Method for producing eurotium cristatum and hippophae rhamnoides leaf tea through solid state fermentation of aspergillus cristatum (MK346334, NCBI) separated from Fuzhuan tea
By using the solid-state fermentation process of Aspergillus cristatus to make sea buckthorn green tea, the problem of low utilization rate of sea buckthorn leaves has been solved, achieving efficient utilization of sea buckthorn resources and improving tea quality.
Patent Information
- Application Number
- CN202511357390.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-22
- Publication Date
- 2025-12-12
AI Technical Summary
The utilization rate of sea buckthorn leaves is low, and their nutritional value has not been fully developed. Existing tea processing technology has failed to effectively utilize the enzymatic action of Aspergillus cristatus, resulting in insufficient utilization of sea buckthorn resources and economic benefits.
The method of solid-state fermentation of sea buckthorn green tea using Aspergillus cristatus (MK346334) includes fixation, rolling, baking, aroma enhancement and sterilization. The tea is then inoculated with a suspension of Aspergillus cristatus spores for 8 days of fermentation, and its extracellular enzymes are used to promote the transformation of substances in the tea leaves.
It improves the nutritional value and economic benefits of sea buckthorn leaves, significantly transforms the substances contained in tea leaves, and greatly improves the flavor and aroma, as well as the color of the tea leaves and the color of the tea liquor.
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Figure CN121101043A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The method for producing sea buckthorn leaf fermented tea by using Aspergillus corallinus isolated from Fu brick tea for solid state fermentation relates to the fields of microbial fermentation and deep processing of tea. BACKGROUND
[0002] Sea buckthorn, also known as acer, is a deciduous shrub of the Elaeagnaceae family. Sea buckthorn is a resource of medicine and food, and different parts are used in traditional Chinese medicine to relieve cough, aid digestion, promote blood circulation, and relieve pain. Pharmacological studies have shown that some crude extracts of sea buckthorn have various health benefits, such as anti-inflammatory, antioxidant, liver protection, anti-cardiovascular disease, blood glucose and lipid lowering, neuroprotection, and antibacterial activity. In recent years, the utilization of sea buckthorn fruit has increased significantly, and sea buckthorn juice is produced as a new type of beverage such as milk tea, yogurt, etc. However, after the sea buckthorn fruit is harvested, the sea buckthorn leaves are often discarded. Sea buckthorn leaves are easy to obtain, have a large quantity and nutritional value, and have great potential for reuse. Therefore, optimizing the traditional tea processing technology and exploring the way of sea buckthorn leaf tea making has great significance for turning waste into treasure and improving market efficiency. Aspergillus corallinus, also known as golden flower fungus, is the dominant fungus in the process of Fu brick tea flower production. Aspergillus corallinus produces and secretes various enzymes, including alpha-amylase, polyphenol oxidase, ferulic acid esterase, pectinase, and protease. During the "flowering" process of Fu brick tea, Aspergillus corallinus becomes the dominant species and proliferates rapidly, releasing various extracellular enzymes while completing metabolism and transformation. These enzymes promote the transformation, degradation, oxidation and polymerization of internal substances in tea leaves, resulting in a series of changes that give tea unique health benefits. Studies have shown that tea fermented by Aspergillus corallinus and its metabolites have health benefits such as antibacterial, blood lipid and glucose lowering, weight loss, antioxidant, and immune enhancement. Currently, the utilization rate of sea buckthorn leaves is low, and their nutritional value has not been fully developed. Therefore, applying Aspergillus corallinus to the solid state fermentation of sea buckthorn green tea can make full use of sea buckthorn resources and improve the utilization rate and economic benefits of sea buckthorn, which is of great significance for the deep processing of sea buckthorn products. SUMMARY
[0003] The present application aims to improve the utilization rate of sea buckthorn resources, explore the nutritional value of sea buckthorn leaves, and improve economic benefits. A process for producing sea buckthorn leaf fermented tea by using Aspergillus corallinus for solid state fermentation is provided, which can be used for preparing golden flower sea buckthorn leaf tea.
