CN102077903A - Method for jointly treating stalks by steam explosion and microorganism fermentation - Google Patents

Abstract

Translated fromChinese

本发明涉及一种蒸汽爆破与微生物发酵联合处理秸秆的方法：先将秸秆进行蒸汽爆破预处理得爆破秸秆；再制备孢子种子液：将米曲霉或康氏木霉接种到PDA培养基中28～32℃静止培养3～5d，配制浓度10⁶～10⁸个/mL孢子种子液；最后进行微生物发酵：取18g爆破秸秆与2g麸皮、30mL矿物元素营养液混匀，用Ca(OH)₂调至pH为7.0，在121℃、0.15MPa压力下灭菌15min得固体发酵培养基a，按2～4%的接种量将孢子种子液接种于a中，28～32℃培养5～7d。采用该方法得到的发酵秸秆中木质素、纤维素和半纤维素含量低，滤纸糖酶、CMC酶、淀粉酶和蛋白酶活力高。The invention relates to a method for combined treatment of straw by steam explosion and microbial fermentation: firstly, the straw is subjected to steam explosion pretreatment to obtain blasting straw; then the spore seed liquid is prepared: inoculating Aspergillus oryzae or Trichoderma konii into PDA medium for 28-28 Cultivate statically at 32°C for 3-5 days, prepare spore seed solution with a concentration of 10⁶ ～10⁸ spores/mL; finally carry out microbial fermentation: take 18g of blasting straw, mix with 2g of bran, and 30mL of mineral element nutrient solution, and use Ca(OH)₂ Adjust the pH to 7.0, sterilize at 121°C and 0.15MPa pressure for 15 minutes to obtain a solid fermentation medium a, inoculate the spore seed solution in a at an inoculum amount of 2-4%, and incubate at 28-32°C for 5-7 days. The content of lignin, cellulose and hemicellulose in the fermented straw obtained by the method is low, and the activities of filter paper carbohydrase, CMC enzyme, amylase and protease are high.

Description

The method of a kind of steam blasting and microbial fermentation Combined Treatment stalk

Technical field

The present invention relates to a kind of processing method of stalk, be specifically related to the method for a kind of steam blasting and microbial fermentation Combined Treatment stalk.

Background technology

A large amount of studies confirm that both at home and abroad utilizes biotechnology to excavate and utilize the potential value of crop stalk fully, is one of effective means of alleviating global grain resource shortage.According to according to a preliminary estimate, if substituting cereals grain, the biofermentation straw with 10～20% is used for pig industry production, annual nearly 3000～5,000 ten thousand tons of the grain of just can saving in the whole nation, can solve the gap problem of the annual 3000 ten thousand tons of cereals feed grains of China fully, have particular importance society and economic implications.

The main component of stalk is a crude fibre; comprise cellulose, hemicellulose and lignin; and the height polymerization of cellulose macromolecule and crystallinity and hemicellulose, lignin and its mutual protective effect of twining; make and utilize methods such as traditional hydrolysis and biofermentation to come degrade coarse fibers, efficient is lower.At present, the preprocess method of stalk has the processing method that physics, chemistry, biology and multiple processing combine.Biologic pretreatment method is because of the utilization ratio that can improve stalk and can not pollute environment, and more and more be subject to people's attention, but there is also shortcoming: i.e. bio-transformation efficient is lower.Traditional physical treatment is adopted minimizing lignocellulosic size and crystal structure to increase specific surface area and is reduced the degree of polymerization of fiber, generally needs much energy consumption.At present aspect physical treatment, the steam blasting preliminary treatment is a kind of the most promising preprocess method of innovation, it utilizes saturated vapor that stalk is heated to certain pressure, high steam infiltrated fiber inside, mode with air-flow discharges from the blind bore crack, make fiber that certain mechanical breaking take place, sudden pressure reduction to atmospheric pressure carries out pretreated a kind of physical means to stalk then.As: can in blasting process, stalk be heated to 0.69～4.83MPa with 160 ℃～260 ℃ saturated vapors; action time be several seconds to a few minutes; sudden pressure reduction is to atmospheric pressure then; cellulose is separated from the structure of complexity; shortened the length of fiber; help follow-up cellulase to cellulosic attack, make easier enzyme and the action of microorganisms of being subjected to of fiber, the production of scale can be saved energy consumption and cost greatly.But the research that the domestic and international at present blasting technique with stalk is applied to animal feed production aspect is less.The alkali preliminary treatment can be carried out at normal temperatures, and reduces the content of lignin in the stalk effectively, but can cause the loss of part fermentable sugars in the process of handling.The acid treatment hemicellulose in the lignocellulose raw material of can degrading makes that cellulose is easier to be utilized by microorganism, but the soda acid of high concentration has increased the cost that reclaims in actual production, be not suitable for industrialized large-scale production.

