A fermented traditional Chinese medicine composition, a preparation method therefor, and an application thereof

Abstract

The application provides a fermented traditional Chinese medicine composition, a preparation method and application, and belongs to the field of traditional Chinese medicines. The fermented traditional Chinese medicine composition is obtained by fermentation of a traditional Chinese medicine component and a probiotic component; the traditional Chinese medicine component comprises angelica sinensis, radix dipsaci, cortex fraxini, radix glechomae, radix aconiti, radix paeoniae and radix alismatis; and the probiotic component comprises lactobacillus plantarum, enterococcus faecalis and enterococcus faecium. The application can be used for treating gout in geese, reducing inflammation, liver damage, kidney damage and intestinal damage caused by gout in geese, increasing the abundance of beneficial bacteria in the intestinal tract, reducing the abundance of harmful bacteria in the intestinal tract, maintaining the health of the intestinal tract, promoting the digestion and absorption of nutrients, and effectively preventing or treating gout in goslings.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine, and relates to a fermented traditional Chinese medicine composition, in particular to a fermented traditional Chinese medicine composition and a preparation method and application thereof. BACKGROUND

[0002] Gout is a common disease of geese, mainly due to excessive urate produced in the body of geese, which causes the geese to be unable to normally discharge, resulting in the deposition of urate in the heart, liver, kidney and joints and causing diseases, which has a great impact on the health and growth of geese. The incidence of gout will cause great economic losses to breeders. For the treatment of gout, drug treatment can be given to enhance the liver and kidney function of geese; nutritional supplements can promote the recovery of sick geese to health; environmental improvement is helpful to alleviate the gout symptoms of geese. Traditional Chinese medicine is often used for the treatment of gout and shows good effects. At present, some traditional Chinese medicine prescriptions have good therapeutic effects on chicken gout, but there are still few traditional Chinese medicine prescriptions for goose gout.

[0003] Xanthine oxidase is a key target for gout treatment, and xanthine oxidase inhibitors have been proved to have a certain therapeutic effect on gout. Allopurinol and other marketed western medicines can reduce the generation of uric acid by competitive inhibition, but there are great side effects in the intake of allopurinol and other drugs, so it has become a research hotspot to explore safe and effective feed additives for inhibiting xanthine oxidase. The application of traditional Chinese medicine with the effect of inhibiting xanthine oxidase to goose gout can solve the problems of great side effects and high residue of western medicine xanthine oxidase inhibitors, and is more green, safe and effective.

[0004] The invention patent with the publication number CN104069391A discloses a traditional Chinese medicine composition for treating gout and hyperuricemia, which is composed of the following drugs: Cortex Fraxini 20-40g, Fructus Mume 5-20g, Lonicerae 5-25g, Smilax Glabra 5-25g, Lysimachia 5-20g, Plantaginis 5-20g, Polygoni Cuspidati 5-20g, Semen Euryale 5-20g, Atractylodis Macrocephalae 5-20g, Disporopsis 5-25g, Clematis 5-25g, Vitis 5-20g, Alisma 5-20g, Lilium 5-20g, Humuli 5-25g, Ostreae 10-30g. Cortex Fraxini is the monarch drug, which can clear heat and dry dampness; Semen Euryale, Fructus Mume, Lysimachia, Plantaginis and Smilax Glabra are the minister drugs, which can strengthen the effect of Cortex Fraxini, and have the effects of invigorating the spleen and draining water, detoxifying, removing dampness and dredging collaterals, among which Semen Euryale and Fructus Mume can purge the kidney of dampness, Clematis, Lysimachia, Plantaginis, Smilax Glabra and the like can promote the transformation of dampness and the discharge of turbidity, and dredge collaterals and resolve nodules; Polygoni Cuspidati, Disporopsis, Atractylodis Macrocephalae, Vitis and Humuli are the assistant drugs, which have the effects of dredging viscera, removing wind and dampness and relieving swelling and pain; Clematis, Alisma, Lilium, Humuli and Ostreae are the ministerial drugs, which can regulate the above drugs. The traditional Chinese medicine composition has the effects of treating gout and hyperuricemia.

[0005] The patent for invention with publication number CN107898978A discloses a traditional Chinese medicine composition containing Herba Lysimachiae and a preparation method thereof, and has the characteristics that it is composed of the following components in parts by weight: Herba Lysimachiae 30-50 parts, Smilax china L. 30-40 parts, Phylllum 20-30 parts, Acanthopanax giraldii Harms 20-30 parts, Radix Anemarrhenae 20-30 parts, Radix Tricampylae 10-20 parts, Fructus Crataegi 10-20 parts, Spatholobus suberectus 10-20 parts, Curcuma longa L. 10-20 parts, Angelica sinensis 10-20 parts, Omphalanthus pescaprae 10-20 parts, Poria cocos 10-20 parts, Radix Astragali 10-15 parts, Ramulus Cinnamomi 10-15 parts, Chaenomeles speciosa 8-12 parts. It has the effects of expelling wind and dampness, relieving arthralgia and pain, and harmonizing the stomach and tonifying the spleen, and has good curative effects on gout and gouty arthritis caused by high uric acid.

