Method for increasing spray drying survival rate of lactic acid bacteria by inducing intracellular protein aggregation of lactic acid bacteria through extracellular pH

Abstract

The invention discloses a method for increasing the spray drying survival rate of lactic acid bacteria by inducing intracellular protein aggregation of the lactic acid bacteria through extracellular pH, and belongs to the technical field of lactic acid bacteria preparations. According to the method for improving the spray drying survival rate of the lactic acid bacteria by inducing the aggregation of the intracellular protein of the lactic acid bacteria through the extracellular pH, disclosed by the invention, the extracellular pH is adjusted to 5.4, the protein in the bacteria is induced to form a protective aggregate, and the drying tolerance is remarkably improved. Compared with a traditional method, the survival rate is increased from 5% to 15% or above, dependence on a high-cost protective agent is not needed, and the method is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid bacteria preparations, and more specifically, to a method for improving the spray-drying survival rate of lactic acid bacteria by inducing intracellular protein aggregation of lactic acid bacteria through extracellular pH. Background Art

[0002] In current solutions for improving the spray-drying survival rate of lactic acid bacteria, enhancing heat resistance by inducing bacterial dormancy, the core technology generally relies on exogenous drug stimulation. Using sub-inhibitory concentration of erythromycin as a dormancy trigger, although the survival rate is increased to 12%-15%, since the inducer itself is included in the "List of Veterinary Drugs Prohibited in Food", the detected residual amount in the final product is 0.01 μg / g, far exceeding the food safety limit standard. By regulating the metabolic stagnation of bacteria through tetracycline gradient stimulation, although a survival rate of 14.2% is achieved at the laboratory stage, the acid-producing ability of the bacteria after induction decreases by more than 40%, and the drug residue removal process increases the production cost by 23%. Although the existing drug-induced dormancy technology can improve the spray-drying resistance of lactic acid bacteria, the dormancy is mainly induced by antibiotic drugs, which is not conducive to use in the food industry.

[0003] In the prior art, the spray drying of lactic acid bacteria mainly relies on adding protectants to improve the spray-drying survival rate, and the possibility of further improving the spray-drying survival rate by strengthening the resistance of lactic acid bacteria is not fully considered.

[0004] Therefore, it is an urgent problem for those skilled in the art to provide a method for improving the spray-drying survival rate of lactic acid bacteria by inducing intracellular protein aggregation of lactic acid bacteria through extracellular pH. Summary of the Invention

[0005] In view of this, the present invention provides a method for improving the spray-drying survival rate of lactic acid bacteria by inducing intracellular protein aggregation of lactic acid bacteria through extracellular pH. By adjusting the extracellular pH to induce intracellular protein aggregation of lactic acid bacteria, the spray-drying survival rate of lactic acid bacteria is significantly improved, which is applicable to industrial production such as probiotic powder and lactic acid bacteria starter. The present invention is a method that can enhance the spray-drying resistance of lactic acid bacteria by inducing intracellular protein aggregation of lactic acid bacteria.

[0006] To achieve the above object, the present invention uses a citric acid potassium hydrogen phosphate buffer solution to adjust the extracellular pH, causing a change in the intracellular pH of lactic acid bacteria. When the intracellular pH reaches the pH for intracellular protein aggregation, the intracellular proteins aggregate and the lactic acid bacteria enter a dormant state. The specific technical solution is as follows:

[0007] (1) Strain activation: Take out the preservation glycerol tube of Lactobacillus delbrueckii subsp. bulgaricus sp1.1 (L. bulgaricus sp1.1) from the -80°C strain preservation library, inoculate it into 1 mL of MRS medium at an inoculation amount of 2% (v / v), and culture it with shaking at 37°C for 24 h until the end of the logarithmic growth phase.

[0008] (2) Subculture: Inoculate the activated Lactobacillus bulgaricus mentioned above into 100 mL of MRS medium. After culturing for 24 h, the viable cell count reaches 10 9 CFU / mL.

[0009] (3) Centrifuge to obtain bacterial sludge: Centrifuge at 6000 r / min for 10 min at 4°C to collect the bacterial cells. After washing the bacteria twice with physiological saline, centrifuge again at 6000 r / min for 10 min at 4°C to collect the bacterial cells, and obtain 2 g of bacterial sludge.

[0010] (4) Resuspend the bacterial sludge with 0.01 M citric acid-potassium hydrogen phosphate buffer solution with a pH of 5.4 that is 10 times the mass of the bacterial sludge, and incubate at 4°C for 1 hour (to induce intracellular pH changes and protein aggregation) to obtain a bacterial suspension;

[0011] (5) Mix the protective agent: Mix the bacterial suspension and 30% w / v maltodextrin at a mass ratio of 5:1.

[0012] (6) Spray drying: The spray drying conditions are as follows: inlet air volume: 380 m 3 / h, inlet air / outlet air temperature: 120 / 70°C, atomization pressure 0.9 m 3 / h, adjust the feed rate so that the inlet and outlet air temperatures meet the preset values.

