Counting Method of Eurotium coronatum in Fuzhuan Tea

Abstract

The present invention discloses a method for counting Eurotium cristatum in Fuzhuan tea. The method comprises the following steps: adding crushed Fuzhuan tea powder to physiological saline at a temperature of 85° C. or higher, soaking the powder for 10 to 30 minutes, beating the powder for 1 to 2 minutes, pouring the powder onto a modified red Bengal culture medium, culturing the powder for 3 to 4 days, and counting the powder. The method for counting Eurotium cristatum in Fuzhuan tea can fully rupture the cleistothecia of the Eurotium cristatum and better release the Eurotium cristatum, thereby truly reflecting the number of Eurotium cristatum in Fuzhuan tea, greatly improving the accuracy and stability of the detection result, and more accurately evaluating the quality of Fuzhuan tea.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a method for counting Eurotium cristatum in Fuzhuan tea. Background Art

[0002] Fuzhuan brick tea has long been an indispensable daily necessity for ethnic minorities in border areas, enjoying the reputation of "rather go without food for three days than go without tea for a day" in these areas. Studies have also found that despite the fact that people in these areas consume beef, mutton, and a variety of high-fat, high-protein foods year-round, their incidence of "rich diseases" such as obesity, hyperlipidemia, and hyperglycemia is not high. Multiple studies have linked this phenomenon to the golden particles found inside or on the surface of Fuzhuan brick tea, a highly beneficial probiotic bacterium named "Eurocystis cristatum." The enzyme it produces has strong effects in lowering blood lipids, regulating intestinal flora, enhancing immunity, and promoting weight loss. This discovery has garnered significant attention and recognition from people in these areas. Fuzhuan brick tea has therefore become a highly recommended way to maintain health, and the development of health foods using Eurocystis cristatum is of great significance.

[0003] Fuzhuan tea is renowned for its unique presence of the fungus Eurotium cristatum, and its abundance is a crucial indicator of tea quality. The current method for enumerating this fungus is based on the first method of GB4789.15-2016, "National Food Safety Standard. Enumeration of Molds and Yeasts in Food Microbiological Examination." However, this method is aimed at counting molds and yeasts in food. The colony morphology of Aspergillus niger on red Bengal medium is not clearly specified during the detection process. Therefore, it is possible to count molds with similar morphology to Aspergillus niger. Not only can it not truly reflect the number of Aspergillus niger, but it may also cause serious safety accidents if toxic molds are mistaken for Aspergillus niger. In addition, in the method of "National Food Safety Standard. Count of Molds and Yeasts in Food Microbiology Examination", the sample adopts the ordinary pretreatment method (five-point sampling method, the dilution temperature is generally at room temperature, and the soaking time is not specified). First, it is difficult to rupture the closed thecoat of Aspergillus niger. Therefore, the colonies formed on potato glucose medium or red Bengal medium may be the growth and development of 2 to 3 or more spores. Second, Aspergillus niger is difficult to release. Third, some fast-growing molds (such as Figure 1 Therefore, the national standard method cannot accurately reflect the number of Eurotium cristatum in Fuzhuan tea.

[0004] In recent years, researchers have made some improvements to the detection process of Eurotium cristatum in Fuzhuan tea, and found that crushing the sample during sample processing can break the closed theca structure of Eurotium cristatum, significantly improving the detection rate. Furthermore, it was found that the key steps in the detection of Eurotium cristatum are: sampling after complete crushing in a universal grinder under sterile conditions, with a sample size of no less than 10g, diluting with physiological saline, beating with a slapping homogenizer for 1 minute, and then performing a coating-type upright culture. However, this improved pre-treatment method still cannot completely release Eurotium cristatum, and it is still possible to count some molds similar to Eurotium cristatum or suppress Eurotium cristatum due to the large-scale reproduction of molds. Therefore, it is still impossible to accurately reflect the number of Eurotium cristatum in Fuzhuan tea.

[0005] Therefore, it is very meaningful to explore a more accurate counting method for Eurotium cristatum. Summary of the Invention

[0006] Based on this, one of the purposes of the present invention is to provide a method for counting Eurotium cristatum in Fu brick tea, which can more accurately reflect the number of Eurotium cristatum in Fu brick tea, thereby more accurately evaluating the quality of Fu brick tea.

