Exocarpium leaf and tremella fermented beverage and preparation method thereof

Abstract

The invention discloses an exocarpium leaf and tremella fermented beverage and a preparation method thereof. Fresh exocarpium leaf is used as a raw material, and the exocarpium leaf and tremella fermented beverage can be prepared through the steps of homogenization, sterilization, tremella fermentation, solid-liquid separation, blending and the like. The obtained fermented beverage is rich in tremella polysaccharide with the effects of tonifying the spleen, appetizing, nourishing yin, moistening the lung, regulating immunity, reducing blood sugar and blood fat and the like, is rich in limonene, naringin and other flavones with the effects of relieving cough, reducing sputum, resisting inflammation, resisting oxidation and the like, is free of bitter taste, has fresh and sweet flower fragrance, and is suitable for being drunk by the majority of people, including diabetics.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a tangerine peel and tremella fermented beverage and a preparation method thereof. Background Art

[0002] Huajuhong (Huajuhong) is the immature or nearly mature outer peel of the Huazhou pomelo (Huazhou pomelo), a Rutaceae plant. It is one of Guangdong Province's "Ten Rare Chinese Medicinal Materials" and is rich in polysaccharides, naringin, naringin, and various flavonoid compounds. Research has shown that, in addition to its well-known cough-relieving and expectorant properties, Huajuhong also possesses anti-inflammatory, antioxidant, anti-thrombotic, anti-fatigue, alcohol-hepatotoxicity-removing, and blood sugar and lipid-lowering properties, making it an increasingly important ingredient in the development of traditional Chinese medicines and health foods. Huajuhong leaves contain β-pinene, β-myrcene, D-limonene, γ-terpinene, and caryophyllene, as well as numerous flavonoid compounds found in Huajuhong, yet these compounds have received relatively little attention from researchers.

[0003] Tremella fuciformis, also known as white fungus or snow fungus, belongs to the Tremellaceae family of the Basidiomycetes class. It is a traditional Chinese medicinal and edible fungus. It contains a variety of ingredients, including polysaccharides, flavonoids, and amino acids. It has the potential to tonify the spleen and appetite, nourish yin and moisten the lungs, regulate immunity, fight tumors, resist oxidation and aging, lower blood sugar and lipids, fight thrombosis, fight ulcers, fight viruses, promote protein synthesis, promote nerve cell growth, and improve memory. In order to promote the comprehensive utilization of tangerine peel resources and increase the content of active ingredients in tangerine peel.

[0004] At present, there is no report on using Tremella to ferment orange peel. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a fermented beverage of tangerine red leaves and tremella.

[0006] Another object of the present invention is to provide a fermented beverage of tangerine peel and tremella obtained by the above-mentioned preparation method.

[0007] The object of the present invention is achieved through the following technical solution: a method for preparing a fermented beverage of tangerine red leaves and tremella, comprising the following steps:

[0008] (1) Fermentation of Citrus aurantium leaves: fresh Citrus aurantium leaves are cleaned, mixed with water, and milk powder, glucose and potassium dihydrogen phosphate are added. After homogenization, sterilization and cooling, Tremella fuciformis seed liquid is added, and fermentation is carried out under temperature control, ventilation and stirring conditions to obtain a fermentation liquid;

[0009] (2) solid-liquid separation: heating and boiling the fermentation liquid obtained in step (1), homogenizing it while hot, and performing solid-liquid separation to obtain a supernatant;

[0010] (3) Preparation: Take the supernatant obtained in step (2) and prepare it to obtain a fermented beverage of tangerine peel and tremella.

[0011] The water described in step (1) is preferably purified water.

[0012] The amount of water used in step (1) is preferably calculated based on a mass ratio of Citrus aurantium to water of 1:(2.8-3.2).

[0013] The milk powder described in step (1) is preferably skimmed milk powder.

[0014] The amount of milk powder used in step (1) is preferably calculated based on 9-11% of the mass of the orange peel leaves.

[0015] The amount of glucose used in step (1) is preferably calculated as 28-32% of the mass of the leaves of Citrus aurantium.

[0016] The potassium dihydrogen phosphate described in step (1) is food grade potassium dihydrogen phosphate; its dosage is preferably calculated based on 0.28-0.32% of the mass of the orange peel leaves.

[0017] The homogenization conditions in step (1) are preferably a rotation speed of 7000 to 8000 r / min and a time of 14 to 16 min.

[0018] The sterilization conditions described in step (1) are preferably sterilized at 121° C. for 16 to 20 minutes.

