CN104431415A - Active microorganism feed for ruminant animals - Google Patents

Abstract

The invention aims at providing an active microorganism fermented feed for ruminant animals. The feed has various coexisting active beneficial microorganisms and can produce various high-activity enzymes; the product has various nutrients and a dual function of active beneficial bacteria and fresh complex enzymes, is easy to digest and absorb, is strong in digestion aiding, is low in cost, and is a novel irreplaceable product in the traditional diets.

Description

A kind of ruminant active microorganism feed

Technical field

The invention belongs to microbiological feed technical field, particularly relate to a kind of ruminant special active fermentative feedstuff of microbe and manufacturing process.

Background technology

The residues such as the fertilizer and pesticide in existing feed cause sub-health state to stomach and intestine, thus occur a series of gastrointestinal disease, and digest and assimilate difference, the absorption rate of feed is low.A large amount of use antibiotic, livestock products medicine is greatly residual; It is single that current active microorganism feed also has beneficial microorganism kind, fills a prescription unreasonable, the problems such as feed price is higher.

Active microorganism feed is in the market carrier mainly with crude fibre materials such as chaff slag, grass meals, and feed nutrition material content is low.The exogenous enzymes preparation added in conventional feed have impact on proenzyme through operations such as purification, dryings mostly activity, and supply ruminant, the activity of enzyme is relatively low.Single thus the enzyme class produced is also few due to probio kind.In addition, existing zymotechnique and environment, equipment need thorough sterilizing, and feed is preserved must seal anaerobism, and yeasting, temperature requirement are stricter.

The present inventor's earlier application number is the Chinese patent application of 201310090163, disclose a kind of ruminant feed additive and preparation method, by weight ratio number be aspergillus group 20 parts, the tunning of water content≤16wt% that obtains after fermenting respectively of the bacterium liquid of saccharomycete group 20 parts, bacillus group 30 parts, compound bacteria group 40 parts combines, preparation process is: preparation culture medium, cooling, the dilution of former bacterium, inoculation, fermentation, dry, mixing, packaging.Feed addictive of the present invention, containing 20 kinds of probios, has and regulates body gut flora balance, improve immunity, strengthen digestive function, the effect of improving food conversion ratio.

The present inventor is on the basis of earlier application, carry out a large amount of experiments and research further, further optimization has been carried out to the kind of bacterial strain, and add trehalose, oligosaccharides in the medium, result prove the feed after optimizing unexpectedly in every respect effect relatively before have great raising, the especially enzyme local flavor of exponential sum milk of living.

Summary of the invention

One is the object of the present invention is to provide to have multiple compatible active beneficial microorganism ruminant active microorganism fermented feed, it can produce the high enzyme of multiple vigor, and many, the double action with active beneficial bacterium and fresh complex enzyme of various nutriment kind in this product, absorption easy to digest, digestion-aid effect is strong, cheap, be the new product that traditional daily ration is not replaced.

The present invention realizes like this, a kind of ruminant special active fermentative feedstuff of microbe, this active microorganism fermented feed AB bacterium liquid used is made up of two parts: A bacterium liquid selects the excellent species of generally acknowledged safe and sanitary, saccharomycete, Bacillus acidi lactici, Corynebacterium glutamicum, bacillus subtilis, bacillus licheniformis, streptococcus fecalis etc.Select suitable condition of culture to expand these bacterium to cultivate.It is 10 that the bacterium liquid be finally prepared into is cultivated in expansion ⁷-10 ⁹bacterial strain/ml.The process of spreading cultivation is generally: slant activation → liquid tube → little triangular flask → large triangular flask → Ka Shi tank (1:10-20) is for subsequent use.

A bacterium liquid culture medium spread cultivation technique composition comprise:

The preparation of A1 yeast liquid culture medium: the saccharification wort getting the large production of 10L, with being heated to 40 DEG C after filter-cloth filtering, adding prior off-the-shelf egg white boils to boiling, start timing, boil 20 minutes again, add appropriate distilled water to add to wort concentration and will control between 10-11 degree, then boil to boiling, cool, filter.PH5.4--5.8 is adjusted with the phosphoric acid of IN, for subsequent use.

A2 Corynebacterium glutamicum, Bacillus acidi lactici culture medium prescription: 2% sucrose 2% yeast extract 10% potato juice is cultivated rear Bacillus acidi lactici and is increased to 1.58*10 ¹¹cfu/ml;

A3 bacillus subtilis bacterium culture medium, glucose 30.0g peptone 10.0g yeast extract 5.0g potassium dihydrogen phosphate 4.5g calcium carbonate 2.0g sterilized water 1000ml

A4 bacillus licheniformis culture medium corn steep liquor 0.45g peptone 1.50g beancake powder soaks the suitableeest kind of 18 hours age of juice 1:15 glucose 1.50g, and inoculum concentration is 8%, initial PH7.5, optimum temperature 37.5 DEG C

A5 streptococcus fecalis culture medium sodium chloride 0.5g peptone 1.0g lactose 2.0g OX-heart soup 100ml PH nature, for activated spawn.Proliferated culture medium (%) peptone 2 beef extract 2 lactose 2PH6.8

B bacterium liquid selects rumen microorganism: Ruminococcus albus, yellow Ruminococcus, bacteroides ruminicola, Selenomonas, butyric acid vibrios, rumen anaerobic fungi;

The mass percent of this biological fermentation feed is further: the certain herbaceous plants with big flowers dregs of rice are 3; The dish dregs of rice are 3; Dregs of beans is 30; Corn is 18; Secondary wheat 9.7; Trehalose 1.8; Oligosaccharides 1.0; Feed-grade calcium phosphate is 1.26; Feed grade calcium carbonate is 1.4; Normal diet additive is 0.84; A bacterium liquid 15, B bacterium liquid 15.。

Described additive is prior art normal diet additive, carries out as required selecting and adding.

