Method for efficiently producing pu'er ripe tea through fermentation under time control

Abstract

The present application belongs to the technical field of tea fermentation, and discloses a method for efficiently producing Pu'er ripe tea through time sequence controlled fermentation, comprising the following steps: (S1) preparing Aspergillus niger and Aspergillus oryzae spore liquid; (S2) preparing a sterilized fermentation tank and putting pretreated sun-dried green tea into the fermentation tank; (S3) starting fermentation after inoculating the Aspergillus oryzae spore liquid, and then inoculating the Aspergillus niger spore liquid when the Aspergillus oryzae is planted on the leaves, and continuing fermentation; and (S4) ending fermentation and putting the tank, thereby obtaining the Pu'er ripe tea product. Through the time sequence controlled fermentation (especially the precise time sequence controlled fermentation), the present application not only significantly improves the quality of the Pu'er ripe tea, but also effectively shortens the fermentation period and improves the production efficiency. In addition, the time sequence controlled technology established by the present application is also applicable to liquid fermentation for producing tea products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of tea fermentation, and more particularly relates to a method for efficiently producing Pu'er ripe tea through time sequence control fermentation. BACKGROUND

[0002] Pu'er tea is a geographical indication product unique to Yunnan. According to the processing technology and quality characteristics of Pu'er tea, Pu'er tea is divided into two types: Pu'er raw tea and Pu'er ripe tea. The traditional Pu'er ripe tea is made from the unique large-leaf species of sun-cured tea in Yunnan through artificial fermentation by piling.

[0003] In recent years, researchers have made many improvements and innovations in the fermentation technology of Pu'er ripe tea:(1) In terms of fermentation form, it has gradually changed from traditional ground fermentation to off-ground small basket fermentation in a clean workshop, which has solved the hygiene problem of Pu'er ripe tea fermentation to some extent. However, in order to further realize controllable fermentation and improve product quality, the use of Pu'er ripe tea fermentation tank has become an extremely innovative fermentation form. It can realize controllable fermentation process temperature and humidity and complete the process of mechanical turning in a fully closed state, ensuring the safety and hygiene of Pu'er ripe tea products, reducing labor costs, and greatly shortening the production cycle. It is the future development trend of Pu'er ripe tea fermentation technology.(2) In terms of fermentation method, it has gradually changed from traditional natural fermentation, which relies on the existing microbial community in the fermentation environment or on the surface of Pu'er ripe tea, to artificial addition of beneficial microorganisms. It focuses on the types and community changes of microorganisms during fermentation. For example, the paper "Multi-omics research on microbial community and protein during Pu'er tea fermentation process" found that the main microorganisms in the early stage of fermentation were Aspergillus niger and Enterobacter; in the middle stage, they were blue fungi and thermophilic filamentous bacteria; and in the late stage, they were Bacillus. The paper "Microbial community structure and changes during solid-state fermentation of Pu'er tea" found that Aspergillus niger was the dominant species in the early stage of fermentation, and Bacillus and Arxula adeninivorans were the main dominant bacterial groups in the late stage of fermentation. It can be seen that during the fermentation process of Pu'er ripe tea, there are mainly three types of microorganisms: molds, bacteria, and yeasts. However, the microorganisms are not completely consistent at different stages of different studies, indicating that the entire fermentation process is extremely complex and important. The type and control of microorganisms will directly affect the conversion process of tea, and then affect the quality of tea, the stability of fermented products, and the fermentation period. However, there is little research on the types of microorganisms required at different stages of Pu'er tea fermentation and how to control them. Chinese patent application 201610097103.3 discloses a method for processing Pu'er tea by batch inoculation of microbial inoculant. It selects Aspergillus niger starter and high-temperature Mucor starter for open fermentation of tea leaves, uses twice-tidal water inoculation method, and controls the fermentation period to 12-15 days. However, it is unknown how the inoculated microorganisms work in this process, as the fermentation parameters are directly related to environmental temperature and humidity, which cannot be controlled. Therefore, it is also unknown how the inoculated microorganisms interact with the microorganisms in the tea leaves and the air. Chinese patent application 201811002772.3 discloses a method for processing Pu'er tea based on batch inoculation of microbial inoculant. It mainly uses batch inoculation of Aspergillus niger, Penicillium, Monascus, and Saccharomyces cerevisiae to establish a phased fermentation process. This method mainly uses the quantity advantage of inoculated microorganisms to improve the quality of Pu'er tea and shorten the period, and to some extent, inhibit the growth of harmful bacteria. However, it is unknown whether other batch inoculation methods have better effects.In addition, the above-mentioned reported technologies still only rely on sensory evaluation for judging the quality of fermentation products, and whether the content of tea polyphenols in Pu'er ripe tea reaches the national standard of less than 15% required by GB / T22111-2008 Geographical Indication Products Pu'er Tea is not given. Therefore, the fermentation effect is unknown, and the fermentation process cannot be accurately controlled. If a time sequence control fermentation technology (especially a precise time sequence control fermentation technology) can be established, it is of great significance to improve the fermentation efficiency and product quality of Pu'er ripe tea. SUMMARY

[0004] In view of the above defects or improvement needs of the prior art, the purpose of the present application is to provide a time sequence control fermentation method for efficiently producing Pu'er ripe tea, wherein the microbial time sequence control fermentation technology is determined by pretreating the sun-dried green tea raw material and combining the material conversion and microbial characteristics of the sun-dried green tea, that is, Aspergillus oryzae is inoculated first, the fermentation parameters suitable for Aspergillus oryzae are controlled, Aspergillus niger is inoculated after Aspergillus oryzae is planted on the leaves, and the fermentation parameters are further precisely controlled. Not only the quality of Pu'er ripe tea is significantly improved, but also the fermentation period is effectively shortened and the production efficiency is improved. The fermentation period is shortened from more than 20 days to 432 hours (i.e. 18 days) or even 384 hours (i.e. 16 days), and the content of theabrownin in the fermentation product is high (in the following examples, the content of theabrownin reaches 11.34-11.60%), which significantly saves time and economic cost and realizes the efficient production of Pu'er ripe tea. Moreover, the method of the present application is suitable for sun-dried green tea raw materials of different grades, and good effects can be achieved.

