CN109504713B - Method for preparing high-ester-bond humic acid preparation by using clostridium beijerinckii and application - Google Patents
Method for preparing high-ester-bond humic acid preparation by using clostridium beijerinckii and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/52—Propionic acid; Butyric acids
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
Abstract
A method for preparing a high-ester-bond humic acid preparation by using clostridium beijerinckii and an application thereof are disclosed, wherein sodium humate is added into a clostridium beijerinckii fermentation system, and butyric acid formed by fermentation of the clostridium beijerinckii and phenolic hydroxyl in the sodium humate are subjected to esterification reaction by lipase, so that the concentration of the butyric acid in fermentation liquor is reduced, and the inhibiting effect of accumulation of the butyric acid on the fermentation process of the clostridium beijerinckii is reduced. The presence of sodium humate prolongs the fermentation time and product accumulation of clostridium beijerinckii. The high ester bond humic acid formed by the sodium humate and the butyric acid can be directly used as an intestinal tract health additive product, and the humic acid and the butyric acid are released under the action of pancreatic lipase after the humic acid enters the intestinal tract to promote the intestinal tract health.
Description
Technical Field
The invention relates to a preparation for preparing high ester bond humic acid by using clostridium beijerinckii and application thereof.
Background
The carbon source is converted into butyric acid in the fermentation process of the clostridium beijerinckii, and the accumulation of the butyric acid can inhibit the fermentation process, so that the fermentation is finished. If the generated butyric acid can be removed in time in the fermentation process, the stable operation of the fermentation process can be ensured, and the efficiency of the fermentation process is improved.
The butyric acid plays an important role in the health of intestinal tracts of livestock and poultry as one of volatile fatty acids, and on one hand, the butyric acid can supply energy to intestinal villi, promote the development of the intestinal villi, and simultaneously can inhibit the growth of harmful bacteria in the intestinal tracts and maintain the microecological balance of the intestinal tracts. However, it is worth noting that a small amount of butyric acid added in the feed can be absorbed by the upper part of the intestinal tract in the using process, so that the butyric acid is difficult to reach the rear end of the intestinal tract to play a role in bacteriostasis and energy supply. Thus, patent application No. 201610999456.2 discloses a micro-encapsulated sodium butyrate and a preparation method thereof, wherein components such as butyric acid and the like are coated by using ethylene glycol. The method is to wrap butyric acid and the like by a simple physical coating method and a coating material. The disadvantage is that the release time and position of the coating in the intestinal tract are not controllable, which may cause the problem that butyric acid can not be released in time in the intestinal tract, or butyric acid can not safely reach the later section of the intestinal tract because of insufficient coating.
Humic acid is a reticular high molecular substance rich in phenolic hydroxyl groups, is insoluble in water and acid, but is soluble in alkali. Humic acid plays a role in astringency in intestinal tracts and promotes intestinal tract injury repair. The method has the advantages that the ester bonds are generated by catalyzing butyric acid and phenolic hydroxyl groups in humic acid by using lipase, so that the inhibition effect of the butyric acid in fermentation liquor in the fermentation process of clostridium beijerinckii is reduced, and the ester bonds can be slowly opened only under the action of pancreatic lipase in intestinal tracts after the butyric acid and sodium humate are subjected to esterification reaction, so that the butyric acid is released; because the ester bond opening process is the normal physiological metabolic process of a living body and is very slow, the high ester bond humic acid continuously moves along with chyme and is continuously opened to release humic acid and butyric acid, the butyric acid in the whole intestinal tract is ensured to exist, and the full intestinal tract bacteriostasis and energy supply function of the butyric acid are realized.
