CN103444988B - Aspergillus oryzae degrading straw produces the method for protein feed - Google Patents
Aspergillus oryzae degrading straw produces the method for protein feed Download PDFInfo
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Abstract
Aspergillus oryzae degrading straw of the present invention produces the method for protein feed, relates to bioengineering field.Relate to and utilize stalk for primary raw material, fermented by aspergillus oryzae (Aspergillus oryzae), produce the method for protein feed, its technological process of production is that stalk is through pretreating process such as pulverizing, prewet, aspergillus oryzae is cultivated through liquid submerged culture, first order seed, is mixed carry out solid state fermentation with stalk, obtains the protein feed of fermented stalk.This zymotechnique is easy, to environment, also can not can not cause waste to resource.Adopt the dietary protein level 15 ~ 30% that the method is obtained, Lignin degradation rate is 34 ~ 76%, feed yield 45 ~ 60%.Feed, due to effect lignin degrading, the cellulose of aspergillus oryzae, thus improves the utilization rate of stalk.
Description
Technical field
The present invention relates to bioengineering field, refer in particular to stalk without high temperature, HIGH PRESSURE TREATMENT by the lignin in aspergillus oryzae degrading straw, cellulose, produce the technological process of protein feed.
Background technology
China is large agricultural country, and annual agricultural crop straw is produced per year and exceeded 600,000,000 tons, and except being used as weaving, paper industry on a small quantity, but major part is with the form impouring environment piled up, burn, and has both wasted resource and has also caused to environment and greatly endanger.
Stalk contains the energy over half that photosynthesis of plant accumulates, be rich in the composition that raw sugar, thick protein etc. are conducive to needed for domestic animals and fowls, as noncompetitive feed resource, as long as can rational Processing farage be carried out, be used for livestock and poultry of feeding, particularly be used for the ruminant domestic animal such as ox, sheep of feeding, likely become the feed resource of high-quality.Meanwhile, China is again the big country of feed (especially protein feeds) critical shortage, needs a large amount of fish meal of import to make up the deficiency of protein feeds every year.Stalk fibre can be converted into animal can utilize and the protein feeds be of high nutritive value, to solve the wretched insufficiency of feed demand, just seem particularly important.
The stalk of plant mainly contains lignin and cellulose is formed.Lignin is the natural polymer with aromatic character that a class is formed by connecting by ehter bond and carbon-carbon bond etc. by phenol and non-phenol construction unit, its diversity in constituent species and connecting key type, mean heterogeneity and the scrambling of its structure, also determine and biodegradable complexity and particularity are carried out to lignin, and after it is combined with covalent bond form with hemicellulose in plant cell wall, cellulosic molecule is embedded in wherein, form the firm natural cover for defense, general microorganism is made to be difficult to enter wherein decomposition of cellulose, therefore, can the degraded of lignin be also the key factor that effectively utilize stalk resource.
Representationally in the main method of current degrading straw comprise 5 kinds of methods such as steam-explosion pretreatment, sulfur acid pretreatment, NaOH pretreatment, pretreatment with agueous Ammonia and Biological Pretreatment.Steam-explosion is then inner with high steam infiltrated fiber, discharge from the space closed in the mode of air-flow, there is certain mechanical breaking in fiber, make most of hemicellulose and a small amount of lignin in raw material, cellulose degradation stripping, the method composition is higher, need large-scale equipment, lignin also needs process; Acidic treatment utilizes sulphuric acid hydrolysis glycosidic bond, ester bond etc., and the network structure of corn stalk fiber is loosened, but the method consumes a large amount of organic acids or inorganic acid, lignin still needs further process, is not suitable with large-scale industrial production.Alkali treatment and WITH AMMONIA TREATMENT utilize lignin to be dissolved in alkaline solution, and the same with acid treatment exist problem of environmental pollution equally.This due to a little method generally all there is the more difficult grasp of reaction condition, reaction temperature is high, the processing time is long, it is large to take up room, treatment effect is undesirable, can not remove the problems such as lignocellulosic.
