Comprehensive evaluation method for nutrition and functional characteristics of edible mushroom protein and application of comprehensive evaluation method

Abstract

The invention provides a comprehensive evaluation method for nutrition and functional characteristics of edible mushroom protein and application of the comprehensive evaluation method, and relates to the technical field of edible mushroom nutrition and physicochemical property evaluation. According to the method, comprehensive analysis is carried out on the edible mushroom protein from the aspects of protein physicochemical properties, amino acid composition, in-vitro simulated digestion and the like, association between functional characteristics and structures of different edible mushroom proteins is established, and a nutritional value evaluation system of the edible mushroom protein is constructed. According to the method, the amino acid score is further optimized through the data of the intestinal terminal digestibility in the in-vitro simulation digestion experiment, and the nutritional value of the edible mushroom protein is better reflected. According to the invention, through data analysis and comprehensive evaluation of the edible mushroom protein, the key effect of the edible mushrooms in mining of novel protein resources and creation of related protein supplement products can be determined, and upgrading of the edible mushrooms from agricultural products to high-value-added deep processing products is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus nutrition evaluation, and in particular relates to a method for comprehensively evaluating the nutrition and functional characteristics of edible fungus protein and an application thereof. Background Art

[0002] Protein is the most important nutrient for humans. It plays a role in forming and repairing human tissues, transporting various substances, maintaining the function of the nervous system and providing energy. However, the existing protein acquisition relies heavily on planting and breeding, is highly dependent on land and water resources, and has serious environmental pollution and is greatly affected by the environment and climate. Therefore, traditional protein production methods cannot meet the future needs of human life in terms of quantity, quality and sustainable supply. Large-scale, low-cost, sustainable and high-quality protein production methods are in urgent need of innovation and development. In response to the growing demand for protein, it is urgent to build a sustainable high-quality protein supply model. The efficient manufacture and large-scale application of microbial protein is an important way to achieve sustainable protein supply. Fungi, algae and other microorganisms use renewable biological matrices as substrates, have the characteristics of low resource consumption and high production efficiency, and are an important source of protein to replace traditional animal proteins.

[0003] The edible fungi industry has the characteristics of a long history of cultivation, high economic benefits and great market potential. With the improvement of the level of edible fungi cultivation, production and processing technology, the edible fungi industry has gradually entered the stage of digital and intelligent development. In addition, edible fungi occupy an important position in modern food culture with their unique nutritional value and flavor. Not only is there the promotion of the healthy diet concept of "one meat, one vegetable and one mushroom", but there are also a variety of categories and processing methods to choose from, which enriches people's taste buds experience. As a green and healthy food, edible fungi have rich nutritional and medicinal value, and their protein content is rich, about 20% to 23%. Edible fungi protein contains a variety of bioactive proteins, which can inhibit the growth of tumor cells and stimulate the expression of immune factors. It plays an important role in anti-tumor, mitogenic, immunomodulatory, antiviral, anti-inflammatory, and antioxidant aspects. Therefore, the development of new meat substitutes based on edible fungi protein and their large-scale application is an important way to achieve sustainable high-quality protein supply, which is in line with the current consumer's pursuit of healthy food with characteristics such as safety, taste, flavor and nutrition.

[0004] The evaluation of protein is mainly carried out from two aspects: functional properties and nutritional properties. The various properties of protein are related to each other and are important references for protein processing. The nutritional evaluation of protein includes biological evaluation and non-biological evaluation. The biological evaluation mainly reflects the digestion and utilization of protein by measuring the parameters of nitrogen metabolism in animal models, but it is costly and time-consuming; the non-biological evaluation standards are also constantly updated. At present, the research on the nutritional components of edible fungi is mainly focused on a single edible fungi species, and there are few comparative analysis studies between different species. There are many types of edible fungi on the market, but the analysis of nutritional components is not comprehensive. Fully understanding the nutritional and functional characteristics of different edible fungi proteins and classifying them from deep structural characteristics can make their nutritional advantages more fully and reasonably utilized. Therefore, it is urgent to provide a scientific, standardized and simple edible fungi protein evaluation method. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a comprehensive evaluation method for the nutritional and functional characteristics of edible fungi protein, which can comprehensively analyze edible fungi and their protein components, and provide a theoretical basis for the evaluation of edible fungi germplasm resources, comprehensive utilization of proteins and high-value development.

[0006] Another object of the present invention is to provide an application of a method for evaluating the nutrition of edible fungus protein.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for comprehensively evaluating the nutritional and functional properties of edible fungus protein, comprising the following steps:

[0009] obtaining an edible fungus protein extract, and determining the physicochemical properties of the edible fungus protein extract;

[0010] Analyzing the amino acid composition of the edible fungus protein extract and calculating the amino acid score of the edible fungus protein extract;

[0011] Determine the in vitro digestibility of the edible fungus protein extract, optimize the amino acid score based on the in vitro digestibility data, and determine the final amino acid score of the edible fungus protein extract;

[0012] The physicochemical properties, amino acid composition and final amino acid score of edible fungus protein extracts were comprehensively analyzed to evaluate the nutritional value and functional properties of edible fungus protein.

[0013] Preferably, the calculation formula of the final amino acid score is: in vitro digestibility×amino acid score=final amino acid score.

[0014] Preferably, the physicochemical properties of the edible fungus protein extract include one or more of solubility, water holding capacity, oil holding capacity, emulsification and foaming properties.

[0015] Preferably, the in vitro digestibility includes one or more of oral digestibility, terminal stomach digestibility and terminal intestinal digestibility.

[0016] Preferably, the edible fungus protein extract is obtained by alkaline extraction and acid precipitation.

[0017] Preferably, the alkali extraction temperature of the alkali extraction and acid precipitation method is 30-65°C, the alkali extraction time is 0.5-3.5h, and the pH value is 10-12; the acid precipitation time of the alkali extraction and acid precipitation method is 2-5h, and the pH value is 3-5.

[0018] Preferably, the method further comprises calculating the amino acid ratio and the amino acid ratio coefficient of the edible fungus protein extract.

[0019] Preferably, the basic composition of edible fungi is determined before obtaining the edible fungi protein extract.

[0020] The present invention also provides an application of the above-mentioned comprehensive evaluation method for nutrition and functional characteristics in assisting the screening of high-quality edible fungi varieties.

[0021] The present invention also provides an application of the above-mentioned comprehensive evaluation method of nutrition and functional characteristics in identifying edible fungi varieties.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a method for comprehensively evaluating the nutritional and functional characteristics of edible fungi proteins and its application. The present invention selects eight common edible fungi, extracts edible fungi proteins by a simple and efficient alkali extraction and acid precipitation method, and further explores the structural characteristics of edible fungi proteins on the basis of analyzing the relevant functional characteristics of edible fungi proteins. The present invention analyzes the secondary structures of the eight edible fungi proteins, preliminarily analyzes the chemical structural characteristics of the eight edible fungi proteins based on Fourier infrared spectroscopy data, and then analyzes the first-order derivative spectrum and second-order derivative spectrum data of the edible fungi proteins, further distinguishes the composition differences of edible fungi proteins, and provides a theoretical basis for identifying and distinguishing different types of edible fungi.

[0024] The present invention also analyzes the amino acid components of eight kinds of edible fungi proteins and obtains that the ratio of essential amino acids to the total amount of amino acids (EAA / TAA) of the eight kinds of edible fungi crude proteins is about 40%, and the EAA / TAA ratios of Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, Pleurotus eryngii, Pleurotus velutipes, Pleurotus eryngii are 42% to 44%; the EAA / TAA ratio of Lentinus edodes is slightly lower, at 37.98%; the essential amino acid content in Agrocybe oleraceus protein is significantly higher, with an EAA / TAA ratio of 47.12%. At the same time, the ratio of essential amino acids to the total amount of amino acids (EAA / TAA) of the eight kinds of edible fungi crude proteins is above 60%, among which the EAA / NEAA value of Agrocybe oleraceus is the highest, at 89.09%, followed by Pleurotus eryngii, at 80.02%. It can be seen that the overall proportion of the eight kinds of edible fungi proteins conforms to the ideal protein model, and the EAA / TAA value and the EAA / NEAA value are close to those of whole egg protein, and are high-quality protein sources. The present invention forms a more systematic evaluation of the nutritional value of edible fungus protein through amino acid analysis, in vitro simulated digestion, etc., providing a theoretical basis for the evaluation, identification, comprehensive utilization and high-value development of edible fungus germplasm resources, helping to establish the key role of edible fungi in the exploration of new protein resources and the creation of related protein supplement products, and promoting the upgrading of edible fungi from agricultural products to high-value-added deep-processed products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 To compare the functional properties of eight edible fungus proteins;

[0026] Figure 2 The infrared spectra of crude protein extracts of eight edible fungi are shown in Figures a to h, respectively, for Lentinula edodes, Agaricus bisporus, Pleurotus ostreatus, Pleurotus ostreatus, Shiitake mushroom, Agrocybe oleraceus, Flammulina velutipes, and Pleurotus eryngii.

