Low-bitterness pericarpium citri reticulatae enzyme and preparation method thereof

Abstract

The application provides a preparation method of low-bitterness pericarpium citri reticulatae enzyme, and comprises the following steps: S1, mixing bitter components containing endogenous enzymes with water, converting bitter substances in the bitter components into non-bitter substances by using the endogenous enzymes, performing enzyme inactivation treatment, and obtaining a mixture; S2, adding fermentation components to the mixture, performing wall breaking treatment, performing first-stage yeast fermentation, monitoring pH value, and performing second-stage lactic acid bacteria fermentation when the pH value decreases to a preset range, so as to obtain fermented material; and S3, performing post-treatment on the fermented material, so as to obtain low-bitterness pericarpium citri reticulatae enzyme, bitter substances are converted by using endogenous enzymes in douchi, and flavor precursors are generated; after the fermentation components are broken, polysaccharides and proteins are degraded by yeast fermentation, and then lactic acid bacteria fermentation is performed to form polysaccharide-saponin complexes to mask the bitter taste of saponin, and the application completely depends on components of raw materials, and does not need to add enzyme preparations or debittering agents.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a low-bitterness tangerine peel enzyme and its preparation method. Background Technology

[0002] Chenpi enzyme is a functional food made by fermenting citrus juice as a starter culture with various medicinal and edible herbs through microbial fermentation. Because chenpi and many of the auxiliary ingredients contain bitter substances such as flavonoids (e.g., naringin and geniposide) and saponins (e.g., ginsenosides and jujube seed saponins), the product has a strong bitter taste and poor palatability, severely impacting consumer acceptance.

[0003] In the food processing field, existing debittering technologies mainly suffer from the following problems: First, the use of exogenous substances such as naringinase, cyclodextrin, and saponins for debittering or masking increases production costs and does not align with the consumption trend of natural ingredients; second, existing technologies are mostly designed for single bitter sources and cannot effectively remove both flavonoid glycosides and saponins simultaneously; third, methods such as physical adsorption, while removing bitterness, non-selectively adsorb flavonoids, saponins, and other functional components, resulting in a loss of product functionality. Therefore, there is an urgent need for a method for preparing tangerine peel enzyme that requires no exogenous additives, can simultaneously process multiple bitter substances, and does not lose functional components. Summary of the Invention

[0004] In view of this, the present invention proposes a low-bitterness tangerine peel enzyme and its preparation method, which solves the technical problems of existing tangerine peel enzymes requiring the addition of exogenous debittering agents and being unable to remove different types of bitterness.

[0005] The technical solution of this invention is implemented as follows: A method for preparing a low-bitterness tangerine peel enzyme includes the following steps: S1, mix the bitter component containing endogenous enzymes with water, use the endogenous enzymes to convert the bitter substances of the bitter component into non-bitter substances, and inactivate the enzymes to obtain the mixture. S2, add fermentation components to the mixture, break the cell walls, carry out the first stage of yeast fermentation, and when the pH value drops to the preset range, carry out the second stage of lactic acid bacteria fermentation to obtain fermented material; S3 involves post-processing the fermented material to obtain a low-bitterness tangerine peel enzyme.

[0006] Based on this technical solution, preferably, the bitter components include fermented black soybeans, dried tangerine peel, gardenia, and mulberry leaves, and step S1 includes: S1.1, Crush dried tangerine peel, gardenia, and mulberry leaves, sieve them, place them in an enzymatic hydrolysis tank, add water at a ratio of 1:0.8-1.2, stir for 20 minutes, then add fermented black soybeans, continue stirring for 15 minutes, heat to 45-55℃, keep warm for 4-8 hours, and use the endogenous enzymes in fermented black soybeans, including α-rhamnosidase and β-glucosidase, to hydrolyze naringin in dried tangerine peel and geniposide in gardenia into non-bitter substances naringenin and genipin, respectively; S1.2 After enzymatic hydrolysis, the enzymatic hydrolysis tank is heated with steam to 85-95℃ and held for 20 minutes to inactivate the endogenous enzymes. Then, it is cooled to 30-40℃ to obtain the mixture.

[0007] Based on this technical solution, preferably, the dried tangerine peel, gardenia, and mulberry leaves are sieved to 40 mesh, and the mass ratio of dried tangerine peel, gardenia, mulberry leaves, and fermented black beans is 10-20:3-8:3-8:5-10.

