A strain of Lactobacillus plantarum INM3121 and its preparation and application

Abstract

The present invention provides a strain of Lactobacillus plantarum INM3121 and its preparation and application, Lactobacillus plantarum INM3121, which was deposited in Guangdong Microbial Culture Collection Center on November 4, 2024, with a deposit number of GDMCC NO: 65410, and a deposit address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province. The advantages are: Lactobacillus plantarum INM3121 can increase the content and flavor of tea polyphenols, theanine and caffeine in tea, and can be used for rice cooking in the later stage, which can effectively improve the product flavor and extend its shelf life.

Description

Technical Field

[0001] The invention belongs to the technical field of industrial microorganisms and relates to a plant lactobacillus INM3121 and a preparation method and application thereof. Background Art

[0002] Rice is the most widely consumed staple food for people in China and Southeast Asia. How to develop this traditional staple food into a new type of convenient rice ball food that can be industrially mass-produced is a topic of great concern to many countries and companies.

[0003] Tea polyphenols are a class of natural products with complex and diverse structures, mainly composed of polyphenolic compounds such as flavanols, flavans, and anthocyanins. Among them, flavanols are the component with the highest content and the strongest biological activity in tea polyphenols, accounting for 60% to 80% of the total tea polyphenols. Flavanol compounds include four main components: epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate. They all have two or three hydroxyl-substituted flavan mother nuclei and are connected to gallic acid through ester bonds. Flavans in tea polyphenols mainly include two structures: flavan-3-ol and flavan-3,4-diol. In summary, tea polyphenols are a class of natural polyphenolic compounds with complex composition and diverse structure. This unique chemical composition gives tea polyphenols a wide range of biological activity and application value.

[0004] At present, the patented method CN202410718705.0 for extracting tea polyphenols uses a supramolecular low eutectic solvent to extract tea polyphenols from tea leaves. The extraction rate of tea polyphenols in the extract is not less than 109.05 mg / g, and the content of tea polyphenols in the extract is not less than 1.67 mg / mL. The extraction rate of tea polyphenols in the present invention is high, the extraction process is simple, and the safety is high, but it is mainly used in the production of tea polyphenol films.

[0005] CN202410462580.X provides an extraction method combining low-temperature ultrasonic technology with pulsed electric field technology. The crushed green tea is placed in an extraction device combining ultrasonic technology and pulsed electric field technology at low temperature. Through the dual technology of discharge and ultrasound, the extraction rate of tea polyphenols is as high as 32% or more, which is suitable for use in animal feed additives.

[0006] CN202410732520.5 uses ethanol aqueous solution to extract tea leaves, and then uses resin adsorption, elution, reduced pressure concentration, extraction and other steps to obtain three products: theanine, quinic acid and tea polyphenols. The extraction method of the present invention is simple and efficient, can effectively avoid the rancidity and fermentation of materials during the production process, and can minimize the introduction of new impurities in the product due to the rancidity and fermentation of materials. The extracted product has good quality, high purity and high yield, but it has not yet been used in food.