[0027] Figure 3 Comparison of secondary structures of proteins from eight edible fungi. (a) shows the infrared spectrum of the amide I band, and (b) shows the percentage of secondary structure.

[0028] Figure 4 The infrared second-order derivative spectra of eight kinds of edible fungi proteins, a is shiitake mushroom; b is Agaricus bisporus; c is white jade mushroom; d is oyster mushroom; e is oyster mushroom; f is Agrocybe fasciata; g is Flammulina velutipes; h is Pleurotus eryngii;

[0029] Figure 5 is the correlation coefficient of the infrared second-order derivative spectra of eight edible fungi proteins;

[0030] Figure 6 This is the principal component analysis of the infrared first-order derivative spectra of eight kinds of edible fungi proteins. In the figure, a is shiitake mushroom; b is Agaricus bisporus; c is white jade mushroom; d is oyster mushroom; e is oyster mushroom; f is Agrocybe fasciata; g is Flammulina velutipes; h is Pleurotus eryngii;

[0031] Figure 7 This is the cluster analysis of infrared first-order derivative spectra of eight kinds of edible fungi proteins. In the figure, a is shiitake mushroom; b is Agaricus bisporus; c is white jade mushroom; d is oyster mushroom; e is oyster mushroom; f is Agrocybe fasciata; g is Flammulina velutipes; h is Pleurotus eryngii;

[0032] Figure 8 The following are scanning electron micrographs of eight edible fungus proteins;

[0033] Fig. 9 In vitro simulated digestibility of eight edible fungus proteins. DETAILED DESCRIPTION

[0034] The present invention provides a method for comprehensively evaluating the nutritional and functional properties of edible fungus protein, preferably comprising the following steps:

[0035] obtaining an edible fungus protein extract, and determining the physicochemical properties of the edible fungus protein extract;

[0036] Analyzing the amino acid composition of the edible fungus protein extract and calculating the amino acid score of the edible fungus protein extract;

[0037] Determine the in vitro digestibility of the edible fungus protein extract, optimize the amino acid score based on the in vitro digestibility data, and determine the final amino acid score of the edible fungus protein extract;

[0038] The physicochemical properties, amino acid composition and final amino acid score of edible fungus protein extracts were comprehensively analyzed to evaluate the nutritional value and functional properties of edible fungus protein.

[0039] In the present invention, the calculation formula of the final amino acid score is preferably: in vitro digestibility × amino acid score = final amino acid score. In the present invention, the calculation formula of the final amino acid score is more preferably: terminal intestinal digestibility × amino acid score = final amino acid score. The present invention has found that the terminal intestinal digestibility of different edible fungi varieties is relatively small. The amino acid score can be further optimized based on the data of terminal intestinal digestibility in in vitro simulated digestion experiments to better reflect the nutritional value of edible fungi protein.

[0040] In the present invention, the physicochemical properties of the edible fungus protein extract include one or more of solubility, water retention, oil retention, emulsification and foaming properties. The present invention can use a measurement method well known in the art to measure the physicochemical properties of the edible fungus protein extract. The present invention also analyzes the secondary structure of the edible fungus protein, preliminarily analyzes the chemical structure characteristics of the edible fungus protein based on Fourier infrared spectroscopy data, and then analyzes the first-order derivative spectrum and second-order derivative spectrum data of the edible fungus protein to further distinguish the composition differences of the edible fungus protein, providing a theoretical basis for identifying and distinguishing different types of edible fungi.

[0041] In the present invention, the in vitro digestibility includes one or more of oral digestibility, end-stomach digestibility and end-intestinal digestibility. In the present invention, the in vitro digestibility of the edible fungus protein extract is preferably detected by an in vitro simulated digestion experiment.

[0042] In the present invention, the edible fungus protein extract is preferably obtained by alkali extraction and acid precipitation. In the present invention, the alkali extraction temperature of the alkali extraction and acid precipitation method is preferably 30-65°C, the alkali extraction time is preferably 0.5-3.5h, and the pH value is preferably 10-12. In the present invention, the acid precipitation time of the alkali extraction and acid precipitation method is preferably 2-5h, and the pH value is preferably 3-5. In a specific embodiment of the present invention, the test parameters of the specific alkali extraction and acid precipitation method need to be adjusted according to different edible fungi varieties. Taking shiitake mushrooms as an example, the preparation method of the shiitake mushroom protein extract includes the following steps: weighing an appropriate amount of shiitake mushroom powder in a beaker, mixing it with deionized water at 1:45 (m / V), and stirring it evenly with a magnetic stirrer at 25°C. Subsequently, the solution pH is adjusted to 10.0 with 1mol / L NaOH, extracted for 3h under 50°C water bath conditions, and centrifuged at 10000r / min for 15min at 4°C to obtain a supernatant. The pH of the supernatant was adjusted to the protein isoelectric point of 4.2 with 1 mol / L HCl, and the crude protein precipitate was obtained by centrifugation at 10000 r / min for 15 min at 4°C, and the precipitate was washed with deionized water for 3 times. Finally, the precipitate was dissolved with a small amount of deionized water, the pH was adjusted to 7.0, and the crude extract powder of Lentinus edodes protein was obtained by freeze-drying and stored at 4°C.

[0043] The composition and proportion of essential amino acids that cannot be synthesized by the human body or the synthesis speed cannot meet the needs of the body and must be ingested through food are important indicators for further evaluating the nutritional value of food protein. The present invention preferably refers to the relevant intake requirements for people over 3 years old in the FAO / WHO model spectrum, calculates the amino acid score (AAS), amino acid ratio (RC) and amino acid ratio coefficient score (SRC) of edible fungus crude protein, and further evaluates the nutritional value of edible fungus protein.

[0044] In the present invention, the basic composition of the edible fungi is preferably determined before obtaining the edible fungi protein extract. The basic composition of the edible fungi preferably includes protein content, fat content, carbohydrate content and water content. In the present invention, the determination of the above basic components can be carried out by a determination method well known in the art. In the present invention, the determination of edible fungi protein refers to the first method of "GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food", using a fully automatic Kjeldahl nitrogen analyzer; the fat content refers to the first method of "GB 5009.6-2016 National Food Safety Standard Determination of Fat in Food" Soxhlet extraction, using a fat analyzer for determination; the carbohydrate content refers to "GB / T 15672-2009 Determination of Total Sugar Content in Edible Fungi", using phenol-sulfuric acid colorimetry for determination; the water content is determined by reference to "GB 5009.3-2016 National Food Safety Standard Determination of Water in Food". Each sample is measured in parallel 3 times, and the results are expressed as "mean ± standard deviation". In a specific embodiment of the present invention, the edible fungi are preferably ground into powder for the determination of the above basic components.

[0045] The present invention also provides an application of the above evaluation method in assisting the screening of high-quality edible fungi varieties, and the application is further preferably to assist in the screening of edible fungi varieties with high-quality protein.

[0046] The present invention also provides an application of the above evaluation method in identifying edible fungi varieties. The present invention analyzes the secondary structures of eight edible fungi proteins, preliminarily analyzes the chemical structure characteristics of the eight edible fungi proteins based on Fourier infrared spectroscopy data, and then analyzes the first-order derivative spectrum and second-order derivative spectrum data of the edible fungi proteins to further distinguish the composition differences of the edible fungi proteins, thereby providing a theoretical basis for identifying and distinguishing different edible fungi species.

[0047] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0049] Example 1

[0050] A method for comprehensively evaluating the nutritional and functional properties of edible fungus protein, the specific steps are as follows:

[0051] (1) Determine the basic composition of edible fungi;

[0052] (2) The edible fungus protein extract was obtained by alkaline extraction and acid precipitation method, and the physical and chemical properties of the edible fungus protein extract were determined, including the solubility, water holding capacity, oil holding capacity, emulsification and foaming properties of the edible fungus protein extract;

[0053] (3) Analyze the secondary structure of edible fungus protein extracts using Fourier transform infrared spectroscopy;

[0054] (4) analyzing the amino acid composition of the edible fungus protein extract, and calculating the amino acid score, amino acid ratio and amino acid ratio coefficient of the edible fungus protein extract;

[0055] (4) determining the in vitro digestibility of the edible fungus protein extract, optimizing the amino acid score based on the in vitro digestibility data, and determining the final amino acid score of the edible fungus protein extract;

[0056] (5) Comprehensively analyze the physicochemical properties, amino acid composition, and final amino acid score of edible fungus protein extracts to evaluate the nutritional value and functional properties of edible fungus protein.

