Method for extracting protein and polysaccharide from dried morchella esculenta step by step

Abstract

The invention belongs to the technical field of deep processing of edible mushrooms and extraction of bioactive substances, and provides a method for extracting protein and polysaccharide from dried morchella esculenta step by step. According to the method, protein is extracted through alkali dissolution and acid precipitation at the pH of 11.0, the extraction rate is improved to 14.0% in cooperation with ultrasonic assistance, the protein content reaches 77.94%, and the solubility is improved to 72.13%; the residues are subjected to synergistic treatment of 0.9 MPa steam explosion and hot water extraction, the polysaccharide yield is increased to 17.42%, and the total sugar content is 46.59%. The process optimizes the pH value of protein precipitation to 4.5, avoids sudden drop of the extraction rate near an isoelectric point, realizes efficient recovery of polysaccharide in the waste material, and improves the resource utilization rate.

Description

A method for stepwise extraction of proteins and polysaccharides from dried morel mushrooms Technical Field

[0001] This invention belongs to the field of edible fungi deep processing and bioactive substance extraction technology, and specifically relates to a method for stepwise extraction of proteins and polysaccharides from dried morel mushrooms. Background Technology

[0002] In the field of food processing and extraction of bioactive substances, morel mushrooms (Morchellaspp.) have attracted much attention due to their rich content of nutrients such as protein and polysaccharides. However, traditional extraction processes have long been hampered by technical bottlenecks that restrict their efficient utilization.

[0003] Existing technologies often employ single extraction methods or simple combinations of processes, which frequently encounter common problems in protein extraction, such as low extraction rates, insufficient protein purity, and poor processing applicability. For example, steam explosion, as a physical cell wall disruption technology, features rapid pressurization, high-temperature steam penetration, and instantaneous pressure release, effectively breaking down fungal cell walls and improving the solubility and extraction efficiency of cell wall polysaccharides. However, if steam explosion is applied directly to the raw material, it can damage the structure of heat-sensitive proteins, hindering protein recycling.

[0004] Meanwhile, traditional polysaccharide extraction often uses hot water extraction or enzymatic hydrolysis, which not only yields less than 15% but also results in the disposal of most of the raw material residues. This not only wastes polysaccharide resources but also fails to achieve efficient utilization of all components of morel mushrooms. In particular, the waste residue after protein extraction still contains a large amount of polysaccharides that have not been effectively recovered. Traditional processes lack in-depth methods for exploring the value of residues. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for the stepwise extraction of proteins and polysaccharides from dried morel mushrooms. This method first involves gentle protein extraction, followed by steam explosion treatment of the residue after protein extraction to improve the polysaccharide extraction rate, thereby achieving full utilization of the morel mushroom raw material.

[0006] The technical solution of the present invention is as follows: The present invention proposes a method for stepwise extraction of protein and polysaccharide from dried morel mushrooms, including the following steps: (1) Protein extraction: After pulverizing the dried morel mushrooms, add water at a material-to-liquid ratio of 1g:(25-35)mL, adjust the pH to 10.0-12.0, extract by ultrasonication, centrifuge, and obtain residue and supernatant; (2) Protein precipitation and preparation: Adjust the pH of the supernatant in (1) to 5.0-5.5 to precipitate the protein for 20-40 minutes, centrifuge to collect the precipitate, adjust the pH of the precipitate to 7.0, and freeze-dry to obtain morel mushroom protein powder; (3) Polysaccharide extraction: Take the residue in (1), after steam explosion treatment, add water at a material-to-liquid ratio of 1g:(25-35)mL, wherein the steam pressure is 0.9-1.2 MPa and the pressure is 90-180. s; Adjust pH to 7.0, extract with hot water, repeat centrifugation twice, and combine the supernatants; (4) Separation and preparation of polysaccharides: Concentrate the supernatant in (3) to 1 / 4 of the original volume, slowly add 3 times the volume of anhydrous ethanol under stirring, let stand at 4℃ for 12 h, centrifuge to collect the precipitate, reconstitute and freeze dry to obtain morel polysaccharide powder.