This active microorganism fermented feed manufacturing process is further:

First this technique choose green material → material storage → lifting → high-efficiency vibration removal of impurities sieve → impurity removing → low pressure dedusting → lifting → magnetic bucket deironing → lifting → batching → pulverizing → lifting → mixing → spray AB bacterium liquid → lifting → loading fermentation tank → loading fermenting cellar → pack → check and dispatch from the factory.

Further, this active microorganism fermented feed adopts temperature control anaerobic fermentation.

Further, this active microorganism fermented feed adopts bioanalysis to remove bad environmental bacterium

Good effect of the present invention is:

1, the beneficial bacterium in this active microorganism fermented feed can suppress or decompose harmful intestinal tract bacteria, decomposes the Harmful Residue such as chemical fertilizer, agricultural chemicals in feed.

The substance decomposition such as the macro-molecular protein in feed, crude fibre, starch can be become the material of the absorptions easy to digest such as micromolecular amino acid, peptide, reduced sugar by the multiple enzyme that 2, active beneficial bacterium and metabolism thereof produce, organic acid, vitamin B complex can be produced simultaneously, but not containing any antibiotic.Abundant raising ruminant digestive utilization ratio, reduces aquaculture cost.

3, this microbiological feed is nutritious, and the beneficial bacterium kind many activity of fresh enzyme class that are many and metabolism generation are high, have high specificity.This product price is cheap.

4, Long-Time Service this product can reduce ight soil ammonia stink, reduces environmental pollution, belongs to environment-protecting feed; Can be and produce nuisanceless green livestock products green feed raw material is provided.

5, the palatability of daily ration is fully improved.

6, this microbiological feed belongs to functional feed, has the irreplaceable advantage of physics and chemistry method, can put forward ruminant height gastrointestinal function and immunity, reduces the generation of disease.

Detailed description of the invention

The production of active microorganism fermented feed of embodiment 1 ruminant:

Embodiments provide a kind of ruminant special active fermentative feedstuff of microbe, this active microorganism fermented feed AB bacterium liquid used is made up of two parts technique: A bacterium liquid selects the excellent species of generally acknowledged safe and sanitary, saccharomycete, Bacillus acidi lactici, Corynebacterium glutamicum, bacillus subtilis, bacillus licheniformis, streptococcus fecalis etc.Select suitable condition of culture to expand these bacterium to cultivate.It is 10 that the bacterium liquid be finally prepared into is cultivated in expansion ⁷-10 ¹¹bacterial strain/ml.The process of spreading cultivation is generally: slant activation → liquid tube → little triangular flask → large triangular flask → Ka Shi tank (1:10-20) A bacterium for subsequent use liquid expands culture medium composition and comprises:

The preparation of A1 yeast liquid culture medium: the saccharification wort getting the large production of 10L, with being heated to 40 DEG C after filter-cloth filtering, adding prior off-the-shelf egg white boils to boiling, start timing, boil 20 minutes again, add appropriate distilled water to add to wort concentration and will control between 10-11 degree, then boil to boiling, cool, filter.PH5.4--5.8 is adjusted with the phosphoric acid of IN, for subsequent use.

A2 Bacillus acidi lactici, Corynebacterium glutamicum culture medium prescription: 2% sucrose 2% yeast extract 10% potato juice is cultivated rear Bacillus acidi lactici and is increased to 1.58*10 ¹¹cfu/ml;

A3 bacillus subtilis bacterium culture medium, glucose 30.0g peptone 10.0g yeast extract 5.0g potassium dihydrogen phosphate 4.5g calcium carbonate 2.0g sterilized water 1000ml;

A4 bacillus licheniformis culture medium corn steep liquor 0.45g peptone 1.50g beancake powder soaks the suitableeest kind of 18 hours age of juice 1:15 glucose 1.50g, and inoculum concentration is 8%, initial PH7.5, optimum temperature 37.5 DEG C;

A5 streptococcus fecalis culture medium sodium chloride 0.5g peptone 1.0g lactose 2.0g OX-heart soup 100ml PH nature, for activated spawn.Proliferated culture medium (%) peptone 2 beef extract 2 lactose 2 PH6.8.

B bacterium liquid is selected:

B bacterium liquid selects rumen microorganism: Ruminococcus albus, yellow Ruminococcus, bacteroides ruminicola, Selenomonas, butyric acid vibrios, rumen anaerobic fungi.

B bacterium liquid selects seed to expand culture medium: according to percentage by weight, corn steep liquor 0.5; Peptone 1.5; Glucose 1.5, all the other are beancake powder juice.

B bacterium liquid only needs once to be separated from cud can carry out expansion cultivation and preserve, and also can extract with additive method, or business is bought, and described bacterial strain is all the conventional known bacterial strain of prior art.

Table 1 is the composition table of the AB bacterium liquid of this biological fermentation feed.