[0008] Based on this technical solution, preferably, the fermentation components include Poria cocos, lily bulb, licorice root, oyster shell, donkey-hide gelatin, longan pulp, perilla leaf, and Xinhui tangerine juice, and step S2 includes: S2.1 First, grind and sieve the Poria cocos, lily bulb, licorice, oyster shell, donkey-hide gelatin, longan pulp, and perilla leaves. Add the ground Poria cocos, lily bulb, licorice, longan pulp, and perilla leaves to the mixture and stir for 20 minutes. Then add the oyster shell and donkey-hide gelatin and continue stirring for 15 minutes. Add the juice of Xinhui tangerine and add water until the material-to-liquid ratio is 1:6-8. Stir and then transfer to a grinder. Control the temperature to be less than 50℃ during grinding and grind until the particle size is <2μm. Transfer to a fermentation tank for fermentation. S2.2, heat the material in the fermenter to 28-32℃, stir, inoculate with brewer's yeast for fermentation, measure the pH value every 2 hours during fermentation, stop stirring when the pH value drops to 5.2-5.5, inoculate with lactic acid bacteria for fermentation, measure the pH value every 4 hours during fermentation, when the pH value drops to 4.8-5.0, cool to room temperature, and let stand for 24-48 hours to obtain the fermented material.

[0009] Based on this technical solution, preferably, in step S2.2, during the fermentation process of inoculating Saccharomyces cerevisiae, sterile air is introduced into the fermenter for 10 minutes every 4 hours, and the stirring speed is increased to 50-80 rpm during the aeration process. During the fermentation process of inoculating Lactobacillus, the fermenter is pressurized to 0.01-0.05 MPa.

[0010] Based on this technical solution, preferably, the mass ratio of Poria cocos, lily bulb, licorice, oyster shell, donkey-hide gelatin, longan pulp, and perilla leaf in the fermentation components is 5-10:5-10:3-8:2-5:2-5:5-10:3-8.

[0011] Based on this technical solution, preferably, the post-processing in step S3 includes solid-liquid separation of the fermented material, collection of the fermentation liquid, and vacuum concentration at ≤60℃ until the soluble solids content is ≥30%, to obtain low-bitter tangerine peel enzyme.

[0012] On the other hand, the present invention provides a low-bitterness tangerine peel enzyme, which is prepared by the method described in any one aspect of the invention, wherein the low-bitterness tangerine peel enzyme has a naringin content ≤100mg / L and a geniposide content ≤50mg / L.

[0013] The low-bitterness tangerine peel enzyme and its preparation method described in this invention have the following advantages over the prior art: By mixing the bitter component containing endogenous enzymes with water, the endogenous enzyme system contained in the bitter component itself is activated, and the bitter substances in the bitter component are enzymatically converted into non-bitter substances. The enzyme inactivation operation precisely terminates the enzymatic hydrolysis reaction, avoiding over-enzymatic hydrolysis that could lead to abnormal flavor. After adding fermentation components to the mixture, cell wall disruption is performed to control the particle size below 2μm, ensuring thorough cell wall breakage of raw materials such as Poria cocos and lily bulbs, allowing large amounts of polysaccharides and other macromolecules to dissolve. The first stage of yeast fermentation then begins, where yeast enzymes secreted at 28-32℃ degrade polysaccharides into oligosaccharides and proteins into peptides and amino acids. When the pH drops to 5.2-5.5, the second stage of lactic acid bacteria fermentation commences. In the weakly acidic environment (pH 4.8-5.0) created by lactic acid bacteria fermentation, the dissolved polysaccharides and saponins form a stable complex structure through hydrogen bonds and hydrophobic interactions. This complex does not dissociate under neutral conditions in the oral cavity, and the bitter saponin molecules are encapsulated within the complex and cannot directly contact the taste buds. At the same time, the generation of organic acids, sweet amino acids, and volatile flavor substances during fermentation further enriches the product's taste. If low-temperature microwave treatment is added before enzyme inactivation, microwave radiation can cause high-frequency vibration of polar molecules inside the cells, resulting in the rupture of the cell wall microstructure at a lower macroscopic temperature. This promotes the release of endogenous enzymes and substrate contact efficiency, and also facilitates the subsequent dissolution of polysaccharides, thereby enhancing the effects of bitter substance conversion and saponin masking.

