Method for improving fermentation of Pu'er ripe tea by introducing black-brown microbial community of Liupao tea

Abstract

The present application relates to a kind of methods for introducing Liupu tea black brown microbial population to improve Pu'er ripe tea pile fermentation, belong to the technical field of tea fermentation, comprising the following steps: S1, screening purification and compound synergic domestication of Liupu tea black brown core microbial population;S2, compound enzymatic hydrolysis pretreatment of Pu'er raw tea, using the compound enzyme solution secreted by domesticated compound functional microbial population to perform mild enzymatic hydrolysis on Yunnan sun-dried raw tea, then adjust moisture content and moisturize tea;S3, tea pile gradient layering targeted inoculation, pile up Pu'er raw tea after pretreatment and divide into pile core, middle layer and surface layer, respectively inoculate gradient compound microbial inoculants adapted to the microenvironment of each part;S4, intelligent dynamic regulation and control of pile microenvironment based on Internet of Things;S5, pile-Liupu tea characteristic basket storage coupled post-ripening;S6, differential post-processing of flavor type.The present application eliminates metabolic competition between strains, and allows the core microbial population to form a stable symbiotic metabolic system.

Description

Technical Field

[0001] This invention relates to the field of tea fermentation technology, and in particular to a method for improving the pile fermentation of ripe Pu-erh tea by introducing black-brown bacteria from Liubao tea. Background Technology

[0002] As a typical representative of post-fermented dark tea, Pu'er ripe tea traditionally adopts a natural large-pile fermentation process, relying on natural microbial flora and human experience to control the fermentation process. This process has problems such as a long fermentation cycle (40-60 days), uneven fermentation of tea piles, and large batch-to-batch quality differences. Moreover, the high temperature and high humidity fermentation environment is prone to the growth of miscellaneous bacteria, resulting in a musty or musty smell in the finished tea. New tea needs to be stored for a long time to "nourish" and dissipate the smell before it can be consumed. At the same time, the fermentation process requires manual turning of the pile 4-6 times, which is labor-intensive and has low production efficiency, becoming the core bottleneck for the modernization of the Pu'er ripe tea industry.

[0003] Existing technologies have attempted to introduce the dark brown microbial community of Liubao tea into the fermentation of ripe Pu'er tea, but most of these methods employ a single-strain screening and mixed one-time inoculation approach. This approach suffers from drawbacks such as low colonization rate of the core microbial community in the Pu'er tea pile, intense metabolic competition among different microbial strains, and difficulty in fully leveraging synergistic effects. Furthermore, the fermentation parameters are mostly fixed and controlled in stages, which cannot match the real-time metabolic patterns of the microbial community, easily leading to local microbial inactivation and insufficient fermentation. At the same time, existing solutions only focus on modifying the fermentation process and do not take into account the post-fermentation advantages of Liubao tea in cellars, resulting in low integration and poor persistence of the distinctive flavors of Liubao tea (areca aroma, fungal aroma) with the aged aroma of ripe Pu'er tea, and also failing to achieve targeted cultivation of the aroma type of ripe Pu'er tea. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the background art by proposing a method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black brown bacteria to eliminate metabolic competition between microbial species and allow the core microbial community to form a stable symbiotic metabolic system.

[0005] The technical solution of this invention: A method for improving the pile fermentation of Pu'er ripe tea by introducing black-brown microorganisms from Liubao tea, comprising the following steps: S1. Screening, purification, and synergistic domestication of the dark brown core microbial community of Liubao tea: First, core strains of Aspergillus niger, Mucor, and Chaetomium globosum were screened and purified from traditional tea cellar / fermentation / cellar-aged tea samples of Liubao tea from Wuzhou, Guangxi. They were then subjected to 10-15 generations of basic co-culture domestication using a mixed extract medium of Pu'er and Liubao tea. Then, based on the aroma cultivation goal, aroma-targeting bacteria were incorporated for 3-5 generations of targeted co-culture domestication to obtain aroma-specific composite functional microbial communities. S2. The compound enzymatic pretreatment of Pu'er tea involves using a compound enzyme solution secreted by domesticated compound functional bacteria to perform mild enzymatic hydrolysis on Yunnan sun-dried green tea, followed by adjusting the moisture content and moistening the tea. S3. Gradient layered targeted inoculation of tea piles: The pre-treated Pu'er tea is piled into three layers: the core, the middle layer, and the surface layer. Gradient compound microbial agents adapted to the microenvironment of each part are inoculated separately. S4. Based on the Internet of Things, intelligent dynamic regulation of the microenvironment of tea piles for fermentation is carried out. Multi-dimensional sensors are arranged in various parts of the tea pile to monitor temperature, humidity, oxygen content and pH value in real time. Combined with the microbial community metabolism law model, the fermentation parameters are dynamically regulated. The entire fermentation process takes 17-25 days and the pile is turned 1-2 times. S5. Fermentation-Liubao Tea Special Basket Storage and Post-Ripe Processing: The fermented tea leaves are packed into bamboo baskets and placed in a micro-environment similar to Liubao tea storage for short-term post-ripening. S6. Aroma Differentiation Post-processing: Low-temperature differentiated drying, grading and sorting, and aroma-fixing aging of the tea leaves after cellar storage to produce finished Pu-erh ripe tea; The aroma-targeting bacteria are Xeromyces dry yeast of the fungal aroma type, or a combination of Wallermi and Debali yeast of the light areca aroma type.

[0006] Preferably, the Pu'er-Liubao tea mixed extract culture medium in step S1 is prepared by mixing Yunnan sun-dried green tea extract and Liubao tea fermentation extract at a volume ratio of 3:1, the pH of the culture medium is adjusted to 5.5-6.0, and the inoculation volume ratio of Aspergillus niger, Mucor, and Chaetomium globosum acclimatum in the basic co-culture is 8:1:1.

