CN118184814A

CN118184814A - Method for extracting pumpkin polysaccharide for seeds by crushing cells at low temperature

Info

Publication number: CN118184814A
Application number: CN202410452856.6A
Authority: CN
Inventors: 胡爱云; 丁健; 王旖璇; 吕成; 陈鹏程; 王若萱
Original assignee: Inner Mongolia Dingshang Jiaren Agricultural Technology Development Co ltd; Jiangnan University; Shazhou Professional Institute of Technology
Current assignee: Inner Mongolia Dingshang Jiaren Agricultural Technology Development Co ltd; Jiangnan University; Shazhou Professional Institute of Technology
Priority date: 2024-04-16
Filing date: 2024-04-16
Publication date: 2024-06-14

Abstract

The invention discloses a method for extracting pumpkin polysaccharide for seeds by crushing cells at low temperature, belonging to the field of food processing. The invention provides a method for extracting pumpkin polysaccharide for seeds by using ultrasonic wave to crush cells at low temperature. The invention can efficiently extract high-quality polysaccharide from the pumpkin for seeds in a short time. In addition, the use of the sodium tartrate solution not only improves the extraction rate of the polysaccharide, but also is beneficial to maintaining the molecular stability of the polysaccharide and reducing the possibility of thermal degradation or acid-base degradation due to the unique chemical property of the polysaccharide. In addition, sodium tartrate is widely accepted as a food-grade additive for safety, making the polysaccharide extracted by the method more suitable for use in the food industry.

Description

Method for extracting pumpkin polysaccharide for seeds by crushing cells at low temperature

Technical Field

The invention relates to the field of food processing, in particular to a method for extracting pumpkin polysaccharide for seeds by crushing cells at low temperature.

Background

Polysaccharides are a class of natural polymeric compounds that are widely distributed in nature and are found in a variety of organisms, including fruits, stems, leaves of plants, and certain microorganisms and animals. For example, mushrooms are a common food material, are rich in polysaccharide content, and exhibit various biological activities such as immunoregulation, antioxidation, anti-inflammatory and the like, so that the mushrooms have wide application prospects in the fields of foods, medicines and health care products. However, polysaccharides often exist in complex forms in organisms, and their extraction process faces technical challenges, mainly because polysaccharides form complex networks in the cellular structure of organisms, are not easily extracted directly, and have large differences in solubility and stability, resulting in the need for efficient and specific methods for extraction. In addition, the selected extraction method also considers the later storage and stability of the polysaccharide, ensures that the polysaccharide maintains ideal chemical and physical properties during long-term storage, so as to adapt to the high standard requirements of industrial application and food processing. Therefore, the development of gentle and efficient extractants and methods is critical for the commercial exploitation of polysaccharides.

The conventional polysaccharide extraction method mainly comprises a hot water extraction method, an alcohol precipitation method and the like, for example, patent literature of CN 108164618A discloses the steps of taking deep sea brown algae as a raw material, preparing stabilized brown algae mixed liquor through hot water extraction, preparing polysaccharide extract, polysaccharide separation liquid, polysaccharide concentrate, polysaccharide filter cake, preparing polysaccharide and the like; CN 117138387a discloses that solid polysaccharide can be obtained by extracting Ganoderma lucidum with hot water, precipitating with ethanol, collecting precipitate, refining, and drying. Although the method can obtain polysaccharide with certain purity, the method has long extraction time, low efficiency, high damage to raw materials, and large amount of waste water and waste residues, which pollute the environment.

In recent years, ultrasonic technology has shown great potential in the field of food processing, especially polysaccharide extraction, due to its cavitation effect, mechanical effect and thermal effect, and the polysaccharide in the cell is released effectively by damaging the plant cell wall. However, conventional ultrasonic extraction has some limitations, such as high temperature and long time extraction in the process are liable to cause partial degradation of polysaccharide molecules, reduce molecular weight, and further affect the functional properties of polysaccharide. In addition, this method mainly dissolves soluble polysaccharides and has limited conversion effects on insoluble polysaccharides.