[0014] In the above steps, the synergistic transformation of endogenous enzymes, the physical masking of polysaccharide-saponin complexes, and the generation of fermentation flavor substances work together to remove bitter substances and mask saponin-based bitterness. All the enzyme systems, polysaccharides, organic acids, and other components that play a role are derived from the bitter components and fermentation components themselves, without the addition of any exogenous enzyme preparations or debittering agents, resulting in a significant reduction in bitterness in the final product. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of the preparation method of the low-bitterness tangerine peel enzyme described in this invention. Detailed Implementation

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

[0018] This invention provides a low-bitterness tangerine peel enzyme and its preparation method. It relies on endogenous enzymes in the raw materials to achieve the directional transformation of bitter substances, and combines two-stage fermentation to form a complex that shields residual bitterness. No exogenous debittering agents, exogenous enzyme preparations, or sweetness-masking substances are added throughout the process. While retaining the effective components of the raw materials, it achieves the technical effect of low bitterness, mellow taste, and stable flavor in the tangerine peel enzyme.

[0019] In one implementation, such as Figure 1 As shown, a method for preparing a low-bitterness tangerine peel enzyme includes the following steps: S1, mix the bitter component containing endogenous enzymes with water, use the endogenous enzymes to convert the bitter substances in the bitter component into non-bitter substances, and inactivate the enzymes to obtain the mixture; Specifically, step S1 includes: S1.1, Crush dried tangerine peel, gardenia, and mulberry leaves, sieve them, and place them in an enzymatic hydrolysis tank. Add water at a material-to-liquid ratio of 1:0.8-1.2, stir for 20 minutes, then add fermented black soybeans and continue stirring for 15 minutes. Heat to 45-55℃ and keep warm for 4-8 hours. Utilize the endogenous enzymes in fermented black soybeans, including α-rhamnosidase and β-glucosidase, to hydrolyze naringin in dried tangerine peel and geniposide in gardenia into non-bitter substances naringenin and genipin, respectively. S1.2, After enzymatic hydrolysis, steam-heat the enzymatic hydrolysis tank to 85-95℃ and maintain for 20 minutes to inactivate the endogenous enzymes. Cool to 30-40℃ to obtain the mixture.

[0020] The bitter components include fermented black soybeans, dried tangerine peel, gardenia, and mulberry leaves; the dried tangerine peel, gardenia, and mulberry leaves are sieved to 40 mesh, and the mass ratio of dried tangerine peel, gardenia, mulberry leaves, and fermented black soybeans is 10-20:3-8:3-8:5-10.

[0021] At 45-55℃, the endogenous enzyme system carried by fermented black soybeans is activated. For naringin in dried tangerine peel, α-L-rhamnosidase in the fermented black soybean endogenous enzyme system first cleaves the terminal rhamnose, and then β-glucosidase cleaves the glucose, a two-step tandem hydrolysis to produce non-bitter naringin. For geniposide in gardenia, whose glycosyl group contains only one glucose molecule, β-glucosidase can directly hydrolyze it to produce genipin. Simultaneously, the endogenous proteases in fermented black soybeans work concurrently, hydrolyzing some proteins in the bitter components into small peptides and amino acids.

[0022] Compared with exogenous single enzyme preparations, the endogenous enzyme system of fermented soybean contains multiple enzyme activities such as β-glucosidase, α-L-rhamnosidase and protease. The synergistic effect of multiple enzymes can simultaneously complete the conversion of two bitter substances with different structures and the accumulation of flavor precursor substances. The enzyme inactivation treatment uses steam heating to 85-95℃ and maintain it for 20 minutes. While terminating the enzymatic hydrolysis reaction, it causes thermal denaturation and precipitation of enzyme proteins, avoiding the excessive hydrolysis of proteins caused by continuous enzymatic hydrolysis, which would produce bitter peptides containing hydrophobic amino acid residues.

[0023] By activating the endogenous enzyme system in the bitter components, without the need for exogenous enzyme preparations such as naringin or β-glucosidase, two typical bitter glycosides, naringin in dried tangerine peel and geniposide in gardenia, can be directionally hydrolyzed into low-bitter aglycones, eliminating bitter precursors at the source. Simultaneously, endogenous proteases are activated, partially hydrolyzing proteins in the raw materials into small peptides and amino acids, enhancing the product's nutritional value while avoiding the astringent taste and aftertaste caused by large protein molecules. Enzyme inactivation treatment terminates the enzymatic hydrolysis reaction while denaturing and precipitating the enzyme proteins, preventing excessive hydrolysis that leads to rancidity, bitterness, and other undesirable flavors, ensuring a controllable and stable enzymatic hydrolysis process.

[0024] S2, add fermentation components to the mixture, break the cell walls, carry out the first stage of yeast fermentation, and when the pH value drops to the preset range, carry out the second stage of lactic acid bacteria fermentation to obtain fermented material; Specifically, the fermentation components include Poria cocos, lily bulb, licorice root, oyster shell, donkey-hide gelatin, longan pulp, perilla leaf, and Xinhui tangerine juice; the mass ratio of Poria cocos, lily bulb, licorice root, oyster shell, donkey-hide gelatin, longan pulp, and perilla leaf in the fermentation components is 5-10:5-10:3-8:2-5:2-5:5-10:3-8.

