A microcapsule wall material, a vitrified probiotic microcapsule prepared based on Maillard reaction products, and a preparation method thereof

Abstract

The present invention provides a microcapsule wall material, a vitrified probiotic microcapsule prepared based on a Maillard reaction product, and a preparation method thereof, relating to the technical field of microcapsules. The preparation method of the microcapsule wall material specifically includes: dissolving whey protein isolate and soybean hull polysaccharide in water; stirring at a constant temperature for a first set time; standing at a second set temperature for a second set time; adjusting the pH of the mixed solution to a first set pH value; performing freeze-drying treatment on the mixed solution after adjusting the pH value; grinding the freeze-dried powder and placing it in a dryer, placing a saturated salt solution at the bottom of the dryer, placing the dryer in an oven, setting the temperature of the oven to a third set temperature and the relative humidity to a first set humidity, and using the graft obtained after the Maillard reaction of whey protein isolate and soybean hull polysaccharide for a first set number of days as the microcapsule wall material. The microcapsule wall material provided by the present invention is beneficial to improving the survival rate of probiotics in high-temperature, gastrointestinal tract environments and during storage.

Description

Technical Field

[0001] The present invention relates to the technical field of microcapsules, and in particular to a microcapsule wall material, a vitrified probiotic microcapsule prepared based on a Maillard reaction product, and a preparation method thereof. Background Art

[0002] The glassy state is crucial for the biological activity and stability of products during freezing, drying, and storage. It is often necessary to add solutes with a high glass transition temperature (T g ) to increase the T g of the matrix, so that drying can be carried out at a higher temperature, and a final dried product with a high T g can be obtained to improve the stability of the sample during storage. Some disaccharides and oligosaccharides are often used as freeze-drying additives because they have a high T g and are easy to form a glassy state. However, sugar alcohols are prone to separate from the frozen solution in the form of crystals, resulting in loss of stability after freeze-drying.

[0003] The physicochemical properties of the microcapsule wall material itself directly affect the stability of the microcapsule, the retention efficiency of the core material, and the shelf life. The wall material must have suitable rheological properties at high concentrations, be able to emulsify the active material, stabilize the produced emulsion, and keep the core material within its structure during processing or storage without being damaged. Whey protein isolate (WPI) is often used as a wall material in the food industry for the encapsulation of probiotics and bioactive substances. However, WPI is vulnerable to processing methods such as high temperature, high acid, and salt during food processing, resulting in unstable product properties, which to a certain extent limits the application of WPI. However, preparing protein-polysaccharide graft products by the Maillard reaction can effectively improve the emulsifying property, thermal stability, antioxidant property, antibacterial property, and water solubility of proteins in a wide pH range. At present, although the graft products of whey protein isolate with maltodextrin, carboxymethyl cellulose, soy soluble polysaccharide, etc. have obtained good protection for bioactive substances or probiotics, the mutual connection between the glass transition of the WPI-polysaccharide graft product as a microcapsule wall material and the stability of the core material during processing or storage has not been analyzed. Therefore, it is necessary to optimize the glass state of the material of the microcapsule wall to meet the use requirements.

[0004] Soy hull polysaccharide (SHP) is a non-linear pectin-like polysaccharide with good stability and gelation properties, and also has a high T g . Covalent grafting with SHP can reduce the environmental sensitivity of WPI and endow the WPI graft with good environmental stability. In addition, soy hull polysaccharide has a larger molecular weight than disaccharides and oligosaccharides, and the WPI-SHP covalent graft has a higher T gTherefore, the microcapsule wall material based on the WPI-SHP Maillard reaction product will have a stronger protective effect on probiotics during processing or storage. By studying the glass transition of the microcapsule wall material, the stability of Lactobacillus paracasei microcapsules during processing and storage was analyzed, in order to provide a theoretical basis for the utilization of Lactobacillus paracasei, thereby strengthening the industrial development and comprehensive utilization of Lactobacillus paracasei. Summary of the Invention

[0005] The object of the present invention is to provide a microcapsule wall material, a glassy probiotic microcapsule prepared based on a Maillard reaction product, and a preparation method thereof, so as to solve the problems of poor stability and weak embedding ability of the wall material of a single material in the prior art, crystallization, aggregation and collapse of the microcapsule caused by instability under high-temperature and high-humidity processing or storage conditions, resulting in damage to probiotics by the outside world.

[0006] In order to achieve the above object, according to the first aspect of the present invention, a preparation method of a microcapsule wall material is provided. The preparation method specifically includes the following steps: dissolving whey protein isolate and soybean hull polysaccharide in water; stirring at a constant temperature for a first set time under a first set temperature condition; standing for a second set time at a second set temperature to obtain a mixed solution; adjusting the pH of the mixed solution to a first set pH value; performing freeze-drying treatment on the mixed solution after adjusting the pH value to obtain a freeze-dried powder; grinding the freeze-dried powder and placing it in a dryer, placing a saturated salt solution at the bottom of the dryer, placing the dryer in a drying oven, setting the temperature of the drying oven to a third set temperature, setting the relative humidity of the drying oven to a first set humidity, and using the graft obtained after the Maillard reaction of whey protein isolate and soybean hull polysaccharide for a first set number of days as the microcapsule wall material.

[0007] In the present invention, whey protein isolate and soybean hull polysaccharide are used in combination. Considering that the mixture obtained by a simple combination is relatively sensitive to the environment, the present invention also covalently modifies whey protein isolate with soybean hull polysaccharide through the Maillard reaction to obtain a Maillard reaction product of whey protein isolate-soybean hull polysaccharide, that is, a whey protein isolate-soybean hull polysaccharide graft. The obtained graft can be used as a microcapsule wall material to embed probiotics. There is a strong binding force between whey protein isolate and probiotics, which can effectively improve the embedding rate. At the same time, polysaccharide molecules can adhere to the surface of the protein core, which helps to improve the stability of the carrier, that is, the microcapsule wall material. And after covalent modification, it is less affected by environmental factors such as pH, ionic strength and temperature, the environmental sensitivity is reduced, and it has good emulsifying properties and a high glass transition temperature, thereby improving the survival rate of probiotics during high temperature, gastrointestinal environment and storage.

[0008] Whey protein isolate (WPI) is a high-quality protein with characteristics such as high nutritional value, easy digestion and absorption, and containing various active ingredients. It has good effects as a wall material in the food industry for the encapsulation of probiotics and bioactive substances. Soy hull polysaccharide (SHP) is a non-linear pectin-like polysaccharide with good stability and gel properties. WPI is easily affected by processing methods such as high temperature, high acid, and salt during food processing, resulting in unstable product properties, which to a certain extent limits the application of WPI. In order to broaden the application scope of WPI, the present invention conducts covalent modification on WPI. The covalent modification method of the present invention utilizes the Maillard reaction to prepare protein-polysaccharide graft products, and the emulsification, thermal stability, antioxidant property, antibacterial property, and water solubility of the improved protein have been greatly improved within a wide pH value range. The present invention applies SHP to the Maillard reaction to improve the functional properties of WPI. Covalent grafting with SHP can reduce the environmental sensitivity of WPI and endow the WPI graft with good environmental stability.

[0009] In the present invention, whey protein isolate and soy hull polysaccharide are selected from numerous proteins and polysaccharides for the Maillard reaction. The obtained graft product has been experimentally verified to have good performance when used as a microcapsule wall material. Further, this microcapsule wall material can be applied to the encapsulation of probiotics.

[0010] Of course, the microcapsule wall material provided by the present invention can also be applied to the encapsulation of other substances.

[0011] Optionally, the water is pure water. Pure water has a high purity and contains extremely few impurities, which is beneficial to the formation of the graft product.

[0012] Optionally, the first set temperature is room temperature, that is, 20°C to 27°C. Too low a temperature will affect the structure and function of the protein; at a higher temperature, the protein structure is damaged due to the increased dissociation time, and the protein activity and biological function will decline accordingly. It has been experimentally verified that when the first set temperature is within the above numerical range, the protein has good activity and biological function and will not change the protein structure.

[0013] Preferably, the first set temperature is 25°C. When the first set temperature is the above value, the protein has better activity, structure, and biological function.

[0014] Optionally, the first set time is greater than or equal to 8 h. Too little stirring time will result in uneven mixing of the protein-polysaccharide. It has been experimentally verified that when the stirring time is greater than or equal to 8 h, uniform mixing can be achieved.

[0015] Preferably, the first set time is 8 h. 8 h can make the protein-polysaccharide stirred and mixed evenly while saving time.

[0016] Preferably, during the constant-temperature stirring, a constant-temperature magnetic stirrer is used for constant-temperature stirring.

[0017] Optionally, the second set temperature is 2-6 °C. Since it needs to be stored overnight at this temperature, too high storage temperature will cause the protein-polysaccharide to deteriorate, and too low temperature will affect its structural function. It has been verified by experiments that when the second set temperature is within the above numerical range, the structural function is not affected while ensuring that the protein-polysaccharide mixture does not deteriorate.

