CN116115662A - Fermentation method of chicory and poria cocos compound - Google Patents
Fermentation method of chicory and poria cocos compound Download PDFInfo
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- CN116115662A CN116115662A CN202211676169.XA CN202211676169A CN116115662A CN 116115662 A CN116115662 A CN 116115662A CN 202211676169 A CN202211676169 A CN 202211676169A CN 116115662 A CN116115662 A CN 116115662A
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- chicory
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- compound
- lactobacillus
- bifidobacterium
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Abstract
The invention relates to the technical field of biological fermentation, in particular to a fermentation method of chicory and poria cocos compounds. The method comprises the following steps: mixing herba Cichorii powder and Poria powder, and decocting twice to obtain extractive solution; adding a fermentation nutrient into the obtained extracting solution and filter residues, and adjusting the pH value to 7.0+/-0.5 to obtain a fermentation substrate; steam sterilizing the fermentation substrate to obtain a chicory compound sterilizing solution; cooling the chicory compound sterilizing liquid, inoculating bifidobacterium and lactobacillus pre-culture bacterial liquid for fermentation, and performing stabilization and inactivation treatment after fermentation to obtain a chicory compound fermentation liquid; filtering the chicory compound fermentation liquor through a screen to remove dregs, and collecting filtrate. The compound probiotics symbiotic fermentation is adopted, so that the traditional Chinese medicine and probiotics are mutually enhanced, the activity and the content of effective substances are increased, the utilization of the effective components of the traditional Chinese medicine is improved, and the bitter taste of the traditional Chinese medicine is lightened. Preventing and treating hyperuricemia and gout.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a fermentation method of chicory and poria cocos compounds.
Technical Field
Uric acid (uric acid) is a final product of purine metabolism in the human body, and excessive production and excretion disorders thereof easily cause hyperuricemia, and further cause gout to affect quality of life. Epidemiological investigation shows that hyperuricemia is often complicated with diseases such as hypertension, coronary heart disease, arteriosclerosis and the like, and simultaneously causes damage to human organ tissues such as kidneys, livers, joints and the like. At present, the treatment of the diseases is mainly carried out in terms of promoting uric acid excretion or controlling uric acid generation or improving inflammatory pain and the like, and traditional medicines such as allopurinol, probenecid, benzbromarone, nonsteroidal anti-inflammatory drugs and the like are mainly used as common means for treating hyperuricemia clinically, but the problems of anaphylactic reaction, toxic and side effects and the like caused by taking the medicines affect the wide use of the medicines. Meanwhile, the therapeutic drugs are also biased, and the therapeutic drugs are used for treating hyperuricemia, uric acid lowering, uric acid generating enzyme inhibiting and acute inflammation onset anti-inflammatory and analgesic only. The single and divided medicine intervention treatment strategy is difficult to be matched with the progressive pathological characteristics of the disease; it is also difficult to block the repetition of the course of the disease. Besides the use of drugs, the intake of purine nucleosides is often reduced by controlling diet, thereby achieving the purpose of preventing hyperuricemia. However, patients may have long caused nutritional imbalance, affecting their daily lives, and thus limiting purine intake is very difficult.
To date, a safe and effective whole-course intervention method capable of controlling hyperuricemia and gout in a long term is not available. Therefore, it is an urgent need for patients to find a treatment that is efficient, accurate and free of side effects.
The traditional Chinese medicine considers that hyperuricemia and gout belong to the categories of consumptive disease, joint, arthromyodynia and the like. The disease is the principal deficiency and excessive syndrome, the spleen and kidney deficiency is the principal, and the phlegm stasis and damp turbidity are the principal. The screened medicinal materials mainly have the functions of strengthening spleen and tonifying kidney, promoting diuresis and expelling turbid urine, conditioning spleen and kidney and restoring transportation and transforming water dampness.
Under the guidance of traditional Chinese medicine theory, the compound dosage form is formed by adopting the principle of combining disease discrimination and syndrome differentiation, the advantages of naturalness, multifunction, safety, environmental protection and the like of the Chinese herbal medicines are fully exerted, and the compound dosage form is developed and developed into safe and effective food which can bring good news to gout patients caused by high uric acid. However, the traditional Chinese medicine decoction has the defects of poor taste, bitter taste, difficult swallowing of patients and influence on treatment effect.
In recent years, the probiotic biological fermentation technology is continuously introduced into the application of traditional Chinese medicine processing, the traditional Chinese medicine fermentation can achieve the effects of attenuation and synergism, the probiotic fermentation can produce unique flavor to improve the bitter taste of the traditional Chinese medicine, the probiotic can produce a plurality of extracellular enzymes in the metabolic process, the extracellular enzymes can degrade components such as cellulose and the like, the structure of plant cell walls is damaged, the curative effect of the traditional Chinese medicine is indirectly improved, and meanwhile, the probiotic metabolites also have the specific effect, and the probiotic metabolites complement each other with the traditional Chinese medicine components, so that the traditional Chinese medicine preparation has multiple purposes.
Therefore, developing a traditional Chinese medicine probiotics fermentation product has important application value; in particular to the 'integrated theory' for treating gout and hyperuricemia with multiple targets and multiple ways by multiple components obtained from the traditional Chinese medicine probiotics fermentation product.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a fermentation method of a chicory and poria cocos compound, which comprises the following steps:
the technical scheme that this application adopted is: a fermentation method of chicory and Poria compound,
step 1): mixing 20-50 parts of chicory powder and 50-80 parts of poria cocos powder according to parts by weight, and decocting twice after mixing to obtain an extracting solution;
step 2): adding a fermentation nutrient into the extracting solution and the filter residue obtained in the step 1), and adjusting the pH to 7.0+/-0.5 to obtain a fermentation substrate;
step 3): steam sterilizing the fermentation substrate to obtain a chicory compound sterilizing solution;
step 4): cooling the chicory compound sterilizing liquid in the step 3), inoculating bifidobacterium and lactobacillus pre-culture bacterial liquid for fermentation, and performing stabilization and inactivation treatment after fermentation is completed to obtain a chicory compound fermentation liquid;
step 5): filtering the chicory compound fermentation liquor through a screen to remove dregs, and collecting filtrate.
