Kluyveromyces marxianus, complex microbial inoculant, fermented tea puree, and preparation method and application of kluyveromyces marxianus

Abstract

The invention belongs to the field of fermentation, and particularly relates to kluyveromyces marxianus, a complex microbial inoculant, fermented tea primary pulp as well as a preparation method and application of the kluyveromyces marxianus. Wherein the Kluyveromyces marxianus is Kluyveromyces marxianus Linzhi.KM-D3, the Kluyveromyces marxianus Linzhi.KM-D3 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the Kluyveromyces marxianus Linzhi.KM-D3 is CCTCC NO: M 20231575. The DPPH free radical scavenging capacity of the fermented tea raw stock obtained through fermentation of the kluyveromyces marxianus is larger than or equal to 140%, the hydroxyl free radical scavenging capacity is larger than 130%, the ABTS free radical scavenging capacity is larger than 80%, and the SOD enzyme activity of each L of the fermented tea raw stock is larger than 1300U. Meanwhile, the pH value of the fermented tea puree provided by the invention is less than or equal to 4.0, the total acid content (in terms of lactic acid) is greater than or equal to 6g / 100g, the ethanol content is less than or equal to 0.5 g / 100g, the sucrose content is less than or equal to 0.5 g / 100g, and the fermented tea puree has no fermentation peculiar smell and light bitter taste in the aspect of sensory evaluation.

Description

Technical Field

[0001] The invention belongs to the field of fermentation, and specifically relates to Kluyveromyces marxianus, a composite bacterial agent, fermented tea pulp, and a preparation method and application thereof. Background Art

[0002] Tea originated in China and has a long history of drinking. The ancient book "Yinshi Zhengyao" once recorded that tea soup "tastes sweet and bitter, is slightly cold and non-toxic, removes phlegm and heat, quenches thirst, promotes urination, helps digestion and relieves gas, and refreshes the mind and reduces sleep." Modern science has confirmed that tea contains many active ingredients, such as tea polyphenols, tea pigments, and theanine, which are beneficial to the human body.

[0003] Fermented tea pulp is prepared by mixing raw tea soup, sugar and microorganisms and then fermenting them. During the fermentation period, raw tea soup can produce flavor substances such as lactic acid and gluconic acid, which makes the final fermented tea pulp taste better. At the same time, the catechins in the fermented tea pulp are reduced, which reduces the bitterness compared with the raw tea soup. Furthermore, microbial fermentation produces phenolic substances, which increases the total phenol content of the fermented tea pulp compared with the total phenol content in the raw tea soup. Since the pH of the fermented tea pulp is low, the stability of phenolic substances can be enhanced, and the antioxidant capacity of the fermented tea pulp can be enhanced; tea pigments can be decomposed into small molecules that are easier for the human body to absorb after fermentation, so the fermented tea pulp is more nutritious and flavorful than the raw tea soup, and is easier for the human body to absorb.

[0004] Fermented tea pulp is usually made from sugar, tea and water through natural fermentation of a variety of microorganisms. It is called Weibao Tea, Kombucha or Haibao Tea in China. Although the fermented tea pulp obtained by traditional fermentation methods has a unique flavor, it has a sharp sour taste and a light tea taste, and is not suitable for direct drinking. At the same time, traditional fermentation methods usually adopt static fermentation, which is easy to form bacterial film, hinder the acid production rate of microorganisms, is not conducive to industrial production, and prolongs the fermentation cycle; natural fermentation causes mixed strains and is not very safe. Summary of the invention

[0005] The problems existing in the prior art are: the preparation method of fermented tea puree in the prior art usually adopts the form of inoculation of bacterial agents for static fermentation, which is easy to form biofilm, hindering the acid production rate of microorganisms, which is not conducive to industrial production and prolongs the fermentation cycle; at the same time, the fermented tea puree in the prior art has the problem of low antioxidant activity.

[0006] In view of the above problems existing in the prior art, the present invention provides a Kluyveromyces marxianus, a composite bacterial agent, a fermented tea puree and a preparation method and application thereof.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a Kluyveromyces marxianus, wherein the Kluyveromyces marxianus is Kluyveromyces marxianus Linzhi.KM-D3, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCCNO:M 20231575.

[0009] Preferably, the ITS gene sequence of Kluyveromyces marxianus is shown as SEQ ID No.3.

[0010] In a second aspect, the present invention provides the use of the Kluyveromyces marxianus in preparing a bacterial agent.

[0011] In a third aspect, the present invention provides the use of the Kluyveromyces marxianus in preparing fermented tea pulp with a SOD enzyme activity greater than 1300 U / L.

[0012] In a fourth aspect, the present invention provides a composite bacterial agent containing the Kluyveromyces marxianus.

[0013] Preferably, the composite bacterial agent also contains Lactobacillus plantarum and Lactobacillus germanica subsp. lactis.

[0014] Preferably, the composite bacterial agent is prepared by a method comprising the following steps: respectively amplifying and culturing Kluyveromyces marxianus, Lactobacillus plantarum and Lactobacillus delbrueckii subsp. lactis to obtain a bacterial solution of Kluyveromyces marxianus, a bacterial solution of Lactobacillus plantarum and a bacterial solution of Lactobacillus delbrueckii subsp. lactis; more preferably, the three bacterial solutions are preserved independently but used in combination; most preferably, the bacterial solution of Kluyveromyces marxianus, the bacterial solution of Lactobacillus plantarum and the bacterial solution of Lactobacillus delbrueckii subsp. lactis are used in combination in a volume ratio of 45-75:65-75:7-12.

[0015] Preferably, the viable bacterial count of Kluyveromyces marxii is 2-3×10 7 cfu / mL, and / or the viable count of Lactobacillus plantarum was 8.5-9.5×10 9 cfu / mL, and / or the viable count of Lactobacillus germanica subsp. lactis was 7.5-8.5×10 9 cfu / mL.

[0016] Preferably, the plant lactobacillus is plant lactobacillus Ali.Plateau.LP.VIII, with a deposit number of CCTCCNO: M 2022068.

[0017] And / or the Lactobacillus delbrueckii subspecies lactis is Lactobacillus delbrueckii subspecies lactis Dangxiong LBⅧ, with the preservation number being CCTCC NO:M 2023396.

[0018] In a fifth aspect, the present invention provides a method for preparing the composite bacterial agent, comprising the following steps: respectively amplifying and culturing the Kluyveromyces marxianus and Lactobacillus plantarum and / or Lactobacillus germanica subsp. lactis as described in claim 1 or 2.

[0019] In a sixth aspect, the present invention provides the use of the composite bacterial agent or the composite bacterial agent prepared by the preparation method in the preparation of fermented tea pulp.

[0020] In a seventh aspect, the present invention provides a fermented tea puree containing the bacterial agent or the bacterial agent prepared by the preparation method.

[0021] Preferably, the fermented tea puree is prepared by mixing tea soup obtained by extracting tea leaves with water with a carbon source, a nitrogen source and the composite bacterial agent to obtain a mixed liquid, subjecting the mixed liquid to intermittent stirring anaerobic fermentation to obtain a fermentation liquid, and mixing the fermentation liquid with a sugar substitute to obtain a fermented tea puree, wherein the SOD enzyme activity of each L of the fermented tea puree is greater than 1300U.

[0022] Preferably, the SOD enzyme activity in each L of fermented tea puree is greater than 1300U and less than or equal to 100000U; preferably, the SOD enzyme activity in each L of fermented tea puree is greater than 1700U and less than or equal to 100000U.

[0023] Preferably, the fermented tea puree has a DPPH free radical scavenging ability greater than or equal to 140%, and / or a hydroxyl free radical scavenging ability greater than 130%, and / or an ABTS free radical scavenging ability greater than 80%.

[0024] Preferably, the DPPH free radical scavenging ability of the fermented tea puree is greater than or equal to 140% and less than or equal to 180%, and / or the hydroxyl free radical scavenging ability is greater than 130% and less than or equal to 160%, and / or the ABTS free radical scavenging ability is greater than 80% and less than or equal to 100%;

[0025] More preferably, the fermented tea puree has a DPPH radical scavenging ability greater than or equal to 165% and less than or equal to 180%, and / or a hydroxyl radical scavenging ability greater than 150% and less than or equal to 160%, and / or an ABTS radical scavenging ability greater than 90% and less than or equal to 100%.

[0026] Preferably, the pH value is less than or equal to 4.0, and / or the sucrose content in every 100g of fermented tea puree is less than 0.5g, and / or the total acid content in terms of lactic acid in every 100g of fermented tea puree is greater than or equal to 6g, and / or the ethanol content in every 100g of fermented tea puree is less than or equal to 0.5g.

