CN103315132B - Method for preparing composite enzyme/probiotic preparation for feed by bioconverting rice straw and castor cake pulp - Google Patents
Method for preparing composite enzyme/probiotic preparation for feed by bioconverting rice straw and castor cake pulp Download PDFInfo
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Abstract
The invention discloses a method for preparing a composite enzyme/probiotic preparation for feed by bioconverting rice straw and castor cake pulp, which comprises the following steps: thoroughly mixing Pu'er tea after pile fermentation, shiitake stick, agrocybe aegerita stick, first rice straw, first castor cake pulp, first wheat bran, first apple pomace and first water, and carrying out acclimatization culture to form a leavening agent; and thoroughly mixing the leavening agent, second rice straw, second castor cake pulp, second wheat bran, second apple pomace and second water, and fermenting to obtain the composite enzyme/probiotic preparation for feed. In the invention, the microecologically-stable food-grade safe florae are utilized to ferment the rice straw and castor cake pulp to prepare the composite enzyme/probiotic preparation for feed with favorable performance indexes, thereby implementing low-cost high-efficiency bioconversion of the rice straw and castor cake pulp. Meanwhile, the method disclosed by the invention has the advantages of easy growth of microorganisms, high enzyme activity and extensive fermentation process, and can easily implement industrialized large-scale production.
Description
Technical field
The invention belongs to microorganism fermentation field, be specifically related to a kind of safe flora fermentation rice straw of food stage and castor bean meal that adopts micro-Ecological Stabilization, prepare the complex enzyme for feed method of probiotics preparation of holding concurrently.
Background technology
China is a populous nation that arable land puts upon the full stretch, and the feedstuff particularly demand of energy feed and protein feed all exists huge breach.
Lignocellulosic is renewable resource abundant, the most cheap on the earth.The process of manufacture of agricultural product and food also produces a large amount of lignocellulose accessory substances.Lignocellulosic is of a great variety, such as various agricultural crop straws, city cellulosic rubbish, processing of farm products accessory substance as corncob, vegetable waste, fruits and vegetables slag etc., the composition of lignocellulosic mainly comprises cellulose, hemicellulose, lignin and pectin etc., because becoming occurring in nature, its large cheapness there is the complex substrate that major application is worth, but due to composition and the complex structure of lignocellulosic, thereby be difficult to by simple stomach animal digestion.Realize the non-grain material high efficiency, low cost bio-transformations such as lignocellulosic, make the cereal such as its replacement of corn as energy feed, by the important growth point that is Feed Industry Development.
Grouts are the rich proteinaceous large agricultural by-products of a class.Fast development along with aquaculture industry, also increasing to protein resource demand, and animality high-quality protein resource (as fish meal) is originated limited, price is in recent years always in quick rise, therefore, take grouts as protein feed, will greatly alleviate the nervous situation of feed protein resource, and can reduce feed cost.Grouts are of a great variety, comprise soybean cake dregs, cottonseed (benevolence) grouts, rapeseed cake dregs, peanut (benevolence) grouts, sunflower (benevolence) grouts, castor bean meal, castor bean meal etc., but in grouts, contain very various toxicant and anti-nutrient substance.Such as sulphur glycosides, decomposite the noxious materials such as isothiocyanates and nitrile having under regimen condition through myrosase, make the enlargement of animal thyroid gland, metabolic disturbance, even causes subcutaneous hemorrhage, also affects the function of the organs such as adrenal cortex, pituitary and liver.Thereby anti-nutrient substance alpha-galactoside compound makes animal intestinal flatulence affect digestive function.And grouts in the proteinaceous while of richness also containing quite high plant fiber component, nonruminant digestion is difficult for.If can eliminate toxicity and the anti-nutrient substance in grouts and carry out bio-transformation in high efficiency, low cost ground, all kinds of grouts will be developed as the high price animal fodder albumen such as protein feed raw material Peru Fish Dietary fully, become another important growth point of future in feed industry development.
