CN103409352B - 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 fermentative feedstuff of microbe technical field, be specifically related to a kind of fermented soybean skin to prepare the complex microbial community of feed.
Background technology
Soybean hulls is the material of the outer bag quilt 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%, calcium 0.53%, and phosphorus 0.18%, content of lignin is lower than 2.0%.As the byproduct of soybean oil-producing technique, soybean hulls can be peeled by the hot method of fats and oils processing or crush screen two kinds of working method gained.
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 and the increase to dregs of beans requirement, the soybean that China needs import a large amount of every year, 2011 ~ 2012 average annual imported soybeans more than 5,700 ten thousand tons, soyabean processing ability is about 6500 ~ 7,800 ten thousand tons/year, estimates the annual soybean hulls producing 500 ~ 5,760,000 tons.And 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/about kg.Therefore, soybean hulls develops as a kind of new feed resource and has Important Economic value and market outlook.
But due to soybean hulls, it contains the antinutritional factor such as a large amount of urases, trypsin ihhibitor, therefore, certain restriction is received at present on livestock and poultry utilize, only be applied in ox, sheep roughage on a small quantity, major part is just used as fertilizer, not by the development and utilization of its science.Further, the processing of current soybean hulls adopts the methods such as traditional baking and high temperature steaming to eliminate soybean hulls antinutritional factor more, cause prepare product power consumption high, palatability is poor.
Along with the continuous progress of biotechnology, the soybean hulls of current use utilize technology will gradually by novel process, new technology replace, technique, towards energy-conservation of consumption reduction, environmental protection future development more, focuses on the comprehensive optimal control of the quality of product, palatability and Biological resources more.Owing to considering the problems such as environmental protection, energy-conservation and cost, according to microbe fermentation method processed soybeans skin, compared with traditional cooking process, the product protein content produced is high, antinutritional factor content is few, be rich in probiotics, environmental pollution is little, is a kind of working method of more science.
But.Up to the present, systematic research is not carried out for soybean hulls processing and utilization both at home and abroad, soybean hulls fermentation technique is not enough maturation also, possessor's still single or two microorganisms fermentation of pilot production to the greatest extent, but the protein increasing amount of soybean hulls leavened prod is few, cellulose degradation rate is low, feed conversion rate variance, causes technique to carry out large-scale promotion.For this reason, further investigation best complex flora fermented soybean skin technology, is worth improving its nutritional utilization further, expands its range of application in feed, substitutes conventional feed have important economy and social value with the fermented feed of cheapness.
Summary of the invention
The object of this invention is to provide a kind of for fermented soybean skin to prepare the multiple of feed and microorganism species, namely adopt complex microorganism method fermented soybean skin, to fill up the blank of this art at present.
Applicant, by for a long time research, through the screening of single factor experiment, determines that the number of various bacterium in the best strain combination of multiple and microbial method fermented soybean skin, flora contrasts and preparation method.
Of the present inventionly prepare the multiple of feed and microorganism species for fermented soybean skin, include milk-acid bacteria, bacillus natto, product Ruan candidiasis and penicillium oxalicum.
In above-mentioned complex microbial community, the bacterial classification number ratio of milk-acid bacteria, bacillus natto, product Ruan candidiasis and penicillium oxalicum is 2:1:2:1;
The best fermented bacterium combination of optimal screening of the present invention, best bacterial classification ratio, its ferment effect is better than traditional single culture and does not have the composite bacteria of proportioning to ferment, can increase percent protein greatly, reduce fibre content, and can effectively shorten the production cycle, reduce production cost, increase production efficiency, improve product quality, improve product nutritional utilization value, meet nowadays feedstuff industry cheapness and demand for security, the system for soybean hulls utilizes the method for the science more that provides.
Accompanying drawing explanation
Fig. 1: the crude protein of single bacterium solid state fermentation test product and fibre content figure;
Fig. 2: the crude protein of hybrid bacterial strain solid state fermentation test product and fibre content figure;
Fig. 3: 36h thalli growth condition diagram;
Fig. 4: different strain inoculative proportion is to the effect diagram of the content of product crude protein and fiber;
Fig. 5: the different vaccination amount of multiculture is to the effect diagram of tunning crude protein and fibre content;
Fig. 6: different wheat bran ratio is to the effect diagram of crude protein in tunning and fibre content.
Embodiment
The present invention have passed through and long-term determined a kind of strain combination for fermented soybean skin, and its complex ferment bacterial strain is made up of penicillium oxalicum, milk-acid bacteria, bacillus natto and product Ruan candidiasis.