[0027] Preferably, the pH value is greater than or equal to 3.0 and less than or equal to 4.0, and / or the sucrose content in every 100g of fermented tea puree is greater than or equal to 0 and less than 0.5g, and / or the total acid content in terms of lactic acid in every 100g of fermented tea puree is greater than or equal to 6g and less than or equal to 10g, and / or the ethanol content in every 100g of fermented tea puree is greater than or equal to 0 and less than or equal to 0.5g;

[0028] More preferably, the sucrose content in every 100g of fermented tea puree is greater than or equal to 0 and less than 0.35g, and / or the total acid content in terms of lactic acid in every 100g of fermented tea puree is greater than or equal to 7g and less than or equal to 10g, and / or the ethanol content in every 100g of fermented tea puree is greater than or equal to 0 and less than or equal to 0.35g.

[0029] Preferably, the inoculation amount of Kluyveromyces marxianus in each mL of tea base liquid is 0.2-0.3×10 6 cfu and / or Lactobacillus plantarum inoculation volume is 100-110×10 6 cfu, and / or Lactobacillus germanica subsp. lactis inoculated at 10-15×10 6 cfu;

[0030] More preferably, the inoculation amount of Kluyveromyces marxianus in each mL of tea base liquid is 0.25-0.3×10 6 cfu, and / or Lactobacillus plantarum inoculation volume is 105-110×10 6 cfu, and / or Lactobacillus delbrueckii subsp. lactis inoculated at 13-15×10 6 cfu.

[0031] Preferably, when the tea leaves are extracted with water, the weight ratio of the tea leaves to the water is 1-2.5:100, preferably, the weight ratio of the tea leaves to the water is 1.5-2.5:100.

[0032] Preferably, the tea leaves include one or more of black tea, oolong tea and dark tea. Preferably, the tea leaves are black tea.

[0033] Preferably, the amount of the carbon source added is 2-6wt% based on the weight of the tea soup, more preferably, the amount of the carbon source added is 4-6wt%; and / or the amount of the nitrogen source added is 0.5-1wt‰ based on the weight of the tea soup, most preferably, the amount of the nitrogen source added is 0.7-1wt‰.

[0034] Preferably, the carbon source includes one or more of sucrose, fructose and glucose, and more preferably, the carbon source is sucrose.

[0035] Preferably, the nitrogen source is yeast extract.

[0036] Preferably, the added amount of the sugar substitute is 0.01-12 wt % based on the weight of the fermentation broth.

[0037] Preferably, the sugar substitute includes one or more of steviol glycoside, erythritol and xylitol. More preferably, the sugar substitute is a combination of steviol glycoside, erythritol and xylitol. Most preferably, based on the weight of the fermentation broth, the amount of steviol glycoside added is 0.01-0.05wt%, and / or the amount of erythritol added is 1wt%-5wt%, and / or the amount of xylitol added is 1wt%-5wt%.

[0038] In an eighth aspect, the present invention provides a method for preparing the fermented tea puree, comprising the following steps: the fermented tea puree is prepared by mixing tea soup obtained by extracting tea leaves with water with a carbon source, a nitrogen source and the composite bacterial agent to obtain a mixed liquid, subjecting the mixed liquid to intermittent stirring anaerobic fermentation to obtain a fermentation liquid, and mixing the fermentation liquid with a sugar substitute to obtain the fermented tea puree.

[0039] Preferably, the preparation method comprises the following steps:

[0040] (1) mixing tea soup obtained by extracting tea leaves with water, a carbon source and a nitrogen source, sterilizing and cooling the mixture to obtain a tea-based liquid;

[0041] (2) mixing the tea-based liquid obtained in step (1) and the composite bacterial agent to obtain a mixed liquid;

[0042] (3) subjecting the mixed solution obtained in step (2) to intermittent stirring anaerobic fermentation to obtain a fermentation liquid;

[0043] (4) The fermentation liquid obtained in step (3) is mixed with the sugar substitute, filtered, filled and sterilized to obtain the fermented tea puree.

[0044] Preferably, the extraction temperature is 95-100°C and / or the extraction time is 5-15 min.

[0045] Preferably, the sterilization temperature in step (1) is 95-115°C, and / or the sterilization temperature in step (3) is 85-95°C.

[0046] Preferably, the sterilization time in step (1) is 10-20 min, and / or the sterilization time in step (3) is 10-20 min.

[0047] Preferably, in step (3), the fermentation is terminated when the pH of the fermentation broth is 3.0-3.5.

[0048] Preferably, in step (3), the frequency of intermittent stirring is stirring for 15-20 minutes and pausing for 40-45 minutes.

[0049] Preferably, the stirring speed before both the yeast and the lactic acid bacteria enter the logarithmic growth phase is 300-500 r / min, and the stirring speed after both the yeast and the lactic acid bacteria enter the logarithmic growth phase is 50-80 r / min.

[0050] Preferably, the stirring temperature is 25-35°C.

[0051] Preferably, in step (3), the filtration is performed by circulating filtration using a 200-300 mesh sieve until the solution is clarified.

[0052] In a ninth aspect, the present invention provides a fermented tea puree, which is a fermented tea puree prepared by the preparation method.

[0053] In a tenth aspect, the present invention provides the use of fermented tea puree in preparing food.

[0054] In an eleventh aspect, the present invention provides a food comprising the fermented tea puree.

[0055] Beneficial effects of the present invention:

[0056] (1) The pH value of the fermented tea puree provided by the present invention is less than or equal to 4.0, the total acid content (calculated as lactic acid) is greater than or equal to 6 g / 100 g, the ethanol content is less than or equal to 0.5 g / 100 g, and the sucrose content is less than or equal to 0.5 g / 100 g. At the same time, the fermented tea puree has no fermented odor and light bitterness in terms of sensory evaluation.

[0057] (2) The SOD enzyme activity of the fermented tea puree provided by the present invention is greater than 1300U / L, and has high antioxidant activity, DPPH free radical scavenging ability is greater than 140%, hydroxyl free radical scavenging ability is greater than 130%, and ABTS free radical scavenging ability is greater than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a microscopic observation picture of Kluyveromyces marxianus Linzhi.KM-D3.

[0059] Figure 2 The catechin content and sucrose content in the fermented tea puree obtained in Example 1, Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3 are shown.

[0060] Figure 3 The ethanol content, sucrose content and total acid (calculated as lactic acid) content in the fermented tea puree obtained in Example 1, Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 and Comparative Example 7 are shown.

[0061] Figure 4The ethanol content, sucrose content and total acid (calculated as lactic acid) content in the fermented tea puree obtained in Example 2, Example 4, Comparative Example 8, Comparative Example 9, Comparative Example 10, Comparative Example 11, Comparative Example 12 and Comparative Example 13.

[0062] Figure 5 The ethanol content, sucrose content and total acid (calculated as lactic acid) content in the fermented tea puree obtained in Example 4, Example 5, Comparative Example 14, Comparative Example 15 and Comparative Example 16.

[0063] Strain deposit information

[0064] The Lactobacillus plantarum Ali. Plateau. LP. VIII used in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on January 13, 2022, with a deposit number of CCTCC NO: M 2022068. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754833. The strain has been recorded in the Chinese patent with publication number CN115838654A.

[0065] The Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII used in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on March 23, 2023, with a deposit number of CCTCC NO: M 2023396, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754833. The strain has been recorded in the Chinese patent publication number CN117562124A.

[0066] The Kluyveromyces marxious Linzhi.KM-D3 used in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with a deposit number of CCTCC NO: M20231575, and a deposit address: Wuhan University, Wuhan, China, Postal Code: 430072; Telephone: 027-68754052.

[0067] The Saccharomyces cerevisiae used in the present invention was deposited in China Center for Type Culture Collection (CCTCC) on September 1, 2009, with a deposit number of CCTCC NO: M209190, deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. The strain has been recorded in the Chinese patent publication number CN103031255A. DETAILED DESCRIPTION

[0068] In order to better understand the above technical solution, the technical solution of the present invention is clearly and completely explained in conjunction with the specific implementation method below. It should be noted that the content in the specific implementation method is only a specific implementation and explanation of the technical solution of the present invention, and should not be understood as limiting the scope of protection of the present invention.

[0069] In some specific embodiments, the present invention provides a fermented tea pulp, wherein the DPPH free radical scavenging capacity of the fermented tea pulp is greater than or equal to 140% and less than or equal to 180%. In some specific embodiments, the SOD enzyme activity of each liter of fermented tea pulp is greater than 1300U and less than or equal to 100000U. Preferably, the SOD enzyme activity of each liter of fermented tea pulp can be 1400U, 1500U, 1600U, 170 0U, 1800U, 1900U, 2000U, 2100U, 2200U, 2300U, 2400U, 2500U, 2600U, 2700U, 2800U, 2900U, 3000U, 3100U, 3200U, 3300U, 3400U, 3500U, 3600U, 3700U, 3800U, 3900U, 4000U, 4100U, 4200 U, 4300U, 4400U, 4500U, 4600U, 4700U, 4800U, 4900U, 5000U, 5100U, 5200U, 5300U, 5400U, 5 500U, 5600U, 5700U, 5800U, 5900U, 6000U, 7000U, 8000U, 9000U, 10000U, 11000U, 12000U, 13 000U, 14000U, 15000U, 16000U, 17000U, 18000U, 19000U, 20000U, 30000U, 40000U, 50000U, 60000U, 70000U, 80000U, 90000U or 100000U, or SOD enzyme activity per L of fermented tea puree within a numerical range consisting of any two of the above specific values ​​as endpoints.