The Chinese medicinal material chicory screened in the invention is medicinal and edible Chinese medicinal material, and has the effects of clearing liver and promoting bile flow, invigorating stomach and promoting digestion, and inducing diuresis and reducing edema. In the uric acid reducing effect of the 38 Chinese herbal medicines, the chicory has the highest inhibition rate on xanthine oxidase; meanwhile, 187 compounds which can act on gout target proteins are screened out from 253 components of chicory, wherein more than 60 percent of the compounds can act on more than two targets, which proves that the chicory can realize the full intervention of various links of inhibiting uric acid generation, promoting uric acid excretion, preventing inflammation recurrence and improving pain when reversing the deposition of uric acid salt and gout attack through the separate or synergistic effect of partial small molecular compounds. Poria is also medicinal and edible Chinese medicinal material, and has effects of promoting diuresis, eliminating dampness, invigorating spleen, and calming heart, and the compatibility of the two materials can be synergistic.
Further, in the step (1), the chicory and the poria cocos are crushed and pass through a 20-60-mesh sieve; mixing 20-50 parts of chicory powder and 50-80 parts of poria cocos powder for 10-40 min to obtain mixed powder;
adding water 10-20 times of the mixed powder, soaking for 1.0 hour, decocting, keeping boiling for 1-3 hours after boiling, and separating out decoction; adding water 8-15 times of the mixed powder, decocting, keeping micro boiling for 1-3 hours after boiling, and separating out decoction again; mixing the decoctions, adding water to constant volume, and mixing the powder and the constant volume decoction at a mass volume ratio of 0.75g/mL.
Further, the addition amount of each component in the fermentation nutrient in the step (2) is as follows: glucose 5-15 g/L, yeast extract 1-4 g/L, yeast powder 0.5-1.5 g/L, peptone 3-10 g/L, nutrient addition iron 3-8 mg/L, zinc 15-25 mg/L, selenium 0.05-0.2 mg/L, magnesium 120-180 mg/L, manganese 3-8 mg/L and potassium 1.0-1.5 g/L.
Further, adding a sweetener before adjusting the pH in the step (2), wherein the sweetener is isomaltooligosaccharide and/or sucralose; the addition amount is 10-80 g/L of isomaltooligosaccharide and 0.01-0.1 g/L of sucralose.
Further, in the step (3), the fermentation substrate is kept at the temperature of 100-115 ℃ for 30-60 min, so as to obtain the chicory compound sterilizing liquid.
Further, the bifidobacterium in the step (4) is selected from one or two of bifidobacterium breve (Bifidobacterium breve), bifidobacterium bifidum (Bifidobacterium bifidum) and bifidobacterium adolescentis (Bifidobacterium adolescentis);
the lactobacillus includes lactobacillus plantarum (Lactobacillus plantarum) and lactobacillus casei (Lactobacillus casei);
and at least one of lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus grignard (Lactobacillus gasseri), lactobacillus delbrueckii subsp bulgaricus (Lactobacillus delbrueckii subsp.
The inoculation amount of the bifidobacterium pre-culture bacterial liquid is 0.1-1.0% of the volume percentage of the chicory compound sterilizing liquid; the inoculation amount of the lactobacillus pre-culture bacterial liquid is 0.2-4% of the volume percentage of the chicory compound sterilizing liquid;
the volume ratio of the bifidobacterium pre-culture bacterial liquid to the lactobacillus pre-culture bacterial liquid is 1:2 to 4; the viable count of the bifidobacterium pre-culture bacterial liquid and the lactobacillus pre-culture bacterial liquid is 2 multiplied by 10 7 CFU·mL -1 。
The screened probiotics are various lactobacillus and bifidobacterium, and have the effect of reducing uric acid through researches; meanwhile, the metabolites of amino acids, biotin, trace elements and the like generated in the process of fermenting the traditional Chinese medicine by probiotics can be reserved in the product, so that richer nutritional support is provided for patients. The preparation method combines a plurality of lactobacillus and bifidobacterium, adopts the symbiotic fermentation of the composite probiotics to obtain the active ingredients of the chicory compound, aims at enhancing the effect of the probiotics on the bioconversion of the chicory, plays a role of multi-strain synergism, enhances the effects of the composite probiotics chicory fermentation product on improving gout and reducing uric acid, and improves the clinical application effect of the composite probiotics chicory fermentation product.
Further, the fermentation temperature in the step (4) is 25-40 ℃, and the total bacterial count of the bifidobacterium and the lactobacillus is kept to be not less than 1.5x10 8 cfu/ml, fermenting for 25-48 h, stopping fermentation when the pH reaches 3.5-4.1; and then sterilizing for 10-40 min at 95-100 ℃ to obtain the chicory compound fermentation liquor.
Further, in the step (5), the chicory compound fermentation liquor is filtered by a screen to remove dregs, and then filtrate is collected and the dregs are washed by water; filtering the water washing liquid again through a screen, collecting filtrate, combining the two filtrates, centrifugally separating, collecting supernatant to obtain clarified chicory compound fermentation filtrate, and filling to obtain a finished product.
