CN103315131A - Preparation method of compound enzymes and probiotics for forage by biotransformation of broccoli rhizome and soybean cake - Google Patents
Preparation method of compound enzymes and probiotics for forage by biotransformation of broccoli rhizome and soybean cake Download PDFInfo
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Abstract
The invention discloses a preparation method of compound enzymes and probiotics for forage by biotransformation of broccoli rhizome and soybean cake, including the steps of fully mixing Pu'er fermented tea, mushroom stick, agrocybe cylindracea stick, first broccoli rhizome, first soybean cake, first wheat bran, first apple slag and first water after pile fermentation and obtaining a starter after habituated culture; and fully mixing the starter, second broccoli rhizome, second soybean cake, second wheat bran, second apple slag and second water and obtaining a compound enzyme and probiotics for forage after fermentation. In the invention, safe floras at food grade with stable microbiology are used for the fermentation of broccoli rhizome and soybean cake in order to prepare compound enzymes and probiotics for forage that has every good performance index, and the biotransformation of broccoli rhizome and soybean cake has low cost and high efficiency. At the same time, in the invention, microbes are easy to grow; the enzyme activity is high; the fermentation is extensive; and no strict aseptic condition is needed, therefore the invention method is suitable for industrial mass production.
Description
Technical field
The invention belongs to the microbial fermentation field, be specifically related to a kind of safe flora of the stable food-grade of little ecology fermentation broccoli rhizome and soybean cake dregs of adopting, prepare feed with the complex enzyme method of probiotics preparation of holding concurrently.
Background technology
China is a populous nation that the arable land puts upon the full stretch, and all there is huge breach in the feedstuff particularly demand of energy feed and protein feed.
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, for example various agricultural crop straws, city cellulosic rubbish, processing of farm products accessory substance such as corncob, vegetables cauline leaf, 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 has the complex substrate that major application is worth, but because composition and the complex structure of lignocellulosic, thereby be difficult to by the simple stomach animal digestion.Realizing non-grain material high efficiency, low cost bio-transformations such as lignocellulosic, make cereal such as its alternative corn as energy feed, will be the important growth point of Feed Industry Development.
Grouts are large agricultural by-products that a class is rich in protein.Fast development along with aquaculture industry, also increasing to the protein resource demand, and animality high-quality protein resource (as fish meal) source is limited, price is in recent years always in quick rise, therefore, be protein feed with grouts, will alleviate the nervous situation of feed protein resource greatly, 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, teaseed cake dregs, sesame cake meal etc., but contain very various toxicant and anti-nutrient substance in the grouts.For example the sulphur glycosides decomposites noxious materials such as isothiocyanates and nitrile through myrosase having under the regimen condition, makes the enlargement of animal thyroid gland, metabolic disturbance, even cause subcutaneous hemorrhage, also influence the function of organs such as adrenal cortex, pituitary and liver.Thereby anti-nutrient substance alpha-galactoside compound then makes animal intestinal flatulence influence digestive function.And grouts also contain quite high plant fiber component when being rich in protein, and nonruminant digestion is difficult for.If can eliminate toxicity and the anti-nutrient substance in the grouts and carry out bio-transformation in high efficiency, low cost ground, then all kinds of grouts will be developed as high price animal fodder albumen such as protein feed raw material and alternative fish meal fully, become another important growth point of following Feed Industry Development.
Microbial 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 basically, grouts are rich in protein, both combinations can be microorganism nutritious, well-proportioned fermentation substrate are provided, and the systems biology theory is then for realizing that by microbial fermentation the low-cost high-efficiency bio-transformation of lignocellulosic and grouts provides New Policy.According to the systems biology 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 evolution naturally 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 as in the same system and environmental factor that shared system provides, effectively conversion of substrate.The little ecological stable equilibrium's who is constituted by many kind different microorganisms at the occurring in nature ubiquity wild microbiologic population, for example 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 because the diversity that its institute's enzyme that produces is can be carried out bio-transformation to complex substrate.Therefore, according to the systems biology theory, by on the basis of natural flora, make up a little ecology of producing enzyme and the powerful and suitable lignocellulosic of fermentability and grouts matrix environment and stablize flora, 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, it has formed the bacterium complicated but stable flora of little ecology mutually in the pile-fermentation process, it is the wet heap flora of Pu'er tea, 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, possessed abundant and powerful enzymatic productivity, fermentation conversion capability and benefit are given birth to effect, are expected to be applied to the bio-transformation of lignocellulosic and puffed soybean agricultural by-products.
