Method for embedding lactobacillus sake by using whey protein isolate and sodium hyaluronate compound

Abstract

The invention relates to a method for embedding lactobacillus sake by using a whey protein isolate and sodium hyaluronate compound, and belongs to the technical field of microorganisms. The invention provides a novel food-grade probiotic delivery system, which is used for carrying out microencapsulation on the easily denatured lactobacillus sake J15 by utilizing the binding capacity between polysaccharide and protein, and is suitable for being directly applied to dairy products. And meanwhile, the safety in the operation process and subsequent application is ensured. The method can be used for subsequent application in food.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a method for embedding Lactobacillus sakei with whey protein isolate and sodium hyaluronate. Background Art

[0002] Microencapsulation of lactic acid bacteria is useful for the nutritional and functional properties of live microorganisms as well as carrier media. In simulated gastrointestinal fluid models, it has been determined that microencapsulation minimizes probiotic cell damage and improves their survival. There are many encapsulation techniques such as spray drying, freeze drying, fluidized bed drying, etc. Freeze drying is the most suitable method for the preparation of heat-sensitive bioactive substances such as probiotics, which can minimize thermal stress and oxidative stress.

[0003] Compared with liquid lactic acid bacteria, microcapsules are easier to transport, control their quality and reduce costs. The selection of materials suitable for microbial cell encapsulation is crucial for the stability and performance of the produced particles. These materials cannot be toxic because it directly affects the morphology, diameter and permeability of the particles. There are also many types of encapsulation materials, mainly polysaccharides and proteins. Among them, the most used materials are whey and soy proteins, alginate, pectin and chitosan. In the past decade, whey protein isolate has been particularly popular and used as an encapsulation material. If a single material is used as a wall material dosage, it does not show effective protection against bacterial cells.

[0004] The microcapsule material directly affects the tolerance of probiotics to heat and gastric conditions and is a key factor in the delivery system. Whey protein isolate (WPI) is a by-product of cheese making and has been widely used as an emulsifier in the food industry. WPI has a high protein content, mainly including bovine serum albumin, β-lactoglobulin, α-lactalbumin and immunoglobulins. WPI has excellent gel properties and is biocompatible with probiotics. In 2021, the National Health Commission officially approved sodium hyaluronate (SH) as a new food ingredient, which can be used in milk and dairy products (up to 0.2g / kg), and it is becoming more and more popular in the food industry. SH has good hygroscopicity, viscoelasticity, plasticity, permeability and biocompatibility.

[0005] Microencapsulated probiotics are often added to foods, and whey protein isolate (WPI) and sodium hyaluronate (SH) are two important food ingredients. Therefore, whey protein isolate and sodium hyaluronate are good candidates for encapsulating probiotic microorganisms and can be directly used as starter cultures for dairy products. Therefore, how to screen out the appropriate proportion of microencapsulation materials is of great significance for the subsequent delivery of microencapsulation in food. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for encapsulating Lactobacillus sakei with whey protein isolate and sodium hyaluronate, wherein the method uses whey protein isolate and sodium hyaluronate as capsule materials to microencapsulate probiotics, thereby stabilizing the activity of probiotics and completing the delivery in food, which is specifically achieved through the following technical solutions:

[0007] A method for embedding Lactobacillus sakei using a whey protein isolate and sodium hyaluronate complex, comprising the following steps:

[0008] 1) Dissolve whey protein isolate in sterile water to prepare a 20 mg / mL solution A; dissolve sodium hyaluronate in sterile water to prepare a 2 mg / mL solution B, and stir magnetically;

[0009] 2) Mixing solution A and solution B in a specific ratio to form a mixed solution with a concentration of 1 mg / mL;

[0010] 3) mixing the mixed solution of step 2) with the treated bacterial suspension of Lactiplantibacillus sakei J15 in a certain ratio to obtain a mixed bacterial solution with a pH value of 7-8;

[0011] 4) After centrifuging the mixed bacterial solution in step 3), taking the precipitate and mixing it evenly with a cryoprotectant in a certain proportion, and then freeze-drying it to obtain a solid powder, namely, microcapsules.