[0006] The patent for invention with publication number CN105056038A discloses a traditional Chinese medicine for treating gout, which is prepared from the following raw medicinal materials in a certain proportion by weight: Radix Aconiti 10-15 g, silkworm excrement 20-30 g, Radix Tricampylae 20-30 g, Rhizoma Typhonii 20-30 g, Radix Stephaniae Tetrandrae 20-30 g, Radix Euphorbiae Humifusae 20-30 g, Semen Plantaginis 20-30 g, Smilax china L. 20-30 g, Cortex Fraxini 20-30 g, Radix Paeoniae Alba 20-30 g, Radix Paeoniae Rubra 15-25 g, Radix et Rhizoma Polygalae 10-20 g, Radix Paeoniae Alba 10-20 g, Radix Paeoniae Rubra 10-20 g, Radix Paeoniae Rubra 5-15 g, Radix Paeoniae Rubra 5-15 g, Radix Paeoniae Rubra 5-15 g. The various raw medicinal materials in the prescription have the synergistic effects of clearing heat and resolving toxins, promoting blood circulation to remove blood stasis, resolving abscess and swelling, dredging collaterals to relieve pain, diuresis and detumescence, invigorating the spleen to remove dampness, relaxing the tendons to relieve arthralgia, promoting the excretion of uric acid in the body, reducing blood uric acid, adjusting the disorder of purine metabolism, and making the patient recover from the disorder of independent metabolism while reducing blood uric acid, and the like. The traditional Chinese medicine has good curative effects on gout, fast-acting, can achieve the effect of treating both the symptoms and the root cause, and is not easy to relapse after recovery.

[0007] Fermented traditional Chinese medicine can be used as an ideal substitute for antibiotics due to its green, non-polluting and non-residual characteristics. At present, there are many studies on fermented traditional Chinese medicine and gout in geese, and the above technologies have not disclosed the use of fermented traditional Chinese medicine for the prevention or treatment of gout in geese, and thus further research is needed. SUMMARY

[0008] The present application provides a fermented traditional Chinese medicine composition, a preparation method and application thereof, aiming at the problems in the prior art. The fermented traditional Chinese medicine composition can alleviate inflammation, liver damage, kidney damage and intestinal damage caused by gout in geese, and can be used for the prevention or treatment of gout in geese.

[0009] To achieve the above-mentioned object, the technical scheme adopted by the present application is as follows:

[0010] In a first aspect, the present application provides a fermented traditional Chinese medicine composition, which is fermented from a traditional Chinese medicine component and a probiotic component; the traditional Chinese medicine component comprises Angelica sinensis, Clematis finetiana, Fructus Forsythiae, Giant Knotweed, Akebia quinata, Ramulus Cinnamomi and Rhizoma Alismatis; the probiotic component comprises Lactobacillus plantarum, Enterococcus faecalis and Enterococcus faecium.

[0011] The mass ratio of the traditional Chinese medicine component to the probiotic component is 100:1-5, preferably 100:3.

[0012] The traditional Chinese medicine component comprises 8-12 parts of Angelica sinensis, 8-12 parts of Clematis finetiana, 18-22 parts of Fructus Forsythiae, 18-22 parts of Giant Knotweed, 13-17 parts of Akebia quinata, 15-20 parts of Ramulus Cinnamomi and 18-22 parts of Rhizoma Alismatis. Preferably, the traditional Chinese medicine component comprises 10 g of Angelica sinensis, 10 g of Clematis finetiana, 20 g of Fructus Forsythiae, 20 g of Giant Knotweed, 15 g of Akebia quinata, 15 g of Ramulus Cinnamomi and 20 g of Rhizoma Alismatis.

[0013] The activity ratio of Lactobacillus plantarum, Enterococcus faecalis and Enterococcus faecium in the probiotic component is 1-2:1-2:1-2, preferably 2:1:1. The strain preservation number of Lactobacillus plantarum is CGMCC 1.557, which is purchased from the China General Microbiological Culture Collection Center. The strain preservation number of Enterococcus faecalis is CICC 6078, which is purchased from the China Industrial Microbial Culture Collection Management Center. The strain preservation number of Enterococcus faecium is CGMCC 1.130, which is purchased from the China General Microbiological Culture Collection Center.

[0014] In a second aspect, the present application provides a preparation method of the fermented traditional Chinese medicine composition, which comprises the following steps:

[0015] 1) extracting the traditional Chinese medicine component to obtain a traditional Chinese medicine extract;

[0016] 2) culturing the probiotic component to obtain a bacterial liquid;

[0017] 3) inoculating the bacterial liquid obtained in step 2) into the traditional Chinese medicine extract obtained in step 1) for fermentation culture to obtain the fermented traditional Chinese medicine composition.

