Preparation process and product of a de-flavored mushroom concentrate based on biotechnology

Abstract

This invention discloses a biotechnology-based process and product for preparing deodorized mushroom concentrate, relating to the field of mushroom concentrate technology. The invention involves taking finely filtered mushroom broth, concentrating it, and controlling the solid content to obtain a mushroom concentrate. The mushroom concentrate is then adjusted to pH using sodium hydroxide or hydrochloric acid, heated, and immobilized bromelain and papain are added, stirred, and then immobilized flavor protease is added, stirred, and filtered to obtain the deodorized mushroom concentrate. Bromelain, papain, and flavor protease are selected. Bromelain and papain are endopeptidases that cleave long protein chains in mushrooms at different sites, providing more ends for the flavor protease. The flavor protease is an exopeptidase that hydrolyzes the protein fragments cleaved by bromelain and papain to form flavor amino acids. During the enzymatic hydrolysis process, the immobilized carriers of the three enzymes remove off-odors through adsorption.

Description

Technical Field

[0001] This invention relates to the field of mushroom concentrate technology, specifically a biotechnology-based deodorized mushroom concentrate preparation process and product. Background Technology

[0002] Button mushrooms have extremely high nutritional value, being rich in amino acids, vitamins, minerals, and bioactive substances beneficial to the human body. When processed into mushroom concentrate, they can be used as a raw material in various foods.

[0003] However, the current industrial process for producing mushroom concentrate—cleaning and removing impurities, cutting, stewing, filtering and clarifying, concentrating, and sterilizing—results in a mushroom concentrate with a strong earthy and lingering odor. Removing the off-flavor from the mushroom concentrate is a problem that needs to be solved in its production.

[0004] To address the aforementioned problems, remove the off-flavor from mushroom concentrate, and increase its saltiness, this invention provides a biotechnology-based process and product for preparing deodorized mushroom concentrate. Summary of the Invention

[0005] The purpose of this invention is to provide a process and product for preparing deodorized mushroom concentrate based on biotechnology, so as to solve the problems raised in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Step 1: Select fresh, disease-free button mushrooms, wash them, slice them to obtain mushroom slices; add water, citric acid, tea tree oil, and rosemary extract to the mushroom slices and extract to obtain mushroom broth; Step 2: Take the mushroom broth and filter it by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it, controlling the solid content in the mushroom concentrate to obtain mushroom concentrate. Step 3: Take the mushroom concentrate, adjust the pH value with sodium hydroxide or hydrochloric acid, heat, add immobilized bromelain and immobilized papain, stir, add immobilized flavor protease, stir, filter, and obtain the deflavored mushroom concentrate.

[0007] A more optimized method for preparing immobilized bromelain is as follows: Add nano-silica to deionized water and sonicate for 25-35 min to obtain a nano-silica dispersion; add tannic acid to Tris-HCl buffer and stir until completely dissolved to obtain a tannic acid-Tris solution; add carboxymethyl chitosan to deionized water and stir until completely dissolved; add bromelain and mix well; add the nano-silica dispersion and mix well; add the tannic acid-Tris solution and react for 4-6 h; filter and wash to obtain immobilized bromelain.

[0008] A more optimized method for preparing immobilized papain is as follows: Papain is added to an aqueous acetic acid solution and stirred until completely dissolved to obtain a papain solution; Quaternary ammonium salt chitosan and bentonite are added to an aqueous acetic acid solution and sonicated for 25-35 min; Papain solution is added and mixed evenly; Sodium alginate solution is added and stirred for 25-35 min; Calcium chloride solution is added and solidified for 1.5-2.5 h; after washing, immobilized papain is obtained.

[0009] A more optimized method for preparing immobilized flavor protease is as follows: hydroxypropyl-β-cyclodextrin and tea polyphenols are added to deionized water and stirred until completely dissolved to obtain a mixed solution; gelatin and vanillin are added to deionized water, heated, and stirred until completely dissolved; flavor protease aqueous solution is added and mixed evenly; the mixed solution is added at a temperature of 45-55℃; the pH value is adjusted to 5.3-5.5 using acetic acid aqueous solution; the mixture is solidified for 55-65 minutes; the mixture is filtered and washed to obtain immobilized flavor protease.

[0010] The optimal mass ratio of immobilized bromelain, immobilized papain, and immobilized flavor protease is (2-3):(2-3):(2-3).

[0011] For optimal results, the pH value should be adjusted to 6.2-6.4.

[0012] In a more optimized manner, in step one, the thickness of the mushroom slices is 1-3 mm; the mass ratio of the mushroom slices, water, citric acid, tea tree oil, and rosemary extract is 10:(150-250):(0.6-2.2):(0.02-0.06):(0.02-0.06).

