CN110036958B

CN110036958B - Breeding method for adjusting intestinal flora ecosystem of procambarus clarkii

Info

Publication number: CN110036958B
Application number: CN201910331409.4A
Authority: CN
Inventors: 魏柯
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-04-24
Filing date: 2019-04-24
Publication date: 2022-09-13
Anticipated expiration: 2039-04-24
Also published as: CN110036958A

Abstract

The invention discloses a culture method for adjusting an ecosystem of intestinal flora of procambarus clarkii, which comprises the steps of selecting healthy crayfish to obtain beneficial flora, selectively culturing the flora, fermenting the beneficial flora and wheat to coat the beneficial flora after the wheat is fermented, so that the beneficial flora can not be dissolved in water, the beneficial flora can effectively enter the intestinal tract through the ingestion of the crayfish, and the ecosystem of the intestinal tract of the crayfish can be adjusted to form dominant flora, thereby improving the resistance of the crayfish to pathogenic microorganisms. The breeding method can reduce the diseases of crayfishes and improve the survival rate.

Description

Breeding method for adjusting intestinal flora ecosystem of procambarus clarkii

Technical Field

The invention relates to the technical field of aquaculture, in particular to a culture method for adjusting an ecosystem of intestinal flora of procambarus clarkia.

Background

Procambarus clarkii (academic name: Procambarus clarkii): also known as crayfish, crayfish and freshwater crayfish. It is shaped like shrimp and hard shell. The length of the formed body is about 5.6-11.9 cm, the formed body is dark red, the crust part is nearly black, and the back of the abdomen is provided with a wedge-shaped stripe. The young shrimp bodies are uniformly gray and sometimes have black ripples. The chelate is long and narrow. The middle part of the crust is not separated by the mesh-shaped gap, and the crust is obviously provided with particles. The frontal sword has lateral spines or the end of the frontal sword has nicks.

The crayfish is a freshwater economic crayfish and is popular with people because of delicious meat taste. Due to the omnivorous property, the fast growth speed and the strong adaptability, the method forms an absolute competitive advantage in the local ecological environment. The food intake range of the feed comprises aquatic weeds, algae, aquatic insects, animal carcasses and the like, and the feed can kill the aquatic weeds, the algae, the aquatic insects, the animal carcasses and the like in case of shortage of food. Crayfish has become an important economic breed in china in recent years. Escape should be strictly prevented in the commercial culture process, especially from escaping into the original ecological water body with rare human beings. Which leads to destructive hazards in terms of ecological competitive advantages for local species.

With the increase of the demand, the artificial crayfish breeding is developed rapidly. The rapid expansion of the density and the area of modern crayfish aquaculture, however, the deterioration of the aquaculture environment and the development of the disease source; abuse of traditional disinfectors, antibiotics and other medicines cannot fully guarantee the smooth breeding of the crayfishes. And the adverse effect of the traditional method on the environment cannot be ignored, which is contradictory to the continuous development.

In order to improve the culture efficiency and reduce the pathogenic harm, beneficial bacteria are adopted to inhibit pathogenic microorganisms in the aquaculture process, so that the method is a feasible method.

However, in the actual culture process, the method for inhibiting pathogenic bacteria by using beneficial bacteria has poor implementation effect, and a large amount of disinfectants and antibiotics can still be used in the culture process. The reason is that: in different water environments and in huge water bodies, dominant bacteria are difficult to form by using beneficial bacteria with common dosage, so that the ecological benefit is low, and if the beneficial bacteria with large dosage are used, the cost is greatly increased.

In order to solve the problems, the feed can be mixed with beneficial bacteria and then fed or the feed is fermented conventionally in the prior art, and the aim is to ensure that the beneficial bacteria are directly taken into intestinal tracts. However, the problem of dissolving can occur through a material mixing mode, namely the feed is immediately dissolved into the water body after being put into use, beneficial bacteria obtained after the crayfish eats are still less, and the effect is reduced. Meanwhile, after the beneficial bacteria and the feed are compounded, the problems of activity maintenance and convenient storage are also faced besides the problem of dissolution.

