Longshan lily bulbil polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses Longshan lily bulbil polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of plant extraction. In the polysaccharide, the total sugar content is greater than 94%, monosaccharide comprises mannose, rhamnose, glucose, galactose and galacturonic acid in a ratio of (5.12 to 5.48): (7.04 to 7.79): (61.55 to 62.38): (17.37 to 18.14): (7.28 to 7.91), and the weight-average molecular weight is 183916 to 184735 Da. The preparation method comprises the following steps: by taking Lilium brownii Bulb as a raw material, carrying out boiling water enzyme deactivation treatment, decolorizing and degreasing, extracting an alcohol insoluble substance with water, synchronously removing starch by using high-temperature-resistant amylase, carrying out microwave digestion on filter residues obtained after filtering by using a sodium hydroxide solution, dialyzing and extracting an extracting solution, and finally preparing the Lilium brownii Bulb polysaccharide with immunocompetence. The method can be applied to food or functional food or medicine, and provides a thought for high-value application of the Longshan lily.

Description

Technical Field

[0001] The invention relates to the technical field of plant extraction, in particular to a Longshan lily bulbil polysaccharide and a preparation method and application thereof. Background Art

[0002] Longshan lily is a species of Lilium lily of the genus Lilium of the family Liliaceae, and is a geographical indication protected product of Longshan County, Hunan Province. Longshan lily is white like jade, slightly bitter in taste, and has high nutritional value. It has enjoyed a high reputation at home and abroad. During the growth process of Longshan lily, a large number of bulbils will be produced. In order to reduce the consumption of nutrients in the plant, it is usually necessary to remove the bulbils in time before the lily is harvested. The bulbils are usually discarded as by-products, resulting in a huge waste of resources. Longshan lily bulbils contain a large amount of polysaccharide components (more than 16%). If the polysaccharide components in Longshan lily bulbils can be effectively developed and utilized, it will greatly promote the comprehensive utilization of Longshan lily by-products, increase the processing added value of lily by-products, and promote the high-quality development of the Longshan lily industry.

[0003] In the prior art, for the extraction of lily polysaccharides, most of them use lily petals, bulbs, etc. as raw materials, and adopt alcohol extraction and water extraction methods to extract water-soluble polysaccharides. For example, Chinese patent CN100572397A reports a method for preparing lily polysaccharides that enhance immunity, and extracts water-soluble polysaccharides with immunity from Lanzhou lily petals. Chinese patent CN114835831A discloses a lily polysaccharide extraction process for Changbai Mountain lily bulbs, which uses ultrasonic treatment + mechanical wall breaking to obtain a higher extraction rate and better maintain the antioxidant activity of polysaccharides. Chinese patent CN112812197A discloses a Lanzhou lily polysaccharide and its preparation method and application. The lily polysaccharide obtained by extracting, separating and purifying Lanzhou lily petals has significant anti-breast cancer and liver cancer activity. Chinese patent CN118530380A discloses a polysaccharide from a lily bulb of Longya lily and its preparation method and application. The lily polysaccharide obtained by ion exchange column purification has the effect of inhibiting the proliferation of harmful bacteria and promoting the proliferation of probiotics. There are few reports on the use of Longshan lily as a raw material for lily polysaccharides. Yang Zhaoxia et al. [Study on Optimization of Ultrasonic Extraction Process of Lily Polysaccharides by Response Surface Methodology, Guangzhou Chemical Industry, 2013, 41(18): 52-55] used ultrasonic technology to extract Longshan lily polysaccharides and optimized the extraction process by response surface methodology.

[0004] At present, there is no report on the extraction of polysaccharides from bulbils of Lilium longshanense. Therefore, it is necessary to provide a method for preparing polysaccharides from bulbils of Lilium longshanense. Summary of the invention

[0005] The object of the present invention is to provide a Longshan lily bulbil polysaccharide, a preparation method and an application thereof. Longshan lily bulbil is used as a raw material, and an alkali-soluble polysaccharide is obtained by boiling water sterilization, alcohol extraction and alkali dissolution. The polysaccharide has higher immune activity than water-soluble polysaccharides.

[0006] The invention provides a Longshan lily bulbil polysaccharide, the total sugar content of which is greater than 94%, the monosaccharide composition is: mannose: rhamnose: glucose: galactose: galacturonic acid = 5.12-5.48: 7.04-7.79: 61.55-62.38: 17.37-18.14: 7.28-7.91, and the weight average molecular weight is 183916-184735Da.

[0007] Preferably, the polysaccharide is extracted from fresh bulbils of Lilium longshanense.

