A low water activity, storage-resistant probiotic microcapsule and its preparation method

Abstract

The present invention discloses a low water activity, storage-resistant probiotic microcapsule and a preparation method thereof. First, a fresh lactobacillus liquid is centrifuged, and then washed with 0.85% physiological saline by mass fraction to obtain a lactobacillus strain suspension; then a capsule wall material emulsion is mixed with the lactobacillus strain suspension and continuously stirred, and low water activity, storage-resistant probiotic microcapsules are obtained by spray drying. The capsule wall material emulsion includes 6-10% (w / v) peanut protein powder, 0-3% (w / v) resistant starch and 0-3% (w / v) beta-cyclodextrin, and the rest is water, and resistant starch and beta-cyclodextrin contain at least one. Resistant starch and beta-cyclodextrin, two polysaccharides, resistant starch and beta-cyclodextrin, cooperate with each other in terms of thermal stability, encapsulation efficiency, water content and survival rate of gastrointestinal digestion, and promote each other to jointly improve the performance of microcapsules.

Description

Technical Field

[0001] The present invention relates to the technical field of microcapsule preparation, and in particular to a low-water-activity, storage-resistant probiotic microcapsule and a preparation method thereof. Background Art

[0002] Probiotics are live bacteria that, when administered in sufficient quantities, can confer health benefits on the host. Extensive evidence demonstrates that probiotics have health benefits for the host, including regulating intestinal flora, improving the intestinal barrier, enhancing immunity, and alleviating a variety of conditions (e.g., colitis, diarrhea, and allergies). Currently, the most popular probiotic strain is Lactobacillus, which is widely used for pharmaceutical purposes. Lactobacillus produces organic acids, specialized enzymes, and acidophilins through fermentation, which possess unique physiological functions. Among these, Lactobacillus plantarum is a particularly promising probiotic. Experimental results have demonstrated that L. plantarum KLDS1.0318 has the ability to regulate intestinal flora composition, repair intestinal mucosal damage, enhance immunity, and possess strong resistance to acid and bile salts. After colonizing and multiplying in the human intestinal tract, Lactobacillus rhamnosus GG simply attaches to the host's intestinal epithelial cells, forming a biological barrier layer in the intestinal mucosa, thereby enhancing the host's intestinal mucosal barrier capacity. Research has found that Lactobacillus reuteri produces reuterin, which inhibits the growth of harmful bacteria such as Escherichia coli and Salmonella, thereby maintaining the intestinal microbiome balance. Furthermore, Lactobacillus reuteri produces lactic acid and various enzymes, such as lipase and bile salt hydrolase, in the animal intestine, which can help improve intestinal pH, inhibit bacterial growth, and improve feed utilization. To fully realize the beneficial effects of probiotics on the host, it is crucial to consume a sufficient amount of live bacteria. The International Dairy Federation recommends a minimum intake of 6 Log CFU / g. However, during the processing, storage, and consumption of probiotic products, various factors can reduce the activity and content of probiotics. First, probiotic products are susceptible to temperature and moisture stress during preparation, resulting in loss of cell viability. Second, probiotics typically undergo a period of storage before oral consumption, and the conditions and duration of storage can affect their survival. Third, probiotics have poor oral stability and are highly sensitive to gastrointestinal environmental stress (such as gastric acid, bile salts, enzymes, etc.), which leads to a decrease in the number and activity of probiotics. Therefore, ensuring the storage vitality of probiotics in food is a serious challenge.

[0003] Microencapsulation has emerged as one of the most promising approaches for protecting probiotic cells from adverse exposure conditions. Microencapsulation is defined as encapsulating a core material containing nutrients or bioactive compounds from food within a polymer matrix shell, protecting the primary components and providing controlled release. Spray drying is one of the most commonly used techniques for microencapsulation due to its short production time, low cost, and excellent final product stability and quality. Compared to other traditional microencapsulation technologies, the spray drying process can be easily scaled up to produce microcapsules in a continuous process.