[0005] To achieve the above-mentioned purpose, according to one aspect of the present application, a time sequence control fermentation method for efficiently producing Pu'er ripe tea is provided, characterized in that it comprises the following steps:

[0006] (S1) preparing Aspergillus niger and Aspergillus oryzae spore solutions;

[0007] (S2) preparing a sterilized fermentation tank;

[0008] (S3) preparing a pretreated sun-dried green tea raw material, transferring the sun-dried green tea raw material into the fermentation tank, and inoculating the Aspergillus oryzae spore solution into the fermentation tank, and then starting fermentation by introducing sterile air; when Aspergillus oryzae is planted on the leaves, inoculating the Aspergillus niger spore solution, and continuing to introduce sterile air for fermentation;

[0009] (S4) when the tea polyphenol content in the Pu'er ripe tea obtained by fermentation in the fermentation tank meets the requirement of less than 15.0wt% in dry basis, the fermentation is ended; then, the moisture content of the Pu'er ripe tea after fermentation is reduced, and the Pu'er ripe tea product is obtained.

[0010] As a further preferred embodiment of the present application, in step (S3), the spore solution of A. niger is accessed, particularly at 49-72 hours after the start of fermentation.

[0011] As a further preferred embodiment of the present application, in step (S3), the fermentation process is maintained at a positive pressure, and the pressure is less than 0.05MPa. During the fermentation process, the humidity in the fermentation tank is 60%-90%. When the moisture content of the fermentation semi-product in the fermentation tank is lower than the preset requirement, sterile water is added to make the moisture content of the semi-product meet the preset requirement. Preferably, the sterile water is added through an atomizing nozzle. During the water adding process, the fermentation tank needs to be kept rotating at a speed of 2-10rpm. After the water adding process, the rotation of the fermentation tank is kept for 5-10min to make the added sterile water mix uniformly with the semi-product for further fermentation.

[0012] The weight of the sun-dried green tea raw material loaded in the fermentation tank with a capacity of 0.5 cubic meters is (2%-10%)*(the volume of the fermentation tank) tons / m 3 The fermentation corresponds to each stage in turn, and the preset requirements of the parameter conditions corresponding to each stage are as follows:

[0013] In the initial stage of fermentation, the temperature in the fermentation tank is controlled at 28-35℃, the aeration amount of sterile air is 0.5-0.8L / min, and the moisture content of the fermentation semi-product in the fermentation tank is 35-37%.

[0014] In the middle stage of fermentation, the temperature in the fermentation tank is controlled at 32-37℃, the aeration amount of sterile air is 2-2.5L / min, and the moisture content of the fermentation semi-product in the fermentation tank is 30-35%.

[0015] In the late stage of fermentation, the temperature in the fermentation tank is controlled at 37-45℃, the aeration amount of sterile air is 2.5-3.0L / min, and the moisture content of the fermentation semi-product in the fermentation tank is 28-30%.

[0016] In the final stage of fermentation, the temperature in the fermentation tank is controlled at 30-37℃, the aeration amount of sterile air is 2-2.5L / min, and the moisture content of the fermentation semi-product in the fermentation tank is 25-28%.

[0017] The initial stage of fermentation lasts for 96-144 hours, the middle stage of fermentation lasts for 96-144 hours, the late stage of fermentation lasts for 48-96 hours, and the final stage of fermentation lasts for 48-144 hours.

[0018] Preferably, the initial stage of fermentation starting after the access of the spore liquid of Aspergillus oryzae is to control the temperature of the fermenter at 28-32℃, then control the temperature of the fermenter at 32-35℃, and finally, after the access of the spore liquid of Aspergillus niger, control the temperature of the fermenter at 28-30℃.

[0019] More preferably, the initial stage of fermentation lasts for 120 hours, the middle stage of fermentation lasts for 120 hours, the late stage of fermentation lasts for 72 hours, and the final stage of fermentation lasts for 72 hours.

[0020] As a further preferred embodiment of the present application, the total fermentation time is not more than 432 hours; preferably, the total fermentation time is not more than 384 hours.

[0021] As a further preferred embodiment of the present application, in step (S3), the sterilization of the sun-dried rough tea material is specifically achieved by ultraviolet pretreatment and / or microwave pretreatment.

[0022] Preferably, the ultraviolet pretreatment is to irradiate the sun-dried rough tea material under an ultraviolet lamp, so that the sun-dried rough tea material meets the requirements of 0-20 MPN / 100g of Escherichia coli, 0-100 cfu / g of total bacteria, and 0-15 cfu / g of mold.

[0023] More preferably, the sun-dried rough tea material is irradiated by an ultraviolet lamp with a power of 30W for 20-30min, the surface is 30-40cm away from the ultraviolet lamp, the thickness is 5-10cm, and the area is 0.5-1m 2 The power of the ultraviolet lamp and the number of ultraviolet lamps are correspondingly set;

[0024] The microwave pretreatment is to treat at a temperature of 70-90℃ for 90-180 seconds.

[0025] Preferably, while the ultraviolet pretreatment or microwave pretreatment is being carried out, the sun-dried rough tea material is also pretreated by ozone.

[0026] As a further preferred embodiment of the present application, in step (S4), the reduction of the water content of the Pu'er tea after the end of fermentation corresponds to a tank discharge treatment, and the specific operation of the tank discharge treatment is as follows: after the end of fermentation, the temperature in the fermenter is controlled at 37-45℃, the sterile air is continuously introduced, the internal pressure of the fermenter is maintained at positive pressure, and the pressure is lower than 0.05MPa.

[0027] As a further preferred embodiment of the present application, in step (S1), the final concentration of each spore liquid meets the requirement of 1x10 6 ~1x10 8 cfu / mL.

[0028] As a further preferred embodiment of the present application, the amount of Aspergillus oryzae spore solution used in step (S3) is 0.05-1.00 mL per gram of sun-dried green tea raw material.

[0029] When the Aspergillus niger spore solution is inoculated, the amount of Aspergillus niger spore solution used is 0.05-1.00 mL per gram of sun-dried green tea raw material.

