CN108634047B - Flowering inducer, application of flowering inducer and flowering process - Google Patents

Flowering inducer, application of flowering inducer and flowering process Download PDF

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CN108634047B
CN108634047B CN201810447590.0A CN201810447590A CN108634047B CN 108634047 B CN108634047 B CN 108634047B CN 201810447590 A CN201810447590 A CN 201810447590A CN 108634047 B CN108634047 B CN 108634047B
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flowering
tea
inducer
eurotium cristatum
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CN108634047A (en
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凌铁军
刘路路
李儒泽
龙雁华
张红
张梁
李大祥
李立祥
张正竹
宛晓春
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Anhui Agricultural University AHAU
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
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    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/123—Bulgaricus
    • A—HUMAN NECESSITIES
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    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/137—Delbrueckii
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/231—Lactis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/513—Adolescentes

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Abstract

The invention relates to a flowering inducer, and application and a flowering process of the flowering inducer. The flowering inducer comprises any one or more of Bacillus strain, Lactobacillus strain, Microbacterium strain, lactococcus strain and Bifidobacterium strain. The tea flowering process using the flowering inducer comprises the following steps: sterilizing tea raw materials; adding eurotium cristatum and a flowering inducer: adding spore suspension of eurotium cristatum and bacterial suspension of a flowering inducer into the tea raw material; the tea leaves are bloomed. The flowering inducer can solve the problems of long flowering period, unstable flowering quality, possibility of harmful bacteria and the like in the processing process of Fuzhuan tea and other flowering tea products, can improve the flowering efficiency and enhance the flowering effect, has definite microbial safety guarantee, and can provide high-quality probiotic Fuzhuan tea containing one or more beneficial microbes and other flowering probiotic tea products.

Description

Flowering inducer, application of flowering inducer and flowering process
Technical Field
The invention belongs to the technical field of application of microorganisms to food processing, and particularly relates to a flowering inducer, and application and a flowering process of the flowering inducer.
Background
Eurotium cristatum (commonly known as "golden flower") is a fungus of the genus Eurotium (Eurotium) of the family Trichocomaceae (Trichocomaceae) of the order Eurotiales (Eurotiales). Research shows that the bacillus subtilis has various health care effects of improving human immunity, regulating human metabolism, reducing blood fat, lowering blood pressure, regulating blood sugar and the like. Fu tea is a special black tea in China. The endoplasmic fungi has flowery flavor, pure and strong fragrance, orange yellow soup color and mellow taste. The most important link in the preparation process is a process commonly called 'flowering', namely, eurotium cristatum is taken as the dominant bacterium and vigorously grows in the raw material of the Fu tea, namely the raw material of the dark raw tea. Therefore, as a kind of microbial fermented tea (i.e. black tea which is one of six major tea types), the unique quality, unique flavor and numerous efficacies of Fu tea are all attributed to eurotium cristatum therein (see the following documents).
[1] Zhengshuan, Shengyao, Ou Xiao, Saiwao, Huangkunlun, Okunju dark tea fragrance and main efficacy research progress, food industry science and technology, 2016, 37 (20): 366-376.
[2] Liulimna, tang rainwei, wang kou, li shibingbing, liu zhong hua, liu courteous, research on fermentation production of indole alkaloids by fuscoporia fusca, tea science, 2017, 37 (5): 503-512.
[3] The research and application progress of the Huangyan, Shirui, Su Di Zheng and Guantu san Saccharum, the biological processing process, 2017, 15 (1): 49-56.
[4] Hou yi xiang, li zong jun, research progress of chemical components and functional characteristics of fu tea, chinese food and nutrition, 2016, 22 (6): 63-67.
The nature of the raw dark green tea is that the green tea is subjected to mild microbial fermentation, the bitter taste is reduced, the flavor is relatively pure and mild, and the raw dark green tea is suitable for being made into various dark green teas.
The traditional process for producing the Fuzhuan tea is to place the raw dark green tea in a flower growing warehouse in a loose tea form or a tea brick form by pressing in advance, and then obtain the finished product of the Fuzhuan tea through a flower growing process for about 2 to 3 weeks under the control of harsh temperature and humidity conditions. However, in both actual production and laboratory, we find that the effect of blossom is not obvious when the eurotium cristatum is inoculated in the raw dark tea, and the high-quality fu tea cannot be produced. Therefore, how to prepare the obvious Fuzhuan tea made of golden flowers becomes a technical problem to be urgently broken through at present.
Disclosure of Invention
In order to solve the technical problems, the invention provides a flowering inducer, and application and a flowering process of the flowering inducer.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a flowering inducer comprising any one or a combination of several of a strain of Bacillus (Bacillus sp.), a strain of Lactobacillus (Lactobacillus sp.), a strain of Microbacterium (Microbacterium sp.), a strain of lactococcus (Lactobacillus sp.), and a strain of Bifidobacterium (Bifidobacterium sp.).
