A strain of Lactococcus lactis from bamboo shoots and its application

Abstract

The present invention belongs to the field of food processing, and relates to a bamboo shoot-derived lactococcus and its application. The present invention discloses a lactococcus, which is Lactococcus lactis (Lactococcus lactis Strain) TJJ-02, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20211689. The present invention uses the bacteria for the fermentation of discarded bamboo shoot liquid in canned bamboo shoot products, which provides a new way to solve the problem of a large amount of bamboo shoot liquid being wasted in canned bamboo shoots, maximizes benefits for enterprises, and is environmentally friendly and resource-intensive for society, and also develops a new bamboo shoot probiotic product.

Description

Technical Field

[0001] The invention belongs to the field of food processing and relates to a bamboo shoot-derived Lactococcus lactis and application thereof. Background Art

[0002] China is located in the center of bamboo distribution in the world, with a bamboo forest area of ​​6.41 million hm2 2 , accounting for about 20% of the world's total bamboo forest area, and there are 39 genera and 837 species of bamboo, accounting for about 50% of the world's total. Therefore, China has rich bamboo shoot resources. Bamboo shoots are the expanded buds and young stems formed by the buds on the bamboo whip or stalk base, also known as bamboo embryos, bamboo meat, etc. Bamboo shoots taste crisp and delicious, and are rich in nutrition.

[0003] According to statistics, about 60% of bamboo shoots are processed in my country every year. The main products include dried bamboo shoots, salted bamboo shoots, water bamboo shoots, seasoned bamboo shoots, etc. Water bamboo shoots are currently the most produced bamboo shoot processed products. They are made by trimming, pre-cooking, canning, soup injection, sterilization, cooling and other processes. They are widely used in catering and bamboo shoot deep processing as raw materials for bamboo shoot deep processing. However, water bamboo shoots are repeatedly heated and boiled during the processing process. Many soluble nutrients in the bamboo shoot raw materials are lost in the soup that is not used by people, causing waste and environmental pollution. According to the current light industry standard QB / T 1406-2014 canned bamboo shoots, the solid content of canned bamboo shoots "should not be less than 55% for superior products and not less than 50% for qualified products", which means that nearly 50% of the bamboo shoot liquid in water cans is wasted.

[0004] At the same time, the study found that bamboo shoots are a healthy food rich in plant polyphenols (PP) and phytosterols (PS). The research team of the inventors measured the total phenol content of secondary metabolites in fresh bamboo shoots to be 48.36±0.18mg / 100g, and the total sterol content to be 74.89±2.77mg / 100g. The total phenol and total sterol contents in the discarded bamboo shoot juice of canned products were 10.11±0.23mg / 100g and 1.05±0.04mg / 100g, respectively.

[0005] As a kind of plant secondary metabolite, plant polyphenols have a basic structure of a phenolic ring structure with one or more hydroxyl groups without nitrogen, and have a wide range of biological activities, such as anti-oxidation, anti-inflammatory, and antibacterial. Phytosterols are a class of steroid compounds in nature, belonging to saturated or unsaturated secondary alcohols. The basic skeleton of the sterol molecule is a cyclopentane polyhydrogen phenanthrene nucleus, which is composed of three six-membered rings and one five-membered ring, with a hydroxyl group at the C3 position, a side chain composed of more than 7 carbon atoms at the C17 position, and a methyl group at each of the C10 and C13 positions. Bamboo shoot sterols include β-sitosterol, which has a relatively high content, and also include cholesterol, ergosterol, brassicasterol, stigmasterol, etc. Phytosterols are a functional component that exists in a free state or in a state combined with sugars. Because it can significantly reduce the body's cholesterol level and has unique important physiological functions such as anti-oxidation, growth promotion, immunomodulation, and hormone-like, phytosterols are called "the key to life" by scientists. The discarded bamboo shoot juice in canned bamboo shoot products is rich in nutrients, containing phenols, sterols, organic acids and free amino acids, so the development and utilization of the discarded bamboo shoot juice is very necessary. Summary of the invention

[0006] To achieve the above-mentioned purpose, the present invention provides a Lactococcus lactis Strain TJJ-02, the preservation number of which in the China Center for Type Culture Collection is CCTCC NO:M 20211689, the preservation address is: Wuhan University, Wuhan, China, and the preservation date is December 29, 2021.

