A lactic acid bacterium with high survival rate in beer

Abstract

The present invention discloses a lactic acid bacterium with high survival rate in beer, belonging to the field of bioengineering technology. The present invention provides a Pediococcus acidilactici ZQ8 with relatively high survival rate in beer. Adding this strain to beer in the form of live bacteria can slow down the aging rate of beer and improve the antioxidant capacity of beer, which is of great significance for the development of new functional beers. The present invention also improves the survival rate of Pediococcus acidilactici ZQ8 in the beer environment by stress pre-treatment of the bacterial cells. The survival rate after standing at 37°C for 12 h can be increased by up to 10 times compared with the original strain, the survival rate after standing at 25°C for 24 h can be increased by up to 2.8 times compared with the original strain, and the survival rate after standing at 4°C for 4 weeks can be increased by up to 2.0 times compared with the original strain.

Description

Technical Field

[0001] The present invention relates to a lactic acid bacterium with high survival rate in beer and belongs to the field of bioengineering technology. Background Art

[0002] Beer is one of the most popular fermented alcoholic beverages in the world, rich in high-value nutritional elements such as protein, minerals, carbohydrates, and vitamins, and can be used as a culture medium for probiotics. Probiotics are "live microorganisms that, when given in sufficient amounts, confer health benefits on the host" and are commonly used to enhance host immunity and maintain the balance of the intestinal flora. Research shows that ingesting 10 8 -10 9 CFU / g of probiotics in the form of food per day can promote physical health. If probiotics can be added to beer, then consumers can obtain a certain amount of probiotics while drinking beer, which is of great benefit to human health. Probiotic beer is one of the innovations of functional beer and can be produced by using probiotics as fermenting microorganisms or directly incorporating live probiotics into the final product. Most probiotic products are dairy-based, and fermented milk is a traditional and popular delivery vehicle for probiotics. There is little research on beer as a probiotic carrier, which creates an opportunity to develop new probiotic alcoholic beverages.

[0003] The decrease in pH value during the growth of probiotics is a limiting factor for fermentation and storage. When probiotics are directly involved in the beer fermentation process as fermenting microorganisms, the production of metabolites (such as organic acids) during fermentation will further lower the pH value. During storage, the pH value will also continue to decline, resulting in over-acidification of the beer. In addition, the pasteurization and filtration procedures used in the conventional beer production process are not suitable for this addition method because probiotics will be killed by heat treatment or removed by filtration. Recently, studies have shown that yeast can improve the survival rate of probiotics. Unfiltered and unpasteurized beer contains yeast, which may become a new carrier for delivering probiotics, but more work is needed to evaluate the viability of probiotics and their possible effects on the beer flavor.

[0004] Directly incorporating live probiotics into finished beer also requires overcoming some difficulties. The most important inhibitory factor for foodborne microorganisms in beer is the hop bitter compound iso-α-acid. As an ionophore for Gram-positive bacteria, iso-α-acid can exchange extracellular H + with intracellular divalent cations and dissipate the ion gradient across the cytoplasmic membrane, reducing the intracellular pH and causing cell inactivation. Mn 2+As an important component of key metabolic enzymes, being excreted outside the cell can also inactivate the key metabolic enzymes of bacteria. The activity of probiotics is the basis for their function. Finding ways to improve the survival rate of lactic acid bacteria in beer and developing probiotics that can adapt to the beer environment and enhance the antioxidant properties of beer are of great significance for the development of new probiotic beers. Summary of the Invention

[0005] The present invention provides a strain of Pediococcus acidilactici ZQ8, which was deposited at the China General Microbiological Culture Collection Center on December 26, 2022, with the deposit number CGMCC No. 26264 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also provides a microbial preparation containing the Pediococcus acidilactici ZQ8.

[0007] In one embodiment, the content of Pediococcus acidilactici ZQ8 in the microbial preparation is ≥ 1×10 7 CFU / mL or 1×10 7 CFU / g.

