Method for aging limequat and application thereof

Abstract

This invention provides a method for aging small green tangerines and its application, belonging to the field of food processing technology. The method includes the following steps: after washing and removing the pulp from the small green tangerine peel, spraying it with inoculated *Pancreatoblasticulata* (a type of bacteria). Pantoea agglomerans The Z05 bacterial suspension was fermented at 25-30℃ for 12-24 hours. After fermentation, the product was rinsed with sterile water, steamed to inactivate, and dried to obtain aged small green tangerine peel. The Z05 bacterial clump is an endophytic bacterium in the small green tangerine peel, with the preservation number CGMCC No. 36512. This invention can complete the aging process that traditional processes require more than one year in 24 hours, increasing the aging efficiency by 300 times. The fermentation treatment significantly reduces the pungent odor of the peel, enhances the aged aroma, promotes the color change of the peel to dark brown, and maintains the flavonoid content and antioxidant activity. This invention has a simple process, short cycle, and stable effects, and is suitable for preparing products such as small green tangerine Pu-erh tea.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for aging small green tangerines and its application. Background Technology

[0002] Xiaoqinggan, the young fruit of the Xinhui tea branch tangerine, is prized for its thin peel, rich aroma, and high content of flavonoids, making it an important raw material in tea products and traditional Chinese medicine in recent years. Xiaoqinggan Pu'er tea, made by combining the peel of Xiaoqinggan with Pu'er tea, is widely favored in the market for its unique flavor and health benefits.

[0003] However, the production process of Xiaoqinggan (small green tangerine) peel faces several technical challenges. Because Xiaoqinggan has a high volatile oil content, its peel is harder and thinner than that of mature tangerines, making aging difficult. Traditional aging process requires a long time, usually more than a year, to achieve good results, and is greatly affected by environmental factors, making it difficult to guarantee the stability of aroma and nutrients. Furthermore, fresh Xiaoqinggan peel has a strong, astringent, and pungent aroma, which needs to be aging to mellow.

[0004] Currently, some studies have attempted to accelerate the aging process of tangerine peel products using microbial fermentation technology. However, the reported strains have limited effects on improving the aroma quality of the peel, shortening the aging period, and stabilizing the content of flavonoids, and cannot yet effectively replace traditional long-term aging processes. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a clump-forming pantothenic bacterium that can be used for the aging of small green tangerines, and this strain can achieve rapid aging of small green tangerine peel in a short period of time.

[0006] The second objective of this invention is to provide the application of the above-mentioned clustered pantothecin in the aging of small green tangerines.

[0007] The third objective of this invention is to provide a method for aging small green tangerines. This method utilizes clustered pan-microbial fermentation, which can complete the aging process that would normally take more than a year in 24 hours, significantly improving aging efficiency while also improving the aroma quality of the peel and maintaining the content of flavonoids and antioxidant activity.

[0008] The fourth objective of this invention is to provide the application of the above-mentioned clump-forming pantothecin or the above-mentioned method in the preparation of small green tangerine Pu-erh tea.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a type of pantothecin bacterium that can be used for the aging of small green tangerines, namely pantothecin bacterium Z05, with the Latin classification name... Pantoea agglomeransThe accession number is CGMCC No.36512.

[0011] This invention also provides the application of the above-mentioned Pantothecin clumps in the aging of small green tangerines.

[0012] The present invention also provides a method for aging small green tangerines, comprising the following steps: inoculating the above-mentioned clump-forming pancreatic bacteria suspension onto the peel of small green tangerines; air-drying and then fermenting; after fermentation, inactivating and drying the peel to obtain aged small green tangerine peel.

[0013] Preferably, the peel of the small green tangerine is washed and the pulp is removed before inoculation.

[0014] Preferably, the fermentation treatment is carried out at a temperature of 25-30°C for 12-24 hours; more preferably, the fermentation treatment is carried out at a temperature of 25°C for 12 hours.

[0015] Preferably, the OD of the bacterial suspension 600 The value is 0.6-1.2, more preferably 1.0.

[0016] Preferably, the bacterial suspension is inoculated onto the outer and / or inner surface of the small green tangerine peel by spraying; the air drying is carried out under ventilated conditions until no liquid droplets remain on the peel surface.

[0017] Preferably, after fermentation, the surface of the fruit peel is rinsed with water before inactivation treatment; the rinsing time is 20-40 seconds, more preferably 30 seconds; the inactivation is performed by steaming for 10-12 minutes, more preferably 10 minutes.

[0018] Preferably, the drying process involves drying at 55-65°C for 4-5 hours, resulting in a moisture content of less than 5% in the peel after drying. More preferably, the drying temperature is 60°C and the drying time is 4 hours.

[0019] This invention also provides the application of the above-mentioned clustered pan-bacteria or the above-mentioned method in the preparation of small green tangerine Pu-erh tea.

[0020] The beneficial effects of this invention are:

[0021] The Pantothecin Z05 strain provided by this invention is isolated from the endophytic bacteria of the peel of small green tangerines. It can quickly achieve the aging effect of the peel of small green tangerines without the need for exogenous strains, thus avoiding the safety hazards that may be caused by exogenous microorganisms.

[0022] The present invention provides a method for aging small green tangerines, which uses Pantothecin Z05 to ferment the peel of small green tangerines. It can complete the aging process that traditional processes require more than one year in 24 hours, improving the aging efficiency by 300 times, greatly shortening the production cycle and reducing storage costs.

[0023] The method provided by this invention yields aged small green tangerine peel with significantly improved aroma quality, a marked reduction in pungent odor, and a significantly enhanced aged aroma. The appearance and color show significant aging, with the peel color transitioning to a dark brown aged state. Functional components remain stable, with no decrease in flavonoid content and stable or significantly increased antioxidant activity. This invention features a simple process, short cycle time, and stable results, making it suitable for industrial production. The resulting product is applicable to the preparation of products such as small green tangerine Pu-erh tea.

[0024] Preservation Instructions

[0025] Pantotheca cum Cuboide Z05 of this invention is deposited at the China General Microbiological Culture Collection Center and classified as Pantotheca cum Cuboide. Pantoea agglomerans The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36512 and deposit date of November 6, 2025. Attached Figure Description

[0026] Figure 1 The clump-forming pantothecin Z05 isolated and purified in Example 1 of this invention ( Pantoea agglomerans Z05) Colony morphology on TSA agar plates.

