙0.24%,溶剂为水,pH7.0±0.2,其配制方法是将各成分混合均匀,灭菌备用。The seed liquid culture medium is: by mass fraction, including glucose 0.5%, soluble starch 1.5%,
2.2产罗伊氏菌素的罗伊氏乳杆菌种子液的制备2.2 Preparation of Lactobacillus reuteri seed solution producing reuterin
2.2.1挑选产罗伊氏菌素的罗伊氏乳杆菌种接种到MRS液体种子培养基中,37±1℃静置培养18h,然后将活化的液体菌种以体积比3%的接种量转接到MRS液体发酵培养基的发酵罐中,发酵罐装液量为60%,发酵温度37±1℃,密闭静置培养,发酵时间为18h,制成种子液;2.2.1 Select the reuterin-producing Lactobacillus reuteri species and inoculate them into the MRS liquid seed medium, culture them statically at 37±1°C for 18 hours, and then inoculate the activated liquid strains with a volume ratio of 3%. Transfer to the fermenter of the MRS liquid fermentation medium, the liquid volume of the fermenter is 60%, the fermentation temperature is 37±1°C, the airtight static culture, the fermentation time is 18h, and the seed liquid is made;
将种子液以体积比3%的接种量转接到含有产甘油脱水酶发酵培养基中进行发酵,发酵罐装液量为50%,发酵温度28℃,微氧培养,转速80rpm,用6mol/LNaOH调节pH,控制整个发酵产酶pH5.5,发酵时间18h后,以变速流加的方式流加已灭菌冷却的甘油(甘油浓度150mmol/L)到菌液中,甘油溶液和菌液体积比:1∶99,酶转化温度:28℃,时间1h,由此制备得到含罗伊氏菌素的罗伊氏乳杆菌菌液。The seed liquid is transferred to the fermentation medium containing glycerol dehydratase with a volume ratio of 3% for fermentation. The liquid volume of the fermentation tank is 50%. Adjust the pH with LNaOH to control the enzyme pH5.5 in the entire fermentation. After 18 hours of fermentation time, add sterilized and cooled glycerin (glycerin concentration 150mmol/L) to the bacterial liquid by feeding at a variable speed. The glycerin solution and the bacterial liquid volume Ratio: 1:99, enzyme conversion temperature: 28° C., and time of 1 hour, thereby preparing Lactobacillus reuteri bacteria liquid containing reuterin.
所述的MRS培养基,种子培养基均为:在1L的体系里面,将蛋白胨10g、酵母粉5g、牛肉膏10g、乙酸钠5g、柠檬酸氢二胺2g、磷酸氢二钾2g、硫酸镁0.2g、硫酸锰0.05g、吐温801mL、碳酸钙10g、加水至1000mL,pH6.8;115℃灭菌30min。The MRS medium and seed medium are: in a 1L system, 10g of peptone, 5g of yeast powder, 10g of beef extract, 5g of sodium acetate, 2g of diamine hydrogen citrate, 2g of dipotassium hydrogen phosphate, and magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 801mL, calcium carbonate 10g, add water to 1000mL, pH6.8; sterilize at 115°C for 30min.
所述的产甘油脱水酶发酵培养基为:在1L的体系里面将糖蜜25g,牛肉膏4g,酵母粉4g,加水至1000mL,pH 6.0,115℃灭菌30min。The fermentation medium for producing glycerol dehydratase is as follows: in a 1L system, add 25g of molasses, 4g of beef extract, and 4g of yeast powder, add water to 1000mL, pH 6.0, and sterilize at 115°C for 30min.
2.3酿酒酵母种子液的制备2.3 Preparation of Saccharomyces cerevisiae seed solution
挑取酿酒酵母斜面菌种接种到PDA液体培养基中,28±1℃摇床活化20h;然后将活化好的菌液按体积比2%接种量接种到种子液体培养基中,28±1℃培养18h得到酿酒酵母种子液。Pick the Saccharomyces cerevisiae slant and inoculate it into the PDA liquid medium, and activate it on a shaking table at 28±1°C for 20 hours; then inoculate the activated bacterial liquid into the seed liquid medium at 28±1°C Cultivate for 18 hours to obtain Saccharomyces cerevisiae seed liquid.
PDA液体培养基配方为:去皮马铃薯200g、切碎煮汁过滤去渣,加入蔗糖20g、蛋白胨10g、琼脂18g,再加水定容至1L,调pH7.0。The formula of PDA liquid medium is: 200g of peeled potatoes, chopped boiled juice, filtered to remove slag, added 20g of sucrose, 10g of peptone, 18g of agar, added water to adjust the volume to 1L, and adjusted the pH to 7.0.