[0057] Example 2

[0058] According to the steps of Example 1, crude protein extracts of 8 edible fungi, including shiitake mushroom, Agaricus bisporus, Pleurotus ostreatus, Pleurotus ostreatus, Shimeji mushroom, Agrocybe tumefaciens, Flammulina velutipes, and Pleurotus eryngii, were analyzed and nutritionally evaluated. The specific test process is as follows:

[0059] (I) Test materials

[0060] Freeze-dried powders of seven kinds of edible fungi, including shiitake mushrooms, button mushrooms, white jade mushrooms, oyster mushrooms, shimeji mushrooms, tea tree mushrooms, and enoki mushrooms, were purchased from Shandong Lvyou Food Technology Co., Ltd., and freeze-dried powder of Pleurotus eryngii was purchased from Xinghua Lianfu Food Co., Ltd. The samples were stored in a cool and dry place for future use.

[0061] (II) Test methods

[0062] 1. Determination of the basic composition of edible fungi

[0063] The determination of protein in edible fungi was carried out according to the first method of "GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food", using a Kjeldahl nitrogen analyzer; the fat content was determined by Soxhlet extraction according to the first method of "GB 5009.6-2016 National Food Safety Standard Determination of Fat in Food", using a fat analyzer; the carbohydrate content was determined according to "GB / T15672-2009 Determination of Total Sugar Content in Edible Fungi", using phenol-sulfuric acid colorimetry; the moisture content was determined according to "GB5009.3-2016 National Food Safety Standard Determination of Moisture in Food". Each sample was measured three times in parallel, and the results were expressed as "mean ± standard deviation".

[0064] 2. Extraction of crude protein from edible fungi

[0065] Taking shiitake mushroom as an example, weigh an appropriate amount of shiitake mushroom powder in a beaker, mix it with deionized water at 1:45 (m / V), and stir it evenly with magnetic force at 25°C. Subsequently, adjust the pH of the solution to 10.0 with 1mol / LNaOH, extract it in a 50°C water bath for 3h, and centrifuge it at 10000r / min for 15min at 4°C to obtain a supernatant. Adjust the pH of the supernatant to 4.2 with 1mol / L HCl, centrifuge it at 10000r / min for 15min at 4°C to obtain a crude protein precipitate, and wash the precipitate with deionized water for 3 times. Finally, dissolve the precipitate with a small amount of deionized water, adjust the pH to 7.0, freeze-dry to obtain a shiitake mushroom protein crude extract powder, and store it at 4°C. According to the above steps, crude protein extracts were prepared according to the extraction conditions of different edible fungi proteins, and the specific conditions are shown in Table 1.

[0066] Table 1 Extraction conditions of crude protein from different edible fungi

[0067]

[0068] The purity of crude protein of edible fungi was determined by Kjeldahl nitrogen determination method, and the protein extraction rate and the weight yield of protein and residue were calculated according to formula 1 to formula 3. Each sample was measured and calculated three times in parallel, and the results were expressed as "mean ± standard deviation".

[0069] Formula 1: Formula 2: Formula 3:

[0070] In the formula, E represents the extraction rate / content extraction rate of edible fungi protein (%); m1 represents the mass of edible fungi crude protein (g); ω1 represents the protein content in edible fungi crude protein (g / 100g); m2 represents the mass of edible fungi powder (g); ω2 represents the protein content in edible fungi powder (g / 100g); WE1 represents the weight extraction rate of edible fungi protein (%); WE2 represents the weight extraction rate of edible fungi residue (%); m3 represents the mass of edible fungi protein alkaline extraction residue (g).

[0071] 3. Determination of physicochemical properties of edible fungus protein

[0072] 3.1 Solubility determination

[0073] Weigh 0.200 g of sample into a 50 mL centrifuge tube, add 20 mL of deionized water to dissolve, adjust the pH to 7.0, vortex for 2 min to mix the sample evenly, let it stand for 1 h, centrifuge at 8000 rpm for 15 min, take the supernatant, determine the water-soluble protein content by BCA method, and calculate the solubility.

[0074]

[0075] 3.2 Determination of water holding capacity

[0076] Weigh 0.100g of edible fungus protein into a centrifuge tube, add 10mL of water, mix well, let stand for 30 minutes, centrifuge at 6000r / min for 15 minutes, remove the upper water. Use filter paper to absorb the excess solution on the inner wall of the protein precipitation tube, weigh the tube and calculate the water holding capacity.

[0077]

[0078] 3.3 Determination of oil holding capacity

[0079] Weigh 0.100g of edible fungus protein into a centrifuge tube, add 10mL of edible oil, mix well, let stand for 1h, centrifuge at 6000r / min for 15min, remove the upper layer of fat, and tilt the centrifuge tube at 45°. Try to remove the fat on the tube wall, and calculate the oil holding capacity after weighing the tube.

[0080]

[0081] 3.4 Emulsification determination

[0082] Refer to the method of Zhu Zengfang and Zhang Y for adjustment, weigh 0.080g edible fungus protein and dissolve it in 40mL water and mix well. Take 15mL of protein solution in a 50mL beaker, add 10mL of edible oil, let it stand for 10min, and homogenize at 10000rpm for 1min. Quickly take 100μL of emulsion from the bottom of the cup and dilute it in 4mL SDS (1mg / mL), and measure the absorbance at 500nm. After the emulsion is allowed to stand for 10min, resample it and measure the absorbance again. Calculate the emulsifying activity index (EAI) and emulsifying stability (ESI) according to the formula.

[0083]

[0084] In the formula, A0 represents the absorbance of the dilution at 500 nm; D represents the dilution factor; c represents the initial concentration of the edible fungus protein; represents the volume fraction of oil used to form the emulsion; ΔA represents the absorbance difference after standing for 10 min; t represents the standing time (10 min).

[0085] 3.5 Foaming property determination

[0086] Weigh 0.500g of edible fungus protein and dissolve it in 25mL of phosphate buffer (0.05M, pH=7.0) at 10000rpm for 2min, and measure the foam volume. After standing for 5min and 10min, measure the foam volume respectively. Calculate the foaming capacity (FC) and foaming stability (FS) according to the formula.

[0087]

[0088]

[0089] In the public notice, V represents the initial volume of the protein solution (25 mL); V0 represents the volume of the foam after homogenization (mL); V5 (or V 10 ) represents the foam volume (mL) after standing for 5 min (or 10 min).

[0090] 3.6 Fourier transform infrared spectroscopy

[0091] The infrared spectrometer PerkinElmer Spectrum 2 was used to analyze the 8 crude protein extracts. The sample powder and potassium bromide were mixed and ground in an agate mortar at a ratio of 1:100 (w / w) and pressed into tablets. 4000~400cm -1 Scanning is performed within the wavenumber range, with a resolution of 4 cm -1 , the scanning times were 32 times, each sample was scanned three times, and the average spectrum was calculated before analysis.

[0092] 3.7 Scanning electron microscopy

[0093] Take a small amount of freeze-dried edible fungus protein crude extract powder, sprinkle it evenly on the double-sided conductive adhesive for gold spraying, adjust the electron microscope to the best shooting field of view, magnify it to 100, 400, and 800 times respectively, observe and take pictures.

[0094] 4. Determination of amino acids in edible fungi protein

[0095] 4.1 Amino acid composition of edible fungus protein

[0096] The amino acid composition and content of edible fungus protein were determined with reference to GB 5009.124-2016 National Food Safety Standard Determination of Amino Acids in Food and the method established in the laboratory in the early stage. The sample mass was calculated according to the purity of the protein in the crude protein of edible fungi, so that the protein content in the sample was within the range of 10-20 mg. Weigh an appropriate amount of sample in a 15mL brown hydrolysis bottle, add 10mL 6M HCl, and hydrolyze at 110℃ for 24h. After the hydrolysis is completed, transfer all the hydrolyzate to a 50mL volumetric flask, and dilute to the scale with deionized water. Pipette 1.5mL of the dilute solution into a small culture dish and evaporate to dryness at 50℃. Then dissolve it with 1mL sodium citrate buffer with pH=2.2, pass the solution through a 0.22μm water system membrane in a 1.5ml injection bottle for testing.

