CN102935095A - Bidirectional solid fermentation irpex cacteus mycoplasm extractive and preparation method and application thereof - Google Patents
Bidirectional solid fermentation irpex cacteus mycoplasm extractive and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a bidirectional solid fermentation irpex cacteus mycoplasm extractive and a preparation method and application thereof and belongs to the field of medical fungi. Irpex cacteus is subjected to liquid culture with stock culture to obtain seeds at logarithmic phase, the seeds are inoculated to a seed bottle containing a barley culture medium under aseptic condition, and seed bottle fermental cultivation and fermentation bottle fermentation are performed to produce, cultivate and extract. The bidirectional solid fermentation irpex cacteus mycoplasm extractive and the preparation method and application thereof can treat metabolin of glomeruli nephritides and simultaneously change original performances of the barley through growth and metabolism of the irpex cacteus.
Description
Technical field
The invention belongs to field of medicinal fungus, refer in particular to a kind of white rake teeth bacterium mycoplasma extract and preparation method and its usage.
Background technology
White rake teeth bacterium
Irpex lacteusFr. belong to a kind of fungus of Basidiomycetes Aphyllophorales hedgehog fungus section rake teeth Pseudomonas, call white bag hole.This culture presevation is at China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: CGMCC No.1016.Its sporophore is born on the dry wood and dead and dying trees of broad leaf tree.Morphological characteristic: basidiocarps (sporophore) is calm, expansion, white, soft leather matter; Sometimes fall to give birth to, be multiple watt shape storied, fan-shaped, narrow and small base portion is arranged; The capping milky, have down, and spun silk sample gloss, have ring grain, and thin edge is lower curved, wavy to shallow splitting.Bacterial context white.Thorn is irpiciform, short, irregular, and white is to yellow-white, the spore ellipse is level and smooth, colourless, cystidium clavate or spindle, draped over one's shoulders crystallization sometimes, mainly is distributed in the provinces and regions such as Forest in Changbai Mountain Forest Region and Heilungkiang, Hebei, Shanxi, Gansu, Jiangxi, Fujian, Yunnan and Tibet.
The medicinal wild sporophore of collection among the people that only limits in history of white rake teeth bacterium, its sporophore is born on the dry wood and dead and dying trees of broad leaf tree.More difficult collecting, so sporophore is more rare, can't collect in a large number.
Chronic glomerulonephritis is caused by the infringement of multiple primary glomerular disease reason, take the class disease that albuminuria, hematuria, edema, hypertension be common clinical manifestation.Kidney section commonly encountered diseases and frequently-occurring disease.Wherein most patients have renal function injury in various degree, and are carrying out property and increase the weight of, the gradual renal failure that transfers to.Thereby treating timely and effectively chronic glomerulonephritis is the important topic of researcher.
The eighties-nineties, professor Song Qiyin of Changchun Traditional Chinese Medical College researchs and develops first the method for the white rake teeth bacteria liquid of application fermentation and produces white rake teeth granulose treatment chronic glomerulonephritis, i.e. Chinese patent medicine " Yi-Shen-Kang " capsule in the market.The Yi-Shen-Kang capsule, Main Ingredients and Appearance is the crude extract of rake teeth granulose.Content is that brown is to auburn powder; Distinguish the flavor of micro-sour and astringent.Effect with eliminating damp-heat.For the burnt damp-heat syndrome person of chronic glomerulonephritis subordinate.But the patient takes accidental stomach discomfort, xerostomia, poor appetite, feel sick.
The production of medicinal fungi at present generally has liquid fermentation and two kinds of forms of solid fermentation.More application liquid fermentation production both at home and abroad is to use for reference antibiotic production technology, and application of fermentation tank advantage is that higher mechanization and automatization are arranged, and production scale is large.But because medicinal fungi self does not generally produce antibiotic, or the antibiotic bacteriostasis produced a little less than, can not be to good antagonisms of other microorganisms such as antibacterials, therefore easy pollution microbes and pouring in down a chimney during the fermentation, cause heavy losses.Its investment of production equipment is huge, consumes energy also more, and production cost is higher.
