Probiotic microcapsule with low water activity and storage resistance and preparation method thereof

Abstract

The preparation method comprises the following steps: firstly, centrifuging a fresh lactobacillus liquid, and then washing with normal saline with the mass fraction of 0.85% to obtain a lactobacillus strain suspension; then, the capsule wall material emulsion and the lactobacillus strain suspension are mixed and continuously stirred, the low-water-activity and storage-resistant probiotic microcapsules are obtained through spray drying, the capsule wall material emulsion comprises 6-10% (w / v) of peanut protein powder, 0-3% (w / v) of resistant starch, 0-3% (w / v) of beta-cyclodextrin and the balance water, and the microcapsule wall material emulsion and the lactobacillus strain suspension are mixed and stirred continuously to obtain the low-water-activity and storage-resistant probiotic microcapsules. At least one of the resistant starch and the beta-cyclodextrin is contained. The two polysaccharides of the resistant starch and the beta-cyclodextrin cooperate with each other from the aspects of thermal stability, entrapment efficiency, water content and gastrointestinal digestion survival rate, and promote each other to jointly improve the performance of the microcapsule.

Description

Technical Field

[0001] The 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 living bacteria that, if given in sufficient amounts, can bring health benefits to the host. A large amount of evidence shows that probiotics have health benefits for the host, including regulating intestinal flora, improving intestinal barriers, enhancing immunity, and improving a variety of diseases (colitis, diarrhea, allergies, etc.). At present, the most popular probiotic strain is Lactobacillus, which has been widely used for pharmaceutical purposes. Organic acids, special enzyme systems, acid bacteria and other substances produced by lactic acid bacteria through fermentation have special physiological functions. Among them, Lactobacillus plantarum is one of the considerable probiotics. Some experimental results show that L. plantarum KLDS1.0318 has the ability to regulate the composition of intestinal flora, repair intestinal mucosal damage, and improve immunity, and has strong resistance to acid and bile salts. After colonization and reproduction in the human intestinal environment, Lactobacillus rhamnosus GG only attaches to the host's intestinal epithelial cells and can become a biological barrier of the intestinal mucosa, thereby enhancing the host's intestinal mucosal barrier capacity. Studies have found that Lactobacillus reuteri can produce reuterin, which can inhibit the growth of harmful bacteria such as Escherichia coli and Salmonella and protect the microecological balance of the intestine. Thirdly, Lactobacillus reuteri can also produce lactic acid and a variety of enzymes in the animal intestine, such as lipase and bile salt hydrolase, which are beneficial to improve the pH of the animal intestine, inhibit bacterial growth, and improve feed utilization. In order to fully utilize the beneficial effects of probiotics on the host, it is important to consume a sufficient amount of live bacteria. The International Dairy Federation recommends at least 6Log CFU / g. However, during the processing, storage and consumption of probiotic products, various factors reduce the activity of probiotics and reduce their content. First, probiotic products are susceptible to temperature stress and water stress during the preparation process, resulting in loss of cell viability. Second, probiotics usually go through a storage period before oral administration, and the conditions and duration of the storage period will affect the survival of probiotics. 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 severe challenge.

[0003] Microencapsulation has emerged as one of the most promising methods to protect probiotic cells from adverse exposure conditions. Microencapsulation is defined as encapsulating nutrients or bioactive compounds from food as core materials within a polymer matrix shell to protect its main components and control its release function. Spray drying is one of the most commonly used techniques in the microencapsulation process due to its short time, low cost, and good stability and quality of the final product. Compared with other traditional microencapsulation technologies, the spray drying process can be easily scaled up to produce microcapsules in a continuous processing operation.

[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 preparing 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 byproduct that is not fully utilized in the processing of peanut oil. For every 1kg of peanut oil squeezed, about 1.25kg 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 cheap 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-resistant probiotic microcapsule based on peanut meal protein and a preparation method thereof. Summary of the invention

[0005] The present invention aims at providing a low-water-activity, storage-resistant probiotic microcapsule and a preparation method thereof. The method uses 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 by spray drying, thereby improving the survival rate of probiotics during storage, solving the disadvantage that the existing probiotic powders are easily inactivated during storage, and the preparation method can improve the spray drying survival rate and encapsulation rate of probiotics, and the probiotic microcapsules prepared by the method 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 and storage-resistant probiotic microcapsules, comprising the following steps:

[0008] (1) centrifuging a fresh lactobacillus liquid, and then washing it with 0.85% saline to obtain a lactobacillus strain suspension;

[0009] (2) After mixing the capsule wall material emulsion with the lactic acid bacillus suspension, the mixture is continuously stirred and spray-dried to obtain low water activity and 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, and the rest is water. In the w / v, w is the solute mass in g, and v is the solvent volume in mL.

