A strain of Lactobacillus buchneri and its application

Abstract

The invention discloses a strain of Lactobacillus parabuchneri, which is named Lentilactobacillus parabuchneri MF2103 and has a preservation number of GDMCC NO: 62118. The Lactobacillus parabuchneri provided by the invention is derived from traditional fermented rice noodles in Guangxi, and metabolites contained in the fermentation liquid of Lentilactobacillus parabuchneri MF2103 are lactic acid and Nisin, which can inhibit bacteria without causing harm to the human body, and can be used to replace traditional preservatives for the preservation of meat products with good preservation effect; further, the application provides that the production cost of the fermentation liquid of Lentilactobacillus parabuchneri MF2103 is low, and can be widely used in the preservation of meat products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus buchneri and application thereof. Background Art

[0002] Food preservation is a great challenge and problem for the whole world. Traditional chemical preservatives will more or less remain in food, causing certain harm to the human body. Traditional plastic packaging materials are difficult to degrade and recycle. Under certain temperature and medium conditions, harmful substances will be dissolved and transferred to food, which is harmful to the human body. Therefore, finding a natural, non-toxic and harmless biomaterial and combining it with other technologies, such as low temperature, dehydration, and radiation, has become a research hotspot in the direction of ensuring food quality and safety and extending its shelf life, and is expected to have good application prospects in future food packaging materials.

[0003] Lactic acid bacteria (LAB) is a general term for bacteria that ferment and decompose carbohydrates and produce a large amount of lactic acid. During the fermentation process, lactic acid bacteria produce many antibacterial substances such as nisin, lactic acid, and acetic acid. These substances can be further processed and modified to replace traditional preservatives for the preservation and storage of fruits, vegetables, and meat products. Guangxi's traditional fermented rice noodles contain rich strain resources and are good raw materials for screening bacteria.

[0004] Chinese application CN110876409A discloses a method for preserving pork with plant lactobacillus fermented liquid, wherein the plant lactobacillus fermented liquid described in the application uses black fungus, garlic, cloves, tea, lemon, lemongrass, and ginger as raw materials, and each raw material extract is obtained by fermentation with probiotics, and then mixed with lactic acid streptococcus and chitosan to obtain pork preserving plant lactobacillus fermented liquid. The fermented liquid substrate of the application is black fungus, etc., and lactic acid streptococcus and chitosan are artificially added, which increases the original cost of the preservative and is not conducive to large-scale industrial production.

[0005] At present, potassium sorbate and nisin are the most commonly used preservatives on the market. Potassium sorbate is a synthetic preservative and a food additive allowed for consumption in my country. It is mainly used in food, beverages, cosmetics, etc. However, if you eat a lot of food with a lot of potassium sorbate for a long time, it will lead to excessive intake of potassium sorbate and accumulation in the body, which can cause certain stimulation to the gastrointestinal mucosa, increase the burden on the gastrointestinal tract and increase the detoxification burden on the liver; excessive intake of potassium sorbate can also have adverse effects on human metabolism and absorption functions, thereby affecting the body's absorption of nutrients such as vitamins and minerals, and even cause damage to liver and kidney functions in severe cases. Nisin is a natural food preservative that is efficient, non-toxic, safe, and has no side effects. However, the production cost of Nisin is very high, and the selling price has always been higher than that of chemical preservatives. Therefore, there is an urgent need for a green, environmentally friendly, and low-cost preservative. Summary of the invention

[0006] The present invention provides a strain of lactobacillus buchneri strain screened from traditional fermented rice noodles in Guangxi, which can solve the problem of food preservation.

[0007] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0008] A strain of Lactobacillus parabuchneri, named Lentilactobacillus parabuchneri MF2103 (abbreviated as L.parabuchneri MF2103), was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 13, 2021. The preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation number is GDMCC NO: 62118. The 16S rDNA gene sequence of the Lactobacillus parabuchneri is shown in SEQ ID NO.1, containing 1376 bases.

[0009] As mentioned above, the application of Lactobacillus buchneri in food preservation.

[0010] Preferably, the Lactobacillus buchneri fermentation broth is used in food preservation.

