CN111513202A

CN111513202A - Microecological preparation and preparation method and application thereof

Info

Publication number: CN111513202A
Application number: CN202010471594.XA
Authority: CN
Inventors: 彭晓培; 吴怡; 胡金华; 张泽宇; 郑美大; 夏黎明; 马曦; 马东立
Original assignee: Zhongnong Jirun Biotechnology Beijing Co ltd
Current assignee: Zhongnong Pet Nutrition Research Institute (Jiangsu) Co.,Ltd.
Priority date: 2020-05-29
Filing date: 2020-05-29
Publication date: 2020-08-11

Abstract

The invention provides a microecological preparation and a preparation method and application thereof. The preparation contains: a probiotic comprising saccharomyces cerevisiae, lactobacillus acidophilus and lactobacillus casei; an amino acid comprising L-arginine and glutamine; prebiotics selected from the group consisting of mannooligosaccharides, kelp hydrolysate, fructose, brown sugar, inulin; a glucose carrier. The invention also provides a method for preparing the probiotic and the use of the probiotic as an additive or supplement to feed, in particular to feed for dogs, such as pet dogs. The probiotics in the microecological preparation can promote each other, has good growth, simple formula, high quality, low price and more importantly, is safe and reliable, can promote the healthy growth of animals, simultaneously has beautiful furs, and has great market prospect in the field of animal health, particularly the health of pet dogs.

Description

Microecological preparation and preparation method and application thereof

Technical Field

The invention relates to the technical field of microecologics, in particular to a microecologics and a preparation method and application thereof.

Background

With the improvement of living standard of people, pets have become indispensable life partners of many families, especially pet dogs with good luck.

The pet dog is a monogastric animal, and the daily ration is mainly meat and lives with human beings. The daily ration mainly based on meat has high protein content, and needs the intestinal health of pet dogs, but the intestinal problem with diarrhea as the main symptom is easily caused. The pet dog living together with human beings has a very sanitary living environment, and forms a living habit of frequent bathing, which also causes the abnormal conditions of single type and small quantity of flora in the intestinal tract of the dog, such as allergic reaction, inflammatory reaction and the like. The intestinal tract has problems, which not only causes diseases such as diarrhea, but also causes the problems of knotted fur, dull and dull light and the like, and seriously reduces the ornamental value of the pet dog. In addition, antibiotics are commonly used in therapeutic regimens for pet dogs, and it is well recognized that antibiotics can cause an imbalance in the intestinal microbial flora. Therefore, whether the dogs are healthy dogs or sick dogs, the improvement of the intestinal health of the pet dogs is the key point for ensuring the health of the pets and accelerating the recovery of the sick dogs. It is well recognized in the industry that the core of gut health is gut flora balance. The maintenance of the balance of the intestinal flora is the most important technical means for preventing and treating diseases and the most important guarantee for the health of animals.

At present, the problem of unbalance of intestinal microecological flora is mainly supplemented by a probiotic preparation, but the common point of the current products is that only one type of beneficial bacteria such as lactobacillus is provided, and the type of the bacteria is single. In fact, healthy intestinal flora is a complex ecosystem with a large number of bacterial species. In addition, the additional supplement of probiotics is only one of the measures for solving the imbalance of the intestinal flora, and the provision of nutrients required for growth and reproduction of the intestinal flora is also a very important aspect. The common denominator of the current products is that only one class of prebiotics, such as fructooligosaccharides, is provided. But the amino acids required for microbial growth are generally ignored or overlooked. The invention provides a plurality of probiotics, not only provides oligosaccharide, but also provides a plurality of amino acids to ensure the intestinal health of pet dogs and assist sick dogs to quickly recover the intestinal flora balance.

Disclosure of Invention

In order to solve one or more of the above technical problems of the prior art, the present invention provides in a first aspect a microecological formulation, wherein the formulation comprises:

(1) a probiotic comprising saccharomyces cerevisiae, lactobacillus acidophilus, and lactobacillus casei;

(2) amino acids comprising L-arginine and glutamine;

(3) prebiotics selected from the group consisting of mannooligosaccharides, kelp hydrolysate, fructose, brown sugar, inulin;

(4) and the carrier is glucose.

In some preferred embodiments, the formulation further comprises a phagostimulant and/or a multivitamin; the phagostimulant can promote food intake, and the compound vitamin can make hair smoother.

In other preferred embodiments, the content of each component in the formulation in parts by weight is as follows:

the saccharomyces cerevisiae of the invention is an aerobic microorganism, and generally can not be independently compounded with lactobacillus acidophilus and lactobacillus casei which are anaerobic microorganisms to form three kinds of composite probiotics which are compounded together to be used as a microecological preparation. But this combination clearly conforms to the structure of the digestive tract of the animal. The upper part of the animal digestive tract, such as oral cavity and stomach, is aerobic, while the lower part of the digestive tract, such as ileum and colon, is strictly anaerobic. Therefore, the microecological preparation compounded by the saccharomyces cerevisiae, the lactobacillus acidophilus and the lactobacillus casei can be mutually matched in the animal digestive tract to complement each other.

In addition, the inventor finds that inulin is originally used for prebiotics of probiotics for human, but is particularly beneficial to the growth of lactobacillus acidophilus and lactobacillus casei. Thus, in other preferred embodiments, the prebiotic is inulin; more preferably, the inulin is present in an amount of 0.1 to 0.3 parts by weight, for example 0.2 parts by weight.

In other preferred embodiments, the phagostimulant is a yeast phagostimulant.