The composition table of the AB bacterium liquid of this biological fermentation feed of table 1

Viable bacteria counting method commonly uses the method for plate culture count, is grow the principle design of a bacterium colony according to the bacterium of each work.Get the bacteria suspension of the active microorganism of certain capacity, do a series of doubling dilution, then quantitative dilution is carried out slat chain conveyor, the viable count in culture can be calculated according to the clump count of turning out.

Mass percent is: the certain herbaceous plants with big flowers dregs of rice are 3; The dish dregs of rice are 3; Dregs of beans is 30; Corn is 19; Wheat 9.7; Trehalose 1.8; Feed-grade calcium phosphate is 1.26; Feed grade calcium carbonate is 1.4; Additive is 0.84; AB bacterium liquid is 30.

This formula records nutrient composition content according to feedstuff to carry out formula Design, meets ruminant feed nutrition and raw material sources, fully meet the growth of multiple beneficial bacterium, breeding, metabolism, produces the active bacteria that vigor is large, selectivity is high and complex enzyme simultaneously.Add the active microorganism fermented feed of (replacement) 10%-30% in ruminant, vigor and the high specificity of active bacteria and enzyme can have been given full play to.

The AB bacterium liquid manufacturing process that this active microorganism fermented feed is used: A bacterium liquid selects the excellent species of generally acknowledged safe and sanitary, saccharomycete, Bacillus acidi lactici, bacillus subtilis, bacillus licheniformis, streptococcus fecalis etc.Select suitable condition of culture to expand these bacterium to cultivate.It is 10 that the bacterium liquid be finally prepared into is cultivated in expansion ⁷-10 ⁹bacterial strain/ml.The process of spreading cultivation is generally: slant activation → liquid tube → little triangular flask → large triangular flask → Ka Shi tank (1:10-20) is for subsequent use.

The culture medium that A bacterium liquid is selected is respectively:

The preparation of A1 yeast liquid culture medium: the saccharification wort getting the large production of 10L, with being heated to 40 DEG C after filter-cloth filtering, adding prior off-the-shelf egg white boils to boiling, start timing, boil 20 minutes again, add appropriate distilled water to add to wort concentration and will control between 10-11 degree, then boil to boiling, cool, filter.PH5.4--5.8 is adjusted with the phosphoric acid of IN, for subsequent use.

A2 Bacillus acidi lactici culture medium prescription: 2% sucrose 2% yeast extract 10% potato juice is cultivated rear Bacillus acidi lactici and is increased to 1.58*10 ¹¹cfu/ml;

A3 bacillus subtilis bacterium culture medium, glucose 30.0g peptone 10.0g yeast extract 5.0g potassium dihydrogen phosphate 4.5g calcium carbonate 2.0g sterilized water 1000ml

A4 bacillus licheniformis culture medium corn steep liquor 0.45g peptone 1.50g beancake powder soaks the suitableeest kind of 18 hours age of juice 1:15 glucose 1.50g, and inoculum concentration is 8%, initial PH7.5, optimum temperature 37.5 DEG C

A5 streptococcus fecalis culture medium sodium chloride 0.5g peptone 1.0g lactose 2.0g OX-heart soup 100ml PH nature, for activated spawn.Proliferated culture medium (%) peptone 2 beef extract 2 lactose 2 PH6.8

The separation that B bacterium liquid is selected and culture medium (rumen fluid, glucose, cellobiose, starch, agar); Seed expands culture medium (corn steep liquor 0.5 peptone 1.5 beancake powder juice 1:1.5 glucose 1.5 rumen microorganism 8%)

This active microorganism fermented feed manufacturing process is: choose green material → material storage → lifting → high-efficiency vibration removal of impurities sieve → impurity removing → low pressure dedusting → lifting → magnetic bucket deironing → lifting → batching → pulverizing → lifting → mixing → spray AB bacterium liquid → lifting → loading fermentation tank → loading fermenting cellar → pack → check.Select raw material all to come from green material planting base and manufacturer, production equipment meets produces green active microorganism fermented feed standard.Workshop, fermentation plant, workman meets production green bio feed condition, produces and detailed record in strict accordance with green bio Feed Manufacturing program.

This active microorganism fermented feed adopts many condition temperature controlled fermentation

In order to fully meet the growth of various active beneficial bacterium, breeding, metabolism, following condition must be provided:

1, moisture: AB bacterium liquid accounts for prescription quality percentage 30%, active microorganism fermented feed contains water inventory not higher than 45%;

2、pH：6.8--7.2

3, fermentation medium: the certain herbaceous plants with big flowers dregs of rice are 3; The dish dregs of rice are 3; Dregs of beans is 30; Corn is 18; Secondary wheat 9.7; Trehalose 1.8; Oligosaccharides 1.0; Feed-grade calcium phosphate is 1.26; Feed grade calcium carbonate is 1.4; Normal diet additive is 0.84; A bacterium liquid 15, B bacterium liquid 15, containing crude protein, crude fibre, starch, carbohydrate, mineral matter etc., not containing other harmful substances.

4, anaerobic environment: use unnecessary oxygen in vavuum pump Suction cop after charging feedstock in fermentation tank, creates anaerobic environment.Ferment after three days, the single air valve on fermentation tank is opened exhaust.

5, temperature: raw material loads after fermentation tank, controls thermostatic in tank in 26-36 DEG C (36 DEG C, first three sky, rear six days 28 DEG C).

6, fermentation time: lucifuge anaerobic fermentation 7-9 days.