[0018] In step 1), the traditional Chinese medicine component is weighed, extracted and concentrated in sequence to obtain the traditional Chinese medicine extract; specifically, 100 g of the traditional Chinese medicine component is weighed, extracted with 1000 mL of water solvent at 100℃ for 0.5 h, filtered, and the filter residue is repeatedly extracted twice, and the extract liquids are combined, concentrated and the traditional Chinese medicine extract is obtained, the relative density of the traditional Chinese medicine extract at 25℃ is 1.1-1.3. In an embodiment, the relative density of the traditional Chinese medicine extract at 25℃ is 1.2.

[0019] The step 2) is: 1g of probiotic components is cultured in MRS liquid medium under anaerobic conditions at 37 DEG C to obtain a bacterial liquid, and the concentration of the bacterial liquid is 3x10 8 -4x10 8 cfu / mL, preferably 3.3x10 8 cfu / mL.

[0020] The step 3) is specifically: the bacterial liquid obtained in step 2) is inoculated into the traditional Chinese medicine extract obtained in step 1) for fermentation culture; the inoculation amount of the bacterial liquid is 1-5%, preferably 3%; the fermentation culture temperature is 35-40 DEG C, preferably 37 DEG C; and the initial pH of the fermentation culture is 6.5-7.5, preferably 7.0. Preferably, after 48h of fermentation culture, a fermented traditional Chinese medicine composition is obtained.

[0021] The fermented traditional Chinese medicine composition provided by the present application can be used for treating gout in geese, reducing inflammation, liver damage, kidney damage and intestinal damage caused by gout in geese, increasing the abundance of beneficial bacteria in the intestinal tract, reducing the abundance of harmful bacteria in the intestinal tract, maintaining the health of the intestinal tract, promoting the digestion and absorption of nutrients, and effectively preventing gout in small geese.

[0022] The fermented traditional Chinese medicine composition provided by the present application can be used for treating gout in geese, reducing inflammation, liver damage, kidney damage and intestinal damage caused by gout in geese, increasing the abundance of beneficial bacteria in the intestinal tract, reducing the abundance of harmful bacteria in the intestinal tract, maintaining the health of the intestinal tract, promoting the digestion and absorption of nutrients, and effectively preventing gout in small geese.

[0023] The fermented traditional Chinese medicine composition provided by the present application can be used for treating gout in geese, reducing inflammation, liver damage, kidney damage and intestinal damage caused by gout in geese, increasing the abundance of beneficial bacteria in the intestinal tract, reducing the abundance of harmful bacteria in the intestinal tract, maintaining the health of the intestinal tract, promoting the digestion and absorption of nutrients, and effectively preventing gout in small geese. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The fermented traditional Chinese medicine composition provided by the present application can be used for treating gout in geese, reducing inflammation, liver damage, kidney damage and intestinal damage caused by gout in geese, increasing the abundance of beneficial bacteria in the intestinal tract, reducing the abundance of harmful bacteria in the intestinal tract, maintaining the health of the intestinal tract, promoting the digestion and absorption of nutrients, and effectively preventing gout in small geese.

[0025] Figure 2 The fermented traditional Chinese medicine composition provided by the present application can be used for treating gout in geese, reducing inflammation, liver damage, kidney damage and intestinal damage caused by gout in geese, increasing the abundance of beneficial bacteria in the intestinal tract, reducing the abundance of harmful bacteria in the intestinal tract, maintaining the health of the intestinal tract, promoting the digestion and absorption of nutrients, and effectively preventing gout in small geese.

[0026] Figure 3 Fig. 2 is a symptom diagram of a gosling gout model; wherein A is a picture of a gosling anus; B is a picture of a gosling standing; C is a gosling kidney dissection diagram; D is a gosling joint dissection diagram; E is a gosling intestinal tract dissection diagram; and F is a gosling liver dissection diagram;

[0027] Figure 4 Fig. 3 is a liver tissue structure of goslings in different treatment groups; wherein A, B, C and D are liver tissue structures of the positive group, the combination group, the traditional Chinese medicine group and the fermentation group, respectively; the red arrow indicates red blood cell accumulation and inflammation; and the blue arrow indicates cell vacuolar degeneration;

[0028] Figure 5 Fig. 4 is a kidney tissue structure of goslings in different treatment groups; wherein E, F, G and H are kidney tissue structures of the positive group, the combination group, the traditional Chinese medicine group and the fermentation group, respectively; the red arrow in E indicates renal tubular epithelial cell exfoliation; the blue arrow in E indicates nuclear condensation; the red arrow in F indicates renal cystic cavity enlargement; and the red arrow in G indicates red blood cell accumulation;

[0029] Figure 6 Fig. 5 is a sample door level intestinal tract flora analysis;

[0030] Figure 7 Fig. 6 is a sample genus level flora analysis. DETAILED DESCRIPTION

[0031] It should be noted that, unless otherwise specified, the raw materials used in the present application are ordinary commercially available products, and their sources are not specifically limited.

[0032] Example 1

[0033] The traditional Chinese medicine components are composed of the following components: Angelica 10 g, Wulingxian 10 g, Qinp 20 g, Huzhang 20 g, Mutong 15 g, Guizhi 15 g, Zexie 20 g.