Edible mushroom is that a big class is the safe macro fungi of food-grade that matrix is carried out growth and breeding with the lignocellulosic, 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), tea tree mushroom (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.Since edible mushroom in the growth and breeding process in the matrix a large amount of lignocellulosic enzymes of secretion, so its matrix to agricultural by-products particularly the lignocellulose agricultural by-products have very strong bio-transformation ability.
Summary of the invention
The invention provides a kind of bio-transformation broccoli rhizome and soybean cake dregs and prepare feed with the complex enzyme method of probiotics preparation of holding concurrently, adopt ferment broccoli rhizome and soybean cake dregs of the safe flora of the stable food-grade of little ecology to prepare feed with the complex enzyme probiotics preparation of holding concurrently, realize the low-cost high-efficiency bio-transformation of broccoli rhizome and soybean cake dregs.
A kind of bio-transformation broccoli rhizome and soybean cake dregs prepare feed with the complex enzyme method of probiotics preparation of holding concurrently, and may further comprise the steps:
1) will fully mix through Pu'er cooked tea, lentinus edodes strain stick, tea tree mushroom bacterium rod, the first broccoli rhizome, first soybean cake, first wheat bran, first pomace and first water of pile-fermentation, after domestication is cultivated, form leavening;
2) leavening in the step 1), the second broccoli rhizome, second largest soybean cake, second wheat bran, second pomace and second water are fully mixed, after fermentation, obtain feed with the complex enzyme 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 the Pu'er tea that forms in the Pu'er cooked tea of pile-fermentation is the safe flora of food-grade, mushroom and tea tree mushroom are the safe macro fungis of daily edible food-grade, contain abundant lignocellulosic enzyme in its bacterium rod.Broccoli rhizome and soybean cake dregs are two kinds of large agricultural by-products.Will be through the Pu'er cooked tea of pile-fermentation, lentinus edodes strain stick, tea tree mushroom bacterium rod and the first broccoli rhizome, first soybean cake, first wheat bran, after matrix such as first pomace and first water are mixed, cultivate by domestication, make the natural flora in the wet heap flora of Pu'er tea and the matrix merge the stable safe flora of food-grade of the little ecology of formation, this flora had both had the abundant and powerful enzymatic productivity of the wet heap flora of Pu'er tea, fermentation conversion capability and benefit are given birth to effect, accommodate substrate environment again, simultaneously, contain the abundant lignocellulosic enzyme that derives from lentinus edodes strain stick and tea tree mushroom bacterium rod in the matrix environment, can carry out the low-cost high-efficiency bio-transformation to broccoli rhizome (i.e. the first broccoli rhizome and the second broccoli rhizome) and soybean cake dregs (being first soybean cake and second largest soybean cake).Therefore, the leavening better performances that obtains.
With the safe flora of the food-grade that little ecology is stable in the leavening broccoli rhizome and soybean cake dregs are fermented, broccoli rhizome and soybean cake dregs are converted into feed with the complex enzyme probiotics preparation of holding concurrently, and the feed that obtains is better with hold concurrently every performance indications of probiotics preparation of complex enzyme.
In leavening preparation and sweat, the broccoli rhizome is as main carbon source, and soybean cake dregs has constituted the basis in the fermentation substrate as main nitrogen.Wheat bran (i.e. first wheat bran and second wheat bran) is rich in vitamin and mineral element, pomace (i.e. first pomace and second pomace) is rich in the fermentable sugar that vitamin and microorganism can utilize fast, adds wheat bran and can be fermentation process with pomace and sufficient growth factor (as the confactor of relevant enzyme) is provided and utilizes carbon source fast.
