CN117981875B - Lactobacillus reuteri Gly LR15 composition with immunity improving function and preparation method thereof - Google Patents
Lactobacillus reuteri Gly LR15 composition with immunity improving function and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a lactobacillus reuteri Glory LR15 composition with immunity improving function and a preparation method thereof, wherein the lactobacillus reuteri Glory LR15 composition consists of probiotic freeze-dried powder and functional extract, the probiotic freeze-dried powder consists of lactobacillus reuteri Glory LR15 freeze-dried powder, lactobacillus paracasei Glory LP16 freeze-dried powder, lactobacillus rhamnosus Glory LG12 freeze-dried powder and lactobacillus grignard freeze-dried powder, and the functional extract consists of cactus extract, acanthopanax extract and oat beta-glucan. The lactobacillus reuteri glass LR15 composition can improve the proliferation of probiotics in intestinal tracts through the synergistic effect between the probiotic freeze-dried powder and the functional extract, regulate the flora in the intestinal tracts, further enhance the immunity of human bodies, and supplement the probiotic freeze-dried powder and the functional extract, so that the lactobacillus reuteri glass LR15 composition enhances the humoral immunity function of mice immunosuppressed by cyclophosphamide, and remarkably improves the immunity of the mice.
Description
Technical Field
The invention belongs to the technical field of microbial agents, and particularly relates to a lactobacillus reuteri Glory LR15 composition with an immunity improving function and a preparation method thereof.
Background
Immunity is the ability of the body to recognize and eliminate foreign invasion foreign bodies (viruses, bacteria, etc.), treat cells such as aging, injury, death, denaturation, mutation, virus infection, etc., and is a physiological response of the body excluding "abnormal", and is also a defense mechanism of the body itself. The normal body is under the action of the immune system, and the mutated cells are inhibited and decomposed. However, in the case of the population with low immunity, the immunocompetent cells cannot recognize the mutant cells, so that the mutant cells are randomly propagated, and finally, the disease is caused. Especially children, because of the imperfect physiological structure and function development, are more easily affected by environmental pathogens and the adverse effects of parents on the use of antibiotics by children in order to prevent infection, and the resistance to certain diseases needs to be improved by means of external immune preparations. Therefore, improving the immunity of the body, preventing the biological factors or allergic infection, finding safer microecological food with less side effects, and becoming a topic for urgent solving at present.
With the acceleration of the life rhythm of people, the phenomena of weak constitution, malnutrition, low immunity and the like occur due to uneven diet, large working pressure, lack of exercise and the like. Treatment is usually carried out by supplementing various vitamins and amino acids, but after the treatment is stopped, the immunity is reduced, and especially for children, the physiological structure and function are not perfect, the children are more vulnerable to pathogenic bacteria, and food allergy is easy to occur.
The immune system of the human body exists in the intestinal tract about 70%, and a large number of immune cells such as T cells, B cells, NK cells and the like of the human body are concentrated in intestinal mucosa, and the intestinal tract is the first defense system of the immunity. The intestinal tract serves as a site for digestion and absorption of nutrients, colonization of microorganisms and aggregation of immune cells, and promotes interaction among the three. The normal intestinal flora plays an important role in promoting the development of the immune system, maintaining normal immune function and synergistically antagonizing invasion of pathogenic bacteria. Therefore, when the intestinal flora is disturbed, the resistance of the human body is weakened and is easy to be attacked by germs, and the prevalence rate of diseases such as influenza, cold, gastroenteritis and the like is increased.
The probiotics can inhibit pathogenic bacteria and toxins from adhering and inhibit or resist the growth of pathogenic bacteria and other microorganisms by occupying effect, nutrition competition, secretion of antibacterial or bactericidal substances, generation of organic acid, stimulation of secretion of secretory immunoglobulin and the like, so as to improve dysbacteriosis. By constituting a biological barrier, the intestinal tract harmful bacteria and endotoxin are prevented from being displaced, and the probiotics can also synthesize more than 130 vitamins required by human body. Probiotics can balance local microbiota by inhibiting the growth of pathogenic microorganisms, or stimulate intestinal mucosa and intestinal epithelial cell metabolism by secreting various enzymes and acids, thereby enhancing local and systemic immune responses. Thus, or licensed to start with intestinal microorganisms, attempts have been made to find new methods of modulating immunity to overcome the drawbacks of the existing therapeutic agents and the side effects of the therapeutic methods.
The Chinese patent application number 202211522999.7 discloses Luo Yishi lactobacillus LL029 for improving immunity and application thereof, and the lactobacillus reuteri LL029 has good gastrointestinal fluid resistance and cholate resistance, can effectively maintain the immune balance of a host organism, improves the immunity of the host, and can be used for preparing foods or medicines for regulating the immunity of the organism. However, there is a limited improvement in immunity, and there is room for further improvement, in which only a single strain, luo Yishi, of Lactobacillus is used.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a lactobacillus reuteri Glory LR15 composition with an immunity improving function and a preparation method thereof.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the lactobacillus reuteri Glory LR15 composition with the immunity improving function comprises probiotic freeze-dried powder and functional extract, wherein the probiotic freeze-dried powder is formed by mixing lactobacillus reuteri Glory LR15 freeze-dried powder, lactobacillus paracasei Glory LP16 freeze-dried powder, lactobacillus rhamnosus Glory LG12 freeze-dried powder and lactobacillus grignard freeze-dried powder, and the functional extract comprises cactus extract, acanthopanax extract and oat beta-glucan.
