Lactoferrin-containing probiotic composition as well as preparation method, product and application thereof

Abstract

The invention provides a lactoferrin-containing probiotic composition as well as a preparation method, a product and application thereof, and belongs to the field of probiotics. The invention particularly provides a probiotic composition containing lactoferrin. The probiotic composition is prepared from bifidobacterium and lactoferrin. Preferably, the use amount ratio of the bifidobacterium to the lactoferrin is 10 < 9 > CFU (colony forming unit): (0.001-10) g. The bifidobacterium and lactoferrin composition disclosed by the invention has a remarkable immunity improving effect in mice, and has a relatively good improving effect on the weight, anti-inflammatory factors and immune proteins of the mice, and the components have a certain synergistic effect. In an immunosuppressive animal model, the invention also shows a relatively good protection effect, which shows that the composition has a very good immune recovery effect.

Description

Technical Field

[0001] The present invention belongs to the field of probiotics, and in particular relates to a probiotic composition containing lactoferrin, a preparation method thereof, a product and an application thereof. Background Art

[0002] Probiotics are active microorganisms that colonize the human body and alter the composition of the host's microbial flora. They promote nutrient absorption and maintain intestinal health by modulating the host's mucosal and systemic immune function or by regulating the balance of intestinal flora, resulting in single microorganisms or a defined mixture of microorganisms with beneficial health effects. Probiotics help boost immunity and enhance disease resistance. Imbalances in the intestinal flora often compromise immunity, and the use of probiotic compositions can restore intestinal flora balance.

[0003] Bifidobacterium is a genus of Gram-positive, non-motile, rod-shaped bacteria that are sometimes forked at one end and strictly anaerobic. It is widely found in the digestive tract, vagina, and oral cavity of humans and animals. Bifidobacterium is an important member of the intestinal flora of humans and animals. On the one hand, Bifidobacterium is a probiotic that helps maintain intestinal health. Healthy intestinal function is very important for the function of the immune system. Bifidobacterium can help regulate the immune system so that it can better respond to pathogens and inflammation, help reduce excessive immune responses, and enhance resistance to pathogens. On the other hand, immunity is a complex system that is affected by many factors, including genes, diet, lifestyle, environment, etc. Bifidobacterium is a part of it, but it is not the only factor.

[0004] Lactoferrin (LF) is an iron-binding glycoprotein in the transferrin family. Found in human milk and various secretions, LF is a core immune protein found in breast milk and particularly high levels in colostrum. It competes with pathogens for iron, resisting their invasion and thus protecting infants from immune damage. Therefore, LF is considered a key ingredient for boosting immunity.

[0005] The Chinese patent application with application number 202211517832.1 discloses a combination of lactoferrin and probiotics. The probiotics are preferably selected from one or more of Bifidobacterium longum infantis subspecies, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium animalis lactis subspecies, Bifidobacterium breve, Lactobacillus acidophilus and Lactobacillus rhamnosus; preferably Bifidobacterium animalis lactis subspecies, more preferably Bifidobacterium animalis lactis subspecies Bb12. The invention also relates to a food comprising the nutritional composition. The present invention also relates to the use of lactoferrin, probiotics, or a nutritional composition or food comprising both for non-therapeutic purposes in promoting the development of the immune system of offspring and improving the intestinal flora of offspring through consumption by human or animal mothers. According to the description of the invention, when lactoferrin and Bb12 are used in combination, the content of IgG can be significantly increased, and there may be a synergistic effect between the two. However, in the data recorded on the serum immunoglobulins of offspring mice, there was a simple additive effect between the two only under some dosage conditions. For example, when the medium dose of lactoferrin was combined with the low dose of Bb12, antagonism was manifested to a certain extent under the condition that both were at high doses. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a probiotic composition containing lactoferrin. By optimizing the ratio of probiotics and lactoferrin, as well as the selection of bacterial species, a composition with better immune enhancement effect is provided.

[0007] In one aspect, the present invention provides a probiotic composition containing lactoferrin.