[0018] Preferably, the second set temperature is 4 °C. It has been verified by experiments that the structural and functional characteristics of the protein-polysaccharide can be better maintained at 4 °C.

[0019] Optionally, the second set time is greater than or equal to 12 h. If the time is shorter, it cannot ensure that the solution is fully hydrated. It has been verified by experiments that when the second set time is greater than or equal to 12 h, it can ensure that the solution is fully hydrated.

[0020] Preferably, the second set time is 12 h. 12 h can ensure that the protein-polysaccharide is evenly mixed and fully hydrated while saving time.

[0021] Optionally, the first set pH value is 6-8. pH has a certain influence on the Maillard reaction. When pH < 6, the Maillard reaction is not obvious, and acidic conditions are not conducive to the Maillard reaction; when pH > 8, the Maillard reaction is significantly accelerated, and when pH > 11, the color change of the Maillard reaction is significantly weakened.

[0022] Preferably, the first set pH value is 7; pH = 7 is the most suitable pH for the Maillard reaction, and the Maillard reaction usually occurs under neutral conditions. When pH > 7, the reaction color substances are formed quickly, and when pH < 7, it is difficult to form pyrazine substances.

[0023] Optionally, the third set temperature is 55-65 °C; the first set humidity is 75-85%. During the Maillard reaction, a saturated salt solution needs to be placed at the bottom of the drying oven to maintain the humidity. Different saturated salt solutions will have different humidities at different temperatures. In addition, the third set temperature will also affect the Maillard reaction.

[0024] Preferably, the drying oven is a forced-air drying oven.

[0025] Preferably, the salt solution is potassium bromide solution (KBr); the third set temperature is 60 °C; the first set humidity is 79%. The Maillard reaction requires a saturated potassium bromide solution to maintain its humidity, and the saturated potassium bromide solution can reach a relative humidity of 79% at 60 °C. Through experimental verification, when the temperature and humidity values are as above, the graft obtained from the Maillard reaction better meets the requirements of the present invention.

[0026] Optionally, the first set number of days is 1 - 6 days to obtain six kinds of grafts, namely WPI - SHP 1d 、WPI - SHP 2d 、WPI - SHP 3d 、WPI - SHP 4d 、WPI - SHP 5d 、WPI - SHP 6d ; One or more of the six kinds of grafts are used as the microcapsule wall material.

[0027] Preferably, the first set number of days is 4 days. Through experimental verification, when the grafting time is 4 days, the obtained graft WPI - SHP 4d has the best emulsifying property and the highest glass transition temperature.

[0028] Optionally, the obtained graft is placed at the fourth set temperature for storage; the fourth set temperature is -20 °C. When the fourth set temperature is the above value, it can avoid the deterioration of the graft and has a good storage effect.

[0029] Preferably, the graft WPI - SHP 4d on the fourth day is used as the microcapsule wall material; the obtained graft is stored in a refrigerator at -20 °C. Through experimental verification, when the parameters are the above values, the performance of the obtained graft is optimal and the storage effect of the graft is optimal.

[0030] Through experimental verification, when each parameter is within the above value range, the performance of the obtained graft is better and the storage effect of the graft is better.

[0031] Among them, the specific method of adjusting the pH of the mixed solution can be achieved by using the existing methods for adjusting pH.

[0032] Optionally, in order to verify the performance of the microcapsule wall material provided by the present invention, the preparation method further includes the following steps: taking the WPI-SHP mixture and the WPI-SHP graft in the reaction process as samples; calculating the grafting degree of the samples; calculating the pH value of the samples; analyzing the emulsifying properties of the samples; analyzing the glass transition temperature of the samples; analyzing the protein molecular weight of the samples; and characterizing the samples. In this way, the preparation method of the present invention also analyzes the performance of the graft and compares it with the mixture without Maillard reaction, so as to verify whether the performance of the graft meets the requirements. The present invention comprehensively analyzes the performance of the graft from multiple aspects and verifies that the performance of the graft is better.

[0033] Optionally, take the WPI-SHP mixture on day 0 during the reaction process and the WPI-SHP graft of WPI-SHP from 1 to 6 days 1d 、WPI-SHP 2d 、WPI-SHP 3d 、WPI-SHP 4d 、WPI-SHP 5d 、WPI-SHP 6d as samples; the method for calculating the grafting degree of the samples specifically includes: weighing 40 mg of o-phthalaldehyde and dissolving it in 1 ml of methanol, adding 2.5 ml of 20% sodium dodecyl sulfate solution, 0.1 mol / L borax solution, and 100 μL of β-mercaptoethanol respectively, mixing evenly and then making up the volume to 50 mL with pure water to prepare o-phthalaldehyde reagent; adding 4 mL of o-phthalaldehyde reagent to 200 μL of the sample and mixing well, heating in a water bath at 35 °C for 2 min; using 200 μL of ultrapure water instead of the sample as a blank control, measuring the absorbance at 340 nm; drawing a standard curve with lysine as the standard and calculating the free amino group content; calculating the grafting degree DG using the following formula:

[0034]

[0035] In the formula, C0 is the free amino group content of the WPI-SHP mixture; C t is the free amino group content of the WPI-SHP graft. In this way, the grafting degree of the graft can be calculated by using the method provided by the present invention, so as to evaluate the performance of the graft.

[0036] Optionally, the method for calculating the pH value of the samples specifically includes: diluting the samples with ultrapure water to a sample solution with a concentration of 2 mg / mL, and measuring the pH value of the sample solution. In this way, the pH value of the graft solution can be calculated by using the method provided by the present invention, so as to evaluate the performance of the graft.

[0037] Optionally, the method for analyzing the emulsifying properties of the graft includes: dissolving the sample in a phosphate buffer solution with a concentration of 0.01 mol / L and a pH of 7.0 to prepare a solution with a protein concentration of 2.0 mg / mL; adding 5 mL of soybean oil to 15 mL of this solution, homogenizing at 13,500 rpm for 2 min, and then taking out 50 μL of the emulsion from the bottom at 0 min and 10 min respectively. Dilute it 100 times with 0.1% sodium dodecyl sulfate solution and measure its absorbance at a wavelength of 500 nm; the emulsifying activity index EAI and the emulsifying stability index ESI are calculated according to formula (2) and formula (3) respectively:

[0038]

[0039]

[0040] In the formula, A0 and A 10 are the absorbance values at 0 min and 10 min after homogenization respectively; C is the protein concentration, with the unit of g / mL; is the oil phase ratio; DF is the dilution factor.

[0041] In this way, the emulsifying properties of the graft can be analyzed by using the method provided by the present invention, so as to evaluate the emulsifying performance of the graft.

[0042] Optionally, the analysis of the glass transition temperature of the sample specifically includes: using a differential scanning calorimeter to measure the glass transition temperature of the sample. Weigh 5 - 7 mg of the sample and seal it in an aluminum crucible, with an empty crucible as a reference; the scanning program is set as follows: heat from room temperature to 180 °C at a rate of 10 °C / min, equilibrate at 180 °C for 5 min, cool to 10 °C at the same rate, and equilibrate at 10 °C for 5 min, and then heat to 220 °C at a rate of 10 °C / min; use the analysis software of the differential scanning calorimeter to analyze and obtain the midpoint within the glass transition temperature range, and use this as T g .

[0043] Optionally, the differential scanning calorimeter is the Q-200 differential scanning calorimeter of TA Instruments, USA, and the analysis software is TA Universal Analysis.

[0044] In this way, the glass transition temperature of the graft can be analyzed by using the method provided by the present invention, so as to evaluate the biological activity and stability of the graft during freezing, drying, and storage.

[0045] It should be noted that the realization of obtaining the glass transition temperature range by software analysis adopts the prior art and will not be elaborated here.

[0046] Optionally, the method for analyzing the protein molecular weight of the sample specifically includes: analyzing the change in protein molecular weight before and after the reaction by high performance size exclusion chromatography; the instrument is LC-10A high performance liquid chromatograph HPLC; mobile phase: sodium chloride solution with a concentration of 0.2 mol / L; chromatographic column: BRT105-103-101 tandem gel column, model 8×300 mm; flow rate: 0.8 ml / min; column temperature: 40 °C; detector: differential refractive index detector RID-10A; analysis time: 60 min; the sample concentration and injection volume are 2 mg / mL and 25 μL respectively. In this way, the high performance size exclusion chromatography elution curve of the sample, that is, the HPSEC elution curve, can be obtained by using the method provided by the present invention, so as to analyze the protein molecular weight of the graft, and the shorter the elution time of the compound with a larger protein molecular weight, and further the performance of the graft can be evaluated to determine whether all WPI are covalently grafted with SHP.

