CN103409352A - Compound microbial community for preparing feed by fermenting soybean hulls - Google Patents
Compound microbial community for preparing feed by fermenting soybean hulls Download PDFInfo
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Abstract
The invention aims to provide a compound microbial community for preparing feed by fermenting soybean hulls, which comprises lactic acid bacteria, bacillus natto, candida utilis and penicillium oxalicum. According to the invention, because an optimum fermentative strain and an optimum strain proportion are optimized and screened, the fermentation effect of the compound microbial community is superior to that of traditional single strain and mixed strain fermentation, so that the protein content can be greatly increased, the fiber content can be reduced, and meanwhile, the production cycle can be effectively shortened, the production cost can be reduced, the production efficiency can be increased, the quality of products can be improved, and the nutrition utilization value of products can be improved; the compound microbial community satisfies the demands on low price and safety in the current feed industry, and provides a more scientific method for the system utilization of soybean hulls.
Description
Technical field
The invention belongs to the fermentative feedstuff of microbe technical field, be specifically related to the complex microbial community that a kind of fermented soybean skin prepares feed.
Background technology
Soybean hulls is the outer coated material of soybean, and color is beige or light yellow, and it accounts for 10% of whole soybean volume, 8% of weight.Research shows that the main component of soybean hulls is cell walls and vegetable fibre, and its crude fiber content is 38.0%, crude protein 10.0%, and calcium 0.53%, phosphorus 0.18%, content of lignin is lower than 2.0%.As the byproduct of soybean oil-producing technique, soybean hulls can be processed hot method decortication or two kinds of working method gained of crushing screen by grease.
China is Soybean production state, and annual production is more than 1,500 ten thousand tons.Along with China's soybean extract oil industrial expansion with to the increase of dregs of beans requirement, China needs a large amount of soybean of import every year, 2011～2012 average annual imported soybeans are more than 5,700 ten thousand tons, and the soyabean processing ability is 6500～7,800 ten thousand ton/years of left and right, estimate the annual soybean hulls that produces 500～5,760,000 tons.And the soybean hulls price is low, be 1.4～1.6 yuan/kg, and corn and Wheat bran price are respectively 2.5 yuan/kg and 1.7 yuan/kg left and right.Therefore, soybean hulls develops and has Important Economic value and market outlook as a kind of new feed resource.
Yet it contains the antinutritional factor such as a large amount of urases, trypsin ihhibitor due to soybean hulls, therefore, on utilizing, livestock and poultry have been subject to certain restriction at present, only be applied on a small quantity in ox, sheep roughage, most of just as fertilizer, not by the development and utilization of its science.And the at present processing of the soybean hulls methods such as traditional baking and high temperature steaming that adopt to be to eliminate the soybean hulls antinutritional factor more, cause that the product prepared power consumption is high, palatability is poor.
Continuous progress along with biotechnology, the soybean hulls of using at present utilizes technology by novel process, new technology, to be replaced gradually, technique, towards energy-conservation of consumption reduction, environmental protection future development more, is focused on the complex optimum utilization of quality, palatability and the Biological resources of product more.Owing to considering the problems such as environmental protection, energy-conservation and cost, if adopt microbe fermentation method processed soybeans skin, with traditional cooking process, to compare, the product protein content of producing is high, antinutritional factor content is few, be rich in probiotics, environmental pollution is little, is a kind of working method of more science.
Yet.Up to the present, for the soybean hulls processing and utilization, do not carry out systematic research both at home and abroad, the soybean hulls fermentation technique is not enough maturation also, to the greatest extent still pilot production of possessor is with single or two strain fermentations, but the protein increasing amount of soybean hulls leavened prod is few, cellulose degradation rate is low, and the feed conversion rate variance causes technique can't carry out large-scale promotion.For this reason, further investigation best complex flora fermented soybean skin technology, be worth its nutritional utilization of further raising, enlarges its range of application in feed, with cheap fermented feed, substitutes conventional feed and have important economy and social value.
Summary of the invention
The purpose of this invention is to provide a kind of multiple and microorganism species for preparing feed for the fermented soybean skin, namely adopt complex microorganism method fermented soybean skin, to fill up the blank of this art at present.
The applicant is by for a long time research, and through the screening of single factor experiment, the number of determining various bacterium in the best strain combination, flora of multiple and microbial method fermented soybean skin contrasts and the preparation method.
Multiple and the microorganism species for preparing feed for the fermented soybean skin of the present invention, include milk-acid bacteria, bacillus natto, product Ruan candidiasis and penicillium oxalicum.
