Lactobacillus paracasei, probiotic preparation beneficial for improving immunity and preparation method of probiotic preparation

Abstract

The invention provides a Lactobacillus paracasei, which has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No.32951. The Lactobacillus paracasei has a preservation number of CGMCC No.32951. The Lactobacillus paracasei has a preservation number of CGMCC No.32951. The lactobacillus paracasei ID001 provided by the invention has an inhibition effect on enteropathogenic bacteria such as salmonella, escherichia coli, staphylococcus aureus and the like. The probiotic preparation is obtained through fermentation and the dendrobium officinale extract, and the probiotic preparation has the immunoregulation effect through performance verification and screening.

Description

Technical Field

[0001] The invention relates to the field of probiotics, and in particular to a Lactobacillus paracasei probiotic preparation that is beneficial for improving immunity and a preparation method thereof. Background Art

[0002] The human body's immunity, as a key force to resist disease and maintain health, relies on a complex system - the immune system for its core operating mechanism. This system is composed of immune organs, immune tissues and immune molecules, which interact with each other to form an indestructible body defense line. Although the scientific community's comprehensive understanding of the immune system is still continuing to deepen, existing research results have revealed an important fact: about 70% of the body's immune cells are gathered in the intestines. More importantly, there is an inextricable connection between the growth and development of the immune system and the flora in the intestines, which influence each other and jointly maintain the health and balance of the human body.

[0003] As people's awareness of health care continues to improve in modern society, the need to prevent "pre-disease" (i.e. potential health problems) is becoming increasingly urgent. By optimizing the intestinal environment, promoting the proliferation of beneficial bacteria, and effectively curbing the growth of harmful bacteria, it can bring many significant positive effects to the body. This concept has been widely accepted and experienced. For people facing the challenge of intestinal flora imbalance, oral probiotic preparations have become a direct and efficient means of regulation.

[0004] At the same time, the effectiveness of probiotics in strengthening the immune system has also been widely recognized. It is worth noting that in addition to the intestines, the spleen and thymus also play an important role in immune regulation. The human digestive tract mucosa is vast, reaching about 300 square meters, which is almost equivalent to 3 / 4 of the area of ​​a basketball court. This makes the digestive tract, under the synergistic effect of lymphoid tissue (GALT), a vital immune defense line in the human body. The number of microorganisms living in the human gastrointestinal tract is even larger, reaching about 10^13, and the number of microbial cells far exceeds that of human cells, reaching ten times as much.

[0005] Every inch of the small intestine is densely packed with cells that produce immunoglobulins, which account for about 80% of all immunoglobulin-producing cells in the human body. Therefore, immune cells in various parts of the digestive tract can trigger a variety of immune responses. Among them, some bacteria of the genera Lactobacillus and Bifidobacterium have shown immune-stimulating effects to varying degrees and are considered to be highly effective immunomodulators, or "biological response modifiers."

[0006] Although probiotics play a key immunomodulatory function through deep interaction with the intestinal immune system, we should not ignore the core role of the spleen and thymus in overall immune regulation. Together, they build a complex and delicate immune network to protect the health of the human body.

[0007] Therefore, immune cells in various parts of the digestive tract can stimulate a variety of immune responses. Among them, some bacteria of the Lactobacillus and Bifidobacterium genera have shown immunostimulatory effects to varying degrees and are considered effective immunomodulators, or "biological response modifiers."

[0008] Probiotics play a key immunomodulatory function through in-depth interaction with the intestinal immune system. The intestinal mucosal immune system is a complex and delicate system, mainly composed of three major barriers: the first is the biological barrier, which is composed of symbiotic bacteria in the intestine; the second is the physical barrier, which is formed by mucin and intestinal epithelial cells; and the last is the immune barrier, which covers the gut-associated lymphoid tissue (GALT) and widely distributed immune cells. In the intestinal mucosal tissue, epithelial cells (IEC) and gut-associated lymphoid tissue (GALT) play a pivotal role. In the area below the epithelial cells, there are antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. These APCs interact with the initial immune cells located in the follicular area and the cells present in the coronary area. This process is crucial for triggering the rapid differentiation of plasma cells in the germinal center into secretory immunoglobulin A (sIgA). Through these complex interactions, probiotics can effectively regulate the function of the intestinal immune system. The interaction between the intestinal mucosal immune system and the intestinal flora is the first line of defense to maintain the body's resistance to infection and its own health.

[0009] Low immunity refers to the weakening of the body's immune system, which reduces its ability to defend against external pathogens (such as bacteria and viruses). Common symptoms include:

[0010] 1. Frequent infections: Low immunity makes the body more vulnerable to invasion by various pathogens, leading to frequent infectious diseases such as colds, flu, and tonsillitis.

[0011] 2. Slow recovery: Once infected, the body takes longer to clear pathogens and recover due to the inefficiency of the immune system. Even a mild illness may last longer.

