CN113583146B - Apricot pectin polysaccharide and preparation method thereof - Google Patents
Apricot pectin polysaccharide and preparation method thereof Download PDFInfo
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Abstract
A preparation method of apricot pectin polysaccharide comprises the steps of pulping apricot fruits and active water according to a material-liquid ratio of 1 (1-3) to obtain slurry, mixing the slurry and Aspergillus niger fermentation liquor for enzymolysis, after enzymolysis is finished, centrifugally concentrating by 2-4 times, adding 2-5 times of volume of absolute ethyl alcohol for alcohol precipitation, and freeze-drying for 12-24 hours to obtain the apricot pectin polysaccharide. The invention greatly simplifies the extraction steps of the natural plant polysaccharide, utilizes the aspergillus niger strains which can be separated and extracted from the nature, greatly reduces the extraction cost, improves the extraction amount and the purity of the natural plant polysaccharide, avoids using toxic and harmful reagents such as ether, chloroform and the like, and is beneficial to the mass application of the natural fruit polysaccharide.
Description
Technical Field
The invention belongs to the field of bioengineering, and relates to apricot pectin polysaccharide and a preparation method thereof.
Background
Apricot, a rose plant (Armeniaca vulgaris Lam). It is sweet, sour, mild in nature and slightly warm, and has the functions of promoting the production of body fluid to quench thirst, moistening lung and relieving asthma. The apricot has the characteristics of common fruits, and also has better development and utilization prospects in the aspect of medicine: the apricot is suitable for patients with hyperlipidemia, acute and chronic tracheitis, cough, lung cancer, nasopharyngeal carcinoma, and breast cancer, and patients after radiotherapy and chemotherapy. The research on the apricot at home and abroad is mostly focused on discussing the biological activity of amygdalin in the apricot, the research on polysaccharide in the apricot pulp is less, and the fruit polysaccharide generally has the biological activities of immunoregulation, anti-tumor, antioxidation, bacteriostasis, antivirus, blood sugar reduction, blood fat reduction and the like.
At present, the common extraction methods of polysaccharide mainly comprise a hot water extraction method, an ultrasonic extraction method, an enzyme extraction method and the like. Different extraction processes have respective advantages and disadvantages, and the yield of the polysaccharide is greatly different. The hot water extraction method has long time consumption and low polysaccharide extraction rate; although the enzyme extraction method can obtain higher polysaccharide yield, the cost of the enzyme is high, which limits the wide application of the enzyme; the ultrasonic extraction method is characterized in that ultrasonic extraction steps are added on the basis of hot water extraction, and the extraction yield is relatively high, so that the ultrasonic extraction method has relative advantages. Pectic polysaccharides are poorly soluble due to their large molecular weight, resulting in limited development.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides a high-purity apricot pectin polysaccharide and a preparation method thereof, which greatly simplifies the steps of extracting and purifying natural plant polysaccharide, improves the extraction efficiency and the purity of the natural plant polysaccharide, and increases the solubility and the thermal stability of the pectin polysaccharide.
The technical scheme is as follows: a preparation method of apricot pectin polysaccharide comprises the following steps: the apricot fruits and the active water are pulped according to a material-liquid ratio of 1 (1-3) to obtain slurry, the material-liquid ratio is a mass-volume ratio, a unit g/mL, the slurry is mixed with Aspergillus niger fermentation liquor for enzymolysis, after the enzymolysis is finished, the slurry is centrifugally concentrated by 2-4 times, 2-5 times of volume of absolute ethyl alcohol is added for alcohol precipitation, and the apricot fruit gum polysaccharide is obtained after freeze drying for 12-24 hours.
The active water is prepared by the following method: the jet discharge mode of the jet type atmosphere low-temperature plasma surface processor is utilized to process 500mL of water for 20-120s at room temperature, the relative humidity of less than 93 percent, the atmospheric pressure of 86-106Kpa and the power of 300-500 w.
Preferably, the inoculation amount of the fermentation enzyme liquid is 20-40% of the mass of the slurry, the enzymolysis temperature is 40-55 ℃, and the enzymolysis time is 6-10 h.