[0013] In a more optimized version, in step two, the concentration process is carried out under the conditions of a temperature of 40-80℃ and a vacuum degree of 0.07-1MPa, and the solid content in the mushroom concentrate is controlled to be 25-35%.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. Prepare mushroom broth by adding citric acid, tea tree oil, and rosemary extract, and then soaking them together with mushroom slices in water. The addition of citric acid serves two purposes: firstly, it lowers the pH, disrupting the mushroom cell walls and facilitating the dissolution of flavor compounds; secondly, it chelates metal ions and, in conjunction with the rosemary extract, inhibits oxidation. The addition of tea tree oil and rosemary extract, rich in terpenoids and polyphenols respectively, allows them to interact and bind with the main off-flavor molecules in button mushrooms—3-octanone, 1-octen-3-one, isovaleraldehyde, and benzyl mercaptan—to remove the off-flavor.

[0015] 2. Preparation of immobilized enzymes to improve enzyme stability, catalytic activity, and recyclability: First, immobilized bromelain is prepared. Carboxymethyl chitosan, which carries a negative charge, binds to the positively charged bromelain through electrostatic interactions. Tannic acid undergoes self-polymerization under alkaline conditions to form polytannic acid. Polytannic acid binds to hydroxymethyl chitosan and nano-silica through interactions, improving the mechanical strength and thermal stability of the immobilized shell. The polytannic acid and hydroxymethyl chitosan in the immobilized shell adsorb odor molecules through their phenolic hydroxyl, amino, and carboxyl groups, further removing odors. Secondly, immobilized papain was prepared. Sodium alginate, which is negatively charged, and chitosan, which is positively charged, are combined through electrostatic interaction to coat papain and bentonite. Then, calcium ions were added and sodium alginate was added to further solidify the immobilized papain through ionic cross-linking. The addition of bentonite not only improved the stability of the immobilized carrier, but also further removed odors by adsorbing odor molecules through its high specific surface area. Finally, immobilized flavor protease was prepared. Gelatin and hydroxypropyl-β-cyclodextrin interacted and precipitated to coat the flavor protease. Vanillin and tea polyphenols were added to crosslink with gelatin through interaction, which improved the stability of the immobilized shell. Among them, hydroxypropyl-β-cyclodextrin and tea polyphenols interacted and bound off-flavor molecules to further remove off-flavors.

[0016] 3. Select bromelain, papain, and flavor protease. Bromelain and papain are endopeptidases that cleave long protein chains in mushrooms at different sites, providing more ends for flavor protease. Flavor protease is an exopeptidase that hydrolyzes the protein fragments cleaved by bromelain and papain to form flavor amino acids. During the enzymatic hydrolysis process, the immobilized carriers of the three enzymes remove off-odors through adsorption. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The sources and types of substances involved in this invention are not particularly limited, and exemplary examples include: The tea tree essential oil, with product code SFF, is supplied by Jiangxi Yisenyuan Plant Fragrance Co., Ltd. The rosemary extract, catalog number 24346576, was provided by Jiangsu Weizhirun Biotechnology Co., Ltd. The nano-silica has a particle size of 100nm, and its product number is XH-SiO2-100. It is provided by Shanghai Xiaohuang Nanotechnology Co., Ltd. The tannic acid, with product number W00529, was supplied by Wuhan Jiyesheng Chemical Co., Ltd. The carboxymethyl chitosan, catalog number FT11, was provided by Shandong Fengtai Biotechnology Co., Ltd. The quaternary ammonium salt chitosan, catalog number 001, was provided by Jiangsu Yihaotian Biotechnology Co., Ltd. The bentonite, with product number 1, was supplied by Wuhan Zero Plus One Chemical Co., Ltd. Sodium alginate, catalog number 354657, is supplied by Jiangsu Weizhirun Biotechnology Co., Ltd. Hydroxypropyl-β-cyclodextrin, catalog number 35, is supplied by Hefei Hongrui Biotechnology Co., Ltd. The tea polyphenols, with product number A00698, were supplied by Wuhan Jiyesheng Chemical Co., Ltd. The gelatin, with product number 546576, was provided by Jiangsu Weizhirun Biotechnology Co., Ltd. The product code for vanillin is 2534546, and it is provided by Jiangsu Weizhirun Biotechnology Co., Ltd. The enzyme activity of bromelain is 50,000-100,000 U / g, provided by Leshengyuan Biotechnology (Nanjing) Co., Ltd. The papain enzyme activity was 100,000 U / g, provided by Chongqing Tianrun Biological Products Co., Ltd. The flavor protease has an enzyme activity of 100,000 U / g and was provided by Zhengzhou Rongyuan Chemical Products Co., Ltd.

