CN117050208A - Young kiwi fruit polysaccharide extract and preparation method and application thereof - Google Patents
Young kiwi fruit polysaccharide extract and preparation method and application thereof Download PDFInfo
- Publication number
- CN117050208A CN117050208A CN202311186897.7A CN202311186897A CN117050208A CN 117050208 A CN117050208 A CN 117050208A CN 202311186897 A CN202311186897 A CN 202311186897A CN 117050208 A CN117050208 A CN 117050208A
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- China
- Prior art keywords
- extraction
- fruit
- polysaccharide
- ethanol
- kiwi fruit
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Links
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Classifications
-
- 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
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Abstract
The invention provides a high-activity kiwi fruit young fruit polysaccharide extract, and a preparation method and application of the extract. The invention fully utilizes resources, and uses the young kiwi fruits obtained by thinning fruits to prepare the polysaccharide. The extraction parameters of extracting kiwi fruit young fruit polysaccharide by using a microwave-assisted eutectic solvent (DES) are optimized through a single factor experiment and a Box-Behnken center combined design-response surface method, and higher extraction rate and shorter extraction time are obtained. And the polysaccharide extracted by the microwave assisted DES has lower molecular weight and higher uronic acid content, antioxidant activity and immunological activity than the polysaccharide extracted by the traditional water extraction. The invention reduces the resource waste of the kiwi fruit industry chain and improves the commercial value of kiwi fruit byproducts (fruit thinning/young fruits).
Description
Technical Field
The invention relates to a kiwi fruit young fruit polysaccharide extract, a preparation method and application thereof.
Background
Kiwi fruit (Actinidia chinensis plane.) is a perennial woody plant of the genus kiwi of the family kiwiaceae, with white villi on the young branches; leaf paper, in the shape of an inverted broad oval; the flower heads of the umbrella are small in flower pieces and oval; the fruit is yellow brown, spherical or inverted oval. The kiwi fruits are rich in taste, sweet and sour, are popular with people, and are widely planted worldwide. The planting area and yield of Chinese kiwi fruits are ranked first in the world. The ripe kiwi fruits are rich in vitamin C, carotenoid, polyphenol, flavone, polysaccharide, protein, amino acid, calcium, selenium, germanium and other microelements beneficial to human bodies. In the related researches, the mature kiwi fruits have the functions of resisting oxidation, resisting inflammation, resisting diabetes, losing weight, treating ulcer and the like, and the functions are indistinguishable from the nutrient substances and the active ingredients which are rich in the kiwi fruits. The polysaccharide is one of important active ingredients in the mature fruit of the kiwi fruit, and research reports on the mature fruit of the kiwi fruit prove that the mature fruit polysaccharide of the kiwi fruit of different varieties and different producing areas have good antioxidant activity, glycosylation resistance and prebiotic activity, but no related research on the young fruit polysaccharide of the kiwi fruit exists at present.
The young kiwi fruits are used as byproducts of the kiwi fruit planting industry and are generally produced during physiological fruit dropping or artificial fruit thinning. Because the natural fruit bearing rate of the kiwi fruit is higher, the fruit is thin and small, the fruit farmers can dredge the fruit about fifteen days after the kiwi fruit flowers, and half of young fruits in one fruit tree can be removed. The young fruits are low in maturity and cannot be eaten normally, and are usually discarded at will in an orchard, so that not only is the valuable resource wasted greatly, but also the young fruits become hosts of pathogenic bacteria, and the propagation of diseases and insect pests of fruit trees is accelerated. However, these young fruits have considerable nutritional value and their functional active substance content is not weaker than that of mature fruits. Application number: CN201811505053.3, name: the application of the kiwi fruit extract in fresh meat preservation discloses the application of the kiwi fruit extract in fresh meat preservation, the kiwi fruit extract and distilled water are uniformly mixed to prepare a preservative, and then the preservative is treated by adopting a spraying mode to treat fresh meat, so that the shelf life of meat products can be prolonged, but no literature report is available at present on active substances in kiwi fruit and the effect of the active substances.
The traditional extraction method of plant polysaccharide is hot water extraction method, but the extraction rate is lower. The eutectic solvent (Deep eutectic solvent, DES) is a green solvent consisting of a hydrogen bond acceptor and a hydrogen bond donor. Compared with the traditional solvent, the polysaccharide extracted by DES has the advantages of high extraction rate, complete structure, high biological activity, certain selective extraction, and the like. In some researches, microwave, ultrasonic or high-pressure homogenization is used for auxiliary extraction, and the microwave-assisted extraction has the advantages of quick temperature rise, short extraction time, high efficiency, energy conservation, easiness in control and the like. Therefore, the high-activity kiwi fruit young fruit polysaccharide can be efficiently prepared by using a microwave-assisted eutectic solvent extraction method.
Disclosure of Invention
The technical scheme of the invention is to provide a high-activity kiwi fruit young fruit polysaccharide extract. The invention also provides a high-efficiency preparation method and application of the kiwi fruit young fruit polysaccharide extract.