The solid fermentation of medicinal fungi sees the production of Chinese medicine " Massa Medicata Fermentata " and " Pinellia leaven " before 2000 the earliest, is to utilize natural mycete to be fermented, and its effect is the function that produces the stomach function regulating that helps digestion.Its technique is used till today always, but the fermentation of " Massa Medicata Fermentata " and " Pinellia leaven " is not pure-blood ferment, the bad control of technique.Along with the development of biotechnology, adopted pure strain inoculation in recent years, the quality supervision system that the science of setting up is controlled, the stability of its sweat has obtained solving well.The Zhuan Yi of Nanjing University of Traditional Chinese Medicine teaches in the research eighties in last century with medicinal fungi " Auricularia Sophorae "
Trametes robiniophilaMurr. be fermented bacterium, carry out solid fermentation take agricultural byproducts on nutrient matrix, its medicinal mycoplasma claims " Trametes robiniophila Murr. ".Develop the new drug " Huaier Granule " with antitumor efficacy, propose the novel amphicheirality's solid fermentation of medicinal fungi engineering theory simultaneously.Its principle of the novel amphicheirality's solid fermentation of medicinal fungi engineering (hereinafter to be referred as " two-way fermentation ") is selected specific fermented bacterium, with specific Chinese crude drug as medicinal mycoplasma, use again agricultural byproducts as nutrient matrix (being collectively referred to as wholeness matrix), both sides form a fermentation combination altogether, carry out under given conditions two-way fermentation, fermented product becomes medicinal fungal substance, and it is a ground zero of biotechnology.It can change performance, the enhancing drug effect of former medicine and enlarge indication.Product after fermentation is larger than the drug effect of the independent use of two kinds of materials that fermented, i.e. " 1+1>2 ".
Fructus Hordei Vulgaris (
Hordeum vulgare) be the seed of grass family annual herb plant, be food and medicine, Compendium of Material Medica record: " Fructus Hordei Vulgaris sweet in the mouth, property put down, have that dietetic therapy is swollen, heat extraction is quenched the thirst, relieves summer heat in removing food stagnancy feed, flat stomach, QI invigorating adjust in, the chest stuffiness relieving atmosphere, bad, the strong blood vessels of tonify deficiency, beneficial color, real the five internal organs, change the merit of paddy food ".Modern study proves, 17 kinds of trace element that Fructus Hordei Vulgaris tea contains needed by human body, and 19 kinds with upper amino acid, is rich in multivitamin and unsaturated fatty acid, protein and dietary fiber.In order to control accumulation of food in the stomach and intes tine due to indigestion, the vexed abdominal distention of gastral cavity, the disease of the weakness of the spleen and stomach such as have loose bowels.
Summary of the invention
The invention provides a kind of amphicheirality's solid fermentation white rake teeth bacterium mycoplasma extract and preparation method and its usage, to solve white rake teeth bacteria liquid, cultivate extract and make that the patient produces stomach discomfort, xerostomia, poor appetite, the problem of the side effect such as feel sick.
The technical scheme that the present invention takes is: the preparation method of the white rake teeth bacterium of amphicheirality's solid fermentation mycoplasma extract is:
(1) white rake teeth bacterium is planted and carries out the seed that liquid culture obtains exponential phase with mother, this seed is inoculated under aseptic condition in the seed bottle of Fructus Hordei Vulgaris culture medium;
(2) seed bottle fermentation culture: at 22 ℃-26 ℃ of temperature, air humidity 65%-70% environment bottom fermentation 7-9 days;
(3) the fermentation flask fermenting and producing is cultivated: 22 ℃-26 ℃ of temperature, air humidity 65%-70% environment bottom fermentation 18-21 days, obtain the mycoplasma of white rake teeth bacterium;
(4) this mycoplasma is with 80 ℃ ~ 90 ℃ hot water extraction 2 ~ 3 times, and 3 hours for the first time, 2 hours for the second time, 1 hour for the third time, merging filtrate, surveyed the clear paste of relative density 1.05 ~ 1.10 while being condensed into 60 ℃, and 50 ℃ ~ 55 ℃ drying under reduced pressure, be ground into fine powder.
Fructus Hordei Vulgaris culture medium of the present invention is: the barley corn that is 95%-97% by weight ratio soaks, and makes water content reach 50-55%, adds the Gypsum Fibrosum powder of weight ratio 1%-2%, the sucrose of weight ratio 2%-3%.
The white rake teeth bacterium mycoplasma extract that preparation method by the white rake teeth bacterium of amphicheirality's solid fermentation mycoplasma extract obtains is applied in the burnt damp-heat syndrome person's of preparation treatment chronic glomerulonephritis subordinate medicine.
The present invention utilizes Fructus Hordei Vulgaris to run concurrently and support substrate (title wholeness matrix) as medicinal mycoplasma, and inoculation has the white rake teeth bacterium for the treatment of glomerulonephritis effect, and both sides form a fermentation combination altogether, carry out under given conditions two-way fermentation, and fermented product becomes medicinal fungal substance.Fructus Hordei Vulgaris can provide the nutritional labeling of white rake teeth bacteria growing, make white rake teeth bacterium mycelial growth breeding, obtain having the brightic metabolite for the treatment of, simultaneously by the metabolism and growth of white rake teeth bacterium, change the original performance of Fructus Hordei Vulgaris, strengthen the function of strengthening the spleen to promote digestion, relax stomach discomfort due to white rake teeth bacterium metabolite, xerostomia, poor appetite, feel sick and wait side effect.