1) character description of bacterial strain is selected:
The penicillium oxalicum selected can produce cellulase and the polygalacturonase of high vigor degraded cellulose; Cellulose degradation in soybean hulls can be sugar by its cellulase, for other growth desired nutritional; The polygalacturonase decomposed is a multienzyme complex system, plant feed (comprising soybean hulls) cell walls mesogloea can be acted on, make cell walls thinning, flanking cell wall disappears, cell walls arrangement is 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 with the semi-lactosi aldehyde radical that α-Isosorbide-5-Nitrae glycosidic link combines in hydrolysis of pectin, produce reducing sugar; Thus improve body to the utilization ratio of the nutritive substances such as fiber, protein, calcium, phosphorus in feed; In addition, the specific absorption of phosphorus increases, and effectively can reduce the excretion of stock-farms phosphorus, reduces environmental pollution, is conducive to protecting ecology.
The bacillus natto selected is the bacterial strain of leavened food level natto, has very strong fermentation capacity to soybean, and has fragrant after fermentation; Bacillus natto has the activity that stronger product amylase, proteolytic enzyme, lipase etc. separate enzyme, also has non-starch polysaccharide enzyme in degrading plant feed simultaneously, as polygalacturonase, dextranase, cellulase etc., promotes animal digesting and assimilating nutritive substance; Bacillus natto can consume a large amount of oxygen, maintains enteron aisle anaerobic environment, suppresses the growth of pathogenic bacterium, maintains the normal eubiosis of enteron aisle; The gemma of bacillus natto can stimulate generation immune response, the immunizing power of enhancing body.
The milk-acid bacteria selected can regulate the palatability of fermented feed; Can form dominant bacteria after milk-acid bacteria enters enteron aisle, thus suppress the breeding of germ, and self can produce multiple antibacterial substance, can suppress or kill pathogenic micro-organism, thus enhancing body immunologic function, increase Abwehrkraft des Koepers, finally improve digestion and the growth performance of animal.
The product Ruan candidiasis selected can rise in value fast in the substratum having sugar, is conventional single-cell protein feed; Yeast is that the length of livestock and poultry animal occupies one of microorganism, is also dominant microflora.In feed, add yeast the quantity of probiotic bacterium to increase in stimulating animal enteron aisle, improve the microbial environment in animal intestinal; Saccharomycetic cell walls is fibrous by special yeast, is mainly made up of mannosans and glucose.Mannosans can strengthen the immunizing power of energy enhancing body; Yeast can suppress the toxication mechanism of aflatoxin, has detoxification to aflatoxin.
The present invention milk-acid bacteria used is lactobacterium acidophilus, its a kind of strain number is ACCC10637, bacillus natto be numbered CGMCC1.1086, produce Ruan's candidiasis be numbered CICC32835.But under invention thinking of the present invention, other bacterial strain can be selected.
For penicillium oxalicum bacterial classification, this bacterium to be separated by high-quality aquatic bird institute of Qingdao Agricultural University and to obtain from goose enteron aisle, have the very high degradation capability of Mierocrystalline cellulose, identify through Institute of Microorganism, Academia Sinica 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 use can maximum raising fermented soybean skin protein content 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 increase fermented feed nutrition content, improve the prebiotic effect in hazardous and noxious substances and good animal productiong in animal palatability and nutrient utilization, soybean hulls of degrading.
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 successively, dipotassium hydrogen phosphate 1.0g, yeast extract powder 2.0g, magnesium sulfate 0.5g, tepor is dissolved, regulate pH value to be about 6.8, boil, after adding glucose 20.0g dissolving, shake up, gauze filtering, is 6.4 ± 0.2 after regulating pH value to make sterilizing, is divided in in each triangular flask, add cotton plug, use pressure kettle sterilizing.Culture temperature is 30 DEG C, and incubation time is 18h ~ 24h, cultivates after terminating, and takes out to be placed in the refrigerator of 4 DEG C and preserve from shaking table.
Milk-acid bacteria amplification culture medium compound method: adding distil water 1000ml in beaker, add peptone 10.0g successively, beef leaching thing (extractum carnis) 10.0g, 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, are divided in in each triangular flask, add cotton plug, use pressure kettle sterilizing.After inoculation, culture temperature is 42 DEG C, amphimicrobian, and culture temperature is 18h ~ 20h, cultivates after terminating, and takes out to be placed in the refrigerator of 4 DEG C and preserve from incubator.
