CN105039459A - Method for extracting beta-glucan in fruiting body of ganoderma lucidum through combination of ethyl alcohol-enzyme system pre-processing and water extraction method - Google Patents
Method for extracting beta-glucan in fruiting body of ganoderma lucidum through combination of ethyl alcohol-enzyme system pre-processing and water extraction method Download PDFInfo
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Abstract
The invention discloses a method for extracting beta-glucan in the fruiting body of ganoderma lucidum through combination of ethyl alcohol-enzyme system pre-processing and a water extraction method. The method comprises the following steps: (1) smashing the fruiting body of ganoderma lucidum, and performing the ethyl alcohol-enzyme system pre-processing; (2) performing centrifugation to obtain filter residues of the fruiting body of ganoderma lucidum after pre-processing; (3) extracting beta-glucan from the filter residues of the fruiting body of ganoderma lucidum after pre-processing with hot water, and performing centrifugation to obtain supernatant containing crude beta-glucan; (4) adding alpha-1,4-glucose hydrolytic enzyme into the supernatant for processing alpha-type polysaccharide; (5) recycling the product: performing alcohol-precipitation on the alpha-1, 4-glucose hydrolytic enzyme treatment liquid, and performing centrifugation, so as to collect sediments, namely the beta-glucan of the ganoderma lucidum. According to the invention, the technical conditions are mild, the process is simple, the yield of ganoderma lucidum beta-glucan as the product is high, and the method is a practical extraction technology for an important polysaccharide component (beta-glucan) of ganoderma lucidum.
Description
Technical field
The present invention relates to medicinal fungi beta-glucan extractive technique field, relate in particular to the method that a kind of ethanol-enzyme system pre-treatment Bound moisture formulation extracts beta-glucan in Ganoderma sporophore.
Background technology
Glossy ganoderma is the red sesame kind Ganodermalucidum of fungi Basidiomycotina Basidiomycotina Hymenomycetes Hymenomycetes Aphyllophorales Polyporales polyporaceae Polyporaceae Ganoderma Ganoderma, and its formal name used at school orders Ganodermalingzhi by name recently.Ganoderma sporophore is conduct rare Chinese medicine simply from ancient times, is once called as in ancient times " celestial grass ", begins to be loaded in Chinese Shennong's Herbal.Ganoderan is one of main composition of glossy ganoderma, and wherein the polysaccharide of glossy ganoderma beta configuration is the composition of most activity in ganoderan, has multiple pharmacological effect such as improving immune, antitumor, anti-oxidant, hypoglycemic and blood fat.
The extractive technique report of existing glossy ganoderma total polysaccharides or Crude polysaccharides, but in glossy ganoderma, the extractive technique report of beta-glucan is less, in glossy ganoderma beta-glucan leaching process, also do not take the report of ethanol-enzyme system preconditioning technique simultaneously.
Document: Li Jing. response surface optimization ganoderan extracts research (foodstuffs industry, 2013) the ultrasonic extraction method technology of polysaccharide in Ganoderma sporophore is reported, but the extractive technique of glossy ganoderma total polysaccharides, not for glossy ganoderma beta-glucan associated extraction technology, more there is no ethanol-enzyme system preconditioning technique report.
Document: Sun little Mei, Dai Jun, Chen Shangwei, Zhu Song. the polysaccharide molecule quality comparation (food and fermentation industries of ganoderma lucidum fruitbody polysaccharide modifiedly extracted method and different sources ganoderma lucidum sporocarp, 2014) report the ultrasonic assistant of polysaccharide in Ganoderma sporophore, microwave-assisted, accelerated solvent extraction and water-bath to reflux 4 kinds of extractive techniques, but are all the extractive techniques for glossy ganoderma total polysaccharides, not for glossy ganoderma beta-glucan associated extraction technology, there is no ethanol-enzyme system preconditioning technique report yet.
