CN103300216A - Method for preparing compound enzyme and probiotic preparation for feed through biotransformation of sorghum straws and rapeseed meals - Google Patents

Abstract

The invention discloses a method for preparing a compound enzyme and probiotic preparation for feed by biotransforming sorghum stalks and rapeseed cakes. The sorghum stalk, the first rapeseed cake, the first wheat bran, the first apple pomace and the first water are fully mixed, and after domestication and cultivation, a starter is formed; the starter, the second sorghum straw, the second rapeseed cake, The second wheat bran, the second apple pomace and the second water are fully mixed and fermented to obtain a compound enzyme and probiotic preparation for feed. In the present invention, the micro-ecologically stable food-grade safe flora is used to ferment sorghum stalks and rapeseed cakes to prepare a compound enzyme and probiotic preparation for feed with better performance indicators, so as to realize the low consumption of sorghum stalks and rapeseed cakes. Cost efficient biotransformation. Simultaneously, the microorganisms in the method of the invention are easy to grow, have high enzyme activity, extensive fermentation process, and easy industrialized large-scale production.

Description

Method for preparing compound enzyme and probiotic preparation for feed by biotransforming sorghum straw and rapeseed cake

technical field

The invention belongs to the field of microbial fermentation, and in particular relates to a method for fermenting sorghum stalks and rapeseed cakes with micro-ecologically stable food-grade safe flora to prepare a compound enzyme and probiotic preparation for feed.

Background technique

my country is a populous country with extremely tight arable land, and there is a huge gap in the demand for feed raw materials, especially energy feed and protein feed.

Lignocellulose is the most abundant and cheapest renewable resource on earth. The production and processing of agricultural products and food also produces a large amount of lignocellulosic by-products. There are many kinds of lignocellulose, such as various crop straws, urban cellulosic waste, agricultural product processing by-products such as corncobs, vegetable stems and leaves, fruit and vegetable residues, etc. The components of lignocellulose mainly include cellulose, hemicellulose, lignin and Pectin, etc., has become a complex substrate with great application value in nature because of its bulk and cheapness. However, due to the complex composition and structure of lignocellulose, it is difficult to be digested by monogastric animals. Realizing high-efficiency and low-cost biotransformation of non-grain substances such as lignocellulose, and making them replace corn and other grains as energy feeds will be an important growth point for the development of the feed industry.

Cakes are a class of bulk agricultural by-products rich in protein. With the rapid development of the breeding industry, the demand for protein resources is also increasing, and the source of high-quality animal protein resources (such as fishmeal) is limited, and the price has been rising rapidly in recent years. It can greatly alleviate the shortage of feed protein resources, and can reduce the cost of feed. There are many kinds of cakes, including soybean cakes, cottonseed (kernel) cakes, rapeseed cakes, peanut (kernel) cakes, sunflower (kernel) cakes, tea seed cakes, sesame cakes, etc., but the cakes Contains a very wide variety of toxic and anti-nutrient substances. For example, in the presence of water, glucosinolates are decomposed by myrosinase to produce toxic substances such as isothiocyanate and nitrile, which can cause animal goiter, metabolic disorders, and even subcutaneous hemorrhage, and also affect the adrenal cortex, pituitary gland, and liver. Function. The anti-nutrient alpha-galactoside compounds cause intestinal gas in animals and affect digestion. Moreover, while the cake is rich in protein, it also contains quite high plant fiber components, which is not easy for monogastric animals to digest. If the toxicity and anti-nutritional substances in the cake can be eliminated efficiently and at low cost and biotransformed, all kinds of cake will be fully developed into protein feed raw materials and replace high-priced animal feed protein such as fish meal, which will become the development of the feed industry in the future. Another important growth point.

Microbial fermentation is an effective way to achieve low-cost and high-efficiency biotransformation of lignocellulose and cake-like agricultural by-products. Lignocellulose is basically carbohydrates, and the cake is rich in protein. The combination of the two can provide microorganisms with a nutrient-rich fermentation substrate with an appropriate ratio. Cost-effective biotransformation offers new strategies. According to the theory of systems biology, the microbial community, its enzyme system and its substrate environment are an inseparable and complete system, and its formation is the result of long-term natural evolution and balance. In this system, the types and activities of enzymes produced by different microorganisms are different. Only when they coexist in the same system and share the environmental factors provided by the system can they effectively transform the substrate. In nature, wild microbial communities with stable microecology and balance composed of many different species of microorganisms are ubiquitous, such as rumen microbial communities, biogas microbial communities, compost microbial communities, feed silage microbial communities, higher animal intestinal microbial communities, plant endogenous bacterial community, white rot fungal community, intestinal endophytic community of wood-eating insects, etc. These wild microbial communities can perform stable mixed fermentation in their specific wild environment, and because of the diversity of enzymes they produce, they can biotransform complex substrates. Therefore, according to the theory of systems biology, by constructing a micro-ecologically stable flora with strong enzyme production and fermentation ability and suitable for lignocellulose and cake substrate environment on the basis of natural flora, it is expected to realize lignocellulose and cake. Low-cost and high-efficiency bioconversion of cakes.