[0070] In some specific embodiments, the DPPH free radical scavenging capacity of the fermented tea puree is greater than or equal to 140% and less than or equal to 180%. Preferably, the DPPH free radical scavenging capacity of the fermented tea puree may be 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%, 201 7%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179% or 180%, or the DPPH free radical scavenging ability of the fermented tea pulp within the numerical range consisting of any two of the above specific numerical values ​​as endpoints.

[0071] In some specific embodiments, the present invention provides a fermented raw tea, the hydroxyl radical scavenging ability of the fermented tea raw pulp is greater than 130% and less than or equal to 160%. Preferably, the hydroxyl radical scavenging ability of the fermented tea raw pulp may be 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159% or 160%, or the hydroxyl radical scavenging ability of the fermented tea raw pulp within the numerical range formed by any two of the above specific numerical values ​​as endpoints.

[0072] In some specific embodiments, the present invention provides a fermented raw tea, the ABTS free radical scavenging ability of the fermented tea raw pulp is greater than 80% and less than or equal to 100%, preferably, the ABTS free radical scavenging ability of the fermented tea raw pulp may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or the ABTS free radical scavenging ability of the fermented tea raw pulp within the numerical range formed by any two of the above specific numerical values ​​as endpoints.

[0073] In some specific embodiments, the present invention provides a fermented tea pulp, which is prepared by the following steps: the fermented tea pulp is prepared by mixing tea soup obtained by extracting tea leaves with water with a carbon source, a nitrogen source and the composite bacterial agent to obtain a mixed solution, the mixed solution is subjected to intermittent stirring anaerobic fermentation to obtain a fermentation liquid, the fermentation liquid is mixed with a sugar substitute to obtain a fermented tea pulp, and the SOD enzyme activity of each liter of the fermented tea pulp is greater than 1300U. In each mL of the tea base liquid, the inoculation amount of Kluyveromyces marxianus is 0.2-0.3×10 6 cfu and / or Lactobacillus plantarum inoculation volume is 100-110×10 6 cfu, and / or Lactobacillus germanica subsp. lactis inoculated at 10-15×10 6 cfu.

[0074] Preferably, the inoculation amount of Kluyveromyces marxianus in each mL of tea base liquid can be 0.20×10 6 cfu, 0.21×10 6 cfu, 0.22×10 6 cfu, 0.23×10 6 cfu, 0.24×10 6 cfu, 0.25×10 6 cfu, 0.26×10 6 cfu, 0.27×10 6 cfu, 0.28×10 6 cfu, 0.29×10 6 cfu or 0.3×10 6 cfu, or the inoculum amount of Kluyveromyces marxianus within a numerical range consisting of any two of the above specific numerical values ​​as endpoints.

[0075] Preferably, the inoculum amount of Lactobacillus plantarum in each mL of tea-based liquid is 100×10 6 cfu, 100.1×10 6 cfu, 100.2×10 6 cfu, 100.3×10 6 cfu, 100.4×10 6 cfu, 100.5×10 6 cfu, 100.6×10 6 cfu, 100.7×10 6 cfu, 100.8×10 6 cfu, 100.9×10 6 cfu, 101.0×10 6 cfu, 101.1×10 6 cfu, 101.2×10 6cfu、101.3×10 6 cfu、101.4×10 6 cfu、101.5×10 6 cfu、101.6×10 6 cfu、101.7×10 6 cfu、101.8×10 6 cfu、101.9×10 6 cfu、102.0×10 6 cfu、102.1×10 6 cfu、102.2×10 6 cfu、102.3×10 6 cfu、102.4×10 6 cfu、102.5×10 6 cfu、102.6×10 6 cfu、102.7×10 6 cfu、102.8×10 6 cfu、102.9×10 6 cfu、103.0×10 6 cfu、103.1×10 6 cfu、103.2×10 6 cfu、103.3×10 6 cfu、103.4×10 6 cfu、103.5×10 6 cfu、103.6×10 6 cfu、103.7×10 6 cfu、103.8×10 6 cfu、103.9×10 6 cfu、104.0×10 6 cfu、104.1×10 6 cfu、104.2×10 6 cfu、104.3×10 6 cfu、104.4×10 6 cfu、104.5×10 6 cfu、104.6×10 6 cfu、104.7×10 6 cfu、104.8×10 6 cfu、104.9×10 6 cfu、105.0×10 6 cfu、105.1×10 6 cfu、105.2×10 6cfu、105.3×10 6 cfu、105.4×10 6 cfu、105.5×10 6 cfu、105.6×10 6 cfu、105.7×10 6 cfu、105.8×10 6 cfu、105.9、106.0、106.1×10 6 cfu、106.2×10 6 cfu、106.3×10 6 cfu、106.4×10 6 cfu、106.5×10 6 cfu、106.6×10 6 cfu、106.7×10 6 cfu、106.8×10 6 cfu、106.9×10 6 cfu、107.0×10 6 cfu、107.1×10 6 cfu、107.2×10 6 cfu、107.3×10 6 cfu、107.4×10 6 cfu、107.5×10 6 cfu、107.6×10 6 cfu、107.7×10 6 cfu、107.8×10 6 cfu、107.9×10 6 cfu、108.0×10 6 cfu、108.1×10 6 cfu、108.2×10 6 cfu、108.3×10 6 cfu、108.4×10 6 cfu、108.5×10 6 cfu、108.6×10 6 cfu、108.7×10 6 cfu、108.8×10 6 cfu、108.9×10 6 cfu、109.0×10 6 cfu、109.1×10 6 cfu、109.2×10 6 cfu、109.3×10 6 cfu、109.4×106 cfu, 109.5×10 6 cfu, 109.6×10 6 cfu, 109.7×10 6 cfu, 109.8×10 6 cfu、109.9×10 6 cfu or 110.0×10 6 cfu, or the inoculum amount of Lactobacillus plantarum within a numerical range consisting of any two of the above specific values ​​as endpoints.

[0076] Preferably, the inoculum amount of Lactobacillus germanica subsp. lactis in each mL of tea base liquid is 10×10 6 cfu, 10.1×10 6 cfu, 10.2×10 6 cfu, 10.3×10 6 cfu, 10.4×10 6 cfu, 10.5×10 6 cfu, 10.6×10 6 cfu, 10.7×10 6 cfu, 10.8×10 6 cfu, 10.9×10 6 cfu, 11.0×10 6 cfu, 11.1×10 6 cfu, 11.2×10 6 cfu, 11.3×10 6 cfu, 11.4×10 6 cfu, 11.5×10 6 cfu, 11.6×10 6 cfu, 11.7×10 6 cfu, 11.8×10 6 cfu, 11.9×10 6 cfu, 12.0×10 6 cfu, 12.1×10 6 cfu, 12.2×10 6 cfu, 12.3×10 6 cfu, 12.4×10 6 cfu, 12.5×10 6 cfu, 12.6×10 6 cfu, 12.7×10 6 cfu, 12.8×10 6 cfu, 12.9×10 6 cfu, 13.0×10 6 cfu, 13.1×106 cfu, 13.2×10 6 cfu, 13.3×10 6 cfu, 13.4×10 6 cfu, 13.5×10 6 cfu, 13.6×10 6 cfu, 13.7×10 6 cfu, 13.8×10 6 cfu, 13.9×10 6 cfu, 14.0×10 6 cfu, 14.1×10 6 cfu, 14.2×10 6 cfu, 14.3×10 6 cfu, 14.4×10 6 cfu, 14.5×10 6 cfu, 14.6×10 6 cfu, 14.7×10 6 cfu, 14.8×10 6 cfu, 14.9×10 6 cfu or 15.0×10 6 cfu, or the inoculum amount of Lactobacillus germanica subsp. lactis within the numerical range formed by any two of the above specific numerical values ​​as endpoints.

