Lentinan-beta glucan complex and application thereof

Abstract

This invention belongs to the field of modified protein technology, and provides a shiitake mushroom protein crude extract-β-glucan complex and its application. The shiitake mushroom variety includes Shenxiang 1513, and the origin of Shenxiang 1513 includes Hubei or Zhejiang. The β-glucan includes at least one of barley β-glucan, oat β-glucan, and yeast β-glucan. This invention found that, compared with shiitake mushroom protein crude extract alone, the shiitake mushroom protein crude extract-β-glucan complex significantly increased the production of short-chain fatty acids and promoted the proliferation of beneficial bacteria. Simultaneously, this invention, through microbiome and metabolomics analysis, identified Bacillus phylum as a key positive regulator and confirmed its association with the upregulation of metabolic pathways such as purines, lipids, bile acids, and amino acids. These findings indicate that the shiitake mushroom protein crude extract-β-glucan complex of this invention has superior prebiotic potential, providing a theoretical basis for the development of functional food ingredients.

Description

Technical Field

[0001] This invention belongs to the field of modified protein technology, specifically relating to a crude extract of shiitake mushroom protein-β-glucan complex and its applications. Background Technology

[0002] β-glucan, a structural non-starch polysaccharide, is a high-molecular-weight polymer composed of glucose linked by glycosidic bonds. Its inherent high viscosity allows it to enhance the emulsion stability and gelation properties of proteins when complexed with them. It also possesses effects such as lowering blood sugar and enhancing immunity, making it suitable for protein glycosylation modification. However, current technologies for protein-β-glucan complexes suffer from low complexation efficiency, structural instability, and unclear digestion and gut microbiota regulation mechanisms, hindering the development and utilization of protein-β-glucan complexes.

[0003] Therefore, there is an urgent need to provide a protein-β-glucan complex that is structurally stable, has high compounding efficiency, is digestible and controllable, has strong intestinal targeting, and has a clear regulatory mechanism, so as to provide a theoretical basis and technical support for protein and β-glucan resources. Summary of the Invention

[0004] The purpose of this invention is to provide a lentinan-beta-glucan complex derived from shiitake mushroom protein and its applications. The lentinan-beta-glucan complex described in this invention can construct a three-layered regulatory network of microorganisms, metabolic pathways, and metabolites, with Bacillus phylum as its core. It possesses multiple advantages, including structural improvement, controllable digestion, intestinal microecological regulation, and metabolic remodeling, exhibiting superior prebiotic potential and providing a theoretical basis for developing functional food ingredients for intestinal health.

[0005] Beneficial Effects: This invention provides a shiitake mushroom protein crude extract-β-glucan complex, wherein the shiitake mushroom variety includes Shenxiang 1513, and the origin of Shenxiang 1513 includes Hubei or Zhejiang; the β-glucan includes at least one of barley β-glucan, oat β-glucan, and yeast β-glucan; the preparation method of the shiitake mushroom protein crude extract includes: mixing shiitake mushrooms with water, adjusting the pH to alkaline, extracting, centrifuging to collect the supernatant after extraction, adjusting the pH to the isoelectric point, centrifuging to obtain the precipitate, adding water to the precipitate to adjust the pH to neutral, collecting the supernatant and freeze-drying to obtain the shiitake mushroom protein crude extract. This invention found that, compared with shiitake mushroom protein crude extract alone, the shiitake mushroom protein crude extract-β-glucan complex significantly increased the production of short-chain fatty acids and promoted the proliferation of beneficial bacteria. The Zhejiang 1513 protein crude extract-yeast βG complex showed the strongest effect in inhibiting dissociation and regulating the microbial community. Meanwhile, this invention identified Bacillus phylum as a key positive regulator through microbiome and metabolomics analysis, and confirmed the upregulation of metabolic pathways related to purines, lipids, bile acids, and amino acids. These findings indicate that the lentinan-beta-glucan complex of shiitake mushroom protein crude extract described in this invention has superior prebiotic potential, providing a theoretical basis for the development of functional food ingredients.

[0006] The shiitake mushroom protein crude extract-β-glucan complex of this invention can alter the conformation of shiitake mushroom protein at the molecular level, forming a stable three-dimensional network structure with covalent and non-covalent bonds. This significantly improves thermal stability, pH adaptability, solubility, water-holding capacity, and emulsification stability, overcoming the defects of natural shiitake mushroom protein, such as easy aggregation, poor stability, and weak functional properties. It can form a spatial barrier in the gastrointestinal tract, precisely inhibiting premature protein enzymatic degradation and rapid release of free amino acids, achieving targeted and sustained protein release in the intestine, significantly improving nutrient utilization and reducing gastrointestinal burden. It can efficiently regulate the intestinal microecological balance, significantly increasing the richness and diversity of the gut microbiota, selectively enriching beneficial bacteria such as Bacillus, Bifidobacterium, Clostridium lacunae, and Bacillus platycos, while inhibiting protein putrefactive bacteria and opportunistic pathogens, shifting intestinal metabolism from harmful protein putrefactive fermentation to a healthy polysaccharide fermentation mode. It can also significantly promote the production of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid. It effectively maintains the acidic environment of the intestine, strengthens intestinal tight junctions, repairs the mucosal barrier, and exerts systemic anti-inflammatory effects. It can also systematically regulate purine metabolism, lipid metabolism, secondary bile acid metabolism, and aromatic amino acid metabolism, promote the accumulation of beneficial metabolites such as ursolic acid, L-tryptophan, and functional peptides, and reduce toxic bile acids, pro-inflammatory lipids, and putrefactive products such as ammonia and indole. It constructs a three-layer positive regulatory network of microorganisms-metabolic pathways-functional metabolites with Bacillus phylum as the core. At the same time, it is the first to clarify the compound differences between crude extracts of shiitake mushroom protein from different origins and β-glucan from different sources, realizing customizable functions and predictable effects. It solves the problems of low compounding efficiency, poor reproducibility, and interference from endogenous polysaccharides in existing technologies. Overall, it has multiple unique advantages such as structural improvement, controllable digestion, prebiotic fortification, metabolic remodeling, and improved processing adaptability, making it more suitable for developing highly stable and highly functional intestinal health foods and special medical foods. Attached Figure Description

[0007] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0008] Figure 1 Figure 1 shows the absorbance changes of crude shiitake mushroom protein extract and its complex; where A represents the absorbance changes of HLEP-0; B represents the absorbance changes of HLEP-BBG; C represents the absorbance changes of HLEP-OBG; D represents the absorbance changes of HLEP-YBG; E represents the absorbance changes of ZLEP-0; F represents the absorbance changes of ZLEP-BBG; G represents the absorbance changes of ZLEP-OBG; H represents the absorbance changes of ZLEP-YBG; and I represents the absorbance changes of the sample after 180 min. Figure 2The graph shows the water-holding capacity (WHC) and thermal stability of crude extract of shiitake mushroom protein and its complex; where A represents the water-holding capacity test result; and B represents the thermal stability test result. Figure 3 The graph shows the results of protein solubility determination of crude extract of shiitake mushroom egg and its complex; where A represents the solubility determination results of HLEP-YBG, HLEP-OBG, HLEP-BBG, HLEP-0 and HLEP; and B represents the solubility determination results of ZLEP-YBG, ZLEP-OBG, ZLEP-BBG, ZLEP-0 and ZLEP. Figure 4 The particle size distribution of crude extract of shiitake mushroom protein and its complex at different pH values ​​is shown in the figure; where A~J are ZLEP, ZLEP-0, ZLEP-BBG, ZLEP-OBG, ZLEP-YBG, HLEP, HLP-0, HLEP-BBG, HLEP-OBG and HLEP-YBG, respectively. Figure 5 The graphs show the zeta potential changes of crude extract of shiitake mushroom protein and its complex at different pH values; where A represents the zeta potential changes of HLEP and HLEP-βG at different pH values; and B represents the zeta potential changes of ZLEP and ZLEP-βG at different pH values. Figure 6 The figure shows the results of emulsifying ability determination of crude extract of shiitake mushroom protein and its complex. In the figure, A is the EAI value of HLEP and HLEP-βG under different pH conditions; B is the EAI value of ZLEP and ZLEP-βG under different pH conditions; C is the ESI value of HLEP and HLEP-βG under different pH conditions; and D is the ESI value of ZLEP and ZLEP-βG under different pH conditions. Figure 7 The graphs show the changes in protein content and free amino content of crude shiitake protein extract and its complex, where A represents the changes in protein content and B represents the changes in free amino content. Figure 8Images show the scanning electron microscopy (SEM) results of crude HLEP protein extract and its complexes from shiitake mushrooms. A is an SEM image of pure HLEP protein in the undigested stage (distance × Magnification: ND 5.3 × 800); B is an SEM image of undigested LEP-0 (distance × Magnification: ND 5.9 × 300); C is an SEM image of undigested HLEP-BBG (distance × Magnification: ND 5.2 × 800); D is an SEM image of undigested HLEP-OBG (distance × Magnification: ND 6.2 × 800); E is an SEM image of undigested HLEP-YBG (distance × Magnification: ND 5.2 × 800); F is an SEM image of pure HLEP protein in the gastric digestion stage (distance × Magnification: ND 5.9 × 800); G is an SEM image of the HLEP-0 control group in the gastric digestion stage (distance × Magnification: ND 5.9 × 800). D 5.9×800; H is the SEM image of the HLEP-BBG complex in the gastric digestion stage, distance × fold: N D 4.7×800; I is the SEM image of the HLEP-OBG complex in the gastric digestion stage, distance × fold: N D 5.1×800; J is the SEM image of the HLEP-YBG complex in the gastric digestion stage, distance × fold: N D 5.3×800; K is the SEM image of pure HLEP protein in the intestinal digestion stage, distance × fold: N D 5.0×800; L is the SEM image of the HLEP-0 control group in the intestinal digestion stage, distance × fold: N D 6.8×800; M is the SEM image of the HLEP-BBG complex in the intestinal digestion stage, distance × fold: N D 6.6×800; N is the SEM image of the HLEP-OBG complex in the intestinal digestion stage, distance × fold: N D6.3×800; O is a SEM image of the HLEP-YBG complex during the intestinal digestion stage, distance × magnification: ND5.4×800; the scale bar in the image is 100µm; Figure 9Images show the scanning electron microscopy (SEM) results of ZLEP crude extract and its complex from shiitake mushrooms. A is the SEM image of pure ZLEP protein in the undigested stage (distance × magnification: ND 5.6 × 900); B is the SEM image of ZLEP-0 in the undigested stage (distance × magnification: ND 5.9 × 600); C is the SEM image of ZLEP-BBG in the undigested stage (distance × magnification: ND 6.2 × 800); D is the SEM image of ZLEP-OBG in the undigested stage (distance × magnification: ND 6.2 × 800); E is the SEM image of ZLEP-YBG in the undigested stage (distance × magnification: ND 5.2 × 800); F is the SEM image of pure ZLEP protein in the gastric digestion stage (distance × magnification: ND 5.5 × 800); G is the SEM image of the ZLEP-0 control group in the gastric digestion stage (distance × magnification: ND 5.5 × 800). D6.6×800; H is the SEM image of the ZLEP-BBG complex during gastric digestion, distance × magnification: N D6.0×800; I is the SEM image of the ZLEP-OBG complex during gastric digestion, distance × magnification: N D4.9×800; J is the SEM image of the ZLEP-YBG complex during gastric digestion, distance × magnification: N D5.1×800; K is the SEM image of pure ZLEP protein during intestinal digestion, distance × magnification: N D5.4×800; L is the SEM image of the ZLEP-0 control group during intestinal digestion, distance × magnification: N D5.3×800; M is the SEM image of the ZLEP-BBG complex during intestinal digestion, distance × magnification: N D5.2×800; N is the SEM image of the ZLEP-OBG complex during intestinal digestion, distance × magnification: N D5.2×800; O is a SEM image of the ZLEP-YBG complex during the intestinal digestion stage, distance × magnification: ND5.1×800; the scale bar in the image is 100µm; Figure 10 Figure 1 shows the secondary structure analysis results of crude lentinan extract and its complex; where A is the FT-IR infrared spectrum of HLEP and its complex in the undigested stage; B is the FT-IR infrared spectrum of ZLEP and its complex in the undigested stage; C is the FT-IR infrared spectrum of HLEP and its complex in the gastric digestion stage; D is the FT-IR infrared spectrum of ZLEP and its complex in the gastric digestion stage; E is the FT-IR infrared spectrum of HLEP and its complex in the intestinal digestion stage; and F is the FT-IR infrared spectrum of ZLEP and its complex in the intestinal digestion stage. Figure 11 Principal component analysis (PCA) diagrams of crude extract of shiitake mushroom protein and its complexes are shown below; where A represents the PCA diagram of all samples in the undigested stage; B represents the PCA diagram of all samples in the gastric digestion stage; and C represents the PCA diagram of all samples in the intestinal digestion stage. Figure 12This is a stacked bar diagram showing the secondary structure composition of protein digestion products; Figure 13 Figure 1 shows the results of tertiary structure analysis of crude shiitake mushroom protein extract and its complex; where A is the endogenous fluorescence spectrum of HLEP and its complex in the undigested stage; B is the endogenous fluorescence spectrum of ZLEP and its complex in the undigested stage; C is the endogenous fluorescence spectrum of HLEP and its complex in the gastric digestion stage; D is the endogenous fluorescence spectrum of ZLEP and its complex in the gastric digestion stage; E is the endogenous fluorescence spectrum of HLEP and its complex in the intestinal digestion stage; F is the endogenous fluorescence spectrum of ZLEP and its complex in the intestinal digestion stage; and G is a radar chart showing the fluorescence intensity changes of each sample at different digestion stages. Figure 14 OD during in vitro fermentation of crude extract of shiitake mushroom protein and its complex 600 The graph shows the changes in pH and OD values; where A represents the OD values ​​during the in vitro fermentation of HLEP and its complexes. 600 The graph shows the changes in pH and pH values; B represents the OD values ​​during the in vitro fermentation of ZLEP and its complexes. 600 Graph showing the changes in pH value; Figure 15 Figure 1 shows the effect of in vitro fermentation of crude shiitake protein extract and its complex on short-chain fatty acids. Figure 2 shows the dynamic changes of acetic acid concentration over time in different treatment groups during fermentation; Figure 3 shows the dynamic changes of propionic acid concentration over time in different treatment groups during fermentation; Figure 4 shows the dynamic changes of butyric acid concentration over time in different treatment groups during fermentation; Figure 5 shows the dynamic changes of isobutyric acid concentration over time in different treatment groups during fermentation; Figure 6 shows the dynamic changes of valeric acid concentration over time in different treatment groups during fermentation; Figure 7 shows the dynamic changes of isovaleric acid concentration over time in different treatment groups during fermentation; Figure 8 shows the dynamic changes of valeric acid concentration over time in different treatment groups during fermentation; Figure 9 shows the dynamic changes of isovaleric acid concentration over time in different treatment groups during fermentation. Figure 16 The results show the effects of in vitro fermentation of shiitake mushroom protein crude extract and its complex on short-chain fatty acids; where A is a pie chart showing the composition ratio of short-chain fatty acids (SCFAs) in the fermentation system of different treatment groups; B is a pie chart showing the composition ratio of branched-chain fatty acids (BCFAs) in the fermentation system of different treatment groups; and C is a heatmap analysis of the concentration of major metabolites in the fermentation system of different treatment groups. Figure 17Figure 1 shows the effects of in vitro fermentation of crude shiitake mushroom protein extract and its complex on gut microbiota. Figure 2 shows the ACE index box plots of gut microbiota in different treatment groups; Figure 3 shows the Chao1 index box plots of gut microbiota in different treatment groups; Figure 4 shows the Shannon index box plots of gut microbiota in different treatment groups; Figure 5 shows the Simpson index box plots of gut microbiota in different treatment groups; Figure 6 shows the principal component analysis (PCA) scores of different treatment groups at the genus level; Figure 7 shows the principal component analysis (PCA) scores of different treatment groups at the genus level; Figure 8 shows the circos diagram of gut microbiota species composition in different treatment groups; Figure 9 shows the heatmap of species composition at the genus level in different treatment groups. Figure 18 The results show the effects of in vitro fermentation of crude shiitake protein extract and its complex on gut microbiota; where A is a bar chart of relative abundance of gut microbiota at the genus level in different treatment groups; B is a heatmap of the correlation between different treatment groups and metabolites / indicators; and C is a heatmap of functional prediction for different treatment groups. Figure 19 The results of in vitro fermentation of crude extract of shiitake mushroom protein and its complex on metabolism are shown in the figure. Among them, A is the principal component analysis (PCA) figure of the metabolome of different treatment groups; B is the principal component analysis (PCA) figure of the metabolome of different treatment groups; C is the principal component analysis score figure of the metabolome of different treatment groups; D is the correlation heatmap between metabolites of different treatment groups; E is the pie chart of the category composition of differential metabolites of different treatment groups. Figure 20 The graph shows the effects of in vitro fermentation of crude extract of shiitake mushroom protein and its complex on metabolism; where A is a heatmap of the abundance of differential metabolites in different treatment groups; and B is a bar chart of the classification statistics of differential metabolites in different treatment groups. Figure 21 Circos diagram showing the correlation between differential metabolites and gut microbiota; Figure 22 A schematic diagram of the key metabolic pathways regulated by different treatment groups. Detailed Implementation