The invention provides a kiwi fruit young fruit polysaccharide extract, which is prepared by extracting and separating kiwi fruit young fruits serving as raw materials, wherein:
every 100mg of kiwi fruit young fruit polysaccharide contains total sugar: (92.53+ -1.29) - (94.28 + -0.72) mg; the esterification degree of the kiwi fruit polysaccharide is as follows: (32.68±0.32)% - (46.29 ±0.35)%;
the molecular weight of the kiwi fruit young fruit polysaccharide is as follows: (1.265.+ -. 0.015). Times.10 5 –(6.081±0.03)×10 5 Da, the polydispersity is 2.312-2.686; the component sugar of the kiwi fruit young fruit polysaccharide mainly comprises the following monosaccharides: galactose, arabinose, galacturonic acid, mannose, glucuronic acid, rhamnose and glucose, and the molar ratio of the monosaccharides is as follows: (2.26-3.33): (0.94-1.99):1.00: (0.16-0.49): (0.12-0.38): (0.29-0.30): (0.21-0.26).
Wherein the extract contains total uronic acid: (23.51 + -0.81) - (30.50+ -1.63) mg; total protein: (3.31.+ -. 0.41) - (3.42.+ -. 0.43) mg; total phenols: (1.01.+ -. 0.01) - (6.64.+ -. 0.26) mg GAE (gallic acid equivalent).
Further preferably, it is prepared by microwave-assisted eutectic solvent extraction;
every 100mg of kiwi fruit young fruit polysaccharide contains total sugar: 92.53+ -1.29 mg; 30.50+ -1.63 mg of total uronic acid; total protein: 3.42.+ -. 0.43mg; total phenols: 6.64.+ -. 0.26mg GAE (gallic acid equivalent);
the molecular weight of the kiwi fruit young fruit polysaccharide is as follows: 1.265×10 5 –1.406×10 5 Da, polydispersity of 2.686-2.947; the component sugar of the kiwi fruit young fruit polysaccharide mainly comprises the following monosaccharides: galactose, arabinose, galacturonic acid, mannose, glucuronic acid, rhamnose and glucose, and the molar ratio of the monosaccharides is as follows: 2.26-2.86:0.94-1.24:1.00:0.16-0.17:0.12-0.13:0.30-0.52:0.26-0.30.
Wherein, the kiwi fruit young fruit is derived from the young fruit of kiwi fruit after thinning.
The invention also provides an extraction method for preparing the kiwi fruit young fruit polysaccharide, which comprises the following steps:
a, freeze-drying young kiwi fruits, pulverizing into powder, and sieving;
b, adding ethanol into the powder, and ultrasonically removing alcohol-soluble components to obtain a precipitate;
c, hot water extraction: b, adding water into the precipitate prepared in the step b, and extracting in a water bath to obtain an extracting solution;
or microwave-assisted eutectic solvent extraction: b, adding the prepared precipitate into a prepared extraction solvent, and carrying out microwave extraction to obtain an extraction solution;
d, adding alpha-amylase and saccharifying enzyme into the concentrated solution to remove starch;
e, adding ethanol into the supernatant, and precipitating the supernatant with ethanol overnight; centrifuging to obtain precipitate, and washing the precipitate with ethanol;
f, adding water for redissolution, dialyzing and drying to obtain the kiwi fruit young fruit polysaccharide.
It is further preferred that the composition comprises,
the freeze drying condition in the step a is that the freeze drying is carried out for 40 to 70 hours at the temperature of-30 to-70 ℃; the number of the screening meshes is 60 to 120 meshes;
b, adding ethanol with the concentration of 20-90% into the mixture; the weight-volume ratio of the powder to the ethanol is as follows: 1: (10-30); the ultrasonic power is 300-800W;
c, the ratio of water to raw materials in the step is 10-60mL/g; extracting in water bath for 2-6 hr;
or microwave-assisted eutectic solvent extraction: the volume ratio of the eutectic solvent to the water is (3-15): (17-5); the ratio of the extraction solvent to the raw materials is 10-60mL/g, the power of microwave extraction is 480-720W, and the extraction time is 6-30min;
the conditions for removing starch by the alpha-amylase and the saccharifying enzyme in the step d are respectively as follows: 5-20 mu/mL alpha-amylase, 60-95 ℃ for 3-12h;5-20 mu/mL saccharifying enzyme, 40-65 ℃ and 10-14h;
and e, in the step of ethanol precipitation, the volume ratio of the ethanol extract to the ethanol is 1: (2-5) ethanol with the concentration of 50-100 percent; the ethanol used for washing the precipitate is 50-95% ethanol;
and f, dialyzing to remove small molecules, wherein the molecular weight cut-off of the small molecules in the step f is 2000-10000Da.
Still further preferably the method comprises the steps of,
step a, freeze drying for 48 hours at the temperature of minus 40 ℃; the sieving number is 80 meshes;
b, adding 80% ethanol in the step; the weight-volume ratio of the powder to the ethanol is 1:10; the ultrasonic power is 640W;
c, the ratio of water to raw materials in the step is 30mL/g; the water bath extraction time is 4 hours;
or microwave-assisted eutectic solvent extraction: the volume ratio of DES to water is 16:9, a step of performing the process; the ratio of the extraction solvent to the raw materials is 30mL/g, the microwave extraction power is 635W, and the extraction time is 17min;
d, adding 5 mu/mL of alpha-amylase in the step, and heating at 80 ℃ for 60 hours; the addition amount of the saccharifying enzyme is 5 mu/mL, the temperature is 59 ℃, and the period of 12 hours is 12 hours;
and e, the volume ratio of the ethanol precipitation is 1:4, a step of; the ethanol is 95% ethanol; the ethanol used for washing the precipitate is 76% ethanol;
the molecular weight cut-off of the small molecules removed by dialysis in the step f is 3500Da.