This active microorganism fermented feed adopts bioanalysis to remove harmful bacteria.

1, workshop before manufacture, first thoroughly cleans up, then within 24 hours, carry out disinfection with Ultraviolet radiation, closes ultraviolet lamp, finally with 10% probiotics bacterial liquid spraying workshop.

2, to production equipment sterilization, first cleaning and disinfecting is carried out with hydrogen peroxide mainly for blending tank and the follow-up equipment be connected.With the probiotics bacterial liquid spraying of 10% after one hour.

3, after feedstuff enters blending tank, spray into active beneficial bacterium bacterium liquid, fully stir, active beneficial bacterium has very strong inhibitory action to the miscellaneous bacteria in feedstuff, enters beneficial bacterium in fermentation tank, quick growth and breeding, thus the miscellaneous bacteria suppressing or kill in raw material.

The mensuration of embodiment 2 enzymatic activity:

The assay method (GB/T23527-2009) of proteinase activity

1 principle

Protease has good hydrolysis to casein, lactalbumin, corn gluten protein etc.Phosphotungstic acid and phosphomolybdic acid mix reagent, i.e. Folin-Phenol reagent, extremely unstable under alkali condition, is easily reduced by phenolic compound and reacts (tungsten orchid and the blue mixture of tungsten) in blue.Due in protein containing the amino acid (tyrosine, tryptophan, phenylalanine) with phenolic group, therefore, protein and hydrolysate thereof also in this reaction.Utilize proteases for decomposing casein (substrate) to generate containing the amino acid whose color reaction of phenolic group, carry out the vigor of indirect determination protease.

2 instrument and equipments

2.1 assay balances: precision 0.0001g

2.2 waters bath with thermostatic control: precision ± 0.2 DEG C

2.3 stopwatch

2.4 spectrophotometer

2.5 boiling water baths

2.6 shaker mixer

2.7pH counts: precision 0.01pH unit

3 reagent and solution

3.1 lactic acid buffer (pH=3.0) are applicable to acid protease

First liquid: take lactic acid (80%-90%) 10.6g, be dissolved in water and be settled to 1000ml.

Second liquid: take sodium lactate (70%) 16g, be dissolved in water and be settled to 1000ml.

Use solution: get first liquid 8ml, add second liquid 1ml, shake up, dilute one times, 0.05mol/L lactic acid cushioning liquid.

The preparation (pH=7.5) of 3.2 phosphate buffers is applicable to neutral proteinase

Accurately take sodium hydrogen phosphate (Na2HPO3.12H2O) 6.02 and sodium dihydrogen phosphate (NaH2PO3.2H2O) 0.5g, add water and be settled to 1000ml

3.3 borate buffers (pH=10.5) are applicable to alkali protease

First liquid: take Boratex 19.08g, is dissolved in water and is settled to 1000ml.

Second liquid: weighing sodium hydroxide 4.0g, is dissolved in water and is settled to 1000ml.

Use solution: get first liquid 500ml, add second liquid 400ml, shake up, be diluted with water to 1L.

3.4 0.4mol/L sodium carbonate liquors:

Accurately take natrium carbonicum calcinatum 42.4g, dissolve with distilled water and be dissolved to 1000ml.

The trichloroacetic acid liquid of 3.5 0.4mol/L:

Accurately take 65.4 trichloroacetic acids, dissolve with distilled water and be dissolved to 1000ml

The NaOH of 3.6 0.5mol/L:

Accurately take 2g NaOH dissolve and determine to 100ml

3.7 10.00mg/ml casein solution

Take casein 1.000g, accurately to 0.001g, soak with the sodium hydroxide solution (if acid protease then drips with dense lactic acid 2-3) of a small amount of 0.5mol/L, add appropriate each suitable buffer solution and be about 80ml, heat while stirring in boiling water bath, until dissolve completely, after cooling, proceed in 100ml volumetric flask, be diluted to scale with suitable buffer solution, this solution is stored in refrigerator, and the term of validity is three days.

3.8 100 μ g/ml tyrosine standard liquids

3.8.1 accurately take the pre-TYR 0.1000g being dried to constant weight prior to 105 DEG C, be settled to 100ml after dissolving with the hydrochloric acid 60ml of 1mol/L, be the tyrosine solution of 1.00mg/ml.

3.8.2 draw 1.00mg/ml tyrosine standard liquid 10.00ml, be settled to 100ml with 0.1mol/L hydrochloric acid, obtain 100.0 μ g/ml TYR standard liquids.

The preparation of 3.9 enzyme samples

Accurately take 1.000g solid enzyme or pipette 1ml liquid enzymes sample, dissolve with a small amount of appropriate buffer and smash with glass bar and grind, then supernatant is poured into 100ml volumetric flask, insert a small amount of buffer solution in sediment again to smash and grind repeatedly, finally all move into volumetric flask, be diluted to scale, by four layers of filtered through gauze.Filtrate can be used as tested enzyme and uses. and this enzyme has diluted 100 times.

4 analytical procedures

The drafting of 4.1 calibration curves

TYR standard liquid according to the form below is prepared.

Table 2

4.2 get each 1.00ml of above-mentioned solution (must do parallel test) respectively, respectively add 0.4mol/L sodium carbonate liquor 5.00ml.Folin reagent uses solution 1.00ml, be placed in 40+0.2 DEG C of water-bath to develop the color 20min, taking-up spectrophotometer is in wavelength 680nm, colorimetric, not contain 0 pipe of tyrosine for blank tube zeroising, measuring its absorbance respectively, take absorbance as ordinate, the concentration of tyrosine is abscissa, drawing standard curve or calculating regression equation.Calculate the amount (μ g) of the tyrosine when OD is 1, be extinction constant K, its K value should within the scope of 95-100.