[0009] This invention provides a crude extract of shiitake mushroom protein-β-glucan complex, wherein the shiitake mushroom variety includes Shenxiang 1513, and the origin of Shenxiang 1513 includes Hubei or Zhejiang; the β-glucan includes at least one of barley β-glucan, oat β-glucan and yeast β-glucan.

[0010] This invention does not have specific requirements for shiitake mushrooms; commercially available ones are acceptable. In this embodiment, the shiitake mushroom is Shenxiang 1513 from Hubei Province, referred to as Hubei 1513. The Hubei 1513 was purchased from Hubei Changjiu Fungi Industry Co., Ltd. and harvested in the winter of 2023. In this embodiment, the shiitake mushroom is Shenxiang 1513 from Zhejiang Province, referred to as Zhejiang 1513. The Zhejiang 1513 was purchased from Zhejiang Xiangmanting Biotechnology Co., Ltd. and harvested in the winter of 2023.

[0011] As one specific embodiment, the method for preparing the crude shiitake mushroom protein extract includes: mixing shiitake mushrooms with water, adjusting the pH to 10 for extraction, centrifuging to collect the supernatant after extraction, adjusting the pH to the isoelectric point 4.2, centrifuging again to obtain a precipitate, adding water to the precipitate to adjust the pH to neutral, and then freeze-drying the supernatant to obtain the crude shiitake mushroom protein extract. As one specific embodiment, the ratio of shiitake mushrooms to water is 1g:20mL; the extraction time is 3 hours, and the extraction temperature is 55℃; after adjusting the pH of the precipitate to neutral, a settling process is also included, with a settling time of 30 minutes and a settling temperature of 25℃.

[0012] In one specific embodiment, the mass ratio of the crude shiitake mushroom protein extract to β-glucan is 2:1. In this invention, the crude shiitake mushroom protein extract is abbreviated as LEP; the crude shiitake mushroom protein extract from Hubei 1513 is abbreviated as HLEP; and the crude shiitake mushroom protein extract from Zhejiang 1513 is abbreviated as ZLEP. The embodiments of this invention show that HLEP and ZLEP are mainly composed of protein and are protein-dominant extracts. HLEP has a slightly higher sugar content than ZLEP, and a slightly lower protein content than ZLEP.

[0013] In one specific implementation, the present invention abbreviates β-glucan as βG; barley β-glucan as BBG; oat β-glucan as OBG; and yeast β-glucan as YBG.

[0014] In one specific embodiment, the present invention abbreviates the lentinan-β-glucan complex prepared from the crude extract of lentinan protein of Hubei 1513 and barley β-glucan as HLEP-BBG; the present invention abbreviates the lentinan-β-glucan complex prepared from the crude extract of lentinan protein of Hubei 1513 and oat β-glucan as HLEP-OBG; the present invention abbreviates the lentinan-β-glucan complex prepared from the crude extract of lentinan protein of Hubei 1513 and yeast β-glucan as HLEP-YBG; the present invention collectively refers to HLEP-BBG, HLEP-OBG and HLEP-YBG as HLEP-βG. In one specific embodiment, the present invention abbreviates the lentinan-beta-glucan complex prepared from the crude extract of lentinan protein of Zhejiang 1513 shiitake mushroom and barley β-glucan as ZLEP-BBG; the lentinan-beta-glucan complex prepared from the crude extract of lentinan protein of Zhejiang 1513 shiitake mushroom and oat β-glucan as ZLEP-OBG; the lentinan-beta-glucan complex prepared from the crude extract of lentinan protein of Zhejiang 1513 shiitake mushroom and yeast β-glucan as ZLEP-YBG; and collectively refers to ZLEP-βG. The present invention also refers to HLEP-BBG and ZLEP-BBG collectively as LEP-BBG; HLEP-OBG and ZLEP-OGB collectively as LEP-OGB; and HLEP-YBG and ZLEP-YBG collectively as LEP-YGB.

[0015] The embodiments of this invention demonstrate that crude protein extracts from different varieties of shiitake mushrooms and β-glucan from different sources exhibit different characteristics. Regarding browning degree, after 180 min of reaction, the browning degree of the LEP-βG group was higher than that of the control group (LEP-0), with yeast β-glucan having the most significant effect on browning. Regarding water-holding capacity, the HLEP-βG complex had a higher water-holding capacity than the ZLEP-βG complex. Yeast β-glucan increased water-holding capacity the most, followed by oat β-glucan, while barley β-glucan decreased water-holding capacity. Regarding thermal stability, HLEP had better thermal stability than ZLEP. For both crude protein extract sources, the thermal stability improvement was most significant in the LEP-OBG and LEP-YBG complexes. Regarding solubility, yeast β-glucan produced the greatest enhancement effect, oat β-glucan produced a moderate effect, and barley β-glucan produced the smallest effect. In terms of particle size and potential determination, lentinan and its complexes exhibited different colloidal behaviors under different pH conditions. This clarified that β-glucan complexes (especially YBG) and heat treatment are effective strategies for improving their stability under neutral to weakly alkaline conditions, providing a theoretical basis for pH control and formulation optimization for their application in food and other fields. Regarding emulsification, different β-glucans interacted with LEP in different ways under different pH conditions, with ZLEP-BBG and ZLEP-YBG showing high stability. In terms of structural characterization during in vitro digestion, HLEP-BBG had significantly fewer free amino acids than the other two HLEP complexes. These results confirm that β-glucan inhibits LEP hydrolysis. Overall, HLEP-BBG and ZLEP-YBG showed the most significant reduction in digestibility. These two β-glucans are highly effective in inhibiting enzymatic proteolysis.

[0016] The preparation method of the shiitake mushroom protein crude extract-β-glucan complex of the present invention includes the following steps: mixing shiitake mushroom protein crude extract, β-glucan and water, and performing composite modification to obtain shiitake mushroom protein crude extract-β-glucan complex.

[0017] The material-to-liquid ratio of the crude shiitake mushroom protein extract to water described in this invention is 1g:150~250mL. As a specific embodiment, the material-to-liquid ratio can be 1g:150mL, 1g:160mL, 1g:170mL, 1g:180mL, 1g:190mL, 1g:200mL, 1g:210mL, 1g:220mL, 1g:230mL, 1g:240mL, or 1g:250mL.