[0012] Furthermore, in step 1), the magnetic stirring condition is: stirring at 37° C. for 30 min.

[0013] Furthermore, in step 2), the mixing ratio of solution A and solution B is 3:1 by mass.

[0014] Furthermore, in step 3), the specific processing steps of the bacterial suspension are:

[0015] The preserved sake Lactobacillus J15 was activated in MRS medium at an addition rate of 1%, and then the activated bacterial solution was inoculated into 50 mL MRS liquid medium at an inoculum rate of 1%. After 18 hours of cultivation, the wet bacteria were collected by centrifugation, and washed twice with sterile saline to prepare a concentrated bacterial suspension.

[0016] Furthermore, the activation temperature is 30°C and the activation time is 24h.

[0017] Furthermore, in step 3), the volume ratio of the mixed solution to the bacterial suspension is 10:1.

[0018] Furthermore, in step 4), the cryoprotectant contains 11.1% trehalose, 9.1% glycerol, 3.5% sodium glutamate and 15.7% skimmed milk powder, and "%" means how many grams of a certain component are contained in 100 mL of the cryoprotectant.

[0019] Furthermore, in step 4), the mass volume ratio of the precipitate to the cryoprotectant is 1:8.

[0020] The preparation method of the present invention uses whey protein isolate and sodium hyaluronate complex as capsule materials to microencapsulate probiotics, thereby stabilizing the activity of the probiotics and completing the delivery in food. It is suitable for direct application in dairy products, while ensuring the safety during operation and subsequent applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Phylogenetic tree of screened strains;

[0022] Figure 2 The growth curves of the strains at different temperatures;

[0023] Figure 3 The growth curves of the strains under different carbohydrates;

[0024] Figure 4 is the differential scanning calorimetry graph of Lactobacillus sake microcapsules;

[0025] Figure 5 This is a scanning electron micrograph of Lactobacillus sake microcapsules;

[0026] Figure 6 is the survival rate of Lactobacillus sakei capsules after simulated digestion in vitro;

[0027] Figure 7 This study is to study the heat resistance of Lactobacillus sake capsules. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific implementations to provide a better understanding of the technical solution.

[0029] The culture medium formula and reagents used for the screening and related verification of Lactobacillus sakei are as follows:

[0030] Culture medium formula:

[0031] The culture medium for isolating high-yield β-galactosidase active lactic acid bacteria: peptone 1%, beef extract 1%, yeast powder 0.5%, K2HPO4 0.2%, diammonium citrate 0.2%, sodium acetate 0.5%, glucose 2%, Tween 80 0.1mL, MgSO4·7H2O 0.058%, MnSO4·4H2O0.025%, agar 2%, X-gal (5-bromo-4-chloro-3-indole β-D-galactoside) 1%. The "%" in the culture medium herein means how many grams of a certain component are contained in 100mL of the culture medium.

[0032] MRS broth medium: peptone 1%, beef extract 1%, yeast powder 0.5%, K2HPO4 0.2%, diammonium citrate 0.2%, sodium acetate 0.5%, glucose 2%, Tween 80 0.1mL, MgSO4·7H2O 0.058%, MnSO4·4H2O 0.025%. The "%" in the medium herein means how many grams of a certain component are contained in 100mL of the medium.

[0033] Phosphate buffer: NaH2PO4 0.265%, Na2HPO4 0.825%. In this article, the "%" in the culture medium means how many grams of a certain component are contained in 100 mL of water.

[0034] Example 1: Preliminary screening of lactic acid bacteria with high β-galactosidase production

[0035] (1) Preliminary screening of lactic acid bacteria in fermented foods

[0036] Select yogurt fermented food, take 100 μL yogurt and add 900 μL 0.85% NaCl solution to dilute to different concentrations (10 -2 ~10 -4 ), spread on MRS solid medium with X-gal added, mark it, and observe and select colonies after culturing at 30°C for 24 hours. As a result, 15 separate colonies were selected on the medium and numbered 1 to 15.

