CN116891810A - Probiotic composition with blood sugar reducing effect and probiotic prebiotic composite preparation - Google Patents
Probiotic composition with blood sugar reducing effect and probiotic prebiotic composite preparation Download PDFInfo
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- CN116891810A CN116891810A CN202211561125.2A CN202211561125A CN116891810A CN 116891810 A CN116891810 A CN 116891810A CN 202211561125 A CN202211561125 A CN 202211561125A CN 116891810 A CN116891810 A CN 116891810A
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- freeze
- lactobacillus fermentum
- dried powder
- bifidobacterium
- probiotic
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Abstract
The invention relates to the field of microorganisms, and discloses a probiotic composition and a probiotic prebiotic composite preparation with a blood sugar reducing effect. The probiotic composition comprises bifidobacterium longum WH2270, bifidobacterium animalis WH2276, lactobacillus fermentum A and lactobacillus fermentum B; the probiotic prebiotic compound preparation comprises bifidobacterium longum WH2270 freeze-dried powder, bifidobacterium animalis WH2276 freeze-dried powder, lactobacillus fermentum A freeze-dried powder, lactobacillus fermentum B freeze-dried powder, fructo-oligosaccharide and galacto-oligosaccharide. Firstly, after four strains are compounded, the strain proliferation and blood sugar reducing effect can be synergistically enhanced, and the blood sugar reducing effect can be remarkably enhanced. Based on the probiotic composition, the galacto-oligosaccharide and the fructo-oligosaccharide are further compounded, the components fully play a synergistic effect, the efficacy is better than that of the independent materials, and the blood sugar reducing efficacy can be further and obviously enhanced.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a probiotic composition with a blood sugar reducing effect and a probiotic prebiotic composite preparation.
Background
Diabetes is a chronic metabolic disease characterized by hyperglycemia, also known as an intangible killer, because hyperglycemia is harmful to the human body, but is difficult to detect. Long-term hyperglycemic symptoms can cause a range of chronic complications, such as retinopathy, nephropathy, foot ulcers, and the like. For the medicine for treating diabetes, the type I diabetes patient injects insulin to control blood sugar; type II diabetics take drugs including biguanides, sulfonylureas, etc., but have side effects such as excessive decrease in blood glucose level, causing hypoglycemia, and also strong hunger sensation, causing failure of diet control, and promoting obesity; and biguanides are prone to lactic acidosis.
There are many reports of probiotics on the study of blood glucose regulation. For example, patent CN202210709617 reports a bifidobacterium longum 070103, and fermented milk prepared by fermenting the strain has the effects of reducing blood sugar and blood fat; patent CN202110685626 reports a bifidobacterium longum MI-186, and a gastric lavage high fat diet induced obese mouse model, and finds that the strain has the effects of reducing blood fat and blood sugar; patent CN202111261188 reports that a bifidobacterium longum NSP008, a gastric lavage high fat diet induced insulin resistant mouse model, can improve glucose tolerance and reduce blood glucose in mice.
Diabetes mellitus often occurs with blood uric acid and dyslipidemia and obesity. There are also many reports on studies of probiotics on blood uric acid regulation, blood lipid regulation and obesity reduction. For example, patent CN202210602874 reports that a bifidobacterium lactis SF-B21 can effectively reduce uric acid content in human body; patent CN202111480237 reports a strain of lactobacillus fermentum which can effectively inhibit the intake of exogenous uric acid and the increase of endogenous uric acid. Patent CN202210611537 reports a strain of lactobacillus plantarum L9 with high cholesterol lowering capacity; patent CN202110965484 reports lactobacillus acidophilus LA85 which can remarkably reduce the blood fat of mice. Patent CN202111476000 reports that lactobacillus reuteri MRD01 has functions of reducing weight and lowering blood sugar; patent CN202111573855 reports that lactobacillus rhamnosus HF01 can reduce the weight of obese mice induced by high fat diet.
