CN107298723B - Preparation method of lentinan and flavor developing substance - Google Patents

Abstract

The invention discloses a preparation method of lentinan and flavor developing substances, which comprises the steps of extracting lentinan fruiting bodies, and performing ultrafiltration on the extracted supernatant by using an ultrafiltration membrane to respectively obtain the lentinan and the flavor developing substances. The method of the invention simultaneously separates micromolecular flavor development substances and polysaccharide in the mushroom, and obviously improves the content of the polysaccharide and the flavor development substances. The extraction process of the invention is easy to realize, and is beneficial to industrial production, compared with the traditional boiling water extraction method, the energy consumption is less, the yield is higher, the content of the obtained mushroom crude polysaccharide is more than 25 percent, and the content of the flavor development substance is more than 50 percent.

Description

Preparation method of lentinan and flavor developing substance

Technical Field

The invention belongs to the technical field of edible fungus deep processing, and particularly relates to a preparation method of lentinan and a flavor developing substance.

Background

Lentinus edodes (lentinus edodes), also known as Lentinus edodes and Lentinus edodes, is a high-fat and low-protein nutritional health food used as both medicine and food. Lentinan is a substance with immunocompetence and anti-tumor functions in the fruiting body of lentinus edodes, the flavor development substances in lentinus edodes mainly comprise amino acids, nucleotides, soluble sugars and the like, and seven of eight amino acids necessary for human bodies are detected in lentinus edodes. In recent years, methods for extracting lentinan and flavor substances mainly comprise a hot water extraction method, a dilute acid dilute alkali extraction method, a compound enzyme method, a microwave ultrasonic assisted extraction method and the like. The common extraction and preparation methods have the disadvantages of complex process, high material and equipment cost, large energy consumption and low crude product yield, and restrict the large-scale preparation of lentinan and flavor substances. Meanwhile, the conventional preparation method only aims at extracting polysaccharide or flavor substances, and few methods aim at simultaneously extracting polysaccharide or flavor substances.

Disclosure of Invention

The invention aims to provide a preparation method of lentinan and a flavor developing substance, which comprises the following steps:

(1) extraction: crushing the fruiting body of the mushroom, adding water according to the material-liquid ratio (weight ratio) of 1:30, soaking for 20min-30min, adding flavor enzyme to react with cellulase for 3-6 h, inactivating the enzyme in water bath at 90-100 ℃ for 15min-20min, filtering, and centrifuging to obtain supernatant;

wherein the addition amount of the flavor enzyme is 0.5 to 2 percent of the weight of the mushroom powder, and the addition amount of the cellulase is 0.5 percent of the weight of the mushroom powder;

wherein the temperature for adding flavor enzyme to react with cellulase is 40-50 ℃;

(2) and (3) ultrafiltration: ultrafiltering the supernatant with a 1KDa roll-type ultrafiltration membrane, and respectively collecting the permeate and the retentate;

wherein the ultrafiltration pressure is 0.2MPa-0.3 MPa;

(3) concentrating and drying the permeate and the retentate respectively to obtain flavor substances and lentinan.

The concentration can be carried out by adopting a rotary evaporator at 60 ℃.

The invention also provides lentinan and a mushroom flavoring substance prepared by the method.

The method of the invention separates micromolecule flavor development substances and polysaccharide in the mushroom, obviously improves the content of the polysaccharide and the flavor development substances, simultaneously reduces the viscosity of a freeze-dried product and is easier to dry. The extraction process of the invention is easy to realize, and is beneficial to industrial production, compared with the traditional boiling water extraction method, the energy consumption is less, the yield is higher, the content of the obtained mushroom crude polysaccharide is more than 27 percent, and the content of the flavor development substance is more than 50 percent. The reaction temperature of the invention is 40-50 ℃, which greatly reduces the energy consumption, saves the energy source, is easy for industrialization and is an effective method for extracting the lentinan. The lentinan prepared by the method has high yield and high content, and simultaneously, the active ingredients are not lost, so the method is an effective method for simultaneously preparing the lentinan and the flavor substances on a large scale.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and do not delimit the invention.