[0019] Example 1: A biotechnology-based process for preparing deodorized mushroom concentrate; Step 1: Prepare the mushroom broth Select fresh, disease-free button mushrooms, wash and slice them to obtain mushroom slices with a thickness of 1 mm; take 10g of mushroom slices and add 150g of water, 0.6g of citric acid, 0.02g of tea tree oil and 0.02g of rosemary extract, and extract at 90℃ for 1 hour to obtain mushroom broth. Step 2: Preparation of mushroom concentrate Take the mushroom broth and filter it through a 200-mesh sieve by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it through a 200-mesh sieve by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it under the conditions of 40℃ and 0.07MPa vacuum, controlling the solid content in the mushroom concentrate to be 25%, to obtain mushroom concentrate. Step 3: Preparation of immobilized enzymes S1: Add 0.3g of nano-silica to 10mL of deionized water and sonicate for 25min to obtain a nano-silica dispersion; add 0.4g of tannic acid to 100mL of pH=8.0 0.05mol / L Tris-HCl buffer and stir until completely dissolved to obtain a tannic acid-Tris solution; add 0.4g of carboxymethyl chitosan to 20mL of deionized water and stir until completely dissolved, add 0.5g of bromelain, mix well, add the nano-silica dispersion, mix well, add the tannic acid-Tris solution, react for 4h, filter, wash to obtain immobilized bromelain; S2: Add 0.5g of papain to 20mL of pH 6.0 acetic acid aqueous solution and stir until completely dissolved to obtain papain solution; add 1.5g of quaternary ammonium salt chitosan and 0.5g of bentonite to 100mL of pH 6.0 acetic acid aqueous solution, sonicate for 25min, add papain solution, mix well, add 100mL of 1% sodium alginate aqueous solution, stir for 25min, add 100mL of 1% calcium chloride aqueous solution, solidify for 1.5h, wash to obtain immobilized papain; S3: Add 1.5g hydroxypropyl-β-cyclodextrin and 0.1g tea polyphenols to 50mL deionized water and stir until completely dissolved to obtain a mixed solution; add 1g gelatin and 0.5g vanillin to 50mL deionized water, heat and stir until completely dissolved, add 10mL of 3% flavor protease aqueous solution, mix well, add the mixed solution at 50℃, adjust the pH to 5.3 with 1% acetic acid aqueous solution, solidify for 1h, filter, wash, and obtain immobilized flavor protease; Step 4: Preparation of Deodorized Mushroom Concentrate Take 200g of mushroom concentrate, adjust the pH to 6.2 using 1mol / L sodium hydroxide or hydrochloric acid, heat, add 2g of immobilized bromelain and 2g of immobilized papain at 50℃, stir for 60min, add 2g of immobilized flavor protease, stir for 30min, filter, and obtain deflavored mushroom concentrate.

[0020] Example 2: A biotechnology-based process for preparing deodorized mushroom concentrate; Step 1: Prepare the mushroom broth Select fresh, disease-free button mushrooms, wash and slice them to obtain mushroom slices with a thickness of 2mm; take 10g of mushroom slices and add 200g of water, 1.4g of citric acid, 0.04g of tea tree oil and 0.04g of rosemary extract, and extract at 94℃ for 1.5h to obtain mushroom broth. Step 2: Preparation of mushroom concentrate Take the mushroom broth and filter it through a 200-mesh sieve by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it through a 200-mesh sieve by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it under the conditions of 60℃ and 0.085MPa vacuum, controlling the solid content in the mushroom concentrate to be 30%, to obtain mushroom concentrate. Step 3: Preparation of immobilized enzymes S1: Add 0.3g of nano-silica to 10mL of deionized water and sonicate for 30min to obtain a nano-silica dispersion; add 0.4g of tannic acid to 100mL of pH=8.0 0.05mol / L Tris-HCl buffer and stir until completely dissolved to obtain a tannic acid-Tris solution; add 0.4g of carboxymethyl chitosan to 20mL of deionized water and stir until completely dissolved, add 0.5g of bromelain, mix well, add the nano-silica dispersion, mix well, add the tannic acid-Tris solution, react for 5h, filter, wash to obtain immobilized bromelain; S2: Add 0.5g of papain to 20mL of pH 6.0 acetic acid aqueous solution and stir until completely dissolved to obtain papain solution; add 1.5g of quaternary ammonium salt chitosan and 0.5g of bentonite to 100mL of pH 6.0 acetic acid aqueous solution, sonicate for 30min, add papain solution, mix evenly, add 100mL of 2% sodium alginate aqueous solution, stir for 30min, add 100mL of 2% calcium chloride aqueous solution, solidify for 2h, wash to obtain immobilized papain; S3: Add 1.5g hydroxypropyl-β-cyclodextrin and 0.1g tea polyphenols to 50mL deionized water and stir until completely dissolved to obtain a mixed solution; add 1g gelatin and 0.5g vanillin to 50mL deionized water, heat and stir until completely dissolved, add 10mL of 3% flavor protease aqueous solution, mix well, add the mixed solution at 50℃, adjust the pH to 5.4 with 1% acetic acid aqueous solution, solidify for 1h, filter, wash, and obtain immobilized flavor protease; Step 4: Preparation of Deodorized Mushroom Concentrate Take 200g of mushroom concentrate, adjust the pH to 6.3 using 1mol / L sodium hydroxide or hydrochloric acid, heat, add 2.5g of immobilized bromelain and 2.5g of immobilized papain at 55℃, stir for 75min, add 2.5g of immobilized flavor protease, stir for 45min, filter to obtain deflavored mushroom concentrate.