Disclosure of Invention

The invention aims to provide a culture method for adjusting an intestinal flora ecosystem of procambarus clarkii, which can ensure that the procambarus clarkii obtains beneficial flora by ingesting fermented wheat feed, adjust the intestinal ecosystem, obtain better resistance to pathogenic microorganisms and have higher culture success rate; meanwhile, the fermented wheat feed can avoid the problem of dissolving, beneficial flora can utilize endosperm, and the beneficial flora can enter the intestinal tract of the crayfish under the condition that wheat bran and the like are wrapped.

In order to achieve the above objects, one embodiment of the present invention provides a method for adjusting ecosystem of intestinal flora of procambarus clarkii, comprising the following steps:

(1) selecting healthy crayfish cultured conventionally, and putting the healthy crayfish in clear water to obtain fresh excrement of the healthy crayfish as a strain source;

(2) respectively adding strains into a plurality of groups of same basic culture media for culturing, detecting whether pathogenic microorganisms exist in the basic culture media after the flora is stable, discarding groups carrying the pathogenic microorganisms, mixing the rest normal groups to obtain normal flora, and adding probiotics into the basic culture media of the normal flora for secondary culture to stabilize the flora; detecting the total number of the floras to obtain normal bacterial liquid;

(3) selecting healthy crayfish, and breeding the crayfish by adopting feed, wherein the feed comprises basic feed and fermented wheat feed; the preparation method of the fermented wheat feed comprises the following steps:

(A) adding a plurality of times of normal bacterial liquid into dry wheat, sealing and fermenting, expanding the wheat during fermentation, filtering after fermentation, washing the expanded wheat with sterile water, and grinding the expanded wheat to obtain a flora which is reserved in wheat bran and can utilize endosperm; then carrying out anaerobic culture on the flora; obtaining zymophyte liquid after the culture is finished;

(B) repeating the step (A) on the flora of the zymophyte liquid for multiple times of stable culture to obtain stable zymophyte liquid containing stable zymophyte flora;

(C) gradually performing amplification culture on the stable zymophyte liquid to obtain an amplification culture bacterial liquid, adding a fixed culture medium into the amplification culture bacterial liquid, uniformly mixing to obtain paste, drying the paste, and crushing into powder to prepare a leavening agent;

(D) adding the leaven into water, uniformly mixing, sealing and culturing for 3-4 days, adding dry wheat after the culture is mature, continuing to seal, fermenting and culturing for 2-4 days to ferment and mature the wheat, and obtaining moist bacteria-rich wheat, namely fermented wheat feed.

Preferably, the basic culture medium comprises 5-15 g/L of brown sugar, 1-3 g/L of beef extract and 1-5 g/L of yeast extract; the basic culture is anaerobic culture, each group of floras are cultured for 5-10 generations, and the total number of the cultured floras is more than 1x10 ⁸ cfu/ml。

Preferably, the probiotics comprise lactobacillus, bacillus subtilis and yeast; continuously culturing for 5-10 generations after adding probiotics to stabilize the flora, wherein the total number of the cultured flora is more than 1x10 ⁸ cfu/ml。

Preferably, the probiotic comprises 1x10 ⁹ 10% cfu/ml lactic acid bacteria; 1x10 ⁹ cfu/ml Bacillus subtilis 5%, 1x10 ⁶ 5% cfu/ml baker's yeast.

Preferably, normal bacteria liquid with 2 times of volume is added into the wheat, and the time of sealed fermentation is 2-4 days.

Preferably, the fermentation flora in step (A) is subjected to two conventional anaerobic cultivations in a basal medium; and (D) repeating the step (A) for 10 times to obtain the stable zymogen liquid.

Preferably, the solid culture medium added into each 100L of the amplification culture bacterial liquid is 10kg of beef extract, 15kg of yeast extract and 50kg of brown sugar; the drying temperature of the paste is 40 ℃.

Preferably, 1kg of leaven and 30kg to 40kg of dry wheat are added to every 100 liters of water in the step (D); the ratio of the basic feed to the fermented wheat feed in the step (3) is 1: 1.

