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 sour tea processing

technical field

The invention belongs to the technical field of food, and relates to plantarum lactobacillus ST strain and its application in sour tea processing.

Background technique

China is the country of tea and the origin of tea culture. The earliest description of tea can be traced back to the Shennong period in ancient times. It is recorded in the contemporary Lu Yu's "Tea Classics": "Tea as a drink originated from Shennong." Drinking tea has a history of thousands of years in my country, and it is a daily beverage that people generally like. It has been called one of the world's three major non-alcoholic beverages together with cocoa and coffee at present. A large number of studies at home and abroad have shown that tea contains many active ingredients such as tea polyphenols, alkaloids, tea polysaccharides, and tea pigments. Among them, tea polyphenols have the functions of scavenging free radicals, anti-cancer, anti-mutation, sterilization, lowering blood pressure, anti-radiation, Prevent cardiovascular disease and diabetes and many other health functions. Catechin compounds are the main components of tea polyphenols, accounting for 65% to 80% of the total amount of tea polyphenols, mainly including catechin (EC), gallocatechin (EGC), catechin gallate ( ECG) and gallocatechin gallate (EGCG) 4 substances.

Yunnan Province is located in the southwest of China. With its unique natural conditions and a long history of tea cultivation, it has bred rich tea tree germplasm resources and is the origin center and origin of tea trees in the world. The ethnic minorities living in various parts of Yunnan grow tea, make tea, and love tea. Almost every ethnic group retains its own unique way of drinking (eating) tea, forming its own unique eating habits and folk health care methods, creating a brilliant ethnic culture.

The De'ang nationality is one of the cross-border ethnic groups in my country. They have lived in remote mountainous areas in Dehong, Baoshan, Lincang and other places in Yunnan for a long time. torture. The De'ang people are known as "ancient tea farmers". In the struggle against diseases, the De'ang people have gradually developed some folk health care methods with distinctive ethnic characteristics, such as sour tea, which have continued to this day. Nowadays, the production process of De'ang sour tea is that in the high temperature and high humidity season in May and June every year, the De'ang people collect the fresh leaves of large-leaved tea trees, dry them in the sun, rinse, steam (boil) tea and knead the tea, and put them into bamboo tubes. Buried underground for 60-70 days of fermentation, brew it with boiling water; it can also be pressed and dried after fermentation to make tea bricks, and brewed with boiling water before drinking. It can be seen from the processing technology of sour tea that the current processing methods of sour tea are still based on traditional experience, and sour tea products generally have disadvantages such as long processing cycle, unstable quality, inconsistent quality, and difficult to guarantee safety. Therefore, standardizing the processing methods of sour tea, improving the quality of sour tea, ensuring stable quality, and making the sour tea of the De'ang ethnic group recognized by the world, which is "hidden in the deep boudoir, slightly sour, slightly bitter and sweet", has broad prospects and great significance.

Contents of the invention

The present invention isolates Lactobacillus plantarum ST from naturally fermented sour tea, inoculates Lactobacillus plantarum ST into the fermentation process of sour tea, can significantly increase the total amount of catechins in sour tea, improve the quality of sour tea, and can shorten the processing cycle at the same time , To ensure product quality.

Technical scheme of the present invention is:

Lactobacillus plantarum ST, the Lactobacillus plantarum was preserved in the General Microbiology Center of China Committee for Culture Collection of Microbial Cultures on April 18, 2017. The preservation number is CGMCC No. 13911, and the classification is named Lactobacillus plantarum ST. General Microbiology Center of China Microbiological Culture Collection Management Committee Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Zip Code: 100101.

The Lactobacillus plantarum has the ability to inhibit the growth of main spoilage bacteria and food-borne pathogenic bacteria and promote the biotransformation of tea polyphenols. Inoculating Lactobacillus plantarum ST into the processing process of sour tea can shorten the processing cycle, ensure product quality, and improve the quality of sour tea. quality.

The method for processing sour tea by using Lactobacillus plantarum ST, the steps are as follows:

(1) Clean the fresh leaves of the tea tree, drain the surface water, and steam the tea leaves after cleaning; the time point of steam killing is when the green grass gas of the fresh tea leaves disappears, and the leaf color turns yellow, which is moderate;

(2) After kneading the tea, put the tea in a glass bottle and press it tightly;

(3) Inoculate Lactobacillus plantarum ST freeze-dried bacterial powder, the inoculation amount is 0.02% of the weight of fresh tea leaves, shake and mix;

(4) Seal the glass bottle, place it in an environment of 25-37°C, and ferment for 15 days;

(5) After draining the surface moisture of the fermented sour tea, heat it to 75°C, pour it into a mold, and press it into shape.