Therefore, there is an urgent need to develop an extraction technique that reduces damage to polysaccharide molecules while ensuring extraction efficiency.

Disclosure of Invention

[ Technical problem ]

The invention aims to solve the problems that insoluble polysaccharide is difficult to extract, the extraction time of a high-temperature extraction technology is long, and partial degradation of polysaccharide molecules is caused.

Technical scheme

In order to solve the problems, the invention provides a method for extracting pumpkin polysaccharide from seeds by crushing cells at low temperature, wherein the crushing of the cells is performed under the ice water bath condition to control the temperature, so as to prevent the degradation of the polysaccharide due to high temperature; meanwhile, sodium tartrate is used as a complexing agent to help destroy plant cell walls and complex metal ions, so that the extraction rate and purity of polysaccharide are improved, and the method comprises the following steps:

S1, pretreatment of raw materials: drying the seeds with pumpkin pulp, grinding, sieving and collecting for later use;

s2, ultrasonic crushing of cells: weighing pumpkin pulp powder for seeds prepared in the step S1, placing the pumpkin pulp powder into a complexing agent solution, and performing ultrasonic extraction by using an ultrasonic cell grinder and ice water bath;

S3, decompression and suction filtration: s2, after ultrasonic extraction of the ice water bath is finished, collecting filtrate through reduced pressure suction filtration, centrifuging the filtrate for three times, and collecting supernatant to obtain crude extract;

s4, concentrating: concentrating the crude extract at 40-80deg.C until the volume is reduced to 1/5-1/3 of the original volume, adding 1-3 times of ethanol into the concentrate, mixing, standing for 6-24 hr, and collecting precipitate;

S5, washing and drying: washing the precipitate with ethanol, and freeze-drying to obtain solid polysaccharide.

In one embodiment of the present invention, the complexing agent in S2 is one or more of sodium tartrate, sodium citrate, and ammonium oxalate solution.

In one embodiment of the invention, the concentration of the complexing agent solution described as S2 is between 0.4% and 1%.

In one embodiment of the invention, the mass of the pumpkin fruit powder of S2: the volume (i.e., feed to liquid ratio) ratio of the complexing agent is 1/(30-50) (g/mL).

In one embodiment of the invention, the operating frequency of the ultrasonic cell disruptor of S2 is 40kHz.

In one embodiment of the invention, the maximum output power of the ultrasonic cell disruptor of S2 is 400W-800W.

In one embodiment of the invention, the time of ultrasound of S2 is 15min-45min.

In one embodiment of the invention, the ethanol content of S4 and S5 is 95%, v/v.

The invention also protects the application of the method in improving the extraction rate of polysaccharide.

[ Advantageous effects ]

1. Under the low-temperature condition, the ultrasonic cell grinder not only can efficiently damage plant cell walls and realize high extraction rate of polysaccharide, but also can keep high integrity of polysaccharide;

2. meanwhile, sodium tartrate is used as a complexing agent to help more efficiently extract polysaccharide from plant materials, and meanwhile, heavy metal ions and other adverse components are prevented from depositing, so that the extraction rate and oxidation resistance of the polysaccharide are improved;

3. The whole process is carried out under the low-temperature condition, which is beneficial to maintaining the natural structure and the characteristics of the polysaccharide, reducing the energy consumption and conforming to the principles of green chemistry and sustainable development.

Drawings

FIG. 1 (1) ultrasonic washer extraction and (2) pumpkin pulp remainder for seed after crushing cells.

Detailed Description

1. The method for detecting and calculating the dry weight yield of polysaccharide comprises the following steps:

the mass m ₁ of the pumpkin pulp for seeds added with the reaction is weighed by an analytical balance, and the polysaccharide yield of the pumpkin pulp for seeds after freeze-drying is calculated according to the formula (1) according to the mass m ₂ of the polysaccharide for seeds:

W＝m₂/m₁×100％ (1)

Wherein: w is polysaccharide yield,%; m ₁ is the mass of the pumpkin pulp added with the reaction, g; m ₂ is the quality of the freeze-dried pumpkin pulp polysaccharide for seeds.