[0027] Figure 2 This is a violin plot showing the sensory evaluation statistics of the fermented group (Z05 treatment) and the control group (CK, unfermented) of small green tangerine peel samples in Experiment Example 1 of this invention. A to G represent the statistical results of scores for color preference, aroma preference, aroma intensity, aroma staleness, aroma irritation, texture preference, and overall acceptability, respectively. The horizontal axis represents the grouping, and the vertical axis represents the sensory score (1-10 points); ns indicates no significant difference between the two groups (P>0.05). This indicates that P < 0.05. This indicates that P < 0.01. This means P < 0.0001.

[0028] Figure 3 This is a statistical graph showing the effect of fermentation treatment on the surface color parameters of the peel of small green tangerines in Experiment Example 2 of this invention. Where A represents L... Value (brightness); B represents a Value (redness / greenness, the higher the value, the redder); C represents b Value (yellowish-blue tint, the higher the value, the more yellow); D represents color saturation (Chroma, C = (The image) This indicates that P < 0.0001 compared to the CK group.

[0029] Figure 4 This is a statistical graph showing the significant changes in the content of volatile organic compounds (VOCs) in the peel of small green tangerines after 12 hours of fermentation in Experiment Example 3 of this invention. In the graph, A represents the change in total VOCs content; B to L represent the changes in the content of D-limonene, γ-terpinene, terpinene, 4-terpenol, α-terpineol, carvacrol, methyl 2-(methylamino)benzoate, caryophyllene, α-rutane, α-farnesene, and hesperidin, respectively. , and The values ​​represent P<0.05, P<0.01, and P<0.001 compared to the CK group, respectively.

[0030] Figure 5 This is a statistical chart showing the significant changes in VOCs content in the final dried fruit peel of Experimental Example 3 of this invention. In the chart, A represents the change in total VOCs content; B to L represent the changes in the content of D-limonene, γ-terpinene, terpinene, 4-terpenol, α-terpineol, carvacrol, methyl 2-(methylamino)benzoate, caryophyllene, α-thiazolinone, α-farnesene, and hesperidin, respectively. , and The values ​​represent P<0.05, P<0.01, and P<0.001 compared to the CK group, respectively.

[0031] Figure 6 This is a graph showing the results of determining the total flavonoid content in the peel of the fermented small green tangerine in Experiment Example 4 of this invention. The vertical axis represents the total flavonoid content (mg / g FW); ns indicates that there is no statistically significant difference between the fermentation group and the control group.

[0032] Figure 7 This figure shows the evaluation results of four antioxidant capacities of the small green tangerine peel extract in Experiment Example 5 of this invention. A represents ABTS free radical scavenging capacity; B represents DPPH free radical scavenging capacity; C represents ferric ion reducing antioxidant capacity (FRAP); and D represents oxygen free radical absorption capacity (ORAC). ns indicates no statistically significant difference between the fermentation group and the control group.

[0033] Figure 8This is a statistical chart (violin diagram) of the sensory evaluation results of the small green tangerine Pu-erh tea soup in Experiment Example 6 of this invention. A to H represent the statistical results of the scores for tea soup color, tea soup purity, fruity aroma, aged aroma, sweetness, astringency, aftertaste, and salivation, respectively. ns indicates P > 0.05. This indicates that P < 0.05. This indicates that P < 0.01. This means P < 0.0001.

[0034] Figure 9 The strain Z05 of this invention in Test Example 7 of this invention is different from other reported endophytic bacteria in dried tangerine peel ( Bacillus gibsonii , Bacillus sp. , Aspergillus sp. The fermentation effects of the tea are compared. A shows the change in total VOCs content after treatment with different strains; B shows the overall sensory evaluation score of the tea infusion after treatment with different strains. This indicates that P < 0.05. Detailed Implementation

[0035] This invention provides a type of pantothecin bacterium that can be used for the aging of small green tangerines, namely pantothecin bacterium Z05, with the Latin classification name... Pantoea agglomerans The accession number is CGMCC No.36512.

[0036] In this invention, the *Pantothecin Z05* strain is an endophytic bacterium isolated from the peel of small green tangerines from Xinhui, Guangdong. During the isolation process, it was found that this strain was the easiest to isolate and had a high concentration, with single colonies accounting for >70%. This invention uses endophytic bacteria to ferment the peel of small green tangerines, eliminating the need for exogenous strains, thus ensuring fermentation effectiveness while avoiding potential safety hazards from exogenous microorganisms.

[0037] In a specific embodiment of the present invention, the method for isolating and screening the clustered pantothecin Z05 is as follows: Small green tangerines from Xinhui, Guangdong Province are collected, and the peel is used for endophytic bacteria isolation. First, the peel sample is immersed in a 1% sodium hypochlorite solution for 5 minutes, then transferred to 70% ethanol for 2 minutes for preliminary surface disinfection, and rinsed three times with sterile water to thoroughly remove residual disinfectant. All of the above processes are performed in a clean bench. The disinfected peel is then ground into small particles in a sterile mortar, and sterile physiological saline (0.9%) is added at a ratio of 1:10 (w / v). NaCl was used to prepare a pericarp homogenate. The homogenate was filtered through sterile double-layer gauze, and the clearer filtrate was retained as the inoculum. 100 μL of the pericarp filtrate was evenly spread on the surface of TSA solid medium and incubated upside down in a constant temperature incubator at 28°C and 70% relative humidity for 48 hours. After incubation, different types of colonies were initially screened based on colony color, edge morphology, and surface smoothness. Continuous purification was performed using the streak plate method, and each candidate colony needed to undergo 2-3 rounds of streak culturing to ensure the acquisition of a single strain. The purified monoclonal strain was inoculated into 5 mL of TSB liquid medium and incubated at 28°C and 180 rpm for 24 hours. The representative endophytic strain obtained from the purification was amplified by PCR and sequenced using the 16S rRNA gene. The sequencing results were compared with BLAST on NCBI, and the identification results showed that the strain was *Pantotheca acuminata* (a type of cloning bacteria). Pantoea agglomerans ), named Z05.

