Nanocapsule for embedding lactic acid bacteria and application thereof

Abstract

The application discloses a kind of nanometer microcapsules for embedding lactic acid bacteria and application thereof, characterized by mixing hyaluronic acid solution, quinoa protein solution and nano zinc oxide solution according to volume ratio 1:1:1, the concentration of hyaluronic acid solution is 1-5g / L, the concentration of quinoa protein solution is 1-3g / L, and the concentration of nano zinc oxide solution is 0.2-1g / L, resuspend lactic acid bacteria in the mixed solution containing 1-5g / L quinoa protein, 1-3g / L hyaluronic acid and 0.2-1g / L nano zinc oxide, which is stirred and crosslinked in advance for 2h, and then incubated at 4℃ overnight. After freeze-drying, the nanometer microcapsules embedding lactic acid bacteria can be obtained. The advantage is to form a protective layer on the surface of a single bacterial body, improve the ability of lactic acid bacteria to resist harsh external environment, prolong the activity of storage period and stability in gastrointestinal transport.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of nanometer microcapsules, especially to a kind of nanometer microcapsules for embedding lactic acid bacteria and its application. BACKGROUND

[0002] Lactic acid bacteria are probiotics beneficial to human body, in the field of food industry, it can produce exocellular polysaccharide, produce acid and aroma, resist pathogenic bacteria, in the field of medicine, lactic acid bacteria and its metabolites have potential of anti-cancer, intervention of immune diseases and anti-obesity.Lactic acid bacteria are susceptible to external environment during production, storage and digestion, such as oxygen and osmotic pressure in fermentation process, high temperature in spray drying, ice crystal and low temperature in freezing process, acidic environment and various digestive enzymes in gastrointestinal environment, bile acid and bile salt in small intestine, etc., resulting in reduced survival rate of lactic acid bacteria cells.In actual production and preservation, dry technology combined with protective agent method is usually used to protect bacterial activity, traditional microcapsule drying technology includes spray drying, fluidized bed drying, vacuum freeze drying, microwave vacuum drying, etc., and in the preparation process, protective agents such as sodium alginate, chitosan and whey protein are mainly used for embedding, but these traditional technologies have disadvantages such as poor stability and low embedding rate, for example, under high temperature and pressure, due to evaporation of free water, bound water in cells is lost, and the state of many hydrophilic macromolecular substances or membrane lipid substances changes, due to loss of cell membrane function, the mortality of bacteria increases greatly;and in the process of low-temperature freezing, ice crystals are formed in extracellular fluid and intracellular fluid, or the concentration of intracellular solution increases, which can cause damage to cells, increase the permeability of cell membrane, cause protein denaturation and salt-solubility reaction, and further cause metabolic damage to cell membrane;in addition, some equipment has large investment, high cost and long production cycle, is limited to small test, and is difficult to realize large-scale production.In order to ensure that lactic acid bacteria are released to a specific site in the gastrointestinal tract in sufficient number of viable bacteria and maintain metabolic activity, protection of individual lactic acid bacteria cells is very important. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a nanometer microcapsule for embedding lactic acid bacteria, which can form a protective layer on the surface of single bacteria, improve the ability of lactic acid bacteria to resist harsh external environment, prolong the activity during storage period and stability during gastrointestinal transport, and its application.

[0004] The technical scheme adopted by the present application to solve the above technical problem is: a nanometer microcapsule for embedding lactic acid bacteria, which is mixed by hyaluronic acid solution, quinoa protein solution and nanometer zinc oxide solution in a volume ratio of 1:1:1.

[0005] Further, the concentration of the hyaluronic acid solution is 1-5 g / L, the concentration of the quinoa protein solution is 1-3 g / L, and the concentration of the nanometer zinc oxide solution is 0.2-1 g / L.

[0006] Preferably, the concentration of the hyaluronic acid solution is 1.87 g / L, the concentration of the quinoa protein solution is 1.49 g / L, and the concentration of the nano-zinc oxide solution is 0.39 g / L.

[0007] Further, the preparation method of the quinoa protein is as follows: quinoa protein powder is dissolved in distilled water at a ratio of 1 g:12 mL, 0.5M NaOH is used to adjust the pH to 10.5, and extraction is performed at a temperature of 46℃ for 2.5h; then, the supernatant is collected by centrifugation at 5000 rpm / min for 10 min at 4℃, and the pH is adjusted to 4.5 using 0.5M HCl; the precipitate is collected by centrifugation at 10000 rpm / min for 15 min at 4℃, and the precipitate is resuspended in distilled water at a ratio of 1 g:12 mL, and washed 2-3 times to remove salt; the resuspension is adjusted to neutral pH, and the quinoa protein is obtained by freeze-drying.