In above-mentioned complex microbial community, the bacterial classification quantity of milk-acid bacteria, bacillus natto, product Ruan candidiasis and penicillium oxalicum is than being 2:1:2:1;
The present invention optimizes best fermented bacterium combination, the best bacterial classification ratio of having screened, its ferment effect is better than traditional single culture and there is no the composite bacteria fermentation of proportioning, can increase percent protein greatly, reduce fibre content, and can effectively shorten the production cycle, reduce production costs, increase production efficiency, improve product quality, improve product nutritional utilization value, meet nowadays feedstuff industry cheapness and demand for security, for the system utilization of soybean hulls provides the method for science more.
The accompanying drawing explanation
Fig. 1: crude protein and the fibre content figure of single bacterium solid state fermentation test product;
Fig. 2: crude protein and the fibre content figure of hybrid bacterial strain solid state fermentation test product;
Fig. 3: 36h thalli growth condition diagram;
Fig. 4: the affect figure of different strain inoculative proportion on the content of product crude protein and fiber;
Fig. 5: the affect figure of the different vaccination amount of multiculture on tunning crude protein and fibre content;
Fig. 6: the affect figure of different wheat bran ratios on crude protein in tunning and fibre content.
Embodiment
The present invention has passed through long-term a kind of strain combination for the fermented soybean skin of having determined, its complex ferment bacterial strain is comprised of penicillium oxalicum, milk-acid bacteria, bacillus natto and product Ruan candidiasis, by the soybean hulls of effectively degrading of the complex role between above-mentioned four kinds of bacterial strains.
1) select the character description of bacterial strain:
The penicillium oxalicum of selecting can produce cellulase and the polygalacturonase of high vigor degraded cellulose; Its cellulase can be sugar by the cellulose degradation in soybean hulls, for other growth desired nutritional; The polygalacturonase decomposed is a multienzyme complex system, can act on plant feed (comprising soybean hulls) cell walls mesogloea, make the cell walls attenuation, the flanking cell wall disappears, cell walls is arranged loose, destroy the structure of cell walls, nutritive substance is fully contacted with digestive ferment, improve the activity of animal endogenous enzyme (as amylase, trypsinase and lipase etc.); And in hydrolysis of pectin with the semi-lactosi aldehyde radical of α-Isosorbide-5-Nitrae glycosidic link combination, produce reducing sugar; Thereby improve the utilization ratio of body to the nutritive substances such as fiber, protein, calcium, phosphorus in feed; In addition, the specific absorption of phosphorus increases, and can effectively reduce the excretion of stock-farms phosphorus, and environmental contamination reduction is conducive to protection ecological.
The bacillus natto of selecting is the bacterial strain that leavened food level natto is used, and soybean is had to very strong fermentation capacity, and after fermentation, fragrant is arranged; Bacillus natto has the activity that stronger product amylase, proteolytic enzyme, lipase etc. are separated enzyme, also has simultaneously non-starch polysaccharide enzyme in the degrading plant forage, as polygalacturonase, dextranase, cellulase etc., promotes animal digesting and assimilating nutritive substance; Bacillus natto can consume a large amount of oxygen, maintains the enteron aisle anaerobic environment, suppresses the growth of pathogenic bacterium, maintains the normal eubiosis of enteron aisle; The gemma of bacillus natto can stimulate the generation immune response, the immunizing power of enhancing body.
The milk-acid bacteria of selecting can be regulated the palatability of fermented feed; Milk-acid bacteria can form dominant bacteria after entering enteron aisle, thereby suppresses the breeding of germ, and self can produce multiple antibacterial substance, can suppress or killing microorganism, thereby the enhancing body immunologic function, increase Abwehrkraft des Koepers, finally improves digestion and the growth performance of animal.
Product Ruan candidiasis of selecting can rise in value fast in sugared substratum is arranged, be the single-cell protein feed of commonly using; Yeast is that the length of livestock and poultry animal occupies one of microorganism, is also dominant microflora.But the quantity of adding probiotic bacterium in yeast stimulating animal enteron aisle in feed increases, and improves the microbial environment in animal intestinal; Saccharomycetic cell walls is fibrous by special yeast, mainly mannosans and glucose, consists of.Mannosans can strengthen the immunizing power of energy enhancing body; Yeast can suppress the toxication mechanism of aflatoxin, and aflatoxin is had to detoxification.
The present invention milk-acid bacteria used is the lactobacterium acidophilus, and its a kind of strain number is the CICC32835 that is numbered that is numbered CGMCC1.1086, product Ruan candidiasis of ACCC10637, bacillus natto.But, under invention thinking of the present invention, can select other bacterial strain.