Microorganism fermentation is the effective way that realizes lignocellulosic and the bio-transformation of puffed soybean agricultural by-products low-cost high-efficiency.Lignocellulosic is carbohydrate substantially, grouts are rich in protein, both combinations can be microorganism nutritious, well-proportioned fermentation substrate are provided, and system biological theory is realized lignocellulosic and grouts low-cost high-efficiency bio-transformation for fermenting by microorganism provides New Policy.According to system biological theory, microbiologic population and its enzyme system with and substrate environment of living in be an indivisible holonomic system, its formation is the result of long-term natural evolution and balance.In this system, kind and the vigor of enzyme that different microorganisms is produced are had nothing in common with each other, and only coexist in same system and environmental factor that shared system provides, effectively conversion of substrate.The wild microbiologic population of the micro-Ecological Stabilization balance being formed by many kind different microorganisms at occurring in nature ubiquity, such as rumen microorganism group, biogas microbiologic population, compost microbe group, feed ensiling microbiologic population, higher mammal intestinal microflora, endophyte of plant group, white-rot fungi group, xylophaga enteron aisle endophyte group etc.These wild micropopulations are dropped in its specific wild environment of living in can stablize mixed culture fermentation, and due to its diversity of the enzyme that produces system, can carry out bio-transformation to complex substrate.Therefore, according to system biological theory, by on the basis of natural flora, build one and produce enzyme and fermentability is powerful and micro-Ecological Stabilization flora of applicable lignocellulosic and grouts matrix environment, be expected to realize the low-cost high-efficiency bio-transformation of lignocellulosic and puffed soybean.
Pu'er tea is a kind of fermented tea, and it has formed the bacterium complicated but flora of micro-Ecological Stabilization mutually in pile-fermentation process, i.e. the wet heap flora of Pu'er tea, and this flora comprises aspergillus (Aspergiltus), rhizopus (Rhizopus), Penicillium (Penicillium), trichoderma (Trichoderma), Candida parapsilosis (C.parapsilosis), candida famata (C.famata), candida ciferrii (C.cifferrii), Crewe takes yeast (Saccharomyces kluyoerii), Cryptococcus laurentii (Cryptococcus laurentii), basidiomycetes (Basidiomycetes), lactobacillus (Lactobacillus), bacillus (Bacillus), brevibacterium (Brevibaterium), Coccus (Staphylococcus), line Pseudomonas (Actinoplanes), streptomyces (Streptomyces) etc., comprising filamentous fungi, yeast and bacterium, existing microbes producing cellulase is profitable probliotics again, has possessed abundant and powerful enzymatic productivity, fermentation conversion capability and prebiotic effect, be expected to be applied to the bio-transformation of lignocellulosic and puffed soybean agricultural by-products.
Edible mushroom is that a large class be take lignocellulosic and carried out the safe macro fungi of food stage of growth and breeding as matrix, and important edible mushroom kind comprises flat mushroom (Pleurotus ostreatus), fan-shaped picking up the ears (Pieurotus flabellatus), lung shape pick up the ears (Pleurotus pulmonarius), phoenix-tail mushroom (Pleurotus sajor-caju), mushroom (Lentinus edobes), Lentinus tigrinus (Lentinus tiginus), asparagus (Flamulina velutipes), glossy ganoderma (Gannodema lucidum), schizophyllum commune (Schizophyllum commune), arteries and veins is penetrated bacterium (Phlebia radiata), rainbow conk (Coriolus versicolor), the yellow mushroom (Plearotus citrinopileatus) of elm, Ji mushroom (Plearotus cornucopiae), pleurotus eryngii (Pleurotus eryngii), auricularia auriculajudae (Auricularia auricular), hickory chick (Morchella esculenta), agrocybe (Agrocybe cylindracea), yellow umbrella (Pholiota adipose), agaricus bisporus (Agaricus bisporus), the beautiful gill fungus (Hypsizigus marmoreus) of spot, Hericium erinaceus (Hercicium erinaceum), Ji Songrong (Agaricus blazei), sliding mushroom (Pholiota nameko) etc.Because edible mushroom secretes a large amount of lignocellulosic enzymes in growth and breeding process in matrix, thus its matrix to agricultural by-products particularly lignocellulose agricultural by-products there is very strong bio-transformation ability.
Summary of the invention
The invention provides a kind of bio-transformation rice straw and castor bean meal and prepare the complex enzyme for feed method of probiotics preparation of holding concurrently, adopt ferment rice straw and castor bean meal of the safe flora of food stage of micro-Ecological Stabilization to prepare the complex enzyme for feed probiotics preparation of holding concurrently, realize the low-cost high-efficiency bio-transformation of rice straw and castor bean meal.
Bio-transformation rice straw and castor bean meal are prepared the complex enzyme for feed method for probiotics preparation of holding concurrently, and comprise the following steps:
1) Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod, the first rice straw, the first castor bean meal, the first wheat bran, the first pomace and the first water are fully mixed, after domestication is cultivated, form leavening;
2) leavening in step 1), the second rice straw, the second castor bean meal, 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.
