A method for expanding the culture and fermentation of red yeast rice fermented summer and autumn tea

Abstract

The present invention discloses a method for expanding the culture of red yeast rice-fermented summer and autumn tea, comprising: preparing a red yeast rice-summer and autumn tea seed solution; preparing a spore suspension; screening conditions for expanding the seed solution culture; and screening conditions for solid-state fermentation. This method utilizes red yeast rice to ferment summer and autumn tea, combining the advantages of red yeast rice to improve the bitter taste of summer and autumn tea. Furthermore, the method determines optimal conditions for expanding the seed solution culture and solid-state fermentation, providing a foundation for expanding fermentation and improving the quality of summer and autumn tea.

Description

Technical Field

[0001] The invention relates to the technical field of red yeast rice fermented summer and autumn tea, in particular to a method for expanding the culture and fermentation of red yeast rice fermented summer and autumn tea. Background Art

[0002] Summer and autumn tea is often discarded due to its bitter taste due to its high phenolamine content. However, its production is far higher than that of spring tea, resulting in significant resource waste. In recent years, with the advent of value-added crop utilization, increasing efforts have been made to improve the utilization rate of summer and autumn tea. In 2014, Liu Shujuan et al. systematically summarized how different processing techniques can be used to reduce the bitterness of summer and autumn tea. In 2022, Shi Daliang et al. inoculated summer and autumn tea with Eurotium cristatum to produce Fuzhuan tea and conducted a sensory evaluation of its flavor, further demonstrating that biotransformation can improve the quality of summer and autumn tea. In 2021, Pan Tianquan et al. used summer and autumn tea and wheat to make distiller's yeast, exploring the koji-making process and providing a research foundation for the cultivation of summer and autumn tea koji. In 2022, Liu Yuchuan et al. used red light withering to improve the flavor and taste of summer and autumn tea. Some researchers have even used summer and autumn tea to improve the immune status of broiler chickens. At present, although some plans for value-added utilization of summer and autumn tea have been proposed, the utilization rate is still low, and there are even fewer plans to improve the quality of summer and autumn tea through biotransformation.

[0003] Monascus is a filamentous fungus primarily used in the food, brewing, and pharmaceutical industries. Although originating in China, Monascus has become widely used worldwide. Currently, its application in the food industry has expanded from its initial use in fermenting rice to fermenting various grains, and even to the feed industry, primarily because the various active substances it produces can effectively enhance the immunity of livestock and poultry. Monascus is also increasingly used in the brewing of baijiu (white liquor), huangjiu (yellow rice wine), sweet rice wine, and fruit and vegetable wines. For example, Li Shaoliang et al. isolated lipidated red yeast rice from brewing koji, resulting in a base liquor with a richer flavor and texture. Wu Yufeng et al. isolated a high-yield lovastatin-producing red yeast rice strain for brewing, significantly increasing the lovastatin content and flavor profile of huangjiu (yellow rice wine). Che Yixin et al. utilized Monascus purpurogenum and Saccharomyces cerevisiae to enhance the flavor and texture of rice wine. Wang Bingying and her colleagues used red yeast rice to ferment highland barley and sweet potatoes to produce low-alcohol purple sweet potato wine. They determined the optimal fermentation process and analyzed the wine's quality, demonstrating its unique flavor and taste. However, to date, no method has been developed to ferment summer and autumn tea using red yeast rice to alter its bitterness, expand its cultivation, and improve its quality.

[0004] In summary, it is necessary to propose a method for expanding the culture and fermentation of red yeast rice fermented summer and autumn tea to solve the above problems. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that, in view of the fact that there is no method for using Monascus to ferment summer and autumn tea to change the bitter taste of summer and autumn tea and to expand the cultivation to improve the quality of summer and autumn tea, a method for expanding the cultivation of summer and autumn tea fermented with Monascus is proposed.