In the prior art, probiotic formulations directed against glycemic control are commonly single probiotic strains, as well as formulations combined with prebiotics or extracts (patent CN202210161883, combination of lactobacillus paracasei LC-37 with dietary fibers). At present, a plurality of probiotic compound preparation products with different functions are lacked, and although a plurality of probiotic vermilions are compounded in some reports, a plurality of probiotic strains are simply added in the formulas, so that the simple superposition of functions is only carried out, and scientific researches on strain compounding are lacked (patent CN202111597046, 1:1:1:1 composite probiotic powder of 5 strains); or the added probiotics strains are all strains with the function of reducing blood sugar (patent CN202011004485, the complex of lactobacillus salivarius AP-32 with the function of reducing blood sugar and lactobacillus reuteri GL-104).
Patent 202110638090 discloses that the blood sugar reducing function of bacillus natto is superior to that of single bacillus natto, but the bacillus natto is not in the list of edible strains, and has safety risks. Patent 202110250523 discloses that after the combination of probiotic compounds (bifidobacterium longum, bifidobacterium lactis, lactobacillus acidophilus, clostridium butyricum and inactivated akkermansia muciniphila) with prebiotics (natto lyophilized powder and chicory powder), the hypoglycemic effect is superior to that of the probiotic compounds, but as such, clostridium butyricum in the formulation is not in the list of edible fungi, and there is a safety risk.
In summary, the prior art has limited effect of singly using the hypoglycemic probiotics, and the multi-strain composite preparation either lacks multifunctional synergy, or lacks scientific basis of strain combination or has safety risk of strain eating.
Disclosure of Invention
In order to solve the technical problems, the invention provides a probiotic composition and a probiotic prebiotic composite preparation with blood sugar reducing effect. Firstly, scientific researches show that after four strains are compounded, the strain proliferation and blood sugar reducing effect can be synergistically enhanced, and the blood sugar reducing effect can be remarkably enhanced. And each strain selected by the invention has no toxic or side effect, can be safely eaten, and does not have safety risk. Secondly, on the basis of the probiotic composition, the invention further compounds prebiotics (galacto-oligosaccharides and fructo-oligosaccharides), and the specific compound probiotic powder and prebiotic combination are adopted, and scientific researches prove that the formula components of the probiotic preparation fully exert synergistic effect, have better efficacy than the independent materials, and can further obviously strengthen the blood sugar reducing efficacy.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides a probiotic composition with hypoglycemic effect, comprising bifidobacterium longum WHH2270, bifidobacterium animalis WHH2276, lactobacillus fermentum a, lactobacillus fermentum B.
The microorganism classification of bifidobacterium longum WHH2270 is named bifidobacterium longum subsp. Bifidobacterium longum subsp.longum; has been deposited at the microorganism strain collection of Guangdong province at 10.18 of 2022, with the deposit address of Hirschner No. 100 in Guangzhou City and the deposit number of GDMCC No:62900.
the microorganism classification of bifidobacterium animalis WHH2276 is designated as bifidobacterium animalis subsp. Bifidobacterium animalis subsp. Has been deposited at the microorganism strain collection of Guangdong province at 10.18 of 2022, with the deposit address of Hirschner No. 100 in Guangzhou City and the deposit number of GDMCC No:62901.
the lactobacillus fermentum A is lactobacillus fermentum WH 2644, and the microorganism classification is named as Limosilobacillus fermentum and is preserved in China general microbiological culture Collection center, with a preservation address: the preservation number of the Beijing city Chaoyang area North Chen Xili No.1 and 3 is CGMCC No.16754.
The lactobacillus fermentum B is lactobacillus fermentum WH3906, and the microorganism classification is named as Limosilobacillus fermentum and is preserved in China general microbiological culture Collection center, with a preservation address: no.1 and No. 3 of North Chen Xili No.1 in the Chaoyang area of Beijing city are preserved with the number of CGMCC No.19472.
