Method for preparing synbiotics microcapsule based on Maillard reaction of protein and oligosaccharide and application of synbiotics microcapsule

Abstract

The invention provides a method for preparing a synbiotics microcapsule based on a Maillard reaction of protein and oligosaccharide and application, and belongs to the technical field of microbial preparations. The preparation method of the synbiotics microcapsule comprises the following steps: mixing galactooligosaccharide and soybean protein isolate to prepare a protein solution, stirring, and hydrating at 4 DEG C overnight; adjusting the hydrated overnight solution to be alkaline, and carrying out wet-process Maillard reaction at 80-90 DEG C to form a protein glycosylation wall material; the method comprises the following steps: drying a protein glycosylation wall material, preparing a wall material solution by using a calcium chloride solution, and mixing a microbial bacterial liquid, D-(+)-gluconic acid delta-lactone and the wall material solution to obtain a mixed solution; mixing soybean oil and span 80 to obtain an oil phase, pouring the mixed solution into the oil phase, stirring to form microspheres, removing the oil phase, and drying to obtain the synbiotics microcapsule. The wall material for preparing the microcapsule is low in raw material cost, mild in reaction condition, suitable for industrial production, high in microorganism embedding rate and capable of adjusting intestinal microecology.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial preparations, and particularly relates to a method for preparing a probiotic microcapsule based on a protein and oligosaccharide Maillard reaction and application thereof. BACKGROUND

[0002] In recent years, microcapsule technology has shown significant advantages in the field of stabilization and controlled release of bioactive substances. Microcapsules prepared based on the Maillard reaction are widely used in the encapsulation and protection of sensitive ingredients such as probiotics due to their simple process, high encapsulation efficiency, and excellent biocompatibility. Probiotics, as active microorganisms beneficial to the health of the host, are easily inactivated by external environments such as high temperature, gastric acid, and oxidative conditions. Therefore, effective encapsulation is the key to expanding the application scenarios of probiotics.

[0003] Currently, more research is focused on binary polymer systems of protein-monosaccharide or protein-polysaccharide, which form covalently cross-linked microcapsule shells through the Maillard reaction. However, the development of protein-oligosaccharide systems in the study of probiotic microcapsules mediated by the Maillard reaction is still in its infancy, and relevant research reports are limited.

[0004] It is worth noting that oligosaccharides, as carbohydrates composed of 2-10 monosaccharides connected by glycosidic bonds, have better biological activity than monosaccharides. Compared with polysaccharides, oligosaccharides have a smaller molecular weight and lower steric hindrance, exhibit a faster reaction rate in the Maillard reaction, and can form stable cross-linked structures in a shorter time. The multiple hydroxyl properties in the molecular structure of oligosaccharides can enhance the hydrophilicity and membrane barrier properties of microcapsules, and theoretically make them more suitable as substitutes for polysaccharides in the Maillard reaction. Therefore, in-depth exploration of the technical path for preparing probiotic microcapsules through protein-oligosaccharide Maillard reaction is of great significance in filling the research gap in this field and promoting the upgraded application of microcapsule technology in probiotic encapsulation. SUMMARY

[0005] In view of the above, one of the purposes of the present application is to provide a preparation method of probiotic microcapsules.

[0006] The second purpose of the present application is to provide probiotic microcapsules prepared by the preparation method.

[0007] The third purpose of the present application is to provide the application of the preparation method or the probiotic microcapsules in the preparation of products for regulating intestinal flora.

[0008] In order to achieve the above-mentioned purposes of the application, the present application provides the following technical solutions:

[0009] A preparation method of probiotic microcapsules, comprising the following steps:

[0010] Mixing oligogalactose and soybean protein isolate to prepare a protein solution, stirring and hydrating overnight at 4℃;

[0011] Adjusting the overnight-hydrated solution to alkaline, and performing a wet Maillard reaction at 80-90℃ to form a protein glycosylation wall material;

[0012] After drying the protein glycosylation wall material, using a calcium chloride solution to prepare a wall material solution, mixing the microbial solution, D-(+)-glucono-δ-lactone and the wall material solution to obtain a mixed solution;

[0013] Mixing soybean oil and Span 80 to obtain an oil phase, pouring the mixed solution into the oil phase, stirring to form microspheres, removing the oil phase, and drying to obtain the synbiotic microcapsules.