[0006] In order to solve the above technical problems, the present invention provides a method for expanding the culture and fermentation of red yeast rice fermented summer and autumn tea. The method comprises the following steps: Step 1, preparing seed liquid: accurately weighing 5 g of raw summer and autumn tea into a teapot, boiling the mixture with 2000 mL of distilled water for 3 minutes, filtering the mixture, and diluting the mixture by 2 times to obtain a standby tea juice; using the tea juice as a base solution, preparing a seed liquid according to the following ratios: 1.141 g / 50 mL of sucrose, 0.712 g / 50 mL of peptone, and 0.048 g / 50 mL of magnesium sulfate; and sterilizing the mixture under high pressure at 121° C. for 20 minutes to obtain the seed liquid;

[0007] Step 2: Preparation of spore suspension: scrape a loopful of cells from an activated red Monascus plate and culture in a PDA dish for 7 days. Use a 7 mm borer to punch three cakes from the activated red Monascus plate into a sterile PDA dish, invert and culture for 5 days, then scrape and wash the cells with 30 mL of sterile water and filter into a sterile Erlenmeyer flask to obtain a spore suspension.

[0008] Step 3: Expand the seed solution: Take a 150 mL Erlenmeyer flask, add the seed solution, adjust the pH, add the spore suspension, place it in a shaker, shake the flask at 180 rpm, and measure the color value and dry weight of the bacteria;

[0009] Step 4, solid-state fermentation culture: accurately weigh 10g of summer and autumn tea raw tea into a 240mL tissue culture bottle, add 3% sucrose water according to the appropriate water content, sterilize at 100℃, inoculate with seed liquid, place in a constant temperature incubator for culture, and measure its color value.

[0010] Among them, the preservation number of Monascus ruber is CGMCC No.3.15746.

[0011] In step 2, the bacteria were scraped and washed with sterile water, filtered into a sterile triangular flask, and counted using a hemocytometer to ensure that the spore concentration reached 10 6 cfu / mL.

[0012] Among them, in step three, the seed liquid expansion culture was carried out with the shaking table temperature, inoculation size, initial pH, liquid volume and culture time as single factors, and the dry weight and color value of the bacteria as indicators to analyze the influence of each factor on the seed liquid expansion culture, determine the factors and levels, and screen out the better seed liquid expansion culture conditions: temperature 28 ° C, inoculation size 4%, initial pH value 5, liquid volume 50 mL, and fermentation time 8 days.

[0013] Among them, in the solid-state fermentation culture in step 4, water content, fermentation temperature, inoculation size and culture time are used as single factors, and color value is used as an indicator to analyze the influence of each factor on solid-state fermentation. On the basis of single-factor experiments, the Box-Benhnken central composite experimental design principle is adopted, and the color value of red yeast fermented summer and autumn tea is used as the response surface value to optimize the fermentation conditions, and the optimal solid-state fermentation conditions are screened out: culture time 7.163d, inoculation size 34.230%, temperature 28.889℃, and water content 42.383%.

[0014] The implementation of the embodiments of the present invention has the following beneficial effects:

[0015] The present method for expanding the culture and fermentation of red yeast rice fermented summer and autumn tea utilizes red yeast rice to ferment summer and autumn tea, combines the advantages of red yeast rice, thereby changing the bitter taste of summer and autumn tea, and determines the conditions for expanding the culture of seed liquid and the optimal solid-state fermentation conditions, thus providing a certain basis for expanding fermentation and improving the quality of summer and autumn tea. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 Schematic diagram of the effect of temperature on seed liquid growth;

[0018] Figure 2 Schematic diagram of the effect of inoculum size on seed liquid growth;

[0019] Figure 3 Schematic diagram of the effect of initial pH value on seed liquid growth;

[0020] Figure 4 Schematic diagram of the effect of liquid volume on seed liquid growth;

[0021] Figure 5 Schematic diagram of the effect of fermentation time on seed liquid growth;

[0022] Figure 6 It is the average value diagram of bacterial dry weight factor level;

[0023] Figure 7 is the average value graph of color valence factor levels;

[0024] Figure 8 Schematic diagram of the effect of water content on solid-state fermentation;

[0025] Figure 9Schematic diagram of the effect of temperature on solid-state fermentation;

[0026] Figure 10 Schematic diagram of the effect of inoculum size on solid-state fermentation;