The apricot pectin polysaccharide prepared by the preparation method.
The obtained pectin has solubility higher than apple pectin by more than 50% in room temperature deionized water, and decomposition temperature not lower than 140 deg.C.
Has the advantages that: the invention provides a preparation method of high-purity apricot pectin polysaccharide, which is characterized in that natural plants are fused with active water, and in the process of mixing and incubating the fused plant sample and an enzyme-producing fermentation culture medium containing cell wall degrading enzymes, the cell wall degrading enzymes can hydrolyze cellulose or lignin rich in plant cell walls, so that the plant cell walls are damaged, and the natural plant polysaccharide located in cells is quickly dissolved out. In the method, the used active water is rich in active ingredients such as Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), and has special physicochemical properties such as low pH, high oxidation-reduction potential and high conductivity; the cell wall degrading enzyme is obtained by directly fermenting the enzyme-producing bacteria, and has good activity and high catalytic efficiency, thereby being beneficial to further improving the extraction efficiency of the natural plant polysaccharide. Meanwhile, the macromolecular polysaccharide is degraded and modified, so that the macromolecular pectin is degraded into micromolecular pectin polysaccharide with lower polymerization degree, and the pectin polysaccharide with higher blood fat reducing activity, blood sugar reducing activity, heat stability activity and purity and more uniformity is obtained. The extraction method greatly simplifies the extraction steps of the natural plant polysaccharide, and utilizes the aspergillus niger strains which can be separated and extracted from the nature, thereby greatly reducing the extraction cost, improving the extraction amount and the purity of the natural plant polysaccharide, avoiding using toxic and harmful reagents such as ether, chloroform and the like, and being beneficial to the mass application of the natural fruit polysaccharide.
Drawings
FIG. 1 is an electron micrograph comparison of polysaccharides from two different preparation methods, wherein a is water extraction and b is microbial fermentation extraction;
FIG. 2 is a comparison of the thermodynamic properties of polysaccharides prepared in two different ways.
Detailed Description
The present invention will be described in detail with reference to specific embodiments, and the exemplary embodiments and descriptions thereof herein are provided to explain the present invention but not to limit the present invention.
In order to further improve the extraction yield, purity and activity of the apricot pectin polysaccharide, the extraction yield and purity of the apricot pectin polysaccharide are improved by adopting an active water assisted microbial fermentation method, and the polysaccharide is degraded by microorganisms while the purity is improved by utilizing consumption of a nitrogen source and a carbon source by microbial fermentation, so that the dissolution of soluble polysaccharide is increased; the modification of the polysaccharide by using active water improves the activity of the polysaccharide. Meanwhile, macromolecular polysaccharide is degraded to be uniform polysaccharide with low polymerization degree.
Content determination of crude polysaccharide in sample
1. The determination method comprises the following steps:
a. preparation of Total sugar Standard Curve
Accurately sucking 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of glucose standard use solution, placing the standard use solution in a 25mL colorimetric tube, adding water to 1mL, adding 1mL of 5 wt.% phenol solution, fully mixing, quickly adding 5mL of concentrated sulfuric acid, standing for 10min, mixing the solution by using a vortex oscillator, placing the test tube in a water bath kettle at 30 ℃ for reaction for 20 min, and measuring the absorbance at 490nm by using a spectrophotometer. And drawing a standard curve by taking the mass of the glucose as an abscissa and the absorbance value as an ordinate.
The equation for the resulting standard curve is: 5.3319x +0.0755, R20.9967, meets the requirements of the standard curve.
b. Determination of total sugar in a sample
Accurately sucking 1mL of polysaccharide solution to be detected and placing the solution in a 25mL colorimetric tube. Then, the absorbance value was measured according to the method of step a. And calculating the glucose quality according to the standard curve.
a. Determination of reducing sugars
Accurately sucking 1.00mg/mL glucose standard solution 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL and 1.20mL, sequentially adding into 25mL colorimetric tube, adding distilled water to 2mL, adding 1.5mL 3, 5-dinitrosalicylic acid solution respectively, shaking, and boiling in boiling water bath for 5min for color development. Taking out, cooling immediately, and adding distilled water to dilute to scale marks. Zero adjustment was performed using tube 0 as a reference. The absorbance of each solution was measured with a spectrophotometer at a wavelength of 540.