[0030] According to another aspect of the present application, a method for time-controlled efficient fermentation for producing a Pu'er tea liquid tea product is provided, which comprises the following steps:

[0031] (S1) preparing Aspergillus spores, and obtaining Aspergillus niger spore solution and Aspergillus oryzae spore solution, respectively;

[0032] (S2) preparing a sterilized fermentation tank and a sterilized TEA liquid medium;

[0033] (S3) inoculating the Aspergillus oryzae spore solution into the TEA liquid medium, and performing fermentation at 100-200 rpm and 28-37℃; when the Aspergillus oryzae spores germinate and mycelium grows, inoculating the Aspergillus niger spore solution, and continuing the fermentation;

[0034] (S4) ending the fermentation when the content of the theabrownin contained in the liquid in the fermentation tank is greater than 1300 μg / mL, thereby obtaining the Pu'er tea liquid tea product;

[0035] The sterilized TEA liquid medium refers to a tea soup obtained by sterilizing a plurality of tea leaves, adding them into boiling distilled water, extracting, and then filtering with gauze; preferably, 1 L of boiling distilled water is used for every 20 g of tea leaves; the sterilization is performed at 121℃ for 20 min; and the extraction time is not less than 2 hours.

[0036] As a further preferred embodiment of the present application, the inoculation of the Aspergillus niger spore solution in step (S3) is performed at 6-24 hours after the start of fermentation.

[0037] Preferably, in step (S4), the fermentation is ended at 36-48 hours after the start of fermentation.

[0038] Compared with the prior art, the present application establishes a time-controlled microbial fermentation technology, inoculates Aspergillus oryzae to sun-dried green tea raw material first, inoculates Aspergillus niger after the Aspergillus oryzae is planted on the leaves, controls the fermentation parameters, and interacts with the two kinds of bacteria, which not only significantly improves the quality of Pu'er tea, but also effectively shortens the fermentation period and improves the production efficiency. In addition, the time-controlled technology is also suitable for fermentation of the tea soup of Pu'er tea, and accordingly can improve the production efficiency of the liquid tea product.

[0039] Taking fermented Pu'er ripe tea as an example, the method of the present application uses microbial time control fermentation technology to add Aspergillus oryzae and Aspergillus niger spore liquid into the fermentation tank at the beginning of fermentation and during the fermentation process, and further controls the corresponding fermentation parameters, so that the corresponding Aspergillus spore liquid of each gram of sun-dried green tea raw material is 0.05-0.1 mL, the final concentration of the Aspergillus oryzae and Aspergillus niger spore liquid for the sun-dried green tea fermentation is 1x10 6 -1x10 8 cfu / mL, the fermentation temperature is controlled according to the growth characteristics of microorganisms in stages, and the tank pressure, humidity and other parameters of the fermentation tank during the entire fermentation process are optimally controlled, and water is supplemented in time to ensure the uniformity, controllability and quality stability of Pu'er tea fermentation. In addition, the present application also pre-treats the sun-dried green tea raw material at the source of fermentation by ultraviolet and microwave, and takes ultraviolet pre-treatment as an example, the raw material can be sterilized and reduced by ultraviolet lamp irradiation (similar to the previous achievement of the inventor), during ultraviolet pre-treatment, the ultraviolet lamp is 30W, during irradiation, the thickness of the Pu'er tea raw material is 5-10cm, the distance between the surface of the sun-dried green tea raw material and the ultraviolet lamp is 30-40cm, the irradiation area is 0.5-1m 2 , the irradiation time is 20-30min, and other power ultraviolet lamps can be used for the same conversion (for example, when the power of the ultraviolet lamp is 60W, the irradiation area is increased by 1 times to 1-2m 2 , and other parameter conditions remain unchanged); in addition to ultraviolet pre-treatment, microwave pre-treatment can also be used, at this time, the sun-dried green tea raw material is transferred into a microwave pre-treatment conveying device, the microwave device treatment temperature is 70-90℃, and the time is about 90-180 seconds (of course, ultraviolet pre-treatment and microwave pre-treatment can also be used together, for example, ultraviolet pre-treatment can be performed first, and then microwave pre-treatment can be performed). Modern biotechnology process is used, especially a horizontal solid fermentation tank designed by the inventor (other types of fermentation tanks for Pu'er tea fermentation are also applicable) is used for fermentation.

[0040] This invention delves into the effects and interrelationships of two microorganisms on the material transformation of Pu-erh tea, establishing a microbial time-controlled fermentation technology that significantly shortens the fermentation cycle of ripe Pu-erh tea and improves the quality of the product. This invention first inoculates with *Aspergillus oryzae*. The growth of *Aspergillus oryzae* in tea leaves and tea infusion is determined by whether it colonizes the leaves or whether the mycelium grows, guiding the precise determination of the inoculation time of *Aspergillus niger*. When *Aspergillus oryzae* has colonized the leaves (or the mycelium is growing), *Aspergillus niger* is then inoculated, achieving precise time-controlled fermentation. This method shortens the fermentation cycle from over 20 days to within 384 hours (i.e., 16 days), and the fermentation product has a high theabrownin content (11.34-11.60% in the examples below), significantly saving time and economic costs, and achieving efficient production of ripe Pu-erh tea. This invention particularly enables the establishment of a precise time-series controlled fermentation technology for microorganisms. After introducing Aspergillus oryzae spore liquid, the fermenter temperature is first controlled at 28℃-32℃ to facilitate spore germination, then at 32℃-35℃ to facilitate spore growth, and finally at 28-30℃ to facilitate enzyme production by Aspergillus oryzae. The Aspergillus niger spore liquid is introduced during the enzyme production stage of Aspergillus oryzae, and the fermenter temperature is controlled at 28-30℃, thus achieving even better precise control.

[0041] Similar to solid-state fermentation of tea, which converts polyphenols and other substances in tea into theabrownins, the timing control technology in this invention is also applicable to liquid fermentation for tea production. By utilizing the interaction between Aspergillus niger and Aspergillus oryzae, fermentation is promoted through sequential inoculation, enabling polyphenols and other substances in the tea soup to be converted into theabrownins efficiently and rapidly, achieving a similar effect.