[0027] Figure 11 Schematic diagram of the effect of fermentation time on solid-state fermentation;

[0028] Figure 12 The response surface and contour lines of the effects of time and inoculum size on color value;

[0029] Figure 13 The response surface and contour lines of the effects of time and temperature on color valence;

[0030] Figure 14 The response surface and contour lines of the effects of time and water content on color value;

[0031] Figure 15 The response surface and contour lines of the effects of inoculum size and temperature on color value;

[0032] Figure 16 The response surface and contour lines of the effects of inoculum size and water content on color value;

[0033] Figure 17 Response surface and contour lines of the effects of temperature and moisture content on color value. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] The expanded culture fermentation method of red yeast rice fermented summer and autumn tea includes:

[0036] Step 1. Preparation of seed liquid: Accurately weigh 5 g of summer and autumn tea raw tea into a teapot, boil it with 2000 mL of distilled water for 3 minutes, filter it, and dilute it 2 times to obtain the standby tea juice; use tea juice as the base solution, prepare seed liquid according to 1.141 g / 50 mL of tea juice of sucrose, 0.712 g / 50 mL of tea juice of peptone, and 0.048 g / 50 mL of tea juice of magnesium sulfate, and sterilize it under high pressure at 121°C for 20 minutes to obtain the seed liquid.

[0037] Step 2: Preparation of spore suspension: scrape a loop of cells from an activated red Monascus (deposit number CGMCC No. 3.15746) plate and culture in a PDA culture dish for 7 days. Use a 7mm punch to punch three cakes from the activated red Monascus plate into a sterile PDA culture dish, invert and culture for 5 days, then use 30 mL of sterile water to scrape and wash the cells, filter into a sterile triangular flask, and count using a hemocytometer to ensure that the spore concentration reaches 10 6 cfu / mL to obtain a spore suspension.

[0038] Step 3, seed liquid expansion culture: take a 150mL triangular flask, fill it with seed liquid, adjust the pH, access the spore suspension, put them in a shaker respectively, shake the flask at 180r / min, and measure the color value and dry weight of the bacteria. Determination of dry weight of bacteria: use a quantitative filter paper dried to constant weight to filter the fermentation liquid, collect the filtrate with a 50mL centrifuge tube for later use, wash the filter residue with ultrapure water and place it at 60°C to dry to constant weight, weigh and calculate the mass difference before and after, and obtain the dry weight of the bacteria. Determination of color value: refer to GB1886.19-2015 to determine the color value of the filtered fermentation liquid and the color value of solid-state fermentation tea.

[0039] The seed liquid expansion culture conditions were screened, with shaking table temperature, inoculation size, initial pH, liquid volume and culture time as single factors, and dry weight and color value of the bacteria as indicators to analyze the effects of each factor on the seed liquid expansion culture and determine the factors and levels.

[0040] Single factor experiment:

[0041] Test ① Temperature: Place 50 mL of seed solution (pH natural) in a 150 mL Erlenmeyer flask. Inoculate the spore suspension at a 2% inoculum concentration. Incubate the flask at 22°C, 25°C, 28°C, 31°C, and 34°C in a shaker at 180 rpm for 5 days. Measure color value and cell dry weight.

[0042] Temperature is one of the important factors affecting the growth of microorganisms. The environmental factors that affect the growth of microorganisms are diverse. It is particularly important to screen out the optimal temperature for the growth of bacteria under specific conditions. Select the common temperature for the growth of Monascus, and judge the growth and reproduction of the bacteria by measuring the dry weight and color value of the bacteria at different temperatures to find the feasible range of temperature. The results are as follows: Figure 1 shown.

[0043] Depend on Figure 1As can be seen, the color value of the seed solution increases between 22°C and 28°C. It decreases when the temperature rises from 28°C to 31°C, with the color value at 31°C being similar to that at 34°C. The color value is lowest at 22°C, at 1.283 μ / g, and highest at 28°C, at 3.422 μ / g. The dry weight of the seed solution is also lowest at 22°C, at 0.038 g. It reaches its highest at 31°C, at 0.176 g. However, the dry weight at 28°C is similar to that at 31°C. Based on this analysis, the temperature of 22°C should be eliminated, and the feasible temperature range should be set between 25°C and 34°C.

