A method for producing probiotic bacterial sludge for relieving sows' constipation by high-concentration enzymatic hydrolysis of traditional Chinese medicine straw

Abstract

The invention discloses a method for producing probiotic bacterial sludge for relieving sows' constipation by high-concentration enzymatic hydrolysis of Chinese medicinal straws, belonging to the technical field of microbial preparations. The method comprises the following steps: acid-treating Chinese medicinal straw powder to obtain a pretreated Chinese medicinal straw powder solution; drying the pretreated Chinese medicinal straw powder solution, adding water, cellulase and maltodextrin for preliminary treatment, and then adding lactic acid for further treatment to obtain a Chinese medicinal straw enzymatic hydrolysate; adding the Chinese medicinal straw enzymatic hydrolysate to a bacterial solution and fermenting to obtain Qiai straw enzymatic hydrolysis fermented bacterial sludge. Through the pretreatment process of composite acid, high-concentration enzymatic hydrolysis process and probiotic fermentation process, the invention produces probiotic bacterial sludge for relieving sows' constipation. The process is unique, and the edible acid is used to treat Chinese medicinal straws, which not only does not pollute the environment, but also can improve the food safety of the straw after pretreatment. Probiotics such as Clostridium butyricum can strengthen intestinal peristalsis and increase intestinal absorption efficiency, and also have a certain effect on relieving constipation.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and more specifically to a method for producing probiotic mud for relieving sows' constipation by high-concentration enzymatic hydrolysis of traditional Chinese medicine straws. Background Art

[0002] After the wormwood is harvested, a large amount of wormwood straws remain. The cellulose crystallinity of the wormwood straws is high and the texture is hard, making it difficult for ruminants to eat. The large amount of waste of wormwood straws causes environmental pollution on the one hand and affects the efficient cultivation of land on the other hand.

[0003] Enzymatic hydrolysis of traditional Chinese medicine straws has the advantages of being green, having a suitable temperature condition, and good feed safety. High-efficiency enzymatic hydrolysis of natural traditional Chinese medicine straws requires pretreatment of the traditional Chinese medicine straws. Methods such as acid hydrolysis and steam explosion have problems such as environmental pollution or high energy consumption.

[0004] During the feeding process of pigs, the constipation problem of sows has always been a stubborn problem. It has now been found that low-degree-of-polymerization fibers have a good effect on relieving sows' constipation. The higher the cellulose crystallinity, the worse the effect of promoting intestinal peristalsis and relieving constipation. Reducing the crystallinity and increasing the fiber pentose at the same time is a feasible method for using cellulose to relieve sows' constipation.

[0005] Therefore, how to provide a method for producing probiotic mud for relieving sows' constipation by straw enzymatic hydrolysis is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for producing probiotic mud for relieving sows' constipation by high-concentration enzymatic hydrolysis of traditional Chinese medicine straws. Through the pretreatment process of composite acid, the high-concentration enzymatic hydrolysis process and the fermentation process of probiotics, probiotic mud for relieving sows' constipation is produced.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A method for producing probiotic mud for relieving sows' constipation by high-concentration enzymatic hydrolysis of traditional Chinese medicine straws, comprising the following steps:

[0009] (1) Acid-treat the traditional Chinese medicine straw powder to obtain a pretreated traditional Chinese medicine straw powder solution;

[0010] (2) Dry the pretreated traditional Chinese medicine straw powder solution, then add water, cellulase and maltodextrin for preliminary treatment, and then add lactic acid for further treatment to obtain a traditional Chinese medicine straw enzymatic hydrolysate;

[0011] (3) Add the traditional Chinese medicine straw enzymatic hydrolysate to the bacterial liquid and ferment to obtain wormwood straw enzymatic hydrolysis fermentation mud.

[0012] Preferably: Step (1) Preparation method of Chinese herbal medicine straw powder: crush the Artemisia argyi straw and pass it through a 100-mesh sieve; acid treatment: treat the Chinese herbal medicine straw powder with acetic acid solution, and add citric acid monohydrate and lactic acid for secondary treatment.

[0013] Preferably: the mass concentration of the acetic acid solution is 30%; the mass ratio of Chinese medicinal straw powder: acetic acid solution is 1:4, the treatment temperature is 30°C, and the treatment time is 18h; the amount of citric acid monohydrate added is 1 / 3 of the mass of acetic acid, and the amount of lactic acid added is 1 / 4 of the mass of acetic acid, and the secondary treatment: 30°C treatment for 24h.

