阅读说明：本技术 一种牛粪纤维素降解菌降解能力的判定方法 (Method for judging degradation capability of cow dung cellulose degradation bacteria ) 是由 马培杰 李亚娇 韩永芬 王小利 牟琼 李娟� 舒健虹 付薇 赵相勇 何静 于 2020-06-16 设计创作，主要内容包括：本发明属于微生物检测技术领域,公开了一种牛粪纤维素降解菌降解能力的判定方法,所述牛粪纤维素降解菌降解能力的判定方法包括进行牛粪的预处理得到牛粪液；将牛粪液与培养基混合,振荡培养；离心,取上清液作为牛粪样品；取培养基置于厌氧试管中,通入氮气填满试管；吸取牛粪样品加入试管,晃匀；恒温水浴培养后冷藏；试管壁上附着物即牛粪纤维素降解菌菌株；将牛粪纤维素降解菌菌株置于发酵培养基中进行发酵培养,离心,取上清液,即为酶液；计算酶液的纤维素酶酶活。本发明对牛粪进行充分处理,最大程度保留牛粪中的纤维素降解菌；在纤维素降解菌提取后进行酶液获取,通过酶液活性实现牛粪纤维素降解菌降解能力判定,判定结果更准确。(The invention belongs to the technical field of microbial detection, and discloses a method for judging the degradation capability of cow dung cellulose degrading bacteria, which comprises the steps of pretreating cow dung to obtain cow dung liquid; mixing the cattle manure liquid with a culture medium, and performing shaking culture; centrifuging, and taking supernatant as a cow dung sample; placing the culture medium in an anaerobic test tube, and introducing nitrogen to fill the test tube; sucking a cow dung sample, adding the cow dung sample into a test tube, and shaking uniformly; culturing in constant temperature water bath, and refrigerating; the attachment on the test tube wall is the cow dung cellulose degrading bacterial strain; placing the cow dung cellulose degrading bacterial strain in a fermentation culture medium for fermentation culture, centrifuging, and taking supernatant fluid, namely enzyme fluid; and calculating the cellulase activity of the enzyme solution. The method fully treats the cow dung, and maximally retains cellulose degrading bacteria in the cow dung; enzyme liquid is obtained after the cellulose degrading bacteria are extracted, the degrading capability of the cow dung cellulose degrading bacteria is judged through the activity of the enzyme liquid, and the judgment result is more accurate.)

1. A method for judging the degradation capability of cow dung cellulose degrading bacteria is characterized by comprising the following steps:

pretreating cow dung to obtain cow dung liquid;

the pretreatment of the cow dung comprises the following steps:

(1.1) removing grass, branches and leaves and silt stained on the surface of the cow dung;

(1.2) measuring the water content of the cow dung, and putting the cow dung with the water content of 65-75% into a beaker;

(1.3) placing the beaker containing the cow dung on a shaking table, and carrying out shaking culture at room temperature to obtain a cow dung solid-liquid mixture;

and (1.4) fully stirring the cow dung sample to be detected until solid and liquid are fused to obtain cow dung liquid.

Step two, preparing a culture medium, mixing the cattle manure liquid and the culture medium according to a certain proportion, and placing the mixture on a shaking table for shake culture;

the culture medium configuration method comprises the following steps:

(2.1) adding 10g of beef extract and 20g of agar into 1000ml of distilled water to obtain a basic culture medium;

(2.2) adding 5-10g of glucose and 5-8g of yeast extract into the basic culture medium, and slowly shaking uniformly to obtain a mixed culture medium;

(2.3) slowly adding a prepared salt solution into the mixed culture medium to obtain a culture medium;

centrifuging the mixture after the shaking culture, and taking the supernatant as a cow dung sample;

the centrifugation method comprises the following steps:

(3.1) placing the mixture after shaking culture in a centrifuge; controlling the change of the centrifugal rotating speed, and stopping increasing the speed after the rotating speed is increased to 2500 rpm;

(3.2) keeping the rotating speed after the speed rising is finished, and centrifuging for 3 min; continuously increasing the rotating speed, controlling the change of the rotating speed, and stopping increasing the rotating speed after the rotating speed is increased to 4000 rpm;

(3.3) keeping the rotating speed after the speed rising is finished, centrifuging for 6min, and finishing centrifuging; controlling the change of the rotating speed, and stopping the speed reduction after the rotating speed is reduced to 3000 rpm;

(3.4) keeping the rotating speed unchanged for a period of time after the speed reduction is finished; continuously reducing the speed, controlling the change of the rotating speed, and stopping reducing the speed after the rotating speed is reduced to 1000 rpm; keeping the rotating speed unchanged for a period of time after the speed reduction is finished;