[0044] Test ② Inoculation: Place 50 mL of seed solution in a 150 mL Erlenmeyer flask at a natural pH. Inoculate the spore suspension at 1%, 2%, 3%, 4%, and 5% inoculum. Incubate in a shaker at 28°C and 180 rpm for 5 days. Measure color value and cell dry weight.

[0045] Inoculation size is also one of the important factors affecting fermentation. Too large an inoculation size may cause insufficient dissolved oxygen and limited substrate concentration, which will restrict the growth and reproduction of bacteria. Too small an inoculation size will increase the process of bacterial reproduction. Therefore, the appropriate inoculation size is crucial in fermentation. Select a commonly used inoculation size range, measure the dry weight and color value of the seed liquid bacteria under different inoculation sizes, and find the feasible range of inoculation size. The results are as follows: Figure 2 shown.

[0046] Depend on Figure 2 As can be seen, when the inoculum size increased from 1% to 4%, the cell dry weight showed an upward trend. When the inoculum size increased from 4% to 5%, the cell dry weight decreased significantly. The cell dry weight was lowest at 1%, at 0.029g, and highest at 4%, at 0.178g. When the inoculum size increased from 1% to 2%, the color value increased significantly. When the inoculum size increased from 2% to 3%, the color value decreased significantly. When the inoculum size increased from 3% to 5%, the color value did not change much overall. The color value was lowest at 1%, at 2.038μ / g, and highest at 2%, at 3.491μ / g. Comprehensive analysis shows that the cell dry weight and color value are both lowest at the 1% inoculum size. The 1% inoculum size should be eliminated, and the feasible inoculum size range should be set between 2% and 5%.

[0047] Test ③ Initial pH: Place 50 mL of seed solution in a 150 mL Erlenmeyer flask and adjust the pH to 3, 4, 5, 6, or 7, respectively. Inoculate the spore suspension at a 2% inoculum level. Incubate the flask at 28°C in a shaker at 180 rpm for 5 days. Measure the color value and cell dry weight.

[0048] Microorganisms can usually grow within a certain pH range, but there is an optimal pH at which enzyme activity is highest. The optimal pH for Monascus is usually between 3.5 and 6. We selected the pH range of commonly used Monascus, measured the dry weight and color value of the seed solution at different initial pH values, and found a feasible initial pH range. The results are as follows: Figure 3 shown.

[0049] Depend on Figure 3 As can be seen, when the initial pH value increased from 3 to 5, the bacterial dry weight showed an upward trend, and when the initial pH value increased from 5 to 7, the bacterial dry weight showed a downward trend. When the initial pH value was 3, the bacterial dry weight was the lowest, 0.048g, and when the initial pH value was 5, the bacterial dry weight was the highest, 0.159g. When the initial pH value increased from 3 to 6, the color value showed an upward trend, and when the initial pH value increased from 6 to 7, the color value showed a downward trend. When the initial pH value was 3, the color value was the lowest, 2.061μ / g, and when the initial pH value was 6, the color value was the highest, 3.368μ / g. Comprehensive analysis shows that the initial pH value of 3 should be eliminated, and the feasible initial pH range should be set to 4-7.

[0050] Test 4: Liquid Volume: Take 150 mL Erlenmeyer flasks and fill them with 20 mL, 30 mL, 40 mL, 50 mL, and 60 mL of seed solution, respectively. Set the pH to natural, inoculate with 2% of the spore suspension, and incubate in a shaker at 28°C and 180 rpm for 5 days. Measure the color value and cell dry weight.

[0051] Liquid volume is also one of the important factors affecting shake flask fermentation. Too much liquid volume will result in a low oxygen transfer coefficient, which will restrict the growth and reproduction of the bacteria. Too little liquid volume will result in less nutrient matrix, which will also reduce the growth and reproduction ability of the bacteria. Therefore, we selected the range of liquid volume of conventional shake flasks, measured the dry weight and color value of the seed liquid bacteria under different liquid volumes, and found the feasible range of liquid volume. The results are as follows: Figure 4 shown.