[0004] The present application is realized by the following technical solutions:
[0005] The preparation of sea buckthorn green tea is as follows:
[0006] 1) Freshly picked sea buckthorn leaves are subjected to fixation (393℃), rolling (58r / min, 12min), baking (125℃)
[0007] and aroma extraction (100℃ to 50℃, 15min) to obtain sea buckthorn green tea;
[0008] 2) Sterilize the tea leaves at 121℃ for 15 minutes to obtain sterile tea leaves.
[0009] Preparation of Aspergillus corntus suspension:
[0010] 1) Inoculate Aspergillus corntus strain (MK346334, NCBI) in potato dextrose medium (PDA) with 15% sucrose as carbon source in a constant temperature incubator at 28℃ for 14 days.
[0011] 2) In a sterile environment, scrape the Aspergillus corntus spores in the PDA plate and transfer them to sterilized pure water. Adjust the spore concentration of the spore suspension to 10 9 -10 11 spores / mL by using a hemocytometer.
[0012] Solid-state fermentation of Aspergillus corntus tea leaves, the steps are as follows:
[0013] 1) Inoculate 8% Aspergillus corntus spore suspension into sterile tea leaves;
[0014] 2) Perform solid-state fermentation at 28℃ for 8 days, and dry to obtain golden flower sea buckthorn leaf tea.
[0015] The beneficial effects of the present application are:
[0016] 1. Sterilize the sea buckthorn green tea before fermentation to ensure single-species fermentation of Aspergillus corntus, which is conducive to improving fermentation efficiency and controlling fermentation conditions.
[0017] 2. The fermentation strain Aspergillus corntus (MK346334, NCBI) is isolated and purified from Fu brick tea in the laboratory, has the characteristics of non-toxicity and safety, secretes multiple extracellular enzymes (polyphenol oxidase, tanninase, and laccase), and can fully utilize the substances in tea leaves for metabolism and promote the transformation of tea leaf contents.
[0018] 3. The sea buckthorn leaves in the present application have a wide source. Using sea buckthorn leaves as raw materials, sea buckthorn green tea is prepared, and then inoculated with Aspergillus corntus for solid-state fermentation, which is conducive to improving the utilization rate of sea buckthorn resources, exploring the nutritional value of sea buckthorn leaves, improving the added value and economic benefits of the product, and has great application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 is the change of water extract content of sea buckthorn leaf tea at different fermentation stages in Example 3.
[0020] Figure 2 is the change of total phenol content of sea buckthorn tea leaves at different fermentation stages in Example 3.
[0021] Figure 3is the total flavonoid content of sea-buckthorn tea leaves at different fermentation stages in Example 3.
[0022] Figure 4 is the total amino acid content of sea-buckthorn tea leaves at different fermentation stages in Example 3.
[0023] Figure 5 is the total sugar content of sea-buckthorn tea leaves at different fermentation stages in Example 3.
[0024] Figure 6 is the radar chart of electronic nose sensor response values and the principal component analysis results of response values in Example 4.
[0025] Figure 7 is a comparison chart of tea leaves before and after solid-state fermentation and tea soup.
[0026] Figure 8 is the radar chart of sensory evaluation aroma in Example 4. DETAILED DESCRIPTION
[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application are further described below in combination with specific examples and the drawings of the specification. The described embodiments are some embodiments of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0028] Example 1: Preparation of Aspergillus coronatus suspension, the steps are as follows:
[0029] The Aspergillus coronatus strain (MK346334, NCBI) that has been isolated, purified and identified is inoculated on PDA medium added with 15% sucrose as a carbon source, and cultured in a 28°C constant temperature incubator for 14 days. The medium is rinsed with sterilized pure water, a small inoculation loop is carefully used to scrape off the yellow cleistothecia, and the scraped cleistothecia are sucked into a sterile container. The spore suspension is adjusted to a concentration of 10 9 -10 11 individuals / mL by using a hemocytometer.