[0007] The Lactococcus lactis Strain TJJ-02 of the present invention has Figure 1 The Gram-positive characteristics are shown in Figure 1. The colony morphology in MRS medium is as follows: Figure 2 The nucleotide sequence of its 16S rRNA is the nucleotide sequence shown in SEQ ID NO.1. The nucleotide sequence of 16S rRNA of Lactococcus lactis strain TJJ-02 was compared with the nucleotide sequence in NCBI. The comparison results were used to construct a phylogenetic tree using the Neighbor-joining algorithm in MEGA software. The phylogenetic tree is shown in Figure 3 shown.

[0008] In order to solve the above technical problems, the present invention also provides a bacterial agent. The bacterial agent contains Lactococcus lactis Strain TJJ-02.

[0009] The preparation method of the liquid proliferation culture medium of the present invention is as follows: weigh 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate [(NH4)2HC6H5O7], and 1.0 g of glucose (C6H 12 O6·H2O) 20.0g, sodium acetate (CH3COONa·3H2O) 5.0g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.0g, magnesium sulfate (MgSO4·7H2O) 0.50g, manganese sulfate (MnSO4·H2O) 0.25g, Tween 80 1.0mL, 500mL of water canned bamboo shoot product waste bamboo shoot liquid added 500mL distilled water.

[0010] The number of Lactococcus lactis strain TJJ-02 in MRS liquid medium was 1.0×10 6 cfu / ml of bacterial solution was added to the high-density proliferation medium and cultured at 37°C for 72 h. The content of Lactococcus lactis Strain TJJ-02 in the liquid culture medium was measured to be 1.0×10 10 cfu / ml. The fermented bacterial solution was centrifuged using an ultracentrifuge at a speed of 4000-6000 r / min for 10 min, and the supernatant was removed to collect the sediment.

[0011] The collected sediment was added to the compound protective agent, the protective agent formula is: trehalose 8%, bamboo shoot powder nanoparticles 2%, bovine serum albumin 6%, skim milk 3%, lysine 1% (the percentage of each component is the mass percentage of the sediment). Mix with an oscillator to make a bacterial suspension. Then put it into a freezing plate, then put it in a 4°C refrigerator for 30 minutes, move it into a -30°C refrigerator for 60 minutes, then put it in a -80°C refrigerator for 60 minutes, and then freeze-dry it in a freeze vacuum dryer for 14 hours (the freeze dryer chamber temperature is -60°C-70°C, the pressure is 0.006Pa), and the live bacterial count of the Lactococcus lactis Strain TJJ-02 freeze-dried powder was measured to be 10 11 cfu / g, and the lyophilized powder was sealed and stored in a 4°C refrigerator for later use.

[0012] The present invention also provides a method for using a bacterial agent containing Lactococcus lactis Strain TJJ-02 and a bacterial agent containing Lactococcus lactis Strain TJJ-02 in waste bamboo shoot liquid of canned bamboo shoot products. The freeze-dried powder of Lactococcus lactis Strain TJJ-02 is inoculated into the waste bamboo shoot liquid after sugar adjustment at an inoculation rate of 1-4%. The method for adjusting the sugar content of the bamboo shoot liquid is to add 1-2% glucose or lactose. The bamboo shoot liquid inoculated with the freeze-dried bacterial powder is fermented at 37°C for 36-72h. Compared with the naturally fermented bamboo shoot liquid after sugar adjustment and the newly opened canned bamboo shoot liquid, the bamboo shoot liquid fermented with the freeze-dried powder of Lactococcus lactis Strain TJJ-02 has a high sterol content, a good taste and a strong antibacterial property.

[0013] Beneficial effects of the present invention:

[0014] The invention uses Hunan naturally fermented sour bamboo shoots as raw materials, screens the dominant bacteria Lactococcus lactis Strain TJJ-02 that can form converted phenols and sterols, and uses the strain to ferment discarded bamboo shoot juice in canned bamboo shoot products. This provides a new way to solve the problem of a large amount of bamboo shoot juice being wasted in canned bamboo shoots, maximizes benefits for enterprises, and achieves environmental friendliness and resource intensiveness for society. At the same time, a new bamboo shoot probiotic product is also developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Gram staining characteristics of Lactococcus lactis Strain TJJ-02 from bamboo shoots;

[0016] Figure 2 Colony morphology of Lactococcus lactis Strain TJJ-02 from bamboo shoots in MRS medium;

[0017] Figure 3 Phylogenetic tree of Lactococcus lactis Strain TJJ-02;

[0018] Figure 4 The sterol content of each example;

[0019] Figure 5 Total phenol content of each example;

[0020] Figure 6 Total acid content of each embodiment;

[0021] Figure 7The diameter (d) of the inhibition zone of each example. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The invention provides a lactococcus lactis strain TJJ-02 screened from naturally fermented sour bamboo shoots in Hunan Province. The preservation number of the strain in China Center for Type Culture Collection is CCTCC NO: M20211689.