Pu'er tea is a kind of drink with a long history, generally regarded as safe, the wet heap flora of Pu'er tea forming in the Pu'er cooked tea of pile-fermentation is the safe flora of food stage, mushroom and agrocybe are the safe macro fungis of daily edible food stage, contain abundant lignocellulosic enzyme in its bacterium rod.Rice straw and castor bean meal are two kinds of large agricultural by-products.By the Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod and the first rice straw, the first castor bean meal, the first wheat bran, after the matrix such as the first pomace and the first water are mixed, by domestication, cultivate, make the natural flora in the wet heap flora of Pu'er tea and matrix merge the safe flora of food stage that forms micro-Ecological Stabilization, this flora had both had the abundant and powerful enzymatic productivity of the wet heap flora of Pu'er tea, fermentation conversion capability and prebiotic effect, accommodate substrate environment again, simultaneously, in matrix environment, contain the abundant lignocellulosic enzyme that derives from lentinus edodes strain stick and agrocybe bacterium rod, can carry out low-cost high-efficiency bio-transformation to rice straw (i.e. the first rice straw and the second rice straw) and castor bean meal (i.e. the first castor bean meal and the second castor bean meal).Therefore the leavening better performances, obtaining.
With the safe flora of food stage of micro-Ecological Stabilization in leavening, rice straw and castor bean meal are fermented, rice straw and castor bean meal are converted into the complex enzyme for feed probiotics preparation of holding concurrently, and the hold concurrently property indices of probiotics preparation of the complex enzyme for feed obtaining is better.
In leavening preparation and sweat, rice straw is as main carbon source, and castor bean meal, as main nitrogen, has formed the basis in fermentation substrate.Wheat bran (i.e. the first wheat bran and the second wheat bran) is rich in vitamin and mineral element, pomace (i.e. the first pomace and the second pomace) is rich in the fermentable sugar that vitamin and microorganism can utilize fast, adds wheat bran and pomace and can be fermentation process and sufficient growth factor (as the confactor of Some Related Enzymes) is provided and utilizes fast carbon source.
In step 1), as preferably, the mass ratio of the described Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod, the first rice straw, the first castor bean meal, 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, under the raw material of above-mentioned mass ratio, be conducive to the wet heap flora of Pu'er tea and natural flora and merge the safe flora of food stage that forms micro-Ecological Stabilization, obtain the better leavening of fermenting property.Further preferably, the mass ratio of the described Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod, the first rice straw, the first castor bean meal, the first wheat bran, the first pomace and the first water is 10:5:5:10:5:2:1:22～25, the leavening of making under the raw material of this mass ratio, its fermenting property is the most excellent.
As preferably, the condition that described domestication is cultivated is to cultivate 24～60 hours 30 ℃～45 ℃ domestications, the natural flora that the condition that this domestication is cultivated is conducive in the wet heap flora of Pu'er tea and matrix merges the safe flora of food stage that forms micro-Ecological Stabilization, prepares the better leavening of fermenting property.Further preferably, the condition that described domestication is cultivated is to cultivate 36～48 hours 35 ℃～40 ℃ domestications, and the condition that this domestication is cultivated can be prepared the most excellent leavening of fermenting property.
Step 2) in, as preferably, the mass ratio of described leavening, the second rice straw, the second castor bean meal, the second wheat bran, the second pomace and the second water is 10:100～800:50～500:10～100:5～60:100～1500, under the raw material of above-mentioned mass ratio, after fermentation, be conducive to obtain the better complex enzyme for feed of the property indices probiotics preparation of holding concurrently.Further preferably, the mass ratio of described leavening, the second rice straw, the second castor bean meal, the second wheat bran, the second pomace and the second water is 10:300～600:150～300:30～60:15～30:400～1000, is conducive to obtain the best complex enzyme for feed of the property indices probiotics preparation of holding concurrently.
As preferably, described fermentation adopts solid state fermentation, and microorganism easily grows, and enzyme activity is high, and preparation is simple, is conducive to industrialization promotion utilization.Further preferred, the condition of described solid state fermentation is: material thickness is 5cm～20cm, and gravity-flow ventilation, 30 ℃～45 ℃ fermentations 2～7 days.This solid state fermentation conditions is conducive to obtain the more excellent complex enzyme for feed of the property indices probiotics preparation of holding concurrently.Further preferably, the condition of described solid state fermentation is: the material thickness of solid state fermentation is 10cm～15cm, gravity-flow ventilation, 35 ℃～40 ℃ fermentations 4～5 days, this solid state fermentation conditions was conducive to obtain the most excellent complex enzyme for feed of the property indices probiotics preparation of holding concurrently.
