CN116439315A - Clostridium butyricum fermentation product of astragalus membranaceus stem and leaf as well as preparation method and application thereof - Google Patents
Clostridium butyricum fermentation product of astragalus membranaceus stem and leaf as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN116439315A CN116439315A CN202310432366.5A CN202310432366A CN116439315A CN 116439315 A CN116439315 A CN 116439315A CN 202310432366 A CN202310432366 A CN 202310432366A CN 116439315 A CN116439315 A CN 116439315A
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- Prior art keywords
- clostridium butyricum
- astragalus
- parts
- fermentation
- leaf
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- C—CHEMISTRY; METALLURGY
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
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Abstract
The invention discloses a clostridium butyricum fermentation product of astragalus stems and leaves, a preparation method and application thereof. The astragalus stem and leaf fermentation medium comprises 6-10 parts of astragalus stem and leaf, 0-4 parts of wheat bran, 5-25 parts of water, 0.25-1.25 parts of yeast extract, 0.125-0.625 parts of peptone, 0.5-2.5 parts of ammonium sulfate, 0.25-1.25 parts of monopotassium phosphate and 0.125-0.625 parts of magnesium sulfate. In the clostridium butyricum fermented product of the astragalus stems and leaves, clostridium butyricum and the astragalus stems and leaves are mutually matched in a synergistic way, so that the growth performance of animals can be effectively improved, and the immunity and intestinal health of the animals can be regulated. The invention can fully utilize natural plant resources of astragalus stem and leaf, improves the feeding value of the astragalus stem and leaf, and provides an effective way for resource utilization of non-medicinal parts of traditional Chinese medicines and development of natural plant feeding tie-down products.
Description
Technical Field
The invention relates to the technical field of natural plant feeding, in particular to a clostridium butyricum fermentation product of astragalus stems and leaves, and a preparation method and application thereof.
Background
In recent years, the abuse of antibiotics in the breeding industry has led to a series of social problems, so the country has greatly advanced the implementation of the policies of "resistance reduction, resistance replacement and no resistance" in the breeding industry. Under the development trend of healthy green cultivation, the demands of the feed and cultivation industry for tibody products are continuously increased. 117 kinds of other natural plants for feeding are recorded in the feed raw materials catalog issued by the agricultural rural department of China, wherein most of the natural plants are medicinal and edible plant traditional Chinese medicine resources. The market demand for traditional Chinese medicine feed products is further improved on the basis of the original traditional Chinese medicine feed products, and the development prospect is wider. However, the traditional Chinese medicine used as a raw material of the feed product has the problems of high cost, poor economic benefit and the like. Therefore, searching for traditional Chinese medicine and natural plant feed raw materials with large resource reserves and low production cost is likely to become a trend in the future.
Astragalus mongholicus has been a common bulk drug in our country since ancient times. A large amount of stem and leaf resources can be generated in the planting and harvesting process of astragalus medicinal materials every year, and the astragalus medicinal materials are not effectively utilized for a long time. Modern researches show that astragalus stem and leaf is rich in amino acids, proteins, saccharides and other nutritional components, and flavonoids, saponins and other functional components, has biological effects of regulating immunity, inhibiting bacteria, resisting oxidation, improving gastrointestinal tract functions and the like, and is a potential antibiotic substitute. However, the application of the stem and leaf of astragalus mongholicus in the animal breeding industry is still in the primary stage, the feeding mode is rough, the palatability is poor, and the utilization rate is low.
Disclosure of Invention
The invention aims to: the invention aims to solve the defects in the prior art. Provides a clostridium butyricum fermentation product of astragalus stems and leaves, a preparation method and application thereof. Clostridium butyricum (Clostridium butyricum) can produce short chain fatty acids such as butyric acid and acetic acid using various carbohydrates, and various digestive enzymes and antioxidases. Researches show that clostridium butyricum can maintain animal intestinal flora and intestinal microecological balance, regulate intestinal mucosa immunity, and therefore play roles in preventing and treating animal diseases and promoting growth. Clostridium butyricum was included in the feed additive catalogue in 2009. In recent years, clostridium butyricum has become a research hotspot in the industry with the implementation of the policies of "resistance reduction" and "resistance replacement" of feeds, and has been widely applied to animal breeding industry. In the fermentation process, the components such as soluble sugar and fiber contained in the astragalus stem and leaf can provide carbon sources for the growth and metabolism of clostridium butyricum, and the components such as short-chain fatty acids and amino acids such as acetic acid and butyric acid generated in the metabolism process of clostridium butyricum can improve the flavor and palatability of the astragalus stem and leaf, and can improve the utilization rate of the astragalus stem and leaf as livestock and poultry feed.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a clostridium butyricum fermentation product of astragalus stems and leaves is obtained by fermenting clostridium butyricum seed liquid in an astragalus stems and leaves fermentation medium.