[0008] The present invention also provides a method for improving the survival rate of Pediococcus acidilactici in beer, which is to pretreat the Pediococcus acidilactici under at least one of the following conditions for a period of time:

[0009] (1) Standing at 35 - 37°C for 4 - 5 h in an environment with a NaCl concentration ≥ 50 g / L;

[0010] (2) Standing for 4 - 5 h in an environment at 4°C - 45°C;

[0011] (3) Standing for 4 - 5 h in an environment with a pH of 4;

[0012] (4) Standing for 5 h in an environment containing 30% - 35% beer by volume;

[0013] (5) Standing for 4 - 5 h in an environment with a hop concentration of 0.1 - 0.2 g / L.

[0014] In one embodiment, the NaCl concentration is 50 - 75 g / L.

[0015] In one embodiment, the environment includes but is not limited to MRS medium.

[0016] In one embodiment, the Pediococcus acidilactici is Pediococcus acidilactici ZQ8.

[0017] The present invention also provides the application of the Pediococcus acidilactici ZQ8 in improving the antioxidant property of beer.

[0018] In one embodiment, the application is to add Pediococcus acidilactici ZQ8 to beer and treat it at 4°C for 4 to 8 weeks.

[0019] The present invention also claims the application of Pediococcus acidilactici ZQ8 in improving the antioxidant property of alcoholic beverages.

[0020] The present invention also claims the application of Pediococcus acidilactici ZQ8 in the preparation of fermented foods.

[0021] In one embodiment, the application includes but is not limited to improving the flavor and quality of fermented foods such as beer, soy sauce, oats, etc.

[0022] Beneficial effects:

[0023] (1) By separating and purifying the microorganisms in the soy sauce mash and testing their survival ability in picric acid, the present invention obtains a Pediococcus acidilactici ZQ8 with a relatively high survival rate in beer and capable of improving the antioxidant ability of beer. Adding this strain to beer will not significantly change the physical and chemical properties of beer, but can slow down the aging rate of beer and improve the antioxidant ability of beer, which is of great significance for the development of new functional beers.

[0024] (2) By subjecting the bacterial cells to stress pretreatment, the survival rate of the strain after standing at 37°C for 12 h can be increased by up to 10 times compared with the original strain, the survival rate after standing at 25°C for 24 h can be increased by up to 2.8 times compared with the original strain, and the survival rate after standing at 4°C for 4 w can be increased by up to 2.0 times compared with the original strain.

[0025] Biological material preservation

[0026] The Pediococcus acidilactici ZQ8 provided by the present invention, classified and named as Pediococcus acidilactici, was deposited on December 26, 2022, at the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 26264 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the drawings

[0027] Figure 1 : Comparison of the survival of 6 lactic acid bacteria under conditions A (4°C) and B (25°C).

[0028] Figure 2 : Comparison of the survival rate of the stressed strain and the original strain at 37°C (12 h).

[0029] Figure 3 : Comparison of the survival rate of the stressed strain and the original strain at 25°C (48 h).

[0030] Figure 4 : Comparison of the survival rates of the stressed strain and the original strain at 4°C (for 4 weeks). Specific implementation manner

[0031] MRS medium (g / L): Peptone 10, Beef extract powder 8, Yeast extract powder 4, Glucose 20, Dipotassium hydrogen phosphate 2, Ammonium citrate dibasic 2, Sodium acetate 5, Magnesium sulfate 0.2, Manganese sulfate 0.04, Tween - 80 1.

[0032] Beer: Heineken beer.

[0033] The strains Pediococcus acidilactici ZQ8, Pediococcus acidilactici WT1, Pediococcus pentosaceus WT6, Weissella paramesenteroides JL - 5, Weissella paramesenteroides LCW - 28, Lactobacillus paracasei XJ01 were respectively obtained from fermented soy mash; among them, Weissella paramesenteroides JL - 5 was disclosed in the patent application document with the publication number CN113913332A; Weissella paramesenteroides LCW - 28 was disclosed in the patent with the publication number CN112852667B. Pediococcus acidilactici ZQ8, Pediococcus acidilactici WT1, and Pediococcus pentosaceus WT6 were all obtained by gradient dilution of the soy mash sample, coating on MRS solid medium, and picking single colonies for screening.