[0025] Step S2 includes: S2.1 First, grind and sieve the Poria cocos, lily bulb, licorice, oyster shell, donkey-hide gelatin, longan pulp, and perilla leaves. Add the ground Poria cocos, lily bulb, licorice, longan pulp, and perilla leaves to the mixture and stir for 20 minutes. Then add the oyster shell and donkey-hide gelatin and continue stirring for 15 minutes. Add the juice of Xinhui tangerine and add water until the material-to-liquid ratio is 1:6-8. Stir and then transfer to a grinder. Control the temperature to be less than 50℃ during grinding and grind until the particle size is <2μm. Transfer to a fermentation tank for fermentation. S2.2, heat the material in the fermenter to 28-32℃, stir, inoculate with brewer's yeast for fermentation, measure the pH value every 2 hours during fermentation, stop stirring when the pH value drops to 5.2-5.5, inoculate with lactic acid bacteria for fermentation, measure the pH value every 4 hours during fermentation, when the pH value drops to 4.8-5.0, cool to room temperature, and let stand for 24-48 hours to obtain the fermented material.

[0026] In step S2.2, during the fermentation process of inoculating Saccharomyces cerevisiae, sterile air is introduced into the fermenter for 10 minutes every 4 hours, and the stirring speed is increased to 50-80 rpm during the aeration process; during the fermentation process of inoculating Lactobacillus, the fermenter is pressurized to 0.01-0.05 MPa.

[0027] The fermentation components undergo low-temperature ultra-micro cell disruption treatment, which fully releases effective ingredients such as polysaccharides, saponins, and amino acids, improving material utilization and fermentation uniformity. Simultaneously, the low-temperature conditions prevent the inactivation or destruction of heat-sensitive active ingredients. The first stage involves facultative anaerobic yeast fermentation, achieving rapid cell proliferation through intermittent aeration. Yeast enzymes degrade polysaccharides into oligosaccharides and proteins into peptides, reducing system viscosity and generating basic flavor compounds. Once the pH drops to 5.2-5.5, the process switches to the second stage of lactic acid bacteria anaerobic fermentation. Lactic acid bacteria produce organic acids, promoting the formation of stable complexes between polysaccharides and saponins in the fermentation components through hydrogen bonding and hydrophobic interactions. These complexes do not dissociate in the oral environment and can encapsulate and shield trace amounts of incompletely converted saponin bitter molecules. Simultaneously, the fermentation process generates natural sweet and umami substances, further balancing the taste and masking residual bitterness. The two-stage fermentation is tightly integrated and under controllable conditions, requiring no exogenous fermentation regulators, thus ensuring the product's natural properties and flavor stability.

[0028] Lactobacillus converts glycyrrhizic acid into glycyrrhetinic acid monoglucuronide, whose sweetness release rate is significantly faster than that of glycyrrhizic acid itself. It also further metabolizes the polypeptides and amino acids produced from the decomposition of proteins in oysters, donkey-hide gelatin, and fermented soybean residues, generating umami substances represented by glutamic acid and aspartic acid. These small-molecule sweet and umami substances dissolve in the oral cavity before the encapsulated saponins, activating sweet and umami receptors and inhibiting the neurotransmission of bitter signals.

[0029] The yeast strains, lactobacillus strains, etc. used in this invention are all existing products, and their usage methods are well known to those skilled in the art.

[0030] S3, post-process the fermented material to obtain low-bitter tangerine peel enzyme; Specifically, step S3 post-processing includes solid-liquid separation of fermentation materials, collection of fermentation broth, and vacuum concentration at ≤60℃ until soluble solids ≥30% to obtain low-bitter tangerine peel enzyme.

[0031] Low-temperature vacuum concentration can improve product concentration and stability while preserving active ingredients and flavor substances, and avoid problems such as browning and flavor loss caused by high-temperature heating.

[0032] On the other hand, the present invention provides a low-bitterness tangerine peel enzyme, which is prepared by any of the above-mentioned methods for preparing low-bitterness tangerine peel enzyme. The low-bitterness tangerine peel enzyme has a naringin content ≤100mg / L, a geniposide content ≤50mg / L, a bitterness score ≤2 points (out of 10), a mellow taste without bitterness, and retains the active ingredients intact.