This comprehensive study is considered biological and physics processing method; stalk is carried out explosion treatment earlier carry out microbial fermentation again; filter out a kind of economical and effective, the straw pretreatment method of safe and feasible is for the scale of stalk resource exploitation and lay the foundation in application in animal husbandry.

Summary of the invention

The object of the invention is to provide the method for a kind of steam blasting and microbial fermentation Combined Treatment stalk, utilizes in the fermented stalk that this method obtains lignin, cellulose and hemicellulose level low, filter paper carbohydrase, CMC enzyme, amylase and prolease activity height.

For achieving the above object, the present invention adopts following technical scheme:

The method of a kind of steam blasting and microbial fermentation Combined Treatment stalk, it comprises the steps:

1. stalk steam blasting preliminary treatment: the stalk that will pulverize 10～20 mesh sieves, moisture content≤15wt% carries out steam blasting to be handled, then it is dried naturally the explosion stalk;

2. prepare aspergillus oryzae/koning trichoderma seed liquor: aspergillus oryzae or koning trichoderma are inoculated in the culture dish of being made by the PDA culture medium, 28～32 ℃ of static cultivation 3～5d, wash culture dish with sterile saline after the spore maturation, spore in the culture dish is transferred in the sterilization triangular flask compound concentration 1 * 10⁶～10⁸The spore suspension of individual/mL is the spore seed liquor; Described PDA culture medium prior to sterilization treatment 15min under 121 ℃, 0.15MPa pressure, is made the culture dish that contains 15～20 mL PDA culture mediums in advance then under aseptic condition;

3. microbial fermentation: get 18g explosion stalk and 2g wheat bran, 30mL mineral element nutrient solution mixes, with Ca (OH)₂Transferring to pH is 7.0, and sterilization 15min gets the solid fermentation culture medium under 121 ℃, 0.15MPa pressure then; By 2～4%(volume/mass percentage) inoculum concentration the spore seed liquor is inoculated in the solid fermentation culture medium, cultivate 5～7d for 28～32 ℃.

Test used aspergillus oryzae and koning trichoderma available from Institute of Microorganism, Academia Sinica, and effluent south agriculture university's Animal nutrition and the preservation of Biotechnology Experiment chamber.

Described stalk can be wheat straw or maize straw etc.

The 1. middle steam blasting of step is treated to: under the condition of steam pressure 1.8～2.5MPa, and release pressure behind pressurize 200～240s.

The step 2. component of middle PDA culture medium is: soluble starch 6 g, peptone 5 g, yeast 2 g, glucose 20 g, KH₂PO₄2 g, MgSO₄7H₂O 0.3 g, agar powder 20 g, distilled water 1000 mL.

The step 3. component of middle mineral element nutrient solution is: (NH₄)₂SO₄1.4 g, KH₂PO₄2.0 g, MgSO₄0.3 g, CaCl₂0.3 g, NaCl 0.5 g, FeSO₄5.0 mg, MnSO₄1.6 mg, ZnCl₂1.7 mg, CoCl₂2.0 mg, distilled water 1000 mL.

