CN103060206B - Fermentation bacteria agent and preparation method and application thereof - Google Patents
Fermentation bacteria agent and preparation method and application thereof Download PDFInfo
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- CN103060206B CN103060206B CN201310009803.9A CN201310009803A CN103060206B CN 103060206 B CN103060206 B CN 103060206B CN 201310009803 A CN201310009803 A CN 201310009803A CN 103060206 B CN103060206 B CN 103060206B
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Abstract
The invention discloses a fermentation bacteria agent and a preparation method and an application thereof. The preparation method comprises the following steps: (1) mixing unsterilized cabbage stems, orange peels and wheat bran according to the weight ratio of (0.8-1.2):(1.5-1.9):(0.3-0.5), and adding water to wet the mixture, thereby obtaining a culturing substrate; (2) inoculating Aspergillus niger, Trichoderma koningii and wheat koji into the culturing substrate to be stirred evenly; and (3) carrying out a stationary culturing, thereby obtaining the fermentation bacteria agent. The invention further provides the fermentation bacteria agent obtained by utilizing the preparation method and the application thereof in the production of a cellulose compound enzyme system. Compared with the prior art, the preparation method provided by the invention has the advantages that microbes for producing enzyme are tamed by utilizing the natural bacteria strains on the unsterilized carriers, so that the obtained fermentation bacteria agent is stable in microbial ecology and is capable of performing a diastatic fermentation on a cellulose substrate to produce the cellulose compound enzyme system.
Description
Technical field
The present invention relates to fibre substrate and utilize field, relate in particular to a kind of fermenting agent and its preparation method and application.
Background technology
Feed is the basic substance of developing animal husbandry, and a large amount of multifilament roughages is inexhaustible, a nexhaustible feed resource in the world.
Cellulosic roughage resource comprises stalk, skin, shell, core, waste wood, sawdust of farm crop etc., and according to statistics, the annual agricultural crop straw output Yue40 Duo Yidun， China in the whole world approximately has 7～800,000,000 tons every year.These resource crude fiber contents are high, and protein is few, and coarse ash is many, and palatability is poor, are unsuitable for directly doing feed, mostly burnt for a long time or part field also, and as feed utilisation still less than 1/5th, resource utilization is low and seriously polluted.
Chinese cabbage is the vegetables of a kind of extensive plantation of China, and its cultivated area and consumption occupy first of all kinds of vegetables in China, at the South and the North, are all seen everywhere.But after Chinese cabbage plantation harvest, all can produce a large amount of vegetable waste every year, account for 30% of its output, not only waste resource, also easy contaminate environment.If concentrate cleaning, cost is also higher.In Chinese cabbage side, fibre content, compared with horn of plenty, accounts for the more than 30% of dry weight, can be used as a kind of good cellulosic feed resource.Therefore, environmental friendliness and effectively utilize discarded Chinese cabbage side and there is great practical significance how.
For many years, many countries are devoted to study several different methods cellulosic roughage are carried out to processing treatment, comprise chemistry, physics, biological method, make it change into the feed that palatability is good, be of high nutritive value.Wherein, the most promising is microbe fermentation method, and the method neither needs too complicated equipment and too much energy expenditure, does not need again the comparatively extreme conditions such as high temperature, high pressure, strong acid, highly basic.
Wherein, micro-storage method is to utilize biological principle after crops stalk crushing, to add microbial strains to carry out biological fermentation processing, utilize enzyme degraded cellulose and the xylogen of various fungies, bacterium secretion, improve digestibility and the nutritive value of material, cellulosic roughage is converted into a kind of method of the feed that is rich in multiple nutritional components.
The at present domestic fermenting agent generally using has two kinds: the single microbial inoculum of purifying and simple mix bacterium agent.The single microbial inoculum of purifying is in physical environment, to screen good product enzyme single strain, then by certain mutagenesis means, obtains the bacterium producing multi enzyme preparation that large-scale industrialization is produced, then be adsorbed onto the microbial inoculum of making on specific support.But in physical environment, to the degraded of the compositions such as Mierocrystalline cellulose, xylogen, be that multiple-microorganism has been worked in coordination with, mutually coordinate, mutually restrict between microorganism, the ratio between the lytic enzyme that makes to produce is in the suitableeest a kind of degraded state.And bacterial classification in the single microbial inoculum of purifying has been owing to having lost coexisting of long-term other bacterium in conspiracy relation under natural condition, its enzymatic productivity reduces greatly, and institute's cellulose complex enzyme that produces is often incomplete, fermentation condition is had relatively high expectations, and vulnerable to pollution.