It is mainly the compounds such as carbohydrate (as starch, cellulose, hemicellulose, pectic substance), protein, heterocyclic and atypia sugar (as lignin, alpha-glucosides class, phytic acid, tannin etc.) that nutritional labeling in feed is divided according to chemical composition.In feed, add the enzyme that these compounds are carried out to predigestion or conversion, for improving livestock and poultry production performance, there is active effects.At present, cellulase, hemicellulase are all widely used in production of fodder as enzyme Preparations Used for Feeds as zytase, amylase, protease, alpha-galactosidase, phytase etc., and in use conventionally combine plurality of enzymes and become complex enzyme formulation and add.The enzyme comprising in complex enzyme formulation is more, and the effect of feed being carried out to predigestion or conversion is higher.The flora that the present invention adopts is the safe flora of food stage that the natural flora in the wet heap flora of Pu'er tea and fermentation substrate merges the micro-Ecological Stabilization forming, microbe species is abundant, can fermenting and producing go out the complex enzyme for feed that kind is very abundant, to the evaluation of its effect by the mensuration of each Some Related Enzymes vigor is carried out.
Probio has remarkable efficacy by conditioning function of intestinal canal to promoting animal health level.Lactic acid bacteria is topmost probio.In the flora that the present invention adopts, topmost source is the wet heap flora of Pu'er tea, wherein contains a large amount of lactic acid bacterias, and these lactic acid bacterias breed in a large number through solid state fermentations, become probiotics preparation, to the evaluation of its effect by the mensuration of lactic acid bacterium number is carried out.
By the measurement result of each Some Related Enzymes vigor and the measurement result of lactic acid bacterium number, known, product prepared by the present invention is suitable as the complex enzyme for feed probiotics preparation of holding concurrently very much.
Compared with prior art, tool of the present invention has the following advantages:
In the present invention, bio-transformation rice straw and castor bean meal are prepared the complex enzyme for feed method of probiotics preparation of holding concurrently, by the Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod and the first rice straw, the first castor bean meal, the first wheat bran, after the matrix such as the first pomace and the first water are mixed, by domestication, cultivate, make the wet heap flora of Pu'er tea and the natural flora in matrix in the Pu'er cooked tea of pile-fermentation merge the safe flora of food stage that forms micro-Ecological Stabilization, the safe flora of food stage of this micro-Ecological Stabilization had both had the abundant and powerful enzymatic productivity of the wet heap flora of Pu'er tea, fermentation conversion capability and prebiotic effect, accommodate substrate environment again, simultaneously, in matrix environment, contain the abundant lignocellulosic enzyme that derives from lentinus edodes strain stick and agrocybe bacterium rod, can carry out low-cost high-efficiency bio-transformation to rice straw and castor bean meal.With the safe flora of food stage of micro-Ecological Stabilization in leavening, rice straw and castor bean meal are fermented, rice straw and castor bean meal are converted into the complex enzyme for feed probiotics preparation of holding concurrently, and the hold concurrently property indices of probiotics preparation of the complex enzyme for feed obtaining is better.
In the present invention, bio-transformation rice straw and castor bean meal are prepared the complex enzyme for feed method of probiotics preparation of holding concurrently, in leavening under the safe flora effect of the food stage of micro-Ecological Stabilization, fermentation can adopt solid state fermentation, microorganism easily grows, enzyme activity is high, sweat is extensive, do not need strict aseptic condition, post processing is easy, non-wastewater discharge, and simple in equipment, small investment, energy consumption are low, easy to operate, be easy to large-scale industrialization and produce, there is good economic benefit and wide application prospect.
The specific embodiment
The commercially available prod that lentinus edodes strain stick in embodiment and agrocybe bacterium rod all adopt the Tianquan Moganshan Mountain, Zhejiang Gu Ye Co., Ltd to produce, rice straw gathers voluntarily from planting site, castor bean meal adopts the commercially available prod of Qingdao Pingdu De Cheng feedstuff business department, the commercially available prod that wheat bran adopts the Jin Maiyuanmai of Dafeng City benevolence factory to produce, the commercially available prod that pomace adopts the two flourish ecological Farming Ltd. in Wanrong County to produce.
Embodiment 1
1) 10kg is fully mixed with 10kg rice straw, 5kg castor bean meal, 2kg wheat bran, 1kg pomace, 22kg water through Pu'er cooked tea, 5kg lentinus edodes strain stick, the 5kg agrocybe bacterium rod of pile-fermentation, 35 ℃ of domestications, cultivate after 48 hours, form leavening;
2) the leavening 20kg in step 1) is fully mixed with 600kg rice straw, 300kg castor bean meal, 60kg wheat bran, 30kg pomace, 800kg water, adopt solid state fermentation, material thickness is 15cm, gravity-flow ventilation, 40 ℃ of fermentations 4 days, obtain the complex enzyme for feed probiotics preparation of holding concurrently.