In a further scheme, the flowering inducer is obtained by freeze drying treatment.
Further, the floral inducer is added with Eurotium cristatum.
In a further scheme, the freeze drying treatment process comprises the following steps: centrifuging milk to remove upper-layer grease, and then sterilizing to obtain a spare protective agent solution; and suspending spores generated by eurotium cristatum growing for 5 to 7 days on a PDA culture medium and a flowering inducer bacterial strain cultured for 48 hours on an LB or MRS culture medium in the protective agent solution respectively, pre-freezing at the temperature of minus 80 ℃ for 2 to 5 hours to be solid, and then putting the solid into a freeze dryer for vacuum freeze drying for 15 to 20 hours.
An application of the flowering inducer in a tea flowering process.
A flowering process using the flowering inducer comprises the following steps:
step 1, adding eurotium cristatum and a flowering inducer: adding the spore suspension of the eurotium cristatum and the bacterial suspension of the flowering inducer into a tea raw material to be flowered;
step 2, flowering: and (3) allowing the tea raw material to bloom for 3-5 days at the temperature of 28-32 ℃ to obtain a blooming tea product.
The preferred scheme is that various tea raw materials are sterilized before the eurotium cristatum and the flowering inducer are added.
In a further scheme, the preparation method of the spore suspension of eurotium cristatum in the step 1 comprises the following steps: firstly activating eurotium cristatum by using a PDA (Potato dextrose agar) culture medium, adding sterile water on the surface of the PDA culture medium, and lightly scraping spores by using a cotton swab to prepare a spore aqueous suspension of the eurotium cristatum with a certain concentration;
the preparation method of the bacterial suspension of the flowering inducer in the step 1 comprises the following steps: activating the flowering inducer by using an LB culture medium or an MRS culture medium, and then uniformly mixing the flowering inducer with sterile water to prepare corresponding bacterial suspension.
Further, in the step 1, the addition amount of the spore suspension of eurotium cristatum in the tea is as follows: adding 1ml of spore suspension of eurotium cristatum into each 28-32 g of tea; the addition amount of the flowering inducer bacterial suspension in the tea is as follows: adding 1ml of bacterial suspension into each 28-32 g of tea leaves.
Further, the tea raw material in the step 1 is any one of green tea, white tea, yellow tea, oolong tea, black tea and raw dark green tea.
The invention has the beneficial effects that:
(1) the invention discovers that a quorum sensing mechanism exists between the eurotium cristatum and the flowering inducer. Namely, the large amount of growth of the blossom inducer leads certain bacterial metabolites to be accumulated in the surrounding environment, and when the concentration of the metabolites in the environment reaches a certain threshold value, the metabolites are identified by the Eurotium cristatum, thereby promoting the vigorous growth of the Eurotium cristatum. Therefore, the flowering effect is greatly improved, and the flowering speed is remarkably accelerated.
The flowering inducer in the invention is a raw material for producing high-quality flowering tea products. The flower-growing inducer can solve the problems that the flower-growing period is long, the flower-growing quality is unstable, harmful bacteria are possibly generated and the like in the processing process of the Fuzhuan tea and other flower-growing tea products, namely the flower-growing inducer can improve the flower-growing efficiency and enhance the flower-growing effect, has definite microbial safety guarantee, and can provide high-quality probiotic Fuzhuan tea containing one or more beneficial microbes and other flower-growing tea products.
(2) The flowering inducer can be prepared into a product in a freeze-dried state for long-term storage, and is convenient to use directly. The product can be directly mixed with sterile water and inoculated into tea for growing flower.
(3) By combining eurotium cristatum and the flowering inducer provided by the invention, the flowering promoting effect can be very good for green tea, white tea, yellow tea, oolong tea, black tea and dark green tea. The invention can further endow the golden flower tea with unique flavor on the basis of keeping the sensory characteristics of various big tea, and various novel flower-shaped tea can be manufactured. The process provides a wide development space for the further development of the prior various large tea products with the functions of safety, high efficiency, benefit and health care.
(4) The flowering process provided by the invention can firstly avoid harmful mixed bacteria in the tea processing process, and secondly can shorten the time for realizing large amount of flowering of tea raw materials from the original 2-3 weeks to within 5 days. Moreover, the flowering inducer is composed of probiotic strains, and can introduce a new health-care effect to tea products. The invention has simple process and can be used for preparing various health tea products with the pattern of growing.
Drawings
FIG. 1 is a comparison of the fifth day of flowering of the radiation sterilized white tea after different microbial additions.