Disclosure of Invention
The invention aims to overcome the defects in the prior art (such as the fermentation process of clostridium beijerinckii is inhibited by product butyric acid, the butyric acid is absorbed by the upper section of an intestinal tract after entering the intestinal tract and cannot reach the rear section of the intestinal tract to play a role, and the traditional coating process cannot ensure that the time and the position of the product released in the intestinal tract are controllable), and provides a method for removing the butyric acid in the fermentation liquid by adding sodium humate in the fermentation process of clostridium beijerinckii, carrying out esterification reaction on the butyric acid and the humic acid under the action of lipase, and simultaneously ensuring the slow release of the butyric acid in the intestinal tract by the generated high ester bond humic acid so as to realize the full intestinal tract bacteriostasis and energy supply of the butyric acid.
The invention also aims to provide a high ester bond humic acid preparation which has important application in the field of livestock and poultry breeding.
A high-ester-bond humic acid preparation is prepared by adding sodium humate into fermentation liquor of clostridium beijerinckii in a certain proportion, catalyzing generated butyric acid in the fermentation process and phenolic hydroxyl in the sodium humate by lipase to form ester bonds, reducing the concentration of free butyric acid in the fermentation liquor, and finally carrying out esterification reaction on humic acid and butyric acid to generate the high-ester-bond humic acid preparation with the component of humic acid butyrate. Specifically, butyric acid formed by fermentation of clostridium beijerinckii and phenolic hydroxyl in sodium humate are subjected to esterification reaction through lipase, so that the concentration of butyric acid in fermentation liquor is reduced, and the inhibiting effect of accumulation of butyric acid on the fermentation process of clostridium beijerinckii is reduced; the high ester bond humic acid formed by humic acid and butyric acid is further used as a feed additive to promote the intestinal health of livestock and poultry.
The purpose of the invention is realized by the following steps: a preparation method of a high ester bond humic acid preparation comprises the following steps:
(1) the seed liquid containing Clostridium beijerinckii was prepared, wherein the concentration of the seed liquid was 0.5X 108-1.0×108CFU/ml；
Seed medium (TYA medium): 2 g of beef extract, 2 g of yeast powder, 6 g of peptone,CH3COONH4 3 g，KH2PO4 0.5 g，MgSO4·7H2O 0.2 g，FeSO4·7H20.01 g of O, 1000 mL of distilled water, pH 6.5, autoclaving at 121 ℃ for 15 min, and blowing with sterile nitrogen for 5 min to drive off oxygen.
(2) Fermentation medium: k2HPO4 0.5 g、KH2PO4 0.5 g 、CH3COONH4 2.2 g、MgSO4·7H2O 0.2 g、MnSO4·H2O 0.01 g、NaCl 0.01 g、FeSO4·7H20.01 g of O, 0.001g of thiamine (thiamine, vitamin B1), 0.00001g of biotin (biotin), 0.001g of p-aminobenzoic acid (p-aminobenzoic acid), and water to 1000 mL.
(3) Adding sodium humate into the fermentation tank according to the proportion of 4-10 g/L.
(4) After the fermentation medium in the fermentation tank is sterilized, cooling to room temperature, and adding lipase into the fermentation tank according to the mass ratio of the lipase to the sodium humate of 1: 1-5.
(5) And (2) adding the seed solution containing clostridium beijerinckii obtained in the step (1) and a fermentation culture medium into a butyric acid production fermentation culture medium by using a peristaltic pump according to the volume ratio of 2%, and stirring and culturing at the temperature of 37-42 ℃ at the speed of 50-120 r/min.
(6) Adding 200g/L xylose into the fermentation tank by using a peristaltic pump, maintaining the concentration of the xylose in the fermentation liquor to be 20g/L, and fermenting for 96 hours.
(7) After fermentation is finished, pH of fermentation liquor is adjusted to 2.5, high ester bond humic acid generated by enzymatic reaction is promoted to be settled, and the high ester bond humic acid preparation is obtained by filter pressing and drying of a plate-and-frame filter press.
The specific characteristics of the scheme are that the lipase is Novozym 435 produced by Novoxin, and the enzyme activity is 10000 PLU/g.