[0007] Theanine, also known as glutamine, is a non-proteinaceous free amino acid in the form of a white powder with unique properties and functions. Theanine accounts for 1% to 2% of the total ingredients in tea, and natural theanine mainly exists in the form of L-theanine. L-theanine is highly soluble in water but insoluble in ethanol. Its aqueous solution is acidic with a melting point of 214-215°C. It is a relatively stable substance. The chemical name of theanine is N-yl-L-glutamine, and its molecular formula is C7H 14 N2O3. Studies have shown that L-theanine is currently the most abundant in green tea. In addition, trace amounts of theanine have only been detected in camellia sasanqua, tea oil, camellia, and mushrooms (fungi), and have not yet been found in other plants. The structure and functional characteristics of theanine are related to its extraction method. At present, there are three main methods for extracting theanine: extraction and separation, chemical synthesis, and biosynthesis. For L-theanine naturally present in tea, the commonly used direct extraction and separation techniques are ultrasonic-assisted extraction, ion exchange chromatography, membrane separation, and chromatographic separation. Ultrasonic-assisted extraction is separation through ultrasonic vibration, and is currently one of the mainstream separation methods used for direct separation of theanine. Long Chengmei et al. used ultrasonic extraction of theanine from Duyun Maojian tea, compared the water bath method and ultrasonic-assisted extraction method, and confirmed that the ultrasonic-assisted extraction method saves time and cost compared to the water bath method. Ultrasonic vibration has strong penetration and vibration stirring effect on the solution. Ultrasonic-assisted extraction can save manpower, improve operating efficiency, and is stable, but it increases technical requirements. Microbial fermentation is the main method for extracting theanine by using the affinity of γ-glutamylmethylamine synthetase (GMAS) and adenosine triphosphate (ATP) to generate theanine. Ji Yuanqing et al. used glucose and ethylamine as raw materials, and the theanine produced by catalytic fermentation of the key enzyme GMAS had a high conversion rate. Zhu Xinya introduced GMAS to act on wild-type E. coli W3110, and found that under the optimal conditions, its raw material conversion rate reached 42%. This discovery has opened up a new path for the synthesis of theanine by microbial fermentation. Microbial raw materials have the advantages of low price, diverse raw material selection, clear substrate fermentation system and simple route, so stable theanine can be synthesized under complete experimental operation. However, the pathway for the synthesis of ethylamine in microorganisms is still unclear and further research is needed.

[0008] Caffeine, also known as 1,3,7-trimethylxanthine, is an important alkaloid compound. Among the plants containing caffeine that have been discovered so far, the content of caffeine in the young branches and leaves of tea trees is the highest, accounting for about 2% to 4% of the dry weight, the content in the seeds of coffee plants is about 0.4% to 2.4%, the content in the fruits and seeds of cocoa trees is about 0.6% to 0.8%, and the content of caffeine in the young leaves of Paraguay tea is 0.8% to 0.9%. Caffeine is a characteristic component of tea leaves. The highest content of caffeine in tea trees is in young buds and leaves, while in other caffeine-containing plants, it is mostly found in seeds or fruits. Therefore, people often use this to identify real and fake tea.

[0009] The most commonly used methods for extracting caffeine are dichloromethane organic solvent extraction and alcohol extraction. The alcohol extraction method is to extract caffeine by using ethanol solution and extractor, or by heating. The former extraction method usually takes 2-3 hours and takes a long time. The latter requires the use of a large amount of organic agents such as dichloromethane, acetone, and chloroform. This agent will cause serious pollution to the human body and the environment, and needs to go through water bath recovery, distillation, filtration and other processes, which is not conducive to daily testing.

[0010] Although there are many studies on the methods of synthetically extracting tea polyphenols, theanine and caffeine, there are many shortcomings in practical applications. From the perspective of environmental protection and health, the current synthetic method has low production safety and heavy environmental pollution. From the perspective of application fields, the application range in food is relatively small, especially in rice products. How to apply tea polyphenols, theanine and caffeine to staple food products is also an urgent problem to be solved. Summary of the invention

[0011] In view of the above problems, the present invention provides a strain of Lactobacillus plantarum INM3121 capable of increasing tea polyphenols, theanine and caffeine, and a preparation method and application thereof.

[0012] The object of the invention is achieved by the following scheme: a strain of Lactobacillus plantarum, Lactobacillus plantarum INM3121, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 4, 2024, with the deposit number: GDMCC NO: 65410, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0013] Furthermore, the colony characteristics of Lactobacillus plantarum are as follows: after culturing on solid MRS plate medium for 48 hours, the colony diameter reaches 1 to 3 mm, the colonies are milky white, round, flat, with a smooth surface and neat colony edges.