The probiotic composition of the invention takes bifidobacterium longum WHH2270 with the blood sugar reducing effect as core bacteria, and then the other three bacteria are compounded around the strain. Wherein:
regarding bifidobacterium longum WHH2270, this strain has the following characteristics: (1) The bifidobacterium longum subspecies of the invention have the function of reducing blood sugar for animals with glucose-lipid disorder of insulin resistance, and can not influence normal blood sugar for healthy animals. Namely, the bifidobacterium longum subspecies longum has the effect of reducing blood sugar only for patients with hyperglycemia after being taken, does not have negative influence on blood sugar of normal people, and does not cause the occurrence of hypoglycemia. Therefore, the preparation method has no side effect and wider application field, and can be applied to not only the medicine field but also the food or health care product field and the like. (2) The bifidobacterium longum subspecies longum can regulate the disturbed intestinal flora and improve the abundance of beneficial bacteria in intestinal tracts. (3) The bifidobacterium longum subspecies longum disclosed by the invention can be used for improving the content of short-chain fatty acid in intestinal tracts, improving the content of acetic acid in serum, stimulating the expression of G protein coupled receptors of the intestinal tracts and pancreas-mediated short-chain fatty acid, and reducing blood sugar. (4) The bifidobacterium longum subspecies are derived from the feces of healthy infants, and the whole genome sequencing analysis shows that the strain has no toxic genes, has no toxic or side effects in animal evaluation, is sensitive to various antibiotics, and has certain advantages compared with the traditional medicines for treating glycolipid metabolic disorders at present.
Regarding the probiotic composition after the compounding according to the invention: interactions between probiotic strains are complex and may be manifested as mutual independence (functional superposition), mutual synergy, mutual competition or functional inhibition. In the multi-strain compound preparation with the blood sugar reducing effect reported in the prior art, the strains are mostly in function superposition, have weak mutual synergistic effect or have safety risk of eating by the strains. In vitro and animal experiments show that the bifidobacterium longum WHH2270 with the function of reducing blood sugar is compounded with other 3 functional strains (lactobacillus fermentum WHH2644 with the function of reducing uric acid, bifidobacterium animalis WHH2276 with the function of reducing blood fat and lactobacillus fermentum WHH3906 with the function of reducing weight), so that the simple functions different from the strains are overlapped, and the proliferation and blood sugar reducing effects of the compounded strains can be synergistically enhanced; through scientific researches, it is determined that the probiotic composition provided by the invention can fully play a synergistic effect between strains, and the blood sugar reducing effect is extremely remarkably enhanced. On the other hand, each strain selected by the invention has no toxic or side effect, can be safely eaten, and does not have safety risk.
Preferably, the bifidobacterium longum WHH2270, bifidobacterium animalis WHH2276, lactobacillus fermentum a and lactobacillus fermentum B are present in the form of a lyophilized powder.
Preferably, the bifidobacterium longum WH2270 freeze-dried powder is 10-15 parts by weight, the bifidobacterium animalis WH2276 freeze-dried powder is 1-4 parts by weight, the lactobacillus fermentum A freeze-dried powder is 10-15 parts by weight, and the lactobacillus fermentum B freeze-dried powder is 1-4 parts by weight.
In a second aspect, the invention provides a probiotic prebiotic composite preparation with blood glucose reducing effect, which comprises bifidobacterium longum WHH2270 freeze-dried powder, bifidobacterium animalis WHH2276 freeze-dried powder, lactobacillus fermentum A freeze-dried powder, lactobacillus fermentum B freeze-dried powder, fructo-oligosaccharide and galacto-oligosaccharide.
Based on the probiotic composition, the invention further compounds prebiotics, namely galacto-oligosaccharides and fructo-oligosaccharides, and scientific researches prove that the formula components of the probiotic preparation provided by the invention fully exert synergistic effect, have better effects than the independent materials, and can further obviously strengthen the blood sugar reducing effect.
Preferably, the bifidobacterium longum WH2270 freeze-dried powder is 10-15 parts by weight, the bifidobacterium animalis WH2276 freeze-dried powder is 1-4 parts by weight, the lactobacillus fermentum A freeze-dried powder is 10-15 parts by weight, the lactobacillus fermentum B freeze-dried powder is 1-4 parts by weight, the fructo-oligosaccharide is 10-20 parts by weight, and the galacto-oligosaccharide is 50-55 parts by weight.