Pu-erh tea is a kind of fermented tea, which forms a complex but micro-ecologically stable flora during the pile fermentation process, that is, the Pu-erh tea pile flora, which includes Aspergiltus (Aspergiltus), Rhizopus ( Rhizopus), Penicillium, Trichoderma, C.parapsilosis, C.famata, C.cifferrii, Gram Saccharomyces kluyoerii, Cryptococcus laurentii, Basidiomycetes, Lactobacillus, Bacillus, Brevibaterium, Staphylococcus , Actinoplanes, Streptomyces, etc., including filamentous fungi, yeast and bacteria, both enzyme-producing microorganisms and probiotics, have rich and powerful enzyme production capabilities, fermentation transformation capabilities and The beneficial effect is expected to be applied to the biotransformation of lignocellulose and cake-like agricultural by-products.

Edible fungi are a large class of food-grade safe large fungi that grow and reproduce on lignocellulose. Important edible fungi include Pleurotus otreatus, Pleurotus flabellatus, Pleurotus pulmonarius, Mushroom (Pleurotus sajor-caju), Shiitake mushroom (Lentinus edobes), Tiger skin mushroom (Lentinus tiginus), Flammulina velutipes (Flamulina velutipes), Ganoderma lucidum (Gannodema lucidum), Schizophyllum commune, Phlebia radiata, cloud Coriolus versicolor, Plearotus citrinopileatus, Plearotus cornucopiae, Pleurotus eryngii, Auricularia auricular, Morchella esculenta, Agrocybecylindracea (Pholiota adipose), Agaricus bisporus, Hypsizigus marmoreus, Hercicium erinaceum, Agaricus blazei, Pholiota nameko, etc. Because edible fungi secrete a large amount of lignocellulosic enzymes into the substrate during the growth and reproduction process, the substrate has a strong biotransformation ability for agricultural by-products, especially lignocellulosic agricultural by-products.

Contents of the invention

The invention provides a method for biotransforming sorghum stalks and rapeseed cakes to prepare a compound enzyme and probiotic preparation for feed, using micro-ecologically stable food-grade safe flora to ferment sorghum stalks and rapeseed cakes to prepare feed compound Enzyme and probiotic preparation to realize low-cost and high-efficiency biotransformation of sorghum straw and rapeseed cake.

A method for preparing compound enzyme and probiotic preparation for feed by biotransforming sorghum stalks and rapeseed cake, comprising the following steps:

1) Fully mix fermented Pu'er cooked tea, shiitake mushroom sticks, tea tree mushroom sticks, the first sorghum straw, the first rapeseed cake, the first wheat bran, the first apple pomace and the first water. After acclimatization and cultivation, a starter culture is formed;

2) Fully mix the starter, the second sorghum stalk, the second rapeseed cake, the second wheat bran, the second apple pomace and the second water in step 1), and after fermentation, a compound enzyme and prebiotic for feed is obtained Bacterial preparations.

Pu-erh tea is a drink with a long history and is recognized as safe. The Pu-erh tea fermented Pu-erh tea flora formed in the fermented Pu-erh tea is food-grade safe flora. Lentinus edodes and tea tree mushrooms are food-grade safe large fungi that are eaten daily. , its fungus sticks are rich in lignocellulosic enzymes. Sorghum straw and rapeseed meal are two bulk agricultural by-products. Mix the fermented Pu'er cooked tea, shiitake mushroom sticks, tea tree mushroom sticks with the first sorghum stalk, the first rapeseed cake, the first wheat bran, the first apple pomace and the first water, etc., through Domesticated and cultivated, the Pu-erh tea pile flora and the natural flora in the substrate are fused to form a micro-ecologically stable food-grade safe flora, which not only has the rich and powerful enzyme-producing ability and fermentation Transformation ability and probiotic effect, and adapt to the substrate environment. At the same time, the substrate environment is rich in lignocellulosic enzymes derived from mushroom sticks and tea tree mushroom sticks, which can treat sorghum straw (namely the first sorghum straw and the second sorghum Straw) and rapeseed cake (that is, the first rapeseed cake and the second rapeseed cake) for low-cost and high-efficiency bioconversion. Therefore, the obtained starter has better performance.