Document: Liu Junfa, Feng Mengying, You Lijun, Luo Wei, Zhao Qiangzhong, Zhao Mouming. ultrasonic and water extraction extracts the structure of ganoderan and comparative studies (the foodstuffs industry science and technology of oxidation-resistance, 2013) ultrasonic and water extraction 2 kinds of extractive techniques of polysaccharide in Ganoderma sporophore are reported, but are all the extractive techniques for glossy ganoderma total polysaccharides, not for glossy ganoderma beta-glucan associated extraction technology, and report in literary composition Ganoderma sporophore pre-treatment adopt REISH add 95% ethanol in the ratio of solid-liquid ratio 1:6 (w/v), reflux 3h under micro-state of boiling, do not relate to ethanol-enzyme system preconditioning technique.
Chinese patent: Academy of Agricultural Sciences, Shanghai City, application number 201410293682, proposes a kind of ganoderan and preparation method thereof.The preparation method of this ganoderan comprises: Ganoderma sporophore carries out water extraction, obtains extracting residue; Extract residue and carry out micronizing, obtain ultrafine powder; Ultrafine powder is through water extract-alcohol precipitation, and the precipitation collected is ganoderan.But research object is still glossy ganoderma total polysaccharides, do not relate to glossy ganoderma beta-glucan.
Chinese patent: Wuhan Su Feima rope Science and Technology Ltd., application number 201310650365.4, proposes a kind of extracting method of ganoderan.After it utilizes Ganoderma sporophore dry powder to remove fat, join in the extraction agent of water and glycerine composition and extract, have and improve extraction efficiency and avoid in traditional method the pollution adopting the organic solvent such as chloroform, formaldehyde or acid base pair environment, but still be the technology extracted for total polysaccharides, not specifically for glossy ganoderma beta-glucan, and do not relate to ethanol-enzyme system preconditioning technique.
We are found by a large amount of experiments, take ethanol-enzyme system pre-treatment effectively can improve the extraction efficiency of glossy ganoderma beta-glucan to technology in glossy ganoderma beta-glucan leaching process.Because beta-glucan does not dissolve in ethanolic soln, therefore its molecule can keep integrity.Ethanol can remove the impurity such as grease, pigment, oligose and small-molecule substance in starting material.In addition, in order to remove this kind of biomacromolecule of protein to the interference of Polyose extraction and raising product purity, in the ethanolic soln of certain mass mark, adding appropriate proteolytic enzyme effectively can remove proteinaceous impurities.Therefore, ethanol-enzyme system pre-treatment has good result for the extraction of glossy ganoderma beta-glucan.By repeatedly testing, obtain a kind of can significantly improve glossy ganoderma beta-glucan extraction efficiency and the easy beta-glucan of production technique extracts pretreatment technology.
Summary of the invention
The object of this invention is to provide the method that a kind of ethanol-enzyme system pre-treatment Bound moisture formulation extracts beta-glucan in Ganoderma sporophore.The method mild condition, technique are simple, the yield of products obtained therefrom glossy ganoderma beta-glucan is high.
Ethanol-enzyme system pre-treatment Bound moisture formulation extracts a method for beta-glucan in Ganoderma sporophore, and the method comprises the steps:
1) Ganoderma sporophore is pulverized, carry out ethanol-enzyme system pre-treatment;
2) the pretreated Ganoderma sporophore filter residue of centrifugal acquisition;
3) hot water extraction beta-glucan is carried out to pretreated Ganoderma sporophore filter residue, through the supernatant liquor of centrifugal acquisition containing thick beta-glucan;
4) in supernatant liquor, add α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme to process α configuration polysaccharide;
5) Product recycling: alcohol is carried out to α-Isosorbide-5-Nitrae-glucose hydrolysis ferment treatment liquid and analyses, be glossy ganoderma beta-glucan through centrifugal collecting precipitation part.
Step 1) in Ganoderma sporophore is crushed to 40-60 order.
Step 1) in ethanol-enzyme system pretreatment reaction system, according to 10-15:1L/kg, Ganoderma sporophore is joined massfraction be in the ethanolic soln of 50-70%, then add trypsinase and carry out enzymolysis: enzyme concentration accounts for the 1.0-2.0% of Ganoderma sporophore quality, enzyme activity 30000-50000u/g.The pretreated pH7.0-9.0 of ethanol-enzyme system, temperature 45-50 DEG C, enzymolysis time 30-50min.Mechanical stirring 20-40min under 100-200rpm/min is added after ethanolic soln in reaction system; Then add trypsinase and carry out enzymolysis.