[0018] The composite modification method includes wet glycosylation composite modification, which includes stirring, low-temperature treatment, and incubation. The stirring temperature is 25-30°C, and the stirring time is 1.5-2.5 h; the low-temperature treatment temperature is 2-6°C, and the low-temperature treatment time is 10-14 h; the incubation temperature is 60-80°C, the incubation speed is 600-800 r / min, and the incubation time is 2-4 h. As a specific embodiment, the stirring temperature can be 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C, or any midpoint between any two values. As a specific embodiment, the stirring time can be 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2.0 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, and 2.5 h, or any midpoint between any two values. In one specific embodiment, the low-temperature treatment temperature can be 2℃, 3℃, 4℃, 5℃, and 6℃, or any midpoint between two such values. In another specific embodiment, the low-temperature treatment time can be 10h, 11h, 12h, 13h, and 14h, or any midpoint between two such values. In another specific embodiment, the incubation temperature can be 60℃, 65℃, 70℃, 75℃, and 80℃, or any midpoint between two such values. In another specific embodiment, the incubation rotation speed can be 600r / min, 650r / min, 700r / min, 750r / min, and 800r / min, or any midpoint between two such values. In yet another specific embodiment, the incubation time can be 2h, 3h, and 4h, or any midpoint between two such values.

[0019] This invention also provides the application of the lentinan-β-glucan complex in regulating the intestinal microbiota, wherein the regulation of the intestinal microbiota includes increasing the abundance of Bacillus phylum and decreasing the abundance of Pseudomonas phylum. Embodiments of this invention show that the lentinan-β-glucan complex can significantly enhance the richness and diversity of the microbiota, effectively enriching short-chain fatty acid-producing bacteria such as Bacillus, Bifidobacterium, Clostridium lacunae, and Bacillus flatulence, while inhibiting protein-putting bacteria and opportunistic pathogens such as Pseudomonas and Clostridium, shifting the intestinal microbial metabolic mode from protein putrefactive fermentation to polysaccharide fermentation metabolism. Among these, Bacillus phylum is the main phylum for positive regulation, being a major decomposer of dietary fiber and a major producer of short-chain fatty acids, playing a crucial role in maintaining intestinal barrier integrity and regulating immune function. Pseudomonas phylum is the core negative regulatory phylum, showing a strong negative correlation with most metabolites. Since this phylum often contains opportunistic pathogens, a decrease in its number is negatively correlated with the abundance of beneficial metabolites. These results indicate that the shift in the gut ecosystem towards a predominantly beneficial bacteria state effectively suppresses the growth of potential pathogens. This microbial shift suggests an overall improvement in gut health and a reduced risk of pathogen-related inflammation.

[0020] This invention also provides the application of the lentinan-β-glucan complex in promoting the production of short-chain fatty acids, wherein the short-chain fatty acids include any one or more of acetic acid, propionic acid, and butyric acid. Examples of this invention show that the contents of acetic acid, propionic acid, butyric acid, and total short-chain fatty acids in the lentinan-β-glucan complex group are significantly higher than those in the single lentinan-β-glucan group and the blank control group. ZLEP-YBG and ZLEP-OBG exhibit a significant synergistic effect in promoting the accumulation of acetic acid, propionic acid, and butyric acid. The increase in these metabolites is important for enhancing intestinal tight junctions. These compounds also help maintain the integrity of the epithelial cell barrier and exert systemic anti-inflammatory effects.

[0021] This invention also provides the application of the lentinan-β-glucan complex in upregulating amino acid metabolism, secondary bile acid biosynthesis, purine metabolism, and / or linoleic acid metabolism pathways. The lentinan-β-glucan complex significantly upregulates pathways such as purine metabolism, lipid metabolism, secondary bile acid metabolism, amino acid metabolism and ABC transporter, and linoleic acid metabolism, promoting the accumulation of beneficial metabolites such as xanthine, functional peptides, ursolic acid, L-tryptophan, and trans-3-indoleacetic acid, while reducing the formation of harmful substances such as deoxycholic acid, pro-inflammatory lipids, and protein putrefaction products. These pathways form a synergistic regulatory network to exert anti-inflammatory effects and support the repair of the intestinal barrier.

[0022] This invention discloses a lentinan-β-glucan complex derived from crude lentinan protein, which reduces the abundance of harmful bacteria and promotes a shift towards a stable microbial structure dominated by beneficial bacteria. These findings confirm the existence of a three-tiered regulatory network comprised of the microbiome, metabolic pathways, and metabolites. This integrated mechanism highlights the potential of lentinan complexes as functional components for enhancing gut health.

[0023] The present invention also provides the application of the aforementioned shiitake mushroom protein crude extract-β-glucan complex in the preparation of weight loss foods and / or foods for maintaining intestinal health.

[0024] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a crude extract of shiitake mushroom protein-β-glucan complex and its applications provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0025] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0026] Example 1: Preparation of protein crude extract-β-glucan complex 1. Extraction of crude protein from shiitake mushrooms Shiitake mushroom ingredients: Hubei 1513 and Zhejiang 1513.

[0027] Referencing the alkali-dissolution and acid-precipitation method, dried shiitake mushrooms were ground into powder using a grinder and passed through an 80-mesh sieve. Distilled water was added at a solid-liquid ratio of 1g:20mL, and the mixture was stirred until homogeneous. The pH of the solution was adjusted to 10 using sodium hydroxide or hydrochloric acid (1mol / L), and extraction was performed at 55℃ for 3 hours. The mixture was then centrifuged at 10000g for 10 minutes, and the supernatant was collected. The pH of the supernatant was adjusted to its isoelectric point (pI LEP = 4.2) using hydrochloric acid (1mol / L), and the mixture was centrifuged at 8000g for 10 minutes, and the precipitate was collected. The precipitate was washed with distilled water at a volume ratio of 1:3 to remove the influence of salt ions on the protein. This process was repeated three times. The precipitate was collected, and three times its volume of distilled water was added. After stirring, the pH was adjusted to 7.0±0.05 using 1mol / L sodium hydroxide, and precipitation was performed at 25℃ for 30 minutes. The supernatant was then freeze-dried to obtain the crude shiitake mushroom protein extract (LEP). HLEP, a crude protein extract from shiitake mushrooms from Hubei, and ZLEP, a crude protein extract from shiitake mushrooms from Zhejiang, were obtained.

[0028] 2. Preparation of protein crude extract-β-glucan complex Raw materials: Crude extracts of shiitake mushroom protein: HLEP crude extract of shiitake mushroom protein from Hubei and ZLEP crude extract of shiitake mushroom protein from Zhejiang obtained in step 1; β-glucan: barley β-glucan (BBG), oat β-glucan (OBG) and yeast β-glucan (YBG).

[0029] The crude extract of shiitake mushroom protein and β-glucan were mixed at a mass ratio of 2:1, and then dissolved in deionized water at a material-to-liquid ratio of 1:200 (w / v). The mixture was stirred magnetically at 27°C for 2 hours. After stirring, the mixture was stored in a refrigerator at 4°C overnight. The fully hydrated mixture was incubated at 80°C with a stirring speed of 800 rpm for 3 hours. After the reaction was completed, the mixture was freeze-dried to obtain the wet-glycosylated shiitake mushroom protein crude extract-β-glucan complex, which was stored at 4°C until analysis. HLEP-BBG, HLEP-OBG, HLEP-YBG, ZLEP-BBG, ZLEP-OBG, and ZLEP-YBG were obtained.

[0030] Example 2: Determination of the content of basic substances 1. Method ZLEP and HLEp obtained in Example 1 were measured.

[0031] (1) Protein content determination According to the kit (Beyotime; P0010) instructions, mix solution A and solution B in a ratio of 50:1 (volume ratio) to prepare BCA working solution, and prepare it fresh for each use. At the same time, prepare bovine serum albumin (BSA) standard, and dilute it with deionized water to a series of concentration gradients: 0, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL.

[0032] Add 20 μL of BSA standard solution of different concentrations to each well of a 96-well plate, followed by 200 μL of BCA working solution. Gently vortex to mix, incubate in a 37°C water bath for 30 min, and after cooling to room temperature, measure the absorbance (OD value) of each well at 562 nm using a microplate reader. Plot a standard curve with standard concentration on the x-axis and corresponding OD value on the y-axis to obtain the regression equation.

[0033] Add 20 μL of sample solution, and then follow the same procedure as for adding BCA working solution, incubation, and OD value measurement as for plotting the standard curve. Finally, calculate the protein content.

[0034] (2) Determination of polysaccharide content The phenol-sulfuric acid method was used for determination, with glucose as the standard.

[0035] Construction of the standard curve: Accurately weigh 3 mg of glucose and dissolve it in 20 mL of ultrapure water to obtain a 0.15 mg / mL glucose standard solution. Sequentially pipette 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, and 1.00 mL of the glucose solution into 2 mL centrifuge tubes, add ultrapure water to bring the volume to 1 mL for dilution, and mix thoroughly. Accurately pipette 0.10 mL of the diluted glucose solution into a new centrifuge tube, add 0.10 mL of 6% phenol solution, mix well, and then quickly add 0.50 mL of concentrated sulfuric acid. Shake well and allow to stand until cooled to room temperature. Measure the absorbance at a wavelength of 490 nm. Construct a standard curve with glucose concentration on the x-axis and absorbance on the y-axis.

[0036] Determination of total sugar yield: Weigh a certain amount of sample powder, centrifuge to obtain the supernatant, accurately pipette 0.1 mL of the supernatant into a 10 mL centrifuge tube, add ultrapure water to make up to 5 mL for dilution and mix thoroughly. Accurately pipette 0.10 mL of the diluted glucose solution into a new centrifuge tube, add 0.10 mL of 6% phenol solution, mix well, and then quickly add 0.50 mL of concentrated sulfuric acid. Shake well, allow to stand and cool, and measure the absorbance. Calculate the mass concentration using a regression equation. The total sugar yield is calculated using the following formula: In the formula: Y is the total sugar yield, %; C is the polysaccharide concentration, mg / mL; N is the dilution factor; V is the total volume of the sample solution, mL; m is the mass of the sample powder, g.

[0037] 2. Results Analysis The protein and polysaccharide contents of ZLEP and HLEP are shown in Table 1. The results show that both ZLEP and HLEP have a protein content of over 64%, while their polysaccharide content is relatively low (9.24%-10.30%), indicating that both are protein-dominant extracts. HLEP has a slightly higher sugar content than ZLEP, and a slightly lower protein content.

[0038] Table 1. Results of protein and polysaccharide content determination for ZLEP and HLEP

[0039] Example 3: Determination of the browning degree of the complex The HLEP-BBG, HLEP-OBG, HLEP-YBG, ZLEP-BBG, ZLEP-OBG and ZLEP-YBG obtained in Example 1 were measured.

[0040] 1. Method Different protein-β-glucan-β-glucan complex sample solutions were tested. After the water bath process, 4 mL of sample solution was taken to measure the change in browning degree. The sample solution was placed in a centrifuge tube and centrifuged at 3500 r / min for 15 min. The supernatant was taken out, and distilled water was used as a blank control. The sample solution was diluted with 0.1% SDS solution, and the absorbance was measured at 420 nm to indicate the change in the degree of browning of the solution.

[0041] 2. Results Analysis See results Figure 1 As shown in Table 2, the degree of browning is reflected by changes in the absorbance of the coloring substances, which reflects the formation of higher-grade products. For example... Figure 1 As shown, the degree of browning fluctuated rapidly in all groups within the first 20 minutes. This fluctuation likely stems from conformational rearrangement during the initial binding of the protein and β-glucan. This rearrangement leads to the exposure of browning-related groups, particularly amino and carbonyl groups. In subsequent periods, the degree of browning showed distinct trends across the groups. OD values ​​for HLEP-0, HLEP-BBG, and HLEP-OBG... 420nm The value remained at a low level of 0.2 to 0.3. HLEP-YBG OD 420nm Slightly higher, but still below 0.5. HLEP-βG showed a lower overall browning degree and a gradual change. ZLEP-0 and ZLEP-BBG maintained low browning levels. In contrast, ZLEP-OBG and ZLEP-YBG showed significantly higher browning degrees. Both the initial and final browning values ​​of ZLEP-βG were higher than those of HLEP-βG. This difference may be due to the higher content of free amino groups in the crude protein extract from Zhejiang. These results indicate that crude protein extracts from different varieties of shiitake mushrooms exhibit different characteristics. After 180 min of reaction, the browning degree of the LEP-βG group was higher than that of the control group (LEP-0). Yeast β-glucan (YBG) had the most significant effect on browning. This result may be due to the β-(1,6) branched structure of YBG. This specific structure increases the exposure of the carbonyl groups at the reducing end of the molecular surface. In addition, the insolubility of YBG promotes the formation of "particulate" dispersions within the complex. This state allows the surface reactive groups to contact the amino groups more frequently, resulting in higher Maillard reactivity. In contrast, barley β-glucan (BBG) and oat β-glucan (OBG) have linear, unbranched structures with β-(1,4)\(1,3) linkages. These linear chains may mask the carbonyl groups at the reducing ends. These highly soluble dextrans readily form encapsulation complexes with proteins. This encapsulation restricts contact with reactive groups. Consequently, these groups exhibit lower Maillard reactivity and less browning.