[0012] 3. Persistent fatigue: The immune system consumes a lot of energy when fighting pathogens. People with low immunity may feel chronic fatigue and lack of energy even in the absence of obvious illness.

[0013] 4. Digestive problems: Immunity is closely related to the health of the digestive system. Low immunity may lead to gastrointestinal dysfunction, manifested as loss of appetite, diarrhea, constipation or food intolerance.

[0014] 5. Skin problems: The skin is the first line of defense of the body. When the immunity is low, it is easily susceptible to external stimuli, resulting in symptoms such as rash, eczema, acne or repeated infections.

[0015] 6. Mood swings and mental health: The immune system interacts with the nervous system. Low immunity may trigger or aggravate emotional problems such as anxiety and depression, and affect mental health.

[0016] 7. Sleep disorders: Abnormalities in the immune system sometimes affect sleep quality, leading to insomnia, nightmares or shallow sleep, further weakening the immune function.

[0017] Low immunity may also cause other symptoms, such as recurrent oral ulcers, swollen lymph nodes, increased risk of autoimmune diseases, etc. These symptoms together affect the patient's daily quality of life and reduce their ability to cope with external challenges.

[0018] On the other hand, a variety of Chinese herbal medicines and foods, such as astragalus, ganoderma lucidum, and dendrobium officinale, are widely believed to have the potential to enhance immunity. According to records in ancient herbal books, Huoshan dendrobium has the best quality and extremely high efficacy. It is an authentic medicinal material of dendrobium, with the effects of thickening the stomach and intestines, calming the mind and eliminating shock, nourishing yin and promoting fluid production, and prolonging life. Due to the mixed varieties of dendrobium officinale on the market, dendrobium officinale and copper-skinned dendrobium with similar appearance are often processed and impersonated as Huoshan dendrobium, which seriously affects the development and application of Huoshan dendrobium. However, studies have found that the efficacy of Huoshan dendrobium and dendrobium officinale is significantly different. Dendrobium officinale has antioxidant, hypoglycemic, hypolipidemic, and immunomodulatory effects. Its main active ingredients are polysaccharides, bibenzyls, flavonoids, phenanthrene, etc. However, the material basis for the high quality and efficacy of Huoshan dendrobium has not yet been clarified.

[0019] Therefore, screening out Chinese herbal medicines that can be effectively fermented and extracting active substances with immunity-enhancing functions has become a research direction for improving human immunity.

[0020] There are already some patents that have been applied for or are being applied for the use of probiotics related to immunity.

[0021] For example, the Chinese invention patent with publication number CN202410469446.2 uses Lactobacillus fermentum ProSci-602 to prepare a drug for allergic reactions induced by cow's milk. It can effectively reduce the level of specific IgE in the serum of people with allergic reactions induced by cow's milk, regulate the gene expression of Th1 and Th2 cytokines in the body, and thus regulate the body's immune function to prevent or treat cow's milk-induced allergic reactions.

[0022] The Chinese invention patent with publication number CN202310603003.3 discovered that Lactobacillus rhamnosus R7041 can be used in oral products for skin allergies, skin allergy-related inflammation, enhancing autoimmunity, regulating human intestinal flora, reducing intestinal epithelial cell damage, etc., to achieve the effect of preventing, alleviating and / or treating skin allergies.

[0023] The Chinese invention patent with publication number CN202410676705.9 discloses that animal Bifidobacterium QC08 can enhance the immune function of cyclophosphamide immunosuppressed mice by improving intestinal function and reducing inflammatory response. It has good application prospects in the field of regulating immune system diseases.

[0024] The Chinese invention patent with publication number CN202410676699.7 found that Lactobacillus rhamnosus TM08 can maintain the basic morphology of colon and jejunal tissues, and has a significant alleviating effect on the increased jejunal permeability caused by β-lactoglobulin-induced food allergy in young mice. TM08 can also inhibit the activation of Th2 and Th17 cells after β-lactoglobulin-induced food allergy in mice, resulting in a significant decrease in the cytokines produced, and increasing the activation of Th1 and Treg cells, thereby alleviating the situation of Th1 / Th2 cell immune deviation. The composite probiotics for improving immunity described in the Chinese invention patent with publication number CN202410239163.9 are composed of animal Bifidobacterium lactis subspecies BLa80 strain, Lactobacillus rhamnosus LRa05 strain, and Lactobacillus acidophilus LA05 strain. The three cooperate with each other, promote each other, and synergize in the effect of improving the body's immunity. When the amount of bacteria is consistent, the combination of the three bacteria is significantly improved in terms of improving the body's immunity compared with the intervention method of the lack of any one of the bacteria. However, none of the above prior arts involve the problem of immune response activators. Summary of the invention

[0025] In order to solve the above technical problems, the present invention is implemented by the following technologies:

[0026] A Lacticaseibacillus paracasei (Lacticaseibacillus paracasei), whose preservation number is CGMCC No. 32951. The fermentation product is obtained by extracting Dendrobium officinale from Lacticaseibacillus paracasei ID001.