[0019] The degree of cooling described in step (1) is to a degree that does not harm the Tremella fuciformis strains; preferably, cooling to below 35°C.

[0020] The Tremella fuciformis strain described in step (1) is any Tremella fuciformis strain; preferably CGMCC5.163 (China General Microorganism Culture Collection Center).

[0021] The Tremella fuciformis seed liquid described in step (1) is preferably prepared by the following steps: inoculating Tremella fuciformis strains into a sterilized Tremella fuciformis strain culture medium, and culturing to a logarithmic growth phase or a stable phase to obtain the Tremella fuciformis seed liquid.

[0022] The inoculation amount is preferably 1 Tremella fuciformis slant strain per 150 mL Tremella fuciformis strain culture medium.

[0023] The composition of the Tremella fuciformis culture medium is preferably as follows: 58-62 g / L of glucose, 9-11 g / L of soybean protein peptide, 4-6 g / L of yeast extract, 0.8-1 g / L of potassium dihydrogen phosphate, 0.4-0.6 g / L of magnesium sulfate, pH 5.8-6.2, and the solvent is water; preferably as follows: 60 g / L of glucose, 10 g / L of soybean protein peptide, 5 g / L of yeast extract, 1 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, pH 6.0, and the solvent is water.

[0024] The culture conditions are preferably 20-28° C., 140-160 rpm shaking culture for 200-230 h; more preferably 23-25° C., 150 rpm shaking culture for 210-222 h.

[0025] The inoculation volume (mL) of the Tremella fuciformis seed solution described in step (1) is preferably 1.6% to 2.4% (v / w) of the mass (g) of the water described in step (1).

[0026] The fermentation conditions described in step (1) are preferably: temperature 23-25°C, stirring speed 70-90r / min, ventilation ratio controlled at 0.24-0.32vvm for fermentation 0-48h, ventilation ratio controlled at 0.32-0.48vvm for fermentation 49-96h, ventilation ratio controlled at 0.48-0.56vvm for fermentation 97-168h, ventilation ratio controlled at 0.56-0.64vvm for fermentation 169-216h, ventilation ratio controlled at 0.48-0.56vvm for fermentation 217h and thereafter, and fermentation period 264-288h.

[0027] The heating and boiling conditions described in step (2) are preferably heated at 100° C. for 8 to 12 minutes.

[0028] The homogenization conditions in step (2) are preferably a rotation speed of 7000 to 8000 r / min and a time of 14 to 16 min.

[0029] The solid-liquid separation method in step (2) is preferably centrifugation.

[0030] The centrifugal conditions are preferably a rotation speed of 6000 to 8000 r / min and a separation time of 18 to 22 min.

[0031] The preparation in step (3) is preferably prepared by adding a sweetener.

[0032] The sweeteners are preferably neomethyl hesperidin dihydrochalcone and maltitol.

[0033] The dosage of the neomethyl hesperidin dihydrochalcone is preferably calculated based on 50 to 60 mg of neomethyl hesperidin dihydrochalcone per liter of supernatant.

[0034] The dosage of the maltitol is preferably calculated based on 80 to 100 g of maltitol per liter of supernatant.

[0035] The above preparation method also includes the following steps: (4) canning and sterilization.

[0036] The sterilization conditions described in step (4) are preferably a temperature of 100° C. and a time of 8 to 10 minutes.

[0037] A fermented beverage of tangerine peel and tremella is obtained by the preparation method.

[0038] The present invention has the following advantages and effects compared to the prior art:

[0039] (1) There is no precedent for using Citrus aurantium leaves to make fermented beverages. The present invention produces a large amount of Tremella fuciformis polysaccharides through liquid fermentation of Tremella fuciformis. Compared with the unfermented material, the polysaccharide content in the fermented beverage is increased by more than 12 g / L, which significantly improves the effects of the polysaccharides in the fermented beverage on tonifying the spleen and appetite, nourishing yin and moistening the lungs, regulating immunity, lowering blood sugar and blood lipids, and promoting the development of Citrus aurantium leaves resources.

[0040] (2) After the red leaves of the tangerine peel were fermented with Tremella fuciformis, there was no loss of naringin, and the precursors of flavonoids were converted into flavonoids, and the precursors of limonene were converted into limonene. Compared with the unfermented materials, the total flavonoid content in the fermented beverage increased by more than 0.1 g / L, and the limonene content increased by more than 1.5 mg / L, which significantly improved the cough and phlegm-relieving, anti-inflammatory, and antioxidant effects of flavonoids and limonene in the fermented beverage.