[0034] The probiotic components are composed of Lactobacillus plantarum, Enterococcus lactis and Enterococcus faecium with a vitality ratio of 2:1:1, wherein the strain preservation number of Lactobacillus plantarum is CGMCC 1.557, purchased from the China General Microbiological Culture Collection Center; the strain preservation number of Enterococcus lactis is CICC 6078, purchased from the China Industrial Microbial Culture Collection Management Center; and the strain preservation number of Enterococcus faecium is CGMCC 1.130, purchased from the China General Microbiological Culture Collection Center.

[0035] The preparation steps of the fermented traditional Chinese medicine composition are as follows:

[0036] Step 1): 100 g of the Chinese medicine component was weighed, and extracted with 1000 g of water solvent at 100°C for 0.5 h, filtered, to obtain an extract and a residue, the residue was repeatedly extracted twice, the extract was combined, concentrated, and a Chinese medicine extract was obtained, the relative density of the Chinese medicine extract at 25°C was 1.2.

[0037] Step 2): 1 g of the probiotic component was cultured in MRS liquid medium at 37°C under anaerobic conditions to obtain a bacterial solution, the concentration of the bacterial solution was 3.3 x 10 8 cfu / mL.

[0038] Step 3): the bacterial solution obtained in step 2) was inoculated into the Chinese medicine extract obtained in step 1) at an inoculation amount of 3%, and fermented at 37°C and pH 7.0 for 48 h to obtain a fermented Chinese medicine composition.

[0039] Example 2

[0040] The extracts of Cortex Fraxini and Polygonum cuspidatum were prepared according to the method of step 1) in Example 1, and the fermented products of Cortex Fraxini and Polygonum cuspidatum were prepared according to steps 1)-3) in Example 1. HPLC detection was performed respectively, and the detection conditions were as follows: the chromatographic column was HegaTechnologie C18 (460 nm x 4.6 mm, 5 μm) column; the mobile phase of oxymatrine, fraxin, and oxymuriine was acetonitrile-0.1% phosphoric acid solution (12:88), isocratic elution; the flow rate was 1 mL / min; the detection wavelength was 334 nm, the column temperature was 30°C, and the injection amount was 10 μL. The mobile phase of polydatin and resveratrol was acetonitrile-0.1% phosphoric acid solution (22:78), isocratic elution; the flow rate was 1 mL / min; the detection wavelength was 306 nm, the column temperature was 30°C, and the injection amount was 10 μL.

[0041] 1. HPLC detection of the content change of the main components of Cortex Fraxini before and after fermentation

[0042] The detection results of the content change of the main components of Cortex Fraxini before and after fermentation are shown in Figure 1 and Table 1. Figure 1 The content change of oxymatrine, fraxin, and oxymuriine before and after fermentation, wherein peak 1 is oxymatrine, peak 2 is fraxin, and peak 3 is oxymuriine; A, B, C, and D are the content changes of oxymatrine, fraxin, and oxymuriine before fermentation, on the 2nd day of fermentation, on the 4th day of fermentation, and on the 7th day of fermentation, respectively.

[0043] Table 1. Content change of oxymatrine, fraxin, and oxymuriine before and after fermentation

[0044]

[0045] Aesculetin is also called Esculetin, and it is a glycoside like Paeoniflorin. The results show that with the change of fermentation time, the contents of Paeoniflorin and Aesculetin show a downward trend with the increase of fermentation days, and the downward speed is faster in the first two days of fermentation, and the downward trend tends to be flat after the fourth day of fermentation. The content of Paeonoside increases gradually with the increase of fermentation days, and it increases faster in the first two days of fermentation, and the upward trend tends to be flat after the fourth day of fermentation, and it is stable at 14.69 μg / mL on the seventh day of fermentation. The contents of Aesculetin, Paeoniflorin and Paeonoside before fermentation are 127.18 μg / mL, 69.07 μg / mL and 12.11 μg / mL respectively, compared with those before fermentation, Aesculetin decreases by 9.2%, Paeoniflorin decreases by 5.73%, and Paeonoside increases by 21.30%. Lactobacillus plantarum is a kind of lactic acid bacteria, and the metabolic products of lactic acid bacteria can hydrolyze the glucose group of Paeoniflorin and Aesculetin, thereby generating Paeonoside, which is the de-glucose group product of Paeoniflorin and Aesculetin. Paeonoside not only has the effect of inhibiting xanthine oxidase, but also has the effects of antibiosis and anti-inflammation.

[0046] 2. Detection of the content change of the main components of Giant Knotweed before and after fermentation by HPLC

[0047] The detection results of the content change of the main components of Giant Knotweed before and after fermentation are shown in Figure 2 and Table 2. Figure 2 The content change of Paeoniflorin and Resveratrol before and after fermentation, wherein peak 1 is Paeoniflorin, and peak 2 is Resveratrol; A, B, C and D are the content changes of Paeoniflorin and Resveratrol before fermentation, on the second day of fermentation, on the fourth day of fermentation and on the seventh day of fermentation respectively.