Step 2) under 2500-4500r/min centrifugal 10-20min obtain pretreated Ganoderma sporophore filter residue.
Step 3) get pretreated Ganoderma sporophore filter residue, add water according to 15-20:1L/kg, lixiviate 1.5-3.5h at 80-90 DEG C of temperature; Collected by centrifugation is containing the supernatant liquor of beta-glucan.After lixiviate under 6000-8000r/min centrifugal 5-15min.
Step 4) in containing the supernatant liquor of beta-glucan, add α-Isosorbide-5-Nitrae-glucose hydrolysis ferment treatment, enzyme activity 40000-60000u/g, enzyme concentration accounts for the 1.0-2.0% of Ganoderma sporophore filter residue quality, enzymolysis pH3.5-4.0, hydrolysis temperature 45-50 DEG C, enzymolysis time 40-60min.
Step 5) in solution after α-Isosorbide-5-Nitrae-glucose hydrolysis ferment treatment, add the industrial alcohol of 3-4 times of volume, at 4-6 DEG C of temperature, place 10-14h carry out alcohol and analyse, then centrifugal 8-12min under 6000-8000r/min, remove supernatant, the precipitation of collection is glossy ganoderma beta-glucan product.
In the present invention, the measuring method of glossy ganoderma beta-glucan content is: congo red method.Accurate pipette samples liquid 1.0mL, be supplemented to 2.0mL with distilled water, then add the Congo red buffered soln of 4.0mL, shake up, at 20 DEG C, accurately act on 10min, then under 544nm wavelength, measure light absorption value, gather content with β-Portugal that beta-glucan sterling is standard substance calculation sample.
Compared with the extractive technique of existing ganoderan, the present invention has following advantage:
1) product object that the present invention extracts is meticulousr product glossy ganoderma beta-glucan (component of the most activity in polysaccharide), and non-generic polysaccharide product, correlation technique is novel.
2) present invention employs a kind of easy, efficient, pretreatment process---ethanol-enzyme system pretreatment process that beta-glucan yield is high, after the method process, extract through water extraction again and can obtain stay-in-grade glossy ganoderma beta-glucan, yield can reach 0.421mg/g, is 2.1 times that traditional method (without ethanol-enzyme system pre-treatment) extracts beta-glucan yield (0.201mg/g).
3) glossy ganoderma beta-glucan extraction yield of the present invention is high, and extraction process is simple, is applicable to suitability for industrialized production.
Embodiment
Be intended to further illustrate the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1:
Ganoderma sporophore: Specialty Co-operative Organization provides purchased from Distributions in Liaocheng of Shandong Province Jing Cai agricultural-food.
Trypsin 40000u/g) and α-Isosorbide-5-Nitrae-glucose hydrolysis ferment treatment (enzyme activity 50000u/g), purchased from Rui Feng bio tech ltd, Shanghai.
Be raw material with Ganoderma sporophore, be ground into 40-60 order powder.Take above-mentioned Ganoderma sporophore powder 1.1kg in 30L mechanical agitator tank, add the ethanolic soln that 15L massfraction is 58% (v/v), 180rpm/min stirs 30min, then 20g trypsinase is added, be 7.8 at pH, enzymolysis 40min under 46 DEG C of conditions, then centrifugal 15min under 3500r/min, obtain Ganoderma sporophore filter residue after pretreatment.
Get above-mentioned pretreated Ganoderma sporophore filter residue 1kg, add 20L tap water, lixiviate 2.5h at 85 DEG C, then centrifugal 10min under 7000r/min, collects extracting solution, is concentrated into 5L, add 16g α-1,4-glucose hydrolysis enzyme, under pH4.0, temperature 45 C, enzymolysis 50min, add the dehydrated alcohol of 4 times of volumes again, at 6 DEG C, place 12h, then under 7000r/min, centrifugal 10min removes supernatant liquor, collecting precipitation and glossy ganoderma beta-glucan product.
The mensuration of beta-glucan content.Accurate pipette samples liquid 1.0mL, be supplemented to 2.0mL with distilled water, then add the Congo red buffered soln of 4.0mL, shake up, at 20 DEG C, accurately act on 10min, then under 544nm wavelength, measure light absorption value, gather content with β-Portugal that beta-glucan sterling is standard substance calculation sample.The yield of gained glossy ganoderma beta-glucan is 0.377mg/g.