[0014] Preferably, the oligogalactose and soybean protein isolate are mixed at a mass ratio of (1:2) to (2:1).

[0015] Preferably, the protein content in the protein solution is 5-10% w / v.

[0016] Preferably, the reaction time of the wet Maillard reaction is 60-180 min.

[0017] Preferably, the concentration of the calcium chloride solution is 13-20 mmol / L, and the protein concentration in the wall material solution is 5-10% w / v.

[0018] Preferably, the volume ratio of the microbial solution to the wall material solution is 1:9, the microbial content in the microbial solution is ≥10 9 / mL, and the content of D-(+)-glucono-δ-lactone in the mixed solution is 0.3-0.7% w / v.

[0019] Preferably, the microorganism in the microbial solution includes Lactobacillus acidophilus.

[0020] Preferably, the content of Span 80 in the oil phase is 0.1-0.5% w / v, and the volume ratio of the oil phase to the mixed solution is 4:1.

[0021] The application also provides the synbiotic microcapsules prepared by the preparation method.

[0022] The application also provides the use of the preparation method or the synbiotic microcapsules in preparing a product for regulating intestinal flora.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The present application provides a preparation method of a synbiotic microcapsule, which uses a Maillard reaction product of oligosaccharide and protein with synbiotic effect as the wall material of probiotic microcapsulation to prepare a novel synbiotic preparation.

[0025] The synbiotic microcapsule prepared by the method of the present application can not only be utilized by the embedded probiotics, but also can regulate intestinal microecology, and the prepared synbiotic microcapsule is a novel synbiotic preparation, which has a broad market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The table shows the analysis results of the Maillard reaction product at different times; wherein, a is the pH value of the reaction product; b is the intermediate index of the reaction product; c is the browning index of the reaction product; d is the GD value of the reaction product;

[0027] Figure 2 The table shows the scanning electron microscope images of the microcapsules; wherein, a is the SPI-embedded microcapsule; b is the SGM-embedded microcapsule;

[0028] Figure 3 The table shows the embedding rates of different microcapsules;

[0029] Figure 4 The table shows the storage stability of different microcapsules;

[0030] Figure 5 The table shows the gastrointestinal fluid tolerance of the microcapsules;

[0031] Figure 6 The table shows the diversity of intestinal microbiota; wherein, a is the sparsity curve; b is the PCA principal component analysis; c is the Simpson index; d is the Shannon index;

[0032] Figure 7 The table shows the analysis of intestinal flora phylum and genus; wherein, a is the phylum difference; b is the genus difference. DETAILED DESCRIPTION

[0033] The application provides a preparation method of a synbiotic microcapsule, comprising the following steps: mixing oligogalactose and soybean protein isolate to prepare a protein solution, stirring, and hydrating overnight at 4 DEG C; adjusting the solution after overnight hydration to be alkaline, performing a wet Maillard reaction at 80-90 DEG C to form a protein glycosylation wall material; after drying the protein glycosylation wall material, using a calcium chloride solution to prepare a wall material solution, mixing microbial liquid, D-(+)-glucono delta-lactone and the wall material solution to obtain a mixed solution; mixing soybean oil and Span 80 to obtain an oil phase, pouring the mixed solution into the oil phase, stirring to form microspheres, removing the oil phase, and drying to obtain the synbiotic microcapsule.