After measured, the hold concurrently performance indications of probiotics preparation of the complex enzyme for feed of the present embodiment are: carboxymethylcelluloenzyme enzyme activity reaches 39.65IU/g; Total fiber element enzyme activity reaches 4.22IU/g; Beta-glucoside enzyme activity reaches 9.85IU/g; Endoglucanase vigor reaches 33.54IU/g; Xylanase activity reaches 38.76IU/g; Beta-xylosidase vigor reaches 3.44IU/g; Polygalacturonase vigor reaches 70.32IU/g; Pectin lyase vigor reaches 65.24IU/g; Laccase activity reaches 88.58IU/g; Mannosan enzyme activity reaches 35.77IU/g; Alpha-galactoside enzyme activity reaches 9.05IU/g; Alpha amylase activity reaches 120.29IU/g; Prolease activity reaches 124335IU/g; Phytase vigor reaches 87.78IU/g; Tannase vigor reaches 1655IU/g; Lactic acid bacteria density reaches 2.54 * 10
8cFU/g.
Embodiment 2
1) 10kg is fully mixed with 10kg rice straw, 5kg castor bean meal, 2kg wheat bran, 1kg pomace, 25kg water through Pu'er cooked tea, 5kg lentinus edodes strain stick, the 5kg agrocybe bacterium rod of pile-fermentation, 35 ℃ of domestications, cultivate after 48 hours, form leavening;
2) the leavening 20kg in step 1) is fully mixed with 600kg rice straw, 300kg castor bean meal, 60kg wheat bran, 30kg pomace, 800kg water, adopt solid state fermentation, material thickness is 15cm, gravity-flow ventilation, 40 ℃ of fermentations 4 days, obtain the complex enzyme for feed probiotics preparation of holding concurrently.
After measured, the hold concurrently performance indications of probiotics preparation of the complex enzyme for feed of the present embodiment are: carboxymethylcelluloenzyme enzyme activity reaches 36.48IU/g; Total fiber element enzyme activity reaches 4.12IU/g; Beta-glucoside enzyme activity reaches 9.56IU/g; Endoglucanase vigor reaches 33.42IU/g; Xylanase activity reaches 37.68IU/g; Beta-xylosidase vigor reaches 3.35IU/g; Polygalacturonase vigor reaches 69.24IU/g; Pectin lyase vigor reaches 64.86IU/g; Laccase activity reaches 81.74IU/g; Mannosan enzyme activity reaches 34.96IU/g; Alpha-galactoside enzyme activity reaches 8.98IU/g; Alpha amylase activity reaches 121.12IU/g; Prolease activity reaches 124426IU/g; Phytase vigor reaches 86.78IU/g; Tannase vigor reaches 1647IU/g; Lactic acid bacteria density reaches 2.53 * 10
8cFU/g.
Embodiment 3
1) 10kg is fully mixed with 10kg rice straw, 5kg castor bean meal, 2kg wheat bran, 1kg pomace, 25kg water through Pu'er cooked tea, 5kg lentinus edodes strain stick, the 5kg agrocybe bacterium rod of pile-fermentation, 40 ℃ of domestications, cultivate after 36 hours, form leavening;
2) the leavening 10kg in step 1) is fully mixed with 600kg rice straw, 300kg castor bean meal, 60kg wheat bran, 30kg pomace, 1000kg water, adopt solid state fermentation, material thickness is 10cm, gravity-flow ventilation, 35 ℃ of fermentations 5 days, obtain the complex enzyme for feed probiotics preparation of holding concurrently.
After measured, the hold concurrently performance indications of probiotics preparation of the complex enzyme for feed of the present embodiment are: carboxymethylcelluloenzyme enzyme activity reaches 42.23IU/g; Total fiber element enzyme activity reaches 4.65IU/g; Beta-glucoside enzyme activity reaches 9.45IU/g; Endoglucanase vigor reaches 33.25IU/g; Xylanase activity reaches 40.43IU/g; Beta-xylosidase vigor reaches 3.75IU/g; Polygalacturonase vigor reaches 64.50IU/g; Pectin lyase vigor reaches 61.12IU/g; Laccase activity reaches 78.57IU/g; Mannosan enzyme activity reaches 30.54IU/g; Alpha-galactoside enzyme activity reaches 7.96IU/g; Alpha amylase activity reaches 115.25IU/g; Prolease activity reaches 118546IU/g; Phytase vigor reaches 98.84IU/g; Tannase vigor reaches 1476IU/g; Lactic acid bacteria density reaches 2.26 * 10
8cFU/g.