[0007] Preferably, the compound enzyme solution in step S2 contains cellulase, pectinase, and hemicellulase, and is sprayed by atomization at 0.5%-1% of the weight of Pu'er tea, with an enzymatic hydrolysis time of 2-3 hours; after enzymatic hydrolysis, the moisture content of the Pu'er tea is adjusted to 28%-30%, and the tea is moistened for 2-3 hours.

[0008] Preferably, the height of the tea pile in step S3 is 0.8-1m, and the thickness of the core, middle layer and surface layer is 20cm, 40-50cm and 20cm respectively; the inoculation amount of the gradient composite microbial agent is 8%-10% of the weight of the tea in the core, 6%-8% in the middle layer and 5%-6% in the surface layer, and ventilation holes are reserved in the core.

[0009] Preferably, in step S4, three sets of multi-dimensional sensors are arranged in the center, middle layer, and surface layer of the tea pile. The microbial community metabolic model divides fermentation into four stages, with the following control parameters for each stage: Establishment period (1-3 days): temperature 28-32℃, oxygen content 15%-20%, pH 5.5-6.0; Propagation period (4-7 days): center temperature 35-38℃, oxygen content 18%-22%, middle / surface layer temperature 30-35℃; Core metabolic period (8-15 days): center temperature 32-35℃, oxygen content 10%-15%, middle layer oxygen content 8%-10%, surface layer 3%-5%, pH 4.5-5.0; Post-metabolic ripening period (16-17 / 25 days): temperature in all parts 25-30℃, oxygen content 3%-5%, humidity 50%-60%.

[0010] Preferably, the triggering condition for intelligent turning of the tea pile in step S4 is that the temperature difference between different parts of the tea pile is ≥5℃, and the intelligent determination criteria for the fermentation endpoint are that the temperature of different parts of the tea pile returns to room temperature, the pH value stabilizes at 4.5-5.0, and the content of aged aroma and characteristic aroma substances reaches a preset threshold.

[0011] Preferably, in step S5, the moisture content of the ripe tea material is adjusted to 20%-25% before cellar storage, and the parameters of the microenvironment for imitating Liubao tea cellar storage are: temperature 22-25℃, relative humidity 65%-70%, and micro-oxygen content 3%-5%; the cellar storage time is 3-5 days for pure aged aroma ripe tea and 7-10 days for fungal aroma and light areca aroma ripe tea.

[0012] Preferably, the inoculation ratio of the aroma-targeted co-culture and domestication microbial groups in step S1 is: 9:1 for the floret aroma-based compound microbial group: Xeromyces dry yeast, and 8.5:1:0.5 for the light areca aroma-based compound microbial group: Wallerian: Debarry yeast; the concentration of the gradient compound microbial agent in step S3 is: 10^7-10^8 CFU / mL for the core layer agent, 10^6-10^7 CFU / mL for the middle layer agent, and 10^5-10^6 CFU / mL for the surface layer agent, and the proportion of aroma-targeted bacteria in the surface layer agent is increased by 10%-15% compared with the basic compound microbial group.

[0013] Preferably, the temperature of the low-temperature differentiated drying in step S6 is: 45-50℃ for pure aged aroma ripe tea, and 40-45℃ for arugula aroma and light areca aroma ripe tea; the moisture content of the tea leaves after drying is controlled to be ≤12% for pure aged aroma and ≤11% for arugula aroma and light areca aroma; the aroma aging time is 3-5 days for pure aged aroma and 7-10 days for arugula aroma and light areca aroma.

[0014] Preferably, the finished Pu-erh ripe tea meets the following quality indicators: microbiological indicators are free of aflatoxin and ochratoxin, pathogenic bacteria such as Salmonella and Staphylococcus aureus, and the total bacterial count is ≤10^4 CFU / g; physicochemical indicators are: pure aged aroma type theabrownin ≥8.0% and tea polyphenols ≤15.0%, fungal aroma type theabrownin ≥8.5% and tea polyphenols ≤14.5%, and light areca aroma type theabrownin ≥9.0% and tea polyphenols ≤14.0%; sensory indicators are free of musty, moldy, and astringent smells.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects: In this invention, the metabolic competition between microbial strains is eliminated through the composite synergistic domestication of Pu'er-Liubao tea mixed extract culture medium, allowing the core microbial community to form a stable symbiotic metabolic system. Combined with gradient stratified targeted inoculation, the colonization rate of the microbial community in the Pu'er tea pile is increased, completely solving the problem of uneven fermentation.

[0016] The compound enzymatic hydrolysis pretreatment accelerates the colonization of microorganisms and the transformation of tea substances. The Internet of Things intelligent dynamic control allows the fermentation parameters to be precisely matched with the metabolic patterns of microorganisms, shortening the fermentation cycle to 17-25 days, reducing the number of turnings to 1-2 times, reducing the intensity of manual labor, and greatly improving the efficiency of industrial production.

[0017] By using aroma-targeted domestication and gradient microbial agent formulation design, three types of Pu-erh ripe tea—pure aged aroma, fungal aroma, and light areca nut aroma—can be precisely produced. These teas have distinct aroma characteristics, meet diverse market demands, and enhance product added value.

[0018] The combination of fermentation and storage in the cellar, a characteristic process of Liubao tea, allows the distinctive flavor of Liubao tea to deeply blend with the aged aroma of Pu'er tea. The finished tea has a full and layered aroma, and the aroma substances are more stable after the secondary metabolism during cellar storage.

