CN112458132A - Method for preparing multi-element antibacterial probiotic by using single raw material - Google Patents
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Abstract
A method for preparing multi-element antibacterial probiotic from single raw material is provided. The invention belongs to the field of extraction and preparation of natural antibacterial probiotics, and discloses a process for producing natural antibacterial probiotics for animal breeding by using barks as raw materials, which mainly comprises the steps of (1) pretreating barks by using a low-temperature hydrothermal method, (2) collecting and concentrating low-temperature hydrothermal extract, (3) pretreating the barks subjected to low-temperature hydrothermal extraction by using high-temperature hydrothermal method, (4) treating the high-temperature hydrothermal extract by using an evaporation concentration method, an acid precipitation method and a xylan enzymolysis method in sequence, (5) mixing the treated high-temperature hydrothermal extract and the low-temperature hydrothermal extract to obtain a tannin-lignin polyphenol-xylooligosaccharide-rich ternary antibacterial probiotic agent, (6) mixing the ternary antibacterial probiotic agent with an acidifier to obtain a tannin-lignin polyphenol-xylooligosaccharide-acidifier-rich quaternary antibacterial probiotic agent. The antibacterial probiotic agent prepared by the invention has the effects of resisting bacteria, maintaining an acidic habitat and promoting the growth of beneficial microorganisms, and can be used as a synthetic antibiotic substitute for animal breeding with low production cost.
Description
Technical Field
The invention relates to the field of extraction and preparation of natural antibacterial probiotics, in particular to a method for preparing a multi-element antibacterial probiotic for replacing antibiotics by using a single raw material.
Background
The long-term use of antibiotics can not only cause animal endogenous infection and superinfection, so that pathogenic bacteria generate drug resistance and reduce the immune function of the pathogenic bacteria, but also the residue of the antibiotics in animal products and ecological environment directly threatens human health and safety. At present, China and European Union have issued regulations to prohibit the addition of antibiotics in feed, so that the development of natural and safe antibiotic substitutes and the development of antibiotic-free breeding become more and more the direction of the vigorous development of China and European countries.
Disclosure of Invention
The invention aims to solve the problem of antibiotic abuse and develop an antibiotic substitute. The invention takes bark as a main raw material, applies a two-stage hydrothermal method to simultaneously extract tannins, lignin polyphenols, oligosaccharides and organic acid from the bark, and prepares a natural antibiotic substitute product with antibacterial and probiotic effects by a simple and low-cost process.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for preparing a multi-element antibacterial probiotic agent from a single raw material comprises the following steps:
A. material pretreatment: selecting barks as raw materials, crushing the barks into slices or threads, continuously crushing the barks into particles after air drying and constant weight, and mixing the particles with iron ion-free water according to a mass ratio of 1: 4-20, uniformly mixing to obtain a first mixed material, and adjusting the pH value of the mixed material to 3-8;
B. low-temperature hydrothermal extraction: transferring the first mixed material into an extraction kettle, heating to 60-100 ℃ under a closed stirring condition, continuously acting for 1-12 hours, then carrying out solid-liquid separation to obtain a solid separation substance and a first supernatant containing a tannin extract, and concentrating the first supernatant by 5-10 times to obtain a concentrated tannin extract and condensed water;
C. mixing the condensed water and the solid-state separator according to a certain solid-liquid ratio, adding new iron-ion-free water according to needs to obtain a second mixed material with the final solid-liquid ratio of 1: 3-20, adjusting the pH value of the second mixed material to 4-5 by phosphoric acid, adding the second mixed material into an extraction kettle, heating to 100 ℃, deflating to no pressure, sealing, continuously heating to 120-150 ℃, preserving heat for 30-60 minutes, deflating, cooling to 20-80 ℃, and performing solid-liquid separation to obtain a liquid extract rich in lignin polyphenol, polysaccharide and oligosaccharide;
D. acid precipitation to remove acid-insoluble lignin: concentrating the liquid extract until the solid content is more than 80% to obtain a concentrated extract, adding sulfuric acid to adjust the pH value of the concentrated extract to 1.0-2.5, standing for 10-60 minutes, performing solid-liquid separation to remove precipitates, and collecting a second supernatant;
E. enzymolysis: adding xylanase into the second supernatant to decompose xylan to obtain an enzymolysis concentrated extract;
F. obtaining the ternary antibacterial probiotic: mixing the concentrated tannin extract and the enzymolysis concentrated extract according to a certain proportion to obtain a liquid ternary antibacterial probiotic; and drying the liquid ternary antibacterial probiotic to obtain the solid ternary antibacterial probiotic.
The bark is the main waste of the paper industry, but the main components of the bark comprise lignin, tannin, hemicellulose and cellulose, and the bark has wide sources and low price. The function of the existing chemical synthetic antibiotics cannot be completely replaced by one antibiotic substitute, the price of the commercialized tannin, lignin polyphenol or oligosaccharide is very expensive, the antibiotic cannot be replaced by a commercialized single-product mixed method, and the method is not feasible. The invention starts from bark, obtains the multi-element antibacterial probiotic agent which is rich in tannin, lignin polyphenol and oligosaccharide, can effectively replace the traditional antibiotics to play a role, and obviously reduces the production cost.