The invention also provides application of the kiwi fruit young fruit polysaccharide extract in preparing health-care food or medicine with an antioxidant function.
The invention also provides application of the kiwi fruit young fruit polysaccharide extract in preparation of health-care food or medicine with the immunity enhancing function.
The invention provides a composition with an antioxidant function or an immunity enhancing function, which comprises the kiwi fruit young fruit polysaccharide extract and a preparation prepared from auxiliary materials or auxiliary components acceptable in foods or medicines.
The kiwi fruit young fruit polysaccharide extract can be used as a prebiotic, and can promote proliferation and activity of beneficial bacteria by providing nutrition and growth environment for the beneficial bacteria, so that the intestinal health is positively influenced.
The invention fully utilizes resources, and uses the young kiwi fruits obtained by thinning fruits to prepare the polysaccharide. The extraction parameters of extracting kiwi fruit young fruit polysaccharide by using the microwave-assisted DES are optimized through a single factor experiment and a Box-Behnken center combined design-response surface method, and higher extraction rate and shorter extraction time are obtained. And the polysaccharide extracted by the microwave assisted DES has higher uronic acid content, antioxidant activity and immune activity and lower molecular weight than the polysaccharide extracted by the traditional water extraction. The invention reduces the resource waste of the kiwi fruit industrial chain and improves the commercial value of kiwi fruit byproducts.
Drawings
FIG. 1A process flow diagram of microwave-assisted eutectic solvent extraction
FIG. 2 is a three-dimensional perspective view of the effect of the water content (A), microwave power (B) and extraction time (C) of the eutectic solvent on the extraction rate of the polysaccharide from the young kiwi fruits by the microwave-assisted eutectic solvent extraction method, and the response surface of each factor (D, E, F) on the extraction rate of the polysaccharide from the young kiwi fruits
FIG. 3 size exclusion chromatogram (A) of the traditional hot water extracted kiwi fruit young fruit polysaccharide (YKP-H) and the microwave assisted eutectic solvent extracted kiwi fruit young fruit polysaccharide (YKP-DM), high performance liquid chromatogram (B) of the constituent monosaccharides and Fourier transform infrared spectrogram (C)
FIG. 4 illustrates the conventional hot water extracted kiwi fruit young fruit polysaccharide (YKP-H) and the microwave-assisted eutectic solvent extracted kiwi fruit young fruit polysaccharide (YKP-DM) 1 H nuclear magnetic resonance spectrum 13 C nuclear magnetic resonance spectrum spectrogram
FIG. 5. Kiwi berry young fruit polysaccharide (YKP-H) and microwave-assisted eutectic solvent extracted Kiwi berry young fruit polysaccharide (YKP-DM) clearance to ABTS (A) and clearance to DPPH (B), and their Total reducing force (C) and Effect on probiotic proliferation (D)
FIG. 6 effects of traditional Hot Water extracted Kiwi berry young fruit polysaccharide (YKP-H) and microwave assisted eutectic solvent extracted Kiwi berry young fruit polysaccharide (YKP-DM) RAW 264.7 macrophage cytotoxicity (A), nitric Oxide (NO) production (B), interleukin-6 (IL-6) production (C) and tumor necrosis factor-alpha (TNF-alpha) production (D)
Detailed Description
Example 1 extraction of kiwi fruit Dioscorea opposite crude polysaccharide (YKP-H) by hot water extraction method of the present invention
Hot water extraction: and freeze-drying the obtained kiwi fruits for 48 hours at the temperature of minus 40 ℃, and then pulverizing the freeze-dried samples by using a pulverizing machine, and sieving the powder with a 80-mesh sieve. Adding 80% ethanol (1:10, W/v) into young kiwi fruit powder, and placing into an ultrasonic cleaning tank under ultrasonic power of 640W for 30min to remove alcohol soluble components. Adding ultrapure water (1:30, w/v) into the precipitate, and extracting in a water bath for 4h. The extract was concentrated to 1/3 of the original volume using a rotary evaporator. High temperature alpha-amylase (5. Mu/mL, 80 ℃,6 h) and saccharifying enzyme (5. Mu/mL, 59 ℃,12 h) were added to remove starch and inactivated at 95℃for 30min. Then four times the volume of 95% ethanol (v\v) was used for the ethanol precipitation at 4℃overnight. And centrifuging to obtain a precipitate, cleaning the precipitate with 76% ethanol, and redissolving the precipitate with ultrapure water. The supernatant was dialyzed to remove small molecules (molecular weight cut-off, 3500 Da). Finally, obtaining the crude polysaccharide (YKP-H) of the young kiwi fruits extracted by water after freeze drying.