In the step 1), as preferably, the mass ratio of described Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, tea tree mushroom bacterium rod, the first broccoli rhizome, first soybean cake, first wheat bran, first pomace and 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 stable safe flora of food-grade of the little ecology of formation, obtain the better leavening of fermenting property.Further preferred, the mass ratio of described Pu'er cooked tea through pile-fermentation, lentinus edodes strain stick, tea tree mushroom bacterium rod, the first broccoli rhizome, first soybean cake, first wheat bran, first pomace and 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, described domestication culture condition is to cultivate 24～60 hours 30 ℃～45 ℃ domestications, the natural flora that this domestication culture condition is conducive in the wet heap flora of Pu'er tea and the matrix merges the stable safe flora of food-grade of the little ecology of formation, the better leavening of preparation fermenting property.Further preferred, described domestication culture condition is to cultivate 36～48 hours 35 ℃～40 ℃ domestications, and this domestication culture condition can prepare the most excellent leavening of fermenting property.
Step 2) in, as preferably, the mass ratio of described leavening, the second broccoli rhizome, second largest soybean cake, second wheat bran, second pomace and 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 feed of every performance indications with the complex enzyme probiotics preparation of holding concurrently.Further preferred, the mass ratio of described leavening, the second broccoli rhizome, second largest soybean cake, second wheat bran, second pomace and second water is 10:300～600:150～300:30～60:15～30:400～1000, is conducive to obtain the best feed of every performance indications with the complex enzyme probiotics preparation of holding concurrently.
As preferably, solid state fermentation is adopted in described fermentation, and microorganism easily grows, the enzyme activity height, and preparation is simple, is conducive to the industrialization promotion utilization.Further preferred, the condition of described solid state fermentation is: material thickness is 5cm～20cm, and gravity-flow ventilation was 30 ℃～45 ℃ fermentations 2～7 days.This solid state fermentation conditions is conducive to obtain the more excellent feed of every performance indications with the complex enzyme probiotics preparation of holding concurrently.Further preferred, 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 feed of every performance indications with the complex enzyme probiotics preparation of holding concurrently.
It mainly is 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 the feed is divided according to chemical composition.In feed, add the enzyme that these compounds are carried out predigestion or conversion, have remarkable effect for improving livestock and poultry production performance.At present, cellulase, hemicellulase such as zytase, amylase, protease, alpha-galactosidase, phytase etc. all are extensive use of in Feed Manufacturing as enzyme Preparations Used for Feeds, and in use make up plurality of enzymes usually and become complex enzyme formulation and add.The enzyme that comprises in the complex enzyme formulation is more many, and the effectiveness of feed being carried out predigestion or conversion is more high.The flora that the present invention adopts is that the natural flora in the wet heap flora of Pu'er tea and the fermentation substrate merges the stable safe flora of food-grade of little ecology that forms, microbe species is abundant, can fermenting and producing go out the profuse feed complex enzyme of kind, to the evaluation of its effect by the mensuration of each relevant enzyme activity is carried out.
Probio has remarkable efficacy by the conditioning function of intestinal canal to promoting the 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 fermentation, become probiotics preparation, and the evaluation of its effect is undertaken by the mensuration to lactic acid bacterium number.
About the measurement result of enzyme activity and the measurement result of lactic acid bacterium number, as can be known, the product of the present invention preparation is suitable as feed with the complex enzyme probiotics preparation of holding concurrently very much by each.
