Zanthoxylum bungeanum polysaccharide for improving reproductive performance of pregnant sows and preparation method thereof

Abstract

The present application relates to a kind of improving the reproductive performance of pregnant sow pepper seed polysaccharide and its preparation method, belong to polysaccharide preparation technical field.The preparation method of pepper seed polysaccharide described in the present application includes organic solvent extraction, microbial fermentation, enzymolysis, impurity removal, alcohol precipitation and the like.Using the pepper seed polysaccharide prepared by the present application for feeding pregnant sow, can significantly improve the live birth rate and healthy birth rate, thereby significantly improving its reproductive performance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polysaccharide preparation, and relates to a Zanthoxylum bungeanum seed polysaccharide for improving the reproductive performance of pregnant sows and a preparation method thereof. BACKGROUND

[0002] Zanthoxylum bungeanum belongs to Rutaceae Zanthoxylum, is a special spice in China, and is also an important economic tree species of oil and traditional Chinese medicinal materials. In China, the planting area of Zanthoxylum bungeanum exceeds 27 million mu, and the yield breaks through 450,000 tons. The planted Zanthoxylum bungeanum is the most complete in the world, and China is the first Zanthoxylum bungeanum producing country in the world. Zanthoxylum bungeanum seed is the Zanthoxylum bungeanum seed obtained after the mature Zanthoxylum bungeanum fruit is harvested and separated from the pericarp, and is one of main by-products of Zanthoxylum bungeanum, accounting for about 60% of the total mass of Zanthoxylum bungeanum. Zanthoxylum bungeanum seed is rich in protein and oil substances, and also contains alkaloids, amides, polyphenols, flavonoids and other medicinal active ingredients, and is a good choice for supplementing animal energy feed raw materials. With the continuous increase of the planting area of Zanthoxylum bungeanum, the yield of Zanthoxylum bungeanum seed also increases, however, the overall utilization efficiency of Zanthoxylum bungeanum seed is low. Therefore, Zanthoxylum bungeanum seed as an unconventional feed resource has the advantages of rich resources, low cost and good feed value, and has broad development potential.

[0003] It is disclosed in the existing literature that adding Poria cocos polysaccharide and Rehmannia glutinosa polysaccharide in the feed of pregnant sows can improve the reproductive performance of sows, reduce the number of stillbirths during delivery, and improve the efficiency of pig production. It is also disclosed in the literature that adding Zanthoxylum bungeanum seed in the diet of growing pigs is beneficial to improving the daily weight gain of growing pigs, improving the fatty acid content of pork, and promoting fat deposition.

[0004] However, there is no research on the reproductive performance of pregnant sows by Zanthoxylum bungeanum seed polysaccharide in the prior art, and the research on Zanthoxylum bungeanum seed in the prior art is mainly on Zanthoxylum bungeanum seed oil and Zanthoxylum bungeanum seed protein, and there is also no research on the preparation method and pharmacological activity of Zanthoxylum bungeanum seed polysaccharide. SUMMARY

[0005] The main purpose of the present application is to provide a Zanthoxylum bungeanum seed polysaccharide for improving the reproductive performance of pregnant sows and a preparation method thereof. The Zanthoxylum bungeanum seed polysaccharide obtained by the preparation method can improve the reproductive performance of pregnant sows, such as improving the live birth rate and improving the healthy birth rate.

[0006] The present application adopts the following technical solutions to achieve the above-mentioned purposes:

[0007] A preparation method of a Zanthoxylum bungeanum seed polysaccharide for improving the reproductive performance of pregnant sows, comprising the following steps:

[0008] Step 1, dry the purchased Zanthoxylum bungeanum Maxim. seed to a water content of 8-10%, and then ultrafine grind at 25-30℃ to obtain Zanthoxylum bungeanum Maxim. seed powder with a particle size of 20-30μm; add the Zanthoxylum bungeanum Maxim. seed powder to a petroleum ether-ethyl acetate mixed solution at a solid-liquid ratio of 1kg / (2-3)L to extract, and obtain the extracted Zanthoxylum bungeanum Maxim. seed powder for later use;

[0009] Step 2, mix the extracted Zanthoxylum bungeanum Maxim. seed powder of step 1 with water at a solid-liquid ratio of 1kg / (1.5-2)L, inoculate with Candida tropicalis seed liquid and Neurospora crassa seed liquid, mix well, and then ferment at 25℃ under aerobic conditions for 6-8h, and then sterilize under a steam pressure of 103.4kPa for 30min to obtain a fermentation mixed solution for later use;

[0010] Step 3, add 1.5-2 times the volume of water to the fermentation mixed solution of step 2, mix well, adjust the pH to 6.5-7.0, add glucose oxidase and papain, and then enzymatically hydrolyze at 60℃ for 2-3h, adjust the pH to 7.5-8.0, and then reflux extract at 70-80℃ for 5-6h, cool to room temperature after boiling, add an adsorbent, stand for 4-6h, filter, and obtain an extract for later use;

[0011] Step 4, concentrate the extract to 1 / 2 of the initial volume, add 2 times the volume of a chloroform-n-butanol mixed solution, shake and then stand to separate the layers, collect the upper layer, remove residual organic solvents, and then concentrate, dry, and obtain a crude polysaccharide; the volume ratio of chloroform to n-butanol is 4:1;

[0012] Step 5, redissolve the crude polysaccharide according to the above step 4 in water at a solid-liquid ratio of 1g / (1-1.5)mL, add anhydrous ethanol, and then adjust the ethanol content to 10%, 30%, 50%, and 80% respectively, stand for 4-5h after each adjustment of the ethanol content, filter, and dry the precipitates obtained at ethanol contents of 30% and 80% to obtain the target Zanthoxylum bungeanum Maxim. seed polysaccharide.