Further, the chicory compound fermentation liquor and the water washing liquor in the step (5) are sequentially screened by a sieve with 100-200 meshes and a sieve with 200-400 meshes.
Further, the rotational speed during the centrifugal separation is 6000-10000rpm/min.
The invention has the advantages that:
1. the fermented chicory and poria cocos compound can strengthen spleen and kidney, promote diuresis and remove turbid urine, achieve the effects of intercepting hyperuricemia and twisting urate deposition and inhibiting acute inflammation, and integrally improve the whole progress process of gout and hyperuricemia.
2. The chicory and poria cocos decoction pieces are crushed, then are subjected to water decoction extraction and then are fermented, and compared with a method for directly fermenting without water decoction after crushing, the extracted fermentation liquor has more dissolved components, and the fermentation strain can effectively ferment and convert traditional Chinese medicine components.
3. The chicory extract is combined with the dregs and fermented, the dregs contain a plurality of nutrient components and active substances, such as acidic polysaccharide which is discarded as the dregs in clinic, and the chicory extract also has the functions of strengthening spleen, promoting diuresis, removing dampness, calming the heart and obviously regulating the level of immune factors, and is a good choice; meanwhile, the dregs can be used as carbon source and nitrogen source of fermentation liquid, and the effective components in chicory and poria cocos can be obtained more comprehensively.
4. And the chicory compound extract is combined with the dregs to be fermented simultaneously, so that various enzymes generated in the fermentation process can degrade the dregs, and meanwhile, the amount of the dregs is reduced by nearly half, so that the chicory compound extract is environment-friendly and economical.
5. The probiotics of various specific types are applied to the fermentation liquor of the chicory compound, and various extracellular enzymes generated in the fermentation process of the probiotics can crack the cell walls of medicinal plants, reduce the wrapping effect of polysaccharide and the like on effective components, separate out more alkaloids, flavone, glycoside and the like, and degrade macromolecular effective active substances which cannot be directly absorbed and utilized by human bodies into small molecules through the fermentation of the probiotics. Greatly improves the utilization rate and the physiological activity of the traditional Chinese medicine components.
5. The chicory compound selects medicinal and edible traditional Chinese medicinal materials, and the probiotics strains are all from the update bulletins of the strain list for food and the strain list for infant food issued by the ministry of health and the ministry of health of 2022, and can be applied to food. In the invention, other auxiliary materials are added, and food raw materials or additives are selected, so that the safety is ensured.
6. The preparation method combines various lactobacillus and bifidobacteria, adopts compound probiotics for symbiotic fermentation, can directionally prevent invasion and colonization of pathogenic bacteria to intestinal tracts after different probiotics are eaten, inhibits pathogenic bacteria from resisting infection, maintains microecological balance of intestinal tracts, prevents and inhibits tumor generation, enhances organism immunity, promotes metabolism and digestion, synthesizes amino acid and vitamin, reduces cholesterol, reduces blood sugar, inhibits production of endotoxin, promotes uric acid excretion, delays aging and radiation and the like. Therefore, the application of symbiotic fermentation of various lactobacillus and bifidobacterium in combination to chicory complex fermentation is more beneficial to human body.
7. The fermentation of the symbiotic application of a plurality of probiotics to chicory liquid has a unique flavor compared with the simple bifidobacterium or lactobacillus, so that the unpleasant bitter taste of chicory compound extract is improved, and the compliance of patients taking the product is improved.
8. The process is simple and easy to operate, and the production time is short.
Drawings
FIG. 1 shows serum uric acid levels of rats in each group according to the experimental example of the invention;
FIG. 2 shows serum xanthine oxidase levels of rats in each group according to the experimental example of the present invention;
FIG. 3 shows serum creatinine levels in groups of rats according to the experimental examples of the present invention;
FIG. 4 shows serum urea nitrogen content of rats in each group according to the experimental example of the present invention;
FIG. 5 shows kidney coefficients of rats in each group according to the experimental example of the present invention;
FIGS. 6-8 show the amounts of serum inflammatory factors IL-1. Beta., IL-6, TNF-alpha. In the rats of each group according to the experimental example of the present invention;
FIGS. 9-11 show joint swelling, gait score and joint temperature change after modeling for gouty arthritis in groups of rats according to the experimental examples of the present invention;
FIG. 12 shows the effect of each group of experimental examples of the present invention on the expression of URAT1 protein in kidney of hyperuricemia rats;
FIG. 13 shows the effect of each group of experimental examples of the present invention on GLUT9 protein expression in the kidney of hyperuricemia rats;
FIG. 14 shows the effect of each group of experimental examples of the present invention on OAT1 protein expression in the kidney of hyperuricemia rats.
Detailed Description
The invention is further described below in connection with the following detailed description.
The bacterial strain adopted by each embodiment of the invention is derived from China Center for Type Culture Collection (CCTCC) (China, wuhan) or China general microbiological culture collection center (CGMCC) (China, beijing) or is separated from feces or intestinal mucosa tissue of normal people.
Example 1: the fermentation method of the chicory and poria cocos compound comprises the following steps:
(1) Raw material extraction pretreatment: pulverizing and pulverizing 100kg of herba Cichorii decoction pieces and 200kg of Poria decoction pieces, sieving with 40 mesh sieve, respectively accurately weighing herba Cichorii powder and Rubi fructus powder according to formula proportion, mixing for 20min to obtain herba Cichorii compound powder, decocting with water, soaking in 12 times of water for 1.0 hr, decocting, boiling for 2 hr, and separating decoction; adding 10 times of water of chicory compound powder, decocting, keeping boiling for 2 hours, and separating decoction again; mixing decoctions, adding water to constant volume, mixing with the mixture powder at a mass/volume ratio of 0.75g/mL, and mixing the residue decoctions to obtain herba Cichorii compound extract.