[0048] Table 2. Content change of Paeoniflorin and Resveratrol before and after fermentation

[0049]

[0050] The results show that the content of Paeoniflorin shows a downward trend with the increase of fermentation days, and it decreases slowly in the first two days of fermentation, and it starts to decrease rapidly after the second day of fermentation. The content of Resveratrol increases continuously with the increase of fermentation days, and it increases slowly in the first two days of fermentation, and it starts to increase rapidly after the second day of fermentation, and it is stable at 3.31 μg / mL on the seventh day of fermentation. The contents of Paeoniflorin and Resveratrol before fermentation are 39.12 μg / mL and 2.03 μg / mL respectively, compared with those before fermentation, the content of Paeoniflorin decreases by 15.54% on the seventh day of fermentation, and the content of Resveratrol increases by 63.05%. Lactobacillus plantarum is a kind of lactic acid bacteria, and the metabolic products of lactic acid bacteria can hydrolyze the glucose group of Paeoniflorin, thereby generating Resveratrol, which is the de-glucose group product of Paeoniflorin. Resveratrol is more easily absorbed by the body than Paeoniflorin, and it also has the effects of antibiosis and anti-inflammation.

[0051] Example 3

[0052] The Chinese medicine extract obtained in step 1) and the fermented Chinese medicine composition obtained in step 3) in Example 1 were used to conduct a prevention test of gout in goslings.

[0053] Establishment of gout model in high calcium and high protein goslings: 1-day-old goslings (ordinary meat geese, body weight 83-87 g) were selected as test animals, and the test goslings were fed with uniform high calcium and high protein feed (containing 28% crude protein and 5.15% calcium) for 14 days. The goslings were allowed to freely eat and drink water every day, and the gout model in goslings was obtained, as shown in Figure 3 Figure 3 A picture of the anus of a gosling, B a picture of a gosling standing, C a gosling kidney dissection diagram, D a gosling joint dissection diagram, E a gosling intestinal tract dissection diagram, and F a gosling liver dissection diagram. Figure 3 It was shown that the gosling anus of the gout model in goslings had white thin feces, was unstable in standing, had white kidney, had urate deposition in the ureter, had joint enlargement with urate deposition, had severe intestinal congestion, and had yellow and brittle liver.

[0054] The test was divided into four different treatment groups, each group having 10 goslings (3 replicates were set in each group): the positive group was fed with high calcium and high protein feed; the combination group was fed with high calcium and high protein feed + 5% of the Chinese medicine and bacteria liquid composition (the bacteria liquid composition was prepared in step 2) of Example 1, and the composition was obtained by inoculating the bacteria liquid obtained in step 1) of Example 1 into the Chinese medicine extract at an inoculation amount of 3%); the Chinese medicine group was fed with high calcium and high protein feed + 5% of the Chinese medicine extract (prepared in step 1) of Example 1); and the fermentation group was fed with high calcium and high protein feed + 5% of the fermented Chinese medicine composition (prepared in step 3) of Example 1). The goslings were fed for 14 days, and were allowed to freely eat and drink water every day.

[0055] 1. Effect of fermented Chinese medicine composition on mortality rate of goslings

[0056] The mental state, defecation, and food intake of the goslings were observed every day, and the number of daily incidence and death was observed and recorded, and the cause of death was recorded. The results are shown in Table 3.

[0057] Table 3 Mortality rate of goslings in different treatment groups

[0058]

[0059] Table 3 shows that after being fed for 14 days, the mortality rate of the Chinese medicine group was 26.7%, which was less than that of the positive group (66.7%). The mortality rate of the goslings in the fermentation group was significantly reduced compared with the positive group, the Chinese medicine group, and the combination group. The mortality rate of the goslings in the fermentation group was reduced by 56.7% compared with the positive group, by 10% compared with the combination group, and by 16.7% compared with the Chinese medicine group, which proves that the addition of the fermented Chinese medicine composition in the diet can reduce the mortality rate of goslings with gout. ​

[0060] 2. Effects of fermented Chinese medicine composition on physiological and biochemical indicators of goslings

[0061] Ten geese were randomly selected from each group on days 6 and 14 of feeding. After fasting for 8 hours, 2 mL of venous blood was collected. The blood was then tilted and allowed to stand at room temperature for 2 hours. The serum was then centrifuged at 4000 rpm for 15 minutes, separated, and stored at -80°C. Serum levels of uric acid, xanthine oxidase, urea nitrogen, creatinine, malondialdehyde, total antioxidant capacity, superoxide dismutase, IgA, and IgG were measured using ELISA kits in each group. The results are shown in Table 4.

[0062] Table 4 Changes in physiological and biochemical indices of goslings in different treatment groups

[0063]

[0064]

[0065] (Note: Mean ± SD; significant difference analysis: P < 0.05; different letters indicate differences between groups)

[0066] The results in Table 4 show that feeding 14

[0067] After 1 day, the serum xanthine oxidase activity, uric acid, and creatinine levels of the goslings in the fermentation group decreased. The xanthine oxidase activity in the fermentation group was 1.13 U / L, significantly lower than that in the positive group, the Chinese medicine group, and the combination group (P < 0.05). The serum uric acid and creatinine levels in the fermentation group were 93.36 μmol / L and 15.05 mmol / mL, respectively, significantly lower than those in the positive group, the Chinese medicine group, and the combination group (P < 0.05).

