Method for improving anti-drying capability of probiotics and application thereof

Abstract

The invention belongs to the technical field of probiotics, and particularly discloses a method for improving the anti-drying capacity of probiotics and application of the method. The preparation method comprises the following steps: S1, inoculating probiotics into a culture medium, adding a fatty acid precursor for culturing, and centrifugally collecting thalli; and S2, washing the thalli collected in the step S1, resuspending the thalli in a protective agent solution, performing spray drying, and performing magnetic stirring in the drying process to obtain dry bacterial powder. The invention discloses a method for improving the anti-drying capacity of probiotics and application of the method, fatty acid precursors are exogenously supplemented in the culture process, the cell membrane lipid composition of the probiotics can be effectively regulated and controlled, the anti-spray-drying capacity of the probiotics is remarkably enhanced, and the key problem that the survival rate of the probiotics is low in the traditional spray drying process is solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of probiotics, and particularly relates to a method for improving the anti-drying capacity of probiotics and application thereof. BACKGROUND

[0002] As a widely used bacteria powder preparation process in the industrialized production of probiotics, spray drying has the advantages of simple operation and large-scale production, but the high-temperature dehydration process can cause multiple damages to the cells of lactic acid bacteria, including cell membrane structure damage, protein denaturation and metabolic function disorder, etc., which leads to a significant decrease in the survival rate of bacteria, and seriously restricts the industrial application of high-activity probiotic preparations.

[0003] At present, the technical means for improving the anti-spray drying capacity of lactic acid bacteria mainly focus on three aspects: first, adding sugar, protein and other protective agents to reduce cell damage during the drying process, but this method requires precise control of the protective agent ratio, and has the problems of complex process, high cost and limited protection effect; second, optimizing the culture conditions such as temperature and pH value, but single condition optimization has a small improvement in the stress resistance of bacteria, which is difficult to meet the needs of industrial production; third, obtaining stress-resistant strains through strain breeding, but the cycle is long, the screening is difficult, and other excellent properties may be degraded. However, there is no related research on the specific improvement of the anti-spray drying capacity of probiotics such as Bifidobacterium lactis by supplementing key fatty acid precursors and directional regulation of membrane lipid composition in the prior art. SUMMARY

[0004] The application aims to provide a method for improving the anti-drying capacity of probiotics and application thereof, which can significantly improve the spray drying capacity of probiotics by regulating the membrane lipid composition of probiotics through exogenous supplementation of key fatty acid precursors, and has important practical application value for realizing the large-scale production of high-activity probiotic powder.

[0005] To solve the above technical problems, the technical scheme adopted by the application is as follows:

[0006] A method for improving the anti-drying capacity of probiotics, comprising the following steps:

[0007] S1, inoculating probiotics into a culture medium, adding a fatty acid precursor for culture, and centrifuging to collect bacteria;

[0008] S2, washing the bacteria collected in S1, resuspending in a protective agent solution, and performing spray drying, and performing magnetic stirring during the drying process to obtain dried bacteria powder.

[0009] Preferably, in S1, the fatty acid precursor is selected from one or more of C16:0, C18:0 and C20:0.

[0010] Preferably, in S1, the concentration of the added fatty acid precursor is 0.25-0.45 M, the added amount is 10-40 μL, and the fatty acid precursor is added into the culture medium in the 0-8 h stage of the culture.

[0011] Preferably, in S1, the culture conditions are as follows: 37℃ for 20 h in an anaerobic environment; and the centrifugation conditions are as follows: 6000 rpm for 15 min at 4℃.

[0012] Preferably, in S2, the bacterial precipitate is washed with sterile NaCl solution for 3 times, and the protective agent solution comprises maltodextrin and isolated whey protein.

[0013] Preferably, in S2, the bacterial precipitate is washed with 0.85% (w / v) sterile NaCl solution for 3 times, and the protective agent comprises 20% (w / v) maltodextrin and 10% (w / v) isolated whey protein.

[0014] Preferably, in S2, the spray drying feed rate is 5 mL / min, the spray drying inlet air temperature is 99-121℃, the outlet air temperature is 59-81℃, and the magnetic stirring speed is 600 rpm.

