CN114190470A

CN114190470A - Novel radix pseudostellariae enzyme, preparation method thereof and application thereof in large yellow croaker culture

Info

Publication number: CN114190470A
Application number: CN202111276484.9A
Authority: CN
Inventors: 缪雄平; 郭团玉; 陈�峰; 陈赋
Original assignee: Xiamen Ocean Vocational College; Ningde Normal University
Current assignee: Xiamen Ocean Vocational College; Ningde Normal University
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2022-03-18

Abstract

The invention belongs to the field of radix pseudostellariae application, and particularly relates to a novel radix pseudostellariae enzyme, a preparation method thereof and application thereof in large yellow croaker culture. The invention provides a preparation method of a molded radix pseudostellariae enzyme, which comprises the following steps: s01, preparing radix pseudostellariae extract and tea extract; s02, preparing a culture medium; s03, inoculating; s04, fermenting. The novel radix pseudostellariae enzyme prepared by the preparation method has a good antibacterial effect, can effectively improve the immunity of the large yellow croaker, reduces the incidence rate of white spot disease of the large yellow croaker, and improves the survival rate of the large yellow croaker.

Description

Novel radix pseudostellariae enzyme, preparation method thereof and application thereof in large yellow croaker culture

Technical Field

The invention belongs to the field of radix pseudostellariae application, and particularly relates to a novel radix pseudostellariae enzyme, a preparation method thereof and application thereof in large yellow croaker culture.

Background

With the rapid development of the large yellow croaker breeding industry, the breeding scale and the breeding area are continuously enlarged, diseases of the large yellow croakers bred in the net cages are continuously generated, and the disease frequency and the breeding scale are in a trend of rising year by year. Among them, the visceral nodulation of large yellow croaker is a common disease of large yellow croaker, which is called white spot disease of large yellow croaker, and is a bacterial disease that seriously harms the cultivation of large yellow croaker. Generally, the body surface of a diseased fish has no obvious symptoms, obvious congestion and fin rot can not be caused, but after dissection, the spleen and the kidney can be seen to have white nodules with the diameter of 0.5-1.0 mm, and the liver becomes white or light yellow. The disease is difficult to find in the early stages, and the disease may be already extensive and widespread when found, causing huge loss. Therefore, the improvement of the immunity of the cultured large yellow croakers, the reduction of the morbidity of white spot diseases and the reduction of the mortality become important research directions.

Disclosure of Invention

The invention provides a novel radix pseudostellariae enzyme, a preparation method thereof and application thereof in large yellow croaker culture, and can effectively solve the problems.

The invention is realized by the following steps:

a novel preparation method of radix pseudostellariae enzyme comprises the following steps:

s01, preparing a radix pseudostellariae extract and a tea leaf extract, wherein the radix pseudostellariae extract is obtained by pretreating radix pseudostellariae, and the tea leaf extract is obtained by pretreating tea leaves;

s02, preparing a culture medium, adding the radix pseudostellariae extract and the white tea extract into a fermentation tank, adding 10% cane molasses, 1% oligosaccharide and distilled water, uniformly mixing, cooking at 100 ℃ for 1h, and cooling to 30 ℃ to obtain a fermentation culture medium;

s03, inoculating, adding 50 parts by weight of yeast, 50 parts by weight of acetic acid bacteria and 50 parts by weight of lactic acid bacteria into the fermentation medium, and uniformly stirring at 25 ℃;

and S04, fermenting, namely introducing compressed air into the fermentation medium, stirring and fermenting, wherein the interval between two times of stirring is 3 days, and the fermentation temperature is within 35 ℃.

In step S01, the pre-treating of radix pseudostellariae includes washing, drying, pulverizing, adding distilled water, decocting at 60 deg.c for 2 hr, and filtering to obtain filtrate of radix pseudostellariae extract;

in step S01, the radix pseudostellariae is pulverized to a fineness of 20-40 meshes, and the weight of the distilled water is 4-6 times of that of the pulverized radix pseudostellariae.