[0077] The invention mixes tea-based liquid, Kluyveromyces marxianus Linzhi.KM-D3, Lactobacillus plantarum Ali.Plateau.LP.VIII and Lactobacillus delbrueckii subspecies Dangxiong LBVIII to obtain a mixed liquid, wherein the live bacteria counts of Kluyveromyces marxianus Linzhi.KM-D3, the live bacteria counts of Lactobacillus plantarum Ali.Plateau.LP.VIII and the live bacteria counts of Lactobacillus delbrueckii subspecies Dangxiong LBVIII exist in the mixed liquid at a live bacteria count ratio of a specific initial fermentation, and then performs anaerobic fermentation. During the anaerobic fermentation process, Kluyveromyces marxianus Linzhi.KM-D3 first converts the polysaccharides in the tea base liquid into monosaccharides, so that Lactobacillus plantarum Ali.Plateau.LP.Ⅷ and Lactobacillus delbrueckii subspecies Dangxiong LBⅧ can further improve the sugar utilization rate. Among them, Lactobacillus plantarum Ali.Plateau.LP.Ⅷ and Lactobacillus delbrueckii subspecies Dangxiong LBⅧ have strong ability to produce lactic acid through synergistic fermentation, and Kluyveromyces marxianus Linzhi.KM-D3 has strong acid resistance. Therefore, the relevant indicators of the fermented tea pulp obtained by the synergistic fermentation of these three specific bacteria are higher than the requirements of QB / T 5323-2018 plant enzymes. Specifically, the pH value of the fermented tea pulp is greater than 3.0 and less than or equal to 4.0, the sucrose content in each 100g of the fermented tea pulp is greater than 0g and less than 0.5g, the total acid (lactic acid) content in each 100g of the fermented tea pulp is greater than or equal to 6g and less than 10g, the ethanol content in each 100g of the fermented tea pulp is greater than 0g and less than or equal to 0.5g, and the SOD enzyme activity in each L of the fermented tea pulp is greater than 1300U and less than 100000U. At the same time, the DPPH free radical scavenging ability of the fermented tea pulp is greater than 140% and less than 180%, the hydroxyl free radical scavenging ability is greater than 130% and less than 160%, and the ABTS free radical scavenging ability is greater than 80% and less than 100%. In addition, the fermented tea pulp has a soft and rich sour taste and a good taste.

[0078] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0079] Unless otherwise specified, the various reagents / instruments used in the examples of the present invention are conventional commercially available products. The sources of experimental materials and instrument information used in the present invention are shown in Table 1:

[0080] Table 1 Reagent / Instrument Information

[0081]

[0082]

[0083] The identification of Kluyveromyces marxianus Linzhi.KM-D3 used in the examples is as follows:

[0084] (1) Isolation of strains: The yogurt pimple samples collected in Linzhi City, Tibet Autonomous Region were diluted 10 times to prepare 10 -5 , 10 -6 The bacterial suspension was spread on a YPD solid medium and cultured at 30°C for 18 hours to obtain a single colony. The solid medium was prepared as follows: 1 g yeast extract, 2 g glucose, 2 g peptone, 2 g agar powder and distilled water were mixed and fixed to 1 L and the pH value was adjusted to 5.5±0.2, followed by sterilization at 115°C for 20 minutes to obtain a solid medium.

[0085] (2) Purification of strains: Pick a single colony in YPD liquid medium and culture it at 30°C for 18 hours to obtain seed liquid. Streak the seed liquid on YPD solid medium and culture it at 30°C for 24 hours to complete the first purification. Perform purification three times in a row.

[0086] (3) Identification of strains: Spread the bacterial suspension of the purified strain on YPD solid medium, incubate at 30°C for 18 h, and observe the colony morphology. Pick a single colony and place it in 5 mL of YPD liquid medium, incubate at 180 rpm and 30°C for 24 h, then pipette 10 μL of the bacterial suspension onto a glass slide and observe the bacterial morphology under an optical microscope at 400x magnification.

[0087] A strain of bacteria was obtained, the colony texture of which was 1-2mm in diameter, round, with neat edges, opaque, grayish white on the front, convex in the middle, smooth and bright on the surface, moist in texture, easy to pick up, and with microscopic morphology of circle, ellipse, oval, etc. Asexual reproduction was budding, with budding at one end, and the cell morphology was consistent with the characteristics of yeast.

[0088] The genome of the bacterium was extracted, and (ITS1 (5'-CCGTAGGTGAACCTGCGG-3', i.e., SEQ ID NO.1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', i.e., SEQ ID NO.2) were used as primers. The PCR program was 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 90 s, 30 cycles; and finally 72°C extension for 10 min. It was used to amplify the ITS gene sequence. After 1% gel electrophoresis detection and sequencing, it was compared with the sequence on GenBank by Blast analysis. The sequence similarity was greater than 99% and it was the same species. The ITS gene sequence of the strain was measured as shown in SEQ ID NO.3:

[0089]

[0090] Combined with morphological analysis and molecular identification, Kluyveromyces marxious Linzhi.KM-D3 was deposited in the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with the deposit number CCTCC NO: M 20231575, deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. Figure 1 Shown is a microscopic observation image of Kluyveromyces marxianus Linzhi.KM-D3.

[0091] The preparation methods of the bacterial suspensions of Kluyveromyces marxianus Linzhi.KM-D3, Lactobacillus plantarum Ali.Plateau.LP.VIII and Lactobacillus germanium subsp. lactis Dangxiong LBVIII used in the examples and comparative examples are as follows:

[0092] (1) A single colony of Kluyveromyces marxianus Linzhi.KM-D3 was selected and placed in YPD liquid medium, cultured at 30°C for 18 h to obtain a bacterial suspension, and the number of viable cells in the bacterial suspension was adjusted to 2.5×10 7 cfu / mL, and obtain the bacterial suspension of Kluyveromyces marxianus Linzhi.KM-D3.

[0093] (2) Pick a single colony of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ and place it in MRS liquid culture medium, culture it at 37°C for 24 h to obtain a bacterial suspension, and adjust the viable bacterial count of the bacterial suspension to 9×10 9 cfu / mL, and obtain the bacterial suspension of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ.

[0094] (3) A single colony of Lactobacillus germanium subspecies Dangxiong LBⅧ was selected and placed in MRS liquid culture medium, cultured at 37°C for 24 h to obtain a bacterial suspension, and the number of viable bacteria in the bacterial suspension was adjusted to 8×10 9 cfu / mL, and obtain the bacterial suspension of Lactobacillus germanium subspecies Dangxiong LBⅧ.

[0095] Example 1

[0096] Example 1 provides a method for preparing fermented tea pulp, and the specific steps of the preparation method are as follows:

[0097] (1) preparing a tea-based liquid: mixing tea leaves and water at a weight ratio of 2.5:100, heating at 100° C. for 15 min, and filtering to obtain tea soup. Then, based on the weight of the tea soup, adding 6 wt % sucrose and 1 wt‰ yeast extract powder to the tea soup, mixing evenly, sterilizing, and cooling to room temperature to obtain 6000 mL of tea-based liquid.

[0098] (2) Preparation of mixed solution: Inoculate 0.3×10 6 cfu of Kluyveromyces marxianus Linzhi.KM-D3, 110×10 6 cfu of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ, and 15×10 6 cfu of Lactobacillus germanium subspecies Dangxiong LBⅧ (i.e., into 6000mL of tea-based liquid, 72mL of Kluyveromyces marxianus Linzhi.KM-D3 bacterial suspension, 73.3mL of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ bacterial suspension, and 11.25mL of Lactobacillus germanium subspecies Dangxiong LBⅧ bacterial suspension are added).

[0099] (3) preparing fermented liquid: the mixed solution obtained in step (2) is subjected to intermittent stirring anaerobic fermentation at 30°C and 500r / min until Kluyveromyces marxianus Linzhi.KM-D3, Lactobacillus plantarum Ali.Plateau.LP.Ⅷ and Lactobacillus delbrueckii lactis subspecies Dangxiong LBⅧ, all of which enter the logarithmic growth phase (the fermentation time at this time is the 18th hour). Then, after the 18th hour, the fermented liquid is subjected to intermittent stirring anaerobic fermentation at 30°C and 50r / min, and the fermentation is stopped when the pH value of the fermented liquid is 3.2 (the fermentation time at this time is the 48th hour), thereby obtaining the fermented liquid. Wherein, the frequency of the intermittent stirring is stirring for 20min and pausing for 40min.

[0100] (5) Preparing fermented tea puree: Based on the weight of the fermented liquid obtained in step (4), add 0.02 wt% stevioside, 3 wt% erythritol and 3 wt% xylitol to the fermented liquid, mix well and filter through a 200-mesh sieve until the solution is clear, then fill and sterilize at 85° C. for 10 min to obtain fermented tea puree.

[0101] Example 2

[0102] Example 2 provides a method for preparing fermented tea pulp, which is different from Example 1 in that: in step (1), tea leaves and water are mixed in a weight ratio of 1:100 and heated at 100°C for 5 minutes and then filtered to obtain tea soup.

[0103] Example 3

[0104] Example 3 provides a method for preparing fermented tea puree. The difference between the preparation method and Example 1 is that in step (1), the amount of sucrose added is 2 wt% and the amount of yeast extract powder added is 0.5 wt‰.