(3.5) closing the centrifugal machine after the speed stabilization is finished, and stopping free inertia of the rotor;

step four, placing the culture medium prepared in the step two in an anaerobic test tube, covering a cover, and introducing nitrogen until the test tube is filled with nitrogen;

step five, sucking a cow dung sample by using a syringe, adding the cow dung sample into a test tube, and shaking uniformly;

placing the test tube in an environment with the temperature of 35-42 ℃, culturing for 4 hours in a constant-temperature water bath, and placing the test tube in a refrigeration state at the temperature of 5-8 ℃ for 3 hours after the culture is finished;

taking out the test tube, opening the cover, and scraping attachments on the wall of the test tube to obtain the cow dung cellulose degrading bacterial strain;

placing the cow dung cellulose degrading bacterial strain in a fermentation culture medium for fermentation culture, centrifuging a fermentation culture solution after the culture is finished, and taking a supernatant, namely an enzyme solution;

and step nine, calculating the cellulase activity of the enzyme solution, namely the degradation capability of the cow dung cellulose degradation bacteria.

2. The method for determining the degrading ability of cow dung cellulose degrading bacteria according to claim 1, wherein in the first step, the oscillation time of cow dung is 2-3 days.

3. The method for determining the degrading ability of cow dung cellulose degrading bacteria according to claim 1, wherein in the second step, the salt solution preparing method comprises: 2g of MnSO were used₄·4H₂O、2g FeSO₄·7H₂O, 2g NaCl, 1000ml saline solution was prepared with distilled water.

4. The method for determining the degrading ability of the cow dung cellulose degrading bacteria as claimed in claim 1, wherein in the second step, the cow dung liquid and the culture medium are mixed according to the mass ratio of 1: 8.

5. The method for determining the degradation capability of the cow dung cellulose degradation bacteria according to claim 1, wherein in the third step, the placing the mixture on a shaking table for shaking culture specifically comprises: the culture temperature was set at 28 ℃, the oscillation frequency was set at 200rpm, and the culture time was set at 25 min.

6. The method for determining the degradation capability of cow dung cellulose-degrading bacteria according to claim 1, wherein in the fourth step, resazurin is added into the test tube before the test tube is filled with nitrogen gas, and when the resazurin becomes colorless, the oxygen in the test tube is exhausted, and the test tube is filled with nitrogen gas.

7. The method for determining the degradation capability of the cow dung cellulose-degrading bacteria according to claim 1, wherein in the step eight, the step of placing the cow dung cellulose-degrading bacteria strain in a fermentation culture medium for fermentation culture specifically comprises: placing the cow dung cellulose degrading bacterial strain in a fermentation culture medium, setting the culture condition to be 37 ℃, culturing at 150rpm, and culturing for 3 days.

8. The method for determining the degrading ability of cow dung cellulose degrading bacteria according to claim 1, wherein in the eighth step, the centrifuging of the fermentation culture solution specifically comprises: the fermentation broth was centrifuged at 3000rpm for 20 min.

9. The method for determining the degrading ability of the cow dung cellulose-degrading bacteria according to claim 1, wherein in the ninth step, before the calculation of the cellulase activity of the enzyme solution, enzyme solution treatment is further performed, and the method comprises:

1) adding enzyme solution into the test tube, shaking, heating in water bath at 60 deg.C for 10 min;

2) dripping 1mL of 2mol/L NaOH solution and 2mL of DNS developer into the test tube, and slowly shaking uniformly;

3) adding the enzyme solution into the test tube again and shaking;

4) heating the test tube in water for 10min, cooling, and diluting with distilled water to 20 ml;

5) and (5) measuring the glucose content.

10. The method for determining the degrading ability of the cow dung cellulose degrading bacteria as claimed in claim 1, wherein in the ninth step, the calculating the cellulase enzyme activity of the enzyme solution specifically comprises:

the enzyme activity is equal to the volume of the glucose solution/10 times the volume of the crude enzyme solution.

Technical Field

The invention belongs to the technical field of microbial detection, and particularly relates to a method for judging the degradation capability of cow dung cellulose degrading bacteria.

