A microcapsule formulation of Lactobacillus plantarum and its application
Microcapsules of *Lactobacillus plantarum* prepared using materials such as alginate and dextrin solve the problem of easy inactivation of probiotics during processing and storage, achieving highly active and stable probiotic products for application in the food and health product fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QINGDAO VLAND BIOTECH GRP CO LTD
- Filing Date
- 2023-12-28
- Publication Date
- 2026-07-17
AI Technical Summary
Probiotics are easily deactivated during processing, transportation and storage, and existing technologies are insufficient to effectively maintain their activity, affecting product quality and efficacy.
Using alginate and dextrin as microcapsule wall materials, microcapsules of *Lactobacillus plantarum* were prepared by combining porous starch solution and gelatin to enhance their tolerance and stability, including concentration control and freeze-drying treatment.
It significantly improved the resistance of Lactobacillus plantarum to gastric acid and choline, maintained a high viable count, enhanced stability during transportation and storage, and improved the intestinal flora regulation effect.
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Figure CN118203115B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of functional probiotic screening and application technology, specifically relating to a microcapsule preparation of Lactobacillus plantarum and its application. Background Technology
[0002] The World Health Organization / Food and Agriculture Organization of the United Nations defines probiotics as: live microorganisms that, when ingested in adequate quantities, provide a health benefit to the host. Clinical studies have demonstrated that probiotics can prevent and improve various health problems in humans, including metabolic diseases, gastrointestinal diseases, and mental illnesses. As a novel dietary supplement, probiotics have attracted widespread consumer favor due to their natural and healthy characteristics. *Lactobacillus plantarum* is an important probiotic flora in the human gut, playing a beneficial role in intestinal health, and is also a commonly used strain in traditional fermented foods and commercial probiotic foods.
[0003] The number of live bacteria is one of the important indicators for measuring the quality and efficacy of probiotic products. However, probiotics are extremely prone to inactivation during processing, transportation, and storage, and microencapsulation technology is undoubtedly a good solution to improve the tolerability of probiotics.
[0004] Microcapsule wall materials provide a physical barrier for probiotics, ensuring their isolation from extreme external environments, enhancing their tolerance, and extending shelf life. Alginate is a common microcapsule wall material, offering advantages such as non-toxicity, low cost, resistance to digestive enzymes, and good biodegradability. Dextrin is used in probiotic microcapsules; its unique network structure facilitates the adsorption and encapsulation of probiotics, thereby increasing the encapsulation rate. Summary of the Invention
[0005] The purpose of this invention is to provide a microcapsule formulation of *Lactobacillus plantarum* and its application. The strain can significantly increase the abundance of Akk bacteria in the intestine, showing broad application prospects and potential for use in various biological products.
[0006] This invention relates to a microcapsule of *Lactobacillus plantarum*, the preparation method of which includes the following steps:
[0007] (1) Mix a porous starch solution with a concentration of 90-100 g / L with a suspension of plant lactobacillus at a volume ratio of 100:1, and shake at 25-35℃ for 30 min to obtain a suspension.
[0008] (2) Add sodium alginate, dextrin, gelatin and skim milk powder to the suspension, stir well to obtain freeze-dried mixture;
[0009] (3) Add a calcium chloride solution with a concentration of 15-20 g / L to the freeze-dried mixture until the freeze-dried mixture solidifies;
[0010] (4) After freeze-drying for 24 hours, pulverize to within 100 mesh to obtain plant lactobacillus microcapsules.
[0011] The plant lactobacillus mentioned in step (1) is strain VHProbi R14, which was deposited on May 10, 2023, at the China Center for Type Culture Collection of Wuhan University, China, with accession number CCTCC NO: M2023721.
[0012] The viable bacteria count in the *Lactobacillus plantarum* suspension is not less than 10. 9 CFU / mL.
[0013] The method for preparing the *Lactobacillus plantarum* suspension includes the following steps:
[0014] (1) Under aseptic conditions, the seed culture of *Lactobacillus plantarum* VHProbi R14 was inoculated into the fermentation medium at a volume ratio of 3%; it was cultured at 37℃ for 24-36 h, with a rotation speed of 50-100 rpm, an aeration rate of 0.3-1 L / min, and a tank pressure of 0.05-0.08 MPa; after fermentation, the fermentation broth of *Lactobacillus plantarum* VHProbi R14 was obtained.