[0024] The present invention also provides a bacterial agent, which is obtained by high-concentration proliferation and cultivation of Lactococcus lactis strain TJJ-02 to reach 1.0×10 10 cfu / mL, then centrifuged, added with protective agent and freeze-dried, the live bacteria content of freeze-dried bacterial powder reached 1.0×10 11 cfu / g.

[0025] The present invention also provides an application method of a lactococcal agent, wherein the above-mentioned Lactococcus lactis strain TJJ-0 freeze-dried powder is inoculated at an inoculation rate of 1-4% into the discarded bamboo shoot liquid after sugar adjustment. The method for adjusting the sugar content of the bamboo shoot liquid is to add 1-2% glucose or lactose. The bamboo shoot liquid inoculated with freeze-dried bacterial powder is fermented at 37°C for 36-72h, and a new type of bamboo shoot beverage product with probiotic properties is developed. The total phenols, total sterols, total acid and antibacterial properties are measured, and the product taste, body, aroma and color are evaluated by sensory evaluation. The sensory evaluation standards are shown in Table 1:

[0026] Table 1 Sensory evaluation reference standards

[0027]

[0028]

[0029] Example 1

[0030] The lyophilized powder of Lactococcus lactis Strain TJJ-02 was inoculated at a rate of 1% into the waste bamboo shoot liquid of sterilized canned bamboo shoot products with 2% lactose added, and fermented at 37°C for 36 hours. The fermented sample was named LS1. The total sterol, total phenol and total acid contents of the sample were determined, and then the antibacterial properties of the sample against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus were tested, and finally sensory evaluation was performed.

[0031] The method for determining the total sterol content is as follows: prepare a standard curve with cholesterol standard, and measure the absorbance of the standard solution at a wavelength of 650nm. Draw a standard curve with cholesterol absorbance as the horizontal axis and cholesterol concentration as the horizontal axis. Take 200mL of bamboo shoot liquid, add n-hexane and ultrasonically extract for 1h, filter to obtain the extract, evaporate the solvent on a rotary evaporator to obtain a sterol sample, and dilute to the scale with chloroform. Add 2mL of acetic anhydride to a 10mL beaker, add a drop of sulfuric acid, and immediately add 1mL of the sample. Vortex oscillate for 1min and let stand for 12min. Detect its absorbance at a wavelength of 650nm. The results are as follows Figure 4 shown.

[0032] The method for determining the total phenol content is as follows: prepare a standard curve with gallic acid standard, measure the absorbance at a wavelength of 765nm, and draw a standard curve with gallic acid absorbance as the horizontal axis and gallic acid concentration as the horizontal axis. Take 200mL of bamboo shoot liquid, add 60% methanol and ultrasonically extract for 1h, filter to obtain the extract, and use a rotary evaporator to evaporate the solvent to obtain a total phenol sample. Add 2mL of Folin-phenol reagent and shake in a 25mL volumetric flask, then add 10mL of 10% sodium carbonate solution by mass, make up to volume with distilled water, and heat in a 50℃ water bath for 1h. Measure the absorbance at 765nm. The results are as follows: Figure 5 shown.

[0033] The total acid was determined according to the method of GB 12456-2021. The results are as follows: Figure 6 The antibacterial properties were measured by the filter paper method, and the results are shown in Figure 7 The taste, body, aroma and color of the product were evaluated by sensory evaluation, and the results are shown in Table 2.

[0034] Example 2

[0035] The lyophilized powder of Lactococcus lactis strain TJJ-02 was inoculated at a rate of 1% into the waste bamboo shoot liquid of sterilized canned bamboo shoot products with 2% lactose added, and fermented at 37°C for 72 hours. The fermented sample was named LS2. The total sterol, total phenol, total acid and antibacterial properties of sample LS2 were determined by the method described in Example 1. The results are as follows: Figure 4-7 The taste, body, aroma and color of the product were evaluated by sensory evaluation, and the results are shown in Table 2.