[0014] A preparation method of Lactobacillus plantarum,

[0015] Step 1: Isolation of lactic acid bacteria:

[0016] Isolate and screen lactic acid bacteria from yogurt samples, add the dairy product into a slurry, add it to sterile water, and dilute it to a concentration of 10 5 -10 6 , the dairy products of different dilution concentrations were evenly spread on the solid MRS medium, and then cultured in an incubator at 37°C for 48 hours. Then, the white colonies on the solid MRS medium and the lactic acid bacteria with the correct morphology were selected for screening;

[0017] Step 2: Lactic acid bacteria screening: The obtained lactic acid bacteria were cultured to the logarithmic growth phase, and then inoculated with 1% of the strain, and the final concentration of the strain was 1×10 6 cfu / mL, inoculated into modified MRS solution, and placed in 37℃ for 3 days to obtain the fermentation liquid.

[0018] Furthermore, the formula of the improved MRS solution is: weigh 10g of peptone, 8g of beef extract powder, 4g of yeast extract powder, 10g of 0.8% tea water, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, and 1g of Tween 80, dissolve in 1000mL of distilled water, and sterilize at 0.1MPa and 121°C for 15min.

[0019] The invention discloses an application of Lactobacillus plantarum, and the application of Lactobacillus plantarum to improve the contents of tea polyphenols, theanine and caffeine in tea water.

[0020] Furthermore, tea water added with Lactobacillus plantarum is used in cooking rice to extend its shelf life.

[0021] Furthermore, 8 g of tea leaves were added to 100 ml of water, and 1 g of lactic acid bacteria was inoculated, and the mixture was fermented at 32° C. for 24 h, and the fermentation liquid was obtained after inactivation.

[0022] Further, the fermentation liquid was added to 900 g of water for standby use, 700 g of rice was washed 3 times and drained, 924 g of the above-mentioned stand-by liquid, 11.2 g of soybean oil, 11.2 g of rice seasoning liquid, and 5.6 g of edible salt were added, and the mixture was stirred evenly and steamed at a cooking temperature of 100° C. for 28.5 min, and the rice was simmered for 15 min.

[0023] Advantages of the present invention: 1. The present invention can provide a preparation method of a plant lactobacillus strain that improves the content and flavor of tea polyphenols, theanine and caffeine. The screening method is universal and can be widely used in the screening of strains for tea polyphenols, theanine and caffeine content and flavor.

[0024] 2. The plant lactobacillus INM3121 provided by the present invention can increase the content and flavor of tea polyphenols, theanine and caffeine in tea, and can be used for rice cooking in the later stage, which can effectively improve the product flavor and extend its shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Flow chart for the preparation of Lactobacillus plantarum.

[0026] Figure 2 This is the morphology of Lactobacillus plantarum culture medium.

[0027] Figure 3 This is a Gram staining microscopic examination of Lactobacillus plantarum. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments:

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0030] Example 1, see attached Figure 1-3 ,

[0031] A strain of Lactobacillus plantarum, Lactobacillus plantarum INM3121, was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 4, 2024, with the deposit number: GDMCC NO: 65410, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0032] A strain of Lactobacillus plantarum, the colony characteristics of Lactobacillus plantarum are as follows: after culturing on solid MRS plate medium for 48 hours, the colony diameter reaches 1-3 mm, the colony is milky white, round, flat, with a smooth surface and neat colony edges.

[0033] A preparation method of Lactobacillus plantarum,

[0034] Step 1: Isolation of lactic acid bacteria:

[0035] Isolate and screen lactic acid bacteria from yogurt samples, add the dairy product into a slurry, add it to sterile water, and dilute it to a concentration of 10 5 -10 6 , the dairy products of different dilution concentrations were evenly spread on the solid MRS medium, and then cultured in an incubator at 37°C for 48 hours. Then, the white colonies on the solid MRS medium and the lactic acid bacteria with the correct morphology were selected for screening;

[0036] Step 2: Lactic acid bacteria screening: The obtained lactic acid bacteria were cultured to the logarithmic growth phase, and then inoculated with 1% of the strain, and the final concentration of the strain was 1×10 6 cfu / mL, inoculated into modified MRS solution, and placed in 37℃ for 3 days to obtain the fermentation liquid.