[0019] The complex and dominant microbial community quickly occupies the ecological niche of the tea pile, effectively inhibiting the growth of miscellaneous bacteria. The finished tea has no piled, musty, or astringent smell. The tea soup is bright red, mellow and smooth, with a significant sweet aftertaste. The content of theaflavins is significantly increased, while the content of irritating substances such as tea polyphenols is reduced. Moreover, all the microbial communities used are food-grade safe strains. No mycotoxins or pathogens were detected in the finished tea, which meets the highest national standards for tea food safety.

[0020] The real-time monitoring by multi-dimensional sensors of the Internet of Things and the dynamic regulation by the microbial community metabolism model have replaced manual experience judgment, reducing the batch-to-batch quality difference rate during mass production and realizing the standardization and intelligentization of Pu'er ripe tea fermentation. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the structure of an embodiment of the present invention. Detailed Implementation

[0023] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0024] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0025] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it an embodiment that is mutually exclusive, either alone or selectively, with other embodiments.

[0026] Example 1 like Figure 1 As shown, the present invention proposes a method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial communities, which includes the following steps: S1. Screening, purification, and synergistic domestication of the dark brown core microbial community of Liubao tea: First, core strains of Aspergillus niger, Mucor, and Chaetomium globosum were screened and purified from traditional tea cellar / fermentation / cellar-aged tea samples of Liubao tea from Wuzhou, Guangxi. They were then subjected to 10-15 generations of basic co-culture domestication using a mixed extract medium of Pu'er and Liubao tea. Then, based on the aroma cultivation goal, aroma-targeting bacteria were incorporated for 3-5 generations of targeted co-culture domestication to obtain aroma-specific composite functional microbial communities. S2. The compound enzymatic pretreatment of Pu'er tea involves using a compound enzyme solution secreted by domesticated compound functional bacteria to perform mild enzymatic hydrolysis on Yunnan sun-dried green tea, followed by adjusting the moisture content and moistening the tea. S3. Gradient layered targeted inoculation of tea piles: The pre-treated Pu'er tea is piled into three layers: the core, the middle layer, and the surface layer. Gradient compound microbial agents adapted to the microenvironment of each part are inoculated separately. S4. Based on the Internet of Things, intelligent dynamic regulation of the microenvironment of tea piles for fermentation is carried out. Multi-dimensional sensors are arranged in various parts of the tea pile to monitor temperature, humidity, oxygen content and pH value in real time. Combined with the microbial community metabolism law model, the fermentation parameters are dynamically regulated. The entire fermentation process takes 17-25 days and the pile is turned 1-2 times. S5. Fermentation-Liubao Tea Special Basket Storage and Post-Ripe Processing: The fermented tea leaves are packed into bamboo baskets and placed in a micro-environment similar to Liubao tea storage for short-term post-ripening. S6. Aroma Differentiation Post-processing: Low-temperature differentiated drying, grading and sorting, and aroma-fixing aging of the tea leaves after cellar storage to produce finished Pu-erh ripe tea; The aroma-targeting bacteria are Xeromyces dry yeast of the fungal aroma type, or a combination of Wallermi and Debali yeast of the light areca aroma type.

[0027] The compound enzyme solution mentioned in step S2 contains cellulase, pectinase, and hemicellulase. It is sprayed via atomization at 0.5%-1% of the weight of the Pu-erh tea, and the enzymatic hydrolysis time is 2-3 hours. After enzymatic hydrolysis, the moisture content of the Pu-erh tea is adjusted to 28%-30%, and the tea is rinsed for 2-3 hours. The Pu-erh tea is then lightly enzymatically hydrolyzed using the compound enzyme solution secreted by the domesticated compound functional microbial community, breaking down the tea cell walls and accelerating microbial colonization and material transformation. Specific operation details are as follows: Enzyme solution preparation: Extract the complex enzyme solution secreted by the domesticated complex functional bacterial community. This enzyme solution contains cellulase, pectinase and hemicellulase, which are the core enzymes for the degradation of tea macromolecules. Enzymatic hydrolysis: Select Yunnan sun-dried green tea that meets the standards, after sieving, removing stems and impurities, dilute the compound enzyme solution at 0.5%-1% of the weight of the Pu'er tea and spray it onto the surface of the tea by atomization, stir evenly, and place it in an environment of 25-30℃ and 60% humidity for 2-3 hours for enzymatic hydrolysis. Moisturizing the tea: After enzymatic hydrolysis, spray the raw tea with clean water to adjust its moisture content to 28%-30% (it should be able to be squeezed into a ball without dripping water and easily crumble when released). Then let the tea stand for 2-3 hours to allow the moisture to be evenly distributed in the tea leaves, thus completing the pretreatment.

[0028] In step S3, the height of the tea pile is 0.8-1m, and the thickness of the core, middle layer and surface layer is 20cm, 40-50cm and 20cm respectively. The inoculation amount of the gradient compound microbial agent is 8%-10% of the weight of the tea in the core, 6%-8% in the middle layer and 5%-6% in the surface layer, and ventilation channels are reserved in the core.

[0029] Based on the microenvironmental characteristics of different parts of the tea pile, a gradient compound microbial agent is inoculated in layers to achieve precise colonization and zoned metabolism of the microbial community. Specific operations are as follows: Tea pile construction: In a clean fermentation workshop, the pre-treated Pu'er tea is piled up in layers to form a fermentation pile with a height of 0.8-1m. The fermentation pile is divided into three areas: the core, the middle layer, and the surface layer, with a thickness of 20cm (core), 40-50cm (middle layer), and 20cm (surface layer), respectively. Gradual inoculation: A layered inoculation method is adopted, inoculating each area of ​​the fermentation pile with a suitable gradient compound microbial agent. The inoculation amount is: 8%-10% of the weight of tea leaves in the core area, 6%-8% in the middle layer, and 5%-6% in the surface layer. During inoculation, the microbial agent is sprayed and stirred at the same time to ensure that the microbial agent and tea leaves are in full contact. Ventilation channels are reserved around the core area to ensure the oxygen supply to the core area. Pile treatment: After inoculation, the fermentation pile is shaped and covered with a breathable palm leaf pad and heat-insulating film. At the same time, three sets of multi-dimensional sensors are placed in the center, middle layer and surface of the tea pile for real-time monitoring of subsequent microenvironment parameters.