Clostridium beijerinckii (C.,)Clostridium beijerinckii) The strain is clostridium beijerinckii SE-2, the preservation number of the strain is CCTCC M2014384, and the preservation date is 2014, 8 and 19 days. Clostridium beijerinckii SE-2, Latin literature name Clostridium beijerinckii SE-2; the strain has been preserved in Chinese typical culture in 2014 8 and 19 daysPreservation center (CCTCC), address: wuhan university, Wuhan City, China, zip code 430072, preservation number CCTCC No. M2014384.
The application of the high ester bond sodium humate preparation is to mix the high ester bond sodium humate preparation with feed in a proportion of 0.2 percent by weight and directly feed livestock and poultry.
The invention has the following positive effects: 1. the lipase is used for catalyzing butyric acid and phenolic hydroxyl in humic acid to generate ester bonds, so that the inhibiting effect of butyric acid in the fermentation process of clostridium beijerinckii is reduced, the smooth proceeding of the fermentation process is ensured, and the fermentation efficiency is improved.
2. After the butyric acid and the sodium humate are subjected to esterification reaction, ester bonds can be slowly opened in the intestinal tract only under the action of pancreatic lipase, so that the butyric acid is released; because the ester bond opening process is very slow, the high ester bond humic acid continuously moves along with the chyme and is continuously opened to release the humic acid and the butyric acid, the butyric acid in the whole intestinal tract is ensured to exist, and the full intestinal tract bacteriostasis and energy supply function of the butyric acid is realized.
3. Humic acid astringes intestinal tract injury, butyric acid can supply energy to intestinal villi, promote intestinal villi development, improve intestinal microecological balance, and the synergistic effect of humic acid and butyric acid provides guarantee for intestinal tract health.
4. After fermentation is finished, the characteristic that humic acid is insoluble in acid and water is utilized, and the pH value is adjusted to promote high ester bond humic acid to form precipitate in fermentation liquor, so that subsequent separation is facilitated, and the separation cost is saved.
According to the invention, the clostridium beijerinckii fermentation product butyric acid and humic acid are enzymatically converted into a stable compound, so that on one hand, the smooth fermentation process is promoted, and meanwhile, the high-ester-bond humic acid can help butyric acid to be slowly released in intestinal tracts, so that the full-intestinal-tract bacteriostasis and energy supply are realized, the effect of the butyric acid on livestock and poultry breeding is enhanced, and the product has stronger competitiveness.
Detailed Description
Example 1: a preparation method of a high ester bond humic acid preparation comprises the following steps:
firstly, preparing seed liquid containing clostridium beijerinckii,wherein the concentration of the bacterial liquid is 0.5 multiplied by 108-1.0×108CFU/ml; the clostridium beijerinckii has a preservation number of CCTCC No. M2014384 clostridium beijerinckii SE-2.
Seed medium (TYA medium): 40 g of glucose, 2 g of beef extract, 2 g of yeast powder, 6 g of peptone and CH3COONH4 3 g，KH2PO4 0.5 g，MgSO4·7H2O 0.2 g，FeSO4·7H20.01 g of O, 1000 mL of distilled water, pH 6.5, autoclaving at 121 ℃ for 15 min, and blowing with sterile nitrogen for 5 min to drive off oxygen.
Step two, preparing a butyric acid production fermentation medium, which specifically comprises the following steps: basic fermentation medium: k2HPO4 0.5 g、KH2PO4 0.5 g 、CH3COONH4 2.2 g、MgSO4·7H2O 0.2 g、MnSO4·H2O 0.01 g、NaCl 0.01 g、FeSO4·7H20.01 g of O, 0.001g of thiamine (thiamine, vitamin B1), 0.00001g of biotin (biotin), 0.001g of p-aminobenzoic acid (p-aminobenzoic acid), and water to 1000 mL.
Thirdly, adding the sodium humate into a fermentation tank according to the proportion of 4 g/L.
And fourthly, after the fermentation medium in the fermentation tank is sterilized, cooling to room temperature, and adding the lipase into the fermentation tank according to the mass ratio of the lipase to the sodium humate of 1: 5.