Embodiment 4
1) 10kg is fully mixed with 5kg rice straw, 2kg castor bean meal, 1kg wheat bran, 0.5kg pomace, 10kg water through Pu'er cooked tea, 1kg lentinus edodes strain stick, the 1kg agrocybe bacterium rod of pile-fermentation, 45 ℃ of domestications, cultivate after 24 hours, form leavening;
2) the leavening 20kg in step 1) is fully mixed with 200kg rice straw, 100kg castor bean meal, 20kg wheat bran, 10kg pomace, 200kg water, adopt solid state fermentation, material thickness is 5cm, gravity-flow ventilation, 45 ℃ of fermentations 2 days, obtain the complex enzyme for feed probiotics preparation of holding concurrently.
After measured, the hold concurrently performance indications of probiotics preparation of the complex enzyme for feed of the present embodiment are: carboxymethylcelluloenzyme enzyme activity reaches 36.15IU/g; Total fiber element enzyme activity reaches 3.86IU/g; Beta-glucoside enzyme activity reaches 8.98IU/g; Endoglucanase vigor reaches 30.66IU/g; Xylanase activity reaches 35.58IU/g; Beta-xylosidase vigor reaches 3.16IU/g; Polygalacturonase vigor reaches 64.12IU/g; Pectin lyase vigor reaches 59.14IU/g; Laccase activity reaches 80.24IU/g; Mannosan enzyme activity reaches 32.23IU/g; Alpha-galactoside enzyme activity reaches 8.42IU/g; Alpha amylase activity reaches 108.48IU/g; Prolease activity reaches 115234IU/g; Phytase vigor reaches 80.12IU/g; Tannase vigor reaches 1332IU/g; Lactic acid bacteria density reaches 2.12 * 10
8cFU/g.
Embodiment 5
1) 10kg is fully mixed with 15kg rice straw, 8kg castor bean meal, 4kg wheat bran, 2kg pomace, 30kg water through Pu'er cooked tea, 10kg lentinus edodes strain stick, the 10kg agrocybe bacterium rod of pile-fermentation, 30 ℃ of domestications, cultivate after 60 hours, form leavening;
2) the leavening 10kg in step 1) is fully mixed with 800kg rice straw, 500kg castor bean meal, 100kg wheat bran, 60kg pomace, 1500kg water, adopt solid state fermentation, material thickness is 20cm, gravity-flow ventilation, 30 ℃ of fermentations 7 days, obtain the complex enzyme for feed probiotics preparation of holding concurrently.
After measured, the hold concurrently performance indications of probiotics preparation of the complex enzyme for feed of the present embodiment are: carboxymethylcelluloenzyme enzyme activity reaches 36.05IU/g; Total fiber element enzyme activity reaches 3.75IU/g; Beta-glucoside enzyme activity reaches 8.86IU/g; Endoglucanase vigor reaches 30.32IU/g; Xylanase activity reaches 35.03IU/g; Beta-xylosidase vigor reaches 3.04IU/g; Polygalacturonase vigor reaches 64.24IU/g; Pectin lyase vigor reaches 59.65IU/g; Laccase activity reaches 80.06IU/g; Mannosan enzyme activity reaches 32.12IU/g; Alpha-galactoside enzyme activity reaches 8.26IU/g; Alpha amylase activity reaches 107.65IU/g; Prolease activity reaches 115634IU/g; Phytase vigor reaches 80.63IU/g; Tannase vigor reaches 1331IU/g; Lactic acid bacteria density reaches 2.08 * 10
8cFU/g.
Various enzyme activities and lactic acid bacteria density inspect method are as follows:
One, the mensuration of the enzyme activity of carboxymethylcelluloenzyme enzyme, total fiber element enzyme and beta-glucuroide
(mensuration that is Filter paper Cellulase (FPase), carboxymethylcelluloenzyme enzyme (CMCase), GBAP vigor is carried out (referring to " Ghose TK; 1987.Measurements of cellulase activities.Pure Appl Chem; 59,257-268. ") according to the established procedure of international pure chemistry and applied chemistry federation (International Union of Pure and Applied Chemistry) to total fiber element enzyme.
Filter paper Cellulase (FPase) unit of activity: the Whatman No.1 filter paper (1.0 * 6.0cm=50.0mg) of take is substrate, at 50 ℃, reaction 60min in 50mM sodium citrate buffer solution (pH=4.8), take release 1 μ mol reduced sugar per minute as an enzyme activity international unit (IU), be expressed as IU/g dry weight.