Aspergillus oryzae can secrete multiple enzyme as protease, amylase, lipase, pectase, phytase, carbohydrase etc., and therefore the application of this bacterium in modern industry is constantly expanded.The people such as Tejomyee S. B. are obtained aspergillus oryzae from being separated Azodrin contaminated soil, and study its biodegradation to agricultural chemicals Azodrin, result of study shows the high efficiency that aspergillus oryzae is degraded to Azodrin in the short period.The people such as Truong Q.T. have studied situation about to contain with aspergillus oryzae process in the tapioca processing waste water of high-density suspended buoyancy aid, by change multiple affect aspergillus oryzae growth because usually improving its degradation efficiency to waste water, its degradation rate can up to 90%.The people such as Xiang J.M. are separated the aspergillus oryzae obtaining nicotine of degrading from tobacco leaf, and the resting cell assays of research and utilization aspergillus oryzae is to the degradation pathway of nicotine.By the analysis to degradation process intermediate product, finally determine the new way utilizing fungus degrading nicotine.The people such as Parshetti G.K. have studied the biodegradation of aspergillus oryzae NCIM-1146 to reactive blue-25.Research finds that the decolouring of reactive blue is then just degraded by the absorption of fungi, has absorb faster and decolour under shaking table condition of culture than quiescent culture.The degraded of aspergillus oryzae to reactive blue is improve after adding glucose.Aspergillus oryzae has the ability of degraded compared with hardly degraded organic substance as can be seen here.
Summary of the invention
The present inventor is through deep research, and finding out a kind of is primary raw material with stalk, by lignin, cellulose in aspergillus oryzae cell solid culture degrading straw, produces the technique of protein feed.This technique method be different from the past be used bacterial classification by Food and Drug Administration (FDA) and U.S. feed Gong Ding association (AAFCO), the identification of the Ministry of Agriculture of China can the aspergillus oryzae bacterial classification of feed level microbe additive of Direct-fed animal, adopt stalk as primary raw material, obtain protein feed by solid state fermentation.
It is bacterial classification with aspergillus oryzae that the object of the invention there is provided a kind of, take stalk as the technique that raw material carries out the production protein feed of solid state fermentation, comprise with aspergillus oryzae is starting strain, carry out test tube and expand cultivation, liquid submerged culture, first order seed cultivation and solid fermentation cultivation, obtain the protein feed of degrading straw.
The present invention's bacterial classification used is aspergillus oryzae any one bacterial classification, as the bacterial classification of China General Microbiological culture presevation administrative center (CCGMC), Chinese agriculture Microbiological Culture Collection administrative center (ACCC), Chinese industrial Microbiological Culture Collection administrative center (CICC), Chinese medicine Microbiological Culture Collection administrative center (CMCC), National Veterinary Culture Collection (CVCC) and antibiotic bacterial classification preservation pipe reason center etc. preservation.
Raw material used in the present invention is any one stalk, agricultural crop straws containing lignin as all in maize straw, rice straw, Wheat Straw, broomcorn straw, soybean stalk, cotton stalk etc., and stalk, first through pulverizing, crosses 20 ~ 100 object sieves.
In zymotechnique of the present invention, it is potato sucrose culture medium that its test tube expands culture medium.