4.3 sample determinations:

4.3.1 first casein solution is put into 40 ± 0.2 DEG C of waters bath with thermostatic control, preheating 5min

4.3.2 get 4 test tubes, respectively add 1ml enzyme liquid

4.3.3 get one as blank tube, add 2ml trichloroacetic acid, other 3 pipes respectively add 1ml casein as testing tube, shake up, 40 DEG C of insulation 10min

4.3.4 take out test tube, respectively add 2ml trichloroacetic acid in 3 testing tubes, in blank tube, add 1ml casein.

4.3.5 10min is left standstill, filtering-depositing

4.3.5 respectively get 1ml filtrate, add the Na2CO35ml of 0.4mol/L, Folin reagent 1ml respectively.At 40 DEG C of colour developing 20min.680nm place surveys OD value.With blank tube zeroising.

Note: 1.398 neutral protein enzyme preparations and 166 neutral protein acid supplements, except reaction and colour temp are except 30 DEG C ± 0.2 DEG C, other operations are the same, and calibration curve does same process.

5. calculate

5.1 enzymes are lived and are defined:

1g solid enzyme powder (or 1ml liquid enzymes), under 40 DEG C of (acid pH=3.0, neutral pH=7.5, alkaline pH=10.5) conditions, it is an enzyme activity unit that 1min hydrolyzed casein produces 1 μ g tyrosine.

5.2 calculate the active unit of enzyme according to following formula

Vigor=A × K × 4/10 × n the U/g (ml) of protease

A: the mean OD value of sample parallel test

K: extinction constant

4: the cumulative volume of reaction reagent

10: the enzyme digestion reaction time

N: enzyme liquid dilution general times

The mensuration of alpha-amylase activity

One principle

AMS can by the α-1 in starch molecular chain, 4 glucoside bonds cut into short chain dextrin different in size, a small amount of maltose and glucose at random, and make starch be that hepatic specific reaction fades away to iodine, in rufous, the speed that its color disappears is relevant with enzyme activity, therefore calculates its enzyme activity by the absorbance after fixation reaction.

Two, reagent and instrument

(1) reagent

1. former iodine liquid

Take iodine (I2) 11g, KI (KI) 22g, makes iodine dissolve completely with a small amount of water, is then settled to 500mL, store in brown bottle.

2. rare iodine liquid

Draw former iodine liquid 2.00mL, add KI 20g, be settled to 500mL by water-soluble solution, store in brown bottle.

3.20g/L soluble starch solution

Take soluble starch (in over dry) 2000g. and be accurate to 0.001g, with water furnishing slurry. under agitation slowly in impouring 70mL boiling water.Then, rinse the beaker of dress starch several times with 30mL moisture, washing lotion is incorporated to wherein, is heated to completely transparent, and cooling, is settled to 100mL.This solution needed prepared the same day.

Note: adopt the soluble starch that Zhejiang Ling Hu food chemistry affiliated company produces.

4. phosphate buffer (pH6.0)

Take sodium hydrogen phosphate (Na2HPO412H2O) 45.23g, citric acid (C6H8O7H2O) 8.07g, be settled to 1000mL by water-soluble solution.Prepare rear pH meter to correct.

(2) instrument

1. spectrophotometer should meet the pertinent regulations of GB 9721

2. stopwatch

3. water bath with thermostatic control (60 ± 0.2) DEG C

4. test tube 25mm × 2000mm

Three, step

1. the preparation of enzyme liquid to be measured

Take enzyme powder l-2g, be accurate to 0.0002g (or imbitition enzyme 100mL), first dissolve with a small amount of phosphate buffer, and smash with glass stirring rod and grind, by careful for supernatant impouring volumetric flask, sediment part adds a small amount of buffer solution again, so smash and grind 3-4 time, in last all immigrations volumetric flask, be settled to scale with buffer solution and (will estimate that enzyme activity is divided by 4, namely enzyme activity should within the scope of 3.7-5.6IU/mL), shake up.By 4 layers of filtered through gauze, filtrate is for mensuration.

2. measure

(1) draw soluble starch solution 20.0mL in test tube, add buffer solution 5.00mL, after shaking up, preheating 5min in (60 ± 0.2) DEG C water bath with thermostatic control.

(2) add the enzyme liquid 1.00mL to be measured diluted, clock at once, shake up, accurate response 5min.

(3) draw reactant liquor 1.00mL immediately in rare iodine liquid 5.00mL, shake up, and do with rare iodine liquid blank, under 660nm wavelength, use 10mm cuvette, its absorbance of rapid test (A).Table look-up according to its absorbance, try to achieve the concentration (C) of tested enzyme liquid.

Four, result arranges

The vigor of sample enzyme is according to following formulae discovery:

X＝c×n

In formula: the enzyme activity [IU/g (IU/mL)] of X-sample

The concentration (IU/mL) of c-sample enzyme liquid

The extension rate of n-sample

Acquired results represents to integer.Parallel laboratory test relative error must not more than 2%.

