CN105777928B - A kind of preparation method of black fruit fructus lycii oligosaccharides - Google Patents
A kind of preparation method of black fruit fructus lycii oligosaccharides Download PDFInfo
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- CN105777928B CN105777928B CN201610273519.6A CN201610273519A CN105777928B CN 105777928 B CN105777928 B CN 105777928B CN 201610273519 A CN201610273519 A CN 201610273519A CN 105777928 B CN105777928 B CN 105777928B
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention discloses a kind of preparation method of black fruit fructus lycii oligosaccharides, this method comprises the following steps：Raw material crushes；Degreasing；Extraction；Except polysaccharide and protein；Decolourize；The separation of oligosaccharides.This method prepares black fruit fructus lycii native oligosaccharides, extraction conditions are gentle, prepared black fruit fructus lycii oligosaccharide structure is able to intact reservation, so that it further has the characteristics of easy to operate, DNA purity is high using this method, the oligosaccharides for preparing gained has immunocompetence, available for health products, or functional food additives, it is adapted to popularization and application.
Description
Technical field
The invention belongs to food and health product technology field, is related to a kind of preparation method of oligosaccharides, specifically, is related to one
The preparation method of kind black fruit fructus lycii oligosaccharides.
Background technology
Black fruit fructus lycii, Latin literary fame：Lycium ruthenicum Murr. Solanaceaes, the more quil shrubs of Lycium, more points
Branch；Branch oblique ascension is lying in ground, white or canescence, hard, and normal zig zag is tortuous, there is irregular vertical stripe, small
Branch top is tapering into echinate, and internode cripetura, berry is spherical, thin skin, atropurpureus after skin is ripe, containing abundant aubergine inside fruit
Element, water is highly soluble in, belongs to natural water-soluble anthocyanin flavonoids.Tibetan medicine consolidates for treating cardiopyretic disease, heart disease, reducing courage
Alcohol, excited cerebral nerve, enhancing immunologic function, anti-curing cancers, anti-aging, beautifying face and moistering lotion, irregular menstruation, menopause etc. and drug effect show
Write.Among the people to make strengthening by means of tonics kidney tonifying, depressor is used.Recorded in《Uygur medicine will》,《Four doctor's allusion quotations》,《Jingzhubencao》Deng Tibetan
In medicine classical works.Lycium ruthenicum is natural basic food, has the function that the acid-base property for nursing one's health human body.
Oligosaccharide is also known as oligosaccharides (English control：oligosaccharide；oligosaccharides；Oligose), it is low
The acquisition of glycan can be generally divided into following 4 kinds：Extraction, solid-state reaction under microwave method, acid-base chemical conversion method, enzyme from natural material
Hydrolyze method etc..Oligosaccharide integrates nutrition, health care, dietotherapy, is widely used in food, health products, beverage, medicine, feed and adds
Add the fields such as agent.It is the new type functional sugar source for substituting sucrose, is Twenty-First Century " future type " effect food of new generation
Product.It is a kind of new product with generally applicable scope and application prospect, it is rather popular in the world in recent years.The U.S., Japan, Europe
There is large-scale production on the ground such as continent, and the development and application of China's oligosaccharide arises from the mid-90, and development was swift and violent in recent years.
At present, function oligosaccharides is prepared generally using polysaccharide as raw material in food, and oligosaccharides is prepared through enzymatic isolation method or acid hydrolyzation, this
The structure of class oligosaccharides and native oligosaccharides has very big difference.Research on black fruit fructus lycii focuses mostly in polysaccharide, to black fruit fructus lycii day
The preparation of right oligosaccharides has no report in the prior art also without a kind of system of black fruit fructus lycii oligosaccharides easy to operate, DNA purity is high
Preparation Method.
The content of the invention
A kind of the defects of it is an object of the invention to overcome above-mentioned technology to exist, there is provided preparation side of black fruit fructus lycii oligosaccharides
Method, this method have the characteristics of easy to operate, DNA purity is high, and the oligosaccharides for preparing gained has immunocompetence, available for health care
Product, or functional food additives, it is adapted to popularization and application.