In a third aspect, the invention provides the use of a probiotic composition or a probiotic prebiotic composite formulation as described above in the preparation of a product having a prophylactic and/or therapeutic effect on diabetes.
Preferably, the product is a tablet, capsule, powder, pill, granule or solution.
Preferably, the product is a pharmaceutical.
Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to scientific researches, the invention discovers that after four specific strains (bifidobacterium longum WHH2270, bifidobacterium animalis WHH2276, lactobacillus fermentum WHH2644 and lactobacillus fermentum WHH 3906) are compounded, the proliferation and blood sugar reducing effects of the strains can be synergistically enhanced, and the blood sugar reducing effect is extremely obviously enhanced. On the other hand, each strain selected by the invention has no toxic or side effect, can be safely eaten, and does not have safety risk.
(2) Based on the probiotic composition, the invention further compounds prebiotics, namely galacto-oligosaccharides and fructo-oligosaccharides, adopts the specific compound probiotic powder and prebiotic combination, and has the formula components of the probiotic preparation fully playing synergistic effect through scientific researches, thereby having better efficacy than the independent materials and extremely remarkably strengthening the blood sugar reducing efficacy.
Drawings
FIG. 1 culture media OD of combination culture of different strains 600 And (5) a value graph.
Detailed Description
The invention is further described below with reference to examples.
General examples
A probiotic composition with blood sugar lowering effect comprises Bifidobacterium longum WH2270 lyophilized powder, bifidobacterium animalis WH2276 lyophilized powder, lactobacillus fermentum WH2644 lyophilized powder, and Lactobacillus fermentum WH3906 lyophilized powder.
Preferably, the bifidobacterium longum WH2270 freeze-dried powder is 10-15 parts by weight, the bifidobacterium animalis WH2276 freeze-dried powder is 1-4 parts by weight, the lactobacillus fermentum A freeze-dried powder is 10-15 parts by weight, and the lactobacillus fermentum B freeze-dried powder is 1-4 parts by weight.
The microorganism classification of bifidobacterium longum WHH2270 is named bifidobacterium longum subsp. Bifidobacterium longum subsp.longum; has been deposited at the microorganism strain collection of Guangdong province at 10.18 of 2022, with the deposit address of Hirschner No. 100 in Guangzhou City and the deposit number of GDMCC No:62900.
the microorganism classification of bifidobacterium animalis WHH2276 is designated as bifidobacterium animalis subsp. Bifidobacterium animalis subsp. Has been deposited at the microorganism strain collection of Guangdong province at 10.18 of 2022, with the deposit address of Hirschner No. 100 in Guangzhou City and the deposit number of GDMCC No:62901.
the lactobacillus fermentum A is lactobacillus fermentum WH 2644, the microorganism classification is Limosilactobacillus fermentum, and the lactobacillus fermentum A is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation address is: the preservation number of the Beijing city Chaoyang area North Chenxi way No.1 and No. 3 is CGMCC No.16754;
the lactobacillus fermentum B is lactobacillus fermentum WH3906, the microorganism classification is Limosilactobacillus fermentum, and the lactobacillus fermentum B is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 3 and 13 days in 2020, and the preservation address is: no.1 and No. 3 of North Chen Xili No.1 in the Chaoyang area of Beijing city are preserved with the number of CGMCC No.19472.
A probiotic prebiotic compound preparation with blood sugar lowering effect comprises Bifidobacterium longum WH2270 lyophilized powder, bifidobacterium animalis WH2276 lyophilized powder, lactobacillus fermentum WH2644 lyophilized powder, lactobacillus fermentum WH3906 lyophilized powder, fructo-oligosaccharide and galacto-oligosaccharide.