In other preferred embodiments, the multivitamin component comprises the following components: vitamin A acetate is more than or equal to 5,000,000IU/kg, vitamin D3 is more than or equal to 500,000IU/kg, tocopherol acetate is more than or equal to 50,000IU/kg, vitamin B1 is more than or equal to 1,600mg/kg, vitamin B2 is more than or equal to 2,200mg/kg, vitamin B6 is more than or equal to 1,600mg/kg, vitamin B12 is more than or equal to 30mg/kg, nicotinamide is more than or equal to 13,000mg/kg, D-calcium pantothenate is more than or equal to 8,000mg/kg, folic acid is more than or equal to 180mg/kg, and biotin is more than or equal to 50 mg/kg.

In other preferred embodiments, the multivitamin component is present in an amount of 0.5 to 1.0 part by weight, for example 0.75 part by weight.

In other preferred embodiments, the phagostimulant is present in an amount of 0.8 to 1.2 parts by weight, for example 1.0 part by weight.

Glucose exists mainly as a carrier and also as an energy substance; in some embodiments, the amount of glucose is the balance of the probiotic other than the listed ingredients. In other words, the probiotic comprises, in addition to the other components listed, the balance glucose.

In other preferred embodiments, the application rate of the probiotic may be as follows: calculating the dosage according to the weight of the dogs, wherein the daily health care dosage is 0.2g/kg of body weight; in special cases, such as diarrhea, the amount can be 0.4g-0.5g/kg body weight.

The present invention provides in a second aspect a method of preparing a formulation according to the first aspect of the invention, wherein the method comprises:

(1) respectively providing saccharomyces cerevisiae bacterial powder, lactobacillus acidophilus bacterial powder and lactobacillus casei inulin;

(2) and (3) uniformly mixing the bacterial powder and other components in an aseptic state to prepare the composite microecological preparation.

In some preferred embodiments, the saccharomyces cerevisiae bacterial powder is prepared by the following steps: (1) activating strains: inoculating Saccharomyces cerevisiae into liquid culture medium, and shake culturing at 26-32 deg.C (such as 30 deg.C) under oscillation parameter of 100-200rpm (such as 150rpm) for 1.5-2.5 hr (such as 2 hr) to obtain activated bacteria solution; (2) fermentation: inoculating the activated bacterial liquid into a culture medium of a fermentation tank for culture, wherein the culture temperature is 26-32 ℃ (such as 30 ℃), the ventilation rate is 0.5-1.6vvm (such as 1.0vvm), the stirrer power is 1-2kwh (such as 1.5kwh) per cubic meter, and the culture time is 7-9h (such as 8h), so as to obtain a saccharomyces cerevisiae fermentation liquid; (3) freeze-drying: and centrifuging the saccharomyces cerevisiae fermentation liquor, removing the supernatant, and freeze-drying the precipitate to obtain saccharomyces cerevisiae bacterial powder.

In other preferred embodiments, the lactobacillus acidophilus powder and the lactobacillus casei powder are independently carried out in the following manner: (1) activating strains: inoculating strain into the MRS culture medium, and performing standing anaerobic culture at 36-38 deg.C (such as 37 deg.C) for 18-30 hr (such as 24 hr) to obtain first activated bacteria solution; inoculating the first activated bacterium liquid into an MRS liquid culture medium, and performing standing anaerobic culture for 18-30h (for example, 24h) at 36-38 ℃ (for example, 37 ℃) to obtain a seed liquid; (2) fermentation: inoculating the seed solution into a fermentation tank culture medium according to the inoculation amount of 4 vol%, and performing constant-temperature anaerobic culture at 36-38 ℃ (such as 37 ℃) and 100rpm for 66-78h (such as 72h) to obtain a fermentation solution; (3) freeze-drying: and centrifuging the fermentation liquor, removing supernatant, and freeze-drying the precipitate to obtain corresponding bacterium powder.

In other preferred embodiments, the Saccharomyces cerevisiae powder has a viable bacteria content of 150-250 million CFU/g (200 million CFU/g). The viable bacteria content of the lactobacillus acidophilus powder is 400-600 million CFU/g (for example, 500-hundred million CFU/g); and/or the viable bacteria content of the lactobacillus casei powder is 800-1200 hundred million CFU/g (1000 hundred million CFU/g).

In other preferred embodiments, the liquid medium is formulated with 1 mass% yeast extract, 2 mass% peptone, and 2 mass% maltose.

In other preferred embodiments, the fermentor medium is made from 0.25 mass% yeast extract, 0.2 mass% maltose, 0.02 mass% sodium chloride, 0.82 mass% sodium acetate.

In other preferred embodiments, the fermentor medium consists of: peptone 1-3%, beef extract 1-3%, yeast extract 1-3%, glucose 6-8%, diammonium citrate 0.3%, K₂HPO₄·7H₂O 0.2％、MgSO₄·7H₂O0.09％、MnSO₄·4H₂O 0.025％、NaAc·3H₂O0.3% and Tween-800.1%.

In a third aspect, the present invention provides the use of a probiotic of the first aspect of the invention or of a probiotic produced by the process of the second aspect of the invention as an additive or supplement to a feed, particularly to a feed for dogs, such as pet dogs.

Compared with the prior art, the invention has the following technical advantages:

(1) the probiotics are capable of promoting each other. The probiotics Saccharomyces cerevisiae, the Lactobacillus acidophilus and the Lactobacillus casei in the microecological preparation can be symbiotic and complement each other, so that the probiotics can be well played together.

(2) The probiotics grew well. Some preferred microecologics of the present invention comprise inulin which significantly promotes the growth of lactobacillus acidophilus and lactobacillus casei.