[0038] In this invention, the colony morphology of the Pantotheca Z05 is generally yellow, with neat edges, a slightly glossy surface, a moist texture, and a stable morphology.

[0039] In this invention, the preservation method of the clump-forming pantothecin Z05 is as follows: after collecting the culture medium, it is mixed with sterile 50% glycerol at a volume ratio of 1:1 and stored at -80℃.

[0040] This invention also provides the application of the aforementioned Pantothecin clumps in the aging of small green tangerines. The Pantothecin Z05 of this invention can rapidly achieve the aging effect of small green tangerine peel, completing the aging process that traditional methods require more than one year within 24 hours, increasing aging efficiency by 300 times.

[0041] The present invention also provides a method for aging small green tangerines, comprising the following steps: inoculating the above-mentioned clump-forming pancreatic bacteria suspension onto the peel of small green tangerines; air-drying and then fermenting; after fermentation, inactivating and drying the peel to obtain aged small green tangerine peel.

[0042] In this invention, the small green tangerine is preferably harvested from young tea-branch tangerines from Xinhui, Guangdong. Before inoculation, the tangerine peel is preferably washed and the pulp removed. Washing is preferably done by spraying the peel with purified water for 30 seconds to remove residual dirt, dust, and other impurities. Pulping is preferably done by making a horizontal cut approximately 1 cm from the stem end to remove the pulp, leaving only the peel. This pulping process allows the bacterial suspension to fully contact the inner and outer surfaces of the peel, improving fermentation efficiency and uniformity.

[0043] In this invention, the method for preparing the bacterial suspension is as follows: the frozen clumps of Pantotheca Z05 are first activated once on TSA solid medium, and then single colonies are picked and cultured in TSB liquid medium until OD. 600 >1; Centrifuge the bacterial culture at 6000 rpm for 10 min, collect the bacterial precipitate, wash twice with sterile water, and resuspend in sterile water to obtain a bacterial suspension. In this invention, the OD of the bacterial suspension is... 600 The preferred value is 0.6-1.2, more preferably 1.0. This invention controls the OD value of the bacterial suspension. 600 Within the above range, sufficient cell density can be ensured for fermentation, while avoiding nutrient competition and accumulation of metabolites caused by excessively high cell concentration.

[0044] In this invention, it is preferable to inoculate the bacterial suspension onto the outer and / or inner surface of the peel of the small green tangerine using a spraying method. The spraying volume is preferably such that water droplets uniformly coat the peel surface. Using a spraying method ensures that the bacterial suspension is evenly distributed on the peel surface, guaranteeing uniform fermentation.

[0045] In this invention, the air-drying process involves air-drying under ventilated conditions until no obvious liquid droplets remain on the surface of the fruit peel. This invention removes excess moisture from the fruit peel surface through air-drying, allowing the microorganisms to better adhere to the peel surface, while simultaneously avoiding the risk of contamination by other microorganisms due to excessive moisture during fermentation.

[0046] In this invention, the fermentation temperature is preferably 25-30℃, more preferably 25℃; the fermentation time is preferably 12-24h, more preferably 12h. By controlling the fermentation temperature, time, and humidity within the above ranges, this invention can ensure that the Pantotheca Z05 clumps are in a suitable growth and metabolic state, fully exerting its aging-promoting effect. Too low a fermentation temperature will lead to slow cell growth and reduced fermentation efficiency, while too high a fermentation temperature may inhibit cell activity or produce undesirable metabolites. Too short a fermentation time will result in insufficient aging, while too long a fermentation time may lead to over-fermentation, affecting the quality of the fruit peel.

[0047] In this invention, after fermentation, it is preferable to first rinse the surface of the fruit peel with water before inactivation treatment. The rinsing is preferably done by spraying sterile water onto the surface of the fruit peel to remove residual bacterial solution; the rinsing time is preferably 20-40 seconds, more preferably 30 seconds. This invention, through rinsing, can remove residual bacteria and metabolic products from the surface of the fruit peel, ensuring the hygiene and safety of the finished product.

[0048] In this invention, the inactivation is preferably carried out by steaming; after the water boils, the fruit peel is placed in a steamer for steaming; the steaming time is preferably 10-12 minutes, more preferably 10 minutes. This invention, through steam inactivation, can effectively kill any remaining live bacteria on the surface of the fruit peel, terminating the fermentation process. Simultaneously, the high-temperature and humid environment during steaming helps to further promote the transformation of aroma substances in the fruit peel, enhancing the aging effect.

[0049] In this invention, the drying process is preferably hot air drying; the drying temperature is preferably 55-65℃, more preferably 60℃; the drying time is preferably 4-5 hours, more preferably 4 hours; the moisture content of the dried peel is preferably less than 5%, and it maintains the integrity of the commercial dried peel. By controlling the drying temperature and time within the above ranges, this invention can ensure that the peel is fully dried while maximizing the preservation of aroma components and active substances in the peel; too low a drying temperature will lead to a prolonged drying time, while too high a drying temperature may damage the volatile aroma components in the peel; a moisture content of less than 5% in the dried peel ensures the storage stability of the product and prevents mold growth.

[0050] This invention also provides the application of the above-mentioned clump-forming bacteria or the above-mentioned method in the preparation of small green tangerine Pu-erh tea. In this invention, the preferred method for preparing the small green tangerine Pu-erh tea is to add Pu-erh tea leaves to the aged small green tangerine peel obtained by the above method, thus obtaining the small green tangerine Pu-erh tea. The aged small green tangerine peel obtained by this invention has a mellow aroma, a deep brown color, and a smooth taste. When combined with Pu-erh tea, the tea soup has a distinct aged aroma, a prominent sweetness, reduced astringency, and a long-lasting aftertaste.

[0051] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Unless otherwise specified, the following embodiments are all conventional methods.

[0053] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0054] Example 1

[0055] Small green tangerines were collected from Xinhui, Guangdong, and their peels were used for endophytic bacteria isolation.