Points for attention during mensuration:

1. concentration of substrate is except the substrate selecting to be applicable to, more in actual applications it is considered that concentration of substrate.Because [S] becomes hyperbolic relation with reaction speed V, when enzyme assay, require that [S] reaches certain level to ensure that enzymatic activity is directly proportional to enzyme amount.[S] scope is typically chosen in 10 ~ 20Km and is advisable, and now reaction speed reaches maximum reaction velocity substantially, and the error of mensuration is at tolerance interval.

2. enzyme concentration is in reaction condition one timing, and enzyme concentration is directly proportional to reaction speed.According to intermediate theory, when only having [S] >> [E], enzyme could be saturated by substrate molecule, and reaction speed just can reach maximum.Therefore, when sample enzyme activity is too high, measured again after sample suitably should being diluted.

3. the enzyme optimum temperature that temperature is different can be different, and the optimum temperature of most enzyme is at 37-40 DEG C, and higher or lower than optimum temperature, enzymatic activity all reduces.At present, the mensuration temperature not yet unification of enzymatic activity, but the many uses of Routine Test Lab 37 DEG C.Temperature represents with temperature coefficient (Q10) usually to the influence degree of enzymatic reaction.Temperature coefficient refers to that temperature often raises 10 DEG C, the multiple that chemical reaction velocity increases.Q10 is generally l-2.Learnt by temperature coefficient, the change of temperature is to enzymatic activity important, and therefore require that enzyme assay will be carried out under constant temperature, temperature fluctuation will control at ± 1 DEG C.

4. ionic strength and pH value are when optimal pH, and the activity of enzyme is the strongest, and higher or lower than optimal pH, the activity of enzyme all reduces, and the optimal pH of most enzyme is between 5-8.When measuring enzymatic activity, require that buffer solution has enough buffer capacities, to make pH value keep stable.Blood plasma or serum specimen contain multiple buffering solute, have stronger buffer capacity.In order to prevent blood plasma or serum specimen buffering solute on the impact of reactant liquor acid-base value, make that pH is unlikely departs from setting value, sample consumption is unsuitable excessive, is advisable in blood plasma or serum specimen volume/reactant liquor volume≤1/10.

5. some metal ion of confactor and vitamins coenzyme are the confactors of desmoenzyme, and such as Zn2+ is the prothetic group of carboxypeptidase, and Mo6+ is the prothetic group of xanthine oxidase, and NADH is the coenzyme of anaerobic dehydrogenase.These enzymes leave their prothetic group or coenzyme just can not show activity, therefore when enzyme assay, will ensure the supply of prothetic group or coenzyme.

6. some enzyme of activator just have when there being activator to exist activity or activity higher, such as Mg2+ is the activator of creatine kinase, and Cl-is diastatic activator.Therefore, when enzyme assay, the needs of enzyme to activator to also be met.

7. the suppression of inhibitor enzyme can be divided into irreversible suppression and reversible inhibition, and the latter can be divided into Reverse transcriptase and Noncompetition inhibition again.Heavy metal ion and arsenide belong to irreversible suppression to the suppression of sulfydryl enzyme, the suppression of organophosphor to hydroxyl enzyme; Malonic acid belongs to Reverse transcriptase to the suppression of succinate dehydrogenase, the suppression of sulfa drugs to dihydrofolate synthetase; The suppression of ouabain to Na+, K+-ATP enzyme belongs to Noncompetition inhibition.Inhibitor makes enzymatic activity reduce, and when measuring enzymatic activity, should avoid the impact of inhibitor.

In sum, when measuring enzymatic activity, the selection of optimum condition should follow the suitableeest concentration of substrate, optimum temperature, optimum pH, the principle that meets confactor and activator, avoid inhibitor.

Cellulase activity assay method---3,5-dinitrosalicylic acid system

1. the unit of activity definition of cellulase:

At 50 DEG C, pH is under 4.8 conditions, per minute from the carboxymethylcellulose sodium solution that concentration is 10mg/mL the enzyme amount of release required for 1 μm of ol reduced sugar be a unit of activity (IU).

2. measuring principle

At 50 DEG C, pH is cellulose hydrolyzation cellulose under 4.8 conditions, and produce polymer and the reduced sugar of molecular weight, the nitroreduction in 3,5-dinitrosalicylic acid can be become orange amino-compound by reduced sugar.Utilize colorimetric method to measure the amount of reduced sugar, thus calculate enzyme activity.

3. reagent and solution

3.110.0mg/mL glucose solution

100mL is settled to after the DEXTROSE ANHYDROUS 1.0000g taken is dissolved in water.

3.20.05mol/L citrate acidic buffer

A, 0.5mol/L citric acid solution

Accurately take 105.0g citric acid to be dissolved in completely in the distilled water of 800mL, be settled to 1000mL, be labeled as solution A;

B, 0.5mol/L citric acid three sodium solution

Accurately take 147.0g trisodium citrate to be dissolved in completely in the distilled water of 800mL, be settled to 1000mL, be labeled as B solution;

The sodium citrate buffer solution of c, 0.5mol/L

The B liquid of the A liquid and 1000mL of getting 600mL fully mixes, and is labeled as C liquid.

The citrate acidic buffer (pH=4.80) of d, 0.05mol/L

Getting C liquid 1000mL joins in the distilled water of 8000mL, pH is adjusted to be 4.80 with the hydrochloric acid solution of 3mol/L or the sodium hydroxide solution of 3mol/L if desired, constant volume 10000mL again, is labeled as the acidic buffer of 0.05mol/L after mixing, with tense marker pH=4.80, preparation date.Preserve in 4 DEG C of refrigerators.