FIG. 2 is a comparison of the fifth day of flowering of the raw dark green tea after irradiation sterilization and treatment with different microbial additives.
FIG. 3 is a comparison of the fifth day of flowering of autoclaved white tea after different microbial additions.
FIG. 4 is a comparison of the fifth day of flowering of autoclaved oolong tea after different microbial additions.
Fig. 5 is a comparison of the fifth day of flowering of autoclaved green tea after different microbial additions.
FIG. 6 is a comparison of the fifth day of flowering of autoclaved yellow tea after different microbial additions.
Figure 7 is a comparison of the fifth day of flowering for autoclaved black tea after different microbial additions.
FIG. 8 is a comparison of the fifth day of flowering of autoclaved raw dark tea after different microorganism addition treatments.
FIG. 9 is a comparison of the fifth day of flowering of autoclaved raw dark tea after the addition of different microbial treatments preserved by freeze-drying.
FIG. 10 is a graph showing the comparison of the blooming of green tea after autoclaving on the fifth day after the addition of the bloom-inducing agent and the Eurotium cristatum at different ratios.
FIG. 11 is a comparison of the fifth day of flowering of the autoclaved oolong tea after the addition of the flowering inducing agent and Eurotium cristatum at different ratios.
FIG. 12 is a comparison of the fifth day of flowering of autoclaved Camellia sinensis after the addition of different proportions of the floral inducer and Eurotium cristatum.
FIG. 13 is a comparison of the blooming of autoclaved Camellia sinensis after the addition of the bloom inducer and Eurotium cristatum in different order.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the microbial strains provided by the invention, eurotium cristatum, bacillus subtilis and microbacterium mothi are all separated from commercial Fuzhuan tea, and the variety identification is based on a DNA comparison result. The other 5 bacterial strains were all commercially available.
Example 1 high-quality Fuzhuan tea and processing technology thereof
The process comprises the following steps:
preparing a tea raw material: firstly, slightly crushing raw dark green tea, and then placing a part of the raw dark green tea under the irradiation measurement of 9K for irradiation sterilization; sterilizing another part of the tea material in a high pressure steam sterilizing pot at 121 deg.C for 20 min, and adding the tea material obtained by the above two sterilization modes into 12-well cell culture plate at an amount of about 0.6g per well under aseptic environment condition for use.
Preparing a eurotium cristatum spore suspension: firstly, the eurotium cristatum is fully activated in a culture dish until a large number of spores are generated, then sterile water is added to the surface of a culture medium, and the spores are scraped by a cotton swab to prepare an eurotium cristatum spore aqueous suspension (600CFU/mL, the concentration does not need to be strictly limited, and any concentration similar to or higher than the value can be a proper concentration) for later use.
Preparing a bacterial suspension of a flowering inducer: respectively activating 7 strains of Bacillus subtilis, Bacillus coagulans, Bacillus methylotrophicus, Lactobacillus delbrueckii subsp bulgaricus, lactococcus lactis, Bifidobacterium adolescentis, and Microbacterium moths, and making into suspension (10) with sterile water6CFU/mL), name calibration, and standby. And respectively taking part of the bacterial suspensions of the 7 bacterial strains and mixing the parts of the bacterial suspensions according to equal proportion to obtain a mixed bacterial suspension, and calibrating the names for later use.
The preparation of the eurotium cristatum and blossom inducer bacterial liquid can also be as follows: the freeze-dried and preserved Eurotium cristatum spores, and strains (about 0.05 g) of bacillus subtilis, Bacillus coagulans, Bacillus methylotrophicus, Lactobacillus delbrueckii subsp bulgaricus of lactobacillus, lactococcus lactis of lactococcus, Bifidobacterium adolescentis of bifidobacterium and Microbacterium moths of Microbacterium are respectively mixed with 1mL of sterile water to prepare corresponding bacterial suspensions of Eurotium cristatum and each florescence inducer freeze-dried powder. In the operation method, the freeze-dried powder of the fungal spores and the bacteria is respectively mixed after being suspended by sterile water, or the freeze-dried powder of the eurotium cristatum and the bacteria is mixed before being suspended by sterile water, and the effect of promoting the flowering after the tea raw material is added is the same.
Adding the prepared Eurotium cristatum spore suspension to the sterile tea raw material of the 12-hole cell culture plate at a volume-to-mass ratio (ml/g) of about 1:30 (which can be adjusted according to practical application in production), and adding the bacterial suspension of single bacteria or mixed bacterial suspension to 106CFU/mL or 105CFU/mL、104CFU/mL、103CFU/mL、102The bacterial concentration of CFU/mL was added to the tea material at a volume to mass ratio (mL/g) of about 1: 30. Then, the inoculated tea raw material is placed in an environment with the temperature of 30 ℃ for about 5 days to finish the flower growing process, and the high-quality Fuzhuan tea is prepared (see figure 2, figure 8, figure 9, figure 12 and figure 13).