Preferably, the mass ratio of the lactobacillus reuteri Glory LR15 freeze-dried powder, the lactobacillus paracasei Glory LP16 freeze-dried powder to the lactobacillus rhamnosus Glory LG12 freeze-dried powder to the lactobacillus gasseri freeze-dried powder is 10-15:5-7:6-8:2-4.
Preferably, the mass ratio of the cactus extract to the acanthopanax extract to the oat beta-glucan is 30-40:20-30:10-20.
Preferably, the preparation method of the cactus extract comprises the following steps:
cleaning radix et caulis Opuntiae Dillenii, mashing, adding deionized water, adding citric acid, extracting with water, filtering to obtain water extractive solution and residue, concentrating the water extractive solution under reduced pressure to 1/3 of the volume, adding 4 times of absolute ethanol for precipitating, centrifuging, removing supernatant, and drying the precipitate to obtain water extract; adding the filter residue into 80% ethanol solution by mass fraction, performing ultrasonic extraction, suction filtering, distilling under reduced pressure to remove the solution, and distilling under reduced pressure at 40kpa-50kpa and 75-80deg.C to obtain ethanol extract; mixing the water extract and the alcohol extract uniformly to obtain the final product.
Preferably, the mass ratio of the cactus to the deionized water to the citric acid is 10:150-200:1-2, the water extraction temperature is 70-80 ℃ and the time is 1-2h; the feed liquid ratio of the filter residue to the ethanol solution is 1:12-18; the temperature of the ultrasonic extraction is 60-70 ℃, the time is 30-50min, and the ultrasonic power is 200-300W; the mass ratio of the water extract to the alcohol extract is 5:1-3.
Preferably, the preparation method of the acanthopanax root extract comprises the following steps:
Pulverizing radix Acanthopanacis Senticosi, sieving with 60 mesh sieve, adding deionized water, adjusting pH to 4.5-5.5, adding cellulase and beta-glucosidase, performing enzymolysis, filtering after enzymolysis, and drying the residue to obtain pretreated radix Acanthopanacis Senticosi powder; adding pretreated acanthopanax powder into ethanol solution with the volume fraction of 70%, then adding lecithin, carrying out alcohol extraction, and filtering after the alcohol extraction is finished to obtain filtrate; concentrating the filtrate under reduced pressure to remove solvent, concentrating under reduced pressure of 40kpa-50kpa at 75-80deg.C.
Preferably, the mass ratio of the acanthopanax to the deionized water is 10:70-100, the dosage of the cellulase is 0.3-0.6% of the mass of the acanthopanax, the dosage of the beta-glucosidase is 0.2-0.4% of the mass of the acanthopanax, and the enzymolysis temperature is 30-35 ℃ and the time is 2-3 hours.
Preferably, the feed liquid ratio of the pretreated acanthopanax powder to the ethanol solution is 1:10-15, the dosage of lecithin is 0.5-1% of the mass of the pretreated acanthopanax powder, the ethanol extraction temperature is 55-65 ℃ and the time is 2-3h.
Preferably, the content of the probiotics freeze-dried powder in the lactobacillus reuteri Glory LR15 composition is 25-35wt%.
The invention also provides an application of the Lactobacillus reuteri Gly LR15 composition with the immunity enhancing function in preparing a product with the immunity enhancing function.
Compared with the prior art, the invention has the following beneficial effects:
(1) The lactobacillus reuteri glass LR15 composition with the immunity improving function is prepared by mixing lactobacillus reuteri glass LR15 freeze-dried powder, lactobacillus paracasei glass LP16 freeze-dried powder, lactobacillus rhamnosus glass LG12 freeze-dried powder and lactobacillus griseus freeze-dried powder, wherein the glass LR15 has better tolerance to gastrointestinal tract environment and has certain tolerance to artificial gastric juice, intestinal juice and bile salt; lactobacillus rhamnosus Glory LP16 has strong survival and reproductive ability under the low pH condition of gastric acid and bile, is easy to colonize, plays a role in intestinal tracts, and has high immune activity; the lactobacillus paracasei Glory LP16 is a strain with probiotic characteristics, and ingestion of the lactobacillus paracasei can achieve the effect of reducing the immune activation of intestinal stress syndrome by regulating the secretion of cytokines and reducing the activity of pro-inflammatory cytokines, and meanwhile, the lactobacillus paracasei can decompose proteins to generate small molecular peptides, thereby playing an important role in reducing blood pressure, resisting cholesterol, resisting oxidization and the like; the four strains act together, so that the probiotic bacterium has better immunity improving effect compared with a single probiotic bacterium, and simultaneously the probiotic bacterium can promote proliferation of the probiotic bacterium, inhibit certain harmful bacteria and improve colonization of the probiotic bacterium in intestinal tracts by assisting with the functional extract, thereby improving human health.
(2) The lactobacillus reuteri Glory LR15 composition with the immunity improving function is added with the cactus extract, and the main substance polysaccharide in the water extract is compounded with the main substances such as the glucosides, the isoquercitrins and the triterpene compounds of isorhamnetin and quercetin in the alcohol extract, so that the lactobacillus reuteri Glory LR15 composition has the effects of immunoregulation, antioxidation, anti-inflammation, liver protection, anti-tumor, blood lipid and blood sugar reduction and the like; the added acanthopanax extract is subjected to enzymolysis treatment, so that the content and the extraction rate of active substances such as acanthopanax polysaccharide, terpenes, lignans, flavonoids, coumarins and the like in the acanthopanax can be effectively improved, the active ingredients can play an anti-inflammatory role while enhancing the immunity of an organism, and lecithin is added as a surface active agent in the extraction process, so that the solubilization effect can be achieved, and the extraction rate of the active substances is further improved; the added oat beta-glucan has various health effects of losing weight, reducing blood sugar, reducing blood fat, improving cardiovascular diseases, resisting tumors, improving immunity and the like, can improve the immunity together with the cactus extract and the acanthopanax extract, and can promote the proliferation of probiotics.