[0037] (2) Rescreening of high-yielding β-galactosidase lactic acid bacteria

[0038] The strain was cultured in MRS broth at 30°C for 24h, centrifuged (7000×g, 15min, 4°C), resuspended in 50mM phosphate buffer (pH 7.0), and broken by ultrasonic treatment. The supernatant was the crude enzyme. The specific activity of β-galactosidase was determined using ONPG (o-nitrophenyl-β-D-pyranogalactoside) as a substrate. The crude enzyme (0.1mL) was mixed with 1mL of substrate (phosphate buffer containing 2mMONPG, pH 7.0) and measured at 405nm using a UV spectrophotometer. One unit of activity is defined as the amount of enzyme required to produce 1μmol o-nitrophenol per minute.

[0039] As shown in Table 1, the β-galactosidase activity of 15 initially screened strains was measured, and it was found that the enzyme activity of strain 15 was the highest, reaching 1.85 mU / mL. The 16s rDNA sequence of the strain was determined to identify the strain.

[0040] Table 1: Comparison of enzyme activities of 15 β-galactosidase producing strains

[0041]

[0042] Example 2: Identification of high-yielding β-galactosidase lactic acid bacteria

[0043] The strain No. 15 obtained by screening was identified by 16s rRNA sequencing. The sequencing sequence results were compared with the homologous sequences in the NCBI database by BLAST to determine the species of the strain. The species can be identified when the homology exceeds 99%. The phylogenetic tree was constructed using the near-joining method in the software MEGA-X for strains with higher homology.

[0044] Results: The 16s rRNA sequencing results of strain No. 15 were as follows.

[0045] The gene sequence of 16s rRNA of screening strain 15 (as shown in SEQ ID NO.1):

[0046] CTCTCGTTTAGATTGAAGGAGCTTGCTCCTGATTGATAAACATTTGAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTAAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAAACCTAACACCGCATGGTGTAGGGTTGAAAGATGGTTTCGGCTATCACTTTAGGATGGACCCGCGGTGCATTAGTTAGTTGGTGAGGTAAAGGCTCACCAAGACCGTGATGCATAGCCGACCTGAGAGGGTAATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTGGAGAAGAATGTATCTGATAGTAACTGATCAGGTAGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGGTAACCCTTCGGGGAGCCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGA

[0047] As shown in Table 2, the screening strain No. 15 was Latilactobacillus sakei, which had 100.00% homology with the standard strain Latilactobacillus sakei ZFM229.

[0048] Table 2: Identification results of strains with high β-galactosidase activity

[0049]

[0050] like Figure 1 As shown, the evolutionary distance between the screened strains Latilactobacillus sakei J15 and Latilactobacillus sakei ZFM229 and Lactiplantibacillus plantarum LG138 was the smallest.

[0051] At the same time, the strain Latilactobacillus sakei J15 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the storage address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The storage name is Lactobacillus sakei J15, the storage time is September 24, 2024, and the strain storage number is CGMCC No.32056.

[0052] Example 3: Growth curves of Latilactobacillus sakei J15 at different temperatures and β-galactosidase activity

[0053] The strains were cultured at 5, 10, 15, 20, 25, 30, and 37°C for 60 h, and the growth curves were measured at 600 nm at 3, 6, 9, 12, 24, 36, 48, and 60 h, respectively. Figure 2 At the same time, the specific enzyme activity of β-galactosidase was determined at different temperatures. The strain was cultured at 30°C for 18 h, and the crude enzyme solution was prepared and measured at 5, 10, 15, and 30°C.

[0054] Test results: Latilactobacillus sakei J15 strain grew better at 25℃ and 30℃, entered the logarithmic growth phase after 3 hours, and the bacteria increased rapidly; it reached the stable growth period at 24-60 hours, when the number of live bacteria remained relatively stable; while at other temperatures (5, 10, 15, 20 and 37℃), it entered the logarithmic growth phase after 9 hours of growth, especially at 5, 10, 15℃, it was still in the logarithmic growth phase until 60 hours.