[0056] Fruit peel samples were immersed in 1% sodium hypochlorite solution for 5 minutes, followed by immersion in 70% ethanol for 2 minutes for surface disinfection. The samples were then rinsed three times with sterile water to thoroughly remove residual disinfectant. All these processes were performed in a clean bench. The disinfected fruit peels were then ground into small particles in a sterile mortar and mixed with sterile physiological saline (0.9% NaCl) at a ratio of 1:10 (w / v) to prepare a fruit peel homogenate. The homogenate was filtered through sterile double-layer gauze, and the clearer filtrate was retained as the inoculum. 100 μL of the fruit peel filtrate was evenly spread on the surface of TSA solid medium and incubated upside down in a constant temperature incubator at 28°C and 70% relative humidity for 48 hours. After incubation, different types of colonies were initially screened based on colony color, edge morphology, and surface smoothness. Continuous purification was performed using the streak plate method, with each candidate colony undergoing three rounds of streak culturing to ensure the acquisition of a single strain. The purified monoclonal strain was inoculated into 5 mL of... The culture was incubated in TSB liquid medium at 28°C and 180 rpm for 24 hours. The purified representative endophytic bacterial strain was amplified by PCR and sequenced using 16S rRNA gene. The sequencing results were compared with BLAST on NCBI. After collecting the culture medium, it was mixed with sterile 50% glycerol at a volume ratio of 1:1 and stored at -80°C.

[0057] During the isolation process, it was found that this strain was the easiest to isolate and had a high concentration, with single colonies accounting for >70%. Figure 1 As shown, the isolated strain exhibits an overall yellow colony morphology with neat edges, a slightly glossy surface, a moist texture, and stable morphology. 16S rRNA sequence BLAST alignment results indicate that this strain is a clustered pantothenic bacterium (Pantotheca acuminata). Pantoea agglomerans ), named Z05.

[0058] The TSA solid culture medium formula is as follows: 15g casein trypsin digest, 5g soybean flour papain digest, 5g sodium chloride, 15g agar, diluted with water to a final volume of 1000mL, and sterilized by steam at 121℃ for 30min. The TSB liquid culture medium formula is as follows: 17g tryptone, 3g soybean peptone, 5g sodium chloride, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, diluted with water to a final volume of 1000mL, and sterilized by steam at 121℃ for 30min.

[0059] Example 2

[0060] A method for aging small green tangerines includes the following steps:

[0061] The frozen clumps of Pantotheca Z05 were first activated and cultured on TSA solid medium at 28°C for 24 hours. Then, single colonies were picked and inoculated into TSB liquid medium and cultured with shaking at 28°C and 180 rpm until OD. 600>1; Centrifuge the bacterial culture at 6000 rpm for 10 min, collect the bacterial precipitate, wash twice with sterile water, resuspend in sterile water, and adjust OD. 600 The value was 0.6, resulting in a suspension of Pantotheca Z05 bacteria in clumps.

[0062] Small green tangerines were collected from Xinhui, Guangdong. The peel was rinsed with purified water for 30 seconds to remove residual dirt, dust, and other impurities. A horizontal cut was made approximately 1 cm from the stem end to remove the pulp, leaving only the peel. The bacterial suspension was evenly inoculated onto the outer and inner surfaces of the tangerine peel using a spray method, with the spray volume sufficient to form uniform water droplets on the peel surface. The peel was then air-dried under ventilated conditions until no obvious droplets remained. The inoculated tangerine peel was then fermented at 25℃ for 12 hours. After fermentation, the peel was rinsed with sterile water for 20 seconds. Once the water boiled, the peel was steamed for 10 minutes to inactivate the bacteria. The inactivated peel was then dried in hot air at 55℃ for 4 hours until the moisture content was reduced to below 5%, while maintaining its intact commercial tangerine peel state, thus obtaining aged small green tangerine peel.

[0063] Example 3

[0064] A method for aging small green tangerines includes the following steps:

[0065] The frozen clumps of Pantotheca Z05 were first activated and cultured on TSA solid medium at 28°C for 24 hours. Then, single colonies were picked and inoculated into TSB liquid medium and cultured with shaking at 28°C and 180 rpm until OD. 600 >1; Centrifuge the bacterial culture at 6000 rpm for 10 min, collect the bacterial precipitate, wash twice with sterile water, resuspend in sterile water, and adjust OD. 600 The value was 1.2, resulting in a clumped Pantotheca Z05 bacterial suspension.

[0066] Small green tangerines were collected from Xinhui, Guangdong. The peel was rinsed with purified water for 30 seconds to remove residual dirt, dust, and other impurities. A horizontal cut was made approximately 1 cm from the stem end to remove the pulp, leaving only the peel. The bacterial suspension was evenly inoculated onto the outer and inner surfaces of the peel using a spray method, with the spray volume sufficient to form uniform water droplets on the peel surface. The peel was then air-dried under ventilated conditions until no obvious droplets remained. The inoculated peel was then fermented at 30℃ for 24 hours. After fermentation, the peel was rinsed with sterile water for 40 seconds. Once the water boiled, the peel was steamed for 12 minutes to inactivate the bacteria. The inactivated peel was then dried in hot air at 65℃ for 5 hours until the moisture content was reduced to below 5%, while maintaining its intact commercial tangerine peel state, thus obtaining aged small green tangerine peel.

[0067] Example 4

[0068] A method for aging small green tangerines includes the following steps:

[0069] The frozen clumps of Pantotheca Z05 were first activated and cultured on TSA solid medium at 28°C for 24 hours. Then, single colonies were picked and inoculated into TSB liquid medium and cultured with shaking at 28°C and 180 rpm until OD. 600 >1; Centrifuge the bacterial culture at 6000 rpm for 10 min, collect the bacterial precipitate, wash twice with sterile water, resuspend in sterile water, and adjust OD. 600 The value was 1.0, resulting in a clumped Pantotheca Z05 bacterial suspension.

[0070] Small green tangerines were collected from Xinhui, Guangdong. The peel was rinsed with purified water for 30 seconds to remove residual dirt, dust, and other impurities. A horizontal cut was made approximately 1 cm from the stem end to remove the pulp, leaving only the peel. The bacterial suspension was evenly inoculated onto the outer and inner surfaces of the peel using a spraying method, with the spray volume sufficient to form uniform water droplets on the peel surface. The peel was then air-dried under ventilated conditions until no obvious droplets remained. The inoculated peel was then fermented at 25℃ for 12 hours. After fermentation, the peel was rinsed with sterile water for 30 seconds. Once the water boiled, the peel was steamed for 10 minutes to inactivate the bacteria. The inactivated peel was then dried in hot air at 60℃ for 4 hours until the moisture content was reduced to below 5%, while maintaining its intact commercial tangerine peel state, thus obtaining aged small green tangerine peel.