[0036] Example 3

[0037] The lyophilized powder of Lactococcus lactis Strain TJJ-02 was inoculated at a rate of 2% into the waste bamboo shoot liquid of sterilized canned bamboo shoot products with 2% lactose added, and fermented at 37°C for 36 hours. The fermented sample was named LS3. The total sterol, total phenol, total acid and antibacterial properties of the sample LS3 were determined by the method described in Example 1. The results are as follows: Figure 4-7 The taste, body, aroma and color of the product were evaluated by sensory evaluation, and the results are shown in Table 2.

[0038] Example 4

[0039] The lyophilized powder of Lactococcus lactis strain TJJ-02 was inoculated at a rate of 2% into the waste bamboo shoot liquid of canned bamboo shoot products with 2% glucose added, and fermented at 37°C for 36 hours. The fermented sample was named LS4. The total sterol, total phenol, total acid and antibacterial properties of sample LS4 were determined by the method described in Example 1. The results are as follows: Figure 4-7 The taste, body, aroma and color of the product were evaluated by sensory evaluation, and the results are shown in Table 2.

[0040] Example 5

[0041] The lyophilized powder of Lactococcus lactis Strain TJJ-02 was inoculated at a rate of 2% into the waste bamboo shoot liquid of sterilized canned bamboo shoot products with 1% glucose and 1% lactose added, and fermented at 37°C for 36 hours. The fermented sample was named LS5. The total sterol, total phenol, total acid and antibacterial properties of the sample LS5 were determined using the method described in Example 1. The results are as follows: Figure 4-7 The taste, body, aroma and color of the product were evaluated by sensory evaluation, and the results are shown in Table 2.

[0042] Example 6

[0043] The lyophilized powder of Lactococcus lactis strain TJJ-02 was inoculated at a rate of 3% into the waste bamboo shoot liquid of sterilized canned bamboo shoot products with 1% glucose and 1% lactose added, and fermented at 37°C for 36 hours. The fermented sample was named LS6. The total sterol, total phenol, total acid and antibacterial properties of sample LS6 were determined by the method described in Example 1. The results are as follows: Figure 4-7 The taste, body, aroma and color of the product were evaluated by sensory evaluation, and the results are shown in Table 2.

[0044] Example 7

[0045] The lyophilized powder of Lactococcus lactis Strain TJJ-02 was inoculated at a rate of 4% into the waste bamboo shoot liquid of sterilized canned bamboo shoot products with 1% glucose and 1% lactose added, and fermented at 37°C for 36 hours. The fermented sample was named LS7. The total sterol, total phenol, total acid and antibacterial properties of the sample LS7 were determined by the method described in Example 1. The results are as follows: Figure 4-7 The taste, body, aroma and color of the product were evaluated by sensory evaluation, and the results are shown in Table 2.

[0046] Example 8

[0047] 1% glucose and 1% lactose were added to the discarded bamboo shoot liquid of canned bamboo shoot products, and then sterilized and placed at 37°C for 36 hours. The sample was named LS8. The total sterol, total phenol, total acid and antibacterial properties of sample LS8 were determined using the method described in Example 1. The results are as follows: Figure 4-7 The taste, body, aroma and color of the product were evaluated by sensory evaluation, and the results are shown in Table 2.

[0048] Example 9

[0049] The discarded bamboo shoot liquid of the canned bamboo shoot product was sterilized and placed at 37°C for 36 hours. The sample was named LS9. The total sterol, total phenol, total acid and antibacterial properties of the sample LS9 were determined by the method described in Example 1. The results are as follows: Figure 4-7 The taste, body, aroma and color of the product were evaluated by sensory evaluation, and the results are shown in Table 2.

[0050] Table 2 Sensory evaluation scores of Examples 1-9

[0051]

[0052] Depend on Figures 4 to 7 It can be seen that when the inoculation amount is 1%, the total sterol and total phenol content of fermentation for 36h is significantly higher than that of fermentation for 72h, indicating that too long fermentation time will reduce the total sterol and total phenol in the discarded bamboo shoot liquid of canned bamboo shoot products, which may be due to the consumption of a certain amount of sterol and phenol in the later stage of microbial growth. However, within 0h-72h, the longer the fermentation time, the higher the total acid content and the stronger the antibacterial ability. The total acid content is proportional to the antibacterial ability, and its inhibitory effect on Escherichia coli is greater than that on Staphylococcus aureus.

