CN105076675A - Novel solid state fermentation technology of corn flour with low moisture - Google Patents
Novel solid state fermentation technology of corn flour with low moisture Download PDFInfo
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Abstract
The invention relates to a novel solid state fermentation technology of corn flour with low moisture. The novel solid state fermentation technology comprises the following steps: (1) taking grinded corn flour, mixing with high-temperature alpha-amylase and part of water, carrying out sterilizing enzymolysis for 20-30 min at 90-100 DEG C, naturally cooling to room temperature, and complementing the rest of water for later use; and (2) inoculating bacillus subtilis, brewer's yeast and enterococcus faecium into the product obtained in the step (1), and fermenting for 48-72 h at 30-40 DEG C, thus preparing the corn flour with low moisture. With the novel technology, the content of soluble sugar in the corn flour is improved to 61.2% from about 12%, and the content of total acids is improved to 1.52%.
Description
Technical field
The invention belongs to corn fermentation technical field, be specifically related to a kind of low moisture solid state fermentation corn new technology.
Technical background
Along with the fast development of Animal Husbandry in China, feed resource shortage becomes the principal element of its development of restriction gradually.The utilization ratio and the new feed resource of exploitation that improve existing feedstuff are the important channels solving feedstuff deficiency.
For a long time, the daily ration structure of corn one bean pulp type is mostly used in livestock and poultry cultivation, using corn as main energy sources.Corn good palatability, amino acid comparatively balance, cultivated area wide, and being therefore used widely in aquaculture, is the main energy sources of weanling pig, accounts for more than 60% of daily ration gross weight.But for piglet, its digestive system development is not yet perfect, digestive ferment hyposecretion is very low to starch utilization ratio.The decline of digestibility and utilization efficiency can cause again the series of problems such as the decline of weanling pig appetite, diarrhoea, growth retardation.
Production utilize mostly extrusion and the broken method of fine powder to improve the utilization ratio of corn, but have and report expanding treatment and the broken digestibility that can not improve cornstarch of fine powder, how improving energy digestibility in corn further becomes and improves dietary digestibility of energy shortage situation and cost-saving key measure.Therefore the method impelling us to find can better to transform corn to make it be conducive to young animal to absorb.
Large quantity research shows: microorganism has good transformation effect to feed.The various enzymes that microorganism produces in growth course can reduce the relative molecular mass of feed Middle nutrition material, make it advantageously digest and assimilate in young animal.Microprotein itself is also a kind of protein resource of high-quality in addition, can improve the content of protein in feed, its amino acid is formed more reasonable.Microorganism can also produce various benefit materials, organic acid as simple in some and some low-molecular-weight antibacterial material.
The application have studied the change of energy matter in corn before and after fermentable, inquire into the transformation effect of microorganism to carbohydrate in corn, for fermentation energy feed provides theoretical foundation, provide the reference of processing method for corn obtains better application on young animal.
Summary of the invention
The object of the invention there are provided a kind of low moisture solid state fermentation corn new technology, it utilizes high-temperatureα-amylase to carry out sterilizing enzymolysis to corn flour in advance, and then inoculate bacillus subtilis, VREF and saccharomyces cerevisiae and ferment, this technique can make the soluble sugar content in corn flour bring up to 61.2% by about 12%, and total acid content brings up to 1.52.
For achieving the above object, the present invention adopts following technical scheme:
A kind of low moisture solid state fermentation corn new technology, it comprises the steps:
1) get the corn flour pulverized, after mixing with high-temperatureα-amylase and part water, in 90-100 DEG C of sterilizing enzymolysis 20-30min, naturally cool to room temperature, supply the water of surplus, for subsequent use;
2) bacillus subtilis, saccharomyces cerevisiae and VREF are seeded in step 1) products therefrom, in 30-40 DEG C of fermentation 48-72h, obtain final product.
Concrete, in step 1), the addition of high-temperatureα-amylase is 10-30U/g corn flour; The weight ratio (i.e. solid-to-liquid ratio or material-water ratio) of corn flour and water is 1:0.50-0.60, and the water yield of first time interpolation accounts for the 50-60% of total water body accumulated amount.