Background

At present: cellulose is a renewable biomass resource with the largest storage capacity on the earth and widely exists in branches, leaves and stalks of plants. The cellulose can be decomposed into related products by microorganisms with cellulose degradation capability, and the method is widely applied to the fields of food, chemical industry, textile, feed, wine brewing, petroleum, exploration, drug extraction and biological energy. The key to cellulose utilization is cellulose-degrading bacteria. Therefore, it is very important to separate and screen cellulose-degrading strains having strong degrading ability from nature. At present, the cellulose degrading bacteria can be obtained by separating and purifying from nature through a primary screening culture medium taking filter paper as a unique carbon source and a method for re-screening a culture medium by using sodium carboxymethyl cellulose. Then, the degradation capability of the separated cellulose degrading bacteria is judged, and strains with wide application prospect and strong degradation capability are screened out. At present, the determination of the degradation capability of cellulose-degrading bacteria is generally carried out by comparing the activity of cellulase in the cellulose-degrading bacteria. The cellulase activity determination method has a plurality of methods, including endoglucanase activity determination, exoglucanase activity determination, beta-glucosidase activity determination and filter paper enzyme activity determination. The cow dung contains a large amount of cellulose decomposing bacteria, and has great development and application prospects. However, the existing method for determining the cellulose-degrading bacteria in the cow dung is very complicated and has a long process, and inconvenience is brought to the judgment of the degrading capability of the cellulose-degrading bacteria in the cow dung.

Through the above analysis, the problems and defects of the prior art are as follows: the existing method for determining the cellulose degrading bacteria in the cow dung is very complicated and has a long process, and inconvenience is brought to the judgment of the degrading capability of the cellulose degrading bacteria in the cow dung.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for judging the degradation capability of cow dung cellulose degrading bacteria.

The invention is realized in such a way that the method for judging the degradation capability of the cow dung cellulose degradation bacteria comprises the following steps:

pretreating cow dung to obtain cow dung liquid;

the pretreatment of the cow dung comprises the following steps:

(1.1) removing grass, branches and leaves and silt stained on the surface of the cow dung;

(1.2) measuring the water content of the cow dung, and putting the cow dung with the water content of 65-75% into a beaker;

(1.3) placing the beaker containing the cow dung on a shaking table, and carrying out shaking culture at room temperature to obtain a cow dung solid-liquid mixture;

and (1.4) fully stirring the cow dung sample to be detected until solid and liquid are fused to obtain cow dung liquid.

Step two, preparing a culture medium, mixing the cattle manure liquid and the culture medium according to a certain proportion, and placing the mixture on a shaking table for shake culture;

the culture medium configuration method comprises the following steps:

(2.1) adding 10g of beef extract and 20g of agar into 1000ml of distilled water to obtain a basic culture medium;

(2.2) adding 5-10g of glucose and 5-8g of yeast extract into the basic culture medium, and slowly shaking uniformly to obtain a mixed culture medium;

and (2.3) slowly adding a prepared salt solution into the mixed culture medium to obtain the culture medium.

Centrifuging the mixture after the shaking culture, and taking the supernatant as a cow dung sample;

the centrifugation method comprises the following steps:

(3.1) placing the mixture after shaking culture in a centrifuge; controlling the change of the centrifugal rotating speed, and stopping increasing the speed after the rotating speed is increased to 2500 rpm;

(3.2) keeping the rotating speed after the speed rising is finished, and centrifuging for 3 min; continuously increasing the rotating speed, controlling the change of the rotating speed, and stopping increasing the rotating speed after the rotating speed is increased to 4000 rpm;

(3.3) keeping the rotating speed after the speed rising is finished, centrifuging for 6min, and finishing centrifuging; controlling the change of the rotating speed, and stopping the speed reduction after the rotating speed is reduced to 3000 rpm;

(3.4) keeping the rotating speed unchanged for a period of time after the speed reduction is finished; continuously reducing the speed, controlling the change of the rotating speed, and stopping reducing the speed after the rotating speed is reduced to 1000 rpm; keeping the rotating speed unchanged for a period of time after the speed reduction is finished;

(3.5) closing the centrifugal machine after the speed stabilization is finished, and stopping free inertia of the rotor;

step four, placing the culture medium prepared in the step two in an anaerobic test tube, covering a cover, and introducing nitrogen until the test tube is filled with nitrogen;

step five, sucking a cow dung sample by using a syringe, adding the cow dung sample into a test tube, and shaking uniformly;

placing the test tube in an environment with the temperature of 35-42 ℃, culturing for 4 hours in a constant-temperature water bath, and placing the test tube in a refrigeration state at the temperature of 5-8 ℃ for 3 hours after the culture is finished;

taking out the test tube, opening the cover, and scraping attachments on the wall of the test tube to obtain the cow dung cellulose degrading bacterial strain;

placing the cow dung cellulose degrading bacterial strain in a fermentation culture medium for fermentation culture, centrifuging a fermentation culture solution after the culture is finished, and taking a supernatant, namely an enzyme solution;

and step nine, calculating the cellulase activity of the enzyme solution, namely the degradation capability of the cow dung cellulose degradation bacteria.