Its concrete technical scheme is：
A kind of preparation method of black fruit fructus lycii oligosaccharides, comprises the following steps：
Step 1. raw material crushes：It is standby that black fruit fructus lycii dry fruit crosses 60 mesh after being crushed with pulverizer；
Step 2. degreasing：100g black fruit fructus lycii powder adds 400mL absolute ethyl alcohol heating and refluxing extraction 2h, filtering, 50 DEG C of bakings
It is dry；
Step 3. is extracted：Black fruit fructus lycii sample 100g after extracting degreasing adds 80 DEG C of extraction 2h of 500mL distilled water, by extract solution
Filtering, residue extract 1h again with 250mL distilled water, merge extract solution twice；
Step 4. removes polysaccharide and protein：After 40 DEG C of vacuum concentrations of extract solution, ethanol degree of thickening is added to be stood to 80%
Precipitate polysaccharides and protein after 12h, filtering, it is oligosaccharides extract solution to take supernatant；
Step 5. is decolourized：The extract solution that step 4 is obtained is concentrated into 20mL, is splined on DEAE Sepharose
Fastflow ion-exchange chromatographies, eluted with 1.0mol/LNaCl solution, according to the measurement result of Phenol sulfuric acid procedure, collection contains
Sugared eluent；
The separation of step 6. oligosaccharides：Separated using activated-charcoal column, by above-mentioned containing after sugared eluent loading, first washed with distillation
Take off to sugar-free, with 40% ethanol elution, collection contains sugared eluent, and after 40 DEG C are concentrated in vacuo, freeze-drying obtains black fruit fructus lycii
Oligosaccharides.
Further, the composition of activated-charcoal column described in step 6 is specially：Activated carbon: diatom=1: 1.
Compared with prior art, beneficial effects of the present invention：
The present invention provides a kind of preparation method of black fruit fructus lycii oligosaccharides, and this method is prepared black fruit fructus lycii native oligosaccharides, carried
Mild condition is taken, prepared black fruit fructus lycii oligosaccharide structure is able to intact reservation, so that it is further applied.With behaviour
Make the characteristics of convenient, DNA purity is high, the oligosaccharides for preparing gained has immunocompetence, available for health products, or functional food
Additive, it is adapted to popularization and application.
Brief description of the drawings
Fig. 1 is the gas chromatogram of six kinds of standard monose；
Fig. 2 is the gas chromatogram of sample；
Fig. 3 is oligosaccharides ESI mass spectrograms.
Embodiment
Technical scheme is described in more detail with specific embodiment below in conjunction with the accompanying drawings.
The preparation method of the black fruit fructus lycii oligosaccharides of embodiment 1
A kind of preparation method of black fruit fructus lycii oligosaccharides, comprises the following steps：
Step 1. raw material crushes：It is standby that black fruit fructus lycii dry fruit crosses 60 mesh after being crushed with pulverizer；
Step 2. degreasing：100g black fruit fructus lycii powder adds 400mL absolute ethyl alcohol heating and refluxing extraction 2h, filtering, 50 DEG C of bakings
It is dry；
Step 3. is extracted：Black fruit fructus lycii sample 100g after extracting degreasing adds 80 DEG C of extraction 2h of 500mL distilled water, by extract solution
Filtering, residue extract 1h again with 250mL distilled water, merge extract solution twice；
Step 4. removes polysaccharide and protein：After 40 DEG C of vacuum concentrations of extract solution, ethanol degree of thickening is added to be stood to 80%
Precipitate polysaccharides and protein after 12h, filtering, it is oligosaccharides extract solution to take supernatant；
Step 5. is decolourized：The extract solution that step 4 is obtained is concentrated into 20mL, is splined on DEAE Sepharose
Fastflow ion-exchange chromatographies, eluted with 1.0mol/LNaCl solution, according to the measurement result of Phenol sulfuric acid procedure, collection contains
Sugared eluent；
The separation of step 6. oligosaccharides：Separated using activated-charcoal column, by above-mentioned containing after sugared eluent loading, first washed with distillation
(except monose and sucrose) is taken off to sugar-free, and with 40% ethanol elution, collection contains sugared eluent, and after 40 DEG C are concentrated in vacuo, freezing is dry
It is dry to obtain black fruit fructus lycii oligosaccharides.
The composition of activated-charcoal column described in step 6 is specially：Activated carbon: diatom=1: 1.