(3) The lactobacillus reuteri Glory LR15 composition with the immunity improving function provided by the invention can improve the proliferation of probiotics in intestinal tracts through the synergistic effect between the probiotics freeze-dried powder and the functional extract, regulate the flora in the intestinal tracts, further strengthen the immunity of human bodies, and the probiotics freeze-dried powder and the functional extract supplement each other, so that the lactobacillus reuteri Glory LR15 composition can strengthen the humoral immunity function of mice immunosuppressed by cyclophosphamide, and remarkably improve the immunity of the mice.
Drawings
FIG. 1 is a graph showing the effect of a Lactobacillus reuteri Glory LR15 composition having an immune enhancing function on the immune organ index of mice in accordance with the present invention; fig. 1A shows thymus index, fig. 1B shows spleen index, P <0.05, P <0.01, compared to the blank; #p <0.05, #p <0.01, compared to model group.
FIG. 2 is a graph showing the effect of a Lactobacillus reuteri Gly LR15 composition having immunity enhancing function on cellular immune function in mice in accordance with the present invention; fig. A2 is the optical density difference, fig. 2B is the plantar thickness, P <0.05, P <0.01, compared to the blank; #p <0.05, #p <0.01, compared to model group.
FIG. 3 is a graph showing the effect of a Lactobacillus reuteri Gly LR15 composition having immunity enhancing function on humoral immunity in mice according to the present invention; fig. 3A shows the number of lysoplaques, fig. 3B shows the number of antibody products, P <0.05, P <0.01, compared to the blank; #p <0.05, #p <0.01, compared to model group.
Fig. 4 shows the effect of lactobacillus reuteri Glory LR15 composition with immunity enhancing function on nonspecific immunity of mice according to the present invention fig. 4A shows NK cell activity, fig. 4B shows carbon clearance phagocytosis index, fig. 4C shows neutral red phagocytosis ability of peritoneal macrophages, fig. 4D shows phagocytosis index, P <0.05, P <0.01, compared to the blank group; #p <0.05, #p <0.01, compared to model group.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The strain provided by the invention is named as lactobacillus reuteri Glory LR15 in a classification way: lactobacillus reuteri Limosilactobacillus reuteri, deposited in the China general microbiological culture collection center (CGMCC), address: the collection date is 2023, 3 and 31 days of the national academy of sciences of China, the Qingyang area North Star, the West way No. 1, the No. 3 and the collection number is CGMCC No.26972.
The strain provided by the invention, lactobacillus rhamnosus Glory LG12, is classified and named as: lactobacillus rhamnosus Lactobacillus rhamnosus, deposited in the China general microbiological culture Collection center (CGMCC), address: north Star Xili No. 1, 3 of the Chaoyang district of Beijing, china academy of sciences microbiological study, 9 months of 2022, and CGMCC No.25668.
The strain provided by the invention, lactobacillus paracasei Glory LP16, is classified and named as: lactobacillus paracasei Lactobacillus paracasei deposited in China general microbiological culture Collection center (CGMCC), address: the collection date is 2022, 9 and 9 days in the China academy of sciences of China, the No. 1 and No. 3 of the West Lu of the Chaoyang district North Star of Beijing, and the collection number is CGMCC No.25669.
The lactobacillus gasseri lyophilized powder is purchased from Guangdong Hongyu biotechnology limited company, and the viable count is more than or equal to 1 multiplied by 10 10 cfu/g.
The following media are referred to as follows: the seed culture medium is as follows: 10g/L of glucose, 50g/L of sucrose, 13g/L of yeast extract, 6g/L of sodium acetate, 5g/L of diammonium hydrogen citrate, 0.5g/L of magnesium sulfate heptahydrate, 0.5g/L of manganese sulfate monohydrate, and pH=6.2; the fermentation medium is as follows: 15g/L of glucose, 75g/L of sucrose, 45g/L of yeast extract, 6g/L of sodium acetate, 4g/L of diammonium hydrogen citrate, 0.6 g/L of magnesium sulfate heptahydrate, 0.3 g/L of manganese sulfate monohydrate and pH=6.2.
The preparation of each probiotic freeze-dried powder in the following examples is as follows:
Preparation of lactobacillus reuteri Glory LR15 freeze-dried powder:
(1) Liquid submerged fermentation culture: inoculating lactobacillus reuteri Glory LR15 deposited strain into 300mL of seed culture medium with an inoculum size of 2%, standing and culturing in a CO 2 incubator at 37 ℃ and 5% for 24 hours, inoculating into 800mL of seed culture medium with an inoculum size of 3%, standing and culturing in a CO 2 incubator at 37 ℃ and 5% for about 10 hours, inoculating into 200mL of fermentation culture medium with an inoculum size of 4%, anaerobic culturing at 35-37 ℃ for 8-12 hours, and collecting fermentation liquor, wherein the thallus density in the fermentation liquor reaches more than 10 10 cfu/mL;
(2) Preparation of a freeze-dried bacterial agent: when the temperature of the lactobacillus reuteri Glory LR15 fermentation liquor is reduced to about 20 ℃, centrifugally collecting bacterial sludge, cooling a centrifugal bacterial body pipeline, obtaining bacterial sludge after centrifugation, adding 250g of skim milk powder, 100g of sucrose and 80g of trehalose as a freeze-drying protective agent, uniformly mixing by 1L of distillation, uniformly spreading the mixture on a tray, placing the tray on a baffle, placing a temperature probe in the material, freeze-drying, collecting bacterial powder, and storing in a refrigerator at the temperature of minus 20 ℃ to obtain the lactobacillus reuteri Glory LR15 freeze-drying bacterial powder, wherein the viable count is 10 10 cfu/g.