And fifthly, adding the clostridium beijerinckii seed liquid and the fermentation culture medium in the first step into the fermentation culture medium according to the volume ratio of 2 percent by using a peristaltic pump, and carrying out stirring culture at 37 ℃ at 50 r/min.
Sixthly, adding 200g/L of xylose into the fermentation tank by using a peristaltic pump, and maintaining the concentration of the xylose in the fermentation liquor to be 20 g/L.
Seventhly, after the fermentation is finished in 96 hours, taking the fermentation liquid to analyze the concentration of butyric acid, wherein the concentration of butyric acid is 1.7 g/L; the total consumption of xylose during fermentation was 118.7 g/L.
And eighthly, after the fermentation is finished, adjusting the pH value of the fermentation liquor to 2.5 to promote the high ester bond humic acid generated by the enzymatic reaction to settle, and performing filter pressing and drying by using a plate and frame filter press to obtain the high ester bond humic acid preparation.
Example 2: a preparation method of a high ester bond humic acid preparation comprises the following steps:
the first step, the second step, is the same as example 1.
Thirdly, adding sodium humate into the fermentation medium according to the proportion of 7 g/L.
And fourthly, after the fermentation medium in the fermentation tank is sterilized, cooling to room temperature, and adding the lipase into the fermentation tank according to the mass ratio of the lipase to the sodium humate of 1: 3.
And fifthly, adding the clostridium beijerinckii seed liquid and the fermentation culture medium in the first step into the fermentation culture medium by a peristaltic pump according to the volume ratio of 2%, and carrying out stirring culture at 40 ℃ at 80 r/min.
Sixthly, adding 200g/L of xylose into the fermentation tank by using a peristaltic pump, and maintaining the concentration of the xylose in the fermentation liquor to be 20 g/L.
Seventhly, after the fermentation is finished in 96 hours, taking the fermentation liquor to analyze the concentration of butyric acid, wherein the concentration of butyric acid is 0.46 g/L; the total consumption of xylose during fermentation was 143.3 g/L.
And eighthly, after the fermentation is finished, adjusting the pH value of the fermentation liquor to 2.5 to promote the high ester bond humic acid generated by the enzymatic reaction to settle, and performing filter pressing and drying by using a plate and frame filter press to obtain the high ester bond humic acid preparation.
Example 3: a preparation method of a high ester bond humic acid preparation comprises the following steps:
the first step, the second step, is the same as example 1.
Thirdly, adding the sodium humate into the fermentation medium according to the proportion of 10 g/L.
And fourthly, after the fermentation medium in the fermentation tank is sterilized, cooling to room temperature, and adding the lipase into the fermentation tank according to the mass ratio of the lipase to the sodium humate of 1: 1.
And fifthly, adding the clostridium beijerinckii seed liquid and the fermentation culture medium in the first step into the fermentation culture medium according to the volume ratio of 2 percent by using a peristaltic pump, and stirring and culturing at 42 ℃ at 100 r/min.
Sixthly, adding 200g/L of xylose into the fermentation tank by using a peristaltic pump, and maintaining the concentration of the xylose in the fermentation liquor to be 20 g/L.
Seventhly, after the fermentation is finished in 96 hours, taking the fermentation liquor to analyze the concentration of butyric acid, wherein the concentration of butyric acid is 0.90 g/L; the total consumption of xylose during fermentation was 129.8 g/L.
And eighthly, after the fermentation is finished, adjusting the pH value of the fermentation liquor to 2.5 to promote the high ester bond humic acid generated by the enzymatic reaction to settle, and performing filter pressing and drying by using a plate and frame filter press to obtain the high ester bond humic acid preparation.
Example 4: a preparation method of a high ester bond humic acid preparation comprises the following steps:
the first step, the second step, is the same as example 1.
Thirdly, adding sodium humate into the fermentation medium according to the proportion of 7 g/L.
Fourthly, adding the clostridium beijerinckii seed liquid and the fermentation culture medium in the first step into the fermentation culture medium according to the volume ratio of 2 percent by using a peristaltic pump, and carrying out stirring culture at 40 ℃ at 80 rpm.