Bacillus natto amplification culture medium compound method: adding distil water 1000ml in beaker, adds sucrose 10g, soy peptone 10g, NaCl5g successively, is placed on the upper heating of fire.Until completely dissolved, with 10% hydrochloric acid or 10% sodium hydroxide adjusted to ph to 7.2 ~ 7.6, be divided in in each triangular flask, add cotton plug, use pressure kettle sterilizing.After inoculation, culture temperature is 37 DEG C, and incubation time is 18h ~ 24h, cultivates after terminating, and takes out to be placed in the refrigerator of 4 DEG C and preserve from shaking table.
Produce Ruan's candidiasis amplification culture medium compound method: get 250g kilned malt, ground by kilned malt, granularity is 100 orders, puts into large beaker, adds 1000ml distilled water, saccharification 3h ~ 4h in 64 DEG C ~ 66 DEG C water-baths.By saccharified liquid 4 ~ 6 layers of filtered through gauze, if filtrate is muddy, clarify with egg albumen.Method is, is taken out by the albumen in an egg and to add water 20ml, mix well till being stirred to raw foam with glass stick, then pours in saccharified liquid after stirring and boiling, by 8 layers of filtered through gauze.Filtrate regulates PH to be 7.0 ~ 7.2, and pol saccharometer is measured and adding distil water is diluted to 5 ° of Be, is divided in in each triangular flask, adds cotton plug, use pressure kettle sterilizing.Culture temperature is 25 DEG C ~ 28 DEG C, and incubation time is 24h, cultivates after terminating, and takes out to be placed in the refrigerator of 4 DEG C and preserve from shaking table.
3) preparation of soybean hulls fermention medium: purchase soybean hulls and wheat bran pulverizer are pulverized, granularity 40 order.Each substratum takes soybean hulls 45g, wheat bran 5g, adds the distilled water of 12.5ml, adds cotton plug, uses pressure kettle sterilizing.
4) substrate is inoculated: by the soybean hulls fermention medium of bacterium of having gone out, be placed on Bechtop, open ultra violet lamp 25min ~ 30min sterilizing.Spirit lamp is lighted after sterilizing terminates, it is 10ml that the bacterium liquid that increased is inoculated into 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, be put into by substratum in incubator, culture temperature is 30 DEG C, and incubation time is 72h.
Cultivation terminates rear substratum to be taken out, tunning is put and dries in an oven, bake out temperature is 35 DEG C, measures the content of CP (crude protein), NDF (neutral detergent fiber), ADF (acid detergent fiber) and CF (robust fibre) after oven dry.
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 pulverizer are pulverized, granularity is 40 orders, be 9:1 in the ratio of soybean hulls and wheat bran, material-water ratio is that 3:1 is mixed to join in 250ml triangular flask, soybean hulls 45g is added, wheat bran 5g, distilled water 12.5ml in each triangular flask, add cotton plug, with pressure kettle 121 DEG C of sterilizing 30min.
1. single strain fermentation test
The penicillium oxalicum increased, milk-acid bacteria, bacillus natto, product Ruan candidiasis bacterium liquid are diluted to 1 × 10 with sterilized water respectively
7individual/ml, is inoculated on fermention medium, each substratum is inoculated respectively the bacterium liquid of 5ml.Add cotton plug after having inoculated, triangle bottleneck kraft paper encases, and is placed in the constant incubator of 30 DEG C and cultivates, and incubation time is 72h.
Test-results shows, four selected 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, improves 8.78% than unfermentable soybean hulls; And robust fibre penicillium oxalicum reduces at most, reduce 33.11% than unfermentable soybean hulls; And urease content is zero, illustrate that four bacterial classifications are all applicable to fermented soybean skin.See accompanying drawing 1.
2. hybrid bacterial strain fermentation test
Two for four bacterial strains bacterium mixing and the mixing of many bacterium are accessed in solid-state fermentation culture medium respectively, 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 penicillium oxalicum, 2 represent milk-acid bacteria, 3 represent bacillus natto, 4 representative product Ruan yeast).The bacterium liquid each substratum being inoculated respectively 5ml carries out mixed fermentation (example: if double-bacterium ferment and individual bacterial classification add bacterium liquid 2.5ml, other the like).Add cotton plug after having inoculated, triangle bottleneck kraft paper encases, and is placed in the constant incubator of 30 DEG C 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 compared with other group content, and improve 32.98% than unfermentable soybean hulls, robust fibre is compared and reduced 41.49%, and urease content is zero.And growth rapidly, can see that after cultivation 36 ~ 48h mycelia covers matrix.See accompanying drawing 2 and accompanying drawing 3.
