CN106244481A - A kind of compound micro-ecological preparation, its preparation method and application processed for farm animal excrement - Google Patents

Abstract

The object of the present invention is to provide a kind of compound probiotics for the treatment of livestock and poultry excrement, its preparation method and application, based on 100g of livestock and poultry excrement, the composite probiotics that needs to be added is prepared by mixing the bacterial liquid of the following volume Ingredients: Lactobacillus casei 0.03~0.10 mL, Enterococcus faecalis 0.03~0.10 mL, Candida utilis 0.05~0.50 mL, Bacillus subtilis 0.03~0.10 mL, said Lactobacillus casei, Enterococcus faecalis, Pseudomonas utilis The effective number of viable bacteria in the bacterial solution of Trichosaccharomyces and Bacillus subtilis was 1.0×10 ⁹ cfu/mL. The present invention uses the compound microecological preparation to ferment livestock and poultry excrement, which can reduce the pH and the number of coliform bacteria (P>0.05), and reduce the content of indole by 58% (P<0.05), which is helpful for eliminating the odor of feces is of great significance.

Description

A compound microecological preparation for treating livestock and poultry excrement, its preparation method and application

technical field

The invention belongs to the technical field of probiotics, and in particular relates to a composite probiotic for treating livestock and poultry excrement, a preparation method and application thereof.

Background technique

According to reports, the annual production of livestock and poultry manure in the country is about 1.9 billion tons, which is 2.4 times that of industrial solid waste. In addition to the discharge of sewage, the waste discharged by the aquaculture industry in my country is as high as more than 6 billion tons every year; livestock and poultry waste Pollution has become one of the main causes of pollution. While the amount of livestock and poultry is kept increasing, the rural construction land reduces the area of farmland that can effectively carry livestock and poultry waste. With the continuous increase of intensive farming models, the output of livestock and poultry waste exceeds the load that local farmland can carry, which has a negative impact on the environment. cause serious pollution.

In 2003, the country formally implemented the Pollutant Discharge Standards for Livestock and Poultry Breeding Industry; on October 8, 2013, the State Council adopted the 26th executive meeting, and in November 2013 promulgated the Regulations on the Prevention and Control of Pollution from Large-scale Livestock and Poultry Breeding. Governments at all levels are required to give strong support to the breeding industry in terms of policies, funds, technology and project construction, take the road of "ecological and welfare" environmental protection, follow the new model of "combination of planting and breeding, ecological environmental protection, circular economy", and pursue innovative Breeding new technology to promote the orderly development of aquaculture and planting.

There are three main methods for the harmless treatment of livestock and poultry waste, namely biological, chemical and physical treatment. The chemical method is a method of using chemical reagents to treat feces. The principle is that the organic matter in the feces reacts with chemical substances. For example, H ₂ S can be oxidized into hydrogen sulfide or sulfur or sulfur dioxide. Due to the unstable effect , making the application of this technique ineffective. Physical methods mainly include thermal spray treatment, high temperature drying, puffing treatment and natural drying, etc. However, there are still certain defects in physical treatment methods. The high-temperature drying method has the disadvantages of large investment in production equipment, high energy consumption, and the discharged gas is likely to cause secondary pollution. Biological method refers to the use of microorganisms to treat livestock and poultry manure in a specific environment. The biological method has the characteristics of high fertilizer efficiency and low cost. In addition, the biological treatment method also has the effect of sterilization and deodorization, which is a relatively reasonable method. Ecological treatment methods.

Contents of the invention

The object of the present invention is to provide a kind of compound probiotics for livestock and poultry excrement treatment, its preparation method and application, utilize this compound probiotics to ferment livestock and poultry excrement, can reduce wherein pH and Escherichia coli quantity (P >0.05), and reduced the content of indole by 58% (P<0.05), which is of great significance for eliminating the odor of feces.