[0015] (2) The fermentation broth of *Lactobacillus plantarum* VHProbi R14 was centrifuged at 4℃ and 8000r / min for 5min. After collecting the cells, the cells were washed twice with sterile physiological saline and resuspended to prepare *Lactobacillus plantarum* bacterial suspension.
[0016] The components and their contents in the fermentation medium are as follows: mannan 35-45 g / L, yeast extract 15-25 g / L, corn peptone 15-25 g / L, ferric sulfate 0.5-1.5 g / L and magnesium sulfate 0.05-0.15 g / L.
[0017] More preferably, the components and their contents in the fermentation medium are as follows: mannan 40 g / L, yeast extract 25 g / L, corn peptone 25 g / L, ferric sulfate 1.5 g / L and magnesium sulfate 0.10 g / L.
[0018] The concentrations of sodium alginate, dextrin, gelatin, and skim milk powder in the freeze-dried mixture are 20-30 g / L, 40-50 g / L, 20-50 g / L, and 50-80 g / L, respectively.
[0019] More preferably, the concentrations of sodium alginate, dextrin, gelatin, and skim milk powder in the freeze-dried mixture are 30 g / L, 40 g / L, 50 g / L, and 80 g / L, respectively.
[0020] The viable bacteria count in the *Lactobacillus plantarum* microcapsules is not less than 10. 8 CFU / g.
[0021] The plant lactobacillus microcapsules provided by this invention have strong resistance to gastric acid and choline. After digestion by artificial gastric juice and artificial intestinal juice, they can still maintain a high number of live bacteria, achieving unexpected technical effects.
[0022] The applicant fed 7-week-old female C57 mice with the probiotic microcapsules provided by this invention for 10 days in advance, and then conducted a diarrhea modeling experiment on the mice to observe intestinal disorders and other indicators. The results showed that, compared with the negative control group, the mice that were given the probiotic microcapsules in advance by gavage had improved intestinal epithelial cell integrity, prolonged time for the first feces excretion, and reduced excessively rapid intestinal peristalsis.
[0023] Seven-week-old female C57 mice were pre-fed the *Lactobacillus plantarum* microcapsules provided by this invention for 10 days before a diarrhea model was established, and changes in their intestinal flora were detected. The results showed that, compared with the negative control group, mice administered *Lactobacillus plantarum* microcapsules by gavage exhibited increased abundance of *Akkermansia* spp. in their intestinal flora, particularly a significant increase in *Akk* spp. abundance.
[0024] The preparation process of the microcapsules of *Lactobacillus plantarum* provided by this invention is simple, the coating material is inexpensive, and the industrialization cost is low. It can effectively maintain the viable count of *Lactobacillus plantarum* VHProbi R14 during transportation, storage and processing, which greatly improves the applicability of *Lactobacillus plantarum* VHProbi R14. Therefore, it can be widely used in food, health products and other fields. Attached Figure Description
[0025] Figure 1 The species composition distribution of mouse fecal microbiota at the genus level;
[0026] Figure 2 Species differences in mouse fecal microbiota at the genus level;
[0027] Figure 3 To analyze species differences in mouse fecal microbiota at the species level. Detailed Implementation
[0028] On May 10, 2023, the applicant deposited strain VHProbi R14 of Lactobacillus plantarum at the China Center for Type Culture Collection (CCTCC) of Wuhan University in Wuhan, China, with accession number CCTCC NO: M2023721.
[0029] The screening method described in this invention is not limited to the embodiments. Any known method capable of achieving the screening purpose can be used. The screening descriptions in the embodiments are merely illustrative of this invention and are not intended to limit the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.
[0030] Example 1: Isolation, screening and identification of Lactobacillus plantarum VHProbi R14
[0031] This invention screened a lactobacillus strain with strong acid resistance from fresh fermented sauerkraut samples. After identification by colony morphology, carbon source metabolism and molecular biology, the strain was identified as a new Lactiplantibacillus plantarum strain, which the applicant named Lactiplantibacillus plantarum VHProbi R14.
[0032] Lactobacillus plantarum VHProbi R14 grows at salt concentrations of 1%-6%, but does not grow at 7% salt concentration. The maximum salt concentration that Lactobacillus plantarum VHProbi R14 can tolerate is 6%. It ferments glucose to produce acid but not gas. It can grow normally at 15-45℃.