[0007] The specific technical solutions for achieving the above-mentioned invention objectives include the following:

[0008] A method for counting Eurotium cristatum in Fuzhuan tea, comprising the following steps:

[0009] (1) Add the crushed Fuzhuan tea powder to physiological saline at 85°C or above and soak for 10 to 30 minutes to obtain a test solution;

[0010] (2) After beating the test solution in step (1) for 1 to 2 minutes, pour the test solution onto a modified red Bengal medium, culture at 26°C to 30°C for 3 to 4 days, and count; the modified red Bengal medium comprises ammonium sulfate, sucrose, and KCl.

[0011] In some embodiments, in step (1), the temperature of the physiological saline is 90°C to 100°C.

[0012] In some embodiments, the temperature of the physiological saline is 95°C to 100°C.

[0013] In some embodiments, in step (1), the soaking time is 10 min to 20 min.

[0014] In some embodiments, in step (2), the improved red Bengal medium comprises the following components: peptone 2.4 g ± 0.2 g, ammonium sulfate 3.6 g ± 0.2 g, magnesium sulfate 1 g ± 0.1 g, potassium dihydrogen phosphate 1 g ± 0.1 g, potassium chloride 40 g ± 2 g, sucrose 40 g ± 2 g, agar powder 20 g ± 1 g, 100 mL ± 10 mL of 1 / 3000 red Bengal solution, 0.1 g ± 0.05 g of chloramphenicol, and 1000 mL ± 10 mL of grade tertiary water.

[0015] In some embodiments, in step (1), the mass volume ratio of the Fuzhuan tea powder to the physiological saline is 1:8-10.

[0016] In some embodiments, the mass volume ratio of the Fuzhuan tea powder to the physiological saline is 1:9-10.

[0017] In some embodiments, in step (1), the mass percentage concentration of the physiological saline is 0.8% to 0.9%.

[0018] In some embodiments, the mass percentage concentration of the physiological saline is 0.85% to 0.9%.

[0019] In some embodiments, in step (2), a beating homogenizer is used for beating for 1.5 min to 2 min.

[0020] In some embodiments, in step (2), the test solution is gradient diluted and then poured onto a modified rose bengal medium.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] In the present invention, the Fu brick tea is first crushed, and then soaked in physiological saline above 85°C for sample preparation, and poured onto a modified red Bengal culture medium for culture and then counting. Under this concept, on the one hand, the cleistothecia of the cristatum can be fully ruptured and the cristatum can be better released. On the other hand, it can avoid to a certain extent the mistaken counting of molds as cristatum (making the counted number more than the actual data), or the suppression of cristatum due to the massive reproduction of molds (making the counted number far less than the actual number). Therefore, the counting method of cristatum in Fu brick tea of ​​the present invention can truly reflect the number of cristatum in Fu brick tea, greatly improve the accuracy and stability of the detection results, and can more accurately evaluate the quality of Fu brick tea. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The growth of Eurotium cristatum after 5 days of culture on Bengal rose medium using the national standard counting method.

[0024] Figure 2 This is the growth of Eurotium cristatum after culturing on the modified Bengal rose medium for 3 days in Test Example 4 of the present invention.

[0025] Figure 3 This is the growth of Eurotium cristatum after culturing on Bengal rose medium for 5 days in Test Example 4 of the present invention. DETAILED DESCRIPTION

[0026] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] Unless otherwise specified, the experimental methods used in the examples of the present invention are all traditional methods in botany, microbiology, plant pathology, etc., and the various reagents and consumables used in the examples are all commercially available products.

[0029] In one aspect of the present invention, a method for counting Eurotium cristatum in Fuzhuan tea is provided, the counting method comprising the following steps:

[0030] (1) Grind the Fuzhuan tea to be tested with a grinder to obtain a sample to be tested;

[0031] Since Fuzhuan tea is a fermented tea, most of the Aspergillus niger is formed inside. It is difficult to sample the whole block of most Fuzhuan tea, and the block sampling is uneven, resulting in large deviations in the results. Therefore, sampling after crushing the Fuzhuan tea can, on the one hand, break the closed theca structure of the Aspergillus niger and significantly improve the detection rate; on the other hand, it can ensure the uniformity of the sample, greatly improve the parallelism of the test results, and will not cause large deviations in the results due to different sampling positions. The deviation is reduced by at least one order of magnitude.