[0030] In step S4, three sets of multi-dimensional sensors are arranged in the center, middle layer, and surface layer of the tea pile. The microbial community metabolic model divides fermentation into four stages, with the following control parameters for each stage: Establishment period (1-3 days): temperature 28-32℃, oxygen content 15%-20%, pH 5.5-6.0; Propagation period (4-7 days): center temperature 35-38℃, oxygen content 18%-22%, middle / surface layer temperature 30-35℃; Core metabolic period (8-15 days): center temperature 32-35℃, oxygen content 10%-15%, middle layer oxygen content 8%-10%, surface layer 3%-5%, pH 4.5-5.0; Post-metabolic ripening period (16-17 / 25 days): temperature in all parts 25-30℃, oxygen content 3%-5%, humidity 50%-60%.

[0031] The intelligent turning of the tea pile in step S4 is triggered when the temperature difference between different parts of the tea pile is ≥5℃. The intelligent determination criteria for the fermentation endpoint are that the temperature of different parts of the tea pile returns to room temperature, the pH value stabilizes at 4.5-5.0, and the content of aged aroma and characteristic aroma substances reaches a preset threshold. The parameters of the tea pile are monitored in real time through IoT sensors, and the fermentation parameters are dynamically adjusted based on the microbial metabolism model, replacing the traditional fixed-stage control, thus realizing intelligent fermentation. Specific operation: Sensor deployment and data acquisition: Three sets of multi-dimensional sensors are deployed in the center, middle layer and surface of the tea pile. The sensors can collect data on temperature, oxygen content, pH value and humidity of the tea pile in real time, and the data is transmitted to the intelligent fermentation control system in real time. The system has a built-in microbial community metabolism model, which divides the fermentation process into four stages: the establishment period, the reproduction period, the core metabolism period and the metabolic ripening period, and presets the parameter control range for each stage.

[0032] Phased intelligent dynamic control: Based on real-time collected parameters of the tea pile, the system automatically adjusts the temperature control equipment, oxygen supply fans, and humidifying spray in the fermentation workshop to ensure that the microenvironment of the tea pile always matches the real-time metabolic needs of the microbial community. The core control parameters for each stage are: Establishment period (1-3 days): Control the temperature of each part of the tea pile to 28-32℃, oxygen content to 15%-20%, and pH value to 5.5-6.0, so that the microbial community can quickly attach to the surface of the tea leaves and complete the establishment. Reproduction period (4-7 days): The temperature of the core pile naturally rises to 35-38℃ due to the heat released by microbial metabolism. The system automatically replenishes oxygen to maintain the oxygen content of the core pile at 18%-22%, and the temperature of the middle / surface layer is maintained at 30-35℃, which promotes the rapid reproduction of microorganisms and makes them the dominant microorganisms in the tea pile. Core metabolic period (8-15 days): The temperature of the core of the pile slowly drops to 32-35℃, the oxygen supply of the system is gradually reduced, the oxygen content of the core is 10%-15%, the middle layer is 8%-10%, and the surface layer is 3%-5%, and the pH value naturally drops to 4.5-5.0, allowing the microbial community to carry out core metabolism, degrade the macromolecular substances of tea, and synthesize the precursors of aged aroma and characteristic aroma. Metabolic ripening period (16-17 / 25 days): The temperature of each part of the tea pile gradually drops to 25-30℃, the oxygen content uniformly drops to 3%-5%, and the humidity is maintained at 50%-60%, allowing the microbial community to transform and accumulate metabolic products and enhance the aroma fusion.

[0033] Intelligent turning: The system judges based on the real-time temperature data of various parts of the tea pile. When the temperature difference between various parts of the tea pile is ≥5℃, it triggers an intelligent turning reminder, and the manual turning operation is performed to fully mix the tea leaves in various parts of the tea pile. After turning, the sensors are rearranged to continue the control. This process only requires turning the pile 1-2 times in the whole process.

[0034] Intelligent determination of fermentation endpoint: When the intelligent fermentation control system detects that the temperature of each part of the tea pile has dropped back to room temperature, the pH value has stabilized at 4.5-5.0, and the content of aged aroma and characteristic aroma substances has reached the preset threshold, it automatically determines that the pile fermentation is complete; at this time, the tea leaves are dark brown, soft to the touch, and have no astringent or piled smell, thus completing the pile fermentation.