As a preferred scheme, the preparation raw materials of the clostridium butyricum fermentation product of the astragalus stems and leaves comprise 6-10 parts of astragalus stems and leaves and 0-4 parts of wheat bran in parts by weight.
As a more preferable scheme, the preparation raw materials of the clostridium butyricum fermentation product of the astragalus stems and leaves further comprise 5-25 parts of water, 0.25-1.25 parts of yeast extract, 0.125-0.625 parts of peptone, 0.5-2.5 parts of ammonium sulfate, 0.25-1.25 parts of monopotassium phosphate and 0.125-0.625 parts of magnesium sulfate.
As a more preferable scheme, the preparation raw materials of the clostridium butyricum fermentation product of the astragalus stems and leaves comprise 7 parts of astragalus stems and leaves and 3 parts of wheat bran in parts by weight.
As a preferable scheme, the preparation raw material of the clostridium butyricum fermentation product of the astragalus stems and leaves has the inoculation amount of clostridium butyricum seed liquid of 10-30%.
As a preferred scheme, the preparation raw material of the clostridium butyricum fermentation product of the stem and leaf of astragalus has the bacterial concentration of 10 6 ～10 8 CFU/mL。
As a more preferable scheme, the preparation raw material of the clostridium butyricum fermentation product of the astragalus stem and leaf is characterized in that the inoculation amount of clostridium butyricum seed liquid is 30%.
In a second aspect, the invention provides a preparation method of clostridium butyricum fermentation product of astragalus stems and leaves, which comprises the following steps:
according to the proportion in the first aspect of the invention, the clostridium butyricum seed liquid is inoculated into the astragalus membranaceus stem and leaf fermentation medium, and then anaerobic solid state fermentation is carried out.
As a preferred scheme, the preparation method of the clostridium butyricum fermentation product of the astragalus stems and leaves is characterized in that the astragalus stems and leaves fermentation medium is subjected to high-temperature wet heat sterilization treatment.
As a preferred scheme, the preparation method of the clostridium butyricum fermentation product of the astragalus stems and leaves is characterized in that clostridium butyricum seed liquid is inoculated into the fermentation medium of the astragalus stems and leaves, and then deoxidization treatment is carried out.
As a preferred scheme, the preparation method of the clostridium butyricum fermentation product of the astragalus stems and leaves is characterized in that the anaerobic solid state fermentation temperature is 35-40 ℃ and the fermentation time is 2-6 d.
As a more preferable scheme, the preparation method of the clostridium butyricum fermentation product of the astragalus stems and leaves is characterized in that the anaerobic solid state fermentation temperature is 37 ℃ and the fermentation time is 5d.
The clostridium butyricum fermented product of astragalus stems and leaves can be used for preparing the feed for regulating animal immunity and intestinal functions. The adding amount of the clostridium butyricum fermentation product of the astragalus stems and leaves is 1-10%.
Compared with the prior art, the clostridium butyricum fermented product of astragalus stems and leaves has the following beneficial effects:
according to the invention, the astragalus stems and leaves are used as main raw materials, clostridium butyricum is utilized to perform anaerobic solid fermentation, and in the fermentation process, the nutrition value of the astragalus stems and leaves is improved, and the flavor and palatability of the astragalus stems and leaves are improved due to the fact that acetic acid, butyric acid and other short-chain fatty acids and amino acids and other components generated by clostridium butyricum are improved. In addition, clostridium butyricum has the functions of maintaining the balance of intestinal flora, regulating the immunity of intestinal mucosa and the like, and can be matched with stem and leaf of astragalus to play a better biological role. The clostridium butyricum fermented product of astragalus stems and leaves can effectively regulate the immunity and intestinal health of animals. The invention fully utilizes the natural plant resource which is not paid attention for a long time, namely the stem and leaf of the astragalus, and provides an effective way for the resource utilization of non-medicinal parts of the traditional Chinese medicine and the development of the natural plant feed-use tiepin product.