3. best strain inoculation ratio test
Accessed respectively in solid-state fermentation culture medium in different ratios by four bacterial classifications, 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 (being followed successively by penicillium oxalicum: milk-acid bacteria: bacillus natto: produce Ruan's yeast).The bacterium liquid each substratum being inoculated respectively 5ml carries out mixed fermentation (example: if strain inoculation ratio is 1:1:1:1, and the bacterium liquid addition of 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 is all diluted to 1 × 10 with sterilized water
7individual/ml.Add cotton plug after having inoculated, triangle bottleneck kraft paper encases, and is placed in the constant incubator of 30 DEG C and cultivates, and incubation time is 72h.
Test-results shows, when the inoculative proportion of bacterial classification is 2:1:2:1, ferment effect is best, and CP content is the highest compared with other group content, and improve 50.44% than unfermentable soybean hulls, robust fibre reduces 39.51%, and urease content is zero.See accompanying drawing 4.
4. optimum inoculation amount test
By four bacterial classifications 2:1:2:1 (penicillium oxalicum × 2: milk-acid bacteria × 1: bacillus natto × 2: produce Ruan yeast × 1, bacterial classification quantity is 2 × 10 in proportion
7: 1 × 10
7: 2 × 10
7: 1 × 10
7) be linked in solid-state fermentation culture medium with the inoculum size of 2%, 5%, 10%, 15%, 20% respectively.Add cotton plug after having inoculated, triangle bottleneck kraft paper encases, and is placed in the constant incubator of 30 DEG C and cultivates, and incubation time is 72h.
Test-results shows, when the inoculum size of bacterial classification is 10%, ferment effect is best, and CP content is the highest compared with other group content, and improve 25.87% than unfermentable soybean hulls, robust fibre reduces 41.49%.See accompanying drawing 5.
Embodiment 2: soybean hulls fermention medium auxiliary material addition is tested
In order to make bacterial classification better grow, in substratum, add a certain amount of wheat bran.Containing abundant VITAMIN and mineral substance in wheat bran, the nutrition of fermented substrate can be regulated.It or one well raising agent, also can be used as supplemental nitrogen source simultaneously.
Four bacterial classifications are fitted in bacterium liquid by optimum proportion, and the inoculum size by 5% is inoculated into different cooperation in 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%.Add cotton plug after having inoculated, triangle bottleneck kraft paper encases, and is placed in the constant incubator of 30 DEG C 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 compared with other group content when the addition of 10%, and improve 25.87% than unfermentable mixed substrates, robust fibre reduces 35.15%.See accompanying drawing 6.
Embodiment 3: compound strain fermented soybean skin Orthogonal Rotational Regressive Tests
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 is all 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%, and found by 5 proof tests, gained CP content improves 50.42%, 51.579%, 50.89%, 51.04%, 49.89% successively than unfermentable soybean hulls under this condition; CF content in turn reduces 39.13%, 39.10%, 39.29%, 38.99,39.30% than unfermentable soybean hulls; Urease 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 double-bacterium ferment method with other single bacterium, and the crude protein of tunning can be made to significantly improve, and robust fibre significantly reduces, and urease content is zero; Considerably increase the nutrition content of soybean hulls, improve safety in utilization and utility value, make it environment-friendly high-efficiency more; It is blank that its good product index has filled up this field; Technique can be widely used in the further exploitation of soybean hulls leavened prod product, has widened the extensive utilization in animal diet, has had the wide prospect of marketing.
Claims (2)
1. for a complex microbial community for fermented soybean skin, it is characterized in that, described complex microbial community is made up of milk-acid bacteria, bacillus natto, product Ruan candidiasis and penicillium oxalicum; Wherein milk-acid bacteria is lactobacterium acidophilus, and it is numbered ACCC10637; Bacillus natto be numbered CGMCC1.1086, produce Ruan candidiasis be numbered CICC32835; Penicillium oxalicum deposit number is CGMCC No.2260; And the number ratio of penicillium oxalicum, milk-acid bacteria, bacillus natto and product Ruan candidiasis is 2:1:2:1.
2. the application of complex microbial community according to claim 1 in fermented soybean skin.
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Inventor after: Wang Baowei Inventor after: Zhang Kailei Inventor after: Ge Wenhua Inventor after: Zhang Mingai Inventor after: Yue Bin Inventor after: Xu Chenchen Inventor before: Wang Baowei Inventor before: Zhang Kailei Inventor before: Ge Wenhua Inventor before: Zhang Mingai Inventor before: Yue Bin Inventor before: Xu Chenchen |
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