This embodiment is further illustrated herein by an analysis of the attached drawings:
firstly, comparing fig. 2 and fig. 8, it can be seen that the raw material of the raw dark green tea sterilized by irradiation or high-pressure steaming can smoothly bloom, and the blooming effect is obviously better than that of the control group without the blooming inducer, so the sterilization method is not limited, that is, various sterilization methods can be used as the pretreatment method of the raw material of the blooming tea.
The types of microorganism addition in each well of the cell culture plate in FIG. 2 are:
c2: eurotium cristatum; c3: no added microorganisms; a1: eurotium cristatum + bacillus subtilis; a2: eurotium cristatum + bacillus methylotrophicus; a3: eurotium cristatum + bacillus coagulans; a4: eurotium cristatum + microbacterium mothi; b1: eurotium cristatum + lactobacillus bulgaricus; b2: the Eurotium cristatum and the above 5 bacterial strains are mixed in equal proportion to form a bacterial suspension.
As can be seen from figure 2, the sterilized raw dark green tea is taken as a tea raw material, and the 5 probiotics flower-growing inducers or the mixed microbial inoculum thereof can obviously promote the eurotium cristatum to flower.
Secondly, comparing fig. 8 and fig. 9, it can be seen that the use of the temporarily activated microbial strains or the use of the microbial strains which are freeze-dried in advance can enable the raw dark green tea to successfully bloom, and the blooming effect is also obviously better than that of the control group without the blooming inducer, so the addition form of the microbial strains is not limited, that is, the addition of the temporarily activated microbial strains or the addition of the freeze-dried and preserved microbial strains can enable the raw dark green tea or other tea materials to successfully bloom.
The types of microorganism addition in each well of the cell culture plate in FIG. 8 are:
c2: eurotium cristatum; c4: no added microorganisms; a1: eurotium cristatum + bacillus subtilis; a3: eurotium cristatum + streptococcus lactis; a4: eurotium cristatum + microbacterium mothi; b1: eurotium cristatum + bacillus methylotrophicus; b2: eurotium cristatum + bacillus coagulans; b4: eurotium cristatum + Lactobacillus bulgaricus.
As can be seen from FIG. 8, the 6 bacterial flowering inducers provided by the invention can obviously promote the flowering of Eurotium cristatum in the raw material of the sterilized raw dark green tea.
The types of microorganism addition in each well of the cell culture plate in FIG. 9 are:
c2: eurotium cristatum; c3: no added microorganisms; a1: eurotium cristatum + bacillus subtilis; a2: eurotium cristatum + bacillus methylotrophicus; a3: eurotium cristatum + bacillus coagulans; a4: eurotium cristatum + microbacterium mothi; b1: eurotium cristatum + streptococcus lactis; b2: eurotium cristatum + bifidobacterium adolescentis; b3: eurotium cristatum + Lactobacillus bulgaricus.
As can be seen from FIG. 9, any of the freeze-dried and preserved flowering revulsants provided by the present invention can significantly promote the flowering of Eurotium cristatum in sterilized raw dark green tea.
Finally, as can be seen from fig. 12 and 13, the respective ratios of the biomass between eurotium cristatum and the bacterial flowering inducer, or the sequence of adding eurotium cristatum and the bacterial flowering inducer, do not significantly affect the flowering effect, and the effect is significantly better than that of the control group without the flowering inducer. This demonstrates that the tea raw material can be added in any order or proportion between eurotium cristatum and bacterial bloom inducer to produce a good blooming effect.
The types of microorganism addition in each well of the cell culture plate in FIG. 12 are:
a1: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)6CFU/mL) A2: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)5CFU/mL); a3: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)4CFU/mL); a4: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)3CFU/mL); b1: eurotium cristatum (600CFU/mL) + Bacillus subtilis (100 CFU/mL); b2: eurotium cristatum (600 CFU/mL); b3: no microorganisms were added.
As can be seen from FIG. 12, the flowering of Eurotium cristatum in the raw material of the sterilized raw dark green tea can be obviously promoted by adding the strain of the flowering inducer provided by the invention and the Eurotium cristatum in any proportion.
The types of microorganism addition in each well of the cell culture plate in FIG. 13 are:
a1: the Eurotium cristatum was added, and 48 hours later, the Lactobacillus bulgaricus strain was added (A1a, A1b, A1c are pictures of the bloom on days 2, 3, and 4, respectively, where the Lactobacillus bulgaricus was added).
