CN107227274A - Lactobacillus plantarum ST and its application in the processing of sour tea - Google Patents

Abstract

The invention belongs to field of food, it is related to Lactobacillus plantarum ST bacterial strains and its application processed in sour tea.Lactobacillus plantarum strain ST, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 18th, 2017, and preserving number is CGMCC No.13911, and Classification And Nomenclature is Lactobacillus plantarum ST.The sour tea of appliable plant lactobacillus ST productions, flow is cleaning fresh leaves of tea plant, drain, steam beating, rub tea, bottling, inoculated plant lactobacillus ST, sealing and fermenting, it is hot-forming, the process such as spontaneously dry.The method can significantly improve master's theine total amount in sour tea, product quality is lifted, it is ensured that product quality, shorten the sour tea production cycle, it is adaptable to the sour tea production of different scales, have broad application prospects.

Description

Lactobacillus plantarum ST and its application in the processing of sour tea

Technical field

The invention belongs to food technology field, it is related to Lactobacillus plantarum ST bacterial strains and its application processed in sour tea.

Background technology

China is the country of tea, is also the area of origin of tea culture, and the description to tea can trace back to the legendary god of farming in ancient times earliest Period, contemporary Lu Yu《Tea is passed through》In just it is on the books：" tea is drink, sends out Shen Nongshi ".Drink tea and had thousands of years in China History, is the daily beverage that people generally like, at present with cocoa, coffee and referred to as one of big non-alcoholic drink in the world three. Domestic and international numerous studies show, the various active compositions, wherein tea such as Tea Polyphenols, alkaloid, tea polysaccharide, Tea Pigment are contained in tealeaves Polyphenol, which has, to be removed free radical, anticancer, anti-mutation, sterilization, hypotensive, radioresistance, prevents that angiocardiopathy and diabetes etc. are more Plant plurality of health care functions.Catechin compounds are the bulk composition of Tea Polyphenols, account for the 65%~80% of Tea Polyphenols total amount, mainly Including catechin (EC), nutgall catechin (EGC), catechin and gallate (ECG) and nutgall catechin gallic acid 4 kinds of materials of ester (EGCG).

Yunnan Province is located in southwest China, advantageous natural conditions and long plant tea history, has bred what is enriched Tea Germplasm, is the centre of origin of tea tree, original producton location in the world.Live in ethnic group's plant teas of Yunnan various regions, tea making, Like tea, almost each nationality maintains the drink differed from one another and (eats) tea mode, form oneself unique eating habit and the people Between health preserving method, create brilliance name race culture.

De'ang nationality is one of cross-border nationality of China, is hung up one's hat in the remote mountain areas on the ground such as Yunnan Dehong, the Baoshan, Lincang, In history because hard to get to, productivity is backward, the low factor of living standard, shortage of medical services and medicines is tortured with a disease again.De'ang nationality's quilt Referred to as " ancient tea grower ", in the fight with disease, long-term practical experience of life is passed through by De'ang nationality, has gradually been pregnant with sour tea etc. Some have the health preserving method among the people of distinct national characters, and continue into the present.Nowadays the sour tea preparation process in Deang be Annual 5, June is in hot and humid time, De'ang nationality's collection large leaf fresh leaves of tea plant, through drying, rinsing, steam (boiling) tea and rub Tea, loads among thick bamboo tube, and after embedment underground is fermented 60-70 days, boiling water is brewed；Repressed again after fermentation it can also be made with dry Into brick tea, boiling water is drunk after brewing.The processing method that can be seen that sour tea at present from the processing technology of sour tea is also only resided within On Conventional wisdom, sour tea product generally existing process-cycle length, unstable quality, quality disunity, security are difficult to ensure that Drawback.Therefore, specification sour tea processing method, improves sour tea quality, it is ensured that steady quality, allow " ensconce boudoir people fail to see, it is slightly sour micro- The sour tea of De'ang nationality of bitter taste sweetness " is approved by the world, with bright prospects and significance.

The content of the invention

Present invention Lactobacillus plantarum ST isolated from natural fermented sourdough tea, is inoculated into sour tea by Lactobacillus plantarum ST and sends out During ferment, catechin total amount in sour tea can be significantly improved, sour tea quality is improved, while the process-cycle can be shortened, it is ensured that Product quality.

The technical scheme is that：

Lactobacillus plantarum ST, the Lactobacillus plantarum is preserved in Chinese microorganism strain preservation management committee on April 18th, 2017 Member's meeting common micro-organisms center, preserving number is CGMCC No.13911, and Classification And Nomenclature is Lactobacillus plantarum ST.China Microbiological bacterium Plant preservation administration committee's common micro-organisms center address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：100101.

The Lactobacillus plantarum, which has, to be suppressed main spoilage organisms and the pathogenic bacteria growing of food source and promotes Tea Polyphenols bioconversion Ability, Lactobacillus plantarum ST is inoculated into sour tea process, can shorten the process-cycle, it is ensured that product quality, raising acid Tea quality.