[0047] Optionally, the method for characterizing the sample specifically includes: characterizing the Maillard reaction product by Fourier transform infrared spectroscopy; mixing the freeze-dried sample with dried potassium bromide at a ratio of 1:100 in an agate mortar and grinding thoroughly, pressing a potassium bromide thin slice of the sample, and using a Fourier transform infrared spectrometer to collect spectra in the wavenumber range of 400 - 4000 cm -1 , with a resolution of 4 cm -1 , and the total number of scans is 32 times. In this way, the spectrum of the graft can be obtained by using the method provided by the present invention to verify whether a covalent grafting reaction occurs between whey protein isolate and soybean hull polysaccharide.

[0048] According to the second aspect of the present invention, a microcapsule wall material is provided. The microcapsule wall material is prepared by the above preparation method, and the microcapsule wall material is a graft obtained from the Maillard reaction product of whey protein isolate - soybean hull polysaccharide. The microcapsule wall material provided by the present invention has good emulsifying property, thermal stability, antioxidant property, antibacterial property, water solubility and storage stability in a relatively wide pH value range.

[0049] According to the third aspect of the present invention, a preparation method of a vitrified probiotic microcapsule prepared based on the Maillard reaction product is provided. The preparation method includes the following steps: activating the probiotic M times in MRS broth medium, culturing for the third set time at the fifth set temperature, centrifuging for the fourth set time at the sixth set temperature, and washing N times with physiological saline to obtain a probiotic suspension; dissolving the above microcapsule wall material in water to obtain a microcapsule wall material solution; mixing the microcapsule wall material solution and the probiotic suspension in a set ratio to obtain a mixture; culturing the mixture in a shaker incubator for the fifth set time, and freezing for the sixth set time at the seventh set temperature; freeze-drying in a freeze dryer to obtain a freeze-dried vitrified probiotic microcapsule.

[0050] Thus, the vitrified probiotic microcapsules obtained by using the preparation method of the vitrified probiotic microcapsules prepared based on the Maillard reaction products provided by the present invention have good embedding rate, survival rate after freeze-drying, thermal stability, gastrointestinal tolerance and storage stability.

[0051] Optionally, the probiotic is Lactobacillus paracasei.

[0052] Optionally, in addition to Lactobacillus paracasei, the microcapsule wall material provided in the present application can also embed other probiotics.

[0053] Optionally, M = 3 - 5; the fifth set temperature is 25 - 37 °C; the third set time is 24 - 48 h; the sixth set temperature is 4 °C; the parameter setting during centrifugation is 8000 rpm; the fourth set time is 5 - 15 min; N = 1 - 3; the set ratio is 2:1 - 6:1; the parameter settings of the shaker incubator are 37 °C and 170 r / min; the fifth set time is 1 - 3 h; the seventh set temperature is -80 °C; the sixth set time is 2 - 4 h. Through experimental verification, when the parameters are within the above numerical range, the performance of the obtained graft is better and the storage effect of the graft is better.

[0054] Preferably, M = 3; the fifth set temperature is 37 °C; the third set time is 48 h; the sixth set temperature is 4 °C; the centrifugation parameter setting is 8000 rpm; the fourth set time is 10 min; N = 2; the set ratio is 4:1; the parameter settings of the shaker incubator are 37 °C and 170 r / min; the fifth set time is 2 h; the sixth set temperature is -80 °C; the sixth set time is 2 h. Through experimental verification, when the parameters are the above values, the performance of the obtained graft is optimal, the storage effect of the graft is optimal, and the efficiency is the highest.

[0055] Optionally, the preparation method of the vitrified probiotic microcapsules prepared based on the Maillard reaction products further includes the following steps: analyzing the embedding rate of the vitrified probiotic microcapsules; analyzing the survival rate after freeze-drying of the vitrified probiotic microcapsules; analyzing the thermal stability of the vitrified probiotic microcapsules; analyzing the gastrointestinal tolerance of the vitrified probiotic microcapsules; analyzing the storage stability of the vitrified probiotic microcapsules. Thus, the present invention comprehensively analyzes the performance of the vitrified probiotic microcapsules in multiple aspects and verifies that the performance of the vitrified probiotic microcapsules provided by the present invention is better.

[0056] Optionally, the analysis of the embedding rate of the vitrified probiotic microcapsules specifically includes;

[0057] The viability of the encapsulated probiotics was determined by the standard plate count method; the vitrified probiotic microcapsules before lyophilization were serially diluted 10-fold in sterile physiological saline at a concentration of 0.85%, and then spread on MRS agar; after incubation at 37 °C for 48 h, the colony-forming units (log CFU) were calculated, that is, the number of viable bacteria in the colonies was calculated, with the unit of log CFU, and the encapsulation efficiency EE was calculated using the following formula:

[0058]

[0059] Among them, EE is the encapsulation efficiency (%), N is the number of viable bacteria encapsulated in the microcapsules, with the unit of log CFU; N1 is the number of viable bacteria added before encapsulation, with the unit of log CFU. In this way, the encapsulation rate of the vitrified probiotic microcapsules can be analyzed by using the method provided by the present invention, so as to evaluate the performance of the vitrified probiotic microcapsules.

[0060] The analysis of the post-lyophilization survival rate of the vitrified probiotic microcapsules specifically includes: determining the viability of the probiotics after lyophilization; dissolving the lyophilized vitrified probiotic microcapsules in 1 mL of phosphate buffer at a concentration of 0.2 mol / L; serially diluting 10-fold in sterile physiological saline at a concentration of 0.85%, and then spreading on MRS agar; incubating at 37 °C for 48 h; counting in units of Log CFU. In this way, the post-lyophilization survival rate of the vitrified probiotic microcapsules can be analyzed by using the method provided by the present invention, so as to evaluate the performance of the vitrified probiotic microcapsules.

[0061] Optionally, the analysis of the thermal stability of the vitrified probiotic microcapsules specifically includes: dissolving the lyophilized vitrified probiotic microcapsules in 1 mL of phosphate buffer at a concentration of 0.2 mol / L, treating at 60 °C for 30 min, and treating at 80 °C for 5 min; calculating the number of viable bacteria of the probiotics after heat treatment; counting in units of Log CFU. In this way, the thermal stability of the vitrified probiotic microcapsules can be analyzed by using the method provided by the present invention, evaluating the tolerance of the probiotics to different temperature conditions, so as to evaluate the performance of the vitrified probiotic microcapsules.

[0062] The sample solution after treatment at 60 °C for 30 min and at 80 °C for 5 min was cooled in an ice-water bath for 10 minutes to terminate the experiment, avoid the loss of activity of the probiotics, and ensure the accuracy of the measured experimental data.

[0063] Optionally, the analysis of the gastrointestinal tolerance of the vitrified probiotic microcapsules specifically includes: Preparation of simulated gastric juice: Dissolve 1 g of pepsin in 50 mL of distilled water, make up the volume to 100 mL, and adjust the pH to 2 with a 1 mol / L hydrochloric acid solution, i.e., HCl solution; Simulated intestinal solution is prepared by mixing solution A and solution B in a ratio of 2:1 (v / v) and adjusting the pH to 8.0; Among them, the preparation of solution A specifically includes: Dissolve 0.1 g of trypsin, 1.1 g of sodium bicarbonate and 0.2 g of sodium chloride in 50 mL of distilled water, make up the volume to 100 mL, and adjust the pH to 8; Among them, the preparation of solution B specifically includes: Weigh 0.9 g of bile salts and place them in a 100 mL beaker, add 50 mL of distilled water to dissolve, and then add water to make up the volume to 100 mL; Weigh 0.05 g of the embedded vitrified probiotic microcapsules, add them to a centrifuge tube containing 500 μL of simulated gastric juice, vortex and mix well, and quickly place them in a shaker incubator for cultivation. The cultivation parameters are 120 r / min and 37 °C; At the 0th, 1st, and 2nd hours of cultivation, quickly take out 100 μL of the solution, put it on ice, and count the viable bacteria count of the probiotic microcapsules in the simulated gastric juice; After culturing in the simulated gastric juice for 2 h, add an equal volume of simulated intestinal fluid to obtain a mixed solution, continue to culture for 3 h, and perform viable bacteria counting at 1 h, 2 h, and 3 h respectively; The counting is expressed in Log CFU units. In this way, the method provided by the present invention can be used to analyze the gastrointestinal tolerance of the probiotic microcapsules, so as to evaluate the performance of the probiotic microcapsules.

[0064] Optionally, the analysis of the storage stability of the vitrified probiotic microcapsules specifically includes: Store the vitrified probiotic microcapsules at 4 °C and 25 °C respectively, and measure the viable bacteria count of the microcapsules at the 0th, 1st, 2nd, 3rd, and 4th weeks; Weigh 10 mg of the vitrified probiotic microcapsule powder into 1 mL of sterile phosphate buffer solution, culture at 37 °C for 30 min, and measure the viable bacteria count by the plate counting method; The counting is expressed in Log CFU units. In this way, the method provided by the present invention can be used to analyze the storage stability of the vitrified probiotic microcapsules, so as to evaluate the performance of the vitrified probiotic microcapsules.