The bifidobacterium longum WH2270 freeze-dried powder comprises, by weight, 10-15 parts of bifidobacterium longum WH2270 freeze-dried powder, 1-4 parts of bifidobacterium animalis WH2276 freeze-dried powder, 10-15 parts of lactobacillus fermentum A freeze-dried powder, 1-4 parts of lactobacillus fermentum B freeze-dried powder, 10-20 parts of fructo-oligosaccharides and 50-55 parts of galacto-oligosaccharides. A product containing the probiotic composition or probiotic prebiotic composite preparation and having the effect of preventing and/or treating diabetes is provided.
Preferably, the product is a tablet, capsule, powder, pill, granule or solution. The product is a medicament. Further preferably, the medicament further comprises pharmaceutically acceptable excipients.
Specific examples the following examples relate to the following methods of preparation of the culture medium:
MRS-Cys Medium (1L): 10g of peptone, 5g of yeast extract powder, 20g of glucose, 10g of beef extract and K HPO 2 42g, diammonium citrate 2g, mgSO 2 -7HO 2 0.58g，MnSO 2 -4HO 2 0.25g of anhydrous sodium acetate 5g, 1mL of Tween 80, 0.5g of cysteine hydrochloride, distilled water to a volume of 1L, adjusting the pH to 6.5, and sterilizing at 121 ℃ for 15min.
The culture medium is selected as a basic culture medium, and is used as a blank control group, and the experimental group replaces glucose with prebiotics on the basis of the basic culture medium.
Example 1 in vitro study of combinations of strains
The interaction between the probiotic strains is complex and may appear to be synergistic, functional stack; competing with each other, inhibiting function; or may be independent of each other. The interaction among different strains can be clearly deconstructed, and a basis can be provided for the combination of the strains in the formula.
4 strains with functions of losing weight, reducing blood fat, reducing blood sugar and reducing blood uric acid respectively are tested by utilizing an in vitro co-culture system to test the growth interaction among the functional strains, so that the optimal combination of the functional strains is obtained.
(1) 4 probiotics (Lactobacillus fermentum WH3906, bifidobacterium animalis WH2276, bifidobacterium longum WH2270, and Lactobacillus fermentum WH2644) were inoculated into 10ml of MRS-Cys medium at an inoculum size of 2%, and cultured in anaerobic bags at 37deg.C for 14 hr.
(2) According to different combinations, 4 strains are inoculated in equal proportion, the total inoculum size is 2 percent, and the strains are inoculated into 10ml of culture medium and are subjected to anaerobic culture for 14 hours at 37 ℃. Taking each culture solution, detecting OD of the culture solution 600 Values.
(3) The detection data show (figure 1), WHH2270 and another 3 strains were mixed and cultured in pairs, and the OD of the culture solution was measured 600 The values were all greater than the culture broth of the single strain, indicating that WHH2270 had growth synergy with the other 3 probiotics. Further, the mixed culture solution of WHH2270, WHH2644 and WHH3906 and OD 600 More than three strains of bacteria are cultured independently. Further, the mixed culture solution of WHH2270 and four strains WHH2644, WHH3906 and WHH2276, OD 600 More than three strains of bacteria mixed culture solution. The results show that the 4 probiotics in the test, when combined together, have no competitive inhibition, but have the effect of growth synergy.
Example 2 in vitro study of Strain combinations and prebiotic formulations
The prebiotics are used as proliferation factors of probiotics and have a synergistic effect on the proliferation of the probiotics. By adding various prebiotics into the culture medium, comparing the proliferation conditions of the strain after culture, the optimal prebiotics and the combination proportion of the prebiotics are screened out.
(1) 8 common prebiotic raw materials including xylo-oligosaccharide, fructo-oligosaccharide, galacto-oligosaccharide, arabinoxylan, resistant dextrin, inulin, polydextrose and chitosan oligosaccharide are selected as single carbon source to replace glucose in MRS-Cys culture medium, and are sterilized at 121deg.C for 15min.
(2) The 4 strains were inoculated into 10ml of the above medium at an inoculum size of 2%, and cultured in an anaerobic bag at 37℃for 14 hours.