所述的种子液体培养基为:按质量分数计,包括葡萄糖2%,蔗糖2%,大豆蛋白胨3%,酵母粉1%,磷酸氢二钾0.5%,尿素0.5%,溶剂为水,pH6.0±0.2,其配制方法是将各成分混合均匀,灭菌备用。The seed liquid culture medium is: by mass fraction, including
3饲料原料粉碎过筛:饲料原料粉碎过40目,按如下质量配比称取:玉米粉10%,豆粕55%,鱼粉25%,麦麸10%,混合均匀,然后加水至水分含量为35%,用混合机混合均匀得到固体培养基。3 Feed raw materials crushing and sieving: Feed raw materials are crushed through 40 meshes, weighed according to the following mass ratio: 10% corn flour, 55% soybean meal, 25% fish meal, 10% wheat bran, mix evenly, and then add water until the moisture content is 35% %, mixed evenly with a mixer to obtain a solid medium.
4接种发酵4 inoculation fermentation
将发酵待用的菌种枯草芽孢杆菌种子液、酿酒酵母种子液、产罗伊氏菌素的罗伊氏乳杆菌菌液按体积比1:1:8比例混合,以固体培养基湿重10%均匀接种到上述固体培养基中,28℃恒温厌氧发酵,发酵时间为72h。Mix the Bacillus subtilis seed liquid, the Saccharomyces cerevisiae seed liquid, and the reuterin-producing Lactobacillus reuteri bacterial liquid in a ratio of 1:1:8 by volume, and use a solid medium with a wet weight of 10 % uniformly inoculated into the above-mentioned solid medium, 28 ℃ constant temperature anaerobic fermentation, the fermentation time is 72h.
以罗伊氏乳杆菌菌液作为对照,以固体培养基湿重10%均匀接种到上述固体培养基中,28℃恒温厌氧发酵,发酵时间为72h。Using the Lactobacillus reuteri bacteria liquid as a control, 10% of the wet weight of the solid medium was evenly inoculated into the above solid medium, and the constant temperature anaerobic fermentation was carried out at 28° C., and the fermentation time was 72 hours.
5厌氧发酵结束后,在厌氧发酵料中添加厌氧发酵料质量占比0.5%加州鲈预混合饲料和厌氧发酵料质量占比0.3%的魔芋精粉,混匀常温制的2.0粒径的软颗粒饲料,即为加州鲈发酵配合饲料(即改善加州鲈糖脂代谢功能性发酵饲料)。5 After the anaerobic fermentation is over, add the anaerobic fermentation material to the anaerobic fermentation material, which accounts for 0.5% of the California perch premix feed and the anaerobic fermentation material, which accounts for 0.3% of the konjac powder, and mix the 2.0 particle size prepared at room temperature The soft pellet feed is the California perch fermented compound feed (that is, the functional fermented feed for improving the glucose and lipid metabolism of California perch).
未发酵加州鲈配合饲料是在固体培养基中添加固体培养基质量占比0.5%加州鲈预混合饲料和固体培养基质量占比0.3%的魔芋精粉,混匀常温制的2.0粒径的软颗粒饲料,即为未发酵加州鲈配合饲料。The unfermented California perch compound feed is made by adding 0.5% California perch premix feed and 0.3% konjac powder to the solid medium, and mixing it at room temperature to make a 2.0-grain-size soft pellet feed , which is the unfermented California perch compound feed.
6检测结果6 test results
分别发酵前后测定加州鲈发酵配合饲料粗蛋白、酸溶蛋白、生物活性肽、游离氨基酸、L-乳酸、罗伊氏菌素的含量及芽孢杆菌、乳酸菌和酵母菌活菌数指标(表1)。The content of crude protein, acid-soluble protein, biologically active peptide, free amino acid, L-lactic acid, reuterin and the number of viable bacteria of Bacillus, lactic acid bacteria and yeast were measured before and after fermentation (Table 1).
表1Table 1
实施例2Example 2
1制备发酵菌种:制备枯草芽孢杆菌、酿酒酵母种子液、产罗伊氏菌素的罗伊氏乳杆菌菌液,其活菌数在1.0×109cfu/mL以上;1 Preparation of fermentation strains: preparation of Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid, the number of viable bacteria of which is above 1.0×10 9 cfu/mL;
1.1枯草芽孢杆菌种子液的制备1.1 Preparation of Bacillus subtilis seed solution
挑取枯草芽孢杆菌接种到LB培养基中,37±1℃摇床活化20h;然后将活化好的菌液按体积比4%接种量接种到种子液体培养基中,37±1℃培养20h得到枯草芽孢杆菌种子液。Pick Bacillus subtilis and inoculate it into LB medium, and activate it on a shaking table at 37±1°C for 20 hours; then inoculate the activated bacterial liquid into the seed liquid medium according to the volume ratio of 4% inoculum, and cultivate it at 37±1°C for 20 hours to obtain Bacillus subtilis seed liquid.