[0004] It is well known that the selection of wall materials is crucial for the microencapsulation of probiotics. For the microencapsulation of probiotics, proteins and polysaccharides are the most commonly used wall materials for the preparation of bacterial microcapsules. Proteins have good film-forming properties, but are easily degraded by pepsin in gastric juice and tend to aggregate. Peanut meal is a by-product that is not fully utilized in the processing of peanut oil. For every 1 kg of peanut oil squeezed, about 1.25 kg of peanut meal will be produced, and its annual output is about 4 million tons in China alone. The product after the peanut meal is crushed is called peanut protein powder, which is inexpensive and abundant in source. Since the protein content in peanut meal is about 50%, it is a high-quality source of plant protein and is considered to be beneficial to the human intestinal flora. To this end, the present invention discloses a low-water-activity, storage-stable probiotic microcapsule based on peanut meal protein and a preparation method thereof. Summary of the Invention

[0005] The present invention aims to provide a low-water-activity, storage-stable probiotic microcapsule and a preparation method thereof. The method utilizes peanut protein powder and two polysaccharides (resistant starch and beta-cyclodextrin) as wall materials to encapsulate probiotics to prepare microcapsules, and produces probiotic microcapsule powders through spray drying, thereby improving the survival rate of probiotics during storage and resolving the shortcoming that existing probiotic powders are easily inactivated during storage. The preparation method can also improve the spray-drying survival rate and encapsulation efficiency of probiotics, and the probiotic microcapsules prepared thereby also have the advantages of low water activity and resistance to gastrointestinal digestion.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for preparing low water activity, storage-stable probiotic microcapsules comprises the following steps:

[0008] (1) centrifuging a fresh Lactobacillus solution, and then washing with 0.85% saline to obtain a Lactobacillus suspension;

[0009] (2) A capsule wall material emulsion is mixed with a Lactobacillus strain suspension, stirred continuously, and spray-dried to obtain low water activity, storage-stable probiotic microcapsules, wherein the capsule wall material emulsion comprises 6-10% (w / v) peanut protein powder, 0-3% (w / v) resistant starch, and 0-3% (w / v) β-cyclodextrin, with the remainder being water. Wherein w is the mass of the solute in g, and v is the volume of the solvent in mL.

[0010] Specifically, the method for culturing the fresh lactobacillus liquid in step (1) is as follows: inoculating the glycerol-preserved lactobacillus into MRS liquid culture medium, culturing at 37°C for 10-12 hours, and then transferring to fresh MRS liquid culture medium and culturing at a constant temperature of 37°C for 16-18 hours.

[0011] Preferably, the capsule wall material emulsion comprises 6-9% (w / v) peanut protein powder and 1.5-3% (w / v) β-cyclodextrin, with the remainder being water.

[0012] β-cyclodextrin has the characteristics of biocompatibility, easy degradation and non-toxicity, and is a widely used polysaccharide wall material. The wall material prepared by combining β-cyclodextrin with peanut protein powder can effectively resist the digestion of gastric juice. However, experimental studies have found that the addition of β-cyclodextrin will lead to an increase in the water content of the peanut protein powder-based wall material, and the microcapsule wall is easily destroyed, affecting the storage stability. To this end, the present invention introduces a new polysaccharide component, resistant starch, on the basis of peanut protein powder and β-cyclodextrin. Resistant starch is divided into resistant starch 1, resistant starch 2, resistant starch 3, resistant starch 4, and resistant starch 5 according to different structures, and resistant starch 3 is preferred. Resistant starch 3 is prepared from a substance rich in starch, contains rich amylose, has good thermal stability, and has good low water retention.

[0013] Preferably, the capsule wall emulsion comprises 6-10% (w / v) peanut protein powder and 1-3% (w / v) resistant starch, with the remainder being water. Protein has excellent film-forming properties, while resistant starch offers maximum resistance to digestion by pancreatic amylase. Therefore, their combined use can produce probiotic microcapsules with a denser structure and enhanced stability.

[0014] Preferably, the capsule wall material emulsion comprises 7-9% (w / v) peanut protein powder, 0.5-1.5% (w / v) resistant starch and 0.5-1.5% (w / v) β-cyclodextrin, and the rest is water. Preferably, the mass ratio of the peanut protein powder to the two polysaccharides of resistant starch and β-cyclodextrin is 3-4:1. More preferably, the capsule wall material emulsion comprises 8% (w / v) peanut protein powder, 1% (w / v) resistant starch and 1% (w / v) β-cyclodextrin, and the rest is water. The capsule wall material prepared from peanut protein powder and the two polysaccharides of resistant starch and β-cyclodextrin not only has excellent heat resistance, which protects probiotics from the effects of high temperature and dehydration stress during spray drying, but also has improved encapsulation efficiency, survival rate and other properties compared to single polysaccharides.

[0015] The lactobacillus includes but is not limited to probiotics having a hydrophobic surface such as Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus fermentum and Lactobacillus paracasei.

[0016] Specifically, in step (2), the capsule wall materials (peanut protein powder, resistant starch and β-cyclodextrin) are added to water and then homogenized on a high-speed disperser at 10,000 rpm for 5 minutes to obtain a completely dispersed emulsion.