Ferment sorghum stalks and rapeseed cakes with micro-ecologically stable food-grade safe bacteria in the starter, and convert sorghum stalks and rapeseed cakes into feed-use compound enzyme and probiotic preparations, and the obtained feed-use compound enzyme and probiotics The performance indexes of the probiotic preparations were better.

During the preparation and fermentation of the starter, sorghum stalks were used as the main carbon source, and rapeseed cake was used as the main nitrogen source, constituting the basic components of the fermentation substrate. Wheat bran (that is, the first wheat bran and the second wheat bran) is rich in vitamins and mineral elements, and apple pomace (that is, the first apple pomace and the second apple pomace) is rich in vitamins and fermentable sugars that can be quickly utilized by microorganisms. Wheat bran and apple pomace can provide sufficient growth factors (such as cofactors of related enzymes) and rapid utilization of carbon source for microbial fermentation process.

In step 1), as a preference, the pile-fermented Pu’er cooked tea, shiitake mushroom sticks, tea tree mushroom sticks, first sorghum straw, first rapeseed cake, first wheat bran, first apple pomace The mass ratio with the first water is 10:1～10:1～10:5～15:2～8:1～4:0.5～2:10～30. Under the above mass ratio of raw materials, it is beneficial to Pu’er tea The fusion of Otto flora and natural flora forms a micro-ecologically stable food-grade safe flora, and a starter with better fermentation performance is obtained. Further preferably, the stack-fermented Pu'er cooked tea, mushroom sticks, tea tree mushroom sticks, the first sorghum straw, the first rapeseed cake, the first wheat bran, the first apple pomace and the first water The mass ratio is 10:5:5:10:5:2:1:22-25, and the starter made from raw materials with this mass ratio has the best fermentation performance.

Preferably, the condition of the domestication culture is 24 to 60 hours at 30°C to 45°C, which is conducive to the fusion of Pu-erh tea stack flora and natural flora in the substrate to form a micro-ecologically stable Food-grade safe flora to prepare a starter with better fermentation performance. Further preferably, the condition of the acclimatization culture is 36-48 hours at 35° C. to 40° C., and the condition of the acclimatization culture can prepare a starter with the best fermentation performance.

In step 2), preferably, the mass ratio of the starter, the second sorghum straw, the second rapeseed cake, the second wheat bran, the second apple pomace and the second water is 10:100-800:50 ～500: 10～100: 5～60: 100～1500, the above mass ratio of raw materials, after fermentation, is conducive to obtaining a compound enzyme and probiotic preparation for feed with better performance indicators. Further preferably, the mass ratio of the starter, the second sorghum straw, the second rapeseed cake, the second wheat bran, the second apple pomace and the second water is 10:300～600:150～300:30～ 60: 15-30: 400-1000, which is conducive to obtaining the compound enzyme and probiotic preparation for feed with the best performance indicators.

Preferably, the fermentation adopts solid-state fermentation, the microorganisms are easy to grow, the enzyme activity is high, and the preparation is simple, which is conducive to industrial promotion and utilization. More preferably, the conditions of the solid-state fermentation are as follows: material thickness is 5cm-20cm, natural ventilation, fermentation at 30°C-45°C for 2-7 days. The solid-state fermentation conditions are conducive to obtaining a compound enzyme and probiotic preparation for feed with more excellent performance indicators. Further preferably, the conditions of the solid-state fermentation are as follows: the material thickness of the solid-state fermentation is 10cm-15cm, naturally ventilated, and fermented at 35°C-40°C for 4-5 days. The solid-state fermentation conditions are conducive to obtaining the most excellent performance indicators. Compound enzyme and probiotic preparation for feed.

According to the chemical composition, the nutrients in the feed are mainly carbohydrates (such as starch, cellulose, hemicellulose, pectin), proteins, heterocyclics and atypical sugars and other compounds (such as lignin, alpha-glycosides, phytic acid, tannins, etc.). Adding enzymes that predigest or convert these compounds to feed has a significant effect on improving livestock performance. At present, cellulase and hemicellulase such as xylanase, amylase, protease, alpha-galactosidase, phytase, etc. are widely used in feed production as feed enzyme preparations, and are usually Combine multiple enzymes and add them as a complex enzyme preparation. The more enzymes contained in the complex enzyme preparation, the higher the efficiency of predigestion or conversion of feed. The flora used in the present invention is a micro-ecologically stable food-grade safe flora formed by the fusion of Pu-erh tea pile flora and natural flora in the fermentation substrate. There are rich types of microorganisms and can be fermented to produce a very rich variety of compound enzymes for feed , the evaluation of its efficacy is carried out by measuring the activities of the relevant enzymes.