In summary, the invention has the following advantages:

1. maintaining the balance of the in-vivo and in-vitro micro-ecosystem of the crayfish: the crayfish has a certain amount of microbial population in intestinal tracts and is in a certain dynamic balance, when the crayfish body is influenced by various adverse factors such as feed change, environment temperature change and long-term use of antibiotics, the balance is lost, the original dominant population is changed, and the resistance of the crayfish body is reduced. At the moment, the bait containing the microecological preparation is mixed and taken, so that beneficial microorganisms are greatly proliferated in the intestinal tract, the pH value of the intestinal tract is reduced by producing metabolites and antibiotic-like substances, and the nutrient is competed with harmful microorganisms, so that the production and the proliferation of pathogenic microorganisms can be resisted, and the balance of the microecological system in the intestinal tract is maintained and restored.

2. Synthetase and vitamins: certain microecological preparations can generate various digestive enzymes in the crayfish body, synthesize various vitamins, amino acids, growth promoting factors and the like, secrete active substances, participate in energy and vitamin metabolism, and promote the crayfish to utilize feed. Some enzymes have low content in intestinal tracts of the crayfish and even cannot be synthesized at all, and beneficial microorganisms producing the enzymes play an important role in ensuring the life activity of the crayfish.

3. Antagonism and protection: some beneficial microorganisms can rapidly propagate in the crayfish intestines, compete with pathogenic microorganisms for intestinal colonization, and inhibit the pathogenic microorganisms from attaching to the intestinal cell wall and having competitive antagonism with the pathogenic microorganisms, so that the balance of intestinal microecological systems is protected.

4. Enhancing the immunity of the organism: some beneficial microorganisms can accelerate the development of immune organs of crayfish bodies and increase the number of TB lymphocytes, thereby improving the humoral immunity and cellular immunity level of crayfish.

5. Biological oxygen deprivation competition: the normal microbial flora in the intestinal tract of the crayfish mainly comprises anaerobic microorganisms, and when certain aerobic beneficial microorganisms enter the digestive tract in a spore state, the aerobic beneficial microorganisms are quickly proliferated, consume a large amount of oxygen in the intestinal tract, reduce the oxygen concentration in the intestinal tract, cause an environment which is beneficial to the growth of the anaerobic microorganisms and is not beneficial to the growth of aerobic pathogenic microorganisms, and simultaneously recover the normal microecological balance in vivo to achieve the purposes of preventing and treating diseases and promoting growth.

6. Reducing the generation of harmful substances: when activities of harmful microorganisms such as escherichia coli and the like in intestinal tracts of crayfish are enhanced, proteins are converted into ammonia, amine and other harmful substances, and the amount of the harmful microorganisms such as escherichia coli, salmonella and the like in the intestinal tracts can be obviously reduced by the microecological preparation, so that excessive production of ammonia and other putrefying substances is reduced, and feces odor is reduced. In addition, the beneficial microorganisms can utilize excessive organic matters in the water environment to synthesize thallus substances, so that the content of harmful substances such as ammonia nitrogen, nitrite nitrogen, hydrogen sulfide and the like in the environment is reduced, and the aquaculture water environment is purified.

Detailed Description

The invention provides a culture method for adjusting an ecosystem of intestinal flora of procambarus clarkii, which comprises the following steps:

(A) adding a plurality of times of normal bacterial liquid into the dried wheat, sealing and fermenting, expanding the wheat in the fermentation process, filtering after the fermentation is finished, washing the expanded wheat with sterile water, and grinding the expanded wheat to obtain a flora which is reserved in wheat bran and can utilize endosperm; then carrying out anaerobic culture on the flora; obtaining zymophyte liquid after the culture is finished;

(C) carrying out stepwise amplification culture on the stable zymophyte liquid to obtain an amplification culture bacterial liquid, adding a fixed culture medium into the amplification culture bacterial liquid, uniformly mixing to prepare paste, drying the paste, and crushing the dried paste into powder to prepare a leavening agent;

The invention comprises two technical contents, one is a preparation method of fermented wheat feed, and the other is a method for breeding crayfish by fermented wheat feed.

Example 1: method for obtaining fermentation strain of fermented wheat feed

(1) Healthy crayfish cultured conventionally is selected and placed in clear water to obtain fresh excrement of the healthy crayfish as a strain source. In the selection process, the crayfish is mainly observed whether the cheek of the crayfish is infected by microorganisms, whether the crayfish has abnormal color, whether the activity is qualified, whether the crayfish eats normally and the like; the healthy crayfishes qualified through multiple tests are used as strain carrying organisms. The conventional culture method of healthy crayfish can adopt the conventional culture method in the prior art, crayfish cultured in a paddy field can be selected, and crayfish cultured in a pond can also be selected; the breeding method has no influence on the selection of the healthy crayfishes, namely, the healthy crayfishes required by the invention can be selected by any conventional breeding method.