Compare beneficial effect of the present invention with prior art:

The present invention at first utilizes the method for steam blasting, and (the steam blasting device explosion pulse width of employing only is 0.00875 S, the abrupt release high density energy is finished explosion to destroy the structure of stalk), make lignin in the straw and cellulose obtain loose and the part degraded, make that microorganism is easier to play a role; Utilize the microorganism that can secrete high vigor cellulase to unite cultivation then, lignin and cellulose in the straw are degraded simultaneously, thereby reach the purpose of thorough degrading straw.The straw that the processing of employing this method obtains also contains a large amount of digestive enzymes (as cellulase, protease and amylase etc.) except that containing a large amount of indigestible carbohydrates.When stalk behind explosion and aspergillus oryzae fermentation process 5～6d, the content of cellulose and hemicellulose has reduced by 27.89% and 64.80% than original stalk respectively, filter paper carbohydrase, CMC enzyme, amylase and prolease activity can reach 335.10,1138.92,32.57 and 201.99 U/g respectively, therefore, stalk after the processing is not only the good feed of herbivore, also can be used as the feed resource of pig fowl, this is for solving the meaning that food shortage that people and animals strive the grain problem and alleviate the China and even the world has particular importance.In addition, this method economical and effective, safe and feasible is for the scale of stalk resource exploitation and lay a good foundation in application in animal husbandry.

Figure of description

Fig. 1 is the glucose calibration curve;

Enzyme is lived over time in Fig. 2 explosion stalk+aspergillus oryzae group sweat;

Soluble sugar content over time in Fig. 3 explosion stalk+aspergillus oryzae group sweat.

The specific embodiment

The present invention is described further by the following examples, but protection scope of the present invention is not limited thereto.

Test material: maize straw comes from energy research key lab of the Ministry of Agriculture of Agricultural University Of He'nan, and after pulverizer was pulverized, it was standby to cross 10～20 mesh sieves.

Testing equipment: stalk steam blasting machine QB-200: produced by Henan Province Hebi City right way heavy-duty machine factory, vapor pressure can reach 6 MPa, and heating power is 8 kW, and effectively the blast chamber volume is 0.405 L.Quick-fried of this vapour adopts gas bullet technology, can finish pressure and discharge in 0.00875 S, is different from the quick-fried mode of so-called vapour (expanded and hot spray) in the past, has realized explosion truly.

Embodiment 1(is an example with the maize straw, and the inventive method is elaborated)

The method of a kind of steam blasting and microbial fermentation Combined Treatment stalk, it comprises the steps:

1. stalk steam blasting preliminary treatment: the maize straw that will pulverize 20 mesh sieves, moisture content 10wt% is under steam pressure 2.5 Mpa conditions, release pressure behind pressurize 200 S, carry out steam blasting and handle, the stalk natural drying after the processing gets the explosion stalk to moisture content≤10wt%, and is standby;

2. prepare the aspergillus oryzae seed liquor: aspergillus oryzae is inoculated in the culture dish of being made by the PDA culture medium, 30 ℃ of static cultivation 3d wash culture dish with sterile saline after the spore maturation, the spore in the culture dish are transferred in the sterilization triangular flask compound concentration 1 * 10⁶The spore suspension of individual/mL is the spore seed liquor; Described PDA culture medium prior to sterilization treatment 15min under 121 ℃, 0.15MPa pressure, is made the culture dish that contains 20mL PDA culture medium in advance then under aseptic condition; Described PDA nutrient media components is: soluble starch 6 g, peptone 5 g, yeast 2 g, glucose 20 g, KH₂PO₄2 g, MgSO₄7H₂O 0.3 g, agar powder 20 g, distilled water 1000 mL;

3. aspergillus oryzae fermentation: (component is: (NH to add 18g explosion stalk, 2 g wheat brans and 30 mL mineral element nutrient solutions in the triangular flask of 500 mL₄)₂SO₄1.4 g, KH₂PO₄2.0 g, MgSO₄0.3 g, CaCl₂0.3 g, NaCl 0.5 g, FeSO₄5.0 mg, MnSO₄1.6 mg, ZnCl₂1.7 mg, CoCl₂2.0 mg, distilled water 1000 mL), after mixing, with Ca (OH)₂Transferring to pH is 7.0, then in 121 ℃, 0.15MPa pressure down sterilization 15min get the solid fermentation culture medium; By 4%(volume/mass percentage) inoculum concentration the spore seed liquor is inoculated in the solid fermentation culture medium, in 30 ℃ constant incubator, cultivate.24h for the first time at interval, every afterwards interval 48h get fermentation from incubator maize straw sample carries out the mensuration of index of correlation.By the index determining result as can be known, generally cultivate 5～7d and just can obtain treatment effect preferably.