[0014] The beneficial effect is that step (1) can effectively reduce the crystallinity of the Chinese medicine straw, and at the same time, acetic acid, lactic acid and citric acid are used in a certain amount ratio, which can effectively reduce the crystallinity without destroying the known or unknown effective ingredients in the Chinese medicine, thereby ensuring the effect.

[0015] Preferably: the drying temperature in step (2) is 60°C and the drying time is 3h; the water is sterile water, and the mass of the sterile water added is the same as the mass of the Chinese medicinal straw powder obtained after drying; the final concentration of cellulase is 0.1g / kg, and the final concentration of maltodextrin is 0.5g / kg; preliminary treatment: 50°C, keep warm for 1h; the amount of lactic acid added is 0.2% of the mass of the Chinese medicinal straw obtained after drying; re-treatment: 50°C, keep warm for 18h.

[0016] The beneficial effect is that in step (2), the crystallinity of the straw is reduced through the action of cellulase, maltodextrin, lactic acid, etc., while the cellulose is not completely degraded into glucose, thereby improving the effect of preventing and treating constipation.

[0017] Preferably: Step (3) bacterial solution: based on the mass of the Chinese medicinal straw hydrolysate, add 1v / w% of saccharomyces cerevisiae solution, 1v / w% of Clostridium butyricum solution, and 1v / w% of Lactobacillus plantarum solution; fermentation: under sealed conditions, 32° C., and fermentation time 40 hours.

[0018] The beneficial effect is that step (3) allows the enzymatically hydrolyzed Chinese medicinal straw to produce a synergistic effect with the bacteria, bacterial metabolites, etc., thereby improving the effect of treating constipation.

[0019] The present invention also provides application of the probiotic sludge prepared by the above method in breeding and feed processing.

[0020] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a method for producing probiotic sludge for relieving sow constipation by high-concentration enzymatic hydrolysis of Chinese medicine straw, and the technical effect achieved is unique process, and the use of edible acid to treat Chinese medicine straw not only does not pollute the environment, but also improves the food safety of straw pretreatment and post-processing. Probiotics such as Clostridium butyricum can strengthen intestinal peristalsis and increase intestinal absorption efficiency, and also have a certain effect on relieving constipation. Detailed implementation mode

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] The embodiments of the present invention disclose a method for producing probiotic bacterial sludge for relieving sows' constipation by high-concentration enzymatic hydrolysis of Chinese medicine straws.

[0023] Sources of some strains, instruments and materials involved in the embodiments:

[0024] Saccharomyces cerevisiae (Blue Ocean Biotech), Clostridium butyricum (Blue Ocean Biotech), Lactobacillus plantarum (Blue Ocean Biotech), cellulase (Xiasheng Group, cellulase CEL-01, 11000U / g), Artemisia argyi straw (Ding'ai Technology), acetic acid (Shandong Jufuxinwang Chemical Co., Ltd.), citric acid monohydrate (Hubei Taiduoyuan Bioengineering Co., Ltd.), lactic acid (Jinan Yuncheng Biotechnology Co., Ltd.), maltodextrin (Jinan Yihewang Chemical Technology Co., Ltd.), starch (Jinan Mingguan Chemical Co., Ltd.), peptone (Jinan Lanjue Trading Co., Ltd.), glucose (Shandong Xujie Chemical Co., Ltd.);

[0025] German Bruker powder X-ray diffractometer (German Bruker Corporation, D8 FOCUS type); TOPAS peak fitting software (German Bruker Corporation, P3 type);

[0026] Culture medium: yeast extract 1% (w / w), peptone 1% (w / w), glucose 4% (w / w); after preparation, sterilize at 115°C for 30 min and cool to room temperature;

[0027] Artemisia argyi straw: Crush it with a pulverizer and sieve it through a 100-mesh sieve.

[0028] Raw materials or equipment not mentioned are all commercially available as usual and will not be elaborated here.

[0029] Example 1

[0030] Activation of strains

[0031] Scrape six loops of Clostridium butyricum strain preserved on the slant with an inoculation loop (loop diameter 0.3 mm), inoculate it into 300 mL of activation medium, place it in a 500 mL Erlenmeyer flask, culture it at 33°C, rotate at 180 r / m, and culture the yeast for 22 h to obtain Clostridium butyricum liquid.