[0013] Preferably, the volume ratio of petroleum ether to ethyl acetate in step 1 is 1:(1-1.5);

[0014] Preferably, the inoculation amount of Candida tropicalis seed liquid in step 2 is 5% (v / v), and the inoculation amount of Neurospora crassa seed liquid is 10% (v / v);

[0015] Preferably, the addition amount of glucose oxidase in step 3 is 8-10g / L, the addition amount of papain is 5-8g / L, and the addition amount of adsorbent is 50-60g / L;

[0016] Further preferably, the preparation method of the adsorbent in step 3 comprises the following steps:

[0017] Step A, peanut shell powder is stirred with 20% NaOH solution at 25℃, and then filtered and washed with water until neutral, and dried to obtain alkaliized peanut shell powder;

[0018] Step B, the alkaliized peanut shell powder of step A is added to distilled water and shaken at 35℃, and then 50% glutaraldehyde solution is added and shaken at 35℃, filtered, washed with distilled water to remove unreacted glutaraldehyde, and dried to obtain glutaraldehyde cross-linked peanut shell powder;

[0019] Step C, succinic anhydride is dissolved in pyridine, and then the glutaraldehyde cross-linked peanut shell powder of step B is added and stirred at 75℃, washed with water, filtered, and dried to obtain modified peanut shell powder, which is then treated with saturated sodium bicarbonate solution, washed, filtered, and dried to obtain the modified peanut shell powder;

[0020] Step D, the modified peanut shell powder is mixed with carboxyl hollow mesoporous silica after the sodium alginate is heated to a molten state, cooled, dried, and ground to obtain the adsorbent.

[0021] Further preferably, the ratio of peanut shell powder to NaOH solution in step A is 10:(250-300) g / mL;

[0022] Further preferably, the mass-volume-volume ratio of alkaliized peanut shell powder, distilled water, and glutaraldehyde solution in step B is 5:(150-175):(5-7.5) g / mL / mL;

[0023] Further preferably, in step C, the mass-volume-mass ratio of succinic anhydride, pyridine, and glutaraldehyde cross-linked peanut shell powder is (9-10):150:3 g / mL / g;

[0024] Further preferably, in step D, the mass ratio of sodium alginate, modified peanut shell powder, and carboxyl hollow mesoporous silica is 100:(30-40):(10-15);

[0025] The application also provides the Zanthoxylum bungeanum seed polysaccharide prepared by the above preparation method.

[0026] The application also provides a specific use of the Zanthoxylum bungeanum seed polysaccharide prepared by the above preparation method, i.e., the Zanthoxylum bungeanum seed polysaccharide can improve the reproductive performance of pregnant sows, and the Zanthoxylum bungeanum seed polysaccharide can be added to the feed of pregnant sows.

[0027] The application has the following beneficial effects:

[0028] 1. The Zanthoxylum bungeanum seed polysaccharide prepared in the application can significantly increase the number of live piglets and healthy piglets born by pregnant sows, thereby improving the survival rate of piglets and reproductive performance;

[0029] 2. The content of crude fat and crude protein in Zanthoxylum bungeanum seeds is relatively high, and the existence of oil will affect the extraction of polysaccharides. Therefore, the oil in the Zanthoxylum bungeanum seeds is removed by adopting the method of ultramicro-grinding and organic solvent extraction, and then the residual oil is decomposed by Candida tropicalis and the Zanthoxylum bungeanum seeds are decomposed by Neocallimastix patricium, and then glucose oxidase and papain are added for further enzymolysis, which is helpful to the dissolution of polysaccharides and improves the extraction rate; after water extraction, a specific adsorbent is added to remove macromolecular impurities such as proteins in the water extract, and then a chloroform-n-butanol mixed solution is used to remove residual small molecular protein impurities, so as to obtain a polysaccharide crude product with high purity. By using the method of ethanol fractionation and alcohol precipitation, the target Zanthoxylum bungeanum seed polysaccharide with obvious physiological activity in the application is obtained. DETAILED DESCRIPTION

[0030] The application will be further illustrated by specific examples, and it should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. After reading the application, those skilled in the art can modify various equivalent forms of the application, which all fall within the scope of the claims of the present application. The microorganisms and enzymes used in the following embodiments are all commercially available products; wherein the seed liquid of Candida tropicalis is obtained by activating the Candida tropicalis strain purchased from Guangdong Microbial Culture Collection Center GDMCC NO.2.6 according to the instructions and then expanding the culture to an OD value of about 0.6; the seed liquid of Neocallimastix patricium is obtained by activating the Neocallimastix patricium strain purchased from China Typical Culture Collection Center CCTCC DF 20081000 according to the instructions and then expanding the culture to an OD value of about 0.8; the activity of glucose oxidase is 100u / mg, and the activity of papain is 800u / mg.

