CN103275875A - Trichoderma koningii, and compound microbial agent composition and application thereof - Google Patents
Trichoderma koningii, and compound microbial agent composition and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microbes, and specifically relates to trichoderma koningii, and a compound microbial agent composition and an application thereof. The invention discloses trichoderma koningii HK43-5 (CGMCC No7422), a compound microbial agent composition composed of a glycosylated bacterial preparation and a forage yeast preparation which are packaged separately, wherein the glycosylated bacterial preparation comprises the trichoderma koningii HK43-5 (CGMCC No7422). Cellulose produced by the trichoderma koningii HK43-5 (CGMCC No7422) has high activity, can be used as an efficient glycosylated bacterial preparation in production of industrial and agricultural residual protein forage. The compound microbial agent composition can efficiently utilize industrial and agricultural residues, particularly potato residues, to produce single cell protein. Crude protein content in fermented mycoprotein is 50.49% which is far superior to that reported in a conventional technology. The industrial and agricultural residues are changed to wealth; and the produced bacterial protein can be applied in the forage to partly substitute soybean meal or fish meal. At the same time, the environment pollution problems resulted from the industrial and agricultural residues are solved.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to a kind of healthy and free from worry wood mould and complex micro organism fungicide combination and purposes.
Background technology
Animal feeding-stuff containing somatic protein is a kind of protein raw materials of biological fermentation, is to utilize various matrix large scale culturing bacteriums, yeast, mould, algae and basidiomycetes and the microbial proteinous (or tropina) that obtains.Tropina claims microbial proteinous or single cell protein (single cell protein is called for short SCP) again, is the key protein source of foodstuffs industry and fodder industry.
The raw material type of manufacture order cell protein is a lot, is divided into 3 classes substantially, (1) industrial effluent class: comprise papermaking wastewater, alcohol slops, monosodium glutamate waste liquid, starch waste liquor, production citric acid waste liquid, molasses waste liquid, wood saccharification waste liquid, bean product waste liquid etc.; (2) industrial or agricultural distillers grains and dregs: comprise that distillers ' grains, brewer's grains, fruit wine slag, vinegar are poor, soy sauce is poor, bean dregs, granulated slag, gluten feed, potato slag, beet pulp, bagasse, pomace, maltose residues etc.; (3) chemical products: comprise oil, paraffin, diesel oil, Sweet natural gas, normal alkane, methyl alcohol, ethanol, acetic acid etc.Except above introduce, agricultural crop straw, batch shell, puffed soybean, feces of livestock and poultry, organic waste, weathered coal etc. also can be used as the raw material production single cell protein.
Be example with the potato residues, potato residues is polysaccharide material such as moisture, pectin, robust fibre mainly, protein content only between 1%～4%, starch content＜1%, nutritive value is lower; Bright potato pulp water divides content up to more than 80%, is not easy to drying and transportation, produces season as if untimely processing, and occupied ground and easily corrupt rotten causes environmental pollution.Potato residues by microbial fermentation, is generated single-cell protein feed, is the main path of potato residues bio-transformation, but the yield of crude protein is on the low side in the fermented feed of existing research report unit weight, can't significantly promote all the time.
Summary of the invention
Technical problem to be solved by this invention provides a kind of healthy and free from worry wood mould and complex micro organism fungicide combination and purposes, solves the yield defective on the low side of tunning crude protein in the prior art.
Above-mentioned technical problem for solving the invention discloses a kind of healthy and free from worry wood mould (Trichoderma koningii) HK43-5, preserving number CGMCC No7422.
The mould HK43-5 of healthy and free from worry wood of the present invention screens acquisition by the healthy and free from worry wood of parental plant mould 40144 behind space flight, in on April 7th, 2013 in China Committee for Culture Collection of Microorganisms common micro-organisms center (China, Beijing) preservation, preserving number CGMCC No7422.