[0032] Scrape six rings of the Saccharomyces cerevisiae strain preserved on the slant with an inoculation loop (loop diameter 0.3 mm), inoculate it into 300 mL of activated medium, place it in a 500 mL Erlenmeyer flask, culture at 33 °C with a rotation speed of 180 r / m, and culture the yeast for 18 h to obtain a Saccharomyces cerevisiae solution.

[0033] Scrape six rings of the Lactobacillus plantarum strain preserved on the slant with an inoculation loop (loop diameter 0.3 mm), inoculate it into 300 mL of activated medium, place it in a 500 mL Erlenmeyer flask, culture at 33 °C with a rotation speed of 180 r / m, and culture the yeast for 18 h to obtain a Lactobacillus plantarum solution.

[0034] A method for producing probiotic mud for alleviating sows' constipation by high-concentration enzymatic hydrolysis of traditional Chinese medicine straws: includes the following steps:

[0035] (1) Acid-treat the traditional Chinese medicine straw powder to obtain a pretreated traditional Chinese medicine straw powder solution;

[0036] (2) Dry the pretreated traditional Chinese medicine straw powder solution, then add water, cellulase, and maltodextrin for preliminary treatment, and then add lactic acid for further treatment to obtain a traditional Chinese medicine straw enzymatic hydrolysate;

[0037] (3) Add the traditional Chinese medicine straw enzymatic hydrolysate to the bacterial solution and ferment to obtain the enzymatic hydrolysis fermentation mud of Artemisia argyi straw.

[0038] To further optimize the technical solution: Preparation method of the traditional Chinese medicine straw powder in step (1): Crush the Artemisia argyi straw and pass it through a 100-mesh sieve to obtain it; Acid treatment: Treat the traditional Chinese medicine straw powder with acetic acid solution, and add citric acid monohydrate and lactic acid for secondary treatment.

[0039] To further optimize the technical solution: The mass concentration of the acetic acid solution is 30%; The mass ratio of the traditional Chinese medicine straw powder to the acetic acid solution is 1:4, the treatment temperature is 30 °C, and the treatment time is 18 h; The addition amount of citric acid monohydrate is 1 / 3 of the mass of acetic acid, and the addition amount of lactic acid is 1 / 4 of the mass of acetic acid. Secondary treatment: Treat at 30 °C for 24 h.

[0040] To further optimize the technical solution: The drying temperature in step (2) is 60 °C and the time is 3 h; The water is sterilized water, and the added mass of the sterilized water is the same as the mass of the traditional Chinese medicine straw powder obtained after drying; The final concentration of cellulase is 0.1 g / kg, and the final concentration of maltodextrin is 0.5 g / kg; Preliminary treatment: Keep warm at 50 °C for 1 h; The addition amount of lactic acid is 0.2% of the mass of the traditional Chinese medicine straw obtained after drying; Further treatment: Keep warm at 50 °C for 18 h.

[0041] To further optimize the technical solution: Bacterial solution in step (3): Based on the mass of the traditional Chinese medicine straw enzymatic hydrolysate, add 1 v / w% of the Saccharomyces cerevisiae solution, 1 v / w% of the Clostridium butyricum solution, and 1 v / w% of the Lactobacillus plantarum solution to the traditional Chinese medicine straw enzymatic hydrolysate; Fermentation: Under sealed conditions, at 32 °C, the fermentation time is 40 h.

[0042] Technical effect:

[0043] The enzyme-hydrolyzed fermentation bacterial sludge of Artemisia argyi Levl. et Vant. straw prepared in Example 1 was diluted 10 times with sterilized ultrapure water, and the cell number of Saccharomyces cerevisiae measured by a hemocytometer was 0.3x10 8 cells / g, and the total cell concentration of Clostridium butyricum and Lactobacillus plantarum was 16.3x10 8 cells / g.

[0044] The enzyme-hydrolyzed fermentation bacterial sludge of Artemisia argyi Levl. et Vant. straw was diluted 20 times with sterilized ultrapure water, shaken well for 15 min after dilution, centrifuged at 5000 r / m for 15 min by a centrifuge, and filtered through a 0.22-μm microporous membrane, and then the content of cellobiose pentaose was measured.

