CN101596220B - Wall-breaking method of ganoderma lucidum spore - Google Patents
Wall-breaking method of ganoderma lucidum spore Download PDFInfo
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Abstract
The invention relates to a wall-breaking technique of ganoderma lucidum spore, in particular to a biologic method for carrying out wall breaking on the ganoderma lucidum spore. The method comprises the following steps of: separating and purifying ganoderma endophytic bacteria from ganoderma; sieving strains; preparing enzyme liquid; carrying out enzymolysis on the ganoderma lucidum spore; and preparing wall-breaking ganoderma lucidum spore powder. The invention utilizes the self symbiotic bacteria of the ganoderma to carry out fermentation on the spore and a fruiting body of the ganoderma, the effective ingredients of the ganoderma more easily escape from cells, a human body can successfully absorb and digest the effective ingredients, and the effective ingredients can be fully utilized. The difficulty that biologic residues caused by introducing foreign bacteria are hard to remove can be eliminated, and the safety of a product is ensured.
Description
(1) technical field:
The present invention relates to the wall breaking technology of Ganoderma spore, particularly Ganoderma spore is carried out breaking cellular wall with biological method.
(2) background technology:
Ganoderma is a kind of very famous and precious medicinal and edible fungi, is commonly called as " Ganoderma lucidum seu Japonicum ", is called " Herba mesonae chinensis " alive for evermore ancient times.Introduce according to China's two medical science monumental work Shennong's Herbals the earliest and " wooden careless detailed outline ", Ganoderma is divided into six kinds, be respectively by color and luster: Ganoderma lucidum (Leyss. Ex Fr.) Karst., Ganoderma lucidum seu Japonicum, Radix Angelicae Dahuricae, Fructificatio Amaurodermatis Rudae, Rhizoma Polygonati, Ganoderma, wherein modal and drug effect is Ganoderma lucidum (Leyss. Ex Fr.) Karst., i.e. Red Ganoderma preferably.China's ganoderma species aboundresources has the honorific title of " Ganoderma kingdom ".Be positioned at the Tibet of southeast of Qinghai-Tibet Plateau, the Ganoderma kind is also many, and the good reputation in " wild Ganoderma native place " is arranged.Here wide ecological environment is the superior place of Ganoderma breeding growth, also is the species resource storehouse of Ganoderma simultaneously.The known Ganoderma in China Tibet reaches 13 kinds, accounts for 15% of the known kind of number in the whole nation.Therefore the exploitation to the Tibet wild Ganoderma has the rich in natural resources advantage.
In the version Pharmacopoeia of the People's Republic of China in 2000 was formally listed the red ganoderma in the Ganoderma (Ganoderma lucidum), Ganoderma sinense Zhao Xu et Zhang (Ganoderma sinense) first in 2000 by China, had affirmed the drug effect of Ganoderma and as legal Chinese medicine medical material.Ministry of Public Health approval Ganoderma is as China's new resource food, and the Ganoderma modern study is used and caused that the Ganoderma drug effect is admitted in domestic and international great attention, the U.S. and Ganoderma listed in " medical herbs pharmacopeia ".Ganoderma with thousands of years cultural connotations with further Application and Development in health food and medicine.
The double-deck outer wall that Ganoderma spore has the extremely difficult chitin that is digested by human body gastric acid of one deck to constitute is only opened this layer outer wall, and the effective ingredient that is wrapped tightly by outer wall could farthest be absorbed by the human body utilization.Human body can improve 45 times more than to the absorbance of Ganoderma spore powder behind the breaking cellular wall, and the content of polysaccharide can improve 1.69 times.
The wall breaking technology of Ganoderma has physics, chemistry, biology and synthetic method at present.