Further, in the step one, the oscillation time of the cow dung is 2-3 days.

Further, in the second step, the saline solution preparation method includes: 2g of MnSO were used₄·4H₂O、2g FeSO₄·7H₂O, 2g NaCl, made up to 1000ml of saline solution with distilled water.

Further, in the second step, the cattle manure liquid and the culture medium are mixed according to the mass ratio of 1: 8.

Further, in the third step, the shaking culture of the mixture on a shaking table specifically comprises: the culture temperature was set at 28 ℃, the oscillation frequency was set at 200rpm, and the culture time was set at 25 min.

Further, in the fourth step, adding resazurin into the test tube before the test tube is filled with nitrogen, wherein oxygen in the test tube is exhausted when the resazurin becomes colorless, and the test tube is filled with nitrogen.

Further, in the eighth step, the step of placing the cow dung cellulose degrading bacterial strain in a fermentation culture medium for fermentation culture specifically comprises: placing the cow dung cellulose degrading bacterial strain in a fermentation culture medium, setting the culture condition to be 37 ℃, culturing at 150rpm, and culturing for 3 days.

Further, in the eighth step, the centrifuging of the fermentation culture solution specifically comprises: the fermentation broth was centrifuged at 3000rpm for 20 min.

Further, in the ninth step, before calculating the cellulase activity of the enzyme solution, enzyme solution treatment is further performed, including:

1) adding enzyme solution into the test tube, shaking, heating in water bath at 60 deg.C for 10 min;

2) dripping 1mL of 2mol/L NaOH solution and 2mL of DNS developer into the test tube, and slowly shaking uniformly;

3) adding the enzyme solution into the test tube again and shaking;

4) heating the test tube in water for 10min, cooling, and diluting with distilled water to 20 ml;

5) and (5) measuring the glucose content.

Further, in the ninth step, the calculating of the cellulase enzyme activity of the enzyme solution specifically comprises:

enzyme activity (u/ml) ═ glucose amount/10 × volume of crude enzyme solution;

wherein u is the amount of enzyme that catalyzes the hydrolysis of cellulose to 1ug glucose per minute; the unit of glucose is mg; and 10 is the holding time.

By combining all the technical schemes, the invention has the advantages and positive effects that: according to the invention, the cow dung is fully treated, the cellulose degrading bacteria in the cow dung are retained to the greatest extent, the damage or insufficient extraction to the cow dung is avoided, and the accuracy of judging the degrading capability of the cow dung cellulose degrading bacteria can be improved; the enzyme liquid is obtained after the cellulose degrading bacteria in the cow dung are extracted, the degrading capability of the cow dung cellulose degrading bacteria is judged through the activity of the enzyme liquid, the number of intervention factors is less than that of intervention factors obtained by directly observing and calculating the degrading capability after strains are screened, and the judgment result is more accurate.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

FIG. 1 is a flowchart of a method for determining the degradation ability of cow dung cellulose-degrading bacteria, which is provided by the embodiment of the present invention.

FIG. 2 is a flow chart of the pretreatment of cow dung provided by the embodiment of the invention.

FIG. 3 is a flow chart of the preparation of a culture medium according to an embodiment of the present invention.

FIG. 4 is a flowchart of the enzyme solution treatment provided in the embodiment of the present invention.

FIG. 5 is a graph comparing the duration of degradation of different cow dung cellulose-degrading bacteria provided by the embodiments of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a method for judging the degradation capability of cow dung cellulose degrading bacteria, and the invention is described in detail with reference to the attached drawings.

As shown in fig. 1, the method for determining the degradation capability of cow dung cellulose-degrading bacteria provided by the embodiment of the present invention includes the following steps:

s101, pretreating cow dung to obtain cow dung liquid;

s102, preparing a culture medium, mixing the cattle manure liquid and the culture medium according to the mass ratio of 1:8, and placing the mixture on a shaking table for shake culture;

s103, centrifuging the mixture after shaking culture, and taking the supernatant as a cow dung sample;

s104, placing the culture medium prepared in the step S102 in an anaerobic test tube, covering a cover, and introducing nitrogen until the test tube is filled with nitrogen;

s105, sucking a cow dung sample by using a syringe, adding the cow dung sample into a test tube, and shaking uniformly;

s106, placing the test tube in an environment with the temperature of 35-42 ℃, culturing for 4 hours in a constant-temperature water bath, and placing the test tube in a refrigerated state at the temperature of 5-8 ℃ for 3 hours after the culture is finished;

s107, taking out the test tube, uncovering the test tube, and scraping attachments on the wall of the test tube to obtain the cow dung cellulose degradation bacterial strain;

s108, placing the cow dung cellulose degrading bacterial strain in a fermentation culture medium for fermentation culture, centrifuging a fermentation culture solution after the culture is finished, and taking a supernatant, namely an enzyme solution;

s109, calculating the cellulase activity of the enzyme solution, namely the degradation capability of the cow dung cellulose degradation bacteria.