[0008] Specifically, the probiotic composition consists of bifidobacteria and lactoferrin.

[0009] Specifically, the bifidobacterium includes but is not limited to any one or more of Bifidobacterium breve, Bifidobacterium longum subsp. infantis, and Bifidobacterium animalis subsp. lactis.

[0010] Preferably, the Bifidobacterium breve includes Bifidobacterium breve HH079, with a deposit number of GDMCC No: 64216; the Bifidobacterium longum subsp. infantis includes Bifidobacterium longum subsp. infantis R0033, with a deposit number of SHBCC D24671; and the Bifidobacterium animalis subsp. lactis includes Bifidobacterium animalis subsp. lactis BB-12, with a deposit number of ATCC27536.

[0011] The Bifidobacterium breve HH079 described in the present invention is a self-screened strain of the present invention, which has been applied for separately. Its preservation number is GDMCC No: 64216, the preservation time is December 29, 2023, the preservation unit is Guangdong Provincial Microbiological Culture Collection Center, the taxonomic name is Bifidobacterium breve, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0012] As preferred, the usage ratio of bifidobacteria and lactoferrin is 10 8 CFU: 0.1-1000mg; or expressed as 10 9 CFU: 1-10000mg, or expressed as 10 9 CFU: 0.001-10g. The dosage ratio of the present invention is intended to emphasize the proportional relationship between bifidobacteria and lactoferrin. When actually applied to products, those skilled in the art may adjust the absolute value of the dosage in consideration of different product dosage forms.

[0013] Preferably, the dosage ratio of bifidobacteria to lactoferrin can be 10 9 CFU: 1-1000mg, 10 9 CFU: 1-5000mg, 10 9 CFU: 1-2000mg, 10 9 CFU: 1-8000mg, 10 9 CFU: 1-10g, 10 9 CFU: 5-8g etc. You can also choose to include but not limited to the following ratio ranges:

[0014] 1×10 8 -2×10 8 CFU: 0.2-100mg;

[0015] 1×10 8 -8×10 8 CFU: 0.8-100mg;

[0016] 2×10 8 -9×10 8 CFU: 0.9-200mg;

[0017] 3×10 8 -7×10 8 CFU: 0.7-300mg;

[0018] 5×10 8 -7×10 8 CFU: 0.7-500mg;

[0019] 6×10 8 -7×10 8 CFU: 0.7-600mg;

[0020] 7×10 8 -9×10 8 CFU: 0.9-700mg;

[0021] 8×10 8 -10×10 8 CFU: 0.1-800mg.

[0022] More preferably, the ratio of bifidobacteria to lactoferrin can be 10 8 CFU: 0.1-100mg.

[0023] When the bifidobacterium in the probiotic composition is Bifidobacterium breve HH079, the present invention provides a reference dosage ratio of bifidobacterium to lactoferrin of 10:1. 8 CFU: 1-100mg, 10 may be selected in specific applications 8 CFU: 1mg, 10 8 CFU: 3mg, 10 8 CFU: 5mg, 10 8 CFU: 6mg, 10 8 CFU: 8mg, 10 8 CFU: 8.2mg, 10 8 CFU: 8.4mg, 10 8 CFU: 8.8mg, 10 8 CFU: 10mg, 10 8 CFU: 12mg, 10 8 CFU: 18mg, 10 8 CFU: 20mg, 10 8 CFU: 24mg, 10 8 CFU: 25mg, 10 8 CFU: 26-30mg, 10 8 CFU: 31mg, 10 8 CFU: 31.4mg, 10 8 CFU: 32.8mg, 10 8 CFU: 33mg, 10 8 CFU: 385mg, 10 8 CFU: 38mg, 10 8 CFU: 50mg, 10 8 CFU: 56mg, 10 8 CFU: 56.2mg, 108 CFU: 56.8mg, 10 8 CFU: 3-100mg, 10 8 CFU: 5-90mg, 10 8 CFU: 20-80mg, 10 8 CFU: 25-72mg, 10 8 CFU: 28-60mg, 10 8 CFU: 30-70mg, 10 8 CFU: 33-67mg, 10 8 CFU: 35-60mg, 10 8 CFU: 38-50mg, 10 8 CFU: 33-67 mg, etc. or any range of these scale points.