[0052] Depend on Figure 4 As can be seen, when the liquid volume increased from 20 mL to 50 mL, the bacterial dry weight showed an upward trend, and when the liquid volume increased from 50 mL to 60 mL, the bacterial dry weight showed a downward trend. The bacterial dry weight was lowest at 20 mL, at 0.042 g, and highest at 50 mL, at 0.143 g. As the liquid volume increased from 20 mL to 60 mL, the color value showed an upward trend, reaching the lowest color value of 2.015 at 20 mL and the highest color value of 3.418 μ / g at 60 mL. Based on this analysis, the 20 mL liquid volume should be eliminated, and the feasible liquid volume range should be set to 30 mL-60 mL.

[0053] Test 5. Duration: Fill a 150 mL Erlenmeyer flask with 50 mL of seed solution at a natural pH, inoculate with a 2% inoculum of the spore suspension, and incubate in a shaker at 28°C and 180 rpm for 4, 5, 6, 7, and 8 days. Measure color value and cell dry weight.

[0054] Different fermentation time is also one of the important factors for the growth and reproduction of the fungus. According to the physiological characteristics of red yeast rice, the commonly used fermentation time range of red yeast rice was selected, the dry weight and color value of the seed liquid at different fermentation times were measured, and the feasible range of fermentation time was found. The results are as follows: Figure 5 shown.

[0055] Depend on Figure 5 It can be seen that both the dry weight and color value of the bacteria show an overall upward trend as the fermentation time increases. When the fermentation time is 4 days, the dry weight of the bacteria is the lowest, at 0.040g, and when the fermentation time is 8 days, the dry weight of the bacteria is the highest, at 0.133μ / g. When the fermentation time is 4 days, the color agent is the lowest, at 2.181μ / g, and when the fermentation time is 8 days, the color value is the highest, at 3.323μ / g. Comprehensive analysis shows that the 4-day fermentation time should be removed and the feasible range of the fermentation time should be set to 5-8 days.

[0056] Orthogonal experiment

[0057] Based on the results of the single-factor experiments, we selected factors and levels to design a five-factor, four-level orthogonal experiment with temperature, inoculum size, initial pH, liquid volume, and time as factors, and cell dry weight and color value as scoring indicators. The factor levels are shown in Table 1. The orthogonal experiment results were systematically analyzed using the SPSSAU scientific analysis platform to identify the optimal seed solution culture protocol.

[0058] Table 1 Orthogonal test factor level table

[0059]

[0060]

[0061] Based on the feasibility range determined by the single-factor experiment on seed liquid expansion culture conditions, an orthogonal experimental factor level table (Table 1) was developed, and the orthogonal experimental results are shown in Table 2. Range analysis was performed using the SPSSAU system, and the range analysis tables for dry cell weight and color value are shown in Tables 3 and 4, respectively.

[0062] Table 2 Orthogonal test results

[0063]

[0064] Table 3 Analysis of the range of bacterial dry weight

[0065]

[0066]

[0067] Note: “-” means there is no data at that level

[0068] The K value in Table 3 is the sum of the test data at a certain level of a factor, the K avg value is the corresponding average value, and the optimal level refers to the level number corresponding to the optimal K avg value of a factor. Figure 6 It can be intuitively concluded that when the temperature is 28°C, the Kavg of factor 1 is the largest, which is 0.14. When the inoculation size is 4%, the Kavg of factor 2 is the largest, which is 0.12. When the initial pH is 5, the Kavg of factor 3 is the largest, which is 0.1225 (≈0.12). When the liquid volume is 50 mL, the Kavg of factor 4 is the largest, which is 0.12. When the fermentation time is 8 days, the Kavg of factor 4 is the largest, which is 0.12. Therefore, it can be determined that when the dry weight of the bacteria is used as the evaluation index, its optimal level is a temperature of 28°C, an inoculation size of 4%, an initial pH of 5, a liquid volume of 50 mL, and a fermentation time of 8 days. R refers to the range of the factor. When this value = the maximum value of Kavg minus the minimum value of Kavg for a certain factor, the advantages and disadvantages of each factor can be compared in combination with the range of the factors. As can be seen from Table 3, the size of R is arranged as factor 1>factor 3>factor 2>factor 4>factor 5. That is, when the dry weight of the bacteria is used as the evaluation index, the factors affecting the growth of the seed liquid are temperature>initial pH>inoculation size>liquid volume>fermentation time.