Drawings
FIG. 1 shows the effect of clostridium butyricum fermentation on the growth performance of mice, wherein A is the weight gain of each group of mice from day 1 to day before molding, and B is the weight change of each group of mice after molding. And (3) injection: compared to the blank control group,: p <0.05,: p <0.01; compared to model group, #: p <0.05, #: p <0.01
FIG. 2 is a spleen tissue section (x 100) of each group of mice.
FIG. 3 shows thymus tissue sections (X200) of mice in each group.
FIG. 4 is a section of small intestine tissue of each group of mice (. Times.100).
Figure 5 is a species composition at the portal level for each group of mice intestinal flora.
FIG. 6 shows species composition at the genus level for each group of mice intestinal flora.
Description of the preferred embodiments
The invention will be better understood from the following examples. It should be noted that the following examples are only for explaining the present invention and are not intended to limit the scope of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the species, materials, reagents, etc. used, unless otherwise specified, are commercially available. The stem and leaf of Astragalus membranaceus used in the following examples are all obtained from Min county, jun xi Shi, gansu province, and dried at 55deg.C. Clostridium butyricum (ATCC 19398) was used as purchased from the Shanghai collection biotechnology center.
Example 1 preparation of Clostridium butyricum fermentation product from Astragalus Stem and leaf
A preparation method of clostridium butyricum fermented product of astragalus stems and leaves comprises the following steps:
(1) Preparing clostridium butyricum seed liquid: inoculating activated clostridium butyricum into LB liquid medium, anaerobic culturing in shaking incubator at 37deg.C for 24 hr to obtain clostridium butyricum seed liquid, and controlling seed liquid concentration to 10 6 CFU/mL。
(2) Pretreatment of fermentation substrate: pulverizing stem and leaf of radix astragali and testa Tritici, respectively, and sieving with 28 mesh sieve.
(3) Weighing a fermentation substrate: and (3) respectively weighing 7 parts of astragalus membranaceus stems and leaves and 3 parts of wheat bran in the step (2) according to parts by weight, and uniformly mixing for later use.
(4) Preparation of fermentation medium: according to the weight portions, 1 portion of yeast extract, 0.5 portion of peptone, 2 portions of ammonium sulfate, 1 portion of monopotassium phosphate, 0.5 portion of magnesium sulfate and 20 portions of water are respectively weighed, fully mixed and dissolved, then evenly mixed with the fermentation substrate in the step (3), placed in a fermentation container, and subjected to high-pressure wet heat sterilization at 121 ℃ for 20 minutes.
(5) And (3) after the fermentation medium in the step (4) is cooled, inoculating the clostridium butyricum seed liquid prepared in the step (1) according to the inoculum size of 30 percent, filling nitrogen into a sealed fermentation container, discharging oxygen, and culturing for 5d at 37 ℃.
(6) And (3) after the step (5) is finished, unsealing the fermentation container, drying the fermentation product at 55 ℃, taking out, slightly crushing, and then sieving with a 28-mesh sieve to obtain the clostridium butyricum fermentation product of the astragalus stems and leaves.
EXAMPLE 2 comparison of chemical Components before and after fermentation of Astragalus Stem and leaf
The unfermented astragalus stem and leaf fermentation substrate prepared according to the methods of step (2) and step (3) in example 1 is a sample before astragalus stem and leaf fermentation, and the clostridium butyricum fermented product of astragalus stem and leaf in example 1 is a sample after astragalus stem and leaf fermentation, and the content changes of crude fiber, total protein, reducing sugar, soluble polysaccharide, total flavone, total saponin, amino acid components and flavonoid components before and after astragalus stem and leaf fermentation are compared. Wherein, the crude fiber measurement refers to national standard GB/T6432-2018, the total protein measurement adopts BCA method, the determination of reducing sugar, soluble polysaccharide, total flavone and total saponin adopts colorimetric method, and the determination of amino acid component and flavonoid component adopts UPLC-TQ-MS method. The results are shown in tables 1 and 2.