(2) And (3) batching: glucose 6kg, yeast extract 1.2kg, yeast powder 0.6kg and peptone 4kg are added into the chicory complex extract. The nutrient compounds are added as follows: ferrous sulfate 7H 2 O8 g, zinc sulfate.7H 2 O35g, sodium selenite.5H 2 O190 mg, magnesium sulfate.7H 2 O560 g, manganese sulfate H 2 O4 g, monopotassium phosphate 800g and dipotassium phosphate 3H 2 O800 g, constant volume to 2000L. And regulating the pH value to 7.0 to obtain the chicory complex probiotics fermented traditional Chinese medicine substrate. Before the pH is adjusted, 40kg of sweetener isomaltooligosaccharide and 50g of sucralose are added.
(3) And (3) sterilization: and (3) preserving the temperature of the chicory compound probiotic fermented traditional Chinese medicine substrate at 100-115 ℃ for 40min to obtain the chicory compound sterilizing liquid.
(4) Fermentation: when the temperature of the chicory compound sterilizing liquid is reduced to 38 ℃, inoculating bifidobacterium adolescentis and bifidobacterium bifidum pre-culture liquid at the same time, wherein the inoculation amount is 0.3 percent of the volume percentage of the chicory compound sterilizing liquid, and culturing for about 6 hours at 35 ℃; and simultaneously inoculating lactobacillus plantarum, lactobacillus casei and lactobacillus acidophilus preculture liquid, wherein the total volume percentage of the preculture liquid inoculated with the preculture probiotics is 1.5% of that of the chicory compound sterilizing liquid, and the volume percentages of the three bacterial liquids are the same. Continuous at 35 DEG CThe fermentation time is 36 hours later. The total bacterial count of the fermentation bacteria detected by a microscope direct counting method is higher than 1.5x10 8 When cfu/ml and pH reaches 4, fermentation is stopped. Sterilizing for 20min at 95-100 ℃ to obtain the chicory compound fermentation liquor.
(5) And (3) clarifying: sieving the chicory compound fermentation liquor with 120 mesh sieve respectively, and collecting filtrate; and washing the filter residue with water with the mass of 3 times of that of the filter residue, passing the water washing solution through a 230-mesh screen, collecting the filtrate, combining the two filtrates, centrifugally separating at 8000rpm/min, and collecting the supernatant to obtain clear chicory compound fermentation filtrate.
(6) And (5) subpackaging: packaging the chicory complex fermentation filtrate into 100ml chicory complex fermentation products under sterile and clean environment.
Example 2: the fermentation method of the chicory and poria cocos compound comprises the following steps:
(1) Raw material extraction pretreatment: pulverizing 90kg of chicory decoction pieces and 210kg of poria cocos decoction pieces, sieving with a 40-mesh sieve, respectively accurately weighing chicory powder and raspberry powder according to the formula proportion, mixing for 20min, decocting the obtained chicory compound powder with water, firstly adding 12 times of water of the chicory compound powder, soaking for 1.0 h, decocting, keeping boiling for 2 h after boiling, and separating out decoction; adding 10 times of water of chicory compound powder, decocting, keeping boiling for 1 hour, and separating decoction again; mixing decoctions, adding water to constant volume, mixing with the mixture powder at a mass/volume ratio of 0.75g/mL, and mixing the residue decoctions to obtain herba Cichorii compound extract.
(2) And (3) batching: glucose 4kg, yeast extract 0.8kg, yeast powder 0.4kg and peptone 3.2kg were added to the chicory complex extract. The nutrient compounds are added as follows: ferrous sulfate 7H 2 O8 g, zinc sulfate.7H 2 O35g, sodium selenite.5H 2 O190 mg, magnesium sulfate.7H 2 O560 g, manganese sulfate H 2 O4 g, monopotassium phosphate 800g and dipotassium phosphate 3H 2 O800 g, constant volume to 2000L. And regulating the pH value to 7.0 to obtain the chicory probiotics fermented traditional Chinese medicine substrate. Before adjusting the pH, 20kg of sweetener isomaltooligosaccharide and 60g of sucralose were added.
(3) And (3) sterilization: and (3) preserving the temperature of the chicory compound probiotic fermented traditional Chinese medicine substrate at 100-115 ℃ for 35min to obtain the chicory compound sterilizing liquid.
(4) Fermentation: when the temperature of the chicory compound sterilizing liquid is reduced to 37 ℃, inoculating bifidobacterium adolescentis and bifidobacterium breve pre-culture liquid, wherein the volume percentages of the bifidobacterium adolescentis and bifidobacterium breve pre-culture liquid are respectively 0.3%, and culturing the chicory compound sterilizing liquid at 30 ℃ for about 6 hours; and simultaneously inoculating lactobacillus plantarum, lactobacillus casei and lactobacillus formans pre-culture solution, wherein the volume percentage of the three probiotic pre-culture solutions inoculated is 0.5 percent of that of the chicory compound sterilizing solution. After a fermentation time of 40h at 35 ℃. The total bacterial count of the fermentation bacteria detected by a microscope direct counting method is higher than 1.5x10 8 Stopping fermentation when cfu/ml and pH reaches 3.5-4.1. Sterilizing for 30min at 95-100 ℃ to obtain the chicory compound fermentation liquor.