[0042] In summary, this invention establishes a time-controlled fermentation method for the efficient production of ripe Pu-erh tea, specifically defining the control technology for the fermentation of sun-dried green tea. The ripe Pu-erh tea produced using this method is of high quality, has a good taste, a bright liquor color, and a brownish-red leaf base, further shortening the fermentation cycle and improving production efficiency. Similarly, this time-controlled technology is also applicable to liquid fermentation production of tea products, achieving similar results. Attached Figure Description

[0043] Figure 1 This is a comparison of the color of Pu-erh tea liquor under different fermentation conditions; among them, Figure 1 The "a" in the text corresponds to the color of the tea liquor obtained from sun-dried green tea (raw material) after the second-grade fresh leaves have undergone sun-drying. Figure 1 The "b" in the text corresponds to the color of the tea liquor obtained from sun-dried green tea (raw material) after 504 hours of conventional fermentation (without time control) of second-grade fresh leaves. Figure 1 The "c" in the text corresponds to the sun-dried green tea (raw material) obtained from second-grade fresh leaves after sun-drying, with precise time-controlled fermentation for 432 hours, resulting in a tea liquor color. Figure 1d corresponds to the soup color of the 384-hour precision timing control fermentation of the sun-cured green tea (raw material) obtained by sun-curing the five-grade fresh leaves.

[0044] Figure 2 is the conventional fermentation and timing control fermentation process diagram compared in the following examples. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0046] Based on the method of the present application, the timing inoculation can be carried out in the following steps to efficiently ferment and produce Pu'er ripe tea:

[0047] (1) Prepare Aspergillus sp.: Under sterile conditions, pick a little Aspergillus niger and Aspergillus oryzae for sun-cured green tea fermentation, inoculate on PDA agar medium, and cultivate at 28°C. After 5-7 days of cultivation of the two Aspergillus, prepare spore solution with appropriate sterile water, so that the final concentration of the spore solution is about 1x10 7 cfu / mL.

[0048] (2) Sterilize the fermentation tank: Sterilize the inside of the fermentation tank and the pipeline with high-temperature steam, and maintain at 121°C for 20 min. The fermentation tank used in the present example is a solid fermentation tank with precise controllable operation parameters developed by the inventors in the early stage (for detailed settings of the components in the fermentation tank, see Chinese patent application CN201920366567.9).

[0049] (3) Raw material pretreatment: According to the existing sun-curing process, sun-cure the Pu'er tea fresh leaves to obtain sun-cured green tea; then take the sun-cured green tea as the raw material, and pretreat 50 kg of sun-cured green tea with ultraviolet light. The ultraviolet lamp is 30W, the number of ultraviolet lamps is 20, the thickness of tea leaves is about 5cm, the distance between tea leaves and ultraviolet lamps is 30cm, the irradiation area of a single ultraviolet lamp is 0.5m 2 , the processing time is 30 min. After ultraviolet treatment, further transfer to a microwave pretreatment conveying device, the microwave device processing temperature is 90°C, and the time is about 90 seconds, so that the raw material contains 0-8 MPN / 100g of Escherichia coli, 0-40 cfu / g of total bacteria, and 0-6 cfu / g of mold.

[0050] (4) Fermentation tank inoculation and fermentation

[0051] Time sequence inoculation fermentation: 50 kg of pre-processed sun-dried raw tea is first transferred into a sterilized 0.5 cubic meter fermentation tank, and then 2.5-5.0 L of Aspergillus oryzae spore solution is introduced into the fermentation tank through a misting nozzle to start fermentation. When the Aspergillus oryzae is colonized on the leaves (generally at 49-72 hours after the start of fermentation, such as at 60 hours), 2.5-5.0 L of Aspergillus niger spore solution is introduced. In actual operation, trypan blue mycelium staining method can also be used to determine the growth of Aspergillus oryzae in the tea leaves and tea soup, to guide the accurate determination of the inoculation time of Aspergillus niger.

[0052] Each stage is fermented by controlling the following parameters, and the tank pressure is maintained as positive pressure during the entire fermentation process, and the pressure is lower than 0.05 MPa.

[0053] The temperature parameter in the fermentation tank during fermentation is controlled as shown in Table 1, the humidity in the fermentation tank is controlled at 60%-90%, the water content in the tea is detected every 48 hours, if the water content in the tea is lower than the water content corresponding to the stage, sterile water is added through a misting nozzle to maintain the water content in the tea at the corresponding stage. The water supplementing process keeps the fermentation tank rotating at a speed of 2 rpm, and after the water supplementing process is completed, the fermentation tank continues to rotate for 3 minutes to mix the added sterile water with the tea in the tank evenly.

[0054] (5) End of fermentation: take tea samples after 313 hours in the fermentation process, detect the content of tea polyphenols, 3 parallel samples are made for each sample, and the average value is taken as the result, when the content of tea polyphenols is <15%, the fermentation is ended.

[0055] (6) Tank discharge: when the fermentation is ended, the temperature in the fermentation tank is controlled at 37-45℃, sterile air is continuously introduced, the internal pressure of the fermentation tank is maintained as positive pressure, and the aeration amount is 5 L / min, when the water content in the tea is ≤15%, the fermented Pu'er ripe tea is discharged from the fermentation tank. The above operation process is shown in Figure 2 .

[0056] Table 1 Parameter control of different fermentation stages in the fermentation tank

[0057]

[0058] Note: "0-120 hours" is divided into 3 sub-stages, mainly considering that the spore germination temperature of Aspergillus oryzae is 28-32℃, the growth temperature is 32-35℃, the optimum enzyme production temperature is 28-30℃, and the growth temperature of Aspergillus niger is about 28℃. Therefore, in order to facilitate the fermentation of Aspergillus oryzae and Aspergillus niger, the initial fermentation stage is further divided into 3 sub-stages for precise timing control. Of course, it can also not be further divided, and the temperature in the fermenter is controlled at 28-35℃, the aeration rate of sterile air is 0.5-0.8 L / min, and the moisture content of the fermentation semi-finished product in the fermenter is 35-37%, and the timing control can be realized.

[0059] Example 1

[0060] This example focuses on the effect of Aspergillus niger and Aspergillus oryzae on material transformation in TEA liquid medium, including the following steps:

[0061] (1) Preparation of Aspergillus spores

[0062] Under sterile conditions, a small amount of Aspergillus niger and Aspergillus oryzae used for fermentation production of Pu'er ripe tea was picked and inoculated into PDA agar medium for 28℃ culture. After 5-7 days of culture of the two Aspergillus, an appropriate amount of sterile water was used to prepare spore solution, so that the final concentration of the spore solution was about 1x10 7 cfu / mL.

[0063] The TEA liquid medium used in the following was prepared as follows: 20g of tea leaves was sterilized at 121℃ for 20min. After sterilization, the tea leaves were added to 1L of boiling distilled water and extracted for 2 hours, then filtered with gauze and divided into triangular bottles. The above operations were all carried out in a clean bench.