[0041] (3) After the red leaves of Citrus aurantium are fermented with Tremella fuciformis, vanillic acid, phenylalanine and other substances can be converted into citronellol and phenylethanol with a sweet rose fragrance. Compared with the unfermented material, the citronellol and phenylethanol in the fermented beverage increased by more than 3 mg / L and more than 5 mg / L respectively. The overall aroma of the liquid changed from a pungent green smell to a sweet rose fragrance, which has the aroma characteristics that people like.

[0042] (4) After the leaves of the Chinese orange peel are fermented with Tremella fuciformis, some substances that block the perception of bitterness are produced, such as polysaccharides and alcohols. Compared with the unfermented material, although the content of naringin does not change much, the bitterness intensity of the fermented beverage is significantly reduced, which is conducive to using a small amount of sweetener to mask the bitterness. DETAILED DESCRIPTION

[0043] The present invention will be described in further detail below, but embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention can be obtained commercially.

[0044] Example 1

[0045] (1) Preparation of Tremella liquid strain

[0046] Tremella liquid culture medium: glucose 60g / L, soy protein peptide 10g / L, yeast extract 5g / L, potassium dihydrogen phosphate (edible grade) 1g / L, magnesium sulfate (edible grade) 0.5g / L, adjust pH to 6.0.

[0047] Prepare 300 mL of culture medium and dispense it into two 1000 mL conical flasks, filling each with 150 mL of liquid. Sterilize at 121°C for 20 min. After cooling, inoculate each bottle with one Tremella fuciformis CGMCC5.163 slant culture (purchased from China General Microbiological Culture Collection) and culture under shaking conditions at 24°C and 150 rpm for 216 h to obtain a Tremella fuciformis liquid culture.

[0048] (2) Fermentation of tangerine peel

[0049] 5 kg of fresh tangerine peel leaves were weighed, cleaned, and placed in a high-speed homogenizer. 15 kg of purified water, 500 g of skim milk powder, 1500 g of glucose, and 15 g of food-grade potassium dihydrogen phosphate were added. The mixture was homogenized at a speed of 7500 r / min for 15 min to obtain material 1. Material 1 was then placed in a 30 L fully automatic fermentation tank and sterilized at 121°C for 18 min.

[0050] After material 1 is cooled, 300mL of the Tremella liquid culture obtained by the above culture is added, sterile air is introduced, and fermentation is started. During the fermentation process, the temperature is controlled at 24°C, the stirring speed is controlled at 80r / min, the ventilation ratio is controlled at 0.24-0.32vvm for fermentation 0-48h, the ventilation ratio is controlled at 0.32-0.48vvm for fermentation 49-96h, the ventilation ratio is controlled at 0.48-0.56vvm for fermentation 97-168h, the ventilation ratio is controlled at 0.56-0.64vvm for fermentation 169-216h, and the ventilation ratio is controlled at 0.48-0.56vvm for fermentation 217h and thereafter. When the fermentation time reaches 272h, the fermentation is terminated to obtain material 2.

[0051] (3) Solid-liquid separation

[0052] After fermentation, Material 2 was heated to 100°C using the fermenter's jacket and maintained at this temperature for 10 minutes. Material 2 was then placed in a high-speed homogenizer and homogenized at 7500 rpm for 15 minutes. The homogenized material was then centrifuged at 7000 rpm for 20 minutes, and 12.4 L of supernatant was collected.

[0053] The naringin content in the supernatant was detected by high performance liquid chromatography ([Reference] Zhou Guifen, Chen Suhong, Lü Guiyuan, Yan Meiqiu. Determination of naringenin content in Dendrobium officinale by high performance liquid chromatography [J]. China Journal of Chinese Materia Medica, 2013, 38(4): 520-523.), and the result was 96.23 mg / L.

[0054] The limonene content in the supernatant was detected by gas chromatography ([Reference] Zhang Jiaying, Yin Yi, Wang Zhe, Sun Qihang, Li Hongjuan, Zhao Juanjuan. Determination of three volatile components in Evodia rutaecarpa by gas chromatography [J]. Chemical Analysis and Meterage, 2021, 30(9): 5-10.), and the result was 3.28 mg / L.

[0055] The citronellol content in the supernatant was detected by gas chromatography ([Reference] Wei Chaojun, Wang Huiwen, Zeng Hui, Shi Shaoran, Yang Aizhen. Analysis of citronellol and geraniol content in health vinegar [J]. Journal of Food Safety and Quality, 2018, 9(17): 4686-4692.), and the result was 8.84 mg / L.