[0008] The application of the above-mentioned nano-microcapsules for embedding lactic acid bacteria is as follows: the lactic acid bacteria are resuspended in a mixed solution containing 1-5 g / L quinoa protein, 1-3 g / L hyaluronic acid, and 0.2-1 g / L nano-zinc oxide, which is stirred and cross-linked for 2h in advance, and then stored at 4℃ overnight; after freeze-drying, the nano-microcapsules for embedding lactic acid bacteria are obtained.

[0009] Further, the freeze-drying conditions are as follows: vacuum degree 0.021 mbar, freeze-drying temperature -55℃, and time 48h.

[0010] Further, the lactic acid bacteria include Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus plantarum, and Lactobacillus casei.

[0011] Compared with the prior art, the application has the advantages that the application discloses a nano microcapsule for embedding lactic acid bacteria and application thereof, and utilizes high-biocompatibility hyaluronic acid HA, green and safe plant-based quinoa protein QA and inorganic nanomaterial zinc oxide ZnO as wall materials for embedding lactic acid bacteria. The hyaluronic acid is an anionic linear glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine and D-glucuronide, has high viscosity, is conducive to crosslinking with other wall materials, and is a good skeleton for loading bioactive substances. The quinoa protein is extracted from quinoa seeds and is a representative of pseudocereal proteins, has high protein content, no gluten, high biological nutritional value and good emulsification stability, mainly contains globulin and albumin and a large amount of amino acids, and especially cysteine, which makes it superior to rice and wheat proteins as wall materials for embedding bioactive substances. The mechanical properties of the natural HA network are relatively weak, and the HA network is dissolved rapidly in water; the quinoa protein is an amphoteric protein, which ensures that the protein is covalently combined with polysaccharides to form a stable condensed layer, and the viscoelastic layer can enhance the stability of the microcapsule structure and intestinal adhesion. The nano zinc oxide can be dissolved in acidic gastric juice to form zinc ions, and interact with sulfur-containing ligands in the protein, and is interpenetrated between the core-shell protective layers through the interaction between the wall materials, thereby stabilizing the structure of the protein and polysaccharides, and better maintaining the probiotic activity of the encapsulated lactic acid bacteria.

[0012] In summary, the nano microcapsule for embedding lactic acid bacteria and application thereof has the advantages that the charge of the wall material quinoa protein changes from positive to negative as the pH is lower than the isoelectric point to higher than the isoelectric point, so that the microcapsule has a certain buffering capacity; the hyaluronic acid is an anionic polysaccharide, and through crosslinking reaction, electrostatic interaction and self-assembly between the wall materials, a viscoelastic protective layer is formed on the surface of a single lactic acid bacterial cell, which makes up for the defect of a large pore size of the protein network, and the inorganic nanomaterial zinc oxide is interpenetrated therein, so that the pore size of the microcapsule is more compact; in the transport process, the cationic groups of the embedding layer polymer network can fully contact with the external anionic groups, slow down the penetration of the external acid or alkali environment, avoid direct contact with the bacterial body, enhance the firmness of the microcapsule structure and intestinal adhesion, improve the stability of the bacterial protein and nucleic acid, increase the survival rate, stress resistance and bioavailability in the transport process of the bacterial body, and prolong the activity of the storage period. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The single-factor experiment of the influence of different materials on the number of live bacteria, wherein A is the influence of different hyaluronic acid concentrations on the number of live bacteria, B is the influence of different zinc oxide concentrations on the number of live bacteria, and C is the influence of different quinoa protein concentrations on the number of live bacteria;

[0014] Figure 2Response surface of the effect of embedding materials on the viable count of L. rhamnosus after freeze-drying, wherein A is the response surface of the effect of hyaluronic acid and zinc oxide on the viable count of L. rhamnosus after freeze-drying; B is the response surface of the effect of hyaluronic acid and quinoa protein on the viable count of L. rhamnosus after freeze-drying; C is the response surface of the effect of zinc oxide and quinoa protein on the viable count of L. rhamnosus after freeze-drying;

[0015] Figure 3 Viable count of the freeze-dried nano-microcapsules;

[0016] Figure 4 Embedding rate of the nano-microcapsules;

[0017] Figure 5 Particle size of the nano-microcapsules;

[0018] Figure 6 Zeta potential of the nano-microcapsules;

[0019] Figure 7 FT-IR diagram of the nano-microcapsules, Quinoa: quinoa protein; ZnO NPS: zinc oxide nanoparticles; HA: hyaluronic acid; LR: L. rhamnosus MC-1; Quinoa LR: quinoa protein-encapsulated LR;

[0020] Figure 8 Morphology of the nano-microcapsules under a scanning electron microscope, magnification 30.00KX, 50.00KX;

[0021] Figure 9 Morphology of the nano-microcapsules under a transmission electron microscope, magnification 7.0K, 10.0K, 10.0K, 7.0K;

[0022] Figure 10 Survival rate of the nano-microcapsules in a simulated gastrointestinal environment (simulated oral environment for 2 min, simulated gastric environment for 2 h, and simulated intestinal environment for 4 h);