Embodiment 2

With reference to embodiment 1, difference is carried out fermentation test for substituting aspergillus oryzae with koning trichoderma.

Embodiment 3

The method of a kind of steam blasting and microbial fermentation Combined Treatment stalk, it comprises the steps:

1. stalk steam blasting preliminary treatment: the stalk that will pulverize 10 mesh sieves, moisture content 15wt% is under the condition of steam pressure 1.8MPa, release pressure behind the pressurize 240s, carrying out steam blasting and handle, is that 15wt% gets the explosion stalk with its natural drying to moisture content then, standby;

2. prepare the koning trichoderma seed liquor: koning trichoderma is inoculated in the culture dish of making by the PDA culture medium, 28 ℃ of static cultivation 5d, wash culture dish with sterile saline after the spore maturation, the spore in the culture dish is transferred in the sterilization triangular flask compound concentration 1 * 10⁸The spore suspension of individual/mL is the spore seed liquor; Described PDA culture medium prior to sterilization treatment 15min under 121 ℃, 0.15MPa pressure, is made the culture dish that contains 15 mL PDA culture mediums in advance then under aseptic condition; Described PDA nutrient media components is: soluble starch 6 g, peptone 5 g, yeast 2 g, glucose 20 g, KH₂PO₄2 g, MgSO₄7H₂O 0.3 g, agar powder 20 g, distilled water 1000 mL;

3. koning trichoderma fermentation: get 18g explosion stalk and 2g wheat bran, 30mL mineral element nutrient solution mixes, usefulness Ca (OH)₂Transferring to pH is 7.0, and sterilization 15min gets the solid fermentation culture medium under 121 ℃, 0.15MPa pressure then; By 2%(volume/mass percentage) inoculum concentration the spore seed liquor is inoculated in the solid fermentation culture medium, cultivate 5～7d for 28 ℃ and get final product.

Use the inventive method to handle the performance measurement test of gained fermented maize stalk.

Experimental design and grouping

It is 6 groups that test is divided into, promptly common stalk group, explosion stalk group, common stalk+aspergillus oryzae group, common stalk+koning trichoderma group, explosion stalk+aspergillus oryzae group and explosion stalk+koning trichoderma group.Every group of 6 repetitions are cultivated in 30 ℃ constant incubator after each winding kind, and the first time is 24h at interval, and every afterwards interval 48h gets the mensuration that part fermented stalk sample carries out index of correlation from incubator.

The physical and chemical index of measuring in the process of the test

2.1 the mensuration of cellulose and content of lignin

Employing Vansoest method (Feng Jihua, etc. use the comparison [J] that Van Soest method and conventional method are measured cellulose and lignin.

Southwest Nationalities College's journal.1994,20 (1): 55-56.) measure of the variation of each processed group respectively at different fermentations time inner cellulose and content of lignin.

2.2 the mensuration of enzyme activity and soluble sugar

Get the fermented maize straw sample of 4.0 g processing gained, add 46 mL physiological saline, soak 2 h after, centrifugal 10 min of 3000 r/min, get supernatant as crude enzyme liquid, measure wherein filter paper carbohydrase, carboxymethylcelluloenzyme enzyme, protease and amylase activity and soluble sugar content.

2.2.1 the mensuration of filter paper carbohydrase (FPU) vigor (Jiang Jiala, etc. the bacterial strain screening of cellulose degradation and utilization thereof [J].The modern biomedical progress.2009,18 (9): 3451-3454.)