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

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

[0012] β-cyclodextrin is a widely used polysaccharide wall material with the characteristics of biocompatibility, easy degradation and non-toxicity. 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 increase the water content of the peanut protein powder-based wall material, and the microcapsule wall is easily damaged, 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, preferably resistant starch 3. Resistant starch 3 is prepared from a starch-rich substance, contains rich amylose, has good thermal stability, and has good low water retention.

[0013] Preferably, the capsule wall material emulsion includes 6-10% (w / v) peanut protein powder and 1-3% (w / v) resistant starch, and the rest is water. Protein has good film-forming properties, and resistant starch can resist digestion by pancreatic amylase to the greatest extent. Therefore, the combination of the two can produce probiotic microcapsules with a denser structure and stronger 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 two polysaccharides of resistant starch and β-cyclodextrin not only has excellent heat resistance, avoiding the probiotics from being affected by high temperature and dehydration stress during spray drying, but also the encapsulation rate, survival rate and other properties are improved compared with 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 material (peanut protein powder, resistant starch and β-cyclodextrin) is 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 lactic acid bacillus 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 preparation method is used to prepare low water activity and storage-resistant probiotic microcapsules.

[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 and two polysaccharides (resistant starch and β-cyclodextrin) as embedding materials, and prepares four wall materials to respectively embed lactobacillus. 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 lactobacillus 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 the peanut meal and effectively reduce the production cost of the microcapsules, which is convenient for 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 microscopy images of microcapsules prepared for PP group, PPS group, PPC group, and PSC group.

[0025] Figure 2 Particle size distribution diagram 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 diagrams 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 through specific embodiments and drawings.

[0031] Example 1

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

[0033] (1) Inoculate glycerol-preserved Lactobacillus plantarum Heal 19 at a 1% inoculum into 50 mL of MRS liquid culture medium, activate at 37°C for 12 h, then transfer to fresh MRS liquid culture medium at a 1% inoculum, and culture at 37°C for 16-18 h to obtain a fresh bacterial liquid. Take the cultured fresh bacterial liquid, centrifuge at 6000 rpm for 12 min, wash the bacteria twice with 0.85% (w / v) physiological 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 prepared four emulsions were homogenized in a high-speed disperser at 10,000 rpm for 5 minutes until the emulsions are completely dispersed and set aside. The harvested strains were suspended in the emulsion at room temperature (25°C). The average bacterial concentration of each bacterial solution was 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 a probiotic suspension encapsulated by different wall materials. The operating conditions were: inlet temperature = 120±1℃; feed flow rate = 7mL / min; air flow rate = 35m 3 / h, compressor pressure = 0.4MPa. During the preparation of microcapsule powder, a magnetic stirrer was used to continuously stir the mixture. After spray drying, the dried powder was collected from the collection tank 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 respectively. The measurement method is as follows:

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

[0038] ② Particle size determination: Take 0.2 g of sample from the microcapsules prepared in the PP group, PPS group, PPC group and PSC group, dissolve in 2 mL of sterile distilled water, and centrifuge at 3000 rpm for 2 min to reduce the influence of insoluble impurities. Use Zetasizer laser diffraction particle size analyzer to analyze the particle size of microcapsule powder. Repeat three times for each sample.

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

[0040] Depend on Figure 1 It can be seen that the SEM image of the spray-dried microcapsule powder shows microspheres of different sizes, ranging from 7-15μm. The surface of the microcapsules is wrinkled and relatively rough, and the microcapsules have a certain degree of collapse. Figure 1 It was found that the microcapsules of the four formulations had different sizes, with spherical microcapsules accounting for the majority, and some microcapsules did not form regular spheres. In addition, the disaccharide component also led to a higher number of spherical microcapsules in the PSC group, indicating that the encapsulation effect of this group of microcapsules was better. The microcapsules of the other three groups had a certain degree of wrinkles, which may be because the droplets formed during spray drying may produce more vaporization, resulting in wrinkles and collapse on the surface of the microcapsules.