Probiotics have a significant effect on improving animal health by regulating intestinal function. Lactic acid bacteria are the most important probiotics. Among the flora used in the present invention, the most important source is Pu-erh tea pile flora, which contains a large number of lactic acid bacteria. These lactic acid bacteria proliferate in large quantities through solid-state fermentation and become probiotic preparations. The evaluation of its efficacy is carried out by measuring the number of lactic acid bacteria. .

Through the measurement results of the relevant enzyme activities and the measurement results of the number of lactic acid bacteria, it can be known that the product prepared by the invention is very suitable as a compound enzyme and probiotic preparation for feed.

Compared with prior art, the present invention has following advantage:

In the present invention, the method of biotransforming sorghum stalks and rapeseed cakes to prepare compound enzymes and probiotic preparations for feed is to mix fermented Pu'er cooked tea, shiitake mushroom sticks, tea tree mushroom sticks with the first sorghum stalks, the second After the rapeseed cake, the first wheat bran, the first apple pomace and the first water are mixed with the substrate, the Pu'er tea fermented flora in the fermented Pu'er ripe tea can be combined with the natural bacteria in the substrate through domestication and cultivation. The micro-ecologically stable food-grade safe flora has the rich and powerful enzyme-producing ability, fermentation transformation ability and probiotic effect of the Pu-erh tea Otto pile flora, and adapts to At the same time, the substrate environment is rich in lignocellulosic enzymes derived from mushroom sticks of shiitake mushrooms and tea tree mushroom sticks, which can perform low-cost and high-efficiency biotransformation of sorghum straw and rapeseed cake. Ferment sorghum stalks and rapeseed cakes with micro-ecologically stable food-grade safe bacteria in the starter, and convert sorghum stalks and rapeseed cakes into feed-use compound enzyme and probiotic preparations, and the obtained feed-use compound enzyme and probiotics The performance indexes of the probiotic preparations were better.

In the present invention, the method of biotransforming sorghum stalks and rapeseed cakes to prepare compound enzymes and probiotic preparations for feed, under the action of microecologically stable food-grade safe flora in the starter, solid-state fermentation can be used for fermentation, and microorganisms are easy to grow , high enzyme activity, extensive fermentation process, no strict sterile conditions, simple post-treatment, no waste water discharge, simple equipment structure, low investment, low energy consumption, easy operation, easy to industrialized large-scale production, and has good economic benefits and Broad application prospects.

Detailed ways

The shiitake mushroom sticks and tea tree mushroom sticks in the examples are all commercially available products produced by Zhejiang Tianquan Moganshan Mushroom Industry Co., Ltd., the sorghum straw is collected from the planting site, and the rapeseed cake is from Sanlihe Zhang’s in Queshan County. For the commercially available products of the Cake Trade Department, wheat bran is commercially available from Dafeng Jinmaiyuan Wheat Kernel Factory, and apple pomace is commercially available from Wanrong County Shuangrong Ecological Agriculture and Animal Husbandry Co., Ltd.

Example 1

1) Fully mix 10kg of fermented Pu-erh cooked tea, 5kg of shiitake mushroom sticks, 5kg of tea tree mushroom sticks, 10kg of sorghum straw, 5kg of rapeseed cake, 2kg of wheat bran, 1kg of apple pomace, and 22kg of water. After 48 hours of acclimatization and cultivation, a leavening agent is formed;

2) Fully mix 20kg of starter in step 1) with 600kg of sorghum stalks, 300kg of rapeseed cake, 60kg of wheat bran, 30kg of apple pomace, and 800kg of water, and use solid-state fermentation with a material thickness of 15cm and natural ventilation at 40°C Ferment for 4 days to obtain the compound enzyme and probiotic preparation for feed.