(2) Respectively adding strains into a plurality of groups of same basic culture media for culturing, detecting whether pathogenic microorganisms exist in the basic culture media after the flora is stable, discarding groups carrying the pathogenic microorganisms, mixing the rest normal groups to obtain normal flora, and adding probiotics into the basic culture media of the normal flora for secondary culture to stabilize the flora; detecting the total number of the floras to obtain normal bacterial liquid.

In the step (2), all microorganisms in the excrement are cultured and screened, the excrement contains intestinal probiotics of the crayfish, the intestinal ecosystem of the healthy crayfish is good, and beneficial bacteria are dominant bacteria, so that the bacterial flora can be obtained more easily by selecting the excrement of the healthy crayfish for culture. In the culture process, proper culture medium or necessary nutrient elements of the microorganism can be added according to the needs, and the culture medium in the prior art can be purchased for culture. The microorganism or flora of the invention can be added with culture solution, pH control and the like according to needs in the culture process, the basic culture medium or the culture medium used by the invention can be replaced by other culture media in the prior art, and the difference of the culture media can have certain influence on the growth rate of the flora.

For example, the basic culture medium comprises 5 g/L-15 g/L brown sugar, 1 g/L-3 g/L beef extract and 1 g/L-5 g/L yeast extract; the basic culture is anaerobic culture, 5-10 generations of each group of floras are cultured, and the total number of the cultured floras is more than 1x10 ⁸ cfu/ml。

Specifically, the basic culture medium can comprise 10g/L of brown sugar, 2g/L of beef extract and 3g of yeast extract; because the beneficial bacteria are anaerobic bacteria, anaerobic culture is also carried out, and the total bacterial population can reach 1x10 after subculture for 5-10 generations ⁸ cfu/ml。

The flora of the culture medium is monitored during the culture process, if a certain group of culture medium contains pathogenic microorganisms, different colonies can be formed or the growth of the probiotic flora is inhibited, and at the moment, the pathogenic microorganisms are discardedGroups of organisms, the remaining groups being mixed together to obtain a normal flora. The invention can avoid the pollution of microorganism and can obtain normal flora at the lowest cost. After the normal flora is combined, the existing other probiotics are added for secondary culture, and a proper amount of culture medium can be added or not added in the process of secondary culture according to the consumption condition of the culture medium. The added probiotics comprise lactobacillus, bacillus subtilis and yeast; after the probiotics are added, the culture is continued for 5-10 generations, so that the flora is stable, and the total number of the cultured flora is still more than 1x10 ⁸ cfu/ml; thus obtaining normal bacterial liquid.

Specifically, the probiotic bacteria include 1x10 ⁹ 10% cfu/ml lactic acid bacteria; 1x10 ⁹ cfu/ml Bacillus subtilis 5%, 1x10 ⁶ 5% cfu/ml baker's yeast. That is, the concentration of the added lactic acid bacteria solution is 1x10 ⁹ cfu/ml. The adding amount is 10% of the volume of the culture medium of the normal flora; the contents of the bacillus subtilis and the bread yeast are added by the method. After the normal bacterial liquid is obtained, the obtained bacterial liquid can be used as a strain for subsequent fermentation and can also be used for mixing materials.

Example 2: preparation method of fermented wheat feed

(A) Adding 2 times of normal bacteria liquid into dry wheat, sealing and fermenting for 3 days, swelling the wheat during fermentation, filtering with gauze after fermentation, washing the swelled wheat with sterile water, and grinding the swelled wheat to obtain a flora which can utilize endosperm and is retained in wheat bran; then carrying out anaerobic culture on the flora twice; and obtaining zymophyte liquid after the culture is finished.

(B) Repeating the step (A) on the flora of the zymophyte liquid for continuous 10 times of stable culture to obtain stable zymophyte liquid containing stable zymophyte flora;

(C) and carrying out stepwise amplification culture on the stable zymophyte liquid to obtain 100L of amplification culture bacteria liquid, adding a fixed culture medium into the 100L of amplification culture bacteria liquid, uniformly mixing to prepare paste, drying the paste, and crushing the dried paste into powder to prepare the leavening agent. Wherein the solid culture medium comprises 10kg of beef extract, 15kg of yeast extract and 50kg of brown sugar; the drying temperature of the paste is 40 ℃.