[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 accordingly. Figure 2 As shown in Table 1, the droplet size of various emulsions ranges from 10 to 1000 nm, showing a unimodal distribution. It is worth noting that if the particle size of the droplets presents 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 polysaccharides 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 by pure peanut protein powder. Therefore, the emulsion prepared by the combination of peanut protein powder and polysaccharides is more uniform, which significantly improves the bioavailability and probiotic effect of probiotics.

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

[0043] First, 1g of spray-dried powder was dissolved in 9mL of 0.85% (w / v) NaCl solution and vortexed for 2min until the powder was completely dissolved, and then the dissolved solution was serially diluted 8 times with 0.85% (w / v) NaCl solution. The diluted bacterial solution was placed in a sterile MRS culture dish and incubated at 37°C for 48h before counting the viable cells. The number of viable cells was expressed as CFU / mL or CFU / g. Each sample was paralleled for a total of 3 times. The encapsulation efficiency (EE) of Lactobacillus plantarum Heal 19 microcapsules was 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 influence 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 disaccharide combination is better than that of the single polysaccharide combination, and the combination of peanut protein powder and disaccharide can better protect the 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 respectively. The measurement method is as follows:

[0048] ① Determination of moisture content: Place the spray-dried microcapsule powder in a drying dish and place it in a conventional hot air oven at 105°C for drying. The microcapsule powder is placed in a dry condition until it reaches a constant weight and then weighed. Each sample has 3 parallels. The moisture content is calculated as follows: Moisture content (%) = (M0-M) / 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 kinds of microcapsule powders was determined at room temperature (25±1°C) using a water activity meter. Each sample was tested three times in parallel.

[0051] The water content of the spray-dried microcapsules is less than 7% (w / w), which is considered to minimize the risk of cell death during storage. In addition, a water content between 4% and 7% can achieve better storage stability of the microcapsules, thereby improving cell viability. From Table 1, it can be seen that the water content of the microcapsules in the present invention varies from 4.82 to 5.26%, which is in line with the above standard range.

[0052] Water activity is another indicator for evaluating the moisture content of microcapsules. It is a good supplement for evaluating the stability of microcapsule powders, because microcapsules with the same water content may show different degrees of perishability. For the food industry, a water activity of less than 0.6 is considered feasible, at which point most microorganisms cannot grow or reproduce. As shown in Table 1, the water activity of the four microcapsules ranged from 0.17 to 0.18, and the water activity values ​​of the microcapsules in the four experimental groups were all lower than 0.6, so they all met the standards of the food industry. Compared with the PP group, the water activity of the PSC group, the PPS group, and the PPC group were all reduced, and the water activity of the PSC group was the lowest at 0.166, indicating that the addition of resistant starch and β-cyclodextrin reduced the possibility of microbial reproduction in the microcapsules, and the simultaneous addition of the two polysaccharides could produce a synergistic effect, further reducing the possibility of microbial reproduction.

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

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

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

[0056] ① Simulated gastric juice digestion: The fresh bacterial liquid and spray-dried powder of Heal 19 were respectively passed through an in vitro digestion model composed of a simulated stomach. 0.56g potassium chloride, 1.0g sodium chloride, 0.055g calcium chloride, 0.2g potassium dihydrogen phosphate, and 0.13g pepsin were added to 500mL sterile deionized water, mixed evenly, and the pH of the solution was adjusted to 1.73 with 1mol / L HCl. After filtering with a 0.22μm membrane filter, simulated gastric juice (SGJ) was prepared for use. Initially, 0.5ml of the fresh bacterial liquid prepared in step 1 or 0.5g of the spray-dried microcapsule powder was mixed with 29.5mL SGJ. Constant temperature culture was carried out at 100rpm and 37℃, and samples were taken every 1h to measure cell viability.

[0057] ②Simulated intestinal fluid digestion: Add 2.5g pancreatic enzyme and 1.3g pig bile salt to 500mL sterile deionized water, mix well, add 1mol / L NaOH to adjust the pH to 7, filter with a 0.22μm membrane filter and prepare simulated intestinal fluid (SIJ) for use. After digesting the Heal 19 fresh bacterial liquid and spray-dried powder with SGJ simulated liquid for 2h, centrifuge the culture liquid at 2370rpm for 15min. Then mix the cell pellet with 29.5mL SIJ. Culture at a constant temperature of 100rpm and 37℃, and take samples every 1h to measure cell viability.