Preparation of lactobacillus rhamnosus Glory LG12 freeze-dried powder:
(1) Liquid submerged fermentation culture: inoculating lactobacillus rhamnosus Glory LG12 deposited strain into 300mL of seed culture medium with an inoculum size of 2%, standing and culturing in a CO 2 incubator at 37 ℃ and 5%, inoculating into 800mL of seed culture medium with an inoculum size of 3% after 24 hours, standing and culturing in a CO 2 incubator at 37 ℃ and 5%, inoculating into 200mL of fermentation culture medium with an inoculum size of 4% after about 10 hours of culture, anaerobic culturing at 35-37 ℃ for 8-12 hours, and collecting fermentation liquor, wherein the thallus density in the fermentation liquor reaches more than 10 10 cfu/mL;
(2) Preparation of a freeze-dried bacterial agent: after the temperature of lactobacillus rhamnosus Glory LG12 fermentation broth is reduced to about 20 ℃, centrifugally collecting bacterial mud, cooling a centrifugal bacterial body pipeline, obtaining bacterial mud after centrifugation, adding 250g of skimmed milk powder, 100g of sucrose and 80g of trehalose as a freeze-drying protective agent, uniformly mixing by distillation with 1L of the solution, uniformly spreading the mixture on a tray, placing the tray on a partition plate, placing a temperature probe in the material, freeze-drying, collecting bacterial powder, and storing in a refrigerator at the temperature of minus 20 ℃ to obtain lactobacillus rhamnosus Glory LG12 freeze-drying bacterial powder, wherein the viable count of the lactobacillus is 10 10 cfu/g.
Preparation of lactobacillus paracasei Glory LP16 lyophilized powder:
(1) Liquid submerged fermentation culture: inoculating lactobacillus paracasei Glory LP16 deposited strain into 300mL of seed culture medium with an inoculum size of 2%, standing and culturing in a CO 2 incubator at 37 ℃ and with an inoculum size of 3% after 24 hours, standing and culturing in a CO 2 incubator at 37 ℃ and with an inoculum size of 5% for about 10 hours, inoculating into 200mL of fermentation culture medium with an inoculum size of 4% after culturing, anaerobic culturing at 35-37 ℃ for 8-12 hours, and collecting fermentation liquor, wherein the thallus density in the fermentation liquor reaches more than 10 10 cfu/mL;
(2) Preparation of a freeze-dried bacterial agent: after the temperature of lactobacillus paracasei Glory LP16 fermentation broth is reduced to about 20 ℃, centrifugally collecting bacterial mud, cooling a centrifugal bacterial body pipeline, obtaining bacterial mud after centrifugation, adding 250g of skimmed milk powder, 100g of sucrose and 80g of trehalose as a freeze-drying protective agent, uniformly mixing by distillation with 1L of the solution, uniformly spreading the mixture on a tray, placing the tray on a partition plate, placing a temperature probe in the material, freeze-drying, collecting bacterial powder, and storing in a refrigerator at the temperature of minus 20 ℃ to obtain lactobacillus paracasei Glory LP16 freeze-dried bacterial powder, wherein the viable count of the bacterial powder is 10 10 cfu/g.
Example 1
A preparation method of a lactobacillus reuteri Glory LR15 composition with immunity improving function, which comprises the following steps:
And uniformly mixing the probiotics freeze-dried powder and the functional extract, wherein the probiotics freeze-dried powder is prepared by mixing 13g of lactobacillus reuteri Glory LR15 freeze-dried powder, 7g of lactobacillus paracasei Glory LP16 freeze-dried powder, 6g of lactobacillus rhamnosus Glory LG12 freeze-dried powder and 2g of lactobacillus grignard freeze-dried powder, and the functional extract consists of 35g of cactus extract, 25g of acanthopanax extract and 15g of oat beta-glucan.
The preparation method of the cactus extract comprises the following steps:
Cleaning 20g of cactus, mashing, adding 350g of deionized water, adding 3g of citric acid, extracting with water at 75 ℃ for 1.5h, filtering after the water extraction is finished to obtain water extract and filter residues, concentrating the water extract under reduced pressure to 1/3 of the volume, adding 4 times of absolute ethyl alcohol for alcohol precipitation, centrifuging, removing supernatant, and drying the precipitate to obtain a water extract; adding 10g of filter residues into 150g of 80% ethanol solution, performing ultrasonic extraction at 65deg.C and ultrasonic power of 250W for 40min, performing suction filtration, distilling under reduced pressure to remove the solution, and performing distillation under reduced pressure of 40kpa at 75deg.C to obtain ethanol extract; mixing 5g of water extract and 2g of alcohol extract uniformly to obtain the final product.
The preparation method of the acanthopanax extract comprises the following steps:
Crushing 10g of acanthopanax, sieving with a 60-mesh sieve, adding 90g of deionized water, adjusting the pH to 5, then adding 0.05g of cellulase and 0.03g of beta-glucosidase, carrying out enzymolysis for 2.5 hours at 33 ℃, filtering after the enzymolysis is finished, and drying filter residues to obtain pretreated acanthopanax powder; adding 10g of pretreated acanthopanax powder into 130g of ethanol solution with the volume fraction of 70%, then adding 0.08g of lecithin, extracting with ethanol at 60 ℃ for 2.5h, and filtering after the ethanol extraction is finished to obtain filtrate; concentrating the filtrate under reduced pressure to remove solvent, concentrating under reduced pressure of 40kpa at 75deg.C.