[0017] Specifically, in step (2), the volume ratio of the capsule wall material emulsion to the Lactobacillus suspension is 4:1.

[0018] Specifically, the spray drying operating conditions in step (2) are: inlet temperature of 110-130°C; feed flow rate of 4-9 mL / min; air flow rate of 30-40 m 3 / h, the compressor air pressure is 0.35-0.45MPa.

[0019] The low water activity and storage-stable probiotic microcapsules prepared by the preparation method.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] (1) The present invention uses peanut protein powder in combination with two polysaccharides (resistant starch and β-cyclodextrin) as encapsulation materials, and prepares four wall materials to encapsulate lactobacilli respectively. On the one hand, resistant starch has low water retention and thermal stability, which effectively improves the survival rate of plant lactobacillus in microcapsules; on the other hand, β-cyclodextrin has a hydrophobic cavity inside and a hydrophilic group outside, which can be tightly connected with lactobacilli with surface hydrophobicity through hydrophobic forces, which can significantly improve the encapsulation rate of probiotic microencapsulation. In addition, the addition of resistant starch and β-cyclodextrin gives the microcapsules the advantage of low water activity; at the same time, the above two polysaccharides can resist gastrointestinal digestion and are both considered to be intestinal prebiotics, which can play a key role in gastrointestinal metabolism and health.

[0022] (2) The present invention adopts a spray drying method to prepare microcapsules, and the preparation process is simple and convenient. The peanut protein powder is prepared by ultrafine grinding of peanut meal. In particular, the peanut meal raw material is cheap and easy to obtain, which can improve the utilization rate of peanut meal and effectively reduce the production cost of microcapsules, facilitating large-scale industrial production and application promotion.

[0023] (3) The two polysaccharides, resistant starch and β-cyclodextrin, synergize with each other in terms of thermal stability, encapsulation efficiency, moisture content, and survival rate of gastrointestinal digestion, promoting each other and jointly improving the performance of microcapsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Scanning electron micrographs of microcapsules prepared for PP group, PPS group, PPC group, and PSC group.

[0025] Figure 2 Particle size distribution of microcapsules prepared for PP group, PPS group, PPC group, and PSC group.

[0026] Figure 3 Survival rate of viable bacteria in in vitro simulated gastrointestinal digestion of microcapsules prepared for PP group, PPS group, PPC group and PSC group.

[0027] Figure 4 Cell viability diagram during storage period of microcapsules prepared for PP group, PPS group, PPC group and PSC group.

[0028] Figure 5 Differential scanning calorimetry (DSC) analysis diagrams of microcapsules prepared for PP group, PPS group, PPC group and PSC group.

[0029] Figure 6 Low-field nuclear magnetic resonance (LF-NMR) images of microcapsules prepared for PP group, PPS group, PPC group and PSC group. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0031] Example 1

[0032] 1: Preparation of Lactobacillus plantarum Heal 19 microcapsules

[0033] (1) Glycerol-preserved Lactobacillus plantarum Heal 19 was inoculated at a 1% inoculum into 50 mL of MRS liquid medium and activated at 37°C for 12 h. Subsequently, the inoculum was transferred to fresh MRS liquid medium at a 1% inoculum and cultured at 37°C for 16-18 h to obtain a fresh bacterial suspension. The fresh bacterial suspension was centrifuged at 6000 rpm for 12 min and washed twice with 0.85% (w / v) saline to obtain a strain suspension for later use.

[0034] (2) Prepare four emulsions: PPS, PPC, PSC and PP. PPS: 8% (w / v) peanut protein powder and 2% (w / v) resistant starch emulsion, PPC: 8% (w / w) peanut protein powder and 2% (w / v) β-cyclodextrin emulsion, PSC: 8% (w / w) peanut protein powder and 1% (w / v) resistant starch and 1% (w / v) β-cyclodextrin emulsion, PP: 10% (w / v) peanut protein emulsion, so that the total polymer concentration is maintained at 10% (w / w), and the ratio of protein to polysaccharide is maintained at 4:1. The four prepared emulsions are homogenized on a high-speed disperser at 10,000 rpm for 5 minutes until the emulsion is completely dispersed and set aside. The harvested strains are suspended in the emulsion at room temperature (25°C). The average bacterial concentration of each bacterial solution is about 10 7 CFU / mL, the emulsion and the strain suspension were mixed at a volume ratio of 4:1, and the mixture was stirred with a magnetic stirrer and spray-dried to prepare probiotic suspensions encapsulated with different wall materials. The operating conditions were: inlet temperature = 120±1℃; feed flow rate = 7mL / min; air flow rate = 35m 3 / h, compressor pressure = 0.4 MPa. During the preparation of the microcapsule powder, the mixture was continuously stirred using a magnetic stirrer. After spray drying, the dried powder was collected from a collection jar and stored at 4°C for further analysis.