Compared with prior art, the present invention has following advantage:
Among the present invention, bio-transformation broccoli rhizome and soybean cake dregs prepare feed with the complex enzyme method of probiotics preparation of holding concurrently, will be through the Pu'er cooked tea of pile-fermentation, lentinus edodes strain stick, tea tree mushroom bacterium rod and the first broccoli rhizome, first soybean cake, first wheat bran, after matrix such as first pomace and first water are mixed, cultivate by domestication, make the wet heap flora of Pu'er tea and the natural flora in the matrix in the Pu'er cooked tea of pile-fermentation merge the stable safe flora of food-grade of the little ecology of formation, the safe flora of the food-grade that this little ecology is stable had both had the abundant and powerful enzymatic productivity of the wet heap flora of Pu'er tea, fermentation conversion capability and benefit are given birth to effect, accommodate substrate environment again, simultaneously, contain the abundant lignocellulosic enzyme that derives from lentinus edodes strain stick and tea tree mushroom bacterium rod in the matrix environment, can carry out the low-cost high-efficiency bio-transformation to broccoli rhizome and soybean cake dregs.With the safe flora of the food-grade that little ecology is stable in the leavening broccoli rhizome and soybean cake dregs are fermented, broccoli rhizome and soybean cake dregs are converted into feed with the complex enzyme probiotics preparation of holding concurrently, and the feed that obtains is better with hold concurrently every performance indications of probiotics preparation of complex enzyme.
Among the present invention, bio-transformation broccoli rhizome and soybean cake dregs prepare feed with the complex enzyme method of probiotics preparation of holding concurrently, under the safe flora effect of the food-grade that little ecology is stable in leavening, fermentation can be adopted solid state fermentation, microorganism easily grows, the enzyme activity height, 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 production, have good economic benefit and wide application prospect.
The specific embodiment
The commercially available prod that lentinus edodes strain stick among the embodiment and tea tree mushroom bacterium rod all adopt the Tianquan Moganshan Mountain, Zhejiang mushroom industry Co., Ltd to produce, the broccoli rhizome is gathered voluntarily from planting site, the commercially available prod that soybean cake dregs adopts Jin Guan Xinda, Zhejiang grain and oil Development Co., Ltd to produce, the commercially available prod that the commercially available prod that wheat bran adopts Dafeng City gold wheat edge Mai Ren factory to produce, pomace adopt Wanrong County two flourish ecological agriculture and animal husbandry Co., Ltd to produce.
Embodiment 1
1) 10kg is fully mixed with 10kg broccoli rhizome, 5kg soybean cake dregs, 2kg wheat bran, 1kg pomace, 22kg water through Pu'er cooked tea, 5kg lentinus edodes strain stick, the 5kg tea tree mushroom bacterium rod of pile-fermentation, after 35 ℃ of domestications are cultivated 48 hours, form leavening;
2) the leavening 20kg in the step 1) is fully mixed with 600kg broccoli rhizome, 300kg soybean cake dregs, 60kg wheat bran, 30kg pomace, 800kg water, adopt solid state fermentation, material thickness is 15cm, gravity-flow ventilation, 40 ℃ of fermentations 4 days, obtain feed with the complex enzyme probiotics preparation of holding concurrently.
After measured, the feed of present embodiment with the hold concurrently performance indications of probiotics preparation of complex enzyme is: carboxymethylcelluloenzyme enzyme activity reaches 65.26IU/g; The plain enzyme activity of total fiber reaches 6.35IU/g; Beta-glucoside enzyme activity reaches 13.33IU/g; The endoglucanase vigor reaches 50.50IU/g; Xylanase activity reaches 55.58IU/g; Beta-xylosidase vigor reaches 5.62IU/g; The polygalacturonase vigor reaches 102.20IU/g; Pectate lyases vigor reaches 98.35IU/g; The laccase vigor reaches 77.44IU/g; The mannosan enzyme activity reaches 54.56IU/g; Alpha-galactoside enzyme activity reaches 12.89IU/g; The alpha amylase activity reaches 178.36IU/g; Prolease activity reaches 178943IU/g; The phytase vigor reaches 128.88IU/g; The tannase vigor reaches 1856IU/g; Lactic acid bacteria density reaches 3.37 * 10
8CFU/g.