As shown in fig. 2, in step S101, the pretreatment of cow dung provided by the embodiment of the present invention includes the following steps:

s201, removing grass, branches and leaves, and silt stained on the surface of the cow dung;

s202, measuring the water content of the cow dung, and putting the cow dung with the water content of 65-75% into a beaker;

s203, placing the beaker containing the cow dung on a shaking table, and carrying out shaking culture at room temperature for 2-3 days to obtain a cow dung solid-liquid mixture;

and S204, fully stirring the cow dung sample to be detected until solid and liquid are fused to obtain cow dung liquid.

As shown in fig. 3, in step S102, the preparing of the culture medium provided by the embodiment of the present invention specifically includes:

s301, adding 10g of beef extract and 20g of agar into 1000ml of distilled water to obtain a basic culture medium;

s302, adding 5-10g of glucose and 5-8g of yeast extract into a basic culture medium, and slowly shaking uniformly to obtain a mixed culture medium;

s303, using 2g MnSO₄·4H₂O、2g FeSO₄·7H₂Preparing 1000ml of salt solution by using distilled water and O and 2g of NaCl;

and S304, slowly adding a salt solution into the mixed culture medium to obtain a culture medium.

In step S102, the step of placing the mixture on a shaking table for shake cultivation provided by the embodiment of the present invention specifically comprises: the culture temperature was set at 28 ℃, the oscillation frequency was set at 200rpm, and the culture time was set at 25 min.

In step S103, the centrifugation method provided in the embodiment of the present invention includes: placing the mixture after shaking culture in a centrifuge; controlling the change of the centrifugal rotating speed, and stopping increasing the speed after the rotating speed is increased to 2500 rpm; keeping the rotating speed after the speed rising is finished, and centrifuging for 3 min; continuously increasing the rotating speed, controlling the change of the rotating speed, and stopping increasing the rotating speed after the rotating speed is increased to 4000 rpm; keeping the rotating speed after the speed rising is finished, centrifuging for 6min, and finishing centrifuging; controlling the change of the rotating speed, and stopping the speed reduction after the rotating speed is reduced to 3000 rpm; keeping the rotating speed unchanged for a period of time after the speed reduction is finished; continuously reducing the speed, controlling the change of the rotating speed, and stopping reducing the speed after the rotating speed is reduced to 1000 rpm; keeping the rotating speed unchanged for a period of time after the speed reduction is finished; closing the centrifugal machine after the speed stabilization is finished, and stopping free inertia of the rotor;

in step S104, adding resazurin into the test tube before filling the test tube with nitrogen gas, where the oxygen in the test tube is exhausted when the resazurin becomes colorless, and the test tube is filled with nitrogen gas.

In step S108, the step of placing the cow dung cellulose degrading bacterial strain in the fermentation medium for fermentation culture provided by the embodiment of the present invention specifically comprises: placing the cow dung cellulose degrading bacterial strain in a fermentation culture medium, setting the culture condition to be 37 ℃, culturing at 150rpm, and culturing for 3 days.

In step S108, the centrifugation of the fermentation culture solution provided by the embodiment of the present invention specifically includes: the fermentation broth was centrifuged at 3000rpm for 20 min.

As shown in fig. 4, in step S109, before calculating the cellulase activity of the enzyme solution, the enzyme solution treatment is further performed, which includes:

s401, adding enzyme liquid into a test tube, shaking, heating in a water bath, and heating for 10min at the temperature of 60 ℃;

s402, dripping 1mL of 2mol/L NaOH solution and 2mL of DNS developer into a test tube, and slowly shaking uniformly;

s403, adding the enzyme solution into the test tube again and shaking;

s404, placing the test tube in water, heating for 10min, taking out, cooling, and fixing the volume to 20ml by using distilled water;

and S405, measuring the glucose content.

In step S109, the calculating of the cellulase enzyme activity of the enzyme solution provided by the embodiment of the present invention specifically includes:

enzyme activity (u/ml) ═ glucose amount/10 × volume of crude enzyme solution;

wherein u is the amount of enzyme that catalyzes the hydrolysis of cellulose to 1ug glucose per minute; the unit of glucose is mg; and 10 is the holding time.

The technical solution of the present invention is further described with reference to the following specific examples.