(5) And (3) clarifying: sieving the chicory compound fermentation liquor with 200 mesh sieve respectively, and collecting filtrate; and washing the filter residue with water with the mass of 4 times of that of the filter residue, sieving the water washing liquid with a 200-mesh sieve, collecting filtrate, combining the two filtrates, centrifugally separating at 8000rpm/min, and collecting supernatant to obtain clear chicory compound fermentation filtrate.
(6) And (5) subpackaging: packaging the chicory complex fermentation filtrate into 250ml chicory complex fermentation products under sterile and clean environment.
Example 3: the fermentation method of the chicory and poria cocos compound comprises the following steps:
(1) Raw material extraction pretreatment: pulverizing 110kg of herba Cichorii decoction pieces and 190kg of Poria decoction pieces, sieving with 40 mesh sieve, respectively accurately weighing herba Cichorii powder and Rubi fructus powder according to formula proportion, mixing for 20min to obtain herba Cichorii compound powder, decocting with water, soaking in 12 times of water for 1.0 hr, decocting, boiling for 3 hr, and separating decoction; adding 10 times of water of chicory compound powder, decocting, keeping boiling for 1 hour, and separating decoction again; mixing decoctions, adding water to constant volume, mixing with the mixture powder at a mass/volume ratio of 0.75g/mL, and mixing the residue decoctions to obtain herba Cichorii compound extract.
(2) And (3) batching: the chicory is compoundedGlucose 5kg, yeast extract 1kg, yeast powder 0.5kg and peptone 4.2kg are added into the extract. The nutrient compounds are added as follows: ferrous sulfate 7H 2 O8 g, zinc sulfate.7H 2 O35g, sodium selenite.5H 2 O190 mg, magnesium sulfate.7H 2 O560 g, manganese sulfate H 2 O4 g, monopotassium phosphate 800g and dipotassium phosphate 3H 2 O800 g, constant volume to 2000L. And regulating the pH value to 7.0 to obtain the chicory probiotics fermented traditional Chinese medicine substrate. Before adjusting the pH, 30kg of sweetener isomaltooligosaccharide and 100g of sucralose were added.
(3) And (3) sterilization: and (3) preserving the temperature of the chicory compound probiotic fermented traditional Chinese medicine substrate at 100-115 ℃ for 40min to obtain the chicory compound sterilizing liquid.
(4) Fermentation: when the temperature of the chicory compound sterilizing liquid is reduced to 39 ℃, inoculating bifidobacterium breve and lactobacillus plantarum pre-culture liquid simultaneously, wherein the volume percentages of the sterilizing liquid of the chicory compound are respectively 0.2 percent, and culturing the chicory compound sterilizing liquid at 37 ℃ for about 4 hours; and simultaneously inoculating lactobacillus casei, lactobacillus delbrueckii and lactobacillus bulgaricus pre-culture liquid, wherein the volume percentage of the three probiotic pre-culture liquids inoculated is 0.4 percent of that of the chicory compound sterilizing liquid. After a fermentation time of 36h at 37 ℃. The total bacterial count of the fermentation bacteria detected by a microscope direct counting method is higher than 1.5x10 8 Stopping fermentation when cfu/ml and pH reaches 3.5-4.1. Sterilizing for 300min at 95-100 ℃ to obtain chicory compound fermentation liquor.
(5) And (3) clarifying: sieving the chicory compound fermentation liquor with 140 mesh sieve respectively, and collecting filtrate; and washing the filter residue with water 3 times of the filter residue, passing the water washing solution through a 325-mesh screen, collecting the filtrate, combining the two filtrates, centrifugally separating at 6000rpm/min, and collecting the supernatant to obtain clear chicory compound fermentation filtrate.
(6) And (5) subpackaging: packaging the chicory complex fermentation filtrate into 100ml chicory complex fermentation products under sterile and clean environment.
Example 4: the fermentation method of the chicory and poria cocos compound comprises the following steps:
(1) Raw material extraction pretreatment: pulverizing 105kg of herba Cichorii decoction pieces and 195kg of Poria decoction pieces, sieving with 40 mesh sieve, respectively accurately weighing herba Cichorii powder and Rubi fructus powder according to formula ratio, mixing for 20min to obtain herba Cichorii compound powder, decocting with water, soaking in 12 times of water for 1.0 hr, decocting, boiling for 1 hr, and separating decoction; adding 10 times of water of chicory compound powder, decocting, keeping boiling for 1 hour, and separating decoction again; mixing decoctions, adding water to constant volume, mixing with the mixture powder at a mass/volume ratio of 0.75g/mL, and mixing the residue decoctions to obtain herba Cichorii compound extract.
(2) And (3) batching: glucose 5.5kg, yeast extract 1.3kg, yeast powder 0.5kg and peptone 3.3kg were added to the chicory complex extract. The nutrient compounds are added as follows: ferrous sulfate 7H 2 O8 g, zinc sulfate.7H 2 O35g, sodium selenite.5H 2 O190 mg, magnesium sulfate.7H 2 O560 g, manganese sulfate H 2 O4 g, monopotassium phosphate 800g and dipotassium phosphate 3H 2 O800 g, constant volume to 2000L. And regulating the pH value to 7.0 to obtain the chicory probiotics fermented traditional Chinese medicine substrate. 80kg of sweetener isomaltooligosaccharide and 40g of sucralose were added before adjusting the pH.
(3) And (3) sterilization: and (3) preserving the temperature of the chicory compound probiotic fermented traditional Chinese medicine substrate at 100-115 ℃ for 40min to obtain the chicory compound sterilizing liquid.