[0058] The results of the survival rate of live bacteria in microcapsules under simulated gastric and intestinal fluids 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 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. +After contact, probiotics must maintain at least 6-7Log CFU / mL of cell viability to reach the intestine and exert their health effects. The cell counts of the PSC, PP, PPS and PPC groups after SGF digestion were 8.55, 6.91, 8.22 and 7.59Log CFU / mL, respectively, and the cell viability of each group was within the food standard range. The reason why the PSC and PPS groups can maintain high cell viability after digestion with simulated gastric and intestinal fluids may be because resistant starch has a special structure of retrograde straight chain starch, which can protect the microcapsules from the action of digestive enzymes and has a very high level of resistance to enzymatic hydrolysis. In addition, resistant starch is an intestinal prebiotic, so microcapsules prepared with resistant starch can enhance the resistance of probiotics to the strong acid environment of the stomach and the digestion of bile salts in the intestine, thereby ensuring that the bacteria reach the gastrointestinal tract to the greatest extent to exert their probiotic effects. β-cyclodextrin has a hydrophobic inner cavity, which can firmly embed the Heal19 strain inside its own structure through hydrophobic action. Due to the characteristics of β-cyclodextrin, the structure of the microcapsules is more compact, reducing the contact between strong acid and pepsin and probiotics. Therefore, compared with the microcapsules encapsulated by pure peanut protein powder, the PPC group greatly improved the survival rate in simulated digestion in vitro. In summary, the combination of peanut protein powder and disaccharide has a significant protective effect on the simulated digestion of Lactobacillus plantarum in vitro, 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 one of the important ways to obtain probiotics. However, the number of probiotics will be reduced by external conditions such as transportation process, storage temperature, storage time, etc. The present invention uses Lactobacillus plantarum Heal 19 as an embedding material to investigate the effect of its microcapsule storage process at 4°C. The determination method is as follows:

[0061] The cell viability of microencapsulated Lactobacillus plantarum was determined during the storage period at 4°C (1, 7, 14, 28, 35, 42, 49, 56 days). 1 g of spray-dried powder was dissolved in 9 mL of 0.85% (w / v) NaCl solution and vortexed for 2 min until the powder was completely dissolved. The dissolved solution was then serially diluted 8 times with 0.85% (w / v) NaCl solution. The diluted bacterial solution was placed in a sterile MRS culture dish and incubated at 37°C for 48 h before counting the viable cells. The number of viable cells was expressed as CFU / mL or CFU / g. A total of 3 parallels were performed for each sample.

[0062] Figure 4The cell viability of each group of microcapsules during storage at 4°C for 56 days was described. The cell viability of the PSC, PP, PPS and PPC groups at the beginning of storage was 9.45, 9.40, 9.41 and 9.44 Log CFU / mL, respectively. After 8 weeks at 4°C, the number of viable cells of the four microcapsules decreased by 1.07, 2.34, 1.36 and 1.54 Log CFU / mL, respectively. With the extension of storage time, the chance of the sample contacting oxygen and water molecules increased, resulting in a downward trend in the cell viability of each group of probiotic strains after 8 weeks of storage. It is worth noting that the dual polysaccharide combination of the PSC group can minimize the loss of cell viability of probiotics within 8 weeks, and its retention effect is the best, followed by the PPS group. In addition to the high initial cell viability of the PSC group, water activity can also affect its cell viability. The water activity of the PSC group is 0.167 (Table 1), which is conducive to it maintaining a relatively high cell viability and storage stability. In addition, the lower water activity of the PPS group (0.169) also resulted in less cell viability loss in the microcapsules during storage, with a reduction of 1.36Log CFU / mL after storage at 4°C for nearly 2 months. In addition, according to the results of LF-NMR, the microcapsule structures of the PSC and PPS groups were more complete and stable, so the probiotics could be well encapsulated in proteins and polysaccharides, making the probiotics in the PSC and PPS groups more stable during storage. In addition, the storage stability of the probiotics in the PP group was the worst. After 8 weeks of storage, the viability of the probiotics in the PP group was 7.06Log CFU / mL, which was lower than that in the PPC group (7.90Log CFU / mL). This may be because β-cyclodextrin contains a hydrophobic interior that can be tightly bound to the probiotics through hydrophobic interaction, making it difficult for the probiotics to contact with external oxygen and water molecules, making the cell viability of the PPC group significantly higher than that of the PP group after storage at 4°C for 56 days. It is worth noting that for probiotics to play a probiotic role, 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 the spray drying process, the probiotic microcapsules are affected by the high temperature at the inlet of the spray dryer, which makes the probiotics vulnerable 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). About 8 mg of sample powder was weighed and placed in an aluminum pot with a lid. Data were collected at a heating rate of 10°C / min, the temperature range was set to -50 to 250°C, and the nitrogen flow rate was set to 50 mL / min.