[0033] Example 1: Weigh out 15 parts dried tangerine peel, 5 parts gardenia, 5 parts mulberry leaves, and 8 parts fermented black soybeans. Crush the dried tangerine peel, gardenia, and mulberry leaves and pass them through a 40-mesh sieve. Place them in an enzymatic hydrolysis tank and add purified water at a material-to-liquid ratio of 1:1. Stir for 20 minutes, add the fermented black soybeans and continue stirring for 15 minutes. Heat to 50°C and keep warm for 6 hours to hydrolyze naringin and gardenin using the endogenous β-glucosidase in the fermented black soybeans. After the enzymatic hydrolysis is completed, heat to 90°C and keep warm for 20 minutes to inactivate the enzyme. Cool to 35°C to obtain the mixture.

[0034] Weigh out 8 parts of Poria cocos, 8 parts of lily bulb, 5 parts of licorice root, 3 parts of oyster shell, 3 parts of donkey-hide gelatin, 8 parts of longan pulp, and 5 parts of perilla leaf. Crush and sieve the mixture. Add Poria cocos, lily bulb, licorice root, longan pulp, and perilla leaf to the mixture and stir for 20 minutes. Then add oyster shell and donkey-hide gelatin and stir for 15 minutes. Add an appropriate amount of Xinhui tangerine juice and add water to make the material-liquid ratio 1:7. Stir evenly and transfer to a grinder. Grind at a temperature of <50℃ until the particle size is <2μm. Transfer to a fermentation tank.

[0035] The material in the fermentation tank was heated to 30°C, and brewer's yeast was inoculated for the first stage of fermentation. Sterile air was introduced for 10 minutes every 4 hours, and the stirring speed was 60 rpm during aeration. When the pH dropped to 5.3, stirring and aeration were stopped, and lactic acid bacteria were inoculated for the second stage of anaerobic fermentation. The fermentation tank was maintained at a pressure of 0.03 MPa. When the pH dropped to 4.9, the material was cooled to room temperature and allowed to stand for 36 hours to obtain the fermented material.

[0036] The fermented material was centrifuged and filtered, and the supernatant was collected and concentrated under vacuum at 55°C until the soluble solids content was ≥30% to obtain low-bitterness tangerine peel enzyme.

[0037] Example 2: Weigh out 10 parts dried tangerine peel, 3 parts gardenia, 3 parts mulberry leaves, and 5 parts fermented black soybeans. Grind them and pass them through a 40-mesh sieve. Add water at a material-to-liquid ratio of 1:0.8, stir and mix, and keep warm at 45℃ for 8 hours for enzymatic hydrolysis. Keep warm at 95℃ for 20 minutes to inactivate the enzymes, and cool to 30℃ to obtain the mixture.

[0038] Weigh out 5 parts of Poria cocos, 5 parts of lily bulb, 3 parts of licorice root, 2 parts of oyster shell, 2 parts of donkey-hide gelatin, 5 parts of longan pulp, and 3 parts of perilla leaf. After crushing and sieving, add the mixture, add Xinhui tangerine juice and add water to make the material-liquid ratio 1:6. Grind at low temperature until the particle size is <2μm and then transfer to a fermentation tank.

[0039] Saccharomyces cerevisiae was inoculated at 32℃ for fermentation, and aeration was carried out for 10 minutes every 4 hours at a speed of 50 rpm. When the pH dropped to 5.2, lactic acid bacteria were inoculated for fermentation and the pressure was maintained at 0.01 MPa. When the pH dropped to 4.8, the mixture was cooled and allowed to stand for 24 hours to obtain the fermented material.

[0040] The fermentation material was subjected to solid-liquid separation, and the fermentation broth was collected. Nitrogen gas was bubbled into the fermentation broth for 30 minutes to remove residual carbon dioxide from the fermentation process, reduce the dissolved oxygen concentration in the system, and reduce the oxidative polymerization of genipin during the subsequent concentration process. After degassing, the fermentation broth was concentrated at -0.09 MPa vacuum and 55°C until the soluble solids content was ≥30%. During the concentration process, the trace amounts of genipin remaining in the fermentation broth underwent mild cross-linking with the small molecule peptides and amino acids produced by the enzymatic hydrolysis of fermented soybeans, further reducing the free genipin content. At the same time, pyrazine derivatives with aroma-enhancing effects were generated. After concentration, a low-bitter tangerine peel enzyme was obtained.

[0041] Example 3: Weigh out 20 parts dried tangerine peel, 8 parts gardenia, 8 parts mulberry leaves, and 10 parts fermented black soybeans. Grind them and pass them through a 40-mesh sieve. Add water at a material-to-liquid ratio of 1:1.2. Incubate at 55℃ for 4 hours for enzymatic hydrolysis. Then, microwave at 45℃ for 15 minutes to inactivate the enzymes. Finally, incubate at 85℃ for 20 minutes to inactivate the enzymes. Cool to 40℃ to obtain the mixture.

