Prebiotic functional wall material prepared based on Maillard reaction and application of prebiotic functional wall material in probiotic microcapsule for producing bile salt hydrolase

Abstract

The invention provides a preparation method of a prebiotic functional wall material prepared based on a Maillard reaction. The preparation method comprises the following steps: dissolving sodium caseinate and prebiotic oligosaccharide in water, carrying out a hydration reaction, adjusting the pH value, carrying out a Maillard reaction, and cooling in an ice bath to obtain a microcapsule wall material solution. The prepared microcapsule wall material is low in cost, simple in preparation method, high in grafting rate, strong in hydrophobicity and high in probiotic activity, and is beneficial to coating the lactic acid bacteria, improving the activity of the lactic acid bacteria and forming the microcapsule with a compact and stable structure; the lactic acid bacteria microcapsule prepared from the microcapsule wall material is compact and stable in structure, can effectively protect lactic acid bacteria, prevents the lactic acid bacteria from being in contact with external adverse conditions, improves the activity of the lactic acid bacteria in gastrointestinal fluid, heat, ions, pH and storage, and has relatively high embedding rate and load rate; the activity of the bile salt hydrolase in the gastrointestinal digestion process of the prepared lactic acid bacteria microcapsule for producing the bile salt hydrolase can be improved, and the reduction of the activity of the bile salt hydrolase is delayed.

Description

Technical Field

[0001] The invention relates to the technical field of microcapsules, and in particular to a prebiotic functional wall material prepared based on the Maillard reaction and application thereof in bile salt hydrolase-producing probiotic microcapsules. Background Art

[0002] Hypercholesterolemia caused by a high-fat diet is affecting people's health and is considered to be one of the main pathogenic factors for the onset of cardiovascular disease. Simvastatin, niacin and fibrate drugs are currently believed to treat hypercholesterolemia by regulating cholesterol metabolism, but these drugs may cause serious adverse reactions. For example, simvastatin is known to cause gastrointestinal discomfort and may cause damage to the nervous system and liver. Bile salt hydrolase-producing lactic acid bacteria have been shown to improve cholesterol metabolism through the bile salt hydrolase they produce. Lactic acid bacteria are easily affected by gastrointestinal fluid, which reduces their efficacy. Microencapsulation technology has been used to improve this problem.

[0003] Microencapsulation technology is an effective way to protect the activity of lactic acid bacteria. The microcapsule wall material entraps the lactic acid bacteria in the middle, forming a physical barrier with the outside world, thereby controlling the release of lactic acid bacteria and improving the biological activity of lactic acid bacteria. Protein and polysaccharide-based wall materials are widely used to encapsulate lactic acid bacteria. However, single protein and polysaccharide or a simple mixture of protein and polysaccharide still has the problem of insufficient stability. The prebiotic properties of the wall material also play a vital role in the efficacy of lactic acid bacteria. In the past, prebiotic wall materials were mainly based on some wall materials with prebiotic functional polysaccharides, but lacked nitrogen sources such as protein, resulting in its prebiotic efficacy being not obvious. The Maillard reaction is usually used to improve the functional properties of proteins. It is an effective way to improve the stability and prebiotic properties of protein and prebiotic oligosaccharide-based wall materials.

[0004] At present, the research on Maillard reaction products as wall materials at home and abroad is mostly focused on single protein polysaccharides or monosaccharides, and the proteins used are mainly whey protein and soy protein, while sodium caseinate has excellent emulsification properties and nutritional value compared with these proteins. The existing Maillard reaction wall materials and microcapsules made from them have the problems of insufficient encapsulation rate and weak protection of lactic acid bacteria under various stress conditions. Summary of the invention

[0005]

Technical issues

[0006] The existing Maillard reaction wall materials and microcapsules made therefrom have the problems of insufficient encapsulation rate and insufficient protection of lactic acid bacteria under various stress conditions.

[0007]

Technical solution

[0008] The present invention uses the Maillard reaction product of sodium caseinate and prebiotic oligosaccharide as the microcapsule wall material. Compared with some common proteins, such as casein, whey protein isolate and soy protein isolate, sodium caseinate has the advantages of good solubility, excellent emulsification performance and strong thermal stability. Encapsulating bile salt hydrolase-producing lactic acid bacteria with the prepared Maillard reaction product wall material improves the encapsulation rate of lactic acid bacteria, cell viability under various stress conditions and enzyme activity of bile salt hydrolase. The encapsulation rate of the sodium caseinate Maillard reaction conjugate reaches more than 90%.

[0009] The first object of the present invention is to provide a method for preparing a microcapsule wall material having a prebiotic function, comprising the steps of:

[0010] (1) dissolving sodium caseinate and prebiotic oligosaccharides in water, stirring and hydrating;

[0011] (2) adjusting the pH of the solution obtained in step (1), performing a Maillard reaction, and cooling the solution in an ice bath to obtain a Maillard conjugate solution;

[0012] (3) Freezing the Maillard conjugate solution and then freeze-drying it to obtain a microcapsule wall material with prebiotic function.

[0013] In one embodiment of the present invention, in step (1), the prebiotic oligosaccharide is one or more of xylooligosaccharide, fructooligosaccharide and inulin.

[0014] In one embodiment of the present invention, in step (1), the concentration of sodium caseinate dissolved in water is 5 to 15 wt %, and the concentration of prebiotic oligosaccharides dissolved in water is 5 to 15 wt %.

[0015] In one embodiment of the present invention, in step (1), the mass ratio of sodium caseinate to prebiotic oligosaccharides is 1:3 to 3:1.

[0016] In one embodiment of the present invention, in step (1), the stirring temperature is 15 to 35° C. and the stirring time is 2 to 8 hours.

[0017] In one embodiment of the present invention, in step (1), the hydration temperature is 0 to 8° C. and the hydration time is 8 to 14 hours.

[0018] In one embodiment of the present invention, in step (2), the pH is adjusted to 8.0-10.0.