(4) Fermentation: inoculating a bifidobacterium bifidum preculture solution when the temperature of the chicory compound sterilizing solution is reduced to 39 ℃, wherein the volume percentage is 0.5%, and culturing for about 6 hours at 28 ℃; and simultaneously inoculating lactobacillus casei, lactobacillus plantarum and lactobacillus rhamnosus pre-culture solution, wherein the volume percentage of the inoculated probiotic pre-culture solution is 0.4% of that of the chicory compound sterilizing solution. After a fermentation time of 40h at 28 ℃. The total bacterial count of the fermentation bacteria detected by a microscope direct counting method is higher than 1.5x10 8 Stopping fermentation when cfu/ml and pH reaches 3.5-4.1. Sterilizing for 20min at 95-100 ℃ to obtain the chicory compound fermentation liquor.
(5) And (3) clarifying: sieving the chicory compound fermentation liquor with 170 mesh sieve respectively, and collecting filtrate; and washing the filter residue with water with the mass of 3 times of that of the filter residue, passing the water washing solution through a 270-mesh screen, collecting the filtrate, combining the two filtrates, centrifugally separating at 6000rpm/min, and collecting the supernatant to obtain clear chicory compound fermentation filtrate.
(6) And (5) subpackaging: and subpackaging the chicory complex fermentation filtrate into 50ml of chicory complex fermentation products under sterile and clean environment.
Experimental example effects on rat hyperuricemia and gouty arthritis
The study selects Potassium Oxazinate (PO) and sodium urate to induce rat hyperuricemia and gout model, and uses benzbromarone and febuxostat as positive control group to more comprehensively evaluate the curative effect of the chicory compound fermentation product of the example 1.
Male SD rats, 70, were randomly divided into 7 groups after 1 week of adaptive feeding, namely, solvent control (Con), hyperuricemia model (model, mod), febuxostat (Feb), benzbromarone (Ben), chicory complex fermented product low dose group (EWPL), chicory complex fermented product medium dose group (EWPM), chicory complex fermented product high dose group (EWPH). The Con group and the Mod group are infused with 0.5% CMC-Na suspension with equal volume, the Feb group is dosed at 13mg/kg, the Ben group is dosed at 15mg/kg, the EWPL group is dosed at 8.3ml/kg, the EWPM group is dosed at 16.7ml/kg, and the EWPH group is dosed at 50mlg/kg. The administration was performed by gavage 1 time a day for 40 days.
Test results:
1. effects on serum uric acid levels in rats
Compared with the model group, the serum uric acid content of rats is obviously reduced (P < 0.01) in the benzbromarone group (Ben), the febuxostat group (Feb), the chicory complex fermentation product low dose group (EWPL), the chicory poria medium dose group (EWPM) and the chicory poria high dose group (EWPH). The results show that the fermentation products of the benzbromarone, febuxostat and chicory compound have the effect of reducing serum uric acid of rats. The uric acid reducing effect of the chicory compound fermentation product is equivalent to that of a positive drug, namely benzbromarone. The results are shown in FIG. 1 (data represented by mean.+ -. Standard error, n=10, n is the number of samples, #P <0.01 compared to the solvent control group; P <0.01 compared to the hyperuricemia model group)
2. Effect on serum xanthine oxidase content in rats
The serum xanthine oxidase content was significantly reduced (P < 0.01) in the febuxostat group, the chicory complex fermented product low dose group, the chicory complex fermented product medium dose group, and the chicory complex fermented product high dose group compared to the model group. The serum of the benzbromarone group rats has no significant change (P > 0.05). Indicating that the febuxostat and chicory complex fermented products have the effect of reducing the serum xanthine oxidase content of rats. The effect of the chicory compound fermentation product for reducing the content of serum xanthine oxidase of rats is stronger than that of the positive drug benzbromarone. The results are shown in fig. 2, (data are represented by mean ± standard error, n=10, # # P <0.01 compared to solvent control group; P <0.01 compared to hyperuricemia model group
3. Effects on serum creatinine levels in rats
Compared with the model group, the serum creatinine content of rats in the phenylbromarone group, the febuxostat group, the chicory compound fermented product low dose group, the chicory compound fermented product medium dose group and the chicory compound fermented product high dose group is obviously reduced (P < 0.01). The serum creatinine-reducing effect of the chicory complex fermentation product is comparable to that of non-b ustat. The results are shown in fig. 3 (data expressed as mean ± standard deviation, n=10. Compared to the solvent control, #p <0.01; compared to the hyperuricemia model, < 0.01)
4. Effects on serum urea nitrogen content in rats
The serum urea nitrogen content was significantly reduced (P < 0.01) in the low dose group of chicory complex fermented products, in the dose group of chicory complex fermented products, and in the high dose group of chicory complex fermented products compared to the model group. The tribromone group and the febuxostat group have no influence on serum urea nitrogen (P is more than 0.05), which indicates that the protective effect of the chicory compound fermentation product on the kidney is superior to that of two positive medicines.