[0055] Although the optimum growth temperature of the strain is 25-30°C, the β-galactosidase activity of the strain at 15°C is the highest, reaching 789.31 mU / mg (Table 3). At 5°C, the strain still maintains a certain enzyme activity (41.08 mU / mg).

[0056] This indicates that Latilactobacillus sakei J15 strain has higher β-galactosidase activity at low temperature.

[0057] Table 3: β-galactosidase activity of Latilactobacillus sakei J15 at different temperatures

[0058]

[0059] Example 4: Ability of Latilactobacillus sakei J15 to utilize 49 carbohydrates

[0060] API 50CH was used to determine the ability of Latilactobacillus sakei J15 to utilize carbohydrates. The strain was activated by culturing in MRS medium for 24 hours, and two McFarland turbidity suspensions were prepared, and then the suspensions were added to 50 microbiological tubes on the reagent strip. When the bacteria can utilize different substances in the substrate as the only carbon source, if the bacteria can utilize or grow, the color changes, which is indicated by positive (+), and the color changes slightly, which is indicated by (±). If the bacteria cannot utilize the carbon source, the color does not change, which is indicated by negative (-).

[0061] Measurement results: The results showed that Latilactobacillus sakei J15 was able to utilize 16 kinds of carbohydrates (Table 4), namely, L-arabinose, D-ribose, galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, arbutin, esculin, salicin, D-maltose, D-lactose, D-melanobiose, cellulose, D-trehalose, and D-toluose.

[0062] Table 4: Fermentation capacity of Latilactobacillus sakei J15 on different carbohydrates and their derivatives

[0063]

[0064] Example 5: Growth curve and enzyme activity of Latilactobacillus sakei J15 in medium containing different carbohydrates

[0065] The strains were cultured in glucose, arabinose, galactose, fructose, sucrose, lactose, and maltose for 36 h, and the growth curves were measured at 600 nm at 3, 6, 9, 12, 24, and 36 h, respectively. Figure 3 ), and at the same time, the β-galactosidase specific enzyme activity was determined (Table 5).

[0066] Test results: Figure 3 As shown, Latilactobacillus sakei J15 strain has a relatively obvious logarithmic growth phase in the medium supplemented with different carbohydrates, except arabinose, in the growth environment of other carbon sources. After 24 hours of cultivation, the OD value reached above 1.0.

[0067] As shown in Table 5, it can be seen that the specific enzyme activity of β-galactosidase of the strain after being cultured in the medium with lactose as the carbon source is much higher than that in other carbon source growth environments, and the specific enzyme activity is as high as 1555.68 mU / mg, indicating that Latilactobacillus sakei J15 can effectively decompose lactose.

[0068] Table 5: Specific enzyme activity of β-galactosidase from Latilactobacillus sakei J15 grown in different carbohydrates

[0069]

[0070] The culture medium and reagents used in the following examples are as follows:

[0071] Culture medium formula:

[0072] MRS medium: peptone 1%, beef extract 1%, yeast powder 0.5%, K2HPO4 0.2%, diammonium citrate 0.2%, sodium acetate 0.5%, glucose 2%, Tween 80 0.1mL, MgSO4·7H20 0.058%, MnSO4·4H2O0.025%, agar 2%. The "%" in the medium herein means how many grams of a certain component are contained in 100mL of the medium.

[0073] Phosphate buffer: NaH2PO4 0.265%, Na2HPO4 0.825%. In this article, the "%" in the culture medium means how many grams of a certain component are contained in 100 mL of water.