[0024] When the bifidobacterium in the probiotic composition is Bifidobacterium longum subspecies infantis R0033, the present invention provides a reference dosage ratio of bifidobacterium to lactoferrin of 10:1. 8 CFU: 0.1-100mg, 10 can be selected in specific applications 8 CFU: 0.1mg, 10 8 CFU: 0.3mg, 10 8 CFU: 0.5mg, 10 8 CFU: 0.8mg, you can also choose: but not limited to the following ratio range: 1×10 8 -2×10 8 CFU: 0.2-100mg; 1×10 8 -8×10 8 CFU: 0.8-100mg; 2×10 8 -9×10 8 CFU: 0.9-200mg; 3×10 8 -7×10 8 CFU: 0.7-300mg; 5×10 8 -7×10 8 CFU: 0.7-500mg; 6×10 8 -7×10 8 CFU: 0.7-600mg; 7×10 8 -9×10 8 CFU: 0.9-700mg; 8×10 8 -10×10 8 CFU: 0.1-800mg. As a preference, 1×10 8 -2×10 8CFU:1-100mg;1×10 8 -2×10 8 CFU:2-100mg;1×10 8 -2×10 8 CFU:3-100mg;1×10 8 -2×10 8 CFU:5-100mg;1.1×10 8 CFU:3-100mg、1.2×10 8 CFU:3-100mg、1.4×10 8 CFU:3-100mg、1.6×10 8 CFU:3-100mg、1.8×10 8 CFU:3-100mg、1.2×10 8 CFU:1mg、1.4×10 8 CFU:3mg、1.6×10 8 CFU:5mg、1.4×10 8 CFU:6mg、1.2×10 8 CFU:8.2mg、1.4×10 8 CFU:8.4mg、1.8×10 8 CFU:8.6mg、1.8×10 8 CFU:10mg、1.2×10 8 CFU:12mg、1.2×10 8 CFU:20mg、1.6×10 8 CFU:24mg、1.8×10 8 CFU:25mg、、10 8 CFU:31mg、10 8 CFU:31.4mg、10 8 CFU:32.8mg、10 8 CFU:33mg、10 8 CFU:385mg、10 8 CFU:38mg、10 8 CFU:50mg、10 8 CFU:56mg、10 8 CFU:56.2mg、10 8 CFU:56.8mg、1.2×10 8 CFU:26-30mg、1.4×10 8 CFU:33-67mg、1.8×10 8CFU: 100-200 mg, etc. or any range of these proportional points.