2. The method for detecting and calculating the total sugar content of the polysaccharide adopts a phenol-sulfuric acid method to measure the glucose content in the polysaccharide, and is used for measuring the total sugar content:

2.1 drawing of a Standard Curve

Preparing 10-70 mug/mL glucose standard solution. Sucking 1.0mL of each in a glass test tube, adding 5.0mL of concentrated sulfuric acid (98%), shaking uniformly, reacting in a boiling water bath for 15min, taking out, and rapidly cooling. 1mL of the prepared phenol solution (6%) was added, and the test tube was continuously shaken to mix them uniformly, and the absorbance at 490nm was measured by using an microplate reader. And drawing a standard curve, wherein the abscissa is the concentration of the glucose solution, and the ordinate is the absorbance value obtained by measurement. The linear regression equation with the best fitting degree obtained by the parallel test is y=0.013dx+0.0558, wherein R ² = 0.9954, which shows that the concentration of the glucose solution is in the range of 0.0-100.0 mg/L, and the linear regression equation has good linear correlation.

2.2 Sample measurement

1ML of the crude polysaccharide extract is diluted by 100 times with deionized water, 0.1mL of the diluted solution is added with water to 1mL, and then concentrated sulfuric acid and phenol solution are added, and the process is the same as standard yeast. Substituting the test result into standard yeast to calculate the total sugar content in the crude extract.

3. The detection and calculation method of pumpkin polysaccharide antioxidant activity (expressed as DPPH clearance rate) comprises the following steps:

100mg of freeze-dried polysaccharide powder is weighed and dissolved in 100mL of water, 200 mu L of polysaccharide solution and 200 mu L of DPPH solution are taken to be evenly vibrated in a test tube, the mixture is reacted for 15min under the dark condition, and the OD value measured at the position of lambda=520 nm is recorded as A ₁. The OD measured for ethanol (70%) instead of the lifting polysaccharide was designated A ₂, and the OD for the blank tube was designated A ₀,S₁ as DPPH radical scavenging rate.

4. Method for testing the molecular weight (M _W) of polysaccharides:

The molecular weight (M _W) of the extracted polysaccharide was determined by Gel Permeation Chromatography (GPC) coupled with a Waters 2414 refractive index detector. Each polysaccharide extract (4-5 mg) was dissolved in 0.02M KH ₂PO₄ buffer (2 mL) and filtered through a 0.45mm membrane. The samples were then separately injected into a series of columns (TSK G-5000, G-3000 PWXL) and eluted with 0.02M KH ₂PO₄ buffer at 35℃at a flow rate of 0.6 mL/min.

Example 1

S1, pretreatment of raw materials: the seeds are dried, grinded and sieved (80 meshes) with pumpkin pulp, and then collected for standby;

S2, ultrasonic crushing of cells: weighing 1g of dried pumpkin pulp powder prepared in S1, wherein the volume of 0.7% sodium tartrate solution is 40mL, the feed-liquid ratio is 1/40 (g/mL), an ultrasonic cell grinder (JR-E2001, guangzhou Jeep ultrasonic instrument Co., ltd.) is adopted, the working frequency is 40kHz, the maximum output power is 600W, an ultrasonic probe is immersed in 2cm deep liquid, and ultrasonic extraction is carried out in an ice-water bath for 30min;

s3, decompression and suction filtration: after the reaction is finished, collecting filtrate through reduced pressure suction filtration, centrifuging the filtrate for three times, and measuring the yield of polysaccharide in pumpkin pulp for seeds by using a phenol-sulfuric acid method, wherein the total sugar content in the extracted polysaccharide is shown in table 1;

S4, concentrating: concentrating the crude extract at 60deg.C until the volume is reduced to 1/5 of the original volume, adding 3 times of ethanol into the concentrate, mixing, standing for 24 hr, and collecting precipitate;

S5, washing and drying: the precipitate was washed with ethanol and finally freeze-dried to give solid polysaccharide, and the antioxidant activity of the polysaccharide was measured as shown in Table 1.