On the other hand, disclosed herein as well is the mould HK43-5 of above-mentioned healthy and free from worry wood as the purposes of saccharification microbial inoculum.
On the other hand, disclosed herein as well is a kind of complex micro organism fungicide combination, it is made up of amylomyces preparation and fodder yeast bacteria preparation, and the two is packing separately, it is characterized in that described amylomyces preparation comprises the mould HK43-5(CGMCC No7422 of healthy and free from worry wood).
In certain embodiments, described amylomyces preparation contains the mould HK43-5 of healthy and free from worry wood and the Aspergillus awamori of mass ratio 1:2-2:1, and wherein preferred described amylomyces preparation is made up of the mould HK43-5 of healthy and free from worry wood and the Aspergillus awamori of mass ratio 1:1.The present invention utilizes Aspergillus awamori can produce polygalacturonase, to be conducive to the saccharification in the fermenting process.
In certain embodiments, described fodder yeast bacteria preparation is candida tropicalis (Candida tropicalis) or Candida utilis (Candida utilis) bacterium, wherein is preferably candida tropicalis (Candida tropicalis) 1779.
In certain embodiments, described fodder yeast bacteria preparation and described amylomyces preparation also comprise the skimming milk 10% of mass percent, trehalose 5%, vitamins C 5%.
On the other hand, the invention also discloses the purposes of described complex micro organism fungicide in the poor residue protein feed of preparation industrial or agricultural.
In certain embodiments, the poor slag of described industrial or agricultural is one of in bean dregs, granulated slag, gluten feed, potato slag, beet pulp, bagasse, pomace or the maltose residues, wherein is preferably potato residues.
In certain embodiments, described purposes comprises the following steps:
A) potato residues and wheat bran add the inorganic nitrogen-sourced of 2-4% with weight part ratio 6:4-9:1 mixing, regulate pH value 3.0-5.0, prepare the fermentation raw material that water content is 60%-80%;
B) amylomyces preparation in the combination of described complex micro organism fungicide is added mixing in the fermentation raw material, the add-on 0.1%-1% that is weight percentage, 30 ℃ of fermentation 12-21h obtain the bed material that ferments;
C) then with mixing in the fodder yeast bacteria preparation inoculation fermentation bed material in the combination of described complex micro organism fungicide, the add-on 0.5-1.5% that is weight percentage continues 30 ℃ and ferments to 90-150h.
The mould HK43-5(CGMCC No.7422 of healthy and free from worry wood of the present invention) the cellulase activity height of Chan Shenging can be used as the efficiently saccharifying microbial inoculum in the poor residue protein fodder production of industrial or agricultural; Described complex micro organism fungicide combination can efficiently utilize the poor slag of industrial or agricultural to generate single cell protein, especially potato residues, and fermentation back tropina crude protein content is 50.49%, is much better than the prior art report.The present invention is not only turned waste into wealth the poor slag of industrial or agricultural, and the tropina that generates can be used for feed, and part substitutes dregs of beans or fish meal, has solved the poor slag of industrial or agricultural simultaneously and has caused problem of environmental pollution.
Description of drawings
The mould HK43-5 cellulase-producing of the healthy and free from worry wood of Fig. 1 is active to be detected.
Fig. 2 different microorganisms preparation combined fermentation potato residues generates crude protein content relatively.
Fig. 3 complex micro organism fungicide is formed fermentation test.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition or the condition of advising according to manufacturer.Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Test materials
Bacterial classification: healthy and free from worry wood is mould 40144, Aspergillus awamori 40499, and candida tropicalis 1779 is available from purchasing in Chinese industrial microbial strains preservation center (CICC); Mutagenic strain: healthy and free from worry wood mould (Trichoderma koningii) HK43-5(CGMCC No7422).
Material: potato residues: from the refined starch factory of Qinan County instrument, dry, pulverize.Wheat bran skin: commercially available.