[0037] The invention discloses a method for preparing plant lactobacillus. The formula of an improved MRS solution is as follows: 10g of peptone, 8g of beef extract powder, 4g of yeast extract powder, 10g of 0.8% tea water, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate and 1g of Tween 80 are weighed, dissolved in 1000mL of distilled water, and sterilized at 0.1MPa and 121°C for 15min.

[0038] A preparation method of Lactobacillus plantarum,

[0039] 1. Processing of raw materials

[0040] Take a yogurt sample and dilute it to 10 with sterile saline. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Concentration, make bacterial dilution, take 0.1mL from each concentration dilution and evenly spread it on MRS (containing 0.015% bromocresol purple) solid culture medium plate, culture at 37℃ for 48h, select colonies that are milky white, round, flat, with smooth surface and neat colony edges. Streak the colonies on MRS solid culture medium for secondary purification, culture at 37℃ for 48h, pick single colonies for glycerol storage, and name them NS3302 to NS3310. The screening process is as follows Figure 1 shown.

[0041] 2. Preliminary screening of bacterial strains

[0042] The 9 strains were inoculated into MRS liquid medium at 1% inoculum and cultured at 37°C for 24 h. Then, they were inoculated into modified MRS medium at 1% inoculum and cultured at 37°C for 24 h. The final cell density reached 10 6 cfu / mL.

[0043] After 24 hours of fermentation, samples were taken from each tube and centrifuged at 10000 r / min for 10 minutes to obtain the supernatant, which was the sample for physical and chemical index testing. The results are shown in Table 1.

[0044] Detection of tea polyphenols, theanine and caffeine content: ① Measure 1.0 mL of gallic acid working solution, water and test solution respectively in graduated test tubes, add 5.0 mL of Folin phenol reagent to each test tube, shake well, react for 3-8 minutes, add 4.0 mL of 7.5% sodium carbonate (Na2CO3) solution, add water to the scale and shake well. Leave at room temperature for 60 minutes. Use a 10 mm cuvette and a spectrophotometer at a wavelength of 765 nm to measure the absorbance (A, A0). According to the absorbance (A) of the gallic acid working solution and the gallic acid concentration of each solution, a standard curve is prepared. The tea polyphenol standard curve is y = 0.0109x + 0.0561, R = 0.9977. ② The determination method of theanine content is carried out in accordance with GB / T23193-2017. Both the standard and the sample are single peaks, and the peak time is about 6.221 minutes. The standard curve of theanine is y=4877.7x+27.82, R2=0.9996. ③ Determination of caffeine content: Chromatographic conditions are as follows:

[0045] —Chromatographic column: S5; 4.6mm idx 150mm

[0046] - Mobile phase: 0.1 vol% H3PO4 / CH3CN = 75 / 25

[0047] —Flow rate: 1mL / min

[0048] —Column temperature: 40℃

[0049] -Injection volume: 5mL

[0050] —Detection wavelength: UV 220nm

[0051] The method of extracting caffeine from the sample is based on the dissolution method of the caffeine standard substance. Both the standard substance and the sample have a single peak, and the peak time is about 4.014mi. The caffeine standard curve is y=23880x+383.18, R2-0.9878.

[0052] Table 1 Analysis of tea polyphenols, theanine and caffeine produced by fermentation

[0053]

[0054]

[0055] Strains NS3302, NS3304 and NS3309 with lower pH and higher contents of tea polyphenols, theanine and caffeine were selected for rescreening.