[0056] The phenylethanol content in the supernatant was detected by gas chromatography ([Reference] Mo Xinliang, Yan Dabao, Teng Mingde. Qualitative and quantitative analysis of 2-phenylethanol in Chinese liquor [J]. Food Science and Technology, 2022, 47(7): 256-260.), and the result was 5.56 mg / L.

[0057] According to the method of the People's Republic of China Entry-Exit Inspection and Quarantine Industry Standard "Determination of Total Flavonoids in Export Food" (SN / T4592-2016), the total flavonoid content in the supernatant was tested, and the result was 0.51 g / L.

[0058] Take 200 mL of supernatant, add 800 mL of purified water, mix well, and perform ultrafiltration using a 360 Da ultrafiltration membrane. When the permeate volume is equal to 800 mL, end the ultrafiltration, take out the retentate, and detect the polysaccharide content in the supernatant in accordance with the Agricultural Industry Standard of the People's Republic of China "Spectrophotometric Method for Determination of Crude Polysaccharides in Edible Fungi" (NY / T 1676-2023). The result is 23.24 g / L.

[0059] A series of concentrations of quinine hydrochloride standard solutions were prepared as controls for bitterness evaluation. A 15-person bitterness evaluation panel evaluated the bitterness of the supernatant. The bitterness of the supernatant was very weak, close to that of a 0.02 mg / L quinine hydrochloride solution.

[0060] (4) Mixing and filling

[0061] Take 11L of supernatant, add 605mg of neomethyl hesperidin dihydrochalcone and 990g of maltitol, stir to dissolve, and then divide into glass bottles, 100mL of liquid in each bottle, seal with an automatic capping machine, put into a sterilizer for heating and sterilization, keep a constant temperature at 100℃ for 9min, and after cooling, you will get the bottled tangerine red leaf and tremella fermented beverage.

[0062] The drink was evaluated by a 15-member sensory evaluation panel, and the results were: the drink exudes a sweet rose aroma, has a pleasant aroma, moderate sweetness, no bitterness, and is suitable for the general taste.

[0063] Example 2

[0064] (1) Preparation of Tremella liquid strain

[0065] Tremella liquid culture medium: glucose 60g / L, soy protein peptide 10g / L, yeast extract 5g / L, potassium dihydrogen phosphate (edible grade) 1g / L, magnesium sulfate (edible grade) 0.5g / L, adjust pH to 6.0.

[0066] Prepare 336 mL of culture medium and distribute it into two 1000 mL conical flasks, filling each with 168 mL of liquid. Sterilize at 121°C for 20 min. After cooling, inoculate each bottle with one Tremella fuciformis CGMCC 5.163 slant culture (purchased from China General Microbiological Culture Collection) and culture under shaking conditions at 23°C and 150 rpm for 222 h to obtain a Tremella fuciformis liquid culture.

[0067] (2) Fermentation of orange peel leaves

[0068] 5 kg of fresh tangerine peel leaves were weighed, cleaned, and placed in a high-speed homogenizer. 14 kg of purified water, 450 g of skim milk powder, 1400 g of glucose, and 14 g of food-grade potassium dihydrogen phosphate were added. The mixture was homogenized at 8000 r / min for 14 min to obtain material 1. Material 1 was then placed in a 30 L fully automatic fermenter and sterilized at 121°C for 20 min.

[0069] After material 1 is cooled, 336mL of the Tremella liquid culture obtained by the above culture is added, sterile air is introduced, and fermentation is started. During the fermentation process, the temperature is controlled at 23°C, the stirring speed is controlled at 90r / min, the ventilation ratio is controlled at 0.24-0.32vvm for fermentation 0-48h, the ventilation ratio is controlled at 0.32-0.48vvm for fermentation 49-96h, the ventilation ratio is controlled at 0.48-0.56vvm for fermentation 97-168h, the ventilation ratio is controlled at 0.56-0.64vvm for fermentation 169-216h, and the ventilation ratio is controlled at 0.48-0.56vvm for fermentation 217h and thereafter. When the fermentation time reaches 288h, the fermentation is terminated to obtain material 2.

[0070] (3) Solid-liquid separation

[0071] After fermentation, Material 2 was heated to 100°C using the fermenter's jacket and maintained at this temperature for 12 minutes. Material 2 was then placed in a high-speed homogenizer and homogenized at 8,000 rpm for 14 minutes. The homogenized material was then centrifuged at 8,000 rpm for 18 minutes, and 11.3 L of supernatant was collected.