Reagent: malt extract, copper sulfate, vitriolate of tartar, ammonium sulfate, sodium hydroxide, boric acid, mixture indicator: methyl red 0.1% ethanolic soln, tetrabromo-mcresolsulfonphthalein 0.5% ethanolic soln, two solution equal-volumes mix.Above reagent is analytical pure.Sulfuric acid, hydrochloric acid, chemical pure.
Test equipment: the sea can full-automatic Kjeldahl determination device K9860, electro-heating standing-temperature cultivator lands constant temperature oscillator.
The crude protein measuring method: Kjeldahl determination, utilize full-automatic Kjeldahl determination device to measure.
1.1 test materials
Raw material: potato residues (the refined starch factory of Qinan County instrument), Testa Tritici (commercially available)
Bacterial classification: healthy and free from worry wood mould (Trichoderma koningii) 40144(purchases in Chinese industrial microbial strains preservation administrative center, and CICC), mutagenic strain: the mould HK43-5(of healthy and free from worry wood screens acquisition by healthy and free from worry wood mould 40144 behind space flight).
Seed culture medium: malt extract 10g, tap water 1000ml, natural pH.
Potato residues solid fermentation substratum: potato residues/wheat bran=9/1(W/W), moisture content 83%, pH nature.
1.2 test method:
1. fermentation test
The mould spore concentration of healthy and free from worry wood that will be in logarithmic phase is adjusted to 108/ml, is inoculated on the solid-state fermentation culture medium according to identical inoculum size, measures cellulase activity variation in the substratum behind the fermentation 72h.
2. leach fermented liquid
Get the bent about 5g of solid state fermentation, add distilled water 50ml, place on 50 ℃ of shaking baths, behind the 150r/min lixiviate 1h, leave standstill a moment, get supernatant, the centrifugal 15min of 10000/min removes spore, measures cellulose enzyme activity in the supernatant.
3. cellulase activity is measured
The cellulase activity unit definition: the 1ml liquid enzymes, at (50 ± 0.1) ℃, pH4.8,1h hydrolyzed carboxymethylcellulo, e sodium substrate produces the reducing sugar amount that is equivalent to 1mg glucose, is 1 enzyme activity unit, represents with u/ml.Be abbreviated as CMCA-DNS.
The CMCA-DNS enzyme activity calculates
X1=A×1/0.5×n×2
In the formula:
The X1---carboxymethylcelluloenzyme enzyme activity, u/ml;
The reducing sugar amount that the A---absorbancy checks at typical curve, mg;
1/0.5---be converted into enzyme liquid 1ml;
The extension rate of n---enzyme sample, (50/ leaven quality);
The 2---time scale factor.
1.3 test-results
Shown in, utilize the mould HK43-5 of healthy and free from worry wood in potato residues solid fermentation substratum, to ferment behind the 72h, the CMC enzyme activity reaches 4.1U/ml, compares with the yield of enzyme 2.1U/ml of contrast (healthy and free from worry wood mould 40144, be called for short CK), and enzymatic productivity has improved nearly one times.Therefore the mould HK43-5 of healthy and free from worry wood can be used as the efficiently saccharifying microbial inoculum in the poor residue protein fodder production of industrial or agricultural.
2.1 amylomyces preparation
Respectively with the mould HK43-5 of healthy and free from worry wood and Aspergillus awamori 40499 inoculation in 1% malt extract substratum; after 200r/min, 30 ℃ of shaking tables are cultivated 60h; the centrifugal 10min of 4000r/min; remove supernatant, obtain the weight in wet base thalline, the two is according to following table 1(mass ratio then) mix; namely obtain the amylomyces preparation; also can add protective material, mix the back freeze-drying, packing.Described protective material can be, and one is the skimming milk 10% that adds mass ratio, glycerine 5%; Another is the skimming milk 10% that adds mass percent, trehalose 5%, vitamins C 5%.