A2: the Eurotium cristatum was added, and the Bacillus subtilis strain (A2a, A2b, A2c are pictures of the blossoming of day 2, day 3, and day 4, respectively, to which Bacillus subtilis was added) was added 48 hours later.
B1: lactobacillus bulgaricus strain is added, and Eurotium cristatum is added after 48 hours (B1a, B1B, B1c are pictures of blooming on days 2, 3, and 4, respectively, where Eurotium cristatum is added).
B2: the Bacillus subtilis strain is added firstly, and the Eurotium cristatum is added after 48 hours (B2a, B2B and B2c are pictures of blossoming on days 2, 3 and 4 respectively).
C1: simultaneously, Eurotium cristatum and Lactobacillus bulgaricus strains (C1a, C1b, C1C are pictures of blossoming on day 4, day 5, day 6, respectively, where the two types of microorganisms were added) were added.
C2: simultaneously, Eurotium cristatum and Bacillus subtilis strains (C2a, C2b, C2C are pictures of blossoming on day 4, day 5, and day 6 of the addition of the two microorganisms, respectively) were added.
B4: only Eurotium cristatum was added (B4a, B4B, B4c are pictures of blossoming on days 2, 3 and 4, respectively, where Eurotium cristatum was added).
C4: no added microorganisms (day 4, day 5, day 6 pictures for C4a, C4b, C4C, respectively).
As can be seen from fig. 13, regardless of the change in the order of addition of eurotium cristatum and bacterial inducer, significant flowering of the tea material occurred approximately 5 days after co-inoculation of eurotium cristatum and flowering inducer, and the flowering effect was significantly better than that of the control group without inducer. Therefore, the strain of the flowering inducer and the eurotium cristatum provided by the invention can obviously promote the flowering of the eurotium cristatum in the raw materials of the sterilized raw dark green tea by adding the strain and the eurotium cristatum in various sequences.
Example 2 high-quality white tea for growing flowers and processing technology thereof
The process comprises the following steps:
preparing a tea raw material: firstly, slightly crushing white tea, and then placing a part of the white tea under the irradiation measurement of 9K for irradiation sterilization; sterilizing another part of the tea material in a high pressure steam sterilizing pot at 121 deg.C for 20 min, and adding the tea material obtained by the above two sterilization modes into 12-well cell culture plate at an amount of about 0.6g per well under aseptic environment condition for use.
Preparing a eurotium cristatum spore suspension: the same as in example 1.
Preparing a bacterial suspension of a flowering inducer: the same as in example 1.
Adding the prepared eurotium cristatum spore suspension to the sterile tea raw material of the 12-hole cell culture plate in a volume-to-mass ratio (milliliter/gram) of about 1:30 (which can be adjusted correspondingly according to practical application in production), adding a single bacterial suspension or a mixed bacterial suspension to the tea raw material in the same ratio as the spore suspension, and then placing the inoculated tea raw material in an environment at 30 ℃ for about 5 days to finish a flowering process to prepare the high-quality flowering white tea (see fig. 1 and 3).
Similarly to the case of example 1, we can also see that the white tea raw material sterilized by irradiation or high-pressure steaming can smoothly bloom and the blooming effect is obviously better than that of the control group without the blooming inducer by analyzing fig. 1 and fig. 3.
The types of microorganism addition in each well of the cell culture plate in FIG. 1 are respectively:
c1: eurotium cristatum; c3: no added microorganisms; a1: eurotium cristatum + bacillus subtilis; a2: eurotium cristatum + bacillus methylotrophicus; a3: eurotium cristatum + bacillus coagulans; a4: eurotium cristatum + microbacterium mothi; b1: eurotium cristatum + lactobacillus bulgaricus; b2: the Eurotium cristatum and the above 5 bacterial strains are mixed in equal proportion to form a bacterial suspension.
As can be seen from figure 1, the 5 bacterial flower-growing inducers and the mixed inocula thereof provided by the invention can obviously promote the growth of Eurotium cristatum in the raw materials of the sterilized white tea (note: a great amount of pekoe which is inherent in the white tea can be seen in C1 and C3, but the pekoe is not 'golden flower')
The types of microorganism addition in each well of the cell culture plate in FIG. 3 are:
c3: eurotium cristatum; c4: no added microorganisms; a1: eurotium cristatum + bacillus subtilis; a3: eurotium cristatum + streptococcus lactis; a4: eurotium cristatum + microbacterium mothi; b1: eurotium cristatum + bacillus methylotrophicus; b2: eurotium cristatum + bacillus coagulans; b3: eurotium cristatum + bifidobacterium adolescentis; b4: eurotium cristatum + lactobacillus bulgaricus; c1: the Eurotium cristatum and the above 7 bacterial strains are mixed in equal proportion to form a bacterial suspension.