[0031] Example 1

[0032] First, the adsorbent is prepared:

[0033] Step A: 10g of peanut shell powder is added with 300mL of 20% NaOH solution, stirred at 25℃ for 16h, filtered and washed with water until neutral, and dried to obtain alkali-treated peanut shell powder;

[0034] Step B: 5g of the above alkali-treated peanut shell powder is added with 175mL of distilled water, shaken at 35℃ for 0.5h, and then 7.5mL of 50% glutaraldehyde aqueous solution is added and shaken in a 35℃ constant temperature water bath for 12h, filtered, and distilled water is used to wash off the unreacted glutaraldehyde, and dried to obtain glutaraldehyde cross-linked peanut shell powder;

[0035] Step C, 10 g succinic anhydride was added to a flask containing 150 mL of pyridine, after the succinic anhydride was completely dissolved, 3 g of the above glutaraldehyde cross-linked peanut shell powder was added, and the reaction was stirred in a 75°C water bath for 24 h, washed with water, suction filtered, and dried to obtain modified peanut shell powder, which was then treated with saturated sodium bicarbonate for 1 h, washed, suction filtered, and dried to obtain the modified peanut shell powder;

[0036] Step D, 100 g of sodium alginate was heated to a molten state, then 40 g of modified peanut shell powder and 10 g of carboxyl hollow mesoporous silica (spherical, 200 nm) were added and mixed, then cooled, dried, and ground to obtain the adsorbent.

[0037] Then the target Zanthoxylum bungeanum seed polysaccharide was prepared:

[0038] Step 1, the purchased Hongpao Zanthoxylum bungeanum seeds were dried in a 50°C oven to a moisture content of 8-10%, then placed in a super micro pulverizer container, set the temperature to 30°C, and the Zanthoxylum bungeanum seeds were super micro-pulverized to obtain Zanthoxylum bungeanum seed powder with a particle size of 20-30 μm; 1 kg of Zanthoxylum bungeanum seed powder was added to 3 L of petroleum ether-ethyl acetate (1:1, v / v) mixed solution, mixed well, and then placed for 4 h of extraction, then filtered, and the residual petroleum ether-ethyl acetate was removed to obtain the extracted Zanthoxylum bungeanum seed powder for use;

[0039] Step 2, the above extracted Zanthoxylum bungeanum seed powder was mixed with water according to the solid-liquid ratio of 1:2, kg / L, then inoculated with Candida tropicalis seed liquid (5%, v / v) and Neurospora crassa seed liquid (10%, v / v), mixed well, then fermented under aerobic conditions at 25°C for 6 h, then sterilized under a steam pressure of 103.4 kPa (about 120°C) for 30 min to obtain a fermentation mixed solution for use;

[0040] Step 3, 2 times the volume of water was added to the above fermentation mixed solution, mixed well, then the pH was adjusted to 7.0, 10 g / L of glucose oxidase and 8 g / L of papain were added, and the enzyme was hydrolyzed at 60°C for 2 h, then the pH was adjusted to 8.0, and the extraction was carried out under reflux at 80°C for 5 h, after boiling, the solution was cooled to room temperature, 60 g / L of adsorbent was added, and the solution was allowed to stand for 4 h, then filtered to obtain an extract for use;

[0041] Step 4, the extract was concentrated to 1 / 2 of the initial volume, 2 times the volume of chloroform-n-butanol (4:1, v / v) mixed solution was added, shaken, and then allowed to stand for layer separation, the upper layer solution was collected, the residual organic solvent was removed, and then concentrated, dried to obtain the crude polysaccharide;

[0042] Step 5, the above polysaccharide crude product was re-dissolved with water according to a solid-liquid ratio of 1:1.5, g / mL, and then anhydrous ethanol was added. The ethanol content was adjusted to 10%, 30%, 50%, and 80%, respectively. After each adjustment of the ethanol content, the mixture was allowed to stand for 5 h, and then the precipitate obtained when the ethanol content was 30% and 80% was dried to obtain the target Zanthoxylum bungeanum seed polysaccharide.

[0043] Example 2

[0044] First, the adsorbent was prepared:

[0045] Step A, 10 g of peanut shell powder was added to 250 mL of a 20% NaOH solution, stirred at 25°C for 16 h, and then filtered and washed with water until neutral. The alkalized peanut shell powder was dried.

[0046] Step B, 5 g of the above alkalized peanut shell powder was added to 150 mL of distilled water, and then stirred at 35°C for 0.5 h. Then, 5 mL of a 50% glutaraldehyde aqueous solution was added, and the mixture was stirred in a 35°C constant-temperature water bath for 12 h. The mixture was filtered, and then washed with distilled water to remove unreacted glutaraldehyde. The glutaraldehyde-crosslinked peanut shell powder was dried.

[0047] Step C, 9 g of succinic anhydride was added to a flask containing 150 mL of pyridine, and then 3 g of the above glutaraldehyde-crosslinked peanut shell powder was added. The mixture was stirred in a 75°C water bath for 24 h, and then washed with water, filtered, and dried to obtain the modified peanut shell powder. The modified peanut shell powder was treated with saturated sodium bicarbonate for 1 h, washed, filtered, and dried to obtain the modified peanut shell powder.

[0048] Step D, 100 g of sodium alginate was heated to a molten state, and then mixed with 30 g of the modified peanut shell powder and 15 g of carboxyl hollow mesoporous silica (spherical, 200 nm) to obtain an adsorbent.

[0049] Then, the target Zanthoxylum bungeanum seed polysaccharide was prepared:

[0050] Step 1, the purchased Zanthoxylum bungeanum seeds were dried in a 50°C oven to a water content of 8-10%, and then subjected to ultrafine grinding in an ultrafine grinder container set to a temperature of 25°C to obtain Zanthoxylum bungeanum seed powder with a particle size of 20-30 μm. 1 kg of the Zanthoxylum bungeanum seed powder was added to 2 L of a petroleum ether-ethyl acetate (1:1.5, v / v) mixed solution, and then allowed to stand for 5 h for extraction. The mixture was filtered, and then the residual petroleum ether-ethyl acetate was removed to obtain the extracted Zanthoxylum bungeanum seed powder for use.