[0042] Table 2. Browning degree of samples after 180 min

[0043] Example 4: Determination of water holding capacity (WHC) and thermal stability The HLEP, HLEP-BBG, HLEP-OBG, HLEP-YBG, ZLEP, ZLEP-BBG, ZLEP-OBG and ZLEP-YBG obtained in Example 1 were measured.

[0044] 1. Method (1) Water-holding capacity test: Weigh the total weight m0 of each group of crude protein extract and crude protein extract-β-glucan complex sample powder, and place it in a 10 mL centrifuge tube. Record the total weight of the centrifuge tube and the sample as m1, add 5 mL of distilled water, and shake thoroughly to mix evenly. Let stand at room temperature for 30 min, centrifuge at 3500 r / min for 20 min, remove the supernatant, and record the total weight of the centrifuge tube and the sample as m2. Repeat the test 3 times. The water-holding capacity is expressed by the following formula: In the formula: m0 is the mass of the sample weighed, g; m1 is the total mass of the sample and centrifuge tube before centrifugation, g; m2 is the total mass of the sample and centrifuge tube after removing the supernatant, g.

[0045] (2) Thermal stability determination: The thermal stability of proteins under different treatment conditions was determined. The concentration of the sample was diluted to 2 mg / mL using 0.01 mol / L PBS (pH 7.0) solution and heated at 90 °C for 1 h. The thermal stability was determined by measuring the UV absorbance of the solution at 500 nm using a UV spectrophotometer.

[0046] 2. Results Analysis See results Figure 2 As shown in Tables A and 3, the water-holding capacity of proteins is a key determinant of their technical functional properties. The water-holding capacities of the two crude shiitake mushroom protein extracts were 6.15±0.02% and 7.20±0.38%, respectively. The water-holding capacity of the HLEP-βG complex was higher than that of the ZLEP-βG complex (…). p<0.05). This difference may be due to the higher initial surface hydrophobicity and conformational flexibility of HLEP, resulting in a stronger and more extensive interaction with β-glucan. In the complex, yeast β-glucan showed the greatest increase in water-holding capacity, followed by oat β-glucan, while barley β-glucan decreased water-holding capacity. Yeast β-glucan, due to its high molecular weight and branched β-(1,6) structure, formed a dense hydrophilic network upon complexation with HLEP, resulting in the greatest increase in water-holding capacity. Oat β-glucan provided a moderate enhancement, consistent with its linear β-(1,3),(1,4) structure and moderate solution viscosity. In contrast, barley β-glucan led to a decrease in water-holding capacity, possibly due to its limited solubility or promotion of tight aggregate formation, thus excluding free water. These results suggest that the intrinsic properties of the protein and the molecular structure of β-glucan jointly determine the microstructure and water-holding behavior of the formed complex.

[0047] See results Figure 2 In Tables B and 3, higher absorbance values ​​indicate higher protein aggregate content and poorer structural integrity. Natural ZLEP exhibits the highest OD value, reflecting its lower thermal stability. The control group (ZLEP-0) shows even lower thermal stability than natural ZLEP. Overall, HLEP demonstrates superior thermal stability compared to ZLEP. The addition of β-glucan effectively reduces the OD value of the protein complex. This reduction indicates that β-glucan provides significant protection and steric hindrance. Notably, the improvement in thermal stability is most significant for the OBG and YBG complexes from both protein sources. A synergistic enhancement exists between the internal structures of these complexes. High molecular weight β-glucan chains form a protective layer on the protein surface. This layer inhibits the vigorous movement of protein molecules and slows down the denaturation rate. Furthermore, the hydrophilic chains of β-glucan bind to protein groups through non-covalent interactions such as hydrogen bonds and hydrophobic forces. These interactions create steric hindrance, preventing further aggregation of denatured proteins. Therefore, the dispersibility and stability of the complex are maintained.

[0048] Table 3. Results of water-holding capacity and thermal stability tests on different substances.

[0049] Example 5: Protein Solubility Determination The HLEP, HLEP-BBG, HLEP-OBG, HLEP-YBG, ZLEP, ZLEP-BBG, ZLEP-OBG and ZLEP-YBG obtained in Example 1 were measured.

[0050] 1. Method A sample solution with a concentration of 0.5 mg / mL was prepared using deionized water, and eight pH gradients were set (4.92, 5.91, 6.47, 6.89, 7, 8.04, 8.67, 9.18). After dissolving at room temperature for 30 min, the sample solution was centrifuged at 4500 g for 10 min (4℃). The protein content in the supernatant was determined using the Bradford method, and the protein solubility of the sample was calculated according to the formula.

[0051] 2. Results Analysis The results are as follows Figure 3 China A and Figure 3As shown in Figure B, the solubility of all samples increased with increasing pH from 4.92 to 9.18. At the lowest pH (4.92), the solubility of both individual proteins and their complexes was lowest. Solubility increased near the neutral range and reached its highest value in the alkaline region. At each pH, ​​the solubility of the LEP-βG complex was higher than that of the corresponding individual protein. Yeast β-glucan produced the greatest enhancement in solubility, oat β-glucan produced a moderate effect, and barley β-glucan produced the smallest effect. Furthermore, HELP-βG showed a greater relative increase in solubility than ZLEP-βG, especially in the pH range near the protein's isoelectric point. The observed pH dependence reflects changes in protein net charge and electrostatic repulsion. Near the isoelectric point, the protein's net charge decreases, tending to aggregate, resulting in the lowest solubility. Moving away from the isoelectric point increases net charge and electrostatic repulsion, thus improving solubility. Firstly, β-glucan increases protein surface hydration through hydrogen bonding between the polysaccharide hydroxyl groups and the protein's polar groups. Secondly, they provide steric stabilization and increase solution viscosity, thereby reducing protein-protein collisions and inhibiting aggregation. Differences among β-glucans likely stem from molecular structure and solution behavior. Yeast β-glucans, with their branched structure and higher molecular weight, offer more accessible hydroxyl groups and a larger fluid volume, which facilitates hydration network formation and stronger steric and hydrogen-bonding stabilization. Oat β-glucans, due to their more linear structure, provide a moderate enhancement. Barley β-glucans appear to have less difficulty forming open hydration networks in the test buffer, thus producing the least enhancement. The difference between HLEP and ZLEP is attributed to the properties of the proteins themselves. The observed greater effect of HLEP suggests that this protein has more accessible binding sites or greater conformational flexibility, allowing for broader protein-polysaccharide interactions and more effective masking of hydrophobic regions. In contrast, ZLEP shows fewer binding sites or a stronger tendency for protein-protein aggregation, which is not entirely inhibited by the added polysaccharides. Overall, there are significant differences in the protein content of different shiitake mushroom varieties, and the addition of β-glucan enhances the stability of shiitake mushroom protein.

[0052] Example 6: Particle size and potential measurement The HLEP, HLEP-BBG, HLEP-OBG, HLEP-YBG, ZLEP, ZLEP-BBG, ZLEP-OBG and ZLEP-YBG obtained in Example 1 were measured.

[0053] 1. Method The zeta potentials of LEP and LEP-βG at different pH values ​​(4.92, 5.91, 6.47, 6.89, 7, 8.04, 8.67, 9.18) were measured using a Malvern zeta potentiometer (Malvern Instruments Ltd., UK). Samples were dissolved in PBS buffer solutions of different pH values ​​to prepare sample solutions with a concentration of 1 mg / ml. Zeta potentials were measured using a Malvern zeta potentiometer at 25°C.

[0054] 2. Results Analysis The changes in particle size at different pH values ​​reflect variations in molecular conformation, surface charge distribution, and aggregation behavior driven by solution acidity and alkalinity. Zeta potentials can illustrate the electrostatic interactions occurring on the protein molecule surface. These measurements directly assess the colloidal stability of the samples.

[0055] Figure 4 and Figure 5 The particle size distribution and zeta potential changes of crude shiitake protein extract and its complexes at different pH values ​​are shown. Near the isoelectric point, the samples exhibited a large average size and a broad multimodal distribution, indicating widespread aggregation. As the pH value moves away from the isoelectric point, most samples become more monodisperse, with a decrease in average diameter. This trend suggests that strong acids promote aggregation, while higher pH values ​​increase solubility and electrostatic repulsion, thus partially depolymerizing the aggregates. At the same pH value, the average size of the β-glucan-protein crude extract complexes was generally smaller than that of the untreated crude protein extract (LEP), exhibiting a single, regular peak shape. In particular, HLEP-YBG and ZLEP-YBG formed small, uniform particles at pH values ​​of 6.5–7.0 and exhibited higher colloidal stability. The untreated crude protein extract, on the other hand, exhibited a multimodal distribution and larger aggregates. The binding of the crude protein extract with β-glucan produces a soluble, hydrated complex, thereby reducing nonspecific aggregation.

[0056] Zeta potential results indicate that ( Figure 5 The crude protein extract of shiitake mushrooms and the LEP-βG complex carry a negative charge. LEP contains many acidic amino acids, and its side chain carboxyl group (-COOH) dissociates into -COO. - And release H +This process generates a negative surface charge. Within the pH range of 6.47–9.18, LEP-YBG, LEP-OBG, and LEP-0 exhibit larger absolute ζ values, indicating stronger electrostatic repulsion and better colloidal stability. In contrast, HLEP and ZLEP have smaller absolute ζ values ​​and poorer stability. These observations suggest that natural LEP possesses a limited number of anionic groups and a low surface charge density. Heat treatment combined with β-glucan increases steric hindrance. It increases the viscosity of the continuous phase and alters interfacial properties. These changes improve the stability of the treated protein. Comprehensive analysis of particle size and ζ value data indicates that samples with small, uniform particles and higher absolute ζ values ​​exhibit stronger stability under neutral and weakly alkaline pH conditions.

[0057] Example 7: Emulsification Test The HLEP, HLEP-BBG, HLEP-OBG, HLEP-YBG, ZLEP, ZLEP-BBG, ZLEP-OBG and ZLEP-YBG obtained in Example 1 were measured.

[0058] 1. Method The sample was dissolved in 0.1 mol / L phosphate buffer solutions at pH 4.92, 5.91, 6.47, 6.89, 7, 8.04, 8.67, and 9.18 to prepare sample solutions with a concentration of 1 mg / mL. Soybean oil was added at an oil:water ratio of 1:3, and the mixture was homogenized (10000 rpm, 1 min). Immediately, 50 μL of the sample was taken from the bottom and added to 5 mL of 0.1% SDS solution. The absorbance was measured at 500 nm and labeled A0. After standing for 10 min, the above experimental procedure was repeated and labeled A10. A 0.1% SDS solution was prepared as a blank control. The formulas for calculating emulsifying property (EAI) and emulsifying stability (ESI) are as follows: In the formula: c is the protein mass concentration, g / ml; This represents the volume fraction of the oil phase. =0.25; A10 is the absorbance value after standing for 10 minutes; A0 is the absorbance value after standing for 0 minutes; t is the standing time, in minutes.

[0059] 2. Results Analysis Emulsifying ability reflects the ability of an emulsifier to adsorb at the oil-water interface and form an interfacial layer to stabilize the oil phase. For LEP-βG complexes, the emulsifying activity index (EAI) and emulsifying stability index (ESI) are the main indicators for determining their functionality as bio-based emulsifiers.