[0025] When the bifidobacterium in the probiotic composition is animal bifidobacterium lactis subspecies Bb12, the present invention provides a reference dosage ratio of bifidobacterium to lactoferrin of 10:1. 8 CFU: 0.1-100mg, 10 can be selected in specific applications 8 CFU: 0.1mg, 10 8 CFU: 0.3mg, 10 8 CFU: 0.5mg, 10 8 CFU: 0.8mg, you can also choose: but not limited to the following ratio range: 1×10 8 -2×10 8 CFU: 0.2-100mg; 1×10 8 -8×10 8 CFU: 0.8-100mg; 2×10 8 -9×10 8 CFU: 0.9-200mg; 3×10 8 -7×10 8 CFU: 0.7-300mg; 5×10 8 -7×10 8 CFU: 0.7-500mg; 6×10 8 -7×10 8 CFU: 0.7-600mg; 7×10 8 -9×10 8 CFU: 0.9-700mg; 8×10 8 -10×10 8 CFU: 0.1-800mg. As a preference, 6×10 8 -7×10 8 CFU: 0.7-600mg, such as 6×10 8 CFU: 0.7mg, 7×10 8 CFU: 600mg, 6.1×10 8 CFU: 0.7-600mg, 6.5×10 8 CFU: 0.7-600mg, 6.8×10 8 CFU: 0.7-600mg, 6.62×10 8 CFU: 0.7-600mg, 6.67×10 8 CFU: 0.7-600mg, 6.6×10 8 CFU: 1-100mg, 6.6×10 8CFU: 1 - 300 mg, 6.6×10 8 CFU: 1 - 600 mg, 6.1×10 8 CFU: 3 - 100 mg, 6.8×10 8 CFU: 100 - 200 mg, 6.6×10 8 CFU: 100 - 200 mg, 6.62×10 8 CFU: 3 - 100 mg, 6.65×10 8 CFU: 3 - 100 mg, 6.67×10 8 CFU: 3 - 100 mg, 6.69×10 8 CFU: 3 - 100 mg, 6.61×10 8 CFU: 100 - 200 mg, 6.63×10 8 CFU: 100 - 200 mg, 6.65×10 8 CFU: 100 - 200 mg, 6.67×10 8 CFU: 100 - 200 mg, 6.69×10 8 CFU: 100 - 200 mg, 6.67×10 8 CFU: 33 - 6​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​8 CFU: 50mg, 10 8 CFU: 56mg, 10 8 CFU: 56.2mg, 10 8 CFU: 56.8mg, 6.6×10 8 CFU: 3mg, 6.6×10 8 CFU: 5mg, 6.6×10 8 CFU: 33mg, 6.6×10 8 CFU: 31.4mg, 6.6×10 8 CFU: 32.8mg, 6.67×10 8 CFU: 3mg, 6.67×10 8 CFU: 6mg, 6.67×10 8 CFU: 8.4mg, 6.67×10 8 CFU: 33mg, 6.67×10 8 CFU: 31.4mg, 6.67×10 8 CFU: 32.8 mg or any range of these scale points.

[0026] In a specific embodiment, the bifidobacterium can be a combination of multiple bacteria. When used in combination, the total bacterial content is the same as that when a single bacteria is used.

[0027] In a specific embodiment, the bifidobacterium is a combination of Bifidobacterium breve HH079 and Bifidobacterium longum subsp. infantis R0033.

[0028] Preferably, the bacterial count ratio of the Bifidobacterium breve HH079 to the Bifidobacterium longum subspecies infantis R0033 may be 1-2:1-2.

[0029] More preferably, the bacterial count ratio of the Bifidobacterium breve HH079 to the Bifidobacterium longum subspecies infantis R0033 may be 1:1.

[0030] In a specific embodiment, the bifidobacterium can be a combination of Bifidobacterium breve HH079 and Bifidobacterium animalis subsp. lactis Bb12.

[0031] Preferably, the bacterial count ratio of the Bifidobacterium breve HH079 to the Bifidobacterium animalis subspecies lactis Bb12 may be 1-2:1-2.

[0032] More preferably, the bacterial count ratio of the Bifidobacterium breve HH079 to the Bifidobacterium animalis subsp. lactis Bb12 may be 1:1.

[0033] Specifically, the bifidobacteria can be any one or more of bacterial liquid, bacterial powder, and bacterial mud.

[0034] In another aspect, the present invention provides a method for preparing the aforementioned probiotic composition.

[0035] Specifically, the preparation method comprises mixing bifidobacteria and lactoferrin according to a usage ratio.

[0036] The preparation method may further include culturing bifidobacteria. As is well known in the art, the culturing may include: (1) inoculation after strain activation; and (2) conditioned culturing.

[0037] In another aspect, the present invention provides use of the aforementioned probiotic composition in preparing a product for regulating immunity.

[0038] The product is used to enhance immunity or restore immunity;

[0039] Or when the product is a medicine, it is used to treat or assist in the treatment of diseases caused by immunosuppression.

[0040] 184 In another aspect, the present invention provides a product of the aforementioned probiotic composition.

[0041] 185 Specifically, the products include food, health products or medicines.