Example 2

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 1, except that the conditions were unchanged by changing the 0.7% sodium tartrate solution in S2 to the 0.7% sodium citrate solution.

Example 3

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 1, with the remaining conditions unchanged, by changing the 0.7% sodium tartrate solution in S2 to a 0.7% ammonium oxalate solution.

TABLE 1 Effect of different complexing agents on polysaccharide extraction

From comparative analysis of table 1, we found that the oxidation resistance of polysaccharide extracted from 0.7% sodium tartrate solution was significantly better than other methods. Sodium tartrate is considered a better complexing agent because of its unique advantages, providing a mildly acidic environment helps to keep the molecular structure of the polysaccharide stable, reduces polysaccharide dissolution losses, maintains a good balance between extraction efficiency and polysaccharide quality, and better maintains the oxidation resistance of the polysaccharide compared to sodium citrate and ammonium oxalate.

Example 4

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 2, except that the conditions were unchanged, by changing the 0.7% sodium tartrate solution in S2 to 0.4% sodium tartrate solution.

Example 5

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 2, with the remaining conditions unchanged, by changing the 0.7% sodium tartrate solution in S2 to 1% sodium tartrate solution.

TABLE 2 Effect of sodium tartrate concentration on polysaccharide extraction

In the extraction process of polysaccharide, the addition amount of sodium tartrate has an important influence on the extraction effect. Initially, as the amount of sodium tartrate added increases, the total sugar content of the extracted polysaccharide increases, because sodium tartrate can form soluble complexes with calcium ions in the pumpkin pulp, thereby destroying the plant cell walls and the middle layers and releasing the polysaccharide. However, when the amount of sodium tartrate added reached a certain level, the polysaccharide extraction amount was reduced. This may be when the concentration of sodium tartrate reaches a certain level and then reaches saturation, all extractable polysaccharides have been extracted, at which time further increases in the amount of sodium tartrate do not further increase the extraction of polysaccharides. At the same time, excess sodium tartrate may form insoluble complexes with the polysaccharide, resulting in precipitation of the polysaccharide, thereby reducing the total sugar content of the solution. In addition, excessive sodium tartrate affects the structural stability of the polysaccharide molecule, or because high concentrations of sodium tartrate interfere with the normal function of the antioxidant substances in the polysaccharide. Too high a concentration of sodium tartrate may cause a pH change in the solution and may also adversely affect the antioxidant components of the polysaccharide.

Example 6

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 3, except that the feed liquid ratio in S2 was changed to 1/40 (g/mL) to 1/30 (g/mL).

Example 7

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 3, except that the feed liquid ratio in S2 was changed to 1/40 (g/mL) to 1/50 (g/mL).

TABLE 3 influence of feed to liquid ratios on polysaccharide extraction

When the feed-liquid ratio is from large to small, initially, the extraction yield of polysaccharide increases with the amount of solvent. This is because more solvent can penetrate the raw material more effectively, dissolving and extracting more polysaccharide. However, when the amount of the solvent reaches a certain level, the extraction rate may start to decrease. This may be due to the fact that when the solvent is excessive, the interaction between the solvent and the polysaccharide molecules becomes saturated, and additional solvent cannot effectively extract more polysaccharide, or because the solvent excessively dilutes the polysaccharide concentration, resulting in a decrease in total sugar content per unit volume.

The oxidation resistance of polysaccharides may also increase initially with increasing liquid to material ratio, as more solvent helps extract polysaccharide molecules with higher oxidation resistance. Antioxidant substances such as polyphenols can be more effectively released during the extraction process. However, when the liquid to material ratio increases to some extent, oxidation resistance may decrease. This reduction may be due to dilution effects of the total sugar concentration caused by excess solvent or because at high liquid to material ratios, more non-specific materials may be extracted during the extraction process which may interact with the antioxidant components of the polysaccharide and affect its antioxidant properties.