After determination, the performance indicators of the compound enzyme and probiotic preparation for feed in this embodiment are: carboxymethyl cellulase activity reaches 77.23IU/g; total cellulase activity reaches 6.88IU/g; beta-glucosidase activity reaches 77.23IU/g; reached 15.56IU/g; endoglucanase activity reached 55.90IU/g; xylanase activity reached 58.57IU/g; beta-xylosidase activity reached 5.64IU/g; polygalacturonase activity reached 77.74IU/g; pectate lyase activity reaches 75.52IU/g; laccase activity reaches 80.61IU/g; mannanase activity reaches 38.16IU/g; alpha-galactosidase activity reaches 14.23IU/g The activity of alpha amylase reaches 150.53IU/g; the activity of protease reaches 188465IU/g; the activity of phytase reaches 114.64IU/g; the activity of tannase reaches ^1435IU /g;

Example 2

1) Fully mix 10kg of fermented Pu-erh tea, 5kg of shiitake mushroom sticks, 5kg of tea tree mushroom sticks, 10kg of sorghum straw, 5kg of rapeseed cake, 2kg of wheat bran, 1kg of apple pomace, and 25kg of water. After 48 hours of acclimatization and cultivation, a leavening agent is formed;

2) Fully mix 20kg of starter in step 1) with 600kg of sorghum stalks, 300kg of rapeseed cake, 60kg of wheat bran, 30kg of apple pomace, and 800kg of water, and use solid-state fermentation with a material thickness of 15cm and natural ventilation at 40°C Ferment for 4 days to obtain the compound enzyme and probiotic preparation for feed.

After determination, the performance indicators of the compound enzyme and probiotic preparation for feed in this embodiment are: carboxymethyl cellulase activity reaches 76.78IU/g; total cellulase activity reaches 6.96IU/g; beta-glucosidase activity reaches 76.78IU/g; reached 15.42IU/g; endoglucanase activity reached 55.46IU/g; xylanase activity reached 58.35IU/g; beta-xylosidase activity reached 5.36IU/g; polygalacturonase activity reached 77.48IU/g; pectate lyase activity reaches 75.48IU/g; laccase activity reaches 80.46IU/g; mannanase activity reaches 37.86IU/g; alpha-galactosidase activity reaches 14.11IU/g The activity of alpha amylase reaches 151.13IU/g; the activity of protease reaches 189775IU/g; the activity of phytase reaches 113.56IU/g; the activity of tannase reaches ^1428IU /g;

Example 3

1) Fully mix 10kg of fermented Pu’er tea, 5kg of shiitake mushroom sticks, 5kg of tea tree mushroom sticks, 10kg of sorghum straw, 5kg of rapeseed cake, 2kg of wheat bran, 1kg of apple pomace, and 25kg of water. After 36 hours of acclimatization and cultivation, a leavening agent is formed;

2) Fully mix 10kg of starter in step 1) with 600kg of sorghum straw, 300kg of rapeseed cake, 60kg of wheat bran, 30kg of apple pomace, and 1000kg of water, and use solid-state fermentation with a material thickness of 10cm, naturally ventilated, and fermented at 35°C After 5 days, a compound enzyme and probiotic preparation for feed was obtained.

After determination, the performance indicators of the compound enzyme and probiotic preparation for feed in this embodiment are: carboxymethyl cellulase activity reaches 74.92IU/g; total cellulase activity reaches 6.14IU/g; beta-glucosidase activity reaches 74.92IU/g; reached 15.51IU/g; endoglucanase activity reached 52.35IU/g; xylanase activity reached 59.66IU/g; beta-xylosidase activity reached 5.88IU/g; polygalacturonase activity reached 78.57IU/g; pectate lyase activity reaches 76.69IU/g; laccase activity reaches 82.22IU/g; mannanase activity reaches 39.23IU/g; alpha-galactosidase activity reaches 13.89IU/g The activity of alpha amylase reaches 154.64IU/g; the activity of protease reaches 190036IU/g; the activity of phytase reaches 115.48IU/g; the activity of tannase reaches ^1397IU /g;

Example 4

1) Fully mix 10kg of fermented Pu’er tea, 1kg of shiitake mushroom sticks, 1kg of tea tree mushroom sticks with 5kg of sorghum straw, 2kg of rapeseed cake, 1kg of wheat bran, 0.5kg of apple pomace, and 10kg of water, and mix them at 45 After 24 hours of domestication and cultivation at ℃, a starter culture is formed;

2) Fully mix 20kg of starter in step 1) with 200kg of sorghum stalks, 100kg of rapeseed cake, 20kg of wheat bran, 10kg of apple pomace, and 200kg of water, and use solid-state fermentation with a material thickness of 5cm, naturally ventilated, at 45°C Ferment for 2 days to obtain a compound enzyme and probiotic preparation for feed.