[0034] Example 1: Comparison of the ability of lactic acid bacteria to tolerate hops

[0035] Single colonies of 6 lactic acid bacteria derived from soy mash were picked from the MRS solid medium and inoculated into liquid MRS medium, and cultured statically at 37°C for 24 h, then inoculated into MRS medium containing 0.3 g / L hops at a ratio of 1% (v / v). After 12 h, the absorbance at 600 nm was measured. As shown in Table 1, the OD of Pediococcus acidilactici ZQ8 600 can grow up to 0.221 at most.

[0036] Table 1 OD of 6 probiotic bacteria in 0.3 g / L hops 600

[0037]

[0038] Example 2: Comparison of the survival of lactic acid bacteria in beer

[0039] Six strains of lactic acid bacteria from moromi were picked from single colonies on MRS solid medium and inoculated into liquid MRS medium. They were statically cultured at 37 °C for 12 h until the logarithmic phase. 10 mL of the bacterial solution was washed with physiological saline and the cells were collected, then added to 10 mL of beer (alcohol content 5.0% VOL, wort concentration 11.4 °P), and stored at low temperature (4 °C) and room temperature (25 °C) respectively to compare the survival situation.

[0040] The results are as Figure 1 , the viable cell counts of Pediococcus acidilactici ZQ8 decreased by 3.36 and 0.51 orders of magnitude at 25 °C and 4 °C respectively.

[0041] Example 3: Pretreatment of cells with salt stress

[0042] (1) Stress pretreatment: 10 mL of the ZQ8 bacterial solution in the stationary phase was centrifuged at 8000 rpm and 4 °C for 2 min, the supernatant was discarded, the cells were collected after washing with physiological saline, and the cells were added to 10 mL of MRS medium containing 25 g / L, 50 g / L, 75 g / L and 100 g / L NaCl respectively, mixed well, and statically cultured at 37 °C for 5 h.

[0043] (2) The cells pretreated with stress in step (1) were centrifuged under the same conditions, the cells were collected after washing with physiological saline, and the cells were transferred to 10 mL of beer. After standing at 4 °C, 25 °C and 37 °C for a period of time respectively, the viable cell counts were calculated by the plate colony counting method, and the survival rate was calculated.

[0044] (3) Results: As Figure 2 shown, after being placed at 37 °C for 12 h, the survival rates of the strains treated with 25 and 100 g / L NaCl stress were lower than those of the control bacteria; the survival rates of the strains treated with 50 and 75 g / L NaCl stress were 6.5 and 7.3 times that of the control bacteria; Figure 3 , after being placed at 25 °C for 48 h, the survival rates of the strains treated with 50 and 75 g / L NaCl stress were 2.3 and 2.7 times that of the control bacteria; Figure 4 , after being placed at 4 °C for 4 weeks, the survival rates of the strains treated with 50 and 75 g / L NaCl stress were 1.8 and 2.0 times that of the control bacteria respectively.

[0045] Example 4: Pretreatment of cells with temperature stress

[0046] (1) Stress pretreatment: Centrifuge 10 mL of ZQ8 bacterial liquid in the stationary phase at 8000 rpm and 4 °C for 2 min, discard the supernatant, wash the cells with physiological saline, collect the cells, add the cells to 10 mL of fresh MRS medium, and let stand at 4 °C and 45 °C for 5 h respectively.

[0047] (2) Centrifuge the cells pretreated by stress in step (1) under the same conditions, wash the cells with physiological saline, collect the cells, add 10 mL of beer to the cells, let stand at 4 °C, 25 °C, and 37 °C for a period of time respectively, then use the plate colony counting method to count the viable bacteria and calculate the survival rate.