[0015] The application also provides an anti-high-temperature-drying probiotic prepared by the method.

[0016] The application also provides a use of the probiotic in the preparation of a probiotic preparation, food or health product.

[0017] Compared with the prior art, the application has the following advantages and technical effects:

[0018] The application discloses a method for improving the anti-drying capability of probiotics and application thereof.

[0019] Compared with the prior art, the application has the following advantages and technical effects:

[0020] The technical solutions of the application will be further described in detail below with reference to the drawings and examples. DESCRIPTION OF DRAWINGS

[0021] Figure 1 Survival rate of B. lactis F1-7 in Example 1-3 and Comparative Example 1;

[0022] Figure 2 Lactate dehydrogenase activity of B. lactis F1-7 in Example 1-3 and Comparative Example 1;

[0023] Figure 3 Na + -K + -ATPase activity of B. lactis F1-7 in Example 1-3 and Comparative Example 1;

[0024] Figure 4 Pathway of regulating the synthesis of phosphatidylinositol (PI) in the membrane of F1-7 after exogenous supplement of C18:0 in Example 1-3;

[0025] Figure 5 Pathway of regulating the synthesis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in the membrane of F1-7 after exogenous supplement of C18:0 in Example 1-3;

[0026] Figure 6 Pathway of regulating the synthesis of phosphatidylglycerol (PG) and cardiolipin (CL) in the membrane of F1-7 after exogenous supplement of C18:0 in Example 1-3;

[0027] Figure 7 Statistical diagram of the length of the acyl chain of the membrane phospholipid after exogenous supplement of C18:0 in Example 1-3, wherein, Figure 7 A in the above formula is the total carbon number of the acyl chain of PG, Figure 7 B in the above formula is the total carbon number of the acyl chain of PE,

[0028] Figure 8 Statistical diagram of the total carbon number of the fatty acyl chain of the membrane as a whole after exogenous supplement of C18:0 in Example 1-3;

[0029] Figure 9 Cluster heat map of the differential lipid molecules containing FA 18:1 after exogenous supplement of C18:0 in Example 1-3;

[0030] Figure 10 Statistical diagram of the relative content of the top 10 differential lipid molecules containing FA 18:1, wherein, Figure 10 A in the above formula is the statistical diagram of the relative content of PE (14:0_18:1), Figure 10 B in the above formula is the statistical diagram of the relative content of PG (23:3_18:1), Figure 10 C in the above formula is the statistical diagram of the relative content of PE (P-18:1_16:1), Figure 10 D in the above formula is the statistical diagram of the relative content of PE (16:1_18:1),Figure 10 E is the relative content statistics chart of DG(18:1_20:2) in F1-7, Figure 10 F is the relative content statistics chart of DG(16:0_18:1) in F1-7, Figure 10 G is the relative content statistics chart of PE(P-18:1_16:0) in F1-7, Figure 10 H is the relative content statistics chart of PE(P-18:0_18:1) in F1-7, Figure 10 I is the relative content statistics chart of PE(P-16:0_18:1) in F1-7, Figure 10 J is the relative content statistics chart of DG(18:0_18:1) in F1-7;

[0031] Figure 11 is the total content statistics chart of F1-7 bacterial membrane lipid molecules containing FA 18:1 after exogenous C18:0 supplement in Examples 1-3;

[0032] Figure 12 is the U- 13 C18:0 stable isotope tracer method verifies that exogenous C18:0 participates in the endogenous lipid biosynthesis pathway, and detects the labeled 13 C18:0 relative abundance, wherein, Figure 12 A is the relative abundance of the detected labeled 13 C18:0, Figure 12 B is the U- 13 C18:0 stable isotope tracer method verifies that exogenous C18:0 participates in the endogenous lipid biosynthesis pathway;

[0033] Figure 13 is the relative abundance of the detected labeled 13 C18:1,

[0034] Figure 14 is the relative abundance of the detected labeled 13 C18:2, 13 C18:3 and other synthesized long-chain fatty acyls;

[0035] Figure 15 is a schematic diagram of exogenous fatty acids participating in the endogenous bacterial type II fatty acid synthesis and elongation pathway. DETAILED DESCRIPTION

[0036] The technical solutions of the present application are further illustrated by the accompanying drawings and examples.