[0042] 186 More specifically, the product contains not less than 10 7 CFU / g or 18710 7 CFU / mL of Bifidobacterium.

[0043] 188 Preferably, the product contains not less than 10 9 CFU / g or 189 10 9 CFU / mL of Bifidobacterium.

[0044] The product described in 190 can be a food, and the food also includes food excipients, and the excipients include but are not limited to: flavoring agents, colorants, fillers, disintegrants, sweeteners, lubricants, adhesives, and pH adjusters.

[0045] The product described in 193 can be a health product, which also includes health product excipients. The excipients 194 include but are not limited to: flavoring agents, colorants, fillers, disintegrants, sweeteners, lubricants, adhesives, and 195 pH regulators.

[0046] The product described in 196 can be a medicine, and the medicine also includes pharmaceutical excipients. The excipients 197 include but are not limited to: excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives, and 198 lubricants.

[0047] 199 Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl 200 cellulose, and mannitol.

[0048] 201 Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0049] 203 Specifically, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, 204 sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglyceryl-3 polyricinoleate.

[0050] 206 Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan 207 ester.

[0051] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose. Specifically, the binder is selected from at least one of ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinyl pyrrolidone.

[0052] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric cyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0053] Specifically, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer

[0054] Beneficial effects of the present invention:

[0055] The composition of bifidobacterium of the present invention and lactoferrin, by adjusting specific dosage ratio, has significant immunity enhancing effect in healthy Kunming mice, has good enhancing effect on mouse body weight, anti-inflammatory factors and immune proteins, and there is certain synergy between components. In immunosuppressive animal models, the present invention has also shown relatively better protective effect, showing that it has good immune restorative effect. It is particularly noted that the breve Bifidobacterium HH079 provided as an example in the bifidobacterium of the present invention is a strain screened by the present invention, which has shown better effect and wider application prospects. DETAILED DESCRIPTION

[0056] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0057] In the present invention, if there is a significant difference mark, it means that compared with the negative control, * means P < 0.05; ** means P < 0.01; *** means P < 0.001.

[0058] In the following embodiments:

[0059] Bifidobacterium breve HH079 is a self-screened strain of the present invention, which has been applied for separately. Its preservation number is GDMCC No: 64216, the preservation time is December 29, 2023, the preservation unit is Guangdong Provincial Microbiological Culture Collection Center, the taxonomic name is Bifidobacterium breve, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0060] Bifidobacterium longum subsp. infantis R0033 (Bifidobacterium longum subsp. Infantis) and Bifidobacterium animalis subsp. lactis Bb-12 (Bifidobacterium animalis subsp. lactis Bb-12) are strains disclosed in the prior art and can be obtained through conventional commercial means. Bifidobacterium longum subsp. infantis R0033 has the accession number SHBCC D24671 and was purchased from Shanghai Collection Biotechnology Center; Bifidobacterium animalis subsp. lactis Bb-12 has the accession number ATCC 27536.

[0061] Basic Example: Preparation of gavage reagents (bacteria strains and lactoferrin)

[0062] After conventional activation of the glycerol-stored strain, inoculate it into MRS solid medium and culture it in an anaerobic incubator at 37°C for 48-72 hours. Pick a single colony and inoculate it into MRS broth medium containing 0.05% L-cysteine ​​hydrochloride. After culture, use the LIVE / DEAD kit BacLight TM The number of viable bacteria was determined by Bacterial Viability and Counting Kit (L34856) and flow cytometry, and the viable bacteria content was adjusted to 10 10 CFU / mL, centrifuge and discard the culture medium, resuspend in PBS and use as the strain test sample for experiment.

[0063] Lactoferrin was administered orally in the form of physiological saline solution at a concentration of 0.6 g / L.

[0064] The present invention screens the combined application of multiple bacterial strains and lactoferrin, and the experimental design scheme is to gavage the bacterial suspension and lactoferrin into mice.