[0023] Figure 11 Survival rate of the nano-microcapsules stored at 4°C for 56 d;

[0024] Figure 12 Survival rate of the nano-microcapsules stored at 25°C for 21 d;

[0025] Figure 13 Viable count of the nano-microcapsules after heat treatment at 72°C for 15, 30, 60, and 180 s;

[0026] Figure 14 Viable count of the nano-microcapsules after heat treatment at 63°C for 10, 20, and 30 min;

[0027] Figure 15 OD of the nano-microcapsules cultured in broth with pH of 2, 4, 6, 8, 10, and 12 for 24 h600 ;

[0028] Figure 16 The growth curve of the nanocapsules over 24 hours is shown. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. I. Specific Implementation Methods

[0031] 1. Strains and experimental materials

[0032] Lactobacillus rhamnosus MC-1, preserved in our laboratory; MRS solid agar plates, MRS broth medium (Qingdao Haibo Biotechnology Co., Ltd.); 90% quinoa protein powder (for quinoa protein extraction) (Xi'an Haotailai Biotechnology Co., Ltd.); 97% hyaluronic acid (Shanghai Maclean Biochemical Technology Co., Ltd.); 50nm 40% nano zinc oxide dispersion (Shanghai Maclean Biochemical Technology Co., Ltd.); 1M NaOH solution, 1M HCl solution, pH 7.0 phosphate buffer, distilled water, glycerol.

[0033] 2. Bacterial cell collection

[0034] Activated and fermented *Lactobacillus rhamnosus* MC-1 was inoculated onto MRS agar plates and incubated at 37°C for 12-16 hours. MRS broth was prepared, and under aseptic conditions in a laminar flow hood, single colonies were picked from the plates using a sterile 10μL pipette tip and inoculated onto the MRS broth. The culture was incubated at 37°C, with OD values ​​measured every hour until the OD value reached the target level. 600 The concentration should be 0.6-0.8. Centrifuge at 4℃ and 6000rpm / min for 5min, collect the bacterial cells, discard the culture medium residue, and repeat the washing 1-2 times until the supernatant after centrifugation is completely clear and transparent. Discard the supernatant and store it in a freezer at -80℃.

[0035] 3. Wall material preparation

[0036] Preparation of zinc oxide solution: Take 5, 10, 15, 20, and 25 μL of the original solution and add 9.995, 9.990, 9.985, 9.980, and 9.975 mL of water, respectively, to prepare gradient dilutions of 0.2, 0.4, 0.6, 0.8, and 1.0 g / L for single-factor experiments. Filter and store at room temperature for later use.

[0037] Preparation of hyaluronic acid solution: 1.0, 2.0, 3.0, 4.0, 5.0 mg of hyaluronic acid was taken respectively and dissolved in 1 mL of water to prepare 1.0, 2.0, 3.0, 4.0, 5.0 g / L solutions for single factor experiment, filtered and stored at 4℃ for standby.

[0038] Preparation of quinoa protein solution: First, quinoa protein was extracted. Specifically, quinoa protein powder was dissolved in distilled water at a ratio of 1 g:12 mL, the pH was adjusted to 10.5 with 0.5 M NaOH, and then extracted at a temperature of 46℃ for 2.5 h. After that, the supernatant was collected by centrifugation at 5000 rpm / min for 10 min at 4℃, and the pH was adjusted to 4.5 with 0.5 M HCl. After centrifugation at 10000 rpm / min for 15 min at 4℃, the precipitate was collected, resuspended in distilled water at a ratio of 1 g:12 mL, and washed 2-3 times to remove salt. The pH of the resuspended solution was adjusted to neutral, and then freeze-dried to obtain quinoa protein. The freeze-drying conditions were vacuum degree 0.021 mbar, freeze-drying temperature -55℃, and time 48 h. 1.0, 1.5, 2.0, 2.5, 3.0 mg of freeze-dried quinoa protein was dissolved in 1 mL of water to prepare 1.0, 1.5, 2.0, 2.5, 3.0 g / L solutions for single factor experiment, filtered and stored at 4℃ for standby.

[0039] 4. Preparation of nano-microcapsules

[0040] 1-5 g / L hyaluronic acid solution, 1-3 g / L quinoa protein solution, and 0.2-1 g / L nano-zinc oxide solution were mixed at a volume ratio of 1:1:1, stirred and cross-linked for 2 h, and stored in a 4℃ refrigerator. Rhamnosus lactis MC-1 was resuspended in the mixed solution, incubated at 4℃ overnight, and then freeze-dried and stored in a 4℃ refrigerator.

[0041] Example 1

[0042] 1.87 g / L hyaluronic acid, 1.49 g / L quinoa protein, and 0.39 g / L zinc oxide solution were mixed at a volume ratio of 1:1:1, stirred and cross-linked for 2 h, and stored in a 4℃ refrigerator. Rhamnosus lactis MC-1 was resuspended in the mixed solution, incubated at 4℃ overnight, and then freeze-dried to obtain nano-microcapsules embedding lactic acid bacteria, which were stored in a 4℃ refrigerator. The nano-microcapsules were named HAQZ.