The present invention adopts following technical scheme:

A compound microecological preparation for the treatment of livestock and poultry excrement. Based on 100g of livestock and poultry excrement, the compound microecological preparation to be added is prepared by mixing the following volumes of bacterial liquid: Lactobacillus casei 0.03~0.10 mL, fecal intestinal Coccus 0.03 ~ 0.10mL, Candida utilis 0.05 ~ 0.50 mL, Bacillus subtilis 0.03 ~ 0.10 mL, the effective effect of the bacterial liquid of Lactobacillus casei, Enterococcus faecalis, Candida utilis and Bacillus subtilis The number of viable bacteria was 1.0×10 ⁹ cfu/mL.

The above-mentioned compound probiotics for the treatment of livestock and poultry excrement, based on 100g of livestock and poultry excrement, the compound probiotics that need to be added are mixed with the following volumes of bacterial liquid: Lactobacillus casei 0.06 mL, Enterococcus faecalis 0.07 mL, Candida utilis 0.15 mL, Bacillus subtilis 0.07 mL, the effective number of viable bacteria of the bacterium liquid of described Lactobacillus casei, Enterococcus faecalis, Candida utilis and Bacillus subtilis is 1.0×10 ⁹ cfu/mL.

A preparation method for a compound probiotics for livestock and poultry excrement treatment, comprising the following steps:

(1) The strains of Lactobacillus casei, Enterococcus faecalis, Candida utilis and Bacillus subtilis were respectively inoculated into MRS liquid medium, MRS liquid medium, YPD liquid medium and LB liquid medium, and cultured to obtain their respective the bacterial liquid;

(2) Then, the obtained bacterial solution of Lactobacillus casei, Enterococcus faecalis, Candida utilis and Bacillus subtilis is mixed according to the volume ratio.

The preparation method of the above-mentioned compound probiotics for livestock and poultry excrement treatment, based on 100g of livestock and poultry excrement, when mixed in step (2), the Lactobacillus casei, Enterococcus faecalis, and Candida utilis The volume ratio of the bacterial liquid of Bacillus subtilis is: Lactobacillus casei 0.03~0.10 mL, Enterococcus faecalis 0.03~0.10 mL, Candida utilis 0.05~0.50 mL, Bacillus subtilis 0.03~0.10 mL, the The effective viable counts of Lactobacillus casei, Enterococcus faecalis, Candida utilis and Bacillus subtilis were all 1.0×10 ⁹ cfu/mL.

The preparation method of the above-mentioned compound probiotics for livestock and poultry excrement treatment, based on 100g of livestock and poultry excrement, when mixed in step (2), the Lactobacillus casei, Enterococcus faecalis, and Candida utilis The volume ratio of the bacterial liquid of Bacillus subtilis is: Lactobacillus casei 0.06 mL, Enterococcus faecalis 0.07 mL, Candida utilis 0.15 mL, Bacillus subtilis 0.07 mL, said Lactobacillus casei, Enterococcus faecalis, The effective number of viable bacteria in the bacterial solution of Candida utilis and Bacillus subtilis was 1.0×10 ⁹ cfu/mL.

For the application of the above-mentioned compound microecological preparation in the treatment of livestock and poultry excrement, livestock and poultry excrement is used as a fermentation medium, and the composite probiotic preparation is inoculated into the fermentation medium, and fermented and cultivated at 35°C~42°C for 100h ~140h.

For the application of the above-mentioned compound microecological preparation in the treatment of livestock and poultry excrement, livestock and poultry excrement is used as a fermentation medium, and the complex probiotic preparation is inoculated into the fermentation medium, and fermented and cultivated at 37°C~42°C for 120h .

The beneficial effects of the present invention are as follows:

The present invention utilizes the composite probiotics to treat the excrement of livestock and poultry. In the present invention, the excrement of livestock and poultry refers to the feces of livestock and poultry. It does not require the addition of chemical substances or the use of excessive equipment, and does not produce harmful gases that pollute the environment, effectively It greatly reduces the processing cost of livestock and poultry excrement, reduces energy consumption, and has a fast processing time. Moreover, using the compound probiotics of the present invention to treat livestock and poultry excrement has both sterilization and deodorizing effects, can reduce the pH and the number of Escherichia coli (P>0.05), and reduce the content of indole by 58% % (P<0.05), it is of great significance to eliminate the odor of feces, and the treated livestock and poultry excrement can be used as fertilizer, which is a more reasonable ecological treatment method.