Example 8

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 4, with the remaining conditions unchanged, by changing the maximum output power in S2 to 600W to 400W.

Example 9

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 4, with the remaining conditions unchanged, by changing the maximum output power in S2 to 600W to 800W.

TABLE 4 influence of ultrasonic Power on polysaccharide extraction

The higher the initial power of wall breaking, the stronger the energy generated, and more cell walls are destroyed, so that the polysaccharide is extracted more thoroughly. At this stage, the increase in energy helps to break up more cell walls and increase the dissolution rate of polysaccharide. However, when the power reaches a certain level, the extraction effect may start to decrease. This may be because too high a power results in disruption of the polysaccharide molecular structure or excessive wall breaking that allows the polysaccharide to mix with other undesirable extraction components. Too strong ultrasonic energy may also cause excessive temperatures that affect the oxidation resistance of the polysaccharide.

Example 10

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 5, with the remaining conditions unchanged, by changing the ultrasonic time in S2 from 30min to 15 min.

Example 11

Referring to example 1, the total sugar content and antioxidant activity of the extracted polysaccharide were shown in Table 5, with the remaining conditions unchanged, by changing the ultrasonic time in S2 from 30min to 45 min.

TABLE 5 influence of ultrasound time on polysaccharide extraction

In the process of extracting polysaccharide, the processing time has a significant effect on the extraction rate. Initially, the extraction yield was relatively low as polysaccharide molecules and other soluble materials began to dissolve out of the plant material. With the extension of time, the solvent goes deep into the raw material to destroy the cell structure and promote the release of more polysaccharide molecules, so the extraction rate is gradually increased. After a certain time, the extraction rate is near maximum, since most of the extractable polysaccharide has been solubilized. However, too long a treatment time increases energy consumption and production costs.

Comparative example 1

The procedure was as in example 1, except that the treatment was carried out by sonication in an ultrasonic cleaner.

S2, ultrasonic extraction: 1g of dried pumpkin pulp powder prepared in S1 is weighed, the volume of 0.7% sodium tartrate solution is 40mL, the feed-liquid ratio is 1/40 (g/mL), an ultrasonic cleaner (KQ-500D, dongguan Kogyo ultrasonic equipment Co., ltd.) is adopted, the working frequency is 40kHz, and the maximum output power is 600W, and ultrasonic extraction is carried out for 30min in a water bath;

S3, decompression and suction filtration: after the reaction is finished, collecting filtrate through reduced pressure suction filtration, centrifuging the filtrate for three times, and measuring the yield of polysaccharide in pumpkin pulp for seeds by using a phenol-sulfuric acid method, wherein the total sugar content in the extracted polysaccharide is shown in Table 6;

s5, washing and drying: washing the precipitate with ethanol, and lyophilizing to obtain solid polysaccharide with antioxidant activity shown in Table 6.

TABLE 6 influence of different ultrasound devices on polysaccharide extraction

Ultrasonic cleaners and cell disruptors, although using ultrasonic technology, are designed and used in a variety of fields.

The ultrasonic wave generated by the ultrasonic cleaning machine is mainly used for generating tiny bubbles which are exploded on the surface of the object, so that the cleaning effect is achieved. The ultrasonic cell grinder is specially used for damaging cell walls or tissue structures, and the ultrasonic intensity of the ultrasonic cell grinder is generally higher than that of the ultrasonic cleaner, so that the ultrasonic cell grinder can damage plant cell walls more effectively and release polysaccharides in cells. At the same power, the extraction effect of the ultrasonic cell grinder on polysaccharide is generally better, because the working principle and design of the ultrasonic cell grinder are used for destroying the cell structure, so that the effective components in the cells are released more effectively. The ultrasonic energy of the ultrasonic cell grinder is concentrated, so that the plant cell walls can be damaged more efficiently, and substances such as polysaccharide in cells can be extracted more easily.