The specific embodiment
White rake teeth bacterium of the present invention
Irpex lacteusFr, this culture presevation is at China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: CGMCC No.1016.
White rake teeth bacterium is planted and carries out liquid culture to obtain the method for seed of exponential phase as follows with mother:
(1) primary inclined plane strain:
Culture medium: glucose 2-3%, Rhizoma Solani tuber osi 20-25%, agar 2.2%, MgSO
40.2%, KH
2PO
40.3%, VB
10.05%;
Slant strains cultivation temperature 25-27 ℃, within 120 hours, mycelia is covered with inclined-plane, places 2-4 ℃ of refrigerator and deposits standby;
(2) secondary shaking flask strain:
Culture medium: glucose 2%, yeast extract 0.5%, KH
2PO
40.2%, NaH
2SO
40.15%, peptone 0.2%, VB
10.01%, pH6.5;
Get the sturdy dense slant strains of mycelia, press the inoculation of 1:4 inoculum concentration under aseptic condition, cultivate 4 ~ 6 days in 26 ± 1 ℃ of shaking tables; Result: bacterium liquid is clear and bright, and yellow has dense fruit aroma, and it is radial that fungus ball is; Microscopy: methylene blue simple stain mycelia color depth, and sturdy, branch is many, and clamp connection is obviously visible; This period is the animated period of hyphal development (exponential phase) just, again without living contaminants, can transfer solid culture to.
The preparation of embodiment 1 capsule of the present invention
(1) white rake teeth bacterium is planted and carries out the seed that liquid culture obtains exponential phase with mother, this seed is inoculated under aseptic condition in the seed bottle of Fructus Hordei Vulgaris culture medium; Described Fructus Hordei Vulgaris culture medium is: the barley corn that is 95% by weight ratio soaks, and makes water content reach 50%, adds the Gypsum Fibrosum powder of weight ratio 1%, the sucrose of weight ratio 2%;
(2) seed bottle fermentation culture: 26 ℃ of temperature, air humidity 70% environment bottom fermentation 7 days;
(3) the fermentation flask fermenting and producing is cultivated: 26 ℃ of temperature, air humidity 70% environment bottom fermentation 18 days, obtain the mycoplasma of white rake teeth bacterium;
(4) this mycoplasma was with 90 ℃ of hot water extraction 3 times, 3 hours for the first time, 2 hours for the second time, 1 hour for the third time, merging filtrate, the clear paste of survey relative density 1.10 while being condensed into 60 ℃, 55 ℃ of drying under reduced pressure, be ground into fine powder, add the adjuvant for preparing capsule, prepare according to a conventional method capsule.
The preparation of embodiment 2 tablets of the present invention
(1) white rake teeth bacterium is planted and carries out the seed that liquid culture obtains exponential phase with mother, this seed is inoculated under aseptic condition in the seed bottle of Fructus Hordei Vulgaris culture medium; Described Fructus Hordei Vulgaris culture medium is: the barley corn that is 96% by weight ratio soaks, and makes water content reach 53%, adds the Gypsum Fibrosum powder of weight ratio 1.5%, the sucrose of weight ratio 2.5%;
(2) seed bottle fermentation culture: 22 ℃ of temperature, air humidity 65% environment bottom fermentation 9 days;
(3) the fermentation flask fermenting and producing is cultivated: 22 ℃ of temperature, air humidity 65% environment bottom fermentation 21 days, obtain the mycoplasma of white rake teeth bacterium;
(4) this mycoplasma is with 80 ℃ of hot water extraction 2 times, and 3 hours for the first time, 2 hours for the second time, merging filtrate, surveyed the clear paste of relative density 1.05 while being condensed into 60 ℃, and 50 ℃ of drying under reduced pressure, be ground into fine powder, adds the adjuvant for preparing tablet, prepares according to a conventional method tablet.