[0027] The fermentation products include immune response activators;

[0028] The immune response activator structural formula is as follows:

[0029]

[0030] A probiotic preparation comprises the Lactobacillus paracasei ID001 and the fermentation product.

[0031] The probiotic preparation also includes a freeze-drying protective agent.

[0032] The freeze-drying protective agent comprises 10-15% skim milk powder, 2-3% trehalose, 0.6-2.5% calcium alginate, 1.5-2.5% sucrose, 1.5-3.5% maltodextrin, 2-4% modified starch, 1-2% inulin, 1.5-1.8% arginine, and the balance is water. The preparation method of the probiotic preparation comprises the steps of fermenting and culturing probiotics to obtain bacterial mud, mixing the bacterial mud with the freeze-drying protective agent, and then freeze-drying.

[0033] The preparation method of the probiotic preparation, the culture medium used in the fermentation culture comprises the following components in percentage by weight:

[0034] Yeast extract powder 0.3-0.8%, beef extract powder 0.4-0.8%, peptone 0.3-0.9%, diammonium hydrogen citrate 0.01-0.2%, dipotassium hydrogen phosphate 0.01-0.3%, MgSO4·7H2O 0.01-0.03%, Tween 80 0.05-0.15%, glucose 0.05-0.3%, sodium acetate 0.01-0.2%, Dendrobium officinale extract 0.3-6%, water as the balance, adjust the pH value to 6.2-6.8.

[0035] The preparation method of the Dendrobium officinale extract is as follows:

[0036] The powder is obtained by crushing Dendrobium officinale, adding 80°C pure water and stirring to extract for 5 hours, centrifuging to obtain two parts: filtrate (part A) and filter residue (part B), extracting the filter residue with 75-95% ethanol, centrifuging at 4000-10000r / min; concentrating at 50-75°C to recover ethanol, and drying the obtained concentrate in a constant temperature drying oven at 50-85°C to obtain the product.

[0037] The ethanol concentration is greater than 90%;

[0038] The solid-liquid ratio of the filter residue B to ethanol is 1:10.

[0039] The ethanol extraction temperature is 70°C;

[0040] The centrifugal speed is 6000r / min;

[0041] The concentration temperature is 60°C;

[0042] The temperature in the drying oven was 70°C.

[0043] The probiotic preparation is used to prepare a drug for improving intestinal immunity; or

[0044] It is used to prepare drugs that inhibit or replace the adsorption of intestinal pathogenic bacteria to intestinal cells.

[0045] The intestinal pathogenic bacteria are Salmonella, Escherichia coli, Staphylococcus aureus or Shigella flexneri.

[0046] The beneficial effects compared to the existing ones are as follows:

[0047] The Lactobacillus paracasei ID001 provided by the present invention has an inhibitory effect on intestinal pathogenic bacteria Salmonella, Escherichia coli, Staphylococcus aureus, etc. The probiotic preparation is obtained by fermentation with Dendrobium candidum extract, and has an immunomodulatory effect through the phagocytic performance screening of macrophage RAW264.7. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The crude extract HPLC chart after elution with macroporous adsorption resin is shown;

[0049] Figure 2 Lactobacillus paracasei ID001 is shown to interfere with macrophages RAW264.7. DETAILED DESCRIPTION

[0050] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0051] The Lactobacillus paracasei ID001 provided by the present invention is separated from traditional kimchi and has a preservation number of CGMCC No. 32951. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 6, 2024, with a preservation unit code of CGMCC, and a preservation unit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101.

[0052] The extraction process of the Dendrobium officinale extract of the present invention is as follows: the leaves of the Dendrobium officinale are crushed into powders of 20-60 meshes, and then the powder is placed in an extraction tank, pure water at 80 DEG C is added, stirred and extracted for 5 hours, and centrifuged to obtain two parts of filtrate (part A) and filter residue (part B), the filter residue is subjected to 75-95% ethanol extraction, and centrifuged at 4000-10000r / min; ethanol is concentrated and recovered at 50-75 DEG C, the obtained concentrate is dried at 50-85 DEG C in a constant temperature drying oven, and the extract is collected for standby use.

[0053] Example 1

[0054] Strain Isolation and Purification Method

[0055] 1. Source of strain:

[0056] Take 2 g of kimchi fermented water, dilute it 10 times with sterile saline, spread it on a plate added with MSR culture medium, place it in an anaerobic incubator, and culture it in a 37°C constant temperature incubator for 24-48 hours.

[0057] After the colonies grow on the plates, observe the single colonies formed on each plate, and select the single colonies that meet the characteristics of lactic acid bacteria (round, milky white or slightly yellow, opaque or translucent, smooth protrusions, and neat edges) according to the shape, size, color, transparency, protrusions, and surface roughness of the colonies. Purify three times until the color, size, transparency, and edge shape of the colonies on the same plate are uniform.

[0058] 2. The purified colonies were subjected to Gram staining for specific detection of Gram-positive bacteria.