Embodiment 2:
The pulverizing of glossy ganoderma sample is with embodiment 1.Take above-mentioned Ganoderma sporophore powder 1.1kg in 30L mechanical agitator tank, add the ethanolic soln that 12L massfraction is 65% (v/v), 200rpm/min stirs 30min, then 18g trypsinase is added, be 7.8 at pH, enzymolysis 40min under 48 DEG C of conditions, then centrifugal 15min under 3500r/min, obtain Ganoderma sporophore filter residue after pretreatment.
Get above-mentioned pretreated Ganoderma sporophore filter residue 1kg, add 18L tap water, lixiviate 2.5h at 85 DEG C, then centrifugal 10min under 7000r/min, collects extracting solution, is concentrated into 5L, add 18g α-1,4-glucose hydrolysis enzyme, at pH4.0, temperature 48 DEG C, enzymolysis 50min, add the dehydrated alcohol of 4 times of volumes again, at 6 DEG C, place 12h, then under 7000r/min, centrifugal 10min removes supernatant liquor, collecting precipitation and glossy ganoderma beta-glucan product.
Beta-glucan assay is with embodiment 1, and the yield preparing gained glossy ganoderma beta-glucan is 0.391mg/g.
Embodiment 3:
The pulverizing of glossy ganoderma sample is with embodiment 1.Take above-mentioned Ganoderma sporophore powder 1.1kg in 30L mechanical agitator tank, add the ethanolic soln that 15L massfraction is 60% (v/v), 180rpm/min stirs 30min, then 20g trypsinase is added, be 7.8 at pH, enzymolysis 40min under 46 DEG C of conditions, then centrifugal 15min under 3500r/min, obtain Ganoderma sporophore filter residue after pretreatment.
Get above-mentioned pretreated Ganoderma sporophore filter residue 1kg, add 20L tap water, lixiviate 2.5h at 85 DEG C, then centrifugal 10min under 7000r/min, collects extracting solution, is concentrated into 5L, add 20g α-1,4-glucose hydrolysis enzyme, under pH3.8, temperature 45 C, enzymolysis 50min, add the dehydrated alcohol of 4 times of volumes again, at 6 DEG C, place 12h, then under 7000r/min, centrifugal 10min removes supernatant liquor, collecting precipitation and glossy ganoderma beta-glucan product.
Beta-glucan assay is with embodiment 1, and the yield preparing gained glossy ganoderma beta-glucan is 0.421mg/g.
Embodiment 4:
The pulverizing of glossy ganoderma sample is with embodiment 1.Take above-mentioned Ganoderma sporophore powder 1.1kg in 30L mechanical agitator tank, add the ethanolic soln that 15L massfraction is 50% (v/v), 180rpm/min stirs 30min, then 18g trypsinase is added, be 7.8 at pH, enzymolysis 40min under 46 DEG C of conditions, then centrifugal 15min under 3500r/min, obtain Ganoderma sporophore filter residue after pretreatment.
Get above-mentioned pretreated Ganoderma sporophore filter residue 1kg, add 20L tap water, lixiviate 2.5h at 85 DEG C, then centrifugal 10min under 7000r/min, collects extracting solution, is concentrated into 5L, add 14g α-1,4-glucose hydrolysis enzyme, under pH3.9, temperature 45 C, enzymolysis 50min, add the dehydrated alcohol of 4 times of volumes again, at 6 DEG C, place 12h, then under 7000r/min, centrifugal 10min removes supernatant liquor, collecting precipitation and glossy ganoderma beta-glucan product.
Beta-glucan assay is with embodiment 1, and the yield preparing gained glossy ganoderma beta-glucan is 0.357mg/g.
Comparative example 1:
The pulverizing of glossy ganoderma sample is with embodiment 1.Take above-mentioned Ganoderma sporophore powder 1.1kg in 30L mechanical agitator tank, without ethanol-enzyme system process, directly add 20L tap water, lixiviate 2.5h at 85 DEG C, below process same embodiment 2.Finally collect glossy ganoderma beta-glucan product.
Beta-glucan assay is with embodiment 1, and the yield preparing gained glossy ganoderma beta-glucan is 0.201mg/g.