[0035] 2: Structural characterization of microcapsules

[0036] The structure, particle size, and polydispersity index (PDI) of the four different Lactobacillus plantarum Heal 19 microcapsules prepared in Example 1 were observed and measured. The measurement method is as follows:

[0037] ① Structural observation: The four microcapsule samples were pasted onto a carbon-and-metal aluminum conductive adhesive. After the samples were sprayed with gold, the morphologies of the four groups of microcapsules were photographed using a SEM.

[0038] ② Particle Size Determination: 0.2 g of microcapsules prepared from the PP, PPS, PPC, and PSC groups were dissolved in 2 mL of sterile distilled water and centrifuged at 3000 rpm for 2 minutes to minimize the effects of insoluble impurities. Particle size analysis of the microcapsule powders was performed using a Zetasizer laser diffraction particle size analyzer. This analysis was repeated three times for each sample.

[0039] ③ PDI determination: The polydispersity index (PDI) of the microcapsule droplets was measured using a Zetasizer laser diffraction particle size analyzer to evaluate the droplet size distribution. Each sample was repeated three times.

[0040] Depend on Figure 1 It can be seen that the SEM images of the spray-dried microcapsule powder show microspheres of different sizes, ranging from 7 to 15 μm. The surface of the microcapsules is wrinkled and relatively rough, and the microcapsules have a certain degree of collapse. By comparison Figure 1 The microcapsules from the four formulations varied in size, with the majority being spherical, and some not forming a regular spherical shape. Furthermore, the presence of the disaccharide component also resulted in a higher number of spherical microcapsules in the PSC group, indicating better encapsulation. The microcapsules from the other three groups exhibited some wrinkling, likely due to the excessive vaporization of the droplets formed during spray drying, which resulted in wrinkles and collapse on the microcapsule surface.

[0041] The average particle size of the microcapsules is shown in Table 1. The average surface diameter of the microcapsules in the PSC group (213.07±19.09nm) was significantly larger than that in the other three groups, the PPS group (133.97±0.29nm), the PPC group (145.37±18.91nm), and the PP group (68.01±1.42nm). This shows that the addition of resistant starch and β-cyclodextrin increased the particle size of the microcapsules. Figure 2 As shown in Table 1, the droplet size of the various emulsions ranges from 10 to 1000 nm, showing a unimodal distribution. It is worth noting that if the droplet size shows a unimodal distribution and the smaller the polymer dispersibility index PDI value (PDI value ≤ 0.3), the more uniform the droplet particles are distributed. As shown in Table 1, the PDI values ​​of the four groups of microcapsules range from 0.21 to 0.29, which confirms that the combination of peanut protein powder and polysaccharide is suitable for preparing emulsions, and the distribution of the emulsions in the PPS group, PPC group, and PSC group is more uniform than that prepared from pure peanut protein powder. Therefore, the emulsion prepared from the combination of peanut protein powder and polysaccharide is more uniform, which significantly improves the bioavailability and prebiotic effect of probiotics.

[0042] 3: Determination of microcapsule cell viability count and encapsulation efficiency (EE)

[0043] First, dissolve 1 g of spray-dried powder in 9 mL of 0.85% (w / v) NaCl solution and vortex for 2 minutes until the powder is completely dissolved. Then, serially dilute the dissolved solution eight-fold with 0.85% (w / v) NaCl solution. Place the diluted bacterial solution in a sterile MRS culture dish and incubate at 37°C for 48 hours before counting viable cells. The number of viable cells is expressed as CFU / mL or CFU / g. Each sample is replicated three times. The encapsulation efficiency (EE) of Lactobacillus plantarum Heal 19 microcapsules is calculated as follows: EE (%) = (E / E0) × 100%.

[0044] Where: E is the total number of viable bacteria embedded in the microcapsules, and E0 is the total number of viable bacteria in the bacterial suspension used to prepare the microcapsules.