The method of the sour tea of appliable plant lactobacillus ST processing, its step is as follows：

(1) fresh leaves of tea plant is cleaned, surface moisture is drained, tealeaves after cleaning is subjected to steam beating；The time of steam beating Point is disappeared with tea fresh leaves green grass gas, and leaf color turns yellow as appropriateness；

(2) rub after tea, tealeaves is placed in vial, compress；

(3) inoculated plant lactobacillus ST freezes bacterium powder, and inoculum concentration is tea fresh leaves weight 0.02%, rocks mixing；

(4) sealed glass jars, are placed in 25-37 DEG C of environment, and ferment 15d；

(5) drain after fermentation after the surface moisture of sour tea, be heated to 75 DEG C, pour into mould, it is compressing.

The present invention can significantly shorten the sour tea production cycle compared with traditional zymotic.Major catechin content in the sour tea of increase, Sour tea quality is improved, and process is controllable, sour tea product uniform quality.

Embodiment

Example 1

Lactobacillus plantarum ST screening, identification

1. Lactobacillus plantarum ST screening

Aseptically, 1g or so sour tea sample is weighed, is added in 10ml PBS, after homogenate 5min, by 1% Ratio be inoculated into containing 0.1%

In ascorbic MRS broth bouillons, 37 DEG C of Anaerobic culturel 18h.By the sterile physiological salt of the nutrient solution after enrichment Water carries out gradient dilution, in calcium carbonate-MRS solid medium bed boards, 37 DEG C of Anaerobic culturel 24h, the molten calcium circle of picking from flat board Big single bacterium colony.

2. Lactobacillus plantarum ST identification

By the isolated doubtful bacterial strain of lactic acid bacteria from sour tea, preserved after expanding culture using glycerol method.And Molecular biology identification is carried out to doubtful bacterial strain, specific method is：(1) genome of the doubtful bacterial strain of lactic acid bacteria is extracted；(2) with just To primers F:5 '-AGAGTTTGATCCTGGCTCAG-3 ' and reverse primer R:5 '-TACGGCTACCTTGTTACGACTT-3 ' enter Row 16S rDNA PCR are expanded.

Table 1PCR amplification systems

Table 1The PCR system for amplification of 16SrDNA

PCR reaction conditions are pre-degeneration (95 DEG C, 4min)；It is denatured (94 DEG C, 30s), anneals (55 DEG C, 30s), extension (72 DEG C, 1.5min), 30 circulations finally extend (72 DEG C, 10.0min)；16S rDNA amplified fragments are carried out with Ago-Gel electricity Swimming, takes pictures, and records testing result；Successful PCR primer ice bag preservation will be expanded and send it to the sequencing of Hua Da scientific ＆ technical corporation, bacterium is measured After strain 16S rDNA sequences, compared on NCBI using blast program, be more than 99% with Lactobacillus plantarum homology, based on all It is that the homology of 16S rDNA variable region sequences is more than 97.5% standard for being just regarded as same kind, the bacterial strain is plant Lactobacillus, is named as Lactobacillus plantarum ST.

Example 2

Lactobacillus plantarum ST antagonistic property

Diffusion method is punched using agar, Lactobacillus plantarum ST is to main spoilage organisms and food-borne pathogens growth and breeding for research Inhibitory action.Lactobacillus plantarum ST is inoculated in MRS broth bouillons, 37 DEG C of Anaerobic culturel 24h, 4 DEG C, 5000rpm, centrifugation 10min, collects supernatant.Supernatant is neutralized to pH 6.5 with sodium hydroxide through 0.22 μm of filtering with microporous membrane.By 20 μ l cultures 24h indicator bacteria bacterium solution (Escherichia coli, salmonella typhimurium, shigella flexneri and unit cell Listeria) is added to thawing And be cooled in 45 DEG C of 1.2%TSA solid mediums, poured into after being vigorously mixed in plate rapidly, after being used after culture medium solidifying Oxford cup punches (diameter 8mm) on culture medium, draws in 90 μ l Lactobacillus plantarum ST nutrient solutions supernatants injection aperture, rhamnose Lactobacillus rhamnosus GG is reference, and the MRS fluid nutrient mediums of sterilizing are blank control, spread after 4h, are placed in 37 DEG C of insulating boxs at room temperature Cultivate after 24h, measure antibacterial circle diameter.Lactobacillus plantarum ST is to shown in the antibacterial activity result table 2 of three plants of pathogenic entero becterias, planting Thing lactobacillus ST can significantly inhibit the growth of Escherichia coli, Salmonella typhimurium, shigella flexneri and unit cell Listeria, resist Bacterium activity is better than with reference to probiotics strain Lactobacillus rhamnosus GG.