Step 2) in bacillus subtilis, saccharomyces cerevisiae and VREF inoculum concentration be respectively 3-5%, 5-7% and 5-9%.Wherein, preferably, the inoculum concentration of bacillus subtilis, saccharomyces cerevisiae and VREF is respectively 5%, 7% and 9%.In the present invention, bacillus subtilis, saccharomyces cerevisiae and VREF are respectively purchased from Chinese industrial Microbiological Culture Collection administrative center, and goods number is respectively CICC20819; CICC1355; CICC6078.
Solid state fermentation refers to not to be had or does not almost have Free water, in the water-insoluble solid state substrate having certain humidity, with a bioprocesses of one or more fermentable.Research shows: fermentable has good transformation effect to feed.Fermentable albumen oil meals, can remove the multiple ANFs in the assorted dregs of rice, improves albumen quality, reduce albumen relative molecular mass, increase little skin material and the microbial metabolic products to animal health.But the research report of fermentable energy feed is less at present.
Compared to the prior art, beneficial effect of the present invention:
The energy feed fermentation technique that the application adopts multiple-microorganism mixed culture fermentation to combine with enzymolysis process, avoids HTHP or other physics modes to the structural damage of corn homenergic raw material, remains its natural structure characteristic; By the fermentation of microorganism, improve the palatability of corn, improve digestibility, microbial cells and the enzyme secreted by growth metabolism process thereof fully contact with raw material during the fermentation simultaneously, the macromolecular structure destroying material surface makes it be easier to digesting and assimilating of animal intestinal, and this also improves capacity usage ratio to a certain extent; Due to the restriction of microbial metabolism rule and the intrinsic shortcoming of solid state fermentation, add certain exogenous amylases class and can make up the not enough or very few problem of the enzyme of microorganism secretion, portion of energy can also be provided for growth of microorganism by hydrolysed corn raw material.The application is that young animal more effectively can utilize seed feed, and the nutritive potential excavating energy feed seeks new techniques and methods, and be fermentable starch based feed, more good utilisation seed feed provides basic basis; Also for compound-feed industries effectively utilizes corn homenergic raw material to provide reference in technology and method.
Detailed description of the invention
The present invention is described further by the following examples, but protection scope of the present invention is not limited thereto.
1 materials and methods
1.1, material
1.1.1, bacterial strain
Bacillus subtilis, saccharomyces cerevisiae and VREF are respectively purchased from Chinese industrial Microbiological Culture Collection administrative center, and goods number is respectively CICC20819; CICC1355; CICC6078.
High-temperatureα-amylase: preference temperature 80-110 DEG C, enzyme 100000U/g alive, purchased from Jining and letter Bioisystech Co., Ltd.
1.1.2, culture medium
LB culture medium: tryptone 1g, dusty yeast 0.5g, sodium chloride 0.5g, water 100ml, pH7.0
YPD culture medium: glucose 2g, dusty yeast 1g, tryptone 2g, water 100ml.
MRS culture medium: glucose 2g, tryptone 1g, beef peptone 1g, dusty yeast 0.5g, crystallization sodium acetate 0.5g, dipotassium hydrogen phosphate 0.2g, Triammonium citrate 0.2g, crystalline sulfuric acid magnesium 0.05g, manganese sulfate 0.02g, Tween 80 0.1ml, water 100ml.
1.1.3, reagent and equipment
1.1.3.1, reagent: anthrone, ethyl acetate, the concentrated sulfuric acid, NaOH, Folin reagent, DNS reagent, iodine liquid etc.
1.1.3.2, equipment: autoclave, centrifuge, spectrophotometer, shaking table, super-clean bench, triangular flask etc.
1.1.4, material
Corn flour (crossing 20 mesh sieves).
2. experimental technique
2.1, Testing index and method
The mensuration of soluble sugar: green onion ketone method
The mensuration of crude protein: triumphant formula nitriding
Total acid assay method; Sodium hydroxide titration
The mensuration of viable count: spread plate
Mycotoxin assay method: liquid chromatogram.
2.2, solid state fermentation conditions optimization
2.2.1, the optimization of compound bacteria-fermented optimum inoculation amount
Design compound bacteria-fermented inoculum concentration orthogonal test.Take corn flour as fermentation substrate, material-water ratio 1:0.45,100 DEG C of sterilizing 20min, fermentation 48h, with the content of soluble sugar after fermenting, acidity for index, comprehensively determines the best inoculative proportion of each bacterium of mixed fungus fermentation.