In zymotechnique of the present invention, its liquid submerged culture base and primary-seed medium are (grams per liter): glucose 5 ~ 30, ammonium tartrate 0.1 ~ 5, phenmethylol 0.2 ~ 3, magnesium sulfate 0.2 ~ 1, Tween 80 0.5 ~ 10, potassium dihydrogen phosphate 2 ~ 9, phthalic acid 3 ~ 18, pH 5 ~ 8,120 ~ 140 DEG C of sterilizings 20 ~ 40 minutes;
Wherein said Shaking culture process conditions are, inoculate a ring aspergillus oryzae test tube slant spore in 16 ~ 48%(volume ratio is housed) in the triangular flask of culture medium, at rotating speed: 150 revs/min, 25 ~ 35 DEG C, cultivate 24 ~ 72 hours;
Wherein said first order seed culture process is, by 1 ~ 20%(volume ratio) inoculum concentration Shaking culture seed liquor is inoculated in primary-seed medium, temperature 25 ~ 35 DEG C, speed of agitator 50 ~ 200 revs/min, throughput 0.2 ~ 2:1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivates 18 ~ 72 hours;
During solid fermentation of the present invention is cultivated, its solid fermentation culture medium consists of: stalk 10 kilograms, 20 ~ 60 kilograms, water, glucose 0.5 ~ 3 kilogram, 0.05 ~ 0.2 kilogram, urea; 5 ~ 15 grams, copper sulphate, 5 ~ 15 grams, ferrous sulfate, manganese sulfate 5 ~ 20 grams, each composition of this culture medium can by this scale amplifying equal;
Wherein said solid fermentation cultivation and fermentation technique is: by 2 ~ 20%(seed liquor volume/fermentation medium weight), compost thickness is 0.1 ~ 0.4 meter, expect wide 0.5 ~ 2 meter, length is not limit, throughput is 0.3 ~ 1.5:1(gas volume/fermentation medium weight)/per minute, incubation time is 3 days ~ 23 days, midway stirring 1 ~ 2 time.
In zymotechnique of the present invention, before carrying out Shaking culture, first cultivate in the potato sucrose culture medium of new for the access of aspergillus oryzae bacterial classification configuration, cultivation temperature 25 ~ 35 DEG C, incubation time 48 ~ 144 hours; 4 ~ 10 DEG C save backup.
Obtain stalk fermentation thing by the present invention and contain a large amount of yellow aspergillus oryzae spores, fermentate chromaticness ecru, to brown, has wine flavour, and Lignin degradation rate is 34 ~ 76%, and protein content is 15 ~ 40%, and feed yield is 45 ~ 60%.
According to fermentating culturing process of the present invention, in conjunction with the ABC of this area, can according to need of production, increase secondary, three grades of even level Four seeding tanks, expand solid state fermentation production scale further, to carry out suitability for industrialized production.Secondary, three grades even level Four seeding tank adopt culture medium identical with primary-seed medium with Shake flask medium, inoculum concentration is 5 ~ 20%, condition of culture be same as first order seed cultivation.
Adopt the advantage of this technique to adopt solid state fermentation, and without high temperature high pressure process, can not environment be given like this, also can not waste resource.
In order to set forth the material and progress involved by technical scheme of the present invention further, give following examples, but these embodiments do not limit the scope of the invention in any form.
Accompanying drawing explanation
Fig. 1 is the process chart that aspergillus oryzae degrading straw of the present invention produces the method for protein feed.
Detailed description of the invention
The method of protein measurement adopted according to this technique is the Coomassie brilliant blue kit measurement method adopting Nanjing to build up bio-engineering corporation to produce.Accurately take 1 gram of protein feed, be dissolved in 50mL water, shaken at room temperature 2 hours, suction filtration, filtrate is settled to 50mL, is sample liquid, for protein determination.Measurement operation step is undertaken by kit description.Protein content is by following formulae discovery:
According to this technique, lignin measures and adopts following method: take sample 100mg and be placed in 15mL reaction bulb, add 3mL72%H
2sO
4, 25 DEG C of hydrolysis 3h, and constantly shake, hydrolysate proceeds in triangular flask, adds the ultrapure H of 190mL
2o is in 121 DEG C of reaction 1h, and cooling, with the core crucible suction filtration of constant weight (W1), filter residue 200mL hot water cyclic washing, filter residue 105 DEG C dries to constant weight, and claims to obtain weight (W2) calculate sour insoluble content of lignin (W2-W1)/W.Filtrate is mended to 500mL, surveys the absorbance of filtrate under 205nm wavelength.When the absorbance (A) of sample solution is between 0.2 ~ 0.7, according to formula (1), calculate acid-soluble content of lignin ρ (mg/g).Each sample repeats 6 times, averages.
In formula, D is the extension rate of sample solution.
Embodiment 1
1, the making of test tube slant bacterial classification
Access one ring aspergillus oryzae bacterial classification (Aspergillus oryzae) in the potato sucrose culture medium (" the industrial microorganism experimental technique handbook " of Zhu Gejian, Wang Zhengxiang chief editor, 1994,367 pages) of new configuration, 26 DEG C, incubation time 50 hours; 4 DEG C save backup.