[0032] (2) Aseptically weigh the sample to be tested (20 g to 30 g, preferably 25 g), add 180 mL to 270 mL (preferably 225 mL) of 0.8% to 0.9% sterile saline (8.0 g to 9.0 g of sodium chloride is weighed and dissolved in 1000 mL of grade tertiary water, and then sterilized at 121°C for 15 min) at 85°C or above (preferably 95°C or above, more preferably 95°C to 100°C) (preferably a concentration of 0.85% to 0.9%), and soak for 10 min to 30 min (preferably 10 min to 20 min);

[0033] In this step, the sample to be tested is soaked in physiological saline above 85°C for 10 to 30 minutes. On the one hand, soaking in a high-temperature environment for a period of time can fully release the Aspergillus niger in the tea leaves. On the other hand, since Aspergillus niger is still active when kept at 120°C for 20 minutes, soaking in a high-temperature environment above 85°C for 10 to 30 minutes can kill most molds (such as Aspergillus flavus, Penicillium, etc.), avoiding mistakenly counting molds as Aspergillus niger, or suppressing Aspergillus niger due to the large-scale reproduction of molds under room temperature conditions, making the counted number far less than the actual number.

[0034] (3) Beat with a slapping homogenizer for 1 min to 2 min (preferably 1.5 min to 2 min), pour (preferably pour after gradient dilution) onto modified red Bengal medium, culture for 3 days, and count. In this step, the modified red Bengal medium includes the following components: peptone 2.4 g ± 0.2 g, ammonium sulfate 3.6 g ± 0.2 g, magnesium sulfate 1 g ± 0.1 g, potassium dihydrogen phosphate 1 g ± 0.1 g, potassium chloride 40 g ± 2 g, sucrose 40 g ± 2 g, agar powder 20 g ± 1 g, 100 mL ± 10 mL of 1 / 3000 red Bengal solution, 0.1 g ± 0.05 g of chloramphenicol, and 1000 mL ± 10 mL of grade tertiary water.

[0035] The improved Rose Bengal culture medium used in the present invention is based on the original Rose Bengal culture medium, and ammonium sulfate is added as a nitrogen source (the combination of organic nitrogen peptone and inorganic nitrogen ammonium sulfate can provide a more sufficient nitrogen source for the Eurotium cristatum than using pure peptone as a nitrogen source); the carbon source glucose is replaced by sucrose (the growth rate of the Eurotium cristatum in sucrose is faster); potassium chloride is added (high salt concentration can inhibit the growth of mold); the growth factor ratio is appropriately adjusted (the sulfate content is increased, so that the pH value of the culture medium is between 5 and 7, which is more suitable for the growth of the Eurotium cristatum); the culture time of the Eurotium cristatum is shortened from 5 days to 3 days, which is beneficial to large-scale detection and Fuzhuan tea production and processing.

[0036] In the following examples, the culture media used are as follows:

[0037] Bengal rose culture medium: commercial culture medium from Guangzhou Huankai Microbiology Technology Co., Ltd. According to the preparation requirements, weigh a certain proportion of dry powder, add grade 3 water in proportion, heat and boil until completely dissolved, sterilize in an autoclave at a temperature of 121°C for 15 minutes, keep warm at 46°C, and set aside.

[0038] Improved Rose Bengal medium: 2.4 g peptone, 3.6 g ammonium sulfate, 1 g magnesium sulfate (MgSO4·7H2O), 1 g potassium dihydrogen phosphate, 40 g potassium chloride, 40 g sucrose, 20 g agar powder, 100 mL 1 / 3000 Rose Bengal solution, 0.1 g chloramphenicol, 1000 mL grade tertiary water; stir and heat until completely dissolved, aliquot into Erlenmeyer flasks, autoclave at 121°C for 15 min, and keep warm at 46°C until ready to use. All reagents were analytical grade; peptone was soy peptone, and agar was technical agar powder.