[0035] In step S5, the moisture content of the ripe Pu-erh tea leaves is adjusted to 20%-25% before aging. The parameters of the simulated Liubao tea aging microenvironment are: temperature 22-25℃, relative humidity 65%-70%, and micro-oxygen content 3%-5%. The aging time is 3-5 days for pure aged aroma ripe Pu-erh tea and 7-10 days for mushroom aroma and light areca aroma ripe Pu-erh tea. Combining the fermentation of ripe Pu-erh tea with the traditional basket aging of Liubao tea allows the microbial community to undergo secondary metabolism, enhancing the layering and persistence of the aroma. Specific operation: Cooling and Humidification: Spread the fermented tea leaves on a clean cooling platform with a thickness of 10-15cm and let them cool naturally for 4-6 hours to remove surface moisture and adjust the moisture content of the fermented tea leaves to 20%-25%. Basket processing: The moistened ripe tea leaves are packed into traditional Liubao tea bamboo baskets, with each basket containing 5-10 kg of tea. The basket opening is sealed with breathable cotton paper to ensure a micro-oxygen environment and avoid contamination by miscellaneous bacteria. Post-fermentation aging: The bamboo baskets containing the tea are placed in a cellar that simulates the microenvironment of Liubao tea. The microenvironmental parameters of this cellar are: temperature 22-25℃, relative humidity 65%-70%, and micro-oxygen content 3%-5%. The walls of the cellar are covered with the dominant microbial community of Liubao tea, which further enhances the flavor integration. The aging time is adjusted according to the aroma type: pure aged aroma ripe tea is aged for 3-5 days, while fungal aroma and light areca nut aroma ripe tea are aged for 7-10 days. Post-aging processing: After aging, take out the tea leaves and spread them out to cool until the surface is dry. At this time, the tea leaves have a rich aged aroma and corresponding characteristic fragrance, without any off-flavors.

[0036] The temperature for low-temperature differentiated drying in step S6 is as follows: 45-50℃ for pure aged aroma ripe tea, and 40-45℃ for arugula aroma and light areca aroma ripe tea; the moisture content of the dried tea is controlled to be ≤12% for pure aged aroma and ≤11% for arugula aroma and light areca aroma; the aroma-fixing aging time is 3-5 days for pure aged aroma and 7-10 days for arugula aroma and light areca aroma; low-temperature differentiated drying: low-temperature drying equipment is used to dry the aged tea, and the drying temperature is adjusted according to the aroma: 45-50℃ for pure aged aroma ripe tea, and 40-45℃ for arugula aroma and light areca aroma ripe tea; the moisture content of the dried tea is strictly controlled: ≤12% for pure aged aroma and ≤11% for arugula aroma and light areca aroma. Lower drying temperatures can effectively preserve characteristic aroma substances. Grading and sorting: The dried ripe tea leaves are sieved and stems are removed according to the conventional Pu-erh tea processing technology. They are graded according to the shape, color and internal quality of the tea leaves, and broken pieces and impurities are removed to ensure the appearance quality of the finished tea. Aroma shaping and aging: The graded tea leaves are placed in a clean, ventilated, and dry shaping chamber for shaping and aging. The aging time is adjusted according to the aroma type: pure aged aroma type ripe tea is aged for 3-5 days, and fungus and light areca aroma type ripe tea is aged for 7-10 days to make the aroma of the tea more stable and harmonious. Finished product processing: After the tea leaves have been shaped and aged, they can be directly pressed into shapes (cakes, bricks, tuocha, etc.) or packaged in bulk to produce finished Pu-erh ripe tea. Ripe tea produced by this process does not require additional storage to dissipate its flavor and can be drunk immediately after brewing.

[0037] The finished Pu-erh ripe tea meets the following quality indicators: Microbiological indicators: free from aflatoxin and ochratoxin, pathogenic bacteria such as Salmonella and Staphylococcus aureus, and total bacterial count ≤10^4 CFU / g; Physicochemical indicators: pure aged aroma type theabrownin ≥8.0% and tea polyphenols ≤15.0%, fungal aroma type theabrownin ≥8.5% and tea polyphenols ≤14.5%, and light areca aroma type theabrownin ≥9.0% and tea polyphenols ≤14.0%; Sensory indicators: free from musty, moldy, and astringent odors.

[0038] In this embodiment, the preparation of light areca nut flavored ripe Pu-erh tea is described. Following step S1, Aspergillus niger, Mucor, Chaetomium globosum, Wallermi, and Debarry yeast were screened from century-old tea cellars in Liubao tea plantations in Wuzhou. After 12 generations of basic co-cultivation and domestication at a ratio of 8:1:1, a targeted domestication was then carried out for 4 generations at a ratio of basic complex microbial community: Wallermi: Debarry yeast = 8.5:1:0.5. Gradient microbial agents were prepared for the core (10^8 CFU / mL), middle layer (10^7 CFU / mL), and surface layer (10^6 CFU / mL), with the proportion of targeted bacteria in the surface layer agent increased by 15%. Take 100kg of Yunnan sun-dried green tea according to step S2, spray with 0.8% compound enzyme solution for enzymatic hydrolysis for 2.5 hours, adjust the moisture content to 29%, and rinse the tea for 2.5 hours; Construct a 0.9m high fermentation pile according to step S3. The thickness of the core, middle layer and surface layer is 20cm, 45cm and 20cm respectively. Inoculate with the corresponding gradient inoculum at inoculation rates of 9%, 7% and 5.5% respectively. Ventilation channels are reserved in the core of the pile. The IoT sensors were deployed according to the S4 steps, and the fermentation was intelligently and dynamically controlled for 22 days. Only on the 10th day, the pile was turned over once due to a temperature difference of 6°C. The system determined that the fermentation was complete after monitoring that the parameters met the standards. Following step S5, spread the ripe tea leaves out to cool for 5 hours, adjust the moisture content to 23%, pack them into bamboo baskets, and store them in a cellar room imitating Liubao tea for 10 days. Following step S6, the tea is dried at a low temperature of 40℃ to a moisture content of 10.5%, then sieved to remove stems, shaped and aged for 10 days, and finally pressed into Pu-erh ripe tea cakes.

[0039] The resulting light areca nut-scented ripe Pu-erh tea has a bacterial colonization rate of 85%, is free of aflatoxin and pathogenic bacteria, and contains 9.2% theabrownin and 13.8% tea polyphenols. Sensoryly, it has a full-bodied aged aroma, with a light areca nut and fruity aroma. The tea soup is bright red, with a mellow and sweet aftertaste and a refreshing sensation, and no off-flavors.