[0037] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs.

[0038] Test material sources:

[0039] Bifidobacterium lactis F1-7 used in the embodiments of the present application is isolated from the intestinal specimens of healthy infants and preserved in China Center for Type Culture Collection (CCTCC M 2020833), stearic acid (C18:0, purity ≥98.5%) is purchased from Sigma-Aldrich Shanghai Trading Co., Ltd., and BS culture medium is purchased from Qingdao Haibo Co., Ltd., 13 C-stearic acid (C18:0, purity ≥98.5%) 13 C 18 H 36 O2, purity ≥98%) is purchased from MedChemExpress Co., Ltd.

[0040] The protective agent solution comprises 20% (w / v) of malt dextrin and 10% (w / v) of isolated whey protein, and the remaining components are F1-7 bacterial slurry obtained after centrifugation of a solution of 200 mL of BS culture bacteria.

[0041] In the present application, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0042] Example 1

[0043] A method for improving the anti-drying capacity of Bifidobacterium lactis F1-7, comprising the following steps:

[0044] S1, inoculate Bifidobacterium lactis F1-7 bacterial solution into 200 mL of sterilized BS culture medium at an inoculation amount of 2%, add 10 μL of 0.395 M C18:0, and add the culture medium at the 0h stage of culture, cultivate in an anaerobic environment at 37°C for 20h, and after the end of culture, centrifuge at 6000 rpm at 4°C for 15 min to collect the bacterial bodies;

[0045] S2, wash the bacterial body precipitate with 0.85% (w / v) of sterile NaCl solution for 3 times, resuspend in the protective agent solution, continuously pump into the spray drying tower for spray drying of the bacterial suspension, set the inlet temperature to 120±1°C, the outlet temperature to 60±1°C, and the feeding speed to 5 mL / min, and continuously homogenize by magnetic stirring at 600 rpm during the drying process to obtain dried bacterial powder.

[0046] Example 2

[0047] A method for improving the anti-drying capacity of Bifidobacterium lactis F1-7, comprising the following steps:

[0048] S1, inoculate Bifidobacterium lactis F1-7 bacterial solution into 200 mL sterilized BS medium at an inoculation amount of 2%, add 20 μL of 0.395 M C18:0, and add the medium at the 0 h stage of culture, cultivate in an anaerobic environment at 37°C for 20 h, and after the end of culture, centrifuge at 6000 rpm for 15 min at 4°C to collect the bacterial bodies;

[0049] S2, wash the bacterial body precipitate 3 times with 0.85% (w / v) sterile NaCl solution, resuspend in a protective agent solution, continuously pump the bacterial suspension into a spray drying tower for spray drying, set the inlet temperature to 120±1°C, the outlet temperature to 60±1°C, and the feeding speed to 5 mL / min, and during the drying process, continuously homogenize by magnetic stirring at 600 rpm to obtain dried bacterial powder.

[0050] Example 3

[0051] A method for improving the anti-drying capacity of Bifidobacterium lactis F1-7, comprising the following steps:

[0052] S1, inoculate Bifidobacterium lactis F1-7 bacterial solution into 200 mL sterilized BS medium at an inoculation amount of 2%, add 20 μL of 0.395 M C18:0, and add the medium at the 0 h stage of culture, cultivate in an anaerobic environment at 37°C for 20 h, and after the end of culture, centrifuge at 6000 rpm for 15 min at 4°C to collect the bacterial bodies;

[0053] S2, wash the bacterial body precipitate 3 times with 0.85% (w / v) sterile NaCl solution, resuspend in a protective agent solution, continuously pump the bacterial suspension into a spray drying tower for spray drying, set the inlet temperature to 120±1°C, the outlet temperature to 60±1°C, and the feeding speed to 5 mL / min, and during the drying process, continuously homogenize by magnetic stirring at 600 rpm to obtain dried bacterial powder.

[0054] Comparative Example 1

[0055] The specific test scheme is the same as that of Example 1, except that C18:0 is not added.