[0064] (2) Aspergillus liquid fermentation

[0065] Aspergillus niger fermentation: 5.0mL of Aspergillus niger spore solution was inoculated into TEA liquid medium and fermented for 48 hours.

[0066] Aspergillus oryzae fermentation: 5.0mL of Aspergillus oryzae spore solution was inoculated into TEA liquid medium and fermented for 48 hours.

[0067] Aspergillus niger and Aspergillus oryzae co-fermentation: 2.5mL of Aspergillus niger and Aspergillus oryzae spore solution was inoculated into TEA liquid medium and fermented for 48 hours.

[0068] The fermentation temperature was 37℃, the rotation speed was 150rpm, 3 parallel samples were prepared for each sample, and the average value was taken.

[0069] Based on the fermentation, tea polyphenols, tea content results as shown in Table 2. Aspergillus fermentation tea soup tea polyphenol content of 214.51 μg / mL, the content of tea theobromine 1319.75 μg / mL; Aspergillus oryzae fermentation tea soup tea polyphenol content of 827.69 μg / mL, the content of tea theobromine 1537.67 μg / mL; Aspergillus niger and Aspergillus oryzae fermentation tea soup tea polyphenol content of 441.98 μg / mL, the content of tea theobromine 1779.12 μg / mL. Aspergillus fermentation process will consume more tea polyphenols, but the synthesis rate of tea theobromine is low; Aspergillus oryzae fermentation process retains more tea polyphenol content, and theobromine conversion synthesis amount is higher; and Aspergillus niger and Aspergillus oryzae together, theobromine conversion synthesis efficiency is the highest, and the consumption of tea polyphenols is between the consumption of Aspergillus niger and Aspergillus oryzae alone.

[0070] Table 2 Comparison of tea polyphenol and theobromine content in liquid fermentation with separate inoculation and mixed inoculation

[0071]

[0072] Note: μg / mL is suitable for the unit indicating the content of substances in liquid, representing the mass of tea polyphenol or theobromine per milliliter of tea soup; of course, their content can also be converted into % unit, i.e. the content of tea polyphenol or theobromine corresponding to each g of tea (conversion formula: 1 μg = 1 (μg / mL) * 1 mL, 1 μg / g = 1 μg / 1 g, 100% = 1 μg / g * 10 -6 % (where 1 μg = 10 -6 g).

[0073] Example 2

[0074] In order to make the fermentation process of Pu'er tea consume as little tea polyphenol as possible while making theobromine conversion synthesis amount large, this embodiment focuses on comparing the process and fermentation effect of time sequence inoculation fermentation technology (including precise time sequence control fermentation technology) and conventional fermentation, including the following steps:

[0075] (1) Prepare Aspergillus spores

[0076] Under sterile conditions, a small amount of Aspergillus niger and Aspergillus oryzae used for fermentation to produce Pu'er ripe tea was picked and inoculated into PDA agar medium for culture at 28°C. After the two Aspergillus were cultured for 5-7 days, an appropriate amount of sterile water was used to prepare spore solution, and the final concentration of the spore solution was about 1x10 7 cfu / mL.

[0077] (2) Sterilize the fermentation tank

[0078] The inside of the Pu'er tea fermentation tank and the pipeline are sterilized by high-temperature steam, and maintained at 121°C for 20 min.

[0079] (3) Raw material pretreatment

[0080] The 50 kg of sun-dried rough tea raw material is subjected to ultraviolet pretreatment, the ultraviolet lamp is 30 W, the number of ultraviolet lamps is 20, the thickness of the tea leaves is about 5 cm, the distance between the tea leaves and the ultraviolet lamp is 30 cm, the irradiation area of a single ultraviolet lamp is 0.5 m 2 , the processing time is 30 min, after ultraviolet treatment, the sun-dried rough tea raw material is further transferred to a microwave pretreatment conveying device, the microwave device processing temperature is 70°C, and the time is about 180 seconds, so that the coliform bacteria in the sun-dried rough tea raw material are 0-8 MPN / 100 g, the total number of bacteria is 0-40 cfu / g, and the mold is 0-6 cfu / g; the sun-dried rough tea obtained by sun-drying the two-stage fresh leaves is used as the raw material in this embodiment (the classification is based on GB / T 22111-2008 “Geographical Indication Product Pu'er Tea”, as shown in the table below) ;

[0081] (4) Fermentation of the fermentation tank

[0082] Conventional fermentation:

[0083] The 50 kg of sun-dried rough tea raw material (water content 10%) is first transferred into the sterilized 0.5 cubic meter fermentation tank, then 14.23 L of sterile water, 2.5 L of Aspergillus oryzae spore solution and 2.5 L of Aspergillus niger spore solution are simultaneously introduced into the fermentation tank through the atomizing nozzle, and mixed uniformly, so that the inoculation amount is 5%, the initial water content is 35%, and the fermentation starts.

[0084] Time sequence inoculation fermentation (including precise time sequence control fermentation): the 50 kg of sun-dried rough tea raw material (water content 10%) is first transferred into the sterilized 0.5 cubic meter fermentation tank, then 16.73 L of sterile water and 2.5 L of Aspergillus oryzae spore solution are introduced into the fermentation tank through the atomizing nozzle, and mixed uniformly, so that the inoculation amount is 2.5%, the initial water content is 35%, and the fermentation starts. The Aspergillus oryzae is planted on the leaves for 49-72 hours (60 hours are used in this embodiment), and then 2.5 L of Aspergillus niger spore solution (inoculation amount 2.5%) is introduced.

[0085] Each stage is fermented by controlling the following parameters, and the tank pressure is maintained at positive pressure during the whole fermentation process, and the pressure is lower than 0.05 MPa;

[0086] The temperature parameters in the fermentation tank during the conventional fermentation, the precise time sequence control fermentation and the time sequence inoculation fermentation process are shown in Table 3, Table 4 and Table 5 respectively, the humidity in the fermentation tank is controlled to be 60%-90%, the water content in the tea is detected by sampling every 48 hours, if the water content in the tea is lower than the water content at the corresponding stage, sterile water is added through the atomizing nozzle, so that the water content in the tea is maintained at the corresponding stage, the fermentation tank is kept rotating during the water supplementing process, the rotating speed is 2 rpm, after the water supplementing process, the fermentation tank is continuously rotated for 3 minutes, so that the added sterile water is uniformly mixed with the tea in the tank;

[0087] (5) Ending fermentation

[0088] The tea samples in the later stage of the fermentation process are taken, the content of tea polyphenols is detected, 3 parallel samples are prepared for each sample, and the average value is taken as the result, when the content of tea polyphenols is <15%, the fermentation is ended.