EXAMPLE 2 preparation of Kiwi berry young fruit crude polysaccharide (YKP-DM) extracted with microwave-assisted eutectic solvent according to the invention
And freeze-drying the obtained kiwi fruits for 48 hours at the temperature of minus 40 ℃, and then pulverizing the freeze-dried samples by using a pulverizing machine, and sieving the powder with a 80-mesh sieve. Adding 20% ethanol (1:10, W/v) into young kiwi fruit powder, and ultrasonic cleaning in an ultrasonic cleaning tank under ultrasonic power of 640W for 30min to remove alcohol soluble components. The precipitate was taken and added with a prepared extraction solvent (DES to water volume ratio of 16:9) and put into a laboratory microwave oven for extraction (extraction conditions refer to example 5). High temperature alpha-amylase (5. Mu/mL, 80 ℃,6 h) and saccharifying enzyme (5. Mu/mL, 59 ℃,12 h) were added to remove starch and inactivated at 95℃for 30min. Then four times the volume of 95% ethanol (v\v) was used for the ethanol precipitation at 4℃overnight. And centrifuging to obtain a precipitate, cleaning the precipitate with 76% ethanol, and redissolving the precipitate with ultrapure water. The supernatant was dialyzed to remove small molecules (molecular weight cut-off, 3500 Da). Finally, obtaining the kiwi fruit young fruit crude polysaccharide (YKP-DM) extracted by the microwave-assisted DES after freeze drying. The process flow diagram is shown in fig. 1.
Example 3 extraction of crude polysaccharide from young kiwi fruits by the Hot Water extraction method of the invention
And freeze-drying the obtained kiwi fruits for 40 hours at the temperature of minus 30 ℃, and then pulverizing the freeze-dried samples by using a pulverizing machine, and sieving the powder by a 60-mesh sieve. Adding 20% ethanol (1:10, W/v) into young kiwi fruit powder, and placing into an ultrasonic cleaning tank under ultrasonic power of 300W for 30min to remove alcohol soluble components. Adding ultrapure water (1:10, w/v) into the precipitate, and extracting in a water bath for 2h. The extract was concentrated to 1/3 of the original volume using a rotary evaporator. High temperature alpha-amylase (5. Mu/mL, 60 ℃ C., 3 h) and saccharifying enzyme (5. Mu/mL, 40 ℃ C., 10 h) were added to remove starch and inactivated at 95 ℃ C. For 30min. The resulting mixture was then subjected to alcohol precipitation at 4℃overnight with a diploid volume of 50% ethanol (v\v). And centrifuging to obtain a precipitate, cleaning the precipitate with 50% ethanol, and redissolving the precipitate with ultrapure water. The supernatant was dialyzed to remove small molecules (molecular weight cut-off, 2000 Da). Finally, obtaining the crude polysaccharide of the young kiwi fruits extracted by water after freeze drying.
Example 4 extraction of crude polysaccharide from young kiwi fruits by the Hot Water extraction method of the invention
And freeze-drying the obtained kiwi fruits for 70 hours at the temperature of-70 ℃, and then pulverizing the freeze-dried samples by using a pulverizing machine, and sieving the powder by a 120-mesh sieve. Adding 90% ethanol (1:30, W/v) into young kiwi fruit powder, and ultrasonic cleaning in an ultrasonic cleaning tank under ultrasonic power of 800W for 30min to remove alcohol soluble components. Adding ultrapure water (1:60, w/v) into the precipitate, and extracting in a water bath for 6h. The extract was concentrated to 1/3 of the original volume using a rotary evaporator. High temperature alpha-amylase (20. Mu/mL, 95 ℃ C., 12 h) and saccharifying enzyme (20. Mu/mL, 65 ℃ C., 14 h) were added to remove starch and inactivated at 95 ℃ C. For 30min. And five volumes of 100% ethanol (v\v) were then ethanol precipitated overnight at 4 ℃. And centrifuging to obtain a precipitate, cleaning the precipitate with 95% ethanol, and redissolving the precipitate with ultrapure water. The supernatant was dialyzed to remove small molecules (molecular weight cut-off, 10000 Da). Finally, obtaining the crude polysaccharide of the young kiwi fruits extracted by water after freeze drying.
EXAMPLE 5 microwave-assisted eutectic solvent extraction Condition parameter screening test of the invention
Microwave-assisted eutectic solvent extraction: the precipitate was added with the prepared extraction solvent (choline chloride: ethylene glycol=1:3) and extracted in a laboratory microwave oven. Other conditions were the same as for the hot water extraction. Then, a single-factor experimental design optimization extraction method is adopted, and the investigation conditions are shown in table 1. In summary, the extracted microwave power and DES water content are determined first, and then the extraction time is optimized. The extraction time and DES moisture content are determined and then the extraction microwave power is optimized. The extraction time and extraction power are determined and then the DES moisture content is optimized.