[0043] Example 2

[0044] 1.0 g / L hyaluronic acid, 1 g / L quinoa protein, and 0.2 g / L zinc oxide solution were mixed at a volume ratio of 1:1:1, stirred and cross-linked for 2 h, and stored in a 4℃ refrigerator. Rhamnosus lactis MC-1 was resuspended in the mixed solution, incubated at 4℃ overnight, and then freeze-dried to obtain nano-microcapsules embedding lactic acid bacteria.

[0045] Example 3

[0046] Mix 5 g / L hyaluronic acid, 3 g / L quinoa protein, 1 g / L zinc oxide solution at a volume ratio of 1:1:1, stir and cross-link for 2 h, and store in a 4°C refrigerator. Resuspend Lactobacillus rhamnosus MC-1 in the mixed solution, incubate at 4°C overnight, and freeze-dry to obtain the lactic acid bacteria-embedded nanometer microcapsules.

[0047] Comparative Example 1

[0048] The same as Example 1 above, except that Lactobacillus rhamnosus MC-1 was resuspended in 1.49 g / L quinoa protein solution, incubated at 4°C overnight, and stored in a 4°C refrigerator after freeze-drying using a freeze-drying machine (Alpha 1-4LD plus, Martin Christ, Germany). The nanometer microcapsules were named QA.

[0049] Comparative Example 2

[0050] The same as Example 1 above, except that 1.49 g / L quinoa protein solution and 1.87 g / L hyaluronic acid solution were mixed at a volume ratio of 1:1, stirred and cross-linked for 2 h, and stored in a 4°C refrigerator. Lactobacillus rhamnosus MC-1 was resuspended in the mixed solution, incubated at 4°C overnight, and stored in a 4°C refrigerator after freeze-drying. The nanometer microcapsules were named HAQ.

[0051] Control group

[0052] Unembedded Lactobacillus rhamnosus MC-1 was named Free LR.

[0053] II. Analysis of experimental results Data are expressed as mean ± standard deviation. SPSS 17.0 software was used for analysis of variance (ANOVA). LSD and Duncan tests were used to determine significant differences between samples at the p<0.05 level.

[0054] 1. Single factor and response surface experiments of wall material ratio of nanometer microcapsules

[0055] Each group of microcapsules was diluted and coated on MRS solid medium by the step-by-step dilution coating method, incubated at 37°C for 24 h, and the number of freeze-dried viable bacteria was calculated by plate coating counting method.

[0056] As shown in Figure 1 A, the concentration of hyaluronic acid was set to 1.0, 2.0, 3.0, 4.0, and 5.0 g / L. With increasing concentration of hyaluronic acid, the number of freeze-dried viable bacteria of the microcapsules showed a trend of first increasing and then decreasing, and the number of viable bacteria reached a maximum when the concentration was 2.0 g / L.

[0057] As shown in Figure 1As shown in B, the concentration of zinc oxide was set to 0.2, 0.4, 0.6, 0.8, 1.0 g / L, and with the increase of zinc oxide concentration, the freeze-dried viable count of microcapsules first increased and then decreased, and the viable count was the highest at 0.4 g / L.

[0058] As shown in Figure 1 C, the concentration of quinoa protein was set to 1.0, 1.5, 2.0, 2.5, 3.0 g / L, and with the increase of quinoa protein concentration, the freeze-dried viable count of microcapsules first increased and then decreased, and the viable count was the highest at 1.5 g / L.

[0059] Taking the viable count as an index, three factors that had a significant effect on the viable count were selected, and three-factor three-level response surface analysis experiments were performed on different concentrations of hyaluronic acid, zinc oxide and quinoa protein. As shown in Figure 2 A, Figure 2 B and Figure 2 C, when the concentration of hyaluronic acid was 1.87 g / L, the concentration of zinc oxide was 0.39 g / L, and the concentration of quinoa protein was 1.49 g / L, the freeze-dried viable count of the prepared microcapsules was the highest, reaching 8.30 log CFU / mL.

[0060] 2. Measurement of freeze-dried viable count and embedding rate of nano-microcapsules

[0061] 1 g of microcapsules was added to 0.1 M of 9 mL sodium dihydrogen phosphate buffer, resuspended for 5 min to dissolve the microcapsules, gradient diluted, and plated for counting to calculate the embedding rate. The embedding rate (%) = (log N / log N0)*100, where N is the viable count released by the embedded microcapsules, and N0 is the initial viable count.