[0039] The present invention is described in detail below with reference to specific embodiments.

[0040] Example 1 A method for counting Eurotium cristatum in Fuzhuan tea

[0041] The method for counting Eurotium cristatum in Fuzhuan tea of ​​the present embodiment comprises the following steps:

[0042] 1. Grind all the Fuzhuan tea to be tested with a grinder and aseptically weigh 25g of Fuzhuan tea powder;

[0043] 2. Prepare 0.85% normal saline solution, autoclave at 121°C for 15 min, cool the saline solution to 95°C, and keep warm;

[0044] 3. Add the Fuzhuan tea powder from step 1 to the normal saline (225 mL) from step 2, maintain the normal saline at 95°C, and soak for 20 minutes;

[0045] 4. After soaking, use a slapping homogenizer to beat for 2 minutes to obtain a homogenous solution of the sample;

[0046] 5. Take 1 mL of the 1:10 sample solution and inject it into a test tube containing 9 mL of 0.85% sterile saline. Vortex and mix well to prepare a 1:100 sample solution. In this way, prepare a 10-fold serial dilution series of sample solutions.

[0047] 6. According to the sample limit estimate, select 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 For each dilution, 1 mL of the sample solution was placed in two sterile plates, and then the modified Bengal rose medium was added.

[0048] 7. After the culture medium solidifies, place the plate upright and culture in an incubator at 28℃±1℃ for 3 days. Count and report the colonies of Eurotium cristatum according to the relevant provisions of GB4789.15 (the colonies of Eurotium cristatum are mostly round or oval, and some are irregular. The thickness of the colony edge is slightly lower than that of the center. The colony edge is yellow, and the center is dark yellow or dark brown. The color of the colony gradually darkens from the edge to the center in concentric circles).

[0049] Example 2 A method for counting Eurotium cristatum in Fuzhuan tea

[0050] The method for counting Eurotium cristatum in Fuzhuan tea of ​​the present embodiment comprises the following steps:

[0051] 1. Grind all the Fuzhuan tea to be tested with a grinder and aseptically weigh 25g of Fuzhuan tea powder;

[0052] 2. Prepare 0.85% normal saline solution, sterilize by autoclaving at 121°C for 15 min, cool the saline solution to 85°C, and keep warm;

[0053] 3. Add the Fuzhuan tea powder from step 1 to the normal saline (225 mL) from step 2, maintain the normal saline at 85°C, and soak for 10 minutes;

[0054] 4. After soaking, beat with a slapping homogenizer for 2 minutes;

[0055] 5. Take 1 mL of the 1:10 sample solution and inject it into a test tube containing 9 mL of 0.85% sterile saline. Vortex and mix well to prepare a 1:100 sample solution. In this way, prepare a 10-fold serial dilution series of sample solutions.

[0056] 6. According to the sample limit estimate, select 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 For each dilution, 1 mL of the sample solution was placed in two sterile plates, and then the modified Bengal rose medium was added.

[0057] 7. After the culture medium solidifies, place the plate upright and incubate it in a 28℃±1℃ incubator for 3 days to count the Eurotium cristatum.

[0058] Example 3 A method for counting Eurotium cristatum in Fuzhuan tea

[0059] The method for counting Eurotium cristatum in Fuzhuan tea of ​​the present embodiment comprises the following steps:

[0060] 1. Grind all the Fuzhuan tea to be tested with a grinder and aseptically weigh 25g of Fuzhuan tea powder;

[0061] 2. Prepare 0.85% normal saline solution, autoclave at 121°C for 15 min, cool the saline solution to 100°C, and keep warm;

[0062] 3. Add the Fuzhuan tea powder from step 1 to the normal saline (225 mL) from step 2, maintain the normal saline at 100°C, and soak for 15 minutes;

[0063] 4. After soaking, beat with a slapping homogenizer for 2 minutes;

[0064] 5. Take 1 mL of the 1:10 sample solution and inject it into a test tube containing 9 mL of 0.85% sterile saline. Vortex and mix well to prepare a 1:100 sample solution. In this way, prepare a 10-fold serial dilution series of sample solutions.