[0065] Optionally, in order to further verify that the vitrified probiotic microcapsules provided by the present invention have better performance, a comparative experiment was conducted. The experimental data shows that the grafted product on the fourth day provided by the present invention has the highest emulsifying property and glass transition temperature. Therefore, the grafted product on the fourth day is preferably used as the microcapsule wall material to encapsulate probiotics. When analyzing the performance of the probiotic microcapsules, the probiotic microcapsules prepared from the grafted product on the fourth day were also selected and compared with the probiotic microcapsules prepared from a mixture of whey protein isolate WPI, soybean hull polysaccharide SHP, and whey protein isolate-soybean hull polysaccharide WPI-SHP, as well as free probiotics, so as to verify the use of the grafted product of whey protein isolate-soybean hull polysaccharide on the 4th day provided by the present invention 4d Whether the prepared vitrified probiotic microcapsules have better performance.

[0066] In the comparative experiment, a whey protein isolate WPI, soybean hull polysaccharide SHP, a mixture of whey protein isolate-soybean hull polysaccharide WPI-SHP, and a grafted product of whey protein isolate-soybean hull polysaccharide on the 4th day WPI-SHP 4d solution were mixed with the suspension of Lactobacillus paracasei at a ratio of 4:1 to obtain four mixtures; the four mixtures were placed in a shaker incubator at 37 °C and 170 r / min for 2 h, and then frozen in a refrigerator at -80 °C for 2 h; freeze-dried in a freeze dryer to obtain four freeze-dried vitrified probiotic microcapsules. Among them, 3.5 g of the grafted product WPI-SHP on the 4th day 4d was dissolved in 100 mL of water to obtain a solution of the grafted product WPI-SHP with a solution ratio of 3.5% 4d solution.

[0067] According to the fourth aspect of the present invention, there is also provided a vitrified probiotic microcapsule prepared based on a Maillard reaction product, and the vitrified probiotic microcapsule is prepared according to the above or the following preparation method.

[0068] Applying the technical solution of the present invention, whey protein isolate and soybean hull polysaccharide are used in combination. Considering that the mixture obtained by simple combination is relatively sensitive to the environment, the present invention covalently modifies whey protein isolate with soybean hull polysaccharide through the Maillard reaction to obtain a Maillard reaction product of whey protein isolate - soybean hull polysaccharide, that is, a whey protein isolate - soybean hull polysaccharide graft. The obtained graft can be used as a microcapsule wall material to encapsulate probiotics. There is a strong binding force between whey protein isolate and probiotics, which can effectively improve the encapsulation rate. At the same time, polysaccharide molecules can attach to the surface of the protein core, helping to enhance the stability of the carrier, that is, the microcapsule wall material. And after covalent modification, it is less affected by environmental factors such as pH, ionic strength, and temperature, reducing the environmental sensitivity, thereby increasing the survival rate of probiotics during high temperature, gastrointestinal environment, and storage processes.

[0069] Based on the implementation manners provided in the above aspects of the present application, further combinations can be made to provide more implementation manners. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0071] Figure 1 Shows the pH change and grafting degree of the WPI - SHP mixture and the WPI - SHP graft;

[0072] Figure 2 Shows the EAI of WPI, the WPI - SHP mixture, and the WPI - SHP graft;

[0073] Figure 3 Shows the ESI of WPI, the WPI - SHP mixture, and the WPI - SHP graft;

[0074] Figure 4 Shows the HPSEC elution curves of WPI, the WPI - SHP mixture, and the WPI - SHP graft;

[0075] Figure 5 Shows the FT - IR spectra of WPI, SHP, the WPI - SHP mixture, and the WPI - SHP graft;

[0076] Figure 6 Shows the encapsulation rate of probiotic microcapsules;

[0077] Figure 7 Shows the survival rate of freeze - dried probiotics;

[0078] Figure 8 shows the viability of free and differently wall - material - embedded Lactobacillus paracasei under simulated pasteurization conditions;

[0079] Figure 9 shows the viability of free and differently wall - material - embedded Lactobacillus paracasei during simulated gastrointestinal digestion;

[0080] Figure 10 shows the viability of free and differently wall - material - embedded Lactobacillus paracasei during storage at 4 °C;

[0081] Figure 11 shows the viability of free and differently wall - material - embedded Lactobacillus paracasei during storage at 25 °C. Detailed implementation mode

[0082] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying 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 of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0083] This application belongs to the field of micro - capsule technology, and specifically relates to the application of the Maillard reaction product of whey protein isolate - soybean hull polysaccharide as a micro - capsule wall material for encapsulating probiotics and improving the survival ability of probiotics during freezing, pasteurization, simulated gastrointestinal digestion, and storage.

[0084] Currently, mostly a single protein or a single polysaccharide is used as the wall material of the micro - capsule, but the single wall material has many disadvantages. Proteins are prone to aggregation and precipitation near the isoelectric point pH or under high ionic strength, and there are often deficiencies in thermal stability; while polysaccharide macromolecules usually have a weak embedding ability for the core material. Therefore, it is necessary to optimize the material of the micro - capsule wall material to meet the usage requirements.

[0085] The glassy state is crucial for the biological activity and stability of products during freezing, drying, and storage. It is often necessary to add solutes with a high glass transition temperature (T g ) to increase the T g of the matrix, so that drying can be carried out at a higher temperature, and a final dried product with a high T g can be obtained to improve the stability of the sample during storage. Some disaccharides and oligosaccharides often have a high T gAnd it is easy to form a glassy state and serve as a lyophilization additive. However, sugar alcohols are prone to separating from the frozen solution in the form of crystals, resulting in their loss of stability after lyophilization. Soybean hull polysaccharide is a non-linear pectin-like polysaccharide with good stability and gelation properties. Soybean hull polysaccharide has a larger molecular weight compared to disaccharides and oligosaccharides and has a higher T g . Therefore, soybean hull polysaccharide as a microcapsule wall material will have a stronger protective effect on probiotics during processing or storage. Currently, there is no technical solution for applying soybean hull polysaccharide in the preparation of microcapsule wall materials.

[0086] The combined use of proteins and polysaccharides can enhance the functional properties of the delivery carrier and improve the embedding and delivery effect. In the protein-polysaccharide composite carrier, the protein has a strong binding force with the core material, which can effectively increase the embedding rate. At the same time, polysaccharide molecules can attach to the surface of the protein core, helping to enhance the stability of the carrier. However, most of the main forces involved in the formation of the protein-polysaccharide biopolymer carrier are affected by environmental factors such as pH, ionic strength, and temperature, resulting in the sensitivity of this system to the environment. Maillard reaction products are a class of complex mixtures prepared by the Maillard reaction, which are the products of the carbonyl-amine reaction between carbonyl compounds (such as reducing sugars) and amino compounds (such as peptides, amino acids, proteins, etc.). Maillard reaction products have good solubility, emulsifying properties, antioxidant properties, and potential probiotic characteristics, and can remain stable within a wide range of pH values, temperatures, and ionic strengths. They are considered good delivery carriers for bioactive substances. Therefore, in the present invention, soybean hull polysaccharide is used to covalently modify whey protein isolate through the Maillard reaction to form a whey protein isolate-soybean hull polysaccharide graft with a higher T g , and this graft is used to encapsulate probiotics, thereby improving the survival rate of probiotics under high temperature, gastrointestinal environment, and storage conditions.

[0087] The covalent modification method adopted in the present invention is to prepare a whey protein isolate-soybean hull polysaccharide graft product using the Maillard reaction. The emulsifying properties, thermal stability, antioxidant properties, antibacterial properties, water solubility, and glass transition temperature of the improved whey protein isolate have been greatly improved within a wide range of pH values. The present invention applies soybean hull polysaccharide to the Maillard reaction to improve the functional properties of WPI. By covalently grafting with SHP, the environmental sensitivity of WPI can be reduced, and good environmental stability can be imparted to the WPI graft. In view of this, the present invention provides a method for preparing a Maillard reaction product of whey protein isolate-soybean hull polysaccharide and applies it to probiotic microcapsules.

[0088] The present invention provides an embodiment 1 for a microcapsule wall material and a preparation method thereof, and an embodiment 2 for a vitrified probiotic microcapsule prepared based on a Maillard reaction product and a preparation method thereof. The embodiments 1 and 2 are described in detail below.

[0089] Embodiment 1

[0090] The invention provides a whey protein isolate-soybean seed coat polysaccharide Maillard reaction product and a preparation method thereof.

[0091] The whey protein isolate used in the present invention is whey protein isolate WPI-90; soybean seed coat polysaccharide is extracted in the laboratory and has a molecular weight of 1.1x10 6 Da.