For the penicillium oxalicum bacterial classification, this bacterium is obtained from separation the goose enteron aisle by high-quality aquatic bird institute of Qingdao Agricultural University, have the very high degradation capability of Mierocrystalline cellulose, through Institute of Microorganism, Academia Sinica, identify and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (microbial preservation numbering: CGMCC No.2260).Above-mentioned 4 kinds of bacterial classification prescriptions are used and can maximumly improve the protein content of fermented soybean skin and reduce content of cellulose, and can degradable antinutritional factor.
Above-mentioned four kinds of fermented bacterium Application of composite that the present invention selects, have the nutrition content that increases fermented feed, improve the prebiotic effect in hazardous and noxious substances in animal palatability and nutrient utilization, degraded soybean hulls and good animal production.
2) preparation of fermented bacterium amplification culture medium:
Penicillium oxalicum amplification culture medium compound method: adding distil water 1000ml in beaker adds peptone 5.0g, dipotassium hydrogen phosphate 1.0g successively, yeast extract powder 2.0g, sal epsom 0.5g, tepor is dissolved, regulate pH value and be about 6.8, boil, after adding glucose 20.0g to dissolve, shake up, the gauze filtering, regulating that pH value makes after sterilizing is 6.4 ± 0.2, be divided in each triangular flask in, add cotton plug, sterilizing with pressure kettle.Culture temperature is 30 ℃, and incubation time is 18h～24h, after cultivating end, from shaking table, taking out in the refrigerator that is placed on 4 ℃ and preserve.
Milk-acid bacteria amplification culture medium compound method: adding distil water 1000ml in beaker adds peptone 10.0g, beef leaching thing (extractum carnis) 10.0g successively, yeast extract (yeast extract paste or yeast powder) 5.0g, glucose 20.0g, sodium acetate 5.0g, citric acid hydrogen diamine 2.0g, tween-80 1.0ml, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, seven water manganous sulfate 0.05g, regulate pH6.2～6.4 with 10% hydrochloric acid, be divided in each triangular flask in, add cotton plug, sterilizing with pressure kettle.After inoculation, culture temperature is 42 ℃, amphimicrobian, and culture temperature is 18h～20h, after cultivating end, from incubator, taking out in the refrigerator that is placed on 4 ℃ and preserve.
Bacillus natto amplification culture medium compound method: adding distil water 1000ml in beaker, add successively sucrose 10g, soy peptone 10g, NaCl5g, be placed on the upper heating of fire.Until completely dissolved, adjust pH value to 7.2～7.6 with the sodium hydroxide of 10% hydrochloric acid or 10%, be divided in each triangular flask in, add cotton plug, sterilizing with pressure kettle.After inoculation, culture temperature is 37 ℃, and incubation time is 18h～24h, after cultivating end, from shaking table, taking out in the refrigerator that is placed on 4 ℃ and preserve.
Produce Ruan's candidiasis amplification culture medium compound method: get the 250g kilned malt, kilned malt is ground, granularity is 100 orders, puts into large beaker, adds 1000ml distilled water, saccharification 3h～4h in 64 ℃～66 ℃ water-baths.Saccharified liquid, by 4～6 layers of filtered through gauze, if filtrate is muddy, is clarified with egg albumen.Method is, the albumen in an egg is taken out and adds water 20ml, till mixing well and being stirred to living foam with glass stick, after then pouring in saccharified liquid and stirring and boil, by 8 layers of filtered through gauze.It is 7.0～7.2 that filtrate is regulated PH, and pol measures with saccharometer and adding distil water is diluted to 5 ° of Be, be divided in each triangular flask in, add cotton plug, sterilizing with pressure kettle.Culture temperature is 25 ℃～28 ℃, and incubation time is 24h, after cultivating end, from shaking table, taking out in the refrigerator that is placed on 4 ℃ and preserve.
3) preparation of soybean hulls fermention medium: will buy soybean hulls and wheat bran is pulverized with pulverizer, granularity 40 orders.Each substratum takes soybean hulls 45g, wheat bran 5g, adds the distilled water of 12.5ml, adds cotton plug, sterilizing with pressure kettle.
4) inoculation substrate: the soybean hulls fermention medium of the bacterium of having gone out, be placed on Bechtop, open ultra violet lamp 25min～30min sterilizing.After sterilizing end, light spirit lamp, it is 10ml that the bacterium liquid that increased is inoculated into to soybean hulls fermention medium inoculum size, and the inoculum size of penicillium oxalicum, milk-acid bacteria, bacillus natto, product Ruan candidiasis is followed successively by 3.33ml, 1.66ml, 3.33ml, 1.66ml.After having inoculated, substratum is put in incubator, culture temperature is 30 ℃, and incubation time is 72h.