[0030] Example 2: A method for producing sea-buckthorn leaf fermented tea by using Aspergillus coronatus solid-state fermentation, the steps are as follows:
[0031] Freshly picked sea buckthorn leaves were subjected to fixation (393 °C), rolling (58 r / min, 10-15 min), baking (125 °C) and aroma extraction (temperature decreased from 100 °C to 50 °C within 15 min), and cooled to obtain sea buckthorn green tea; in the sea buckthorn green tea, pure water was added to the tea leaves to a water content of 45%, and sterilized at 121 °C for 20 min. After cooling to room temperature, inoculate the spore suspension, and incubate in a 28 °C incubator for 8 days. Take samples every 2 days, place them in a 70 °C oven for 2 h, and perform subsequent analysis. Dry the fermented tea leaves, cool them to room temperature, and store them in a package.
[0032] Example 3: Chemical composition analysis of fermented tea leaves, the steps of which are as follows:
[0033] Experiment 1: Determination of tea water extract content at different fermentation stages. Refer to GB / T 8305-2013 Tea Water Extract Determination.
[0034] Experiment 2: Total phenolic and flavonoid content determination at different fermentation stages. 0.2 g of ground tea sample was placed in a 50 mL centrifuge tube, and 14 mL of 50% ethanol was added to the tube according to a solid-to-liquid ratio of 1:70. The mixture was shaken well and extracted at 70°C for 10 min. After extraction, the solution was allowed to cool to room temperature, and then centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter and used for total phenolic and flavonoid content determination. For total phenolic content determination, 0.1, 0.15, 0.2, 0.25, and 0.3 mg / mL gallic acid standard solutions were prepared. 100 μL of each standard solution was added to 500 μL of 10% Folin-Ciocalteu reagent, and the mixture was allowed to stand for 5 min. Then, 400 μL of 7.5% (w / v) sodium carbonate solution was added, and the mixture was allowed to stand in the dark for 60 min. The absorbance at 765 nm was measured, and a standard curve was prepared. The sample was diluted to an appropriate concentration, and 100 μL of the diluted sample was added to the centrifuge tube. The absorbance was measured according to the standard curve, and the total phenolic content was calculated. The unit was mg GAE / g DW. For total flavonoid content determination, 0.1, 0.2, 0.4, 0.6, and 0.8 mg / mL rutin standard solutions were prepared. 200 μL of each standard solution was added to a 2 mL centrifuge tube, and 100 μL of 5% (w / v) NaNO2 solution was added. After 6 min, 100 μL of 10% (w / v) Al(NO3)3 solution was added, and the mixture was allowed to stand for 6 min. Then, 800 μL of 4% (w / v) NaOH solution was added, and the mixture was mixed well. The absorbance at 510 nm was measured, and a standard curve was prepared. The sample was diluted to an appropriate concentration, and 200 μL of the diluted sample was added to the centrifuge tube. The absorbance was measured according to the standard curve, and the total flavonoid content was calculated. The unit was Rutin equivalents (mg rutin / g DW).
[0035] Experiment 3: Determination of free amino acids in tea at different fermentation stages. 0.3 g of ground tea sample was weighed into a 15 mL centrifuge tube, 12 mL of boiling water was accurately measured into the centrifuge tube according to a solid-liquid ratio of 1:40, and the centrifuge tube was shaken well. The sample was extracted in a boiling water bath for 30 min, and then centrifuged at 8000 rpm for 5 min. The supernatant was filtered with a 0.45 μm water filter membrane for use. The content of total free amino acids was determined by the ninhydrin colorimetric method. The concentration gradient of theanine standard solution was 0, 0.13, 0.16, 0.19, 0.22, and 0.25 mg / mL. 300 μL of the above standard solution was taken into a 2 mL centrifuge tube, and 300 μL of 0.01 M PBS buffer (pH 7.4) and 300 μL of 2% ninhydrin solution were added in turn. After boiling in a water bath for 15 min, the sample was naturally cooled to room temperature, and the absorbance at 570 nm was measured. The standard curve was drawn. The sample was diluted to the appropriate concentration, and 300 μL of the sample was taken into a 2 mL centrifuge tube. The absorbance was measured according to the method of the standard curve, and the data was substituted into the standard curve to calculate the content of free amino acids, which was expressed in mg Thea / g DW.