[0011] Preferably, the method for preparing the fermentation broth of Lactobacillus parabuchneri is as follows: the Lactobacillus parabuchneri MF2103 is activated with MRS liquid culture medium, inoculated into MRS-S liquid culture medium at an inoculum amount of 5% (v / v), and cultured under shaking conditions of 37° C. and 120 rpm for 24 hours to obtain a crude fermentation broth, the obtained crude fermentation broth is centrifuged to remove bacterial precipitates, and then filtered with a 0.22 μm water-based sterile filter membrane to obtain a fermentation broth.

[0012] Preferably, the MRS liquid culture medium contains 10g of peptone, 10g of beef extract, 10g of yeast powder, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 20g of glucose per liter of MRS liquid culture medium; the MRS-S liquid culture medium contains 10g of peptone, 10g of beef extract, 10g of yeast powder, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 5g of glucose and 50g of sucrose per liter of MRS-S liquid culture medium.

[0013] Compared with the prior art, the present invention has the following beneficial technical effects:

[0014] The lactobacillus parabuchneri MF2103 provided by the present invention is derived from traditional fermented rice noodles in Guangxi. The metabolites contained in the fermentation liquid of the Lentilactobacillus parabuchneri MF2103 are lactic acid and Nisin. These substances can inhibit bacteria and do not cause harm to the human body. The Lentilactobacillus parabuchneri MF2103 can be used to replace traditional preservatives for the preservation of meat products with good preservation effect. Furthermore, the present application adopts MRS-S culture medium to prepare the fermentation liquid of the Lentilactobacillus parabuchneri MF2103. The culture medium is cheap and easy to obtain, which greatly reduces the production cost of the fermentation liquid of the Lentilactobacillus parabuchneri MF2103, and can be widely used in the preservation of meat products.

[0015] Storage Information

[0016] Lactobacillus buchneri was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on December 13, 2021, with the deposit number GDMCC NO: 62118. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figures show the growth state (a) and colony state (b) of Lentilactobacillus parabuchneri MF2103 of the present invention in MRS culture medium.

[0018] Figure 2 The Gram-stained microscopic photographs of Lentilactobacillus parabuchneri MF2103 of the present invention (40×(a) and 100×(b)).

[0019] Figure 3The antibacterial results of the fermentation broth of Lentilactobacillus parabuchneriMF2103 of the present invention (left: S. aureus; right: E. coli).

[0020] Figure 4 It refers to the surface state of pork chunks during the storage period of 0-10 days.

[0021] Figure 5 It is the total number of colonies in pork chunks during storage.

[0022] Figure 6 is the pH value of pork chunks during storage.

[0023] Figure 7 The effects of different treatments on TVB-N.

[0024] Figure 8 The effects of different treatments on GLOB. DETAILED DESCRIPTION

[0025] The following is a detailed description of the specific embodiments in conjunction with the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. The raw materials and reagents used in the examples are all commercially available unless otherwise specified. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged. The E. coli and S. aureus used in the examples were donated by Professor Li Shubo's research group of Guangxi University.

[0026] In the following examples, the culture media used in the examples are composed of:

[0027] The composition of 1L MRS solid culture medium is: peptone 10g, beef extract 10g, yeast powder 10g, Tween 80 1mL, dipotassium hydrogen phosphate 2g, sodium acetate 5g, triammonium citrate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, glucose 20g, agar 15g.

[0028] The composition of 1L MRS liquid culture medium is: peptone 10g, beef extract 10g, yeast powder 10g, Tween 80 1mL, dipotassium hydrogen phosphate 2g, sodium acetate 5g, triammonium citrate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, glucose 20g.

[0029] MRS-S liquid culture medium: replace 20 g glucose in the MRS liquid culture medium with 5 g glucose and 50 g sucrose, while keeping other components unchanged.

[0030] 1L LB liquid medium: 10g tryptone, 5g yeast extract powder, 10g sodium chloride.

[0031] 1L LB solid medium: 10g tryptone, 5g yeast extract powder, 10g sodium chloride, and 15g agar.