Description of drawings

Fig. 1 is the standard curve of indole content;

The influence of Fig. 2 Lactobacillus casei and Enterococcus faecalis on indole content;

The influence of Fig. 3 Lactobacillus casei and Candida utilis on indole content;

The influence of Fig. 4 Lactobacillus casei and Bacillus subtilis on indole content;

The influence of Fig. 5 Enterococcus faecalis and Candida utilis on indole content;

The influence of Fig. 6 Bacillus subtilis and Enterococcus faecalis on indole content;

Figure 7 Effect of Bacillus subtilis and Candida utilis on indole content.

detailed description

In order to make the technical objectives, technical solutions and beneficial effects of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments are intended to explain the present invention, and cannot be interpreted as explanations for the present invention. Limitation of the invention, if the specific techniques or conditions are not indicated in the examples, it shall be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions, and the reagents or instruments used shall not indicate the manufacturer, all of which can be obtained through the market. conventional products purchased.

Example 1

1. Test materials and methods

1.1 Test material

1.1.1 Strain selection

Lactobacillus casei (CGMCC1.62) Lactobacillus casei , Candida utilis (CGMCC2.1004) Candida utilis , Enterococcus faecalis (CGMCC1.101) Enterococcus faecalis , Bacillus subtilis (CGMCC1.504) Bacillus subtilis were selected, and the above All strains were purchased from China General Microorganism Culture Collection and Management Center.

1.1.2 Instruments and equipment

Electric constant temperature water-proof incubator (Shanghai Yuejin Medical Machinery Factory);

Vertical high-pressure steam sterilizer (Shanghai Shen'an Medical Instrument Co., Ltd.);

BCM-1000 biological purification workbench (Suzhou Purification Equipment Co., Ltd.);

Double-layer steam bath oscillator (Jiangsu Jintan Jerry Electric Co., Ltd.);

PHS-2C acidity meter (Tianjin Saidelis Experimental Analysis Instrument Factory);

79-1 magnetic stirrer (Jintan Zhongda Instrument Factory);

High-speed refrigerated centrifuge;

UV spectrophotometer;

electronic analytical balance;

triangular flask;

petri dish.

1.1.3 Bacteria culture

LB liquid medium for cultivating Bacillus subtilis: tryptone 10 g, yeast extract powder 5 g, sodium chloride 10 g, dilute to 1 L with distilled water, sterilize at 121 °C, 0.15 MPa high-pressure steam for 20 min; solid Add 1.5% agar to the medium.

After Bacillus subtilis was cultured, screened and purified in LB solid medium, it was inoculated into LB liquid medium for expansion. The culture conditions were 37°C, 200 r/min, and the culture time was 24 h to obtain the bacterial liquid of Bacillus subtilis; the obtained subtilis The effective number of viable bacteria in the bacterial solution of Bacillus was adjusted to 1.0×10 ⁹ cfu/mL.

MRS liquid medium for cultivating Lactobacillus casei and Enterococcus faecalis: tryptone 15 g, yeast extract 10 g, glucose 20 g, Tween-80 1 mL, dipotassium hydrogen phosphate 2 g, sodium acetate 2 g, citric acid Ammonium 2 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, dilute to 1 L with distilled water, sterilize at 121 °C, 0.15 MPa high-pressure steam for 20 min for later use; add 1.5% agar to the solid medium.

Lactobacillus casei and Enterococcus faecalis were cultured, screened and purified on MRS solid medium, respectively, and then inoculated into MRS liquid medium for expansion. The culture conditions were 37°C and static culture for 24 hours to obtain Lactobacillus casei and Enterococcus faecalis, respectively. The bacterial liquid of Lactobacillus casei and Enterococcus faecalis obtained was adjusted to 1.0×10 ⁹ cfu/mL.