Bioanalysis: comprise enzymatic isolation method, the molten method of bacterium and activate spore.Enzymatic isolation method: use cellulase, hemicellulase, protease, pectase, lysozyme, Snailase, chitinase to make the degraded of Ganoderma spore wall, reach the purpose of breaking cellular wall; The molten method of bacterium: that uses that liquor-saturated female bacterium etc. carries out Ganoderma spore sends out processing liquor-saturated, destroys the Ganoderma spore wall; Activate spore: utilize spore germination power to destroy the Ganoderma spore wall.The advantage of these class methods is that energy expenditure is little, crushing effect good, and shortcoming is that action time is very long, uses the enzyme and the breaking cellular wall bacterium of external source, and the enzyme or the bacterium of removing in the product are very difficult.
Chemical method: comprise methods such as solvent soaking, acid degradation, alkaline degradation.The advantage and the bioanalysis of this method are similar, and shortcoming is often to cause the effective ingredient degeneration, and the hazard residue in the product is higher and be difficult to remove very difficult effectiveness and the safety that guarantees product.
Physics method: use physical actions such as low temperature, supercritical, homogenate, freezing (embrittlement) ultrasound wave, microwave to destroy the Ganoderma spore wall.In general, this class methods sporoderm-broken rate is not high, and perhaps equipment investment is bigger.
Mechanical Method: destroy the Ganoderma spore wall by rolling, push, spray mechanisms such as pulverizing, comminution by gas stream bump.Advantage is simple, and shortcoming is plant equipment complex structure, price height, operating cost height.
Synthetic method: comprehensive above the whole bag of tricks is used.
(3) summary of the invention:
The problem to be solved in the present invention is exactly to provide a kind of as the molten wall bacterium of Ganoderma Ganoderma to be carried out method effective, safe breaking cellular wall with the Ganoderma endophyte at above deficiency after separating the Ganoderma endophyte.It may further comprise the steps:
1) separation, purification of ganoderma lucidum endogenetic bacteria from Ganoderma;
2) screening bacterial strain;
3) preparation enzyme liquid;
4) enzyme liquid is added in the powdery Ganoderma spore of the bacterium of having gone out, 35～37 ℃ were descended static enzymolysis 2～3 days;
5) will filter through the powdery Ganoderma spore of enzymolysis, filtrate concentrates, and is spray dried to powder; With the precipitation drying after filtering, the powder that is dried to filtrate merges again, makes Ganoderma spore powder.
Separation, the screening technique of Ganoderma endogenetic bacteria may further comprise the steps:
1) separate endogenetic bacteria:
With fresh Tibet wild Ganoderma surface sterilizing, being cut into segment sterilizes once more, to organize epidermis to prune to be cut into small pieces to paste with sterile razor blade then and be put on the culture medium, put in 35～37 ℃ of calorstats and cultivated 3～20 days, the picking marginal portion is transferred on the new culture medium after treating to grow bacterium colony around each plant tissue on the culture medium, continues to put in 35～37 ℃ of calorstats and cultivates 3-7 days;
2) purification of bacterial strain:
Adopt the picking method to institute's isolating endogenetic bacteria line purification, the single bacterium colony of picking is put in 35～37 ℃ of calorstats and is cultivated, again picking a little put microscopically and observe, the single purification bacterium colony of picking is preserved, the bacterial strain of purification is transferred to respectively on the slant medium, and two inclined-planes are preserved in every strain, cultivate 3-7 days in 35～37 ℃ of calorstats, when treating that strain growth is vigorous, 4 ℃ of preservations are put in taking-up, and switching is gone on the new slant medium after 30～60 days, and inoculation is subculture for several times.