[0065] 6. According to the sample limit estimate, select 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 For each dilution, 1 mL of the sample solution was placed in two sterile plates, and then the modified Bengal rose medium was added.

[0066] 7. After the culture medium solidifies, place the plate upright and incubate it in a 28℃±1℃ incubator for 3 days to count the Eurotium cristatum.

[0067] Experimental Example 1 Effect of Temperature on the Detection Results of Eurotium cristatum in Fuzhuan Tea

[0068] This test example investigated the effect of different temperatures on the count of Eurotium cristatum in Fuzhuan tea, and specifically included the following steps:

[0069] 1. After the same Fuzhuan tea to be tested was crushed according to the method of Example 1, it was soaked in 0.85% physiological saline for 20 min at 25°C, 45°C, 65°C, 75°C, 85°C, 95°C, and 100°C (each temperature was tested 6 times in parallel); after beating for 1-2 min, it was placed in a sterile culture dish; and then the modified Bengal rose medium was poured in;

[0070] 2. Cultivate according to the method of Example 1. After 3 days, count the number of Eurotium cristatum.

[0071] The results are shown in Table 1.

[0072] Table 1 Effects of different temperatures on the count of Eurotium coronatum in Fuzhuan tea

[0073]

[0074] As can be seen from Table 1, with the increase of temperature, the number of Eurotium cristatum in Fu brick tea also increases. When soaked at a temperature above 85℃ for 20 minutes, the number of Eurotium cristatum increases significantly compared with room temperature. When the soaking temperature is between 95℃ and 100℃, the number of Eurotium cristatum reaches the highest value. Therefore, the increase in temperature can fully release the Eurotium cristatum, thereby being able to more accurately and truly reflect the number of Eurotium cristatum in Fu brick tea.

[0075] Experimental Example 2 Effect of Soaking Time on the Detection Results of Eurotium cristatum in Fuzhuan Tea

[0076] This test example investigated the effects of different soaking times on the count of Eurotium cristatum in Fuzhuan brick tea, and specifically included the following steps:

[0077] 1. After the same Fuzhuan tea to be tested was crushed according to the method of Example 1, it was soaked in 95°C 0.85% physiological saline for 5 min, 10 min, 20 min and 30 min respectively (each time was tested in parallel 6 times); after beating for 1-2 min, it was placed in a sterile culture dish; and then the modified Bengal rose medium was poured in;

[0078] 2. Cultivate according to the method of Example 1. After 3 days, count the number of Eurotium cristatum.

[0079] The results are shown in Table 2.

[0080] Table 2 Effects of different soaking times on the count of Eurotium coronatum in Fuzhuan tea

[0081]

[0082] As shown in Table 2, the soaking time also affects the release of Eurotium cristatum from Fuzhuan tea. After soaking at 95°C for 10 minutes, the number of Eurotium cristatum released was 7.5 times that of the 5-minute soaking at 95°C. However, the number of Eurotium cristatum did not increase consistently with increasing soaking time. Soaking for 10 to 30 minutes can fully release Eurotium cristatum, with 10 minutes being the optimal time.

[0083] Experimental Example 3 Effect of Sample Preparation Method on the Detection Results of Eurotium cristatum in Fuzhuan Tea

[0084] This test investigated the effects of different sample preparation methods on the count of Eurotium cristatum in the same Fuzhuan brick tea. The two sample preparation methods were as follows:

[0085] Sample preparation method 1: After crushing the Fuzhuan tea to be tested according to the method of Example 1, take 25g and add it into 225mL 95℃ 0.85% physiological saline, soak for 20min; then pat for 1-2min (parallel measurement six times).

[0086] Sample preparation method 2: After crushing the Fuzhuan tea to be tested according to the method of Example 1, take 25g and add it into 225mL 0.85% saline; then beat it for 1-2 minutes (parallel measurement six times).

[0087] The samples prepared by sample preparation method 1 and sample preparation method 2 were placed in a sterile culture dish; then, modified rose Bengal culture medium was poured in; and the culture was carried out according to the method of Example 1. After 3 days, the number of Eurotium cristatum was counted.

[0088] The results are shown in Table 3.