(D) Adding 0.7kg of leaven into 70L of water, uniformly mixing, sealing and culturing for 3-4 days, adding 25kg of dry wheat after the culture is mature, continuing to seal, fermenting and culturing for 2-4 days to ferment and mature the wheat, and obtaining about 50kg of moist bacteria-rich wheat, namely fermented wheat feed.

Example 3: ecological breeding of crayfish

Selecting healthy crayfish, soaking the healthy crayfish in normal bacterial liquid diluted by 1000 times, then putting the crayfish into a culture pond for isolated culture for 1 month, observing whether the crayfish has pathological changes in the whole process, discarding the crayfish with pathological changes, and culturing the crayfish without pathological changes by adopting feed, wherein the feed comprises basic feed and fermented wheat feed. The crayfish is soaked in the normal bacterial liquid for the purpose of detecting whether the bacterial liquid contains pathogenic microorganisms or not, and the secondary step can be omitted. The ratio of basic feed to fermented wheat feed is 1: 1. The basal feed can be purchased from conventional crayfish feed known in the art.

Test example 1: immunological competence test

The test method comprises the following steps: in the same farm, different adjacent ponds with similar pond environments are selected, the pond numbers are 1-10 in sequence, different methods are used at intervals for feeding, and a single pond is completely fed with compound feed, namely basic feed. 50% of compound feed and 50% of fermented wheat feed are used in the pond of the second generation. The feeding amount and the culture process of each pond are the same.

The test is started from the beginning of 3 months at the same time, the bottom of 4 months is supplemented properly, so that the crayfish in all the test ponds can be stored by about 150 jin/mu, and the disease peak period of 5-7 months can be reached at high density.

And (3) test results: and detecting the crayfish culture condition of each pond. Over 7 consecutive days, the odd ponds have different degrees of obvious death; no. two ponds have no obvious disease. The result shows that the method has obvious prevention and treatment effect on the bacterial infection of the crayfish and can improve the survival rate of the crayfish.

In the practical application of the invention, the fermented wheat feed has obvious food calling effect on crayfishes, and the use ratio of wheat and the compound feed can be comprehensively considered and adjusted according to the practical situation.

The crayfish fed with the feed prepared by the method can obviously cure tail fan edema disease and liver erosion diseases of the crayfish, and when the crayfish is fed with the feed by using the method for a long time, the crayfish can pass through the bacterial disease peak period by reasonably controlling the density and the water quality, is produced safely throughout the year, and has no early-stage characteristic of the bacterial disease. Meanwhile, the crayfish is bred by using the method, any disinfectant and antibiotic are not used in the breeding process, the produced crayfish is safer, and the breeding cost is reduced; the growth speed of the crayfish and the specification of the finished product are not reduced, and side effects are avoided.

The invention has the following optimization design scheme in the preparation process of fermented wheat feed:

example 4:

the optimized preparation method of the fermented wheat feed comprises the following steps:

(D) adding a leavening agent into water, uniformly mixing, performing sealed culture for 3-4 days, adding dry wheat after the culture is mature, continuing to perform sealed fermentation culture for 2-4 days to make the wheat mature, obtaining moist bacteria-rich wheat, taking out the moist bacteria-rich wheat, then placing the moist bacteria-rich wheat into an anti-dispersion solution, soaking for 5-10 min, and taking out the wheat with water after soaking, thus obtaining the fermented wheat feed. Wherein the anti-dissolving liquid comprises 100 parts by weight of water and 5-8 parts by weight of guar gum; 3-5 parts of hydroxypropyl cellulose; 1-3 parts of sodium caseinate.

The anti-dissolving liquid is designed and used for treating fermented wheat, a large amount of beneficial bacteria are adhered to the surface of the fermented wheat, the wheat is put into the anti-dissolving liquid for treatment, so that the anti-dissolving liquid can be adhered to the surface of the wheat to wrap the beneficial bacteria, and the beneficial bacteria are prevented from being dissolved by water and entering the water body and not entering the crayfish after the wheat is put into the culture pond. Meanwhile, the beneficial bacteria are reduced from being blended into the anti-dissolving liquid; the dual guarantee can improve the acquisition dosage of beneficial bacteria after crayfish ingests.