[0056] 3. Strain rescreening

[0057] The lactic acid bacteria strains NS3302, NS3304 and NS3309 selected in the initial screening were reactivated and cultured at a bacterial concentration of 10 6 cfu / mL The medium was prepared according to the national standard GB / T14612-2008 method, and the fermentation liquid was obtained by adding it to tea juice, and then made into rice for sensory evaluation. The rice cooking method was referenced to the technology. The results are shown in Table 2

[0058] Table 2 Sensory analysis of rice

[0059] Species name Appearance taste Average score Tea polyphenols Theanine Caffeine NS3302 4.4 4.8 4.6 0.469 0.037 0.375 NS3304 4.5 4.5 4.5 0.461 0.033 0.371 NS3309 4.7 4.4 4.55 0.452 0.032 0.370

[0060] Note: The maximum score for each indicator is 5 points, and the higher the score, the better the result.

[0061] From the results in Table 2, we can see that NS3302 has the best sensory evaluation result. As the target strain for rescreening, the morphological observation of this strain on the solid MRS plate medium is as follows: Figure 2 As shown, the colony diameter is 1-3 mm, the colony is milky white, round, flat, with a smooth surface and neat colony edges.

[0062] After Gram staining, the results were observed under a microscope. Figure 3 shown.

[0063] The NS3302 obtained by the above screening was subjected to 16SrDNA sequencing and identification. After comparative analysis, the 16S region sequence obtained from the strain of the present invention was 99.86% similar to that of Lactobacillus plantarum. It can be determined that the strain is a strain of Lactobacillus plantarum. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 4, 2024. Lactobacillus plantarum can be used in the food fermentation industry. It is named INM3121, and the deposit number is: GDMCC No: 65410. Therefore, INM3121 and NS3302 refer to the same strain in this application. The NS3302 strain of the present invention has been genetically identified, and its gene sequence is as follows:

[0064] GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGAT

[0065] TGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAAC

[0066] CTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCG

[0067] CATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGC

[0068] TAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGC

[0069] CACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAA

[0070] TGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCT

[0071] GTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAG

[0072] CCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTA

[0073] TTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGA

[0074] AGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGC

[0075] GGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTG

[0076] ACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAA

[0077] ACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCAT

[0078] TCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGC

[0079] GGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATG

[0080] CAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTC

[0081] AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCC

[0082] AGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG

[0083] TCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGT

[0084] TGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAA

[0085] CTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTC

[0086] CCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGT

[0087] AACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGT

[0088] Detection of genetic stability of Lactobacillus plantarum INM3121 in Example 2

[0089] The plant lactobacillus INM3121 in Example 1 was continuously propagated for 10 generations on the modified MRS liquid medium, and the 1st, 5th and 10th generation samples were centrifuged at 10,000 r / min for 10 min to obtain the supernatant, which was the sample, and the physical and chemical indexes were tested. The method for testing the content of tea polyphenols, theanine and caffeine was referenced to Example 1. At the same time, sensory evaluation was performed to determine its stability of propagation, and the specific data are shown in Table 3.

[0090] Table 3 Results of the stability test for passage

[0091]

[0092]

[0093] The results in Table 3 show that the fermentation performance of Lactobacillus plantarum INM3121 of different generations is normal, the content of tea polyphenols, theanine and caffeine varies within 10%, the strain has good genetic stability and meets the requirements for production and use.

[0094] Example 3: An application of Lactobacillus plantarum to increase the content of tea polyphenols, theanine and caffeine in tea water.

[0095] The invention discloses an application of plant lactobacillus, wherein tea water added with plant lactobacillus is applied to steaming rice to extend the shelf life of rice.

[0096] The invention discloses an application of plant lactobacillus. The invention comprises the following steps: adding 8g of tea leaves into 100ml of water, inoculating 1g of lactic acid bacteria, fermenting at 32°C for 24h, and obtaining fermentation liquid after inactivation.