In step S01, the dried tea is dried white tea, and the pre-treating the dried white tea comprises pulverizing the dried white tea, adding distilled water, steaming at 90 deg.c for 0.5 hr, and filtering to obtain filtrate of white tea extract;

in step S01, the dried white tea is pulverized to a fineness of 20-40 meshes, and the weight of the distilled water is 4-6 times of that of the pulverized dried white tea.

In step S04, when the fermentation temperature is higher than 35 ℃, the temperature is reduced by jacket cooling water.

In step S04, the fermentation period of the fermentation medium is 60 days, and after one fermentation period is finished, the pH value of the fermentation liquid in the fermentation medium reaches within 4.0, so as to obtain the radix pseudostellariae enzyme.

The invention also discloses the radix pseudostellariae enzyme prepared by the preparation method.

The invention also discloses an application of the radix pseudostellariae enzyme in large yellow croaker culture.

The invention has the beneficial effects that:

after the radix pseudostellariae and the tea are respectively pretreated, enzyme strains are directly added for inoculation and fermentation treatment without separation, so that the processing cost is reduced; the subsequent fermentation process can convert the macromolecular substances of the effective components in the radix pseudostellariae and the tea leaves into the micromolecular substances, and the micromolecular substances have higher pharmacological activity, are quick to take effect and are easier to be absorbed by animal organisms, so that the effective absorption and utilization of the radix pseudostellariae enzymes by the animal organisms are facilitated, and the good curative effect is achieved; meanwhile, substances such as bad substances, taste and peculiar smell in the radix pseudostellariae can be degraded in the fermentation process, and the defects of low mouthfeel, low absorption and utilization and the like of the conventional radix pseudostellariae are overcome. The ferment can remarkably enhance the immunity of the large yellow croaker, reduce the morbidity and improve the survival rate.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

Fig. 1 is a flow chart of a preparation method of radix pseudostellariae enzyme in the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.

Referring to fig. 1, a novel preparation method of radix pseudostellariae enzyme includes the following steps:

the step of pretreating the radix pseudostellariae comprises the steps of cleaning, drying and crushing the radix pseudostellariae, adding distilled water, cooking for 2 hours at the temperature of 60 ℃, and filtering to obtain filtrate of the radix pseudostellariae extract; the radix pseudostellariae is smashed to the fineness of 20-40 meshes, and the weight of the distilled water is 4-6 times that of the smashed product of the radix pseudostellariae;

the dried tea is preferably a dried white tea, and the pretreatment of the dried white tea comprises crushing the dried white tea, adding distilled water, cooking at 90 ℃ for 0.5h, and filtering to obtain a filtrate of a white tea extract; the dried white tea is crushed to the fineness of 20-40 meshes, and the weight of the distilled water is 4-6 times of that of the crushed product of the dried white tea.

S02, preparing a culture medium, adding the radix pseudostellariae extract and the white tea extract into a fermentation tank, adding 10% cane molasses, 1% oligosaccharide and distilled water, uniformly mixing, cooking at 100 ℃ for 1h, and cooling to 30 ℃ to obtain the fermentation culture medium.

S03, inoculating, adding 50 parts by weight of yeast, 50 parts by weight of acetic acid bacteria and 50 parts by weight of lactic acid bacteria into the fermentation medium, and uniformly stirring at 25 ℃.

And S04, fermenting, namely introducing compressed air into the fermentation medium, stirring and fermenting, wherein the interval of stirring for two times is 3 days, the fermentation temperature is within 35 ℃, and when the fermentation temperature is higher than 35 ℃, cooling by cooling water through a jacket. The fermentation period of the fermentation medium is 60 days, and after one fermentation period is finished, the pH value of fermentation liquor in the fermentation medium is within 4.0, so that the radix pseudostellariae enzyme is obtained.

