Fermented sour tea and preparation method thereof

Abstract

The invention belongs to the field of tea making methods, and discloses a novel fermented sour tea and a preparation method thereof. The invention uses Aspergillus niger RAF106 strain to make lightly fermented sour tea, and the method is short in time and simple in method. The free amino acid content of tea leaves obtained by fermentation by the method is significantly increased, the gallic acid content is increased and the tea leaves are rich in non-ester catechins, and the tea soup is weakly acidic and has obvious sweet aftertaste.

Description

Technical Field

[0001] The invention belongs to the field of tea making methods, and in particular relates to fermented sour tea and a preparation method thereof. Background Art

[0002] In China, sour tea is a fermented tea with ethnic characteristics in the minority areas of Yunnan Province, such as Bulang sour tea and Deang sour tea. Sour tea has the quality characteristics of golden soup color, fresh fragrance, and slightly sour taste. Sour tea contains catechins, amino acids, organic acids and other active substances, which make sour tea have physiological functions such as thirst quenching, heat relief, digestion, etc., as well as antioxidant, anti-anxiety, anti-tumor, anti-aging and immune regulation. The traditional production process of traditional sour tea is mainly earth pit production method and bamboo tube production method, which adopts anaerobic fermentation process. The production method is complicated and the contents of tea are dissolved more during the fermentation process; the fermentation time required for sour tea production is relatively long, generally ranging from 1 to 3 months, and natural fermentation and room temperature fermentation are adopted. Environmental factors have a great influence on the fermentation effect of sour tea, affecting the stability of its quality. These factors affect the industrialization and promotion of sour tea.

[0003] The dominant microorganisms lactic acid bacteria and yeast in the anaerobic fermentation process of sour tea produce a large amount of organic acids, which promote the sour aroma of sour tea. The main organic acids in sour tea are lactic acid, succinic acid, and citric acid. The increase in organic acid content can improve the antioxidant capacity of sour tea. On the other hand, the increase in organic acids can reduce the pH value of tea leaves. The acidic environment helps to improve the stability of polyphenols. At the same time, anaerobic fermentation contains a variety of gallic acid (GA) synthesis pathways, and the gallic acid content increases after fermentation. Aspergillus niger is a common species in the microbial flora of tea fermentation. It also has the ability to decompose organic matter to produce a variety of organic acids, such as citric acid, gallic acid, etc.

[0004] In addition to organic acids, catechins and free amino acids are also important flavor substances and bioactive substances in sour tea. Catechins are divided into non-ester catechins and ester catechins. Non-ester catechins include catechin (C), epicatechin (EC), and epigallocatechin (EGC). Ester catechins mainly include epicatechin gallate (ECG) and epigallocatechin gallate (EGCG). Ester catechins have stronger astringency and bitter taste than non-ester catechins, while non-ester catechins are easier to be absorbed by the human body. Free amino acids make the tea soup taste fresh, but the free amino acid content of tea generally decreases or only increases slightly after microbial fermentation. For example, a study used different Aspergillus and Penicillium to ferment tea, and the free amino acid decreased by 45% to 58% after the fermentation. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies in the prior art, the primary purpose of the present invention is to provide a method for preparing fermented sour tea; the method is to prepare fermented sour tea by using Aspergillus niger RAF106 strain, the method is short in time and simple in method, and the tea obtained by fermentation by the method has significantly increased organic acid and gallic acid content, significantly increased free amino acid content, and is rich in non-ester catechins, the tea soup has a slightly sour taste, and a distinct sweet aftertaste.

[0006] Another object of the present invention is to provide a fermented sour tea prepared by the above preparation method.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for preparing fermented sour tea, which uses Aspergillus niger strains to perform solid-state aerobic light fermentation, so that the contents of gallic acid and non-ester catechins in the tea are increased, the content of ester catechins is reduced, and the content of free amino acids is increased.

[0009] The method for preparing the fermented sour tea specifically comprises the following steps:

[0010] (1) adding a spore suspension of Aspergillus niger to distilled water used for tea leaves, wherein the amount of tea leaves is 30% to 45%, and then spraying the distilled water added with the spore suspension of Aspergillus niger evenly on the Yunnan large-leaf sun-dried green raw tea, using 1 mL of the spore suspension of Aspergillus niger for every 100 g of the Yunnan large-leaf sun-dried green raw tea on average, mixing evenly after spraying, and placing the mixture into a fermentation bag;

[0011] (2) placing the fermentation bag containing the Yunnan large-leaf sun-dried green tea in step (1) in a fermentation box set at a temperature of 40° C. to 45° C., and fermenting for 2 to 6 days under moisturizing conditions; taking out the fermented sun-dried green tea and drying it at 80° C. until the water content is less than 5%, thereby obtaining a lightly fermented tea rich in organic acids and with significantly increased amino acid content, wherein the tea soup is weakly acidic and has a distinct sweet aftertaste.