[0071] Example 5

[0072] The application of the method described in Example 4 in the preparation of small green tangerine Pu-erh tea includes the following steps:

[0073] Adding Pu-erh tea leaves to the aged small green tangerine peel prepared in Example 4 yields small green tangerine Pu-erh tea.

[0074] Brewing method: First, add a small amount of boiling water and pour out the first brew. Then, add boiling water again, steep for 60 seconds, and pour into a teacup. Drink after the tea has cooled slightly. The resulting tea has a distinct aged aroma, prominent sweetness, reduced astringency, and a long-lasting aftertaste.

[0075] Comparative Example 1

[0076] A method for aging small green tangerines includes the following steps:

[0077] The difference from Example 4 is that sterile water is used instead of bacterial suspension for inoculation, while the other steps and parameters are the same as in Example 4.

[0078] Small green tangerines were collected from Xinhui, Guangdong. The peel was rinsed with purified water for 30 seconds to remove residual dirt, dust, and other impurities. A horizontal cut was made approximately 1 cm from the stem end to remove the pulp, leaving only the peel. Sterile water was sprayed evenly onto both the outer and inner surfaces of the peel, with the amount of water enough to form uniform droplets on the surface. The peel was then air-dried under ventilation until no obvious droplets remained. The peel was then left to stand at 25℃ for 12 hours. After standing, the peel was rinsed with sterile water for 30 seconds. Once the water boiled, the peel was steamed for 10 minutes. The steamed peel was then dried in hot air at 60℃ for 4 hours until the moisture content decreased to below 5%, resulting in the control group of small green tangerine peel.

[0079] Comparative Example 2

[0080] A method for aging small green tangerines includes the following steps:

[0081] The difference from Example 4 is that: the endophytic bacteria reported to accelerate the aging of dried tangerine peel were inoculated separately. Bacillus gibsonii , Bacillus sp. , Aspergillus sp. The bacterial suspension was prepared, and the remaining steps and parameters were the same as in Example 4.

[0082] The Bacillus gibsonii , Bacillus sp. , Aspergillus sp. All strains were purchased from Biobw.

[0083] Bacillus gibsonii bacterial suspension and Bacillus sp. The preparation method of the bacterial suspension is the same as in Example 4: the frozen strain was first activated and cultured on TSA solid medium at 28°C for 24 hours, then a single colony was picked and inoculated into TSB liquid medium, and cultured with shaking at 28°C and 180 rpm until OD. 600 >1; Centrifuge the bacterial culture at 6000 rpm for 10 min, collect the bacterial precipitate, wash twice with sterile water, resuspend in sterile water, and adjust OD. 600 The value is 1.0, and the following are obtained: Bacillus gibsonii bacterial suspension and Bacillus sp. bacterial suspension.

[0084] Aspergillus sp. The spore suspension was inoculated into PDB liquid medium and cultured. The PDB liquid medium was purchased from Solarbio.

[0085] Small green tangerines were collected from Xinhui, Guangdong. The peel was rinsed with purified water for 30 seconds to remove residual dirt, dust, and other impurities. A horizontal cut was made approximately 1 cm from the stem end to remove the pulp, leaving only the peel. The peel was then rinsed using a spraying method. Bacillus gibsonii bacterial suspension, Bacillus sp. bacterial suspension, Aspergillus sp. The spore suspension was evenly inoculated onto the outer and inner surfaces of the peel of the small green tangerine. The spraying volume was sufficient to form uniform water droplets on the peel surface. The peel was then air-dried under ventilated conditions until no obvious droplets remained. The inoculated peel was then fermented at 25℃ for 12 hours. After fermentation, the peel surface was rinsed with sterile water for 30 seconds. After the water boiled, the peel was steamed for 10 minutes to inactivate the spores. The inactivated peel was then dried in hot air at 60℃ for 4 hours until the moisture content was reduced to below 5%, yielding the desired results. Bacillus gibsonii Fermentation group Bacillus sp. Fermentation group Aspergillus sp. Fermented small green tangerine peel.

[0086] Experimental Example 1

[0087] Sensory evaluations were performed on the aged green tangerine peel (fermentation group) prepared in Example 4 and the green tangerine peel (control group) prepared in Comparative Example 1.

[0088] Three fermentation groups were randomly selected, and their fruit peels were placed in 100mL sealed bags. To prevent odor loss due to repeated opening and closing of the bags, each bag was only used for one person's sniffing. A total of 30 sealed bags were sealed. The control group was sealed in the same way, with 30 bags numbered A1-A30, and the fermentation groups were numbered B1-B30. Thirty healthy adult volunteers (25-35 years old) were invited to conduct a sensory evaluation by smelling the bags. Each volunteer randomly selected one bag from the control group and one from the fermentation group to smell and filled out a sensory evaluation form. The sensory evaluation form included seven indicators: color preference, aroma preference, aroma intensity, aroma staleness, odor irritation, texture preference, and overall acceptability. Each indicator was scored on a scale of 1-10, with 1 being the lowest and 10 being the highest.

[0089] Depend on Figure 2 As shown in A, the color preference of the fermented group was significantly higher than that of the control group (p<0.05), indicating that the color of the peel after fermentation is deeper and more natural, conforming to the traditional visual perception of aged tangerine peel and making it more acceptable to consumers. Figure 2 As can be seen from B and C, there was no significant difference in aroma preference and aroma concentration scores between the two groups, indicating that the fermentation treatment achieved sensory improvement while maintaining the original basic aroma characteristics of the small green tangerine. Figure 2 D in the figure represents the aroma aging assessment. It can be seen that the score of the fermentation group is significantly higher (p<0.01), indicating that fermentation accelerates the formation of the characteristic aroma of tangerine peel, making it present a more mellow and mature aroma. Figure 2E represents the irritation rating, with the fermentation group showing a significantly lower rate than the control group (p<0.0001). Combined with open-ended descriptions from volunteers, the fermented samples exhibited a significant reduction in irritating odors such as "raw green taste" and "spicy throat sensation," resulting in a milder and more pleasant aroma overall. This clearly demonstrates that fermentation helps to weaken the initial pungent and astringent characteristics of young green tangerines, achieving a gentler optimization of aroma quality. Figure 2 The F in the figure shows that there was no significant difference in texture score between the two groups, indicating that the fermentation treatment preserved the physical properties of the peel. Figure 2 G represents overall acceptability. The fermentation group had a slightly higher acceptance rate than the control group, but the difference was not significant, suggesting that this indicator is greatly influenced by individual subjective preferences. The results indicate that short-term fermentation treatment significantly improved the sensory characteristics of Xiaoqinggan tangerine peel, including enhancing the aged flavor, reducing the pungent odor, and making the aroma more mature and mellow, consistent with the characteristics of traditional tangerine peel, while other sensory attributes remained basically stable.