The tannin is a water-soluble phenolic compound with the molecular weight of 500-3000 Da. Mainly comprises hydrolyzed tannin (including tannic acid, gallic acid and ellagic acid) with gallic acid as basic constituent unit and condensed tannin (anthocyanidin, procyanidin) with flavanol as basic constituent unit. Researches find that the tannin has strong protein complexing ability, metal binding ability, oxidation resistance and bacteriostasis.
Lignin polyphenols are a hydrolyzed lignin, the basic raw material constituting lignin is monolignol, and the starting material for producing monolignol is the amino acid phenylalanine. The lignin polyphenol comprises phenolic acid (ferulic acid, syringic acid, etc.) and phenol such as phenol alcohol. It has multiple "strategic" defense functions, such as antioxidant, antibacterial, antitoxin, and antiviral effects.
Xylanases break down xylans to obtain oligosaccharides, which utilize the prebiotic effect of natural compounds, i.e. by adding nutrient components required for beneficial microbial growth to promote microbial growth. The oligosaccharide mainly comprises water-soluble polysaccharide and oligosaccharide, and the oligosaccharide is a compound consisting of 2-10 monosaccharides and mainly comprises xylooligosaccharide, lactooligosaccharide and the like. Although animals cannot directly utilize the large molecular oligosaccharides, microorganisms in the intestinal tract can utilize these oligosaccharides and produce short chain fatty acids such as acetic acid, propionic acid, and butyric acid. Thus, oligosaccharides can promote the growth of beneficial microorganisms and maintain a healthy intestinal environment. The xylo-oligosaccharide is mainly obtained by the process, and the prebiotic effect of the xylo-oligosaccharide is obviously stronger than that of other prebiotics such as lacto-oligosaccharide, fructo-oligosaccharide and the like.
The invention combines a low-temperature hydrothermal method, a high-temperature hydrothermal method and active enzyme catalytic decomposition, simultaneously obtains tannin, lignin polyphenol and oligosaccharide from a single material of bark at low cost, prepares the multi-element antibacterial probiotic agent, and integrates multiple functions of resisting bacteria and diminishing inflammation, protecting the liver of animals, promoting the growth of the animals and the like.
After the low-temperature hydrothermal treatment, the high-temperature hydrothermal extraction is carried out, and the treatment temperature and the treatment time in the high-temperature hydrothermal stage can be effectively reduced. And D, after the low-temperature hydrothermal extraction in the step B is finished, the natural structure of the bark is damaged to a certain degree, and the obtained solid isolate mainly comprises lignin, hemicellulose (mainly xylan), polyphenol and cellulose. At this point, the step C of high temperature hydrothermal process is performed, and since the natural structure of the bark is destroyed, a lower treatment temperature and a shorter treatment time can be used. After the high-temperature hydrothermal treatment in the step C, the hemicellulose and the lignin polyphenol are dissolved in a liquid phase, and acid-insoluble lignin and cellulose are contained in solid-phase residues.
Preferably, the method further comprises the following steps:
G. obtaining the quaternary antibacterial probiotic: mixing the liquid ternary antibacterial probiotic agent with a liquid acidifier according to any proportion to obtain a liquid quaternary antibacterial probiotic agent; mixing the liquid acidifier with a silicon dioxide carrier to obtain a solid acidifier, and mixing the solid ternary antibacterial probiotic agent with the solid acidifier in any proportion to obtain the solid quaternary antibacterial probiotic agent.
On the basis of the ternary antibacterial probiotic, acidic substances which can reduce the pH value of the environment, also called acidifying agents, such as organic acid and inorganic acid, are directly added to obtain the quaternary antibacterial probiotic. The growth rate of beneficial microorganisms in the intestinal tract is obviously higher than that of harmful microorganisms under the acidic condition, and the growth of the harmful microorganisms can be completely inhibited even under the acidic condition. Although beneficial microorganisms in the gut may also rely on their own production of short chain organic acids to lower the environmental pH, the mass production of beneficial microorganisms requires reliance on good initial nutrition and environmental conditions. Thus, in addition to providing xylooligosaccharides, it is also desirable to provide a stable acidic environment to facilitate the growth of beneficial microorganisms into a superior flora.
The product of the invention can inhibit harmful bacteria, provide nutrient substances for beneficial bacteria and provide a good growth environment, and can achieve the purposes of reducing the growth of harmful microorganisms, maintaining the health of organisms, reducing the morbidity, reducing the utilization of chemically synthesized antibiotics, reducing environmental pollution and keeping environmental safety.