[0012] Preferably, the Aspergillus niger spore suspension in step (1) is prepared according to the following steps:

[0013] (a) 10 g of sun-dried green tea is added to 100 ml of boiling water, boiled for 10 min, filtered and diluted to 200 ml, 4.5 g of potato extract powder, 0.3 g of sucrose and 4 g of agar powder are added to the diluted tea water, and sterilized by high pressure steam at 121° C. for 15 min to obtain a modified potato dextrose agar medium;

[0014] (b) taking out the glycerol tube of Aspergillus niger RAF106 strain stored at -20°C, using a pipette to draw the Aspergillus niger spore suspension and applying it on a modified potato dextrose agar medium, or using an inoculation loop to spot the Aspergillus niger spore suspension on a modified potato dextrose agar medium, and culturing and activating it at a constant temperature of 30°C for 4 to 5 days;

[0015] (c) After the modified potato dextrose agar medium is full of spores, sterile water with a mass concentration of 0.1% Tween 80 is drawn and added to the modified potato dextrose agar medium to wash off the surface spores, and the washed spore suspension is drawn and transferred into a sterile centrifuge tube. The spores on the modified potato dextrose agar medium are washed several times; the spore concentration is counted using a hemocytometer, and the spore concentration is adjusted to 1 to 2×10 with sterile water with a mass concentration of 0.1% Tween 80. 8 / mL to obtain Aspergillus niger spore suspension.

[0016] Preferably, the Aspergillus niger spore suspension in step (1) is prepared according to the following steps: sterilizing the sun-dried green tea with high-pressure steam at 121° C. for 15 min, adding sterile water after cooling to rehydrate the tea to 35% to 45%, and using the improved Aspergillus niger spore enrichment medium as the medium; taking out the Aspergillus niger spore suspension in the glycerol tube of Aspergillus niger RAF106 strain stored at -20° C., inoculating it into the improved Aspergillus niger spore enrichment medium, and culturing it at a constant temperature of 30° C. for 1 to 2 weeks, turning the leaves every day for the first four days during this period, collecting and counting the spore powder after the cultivation, and then using Tween 80 with a mass concentration of 0.1% to prepare a spore suspension with a concentration of 1 to 2×10 8 / mL of Aspergillus niger spore suspension.

[0017] The sealing in step (1) is performed using six layers of gauze and a rubber band.

[0018] During the fermentation process of step (2), the tea leaves need to be turned over every day.

[0019] A fermented acid tea prepared by the above preparation method is characterized in that: the fermented acid tea is rich in organic acid content, and the amino acid content is increased by more than 14.9% compared with the amino acid content before fermentation, the gallic acid content is increased by more than 15 times compared with that before fermentation, and the content of non-ester catechins is increased by more than twice compared with the non-ester catechins content before fermentation.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] (1) The method of the present invention adopts an aerobic fermentation method, which is different from the anaerobic fermentation method of traditional sour tea production, and the fermentation time is shortened and the environmental requirements are reduced;

[0022] (2) The free amino acid content in the tea leaves obtained by the present invention is significantly increased, the tea soup is weakly acidic, the sweet taste of the tea soup is improved, the aftertaste is obvious, and the freshness is improved;

[0023] (3) After fermentation by the method of the present invention, the content of non-ester catechins in tea leaves is greatly increased, which is easier to absorb than ester catechins, thereby improving the bioavailability of catechins;

[0024] (4) The present invention can ferment summer and autumn tea to improve its flavor and quality, and the fermentation process is simple, the time used is short, and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the change chart of the types and contents of organic acids in tea leaves before and after fermentation;

[0026] Figure 2 This is a graph showing the changes in total phenol content (left) and amino acid content (right) in tea during the fermentation process. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0028] Embodiment 1:

[0029] 1. Preparation of improved PDA medium: Weigh 10g of sun-dried green tea and boil it in boiling water for 10 minutes. After cooling, dilute the volume to 200ml, add 4.5g of potato extract powder, 0.3g of sucrose, and 4g of agar powder, and sterilize it with high-pressure steam at 121℃ for 15min to prepare 200ml of improved PDA plate medium.