[0059] 3. The results of 16s rDNA sequencing (SEQ ID NO: 1) of the bacterium are as follows:

[0060] GTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGT

[0061] GGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGG

[0062] AAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGG

[0063] CGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGG

[0064] CTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACT

[0065] GAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACG

[0066] CAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCT

[0067] GTtGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCA

[0068] GAAAGcCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTT

[0069] ATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAG

[0070] CCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGG

[0071] ACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGG

[0072] CGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAA

[0073] CAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGG

[0074] TTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGAC

[0075] CGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGT

[0076] GGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTG

[0077] AGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAG

[0078] CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTT

[0079] GCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTG

[0080] GGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGG

[0081] ATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAG

[0082] TTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGAT

[0083] CAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATG

[0084] AGAGTTTGTAACACCCGAAGCCGGTGG

[0085] The strain ID001 was identified as Lactobacillus paracasei, and its Latin name is Lacticaseibacillus paracasei. The deposit number of the strain is CGMCC No.32951. The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 6, 2024. The depository code is CGMCC. The depository address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the deposit number is CGMCC No.32951.

[0086] Example 2

[0087] Evaluation of probiotic properties of strains in vitro

[0088] 1. Gastric acid and bile salt tolerance test

[0089] Prepare artificial gastric juice (acid): add 0.06 g pepsin (3 g / L) to 20 ml of sterilized normal saline, adjust the pH to 3.0 with 0.1 mol / L HCl, and filter with a 0.22 μm filter membrane into a sterilized empty conical flask for later use.

[0090] Preparation of artificial intestinal fluid (bile salt): add 0.02g trypsin (1g / L) and 0.06g bile salt (0.3%) to 20ml sterilized physiological saline, adjust pH to 8.0 with 0.1mol / L NaOH, filter into a sterilized empty conical bottle with a 0.22 microfiltration membrane, and set aside. Test of tolerance to artificial gastric fluid: inoculate the treated bacterial fluid into artificial gastric fluid with pH 3.0, place in a 37℃ water bath and culture for 3h, then determine the number of viable bacteria and calculate the survival rate.

[0091] Artificial intestinal fluid tolerance test: After the bacteria with high survival rate were treated in artificial gastric fluid for 3 hours, they were inoculated into artificial intestinal fluid (containing 0.3% bile salt) and cultured in a 37°C water bath for 6 hours. After that, the number of live bacteria was determined and the survival rate was calculated.

[0092] 2. Determination of hydrophobicity (hydrocarbon compound adhesion method) and self-aggregation

[0093] Take the cultured bacterial solution, centrifuge at 6000r / min for 10min, and collect the bacteria at the bottom. Wash the bacteria twice with PBS buffer. The blank control is PBS buffer, and the concentration of the bacteria is adjusted with PBS buffer so that its OD600 nm value is about 1.00 (A0). Pipette 3mL of the adjusted bacterial solution into a 10mL sterile centrifuge tube, add 1mL of xylene, shake for 2min, and let it stand at 37℃ for 1h. Pipette the lower aqueous phase, use PBS buffer as the blank control, and measure the OD600 nm value (A). The calculation formula is:

[0094]

[0095] Take the cultured bacterial solution and centrifuge at 4000r / min for 10min to collect the bottom bacteria. Wash the bacteria twice with PBS buffer. The blank control is PBS buffer, and the bacterial concentration is adjusted with PBS buffer to make its OD600 nm value about 1.00 (A0). Stand at 37℃, take samples every 1h to measure the optical density (At) of the supernatant of the samples incubated for different times at 600nm until the end of 6h. The calculation formula is:

[0096]

[0097] The specific screening results are shown in Table 1 below.

[0098] Table 1

[0099] strain Gastric acid tolerance Bile salt tolerance Hydrophobicity Self-aggregation GG (Lactobacillus rhamnosus) 87.35 97.52 74.27 37.31 Lactobacillus paracasei ID001 >100 94.29 76.56 88.73

[0100] Screen out strains with better tolerance to gastric acid and bile salts, so that the strains can pass through the human body's gastric acid and intestinal fluid during consumption. Next, use the two characteristics of the strains, hydrophobicity and self-aggregation, to preliminarily predict their ability to colonize in the intestine. Strains with high hydrophobicity can usually attach better to the intestinal mucosa, while strains with strong self-aggregation indicate that they have strong interactions, which helps to form a stable flora in the intestine.

[0101] Through such a screening and evaluation process, strains that can remain active in the human digestive system and effectively colonize in the intestines can be selected, thus providing a solid foundation for the subsequent development of probiotic products.