Compared with traditional fermentation, the invention can significantly shorten the production cycle of sour tea. The main catechin content in the sour tea is increased, the quality of the sour tea is improved, and the processing process is controllable, so that the quality of the sour tea product is consistent.

detailed description

Example 1

Screening and identification of Lactobacillus plantarum ST

1. Screening of Lactobacillus plantarum ST

Under sterile conditions, weigh about 1 g of sour tea sample, add it to 10 ml of PBS, homogenize for 5 minutes, and inoculate at a rate of 1% to contain 0.1%

In the MRS broth medium of vitamin C, culture anaerobically at 37°C for 18h. The enriched culture solution was serially diluted with sterile physiological saline, plated on calcium carbonate-MRS solid medium, cultured anaerobically at 37°C for 24 hours, and a single colony with a large calcium-dissolving circle was picked from the plate.

2. Identification of Lactobacillus plantarum ST

The suspected strains of lactic acid bacteria isolated from sour tea were preserved by glycerin method after expanded culture. And carry out molecular biology identification to suspected bacterial strain, specific method is: (1) extract the genome of lactic acid bacteria suspected bacterial strain; -3' for 16S rDNA PCR amplification.

Table 1 PCR amplification system

Table 1The PCR system for amplification of 16SrDNA

The PCR reaction conditions were pre-denaturation (95°C, 4min); denaturation (94°C, 30s), annealing (55°C, 30s), extension (72°C, 1.5min), 30 cycles, and finally extension (72°C, 10.0min ); perform agarose gel electrophoresis on the amplified 16S rDNA fragment, take pictures, and record the test results; store the successfully amplified PCR product in an ice pack and send it to Huada Technology Company for sequencing. Using the BLAST program, the homology with Lactobacillus plantarum is greater than 99%. Based on the standard that the homology of the 16S rDNA variable region sequence is greater than 97.5%, it can be considered as the same species. The strain is Lactobacillus plantarum. Named as Lactobacillus plantarum ST.

Example 2

Bacteriostatic properties of Lactobacillus plantarum ST

The inhibitory effect of Lactobacillus plantarum ST on the growth and reproduction of main spoilage bacteria and food-borne pathogenic bacteria was studied by agar punching diffusion method. Lactobacillus plantarum ST was inoculated in MRS broth medium, cultured anaerobically at 37°C for 24h, centrifuged at 5000rpm at 4°C for 10min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane, and neutralized to pH 6.5 with sodium hydroxide. Add 20 μl of indicator bacteria solution (Escherichia coli, Salmonella typhimurium, Shigella flexneri and Listeria unicellulare) cultured for 24 hours into the 1.2% TSA solid medium that was melted and cooled to 45°C, and after vigorous mixing Quickly pour it into a plate, and after the medium is solidified, use an Oxford cup to punch a hole (diameter 8mm) on the medium, absorb 90 μl of the supernatant of the culture medium of Lactobacillus plantarum ST and inject it into the small hole, and use Lactobacillus rhamnosus GG as a reference. The MRS liquid culture medium of the bacteria was used as the blank control. After diffusion at room temperature for 4 hours, it was cultured in a 37°C incubator for 24 hours, and the diameter of the inhibition zone was measured. The results of the antibacterial activity of Lactobacillus plantarum ST on three strains of intestinal pathogenic bacteria are shown in Table 2. Lactobacillus plantarum ST can significantly inhibit the growth of Escherichia coli, Salmonella typhimurium, Shigella flexneri and Listeria monocytogenes , the antibacterial activity was superior to that of the reference probiotic strain Lactobacillus rhamnosus GG.

Table 2 Inhibitory effect of Lactobacillus plantarum ST on intestinal pathogenic bacteria

Note: -, no zone of inhibition; +, diameter of zone of inhibition <3mm; ++, diameter of zone of inhibition between 3-6mm; +++, diameter of zone of inhibition> 6mm

The tea polyphenol biotransformation ability of example 3 lactobacillus plantarum ST

Plantarum Lactobacillus ST was inoculated into the water extract of sun-dried green hair tea, and after anaerobic fermentation at 37°C for 48 hours, centrifuged at 10,000g for 10 minutes, collected the supernatant, filtered through a 0.22μm sterile filter membrane, and detected EGC, EC by HPLC-DAD method , EGCG, ECG content. The results are shown in Table 3. Compared with the tea extract, the total amount of main catechins in Lactobacillus plantarum ST fermented yogurt increased by 28.4% (P<0.05), and the EGC content increased from the original undetected to 95.2μg/ml (P<0.05), the EC content increased by 600% (P<0.05), the EGCG content decreased by 40.8% (P<0.05), and the ECG content decreased by 39.5% (P<0.05). The results showed that Lactobacillus plantarum ST liquid fermented yogurt promoted the biotransformation of catechins, and the contents of EGC and EC increased significantly.