Claims (10)
1. ethanol-enzyme system pre-treatment Bound moisture formulation extracts a method for beta-glucan in Ganoderma sporophore, and it is characterized in that, the method comprises the steps:
1) Ganoderma sporophore is pulverized, carry out ethanol-enzyme system pre-treatment;
2) the pretreated Ganoderma sporophore filter residue of centrifugal acquisition;
3) hot water extraction beta-glucan is carried out to pretreated Ganoderma sporophore filter residue, through the supernatant liquor of centrifugal acquisition containing thick beta-glucan;
4) in supernatant liquor, add α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme to process α configuration polysaccharide;
5) Product recycling: alcohol is carried out to α-Isosorbide-5-Nitrae-glucose hydrolysis ferment treatment liquid and analyses, be glossy ganoderma beta-glucan through centrifugal collecting precipitation part.
2. method according to claim 1, is characterized in that: step 1) in Ganoderma sporophore is crushed to 40-60 order.
3. method according to claim 1, it is characterized in that: step 1) in ethanol-enzyme system pretreatment reaction system, according to 10-15:1L/kg, Ganoderma sporophore is joined massfraction be in the ethanolic soln of 50-70%, then add trypsinase and carry out enzymolysis: enzyme concentration accounts for the 1.0-2.0% of Ganoderma sporophore quality, enzyme activity 30000-50000u/g.
4. method according to claim 3, is characterized in that: the pretreated pH7.0-9.0 of ethanol-enzyme system, temperature 45-50 DEG C, enzymolysis time 30-50min.
5. method according to claim 3, is characterized in that: in reaction system, to add after ethanolic soln mechanical stirring 20-40min under 100-200rpm/min; Then add trypsinase and carry out enzymolysis.
6. method according to claim 1, is characterized in that: step 2) under 2500-4500r/min centrifugal 10-20min obtain pretreated Ganoderma sporophore filter residue.
7. method according to claim 1, is characterized in that: step 3) get pretreated Ganoderma sporophore filter residue, add water according to 15-20:1L/kg, lixiviate 1.5-3.5h at 80-90 DEG C of temperature; Collected by centrifugation is containing the supernatant liquor of beta-glucan.
8. method according to claim 7, is characterized in that: after lixiviate under 6000-8000r/min centrifugal 5-15min.
9. method according to claim 1, it is characterized in that: step 4) in containing the supernatant liquor of beta-glucan, add α-1,4-glucose hydrolysis ferment treatment, enzyme activity 40000-60000u/g, enzyme concentration accounts for the 1.0-2.0% of Ganoderma sporophore filter residue quality, enzymolysis pH3.5-4.0, hydrolysis temperature 45-50 DEG C, enzymolysis time 40-60min.
10. method according to claim 1, it is characterized in that: step 5) at α-1, in solution after 4-glucose hydrolysis ferment treatment, add the industrial alcohol of 3-4 times of volume, at 4-6 DEG C of temperature, place 10-14h carry out alcohol and analyse, then centrifugal 8-12min under 6000-8000r/min, remove supernatant, the precipitation of collection is glossy ganoderma beta-glucan product.
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| CN115558037A (en) * | 2022-10-19 | 2023-01-03 | 陕西科技大学 | Ganoderma lucidum beta-glucan extract and preparation method and detection method thereof |
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| CN101805414A (en) * | 2010-04-06 | 2010-08-18 | 无限极（中国）有限公司 | Preparation method of ganoderma lucidum polysaccharide with high yield |
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| CN101805414A (en) * | 2010-04-06 | 2010-08-18 | 无限极（中国）有限公司 | Preparation method of ganoderma lucidum polysaccharide with high yield |
| CN102766221A (en) * | 2012-08-15 | 2012-11-07 | 苏州菩芸生物科技有限公司 | Extraction method of ganoderan |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115558037A (en) * | 2022-10-19 | 2023-01-03 | 陕西科技大学 | Ganoderma lucidum beta-glucan extract and preparation method and detection method thereof |
| CN115558037B (en) * | 2022-10-19 | 2024-02-06 | 陕西科技大学 | Ganoderma lucidum beta-glucan extract and preparation method and detection method thereof |
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