[0065] The experimental animals were healthy female SPF Kunming mice, weighing 20 ± 2 g, and housed in a standard manner. After 2 weeks of adaptive feeding, the mice were randomly divided into groups (n = 7). In addition to the experimental group, blank control, negative control, and single-ingredient control were set up. The grouping can be based on the following format:

[0066] Blank control: equal volume of pure water was administered orally;

[0067] Negative control: gavage with equal volume of PBS + saline;

[0068] Experimental group (multiple groups): oral administration of bacterial suspension and lactoferrin;

[0069] Single component control 1 (multiple groups): oral administration of bacterial suspension + normal saline;

[0070] Single component control 2: intragastric administration of PBS + lactoferrin.

[0071] The animals in each group were cultured under the same conditions.

[0072] Specific administration method: All groups were given oral gavage intervention treatment every day for 3 consecutive weeks. 12 hours after the last oral gavage, the mice were anesthetized and killed by overdose of sodium pentobarbital, and samples were collected.

[0073] Detection indicators and corresponding detection methods:

[0074] (1) Monitor the body weight of mice every 7 days and calculate the weight change: the last weight minus the first weight.

[0075] (2) Liver indices: Mouse livers were harvested, rinsed, and then sterile 0.9% saline solution was added to prepare a 10% (w / v) homogenate. Commercial kits were used to detect liver antioxidant indices including total antioxidant capacity (TAOC, ELISA, ml093083), glutathione peroxidase (GSH PX, ELISA, ml092941), and superoxide dismutase (SOD, ELISA, ml092619).

[0076] (2) Serum indicators: Blood was collected from the mouse orbits and centrifuged at 2000 g for 15 min. The supernatant was used as a sample and quantitatively detected using commercial kits for IL-6 (Solarbio, SEKM-0007), TNF-α (Solarbio, SEKM-0034), immunoglobulin IgG (ELISA, ml037601), and immunoglobulin IgA (ELISA, ml037606). Serum indicators IL-6 and TNF-α are anti-inflammatory factor indicators, while immunoglobulin IgG and immunoglobulin IgA are immunoglobulin indicators.

[0077] After screening a large amount of data in the early stage, it was found that the combination of Bifidobacterium + Lactoferrin showed better effects in the immunity of mouse models, thus obtaining the technical solution of the present invention.

[0078] The exemplary verification data of the technical solution of the present invention are shown in the examples.

[0079] For the blank control, negative control, and single component control 2, the corresponding data in the embodiments of the present invention are the same conditions, as follows:

[0080]

[0081] Example 1 Verification of the efficacy of Bifidobacterium breve HH079 and lactoferrin in mouse immunity

[0082] Gavage volume: 1 mL of bacterial suspension + 1 mL of lactoferrin solution.

[0083] Here are the results:

[0084]

[0085] By comparing with the control group, it can be seen that Bifidobacterium breve HH079 can effectively enhance the immunity of mice, and the effect is better when used in combination with lactoferrin, showing a weak synergistic effect.

[0086] Example 2 Verification of the efficacy of Bifidobacterium longum subsp. infantis R0033 and lactoferrin in mouse immunization. Oral administration volume: 1 mL of bacterial suspension + 1 mL of lactoferrin solution.

[0087] Here are the results:

[0088]

[0089]

[0090] By comparing with the control group, it can be seen that Bifidobacterium longum subspecies infantis R0033 can enhance the immunity of mice to a certain extent, and the effect is better when used in combination with lactoferrin, showing a good synergistic effect.

[0091] Example 3 Verification of the efficacy of animal Bifidobacterium lactis subsp. Bb12 and lactoferrin in mouse immunity Oral administration volume: 1 mL of bacterial suspension + 1 mL of lactoferrin solution.

[0092] Here are the results:

[0093]

[0094] By comparing with the control group, it can be seen that animal Bifidobacterium lactis subspecies Bb12 can enhance the immunity of mice to a certain extent, and the effect is better when used in combination with lactoferrin, showing a good synergistic effect.