The preparation of embodiment 3 granules of the present invention
(1) white rake teeth bacterium is planted and carries out the seed that liquid culture obtains exponential phase with mother, this seed is inoculated under aseptic condition in the seed bottle of Fructus Hordei Vulgaris culture medium; Described Fructus Hordei Vulgaris culture medium is: the barley corn that is 97% by weight ratio soaks, and makes water content reach 55%, adds the Gypsum Fibrosum powder of weight ratio 2%, the sucrose of weight ratio 3%;
(2) seed bottle fermentation culture: 24 ℃ of temperature, air humidity 67% environment bottom fermentation 8 days;
(3) the fermentation flask fermenting and producing is cultivated: 24 ℃ of temperature, air humidity 67% environment bottom fermentation 20 days, obtain the mycoplasma of white rake teeth bacterium;
(4) this mycoplasma was with 85 ℃ of hot water extraction 3 times, 3 hours for the first time, 2 hours for the second time, 1 hour for the third time, merging filtrate, the clear paste of survey relative density 1.08 while being condensed into 60 ℃, 52 ℃ of drying under reduced pressure, be ground into fine powder, add the adjuvant for preparing granule, prepare according to a conventional method granule.
The curative effect report of the white rake teeth fungus extract of solid fermentation of the present invention preparation for treating chronic glomerulonephritis
Clinical dataAll have a chronic renal glomerulonephritis medical history, between 24h urine protein quantitation 0. 3～3g, Scr<350umo/ L person classifies object of study as, observe altogether 264 examples, inpatient's 120 examples wherein, outpatient's 144 examples, according to its medical and the order of being in hospital, be divided at random treatment group 132 examples, matched group 132 examples.Exclusion standard: (1) non-gravid woman, non-allergic constitution and to this medicine without allergy sufferers; (2) the serious primary disease person such as nonjoinder cardiovascular, liver, hemopoietic system; (3) other than psychotic patient; (4) person that adheres to the medication treatment.This problem is by above-mentioned Standard Selection 264 examples, treatment group 132 examples, and male's 64 examples wherein, women's 68 examples, the age, the course of disease was between April～8 year between 14～64 years old.Merge creatinine, abnormal person's 56 examples of blood urea nitrogen; Hematochrome reduction person 62 examples; Hyperpietic's 33 examples.Matched group 132 examples, male's 62 examples wherein, women's 70 examples, the age, the course of disease was between 6 months～7 years between 15～68 years old.Merge creatinine, abnormal person's 44 examples of blood urea nitrogen; Hematochrome reduction person 50 examples; Hyperpietic's 30 examples.Two groups of cases are statistically without significant difference.
Therapeutic Method
Primary Care: (1) infection control.(2) control blood pressure.(3) person gives the correction acidosis to have the acidosis.
Treatment group: take the capsule that the white rake teeth bacterium of amphicheirality's solid fermentation mycoplasma extract is made, each 2 every day 3 times, 30 days is 1 course for the treatment of, treats and adds up curative effect 3 courses for the treatment of.
Matched group: taking the liquid fermentation preparation is each 2 of Yi-Shen-Kang capsule (production of Changchun Yi-Shen-Kang Biology Pharmacy Co., Ltd), every day 3 times, within 30 days, is 1 course for the treatment of, treats and adds up curative effect 3 courses for the treatment of.
Observation index: cardinal symptom changes: comprise soreness of the waist and knees, and fatigue and weakness, edema, dizzy etc.Situation appears in side effect: comprise stomach discomfort, and xerostomia, poor appetite, feel sick etc.Lab index: do the inspections such as 24h urine protein quantitation, serum creatinine, blood urea nitrogen, hemoglobin before and after treatment.
Statistical procedures: measurement data adopts the T check, and enumeration data adopts x
2Check.
Criterion of therapeutical effect recovery from illness: the symptom such as edema and sign disappear fully, and it is negative that urine protein examination continues, or the 24h urine protein quantitation continues to be less than 200mg, and under high power lens, urine erythrocyte disappears.The Urine sediments analyzer counting is normal, and graft function is normal.Produce effects: the symptom such as edema and sign disappear substantially, and the urine protein examination continuous decrease is more than 50%, and under high power lens, urine erythrocyte is no more than 3, and the Urine sediments analyzer counting approaches normal.Normal renal function or normal, differ and be no more than 15% with normal value.Effectively: the symptom such as edema and sign are clearly better, 1 of urine protein examination continuous decrease+or 24h urine protein quantitation continuous decrease more than 25%, and under high power lens, urine erythrocyte is no more than 5, normal renal function or be improved.Invalid: clinical manifestation and above-mentioned lab testing all are not improved or add severe one.
Therapeutic outcome
1, between two groups curative effect relatively this organizes 264 examples, treatment group: 132 examples, 16 examples of fully recovering, produce effects 44 examples, effective 66 examples, invalid 6 examples, total effective rate 95.4%.Matched group: 132 examples, 14 examples of fully recovering, produce effects 40 examples, effective 69 examples, invalid 9 examples, total effective rate 93. 1%.Two groups of total effective rate comparison P values are greater than 0. 05, and difference is not very significant.