[0007] Preferably, in (1), the pH is adjusted to 10.0-12.0 using 1 mol / L NaOH solution, and the mixture is extracted by ultrasonication at 200 W for 30 min and centrifuged at 4000 r / min for 25 min.

[0008] As a further preferred embodiment, in (1), the pH is adjusted to 11.0 using a 1 mol / L NaOH solution.

[0009] Preferably, in (2), the pH is adjusted to 5.0-5.5 using 1 mol / L HCl solution for 20-40 min to precipitate the protein, and the pH is adjusted to 7.0 using 0.05 mol / L NaOH solution.

[0010] As a further preferred option, in (2), the pH is adjusted to 4.5 using 1 mol / L HCl solution for 30 min to precipitate the protein.

[0011] Preferably, in (3), the conditions for hot water extraction are: temperature 90℃, time 160 min; the conditions for centrifugation are: speed 4000 r / min, time 15 min.

[0012] This invention achieves efficient separation and nutritional value enhancement of morel mushroom protein and polysaccharide through a stepwise extraction process. Compared with the existing technology, it has the following advantages and effects: (1) In terms of protein extraction, the extraction rate reaches 14.0% and the protein content reaches 77.94% with ultrasonic assistance under pH 11.0 alkaline conditions. The solubility reaches 72.13%, which improves the processing applicability. Amino acid analysis shows that the total amino acid content of the protein extracted at pH 11.0 is 51.10 g / 100g, and the essential amino acid content is 21.25 g / 100g, accounting for 41.59%. Among them, threonine, phenylalanine + tyrosine scores are over 100%. Although there are gaps in lysine and methionine + cysteine, the overall nutritional value is better than that of the protein obtained by traditional extraction process; (2) Polysaccharide extraction achieves efficient utilization of waste through steam explosion combined with hot water extraction: the residue after protein extraction is subjected to 0.9 MPa steam pressure and 150 With prolonged steam pressure treatment, the polysaccharide yield increased to 17.42%, 1.05 times higher than that without explosion, and the total sugar content reached 46.59%, close to the 51.19% of the morel powder raw material. Although the polysaccharide yield of the waste material was 8.50%, slightly lower than that of the raw material, the difference in total sugar content was small, verifying the recycling value of the waste material and reducing resource waste. In terms of precise optimization of process parameters, the optimal pH for protein precipitation was 4.5, avoiding a sharp drop in extraction rate near the isoelectric point. The synergistic effect of steam explosion pressure and prolonged steam pressure treatment significantly promoted polysaccharide dissolution, solving the problem of low yield in traditional extraction methods. Attached Figure Description

[0013] Figure 1 shows the crude protein extraction rate under different dissolution pH conditions; Figure 2 shows the protein extraction rate under different precipitation pH conditions; Figure 3 shows the SDS-PAGE spectrum, where 1 represents non-reduced protein at pH 8.0, 2 represents reduced protein at pH 8.0, 3 represents non-reduced protein at pH 11.0, and 4 represents reduced protein at pH 11.0; Figure 4 shows the effect of steam explosion pressure on polysaccharide yield; Figure 5 shows the effect of steam explosion pressure maintenance time on polysaccharide yield; Figure 6 is a process flow diagram of this invention. Detailed Implementation

[0014] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0015] Example 1: A method for stepwise extraction of protein and polysaccharide from dried morel mushrooms. The flowchart of the method is shown in Figure 6. The specific steps include: (1) Protein extraction: After crushing the dried morel mushrooms, add water at a ratio of 1 g: 30 mL. Adjust the pH to 11.0 using 1 mol / L NaOH solution. Extract under 200 W ultrasonic conditions for 30 min. Centrifuge (4000 r / min, 25 min) to obtain residue and supernatant.