[0019] In one embodiment of the present invention, in step (2), the pH is adjusted with a solution of one or more of NaOH, HCl, and citric acid, with a concentration of 0.1 to 5 M.

[0020] In one embodiment of the present invention, in step (2), the temperature of the Maillard reaction is 80-90° C., and the reaction time is 2-5 h.

[0021] In one embodiment of the present invention, in step (2), the temperature is cooled in an ice bath to 15-35°C.

[0022] In one embodiment of the present invention, in step (3), the freezing temperature is -80 to -20°C, and the freezing time is 24 to 48 hours.

[0023] In one embodiment of the present invention, in step (3), the freeze-drying temperature is -40 to -60°C, the pressure is 10 to 50 Pa, and the time is 72 to 96 hours.

[0024] The second object of the present invention is to provide a microcapsule wall material prepared by the above preparation method.

[0025] The third object of the present invention is to provide the use of the above microcapsule wall material in probiotic microcapsules.

[0026] A fourth object of the present invention is to provide a method for preparing lactic acid bacteria microcapsules using the above-mentioned microcapsule wall material, comprising the steps of:

[0027] (1) dissolving the microcapsule wall material in water to prepare a solution, mixing the solution with the lactic acid bacteria suspension, adding gluconolactone and stirring to dissolve, thereby obtaining an aqueous phase;

[0028] (2) The oil phase and the water phase are mixed, stirred, and filtered to obtain a precipitate, and the precipitate is washed to obtain lactic acid bacteria microcapsules.

[0029] In one embodiment of the present invention, in step (1), the concentration of the microcapsule wall material in the solution of the microcapsule wall material is 8 to 12 wt %.

[0030] In one embodiment of the present invention, in step (1), the lactic acid bacteria are bile salt hydrolase-producing lactic acid bacteria.

[0031] In one embodiment of the present invention, in step (1), the lactic acid bacteria is bile salt hydrolase-producing lactic acid bacteria Q11 (GDMCC 65303). Bile salt hydrolase-producing lactic acid bacteria Q11 was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 21, 2024, and the address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0032] In one embodiment of the present invention, in step (1), the cell concentration of the lactic acid bacteria suspension is 10 9 ~10 11 CFU / mL.

[0033] In one embodiment of the present invention, in step (1), the solution of microcapsule wall material and the lactic acid bacteria suspension are mixed in a volume ratio of 1:9 to 2:8.

[0034] In one embodiment of the present invention, in step (1), the amount of gluconolactone added is 1.5-3 wt % of the mixture of the microcapsule wall material solution and the lactic acid bacteria suspension.

[0035] In one embodiment of the present invention, in step (2), the oil phase is one or more of soybean oil, corn oil, olive oil, and sunflower oil.

[0036] In one embodiment of the present invention, in step (2), the volume of the oil phase is 3 to 5 times that of the water phase.

[0037] In one embodiment of the present invention, in step (2), the stirring temperature is 30 to 45° C., and the stirring time is 2 to 5 hours.

[0038] In one embodiment of the present invention, in step (2), the filtration is suction filtration.

[0039] In one embodiment of the present invention, in step (2), washing is performed with physiological saline for 1 to 5 times.

[0040] The fifth object of the present invention is to provide lactic acid bacteria microcapsules prepared by the above preparation method.

[0041] The sixth object of the present invention is to provide application of the lactic acid bacteria microcapsules in the food field.

[0042] Beneficial effects:

[0043] (1) The prepared microcapsule wall material has low cost and simple preparation method, and is suitable for industrial preparation.

[0044] (2) The prepared microcapsule wall material has a high grafting rate, excellent emulsification, solubility and gelation, strong hydrophobicity, high beneficial activity, and is easy to form an amorphous material, which is beneficial for encapsulating lactic acid bacteria, improving their activity, forming a compact and stable microcapsule, and facilitating the colonization of lactic acid bacteria in the intestine.

[0045] (3) The structure of the prepared lactic acid bacteria microcapsules is tight and stable, which can effectively protect the lactic acid bacteria, prevent the lactic acid bacteria from contacting with adverse external conditions, and improve the activity of the lactic acid bacteria under conditions such as gastrointestinal fluid, heat, ions, pH and storage.

[0046] (4) The prepared lactic acid bacteria microcapsules have a compact structure and a high embedding rate and loading rate. The embedding rate is above 90%, which greatly improves the application value of bile salt hydrolase-producing lactic acid bacteria and can be used in the food industry.

[0047] (5) The prepared lactic acid bacteria microcapsules significantly increased the activity of bile salt hydrolase during gastrointestinal digestion and significantly delayed the decline of bile salt hydrolase activity under the conditions of gastrointestinal fluid, heat, ions, pH and storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The grafting rate of NaCas-OS (OS represents oligosaccharide) is shown.

[0049] Figure 2 Shown is the endogenous fluorescence spectroscopy analysis of NaCas-OS.

[0050] Figure 3 The prebiotic activity analysis of NaCas-OS is shown, the left picture is before simulated digestion, and the right picture is after simulated digestion.

[0051] Figure 4 An analysis of the emulsification properties of NaCas-OS is shown.

[0052] Figure 5 The encapsulation rate and loading rate of lactic acid bacteria microcapsules are shown.

[0053] Figure 6 A scanning electron microscope image of lactic acid bacteria microcapsules is shown.

[0054] Figure 7 X-ray diffraction analysis of lactic acid bacteria microcapsules is shown.

[0055] Figure 8 The cell viability of lactic acid bacteria microcapsules under gastrointestinal fluid stress is shown.

[0056] Fig. 9 The cell viability of lactic acid bacteria microcapsules under heat stress is shown.

[0057] Fig.10 The cell viability of lactic acid bacteria microcapsules under acid-base stress and ion stress is shown.