(3) Simple formula, high quality and low price. The microecological preparation of the invention has relatively few components and relatively low raw material price.

(4) Promoting the healthy growth of animals. The probiotics of the invention are particularly capable of promoting the growth of animals and can therefore be used as feed additives or supplements.

(5) Has the function of beautifying the skin. The beautiful woollen coat is particularly important for pet dogs, and the microecological preparation of the invention ensures that the woollen coat is more smooth and glossy.

(6) Is safe and reliable. Tests prove that the microecological preparation is safe and reliable.

Drawings

FIG. 1 is a lactic acid bacteria growth curve. 1.2, 3 and 4 are only numbers (same below) in the figure legend, i.e., curve 1 represents cheese 4%, curve 2 represents cheese 3%, curve 3 represents acidophilic 4%, and curve 4 represents acidophilic 3%.

FIG. 2 is a pH profile of a culture medium for lactic acid bacteria.

FIG. 3 is a growth curve at different pH. Legend numbers are first row legends (fig. a-c) and then second row legends (d-f).

FIG. 4 is a graph comparing the effect of culturing two lactobacilli in different media. The bars within the same group are arranged as shown in the figure (same below).

FIG. 5 shows the OD 600 values of co-culture of two species of Lactobacillus.

FIG. 6 is a diagram showing the viable cells of Lactobacillus casei co-cultured with Lactobacillus acidophilus and stained with methylene blue (see FIG. 6).

FIG. 7 is a graph showing the selection results of the co-culture medium.

FIG. 8 is a graph showing the acid production characteristics of Lactobacillus casei and Lactobacillus acidophilus.

FIG. 9 is the effect of inulin and kelp zymolyte on the growth of Lactobacillus acidophilus.

FIG. 10 is a graph showing the effect of inulin and kelp zymolyte on the growth of Lactobacillus casei (the sequence of bars is the wet weight of bacteria at the front and the dry weight of bacteria at the back).

FIG. 11 shows the effect of inulin on the growth (dry weight) of Lactobacillus acidophilus.

FIG. 12 shows the effect of inulin on growth (dry weight) of Lactobacillus casei.

Figure 13 is a comparison of coat condition of groups of dogs at different times during the test period.

Detailed Description

The present invention will be further illustrated by the following examples, but the scope of the invention as claimed is not limited to these examples. The starting materials used in the examples are, unless otherwise indicated, conventional starting materials which are commercially available.

The main material sources used in the examples are shown in the table below.

TABLE 1 sources of the principal materials used in the examples

Composition (I)	Origin of origin
		Saccharomyces cerevisiae	Angel Yeast Co Ltd
Lactobacillus acidophilus	Dalian Suxin Biotech Co Ltd
		Lactobacillus casei	Dalian Suxin Biotech Co Ltd
L-arginine	Jiangsu and Whole food ingredients Limited
		Glutamine	Jiangsu and Whole food ingredients Limited
Inulin powder	Hebei Fengning safety high practice Co Ltd
		Yeast phagostimulant	FUJIAN LUODONG BIO-TECHNOLOGY Co.,Ltd.
Glucose (analytically pure)	SINOPHARM CHEMICAL REAGENT Co.,Ltd.

Example 1

Through preliminary screening tests, the inventor takes lactobacillus casei and lactobacillus acidophilus as candidate probiotics and further tests are carried out on the two probiotics. The test procedure was as follows:

(1) respectively purifying lactobacillus casei and lactobacillus acidophilus powder for 3 times to obtain strains, and determining the activated strains to be gram-positive bacilli and pure bacilli by gram staining.

(2) Carrying out a thallus growth curve preliminary experiment: lactobacillus casei and Lactobacillus acidophilus were inoculated at 3% and 4% respectively in MRS medium with initial pH of 5.9, and growth curves were plotted.

From the results it can be seen that: the method is characterized in that the method enters a plateau stage after 14h of the bacterial biomass of lactobacillus casei reaches the maximum value, and enters a plateau stage after 16h of the bacterial biomass of lactobacillus acidophilus reaches the maximum value (see figure 1).

(3) Since lactobacillus acidophilus grows slowly and is presumed to be more sensitive to oxygen concentration, after the lactobacillus acidophilus and the lactobacillus casei are respectively inoculated into an MRS culture medium and sealed by paraffin oil, the lactobacillus acidophilus grows fast and the lactobacillus casei grows slowly, which indicates that the lactobacillus acidophilus has more strict requirements on anaerobic conditions and the lactobacillus casei is more resistant to oxygen.

(4) The acid production characteristics of lactic acid bacteria were measured, and the results are shown in FIG. 2. As can be seen from the figure, the pH of both lactobacilli was decreasing slowly for 20 h.

(5) The lactic acid bacteria culture medium component simplification and culture pH optimization experiment: adjusting the pH of a simple culture medium (glucose 2%, protein powder 1% and yeast extract 1%) to pH 3, pH 4 and pH 5, inoculating lactobacillus casei and lactobacillus acidophilus respectively, sampling every 2 hours to measure OD 600 value and pH, and freezing and storing the test tube to measure the content of lactic acid. As a result of the experiment, it was found that the two lactic acid bacteria grew slowly under the culture conditions (as shown in FIG. 3).

(6) MRS is used as a control culture medium, 2X and 3X culture media are prepared on the basis of a simple culture medium (2% of glucose, 1% of protein powder and 1% of yeast extract), and after inoculating 3% of lactobacillus casei and lactobacillus acidophilus for overnight culture, OD 600 is measured at 4h, 11h, 24h and 32 h. As can be seen from fig. 4, lactobacillus casei grew faster in 1X medium and lactobacillus acidophilus in 3X medium, but the overall growth rate was slow compared to MRS medium, indicating that two lactic acid bacteria were not suitable for growth in this medium.