Preferably, the liquid acidifying agent is a mixture of an organic acid and an inorganic acid mixed according to any proportion. The organic acid comprises one or more of formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid, L-lactic acid, fumaric acid, sorbic acid, tartaric acid, benzoic acid and malic acid; the inorganic acid comprises one or more of hydrochloric acid and phosphoric acid.
On one hand, the action speed of the inorganic acid is high, and the alkali in the feed can be neutralized quickly; on the other hand, the inorganic acid is beneficial to maintaining the non-ionization state of the organic acid, so that the organic acid is easily utilized by beneficial microorganisms, and the combination effect of the organic acid and the beneficial microorganisms is good.
Preferably, in the step A, the bark includes one or more of cortex myricae rubra, cortex Querci Acutissimae, cortex Castaneae, Ephedra sinica, cortex Querci Densiflorae, leaves of Rhus verniciflua, bark of spruce, bark of larch, bark of Chinese orange, bark of negundo chastetree, bark of acacia; in step E, the xylanase is an endo-1, 4-beta xylanase (enzyme No. 3.2.1.8).
Preferably, in the step E, the endo-1, 4-beta-xylanase comprises a glycosideOne or more of hydrolase GH10 family, glycoside hydrolase GH11 family; the ratio of the xylanase to the bark is 1 × 106IU total enzyme activity: 1X 102～1×105g。
Glycoside hydrolase GH10, GH11 families have good endonuclease activity, and can catalyze and decompose xylan well.
Preferably, in the step E, the enzymolysis reaction is carried out in an aqueous environment, the hydrolysis temperature is 20-90 ℃, the pH value is 3-8, and the hydrolysis time is 1-96 hours.
The xylanase is added in an amount effective to hydrolyze hemicellulose, e.g., per 1X 102To 1X 105Adding 1g xylanase per gram dry plant biomass material, or every 1 × 102To 1X 105Adding 1X 10 total enzyme activity into gram dry plant biomass material6IU xylanase.
Preferably, in the step B and the step C, the solid-liquid separation method comprises one or more of plate-and-frame filter press, belt filter press, spiral press and butterfly-type centrifugal separation; in the step B and the step D, the concentration method comprises one or more of a concentration tank, a multifunctional scraper concentration tank, a rotary film evaporator, a single-effect, multi-effect rising film type or falling film type evaporator, an external circulation one-effect, two-effect and three-effect evaporator and a forced circulation type evaporator.
Preferably, in the step F, the ratio of the concentrated tannin extract to the enzymatic hydrolysis concentrated extract is 10: 1-1: 10; the drying method comprises one or more of freeze drying, vacuum drying, hot air drying and spray drying.
A multi-element antibacterial probiotic agent prepared by the method for preparing the multi-element antibacterial probiotic agent by using single raw material.
The application of the multi-element antibacterial probiotic is characterized in that the multi-element antibacterial probiotic is applied to feed additives, water quality regulators and skin spraying agents of livestock and poultry and aquaculture animals.
The livestock and poultry comprise cattle, pigs, chickens, ducks, geese and the like; the aquatic animals include shrimp, fish, crab, amphibian bullfrog, American frog, tortoise, and turtle.
In the application of the multi-element antibacterial probiotic composition as a feed additive, the acidifier in the multi-element antibacterial probiotic composition can maintain the intestinal health of animals and maintain a stable pH environment, phenols, organic acids and tannins can directly inhibit the growth of harmful microorganisms, and oligosaccharides can promote the proliferation of beneficial microorganisms. The multi-component synergistic interaction can achieve the effects of inhibiting bacteria and promoting growth, and is suitable for feed additives of livestock, poultry and aquatic animals.
In the application as a water quality regulator, the acidifying agent in the components of the multi-element antibacterial probiotic can regulate the pH value of pool water, and polyphenol and tannin can directly inhibit the proliferation of harmful microorganisms in the pool water; the tannin can also be used as a flocculating agent to precipitate suspended particles and inhibit floating of bottom mud floating pathogenic bacteria, so that the number of harmful microorganisms in water and feed residues are reduced; the xylo-oligosaccharide can be used as a carbon source of beneficial microorganisms in a water body, promotes the propagation of the beneficial microorganisms, and is suitable for water quality regulation of aquaculture animals.
In the application as a skin spray agent, the multi-element antibacterial probiotic agent has remarkable effect on protecting and treating the skin and skin diseases of amphibian animals, particularly economic animals such as bullfrogs, American frogs, turtles and the like. The skin of amphibians is an important immune and respiratory organ, which is very important for animal growth, however skin diseases such as putrid skin disease often occur due to high-density breeding. The multi-element antibacterial probiotic can be directly sprayed or coated on the skin ulceration for local treatment, and has the effects of inhibiting bacteria, drying, astringing skin and promoting rapid healing of the skin. In addition, for the individuals with intact skin, the multi-element antibacterial probiotic also can play a role in maintaining the health of skin microbiome, so that the damage of germs is prevented, and the skin health-care composition is suitable for skin health care and disease treatment of amphibian aquatic animals.