[0042] Weigh out 10 parts of Poria cocos, 10 parts of lily bulb, 8 parts of licorice root, 5 parts of oyster shell, 5 parts of donkey-hide gelatin, 10 parts of longan pulp, and 8 parts of perilla leaf. After crushing and sieving, add the mixture, add Xinhui tangerine juice and add water to make the material-liquid ratio 1:8. Grind at low temperature until the particle size is <2μm and then transfer to a fermentation tank.

[0043] Fermentation was carried out by inoculating with brewer's yeast at 28℃, aerating for 10 minutes every 4 hours at a speed of 80 rpm; when the pH dropped to 5.5, lactic acid bacteria were inoculated for fermentation, and the pressure was maintained at 0.05 MPa; when the pH dropped to 5.0, the mixture was cooled and allowed to stand for 48 hours. The fermented material was then separated into solid and liquid components and concentrated under vacuum at ≤60℃ until the soluble solids content was ≥30%, resulting in low-bitterness tangerine peel enzyme.

[0044] Comparative Example 1: The difference from Example 1 is that: in step S1, fermented black soybeans are not added, and exogenous β-glucosidase is added directly for enzymatic hydrolysis. The remaining steps and parameters are exactly the same.

[0045] Comparative Example 2: The difference from Example 1 is that the two-stage fermentation is cancelled and only single yeast fermentation is used, while the remaining steps and parameters are exactly the same.

[0046] Comparative Example 3: The difference from Example 1 is that step S2 does not involve low-temperature cell disruption, while the remaining steps and parameters are exactly the same.

[0047] Comparative Example 4: The difference from Example 1 is that in step S1.2, the enzyme inactivation treatment with steam is not performed, and the mixture is directly cooled to 35°C after enzymatic hydrolysis to obtain the mixture. The other steps are exactly the same as in Example 1.

[0048] The low-bitterness tangerine peel enzymes obtained in the examples and comparative examples were subjected to relevant performance tests. The test contents and methods are as follows: Determination of naringin and geniposide content: High performance liquid chromatography (HPLC) was used. After appropriate dilution and filtration, the samples were eluted using a C18 column with gradient elution of acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase. The detection wavelengths were 283 nm and 238 nm, respectively, and the content was calculated by external standard method.

[0049] Sensory evaluation: An evaluation panel of 10 trained sensory evaluators scored the bitterness intensity of the samples using a scale of 0-9, where 0 indicates no bitterness and 9 indicates extreme bitterness. Each sample was evaluated three times, and the average score was taken.

[0050] Soluble solids content determination: The Abbe refractometer was used to directly measure the content at 20℃.

[0051] Table 1: Performance tests of the low-bitterness tangerine peel enzyme obtained from the examples and comparative examples.

[0052] As shown in Table 1, the performance test results of the tangerine peel enzyme obtained in Examples 1-3 were better than those in Comparative Examples 1-4. The conversion of bitter substances was better. Compared with Examples 1 and 3, the sensory score of Example 2 was the lowest at 0.9 points, and the residual amounts of naringin and geniposide were reduced to 52 mg / L and 22 mg / L, respectively, showing the best overall performance.

[0053] The reason is that Example 2 added a low-temperature vacuum degassing and chitosan adsorption treatment step in the post-processing stage. The fermented material was first degassed for 15-20 minutes at 30-35℃ and -0.06~-0.08MPa. The degassing operation removed residual carbon dioxide from the fermentation broth and reduced the dissolved oxygen concentration of the system. Genipin, the hydrolysis product of genipin, is easily oxidized and polymerized by dissolved oxygen in its free state, generating blue-black pigments and oligomers. Although these polymers do not constitute a strong bitter taste, they will produce an astringent feeling and unpleasant aftertaste in the mouth. The degassing treatment inhibited the oxidative polymerization pathway of genipin by reducing dissolved oxygen. Subsequently, 0.1%-0.3% of food-grade chitosan by mass of the material is added for adsorption treatment. The free amino groups on the chitosan molecular chain are protonated under weakly acidic conditions, which can bind with residual bitter substances and genipin oxidation intermediates through electrostatic adsorption and hydrogen bonding, further reducing the concentration of free bitter substances in the system. In both steps, degassing inhibits genipin oxidation from the source, while chitosan adsorption captures the trace amounts of bitter substances and oxidation intermediates that have already been generated, resulting in a significant decrease in the bitterness sensory score of the final product.