[0048] (3) Results: As Figure 2 shown, after being placed at 37 °C for 12 h, the survival rates of the strains treated by stress at 4 °C and 45 °C are 10 and 6.5 times that of the control bacteria respectively; Figure 3 , after being placed at 25 °C for 48 h, the survival rates of the strains treated by stress at 4 °C and 45 °C are 1.1 and 2.8 times that of the control bacteria respectively; Figure 4 , after being placed at 4 °C for 4 weeks, the survival rate of the strain treated by temperature stress is lower than that of the control bacteria.

[0049] Example 5: Pretreatment of cells with hop stress

[0050] (1) Stress pretreatment: Centrifuge 10 mL of ZQ8 bacterial liquid in the stationary phase at 8000 rpm and 4 °C for 2 min, discard the supernatant, wash the cells with physiological saline, collect the cells, add the cells to 10 mL of MRS medium containing 0.1%, 0.2%, and 0.3 g / L of hops respectively, mix well, and let stand at 37 °C for 5 h.

[0051] (2) Centrifuge the cells pretreated by pre-stress in step (1) under the same conditions, wash the cells with physiological saline, collect the cells, add 10 mL of beer to the cells, let stand at 4 °C, 25 °C, and 37 °C for a period of time respectively, then use the plate colony counting method to count the viable bacteria and calculate the survival rate.

[0052] (3) Results: As Figure 2 shown, after being placed at 37 °C for 12 h, the survival rates of the strains treated by stress with 0.2% and 0.3 g / L of hops are lower than that of the control bacteria, so this condition will not be considered later. The survival rate of the strain treated by stress with 0.1 g / L of hops is 2.9 times that of the control bacteria; Figure 3 , after being placed at 25 °C for 48 h, the survival rate of the strain treated by stress with 0.1 g / L of hops is 1.3 times that of the control bacteria; Figure 4 , after being placed at 4 °C for 4 w, the survival rate of the strain treated by stress with 0.1 g / L of hops is 1.6 times that of the control bacteria.

[0053] Example 6: Pretreatment of cells with acid stress

[0054] (1) Stress pretreatment: 10 mL of ZQ8 bacterial culture grown to the stationary phase was centrifuged at 8000 rpm and 4°C for 2 min, the supernatant was discarded, and the cells were washed with physiological saline and collected. The cells were added to 10 mL of MRS medium at pH 4 and pH 5, respectively, mixed thoroughly, and allowed to stand at 37°C for 5 h.

[0055] (2) The bacteria after pre-stress treatment in step (1) were centrifuged under the same conditions, washed with physiological saline, and collected. 10 mL of beer was added to the bacteria, and the bacteria were allowed to stand for a period of time at 4°C, 25°C, and 37°C, respectively. The number of live bacteria was counted using the plate colony count method, and the survival rate was calculated.

[0056] (3) Results: Figure 2 As shown in the figure, the survival rate of the strain treated with pH 5 after being placed at 37℃ for 12 hours was lower than that of the control bacteria. This condition was not examined in the subsequent study. The survival rate of the strain treated with pH 4 was 4.7 times that of the control bacteria. Figure 3 , placed at 25℃ for 48h, the survival rate of the strain treated with pH 4 stress was 1.5 times that of the control strain; Figure 4 , placed at 4℃ for 4 weeks, the survival rate of the strain treated with pH4 stress was 1.4 times that of the control bacteria.

[0057] Example 7: Low-concentration beer stress pretreatment of bacterial cells

[0058] (1) Stress pretreatment: 10 mL of ZQ8 bacterial liquid grown to the stable phase was centrifuged at 8000 rpm and 4°C for 2 min, the supernatant was discarded, and the cells were collected after washing with physiological saline. The cells were added to 10 mL of MRS medium containing 33.3% and 66.6% (by volume percentage) beer (alcohol content 5.0% VOL, malt wort concentration 11.4°P), respectively, mixed thoroughly, and allowed to stand at 37°C for 5 h.