[0033] Lactobacillus plantarum VHProbi R14 does not produce hemolysin and cannot lyse blood cells; Lactobacillus plantarum VHProbi R14 is sensitive to common antibiotics such as erythromycin, clindamycin, and ampicillin, and has good biocompatibility; the cell surface hydrophobicity is 8.02%.
[0034] Example 2: Tolerance of *Lactobacillus plantarum* VHProbi R14 to artificial gastric and intestinal fluids
[0035] 1. Preparation of bacterial culture:
[0036] The cryopreserved *Lactobacillus plantarum* strain VHProbi R14 was streaked onto MRS solid medium and cultured at 37°C for 24–48 h. After one subculture on MRS liquid medium, *Lactobacillus plantarum* strain VHProbi R14 was inoculated into fresh MRS liquid medium at a 5% inoculum and cultured at 40°C with shaking for 24–48 h to obtain fresh bacterial culture.
[0037] 2. Preparation of artificial gastric juice
[0038] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, and 2g of NaCl, add them to 1000ml of distilled water, adjust the pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. Before use, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and incubate in a 37℃ water bath shaker for 1h to simulate human body temperature.
[0039] 3. Preparation of artificial intestinal fluid
[0040] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH₂PO₄, and 3.0g of ox bile salts, respectively. Add them to 77ml of 0.2mol / L NaOH solution, and bring the volume to 1000ml. Adjust the pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution, and sterilize at 115℃ for 20min. Before use, add 1g of trypsin, shake well to dissolve, and incubate in a 37℃ water bath for 1h to simulate human body temperature.
[0041] 4. Test methods
[0042] Take 2 mL of fresh bacterial culture, centrifuge at 5000 rpm for 5 min to collect bacterial cells, wash the cells three times with physiological saline, and then resuspend them in 2 mL of physiological saline as the inoculum. Take 1 mL of the inoculum and add it to 9 mL of artificial gastric fluid that has been warmed for 1 h. Place the mixture in a 37℃ water bath shaker at 200 rpm for 2 h. Take 1 mL samples at 0 h and 2 h to detect the viable bacterial count. Then take 1 mL of artificial gastric fluid after 2 h of digestion and add it to 24 mL of artificial intestinal fluid. Place the mixture in a 37℃ water bath shaker (200 rpm) for 3 h. Take 1 mL samples to detect the viable bacterial count.
[0043] The viable count method was determined according to the national standard GB4789.35-2016-Food Microbiology Examination - Lactic Acid Bacteria Examination. The LOG (CFU / mL) of the viable count of this strain after artificial gastric fluid and artificial intestinal fluid are shown in Table 1.
[0044] Table 1. Viable bacterial count after digestion in artificial gastrointestinal tract
[0045]
[0046] As shown in Table 1, the viable count of *Lactobacillus plantarum* VHProbi R14 decreased only slightly after digestion with artificial gastric and intestinal fluids, indicating that this strain has strong tolerance to artificial gastric and intestinal fluids.
[0047] Example 3: Determination of the antioxidant function of Lactobacillus plantarum VHProbi R14
[0048] 1. Determination of the strain's ability to scavenge DPPH and hydroxyl radicals (HRS)
[0049] 1) Preparation of bacterial suspension
[0050] A single colony of *Lactobacillus plantarum* VHProbi R14 in excellent growth condition was inoculated into 3 ml of MRS liquid medium and cultured at 37°C for 18-20 h. Using this culture as the inoculum, 2% of the colony was inoculated into 50 ml of MRS liquid medium and incubated statically for 18 h to obtain *Lactobacillus plantarum* VHProbi R14 bacterial suspension. 1 mL of the bacterial suspension was collected by centrifugation, and the bacterial cells were washed twice with 1 mL of PBS buffer, then resuspended in 2 mL of PBS solution for later use.
[0051] 2) Determination of the strain's ability to scavenge DPPH free radicals
[0052] Take 1 mL of *Lactobacillus plantarum* VHProbi R14 bacterial suspension, add 1 mL of 0.4 mM freshly prepared DPPH free radical solution, mix well, and then incubate at room temperature in the dark for 30 min. Then measure the absorbance A of the sample at a wavelength of 517 nm. 样本 The test was performed in triplicate. The control group sample was prepared with an equal volume of PBS solution and DPPH-ethanol mixture, and the blank was zeroed with an equal volume of Lactobacillus plantarum VHProbi R14 bacterial suspension and ethanol mixture.