[0097] The invention discloses an application of plant lactobacillus. The fermentation liquid is added into 900g water for standby use. 700g rice is washed three times and then drained. Then 924g of the standby liquid, 11.2g of soybean oil, 11.2g of rice seasoning liquid and 5.6g of edible salt are added. The mixture is evenly stirred and then steamed. The steaming temperature is 100°C, the steaming time is 28.5min, and the rice is stewed for 15min.

[0098] An application of plant lactobacillus, tea polyphenols, theanine and caffeine fermentation liquid is applied to rice cooking, the rice cooking method is: plant lactobacillus INM3121 is inoculated into tea water, the inoculation bacteria concentration is 10 4 -10 8CFU / mL, add 100mL of water, ferment at 32°C for 24h, inactivate, obtain fermentation broth, add 10% of fermentation broth for rice cooking, rice cooking method reference technology, detection method of tea polyphenols, theanine and caffeine content in fermentation broth reference Example 1, detection of tea polyphenols, theanine and caffeine content in rice, first add distilled water to the rice 1 to be tested, extract at 100°C for 1h, add 50% methanol-water solution to the rice 2 and 3 to be tested, extract at 100°C for 20min, centrifuge at 10000r / min for 10min, take supernatant, detection method reference Example 1, the results are shown in Table 4. The sensory evaluation of rice is shown in Table 5.

[0099] Table 4 Contents of tea polyphenols, theanine and caffeine in fermented liquid and rice

[0100]

[0101] Table 5 Sensory evaluation results of rice

[0102] Grouping Inoculation volume cfu / mL Appearance taste Taste Overall Rating Experimental Group 1 <![CDATA[1.0×10 4 ]]> 6.7 7.1 7.4 21.2 Experimental Group 2 <![CDATA[1.0×10 5 ]]> 7.1 8.0 7.3 22.4 Experimental Group 3 <![CDATA[1.0×10 6 ]]> 8.3 7.9 8.1 24.3 Experimental Group 4 <![CDATA[1.0×10 7 ]]> 9.5 9.3 9.1 27.9 Experimental Group 5 <![CDATA[1.0×10 8 ]]> 8.5 8.3 8.6 25.4

[0103] Note: Each item has a full score of 10 points, with a scoring interval of 0.1, and the comprehensive score is the sum of the values ​​of each item.

[0104] From the results in Tables 4 and 5, we can see that Lactobacillus plantarum INM3121 in the fermentation broth can significantly increase the content of tea polyphenols, theanine and caffeine in tea, and the more inoculation amount, the higher the content; however, too high or too low inoculation concentration of Lactobacillus plantarum INM3121 will affect the appearance, taste and mouthfeel of rice. The most suitable inoculation concentration is 1.0×10 7 CFU / mL, this concentration can provide higher content of tea polyphenols, theanine and caffeine, and improve the flavor of rice.

[0105] Example 4: Test of shelf life of different rice

[0106] The rice prepared from the experimental group 4 and the control group (unfermented tea) in Example 3 was used for shelf life detection, and the rice category was rice, black rice included black rice, polished rice, polished glutinous rice, and quinoa rice included rice and quinoa. The detection method was to place the product in a constant temperature box at 2-6°C, and detect the physical and chemical indicators and microbiological indicators of the product every day. The moisture content is tested according to the first method of GB 5009.3-2016, the acid value is tested according to the second method of GB 5009.229-2016, the peroxide value is tested according to the first method of GB 5009.227-2023, the total colony count is tested according to GB 4789.2-2022, the coliform group is tested according to the second method of GB 4789.3-2016, the salmonella is tested according to GB 4789.4-2016, the Staphylococcus aureus is tested according to the second method of GB 4789.10-2016, the monocytogenes Listeria is tested according to the first method of GB 4789.30-2016, and the technical requirements are tested according to QZYM0002S. The specific results are shown in Table 6 for the shelf life test of rice, Table 7 for the shelf life test of black rice, and Table 8 for the shelf life test of quinoa rice