YPD liquid medium for cultivating Candida utilis: yeast extract powder 10 g, peptone 20 g, glucose 20 g, dilute to 1 L with distilled water, sterilize at 121°C, 0.15 MPa high-pressure steam for 20 min; solid Add 1.5% agar to the medium.

After Candida utilis was screened and purified by YPD solid medium culture, it was inoculated into YPD liquid medium for expansion culture. The culture conditions were 28°C, 200 r/min, and the culture time was 24 h to obtain Candida utilis The bacterial liquid of Candida utilis obtained was adjusted to 1.0×10 ⁹ cfu/mL for effective viable count.

Eosin methylene blue medium for cultivating Escherichia coli: (EMB, Aoboxing, Beijing): Weigh 42.5 g of EMB, dilute to 1 L with distilled water, and sterilize at 121°C, 0.15 MPa high-pressure steam for 20 min for later use. The culture conditions were 37°C, static culture for 48 h.

When using Lactobacillus casei, Candida utilis, Enterococcus faecalis and Bacillus subtilis in the following single factor test and response surface design test, each bacterial solution obtained above was used.

1.2 Test method

1.2.1 Single factor test

The bacterial solutions of Lactobacillus casei, Candida utilis, Enterococcus faecalis and Bacillus subtilis obtained in 1.1.3 were used to ferment livestock and poultry excrement respectively, and a single factor test was carried out.

Taking Lactobacillus casei as an example to illustrate the single factor test process, the single factor test process of other strains is the same:

(1) Firstly, the water content in livestock and poultry excreta was determined to be 69.69%, and five 250 mL Erlenmeyer flasks were taken, and 200 g of livestock and poultry excreta were added to each Erlenmeyer flask as a fermentation medium;

(2) The above five Erlenmeyer flasks were used as a control group and four test groups, respectively, and were inoculated with the 1.1.3 strain culture at different gradient volume-to-weight ratios of 0, 0.05%, 0.1%, 0.5% and 1%. The bacterial solution of Lactobacillus casei obtained in the above method (for example, the volume-to-weight ratio of 0.05% means that 100g of livestock and poultry excrement is added to 0.05mL of the bacterial solution), and the bacterial solutions with different gradient volume-to-weight ratios are diluted to the same volume with the corresponding medium ; So get the bacterium liquid of 0mL, 0.1mL, 0.2mL, 1.0mL and 2mL of Lactobacillus casei respectively and be diluted to 2mL with LB liquid culture medium, then add to control group and test group successively;

(3) Both the control group and the test group were fermented and cultured in a 37°C incubator for 120 h, and then samples were taken to measure water content, dry matter, total viable bacteria count, E. coli count, pH value, nitrogen and indole content and other indicators.

1) Determination of water content: adopt national standard GB/T6435-1986;

2) Total number of viable bacteria: LB agar plate coating method;

3) Escherichia coli count: eosin methylene blue plate coating method;

4) pH value: pH S-2C acidity meter (Tianjin Saidelis experimental analysis instrument);

5) N content: GB/T6432-94;

6) The determination method of indole acetic acid (IAA) content is as follows:

a. Drawing of IAA standard curve

Preparation of IAA standard solution: Accurately weigh 10 g of IAA (A.R.), dissolve it with a small amount of ethanol, and then dilute to 100 mL with distilled water (the concentration is 100 mg/mL) as a stock solution; then use the stock solution to make 0 mg/mL (blank), 0.5 mg/mL, 1.0 mg/mL, 5.0 mg/mL, 10.0 mg/mL, 15.0 mg/mL, 20.0 mg/mL, 25.0 mg/mL serial concentration standard solution match.

Reagent A: Prepared from 15 mL of 0.5 mol/L FeCl ₃ solution, 300 mL of concentrated H ₂ SO ₄ (specific gravity 1.84), and 500 mL of distilled water, mix well before use, and store away from light.