LB培养基(Luria-Bertani培养基)配方为:将胰蛋白胨10g、酵母粉5g、氯化钠10g加入到1L蒸馏水中,调pH7.0,灭菌备用。The formula of LB medium (Luria-Bertani medium) is: add 10 g of tryptone, 5 g of yeast powder, and 10 g of sodium chloride into 1 L of distilled water, adjust the pH to 7.0, and sterilize for later use.
所述的种子液体培养基为:按质量分数计,包括葡萄糖0.5%,可溶性淀粉1.5%,蛋白胨2%,酵母粉0.5%,硫酸镁0.2%,磷酸氢二钾0.2%,硫酸锰,0.03%,碳酸钙0.24%,pH7.0±0.2,其配制方法是将各成分混合均匀,灭菌备用。The seed liquid culture medium is: by mass fraction, including glucose 0.5%, soluble starch 1.5%, peptone 2%, yeast powder 0.5%, magnesium sulfate 0.2%, dipotassium hydrogen phosphate 0.2%, manganese sulfate, 0.03% , calcium carbonate 0.24%, pH7.0±0.2, its preparation method is to mix each component uniformly, and sterilize for later use.
2.2产罗伊氏菌素的罗伊氏乳杆菌种子液的制备2.2 Preparation of Lactobacillus reuteri seed solution producing reuterin
2.2.1挑选产罗伊氏菌素的罗伊氏乳杆菌种接种到MRS液体种子培养基中,37±1℃静置培养22h,然后将活化的液体菌种以体积比4%的接种量转接到MRS液体发酵培养基的发酵罐中,发酵罐装液量为70%,发酵温度37±1℃,密闭静置培养,发酵时间为22h,制成种子液;2.2.1 Select the reuterin-producing Lactobacillus reuteri species and inoculate them into the MRS liquid seed medium, culture them statically at 37±1°C for 22 hours, and then inoculate the activated liquid strains with a volume ratio of 4%. Transfer to the fermenter of the MRS liquid fermentation medium, the liquid volume of the fermenter is 70%, the fermentation temperature is 37±1°C, the airtight static culture, the fermentation time is 22h, and the seed liquid is made;
将种子液以体积比4%的接种量转接到含有产甘油脱水酶发酵培养基中进行发酵,发酵罐装液量为58%,发酵温度30℃,微氧培养,转速90rpm,用6mol/LNaOH调节pH,控制整个发酵产酶pH6.0,发酵时间21h后,以变速流加的方式流加已灭菌冷却的甘油(甘油浓度300mmol/L)到菌液中,甘油溶液和菌液体积比:2∶98,酶转化温度:30℃,时间2.5h,由此制备得到含罗伊氏菌素的罗伊氏乳杆菌菌液。The seed liquid is transferred to the fermentation medium containing glycerol dehydratase with a volume ratio of 4% for fermentation. The liquid volume of the fermentation tank is 58%. Adjust the pH with LNaOH to control the pH of the entire fermentation to produce enzymes at pH 6.0. After 21 hours of fermentation time, add sterilized and cooled glycerin (glycerin concentration 300mmol/L) into the bacterial solution by feeding at a variable speed. The glycerol solution and bacterial solution volume Ratio: 2:98, enzyme conversion temperature: 30° C., time 2.5 hours, thus preparing Lactobacillus reuteri bacteria liquid containing reuterin.
所述的MRS液体发酵培养基,种子培养基均为:在1L的体系里面,将蛋白胨10g、酵母粉5g、牛肉膏10g、乙酸钠5g、柠檬酸氢二胺2g、磷酸氢二钾2g、硫酸镁0.2g、硫酸锰0.05g、吐温80 1mL、碳酸钙10g,加水至1000mL,pH6.8;115℃灭菌30min。The MRS liquid fermentation medium and the seed medium are: in a 1L system, 10g of peptone, 5g of yeast powder, 10g of beef extract, 5g of sodium acetate, 2g of diamine hydrogen citrate, 2g of dipotassium hydrogen phosphate, Magnesium sulfate 0.2g, manganese sulfate 0.05g,
所述的产甘油脱水酶发酵培养基为:在1L的体系里面,将糖蜜25g、牛肉膏4g、酵母粉4g,pH6.0加水至1000mL,115℃灭菌30min。The fermentation medium for producing glycerol dehydratase is as follows: in a 1L system, add molasses 25g, beef extract 4g, yeast powder 4g, pH 6.0, add water to 1000mL, and sterilize at 115°C for 30min.