[0054] In addition, compared with Example 1, Comparative Example 1, which replaced fermented soybean with exogenous β-glucosidase, had residual levels of naringin and geniposide of 78 mg / L and 38 mg / L, respectively, which were slightly higher than those in Example 1. This indicates that the exogenous enzyme has the ability to hydrolyze bitter substances, but its efficiency is slightly lower than that of the endogenous enzyme system in fermented soybean. However, its sensory score rose to 3.2 points. The main reason is that the exogenous enzyme has a single component and lacks the synergistic effect of endogenous proteases. The protein was not effectively hydrolyzed into flavor peptides and amino acids, resulting in insufficient flavor precursors during the fermentation stage and a lack of competitive masking of bitter receptors by peptides.

[0055] Comparative Example 2 eliminated the two-stage fermentation process and only underwent yeast fermentation. Its naringin and geniposide content was similar to that of Example 1, indicating that single yeast fermentation had no significant impact on the transformed bitter substances. However, this comparative example had a high bitterness sensory score of 5.5, indicating the presence of bitterness sources in the system that were not eliminated through enzymatic hydrolysis. According to the aforementioned mechanism, saponins in the fermentation components cannot be effectively masked during yeast fermentation. They require a slightly acidic environment (pH 4.8-5.0) created by lactic acid bacteria fermentation to form stable complexes with polysaccharides released from Poria cocos, lily bulbs, etc., through hydrogen bonds and hydrophobic interactions. The final pH of Comparative Example 2's fermentation failed to reach this range, preventing the formation of polysaccharide-saponin complexes. Saponin molecules directly contacted taste buds in the mouth, leading to a significantly enhanced bitterness perception. Furthermore, the lack of organic acids and flavor substances generated during lactic acid bacteria fermentation also weakened the overall flavor harmony of the product.

[0056] Comparative Example 3 did not undergo low-temperature cell wall disruption in step S2; the components were simply mixed and fermented directly. Data showed that its naringin and geniposide contents were 72 mg / L and 33 mg / L, respectively, which were not significantly different from Example 1. However, the bitterness sensory score increased to 4.8. The role of low-temperature cell wall disruption is to reduce the particle size of the material to below 2 μm, fully destroying the plant cell wall structure and releasing a large amount of intracellular contents. Without cell wall disruption, polysaccharide molecules in raw materials such as Poria cocos and lily bulbs are mostly bound within the intact cell walls and cannot be fully dissolved to participate in the subsequent complex formation process. Insufficient polysaccharide dissolution directly affects the formation efficiency of polysaccharide-saponin complexes, resulting in a decrease in the masking effect of saponin-based bitter substances. This result indicates that the enzymatic transformation of bitter substances and the complex masking of saponins are two relatively independent debittering pathways. The former mainly depends on enzyme activity, while the latter is limited by the degree of dissolution of macromolecules.

[0057] Comparative Example 4 omitted the steam inactivation treatment and proceeded directly to subsequent steps after enzymatic hydrolysis. The residual amounts of naringin and geniposide in this example were 42 mg / L and 18 mg / L, respectively, the lowest among all groups. This is because the enzymatic hydrolysis continued during subsequent fermentation, converting more bitter substances into non-bitter products. However, its bitterness sensory score remained at 3.9 points, not decreasing synchronously with the reduction in bitter substance content, but instead higher than Example 1. During continuous enzymatic hydrolysis, the endogenous proteases in the fermented soybeans remained active, excessively hydrolyzing the raw material protein into small peptides containing hydrophobic amino acid residues. Some of these hydrophobic peptides themselves are bitter, and the excessive fragmentation of the peptide structure weakens their ability to participate in flavor reshaping during fermentation. The role of the enzyme inactivation treatment is to precisely control the end point of enzymatic hydrolysis, converting the original bitter substances while avoiding excessive protein degradation that produces secondary bitter substances. This result indicates that obtaining low-bitter products requires not only the removal of original bitter components but also the control of the generation of new bitter substances during processing.

[0058] In summary, the low-bitterness tangerine peel enzyme and its preparation method provided by this invention successfully achieve efficient conversion of bitter substances in raw materials such as tangerine peel and gardenia, and effective masking of saponin bitterness by utilizing endogenous enzymatic hydrolysis, two-stage fermentation, and cell wall breaking treatment. This method utilizes the endogenous enzyme system and fermentation metabolites of the raw materials themselves, without the need to add any exogenous debittering agents or enzyme preparations. While significantly reducing the bitterness of the product and improving the taste, it retains the effective components of the raw materials to the greatest extent. The resulting tangerine peel enzyme has a mellow taste and pleasant flavor.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a low-bitterness tangerine peel enzyme, characterized in that, Includes the following steps: S1, mix the bitter component containing endogenous enzymes with water, use the endogenous enzymes to convert the bitter substances of the bitter component into non-bitter substances, and inactivate the enzymes to obtain the mixture. S2, add fermentation components to the mixture, break the cell walls, carry out the first stage of yeast fermentation, and when the pH value drops to the preset range, carry out the second stage of lactic acid bacteria fermentation to obtain fermented material; S3 involves post-processing the fermented material to obtain a low-bitterness tangerine peel enzyme.