3.31.0%CMC substrate

Precision takes sodium carboxymethylcellulose 1.0000g and is dissolved in the acidic buffer of 80mL0.1mol/L, and room temperature lower magnetic force is stirred to and dissolves (more than 3 hours) completely.Be 4.80 with the sodium hydroxide solution adjust pH of 3mol/L hydrochloric acid solution or 3mol/L, then use the acidic buffer constant volume of 0.05mol/L in the volumetric flask of 100mL.Be labeled as 1.0% acid carboxymethyl cellulose sodium solution, with tense marker pH=4.80, preparation date.Be kept in the refrigerator of 4 DEG C.Return to room temperature before using and shake all.

3.4DNS reagent

Dissolve 10.0g 3,5-dinitrosalicylic acid, 16g NaOH and 300g sodium potassium tartrate tetrahydrate, in about 700mL distilled water, add thermal agitation and make to be dissolved to clarification completely, put to normal temperature and be settled to 1000mL, put in brown reagent bottle, dark place uses after preserving a week.

4. instrument and equipment

4.1 assay balances: sensibility reciprocal 0.0001g

4.2 accurate pH meters: be accurate to 0.01

4.3 magnetic force heating stirrers

4.4 spectrophotometers: GB9721 pertinent regulations should be met, 5mm cuvette

4.5 electric-heated thermostatic water baths: 30-100 DEG C, ± 0.1 DEG C

4.6 timers: error per hour is no more than five seconds

4.7 pipettors: 100-1000 μ l

4.8 micro-sample input devices: 0-50 μ l

5. the drafting of calibration curve

Get 8 test tube accordings to the form below and add reagent, dilution glucose standard; Add 3mL DNS reagent again.Abundant mixing is put in boiling water and is boiled 10min, after water-bath cooling, does the absorbance to surveying other test solution under impinging upon 540nm with No. 0 test tube.Being ordinate with absorbance, take glucose content as abscissa drawing standard curve.

6, the preparation of enzyme liquid to be measured

Precision takes solid protoenzyme powder 1.0000g and is dissolved in (extension rate≤100 times direct buffer solution) in 80mL distilled water; Room temperature lower magnetic force stirs more than 15min, with 100mL volumetric flask constant volume, gets supernatant buffer solution and dilute after centrifugal, make its absorbance between 0.2-0.25.

7. level controls the preparation of liquid

Draw 1.0mL cellulase titer, (sign enzyme activity is 2500IU/Ml), dilutes 10000 times with the buffer solution of the citrate acidity (pH=4.80) of 0.05mol/L.Fresh level controls liquid to be needed now with the current.

8. determination step

8.1 get three test tubes respectively adds 0.5mL substrate, together with enzyme liquid to be measured in 50 DEG C of (± 1 DEG C) water-baths preheating 5min.

The 8.2 enzyme liquid to be measured respectively adding 0.5mL in first and second test tube, react 10min in 50 DEG C of water-baths.

The 8.3 DNS reagent respectively adding 3mL in three test tubes, then add the enzyme liquid to be measured of 0.5mL in the 3rd test tube.

8.4 shake up three test tubes after, in boiling water bath, boil 10min.

After 8.5 water-baths are cooled to room temperature, be the light absorption value to surveying first and second test tube under impinging upon 540nm condition with the 3rd test tube.Light absorption value, to be advisable between 0.2-0.25, is reformed if can not change extension rate within the scope of this.

9. result calculates

Enzyme activity (IU/g or IU/mL)=(value/180/10/0.5 such as glucose) × n

In formula: 180 refer to that glucose is converted into micromolar number from microgram

The reaction time of 10 finger liquid to be measured and low thing

0.5 refers to the enzyme liquid measure to be measured that low thing reacts

N refers to the extension rate of original enzyme liquid

10. allow analysis result to accept standard:

10.1 allow bulk analysis error ranges, two sub-samplings and testing result relative error is less than 10%.

10.2 levels control detection enzyme activity and standard enzyme vigor error is less than 10%

10.3 calibration curves at least form by 5

Ruminant special active fermentative feedstuff of microbe active bacteria of the present invention and enzyme measurement result as shown in table 3:

Table 3

And control group, priority of use application number is the feed of the technical scheme production of 201310090163, and its enzymatic activity/kg is protease <2100u, amylase <800u, cellulase <1600u.As can be seen here, the feed of the present invention after optimization, unexpected have high enzyme work.

By testing to the composition of active microorganism feed; See that inspection report No.WS 12-145 raises in feed grass seeds inspection point, Inner Mongolia Autonomous Region; Baotou product quality gauge check institute survey report No. (2012) L801, all indexs all reach regulation.

Embodiment 3 milking cow feeding experiment is reported:

The absorption easy to digest of this active microorganism fermented feed, reduces enterogastric diseases, improves efficiency of feed utilization.