[0058] Fig.11 The cell viability of lactic acid bacteria in microcapsule storage is shown. DETAILED DESCRIPTION

[0059] Test methods for microcapsule wall materials

[0060] Determination of grafting rate:

[0061] Prepare 500mL of o-phthalaldehyde (OPA) working solution: mix 20mL of 40mg / mL OPA anhydrous ethanol solution, 25ml of 20% SDS solution, 250mL of 0.1mol / L sodium tetraborate solution and 2mL of β-mercaptoethanol solution, and dilute to 500mL with deionized water. OPA working solution needs to be prepared and used immediately. Mix 200μL of sodium caseinate solution (5mg / mL) or the microcapsule wall material solution (5mg / mL) prepared in step (2) with 4mL of OPA working solution, react at room temperature for 5min, generate a standard curve using 0.5-3mM L-lysine, and calculate the content of free amino acids in the microcapsule wall material solution based on the standard curve.

[0062] Determination of intrinsic fluorescence spectrum:

[0063] The microcapsule wall material is taken to prepare a microcapsule wall material solution with a concentration of 1 mg / mL, and then a fluorescence spectrum scan is performed with an excitation wavelength of 290 nm and an emission wavelength ranging from 300 to 450 nm.

[0064] Determination of prebiotic activity:

[0065] Simulated gastric fluid: Use sterile water to prepare 2 g / L NaCl solution, use 1 M HCl solution to adjust the pH to 2.0, add 3 g / L pepsin, and after fully dissolving, use a 0.22 μm sterile organic filter membrane to filter and sterilize to obtain simulated gastric fluid.

[0066] Conduct simulated gastric digestion experiment: add 0.5g microcapsule wall material to 5mL simulated gastric juice and incubate in a shaker incubator for 2h (200rpm 37℃). After incubation, add 100μL 1M Na2CO3 and adjust the pH to 7.0 with 5M NaOH solution to terminate the reaction. Conduct simulated gastric digestion experiment: then add 1g / L trypsin, 5g / L bile salt and 6.8g / L potassium dihydrogen phosphate, incubate in a shaker incubator for 2h (200rpm 37℃) after complete dissolution, heat at 100℃ for 5min to terminate the reaction, then cool to room temperature, place in a -80℃ refrigerator overnight, and freeze-dry for 72~96h the next day at a pressure of 10~50Pa.

[0067] The microcapsule wall materials before and after the simulated gastrointestinal digestion experiment were used as nitrogen and carbon sources to prepare a modified MRS medium (45 g / L microcapsule wall material, 2 g / L diammonium hydrogen citrate, 1 mL Tween 80, 5 g / L sodium acetate trihydrate, 2 g / L dipotassium hydrogen phosphate trihydrate, 0.58 g / L magnesium sulfate heptahydrate and 0.25 g / L manganese sulfate monohydrate). The inoculation amount was 2% (200 μL of bacterial solution was inoculated in 10 mL of liquid culture medium, and the cell concentration was 10 10CFU / mL) was inoculated with fermented Lactobacillus Q11 and placed in an anaerobic incubator. After anaerobic culture for 24 h, OD600nm was measured, and the culture medium before culture was used as a blank control.

[0068] Determination of emulsifying activity:

[0069] Prepare a 5 mg / mL protein solution, mix 2 mL of soybean oil solution with 8 mL of sample solution, and then homogenize using a high-speed homogenizer (12000 rpm for 2 min). At 0 min and 10 min after homogenization, take 50 μL of the bottom solution and add it to 2.45 mL of 0.1% (w / v) SDS solution. After mixing evenly using a vortex instrument, measure the absorbance at 500 nm. EAI (m 2 / g) and ESI(min) are calculated by the following two formulas:

[0070]

[0071] Where EAI is the emulsifying activity index, c is the protein concentration 5 mg / mL, is the volume fraction of the oil phase 0.2, A0 is the absorbance value after standing for 0 min, N is the emulsion dilution factor 50, ESI is the emulsion stability index, A 10 It is the absorbance value after standing for 10 minutes.

[0072] Testing methods for lactic acid bacteria microcapsules

[0073] Determination of embedding rate and loading rate:

[0074] 0.5 g of lactic acid bacteria microcapsules were dispersed in 5 mL of 0.9% sterile saline. The lactic acid bacteria microcapsules were broken using a high-speed homogenizer (10,000 rpm for 60 s) to release the fermented lactobacillus, and the live bacteria were counted. The encapsulation efficiency and loading capacity were calculated using the following two formulas:

[0075] Embedding rate (%) = (LogN / LogN0) × 100

[0076] Loading amount (LogCFU / g) = LogN / G

[0077] Wherein, N is the total number of colonies in the microcapsules after encapsulation, N0 is the total number of colonies added to prepare the microcapsules before encapsulation, LogCFU / g is the total number of microbial colonies contained in each gram of sample, and G is the weight of the microcapsules (g).

[0078] Scanning electron microscope test:

[0079] The freshly prepared lactic acid bacteria microcapsules were frozen in a -80°C refrigerator for 6 h, and freeze-dried for 12 h using a freeze dryer at a temperature of 40-60°C and a pressure of 50-100 Pa. After gold spraying, the morphology of the microcapsules was observed under a scanning electron microscope.

[0080] X-ray diffraction measurement:

[0081] The 2θ crystal structure of the microcapsule wall material or the lactic acid bacteria microcapsule at 5 to 90° was tested by XRD.

[0082] Determination of heat stress:

[0083] Add 0.5g lactic acid bacteria microcapsules and 5mL sterile PBS or 5mL bacterial suspension to a sterile centrifuge tube. After uniform dispersion, incubate in a water bath at 50℃, 60℃ and 70℃ for 10min. After the water bath, quickly cool to room temperature in running water and count the cells.

[0084] Determination of gastrointestinal fluid stress:

[0085] Simulated saliva: Use sterile water to prepare 1 g / L NaCl solution, use 1 M HCl solution to adjust the pH to 6.9, add 0.86 g / L α-amylase, and after fully dissolving, use a 0.22 μm sterile organic filter membrane to filter and sterilize to obtain simulated saliva.