[0016] (2) Protein precipitation and preparation: The supernatant in (1) was adjusted to pH 4.5 with 1 mol / L HCl solution to precipitate the protein for 30 min. The precipitate was collected by centrifugation. The pH was adjusted to 7.0 with 0.05 mol / L NaOH solution and then freeze-dried to obtain morel protein powder.

[0017] (3) Steam explosion-assisted polysaccharide extraction: The residue in (1) was subjected to steam explosion treatment with a steam pressure of 1.0 MPa and a pressure of 100 s. After explosion, water was added at a material-to-liquid ratio of 1g:30mL, the pH was adjusted to 7.0, and the mixture was extracted with hot water at 90℃ for 160 min. The mixture was then centrifuged twice (4000 r / min, 15 min) and the supernatant was combined.

[0018] (4) Separation and preparation of polysaccharides: The supernatant obtained in step (3) is concentrated to 1 / 4 of the original volume, and 3 times the volume of anhydrous ethanol is slowly added under stirring. The mixture is then allowed to stand at 4°C for 12 h. The precipitate is collected by centrifugation, reconstituted, and freeze-dried to obtain morel polysaccharide powder.

[0019] Example 2: The steps of the method in Example 1 are different from those in Example 1. (1) Protein extraction: After the dried morel mushrooms are crushed, water is added at a ratio of 1 g: 35 mL and the pH is adjusted to 8.0 using 1 mol / L NaOH solution.

[0020] (2) Protein precipitation and preparation: The supernatant in (1) was adjusted to pH 5.5 with 1 mol / L HCl solution to induce protein precipitation for 40 min. The precipitate was collected by centrifugation. The pH was then adjusted to 7.0 with 0.05 mol / L NaOH solution.

[0021] (3) Steam explosion-assisted polysaccharide extraction: The residue in (1) was subjected to steam explosion treatment. The steam pressure was 1.5 MPa and the pressure was maintained for 30 s. After the explosion, water was added at a material-to-liquid ratio of 1 g: 35 mL.

[0022] (4) Same as Example 1.

[0023] Example 3: (1) Protein extraction: After pulverizing dried morel mushrooms, add water at a ratio of 1 g: 25 mL.

[0024] (2) Protein precipitation and preparation: The supernatant from (1) was adjusted to pH 4.5 with 1 mol / L HCl solution to precipitate the protein for 20 min.

[0025] (3) Steam explosion-assisted polysaccharide extraction: The residue in (1) was subjected to steam explosion treatment with a steam pressure of 0.3 MPa and a pressure of 180 s. After the explosion, water was added at a material-to-liquid ratio of 1g:25mL.

[0026] (4) Same as Example 1.

[0027] Experimental Example 1: 1. Effect of pH on Morel Protein Extraction This experimental example investigated the effect of pH on the protein extraction rate at different stages of Morel protein extraction.

[0028] 1.1 Crude Protein Extraction Rate under Different Dissolution pH Conditions Table 1 Crude protein extraction rate data under different dissolution pH conditions pH Extraction rate (%) 8.0 10.7 9.0 10.4 10.0 12.3 11.0 14.0 12.0 12.2 surface

[0029] The protein extraction pH in step (1) of Example 1 was changed, and the effect of different dissolution pH conditions on the protein extraction rate was compared. The pH value has a significant impact on the crude protein extraction rate. The data in Table 1 and Figure 1 show that within the experimental pH range of 8.0 to 12.0, the extraction rate first increases and then decreases. When the pH rises to 11.0, the extraction rate reaches the highest experimental value of 14.0%. When the pH is further increased to 12.0, the extraction rate drops back to 12.2%. Selecting a dissolution pH of 11.0 is more reasonable.