[0030] 2. Pure culture of Aspergillus niger: Take out the Aspergillus niger RAF106 strain stored at -20°C (the strain is disclosed in the patent No.: 201510085412.4, invention name "Method for producing epigallocatechin and gallic acid by transformation of Aspergillus niger whole cells") glycerol tube, use a pipette to draw 100 μl of Aspergillus niger spore suspension and spread it on the modified PDA plate culture medium, and culture and activate it at a constant temperature of 30°C for 4 to 5 days, and use it after the plate is full of spores.

[0031] 3. Preparation of Aspergillus niger spore suspension: Take out the plate full of spores and operate in a clean bench. Use a pipette to draw 0.1% Tween 80 on the plate, use a coating stick to scrape off the spores on the plate, and transfer the spore suspension to a 10ml centrifuge tube with a pipette. Shake evenly to obtain a spore suspension. Use a hemocytometer to count the spores and adjust the spore concentration to obtain a concentration of 1.18×10 8 / ml of Aspergillus niger spore suspension.

[0032] 4. The spore suspension is mixed with sun-dried green tea in a certain proportion: 500 g of Yunnan large-leaf sun-dried green tea (from Menghai County, Yunnan Province) is weighed. 225 ml of distilled water is needed to rehydrate the tea to 45%. Therefore, 5 ml of the Aspergillus niger spore suspension obtained in step 3 above is mixed with 220 ml of distilled water and added to a spray pot. After shaking evenly, the mixture is sprayed on the Yunnan large-leaf sun-dried green tea. After spraying, the bag mouth is covered with six layers of gauze and wrapped with a rubber band.

[0033] 5. High temperature fermentation: Put the fermentation bag containing Yunnan large-leaf sun-dried green tea into a fermentation incubator at 42°C, and place a fresh-keeping box filled with water under the fermentation bag to maintain the humidity inside the fermentation box. Turn the leaves every day and ferment for 6 days.

[0034] 6. Drying and packaging: Take out the fermented tea leaves, spread them out on a tray, untie the tea leaves entangled by the growth of mycelium to make them loose, put them into an 80℃ oven and dry them for 1 to 2 hours, turning them over every half an hour. The moisture content of the obtained tea leaves is less than 5%, and fermented acid tea is obtained.

[0035] Example 2

[0036] The fermented sour tea obtained according to the above Example 1 is weakly acidic, and the organic acid content is significantly increased. The pH value and organic acid content of this example were measured.

[0037] Method for determining the pH value of fermented acid tea: Take 3.0 g of tea sample with a tea-to-water ratio (mass-to-volume ratio) of 1:50, place in a 250 mL beaker, inject 150 mL of 80°C-90°C hot water, brew for 5 minutes, and take 5 mL of the tea soup for determination in a pH meter.

[0038] Determination of organic acid content:

[0039] The extraction was carried out according to the extraction method in GB / T8314-2013.

[0040] Flow rate: 0.8 mL / mL, column temperature 27.5°C, 210 nm

[0041] Table 1. HPLC determination of organic acids

[0042]

[0043] The pH measurement results are shown in Table 2. After fermentation, the pH of the tea soup decreased and became weakly acidic. Figure 1 It can be seen that the content of organic acids in tea leaves increased significantly by about 210 mg / g after fermentation. Among them, the content of lactic acid, acetic acid and citric acid increased greatly after fermentation.

[0044] Table 2. Changes in pH value of tea soup before and after fermentation

[0045]

[0046] Example 3

[0047] The fermented sour tea obtained in Example 1 has a bright yellow tea soup with a long aftertaste. The internal components thereof were determined to show that the total phenol and amino acid contents increased significantly after fermentation, the EC and EGC contents increased significantly, and the total amount of non-ester catechins increased significantly.

[0048] The determination of the total phenolic and catechin content of free amino acids was carried out according to GB / T8314-2013 "Determination of the total amount of free amino acids in tea" on the amino acid content of the samples before and after fermentation. The total phenolic content and catechin content were determined according to GB8313-2018 "Determination of the content of tea polyphenols and catechins in tea", and some improvements were made to the HPLC method as shown in Tables 3 and 4:

[0049] HPLC chromatographic conditions: chromatographic column: Waters XBridge C18; column temperature: 25°C; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 280 nm.