[0089] Table 3 Effects of different sample preparation methods on the count of Eurotium coronatum in Fuzhuan tea

[0090] Sample preparation method Sample preparation method 1 Sample preparation method 2 Average number of Eurotium cristatum CFU / g <![CDATA[6.7×10 6 ]]> <![CDATA[7.0×10 5 ]]>

[0091] As shown in Table 3, the number of Eurotium cristatum counted by sample preparation method 1 is about 10 times that of Eurotium cristatum counted by sample preparation method 2. This shows that the change of sample preparation method will significantly affect the number of Eurotium cristatum, from hundreds of thousands to millions.

[0092] Experimental Example 4: Effect of culture medium on the detection results of Eurotium cristatum in Fuzhuan tea

[0093] This test example investigated the effects of different culture media on the count of Eurotium cristatum in Fuzhuan tea, and specifically included the following steps:

[0094] 1. After crushing the same Fuzhuan tea to be tested according to the method of Example 1, soak it in 95℃ 0.85% physiological saline for 20 minutes; beat it for 1-2 minutes; and then place it in a sterile culture dish (parallel measurement six times);

[0095] 2. Pour Red Bengal medium and modified Red Bengal medium into culture dishes respectively;

[0096] 3. Cultivate according to the method of Example 1. On the 3rd and 5th days, count the number of Eurotium cristatum.

[0097] The results are as follows Figures 2-3 and as shown in Table 4.

[0098] Table 4 Effects of different culture media on the count of Eurotium coronatum in Fuzhuan tea

[0099]

[0100]

[0101] from Figures 2-3 As shown in Table 4, the improved Bengal red medium can provide better growth conditions for Eurotium cristatum ( Figure 2 ), after 3 days of culture, the number of Eurotium cristatum had reached 6.7×10 6 , while after 5 days of culture with Bengal red medium ( Figure 3 ), the number of Eurotium cristatum was only 9.2×10 5 , which is about 1 / 7 of the 3-day culture time of the modified Bengal rose medium. This shows that the improved medium not only provides favorable growth conditions, but also shortens the culture time and saves costs.

[0102] Test Example 5 Stability test of the method of the present invention

[0103] This test example uses Fuzhuan tea produced by different manufacturers (Shaanxi Daqin Tea Co., Ltd., Shaanxi Yunshui Fuxiang Tea Manufacturing Co., Ltd., Shaanxi Jingyang Yuanshun Tea Co., Ltd., Shaanxi Gudu Fuzhuan Tea Co., Ltd., Xianyang Jinghe Fuzhuan Tea Co., Ltd., Shaanxi Jingyang Baifu Fuzhuan Tea Co., Ltd., each manufacturer is tested in parallel 6 times, and the average result is taken) and Fuzhuan tea produced by the same manufacturer (Shaanxi Yunshui Fuxiang Tea Manufacturing Co., Ltd.) as raw materials, and investigates the stability of the counting method of Eurotium coronatum in Fuzhuan tea of ​​the present invention, specifically comprising the following steps:

[0104] 1. According to the method of Example 1, Fuzhuan tea produced by different manufacturers and Fuzhuan tea produced by the same manufacturer were crushed, soaked in 95°C 0.85% physiological saline for 20 minutes, patted for 1-2 minutes, and then placed in a sterile culture dish; modified Bengal red medium was poured in; the control group was: Fuzhuan tea was crushed, 0.85% sterile physiological saline was added, patted for 1-2 minutes, and then placed in a sterile culture dish; modified Bengal red medium was poured in;

[0105] 2. Cultivate according to the method of Example 1. On the third day, count the number of Eurotium cristatum.

[0106] The results are shown in Tables 5 and 6.

[0107] Table 5 Test results of Fuzhuan tea from different manufacturers

[0108]

[0109]

[0110] As can be seen from Table 5, the number of Eurotium cristatum counted in the experimental group differed from that in the control group by at least one order of magnitude, which is consistent with the results of Experimental Example 3.

[0111] Table 6 Test results of Fuzhuan tea (3 parallels) from the same manufacturer

[0112]

[0113] From the results in Table 6, we can see that in the repeatability test of the same manufacturer, the relative error of the experimental group is 1.3%, and the relative error of the control group is 9.7%. The parallel stability of the results of the experimental group is higher than that of the control group, which is consistent with the results of Experimental Example 3.