Simple mix bacterium agent is that two or more zymogenic bacteria kind is mixed according to a certain percentage, is adsorbed on suitable carrier and makes.Simply mix artificially, between various bacterium, whether there is conspiracy relation, whether produce antagonistic action and need to confirm.
Summary of the invention
The preparation method who the invention provides a kind of fermenting agent, the method is easy and simple to handle, the micro-Ecological Stabilization of fermenting agent of acquisition.
A preparation method for fermenting agent, comprising:
(1) Chinese cabbage side, tangerine peel, the wheat bran naturally chosen are mixed in proportion, add water and soak, obtain culture medium;
(2) in described culture medium, access aspergillus niger (Aspergillus niger), koning trichoderma (Trichoderma koningii), wheat koji, stir;
(3) standing cultivation, obtains described fermenting agent.
The present invention is usingd the Chinese cabbage side that naturally chooses, tangerine peel, wheat bran as carrier, maintains the mutualism relation of each bacterial classification in natural flora that is grown on described carrier; And on the basis of natural flora, microbes producing cellulase is inoculated in strengthening, forms the fermenting agent of micro-Ecological Stabilization by domestication.
In Chinese cabbage side and tangerine peel, water content is larger, is unfavorable for microbes producing cellulase growth.Therefore, described Chinese cabbage side, tangerine peel be through being crushed to 1～2mm, toasts remix after 2.5～3.5h at 60 ℃.
Chinese cabbage side, tangerine peel, wheat bran are preferably with weight ratio 0.8～1.2: mix at 1.5～1.9: 0.3～0.5, then after adding water and soak in the ratio of 0.4～0.6mL/g, access microbes producing cellulase.
Aspergillus niger of the present invention (Aspergillus niger) can be selected aspergillus niger (Aspergillus niger) ZJU-RYD1; Described koning trichoderma (Trichoderma koningii) can be selected koning trichoderma (Trichoderma koningii) ZJU-RDY2; Be Zhejiang University's Food science and the preservation of fermentation engineering institute.Also can use other can fermentative production cellulose complex enzyme aspergillus niger (Aspergillus niger) or the koning trichoderma (Trichoderma koningii) of one or more enzymes in system.
After described aspergillus niger (Aspergillus niger) and koning trichoderma (Trichoderma koningii) are activated, be mixed with 1 * 10
8～10
9the spore suspension of spores/mL accesses in culture medium again; Inoculum size is 0.8～1.2mL/100g culture medium.
Preferably, the described wheat koji wheat koji of making a living, by Anji, crow beaver brewery provides.Compare with ripe wheat koji, in raw wheat koji, microbe species, compared with horn of plenty, can be secreted more abundanter enzymes.
The weight ratio of described raw wheat koji and culture medium is preferably 0.8～1.2: 10.
Microbes producing cellulase inoculation is complete, stir after, culture medium tiling is scattered, stromal thickness is controlled at 2～3cm, to increase Gas permeability, accelerates the growth of natural flora and microbes producing cellulase.
During standing cultivation, culture temperature is 28～31 ℃, and culture environment humidity is controlled at 86～94%, and incubation time is 3～4 days.
The present invention also provides a kind of fermenting agent, and this microbial inoculum is to utilize described preparation method to be prepared from.The micro-Ecological Stabilization of described fermenting agent, can carry out diastatic fermentation to fibre substrate and produce cellulose complex enzyme system, utilizing this micro-synusium is to carry out comparatively extensive fermentation, needn't worry pollution problem, realize the low-cost high performance-price ratio bio-transformation to fibre substrate.
Cellulose complex enzyme system described in the present invention comprises cellulase, polygalacturonase, amylase and zytase.
The present invention also provides the application of described fermenting agent in production of cellulose prozyme system, comprising:
Described fermenting agent is joined in fibre substrate, stir, standing for fermentation, obtains described cellulose complex enzyme system.
This cellulose complex enzyme system is mixed with suitable carrier, also be can be made into cellulase preparation, as fodder additives, improve efficiency of feed utilization, improve feed quality.
Described fibre substrate is also preferably to be crushed to after 1～2mm and re-uses, if water content is larger, uses after also should toasting 2.5～3.5h at 60 ℃.The addition of described fermenting agent is preferably 8～12%.
The condition optimization of described standing for fermentation is: leavening temperature is 28～31 ℃, and yeasting humidity is controlled at 86～94%, and fermentation time is 3～6 days.The gauge control of fermented substrate is at 2～3cm, to increase air penetrability, accelerate growth of microorganism.
After having fermented, fermented product is mixed with appropriate damping fluid, carry out the extraction of enzyme liquid.