[0092] First, the whey protein isolate-soybean seed coat polysaccharide Maillard reaction product was prepared:

[0093] Dissolve whey protein isolate and soybean seed coat polysaccharide in ultrapure water at a ratio of 1:1 (m / m), adjust the ratio to 1%, wherein 1% means that the concentration of whey protein isolate and soybean seed coat polysaccharide accounts for 1% of pure water, stir fully at room temperature of 25°C for 8h, and keep overnight at 4°C in a constant temperature magnetic stirrer, wherein overnight means more than 12h, to ensure that it is fully hydrated, adjust the solution pH to 7.0, preferably, freeze-dry after fully mixing, grind the freeze-dried powder fully and place it in a dryer (a saturated salt solution is placed at the bottom of the container, and the saturated salt solution is a KBr solution), place the dryer in a forced air drying oven and adjust the temperature to 60°C to keep the relative humidity in the dryer at 79%, and react for 1-6 days to obtain the grafted product WPI-SHP 1d 、WPI-SHP 2d 、WPI-SHP 3d 、WPI-SHP 4d 、WPI-SHP 5d 、WPI-SHP 6d The obtained product was stored in a -20°C refrigerator.

[0094] Secondly, the grafting degree and pH value of the Maillard reaction product of whey protein isolate-soybean seed coat polysaccharide were analyzed.

[0095] Weigh 40 mg of o-phthalaldehyde (OPA) and dissolve it in 1 ml of methanol. Then add 2.5 ml of 20% sodium dodecyl sulfate (SDS) solution, 0.1 mol / L borax solution, and 100 μL of β-mercaptoethanol. After mixing evenly, make up the volume to 50 mL with pure water to prepare the OPA reagent. Add 4 mL of the OPA reagent to 200 μL of the sample and mix well. Incubate in a water bath at 35 °C for 2 min. Here, the sample refers to the WPI-SHP mixture on day 0 during the reaction process and the grafted products from days 1 to 6 as samples. Use 200 μL of ultrapure water to replace the sample as a blank control, and measure the absorbance at 340 nm. Draw a standard curve with lysine as the standard and calculate the free amino group content. The calculation formula for the degree of grafting DG is as follows:

[0096]

[0097] In formula (1), the meaning of C0 is the free amino group content of the WPI-SHP mixture, and C t means the free amino group content of the WPI-SHP grafted product.

[0098] Take the samples from days 0 to 6 during the reaction process, and dilute the samples with ultrapure water to a concentration of 2 mg / mL to measure the pH value of the system. The analysis results of the degree of grafting and pH value obtained in this example are as Figure 1 shown, Figure 1 where different lowercase letters indicate significant differences (p < 0.05).

[0099] Specifically, Figure 1 shows the pH changes and degree of grafting of the WPI-SHP mixture and the WPI-SHP grafted product. According to Figure 1 it can be seen that the degree of grafting of the WPI-SHP grafted product gradually increases with the prolongation of the reaction time. In the first 2 days of the reaction, the degree of grafting rapidly increases to 25.29%, indicating that the grafted products are continuously formed during the dry-heat reaction. In the following days, the degree of grafting increases slowly because during the grafting reaction, the reactive groups in the polysaccharide and protein can only undergo covalent bonding when they come into contact. As the reaction continues, the protein molecular chain gradually unfolds, and more free amino groups are exposed, thus increasing the contact with the polysaccharide and the degree of grafting. The slow increase in the degree of grafting in the later stage indicates that most of the reactive groups in the protein have been occupied by the polysaccharide molecules. The degree of grafting directly reflects the glycosylation degree of the protein during the Maillard reaction. The higher the degree of grafting, the better the covalent grafting degree of the Maillard reaction products.

[0100] The pH changes of the WPI-SHP grafted product at different reaction times are as Figure 1As shown. As the reaction time extended, the pH showed a downward trend. In the first day of the reaction, the pH of the system decreased rapidly. Thereafter, the pH decreased slowly until the end of the reaction, and the pH decreased from 6.92 to 6.18. There were two reasons for the decrease in pH: the first reason was that sugars were degraded into acidic substances such as formic acid, acetic acid, and hydroxymethylfurfural, etc. At the same time, the acidic substances rapidly produced in the initial stage of the reaction also promoted the degradation of sugars; the second reason was that the Maillard reaction was actually a carbonyl-amine condensation reaction, and the basic group of amino acids - amino continuously combined with the carboxyl group of reducing sugars, thus causing the pH value of the reaction system to decrease. The occurrence of the Maillard reaction would lead to a decrease in pH, and the decrease in pH would enable the product to form good aroma and flavor.

[0101] Then, the emulsifying properties of the Maillard reaction product of whey protein isolate - soybean hull polysaccharide were analyzed.

[0102] The sample was dissolved in 0.01 M phosphate buffer solution (PBS) (pH 7.0) to prepare a solution with a protein concentration of 2.0 mg / mL. 5 mL of soybean oil was added to 15 mL of this solution. After homogenization at 13500 rpm for 2 min, 50 μL of the emulsion was taken from the bottom at 0 min and 10 min respectively, diluted 100 times with 0.1% (w / v) SDS solution, and its absorbance was measured at a wavelength of 500 nm. The emulsifying activity index (EAI) and emulsion stability index (ESI) were calculated according to formula (2) and formula (3) respectively:

[0103]

[0104]

[0105] where A0 and A 10 were the absorbance values at 0 min and 10 min after homogenization respectively; C was the protein concentration (g / mL); φ was the oil phase ratio; DF was the dilution factor.

[0106] The results obtained in this example were as Figure 2 and Figure 3 shown, Figure 2 showing the EAI of WPI, the WPI - SHP mixture, and the WPI - SHP graft. Figure 3 showing the ESI of WPI, the WPI - SHP mixture, and the WPI - SHP graft. In the figure, different lowercase letters indicate significant differences (p < 0.05).

[0107] For protein-polysaccharide grafts, their protein hydrophobic groups can adsorb onto the oil phase, while their strongly hydrophilic polysaccharide chains can dissolve in the aqueous phase. Therefore, one of the most remarkable characteristics of glycated proteins is their excellent emulsifying properties. As Figure 2 shown, as the grafting time extends, the EAI value shows a trend of first increasing and then decreasing. Compared with WPI and the WPI-SHP mixture, the ESI values of all WPI-SHP grafts are significantly improved, and both EAI and ESI reach the highest at a grafting time of 4 days. It can be seen that WPI-SHP 4d has the optimal emulsifying performance. This is because after SHP covalently binds to WPI, it can improve its emulsifying performance through steric stabilization and the formation of a macromolecular stable layer around the oil droplets. In addition, the better solubility of the graft will also have a positive impact on its emulsifying performance. However, the emulsifying property of the graft begins to decrease after the reaction time exceeds 5 days. This is because the solubility and surface hydrophobicity of the graft decrease after long-term heat treatment. Therefore, an appropriate balance between the hydrophobicity and hydrophilicity of the protein-polysaccharide conjugate is the key to its excellent emulsifying performance.

[0108] After that, the glass transition temperature of the Maillard reaction product of whey protein isolate-soybean hull polysaccharide was analyzed.

[0109] The differential scanning calorimeter was used to determine the glass transition temperature of the samples. Weighed 5-7 mg of the samples and sealed them in an aluminum crucible, using an empty crucible as a reference. The scanning program was set as follows: heated from room temperature to 180 °C at a rate of 10 °C / min, equilibrated at 180 °C for 5 min, cooled to 10 °C at the same rate, and equilibrated at 10 °C for 5 min, and then heated to 220 °C at a rate of 10 °C / min. The instrument's built-in software was used to analyze and obtain the midpoint within the glass transition temperature range, and this was used as T g . The results are shown in Table 1.

[0110] Table 1

[0111]

[0112] Table 1 shows the onset temperature (T0), glass transition temperature (T g ), and end temperature (T e ) of WPI, SHP, the WPI-SHP mixture, and WPI-SHP grafts. In Table 1, different lowercase letters indicate significant differences (p < 0.05).

[0113] During the freeze-drying and storage of probiotic microcapsules, T g plays an important role in the survival rate of the microencapsulated probiotics. Generally speaking, a higher T gThe wall material can inhibit the formation of large ice crystals in the liquid during the pre-freezing process, accelerate the sublimation process, and thus reduce the damage to probiotics. In the glassy state, in addition to restricting the molecular mobility, a higher T g The wall material can slow down the transition of the microcapsules from the glassy state to the rubbery state during storage. Therefore, the crystallization, aggregation, and collapse of the microcapsules caused by external factors such as moisture and air are reduced, thereby reducing the damage to probiotics from the outside. As can be seen from Table 1, the T g of whey protein isolate and soybean hull polysaccharide are 65.92 and 120.08 °C respectively. The T g of the graft products are all higher than those of whey protein isolate and soybean hull polysaccharide, and the graft product obtained by dry-heat graft reaction for 4 days has the highest T g of 140.51 °C. The graft products produced by long-term Maillard reaction have a higher T g , which has a good effect on the graft product as an embedding wall material, and SHP has good compatibility. The Maillard reaction is a complex process. With the extension of the dry-heat reaction time, graft products with larger molecular weights are formed, which is the reason for the increase in T g , which is beneficial to the preparation and storage of Lactobacillus paracasei microcapsules.