Get crude enzyme liquid and boil each 0.5 mL of crude enzyme liquid of 10min deactivation, the citrate buffer solution and the 50 mg filter paper bars that add 1.5 mL concentration, 0.05 mol/L, pH4.5 respectively, in 50 ℃ of water bath heat preservation l h, add 1.5 mL 3 then respectively, 5-dinitrosalicylic acid (DNS), boiling 5 min, is contrast with the crude enzyme liquid group of deactivation, measures absorbance at wavelength 550 nm places.Enzyme activity unit definition: with hydrolysis per hour the catalytic substrate hydrolysis enzyme amount that forms 1 μ mol glucose be a unit.

2.2.2 the mensuration of carboxymethylcelluloenzyme enzyme (CMCase) vigor (Jiang Jiala, etc. the bacterial strain screening of cellulose degradation and utilization thereof [J].The modern biomedical progress.2009,18 (9): 3451-3454.)

Get crude enzyme liquid and boil each 0.5 mL of crude enzyme liquid of 10min deactivation, add 1.5 mL, 0.51%CMC citrate buffer solution respectively, in 50 ℃ of thermostat water baths behind enzymolysis 30 min, from water-bath, take out, add 1.5 mL DNS colour developing liquid cessation reaction then respectively, behind the abundant mixing, boiling water bath 15 min, crude enzyme liquid group with deactivation is contrast, measures absorbance at wavelength 550nm place.Enzyme activity unit definition: with hydrolysis per hour the catalytic substrate hydrolysis enzyme amount that forms 1 μ mol glucose be a unit.

2.2.3 the mensuration of amylase activity (Yasser B, et al. Isolation and identification of a new fungal strain for amylase biosynthesis[J].Polish Journal of Microbiology. 2009,58 (3): 269-273.)

Get the starch of 1.25 mL 1%, 0.25 mL acetate buffer solution (0.1mol/L, pH5.0), 0.25 mL deionized water, 0.25 the crude enzyme liquid of mL adds 1.5 mL DNS, boiling water bath 15 min behind 50 ℃ of insulation 10 min, crude enzyme liquid group with deactivation is contrast simultaneously, measures absorbance at wavelength 550 nm places.The enzyme activity unit definition: the enzyme amount that forms 1 μ mol glucose with the hydrolysis of hydrolysis per minute catalytic substrate is a unit.

2.2.4 the mensuration of prolease activity:

The vigor of employing Folin one phenol method mensuration protease (Wang Pengpeng, etc. protease and amylase produce screening and the zymologic property analysis and research [J] of bacterium.China's herding magazine. 2009, (21): 48-51.).

2.2.5 the mensuration of soluble sugar:

Adopt 3, content of reducing sugar in the 5-dinitrosalicylic acid colorimetric method for determining hydrolyzate (Wang Ping, etc. the research [J] that reaches the maize straw degradation effect is identified in the separation of aspergillus oryzae in the bovine rumen.Agricultural University Of He'nan's journal, 2010, (44) 3:295-299.).

Accurately take by weighing DEXTROSE ANHYDROUS 1.000 g of drying to constant weight, add 100 mL water, be made into the standard glucose liquid of 10 mg/mL concentration through 105 ℃.Draw the standard glucose liquid 1.0,2.0,3.0,4.0 of 10 mg/mL concentration respectively, 5.0,6.0 mL are settled to 50 mL with distilled water in the volumetric flask of 50 mL, are made into 200,400,600,800, the standard liquid of 1000,1200 μ g/mL.Get variable concentrations standard liquid 1mL in test tube, add 3 mL DNS reagent and react 15 min in boiling water, cooling under 550 nm, with spectrophotometric instrumentation absorbance, is an ordinate with the absorbance, and glucose amount is an abscissa, the drawing standard curve.The blank standard glucose that replaces 1 mL with 1 mL distilled water.The mark curve is seen Fig. 1.