TABLE 1 Single factor experiment design Table
And secondly, adopting a three-factor response surface design to further optimize the influence of each extraction parameter on the extraction rate of the kiwi fruit polysaccharide. The argument includes the extraction time (X 1 12, 18, 24 min), extract power (X 2 560, 640, 720 mL/g), DES moisture content (X 3 15, 30, 45%). The study designed 17 experiments, the response surface factor codes are shown in Table 2, and then the response surface experimental data were analyzed by Design-Expert. The resulting data were fitted by a second order polynomial model as in table 2, fig. 2:
TABLE 2 response surface Experimental design and results
Compared with the traditional extraction method, the microwave has the advantages of high efficiency, energy saving, no toxicity, no harm and the like in extracting polysaccharide. Therefore, the invention utilizes microwaves and DES as extraction solvent to extract the kiwi fruit young fruit polysaccharide. The water content of the DES, the extraction power and the extraction time can obviously influence the extraction rate of extracting the kiwi fruit young fruit polysaccharide by the microwave-assisted DES. When DES is high in water, interaction of DES with polysaccharide is affected. When the water content is low, DES is too viscous as a solvent. The optimal condition for the final optimization is 30%. The higher the power of the microwaves, the higher the penetration of the microwaves, and the faster the sample will heat up. So that the polysaccharide extraction rate gradually increases as the microwave power is from 400W to 640W. But the greater the microwave power, the more likely the polysaccharide will be degraded. So that the extraction rate of polysaccharide is lowered when the power is increased to 720W. As the extraction time increased from 6min to 18min, the YKP-DM extraction rate increased. The polysaccharide is gradually dissolved out of the sample as the microwave time is prolonged. And after 18min, the extraction rate gradually decreases. It is possible that microwaves may cause degradation of polysaccharide as the extraction time is prolonged.
And further adopting a Box-Behnken experimental design to optimize the extraction rate of YKP-DM according to the single-factor result. The final BBD experimental data are shown in Table 3. And the resulting second order polynomial equation is as follows:
wherein Y represents the extraction rate; x is X 1 ，X 2 And X 3 The extraction time (min), microwave power (W) and DES moisture (%), respectively.
TABLE 3 analysis of variance of regression model of microwave-assisted eutectic solvent extraction
Note that: x is X 1 Extraction time (min); x is X 2 Feed-to-liquid ratio (mL/g); x is X 3 DES moisture content (%, v/v);
MDE,R 2 ＝0.9808,R 2 adj 0.9561, coefficient of Variation (CV) =2.88%, and adeq.precision= 21.4226.
* Significant difference (p)<0.05)， ** Extremely significant difference (p)<0.01)。
According to table 3, one-way analysis of variance (ANOVA) was used to evaluate the effect of extraction parameters on kiwi fruit young fruit polysaccharide extraction rate and the effectiveness of the fitted model. The fitted model was very significant based on P values (< 0.0001) and F values (39.24). In addition, the model is proved to have higher fitting degree according to the values of the mismatch term, the decision coefficient and the correction decision coefficient. In addition, the fitting model is proved to have good repeatability and reliability according to the variation coefficient and the value of moderate precision. In addition, the p values of the linear coefficient, the interaction coefficient and the quadratic term coefficient of all the extraction factors are smaller than 0.05, which shows that all the extraction parameters can obviously influence the extraction rate of the kiwi fruit young fruit polysaccharide. In fact, the three-dimensional response surface plot also shows that the interaction between the extraction time and the extraction power, the extraction time and the DES water content, and the extraction power and the DES water content is significant. All these data indicate that extraction time, microwave power and DES water content are important parameters affecting kiwi fruit young fruit polysaccharide extraction.
According to analysis of experimental results, the predicted optimal extraction conditions are determined as follows: the extraction time is 17min, the extraction power is 635W, and the DES water content is 35%. Verification experiments were performed under this condition, with an actual extraction of 10.04% ± 0.16%, very close to the predicted value 9.991%. Compared with the traditional water extraction method, the optimized MDE extraction rate is higher in extraction rate of kiwi fruit young fruit polysaccharide, and the extraction time is shorter. In addition, the content of polysaccharide in kiwi fruits is higher than that of mature kiwi fruits, which indicates that kiwi fruits are good resources for providing polysaccharide. Overall, these results indicate that MDE can be used as an efficient method for preparing polysaccharides from kiwi fruit kids in the food industry.
EXAMPLE 6 structural characterization experiments of the Young kiwi fruit polysaccharide prepared by the invention
And detecting total sugar, total uronic acid, total phenol and total protein of the kiwi fruit young fruit polysaccharide by adopting a colorimetric method. The molecular weight and dispersibility of YKP-H and YKP-DM were measured using SEC-MALLS-RID (Wyatt Technology Co., santa Barbara, calif., USA). The monosaccharide composition of the polysaccharide samples was determined by high performance liquid chromatography (L-20A, shimadzu, japan) combined with 1-phenyl-3-methyl-5-pyrazolone (1-phenyl-3-methyl-5-pyrazolone, PMP) pre-column derivatization. Samples were analyzed for functional groups and degree of esterification by fourier transform infrared spectroscopy (PerkinElmer, waltham, MA, USA). The glycosidic linkages of the polysaccharide were analyzed by nuclear magnetic resonance spectroscopy (Bruker, rheinstetten, germany). The specific method is as follows: the authors: ding-Tao Wu, men-Xi Fu, huan Guo, yi-Chen Hu, xiao-Qin Zheng, ren-You Gan and Liang Zou; title: microwave-Assisted Deep Eutectic Solvent Extraction, structural Characteristics, and Biological Functions of Polysaccharides from Sweet Tea (Lithocarpus litseifolius) Leaves; journal name: ANTIOXIDANTS, volume 11, phase 8; DOI:10.3390/antiox11081578.
TABLE 4 chemical composition, molecular weight and monosaccharide composition of kiwi fruit Young fruit polysaccharide extracted by different extraction methods
YKP-H and YKP-DM respectively represent kiwi fruit young fruit polysaccharide extracted by hot water extraction and microwave-assisted eutectic solvent; the superscript (a-b) between YKP-H and YKP-DM indicates that the difference between the two is significant (p < 0.05).