2.3酿酒酵母种子液的制备2.3 Preparation of Saccharomyces cerevisiae seed solution
挑取酿酒酵母斜面菌种接种到PDA液体培养基中,28±1℃摇床活化22h;然后将活化好的菌液按体积比3%接种量接种到种子液体培养基中,28±1℃培养22h得到酿酒酵母种子液。Pick the Saccharomyces cerevisiae slant and inoculate it into the PDA liquid medium, and activate it on a shaking table at 28±1°C for 22 hours; then inoculate the activated bacterial liquid into the seed liquid medium at 28±1°C Cultivate for 22 hours to obtain Saccharomyces cerevisiae seed liquid.
PDA液体培养基配方为:去皮马铃薯200g、切碎煮汁过滤去渣,加入蔗糖20g、蛋白胨10g、琼脂18g,再加水定容至1L,调pH7.0,灭菌备用。The formula of PDA liquid medium is: 200g of peeled potatoes, chopped boiled juice, filtered to remove slag, added 20g of sucrose, 10g of peptone, 18g of agar, added water to make the volume to 1L, adjusted to pH 7.0, and sterilized for later use.
所述的种子液体培养基为:按质量分数计,包括葡萄糖2%,蔗糖2%,大豆蛋白胨3%,酵母粉1%,磷酸氢二钾0.5%,尿素0.5%,pH6.0±0.2,其配制方法是将各成分混合均匀,灭菌备用。The seed liquid medium is: by mass fraction, including
3饲料原料粉碎过筛:饲料原料粉碎过40目,按如下质量配比称取:玉米粉20%,豆粕30%,鱼粉45%,麦麸5%,加水至水份含量为35%,用混合机混合均匀得到固体培养基。3 Feed raw material crushing and sieving: Feed raw material is crushed through 40 meshes, weighed according to the following mass ratio: 20% corn flour, 30% soybean meal, 45% fish meal, 5% wheat bran, add water until the water content is 35%, and use The mixer mixes evenly to obtain a solid medium.
4接种发酵4 inoculation fermentation
将发酵待用的菌种枯草芽孢杆菌、酿酒酵母种子液、产罗伊氏菌素的罗伊氏乳杆菌菌液按体积比2:2:6比例,以固体培养基湿重15%均匀接种到上述固体培养基中,32℃恒温厌氧发酵,发酵时间为84h。Inoculate Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid in a volume ratio of 2:2:6, and uniformly inoculate them with a solid medium with a wet weight of 15%. Into the above solid medium, 32 ° C constant temperature anaerobic fermentation, the fermentation time is 84h.
5厌氧发酵结束后,在厌氧发酵料中添加厌氧发酵料重量0.75%加州鲈预混合饲料和厌氧发酵料重量0.1%的魔芋精粉,混匀常温制得2.0粒径的软颗粒饲料(加州鲈发酵配合饲料,改善加州鲈糖脂代谢功能性发酵饲料)。5. After the anaerobic fermentation, add anaerobic fermentation material weight 0.75% California perch premixed feed and anaerobic fermentation material weight 0.1% konjac fine powder in the anaerobic fermentation material, and mix it at room temperature to make a soft pellet feed with a particle size of 2.0 (California perch fermented compound feed, functional fermented feed for improving glucose and lipid metabolism of California perch).
未发酵加州鲈配合饲料是在固体培养基中添加固体培养基质量占比0.75%加州鲈预混合饲料和固体培养基质量占比0.1%的魔芋精粉,混匀常温制的2.0粒径的软颗粒饲料,即为未发酵加州鲈配合饲料。The unfermented California perch compound feed is made by adding 0.75% California perch premix feed and 0.1% konjac fine powder to the solid medium, and mixing it at room temperature to make soft pellet feed with a particle size of 2.0 , which is the unfermented California perch compound feed.
6检测结果6 test results
分别发酵前后测定加州鲈发酵配合饲料粗蛋白、酸溶蛋白、生物活性肽、游离氨基酸、L-乳酸、罗伊氏菌素的含量及芽孢杆菌、乳酸菌和酵母菌活菌数指标(表2)。The content of crude protein, acid-soluble protein, biologically active peptide, free amino acid, L-lactic acid, reuterin and the number of live bacteria of Bacillus, lactic acid bacteria and yeast were measured before and after fermentation (Table 2).
表2Table 2
实施例3Example 3
1制备发酵菌种:制备枯草芽孢杆菌、酿酒酵母种子液、产罗伊氏菌素的罗伊氏乳杆菌菌液,其活菌数在1.0×109cfu/mL以上;1 Preparation of fermentation strains: preparation of Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid, the number of viable bacteria of which is above 1.0×10 9 cfu/mL;
1.1枯草芽孢杆菌种子液的制备1.1 Preparation of Bacillus subtilis seed solution
挑取枯草芽孢杆菌接种到LB培养基中,37±1℃摇床活化24h;然后将活化好的菌液按体积比6%接种量接种到种子液体培养基中,37±1℃培养24h得到枯草芽孢杆菌种子液。Pick Bacillus subtilis and inoculate it into LB medium, and activate it on a shaking table at 37±1°C for 24 hours; then inoculate the activated bacterial liquid into the seed liquid medium according to the volume ratio of 6% inoculum, and cultivate it at 37±1°C for 24 hours to obtain Bacillus subtilis seed liquid.