[0089] (6) Tank discharge

[0090] When the fermentation is ended, the temperature in the fermentation tank is controlled to be 37-45℃, sterile air is continuously introduced, the internal pressure of the fermentation tank is maintained as positive pressure, and the air flow is 5 L / min, when the water content in the tea is ≤15%, the fermented Pu'er ripe tea is discharged from the fermentation tank.

[0091] Table 3 Temperature control at different fermentation stages during conventional fermentation

[0092]

[0093] Table 4 Temperature control at different fermentation stages during precise time sequence control fermentation

[0094]

[0095] Table 5 Temperature control at different fermentation stages during time sequence inoculation fermentation

[0096]

[0097] Based on the above inoculation fermentation production method, the contents of tea polyphenols and theabrownins during the conventional fermentation and the precise time sequence control fermentation are shown in Table 6. During the conventional fermentation for producing Pu'er ripe tea, the content of tea polyphenols in the sun-dried green tea raw material (the soup color is shown as a in Figure 1 ) is reduced from 23.10% to 14.91%, the content of theabrownins is increased from 1.64% to 7.29%, and the fermentation period is 504 hours (i.e. 21 days, the soup color is shown as b in Figure 1 ). During the precise time sequence control fermentation for 432 hours (i.e. 18 days), the content of tea polyphenols in the Pu'er ripe tea is 14.78%, and the content of theabrownins is 11.60% (the soup color is shown as c in Figure 1 ).

[0098] The fermentation was controlled by time sequence for 480 hours (i.e. 20 days), and the content of tea polyphenol in Pu'er ripe tea was 14.85%, and the content of theabrownin was 9.37%. High-quality Pu'er ripe tea was obtained under the three conditions, without stacky taste, good taste, bright soup color, and brownish red leaf bottom, which was significantly higher than the national standard, but through the fermentation controlled by precise time sequence, the conversion efficiency of theabrownin was higher, and the fermentation period was shorter.

[0099] Table 6 Comparison of tea polyphenol and theabrownin contents in mixed inoculation and time sequence inoculation solid-state fermentation

[0100]

[0101] Example 3

[0102] The present embodiment is a method for efficiently producing Pu'er ripe tea based on precise time sequence controlled fermentation, comprising the following steps:

[0103] (1) Preparation of Aspergillus sp.

[0104] Under sterile conditions, a small amount of Aspergillus niger and Aspergillus oryzae used for fermentation to produce Pu'er ripe tea was picked and inoculated on PDA agar medium for culture at 28°C. After the two kinds of Aspergillus were cultured for 5-7 days, a certain amount of sterile water was used to prepare spore solution, and the final concentration of the spore solution was about 1x10 7 cfu / mL.

[0105] (2) Sterilization of fermentation tank

[0106] The inside of the fermentation tank for producing Pu'er ripe tea and the pipeline were sterilized by high-temperature steam, and maintained at 121°C for 20 min.

[0107] (3) Pretreatment of raw materials

[0108] The 50 kg of sun-dried green tea raw material was subjected to ultraviolet pretreatment, the ultraviolet lamp was 30 W, the number of ultraviolet lamps was 20, the thickness of the tea leaves was about 5 cm, the distance between the tea leaves and the ultraviolet lamp was 30 cm, the irradiation area of a single ultraviolet lamp was 0.5 m 2 , the treatment time was 30 min, after ultraviolet treatment, the raw material was further transferred to a microwave pretreatment conveying device, the microwave device treatment temperature was 70°C, and the time was about 180 seconds, so that the raw material had 0-8 MPN / 100 g of E. coli, 0-40 cfu / g of total bacteria, and 0-6 cfu / g of mold. The sun-dried green tea obtained by five-stage fresh leaves after sun-drying treatment was used as the raw material in the present embodiment;

[0109] (4) Inoculation and fermentation of fermentation tank

[0110] Precise timing control fermentation: 50 kg of pretreated sun-dried green tea raw materials (moisture content 10%) were first transferred into a sterilized 0.5 cubic meter fermentation tank, then 16.73 L of sterile water and 2.5 L of Aspergillus oryzae spore solution were connected to the fermentation tank through a misting nozzle, mixed evenly, so that the inoculation amount was 2.5%, the initial moisture content was 35%, and the fermentation started. When Aspergillus oryzae was cultured for 49 hours and colonized on the leaves, 2.5 L of Aspergillus niger spore solution was connected (inoculation amount 2.5%).

[0111] Each stage was fermented by controlling the following parameters, and the tank pressure was maintained at positive pressure during the entire fermentation process, and the pressure was lower than 0.05 MPa;

[0112] During the precise timing control fermentation process, the parameters in the fermentation tank were controlled as shown in Table 7, the humidity in the fermentation tank was controlled at 60%-90%, the moisture content in the tea leaves was detected every 48 hours, if the moisture content in the tea leaves was lower than the corresponding stage, sterile water was added through the misting nozzle, so that the moisture content in the tea leaves was maintained at the corresponding stage, the fermentation tank was kept rotating during the water supplementing process, the rotating speed was 2 rpm, after the water supplementing process was completed, the fermentation tank was continued to rotate for 3 minutes, so that the added sterile water was mixed evenly with the tea leaves in the tank;

[0113] (5) End of fermentation

[0114] Tea samples were taken at the later stage of the fermentation process, the content of tea polyphenols was detected, 3 parallel samples were prepared for each sample, and the average value was taken as the result, when the content of tea polyphenols was <15%, the fermentation was ended.

[0115] (6) Tank discharge

[0116] When the fermentation was completed, the temperature in the fermentation tank was controlled at 37-45℃, sterile air was continued to be introduced, the internal pressure of the fermentation tank was maintained at positive pressure, and the aeration amount was 5 L / min, when the moisture content in the tea leaves was ≤15%, the tank was discharged.