Compared with the prior art, the implementation of the invention has the following beneficial effects:
1. the probiotic-antibacterial agent production process developed by the invention innovatively combines a low-temperature hydrothermal method and a high-temperature hydrothermal method to treat the bark, extracts tannin substances and lignin polyphenol in the bark at the same time with low cost, and fully utilizes the antibacterial potential of the bark component. In addition, the probiotic-antibacterial agent can protect the liver of animals and promote the growth of the animals besides resisting bacteria and diminishing inflammation.
2. The invention utilizes xylanase to enzymolyze bark low-temperature and high-temperature hydrothermal extraction liquid to hydrolyze xylan in the treatment liquid into prebiotics-xylooligosaccharide which can promote the growth of beneficial microorganisms in intestinal tracts.
3. The quaternary antibacterial probiotic prepared by the invention contains the acidifier, so that the pH value of animal intestinal tracts, skins and aquaculture water can be reduced, an acidic environment is maintained, beneficial microorganisms are promoted, harmful microorganisms are inhibited from growing, a healthy microbial flora structure is maintained, and the disease incidence of the aquaculture animals and the use of antibiotics can be reduced.
4. The antibacterial probiotic agent prepared by the invention combines the advantages of tannin, lignophenol, xylo-oligosaccharide and acidifier, maintains the healthy microbial community structure of intestinal tracts, skins and water bodies of cultured animals by directly inhibiting the growth of harmful microorganisms, providing a growth carbon source for beneficial microorganisms and creating a mechanism which is beneficial to the growth of the beneficial microorganisms, thereby improving the disease resistance of the animals, reducing the disease incidence and the usage amount of antibiotics, finally producing healthy animal products and reducing the environmental pollution.
Drawings
FIG. 1 is a process flow diagram of the present invention;
fig. 2 is a bar graph showing the effect example 1 of the invention that the ternary antibacterial probiotic and the amoxicillin for animals inhibit the growth of microorganisms in the water of a frog pond, wherein AMCL is the amoxicillin for animals, and TE is the ternary antibacterial probiotic;
FIG. 3 is a graph showing the healing effect of skin of Rana temporaria chensinensis David after the treatment in the experimental group (example 1);
FIG. 4 is a graph showing the healing effect of skin of 2 nd day frog in the blank group.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Example 1
As shown in figure 1, air-dried constant-weight Marina abrus bark particles and water are mixed according to a ratio of 1:6, poured into a high-pressure hydrothermal reaction kettle, covered with a reaction kettle cover, connected with a vacuum pump through an air outlet above the liquid level, and vacuumized for 5 minutes to remove oxygen. Then, the outlets of the reaction kettle are sealed, the temperature is increased to 80 ℃, and then low-temperature hydrothermal pretreatment is started. The hydrothermal pretreatment condition is that the temperature is 80 ℃, the treatment time is 12 hours, the stirring speed is 30 r/min, and the initial pH value is 7. After the treatment is finished, the temperature is quickly reduced to 40 ℃, after solid-liquid separation, the liquid phase is concentrated by a triple-effect evaporator to obtain concentrated tannin mixed liquor, the solid phase part and condensed water obtained by triple-effect evaporation are mixed according to the proportion of 1:4, the mixture is poured into a high-pressure hydrothermal reaction kettle, a reaction kettle cover is covered, a vacuum pump is connected with an air outlet above the liquid level, and the vacuum pump is vacuumized for 5 minutes to remove oxygen. Then, the outlets of the reaction kettle are sealed, the temperature is increased to 100 ℃, and the high-temperature hydrothermal pretreatment is started after the cover is opened and the gas is released. The hydrothermal pretreatment condition is that the temperature is 150 ℃, the treatment time is 30 minutes, the stirring speed is 30 r/min, the pH value is adjusted to 5 by phosphoric acid, the temperature is rapidly reduced to 80 ℃ after the treatment is finished, after solid-liquid separation, the liquid phase is concentrated by a triple-effect evaporator to obtain the concentrated high-temperature hydrothermal extraction liquid rich in lignin polyphenol and polysaccharide. Adjusting pH of concentrated high temperature hydrothermal extract to 5.0 and temperature to 60 deg.C, and adjusting the temperature to 1 × 104Air-dried constant-weight 1 x 10 bark of acacia6Adding xylanase into IU, stirring at constant temperature for enzymolysis for 12 hours, fully mixing the concentrated high-temperature hydrothermal extract after enzymolysis with the concentrated tannin mixed solution to obtain a liquid product A rich in tannin, lignin polyphenol and xylo-oligosaccharide, and spray-drying the product A to obtain a solid product B. Mixing the liquid product A with a liquid acidulant containing formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid and L-lactic acid according to a volume ratio of 10:1 to prepare a product C; and mixing the solid product B with a solid acidifying agent containing formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid and L-lactic acid according to the mass ratio of 5:1 to prepare a solid product D.