[0045] The results of cell viability and encapsulation efficiency are shown in Table 1. The cell viability of the four groups of microcapsules decreased due to the effect of spray drying. The cell viability of the PP group after spray drying was 0.83×10 7 log CFU / mL, the encapsulation efficiency was only 77.8%. However, the cell viability increased significantly after adding resistant starch and β-cyclodextrin. The cell viability of the PPS group and the PPC group after spray drying was 1.81×10 7 log CFU / mL and 1.69×10 7 log CFU / mL, and the encapsulation efficiency was 87.91% and 85.38%, respectively. This indicates that the microcapsules of protein combined with a polysaccharide combination (PPS group and PPC group) also have sufficient protective effects on probiotics. The number of probiotic strains in the PSC group was the largest, at 2.55×10 7 log CFU / mL, the microencapsulation efficiency is close to 94%, the protection effect of the microcapsules of the dual polysaccharide combination is better than that of the single polysaccharide combination, and the combination of peanut protein powder and dual polysaccharide can better protect probiotics from high temperature and dehydration stress during spray drying.

[0046] 4: Determination of water content and water activity (aw) of microcapsules

[0047] The water content and water activity of the four different Lactobacillus plantarum Heal 19 microcapsules prepared in Example 1 were measured. The measurement method is as follows:

[0048] ① Moisture Content Determination: Place the spray-dried microcapsule powder in a drying dish and dry it in a conventional hot air oven at 105°C. Allow the microcapsule powder to dry until it reaches a constant weight, then weigh it. Three replicates were performed for each sample. Moisture content was calculated as follows: Moisture (%) = (M0 - M0) / M0 × 100%.

[0049] Wherein: M0 and M represent the initial weight of the powder before drying and the final weight of the powder after drying, respectively.

[0050] ② Water activity determination: The water activity of the four microcapsule powders was measured at room temperature (25±1°C) using a water activity meter. Each sample was run three times in parallel.

[0051] A water content of less than 7% (w / w) after spray drying is believed to minimize the risk of cell death during storage. Furthermore, a water content between 4% and 7% can achieve better storage stability, thereby enhancing cell viability. As can be seen from Table 1, the water content of the microcapsules in the present invention ranged from 4.82% to 5.26%, both within the aforementioned standard range.

[0052] Water activity is another indicator for assessing the moisture content of microcapsules. It is a useful supplement to evaluating the stability of microcapsule powders, as microcapsules with the same moisture content can exhibit varying degrees of perishability. For the food industry, a water activity of less than 0.6 is considered acceptable, as most microorganisms cannot grow or reproduce at this level. As shown in Table 1, the water activity of the four microcapsules ranged from 0.17 to 0.18, with all four experimental groups achieving water activity values ​​below 0.6, thus meeting food industry standards. Compared to the PP group, the water activity of the PSC, PPS, and PPC groups decreased, with the PSC group achieving the lowest water activity of 0.166. This suggests that the addition of resistant starch and β-cyclodextrin reduces the likelihood of microbial growth in the microcapsules, and that the simultaneous addition of the two polysaccharides produces a synergistic effect, further reducing the likelihood of microbial growth.

[0053] It is generally recommended that the moisture content and water activity should be below 7% and 0.25 during long-term storage. As shown in Table 1, the moisture content and water activity of probiotic microcapsules prepared using different encapsulant formulations (PSC, PP, PPS, and PPC) all meet the standard acceptance values ​​for probiotic products. This is beneficial for probiotic microcapsules that require long-term storage. By selecting the appropriate encapsulant during production to control the water activity of probiotic microcapsules, their stability during storage can be improved.

[0054] 5: Survival rate of live bacteria in microcapsules during in vitro simulated gastrointestinal digestion

[0055] The method for determining the viable bacterial survival rate of Lactobacillus plantarum microcapsules in simulated gastric and intestinal fluids is as follows:

[0056] ① Simulated gastric fluid digestion: Fresh Heal 19 bacterial suspension and spray-dried powder were passed through an in vitro digestion model consisting of a simulated stomach. Add 0.56g potassium chloride, 1.0g sodium chloride, 0.055g calcium chloride, 0.2g potassium dihydrogen phosphate, and 0.13g pepsin to 500mL of sterile deionized water and mix well. Adjust the pH of the solution to 1.73 with 1mol / L HCl and filter through a 0.22μm membrane filter to prepare simulated gastric fluid (SGJ) for later use. Initially, mix 0.5ml of the fresh bacterial suspension prepared in step 1 or 0.5g of the spray-dried microcapsule powder with 29.5mL of SGJ. Incubate at 100rpm and 37°C, and sample every 1h to measure cell viability.