[0021] Example 3: A biotechnology-based process for preparing deodorized mushroom concentrate; Step 1: Prepare the mushroom broth Select fresh, disease-free button mushrooms, wash and slice them to obtain mushroom slices with a thickness of 3mm; take 10g of mushroom slices and add 250g of water, 2.2g of citric acid, 0.06g of tea tree oil and 0.06g of rosemary extract, and extract at 98℃ for 2 hours to obtain mushroom broth. Step 2: Preparation of mushroom concentrate Take the mushroom broth and filter it through a 200-mesh sieve by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it through a 200-mesh sieve by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it under the conditions of 80℃ and 0.1MPa vacuum, controlling the solid content in the mushroom concentrate to be 35%, to obtain mushroom concentrate. Step 3: Preparation of immobilized enzymes S1: Add 0.3g of nano-silica to 10mL of deionized water and sonicate for 35min to obtain a nano-silica dispersion; add 0.4g of tannic acid to 100mL of pH=8.0 0.05mol / L Tris-HCl buffer and stir until completely dissolved to obtain a tannic acid-Tris solution; add 0.4g of carboxymethyl chitosan to 20mL of deionized water and stir until completely dissolved, add 0.5g of bromelain, mix well, add the nano-silica dispersion, mix well, add the tannic acid-Tris solution, react for 6h, filter, wash to obtain immobilized bromelain; S2: Add 0.5g of papain to 20mL of pH 6.0 acetic acid aqueous solution and stir until completely dissolved to obtain papain solution; add 1.5g of quaternary ammonium salt chitosan and 0.5g of bentonite to 100mL of pH 6.0 acetic acid aqueous solution, sonicate for 35min, add papain solution, mix well, add 100mL of 3% sodium alginate aqueous solution, stir for 35min, add 100mL of 3% calcium chloride aqueous solution, solidify for 2.5h, wash to obtain immobilized papain; S3: Add 1.5g hydroxypropyl-β-cyclodextrin and 0.1g tea polyphenols to 50mL deionized water and stir until completely dissolved to obtain a mixed solution; add 1g gelatin and 0.5g vanillin to 50mL deionized water, heat and stir until completely dissolved, add 10mL of 3% flavor protease aqueous solution, mix well, add the mixed solution at 50℃, adjust the pH to 5.5 with 1% acetic acid aqueous solution, solidify for 1h, filter, wash, and obtain immobilized flavor protease; Step 4: Preparation of Deodorized Mushroom Concentrate Take 200g of mushroom concentrate, adjust the pH to 6.4 using 1mol / L sodium hydroxide or hydrochloric acid, heat, add 3g of immobilized bromelain and 3g of immobilized papain at 60℃, stir for 90min, add 3g of immobilized flavor protease, stir for 60min, filter, and obtain deflavored mushroom concentrate.

[0022] Comparative Example 1: No immobilization was performed; all other aspects are the same as in Example 2. Step 1: Prepare the mushroom broth Select fresh, disease-free button mushrooms, wash and slice them to obtain mushroom slices with a thickness of 2mm; take 10g of mushroom slices and add 200g of water, 1.4g of citric acid, 0.04g of tea tree oil and 0.04g of rosemary extract, and extract at 94℃ for 1.5h to obtain mushroom broth. Step 2: Preparation of mushroom concentrate Take the mushroom broth and filter it through a 200-mesh sieve by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it through a 200-mesh sieve by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it under the conditions of 60℃ and 0.085MPa vacuum, controlling the solid content in the mushroom concentrate to be 30%, to obtain mushroom concentrate. Step 3: Preparation of deodorized mushroom concentrate Take 200g of mushroom concentrate, adjust the pH to 6.3 using 1mol / L sodium hydroxide or hydrochloric acid, heat, add 2.5g of bromelain and 2.5g of papain at 55℃, stir for 75min, add 2.5g of flavor protease, stir for 45min; inactivate the enzyme at 95℃ for 37.5min to obtain deflavored mushroom concentrate.