[0068] In terms of antioxidant indicators, the malondialdehyde level in the serum of geese in the fermentation group was 1.89 nmol / mL, which was significantly lower than that in the positive group, Chinese medicine group, and combination group (P<0.05). The total antioxidant capacity and SOD (superoxide dismutase) levels of geese in the fermentation group were 5.71 U / mL and 3.93 ng / mL, respectively, which were significantly higher than those in the positive group, Chinese medicine group, and combination group (P<0.05), proving that adding fermented Chinese medicine compositions to the diet can improve the antioxidant level of geese.

[0069] In terms of immunoglobulins, the IgA (immunoglobulin A) level in the serum of geese in the fermentation group was 3.62 μg / mL, which was significantly higher than that in the positive group (P<0.05), but there was no significant difference between the fermentation group and the Chinese medicine group and the combination group (P>0.05). The IgG (immunoglobulin G) level in the serum of geese in the fermentation group was 17.98 μg / mL, which was significantly higher than that in the positive group, the Chinese medicine group and the combination group (P<0.05), proving that the fermented Chinese medicine composition can increase the level of immunoglobulin IgG in goose serum.

[0070] 3. Effects of fermented Chinese medicine composition on liver, kidney and intestinal tissues of goslings

[0071] After feeding for 14 days, one gosling was randomly selected from each replicate in each treatment group, and a total of 3 goslings were killed in each group. The liver, kidney and intestinal tissues of the goslings were collected under sterile conditions and immersed in 10% formaldehyde solution for 2 weeks in the dark. The solution was replaced daily to observe turbidity. After complete fixation, the tissues were sectioned.

[0072] The fixed tissues were first removed and cut into 1x1 cm tissues on a clean bench.

[0073] (2) Dehydration and wax immersion

[0074] The tissues were gradually dehydrated in gradient alcohol, with the following dehydration process: 75% (1.5 h)→85% (1.5 h)→90% (1.5 h)→95% I (1.5 h)→95% II (1.5 h)→100% I (5 min)→100% II (5 min). The dehydrated tissues were placed in xylene for transparency. Before transparency, the edges of the tissues were gently clamped with tweezers, and the original transparency of the tissues was observed under light. The transparency of the tissues was observed under light during the transparency process, and the transparency time was determined according to the size and properties of the tissues. The transparency process was as follows: xylene I (15 min)→xylene II (self-controlled time). The transparent tissues were placed in a wax bath to completely immerse the wax into the tissues. The wax immersion process was as follows: wax I (1 h)→wax II (1 h)→wax III (1 h)→embedding machine wax bath (3 h).

[0075] (3) Embedding

[0076] Liquid paraffin was introduced into the embedding holder, and the wax-embedded tissues were placed in the embedding holder (in the same direction as the embedding box). The cover of the embedding box was removed, and the outer bottom surface was slowly covered on the embedding holder from one side. The -20°C cold table was cooled for 30 min, and after the wax was completely solidified, the sample was removed for block trimming.

[0077] (4) Sectioning

[0078] After block trimming, the tissues were again placed in a -20°C freezing table and cooled for 30 min. After cooling, the tissues were sectioned in a microtome with a thickness of 3 μm. The edges of the paraffin sections were gently clamped with tweezers, and the sections were placed in a 37°C water bath for spreading. The spread tissues were removed with a glass slide, and the tissue information was written on the glass slide before being placed in a baking rack or oven for 2 h at 60°C.

[0079] (5) Staining

[0080] ① Dewaxing to water: xylene I 10 min → xylene II 10 min → 100% alcohol solution I → 5 min → 100% alcohol solution II 3 min → 95% alcohol solution 3 min → 90% alcohol solution 3 min → 80% alcohol solution 3 min → 70% alcohol solution 3 min.

[0081] ②Hematoxylin staining: distilled water for 3 minutes → hematoxylin for 4-6 minutes → distilled water to wash away excess stain → hydrochloric acid differentiation solution for 10-15 seconds → blueing in weak alkaline tap water for 15 minutes.

[0082] ③ Eosin staining: 70% alcohol solution for 2 minutes → 80% alcohol solution for 2 minutes → 90% alcohol solution for 2 minutes → Eosin staining for 40-1 minute → wash away excess stain with distilled water → decolorize with 95% alcohol solution for 15 seconds.

[0083] The slides were sealed and observed under scanning electron microscope.

[0084] Liver pathological section (400X) Figure 4 As shown, Figure 4 The liver tissue structure of goslings in different treatment groups, among which A, B, C, and D are the liver tissue structures of the positive group, combination group, Chinese medicine group, and fermentation group, respectively. The red arrows indicate red blood cell accumulation and inflammation; the blue arrows indicate cell vacuolar degeneration.