Reagent B: Prepared from 10 mL of 0.5 mol/L FeCl ₃ solution and 500 mL of 35% perchloric acid, mix and shake well before use, and store in the dark.

1 mL of test solution needs to add 4 mL of reagent A or 2 mL of reagent B, either of reagent A and reagent B can be selected (reagent B is more sensitive than reagent A)

Take 8 test tubes (marked as No. 0-7), add 2 mL of IAA series concentration standard solution in sequence, and add 4 mL of reagent B (or 8 mL of reagent A) respectively, and incubate in the dark at 40°C for 30 min (accelerated color development). reaction); colorimetric at 530 nm, with the OD value as the ordinate and the IAA concentration (mg/mL) as the abscissa, draw a standard curve, as shown in Figure 1.

a. Determination of sample IAA content

Weigh 10 g of livestock and poultry excrement fermented and cultured samples into a beaker, add 40 mL of 0.1 mol/L NaOH solution, boil in a water bath at 100°C for 15 min (cover the top to prevent the water from evaporating to dryness), take out the beaker and then Add 25 mL of 0.1 mol/L NaOH solution to it, shake well, then dilute to 100 mL with methanol, let stand for 30 min, and centrifuge for 30 min to get the supernatant, which is the IAA extract of the sample.

Take two test tubes, add 2 mL of the IAA extract of the above sample and 8 mL of reagent A to both test tubes, develop the color in a 40°C incubator for 30 min, and measure the color at 530 nm (set to zero with double distilled water) , record the OD value. Calculate the indole content in the sample according to the following formula (1):

Sample indole content (mg/g)=(A×V1)/(W×V2) (1)

In the formula: A—the concentration of IAA found on the standard curve (mg/mL);

V1—the volume of the IAA extract of the sample (mL);

W - weighed sample weight (g);

V2—the total volume of the sample reaction solution (mL), the total volume of the sample reaction solution refers to the total volume of the sample IAA extract and reagent A.

1.2.2 Single factor test results

According to the method described in the single factor test in 1.2.1, the fermentation culture test of the bacterial liquid of Lactobacillus casei, Candida utilis, Enterococcus faecalis and Bacillus subtilis on livestock and poultry excrement was carried out respectively, and a control group was set up , the treatment process of the control group is the same as that of the test groups, the only difference is that bacteria are not added, that is, the amount of bacteria added is 0%, and the detection of each index is carried out after the treatment.

1) Data statistics and analysis

The data obtained from the experiment were analyzed by ANOVA using SPSS software, and the results were expressed as "mean ± standard deviation", and P<0.05 indicated a significant difference. The obtained data are shown in Table 1 to Table 4.

Table 1 Single factor test of Lactobacillus casei fermented livestock and poultry excrement

Note: The same uppercase letters in the same row indicate no significant difference (P > 0.05), and different ones indicate significant difference (P < 0.05).

It can be seen from Table 1 that, compared with the control group, adding Lactobacillus casei during the fermentation of livestock and poultry excrement can increase the fermentation temperature to varying degrees, significantly reduce the nitrogen content, the total number of viable bacteria, the number of E. Material loss rate and indole content (P<0.05); when the addition of lactic acid bacteria was 0.5%, the temperature value was the highest, and the dry matter loss rate and indole content were the lowest. Since 0.05% and 0.5% of Lactobacillus casei had no significant difference (P>0.05) in increasing the fermentation temperature and reducing the indole content, the central dosage of Lactobacillus casei was 0.05% in the next response surface design experiment.

Table 2 Single factor test of Candida utilis fermentation of livestock and poultry excrement

Note: The same uppercase letters in the same row indicate no significant difference (P > 0.05), and different ones indicate significant difference (P < 0.05).

As can be seen from Table 2, the temperature value, nitrogen content, pH value, total number of live bacteria, number of Escherichia coli, dry matter loss rate and indole content in the single factor test of Candida utilis have significant differences between the control group and each test group (P<0.05); the temperature value was the highest and the indole content was the lowest when the addition of Candida utilis was 0.1% (P<0.05), and the number of E. coli and the loss rate of dry matter were the lowest when the amount of Candida utilis was 0.5% (P<0.05). Focusing on the indole content factor, 0.1% Candida utilis was used as the central dose of the next response surface design experiment.