[0050] Table 3. HPLC mobile phase preparation

[0051]

[0052] Table 4. HPLC elution program

[0053]

[0054] The results of the test are as follows Figure 1 , and as shown in Tables 5 and 6, Figure 2 It can be seen that the total phenol content increased slightly after fermentation, while the free amino acid content increased by 77.28% compared with before fermentation, and the phenol-ammonia ratio decreased. Generally speaking, the lower the phenol-ammonia ratio, the fresher the taste of the tea soup. This may be the reason for the delicious and long-lasting taste of the tea soup after fermentation. According to Tables 5 and 6, the gallic acid content increased greatly, and the content after fermentation was more than 21 times higher than before fermentation. EGC and EC increased significantly after fermentation. From the results, it can be seen that during the constant high temperature fermentation process, the total amount of non-ester catechins increased by 7.131% after fermentation, and the total amount of ester catechins decreased by 4.223%.

[0055] Table 5. Changes in tea catechin content during fermentation

[0056]

[0057] Table 6. Comparison of the contents of two types of catechins before and after fermentation

[0058]

[0059] Example 4

[0060] The fermentation method of this embodiment refers to that of embodiment 1, except that the high temperature fermentation time is 2 days.

[0061] According to the preparation method of Aspergillus niger spore suspension, the concentration of spore suspension was 1.56×10 8 Pieces / ml.

[0062] Example 5

[0063] The fermented tea leaves obtained in Example 4 were subjected to the determination of catechins and free amino acids according to the determination method in Example 3.

[0064] The results are shown in Tables 7, 8 and 9. It can be seen from Tables 7 and 8 that the GA content in the tea leaves after fermentation for 2 days increased by about 15 times, the EGC and EC contents increased by about 2 times compared with the contents before fermentation, and the non-ester catechins and the total amount of catechins after fermentation increased significantly compared with the contents before fermentation. It can be seen from Table 9 that the free amino acid content of the tea leaves increased significantly. The free amino acid content of the tea leaves after fermentation was 5.321%, an increase of 33.16% compared with the contents before fermentation.

[0065] Table 7. Changes in tea catechin content during fermentation

[0066]

[0067] Table 8. Comparison of the contents of two types of catechins before and after fermentation

[0068]

[0069] Table 9. Comparison of free amino acid content in tea leaves before and after fermentation

[0070]

[0071] Example 6

[0072] The fermentation method of this embodiment refers to that of embodiment 1, except that the tea leaves used are sun-dried green tea leaves from large, old, or ancient tea trees, and the spore suspension concentration is 1.13×10 8 Pieces / ml.

[0073] Example 7

[0074] The fermentation method of this embodiment refers to Example 1, except that when the sun-dried green tea is mixed with the spore suspension, 200 ml of distilled water is needed to rehydrate the tea to 35%, so 4 ml of Aspergillus niger spore suspension is mixed with 196 ml of distilled water and added to a spray pot, shaken evenly and then sprayed onto the Yunnan large-leaf sun-dried green tea;

[0075] According to the preparation method of Aspergillus niger spore suspension, the concentration of spore suspension was 1.18×10 8 Pieces / ml.

[0076] Example 8

[0077] The free amino acid content of the fermented tea leaves obtained in Example 7 was determined according to the determination method in Example 3.

[0078] The results are shown in Table 10. The free amino acid content of tea leaves increased significantly after fermentation. The free amino acid content of tea leaves after fermentation was 7.477%, an increase of 36.89% compared with that before fermentation.

[0079] Table 10. Comparison of free amino acid content in tea leaves before and after fermentation

[0080]

[0081] Example 9

[0082] The fermentation method of this embodiment refers to that of embodiment 1, except that the tea leaves are placed in a fermentation box at 45°C for high temperature fermentation. The catechin and free amino acid contents are determined according to the method described in embodiment 3.

[0083] According to Tables 11 and 12, under high temperature fermentation at 45°C, the GA content after fermentation increased by about 39 times compared with that before fermentation, while the EGC and EC contents increased by 2 to 3 times compared with that before fermentation. The non-ester catechin content increased by about 3 times compared with that before fermentation. As shown in Table 13, the free amino acid content in tea leaves increased after fermentation, which increased by 14.98% compared with that before fermentation.

[0084] Table 11. Changes in catechin content in tea leaves before and after fermentation

[0085]

[0086] Table 12. Comparison of the contents of two types of catechins before and after fermentation

[0087]

[0088] Table 13. Free amino acid content of tea leaves before and after fermentation

[0089]

[0090] Example 10

[0091] The tea soup of Example 9 above was bright yellow in color, slightly sour in taste, and had a distinct sweet aftertaste. A sensory evaluation was conducted on this example.

[0092] Sensory evaluation method:

[0093] Take 3.0g of tea sample with a tea-to-water ratio of 1:50 (mass to volume ratio), place in a corresponding tea evaluation cup, fill with 80℃~90℃ hot water, cover, start timing, brew for 5 minutes, filter out the tea soup at an equal speed, leaving the tea leaves in the cup, and evaluate each item in the order of soup color, aroma, and taste.