TABLE 1 nutrient content comparison before and after fermentation of Astragalus stems and leaves
Note that: post-fermentation compared to pre-fermentation, represents significant difference (P < 0.05) and represents very significant difference (P < 0.01).
TABLE 2 comparison of the content of functional Components before and after fermentation of Astragalus Stem and leaf
Note that: post-fermentation compared to pre-fermentation, represents significant difference (P < 0.05) and represents very significant difference (P < 0.01).
From tables 1 and 2, the content of crude fiber and reducing sugar in the stem and leaf of astragalus root after fermentation is obviously reduced, and the content of soluble polysaccharide is obviously improved. Presumably, this is due to the consumption of glucose or xylose in the stem and leaf of astragalus membranaceus by clostridium butyricum during growth and metabolism; at the same time, the cellulase produced by the method degrades part of crude fiber and converts the crude fiber into polysaccharide with smaller molecular weight. The total protein content of the fermented product of the stems and leaves of the astragalus membranaceus is obviously increased compared with that before fermentation, one part of the reasons are that clostridium butyricum generates various enzymes in the growth metabolism process, the other part of the reasons are that a certain amount of yeast extract and peptone are added into a fermentation substrate to cause interference to protein content measurement when clostridium butyricum ferments the stems and leaves of the astragalus membranaceus. Compared with the stem and leaf of astragalus membranaceus before fermentation, the stem and leaf of astragalus membranaceus has obviously improved contents of various amino acids and partial nucleosides. The results show that after clostridium butyricum ferments the astragalus stems and leaves, the nutrition value and flavor of the astragalus stems and leaves are effectively improved. In addition, after fermentation, the composition and content of the secondary metabolites in the stem and leaf of astragalus membranaceus are also changed. In the previous research, the flavonoids in the stems and leaves of astragalus root are mainly composed of kaempferol, kaempferide, quercetin and isorhamnetin as parent nuclei, and after clostridium butyricum fermentation, the contents of the 4 flavonoid aglycones are obviously improved, and the contents of the corresponding flavonoid aglycones are reduced to different degrees. The membrane permeability of the flavonoid aglycone is stronger than that of the flavonoid glycoside, so that the biological activity of the stem and leaf of astragalus membranaceus can be improved after fermentation.
Example 3 influence of Clostridium butyricum fermentation of Astragalus Stem and leaf on mouse immune function and intestinal health
SPF grade ICR healthy mice (body weight 15-18 g) used in this example were purchased from Shanghai Laek laboratory animal Limited, animal license number: SCXK (Shanghai) 2022-0004. Mice were kept in the university of chinese medicine, south kyo, drug safety evaluation research center. The test scheme is approved by the ethical committee of animal experiments of Nanjing university of traditional Chinese medicine, and the ethical license number is fed: 202206A010. The eucommia ulmoides element used in this example was purchased from Zhangjiu Hexing Biotechnology Co.
80 SPF grade ICR healthy mice were selected, male and female halves. After SPF environmental adaptation feeding for 3 days, mice were randomly divided into 5 groups, which are respectively a blank group (control), a model group (model), a positive control group (YX, 1% eucommia ulmoides element was added to the daily maintenance feed for mice), a low dose group of clostridium butyricum fermented product of astragalus mongholicus stems and leaves (CB-L, 2% clostridium butyricum fermented product of astragalus mongholicus stems and leaves was added to the daily maintenance feed for mice), and a high dose group of clostridium butyricum fermented product of astragalus mongholicus stems and leaves (CB-H, 4% clostridium butyricum fermented product of astragalus mongholicus stems and leaves was added to the daily maintenance feed for mice), each group was fed in 16 cages, and 4 mice were fed in 4 cages. Mice in the blank group and the model group are fed with conventional maintenance feed every day, and other mice in each group are respectively fed with test feed containing corresponding doses and are continuously fed for 25 days. Starting from the 22 th day of feeding, the mice in the blank group are intraperitoneally injected with physiological saline every day, and the other mice in each group are intraperitoneally injected with cyclophosphamide 50mg/kg every day for 3 days, so that an immunocompromised model is established. If the mice have serious unhairing, appetite reduction, listlessness, spleen atrophy, liver blushing, thymus damage and the like, the establishment of the model of the immune deficiency is successful.