[0008] The present invention also provides a method for preparing the above-mentioned Longshan lily bulbil polysaccharide, comprising the following steps:

[0009] S1, taking Lilium bulbils, sterilizing and inactivating enzymes with boiling water, drying and crushing, and passing through a 300-500 mesh sieve to obtain Lilium bulbils powder;

[0010] S2, adding 5 to 10 times the amount of ethanol solution to the Longshan lily bulbil powder, decolorizing and defatting, filtering, and drying the filter residue to obtain Longshan lily bulbil alcohol insoluble matter;

[0011] S3, adding water and thermostable amylase to the bulbil insoluble matter of Longshan lily, extracting at 75-85° C. for 100-200 minutes, filtering to obtain a residue and a filtrate, and setting the residue aside;

[0012] S4, adding sodium hydroxide solution to the filter residue obtained in step S3, performing microwave digestion, concentrating the filtrate by rotary evaporation and then dialyzing the dialysate for later use;

[0013] S5. After the dialysate is concentrated, it is extracted with a mixture of chloroform and n-butanol, the upper aqueous solution is collected, anhydrous ethanol is added and allowed to stand, centrifuged to obtain a precipitate, and dried to obtain Longshan lily bulbil polysaccharide.

[0014] Preferably, in step S1, the time for sterilizing the enzyme is 90 to 120 seconds; preferably, the pulverization is performed in a low-temperature ultrafine pulverizer.

[0015] Preferably, the ethanol solution is an ethanol aqueous solution with a mass fraction of 95%.

[0016] Preferably, the decolorization and degreasing specifically comprises: treating at 40-50° C. for 2-6 hours, and repeating 3-6 times.

[0017] Preferably, in step S3, the volume of water added is 10-20 times the mass of the bulbilol-insoluble matter of Lilium longshanense, and the amount of the thermostable amylase used is 0.2% to 1% of the mass of the bulbilol-insoluble matter of Lilium longshanense.

[0018] Preferably, in step S4, the volume of the sodium hydroxide solution added is 15-30 times the mass of the filter residue, wherein the concentration of the sodium hydroxide solution is 3wt% to 6wt%.

[0019] Preferably, in step S4, the microwave digestion is specifically: extracting 2 to 4 times at a power of 400 to 800 W, each time for 20 to 40 minutes, cooling to room temperature, and filtering.

[0020] Preferably, in step S5, the volume ratio of chloroform to n-butanol in the chloroform and n-butanol mixture is 1:3-5.

[0021] Preferably, the volume proportion of anhydrous ethanol added to the solution is 75% to 80%.

[0022] Preferably, the standing temperature is 4° C. and the standing time is 8 to 12 hours.

[0023] Preferably, the drying temperature is 40-50°C.

[0024] The Longshan lily bulbil polysaccharide prepared by the present invention has good immune activity. Therefore, the present invention also provides the application of the Longshan lily bulbil polysaccharide in food, functional food or medicine.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention uses Longshan lily bulbils as raw materials, firstly uses boiling water to blanch the bulbils for a short time to inactivate enzymes such as oxidase in the bulbils, prevents enzymatic browning and other reactions from occurring during the drying process to generate melanin and other substances, thereby requiring prolonged ethanol decolorization time, and prevents changes in polysaccharide structural characteristics that may be caused by enzyme catalysis.

[0027] 2. The present invention uses microwave low-temperature drying to dry the Longshan lily bulbils, and uses a low-temperature ultrafine grinder to grind them, which can prevent the structural characteristics of the polysaccharide from changing due to high-temperature drying or grinding. At the same time, the lily bulbil powder prepared by ultrafine grinding has small particles, which is more conducive to the full dissolution of the polysaccharide.

[0028] 3. The present invention uses a thermostable amylase for simultaneous treatment in the water extraction step, which can effectively remove starch in the lily bulbils by enzymatic hydrolysis and improve the purity of the polysaccharide; on the other hand, simultaneous starch removal can also save extraction time and improve extraction efficiency.

[0029] 4. The present invention first uses water for extraction to extract water-soluble polysaccharides, and then uses an alkaline solution combined with microwaves to extract alkali-soluble polysaccharides to prepare Longshan lily bulbil polysaccharides, and its immune activity is enhanced. The polysaccharide can be applied to food and functional food, which helps to achieve high-value utilization of lily bulbils. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the promoting effect of the Longshan lily bulbil polysaccharide prepared in Examples 1-3 of the present invention and the control group 1 on the secretion of NO by macrophages.