[0030] 1.2 Protein Extraction Rate under Different Precipitation pH Conditions Table 2 Protein Extraction Rate under Different Precipitation pH Conditions pH Protein Extraction Rate (%) 4.5 1 4.0 5.0 3.2 5.5 1.5 8 surface

[0031] The protein extraction pH in step (2) of Example 1 was changed. Albumin accounts for 54% of the total protein in the morel fruiting body, and the isoelectric point of morel albumin is pH 5.0. Therefore, the effect of different precipitation pH values ​​(4.5, 5.0, and 5.5) on the morel protein extraction rate was compared. As shown in Figure 2, under the condition of extraction pH 11.0, as the precipitation pH value increased in the range of 4.5-5.5, the morel protein extraction rate gradually decreased from 14% to 1.58% (Table 2). The optimal precipitation pH value for extracting morel protein may be affected by the differences in protein composition from different sources of morel; therefore, the optimal precipitation pH was determined to be 4.5.

[0032] 1.3 Effect of Different Dissolution pH Conditions on Protein Content of Morel Extract Table 3 Protein content of Morel extract extracted under different dissolution pH conditions Dissolution pH Protein content (%) 8.06 4.93±3.93 11.07 7.94±2.75 surface

[0033] The protein content in crude morel protein was determined using the Kjeldahl method. Table 3 shows that, at a precipitation pH of 4.5, the protein extract obtained at pH 11.0 contained 77.94% protein, which is higher than the extraction purity at pH 8.0 (64.93%).

[0034] Table 4. Solubility of Morel Protein Extracts Extracted Under Different Solubility pH Conditions Solubility (%) 8.03 5.75±0.24 11.07 2.13±0.18 surface

[0035] The protein extract obtained at pH 8.0 had a solubility of 35.75%, which was higher than the solubility of 72.13% obtained at pH 11.0.

[0036] Figure 3 shows the SDS-PAGE spectrum, where 1 is the non-reduced protein at pH 8.0, 2 is the reduced protein at pH 8.0, 3 is the non-reduced protein at pH 11.0, and 4 is the reduced protein at pH 11.0.

[0037] As shown in Figure 3, the non-reduced crude morel protein extracted at pH 8.0 and 11.0 both contained bands with a molecular weight greater than 250 kDa, and these bands constituted a larger proportion. This indicates that during the ultrasound-assisted alkali dissolution and acid precipitation process, some morel protein may undergo cross-linking to form aggregates. Compared to pH 8.0, the aggregate bands at pH 11.0 were weaker, possibly because the protein extracted at pH 8.0 had higher solubility and thus higher protein content at the corresponding bands. However, the morel protein extracted at pH 11.0 showed more pronounced bands at 60 kDa, 30 kDa, and 15 kDa compared to that extracted at pH 8.0, indicating that the morel protein possesses multiple subunit bands at these values. It is noteworthy that the electrophoretic bands of morel proteins extracted at pH 11.0 after reduction with β-mercaptoethanol showed no significant change, indicating that the extracted morel protein aggregates are mainly cross-linked by covalent bonds other than disulfide bonds. However, at pH 8.0, the 65 kDa and 30 kDa bands were more prominent than before reduction, suggesting that under these conditions, the 65 kDa and 30 kDa subunits participated in the formation of morel protein aggregates via disulfide bonds.

[0038] Table 5. Amino acid content of morel protein extracted under different dissolution conditions (g / 100g protein)

[0039] Table 6. Amino acid scores (%) of morel protein extracted under different dissolution conditions. Essential amino acids FAO / WHO reference values. pH 11. AAS (%) of extracted morel protein. 8. Extracted Morel Protein AAS (%) Whole Egg Protein AAS (%) Methionine + Cysteine ​​2.26 0.00 4 1.36 14 1.82 Phenylalanine + Tyrosine 3.81 2 0.26 7 9.21 24 3.42 Isoleucine 3 10 5.67 7 4.67 17 4.00 Leucine 5.97 5.93 5 1.36 14 5.08 Valine 3.98 9.23 6 3.08 16 9.49 Histidine 1.56 8.00 5 4.00 15 1.33 Threonine 2.31 2 7.39 9 1.30 19 4.35 Lysine 4.54 9.11 4 1.11 15 6.00 Tryptophan 0.69 8.33 7 1.67 26 0.00 surface