[0117] Table 7 Parameter control of different fermentation stages in the fermentation tank

[0118]

[0119] Based on the above inoculation fermentation production method, the contents of tea polyphenols and theabrownine were as shown in Table 8. When the precise timing control fermentation was carried out for 384 hours (i.e. 16 days), the content of tea polyphenols in the Pu'er ripe tea was reduced from 21.16% in the sun-dried green tea raw materials to 14.05%, and the content of theabrownine was increased from 2.59% to 11.34% (the color of the soup was as shown in d of Figure 1 ).

[0120] Table 8 Contents of tea polyphenols and theabrownine in precise timing control fermentation

[0121]

[0122] It can be seen that the method for producing Pu'er ripe tea by precise timing control fermentation can efficiently convert tea polyphenols in raw materials of different grades.

[0123] Example 4

[0124] The present embodiment is a method for efficiently producing Pu'er ripe tea based on precise timing control fermentation, comprising the following steps:

[0125] (1) Preparation of Aspergillus sp.

[0126] Under sterile conditions, a small amount of Aspergillus niger and Aspergillus oryzae used for fermentation to produce Pu'er ripe tea was picked and inoculated into PDA agar medium for culture at 28°C. After 5-7 days of culture of the two Aspergillus, a spore solution was prepared by adding an appropriate amount of sterile water, so that the final concentration of the spore solution was about 1x10 7 cfu / mL.

[0127] (2) Sterilization of the fermentation tank

[0128] The inside of the fermentation tank for producing Pu'er tea and the pipelines were sterilized by high-temperature steam, and maintained at 121°C for 20 min.

[0129] (3) Pretreatment of raw materials

[0130] The 50 kg of sun-dried raw tea was subjected to ultraviolet pretreatment, with 30 W ultraviolet lamps, 20 ultraviolet lamps, a tea thickness of about 5 cm, a distance between the tea and the ultraviolet lamps of 30 cm, an irradiation area of 0.5 m 2 per ultraviolet lamp, and a treatment time of 30 min. After ultraviolet treatment, the material was further transferred to a microwave pretreatment conveying device, and the microwave device was operated at a temperature of 70°C for about 180 seconds, so that the E. coli count was 0-8 MPN / 100 g, the total bacterial count was 0-40 cfu / g, and the mold count was 0-6 cfu / g. In the present embodiment, sun-dried raw tea obtained by sun-drying of five levels of fresh leaves was used as the raw material.

[0131] (4) Inoculation and fermentation in the fermentation tank

[0132] Precise timing control fermentation: 50 kg of pretreated sun-dried raw tea (moisture content 10%) was first transferred into a sterilized 0.5 cubic meter fermentation tank, and then 16.73 L of sterile water and 2.5 L of Aspergillus oryzae spore solution were inoculated into the fermentation tank through a misting nozzle, so that the inoculation amount was 2.5% and the initial moisture content was 35%. The fermentation was started. When the Aspergillus oryzae was planted on the leaves for 72 hours, 2.5 L of Aspergillus niger spore solution was inoculated (inoculation amount 2.5%).

[0133] Each stage is fermented by controlling the following parameters, and the tank pressure is maintained as positive pressure during the whole fermentation process, and the pressure is lower than 0.05 MPa.

[0134] The parameters in the fermentation tank during the time-sequential inoculation fermentation process are controlled as shown in Table 9, the humidity in the fermentation tank is controlled to be 60%-90%, the water content in the tea is detected by sampling every 48 hours, if the water content in the tea is lower than the water content corresponding to the stage, sterile water is added through a misting nozzle, so that the water content in the tea is maintained at the corresponding stage water content, the water supplement process makes the fermentation tank keep rotating, the rotating speed is 2 rpm, after the water supplement is completed, the fermentation tank is continuously rotated for 3 min, so that the added sterile water is uniformly mixed with the tea in the tank;

[0135] (5) Ending fermentation

[0136] Tea samples in the later stage of the fermentation process are taken, the content of tea polyphenols is detected, 3 parallel samples are prepared for each sample, and the average value is taken as the result, when the content of tea polyphenols is <15%, the fermentation is ended.

[0137] (6) Tank discharge

[0138] When the fermentation is ended, the temperature in the fermentation tank is controlled to be 37-45℃, sterile air is continuously introduced, the internal pressure of the fermentation tank is maintained as positive pressure, and the aeration amount is 5 L / min, when the water content in the tea is ≤15%, the fermented Pu'er ripe tea is discharged from the fermentation tank.

[0139] Table 9 Parameter control of different fermentation stages in the fermentation tank

[0140]

[0141]

[0142] Based on the above inoculation fermentation production method, the contents of tea polyphenols and theabrownine are as shown in Table 10. When the time-sequential inoculation fermentation is performed for 384 hours (i.e. 16 days), the content of tea polyphenols in the Pu'er ripe tea is reduced from 23.99% in the sun-dried green tea to 13.72%, and the content of theabrownine is increased from 2.01% to 10.55%.

[0143] Table 10 Contents of tea polyphenols and theabrownine in the precise time-sequential control solid-state fermentation

[0144]

[0145] It can be seen that the method for efficiently producing Pu'er ripe tea by precise time-sequential control fermentation in the application is suitable for a fermentation process with controllable temperature parameters.

[0146] In addition to the solid fermentation tank used in the above-mentioned embodiments, the timing control technology of the present application is also applicable to the production of tea products by liquid fermentation, in which case a liquid fermentation tank can be used. The essence of the solid-state fermentation process of tea leaves is that the extracellular enzymes (such as polyphenol oxidase, peroxidase, etc.) secreted by microorganisms convert the internal substances such as polyphenols in tea leaves, ultimately generating substances such as theabrownine. Similarly, the liquid-state fermentation process of tea soup is similar to the solid-state fermentation process, both of which are the conversion of polyphenols and other substances into theabrownine by extracellular enzymes secreted by microorganisms, and microorganisms are more likely to play a role in a liquid system. Therefore, the timing control technology of the present application is also applicable to the production of tea products by liquid fermentation.

[0147] In addition, the grading indicators of the fermented fresh leaves are shown in the following table.