2, the making of liquid submerged culture bacterial classification
Accurately take glucose 5 grams, ammonium tartrate 0.1 gram, phenmethylol 0.2 gram, 0.2 gram, magnesium sulfate, Tween 80 0.5 gram, potassium dihydrogen phosphate 2 grams, phthalate buffer 3 grams, water 1000 mL, pH 5, be sub-packed in 250mL triangular flask, 50 grams every bottle, totally 20 bottles, 120 DEG C of sterilizings 40 minutes; After cooling, every bottle graft enters the aspergillus oryzae bacterial classification that 4 DEG C, a ring is preserved, and at 25 DEG C, 150 revs/min, cultivates 72 hours;
3, the making of level liquid bacterial classification
Accurately take glucose 50 grams, ammonium tartrate 1 gram, phenmethylol 2 grams, 2 grams, magnesium sulfate, Tween 80 5 grams, potassium dihydrogen phosphate 20 grams, phthalic acid 30 grams, water 10L, is loaded in the seeding tank of 15L, 120 DEG C of sterilizings 20 minutes; After sterilizing cooling, by the inoculum concentration access aforesaid liquid shaking flask bacterial classification of 20%, temperature 25 DEG C, speed of agitator 50 revs/min, throughput 0.2: 1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivates 72 hours;
4, solid fermentation protein feed
Rice straw is pulverized, and crosses 20 mesh sieves, takes this powder of straw 750 kilograms, 1500 kilograms, water, glucose 37.5 kilograms, 3.75 kilograms, urea; 375 grams, copper sulphate, 375 grams, ferrous sulfate, manganese sulfate 375 grams, fully mix thoroughly, the inoculum concentration by 20% accesses above-mentioned first order seed nutrient solution, mixes, making compost thickness is 0.1 meter, expect wide 0.5 meter, the stockpile that length is 37.5 meters, control temperature 25 DEG C is cultivated 23 days, midway stirring 2 times,, cultivate and terminate the oven dry of after fermentation thing, pulverize and be protein feed.
Obtain stalk fermentation thing and contain a large amount of yellow aspergillus oryzae spores, fermentate orange colour, has wine flavour.Lignin degradation rate 42.2%, protein content 16.7%, feed yield is 49.1%.
Embodiment 2
1, the making of test tube slant bacterial classification
Access one ring aspergillus oryzae bacterial classification (Aspergillus oryzae) in the potato sucrose culture medium (" the industrial microorganism experimental technique handbook " of Zhu Gejian, Wang Zhengxiang chief editor, 1994,367 pages) of new configuration, 30 DEG C, incubation time 100 hours; 7 DEG C save backup.
2, the making of liquid submerged culture bacterial classification
Accurately take glucose 150 grams, ammonium tartrate 250 grams, phenmethylol 15 grams, 0.4 gram, magnesium sulfate, Tween 80 4 grams, potassium dihydrogen phosphate 5 grams, O-phthalic acid buffering 20 grams, water 10 L, pH 6, be sub-packed in 250mL triangular flask, 100 grams every bottle, totally 100 bottles, 130 DEG C of sterilizings 30 minutes; After cooling, every bottle graft enters the aspergillus oryzae bacterial classification that 7 DEG C, a ring is preserved, and at 30 DEG C, 150 revs/min, cultivates 50 hours;
3, the making of level liquid bacterial classification
Accurately take glucose 2000 grams, ammonium tartrate 200 grams, phenmethylol 150 grams, 60 grams, magnesium sulfate, Tween 80 60 grams, potassium dihydrogen phosphate 400 grams, phthalic acid 1000 grams, water 100L, is loaded in the seeding tank of 130L, 130 DEG C of sterilizings 30 minutes; After cooling, be the ratio access aforesaid liquid shaking flask bacterial classification of 10% by volume, temperature 30 DEG C, speed of agitator 125 revs/min, throughput 1:1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivates 50 hours;
4, solid fermentation protein feed
Corn straw smashing, crosses 60 mesh sieves, takes this powder of straw 1000 kilograms, 3000 kilograms, water, glucose 150 kilograms, 10 kilograms, urea; 600 grams, copper sulphate, 600 grams, ferrous sulfate, manganese sulfate 1000 grams, fully mix thoroughly, by 10%(volume/weight) access above-mentioned first order seed nutrient solution, mix, making compost thickness is 0.2 meter, expect wide 1 meter, the stockpile that length is 225 meters, control temperature 30 DEG C is cultivated 15 days, midway stirring 2 times, cultivate and terminate the oven dry of after fermentation thing, pulverize and be protein feed.