And drawing a standard curve by taking the glucose content (microgrammes) as an abscissa and the absorbance A as an ordinate, wherein the equation of the obtained standard curve is as follows: 0.5918x +0.0283, R20.9996, meets the requirements of the standard curve.
b. Determination of reducing sugar content of sample
2mL of the polysaccharide solution to be measured was taken out and placed in a 25mL colorimetric tube, and 1.5mL of 3, 5-dinitrosalicylic acid solution was added thereto, and the absorbance was measured at a maximum absorption wavelength of 540nm by the same method. The content of reducing sugar in the sample can be determined by using a linear equation of a standard curve.
Content of polysaccharides in sample
Since the sample crude polysaccharide may contain a certain amount of reducing sugar, which may interfere with the final result, the content of the reducing sugar is subtracted from the mass of the sample crude polysaccharide, i.e. the content of the sample polysaccharide.
Hot water extraction method for preparing apricot pectin polysaccharide
Cleaning and pitting fructus Pruni, mixing fructus Pruni and distilled water at a ratio of (w/v, g/mL)1:1 with a high pressure homogenizer to obtain slurry, mixing about 10g fructus Pruni slurry with distilled water at a ratio of (w/v, g/mL)1:10, and extracting with 80 deg.C hot water under stirring for 4 hr. Centrifuging the filtrate at 4000r/min for 15min, pouring out supernatant, and metering to 100 mL. Concentrating by rotary evaporation under reduced pressure, pouring out, adding anhydrous ethanol until the mass fraction of ethanol is 80%, standing overnight, and pouring out the supernatant. Washing the lower layer precipitate with anhydrous ethanol, volatilizing ethanol, re-dissolving in water, decolorizing with macroporous resin, and freeze drying to obtain apricot pectin polysaccharide.
Preparation of apricot pectin polysaccharide by microbial fermentation
Culture of Strain a
Inoculating the strain in the glycerol tube stored in a refrigerator at-80 deg.C to PDA culture medium, sucking 200 μ L of bacterial liquid from each culture medium, uniformly coating on the culture medium, and culturing for 7 d.
b preparation of spore liquid
Washing spores with sterile water into 150mL Erlenmeyer flasks and adjusting the concentration to 1.55X 10 with sterile water7one/mL.
c preparation of fermentation Medium
Weighing 10g of apricot pulp without adding active water homogenate, adding 50mL of distilled water into a 250mL conical flask, inoculating Aspergillus niger spore liquid into a fermentation culture medium according to 6% of inoculation amount, adjusting the pH value to 6.0, adjusting the pH value to 121 ℃, keeping for 15min for later use, and incubating for 5 days at 140rpm to obtain fermentation enzyme liquid, wherein the other components of the culture medium are 3g/L of monopotassium phosphate, 1.5g/L of magnesium sulfate, 10g/L of CMC-Na and 0.5g/L of peptone.
Example 1 preparation of apricot pectin polysaccharides by microbial fermentation
Apricot pulp preparation: a500 mL volume of distilled water was treated for 30s with a low temperature plasma jet discharge mode at a power of 350 w. The homogenate was mixed at a ratio of 1:1 of the feed-to-liquid ratio (w/v, g/mL).
Weighing 10g of apricot pulp, adding distilled water into a conical flask according to the ratio of material to liquid (w/v, g/mL) of 1:10, adjusting the pH value to be 6.0, adding Aspergillus niger fermentation enzyme liquid according to the amount of 20% of the mass of the apricot pulp, uniformly mixing, and incubating at 40 ℃ and 120rpm for 6h to obtain enzymatic hydrolysate.