As can be seen from figure 3, the flower-growing inducer and the mixed microbial inoculum thereof can obviously promote the growth of Eurotium cristatum in the raw materials of the sterilized white tea (note: a great amount of pekoe inherent in the white tea can be seen in C3 and C4, which are not 'golden flower')
Examples 1 and 2 demonstrate that various sterilization methods should be used as a pretreatment method for the starting material of the scented tea.
Example 3 high quality flowering oolong tea and processing technology thereof
The process comprises the following steps:
preparing a tea raw material: oolong tea was first slightly pulverized, then placed in an autoclave at 121 ℃ for 20 minutes, and then added to a 12-well cell culture plate in an amount of about 0.6g per well under aseptic conditions for future use.
Preparing a eurotium cristatum spore suspension: the same as in example 1.
Preparing a bacterial suspension of a flowering inducer: the same as in example 1.
Adding the prepared eurotium cristatum spore suspension to the sterile tea raw material of the 12-hole cell culture plate at a volume-to-mass ratio (ml/g) of about 1:30 (which can be adjusted according to practical application in production), and adding the single bacterial suspension or the mixed bacterial suspension to the sterile tea raw material of the 12-hole cell culture plate at a ratio of 106CFU/mL or 105CFU/mL、104CFU/mL、103CFU/mL、102The bacterial concentration of CFU/mL is added to the tea material at a ratio of about 1:30 in terms of volume to mass (mL/g), and then the inoculated tea material is placed in an environment at 30 ℃ for about 5 days to complete the flowering process, thereby producing high-quality, flowering oolong tea (see FIGS. 4 and 11).
The types of microorganism addition in each well of the cell culture plate in FIG. 4 are:
c2: eurotium cristatum; c4: no added microorganisms; a1: eurotium cristatum + bacillus subtilis; a3: eurotium cristatum + streptococcus lactis; a4: eurotium cristatum + microbacterium mothi; b1: eurotium cristatum + bacillus methylotrophicus; b2: eurotium cristatum + bacillus coagulans; b4: eurotium cristatum + lactobacillus bulgaricus; c1: bacterial suspension prepared by mixing eurotium cristatum and more than 6 bacterial strains in equal proportion
As can be seen from FIG. 4, the 6 bacterial flowering inducers and the mixed microbial inoculum thereof provided by the invention can obviously promote the flowering of Eurotium cristatum in the raw material of the sterilized oolong tea.
The types of microorganism addition in each well of the cell culture plate in FIG. 11 are:
a1: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)6CFU/mL); a2: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)5CFU/mL); a3: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)4CFU/mL); a4: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)3CFU/mL); b1: eurotium cristatum (600CFU/mL) + Bacillus subtilis (100 CFU/mL); b2: eurotium cristatum (600 CFU/mL); b3: no microorganisms were added.
As can be seen from FIG. 11, the bloom inducer strain provided by the present invention can significantly promote the bloom of Eurotium cristatum in the raw material of sterilized oolong tea when added in various proportions.
Example 4 high quality hairy Green tea and Process for producing the same
The process comprises the following steps:
preparing a tea raw material: the green tea was first lightly pulverized, then placed in an autoclave at 121 ℃ for 20 minutes, sterilized, and then added to a 12-well cell culture plate in an amount of about 0.6g per well under aseptic conditions for future use.
Preparing a eurotium cristatum spore suspension: the same as in example 1.
Preparing a bacterial suspension of a flowering inducer: the same as in example 1.
Adding the prepared eurotium cristatum spore suspension to the sterile tea raw material of the 12-hole cell culture plate at a volume-to-mass ratio (ml/g) of about 1:30 (which can be adjusted according to practical application in production), and adding the single bacterial suspension or the mixed bacterial suspension of the flowering inducer at 10%6CFU/mL or 105CFU/mL、104CFU/mL、103CFU/mL、102The bacterial concentration of CFU/mL is added to the tea material at a ratio of about 1:30 in terms of volume to mass (mL/g), and then the inoculated tea material is placed in an environment at 30 ℃ for about 5 days to complete the flowering process, thereby producing high-quality flowering green tea (see FIG. 5 and FIG. 10).
The types of microorganism addition in each well of the cell culture plate in FIG. 5 are:
c3: eurotium cristatum; c4: no added microorganisms; a1: eurotium cristatum + bacillus subtilis; a3: eurotium cristatum + streptococcus lactis; a4: eurotium cristatum + microbacterium mothi; b1: eurotium cristatum + bacillus methylotrophicus; b2: eurotium cristatum + bacillus coagulans; b3: eurotium cristatum + bifidobacterium adolescentis; b4: eurotium cristatum + lactobacillus bulgaricus; c1: the Eurotium cristatum and the above 7 bacterial strains are mixed in equal proportion to form a bacterial suspension.