(7) Co-culturing lactobacillus casei and lactobacillus acidophilus: the growth became faster after co-culturing lactobacillus casei and lactobacillus acidophilus (as shown in fig. 5), and was confirmed by the increase in viable count by the melan staining (as shown in fig. 6).

(8) Study of selection of co-culture medium: in order to verify the requirements of co-culture on the culture medium, MRS is used as a control culture medium, 2X and 3X culture media are prepared on the basis of a simple culture medium (glucose 2% protein powder 1% yeast extract 1%), 3% lactobacillus casei and lactobacillus acidophilus are inoculated for overnight culture, and then OD 600 is measured at 7h, 20h and 28 h. As can be seen from fig. 7, MRS far outperformed the simplified medium as a co-culture medium.

(9) Study of the co-culture acid production characteristics: MRS is used as a control culture medium, 2X culture mediums and 3X culture mediums are respectively prepared on the basis of a simple culture medium (glucose 2% protein powder 1% yeast extract 1%), three treatments are carried out in each culture medium, namely, a mixed solution of 3% lactobacillus casei, 3% lactobacillus acidophilus, 1.5% lactobacillus casei and 1.5% lactobacillus acidophilus is inoculated, overnight culture is carried out, the pH value of the culture medium is measured after 36h culture is finished, and the acid production performance of independent culture and co-culture in different culture mediums is tested. As can be seen from FIG. 8, the pH was lowest and acid production was highest in the co-cultured cultures in all media except the 3X medium.

Example 2

1 test name: effect of inulin and Zosterase decomposition products on the growth of lactic acid bacteria

2, test method:

taking MRS culture medium as a reference culture medium, adding inulin and laminaria enzyme decomposition products in different proportions, and determining the influence of the inulin and the laminaria enzyme decomposition products on the growth of the lactic acid bacteria by comparing the biomass of the lactic acid bacteria and the acid yield.

3 test procedure

3.1 Effect of inulin on growth of lactic acid bacteria: 4.83g of MRS culture medium is added with 100ml of distilled water to prepare a liquid culture medium; wherein "0.5 g of sugar is substituted" means "a liquid medium prepared by replacing 0.5g of glucose in 4.83g of MRS medium with 0.5g of inulin", and the like.

3.2 influence of Zosterase decomposition products on lactic acid bacteria growth: 4.83g of MRS culture medium is added with 100ml of distilled water to prepare a liquid culture medium; adding the supernatant of the kelp zymolyte (abbreviated as kelp clear) according to the proportion of 5 percent (volume ratio).

The tube was 50mL orange centrifuge tube and 50mL liquid medium, and the tube was placed on a centrifuge tube rack, allowed to stand, and incubated at 37 ℃ for 48 hours. The inoculum size was the same in all media (200 uL). After the culture is finished, centrifuging to obtain wet weight of the strain, and drying for 8 hours at 65 ℃ to obtain dry weight. The Aohaus company ST3100pH measures pH, and the content of lactic acid was measured by a lactic acid test kit from Nanjing institute of construction.

As can be seen from FIG. 9, replacing glucose in MRS medium with inulin, 0.5g and 1.0g of inulin had good growth promoting effects on Lactobacillus acidophilus, with 0.5g of inulin being the best. 5% Laminarin did not show growth promoting effect on Lactobacillus acidophilus. Therefore, compared with inulin, the growth promoting effect of inulin on lactobacillus acidophilus is better. FIG. 10 shows that the wet weight and dry weight of bacteria in the 0.5g sugar group and the 5% kelp clear group are both higher than those in the MRS group, i.e. equivalent glucose in the MRS culture medium is replaced by 0.5g inulin, or 5% kelp zymolyte supernatant is added in the MRS culture medium, so that the growth promoting effect on lactobacillus casei is good.

Example 3

The test name is: effect of inulin on the growth of lactic acid bacteria

The test method comprises the following steps: adding inulin into MRS culture medium as reference culture medium according to different proportions, and comparing the yield (quality) of lactobacillus to determine the influence of inulin on the growth of lactobacillus.

The test process comprises the following steps: 4.83g of MRS culture medium is added with 100ml of distilled water to prepare a liquid culture medium; wherein, the generation of 0.5g of sugar refers to the generation of a liquid culture medium prepared by replacing 0.5g of glucose in 4.83g of MRS culture medium with 0.5g of inulin, and the rest is analogized; "Add 0.5g inulin" means "0.5 g inulin +4.83g MRS medium prepared into liquid medium"; and the rest is analogized. The inoculum size was the same in all media. After 24h of culture, the wet weight of the strain is obtained by centrifugation, and the dry weight is obtained after drying for 8h at 55 ℃.

As a result: as can be seen from FIG. 11, for Lactobacillus acidophilus, inulin, whether used to replace glucose in MRS medium or added directly to MRS medium, can promote the growth of flora. Wherein the dry weight of Lactobacillus acidophilus produced in the culture medium substituted by 0.5g of sugar is the largest. As can be seen from FIG. 12, for Lactobacillus casei, the replacement of glucose in MRS medium by inulin and the growth of flora were promoted by the addition of more inulin in MRS medium. Wherein the dry weight of Lactobacillus casei producing medium with "1.5 g inulin" is the largest.