And (3) extracting polysaccharide: inactivating enzyme of the fermentation liquor at 90 deg.C for 10min, centrifuging, collecting supernatant, and diluting to 100 mL. Concentrating to 1/2 volume at 45 deg.C with rotary steaming apparatus, precipitating with 3 times volume of anhydrous ethanol, standing at 4 deg.C overnight, centrifuging the obtained precipitate, washing with anhydrous ethanol for 3 times, volatilizing ethanol, redissolving in water, performing static adsorption and decolorization with macroporous resin, and freeze drying for 12 hr to obtain pale grey white apricot polysaccharide. The total sugar content of the apricot polysaccharide in the constant volume of the effusion is determined by a phenol-sulfuric acid method, and the reducing sugar content is determined by a 3, 5-dinitrosalicylic acid method. The polysaccharide sugar content (mg/g) is (total sugar-reducing sugar)/10 g by fermentation method. The extraction rate of polysaccharide can reach 5.0 percent, the purity can reach 69.3 percent, the solubility is 85.0 percent, the alpha-glycosidase inhibition rate is 70.8 percent, and the cholate binding rate is 60.3 percent.
Example 2 preparation of apricot pectin polysaccharide by microbial fermentation
Apricot pulp preparation: a500 mL volume of distilled water was treated for 60s with a power of 400w in a low temperature plasma jet discharge mode. According to the feed-liquid ratio (w/v, g/mL) of 1: 2 to mix the homogenate.
Weighing 10g of apricot pulp, adding distilled water into a conical flask according to the ratio of material to liquid (w/v, g/mL) of 1:10, adjusting the pH value to be 6.0, adding Aspergillus niger fermentation enzyme liquid by 30% of the mass of the apricot pulp, uniformly mixing, and incubating at 50 ℃ and 100rpm for 8 hours to obtain enzymatic hydrolysate.
And (3) extracting polysaccharide: inactivating enzyme of the fermentation liquid at 90 deg.C for 10min, centrifuging, collecting supernatant, and fixing volume to a certain volume. Concentrating at 45 deg.C with rotary evaporator to 1/3 volume, precipitating with 4 times volume of anhydrous ethanol, standing at 4 deg.C overnight, centrifuging the obtained precipitate, washing with anhydrous ethanol for 3 times, volatilizing ethanol, redissolving in water, performing static adsorption decolorizing with macroporous resin, volatilizing ethanol, and freeze drying for 18 hr to obtain pale-gray white apricot polysaccharide. The total sugar content of the apricot polysaccharide in the constant volume of the effusion is determined by a phenol-sulfuric acid method, and the reducing sugar content is determined by a 3, 5-dinitrosalicylic acid method. The polysaccharide sugar content (mg/g) is (total sugar-reducing sugar)/10 g by fermentation method. The calculated polysaccharide extraction rate can reach 6.1%, the purity can reach 71.5%, the solubility is 86.3%, the alpha-glycosidase inhibition rate is 65.6%, and the cholate binding rate is 55.5%. .
Example 3 preparation of apricot pectin polysaccharide by microbial fermentation
Apricot pulp preparation: a volume of 500mL of distilled water was treated for 90s with a low temperature plasma jet discharge mode at a power of 450 w. According to the feed-liquid ratio (w/v, g/mL) of 1: 3 ratio of blending homogenate
Weighing 10g of apricot pulp, adding distilled water into a conical flask according to the ratio of material to liquid (w/v, g/mL) of 1:10, adjusting the pH value to be 6.0, adding Aspergillus niger fermentation enzyme liquid according to 40% of the mass of the apricot pulp, uniformly mixing, and incubating at 55 ℃ and 100rpm for 10 hours to obtain enzymatic hydrolysate.
And (3) extracting polysaccharide: inactivating enzyme of the fermentation liquid at 90 deg.C for 10min, centrifuging, collecting supernatant, and metering to a certain volume. Concentrating to 1/4 volume at 45 deg.C with rotary steaming apparatus, precipitating with 5 times volume of anhydrous ethanol, standing at 4 deg.C overnight, centrifuging the obtained precipitate, washing with anhydrous ethanol for 3 times, volatilizing ethanol, redissolving in water, performing static adsorption and decolorization with macroporous resin, and freeze drying for 24 hr to obtain pale grey white apricot pectin polysaccharide. The total sugar content of the apricot polysaccharide in the constant volume of the effusion is determined by a phenol-sulfuric acid method, and the reducing sugar content is determined by a 3, 5-dinitrosalicylic acid method. The polysaccharide sugar content (mg/g) is (total sugar-reducing sugar)/10 g by fermentation method. The calculated polysaccharide extraction rate can reach 5.2%, the purity can reach 64.6%, the solubility is 90.5%, the alpha-glycosidase inhibition rate is 50.2%, and the cholate binding rate is 92.6%.