[0023] Comparative Example 2: Rosemary extract was not added; otherwise, refer to Example 2. Step 1: Prepare the mushroom broth Select fresh, disease-free button mushrooms, wash and slice them to obtain mushroom slices with a thickness of 2mm; take 10g of mushroom slices, add 200g of water, 1.4g of citric acid and 0.04g of tea tree oil, and extract at 94℃ for 1.5h to obtain mushroom broth. Step 2: Preparation of mushroom concentrate Take the mushroom broth and filter it through a 200-mesh sieve by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it through a 200-mesh sieve by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it under the conditions of 60℃ and 0.085MPa vacuum, controlling the solid content in the mushroom concentrate to be 30%, to obtain mushroom concentrate. Step 3: Preparation of immobilized enzymes S1: Add 0.3g of nano-silica to 10mL of deionized water and sonicate for 30min to obtain a nano-silica dispersion; add 0.4g of tannic acid to 100mL of pH=8.0 0.05mol / L Tris-HCl buffer and stir until completely dissolved to obtain a tannic acid-Tris solution; add 0.4g of carboxymethyl chitosan to 20mL of deionized water and stir until completely dissolved, add 0.5g of bromelain, mix well, add the nano-silica dispersion, mix well, add the tannic acid-Tris solution, react for 5h, filter, wash to obtain immobilized bromelain; S2: Add 0.5g of papain to 20mL of pH 6.0 acetic acid aqueous solution and stir until completely dissolved to obtain papain solution; add 1.5g of quaternary ammonium salt chitosan and 0.5g of bentonite to 100mL of pH 6.0 acetic acid aqueous solution, sonicate for 30min, add papain solution, mix evenly, add 100mL of 2% sodium alginate aqueous solution, stir for 30min, add 100mL of 2% calcium chloride aqueous solution, solidify for 2h, wash to obtain immobilized papain; S3: Add 1.5g hydroxypropyl-β-cyclodextrin and 0.1g tea polyphenols to 50mL deionized water and stir until completely dissolved to obtain a mixed solution; add 1g gelatin and 0.5g vanillin to 50mL deionized water, heat and stir until completely dissolved, add 10mL of 3% flavor protease aqueous solution, mix well, add the mixed solution at 50℃, adjust the pH to 5.4 with 1% acetic acid aqueous solution, solidify for 1h, filter, wash, and obtain immobilized flavor protease; Step 4: Preparation of Deodorized Mushroom Concentrate Take 200g of mushroom concentrate, adjust the pH to 6.3 using 1mol / L sodium hydroxide or hydrochloric acid, heat, add 2.5g of immobilized bromelain and 2.5g of immobilized papain at 55℃, stir for 75min, add 2.5g of immobilized flavor protease, stir for 45min, filter to obtain deflavored mushroom concentrate.

[0024] Comparative Example 3: No hydroxymethyl chitosan or nano-silica were added; all other aspects were the same as in Example 2. Step 1: Prepare the mushroom broth Select fresh, disease-free button mushrooms, wash and slice them to obtain mushroom slices with a thickness of 2mm; take 10g of mushroom slices and add 200g of water, 1.4g of citric acid, 0.04g of tea tree oil and 0.04g of rosemary extract, and extract at 94℃ for 1.5h to obtain mushroom broth. Step 2: Preparation of mushroom concentrate Take the mushroom broth and filter it through a 200-mesh sieve by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it through a 200-mesh sieve by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it under the conditions of 60℃ and 0.085MPa vacuum, controlling the solid content in the mushroom concentrate to be 30%, to obtain mushroom concentrate. Step 3: Preparation of immobilized enzymes S1: Take 0.4g of tannic acid and add it to 100mL of pH=8.0 0.05mol / L Tris-HCl buffer. Stir until completely dissolved to obtain tannic acid-Tris solution. Take 20mL of deionized water, add 0.5g of bromelain, mix well, add 10mL of deionized water, mix well, add tannic acid-Tris solution, react for 5h, filter, wash, and obtain immobilized bromelain. S2: Add 0.5g of papain to 20mL of pH 6.0 acetic acid aqueous solution and stir until completely dissolved to obtain papain solution; add 1.5g of quaternary ammonium salt chitosan and 0.5g of bentonite to 100mL of pH 6.0 acetic acid aqueous solution, sonicate for 30min, add papain solution, mix evenly, add 100mL of 2% sodium alginate aqueous solution, stir for 30min, add 100mL of 2% calcium chloride aqueous solution, solidify for 2h, wash to obtain immobilized papain; S3: Add 1.5g hydroxypropyl-β-cyclodextrin and 0.1g tea polyphenols to 50mL deionized water and stir until completely dissolved to obtain a mixed solution; add 1g gelatin and 0.5g vanillin to 50mL deionized water, heat and stir until completely dissolved, add 10mL of 3% flavor protease aqueous solution, mix well, add the mixed solution at 50℃, adjust the pH to 5.4 with 1% acetic acid aqueous solution, solidify for 1h, filter, wash, and obtain immobilized flavor protease; Step 4: Preparation of Deodorized Mushroom Concentrate Take 200g of mushroom concentrate, adjust the pH to 6.3 using 1mol / L sodium hydroxide or hydrochloric acid, heat, add 2.5g of immobilized bromelain and 2.5g of immobilized papain at 55℃, stir for 75min, add 2.5g of immobilized flavor protease, stir for 45min, filter to obtain deflavored mushroom concentrate.