[0051] Step 2, the above-mentioned after leaching of Zanthoxylum bungeanum seed powder was mixed with water according to the ratio of 1:1.5, kg / L, then inoculated with Candida tropicalis seed liquid (5%, v / v) and Neurospora crassa seed liquid (10%, v / v), mixed, and then fermented under aerobic conditions at 25°C for 8h, and then sterilized under the steam pressure of 103.4kPa (about 120°C) for 30min to obtain a fermentation mixed solution for standby;

[0052] Step 3, 1.5 times the volume of water was added to the above-mentioned fermentation mixed solution, mixed, the pH was adjusted to 6.5, 8g / L glucose oxidase and 5g / L papain were added, and then enzymolysis was carried out at 60°C for 3h, then the pH was adjusted to 7.5, and then reflux extraction was carried out at 70°C for 6h, after boiling, the temperature was cooled to room temperature, 50g / L adsorbent was added, and then static standing was carried out for 6h, and then filtration was carried out to obtain an extract solution for standby;

[0053] Step 4, the extract solution was concentrated to 1 / 2 of the initial volume, 2 times the volume of chloroform-n-butanol (4:1, v / v) mixed solution was added, after oscillation, static standing was carried out for layering, the upper layer solution was collected, after removing the residual organic solvent, concentration, drying was carried out to obtain a crude polysaccharide product;

[0054] Step 5, the above-mentioned crude polysaccharide product was redissolved according to the ratio of 1:1, g / mL, and then anhydrous ethanol was added, and the ethanol content was adjusted to 10%, 30%, 50% and 80% respectively, after adjusting the ethanol content each time, static standing was carried out for 4h, and then filtration was carried out, and the precipitates obtained by adjusting the ethanol content to 30% and 80% were dried to obtain the target Zanthoxylum bungeanum seed polysaccharide.

[0055] Example 3

[0056] Firstly, the adsorbent was prepared:

[0057] Step A, 10g of peanut shell powder was taken, 320mL of 20% NaOH solution was added, stirring was carried out at 25°C for 16h, after suction filtration, water washing was carried out until neutral, and then drying was carried out to obtain alkaliized peanut shell powder;

[0058] Step B, 5g of the above-mentioned alkaliized peanut shell powder was taken, 160mL of distilled water was added, oscillation was carried out at 35°C for 0.5h, then 6mL of 50% glutaraldehyde aqueous solution was added, and then oscillation was carried out in a 35°C constant temperature water bath for 12h, filtration was carried out, and then distilled water was used to wash the unreacted glutaraldehyde, and then drying was carried out to obtain glutaraldehyde crosslinked peanut shell powder;

[0059] Step C, 10g of succinic anhydride was taken, and then placed in a flask containing 150mL of pyridine, after the succinic anhydride was completely dissolved, 3g of the above-mentioned glutaraldehyde crosslinked peanut shell powder was added, and then stirring was carried out in a 75°C water bath for 24h, water washing was carried out, suction filtration was carried out, and then drying was carried out to obtain modified peanut shell powder, and then the modified peanut shell powder was treated with saturated sodium bicarbonate for 1h, washed, suction filtered, and then dried to obtain the modified peanut shell powder;

[0060] Step D, after sodium alginate 100 g is heated to a molten state, 35 g of modified peanut shell powder, 13 g of carboxyl hollow mesoporous silica (spherical, 200 nm) are added and mixed, then cooled, dried and ground to obtain the adsorbent.

[0061] Then the target Zanthoxylum bungeanum seed polysaccharide is prepared:

[0062] Step 1, the purchased Hongpao Zanthoxylum bungeanum seeds are dried in a 50℃ oven to a moisture content of 8-10%, then placed in a super micro grinder container, set the temperature to 25℃, and the Zanthoxylum bungeanum seeds are super micro ground to obtain Zanthoxylum bungeanum seed powder with a particle size of 20-30μm; take 1kg of Zanthoxylum bungeanum seed powder and add 3L of petroleum ether-ethyl acetate (1:1.5, v / v) mixed solution, mix well and soak for 4h, then filter and remove the residual petroleum ether-ethyl acetate to obtain the soaked Zanthoxylum bungeanum seed powder for use;

[0063] Step 2, the above soaked Zanthoxylum bungeanum seed powder is mixed with water according to the solid-liquid ratio of 1:2, kg / L, then inoculated with Candida tropicalis seed liquid (5%, v / v) and Neurospora crassa seed liquid (10%, v / v), mixed well, and then fermented under aerobic conditions at 25℃ for 7h, then sterilized under steam pressure of 103.4kPa (about 120℃) for 30min to obtain a fermentation mixed solution for use;

[0064] Step 3, add 2 times the volume of water to the above fermentation mixed solution, mix well, adjust the pH to 7.0, add glucose oxidase 9g / L and papain 7g / L, and then enzymatically hydrolyze at 60℃ for 2.5h, then adjust the pH to 8.0 and extract by refluxing at 80℃ for 5h, after boiling, cool to room temperature, add adsorbent 55g / L, stand for 5h, filter to obtain the extract for use;

[0065] Step 4, concentrate the extract to 1 / 2 of the initial volume, add 2 times the volume of chloroform-n-butanol (4:1, v / v) mixed solution, shake and stand for layering, collect the upper layer solution, remove the residual organic solvent, and then concentrate and dry to obtain the crude polysaccharide;

[0066] Step 5, add anhydrous ethanol to the above crude polysaccharide according to the solid-liquid ratio of 1:1.3, g / mL, and then adjust the ethanol content to 10%, 30%, 50% and 80% respectively, stand for 5h after each adjustment of ethanol content, filter, and dry the precipitates obtained by adjusting the ethanol content to 30% and 80% respectively to obtain the target Zanthoxylum bungeanum seed polysaccharide.