[0053] At the same time, within the range of 1%-4% inoculation, the total sterol and total phenol content in the fermentation broth first increased and then decreased, both reaching the highest level when the inoculation amount was 2%. However, as the inoculation amount of the bacteria increased, its total acid content continued to increase, and its antibacterial ability also increased with the increase of inoculation amount.

[0054] The types of sugar adjustment, lactose and glucose, had no significant effect on antibacterial ability, total phenols and total acids, but at 2% inoculation, the LS5 sample with 1% lactose and 1% glucose had the highest sterol content, followed by the LS3 sample with 2% lactose, and finally the LS4 sample with 2% glucose. Sugar adjustment had a significant effect on the sensory evaluation score, and the discarded bamboo shoot liquid of the clear water canned bamboo shoot product without sugar adjustment scored the lowest.

[0055] From the results obtained in the examples, it can be seen that the Lactococcus lactis Strain TJJ-02 can significantly increase the total sterol, total phenol and total acid contents in the discarded bamboo shoot liquid of canned bamboo shoot products, and can also significantly improve the antibacterial performance; fermentation with the bacteria after sugar adjustment can also significantly improve the sensory quality of the fermentation liquid.

Claims

1. A strain of Lactococcus lactis, characterized in that The lactococcus lactis is Lactococcus lactis ( Lactococcus lactis )TJJ-02, deposited in China Center for Type Culture Collection with the accession number CCTCC NO:M 20211689.

2. A bacterial agent, characterized in that: The bacterial agent contains the Lactococcus lactis according to claim 1 ( Lactococcus lactis )TJJ-02.

3. The bacterial agent according to claim 2, characterized in that The bacterial preparation also contains a culture medium.

4. The bacterial agent according to claim 3, characterized in that The culture medium includes a liquid proliferation medium or a lyoprotectant.

5. The bacterial agent according to claim 4, characterized in that The Lactococcus lactis ( Lactococcus lactis )The content of TJJ-02 in liquid proliferation medium is 1.0×10 6 -10 10 CFU / mL; the Lactococcus lactis ( Lactococcuslactis )The content of TJJ-02 in the freeze-dried protective agent is 1.0×10 11 CFU / mL.

6. The bacterial agent according to claim 4 or 5, characterized in that The preparation method of the liquid proliferation medium is as follows: weigh 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 0.50 g of magnesium sulfate, 0.25 g of manganese sulfate, and 1.0 mL of Tween 80, add 500 mL of waste bamboo shoot liquid from canned bamboo shoot products, and then add 500 mL of distilled water.

7. The bacterial agent according to claim 6, characterized in that The formula of the freeze-dried protective agent is trehalose 8%, bamboo shoot powder nanoparticles 2%, bovine serum albumin 6%, skim milk 2%, lysine 1%; the percentage of each component is the liquid proliferation culture medium in claim 6 for culturing Lactococcus lactis ( Lactococcus lactis ) The mass percentage of the sediment obtained by centrifugation after TJJ-02.

8. The Lactococcus lactis according to claim 1 ( Lactococcus lactis ) Use of the bacterial agent described in TJJ-02 or any one of claims 2-5 in fermenting waste bamboo shoot liquid.

9. A method for preparing a bamboo shoot beneficial beverage product, characterized in that: The method comprises the following steps: (1) sugar adjustment: adding 1-2% by weight of sugar to the waste bamboo shoot liquid of the sterilized water canned bamboo shoot product to obtain the waste bamboo shoot liquid of the sterilized water canned bamboo shoot product after sugar adjustment, wherein the sugar is at least one of glucose or lactose; (2) Fermentation: inoculating the Lactococcus lactis according to claim 1 into the waste bamboo shoot liquid of the sterilized canned bamboo shoot product after sugar adjustment ( Lactococcus lactis ) TJJ-02 or the bacterial agent described in claim 2, with an inoculation amount of 1-4%, and fermentation at 37°C for 36-72h to obtain the bamboo shoot probiotic beverage product.

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