Table 3 Single factor test of Enterococcus faecalis fermented livestock and poultry excrement

Note: The same uppercase letters in the same row mean no significant difference (P > 0.05), different means significant difference (P < 0.05)

It can be seen from Table 3 that in the single factor test of Enterococcus faecalis, the temperature value, pH value, total number of viable bacteria, number of Escherichia coli, dry matter loss rate and indole content were significantly different between the control group and each test group (P<0.05) ; The temperature value of Enterococcus faecalis was the highest at 0.05%, the dry matter loss rate was the lowest, the nitrogen content was relatively high at 1%, the indole content was the smallest, and the number of E. coli was the lowest at 0.5%. Considering comprehensively, 0.05% Enterococcus faecalis was used as the central dose of the next response surface design experiment.

Table 4 Single factor test of Bacillus subtilis fermented livestock and poultry excrement

Note: The same uppercase letters in the same row mean no significant difference (P > 0.05), different means significant difference (P < 0.05)

As can be seen from Table 4, the temperature value, nitrogen content, total viable count, dry matter loss rate and indole content in the Bacillus subtilis single factor test were significantly different between the control group and each test group (P<0.05); The dry matter loss rate was the lowest when the addition amount was 0.05%, the temperature value was the highest when the addition amount was 0.5%, and the indole content was the smallest when the addition amount was 1%. Considering comprehensively, 0.05% Bacillus subtilis was used as the central dose of the next response surface design experiment.

Example 2

2.1 Compound probiotics

According to the single factor test results in Example 1, the bacterial liquid of Lactobacillus casei, Enterococcus faecalis, Candida utilis and Bacillus subtilis fermented and cultivated livestock and poultry excrement, all of which can reduce Escherichia coli therein to varying degrees. It can reduce the number and indole content, eliminate the odor of livestock and poultry excrement, and play an important role in the harmless treatment of livestock and poultry excrement.

In this example, based on 100 g of livestock and poultry excrement, the following volume ranges of bacterial liquids were used: Lactobacillus casei 0.03-0.10 mL, Enterococcus faecalis 0.03-0.10 mL, Candida utilis 0.05-0.50 mL, Bacillus subtilis Bacillus 0.03~0.10 mL, take different volume values to combine (see Table 5 below), mix well to get multiple combinations of compound probiotics, and use the compound probiotics for fermentation and culture tests of livestock and poultry excrement , the test method of fermentation culture is the same as the method in 1.2.1, that is, a 250 mL Erlenmeyer flask is used, and the sampling volume of livestock and poultry excrement is 200 g as the fermentation medium, and the obtained compound microecological preparation is inoculated according to the ratio, and cultured at 37 °C box, take samples after 120 hours, and test related indicators (see Table 6 below).

2.2 Response surface regression design of optimal compound microecological preparation

2.2.1 Experimental design

According to the results of the single factor test, Lactobacillus casei ( Lactobacillus casei ): 0.05%, Enterococcus faecalis ( Enterococcus faecalis ): 0.05%, Candida utilis ( Candida utilis ): 0.10 %, Bacillus subtilis ( Bacillus subtilis ): 0.05% was used as four factors for response surface regression design, Table 5 is the level code of the experimental factors; the indole content was taken as the response value R1, Table 6 is the experimental design and results. Design-expert software was used for experimental design and analysis.

The regression equation obtained from the experiment is: R1=0.12 + 0.007275A + 0.013B + 0.010C - 0.011D+ 0.027AB - 0.00285AC - 0.007625AD - 0.011BC + 0.0041BD + 0.008025CD ⁺ 0.009164A ² + 0.07 -02 C ² + 0.016D ² .