Table 1
| ? | |
|
Prescription 3 |
| The mould HK43-5 of healthy and free from |
2 | 1 | 1 |
| Aspergillus awamori 40499 | 1 | 1 | 2 |
2.2 the yeast preparation,
Candida tropicalis 1779 inoculation in 1% malt extract substratum, after 200r/min, 30 ℃ of shaking tables are cultivated 60h, the centrifugal 10min of 4000r/min; remove supernatant, obtain the weight in wet base thalline, namely obtain the amylomyces preparation; also can add protective material, mix the back freeze-drying, packing.Described protective material can be, and one is the skimming milk 10% that adds mass ratio, glycerine 5%; Another is the skimming milk 10% that adds mass ratio, trehalose 5%, vitamins C 5%.
Embodiment 3, complex micro organism fungicide fermentation
3.1 fermentation condition optimization
According to the level of each definite factor of single factor experiment, adopt Plackett-Burman design screening from 8 influence factors to have the factor of remarkably influenced.Each factor is got 2 levels: i.e. high-level (+1) and low-level (1).As shown in table 2.
Table 2.Plackett-Burman designs each level of factor and span
Result and analysis
1.Plackett-Burman design result such as table 3.
Table 3.Plackett-Burman design result
Above result is carried out variance analysis, the results are shown in Table 4.
Table 4.Plackett-Burman design result is analyzed
Annotate: * representative influence is remarkable, and * * representative influence is extremely remarkable
According to Principle of Statistics, significance level reaches more than 95%, thinks that then this factor is remarkable to the influence of response value.As shown in Table 4, the overall significance level of model is＜0.0001, show that model is extremely remarkable, it is B, C, D, G, H that response value is influenced bigger factor, be respectively urea, water content, pH value, candiyeast inoculation time and saccharifying enzyme inoculum size, wherein urea, water content, ph value and saccharifying enzyme inoculum size reach utmost point conspicuous level to the influence of protein content in the fermention medium.
The equation of linear regression of this model is
R=+29.83+0.035*A+6.16*B+3.61*C-2.39*D+0.51*E+0.26*F+1.46*G-2.56*H
In the formula, R represents the encoded radio of crude protein content, and A, B, C, D, E, F, G, H represent the encoded radio of wheat bran ratio, urea, water content, initial pH, saccharification bacterial classification ratio, candiyeast inoculum size, candiyeast inoculation time, saccharifying enzyme inoculum size respectively.
Accordingly, equation of linear regression can be provided by the actual value of each factor:
y=+7.15038+0.027824x
1+6.15992x
2+0.36130x
3-2.39130x
4+0.67753x
5+0.52338x
6+0.48703x
7-5.11124x
8
In the formula, y represents the actual value of crude protein content, x
1, x
2, x
3, x
4, x
5, x
6, x
7, x
8Represent the actual value of wheat bran ratio, urea, water content, initial pH, amylomyces bacterial classification ratio, candiyeast inoculum size, candiyeast inoculation time, amylomyces inoculum size respectively.
The influence factor that is obtained by model is urea, water content, initial pH value, candiyeast inoculation time and amylomyces inoculum size, wherein urea content necessarily is directly proportional with crude protein content, can not be as influence factor, water content can be calculated by model, and therefore the attention influence factor that finally obtains is pH value, candiyeast inoculation time and amylomyces inoculum size.The theoretical maximum crude protein content that obtains of model prediction is 52.2498% thus, and this moment, the value of each major influence factors was respectively: pH3.0, candiyeast inoculation time are that 21h, amylomyces inoculum size are 0.1%.
3.2 proof test
For the validity of verification model, 6 group model confirmatory experiments have been carried out.The proof test fermentation condition is: wheat bran is than (potato residues: wheat bran) for 7:3, urea content are 3%, water content is 60%, bacterial classification is than (healthy and free from worry wood is mould: Aspergillus awamori) for 1:1, candiyeast inoculum size are 1%, pH3.00, candiyeast inoculation time are that 21h, amylomyces inoculum size are 0.1%, 30 ℃ of fermentation 120h.The results are shown in Table 5: maximum crude protein content predictor is close with measured value, and average relative error is 3.23%, proves that the potato residues tropina of this explained hereafter has circulation ratio preferably.