[0095] Example 4 Verification of Bifidobacterium HH079 Dosage and Lactoferrin Dosage Adjustment

[0096] The dosage of Bifidobacterium breve HH079 and lactoferrin in the combination of Example 1 was adjusted to observe their efficacy in a mouse model.

[0097] Example 1 Bacterial suspension 1mL contains 10 10 CFU, 1 mL of lactoferrin solution contains 0.6 g of lactoferrin.

[0098] Dosage adjustment group:

[0099] (1) 1 mL of bacterial suspension contains 10 10 CFU, 1 mL of lactoferrin solution contains 0.3 g of lactoferrin;

[0100] (2) 1 mL of bacterial suspension contains 10 10 CFU, 1 mL of lactoferrin solution contains 0.84 g of lactoferrin;

[0101] (3) 1 mL of bacterial suspension contains 10 11 CFU, 1 mL of lactoferrin solution contains 0.6 g of lactoferrin;

[0102] (4) 1 mL of bacterial suspension contains 10 11 CFU, 1 mL of lactoferrin solution contains 0.84 g of lactoferrin;

[0103] (5) 1 mL of bacterial suspension contains 10 8 CFU, 1 mL of lactoferrin solution contains 0.6 g of lactoferrin;

[0104] (6) 1 mL of bacterial suspension contains 10 8 CFU, 1 mL of lactoferrin solution contains 0.84 g of lactoferrin;

[0105] (7) 1 mL of bacterial suspension contains 10 10 CFU, 1 mL of lactoferrin solution contains 6.7 g of lactoferrin;

[0106] Here are the results:

[0107]

[0108] The above results show that in 10 9 CFU: Within the dosage range of 1-10 g, the composition has a better effect.

[0109] Example 5 Adjustment of the dosage of Bifidobacterium longum subsp. infantis R0033, Bifidobacterium animalis subsp. lactis Bb12 and lactoferrin

[0110] Referring to Examples 2-3, this Example set up two groups of experiments, and adjusted the combined dosage of Bifidobacterium longum subsp. infantis R0033 and lactoferrin (8) and the combined dosage of Bifidobacterium longum subsp. infantis R0033 and lactoferrin (9), respectively. The specific differences are as follows:

[0111] (8) Referring to Example 2, the dosage concentration of Bifidobacterium longum subsp. infantis R0033 was adjusted to 1.40×10 10 CFU / mL;

[0112] (9) Referring to Example 3, the dosage concentration of Bifidobacterium animalis subsp. lactis Bb12 was adjusted to 6.67×10 10 CFU / mL;

[0113] Other conditions remain unchanged.

[0114] Here are the results:

[0115]

[0116] According to Example 2 and (8) of this example, it can be seen that the combination of Bifidobacterium longum subsp. infantis R0033 and lactoferrin is 1-1.40×10 10 CFU: Within the dosage ratio of 0.6g, the probiotic composition has a good effect.

[0117] According to Example 3 and (9) of this example, it can be seen that the combination of animal Bifidobacterium lactis subspecies Bb12 and lactoferrin is 1-6.67×10 10 CFU: Within the range of 0.6 g, the probiotic compositions all had good effects.

[0118] Comparative Example 1: Verification of the efficacy of Lactobacillus acidophilus and lactoferrin in mouse immunity

[0119] Referring to Example 1, Bifidobacterium breve HH079 was replaced by Lactobacillus acidophilus ATCC 4356.

[0120] Here are the results:

[0121]

[0122] Comparative Example 2: Verification of the efficacy of Lactobacillus plantarum and lactoferrin in mouse immunity

[0123] Referring to Example 1, Bifidobacterium breve HH079 was replaced by Lactobacillus plantarum ATCC 8014.

[0124] Here are the results:

[0125]

[0126] From Comparative Examples 1-2, it can be seen that after replacing with other types of probiotics, the performance of the composition in improving the immune function of mice deteriorates, indicating that the combination of bifidobacteria and lactoferrin has a better effect on immunity enhancement.