Embodiment 2
1) 10kg is fully mixed with 10kg broccoli rhizome, 5kg soybean cake dregs, 2kg wheat bran, 1kg pomace, 25kg water through Pu'er cooked tea, 5kg lentinus edodes strain stick, the 5kg tea tree mushroom bacterium rod of pile-fermentation, after 35 ℃ of domestications are cultivated 48 hours, form leavening;
2) the leavening 20kg in the step 1) is fully mixed with 600kg broccoli rhizome, 300kg soybean cake dregs, 60kg wheat bran, 30kg pomace, 800kg water, adopt solid state fermentation, material thickness is 15cm, gravity-flow ventilation, 40 ℃ of fermentations 4 days, obtain feed with the complex enzyme probiotics preparation of holding concurrently.
After measured, the feed of present embodiment with the hold concurrently performance indications of probiotics preparation of complex enzyme is: carboxymethylcelluloenzyme enzyme activity reaches 64.18IU/g; The plain enzyme activity of total fiber reaches 6.19IU/g; Beta-glucoside enzyme activity reaches 13.14IU/g; The endoglucanase vigor reaches 50.24IU/g; Xylanase activity reaches 55.37IU/g; Beta-xylosidase vigor reaches 5.31IU/g; The polygalacturonase vigor reaches 101.24IU/g; Pectate lyases vigor reaches 99.14IU/g; The laccase vigor reaches 76.88IU/g; The mannosan enzyme activity reaches 54.34IU/g; Alpha-galactoside enzyme activity reaches 12.54IU/g; The alpha amylase activity reaches 178.18IU/g; Prolease activity reaches 178764IU/g; The phytase vigor reaches 126.56IU/g; The tannase vigor reaches 1848IU/g; Lactic acid bacteria density reaches 3.34 * 10
8CFU/g.
Embodiment 3
1) 10kg is fully mixed with 10kg broccoli rhizome, 5kg soybean cake dregs, 2kg wheat bran, 1kg pomace, 25kg water through Pu'er cooked tea, 5kg lentinus edodes strain stick, the 5kg tea tree mushroom bacterium rod of pile-fermentation, after 40 ℃ of domestications are cultivated 36 hours, form leavening;
2) the leavening 10kg in the step 1) is fully mixed with 600kg broccoli rhizome, 300kg soybean cake dregs, 60kg wheat bran, 30kg pomace, 1000kg water, adopt solid state fermentation, material thickness is 10cm, gravity-flow ventilation, 35 ℃ of fermentations 5 days obtain feed with the complex enzyme probiotics preparation of holding concurrently.
After measured, the feed of present embodiment with the hold concurrently performance indications of probiotics preparation of complex enzyme is: carboxymethylcelluloenzyme enzyme activity reaches 59.60IU/g; The plain enzyme activity of total fiber reaches 6.22IU/g; Beta-glucoside enzyme activity reaches 12.94IU/g; The endoglucanase vigor reaches 45.85IU/g; Xylanase activity reaches 54.80IU/g; Beta-xylosidase vigor reaches 5.32IU/g; The polygalacturonase vigor reaches 113.15IU/g; Pectate lyases vigor reaches 90.94IU/g; The laccase vigor reaches 72.23IU/g; The mannosan enzyme activity reaches 51.27IU/g; Alpha-galactoside enzyme activity reaches 13.32IU/g; The alpha amylase activity reaches 186.25IU/g; Prolease activity reaches 187765IU/g; The phytase vigor reaches 130.24IU/g; The tannase vigor reaches 1965IU/g; Lactic acid bacteria density reaches 3.46 * 10
8CFU/g.