According to composition of raw materials, this fermentative feedstuff of microbe can find out that nutritional labeling is higher, the amount reproduction of active beneficial bacterium and the fresh complex enzyme Direct-fed ruminant of generation thereof give full play to the double action of active beneficial bacterium and high vigor complex enzyme during the fermentation.In the cultivation of cattle and sheep etc., the main component in its feed is all holophytic nutrition material.And the cell membrane that plant cell outermost is all made up of cellulosic molecule one deck, this hinders the release of nutriment in plant cell greatly, and makes the cellulose family nutriment in feed cause waste.Utilize the cellulase that microbe fermentation method produces, plant cellulose can be decomposed into glucose, eliminate the anti-oxidant action of SNSP in feed, cellulose is absorbed and used with the form of glucose.In addition, cellulase divides the cellulose taken off in animal intestinal, can reduce the viscosity of intestinal contents to a great extent, increases the touch opportunity of the digestive ferment in feed Middle nutrition material and gastric juice, digestibility and the utilization rate of feed can be significantly improved, promote growth of animal.Utilize microbe fermentation method, due to the multiple protein catabolic enzyme that protein feeds raw material different beneficial bacterium kind fecund is raw, effectively multiple crude protein can be resolved into amino acid, peptide that Small molecular easily absorbs.

Multiple beneficial bacterium in active microorganism fermented feed is growth and breeding metabolism in the raw material commonly used in feed formula, its fresh complex enzyme produced can give full play to enzyme activity, these features of high specificity, effectively prevent the harmful effects such as exogenous enzyme antagonism, vigor be low.

Milking cow feeding experiment report (foreign exchange original seed cultivation Co., Ltd of Wulate Front Banner tests):

504 milk cow in lactation period are divided into 2 groups according to the output of milk, parity by this test, and the smart feed supplement of 10% is replaced to active microorganism fermented feed by test group, and control group, by former Diet, is fed 30 days.As a result, the per day output of milk of test group 34.2 kilograms, the per day output of milk of control group 32.1 kilograms.Through inspection, the output of milk improves 6.9% than control group, and obviously (P < 0.5), test group gives milk the diarrhea of dairy cow incidence of disease than control group reduction by 80% to difference.Test group every milk cow in lactation period average control group profit 315 yuan more than control group.(milk unit price is by 5 yuan every kilogram)

This active microorganism fermented feed price is low:

Protein content is the milk cow essence feed supplement of 18%, the average price per ton 3100 yuan of market price, the milk cow special active microbiological feed of protein content 18% on average 2800 yuan per ton.Market complex enzyme preparation for feeding per ton 8000-60000 yuan not etc.

As can be seen here, provided by the invention ruminating uses active microorganism fermented feed, have very outstanding index performance, and each side index all reaches National Performance Criteria.

The above; be only the present invention's preferably detailed description of the invention; but protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to according to technical scheme of the present invention and inventive concept thereof and replace or change, all should be encompassed within protection scope of the present invention.

Claims (2)

1. a ruminant special active microorganism biological fermented feed, is characterized in that: this active microorganism fermented feed AB bacterium liquid used is made up of two parts:

A bacterium liquid: saccharomycete, Bacillus acidi lactici, Corynebacterium glutamicum, bacillus subtilis, bacillus licheniformis, streptococcus fecalis etc., select suitable condition of culture to expand these bacterium and cultivate, it is 10 that the bacterium liquid be finally prepared into is cultivated in expansion ⁷-10 ⁹bacterial strain/ml;

B bacterium liquid selects rumen microorganism: Ruminococcus albus, yellow Ruminococcus, bacteroides ruminicola, Selenomonas, butyric acid vibrios, rumen anaerobic fungi, and it is 10 that the bacterium liquid be finally prepared into is cultivated in expansion ⁷-10 ⁹bacterial strain/ml;

Then AB bacterium liquid is carried out fermented and cultured, the mass percent of described fermentation medium is: the certain herbaceous plants with big flowers dregs of rice are 3; The dish dregs of rice are 3; Dregs of beans is 30; Corn is 18; Secondary wheat 9.7; Trehalose 1.8; Oligosaccharides 1.0; Feed-grade calcium phosphate is 1.26; Feed grade calcium carbonate is 1.4; Normal diet additive is 0.84; A bacterium liquid 15, B bacterium liquid 15.

2. ruminant special active microorganism biological fermented feed according to claim 1, wherein expands culture medium and consists of:

The preparation of A1 yeast liquid culture medium: the saccharification wort getting the large production of 10L, with being heated to 40 DEG C after filter-cloth filtering, adding prior off-the-shelf egg white boils to boiling, start timing, then boil 20 minutes, add appropriate distilled water and add to wort concentration and will control between 10-11 degree, boil again to boiling, cool, filter, adjust PH5.4--5.8 with the phosphoric acid of IN, for subsequent use;

A2 Corynebacterium glutamicum, Bacillus acidi lactici culture medium prescription: 2% sucrose 2% yeast extract 10% potato juice is cultivated rear Bacillus acidi lactici and is increased to 1.58*10 ¹¹cfu/ml;

A3 bacillus subtilis bacterium culture medium, glucose 30.0g peptone 10.0g yeast extract 5.0g potassium dihydrogen phosphate 4.5g calcium carbonate 2.0g sterilized water 1000ml;

A4 bacillus licheniformis culture medium corn steep liquor 0.45g peptone 1.50g beancake powder soaks the suitableeest kind of 18 hours age of juice 1:15 glucose 1.50g, and inoculum concentration is 8%, initial PH7.5, optimum temperature 37.5 DEG C;

A5 streptococcus fecalis culture medium sodium chloride 0.5g peptone 1.0g lactose 2.0g OX-heart soup 100ml PH nature, for activated spawn, proliferated culture medium (%) peptone 2 beef extract 2 lactose 2 PH6.8.