LB培养基(Luria-Bertani培养基)配方为:将胰蛋白胨10g、酵母粉5g、氯化钠10g加入到1L蒸馏水中,调pH7.0,灭菌备用。The formula of LB medium (Luria-Bertani medium) is: add 10 g of tryptone, 5 g of yeast powder, and 10 g of sodium chloride into 1 L of distilled water, adjust the pH to 7.0, and sterilize for later use.
所述的种子液体培养基为:按质量分数计,包括葡萄糖0.5%,可溶性淀粉1.5%,蛋白胨2%,酵母粉0.5%,硫酸镁0.2%,磷酸氢二钾0.2%,硫酸锰,0.03%,碳酸钙0.24%,pH7.0±0.2,其配制方法是将各成分混合均匀,灭菌备用。The seed liquid culture medium is: by mass fraction, including glucose 0.5%, soluble starch 1.5%, peptone 2%, yeast powder 0.5%, magnesium sulfate 0.2%, dipotassium hydrogen phosphate 0.2%, manganese sulfate, 0.03% , calcium carbonate 0.24%, pH7.0±0.2, its preparation method is to mix each component uniformly, and sterilize for later use.
2.2产罗伊氏菌素的罗伊氏乳杆菌种子液的制备2.2 Preparation of Lactobacillus reuteri seed solution producing reuterin
2.2.1挑选产罗伊氏菌素的罗伊氏乳杆菌种接种到MRS液体种子培养基中,37±1℃静置培养26h,然后将活化的液体菌种以体积比6%的接种量转接到MRS液体发酵培养基的发酵罐中,发酵罐装液量为80%,发酵温度37±1℃,密闭静置培养,发酵时间为26h,制成种子液;2.2.1 Select the reuterin-producing Lactobacillus reuteri species and inoculate them into the MRS liquid seed medium, culture them statically at 37±1°C for 26 hours, and then inoculate the activated liquid strains with a volume ratio of 6%. Transfer to the fermenter of the MRS liquid fermentation medium, the liquid volume of the fermenter is 80%, the fermentation temperature is 37±1°C, the airtight static culture, the fermentation time is 26h, and the seed liquid is made;
将种子液以体积比6%的接种量转接到含有产甘油脱水酶发酵培养基中进行发酵,发酵罐装液量为65%,发酵温度32℃,微氧培养,转速100rpm,用6mol/LNaOH调节pH,控制整个发酵产酶pH7.0,发酵时间25h后,以变速流加的方式流加已灭菌冷却的甘油(甘油浓度450mmol/L)到菌液中,甘油溶液和菌液体积比:5∶95,酶转化温度:32℃,时间5h,由此制备得到含罗伊氏菌素的罗伊氏乳杆菌菌液。The seed liquid is transferred to the fermentation medium containing glycerol dehydratase with a volume ratio of 6% for fermentation. The liquid volume of the fermentation tank is 65%. Adjust the pH with LNaOH to control the pH of the entire fermentation enzyme production to 7.0. After 25 hours of fermentation time, add sterilized and cooled glycerin (glycerin concentration 450mmol/L) into the bacterial solution by feeding at a variable speed. The glycerin solution and the bacterial solution volume Ratio: 5:95, enzyme conversion temperature: 32° C., time 5 h, thus preparing Lactobacillus reuteri bacteria liquid containing reuterin.
所述的MRS液体发酵培养基,种子培养基均为:在1L的体系里面将蛋白胨10g、酵母粉5g、牛肉膏10g、乙酸钠5g、柠檬酸氢二胺2g、磷酸氢二钾2g、硫酸镁0.2g硫酸锰0.05g、吐温80 1mL、碳酸钙10g,加水至1000mL,pH6.8;115℃灭菌30min。The MRS liquid fermentation medium and the seed medium are: 10 g of peptone, 5 g of yeast powder, 10 g of beef extract, 5 g of sodium acetate, 2 g of diamine hydrogen citrate, 2 g of dipotassium hydrogen phosphate, and 2 g of sulfuric acid in a 1 L system. Magnesium 0.2g manganese sulfate 0.05g,
所述的产甘油脱水酶发酵培养基为:在1L的体系里面,将糖蜜25g、牛肉膏4g、酵母粉4g,pH6.0加水至1000mL,115℃灭菌30min。The fermentation medium for producing glycerol dehydratase is as follows: in a 1L system, add molasses 25g, beef extract 4g, yeast powder 4g, pH 6.0, add water to 1000mL, and sterilize at 115°C for 30min.