The results are shown in fig. 4, (data are represented by mean ± standard error, n=10. Compared to solvent control, #p <0.01; compared to hyperuricemia model group, < 0.01:)
5. Effects on rat kidney coefficients
Compared with the model group, the kidney coefficient of rats in the febuxostat group, the chicory compound fermentation product low dose group, the chicory poria medium dose group and the chicory poria high dose group is obviously reduced (P < 0.01). The chicory complex fermentation product has better effect than benzbromarone and is equivalent to non-bestatin. The results are shown in fig. 5 (data expressed as mean ± standard deviation, n=10. Compared to the solvent control, #p <0.01; compared to the hyperuricemia model, < 0.01)
6. Effects on rat serum inflammatory factors IL-1 beta, IL-6, TNF-alpha
Compared with the model group, the inflammatory factors IL-1 beta, IL-6 and TNF-alpha in the serum of rats of the low dose group of the phenylbromarone group, the febuxostat group, the low dose group of the chicory compound fermentation product, the medium dose group of the chicory poria cocos and the high dose group of the chicory poria cocos are all obviously reduced (P < 0.01). Indicating that the febuxostat, benzbromarone and chicory compound fermented products have the functions of reducing the serum inflammatory factors IL-1 beta, IL-6 and TNF-alpha of rats. The results are shown in fig. 6-8, (data expressed as mean ± standard deviation, n=10. Compared to the solvent control, #p <0.01; compared to the hyperuricemia model, < 0.01:
7. influence on joint swelling degree, gait score and joint temperature change after modeling of gouty arthritis in rats
On day 37 of administration, the right ankle joint cavities of the rats of each group were each injected with 0.1mL of MSU solution (50 mg/mL) except for the solvent control group, which was injected with 0.1mL of physiological saline. The joint swelling, walking gait score and joint temperature changes of each group of rats were measured before and after MSU injection for 4h, 8h, 16h, 24h, 48h, respectively. The results are shown in FIGS. 9-11.
7.1 Effect on joint swelling degree after modeling of gouty arthritis in rats
Administration of Ben, feb, EWP treatment improved joint swelling in rats and each dose group of EWP worked most rapidly compared to model group rats, and significantly reduced joint swelling (P < 0.01) from 4 h. Ben and Feb act slowly, have no significant change (P > 0.05) at 4h and 8h, and can significantly reduce the joint swelling degree of rats (P < 0.01) at 16 h. The onset time of the EWP on the treatment of the joint swelling degree of the gouty arthritis rat is earlier than that of Ben and Feb. As shown in fig. 9. ( Data are expressed as mean ± standard error, n=10. # P <0.01 compared to the solvent control group; p <0.01 compared to gouty arthritis model group )
7.2 Effect on gait score after modeling gouty arthritis in rats
The Ben, feb, EWP treatment can improve the lameness gait of the rat, compared with the model group of rats, the Ben and EWP dose groups can obviously reduce the gait score of the rat (P < 0.01) in 16h, 24h and 48h, and the Feb group obviously reduces the gait score of the rat (P < 0.01) in 24h and 48 h. As shown in fig. 10. ( Data are expressed as mean ± standard error, n=10. # P <0.01 compared to the solvent control group; p <0.01 compared to gouty arthritis model group )
7.3 Effect on joint temperature Change after modeling of gouty arthritis in rats
Administration of Ben, feb, EWP treatment reduced joint temperature in rats, ben had the fastest onset and significantly reduced joint temperature (P < 0.01) at the beginning of 8 h. The EWP dose groups significantly reduced the joint temperature of rats (P < 0.01) from 16 h. The Feb group had the slowest onset, significantly lowering the rat joint temperature from 24h (P < 0.01). As shown in fig. 11. ( Data are expressed as mean ± standard error, n=10. # P <0.01 compared to the solvent control group; p <0.01 compared to gouty arthritis model group )
8 effects on the expression of the rat kidney URAT1, GLUT9 and OAT1 proteins with hyperuricemia
8.1 Effect on the expression of the rat kidney URAT1 protein with hyperuricemia
URAT1 protein expression was significantly reduced in the kidneys of rats in each of the Feb, EWP groups compared to the hyperuricemia model group (P <0.05, P < 0.01). Ben has no effect on kidney URAT1 protein expression (P > 0.05). The URAT1 protein has the main function of mediating the reabsorption of urinary catheter acid salt from the renal tubule cavity to blood, and the chicory compound fermented product can inhibit the expression of URAT1 to reduce the uric acid content in serum. The results are shown in fig. 12, (data expressed as mean ± standard deviation, n=3. Compared to the solvent control group, #p <0.05; compared to the hyperuricemia model group, <0.05, < P < 0.01)
8.2 Effect on GLUT9 protein expression in the kidney of hyperuricemia rats
GLUT9 protein expression was significantly reduced in rat kidney tissue from each group Ben, feb, EWP compared to hyperuricemia model group (P <0.01, P < 0.05). The effect of EWP is better than Ben. GLUT9 protein mainly plays a role in mediating reabsorption of urinary catheter acid salt from the renal tubule cavity to blood, and the chicory complex fermentation product can play a role in reducing serum uric acid content by inhibiting GLUT9 protein expression. The experimental results are shown in FIG. 13. ( Data are expressed as mean ± standard error, n=3. # P <0.01 compared to the solvent control group; p <0.05, P <0.01 compared to hyperuricemia model group )
8.3 Effect on the expression of OAT1 protein in the kidney of hyperuricemia rats
Compared with the hyperuricemia model group, the OAT1 protein expression in the kidney of rats in the chicory Poria cocos dose group and the chicory Poria cocos high dose group is significantly increased (P <0.01, P < 0.05). OAT1 protein belongs to uric acid excretion transporter, and experimental results suggest that chicory complex fermentation products can accelerate uric acid excretion by promoting OAT1 protein expression. The results are shown in fig. 14, (data are represented by mean ± standard error, n=3, #p <0.05 compared to the solvent control group, #p <0.05, < P <0.01 compared to the hyperuricemia model group)
Conclusion of experiment:
(1) The chicory complex fermentation product has therapeutic effect on rat hyperuricemia.
(2) The chicory compound fermentation product has a protective effect on kidney injury caused by hyperuricemia of rats.
(3) The chicory complex fermentation product has therapeutic effect on acute gouty arthritis in rats.