[0032] Example 1

[0033] Isolation and identification of strains

[0034] (1) Sample collection: Samples were collected from fermented rice noodles in Dashatian, Liangqing District, Nanning City, Guangxi Zhuang Autonomous Region, sealed in sterile sealed bags and stored in a -20°C refrigerator;

[0035] (2) Dilution and coating: Take 1g of fermented rice flour and add 9mL of sterile saline, homogenize, and dilute the homogenate 10 times in a gradient manner. -4 , 10 -5 , 10 -6 The diluted homogenate was spread on MRS solid medium and cultured at 37°C for 48h;

[0036] (3) Isolating single colonies: Pick out colonies with different morphologies after culturing on MRS solid medium for 48 h in step (2), streak them on new MRS solid medium, and culture them at 37° C. for 48 h. Repeat this process three or more times until pure colonies are cultured;

[0037] (4) Colony morphology observation and Gram staining: The pure colony numbered MF2103 in step (3) was picked and placed in a test tube containing MRS liquid culture medium and cultured at 37°C for 15 h. There was obvious bacterial deposition at the bottom of the test tube (e.g. Figure 1 (a)); then the pure strain MF2103 obtained in step (3) was streaked on MRS solid medium (37°C, 15h), and the colonies were observed to be round, white, smooth, soft, and easy to pick up. Figure 1 (b) As shown; MF2103 was selected for Gram staining, and the steps were as follows: the smear was fixed, stained with crystal violet for 1 minute, rinsed with tap water, and the water was removed; iodine solution was added to cover the smear and stained for about 1 minute, washed with water, and the water was removed with absorbent paper; a few drops of 95% alcohol were added and gently shaken for decolorization, washed with water after 20 seconds, and the water was removed; after staining with safranin dye solution for 1 minute, the smear was rinsed with tap water, dried, and examined under a microscope; the bacteria were observed to be rod-shaped and purple (such as Figure 2 As shown), it was determined to be Gram-positive bacteria;

[0038] (5) Strain identification: The pure bacteria obtained in step (3) were selected and sent for 16S rDNA detection. The 16S rDNA gene sequence was shown in SEQ ID NO. 1. The detection result was compared with the bacterial 16S rDNA sequence in the database using the Blast program in NCBI. The comparison result showed that the MF2103 strain was Lentilactobacillus parabuchneri and was named Lentilactobacillus parabuchneri MF2103.

[0039] Example 2

[0040] Preparation of fermentation broth of L. parabuchneri MF2103 and its antibacterial activity

[0041] (1) Preparation of fermentation broth: The L. parabuchneri MF2103 identified above was activated three times with MRS liquid medium, and then inoculated into MRS-S liquid medium at an inoculum amount of 5% (v / v), and cultured with shaking at 37° C. and 120 rpm for 24 h to obtain a crude fermentation broth. The obtained crude fermentation broth was centrifuged at 4° C. and 8000×g for 20 min to remove bacterial precipitates, and then filtered with a 0.22 μm water-based sterile filter membrane to obtain a fermentation broth for later use;

[0042] (2) Activation of bacterial strains: Activate frozen E. coli and S. aureus in LB liquid medium;

[0043] (3) Antibacterial experiment: The bacterial solution activated in step (2) was diluted to OD 600nm The value is about 0.5, and 200 μL of the diluted bacterial solution is transferred to the cooled LB solid culture medium. An Oxford cup is placed vertically on the surface of the culture medium using sterile operation. 100 μL of the fermentation liquid reserved in step (1) is added to the Oxford cup. Sterile water and MRS culture medium are used as negative controls, and the culture is incubated at 37°C overnight.

[0044] After experimental determination, the fermentation broth of the present invention showed a relatively obvious inhibition zone (such as Figure 3 ), and its inhibitory effect on S. aureus was higher than that on E. coli, that is, the fermentation broth had certain antibacterial activity.