3) screening of bacterial strain:
With the Ganoderma endogenetic bacteria of purification, be inoculated in the chitin screening culture medium, put in 35～37 ℃ of calorstats and cultivated 3～7 days, according to having or not of transparent circle, screen corresponding endogenetic bacteria;
With the Ganoderma endogenetic bacteria of purification, be inoculated in the cellulose screening culture medium, put in 35～37 ℃ of calorstats and cultivated 3～7 days, after the dyeing,, screen corresponding wall breaking enzyme according to having or not of transparent circle;
The preparation method of enzyme liquid is:
The good Ganoderma endogenetic bacteria of inoculation purification is inoculated in the culture medium, puts 35～37 ℃ of shaking tables, 200～300 rev/mins, shake OD value 0.5～0.7, get bacterium liquid, the bacterium liquid that shakes is centrifugal, get supernatant liquid filtering, promptly get enzyme liquid.
With the isolated Ganoderma endogenetic bacteria of the inventive method is series bacillus in the Ma Kuo (Paenibacillus macquariensis)-B22 CGMCC No:2918.Now be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), preservation date is on February 23rd, 2009, and deposit number is CGMCC No:2918.
Be shaft-like with the isolated Ganoderma endogenetic bacteria of the inventive method cell, Gram-positive, feminine gender or variable are moved with peritrichous.Oval spore is arranged in the cyst expanding, on Nutrient agar, do not have soluble pigment.Amphimicrobian or strict aerobic.Except two subspecies of Paenibacillus larvae, nearly all kind catalase is positive.Oxydase reaction is variable.
Compared with prior art the present invention has following beneficial effect:
1, the present invention is according to symbiosis theory, from the new fresh sporophore of Tibet wild Ganoderma and host's thereof microecological environment, separate can biological wall breaking fungal component (molten wall bacterium).The fungal component that utilizes Ganoderma itself is fermented to spore and the sporophore of Ganoderma, and the active ingredient of Ganoderma is overflowed from born of the same parents Nei Gengyi, and human body can absorb smoothly, digest, and active ingredient can make full use of.Because molten wall bacterium from Ganoderma itself, can be eliminated because of drawing as being difficult to of bringing of foreign bacteria and be removed biological residual difficulty, guarantee security of products.
2, the present invention utilizes vitro enzyme that the wall breaking enzyme in the metabolite of molten wall bacterium is also screened simultaneously, the present invention integrates the molten breaking cellular wall of bacterium, breaking cellular wall with enzyme technology, improved the extraction yield (recording Ganoderma dry extract polyoses content) of ganoderma spore polysaccharide, reduced or to have stopped harmful organism residual 0.9%.
3, do not contact technical process nature, environmental protection with organic reagent.
4, this law power consumption is low, and equipment is simple, and is easy and simple to handle, environmental protection, sustainable use.
5, directly obtain effective ingredient, shortened process.
(4) description of drawings:
Fig. 1 is process chart of the present invention.
(5) specific embodiment:
Referring to Fig. 1, the inventive method may further comprise the steps:
1, separation, purification of ganoderma lucidum endogenetic bacteria from Ganoderma:
1) separates the Ganoderma endogenetic bacteria: with tap water fresh Ganoderma surface is cleaned earlier, soaked 3 minutes with mercuric chloride earlier, then with tap water flushing sterilization, the segment that is cut into suitable length after dried slightly soaks 3～5min in 75% ethanol, with 3～4 sterilizations once more of aseptic water washing, aseptic filter paper blots, the fritter that to organize epidermis to prune to be cut into 0.5cm * 0.5cm * 0.1cm with sterile razor blade pastes and is put on the beef-protein medium then, put in 35～37 ℃ of calorstats and cultivated 3～20 days, the picking marginal portion is transferred on the new beef extract-peptone plating medium after treating to grow mycelia around each plant tissue on the culture medium, continues to put in 35～37 ℃ of calorstats and cultivates 3-7 days.
2) purification of bacterial strain: adopt the picking method to institute's isolating endogenetic bacteria line purification, the single bacterium colony of picking, put in 35～37 ℃ of calorstats and cultivate, again picking a little put microscopically and observe, the single purification bacterium colony of picking is preserved, the bacterial strain of purification is transferred to respectively on the beef extract-peptone slant medium, two inclined-planes are preserved in every strain, cultivated 3-7 days in 35～37 ℃ of calorstats, when treating that strain growth is vigorous, 4 ℃ of preservations are put in taking-up, and switching is gone on the new beef extract-peptone slant medium after 30～60 days, and inoculation is subculture for several times.