[0045] The measurement method was based on the reference literature (Peng Hong, Lin Lu, Liu Yuhuan, Ruan Rongsheng. Study on the hydrolysis of cellooligosaccharides by cellulase, Food Science, 2009, 30(13): 218-222), and the content of cellobiose pentaose measured was 8.1 g / L.

[0046] The enzyme-hydrolyzed fermentation bacterial sludge of Artemisia argyi Levl. et Vant. straw was dried at 60 °C for 3 h, and then the crystallinity was measured to be 25.71±6.71%.

[0047] Comparative experiment 1

[0048] Measure the crystallinity of cellulose.

[0049] Control group: The crystallinity of the crushed Artemisia argyi Levl. et Vant. straw without acid treatment was directly measured.

[0050] Experimental group: The Artemisia argyi Levl. et Vant. straw solution treated with acid in step (1) of Example 1 was dried at 60 °C for 3 h, and then the crystallinity was measured.

[0051] The measurement method was the method in the literature by Liu Zhigang, Gao Yan, Jin Hua, Yu Shihua, etc. (Study on the determination of the crystallinity of natural cellulose by XRD peak separation method, China Measurement & Test, 2015, 41(2): 38-41). The measurement conditions were: the fitting function was pV, the position of the amorphous peak was selected at 18°, and the scanning range was 7° to 40°.

[0052] Measurement results: The crystallinity of the crushed Artemisia argyi Levl. et Vant. straw in the control group was 88.71±6.71%, and the crystallinity after treatment in the experimental group was 55.71±6.71%.

[0053] Comparative experiment 2

[0054] Effect of relieving sows' constipation

[0055] Two hundred Duroc×Landrace×Yorkshire sows at 60 days of pregnancy with similar health conditions and parities were selected and randomly divided into 2 treatment groups, with 100 replicates in each treatment group and one sow in each replicate.

[0056] Control group: Sows were fed a basal diet.

[0057] Experimental group: Fed a basal diet + 2% probiotic mud prepared in Example 1.

[0058] The experimental period was 13 days. The composition of the basal diet was corn: soybean meal: concentrated feed (Baiyi Yunzhu concentrated feed, Hunan Baiyi Yunshang Technology Co., Ltd.) at 64:24:12.

[0059] The statistical data standard was as follows: Severe constipation: Difficult defecation in pigs, with feces in granular form and hard texture.

[0060] Mild constipation: Feces in cylindrical shape, or with some particles, and hard when stepped on.

[0061] Appropriate feces: Easy defecation in sows, with feces soft and able to form and disperse when landing.

[0062] The statistical results were based on the average over 13 days.

[0063] Number of heads = sum of the number of sows with constipation per day within 13 days / 13. The results are shown in Table 1:

[0064] Table 1

[0065] Project Number of animals with severe constipation Number of animals with mild constipation Number of animals with moderate feces Control group 45.6 42.4 12 Experimental group 13.8 27.1 60.1

[0066] Comparative experiment 3

[0067] Experimental group: Except that acetic acid of equal mass was used to replace citric acid monohydrate and lactic acid in the acid treatment of Artemisia argyi straw, the rest was the same as in Example 1. The crystallinity of the enzymatically hydrolyzed and fermented Artemisia argyi mud was measured to be 31.71 ± 6.76%, and the content of cellopentaose was measured to be 7.2 g / L.

[0068] Sow constipation test:

[0069] Control group: The same as the control group in Comparative experiment 2.

[0070] Experimental group: Except for the above process changes, the rest was the same as the experimental group in Comparative experiment 2.

[0071] The results of the sow constipation test are shown in Table 2:

[0072] Table 2

[0073] Project Number of animals with severe constipation Number of animals with mild constipation Number of animals with moderate feces Control group 45.6 42.4 12 Experimental group 16.8 25.3 58.9

[0074] Comparative test 4

[0075] Experimental group: Except that dextrin was not added in the enzymatic hydrolysis treatment, the rest was the same as in Example 1. The crystallinity of the enzymatically hydrolyzed and fermented Artemisia argyi mud was measured to be 23.71 ± 6.71%. The content of cellopentaose was measured to be 5.1 g / L.

[0076] Sow constipation test:

[0077] Control group: The same as the control group in Comparative Experiment 2.

[0078] Experimental group: Except for the above process changes, the same as the experimental group in Comparative Experiment 2.