[0057] ② Simulated intestinal fluid digestion: Add 2.5g of pancreatic enzyme and 1.3g of porcine bile salt to 500mL of sterile deionized water, mix thoroughly, and adjust the pH to 7 with 1mol / L NaOH. Filter through a 0.22μm membrane filter to prepare simulated intestinal fluid (SIJ). Digest Heal 19 fresh bacterial culture and spray-dried powder in SGJ simulated fluid for 2h. Centrifuge the culture at 2370rpm for 15min. Then, mix the cell pellet with 29.5mL of SIJ. Incubate at 100rpm and 37°C. Samples are taken every 1h to measure cell viability.

[0058] The results of the survival rate of live bacteria in microcapsules under simulated gastric fluid and intestinal fluid are as follows Figure 3 As shown (G1 and G2 represent 1h and 2h of simulated gastric juice digestion, respectively; I1 and I2 represent 1h and 2h of simulated intestinal juice digestion, respectively). After 2h of SGF (pH 1.73) digestion, the cell viability of Heal19 strain in PSC, PPS, PPC and PP groups decreased by 1.35, 1.60, 2.12 and 2.83Log CFU / mL, respectively. After 2h of SIJ (pH 7.0) digestion, the cell viability of Heal19 strain in PSC, PPS, PPC and PP groups decreased by 2.41, 2.72, 3.08 and 4.92Log CFU / mL, respectively. Compared with the high concentration of SGF, the Heal19 strain in PSC, PPS, PPC and PP groups decreased significantly. +After contact, probiotics must maintain a cell viability of at least 6-7 Log CFU / mL to reach the intestine and exert their beneficial effects. After SGF digestion, the cell counts of the PSC, PP, PPS, and PPC groups were 8.55, 6.91, 8.22, and 7.59 Log CFU / mL, respectively. The cell viability of each group is within the food standard range. The high cell viability of the PSC and PPS groups after digestion in simulated gastric and intestinal fluids may be due to the unique structure of resistant starch, which protects the microcapsules from digestive enzymes and provides a very high level of resistance to enzymatic degradation. Furthermore, resistant starch is a prebiotic in the intestine. Therefore, microcapsules prepared with resistant starch can enhance the probiotic's resistance to the acidic environment of the stomach and the digestion of bile salts in the intestine, thereby ensuring that the bacteria reach the gastrointestinal tract and exert their beneficial effects to the greatest extent possible. β-cyclodextrin has a hydrophobic inner cavity, which can firmly encapsulate the Heal19 strain within its structure through hydrophobic interactions. Due to the properties of β-cyclodextrin, the microcapsule structure is more compact, reducing the contact between strong acid and pepsin and the probiotics. Therefore, compared with the microcapsules encapsulated with pure peanut protein powder, the PPC group greatly improved the survival rate in simulated in vitro digestion. In summary, the combination of peanut protein powder and disaccharide has a significant protective effect on the simulated in vitro digestion of Lactobacillus plantarum, indicating that the combination of protein and polysaccharide is suitable for the preparation of microcapsules, and the protective effect of peanut protein powder combined with resistant starch and β-cyclodextrin is the best.

[0059] 6: Determination of cell viability during storage of microcapsules

[0060] Fermented dairy products are an important way to obtain probiotics. However, the number of probiotics can be reduced by external conditions such as transportation, storage temperature, and storage time. This study used Lactobacillus plantarum Heal 19 as an embedding material to investigate the effects of storage at 4°C on the microcapsules. The determination method is as follows:

[0061] Cell viability of microencapsulated Lactobacillus plantarum was determined during storage at 4°C for 1, 7, 14, 28, 35, 42, 49, and 56 days. One gram of spray-dried powder was dissolved in 9 mL of 0.85% (w / v) NaCl solution and vortexed for 2 minutes until completely dissolved. The resulting solution was then serially diluted eight-fold with 0.85% (w / v) NaCl. The diluted solution was placed in a sterile MRS culture dish and incubated at 37°C for 48 hours before viable cell counts were performed. Viable cell counts were expressed as CFU / mL or CFU / g. Each sample was replicated in triplicate.