After determination, the performance indicators of the compound enzyme and probiotic preparation for feed in this embodiment are: carboxymethyl cellulase activity reaches 68.86IU/g; total cellulase activity reaches 5.66IU/g; beta-glucosidase activity reaches 68.86IU/g; reached 14.11IU/g; endoglucanase activity reached 47.28IU/g; xylanase activity reached 54.87IU/g; beta-xylosidase activity reached 5.35IU/g; polygalacturonase activity reached 71.27IU/g; pectate lyase activity reaches 70.12IU/g; laccase activity reaches 74.35IU/g; mannanase activity reaches 35.12IU/g; alpha-galactosidase activity reaches 12.57IU/g The activity of alpha amylase reaches 141.45IU/g; the activity of protease reaches 173028IU/g; the activity of phytase reaches 104.24IU/g; the activity of tannase reaches ^1278IU /g;

Example 5

1) Fully mix 10kg of fermented Pu-erh tea, 10kg of shiitake mushroom sticks, 10kg of tea tree mushroom sticks, 15kg of sorghum straw, 8kg of rapeseed cake, 4kg of wheat bran, 2kg of apple pomace, and 30kg of water, and mix them at 30°C After 60 hours of acclimatization and cultivation, a leavening agent is formed;

2) Fully mix 10kg of starter in step 1) with 800kg of sorghum stalks, 500kg of rapeseed cake, 100kg of wheat bran, 60kg of apple pomace, and 1500kg of water, and use solid-state fermentation with a material thickness of 20cm, naturally ventilated, and fermented at 30°C After 7 days, a compound enzyme and probiotic preparation for feed was obtained.

After determination, the performance indicators of the compound enzyme and probiotic preparation for feed in this embodiment are: carboxymethyl cellulase activity reaches 67.86IU/g; total cellulase activity reaches 5.72IU/g; beta-glucosidase activity reaches 5.72IU/g; reached 14.24IU/g; endoglucanase activity reached 47.56IU/g; xylanase activity reached 54.36IU/g; beta-xylosidase activity reached 5.57IU/g; polygalacturonase activity reached 71.52IU/g; pectate lyase activity reaches 71.09IU/g; laccase activity reaches 74.13IU/g; mannanase activity reaches 34.92IU/g; alpha-galactosidase activity reaches 12.64IU/g The activity of alpha amylase reaches 141.43IU/g; the activity of protease reaches 174828IU/g; the activity of phytase reaches 105.35IU/g; the activity of tannase reaches ^1236IU /g;

Various enzyme activities and lactic acid bacteria density assay methods are as follows:

1. Determination of enzyme activity of carboxymethyl cellulase, total cellulase and beta-glucosidase

The determination of total cellulase (i.e. filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and beta-glucosidase activity is in accordance with the regulations of the International Union of Pure and Applied Chemistry (International Union of Pure and Applied Chemistry) procedure (see "Ghose TK, 1987. Measurements of cellulase activities. Pure Appl Chem, 59, 257-268." for details).

Filter paper cellulase (FPase) activity unit: take Whatman No.1 filter paper (1.0×6.0cm=50.0mg) as substrate, react in 50mM sodium citrate buffer (pH=4.8) for 60min at 50°C, The release of 1 μmol reducing sugar per minute is an international unit (IU) of enzyme activity, expressed as IU/g dry weight.

Carboxymethyl cellulase (CMCase) activity unit: take 2% (w/v) carboxymethyl cellulose as substrate, react in 50mM sodium citrate buffer (pH=5.5) for 30min at 50°C, The release of 1 μmol reducing sugar per minute is an international unit (IU) of enzyme activity, expressed as IU/g dry weight.

Beta glucosidase activity unit: add 1mL p-nitrophenol-β-glucoside (p-nitrophenyl glucopyranoside, pNPG) (1mM) (1mM) to 2mL acetate buffer as a substrate, react at 50°C for 30min, and measure it colorimetrically at 430nm , with the release of 1 μmol p-nitrophenol (p-nitrophenol) (pNP) per minute as an international unit (IU) of enzyme activity, expressed as IU/g dry weight.