2.3酿酒酵母种子液的制备2.3 Preparation of Saccharomyces cerevisiae seed solution
挑取酿酒酵母斜面菌种接种到PDA液体培养基中,28±1℃摇床活化24h;然后将活化好的菌液按体积比5%接种量接种到种子液体培养基中,28±1℃培养24h得到酿酒酵母种子液。Pick the Saccharomyces cerevisiae slant and inoculate it into the PDA liquid medium, and activate it on a shaking table at 28±1°C for 24 hours; then inoculate the activated bacterial liquid into the seed liquid medium at 28±1°C Cultivate for 24 hours to obtain Saccharomyces cerevisiae seed liquid.
PDA液体培养基配方为:去皮马铃薯200g、切碎煮汁过滤去渣,加入蔗糖20g、蛋白胨10g、琼脂18g,再加水定容至1L,调pH7.0,灭菌备用。The formula of PDA liquid medium is: 200g of peeled potatoes, chopped boiled juice, filtered to remove slag, added 20g of sucrose, 10g of peptone, 18g of agar, added water to make the volume to 1L, adjusted to pH 7.0, and sterilized for later use.
所述的种子培养基为:按质量分数计,包括葡萄糖2%,蔗糖2%,大豆蛋白胨3%,酵母粉1%,磷酸氢二钾0.5%,尿素0.5%,pH6.0±0.2,其配制方法是将各成分混合均匀,灭菌备用。The seed medium is: by mass fraction, including 2% glucose, 2% sucrose, 3% soybean peptone, 1% yeast powder, 0.5% dipotassium hydrogen phosphate, 0.5% urea, pH6.0±0.2, and The preparation method is to mix all components uniformly, and sterilize for standby use.
3饲料原料粉碎过筛:饲料原料粉碎过40目,按如下质量配比称取:玉米粉15%,豆粕43%,鱼粉35%,麦麸7%,加水至水份含量为35%,用混合机混合均匀得到固体培养基。3 Feed raw material crushing and sieving: Feed raw material is crushed through 40 meshes, weighed according to the following mass ratio: 15% corn flour, 43% soybean meal, 35% fish meal, 7% wheat bran, add water until the water content is 35%, use The mixer mixes evenly to obtain a solid medium.
4接种发酵4 inoculation fermentation
将发酵待用的菌种枯草芽孢杆菌、酿酒酵母种子液、产罗伊氏菌素的罗伊氏乳杆菌菌液按体积比3:3:4比例,以固体培养基湿重20%均匀接种到上述固体培养基中,35℃恒温厌氧发酵,发酵时间为96h。Inoculate Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid in a volume ratio of 3:3:4, and uniformly inoculate the solid medium with a wet weight of 20% Into the above solid medium, 35 ° C constant temperature anaerobic fermentation, the fermentation time is 96h.
以罗伊氏乳杆菌菌液作为对照,以固体培养基湿重20%均匀接种到上述固体培养基中,35℃恒温厌氧发酵,发酵时间为96h。Lactobacillus reuteri bacteria liquid was used as a control, and 20% of the wet weight of the solid medium was evenly inoculated into the above-mentioned solid medium, and the anaerobic fermentation was carried out at a constant temperature of 35° C., and the fermentation time was 96 hours.
5厌氧发酵结束后,在厌氧发酵料中添加厌氧发酵料重量1.0%加州鲈预混合饲料和0.5%的魔芋精粉,混匀常温制的2.0粒径的软颗粒饲料(加州鲈发酵配合饲料,即改善加州鲈糖脂代谢功能性发酵饲料)。5. After the anaerobic fermentation finishes, add anaerobic fermentation material weight 1.0% California perch premixed feed and 0.5% konjac powder in the anaerobic fermentation material, mix the soft pellet feed of 2.0 grain diameters (California perch fermentation compound feed , that is, functional fermented feed for improving glucose and lipid metabolism in California perch).
未发酵加州鲈配合饲料是在固体培养基中添加固体培养基质量占比1%加州鲈预混合饲料和固体培养基质量占比0.5%的魔芋精粉,混匀常温制的2.0粒径的软颗粒饲料,即为未发酵加州鲈配合饲料。The unfermented California perch compound feed is a 2.0-grain-size soft pellet feed prepared by adding 1% California perch premix feed and 0.5% solid medium mass to the solid medium and mixing them at room temperature. , which is the unfermented California perch compound feed.