[0114] The state of a substance between the solid state and the liquid state is called the amorphous state, which generally includes three stages: the glassy state, the rubbery state, and the viscous flow state. As the heat for molecular motion increases, the substance gradually changes from the glassy state to the rubbery state. When the heat increases to a state where the substance can flow and has viscosity, its state becomes the viscous flow state. It is found that the process of the substance changing from the glassy state to the rubbery state is considered to be of great significance to the stability of the food matrix, that is, the glass transition, and the environmental temperature corresponding to this transition is called the glass transition temperature (glass transition temperature, T g ). Research shows that the glassy state is crucial for the biological activity and stability of products during freezing, drying, and storage. For example, it is often necessary to add solutes with a high T g to increase the T g of the matrix, so as to achieve drying at a higher temperature and obtain a final dried product with a high T g to improve the stability of the sample during storage. Some disaccharides and oligosaccharides are often used as freeze-drying additives because they have a high T g and are easy to form a glassy state. However, sugar alcohols are prone to separate from the frozen solution in the form of crystals, resulting in the loss of their stability after freeze-drying.

[0115] In view of this, the present invention provides a method for preparing a Maillard reaction product of whey protein isolate and soybean hull polysaccharide. The Maillard reaction product prepared by this method has good emulsifying properties and a relatively high glass transition temperature, and this product is used as an embedding wall material for probiotics to protect the activity of probiotics under conditions such as high temperature, gastrointestinal tract, and storage.

[0116] After that, the Maillard reaction product of whey protein isolate - soybean hull polysaccharide was characterized.

[0117] The Maillard reaction product of whey protein isolate - soybean hull polysaccharide was characterized by high performance size exclusion chromatography (HPSEC) and Fourier transform infrared spectroscopy (FT-IR).

[0118] The change in protein molecular weight before and after the reaction was analyzed by high performance size exclusion chromatography. The instrument used was an LC-10A high performance liquid chromatography (HPLC). Mobile phase: 0.2 M sodium chloride solution; Chromatographic column: BRT105-103-101 tandem gel column (8×300 mm); Flow rate: 0.8 ml / min; Column temperature: 40 °C; Detector: differential refractive index detector RID-10A; Analysis time: 60 min. The sample concentration and injection volume were 2 mg / mL and 25 μL, respectively.

[0119] Figure 4 The HPSEC elution curves of WPI, WPI-SHP mixture, and WPI-SHP graft were shown. According to Figure 4 It can be seen that there is only one main peak in the WPI elution curve at about 37 min. For the WPI-SHP mixture, an elution peak can be observed at 31 min. This is because there is a small amount of protein in SHP. The elution curve of the WPI-SHP graft is similar in shape to that of the WPI-SHP mixture, but with the extension of the reaction time, the second elution peak decreases significantly. In HPSEC analysis, the larger the molecular weight of the compound, the shorter the elution time. Therefore, it indicates the formation of a high molecular weight WPI-SHP graft and its content increases with the extension of the reaction time. However, the subsequent elution peak of WPI did not completely disappear, meaning that not all WPI was covalently grafted with SHP.

[0120] The Maillard reaction product was characterized by Fourier transform infrared spectroscopy. The freeze-dried sample was mixed with dried potassium bromide at a ratio of 1:100 in an agate mortar and ground thoroughly, and a potassium bromide thin slice of the sample was pressed. The spectrum was collected using a Fourier transform infrared spectrometer in the wavenumber range of 400 - 4000 cm -1 with a resolution of 4 cm -1 , and the total number of scans was 32 times.

[0121] Figure 5 The FT-IR spectra of WPI, SHP, the WPI-SHP mixture, and the WPI-SHP graft are shown. According to Figure 5 it can be seen that the absorption peak of whey protein isolate in the amide I band is located at 1649 cm -1 , corresponding to the C=O stretching vibration; the amide II band is located at 1531 cm -1 , corresponding to the N-H bending vibration, and the absorption peak at a wavenumber of 1246 cm -1 represents the amide III band, corresponding to the C-N stretching vibration and the N-H deformation vibration. The polysaccharide has a strong absorption peak at 3414 cm -1 , which is caused by the O-H vibration of the hydroxyl group. The Maillard reaction occurs between whey protein isolate and soybean seed coat polysaccharide, and the polysaccharide consumes some carbonyl and amino groups, and generates Amadori compounds (C-O), Schiff bases (C-N), and pyrazines (CN), resulting in changes in peak intensity and position. The wavenumber of whey protein isolate at 1649 cm -1 shifts to 1652 cm -1 during the progress of the Maillard reaction, which represents the formation of Schiff base with a C-N structure. After glycosylation, the absorption peak of whey protein isolate at 1531 cm -1 shifts to 1537 cm -1 , indicating the depletion of amino acid residues during the Maillard reaction. The above results all prove the occurrence of the covalent grafting reaction between whey protein isolate and soybean seed coat polysaccharide.

[0122] The occurrence of the Maillard reaction was proved by the grafting degree, high performance size exclusion chromatography, and Fourier transform infrared spectroscopy. The graft of whey protein isolate-soybean seed coat polysaccharide for 4 days has the highest emulsifying property and glass transition temperature. Based on this, the graft of the Maillard reaction of whey protein isolate-soybean seed coat polysaccharide for 4 days is preferably used to encapsulate Lactobacillus paracasei.

[0123] Example 2

[0124] Example 2 elaborates in detail on the glassy probiotic microcapsules prepared based on the Maillard reaction product and its preparation method. In this example, the probiotic is Lactobacillus paracasei. In other alternative examples, other types of probiotics can also be used.

[0125] First, culture Lactobacillus paracasei:

[0126] Lactobacillus paracasei was activated twice in MRS broth medium. After culturing at 37 °C for 48 h, it was centrifuged at 8000 rpm for 10 min at 4 °C and then washed twice with physiological saline.

[0127] Then, prepare the probiotic microcapsules:

[0128] The whey protein isolate (WPI), soybean hull polysaccharide (SHP), whey protein isolate - soybean hull polysaccharide mixture (WPI - SHP), and whey protein isolate - soybean hull polysaccharide grafted product after 4 days of Maillard reaction (WPI - SHP 4d ) solutions (3.5%, w / v) were respectively mixed with the suspension of Lactobacillus paracasei at a ratio of 4:1. Subsequently, the mixture was cultured in a shaking incubator at 37°C and 170 r / min for 2 h, and then frozen overnight in a refrigerator at -80°C. The samples were freeze-dried in a freeze dryer to obtain four kinds of freeze-dried probiotic microcapsules.

[0129] In this example, whey protein isolate, soybean hull polysaccharide, whey protein isolate - soybean hull polysaccharide mixture, and the grafted product of whey protein isolate - soybean hull polysaccharide after 4 days of Maillard reaction were selected to encapsulate Lactobacillus paracasei, obtaining four kinds of probiotic microcapsules. Thus, by analyzing the performance of the four kinds of probiotic microcapsules subsequently, it was verified that the performance of the probiotic microcapsules provided by the present invention was better.

[0130] First, the encapsulation rate of the probiotic microcapsules was analyzed:

[0131] The viability of Lactobacillus paracasei encapsulated in the probiotic microcapsules was determined by the standard plate counting method. The probiotic microcapsules before freeze-drying were serially diluted 10-fold in sterile normal saline (0.85%, w / v), and then spread on MRS agar. After incubation at 37°C for 48 h, the colony-forming units (CFU) were calculated. The calculation formula for the encapsulation efficiency (EE) is as follows. (The results are shown in Figure 5 )

[0132]

[0133] Among them, EE is the encapsulation efficiency (%), N is the number of viable bacteria encapsulated in the microcapsules (log CFU), and N1 is the number of viable bacteria added before encapsulation (log CFU).

[0134] Figure 6 The encapsulation rate of the probiotic microcapsules is shown, Figure 6 in which different lowercase letters indicate significant differences (p < 0.05). According to Figure 6 it can be seen that the high encapsulation rate of probiotics in the product after 4 days of Maillard reaction is 94.01% ± 2.66%. Compared with the other three kinds of probiotics, WPI - SHP 4d has the best encapsulation effect.

[0135] After that, the survival rate of the probiotic microcapsules after freeze-drying was analyzed:

[0136] Determine the viability of Lactobacillus paracasei before and after lyophilization. The obtained microcapsules were dissolved in 1 mL of phosphate buffer (0.2 mol / L). Serial dilutions of 10-fold were made in sterile saline (0.85%, w / v), and then plated on MRS agar. Incubate at 37 °C for 48 h. The count was expressed in Log CFU units.