Example 2
As shown in figure 1, air-dried cortex Myricae Rubrae with equal mass ratio and constant weight is preparedMixing the mixture of oak bark, chestnut bark and casuarina equisetifolia bark with water according to the mass ratio of 1:4, pouring the mixture into a high-pressure hydrothermal reaction kettle, covering a reaction kettle cover, connecting a vacuum pump through an air outlet above the liquid level, and vacuumizing for 5 minutes to remove oxygen. Then, the outlets of the reaction kettle are sealed, heated to 70 ℃, and then low-temperature hydrothermal pretreatment is started. The hydrothermal pretreatment condition is 70 ℃, the treatment time is 18 hours, the stirring speed is 50 r/min, and the initial pH value is 6. After the treatment is finished, the temperature is rapidly reduced to 50 ℃, after solid-liquid separation, the liquid phase is concentrated by a triple-effect evaporator to obtain concentrated tannin mixed liquid, the solid phase part and condensed water obtained by triple-effect evaporation are mixed according to the proportion of 1:5, the mixture is poured into a high-pressure hydrothermal reaction kettle, a reaction kettle cover is covered, a vacuum pump is connected with an air outlet above the liquid level, and the vacuum pump is vacuumized for 5 minutes to remove oxygen. Then, the outlets of the reaction kettle are sealed, the temperature is raised to 100 ℃, and after the cover is opened and the air is released, the high-temperature hydrothermal pretreatment is started. The hydrothermal pretreatment condition is that the temperature is 120 ℃, the treatment time is 60 minutes, the stirring speed is 50 r/min, and the pH value is adjusted to 5 by phosphoric acid. After the treatment is finished, the temperature is rapidly reduced to 70 ℃, after solid-liquid separation, the liquid phase is concentrated by a triple-effect evaporator to obtain a concentrated high-temperature hydrothermal extraction liquid rich in lignin polyphenol and polysaccharide. Adjusting pH of the concentrated hot water extractive solution to 5.5 and temperature to 55 deg.C, and adjusting the temperature to 1 × 105Bark of Keshu 5X 106Adding xylanase into IU, stirring at constant temperature for enzymolysis for 8 hours, fully mixing the concentrated high-temperature hydrothermal extract after enzymolysis with the concentrated tannin mixed solution to obtain a liquid product A rich in tannin, lignin polyphenol and xylo-oligosaccharide, and spray-drying the product A to obtain a solid product B. Mixing the liquid product A with a liquid acidulant containing succinic acid, citric acid, L-lactic acid, fumaric acid and sorbic acid at a volume ratio of 15:1 to prepare a product C; and mixing the solid product B with a solid acidulant containing succinic acid, citric acid, L-lactic acid, fumaric acid and sorbic acid according to the enzyme ratio of 6:1 to prepare a solid product D.
Example 3
As shown in figure 1, air-dried constant weight oak bark, sumac leaf, spruce bark, larch bark, mandarin orange bark and black wattle bark in equal mass ratio are mixedMixing the mixture and water according to the mass ratio of 1:5, pouring the mixture into a high-pressure hydrothermal reaction kettle, covering a reaction kettle cover, connecting a vacuum pump through an air outlet above the liquid level, vacuumizing for 4 minutes to remove oxygen, then sealing each outlet of the reaction kettle, heating to 80 ℃, and then starting low-temperature hydrothermal pretreatment. The hydrothermal pretreatment condition is that the temperature is 80 ℃, the treatment time is 24 hours, the stirring speed is 40 r/min, and the initial pH value is 7. After the treatment is finished, the temperature is quickly reduced to 50 ℃, after solid-liquid separation, the liquid phase is concentrated by a triple-effect evaporator to obtain a concentrated tannin mixed solution, the solid phase part and condensed water obtained by triple-effect evaporation are mixed according to the proportion of 1:6, the mixture is poured into a high-pressure hydrothermal reaction kettle, a reaction kettle cover is covered, a vacuum pump is connected with an air outlet above the liquid level, and the vacuum pump is vacuumized for 5 minutes to remove oxygen. Then, the outlets of the reaction kettle are sealed, the temperature is raised to 100 ℃, the cover is opened, the air is released for 5 minutes, and then the high-temperature hydrothermal pretreatment is started. The hydrothermal pretreatment condition is that the temperature is 130 ℃, the treatment time is 45 minutes, the stirring speed is 60 r/min, and the pH value is adjusted to 4 by phosphoric acid. After the treatment is finished, the temperature is rapidly reduced to 70 ℃, after solid-liquid separation, the liquid phase is concentrated by a triple-effect evaporator to obtain a concentrated high-temperature hydrothermal extraction liquid rich in lignin polyphenol and polysaccharide. Adjusting pH of concentrated high temperature hydrothermal extract to 5 and 65 deg.C, and adjusting the temperature to 1 × 102Bark of Keshu 1X 106Adding xylanase into IU, stirring at constant temperature for enzymolysis for 12 hours, fully mixing the concentrated high-temperature hydrothermal extract after enzymolysis with the concentrated tannin mixed solution to obtain a liquid product A rich in tannin, lignin polyphenol and xylo-oligosaccharide, and spray-drying the product A to obtain a solid product B. Mixing the liquid product A with a liquid acidulant containing L-lactic acid and malic acid at a volume ratio of 15:1 to prepare a product C; and mixing the solid product B with a solid acidulant containing L-lactic acid and malic acid according to the enzyme ratio of 6:1 by mass to prepare a solid product D.