The preparation method of above-mentioned beef-protein medium is: Carnis Bovis seu Bubali cream 3g, peptone 10g, NaCl5g, agar 15～20g are added in the 1000ml distilled water mix well, transfer pH 7.2～7.6.
3) screening of bacterial strain:
With the Ganoderma endogenetic bacteria of purification, under aseptic condition, be inoculated in the chitin screening culture medium, to put in 35～37 ℃ of calorstats and cultivated 3～7 days, antibacterial secretion chitinolytic enzyme can dissolve the chitin of periphery of bacterial colonies and is transparent round.According to having or not of transparent circle, screen corresponding endogenetic bacteria.The preparation method of chitin screening culture medium is that Carnis Bovis seu Bubali cream 3g, peptone 10g, NaCl 5g, agar 15～20g, chitin 2g (chitin is worn into behind the fine powder with little acetic acid or hydrochloric acid hydrotropy) are added in the 1000ml distilled water and make, and transfers pH 7.2～7.6.
With the Ganoderma endogenetic bacteria of purification, under aseptic condition, be inoculated in the cellulose screening culture medium, put in 35～37 ℃ of calorstats and cultivated 3～7 days, behind 5mg/l congo red staining 5min, the dye liquor that inclines according to having or not of transparent circle, screens corresponding wall breaking enzyme, having transparent circle just to show has molten wall activity, can preserve and continue to employ.The method is the wall breaking enzyme in the screening Ganoderma endogenetic bacteria metabolite.The preparation method of cellulose screening culture medium is with CMC-Na 10g, (NH
4)
2SO
42g, KH
2PO
40.5g, MgSO
47H
2O 0.5g makes in peptone 1g, the agar 15～20g adding 1000ml distilled water, and heating for dissolving is mixed well.
2, the preparation of enzyme liquid:
The Ganoderma endogenetic bacteria that purification is good is inoculated in the beef extract-peptone fluid medium, put 35～37 ℃ of shaking tables, 200～300 rev/mins, shake OD value 0.5～0.7, bacterium liquid, with the bacterium liquid that shakes through 12000 rev/mins centrifugal 5 minutes, abandon precipitation, get supernatant and under 0.22 μ m condition, filter, promptly get enzyme liquid.The preparation method of beef extract-peptone fluid medium is: Carnis Bovis seu Bubali cream 3g, peptone 10g, NaCl 5g are added in the 1000ml distilled water, transfer pH 7.2～7.6, heating for dissolving is mixed well.
3, to breaking trachytectum of glossy ganoderma: the powdery Ganoderma spore of the bacterium of will having gone out adds enzyme liquid by 10% (w/V), and after shaking up gently, 35～37 ℃ were descended static enzymolysis 2～3 days.
4, will filter through the powdery Ganoderma spore of enzymolysis, filtrate concentrates, and is spray dried to powder; With the precipitation drying after filtering, the powder that is dried to filtrate merges again, makes the Ganoderma spore powder of breaking cellular wall.
Ganoderma dry extract polysaccharide determination:
Precision is measured reference substance solution 0.2,0.4,0.6,0.8,1.0 respectively, 1.2ml places in the 10ml tool plug test tube, adds the water standardize solution to 2ml.The accurate sulphuric acid anthrone solution 6ml that adds, shake up rearmounted heating in water bath 15min after, take out ice bath 15min.With the reagent corresponding is blank, uses UV spectrophotometer measuring.Measure absorbance at wavelength 625nm place.The drawing standard curve.