Table 5 Factor level coding

Note: The amount of each bacterial solution listed in Table 5 is based on 100g of livestock and poultry excrement.

Table 6 Experimental design and results

The experimental design and results are shown in Table 6, combined with the response surface analysis results shown in Figures 2 to 7 below:

It can be seen from Figure 2 that the interaction between Lactobacillus casei and Enterococcus faecalis has a significant effect on reducing the indole content in livestock and poultry manure. When Enterococcus faecalis is at 0.07% and Lactobacillus is at about 0.06%, the indole content is the smallest.

It can be seen from Figure 3 that the interaction between Candida utilis and Lactobacillus casei has a certain effect on reducing the indole content in livestock and poultry manure. Candida utilis had the lowest indole content at 0.1% and Lactobacillus casei at about 0.06%.

It can be seen from Figure 4 that the interaction between Lactobacillus casei and Bacillus subtilis can significantly reduce the content of indole in livestock and poultry manure.

It can be seen from Figure 5 that the interaction between Enterococcus faecalis and Candida utilis has a significant effect on reducing the content of indole in livestock and poultry manure. When Enterococcus faecalis is 0.07% and Candida utilis is at 0.15%, indole The content is lower.

It can be seen from Figure 6 that the interaction between Enterococcus faecalis and Bacillus subtilis can significantly reduce the content of indole in livestock and poultry manure, and the indole content is the lowest when the two are around 0.07%.

It can be seen from Figure 7 that the interaction between Candida utilis and Bacillus subtilis is more significant in reducing the content of indole in livestock and poultry manure. Low.

Therefore, when the volume-to-weight ratio of the compound microecological preparation to livestock and poultry excrement is the following value, the treatment effect is the best: Lactobacillus casei 0.06%, Enterococcus faecalis 0.07%, Candida utilis 0.15%, Bacillus subtilis 0.07% %; that is, the optimal ratio of each bacterial solution included in the compound microecological preparation is: based on 100g of livestock and poultry excrement, Lactobacillus casei 0.06 mL, Enterococcus faecalis 0.07 mL, Candida utilis 0.15 mL, Bacillus subtilis Bacillus 0.07 mL.

2.2.2 Response Surface Regression Design Analysis of Variance

From the variance analysis results of the response surface regression equation coefficients in Table 7, it can be seen that the model Prob>F value is less than 0.01, indicating that the model has high reliability, so the obtained optimal ratio of the compound microecological preparation has high reliability.

Table 7 Analysis of variance of coefficients of response surface regression equation

Note: Prob>F less than 0.05 indicates that the model or factors under investigation have a significant impact, and Prob>F less than 0.01 indicates that the impact is extremely significant.

Example 3: Effects of compound probiotics on Escherichia coli and odor from livestock and poultry excrement

The compound microecological preparation with the optimal proportion obtained in Example 2 is carried out the fermentation culture experiment of livestock and poultry excrement, counts 100g of livestock and poultry excrement, the bacterium liquid of following volume: Lactobacillus casei 0.06 mL, Enterococcus faecalis 0.07 mL, 0.15 mL of Candida utilis, and 0.07 mL of Bacillus subtilis were mixed to obtain a compound microecological preparation, and the compound microecological preparation was used as a fermentation test of livestock and poultry excrement. The test method of fermentation culture was the same as that in 2.1. The method in the article is to use a 250 mL Erlenmeyer flask with a sampling volume of 200 g, inoculate the obtained compound microecological preparation according to the ratio, place it in a 37°C incubator, and take a sample after 120 hours to detect related indicators.

Table 8 Changes of pH value, indole content and Escherichia coli number in the excrement of livestock and poultry fermented by compound microbial preparation (n=6)

It can be seen from Table 8 that the use of the optimal ratio of compound probiotics to ferment livestock and poultry excrement can reduce the pH and the number of Escherichia coli (P>0.05), and reduce the content of indole by 58% (P<0.05) , It is of great significance to eliminate the odor of feces.