As can be seen from FIG. 5, any one of the flowering inducers and the mixed microbial inoculum thereof provided by the invention can obviously promote the flowering of Eurotium cristatum in sterilized green tea raw materials.
The types of microorganism addition in each well of the cell culture plate in FIG. 10 are:
a1: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)6CFU/mL); a2: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)5CFU/mL); a3: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)4CFU/mL); a4: eurotium cristatum (600CFU/mL) + Bacillus subtilis (10)3CFU/mL); b1: eurotium cristatum (600CFU/mL) + Bacillus subtilis (100 CFU/mL); b2: eurotium cristatum (600 CFU/mL); b3: no microorganisms were added.
As can be seen from FIG. 10, the bloom of Eurotium cristatum in sterilized green tea materials can be significantly promoted by adding the strain of the floral inducer provided by the present invention in various proportions to Eurotium cristatum.
By analyzing fig. 10, fig. 11 and fig. 12, we found that several tea raw materials can obviously bloom under 5 biomass ratios with large span between eurotium cristatum and bacterial bloom inducer, and the effect of the tea raw materials is remarkably superior to that of the control group without the bloom inducer. Therefore, the tea raw materials can be added into the eurotium cristatum and the bacterial flowering inducer in any proportion to produce good flowering effect.
Example 5 high quality flowering yellow tea and processing technology thereof
The process comprises the following steps:
preparing a tea raw material: the yellow tea is firstly slightly crushed, then is put into an autoclave for sterilization at the temperature of 121 ℃ for 20 minutes, is cooled to the room temperature after sterilization, and is added into a 12-hole cell culture plate in an amount of about 0.6g per hole under the aseptic environment condition for standby.
Preparing a eurotium cristatum spore suspension: the same as in example 1.
Preparing a bacterial suspension of a flowering inducer: the same as in example 1.
Adding the prepared eurotium cristatum spore suspension to the sterile tea raw material of the 12-hole cell culture plate in a volume-to-mass ratio (milliliter/gram) of about 1:30 (which can be adjusted correspondingly according to practical application in production), adding a single bacterial suspension or a mixed bacterial suspension to the tea raw material in the same ratio as the spore suspension, and then placing the inoculated tea raw material in an environment at 30 ℃ for about 5 days to finish a flowering process to prepare the high-quality flowering yellow tea (see figure 6).
The types of microorganism addition in each well of the cell culture plate in FIG. 6 are:
c3: eurotium cristatum; c4: no added microorganisms; a1: eurotium cristatum + bacillus subtilis; a3: eurotium cristatum + streptococcus lactis; a4: eurotium cristatum + microbacterium mothi; b1: eurotium cristatum + bacillus methylotrophicus; b2: eurotium cristatum + bacillus coagulans; b3: eurotium cristatum + bifidobacterium adolescentis; b4: eurotium cristatum + lactobacillus bulgaricus; c1: the Eurotium cristatum and the above 7 bacterial strains are mixed in equal proportion to form a bacterial suspension.
As can be seen from FIG. 6, any one of the flower-growing inducers and the mixed microbial inoculum thereof provided by the invention can obviously promote the flower growing of the eurotium cristatum in the raw material of the sterilized yellow tea.
Example 6 high-quality sparkling black tea and processing technology thereof
The process comprises the following steps:
preparing a tea raw material: the black tea is firstly slightly crushed, then is put into an autoclave for sterilization at 121 ℃ for 20 minutes, is cooled to room temperature after sterilization, and is added into a 12-hole cell culture plate in an amount of about 0.6g per hole under the aseptic environment condition for standby.
Preparing a eurotium cristatum spore suspension: the same as in example 1.
Preparing a bacterial suspension of a flowering inducer: the same as in example 1.
Adding the prepared eurotium cristatum spore suspension to the sterile tea raw material of the 12-hole cell culture plate in a volume-to-mass ratio (milliliter/gram) of about 1:30 (which can be adjusted correspondingly according to practical application in production), adding a single bacterial suspension or a mixed bacterial suspension to the tea raw material in the same ratio as the spore suspension, and then placing the inoculated tea raw material in an environment at 30 ℃ for about 5 days to finish a flowering process to prepare the high-quality flowering black tea (see figure 7).
The types of microorganism addition in each well of the cell culture plate in FIG. 7 are:
c2: eurotium cristatum; c4: no added microorganisms; a1: eurotium cristatum + bacillus subtilis; a2: eurotium cristatum + bacillus methylotrophicus; a3: eurotium cristatum + bacillus coagulans; a4: eurotium cristatum + microbacterium mothi; b1: eurotium cristatum + lactobacillus bulgaricus; b2: eurotium cristatum + streptococcus lactis; b3: eurotium cristatum + bifidobacterium adolescentis; b4: the Eurotium cristatum and the above 7 bacterial strains are mixed in equal proportion to form a bacterial suspension.