[0097] The 7 essential amino acids (EAA) such as threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine and the 10 non-essential amino acids (NEAA) such as aspartic acid, serine, glutamic acid, glycine, alanine, cysteine, tyrosine, histidine, arginine, and proline were measured by the fully automatic amino acid analyzer L-8900. Each sample was measured three times in parallel, and the results were expressed as "mean ± standard deviation".

[0098] 4.2 Nutritional evaluation of edible fungus protein

[0099] Referring to the model for evaluating the nutritional value of protein proposed by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) and revised in 2013, and considering that the final product is mainly aimed at adolescents and adults, the relevant indicators for "children over 3 years old, adolescents and adults" in the reference model spectrum were used. According to formulas 4 to 10, the amino acid score (Amino acid score, AAS), amino acid ratio coefficient (Ratio of amino acid coefficient, RC) and amino acid ratio coefficient score (Score of ratio coefficient of amino acid, SRC) of crude protein of eight edible fungi were calculated. In addition, eggs have been regarded as a high-quality protein source due to their rich protein content and appropriate amino acid ratio, and are often used as reference proteins. The amino acid chemical score (Chemical score, CS) and essential amino acid index (Essential amino acid index, EAAI) of crude protein of edible fungi were calculated with reference to whole egg protein.

[0100] Formula 4: Formula 5: Formula 6:

[0101] Formula 7: SRC = 100-CV × 100; Formula 8:

[0102] Formula 9: Formula 10:

[0103] In the above formula, TP represents the content of a certain amino acid in the crude protein of edible fungi (mg / g); RP represents the corresponding amino acid content in the WHO / FAO model spectrum (mg / g); RAA represents the amino acid ratio; CV represents the coefficient of variation of RV; rp represents the corresponding amino acid content in whole eggs (mg / g).

[0104] 5. In vitro digestion of edible fungus protein

[0105] (1) In vitro digestion test method of edible fungus protein

[0106] The electrolyte solution was prepared according to the method of Brodkorb et al., and an in vitro simulated digestion experiment was carried out. The digestion was divided into three stages: oral cavity, stomach and intestine. The composition and concentration of the electrolyte solution of simulated saliva (Simulated salivary fluid, SSF), simulated gastric fluid (Simulated gastric fluid, SGF) and simulated intestinal fluid (SIF) are shown in Table 2.

[0107] Table 2 Composition and concentration of digestion electrolyte solution

[0108] M SSF pH = 7 (mM) SGF pH = 3 (mM) SIF pH = 7 (mM) NaCl 2 - 47.2 38.4 KCl 0.5 15.1 6.9 6.8 <![CDATA[KH2PO4]]> 0.5 3.7 0.9 0.8 <![CDATA[NaHCO3]]> 1 13.6 25 85 <![CDATA[MgCl2(H2O)6]]> 0.15 0.15 0.12 0.33 <![CDATA[(NH4)2CO3]]> 0.5 0.06 0.5 - HCl 6 1.1 15.6 0.4 <![CDATA[CaCl2(H2O)2]]> 0.3 1.5 2.5 0.6

[0109] Note: CaCl2 should be added just before use to avoid coagulation.

[0110] Weigh 0.200g of edible fungus protein sample and add 10mL of deionized water to fully dissolve. Salivary protease is dissolved with oral electrolyte to an activity of 75U / mL, pepsin is dissolved with gastric electrolyte to an activity of 2000U / mL, trypsin is dissolved with intestinal electrolyte to an activity of 100U / mL, and ox bile salt is dissolved with intestinal electrolyte to a concentration of 10mM.

[0111] Simulated oral digestion: Add 10 mL of simulated saliva (8 mL of oral electrolytes, 50 μL CaCl2 (0.3 M), 1500 μL of salivary amylase solution, 450 μL H2O) to 10 mL of sample solution, pH 7, and shake at 37°C for 5 min. Take 2 mL of the digestion solution after simulated oral reaction, add 100 μL of HCl (1 M) to terminate the reaction, and store at -20°C.

[0112] Simulated gastric digestion: Take 14 mL of oral digestive fluid and add 12.5 mL of simulated gastric fluid (10 mL of gastric electrolytes, 6.25 μL of 0.3 M CaCl2, 1605 μL of pepsin solution, 723 μL of H2O, 465 μL of 6 M HCl). Use 1 mol / L HCl to adjust the pH of the mixed system to 1.5, simulate digestion at 37°C for 2 h, take samples every 30 min (2 mL, 4 times in total), add 100 μL of 1 M NaOH to terminate the reaction, and store at -20°C.

[0113] Simulated intestinal digestion: 20 mL of gastric digestion fluid was added with 20 mL of simulated intestinal fluid (8 mL of intestinal electrolytes, 40 μL of 0.3 M CaCl2, 5050 μL of trypsin solution, 3083 μL of ox bile salt solution, 3310 μL of H2O, 517 μL of 6 M NaOH). The pH value of the mixed system was adjusted to 7.5 using 1 mol / L NaOH. Finally, the mixed system was placed at 37°C for 2 h of simulated digestion, and samples were taken every 30 min (3 mL, 4 times in total) and 45 μL of 1 mg / mL trypsin inhibitor was added to terminate the reaction, and stored at -20°C.

[0114] (2) Analysis of digestibility and utilization of edible fungus protein

[0115] After thawing the digestion products from the oral cavity, stomach and intestine, centrifuge at 6000 rpm for 20 min at 4°C, take the supernatant, dilute it at 1:9 (V / V), 1:3 (V / V) and 1:1 (V / V), and measure the protein concentration of the supernatant. Calculate the protein digestibility according to formula (2-18).

[0116] 6. Data collation and statistical analysis

[0117] Excel 2016 was used to organize the data, OriginPro 2021 was used to draw relevant graphs, and SpssStatistics was used to perform difference and significance analysis on the relevant data. p<0.05 indicated a significant difference.

[0118] (III) Results analysis

[0119] 1. Analysis of nutritional composition and content of edible fungi

[0120] The contents of protein, fat, carbohydrates and water in eight kinds of edible fungi (shiitake mushroom, button mushroom, white jade mushroom, oyster mushroom, oyster mushroom, oyster mushroom, tea tree mushroom, enoki mushroom and king oyster mushroom) are shown in Table 3. It can be seen from the table that there are certain differences among the nutritional components of the eight kinds of edible fungi powder. Carbohydrates are the main component of edible fungi, which is 35% to 85%. Among them, the carbohydrate content in enoki mushroom and shiitake mushroom is relatively high, which is 83.03% and 71.53% respectively, which is about twice that of button mushroom. Secondly, the protein content in edible fungi is rich, about 20%, the protein content in tea tree mushroom is as high as 27.20%, and the protein content in button mushroom and oyster mushroom is 23.70% and 23.87% respectively, but the protein content in enoki mushroom is only 6.10%, which is about 1 / 4 of that in tea tree mushroom. The moisture content in edible fungus powder is low, 3% to 8%. Although its moisture content is slightly higher than the moisture content test at the factory, the moisture content of Flammulina velutipes has increased significantly, and the moisture absorption is slightly different, which may be due to the high polysaccharide content in Flammulina velutipes; the fat content in edible fungi is extremely low, accounting for only about 1%, which can effectively reduce fat intake. In addition, the carbohydrates in edible fungi are mainly insoluble dietary fiber, which can promote intestinal peristalsis and promote digestion synergistically with protein; at the same time, studies have shown that the hygroscopicity and water retention of dietary fiber can improve the elasticity, texture and juiciness of the meat-like products added with edible fungus powder. It can be seen that high-carb, high-protein and low-fat edible fungi can be used as high-quality raw materials for plant-based meat substitutes.