[0069] Table 4 Color Price Range Analysis

[0070]

[0071] As shown in Table 4 and Figure 7As shown in Table 4, when the temperature is 28°C, the K avg of factor 1 is maximum, at 2.68. When the inoculum size is 2%, the K avg of factor 2 is maximum, at 2.65. When the initial pH is 5, the K avg of factor 3 is maximum, at 2.63. When the liquid volume is 50 mL, the K avg of factor 4 is maximum, at 2.63. When the fermentation time is 8 days, the K avg of factor 4 is maximum, at 2.63. Therefore, it can be determined that the optimal level for color value evaluation is a temperature of 28°C, an inoculum size of 2%, an initial pH of 5, a liquid volume of 50 mL, and a fermentation time of 8 days. As shown in Table 4, the order of R is factor 2 > factor 5 > factor 1 > factor 4 > factor 3. In other words, when color value is used as the evaluation indicator, the factors affecting seed liquid growth are in the order of inoculum size > fermentation time > temperature > liquid volume > initial pH. The optimal conditions obtained by using color value and dry weight of bacteria as evaluation indicators were basically the same, but when dry weight of bacteria was used as the evaluation indicator, the optimal inoculation size was 4%, and when color value was used as the evaluation indicator, the optimal inoculation size was 2%. Therefore, the fermentation temperature was kept at 28°C, the initial pH value was 5, the liquid volume was 50mL, and the fermentation time was 8 days. Verification tests were carried out with inoculation sizes of 2% and 4%, respectively. When the inoculation size was 2%, the dry weight of bacteria was 0.25 and the color value was 3.61. When the inoculation size was 4%, the dry weight of bacteria was 0.31 and the color value was 3.88. Therefore, the optimal seed liquid culture conditions were determined to be temperature 28°C, inoculation size 4%, initial pH value 5, liquid volume 50mL, and fermentation time 8 days.

[0072] Step 4, solid-state fermentation culture: accurately weigh 10g of summer and autumn tea raw tea into a 240mL tissue culture bottle, add 3% sucrose water according to the appropriate water content, sterilize at 100℃, inoculate with seed liquid, place in a constant temperature incubator for culture, and measure its color value.

[0073] Solid-state fermentation culture was carried out with water content, fermentation temperature, inoculation size and culture time as single factors, and color value as an indicator to analyze the influence of each factor on solid-state fermentation. On the basis of single factor experiment, the Box-Benhnken central composite experimental design principle was adopted, and the color value of red yeast rice fermented summer and autumn tea was used as the response surface value to optimize the fermentation conditions.

[0074] Single-factor experiment

[0075] Test ① Water content: Accurately weigh 10g of summer and autumn tea raw tea into a 240mL tissue culture bottle, add 3% sucrose water at 10%, 20%, 30%, 40%, and 50% water content respectively, sterilize at 100℃, inoculate with seed liquid at a rate of 30% (g / mL), place in a constant temperature incubator at 28℃ and culture for 9 days, and measure its color value.

[0076] Water is one of the six nutrients required for microbial growth. Appropriate water content is essential for solid-state fermentation. The commonly used water content range for solid-state fermentation is selected, and the degree of solid-state fermentation is evaluated by measuring the color value to determine the level of response surface experiment. The results are as follows: Figure 8 As shown in the figure, color value increases with increasing moisture content, particularly when the moisture content rises from 30% to 40%, where the color value rises sharply. The color value is lowest at 10% moisture content, at 14.93 μ / g, and highest at 50% moisture content, at 21.20 μ / g. The color value is low at both 10% and 20% moisture contents, establishing the moisture content levels for the response surface experiment as 30%, 40%, and 50%.