The experimental results show that the total sugar content of YKP-H and YKP-DM is 94.28mg/100mg and 92.53mg/100mg respectively, and the uronic acid content is 23.51mg/100mg and 30.50mg/100mg respectively, which indicates that the sample extracted by the method of the invention takes polysaccharide as the main component and is rich in pectin polysaccharide. The content of aldonic acid can react the results and activities of the polysaccharide to some extent, and in general, the higher the content of aldonic acid, the better the biological activity of the polysaccharide. Protein was one of the polysaccharide impurities, and the protein content was not large in the samples extracted by the two methods, indicating that the effect of protein on the samples was not large. Furthermore, the higher polyphenol content in YKP-DM compared to YKP-H may be due to the higher solubility of DES for polyphenols than water. Polyphenols are a class of active substances with antioxidant and hypoglycemic effects. In general, the higher the polyphenol content in the polysaccharide, the better its activity.
The molecular weight also has a certain correlation with the biological activity of the polysaccharide. The molecular weight of YKP-DM as shown in Table 4 (1.267X10 5 Da) is lower than YKP-H (6.081 ×10) 5 Da). It is explained that different extraction methods affect the molecular weight of the sample. In addition, generally the lower the molecular weight, the better the activity of the sample.
To further understand the chemical structures of YKP-H and YKP-DM, their monosaccharide composition, functional groups and glycosidic linkages were studied by high performance liquid chromatography, fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The monosaccharide composition types of YKP-H and YKP-DM are similar, and the main monosaccharides are galactose, arabinose and galacturonic acid, which are similar to the polysaccharide monosaccharide composition in the mature fruit of Chinese goosebeery. Based on the molar ratio of monosaccharide composition, it is presumed that polysaccharide (HG) composed of galactose, polysaccharide (RGI) composed of rhamnose acid and galactose monosaccharides, and polysaccharide (AG) composed of arabinose and galactose monosaccharides may be present in the kiwi fruit polysaccharide. Based on the ratio of galacturonic acid/rhamnose (MR 1 ratio), the ratio of HG and RGI pectin domains can be revealed, and kiwi fruit young fruit polysaccharide is found to be rich in HG pectin domains. In addition, mannose and glucose are the basic monosaccharide components of hemicellulose, so small amounts of galactomannans may also be present in kiwi fruit young fruit polysaccharides.
FT-IR spectra of YKP-H and YKP-DM are similar, with typical signals for pectic polysaccharides, including 3415, 2932, 1743, 1636, 1437, 1249, 1037 and 1019cm -1 Indicating that their functional groups are similar. In particular 1743cm -1 The absorption peak at which corresponds to the asymmetric stretching vibration of the esterified carboxyl group (c=o), but 1636cm -1 The strong absorption peak at this point is then due to the asymmetric stretching vibration of the free carboxyl group. Taken together, these results indicate that uronic acid is present in kiwi fruit puree, which contains pectic polysaccharide. Furthermore, according to 1743cm -1 And 1636cm -1 The signal intensity was calculated to show that the degrees of esterification of YKP-H and YKP-DM were 46.29% and 32.68%, respectively, indicating that MDE can significantly reduce the degree of esterification of young kiwi fruit polysaccharide (see FIG. 3 for results).
The NMR spectra of YKP-H and YKP-DM were also similar, further confirming their major chemical structures. And from 1 Typical signals for pectic polysaccharides, including HG, RGI and AG, are observed by H NMR, e.g., in the range of 5.10 to 5.44ppm 1 H NMR signals are due to the alpha-L-Araf and alpha-L-Rhap residues, 1H NMR signals in the range of 4.40 to 4.53ppm are due to the beta-D-Galp residues, and 3.81ppm and 4.97ppm 1 The H NMR signal is due to the GalAMe residues. The signals at 4.97ppm, 170.59ppm and 100.35ppm indicate the presence of 1, 4-alpha-D-GalAMep, whereas GalA-OCH 3 Is measured at 3.81ppm and 52.78 ppm. The signals at 2.08ppm and 19.87ppm indicate the presence of O-acetyl. The signals at 5.15ppm and 107.38ppm indicateThe presence of T-alpha-L-Araf, while signals at 5.10ppm, 5.44ppm and 5.24ppm indicate the presence of 1, 5-alpha-L-Araf, 1, 3-alpha-L-Araf and 1,3, 5-alpha-L-Araf, respectively. In fact, the signal at 109.30ppm also indicates the presence of 1,3- α -L-Araf and 1,5- α -L-Araf. Signals at 5.32/1.25ppm and 5.27/1.31ppm indicate the presence of 1,2- α -L-Rhap and 1,2,4- α -L-Rhap. Signals at 4.53ppm and 103.10ppm indicate the presence of 1,3,6- α0-D-Galp, signals at 4.46ppm and 103.22ppm indicate the presence of 1,3- β -D-Galp, and signals at 4.40ppm indicate the presence of T- β -D-Galp. Based on monosaccharide composition and NMR analysis results, it was found that HG, RG I and AG were indeed present in YKP-H and YKP-DM (see FIG. 4 for the results). Studies have also shown that polysaccharides extracted from mature kiwi fruits also contain HG, RG I and AG.
The beneficial effects of the invention are demonstrated by the following efficacy tests.