Data statistics and analysis

All (mean ± represent that SD) result carries out variance analysis with One-way ANOVA process in 16.0 editions statistical softwares of SPSS, and carries out the DuncanShi multiple ratio, P＜0.05 is a significant difference to test data with " mean+SD ".

Result and analysis

4.1 embodiment 1 method is handled the variation of gained maize straw chemical composition with fermentation time

As shown in Table 2, adopt embodiment 1 method to handle the prolongation of the maize straw of (being explosion stalk+aspergillus oryzae group) along with fermentation time, the content of the neutral detergent fiber in the fermented stalk, acid detergent fiber, cellulose and hemicellulose is downward trend.At the 1st d of aspergillus oryzae fermentation, cellulose in the maize straw and hemicellulose level promptly have obvious reduction (p＜0.05); At the 5th d of fermentation, the hemicellulose level in the sample significantly is lower than the 1st d and the 3rd d, reduces 39.11%(p＜0.05 during than 0 d); And content of cellulose and the 3rd d difference not significantly (p＞0.05), but reduce 17.35%(p＜0.05 during than 0 d).Consider that from stalk composition degraded situation the effect of 5 d that ferment is better than preceding 3 d.In addition, most of indexs of the 5th d that ferments do not have significant difference with fermentation the 7th d, thereby consider that from economic angle selecting the suitable fermentation time of aspergillus oryzae is 5 d.

The aspergillus oryzae fermentation can not reduce the content of lignin in the explosion stalk during the fermentation, make content of lignin rise to some extent (p＞0.05) on the contrary, mainly be because microbial fermentation increases the solable matter content in the fermented stalk, in measuring the content of lignin process, because the loss of solable matter, the gross weight of the final dry of fermented stalk is reduced, cause the relative rising (table 3 has also obtained similar result) of content of lignin.

From the above, when fermentation 5 d, each performance indications of maize straw promptly obtain effect preferably.Therefore, when below getting fermentation time and being 5 d, the chemical composition of maize straw respectively organized in record.

4.2 the different disposal method is in fermentation influence to maize straw degradation rate during 5 d

The chemical composition (based on dry) of different disposal group maize straw during table 3 fermentation 5d

Group is formed	Cellulose %	Neutral detergent fiber %	Acid detergent fiber %	Hemicellulose %	Lignin %
						Common stalk group	41.30±2.31A	79.26±1.31A	59.23±2.00A	20.02±1.18A	12.58±1.07A
Explosion stalk group	37.80±0.40B	60.94±0.34C	51.12±1.12B	9.92±0.86C	7.97±1.21B
						Common stalk+aspergillus oryzae group	33.77±1.23C	71.78±1.27B	52.19±0.60B	19.59±1.02A	14.58±2.18A
Common stalk+koning trichoderma group	34.02±1.74C	72.66±0.24B	53.62±1.69B	14.76±2.40B	12.44±0.90A
						Explosion stalk+koning trichoderma group	33.33±1.10C	60.42±1.41C	52.31±0.89B	7.30±0.54C	10.07±0.22B
Explosion stalk+aspergillus oryzae group	29.78±2.92D	52.76±2.36D	47.28±3.75C	7.06±1.03C	9.54±0.97B

As shown in Table 3, explosion and various microbial fermentation are handled and have all been reduced the content of cellulose in the stalk (p＜0.05) significantly, and common stalk and explosion stalk are after aspergillus oryzae and koning trichoderma fermentation, its content of cellulose all significantly is lower than explosion stalk group (p＜0.05), illustrates that the sweat of microorganism has utilized the cellulose components in the maize straw.Wherein, stalk is behind the inoculation aspergillus oryzae (being explosion stalk+aspergillus oryzae group) after the explosion, its content of cellulose significantly is lower than explosion stalk+koning trichoderma group (p＜0.05), illustrated that aspergillus oryzae utilizes the efficient aspect the explosion stalk cellulose to be higher than koning trichoderma in decomposition.Therefore, when next step measures the variation that various enzymes are lived in the fermented stalk sample, be main reference object with " explosion stalk+aspergillus oryzae group ".Explosion treatment has significantly reduced the content of lignin in the stalk (p＜0.05), but aspergillus oryzae and koning trichoderma fermentation all do not have remarkable influence (p＞0.05) to the lignin in common stalk and the explosion stalk, and this does not secrete the cause of lignin-degrading enzymes mainly due to aspergillus oryzae and koning trichoderma.