[0105] Example 4

[0106] Example 4 provides a method for preparing fermented tea pulp, which is different from Example 1 in that: in step (1), tea leaves and water are mixed in a weight ratio of 1:100 and heated at 100°C for 5 minutes and then filtered to obtain tea soup.

[0107] In step (2), 0.2×10 6 cfu of Kluyveromyces marxianus Linzhi.KM-D3, 100×10 6 cfu of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ, and 10×10 6 cfu of Lactobacillus germanium subspecies Dangxiong LBⅧ (i.e., into 6000mL of tea-based liquid, 48mL of Kluyveromyces marxianus Linzhi.KM-D3 bacterial suspension, 66.7mL of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ bacterial suspension, and 7.5mL of Lactobacillus germanium subspecies Dangxiong LBⅧ bacterial suspension were added).

[0108] In step (3), the mixed solution obtained by step (2) is subjected to intermittent stirring anaerobic fermentation under the condition of 30 ℃, 300r / min to Kluyveromyces marxianus Linzhi.KM-D3, Lactobacillus plantarum Ali.Plateau.LP.Ⅷ and Lactobacillus delbrueckii lactis subspecies Dangxiong LBⅧ, and these three kinds of bacteria all enter the logarithmic growth phase (fermentation time at this moment is the 18th hour). Then after the 18th hour, the fermented liquid is subjected to intermittent stirring anaerobic fermentation under the condition of 30 ℃, 50r / min, and fermentation is stopped when the fermented liquid pH value is 3.2 (fermentation time at this moment is the 48th hour), to obtain the fermented liquid. Wherein, the frequency of intermittent stirring is to stir 20min and suspend 40min.

[0109] Example 5

[0110] Example 5 provides a method for preparing fermented tea pulp, which is different from Example 1 in that: in step (1), tea leaves and water are mixed in a weight ratio of 1:100 and heated at 100°C for 5 minutes and then filtered to obtain tea soup.

[0111] In step (2), 0.2×10 6 cfu of Kluyveromyces marxianus Linzhi.KM-D3, 100×10 6 cfu of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ, and 10×10 6 cfu of Lactobacillus germanium subspecies Dangxiong LBⅧ. (That is, 48 ​​mL of Kluyveromyces marxianus Linzhi.KM-D3 suspension, 66.7 mL of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ suspension, and 7.5 mL of Lactobacillus germanium subspecies Dangxiong LBⅧ suspension were added to 6000 mL of tea-based liquid).

[0112] Comparative Example 1

[0113] Comparative Example 1 provides a method for preparing fermented tea puree, which is different from Example 1 in that: in step (1), tea leaves and water are mixed in a weight ratio of 3:100.

[0114] Comparative Example 2

[0115] Comparative Example 1 provides a method for preparing fermented tea puree, which is different from Example 1 in that: in step (1), tea leaves and water are mixed and heated at 100° C. for 17 minutes.

[0116] Comparative Example 3

[0117] Comparative Example 3 provides a method for preparing fermented tea puree, which is different from Example 1 in that: in step (1), tea leaves and water are mixed in a weight ratio of 0.5:100.

[0118] Comparative Example 4

[0119] Comparative Example 4 provides a method for preparing fermented tea puree, which is different from Example 1 in that: in step (1), the amount of sucrose added is 7 wt %.

[0120] Comparative Example 5

[0121] Comparative Example 5 provides a method for preparing fermented tea puree, which is different from Example 1 in that: in step (1), the amount of yeast extract powder added is 1.5 wt‰.

[0122] Comparative Example 6

[0123] Comparative Example 6 provides a method for preparing fermented tea puree, which is different from Example 3 in that: in step (1), the amount of sucrose added is 1 wt%.

[0124] Comparative Example 7

[0125] Comparative Example 7 provides a method for preparing fermented tea puree, which is different from Example 3 in that: in step (1), no yeast extract powder is added.

[0126] Comparative Example 8

[0127] Comparative Example 8 provides a method for preparing fermented tea pulp. The difference between the preparation method and Example 2 is that in step (2), 0.4×10 6 cfu of Kluyveromyces marxianus Linzhi.KM-D3 (i.e., 96 mL of Kluyveromyces marxianus Linzhi.KM-D3 bacterial suspension was added to 6000 mL of tea-based liquid). The inoculation amount of other strains and the added volume of bacterial suspension were the same as those in Example 2.

[0128] Comparative Example 9

[0129] Comparative Example 9 provides a method for preparing fermented tea puree. The difference between the preparation method and Example 2 is that: in step (2), 115×10 6 cfu of Lactobacillus plantarum Ali. Plateau. LP. VIII, (ie, 76.7 mL of bacterial suspension of Lactobacillus plantarum Ali. Plateau. LP. VIII was added to 6000 mL of tea-based liquid). The inoculation amount of other strains and the added volume of bacterial suspension were the same as those in Example 2.

[0130] Comparative Example 10

[0131] Comparative Example 10 provides a method for preparing fermented tea pulp, which is different from that of Example 2 in that: in step (2), 20×10 6 cfu of Lactobacillus germanium subspecies Dangxiong LBⅧ (i.e., 15 mL of bacterial suspension of Lactobacillus germanium subspecies Dangxiong LBⅧ was added to 6000 mL of tea-based liquid). The inoculation amount of other strains and the added volume of bacterial suspension were the same as those in Example 2.

[0132] Comparative Example 11

[0133] Comparative Example 11 provides a method for preparing fermented tea puree. The difference between the preparation method and Example 4 is that in step (2), 0.1×10 6cfu of Kluyveromyces marxianus Linzhi.KM-D3 (i.e., 24 mL of Kluyveromyces marxianus Linzhi.KM-D3 bacterial suspension was added to 6000 mL of tea-based liquid). The inoculation amount of other strains and the added volume of bacterial suspension were the same as those in Example 4.

[0134] Comparative Example 12

[0135] Comparative Example 12 provides a method for preparing fermented tea pulp. The difference between the preparation method and Example 4 is that in step (2), 90×10 6 cfu of Lactobacillus plantarum Ali. Plateau. LP. VIII, (ie, 60 mL of a bacterial suspension of Lactobacillus plantarum Ali. Plateau. LP. VIII was added to 6000 mL of tea-based liquid). The inoculation amount of other strains and the added volume of the bacterial suspension were the same as those in Example 4.

[0136] Comparative Example 13

[0137] Comparative Example 13 provides a method for preparing fermented tea puree. The difference between the preparation method and Example 4 is that in step (2), 5×10 6 cfu of Lactobacillus germanium subspecies Dangxiong LBⅧ (i.e., 3.75 mL of bacterial suspension of Lactobacillus germanium subspecies Dangxiong LBⅧ was added to 6000 mL of tea-based liquid). The inoculation amount of other strains and the added volume of bacterial suspension were the same as those in Example 4.

[0138] Comparative Example 14

[0139] Comparative Example 14 provides a method for preparing a fermented tea pulp, and the difference between this method and Example 4 is that: in step (3), the mixed solution obtained in step (2) is subjected to intermittent stirring anaerobic fermentation at 30°C and 200r / min to Kluyveromyces marxianus Linzhi.KM-D3, Lactobacillus plantarum Ali.Plateau.LP.Ⅷ and Lactobacillus delbrueckii lactis subspecies Dangxiong LBⅧ, and these three bacteria all enter the logarithmic growth phase (the fermentation time at this time is the 18th hour). Then after the 18th hour, the fermented liquid is subjected to intermittent stirring anaerobic fermentation at 30°C and 50r / min, and the fermentation is stopped when the pH value of the fermented liquid is 3.2 (the fermentation time at this time is the 48th hour), to obtain the fermented liquid. Wherein, the frequency of intermittent stirring is stirring for 20min and pausing for 40min.

[0140] Comparative Example 15

[0141] Comparative Example 15 provides a method for preparing a fermented tea pulp, and the difference between this method and Example 5 is that: in step (3), the mixed solution obtained in step (2) is subjected to intermittent stirring anaerobic fermentation at 30°C and 600r / min to Kluyveromyces marxianus Linzhi.KM-D3, Lactobacillus plantarum Ali.Plateau.LP.Ⅷ and Lactobacillus delbrueckii lactis subspecies Dangxiong LBⅧ, and these three bacteria all enter the logarithmic growth phase (the fermentation time at this time is the 18th hour). Then after the 18th hour, the fermented liquid is subjected to intermittent stirring anaerobic fermentation at 30°C and 50r / min, and the fermentation is stopped when the pH value of the fermented liquid is 3.2 (the fermentation time at this time is the 48th hour), to obtain the fermented liquid. Wherein, the frequency of intermittent stirring is stirring for 20min and pausing for 40min.