[0025] Comparative Example 4: No bentonite was added; otherwise, refer to Example 2. Step 1: Prepare the mushroom broth Select fresh, disease-free button mushrooms, wash and slice them to obtain mushroom slices with a thickness of 2mm; take 10g of mushroom slices and add 200g of water, 1.4g of citric acid, 0.04g of tea tree oil and 0.04g of rosemary extract, and extract at 94℃ for 1.5h to obtain mushroom broth. Step 2: Preparation of mushroom concentrate Take the mushroom broth and filter it through a 200-mesh sieve by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it through a 200-mesh sieve by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it under the conditions of 60℃ and 0.085MPa vacuum, controlling the solid content in the mushroom concentrate to be 30%, to obtain mushroom concentrate. Step 3: Preparation of immobilized enzymes S1: Add 0.3g of nano-silica to 10mL of deionized water and sonicate for 30min to obtain a nano-silica dispersion; add 0.4g of tannic acid to 100mL of pH=8.0 0.05mol / L Tris-HCl buffer and stir until completely dissolved to obtain a tannic acid-Tris solution; add 0.4g of carboxymethyl chitosan to 20mL of deionized water and stir until completely dissolved, add 0.5g of bromelain, mix well, add the nano-silica dispersion, mix well, add the tannic acid-Tris solution, react for 5h, filter, wash to obtain immobilized bromelain; S2: Add 0.5g of papain to 20mL of pH 6.0 acetic acid aqueous solution and stir until completely dissolved to obtain papain solution; add 1.5g of quaternary ammonium salt chitosan to 100mL of pH 6.0 acetic acid aqueous solution, sonicate for 30min, add papain solution, mix well, add 100mL of 2% sodium alginate aqueous solution, stir for 30min, add 100mL of 2% calcium chloride aqueous solution, solidify for 2h, wash to obtain immobilized papain; S3: Add 1.5g hydroxypropyl-β-cyclodextrin and 0.1g tea polyphenols to 50mL deionized water and stir until completely dissolved to obtain a mixed solution; add 1g gelatin and 0.5g vanillin to 50mL deionized water, heat and stir until completely dissolved, add 10mL of 3% flavor protease aqueous solution, mix well, add the mixed solution at 50℃, adjust the pH to 5.4 with 1% acetic acid aqueous solution, solidify for 1h, filter, wash, and obtain immobilized flavor protease; Step 4: Preparation of Deodorized Mushroom Concentrate Take 200g of mushroom concentrate, adjust the pH to 6.3 using 1mol / L sodium hydroxide or hydrochloric acid, heat, add 2.5g of immobilized bromelain and 2.5g of immobilized papain at 55℃, stir for 75min, add 2.5g of immobilized flavor protease, stir for 45min, filter to obtain deflavored mushroom concentrate.