[0074] Example 6: Encapsulation efficiency of Lactobacillus sakei microencapsulation in different ratios of whey protein isolate and sodium hyaluronate

[0075] (1) Activation of Lactobacillus sakei

[0076] The strain Lactiplantibacillus sakei J15 was inoculated into MRS liquid medium at a 10% inoculum for activation, and then spread on an MRS plate for line culture. After culturing at 30°C for 48 hours, a single colony was picked and inoculated into 5 mL of MRS liquid medium, and cultured at 30°C for 24 hours. Then, it was inoculated into 5 mL of MRS liquid medium at a 1% inoculum. After 18 hours of culture, the bacterial sludge was collected by centrifugation, washed twice with sterile saline, and a concentrated bacterial solution was prepared. At this time, the bacterial solution concentration was about 10 10 CFU / mL, and the concentrated bacterial solution was spread and counted.

[0077] (2) Preparation of probiotic microcapsules

[0078] After the whey protein isolate and sodium hyaluronate were mixed evenly in a ratio of 3:1, 1:1, and 1:3, the concentrated bacterial solution was added to the mixed solution at a ratio of 1:10, stirred for 30 minutes, and the mixed solution was centrifuged at 4000g, 4°C, 20 minutes to obtain the precipitate, which was then mixed with the pre-sterilized cryoprotectant at a ratio of 1:8. The mixed solution was pre-frozen (-20°C for 24 hours, -80°C for 48 hours) and then freeze-dried.

[0079] (3) Determination of embedding rate

[0080] Take 0.1 g of microcapsule powder and add it into 5 mL of phosphate buffered saline (PBS) (0.2 M, pH = 7.2).

[0081] Then shake in a constant temperature shaker at 37°C until it is completely dissolved, and finally dilute and spread to count. The calculation formula is as follows:

[0082] Embedding rate (%) = N / N0 × 100%

[0083] Where: N——concentration of live bacteria embedded in microcapsules (log10CFU / g);

[0084] N0——Concentration of live bacteria added during microencapsulation (log10CFU / g).

[0085] Measurement results: The results are shown below.

[0086] The embedding rate is shown in Table 6. Under different ratios of WPI to SH, the survival rate of Lactiplantibacillus sakei J15 was greater than 90%, indicating that the complex of whey protein isolate and sodium hyaluronate had a good embedding effect.

[0087] Table 6: Microcapsule embedding efficiency results of Lactobacillus sakei

[0088]

[0089] Different letters indicate significant differences between different WPI and SH ratios of the same strain (P<0.05)

[0090] Example 7: Determination of particle size of Lactobacillus sake microcapsules

[0091] The diameter and dispersibility after microencapsulation were observed by measuring the particle size. Before freeze drying, one milliliter of the mixture was analyzed using a particle size analyzer (Malvern Instruments, UK, Malvern DTS0012) without mixing with cryoprotectant.

[0092] Measurement results: As shown in Table 7, when the ratio of whey protein isolate to sodium hyaluronate is 3:1, the particle size of the microcapsule is the smallest (1428 nm) and the absolute value of the polymer dispersibility index (PDI) is also the smallest (0.411).

[0093] Table 7: Particle size and PDI results of Lactobacillus sake microcapsules

[0094]

[0095] Different letters indicate significant differences between different WPI and SH ratios of the same strain (P<0.05)

[0096] Example 8: Study on the thermal stability of Lactobacillus sake microcapsules

[0097] Differential scanning calorimetry was used to determine the thermal stability of microcapsules embedded with different ratios of whey protein isolate and sodium hyaluronate complexes. The operation was as follows:

[0098] Accurately weigh 5-10 mg of microcapsule powder into a sealed aluminum box and place the aluminum box in a differential scanning calorimeter. Set the program to heat the sample from 25°C to 200°C at a rate of 10°C / min. Use a blank aluminum box as a control and plot the thermal property curve using the heat flow difference and temperature ( Figure 4 ).