The preparation of need testing solution:
Get product Ganoderma spore powder with cellular wall broken of the present invention end 2g, the accurate title, decide, and puts into apparatus,Soxhlet's and add water 90ml, and heating and refluxing extraction is to colourless.Extracting solution is transferred in the 100ml measuring bottle, adds the water standardize solution, shakes up, and precision is measured 10ml, adds ethanol 150ml, shakes up.Place 12h at 4 ℃, take out, centrifugal, the tipping supernatant, precipitation is dissolved in water and transfers in the 50ml measuring bottle, and thin up to scale both shook up and got.
Assay method:
Precision is measured test sample 2ml, is placed in the 10ml tool plug test tube, and operating process is operated after adding sulphuric acid anthrone solution with reference substance.Measure absorbance in accordance with the law.
Record test sample Ganoderma dry extract polyoses content 0.9%.
Claims (5)
1. the wall-breaking method of a Ganoderma spore is characterized in that may further comprise the steps:
1) from Ganoderma, separate, series bacillus (Paenibacillus macquariensis)-B22 CGMCC No:2918 in the purification of ganoderma lucidum endogenetic bacteria Ma Kuo;
2) screening bacterial strain;
3) preparation enzyme liquid;
4) enzyme liquid is added in the powdery Ganoderma spore of the bacterium of having gone out, 35～37 ℃ were descended static enzymolysis 2～3 days;
5) will filter through the powdery Ganoderma spore of enzymolysis, filtrate concentrates, and is spray dried to powder; With the precipitation drying after filtering, the powder that is dried to filtrate merges again, makes Ganoderma spore powder.
2. according to the wall-breaking method of the described Ganoderma spore of claim 1, it is characterized in that:
Separation, the screening technique of series bacillus in the Ganoderma endogenetic bacteria Ma Kuo (Paenibacillus macquariensis)-B22 CGMCC No:2918 may further comprise the steps:
1) separate series bacillus (Paenibacillus macquariensis)-B22 CGMCC No:2918 in the endogenetic bacteria Ma Kuo:
With fresh Tibet wild Ganoderma surface sterilizing, being cut into segment sterilizes once more, to organize epidermis to prune to be cut into small pieces to paste with sterile razor blade then and be put on the culture medium, put in 35～37 ℃ of calorstats and cultivated 3～20 days, the picking marginal portion is transferred on the new culture medium after treating to grow bacterium colony around each plant tissue on the culture medium, continues to put in 35～37 ℃ of calorstats and cultivates 3-7 days;
2) purification of bacterial strain:
Adopt the picking method to the purification of ruling of series bacillus (Paenibacillus macquariensis)-B22 CGMCC No:2918 in the isolating endogenetic bacteria Ma Kuo of institute, the single bacterium colony of picking, put in 35～37 ℃ of calorstats and cultivate, again picking a little put microscopically and observe, the single purification bacterium colony of picking is preserved, be transferred to the bacterial strain of purification on the slant medium respectively, two inclined-planes are preserved in every strain, cultivated 3-7 days in 35～37 ℃ of calorstats, when treating that strain growth is vigorous, 4 ℃ of preservations are put in taking-up, and switching is gone on the new slant medium after 30～60 days, and inoculation is subculture for several times.
3) screening of bacterial strain:
With series bacillus (Paenibacillus macquariensis)-B22CGMCC No:2918 in the Ganoderma endogenetic bacteria Ma Kuo of purification, be inoculated in the chitin screening culture medium, put in 35～37 ℃ of calorstats and cultivated 3～7 days,, screen corresponding endogenetic bacteria according to having or not of transparent circle;
With series bacillus (Paenibacillus macquariensis)-B22CGMCC No:2918 in the Ganoderma endogenetic bacteria Ma Kuo of purification, be inoculated in the cellulose screening culture medium, put in 35～37 ℃ of calorstats and cultivated 3～7 days, after the dyeing, according to having or not of transparent circle, screen corresponding wall breaking enzyme;
The preparation method of enzyme liquid is:
Series bacillus (Paenibacillus macquariensis)-B22 CGMCC No:2918 is inoculated in the culture medium in the Ganoderma endogenetic bacteria Ma Kuo that the inoculation purification is good, put 35～37 ℃ of shaking tables, 200～300 rev/mins, shake OD value 0.5～0.7, get bacterium liquid, the bacterium liquid that shakes is centrifugal, get supernatant liquid filtering, promptly get enzyme liquid.