The monosaccharide composition analysis of the oligosaccharides of embodiment 2
By isolated oligosaccharide sample after 2mol/L TFA hydrolysis, NaBH4 reduction, acetylation generate corresponding sugar alcohol
Acetyl ester derivant, acetylate dissolve laggard promoting the circulation of qi analysis of hplc through dichloromethane.Six kinds of monose hybrid standard product with
And gas chromatographic analysis result of the oligosaccharide sample hydrolysate after sugar alcohol acetonyl ester derivatization is as shown in Fig. 1 and table 1, table 2.
The sugar alcohol acetyl ester derivant of the lower six kinds of monose standard items of this chromatographic condition can reach separation, according to retention time and standard
Collection of illustrative plates contrast can determine whether the monose type in sample, while the response factor of each monose can be calculated according to standard diagram, by
This obtains the mol ratio of monose composition.
Table 1：The gas chromatograph results of six kinds of standard monose
The gas chromatograph results of the sample of table 2
Understand that sample is by arabinose and glucose group by the calculating to sample and standard items appearance result and contrast
Into wherein arabinose accounts for 27%, and glucose accounts for 73%.
The mass spectral analysis of the oligosaccharides of embodiment 3
Take 1mg oligosaccharide samples to add 1ml water to dissolve with general C18 pillars, loading 5ml water elutions, carry out matter after filter membrane
Spectrum analysis.Sample is subjected to ESI-MS analyses, as a result as shown in Figure 3.Oligose fragment is carried out according to monose composition and molecular weight to return
Category.Mass-to-charge ratio (m/z) is [M+Na] that 305.3,437.3,569.3 peak is respectively the arabinose of the degree of polymerization 2~4 in figure+Peak,
Show the AOS that low polymerization degree be present.Separately there are 3 peaks to be attributed to [M+K] in mass spectrogram+, i.e., mass-to-charge ratio 381.3,
483.2 and 543.2, Glc is attributed to respectively2、GlcAra2And Glc3。
Finishing analysis result can draw table 3：
The mass spectrometry results of the oligosaccharides of table 3
Note：Ara：Arabinose Glc：Glucose
The foregoing is only a preferred embodiment of the present invention, protection scope of the present invention not limited to this, any ripe
Those skilled in the art are known in the technical scope of present disclosure, the letter for the technical scheme that can be become apparent to
Altered or equivalence replacement are each fallen within protection scope of the present invention.
Claims (1)
1. a kind of preparation method of black fruit fructus lycii oligosaccharides, it is characterised in that comprise the following steps：
Step 1. raw material crushes：It is standby that black fruit fructus lycii dry fruit crosses 60 mesh after being crushed with pulverizer；
Step 2. degreasing：100g black fruit fructus lycii powder adds 400mL absolute ethyl alcohol heating and refluxing extraction 2h, filtering, 50 DEG C of drying；
Step 3. is extracted：Black fruit fructus lycii sample 100g after extracting degreasing adds 80 DEG C of extraction 2h of 500mL distilled water, by extract solution mistake
Filter, residue extract 1h again with 250mL distilled water, merge extract solution twice；
Step 4. removes polysaccharide and protein：After 40 DEG C of extract solution is concentrated in vacuo, add ethanol degree of thickening to 80%, after standing 12h
Precipitate polysaccharides and protein, filtering, it is oligosaccharides extract solution to take supernatant；
Step 5. is decolourized：The extract solution that step 4 is obtained is concentrated into 20mL, be splined on DEAE Sepharose fastflow from
Sub- exchange chromatography post, eluted with 1.0mol/L NaCl solutions, according to the measurement result of Phenol sulfuric acid procedure, collection contains sugared eluent；
The separation of step 6. oligosaccharides：Separated using activated-charcoal column, by above-mentioned containing after sugared eluent loading, be first eluted to distilled water
After sugar-free, with 40% ethanol elution, collection contains sugared eluent, and after 40 DEG C are concentrated in vacuo, freeze-drying obtains black fruit fructus lycii oligosaccharides.
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| CN103172759B (en) * | 2013-03-18 | 2015-06-03 | 中国科学院过程工程研究所 | Method for preparing functional polysaccharide and oligosaccharide with low molecular weights by using lyceum-barbarum polysaccharide |
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