[0056] The dried bacterial powder provided in Examples 1-3 above is verified for effect through the following test.

[0057] 1. Plate counting method is used to count live cells

[0058] Take 100 mg of spray-dried bacterial powder, reconstitute with 1 mL of NaCl solution and gradient dilute. The survival rate of bacteria after spray drying is calculated according to the following formula:

[0059]

[0060] Results are shown in Figure 1 .

[0061] As shown in Figure 1 , the survival rate of F1-7 strain was significantly improved by adding C18:0, 10, 20 and 40 μL of C18:0 in BS medium, and the survival rate was increased from 13.38% ± 5.58% to 18.80% ± 2.11%, 26.07% ± 3.55% and 33.36% ± 5.63%, respectively. In addition, the survival rate of F1-7 strain was not significantly improved by adding 20 and 40 μL of C18:0, indicating that the exogenous addition amount may have reached the upper limit. In summary, the maximum increase in survival rate of F1-7 was 2.4-fold, and C18:0 changed the composition of the bacterial membrane by participating in the synthesis of membrane phospholipids.

[0062] 2. Lactate dehydrogenase (LDH) activity assay

[0063] The spray-dried B. lactis F1-7 and F1-7-C18:0 powder prepared in Example 1 (0.016 g) were dissolved in 1 mL of ultrapure water, and centrifuged at 10,000 g for 10 min. An equal amount of supernatant was taken, and the LDH activity leaked from the cell membrane was determined using an LDH detection kit (Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd., China) according to the kit instructions.

[0064] The absorbance of the samples was repeatedly measured at 440 nm using a full-automatic enzyme marker (Thermofisher Scientific, Multiskan FC, MA, USA), and the measurement was repeated three times. Each enzyme unit was defined as: 1 μmol of pyruvic acid was generated in the reaction system per gram of tissue protein and substrate at 37°C for 15 min, and the results are shown in Figure 2 .

[0065] As shown in Figure 2 , the extracellular LDH enzyme activity of F1-7 strain treated with 20 and 40 μL of C18:0 was significantly reduced, while there was no significant change in enzyme activity when treated with 10 μL dose. In addition, F1-7 added with 40 μL of C18:0 showed the least LDH leakage, and the measured extracellular LDH activity was the lowest at 48.89 U / L, indicating that the addition of 40 μL of C18:0 played an important role in maintaining the integrity of the bacterial membrane after drying.

[0066] 3. Ultra-trace Na + -K + -ATPase activity assay

[0067] Ultra-trace Na + -K +- ATPase kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., China) to determine Na + -K + -ATPase activity according to the kit instructions by measuring the release of inorganic phosphate (Pi) ATP. Methods: 0.01 g of F1-7 and F1-7-C18:0 powder was re-dissolved in 1 mL of ultrapure water, centrifuged at 10,000 g for 10 min, and the supernatant was taken. The absorbance of ATPase at 636 nm was measured using a full-automatic microplate reader (Thermofisher Scientific, Multiskan FC, MA, USA) in triplicate. The ATPase activity unit was determined as the amount of ATPase that decomposes 1 pmol of Pi in the tissue per hour per mg of tissue protein, in units of micromoles of phosphate per milligram of protein per hour (pmol Pi / mg prot / hour), and the results are shown in Figure 3

[0068] As can be seen from Figure 3 , only after the addition of 40 pL of C18:0, the extracellular Na + -K + -ATPase activity of F1-7 was significantly reduced, and there was no significant difference in enzyme activity after the addition of 10 pL and 20 pL of C18:0 compared with the control group, indicating that the addition of 40 pL of C18:0 had the most significant effect on the improvement of F1-7 membrane integrity. The addition of 40 pL of C18:0 was selected for subsequent membrane lipid determination.