[0062] Figure 4Cell viability of each microcapsule group during 56 days of storage at 4°C was described. Initial cell viability in the PSC, PP, PPS, and PPC groups was 9.45, 9.40, 9.41, and 9.44 Log CFU / mL, respectively. After 8 weeks at 4°C, the viable cell counts of the four microcapsules decreased by 1.07, 2.34, 1.36, and 1.54 Log CFU / mL, respectively. With increased storage time, the samples' exposure to oxygen and water molecules increased, resulting in a downward trend in cell viability for each probiotic strain after 8 weeks of storage. Notably, the dual polysaccharide combination in the PSC group minimized the loss of probiotic cell viability over 8 weeks, achieving the best retention, followed by the PPS group. In addition to the high initial cell viability of the PSC group, water activity also influenced its viability. The water activity of the PSC group was 0.167 (Table 1), which contributes to its relatively high cell viability and storage stability. Furthermore, the lower water activity of the PPS group (0.169) also resulted in less cell viability loss during storage, with a decrease of 1.36 Log CFU / mL after nearly two months of storage at 4°C. Furthermore, LF-NMR results showed that the microcapsules in the PSC and PPS groups had more complete and stable structures, allowing the probiotics to be well encapsulated within the protein and polysaccharide, resulting in greater storage stability. Furthermore, the PP group exhibited the poorest storage stability. After eight weeks of storage, the viability of the probiotics in the PP group was 7.06 Log CFU / mL, lower than that of the PPC group (7.90 Log CFU / mL). This may be because β-cyclodextrin contains a hydrophobic interior that tightly binds to the probiotics through hydrophobic interactions, making it difficult for the probiotics to come into contact with external oxygen and water molecules. This resulted in significantly higher cell viability in the PPC group after 56 days of storage at 4°C than in the PP group. It is worth noting that for probiotics to exert their probiotic effects, the cell viability must be above 6Log CFU / mL. Therefore, it is believed that both microcapsules encapsulated with pure peanut protein powder and microcapsules encapsulated with protein combined with polysaccharides meet the standards of the food industry.

[0063] 7: Differential Scanning Calorimetry (DSC) Analysis

[0064] During spray drying, probiotic microcapsules are affected by the high temperature at the inlet of the spray dryer, which makes the probiotics susceptible to high temperature stress and dehydration stress and inactivation. Therefore, it is necessary to analyze the moisture content and thermal stability of the microcapsules. The thermal stability determination method is as follows:

[0065] DSC analysis was performed using a DSC analyzer (TADSC250, USA). Approximately 8 mg of sample powder was weighed into a covered aluminum pan. Data were collected at a heating rate of 10°C / min, with a temperature range of -50 to 250°C and a nitrogen flow rate of 50 mL / min.

[0066] The experimental results of DSC are crucial for analyzing the survival of microcapsules under different encapsulating agents and evaluating their stability during storage. Figure 5 The DSC heat flow diagrams corresponding to the four groups of microcapsules PSC, PP, PPS and PPC. Compared with the microcapsules embedded in pure peanut protein powder, the thermodynamic properties of the microcapsules prepared by resistant starch and β-cyclodextrin have changed. A clear broad endothermic peak was detected at 127.20℃, 127.52℃, 134.57℃ and 136℃ (peak) for PP, PPS, PPC and PSC microcapsules, respectively. Figure 5 ). The temperature corresponding to the peak represents the melting temperature, and the higher the melting temperature, the better the thermal stability of the microcapsule. The PP group had the lowest melting temperature, indicating that the microcapsules prepared from peanut protein powder had limited ability to protect the stability of probiotics. The melting temperature increased significantly after adding β-cyclodextrin, which was attributed to the hydrophilicity of the β-cyclodextrin surface, thereby enhancing the melting temperature of the microcapsule powder. The melting temperature also increased after adding resistant starch, because the special structure of resistant starch itself (retrograde amylose crystals) makes it stable during high-temperature heating. The melting temperature of the microcapsules in the PSC group was the highest, reaching 136°C, indicating that the thermal stability of the added disaccharide is better than that of the monosaccharide group.

[0067] 8. Moisture status of microcapsules

[0068] LF-NMR can quickly analyze the moisture distribution in the powder without destroying the microcapsule powder structure. The LF-NMR measurement method is as follows:

[0069] The moisture distribution of the microcapsule powder was analyzed using a low-field nuclear magnetic resonance analyzer (VTMR20-010V-I). The transverse relaxation time (T2) of the microcapsule powder was determined using a Carr–Purcell–Meiboom–Gill (CPMG) sequence. The parameters were set as follows: SF = 21 MHz, 90° pulse width (P1) = 3.2 μs, 180° pulse width (P2) = 5.36 μs, wait time (TW) = 2000 ms, number of scans (NS) = 64, number of echoes (NECH) = 10,000, echo time (TE) = 0.06 ms, and repetition number (NS) = 64. The resulting image was inverted to obtain a relaxation time spectrum, and the relaxation time, peak area, and peak area ratio were recorded.