Embodiment 4
1) 10kg is fully mixed with 5kg broccoli rhizome, 2kg soybean cake dregs, 1kg wheat bran, 0.5kg pomace, 10kg water through Pu'er cooked tea, 1kg lentinus edodes strain stick, the 1kg tea tree mushroom bacterium rod of pile-fermentation, after 45 ℃ of domestications are cultivated 24 hours, form leavening;
2) the leavening 20kg in the step 1) is fully mixed with 200kg broccoli rhizome, 100kg soybean cake dregs, 20kg wheat bran, 10kg pomace, 200kg water, adopt solid state fermentation, material thickness is 5cm, gravity-flow ventilation, 45 ℃ of fermentations 2 days, obtain feed with the complex enzyme probiotics preparation of holding concurrently.
After measured, the feed of present embodiment with the hold concurrently performance indications of probiotics preparation of complex enzyme is: carboxymethylcelluloenzyme enzyme activity reaches 54.32IU/g; The plain enzyme activity of total fiber reaches 5.64IU/g; Beta-glucoside enzyme activity reaches 11.62IU/g; The endoglucanase vigor reaches 41.22IU/g; Xylanase activity reaches 49.84IU/g; Beta-xylosidase vigor reaches 4.84IU/g; The polygalacturonase vigor reaches 105.32IU/g; Pectate lyases vigor reaches 81.88IU/g; The laccase vigor reaches 68.11IU/g; The mannosan enzyme activity reaches 46.86IU/g; Alpha-galactoside enzyme activity reaches 11.84IU/g; The alpha amylase activity reaches 170.22IU/g; Prolease activity reaches 171278IU/g; The phytase vigor reaches 117.22IU/g; The tannase vigor reaches 1786IU/g; Lactic acid bacteria density reaches 3.18 * 10
8CFU/g.
Embodiment 5
1) 10kg is fully mixed with 15kg broccoli rhizome, 8kg soybean cake dregs, 4kg wheat bran, 2kg pomace, 30kg water through Pu'er cooked tea, 10kg lentinus edodes strain stick, the 10kg tea tree mushroom bacterium rod of pile-fermentation, after 30 ℃ of domestications are cultivated 60 hours, form leavening;
2) the leavening 10kg in the step 1) is fully mixed with 800kg broccoli rhizome, 500kg soybean cake dregs, 100kg wheat bran, 60kg pomace, 1500kg water, adopt solid state fermentation, material thickness is 20cm, gravity-flow ventilation, 30 ℃ of fermentations 7 days obtain feed with the complex enzyme probiotics preparation of holding concurrently.
After measured, the feed of present embodiment with the hold concurrently performance indications of probiotics preparation of complex enzyme is: carboxymethylcelluloenzyme enzyme activity reaches 53.75IU/g; The plain enzyme activity of total fiber reaches 5.46IU/g; Beta-glucoside enzyme activity reaches 11.58IU/g; The endoglucanase vigor reaches 41.14IU/g; Xylanase activity reaches 49.69IU/g; Beta-xylosidase vigor reaches 4.78IU/g; The polygalacturonase vigor reaches 104.68IU/g; Pectate lyases vigor reaches 80.96IU/g; The laccase vigor reaches 67.76IU/g; The mannosan enzyme activity reaches 46.62IU/g; Alpha-galactoside enzyme activity reaches 11.76IU/g; The alpha amylase activity reaches 170.12IU/g; Prolease activity reaches 172362IU/g; The phytase vigor reaches 115.78IU/g; The tannase vigor reaches 1798IU/g; Lactic acid bacteria density reaches 3.16 * 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, the plain enzyme of total fiber and beta-glucuroide
(mensuration that is the plain enzyme (FPase) of filter paper fibre, carboxymethylcelluloenzyme enzyme (CMCase), beta glucoside enzyme activity is carried out (seeing " Ghose TK; 1987.Measurements of cellulase activities.Pure Appl Chem; 59,257-268. " for details) according to the established procedure of international pure chemistry and applied chemistry federation (International Union of Pure and Applied Chemistry) to the plain enzyme of total fiber.
Plain enzyme (FPase) unit of activity of filter paper fibre: (1.0 * 6.0cm=50.0mg) is substrate with Whatman No.1 filter paper, under 50 ℃, reaction 60min in the 50mM sodium citrate buffer solution (pH=4.8), discharging 1 μ mol reduced sugar with per minute is an enzyme activity international unit (IU), is expressed as the IU/g dry weight.