Comparative example 1
Mixing air-dried constant-weight Marina abrus bark particles with water in a ratio of 1:6, pouring into a high-pressure hydrothermal reaction kettle, covering a reaction kettle cover, connecting a vacuum pump through an air outlet above the liquid level, and vacuumizing for 5 minutes to remove oxygen. Then pouring the mixture into a high-pressure hydrothermal reaction kettle, and covering the reaction kettle coverAnd the air outlet above the liquid level is connected with a vacuum pump, and the vacuum pump is vacuumized for 5 minutes to remove oxygen. Then, the outlets of the reaction kettle are sealed, the temperature is raised to 100 ℃, and after the cover is opened and the gas is released, the high-temperature hydrothermal pretreatment is started. The hydrothermal pretreatment condition is that the temperature is 150 ℃, the treatment time is 30 minutes, the stirring speed is 30 r/min, and the pH value is adjusted to 5 by phosphoric acid. After the treatment is finished, the temperature is rapidly reduced to 80 ℃, after solid-liquid separation, the liquid phase is concentrated by a triple-effect evaporator to obtain concentrated high-temperature hydrothermal extraction liquid rich in lignin polyphenol and polysaccharide. Adjusting pH of concentrated high temperature hydrothermal extract to 5.0 and temperature to 60 deg.C, and adjusting the temperature to 1 × 104Air-dried constant-weight 1 x 10 bark of acacia6Adding xylanase into IU dosage, stirring at constant temperature for enzymolysis for 12 hours to obtain a liquid product A, and spray drying the product A to obtain a solid product B. Mixing the liquid product A with a liquid acidulant containing formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid and L-lactic acid according to a volume ratio of 10:1 to prepare a product C; and mixing the solid product B with a solid acidifier containing formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid and L-lactic acid according to an enzyme ratio of 5:1 by mass to prepare a solid product D.
Comparative example 2
Mixing air-dried constant-weight Marina abrus bark particles with water in a ratio of 1:6, pouring into a high-pressure hydrothermal reaction kettle, covering a reaction kettle cover, connecting a vacuum pump through an air outlet above the liquid level, and vacuumizing for 5 minutes to remove oxygen. Then, the outlets of the reaction kettle are sealed, heated to 80 ℃, and then low-temperature hydrothermal pretreatment is started. The hydrothermal pretreatment condition is that the temperature is 80 ℃, the treatment time is 12 hours, the stirring speed is 30 r/min, and the initial pH value is 7. Rapidly cooling to 40 deg.C after treatment, separating solid and liquid, concentrating the liquid phase with triple effect evaporator to obtain concentrated tannin mixed solution with pH value of 5.0 and temperature of 60 deg.C, and adding tannin at a ratio of 1 × 104Air-dried constant-weight 1 x 10 bark of acacia6Adding xylanase into IU dosage, stirring at constant temperature for enzymolysis for 12 hours to obtain a liquid product A, and spray drying the product A to obtain a solid product B. Mixing the liquid product A with a solution containing formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid and L-lactic acidMixing the state acidifying agents according to the volume ratio of 10:1 to prepare a product C; and mixing the solid product B with a solid acidifier containing formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid and L-lactic acid according to an enzyme ratio of 5:1 by mass to prepare a solid product D.
Comparative example 3
Mixing air-dried constant-weight Marina abrus bark particles with water in a ratio of 1:6, pouring into a high-pressure hydrothermal reaction kettle, covering a reaction kettle cover, connecting a vacuum pump through an air outlet above the liquid level, and vacuumizing for 5 minutes to remove oxygen. Then, the outlets of the reaction kettle are sealed, heated to 80 ℃, and then low-temperature hydrothermal pretreatment is started. The hydrothermal pretreatment condition is that the temperature is 80 ℃, the treatment time is 12 hours, the stirring speed is 30 r/min, and the initial pH value is 7. After the treatment is finished, the temperature is quickly reduced to 40 ℃, after solid-liquid separation, the liquid phase is concentrated by a triple-effect evaporator to obtain concentrated tannin mixed liquor, the solid phase part and condensed water obtained by triple-effect evaporation are mixed according to the proportion of 1:4, the mixture is poured into a high-pressure hydrothermal reaction kettle, a reaction kettle cover is covered, a vacuum pump is connected with an air outlet above the liquid level, and the vacuum pump is vacuumized for 5 minutes to remove oxygen. Then, the outlets of the reaction kettle are sealed, the temperature is raised to 100 ℃, and after the cover is opened and the air is released, the high-temperature hydrothermal pretreatment is started. The hydrothermal pretreatment condition is that the temperature is 150 ℃, the treatment time is 30 minutes, the stirring speed is 30 r/min, and the pH value is adjusted to 5 by phosphoric acid. After the treatment is finished, the temperature is rapidly reduced to 80 ℃, after solid-liquid separation, the liquid phase is concentrated by a triple-effect evaporator to obtain a concentrated high-temperature hydrothermal extraction liquid rich in lignin polyphenol and polysaccharide. And fully mixing the concentrated high-temperature hydrothermal extraction liquid with the concentrated tannin mixed liquid to obtain a liquid product A, and spray-drying the product A to obtain a solid product B. Mixing the liquid product A with a liquid acidulant containing formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid and L-lactic acid according to a volume ratio of 10:1 to prepare a product C; and mixing the solid product B with a solid acidifier containing formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid and L-lactic acid according to an enzyme ratio of 5:1 by mass to prepare a solid product D.