[0067] Comparative Example 1

[0068] First, the adsorbent is prepared:

[0069] Step A, take 10 g of peanut shell powder, add 320 mL of 20% NaOH solution, stir at 25°C for 16 h, filter and wash with water until neutral, dry to obtain alkaliized peanut shell powder;

[0070] Step B, take 5 g of the above alkaliized peanut shell powder, add 160 mL of distilled water, shake at 35°C for 0.5 h, then add 6 mL of 50% glutaraldehyde solution and shake in a 35°C constant temperature water bath for 12 h, filter, wash with distilled water to remove unreacted glutaraldehyde, dry to obtain glutaraldehyde cross-linked peanut shell powder;

[0071] Step C, take 10 g of succinic anhydride and place it in a flask containing 150 mL of pyridine, after the succinic anhydride is completely dissolved, add 3 g of the above glutaraldehyde cross-linked peanut shell powder, stir in a 75°C water bath for 24 h, wash with water, filter and dry to obtain modified peanut shell powder, then treat with saturated sodium bicarbonate for 1 h, wash, filter and dry to obtain the modified peanut shell powder;

[0072] Step D, melt 100 g of sodium alginate, add 35 g of modified peanut shell powder and 13 g of carboxyl hollow mesoporous silica (spherical, 200 nm), mix well, cool and dry, then grind to obtain the adsorbent.

[0073] Then prepare the target Zanthoxylum bungeanum seed polysaccharide:

[0074] Step 1, dry the purchased Zanthoxylum bungeanum seeds in a 50°C oven to a moisture content of 8-10%, then place them in a super micro grinder container, set the temperature to 25°C, and super micro grind the Zanthoxylum bungeanum seeds to obtain Zanthoxylum bungeanum seed powder with a particle size of 20-30 μm; take 1 kg of Zanthoxylum bungeanum seed powder and add 3 L of petroleum ether-ethyl acetate (1:1.5, v / v) mixed solution, mix well, then soak for 4 h, then filter and remove the residual petroleum ether-ethyl acetate to obtain the soaked Zanthoxylum bungeanum seed powder for use;

[0075] Step 2, take the above soaked Zanthoxylum bungeanum seed powder, add water to make the solid-liquid ratio 1:2, kg / L, mix well, then inoculate Candida tropicalis seed liquid (5%, v / v) and Neurospora crassa seed liquid (10%, v / v), mix well, then ferment under aerobic conditions at 25°C for 7 h, then sterilize under a steam pressure of 103.4 kPa (about 120°C) for 30 min to obtain a fermentation mixed solution for use;

[0076] Step 3, add 2 times the volume of water to the above fermentation mixed solution, mix well, adjust the pH to 7.0, add glucose oxidase 9 g / L and papain 7 g / L, then enzymatically hydrolyze at 60°C for 2.5 h, then adjust the pH to 8.0, reflux extract at 80°C for 5 h, after boiling, cool to room temperature, add adsorbent 55 g / L, stand for 5 h, filter to obtain the extract for use;

[0077] Step 4, the extract was concentrated to 1 / 2 of the initial volume, 2 volumes of chloroform-n-butanol (4:1, v / v) mixed solution was added, after oscillation, the upper layer was collected after standing, and the residual organic solvent was removed, and then concentrated, dried to obtain the crude polysaccharide;

[0078] Step 5, the above crude polysaccharide was redissolved with water according to the feed liquid ratio of 1:1.3, g / mL, then anhydrous ethanol was added, and the ethanol content was adjusted to 10%, 30%, 50%, and 80%, respectively. After adjusting the alcohol content each time, it was placed for 5h, and then filtered. The precipitate obtained by adjusting the alcohol content to 10% and 50% was dried to obtain the target Zanthoxylum bungeanum seed polysaccharide.

[0079] Comparative Example 2

[0080] Step 1, the purchased Zanthoxylum bungeanum seeds were dried in a 50℃ oven to a water content of 8-10%, then placed in a ultrafine grinder container, and the temperature was set to 25℃. The Zanthoxylum bungeanum seeds were ultrafine ground to obtain Zanthoxylum bungeanum seed powder with a particle size of 20-30μm. 1kg of Zanthoxylum bungeanum seed powder was added to 3L of petroleum ether-ethyl acetate (1:1.5, v / v) mixed solution, mixed and placed for 4h for extraction, then filtered, and the residual petroleum ether-ethyl acetate was removed to obtain the extracted Zanthoxylum bungeanum seed powder for use;

[0081] Step 2, the above extracted Zanthoxylum bungeanum seed powder was added with water according to the feed liquid ratio of 1:5, kg / L, mixed, and the pH was adjusted to 7.0. Glucose oxidase 9g / L and papain 7g / L were added, and enzyme hydrolysis was carried out at 60℃ for 2.5h. Then the pH was adjusted to 8.0, and reflux extraction was carried out at 80℃ for 5h. After boiling, it was cooled to room temperature, placed for 5h, filtered, and the extract was obtained for use;

[0082] Step 3, the extract was concentrated to 1 / 2 of the initial volume, 2 volumes of chloroform-n-butanol (4:1, v / v) mixed solution was added, after oscillation, the upper layer was collected after standing, and the residual organic solvent was removed, and then concentrated, dried to obtain the crude polysaccharide;