[0069] 4. Remodeling of membrane lipid composition by exogenous supplementation of key fatty acid precursor C18:0

[0070] (1) Membrane lipid extraction

[0071] For strains that were not spray-dried, the slurry was collected and freeze-dried, and 20 mg of powder was weighed; for spray-dried bacterial powder, 20 mg of powder was also weighed. Then, 190 pL of a solution of butanol-methanol and 10 mM ammonium formate (prepared at a ratio of 1 : 1) and 10 pL of an internal standard were mixed, respectively. The mixture was homogenized for 5 min, vortexed for 30 s, then ultrasonically treated at room temperature for 1 h, and finally centrifuged at 13,000 g for 10 min to obtain the supernatant.

[0072] (2) UHPLC-MS / MS analysis of membrane composition

[0073] ​Lipids were separated using a Nexera X2 LC-30AD ultra-high pressure liquid chromatography system (SHIMADZU, Japan) equipped with an Acquity UPLC BEH HILIC column (1.7 pm, 2.1 x 100 mm; Waters, USA). The mobile phase composition was as follows: Solvent A: 50% acetonitrile (containing 10 mM ammonium acetate, pH 8.0); Solvent B: 100% acetonitrile. The gradient elution program was as follows: 0-0.1 min: 90% B; 0.1-5 min: B linearly decreased from 90% to 65%; 5-5.3 min: B linearly decreased from 65% to 0%; 5.3-7.2 min: 0% B; 7.2-7.4 min: B linearly increased from 0% to 90%; 7.4-10 min: 90% B; Chromatographic conditions: column temperature 35 °C, flow rate 300 pL / min, 6 pL injection volume per needle.

[0074] Mass spectrometry analysis was performed using a Triple Quad 6500+ mass spectrometer (AB SCIEX) in positive and negative ion modes, respectively. Ion source parameters were set as follows: Ion spray voltage: +5500 V (positive ion mode) / -4500 V (negative ion mode); Ion source temperature: 400 °C; Ion source gas 1: 55 psi; Ion source gas 2: 60 psi; Curtain gas: 35 psi; Collision gas: 8 psi; Potential and collision energy parameters were optimized for different lipid classes to ensure detection sensitivity.

[0075] Chromatographic peak areas were extracted and integrated using Multiquant software. Lipid identification and quantitative analysis were performed by internal standard method by comparing the retention time of lipid standards. The formula for calculating the concentration of lipids was as follows:

[0076]

[0077] Where: C: lipid concentration; R: sample to internal standard peak area ratio; MIS: amount of internal standard added; V: sample volume. All statistical analysis and bioinformatics processing were completed using R programming language. Lipid nomenclature followed the LIPID MAPS rules (https: / / doi.org / 10.1194 / jlr.S120001025).

[0078] Exogenous fatty acids are integrated into the phospholipid synthesis pathway of Gram-positive bacteria through the FASII system. This process begins with the FakA / B-mediated phosphorylation of fatty acids to generate acyl-phosphates, which are then converted to acyl-ACPs by PlsX. This key intermediate is then recycled by the FASII system to regenerate acyl-phosphates, or is condensed with 3-phosphoglycerol to generate phosphatidic acid (PA) catalyzed by PlsC, and the generated CDP-DAG is then converted to the corresponding phospholipids through three different pathways.

[0079] The first pathway leads to the synthesis of phosphatidylinositol (PI), which is catalyzed by Figure 4 It can be seen that the PI content is significantly reduced after exogenous supplementation of C18:0. The second pathway leads to the synthesis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), as shown in Figure 5 The PE content is significantly increased from 4.20 nmol / mg to 16.89 nmol / mg by adding C18:0, but the PC content is not affected. The third pathway leads to the synthesis of phosphatidylglycerol (PG) and cardiolipin (CL), in which the PG content is increased by 5.46 nmol / mg and the CL is reduced by 3.99 nmol / mg, which is closely related to the metabolic process of PG forming CL catalyzed by cardiolipin synthase (as shown in Figure 6 PG is the main component of bacterial membranes, generally accounting for about 25% of membrane lipids, but in S. aureus it can reach 80%, and PG also plays a key role in maintaining membrane stability by inhibiting lipid desorption and reducing membrane permeability.