[0059] (2) The stressed bacteria were centrifuged under the same conditions, washed with physiological saline, and collected. 10 mL of beer was added to the bacteria. After standing for a period of time at 4°C, 25°C, and 37°C, the number of viable bacteria was calculated using the plate colony counting method, and the survival rate was calculated.

[0060] (3) Results: Figure 2 As shown in the figure, when placed at 37℃ for 12h, the survival rate of the strain treated with beer stress was 66.6% lower than that of the control bacteria. In the subsequent experiment, this condition was not examined, and the survival rate of the strain treated with beer stress was 33.3% which was 6.7 times that of the control bacteria. Figure 3 , placed at 25℃ for 48h, the survival rate of the strain treated with 33.3% beer stress was 1.4 times that of the control strain; Figure 4, when placed at 4°C for 4 weeks, the survival rate of the strain treated with 33.3% beer stress is 1.4 times that of the control bacteria.

[0061] Example 8: Changes in the physical and chemical properties and antioxidant capacity of beer

[0062] Add Pediococcus acidilactici to beer with physical and chemical properties (alcohol content 5.0% VOL, wort concentration 11.4°P) at an addition amount of 1×10 9 CFU / mL, and store it in the dark at 4°C, 25°C, and 37°C for a certain period of time, and detect the following indicators:

[0063] (1) pH: Measured directly by a pH meter.

[0064] (2) Ethanol concentration: Determined by high performance liquid chromatography. After centrifuging the sample, filter it through a water-based filter membrane with a pore size of 0.22 μm and take 1 mL for testing. Chromatographic conditions: Liquid phase system: Agillent 1260; Chromatographic column: Organic acid column; Differential refractive index detector; Injection volume: 10 μL; Mobile phase: 5 mmol / L dilute sulfuric acid; Column temperature: 40°C; Elution speed: 0.6 mL / min; Separation time: 20 min.

[0065] (3) Degree of beer aging (TBA): Take 5 mL of beer and place it in a 15 mL stoppered test tube. Add 2 mL of 0.33% TBA solution (using 50% acetic acid solution as the solvent, prepared freshly before use), shake well, place the test tube in a 60°C water bath for 60 min, then quickly cool the test tube, and measure its absorbance at a wavelength of 530 nm.

[0066] (4) Antioxidant capacity (DPPH): Take 200 μL of beer, add 3.8 mL of methanol solution containing 0.1 mmol / L DPPH free radicals, vortex and mix well, then let it stand in the dark for 30 min to react, measure the absorbance at 517 nm, and calculate the degradation percentage.

[0067] (5) Results: When stored under different temperature conditions, adding probiotics did not significantly change the pH and ethanol concentration of beer, and could reduce the degree of beer aging by up to 25.7% and improve the antioxidant capacity of beer by 15.2%.

[0068] Table 2 Changes in the physical and chemical properties and antioxidant capacity of beer

[0069]

[0070] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A strain of Pediococcus acidilactici ( Pediococcus acidilactici ) ZQ8, which was deposited at the China General Microbiological Culture Collection Center on December 26, 2022, with the deposit number CGMCC No. 26264.

2. A microbial preparation containing the Pediococcus acidilactici ZQ8 according to claim 1.

3. The microbial agent according to claim 2, characterized in that, The content of Pediococcus acidilactici ZQ8 in the microbial agent is ≥ 1×10 7 CFU / mL or 1×10 7 CFU / g.

4. Use of the Pediococcus acidilactici ZQ8 according to claim 1 or the microbial preparation according to any one of claims 2 to 3 in improving the antioxidant properties of beer.

5. A method for improving the antioxidant property of beer, characterized in that, The Pediococcus acidilactici ZQ8 according to claim 1 is added to beer and treated at 4° C. for at least 4 weeks.

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