Table 5 potato residues tropina fermentation model predication value and experimental value
Embodiment 4, the fermentation of initial mixing bacterium and mutagenesis mixed fermentation test contrast
Composite fungus agent 1: mould 40144, the Aspergillus awamori 40499 of healthy and free from worry wood.
Composite fungus agent 2: the mould HK43-5 of healthy and free from worry wood; Aspergillus awamori 40499.
Yeast microbial inoculum: candida tropicalis 1779.
Material: potato residues: from the refined starch factory of Qinan County instrument, dry, pulverize.Wheat bran skin: commercially available.
Reagent: copper sulfate, vitriolate of tartar, ammonium sulfate, sodium hydroxide, boric acid, mixture indicator: methyl red 0.1% ethanolic soln, tetrabromo-mcresolsulfonphthalein 0.5% ethanolic soln, two solution equal-volumes mix.Above reagent is analytical pure.Sulfuric acid, hydrochloric acid, chemical pure.
Test equipment: the sea can full-automatic Kjeldahl determination device K9860, electro-heating standing-temperature cultivator lands constant temperature oscillator.
Test method
Zymotechnique: the zymotechnique of two kinds of microbial inoculums all is optimized definite by single factor experiment, Plackett-Burman test and response surface test.
Fermention medium is formed: potato residues: wheat bran is 7:3; Add urea content 3.6%; Water content 63%; Regulate medium pH to 7.0.
Fermentation condition: the amylomyces bacterial classification is than (healthy and free from worry wood is mould: be the 1%(mass percent for 1:1, inoculum size Aspergillus awamori)), 30 ℃ cultivate 12h after, insert the 1%(mass percent) candiyeast, 30 ℃ ferment to 120h.
Fermention medium is formed: potato residues: wheat bran is 7:3, add that urea content is 3%, water content is 60%, regulates medium pH to 3.0.
Fermentation condition: the amylomyces bacterial classification is than (healthy and free from worry wood is mould: be the 0.1%(mass percent for 1:1, inoculum size Aspergillus awamori)), and 30 ℃ are inserted the 1%(mass percent after cultivating 21h) candiyeast, 30 ℃ ferment to 120h.
The test of complex micro organism fungicide proportion of composing:
Healthy and free from worry wood is mould in the change composite fungus agent 2 carries out fermentation test with mass percent Aspergillus awamori, and both ratios are got 1:2,1:1,2:1 respectively, and fermentation result and composite fungus agent 1 fermentation result compare.
The crude protein measuring method: Kjeldahl determination, utilize full-automatic Kjeldahl determination device to measure.
Test-results:
As shown in Figure 2, use composite fungus agent 1, be healthy and free from worry wood mould 40414 and Aspergillus awamori 40499 composite fungus agents and candida tropicalis 1779 microbial inoculums under the zymotechnique of optimizing, crude protein content reaches maximum value 22.52% at 120h in the potato residues animal feeding-stuff containing somatic protein of fermentative production.Use composite fungus agent 2, namely the mould HK43-5 of healthy and free from worry wood and Aspergillus awamori 40499 composite fungus agents and candida tropicalis 1779 microbial inoculums are under the zymotechnique of optimizing, and crude protein content reaches maximum value 49.18% at 96h in the potato residues animal feeding-stuff containing somatic protein of fermentative production.Therefore use composite fungus agent 2 and the asynchronous zymotechnique of yeast microbial inoculum, can efficiently utilize potato residues to generate single-cell protein feed, crude protein content significantly improves in the animal feeding-stuff containing somatic protein of gained, and saccharification microbial inoculum usage quantity is few, fermentation period is short, only needs degraded and the conversion that can finish potato residues in 96 hours.