[0053] The clearance rate is calculated using the following formula:
[0054] Clearance rate % = [1-(A 样品 -A 空白 ) / A 对照 ×100%.
[0055] The results showed that the *Lactobacillus plantarum* VHProbi R14 provided by this invention had a DPPH free radical scavenging rate of up to 19.52% with a standard deviation of 0.91%.
[0056] 3) Determination of the strain's ability to scavenge hydroxyl radicals (HRS)
[0057] Mix 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, 500 μL of deionized water, and 200 μL of *Lactobacillus plantarum* VHProbi R14 suspension, then add 100 μL of 3 mM hydrogen peroxide solution. After incubating in a water bath at 37°C for 15 min, measure the absorbance of the sample at a wavelength of 510 nm.
[0058] The hydroxyl radical scavenging rate is calculated using the following formula:
[0059] Clearance rate % = (A 样品 -A 控制 ) / (A 空白 -A 控制 )×100%.
[0060] Where A 控制 A was used as a substitute for deionized water in the sample. 空白 Deionized water was used to replace the sample and H2O2.
[0061] The results showed that the supernatant of *Lactobacillus plantarum* VHProbi R14 fermentation provided by this invention had a scavenging rate of up to 83.89% for HRS free radicals, with a standard deviation of 0.42%; and its bacterial suspension had a scavenging rate of up to 25.36% for HRS free radicals, with a standard deviation of 3.06%.
[0062] 2. Determination of the strain's resistance to lipid peroxidation
[0063] 1) Preparation of bacterial strain culture and fermentation supernatant, bacterial cells, and intracellular extracts:
[0064] The strain was cultured in MRS liquid medium at 37°C for 24 h, and after 3 passages, it was centrifuged at 6000 rpm for 10 min at 4°C, and the supernatant was collected as the fermentation supernatant. The collected bacterial cells were washed three times by centrifugation at 6000 rpm for 10 min in PBS buffer (pH 7.4). The bacterial cells were then resuspended in PBS buffer to adjust the bacterial concentration to 1.0 × 10⁻⁶ cells / mL. 9 The bacterial suspension was obtained by measuring cells / mL.
[0065] 2) Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water.
[0066] 3) Add 1 mL of linoleic acid emulsion and 1 mL of FeSO4 (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of sample. Incubate at 37°C for 1.5 h. Add 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) to the mixture, incubate at 100°C for 30 min, cool rapidly, centrifuge at 4000 r / min for 15 min, and collect the supernatant. 532 nm The absorbance measured is A; the control group is represented by 0.5 mL of distilled water instead of the sample, which is A0. Inhibition rate / % = (A0 - A) / A0 × 100.
[0067] The results showed that the supernatant of *Lactobacillus plantarum* VHProbi R14 provided by this invention had an anti-lipid peroxidation inhibition rate of 35.81% with a standard deviation of 0.18%; the bacterial cell anti-lipid peroxidation inhibition rate was 32.47% with a standard deviation of 0.21%; and the intracellular extract had an anti-lipid peroxidation inhibition rate of 27.43% with a standard deviation of 0.29%.
[0068] Example 4: In vitro cholesterol degradation assay using *Lactobacillus plantarum* VHProbi R14
[0069] 1. Preparation of cholesterol micelle solution: Accurately weigh 1g of cholesterol, dissolve it in anhydrous ethanol, and make up to 100mL. Filter the solution under sterile conditions using a 0.22μm microporous membrane.
[0070] 2. Weigh out 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 2.0g diammonium citrate, 20.0g glucose, 1.0mL Tween 80, 5.0g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 2.0g dipotassium hydrogen phosphate, and 1000ml distilled water. Adjust the pH to 7.3, sterilize at 115℃ for 30min, then add cholesterol solution to bring the final cholesterol concentration to 0.1%. Inoculate fresh bacterial culture at a rate of 0.1%, incubate at 37℃ for 48 hours, then take 0.2ml of bacterial culture, add 1.8ml anhydrous ethanol, mix well, let stand for 10 minutes, centrifuge at 3000 rpm for 5 minutes, and use the supernatant to determine the cholesterol content. The cholesterol determination method follows GB / T5009.128-2003 "Determination of Cholesterol in Food".