2. Determination of enzyme activity of endoglucanase, xylanase, beta-xylosidase, polygalacturonase and pectate lyase

The activities of endoglucanase, xylanase, polygalacturonase and pectate lyase were determined by carboxymethylcellulose, birch wood xylanase, polygalacturonic acid and pectate As a substrate, according to relevant literature (Cheilas T, Stoupis T, Christakopoulos P, Katapodis P, Mamma D, Hatzinikolaou DG, Kekos D, Macris BJ, 2000. Hemicellulolytic activity of Fusarium oxysporum grown on sugar beet cheese pulp. , 35, 557-561; Jayani RS, Saxena S, Gupta R, 2005. Microbial pectinolytic enzymes: a review. Process Biochem, 40, 2931-2944.). The release of 1 μmol reducing sugar per minute is an international unit (IU) of enzyme activity, expressed as IU/g dry weight.

The determination of beta-xylosidase activity uses p-nitrophenyl glycoside as the substrate, specifically according to the relevant literature (Mamma D, Koullas D, Fountoukidis G, Kekos D, MacrisBJ, Koukios E, 1996. Bioethanol from sweet sorghum : Simultaneous saccharification and fermentation of carbohydrates by a mixed microbial culture. Process Biochem, 31, 377-381.). The release of 1 μmol p-nitrophenol per minute is one international unit (IU) of enzyme activity, expressed as IU/g dry weight.

3. Determination of enzyme activity of laccase

Laccase activity was determined by the oxidation of ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonicacid), the reaction system was adding 100μL of 20mM ABTS to sodium citrate buffer (pH=3.0) and then using Sodium citrate buffer (pH=3.0) was adjusted to 1mL, and the oxidation rate of ABTS was determined by the absorbance at 420nm, according to relevant literature (Susan Grace Karp, Vincenza Faraco, Antonella Amore, Leila Birolo, Chiara Giangrande, Vanete ThomazSoccol, Ashok Pandey , Carlos Ricardo Soccol. Characterization of laccaseisoforms produced by Pleurotus otreatus in solid state fermentation of sugarcane bagasse. Bioresource Technology, 2012, 114, 735-739.). The oxidation of 1 μmol ABTS per minute is one international unit (IU) of enzyme activity, expressed as IU/g dry weight.

4. Determination of Enzyme Activity of Mannanase

The activity of mannanase was measured with carob gum as the substrate, and 1 g of enzyme powder was hydrolyzed at 40°C and pH 5.0 for 1 min to produce a reducing substance equivalent to 1 μmol of mannose, which was 1 unit of enzyme activity. Expressed as IU/g dry weight.

5. Determination of the enzyme activity of alpha-galactosidase

The alpha-galactosidase activity assay system is "0.1mL enzyme solution + 0.8mL 0.2M acetate buffer (pH4.8) + 0.1mL 2mM pNPG", after reacting at 50°C for 15min, add 3mL0.2MNa ₂ CO ₃ to stop For the reaction, the absorbance value was measured at 405 nm, specifically according to the relevant literature (Dey PM, Pridham JB, 1972. Biochemistry of alpha-galactosidase. Adv Enzymol, 36, 911-930.). Release 1 μmol of paranitrophenol per minute as an international unit of enzyme activity (IU), expressed as IU/g dry weight.

6. Determination of enzyme activity of alpha-amylase

The alpha-amylase activity assay system is "150μL enzyme solution + 200μL 0.2% soluble starch, using 0.1M Tris-HCl buffer (pH=7.0) as the solution system", after reacting at 37°C for 30min, add 400μl 3,5-dinitro Salicylic acid terminated the reaction and kept in a boiling water bath for 5 minutes, cooled to room temperature at 25°C, diluted with 8 mL of distilled water, and measured the absorption value at 489 nm, specifically according to the relevant literature (BernfeldP (1955) Amylases, alpha and beta. eds) Methods in enzymemology, Vol1. Academic, New York, pp149-154.). The release of 1 μmol reducing sugar per minute is an international unit (IU) of enzyme activity, expressed as IU/g dry weight.

7. Determination of enzyme activity of protease

Protease activity was determined using sulfanilamide azocasein as a substrate, and the reaction system was 250 μL of 0.1M phosphate buffer (pH 8.5) containing 0.5% azocasein (w/v), Add 150 μL of enzyme solution, react at 37°C for 30 minutes, add 1.2 mL of trichloroacetic acid solution (10%, w/v) to inactivate the enzyme, add 800 μL of1.8N NaOH for neutralization, and measure the absorbance at 420 nm Specifically according to relevant literature (Leighton TJ, Doi RH, Warren RAJ, Kelln RA (1973) The relationship of serine protease activity to RNApolymerase modification and sporulation in Bacillus subtilis.J MolBiol, 76:103-122.). Release 1 μg of even per minute Azocasein is an international unit (IU) of enzyme activity, expressed as IU/g dry weight.