[0121] Table 3 Content and comparative analysis of main nutrients in different edible fungi (g / 100g)

[0122] Types of Edible Fungi protein Fat carbohydrate Moisture mushroom <![CDATA[18.54±0.86 f ]]> <![CDATA[0.49±0.03 c ]]> <![CDATA[71.53±0.16 b ]]> <![CDATA[5.05±0.45 c ]]> Agaricus bisporus <![CDATA[23.70±0.28 b ]]> <![CDATA[1.08±0.06 ab ]]> <![CDATA[37.7±0.09 h ]]> <![CDATA[3.87±0.24 de ]]> White jade mushroom <![CDATA[21.07±0.05 c ]]> <![CDATA[1.13±0.05 ab ]]> <![CDATA[50.45±0.13 e ]]> <![CDATA[2.93±0.27 f ]]> Oyster Mushroom <![CDATA[23.87±0.23 b ]]> <![CDATA[1.11±0.05 ab ]]> <![CDATA[60.08±0.48 c ]]> <![CDATA[3.60±0.21 def ]]> Oyster Mushroom <![CDATA[19.14±0.14 e ]]> <![CDATA[1.19±0.03 ab ]]> <![CDATA[52.73±0.37 d ]]> <![CDATA[3.25±0.19 ef ]]> Agrocybe <![CDATA[27.20±0.1 a ]]> <![CDATA[1.55±0.08 a ]]> <![CDATA[47.69±0.39 g ]]> <![CDATA[4.10±0.38 d ]]> Flammulina velutipes <![CDATA[6.10±0.01 g ]]> <![CDATA[0.81±0.06 bc ]]> <![CDATA[83.03±0.90 a ]]> <![CDATA[7.77±0.48 a ]]> King Oyster Mushroom <![CDATA[19.79±0.07 d ]]> <![CDATA[1.26±0.68 ab ]]> <![CDATA[48.36±0.42 f ]]> <![CDATA[6.81±0.59 b ]]>

[0123] 2. Extraction of edible fungus protein and analysis of protein content

[0124] The purity of crude protein of eight edible fungi was measured by Kjeldahl nitrogen determination method as shown in Table 4. The protein extraction rate and weight yield were calculated at the same time. The residue was the precipitate during alkali extraction and could be used as a recombinant material for further processing, but the specific composition was yet to be determined.

[0125] There are obvious differences in the purity and protein extraction rate of edible fungi crude protein extracted by the relatively simple, more economical and efficient alkaline extraction and acid precipitation method. The purity of the eight edible fungi proteins ranged from 30% to 80%, and the extraction rate was 16.36% to 65.53%. Among them, the purity of the crude protein of shiitake mushrooms, Agaricus bisporus, Pleurotus ostreatus and Pleurotus eryngii was above 50%, which is a high-quality source of protein; the extraction rate of shiitake mushrooms, Pleurotus ostreatus and Agrocybe edulis proteins was above 50%, indicating that compared with other edible fungi, alkaline extraction and acid precipitation is a more effective extraction method for them.

[0126] The purity and extraction rate of shiitake edodes crude protein are very high, 79.20% and 63.25% respectively, and the extraction effect is significantly better than that of the other seven edible fungi. The second is oyster mushroom protein, with a purity and extraction rate of 56.07% and 52.76% respectively. The purity and extraction rate of Flammulina velutipes protein are very low, 33.62% and 16.36% respectively, which may be because Flammulina velutipes itself has a low protein content, a high polysaccharide content, and good protein solubility, resulting in a large loss during the separation process. In addition, the protein purity of Agaricus bisporus and Pleurotus eryngii proteins is high, but the extraction rate is relatively low.

[0127] Table 4 Protein content and extraction rate of crude protein from different edible fungi

[0128]

[0129] 3. Analysis of the physical and chemical properties of edible fungus protein

[0130] 3.1 Analysis of solubility, water holding capacity and oil holding capacity

[0131] Solubility, water holding capacity and oil holding capacity of eight edible fungus proteins Figure 1 (a) As shown. The solubility of edible fungus protein ranges from 38.66% to 104.22%, and there are significant differences among the eight proteins (p<0.05). Solubility is an important basis for the analysis of protein functional properties and is related to other functional properties such as emulsification and foaming. The solubility of Flammulina velutipes and Pleurotus eryngii proteins is 104.22% and 99.71%, respectively, which are almost completely dissolved; the solubility of Oyster mushroom protein (95.20%) and Pleurotus ostreatus protein (86.26%) is also above 80%; the solubility of Lentinus edodes and Agaricus bisporus proteins is poor, at 38.66% and 47.38%, respectively. It may be because during the protein extraction process, the different temperatures and pH lead to different degrees of opening of the protein structure. The structure of Flammulina velutipes, Pleurotus eryngii, and Oyster mushroom proteins may be more opened, more hydrophilic sites are exposed, and there are more disordered protein structures, so the solubility is better. In addition, if the proportion of hydrophilic amino acids in the protein is high, it will also show better solubility.

[0132] Water-binding capacity and oil-binding capacity are important properties that need to be considered during protein processing. Figure 1As shown in (a), the water holding capacity of Agrocybe aegerita protein is 58.10%, which is better than other proteins (p<0.05), close to that of flaxseed protein (59.83%), and better than soy protein (about 45%) and rapeseed protein (about 40%). The water holding capacity of Oyster mushroom, Pleurotus eryngii, and Oyster mushroom is relatively close, ranging from 45% to 50%. In addition, the lower water holding capacity of shiitake mushroom protein (37.10%) and Agaricus bisporus (39.55%) may be due to their tight structure, making it difficult for water molecules to enter the protein; while the water holding capacity of Flammulina velutipes (35.00%) is poor under this determination method, which may be due to its good solubility and difficulty in separation during centrifugation. In addition, raw materials with better water holding capacity can make imitation meat products more elastic and juicy in the post-ripening stage, improving the quality and taste of the products.

[0133] The oil holding capacity of eight edible fungi proteins ranged from 31.40% to 79.00%, among which Lentinus edodes protein and Pleurotus eryngii protein had stronger oil holding capacity, which were 79.00% and 78.30% respectively, and there was no significant difference between the two; Agrocybe agrocybe and Agaricus bisporus followed closely, which were 75.55% and 71.05% respectively; Flammulina velutipes protein had poor oil holding capacity, which was only 31.40%. The difference in oil holding capacity may be related to the purity of the protein and the type and number of lipophilic groups on the surface of the protein molecules. If the types of lipophilic groups are rich and the proportion is high, the protein has a better ability to bind fat and shows better oil holding capacity. The fat content in edible fungi is low, only about 2%. If the edible fungi protein has better oil holding capacity, on the one hand, the content of high-quality fat can be increased in the reconstructed system, making the nutrition more balanced, and the oil has better stability in the entire compound system. On the other hand, the addition of oil can make the flavor richer.

[0134] 3.2 Analysis of emulsification and emulsion stability

[0135] The emulsification and emulsification stability of eight edible fungus proteins showed some differences. Figure 1 As shown in (b), the emulsification of protein is 9.20m 2 / g~15.00m 2 / g, from high to low, Oyster mushroom (15.00m 2 / g), white jade mushroom (13.7m 2 / g), Flammulina velutipes (13.16m 2 / g), Agaricus bisporus (11.26m 2 / g), Oyster mushroom (10.23m 2 / g), Agrocybe aegerita(9.59m 2 / g), Pleurotus eryngii (9.20m 2 / g), shiitake mushroom (4.03m 2 / g), usually, the strength of emulsification is positively correlated with the solubility of protein. The better the solubility of protein, the easier it is to diffuse to the water-oil interface and form a relatively stable system. The emulsification stability of edible fungus protein is between 18min and 55min. Among them, the emulsification stability of Agaricus bisporus is relatively strong, at 53.26min; followed by white jade mushroom and shimeji protein, about 35min; shiitake mushroom, oyster mushroom, enoki mushroom and tea tree mushroom, about 24min; the emulsification stability of Pleurotus eryngii protein is relatively poor, only 18.17min. In summary, white jade mushroom, shimeji and Agaricus bisporus protein have good emulsification properties.

[0136] 3.3 Analysis of foaming properties and foaming stability

[0137] The foaming properties of edible fungus protein vary slightly. Figure 1 As shown in (c), the foaming ability of Pleurotus ostreatus and Pleurotus eryngii proteins is strong, with foaming abilities of 169.66% and 164.57% respectively. The foaming abilities of Lentinus edodes, Oyster mushroom, and Agrocybe oleraceus proteins are close, about 145%. The foaming abilities of Agaricus bisporus and Agrocybe edulis proteins are about 130%. The foaming ability of Flammulina velutipes protein is significantly weaker, only 78.15%, which may be due to its low purity, resulting in a low protein molecular weight content in the solution. As the standing time goes by, the foam gradually decreases. After standing for 5 minutes, the foam stability of Lentinus edodes, Agrocybe edulis, Pleurotus ostreatus, Oyster mushroom, Flammulina velutipes, and Pleurotus eryngii is above 60%, while the foam stability of Agaricus bisporus and Agrocybe oleraceus proteins is about 40%, and there is a significant difference between the two groups. After standing for 10 minutes, the foam stability of Pleurotus eryngii protein still remained at about 60%, the foam stability of Lentinus edodes, Pleurotus ostreatus, Pleurotus ostreatus and Oyster mushroom protein was about 45%, and the foam stability of Agaricus bisporus and Agrocybe aegerita was about 30%. In summary, Pleurotus eryngii, Pleurotus ostreatus, Pleurotus ostreatus and Oyster mushroom protein had better foaming performance.