The raw material sources are as follows:

mushroom: shiitake variety 808 cultivated in Shanghai Jinshan region

Flavor enzyme, cellulase: pompe bioengineering Co., Ltd

Roll-type ultrafiltration membrane: ande Membrane separation engineering Co Ltd

Determination of amino acid content

Spectrophotometry: making an L-phenylalanine standard curve, precisely sucking 0.0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of standard solution, respectively placing the standard solution in a 10mL volumetric flask, adding 70% ethanol to 1.0mL, adding 0.5mL of ninhydrin reagent, shaking up, placing in a 65 ℃ water bath, keeping the temperature for 30min, taking out, cooling to room temperature at constant speed, adding 70% ethanol to a constant volume to scale, shaking up, and measuring the absorbance at a wavelength of 570 nm. And (5) taking the absorbance value as a vertical coordinate and the concentration as a horizontal coordinate to prepare a standard curve. Taking 0.5mL of a sample to be detected, adding 70% ethanol to 1.0mL, adding 0.5mL of ninhydrin reagent, and determining according to the steps. And substituting the measured light absorption value of the amino acid into a standard curve, and calculating the concentration of the amino acid in the extracting solution.

Determination of nucleotide content

Ultraviolet spectrophotometry: a5' -guanylic acid standard curve is prepared, 0.0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of the standard solution are precisely absorbed and respectively placed in a glass test tube, distilled water is added to the glass test tube to fix the volume to 5.0mL, the glass test tube is shaken up, and the absorbance is measured at the wavelength of 260 nm. And (5) taking the absorbance value as a vertical coordinate and the concentration as a horizontal coordinate to prepare a standard curve. Taking 0.2mL of sample to be detected, adding distilled water to 5.0mL, and determining according to the steps. And (4) bringing the measured nucleotide light absorption value into a standard curve, and calculating the nucleotide concentration in the extracting solution.

High performance liquid chromatography analysis of polysaccharide molecular weight distribution

A Waters e2695 high performance liquid chromatography system is adopted to be matched with gel chromatographic columns TSK PWXL6000 and TSKPWXL4000 to be connected in series, 2414 differential refraction detector and DAWN8 laser detector are used for analyzing the molecular weight distribution of polysaccharide, and ASTRA6.1 data analysis software is used for collecting and analyzing light scattering data and calculating the molecular mass of the polysaccharide.

Example 1

Drying the fruiting body of the shiitake mushroom at 50 ℃ to constant weight, and crushing to 60 meshes to obtain 5 g;

adding distilled water according to a material-liquid ratio of 1:30, fully soaking for 30min, adding 0.5% of cellulase and 1% of flavor enzyme by weight of mushroom powder, reacting at 40 ℃ for 6 h, inactivating enzyme by boiling water for 20min, centrifuging at 10000rpm for 10min, collecting filtrate, cooling, fixing volume, diluting the filtrate to 6 times of original volume, performing ultrafiltration by using a 1KDa roll-type ultrafiltration membrane under the pressure of 0.2MPa, concentrating the filtrate under reduced pressure to obtain permeate and retentate, performing vacuum freeze drying to obtain flavor development substances and polysaccharide, and measuring the content of polysaccharide and amino acid. The total sugar content was measured by phenol-sulfuric acid method, wherein the amount of free amino acids was determined by ninhydrin colorimetry to obtain polysaccharide content of 26.17%, amino acid content of 48.16%, and nucleotide content of 6.13%.