Fifthly, adding 200g/L xylose into the fermentation tank by using a peristaltic pump, and maintaining the concentration of the xylose in the fermentation liquor to be 20 g/L.
Sixthly, after the fermentation is finished in 96 hours, taking the fermentation liquor to analyze the concentration of butyric acid, wherein the concentration of butyric acid is 21.02 g/L; the total consumption of xylose during fermentation was 33.2 g/L.
And seventhly, after the fermentation is finished, the pH value of the fermentation liquor is adjusted to 2.5, the high ester bond humic acid generated by the enzymatic reaction is promoted to be settled, and the high ester bond humic acid preparation is obtained by filter pressing and drying through a plate and frame filter press.
Example 4 compared to example 2, only no lipase was added, and due to the lack of lipase, the esterification reaction of butyric acid and humic acid was catalyzed, so butyric acid in the fermentation broth accumulated, and the xylose utilization amount was very low, only 23.2% of example 2, indicating that the presence of butyric acid resulted in the unexpected termination of the fermentation process. Meanwhile, the two are calculated by xylose consumption, and the butyric acid amount generated in the whole process is easy to be greatly different.
Example 5: a preparation method of a high ester bond humic acid preparation comprises the following steps:
the first step, the second step, is the same as example 1.
Thirdly, adding sodium humate into the fermentation medium according to the proportion of 7 g/L.
And fourthly, adding the clostridium beijerinckii seed liquid and the fermentation culture medium in the first step into the fermentation culture medium by using a peristaltic pump according to the volume ratio of 2%, and stirring and culturing at 40 ℃ at 80 r/min.
Fifthly, adding 200g/L xylose into the fermentation tank by using a peristaltic pump, and maintaining the concentration of the xylose in the fermentation liquor to be 20 g/L.
Sixthly, after the fermentation is finished in 96 hours, taking the fermentation liquor to analyze the concentration of butyric acid, wherein the concentration of butyric acid is 21.02 g/L; the total consumption of xylose during fermentation was 33.2 g/L.
And seventhly, adding the lipase into the fermentation tank according to the mass ratio of the lipase to the sodium humate of 1:3, stirring and reacting at 40 ℃ at 80 rpm for 12 hours, wherein the concentration of butyric acid in the fermentation liquor is 0.19 g/L.
And eighthly, after the reaction is finished, adjusting the pH value of the fermentation liquor to 2.5 to promote the high ester bond humic acid generated by the enzymatic reaction to settle, and performing filter pressing and drying by using a plate-and-frame filter press to obtain a high ester bond humic acid product.
In example 5, compared with example 4, lipase is added after the fermentation is finished to promote esterification reaction between sodium humate and generated butyric acid to form high ester bond humic acid.
Example 6: a preparation method of humic acid preparation comprises the following steps:
the first to sixth steps are the same as in example 5;
and seventhly, after the reaction is finished, adjusting the pH value of the fermentation liquor to 2.5 to promote the humic acid generated by the enzymatic reaction to settle, and performing filter pressing and drying by using a plate and frame filter press to obtain the humic acid preparation.
And step eight, adding 21.02 g/L butyric acid into the humic acid obtained in the step seven according to the fermentation volume to obtain a final product.
Example 6 is different from example 5 in that butyric acid in example 5 is ester-bonded to humic acid, whereas butyric acid in example 6 is present as a butyric acid molecule alone.
Example 7: the application of the high ester bond sodium humate preparation is that the high ester bond sodium humate preparation obtained in the example 2 is mixed with feed by 0.2 weight percent and directly fed.
And (3) test results:
the experimental subject is a piglet born by a certain commercial pig.
Experimental groups: the newborn piglets are fed with the feed added with the high ester bond humic acid preparation, the prevention and control of methods such as the administration of an antibiotic injection and the like are not carried out, and other feeding management is carried out according to the conventional method of a pig farm.