1. Influence on the growth performance of mice
The weight of the mice was weighed and recorded daily, and the weight gain from day 1 to day before molding was calculated for each group of mice, and the weight change for each group of mice after molding was calculated, and the results are shown in fig. 1.
Before molding, the average daily gain of mice fed with the clostridium butyricum fermented feed containing the astragalus stems and leaves prepared in example 1 was significantly increased (P < 0.05) compared with mice fed with daily maintenance feed. After 3 days of continuous intraperitoneal injection of cyclophosphamide, the mice in the model group had significantly reduced body weight (P < 0.01) compared to the control group; compared with the model group, the body weight reduction degree of the female mice in the positive control group and the clostridium butyricum ferment of the astragalus stem and leaf is obviously reduced (P < 0.01) in the male mice in the high-dose group; the results indicate that the clostridium butyricum fermented product of astragalus stem and leaf not only can improve the growth performance of normal mice, but also can effectively restore the weight of immunocompromised mice.
2. Effects on immune function in mice
After 12h of last feeding, the weight of the mice was weighed, then the orbital veins were bled and rapidly transferred to a 2mL EP centrifuge tube for determination of IL-2, IL-6, TNF- α and IgA; the mice were then sacrificed by cervical dislocation, immediately removed spleen and thymus tissues, washed clean with physiological saline, spleen and thymus mass were weighed, and immune organ indices were calculated. And subpackaging the collected tissue samples, respectively placing the tissue samples into 4% paraformaldehyde fixing solution for fixing, performing trimming, dehydration, embedding, slicing and dyeing treatment, and observing the morphology under an optical microscope. The mouse immune organ index is shown in table 3, the cytokine and immunoglobulin levels are shown in table 4, and spleen and thymus tissue sections are shown in fig. 2 and 3, respectively.
TABLE 3 immune organ index for mice of each group
Note that: compared to the blank control group,: p <0.05,: p <0.01; compared to model group, #: p <0.05, #: p <0.01.
TABLE 4 serum cytokines and immunoglobulin content measurements for groups of mice
Note that: compared to the blank control group,: p <0.05,: p <0.01; compared to model group, #: p <0.05, #: p <0.01.
As can be seen from table 3, after 3 days of continuous intraperitoneal injection of cyclophosphamide, both spleen index and thymus index were significantly reduced (P < 0.01) in the mice of the model group compared to the blank group; compared with a model group, the positive control group and the clostridium butyricum fermented product of the astragalus stems and leaves prepared in the embodiment 1 of the invention have low spleen index and thymus index descending trend of mice in a high-dose group obviously reduced, and the preliminary indication that the clostridium butyricum fermented product of the astragalus stems and leaves can effectively improve the spleen and thymus injuries of immunocompromised mice.
As can be seen from table 4, the serum IL-2, IL-6, TNF- α and IgA levels were significantly reduced in the mice of the model group compared to the blank group (P < 0.01); compared with a model group, the low-dose group mice IL-6, TNF-alpha and IgA content of the clostridium butyricum fermented product of the astragalus stems and leaves prepared by the embodiment 1 of the invention are obviously increased (P < 0.01), and the low-dose group mice IL-2 of the clostridium butyricum fermented product of the astragalus stems and leaves are obviously increased (P < 0.05).
The results indicate that the clostridium butyricum fermented product of astragalus stem and leaf can effectively regulate the serum cytokine and immunoglobulin level of the immunosuppressive mouse caused by cyclophosphamide.
As can be seen from fig. 2, the spleen red marrow and white marrow of the mice in the blank group are clearly demarcated; the lymphocyte arrangement is compact; the spleen red marrow and white marrow of the mice in the model group are fuzzy in demarcation, the cell arrangement is disordered, and the marginal area is obviously widened; the area of the white marrow area is obviously reduced; compared with the model group, the low-dosage group mice with low-dosage clostridium butyricum ferment of astragalus stem and leaf prepared in the example 1 tend to be tidy and compact in arrangement of spleen cells, and the red marrow and white marrow are obviously and clearly demarcated.