Obtain stalk fermentation thing and contain a large amount of yellow aspergillus oryzae spores, fermentate yellowish-brown, has wine flavour.Lignin degradation rate 58.4%, protein content 31.2%, feed yield is 59.2%.
Embodiment 3
1, the making of test tube slant bacterial classification
Access one ring aspergillus oryzae bacterial classification (Aspergillus oryzae) in the potato sucrose culture medium (" the industrial microorganism experimental technique handbook " of Zhu Gejian, Wang Zhengxiang chief editor, 1994,367 pages) of new configuration, 35 DEG C, incubation time 144 hours; 10 DEG C save backup.
2, the making of liquid submerged culture bacterial classification
Accurately take glucose 300 grams, ammonium tartrate 50 grams, phenmethylol 30 grams, 10 grams, magnesium sulfate, Tween 80 100 grams, potassium dihydrogen phosphate 90 grams, phthalate buffer 180 grams, water 10L, pH7.5, be sub-packed in 250mL triangular flask, every bottle of 120 mL, totally 80 bottles, 140 DEG C of sterilizings 20 minutes; After cooling, every bottle graft enters the aspergillus oryzae bacterial classification that 10 DEG C, a ring is preserved, and at 35 DEG C, 150 revs/min, cultivates 72 hours;
3, the making of level liquid bacterial classification
Accurately take glucose 1500 grams, ammonium tartrate 250 grams, phenmethylol 150 grams, 50 grams, magnesium sulfate, Tween 80 500 grams, potassium dihydrogen phosphate 450 grams, phthalate buffer 900 grams, water 50L, is loaded in the first class seed pot of 70L, 140 DEG C of sterilizings 40 minutes; After sterilizing cooling, by the inoculum concentration access aforesaid liquid shaking flask bacterial classification of 5%, temperature 35 DEG C, speed of agitator 200 revs/min, throughput 2:1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivates 72 hours;
4, the making of second-class liquid isolate
Accurately take glucose 15000 grams, ammonium tartrate 2500 grams, phenmethylol 1500 grams, 500 grams, magnesium sulfate, Tween 80 5000 grams, potassium dihydrogen phosphate 4500 grams, phthalate buffer 9000 grams, water 500L, is loaded in the first class seed pot of 700L, 140 DEG C of sterilizings 40 minutes; After sterilizing cooling, the ratio in 5% accesses above-mentioned level liquid bacterial classification, temperature 35 DEG C, and speed of agitator 200 revs/min, throughput 2:1 volume/volume/per minute (ventilation gas volume/fermentating liquid volume/per minute), cultivates 18 hours;
5, solid fermentation protein feed
Wheat straw waste is pulverized, and crosses 100 mesh sieves, takes this powder of straw 3500 kilograms, 21000 kilograms, water, glucose 525 kilograms, 52.5 kilograms, urea; 5000 grams, copper sulphate, 5000 grams, ferrous sulfate, manganese sulfate 6000 grams, fully mix thoroughly, ratio in 5% accesses above-mentioned secondary seed nutrient solution, mixes, and making compost thickness is 0.3 meter, expect wide 2 meters, the stockpile that length is 300 meters, control temperature 35 DEG C is cultivated 6 days, midway stirring 1 time, cultivate and terminate the oven dry of after fermentation thing, pulverize and be protein feed.
Obtain stalk fermentation protein feed and contain a large amount of yellow aspergillus oryzae spores, fermentate brown, has wine flavour, Lignin degradation rate 69.7%, and protein content is 37.3%, and feed yield is 51.6%.