[0080] Membrane fluidity is regulated by a variety of interdependent factors, among which the packing of acyl chains plays a key role. The melting point of long-chain fatty acids is higher than that of short-chain fatty acids, because the extension of the carbon-hydrogen chain enables it to form a bilayer membrane and enhance the van der Waals interaction between chains, thereby promoting the packing of acyl chains. In the present invention, the long-chain fatty acyl content of F1-7 membrane lipids is significantly increased by adding C18:0 to Lactobacillus lactis F1-7, especially the content of 32 and 34 carbon acyl chains (A in Figure 7 ) in PG and 34 carbon acyl chains (B in Figure 7 ) in PE.

[0081] In addition, Figure 8 The results of total fatty acyl groups of membrane lipids show that the content of long acyl chains of eicosanoic acid is significantly increased in the F1-7 strain with added C18:0, indicating that longer acyl chains make the acyl chains in the bacterial membrane more closely arranged, and the increase in membrane rigidity leads to an improvement in its ability to cope with heat drying.

[0082] The F1-7 strain with added C18:0 provided in Example 1 was subjected to cluster analysis, and the results are shown in Figure 9 .

[0083] It can be seen from Figure 9 that the cluster heat map shows that the abundance of 46 kinds of F1-7-C18:0 is significantly higher than that of phospholipid molecules containing FA 18:1 in F1-7.

[0084] According to the P<0.05 and FC value ranking, the top 10 lipid molecules were identified, and the results are shown in Figure 10 .

[0085] It can be seen from Figure 10It can be seen that the top 10 lipid molecules include PE (14:0_18:1), PG (23:3_18:1), PE (P-18:1_16:1), PE (16:1_18:1), DG (18:1_20:2), DG (16:0_18:1), PE (P-18:1_16:0), PE (P-18:0_18:1), PE (P-16:0_18:1), and DG (18:0_18:1), of which 6 are PE molecules.

[0086] The total content of FA 18:1 in the lipid molecules of F1-7 bacterial membranes after exogenous supplementation with C18:0 was determined, and the results are as follows: Figure 11 As shown.

[0087] Depend on Figure 11 It can be seen that after the addition of C18:0, the total lipid molecule content containing FA 18:1 increased to 1.83 mmol / mg, indicating that exogenous fatty acid C18:0 participates in the synthesis of endogenous phospholipids in the bacterial film, regulates the composition of bacterial film phospholipids, thereby increasing the lipid content containing FA 18:1 in the phosphatidyl chain, and improving the strain's tolerance to spray drying by increasing lipid accumulation.

[0088] 5.[U- 13 C] Stearic acid isotope labeling tracer

[0089] 20 μL 80 mM [U- 13 C] Stearic acid (purity ≥98%, Sigma-Aldrich) was mixed with 200 mL of medium containing 2% (v / v) BS, and inoculated with F1-7 strain. The mixture was then anaerobically cultured until the stationary phase. After culture, the bacterial pellet was collected by centrifugation at 6000 rpm for 15 minutes and spray-dried to obtain bacterial powder. Approximately 20 mg of F1-7-[U- 13 [C] Stearic acid bacteria powder was mixed with pre-cooled methanol, vortexed, and then allowed to stand at -20°C for 30 min. Subsequently, fractional centrifugation was performed: first centrifuged at 1500 rpm for 30 min at 4°C, then centrifuged again at 12,000 rpm for 10 min at the same temperature. The supernatant was collected and dried using a SpeedVac vacuum dryer. The resulting extract was redissolved in methanol and analyzed by LC-MS.

[0090] The analysis was performed on an Agilent 1290 II UPLC system coupled with Sciex 5600+ quadrupole-time-of-flight mass spectrometer. Waters ACQUITY HSS-T3 column (3.0 x 100 mm, 1.8 μm) was used for chromatographic separation. The mass spectrometry detection parameters were set as follows: negative ion mode ESI source voltage -4.5 kV, ion source temperature 500 °C, dry gas (N2) and nebulizing gas (N2) pressure both 50 psi, curtain gas (N2) pressure 35 psi. MS / MS analysis was performed in information-dependent acquisition (IDA) mode, scan range 60-800 m / z, collision energy -35 ± 15 eV. Data acquisition and processing were performed using TF1.7.1 software, and all detected ions were extracted by MarkerView 1.3 software and exported to Excel in a two-dimensional matrix format (including m / s, retention time and peak area). MS / MS data extraction and metabolite annotation were performed using PeakView 2.2 software, combined with AB Sciex metabolite database, HMDB database and standard references for comparison. For relative quantitative analysis, all peak areas were normalized using 19:0-FA (free fatty acid) as an internal standard.