2. The method for preparing low-bitterness tangerine peel enzyme as described in claim 1, characterized in that, The bitter components include fermented black soybeans, dried tangerine peel, gardenia, and mulberry leaves. Step S1 includes: S1.1, Crush dried tangerine peel, gardenia, and mulberry leaves, sieve them, place them in an enzymatic hydrolysis tank, add water at a ratio of 1:0.8-1.2, stir for 20 minutes, then add fermented black soybeans, continue stirring for 15 minutes, heat to 45-55℃, keep warm for 4-8 hours, and use the endogenous enzymes in fermented black soybeans, including α-rhamnosidase and β-glucosidase, to hydrolyze naringin in dried tangerine peel and geniposide in gardenia into non-bitter substances naringenin and genipin, respectively; S1.2 After enzymatic hydrolysis, the enzymatic hydrolysis tank is heated with steam to 85-95℃ and held for 20 minutes to inactivate the endogenous enzymes. Then, it is cooled to 30-40℃ to obtain the mixture.

3. The method for preparing low-bitterness tangerine peel enzyme as described in claim 2, characterized in that, The dried tangerine peel, gardenia, and mulberry leaves were sieved to 40 mesh, and the mass ratio of dried tangerine peel, gardenia, mulberry leaves, and fermented black beans was 10-20:3-8:3-8:5-10.

4. The method for preparing low-bitterness tangerine peel enzyme as described in claim 1, characterized in that, The fermentation components include Poria cocos, lily bulb, licorice root, oyster shell, donkey-hide gelatin, longan pulp, perilla leaf, and Xinhui tangerine juice. Step S2 includes: S2.1 First, grind and sieve the Poria cocos, lily bulb, licorice, oyster shell, donkey-hide gelatin, longan pulp, and perilla leaves. Add the ground Poria cocos, lily bulb, licorice, longan pulp, and perilla leaves to the mixture and stir for 20 minutes. Then add the oyster shell and donkey-hide gelatin and continue stirring for 15 minutes. Add the juice of Xinhui tangerine and add water until the material-to-liquid ratio is 1:6-8. Stir and then transfer to a grinder. Control the temperature to be less than 50℃ during grinding and grind until the particle size is <2μm. Transfer to a fermentation tank for fermentation. S2.2, heat the material in the fermenter to 28-32℃, stir, inoculate with brewer's yeast for fermentation, measure the pH value every 2 hours during fermentation, stop stirring when the pH value drops to 5.2-5.5, inoculate with lactic acid bacteria for fermentation, measure the pH value every 4 hours during fermentation, when the pH value drops to 4.8-5.0, cool to room temperature, and let stand for 24-48 hours to obtain the fermented material.

5. The method for preparing low-bitterness tangerine peel enzyme as described in claim 4, characterized in that, In step S2.2, during the fermentation process of inoculating Saccharomyces cerevisiae, sterile air is introduced into the fermenter for 10 minutes every 4 hours, and the stirring speed is increased to 50-80 rpm during the aeration process. During the fermentation process of inoculating Lactobacillus, the fermenter is pressurized to 0.01-0.05 MPa.

6. The method for preparing low-bitterness tangerine peel enzyme as described in claim 4, characterized in that, The mass ratio of Poria cocos, lily bulb, licorice, oyster shell, donkey-hide gelatin, longan pulp, and perilla leaf in the fermentation components is 5-10:5-10:3-8:2-5:2-5:5-10:3-8.

7. The method for preparing low-bitterness tangerine peel enzyme as described in claim 1, characterized in that, Step S3 post-processing includes solid-liquid separation of fermentation materials, collection of fermentation broth, and vacuum concentration at ≤60℃ until soluble solids ≥30% to obtain low-bitter tangerine peel enzyme.

8. A low-bitterness tangerine peel enzyme, prepared by the method for preparing low-bitterness tangerine peel enzyme according to any one of claims 1-7, characterized in that, The low-bitterness tangerine peel enzyme contains naringin ≤100mg / L and geniposide ≤50mg / L.

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