[0114] Test Example 6 Accuracy Test of the Method of the Present Invention

[0115] This test example adopts the method of Example 1 of the present invention, and the bacterial suspension (concentration of 1×10 8 CFU / mL), and 6 determinations were performed. Specifically, the following steps were included:

[0116] 1. Take 1mL of Eurotium cristatum suspension (concentration is 1×10 8 / mL), add 9mL95℃0.85% sterile saline, and soak at 95℃ for 20min; the control group: take 1mL of Eurotium cristatum suspension (concentration of 1×10 8 CFU / mL), add 9 mL of 0.85% sterile saline;

[0117] 2. After using a slapping homogenizer for 2 minutes, pipette 1 mL of the dilution into two sterile culture dishes, pour in modified Bengal rose medium, and culture and count.

[0118] 3. Cultivate according to the method of Example 1. On the third day, count the number of Eurotium cristatum.

[0119] The results are shown in Table 7.

[0120] Table 7 Accuracy test results of the method of the present invention

[0121] Group 1 2 3 4 5 6 Number of Eurotium cristatum (experimental group) <![CDATA[1.2×10 8 ]]> <![CDATA[1.1×10 8 ]]> <![CDATA[1.1×10 8 ]]> <![CDATA[1.0×10 8 ]]> <![CDATA[1.1×10 8 ]]> <![CDATA[1.0×10 8 ]]> Number of Eurotium cristatum (control group) <![CDATA[8.3×10 7 ]]> <![CDATA[7.6×10 7 ]]> <![CDATA[9.3×10 7 ]]> <![CDATA[8.8×10 7 ]]> <![CDATA[9.0×10 7 ]]> <![CDATA[9.1×10 7 ]]>

[0122] From Table 7, we can see that the average number of Eurotium cristatum in the experimental group is 1.1×10 8 CFU / mL, while the average number of Eurotium cristatum in the control group was 8.6×10 7 CFU / mL, therefore, the results of the experimental group are closer to the bacterial liquid concentration of Eurotium cristatum (true value), while the control group has a large deviation from the true value due to insufficient release of Eurotium cristatum spores. This proves that the method of the present invention has high accuracy and can more truly reflect the number of Eurotium cristatum in Fuzhuan tea.

[0123] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0124] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for counting Eurotium cristatum in Fuzhuan tea, characterized in that: The counting method comprises the following steps: (1) Add the crushed Fuzhuan tea powder to 95℃~100℃ physiological saline and soak for 10min~20min to obtain the test solution; (2) After beating the test solution in step (1) for 1 to 2 minutes, pour the test solution onto a modified red Bengal medium, culture at 26°C to 30°C for 3 days, and count; the modified red Bengal medium is composed of the following components: peptone 2.4g±0.2g, ammonium sulfate 3.6g±0.2g, magnesium sulfate 1g±0.1g, potassium dihydrogen phosphate 1g±0.1g, potassium chloride 40g±2g, sucrose 40g±2g, agar powder 20g±1g, 100mL±10mL of 1 / 3000 red Bengal solution, chloramphenicol 0.1g±0.05g, and grade tertiary water 1000mL±10mL.

2. The method for counting Eurotium cristatum in Fuzhuan tea according to claim 1, wherein In step (1), the mass volume ratio of the Fuzhuan tea powder to the physiological saline is 1:8-10.

3. The method for counting Eurotium cristatum in Fuzhuan tea according to claim 2, wherein The mass volume ratio of the Fuzhuan tea powder to the physiological saline is 1:9-10.

4. The method for counting Eurotium cristatum in Fuzhuan tea according to claim 1, wherein In step (1), the mass percentage concentration of the physiological saline is 0.8% to 0.9%.

5. The method for counting Eurotium cristatum in Fuzhuan tea according to claim 1, wherein In step (2), a slapping homogenizer is used to slap for 1.5 min to 2 min.

6. The method for counting Eurotium cristatum in Fuzhuan tea according to claim 1, wherein In step (2), the solution to be tested is gradiently diluted and then poured onto a modified rose Bengal medium.

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