[0090] Experimental Example 2

[0091] The color of the aged green tangerine peel (fermentation group) prepared in Example 4 and the green tangerine peel (control group) prepared in Comparative Example 1 were measured.

[0092] Ten finished fruit peel samples were randomly selected from the fermentation group, and the samples were measured using a HunterLab colorimeter. Four points were selected on the front, back, left, and right sides of each circular fruit peel sample for measurement, resulting in a total of 40 measurement points. The control group underwent the same testing method as the fermentation group. Indicates lightness, ranging from 0 (black) to 100 (white). Decreasing lightness results in a darker surface and deeper aging. Indicating a range of hues from green to red, a <0 is green, a >0 is red, a The higher the value, the less green there is, and the deeper the aging process; b b represents a hue ranging from blue to yellow. <0 is blue, b >0 is yellow, b The lower the value, the less yellow there is, and the deeper the aging. Color saturation (Chroma, C )= The intensity of a color is characterized by its "vividness," while saturation decreases (C). A decrease in color indicates a duller appearance, which aligns with the trend of increased "aging" of dried tangerine peel.

[0093] Depend on Figure 3 The A in the figure can be seen to indicate the L of the fermentation group sample. The value was significantly lower than that of the control group, indicating that the overall color of the peel surface had darkened, the degree of blackening had increased, and the aged appearance had intensified. Figure 3B in the text indicates that fermentation group a The value decreased significantly, indicating a further reduction in the green component. (From...) Figure 3 C in the text indicates that the fermentation group b The value also decreased significantly, indicating a weakening of the yellow hue, and the surface color gradually changed from yellowish-green to dark brown. Figure 3 The value of D in the figure indicates the color saturation of the fermentation group. The significant reduction reflects a decrease in the vibrancy of the peel color, resulting in a more subdued and profound visual appearance. The results indicate that fermentation treatment can effectively accelerate the transition of the peel color of the small green tangerine from bright green to a dark brown, aged state.

[0094] Experimental Example 3

[0095] The volatile aroma components of the aged green tangerine peel (fermentation group) prepared in Example 4 and the green tangerine peel (control group) prepared in Comparative Example 1 were analyzed.

[0096] Volatile organic compounds (VOCs) in fermented and finally dried tangerine peels were analyzed using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME / GC-MS). Each sample, after being ground in liquid nitrogen, was accurately weighed at 0.04 g and transferred to a 20 mL headspace vial with a screw cap. Then, 2 mL of saturated sodium chloride solution and 20 μL of 0.1% (v / v) 1-hexanol (as an internal standard) were added. The vials were sealed with PTFE / silicone stoppers and magnetic screw caps. Sample pretreatment was performed using an Agilent PAL autosampler. The headspace vial was shaken at 45°C and 300 rpm for 30 min, and then a 50 / 30 μm DVB / CAR / PDMS fiber (Supelco, USA) was inserted for adsorption of volatiles. The puncture depth was 22 mm and the adsorption time was 30 min. After extraction, the fiber was immediately transferred to the injection port for thermal desorption at 250°C for 5 min (puncture depth: 54 mm; carrier gas pressure: 7.1 psi; plug flushing flow rate: 3 mL / min). The gas chromatography system used was an Agilent 7890A gas chromatograph coupled with a 5975C mass spectrometer with a selective detector (Agilent Technologies, USA). Volatile organic compounds were separated using an HP-5 capillary column (30 m × 0.25 mm, 0.25 μm) with helium as the carrier gas at a constant flow rate of 1.0 mL / min (column pressure: 7.1 psi). The temperature program was as follows: initial temperature 40 °C, hold for 3 min, then ramp to 70 °C at a rate of 3 °C / min (hold for 0 min), then ramp to 130 °C at a rate of 1 °C / min (hold for 0 min), and finally ramp to 230 °C at a rate of 15 °C / min (hold for 0 min). Mass spectrometry was performed in electron ionization (EI) mode with an ionization energy of 70 eV. The interface and ion source temperatures were both set to 230 °C. Full scan mass spectrometry was performed in the m / z range of 20–500, with a scan interval of 5 s.

[0097] Figure 4 The VOCs that showed significant changes after 12 hours of fermentation of small green tangerine peel were analyzed. Figure 5 These are the VOCs that showed significant changes in the peel of the small green tangerine after final drying. Figure 4 and Figure 5It can be seen that a total of 11 VOCs underwent significant changes, and their content changes after 12 hours of fermentation were basically consistent with those of the final dried peel. After 12 hours of fermentation, the total VOC content in the peel decreased, which in turn led to a decrease in the total VOC content of the final peel. Among them, the content of 7 VOCs decreased significantly, including: D-limonene (citrus aroma), γ-terpinene (citrus and lemon aroma), terpinene (citrus herbal aroma), 4-terpene alcohol (peppery woody aroma), α-terpineol (woody floral aroma), carvacrol (camphor and thymol aroma), and methyl 2-(methylamino)benzoate (floral and fruity aroma). By reviewing the aroma descriptions of these 7 VOCs, it was found that the fermentation treatment by bacteria can reduce the citrus aroma, floral aroma, and some pungent woody aroma of the small green tangerine peel. In addition, fermentation treatment increased the content of four VOCs: caryophyllene (sweet woody aroma), α-ursulphene (woody aroma), α-farnesene (citrus herbal woody aroma), and sweet orange aldehyde. The significant increases in caryophyllene, α-ursulphene, and α-farnesene added a mellow woody aroma to the peel of the small green tangerine, enhancing its aged flavor. The results showed that fermentation treatment significantly reduced the total VOC content, specifically reducing fruity, floral, and some woody aromas to remove pungent odors; simultaneously, the increase in some mild woody aromas added a mellow aged aroma to the peel.