[0031] Figure 2 This is the promoting effect of the Longshan lily bulbil polysaccharide prepared in Examples 1-3 of the present invention and the control group 1 on the secretion of TNF-α by macrophages.

[0032] Figure 3 This is the promoting effect of the Longshan lily bulbil polysaccharide prepared in Examples 1-3 of the present invention and the control group 1 on the secretion of IL-2 by macrophages. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] Example 1

[0035] A method for preparing longshan lily bulbil polysaccharide comprises the following steps:

[0036] (1) Collect fresh bulbils of Lilium longituba, cut them in half, blanch them in boiling water for 90 seconds to inactivate enzymes, remove them and drain them, dry them in a microwave dryer at 42° C. for 45 hours, and then grind them in a low-temperature ultrafine grinder at 25° C., and pass them through a 300-mesh sieve to obtain bulbil powder of Lilium longituba;

[0037] (2) placing the bulbil powder of Lilium longituba in a bottle, adding 5 times (volume-to-mass ratio, the same below) of 95% ethanol solution, decolorizing and defatting at 40° C. for 6 hours, treating for 3 times in total, filtering, evaporating the ethanol from the residue, and drying at 40° C. to prepare bulbil alcohol-insoluble matter of Lilium longituba;

[0038] (3) adding 10 times the amount of distilled water and 0.3% of thermostable amylase to the alcohol-insoluble matter obtained in step (2), extracting at 75° C. for 200 minutes, filtering, and setting aside the filter residue; after the filtrate is concentrated, extracting three times with a mixture of chloroform and n-butanol in a volume ratio of 1:4, collecting the upper aqueous solution, adding anhydrous ethanol until the volume ratio of ethanol in the solution is 75%, standing at 4° C. for 8 hours, centrifuging to obtain a precipitate, evaporating the ethanol from the precipitate and drying at 40° C. to obtain substance A;

[0039] (4) Add 15 times the amount of 3% sodium hydroxide solution to the filter residue obtained in step (3), place it in a microwave digestion instrument, extract it 4 times at a power of 400 W, each time for 20 minutes, cool it to room temperature, filter it, and concentrate the filtrate by rotary evaporation at 45° C., pour it into a dialysis membrane (molecular weight cutoff 5 kDa), and dialyze it in distilled water for 48 hours. The dialysate is used for standby use.

[0040] (5) extracting the dialysate obtained in step (4) three times with a mixture of chloroform and n-butanol in a volume ratio of 1:3, collecting the upper aqueous solution, adding anhydrous ethanol until the volume ratio of ethanol in the solution is 75%, standing at 4° C. for 8 hours, centrifuging to obtain a precipitate, and drying at 40° C. for 20 hours to obtain lily bulbil polysaccharide.

[0041] Example 2

[0042] A method for preparing longshan lily bulbil polysaccharide comprises the following steps:

[0043] (1) Collect fresh bulbils of Lilium longituba, cut them in half, blanch them in boiling water for 100 seconds to inactivate enzymes, remove them and drain them, dry them in a microwave dryer at 45° C. for 40 hours, and then grind them in a low-temperature ultrafine grinder at 30° C., and pass them through a 400-mesh sieve to obtain bulbil powder of Lilium longituba;

[0044] (2) placing the bulbil powder of Lilium longituba in a bottle, adding 8 times the amount of 95% ethanol solution, decolorizing and defatting at 45° C. for 4 hours, treating for 5 times in total, filtering, evaporating the ethanol from the residue, and drying at 40° C. to prepare bulbil alcohol-insoluble matter of Lilium longituba;

[0045] (3) adding 15 times the amount of distilled water and 0.8% of thermostable amylase to the alcohol-insoluble matter obtained in step (2), extracting at 78° C. for 150 minutes, filtering, and reserving the residue for later use;

[0046] (4) Add 20 times the amount of 5% sodium hydroxide solution to the filter residue obtained in step (3), place it in a microwave digestion instrument, extract it 3 times at 600 W power, each time for 30 minutes, cool it to room temperature, filter it, and concentrate the filtrate by rotary evaporation at 45°C, pour it into a dialysis membrane (molecular weight cutoff 5 kDa), dialyze it in distilled water for 48 hours, and use the dialysate for later use.

[0047] (5) extracting the dialysate obtained in step (4) four times with a mixture of chloroform and n-butanol in a volume ratio of 1:4, collecting the upper aqueous solution, adding anhydrous ethanol until the volume ratio of ethanol in the solution is 78%, standing at 4° C. for 10 hours, centrifuging to obtain a precipitate, and drying at 45° C. for 18 hours to obtain lily bulbil polysaccharide.