In step S01, radix pseudostellariae and tea leaves with a certain weight are pulverized to a fineness of 20-40 meshes, the tea leaves can be one or more of white tea, green tea, black tea and the like, and distilled water with a weight 4-6 times of that of the sample is added to the tea leaves to be steamed and boiled under corresponding temperature conditions, so as to obtain extracts of the radix pseudostellariae and the tea leaves. When the cooking temperature is too high, although the internal components can be quickly cooked, the effective components are easily damaged. Therefore, the effective components of the radix pseudostellariae and the tea can be quickly cooked out by selecting the temperature under the precondition that the effective components are not damaged.

In step S02, the oligosaccharide is one or more of xylooligosaccharide, mannooligosaccharide, soy oligosaccharide, fructooligosaccharide, inulin, galactooligosaccharide, etc. Mixing cane molasses, oligosaccharide and tap water, keeping the temperature for a certain time at a specified temperature, and cooling to room temperature of 30 ℃ to obtain the fermentation medium. The bacteria in the mixture can be completely killed by the sterilization treatment.

In step S03, a certain amount of yeast, acetic acid bacteria, lactic acid bacteria are added to the culture medium prepared in the fermentation tank, and then the mixture is cultured for 60 days at room temperature of 30 ℃ by introducing compressed air of 0.1 to 0.3VVM, so as to obtain the radix pseudostellariae enzyme.

The first embodiment is as follows:

selecting 10g of fresh, impurity-free and mildew-free high-quality radix pseudostellariae, cleaning, drying, crushing into 20-40-mesh radix pseudostellariae powder, adding 50g of distilled water into the radix pseudostellariae powder, cooking for 2 hours at 60 ℃, and filtering to obtain filtrate, namely the radix pseudostellariae extract; selecting 10g of dried white tea which is produced in the current year, fresh, free of impurities and free of mildew, crushing the dried white tea into white tea powder with the fineness of 20-40 meshes, adding 50g of distilled water into the white tea powder, cooking the white tea powder at 90 ℃ for 0.5h, and filtering the mixture to obtain filtrate, namely the white tea extract.

Adding the radix pseudostellariae extract and the white tea extract into a fermentation tank, adding 10% cane molasses and 1% oligosaccharide, finally adding distilled water to enable the total amount of the mixed solution to reach 1L, uniformly mixing the solutions, then cooking for 1h at 100 ℃, and then cooling to 30 ℃ to obtain the fermentation culture medium.

Adding 50 parts by weight of yeast, 50 parts by weight of acetic acid bacteria and 50 parts by weight of lactic acid bacteria into the fermentation medium, controlling the initial temperature to be 25 ℃, uniformly stirring the mixture in the fermentation medium, and introducing compressed air for stirring and fermenting.

In this embodiment, the step of introducing compressed air for stirring fermentation includes introducing 0.1VVM compressed air, and stirring once every 3 days; the temperature is controlled within 35 ℃ in the fermentation process, and when the temperature is too high, jacket cooling water can be adopted for cooling.

The whole fermentation period is about 60 days, after the fermentation is successful, the fermentation liquor has the acid fragrance of liquid fermentation, the pH value reaches within 4.0, the fermentation liquor is dark, at the moment, the fermentation is stopped, the fermentation liquor is stirred and canned, and the radix pseudostellariae enzyme is subjected to aseptic sealed filling, so that the radix pseudostellariae enzyme is obtained.

Example two:

the invention further discloses the radix pseudostellariae enzyme prepared by the preparation method, and the antibacterial activity of the radix pseudostellariae enzyme is tested by adopting an oxford cup method.