[0138] 3.4 Analysis of secondary structure of edible fungus proteins

[0139] The infrared determination results of eight different edible fungi proteins are as follows Figure 2 As shown in the figure, a~h are infrared spectra of Lentinus edodes, Agaricus bisporus, Pleurotus ostreatus, Pleurotus ostreatus, Pleurotus eryngii, Agrocybe chrysogenum, Agrocybe chinensis, Flammulina velutipes, and Pleurotus eryngii, respectively. By comparing the wave number position, shape and intensity of the Fourier transform infrared spectrum absorption peaks of each edible fungus protein in the figure, it is found that the wave number position, peak shape and absorption intensity of their absorption peaks are highly similar, indicating the similarity of the chemical structures of the crude extracts of the eight edible fungus proteins.

[0140] It can be seen from the figure that the eight kinds of edible fungus protein crude extracts have five obvious main protein identification peaks, namely 3400cm -1 、1650cm -1 、1545cm -1 、1400cm-1 、1240cm -1 Among them, 3500~3200cm -1 The interval is 3380cm -1 The broad and strong absorption peak centered on is the mixed superposition absorption peak of the stretching vibration of hydroxyl OH and amino NH; 3000~2850cm -1 The range is CH3 and CH2 stretching oscillation peaks, 1600-1680cm -1 The absorption peak at 1560-1500cm is mainly related to the stretching vibration of carbonyl C=O, the angular vibration of amino NH and the bending vibration of hydroxyl OH, corresponding to the characteristic absorption peak of protein amide I band; -1 The characteristic absorption peak of the amide II band is related to the deformation vibration of the amino group NH and the stretching vibration of CN, and is affected by the amide I band. The absorption peaks of these two places are very strong, indicating that the crude protein extracts of these eight edible fungi contain a large amount of protein. -1 It is the mixed vibration absorption area of ​​protein and polysaccharide, 1455~1400cm -1 The weak absorption peak at 1420-1400cm is related to the symmetrical contraction of COO, which is also a protein absorption peak. -1 The characteristic absorption peak of amide III band was also observed, which was related to the stretching vibration of NH and CN, and the influence of C=O intra-plane bond and CC stretching vibration was very small.

[0141] The secondary structure determination results of eight edible fungi proteins are as follows Figure 3 As shown in (a), the amide I band (1700-1600 cm -1 ) spectra were deconvoluted and Gaussian fitted to calculate the percentage of secondary structure. -1 β-fold, 1650~1640cm -1 Irregular curl, 1660~1650cm -1 α-helix, 1700~1670cm -1 The β-turn angle is a quantitative indicator, and the calculation results are shown in Table 5. Figure 3 (b) as shown.

[0142] Table 5 Secondary structure content of crude protein in different edible fungi

[0143]

[0144]

[0145] Figure 3(b) is the secondary structure analysis result of eight edible fungi proteins based on Fourier infrared spectroscopy data. β-folds and α-helices in protein molecules can form a compact cavity-free structure, which has stronger conformational stability and compactness than random coils. Among them, the total amount of α-helices and β-folds in the proteins of Lentinus edodes, Agaricus bisporus, Agaricus leucorus, Pleurotus ostreatus, Oyster mushroom, Agrocybe fasciatus, Agrocybe tumefaciens, Flammulina velutipes, and Pleurotus eryngii are 57.83%, 58.34%, 51.95%, 57.91%, 57.81%, 60.08%, 42.10%, and 49.33%, respectively. Therefore, Agrocybe tumefaciens protein has a relatively more stable structure, followed by Agaricus bisporus, Pleurotus ostreatus, Oyster mushroom, and Lentinus edodes protein, while the stability of Flammulina velutipes protein is poor.

[0146] 3.5 Correlation and principal component analysis of edible fungus protein structures

[0147] Since the Fourier transform infrared spectra of the eight edible fungi proteins were highly similar, it was difficult to conduct a similarity and difference analysis as a whole. Therefore, the Savitsky-Golay second-order derivative spectrum (1800-700 cm -1 ) to distinguish, the result is as follows Figure 4 As shown, the second-order derivative spectrum is consistent with the original infrared data in overall trend, but can improve the resolution of the spectrum and provide more molecular information.

[0148] The second-order derivative infrared spectrum shows that the spectra of the eight edible fungi proteins are generally consistent, but there are some differences, mainly concentrated in the range of 1760-1710 cm -1 1600~1480cm -1 1320~1290cm -1 1140~1100cm -1 1060~980cm -1 Among them, 1745cm -1 The absorption peak near 1600-1550cm is mainly from the stretching vibration of lipid C=O. The proteins of Pleurotus eryngii, Oyster mushroom, Agrocybe tumefaciens and Pleurotus eryngii have obvious absorption peaks here. -1 The absorption peaks are mainly the vibration of the amino NH angle and the vibration of the aromatic ring skeleton. It can be found that the Agaricus bisporus and Agrocybe aegerita proteins are highly similar in this structure. 1500~1200cm -1 It is the mixed vibration absorption area of ​​protein and polysaccharide; 1315~1300cm -1 The absorption peak at 1050.17 cm is mainly due to the deformation vibration of amino NH and the stretching vibration of CN; the absorption peak of Flammulina velutipes protein at 1050.17 cm -1 、1021.89cm -1 、993.28cm -1There is an obvious absorption peak at 1200-960cm -1 It is the main absorption area of ​​polysaccharides, 1060~1050cm -1 The absorption peaks nearby are mainly caused by OH bending vibration and COC antisymmetric stretching vibration, which corresponds to the low purity of Flammulina velutipes crude protein.

[0149] Correlation analysis can measure the degree of linear correlation between variables. The closer the similarity value is to 1, the higher the correlation is, and the smaller the difference between samples is; the closer the similarity value is to 0, the lower the correlation is, indicating that the difference between samples is greater. Correlation analysis of infrared second-order derivative spectra is performed, and the results are as follows: Figure 5 As shown, the correlation coefficients of each sample are between 0.5246 and 0.9375, showing a strong correlation. Except for Flammulina velutipes protein, the correlation coefficients of other proteins are all above 0.7, indicating that the structural differences of these seven edible fungi proteins are not large. Among them, the smallest correlation is 0.5246 for Lentinus edodes protein and Flammulina velutipes protein; the largest correlation is Lentinus edodes protein and Pleurotus eryngii protein. In addition, it can be seen that the correlation between Lentinus edodes protein and Flammulina velutipes protein and other samples is relatively low, indicating that the chemical composition and proportion of these two edible fungi proteins are somewhat different from other samples, which can be further observed in the microstructure determination.

[0150] The comparison shows that the principal component division of the first-order derivative spectrum data is clearer than that of the second-order derivative data, and the classification effect is better. Therefore, the Savitsky-Golay first-order derivative spectrum (1800~700cm -1 ) data were subjected to principal component analysis (PCA), and the results are as follows Figure 6 As shown. The contribution rates of PC1, PC2, and PC3 are 36.04%, 22.24%, and 13.99%, respectively, and the cumulative contribution rate is 72.27%. According to the principal component analysis diagram, it can be found that the scatter points of Flammulina velutipes protein and the other seven proteins are far apart, and the differences are significant. Secondly, although the scatter points of Lentinus edodes protein are close to the center, they also show certain differences; the confidence intervals of Oyster mushroom protein and Agrocybe oleracea protein have almost no intersection, that is, there are relatively obvious structural differences between the two. The results are basically consistent with the correlation analysis.

[0151] For eight kinds of edible fungus protein 1800~700cm -1 Cluster analysis was performed on the first-order derivative spectral data within the range of Figure 7As shown, at a distance of about 5, they can be divided into four categories. Agaricus bisporus and Agrocybe aegerita proteins are in one category, which is consistent with the highly similar results of the two in the previous infrared second-order derivative spectrum analysis; Pleurotus ostreatus, Pleurotus eryngii, Pleurotus eryngii and Oyster mushroom proteins are in one category, and Lentinus edodes proteins and Flammulina velutipes proteins are in one category each, which is consistent with the results of principal component analysis. At a distance of about 10, Lentinus edodes proteins can be classified into one category with five proteins including Pleurotus ostreatus proteins. At a distance of about 15, Flammulina velutipes proteins are in a separate category, and other edible fungi proteins are in one category. The compositional differences of edible fungi proteins with similar structures under higher resolution conditions are more clearly presented.