Carboxymethylcelluloenzyme enzyme (CMCase) unit of activity: 2% (w/v) carboxymethyl cellulose of take is substrate, at 50 ℃, reaction 30min in 50mM sodium citrate buffer solution (pH=5.5), take release 1 μ mol reduced sugar per minute as an enzyme activity international unit (IU), be expressed as IU/g dry weight.
GBAP unit of activity: add 1mL paranitrophenol-beta-glucosidase (p-nitrophenyl glucopyranoside in 2mL acetate buffer, pNPG) (1mM) as substrate, at 50 ℃, react 30min, 430nm colorimetric estimation, take release 1 μ mol paranitrophenol (p-nitrophenol) per minute (pNP) as an enzyme activity international unit (IU), be expressed as IU/g dry weight.
Two, the mensuration of the enzyme activity of endoglucanase, zytase, beta-xylosidase, polygalacturonase and Pectin lyase
Endoglucanase, zytase, polygalacturonase and Pectin lyase vitality test are respectively with carboxymethyl cellulose, birch xylan enzyme, polygalacturonic acid and pectic acid are substrate, specifically according to related documents (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 pulp.Production of extracellular arabinanase.Process Biochem, 35, 557-561, Jayani RS, Saxena S, Gupta R, 2005.Microbial pectinolytic enzymes:a review.Process Biochem, 40,2931-2944.).Release 1 μ mol reduced sugar per minute is an enzyme activity international unit (IU), is expressed as IU/g dry weight.
It is substrate that beta-xylosidase vitality test be take nitre phenyl glucosides (p-nitrophenyl glycoside), specifically according to related documents (Mamma D, Koullas D, Fountoukidis G, Kekos D, Macris BJ, Koukios E, 1996.Bioethanol from sweet sorghum:Simultaneous saccharification and fermentation of carbohydrates by a mixed microbial culture.Process Biochem, 31,377-381.).Release 1 μ mol p-nitrophenol per minute is an enzyme activity international unit (IU), is expressed as IU/g dry weight.
Three, the mensuration of the enzyme activity of laccase
Laccase activity passes through ABTS (2, 2 '-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) oxidation and measuring, reaction system is in sodium citrate buffer solution (pH=3.0), to add the 20mM ABTS of 100 μ L to use sodium citrate buffer solution (pH=3.0) to be settled to 1mL again, ABTS oxygenation efficiency is determined by 420nm absorption value, specifically according to related documents (Susan Grace Karp, Vincenza Faraco, Antonella Amore, Leila Birolo, Chiara Giangrande, Vanete Thomaz Soccol, Ashok Pandey, Carlos Ricardo Soccol.Characterization of laccase isoforms produced by Pleurotus ostreatus in solid state fermentation of sugarcane bagasse.Bioresource Technology, 2012, 114, 735-739.).Oxidation 1 μ mol ABTS per minute is an enzyme activity international unit (IU), is expressed as IU/g dry weight.
Four, the mensuration of the enzyme activity of mannase
The vitality test of mannase be take carob as substrate, and 1g enzyme powder is under 40 ℃, pH5.0 condition, and 1min hydrolysis carob generates and is equivalent to 1 μ mol mannose reducing substances, is 1 enzyme activity unit, is expressed as IU/g dry weight.
Five, the mensuration of the enzyme activity of alpha-galactosidase
Alpha-galactoside enzyme activity determination system is " 0.1mL enzyme liquid+0.8mL0.2M acetate buffer (pH4.8)+0.1mL2mM pNPG ", after 50 ℃ of reaction 15min, adds 3mL0.2MNa
2cO
3cessation reaction, 405nm place surveys absorption value, specifically according to related documents (Dey PM, Pridham JB, 1972.Biochemistry of alpha-galactosidase.Adv Enzymol, 36,911-930.).Release 1 μ mol paranitrophenol (paranitrophenol) per minute is an enzyme activity international unit (IU), is expressed as IU/g dry weight.
Six, the mensuration of the enzyme activity of AMY
AMY vitality test system is " 150 μ L enzyme liquid+200 μ L0.2% soluble starches, the 0.1M Tris-HCl buffer solution (pH=7.0) of take is solution system ", after 37 ℃ of reaction 30min, add 400 μ l3, 5-dinitro salicylic acid cessation reaction boiling water bath keep 5min, after being cooled to 25 ℃ of room temperatures, add 8mL distilled water diluting, 489nm place surveys absorption value, specifically according to related documents (Bernfeld P (1955) Amylases, alpha and beta.In:Colowick SP, Kaplan NO (eds) Methods in enzymology, Vol1.Academic, New York, pp149-154.).Release 1 μ mol reduced sugar per minute is an enzyme activity international unit (IU), is expressed as IU/g dry weight.