[0098] Test Example 4

[0099] The total flavonoid content of aged green tangerine peel (fermentation group) prepared in Example 4 and green tangerine peel (control group) prepared in Comparative Example 1 was determined.

[0100] All samples were first flash-frozen in liquid nitrogen and then ground into a fine powder using a sample grinder. 0.1 g of fresh sample was accurately weighed and extracted with 1 mL of 70% (v / v) methanol at 25°C under ultrasonic-assisted conditions for 30 min. After centrifugation at 12000 rpm for 20 min, the supernatant was collected. Then, 1 mL of 70% methanol was added to re-extract the sample, and the process was repeated once. The two supernatants were mixed to achieve a final material-to-solvent ratio of 1:20 (w / v) and stored at 4°C for further analysis. An Agilent HPLC system was used with a Waters ODS C18 column (Sunfire 5μm, 4.6×250mm) as the stationary phase. The mobile phase consisted of chromatographic acetonitrile (solution A) and ultrapure water containing 0.1% formic acid (solution B). A linear gradient elution was employed, with the following gradients: 0–5 min, solution A 20%; 5–10 min, solution A 20%–27%; 10–15 min, solution A 27%; 15–25 min, solution A 27%–40%; 25–35 min, solution A 40%–60%; 35–40 min, solution A 60%–80%; 40–42 min, solution A 80%–100%; 42–45 min, solution A 100%–20%; 45–52 min, solution A 20%. Detection wavelengths: 280 nm and 330 nm; column temperature: 25 °C; injection volume: 10 μL; flow rate: 1 mL / min.

[0101] The results are as follows Figure 6 As shown, by Figure 6 It can be seen that there was no significant difference in the total flavonoid content between the fermentation group and the control group. The results indicate that the microbial fermentation treatment has no effect on the total flavonoid content of the small green tangerine, and while reducing the irritation of the small green tangerine peel, it does not lead to the loss of flavonoid nutrients in the peel.

[0102] Experimental Example 5

[0103] The antioxidant activity of aged green tangerine peel (fermentation group) prepared in Example 4 and green tangerine peel (control group) prepared in Comparative Example 1 was determined.

[0104] The antioxidant capacity of different extracts was evaluated by measuring four antioxidant indicators: ABTS free radical scavenging activity, DPPH free radical scavenging capacity, iron ion reducing antioxidant capacity (FRAP), and oxygen free radical absorption capacity (ORAC).

[0105] ABTS free radical scavenging activity assay: Prepare 7 mM ABTS and 2.6 mM potassium persulfate (K2S2O8) solution, mix them in a 1:1 ratio, and incubate at 25°C in the dark for 12 hours. Then dilute the resulting ABTS working solution with solvent to OD. 734nm≈0.63. A suitable amount of sample (10 μL) was mixed with 200 μL of ABTS working solution, and after reacting for 5 minutes, the absorbance was measured at a wavelength of 734 nm. The ABTS radical scavenging capacity was calculated using the Trolox standard curve and expressed in Trolox equivalents (TE) per gram.

[0106] DPPH radical scavenging capacity determination: Take an appropriate amount of diluted sample (2 μL) and add 198 μL of freshly prepared 60 μM DPPH solution. After reacting the mixture at 25°C in the dark for 2 hours, measure the absorbance at 517 nm using a microplate reader. Calculate the DPPH radical scavenging capacity using the Trolox standard curve and express it in Trolox equivalents (TE) per gram.

[0107] Ferrous Reduction Antioxidant Capacity (FRAP) Determination: Prepare the FRAP working solution by mixing 300 mM pH 3.6 sodium acetate buffer, 10 mM TPTZ solution, and 20 mM FeCl3 solution in a 10:1:1 ratio. Take an appropriate amount of the diluted sample (20 μL) and mix it with 180 μL of the FRAP working solution. After reacting for 5 minutes, measure the absorbance at 593 nm. Calculate the FRAP value using the Trolox standard curve and express it in Trolox equivalents (TE) per gram.

[0108] Oxygen radical absorbance capacity (ORAC) determination: A suitable amount of diluted sample (25 μL) was added to 150 μL of 40 nM fluorescein sodium solution. After reacting for 10 minutes, 150 mM AAPH solution (25 μL) was added. Fluorescence intensity was measured by excitation at 485 nm and emission at 535 nm, recorded every 2 minutes for 2 hours. The net area under the fluorescence decay curve of the sample and AAPH was calculated (net AUC), and the ORAC value was calculated using the Trolox standard curve, expressed in Trolox equivalents (TE) per gram.

[0109] Figure 7 This study demonstrates the performance of the small green tangerine peel extract in four methods for assessing antioxidant capacity, among which... Figure 7 In this context, A represents the ABTS free radical scavenging ability. Figure 7 The B in the text represents the DPPH free radical scavenging ability. Figure 7 The C in the figure represents the iron ion reducing antioxidant capacity and Figure 7In this context, D represents the oxygen free radical absorption capacity. It can be seen that, among the four methods, the differences in antioxidant capacity between the control group and the fermentation group did not reach statistical significance (p > 0.05). The results indicate that fermentation treatment had no significant effect on the antioxidant capacity of the small green tangerine peel extract. The small green tangerine peel extract possesses antioxidant capacity, and the antioxidant capacity of the fermentation group remained stable compared to the control group, consistent with the finding that the total flavonoid content did not change significantly in Experiment 4.

[0110] Experimental Example 6

[0111] Sensory evaluations were performed on the small green tangerine Pu-erh tea (fermentation group) prepared in Example 5 and the small green tangerine Pu-erh tea (control group) prepared from the peel of small green tangerines obtained in Comparative Example 1.