[0072] The naringin content in the supernatant was detected by high performance liquid chromatography ([Reference] Zhou Guifen, Chen Suhong, Lü Guiyuan, Yan Meiqiu. Determination of naringenin in Dendrobium officinale by high performance liquid chromatography [J]. China Journal of Chinese Materia Medica, 2013, 38(4): 520-523.), and the result was 103.86 mg / L.

[0073] The limonene content in the supernatant was detected by gas chromatography ([Reference] Zhang Jiaying, Yin Yi, Wang Zhe, Sun Qihang, Li Hongjuan, Zhao Juanjuan. Determination of three volatile components in Evodia rutaecarpa by gas chromatography [J]. Chemical Analysis and Meterage, 2021, 30(9): 5-10.), and the result was 3.66 mg / L.

[0074] The citronellol content in the supernatant was detected by gas chromatography ([Reference] Wei Chaojun, Wang Huiwen, Zeng Hui, Shi Shaoran, Yang Aizhen. Analysis of citronellol and geraniol content in health vinegar [J]. Journal of Food Safety and Quality, 2018, 9(17): 4686-4692.), and the result was 9.72 mg / L.

[0075] The phenylethanol content in the supernatant was detected by gas chromatography ([Reference] Mo Xinliang, Yan Dabao, Teng Mingde. Qualitative and quantitative analysis of 2-phenylethanol in Chinese liquor [J]. Food Science and Technology, 2022, 47(7): 256-260.), and the result was 5.84 mg / L.

[0076] According to the method of the entry-exit inspection and quarantine industry standard of the People's Republic of China "Determination of Total Flavonoids in Export Food" (SN / T4592-2016), the total flavonoid content in the supernatant was tested, and the result was 0.57 g / L.

[0077] Take 200 mL of supernatant, add 800 mL of purified water, mix well, and perform ultrafiltration using a 360 Da ultrafiltration membrane. When the permeate volume is equal to 800 mL, end the ultrafiltration, take out the retentate, and detect the polysaccharide content in the supernatant in accordance with the "Spectrophotometric Method for Determination of Crude Polysaccharides in Edible Fungi" (NY / T 1676-2023), the agricultural industry standard of the People's Republic of China. The result is 25.48 g / L.

[0078] A series of concentrations of quinine hydrochloride standard solutions were prepared as controls for bitterness evaluation. A 15-person bitterness evaluation panel evaluated the bitterness of the supernatant. The bitterness of the supernatant was very weak, close to that of a 0.02 mg / L quinine hydrochloride solution.

[0079] (4) Mixing and filling

[0080] Take 10L of supernatant, add 500mg of neomethyl hesperidin dihydrochalcone and 1000g of maltitol, stir to dissolve, and then divide into glass bottles, 100mL of liquid in each bottle, seal with an automatic capping machine, put into a sterilizer for heating and sterilization, keep a constant temperature at 100℃ for 10min, and after cooling, you will get the bottled tangerine red leaf and tremella fermented beverage.

[0081] The drink was evaluated by a 15-member sensory evaluation panel, and the results were: the drink exudes a sweet rose aroma, has a pleasant aroma, moderate sweetness, no bitterness, and is suitable for the general taste.

[0082] Example 3

[0083] (1) Preparation of Tremella liquid strain

[0084] Tremella liquid culture medium: glucose 60g / L, soy protein peptide 10g / L, yeast extract 5g / L, potassium dihydrogen phosphate (edible grade) 1g / L, magnesium sulfate (edible grade) 0.5g / L, adjust pH to 6.0.

[0085] Prepare 256 mL of culture medium and dispense it into two 1000 mL conical flasks, filling each with 128 mL of liquid. Sterilize at 121°C for 20 min. After cooling, inoculate each bottle with one Tremella fuciformis CGMCC5.163 slant culture (purchased from China General Microbiological Culture Collection) and culture under shaking conditions at 25°C and 150 rpm for 210 h to obtain a Tremella fuciformis liquid culture.

[0086] (2) Fermentation of orange peel leaves

[0087] 5 kg of fresh tangerine peel leaves were weighed, cleaned, and placed in a high-speed homogenizer. 16 kg of purified water, 550 g of skim milk powder, 1600 g of glucose, and 16 g of food-grade potassium dihydrogen phosphate were added. The mixture was homogenized at a speed of 7000 r / min for 16 min to obtain material 1. Material 1 was then placed in a 30 L fully automatic fermenter and sterilized at 121°C for 16 min.