[0152] 3.6 Analysis of the microstructure of edible fungus protein

[0153] Scanning electron microscopy images of crude protein extracts of eight edible fungi Figure 8 As shown. In order to better observe the microstructural differences between the sample components, images at different magnifications (100x, 400x and 800x) were taken. Comparing all the images, Agaricus bisporus (b) and Agrocybe oleracea (f) proteins have large-area flaky structures, smooth surfaces, good spatial ductility of the proteins, forming a stable structure with large gaps, and a dense fracture structure; Lentinus edodes (a) and Pleurotus eryngii (h) proteins have slightly lower relative strength, and are basically irregular block structures, but also have smooth surfaces and dense internal structures. A large number of extended secondary structures can be clearly seen on the surface of Pleurotus ostreatus (d), Pleurotus eryngii (c) and Pleurotus ostreatus (e) proteins, with smaller thickness and slightly weaker strength. The exposed parts are easily broken (d-3, e-3), but also form a pore structure with a more complex internal spatial structure (c-3). Although a large number of secondary structures are also formed on the surface of Flammulina velutipes protein, the strength is obviously very low, and the structure is stacked (g-2). It is generally consistent with the previous analysis results of the secondary structure of edible fungi crude protein. The proteins of Agaricus bisporus and Agrocybe oleracea, which have a higher proportion of β-fold and α-helix in the secondary structure of protein molecules, form a compact cavity-free structure with strong spatial ductility and stability. Pleurotus ostreatus, Oyster mushroom and Pleurotus eryngii have a certain degree of spatial ductility and are more flexible; in addition, Oyster mushroom and Pleurotus eryngii also show a high degree of similarity in microstructure.

[0154] 4. Analysis of amino acid composition of edible fungus protein

[0155] 4.1 Amino acid composition of edible fungus protein

[0156] The nutritional value of protein mainly depends on the type, quantity and composition ratio of amino acids. The amino acid composition of eight edible fungi proteins is shown in Table 6. It can be seen that the amino acid types in edible fungi crude protein are complete, containing 7 essential amino acids and 10 non-essential amino acids. The total amino acid (Total amino acid, TAA) is between 300 mg / g and 800 mg / g. Among them, the total amino acid of shiitake mushroom protein is the highest, which is 807.78 mg / g, followed by oyster mushroom protein and Agaricus bisporus protein, which are 543.58 mg / g and 525.13 mg / g respectively. The total amino acid of Flammulina velutipes is the lowest, which is only 298.26 mg / g, which is about 0.35 of that of shiitake mushroom protein. However, the results of the determination of the total amino acid content and protein content are basically consistent. According to the total amino acid content, they are ranked as follows: shiitake mushroom> oyster mushroom> Agaricus bisporus> Pleurotus eryngii> Oyster mushroom> Agrocybe tumefaciens> Agrocybe glabra> Flammulina velutipes.

[0157] The content of essential amino acids (EAA) ranged from 125mg / g to 305mg / g, of which the content of essential amino acids in shiitake mushroom protein was the highest, at 306.76mg / g, followed by oyster mushroom protein and Agaricus bisporus protein, at 232.95mg / g and 231.19mg / g, respectively. The non-essential amino acids also showed similar results, with the content of non-essential amino acids (NEAA) ranging from 170mg / g to 500mg / g, and the content of non-essential amino acids in shiitake mushroom was the highest, at 501.01mg / g, followed by oyster mushroom protein (310.63mg / g) and Agaricus bisporus protein (293.94mg / g).

[0158] When evaluating protein quality, not only the amino acid content is considered, but also the composition and ratio of amino acids. The ideal protein model proposed by FAO / WHO points out that the amino acid composition of high-quality protein is that the ratio of essential amino acids to total amino acids (EAA / TAA) is about 40%, and the ratio of essential amino acids to non-essential amino acids (E / N) is above 60%. The EAA / TAA ratios of the crude proteins of eight edible fungi are all around 40%, and the EAA / TAA ratios of Agaricus bisporus, Agaricus leuconi, Agaricus oyster, Oyster mushroom, Enoki mushroom, and Pleurotus eryngii are between 42% and 44%; the EAA / TAA ratio of Lentinus edodes is slightly lower, at 37.98%; the essential amino acid content in the protein of Agrocybe oleraceus is significantly higher, with an EAA / TAA ratio of 47.12%. At the same time, the EAA / NEAA values ​​of the crude proteins of the eight edible fungi are all above 60%, among which the EAA / NEAA value of Agrocybe oleraceus is the highest, at 89.09%, followed by Oyster mushroom, at 80.02%. It can be seen that the overall proportion of proteins in these eight edible fungi conforms to the ideal protein model, and the EAA / TAA values ​​and EAA / NEAA values ​​are close to those of whole egg proteins, making them high-quality protein sources.

[0159] Table 6 Amino acid composition of different edible fungus proteins (mg / g)

[0160]

[0161]

[0162] Note: * indicates essential amino acids

[0163] 4.2 Evaluation of nutritional value of edible fungus protein

[0164] The composition and proportion of essential amino acids that the human body cannot synthesize or the synthesis rate cannot meet the body's needs and must be ingested through food are important indicators for further evaluating the nutritional value of food protein. Referring to the relevant intake requirements for people over 3 years old in the FAO / WHO model spectrum, the amino acid score (AAS), amino acid ratio (RC) and amino acid ratio coefficient score (SRC) of edible fungi crude protein are calculated, as shown in Tables 7 and 8.

[0165] The closer the amino acid score is to 100, the closer it is to the pattern spectrum requirements, and the better the protein quality. The limiting amino acid score is less than 100, and the lowest score is the first limiting amino acid, which will affect the body's utilization of protein. As shown in Table 7, the amino acid scores of Tyr+Phe in the eight edible fungi proteins are all greater than 100, which is better than the ideal model proposed by FAO / WHO. The content of Tyr+Phe in shiitake mushroom protein is 2.48 times the recommended amount of the pattern spectrum, which has a clear content advantage. The amino acid scores of His, Thr, Leu and Lys in shiitake mushroom protein are all greater than 100, which are 148.42, 103.52, 123.74 and 132.97 respectively. The amino acid score of Ile is 91.31, which is close to 100. It can be seen that shiitake mushroom protein is an ideal protein source. And its essential amino acid score, except Thr, is the highest among the eight edible fungi proteins. In addition, the amino acid scores of His, Thr and Leu in Agaricus bisporus, Pleurotus ostreatus and Pleurotus eryngii proteins are also close to 100. Val, Met+Cys, and Ile are the limiting amino acids for all edible fungi proteins, among which Met+Cys is the first limiting amino acid. Among them, methionine plays an important role in participating in protein synthesis, anti-aging, and promoting muscle growth; it can also increase the synthesis of the neurotransmitter acetylcholine, which helps to improve memory and cognitive function. Cysteine ​​is a semi-essential amino acid that plays an important role in scavenging free radicals in the body, enhancing antioxidant and immunity. Phenylalanine plays an important regulatory role in the body's metabolism, can promote blood circulation, and effectively prevent brain dullness. Lack of phenylalanine may lead to malnutrition and brain dysfunction.

[0166] Therefore, among all the edible fungi crude proteins, the protein composition of shiitake mushroom protein is closest to the ideal model, followed by Agaricus bisporus, Oyster mushroom and Pleurotus eryngii proteins, and Flammulina velutipes protein is the most different. In subsequent applications, it is necessary to consider complementing with other proteins to make up for the lack of methionine and cysteine.

[0167] The amino acid ratio (RC) and amino acid ratio coefficient score (SRC) not only consider the degree of proximity of the amino acid composition of the protein to the ideal model, but also measure the balance between its own amino acids. The closer the RC value is to 1, the smaller its coefficient of variation is, the closer the SRC is to 100, the balanced amino acid composition of the protein is, and the higher the nutritional value is. When RC>1, the corresponding amino acid is relatively surplus; otherwise, it is relatively insufficient. As shown in Table 8, the amino acid ratio coefficient score of Pleurotus eryngii is the highest, which is 64.20, followed by Pleurotus ostreatus (63.51) and Agaricus bisporus (62.21); the amino acid ratio coefficient scores of Lentinus edodes and Flammulina velutipes are relatively low, which are 54.94 and 51.51 respectively, mainly because the relative content of Met+Cys in Lentinus edodes protein and Lys in Flammulina velutipes is very low, while the relative content of Tyr+Phe is very high, and the amino acid composition is significantly different, which is quite different from the model amino acid.