Example 5

(A) Adding 2 times volume of normal bacteria liquid into dry wheat, sealing and fermenting for 3 days, swelling the wheat during fermentation, filtering with gauze after fermentation, washing the swelled wheat with sterile water, and grinding the swelled wheat to obtain a flora which is retained in wheat bran and can utilize endosperm; then carrying out anaerobic culture on the flora twice; and obtaining zymophyte liquid after the culture is finished.

(D) Adding 0.7kg of leaven into 70L of water, uniformly mixing, sealing and culturing for 3-4 days, adding 25kg of dry wheat and an anti-dissolving solution after the culture is mature, continuing to seal, fermenting and culturing for 2-4 days to ferment and mature the wheat, and obtaining about 50kg of moist bacteria-rich wheat, namely the fermented wheat feed. Wherein the anti-dispersion liquid comprises 100 parts by weight of water and 6 parts by weight of guar gum; 4 parts of hydroxypropyl cellulose; and 2 parts of sodium caseinate.

Example 6

(D) Adding 0.7kg of leaven into 70L of water, uniformly mixing, sealing and culturing for 3-4 days, adding 25kg of dry wheat after the culture is mature, continuing to seal, fermenting and culturing for 2-4 days to ensure that the wheat is mature, obtaining about 50kg of moist and bacteria-rich wheat, taking out the moist and bacteria-rich wheat, placing the moist and bacteria-rich wheat into an anti-dissolving solution, soaking for 60min, and taking out the wheat with water after the soaking is finished, thus obtaining the fermented wheat feed. Wherein the anti-dissolving liquid comprises 100 parts by weight of water and 6 parts by weight of guar gum; 4 parts of hydroxypropyl cellulose; and 2 parts of sodium caseinate.

Example 7

Experiment of anti-dissolving liquid on dissolving condition of fermented wheat feed bacteria in water

The experimental method comprises the following steps: soaking 100g of fermented wheat feed in 1L of sterile water for 1 h; and after soaking, diluting the soaking solution by 100 times, and then detecting the total number of bacteria in the diluted soaking solution. The detection method adopts a microorganism detection method specified in GB 4789.2-2016. The method can be used for detecting how many bacteria are dissolved into water after the fermented wheat feed is contacted with the water in the culture pond for 1 hour; namely, the dissolving degree of the flora on the fermented wheat is detected.

The experimental contents are as follows: the fermented wheat feeds of example 2 and example 5, example 6 were compared and the results are shown in table 1:

table 1: test results of example 2 and example 5

Examples	Number of colonies (pieces/ml)	Total number of bacteria (one/ml)
			Example 2	1643	205000
Example 5	1620	196000
			Example 6	286	34700

As can be seen from Table 1, the colonies contained in examples 2 and 5 were larger than those in example 6 at the same dilution and dissolution time of 1 hour, which indicates that the colonies on the surface of the fermented wheat of examples 2 and 5 were more dispersed into water.

Example 2 and example 6 two comparative experiments show that the total number of bacteria and colony number dissolved out in the sterile water after the fermented wheat of example 6 is treated by soaking in the anti-dissolution solution is much lower than that of example 2, which indicates that the bacteria can be dissolved into the water from the surface of the wheat after the fermented wheat is treated. Since example 2 is different from example 6 only in that example 6 is subjected to the treatment of the anti-dissociable solution, it can be seen that the anti-dissociable solution can reduce the dissociable microorganisms from the surface of the fermented wheat to the water, when the dissociable microorganisms are reduced; the anti-ravel formulation of example 6 increased the number of beneficial bacteria that crayfish could ingest fermented wheat.