[0088] After material 1 is cooled, 256mL of the Tremella liquid culture obtained by the above culture is added, sterile air is introduced, and fermentation is started. During the fermentation process, the temperature is controlled at 25°C, the stirring speed is controlled at 70r / min, the ventilation ratio is controlled at 0.24-0.32vvm for fermentation 0-48h, the ventilation ratio is controlled at 0.32-0.48vvm for fermentation 49-96h, the ventilation ratio is controlled at 0.48-0.56vvm for fermentation 97-168h, the ventilation ratio is controlled at 0.56-0.64vvm for fermentation 169-216h, and the ventilation ratio is controlled at 0.48-0.56vvm for fermentation 217h and thereafter. When the fermentation time reaches 264h, the fermentation is terminated to obtain material 2.

[0089] (3) Solid-liquid separation

[0090] After fermentation, Material 2 was heated to 100°C using the fermenter's jacket and maintained at this temperature for 8 minutes. Material 2 was then placed in a high-speed homogenizer and homogenized at 7000 rpm for 16 minutes. The homogenized material was then centrifuged at 6000 rpm for 22 minutes, and 13.2 L of supernatant was collected.

[0091] The naringin content in the supernatant was detected by high performance liquid chromatography ([Reference] Zhou Guifen, Chen Suhong, Lü Guiyuan, Yan Meiqiu. Determination of naringenin content in Dendrobium officinale by high performance liquid chromatography [J]. China Journal of Chinese Materia Medica, 2013, 38(4): 520-523.), and the result was 91.18 mg / L.

[0092] The limonene content in the supernatant was detected by gas chromatography ([Reference] Zhang Jiaying, Yin Yi, Wang Zhe, Sun Qihang, Li Hongjuan, Zhao Juanjuan. Determination of three volatile components in Evodia rutaecarpa by gas chromatography [J]. Chemical Analysis and Meterage, 2021, 30(9): 5-10.), and the result was 3.19 mg / L.

[0093] The citronellol content in the supernatant was detected by gas chromatography ([Reference] Wei Chaojun, Wang Huiwen, Zeng Hui, Shi Shaoran, Yang Aizhen. Analysis of citronellol and geraniol content in health vinegar [J]. Journal of Food Safety and Quality, 2018, 9(17): 4686-4692.), and the result was 8.32 mg / L.

[0094] The phenylethanol content in the supernatant was detected by gas chromatography ([Reference] Mo Xinliang, Yan Dabao, Teng Mingde. Qualitative and quantitative analysis of 2-phenylethanol in Chinese liquor [J]. Food Science and Technology, 2022, 47(7): 256-260.), and the result was 5.41 mg / L.

[0095] According to the method of the People's Republic of China Entry-Exit Inspection and Quarantine Industry Standard "Determination of Total Flavonoids in Export Food" (SN / T4592-2016), the total flavonoid content in the supernatant was tested, and the result was 0.49 g / L.

[0096] Take 200 mL of supernatant, add 800 mL of purified water, mix well, and perform ultrafiltration using a 360 Da ultrafiltration membrane. When the permeate volume is equal to 800 mL, end the ultrafiltration, take out the retentate, and detect the polysaccharide content in the supernatant in accordance with the "Spectrophotometric Method for Determination of Crude Polysaccharides in Edible Fungi" (NY / T 1676-2023), the agricultural industry standard of the People's Republic of China. The result is 21.31 g / L.

[0097] A series of concentrations of quinine hydrochloride standard solutions were prepared as controls for bitterness evaluation. A 15-person bitterness evaluation panel evaluated the bitterness of the supernatant. The bitterness of the supernatant was very weak, close to that of a 0.02 mg / L quinine hydrochloride solution.

[0098] (4) Mixing and filling

[0099] Take 12L of supernatant, add 720mg of neomethyl hesperidin dihydrochalcone and 960g of maltitol, stir to dissolve, and then divide into glass bottles, 100mL of liquid in each bottle, seal with an automatic capping machine, put into a sterilizer for heating and sterilization, keep a constant temperature at 100℃ for 8min, and after cooling, you will get the bottled tangerine red leaf and tremella fermented beverage.

[0100] The drink was evaluated by a 15-member sensory evaluation panel, and the results were: the drink exudes a sweet rose aroma, has a pleasant aroma, moderate sweetness, no bitterness, and is suitable for the general taste.