[0107] Table 6 Test results of rice shelf life

[0108]

[0109] Table 7 Black rice shelf life test results

[0110]

[0111]

[0112] Table 8 Test results of shelf life of quinoa rice

[0113]

[0114] From the results of Tables 6, 7 and 8, it can be seen that after inoculating Lactobacillus plantarum INM3121 in tea water for rice cooking, the storage time of rice at 2-6°C can be extended. From the perspective of physical and chemical properties, the moisture content of rice decreased during storage, the acid value and peroxide value indexes did not change much, the total colony count gradually increased during storage, the number of coliform bacteria did not change much, and Salmonella, Staphylococcus aureus and Listeria monocytogenes were all within the standard. The experimental group was compared with the control group. The shelf life of rice in the experimental group was 5 days, and that in the control group was 3 days, indicating that Lactobacillus plantarum INM3121 can extend the shelf life of rice after being used for rice cooking.

[0115] Based on the results of the above embodiments, the plant lactobacillus INM3121 provided by the present invention is applied to rice cooking. On the basis of not changing the existing process, the content of tea polyphenols, theanine and caffeine in tea fermentation broth can be significantly increased, while the aroma and taste of the rice product can be improved, and the shelf life of the rice product can be extended.

[0116] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A plant lactobacillus, characterized in that: Lactobacillus plantarum INM3121, the strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 4, 2024, with the deposit number: GDMCC NO: 65410, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

2. A plant lactobacillus according to claim 1, characterized in that: The colony characteristics of Lactobacillus plantarum are as follows: after 48 hours of culture on solid MRS plate medium, the colony diameter reaches 1-3 mm, the colonies are milky white, round, flat, with a smooth surface and neat colony edges.

3. A method for preparing plant lactobacillus according to claim 1, characterized in that: Step 1: Isolation of lactic acid bacteria: Isolate and screen lactic acid bacteria from yogurt samples, add the dairy product into a slurry, add it to sterile water, and dilute it to a concentration of 10 5 -10 6 , the dairy products of different dilution concentrations were evenly spread on the solid MRS medium, and then cultured in an incubator at 37°C for 48 hours. Then, the white colonies on the solid MRS medium and the lactic acid bacteria with the correct morphology were selected for screening; Step 2: Lactic acid bacteria screening: The obtained lactic acid bacteria were cultured to the logarithmic growth phase, and then inoculated with 1% of the strain, and the final concentration of the strain was 1×10 6 cfu / mL, inoculated into modified MRS solution, and placed in 37℃ for 3 days to obtain the fermentation liquid.

4. The preparation method of plant lactobacillus according to claim 3, wherein: The formula of the improved MRS solution is: weigh 10g of peptone, 8g of beef extract powder, 4g of yeast extract powder, 10g of 0.8% tea water, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, and 1g of Tween 80, dissolve in 1000mL of distilled water, and sterilize at 0.1MPa and 121℃ for 15min.

5. An application of Lactobacillus plantarum according to claim 1, characterized in that: Application of Lactobacillus plantarum to increase the content of tea polyphenols, theanine and caffeine in tea water.

6. The application of plant lactobacillus according to claim 5, characterized in that: Tea water with added Lactobacillus plantarum can be used in rice cooking to extend its shelf life.

7. The application of plant lactobacillus according to claim 6, characterized in that: Add 8g of tea leaves to 100ml of water and inoculate 1g of lactic acid bacteria, ferment at 32°C for 24h, and obtain the fermentation liquid after inactivation.

8. The application of plant lactobacillus according to claim 7, characterized in that: Add the fermentation liquid to 900g of water for standby use, wash 700g of rice three times and drain the water, add 924g of the above-mentioned stand-by liquid, 11.2g of soybean oil, 11.2g of rice seasoning liquid, and 5.6g of edible salt, stir well and steam at a cooking temperature of 100°C for 28.5min and simmer the rice for 15min.

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