TABLE 1 comparison of the indices of two different preparations
In conclusion, the extraction rate of the apricot pectin polysaccharide is increased by 25% compared with the extraction rate of the apricot pectin polysaccharide by a water method by adopting a microbial fermentation method, and the polysaccharide structure is more uniform as shown by an electron microscope picture. Meanwhile, the apricot polysaccharide mainly comprises pectin, and the pectin is degraded in the microbial fermentation process by measuring the content of galacturonic acid, so that the solubility of the pectin polysaccharide is increased. Compared with apple pectin, the polysaccharide is easy to dissolve in normal-temperature deionized water by a fermentation method, while the apple pectin is difficult to dissolve in the room-temperature deionized water, and the solubility of the apple pectin is higher than that of the apple polysaccharide by more than 50%. The research on the hypoglycemic activity and the hypolipidemic activity of the polysaccharides with two different extraction modes shows that the polysaccharides have the hypoglycemic activity and the hypolipidemic activity with different degrees. Finally, the thermodynamic property of the polysaccharides obtained by two different extraction modes is researched, the polysaccharides obtained by the microbial fermentation method start to be decomposed at about 150 ℃, the initial decomposition temperatures of the polysaccharides obtained by the hot water extraction method are respectively 140 ℃, the residual masses of the polysaccharides obtained by the microbial fermentation method and the hot water extraction method are respectively 38% and 31% under the heating of 600 ℃, and the temperatures corresponding to the stages with the most mass loss are respectively 248 ℃ and 215 ℃, so that the polysaccharides obtained by the microbial fermentation method have good water retention and thermal stability, and compared with apple pectin, the initial decomposition temperature of the apple pectin is 90 ℃, and the temperature with the most mass loss is about 230 ℃. Compared with apple pectin comprehensively, the polysaccharide obtained by a microbial fermentation method has the potential of serving as a novel heat-resistant material.
Claims (3)
1. The preparation method of the apricot pectin polysaccharide is characterized by comprising the following steps: pulping apricot fruits and active water according to a material-liquid ratio of 1 (1-3) to obtain slurry, wherein the material-liquid ratio is a mass-volume ratio and a unit g/mL, mixing the slurry with Aspergillus niger fermentation liquor for enzymolysis, centrifugally concentrating the mixture 2-4 times after the enzymolysis is finished, adding 2-5 times of absolute ethyl alcohol for alcohol precipitation, and freeze-drying the mixture for 12-24 hours to obtain apricot pectin polysaccharide; the active water is prepared by the following method: the jet discharge mode of the jet type atmosphere low-temperature plasma surface processor is utilized to process 500mL of water for 20-120s at room temperature, the relative humidity of less than 93 percent, the atmospheric pressure of 86-106Kpa and the power of 300-500 w.
2. The method for preparing apricot pectin polysaccharide according to claim 1, wherein the inoculation amount of the fermentation enzyme solution is 20% -40% of the slurry mass, the enzymolysis temperature is 40-55 ℃, and the enzymolysis time is 6-10 h.
3. Apricot pectin polysaccharides obtained by the production method according to claim 1 or 2.
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| AU4881002A (en) * | 2002-06-17 | 2003-12-18 | SweetLife Australia Pty Ltd | Composition containing xylitol gum and fibre |
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| AU4881002A (en) * | 2002-06-17 | 2003-12-18 | SweetLife Australia Pty Ltd | Composition containing xylitol gum and fibre |
| CN104974273A (en) * | 2014-04-08 | 2015-10-14 | 武永福 | Extraction process of apricot pectin and physicochemical property study thereof |
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| 曾献春等."杏多糖粗提物对荷瘤小鼠免疫功能的影响".《中国临床康复》.2005,第9卷(第18期),第142-143页. * |
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