[0066] The experimental results of DSC are crucial to analyze the survival of microcapsules under different encapsulating agents and evaluate 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. An obvious 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 was 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 determination method is as follows:

[0069] The moisture distribution of microcapsule powder was analyzed by low-field nuclear magnetic resonance analyzer (VTMR20-010V-I). The Carr–Purcell–Meiboom–Gill (CPMG) sequence was selected to determine the transverse relaxation time (T2) of microcapsule powder. The parameters were set as follows: SF = 21 MHz, 90° pulse width (P1) = 3.2 us, 180° pulse width (P2) = 5.36 us, waiting time (TW) = 2000 ms, number of scans (NS) = 64, number of echoes (NECH) = 10000, echo time (TE) = 0.06 ms, and number of repeated samplings (NS) = 64. The generated image was inverted to obtain the 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 ). 21 Represents bound water firmly bound to macromolecules, T 22 represents the immobile water fixed in the cytoplasm, T 23 It represents the free water outside the sample that is easily removed. 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 that of the PP group and the PPC group, so the area of ​​the signal peak represents the moisture content. In order to evaluate the moisture 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). 23 ) is the highest, followed by bound water (R 21 ) and immovable water (R 22 ). The bound water content of the PPC group and the PP group was 40.8% and 44.1%, respectively, while the free water content was 54.6% and 50.8%, respectively. In the PSC group and the PPS group, 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 group and the PPS group 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 group and the PP group may be damaged due to the unstable microcapsule structure, and part 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 group and the PPS group was relatively high, while the free water content was relatively low, which indicates that the protein and the 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 group and the PPS group provides an environment that is not suitable for the growth and reproduction of microorganisms, which is conducive to the long-term storage of 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). The means with different superscript lowercase letters in the same column indicate statistical differences (p < 0.05).

[0074] Table 2

[0075]

[0076] ad Mean values ​​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-resistant probiotic microcapsules, characterized in that: The following steps are involved: (1) centrifuging a fresh lactobacillus liquid, and then washing it with 0.85% saline to obtain a lactobacillus strain suspension; (2) After mixing the capsule wall material emulsion with the lactic acid bacillus strain suspension and continuously stirring, the mixture is 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, and the rest is water, and at least one of the resistant starch and β-cyclodextrin is contained.

2. The method for preparing low water activity and 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: inoculate the lactobacillus into MRS liquid culture medium, culture it at 37° C. for 10-12 h, then transfer it to fresh MRS liquid culture medium, and culture it at a constant temperature of 37° C. for 16-18 h.

3. The method for preparing low water activity and storage-stable probiotic microcapsules according to claim 1, characterized in that: The capsule wall material emulsion comprises 6-9% (w / v) peanut protein powder and 1.5-3% (w / v) beta-cyclodextrin, and the rest is water.

4. The method for preparing low water activity, storage-stable probiotic microcapsules according to claim 1, characterized in that: The capsule wall material emulsion comprises 6-10% (w / v) peanut protein powder and 1-3% (w / v) resistant starch, and the rest is water.

5. The method for preparing low water activity, storage-stable probiotic microcapsules according to claim 1, characterized in that: 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) beta-cyclodextrin, and the rest is water.

6. The method for preparing low water activity, storage-stable probiotic microcapsules according to claim 5, characterized in that: The mass ratio of the peanut protein powder to the two polysaccharides of resistant starch and beta-cyclodextrin is 3-4:

1.

7. The method for preparing low water activity and storage-stable probiotic microcapsules according to claim 5, characterized in that: 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.

8. The method for preparing low water activity and 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.

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

10. The low water activity and storage-stable probiotic microcapsules prepared by the preparation method according to any one of claims 1 to 9.

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