(1) Pathogenic bacteria expanding culture

Selecting fresh slant for preserving pathogenic bacteria 1 ring in a clean bench, inoculating into 100ml sterile nutrient broth culture medium, fixing the triangular flask in a shaking table, mixing at 37 deg.C and 200r min-1, and culturing for 16-20 h. Diluting the cultured pathogenic bacteria suspension by 10 times with a blank nutrient broth culture medium, and if the OD600 is between 0.30 and 0.36, the pathogenic bacteria suspension is an effective pathogenic bacteria suspension, and the content of pathogenic bacteria in the stock solution of the pathogenic bacteria suspension is about 109CFU & mL & lt-1 & gt.

(2) Preparation of bacteriostatic substance

a. Antibiotics: diluting antibiotics or other antibiotics with sterile water;

b. expanding culture of lactic acid bacteria: weighing lactobacillus powder with the amount equivalent to about 10 hundred million CFU (millibar CFU) under the aseptic condition (if the coating product needs to be ground under the aseptic condition before weighing to release lactobacillus), inoculating the powder into 100ml of MRS liquid culture medium, and standing and culturing at 37 ℃ for 24-48 h to obtain the expanded culture lactobacillus suspension;

c. expanding and culturing spore bacteria: weighing spore bacteria powder with the amount equivalent to about 10 hundred million CFU under the aseptic condition, inoculating the spore bacteria powder into 100ml of nutrient broth culture medium, after inoculation, placing a triangular flask in a shaking table for fixation, controlling the rotating speed to be 200 r.min < -1 > under the temperature environment of 37 ℃, and culturing for 14-18 h to obtain the expanded spore bacteria suspension.

(3) Preparation of double-layer plate

And (3) pouring 15-18 ml of nutrient agar culture medium into each plate, uniformly spreading the nutrient agar culture medium in the plate, placing the plate on a horizontal table to solidify the nutrient agar culture medium to serve as a bottom layer, inverting the plate, averagely dividing the area, and marking additives. And cooling a proper amount of the semi-solid nutrient agar culture medium to 48-50 ℃, diluting the stock solution of the expanded pathogenic bacteria suspension to about 106 CFU.mL < -1 >, adding 0.5mL of the semi-solid nutrient agar culture medium into 4.5mL of the semi-solid nutrient agar culture medium, pouring the mixture into each flat dish, and uniformly spreading the mixture on the bottom layer to form a bacteria layer. After being placed on a horizontal table for cooling, 4 oxford cups are uniformly arranged in each 1 plate in the divided areas at equal intervals for later use.

(4) Bacteriostasis test

And (3) taking the prepared antibacterial substances, respectively dripping 200 mu L of the antibacterial substances into the Oxford cup in each double-layer plate, performing static culture at 37 ℃ for 16-20h, and measuring the diameter (or area) of each antibacterial zone.

Table 1 shows the bacteriostatic effect of the prepared radix pseudostellariae ferment on Escherichia coli. The bacteriostatic circle refers to the whole diameter of the circular transparent circle, and the larger the numerical value is, the better the bacteriostatic effect is; the analysis by combining the data in the table 1 shows that the radix pseudostellariae enzyme has obvious inhibition effect on various bacteria, and the inhibition diameter is larger than 15mm, which indicates that the inhibition effect of the enzyme is high inhibition.

TABLE 1 inhibitory Effect of Pseudostellaria ferment on various bacteria

Example three:

And mixing and stirring the prepared radix pseudostellariae enzyme and the feed for breeding the large yellow croakers, and performing a feeding test. The tested fish species are divided into 2 types of large and small specifications: the large specification means that the average body length is 14.2cm, the weight is about 50g, and the fish seeds are continuously cultured in the current year in the previous year; the small size is fish breed bred in the current year, the average body length is 4.4cm, and the body weight is about 1.5 g.

The experiments were divided into 2 groups: (1) control group: feeding by conventional methods; (2) test groups: 5% of radix pseudostellariae enzyme is added into conventional bait. When the frozen fresh is adopted for feeding, the radix pseudostellariae enzyme is added into the frozen fresh for homogenization, and after the homogenate is placed for 30min, the concentrated feeding is carried out; when the compound feed is adopted, the radix pseudostellariae enzyme is added into the compound feed to be soaked for 2 hours, and then the feed is intensively fed.