[0040] Preparation of Pu-erh Ripe Tea with Floral Fragrance Following step S1, Aspergillus niger, Mucor, Chaetomium globulus, and Xeromyces dry yeast were screened from traditional tea cellars in Wuzhou Liubao tea plantations. After 10 generations of basic co-cultivation and domestication at a ratio of 8:1:1, a targeted domestication was carried out for 3 generations at a ratio of basic compound microbial community to Xeromyces dry yeast of 9:1. Gradient microbial agents were prepared for the core (10^7 CFU / mL), middle layer (10^6 CFU / mL), and surface layer (10^5 CFU / mL), with the proportion of targeted bacteria in the surface layer agent increased by 10%. Take 100kg of Yunnan sun-dried green tea according to step S2, spray with 0.6% compound enzyme solution for 2 hours of enzymatic hydrolysis, adjust the moisture content to 28%, and rinse the tea for 2 hours; Construct a 0.8m high fermentation pile according to step S3. The thickness of the core, middle layer and surface layer is 20cm, 40cm and 20cm respectively. Inoculate with the corresponding gradient inoculum at inoculation rates of 8%, 6% and 5% respectively. Leave ventilation channels in the core of the pile. The IoT sensors are deployed according to the S4 steps, and the fermentation is intelligently and dynamically controlled for 18 days without turning the pile. The system determines that the fermentation is complete after monitoring that the parameters meet the standards. Following step S5, spread the ripe tea leaves out to cool for 4 hours, adjust the moisture content to 20%, pack them into bamboo baskets, and store them in a cellar room imitating Liubao tea for 7 days. Following step S6, the tea is dried at a low temperature of 42℃ to a moisture content of 11%, then sieved to remove stems, shaped and aged for 7 days, and finally pressed into Pu-erh ripe tea cakes.

[0041] The resulting Pu-erh ripe tea with a fungal aroma has a fungal colonization rate of 82%, is free of aflatoxin and pathogenic bacteria, and contains 8.8% theabrownin and 14.2% tea polyphenols. Sensoryly, it has a mellow and aged aroma, with an elegant fungal and honey fragrance. The tea soup is bright red, and the taste is smooth, sweet, and has a rapid aftertaste without any off-flavors.

[0042] In this invention, the metabolic competition between microbial strains is eliminated through the composite synergistic domestication of Pu'er-Liubao tea mixed extract culture medium, allowing the core microbial community to form a stable symbiotic metabolic system. Combined with gradient stratified targeted inoculation, the colonization rate of the microbial community in the Pu'er tea pile is increased, completely solving the problem of uneven fermentation.

[0043] The compound enzymatic hydrolysis pretreatment accelerates the colonization of microorganisms and the transformation of tea substances. The Internet of Things intelligent dynamic control allows the fermentation parameters to be precisely matched with the metabolic patterns of microorganisms, shortening the fermentation cycle to 17-25 days, reducing the number of turnings to 1-2 times, reducing the intensity of manual labor, and greatly improving the efficiency of industrial production.

[0044] By using aroma-targeted domestication and gradient microbial agent formulation design, three types of Pu-erh ripe tea—pure aged aroma, fungal aroma, and light areca nut aroma—can be precisely produced. These teas have distinct aroma characteristics, meet diverse market demands, and enhance product added value.

[0045] The combination of fermentation and storage in the cellar, a characteristic process of Liubao tea, allows the distinctive flavor of Liubao tea to deeply blend with the aged aroma of Pu'er tea. The finished tea has a full and layered aroma, and the aroma substances are more stable after the secondary metabolism during cellar storage.

[0046] The complex and dominant microbial community quickly occupies the ecological niche of the tea pile, effectively inhibiting the growth of miscellaneous bacteria. The finished tea has no piled, musty, or astringent smell. The tea soup is bright red, mellow and smooth, with a significant sweet aftertaste. The content of theaflavins is significantly increased, while the content of irritating substances such as tea polyphenols is reduced. Moreover, all the microbial communities used are food-grade safe strains. No mycotoxins or pathogens were detected in the finished tea, which meets the highest national standards for tea food safety.

[0047] The real-time monitoring by multi-dimensional sensors of the Internet of Things and the dynamic regulation by the microbial community metabolism model have replaced manual experience judgment, reducing the batch-to-batch quality difference rate during mass production and realizing the standardization and intelligentization of Pu'er ripe tea fermentation.

[0048] Example 2 like Figure 1 As shown, the present invention proposes a method for improving the fermentation of Pu'er ripe tea by introducing black-brown bacteria from Liubao tea. Compared with Example 1, in this example, the Pu'er-Liubao tea mixed extract culture medium in step S1 is prepared by mixing Yunnan sun-dried green tea extract and Liubao tea fermentation extract at a volume ratio of 3:1. The pH of the culture medium is adjusted to 5.5-6.0, and the inoculation volume ratio of Aspergillus niger, Mucor, and Chaetomium globosum acclimatized in the basic co-culture is 8:1:1. The inoculation ratio of the aroma-targeted co-culture and domestication microbial groups in step S1 is as follows: the basic compound microbial group for floret aroma is Xeromyces dry yeast = 9:1, and the basic compound microbial group for light areca nut aroma is Wallerian: Debali yeast = 8.5:1:0.5. The concentration of the gradient compound microbial agent in step S3 is as follows: 10^7-10^8 CFU / mL for the core layer agent, 10^6-10^7 CFU / mL for the middle layer agent, and 10^5-10^6 CFU / mL for the surface layer agent. The proportion of aroma-targeted bacteria in the surface layer agent is increased by 10%-15% compared with the basic compound microbial group.