[0099] Measurement results: Figure 4As shown, it can be seen that the denaturation temperature (Ts) of Lactobacillus sake is 141.38°C (ΔH = 142.07 J / g). In the Lactobacillus sake microcapsules, when WPI:SH = 3:1, Ts is 142.96°C (ΔH = 140.25 J / g); when WPI:SH = 1:1, Ts is 153.33°C (ΔH = 124.26 J / g); when WPI:SH = 1:3, Ts is 145.20°C (ΔH = 142.75 J / g). In general, the Ts of Lactobacillus sake microcapsules of all proportions are higher than those of strains that are not microencapsulated, indicating that the presence of the WPI-SH complex improves the anti-disintegration performance of bacterial cells. The higher the melting temperature, the more stable the material, and the higher its thermal stability.

[0100] Example 9: Study on the morphology of Lactobacillus sake microcapsules

[0101] The morphology of the microcapsules was analyzed using a scanning electron microscope as follows:

[0102] A carbon adhesive label was attached to an aluminum sheet, and then an appropriate amount of microcapsule powder was spread on the carbon adhesive label. The sample not adhering to the surface was removed with tweezers, and then the sample was placed in a SU8010 scanning electron microscope at an accelerating voltage of 5 kV to obtain a scanning electron microscope image ( Figure 5 ).

[0103] Measurement results: Figure 5 As shown, the fiber-like filaments decreased with the decrease of SH concentration and increased with the increase of WPI concentration. The effect of adding WPI and SH showed that the fiber-like filaments increased and the fiber-like filaments structure was more compact. According to the morphological analysis, it can be seen that when the ratio of WPI to SH is 3:1, the binding state of the strain and the WPI-SH complex is appropriate. This dense structure can prevent bacterial cells from entering the cavity.

[0104] Example 10: In vitro simulation study of Lactobacillus sake microcapsules

[0105] Add 0.1 g of microcapsule powder to 5 mL of sterile saline, take a sample, apply it, and calculate the preparation of in vitro simulated liquid. The operation is as follows:

[0106] A simulated oral solution (0.896 mg / mL KCl solution, 0.298 mg / mL NaCl solution, and 0.6 mg / mL α-amylase solution) was prepared, and the oral solution was mixed with the sample in a ratio of 1:1. The pH value of the reaction system was adjusted to 6.8, and the mixture was stirred and digested in a magnetic stirring pot at 37°C for 10 min. The samples were then taken, coated, and counted. A simulated gastric fluid (2 mg / mL NaCl solution and 3.2 mg / mL pepsin solution) was prepared, heated to 37°C in advance, and mixed with the sample after oral digestion in a ratio of 1:1. The pH value of the reaction system was then adjusted to 2.0 with 0.1 M HCl, and the mixture was stirred and digested in a magnetic stirring pot at 37°C for 180 min. The samples were then taken, coated, and counted. The previously digested sample was adjusted to pH 7.0 with 0.1M NaOH solution and mixed with the prepared simulated intestinal fluid (36.7mg / mL CaCl2, 218.7mg / mL NaCl solution, 24mg / mL trypsin solution and 0.3mg / mL bile) in a 1:1 ratio. Then, the sample was stirred and digested for 8h at 37°C and the survival rate was calculated ( Figure 6 )

[0107] Test results: Probiotic microcapsules with different ratios showed different survival characteristics. After digestion, the survival rate of free sake Lactobacillus J15 was 16.55%. After microencapsulation, the survival rates of microcapsule strains encapsulated by whey protein isolate and sodium hyaluronate complexes of all different ratios were higher than those of free strains in the stomach and intestines. Among them, the microcapsule ratio with the highest survival rate was WPI:SH=3:1, which was 45.39%.

[0108] Example 11: Study on the heat resistance of Lactobacillus sake microcapsules

[0109] Heat resistance study, the operation is as follows:

[0110] 0.5 g wet microcapsules or 0.5 mL probiotic suspension were added to a centrifuge tube and placed in a water bath at different temperatures (40, 50, 60, and 70°C) for 30 min. After heat treatment, 4.5 mL PBS was added and incubated at 37°C in a shaker for 2 h. Then, the counts were spread ( Figure 7 ).