[0086] Simulated gastric fluid: Use sterile water to prepare 2 g / L NaCl solution, use 1 M HCl solution to adjust the pH to 2.0, add 3 g / L pepsin, and after fully dissolving, use a 0.22 μm sterile organic filter membrane to filter and sterilize to obtain simulated gastric fluid.

[0087] Simulated intestinal fluid: Use sterile water to prepare 6.8 g / L potassium dihydrogen phosphate solution, use 5 M NaOH solution to adjust the pH to 7.5, add 5 g / L bile salts and 1 g / L trypsin, and after fully dissolving, use a 0.22 μm sterile organic filter membrane to filter and sterilize to obtain simulated intestinal fluid.

[0088] 0.5g microcapsules or 10 9CFU of fermented Lactobacillus was resuspended in 5 mL of simulated saliva. After digestion for 5 minutes, the supernatant was discarded after centrifugation (4°C, 10,000 × g, 2 minutes), and the precipitate was resuspended in 5 mL of simulated gastric juice for simulated gastric juice digestion. After gastric juice digestion for 2 hours, the supernatant was discarded after centrifugation (4°C, 10,000 × g, 2 minutes), and the precipitate was resuspended in 5 mL of simulated intestinal juice for simulated intestinal juice digestion for 2 hours. The entire simulation process was carried out in a shaking incubator at 100 rpm and 37°C. The colony counts were performed at 5 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, and 240 minutes.

[0089] Determination of acid-base stress and ion stress:

[0090] Add 0.5 g lactic acid bacteria microcapsules and 5 mL sterile PBS or 5 mL bacterial suspension to a sterile centrifuge tube. Then adjust the pH to 2, 4, 6, 8 and 10 respectively. After uniform dispersion, incubate in a 37°C water bath for 4 h, quickly cool to room temperature in running water, and count the cells.

[0091] Add 0.5g lactic acid bacteria microcapsules to a sterile centrifuge tube, add 5mL sterile PBS or 5mL bacterial suspension, and then add NaCl to make the concentration of NaCl 10g / L, 15g / L, 20g / L, 25g / L and 30g / L. After uniform dispersion, incubate in a 37℃ water bath for 4h, quickly cool to room temperature in running water, and count the cells.

[0092] Determination of storage stability:

[0093] The lactic acid bacteria microcapsules were stored at 25°C and 4°C, and the cells were counted every 7 days.

[0094] Determination of bile salt hydrolase activity:

[0095] Lactobacillus microcapsules or lactobacilli were mixed with 100-200 μL dithiothreitol and crushed using an ultrasonic cell crusher. The crushing power was 300 W, the operation time was 3 s, the interval time was 2 s, and the total time was 10 min. After crushing, the supernatant was centrifuged at 12000 g for 10 min at 4 ° C in an STplus centrifuge. The protein content in the centrifuged supernatant was quantified using a BCA kit. The supernatant was further mixed with TDCA or GDCA. After incubation at 37 ° C for 30 min, 1 ml of 15% trichloroacetic acid was added to precipitate and remove the protein. Then, the mixture was centrifuged at 12000 g for 5 min at 4 ° C. Next, 1 mL of the reaction supernatant was mixed with 200 μL ninhydrin reagent, heated for 30 min, and then cooled to room temperature. The absorbance was measured at 570 nm using an xMark microplate reader.

[0096] The specific activity is defined as the amount of product (glycine or taurine) produced per minute per milligram of protein (U / mg), calculated using the respective standard curves. Blank microcapsules were used as blank controls. The protein content of lactic acid bacteria = the protein content of microcapsules containing bacteria - the protein content of microcapsules without bacteria. The amount of amino acids produced = the amount of amino acids in microcapsules containing bacteria - the amount of amino acids in microcapsules without bacteria. BSH-G is the enzyme activity (U / mg) for hydrolysis of sodium taurodeoxycholate, and BSH-T is the enzyme activity (U / mg) for hydrolysis of sodium glycodeoxycholate.

[0097] Screening of lactic acid bacteria Q11 producing bile salt hydrolase

[0098] Bile salt hydrolase-producing lactic acid bacteria Q11 (LfQ11) was screened from infant feces. The screening method was based on Screening of bile salt hydrolase-producing lactic acid bacteria and evaluation of cholesterol-lowering activity in vitro, Food Bioscience (https: / / doi.org / 10.1016 / j.fbio.2024.105338).

[0099] Example 1

[0100] A method for preparing a microcapsule wall material with prebiotic function comprises the steps of:

[0101] (1) Sodium caseinate and xylo-oligosaccharide were mixed in a mass ratio of 1:1, deionized water was added, and the mixture was stirred at 500 rpm at room temperature until completely dissolved to prepare a solution with a sodium caseinate concentration of 10 wt% and a prebiotic oligosaccharide concentration of 10 wt%, stirred at room temperature for 2 h, and hydrated at 4°C for 12 h;

[0102] (2) adjusting the pH of the solution obtained in step (1) to 9.0 using 1 M HCl and 5 M NaOH, heating in a water bath at 85° C. for 3 h to carry out the Maillard reaction, and cooling in an ice bath to room temperature to obtain a Maillard conjugate solution;

[0103] (3) The Maillard conjugate solution was frozen at -80°C and then freeze-dried at -40°C and 50 Pa pressure for 72 h to obtain the microcapsule wall material.

[0104] Example 2

[0105] A method for preparing a microcapsule wall material with prebiotic function, which differs from Example 1 in that xylooligosaccharide is replaced with fructo-oligosaccharide; and the Maillard reaction time in step (2) is 4 hours.

[0106] Example 3

[0107] A method for preparing a microcapsule wall material with prebiotic function, which differs from Example 1 in that: xylo-oligosaccharide is replaced with inulin; and the Maillard reaction time in step (2) is 4 hours.