[0101] Comparative Example 1

[0102] (1) Preparation of Tremella liquid culture medium

[0103] Tremella liquid culture medium: glucose 60g / L, soy protein peptide 10g / L, yeast extract 5g / L, potassium dihydrogen phosphate (edible grade) 1g / L, magnesium sulfate (edible grade) 0.5g / L, adjust pH to 6.0.

[0104] Prepare 256 mL of culture medium, put it into a 1000 mL conical flask, sterilize it at 121°C for 20 min, cool it down and set aside.

[0105] (2) Treatment of tangerine peel

[0106] 5 kg of fresh tangerine peel leaves were weighed, cleaned, and placed in a high-speed homogenizer. 16 kg of purified water, 550 g of skim milk powder, 1600 g of glucose, and 16 g of food-grade potassium dihydrogen phosphate were added. The mixture was homogenized at a speed of 7000 r / min for 16 min to obtain material 1. Material 1 was then placed in a 30 L fully automatic fermenter and sterilized at 121°C for 16 min.

[0107] After material 1 has cooled, add 256 mL of the Tremella fuciformis liquid culture medium prepared above, introduce sterile air, and start stirring. Control the temperature to 25°C, the stirring speed to 70 r / min, the ventilation ratio to 0.24-0.32 vvm for 0-48 hours, the ventilation ratio to 0.32-0.48 vvm for 49-96 hours, the ventilation ratio to 0.48-0.56 vvm for 97-168 hours, the ventilation ratio to 0.56-0.64 vvm for 169-216 hours, and the ventilation ratio to 0.48-0.56 vvm for 217 hours and thereafter. When the ventilation and stirring time reaches 264 hours, end the operation to obtain material 2.

[0108] (3) Solid-liquid separation

[0109] Using the fermenter's jacket, heat Material 2 to 100°C and maintain this temperature for 8 minutes. Then, place Material 2 in a high-speed homogenizer and homogenize at 7000 rpm for 16 minutes. Centrifuge the homogenized material at 6000 rpm for 22 minutes, collecting 13 L of supernatant.

[0110] The naringin content in the supernatant was detected by high performance liquid chromatography ([Reference] Zhou Guifen, Chen Suhong, Lü Guiyuan, Yan Meiqiu. Determination of naringenin content in Dendrobium officinale by high performance liquid chromatography [J]. China Journal of Chinese Materia Medica, 2013, 38(4): 520-523.), and the result was 88.65 mg / L.

[0111] The limonene content in the supernatant was detected by gas chromatography ([Reference] Zhang Jiaying, Yin Yi, Wang Zhe, Sun Qihang, Li Hongjuan, Zhao Juanjuan. Determination of three volatile components in Evodia rutaecarpa by gas chromatography [J]. Chemical Analysis and Meterage, 2021, 30(9): 5-10.), and the result was 1.62 mg / L.

[0112] The citronellol content in the supernatant was detected by gas chromatography ([Reference] Wei Chaojun, Wang Huiwen, Zeng Hui, Shi Shaoran, Yang Aizhen. Analysis of citronellol and geraniol content in health vinegar [J]. Journal of Food Safety and Quality, 2018, 9(17): 4686-4692.), and the result was 4.99 mg / L.

[0113] The phenylethanol content in the supernatant was detected by gas chromatography ([Reference] Mo Xinliang, Yan Dabao, Teng Mingde. Qualitative and quantitative analysis of 2-phenylethanol in Chinese liquor [J]. Food Science and Technology, 2022, 47(7): 256-260.), and the result was 0.26 mg / L.

[0114] According to the method of the entry-exit inspection and quarantine industry standard of the People's Republic of China "Determination of Total Flavonoids in Export Food" (SN / T4592-2016), the total flavonoid content in the supernatant was tested, and the result was 0.38 g / L.

[0115] Take 200 mL of supernatant, add 800 mL of purified water, mix well, and perform ultrafiltration using a 360 Da ultrafiltration membrane. When the permeate volume is equal to 800 mL, end the ultrafiltration, take out the retentate, and detect the polysaccharide content in the supernatant in accordance with the "Spectrophotometric Method for Determination of Crude Polysaccharides in Edible Fungi" (NY / T 1676-2023), the agricultural industry standard of the People's Republic of China. The result is 9.12 g / L.

[0116] A series of concentrations of quinine hydrochloride standard solutions were prepared as controls for bitterness evaluation. A 15-person bitterness evaluation panel evaluated the bitterness of the supernatant. The supernatant had a strong bitterness, close to that of a 0.045 mg / L quinine hydrochloride solution.