[0077] Test ② Temperature: Accurately weigh 10g of summer and autumn tea raw tea into a 240mL tissue culture bottle, add 3% sucrose water according to 30% water content, sterilize at 100℃, inoculate with seed liquid at a rate of 30% (g / mL), and culture in a constant temperature incubator at 20℃, 24℃, 28℃, 32℃, and 36℃ for 9 days, and measure its color value.

[0078] Whether it is dark tea pile fermentation or black tea fermentation, temperature is crucial. Figure 9 As shown in the figure, the color value is the lowest at 2.11 μ / g when the fermentation temperature is 20°C, the color value is also low at 9.99 μ / g when the fermentation temperature is 36°C, and the color value is the highest at 21.8 μ / g when the fermentation temperature is 28°C. Therefore, the fermentation temperatures of 20°C and 36°C should be eliminated, and the solid-state fermentation temperatures for the response surface experiment should be determined as 24°C, 28°C, and 32°C.

[0079] ③Inoculation amount: Accurately weigh 10g of summer and autumn tea raw tea into a 240mL tissue culture bottle, add 3% sucrose water according to 30% water content, sterilize at 100℃, access the seed liquid at 10%, 20%, 30%, 40%, and 50% (g / mL) inoculation amounts respectively, place it in a constant temperature incubator at 28℃ and culture for 9 days, and measure its color value.

[0080] Like seed liquid shake flask fermentation, inoculation amount is also one of the important factors affecting solid-state fermentation. Figure 10 As shown, color values were similar at inoculum concentrations of 10% and 20%, but slightly higher at 10% than at 20%. The highest color value, 20.77 μ / g, was achieved at 30%. Color values were similar at inoculum concentrations of 40% and 50%, but slightly higher at 50%. Therefore, the inoculum concentration levels for the response surface experiment were determined to be 10%, 30%, and 50%.

[0081] ④ Time: Accurately weigh 10g of summer and autumn tea raw tea into a 240mL tissue culture bottle, add 3% sucrose water according to 30% water content, sterilize at 100℃, inoculate with seed liquid at a rate of 30% (g / mL), place in a constant temperature incubator at 28℃ and culture for 5, 7, 9, 11 and 13 days respectively, and measure its color value.

[0082] Solid-state fermentation has its optimal fermentation cycle. A cycle that is too long or too short will reduce the fermentation quality. Figure 11 As shown in the figure, the color value reached its highest value of 22.93 μ / g at 7 days of fermentation. When the fermentation time was extended to 9 days, the color value decreased slightly. However, when the fermentation time was further extended to 11 days and 13 days, the color value dropped sharply and was lower than the color value at 5 days of fermentation. Therefore, the fermentation times of 11 days and 13 days should be eliminated, and the solid-state fermentation times for the response surface experiment were determined to be 5 days, 7 days, and 9 days.

[0083] Response surface optimization experiment

[0084] On the basis of the single-factor experiment, time, inoculation size, temperature and water content were used as independent variables, three levels were designed for each factor, and color value was used as the response index (Y). The Box-Benhnken method of Design-Expert 10 software was used for experimental design to determine the optimal conditions for solid-state fermentation. Each treatment was repeated three times. The experimental factor levels are shown in Table 5.

[0085] Table 5 Factors and levels of response surface experiment

[0086]

[0087] The experimental design and results are shown in Table 6.

[0088] Table 6 Response surface experimental design and results

[0089]

[0090]

[0091] Based on the experimental results in Table 6, the results were regressed and fitted to obtain the following regression equation: Color value = 23.50-0.18A+1.52B+0.54C+1.10D+0.17AB-0.57AC+0.13AD-0.49BC+0.82BD+2.39CD-3.11A2-3.82B2-2.15C2-3.81D2. As shown in Table 7, the model P<0.0001, indicating that the model is extremely significant. The model correlation coefficient R2 = 0.9794, indicating that the model fits well. The P value of the model lack of fit term is 0.1102, and the difference is not significant, indicating that the model is consistent with the actual situation. From the significance test of the regression equation coefficient, we can see that:

[0092] Table 7 Analysis of variance of regression model

[0093]

[0094]

[0095] The color-valence response surface diagram was drawn using Design-expert10.0 software. Figures 12 to 17 , and conduct visual analysis of the test results.