In the preprocessing process of stalk resource, the most important thing is to reduce the ratio of lignin in the stalk, and avoid cellulosic degraded as far as possible.Under these trial shots condition, blasting process has significantly reduced the content (p＜0.05) of the cellulose in the stalk, hemicellulose and lignin, make that cellulose degradation rate is that 8.47%(significantly is lower than common stalk+aspergillus oryzae group p＜0.05 in the stalk), the degradation rate of hemicellulose is 50.45%, and the degradation rate of lignin reaches 36.65%.This explanation explosion treatment has a significant effect aspect cellulose, hemicellulose and the lignin in degrading straw.Lignin is the barrier that straw biological utilizes, and is accompanied by the degraded of hemicellulose and lignin in the blasting process of stalk, and the structural lignin in the stalk is damaged, thereby more helps the effect of microorganism and enzyme.Studies show that of Moyson (Moyson E, Verachtert H. Growth of higher fungi on wheat straw and their impact on the digestibility of the substrate[J].Appl. Microbial. Biotechnol. 1991,36:421-424.), animal increases along with the reduction of lignin in the stalk the digestibility of maize straw, and this illustrates simultaneously that also this explosion treatment has great importance to the utilization of straw feed resource.

Common stalk and explosion stalk are after the aspergillus oryzae fermentation, and its cellulosic content all significantly is lower than explosion stalk group (p＜0.05), illustrates that the process of microbial fermentation has been utilized the cellulose components in the stalk.Explosion stalk wherein neutral detergent fiber and acid detergent fiber content after aspergillus oryzae fermentation all significantly reduces (p＜0.05), illustrate that aspergillus oryzae has obvious facilitation to the conversion of stalk insoluble fibre composition, stalk after the explosion can promote nutrition and the part active material that it is converted into thalline self through the fermentation process of aspergillus oryzae, animal is produced have certain facilitation.

4.3 explosion stalk+aspergillus oryzae group maize straw is the variation of enzyme activity and soluble sugar during the fermentation

In the aspergillus oryzae sweat, Fig. 2 and Fig. 3 are seen in the variation of filter paper carbohydrase, CMC enzyme, amylase, protease and soluble sugar respectively in the maize straw sample.

The filter paper carbohydrase is with the index utilizing cellulose total enzyme alive height of filter paper as reaction substrate.As shown in Figure 2, in aspergillus oryzae fermentation explosion stalk process, the enzyme in early stage is lived lower, raises gradually behind the 2nd d, and the 6th d is significantly higher than point (p＜0.05) At All Other Times.

The work of CMC enzyme is an index of reacting cellulase restriction endonuclease height, the CMC enzyme of aspergillus oryzae fermentation explosion stalk is lived, promptly reach the highest at 1d, this may with the stalk blasting process in to produce the carbohydrate of more short chain fiber and short chain relevant, these materials can impel aspergillus oryzae to secrete more restriction endonuclease and promote its utilization.The 2nd d reduces gradually afterwards, and 4d rises to the highest again, and the 7th d enzyme of fermentation is lived and significantly reduced (p＜0.05).Many studies show that, the explosion treatment of stalk helps to weaken the structure of lignocellulosic, makes it be easy to be subjected to the attack of enzyme and microorganism.This result illustrates that also the structure of the stalk lignocellulosic after the explosion is damaged, and makes that wherein short chain cellulose is easier to be utilized by microorganism.

The diastatic activity of explosion stalk is from the remarkable rising of 2d of fermentation, and the fermentation back all kept higher level in several days.The protease activity of explosion stalk presents the variation tendency that raises and afterwards descend earlier, and the enzyme work of ferment the 3rd d and the 4th d reaches the highest, and the 7th d enzyme work of fermenting is reduced to minimum.