Example 2
A preparation method of a lactobacillus reuteri Glory LR15 composition with immunity improving function, which comprises the following steps:
And uniformly mixing the probiotics freeze-dried powder and the functional extract, wherein the probiotics freeze-dried powder is prepared by mixing 10g of lactobacillus reuteri Glory LR15 freeze-dried powder, 5g of lactobacillus paracasei Glory LP16 freeze-dried powder, 8g of lactobacillus rhamnosus Glory LG12 freeze-dried powder and 2g of lactobacillus grignard freeze-dried powder, and the functional extract consists of 30g of cactus extract, 20g of acanthopanax extract and 20g of oat beta-glucan.
The preparation method of the cactus extract comprises the following steps:
Cleaning 20g of cactus, mashing, adding 300g of deionized water, adding 2g of citric acid, extracting with water at 80 ℃ for 1h, filtering after the water extraction is finished to obtain water extract and filter residues, concentrating the water extract under reduced pressure to 1/3 of the volume, adding 4 times of absolute ethyl alcohol for alcohol precipitation, centrifuging, removing supernatant, and drying the precipitate to obtain a water extract; adding 10g of filter residues into 120g of 80% ethanol solution, performing ultrasonic extraction at 60 ℃ and ultrasonic power of 300W for 50min, performing suction filtration, and performing reduced pressure distillation to remove the solution, wherein the reduced pressure distillation pressure is 50kpa, and the temperature is 80 ℃ to obtain an ethanol extract; mixing 5g of water extract and 1g of alcohol extract uniformly to obtain the final product.
The preparation method of the acanthopanax extract comprises the following steps:
Crushing 10g of acanthopanax, sieving with a 60-mesh sieve, adding 70g of deionized water, adjusting the pH to 4.5, adding 0.03g of cellulase and 0.04g of beta-glucosidase, carrying out enzymolysis for 3 hours at 30 ℃, filtering after the enzymolysis is finished, and drying filter residues to obtain pretreated acanthopanax powder; adding 10g of pretreated acanthopanax powder into 100g of ethanol solution with the volume fraction of 70%, then adding 0.05g of lecithin, carrying out alcohol extraction for 3h at 55 ℃, and filtering after the alcohol extraction is finished to obtain filtrate; concentrating the filtrate under reduced pressure to remove solvent, concentrating under reduced pressure of 40kpa at 75deg.C.
Example 3
A preparation method of a lactobacillus reuteri Glory LR15 composition with immunity improving function, which comprises the following steps:
And uniformly mixing the probiotics freeze-dried powder and the functional extract, wherein the probiotics freeze-dried powder is prepared by mixing 15g of lactobacillus reuteri Glory LR15 freeze-dried powder, 5g of lactobacillus paracasei Glory LP16 freeze-dried powder, 8g of lactobacillus rhamnosus Glory LG12 freeze-dried powder and 4g of lactobacillus grignard freeze-dried powder, and the functional extract consists of 40g of cactus extract, 30g of acanthopanax extract and 10g of oat beta-glucan.
The preparation method of the cactus extract comprises the following steps:
cleaning 20g of cactus, mashing, adding 400g of deionized water, adding 4g of citric acid, extracting with water at 70 ℃ for 2 hours, filtering after the water extraction is finished to obtain water extract and filter residues, concentrating the water extract under reduced pressure to 1/3 of the volume, adding 4 times of absolute ethyl alcohol for alcohol precipitation, centrifuging, removing supernatant, and drying the precipitate to obtain a water extract; adding 10g of filter residues into 180g of 80% ethanol solution, performing ultrasonic extraction at 70deg.C and ultrasonic power of 200W for 30min, performing suction filtration, and distilling under reduced pressure to remove the solution at 50kpa and 80deg.C to obtain ethanol extract; mixing 5g of water extract and 3g of alcohol extract uniformly to obtain the final product.
The preparation method of the acanthopanax extract comprises the following steps:
Crushing 10g of acanthopanax, sieving with a 60-mesh sieve, adding 100g of deionized water, adjusting the pH to 5.5, adding 0.06g of cellulase and 0.02g of beta-glucosidase, carrying out enzymolysis for 2 hours at 35 ℃, filtering after the enzymolysis is finished, and drying filter residues to obtain pretreated acanthopanax powder; adding 10g of pretreated acanthopanax powder into 150g of ethanol solution with the volume fraction of 70%, then adding 0.1g of lecithin, carrying out alcohol extraction for 2 hours at 65 ℃, and filtering after the alcohol extraction is finished to obtain filtrate; concentrating the filtrate under reduced pressure to remove solvent, concentrating under reduced pressure of 40kpa at 75deg.C.
Comparative example 1
A preparation method of a lactobacillus reuteri Glory LR15 composition with immunity improving function, which comprises the following steps:
And uniformly mixing 13g of lactobacillus reuteri Glory LR15 freeze-dried powder, 7g of lactobacillus paracasei Glory LP16 freeze-dried powder, 6g of lactobacillus rhamnosus Glory LG12 freeze-dried powder and 2g of lactobacillus gasseri freeze-dried powder.
Comparative example 2
A preparation method of a composition with the function of improving immunity comprises the following steps:
Uniformly mixing the probiotics freeze-dried powder and the functional extract, wherein the probiotics freeze-dried powder is prepared by mixing 20g of lactobacillus paracasei Glory LP16 freeze-dried powder and 8g of lactobacillus grignard freeze-dried powder, and the functional extract consists of 35g of cactus extract, 25g of acanthopanax extract and 15g of oat beta-glucan.