[0112] First, add a small amount of boiling water, pour out the first brew, add boiling water again, steep for 60 seconds, then pour into a teacup. Drink the tea after it has cooled slightly. The control group (Group A) brewed the tea using the same method as the fermentation group (Group B). Each group brewed 30 cups, which were then consumed by 30 volunteers, who completed a sensory evaluation form. The sensory evaluation form included eight indicators: appearance (tea color, tea purity), aroma (fruity, aged aroma), taste (sweet, astringent), and aftertaste (lingering sweetness, salivation). Each indicator was scored on a scale of 1 to 10, with 1 being the lowest and 10 the highest.

[0113] Figure 8 The middle section shows the sensory differences between the control group and the fermented group in terms of tea soup appearance, aroma characteristics, taste, and aftertaste of the small green tangerine Pu'er tea. Figure 8 In the figure, A and B are the scores for tea soup color and purity, respectively. As can be seen from the figure, the difference between the two is not significant (ns), indicating that the fermentation process did not significantly change the basic properties of the tea soup appearance and maintained its original visual stability. Figure 8 C and D in the figure represent the scores for fruity aroma and aged aroma, respectively. The figure shows that the fermented group significantly outperformed the control group in terms of aged aroma (p<0.01), indicating that fermentation effectively promoted the formation of the unique aged flavor of the small green tangerine, resulting in a more mellow and mature tea aroma, consistent with the aroma characteristics of traditional aged tangerine peel tea. The fruity aroma score was also slightly higher than the control group (p<0.05), indicating that fermentation did not weaken the fruity aroma base but rather enhanced the overall aroma complexity. The most significant differences were in taste. Figure 8 The E value in the figure shows a significant increase in the sweetness score of the fermentation group (p<0.01), which is due to... Figure 8 The F value shows a significant decrease in astringency (p<0.0001), indicating that the fermentation process reduced bitterness and astringency while enhancing the sweetness and smoothness of the tea soup. In the aftertaste index, Figure 8 G represents the aftertaste score. The fermented group was significantly better than the control group (p<0.01), indicating that it can leave a lasting sweet experience after drinking and has a better aftertaste. Figure 8 H represents the salivation score, and the difference between the two groups was not significant, suggesting that fermentation treatment had a limited impact on this characteristic. The results indicate that short-term fermentation treatment significantly improved the aroma mellowness and taste smoothness of Xiaoqinggan Pu'er tea, especially in reducing astringency and enhancing sweetness and aged aroma, while maintaining stable appearance.

[0114] Experimental Example 7

[0115] The aged green tangerine peel prepared in Example 4 (Z05 fermentation group), the green tangerine peel prepared in Comparative Example 1 (control group), and the peel prepared in Comparative Example 2 were compared. Bacillus gibsonii Fermentation group Bacillus sp. Fermentation group Aspergillus sp. A comparison of the fermentation effects of small green tangerine peels in the fermentation group.

[0116] The aroma determination method was the same as in Experiment 3, and the sensory evaluation method for the tea soup was the same as in Experiment 6.

[0117] The results are as follows Figure 9 As shown, by Figure 9 As can be seen from A in the figure, compared with the control group, the Z05 fermentation group significantly reduced the total VOCs content (p<0.05), while Bacillus gibsonii Fermentation group Bacillus sp. Fermentation group Aspergillus sp. The fermentation group had no significant effect on the total amount of VOCs (ns). Figure 9 In the figure, B represents the comprehensive sensory score of the Xiaoqinggan Pu'er tea soup. As shown in the figure, the comprehensive score of the Z05 treatment group was significantly higher than that of the control group (p<0.05), specifically manifested as decreased astringency, higher sweetness, and better aftertaste. The other fermentation treatment groups showed limited improvement compared to the control group, and none were significant (ns). The results indicate that under the conditions of this fermentation process, compared to previously reported… Bacillus gibsonii , Bacillus sp. , Aspergillus sp. With endophytic bacteria, Z05 exhibits a superior effect in reducing irritation, resulting in better overall sensory acceptance when brewing small green tangerine Pu-erh tea.

[0118] The above results demonstrate that the Pantothenic Acid Z05 and the aging method for small green tangerines provided by this invention can complete the aging process that traditional processes require more than one year within 24 hours, increasing the aging efficiency by more than 300 times. After fermentation, the pungent odor of the small green tangerine peel is significantly reduced, the aged aroma is significantly enhanced, and the appearance color changes to a dark brown aged state, while the total flavonoid content and antioxidant activity remain stable. Compared with other reported endophytic bacteria, the Pantothenic Acid Z05 of this invention performs superiorly in improving sensory quality. This invention has a simple process, short cycle, and stable effects, making it suitable for industrial production and showing good application prospects.

[0119] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of pantothenic fungus that can be used for the aging of small green tangerines, characterized in that, It is a cluster of pantothecin Z05, with the Latin classification name... Pantoea agglomerans, It was deposited on November 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36512.

2. The application of the Pantothecin clumps as described in claim 1 in the aging of small green tangerines.

3. A method for aging small green tangerines, characterized in that, Includes the following steps: The bacterial suspension of Pantothecin as described in claim 1 was inoculated onto the peel of a small green tangerine; after air drying, it was fermented; after fermentation, the peel was inactivated and dried to obtain aged small green tangerine peel.

4. The method according to claim 3, characterized in that, Before inoculation, the peel of the small green tangerines should be washed and the pulp removed.

5. The method according to claim 3, characterized in that, The OD of the bacterial suspension 600 The value is 0.6-1.

2.

6. The method according to claim 3, characterized in that, The bacterial suspension is inoculated onto the outer and / or inner surface of the peel of the small green tangerine by spraying; the air drying is carried out under ventilated conditions until no liquid droplets remain on the surface of the peel.

7. The method according to claim 3, characterized in that, The fermentation process is carried out at a temperature of 25-30℃ for 12-24 hours.

8. The method according to claim 3, characterized in that, After fermentation, the surface of the fruit peel is first rinsed with water, and then inactivated. The rinsing time is 20-40 seconds. The inactivation is carried out by steaming for 10-12 minutes.

9. The method according to claim 3, characterized in that, The drying process involves drying at 55-65℃ for 4-5 hours, resulting in a fruit peel moisture content of less than 5%.

10. The application of the Pantothecin bacteria of claim 1 or the method of any one of claims 3-9 in the preparation of small green tangerine Pu-erh tea.

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