[0168] Table 7 Amino acid scores of different edible fungi proteins

[0169]

[0170] Note: The amino acid scoring model recommended by FAO / WHO for children over 3 years old, adolescents and adults is: histidine (His) = 16, threonine (Thr) = 25, valine (Val) = 40, methionine + cysteine ​​(Met + Cys) = 23, isoleucine (Ile) = 30, leucine (Leu) = 61, tyrosine + phenylalanine (Tyr + Phe) = 41, lysine (Lys) = 48

[0171] Table 8 Amino acid ratios, amino acid ratio coefficients and amino acid ratio coefficients of different edible fungi proteins

[0172]

[0173]

[0174] The amino acid composition of egg protein is rich and complete, and its essential amino acid composition is basically similar to that of the human body. It has outstanding biological value and is therefore often used as a reference protein to measure the nutritional value of other proteins. The amino acid chemical score (CS) and essential amino acid index (EAAI) of edible fungus crude protein were calculated based on the whole egg protein model, as shown in Table 9. The closer the EAAI value is to 100, the closer the amino acid composition of edible fungus protein is to eggs, the more balanced the overall nutrition is, and the higher the nutritional value of the protein is.

[0175] The amino acid chemical scores of His (107.94), Tyr+Phe (109.50), and Lys (91.18) in shiitake mushroom protein are above 90, which is close to the composition of essential amino acids in whole egg protein. Among the other seven edible fungi proteins, the amino acid chemical scores of His (74.28, 71.45, 66.07) and Tyr+Phe (78.46, 78.57, 70.99) in Agaricus bisporus, Pleurotus ostreatus, and Pleurotus eryngii are above 70, and the chemical scores of the remaining amino acids are all low. It can be seen that the essential amino acid composition of edible fungi crude protein is still significantly insufficient compared to the ideal protein source, eggs.

[0176] The essential amino acid index (EAAI) of eight edible fungi proteins ranged from 28.57 to 66.31, with shiitake protein having the highest EAAI and Flammulina velutipes protein having the lowest EAAI. The ranking of their scores was as follows: shiitake (66.31) > oyster mushroom (52.54) > Agaricus bisporus (52.16) > Pleurotus eryngii (48.44) > Oyster mushroom (40.55) > Agrocybe aegerita (39.30) > Oyster mushroom (36.59) > Flammulina velutipes (28.57). Among them, the EAAI of shiitake, oyster mushroom, and Agaricus bisporus proteins was similar to that of wheat protein (64.83) and rice protein (66.47), and lower than that of corn protein (70.90). In addition, Thr, Tyr+Phe are limiting amino acids of many cereal proteins, which can complement edible fungi proteins and better exert the nutritional value of proteins.

[0177] Table 9 Amino acid chemical scores and essential amino acid index of different edible fungi proteins

[0178]

[0179]

[0180] Note: FAO / WHO pointed out that the amino acid score pattern of whole eggs is: histidine (His) = 22, threonine (Thr) = 47, valine (Val) = 66, methionine + cysteine ​​(Met + Cys) = 57, isoleucine (Ile) = 54, leucine (Leu) = 86, tyrosine + phenylalanine (Tyr + Phe) = 93, lysine (Lys) = 70

[0181] 5. Analysis of protein digestibility of edible fungi

[0182] The in vitro simulated digestibility can be used to preliminarily determine the body's digestion and utilization of protein. The digestibility of eight edible fungus proteins is as follows: Fig. 9 As shown. The oral digestibility of the eight edible fungi proteins showed significant differences (p<0.05), ranging from 23% to 66%. Among them, the oral digestibility of Agaricus bisporus protein was lower, at 22.89%, which may be related to the purity and solubility of the protein. The protein has better solubility and more exposed binding sites, so its digestibility is higher in the initial stage of digestion. The digestibility of the eight edible fungi proteins at the end of the stomach was relatively small, ranging from 62% to 78%, among which the digestibility of white jade mushroom protein was significantly higher, at 78.06%, followed by Pleurotus eryngii, Flammulina velutipes and Oyster mushroom protein, which were 74.48%, 73.56% and 72.09% respectively. Among them, the purity of Pleurotus eryngii, white jade mushroom and Oyster mushroom protein was 51.71%, 40.84% ​​and 43.78% respectively, and showed a strong correlation in secondary structure and microstructure. The white jade mushroom, Oyster mushroom and Oyster mushroom protein had good solubility (more than 95%). The digestibility of eight kinds of edible fungus proteins at the end of the intestine ranged from 65% to 90%, with certain differences (p<0.05), and was higher than the in vitro digestibility of wheat protein, corn protein, and chickpea protein. This may be because the extraction method of edible fungus protein is relatively simple, without using ultrasound and other methods, so the protein structure is more unfolded and has a higher water solubility. Under the action of the enzyme system, the protein structure is further unfolded. In addition, the digestibility at the end of gastric digestion and the end of intestinal digestion is not much different, indicating that these edible fungus proteins are proteins that can be quickly digested and absorbed, and are an excellent choice for rapid protein supplementation. Among them, the digestibility of white jade mushroom protein at the end of the intestine is significantly higher, at 89.59%, higher than the digestibility of yeast protein concentrate (75.12%), and close to the digestibility of whey protein concentrate (95.2%).

[0183] At present, due to the certain errors in the digestibility determination of the in vivo model and the obvious differences in the fecal digestibility of different edible fungi proteins, it is difficult to conduct a standardized evaluation of edible fungi proteins from the perspective of ileal digestibility, and its evaluation focuses on non-biological evaluation methods. According to the data of the terminal digestibility in the in vitro simulated digestion experiment, the amino acid score can be further optimized to better reflect the nutritional value of edible fungi proteins. The calculation results are shown in Table 10. It can be seen that the corrected scores of Tyr+Phe in almost all edible fungi proteins are above 100, that is, the content of these two amino acids is above the standard value. The corrected amino acid scores of His, Ile, Leu, Tyr+Phe, and Lys in shiitake mushroom protein are the highest among the eight edible fungi proteins, followed by the overall higher scores of oyster mushroom, Agaricus bisporus, and Pleurotus eryngii proteins.

[0184] Table 10 Digestibility-corrected amino acid scores of different edible fungi proteins

[0185]

[0186]

[0187] Note: The amino acid scoring model recommended by FAO / WHO for children over 3 years old, adolescents and adults is: histidine (His) = 16, threonine (Thr) = 25, valine (Val) = 40, methionine + cysteine ​​(Met + Cys) = 23, isoleucine (Ile) = 30, leucine (Leu) = 61, tyrosine + phenylalanine (Tyr + Phe) = 41, lysine (Lys) = 48

[0188] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for comprehensive evaluation of nutritional and functional properties of edible fungus protein, characterized in that: The steps include: obtaining an edible fungus protein extract, and determining the physicochemical properties of the edible fungus protein extract; Analyzing the amino acid composition of the edible fungus protein extract and calculating the amino acid score of the edible fungus protein extract; Determine the in vitro digestibility of the edible fungus protein extract, optimize the amino acid score based on the in vitro digestibility data, and determine the final amino acid score of the edible fungus protein extract; The physicochemical properties, amino acid composition and final amino acid score of edible fungus protein extracts were comprehensively analyzed to evaluate the nutritional value and functional properties of edible fungus protein.

2. The method for comprehensive evaluation of nutrition and functional properties according to claim 1, characterized in that: The calculation formula of the final amino acid score is: in vitro digestibility×amino acid score=final amino acid score.

3. The method for comprehensive evaluation of nutrition and functional properties according to claim 1, characterized in that: The physicochemical properties of the edible fungus protein extract include one or more of solubility, water holding capacity, oil holding capacity, emulsification and foaming properties.

4. The method for comprehensive evaluation of nutrition and functional properties according to claim 1 or 2, characterized in that: The in vitro digestibility includes one or more of oral digestibility, terminal stomach digestibility and terminal intestinal digestibility.

5. The method for comprehensive evaluation of nutrition and functional properties according to claim 1, characterized in that: The edible fungus protein extract is obtained by extracting with an alkali extraction and acid precipitation method.

6. The method for comprehensive evaluation of nutrition and functional properties according to claim 5, characterized in that: The alkali extraction temperature of the alkali extraction and acid precipitation method is 30-65°C, the alkali extraction time is 0.5-3.5h, and the pH value is 10-12; the acid precipitation time of the alkali extraction and acid precipitation method is 2-5h, and the pH value is 3-5.

7. The method for comprehensive evaluation of nutrition and functional properties according to claim 1, characterized in that: The method also includes calculating the amino acid ratio and amino acid ratio coefficient of the edible fungus protein extract.

8. The method for comprehensive evaluation of nutrition and functional properties according to claim 1, characterized in that: Before obtaining the edible fungus protein extract, the basic composition of the edible fungus is determined.

9. Application of the comprehensive evaluation method of nutritional and functional characteristics as described in any one of claims 1 to 8 in assisting the screening of high-quality edible fungi varieties.

10. Use of the comprehensive evaluation method of nutritional and functional characteristics according to any one of claims 1 to 8 in identifying edible fungi varieties.

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