[0083] Comparative Example 3

[0084] Step 1, the purchased Zanthoxylum bungeanum seeds were dried in a 50℃ oven to a water content of 8-10%, then placed in a ultrafine grinder container, and the temperature was set to 25℃. The Zanthoxylum bungeanum seeds were ultrafine ground to obtain Zanthoxylum bungeanum seed powder with a particle size of 20-30μm. 1kg of Zanthoxylum bungeanum seed powder was added to 3L of petroleum ether-ethyl acetate (1:1, v / v) mixed solution, mixed and placed for 4h for extraction, then filtered, and the residual petroleum ether-ethyl acetate was removed to obtain the extracted Zanthoxylum bungeanum seed powder for use;

[0085] Step 2, the above-mentioned after leaching of Zanthoxylum bungeanum seed powder was mixed with water according to the ratio of material to liquid 1:2, kg / L, then inoculated with Candida tropicalis seed liquid (5%, v / v) and Neurospora crassa seed liquid (10%, v / v), mixed, and then fermented under aerobic conditions at 25°C for 6h, and then sterilized under the steam pressure of 103.4kPa (about 120°C) for 30min to obtain a fermentation mixed solution for standby;

[0086] Step 3, 2 times the volume of water was added to the above-mentioned fermentation mixed solution, mixed, the pH was adjusted to 7.0, 10g / L glucose oxidase and 8g / L papain were added, and then enzymolysis was carried out at 60°C for 2h, then the pH was adjusted to 8.0, and then reflux extraction was carried out at 80°C for 5h, after boiling, the temperature was cooled to room temperature, and then the solution was left to stand for 4h, filtered, and then an extract was obtained for standby;

[0087] Step 4, the extract was concentrated to 1 / 2 of the initial volume, 2 times the volume of chloroform-n-butanol (4:1, v / v) mixed solution was added, shaken and then left to stand for stratification, the upper layer solution was collected, residual organic solvent was removed, and then concentrated, dried, and then a crude polysaccharide was obtained;

[0088] Step 5, the above-mentioned crude polysaccharide was redissolved with water according to the ratio of material to liquid 1:1.5, g / mL, and then anhydrous ethanol was added, and the ethanol content was adjusted to 10%, 30%, 50% and 80% respectively, each time after adjusting the ethanol content, the solution was left to stand for 5h, filtered, and then the precipitates obtained by adjusting the ethanol content to 30% and 80% were dried, and then the target Zanthoxylum bungeanum seed polysaccharide was obtained.

[0089] Comparative Example 4

[0090] Firstly, the adsorbent was prepared:

[0091] Step A, 10g of peanut shell powder was added into 300mL of 20% NaOH solution, stirred at 25°C for 16h, filtered and then washed with water until neutral, and then dried to obtain alkali-treated peanut shell powder;

[0092] Step B, 5g of the above-mentioned alkali-treated peanut shell powder was added into 175mL of distilled water, shaken at 35°C for 0.5h, and then 7.5mL of 50% glutaraldehyde aqueous solution was added and shaken in a 35°C constant temperature water bath for 12h, filtered, washed with distilled water to remove unreacted glutaraldehyde, and then dried to obtain glutaraldehyde cross-linked peanut shell powder;

[0093] Step C, 10g of succinic anhydride was added into a flask containing 150mL of pyridine, and after the succinic anhydride was completely dissolved, 3g of the above-mentioned glutaraldehyde cross-linked peanut shell powder was added, stirred in a 75°C water bath for 24h, washed with water, filtered, and then dried to obtain modified peanut shell powder, which was treated with saturated sodium bicarbonate for 1h, washed, filtered, and then dried to obtain the modified peanut shell powder;

[0094] Step D, after sodium alginate 100 g is heated to a molten state, modified peanut shell powder 40 g, 10 g carboxyl hollow mesoporous silica (spherical, 200 nm) is added and uniformly mixed, then cooled, dried and ground to obtain the adsorbent.

[0095] Then the target Zanthoxylum bungeanum seed polysaccharide is prepared:

[0096] Step 1, the purchased Zanthoxylum bungeanum seeds are dried in a 50℃ oven to a moisture content of 8-10%, crushed, and Zanthoxylum bungeanum seed powder is obtained for use.

[0097] Step 2, the above Zanthoxylum bungeanum seed powder is mixed with water according to a solid-liquid ratio of 1:5, kg / L, the pH is adjusted to 7.0, 10 g / L glucose oxidase and 8 g / L papain are added, and enzymatic hydrolysis is carried out at 60℃ for 2 h. Then the pH is adjusted to 8.0, and reflux extraction is carried out at 80℃ for 5 h. After boiling, the temperature is cooled to room temperature, 60 g / L of adsorbent is added, and the mixture is allowed to stand for 4 h. Then filtration is performed to obtain an extract for use.

[0098] Step 3, the extract is concentrated to 1 / 2 of the initial volume, 2 volumes of a chloroform-n-butanol (4:1, v / v) mixed solution are added, the mixture is shaken and then allowed to stand to separate the layers. The upper layer solution is collected, residual organic solvent is removed, and the solution is concentrated and dried to obtain a crude polysaccharide.

[0099] Step 4, the above crude polysaccharide is redissolved with water according to a solid-liquid ratio of 1:1.5, g / mL, and then anhydrous ethanol is added. The ethanol content is adjusted to 10%, 30%, 50% and 80% respectively, and the mixture is allowed to stand for 5 h after each adjustment. The precipitates obtained by adjusting the ethanol content to 30% and 80% are dried to obtain the target Zanthoxylum bungeanum seed polysaccharide.