[0034] In the preparation method of the synbiotic microcapsule, the oligogalactose and the soybean protein isolate are preferably mixed at a mass ratio of (1:2) to (2:1), and in some examples, the mass ratio can be 1:2 or 2:1. The solution after overnight hydration is preferably adjusted to be alkaline by using a 6M NaOH solution, and the alkalinity is preferably that the pH value of the solution is 8-11. The protein content in the protein solution is preferably 5%-10% w / v, and in some examples, the protein content can be 5% w / v, 6% w / v, 7% w / v, 8% w / v, 9% w / v or 10% w / v. In some examples, the wet Maillard reaction is preferably performed at 80 DEG C, 85 DEG C or 90 DEG C; the reaction time of the wet Maillard reaction is preferably 60-180 min, more preferably 100-160 min, and further preferably 150 min. The concentration of the calcium chloride solution is preferably 13-20 mmol / L, and more preferably 15 mmol / L; the protein concentration in the wall material solution is 5%-10% w / v, and in some examples, the protein concentration can be 5% w / v, 6% w / v, 7% w / v, 8% w / v, 9% w / v or 10% w / v. The volume ratio of the microbial liquid to the wall material solution is preferably 1:9; the microbial content in the microbial liquid is greater than or equal to 10 9 / mL; the content of D-(+)-glucono delta-lactone in the mixed solution is preferably 0.3%-0.7% w / v, and more preferably 0.5% w / v; the microorganism in the microbial liquid includes Lactobacillus acidophilus. The content of Span 80 in the oil phase is preferably 0.1%-0.5% w / v, and more preferably 0.2% w / v; the volume ratio of the oil phase to the mixed solution is preferably 4:1.

[0035] In the application, the protein-oligosaccharide Maillard reaction product is used as a probiotic embedding material, the low digestibility of the Maillard reaction product allows the microcapsule to enter the colon completely, and the Maillard reaction product in the application includes a carbohydrate and a protein substrate, which can be utilized by intestinal microorganisms to affect the health of a host.

[0036] The application further provides the synbiotic microcapsule prepared by the preparation method, and the synbiotic microcapsule prepared by the application has high embedding rate, can well preserve the activity of microorganisms, has high resistance to gastrointestinal juice digestion, and can significantly regulate the diversity of intestinal flora.

[0037] The application further provides application of the preparation method or the synbiotic microcapsule in preparation of a product for regulating intestinal flora, and the product includes food and medicine.

[0038] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0039] Example 1

[0040] A preparation method of a synbiotic microcapsule.

[0041] Preparation of a synbiotic wall material:

[0042] Soybean protein isolate (SPI) and galacto-oligosaccharide (GOS) are dissolved in sterile deionized water at a mass ratio of 1:2, the SPI content in the dissolved solution is 5% (w / v), and the GOS content is 10% (w / v), and the mixture is mixed overnight in an environment at 4°C. The pH of the solution is adjusted to 8.0 by using a 6M NaOH solution. Then, the solution is placed in a 90°C water bath for 150 min to obtain a SPI-GOS Maillard (SGM) conjugate, which is freeze-dried into a powder (i.e., a wall material powder) for standby.

[0043] Preparation of a synbiotic microcapsule:

[0044] The freeze-dried wall material powder is fully dissolved in a 15mmol / L CaCl2 solution to prepare a wall material solution with a protein concentration of 8% (w / v).

[0045] Lactobacillus acidophilus (LAC) cultured for 24h is centrifuged (5000rpm) at 4°C, the supernatant is discarded, and the bacteria are resuspended in normal saline to adjust the final concentration to 1×10 9 CFU / mL. 1mL of the resuspended bacteria solution is taken and added to 9mL of the wall material solution together with D-(+)-glucono-δ-lactone to make the concentration of D-(+)-glucono-δ-lactone in the solution 0.5% (w / v), and the mixture is fully stirred at a speed of 300rpm for 1h to obtain a mixed solution. 0.2% (w / v) of Span 80 is added to 40mL of soybean oil, and the mixture is stirred at a speed of 800rpm for 30min to obtain an oil phase. Then, 10mL of the mixed solution is slowly poured into the oil phase during stirring, and the mixture is stirred at a speed of 800rpm for 3h to form microspheres. Finally, the upper oil phase is decanted, and the remaining oil phase is removed by repeatedly washing with sterile water to obtain the microcapsule of LAC, which is freeze-dried to obtain a microcapsule powder.

[0046] In this embodiment, the concentration of each reagent is its working concentration.

[0047] Example 2

[0048] Characterization of Biostime microcapsules.

[0049] 1. Synbiotic wall material.