[0096] The three-dimensional response surface plot shows that the parabola of the color value equation opens downward, indicating that this equation has a maximum. Software analysis shows that the optimal solid-state fermentation conditions for red yeast rice are 7.163 days, an inoculum size of 34.230%, a temperature of 28.889°C, and a moisture content of 42.383%. The color value of red yeast rice is 23.809 u / g.

[0097] To verify the reliability of the results obtained by response surface methodology, solid-state fermentation was carried out according to the factors determined by the response surface methodology. After fermentation, the color value of red yeast rice was determined to be 23.791u / g, which was close to the model prediction value.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for expanding the culture and fermentation of red yeast rice fermented summer and autumn tea, characterized in that: The following steps are involved: Step 1, preparation of seed solution: accurately weigh 5 g of summer and autumn tea raw tea into a teapot, boil 2000 mL of distilled water for 3 minutes, filter, and dilute 2 times to obtain the standby tea juice; using the tea juice as the base solution, prepare the seed solution according to the following ratios: 1.141 g / 50 mL of tea juice of sucrose, 0.712 g / 50 mL of tea juice of peptone, and 0.048 g / 50 mL of tea juice of magnesium sulfate, and sterilize under high pressure at 121°C for 20 minutes to obtain the seed solution; Step 2, preparation of spore suspension: scrape a loop of cells from an activated red Monascus plate and culture in a PDA culture dish for 7 days, use a 7mm punch to punch three cakes from the activated red Monascus plate in a sterile PDA culture dish, culture inverted for 5 days, then use 30 mL of sterile water to scrape and wash the cells, filter into a sterile Erlenmeyer flask, and obtain a spore suspension; the deposit number of the red Monascus is CGMCC No. 3.15746; Step 3: Expand the seed solution: Take a 150 mL Erlenmeyer flask, add the seed solution, adjust the pH, add the spore suspension, place it in a shaker, shake the flask at 180 rpm, and measure the color value and dry weight of the bacteria; Step 4, solid-state fermentation culture: accurately weigh 10g of summer and autumn tea raw tea into a 240mL tissue culture bottle, add 3% sucrose water according to the appropriate water content, sterilize at 100℃, inoculate the seed liquid after expanded culture, place it in a constant temperature incubator for culture, and measure its color value.

2. The method for expanding the culture and fermentation of red yeast rice fermented summer and autumn tea according to claim 1, characterized in that: In step 2, the bacteria were scraped and washed with sterile water, filtered into a sterile triangular flask, and counted using a hemocytometer to ensure that the spore concentration reached 10 6 cfu / mL.

3. The method for expanding the culture and fermentation of red yeast rice fermented summer and autumn tea according to claim 1, characterized in that: In the step 3, the seed liquid expansion culture was performed with the shaker temperature, inoculation size, initial pH, liquid volume and culture time as single factors, and the dry weight and color value of the bacteria as indicators to analyze the influence of each factor on the seed liquid expansion culture, determine the factors and levels, and screen out the optimal seed liquid expansion culture conditions: temperature 28°C, inoculation size 4%, initial pH value 5, liquid volume 50 mL, and fermentation time 8 days.

4. The method for expanding the culture and fermentation of red yeast rice fermented summer and autumn tea according to claim 1, characterized in that: In the solid-state fermentation culture in step 4, water content, fermentation temperature, inoculation size and culture time are used as single factors, and color value is used as an indicator to analyze the influence of each factor on solid-state fermentation. On the basis of the single factor experiment, the Box-Benhnken central composite experimental design principle is adopted, and the color value of red yeast rice fermented summer and autumn tea is used as the response surface value to optimize the fermentation conditions, and the optimal solid-state fermentation conditions are screened out: culture time 7.163 days, inoculation size 34.230%, temperature 28.889°C, and water content 42.383%.

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