[0108] Example 4

[0109] A method for preparing bile salt hydrolase-producing lactic acid bacteria microcapsules comprises the following steps:

[0110] (1) The fermented Lactobacillus Q11 bacterial solution was inoculated into the MRS liquid medium at a 2% inoculation amount (200 μL of bacterial solution was inoculated into 10 mL of liquid medium), and the culture was subcultured once every 18 hours, which was recorded as one activation. After activation twice, the fermented Lactobacillus Q11 was inoculated into the MRS liquid medium at a 2% inoculation amount. When the culture was cultured for 16 hours, the cell concentration of the fermented Lactobacillus was measured by the dilution coating method to be 10 10 CFU / mL, collect the precipitate by centrifugation, and resuspend it in physiological saline to obtain a cell concentration of 10 10 CFU / mL of Lactobacillus fermentum Q11 working solution.

[0111] (2) preparing a 10 wt % microcapsule wall material solution of the microcapsule wall material prepared in Example 1, mixing the solution with a Lactobacillus fermentum Q11 working solution at a volume ratio of 1:9, adding 1.5 wt % gluconolactone to the mixture of the microcapsule wall material solution and the Lactobacillus fermentum Q11 working solution, and rapidly stirring to dissolve to obtain an aqueous phase;

[0112] (3) Soybean oil and water were mixed in a volume ratio of 3:1, incubated in a water bath at 37°C and 500 rpm for 3 h, allowed to stand for stratification, the upper soybean oil was removed, the remaining soybean oil in the lower precipitate was removed using a vacuum filter, and the mixture was rinsed three times with saline to remove the fermented Lactobacillus Q11 that was not embedded, thereby obtaining lactic acid bacteria microcapsules.

[0113] Example 5

[0114] A method for preparing bile salt hydrolase-producing lactic acid bacteria microcapsules, which differs from Example 4 in that the microcapsule wall material prepared in Example 1 is replaced with the microcapsule wall material prepared in Example 2.

[0115] Example 6

[0116] A method for preparing bile salt hydrolase-producing lactic acid bacteria microcapsules, which differs from Example 4 in that the microcapsule wall material prepared in Example 1 is replaced with the microcapsule wall material prepared in Example 3.

[0117] Comparative Example 1

[0118] A method for preparing bile salt hydrolase-producing lactic acid bacteria microcapsules, which differs from Example 4 in that the wall material of the microcapsules prepared in Example 1 is replaced with sodium caseinate.

[0119] The grafting rates of the test examples 1 to 3 when the Maillard reaction was carried out for 0 to 5 hours were as follows: Figure 1 As shown in the figure, the grafting rates of the Maillard reaction conjugate of sodium caseinate-xylo-oligosaccharide (NaCas-XOS, Example 1), the Maillard reaction conjugate of sodium caseinate-fructose oligosaccharide (NaCas-FOS, Example 2) and the Maillard reaction conjugate of sodium caseinate-inulin (NaCas-GFN, Example 3) reached the maximum values ​​at 3h, 4h and 4h of Maillard reaction, which were 24.97%, 22.33% and 18.53%, respectively. The results show that XOS (xylo-oligosaccharide) is easier to graft with sodium caseinate (NaCas), and the higher the grafting rate, the better the emulsification, solubility and gelation of the Maillard reaction product. Therefore, NaCas-XOS 3h 、NaCas-FOS 4h 、NaCas-GFN 4h Can be used as potential embedding wall material.

[0120] Figure 2 The endogenous fluorescence spectra of the Maillard reaction for 0 to 5 hours for Examples 1 to 3 are shown. The reduction in fluorescence intensity may be attributed to the shielding of tryptophan residues by sugars and the conversion of some fluorescent substances into other substances during the Maillard reaction. The fluorescence intensity of NaCas-XOS is more reduced than that of NaCas-FOS and NaCas-GFN, which may be attributed to the fact that XOS is more easily reacted with NaCas and has a higher grafting rate. As the Maillard reaction proceeds, the maximum absorption wavelength (λmax) of NaCas-XOS, NaCas-FOS, and NaCas-GFN changes from 347nm (NaCas) to 351nm, 350.6nm, and 350.6nm. The increase in the maximum absorption wavelength indicates that the structure of NaCas unfolds during the Maillard reaction, which is beneficial for NaCas to expose more hydrophobic groups and increase the hydrophobic interaction when preparing microcapsules.

[0121] The Maillard reaction conjugates of sodium caseinate and oligosaccharides before and after simulated digestion were used as carbon and nitrogen sources for LfQ11 culture. Figure 3 As shown ( Figure 3 The left is before simulated digestion. Figure 3The right side is after simulated digestion). The Maillard reaction conjugate (Maillard reaction time 1-5h) can significantly promote the growth of LfQ11 compared with the sodium caseinate-oligosaccharide mixture (Maillard reaction time 0h). The OD600nm of NaCas-XOS, NaCas-FOS, and NaCas-GFN reached the maximum value of 0.485, 0.406, and 0.506 at the Maillard reaction time of 3h, 4h, and 3h, respectively, and there was no significant change with the extension of reaction time. This may be because the Maillard reaction produces a certain product, which is a good prebiotic, but as the Maillard reaction reaches a certain extent, this product no longer increases. After simulated gastrointestinal fluid digestion, the Maillard reaction conjugate has a significantly higher promoting effect on the growth of LfQ11 than the undigested one. This may be because the Maillard reaction conjugate produces some small molecular peptides and proteins after digestion, which is conducive to the growth of LfQ11. After simulated digestion, NaCas-XOS, NaCas-FOS, and NaCas-GFN reached the maximum OD600nm values ​​of 1.487, 1.364, and 1.512 at Maillard reaction times of 3h, 3h, and 3h, respectively. It is worth noting that the prebiotic activity of NaCas-XOS after 4h of Maillard reaction was significantly reduced compared with 3h, which further shows that the prebiotic active substances produced by the Maillard reaction do not increase with the reaction time. Therefore, in addition to playing a protective role, the use of Maillard reaction conjugates to encapsulate fermented Lactobacillus Q11 can also serve as a nutrient for the growth of fermented Lactobacillus Q11. In particular, the Maillard reaction conjugate after simulated gastrointestinal fluid digestion has stronger prebiotic activity than the Maillard reaction conjugate before digestion, which will facilitate the colonization of LfQ11 in the intestine.