[0060] like Figure 6China A and Figure 6 As shown in Figure B, the EAI values ​​of HLEP-βG and ZLEP-βG initially increased and then decreased with increasing pH. At the isoelectric point (pH 4.92), the EAI of all samples reached its lowest value. This is because low solubility and protein aggregation limited interfacial adsorption. As the pH transitioned towards the neutral and alkaline range, LEP-BBG and LEP-OBG exhibited higher EAIs than LEP. Furthermore, LEP-YBG showed good emulsifying activity under alkaline conditions. These results indicate that different β-glucans interact with LEP in different ways under different pH conditions. Under neutral conditions, ZLEP maintained a high EAI level. This suggests that ZLEP possesses intrinsic interfacial activity. Although protein subunits can rapidly form films through dissociation, large glycan chains in the complex may create steric hindrance. Heat treatment also affected the EAI. It promoted the exposure of hydrophobic residues, thereby facilitating protein adsorption at the oil-water interface.

[0061] ESI of ZLEP-βG and HLEP-βG, such as Figure 6 C and Figure 6 As shown in Figure D, the ESI initially decreases and then increases with rising pH. These trends are consistent with the solubility data. ZLEP maintains a high ESI at the optimal pH level. Its structure forms a stable adsorption layer, effectively preventing oil droplet aggregation. Notably, ZLEP-BBG and ZLEP-YBG exhibit high stability. In contrast, HLEP-βG shows lower emulsification stability than ZLEP-βG. The control group exhibits poor ESI. This may be due to changes in molecular structure and charge distribution caused by heat treatment, which hinders film formation.

[0062] The synergistic effect of heat treatment and β-glucan determined the emulsification differences. Long-chain β-glucan formed a dense physical coating on the oil droplet surface. This layer had a strong steric hindrance effect and increased the viscosity of the continuous phase. These molecules also promoted hydrophobic interactions, resulting in a viscoelastic interfacial film. Overall, the enhancing effect of β-glucan on electrostatic stabilization was stronger than that on electrostatic adsorption. The combination of electrostatic repulsion and steric hindrance effectively stabilized the shiitake protein emulsion. ZLEP-BBG and ZLEP-YBG became the most effective samples, providing superior long-term stability in neutral and alkaline environments.

[0063] Example 8: Structural characterization of in vitro digestion 1. Method 1.1 Protein content in supernatant Digestion in the stomach and small intestine was stopped by adjusting the pH and adding trypsin inhibitors, respectively. After centrifugation, the supernatant was obtained, and the protein content in the supernatant was determined using a BCA kit.

[0064] 1.2 Free amino Mix 1 mL of OPA working solution with 50 μL of sample solution thoroughly, incubate the mixture at 37 °C for 2 min, measure the absorbance at 340 nm using an ELISA reader, and plot a standard curve using L-leucine.

[0065] 1.3 Fourier Transform Infrared Spectroscopy Weigh 2 mg of lyophilized sample, add 200 mg of high-temperature dried potassium bromide powder, and grind clockwise at a uniform speed for 5 minutes until a homogeneous powder is formed. Press the powder into thin tablets using a tablet press, and scan the wavelength range 4 times using a Fourier transform infrared spectroscopy (FTIR) spectrometer, with a scanning wavenumber range of 500 cm⁻¹. -1 -4000cm -1 After the scan is completed, PeakFit software is used to process the data.

[0066] 1.4 Fluorescence spectrum Fluorescence emission spectra were measured using a fluorescence spectrophotometer. Samples were diluted to 0.25 mg / mL with 0.1 M phosphate buffer. Emission spectra were scanned for each sample, with the excitation wavelength set to 280 nm. Emission was monitored at a fixed slit width of 5 nm in the range of 300–500 nm.

[0067] 1.5 Surface hydrophobicity analysis Sample solutions with concentrations of 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, and 0.16 mg / mL were prepared using 0.01 mol / L pH 7 phosphate buffer. The initial concentration of 8-aniline-1-naphthalenesulfonic acid (ANS) solution was 8 mmol / L. 4.0 mL of sample solution was added to 20 μL of ANS phosphate buffer, and the reaction was carried out in the dark for 5 min. Immediately afterward, fluorescence spectroscopy was used for measurement. The excitation wavelength was set to 390 nm, the emission wavelength to 470 nm, and the slit width to 5 nm. Using the phosphate buffer as a blank control group, a curve was plotted between fluorescence intensity and sample concentration; the slope of the straight line represents the surface hydrophobicity of the sample.

[0068] 1.6 Scanning electron microscopy observation The surface morphology of crude shiitake protein extract and crude shiitake protein extract-β-glucan complex samples was observed using cold field emission scanning electron microscopy. The samples were fixed to conductive adhesive on the sample stage with double-sided tape, then gold was sprayed, a vacuum was applied, and an accelerating voltage of 15 kV was used.

[0069] 2. Results Analysis 2.1 Changes in protein content and free amino acid content The determination of protein and free amino acid content in the supernatant indicates the degree of enzymatic hydrolysis. These indicators provide a multifaceted assessment of protein digestibility. Figure 7 As shown, the heat-treated groups (ZLEP-0 and HLEP-0) reached peak levels of protein release and free amino acid content. Heat denaturation promoted protein solubility and enzyme sensitivity. Throughout the digestion process, ZLEP-YBG showed significantly lower values ​​for both metrics than ZLEP. ZLEP-BBG and ZLEP-OBG reduced protein release, but their amino acid content remained stable. Regarding HELP-βG, all three complexes slightly reduced protein content. Notably, HLEP-BBG had significantly fewer free amino acids than the other two HLEP complexes. These results confirm that β-glucan inhibits LEP hydrolysis. This interaction reduces the breakdown of large peptides during digestion. Therefore, these complexes form a compact structural barrier. This barrier effectively prevents premature degradation of shiitake mushroom proteins in the gastrointestinal tract. Furthermore, HLEP-βG exhibits stronger resistance to pepsin and trypsin than ZLEP-βG. This difference likely stems from the intrinsic properties of proteins from different sources.

[0070] Overall, HLEP-BBG and ZLEP-YBG showed the most significant reductions in digestibility. These two β-glucans are highly effective in inhibiting enzymatic proteolysis. The tight complexation between protein and β-glucan alters the spatial structure. Therefore, digestive enzymes find it difficult to interact with proteolytic sites. The β-glucan coating shields peptide bonds on the protein surface through steric hindrance. This mechanism inhibits the hydrolysis process and minimizes the release of free amino acids.

[0071] 2.2 Scanning Electron Microscope See results Figure 8 and Figure 9 Scanning electron microscopy (SEM) was used to evaluate lentinan proteins and their complexes at various digestion stages. These images clearly revealed significant differences in microstructure and surface morphology. The results indicate that β-glucan successfully altered the conformation of the proteins during in vitro processes. In the undigested state (… Figure 8 AE and Figure 9(Amino Acids), natural proteins exhibit unique characteristics related to their source. HLEP exhibits dense aggregates with smooth edges. This compact structure indicates the presence of strong intermolecular interactions. In contrast, ZLEP particles are relatively loose. These particles also have characteristic curled edges and fibrous texture. The introduction of β-glucan successfully reorganized the monolayer protein structure into a complex three-dimensional network. ZLEP-OBG forms a uniform honeycomb scaffold with large pores. This high porosity effectively increases the surface area exposed to the substrate. Therefore, this structure indicates superior adsorption potential. In contrast, ZLEP-YBG exhibits a continuous and dense surface. This low-porosity morphology reflects a degree of mechanical integrity. HLEP-βG complexes generally appear more robust. Notably, HLEP-YBG has almost no detectable surface pores. These morphological changes indicate the role of β-glucan as a linker or template. They promote multi-point entanglement and cross-linking of protein fibers through hydrogen bonding, electrostatic interactions, and hydrophobic interactions.

[0072] The synergistic effect of acids and enzymes in the digestive fluid altered the morphology of the sample. ZLEP-OBG transformed into loose particles with a diameter of less than 5 micrometers. This change significantly increased the contact area between the substrate and the digestive enzymes. Consequently, ZLEP-OBG exhibited high enzyme sensitivity and digestion efficiency. In contrast, ZLEP-YBG resisted proteolysis through its dense encapsulation structure. ZLEP-YBG utilizes its dense coating to prevent protein breakdown. It possesses a strong ability for controlled release. This structure allows the bioactive components to pass through the stomach and enter the next digestion. Notably, the HLEP-βG complex remained as long fibers or bulky particles at the end of digestion. This morphological structure may have protected the microenvironment containing the aromatic groups to some extent, consistent with the observed slight decrease in fluorescence. These results demonstrate the inherent structural stability of the HLEP group.

[0073] In summary, the ZLEP-OBG combination significantly improved the in vitro bioavailability of the matrix. Conversely, the HLEP-YBG complex conferred high enzyme resistance to the system. This complex also enhanced targeted release capability. Experimental data confirmed a key point: the morphological differences of shiitake mushroom proteins from different origins are a major factor in their function. Therefore, dextran complexation can precisely modulate protein metabolic patterns. This work lays the structural foundation for creating gut health foods with specific release targets.

[0074] 2.3 Secondary Structure Analysis Figure 10 The AF shows the spectra of all groups, which are commonly used to characterize the secondary structure of proteins. Figure 10 China A and Figure 10In the undigested stage, LEP and the LEP–βG complex showed significant differences in peak shape. This difference may be due to changes in the intensity of the characteristic peak after the addition of β-glucan, indicating that β-glucan affects the structure of LEP. The key absorption band is located in the amide I region (1600-1700 cm⁻¹). -1 This is related to the C=O vibration; amide II band (1475-1575 cm⁻¹) -1 ), mainly generated by N–H Schottky vibrations and CN vibrations; amide III band (1225-1330 cm) -1 ), representing CN and NH vibrations; and the β-glucan characteristic region (1000-1200 cm⁻¹). -1 This is due to COC vibration.

[0075] Further analysis of the amide I band helps characterize changes in protein secondary structure. The spectrum shows that at 1600-1700 cm⁻¹... -1 In the region, characteristic peaks shifted in different treatment groups (LEP and LEP-βG). These shifts were related to changes in the C=O and N–H oscillation frequencies, which were caused by changes in the bending and vibrational frequencies of intramolecular and intermolecular hydrogen bonds, indicating the presence of hydrophobic interactions in the complex. In the undigested stage, the amide peak was stable and sharp, reflecting intact peptide chain chemical bonds and a complete hydrogen bond network. β-glucan binds to the protein primarily through non-covalent interactions and does not disrupt the initial structure.

[0076] The secondary structure spectra of the two shiitake mushroom-derived proteins showed significant differences, indicating a substantial structural difference between the two LEPs. The effects of different β-glucans on protein processing also varied. However, during gastric digestion, the amide I band broadened, shifted towards the random coil region, and weakened in intensity. Differences between the LEP and LEP-βG groups began to emerge. The LEP-βG spectrum was flatter, with smaller peak intensity variations than LEP. This suggests that the acidic environment and pepsin induce protein unfolding and proteolysis, thereby weakening hydrogen bonds associated with C=O and NH vibrations. However, β-glucans inhibited excessive protein denaturation and secondary structure disruption through steric hindrance and hydrogen bonding, making the complex more structurally stable than the individual proteins. During intestinal digestion, the amide peak further weakened. The complex group showed a peak intensity of 1000–1200 cm⁻¹. -1 The region showed a stronger signal, and the shape of the amide band was more stable. This indicates that trypsin broke down the protein, but β-glucan acted as a protective barrier, delaying protein proteolysis. Simultaneously, the β-glucan polysaccharide chains formed new complexes with protein degradation products, retaining some residual secondary structures, while the secondary structure of LEP was largely destroyed. Figure 11The AC diagram shows principal component analysis (PCA) plots for different digestion stages. PCA results indicate that the rearrangement of secondary structures induced by digestion is the main source of variation, with PC1 accounting for approximately 50%-70% of the total variance. Samples from different digestion stages were completely separated in the PCA space. Within the same stage, the LEP and LEP-βG groups gradually differentiated as digestion progressed, confirming that β-glucan regulates the digestive behavior of LEP. Figure 12 This diagram shows a stacked bar graph illustrating the secondary structure composition of protein digestion products. In the undigested stage, α-helices and β-sheets are the predominant ordered structures. As digestion progresses, the content of α-helices decreases significantly. p <0.05), while the proportion of random curls increased significantly ( p <0.01). These findings indicate protein denaturation and disruption of the hydrogen bond network.