As can be seen from fig. 3, the thymus lobule structure of the mice in the blank group is obvious, the cortical medulla is clearly divided, the small lymphocytes in the cortex are abundant in quantity, the morphological structure is normal, the medulla is mainly large lymphocytes, and the thymus bodies are distributed in a dispersed manner, so that no obvious abnormality is seen; the thymus tissue of the mice in the model group is obviously atrophic, the boundary between the cortex and the medulla is not clear, the cortex layer is obviously reduced, the lymphocyte number is severely reduced, and the thymus body number in the medulla is reduced; compared with a model group, the low-dosage group mice with low-dosage clostridium butyricum fermented product of astragalus stems and leaves prepared in the embodiment 1 have obviously improved thymus injury degree, increased cortical layer ratio and obviously increased lymphocyte number. The results further indicate that the clostridium butyricum fermented product of astragalus stem and leaf can effectively improve spleen and thymus tissue damage caused by cyclophosphamide to mice.
3. Effects on intestinal health in mice
Before final feeding, the abdomen of the mice is gently rubbed in an aseptic environment, the fresh colon content is taken out, and the mice are put into a 2mL aseptic EP tube, quickly placed into liquid nitrogen for freezing and preserving, and used for sequencing intestinal flora. After killing the mice by cervical dislocation, rapidly cutting off a jejunum section which is approximately 2cm close to the duodenum, washing with normal saline, fixing in 4% paraformaldehyde fixing solution, trimming, dehydrating, embedding, slicing and dyeing, and observing the shape of small intestine slices under an optical microscope. The small intestine sections are shown in fig. 4 and the intestinal flora species composition is shown in fig. 5 and 6.
As can be seen from fig. 4, the mice in the blank group have intact intestinal mucosa structure, intact intestinal villi, clear outline, tight and orderly arrangement, and continuous and intact villi epithelial cells. The mice in the model group have serious damage to the intestinal mucosa, so that villus epithelial cells can be obviously shed, and meanwhile, the intestinal villus structure is damaged, and the arrangement is obviously loose and disordered; the intestinal villi is obviously shortened, and the crypt is obviously shallowed. Compared with a model group, the clostridium butyricum fermented product of astragalus stems and leaves, which is prepared in the embodiment 1, has low damage degree to the small intestinal mucosa of mice in a high-dose group, the length of intestinal villi is obviously increased, and the arrangement tends to be orderly. The results indicate that the clostridium butyricum fermented product of the stem and leaf of the astragalus can effectively improve the damage of cyclophosphamide to intestinal mucosa of mice and repair the mechanical barrier effect of the intestinal canal.
As can be seen from fig. 5 and 6, at the phylum level, each group of mouse intestinal flora is mainly composed of Firmicutes, bacteroides (bacterioides), actinomycetes (actionobacteria), proteus (Proteobacteria) and the like, wherein Firmicutes and bacteroides are in front of two in proportion. Compared with a blank control group, the proportion of the firmicutes in the intestinal flora of the mice in the model group is obviously increased (P < 0.05), and the proportion of the bacteroides is obviously reduced (P < 0.05); compared with the model group, the ratio of the clostridium butyricum fermented product group of the astragalus stems and leaves prepared in the embodiment 1 to the positive medicine group of the thick-walled fungus door and the bacteroides door tends to the blank control group. At the genus level, the intestinal flora of mice in each group is mainly Lactobacillus (Lactobacillus), norank_f_Muribacuerae, enterobacter (Enterohabdus), alistipes, etc.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (9)
1. A clostridium butyricum ferment of astragalus stems and leaves is characterized in that: it is obtained by fermenting clostridium butyricum seed liquid in a astragalus stem and leaf fermentation medium.
2. The clostridium butyricum ferment of claim 1, wherein the clostridium butyricum ferment is characterized in that: the astragalus stem leaf fermentation medium comprises 6-10 parts of astragalus stem leaf and 0-4 parts of wheat bran.
3. The clostridium butyricum ferment of claim 2, wherein the clostridium butyricum ferment is characterized in that: the astragalus stem and leaf fermentation medium also comprises 5 to 25 parts of water, 0.25 to 1.25 parts of yeast extract, 0.125 to 0.625 parts of peptone, 0.5 to 2.5 parts of ammonium sulfate, 0.25 to 1.25 parts of monopotassium phosphate and 0.125 to 0.625 parts of magnesium sulfate.