[0070] The transverse relaxation time T2 is generally used to evaluate the migration of hydrogen molecules. Therefore, LF-NMR scanning is performed on microcapsules encapsulated with different wall materials to obtain the inversion spectrum of the T2 transverse relaxation time ( Figure 6 ), the horizontal axis represents the transverse relaxation time T2, and the vertical axis represents the signal amplitude. Figure 6 As shown, the probiotic microcapsules have three transverse relaxation peaks (T 21 、T 22 、T 23 ). T 21 Represents bound water firmly bound to macromolecules, T 22 represents the immobile water fixed in the cytoplasmic structure, T 23 It is worth noting that the contents of bound water, immobile water and free water in the sample are respectively related to T 21 、T 21 and T 23 The peak ratio of the signal peak is positively correlated with the water content. Therefore, the area of ​​the signal peak represents the water content. In order to evaluate the water state in the microcapsule powder, the relaxation peak time T2 and peak ratio R2 of the samples were analyzed, as shown in Table 2 (p < 0.05). The free water content (R2) in the microcapsule samples of the PP group and the PPC group was significantly higher than that in the control group. 23 ) is the highest, followed by bound water (R 21 ) and immovable water (R 22 ). The bound water content of the PPC and PP groups was 40.8% and 44.1%, respectively, while the free water content was 54.6% and 50.8%, respectively. In the PSC and PPS groups, the bound water content in the microcapsule powder was the highest, followed by free water and immobile water. The bound water content of the PSC and PPS groups was 51.7% and 54.8%, respectively, while the free water content was 44.6% and 43.6%, respectively. This phenomenon indicates that the PPC and PP groups may have been damaged due to the unstable microcapsule structure, and some of the bound water inside the sample migrated and converted into free water, resulting in a higher free water content. The bound water content of the microcapsules in the PSC and PPS groups was relatively high, while the free water content was relatively low, which indicates that the protein and polysaccharide are tightly bound, making the pore size of the microcapsules smaller and the structure more dense and uniform. At the same time, the lower free water content of the microcapsules in the PSC and PPS groups provides an environment that is not suitable for the growth and reproduction of microorganisms, which is conducive to the long-term storage of the microcapsules. All the above results indicate that the combination of resistant starch and β-cyclodextrin combined with peanut protein powder can form a dense, uniform and complete microcapsule system, which creates a stable environment for the long-term storage of probiotics.

[0071] Table 1

[0072]

[0073] The results are expressed as mean ± standard deviation (n = 3). Means with different superscript lowercase letters in the same column ad indicate statistically significant differences (p < 0.05).

[0074] Table 2

[0075]

[0076] ad Means with different superscript lowercase letters in the same column indicate statistically significant differences (p<0.05).

Claims

1. A method for preparing low water activity, storage-stable probiotic microcapsules, characterized in that: The following steps are involved: (1) Centrifuge the fresh Lactobacillus liquid and then wash it with 0.85% saline to obtain a Lactobacillus strain suspension; (2) The capsule wall material emulsion is mixed with the Lactobacillus strain suspension and continuously stirred, and low water activity, storage-stable probiotic microcapsules are obtained by spray drying. The capsule wall material emulsion includes 8% (w / v) peanut protein powder, 1% (w / v) resistant starch and 1% (w / v) β-cyclodextrin, and the rest is water.

2. The method for preparing low water activity, storage-stable probiotic microcapsules according to claim 1, characterized in that: The method for culturing the fresh lactobacillus liquid in step (1) is as follows: inoculating the lactobacillus into MRS liquid culture medium, culturing at 37°C for 10-12 hours, and then transferring the culture into fresh MRS liquid culture medium and culturing at a constant temperature of 37°C for 16-18 hours.

3. The method for preparing low water activity, storage-stable probiotic microcapsules according to claim 1, characterized in that: The mass ratio of the peanut protein powder to the two polysaccharides of resistant starch and beta-cyclodextrin is 4:

1.

4. The method for preparing low water activity, storage-stable probiotic microcapsules according to claim 1, characterized in that: The lactobacillus is Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus fermentum or Lactobacillus paracasei.

5. The method for preparing low water activity, storage-stable probiotic microcapsules according to claim 1, characterized in that: In step (2), peanut protein powder, resistant starch and β-cyclodextrin are added to water and homogenized on a high-speed disperser at 10,000 rpm for 5 minutes to obtain a capsule wall material emulsion.

6. Low water activity, storage-stable probiotic microcapsules prepared by the method according to any one of claims 1 to 5.

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