[0077] During gastric digestion, the LEP-βG complex retained 8%–12% more ordered structure than LEP alone, indicating that β-glucan inhibits protein unfolding and proteolysis through steric hindrance. Specifically, HLEP-YBG, ZLEP-OBG, and ZLEP-YBG exhibited lower random structure content, suggesting that disordered regions are more likely to be cleaved into peptides. These cross-linked structures enhance resistance to enzymatic degradation and acidic environments. In the intestinal stage, LEP-OBG and LEP-YBG maintained significantly higher α-helical content, confirming their superior structural stability. Furthermore, the surface hydrophobicity of the complex group correlated with the proportion of random coils, supporting the role of β-glucan in stabilizing the protein backbone and hydrogen bonds through non-covalent interactions and steric effects. This structural protection may influence the subsequent release of nutrients and the bioactivity of the resulting peptides.

[0078] 2.4 Three-level structure analysis Intrinsic fluorescence primarily originates from aromatic amino acids in proteins, such as tryptophan and tyrosine. Typically, the maximum fluorescence emission wavelength is around 340-350 nm, used to determine the tertiary structure of proteins. A redshift in emission wavelength may indicate increased protein unfolding or exposure of residues to polar environments. Conversely, a blueshift indicates a more compact structure.

[0079] In the undigested stage ( Figure 13The native protein samples (AB) showed the highest fluorescence intensity. The peak emission wavelength (λmax) was approximately 330 nm. The fluorescence intensity of all treated groups was significantly lower than that of the LEP-0 group. These samples also exhibited varying degrees of redshift. These results indicate that heat treatment promotes the migration of tryptophan and tyrosine residues. These residues move from the hydrophobic interior to a more polar, solvent-exposed environment. However, the addition of β-glucan resulted in a more compact protein structure. The glycan may form covalent bonds with the amino side chains of the protein. This interaction alters the polarity of the tryptophan microenvironment and generates a non-fluorescent complex. Furthermore, β-glucan acts as a molecular scaffold. It induces a rearrangement of protein conformation through non-covalent forces such as hydrogen bonding and hydrophobic interactions. This process buries the fluorophore within the hydrophobic core of the complex. These findings are consistent with previous reports. The addition of β-glucan and heat treatment significantly altered the intrinsic fluorescence and enhanced the protective effect of tryptophan.

[0080] During the digestive stages in the stomach and intestines ( Figure 13 In the intestinal tract (CIC) digestion, the λmax of all samples changed significantly, shifting from 330 nm to 360 nm. Simultaneously, fluorescence intensity gradually recovered as digestion progressed. The synergistic effect of the acidic environment and enzymes triggered protein unfolding. Tryptophan residues previously buried internally were exposed to the polar solvent, resulting in a redshift. Notably, ZLEP-YBG and ZLEP-OBG exhibited larger redshifts during these stages. Their fluorescence recovery rates at the end of the intestinal stage were also quite high. This indicates a more extensive complexation between β-glucan and ZLEP. These structures completely collapsed under enzymatic action, resulting in complete release of nutrients. Conversely, HLEP-BBG and HLEP-YBG showed smaller redshifts. This suggests that β-glucan helps maintain the conformation of native proteins. These complexes remained structurally compact even under the influence of pepsin and trypsin. The variations in tryptophan content and residue distribution between Zhejiang and Hubei proteins are likely the underlying cause. These factors led to different responses under the same treatment conditions.

[0081] Surface hydrophobicity (H0) reflects the exposure of nonpolar residues on the protein surface and affects its functionality. Figure 13 (G). Initially, the H0 in the HLEP-βG group was significantly higher than that in the ZLEP-βG group (G). p<0.05). Complexes with various β-glucans resulted in a decrease in H0, with yeast β-glucan showing the most significant effect, followed by oat and barley varieties. During gastric digestion, the H0 value of the control group transiently increased, consistent with acid-induced structural unfolding and exposure of internal hydrophobic regions. Conversely, the surface hydrophobicity of most LEP-βG complexes was significantly reduced, suggesting that polysaccharide coating and partial proteolysis masked hydrophobic sites. In the intestinal stage, the H0 value of all samples further decreased, especially in the complexes. This decrease is attributed to extensive proteolysis into hydrophilic peptides and more complete coverage of the remaining hydrophobic regions by β-glucan. Notably, HLEP-βG retained a relatively higher H0 value than ZLEP-βG throughout digestion, reflecting differences in the proteins themselves.

[0082] Example 9: In vitro fermentation 1. Method 1.1 Preparation of in vitro fermentation culture medium A 1L culture medium was prepared, comprising: 2g peptone, 2g yeast extract, 0.1g sodium chloride, 0.04g dipotassium hydrogen phosphate, 0.04g diammonium hydrogen phosphate, 0.01g magnesium sulfate hexahydrate, 0.01g calcium chloride hexahydrate, 2g sodium bicarbonate, 2mL Tween-80, 0.5g bile salts, and 0.5g cysteine, with the pH adjusted to 6.8. Before sterilization at 121℃, 1mg of roxazuline was added as an anaerobic indicator. After sterilization, 0.02g hemoglobin, 0.5mg vitamin K1, and 0.5g cysteine ​​were added to the sterilized culture medium.

[0083] 1.2 Collection of fecal samples Fresh stool samples were collected from six volunteers (three men and three women, aged 20 to 25 years) who had no digestive system diseases, maintained healthy eating habits, and had not used antibiotics in the three months prior to the study. Equal volumes of stool samples from all six individuals were thoroughly mixed, and the mixed stool samples were then diluted with D-PBS to a concentration of 12.5% ​​(w / v). After filtration through two layers of sterile gauze, the filtrate was collected for subsequent experiments.

[0084] 1.3 Simulated in vitro fermentation Following in vitro digestion, the digestion products were collected and further used in in vitro fermentation experiments. The fermentation system contained 4 mL of fecal filtrate and 36 mL of basal medium, with various digestion substrates added to form eight different experimental groups. These groups included: a control group (blank medium); HLEP and ZLEP groups (with digestion products from 400 mg LEP); HLEP-0 and ZLEP-0 groups (using products from 600 mg LEP-0); and the LEP-βG complex group (HLEP-BBG, OBG, YBG and ZLEP-BBG, OBG, YBG, using products from 600 mg LEP-βG). Incubation was conducted in an anaerobic chamber at 37°C. Fermentation broth was collected at specified time intervals (0 h, 6 h, 12 h, 24 h, and 48 h) for subsequent analysis.

[0085] Example 10: Analysis of in vitro fermentation characteristics The in vitro fermentation sample from Example 9 was used for the experiment.

[0086] 1. Method 1.1 pH value During the sample fermentation process, each collected fermentation sample was kept in an ice-water bath for more than 20 minutes to prevent the fermentation reaction from continuing, and the pH value of each sample solution in each group was measured in 3 parallel samples using a pH meter.

[0087] 1.2 Short-chain fatty acids The supernatant was centrifuged. 80 μL of 50% sulfuric acid (0.4 mL) was added to the supernatant, and the mixture was vortexed for 10 min, followed by acidification at 4 °C for 1 h. The supernatant was then added to 0.4 mL of ethyl acetate, and the mixture was vortexed for 5 min, followed by complete extraction at 4 °C for 10 min. The supernatant was centrifuged at 13,000 g for 5 min at 4 °C, and the upper layer was filtered through a 0.22 μm organic filter membrane and stored at -80 °C. Short-chain fatty acids in the sample were analyzed by gas chromatography using an HP-5 column (30 m × 0.32 mm × 0.25 µm), a sample volume of 1 μL, and a split ratio of 1:10. The injection temperature was 200 °C, and the FID detector temperature was 250 °C. The gradient heating conditions are as follows: initial temperature is 105℃, held for 3 min; heating rate is 10℃ / min, heated to 170℃; heating rate is 70℃, heated to 240℃, held for 2 min.

[0088] 1.3 16S rDNA gene amplification and sequencing: After 48 hours of fermentation, the fermentation broth was rapidly removed to terminate the reaction, followed by centrifugation and storage at -80°C. Samples were sent to Meiji Biotechnology Co., Ltd. Original sequences were assigned to samples based on barcode sequences. Length filtering and orientation correction were performed to retain sequences of 1000–1800 base pairs (16S) or 300–900 base pairs (ITS). The optimized sequences were denoised using the DADA2_CCS plugin within the QIIME2 pipeline to generate amplicon sequence variants (ASVs). The classification of sequences was determined using the classify-consensus-blast tool in the bacterial database. Sequences annotated as chloroplasts or mitochondria were excluded from analysis. To minimize the impact of sequencing depth, all samples were diluted to 3669 sequences per sample, resulting in 901 ASVs. Alpha and beta diversity indices were calculated using Mothur (v1.30.2) and visualized using R (v3.3.1). Principal coordinate analysis (PCoA) based on Bray-Curtis distance, combined with the ANOSIM / Adonis test, was used to assess the overall variability in bacterial community structure. Venn diagrams (v2.4.3) were used to identify unique and shared species among groups at different time points. Community bar plots and heatmaps were generated to visualize the distribution of microbial species. Kruskal-Wassirer or Wilcoxon tests were used in R to assess significant differences between groups. LEfSe (Linear Discriminant Analysis Effect Size) was applied to identify species contributing to significant inter-group differences. Correlation between microbial species and physicochemical indicators was also analyzed. Based on these candidates, a random forest machine learning algorithm was used to identify potential biomarkers for group differentiation. The relationship between microbial communities and physicochemical factors was investigated using RDA or CCA. Analysis of variance (VPA) was performed using the “vegan” package in R to reveal the main environmental drivers of community change. Finally, PICRUSt2 (v2.2.0) was used to predict microbial function. All data analyses were performed on the Majorbio cloud platform.

[0089] 1.4 Metabolite Analysis After 48 hours of fermentation, the fermentation broth was rapidly removed to terminate the reaction, followed by centrifugation and storage at -80°C. Samples were sent to Meiji Biotechnology Co., Ltd. Samples underwent LC-MS / MS analysis. After data acquisition, raw data were processed using Waters' Progenesis QI software (located in Milford, USA), including peak detection, extraction, alignment, and integration. Metabolite annotation was performed using HMDB, METLIN, and Majorbio's internal databases. Quantitative data were preprocessed to reduce experimental and analytical errors. Features with missing values ​​>20% in any group were excluded, and the remaining missing values ​​were filled using the minimum value from all samples. Mass spectrometry peak response intensities were standardized using summation normalization. Furthermore, variables with a relative standard deviation (RSD) >30% in QC samples were removed. The final data matrix was obtained after a logarithmic 10 transformation. Statistical analyses, including principal component analysis (PCA) and partial least squares discriminant analysis (OPLS-DA), were performed using the "ropls" package (version 1.6.2) in R. Metabolite annotation was also supported by the HMDB and KEGG databases. Finally, pathway enrichment analysis was performed using the scipy.stats package in Python.

[0090] 2. Results Analysis 2.1 OD during in vitro fermentation 600 Changes in value and pH value OD 600 Values ​​and pH are key indicators for assessing the utilization of nutrients by the gut microbiota. These parameters can reflect the fermentation performance of the LEP-βG complex and its potential health effects. Figure 14 AB shows the OD at different time intervals. 600 OD values ​​and pH values. Within the first 6 hours, the OD values ​​of all samples increased significantly and reached a peak. This trend indicates that the microorganisms entered a rapid logarithmic growth phase. Notably, ZLEP-YBG and HLEP-YBG exhibited the highest fermentation capacity. Correspondingly, the pH values ​​of all groups dropped sharply from 8.5 to approximately 7.0 in the early stages. However, significant differences emerged between the samples after 12 hours. The pH values ​​of the LEP and LEP-0 groups rebounded sharply. After 48 hours of fermentation, their pH values ​​rose to 8.5. In contrast, all LEP-βG complex groups maintained lower and more stable pH values ​​during fermentation. Specifically, HLEP-YBG, ZLEP-OBG, and ZLEP-YBG showed the lowest pH values. The moderate pH reduction promoted the proliferation of beneficial bacteria and inhibited harmful pathogens. These results suggest that β-glucan can effectively regulate the metabolic processes of gut microbiota.