Effect example 1
Taking bullfrog culture pond water with the incidence rate of the wry head disease being up to 30%, inoculating the water into an YPD culture medium, respectively adding veterinary amoxicillin (the final concentration is 0.1 and 0.2g/L) and ternary antibacterial probiotic (the final concentration is 0.1 and 0.5g/L) into an aerobic culture bottle, taking a blank as a control group, measuring the cell optical density (OD700) at 12, 24 and 48 hours respectively under the culture conditions of 27 ℃ and 300rpm of a shaking table, taking OD700 of the control group as a standard, and measuring the growth inhibition rate, wherein the test results are shown in Table 1.
TABLE 1 comparison of the effect of the ternary antibacterial probiotic agent and amoxicillin for veterinary use in inhibiting the growth of microorganisms in the water of the frog pond (inhibition rate,%)
The table is drawn into a bar chart, as shown in figure 2, the result shows that 0.1g/L of ternary antibacterial prebiotics can replace 0.2g/L of amoxicillin for animals.
Effect example 2
Taking bullfrog culture pond water with the incidence rate of the wry head disease being up to 30%, inoculating the water in a YPD culture medium, adding veterinary amoxicillin (the final concentration is 0.1 and 0.2g/L) and quaternary antibacterial probiotic (the final concentration is 0.1 and 0.5g/L) into an aerobic culture bottle respectively, taking a blank as a control group, measuring the cell optical density (OD700) at 12, 24 and 48 hours respectively under the culture conditions of 27 ℃ and 300rpm of a shaking table, measuring the growth inhibition rate by taking the OD700 of the control group as a standard, and measuring results as shown in Table 2.
Table 2. quaternary antibacterial probiotic agent compared with veterinary amoxicillin in inhibiting the growth of microorganisms in the water of cow frog's pool (inhibition rate,%)
The results show that the effect of the 0.1g/L quaternary antibacterial prebiotics is basically better than that of the 0.2g/L veterinary amoxicillin.
Effect example 3
Taking 40 bullfrogs which have ulcerated skin on the head and the back and the weight of about 100g, averagely dividing into 2 groups, respectively culturing in a white 300L culturing barrel, and adding tap water to the bottom of the barrel, wherein the water is preferably used for submerging the abdomen of the bullfrogs. The experimental group was coated with the solid product D obtained in examples 1 to 3 every day, the control group was coated with the solid product D obtained in comparative examples 1 to 3, respectively, and the blank group was not coated with any drug. Food was taken twice daily and mortality and skin healing status were recorded. The test results are shown in fig. 3, fig. 4 and table 3.
TABLE 3 Effect of Quaternary antimicrobial Probiotics as skin spray
The results show that the quaternary antibacterial probiotic agent obtained by the process can well treat skin wounds of amphibian economic animals such as bullfrogs and the like after being applied, and the survival rate of the bullfrogs is improved. In contrast, comparative example 1, which lacks the extraction of tannin components, showed a significant decrease in both cure rate and survival rate, indicating that tannin components play an important role in the skin healing process. The comparative examples 2 and 3 lack the steps of extracting lignin polyphenol and obtaining oligosaccharide respectively, the cure rate and the survival rate are reduced to a certain degree, and the results show that the lignin polyphenol and the oligosaccharide play a role in synergistic enhancement in the skin healing process.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the appended claims are to be accorded the full scope of the invention.