[0117] (4) Mixing and filling

[0118] Take 12L of supernatant, add 720mg of neomethyl hesperidin dihydrochalcone and 960g of maltitol, stir to dissolve, and then divide into glass bottles, 100mL of liquid in each bottle, seal with an automatic capping machine, put into a sterilizer for heating and sterilization, keep a constant temperature at 100℃ for 8min, and after cooling, you will get the bottled tangerine red leaf and tremella fermented beverage.

[0119] The beverage was evaluated by a 15-member sensory evaluation panel, and the results showed that the beverage had a pungent green smell, a distinct bitter taste and a sweet aftertaste, and most people did not like its aroma and taste.

[0120] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a fermented beverage of tangerine peel and tremella, characterized in that The steps include: (1) Fermentation of Citrus aurantium leaves: fresh Citrus aurantium leaves are cleaned, mixed with water, and milk powder, glucose and potassium dihydrogen phosphate are added. After homogenization, sterilization and cooling, Tremella fuciformis seed liquid is added, and fermentation is carried out under temperature control, ventilation and stirring conditions to obtain a fermentation liquid; (2) solid-liquid separation: heating and boiling the fermentation liquid obtained in step (1), homogenizing it while hot, and performing solid-liquid separation to obtain a supernatant; (3) Preparation: Take the supernatant obtained in step (2) and prepare it to obtain a fermented beverage of tangerine peel and tremella.

2. The preparation method according to claim 1, wherein: The water described in step (1) is purified water; The milk powder described in step (1) is skimmed milk powder; The Tremella fuciformis strain described in step (1) is CGMCC5.

163.

3. The preparation method according to claim 1, wherein: The amount of water in step (1) is calculated based on a mass ratio of Citrus aurantium to water of 1:(2.8-3.2); The amount of milk powder in step (1) is calculated based on 9-11% of the mass of the tangerine peel; The amount of glucose in step (1) is calculated based on 28-32% of the mass of the tangerine peel; The amount of potassium dihydrogen phosphate in step (1) is calculated based on 0.28-0.32% of the mass of the tangerine peel; The inoculation volume of the Tremella fuciformis seed liquid described in step (1) is 1.6% to 2.4% of the mass of the water described in step (1).

4. The preparation method according to claim 1, wherein: The homogenization conditions in step (1) are a rotation speed of 7000-8000 r / min and a time of 14-16 min; The sterilization conditions described in step (1) are 121° C. for 16 to 20 minutes; The heating and boiling conditions in step (2) are heating at 100° C. for 8 to 12 minutes; The homogenization conditions in step (2) are a rotation speed of 7000-8000 r / min and a time of 14-16 min; The solid-liquid separation method in step (2) is centrifugation.

5. The preparation method according to claim 1, wherein: The Tremella fuciformis seed liquid described in step (1) is prepared by the following steps: inoculating Tremella fuciformis strains into a sterilized Tremella fuciformis strain culture medium, and culturing the Tremella fuciformis strains to a logarithmic growth phase or a stable phase to obtain the Tremella fuciformis seed liquid.

6. The preparation method according to claim 1, wherein: The fermentation conditions described in step (1) are: temperature 23-25°C, stirring speed 70-90r / min, ventilation ratio controlled at 0.24-0.32vvm for 0-48h of fermentation, ventilation ratio controlled at 0.32-0.48vvm for 49-96h of fermentation, ventilation ratio controlled at 0.48-0.56vvm for 97-168h of fermentation, ventilation ratio controlled at 0.56-0.64vvm for 169-216h of fermentation, ventilation ratio controlled at 0.48-0.56vvm for 217h and thereafter, and fermentation period 264-288h.

7. The preparation method according to claim 1, wherein: The preparation in step (3) is prepared by adding a sweetener.

8. The preparation method according to claim 7, characterized in that: The sweeteners are neomethyl hesperidin dihydrochalcone and maltitol; The dosage of the neomethyl hesperidin dihydrochalcone is calculated based on a ratio of 50 to 60 mg of neomethyl hesperidin dihydrochalcone per liter of supernatant; The dosage of the maltitol is calculated based on 80-100 g of maltitol per liter of supernatant.

9. The preparation method according to any one of claims 1 to 8, characterized in that The method further comprises the following steps: (4) canning and sterilization.

10. A fermented beverage of tangerine peel and tremella, characterized by: The method is obtained by the preparation method according to any one of claims 1 to 9.