(3) Taking each culture solution, detecting OD 600 。
MRS-Cys culture medium is used as blank control, OD 600 The results are shown in Table 1. The data show that growth of WHH2644 prefers fructooligosaccharides, growth of WHH3906 prefers xylo-and fructooligosaccharides, and growth of WHH2270 and WHH2276 prefers galactooligosaccharides. Fructo-oligosaccharides and galacto-oligosaccharides are widely used as prebiotic combinations in food products, especially infant food products. Further, the ratio of fructo-oligosaccharide to galacto-oligosaccharide was investigated and the results are shown in Table 2. The data show that fructo-and galacto-oligosaccharides are present at 1:6, optimal for growth of the probiotic combinations of the present invention.
TABLE 1 culture media OD of different prebiotic Medium for growth of probiotic strains 600 Value of
TABLE 2 culture media OD of different fructo-and galacto-oligosaccharide ratio Medium for Combined growth of probiotics 600 Value of
Example 3: bifidobacterium longum WHH2270 formulations
Live bifidobacterium longum WHH2270 formulationThe bacterial count is 5 multiplied by 10 10 cfu/g, comprising the following components: bifidobacterium longum WHH2270 lyophilized powder and maltodextrin. Wherein the mass percentage of the bifidobacterium longum WH2270 freeze-dried powder is 50 percent, and the mass percentage of the maltodextrin is 50 percent.
The preparation method of the bifidobacterium longum WHH2270 preparation comprises the following steps:
(1) Preparation of bifidobacterium longum WHH2270 lyophilized powder:
(1.1) Strain activation: taking a bifidobacterium longum WH2270 freezing tube, inoculating 2% of the inoculating amount into a liquid MRS-Cys culture medium, and performing anaerobic culture at 37 ℃ overnight to obtain a generation-1 activated seed liquid;
(1.2) spread cultivation: inoculating the 1 generation of activated seed liquid with 2% inoculum size into a liquid MRS-Cys culture medium, and performing anaerobic culture at 37 ℃ overnight to obtain 2 generation of activated seed liquid;
(1.3) fermentation: inoculating 2 generation of activated seed solution with 5% inoculum size into MRS-Cys culture medium, controlling pH to 5.5, 37 deg.C, introducing CO 2 Culturing in a fermentation tank for 16h to obtain a fermented product;
(1.4) bacterial sludge collection and emulsification: placing the fermented product at 8000rpm, centrifuging for 10min, discarding supernatant, collecting bacterial mud, and mixing the bacterial mud with the mass ratio of 1:1, adding bacterial mud into a freeze-drying protective agent, and emulsifying for 15min at 180rpm to obtain emulsion; the preparation of the lyoprotectant is as follows: uniformly mixing 1.3wt% of glycerin, 24.2wt% of skim milk powder, 1.0wt% of sodium glutamate, 16.1wt% of lactose and the balance of deionized water, and sterilizing at 105 ℃ for 10 min;
(1.5) freeze-drying and crushing: freezing the emulsion at-80deg.C, drying at-50deg.C under vacuum degree of 0.2mbar, sieving with 20 mesh sieve to obtain lyophilized powder of Bifidobacterium longum WHH2270 with viable count of 1×10 11 cfu/g。
(2) And weighing maltodextrin and bifidobacterium longum WHH2270 freeze-dried powder, and uniformly mixing the maltodextrin and the bifidobacterium longum WHH2270 freeze-dried powder to prepare powder.
Example 4: bifidobacterium animalis WHH2276 preparation
The viable count of the preparation of the bifidobacterium animalis WHH2276 is 5 multiplied by 10 10 cfu/g, comprising the following components: bifidobacterium animalis WH2276 freeze-dried powder and wheatAnd (3) maltodextrin. Wherein the mass percentage of the bifidobacterium animalis WH2276 freeze-dried powder is 10 percent, and the mass percentage of maltodextrin is 90 percent.