[0048] Example 3

[0049] A method for preparing longshan lily bulbil polysaccharide comprises the following steps:

[0050] (1) Collect fresh bulbils of Lilium longituba, cut them in half, blanch them in boiling water for 120 seconds to inactivate enzymes, remove them and drain them, dry them in a microwave dryer at 48° C. for 30 hours, and then grind them in a low-temperature ultrafine grinder at 35° C., and pass them through a 500-mesh sieve to obtain bulbil powder of Lilium longituba;

[0051] (2) placing the bulbil powder of Lilium longituba in a bottle, adding 10 times the amount of 95% ethanol solution, decolorizing and defatting at 50° C. for 2 hours, treating for 6 times in total, filtering, evaporating the ethanol from the residue, and drying at 40° C. to prepare bulbil alcohol-insoluble matter of Lilium longituba;

[0052] (3) adding 20 times the amount of distilled water and 1.2% of thermostable amylase to the alcohol-insoluble matter obtained in step (2), extracting at 85° C. for 100 minutes, filtering, and reserving the residue for later use;

[0053] (4) Add 30 times the amount of 6% sodium hydroxide solution to the filter residue obtained in step (3), place it in a microwave digester, extract it twice at 800 W, each time for 40 minutes, cool it to room temperature, filter it, concentrate the filtrate by rotary evaporation at 45°C, pour it into a dialysis membrane (molecular weight cutoff 5 kDa), dialyze it in distilled water for 48 hours, and use the dialysate for later use.

[0054] (5) extracting the dialysate obtained in step (4) five times with a mixture of chloroform and n-butanol in a volume ratio of 1:5, collecting the upper aqueous solution, adding anhydrous ethanol until the volume ratio of ethanol in the solution is 80%, standing at 4° C. for 12 hours, centrifuging to obtain a precipitate, and drying at 50° C. for 15 hours to obtain lily bulbil polysaccharide.

[0055] Control group 1

[0056] On the basis of Example 1, the step (4) of "placing in a microwave digestion instrument and extracting 4 times at a power of 400 W, each time for 20 minutes" was changed to "placing in a water bath at 95°C and extracting 4 times, each time for 20 minutes", and the other steps were consistent with Example 1. The prepared Longshan lily bulbil polysaccharide was the control group 1.

[0057] Experimental Example 1

[0058] The physicochemical properties of substance A and Longshan lily bulbil polysaccharide were analyzed, including the determination of total polysaccharide content using phenol-sulfuric acid, the analysis of monosaccharide composition and ratio using ion chromatography, and the determination of weight-average molecular weight using gel permeation chromatography-laser light scattering.

[0059] The results of the determination of the physicochemical properties of the Longshan lily bulbil polysaccharide in Examples 1 to 3 and Control Group 1 are shown in Table 1.

[0060] Table 1

[0061]

[0062] It can be seen from the above experimental results that the total sugar content of the Longshan lily bulbil polysaccharides prepared in Examples 1 to 3 is not significantly different. The total sugar content of the (alkali-soluble) Longshan lily bulbil polysaccharide prepared in Example 1 is significantly higher than that of substance A (Longshan lily bulbil water-soluble polysaccharide); compared with the control group 1, the total sugar content of the Longshan lily bulbil polysaccharide prepared in Example 1 after microwave treatment is significantly improved.

[0063] The monosaccharide components of the Longshan lily bulbil polysaccharides prepared in Examples 1 to 3 all include xylose, rhamnose, glucose, galactose, and galacturonic acid, and the percentages of the five monosaccharides are not much different. There are large differences in the monosaccharide components of the Longshan lily bulbil polysaccharides prepared by substance A and Example 1, which are manifested in that substance A contains mannose but does not contain xylose, and the proportion of glucose and galacturonic acid is lower than that of the polysaccharides in Example 1, and the proportion of galactose is higher than that of the polysaccharides in Example 1; compared with the control group 1, the monosaccharide components of the Longshan lily bulbil polysaccharides prepared by microwave treatment in Example 1 did not change, but the percentages between the components changed, especially the proportion of rhamnose and glucose decreased, and the proportion of galactose and galacturonic acid increased.

[0064] The weight average molecular weights of the Longshan lily bulbil polysaccharides prepared in Examples 1 to 3 are relatively close. The molecular weight of substance A is significantly lower than that of Example 1, and the molecular weight of the polysaccharide in control group 1 is significantly higher than that of Example 1.