[0050] Soy protein isolate (SPI) and galactooligosaccharides (GOS) were dissolved in sterile deionized water at a mass ratio of 1:2. The resulting solution contained 5% (w / v) SPI and 10% (w / v) GOS. The mixture was incubated overnight at 4°C. The pH of the solution was adjusted to 8.0 with 6M NaOH solution. The solution was then placed in a 90°C water bath for 180 min. During heating, samples were collected every 30 min and immediately transferred to an ice-water bath to stop the reaction. The content of intermediate products, the degree of browning, and the grafting degree were measured to determine the extent of the Maillard reaction. The results are as follows: Figure 1 As shown.

[0051] according to Figure 1 The results show that after 150 min of reaction, the content of intermediate products and the degree of browning both reach their first maximum values. At this time, the grafting degree value (GD value) of SGM also reaches its maximum value of 18.1 ± 0.73%. Therefore, 150 min is the optimal reaction time for microcapsule wall materials.

[0052] 2. Biostime Microcapsules.

[0053] Synbiotic microcapsules (denoted as SGM-encapsulated microcapsules) were prepared using the method of Example 1. Microcapsules prepared using a single soybean protein (SPI) as the wall material were used as the control group (denoted as SPI-encapsulated microcapsules). The steps for encapsulating microorganisms were the same as in Example 1.

[0054] After freeze-drying, the microcapsule powder was attached to the sample stage using conductive adhesive, sputter-coated with gold, and then the morphology of the microcapsules was observed using a scanning electron microscope at an accelerating voltage of 15 kV. The results are as follows. Figure 2 As shown.

[0055] according to Figure 2 The results show that the microcapsules prepared in Example 1 of the present invention do not present a smooth granular shape, but rather an irregular blocky structure. This appearance is due to the fact that some of the wall material has not undergone sufficient Maillard reaction. This morphology is similar to the Maillard reaction microcapsules reported in the prior art. Some studies have used the Maillard reaction products of gelatin / gum arabic to prepare pine nut oil microcapsules, which also present an irregular sheet-like structure.

[0056] Then, 500 mg of the two types of microcapsules prepared above were added to 5 mL of sterile physiological saline, mixed thoroughly, and homogenized for 45 s using a high-speed homogenizer to break the microcapsules and release free LAC. The encapsulation efficiency of the microcapsules obtained by coating and counting the gradient dilution lysate is as follows: Figure 3 As shown. By Figure 3 It can be seen that the encapsulation rate of the single SPI wall material is 53.0% ± 3.1%, while SGM significantly improved the encapsulation rate of LAC to 66.7% ± 3.7%, indicating that the Maillard reaction can improve the encapsulation rate of microorganisms.

[0057] Example 3

[0058] Storage stability determination of microcapsules.

[0059] Lactobacillus acidophilus (FREE), the lyophilized powder of microcapsules prepared in Example 1 (SPI-GOS), and the lyophilized powder of microcapsules prepared in Example 2 using SPI as the wall material (SPI) were respectively placed into sterile centrifuge tubes and placed in a desiccator at room temperature (25°C) for one month. Samples were taken and counted on day 0, day 10, day 20, and day 30.

[0060] The sampling and counting method was as follows: 10 mg of lyophilized LAC powder and 500 mg of microcapsule powder were added separately to 5 mL of sterile physiological saline, serially diluted, and then counted using the spread count method. The results are as follows. Figure 4 As shown.

[0061] according to Figure 4 The results showed that during long-term storage, the survival rate of LAC, whether free or in both types of microcapsules, decreased over time. This is closely related to oxygen-mediated oxidative damage: dissolved oxygen in the environment triggers a chain reaction of lipid peroxidation in the phospholipid bilayer of bacterial cell membranes, leading to an abnormal increase in membrane permeability and ultimately causing irreversible death of the strains.

[0062] During storage, the activity of freeze-dried free LAC decreased significantly, from an initial value of 1.76 × 10⁻⁶. 8 By day 30, the viable bacterial count (CFU / mL) approached 0 CFU / mL, indicating that free LAC could not be preserved for long periods at room temperature, and prolonged storage at room temperature would lead to loss of bacterial viability. Both microcapsule encapsulation methods effectively preserved LAC viability in a short time, with no significant difference between the two (P > 0.05).