The optimization orthogonal test factor level table of table 1 compound bacteria-fermented optimum inoculation amount
Note: in table, numeral 5,7,9 represents percentage composition, and namely the amount of accessed seed liquor accounts for the percent by volume of institute's access system.
2.2.2 the optimization of compound bacteria-fermented condition
On the basis of compound bacteria-fermented inoculative proportion optimum results, orthogonal test is adopted to be optimized mixed fungus fermentation condition.
The optimization orthogonal test factor level table Lg (3 of table 2 compound bacteria-fermented condition
3)
2.3, exogenous amylases is on the impact of composite fermentation
2.3.1, the determination of the suitableeest addition of exogenous enzymes
Take corn flour as culture matrix, 100 DEG C of high-temperature sterilization 20min, natural pH, material-water ratio 1:0.45, high temperature alpha amylase addition is 10,20,30U/g corn flour; Often group three is parallel, and cultivate 48h, dry, pulverized 60 mesh sieves in sample 65 DEG C of baking ovens for 35 DEG C, detecting the content of soluble sugar, take soluble sugar content as index.
2.3.2, the selection of enzymolysis process is on the impact of compound bacteria-fermented
Consider that the optimum temperature of the alpha-amylase that 1.2.3.2 selects is all higher than 70 degree, combining with fermentation technique, elitely select the sterilization process of enzymolysis process in conjunction with solid medium.
3, result
3.1 obtain a result as follows by 2.2.1 method
The optimum results of table 3-1 compound bacteria-fermented optimum inoculation amount
Note: in table, A, B, C represent experimental factor, namely represents bacillus subtilis respectively, saccharomyces cerevisiae, VREF; In table, numeral 1,2,3 represents the inoculum concentration of A, B, C tri-kinds of factors, and namely 1,2,3 to represent inoculum concentration be respectively 5%, 7%, 9%.
Influence factor to total acid: VREF > saccharomyces cerevisiae > bacillus subtilis, the optimum combination that system provides is A
1b
2c
3, that is: bacillus subtilis inoculum concentration 5%, saccharomyces cerevisiae inoculum concentration 7%, VREF kind amount are 9%.
The optimum results of table 3-2 compound bacteria-fermented optimum inoculation amount
Note: in table, A, B, C represent experimental factor, namely represents bacillus subtilis respectively, saccharomyces cerevisiae, VREF; In table, numeral 1,2,3 represents the inoculum concentration of A, B, C tri-kinds of factors, and namely 1,2,3 to represent inoculum concentration be respectively 5%, 7%, 9%.
Influence factor to soluble sugar content: saccharomyces cerevisiae > VREF > bacillus subtilis, the optimum combination that system provides is A
1b
2c
1, that is: bacillus subtilis inoculum concentration 5%, saccharomyces cerevisiae inoculum concentration 7%, VREF inoculum concentration are 5%.
Analyze Orthogonal experiment results and show that several groups are better combined: A
3b
1c
3, A
1b
2c
3, A
1b
2c
2, A
1b
2c
1carry out repeating test.
According to Orthogonal experiment results, carrying out Latin square experiment to testing the four kinds of assembled schemes drawn respectively, finding and A
1b
2c
3group is compared, A
3b
1c
3group due to the yeast-inoculated amount affecting sugared content factor maximum less, and bacillus subtilis inoculum concentration is excessive forms certain competitive relation with yeast, causes yeast can better not grow and soluble sugar content is reduced; A
1b
2c
2with A
1b
2c
1, because VREF inoculum concentration is less, breed the growth of the pH suppression bacillus subtilis failing to change rapidly environment more slowly in the fermentation middle and later periods, thus promote that the growth of saccharomyces cerevisiae causes sugared content to reduce.So determine sour optimal set and bacillus subtilis inoculum concentration 5%, saccharomyces cerevisiae inoculum concentration 7%, VREF inoculum concentration be 9% be best experimental program.
3.2, composite fermentation condition optimizing result of the test
Table 4-1 composite fermentation condition optimizing result of the test-soluble sugar
Note: in table, A, B, C represent experimental factor, namely represents time, temperature, moisture respectively; D be confactor on analysis result without impact; Numeral 1 in table
a, 2
a, 3
athe fermentation time that expression three kinds is different respectively, i.e. 48h, 60h, 72h.Numeral 1 in table
b, 2
b, 3
bbetween the fermentation temperature that expression three kinds is different respectively, namely 30 DEG C, 35 DEG C, 40 DEG C.Numeral 1 in table
c, 2
c, 3
cmaterial and moisture content, i.e. 1:0.45,1:0.50,1:0.55 needed for the fermentation that expression three kinds is different respectively.