As can be seen from FIG. 7, any one of the flowering inducers and the mixed microbial inoculum thereof provided by the invention can obviously promote the flowering of the Eurotium cristatum in the sterilized black tea raw material.
The temperature of the process is set under laboratory conditions, and can be correspondingly adjusted according to actual environmental conditions during production.
Combining the above examples, it was found that: the bacillus bacterial strain, the lactobacillus bacterial strain, the microbacterium bacterial strain, the lactococcus bacterial strain and the bifidobacterium bacterial strain can be respectively used as a flowering inducer and the eurotium cristatum in a form of being singly or mixed in any proportion to be used together, and the method can be applied to the field of tea fermentation processing, can promote the efficient flowering of the eurotium cristatum, greatly improves the production efficiency of the flowering tea product and improves the integral flavor of the finished tea. In addition, the tea raw material is sterilized, and the inducer is a probiotic bacterial strain, so that the safety of the tea fermentation process and the health-care effect of the tea product are obviously improved.

Claims (9)

1. The application of a flowering inducer in a tea flowering process is characterized in that: the flowering inducer comprises a strain of Microbacterium and/or a strain of lactococcus, wherein the strain of Microbacterium is Microbacterium mothecium, and the strain of lactococcus is lactococcus lactis; the flowering inducer is used for promoting the flowering of eurotium cristatum.
2. The use of the agent for inducing blooming in tea leaves according to claim 1, wherein: the floral inducer is added with Eurotium cristatum.
3. Use of a flowering inducer according to claim 1 or 2 in a tea leaf flowering process, wherein: the flowering inducer is obtained by freeze drying treatment.
4. The use of the agent for inducing blooming in tea leaves as claimed in claim 3, wherein: the freeze drying process comprises the following steps: centrifuging milk to remove upper-layer grease, and then sterilizing to obtain a spare protective agent solution; and suspending spores generated by eurotium cristatum growing for 5 to 7 days on the PDA culture medium and a flowering inducer bacterial strain growing for 48 hours on the LB or MRS culture medium in the protective agent solution respectively, pre-freezing at the temperature of minus 80 ℃ for 2 to 5 hours to be solid, and then putting the solid into a freeze dryer for vacuum freeze drying for 15 to 20 hours.
5. A flowering process using the flowering inducer of claim 1, characterized by comprising the steps of:
step 1, adding eurotium cristatum and a flowering inducer: adding the spore suspension of the eurotium cristatum and the bacterial suspension of the flowering inducer into a tea raw material to be flowered;
step 2, flowering: and (3) allowing the tea raw material to bloom for 3-5 days at the temperature of 28-32 ℃ to obtain a blooming tea product.
6. A flowering process by a flowering inducer as defined in claim 5, wherein: before adding eurotium cristatum and a flowering inducer, various tea raw materials are sterilized.
7. A flowering process by a flowering inducer as defined in claim 5, wherein:
the preparation method of the spore suspension of eurotium cristatum in the step 1 comprises the following steps: firstly activating eurotium cristatum by using a PDA (Potato dextrose agar) culture medium, adding sterile water on the surface of the PDA culture medium, and lightly scraping spores by using a cotton swab to prepare a spore aqueous suspension of the eurotium cristatum with a certain concentration;
the preparation method of the bacterial suspension of the flowering inducer in the step 1 comprises the following steps: activating the flowering inducer by using an LB culture medium or an MRS culture medium, and then uniformly mixing the flowering inducer with sterile water to prepare corresponding bacterial suspension.
8. A flowering process by a flowering inducer as defined in claim 5, wherein: the addition amount of the spore suspension of eurotium cristatum in the tea in the step 1 is as follows: adding 1ml of spore suspension of eurotium cristatum into each 28-32 g of tea; the addition amount of the flowering inducer bacterial suspension in the tea is as follows: adding 1ml of bacterial suspension into each 28-32 g of tea leaves.
9. A flowering process by a flowering inducer as defined in claim 5, wherein: the raw material of the flowering tea in the step 1 is any one of green tea, white tea, yellow tea, oolong tea, black tea and dark green tea.
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CN103843919A (en) * 2014-02-25 2014-06-11 湖南农业大学 High-quality dark green tea and processing technique thereof
CN104222352A (en) * 2014-07-25 2014-12-24 周壮生 Method for culturing eurotium cristatum of loose black tea and temperature and humidity controlled incubator applied to process of culturing eurotium cristatum of loose black tea
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