After cultivate finishing, substratum is taken out, tunning is placed in baking oven and dries, bake out temperature is 35 ℃, measures the CP(crude protein after drying), the NDF(neutral detergent fiber), the ADF(acid detergent fiber) and the CF(robust fibre) content.
Below in conjunction with case study on implementation, method of the present invention is described in detail.
Embodiment 1: the shaker test of soybean hulls fermented bacterium
Raw material disposal: commercially available soybean hulls and wheat bran are pulverized with pulverizer, granularity is 40 orders, ratio in soybean hulls and wheat bran is 9:1, material-water ratio is that 3:1 is mixed to join in the 250ml triangular flask, in each triangular flask, add soybean hulls 45g, wheat bran 5g, distilled water 12.5ml, add cotton plug, with 121 ℃ of sterilizing 30min of pressure kettle.
1. single strain fermentation test
The penicillium oxalicum, milk-acid bacteria, bacillus natto, the product Ruan candidiasis bacterium liquid that have increased are diluted to 1 * 10 with sterilized water respectively
7Individual/ml, be inoculated on fermention medium, on each substratum, inoculates respectively the bacterium liquid of 5ml.After having inoculated, add cotton plug, the triangle bottleneck encases with kraft paper, is placed in the constant incubator of 30 ℃ and cultivates, and incubation time is 72h.
Test-results shows, selected four bacterial classifications all can improve crude protein content, reduces crude fiber content;
The albumen of the tunning crude protein content milk-acid bacteria group of four bacterial classifications improves at most, has improved 8.78% than unfermentable soybean hulls; And the robust fibre penicillium oxalicum reduces at most, reduced by 33.11% than unfermentable soybean hulls; And urase content is zero, illustrate that four bacterial classifications all are applicable to the fermented soybean skin.See accompanying drawing 1.
2. hybrid bacterial strain fermentation test
The two bacterium mixing of four bacterial strains and the mixing of many bacterium are accessed respectively in solid-state fermentation culture medium, and strain combination is: 1+2,1+3,1+4,2+3,2+4,3+4,1+2+3,1+2+4,1+3+4,2+3+4,1+2+3+4(1 represent that penicillium oxalicum, 2 represents that milk-acid bacteria, 3 represents bacillus natto, 4 representative product Ruan yeast).The bacterium liquid of inoculating respectively 5ml on each substratum carry out mixed fermentation (example: if double-bacterium ferment and a bacterial classification add bacterium liquid 2.5ml, other the like).After having inoculated, add cotton plug, the triangle bottleneck encases with kraft paper, is placed in the constant incubator of 30 ℃ and cultivates, and incubation time is 72h.
Test-results shows, the product C P content of fermented by mixed bacterium ferments apparently higher than single bacterium, and three bacterium are higher than two bacterium, and four bacterium are again higher than three bacterium.Wherein the CP content of 1+2+3+4 strain combination tunning is the highest than other group content, has improved 32.98% than unfermentable soybean hulls, and robust fibre is compared and reduced by 41.49%, and urase content is zero.And growth rapidly, after cultivating 36～48h, can see that mycelia covers matrix.See accompanying drawing 2 and accompanying drawing 3.
3. best bacterial classification inoculative proportion test
Four bacterial classifications are accessed respectively in solid-state fermentation culture medium in different ratios, and the inoculative proportion of bacterial classification is:
1:1:1:1,1:1:1:2,1:1:2:1,2:1:1:2,1:2:1:2,2:1:2:1,2:1:1:1,1:1:2:2,2:2:1:1,1:2:2:1,1:2:2:2,2:2:2:1(are followed successively by penicillium oxalicum: milk-acid bacteria: bacillus natto: produce Ruan's yeast).The bacterium liquid of inoculating respectively 5ml on each substratum carries out mixed fermentation, and (example: if the bacterial classification inoculative proportion is 1:1:1:1, the bacterium liquid addition that reaches each bacterial classification is 1.25ml, and bacterial classification quantity is 2 * 10
7: 1 * 10
7: 2 * 10
7: 1 * 10
7, the concentration of four bacterium liquid all is diluted to 1 * 10 with sterilized water
7Individual/ml.After having inoculated, add cotton plug, the triangle bottleneck encases with kraft paper, is placed in the constant incubator of 30 ℃ and cultivates, and incubation time is 72h.