[0062] As shown in Figure 3 , the freeze-dried viable count of microcapsule HAQZ was 8.30 log CFU / mL, which was significantly higher than that of other microcapsule groups (p<0.05), and compared with unembedded LR, it increased by 1 log CFU / mL in each group of microcapsules, and the viable count of QA microcapsules embedded with quinoa protein alone increased by only 0.5 log CFU / mL, indicating that the addition of hyaluronic acid and zinc oxide was beneficial to LR to resist low temperature and ice crystal damage, and increased the freeze-dried viable count. As shown in Figure 4 , the embedding rates of HAQZ and HAQ microcapsules were both above 90%, and the embedding rate of HAQZ (92.27%) was increased by 11.12% compared with that of unembedded LR (81.15%), although the embedding of QA quinoa protein microcapsules could increase the embedding rate to a certain extent, but due to the large pore structure formed after protein freeze-drying, part of the bacteria was exposed outside, and the addition of hyaluronic acid increased the thickness of the protective layer, and nano-zinc oxide made the network structure more compact, which could embed most of the bacteria, indicating that this HAQZ microcapsule matrix was helpful to improve the embedding rate of freeze-dried bacteria.

[0063] 2. Particle size and potential determination

[0064] Microcapsule potential and particle size were determined using a particle size analyzer (Zetasizer Lab, Malvern Panaco Ltd.).

[0065] like Figure 5 As shown, the unencapsulated LR particles had a size of 1.02 μm, while the particle size of the microcapsules HAQZ increased to 2.87 μm after the addition of three wall materials.

[0066] like Figure 6 As shown, the microcapsules have a negative charge, and the potential differences among the groups are significant (p<0.05). The addition of quinoa protein, anionic polysaccharide hyaluronic acid, and nano zinc oxide makes the HAQZ potential of the microcapsules even more negative, at -11.5mV. The differences in interface composition suggest that the encapsulation process may be related to the electrostatic interaction between materials.

[0067] 3. Fourier transform infrared spectroscopy characterization

[0068] Fourier transform infrared spectrometry (FT-IR) was performed using a Nicolet iS20 (USA).

[0069] like Figure 7 As shown, the FT-IR image reveals that LF exhibits characteristic amide I (1650 cm⁻¹) from the protein. -1 ) and amide II (1525cm) -1 ) area, at 1228cm -1 The peak value is due to the asymmetric tensile vibration of amide III or phosphate, at 2973 cm⁻¹. -1 The peak value may be due to the symmetric and asymmetric CH2 stretching vibrations of the lipid membrane. At 450 cm⁻¹ -1 The broad peak nearby is a characteristic peak of the Zn-O bond. The peak value of the QA microcapsule in the amide I region is 1668 cm⁻¹. -1 Primarily caused by C=O stretching and HOH bending vibrations, changes in protein secondary structure, such as α-helices, β-sheets, β-turns, and random coils, can all be reflected in this region. HAQZ at 1115 cm⁻¹ -1 The peak value indicates the presence of C=O and CO, 1650 cm⁻¹ -1 This corresponds to the amide I band from the protein, 2963 cm. -1 The length is 3420cm due to the stretching of CH. -1 The stretching of -OH was confirmed. The amide II region can reflect the NH bending of proteins, and the position of the amide II peak changed, with LF changing from 1554 cm⁻¹. -11543 cm -1 HAQZ, indicating that the hyaluronic acid, quinoa protein and zinc oxide might interact with the bacterial protein and change the protein structure. The hydroxyl stretching region between 3650-3100 cm -1 indicates the formation of hydrogen bonds between different materials. Compared with HAQ, the red shift of HAQZ from 3306 cm -1 to 3420 cm -1 may be the result of hydrogen bonding between zinc oxide and hyaluronic acid, quinoa protein.

[0070] 4. Scanning electron microscopy, transmission electron microscopy observation of structure

[0071] The freeze-dried microcapsules were pasted on conductive glue and gold was sprayed on the surface. The appearance was photographed by scanning electron microscope (Hitachi S-3400, Japan). The microcapsules were fixed with glutaraldehyde and dehydrated in alcohol gradient. 10.0 μL of sample was coated on a copper mesh and air dried, and observed using transmission electron microscope (Hitachi HT-7800, Japan).

[0072] As shown in Figure 8 , the LR was observed as a rod by scanning electron microscopy. The surface of the single bacterial body without embedding was wrinkled after freeze-drying due to water loss. The surface of the QA microcapsule showed a porous morphology due to the presence of quinoa protein. After adding hyaluronic acid and zinc oxide, the surface of the single bacterial body of the HAQ microcapsule became smooth. There was no aggregation phenomenon in the HAQZ microcapsule, and the overall morphology was uneven, indicating that fewer bacteria were exposed outside the microcapsule, and most of the bacterial bodies were wrapped in the wall material.

[0073] As shown in Figure 9 , transmission electron microscopy can observe that the surface of the unembedded LR bacterial body has no embedding layer. With the addition of quinoa protein and hyaluronic acid, the embedding layer gradually becomes obvious. The surface of the bacterial body of the HAQZ microcapsule is wrapped in a thin protective layer, and nano zinc oxide particles are interspersed therein. The thickness of the embedding layer is about 100 nm, indicating that the LR is successfully encapsulated.