Carboxymethylcelluloenzyme enzyme (CMCase) unit of activity: be substrate with 2% (w/v) carboxymethyl cellulose, under 50 ℃, reaction 30min in the 50mM sodium citrate buffer solution (pH=5.5), discharging 1 μ mol reduced sugar with per minute is an enzyme activity international unit (IU), is expressed as the IU/g dry weight.
Beta glucoside enzyme activity unit: add 1mL paranitrophenol-beta-glucosidase (p-nitrophenyl glucopyranoside in the 2mL acetate buffer, pNPG) (1mM) as substrate, 50 ℃ are reacted 30min down, the 430nm colorimetric estimation, discharging 1 μ mol paranitrophenol (p-nitrophenol) with per minute is an enzyme activity international unit (IU) (pNP), is expressed as the IU/g dry weight.
Two, the mensuration of the enzyme activity of endoglucanase, zytase, beta-xylosidase, polygalacturonase and pectate lyases
Endoglucanase, zytase, polygalacturonase and pectate lyases vitality test are respectively with carboxymethyl cellulose, the birch xylan enzyme, polygalacturonic acid and pectic acid are substrate, specifically according to relevant document (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.).It is an enzyme activity international unit (IU) that per minute discharges 1 μ mol reduced sugar, is expressed as the IU/g dry weight.
Beta-xylosidase vitality test is substrate with nitre phenyl glucosides (p-nitrophenyl glycoside), specifically according to relevant document (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.).It is an enzyme activity international unit (IU) that per minute discharges 1 μ mol p-nitrophenol, is expressed as the IU/g dry weight.
Three, the mensuration of the enzyme activity of laccase
The laccase vigor passes through ABTS (2,2 '-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) oxidation and measuring, reaction system is that the 20mM ABTS that adds 100 μ L in the sodium citrate buffer solution (pH=3.0) uses sodium citrate buffer solution (pH=3.0) to be settled to 1mL again, the ABTS oxygenation efficiency is determined by the 420nm absorption value, specifically according to relevant document (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.).Per minute oxidation 1 μ mol ABTS is an enzyme activity international unit (IU), is expressed as the IU/g dry weight.
Four, the mensuration of the enzyme activity of mannase
The vitality test of mannase is substrate with the carob, 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 the 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 ", behind 50 ℃ of reaction 15min, adds 3mL0.2M Na
2CO
3Cessation reaction, the 405nm place surveys absorption value, specifically according to relevant document (Dey PM, Pridham JB, 1972.Biochemistry of alpha-galactosidase.Adv Enzymol, 36,911-930.).It is an enzyme activity international unit (IU) that per minute discharges 1 μ mol paranitrophenol (paranitrophenol), is expressed as the IU/g dry weight.
Six, the mensuration of the diastatic enzyme activity of alpha-
Alpha-amylase activity mensuration system is " 150 μ L enzyme liquid+200 μ L0.2% soluble starches; be solution system with 0.1M Tris-HCl buffer solution (pH=7.0) ", behind 37 ℃ of reaction 30min, add 400 μ l3,5-dinitro salicylic acid cessation reaction and boiling water bath keep 5min, add the 8mL distilled water diluting after being cooled to 25 ℃ of room temperatures, the 489nm place surveys absorption value, specifically according to relevant document (Bernfeld P (1955) Amylases, alpha and beta.In:Colowick SP, Kaplan NO (eds) Methods in enzymology, Vol1.Academic, New York, pp149-154.).It is an enzyme activity international unit (IU) that per minute discharges 1 μ mol reduced sugar, is expressed as the IU/g dry weight.