4. The clostridium butyricum ferment of claim 1, wherein the clostridium butyricum ferment is characterized in that: the inoculation amount of clostridium butyricum seed liquid is 10-30%.
5. The clostridium butyricum ferment of claim 1, wherein the clostridium butyricum ferment is characterized in that: the bacterial concentration of the clostridium butyricum seed liquid is 10 6 ～10 8 CFU/mL。
6. A preparation method of clostridium butyricum fermentation product of astragalus stems and leaves is characterized in that: the method comprises the following steps:
(1) Preparing clostridium butyricum seed liquid: inoculating activated clostridium butyricum into LB liquid culture medium, performing anaerobic culture in shaking incubator to prepare clostridium butyricum seed liquid, and controlling the concentration of seed liquid to be 10 6 ～10 8 CFU/mL；
(2) Pretreatment of fermentation substrate: pulverizing radix astragali stem and leaf and testa Tritici, respectively, and sieving;
(3) Weighing a fermentation substrate: weighing a certain amount of astragalus root stems and leaves and wheat bran in the step (2) respectively according to parts by weight, and uniformly mixing for later use;
(4) Preparation of fermentation medium: weighing a certain amount of yeast extract, peptone, ammonium sulfate, monopotassium phosphate, magnesium sulfate and water according to parts by weight, fully mixing, dissolving, uniformly mixing with the fermentation substrate in the step (3), placing in a fermentation container, and sterilizing under high pressure and moist heat;
(5) After the fermentation medium in the step (4) is cooled, inoculating the clostridium butyricum seed liquid prepared in the step (1), filling nitrogen into a sealed fermentation container, discharging oxygen, and fermenting for 2-6 d at 35-40 ℃;
(6) And (3) after the step (5) is finished, unsealing the fermentation container, drying the fermentation product, taking out, slightly crushing, and screening to obtain the clostridium butyricum fermentation product of the astragalus stems and leaves.
7. The method for preparing clostridium butyricum fermented product of astragalus stem and leaf according to claim 6, wherein the method is characterized in that: the method comprises the following steps:
(1) Preparing clostridium butyricum seed liquid: inoculating activated clostridium butyricum into LB liquid medium, anaerobic culturing in shaking incubator at 37deg.C for 24 hr to obtain clostridium butyricum seed liquid, and controlling seed liquid concentration to 10 6 CFU/mL；
(2) Pretreatment of fermentation substrate: pulverizing radix astragali stem and leaf and testa Tritici, respectively, and sieving with 28 mesh sieve;
(3) Weighing a fermentation substrate: respectively weighing 7 parts of astragalus membranaceus stems and leaves and 3 parts of wheat bran in the step (2) according to parts by weight, and uniformly mixing for later use;
(4) Preparation of fermentation medium: weighing 1 part of yeast extract, 0.5 part of peptone, 2 parts of ammonium sulfate, 1 part of monopotassium phosphate, 0.5 part of magnesium sulfate and 20 parts of water according to parts by weight, fully mixing and dissolving, uniformly mixing with the fermentation substrate in the step (3), placing in a fermentation container, and sterilizing at 121 ℃ under high pressure and humidity for 20min;
(5) Inoculating the clostridium butyricum seed liquid prepared in the step (1) according to 30% of inoculum size after the fermentation medium in the step (4) is cooled, filling nitrogen into a sealed fermentation container, discharging oxygen, and culturing for 5d at 37 ℃;
(6) And (3) after the step (5) is finished, unsealing the fermentation container, drying the fermentation product at 55 ℃, taking out, slightly crushing, and then sieving with a 28-mesh sieve to obtain the clostridium butyricum fermentation product of the astragalus stems and leaves.
8. Use of clostridium butyricum fermented product of astragalus stem and leaf according to any one of claims 1 to 5 for preparing a feed additive for regulating animal immunity and intestinal function.
9. The use according to claim 8, characterized in that: the adding amount of the clostridium butyricum fermentation product of the stem and leaf of the astragalus is 1-10 percent.
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