Result of the test-the acid of table 4-2 composite fermentation condition optimizing
Note: in table, A, B, C represent experimental factor, namely represents time, temperature, moisture respectively; D be confactor on analysis result without impact; Numeral 1 in table
a, 2
a, 3
athe fermentation time that expression three kinds is different respectively, i.e. 48h, 60h, 72h.Numeral 1 in table
b, 2
b, 3
bbetween the fermentation temperature that expression three kinds is different respectively, namely 30 DEG C, 35 DEG C, 40 DEG C.Numeral 1 in table
c, 2
c, 3
cmaterial and moisture content, i.e. 1:0.45,1:0.50,1:0.55 needed for the fermentation that expression three kinds is different respectively.
According to analysis result, as can be seen from extreme difference, optimum combination fermentation is 72h, 35 DEG C, material-water ratio is 1:0.55; To the influence factor mainly fermentation time of soluble sugar content, be secondly moisture, and temperature impact is minimum.
Drawn by above data, the influence factor to total acid: temperature > time > moisture, optimal conditions of fermentation be 60h, 35 DEG C, material-water ratio is 1:0.55; And soluble sugar content is not high only has 39.7%.
According to Orthogonal experiment results, show that optimal conditions of fermentation is fermentation 72h, fermentation temperature 35 DEG C, material-water ratio is 1:0.55, and tunning soluble sugar content reaches 48.6% under this condition, and total acid content can reach 1.52%.Analyzing reason is under anaerobic carried out again a large amount of propagation with the prolongation of fermentation time to sugared content and the larger VREF of Effect of Acidity On Absorption, initial temperature shortens the resting stage of growth, can conform rapidly and carry out proliferation and metabolism, in the surrounding environment simultaneously causing the carbohydrate in saccharomyces cerevisiae Mortality cell lysis to be discharged into along with the rising of temperature in environment.
4, amylase result that composite fermentation is affected
4.1 obtain a result through method 2.3.1, as can be seen from Table 5, addition is when 10-20U/g corn flour, soluble sugar content reduces along with the addition increase of high-temperatureα-amylase, when 20-30U/g corn flour, soluble sugar content raises, integrated economics angle along with the addition increase of high-temperatureα-amylase, and high-temperatureα-amylase addition elects 10U/g corn flour as.
Table 5 high-temperatureα-amylase addition is on the impact of soluble sugar content
4.2, for improving test effect, make aimed strain can adapt to rapidly yeasting, fast breeding, on the basis of composite bacteria fermentation, in conjunction with optimal conditions of fermentation be 72h, 35 DEG C, material-water ratio is 1:0.55; Consider to add amylase, the amylase of interpolation is high-temperatureα-amylase, and addition is 10U/g corn flour, and sterilization method adopts 100 DEG C of 20min, and testing program has three:
Scheme one: add fermentable (namely the inoculum concentration of bacillus subtilis, saccharomyces cerevisiae and VREF is respectively 5%, 7% and 9%, 35 DEG C of fermentation 72h) according to the result of 3.2 after sterilizing;
Scheme two: add high-temperatureα-amylase 10U/g corn flour after sterilizing;
Scheme three: add high-temperatureα-amylase 10U/g corn flour before sterilizing, and the water yield that first time adds accounts for 55% of total water body accumulated amount, then mixes sterilizing, supplies moisture after being cooled to room temperature, then add fermentable according to the result of 3.2;
Table 6 exogenous amylases is in conjunction with fermentable effect
Interpretation of result: as can be seen from the change of soluble sugar content in three kinds of schemes, scheme 1 is pure fermentable, scheme 2 is pure enzymolysis, soluble sugar content soluble sugar content brings up to 32.3% and 37.8% by 11.5% of corn flour respectively, in scheme 3, it is about 90 DEG C according to alpha-amylase optimum temperature, in conjunction with sterilizing and cooling procedure, the activity of enzyme can be given full play to, therefore add the high-temperatureα-amylase of 10U/g corn flour before sterilization, best ferment effect can be reached, fermentable is in conjunction with enzymolysis process, not only the cornstarch overwhelming majority is decomposed, for growth of microorganism provides necessary carbon source, turn avoid the waste of simple sterilizing heat in temperature-rise period and temperature-fall period.