Seven, the mensuration of the enzyme activity of protease
Prolease activity measures that to take sulphanilamide azo-casein (sulphanilamide azocasein) be substrate, reaction system is to contain 0.5% azo-casein (azocasein) (w/v) in 250 μ L0.1M phosphate buffers (pH8.5), add again 150 μ L enzyme liquid, after 37 ℃ of reaction 30min, add 1.2mL trichloroacetic acid solution (trichloroacetic acid solution) (10%, w/v) enzyme that goes out, add again 800 μ L of1.8N NaOH neutralizations, 420nm place surveys absorption value, specifically according to related documents (Leighton TJ, Doi RH, Warren RAJ, Kelln RA (1973) The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis.J Mol Biol, 76:103-122.). release 1 μ g azo-casein (azocasein) per minute is an enzyme activity international unit (IU), be expressed as IU/g dry weight.
Eight, the mensuration of the enzyme activity of phytase and tannase
It is substrate that phytase vitality test be take para-nitro-pheneye phosphate (p-nitrophenylphosphate), specifically according to related documents (Stockmann C, Losen M, Dahlems U, Knocke C, Gellissen G, Buchs J (2003) .Effect of oxygen supply on passaging, stabilizing and screening of recombinant Hansenula polymorpha production strains in test tube cultures.FEMS Yeast Res, 4 (2): 195-205.).Release 1 μ mol p-nitrophenol (p-nitrophenol) per minute is an enzyme activity international unit (IU), is expressed as IU/g dry weight.
Tannase vitality test be take tannic acid as substrate, specifically according to related documents (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.).Conversion 1 μ mol tannic acid per minute is an enzyme activity international unit (IU), is expressed as IU/g dry weight.
Nine, the mensuration of lactic acid bacteria density
Man Rogosa Sharpe (MRS) culture medium is that lactic acid bacteria is selected culture medium, measures the total plate count on Man Rogosa Sharpe (MRS) culture medium, can be scaled lactic acid bacteria density.Total plate count is measured and is carried out (Song Y according to related documents, 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 Food Agric, 92 (1), 197-202.).
Claims (5)
1. bio-transformation rice straw and castor bean meal are prepared the complex enzyme for feed method for probiotics preparation of holding concurrently, and it is characterized in that, comprise the following steps:
1) Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod, the first rice straw, the first castor bean meal, the first wheat bran, the first pomace and the first water are fully mixed, after domestication is cultivated, form leavening;
The mass ratio of the described Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod, the first rice straw, the first castor bean meal, 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;
The condition that described domestication is cultivated is to cultivate 24～60 hours 30 ℃～45 ℃ domestications;
2) by step 1) in leavening, the second rice straw, the second castor bean meal, the second wheat bran, the second pomace and the second water fully mix, after fermentation, obtain the complex enzyme for feed probiotics preparation of holding concurrently;
The mass ratio of described leavening, the second rice straw, the second castor bean meal, the second wheat bran, the second pomace and the second water is 10:100～800:50～500:10～100:5～60:100～1500;
Described fermentation adopts solid state fermentation, and the condition of described solid state fermentation is: material thickness is 5cm～20cm, and gravity-flow ventilation, 30 ℃～45 ℃ fermentations 2～7 days.
2. bio-transformation rice straw according to claim 1 and castor bean meal are prepared the complex enzyme for feed method of probiotics preparation of holding concurrently, it is characterized in that, step 1), in, the mass ratio of the described Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, agrocybe bacterium rod, the first rice straw, the first castor bean meal, the first wheat bran, the first pomace and the first water is 10:5:5:10:5:2:1:22～25.
3. bio-transformation rice straw according to claim 1 and castor bean meal are prepared the complex enzyme for feed method of probiotics preparation of holding concurrently, and it is characterized in that step 1) in, the condition that described domestication is cultivated be 35 ℃～40 ℃ domestication cultivations 36～48 hours.
4. bio-transformation rice straw according to claim 1 and castor bean meal are prepared 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 rice straw, the second castor bean meal, the second wheat bran, the second pomace and the second water is 10:300～600:150～300:30～60:15～30:400～1000.
5. bio-transformation rice straw according to claim 1 and castor bean meal are prepared the complex enzyme for feed method of probiotics preparation of holding concurrently, it is characterized in that, step 2) in, the condition of described solid state fermentation is: the material thickness of solid state fermentation is 10cm～15cm, gravity-flow ventilation, 35 ℃～40 ℃ fermentations 4～5 days.
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