Inhibitory action of the Lactobacillus plantarum ST of table 2 to pathogenic entero becteria

Note：-, no inhibition zone；+, antibacterial circle diameter<3mm；++, antibacterial circle diameter is between 3-6mm；+++, inhibition zone is straight Footpath>6mm

The Lactobacillus plantarum ST of example 3 Tea Polyphenols biotransformation capacity

By inoculated plant lactobacillus ST in solar dried green tea water extract, 37 DEG C of anaerobic fermentations are after 48 hours, 10000g, centrifugation 10 minutes, supernatant is collected, 0.22 μm of sterilised membrane filter filtering, HPLC-DAD methods detect EGC, EC, EGCG, ECG content.Knot As shown in table 3, compared with gross tea leaching liquor, the major catechin total amount in the sour tea of Lactobacillus plantarum ST fermentations is improved fruit 28.4% (P<0.05), EGC contents increase to 95.2 μ g/ml (P from original not detecting<0.05), EC contents add 600% (P<0.05), EGCG content reduces 40.8% (P<0.05), ECG contents reduce 39.5% (P<0.05).As a result show, plant Thing lactobacillus ST liquid state fermentations acid tea promotes the bioconversion of catechin, EGC and EC contents are dramatically increased.

The sour tea experiment 1 of the inoculated plant lactobacillus ST of example 4 productions

Fresh leaves of tea plant is plucked, is cleaned up with running water, drains surface moisture, through steam beating and rubbing tea,

The sour tea plant lactobacillus liquid state fermentation of table 3 is to catechin content in sour tea

Note：Significant difference (P is represented with a line shoulder mark difference<0.05).

It is placed in sterile glass vials, weighing Lactobacillus plantarum ST by the 0.02% of tea fresh leaves weight freezes bacterium powder, with a small amount of After sterilized water mixing, tealeaves surface is uniformly sprayed onto, tea fresh leaves are compressed, inoculated plant lactobacillus ST groups are not control.Seal glass Glass bottle, 37 DEG C of fermentation 15d.Drain after fermentation after the surface moisture of sour tea, be heated to 75 DEG C, pour into mould, be pressed into brick tea.Adopt Tea Polyphenols weight, HPLC methods detection catechin content are detected with ferrous tartrate method.The testing result such as institute of table 4 Show, compared with natural fermented sourdough tea, the Tea Polyphenols total amount in the sour tea of Lactobacillus plantarum ST fermentations is without significant changes, catechin total amount Improve 30.8% (P<0.05), EGC contents add 70% (P<0.05), EC contents add 78.8% (P<0.05), GCG With DL-C contents without significant changes, EGCG content reduces 39.8% (P<0.05), EGC contents reduce 39.4% (P< 0.05)。

The sour tea of the Lactobacillus plantarum ST of table 4 fermentations is compared with natural fermented sourdough tea tea main chemical compositions

Note：Significant difference (P is represented with a line shoulder mark difference<0.05).

Example 5

The sour tea experiment 2 of inoculated plant lactobacillus ST productions

Fresh leaves of tea plant is plucked, is cleaned up with running water, drains surface moisture, through steam beating and rubbing tea, is placed in sterile In vial, weigh Lactobacillus plantarum ST by the 0.02% of tea fresh leaves weight and freeze bacterium powder, after being mixed with a small amount of sterilized water, uniformly Tealeaves surface is sprayed onto, tea fresh leaves are compressed, inoculated plant lactobacillus ST groups are not control.Sealed glass jars, 37 DEG C of fermentation 15d. Drain after fermentation after the surface moisture of sour tea, be heated to 75 DEG C, pour into mould, be pressed into brick tea.Using ferrous tartrate light splitting Photometry detects Tea Polyphenols weight, HPLC methods detection catechin content.Testing result is as shown in table 5, with natural fermented sourdough Tea is compared, and the Tea Polyphenols total amount in the sour tea of Lactobacillus plantarum ST fermentations is without significant changes, and catechin total amount improves 29.8% (P< 0.05), EGC contents add 72.1% (P<0.05), EC contents add 75.8% (P<0.05), GCG and DL-C contents without Significant changes, EGCG content reduces 40.9% (P<0.05), EGC contents reduce 40.5% (P<0.05).

The sour tea of the Lactobacillus plantarum ST of table 5 fermentations is compared with natural fermented sourdough tea tea main chemical compositions

Note：Significant difference (P is represented with a line shoulder mark difference<0.05).

Claims (3)

1. Lactobacillus plantarum ST, it is characterised in that the Lactobacillus plantarum is preserved in Chinese microorganism strain guarantor on April 18th, 2017 Administration committee's common micro-organisms center is hidden, preserving number is CGMCC No.13911, and Classification And Nomenclature is Lactobacillus plantarum ST.

2. the Lactobacillus plantarum ST described in claim 1, with the main spoilage organisms of suppression and the pathogenic bacteria growing of food source and promotion tea The ability of polyphenol bioconversion.

3. the method for the sour tea of Lactobacillus plantarum ST processing described in application claim 1, its step is as follows：

(1) fresh leaves of tea plant is cleaned, surface moisture is drained, tealeaves after cleaning is subjected to steam beating；The time point of steam beating with Tea fresh leaves green grass gas disappears, and leaf color turns yellow as appropriateness；

(2) rub after tea, tealeaves is placed in vial, compress；

(3) inoculated plant lactobacillus ST freezes bacterium powder, and inoculum concentration is tea fresh leaves weight 0.02%, rocks mixing；

(4) sealed glass jars, are placed in 25-37 DEG C of environment, and ferment 15d；

(5) drain after fermentation after the surface moisture of sour tea, be heated to 75 DEG C, pour into mould, it is compressing.