[0142] Comparative Example 16

[0143] Comparative Example 16 provides a method for preparing fermented tea puree, which is different from that of Example 4 in that: in step (3), anaerobic static fermentation is carried out at 30°C for 48 hours.

[0144] Comparative Example 17

[0145] Comparative Example 17 provides a method for preparing fermented tea puree, which is different from Example 2 in that: the Kluyveromyces marxianus Linzhi.KM-D3 is replaced with Saccharomyces cerevisiae with a preservation number of CCTCC NO: M 209190, wherein the number of viable bacteria in the bacterial suspension of the Saccharomyces cerevisiae is the same as that in the bacterial suspension of Kluyveromyces marxianus Linzhi.KM-D3.

[0146] Comparative Example 18

[0147] Comparative Example 18 provides a method for preparing fermented tea puree, which is different from Example 2 in that Lactobacillus plantarum Ali.Plateau.LP.Ⅷ is replaced by fermented Lactobacillus mucilaginosus CQPC04, wherein the number of viable bacteria in the bacterial suspension of fermented Lactobacillus mucilaginosus CQPC04 is the same as that in the bacterial suspension of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ.

[0148] Comparative Example 19

[0149] Comparative Example 19 provides a method for preparing fermented tea puree, which is different from Example 2 in that Lactobacillus germanium subspecies Dangxiong LBⅧ is replaced by Lactobacillus acidophilus LA-10A, wherein the number of live bacteria in the bacterial suspension of Lactobacillus acidophilus LA-10A is the same as that in the bacterial suspension of Lactobacillus germanium subspecies Dangxiong LBⅧ.

[0150] Technical effect evaluation

[0151] 1. The fermented tea pulp obtained in Examples 1-5 and Comparative Examples 1-16 was tested for catechin content, total acid content (calculated as lactic acid), pH value, ethanol content and sucrose content. The test results are shown in Table 2. Figure 2 , Figure 3 , Figure 4 and Figure 5 As shown,

[0152] Catechin content detection method: according to the method described in the national standard GB 5009 157.

[0153] Total acid content detection method: detection is carried out according to the method described in national standard GB 5009 157.

[0154] Ethanol content detection method: Detection is carried out according to the method described in the national standard GB / T 12143.

[0155] Sucrose content detection method: detection is carried out according to the method described in the national standard GB 5009.8-2016.

[0156] pH value detection method: Detection is carried out according to the method described in the national standard GB / T 10468.

[0157] Table 2. Ethanol content, total acid content, pH value and sucrose content in the fermented tea pulp obtained in Examples 1-5 and Comparative Examples 1-16

[0158]

[0159]

[0160] 2. Fifty people were randomly selected to conduct sensory evaluation on the fermented tea purees obtained in Examples 1-5 and Comparative Examples 1-16 according to the sensory evaluation indicators shown in Table 3. The specific sensory evaluation results are shown in Table 4.

[0161] Table 3. Sensory evaluation indexes

[0162]

[0163]

[0164] Table 4. Sensory evaluation results

[0165]

[0166]

[0167] As shown in Table 2 and Figure 2 As shown, Figure 2The catechin content and sucrose content in the fermented tea pulp obtained in Example 1, Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3 are shown. The results show that in terms of extraction process, as the proportion of tea leaves increases and the extraction time increases, the catechin content in the tea soup is higher. However, too much tea leaves are added during extraction in Comparative Example 1 and the extraction time is too long in Comparative Example 2, resulting in too high catechin content affecting microbial growth, low utilization rate of sucrose by microorganisms, and sucrose content greater than 0.5g / 100g; too little tea leaves are added during extraction in Comparative Example 3, resulting in too low dissolution of nutrients in the tea leaves, affecting the demand of microorganisms for nutrients, resulting in insufficient fermentation power, low utilization rate of sucrose by microorganisms, and sucrose content greater than 0.5g / 100g.

[0168] As shown in Table 2 and Figure 3 As shown, Figure 3 The ethanol content, sucrose content and total acid (calculated as lactic acid) content in the fermented tea puree obtained in Example 1, Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 and Comparative Example 7 are shown. The results show that, as shown in Example 1 and Example 3, adding a specific ratio of carbon source (i.e. sucrose) and nitrogen source (i.e. yeast extract) can promote the growth of microorganisms.

[0169] It can be seen from Comparative Example 4 that the addition of excessive carbon source (i.e., sucrose) results in the ethanol content in the fermented tea pulp prepared in Comparative Example 4 being greater than 0.5 g / 100 g.

[0170] It can be seen from Comparative Example 5 that the fermented tea pulp prepared by adding an excessive amount of nitrogen source (i.e., yeast extract powder) meets the requirements in terms of ethanol content, total acid content, pH value and sucrose content. However, it can be seen from Table 4 that the fermented tea pulp prepared in Comparative Example 5 has a fermentation odor, which indicates that a high addition amount of nitrogen source (i.e., yeast extract powder) will cause an odor in the fermentation liquid, affecting the sensory flavor of the fermented tea pulp.

[0171] It can be seen from Comparative Examples 6 and 7 that adding too little carbon source (i.e. sucrose) or not adding nitrogen source (i.e. yeast extract powder) will lead to insufficient growth power of microorganisms, resulting in low utilization rate of sucrose by microorganisms, so that the sucrose content in the fermented tea puree prepared in Comparative Examples 6 and 7 is greater than 0.5g / 100g;

[0172] As shown in Table 2 and Figure 4 As shown, Figure 4The ethanol content, sucrose content and total acid (calculated as lactic acid) in the fermented tea puree obtained in Example 2, Example 4, Comparative Example 8, Comparative Example 9, Comparative Example 10, Comparative Example 11, Comparative Example 12 and Comparative Example 13. The results show that: from Example 2 and Example 4, when Kluyveromyces marxianus Linzhi.KM-D3, Lactobacillus plantarum Ali.Plateau.LP.Ⅷ and Lactobacillus delbrueckii lactis subsp. Dangxiong LBⅧ are added to the tea base liquid in a specific inoculation amount, the synergistic symbiosis between the bacteria can be promoted.

[0173] From Comparative Examples 8-13, it can be seen that adjusting the inoculation amount of a certain bacteria in the tea-based liquid alone will have different effects on the fermentation results. Specifically, from Comparative Example 8, it can be seen that Comparative Example 8 increases the inoculation amount of Kluyveromyces marxianus Linzhi.KM-D3 in the tea-based liquid, so that the ethanol content in the fermented tea pulp is greater than 0.5g / 100g.

[0174] It can be seen from Comparative Examples 9 and 10 that Comparative Example 9 increases the inoculation amount of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ in the tea-based liquid, and Comparative Example 10 increases the inoculation amount of Lactobacillus Germanus subspecies Dangxiong LBⅧ in the tea-based liquid. Although the ethanol content, total acid content, pH value and sucrose content all meet the requirements, it can be seen from Table 4 that the fermented tea puree prepared in Comparative Examples 9 and 10 has a fermentation odor, which indicates that the inoculation amount of lactic acid bacteria in the tea-based liquid is too high, which will cause a peculiar smell in the fermentation liquid and affect the sensory flavor of the fermented tea puree.

[0175] It can be seen from Comparative Example 11 that Comparative Example 11 reduces the inoculation amount of Kluyveromyces marxianus Linzhi.KM-D3, resulting in weak synergistic ability of the microbial flora and low acid production during the fermentation process, resulting in a sucrose content in the fermented tea pulp greater than 0.5g / 100g.

[0176] It can be seen from Comparative Examples 12 and 13 that Comparative Example 12 reduces the inoculation amount of Lactobacillus plantarum Ali.Plateau.LP.Ⅷ, and Comparative Example 13 reduces the inoculation amount of Lactobacillus germanium subspecies Dangxiong LBⅧ, resulting in weak synergistic ability of the microbial flora during the fermentation process, resulting in a sucrose content in the obtained fermented tea pulp greater than 0.5g / 100g.

[0177] As shown in Table 2 and Figure 5 As shown, Figure 5The ethanol content, sucrose content and total acid (in terms of lactic acid) content in the fermented tea pulp obtained by Example 4, Example 5, Comparative Example 14, Comparative Example 15 and Comparative Example 16. The results show that in the anaerobic fermentation, it can be seen from Examples 4 and 5 that intermittent stirring can make the microorganisms fully contact with the nutrients, making the fermentation more thorough and the sucrose content low. It can be seen from Comparative Examples 14 and 15 that the speed of intermittent stirring is too low, which is not conducive to the utilization of nutrients by microorganisms, and the speed of intermittent stirring is too high, which is not conducive to the growth of microorganisms, and both will lead to the sucrose content in the fermented tea pulp being greater than 0.5g / 100g; Comparative Example 16 directly adopts static anaerobic fermentation, and the fermented tea pulp obtained by this method has low acid production and sucrose content greater than 0.5g / 100g, which is higher than other embodiments and comparative examples. It can be obtained that intermittent stirring anaerobic fermentation can make the utilization rate of nutrients by microorganisms higher and the fermentation more thorough.