Seven, the mensuration of the enzyme activity of protease
It is substrate that prolease activity is measured with sulphanilamide azo-casein (sulphanilamide azocasein), reaction system is to contain 0.5% azo-casein (azocasein) (w/v) in the 250 μ L0.1M phosphate buffers (pH8.5), add 150 μ L enzyme liquid again, behind 37 ℃ of reaction 30min, add 1.2mL trichloroacetic acid solution (trichloroacetic acid solution) (10%, w/v) enzyme that goes out, add 800 μ L of1.8N NaOH neutralization again, the 420nm place surveys absorption value, specifically according to relevant document (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.). it is an enzyme activity international unit (IU) that per minute discharges 1 μ g azo-casein (azocasein), is expressed as the IU/g dry weight.
Eight, the mensuration of the enzyme activity of phytase and tannase
The phytase vitality test is substrate with para-nitro-pheneye phosphate (p-nitrophenylphosphate), specifically according to relevant document (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.).It is an enzyme activity international unit (IU) that per minute discharges 1 μ mol p-nitrophenol (p-nitrophenol), is expressed as the IU/g dry weight.
The tannase vitality test is substrate with the tannic acid, specifically according to relevant document (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.).It is an enzyme activity international unit (IU) that per minute transforms 1 μ mol tannic acid, is expressed as the 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, Luo Y, You J, Shen H ， ﹠amp according to relevant document; 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 (10)
1. a bio-transformation broccoli rhizome and soybean cake dregs prepare feed with the complex enzyme 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, tea tree mushroom bacterium rod, the first broccoli rhizome, first soybean cake, first wheat bran, first pomace and first water of pile-fermentation, after domestication is cultivated, form leavening;
2) leavening in the step 1), the second broccoli rhizome, second largest soybean cake, second wheat bran, second pomace and second water are fully mixed, after fermentation, obtain feed with the complex enzyme probiotics preparation of holding concurrently.
2. bio-transformation broccoli rhizome according to claim 1 and soybean cake dregs prepare feed with the complex enzyme 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, tea tree mushroom bacterium rod, the first broccoli rhizome, first soybean cake, first wheat bran, first pomace and first water is 10:1～10:1～10:5～15:2～8:1～4:0.5～2:10～30.
3. bio-transformation broccoli rhizome according to claim 2 and soybean cake dregs prepare feed with the complex enzyme 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, tea tree mushroom bacterium rod, the first broccoli rhizome, first soybean cake, first wheat bran, first pomace and first water is 10:5:5:10:5:2:1:22～25.
4. bio-transformation broccoli rhizome according to claim 1 and soybean cake dregs prepare feed with the complex enzyme method of probiotics preparation of holding concurrently, and it is characterized in that, in the step 1), described domestication culture condition is to cultivate 24～60 hours 30 ℃～45 ℃ domestications.
5. bio-transformation broccoli rhizome according to claim 4 and soybean cake dregs prepare feed with the complex enzyme method of probiotics preparation of holding concurrently, and it is characterized in that, described domestication culture condition is to cultivate 36～48 hours 35 ℃～40 ℃ domestications.
6. bio-transformation broccoli rhizome according to claim 1 and soybean cake dregs prepare feed with the complex enzyme method of probiotics preparation of holding concurrently, it is characterized in that, step 2) in, the mass ratio of described leavening, the second broccoli rhizome, second largest soybean cake, second wheat bran, second pomace and second water is 10:100～800:50～500:10～100:5～60:100～1500.
7. bio-transformation broccoli rhizome according to claim 6 and soybean cake dregs prepare feed with the complex enzyme method of probiotics preparation of holding concurrently, it is characterized in that the mass ratio of described leavening, the second broccoli rhizome, second largest soybean cake, second wheat bran, second pomace and second water is 10:300～600:150～300:30～60:15～30:400～1000.
8. bio-transformation broccoli rhizome according to claim 1 and soybean cake dregs prepare feed with the complex enzyme 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 broccoli rhizome according to claim 8 and soybean cake dregs prepare feed with the complex enzyme 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 broccoli rhizome according to claim 9 and soybean cake dregs prepare feed with the complex enzyme 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.
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