Test-results shows, when the inoculative proportion of bacterial classification was 2:1:2:1, ferment effect was best, and CP content is the highest than other group content, has improved 50.44% than unfermentable soybean hulls, and robust fibre has reduced by 39.51%, and urase content is zero.See accompanying drawing 4.
4. optimum inoculation amount test
By four bacterial classifications 2:1:2:1(penicillium oxalicum * 2 in proportion: milk-acid bacteria * 1: bacillus natto * 2: produce Ruan's yeast * 1, bacterial classification quantity is 2 * 10
7: 1 * 10
7: 2 * 10
7: 1 * 10
7) with 2%, 5%, 10%, 15%, 20% inoculum size, be linked in solid-state fermentation culture medium respectively.After having inoculated, add cotton plug, the triangle bottleneck encases with kraft paper, is placed in the constant incubator of 30 ℃ and cultivates, and incubation time is 72h.
Test-results shows, when the inoculum size of bacterial classification was 10%, ferment effect was best, and CP content is the highest than other group content, has improved 25.87% than unfermentable soybean hulls, and robust fibre has reduced by 41.49%.See accompanying drawing 5.
Embodiment 2: the test of soybean hulls fermention medium auxiliary material addition
For bacterial classification is better grown, add a certain amount of wheat bran in substratum.In wheat bran, contain abundant VITAMIN and mineral substance, can regulate the nutrition of fermented substrate.It or a kind of good raising agent, also can be used as auxiliary nitrogenous source simultaneously.
Four bacterial classifications are fitted in to bacterium liquid by optimum proportion, and the inoculum size by 5% is inoculated into different the cooperation on solid-state fermentation culture medium.The mixing ratio of solid-state fermentation culture medium is: wheat bran 5%, soybean hulls 95%; Wheat bran 10%, soybean hulls 90%; Wheat bran 15%, soybean hulls 85%; Wheat bran 20%, soybean hulls 80%.After having inoculated, add cotton plug, the triangle bottleneck encases with kraft paper, is placed in the constant incubator of 30 ℃ and cultivates, and incubation time is 72h.
Test-results shows, ferment effect is first increases and then decreases along with the increase of wheat bran addition, and CP content is the highest than other group content when 10% addition, has improved 25.87% than unfermentable mixed substrates, and robust fibre has reduced by 35.15%.See accompanying drawing 6.
Embodiment 3: compound strain fermented soybean skin Orthogonal Rotational Regressive Tests
The employing compound strain is respectively: penicillium oxalicum, milk-acid bacteria, bacillus natto, product Ruan candidiasis; Its bacterial classification quantity is 2 * 10
7: 1 * 10
7: 2 * 10
7: 1 * 10
7, the concentration of four bacterium liquid all is diluted to 1 * 10 with sterilized water
7Individual/ml; Its optimum inoculation amount according to weight fraction is 10%; The adding proportion of solid-state fermentation culture medium wheat bran is 10%, finds by 5 proof tests, and gained CP content has improved 50.42%, 51.579%, 50.89%, 51.04%, 49.89% successively than unfermentable soybean hulls under this condition; CF content has reduced by 39.13%, 39.10%, 39.29%, 38.99,39.30% successively than unfermentable soybean hulls; Urase content is zero.Basically identical with the result of case study on implementation 2; Show that complex microbial community of the present invention has good ferment effect for soybean hulls.
The screening method of fermented soybean skin compound strain of the present invention is compared with the double-bacterium ferment method with other single bacterium, and the crude protein of tunning is significantly improved, and robust fibre significantly reduces, and urase content is zero; Greatly increase the nutrition content of soybean hulls, improved safety in utilization and utility value, made it environment-friendly high-efficiency more; Its good product index has been filled up this field blank; Technique can be widely used in the further exploitation of soybean hulls leavened prod product, has widened the extensive utilization in the animal diet, has the wide prospect of marketing.
Claims (4)
1. a multiple and microorganism species, is characterized in that, described complex microbial community includes milk-acid bacteria, bacillus natto, product Ruan candidiasis and penicillium oxalicum.
2. multiple and microorganism species as claimed in claim 1, is characterized in that, described complex microbial community is comprised of milk-acid bacteria, bacillus natto, product Ruan candidiasis and penicillium oxalicum.
3. multiple and microorganism species as claimed in claim 2, is characterized in that the quantity of described milk-acid bacteria, bacillus natto, product Ruan candidiasis and penicillium oxalicum is than being 2:1:2:1.
4. the described multiple and application of microorganism species in the fermented soybean skin of claim 1-3 any one.
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