[0071] The results showed that the *Lactobacillus plantarum* VHProbi R14 provided by this invention had a cholesterol degradation rate as high as 21.24%.
[0072] Example 5: Effect of Lactobacillus plantarum VHProbi R14 on increasing beneficial bacteria in the mouse gut.
[0073] 1. Laboratory animals
[0074] C57 mice, SPF grade, male, 7 weeks old, 20 mice, weighing 19–25g. Environmental conditions for animal husbandry: room temperature 20–26℃, daily temperature difference ≤4℃, relative humidity 40–70%, light / dark cycle 12 / 12h. Animals were housed in standard mouse cages, 10 mice per cage. Feed and water: ad libitum. Feed consisted of SPF grade rat and mouse growth and breeding feed. Drinking water was sterilized municipal tap water.
[0075] 2. Experimental Methods
[0076] After 7 days of acclimatization, mice were randomly divided into a blank group and a probiotic group, with 10 mice in each group. The probiotic group was administered *Lactobacillus plantarum* VHProbi R14 bacterial suspension (0.2 mL / 10 g) via gavage. 9 Mice in the control group were administered an equal volume of physiological saline by gavage for 60 days. Mice were placed in clean, empty cages, and after a period of time, 3-5g of feces were collected using sterile forceps, placed in a 1.5mL tube, flash-frozen in liquid nitrogen, and stored at -80°C.
[0077] 3. 16S amplicon sequencing protocol for fecal samples
[0078] 30 ng of qualified fecal DNA sample and corresponding fusion primers were used to prepare the PCR reaction system. PCR reaction parameters were set for PCR amplification. The PCR amplification product was purified using Agencourt AMPure XP magnetic beads and dissolved in Elution Buffer. Labeling was then performed to complete library construction. The fragment range and concentration of the library were detected using an Agilent 2100 Bioanalyzer. Libraries that passed the detection were sequenced using the HiSeq platform according to the insert fragment size.
[0079] After the data is filtered, the remaining high-quality clean data is used for later analysis; the reads are spliced into tags by the overlap relationship between the reads; the tags are clustered into OTUs and compared with the database, and species are annotated; sample species analysis is performed based on OTUs and annotation results, including intergroup species difference analysis at the genus and species levels, and species composition histogram analysis.
[0080] 4. Sequencing results
[0081] like Figure 1 As shown, at the genus level, the dominant genera in the feces of the blank group mice included *Lactobacillus*, *Bacteroides*, *Alloprevotella*, *Barnesiella*, *Clostridium XlVb*, and *Prevotella*. The dominant genera in the feces of the probiotics group mice were *Lactobacillus*, *Alloprevotella*, *Barnesiella*, *Akkermansia*, *Clostridium XlVb*, and *Prevotella*. Figure 1 The area framed in the image represents Akkermansia. It can be seen that, at the genus level, the abundance of Akkermansia in the fecal microbiota of mice in the probiotic group that were administered Lactobacillus plantarum VHProbi R14 via gavage was significantly higher than that in the control group.
[0082] like Figure 2As shown, compared with the blank group mice, at the genus level, the levels of Prevotella, Lachnospiraceae incertae sedis, Mucispirillum, and Clostridium were downregulated in the feces of mice in the probiotics group (which were administered Lactobacillus plantarum VHProbiR14 via gavage), while Akkermansia was significantly upregulated.
[0083] like Figure 3 As shown, at the species level, the abundance of Akkermansia muciniphila in the feces of mice in the probiotics group, which were administered Lactobacillus plantarum VHProbiR14 via gavage, was significantly upregulated compared with the blank group mice.
[0084] Combination Figure 1 , 2 As shown in section 3, gavage administration of the *Lactobacillus plantarum* VHProbi R14 bacterial solution provided by this invention to normal mice significantly increased the abundance of *Akkermansia* spp. in their feces, particularly *Akkermansia myxophilus*. *Akkermansia myxophilus* plays an important role in regulating intestinal and host health and has been shown to improve metabolic diseases such as obesity, diabetes, and cardiovascular disease. Numerous studies have demonstrated that *Akkermansia myxophilus* has positive effects in reducing fat accumulation, alleviating inflammation, improving insulin resistance, and improving glucose homeostasis, and is considered a promising "second-generation probiotic." Therefore, the *Lactobacillus plantarum* VHProbi R14 provided by this invention can significantly increase the abundance of *Akkermansia* spp. in the mouse intestine, which is of great significance for maintaining intestinal flora health and host health.