In conclusion, inulin instead of glucose in MRS medium is beneficial for the growth of lactobacillus acidophilus and lactobacillus casei, wherein lactobacillus casei is more affected; and the inulin is used for replacing glucose, so that the effect is better than that of directly adding the inulin into the culture medium. When inulin replaced 1.0g of glucose, the amount of lactobacillus casei (wet weight) (0.93g) was 2.6 times the amount of bacteria in MRS medium (0.35 g); whereas when inulin was substituted for 0.5g of glucose, the amount of Lactobacillus acidophilus (wet weight) (0.5g) was 1.4 times the amount of bacteria in MRS medium (0.35 g).

Example 4: effect of different polysaccharides on fermentation

This example compares the effect of brown sugar, fructooligosaccharide, inulin, and kelp enzymatic hydrolysis granules on the fermentation material.

Mixing 2.5kg of soybean meal, 2.5kg of wheat bran and 2kg of water as a fermentation substrate, respectively adding 200g of different polysaccharides, and uniformly mixing. Then inoculating yeast liquid, acidophilic lactobacillus liquid and cheese lactobacillus liquid. Then, the cells were distributed in 7 breathing bags and cultured at 30 ℃ for 4 days.

After the fermentation was completed, pH was measured using ST3100pH, ohauss corporation, and lactic acid content was measured using a lactic acid test kit of the institute of tokyo, and lactic acid bacteria were counted.

3 results of the test

TABLE 2 Effect of different polysaccharides on fermentation

Polysaccharide type	Brown sugar	Inulin powder	Fructose	Sea tangle
					pH	6.12	6.03	6.05	5.43
Lactic acid/mmol/g	0.022	0.035	0.032	0.111
					Lactobacillus/CFU/g	3.40×10⁷	5.28×10⁷	5.28×10⁷	2.78×10⁸

As can be seen from the above table, the pH value of the brown sugar fermentation material is the highest, and the yield of lactic acid and the number of lactic acid bacteria are the lowest. The pH value of the kelp fermented material is the lowest, and the lactic acid yield and the lactic acid bacteria quantity are the highest. The values of the inulin fermentation material and the fructose fermentation material are similar. Therefore, the kelp enzymolysis particles have the best fermentation promoting effect on the fermentation material, and the brown sugar is the worst.

Preparation example: preparation of microecological preparation

Preparing saccharomyces cerevisiae powder:

activating strains: preparing liquid culture medium according to the proportion of 1% yeast extract, 2% peptone and 2% maltose, and autoclaving at 121 deg.C for 30 min. Under the aseptic environment, the saccharomyces cerevisiae strain is inoculated in the culture medium and is cultured by a shaking table, the culture temperature is 26-32 ℃, the oscillation parameter is 100-200rpm, and the culture time is 2 hours, so as to obtain the activated bacterial liquid. In an aseptic environment, samples were taken from the activated bacteria solution, and the resultant was stained with methylene blue, and microscopic examination was performed to confirm that the cells were pure.

Fermentation: inoculating the activated liquid of the saccharomyces cerevisiae into a fermentation tank culture medium according to the proportion of 2-5% for culture. The volume of the culture medium in the fermentation tank is not more than 2/3 of the total volume of the fermentation tank, the culture temperature is 26-32 ℃, the ventilation rate is 0.5-1.6vvm (the ratio of the ventilation rate per minute to the volume of the culture medium), the power of the stirrer is 1-2kwh per cubic meter, and the culture time is 8h, so that the saccharomyces cerevisiae fermentation liquor is obtained. The formula of the fermentation tank culture medium is as follows: 0.25% yeast extract, 0.2% maltose, 0.02% sodium chloride, 0.82% sodium acetate, and autoclaving at 121 deg.C for 30 min.

Freeze-drying: centrifuging the Saccharomyces cerevisiae fermentation liquid, removing supernatant, pre-freezing at (-40 deg.C) for more than 5h, and freeze-drying in vacuum freeze-dryer to obtain Saccharomyces cerevisiae powder. The content of the live bacteria of the saccharomyces cerevisiae is 200 hundred million CFU/g.

Preparing lactobacillus acidophilus powder:

(1) activating strains: adding 48.3g MRS culture medium (Beijing Oborg Star) into 1000ml distilled water, boiling to dissolve, packaging, and autoclaving at 110 deg.C for 15min to obtain MRS liquid culture medium. Inoculating Lactobacillus acidophilus strain into the above culture, standing at 36-38 deg.C, and anaerobically culturing for 24 hr to obtain first activated bacteria solution. In an aseptic environment, samples were taken from the first activated cell suspension, and gram-stained and microscopic examination was performed to confirm that the cells were pure. Inoculating the first activated bacterium liquid into an MRS liquid culture medium, standing at 36-38 ℃ for anaerobic culture for 24h to obtain a seed liquid.

(2) Fermentation: inoculating the prepared lactobacillus acidophilus seed solution into a fermentation tank culture medium according to the inoculation amount of 4%, and carrying out constant-temperature anaerobic culture at 36-38 ℃ and 100rpm for 72h to obtain a fermentation liquid. Wherein the culture medium of the fermentation tank comprises: peptone 1-3%, beef extract 1-3%, yeast extract 1-3%, glucose 6-8%, diammonium citrate 0.3%, K₂HPO₄·7H₂O 0.2％、MgSO₄·7H₂O0.09％、MnSO₄·4H₂O 0.025％、NaAc·3H₂O0.3% and Tween-800.1%, dissolving in 1000ml distilled water, and autoclaving at 121 deg.C for 30 min.