The preparation method of the cactus extract comprises the following steps:
cleaning 20g of cactus, mashing, adding 350g of deionized water, adding 3g of citric acid, extracting with water at 75 ℃ for 1.5h, filtering after the water extraction is finished to obtain water extract and filter residues, concentrating the water extract under reduced pressure to 1/3 of the volume, adding 4 times of absolute ethyl alcohol for alcohol precipitation, centrifuging, removing supernatant, and drying the precipitate to obtain a water extract; adding 10g of filter residues into 150g of 80% ethanol solution, performing ultrasonic extraction at 65deg.C and ultrasonic power of 250W for 40min, performing suction filtration, distilling under reduced pressure to remove the solution, and performing distillation under reduced pressure of 50kpa at 80deg.C to obtain ethanol extract; mixing 5g of water extract and 2g of alcohol extract uniformly to obtain the final product.
The preparation method of the acanthopanax extract comprises the following steps:
Crushing 10g of acanthopanax, sieving with a 60-mesh sieve, adding 90g of deionized water, adjusting the pH to 5, then adding 0.05g of cellulase and 0.03g of beta-glucosidase, carrying out enzymolysis for 2.5 hours at 33 ℃, filtering after the enzymolysis is finished, and drying filter residues to obtain pretreated acanthopanax powder; adding 10g of pretreated acanthopanax powder into 130g of ethanol solution with the volume fraction of 70%, then adding 0.08g of lecithin, extracting with ethanol at 60 ℃ for 2.5h, and filtering after the ethanol extraction is finished to obtain filtrate; concentrating the filtrate under reduced pressure to remove solvent, concentrating under reduced pressure of 40kpa at 75deg.C.
Comparative example 3
A preparation method of a lactobacillus reuteri Glory LR15 composition with immunity improving function, which comprises the following steps:
and uniformly mixing the probiotics freeze-dried powder and the functional extract, wherein the probiotics freeze-dried powder is prepared by mixing 20g of lactobacillus reuteri Gly LR15 freeze-dried powder, 6g of lactobacillus rhamnosus Gly 12 freeze-dried powder and 2g of lactobacillus grignard freeze-dried powder, and the functional extract consists of 60g of cactus extract and 15g of oat beta-glucan.
The preparation method of the cactus extract comprises the following steps:
cleaning 20g of cactus, mashing, adding 350g of deionized water, adding 3g of citric acid, extracting with water at 75 ℃ for 1.5h, filtering after the water extraction is finished to obtain water extract and filter residues, concentrating the water extract under reduced pressure to 1/3 of the volume, adding 4 times of absolute ethyl alcohol for alcohol precipitation, centrifuging, removing supernatant, and drying the precipitate to obtain a water extract; adding 10g of filter residues into 150g of 80% ethanol solution, performing ultrasonic extraction at 65deg.C and ultrasonic power of 250W for 40min, performing suction filtration, distilling under reduced pressure to remove the solution, and performing distillation under reduced pressure of 50kpa at 80deg.C to obtain ethanol extract; mixing 5g of water extract and 2g of alcohol extract uniformly to obtain the final product.
The lactobacillus reuteri Glory LR15 compositions prepared in examples 1-3 and comparative examples 1-3 were subjected to a mouse test, specifically as follows:
80 female BALB/c mice were randomly divided into 8 groups of 10. BALB/c mice, 6-8 weeks old, 18-22 g body weight, supplied by Liaoning Changsheng Biotechnology Co., ltd., animal license number: SCXK (Liao) 2020-0001, the grouping is shown in Table 1. The experimental animals are fed in an environment-shielded animal room at 22 ℃ and humidity of 40-70%, light and dark are alternated for 12 hours, and the experimental animals are fed by standard feed and are fed for 1 week in an adaptive way, and are free to drink water. The blank group and the model group are filled with normal saline for 30 days continuously; the lactobacillus reuteri Glory LR15 composition of example 1, the lactobacillus reuteri Glory LR15 composition of example 2, the lactobacillus reuteri Glory LR15 composition of example 3, the lactobacillus reuteri Glory LR15 composition of comparative example 1, the lactobacillus reuteri Glory LR15 composition of comparative example 2, the lactobacillus reuteri Glory LR15 composition of comparative example 3, and the bolus dose of 500 mg/kg/day for 30d; on days 1-3, except for the blank group, 100. Mu.L cyclophosphamide (40 mg/kg) was intraperitoneally injected into each group of mice, respectively, to establish an immunocompromised mouse model. After the mice were drenched for the last 12 hours, weight cervical dislocation was recorded for sacrifice, thymus and spleen were aseptically removed and weights recorded.
TABLE 1 Experimental fractionation scenario
Immune function index determination
Immune function index determination referring to "health food inspection and evaluation technical Specification" (2003 edition), determination of immune function mainly includes four aspects of cellular immunity, humoral immunity, monocyte-macrophage function and natural killer cell (NK cell) activity. The method for determining the immune function of mice in this experiment is as follows.
1. Cellular immune function assay
(1) Spleen lymphocyte transformation experiments ConA-induced proliferation of spleen lymphocytes was observed.
(2) Delayed type allergy test the delayed-TYPE HYPERSENSITIVITY (DTH) response was evaluated by measuring the change in plantar thickness of the mouse foot.
(3) Antibody-producing cell detection, namely, the capacity of spleen cells to prepare the SRBC antibody is detected by adopting a Jerne method.
2. Humoral immune function assay
(1) Serum hemolysin level assay the ability of mice to produce antibodies to SRBC was determined.
(2) Antibody-producing cell detection was as above.
3. Monocyte-macrophage function assay
(1) And (3) a mouse carbon powder cleaning experiment, namely evaluating phagocytic function of the RET system.