Control group: after the piglets have diarrhea, antibiotics and other medicines are injected, and certain other medicines are given for treatment in combination with the treatment management of a pig farm, and the daily ration, the nutrient components, the feeding management and the like are completely the same as those of the experimental group. The test results are shown in table 1:
TABLE 1
The results show that: compared with a control group, the high ester bond humic acid preparation can effectively prevent diarrhea of piglets and reduce the medication cost.
Example 8: the application of the high ester bond sodium humate preparation is that the humic acid preparation obtained in the example 4 is mixed with feed by 0.2 weight percent and directly fed.
And (3) test results:
the experimental subject is a piglet born by a certain commercial pig.
Experimental groups: the newborn piglets are fed with the feed added with the high ester bond humic acid preparation, the prevention and control of methods such as the administration of antibiotic injection and the like are not carried out, and other feeding management is carried out according to the conventional method of a pig farm.
Control group: after the piglets have diarrhea, antibiotics and other medicines are injected, and certain other medicines are given for treatment in combination with the treatment management of a pig farm, and the daily ration, the nutrient components, the feeding management and the like are completely the same as those of the experimental group. The test results are shown in table 2:
TABLE 2
The results show that: compared with the control group, the experimental group can prevent the diarrhea of piglets, but compared with the example 7, the product (example 4) used in the example 8 does not contain lipase, so the sodium humate only plays a certain role in intestinal tracts finally and cannot play a role in the fermentation process; the butyric acid is dissolved in the fermentation broth, so the final humic acid preparation does not contain butyric acid, and the final example 8 (example 4) is different from the example 7 (example 2) only in lipase, but the product has a very poor prevention effect on piglet diarrhea.
Example 9: the application of the high ester bond sodium humate preparation is that the humic acid preparation obtained in the example 5 is mixed with feed by 0.2 weight percent and directly fed.
And (3) test results:
the experimental subject is a piglet born by a certain commercial pig.
Experimental groups: the newborn piglets are fed with the feed added with the high ester bond humic acid preparation, the prevention and control of methods such as the administration of an antibiotic injection and the like are not carried out, and other feeding management is carried out according to the conventional method of a pig farm.
Control group: after the piglets have diarrhea, antibiotics and other medicines are injected, and certain other medicines are given for treatment in combination with the treatment management of a pig farm, and the daily ration, the nutrient components, the feeding management and the like are completely the same as those of the experimental group. The test results are shown in table 3:
TABLE 3
Example 10: the application of the sodium humate preparation is that the humic acid preparation obtained in the example 6 is mixed with feed by 0.2 weight percent and directly fed.
And (3) test results:
the experimental subject is a piglet born by a certain commercial pig.
Experimental groups: the newborn piglets are fed with the feed added with the high ester bond humic acid preparation, the prevention and control of methods such as the administration of an antibiotic injection and the like are not carried out, and other feeding management is carried out according to the conventional method of a pig farm.
Control group: after the piglets are diarrhea, antibiotics and other medicines are injected, certain treatment of other medicines is given in combination with the treatment management of the pig farm, and the daily ration, the nutrient components, the feeding management and the like are completely the same as those of the experimental group.
The test results are shown in Table 4:
TABLE 4
The results show that: compared with a control group, the experimental group can prevent diarrhea of piglets, although the contents of humic acid and butyric acid in the products of examples 5 and 6 are the same, butyric acid and the humic acid are connected by ester bonds in example 5, and the butyric acid in example 6 exists in the form of butyric acid molecules, so that the butyric acid in example 10 cannot be slowly released like the butyric acid in example 9, and the effects of inhibiting bacteria in the whole intestinal tract and promoting intestinal villus development are achieved, and finally, the diarrhea rate of piglets in example 10 is higher than that in example 9. The results further demonstrate that enzymatic attachment of butyric acid to humic acid by lipase, by addition of sodium humate, is essential to improve the effect of the product in the intestinal tract.