(3) Freeze-drying: centrifuging the lactobacillus acidophilus fermentation liquid, removing supernatant, adding the composite protective agent into the lower layer bacterial mud, mixing uniformly, pre-freezing at (-40 ℃) for more than 6h, and freeze-drying in a vacuum freeze dryer to obtain lactobacillus acidophilus powder. The content of Lactobacillus acidophilus is 500 hundred million CFU/g.

Preparation of lactobacillus casei powder:

lactobacillus casei inulin is prepared in exactly the same manner as Lactobacillus acidophilus powder, and the content of Lactobacillus casei is 1000 hundred million CFU/g.

Preparing a finished product of the microecological preparation:

1kg of microecological preparation finished product is prepared according to the proportion shown in the following table. The preparation method comprises the following steps: and weighing the saccharomyces cerevisiae powder, the lactobacillus acidophilus powder, the lactobacillus casei powder and other components listed in the table, uniformly mixing in an aseptic state, and bagging to obtain a microecological preparation finished product.

TABLE 4 composition of the different formulations (parts by weight)

Components	Preparation	1	Preparation 2	Preparation 3	Preparation 4	Preparation 5	Preparation 6
								Saccharomyces cerevisiae	0.1	0.2	0.2	0.2	0.2	0.1
Lactobacillus acidophilus	0.1	0.01	0.1	0.1	0.1	0.1
							Lactobacillus casei	0.01	0.1	0.1	0.1	0.1	0.01
L-arginine	0.01	0.1	0.1	0.1	0.1	0.01
							Glutamine	0.1	0.5	0.5	0.5	0.5	0.1
Inulin powder	0.1	0.3	0.3	0.3	0.3	0.1
							Compound vitaminComposition (I)	0.5	1	0	1	0	0.5
Yeast phagostimulant	0.6	1.2	0	0	1.2	0.6
							Glucose (analytically pure)	8.48	6.59	8.7	7.7	7.5	8.48

Note: the composition of the vitamin complex components is as follows: vitamin A acetate was 5,000,000IU/kg, vitamin D3 was 500,000IU/kg, tocopherol acetate was 50,000IU/kg, vitamin B1 was 1,600mg/kg, vitamin B2 was 2,200mg/kg, vitamin B6 was 1,600mg/kg, vitamin B12 was 30mg/kg, nicotinamide was 13,000mg/kg, calcium D-pantothenate was 8,000mg/kg, folic acid was 180mg/kg, biotin was 50 mg/kg.

Test examples

1. The purpose of the test is as follows: this example demonstrates the safety and probiotic effect of the probiotic of the present invention using a beagle dog as the animal.

2. Materials and methods

2.1 probiotic preparation: explanation will be made by taking the probiotic 3 (hereinafter sometimes referred to as "probiotic") prepared in the preparation examples as an example; the positive control is Maillard pet nutritional supplement (gastrointestinal probiotic bacteria, sometimes called Maillard probiotic bacteria hereinafter) (mainly comprising Pediococcus acidilactici, Lactobacillus acidophilus, fructooligosaccharide, xylanase, protease, amylase, lipase, tocopherol acetate, vitamin D3, vitamin A acetate, vitamin B1, vitamin B2, vitamin B6, vitamin B12, nicotinamide, calcium pantothenate, biotin, folic acid, glucose, and Yucca powder).

2.2 Experimental animals: 20 beagle dogs, including 10 senior dogs and 10 juvenile dogs, each half of the male and female dogs at each stage have similar initial body weights.

2.3 experimental design: according to the principle that the weight and sex ratio are similar, the test is divided into 3 groups, 8 blank control groups, 8 test groups and 4 positive control groups. The blank control group is fed with basic full-value dog food, the test group is fed with the basic full-value dog food plus 5g of the microecological preparation, and the positive control group is fed with the basic full-value dog food (ROYAL CANIN Royal dog food) +4g of Maide probiotics. See table 5 below.

TABLE 5 test grouping

Note: the dosage of the microecological preparation is calculated according to the average weight of 10kg of old dogs and 5kg of young dogs.

2.4 Breeding management

The test was conducted in Shanxi Lanling Biotechnology, Inc., located in Yao village of Yixing town, Shanxi province and Shunxian county. The test dogs are fed in a single cage, the test dogs are fed with 250g of feed every day, old dogs are fed with 200g of feed every day. The feed is fed for 1 time respectively in the morning and afternoon, and the water is freely drunk. Exercise time is available every day. Feces are cleared every day. The test started on day 25, month 2 of 2020 and lasted for 30 days.

2.5 sampling index and method

2.5.1 weight: the body weight was weighed on days 0, 7, 14, 21, 30 at the start of the test.

2.5.2 feed intake: feed intake was recorded 1 total per dog daily.

2.5.3 case of pile: the condition of the hair of each dog was recorded 1 time each morning round by taking a photograph, which required that the smoothness and gloss of the hair be clearly seen.

2.5.4 skin condition: the skin state of each dog is recorded 1 time every morning round, and the back, chest and face of the dog are pulled out each time to observe whether the skin has erythema, erythra, dandruff and other abnormal conditions.

2.5.5 defecation condition: feces were recorded for each dog 1 time daily in the morning at the time of feces clearance.

2.5.6 mental state: the mental state of each dog was recorded 2 times each day during morning and evening rounds and scored.

3. Results

3.1 body weight

The test dogs were scored for a total of five body weights during the test period and the results are shown in table 6. The body weight changes on days 7, 14, 21 and 30 of the test as compared with those before the start of the test (D0) are shown in table 7, and the body weight changes on each week of the test are shown in table 7. The body weight analysis of the test dogs is shown in tables 8, 9 and 10.