[0040] This invention determined the amino acid composition of morel protein extracted at different dissolution pH levels using acid hydrolysis and alkaline hydrolysis pretreatments. The results are shown in Tables 5 and 6. The essential amino acid and total amino acid content of the protein extracted at pH 8.0 were lower than those extracted at pH 11.0, indicating that the protein components in the experimental morel mushrooms could not be completely extracted under pH 8.0 conditions. The essential amino acid contents of the morel protein extracted at pH 8.0 and pH 11.0 were 16.84 g / 100g and 21.25 g / 100g, respectively, accounting for 45.42% and 41.59% of the total amino acid content. The content of all essential amino acids in the pH 11.0 extract was significantly higher than that in the pH 8.0 extract, further demonstrating that pH 11.0 can extract more more morel protein components and further enhance the nutritional value.

[0041] The total amino acid content of the extract at pH 11.0 (51.10 g / 100g) increased by 37.8% compared to that at pH 8.0 (37.08 g / 100g), but the proportion of essential amino acids decreased from 45.42% to 41.59%, indicating that alkaline extraction increased the proportion of non-essential amino acids.

[0042] The amino acid score (AAS) of all essential amino acids in whole egg protein far exceeded 100% (average 186%), representing the gold standard for high-quality protein. Morel protein extracted at pH 11.0 showed the best performance in threonine (127.39%) and phenylalanine + tyrosine (120.26%), but was severely deficient in lysine (49.11%) and methionine + cysteine ​​(60%). Morel protein extracted at pH 8.0 had even lower amino acid scores, with more significant deficiencies in lysine (41.11%) and sulfur-containing amino acids (41.36%).

[0043] 2. Factors Affecting Morel Polysaccharide Extract 2.1 Effect of Raw Materials on Morel Polysaccharide Extract Table 7: Yield and Total Sugar Content of Morel Polysaccharide Extract from Different Raw Materials Raw Material Condition Yield (%) Total Sugar Content (%) Morel Powder 10.33±2.45 51.19±4.70 Waste Material After Protein Extraction from Morel 8.50±1.49 46.59±3.28 surface

[0044] As shown in Table 7, the yield of morel polysaccharides extracted from the waste after protein extraction from morel mushrooms was 8.50%. Based on previous experimental results, the lower yield of morel polysaccharides from the waste may be due to some polysaccharides being extracted along with the protein during the alkaline dissolution and acid precipitation extraction process, resulting in a lower polysaccharide yield from the waste compared to the morel powder. The total sugar content of the polysaccharides directly extracted from morel mushrooms, determined by the phenol-sulfuric acid method, was 51.19±4.70%, indicating that the extraction process effectively enriched the polysaccharide components. The total sugar content of the polysaccharides extracted from the waste was 46.59±3.28%, slightly lower than that extracted from the morel powder raw material, indicating that protein extraction caused less loss of polysaccharides from the morel mushrooms. Although the polysaccharide yield and total sugar content extracted from the waste were slightly lower, they still have recycling value.

[0045] 2.2 Effect of Steam Explosion Process Parameters on Morel Polysaccharide Extract Table 8 Effect of Steam Explosion Pressure on Polysaccharide Yield Data Steam Explosion Pressure / MPa Polysaccharide Yield / % Blank 8.5 0.3 8.7 0.6 9.5 0.9 15.7 81.2 15.92 surface

[0046] As shown in Figure 4 and Table 8, steam explosion treatment of the waste material after protein extraction from morel mushrooms showed no significant improvement in polysaccharide yield when the steam explosion pressure was below 0.9 MPa. However, when the steam explosion pressure was 0.9 MPa, the polysaccharide yield increased to 15.78%. Further increasing the steam explosion pressure did not significantly improve the polysaccharide yield. Therefore, 0.9 MPa was selected as the steam explosion pressure.