[0074] 5. Survival rate in simulated gastrointestinal tract

[0075] Prepare 20 mL simulated saliva (SSF) in advance: KCl 0.604 g, KH2PO4 0.148 g, NaHCO3 0.272 g, NaCl 2.34 g, MgCl2(H2O)6 0.02 g, (NH4)2CO3 0.0024 g, dissolved in water. 20 ml simulated gastric fluid (SGF): KCl 0.276 g, KH2PO4 0.036 g, NaHCO3 0.5 g, NaCl 0.472 g, MgCl2(H2O)6 0.016 g, (NH4)2CO3 0.02 g, dissolved in water. 20 mL simulated intestinal fluid (SIF): KCl 0.272 g, KH2PO4 0.032 g, NaHCO3 1.7 g, NaCl 0.384 g, MgCl2(H2O)6 0.044 g, dissolved in water. Disperse 1.0 g of microcapsules in 3.5 mL of preheated SSF and shake for 5 s, then add 0.5 mL of saliva alpha-amylase solution, 25 μL of CaCl2(0.3 M) and 0.975 mL of distilled water, and shake at 100 rpm, 37°C for 2 min. Mix the digested sample with simulated gastric fluid (1:1 v / v) of pepsin (3.2 mg / mL), NaCl (2 mg / mL), HCl (7 ml / L), adjust the pH of the mixture to 2.0, and shake the sample at 100 rpm, 37°C for 2 h. Adjust the pH to 7.0 after simulated gastric fluid digestion, add 2.5 mL of trypsin, 2.5 mL of lipase, 3.5 mL of bile salts (all dissolved in 5 mM phosphate buffer, pH 7.0) and 1.5 mL of simulated intestinal fluid (containing 36.7 mg / mL CaCl2, 218.7 mg / mL NaCl), and shake at 100 rpm, 37°C for 4 h.

[0076] As Figure 10As shown, the initial viable cell count under simulated gastrointestinal conditions was 9.00 log CFU / mL. After the microcapsules were exposed to simulated oral cavity for 2 min, the viable cell count remained at about 8.30 log CFU / mL, and there was no difference in the viable cell count of the microcapsules in each group, indicating that the microcapsules in each group could stably exist in the simulated oral cavity. After 2 h of digestion in simulated gastric juice, the viable cell count of the unembedded LR decreased to about 6.60 log CFU / mL, indicating that the acidic environment in the gastric juice easily caused damage to the bacterial cells. The HAQZ microcapsules decreased by 1.41 log CFU / mL, the HAQ decreased by 1.53 log CFU / mL, and the QA decreased by 1.81 log CFU / mL, indicating that HAQZ could improve the resistance of bacterial cells in the simulated gastric environment. After 4 h in the simulated intestinal environment, the viable cell counts of the free, QA, HAQ, and HAQZ microcapsules were 5.17, 6.15, 6.35, and 6.75 log CFU / mL, respectively. Due to the damage to the cell membrane caused by bile salts in the intestinal environment, the viable cell count of the unembedded LR was significantly lower than that of each embedded group, and the viable cell counts of the remaining microcapsule groups were all greater than 6.00 log CFU / mL, indicating that the wall material ensured that a sufficient number of viable cells reached the gastrointestinal tract to exert an effect. Although quinoa protein had a certain protective effect on bacterial cells, after the addition of hyaluronic acid, the viscoelastic layer formed by HAQ could block more bile salts from penetrating into the microcapsule interior, and the viable cell count was higher than that of the QA microcapsule. The decrease in the viable cell count of HAQZ was the lowest at 2.28 log CFU / mL, and the decrease in the viable cell count of the free bacteria was the highest at 3.83 log CFU / mL. HAQZ was improved by 1.58 log CFU / mL compared to the unembedded bacteria, and the viable cell count of the HAQZ microcapsule was significantly higher than that of the other microcapsule groups after simulated gastrointestinal digestion (p<0.05). The addition of zinc oxide may have made the crosslinking between hyaluronic acid and quinoa protein more compact, the cationic groups of the microcapsule could fully contact the external anionic groups, thereby delaying the release time of the bacterial cells and making them more resistant to the bile salt environment, so that more bacterial cells could maintain activity to reach the simulated intestinal tract. The HAQZ encapsulation matrix has the potential to deliver a sufficient number of bacterial cells to the gastrointestinal tract.

[0077] 6. Storage stability

[0078] The microcapsules after freeze-drying were stored at 4°C to determine the activity at 0, 7, 14, 21, 28, 35, 42, 49, and 56 d, and at room temperature 25°C to determine the activity at 0, 7, 14, and 21 d. The viable cell count was determined by plate colony counting.