(4) Chicory complex fermented preparations treat hyperuricemia by affecting URAT1, GLUT9, and OAT1 protein expression.
Efficacy tests of rat hyperuricemia and gouty arthritis further prove that the whole chicory compound fermentation product improves the whole progress process of gout and hyperuricemia.
The present invention is not limited to the above-described embodiments, and any modifications, improvements, substitutions, and the like, which may occur to those skilled in the art, fall within the scope of the present invention without departing from the spirit of the invention.
Claims (10)
1. A fermentation method of chicory and poria cocos compound is characterized by comprising the following steps of:
step 1): mixing 20-50 parts of chicory powder and 50-80 parts of poria cocos powder according to parts by weight, and decocting twice after mixing to obtain an extracting solution;
step 2): adding a fermentation nutrient into the extracting solution and the filter residue obtained in the step 1), and adjusting the pH to 7.0+/-0.5 to obtain a fermentation substrate;
step 3): steam sterilizing the fermentation substrate to obtain a chicory compound sterilizing solution;
step 4): cooling the chicory compound sterilizing liquid in the step 3), inoculating bifidobacterium and lactobacillus pre-culture bacterial liquid for fermentation, and performing stabilization and inactivation treatment after fermentation is completed to obtain a chicory compound fermentation liquid;
step 5): filtering the chicory compound fermentation liquor through a screen to remove dregs, and collecting filtrate.
2. The method for fermenting chicory, poria complex according to claim 1, characterized in that:
in the step (1), the chicory and the poria cocos are crushed and pass through a 20-60-mesh sieve; mixing 20-50 parts of chicory powder and 50-80 parts of poria cocos powder for 10-40 min to obtain mixed powder;
adding water 10-20 times of the mixed powder, soaking for 1.0 hour, decocting, keeping boiling for 1-3 hours after boiling, and separating out decoction; adding water 8-15 times of the mixed powder, decocting, keeping micro boiling for 1-3 hours after boiling, and separating out decoction again; mixing the decoctions, adding water to constant volume, and mixing the powder and the constant volume decoction at a mass volume ratio of 0.75g/mL.
3. The method for fermenting chicory, poria complex according to claim 1, characterized in that: the addition amount of each component in the fermentation nutrient in the step (2) is as follows: glucose 5-15 g/L, yeast extract 1-4 g/L, yeast powder 0.5-1.5 g/L, peptone 3-10 g/L, and nutrients including iron 3-8 mg/L, zinc 15-25 mg/L, selenium 0.05-0.2 mg/L, magnesium 120-180 mg/L, manganese 3-8 mg/L, and potassium 1.0-1.5 g/L.
4. The method for fermenting chicory, poria complex according to claim 1, characterized in that:
adding a sweetener before adjusting the pH in the step (2), wherein the sweetener is isomaltooligosaccharide and/or sucralose; the addition amount is 10-80 g/L of isomaltooligosaccharide and 0.01-0.1 g/L of sucralose.
5. The method for fermenting chicory, poria complex according to claim 1, characterized in that:
in the step (3), the fermentation substrate is kept at the temperature of 100-115 ℃ for 30-60 min, and the chicory compound sterilizing liquid is obtained.
6. The method for fermenting chicory, poria complex according to claim 1, characterized in that: the bifidobacterium in the step (4) is selected from one or two of bifidobacterium breve (bifidobacterium breve), bifidobacterium bifidum (bifidobacterium bifidum) or bifidobacterium adolescentis (Bifidobacterium adolescentis);
the lactobacillus includes lactobacillus plantarum (Lactobacillus plantarum) and lactobacillus casei (Lactobacillus casei);
and at least one of Lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus gasseri, and Lactobacillus delbrueckii subsp.
The inoculation amount of the bifidobacterium pre-culture bacterial liquid is 0.1-1.0% of the volume percentage of the chicory compound sterilizing liquid; the inoculation amount of the lactobacillus pre-culture bacterial liquid is 0.2-4% of the volume percentage of the chicory compound sterilizing liquid;
the volume ratio of the bifidobacterium pre-culture bacterial liquid to the lactobacillus pre-culture bacterial liquid is 1:2 to 4; the viable count of the bifidobacterium pre-culture bacterial liquid and the lactobacillus pre-culture bacterial liquid is 2 multiplied by 10 7 CFU·mL -1 。
7. A fermentation process of chicory, poria cocos complexes according to claim 1 or 6, characterized in that: the fermentation temperature in the step (4) is 25-40 ℃, and the total bacterial count of the bifidobacterium and the lactobacillus is kept to be not less than 1.5x10 8 cfu/ml, fermenting for 25-48 h, stopping fermentation when the pH reaches 3.5-4.1; and then sterilizing for 10-40 min at 95-100 ℃ to obtain the chicory compound fermentation liquor.
8. The method for fermenting chicory, poria complex according to claim 1, characterized in that:
in the step (5), the chicory compound fermentation liquor is filtered by a screen to remove dregs, and then filtrate is collected and the dregs are washed by water; filtering the water washing liquid again through a screen, collecting filtrate, combining the two filtrates, centrifugally separating, collecting supernatant to obtain clarified chicory compound fermentation filtrate, and filling to obtain a finished product.
9. A fermentation process of chicory, poria cocos complex as claimed in claim 8, wherein:
and (3) sequentially passing the chicory compound fermentation liquor and the water washing liquor through 100-200 meshes and 200-400 meshes of screens in the step (5).
10. A fermentation process of chicory, poria cocos complex as claimed in claim 8, wherein: the rotational speed during centrifugal separation is 6000-10000rpm/min.
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