[0036] Experiment 4: Determination of total sugar content at different fermentation stages. 0.2 g of ground tea was weighed into a 15 mL centrifuge tube, 8 mL of boiling water was added according to a solid-liquid ratio of 1:40, and the sample was shaken well. The sample was extracted in a boiling water bath for 30 min, and the extraction was repeated three times. The three extraction solutions were combined and filtered into a 50 mL centrifuge tube using a small funnel for determination of total sugar. The total sugar was determined by the phenol-sulfuric acid method. 2 mL of the diluted extraction solution was taken into a test tube, 1 mL of 5% phenol solution was added, and then 5 mL of concentrated sulfuric acid solution was slowly added with a pipette. The reaction system was shaken well after standing for 10 min. The absorbance at 490 nm was measured. D(+)-anhydrous glucose was used as the standard, and the standard curve of absorbance vs. concentration was drawn by preparing standard solutions with different concentration gradients. The absorbance of the sample was substituted into the standard curve to calculate the total sugar content, which was finally expressed in mg DE / g DW.
[0037] Example 4: Electronic nose analysis and sensory evaluation, the steps are as follows:
[0038] Experiment 1: 2.0 g of crushed tea was weighed into a 20 mL headspace bottle, 10 mL of boiling water was added, and the sample was equilibrated in a 65°C oven for 2 h before detection. The experimental conditions were as follows: sample washing time was 120 s, automatic zeroing time was 5 s, sample preparation time was 5 s, sensor and internal flow rates were 0.4 L / min and 0.15 L / min, respectively, sampling time was 100 s, each fermentation sample was repeated 3 times, and the volatile components of the tea were analyzed.
[0039] Experiment 2: Based on GB / T 23776-2018 Sensory Evaluation Methods for Tea and GB / T 14487-2017 Sensory Evaluation Terminology for Tea, and considering the aroma characteristics of fermented tea, six aroma attributes of Aspergillus cristatus fermented tea were selected: floral, woody, grassy, fungal-floral, fruity, and sweet. Evaluators independently scored the aroma intensity of the six tea samples on a 0-6 intensity scale (0 = not detected, 6 = very strong). The scoring table is shown in Table 1.
[0040]
[0041] The sensory evaluation panel consisted of eight members, aged between 20 and 30, all with extensive experience in tea evaluation. The scoring sheet is shown in Table 2.
[0042]
[0043] like Figure 1 As shown, in the early stages of fermentation, *Aspergillus cristatus* is still in the adaptation phase, secreting a small amount of extracellular enzymes to begin degrading some insoluble substances in the tea leaves (such as cellulose and pectin), resulting in a slight increase in the water extract content. From day 4 to day 6 of fermentation, vigorous growth of *Aspergillus cristatus* caused the water extract content to drop to 33.56%. Afterward, *Aspergillus cristatus* may enter a secondary metabolic phase, secreting more complex enzyme systems to further decompose recalcitrant substances and release new soluble components, leading to a slight recovery in the water extract content to 34.80% on day 8 of fermentation. Overall, the change in the water extract content of the tea leaves was not significant during the 8-day fermentation process.
[0044] like Figure 2 As shown, the total phenol content first decreases and then increases during fermentation. In the early stages of fermentation, the degradation of total phenols may be caused by moisture and heat reactions. The sharp increase in total phenol content from day 4 to day 6 of fermentation is closely related to the growth of microorganisms. *Aspergillus cristatus* proliferates rapidly around day 5 of fermentation. Its pectinase, tanninase, and glycosidase enzymes can act on sea buckthorn leaves to release bound phenolic substances from the leaf wall, converting conjugated phenols into free phenols, thereby improving the nutritional value of the tea. Figure 3 As shown, the total flavonoid content did not change significantly before the large-scale growth of Aspergillus cristatus, with only small fluctuations within the range of 1 mg Rutin / g DW. From day 4 to day 8 of fermentation, the flavonoid content increased by 5.5 mg Rutin / g DW.
[0045] like Figure 4 As shown, the total free amino acids decreased rapidly from day 0 to day 2 of fermentation, and showed no significant change after day 6 of fermentation, and began to stabilize. This is actually the result of the dynamic balance between the production and metabolism of free amino acids: in the Aspergillus cristatus fermentation system, amino acids are an indispensable nitrogen source, providing continuous energy for the growth and metabolic activities of the cells. During this process, protein degradation reactions occur simultaneously.