When the ultrasonic cell disruption operation is performed, low temperature conditions (such as ice water bath) are often required to avoid the loss of active ingredients caused by heating the sample, because the stability of the sample may be affected by the heat generated by the ultrasonic action. In contrast, the heat typically generated by ultrasonic cleaners during extraction causes the system to rise in temperature, which can lead to degradation of polysaccharide molecules.

Comparative example 2

S2, high-temperature extraction: weighing 1g of dried pumpkin pulp powder prepared in S1, wherein the volume of 0.7% sodium tartrate solution is 40mL, the feed-liquid ratio is 1/40 (g/mL), the pH value of the solution is regulated to be 2 by hydrochloric acid, and stirring the solution in a water bath kettle at 90 ℃ for 90min;

S3, decompression and suction filtration: after the reaction is finished, collecting filtrate through reduced pressure suction filtration, centrifuging the filtrate for three times, and measuring the yield of polysaccharide in pumpkin pulp for seeds by using a phenol-sulfuric acid method, wherein the total sugar content of the extracted polysaccharide is shown in Table 7;

S5, washing and drying: the precipitate was washed with ethanol and finally freeze-dried to give a solid polysaccharide, the antioxidant activity of which is shown in Table 7.

Comparative example 3

Referring to comparative example 2, the temperature of stirring in comparative example 2 was changed to 25℃and the remaining conditions were unchanged, and the total sugar content, antioxidant activity and molecular weight in the extracted polysaccharide were as shown in Table 7.

TABLE 7 influence of different methods on polysaccharide extraction

High temperature extraction may result in loss of certain nutrients and bioactive components in the polysaccharide, such as vitamins and antioxidants. The low temperature extraction method in the ice-water bath helps to retain these beneficial ingredients, thereby providing the polysaccharide with a higher oxidation resistance. High temperature strong acid treatment may lead to partial degradation of polysaccharide molecules, thereby reducing molecular weight. It can also be seen from the results that at the same pH, the polysaccharide degraded much slower at normal temperature than at high temperature.

The above examples are not intended to limit the scope of the invention nor the order of execution of the steps described. The present invention is obviously modified by a person skilled in the art in combination with the prior common general knowledge, and falls within the scope of protection defined by the claims of the present invention.

Claims

1. A method for extracting pumpkin polysaccharide for seeds by ultrasonic wave cell disruption, which is characterized by comprising the following steps:

2. The method for extracting pumpkin polysaccharide from seeds by ultrasonic crushed cells according to claim 1, wherein the complexing agent in S2 is one or more of sodium tartrate, sodium citrate and ammonium oxalate solution.

3. The method for extracting pumpkin polysaccharide from seeds by ultrasonic crushing cells according to claim 1 or 2, wherein the concentration of the complexing agent solution of S2 is 0.4% -1%.

4. The method for extracting pumpkin polysaccharide for seeds by ultrasonic wave crushed cells according to claim 1, wherein the mass of the pumpkin fruit powder of S2 is: the volume ratio of the complexing agent solution is 1/(30-50) (g/mL).

5. The method for extracting pumpkin polysaccharide from seed by ultrasonic cell disruption according to claim 1, wherein the operating frequency of the ultrasonic cell disruption machine of S2 is 40kHz.

6. The method for extracting pumpkin polysaccharide from seed by ultrasonic-crushing cells according to claim 1, wherein the maximum output power of the ultrasonic-crushing machine for S2 is 400W-800W.

7. The method for extracting pumpkin polysaccharide from seeds by ultrasonic pulverization of cells according to claim 1, wherein the ultrasonic time of S2 is 15min to 45min.

8. The method for extracting pumpkin polysaccharide from seed according to claim 1, wherein the volume concentration of ethanol used for S4 and S5 is 95%, v/v.

9. A polysaccharide prepared according to the method of any one of claims 1 to 8.

10. Use of the polysaccharide prepared according to the method of any one of claims 1 to 8 in the food industry.