[0063] Example 4

[0064] The ability of microcapsules to withstand digestion by gastrointestinal fluids.

[0065] Samples to be tested: Lactobacillus acidophilus (FREE), lyophilized powder of microcapsules prepared in Example 1 (SPI-GOS), and lyophilized powder of microcapsules prepared in Example 2 using SPI as the wall material (SPI).

[0066] Prepare 500 mL of simulated gastric digestive juice (SGF) according to Table 1.

[0067] Table 1. Formula for simulated gastric digestive juices

[0068] Ingredients Concentration (mmol / L) Potassium chloride 6.9 Potassium dihydrogen phosphate 0.9 Sodium bicarbonate 25.0 Sodium chloride 47.2 Magnesium chloride hexahydrate 0.1 Ammonium carbonate 0.5

[0069] Add 7.5 mL of SGF to a test tube, then add 5 μL of 0.3 M calcium chloride and 0.695 mL of deionized water. Adjust the pH to 3 with 1 M HCl, and place the tube in a 37°C constant temperature shaker. Finally, add 13.33 mg of pepsin to 1.6 mL of simulated gastric digestion fluid, along with LAC and two types of microcapsules, to control the initial viable bacteria count in each group to 1 × 10⁻⁶. 8 The LAC was digested at CFU / mL and shaken in a constant temperature shaker at 37℃ for 2 hours. The digested LAC and the two microcapsules were then serially diluted and plated for counting.

[0070] Prepare 500 mL of simulated small intestinal digestive fluid (SIF) according to Table 2.

[0071] Table 2 Formulas of Simulated Small Intestinal Digestive Fluids

[0072] Ingredients Concentration (mmol / L) Potassium chloride 6.8 Potassium dihydrogen phosphate 0.8 Sodium bicarbonate 85.0 Sodium chloride 34.8 Magnesium chloride hexahydrate 0.33 Ammonium carbonate 0.5

[0073] 11.0 mL of SIF was added to a test tube, followed by 0.04 mL of 0.3 M calcium chloride, 2.5 mL of bile salts, and 1.31 mL of deionized water. The pH was adjusted to 7 with 3 M NaOH, and the mixture was shaken in a constant temperature shaker at 37°C. Finally, 40 mg of pancreatic enzyme was added to 5.0 mL of simulated small intestinal digestion solution, and the sample after simulated gastric digestion was added. The mixture was then shaken and digested in a constant temperature shaker at 37°C for 2 hours. The digested LAC and microcapsules were serially diluted and then plated for counting. The results are as follows. Figure 5 As shown.

[0074] Depend on Figure 5 It can be seen that free LACs have poor tolerance to the complex environment of gastrointestinal fluid. After 4 hours of simulated digestion, the viable bacterial count decreased from the initial 1.76 × 10⁻⁶. 8CFU / mL decreased to the last approach to 0 CFU / mL. Single protein (SPI) prepared microcapsules can play a protective role for LAC, but the effect is not as good as SGM microcapsules. After 4 hours of simulated digestion, the number of live LAC protected by SGM microcapsules is 10 times higher than that of SPI microcapsules, because single protein microcapsules can be decomposed by protease in the gastrointestinal fluid, leading to the collapse of the structure. While SGM microcapsules can form a more compact embedding structure through the Maillard reaction, thereby better resisting the decomposition of protease.

[0075] Example 5

[0076] Evaluation of the efficacy of the synbiotic microcapsules.

[0077] Test samples: Lactobacillus acidophilus (FREE), freeze-dried powder of microcapsules prepared in Example 1 (SPI-GOS), and freeze-dried powder of microcapsules prepared in Example 2 with SPI as the wall material (SPI).

[0078] (1) Colonization flora extraction:

[0079] The feces of 3 healthy volunteers (aged 25-30 years old) were collected, and there was no history of antibiotic use within 30 days. 15 g of feces was weighed and dissolved with 120 mL of sterile PBS (pH 6.8). The solid-free filtrate was obtained by filtering with three layers of sterile gauze, and the filtrate was used as the original solution of the colon flora and quickly placed in an anaerobic bag for subsequent use.