[0122] The emulsifying ability of protein is the ability of protein to form a stable emulsion. The excellent emulsifying ability of protein is very important for the preparation of microcapsules by emulsification method. The emulsifying activity (EAI) and emulsifying stability index (ESI) reflect the emulsifying properties of protein. Figure 4 As shown in the figure, the EAI and ESI of NaCas-XOS, NaCas-FOS, and NaCas-GFN reached their maximum values ​​at 3h, 4h, and 4h of Maillard reaction, which were 13.38 m 2 / g and 28.23min, 13.26m 2 / g and 27.50min, 11.76m 2 / g and 22.31min. However, the EAI and ESI of NaCas did not gradually increase with the heating time. On the contrary, in the later stage of heating, the emulsifying properties of the Maillard reaction conjugate decreased significantly. This may be because the formation of products such as melanoidins in the later stage of the Maillard reaction is not conducive to the emulsifying activity and stability of the protein, which is consistent with previous studies. The results show that the glycosylation of oligosaccharides can enhance the emulsifying properties of sodium caseinate, thereby potentially improving the properties of sodium caseinate microcapsules.

[0123] like Figure 5 As shown, the embedding rate and loading capacity of LfQ11 microcapsules made of sodium caseinate-oligosaccharide Maillard reaction conjugates are significantly higher than those of LfQ11 microcapsules made of sodium caseinate, and the higher the degree of glycosylation, the higher the embedding rate and loading capacity. The embedding rates of the three sodium caseinate-oligosaccharide Maillard reaction conjugates are all above 90%, namely 92.17% (LfQ11@NaCas-XOS, Example 4), 91.72% (LfQ11@NaCas-FOS, Example 5) and 91.05% (LfQ11@NaCas-GFN, Example 6), respectively. The embedding rate of fermented Lactobacillus Q11 microcapsules (LfQ11@NaCas, Comparative Example 1) made of sodium caseinate is only 87.26%. The loading rates of LfQ11@NaCas, LfQ11@NaCas-XOS, LfQ11@NaCas-FOS and LfQ11@NaCas-GFN were 9.02LogCFUg -1 、9.17LogCFUg -1 、9.13LogCFUg -1 and 9.07LogCFUg -1 The results showed that glycosylation improved the coagulation performance of sodium caseinate, making it easier to coagulate and embed the probiotics under the action of gluconolactone. The LfQ11 microcapsules made of sodium caseinate-xylo-oligosaccharide with a higher degree of glycosylation had a higher embedding rate and loading capacity.

[0124] The surface morphology of the microcapsules was observed using a scanning electron microscope. Figure 6The microcapsules of the four wall materials all showed some small protrusions, indicating that the four wall materials had film-forming properties and wrapped LfQ11 inside, and LfQ11@NaCas-XOS and LfQ11@NaCas-FOS had more protrusions, indicating that LfQ11@NaCas-XOS and LfQ11@NaCas-FOS had better film-forming properties and embedded more LfQ11 inside. The microcapsule surfaces of the four wall materials all showed different degrees of depression and wrinkles. LfQ11@NaCas had the most wrinkles, while LfQ11@NaCas-OS had fewer wrinkles than LfQ11@NaCas. The reduction in wrinkles may make it difficult for LfQ11 to be released, thus better protecting LfQ11 in the microcapsule. The surfaces of LfQ11@NaCas-XOS and LfQ11@NaCas-FOS were denser and smoother, indicating that LfQ11@NaCas-XOS and LfQ11@NaCas-FOS could provide better protection for LfQ11. In addition, there are some honeycomb structures on the surface of LfQ11@NaCas, which will expose LfQ11 to the external environment more, which is not conducive to the protection of LfQ11.

[0125] like Figure 7As shown in the figure, after adding gluconolactone (GDL) cross-linking, the -OH peaks of the microcapsule wall materials NaCas (LfQ11-free), NaCas-XOS (LfQ11-free), NaCas-FOS (LfQ11-free) and NaCas-GFN (LfQ11-free) shifted from 3402, 3420, 3411 and 3400 to 3400, 3415, 3408 and 3402, respectively, indicating that the three wall materials formed hydrogen bond interactions. The shift of NaCas-XOS (LfQ11-free) is greater than that of the other three groups, indicating that due to the presence of oligoxylose, it has produced a lot of hydrogen bond interactions with proteins. However, when LfQ11 is added, the maximum absorption wavelength of the -OH peak increases, indicating that lactic acid bacteria reduce the hydrogen bond interactions between wall materials. After GDL cross-linking, the peaks of NaCas (LfQ11-free), NaCas-XOS (LfQ11-free), NaCas-FOS (LfQ11-free) and NaCas-GFN (LfQ11-free) at 1655, 1657, 1655 and 1656 shifted to. This indicates that after the addition of GDL, electrostatic interactions occurred between the wall materials of the prepared microcapsules. The shift of sodium caseinate-oligosaccharide Maillard reaction conjugate was larger, indicating that sodium caseinate-oligosaccharide Maillard reaction conjugate produced a greater electrostatic interaction. After the addition of LfQ11, further shifts occurred, indicating that LfQ11 can also have electrostatic interactions with the wall materials of the Maillard reaction conjugate. In general, the Maillard reaction conjugate can enhance the hydrogen bond interaction and electrostatic interaction of the microcapsules, and LfQ11 can have electrostatic interactions with the wall materials. These interactions make the structure of the microcapsules more compact, forming a physical barrier to protect LfQ11.