[0111] Test results: Figure 7As shown in the figure, at 40℃, the survival rates of free Lactobacillus sakei J15 and after microencapsulation were both over 70%. The survival rates of free Lactobacillus sakei J15 at 50℃, 60℃ and 70℃ were 11.91%, 5.95% and 0%, respectively, and higher temperatures would reduce the survival rate. Microencapsulation has a very significant protective effect on cells. After encapsulation with WPI-SH complex, when WPI:SH=3:1, the survival rates at 50℃, 60℃ and 70℃ were 48.39%, 33.93% and 16.97%, respectively; when WPI:SH=1:1, the survival rates at 50℃, 60℃ and 70℃ were about 37.72%, 33.93% and 16.97%, respectively; when WPI:SH=1:3, the survival rates at 50℃, 60℃ and 70℃ were about 43.79%, 33.73% and 31.95%, respectively. Among them, the microcapsules with a ratio of WPI:SH=3:1 have higher thermal stability than those with other ratios at 50°C and 60°C.

[0112] Finally, in summary, microcapsules with WPI:SH=3:1 were shown to be most effective in protecting the survival of bacteria, and the whey protein isolate and sodium hyaluronate complex has great potential as a new delivery vehicle for probiotics and bioactive compounds.

Claims

1. A method for encapsulating Lactobacillus sakei using a whey protein isolate and sodium hyaluronate complex, characterized in that The following steps are involved: 1) Dissolve whey protein isolate in sterile water to prepare a 20 mg / mL solution A; dissolve sodium hyaluronate in sterile water to prepare a 2 mg / mL solution B, and stir magnetically; 2) Mixing solution A and solution B in a specific ratio to form a mixed solution with a concentration of 1 mg / mL; 3) mixing the mixed solution of step 2) with the treated bacterial suspension of Lactiplantibacillus sakei J15 in a certain ratio to obtain a mixed bacterial solution with a pH value of 7-8; 4) After centrifuging the mixed bacterial solution in step 3), taking the precipitate and mixing it evenly with a cryoprotectant in a certain proportion, and then freeze-drying it to obtain a solid powder, namely, microcapsules.

2. A method for embedding Lactobacillus sakei using a whey protein isolate and sodium hyaluronate complex as claimed in claim 1, characterized in that In step 1), the magnetic stirring condition is: stirring at 37° C. for 30 min.

3. A method for embedding Lactobacillus sakei using a whey protein isolate and sodium hyaluronate complex as claimed in claim 1, characterized in that In step 2), the mixing ratio of solution A and solution B is 3:1 by mass.

4. A method for embedding Lactobacillus sakei using a whey protein isolate and sodium hyaluronate complex as claimed in claim 1, characterized in that In step 3), the specific processing steps of the bacterial suspension are: The preserved sake Lactobacillus J15 was activated in MRS medium at an addition rate of 1%, and then the activated bacterial solution was inoculated into 50 mL MRS liquid medium at an inoculum rate of 1%. After 18 hours of cultivation, the wet bacteria were collected by centrifugation, and washed twice with sterile saline to prepare a concentrated bacterial suspension.

5. A method for embedding Lactobacillus sakei using a whey protein isolate and sodium hyaluronate inclusion complex as claimed in claim 4, characterized in that The activation temperature is 30°C and the activation time is 24h.

6. A method for embedding Lactobacillus sakei using a complex of whey protein isolate and sodium hyaluronate as claimed in claim 1, characterized in that In step 3), the volume ratio of the mixed solution to the bacterial suspension is 10:

1.

7. A method for encapsulating Lactobacillus sakei using a complex of whey protein isolate and sodium hyaluronate as claimed in claim 1, characterized in that In step 4), the cryoprotectant contains 11.1% trehalose, 9.1% glycerol, 3.5% sodium glutamate and 15.7% skimmed milk powder, and "%" means how many grams of a certain component are contained in 100 mL of the cryoprotectant.

8. A method for embedding Lactobacillus sakei using a complex of whey protein isolate and sodium hyaluronate as claimed in claim 1, characterized in that In step 4), the mass volume ratio of the precipitate to the cryoprotectant is 1:8.

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