[0026] Comparative Example 5: Vanillin and tea polyphenols were not added; otherwise, refer to Example 2. Step 1: Prepare the mushroom broth Select fresh, disease-free button mushrooms, wash and slice them to obtain mushroom slices with a thickness of 2mm; take 10g of mushroom slices and add 200g of water, 1.4g of citric acid, 0.04g of tea tree oil and 0.04g of rosemary extract, and extract at 94℃ for 1.5h to obtain mushroom broth. Step 2: Preparation of mushroom concentrate Take the mushroom broth and filter it through a 200-mesh sieve by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it through a 200-mesh sieve by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it under the conditions of 60℃ and 0.085MPa vacuum, controlling the solid content in the mushroom concentrate to be 30%, to obtain mushroom concentrate. Step 3: Preparation of immobilized enzymes S1: Add 0.3g of nano-silica to 10mL of deionized water and sonicate for 30min to obtain a nano-silica dispersion; add 0.4g of tannic acid to 100mL of pH=8.0 0.05mol / L Tris-HCl buffer and stir until completely dissolved to obtain a tannic acid-Tris solution; add 0.4g of carboxymethyl chitosan to 20mL of deionized water and stir until completely dissolved, add 0.5g of bromelain, mix well, add the nano-silica dispersion, mix well, add the tannic acid-Tris solution, react for 5h, filter, wash to obtain immobilized bromelain; S2: Add 0.5g of papain to 20mL of pH 6.0 acetic acid aqueous solution and stir until completely dissolved to obtain papain solution; add 1.5g of quaternary ammonium salt chitosan and 0.5g of bentonite to 100mL of pH 6.0 acetic acid aqueous solution, sonicate for 30min, add papain solution, mix evenly, add 100mL of 2% sodium alginate aqueous solution, stir for 30min, add 100mL of 2% calcium chloride aqueous solution, solidify for 2h, wash to obtain immobilized papain; S3: Add 1.5g of hydroxypropyl-β-cyclodextrin to 50mL of deionized water and stir until completely dissolved to obtain a mixed solution; add 1g of gelatin to 50mL of deionized water, heat and stir until completely dissolved, add 10mL of 3% flavor protease aqueous solution, mix well, add the mixed solution at 50℃, adjust the pH to 5.4 with 1% acetic acid aqueous solution, solidify for 1h, filter, wash, and obtain immobilized flavor protease; Step 4: Preparation of Deodorized Mushroom Concentrate Take 200g of mushroom concentrate, adjust the pH to 6.3 using 1mol / L sodium hydroxide or hydrochloric acid, heat, add 2.5g of immobilized bromelain and 2.5g of immobilized papain at 55℃, stir for 75min, add 2.5g of immobilized flavor protease, stir for 45min, filter to obtain deflavored mushroom concentrate.

[0027] experiment: The deodorized mushroom concentrates prepared in Examples 1-3 and Comparative Examples 1-5 were subjected to electronic tongue analysis. Fresh, disease-free button mushrooms were selected, washed, and sliced ​​to obtain mushroom slices with a thickness of 2 mm. 10 g of mushroom slices were added to 200 g of water and 1.4 g of citric acid, and extracted at 94℃ for 1.5 h to obtain mushroom broth. The mushroom broth was then filtered through a 200-mesh sieve using vibration to obtain coarsely filtered mushroom broth. The coarsely filtered mushroom broth was then filtered through a 200-mesh sieve using pressure to obtain finely filtered mushroom broth. The finely filtered mushroom broth was concentrated at 60℃ and a vacuum of 0.085 MPa, controlling the solid content in the concentrated mushroom broth to 30%, to obtain untreated mushroom concentrate. 20g of untreated mushroom concentrate and deodorized mushroom concentrate were taken separately, added to 180g of pure water, and stirred evenly to obtain samples. The sensory quality was analyzed using an INSENT SA402B Controller taste analysis system that simulated human taste with an electronic tongue. After the reference electrode of the electronic tongue and the five sensors for sour, bitter, astringent, umami, and salty tastes were activated for more than 24 hours, the samples were placed in the instrument. After the instrument underwent self-testing and calibration, a two-step cleaning program was selected for measurement. The sweetness sensor was activated for more than 48 hours in advance and measured using the sweetness measurement program. The sampling time was 120s. Each group of samples was repeated 4 times, and the last 3 data were analyzed. The sweetness data were repeated 5 times, and the last 4 data were analyzed. The above experiments were completed, and the results are shown in Table 1 below: Table 1

[0028] Conclusion: Based on the analysis of the above experimental data, the deodorized mushroom concentrate prepared in Examples 1-3 of this invention has a milder sour, bitter, and astringent taste, a stronger salty taste, and a better overall flavor; while the deodorized mushroom concentrate prepared in Comparative Examples 1-5 has a stronger sour, bitter, and astringent taste, and a poorer overall flavor.

[0029] Comparative analysis of Comparative Example 1 (without immobilization) and Example 2 shows that bromelain, papain, and flavor protease were selected. Bromelain and papain are endonucleases that cleave long protein chains in mushrooms at different sites, providing more ends for flavor protease. Flavor protease is an exonuclease that hydrolyzes the protein fragments cleaved by bromelain and papain to form flavor amino acids. During the enzymatic hydrolysis process, the immobilization carriers of the three enzymes remove off-odors through adsorption, and immobilization improves the stability, catalytic activity, and recyclability of the enzymes.

[0030] Comparative analysis of Comparative Example 2 (without rosemary extract) and Example 2 showed that rosemary extract is rich in polyphenolic compounds, which interact with the main odor molecules in button mushrooms, such as 3-octanone, 1-octen-3-one, isovaleraldehyde, and benzyl thiol, to remove odors.

[0031] Comparative analysis of Comparative Example 3 (without hydroxymethyl chitosan and nano-silica) and Example 2 shows that carboxymethyl chitosan, being negatively charged, binds to positively charged bromelain through electrostatic interaction. Tannic acid undergoes self-polymerization under alkaline conditions to form polytannic acid. Polytannic acid binds to hydroxymethyl chitosan and nano-silica through interaction, improving the mechanical strength and thermal stability of the immobilized shell. The polytannic acid and hydroxymethyl chitosan in the immobilized shell adsorb odor molecules through their phenolic hydroxyl, amino, and carboxyl groups, further removing odors.