[0126] The number of lactic acid bacteria after digestion by gastrointestinal fluid determines its efficacy. Figure 8As shown. 0-5min is the saliva digestion stage, and the survival rate is above 96%, indicating that lactic acid bacteria can resist the damage of saliva very well. 5-120min is the gastric juice digestion stage, and the survival rate of free lactic acid bacteria after 120min digestion is 71.08%. This may be because the pepsin and hydrogen ions in the gastric juice damage the physiological functions of lactic acid bacteria such as cell membrane integrity and osmotic pressure, reducing the vitality of lactic acid bacteria. The survival rate of lactic acid bacteria in gastric juice has been significantly improved after embedding. The survival rate of LfQ11@NaCas (90.32%) is significantly lower than that of sodium caseinate-oligosaccharide conjugate. In addition, the survival rate of lactic acid bacteria in LfQ11@NaCas-XOS (92.55%) is significantly higher than that in LfQ11@NaCas-FOS (91.41%) and LfQ11@NaCas-GFN (91.31%). It is worth noting that sodium caseinate-oligosaccharide microcapsules have stronger hydrophobic interactions and hydrogen bonds than sodium caseinate microcapsules, and the surface is smoother, which prevents the invasion and digestion of gastric juice. In addition, the stronger hydrophobic interactions and hydrogen bonds in LfQ11@NaCas-XOS lead to its better cell viability. During the 120-240min intestinal digestion stage, the survival rate of lactic acid bacteria after intestinal digestion was 51.52%. The survival rate of lactic acid bacteria in intestinal juice after embedding was significantly improved. The survival rate of LfQ11@NaCas was 63.71%, which was significantly lower than that of sodium caseinate-oligosaccharide conjugates. In addition, the survival rate of lactic acid bacteria in LfQ11@NaCas-XOS (82.46%) was significantly higher than that in LfQ11@NaCas-FOS (81.38%) and LfQ11@NaCas-GFN (81.09%). In general: Maillard reaction conjugates improve the stability of microcapsules in gastrointestinal fluid, increase the survival rate of lactic acid bacteria, and enable them to better exert their efficacy.

[0127] Table 1 shows the changes in bile salt hydrolase activity of lactic acid bacteria microcapsules in the process of simulating gastrointestinal fluid. After the gastric juice digestion stage, the loss of BSH-G and BSH-T in sodium caseinate-oligosaccharide microcapsules was less than that in LfQ11@NaCas, indicating that sodium caseinate-oligosaccharide microcapsules have better protection and probiotic effects on LfQ11, and the survival rate of lactic acid bacteria is higher. After the intestinal juice digestion stage, the BSH-G and BSH-T of LfQ11 and LfQ11@NaCas decreased significantly, but the BSH-G and BSH-T of sodium caseinate-oligosaccharide microcapsules increased instead. The reason is that the survival rate of LfQ11 protected by sodium caseinate-oligosaccharide microcapsules is higher, and more bile salt hydrolase is released after being stimulated by bile salts in the intestinal juice. However, the survival rate of LfQ11 in samples LfQ11 and LfQ11@NaCas is too low, and sufficient bile salt hydrolase cannot be released after being stimulated by bile salts in the intestinal juice.

[0128] Table 1 Changes in bile salt hydrolase activity of lactic acid bacteria microcapsules in simulated gastrointestinal fluid

[0129]

[0130]

[0131] Heat stress is one of the important factors affecting cell viability. Lactobacillus cells are very sensitive to heat. LfQ11 microcapsules are used to extend the heat conduction path to maintain the cell viability of lactobacillus. The cell viability of free lactobacillus and microcapsules at different temperatures (50℃, 60℃ and 70℃) is shown in Figure 2. Fig. 9 As shown in Figure 2, as the temperature increases, the cell viability of lactic acid bacteria decreases. At 70°C, the cell viability of free lactic acid bacteria dropped to 1.11LogCFUmL. -1 The microcapsules significantly improved the activity of lactic acid bacteria at different temperatures. At 50°C, there was no significant difference in the cell activity of the three sodium caseinate-oligosaccharide microcapsules (7.57LogCFUmL -1 、7.53LogCFUmL -1 and 7.40LogCFUmL -1 ). At 60℃ and 70℃, the survival rate of LfQ11@NaCas-XOS was significantly higher than that of LfQ11@NaCas-FOS and LfQ11@NaCas-GFN. This may be because LfQ11@NaCas-XOS has more grafted oligosaccharides, stronger interaction forces, and a tighter structure, which delays the transfer of temperature. These results show that oligosaccharides with higher grafting rates can produce more interaction forces, encapsulating lactic acid bacteria in microcapsules to maintain their vitality. Table 2 shows the changes in the activity of bile salt hydrolase in lactic acid bacteria microcapsules under heat stress. Since the cell viability of sodium caseinate-oligosaccharide microcapsules is higher than that of LfQ11 and LfQ11@NaCas, its bile salt hydrolase activity is also higher under heat stress.

[0132] Table 2 Changes in the activity of lactic acid bacteria microcapsule bile salt hydrolase under heat stress

[0133]

[0134]

[0135] The protective effects of different microcapsules on the survival of lactic acid bacteria at different pH values ​​(2.0, 4.0, 6.0) and different NaCl levels (10 g / L, 15 g / L, 20 g / L, 25 g / L and 30 g / L) are shown in Figure 2. Fig.10The bile salt hydrolase activity is shown in Tables 3 and 4. The cell viability of free lactic acid bacteria is lower under extreme pH conditions, and the cell viability of lactic acid bacteria decreases with increasing NaCl concentration. At pH 2, the cell viability of free lactic acid bacteria is the lowest, which is 7.25LogCFUmL -1 When NaCl was 30 g / L, the cell activity of free lactic acid bacteria was 6.07 Log CFU mL -1 The microcapsules significantly increased the activity of lactic acid bacteria and bile salt hydrolase at extreme pH and high NaCl concentrations. In addition, at different pH values ​​and NaCl concentrations, there was no significant difference in the cell viability of the three sodium caseinate-oligosaccharide microcapsules, and the cell viability was all within 6.9LogCFUmL -1 The three sodium caseinate-oligosaccharide microcapsules have the same protective effect on lactic acid bacteria under pH and ion stress.