[0091] UHPLC-MS / MS analysis was performed on an Agilent 1290 II UPLC system coupled with Sciex 5600+ quadrupole-time-of-flight mass spectrometer. Waters ACQUITY HSS-T3 column (3.0 x 100 mm, 1.8 μm) was used for chromatographic separation. The mass spectrometry detection parameters were set as follows: negative ion mode ESI source voltage -4.5 kV, ion source temperature 500 °C, dry gas (N2) and nebulizing gas (N2) pressure both 50 psi, curtain gas (N2) pressure 35 psi. MS / MS analysis was performed in information-dependent acquisition (IDA) mode, scan range 60-800 m / z, collision energy -35 ± 15 eV. Data acquisition and processing were performed using 13 C18:0 isotopic tracing technology was used to explore the mechanism of exogenous C18:0 participating in the synthesis of endogenous membrane lipids in F1-7 strain, and the results are shown in Figures 12-15

[0092] As can be seen from Figure 12 , the content of [M+18] FA 18:0 in the phosphatidyl group significantly increased, confirming that exogenous C18:0 directly participated in the synthesis of bacterial membrane lipids.

[0093] As can be seen from Figure 13 , [M+4] FA 18:1 was detected, which was confirmed by UHPLC-MS / MS analysis results, indicating that C18:0 promoted the synthesis of FA 18:1 acyl chain.

[0094] As can be seen from Figure 14 , in addition to [M+4] FA 18:1, [M+4] FA 18:2, [M+4] FA 18:3 and a series of long-chain fatty acids ([M+20] FA 20:0 to [M+26] FA 26:0) were also detected.

[0095] As can be seen from Figure 15 , all detected 13 ​The C-labeled fatty acids all showed the increment of two carbon atoms, which was consistent with the mechanism of FabF-mediated malonyl-ACP condensation reaction in FASII pathway. The above results fully demonstrated that the exogenous C18:0 not only participated in the membrane lipid remodeling, but also promoted the synthesis of phospholipids containing FA 18:1.

[0096] Finally, it should be noted that the above examples are intended to illustrate the technical solutions of the present application rather than limit them. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or equivalently replaced, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method of improving the dry robustness of probiotic bacteria, characterized in that, The method comprises the following steps: S1, inoculating probiotics into culture medium, adding fatty acid precursors for culture, and collecting bacterial bodies by centrifugation; S2, washing the bacterial bodies collected in S1, resuspending in a protective agent solution, and performing spray drying, and performing magnetic stirring during the drying process to obtain dried bacterial powder.

2. The method of claim 1, wherein, In S1, the fatty acid precursors are selected from one or more of C16:0, C18:0 and C20:

0.

3. The method of claim 1, wherein, In S1, the addition concentration of the fatty acid precursors is 0.25-0.45 M, the addition amount is 10-40 μL, and the fatty acid precursors are added into the culture medium at a culture stage of 0-8 h.

4. The method of claim 1, wherein, In S1, the culture conditions are as follows: culturing at 37℃ for 20 h in an anaerobic environment; and the centrifugation process conditions are as follows: centrifuging at 6000 rpm for 15 min at 4℃.

5. The method of claim 1, wherein, In S2, the bacterial body precipitate is washed with sterile NaCl solution for 3 times, and the protective agent solution comprises maltodextrin and separated whey protein.

6. The method of claim 1, wherein, In S2, the spray drying feeding rate is 5 mL / min, the spray drying inlet air temperature is 99-121℃, the outlet air temperature is 59-81℃, and the magnetic stirring speed is 600 rpm.

7. The high-temperature resistant dried probiotics prepared by the method according to any one of claims 1-6.

8. The probiotics according to claim 7 for use in the preparation of probiotic preparations, food or health products.

Images