[0079] As shown in Table 6, the viable cell count of the free bacteria decreased by 3.83 log CFU / mL, and the viable cell count of the QA microcapsules decreased by 1.81 log CFU / mL. The viable cell count of the HAQ microcapsules decreased by 1.53 log CFU / mL, and the viable cell count of the HAQZ microcapsules decreased by 2.28 log CFU / mL. The decrease in the viable cell count of the HAQZ microcapsules was the lowest, and the decrease in the viable cell count of the free bacteria was the highest. The viable cell count of the HAQZ microcapsules was significantly higher than that of the other microcapsule groups (p<0.05), indicating that the HAQZ microcapsules had the best storage stability. Figure 11As shown, the viable cell count of the unembedded bacteria stored at 4°C for 7 days decreased rapidly to 7.36 log CFU / mL, and at 28 and 56 days, it was 6.44 and 5.98 log CFU / mL, respectively, which decreased by 3.02 log CFU / mL during the storage period. The decrease in the viable cell count of the other microcapsules was slower than that of the unembedded group. At 56 days, the viable cell count of the QA, HAQ, and HAQZ microcapsules was 6.48, 6.96, and 7.44 log CFU / mL, respectively, and the viable cell count of the HAQZ microcapsules was significantly higher than that of the other groups (p < 0.05), which was 1.46 log CFU / mL higher than that of the unembedded group. The viable cell count of the HAQZ microcapsules was stable during the storage period, and the decrease in the viable cell count was slower with an increase in the number of embedding layers.

[0080] As shown, Figure 12 During storage at room temperature (25°C), the viable cell count of the unembedded microcapsules was lower than 6.0 log CFU / mL at 14 days, and the viable cell count of the QA and HAQ microcapsules was only 5.17 and 6.18 log CFU / mL at 21 days, respectively. The decrease in the viable cell count of the HAQZ microcapsules was slower than that of the other groups, and the viable cell count was 6.33 log CFU / mL at 21 days, which was significantly higher than that of the unembedded group (p < 0.05). Lactic acid bacteria are susceptible to changes in external conditions such as temperature, oxygen, and moisture during long-term storage, and the viable cell count is significantly reduced. The determination of the viable cell count during storage is an important indicator for evaluating the quality of microcapsules. Proteins are usually used as cryoprotective agents for microcapsules, but the effect of single quinoa protein may not be sufficient to resist the influence of changes in external environmental temperature on bacterial cells. The hyaluronic acid protective layer may help to stabilize the cell membrane and components of the freeze-dried microcapsules during low-temperature storage. The addition of zinc oxide reduces the pore size, which shows better protection effect at low temperature. After 8 weeks of low-temperature storage, the viable cell count remained at about 7.44 log CFU / mL, which met the requirement of a viable cell count of more than 6.0 log CFU / mL. Low-temperature storage microcapsules can better maintain the viable cell count, and HAQZ microcapsules may help to improve the stability of bacterial cells during long-term low-temperature storage.

[0081] 7. Heat stability

[0082] The microcapsules were heat-treated at 65°C for 10, 20, and 30 min, and at 72°C for 15, 30, 60, and 180 s, respectively. After heat treatment, the samples were immediately placed in an ice bath, and the viable cell count was determined by plate colony counting.

[0083] As shown, Figure 13 and Figure 14As shown, the viable cell counts of HAQZ microcapsules after treatment at 63℃ for 30 min and 72℃ for 180 s were 7.44 and 7.54 log CFU / ml, respectively, which were 1.72 and 0.97 log CFU / ml higher than those of unencapsulated bacteria. Unencapsulated bacteria were more sensitive to high temperatures, and the bacteria died continuously with prolonged heating time and temperature. The viable cell count of HAQZ microcapsules was significantly higher than that of the control group (p<0.05). In the production process, pasteurization is an essential heat treatment process. After high-temperature and long-term heat treatment, the bacteria are more easily inactivated. Quinoa protein has a large pore size, and some bacteria cannot be encapsulated. The cross-linking layer of hyaluronic acid and zinc oxide can more tightly encapsulate the bacteria, reducing the number of exposed bacteria. The viable cell count of HAQZ microcapsules after heat treatment was higher than that of HAQ, which improved the thermal stability of LR.

[0084] 8. Acid resistance and growth curve

[0085] The optical density (OD) values ​​of the microcapsules at 600 nm were determined over 24 hours in MRS broth at pH 2, 4, 6, 8, 10, and 12. 600 1.0 g of microcapsules were cultured in MRS broth, and the OD of the microcapsules was measured after 24 h. 600 .

[0086] like Figure 15 As shown, after culturing HAQZ microcapsules in broths of different pH values ​​for 24 hours, except for poor cell activity under extremely acidic and extremely alkaline conditions, the OD values ​​of HAQZ microcapsules at pH 4, 6, and 8 were [not specified]. 600 The levels were significantly higher than the control group (p<0.05), and the bacteria could grow normally.