[0047] Table 9. Effect of Steam Explosion Holding Time on Polysaccharide Yield Data: Steam Explosion Holding Time / s, Polysaccharide Yield / % 308.96, 010.56, 9014.89, 12015.78, 15017.42, 18017.31 surface

[0048] As shown in Figure 5 and Table 9, when the steam explosion pressure maintenance time is less than 90 s, the improvement in polysaccharide yield is not significant. However, when the steam explosion pressure maintenance time exceeds 90 s, the polysaccharide yield is significantly improved. At a pressure maintenance time of 150 s, the polysaccharide yield reaches its maximum value of 17.42%, representing a 1.05-fold increase. This indicates that steam explosion, as a waste treatment method, has a significant advantage in promoting polysaccharide dissolution.

[0049] In summary, the stepwise extraction method provided by this invention achieves efficient protein extraction (extraction rate 14.0%, content 77.94%) through the synergistic effect of pH 11.0 alkali dissolution and acid precipitation with ultrasound. The pH 4.5 precipitation optimization prevents a sharp drop in extraction rate. The residue is treated with 0.9 MPa steam explosion for 150 s, increasing the polysaccharide yield to 17.42% and the total sugar content to 46.59%, thus realizing the resource utilization of waste materials. This method significantly improves the extraction efficiency, purity, and nutritional value of morel mushroom protein and polysaccharides. Simultaneously, precise control of process parameters (such as pH, pressure, and time) enhances resource utilization, providing a new technical solution for the deep processing of edible fungi and the development of functional foods, with significant economic and nutritional advantages.

[0050] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for stepwise extraction of protein and polysaccharides from dried morel mushrooms, characterized in that, The steps include: (1) Protein extraction: After pulverizing dried morel mushrooms, add water at a ratio of 1g:(25-35)mL, adjust the pH to 10.0-12.0, extract by ultrasonication, centrifuge, and obtain residue and supernatant; (2) Protein precipitation and preparation: Adjust the pH of the supernatant in (1) to 4.5-5.5 to precipitate the protein for 20-40 min, collect the precipitate by centrifugation, adjust the pH of the precipitate to 7.0, and freeze-dry to obtain morel mushroom protein powder; (3) Polysaccharide extraction: Take the residue in (1), after steam explosion treatment, add water at a ratio of 1g:(25-35)mL, wherein the steam pressure is 0.9-1.2 MPa and the pressure is 90-180. s; Adjust pH to 7.0, extract with hot water, repeat centrifugation twice, and combine the supernatants; (4) Separation and preparation of polysaccharides: Concentrate the supernatant in (3) to 1 / 4 of the original volume, slowly add 3 times the volume of anhydrous ethanol under stirring, let stand at 4℃ for 12 h, centrifuge to collect the precipitate, reconstitute and freeze dry to obtain morel polysaccharide powder.

2. The method as described in claim 1, characterized in that, In (1), the pH was adjusted to 10.0-12.0 using 1 mol / L NaOH solution, and the mixture was extracted by sonication at 200 W for 30 min and centrifuged at 4000 r / min for 25 min.

3. The method as described in claim 1, characterized in that, In (1), the pH was adjusted to 11.0 using 1 mol / L NaOH solution.

4. The method as described in claim 1, characterized in that, (2) Use 1 mol / L HCl solution to adjust the pH to 5.0-5.5 to precipitate the protein for 20-40 min, and then use 0.05 mol / L NaOH solution to adjust the pH to 7.

0.

5. The method as described in claim 1, characterized in that, In (2), the pH was adjusted to 4.5 using 1 mol / L HCl solution to precipitate the protein for 30 min.

6. The method as described in claim 1, characterized in that, (3) The conditions for hot water extraction are: temperature 90℃, time 160 min; the conditions for centrifugation are: speed 4000 r / min, time 15 min.

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