[0085] Example 6: A microcapsule of *Lactobacillus plantarum* and its preparation method
[0086] 1. Preparation of *Lactobacillus plantarum* fermentation broth
[0087] Under aseptic conditions, *Lactobacillus plantarum* VHProbi R14 seed culture was inoculated into a fermentation medium at a volume ratio of 3%. The components and their contents in the fermentation medium were as follows: mannan 45 g / L, yeast extract 15 g / L, corn peptone 15 g / L, ferric sulfate 0.5 g / L, and magnesium sulfate 0.05 g / L. The culture was carried out at 37°C for 24 h, with a rotation speed of 50 rpm, an aeration rate of 0.3 L / min, and a tank pressure of 0.05 MPa. After fermentation, *Lactobacillus plantarum* VHProbi R14 fermentation broth was obtained, with a viable cell count of 1.8 × 10⁻⁶. 9 -2.5×10 10 CFU / mL;
[0088] 2. Preparation of *Lactobacillus plantarum* microcapsules
[0089] The fermentation broth of *Lactobacillus plantarum* VHProbi R14 was centrifuged at 4℃ and 8000 r / min for 5 min. After collecting the cells, the cells were washed twice with sterile physiological saline and resuspended to prepare a bacterial suspension.
[0090] A porous starch solution of 100 g / L was mixed with the bacterial suspension at a volume ratio of 100:1, and the pH was adjusted to 6.5. The mixture was then shaken at 25-35℃ for 30 min. Sodium alginate, dextrin, gelatin, and skim milk powder were added sequentially and stirred until homogeneous to obtain a freeze-dried mixture. The concentrations of sodium alginate, dextrin, gelatin, and skim milk powder were 20 g / L, 40 g / L, 20 g / L, and 50 g / L, respectively. A calcium chloride solution of 15 g / L was added dropwise to the freeze-dried mixture until it solidified. After freeze-drying for 24 h, the mixture was pulverized to a mesh size of less than 100 to obtain *Lactobacillus plantarum* microcapsules.
[0091] The viable count of *Lactobacillus plantarum* VHProbi R14 in the *Lactobacillus plantarum* microcapsules was 3.7 × 10⁻⁶. 9 CFU / g.
[0092] Example 7: A microcapsule of *Lactobacillus plantarum* and its preparation method
[0093] 1. Preparation of *Lactobacillus plantarum* fermentation broth
[0094] Under aseptic conditions, *Lactobacillus plantarum* VHProbi R14 seed culture was inoculated into a fermentation medium at a volume ratio of 3%–10%. The components and their contents in the fermentation medium were as follows: mannan 35 g / L, yeast extract 20 g / L, corn peptone 20 g / L, ferric sulfate 1 g / L, and magnesium sulfate 0.15 g / L. The culture was carried out at 37°C for 36 h, with a rotation speed of 150 rpm, an aeration rate of 1 L / min, and a tank pressure of 0.08 MPa. After fermentation, *Lactobacillus plantarum* VHProbi R14 fermentation broth was obtained, with a viable cell count of 8.0 × 10⁻⁶. 8 -2.4×10 9 CFU / mL;
[0095] 2. Preparation of *Lactobacillus plantarum* microcapsules
[0096] The fermentation broth of *Lactobacillus plantarum* VHProbi R14 was centrifuged at 4℃ and 8000 r / min for 5 min. After collecting the cells, the cells were washed twice with sterile physiological saline and resuspended to prepare a bacterial suspension.
[0097] A porous starch solution of 90 g / L was mixed with the bacterial suspension at a volume ratio of 100:1, and the pH was adjusted to 6.5. The mixture was then shaken at 25-35℃ for 30 min. Sodium alginate, dextrin, gelatin, and skim milk powder were added sequentially and stirred until homogeneous to obtain a freeze-dried mixture. The concentrations of sodium alginate, dextrin, gelatin, and skim milk powder were 25 g / L, 50 g / L, 30 g / L, and 55 g / L, respectively. A calcium chloride solution of 20 g / L was added dropwise to the freeze-dried mixture until it solidified. After freeze-drying for 24 h, the mixture was pulverized to a mesh size of less than 100 to obtain *Lactobacillus plantarum* microcapsules.