[0080] (2) Simulated intestinal fermentation:

[0081] Dissolve 2.0 g of tryptone, 2.0 g of yeast extract powder, 0.1 g of NaCl, 0.04 g of KH2PO4, 0.04 g of K2HPO4, 0.01 g of MgSO4·7H2O, 0.01 g of CaCl2·6H2O, 2.0 g of NaHCO3, and 2.0 mL of Tween 80 in 1 L of water, and adjust the pH to 6.0 with 1 M HCl solution. After sterilization, add 0.02 g of hematin, 10 mL of vitamin k1, 0.5 g of cholate, and 0.5 g of cysteine hydrochloride to prepare simulated colon fluid (SCF). Add 27 mL of SCF and 3 mL of the original solution containing colon flora to a sterile shaking tube and shake well in an anaerobic environment. Place the shaking tube in a 37°C constant temperature incubator for 72 h, and take 3 mL of fermentation liquid from each shaking tube. Then, add 3 mL of the test sample dissolved in water to each shaking tube, and control the number of live bacteria in each group of samples to be 1×10 10CFU / mL. The whole operation process was carried out under anaerobic conditions. During the whole in vitro intestinal simulation process, the bacterial shaking tube was placed in a constant temperature shaker at 37°C, and the shaking frequency was 200 times / min. Every 24 hours, 3 mL of sample was taken and frozen at -20°C for sequencing analysis. The results are shown in Figure 6 and Figure 7 .

[0082] As shown in Figure 6 , the addition of synbiotic microcapsules can significantly regulate the diversity of intestinal flora. And as shown in Figure 7 , the addition of synbiotic microcapsules can increase the relative abundance of beneficial bacteria in the intestine, which is manifested as a significant increase in beneficial bacteria in the phylum Actinobacteria (such as Bifidobacterium and Prevotella_9 in the phylum Firmicutes). At the same time, the addition of SGM microcapsules reduces the relative abundance of harmful bacteria in the intestine (Desulfovibrio and Escherichia_Shigella in the phylum Proteobacteria, Eggerthella and Actinomyces in the phylum Actinobacteria).

[0083] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A method for preparing synbiotic microcapsules, characterized in that, Includes the following steps: Galacto-oligosaccharides and soy protein isolate were mixed to prepare a protein solution, which was then stirred and hydrated overnight at 4°C. The solution after overnight hydration is adjusted to alkalinity and subjected to a wet Maillard reaction at 80–90°C to form a protein glycosylated wall material. After drying the protein glycosylated wall material, a wall material solution was prepared using calcium chloride solution. The microbial inoculum, D-(+)-gluconolactone, and the wall material solution were then mixed to obtain a mixed solution. Soybean oil and Span 80 were mixed to obtain an oil phase. The mixed solution was poured into the oil phase and stirred to form microspheres. The oil phase was removed and dried to obtain Synbiotic microcapsules.

2. The preparation method according to claim 1, characterized in that, Galacto-oligosaccharides and soy protein isolate were mixed at a mass ratio of (1:2) to (2:1).

3. The preparation method according to claim 1, characterized in that, The protein solution contains 5% to 10% w / v of protein.

4. The preparation method according to claim 1, characterized in that, The reaction time for the wet Maillard reaction is 60–180 min.

5. The preparation method according to claim 1, characterized in that, The concentration of the calcium chloride solution is 13–20 mmol / L, and the protein concentration in the wall material solution is 5%–10% w / v.

6. The preparation method according to claim 1, characterized in that, The volume ratio of microbial inoculum solution to wall material solution is 1:9, and the microbial inoculum solution contains ≥10 microorganisms. 9 / mL, the content of D-(+)-gluconic acid δ-lactone in the mixed solution is 0.3% to 0.7% w / v.

7. The preparation method according to claim 6, characterized in that, The microorganisms in the microbial solution include Lactobacillus acidophilus.

8. The preparation method according to claim 1, characterized in that, The oil phase contains 0.1% to 0.5% Span 80 w / v; the volume ratio of the oil phase to the mixed solution is 4:

1.

9. Synbiotic microcapsules prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the preparation method according to any one of claims 1 to 8 or the synbiotic microcapsules according to claim 9 in the preparation of products that regulate intestinal flora.

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