[0127] Experimental Example Effect of Lactoferrin-Containing Probiotic Composition on Immune Recovery

[0128] It is well known to those skilled in the art that DNCB can induce immune inflammatory response. Based on the prior art, an immunosuppression model was constructed to verify the effect of the composition of the present invention on immune recovery.

[0129] Eight-week-old female C57BL / 6 mice were used as experimental animals and administered via gavage. The day of gavage initiation was designated as D0. An immunosuppressive model was established based on the administration method. The experimental grouping was as follows:

[0130] Blank group: normal culture, daily gavage with normal saline during the experiment.

[0131] Model group: 1% DNCB (dinitrochlorobenzene) 100 μL was injected subcutaneously on D0, and 200 mg / kg cyclophosphamide (CTX) was injected intraperitoneally; 1% DNCB 10 μL was applied to both ears for sensitivity enhancement on D1; 2% DNCB 20 μL was applied to the right ear on D4; normal saline was administered daily during the experiment.

[0132] Experimental group: Modeling was performed with reference to the model group, and the normal saline was replaced by the compositions of Examples 1-3 and Comparative Examples 1-2.

[0133] Single component control 1: oral administration of Bifidobacterium HH079 suspension + normal saline;

[0134] Single component control 2: intragastric administration of PBS + lactoferrin.

[0135] Unless otherwise specified, the dosage volume was kept consistent across all groups.

[0136] On the 6th day after administration, the mice were killed, and ear pieces from the same part of both ears were taken and weighed. The weight difference was used to calculate the protection rate, protection rate = (mean value of the drug administration group - mean value of the model group) / mean value of the model group × 100%.

[0137] The statistical results are as follows:

[0138]

[0139]

[0140] The above results show that the composition of the present invention has a better protective effect on immunosuppression recovery than the combination containing other strains (Comparative Examples 1-2), and the combination of Bifidobacterium and lactoferrin has a certain synergistic effect on immunosuppression recovery in mice.

[0141] The above embodiments are only used to illustrate the present invention and are not used to limit the present invention. Technical solutions obtained by replacing and optimizing conventional means performed by those skilled in the art on the basis of the present invention are also within the scope of protection of the present invention.

Claims

1. A probiotic composition containing lactoferrin, characterized in that Composed of bifidobacteria and lactoferrin.

2. The probiotic composition according to claim 1, characterized in that The bifidobacterium includes any one or more of Bifidobacterium breve, Bifidobacterium longum subsp. infantis, and Bifidobacterium animalis subsp. lactis.

3. The probiotic composition according to claim 2, characterized in that The Bifidobacterium breve includes Bifidobacterium breve HH079, with a preservation number of GDMCC No: 64216; the Bifidobacterium longum subsp. infantis includes Bifidobacterium longum subsp. infantis R0033, with a preservation number of SHBCC D24671; and the Bifidobacterium animalis subsp. lactis includes Bifidobacterium animalis subsp. lactis Bb-12, with a preservation number of DSM15954.

4. The probiotic composition according to claim 1, characterized in that The dosage ratio of bifidobacteria and lactoferrin is 10 9 CFU: 0.001-10g.

5. The probiotic composition according to claim 4, characterized in that The dosage ratio of bifidobacteria and lactoferrin is 10 8 CFU: 1-100mg.

6. The probiotic composition according to claim 1, characterized in that The bifidobacterium is any one or more of bacterial liquid, bacterial powder and bacterial mud.

7. The method for preparing the probiotic composition according to any one of claims 1 to 6, characterized in that: The method comprises mixing bifidobacteria and lactoferrin according to a usage ratio.

8. Use of the probiotic composition according to any one of claims 1 to 6 in the preparation of a product for regulating immunity.

9. The use according to claim 8, characterized in that The product is used to improve immunity or immune recovery; or when the product is a medicine, it is used to treat or assist in the treatment of diseases caused by reduced immunity.

10. A product comprising the probiotic composition according to any one of claims 1 to 6, characterized in that The products include food, health products or medicines.

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