The preparation method of the bifidobacterium animalis WHH2276 preparation comprises the following steps:
(1) Preparation of bifidobacterium animalis WHH2276 lyophilized powder:
(1.1) Strain activation: taking a bifidobacterium animalis WH2276 freezing tube, inoculating 2% of the inoculating amount into a liquid MRS-Cys culture medium, and performing anaerobic culture at 37 ℃ for overnight to obtain a generation-1 activated seed liquid;
(1.2) spread cultivation: inoculating the 1 generation of activated seed liquid with 2% inoculum size into a liquid MRS-Cys culture medium, and performing anaerobic culture at 37 ℃ overnight to obtain 2 generation of activated seed liquid;
(1.3) fermentation: inoculating 2 generation of activated seed solution with 5% inoculum size into MRS-Cys culture medium, controlling pH to 5.5, 37 deg.C, introducing CO 2 Culturing in a fermentation tank for 16h to obtain a fermented product;
(1.4) bacterial sludge collection and emulsification: placing the fermented product at 8000rpm, centrifuging for 10min, discarding supernatant, collecting bacterial mud, and mixing the bacterial mud with the mass ratio of 1:1, adding bacterial mud into a freeze-drying protective agent, and emulsifying for 15min at 180rpm to obtain emulsion; the preparation of the lyoprotectant is as follows: uniformly mixing 1.3wt% of glycerin, 24.2wt% of skim milk powder, 1.0wt% of sodium glutamate, 16.1wt% of lactose and the balance of deionized water, and sterilizing at 105 ℃ for 10 min;
(1.5) freeze-drying and crushing: freezing the emulsion at-80deg.C, drying at-50deg.C under vacuum degree of 0.2mbar, sieving with 20 mesh sieve to obtain lyophilized powder of animal bifidobacterium WH2276 with viable count of 5×10 11 cfu/g。
(2) And weighing maltodextrin and bifidobacterium animalis WH2276 freeze-dried powder, and uniformly mixing the maltodextrin and the bifidobacterium animalis WH2276 freeze-dried powder to prepare powder.
Example 5: lactobacillus fermentum WH2644 preparation
The viable count of the lactobacillus fermentum WH2644 preparation is 5×10 10 cfu/g, comprising the following components: lactobacillus fermentum WHH2644 lyophilized powder and maltodextrin. Wherein the mass percentage of the lactobacillus fermentum WH2644 freeze-dried powder is 50 percent, and the weight percentage of the lactobacillus fermentum WH2644 freeze-dried powder is wheatThe mass percentage of the maltodextrin is 50%.
The preparation method of the lactobacillus fermentum WH2644 preparation comprises the following steps:
(1) Preparation of lactobacillus fermentum WHH2644 lyophilized powder:
(1.1) Strain activation: inoculating lactobacillus fermentum WH2644 cryopreservation tube with 2% inoculum size into liquid MRS culture medium, and culturing at 37deg.C overnight to obtain 1 generation activated seed solution;
(1.2) spread cultivation: inoculating the 1-generation activated seed liquid with 2% inoculum size in a liquid MRS culture medium, and culturing overnight at 37 ℃ to obtain a 2-generation activated seed liquid;
(1.3) fermentation: inoculating 2 generation of activated seed solution with 5% inoculum size into MRS culture medium, controlling pH to 5.5, and introducing N at 37deg.C 2 Culturing for 16h overnight to obtain fermented product;
(1.4) bacterial sludge collection and emulsification: placing the fermented product at 8000rpm, centrifuging for 10min, discarding supernatant, collecting bacterial mud, and mixing the bacterial mud with the mass ratio of 1:1, adding bacterial mud into a freeze-drying protective agent, and emulsifying for 30min at 200rpm to obtain emulsion; the preparation of the lyoprotectant is as follows: uniformly mixing 1.3wt% of glycerin, 24.2wt% of skim milk powder, 1.0wt% of sodium glutamate, 16.1wt% of lactose and the balance of deionized water, and sterilizing at 105 ℃ for 10 min;
(1.5) freeze-drying and crushing: freezing the emulsion at-80deg.C, drying at-50deg.C under vacuum degree of 0.2mbar, sieving with 20 mesh sieve to obtain lyophilized powder of Lactobacillus fermentum WHH2644 with viable count of 1×10 11 cfu/g。
(2) And weighing maltodextrin and lactobacillus fermentum WH2644 freeze-dried powder, and uniformly mixing the maltodextrin and the lactobacillus fermentum WH2644 freeze-dried powder to prepare powder.