conclusion
Optimal culture condition and the technological condition for fermentation of test by having determined mixed culture solid state fermentation corn flour, the soluble sugar content in corn flour is made to bring up to 32.3% by original 11.5%, amount of increase reaches 181%, probe into further again and combine with biological enzymolysis technique simultaneously, make soluble sugar content turn improve more than 89%, total acid content reaches 1.5%.
Research finally determines best enzymolysis process and is: the high-temperatureα-amylase adding 10U/g corn flour before sterilizing, and the water yield of first time interpolation accounts for the 50-60% of total water body accumulated amount, mixes rear 100 DEG C of sterilizing enzymolysis 20min, supplies residual moisture after being cooled to room temperature; Best zymotechnique is: bacillus subtilis inoculum concentration is 5%, saccharomyces cerevisiae inoculum concentration is 7%, VREF inoculum concentration is 9%, and technological condition for fermentation is: 35 DEG C of fermentation 72h, material-water ratio 1; 0.55.
Combined with biological enzymolysis by fermentable, researched and solved that the solid state fermentation nutriment that especially cornstarch is intrinsic is single, low, the tunning of fermentation utilization rate exists the not tempting shortcoming of sense organ difference, smell.Not only make the bulky grain starch degradation in corn flour be the soluble sugar such as glucose, maltose, dextrin easily absorbed, improve corn digestibility, additionally reduce the impact of external source bacterial classification on fermentation system, improve the stability that product quality in turn ensure that product.Not only improved on the other hand the palatability of corn by saccharomyces cerevisiae and the growing multiplication of VREF, improve capacity usage ratio, probio contained in product improves animal intestinal environment to a certain extent, promotion microbial growth.
Claims (3)
1. a low moisture solid state fermentation corn new technology, is characterized in that, comprise the steps:
1) get the corn flour pulverized, after mixing with high-temperatureα-amylase and part water, in 90-100 DEG C of sterilizing enzymolysis 20-30min, naturally cool to room temperature, supply the water of surplus, for subsequent use;
2) bacillus subtilis, saccharomyces cerevisiae and VREF are seeded in step 1) products therefrom, in 30-40 DEG C of fermentation 48-72h, obtain final product.
2. low moisture solid state fermentation corn new technology as claimed in claim 1, it is characterized in that, in step 1), the addition of high-temperatureα-amylase is 10-30U/g corn flour; The weight ratio of corn flour and water is 1:0.50-0.60, and the water yield of first time interpolation accounts for the 50-60% of total water body accumulated amount.
3. low moisture solid state fermentation corn new technology as claimed in claim 1, is characterized in that, step 2) in bacillus subtilis, saccharomyces cerevisiae and VREF inoculum concentration be respectively 3-5%, 5-7% and 5-9%.
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Citations (4)
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|---|---|---|---|---|
| CN101253934A (en) * | 2008-04-07 | 2008-09-03 | 中国农业科学院北京畜牧兽医研究所 | Composite microorganism additive agent for milk cattle feed stuff and method of preparing the same |
| CN102796787A (en) * | 2012-09-07 | 2012-11-28 | 吉林中粮生化科技有限公司 | Novel preparation method of corn starch sugar |
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| CN103564199A (en) * | 2012-07-31 | 2014-02-12 | 黑龙江省轻工科学研究院 | Method for producing cow feed by multi-strain mixing solid-state fermentation |
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Patent Citations (4)
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| CN101253934A (en) * | 2008-04-07 | 2008-09-03 | 中国农业科学院北京畜牧兽医研究所 | Composite microorganism additive agent for milk cattle feed stuff and method of preparing the same |
| CN103564199A (en) * | 2012-07-31 | 2014-02-12 | 黑龙江省轻工科学研究院 | Method for producing cow feed by multi-strain mixing solid-state fermentation |
| CN102870977A (en) * | 2012-08-14 | 2013-01-16 | 郑州市大北农饲料科技有限公司 | Wheat middling-corn type compound feed for lactating sow and preparation method of compound feed |
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