Claims (4)
1. The preparation method of the high-ester-bond humic acid preparation is characterized by comprising the following steps of:
(1) preparation of seed solution containing Clostridium beijerinckii, wherein the concentration of the seed solution is 0.5 × 108-1.0×108CFU/ml；
Seed culture medium: 2 g of beef extract, 2 g of yeast powder, 6 g of peptone and CH3COONH4 3 g，KH2PO4 0.5 g，MgSO4·7H2O 0.2 g，FeSO4·7H20.01 g of O, 1000 mL of distilled water, pH 6.5, and autoclaving at 121 DEG CSterilizing for 15 min, and blowing with sterile nitrogen for 5 min to remove oxygen; the Clostridium beijerinckii strain is Clostridium beijerinckii SE-2, Latin literature name Clostridium beijerinckii SE-2; the strain is preserved in China Center for Type Culture Collection (CCTCC) at 8-19 th month in 2014, and the address is as follows: wuhan university in Wuhan, China, with a preservation number of CCTCC No. M2014384;
(2) fermentation medium: k2HPO4 0.5 g、KH2PO4 0.5 g 、CH3COONH4 2.2 g、MgSO4·7H2O 0.2 g、MnSO4·H2O 0.01 g、NaCl 0.01 g、FeSO4·7H20.01 g of O, 10.001g of thiamine vitamin B, 0.00001g of biotin and 0.001g of p-aminobenzoic acid, and adding water to 1000 mL;
(3) adding sodium humate into a fermentation tank according to the proportion of 4-10 g/L;
(4) after the fermentation medium in the fermentation tank is sterilized, cooling to room temperature, and adding lipase into the fermentation tank according to the mass ratio of 1:1-5 of lipase to sodium humate; the lipase is Novozym 435 produced by Novoxin;
(5) adding the seed solution containing clostridium beijerinckii obtained in the step (1) and a fermentation culture medium into a fermentation tank by using a peristaltic pump according to the volume ratio of 2%, and carrying out stirring culture at the temperature of 37-42 ℃ at 50-120 r/min;
(6) adding 200g/L xylose into a fermentation tank by using a peristaltic pump, maintaining the concentration of the xylose in the fermentation liquor to be 20g/L, and fermenting for 96 hours;
(7) after fermentation is finished, pH of fermentation liquor is adjusted to 2.5, high ester bond humic acid generated by enzymatic reaction is promoted to be settled, and the high ester bond humic acid preparation is obtained by filter pressing and drying of a plate-and-frame filter press.
2. The method for preparing a high ester bond humic acid preparation as claimed in claim 1 wherein the lipase Novozym 435 has an enzyme activity of 10000 PLU/g.
3. A humic acid preparation with high ester bond obtained by the preparation method of claim 1, which is characterized in that sodium humate is added into the fermentation liquor of clostridium beijerinckii according to a certain proportion, butyric acid generated in the fermentation process and phenolic hydroxyl in the sodium humate form ester bond through lipase catalysis, the concentration of free butyric acid in the fermentation liquor is reduced, and finally humic acid and butyric acid generate the humic acid preparation with high ester bond of which the component is humic acid butyrate through esterification reaction.
4. The use of the humic acid preparation with high ester bond as claimed in claim 3, wherein the humic acid preparation with high ester bond is mixed with feed by 0.2 percent by weight and is directly fed to livestock and poultry.
Priority Applications (1)
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| WO2000016785A2 (en) * | 1998-09-23 | 2000-03-30 | Enerkom (Proprietary) Limited | Humic acid and its use in the treatment of various conditions |
| WO2007036363A2 (en) * | 2005-09-28 | 2007-04-05 | Südzucker Aktiengesellschaft Mannheim/Ochsenfurt | Compositions containing butyric acid ester of carbohydrates and carbohydrate polyols |
| CN104673712A (en) * | 2015-01-16 | 2015-06-03 | 山东省科学院能源研究所 | Bacterial strain for producing alcohol fuels by synchronously utilizing glucose and xylose and application of bacterial strain |
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| WO2000016785A2 (en) * | 1998-09-23 | 2000-03-30 | Enerkom (Proprietary) Limited | Humic acid and its use in the treatment of various conditions |
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