Claims (1)
1. aspergillus oryzae degrading straw produces the method for protein feed, it is characterized in that carrying out according to following step: take stalk as the technique that raw material carries out the production protein feed of solid state fermentation, comprise with aspergillus oryzae is starting strain, carry out test tube and expand cultivation, liquid submerged culture, first order seed cultivation and solid fermentation cultivation, obtain the protein feed of degrading straw;
Bacterial classification used is aspergillus oryzae any one bacterial classification;
The raw material used is any one stalk, and stalk is first through pulverizing, and crosses 20 ~ 100 object sieves;
It is potato sucrose culture medium that described test tube expands culture medium;
In described zymotechnique, its liquid submerged culture base and primary-seed medium are: glucose 5 ~ 30 grams per liter, ammonium tartrate 0.1 ~ 5 grams per liter, phenmethylol 0.2 ~ 3 grams per liter, magnesium sulfate 0.2 ~ 1 grams per liter, Tween 80 0.5 ~ 10 grams per liter, potassium dihydrogen phosphate 2 ~ 9 grams per liter, phthalic acid 3 ~ 18 grams per liter, pH 5 ~ 8,120 ~ 140 DEG C of sterilizings 20 ~ 40 minutes;
Wherein said Shaking culture process conditions are, inoculate a ring aspergillus oryzae test tube slant spore in the triangular flask that volume ratio 16 ~ 48% culture medium is housed, and at rotating speed: 150 revs/min, 25 ~ 35 DEG C, cultivate 24 ~ 72 hours;
Wherein said first order seed culture process is, Shaking culture seed liquor is inoculated in primary-seed medium by the inoculum concentration of 1 ~ 20% by volume, temperature 25 ~ 35 DEG C, speed of agitator 50 ~ 200 revs/min, throughput 0.2 ~ 2:1 ventilation gas volume/fermentating liquid volume/per minute, cultivates 18 ~ 72 hours;
During described solid fermentation is cultivated, its solid fermentation culture medium consists of: stalk 10 kilograms, 20 ~ 60 kilograms, water, glucose 0.5 ~ 3 kilogram, 0.05 ~ 0.2 kilogram, urea; 5 ~ 15 grams, copper sulphate, 5 ~ 15 grams, ferrous sulfate, manganese sulfate 5 ~ 20 grams, each composition of this culture medium can by this scale amplifying equal;
Wherein said solid fermentation cultivation and fermentation technique is: be 2 ~ 20% by the ratio of seed liquor volume/fermentation medium weight, compost thickness is 0.1 ~ 0.4 meter, expect wide 0.5 ~ 2 meter, length is not limit, throughput is 0.3 ~ 1.5:1(gas volume/fermentation medium weight)/per minute, incubation time is 3 days ~ 23 days, midway stirring 1 ~ 2 time; Described test tube expands cultivation, before carrying out Shaking culture, first cultivates in the potato sucrose culture medium of new for the access of aspergillus oryzae bacterial classification preparation, cultivation temperature 25 ~ 35 DEG C, incubation time 48 ~ 144 hours; 4 ~ 10 DEG C save backup.
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| CN104531640B (en) * | 2015-01-08 | 2018-08-28 | 北京绿科天成生物科技有限公司 | A kind of aspergillus oryzae zymotic fluid, the maize straw liquid glucose prepared by the zymotic fluid and preparation method and purposes |
| CN105039286B (en) * | 2015-02-06 | 2018-11-06 | 江苏一鸣生物股份有限公司 | Liquid glucose and preparation method and purposes prepared by aspergillus oryzae zymotic fluid, the zymotic fluid degradation of rice straw powder |
| FR3078976B1 (en) * | 2018-03-13 | 2023-04-14 | Commissariat Energie Atomique | SEQUENTIAL PROCESS FOR FUNGAL FERMENTATION OF WOOD RESOURCES |
| CN111909857B (en) * | 2020-08-21 | 2023-08-08 | 四川润格生物科技有限公司 | Technical method for producing aspergillus oryzae by using white spirit vinasse |
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