The anti-dispersion liquid substance of the invention is also used in other feeds, but the inventor finds that part of the components of the anti-dispersion liquid are only used as one of the raw materials of the feed in the prior art, namely as one of the nutrient elements of the feed, and does not disclose the application of guar gum and hydroxypropyl cellulose in the anti-dispersion liquid. In addition, the fermented feed of the prior art is prepared by uniformly fermenting all the raw materials, for example, guar gum or hydroxypropyl cellulose is added to a fermentation substrate to perform co-fermentation, and the mixture is used as one of the fermentation raw materials in reference example 5. It was verified that the experimental results of comparative example 5 and example 6, when guar gum, hydroxypropyl cellulose, etc. were added to the fermentation substrate for fermentation, they could not play a significant role in the anti-dissolution property.

Example 8: preparation method of fermented wheat feed

A) Adding 2 times of normal bacteria liquid into dry wheat, sealing and fermenting for 3 days, swelling the wheat during fermentation, filtering with gauze after fermentation, washing the swelled wheat with sterile water, and grinding the swelled wheat to obtain a flora which can utilize endosperm and is retained in wheat bran; then carrying out anaerobic culture on the flora twice; and obtaining zymophyte liquid after the culture is finished.

(D) Adding 0.7kg of leaven into 70L of water, uniformly mixing, sealing and culturing for 3-4 days, adding 25kg of dry wheat after the culture is mature, continuing to seal, fermenting and culturing for 2-4 days to ensure that the wheat is mature, obtaining about 50kg of moist and bacteria-rich wheat, taking out the moist and bacteria-rich wheat, placing the moist and bacteria-rich wheat into an anti-dissolving solution, soaking for 60min, and taking out the wheat with water after the soaking is finished, thus obtaining the fermented wheat feed. After soaking, in order to avoid the pollution of the strains of the next batch or the pathogenic microorganisms contained in the strains of the previous batch, the soaked anti-dissolving liquid can be sterilized and then reused, namely, reused for soaking. The anti-dissolving liquid comprises 100 parts by weight of water and 6 parts by weight of guar gum; 4 parts of hydroxypropyl cellulose; and 2 parts of sodium caseinate. Wherein the anti-blooming liquid in each experimental group of example 8 includes 100 parts by weight of water plus the components in Table 2.

First, the relationship between the solubility of the flora in the anti-dissolving liquid and the components of the anti-dissolving liquid

The fermented wheat feed is prepared by directly soaking the wet bacterium-rich wheat obtained by fermentation in the anti-dispersion liquid, so that a part of strains can be dissolved in the anti-dispersion liquid. When the strains are dissolved into the anti-dissolving liquid more, the number of the strains carried by the fermented wheat feed is reduced. After each batch of the fermented wheat feed of example 8 is soaked, the colony count and the total number of bacteria in the anti-dissolving liquid are detected by the following detection method:

taking the anti-dissolving liquid after soaking the fermented wheat feed for 1 hour, diluting the anti-dissolving liquid by 100 times, and then detecting the total number of bacteria in the diluted anti-dissolving liquid. The detection method adopts a microorganism detection method specified in GB 4789.2-2016. The method can be used for detecting how many bacteria are dissolved into the anti-dissolving liquid after the fermented wheat feed is contacted with the anti-dissolving liquid for 1 hour; namely, the degree of the bacterial flora in the anti-dissolving liquid on the fermented wheat was detected, and the detection results are shown in table 3.

Secondly, the relationship between the solubility of the flora in water and the components of the anti-dissolving liquid

The experimental groups in table 2 were tested according to the method of example 7, and the degree of dissolution of the fermented wheat feed in water was measured, and the test results are shown in table 3.

Table 2: formulation of each of the examples in example 8

Group of	Guar gum	Hydroxypropyl cellulose	Sodium caseinate
				Example 2	-	-	-
Example 6	6 portions of	4 portions of	2 portions of
				Group A	6 portions of
Group B		4 portions of
				Group C			2 portions of

The results of dissolution in the anti-dispersion liquid and in water of each example 8 are shown in table 3.

Table 3: results of tests conducted in each of the examples 8

As can be seen from the test results in table 3:

in example 2, the anti-disslovant solution is not used for treatment, so the detection result in water is far greater than that of other components, which shows that more strains of the fermented wheat feed in example 2 are disslovated into water, the total number of floras of the fermented wheat feed is the lowest, and the total number of crayfish feeding floras is low. Guar gum was included in group a, with the total number of bacteria in the anti-dispersion solution in group a being comparable to example 6, whereas guar gum was not included in groups B and C, with more bacteria in groups B and C being dispersed into the anti-dispersion solution, indicating that guar gum is capable of inhibiting bacterial dissolution from the surface of fermented wheat feed into the anti-dispersion solution.