[0148] Table: Fresh leaf grading indicators stipulated in GB / T 22111-2008 "Geographical Indication Product Pu'er Tea"

[0149] Grade Ratio of bud leaves Superior grade More than 70% of one bud and one leaf, and 30% or less of one bud and two leaves First grade More than 70% of one bud and two leaves, and 30% or less of other bud leaves of the same tenderness Second grade More than 60% of one bud and two or three leaves, and 40% or less of other bud leaves of the same tenderness Third grade More than 50% of one bud and two or three leaves, and 50% or less of other bud leaves of the same tenderness Fourth grade More than 70% of one bud and three or four leaves, and 30% or less of other bud leaves of the same tenderness Fifth grade More than 50% of one bud and three or four leaves, and 50% or less of other bud leaves of the same tenderness

[0150] The details of the present application not described in detail can be referred to the prior art (for example, can refer to Chinese patent 201810064877.5 "A stable and controllable high-quality pu'er tea and its fermentation production method").

[0151] Those skilled in the art will readily understand that the above description is only the preferred embodiments of the present application and is not intended to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for efficiently producing Pu'er ripe tea by controlling the fermentation time, characterized in that, The method comprises the following steps: (S1) preparing Aspergillus spores, respectively obtaining Aspergillus niger spore solution and Aspergillus oryzae spore solution; (S2) preparing a sterilized fermentation tank; (S3) preparing a pre-processed green tea raw material, transferring the green tea raw material into the fermentation tank, and introducing the Aspergillus oryzae spore solution into the fermentation tank, and introducing sterile air to start fermentation; when the Aspergillus oryzae is planted on the leaves, the Aspergillus niger spore solution is introduced, and the sterile air is continuously introduced for fermentation; (S4) when the tea polyphenol content in the fermented Pu'er tea obtained in the fermentation tank is less than 15.0wt%, the fermentation is ended; then, the water content of the fermented Pu'er tea after the fermentation is ended is reduced, and a Pu'er tea product is obtained; wherein, in step (S1), the final concentration of each spore solution satisfies 1x10 6 ~1x10 8 cfu / mL; In step (S3), the amount of Aspergillus oryzae spore solution corresponding to each gram of green tea raw material is 0.05-1.00mL; When the Aspergillus niger spore solution is introduced, the amount of Aspergillus niger spore solution corresponding to each gram of green tea raw material is 0.05-1.00mL; In step (S3), the fermentation process is maintained at a positive pressure, and the pressure is lower than 0.05MPa; during the fermentation process, the humidity in the fermentation tank is 60%-90%; when the water content of the fermentation semi-product in the fermentation tank is lower than the preset requirement, sterile water needs to be added to make the water content of the semi-product meet the preset requirement, The weight of the sun-dried rough tea raw material loaded in the fermenter according to the scale of 0.5 cubic meter capacity is (2%-10%)*(fermenter capacity volume) tons / m 3 ; the fermentation corresponds to each stage in turn and the parameter condition preset requirements of each stage are as follows: In the early stage of fermentation, the temperature in the fermentation tank is controlled to be 28-35℃, the ventilation amount of sterile air is 0.5-0.8L / min, and the water content of the fermentation semi-product in the fermentation tank is 35-37%; In the middle stage of fermentation, the temperature in the fermentation tank is controlled to be 32-37℃, the ventilation amount of sterile air is 2-2.5L / min, and the water content of the fermentation semi-product in the fermentation tank is 30-35%; In the late stage of fermentation, the temperature in the fermentation tank is controlled to be 37-45℃, the ventilation amount of sterile air is 2.5-3.0L / min, and the water content of the fermentation semi-product in the fermentation tank is 28-30%; In the final stage of fermentation, the temperature in the fermentation tank is controlled to be 30-37℃, the ventilation amount of sterile air is 2-2.5L / min, and the water content of the fermentation semi-product in the fermentation tank is 25-28%; The early stage of fermentation lasts for 96-144 hours, the middle stage of fermentation lasts for 96-144 hours, the late stage of fermentation lasts for 48-96 hours, and the final stage of fermentation lasts for 48-144 hours; The total fermentation time is not more than 432 hours; in the early stage of fermentation after the introduction of the Aspergillus oryzae spore solution, the temperature in the fermentation tank is first controlled to be 28-32℃, then controlled to be 32-35℃, and finally, after the introduction of the Aspergillus niger spore solution, the temperature in the fermentation tank is controlled to be 28-30℃; at the end of fermentation, the content of theabrownin in the fermentation product is 11.34-11.60%.

2. The method of claim 1, wherein, In step (S3), the introduction of the Aspergillus niger spore solution is specifically performed at 49-72 hours after the start of fermentation.

3. The method of claim 1, wherein, The sterile water is added through an atomizing nozzle, and the fermenter needs to be kept rotating during the water adding process, with a rotating speed of 2-10 rpm. After the water adding process, the fermenter needs to be kept rotating for 5-10 min to make the added sterile water mix with the semi-finished product uniformly and continue to ferment.

4. The method of claim 1, wherein, The initial fermentation stage lasts for 120 hours, the middle fermentation stage lasts for 120 hours, the late fermentation stage lasts for 72 hours, and the final fermentation stage lasts for 72 hours.

5. The method of claim 1, wherein, The total fermentation time is not more than 384 hours.

6. The method of claim 1, wherein, In step (S3), the sterilization of the sun-dried rough tea material is achieved through ultraviolet pretreatment and / or microwave pretreatment. The ultraviolet pretreatment is to irradiate the sun-dried rough tea material under an ultraviolet lamp, so that the sun-dried rough tea material meets the requirements of 0-20 MPN / 100g of E. coli, 0-100 cfu / g of total bacteria, and 0-15 cfu / g of mold. The microwave pretreatment is to treat the sun-dried rough tea material at a temperature of 70-90℃ for 90-180 seconds.

7. The method of claim 6, wherein, The sun-dried rough tea material is also pretreated with ozone while the ultraviolet pretreatment or microwave pretreatment is carried out.

8. The method of claim 6, wherein, The UV pretreatment is irradiating the raw material of green tea 20-30 min by a UV lamp with a power of 30 W, the surface is 30-40 cm away from the UV lamp, the thickness is 5-10 cm, and the area is 0.5-1 m 2 The power and the number of the UV lamps are set correspondingly.

9. The method of claim 1, wherein, In step (S4), the reduction of the water content of the Pu'er tea after the fermentation is ended corresponds to a tank discharge treatment, and the specific operation of the tank discharge treatment is as follows: after the fermentation is ended, the temperature in the fermenter is controlled to be 37-45℃, the sterile air is continuously introduced, the internal pressure of the fermenter is maintained to be positive, and the pressure is lower than 0.05 MPa.

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