[0049] In this embodiment, the strains were screened and purified: samples were collected from traditional Liubao tea cellars, late-stage fermentation tea samples, and basket-packed aged tea samples. The strains were isolated using the dilution plating method. Single colonies of Aspergillus niger (blackish-brown colonies), Mucor (white fluffy colonies), and Chaetomium globulus (blackish-brown flocculent colonies) were selected and purified by multiple streak plating to obtain pure strains. At the same time, aroma-targeting bacteria were isolated. The mycelium aroma type was identified as Xeromyces dry yeast, and the light areca nut aroma type was identified as Wallermi and Debarry yeast. After the species were identified by ITS gene sequence, strains with slow growth rate and low enzyme production capacity were eliminated, and high-activity strains were retained.

[0050] Culture medium preparation: A mixed extract culture medium of Pu'er and Liubao tea was prepared. This culture medium was prepared by mixing Yunnan sun-dried green tea extract and Liubao fermented tea extract at a volume ratio of 3:1, with the addition of 10 g / L glucose, 5 g / L peptone, 1 g / L KH2PO4, and 0.5 g / L MgSO4·7H2O. 20 g / L agar was added to the solid culture medium, while no agar was added to the liquid culture medium. The pH of the culture medium was adjusted to 5.5-6.0, and the medium was autoclaved at 121℃ for 20 min before use. This culture medium simulates the fermentation substrate characteristics of Pu'er ripe tea, allowing the microbial community to adapt to the Pu'er raw tea environment from the initial acclimatization stage.

[0051] Basic co-cultivation and domestication: Activated Aspergillus niger, Mucor, and Chaetomium globosum seed cultures were inoculated into the above liquid culture medium at an inoculation volume ratio of 8:1:1, with a 5% inoculation amount. The culture was carried out at 28℃ and 120 rpm for 72 hours to complete the primary culture. Subsequently, 10-15 generations of continuous subculturing were carried out. After each generation, the bacterial culture was transferred to fresh culture medium at a 5% inoculation amount. After the 5th generation, the culture temperature was gradually increased to 30-32℃ and the shaking speed was reduced to 100 rpm to simulate the mesophilic and microaerobic environment of Pu'er fermentation. After each generation of domestication, enzyme activity, synergistic metabolism, and colonization ability indicators were tested. Unqualified bacterial groups were eliminated, and finally, a basic complex bacterial group with a stable symbiotic metabolic system was obtained.

[0052] Flavor-targeted co-cultivation and domestication: Using a basic compound microbial community as a base, flavor-targeting bacteria are introduced according to the flavor cultivation goals for 3-5 generations of short-cycle domestication. The inoculum size is 5%, and the culture conditions are 30℃, 90r / min shaker (micro-aerobic) culture for 72h. Among them, the inoculum ratio for the floret flavor is basic compound microbial community: Xeromyces dry yeast = 9:1, and the inoculum ratio for the light areca flavor is basic compound microbial community: Wallerian: Debali yeast = 8.5:1:0.5. In addition to basic indicators, characteristic flavor-producing indicators are also tested in each generation. The floret flavor requires fungal alcohol ≥0.1mg / L, and the light areca flavor requires pine smoke volatile matter ≥0.08mg / L. Finally, flavor-specific compound functional microbial communities are obtained, including the basic compound microbial community for pure aged flavor, the floret flavor compound microbial community, and the light areca flavor compound microbial community.

[0053] Preparation of gradient compound microbial agents: The above-mentioned aroma-specific compound functional microbial groups were propagated separately to prepare gradient compound microbial agents suitable for different parts of the tea pile. The concentration of the microbial agents was as follows: 10^7-10^8 CFU / mL for the core layer, 10^6-10^7 CFU / mL for the middle layer, and 10^5-10^6 CFU / mL for the surface layer. The proportion of aroma-targeting bacteria in the surface layer microbial agent was increased by 10%-15% compared with the basic compound microbial group, which is suitable for the low-temperature and micro-oxygen aroma-producing environment on the surface of the tea pile. The microbial agents were prepared and used immediately to ensure their activity.

[0054] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited thereto. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. A method for improving the pile fermentation of ripe Pu-erh tea by introducing blackish-brown microorganisms from Liubao tea, characterized in that, Includes the following steps: S1. Screening, purification, and synergistic domestication of the dark brown core microbial community of Liubao tea: First, core strains of Aspergillus niger, Mucor, and Chaetomium globosum were screened and purified from traditional tea cellar / fermentation / cellar-aged tea samples of Liubao tea from Wuzhou, Guangxi. They were then subjected to 10-15 generations of basic co-culture domestication using a mixed extract medium of Pu'er and Liubao tea. Then, based on the aroma cultivation goal, aroma-targeting bacteria were incorporated for 3-5 generations of targeted co-culture domestication to obtain aroma-specific composite functional microbial communities. S2. The compound enzymatic pretreatment of Pu'er tea involves using a compound enzyme solution secreted by domesticated compound functional bacteria to perform mild enzymatic hydrolysis on Yunnan sun-dried green tea, followed by adjusting the moisture content and moistening the tea. S3. Gradient layered targeted inoculation of tea piles: The pre-treated Pu'er tea is piled into three layers: the core, the middle layer, and the surface layer. Gradient compound microbial agents adapted to the microenvironment of each part are inoculated separately. S4. Based on the Internet of Things, intelligent dynamic regulation of the microenvironment of tea piles for fermentation is carried out. Multi-dimensional sensors are arranged in various parts of the tea pile to monitor temperature, humidity, oxygen content and pH value in real time. Combined with the microbial community metabolism law model, the fermentation parameters are dynamically regulated. The entire fermentation process takes 17-25 days and the pile is turned 1-2 times. S5. Fermentation-Liubao Tea Special Basket Storage and Post-Ripe Processing: The fermented tea leaves are packed into bamboo baskets and placed in a micro-environment similar to Liubao tea storage for short-term post-ripening. S6. Aroma Differentiation Post-processing: Low-temperature differentiated drying, grading and sorting, and aroma-fixing aging of the tea leaves after cellar storage to produce finished Pu-erh ripe tea; The aroma-targeting bacteria are Xeromyces dry yeast of the fungal aroma type, or a combination of Wallermi and Debali yeast of the light areca aroma type.