[0100] Performance test

[0101] I. Pre-test: Effect of Zanthoxylum bungeanum seed polysaccharide on the reproductive performance of sows in the late gestation period.

[0102] 1. Experimental design

[0103] Sixteen multiparous sows with similar body weight, 2-3 parity, and similar estrus time were selected and randomly divided into 8 groups, 2 sows per group. The control group was fed with a basic diet, and the experimental groups 1-7 were fed with the basic diet supplemented with Zanthoxylum bungeanum seed polysaccharide prepared in Examples 1-3 and Comparative Examples 1-4 at a rate of 2.0‰. The experiment started when the sows were about 90 days pregnant and ended 5 days after delivery. The pre-feeding period was 5 days. The feeding management and epidemic prevention system were the same, and the pig house was kept clean, ventilated and dry. The temperature in the pig house during pregnancy was 18-25℃, and the temperature during lactation was 28-32℃. The pregnant sows were fed twice a day, and the lactating sows were free to eat.

[0104] 2. Detection index

[0105] Record the number of piglets per sow per group, the number of live piglets, the number of healthy piglets, the number of weak piglets, the number of stillborn piglets, and determine the average number of piglets per sow, the average number of live piglets, the average number of healthy piglets, the average number of weak piglets, and the average number of stillborn piglets.

[0106] Among them, healthy piglets are piglets with a body weight of ≥0.8 kg and no deformity; weak piglets are piglets with a body weight of <0.8 kg.

[0107] 3. Results and analysis

[0108] From the results in Table 1, it can be seen that the number of piglets per sow in each group is basically the same, and there is no significant difference; however, the addition of the Zanthoxylum bungeanum seed polysaccharide prepared in Examples 1-3 and Comparative Example 3 in the feed can significantly increase the number of live piglets and the number of healthy piglets, and the live piglet rate and the healthy piglet rate of the sows in Pre-experiments 1-3 and 6 are much higher than those of the control group and also higher than those of the sows in Experiments 4-5 and 7, indicating that the Zanthoxylum bungeanum seed polysaccharide can significantly improve the live piglet rate and the healthy piglet rate of sows in the late pregnancy, improve the survival rate of piglets, and thus improve the reproductive performance.

[0109] Table 1 Effect of different Zanthoxylum bungeanum seed polysaccharides on the reproductive performance of sows in the late pregnancy

[0110]

[0111] Note: * indicates P<0.05 compared with the control group.

[0112] II. Effect of Zanthoxylum bungeanum seed polysaccharide on the reproductive performance of sows

[0113] 1. Experimental design

[0114] In this experiment, 20 multiparous sows with the third parity, basically consistent estrus time, and about 70 days of pregnancy were selected and randomly divided into 4 groups, namely a blank control group (fed with a basic feed), a test group 1 (basic feed + 2.0‰ Zanthoxylum bungeanum seed polysaccharide of Example 1), a test group 2 (basic feed + 2.0‰ Zanthoxylum bungeanum seed polysaccharide of Comparative Example 1), and a test group 3 (basic feed + 2.0‰ Zanthoxylum bungeanum seed polysaccharide of Comparative Example 2), and the experiment ended 20 days after the birth of piglets, and the piglets were weaned at 21 days of age.

[0115] 2. Detection index

[0116] Record the number of piglets per sow per group, the number of live piglets, the number of healthy piglets (body weight >0.8 kg), the birth weight of piglets, the number of sows in estrus 7 days after weaning, calculate the average number of piglets, the average number of live piglets, the average number of healthy piglets, the average weight of piglets, the average weight of healthy piglets, and the 7-day estrus rate of sows.

[0117] 3. Results and analysis

[0118] From the results in Table 2, it can be seen that the number of piglets born by each group of sows is basically the same, and there is no significant difference; but adding the Zanthoxylum bungeanum seed polysaccharide prepared in Examples 1-3 of the present application in the feed can obviously improve the number of live piglets and the number of healthy piglets born by sows, the live piglet rate of the sows in Test 1 group is 98.6%, which is much higher than the live piglet rate 92.2% of the blank control group, and also higher than 95.4% of Test 2 group and 92.5% of Test 3 group; the healthy piglet rate of the sows in Test 1 group is 96.9%, which is much higher than 91.3% of the control group, and also higher than 95.4% of Test 2 group and 93.4% of Test 3 group, indicating that the Zanthoxylum bungeanum seed polysaccharide of the present application can obviously improve the live piglet rate and the healthy piglet rate of sows in the late pregnancy, improve the survival rate of piglets and thus improve the reproductive performance.

[0119] Table 2 Effects of different Zanthoxylum bungeanum seed polysaccharides on the reproductive performance of sows in the late pregnancy

[0120]

[0121] Note: * indicates P<0.05 compared with the control group.

[0122] From the results in Table 2, it can be seen that the average weight of piglets born in Test 1 group is higher than that of the blank control group and Test 2-3 groups, and the estrus rate of sows after weaning is also higher.

[0123] III. Effects of different preparation methods of Zanthoxylum bungeanum seed polysaccharide on the extraction rate

[0124] Take the polysaccharide crude product obtained in step 4 in Examples 1-3, the polysaccharide crude product in step 4 in Comparative Example 1, the Zanthoxylum bungeanum seed polysaccharide obtained in Comparative Example 2, the polysaccharide crude product in step 4 in Comparative Example 3, and the polysaccharide crude product in step 3 in Comparative Example 4, weigh them, calculate the polysaccharide yield, and detect the purity of the polysaccharide.