[0178] In summary, controlling the tea extraction process, the amount of carbon source added, the amount of nitrogen source added, the inoculation amount of the bacterial species in the tea-based liquid and the parameters of intermittent stirring anaerobic fermentation can improve the utilization rate of microbial fermentation, so that the pH value of the obtained fermented tea puree is less than or equal to 4.0, the total acid content (calculated as lactic acid) is greater than or equal to 6g / 100g, the ethanol content is less than or equal to 0.5g / 100g, the sucrose content is less than or equal to 0.5g / 100g, and the fermented tea puree has no fermentation odor and light bitterness in terms of sensory evaluation.

[0179] 3. The fermented tea pulp obtained in Examples 1-5 and Comparative Examples 17-19 was tested for SOD enzyme activity, DPPH free radical scavenging ability, hydroxyl free radical scavenging ability and ABTS free radical scavenging ability. The test results are shown in Table 5, wherein:

[0180] (1) The SOD enzyme activity was detected according to the first method of national standard GB / T 5009.171.

[0181] (2) The detection method of DPPH free radical scavenging ability is as follows: the fermented tea pulp obtained in Examples 1-5 and Comparative Examples 17-19 is diluted with deionized water to obtain a dilution with a concentration of 1 mg / mL (i.e., there is 1 mg of fermented tea pulp in each mL of dilution). Take 1 mL of the dilution, add 1 mL of 0.1 mmol / L DPPH-anhydrous ethanol solution thereto, mix well, react for 30 minutes at room temperature and in the dark, obtain a sample solution, measure its absorbance at a wavelength of 517 nm (denoted as A1), use anhydrous ethanol to replace the sample solution in the blank group to determine its absorbance (denoted as A0), and use anhydrous ethanol to replace the DPPH-anhydrous ethanol solution in the control group to determine the absorbance of the solution (denoted as A2). The DPPH free radical scavenging rate is calculated according to the formula: DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0]×100, and the detection results are shown in Table 5.

[0182] (3) The method for detecting the hydroxyl radical scavenging ability is as follows: the fermented tea pulp obtained in Examples 1-5 and Comparative Examples 17-19 is diluted to 1 mg / mL with deionized water to obtain a dilution solution, 1 mL of the dilution solution is taken, 1 mL of a 6 mmol / L FeSO4 solution and 1 mL of a 2.4 mmol / L H2O2 solution are added thereto respectively, mixed, reacted for 10 min, and then 1 mL of a 6 mmol / L salicylic acid solution is added, reacted at 37°C for 30 min, and a sample solution is obtained, and its absorbance value (A1) is measured at a wavelength of 510 nm. The blank group uses distilled water instead of the sample solution to determine its absorbance value (A0), and the control group uses distilled water instead of the H2O2 solution to determine its absorbance value (A2) after the reaction. The hydroxyl radical scavenging rate is calculated according to the formula: hydroxyl radical scavenging rate (%) = [1-(A1-A2) / A0] × 100, and the test results are shown in Table 5.

[0183] (4) The detection method of ABTS free radical scavenging ability is as follows: the fermented tea pulp obtained in Examples 1-5 and Comparative Examples 17-19 is diluted to 1 mg / mL with deionized water to obtain a dilution solution, 1 mL of ABTS solution with a concentration of 7.4 mmol / L is mixed with 1 mL of K2ALI.LP-VIIIO4 with a concentration of 2.6 mmol / L, and the mixture is kept in a dark place for 16 hours to obtain an ABTS+ stock solution; before use, the ABTS+ stock solution is appropriately diluted with anhydrous ethanol solution so that the absorbance range of the mixed solution at 734 nm is 0.7±0.02 to obtain an ABTS working solution. 1.980 mL of ABTS working solution is mixed with 20 μL of sample diluent, reacted at 30° C. for 10 minutes, and the absorbance value (A1) is measured at 734 nm; the control group is mixed with distilled water and ethanol to measure the absorbance value (A2); the blank group is replaced with distilled water to measure the absorbance value (A0). The ABTS free radical scavenging ability was calculated according to the formula: ABTS free radical scavenging rate (%) = [1–(A1–A2) / A0] × 100. The test results are shown in Table 5.

[0184] Table 5. Test results of DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, ABTS free radical scavenging rate and SOD enzyme activity

[0185]

[0186] As shown in Table 5, the results show that compared with Comparative Examples 17, 18, and 19, Examples 1-5 have higher SOD enzyme activity, and the scavenging rates of DPPH, ABTS, and hydroxyl radicals are also higher. It can be seen that the fermented tea puree of Kluyveromyces marxianus Linzhi.KM-D3, Lactobacillus plantarum Ali.Plateau.LP.Ⅷ, and Lactobacillus delbrueckii lactis subspecies Dangxiong LBⅧ has antioxidant activity. And combined with the above-mentioned technical effect evaluation (I) and technical effect evaluation (II), it can be obtained that in the process of preparing fermented tea puree, there is a synergistic symbiotic effect between Kluyveromyces marxianus Linzhi.KM-D3, Lactobacillus plantarum Ali.Plateau.LP.Ⅷ, and Lactobacillus delbrueckii lactis subspecies Dangxiong LBⅧ.

[0187] In summary, the relevant indicators of the fermented tea pulp prepared by the preparation method of Examples 1-5 of the present invention are higher than the provisions of QB / T 5323-2018 Plant Enzyme. Specifically, the pH value of the fermented tea pulp is greater than 3.0 and less than or equal to 4.0, the sugar content in each 100g of fermented tea pulp is greater than 0g and less than 0.5g, the total acid content (calculated as lactic acid) in each 100g of fermented tea pulp is greater than or equal to 6g and less than 10g, the ethanol content in each 100g of fermented tea pulp is greater than 0g and less than or equal to 0.5g, and the SOD enzyme activity in each L of fermented tea pulp is greater than 1300U and less than 100000U.

[0188] At the same time, the fermented tea puree prepared by the preparation method of Examples 1-5 has a DPPH free radical scavenging capacity greater than 140% and less than 180%, a hydroxyl free radical scavenging capacity greater than 130% and less than 160%, and an ABTS free radical scavenging capacity greater than 80% and less than 100%. In addition, the fermented tea puree prepared by the preparation method of Examples 1-5 has a soft and rich sour taste and a good taste.

[0189] The above embodiments are only for further explanation and understanding of the technical solution of the present invention, and are not limitations of the present invention. Any non-prominent substantial features and non-significant improvements made by those skilled in the art on this basis should fall within the scope of protection of the present invention.

Claims

1. A Kluyveromyces marxianus, characterized in that The Kluyveromyces marxianus is Kluyveromyces marxianus Linzhi.KM-D3, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO:M 20231575.

2. The Kluyveromyces marxianus according to claim 1, characterized in that The ITS gene sequence of Kluyveromyces marxianus is shown in SEQ ID No.

3.

3. Use of the Kluyveromyces marxianus according to claim 1 or 2 in the preparation of a bacterial agent.

4. Use of the Kluyveromyces marxianus according to claim 1 or 2 in preparing fermented tea pulp with a SOD enzyme activity of more than 1300 U / L.

5. A composite bacterial agent, characterized in that: The method comprises the Kluyveromyces marxianus according to claim 1 or 2.

6. The composite bacterial agent according to claim 5, characterized in that: Also contains Lactobacillus plantarum and / or Lactobacillus germanica subsp. lactis.

7. The composite bacterial agent according to claim 6, characterized in that: The method is prepared by a method comprising the following steps: respectively amplifying and culturing Kluyveromyces marxianus, Lactobacillus plantarum and Lactobacillus delbrueckii subsp. lactis to obtain a bacterial solution of Kluyveromyces marxianus, a bacterial solution of Lactobacillus plantarum and a bacterial solution of Lactobacillus delbrueckii subsp. lactis. Preferably, the three bacterial solutions are preserved independently but used in combination. More preferably, the bacterial solution of Kluyveromyces marxianus, the bacterial solution of Lactobacillus plantarum and the bacterial solution of Lactobacillus delbrueckii subsp. lactis are used in combination in a volume ratio of 45-75:65-75:7-12.

8. The composite bacterial agent according to claim 7, wherein the number of viable bacteria of Kluyveromyces marxense is 2-3×10 7 cfu / mL, and / or the viable count of Lactobacillus plantarum was 8.5-9.5×10 9 cfu / mL, and / or the viable count of Lactobacillus germanica subsp. lactis was 7.5-8.5×10 9 cfu / mL.

9. The composite bacterial agent according to any one of claims 5 to 8, wherein the plant lactobacillus is plant lactobacillus Ali. Plateau. LP. VIII, with a deposit number of CCTCC NO: M 2022068, And / or the Lactobacillus delbrueckii subspecies lactis is Lactobacillus delbrueckii subspecies lactis Dangxiong LBⅧ, with the deposit number being CCTCCNO:M 2023396.