[0091] The initial pH drop was closely associated with the rapid production of short-chain fatty acids. The pH rebound in the single-protein group indicated potential over-fermentation. This process typically produces alkaline, harmful metabolites such as ammonia and indole. However, complexation with β-glucan effectively inhibited this phenomenon. β-glucan serves as a preferred carbon source, guiding the microorganisms from protein metabolism to polysaccharide metabolism. An acidic environment was maintained through the continuous production of short-chain fatty acids. The fermentation activity of the ZLEP group was significantly stronger than that of the HLEP group. This result is attributed to the intrinsic structure and higher bioavailability of ZLEP, consistent with previous findings.

[0092] 2.2 Short-chain fatty acid analysis like Figure 15 As shown in Figure AF, the concentrations of short-chain fatty acids (SCFAs) increased significantly in all experimental groups during the 48-hour in vitro fermentation process. The production of various SCFAs in the LEP-βG group was significantly higher than that in the single LEP group and the control group (CON group). These results indicate that the protein-polysaccharide complex system is more effectively broken down by gut microbes. Furthermore, these complexes are more efficiently converted into microbial metabolites compared to other groups.

[0093] Experimental results showed that different types of lentinan significantly affected the in vitro fermentation mode of the complex. Compared with HLEP, ZLEP exhibited stronger fermentation activity. ZLEP showed a significant advantage in the production of total short-chain fatty acids and total branched-chain fatty acids (BCFAs). Figure 16 (AB). Therefore, ZLEP-OBG and ZLEP-YBG exhibit superior functional properties compared to HLEP-βG in promoting the accumulation of probiotic metabolites. This difference may be attributed to the unique amino acid composition of ZLEP. The specific spatial configuration formed after binding with β-glucan also contributes to this effect. The substrate specificity of the short-chain fatty acid components significantly affects their bioavailability. Among the different complexes, the complexes formed with OBG and YBG promote the accumulation of short-chain fatty acids more effectively than the BBG group. This is not only due to the interaction between the protein and the specific β-glucan structure. These interactions promote the co-utilization of carbon and nitrogen sources. Furthermore, this effect may be related to the structural differences in β-glucan. These structural changes lead to the specific enrichment of core acid-producing bacteria in the gut.

[0094] Short-chain fatty acid heatmap ( Figure 16Figure C) provides a more intuitive assessment of the differences in metabolite distribution among the groups. The heatmap shows that the ZLEP-βG group exhibits a denser and deeper red feature. This finding further confirms their stronger acid-producing potential. Specifically, ZLEP-YBG and ZLEP-OBG show significant synergistic effects in promoting the accumulation of acetic acid, propionic acid, and butyric acid. The increase in these metabolites is important for enhancing intestinal tight junctions. These compounds also help maintain the integrity of the epithelial cell barrier and exert systemic anti-inflammatory effects. The ZLEP-YBG and ZLEP-OBG complexes show remarkable potential in promoting gut health by optimizing the metabolic profile of the gut microbiota. These protein-polysaccharide structures effectively maintain metabolic homeostasis by inhibiting harmful proteolytic degradation and shifting microbial pathways to beneficial carbohydrate fermentation. Furthermore, the elevated valeric acid levels highlight the potential of the lentinan complex to regulate neurological health and systemic homeostasis. These findings suggest that glycosylated lentinan is an effective functional component for regulating gut health.

[0095] 2.3 Gut microbiota analysis To assess the ecological changes induced by LEP and protein-β-glucan complex after 48 h of in vitro fermentation, the α-diversity of the gut microbiota was rigorously measured. Figure 17 (AD). ACE and Chao1 indices reflect the richness of the gut microbiota, while Shannon and Simpson indices reflect community diversity. Based on ACE and Chao1 indices, LEP from a single source significantly reduced microbial richness compared to the control group (CON group). p <0.05). The ACE and Chao1 indices of HLEP were significantly lower than those of other groups, indicating that the species' ability to utilize HLEP rapidly increased, leading to their dominance and a decrease in the richness and diversity of the gut microbiota. The Shannon and Simpson indices of the LEP-0 group increased, indicating that the inhibition of community evenness after heat treatment had been alleviated, but species richness remained low. OBG and YBG effectively promoted a balanced and diverse gut microbiota. Specifically, the species richness and evenness of the ZLEP-βG group remained at a high level. These complexes effectively optimized the overall structure of the microbial community. However, significant differences in microbial composition were observed among the samples in the ZLEP-βG group. This large difference suggests low selectivity of the microbes for ZLEP, resulting in significant differences in microbial community responses among different samples.

[0096] The β diversity index was used to assess the differences in species composition in post-fermentation samples. Principal component analysis at the genus level showed significant segregation of the gut microbiota under different treatment conditions. Figure 17(EG). In complex populations, the confidence intervals for the LEP-βG and LEP groups partially overlapped. However, a clear trend of separation was also evident. This pattern suggests that the introduction of β-glucan alters the microbial community structure. This structural change further alters the way the gut microbiota metabolizes and utilizes LEP. Notably, OBG and YBG show the greatest potential for optimizing the composition of the gut microbiota.

[0097] Taxonomic distribution was analyzed to clarify the regulatory role of the LEP-βG complex in microbial communities. This study focused particularly on phylum-level community composition. Circular plots clearly illustrate the differences in community composition at the genus level among different treatment groups. Figure 17 As shown in Figure H, the bacterial communities at the phylum level mainly include Bacillus, Pseudomonas, Thermodesulfobacterium, Verrucous Microbes, Bacteroidetes, and Actinomycetes. Bacillus and Bacteroidetes are major decomposers of dietary fiber and major producers of short-chain fatty acids, playing crucial roles in maintaining intestinal barrier integrity and regulating immune function. Verrucous Microbes are associated with mucosal health and a reduced risk of metabolic diseases. Actinomycetes primarily participate in the breakdown of complex carbohydrates. However, it is important to note that Pseudomonas is identified as a major protein decomposer in the gut microbiota and possesses certain pathogenic potential.

[0098] At the genus level, different fermentation samples exhibit different microbial characteristics. Figure 17 (I). Although *Clostridium* is an opportunistic pathogen that thrives under high-protein conditions, its numbers were significantly reduced in the complex-treated groups. This reduction indicates that β-glucan effectively inhibited the overgrowth of proteolytic bacteria. Conversely, the complex promoted the growth of beneficial bacteria, with a significant increase in *Bifidobacterium*, particularly in the LEP-YBG group. Furthermore, *Clostridium lacunae* abundance was increased in specific groups (HLEP-BBG, HLEP-OBG, ZLEP-OBG, and ZLEP-YBG), suggesting increased production of acetic and butyric acids. These short-chain fatty acids are crucial for maintaining epithelial cell integrity and supporting anti-inflammatory responses. Additionally, *Bacillus platycos* abundance remained high, contributing to the conversion of succinate to propionic acid and promoting a healthier fermentation pattern. Taken together, these results suggest that the LEP-βG complex serves as both an important nutrient source and an effective metabolic buffer in the gut. LEfSe analysis ( Figure 18 (A) indicates that the HLEP and ZLEP groups are characterized by bacilli (Bacteria phylum). BacillotaThe HLEP-BBG group was primarily composed of proteolytic bacteria such as Enterococcus and Clostridium. These genera proliferated excessively in a protein-rich environment, leading to decreased α-diversity and promoting putrefactive fermentation, potentially producing harmful metabolites such as ammonia and short-chain fatty acids (BCFAs). Conversely, all complexes significantly enriched beneficial microbial communities. The HLEP-BBG group was dominated by Bifidobacteria (Bacteria phylum...). Actinomycetota Characterized by [characteristic name missing], this is a key genus in carbohydrate metabolism. Other complexes, including HLEP-OBG, HLEP-YBG, ZLEP-OBG, and ZLEP-YBG, showed enrichment in *Alternaria*, *Clostridium lacunae*, and *Broiia*. These genera were identified as primary drivers of dietary fiber fermentation and short-chain fatty acid (SCFA) production. These findings suggest that the complexation of β-glucan effectively shifts the microbial structure from a putrefactive, proteolytic approach to a beneficial glycolytic fermentation approach.

[0099] The functional prediction results are highly consistent with the changes in microbial community structure. Figure 18 (B) KEGG module heatmaps showed that the abundance of modules associated with amino acid metabolic pathways (M00002, M00004) was significantly increased in the pure proteome, reflecting a metabolic shift towards protein putrefaction and fermentation. In contrast, the expression levels of modules associated with glycolysis (M00001) and short-chain fatty acid synthesis (particularly propionic acid (M00121) and butyric acid (M00009) pathways) were significantly increased in the complex group, which is consistent with... Phascolarctobacterium and Blautia The function of these strains is directly related.

[0100] COG Functional Classification Diagram ( Figure 18 Further analysis (C) confirmed that the amino acid transport and metabolism (E) functions of the pure proteome were significantly higher than those of the complex group, while the carbohydrate transport and metabolism (G) functions of the complex group were significantly increased. This indicates that the presence of the complex leads to a significant increase in the gut microbiota, enabling it to degrade polysaccharides and convert them into energy. The more balanced E to G ratio in the complex suggests that the addition of β-glucan effectively modulates the carbon-nitrogen ratio (C / N), shifting microbial metabolism from putrefactive protein fermentation to glycolytic fermentation, thereby mitigating the potential risks of excessive protein fermentation.

[0101] 2.4 Metabolomics Analysis A total of 2799 metabolites were identified. Principal component analysis (PCA) was used to evaluate the metabolite profiles of different lentinan and complex samples. Based on the results of the principal component analysis (… Figure 19 AC) and sample-related heatmaps ( Figure 19The reliability of the data was assessed using the PCA method (PCA). The x-axis (PC1) represents the explanatory power of the first principal component, while the y-axis (PC2) represents the explanatory power of the second principal component. The results show that the quality control (QC) samples exhibit excellent clustering consistency in the PCA score plots comparing the three sample categories. The sample correlation heatmap shows that the correlation coefficients between samples within the QC group tend to be 1.0, forming a highly compact, dark red block region, indicating the stability of the mass spectrometry system and the reproducibility of the experimental data. Furthermore, the PCA results showed significant inter-group differences. Figure 19 As shown in Figure A, PC1 and PC2 accounted for 40.20% and 28.90% of the total variation, respectively, indicating that the CON, HLEP, and ZLEP groups were completely spatially separated and exhibited significant differences in their metabolic characteristics. These findings suggest that gut microbiota utilize LEP as a fermentation substrate to significantly reshape the metabolic network structure, and that LEP from different sources plays different roles in driving gut metabolic differentiation.

[0102] exist Figure 19 In group B, the various complexes exhibit a distribution pattern that changes along a gradient, which is represented by a gradual color transition in the heatmap. This indicates that the interaction between HLEP and different β-glucans is highly systematic and continuous. Conversely, ZLEP-YBG ( Figure 19 In sample C), the significant deviation from the principal component analysis plot and low correlation with other ZLEP groups indicate that YBG exerts a highly specific metabolic regulatory effect on ZLEP. The heatmap and principal component analysis results are highly consistent; the red clusters along the diagonal indicate strong intra-group correlations among biological replicates. The red clusters along the diagonal in the heatmap demonstrate high internal correlations among most biological replicate experimental groups. Protein substrates determine the main direction of metabolic evolution, while complexes achieve targeted modification of metabolite properties through specific interactions.