[0045] Example 3

[0046] Preparation of Extracellular Polysaccharide (EPS) from L. parabuchneri MF2103

[0047] (1) Alcohol precipitation: add 4 times the volume of 95% (V / V) ethanol to the fermentation broth reserved in step (1) of Example 2, stir evenly, stand at 4°C overnight to precipitate polysaccharides with alcohol, and centrifuge at 8000×g for 20 min to obtain a polysaccharide precipitate;

[0048] (2) The polysaccharide precipitate obtained in step (1) was redissolved in a certain amount of deionized water, 80% (W / V) trichloroacetic acid was added to a final concentration of 4% (W / V) and stirred evenly, and the mixture was allowed to stand at 4°C for 10 h and then centrifuged at 8000 × g for 20 min to remove protein, thereby obtaining a supernatant;

[0049] (3) Secondary alcohol precipitation: add 4 volumes of 95% (V / V) ethanol to the supernatant obtained in step (2), stir evenly, stand at 4°C overnight to precipitate the polysaccharide, and centrifuge at 8000×g for 20 min to remove the trichloroacetic acid added in step (2) to obtain a polysaccharide precipitate;

[0050] (4) Dialysis: The polysaccharide precipitate obtained in step (1) was redissolved in a certain amount of deionized water, placed in a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed at 4° C. for 48 h, with the water being changed every 8 h to obtain a polysaccharide solution;

[0051] (5) Freeze drying: The polysaccharide solution obtained in step (4) was frozen at -20°C for 48 hours, and then placed in a vacuum freeze dryer for freeze drying for 48 hours to obtain a crude polysaccharide powder (Exopolysaccharides, EPS).

[0052] Example 4

[0053] Application of L.parabuchneri MF2103 fermentation liquid in pork preservation

[0054] (1) Preparation of 0.5% EPS: 0.5 g of the crude polysaccharide powder obtained in Example 3 was dissolved in 100 mL of deionized water to obtain a 0.5% EPS solution. The 0.5% EPS solution was filtered through a 0.22 μm water-based sterile filter membrane to obtain a sterile 0.5% EPS solution for later use.

[0055] (2) Pretreatment of pork: Fresh pork was cut into 2.5 g meat blocks of uniform thickness under sterile conditions. The meat blocks were randomly divided into 6 groups, each of which was randomly divided into 4 small groups. Three parallel groups were set up in each group and treated according to Table 1. The treated pork blocks were placed in sterile culture dishes, sealed with PE plastic wrap, and stored at 4°C.

[0056] Table 1 Groups with different treatments

[0057]

[0058] (3) Surface conditions during storage: During storage, the pork blocks were photographed on days 0, 2, 4, 6, 8, and 10 to record their surface conditions; Figure 4 The figure shows the surface state of pork during storage. During storage, the pork treated with the fermentation liquid (F) had no water on the surface and maintained a good external state (lighter color). On the 6th day, the pork in groups E and W had more water and was sticky. At the same time, the pork in groups E and W had obvious yellowing. The pork in the control group was dark red and dull. It was preliminarily determined that the pork in these three groups had been corrupted and deteriorated. On the 8th day, except for group F, the pork in other groups emitted obvious corrupt odor. On the 10th day, the pork in group F still maintained a good appearance and had no adverse phenomena, while the pork samples in the other three groups had a very strong odor. Therefore, the fermentation liquid of L. parabuchneri MF2103 can maintain the surface state of pork, has a certain preservation effect on pork, and can extend the shelf life of pork.

[0059] (4) Determination of total colony count (CFU) of pork samples: Referring to GB4789.2-2016 “Determination of total colony count in food microbiological examination”, on days 0, 2, 4, 6, and 8, take 2.5 g of the meat after observation in step (3) under sterile conditions, chop it thoroughly with scissors, add sterile physiological saline at a solid-liquid ratio of 1:10 (W / V), mix thoroughly, and obtain a 1:10 broth dilution. Dilute it in sequence, and pipette 1 mL of the dilution at the appropriate dilution into a sterile culture dish, and culture it in a 37°C incubator for 48 h. The experimental results are as follows: Figure 5 As shown in the figure, the total colony count of group F was always lower than that of the other three groups (p<0.05). After 8 days of storage, the total colony counts of groups E, W, and Control reached 7.64±0.02Lg (CFU / g), 7.39±0.24Lg (CFU / g), and 7.59±0.00Lg (CFU / g), respectively. In comparison, the total colony count of group F was only 5.70±0.02Lg (CFU / g), which was 2 orders of magnitude lower, with a significant difference (p<0.05). During the storage period, the total colony count of group F was always lower than 6Lg (CFU / g), which was first-class fresh meat. Therefore, the fermentation liquid of L. parabuchneri MF2103 played a good bactericidal role in the storage process of pork, which could delay the corruption and deterioration of pork and achieve the effect of preservation.