[0098] The viable count of *Lactobacillus plantarum* VHProbi R14 in the *Lactobacillus plantarum* microcapsules was 1.02 × 10⁻⁶. 9 CFU / mL.
[0099] Example 8: A microcapsule of *Lactobacillus plantarum* and its preparation method
[0100] 1. Preparation of *Lactobacillus plantarum* fermentation broth
[0101] Under aseptic conditions, *Lactobacillus plantarum* VHProbi R14 seed culture was inoculated into a fermentation medium at a volume ratio of 3%–10%. The components and their contents in the fermentation medium were as follows: mannan 40 g / L, yeast extract 25 g / L, corn peptone 25 g / L, ferric sulfate 1.5 g / L, and magnesium sulfate 0.10 g / L. The culture was carried out at 37°C for 48 h, with a rotation speed of 200 rpm, an aeration rate of 0.5 L / min, and a tank pressure of 0.07 MPa. After fermentation, *Lactobacillus plantarum* VHProbi R14 fermentation broth was obtained, with a viable cell count of 5.6 × 10⁻⁶. 9 -1.5×10 10 CFU / mL;
[0102] 2. Preparation of *Lactobacillus plantarum* microcapsules
[0103] The fermentation broth of *Lactobacillus plantarum* VHProbi R14 was centrifuged at 4℃ and 8000 r / min for 5 min. After collecting the cells, the cells were washed twice with sterile physiological saline and resuspended to prepare a bacterial suspension.
[0104] A porous starch solution of 90 g / L was mixed with the bacterial suspension at a volume ratio of 100:1, and the pH was adjusted to 6.0. The mixture was then shaken at 25-35℃ for 30 min. Sodium alginate, dextrin, gelatin, and skim milk powder were added sequentially and stirred until homogeneous to obtain a freeze-dried mixture. The concentrations of sodium alginate, dextrin, gelatin, and skim milk powder were 30 g / L, 40 g / L, 50 g / L, and 80 g / L, respectively. A calcium chloride solution of 15 g / L was added dropwise to the freeze-dried mixture until it solidified. After freeze-drying for 24 h, the mixture was pulverized to a mesh size of less than 100 to obtain *Lactobacillus plantarum* microcapsules.
[0105] The viable count of *Lactobacillus plantarum* VHProbi R14 in the *Lactobacillus plantarum* microcapsules was 9.8 × 10⁻⁶. 10 CFU / mL.
[0106] Example 9: Stress resistance detection of *Lactobacillus plantarum* microcapsules
[0107] 1. Preparation of artificial gastric juice
[0108] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, and 2g of NaCl, add them to 1000mL of distilled water, adjust the pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. Before use, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.
[0109] 2. Preparation of intestinal fluid
[0110] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH₂PO₄, and 3.0g of ox bile salts, respectively. Add them to 77mL of 0.2mol / L NaOH solution, and bring the volume to 1000mL. Adjust the pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution, and sterilize at 115℃ for 20min. Before use, add 1g of trypsin, shake well to dissolve, and place in a 37℃ water bath for 1h to simulate human body temperature.
[0111] 3. Test methods
[0112] Take 2g of *Lactobacillus plantarum* microcapsules prepared in Examples 6-8, resuspend them in 2mL of physiological saline, and use them as inoculum. Take 1mL of the inoculum and add it to 9mL of artificial gastric fluid that has been preheated by a water bath for 1 hour. Place the mixture in a 37℃ water bath shaker at 200rpm / min for 2 hours. Take 1mL of the mixture at 0h and 2h to detect the viable bacterial count. Then, take 1mL of the artificial gastric fluid after 2 hours of digestion and add it to 24mL of artificial intestinal fluid. Place the mixture in a 37℃ water bath shaker (200rpm / min) for 3 hours. Take 1mL of the mixture to detect the viable bacterial count. The viable bacterial count was determined according to the national standard GB4789.35-2016-Food Microbiology Examination - Lactic Acid Bacteria Examination. The viable bacterial count (Log CFU / mL) of the probiotic microcapsules prepared in Examples 13-15 after digestion in artificial gastric fluid and artificial intestinal fluid is shown in Table 2.
[0113] Table 2. Tolerance effects of *Lactobacillus plantarum* microcapsules on simulated gastric and intestinal fluids.