[0102] 3. Evaluation of the performance of inhibiting pathogenic bacteria: The inhibitory effect of Lactobacillus paracasei ID001 on Salmonella, Escherichia coli, Staphylococcus aureus and Shigella flexneri was studied. The conditional pathogens and test strains were activated according to the corresponding culture conditions until OD600nm = 2. 100ul of the bacterial solution of the conditional pathogen was applied to the MRS culture medium with glass beads. After the bacterial solution was absorbed (30min), the Oxford cup was placed on the plate with sterile tweezers, and then 100ul of the test strain was added to the Oxford cup. Physiological saline and antibiotics were used as controls. The plates were placed upright and sealed, soaked at 4°C for 2h, and cultured at 37°C for 24h. The diameter of the inhibition zone of the test strain was measured and recorded with a ruler.

[0103] strain Escherichia coli salmonella Shigella flexneri Staphylococcus aureus GG (Lactobacillus rhamnosus) 19.98 14.86 14.25 15.33 Lactobacillus paracasei ID001 27.57 16.16 27.35 20.33

[0104] By measuring the diameter of the inhibition zone, we can quantitatively evaluate the inhibitory effect of the test strain on each conditional pathogen. The larger the diameter of the inhibition zone, the stronger the inhibitory effect.

[0105] Example 3 Identification of immune response activators from extracts of Dendrobium officinale fermented with Lactobacillus paracasei

[0106] 1. Grind the Dendrobium officinale to obtain powder, add 80°C pure water and stir to extract for 5 hours, centrifuge to obtain a filtrate (part A) and a filter residue (part B), extract the filter residue with 90% ethanol, and centrifuge at 6000r / min; concentrate at 60°C to recover ethanol, and dry the concentrate at 70°C in a constant temperature drying oven to obtain the Dendrobium officinale extract.

[0107] 2. Using culture medium: yeast extract powder 0.5%, beef extract powder 0.5%, peptone 0.5%, diammonium hydrogen citrate 0.02%, dipotassium hydrogen phosphate 0.01%, MgSO4·7H2O 0.01%, Tween 80 0.05%, glucose 0.1%, sodium acetate 0.1%, Dendrobium officinale extract 2%, water balance, adjust pH value to 6.2-6.8, ferment at 37°C for 24 hours, wherein the bacterial inoculation amount is 2%.

[0108] In order to extract, separate and identify the activator that can act on the immune response from the fermentation broth, the present invention purifies the fermentation broth. The specific steps are as follows:

[0109] After fermentation, the fermentation broth was processed according to the following steps: First, the fermentation broth was centrifuged to separate the bacterial sludge. Then, this part of the bacterial sludge was soaked in 95% ethanol. After soaking, it was centrifuged again, and the bacterial residue was discarded, leaving only the clear liquid obtained by ethanol soaking. Then, this clear liquid was slowly added to the column containing HP20 macroporous adsorption resin at a rate of 0.5 volumes per hour (BV / h) for adsorption treatment. Then, we used 6 volumes of pure water and 50% ethanol aqueous solution at a flow rate of 1.5 volumes per hour (BV / h) to elute the resin column. The purpose of these two elution steps is to remove some impurities and pigments adsorbed on the resin to ensure the purity of subsequent extraction. Finally, we used 4 volumes of 95% ethanol for elution, collected this part of the eluate, and concentrated it to obtain a paste-like crude extract.

[0110] 3. The collected eluate was tested for effective components (such as Figure 1 ), combine the same components and concentrate under vacuum, collect the concentrate, dissolve it in DMSO and load it on the DAC dynamic axial compression system, combine the target product after HPLC detection, and obtain the pure compound after vacuum drying. HPLC analysis conditions: mobile phase acetonitrile (A)-formic acid water 0.1% (B) as mobile phase: 0min (2% A)-5min (2% A)--10min (20% A)--15min (30% A)--20min (50% A)--25min (50% A)--30min (60% A)--35min (80% A)--40min (90% A)--45min (2% A). Chromatographic column: ZORBAX SB-C18 (250×4.6 mm, 25 μm); flow rate: 1 mL / min; column temperature: 25°C; wavelength: 270 nm; injection volume: 10 μL; the DAC dynamic axial compression system, the filler is C18, the mobile phase is 85% acetonitrile-water solution, the flow rate is controlled at 10 ml / min, and the detection wavelength is 270 nm.

[0111] Among the substances extracted by the above method, the following compounds showed the effect of immune response activator

[0112]

[0113] The compound's hydrogen spectrum 1H-NMR (600 MHz, CDCl3) and carbon spectrum 13C-NMR (600 MHz, CDCl3) were measured by nuclear magnetic resonance to identify its chemical structure.

[0114] 9.48(2H in-OH),7.21-6.52(6H in benzene),5.82-4.88(3H in ethylene),3.61(1H in methine),3.59-3.39(2H in methylene),3.04-2.91(2H in methine),2.85(1H in methylene),2.785-2.68(3H in methine),2.59-2.53(2H in methylene),2.485(1H in methine),2.19-1.79(2H in methylene),1.11(1H in methyl),1.07(2H in-NH-).

[0115] 207.1,151.3,145.5,144.5135.8,135.7,131.0,127.8,123.3,127.2,120.4,120.3116.8,116.4,115.8,76.6,63.6,57.4,52.7,50.5,49.1,44.3,37.8,35.9,32.0,31.2,14.9.