6检测结果6 test results
分别发酵前后测定加州鲈发酵配合饲料粗蛋白、酸溶蛋白、生物活性肽、游离氨基酸、L-乳酸、罗伊氏菌素的含量及芽孢杆菌、乳酸菌和酵母菌活菌数指标(表3)。The content of crude protein, acid-soluble protein, biologically active peptide, free amino acid, L-lactic acid, reuterin and the number of viable bacteria of Bacillus, lactic acid bacteria and yeast were measured before and after fermentation (Table 3).
表3table 3
实施例4Example 4
将上述实施例1发酵制得的加州鲈发酵配合饲料饲喂8g左右的加州鲈为试验组Ⅱ,记为Ⅱ,同时以未发酵的加州鲈配合饲料为空白对照组,记为Ctr0,单一罗伊氏乳杆菌发酵的加州鲈发酵配合饲料为试验组Ⅰ,记为Ⅰ,饲喂8周,每个试验设3重复,每个重复30尾鱼。试验结果如图1,试验组Ⅰ加州鲈饲料脂肪和总糖表观消化率分别比对照组提高了7.93%和14.97%。试验组Ⅱ加州鲈饲料脂肪和总糖表观消化率分别比对照组提高了26.80%和39.19%,显著高于Ctr0(P<0.05),比试验组Ⅰ提高了17.48%和21.07%,显著高于试验组Ⅰ(P<0.05)。结果显示,本发明制备的加州鲈发酵配合饲料,可显著提高加州鲈对饲料糖脂利用率,其效果优于未发酵的加州鲈配合饲料和单一罗伊氏乳杆菌发酵的加州鲈发酵配合饲料。About 8g of California perch fed with the California perch fermented compound feed obtained by the fermentation of the above-mentioned Example 1 was regarded as test group II, denoted as II, and at the same time, the unfermented California perch compound feed was used as a blank control group, denoted as Ctr0, and single Roy's milk The California perch fermented compound feed fermented by Bacillus was the experimental group Ⅰ, denoted as Ⅰ, fed for 8 weeks, and each experiment was repeated 3 times, with 30 fish in each repetition. The test results are shown in Figure 1. The apparent digestibility of dietary fat and total sugar in test group I increased by 7.93% and 14.97% respectively compared with the control group. Compared with the control group, the apparent digestibility of fat and total sugar in the experimental group II increased by 26.80% and 39.19%, significantly higher than Ctr0 (P<0.05), and increased by 17.48% and 21.07% compared with the experimental group I, significantly higher than Test group I (P<0.05). The results show that the California perch fermented compound feed prepared by the invention can significantly improve the glycolipid utilization rate of the California perch, and its effect is better than that of the unfermented California perch compound feed and the California perch fermented compound feed fermented by a single Lactobacillus reuteri.
实施例5Example 5
将上述实施例2发酵制得的加州鲈发酵配合饲料饲喂25g左右的加州鲈为试验组,记为Ⅰ,同时以未发酵的加州鲈配合饲料为空白对照组,记为Ctr0,饲喂8周,每个试验设3重复,每个重复20尾鱼。试验结束后,禁食24h后,每缸随机取10尾鱼,用干净纱布擦干鱼体表面,在冰盘上解剖取肠道放入冷冻管中,液氮速冻后,-80℃超低温冰箱保存。加州鲈肠道样品,冷冻干燥后,采用气相色谱测定乙酸等短链脂肪酸含量,生物胺含量采用高效液相色谱检测。The California perch fermented compound feed obtained by the above-mentioned
试验结果如图2和图3:试验组Ⅰ肠道代谢物组胺、尸胺和腐胺含量与对照组相比,降低了67.84%、89.36%和84.53%,显著低于对照组(P<0.05)。试验组Ⅰ肠道代谢物乙酸、丙酸和丁酸的含量分别比对照组提高了119.26%、340.21%和426.90%。结果显示:饲喂本发明的加州鲈发酵配合饲料显著降低了加州鲈肠道代谢物组胺、尸胺和腐胺生物胺的含量,提高了加州鲈肠道代谢物乙酸、丙酸和丁酸短链脂肪酸的含量。饲喂本发明的加州鲈发酵配合饲料调控肠道代谢物短链脂肪酸,促进和提高加州鲈肠道营养物质的消化吸收。饲喂本发明的加州鲈发酵配合饲料调控肠道代谢物生物胺水平,一定程度可保护肠道因糖脂代谢紊乱导致的肠道损伤。The test results are shown in Figure 2 and Figure 3: compared with the control group, the contents of the intestinal metabolites histamine, cadaverine and putrescine in the test group I decreased by 67.84%, 89.36% and 84.53%, which were significantly lower than the control group (P< 0.05). The contents of intestinal metabolites acetic acid, propionic acid and butyric acid in the test group Ⅰ were increased by 119.26%, 340.21% and 426.90% respectively compared with the control group. The results show: feeding the California perch fermented compound feed of the present invention significantly reduces the content of the intestinal metabolites histamine, cadaverine and putrescine biogenic amine in the California perch, and improves the intestinal metabolites acetic acid, propionic acid and butyric short-chain fatty acids in the California perch. content. Feeding the California perch fermented compound feed of the present invention regulates short-chain fatty acids of intestinal metabolites, and promotes and improves the digestion and absorption of intestinal nutrients of California perch. Feeding the California perch fermented compound feed of the present invention regulates the level of intestinal metabolite biogenic amine, and can protect the intestinal tract from intestinal damage caused by glucose and lipid metabolism disorders to a certain extent.