Example 2

Drying the mushroom fruiting body at 50 ℃ to constant weight, crushing to 60 meshes, taking 5 g, adding distilled water according to the material-liquid ratio of 1:30, fully soaking for 30min, wherein the addition amount of cellulase is 0.5% of the weight of mushroom powder, the addition amount of flavor enzyme is 0.5% -2% of the weight of mushroom powder (0.5%, 1%, 1.5% and 2% are respectively taken), naturally adjusting the pH value, reacting for 6 h at 40 ℃, inactivating the enzyme in 90 ℃ water bath for 20min, centrifugally filtering, collecting filtrate, cooling, fixing the volume, selecting a 1KDa roll-type ultrafiltration membrane, carrying out ultrafiltration at the pressure of 0.2MPa, concentrating the obtained permeate and retentate of ultrafiltration under reduced pressure, respectively carrying out vacuum freeze drying, and measuring the content of polysaccharide and amino acid. Specific results are shown in table 1:

TABLE 1 comparison of Total sugar content and amino acid content at different flavor enzyme concentrations

Example 3

Oven drying dried Lentinus Edodes fruiting body, pulverizing, sieving with sixty mesh sieve, weighing 600 g, adding distilled water according to the material-liquid ratio of 1:30, soaking for 30min, adding flavor enzyme 1.5 wt% of Lentinus Edodes powder, adding cellulase 0.5 wt% of Lentinus Edodes powder, reacting at 50 deg.C for 6 hr, deactivating enzyme in 90 deg.C water bath for 20min, centrifuging, filtering, concentrating, cooling, centrifuging, filtering, diluting, cooling, quartering, selecting 1KDa roll type ultrafiltration membrane, ultrafiltering at 0.2 and 0.3MPa, concentrating under reduced pressure to obtain filtrate and retentate, vacuum freeze drying, and measuring polysaccharide and amino acid content. The total sugar content was measured by the phenol-sulfuric acid method and the amount of amino acids was measured by the ninhydrin colorimetry. Specific results are shown in table 2 below.

TABLE 2 comparison of Total sugar content and amino acid content

Example 4

Oven drying dried Lentinus Edodes fruiting body, pulverizing, sieving with sixty mesh sieve, weighing 400 g, adding distilled water according to the material-liquid ratio of 1:30, soaking for 30min, adding flavor enzyme 2% of Lentinus Edodes powder, adding cellulase 0.5% of Lentinus Edodes powder, reacting at 45 deg.C for 6 hr, deactivating enzyme in 90 deg.C water bath for 20min, centrifuging, filtering, concentrating, cooling, centrifuging, diluting, cooling, selecting 1KDa roll-type ultrafiltration membrane, ultrafiltering under 0.3MPa, concentrating the filtrate and retentate under reduced pressure, vacuum freeze drying, and measuring the content of polysaccharide, amino acids and nucleotides. Measuring total sugar content with phenol-sulfuric acid method, measuring amino acid content with ninhydrin colorimetric method, and measuring nucleotide content with ultraviolet spectrophotometry. The specific method and results are shown as follows:

sample preparation and treatment: scale 5mg of a sample of the lyophilized retentate dissolved in 1ml of a mobile phase containing 0.05mol/L NaH₂PO₄·2H₂O and 0.15mol/L NaNO₃And (3) solution. After centrifugation for 20min, the supernatant was collected and filtered through a 0.22 μm aqueous microporous membrane for analysis.

The experimental result shows that after the ultrafiltration treatment, the amino acid content in the permeate is 53%, the amino acid content is obviously improved, the polysaccharide content is 9%, the nucleotide content is 7%, the polysaccharide content in the retentate is 28%, the polysaccharide content is obviously improved, the amino acid content is 21%, the nucleotide content is 2%, and the polysaccharide molecular weight distribution of the retentate subjected to the ultrafiltration treatment is 5.2 × 10³(+ -16.1%) g/mol 91.3%, 3.679 × 10³(+ -10.3%) g/mol makes up 8.7%. It can be seen that the purity of the polysaccharide is obviously improved after the ultrafiltration treatment, which also indicates that the ultrafiltration technology has the effect of separating the flavor development micromolecule substances from the polysaccharide.