(2) Abdominal macrophage phagocytosis experiment, which is to observe the ability of macrophages to phagocytose chicken erythrocytes.
4. NK cell Activity assay
Killing activity of spleen cells against tumor target cells (YAC-1).
The effect of different groups of treatments on mouse body weight is shown in table 2:
TABLE 2 weight variation in mice
Note that: * P <0.05, P <0.01, compared to the blank group; # P <0.05, # P <0.01, compared to model group
From table 2 above, the weight of each group was increased, but the weight increase of the model group was significantly lower than that of the blank group, indicating that immunocompromised mice were molded, while the weight increase of G3-G5 was significantly higher (P < 0.05) than that of the model group, and was extremely significant (P < 0.01), indicating that lactobacillus reuteri Glory LR15 composition prepared according to the present invention had a better effect on weight recovery and growth of immunocompromised mice, while the G3-G5 group was superior to the G6-G8 group, indicating that there was a synergistic effect between probiotic lyophilized powder and functional extract in lactobacillus reuteri Glory LR15 composition.
Thymus and spleen are important immune organs, and thymus index and spleen index can be calculated according to the development condition. The thymus index and the spleen index can indirectly reflect the whole immunity level of the organism, and have important significance in immune function evaluation.
As shown in fig. 1, the thymus and spleen index of the mice in the model group were significantly reduced compared to the normal control group, and had a very significant (P < 0.01), indicating a decrease in immune function. The thymus and spleen indexes of the G3-G8 group are obviously improved compared with those of the model group, particularly the G3-G5 group, and the differences are extremely obvious compared with the model group (P < 0.01). This shows that the Lactobacillus reuteri Glory LR15 composition prepared by the invention can promote the development of thymus and spleen of mice with low immune function, and increase the quality of immune organs, thereby improving the immune state of organisms. Meanwhile, the group G3-G5 is better than the group G6-G8, which shows that the probiotic freeze-dried powder and the functional extract in the lactobacillus reuteri Glory LR15 composition have a synergistic effect.
The mouse spleen lymphocyte transformation experiment can evaluate the change of the cell immune function. As can be seen from FIG. 2A, the absorbance of the model group mice after CTX treatment was significantly reduced (P < 0.01) compared with the normal control group, indicating that CTX inhibited the proliferation activity of spleen lymphocytes, resulting in reduced cellular immune function. The absorption value of the spleen lymphocytes in the group G3-G8 is improved compared with that in the group G3-G5, and particularly, the improvement is obvious, compared with the group G3-G5, the difference is extremely obvious (P is less than 0.01), so that the Lactobacillus reuteri Gly LR15 composition can improve the cellular immune function. Meanwhile, the group G3-G5 is better than the group G6-G8, which shows that the probiotic freeze-dried powder and the functional extract in the lactobacillus reuteri Glory LR15 composition have a synergistic effect.
Delayed type allergy experiments can evaluate the enhanced hypersensitivity of the body. As can be seen from fig. 2B, the thickness of the plantar region of the mice in the model group was significantly reduced compared with that in the normal control group, and the difference was very significant (P < 0.01). After administration of lactobacillus reuteri Glory LR15 compositions, the G3-G8 groups had a higher degree of plantar thickening than the model group, and in particular the G3-G5 groups had a more pronounced elevation with a very pronounced difference (P < 0.01). This indicates that the lactobacillus reuteri Glory LR15 composition of the present invention can enhance delayed type allergy of the body and enhance cellular immune function. Meanwhile, the group G3-G5 is better than the group G6-G8, which shows that the probiotic freeze-dried powder and the functional extract in the lactobacillus reuteri Glory LR15 composition have a synergistic effect.
Antibody-producing cell detection (Jerne modified slide method) can be used to detect and enumerate antibody-producing cells (plasma cells) that produce IgM and other types of immunoglobulins. As can be seen from fig. 3A, the model group had a significant difference in the number of lysoplaques compared to the blank group (P < 0.01). The number of hemolytic plaques was increased in the G3-G8 group compared to the model group, with the G3-G5 group having significantly higher number of hemolytic plaques than in the model group (P < 0.05). As can be seen from fig. 3B, the model group G2 has a very significant difference in the number of antibody products (P < 0.01) compared to the blank group G1. The G3-G8 groups increased the antibody product number compared to the model group, with the G3-G5 groups having significantly higher antibody product numbers than the model group and significantly different (P < 0.01). The lactobacillus reuteri Glory LR15 composition prepared by the invention can enhance the humoral immunity function of a mouse with CTX inhibition, and has a certain immunity enhancing function. Meanwhile, as shown in fig. 3A, the average value of the group G2 is 26, the average value of the group G3 is 35.8, the average value of the group G6 is 29.4, the average value of the group G7 is 30, the average value of the group G8 is 30.8, and the group G3 significantly improves the number of hemolytic plaques compared with the group G6-G8, which indicates that the probiotic freeze-dried powder and the functional extract in the lactobacillus reuteri Glory LR15 composition have a synergistic effect.