[0114]
[0115] As can be seen from the results in Table 2, the plant lactobacillus microcapsules provided by the present invention can still maintain a high number of live bacteria after being digested by artificial gastric juice and artificial intestinal juice, and have a strong ability to resist gastric acid and choline, achieving unexpected technical effects.
[0116] The applicant fed 7-week-old female C57 mice with the probiotic microcapsules provided by this invention for 10 days in advance, and then conducted a diarrhea modeling experiment on the mice to observe intestinal disorders and other indicators. The results showed that, compared with the negative control group, the mice that were given the probiotic microcapsules in advance by gavage had improved intestinal epithelial cell integrity, prolonged time for the first feces excretion, and reduced excessively rapid intestinal peristalsis.
[0117] Seven-week-old female C57 mice were pre-fed the *Lactobacillus plantarum* microcapsules provided by this invention for 10 days before a diarrhea model was established, and changes in their intestinal flora were detected. The results showed that, compared with the negative control group, mice administered *Lactobacillus plantarum* microcapsules by gavage exhibited increased abundance of *Akkermansia* spp. in their intestinal flora, particularly a significant increase in *Akk* spp. abundance.
[0118] The preparation process of the microcapsules of *Lactobacillus plantarum* provided by this invention is simple, the coating material is inexpensive, and the industrialization cost is low. It can effectively maintain the viable count of *Lactobacillus plantarum* VHProbi R14 during transportation, storage and processing, which greatly improves the applicability of *Lactobacillus plantarum* VHProbi R14. Therefore, it can be widely used in food, health products and other fields.
Claims
1. A microcapsule of *Lactobacillus plantarum*, characterized in that, The method for preparing the microcapsules includes the following steps: (1) Mix a porous starch solution with a concentration of 90-100 g / L with a viable bacterial count of not less than 10 g / L. 9 The CFU / mL *Lactobacillus plantarum* CCTCC NO: M2023721 bacterial suspension was mixed at a volume ratio of 100:1 and shaken at 25-35℃ for 30 min to obtain the suspension. (2) Add sodium alginate, dextrin, gelatin and skim milk powder to the suspension and stir evenly to obtain a freeze-dried mixture; the concentrations of sodium alginate, dextrin, gelatin and skim milk powder in the freeze-dried mixture are 20-30 g / L, 40-50 g / L, 20-50 g / L and 50-80 g / L, respectively. (3) Add a calcium chloride solution with a concentration of 15-20 g / L to the freeze-dried mixture until the freeze-dried mixture solidifies; (4) After freeze-drying for 24 hours, pulverize to within 100 mesh to obtain plant lactobacillus microcapsules.
2. The microcapsule as described in claim 1, characterized in that, The method for preparing the *Lactobacillus plantarum* suspension includes the following steps: (1) Under aseptic conditions, the seed culture of *Lactobacillus plantarum* was inoculated into the fermentation medium at a volume ratio of 3%; cultured at 37℃ for 24-36 h, with a rotation speed of 50-100 rpm, an aeration rate of 0.3-1 L / min, and a tank pressure of 0.05-0.08 MPa; after fermentation, *Lactobacillus plantarum* fermentation broth was obtained. (2) Centrifuge the fermentation broth of *Lactobacillus plantarum* at 4℃ and 8000r / min for 5 min, collect the cells, wash twice with sterile physiological saline, resuspend the cells, and prepare *Lactobacillus plantarum* bacterial suspension.
3. The microcapsule as described in claim 2, characterized in that, The components and their contents in the fermentation medium are as follows: mannan 35-45 g / L, yeast extract 15-25 g / L, corn peptone 15-25 g / L, ferric sulfate 0.5-1.5 g / L and magnesium sulfate 0.05-0.15 g / L.
4. The microcapsule as described in claim 3, characterized in that, The components and their contents in the fermentation medium are as follows: mannan 40 g / L, yeast extract 25 g / L, corn peptone 25 g / L, ferric sulfate 1.5 g / L and magnesium sulfate 0.10 g / L.
5. The microcapsule as described in claim 4, characterized in that, The concentrations of sodium alginate, dextrin, gelatin, and skim milk powder in the freeze-dried mixture were 30 g / L, 40 g / L, 50 g / L, and 80 g / L, respectively.
6. The microcapsule according to any one of claims 1-5, characterized in that, The microcapsules contain no less than 10 live bacteria. 8 CFU / g.