Described damping fluid is preferably the citric acid sodium solution (pH 4.8) of 0.5M, with fermented product with 6～8: the ratio of 1 (v/w) is mixed, centrifugal after 30 ℃ of water-bath 55～65min, and supernatant liquor is required enzyme liquid.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention utilizes the natural flora on sterilizing carrier not to tame microbes producing cellulase, the fermenting agent obtaining is micro-Ecological Stabilization not only, and can carry out diastatic fermentation production of cellulose prozyme system to fibre substrate, can realize the conversion to the low-cost high performance-price ratio of fibre substrate;
(2) fermenting agent of the present invention is solid fungicide, is convenient to transportation and uses; And the processing such as dry, freezing can not reduce quantity and the activity of viable bacteria in fermenting agent, long quality-guarantee period.
Accompanying drawing explanation
Fig. 1 is fermenting agent stability test result figure of the present invention.
Embodiment
1 substrate preparation
Help waste, orange meal to be broken to 1-2mm Chinese cabbage, under 60 ℃ of conditions, dry 3h, standby.Wheat bran is provided by Anji crow beaver brewery.
2 fermenting agent preparations
Aspergillus niger (Aspergillus niger) ZJU-RYD1 and koning trichoderma (Trichodermakoningii) ZJU-RDY2 are Zhejiang University's Food science and the preservation of fermentation engineering institute.Raw wheat koji is provided by Anji crow beaver brewery.
Aspergillus niger (Aspergillus niger) ZJU-RYD1 and koning trichoderma (Trichodermakoningii) ZJU-RDY2 are inoculated into respectively to PDA medium slant, under 28 ℃ of conditions, cultivate 3-4d and activate.
PDA culture medium prescription is: 30min is boiled in potato 200g chopping, and eight layers of filtered through gauze are got filtrate; Glucose 20g, agar 15g, water 1000mL, pH nature.
Obtain the aspergillus niger after activation, the spore of koning trichoderma, with distilled water, making concentration is 1 * 10
8the spore suspension of spores/mL, standby.
To through the Chinese cabbage of sterilizing, not help waste, tangerine peel and wheat bran in the ratio of 1: 1.7: 0.4 (w/w/w), to be mixed into the culture medium of 100g, culture medium adds water in the ratio of 0.5mL/g and soaks; Inoculate respectively again aspergillus niger, the koning trichoderma spore suspension of 1mL, and the abundant stirring and evenly mixing of raw wheat koji of 1g, standing cultivation 3d.Culture medium is controlled at the thickness of 2cm left and right, and culture temperature is controlled at 30 ± 1 ℃, and ambient moisture is controlled at 90 ± 4%.Final acquisition fermenting agent of the present invention.
3 solid fermentations
The Chinese cabbage of take below side waste is example as fermentation substrate, illustrates the application of fermenting agent of the present invention in preparing cellulose complex enzyme system.
The above-mentioned fermenting agent making is joined to 1kg in being crushed to the Chinese cabbage side waste of 1～2mm and oven dry by 10% addition, be placed in iron pan and carry out standing for fermentation, fermented substrate gauge control is in 2cm left and right, leavening temperature is controlled at 30 ± 1 ℃, ambient moisture is controlled at 90 ± 4%, ferments 5～6 days.
4 enzyme liquid extract and enzyme activity determination
Every 24h, measure the enzyme activity of fermented product cellulase (endoglucanase), polygalacturonase, amylase zytase.Each detection made 3 parallel tests, and calculates last mean value.
The citric acid sodium damping fluid (pH 4.8) of getting fermented product and 0.5M is (w/v=1: 7), after mixing, put into 30 ℃ of water-bath 60min, then 3000rpm centrifuging and taking supernatant liquor at room temperature, obtains required enzyme liquid in proportion.
(1) cellulase (endoglucanase) determination of activity
Adopt DNS method to measure.Add 0.5mL enzyme liquid in test tube, then add Xylo-Mucine (CMC-Na) solution (1g CMC-Na is dissolved in the 0.5mol/L sodium citrate buffer of 100mL pH 4.8) of 0.5mL 1%, 50 ℃ of insulation 30min.Add 0.75mL DNS reagent, boiling water bath heating 5min, is cooled to room temperature, adds 10mL distilled water, measures the reducing sugar amount of its generation.
(2) pectinase activity measuring method
Adopt DNS method to measure.Add 0.5mL enzyme liquid in test tube, then add the pectin solution (1g pectin is dissolved in the 0.1mol/L sodium citrate buffer of 100mL pH 5.0) of 0.5mL 1%, 50 ℃ of insulation 10min.Add 0.75mL DNS reagent, boiling water bath heating 5min, is cooled to room temperature, adds 10mL distilled water, measures the reducing sugar amount of its generation, then converts with galacturonic acid.