As can be seen from fig. 4A, the NK cell activity was extremely significantly reduced (P < 0.01) in the model group compared to the blank group G1; compared with the model group G2, the NK cell activity can be enhanced by the G3-G5. As can be seen from fig. 4B, the carbon clearance phagocytosis index of mice in the model group was extremely significantly reduced (P < 0.01) compared to the blank group; compared with the model group G2, the carbon clearance phagocytosis index (P is less than 0.01) can be remarkably improved by the G3-G5. As can be seen from fig. 4C, D, the model group mice showed extremely significant decrease in the phagocytic index α and the phagocytic neutral red rate of macrophages (P < 0.01) compared to the blank group G1; compared with the model group G2, the G3-G5 can remarkably improve the neutral red phagocytosis rate and the phagocytosis index alpha (P is less than 0.01) of macrophages, and improves the Lactobacillus reuteri Glory LR15 composition prepared by the invention, so that the nonspecific immunity of mice can be improved to a certain extent. Meanwhile, as shown in fig. 4D, the average value of the group G2 is 0.3, the average value of the group G3 is 0.406, the average value of the group G6 is 0.326, the average value of the group G7 is 0.332, the average value of the group G8 is 0.4, and the group G3 remarkably improves the phagocytic index α compared with the group G6-G8, which indicates that the probiotic freeze-dried powder and the functional extract in the lactobacillus reuteri Glory LR15 composition have a synergistic effect.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. The lactobacillus reuteri Glory LR15 composition with the immunity improving function is characterized by comprising probiotic freeze-dried powder and functional extract, wherein the probiotic freeze-dried powder is formed by mixing lactobacillus reuteri Glory LR15 (Limosilactobacillus reuteri) freeze-dried powder, lactobacillus paracasei Glory LP16 (Lactobacillus paracasei) freeze-dried powder, lactobacillus rhamnosus Glory LG12 (Lactobacillus rhamnosus) freeze-dried powder and lactobacillus grignard freeze-dried powder, and the functional extract is formed by cactus extract, acanthopanax senticosus extract and oat beta-glucan;
Wherein, the Lactobacillus reuteri Glory LR15 is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2023, 3 and 31 days, and the preservation number is CGMCC No.26972; the Lactobacillus paracasei Glory LP16 is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2022, 9 months and 9 meshes, and the preservation number is CGMCC No.25669; the lactobacillus rhamnosus Gly 12 is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2022, 9 months and 9 meshes, and the preservation number is CGMCC No.25668;
The preparation method of the cactus extract comprises the following steps: cleaning radix et caulis Opuntiae Dillenii, mashing, adding deionized water, adding citric acid, extracting with water, filtering to obtain water extractive solution and residue, concentrating the water extractive solution under reduced pressure to 1/3 of the volume, adding 4 times of absolute ethanol for precipitating, centrifuging, removing supernatant, and drying the precipitate to obtain water extract; adding the filter residue into 80% ethanol solution by mass fraction, performing ultrasonic extraction, suction filtering, distilling under reduced pressure to remove the solution, and distilling under reduced pressure at 40kpa-50kpa and 75-80deg.C to obtain ethanol extract; mixing the water extract and the alcohol extract uniformly to obtain the product;
The preparation method of the acanthopanax extract comprises the following steps: pulverizing radix Acanthopanacis Senticosi, sieving with 60 mesh sieve, adding deionized water, adjusting pH to 4.5-5.5, adding cellulase and beta-glucosidase, performing enzymolysis, filtering after enzymolysis, and drying the residue to obtain pretreated radix Acanthopanacis Senticosi powder; adding pretreated acanthopanax powder into ethanol solution with the volume fraction of 70%, then adding lecithin, carrying out alcohol extraction, and filtering after the alcohol extraction is finished to obtain filtrate; concentrating the filtrate under reduced pressure to remove solvent, concentrating under reduced pressure of 40kpa-50kpa at 75-80deg.C.
2. The lactobacillus reuteri Glory LR15 composition with immunity-enhancing function according to claim 1, wherein the mass ratio of lactobacillus reuteri Glory LR15 freeze-dried powder, lactobacillus paracasei Glory LP16 freeze-dried powder, lactobacillus rhamnosus Glory LG12 freeze-dried powder and lactobacillus griseus freeze-dried powder is 10-15:5-7:6-8:2-4.
3. The lactobacillus reuteri Glory LR15 composition with immunity-enhancing function according to claim 1, wherein the mass ratio of the cactus extract, acanthopanax extract and oat β -glucan is 30-40:20-30:10-20.
4. The lactobacillus reuteri Glory LR15 composition with immunity-improving function according to claim 1, wherein the mass ratio of the cactus to the deionized water to the citric acid is 10:150-200:1-2, the water extraction temperature is 70-80 ℃ and the time is 1-2h; the feed liquid ratio of the filter residue to the ethanol solution is 1:12-18; the temperature of the ultrasonic extraction is 60-70 ℃, the time is 30-50min, and the ultrasonic power is 200-300W; the mass ratio of the water extract to the alcohol extract is 5:1-3.
5. The lactobacillus reuteri Glory LR15 composition with the immunity improving function according to claim 1, wherein the mass ratio of the acanthopanax and the deionized water is 10:70-100, the dosage of the cellulase is 0.3-0.6% of the mass of the acanthopanax, the dosage of the beta-glucosidase is 0.2-0.4% of the mass of the acanthopanax, and the enzymolysis temperature is 30-35 ℃ for 2-3 hours.
6. The lactobacillus reuteri Glory LR15 composition with immunity-improving function according to claim 1, wherein the feed liquid ratio of the pretreated acanthopanax powder to the ethanol solution is 1:10-15, the amount of lecithin is 0.5-1% of the mass of the pretreated acanthopanax powder, the ethanol extraction temperature is 55-65 ℃ and the time is 2-3h.
7. The lactobacillus reuteri Glory LR15 composition with immunity enhancing function according to claim 1, wherein the content of the probiotic freeze-dried powder in the lactobacillus reuteri Glory LR15 composition is 25-35wt%.
8. Use of a lactobacillus reuteri Glory LR15 composition having an immunity enhancing function according to any of claims 1 to 7 in the manufacture of a product having an immunity enhancing function.
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