[0116] Example 4 Compounds and cultured probiotic preparations for immune response activation test

[0117] 1. Construct a macrophage RAW264.7 screening model, and verify the immune function of the probiotic preparation of immune response activator by macrophage RAW264.7 cell proliferation test, phagocytosis test, and macrophage secretion of immune active molecules (NO level). Adjust the bacterial suspension density to 1*10 9 cfu / mL, compound 50ug / mL.

[0118] RAW264.7 macrophages were routinely cultured in an incubator at 37°C and 5% CO2 in a medium containing DMEM (containing 10% fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin). When the cells were confluent, they were digested with 0.25% trypsin and then terminated with DMEM containing 10% fetal bovine serum. The cell concentration was adjusted to 1*10 6 cfu / mL.

[0119] 2. Cell proliferation assay

[0120] The cell suspension was transferred to a 24-well plate and grown to a monolayer. The plate was washed twice with DMEM and 50uL of the strain suspension to be tested and 950uL of DMEM medium containing 10% fetal bovine serum were added to make the amount of the two bacteria (ID001 and GG) 1*10 9 cfu / mL, so that the amount of immune response activator added is 100uL. Continue to incubate in a carbon dioxide incubator for 12 hours. Figure 2 ), remove the supernatant from each well, and wash the cells three times with 2 ml PBS to remove unbound bacteria and other impurities.

[0121] Add CCK-8 reagent: add 100 μL complete medium to each well, add 10 μL CCK-8 reagent to each well. After incubation for 2 hours, take three wells from each group for measurement. Measure the OD value at a wavelength of 450 nm.

[0122] Control group setting: The blank control group represents cells that have not been treated in any way. Lactobacillus rhamnosus GG is used as a positive control. Cell activity is calculated by the following method:

[0123] Cell activity = (OD value of experimental group - OD value of blank control group) / (OD value after mixing cells and culture medium - OD value of blank control group) * 100%

[0124] project Cell proliferation activity GG (Lactobacillus rhamnosus) 128.35％ Lactobacillus paracasei ID001 127.57％ Immune response activators 118.33％

[0125] 3. Phagocytosis Experiment

[0126] The neutral red uptake method was used to detect the effects of Lactobacillus paracasei ID001 and immune response activator on the phagocytic activity of RAW264.7 cells. Phagocytosis was evaluated using a phagocytosis assay kit. RAW264.7 cells were cultured at 5*10 6 Then, RAW264.7 cells were cultured with different strains / compounds (OD600nm=0.8) for 12 h, so that the amount of bacteria added was 1*10 9 cfu / mL, so that the amount of compound added is 100uL. Subsequently, the culture medium was removed, 100μl neutral red solution was added, and incubated for 2h. The supernatant was discarded and washed 3 times with PBS to remove the neutral red that was not phagocytosed by RAW264.7 cells. Neutral red was extracted from RAW264.7 cells using lysis buffer (1% acetic acid: 50% ethanol = 1:1). The absorbance was measured at 540nm using a full-wavelength enzyme-linked immunosorbent assay. The phagocytic activity was calculated as follows.

[0127] Phagocytic activity = As is the absorbance of the test sample at 540 nm / Ac is the absorbance of the control sample at 540 nm * 100%.

[0128] project Cell proliferation activity Control group 100％ GG (Lactobacillus rhamnosus) 128.35％ Lactobacillus paracasei ID001 127.57％ Immune response activators 109.25％

[0129] 4. Effects on NO production by macrophages

[0130] Drawing of standard curve: Accurately weigh 69 mg of NaNO2 into a volumetric flask, add double distilled water to make up to 1L, and prepare 1mmol / L NaNO2 solution. Pipette 0.05, 0.1, 0.2, 0.4, 0.6, and 1.0 ml of 1mmol / L NaNO2 solution into 10ml centrifuge tubes respectively, add double distilled water to 10ml, and you will get NO2 solutions with concentrations of 5, 10, 20, 40, 60, and 100umol / L. Then take 100ul of each of the above concentration solutions into a 96-well plate, add an equal volume of Griess reagent, and shake gently at room temperature for 10 minutes. NO2 reacts with Griess reagent to generate a pink compound. The absorbance is read at a wavelength of 540nm by an enzyme reader. The standard curve is drawn with the NO2 molar concentration as the horizontal coordinate and the corresponding absorbance as the vertical coordinate. Y=0.4441x+0.0777, R 2 =0.9994

[0131] RAW264.7 macrophages were cultured at 2*10 6 The concentration of each bacterium was inoculated into a 24-well culture plate. After 24 hours, the culture medium was replaced and different substances were added to make the concentration of the two bacteria 1*10 9 cfu / mL, so that the amount of immune response activator added is 100uL. NO is a free radical with active chemical properties and a half-life of about 30s in the body, so it is difficult to detect. This experiment uses the Griess method to detect NO in the culture supernatant to indirectly reflect the production of NO. After 24h of culture, take 200ml of each group of culture fluid in an Eppendorf centrifuge tube, add 300g / LZnS0410ul to remove protein, centrifuge at 9000r / min for 10min, then take 100ul of supernatant from each tube and place it in a 96-well plate, add an equal volume of Griess reagent, shake gently at room temperature for 10min, read the absorbance at a wavelength of 540nm on an enzyme reader, and calculate the NO in each group of culture fluid using a standard curve.