[0079] The results of the sow constipation test are shown in Table 3:

[0080] Table 3

[0081] Project Number of animals with severe constipation Number of animals with mild constipation Number of animals with moderate feces Control group 45.6 42.4 12 Experimental group 18 26 56

[0082] Comparative Test 5

[0083] Experimental group: Except that maltodextrin is replaced with starch of equal amount for enzymatic hydrolysis, the others are the same as in Example 1. The crystallinity of the fermented mud of Artemisia argyi after enzymatic hydrolysis is measured to be 26.12 ± 6.66%, and the content of cellopentaose is measured to be 4.9 g / L.

[0084] Sow constipation test:

[0085] Control group: The same as the control group in Comparative Experiment 2.

[0086] Experimental group: Except for the above process changes, the same as the experimental group in Comparative Experiment 2.

[0087] The results of the sow constipation test are shown in Table 4:

[0088] Table 4

[0089] Project Number of animals with severe constipation Number of animals with mild constipation Number of animals with moderate feces Control group 45.6 42.4 12 Experimental group 19 29 52

[0090] Comparative Experiment 6

[0091] Experimental group: Except that the concentration of cellulase in the enzymatic hydrolysis treatment is 0.15 g / L, the others are the same as in Example 1. The crystallinity of the fermented mud of Artemisia argyi after enzymatic hydrolysis is measured to be 16.18 ± 6.66%, and the content of cellopentaose is measured to be 2.9 g / L.

[0092] Sow constipation test:

[0093] Control group: The same as the control group in Comparative Experiment 2.

[0094] Experimental group: Except for the above process changes, the same as the experimental group in Comparative Experiment 2.

[0095] The results of the sow constipation test are shown in Table 5:

[0096] Table 5

[0097] Project Number of animals with severe constipation Number of animals with mild constipation Number of animals with moderate feces Control group 45.6 42.4 12 Experimental group 20 28 52

[0098] In the present specification, the various embodiments are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference may be made to each other.

[0099] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for producing probiotic bacterial sludge that relieves sows' constipation by high-concentration enzymatic hydrolysis of traditional Chinese medicine straws, characterized in that, The following steps are involved: (1) treating the Chinese medicinal straw powder with acid to obtain a pretreated Chinese medicinal straw powder solution; (2) drying the pretreated Chinese medicine straw powder solution, adding water, cellulase and maltodextrin for preliminary treatment, and then adding lactic acid for further treatment to obtain a Chinese medicine straw enzymatic hydrolyzate; (3) adding the Chinese medicine straw enzymatic hydrolyzate to the bacterial solution, and fermenting to obtain the Qi Ai straw enzymatic hydrolyzed fermented bacterial mud; The preparation method of the Chinese medicine straw powder in step (1) is as follows: the Chinese medicine straw powder is crushed and passed through a 100-mesh sieve; the acid treatment is as follows: the Chinese medicine straw powder is treated with an acetic acid solution, and then treated with citric acid monohydrate and lactic acid for a second time; The mass concentration of the acetic acid solution is 30%; the mass ratio of the Chinese medicinal straw powder to the acetic acid solution is 1:4, the treatment temperature is 30°C, and the treatment time is 18h; the amount of citric acid monohydrate added is 1 / 3 of the mass of acetic acid, and the amount of lactic acid added is 1 / 4 of the mass of acetic acid, and the secondary treatment: 30°C for 24h; Step (2) The drying temperature is 60°C and the time is 3h; the water is sterile water, and the mass of the sterile water added is the same as the mass of the Chinese medicinal straw powder obtained after drying; the final concentration of the cellulase is 0.1g / kg, and the final concentration of the maltodextrin is 0.5g / kg; the preliminary treatment is 50°C and kept warm for 1h; the amount of lactic acid added is 0.2% of the mass of the Chinese medicinal straw obtained after drying; the re-treatment is 50°C and kept warm for 18h; The bacterial solution in step (3) is as follows: based on the mass of the Chinese medicinal straw hydrolysate, 1v / w% of saccharomyces cerevisiae solution, 1v / w% of Clostridium butyricum solution, and 1v / w% of Lactobacillus plantarum solution are added to the Chinese medicinal straw hydrolysate; the fermentation is carried out under sealed conditions at 32° C. for 40 hours.

2. Use of the probiotic slurry prepared by the method according to claim 1 in the preparation of feed additives.