Example 5

In vitro stimulation macrophage release NO activity assay

In this example, RAW264.7 bone marrow macrophage cell line was purchased from the American national culture Collection (ATCC number TIB-71)^TM)；

DMEM and RPMI1640 were purchased from GIBCO in this example.

Preparation of test samples

A sample of the crude lentinan prepared in example 4 was accurately weighed into a sterilized eppendorf tube and prepared to a concentration of 5mg/mL using PBS buffer. After fully dissolving, centrifuging at 15000rpm/min for 30min, transferring to a new sterile eppendorf tube under aseptic condition, and diluting the sample to 50, 200 and 500. mu.g/mL for standby.

Preparation of mouse RAW264.7 macrophage

DMEM medium at 37 deg.C and 5% CO₂After subculture under the conditions, digestion was performed with 0.25% trypsin, 300 × g of the suspension was centrifuged for 3min to collect cells, and the cells were diluted to a certain concentration with colorless RPMI1640 medium for use.

Determination of NO releasing Activity of macrophages

Because NO is extremely unstable, nitrous oxides are generated in vivo quicklyAcid radical (NO 2)^-) And a nitrate group (NO 3)^-) Therefore, the Griess method is adopted to determine NO2 in the sample^-/NO3^-As a measure of NO levels.

(1) Standard curve with sodium nitrite:

preparing sodium nitrite solutions with different concentrations, wherein the concentration gradients are 9 concentration gradients of 0, 5, 10, 15, 20, 25, 30, 35 and 40 mu M; and (3) putting 100 mu L of the solution in a 96-well plate, adding 50 mu L of Griess reagent, measuring the absorbance value of 543nm, repeating the standard curve at each concentration by 3 times, and drawing the standard curve.

(2) Determination of NO production

After the macrophage culture solution is digested, the cells are diluted to 5 × 10 by colorless RPMI1640 culture medium (containing 10% fetal calf serum and 1% antibiotic liquid)⁵and/mL, adding 180 mu L of each well into a 96-well plate, then adding 20 mu L of samples to be tested with each concentration and negative (PBS) and positive (LPS, 10 mu g/mL) control, culturing for 48 hours at 37 ℃ under the condition of containing 5% CO2, taking 100 mu L of culture supernatant into the 96-well plate, adding 50 mu L of Griess reagent, developing for 10min, then measuring absorbance at the position of 543nm of wavelength, and calculating the corresponding NO amount according to a standard curve.

The experimental results are as follows:

the crude lentinan stimulates macrophage to produce NO (unit: mu moL/5.0 × 10) at a concentration of 50 μ g/mL⁵cells) are: 10-50 parts of; at a concentration of 200. mu.g/mL, the amount of NO produced is 10 to 50; at a concentration of 500. mu.g/mL, the amount of NO produced was 10 to 50, and as the concentration of the polysaccharide sample increased, it showed good NO production stimulating activity by macrophages.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (3)

1. A preparation method of lentinan and flavor developing substances comprises the following steps:

(1) extraction: crushing the fruiting body of the mushroom, and mixing the crushed materials according to a material-liquid ratio of 1:30 adding water, soaking for 20min-30min, adding flavor enzyme and cellulase, reacting for 3-6 hr, inactivating enzyme in 90-100 deg.C water bath for 15min-20min, filtering, and centrifuging to obtain supernatant;

wherein the addition amount of the flavor enzyme is 1.5 percent of the weight of the mushroom powder, and the addition amount of the cellulase is 0.5 percent of the weight of the mushroom powder;

wherein the temperature for adding flavor enzyme to react with cellulase is 40-50 ℃;

(2) and (3) ultrafiltration: ultrafiltering the supernatant with a 1KDa roll-type ultrafiltration membrane, and respectively collecting the permeate and the retentate;

wherein the ultrafiltration pressure is 0.2MPa-0.3 MPa;

(3) concentrating and drying the permeate and the retentate respectively to obtain flavor substances and lentinan.

2. Lentinan prepared by the process of claim 1.

3. A flavor developing material of shiitake mushroom prepared by the method of claim 1.