[0032] Comparative analysis of Comparative Example 4 (without bentonite) and Example 2 shows that the addition of bentonite improves the stability of the immobilized carrier and further removes odors by adsorbing odor molecules through its high specific surface area.

[0033] Comparative analysis of Comparative Example 5 (without vanillin and tea polyphenols) and Example 2 shows that the addition of vanillin and tea polyphenols enhances the stability of the immobilized shell through cross-linking with gelatin. In particular, tea polyphenols further remove odors by binding odor molecules through interaction.

[0034] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A process for preparing a biotechnologically based deodorized mushroom concentrate, characterized in that: Includes the following steps: Step 1: Select fresh, disease-free button mushrooms, wash them, slice them to obtain mushroom slices; add water, citric acid, tea tree oil, and rosemary extract to the mushroom slices and extract to obtain mushroom broth; Step 2: Take the mushroom broth and filter it by vibration to obtain coarsely filtered mushroom broth; take the coarsely filtered mushroom broth and filter it by pressure to obtain finely filtered mushroom broth; take the finely filtered mushroom broth and concentrate it, controlling the solid content in the mushroom concentrate to obtain mushroom concentrate. Step 3: Take the mushroom concentrate, adjust the pH value with sodium hydroxide or hydrochloric acid, heat, add immobilized bromelain and immobilized papain, stir, add immobilized flavor protease, stir, filter, and obtain the deflavored mushroom concentrate.

2. The process for preparing a de-flavoured mushroom concentrate based on biotechnology according to claim 1, characterized in that: The method for preparing the immobilized bromelain is as follows: Add nano-silica to deionized water and sonicate for 25-35 minutes to obtain a nano-silica dispersion; add tannic acid to Tris-HCl buffer and stir until completely dissolved to obtain a tannic acid-Tris solution; add carboxymethyl chitosan to deionized water and stir until completely dissolved; add bromelain and mix well; add the nano-silica dispersion and mix well; add the tannic acid-Tris solution and react for 4-6 hours; filter and wash to obtain the immobilized bromelain.

3. The process for preparing deodorized mushroom concentrate based on biotechnology according to claim 1, characterized in that: The method for preparing the immobilized papain is as follows: Papain is added to an acetic acid aqueous solution and stirred until completely dissolved to obtain a papain solution; Quaternary ammonium salt chitosan and bentonite are added to an acetic acid aqueous solution and sonicated for 25-35 min; Papain solution is added and mixed evenly; Sodium alginate aqueous solution is added and stirred for 25-35 min; Calcium chloride aqueous solution is added and solidified for 1.5-2.5 h; and the mixture is washed to obtain immobilized papain.

4. The process for preparing deodorized mushroom concentrate based on biotechnology according to claim 1, characterized in that: The method for preparing the immobilized flavor protease is as follows: hydroxypropyl-β-cyclodextrin and tea polyphenols are added to deionized water and stirred until completely dissolved to obtain a mixed solution; gelatin and vanillin are added to deionized water, heated, and stirred until completely dissolved; flavor protease aqueous solution is added and mixed evenly; the mixed solution is added at a temperature of 45-55℃; the pH value is adjusted to 5.3-5.5 using acetic acid aqueous solution; the mixture is solidified for 55-65 minutes; the mixture is filtered and washed to obtain the immobilized flavor protease.

5. The process for preparing deodorized mushroom concentrate based on biotechnology according to claim 1, characterized in that: The mass ratio of the immobilized bromelain, immobilized papain, and immobilized flavor protease is (2-3):(2-3):(2-3).

6. The process for preparing deodorized mushroom concentrate based on biotechnology according to claim 1, characterized in that: The pH value is adjusted to 6.2-6.

4.

7. The process for preparing deodorized mushroom concentrate based on biotechnology according to claim 1, characterized in that: In step one, the thickness of the mushroom slices is 1-3 mm; the mass ratio of the mushroom slices, water, citric acid, tea tree oil, and rosemary extract is 10:(150-250):(0.6-2.2):(0.02-0.06):(0.02-0.06).

8. The process for preparing deodorized mushroom concentrate based on biotechnology according to claim 1, characterized in that: In step two, the concentration process is as follows: concentration is carried out under the conditions of temperature 40-80℃ and vacuum degree 0.07-1MPa, and the solid content in the mushroom concentrate is controlled to be 25-35%.

9. The product prepared by the biotechnology-based deodorized mushroom concentrate preparation process according to any one of claims 1-8.

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