[0085] Figure 4 The results showed that the renal tubular epithelium in the positive group showed large areas of nuclear condensation, vacuolation, cell disintegration, and some detachment from the basement membrane, indicating that the renal tubules were severely damaged, the renal capsules were swollen, and the renal capsule cavity was enlarged. The combination group also had large areas of nuclear condensation, but no vacuolation, the renal capsule was slightly enlarged, and the renal capsule cavity was slightly enlarged. The renal tubular epithelial cells in the Chinese medicine group showed nuclear condensation and vacuolation, with more lesions, and slight detachment of renal tubular epithelial cells. In addition, red blood cells accumulated in the renal tubular spaces, the renal capsule cavity was slightly enlarged, and the renal tubular structure was basically intact. The renal tubular epithelial cells in the fermentation group had only a small amount of nuclear condensation, no vacuolation, and the renal tubular structure was intact with fewer lesions. The above results show that the high-calcium and high-protein diet caused significant damage to the kidneys of goslings, and the fermented Chinese medicine combination can effectively alleviate this damage.

[0086] Kidney pathology section (400X) Figure 5 As shown, Figure 5 The kidney tissue structure of goslings in different treatment groups; among them, E, F, G, and H are the kidney tissue structures of the positive group, combination group, Chinese medicine group, and fermentation group, respectively; the red arrow in E indicates the shedding of renal tubular epithelial cells, the blue arrow indicates nuclear condensation, the red arrow in F indicates the enlargement of the renal cyst cavity, and the red arrow in G indicates the accumulation of red blood cells.

[0087] Figure 5The results showed that the positive group had large area of nuclear condensation, vacuolization, cell disintegration, some of which were separated from the basement membrane, indicating that the renal tubular damage was serious, the renal capsule was swollen, and the renal capsule cavity was enlarged. The combination group also had large area of nuclear condensation, but no vacuolization, the renal capsule was slightly enlarged, and the renal capsule cavity was slightly enlarged. The traditional Chinese medicine group had more lesions of nuclear condensation and vacuolization of renal tubular epithelial cells, and the renal tubular epithelial cells were slightly shed. In addition, there were red blood cell accumulations in the renal tubular space, the renal capsule cavity was slightly enlarged, and the structure of the renal tubule was basically complete. The fermentation group had only a small amount of nuclear condensation, no vacuolization, and the structure of the renal tubule was complete, with fewer lesions. The above results showed that the high calcium and high protein diet caused great damage to the kidneys of goslings, and the fermented traditional Chinese medicine combination could effectively reduce this damage.

[0088] The results of intestinal pathological sections are shown in Table 5.

[0089] Table 5 Changes in the intestinal tissues of goslings in different treatment groups

[0090]

[0091] (Note: mean ± standard deviation; significant difference analysis: P < 0.05; different letters indicate differences between groups)

[0092] Table 5 shows that the positive group had shorter intestinal villus height and deeper crypt depth, the combination group still had shorter intestinal villus height and deeper crypt depth, the traditional Chinese medicine group had longer intestinal villus length but deeper crypt depth, and the fermentation group had improved intestinal villus height and crypt depth compared to the positive group. Compared to the positive group, the traditional Chinese medicine group, and the combination group, the intestinal villus height of the fermentation group was increased by 25.29%, 3.9%, and 14.15% compared to the positive group, the combination group, and the traditional Chinese medicine group, respectively, with significant differences (P < 0.05) compared to the positive group and the traditional Chinese medicine group, and no significant difference (P > 0.05) compared to the combination group. The crypt depth was decreased by 36.49%, 37.33%, and 40.25% compared to the positive group, the combination group, and the traditional Chinese medicine group, respectively, with significant differences (P < 0.05). The V / C (specifically intestinal villus height / crypt depth) was increased by 81.71%, 68.65%, and 84.58%, respectively, with significant differences (P < 0.05).

[0093] 4. Effect of fermented traditional Chinese medicine combination on intestinal flora of goslings

[0094] After 6 days and 14 days of feeding, respectively, one gosling was randomly selected from each repeat in each treatment group, and a total of 3 goslings were selected from each group for slaughter. After death, the cecal feces was collected under sterile conditions and placed in a 2 mL sterile tube, which was sent to a biological company (Shanghai Genechem Biotech Co., Ltd.) for intestinal flora high-throughput sequencing.

[0095] The results are shown in Tables 6-7 and Figures 6-7Table 6 shows the cecum Alpha diversity index, and Table 7 shows the significant analysis of intestinal microbial abundance at the phylum and genus level. Figure 6 Table 6 shows the cecum Alpha diversity index, and Table 7 shows the significant analysis of intestinal microbial abundance at the phylum and genus level. Figure 7 Table 6 shows the cecum Alpha diversity index, and Table 7 shows the significant analysis of intestinal microbial abundance at the phylum and genus level. Figure 6 Table 6 shows the cecum Alpha diversity index, and Table 7 shows the significant analysis of intestinal microbial abundance at the phylum and genus level. Figure 7 In Table 7, 7A1-7A3 represents the intestinal flora of geese fed with traditional Chinese medicine for 6 days, 7B1-7B3 represents the intestinal flora of geese fed with combination for 6 days, 7C1-7C3 represents the intestinal flora of geese fed with fermentation for 6 days, and 7D1-7D2 represents the intestinal flora of geese fed with positive control for 6 days. In Table 7, 14A1-14A3 represents the intestinal flora of geese fed with traditional Chinese medicine for 14 days, 14B1-14B3 represents the intestinal flora of geese fed with combination for 14 days, 14C1-14C3 represents the intestinal flora of geese fed with fermentation for 14 days, and 14D1-14D2 represents the intestinal flora of geese fed with positive control for 14 days.