Finally, it should be noted that: the above-mentioned embodiments are only used to illustrate and not limit the technical solutions of the present invention, and any equivalent replacements to the present invention and modifications or partial replacements that do not depart from the spirit and scope of the present invention shall be covered by this disclosure. within the scope of protection of the invention claims.

Claims (7)

1. the compound micro-ecological preparation processed for farm animal excrement, it is characterised in that in terms of 100g farm animal excrement, needs Compound micro-ecological preparation to be added is mixed by the bacterium solution of volumes below: lactobacillus casei 0.03 ~ 0.10 mL, excrement intestinal ball Bacterium 0.03 ~ 0.10 mL, Candida utilis 0.05 ~ 0.50 mL, bacillus subtilis 0.03 ~ 0.10 mL, described cheese The living bacteria count of the bacterium solution of lactobacillus, enterococcus faecalis, Candida utilis and bacillus subtilis is 1.0 × 10⁹ cfu/mL。

The compound micro-ecological preparation processed for farm animal excrement the most according to claim 1, it is characterised in that with 100g Farm animal excrement meter, needs the compound micro-ecological preparation added to be mixed by the bacterium solution of volumes below: lactobacillus casei 0.06 ML, enterococcus faecalis 0.07 mL, Candida utilis 0.15 mL, bacillus subtilis 0.07 mL, described cheese milk bar The living bacteria count of the bacterium solution of bacterium, enterococcus faecalis, Candida utilis and bacillus subtilis is 1.0 × 10⁹ cfu/ mL。

3. the preparation method of the compound micro-ecological preparation processed for farm animal excrement, it is characterised in that include following step Rapid:

(1) bacterial strain of lactobacillus casei, enterococcus faecalis, Candida utilis and bacillus subtilis is inoculated into MRS respectively Fluid medium, MRS fluid medium, YPD fluid medium and LB fluid medium, cultivate and obtain respective bacterium solution；

(2) then by the bacterium solution of gained lactobacillus casei, enterococcus faecalis, Candida utilis and bacillus subtilis by volume Proportioning mixes and get final product.

The preparation method of the compound micro-ecological preparation processed for farm animal excrement the most according to claim 3, its feature It is, in terms of 100g farm animal excrement, in step (2) during mixing, described lactobacillus casei, enterococcus faecalis, Candida utilis The volume proportion of the bacterium solution of bacterium and bacillus subtilis is: lactobacillus casei 0.03 ~ 0.10 mL, enterococcus faecalis 0.03 ~ 0.10 ML, Candida utilis 0.05 ~ 0.50 mL, bacillus subtilis 0.03 ~ 0.10 mL, described lactobacillus casei, excrement intestinal ball The living bacteria count of the bacterium solution of bacterium, Candida utilis and bacillus subtilis is 1.0 × 10⁹ cfu/mL。

The preparation method of the compound micro-ecological preparation processed for farm animal excrement the most according to claim 4, its feature It is, in terms of 100g farm animal excrement, in step (2) during mixing, described lactobacillus casei, enterococcus faecalis, Candida utilis The volume proportion of the bacterium solution of bacterium and bacillus subtilis is: lactobacillus casei 0.06 mL, enterococcus faecalis 0.07 mL, product protein are false Silk yeast 0.15 mL, bacillus subtilis 0.07 mL, described lactobacillus casei, enterococcus faecalis, Candida utilis and The living bacteria count of the bacterium solution of bacillus subtilis is 1.0 × 10⁹ cfu/mL。

The compound micro-ecological preparation the most according to claim 1 and 2 application when farm animal excrement processes, its feature exists In: take farm animal excrement as fermentation medium, described compound micro-ecological preparation is seeded in described fermentation medium, 35 DEG C ~ 42 DEG C of fermentation culture 100h ~ 140h.

The compound micro-ecological preparation the most according to claim 4 application when farm animal excrement processes, it is characterised in that: take Described compound micro-ecological preparation, as fermentation medium, is seeded in described fermentation medium by farm animal excrement, 37 DEG C ~ 42 DEG C Fermentation culture 120h.