[0046] As Figure 5 shown, in the early fermentation stage (0-4 days), G. coronatum decomposes cellulose, hemicellulose, pectin, etc. to generate soluble sugars, and the extracellular polysaccharides (such as β-glucan) synthesized by the microorganism in the early stage contribute to the total sugar increment, so the total sugar content accumulates and reaches the highest level (126.25 mg DE / g DW) on the 4th day. Subsequently, due to the rapid proliferation of G. coronatum around the 5th day of fermentation, a large amount of carbon source is needed, and the total sugar decreases to (91.78 mg DE / g DW), and the water-insoluble cellulose in tea is decomposed into water-soluble monosaccharides or disaccharides, polysaccharides, etc., making the tea soup more sweet and mellow.
[0047] As Figure 6 shown, the response values on the 0th day and the 8th day of fermentation are different, indicating that there is a certain difference in the odor of the two, mainly reflected in the W1W and W2W sensors. The W1W sensor is sensitive to inorganic sulfides, and the W2W sensor is sensitive to aromatic compounds and organic sulfides. The results show that fermentation reduces the content of inorganic sulfides in tea and also reduces the content of organic sulfides or aromatic compounds, which may be due to the removal of green smell after fermentation and the production of fungus smell and stimulating metabolites. As Figure 6 shown in B, the contribution rates of PC1 and PC2 are 70.7% and 17.9%, respectively, and the sum of the contribution rates is 88.6%, which can effectively represent the flavor information of fermented tea.
[0048] The finished product of fermentation is shown in Figure 7 G. coronatum fermentation deepens the color of tea leaves, and the leaf surface is full of golden flowers with obvious fungus smell. The tea soup changes from yellow to orange yellow.
[0049] As Figure 8 shown in the sensory evaluation aroma radar chart, the woody aroma and fungus flower aroma in the aroma of fermented tea are relatively obvious. The comprehensive score of tea is 89.86, the sand pear leaf fermented tea is uniform, and the golden flower is obvious; the tea soup is orange yellow, clear and bright; the entry is mellow and smooth, and the fungus smell is rich. Fermentation effectively improves the aroma and flavor of sand pear leaf. The specific scores are shown in Table 3:
[0050]
Claims
1. A process for producing golden flower sea buckthorn leaf tea by solid state fermentation using Aspergillus Caudatus (MK346334, NCBI) isolated from Fuzhuan tea, comprising: 1) Freshly picked sea buckthorn leaves are subjected to steps of fixation, rolling, baking and aroma extraction to obtain sea buckthorn green tea; 2) The tea leaves are sterilized at 121℃ for 15 min to obtain sterile tea leaves; 3) 8% Aspergillus Caudatus spore suspension is inoculated into the sterile tea leaves; 4) Solid state fermentation is carried out at 28℃ for 8 days, and the golden flower sea buckthorn leaf tea is obtained after drying.
2. The preparation of sea buckthorn green tea as claimed in claim 1, wherein Step 1) Fresh sea buckthorn leaves are fixed at 393℃, the rolling machine rotates at 58r / min for 12 min, the tea leaves are baked at 125℃, and the temperature should be reduced from 100℃ to 50℃ within 15 min during the aroma extraction process.
3. The method for producing sea buckthorn leaf tea by solid-state fermentation with Aspergillus cristatus as described in claim 1, characterized in that... Step 2) After weighing the sea buckthorn green tea, add pure water to the tea leaves to a moisture content of 45%, then sterilize at 121℃ for 15 min, and cool to room temperature in a sterile environment.
4. The preparation of the conidial suspension of G. coronata (MK346334, NCBI) as claimed in claim 1, wherein Step 3) The G. fimbriatum strain was inoculated on potato dextrose medium (PDA) with 15% sucrose as carbon source and incubated in a constant temperature incubator at 28°C for 14 days. The spores of G. fimbriatum on PDA plates were scraped and transferred into sterilized pure water in a clean bench. The spore concentration of the spore suspension was adjusted to 10 9 -1011spores / mL by using a hemocytometer.
5. The fermented tea leaves of claim 1 are taken every two days, placed in a 70℃ oven for 2h, and used for subsequent chemical substance determination.