Feeding is carried out according to the requirements of 'four definite', the feeding is carried out once every morning and evening, and the feeding amount in the evening is more than that in the morning. In the process of gradually rising and falling the water temperature in spring beginning and late autumn, the feeding amount is correspondingly and gradually increased and decreased. The using condition of the bait is recorded every day, the feeding amount is adjusted every 7-10 days, and the feeding amount is accumulated once a month every month.

And measuring the growth condition of the fish once per month, randomly sampling 20-25 fish from different feeding boxes according to the fish species specification, and measuring the body length and the weight of the fish, wherein the length unit is accurate to mm, and the weight weighing is accurate to 0.1 g. Timely checking and diagnosing diseased fish, timely preventing and treating diseases by using drugs, and timely fishing out dead fish. And (5) timely recording feeding, water temperature, medication condition, death condition and the like, and weighing and recording the dead fishes after the dead fishes are fished out. And weighing and recording the sold fish, and finally counting the survival rate.

As can be seen from Table 2, the average weight at the end, the average body length at the end, the daily gain, the daily growth and the average survival rate of the test group are greatly improved compared with those of the control group in the case of the large or small-sized large yellow croaker culture. The fermentation process can convert macromolecular substances of effective components in the pseudostellaria root and the tea into micromolecular substances, and the micromolecular substances have higher pharmacological activity, are quick to take effect and easier to be absorbed by animal bodies, are beneficial to the effective absorption and utilization of pseudostellaria root enzymes by the animal bodies, obviously enhance the immunity of the pseudosciaena crocea, reduce the morbidity and improve the survival rate.

TABLE 2 evaluation data for large yellow croaker farming applications

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The preparation method of the novel radix pseudostellariae enzyme is characterized by comprising the following steps:

s02, preparing a culture medium, adding the radix pseudostellariae extract and the tea extract into a fermentation tank, adding 10% cane molasses, 1% oligosaccharide and distilled water, uniformly mixing, cooking at 100 ℃ for 1h, and cooling to 30 ℃ to obtain a fermentation culture medium;

2. The preparation method of novel radix pseudostellariae enzyme according to claim 1, wherein the step of pretreating radix pseudostellariae in step S01 comprises washing radix pseudostellariae, oven drying, pulverizing, adding distilled water, decocting at 60 ℃ for 2h, and filtering to obtain a filtrate of radix pseudostellariae extract.

3. The preparation method of the novel radix pseudostellariae enzyme according to claim 2, wherein in step S01, the radix pseudostellariae is pulverized to a fineness of 20-40 meshes, and the weight of the distilled water is 4-6 times of the pulverized radix pseudostellariae.

4. The method for preparing radix pseudostellariae enzyme according to claim 1, wherein in step S01, the dried tea is dried white tea, and the pre-treatment of the dried white tea comprises pulverizing the dried white tea, adding distilled water, steaming at 90 ℃ for 0.5h, and filtering to obtain a filtrate of white tea extract.

5. The method for preparing the novel radix pseudostellariae enzyme according to claim 4, wherein in step S01, the dried white tea is pulverized to a fineness of 20-40 meshes, and the weight of the distilled water is 4-6 times of the pulverized dried white tea.

6. The method for preparing radix pseudostellariae enzyme according to claim 1, wherein the temperature of fermentation is higher than 35 ℃ in step S04, and is reduced by cooling water with a jacket.

7. The method of claim 1, wherein in step S04, the fermentation period of the fermentation medium is 60 days, and after a fermentation period is completed, the pH of the fermentation liquid in the fermentation medium is within 4.0, so as to obtain the radix pseudostellariae enzyme.

8. Radix pseudostellariae enzyme prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the radix pseudostellariae enzyme according to claim 8 in large yellow croaker farming.