[0137] Figure 7 The survival rate of probiotics after lyophilization is shown. Figure 7 In it, different lowercase letters indicate significant differences (p < 0.05). Figure 7 It can be seen that the survival rates of Lactobacillus paracasei under the protection of WPI, SHP, WPI-SHP, and WPI-SHP 4d were 53.99% ± 0.19%, 63.88% ± 0.34%, 76.48% ± 1.01%, and 87.35% ± 1.73% respectively. The survival rate of free Lactobacillus paracasei (naked) was only 24.95% ± 0.55%. Compared with single-wall materials and WPI-SHP mixtures, the encapsulation rate and survival rate after freeze-drying of Lactobacillus paracasei embedded with Maillard reaction products for 4 days were higher. This is because covalent bonds are formed between whey protein isolate and soy chitosan, resulting in a larger molecular weight of the Maillard reaction product and a higher T g which provides better protection for Lactobacillus paracasei. In addition, as a probiotic factor, the Maillard reaction product can be embedded in Lactobacillus paracasei to play a protective role and can also provide nutrients for its growth and reproduction.

[0138] After that, the thermal stability of the probiotic microcapsules was analyzed:

[0139] The microcapsules after freeze-drying were dissolved in 1 mL of phosphate buffer (0.2 mol / L), treated at 60 °C for 30 min, and at 80 °C for 5 min. Then, the samples were cooled in an ice-water bath for 10 minutes to evaluate the tolerance of free and encapsulated Lactobacillus paracasei to different temperature conditions. Calculate the viable counts of free and encapsulated Lactobacillus paracasei after heat treatment. The count was expressed in Log CFU units.

[0140] Figure 8 The survival ability of free and Lactobacillus paracasei encapsulated with different wall materials under simulated pasteurization conditions is shown. Figure 8 In it, different lowercase letters indicate significant differences (p < 0.05). According to Figure 8 It can be seen that after heat treatment at 60 °C for 30 min, the free Lactobacillus paracasei decreased from the initial 10.94 log CFU / mL to 9.13 log CFU / mL. The 4 kinds of microcapsules also had different protective effects on Lactobacillus paracasei, among which WPI-SHP 4dIt has the best protective effect on Lactobacillus paracasei during the entire heat treatment process. The viable cell count only decreases by 0.18 log CFU / mL. After heat treatment at 80 °C for 5 min, the free Lactobacillus paracasei decreases by 6.57 log CFU / mL. However, the Maillard reaction products significantly improve the survival rate of probiotics. After heat treatment for 5 min, the viable cell count of Lactobacillus paracasei remains at 6.19 log CFU / mL.

[0141] After that, the gastrointestinal tolerance of the probiotic microcapsules was analyzed:

[0142] Preparation of simulated gastric juice: Dissolve 1 g of pepsin in 50 mL of distilled water, make up the volume to 100 mL, and adjust the pH to 2 with 1 mol / L hydrochloric acid. The simulated intestinal solution is prepared by mixing solution A and solution B at a ratio of 2:1 (v / v) and adjusting the pH to 8.0. Preparation of solution A: Dissolve 0.1 g of trypsin, 1.1 g of sodium bicarbonate, and 0.2 g of sodium chloride in 50 mL of distilled water, make up the volume to 100 mL, and adjust the pH to 8. Preparation of solution B: Weigh 0.9 g of bile salt into a 100 mL beaker, add 50 mL of distilled water to dissolve it, and then make up the volume to 100 mL with water.

[0143] Weigh 0.05 g of Lactobacillus paracasei microcapsules encapsulated with WPI, SHP, WPI-SHP, WPI-SHP 4d and add them to a centrifuge tube containing 500 μL of simulated gastric juice. Vortex and mix well, and quickly place it in a shaking incubator for cultivation. The cultivation parameters are 120 r / min and 37 °C. At 0, 1, and 2 h of cultivation, quickly take out 100 μL of the solution, put it on ice, and count the viable cell count of the probiotic microcapsules in the simulated gastric juice. After culturing in the simulated gastric juice for 2 h, add an equal volume of simulated intestinal juice to obtain a mixed solution, continue to culture for 3 h, and perform viable cell counting at 1, 2, and 3 h respectively. The counting is expressed in Log CFU units.

[0144] Figure 9 Shows the survival ability of free and Lactobacillus paracasei encapsulated with different wall materials during simulated gastrointestinal digestion.

[0145] According to Figure 9It can be seen that the viable count of free *Lactobacillus paracasei* was 9.91 log CFU / mL at 0 h in simulated gastric juice, and decreased to 9.66 and 8.39 log CFU / mL at 1 h and 2 h, respectively. Within 3 hours after *Lactobacillus paracasei* entered the simulated intestinal fluid, the viable count decreased from 8.39 log CFU / mL to 6.74 log CFU / mL. In addition, after the *Lactobacillus paracasei* encapsulated with different wall materials was digested by the gastrointestinal tract, the difference in the viable count among different samples was not significant. During the whole digestion process, the viable count of free probiotics decreased by 3.17 log CFU / mL, and the survival rate of probiotics after being encapsulated with different wall materials was greater than 7 log CFU / mL. The in vitro digestion results showed that microencapsulation could improve the resistance of probiotics to gastrointestinal diseases, enabling sufficient probiotics to enter the human body and be slowly released in the intestine. Microencapsulation improved the survival rate of probiotics under acidic conditions because the wall material of the microcapsule hindered the diffusion of acid into the interior of the microcapsule, making the internal pH value of the microcapsule higher than the external pH value. In addition, the interaction between probiotics and the wall material could improve the tolerance of probiotics to gastric juice. The presence of microcapsules slowed down the diffusion of bile salts, thereby slowing down the damage of bile salts to the cell wall of probiotics and protecting the probiotics.

[0146] After that, the storage stability of the probiotic microcapsules was analyzed:

[0147] The freeze-dried microcapsules were stored at 4 °C and 25 °C respectively, and the viable count of the microcapsules was measured at 0, 1, 2, 3, and 4 weeks. Weighed 10 mg of microcapsule powder into 1 mL of sterile phosphate buffer solution, cultured at 37 °C for 30 min, and the viable count was measured by the plate counting method. The counting was expressed in Log CFU units.

[0148] Figure 10 Shows the viability of free and *Lactobacillus paracasei* encapsulated with different wall materials during storage at 4 °C.

[0149] According to Figure 10 It can be seen that with the extension of the storage time, the viability of *Lactobacillus paracasei* gradually decreased. After being stored at 4 °C for 4 weeks, the viable count of free probiotics decreased from 10.41 log CFU / mL to 8.10 log CFU / mL. And compared with the single wall material and the mixture, the Maillard reaction products had a stronger protective effect on probiotics, and its viable count was still > 10 log CFU / mL after being stored for 4 weeks, meeting the requirements of probiotic products in terms of viable count and health benefits.

[0150] Figure 11 Shows the viability of free and *Lactobacillus paracasei* encapsulated with different wall materials during storage at 25 °C.

[0151] According to Figure 11It can be seen that after storage at 25 °C for 4 weeks, the free Lactobacillus paracasei decreased to 4.2 log CFU / mL. The encapsulated probiotics showed higher survival rates than the free probiotics, and the viable cell numbers were all > 6 log CFU / mL. And the T of the wall material g plays a crucial role in the protection of probiotics. The Maillard reaction products generated by the reaction have a larger molecular weight and a higher T g . The molecular mobility of the microcapsule wall material decreases, the molecular movement is restricted, and the glassy state of the microcapsule is more stable, providing more effective protection for Lactobacillus paracasei.

[0152] In summary, the probiotic microcapsules prepared from the products of the Maillard reaction for 4 days can achieve a high encapsulation rate of probiotics, enhance the resistance of probiotics to high temperature and gastrointestinal environment, ensure that a sufficient number of probiotics reach the target site, and improve their storage stability.

[0153] In the present invention, Maillard reaction products are prepared from whey protein isolate - soybean hull polysaccharide, and the structures of the grafts are characterized by grafting degree, high performance size exclusion chromatography, and Fourier transform infrared spectroscopy; the physicochemical properties of the grafts are characterized by emulsifying properties and glass transition temperature. Lactobacillus paracasei is encapsulated using whey protein isolate, soybean hull polysaccharide, a mixture of whey protein isolate - soybean hull polysaccharide, and a whey protein isolate - soybean hull polysaccharide graft, respectively, and their encapsulation rates, survival rates after freeze-drying, thermal stability, gastrointestinal tolerance, and storage stability are analyzed.

[0154] This application belongs to the technical field of microcapsules, and specifically relates to the application of Maillard reaction products of whey protein isolate - soybean hull polysaccharide as microcapsule wall materials for encapsulating probiotics and improving the survival ability of probiotics in freezing, pasteurization, simulated gastrointestinal digestion, and storage.