As shown in Figure 3, use and to be undertaken by the mould composite fungus agent of forming with Aspergillus awamori of the healthy and free from worry wood of different ratios that crude protein content does not have significant difference in the animal feeding-stuff containing somatic protein of fermentation test gained, but compare significant difference with composite fungus agent 1 result of fermenting.
Scope of the present invention is not subjected to the restriction of described specific embodiments, and described embodiment also comprises method and the component of functional equivalent only as the single example of illustrating all respects of the present invention in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to above description and accompanying drawing.Described improvement also falls within the scope of appended claims.Every piece of reference mentioned above is listed this paper in as a reference all in full.
Claims (10)
1. mould HK43-5(CGMCC No7422 of healthy and free from worry wood).
2. the mould HK43-5(CGMCC No7422 of the described healthy and free from worry wood of claim 1) as the purposes of saccharification microbial inoculum.
3. complex micro organism fungicide combination, it is made up of amylomyces preparation and fodder yeast bacteria preparation, the two is packing separately, it is characterized in that described amylomyces preparation comprises the mould HK43-5(CGMCC No7422 of healthy and free from worry wood).
4. as complex micro organism fungicide combination as described in the claim 3, it is characterized in that described amylomyces preparation comprises the mould HK43-5(CGMCC No7422 of healthy and free from worry wood of mass ratio 2:1-1:2) and Aspergillus awamori.
5. as complex micro organism fungicide combination as described in the claim 3, it is characterized in that described amylomyces preparation comprises the mould HK43-5(CGMCC No7422 of healthy and free from worry wood of mass ratio 1:1) and Aspergillus awamori.
6. as complex micro organism fungicide combination as described in the claim 3, it is characterized in that described fodder yeast bacteria preparation is candida tropicalis or Candida utilis bacterium.
7. complex micro organism fungicide combination according to claim 1 is characterized in that described fodder yeast bacteria preparation is candida tropicalis.
8. each described complex micro organism fungicide is combined in the purposes for preparing in the poor residue protein feed of industrial or agricultural among the claim 3-7.
9. purposes as claimed in claim 8 is characterized in that the poor slag of described industrial or agricultural is one of in bean dregs, granulated slag, gluten feed, potato slag, beet pulp, bagasse, pomace or the maltose residues.
10. purposes as claimed in claim 8 is characterized in that it comprises the following step:
A) potato residues and wheat bran add the inorganic nitrogen-sourced of 2-4% with weight part ratio 6:4-9:1 mixing, regulate pH value 3.0-5.0, prepare the fermentation raw material that water content is 60%-80%;
B) amylomyces preparation in the combination of described complex micro organism fungicide is added mixing in the fermentation raw material, the add-on 0.1%-1% that is weight percentage, 30 ℃ of fermentation 12-21h obtain the bed material that ferments;
C) then with mixing in the fodder yeast bacteria preparation inoculation fermentation bed material in the combination of described complex micro organism fungicide, the add-on 0.5-1.5% that is weight percentage continues 30 ℃ and ferments to 90-150h.
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| CN106047954B (en) * | 2016-07-29 | 2020-01-07 | 河南金丹乳酸科技股份有限公司 | Method for producing lactic acid and co-producing protein feed through circulating fermentation |
| CN108157588A (en) * | 2017-12-23 | 2018-06-15 | 董晓 | A kind of preparation method of protein feed |
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| CN111777437B (en) * | 2020-07-13 | 2022-07-05 | 广西壮族自治区农业科学院 | Trichoderma asperellum bacterial fertilizer and preparation method thereof |
| CN114015578B (en) * | 2020-11-24 | 2023-06-16 | 南昌师范学院 | Trichoderma koningii and fermentation product thereof, composition containing trichoderma koningii and application of trichoderma koningii and fermentation product |
| CN113678944A (en) * | 2021-07-30 | 2021-11-23 | 西吉县万里淀粉有限公司 | Production process for producing cattle protein feed by using potato powder residues |
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