[0132] project NO concentration produced by macrophages (umol / L) GG (Lactobacillus rhamnosus) 23.25 Lactobacillus paracasei ID001 22.58 Immune response activators 19.87

[0133] Macrophages play an important role in preventing infection, maintaining self-stability and immune surveillance. In addition to directly killing pathogenic microorganisms and clearing apoptotic and mutant cells, activated macrophages secrete various immune active molecules, such as NO, which play an irreplaceable role in innate immune prevention and acquired immune response. NO is regarded as one of the effectors of macrophages in exerting immune effects. On the one hand, the cytotoxic effect of macrophages on tumor cells can be blocked by inhibitors that inhibit NO synthesis. On the other hand, the cytotoxic effect of macrophages on tumor cells is positively correlated with the production of NO, and its series of oxidation products can also enhance the host's resistance to bacteria, fungi and parasites. By studying the partial mechanism of probiotic preparations and compounds after fermentation of Lactobacillus paracasei ID001 on macrophages, the results showed that Lactobacillus paracasei ID001 and compounds can activate macrophages and thus play a role in regulating immunity.

[0134] Example 5 Animal Experiment Design

[0135] 1. Mouse modeling and grouping

[0136] 40 female BALB / c mice aged 6-8 weeks were randomly selected as the normal control group after one week of adaptive feeding, and the remaining 32 mice were intraperitoneally injected with cyclophosphamide (60 mg / kg) for 3 consecutive days, and the food and water intake were maintained at normal levels to establish an immune-compromised model. The mice with symptoms such as body weight loss, large hair loss, and lethargy were used as the standard for successful modeling.

[0137] After successful modeling, the mice were randomly divided into 3 groups, 10 in each group, and raised in an automatic air supply system at 25°C. The drinking water was boiled and disinfected, and the mice were free to eat. 9 CFU / mL), medium dose group (5*10 8 CFU / mL). The mice in the four groups were gavaged once a day, with 200uL each time. The indicators to be tested were tested on the 0th, 14th and 28th days for all groups of mice.

[0138] 2. Testing indicators and methods

[0139] ① Determination of immune organ index, mouse thymus and spleen organ index determination experiment Record the weight of mice after the last administration, take the spleen and thymus, test their weights, and calculate the organ index according to the following formula:

[0140] Mouse thymus index = thymus weight (mg) / mouse body weight (g)

[0141] Mouse spleen index = spleen weight (mg) / mouse body weight (g).

[0142] Mouse weight changes

[0143] Group Mouse weight (g) Thymus index (mg / g) Spleen index (mg / g) Normal control group 26.37±0.21 3.15±0.13 4.05±0.12 Blank control group 20.73±0.19 2.09±0.17 2.17±0.21 High dose group 26.50±0.35 3.59±0.22 4.57±0.43 Medium dose group 24.77±0.44 3.01±0.14 4.07±0.32

[0144] Compared with the blank control group (cyclophosphamide model group), the spleen and thymus index levels of mice in the high-dose group and the medium-dose group were significantly increased, indicating that the probiotic preparation of Lactobacillus paracasei ID001 can increase the spleen and thymus index of mice with low immunity induced by cyclophosphamide; compared with the normal control group, the spleen index and thymus of mice in the high-dose group were higher than those in the normal control group, indicating that the probiotic preparation in the high-dose group can increase the weight of the spleen and thymus of mice. The spleen is the center of the body's cellular immunity and humoral immunity, containing a large number of lymphocytes and macrophages. The spleen index can reflect the body's immune function, which further proves that the probiotic preparation can play an immunomodulatory role by increasing the spleen index of mice. The thymus plays an important role as a central immune organ, and is filled with developing T cell precursors and mature T cells. The level of the thymus index can intuitively reflect the developmental state of the thymus and the richness of the T cell pool, thus becoming an important indicator for measuring the body's cellular immune function. Therefore, probiotic preparations effectively promoted the generation and maturation of T cells by increasing the thymus index of mice, thereby enhancing the body's cellular immune response.