[0087] like Figure 16 As shown, the bacterial cells encapsulated with quinoa protein, hyaluronic acid, and zinc oxide were able to grow stably in MRS broth. Compared with the unencapsulated LR, the bacterial cells encapsulated with HAQ and HAQZ microcapsules reached the stationary phase later. The HAQZ microcapsules had a higher optical density value at the same time, which may be due to the prebiotic function of quinoa protein and zinc oxide synergistically with hyaluronic acid, which helps the bacterial cells grow.

[0088] In actual production and oral delivery process, when single bacterial LR is exposed to the environment with variable temperature or gastrointestinal salt environment, the cell membrane may be damaged, the protein and nucleic acid may be denatured, and the biochemical pathway of the bacterial body is disturbed and inactivated. The single quinoa protein network has a larger pore size, which has a certain protective effect on the bacterial body, but hyaluronic acid is an anionic polysaccharide, which can form a layer of viscoelastic layer on the surface of the bacterial body, and the amphoteric property of quinoa protein makes it easier to combine with polysaccharide, and the protective effect is better than that of the single functional polymer network. The particle size of nano-zinc oxide particles is small, which can penetrate into the protein and hyaluronic acid network, so that the protective layer is more compact. The protective network with buffering capacity improves the stability of the bacterial protein and nucleic acid, reduces the influence of the external environment on the bacterial body under high-temperature heat treatment and low-temperature storage conditions, and improves the stress resistance of the bacterial body. In addition, hyaluronic acid, quinoa protein and zinc oxide embed more bacterial bodies, when the microcapsule is exposed to the digestive environment, there are fewer unembedded bacterial bodies, and the cationic groups on the surface of the embedded bacterial bodies can fully contact the external anions, prolong the dissolution time of the microcapsule, and enhance the intestinal adhesion and stability of the microcapsule.

[0089] In summary, the nano-microcapsule of the present application embeds rhamnose lactobacillus MC-1 by quinoa protein, hyaluronic acid and nano-zinc oxide to improve the stability and activity of the bacterial body, which is beneficial to improve the freeze-drying survival rate and embedding rate, increase the number of viable bacteria in simulated gastrointestinal digestion, maintain the stability of long-term storage at low temperature or room temperature, improve the heat resistance, acid and alkali resistance of the bacterial body, has the potential to protect and deliver the bacterial body to the gastrointestinal tract, and avoid the loss of viable bacteria during processing and storage.

[0090] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary skilled in the art within the essential scope of the present application shall also belong to the protection scope of the present application.

Claims

1. A nanocapsule for embedding lactic acid bacteria, characterized by: Mix 1-5 g / L hyaluronic acid solution, 1-3 g / L quinoa protein solution and 0.2-1 g / L nano zinc oxide solution in a volume ratio of 1:1:1, obtain a mixed solution after stirring and cross-linking for 2 h, resuspend lactic acid bacteria in the mixed solution, and then perform overnight incubation at 4 DEG C, and then obtain the nano microcapsule embedding lactic acid bacteria after freeze-drying.

2. The nanocapsule for embedding lactic acid bacteria according to claim 1, wherein: The concentration of the hyaluronic acid solution is 1.87 g / L, the concentration of the quinoa protein solution is 1.49 g / L, and the concentration of the nano zinc oxide solution is 0.39 g / L.

3. The nanocapsule for embedding lactic acid bacteria according to claim 1, wherein: The preparation method of the quinoa protein is as follows: dissolve quinoa protein powder in distilled water at a ratio of 1 g:12 mL, adjust the pH to 10.5 with 0.5M NaOH, extract at a temperature of 46 DEG C for 2.5 h, centrifuge at 5000 rpm for 10 min at 4 DEG C, collect the supernatant, adjust the pH to 4.5 with 0.5M HCl, centrifuge at 10000 rpm for 15 min at 4 DEG C to collect the precipitate, resuspend the precipitate in distilled water at a ratio of 1 g:12 mL, wash 2-3 times to remove salt, adjust the pH of the resuspension to neutral, and then obtain the quinoa protein by freeze-drying.

4. A method for producing the nanocapsule for embedding lactic acid bacteria according to any one of claims 1 to 3, characterized by: Mix hyaluronic acid solution, quinoa protein solution and nano zinc oxide solution in a volume ratio of 1:1:1, obtain a mixed solution after stirring and cross-linking for 2 h, resuspend lactic acid bacteria in the mixed solution, and then perform overnight incubation at 4 DEG C, and then obtain the nano microcapsule embedding lactic acid bacteria after freeze-drying.

5. The method for preparing nanocapsules for embedding lactic acid bacteria according to claim 4, wherein: The freeze-drying conditions are as follows: vacuum degree 0.021 mbar, freeze-drying temperature-55 DEG C, and time 48 h.

6. The method for preparing nanocapsules for embedding lactic acid bacteria according to claim 4, wherein: The lactic acid bacteria include Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus casei.

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