Table 6 test dogs body weight recording units: kg of

Description of the drawings: d0: the day before the start of the test; d7: day 7 of start of experiment; d14: day 14 after the start of the test, and so on.

Table 7. test dog body weight change units: kg of

Table 8 mean body weight units of test dogs at different test stages: kg of

Group of	D0	D7	D14	D21	D30
						Blank control group	8.02	7.96	7.97	8.24	8.69
Test group	7.67	7.94	8.11	8.35	8.84
						Positive control group	8.16	8.39	8.50	8.73	8.84

Table 9 mean weight gain in units of test dogs at different test stages compared to initial body weight: kg of

Group of	D7-D0	D14-D0	D21-D0	D30-D0 weight gain
					Blank control group	-0.06	-0.05	0.22	0.68
Test group	0.27	0.44	0.68	1.17
					Positive control group	0.23	0.34	0.56	0.68

Table 10 average weight gain per week units for test dogs: kg of

Group of	D7-D0	D14-D7	D21-14	D30-21
					Blank control group	-0.06	0.01	0.27	0.46
Test group	0.27	0.16	0.24	0.49
					Positive control group	0.23	0.11	0.22	0.11

As can be seen from table 8, the body weight of the blank control group dogs was greater at the beginning of the test than in the test group, but the body weight did not rise and fall for the next 2 weeks, and began to increase after 2 weeks, but at the end of the test, the average body weight of this group was the lowest of the three groups. Table 10 shows that the weight gain of the blank control group dogs was worst and even negative within the first 2 weeks, but the dogs in this group had the fastest weekly weight gain at week 3, and the weekly weight gain at week 4 was lower than that in the test group but higher than that in the positive control group, and from Table 9, the average weight gains of the dogs in the blank control group and the positive control group were the same throughout the test period

From the body weight data, the test group performed best. Table 8 shows that at the beginning of the test, the test dogs had the lowest body weight, but at the end of the test period, the group was higher than the blank control group and the same as the positive control group. The data in Table 9 show that the dogs in the test group increased their weight throughout the test period, and at the end of the test, the average weight gain of the dogs in this group was 1.11 kg/dog, which was the highest cumulative weight gain in the three groups. Furthermore, table 10 shows that the weekly weight gain of the test groups was always the highest in the three groups. This indicates that the probiotic effect of the probiotic formulation is very significant.

3.2 ingestion

From the record table, 250g of the daily diet is taken by one big dog and 200g of the daily diet is provided for puppies, and the puppies are fed cleanly every day without residue.

3.3 case of duds

See table 11. There was a slight dehairing in each group of dogs. In addition, the blank test group has the most abnormal hair in the whole test period, the positive control group has only 1 dark and dull hair, and the test group has no abnormality. This is sufficient to indicate that the present probiotics are not harmful to beagle dogs.

At the beginning of the test, the fur of each group of dogs is flat, bright and upright, and no unhairing phenomenon occurs. Light depilation appeared in each group starting on day 4, but the blank group had 4 dogs with rough, dull, soft and collapsed fur for 5 days. The 9 th sky white control group had only 2 dogs with poor hair and persisted until day 17. On day 23, one dog was dull with hair in each of the blank and positive control groups. In other test periods, the fur of each group of dogs appears without abnormal condition. It was observed that the sheds of the homemade microbiota were the most smooth (see fig. 13).

TABLE 11 abnormal statistics of experimental dog fur conditions

Note: the coat status of one dog was scored as 1 dog per day.

3.4 skin Condition

From the recorded table, the skin was free of abnormalities except for the presence of hair loss. The microecological preparation of the invention is harmless to dogs and does not cause skin abnormality.

At the beginning of the test, the skin of each group of dogs was free of abnormal conditions. From day 4 onwards, the dogs in each group showed a slight loss of hair, but no other skin abnormalities. This continued until the end of the test period, where no dandruff, red rash, redness and other abnormalities were present on the skin of the test dogs in each group, except for mild alopecia.

3.5 fecal Condition

The excrement conditions of the test dogs in each group are summarized in a table 12, 7 dog hard stools and 4 dog brown stools appear in a blank control group, 1 dog hard stool and 1 dog brown stool appear in a positive control group in the whole test period, and the whole test period of the test group is not abnormal. This indicates that the present probiotics are safe and harmless to beagle dogs.

TABLE 12 statistics of abnormal stool status of test dogs

Note: fecal status of one dog a day was scored as 1 dog.

At the beginning of the test, the feces of each group of dogs were yellow and soft stools. On day 22, 3 hard stools appeared in the blank group. On day 23, 2 brown hard stools appeared in the blank group and continued until day 24 and disappeared on day 25. On day 24, 1 brown hard stool appeared in the positive control group and disappeared on day 25. The test group did not show loose stool, soft stool or hard stool, abnormal stool color and stool residue.

3.6 mental states

See table 13. The blank dogs showed the most psychiatric abnormalities, followed by the test group and the lowest positive control group. These abnormalities appeared within 18 days after the start of the test, and no abnormalities appeared in each group from day 19 to the end of the test. This indicates that the present probiotic is not harmful to the beagle.

TABLE 13 Experimental dog statistics of mental state abnormalities

Specifically, at the beginning of the test, all groups of dogs were active, i.e., active activities such as barking, walking, tail-waving, forepaw-stretching, and hurdling were all observed, except blinking. On day 4, the activity of 4 dogs in the blank control group decreased without climbing the fence and reaching the forepaw, and this status continued until day 8. On day 9, 2 dogs in the blank control group had low activity and appeared in a lying-in-sitting state, while the activity of the test group and the positive control group was not abnormal. Recovering the liveness of the blank control group dogs on the 10 th day; the test group and the positive control group have no abnormal activity. In addition, one sitting and lying down occurred in each of the blank control group and the positive control group on day 15. This condition continued until day 17. Starting on day 18, there were no abnormalities in each group.