[0060] (5) Determination of pH value of pork sample: Use a pH meter (calibrated in advance) to measure the 1:10 dilution of broth in step (4). The result is as follows: Figure 6As shown; on the 6th day, except for the pH value of group F of 5.37±0.03, the pH values ​​of samples in other groups reached above 6.70, which was judged to be spoiled; when it reached the 10th day, compared with group E (7.48±0.01), group W (7.52±0.04) and control group (7.43±0.01), the pH value of samples in group F was 5.65±0.39, which was still in a relatively acidic state and was judged to be fresh; therefore, the fermentation broth of L. parabuchneri MF2103 can inhibit the decomposition of protein by microorganisms to a certain extent, thereby delaying the rise of pH value;

[0061] (6) Determination of volatile basic nitrogen (TVB-N) in pork samples: The determination was performed using a volatile basic nitrogen rapid test kit. The specific steps are as follows: 1 mL of the broth dilution obtained in step (4) was placed in a test tube, and 2 drops of the test solution provided in the rapid test kit were added to the test tube and shaken well; Figure 7 The TVB-N content of pork treated according to Table 1 during storage was shown in Table 1. The results showed that the change in group F was the slowest. From the 6th day, groups E, W and Control began to show obvious yellow reaction, while group F did not show a slight yellowish color until the 10th day, indicating that the TVB-N content of its pork was low, indicating that the fermentation liquid of L. parabuchneri MF2103 could significantly inhibit the decomposition of endogenous protein in pork by microorganisms, reduce the content of volatile basic nitrogen, and thus delay the corruption of pork.

[0062] (7) Determination of meat globulin (GLOB) in pork samples: The determination was performed using a meat globulin rapid test kit, with water as the control. The specific steps are as follows: 1 mL of the broth dilution obtained in step (4) was placed in a test tube, and 2 drops of the test solution provided in the rapid test kit were added to the test tube and shaken well; the reaction solution of fresh meat appeared light blue and completely transparent or slightly turbid, while the reaction solution of non-fresh meat appeared obviously turbid, with obvious floccules or white precipitates (such as Figure 8 The results on day 0 showed that the reaction liquid was light blue and completely transparent, indicating that the meat was fresh; Group F showed only slight turbidity on day 10, while this phenomenon began to appear in the other three groups from day 2, and as the number of storage days increased, the reaction results became increasingly turbid, and there was no significant difference among the three groups.

[0063] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A strain of Lactobacillus buchneri ( Lentilactobacillus parabuchneri ) MF2103, characterized in that: The Lactobacillus buchneri deposit number is GDMCC NO: 62118, and the 16S rDNA gene sequence of the Lactobacillus buchneri is shown in SEQ ID NO.

1.

2. Use of the fermentation liquid of the Lactobacillus buchneri strain according to claim 1 in food preservation, characterized in that: The food preservation refers to pork preservation.

3. The use of the fermentation liquid of the Lactobacillus buchneri-like substance in food preservation according to claim 2, characterized in that: The preparation method of the fermentation liquid of Lactobacillus brucei is as follows: activating the Lactobacillus brucei MF2103 with an MRS liquid culture medium, inoculating it into an MRS-S liquid culture medium with an inoculum amount of 5%, shaking and culturing it at 37°C and 120 rpm for 24 hours to obtain a crude fermentation liquid, centrifuging the obtained crude fermentation liquid, and then filtering it with a 0.22 μm water system sterile filter membrane to obtain a fermentation liquid; the MRS liquid culture medium contains 10g of peptone, 10g of beef extract, 10g of yeast powder, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 20g of glucose per liter of the MRS liquid culture medium; the MRS-S liquid culture medium contains 10g of peptone, 10g of beef extract, 10g of yeast powder, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 20g of glucose per liter of the MRS-S liquid culture medium. 1mL, dipotassium hydrogen phosphate 2g, sodium acetate 5g, triammonium citrate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, 5g glucose and 50g sucrose.

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