3. according to the wall-breaking method of the described Ganoderma spore of claim 2, it is characterized in that culture medium is a beef-protein medium, its raw material comprises: Carnis Bovis seu Bubali cream 3g, peptone 10g, NaCl 5g, agar 15～20g, distilled water 1000ml, pH 7.2～7.6.
4. according to the wall-breaking method of the described Ganoderma spore of claim 2, it is characterized in that chitin screening culture medium raw material comprises Carnis Bovis seu Bubali cream 3g, peptone 10g, NaCl 5g, agar 15～20g, chitin 2g, distilled water 1000ml, pH 7.2～7.6.
5. according to the wall-breaking method of the described Ganoderma spore of claim 2, it is characterized in that cellulose screening culture medium raw material comprises CMC-Na 10g, (NH
4)
2SO
42g, KH
2PO
40.5g, MgSO
47H
2O 0.5g, peptone 1g, agar 15～20g, distilled water 1000ml.
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| CN102138679A (en) * | 2010-11-30 | 2011-08-03 | 山东海之宝海洋科技有限公司 | Technique for processing and preparing kelp seaweed spore powder |
| CN102091102B (en) * | 2011-02-17 | 2012-09-05 | 吴新芳 | Glossy ganoderma extract and pharmaceutical application thereof |
| CN102498946A (en) * | 2011-11-03 | 2012-06-20 | 杨毅 | New technology for fermenting glossy ganoderma spores by edible and medicinal fungi biologically |
| CN102600213A (en) * | 2012-04-01 | 2012-07-25 | 重庆邮电大学 | Effective antioxidation method for ganoderma lucidum spore powder |
| CN103340909B8 (en) * | 2013-06-07 | 2022-07-29 | 浙江五养堂药业有限公司 | Biological wall breaking method for ganoderma lucidum spore powder |
| CN104286827B (en) * | 2014-09-16 | 2017-01-18 | 浙江亚林生物科技股份有限公司 | Method for fermenting ganoderma lucidum spore powder by using probiotics |
| CN106138116B (en) * | 2015-04-09 | 2019-07-23 | 西藏圣龙实业有限公司 | Lucidum spore powder based on Ganoderma lucidum mycelium secretase optimizes wall-breaking method |
| CN107998684A (en) * | 2016-11-02 | 2018-05-08 | 欧思佛生物科技股份有限公司 | The method of active ingredient in ferment broken wall vacuum ultrasonic low-temperature extraction plant |
| CN108212314B (en) * | 2018-01-15 | 2023-10-27 | 吉林大学 | Western medicine reducing mechanism is used in medical treatment |
| CN108543576B (en) * | 2018-05-04 | 2020-01-31 | 北京中科泰昌生物科技有限公司 | Ganoderma spore powder wall breaking equipment, wall breaking method, and application method thereof |
| CN108938681A (en) * | 2018-07-25 | 2018-12-07 | 王碧光 | It is a kind of to have both the antitumor soft wall-breaking method of lucidum spore powder enzyme fermentation of anti-aging anticancer |
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Address after: 850000 Lhasa economic and Technological Development Zone, Tibet City, Lhasa Road, No. 9 Patentee after: TIBET YUEWANG DRUGSTORE ECOLOGICAL TIBETAN MEDICINE TECHNOLOGY Co.,Ltd. Address before: Lhasa City, Tibet autonomous region 850000 empty road No. 1 cefn Druids District Patentee before: TIBET YUEWANG BIOLOGIC TECHNETRONIC Co.,Ltd. |