[0094] Table 14. Sensory evaluation table

[0095]

[0096]

[0097] The sensory evaluation results of tea soup are shown in Table 15. It can be seen from the table that after fermentation, the tea soup, aroma and taste are all improved, the bitterness of the tea soup is reduced, the tea soup has a sour taste, and the sweetness lasts for a long time.

[0098] Table 15. Sensory evaluation results

[0099]

[0100] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing fermented sour tea, characterized in that: The method uses Aspergillus niger strains to perform solid-state aerobic light fermentation, so that the content of gallic acid and non-ester catechins in tea leaves increases, the content of ester catechins decreases, and the content of free amino acids increases; The method specifically follows the following steps: (1) adding a spore suspension of Aspergillus niger to distilled water used for tea leaves, wherein the amount of tea leaves is 30% to 45%, and then spraying the distilled water added with the spore suspension of Aspergillus niger evenly on the Yunnan large-leaf sun-dried green raw tea, using 1 mL of the spore suspension of Aspergillus niger for every 100 g of the Yunnan large-leaf sun-dried green raw tea on average, mixing evenly after spraying, and placing the mixture into a fermentation bag; (2) placing the fermentation bag containing the Yunnan large-leaf sun-dried green tea in step (1) in a fermentation box set at a temperature of 45° C., and fermenting for 2 to 6 days under moisturizing conditions; taking out the fermented sun-dried green tea and drying it at 80° C. until the water content is less than 5%, thereby obtaining a lightly fermented tea rich in organic acids and with a significantly increased free amino acid content, wherein the tea soup is weakly acidic and has a distinct sweet aftertaste; The Aspergillus niger strain is Aspergillus niger RAF106; The concentration of the Aspergillus niger spore suspension is 1 to 2×10 8 Pieces / mL.

2. The preparation method according to claim 1, characterized in that: The Aspergillus niger spore suspension in step (1) is prepared according to the following steps: (a) 10 g of sun-dried green tea is added to 100 ml of boiling water, boiled for 10 min, filtered and diluted to 200 ml, 4.5 g of potato extract powder, 0.3 g of sucrose and 4 g of agar powder are added to the diluted tea water, and sterilized by high pressure steam at 121° C. for 15 min to obtain a modified potato dextrose agar medium; (b) taking out the glycerol tube of Aspergillus niger RAF106 strain stored at -20°C, using a pipette to draw the Aspergillus niger spore suspension and applying it on a modified potato dextrose agar medium, or using an inoculation loop to spot the Aspergillus niger spore suspension on a modified potato dextrose agar medium, and culturing and activating it at a constant temperature of 30°C for 4 to 5 days; (c) After the modified potato dextrose agar medium is full of spores, Tween 80 with a mass concentration of 0.1% is pipetted and added to the modified potato dextrose agar medium to wash off the surface spores, and the washed spore suspension is pipetted and transferred into a sterile centrifuge tube. The spores on the modified potato dextrose agar medium are washed several times; the spore concentration is counted using a hemocytometer, and the spore concentration is adjusted to 1 to 2×10 8 / mL to obtain Aspergillus niger spore suspension.

3. The preparation method according to claim 1, characterized in that: The Aspergillus niger spore suspension in step (1) is prepared according to the following steps: sterilizing the sun-dried green tea with high-pressure steam at 121° C. for 15 min, adding sterile water to rehydrate the tea to 35% to 45% after cooling, and using the improved Aspergillus niger spore enrichment medium as the medium; taking out the Aspergillus niger spore suspension in a glycerol tube of Aspergillus niger RAF106 strain stored at -20° C., inoculating it into the improved Aspergillus niger spore enrichment medium, and culturing it at a constant temperature of 30° C. for 1 to 2 weeks, turning the leaves every day for the first four days during this period, collecting and counting the spore powder after the cultivation, and then using Tween 80 with a mass concentration of 0.1% to prepare a spore suspension with a concentration of 1 to 2×10 8 / mL of Aspergillus niger spore suspension.

4. The preparation method according to claim 1, characterized in that: During the fermentation process of step (2), the tea leaves need to be turned over every day.

5. A fermented sour tea prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The fermented acid tea is rich in organic acid, and the free amino acid content is increased by more than 14.9% compared with the free amino acid content before fermentation, the gallic acid content is increased by more than 15 times compared with that before fermentation, and the non-ester catechin content is increased by more than twice compared with that before fermentation.

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