[0065] Experimental Example 2

[0066] With reference to the literature “Xiaona Yu, Xiushi Yang, Bo Cui, Lijun Wang, Guixing Ren. Antioxidant and immunoregulatory activity of alkali-extractable polysaccharides from North American ginseng. International Journal of Biological Macromolecules. 2014, 65: 357-361.”, the immunoenhancing activity of the Longshan lily bulbil polysaccharide of the present invention was evaluated by measuring the secretion of macrophage immune factors NO, TNF-α, and IL-2.

[0067] Depend on Figure 1 , Figure 2 , Figure 3 It can be seen that the Longshan lily bulbil polysaccharides prepared in Examples 1, 2 and 3 can promote macrophages to secrete NO, TNF-α and IL-2, thereby showing an immunoenhancing effect, and the immunoenhancing effects of the polysaccharides in the three examples are relatively similar.

[0068] The secretion of the three immune factors in Example 1 at each concentration was higher than that of substance A. Compared with the control group 1, the secretion of the three immune factors in Example 1 at each concentration was higher than that of the control group 1, which indicates that the immunoenhancing effect of the polysaccharide extracted by microwave in Example 1 is significantly higher than that of the control group 1.

[0069] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A Longshan lily bulbil polysaccharide, characterized in that: The total sugar content is greater than 94%, and the monosaccharide composition is: mannose: rhamnose: glucose: galactose: galacturonic acid = 5.12-5.48: 7.04-7.79: 61.55-62.38: 17.37-18.14: 7.28-7.91, and the weight average molecular weight is 183916-184735 Da.

2. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that: The polysaccharide is extracted from fresh bulbils of Longshan lily.

3. The method for preparing the Longshan lily bulbil polysaccharide according to claim 1 or 2, characterized in that: The steps include: S1, taking Lilium bulbils, sterilizing and inactivating enzymes with boiling water, drying and crushing, and passing through a 300-500 mesh sieve to obtain Lilium bulbils powder; S2, adding ethanol solution to the Longshan lily bulbil powder, decolorizing and defatting, filtering, and drying the filter residue to obtain Longshan lily bulbil alcohol insoluble matter; S3, adding water and thermostable amylase to the bulbil insoluble matter of Longshan lily, extracting at 75-85° C. for 100-200 minutes, filtering to obtain a residue and a filtrate, and setting the residue aside; S4, adding sodium hydroxide solution to the filter residue obtained in step S3, performing microwave digestion, concentrating the filtrate by rotary evaporation and then dialyzing the dialysate for later use; S5. After the dialysate is concentrated, it is extracted with a mixture of chloroform and n-butanol, the upper aqueous solution is collected, anhydrous ethanol is added and allowed to stand, and the precipitate is separated by centrifugation and dried to obtain Longshan lily bulbil polysaccharide.

4. The preparation method according to claim 3, characterized in that: In step S1, the time for sterilizing the enzyme is 90 to 120 seconds; preferably, the pulverization is performed in a low-temperature ultrafine pulverizer.

5. The preparation method according to claim 3, characterized in that: In step S2, the added volume of the ethanol solution is 5 to 10 times the mass of the Longshan lily bulbil powder; the ethanol solution is an ethanol aqueous solution with a mass fraction of 95%; preferably, the decolorization and defatting specifically includes: treating at 40 to 50° C. for 2 to 6 hours, repeated 3 to 6 times.

6. The preparation method according to claim 3, characterized in that: In step S3, the volume of water added is 10-20 times the mass of the bulbilol-insoluble matter of Lilium longshanense, and the amount of the thermostable amylase used is 0.2% to 1% of the mass of the bulbilol-insoluble matter of Lilium longshanense.

7. The preparation method according to claim 3, characterized in that: In step S4, the volume of the sodium hydroxide solution added is 15-30 times the mass of the filter residue, wherein the concentration of the sodium hydroxide solution is 3wt% to 6wt%.

8. The preparation method according to claim 3, characterized in that: In step S4, the microwave digestion is specifically as follows: extracting 2 to 4 times at a power of 400 to 800 W, each time for 20 to 40 minutes, cooling to room temperature, and filtering.

9. The preparation method according to claim 3, characterized in that: In step S5, the volume ratio of chloroform to n-butanol in the chloroform and n-butanol mixture is 1:3-5; preferably, anhydrous ethanol is added to the solution so that the volume ratio of anhydrous ethanol is 75%-80%; preferably, the standing temperature is 4°C and the standing time is 8-12 hours; preferably, the drying temperature is 40-50°C.

10. Use of the Longshan lily bulbil polysaccharide according to any one of claims 1 or 2 in food, functional food or medicine.

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