10. A method for preparing the composite bacterial agent according to any one of claims 5 to 9, characterized in that: The method comprises the following steps: respectively amplifying and culturing the Kluyveromyces marxianus and Lactobacillus plantarum and / or Lactobacillus germanica subsp. lactis as claimed in claim 1 or 2.

11. Use of the composite bacterial agent according to any one of claims 5 to 9 or the composite bacterial agent prepared by the preparation method according to claim 10 in preparing fermented tea puree.

12. A fermented tea puree, characterized in that: A composite bacterial agent comprising any one of claims 5 to 9 or a composite bacterial agent prepared by the preparation method according to claim 10.

13. The fermented tea puree according to claim 12, characterized in that: The fermented tea pulp is prepared by mixing tea soup obtained by extracting tea leaves with water with a carbon source, a nitrogen source and the composite bacterial agent to obtain a mixed liquid, subjecting the mixed liquid to intermittent stirring anaerobic fermentation to obtain a fermentation liquid, and mixing the fermentation liquid with a sugar substitute to obtain the fermented tea pulp, wherein the SOD enzyme activity of each liter of the fermented tea pulp is greater than 1300U.

14. The fermented tea puree according to claim 13, characterized in that: The SOD enzyme activity in each L of fermented tea puree is greater than 1300U and less than or equal to 100000U; preferably, the SOD enzyme activity in each L of fermented tea puree is greater than 1700U and less than or equal to 100000U.

15. The fermented tea puree according to claim 13 or 14, wherein The fermented tea pulp has a DPPH free radical scavenging ability greater than or equal to 140%, and / or a hydroxyl free radical scavenging ability greater than 130%, and / or an ABTS free radical scavenging ability greater than 80%.

16. The fermented tea puree according to any one of claims 12 to 15, characterized in that: The fermented tea puree has a DPPH free radical scavenging capacity greater than or equal to 140% and less than or equal to 180%, and / or a hydroxyl free radical scavenging capacity greater than 130% and less than or equal to 160%, and / or an ABTS free radical scavenging capacity greater than 80% and less than or equal to 100%; Preferably, the fermented tea puree has a DPPH radical scavenging capacity greater than or equal to 165% and less than or equal to 180%, and / or a hydroxyl radical scavenging capacity greater than 150% and less than or equal to 160%, and / or an ABTS radical scavenging capacity greater than 90% and less than or equal to 100%.

17. The fermented tea puree according to any one of claims 12 to 16, characterized in that: The pH value is less than or equal to 4.0, and / or the sucrose content in every 100g of fermented tea puree is less than 0.5g, and / or the total acid content in terms of lactic acid in every 100g of fermented tea puree is greater than or equal to 6g, and / or the ethanol content in every 100g of fermented tea puree is less than or equal to 0.5g.

18. The fermented tea puree according to any one of claims 12 to 17, characterized in that: The pH value is greater than or equal to 3.0 and less than or equal to 4.0, and / or the sucrose content in each 100g of fermented tea puree is greater than or equal to 0 and less than 0.5g, and / or the total acid content in terms of lactic acid in each 100g of fermented tea puree is greater than or equal to 6g and less than or equal to 10g, and / or the ethanol content in each 100g of fermented tea puree is greater than or equal to 0 and less than or equal to 0.5g; Preferably, the sucrose content in every 100g of fermented tea puree is greater than or equal to 0 and less than 0.35g, and / or the total acid content in terms of lactic acid in every 100g of fermented tea puree is greater than or equal to 7g and less than or equal to 10g, and / or the ethanol content in every 100g of fermented tea puree is greater than or equal to 0 and less than or equal to 0.35g.

19. The fermented tea puree according to any one of claims 12 to 18, characterized in that: The inoculation amount of Kluyveromyces marxianus in each mL of tea base liquid is 0.2-0.3×10 6 cfu, and / or Lactobacillus plantarum inoculation volume is 100-110×10 6 cfu, and / or Lactobacillus germanica subsp. lactis inoculated at 10-15×10 6 cfu; Preferably, the inoculation amount of Kluyveromyces marxianus in each mL of tea base liquid is 0.25-0.3×10 6 cfu, and / or Lactobacillus plantarum inoculation volume is 105-110×10 6 cfu, and / or Lactobacillus delbrueckii subsp. lactis inoculated at 13-15×10 6 cfu.

20. The fermented tea puree according to claim 13, characterized in that: When the tea leaves are extracted with water, the weight ratio of the tea leaves to the water is 1-2.5:100, preferably, the weight ratio of the tea leaves to the water is 1.5-2.5:

100.

21. The fermented tea puree according to claim 20, characterized in that: The tea leaves include one or more of black tea, oolong tea and dark tea. Preferably, the tea leaves are black tea.

22. The fermented tea puree according to claim 13, characterized in that: Based on the weight of the tea soup, the added amount of the carbon source is 2-6wt%, preferably, the added amount of the carbon source is 4-6wt%; and / or based on the weight of the tea soup, the added amount of the nitrogen source is 0.5-1wt‰, preferably, the added amount of the nitrogen source is 0.7-1wt‰.

23. The fermented tea puree according to claim 22, characterized in that: The carbon source includes one or more of sucrose, fructose and glucose. Preferably, the carbon source is sucrose.

24. The fermented tea puree according to claim 13, characterized in that: The nitrogen source was yeast extract powder.

25. The fermented tea puree according to claim 13, characterized in that: The added amount of the sugar substitute is 0.01-12 wt % based on the weight of the fermentation liquid.

26. The fermented tea puree according to claim 25, characterized in that: The sugar substitute includes one or more of steviol glycoside, erythritol and xylitol. Preferably, the sugar substitute is a combination of steviol glycoside, erythritol and xylitol; more preferably, based on the weight of the fermentation liquid, the amount of steviol glycoside added is 0.01-0.05wt%, and / or the amount of erythritol added is 1wt%-5wt%, and / or the amount of xylitol added is 1wt%-5wt%.

27. A method for preparing fermented tea puree according to any one of claims 12 to 26, characterized in that: The method comprises the following steps: the fermented tea pulp is prepared by mixing tea soup obtained by extracting tea leaves with water with a carbon source, a nitrogen source and the composite bacterial agent to obtain a mixed liquid; the mixed liquid is subjected to intermittent stirring anaerobic fermentation to obtain a fermented liquid; and the fermented liquid is mixed with a sugar substitute to obtain the fermented tea pulp.

28. The preparation method according to claim 27, characterized in that: The steps include: (1) mixing tea soup obtained by extracting tea leaves with water, a carbon source and a nitrogen source, sterilizing and cooling the mixture to obtain a tea-based liquid; (2) mixing the tea-based liquid obtained in step (1) and the composite bacterial agent to obtain a mixed liquid; (3) subjecting the mixed solution obtained in step (2) to intermittent stirring anaerobic fermentation to obtain a fermentation liquid; (4) The fermentation liquid obtained in step (3) is mixed with the sugar substitute, filtered, filled and sterilized to obtain the fermented tea puree.

29. The preparation method according to claim 27 or 28, characterized in that: The extraction temperature is: 95-100°C and / or the extraction time is: 5-15min.

30. The preparation method according to claim 28, characterized in that: The sterilization temperature in step (1) is 95-115°C, and / or the sterilization temperature in step (3) is 85-95°C.

31. The preparation method according to claim 28, characterized in that: The sterilization time in step (1) is 10-20 min, and / or the sterilization time in step (3) is 10-20 min.

32. The preparation method according to claim 28, characterized in that: In step (3), the fermentation is terminated when the pH of the fermentation liquid is 3.0-3.

5.

33. The preparation method according to claim 28, characterized in that: In step (3), the frequency of intermittent stirring is stirring for 15-20 minutes and pausing for 40-45 minutes.

34. The preparation method according to any one of claims 27 to 33, characterized in that: The stirring speed is 300-500 r / min before both the yeast and the lactic acid bacteria enter the logarithmic growth phase, and the stirring speed is 50-80 r / min after both the yeast and the lactic acid bacteria enter the logarithmic growth phase.

35. The preparation method according to any one of claims 27 to 34, characterized in that: The stirring temperature is 25-35°C.

36. The preparation method according to any one of claims 27 to 35, characterized in that: In step (3), the filtration is performed by circulating filtration using a 200-300 mesh sieve until the solution is clarified.

37. A fermented tea puree, characterized in that: The fermented tea puree is prepared by the preparation method according to any one of claims 27 to 36.

38. Use of the fermented tea puree according to any one of claims 12 to 26 or claim 37 in preparing food.

39. A food, characterized in that Contains the fermented tea puree according to any one of claims 12 to 26 or claim 37.

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