Test example 1 antioxidant Activity of the kiwi fruit Young fruit polysaccharide of the present invention
The antioxidant activity of kiwi fruits extracted by different methods is evaluated by the ABTS, DPPH free radical scavenging rate and total reducing power. Experiments are carried out by YKP-H and YKP-DM with different concentrations, and finally IC is obtained 50 Values. And VC and BHT were used as positive controls.
As shown, the antioxidant activity of YKP-DM is much higher than that of YKP-H. IC of ABTS and DPPH radical scavenging ability of YKP-DM 50 The values were 0.285mg/mL and 0.351mg/mL, respectively, well below the value of YKP-H (ABTS, 1.741mg/mL; DPPH,2.507 mg/mL). In addition, the value of the total reducing force of YKP-DM is also much higher than that of YKP-H (see FIG. 5 for the results).
Test example 2 promotion of proliferation of probiotics by the kiwi fruit Young fruit polysaccharide of the present invention
Subsequently, using 4 probiotics of lactobacillus fermentum (CGMCC 1.15608), lactobacillus plantarum (CGMCC 1.12974), lactobacillus rhamnosus (ATCC 53103) and bifidobacterium adolescentis (ATCC 15703), an in vitro fermentation model was established and the effect of YKP-H and YKP-DM on the proliferation of the probiotics was investigated with inulin (FOS) as positive control.
Research shows that plant polysaccharide can be used as prebiotics, and can promote proliferation and activity of beneficial bacteria by providing nutrition and growth environment for the beneficial bacteria, thereby positively affecting intestinal health. We studied the effect of YKP-H and YKP-DM on the growth of Lactobacillus fermentum (CGMCC 1.15608), lactobacillus plantarum (CGMCC 1.12974), lactobacillus rhamnosus (ATCC 53103) and Bifidobacterium (ATCC 15703). As shown in fig. 5, the kiwi fruit young fruit polysaccharide extracted by different methods can promote the proliferation of probiotics. Thus, the kiwi fruit young fruit polysaccharide has potential prebiotic activity (the result is shown in figure 5).
Test example 3 immunostimulation of the young fruit polysaccharide of Kiwi berry of the present invention
Models were created by culturing RAW 264.7 macrophages in RPMI-1640 medium. Subsequently, the effect of YKP-H and YKP-DM on macrophage proliferation was determined by MTT colorimetry. The amount of NO released by macrophages was measured using Griess reagent. Finally, secretion of leukocyte-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) by macrophages was determined using ELISA kits.
The results of the cellular immunocompetence experiments show that at concentrations ranging from 12.5 mug/mL to 50.0 mug/mL, YKP-H and YKP-DM have no cytotoxic effect on RAW 264.7 macrophages. In addition, samples stimulated RAW 264.7 macrophages to produce NO, IL-6 and TNF- α at various concentrations. The immunomodulatory effect of YKP-DM on RAW 264.7 macrophages is greater than that of YKP-H, similar to previous studies. Indeed, many experimental results indicate that the physical and chemical properties of pectic polysaccharides can influence their immunomodulatory effects. Thus, the observed differences in the immunomodulatory effects of YKP-H and YKP-DM may be due to their different structures. The higher aldehyde content and lower molecular weight of YKP-DM may also be factors that result in a stronger immunomodulatory effect (see FIG. 6 for results).
In conclusion, the method recycles the byproduct of the kiwi fruit planting industry, namely kiwi fruit young fruits. And optimizes the method for extracting polysaccharide from the polysaccharide, and the activity of YKP-DM extracted by the method is higher than that of YKP-H extracted by the traditional water extraction method. The research reduces the resource waste of the kiwi fruit industrial chain, improves the commercial value of kiwi fruits, and provides a reference for developing products by using kiwi fruit young fruits subsequently.
Claims (10)
1. The kiwi fruit young fruit polysaccharide extract is characterized in that: the kiwi fruit polysaccharide is obtained by extracting and separating kiwi fruit as a raw material, wherein:
every 100mg of kiwi fruit young fruit polysaccharide contains total sugar: (92.53+ -1.29) - (94.28 + -0.72) mg; the esterification degree of the kiwi fruit polysaccharide is as follows: (32.68±0.32)% - (46.29 ±0.35)%;
the molecular weight of the kiwi fruit young fruit polysaccharide is as follows: (1.265.+ -. 0.015). Times.10 5 –(6.081±0.03)×10 5 Da, the polydispersity is 2.312-2.686; the component sugar of the kiwi fruit young fruit polysaccharide mainly comprises the following monosaccharides: galactose, arabinose, galacturonic acid, mannose, glucuronic acid, rhamnose and glucose, and the molar ratio of the monosaccharides is as follows: (2.26-3.33): (0.94-1.99):1.00: (0.16-0.49): (0.12-0.38): (0.29-0.30): (0.21-0.26).
2. The kiwi fruit young fruit polysaccharide extract according to claim 1, wherein: the extract contains total uronic acid: (23.51 + -0.81) - (30.50+ -1.63) mg; total protein: (3.31.+ -. 0.41) - (3.42.+ -. 0.43) mg; total phenols: (1.01.+ -. 0.01) - (6.64.+ -. 0.26) mg GAE (gallic acid equivalent).