[0013] (7) After all the materials are dried, collect the bacterial powder to obtain the starter culture, and measure the viable cell count of the starter culture.

[0014] (8) After dormancy induction, the survival rate of the bacterial powder is increased to 15.11%, which is nearly 3 times higher than that of the control group.

[0015] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a method for improving the spray drying survival rate of lactic acid bacteria by inducing intracellular protein aggregation of lactic acid bacteria through extracellular pH. Without changing the protective agent (maltodextrin), intracellular protein aggregation can be induced by adjusting the extracellular pH, and the spray drying survival rate is increased from 5% of the traditional method (without adjusting the extracellular pH) to more than 15%. Description of the Drawings

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0017] Figure 1 The attached drawing is the solubility of intracellular proteins of Lactobacillus delbrueckii subsp. bulgaricus of the present invention at different pH values;

[0018] Figure 2 The attached drawing is the standard curve of the fluorescence intensity ratio of the present invention with respect to intracellular pH. Detailed implementation manners

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0020] Example 1

[0021] By adjusting the extracellular pH, its protective effect on Lactobacillus delbrueckii subsp. bulgaricus sp1.1

[0022] (L.bulgaricus sp1.1) was explored. The activated L.bulgaricus sp1.1 was inoculated into MRS medium and cultured at 37 °C for 24 hours until the end of the logarithmic growth phase. After the bacterial cells were collected by centrifugation (4 °C, 6000 rpm, 10 min), they were washed twice with physiological saline to obtain bacterial mud.

[0023] According to Figure 1 the solubility of intracellular proteins of Lactobacillus delbrueckii subsp. bulgaricus sp1.1 at different pH values, when the intracellular pH is 6.0, the protein solubility is the lowest and aggregation occurs; further according to Table 1 showing the correspondence between intracellular and extracellular pH of Lactobacillus delbrueckii subsp. bulgaricus, when the extracellular pH is 5.4, the intracellular pH is 6.0. Therefore, the bacterial mud was resuspended with a citric acid-potassium hydrogen phosphate buffer solution of pH 5.4 with a mass 10 times that of the bacterial mud and incubated at 4 °C for 1 hour to obtain a bacterial suspension. Subsequently, the bacterial suspension was mixed with 30% (w / v) maltodextrin at a mass ratio of 5:1 and spray-dried (inlet air volume 380 m 3 / h, inlet air temperature 120 °C, outlet air temperature 70 °C, atomization pressure 0.9 m 3 / h).

[0024] Table 1 Corresponding Table of Intracellular and Extracellular pH of Lactobacillus delbrueckii subsp. bulgaricus

[0025] Intracellular pH Extracellular pH 5.0 4.15 6.0 5.41 6.5 6.13 7.5 7.97

[0026] Method for measuring intracellular pH: Use BCECF-AM fluorescent dye to measure the intracellular pH of L. bulgaricus sp1.1. Add BCECF-AM dye solution to make its final concentration reach 1.25 μM, stain for 0.5 h, wash the bacteria twice with citric acid-dipotassium hydrogen phosphate buffer solution with corresponding pH, and then use a flow cytometer equipped with 488 nm and 405 nm excitation lights to measure cell fluorescence. Obtain the signals in 30,000 cells, use Flow Jo software to calculate the average fluorescence intensity of each group, and calculate the intracellular pH according to the standard curve of the fluorescence intensity after excitation at 488 nm and 405 nm and the intracellular pH ( Figure 2 ).

[0027] The survival rate results of the dried bacterial powder are shown in Table 2.

[0028] Table 2 Spray-drying Survival Rate of Lactic Acid Bacteria under Different Extracellular pH Treatments

[0029]

[0030] As can be seen from Table 2, the survival rate of the group treated with citric acid-dipotassium hydrogen phosphate buffer solution is nearly 3 times higher than that of the control group, indicating that the formation of intracellular protein aggregates significantly enhances the drying tolerance of the bacteria.

[0031] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for improving the spray-drying survival rate of lactic acid bacteria by inducing intracellular protein aggregation in lactic acid bacteria through extracellular pH, characterized in that, The specific steps are as follows: (1) Cultivate lactic acid bacteria in a culture medium until the late logarithmic phase and the early stationary phase; (2) Centrifuge to collect the bacterial cells, and rinse the bacterial cells twice with physiological saline or 0.01 M phosphate buffer solution; centrifuge again to collect the bacterial cells and obtain bacterial sludge; (3) Resuspend the bacterial sludge with 0.01 M citric acid-disodium hydrogen phosphate buffer solution with a pH of 5.4 and a mass 10 times that of the bacterial sludge, and incubate at 4°C for 1 hour to obtain a bacterial suspension; (4) Mix the bacterial suspension with 30% w / v maltodextrin at a mass ratio of 5:1, and spray-dry to make bacterial powder.

2. A method for improving the spray-drying survival rate of lactic acid bacteria by inducing intracellular protein aggregation of lactic acid bacteria through extracellular pH according to claim 1, characterized in that The centrifugation in step (2) is carried out at 4°C and 6000 r / min for 10 min.

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