2, between two groups, treatment front and back lab index situation of change is shown in (table one)
Before and after table 2 liang group patient treatment, lab index changes
Annotate: with compare * P<0. 01 * * P<0. 05 and compare △ P>0. 05 after treatment of control group before this group is treated
3 routine stomach discomfort appear after the capsule that 3, side effect treatment group is made with solid fermentation, 6 routine xerostomias, and poor appetite appears in 2 examples, the nauseating symptom of 0 example.The capsule that the matched group liquid fermentation is made (Yi-Shen-Kang) 40 routine stomach discomfort, 32 routine xerostomias, poor appetite appears in 20 examples, the nauseating symptom of 12 example.
The white rake teeth bacterium of conclusion amphicheirality's solid fermentation mycoplasma extract and liquid fermentation production preparation all have curative effect preferably to glomerulonephritis, but the liquid fermentation production side effect is more, and the side effect of the white rake teeth bacterium of amphicheirality's solid fermentation mycoplasma extract is less, so the white rake teeth bacterium of amphicheirality's solid fermentation mycoplasma extract formulation is better than the liquid fermentation preparation.
Claims (5)
1. the white rake teeth bacterium of an amphicheirality's solid fermentation mycoplasma extract is characterized in that it is obtained by following method:
(1) white rake teeth bacterium is planted and carries out the seed that liquid culture obtains exponential phase with mother, this seed is inoculated under aseptic condition in the seed bottle of Fructus Hordei Vulgaris culture medium;
(2) seed bottle fermentation culture: at 22 ℃-26 ℃ of temperature, air humidity 65%-70% environment bottom fermentation 7-9 days;
(3) the fermentation flask fermenting and producing is cultivated: 22 ℃-26 ℃ of temperature, air humidity 65%-70% environment bottom fermentation 18-21 days, obtain the mycoplasma of white rake teeth bacterium;
(4) this mycoplasma is with 80 ℃ ~ 90 ℃ hot water extraction 2 ~ 3 times, and 3 hours for the first time, 2 hours for the second time, 1 hour for the third time, merging filtrate, surveyed the clear paste of relative density 1.05 ~ 1.10 while being condensed into 60 ℃, and 50 ℃ ~ 55 ℃ drying under reduced pressure, be ground into fine powder.
2. the white rake teeth bacterium of amphicheirality's solid fermentation according to claim 1 mycoplasma extract, it is characterized in that: described Fructus Hordei Vulgaris culture medium is: the barley corn that is 95%-97% by weight ratio soaks, make water content reach 50-55%, add the Gypsum Fibrosum powder of weight ratio 1%-2%, the sucrose of weight ratio 2%-3%.
The white rake teeth bacterium of amphicheirality's solid fermentation as claimed in claim 1 mycoplasma extract preparation method, comprise the following steps:
(1) white rake teeth bacterium is planted and carries out the seed that liquid culture obtains exponential phase with mother, this seed is inoculated under aseptic condition in the seed bottle of Fructus Hordei Vulgaris culture medium;
(2) seed bottle fermentation culture: at 22 ℃-26 ℃ of temperature, air humidity 65%-70% environment bottom fermentation 7-9 days;
(3) the fermentation flask fermenting and producing is cultivated: 22 ℃-26 ℃ of temperature, air humidity 65%-70% environment bottom fermentation 18-21 days, obtain the mycoplasma of white rake teeth bacterium;
(4) this mycoplasma is with 80 ℃ ~ 90 ℃ hot water extraction 2 ~ 3 times, and 3 hours for the first time, 2 hours for the second time, 1 hour for the third time, merging filtrate, surveyed the clear paste of relative density 1.05 ~ 1.10 while being condensed into 60 ℃, and 50 ℃ ~ 55 ℃ drying under reduced pressure, be ground into fine powder.
4. the preparation method of the white rake teeth bacterium of amphicheirality's solid fermentation according to claim 2 mycoplasma extract, it is characterized in that: described Fructus Hordei Vulgaris culture medium is: the barley corn that is 95%-97% by weight ratio soaks, make water content reach 50-55%, the Gypsum Fibrosum powder that adds weight ratio 1%-2%, the sucrose of weight ratio 2%-3%.
5. the white rake teeth bacterium of amphicheirality's solid fermentation as claimed in claim 1 mycoplasma extract is applied in the burnt damp-heat syndrome person's of preparation treatment chronic glomerulonephritis subordinate medicine.
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| CN114097906A (en) * | 2020-08-28 | 2022-03-01 | 广西民族大学 | Rapex cacteus fermented tea and preparation method thereof |
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