[0125] From the results in Table 1, it can be seen that the extraction rate of the Zanthoxylum bungeanum seed polysaccharide prepared by the method of the present application is higher, and the purity of the polysaccharide is higher; in Comparative Example 2, the polysaccharide is extracted by the ordinary extraction and enzymolysis method, and the extraction rate and purity of the polysaccharide are lower; in Comparative Example 3, no adsorbent is added, the extraction rate is higher, but the purity is lower; in Comparative Example 4, no microbial fermentation step is carried out, and the extraction rate and purity are low.

[0126] Table 3 Polysaccharide yield and purity in different embodiments

[0127]

Claims

1. A preparation method of Zanthoxylum bungeanum seed polysaccharide, characterized in that, Comprise the following steps: Step 1, the purchase of big red pepper seed is dried to the moisture content of 8-10%, under the condition of 25-30℃, the ultrafine grinding is obtained, the particle size of the pepper seed powder is 20-30μm; Pepper seed powder is added to petroleum ether-ethyl acetate mixed solution according to the ratio of 1kg / (2-3)L, and the extraction of the pepper seed powder is obtained; Step 2, the extraction of the pepper seed powder in step 1 is mixed with water according to the ratio of 1kg / (1.5-2)L, and then inoculated with candida tropicalis seed liquid and neurospora crassa seed liquid; After mixing, it is fermented under aerobic condition at 25℃ for 6-8h, and then sterilized under the steam pressure of 103.4kPa for 30min, and the fermentation mixed solution is obtained for standby; Step 3, 1.5-2 times volume of water is added to the fermentation mixed solution in step 2, mixed, and then the pH is adjusted to 6.5-7.0; Glucose oxidase and papain are added, and enzymolysis is carried out at 60℃ for 2-3h; Then the pH is adjusted to 7.5-8.0, and reflux extraction is carried out at 70-80℃ for 5-6h; After boiling, it is cooled to room temperature, and adsorbent is added; After standing for 4-6h, filtration is carried out, and the extraction liquid is obtained for standby; Step 4, the extraction liquid is concentrated to 1 / 2 of the initial volume, and 2 times volume of chloroform-n-butanol mixed solution is added; After oscillation, it is layered, and the upper solution is collected; After removing the residual organic solvent, concentration, drying, the crude polysaccharide is obtained; The volume ratio of chloroform-n-butanol is 4:1; Step 5, the crude polysaccharide is dissolved in water according to the ratio of 1g / (1-1.5)mL, and then anhydrous ethanol is added; The ethanol content is adjusted to 10%, 30%, 50% and 80% respectively; After adjusting the alcohol content each time, it is placed for 4-5h, and then filtered; The precipitates obtained by adjusting the alcohol content to 30% and 80% are dried, and the target pepper seed polysaccharide is obtained. The volume ratio of petroleum ether-ethyl acetate in step 1 is 1:(1-1.5). The inoculation amount of candida tropicalis seed liquid in step 2 is 5%, v / v; The inoculation amount of neurospora crassa seed liquid is 10%, v / v. The addition amount of glucose oxidase in step 3 is 8-10g / L, the addition amount of papain is 5-8g / L, and the addition amount of adsorbent is 50-60g / L. The preparation method of the adsorbent in step 3 comprises the following steps:

2. The production method according to claim 1, wherein Step A, peanut shell powder is stirred with 20% NaOH solution at 25℃, and then filtered and washed with water until neutral; After drying, the alkaliized peanut shell powder is obtained; Step B, the alkaliized peanut shell powder in step A is added to distilled water and shaken at 35℃; Then 50% glutaraldehyde aqueous solution is added and shaken at 35℃; After filtration, the unreacted glutaraldehyde is washed away with distilled water, and the glutaraldehyde crosslinked peanut shell powder is obtained by drying; Step C, succinic anhydride is dissolved in pyridine, and then the glutaraldehyde crosslinked peanut shell powder in step B is added; Stirring reaction is carried out at 75℃, and then washed with water; After filtration and drying, the modified peanut shell powder is obtained; After treatment with saturated sodium bicarbonate and washing, the modified peanut shell powder is obtained by filtration and drying.

3. The production method according to claim 1, wherein ​ 4. The production method according to claim 1, wherein ​ 5. The production method according to claim 1, wherein ​ ​ ​ ​ Step D, after heating sodium alginate to a molten state, adding modified peanut shell powder, carboxyl hollow mesoporous silica, mixing, cooling, drying, grinding, the adsorbent is obtained.

6. The production method according to claim 5, wherein The solid-liquid ratio of peanut shell powder to NaOH solution in step A is 10:(250-300) g / mL.

7. The production method according to claim 5, wherein The mass-volume-volume ratio of alkaliized peanut shell powder, distilled water, and glutaraldehyde aqueous solution in step B is 5:(150-175):(5-7.5) g / mL / mL.

8. The production method according to claim 5, wherein In step C, the mass-volume-mass ratio of succinic anhydride, pyridine, and glutaraldehyde crosslinked peanut shell powder is (9-10):150:3 g / mL / g.

9. The production method according to claim 5, wherein In step D, the mass ratio of sodium alginate, modified peanut shell powder, and carboxyl hollow mesoporous silica is 100:(30-40):(10-15).

10. The use of Zanthoxylum bungeanum seed polysaccharide prepared by the preparation method of any one of claims 1-9 in improving the reproductive performance of pregnant sows.

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