[0096] Alpha diversity refers to the diversity within a specific area or ecosystem, reflecting the number of species and their relative abundance within the community. By comparing the Alpha diversity index of different samples, we can determine the differences in diversity among different samples. Chao index is a diversity estimation index based on the abundance distribution of species individuals in the sample. The larger the Chao index, the more species the sample has. Simpson index is a measure of microbial diversity that takes into account both the abundance of species and the number of species. The larger the Simpson index value, the lower the community diversity. Shannon index is a measure of species richness and evenness in an ecosystem. The more evenly distributed the relative abundance of species, the higher the Shannon index. ACE index is a measure of sample coverage, used to assess the diversity and stability of species in an ecosystem. The larger the ACE index, the greater the number of species in the community.

[0097] As shown in Table 6, after 14 days of testing, the Chao index (453.48+29.95), Shannon index (2.26±0.03), and ACE index (387.98+67.07) of the fermentation group were significantly higher than those of the combination group, traditional Chinese medicine group, and positive control group (P<0.05), indicating that the flora was more diverse.

[0098] The results of Table 7 show that after 14 days of the test, it is found from the phylum level analysis that the abundance of Proteobacteria (40.1%) in the fermentation group is significantly lower than that in the combination group, the positive group and the traditional Chinese medicine group (P<0.05), and the abundance of Firmicutes (59.2%) is the highest; it is found from the genus level analysis that the abundance of Salmonella and Escherichia-Shigella in the fermentation group is the lowest, which is 9.54% and 3.01% respectively, the abundance of Lactobacillus (10.56%) is significantly higher than that in the combination group, the positive group and the traditional Chinese medicine group (P<0.05), and the abundance of Enterococcus (18.21%) is significantly higher than that in the combination group, the positive group and the traditional Chinese medicine group (P<0.05). The results show that the fermentation traditional Chinese medicine group can change the intestinal flora, increase the abundance of beneficial bacteria in the intestine, reduce the abundance of harmful bacteria in the intestine, maintain the health of the intestine and promote the digestion and absorption of nutrients.

[0099] Table 6 Alpha diversity index of cecum

[0100]

[0101] (Note: mean ± standard deviation; significant difference analysis: P<0.05; different letters represent significant differences between groups)

[0102] Table 7 Significant analysis of the abundance of intestinal microorganisms at the phylum and genus levels

[0103]

[0104]

[0105] (Note: mean ± standard deviation; significant difference analysis: P<0.05; different letters represent significant differences between groups)

[0106] The above results show that the new functional feed additive prepared in the present study has excellent effect in preventing gout in small geese, and can be further popularized and applied.

[0107] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A fermented Chinese medicine composition, characterized in that: Obtained by fermentation of traditional Chinese medicine components and probiotic components; The Chinese medicinal components, by weight, are: 8-12 parts of angelica sinensis, 8-12 parts of clematis root, 18-22 parts of frassinus root, 18-22 parts of polygonum cuspidatum, 13-17 parts of akebia, 15-20 parts of cassia twig, and 18-22 parts of oriental rhizome; The probiotic components are: Lactobacillus plantarum, Enterococcus lactis and Enterococcus faecium; The mass ratio of the traditional Chinese medicine component to the probiotic component is 100:1-5.

2. The fermented Chinese medicine composition according to claim 1, characterized in that The strain collection number of the Lactobacillus plantarum is CGMCC 1.557; the strain collection number of the Enterococcus lactis is CICC 6078; and the strain collection number of the Enterococcus faecium is CGMCC 1.

130.

3. The method for preparing the fermented Chinese medicine composition according to any one of claims 1 to 2, characterized in that: The following steps are involved: 1) Extracting the components of the traditional Chinese medicine to obtain a traditional Chinese medicine extract; 2) Cultivating the probiotic components to obtain a bacterial solution; 3) The bacterial solution obtained in step 2) is inoculated into the Chinese medicine extract obtained in step 1) for fermentation to obtain a fermented Chinese medicine composition.

4. The preparation method according to claim 3, characterized in that The concentration of the bacterial solution in step 2) is 3×10 8 -4×10 8 cfu / mL.

5. The preparation method according to claim 3, characterized in that: The inoculation amount of the bacterial solution in step 3) is 1-5%.

6. The preparation method according to claim 3, characterized in that: In step 3), the fermentation temperature is 35-40° C.; the initial pH of the fermentation is 6.5-7.

5.

7. Use of the fermented Chinese medicinal composition according to any one of claims 1 to 2 or the fermented Chinese medicinal composition obtained by the preparation method according to any one of claims 3 to 6 in the preparation of a medicament for preventing or treating gout.

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