(3) Method for Determining Amylase Activity
Adopt DNS method to measure.Add 0.5mL enzyme liquid in test tube, then add the starch solution (1g Zulkovsky starch is dissolved in the 0.1mol/L sodium citrate buffer of 100mL pH 5.0) of 0.5mL 1%, 50 ℃ of insulation 10min.Add 0.75mL DNS reagent, boiling water bath heating 5min, is cooled to room temperature, adds 10mL distilled water, measures the reducing sugar amount of its generation.
For above three kinds of enzymes, an enzyme activity unit is defined as: under condition determination, per minute discharges the required enzyme amount of 1 μ mol glucose.
(4) xylanase activity measuring method
Adopt DNS method to measure, add 0.5mL enzyme liquid in test tube, then add the xylan solution (1g xylan is dissolved in the 0.1mol/L sodium citrate buffer of 100mLpH 5.0) of 0.5mL 1%, 50 ℃ of insulation 10min.Add 0.75mL DNS reagent, boiling water bath heating 5min, is cooled to room temperature, adds 10mL distilled water, measures the reducing sugar amount of its generation.
For zytase, an enzyme activity unit is defined as: under condition determination, per minute discharges the required enzyme amount of 1 μ moL wood sugar.
In fermenting process, in fermented product, the enzyme activity of four kinds of enzymes is as shown in table 1.
The enzyme activity of four kinds of enzymes in table 1 different fermentations time bottom fermentation thing
From table 1, in the Chinese cabbage side waste fermented product that utilizes fermenting agent of the present invention to ferment to obtain, cellulase (endoglucanase), polygalacturonase, amylase and xylanase activity are higher, and enzyme is abundant; The enzyme activity of four kinds of enzymes the 5th day time that ferments all reaches the highest.
5 fermenting agent Stability Determinations
The fermenting agent of placing at normal temperatures after 8 months is carried out to solid state fermentation and enzyme activity determination by above-mentioned method, and compare with the measurement result before 8 months.Measurement result demonstration, each enzyme enzyme ratio alive that the enzyme work of cellulase (endoglucanase), polygalacturonase, amylase and the zytase of secondary fermentation microbial inoculum generation in 8 months accounted for before 8 months is respectively: 68.32%, 98.17%, 76.90%, 68.03%.Comparative result as shown in Figure 1.
As seen from Figure 1, the micro-Ecological Stabilization of fermenting agent of the present invention, long quality-guarantee period.
The foregoing is only better implementation example of the present invention, be not limited to the present invention, all within the present invention spirit and principle, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (4)
1. a preparation method for fermenting agent, is characterized in that, comprising:
(1) Chinese cabbage side, tangerine peel, the wheat bran naturally chosen are mixed in proportion, add water and soak, obtain culture medium;
(2) in described culture medium, access aspergillus niger (Aspergillus niger), koning trichoderma (Trichoderma koningii), wheat koji, stir;
(3) standing cultivation, obtains described fermenting agent;
Described Chinese cabbage side, tangerine peel be through being crushed to 1～2mm, toasts remix after 2.5～3.5h at 60 ℃;
Described Chinese cabbage side, tangerine peel, wheat bran mix with weight ratio 0.8～1.2:1.5～1.9:0.3～0.5.
2. preparation method as claimed in claim 1, is characterized in that, after described aspergillus niger (Aspergillus niger) and koning trichoderma (Trichoderma koningii) are activated, is mixed with 1 * 10
8～10
9the spore suspension of spores/mL is inoculated again; Inoculum size is 0.8～1.2mL/100g culture medium.
3. preparation method as claimed in claim 1, is characterized in that, the weight ratio of described wheat koji and culture medium is 0.8～1.2:10.
4. preparation method as claimed in claim 1, is characterized in that, the temperature of described standing cultivation is 28～31 ℃, and culture environment humidity is controlled at 86～94%, and incubation time is 3～4 days.
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| CN103468629B (en) * | 2013-09-11 | 2015-04-08 | 中国科学院南京土壤研究所 | Production process for fermenting trichoderma spp by taking orange peels as raw materials |
| CN105018445A (en) * | 2015-08-05 | 2015-11-04 | 吉林市新陆生态农业科技有限公司 | Cellulase preparation for composite biological fungus fertilizer and preparation method thereof |
| CN106588467A (en) * | 2016-12-09 | 2017-04-26 | 兴业县保旺生物肥有限公司 | Biological fertilizer special for radix sophorae subprostratae and preparation method thereof |
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