Example 4 Inoculation of Lactobacillus plantarum ST to produce sour tea experiment 1

Pick the fresh leaves of tea tree, wash them with tap water, drain the surface water, kill them by steam and knead the tea,

Table 3 Effect of liquid fermentation of Lactobacillus plantarum on catechin content in sour tea

Note: Different shoulder labels in the same row indicate significant difference (P<0.05).

Put it in a sterile glass bottle, weigh 0.02% of the weight of the fresh tea leaves, and weigh the Lactobacillus plantarum ST freeze-dried bacteria powder, mix it with a small amount of sterile water, spray it evenly on the surface of the tea leaves, and press the fresh tea leaves tightly without inoculating plants Lactobacillus ST group was used as control. Seal the glass bottle and ferment at 37°C for 15 days. After draining the surface moisture of the fermented sour tea, heat it to 75°C, pour it into a mold, and press it into tea bricks. The weight of tea polyphenols was detected by ferrous tartrate spectrophotometry, and the content of catechins was detected by HPLC. The test results are shown in Table 4. Compared with naturally fermented yogurt, the total amount of tea polyphenols in yogurt fermented by Lactobacillus plantarum ST has no significant change, and the total amount of catechins has increased by 30.8% (P<0.05). content increased by 70% (P<0.05), EC content increased by 78.8% (P<0.05), GCG and DL-C content did not change significantly, EGCG content decreased by 39.8% (P<0.05), EGC content decreased by 39.4 % (P<0.05).

Table 4 Comparison of main chemical components between Lactobacillus plantarum ST fermented yogurt tea and natural fermented yogurt tea

Note: Different shoulder labels in the same row indicate significant difference (P<0.05).

Example 5

Sour tea production experiment 2 by inoculating Lactobacillus plantarum ST

Pick the fresh leaves of tea tree, wash them with tap water, drain the surface water, put them in a sterile glass bottle after steaming and kneading the tea, and weigh 0.02% of the weight of the fresh tea leaves. After mixing a small amount of sterile water, spray it evenly on the surface of the tea leaves, compact the fresh tea leaves, and the group without Lactobacillus plantarum ST was used as the control. Seal the glass bottle and ferment at 37°C for 15 days. After draining the surface moisture of the fermented sour tea, heat it to 75°C, pour it into a mold, and press it into tea bricks. The weight of tea polyphenols was detected by ferrous tartrate spectrophotometry, and the content of catechins was detected by HPLC. The test results are shown in Table 5. Compared with naturally fermented yogurt, the total amount of tea polyphenols in yogurt fermented by Lactobacillus plantarum ST has no significant change, and the total amount of catechins has increased by 29.8% (P<0.05). content increased by 72.1% (P<0.05), EC content increased by 75.8% (P<0.05), GCG and DL-C content did not change significantly, EGCG content decreased by 40.9% (P<0.05), EGC content decreased by 40.5 % (P<0.05).

Table 5 Comparison of main chemical components between Lactobacillus plantarum ST fermented yogurt tea and natural fermented yogurt tea

Note: Different shoulder labels in the same row indicate significant difference (P<0.05).

Claims (3)

1. Lactobacillus plantarum ST, which is characterized in that the Lactobacillus plantarum was preserved in the General Microbiology Center of China Microbial Culture Collection Management Committee on April 18, 2017. The preservation number is CGMCC No. 13911, and the classification is named Lactobacillus plantarum ST. 2. The Lactobacillus plantarum ST described in claim 1 has the ability to inhibit the growth of main spoilage bacteria and food-borne pathogenic bacteria and to promote the biotransformation of tea polyphenols. 3. apply the method for processing acid tea with lactobacillus plantarum ST described in claim 1, its steps are as follows: (1) Clean the fresh leaves of the tea tree, drain the surface water, and steam the tea leaves after cleaning; the time point of steam killing is when the green grass gas of the fresh tea leaves disappears, and the leaf color turns yellow, which is moderate; (2) After kneading the tea, put the tea in a glass bottle and press it tightly; (3) Inoculate Lactobacillus plantarum ST freeze-dried bacterial powder, the inoculation amount is 0.02% of the weight of fresh tea leaves, shake and mix; (4) Seal the glass bottle, place it in an environment of 25-37°C, and ferment for 15 days; (5) After draining the surface moisture of the fermented sour tea, heat it to 75°C, pour it into a mold, and press it into shape.