[0103] HMDB classification data shows that carboxylic acids and their derivatives are the main fermentation products, accounting for approximately 32.8% of the total products. Figure 19The most abundant compounds were fatty acyl groups (9.6%), organic oxidized compounds (9.5%), steroids and steroid derivatives (6.0%), and pregnenol esters (5.8%). The remaining compounds accounted for relatively small proportions. The high content of carboxylic acids indicates the presence of a diverse range of organic acids in the fermentation system, reflecting the strong degradation capacity of the gut microbiota for LEP. During the 48-hour fermentation process, proteins were hydrolyzed into peptides and amino acids by microbial proteases, subsequently converted into various organic acids through deamination and decarboxylation. These findings are consistent with previous results, indicating that the introduction of protein substrates thoroughly activates the proteolytic metabolic pathways in the gut. This observation is also consistent with the biological characteristics of LEP as a major nitrogen source. The lipid content in the samples was low (approximately 2.8%–8.0%), but rich in high-quality fatty acids. It is well known that edible fungi can produce lipases that catalyze the hydrolysis of fatty acids. Fatty acyl groups and organic oxidized compounds are closely related to lipid and carbohydrate metabolism. These organic oxidized compounds mainly originate from β-glucan glycolysis and its intermediates. Fatty acyl groups may include short-chain fatty acids and their precursors produced during fermentation. Other metabolites, such as indoles and flavonoids, are present in smaller quantities but are still important for gut health. These compounds are involved in the microbial metabolism of tryptophan and tyrosine. They function as ligands for aryl hydrocarbon receptors, helping to strengthen the intestinal barrier and possessing anti-inflammatory effects. The metabolite profile is consistent with microbial data. These results indicate that different LEPs and their complexes drive the metabolism of carbon and nitrogen substrates. This process leads to significant differences in the types of metabolites produced. To further reveal the dynamic changes in metabolites among different shiitake proteins and complexes, hierarchical clustering analysis (HCA) was performed, and the results are as follows: Figure 20 As shown in Figure A, columns represent two groups of shiitake mushroom proteins and six complexes, with each row representing the top 50 differentially expressed metabolites across all studied samples. A color-coded scale from red to blue represents the relative abundance of metabolites from high to low. All samples showed significant differences from the control group (CON group). Significant differences in metabolites were also observed between the two types of LEP. These results confirm that LEP influences intestinal metabolism. ZLEP is primarily composed of amino acids, bile acids, and fatty acids. This suggests that ZLEP fermentation may have a stronger ability to reduce inflammation and maintain intestinal homeostasis. In contrast, aromatic amino acids were significantly upregulated in the HLEP group. In the complex group, ZLEP-YBG and HLEP-YBG showed significant upregulation in subclusters 11-13. Overall, β-glucan is a key factor regulating the depth of protein fermentation metabolism.

[0104] For different classes of substances, amino acids and peptides break down substrates and interact with carbon sources. These compounds are widely enriched in subclusters 1 through 8 and exhibit high expression levels across all protein and complex groups. L-tryptophan and L-tyrosine are aromatic amino acids and core precursors of essential neurotransmitters and signaling molecules. Indole-3-acetic acid is an indole derivative and a common gut microbial metabolite of L-tryptophan. Studies have shown that this compound functions as an aryl hydrocarbon receptor (AhR) agonist. It stimulates intestinal epithelial cells to secrete mucin, thereby strengthening the intestinal barrier and exhibiting anti-inflammatory effects.

[0105] The extent to which bile acid metabolites varied across different samples. The control group (CON group) accumulated high levels of deoxycholic acid (DCA), a hydrophobic secondary bile acid with potential cytotoxicity. In contrast, the dextran-containing complex group shifted towards hepatoprotective bile acid derivatives, such as ursocolic acid. These results indicate that these complexes specifically modulate the activities of microbial bile salt hydrolase (BSH) and hydroxysteroid dehydrogenase (HSDH). This modulation may contribute to suppressing intestinal inflammation and repairing the intestinal mucosa.

[0106] Nucleoside metabolic intermediates serve as indicators of rapid microbial proliferation. These metabolites are primarily concentrated in subcluster 12 and show a high correlation with the HELP-OBG, ZLEP-YBG, and HELP-YBG groups. According to KEGG pathway results, increased purine metabolites, such as xanthine, indicate higher levels of microbial DNA synthesis and cell division. These findings suggest that the LEP-OBG and LEP-YBG complexes function as mixed carbon and nitrogen sources, stimulating the proliferation of beneficial bacteria. Long-chain lipid oxides, such as 12,13-dihydrolactone and 3-oxodeoxycholic acid, form subclusters 14-15. These metabolites are abundant in the CON group but significantly inhibited in other groups. This suggests that shifts in metabolic substrates reduce potential pathogenicity and suppress pro-inflammatory metabolic pathways.

[0107] Analysis of differentially metabolites revealed two distinct metabolic trends in LEP and its complexes. The proteome was significantly enriched with amino acids, short peptides, and purine intermediates, including inosine, deoxyinosine, hypoxanthine, L-proline, and DL-phenylalanine. In contrast, the complexes were characterized by aromatic amino acids and functional short peptides. These complexes upregulated the tryptophan metabolic pathway and optimized the intestinal bile acid pool. They also synergistically promoted microbial growth and showed a trend toward optimizing short-chain fatty acid production.

[0108] 2.5 Correlation Analysis The top 10 most abundant metabolites were selected and their correlation with the dominant gut microbiota (at the phylum level) was analyzed. (Pie chart) Figure 21 The diagrams illustrate specific patterns. Four metabolic pathway flowcharts clearly demonstrate the interactions between metabolites and the microbiome. Figure 22 The results showed that Bacillus was the dominant phylum for positive regulation. As the predominant beneficial flora, Bacillus activated core pathways, including amino acid metabolism, secondary bile acid biosynthesis, purine metabolism, and linoleic acid metabolism. Bacillus was identified as a key regulator of functional metabolite accumulation, showing a significant positive correlation with all bile acid metabolites. This confirms its crucial role in secondary bile acid biosynthesis and anti-inflammatory signaling pathways. Furthermore, as a major driver of branched-chain and aromatic amino acid metabolism, Bacillus promoted these metabolic processes by catalyzing key steps from production to transport. Therefore, this phylum directly regulated the levels of specific metabolites, including L-taurine and 2-hydroxy-2-phenylpropionamide. Pseudomonas was the core negative regulatory phylum, showing a strong negative correlation with most metabolites. Since this phylum often contains opportunistic pathogens, its decrease was negatively correlated with the abundance of beneficial metabolites. These results indicate that a shift in the gut ecosystem towards a predominantly beneficial bacteria state effectively inhibits the growth of potential pathogens. This microbial shift indicates an overall improvement in gut health and a reduced risk of pathogen-associated inflammation. KEGG pathway enrichment analysis further confirmed these findings. Differential metabolites were closely associated with purine metabolism, secondary bile acid synthesis, linoleic acid metabolism, and the ABC transporter pathway. These pathways form a synergistic regulatory network. They collectively exert anti-inflammatory effects and support intestinal barrier repair. Thermosulfur bacteria, acting as co-regulators of secondary metabolites, showed positive correlations with various functional compounds. This community participates in lipid and bile acid metabolism, enhancing anti-inflammatory pathways and promoting lipid barrier repair, synergistically with spore-forming bacteria. Furthermore, thermosulfur bacteria regulate H2S signaling by degrading sulfur-containing amino acids, thereby maintaining intestinal redox balance and indirectly influencing spore-forming bacteria-mediated amino acid metabolism. Overall, the LEP-βG complex reduced the abundance of harmful bacteria and promoted a shift towards a stable microbial structure dominated by beneficial bacteria. These findings confirm the existence of a three-tiered regulatory network consisting of the microbiome, metabolic pathways, and metabolites. This integrated mechanism highlights the potential of lentinan complex as a functional component for enhancing gut health.

[0109] Therefore, the shiitake mushroom protein crude extract-β-glucan complex of the present invention can change the conformation of shiitake mushroom protein at the molecular level, forming a stable three-dimensional network structure with covalent and non-covalent bonds, significantly improving thermal stability, pH adaptability, solubility, water holding capacity, and emulsification stability, overcoming the defects of natural shiitake mushroom protein such as easy aggregation, poor stability, and weak functional properties; it can form a spatial barrier in the gastrointestinal tract, precisely inhibiting premature protein enzymatic hydrolysis and rapid release of free amino acids, achieving targeted and sustained protein release in the intestine, significantly improving nutrient utilization and reducing gastrointestinal burden; it can efficiently regulate the intestinal microecological balance, significantly improve the richness and diversity of the flora, selectively enrich beneficial bacteria such as Bacillus, Bifidobacterium, Clostridium lacunae, and Bacillus platycos, inhibiting protein putrefactive bacteria and conditionally pathogenic bacteria, shifting intestinal metabolism from harmful protein putrefactive fermentation to a healthy polysaccharide fermentation mode; it can significantly promote the production of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid. This product effectively maintains the acidic environment of the intestine, strengthens intestinal tight junctions, repairs the mucosal barrier, and exerts systemic anti-inflammatory effects. It also systematically regulates purine metabolism, lipid metabolism, secondary bile acid metabolism, and aromatic amino acid metabolism, promoting the accumulation of beneficial metabolites such as urocholic acid, L-tryptophan, and functional peptides, while reducing toxic bile acids, pro-inflammatory lipids, and putrefactive products such as ammonia and indole. It constructs a three-layered positive regulatory network centered on Bacillus phylum: microorganisms, metabolic pathways, and functional metabolites. Furthermore, it clarifies for the first time the complex differences between crude extracts of shiitake mushroom protein from different origins and β-glucan from different sources, enabling customizable functions and predictable effects. This solves the problems of low compounding efficiency, poor reproducibility, and interference from endogenous polysaccharides in existing technologies. Overall, it possesses multiple unique advantages, including structural improvement, controllable digestion, prebiotic fortification, metabolic remodeling, and enhanced processing adaptability, making it more suitable for developing highly stable and functional intestinal health foods and special medical foods.

[0110] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A crude extract of shiitake mushroom protein-β-glucan complex, characterized in that, The shiitake mushroom variety includes Shenxiang 1513, and the place of origin of Shenxiang 1513 includes Hubei or Zhejiang; the β-glucan includes at least one of barley β-glucan, oat β-glucan and yeast β-glucan; The method for preparing the crude extract of shiitake mushroom protein includes: mixing shiitake mushrooms with water and adjusting the pH value to alkaline for extraction; centrifuging to collect the supernatant after extraction; adjusting the pH to the isoelectric point and centrifuging to obtain the precipitate; adding water to the precipitate to adjust the pH to neutral; and then freeze-drying the supernatant to obtain the crude extract of shiitake mushroom protein.

2. The shiitake mushroom protein crude extract-β-glucan complex according to claim 1, characterized in that, The mass ratio of the crude extract of shiitake mushroom protein to β-glucan is 2:

1.

3. The shiitake mushroom protein crude extract-β-glucan complex according to claim 1 or 2, characterized in that, The preparation method of the shiitake mushroom protein crude extract-β-glucan complex includes the following steps: The crude extract of shiitake mushroom protein, β-glucan, and water were mixed and subjected to composite modification to obtain the shiitake mushroom protein crude extract-β-glucan complex.

4. The shiitake mushroom protein crude extract-β-glucan complex according to claim 3, characterized in that, The ratio of the crude extract of shiitake mushroom protein to water is 1g:150~250mL.

5. The shiitake mushroom protein crude extract-β-glucan complex according to claim 3, characterized in that, The method of composite modification includes wet glycosylation composite modification.

6. The shiitake mushroom protein crude extract-β-glucan complex according to claim 5, characterized in that, The wet glycosylation composite modification includes stirring, low-temperature treatment, and incubation.

7. The application of the shiitake mushroom protein crude extract-β-glucan complex according to any one of claims 1 to 6 in regulating intestinal microbiota, characterized in that, The regulation of the intestinal microbiota includes increasing the abundance of Bacillus phylum and decreasing the abundance of Pseudomonas phylum.

8. The application of the shiitake mushroom protein crude extract-β-glucan complex according to any one of claims 1 to 6 in promoting the production of short-chain fatty acids, characterized in that, The short-chain fatty acids include any one or more of acetic acid, propionic acid, and butyric acid.

9. The use of the lentinan-β-glucan complex of shiitake mushroom protein crude extract according to any one of claims 1 to 6 in upregulating amino acid metabolism, secondary bile acid biosynthesis, purine metabolism and / or linoleic acid metabolism pathways.

10. The use of the shiitake mushroom protein crude extract-β-glucan complex according to any one of claims 1 to 6 in the preparation of weight-loss foods and / or foods for maintaining intestinal health.

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