Claims (10)
1. A method for preparing a multi-element antibacterial probiotic agent from a single raw material is characterized by comprising the following steps:
A. material pretreatment: selecting barks as raw materials, crushing the barks into slices or threads, continuously crushing the barks into particles after air drying and constant weight, and mixing the particles with iron ion-free water according to a mass ratio of 1: 4-20, uniformly mixing to obtain a first mixed material, and adjusting the pH value of the mixed material to 3-8;
B. low-temperature hydrothermal extraction: transferring the first mixed material into an extraction kettle, heating to 60-100 ℃ under a closed stirring condition, continuously acting for 1-12 hours, then carrying out solid-liquid separation to obtain a solid separation substance and a first supernatant containing a tannin extract, and concentrating the first supernatant by 5-10 times to obtain a concentrated tannin extract and condensed water;
C. mixing the condensed water and the solid-state separator according to a certain solid-liquid ratio, adding new iron-ion-free water according to needs to obtain a second mixed material with the final solid-liquid ratio of 1: 3-20, adjusting the pH value of the second mixed material to 4-5 by using phosphoric acid, adding the second mixed material into an extraction kettle, heating to 100 ℃, deflating to no pressure, sealing, continuously heating to 120-150 ℃, preserving heat for 30-60 minutes, deflating, cooling to 20-80 ℃, and performing solid-liquid separation to obtain a liquid extract rich in lignin polyphenol, polysaccharide and oligosaccharide;
D. acid precipitation to remove acid-insoluble lignin: concentrating the liquid extract until the solid content is more than 80% to obtain a concentrated extract, adding sulfuric acid to adjust the pH value of the concentrated extract to 1.0-2.5, standing for 10-60 minutes, performing solid-liquid separation to remove precipitates, and collecting a second supernatant;
E. enzymolysis: adding xylanase into the second supernatant to decompose xylan to obtain an enzymolysis concentrated extract;
F. obtaining the ternary antibacterial probiotic: mixing the concentrated tannin extract and the enzymolysis concentrated extract according to a certain proportion to obtain a liquid ternary antibacterial probiotic; and drying the liquid ternary antibacterial probiotic to obtain the solid ternary antibacterial probiotic.
2. The method for preparing the multi-element antibacterial and probiotic agent from a single raw material according to claim 1, characterized by further comprising the following steps:
G. obtaining the quaternary antibacterial probiotic: mixing the liquid ternary antibacterial probiotic with a liquid acidifier according to any proportion to obtain a liquid quaternary antibacterial probiotic; mixing the liquid acidifier with a silicon dioxide carrier to obtain a solid acidifier, and mixing the solid ternary antibacterial probiotic agent with the solid acidifier in any proportion to obtain the solid quaternary antibacterial probiotic agent.
3. The method for preparing the multi-element antibacterial and probiotic agent from a single raw material as claimed in claim 2, characterized in that the liquid acidifier is a mixture of organic acid and inorganic acid mixed according to any proportion; the organic acid comprises one or more of formic acid, acetic acid, propionic acid, butyric acid, succinic acid, citric acid, L-lactic acid, fumaric acid, sorbic acid, tartaric acid, benzoic acid and malic acid; the inorganic acid comprises one or more of hydrochloric acid and phosphoric acid.
4. The method for preparing multi-component antibacterial and probiotic agent from a single raw material as claimed in claim 1, wherein, in the step A, the bark includes one or more of cortex myricae, oak, chestnut, casuarina, oak, sumac leaves, spruce, larch, mandarin orange, negundo, acacia; in said step E, said xylanase is an endo-1, 4-beta xylanase.
5. The method for preparing the multi-element antibacterial probiotic agent from a single raw material according to claim 1, characterized in that in the step E, the endo-1, 4-beta xylanase comprises one or more of glycoside hydrolase GH10 family, glycoside hydrolase GH11 family; the ratio of the xylanase to the bark is 1 × 106IU total enzyme activity: 1X 102～1×105g。
6. The method for preparing the multi-element antibacterial probiotic agent from a single raw material according to claim 1, wherein in the step E, the enzymolysis reaction is carried out in an aqueous environment, the hydrolysis temperature is 20-90 ℃, the pH is 3-8, and the hydrolysis time is 1-96 hours.
7. The method for preparing the multi-element antibacterial probiotic agent from a single raw material according to claim 6, characterized in that in the step B and the step C, the solid-liquid separation method comprises one or more of plate-and-frame filter press, belt filter press, spiral press and butterfly centrifugal; in the step B and the step D, the concentration method comprises one or more of a concentration tank, a multifunctional scraper concentration tank, a rotary film evaporator, a single-effect, multi-effect rising film type or falling film type evaporator, an external circulation one-effect, two-effect and three-effect evaporator and a forced circulation type evaporator.
8. The method for preparing the multi-element antibacterial probiotic agent from a single raw material as claimed in claim 1, wherein in the step F, the ratio of the concentrated tannin extract to the enzymolysis concentrated extract is 10: 1-1: 10; the drying method comprises one or more of freeze drying, vacuum drying, hot air drying and spray drying.
9. A multi-antibacterial probiotic prepared by the process of preparing a multi-antibacterial probiotic from a single raw material according to any one of claims 1 or 2.
10. The use of the multi-element antibacterial probiotic according to claim 9, characterized in that it is applied to feed additives, water quality regulators and skin sprays for livestock, special farm animals and aquaculture animals.
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