Because of the nitrogenous source that contains in the fermentation medium less relatively, main cellulolytic filter paper carbohydrase reaches at the 6th d and is up to 335.10 U/g, the CMC of the 6th d is relative with diastatic activity higher, is respectively 1138.92 U/g and 32.57 U/g, and protease activity is 201.99 U/g.6 d that can consider to ferment in explosion stalk microbe trans-utilization process effectively bring into play the effect that it utilizes stalk.

As shown in Figure 3, aspergillus oryzae in fermentation explosion stalk process along with the increase of fermentation time, soluble sugar in the maize straw sample reduces gradually, be that explosion stalk soluble sugar content when fermentation the 1st～2 d is the highest, significantly descend subsequently, maintain a lower level, this is relevant with the soluble sugar that microorganism has utilized explosion to produce during the fermentation.

Conclusion

The explosion preliminary treatment of this research (pressurize 200s under the 2.5MPa pressure) makes the content of cellulose in the maize straw, hemicellulose and lignin reduce by 8.47%, 50.45% and 36.65% respectively.Behind aspergillus oryzae fermentation 5～6 d, the cellulose wherein and the content of hemicellulose have reduced by 27.89% and 64.80% than original stalk respectively to the explosion stalk, have reduced by 21.17% and 28.28% than steam blasting stalk again.Filter paper carbohydrase in the fermented stalk, CMC enzyme, amylase and prolease activity reach 335.10,1138.92,32.57 and 201.99 U/g respectively.This proves absolutely maize straw after explosion and microbial fermentation Combined Treatment, and its degradation rate and nutritive value have all obtained increasing substantially, for the development and use of agricultural crop straw are laid a good foundation.

Claims (5)

1. the method for steam blasting and microbial fermentation Combined Treatment stalk is characterized in that, comprises the steps:

1. stalk steam blasting preliminary treatment: the stalk that will pulverize 10～20 mesh sieves, moisture content≤15wt% carries out steam blasting to be handled, then it is dried naturally the explosion stalk;

2. prepare aspergillus oryzae/koning trichoderma seed liquor: aspergillus oryzae or koning trichoderma are inoculated in the culture dish of being made by the PDA culture medium, 28～32 ℃ of static cultivation 3～5d, wash culture dish with sterile saline after the spore maturation, spore in the culture dish is transferred in the sterilization triangular flask compound concentration 1 * 10⁶～10⁸The spore suspension of individual/mL is the spore seed liquor;

3. microbial fermentation: get 18g explosion stalk and 2g wheat bran, 30mL mineral element nutrient solution mixes, with Ca (OH)₂Transferring to pH is 7.0, and sterilization 15min gets the solid fermentation culture medium under 121 ℃, 0.15MPa pressure then; Inoculum concentration by 2～4% is inoculated in the spore seed liquor in the solid fermentation culture medium, cultivates 5～7d for 28～32 ℃.

2. handle the method for stalk according to claim 1, it is characterized in that, described stalk is wheat straw or maize straw.

3. handle the method for stalk according to claim 1, it is characterized in that, the 1. middle steam blasting of described step is treated to: under the condition of steam pressure 1.8～2.5MPa, and release pressure behind pressurize 200～240s.

4. handle the method for stalk according to claim 1, it is characterized in that, the 2. middle PDA nutrient media components of described step is: soluble starch 6 g, peptone 5 g, yeast 2 g, glucose 20 g, KH₂PO₄2 g, MgSO₄7H₂O 0.3 g, agar powder 20 g, distilled water 1000 mL.

5. handle the method for stalk according to claim 1, it is characterized in that, the 3. middle mineral element nutrient solution prescription of described step is: (NH₄)₂SO₄1.4 g, KH₂PO₄2.0 g, MgSO₄0.3 g, CaCl₂0.3 g, NaCl 0.5 g, FeSO₄5.0 mg, MnSO₄1.6 mg, ZnCl₂1.7 mg, CoCl₂2.0 mg, distilled water 1000 mL.