[0136] Table 3 Changes in bile salt hydrolase activity of lactic acid bacteria microcapsules under acid stress

[0137]

[0138] Table 4 Changes in the activity of lactic acid bacteria microcapsule bile salt hydrolase under ion stress

[0139]

[0140] The efficacy of lactic acid bacteria is closely related to the number of live bacteria, so it is necessary to ensure that the lactic acid bacteria have a sufficient number of live bacteria during storage and transportation. It is very important for the packaging material to maintain the cell viability of lactic acid bacteria during storage, so the changes in the cell viability of lactic acid bacteria in microcapsules at room temperature (25°C) and under fresh-keeping conditions (4°C) were measured. Fig.11 As shown in Figure 2, under room temperature and fresh storage, the activity of free lactic acid bacteria was 4.21LogCFUmL after 28 days. -1 and 5.86LogCFUmL -1 The four microcapsules significantly increased the cell viability of lactic acid bacteria stored at room temperature. LfQ11@NaCas (6.02LogCFUmL -1 and 7.31LogCFUmL -1 ) had a significantly lower cell viability than sodium caseinate-oligosaccharide microcapsules. LfQ11@NaCas-XOS (7.42LogCFUmL -1 and 7.71LogCFUmL -1 ) was significantly lower than LfQ11@NaCas-FOS (7.35LogCFUmL -1 and 7.65LogCFUmL -1 ) and LfQ11@NaCas-GFN (7.27LogCFUmL-1 and 7.57LogCFUmL -1 ). This may be due to the fact that sodium caseinate-oligosaccharide microcapsules contain more bound water content and a higher glass transition temperature, and have higher storage stability. Secondly, the hydrogen bonds and hydrophobic interactions in sodium caseinate-oligosaccharide microcapsules, especially LfQ11@NaCas-XOS, are stronger, making their structure more compact and avoiding the contact of lactic acid bacteria with adverse external conditions. Table 5 shows the changes in the activity of bile salt hydrolase in lactic acid bacteria microcapsules under room temperature storage conditions. Since the LfQ11 cell activity in sodium caseinate-oligosaccharide microcapsules is higher, its bile salt hydrolase activity is also higher under room temperature storage conditions.

[0141] Table 5 Changes in bile salt hydrolase activity of lactic acid bacteria microcapsules under room temperature storage conditions

[0142]

[0143]

[0144] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a microcapsule wall material having a prebiotic function, characterized in that: Includes steps: (1) dissolving sodium caseinate and prebiotic oligosaccharides in water, stirring and hydrating; (2) adjusting the pH of the solution obtained in step (1), performing a Maillard reaction, and cooling the solution in an ice bath to obtain a Maillard conjugate solution; (3) Freezing the Maillard conjugate solution and then freeze-drying it to obtain a microcapsule wall material with prebiotic function.

2. The preparation method according to claim 1, characterized in that: In step (1), the prebiotic oligosaccharide is one or more of xylooligosaccharide, fructooligosaccharide and inulin; the concentration of sodium caseinate dissolved in water is 5-15wt%, and the concentration of prebiotic oligosaccharide dissolved in water is 5-15wt%; the mass ratio of sodium caseinate to prebiotic oligosaccharide is 1:3-3:1; the stirring temperature is 15-35°C and the time is 2-8h; the hydration temperature is 0-8°C and the time is 8-14h; In step (2), the pH is adjusted to 8.0-10.0; the pH is adjusted with a solution of one or more of NaOH, HCl, and citric acid, with a concentration of 0.1-5 M; the temperature of the Maillard reaction is 80-90° C., and the reaction time is 2-5 h; the temperature is cooled in an ice bath to 15-35° C.; In step (3), the freezing temperature is -80 to -20°C, and the freezing time is 24 to 48 hours; the freeze-drying temperature is -40 to -60°C, the pressure is 10 to 50 Pa, and the time is 72 to 96 hours.

3. The microcapsule wall material prepared by the preparation method according to claim 1.

4. Use of the microcapsule wall material according to claim 3 in probiotic microcapsules.

5. A method for preparing lactic acid bacteria microcapsules using the microcapsule wall material according to claim 3, characterized in that: Includes steps: (1) dissolving the microcapsule wall material in water to prepare a solution, mixing the solution with the lactic acid bacteria suspension, adding gluconolactone and stirring to dissolve, thereby obtaining an aqueous phase; (2) The oil phase and the water phase are mixed, stirred, and filtered to obtain a precipitate, and the precipitate is washed to obtain lactic acid bacteria microcapsules.

6. The preparation method according to claim 5, characterized in that: In step (1), the concentration of the microcapsule wall material solution is 8-12 wt %; the lactic acid bacteria are bile salt hydrolase-producing lactic acid bacteria; the cell concentration of the lactic acid bacteria suspension is 10 9 ~10 11 CFU / mL; the microcapsule wall material solution and the lactic acid bacteria suspension are mixed in a volume ratio of 1:9 to 2:8; the amount of gluconolactone added is 1.5 to 3 wt % of the mixture of the microcapsule wall material solution and the lactic acid bacteria suspension.

7. The preparation method according to claim 5, characterized in that: In step (2), the oil phase is one or more of soybean oil, corn oil, olive oil, and sunflower oil; the volume of the oil phase is 3 to 5 times that of the water phase; the stirring temperature is 30 to 45° C., and the stirring time is 2 to 5 hours; and the mixture is washed with physiological saline for 1 to 5 times.

8. The preparation method according to claim 5, characterized in that: The lactic acid bacteria were bile salt hydrolase-producing lactic acid bacteria Q11 (GDMCC 65303).

9. The lactic acid bacteria microcapsules prepared by the preparation method according to claim 5.

10. Use of the lactic acid bacteria microcapsule according to claim 9 in the food field.

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