[0145] ② Determination of the phagocytic index of phagocytes in the peritoneal cavity. 20% Indian ink (10mL-1kgBW) was injected intravenously into the tail of mice 24 hours after the last administration. 20uL of blood was collected from the orbital vein 2 minutes (T1) and 7 minutes (T2), respectively. Each was added to 2mL of 0.1% Na2CO3 solution, mixed, and the absorbance values ​​of T1 and T2 were measured at 680nm, recorded as A1 and A2, respectively. The carbon clearance index K was calculated by the formula: Carbon clearance index K = (lgA1-lgA2) / (T2-T1)

[0146] The mice were killed by cervical dislocation, and the spleen and liver were removed by dissection. After washing and drying, they were weighed separately. The phagocytic index α was calculated according to the formula: phagocytic index α = M body weight / (M liver weight + M spleen weight) × K1 / 3

[0147] Group Phagocytic index Normal control group 5.33±0.23 Blank control group 4.23±0.11 High dose group 5.21±0.23 Medium dose group 4.92±0.14

[0148] Macrophages include mononuclear macrophages and neutrophils, which can engulf and digest aging and dead cells and foreign bodies in the body. They are an important mechanism of the body's natural defense. By monitoring the ability of neutrophils and macrophages to engulf foreign microorganisms and other particles, the body's natural immune defense function can be reflected. The results of the phagocytic index of probiotic preparations showed that the phagocytic index of the high-dose group was significantly higher than that of the blank control group (cyclophosphamide model group), and there was no statistical difference between the phagocytic index of the high-dose group and the normal group. The above results show that high-dose probiotic preparations can enhance the phagocytic ability of mouse macrophages and have the effect of promoting and enhancing the immune function of mice.

[0149] ③ Detection of cytokines in serum: 24 hours after the last administration, blood was collected from the eye sockets of mice, centrifuged at 3000g for 10 minutes at low temperature, and the serum was retained. According to the instructions of the enzyme-linked immunosorbent assay kit, the contents of IL-10, IFN-γ, and TNF-α in the serum were determined.

[0150] Group IL-10 IFN-γ TNF-α Normal control group 47.17±0.12 30.12±0.15 26.33±0.23 Blank control group 35.12±0.20 20.07±0.33 18.71±0.13 High dose group 46.83±0.23 29.31±0.18 25.38±0.11 Medium dose group 39.79±0.31 28.17±0.25 24.27±0.05

[0151] Compared with the normal control group, the levels of IL-10, IFN-γ, and TNF-α in the high-dose group and the medium-dose group did not change significantly. Compared with the blank control group (cyclophosphamide model group), the levels of IL-10, IFN-γ, and TNF-α in the serum of the high-dose group and the medium-dose group increased significantly. The above results show that the probiotic preparation fermented by Lactobacillus paracasei ID001 can effectively enhance the immune response activation ability of cells, promote the secretion level of IL-10, IFN-γ, and TNF-α cytokines in immune-damaged mice, and enhance the cellular and humoral immunity levels of mice.

Claims

1. A Lactobacillus paracasei (Lacticaseibacillus paracasei) ID001, characterized in that: Its accession number is CGMCC No.32951.

2. A fermentation product obtained by extracting Dendrobium officinale by fermenting Lactobacillus paracasei ID001 according to claim 1.

3. The fermentation product according to claim 1, characterized in that: The fermentation products include immune response activators; The immune response activator structural formula is as follows:

4. A probiotic preparation, characterized in that: The method comprises the Lactobacillus paracasei ID001 as claimed in claim 1 and the fermentation product as claimed in claim 2.

5. The probiotic preparation according to claim 3, characterized in that: Lyoprotectants are also included.

6. The probiotic preparation according to claim 4, characterized in that: The freeze-drying protective agent comprises 10-15% skim milk powder, 2-3% trehalose, 0.6-2.5% calcium alginate, 1.5-2.5% sucrose, 1.5-3.5% maltodextrin, 2-4% modified starch, 1-2% inulin, 1.5-1.8% arginine and the balance water.

7. The method for preparing the probiotic preparation according to claim 4, characterized in that: The method comprises the steps of fermenting and culturing probiotics to obtain bacterial mud, mixing the bacterial mud with a freeze-drying protective agent, and then freeze-drying the mixture.

8. The method for preparing the probiotic preparation according to claim 7, characterized in that: The culture medium used in the fermentation culture includes the following components in percentage by weight: Yeast extract powder 0.3-0.8%, beef extract powder 0.4-0.8%, peptone 0.3-0.9%, diammonium hydrogen citrate 0.01-0.2%, dipotassium hydrogen phosphate 0.01-0.3%, MgSO4·7H2O 0.01-0.03%, Tween 80 0.05-0.15%, glucose 0.05-0.3%, sodium acetate 0.01-0.2%, Dendrobium officinale extract 0.3-6%, water as the balance, adjust the pH value to 6.2-6.

8.

9. The method for preparing the probiotic preparation according to claim 7, characterized in that: The preparation method of the Dendrobium officinale extract is as follows: The powder is obtained by crushing the Dendrobium officinale, adding 80°C pure water and stirring for extraction for 5 hours, centrifuging to obtain a filtrate and a filter residue, extracting the filter residue with 75-95% ethanol, and centrifuging at 4000-10000r / min; concentrating at 50-75°C to recover ethanol, and drying the obtained concentrate at 50-85°C in a constant temperature drying oven to obtain the product.

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