The detection results of the anti-dissolving liquid of the groups B and C are equivalent, the total number of bacterial colonies is 18000 to 17660, and no significant difference exists, which indicates that neither the hydroxypropyl cellulose nor the sodium caseinate can inhibit the bacteria from dissolving from the surface of the fermented wheat feed into the anti-dissolving liquid.

However, the results of the tests in water for groups B and C were much lower than those for group A, indicating that groups B and C were able to inhibit the bacteria from dissolving from the surface of the fermented wheat feed into water, i.e., both hydroxypropyl cellulose and sodium caseinate were able to inhibit the bacteria from dissolving from the surface of the fermented wheat feed into water.

Furthermore, the detection results of the groups B and C in water are lower than that of the example 6 because guar gum is not added in the groups B and C, the flora in the fermented wheat feed is partially dissolved in the anti-dissolving liquid, so that the number of the flora carried by the fermented wheat feed is reduced, and the total number of naturally dissolved bacteria in water is lower than that of the example 6.

Thus, it can be seen from the verification of example 8 that guar gum can inhibit bacterial dissolution from the surface of fermented wheat feed into the anti-dissolution solution; neither hydroxypropyl cellulose nor sodium caseinate is able to inhibit bacterial lysis from the surface of fermented wheat feed into the anti-lysis solution; but both hydroxypropyl cellulose and sodium caseinate inhibited bacterial dissolution from the surface of the fermented wheat feed into the water.

While the specification describes in detail certain embodiments of the invention, this is not to be considered as a limitation on the scope of this patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.

Claims

1. A culture method for adjusting an ecosystem of intestinal flora of procambarus clarkii is characterized by comprising the following steps:

(D) adding a leavening agent into water, uniformly mixing, sealing and culturing for 3-4 days, adding dry wheat after the culture is mature, continuing to seal, fermenting and culturing for 2-4 days to make the wheat mature, obtaining moist bacteria-rich wheat, taking out the moist bacteria-rich wheat, soaking the moist bacteria-rich wheat in an anti-dissolving solution for 5-10 min, and taking out the wheat with water after the soaking is finished, thus obtaining the fermented wheat feed; wherein the anti-dissolving liquid comprises 100 parts by weight of water and 5-8 parts by weight of guar gum; 3-5 parts of hydroxypropyl cellulose; 1-3 parts of sodium caseinate.

2. The culture method according to claim 1, wherein: the basic culture medium comprises 5-15 g/L of brown sugar, 1-3 g/L of beef extract and 1-5 g/L of yeast extract; the basic culture is anaerobic culture, 5-10 generations of each group of floras are cultured, and the total number of the cultured floras is more than 1x10 ⁸ cfu/ml。

3. The culture method according to claim 1, wherein: the probiotics comprise lactic acid bacteria, bacillus subtilis and saccharomycetes; continuously culturing for 5-10 generations after adding probiotics to stabilize the flora, wherein the total number of the cultured flora is more than 1x10 ⁸ cfu/ml。

4. The culture method according to claim 1, wherein: the probiotic comprises 1x10 ⁹ 10% cfu/ml lactic acid bacteria; 1x10 ⁹ cfu/ml Bacillus subtilis 5%, 1x10 ⁶ 5 percent of cfu/ml baker's yeast.

5. The culture method according to claim 1, wherein: and adding 2 times of normal bacterial liquid into the wheat, and sealing and fermenting for 2-4 days.

6. The culture method according to claim 1, wherein: performing conventional anaerobic culture twice on the fermentation flora in the step (A) in a basal culture medium; and (D) repeating the step (A) for 10 times to obtain the stable zymogen liquid.

7. The culture method according to claim 1, wherein: the solid culture medium added into each 100L of the expanded culture bacterial liquid is 10kg of beef extract, 15kg of yeast extract and 50kg of brown sugar; the drying temperature of the paste is 40 ℃.

8. The culture method according to claim 1, wherein: adding 1kg of leaven and 30 kg-40 kg of dried wheat into every 100 liters of water in the step (D); the ratio of the basic feed to the fermented wheat feed in the step (3) is 1: 1.