实施例6Example 6
将上述实施例3发酵制得的加州鲈发酵配合饲料饲喂40g左右的加州鲈试验组Ⅱ,记为Ⅱ,同时以未发酵的加州鲈配合饲料为空白对照组,记为Ctr0,单一罗伊氏乳杆菌发酵的加州鲈发酵配合饲料为试验组Ⅰ,记为Ⅰ,饲喂8周,每个试验设3重复,每个重复20尾鱼。试验结束后,禁食24h后,每缸随机取5尾鱼,用干净纱布擦干鱼体表面,尾静脉采血,在3000rpm4℃离心10min,分离血清,液氮速冻后,-80℃超低温冰箱保存用于血清糖脂代谢相关指标分析。About 40g of California perch test group II was fed with the California perch fermented compound feed obtained by the fermentation in Example 3 above, which was designated as II. At the same time, the unfermented California perch compound feed was used as a blank control group, which was designated as Ctr0. A single Lactobacillus reuteri The fermented California perch fermented compound feed was the test group Ⅰ, denoted as Ⅰ, fed for 8 weeks, and each experiment was repeated 3 times, with 20 fish in each repetition. After the test, after fasting for 24 hours, randomly select 5 fish from each tank, dry the surface of the fish with clean gauze, collect blood from the tail vein, centrifuge at 3000rpm at 4°C for 10min, separate the serum, freeze it in liquid nitrogen, and store it in a -80°C ultra-low temperature refrigerator Used for the analysis of related indicators of serum glucose and lipid metabolism.
试验结果如图4和图5所示。试验组Ⅰ加州鲈血清甘油三脂、总胆固醇、游离脂肪酸和低密度脂蛋白胆固醇含量分别比对照组降低了9.19%、10.60%、8.49%和10.63%。试验组Ⅰ加州鲈血清丙酮酸激酶和葡糖糖激酶分别比对照组提高了8.85%和9.33%。试验组Ⅱ加州鲈血清甘油三脂、总胆固醇和游离脂肪酸含量分别比对照组降低了41.84%、40.63%、36.39%和31.50%,显著低于对照组(P<0.05),比试验组Ⅰ分别降低了35.95%、33.58%、30.49%和23.35%,显著低于试验组Ⅰ(P<0.05)。试验组Ⅱ加州鲈血清丙酮酸激酶和葡糖糖激酶分别比对照组提高了34.07%和39.39%,显著高于对照组(P<0.05),试验组Ⅰ相比,分别提高了23.17%和27.49%,显著高于试验组Ⅰ(P<0.05)。结果显示,饲喂本发明的加州鲈发酵配合饲料可显著提高加州鲈糖脂代谢能力,其效果优于常规加州鲈配合饲料和单一罗伊氏乳杆菌发酵的加州鲈发酵配合饲料。The test results are shown in Figure 4 and Figure 5. The content of serum triglyceride, total cholesterol, free fatty acid and low-density lipoprotein cholesterol in test group Ⅰ California perch decreased by 9.19%, 10.60%, 8.49% and 10.63% respectively compared with the control group. Serum pyruvate kinase and glucokinase of California perch in test group Ⅰ increased by 8.85% and 9.33% respectively compared with the control group. The content of serum triglyceride, total cholesterol and free fatty acid in California perch in test group Ⅱ was 41.84%, 40.63%, 36.39% and 31.50% lower than that of control group, significantly lower than that of control group (P<0.05), and lower than that of test group Ⅰ. 35.95%, 33.58%, 30.49% and 23.35%, significantly lower than the test group I (P <0.05). Compared with the control group, the serum pyruvate kinase and glucokinase of California perch in the test group II increased by 34.07% and 39.39%, which were significantly higher than the control group (P<0.05). Compared with the test group I, they increased by 23.17% and 27.49% respectively , significantly higher than that of the experimental group I (P<0.05). The results show that feeding the California perch fermented compound feed of the present invention can significantly improve the California perch's glycolipid metabolism, and its effect is better than that of the conventional California perch compound feed and the California perch fermented compound feed fermented by a single Lactobacillus reuteri.
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