2. The method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial community according to claim 1, characterized in that, The Pu'er-Liubao tea mixed extract culture medium mentioned in step S1 is prepared by mixing Yunnan sun-dried green tea extract and Liubao tea fermentation extract at a volume ratio of 3:

1. The pH of the culture medium is adjusted to 5.5-6.0, and the inoculation volume ratio of Aspergillus niger, Mucor, and Chaetomium globosum, which are domesticated by basic co-culture, is 8:1:

1.

3. The method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial community according to claim 2, characterized in that, The compound enzyme solution mentioned in step S2 contains cellulase, pectinase, and hemicellulase. It is sprayed by atomization at 0.5%-1% of the weight of Pu'er tea, and the enzymatic hydrolysis time is 2-3 hours. After enzymatic hydrolysis, the moisture content of Pu'er tea is adjusted to 28%-30%, and the tea is moistened for 2-3 hours.

4. The method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial community according to claim 3, characterized in that, In step S3, the height of the tea pile is 0.8-1m, and the thickness of the core, middle layer and surface layer is 20cm, 40-50cm and 20cm respectively. The inoculation amount of the gradient compound microbial agent is 8%-10% of the weight of the tea in the core, 6%-8% in the middle layer and 5%-6% in the surface layer, and ventilation channels are reserved in the core.

5. The method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial community according to claim 4, characterized in that, In step S4, three sets of multi-dimensional sensors are arranged in the center, middle layer, and surface layer of the tea pile. The microbial community metabolic model divides fermentation into four stages, with the following control parameters for each stage: Establishment period (1-3 days): temperature 28-32℃, oxygen content 15%-20%, pH 5.5-6.0; Propagation period (4-7 days): center temperature 35-38℃, oxygen content 18%-22%, middle / surface layer temperature 30-35℃; Core metabolic period (8-15 days): center temperature 32-35℃, oxygen content 10%-15%, middle layer oxygen content 8%-10%, surface layer 3%-5%, pH 4.5-5.0; Post-metabolic ripening period (16-17 / 25 days): temperature in all parts 25-30℃, oxygen content 3%-5%, humidity 50%-60%.

6. The method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial community according to claim 5, characterized in that, The triggering condition for intelligent turning of the tea pile in step S4 is that the temperature difference between different parts of the tea pile is ≥5℃. The intelligent determination criteria for the end of fermentation are that the temperature of different parts of the tea pile drops back to room temperature, the pH value stabilizes at 4.5-5.0, and the content of aged aroma and characteristic aroma substances reaches the preset threshold.

7. The method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial community according to claim 6, characterized in that, In step S5, the moisture content of the ripe tea material is adjusted to 20%-25% before cellaring. The parameters of the microenvironment for imitating Liubao tea cellaring are: temperature 22-25℃, relative humidity 65%-70%, and micro-oxygen content 3%-5%. The cellaring time is 3-5 days for pure aged aroma ripe tea and 7-10 days for fungal aroma and light areca aroma ripe tea.

8. The method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial community according to claim 7, characterized in that, The inoculation ratio of the aroma-targeted co-culture and domestication microbial groups in step S1 is as follows: the basic compound microbial group for floret aroma is Xeromyces dry yeast = 9:1, and the basic compound microbial group for light areca nut aroma is Wallerian: Debali yeast = 8.5:1:0.

5. The concentration of the gradient compound microbial agent in step S3 is as follows: 10^7-10^8 CFU / mL for the core layer agent, 10^6-10^7 CFU / mL for the middle layer agent, and 10^5-10^6 CFU / mL for the surface layer agent. The proportion of aroma-targeted bacteria in the surface layer agent is increased by 10%-15% compared with the basic compound microbial group.

9. The method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial community according to claim 8, characterized in that, The temperature for low-temperature differentiated drying in step S6 is as follows: 45-50℃ for pure aged aroma ripe tea, and 40-45℃ for arugula aroma and light areca aroma ripe tea; the moisture content of the tea leaves after drying is controlled to be ≤12% for pure aged aroma and ≤11% for arugula aroma and light areca aroma; the aroma aging time is 3-5 days for pure aged aroma and 7-10 days for arugula aroma and light areca aroma.

10. The method for improving the pile fermentation of Pu'er ripe tea by introducing Liubao tea black-brown microbial community according to claim 8, characterized in that, The finished Pu-erh ripe tea meets the following quality indicators: Microbiological indicators: free from aflatoxin and ochratoxin, pathogenic bacteria such as Salmonella and Staphylococcus aureus, and total bacterial count ≤10^4 CFU / g; Physicochemical indicators: pure aged aroma type theabrownin ≥8.0% and tea polyphenols ≤15.0%, fungal aroma type theabrownin ≥8.5% and tea polyphenols ≤14.5%, and light areca aroma type theabrownin ≥9.0% and tea polyphenols ≤14.0%; Sensory indicators: free from musty, moldy, and astringent odors.

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