[0155] The physical and chemical properties of the microcapsule wall material directly affect the stability of the microcapsules, the retention efficiency of the core material, and the shelf life. The wall material must have suitable rheological properties at high concentrations and be able to emulsify the active material, stabilize the produced emulsion, and keep the core material within its structure during processing or storage without being damaged. Whey protein isolate is often used as a wall material in the food industry for the encapsulation of probiotics and bioactive substances. However, WPI is vulnerable to processing methods such as high temperature, high acid, and salt during food processing, resulting in unstable product properties, which to a certain extent limits the application of WPI. However, the preparation of protein-polysaccharide graft products by the Maillard reaction can effectively improve the emulsifying property, thermal stability, antioxidant property, antibacterial property, and water solubility of proteins within a wide pH range. At present, although the graft products of whey protein isolate with maltodextrin, carboxymethyl cellulose, soy soluble polysaccharide, etc. have obtained a high encapsulation rate and good protection for bioactive substances or probiotics, no study has been conducted on the interrelationship between the glass transition of WPI-polysaccharide graft products as microcapsule wall materials and the stability of the core material during processing or storage. Soy hull polysaccharide (SHP) is a non-linear pectin-like polysaccharide with good stability and gelation properties, and is widely used in many fields such as food and medicine. Covalent grafting with SHP can reduce the environmental sensitivity of WPI and endow the WPI graft with good environmental stability. In addition, soy hull polysaccharide has a larger molecular weight than disaccharides and oligosaccharides, and the WPI-SHP covalent graft has a higher T g . Therefore, the microcapsule wall material based on the Maillard reaction product of WPI-SHP will have stronger protection for probiotics during processing or storage. By studying the glass transition of the microcapsule wall material, the stability of Lactobacillus paracasei microcapsules during processing and storage was studied, in order to provide a theoretical basis for the utilization of Lactobacillus paracasei, thereby strengthening the industrial development and comprehensive utilization of Lactobacillus paracasei.

[0156] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the said claims.

[0157] Unless otherwise defined, technical terms or scientific terms used in this disclosure shall have the ordinary meanings as understood by those of ordinary skill in the art to which this disclosure pertains. The "first", "second" and similar terms used in this disclosure do not denote any order, quantity or importance, but are merely used to distinguish different components. Similarly, words such as "a", "an" or "the" do not denote a quantity limitation, but rather denote the presence of at least one. Words such as "comprising" or "including" mean that the elements or items appearing before this word cover the elements or items listed after this word and their equivalents, without excluding other elements or items. Words such as "connected" or "coupled" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Upper", "lower", "left", "right", etc. are only used to indicate relative positional relationships, and when the absolute position of the object being described changes, the relative positional relationship also changes accordingly.

Claims

1. A preparation method of vitrified probiotic microcapsules prepared based on Maillard reaction products, using a microcapsule wall material. The preparation method of the microcapsule wall material specifically includes the following steps: Dissolve whey protein isolate and soybean hull polysaccharide in water; stir at a constant temperature for a first set time under a first set temperature condition; let stand for a second set time at a second set temperature to obtain a mixed solution; Adjust the pH of the mixed solution to a first set pH value; Perform freeze-drying treatment on the mixed solution after adjusting the pH value to obtain a freeze-dried powder; Grind the freeze-dried powder and place it in a dryer. Place a saturated salt solution at the bottom of the dryer. Place the dryer in a drying oven. Set the temperature of the drying oven to a third set temperature and the relative humidity of the drying oven to a first set humidity. The graft obtained after the Maillard reaction of whey protein isolate and soybean hull polysaccharide for a first set number of days is used as the microcapsule wall material; The microcapsule wall material is a graft obtained from the Maillard reaction product of whey protein isolate - soybean hull polysaccharide; A preparation method of vitrified probiotic microcapsules prepared based on Maillard reaction products, characterized in that the preparation method includes the following steps: Activate the probiotics M times in MRS broth medium, culture for a third set time at a fifth set temperature, then centrifuge for a fourth set time at a sixth set temperature, and wash N times with physiological saline to obtain a probiotic suspension; Dissolve the microcapsule wall material in water to obtain a microcapsule wall material solution; mix the microcapsule wall material solution and the probiotic suspension in a set ratio to obtain a mixture; Cultivate the mixture in a shaking incubator for a fifth set time, and then freeze at a seventh set temperature for a sixth set time; Perform freeze-drying in a freeze-dryer to obtain freeze-dried vitrified probiotic microcapsules.

2. According to the preparation method described in claim 1, characterized in that, M=3-5； The fifth set temperature is 25 - 37 °C; The third set time is 24 - 48 h; The sixth set temperature is 4 °C; The parameter setting for centrifugation is 8000 rpm; The fourth set time is 5 - 15 min; N=1-3； The set ratio is 2:1 - 6:1; The parameter settings of the shaking incubator are 37 °C and 170 r / min; The fifth set time is 1 - 3 h; The seventh set temperature is -80 °C; The sixth set time is 2 - 4 h.

3. The preparation method according to claim 1 or 2, characterized in that, The preparation method further includes the following steps: Analyze the encapsulation rate of the vitrified probiotic microcapsules; Analyze the survival rate after freeze-drying of the vitrified probiotic microcapsules; Analyze the thermal stability of the vitrified probiotic microcapsules; Analyze the gastrointestinal tolerance of the vitrified probiotic microcapsules; Analyze the storage stability of the vitrified probiotic microcapsules.

4. According to the preparation method described in claim 3, characterized in that, The analysis of the encapsulation rate of the vitrified probiotic microcapsules specifically includes; The viable count of probiotics after encapsulation was determined by the standard plate count method; the vitrified probiotic microcapsules before freeze-drying were serially diluted 10-fold in sterile physiological saline at a concentration of 0.85%, and then spread on MRS agar; after incubation at 37 °C for 48 h, the number of viable bacteria in the colonies was counted, with the unit of log CFU, and the encapsulation efficiency was calculated using the following formula : （4）； where EE is the encapsulation efficiency (%), is the number of viable bacteria encapsulated in the microcapsules, in units of log CFU; is the number of viable bacteria added before encapsulation, in units of log CFU; The analysis of the survival rate after freeze-drying of the vitrified probiotic microcapsules specifically includes: Determine the viability of freeze-dried probiotics; dissolve the freeze-dried vitrified probiotic microcapsules in 1 mL of phosphate buffer solution with a concentration of 0.2 mol / L; serially dilute 10-fold in sterile physiological saline with a concentration of 0.85%, and then spread on MRS agar; incubate at 37 °C for 48 h; count in Log CFU units; The analysis of the thermal stability of the vitrified probiotic microcapsules specifically includes: Dissolve the freeze-dried vitrified probiotic microcapsules in 1 mL of phosphate buffer solution with a concentration of 0.2 mol / L, treat at 60 °C for 30 min, and treat at 80 °C for 5 min; calculate the viable count of probiotics after heat treatment; count in Log CFU units; The analysis of the gastrointestinal tolerance of the vitrified probiotic microcapsules specifically includes: Preparation of simulated gastric juice: Dissolve 1 g of pepsin in 50 mL of distilled water, make up the volume to 100 mL, and adjust the pH to 2 with a 1 mol / L hydrochloric acid solution; Simulated intestinal solution is prepared by mixing solution A and solution B in a ratio of 2:1 and adjusting the pH to 8.0; among them, the preparation of solution A specifically includes: dissolve 0.1 g of trypsin, 1.1 g of sodium bicarbonate and 0.2 g of sodium chloride in 50 mL of distilled water, make up the volume to 100 mL, and adjust the pH to 8; among them, the preparation of solution B specifically includes: weigh 0.9 g of bile salt in a 100 mL beaker, add 50 mL of distilled water to dissolve, and then add water to make up the volume to 100 mL; Weigh 0.05 g of the embedded vitrified probiotic microcapsules, add them to a centrifuge tube containing 500 μL of simulated gastric juice, vortex and mix well, quickly place them in a shaking incubator for cultivation, and the cultivation parameters are 120 r / min, 37 °C; at 0, 1, and 2 h of cultivation, quickly take out 100 μL of the solution, put it on ice, and count the viable count of the probiotic microcapsules in the simulated gastric juice; after culturing in the simulated gastric juice for 2 h, add an equal volume of simulated intestinal juice to obtain a mixed solution, continue to culture for 3 h, and perform viable count at 1, 2, and 3 h respectively; count in Log CFU units; The analysis of the storage stability of the vitrified probiotic microcapsules specifically includes: Store the vitrified probiotic microcapsules at 4 °C and 25 °C respectively, and measure the viable count of the microcapsules at 0, 1, 2, 3, and 4 weeks; weigh 10 mg of the vitrified probiotic microcapsule powder in 1 mL of sterile phosphate buffer solution, culture at 37 °C for 30 min, and determine the viable count by the plate counting method; count in Log CFU units.

5. A capsule prepared by the method for preparing a vitrified probiotic microcapsule according to claim 1 based on a Maillard reaction product, characterized in that, The vitrified probiotic microcapsules are prepared according to the preparation method described in claim 1 or 2.

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