8. Determination of Enzyme Activity of Phytase and Tannase

Phytase activity was measured with p-nitrophenylphosphate (p-nitrophenylphosphate) as substrate, specifically according to relevant literature (Stockmann C, Losen M, Dahlems U, Knocke C, Gellissen G, Buchs J(2003).Effect of oxygen supply on passing, stabilizing and screening of recombinant Hansenula polymorpha production strains in test tube cultures. FEMS Yeast Res, 4(2):195-205.). Release 1 μmol of p-nitrophenol (p-nitrophenol) per minute as an international unit of enzyme activity (IU), expressed as IU/g dry weight.

Tannase activity was determined with tannic acid as the substrate, specifically according to the relevant literature (Mondal KC, Banerjee D, Jana M, Pati BR (2001). Colorimetric assay method for determination of the tannin acyl hydrolase (EC3.1.1.20) activity . Anal Biochem, 295(2):168-171.). Convert 1 μmol of tannic acid into one international unit (IU) of enzyme activity per minute, expressed as IU/g dry weight.

9. Determination of the density of lactic acid bacteria

Man Rogosa Sharpe (MRS) medium is a selective medium for lactic acid bacteria. The total number of colonies on ManRogosa Sharpe (MRS) medium can be measured and converted to the density of lactic acid bacteria. The total number of colonies was determined according to the relevant literature (Song Y, Luo Y, You J, Shen H, & Hu S. (2012). Biochemical, sensory and microbiological attributes of bream (Megalobrama amblycephala) during partial freezing and chilled storage. J Sci FoodAgric, 92 (1), 197-202.).

Claims (10)

1. a bio-transformation broomcorn straw and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, and it is characterized in that, may further comprise the steps:

1) will fully mix through Pu'er cooked tea, lentinus edodes strain stick, agrocybe bacterium rod, the first broomcorn straw, the first rapeseed cake dregs, the first wheat bran, the first pomace and first water of pile-fermentation, after domestication is cultivated, form leavening;

2) leavening in the step 1), the second broomcorn straw, the second rapeseed cake dregs, the second wheat bran, the second pomace and the second water are fully mixed, after fermentation, obtain the complex enzyme for feed probiotics preparation of holding concurrently.

2. bio-transformation broomcorn straw according to claim 1 and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, it is characterized in that, in the step 1), the mass ratio of described Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod, the first broomcorn straw, the first rapeseed cake dregs, the first wheat bran, the first pomace and the first water is 10:1～10:1～10:5～15:2～8:1～4:0.5～2:10～30.

3. bio-transformation broomcorn straw according to claim 2 and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, it is characterized in that, the mass ratio of described Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod, the first broomcorn straw, the first rapeseed cake dregs, the first wheat bran, the first pomace and the first water is 10:5:5:10:5:2:1:22～25.

4. bio-transformation broomcorn straw according to claim 1 and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, and it is characterized in that, in the step 1), the condition that described domestication is cultivated be 30 ℃～45 ℃ domestication cultivations 24～60 hours.

5. bio-transformation broomcorn straw according to claim 4 and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, and it is characterized in that, the condition that described domestication is cultivated be 35 ℃～40 ℃ domestication cultivations 36～48 hours.

6. bio-transformation broomcorn straw according to claim 1 and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, it is characterized in that, step 2) in, the mass ratio of described leavening, the second broomcorn straw, the second rapeseed cake dregs, the second wheat bran, the second pomace and the second water is 10:100～800:50～500:10～100:5～60:100～1500.

7. bio-transformation broomcorn straw according to claim 6 and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, it is characterized in that, the mass ratio of described leavening, the second broomcorn straw, the second rapeseed cake dregs, the second wheat bran, the second pomace and the second water is 10:300～600:150～300:30～60:15～30:400～1000.

8. bio-transformation broomcorn straw according to claim 1 and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, and it is characterized in that step 2) in, solid state fermentation is adopted in described fermentation.

9. bio-transformation broomcorn straw according to claim 8 and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, it is characterized in that, the condition of described solid state fermentation is: material thickness is 5cm～20cm, and gravity-flow ventilation was 30 ℃～45 ℃ fermentations 2～7 days.

10. bio-transformation broomcorn straw according to claim 9 and rapeseed cake dregs prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, it is characterized in that, the condition of described solid state fermentation is: the material thickness of solid state fermentation is 10cm～15cm, gravity-flow ventilation was 35 ℃～40 ℃ fermentations 4～5 days.