3. The kiwi fruit young fruit polysaccharide extract according to claim 2, wherein: the method is prepared by adopting microwave-assisted eutectic solvent extraction;
every 100mg of kiwi fruit young fruit polysaccharide contains total sugar: 92.53+ -1.29 mg; 30.50+ -1.63 mg of total uronic acid; total protein: 3.42.+ -. 0.43mg; total phenols: 6.64.+ -. 0.26mg GAE (gallic acid equivalent);
the molecular weight of the kiwi fruit young fruit polysaccharide is as follows: 1.265×10 5 –1.406×10 5 Da, polydispersity of 2.686-2.947; the component sugar of the kiwi fruit young fruit polysaccharide mainly comprises the following monosaccharides: galactose, arabinose, galacturonic acid, mannose, glucuronic acid, rhamnose and glucose, and the molar ratio of the monosaccharides is as follows: 2.26-2.86:0.94-1.24:1.00:0.16-0.17:0.12-0.13:0.30-0.52:0.26-0.30.
4. A kiwi fruit young fruit polysaccharide extract according to any one of claims 1-3, characterized in that: the kiwi fruit young fruit is derived from young fruit of kiwi fruit after thinning.
5. An extraction method for preparing the kiwi fruit young fruit polysaccharide according to any one of claims 1 to 4, which is characterized in that: it comprises the following steps:
a, freeze-drying young kiwi fruits, pulverizing into powder, and sieving;
b, adding ethanol into the powder, and ultrasonically removing alcohol-soluble components to obtain a precipitate;
c, hot water extraction: b, adding water into the precipitate prepared in the step b, and extracting in a water bath to obtain an extracting solution;
or microwave-assisted eutectic solvent extraction: b, adding the prepared precipitate into a prepared extraction solvent, and carrying out microwave extraction to obtain an extraction solution;
d, adding alpha-amylase and saccharifying enzyme into the concentrated solution to remove starch;
e, adding ethanol into the supernatant, and precipitating the supernatant with ethanol overnight; centrifuging to obtain precipitate, and washing the precipitate with ethanol;
f, adding water for redissolution, dialyzing and drying to obtain the kiwi fruit young fruit polysaccharide.
6. The method for preparing the kiwi fruit young fruit polysaccharide extract according to claim 5, wherein:
the freeze drying condition in the step a is that the freeze drying is carried out for 40 to 70 hours at the temperature of-30 to-70 ℃; the number of the screening meshes is 60 to 120 meshes;
b, adding ethanol with the concentration of 20-90% into the mixture; the weight-volume ratio of the powder to the ethanol is as follows: 1: (10-30); the ultrasonic power is 300-800W;
c, the ratio of water to raw materials in the step is 10-60mL/g; extracting in water bath for 2-6 hr;
or microwave-assisted eutectic solvent extraction: the volume ratio of the eutectic solvent to the water is (3-15): (17-5); the ratio of the extraction solvent to the raw materials is 10-60mL/g, the power of microwave extraction is 480-720W, and the extraction time is 6-30min;
the conditions for removing starch by the alpha-amylase and the saccharifying enzyme in the step d are respectively as follows: 5-20 mu/mL alpha-amylase, 60-95 ℃ for 3-12h;5-20 mu/mL saccharifying enzyme, 40-65 ℃ and 10-14h;
and e, in the step of ethanol precipitation, the volume ratio of the ethanol extract to the ethanol is 1: (2-5) ethanol with the concentration of 50-100 percent; the ethanol used for washing the precipitate is 50-95% ethanol;
and f, dialyzing to remove small molecules, wherein the molecular weight cut-off of the small molecules in the step f is 2000-10000Da.
7. The method for preparing the kiwi fruit young fruit polysaccharide extract according to claim 6, wherein:
step a, freeze drying for 48 hours at the temperature of minus 40 ℃; the sieving number is 80 meshes;
b, adding 80% ethanol in the step; the weight-volume ratio of the powder to the ethanol is 1:10; the ultrasonic power is 640W;
c, the ratio of water to raw materials in the step is 30mL/g; the water bath extraction time is 4 hours;
or microwave-assisted eutectic solvent extraction: the volume ratio of DES to water is 16:9, a step of performing the process; the ratio of the extraction solvent to the raw materials is 30mL/g, the microwave extraction power is 635W, and the extraction time is 17min;
d, adding 5 mu/mL of alpha-amylase in the step, and 80 ℃ for 6 hours; the addition amount of the saccharifying enzyme is 5 mu/mL, the temperature is 59 ℃, and the period of 12 hours is 12 hours;
and e, the volume ratio of the ethanol precipitation is 1:4, a step of; the ethanol is 95% ethanol; the ethanol used for washing the precipitate is 76% ethanol;
the molecular weight cut-off of the small molecules removed by dialysis in the step f is 3500Da.
8. Use of the kiwi fruit young fruit polysaccharide extract according to any one of claims 1 to 4 for preparing a health food or a medicine having an antioxidant function.
9. Use of the kiwi fruit young fruit polysaccharide extract of any one of claims 1 to 4 for preparing a health food or a medicine having an enhanced immune function.
10. A composition having an antioxidant function or an immunity enhancing function, characterized in that: comprising the kiwi fruit young fruit polysaccharide extract according to any one of claims 1 to 4, and an acceptable auxiliary material or auxiliary ingredient in food or medicine.
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