From the aspects of weight, ingestion, hair, skin, feces and mental conditions, the microecological preparation is harmless to dogs and can be eaten for a long time. The feeding amount of the microecological preparation can be lower than 0.5g/kg body weight, namely less than the dosage of the probiotics of Maidred. The growth promoting effect of the probiotic of the invention is best seen in the weight gain of the test dogs. The formula of the microecological preparation not only is added with prebiotics such as oligosaccharide, but also is added with amino acid for promoting the growth of intestinal microorganisms. From the smoothness of the hair, the microecological preparation is obviously superior to a positive control group. In conclusion, the microecological preparation is harmless to dogs, has no abnormal expression when being continuously fed for 30 days, has good weight increasing effect, and obviously improves the smoothness of hairs.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A microecological formulation, which comprises:

(2) amino acids comprising L-arginine and glutamine;

(4) and the carrier is glucose.

2. The formulation of claim 1, wherein:

the preparation also comprises a phagostimulant and/or a vitamin complex.

3. The method according to claim 1 or 2, characterized in that the content of the components in the formulation in parts by weight is as follows:

4. the formulation according to any one of claims 1 to 3, characterized in that:

the prebiotics are inulin; preferably, the inulin is present in an amount of 0.1 to 0.3 parts by weight.

5. The formulation according to any one of claims 1 to 4, characterized in that:

the phagostimulant is a yeast phagostimulant; preferably, the content of the phagostimulant is 0.8-1.2 parts by weight;

the vitamin complex component comprises the following components: vitamin A acetate is more than or equal to 5,000,000IU/kg, vitamin D3 is more than or equal to 500,000IU/kg, tocopherol acetate is more than or equal to 50,000IU/kg, vitamin B1 is more than or equal to 1,600mg/kg, vitamin B2 is more than or equal to 2,200mg/kg, vitamin B6 is more than or equal to 1,600mg/kg, vitamin B12 is more than or equal to 30mg/kg, nicotinamide is more than or equal to 13,000mg/kg, D-calcium pantothenate is more than or equal to 8,000mg/kg, folic acid is more than or equal to 180mg/kg, and biotin is more than or equal to 50 mg/kg; preferably, the content of the multivitamin component is 0.5 to 1.0 part by weight.

6. A method of making the formulation of any one of claims 1 to 5, comprising:

7. The method of claim 6, wherein:

the saccharomyces cerevisiae bacterial powder is prepared by adopting the following steps: (1) activating strains: inoculating saccharomyces cerevisiae into a liquid culture medium for shake cultivation, wherein the cultivation temperature is 26-32 ℃, the oscillation parameter is 100-200rpm, and the cultivation time is 1.5-2.5h, so as to obtain activated bacterial liquid; (2) fermentation: inoculating the activated bacterial liquid into a culture medium of a fermentation tank for culture, wherein the culture temperature is 26-32 ℃, the ventilation quantity is 0.5-1.6vvm, the power of a stirrer is 1-2kwh per cubic meter, and the culture time is 7-9h, so as to obtain a saccharomyces cerevisiae fermentation liquid; (3) freeze-drying: centrifuging the saccharomyces cerevisiae fermentation liquor, removing supernatant, and freeze-drying the precipitate to obtain saccharomyces cerevisiae bacterial powder;

the lactobacillus acidophilus powder and the lactobacillus casei powder are independently carried out in the following modes: (1) activating strains: inoculating strains into the MRS culture medium, standing at 36-38 deg.C, and anaerobically culturing for 18-30h to obtain first activated bacteria liquid; inoculating the first activated bacterium liquid into an MRS liquid culture medium, standing at 36-38 ℃ for anaerobic culture for 18-30h to obtain a seed liquid; (2) fermentation: inoculating the seed solution into a fermentation tank culture medium according to the inoculation amount of 4%, carrying out constant-temperature anaerobic culture at 36-38 ℃ and 100rpm for 66-78h to obtain a fermentation liquid; (3) freeze-drying: and centrifuging the fermentation liquor, removing supernatant, and freeze-drying the precipitate to obtain corresponding bacterium powder.

8. The method of claim 7, wherein:

the viable bacteria content of the saccharomyces cerevisiae inulin is 150-250 hundred million CFU/g;

the viable bacteria content of the lactobacillus acidophilus powder is 400-600 hundred million CFU/g; and/or

The viable bacteria content of the lactobacillus casei powder is 800-1200 hundred million CFU/g.

9. The method according to claim 7 or 8, characterized in that:

the liquid medium was formulated with 1 mass% yeast extract, 2 mass% peptone and 2 mass% maltose;

the fermentation tank culture medium is prepared from 0.25 mass% of yeast extract, 0.2 mass% of maltose, 0.02 mass% of sodium chloride and 0.82 mass% of sodium acetate; and/or

The culture medium of the fermentation tank comprises the following components: peptone 1-3%, beef extract 1-3%, yeast extract 1-3%, glucose 6-8%, diammonium citrate 0.3%, K₂HPO₄·7H₂O 0.2％、MgSO₄·7H₂O 0.09％、MnSO₄·4H₂O 0.025％、NaAc·3H₂O0.3% and Tween-800.1%.

10. Use of a probiotic according to any one of claims 1 to 5 or prepared by a process according to any one of claims 6 to 9 as an additive or supplement to a feed, in particular to a feed for dogs, such as pet dogs.