Example 6: lactobacillus fermentum WH3906 preparation
The viable count of the lactobacillus fermentum WHH3906 preparation is 5×10 10 cfu/g, comprising the following components: lactobacillus fermentum WHH3906 lyophilized powder and maltodextrin. Wherein the mass percentage of the lactobacillus fermentum WH3906 freeze-dried powder is 10 percent, and the mass percentage of the maltodextrin is 90 percent.
The preparation method of the lactobacillus fermentum WH3906 preparation comprises the following steps:
(1) Preparation of lactobacillus fermentum WHH3906 lyophilized powder:
(1.1) Strain activation: inoculating lactobacillus fermentum WH3906 in a freezing tube with an inoculum size of 2% in a liquid MRS culture medium, and culturing overnight at 37 ℃ to obtain 1 generation of activated seed liquid;
(1.2) spread cultivation: inoculating the 1-generation activated seed liquid with 2% inoculum size in a liquid MRS culture medium, and culturing overnight at 37 ℃ to obtain a 2-generation activated seed liquid;
(1.3) fermentation: inoculating 2 generation of activated seed solution with 5% inoculum size into MRS culture medium, controlling pH to 5.5, and introducing N at 37deg.C 2 Culturing for 16h overnight to obtain fermented product;
(1.4) bacterial sludge collection and emulsification: placing the fermented product at 8000rpm, centrifuging for 10min, discarding supernatant, collecting bacterial mud, and mixing the bacterial mud with the mass ratio of 1:1, adding bacterial mud into a freeze-drying protective agent, and emulsifying for 30min at 200rpm to obtain emulsion A; the preparation of the lyoprotectant is as follows: uniformly mixing 1.3wt% of glycerin, 24.2wt% of skim milk powder, 1.0wt% of sodium glutamate, 16.1wt% of lactose and the balance of deionized water, and sterilizing at 105 ℃ for 10 min;
(1.5) freeze-drying and crushing: freezing the emulsion at-80deg.C, drying at-50deg.C under vacuum degree of 0.2mbar, sieving with 20 mesh sieve to obtain lyophilized powder of Lactobacillus fermentum WHH3906 with viable count of 5×10 11 cfu/g。
(2) And weighing maltodextrin and lactobacillus fermentum WH3906 freeze-dried powder, and uniformly mixing the maltodextrin and the lactobacillus fermentum WH3906 freeze-dried powder to prepare powder.
Example 7: probiotic composition formulation A
Probiotic composition preparation A with viable count of 5×10 10 cfu/g, consisting of the following components: bifidobacterium longum WH2270 freeze-dried powder, bifidobacterium animalis WH2276 freeze-dried powder, lactobacillus fermentum WH2644 freeze-dried powder, lactobacillus fermentum WH3906 freeze-dried powder and maltodextrin. Wherein the mass percentage of the bifidobacterium longum WHH2270 freeze-dried powder is 12.5 percent, the mass percentage of the bifidobacterium animalis WHH2276 freeze-dried powder is 2.5 percent, the mass percentage of the lactobacillus fermentum WHH2644 freeze-dried powder is 12.5 percent, and the lactobacillus fermentum WHH3906 freeze-dried powderThe mass percentage of the powder is 2.5 percent, and the mass percentage of maltodextrin is 70 percent.
The preparation method of the probiotic composition preparation A comprises the following steps:
(1) Maltodextrin and bifidobacterium longum WHH2270 freeze-dried powder, bifidobacterium animalis WHH2276 freeze-dried powder, lactobacillus fermentum WHH2644 freeze-dried powder and lactobacillus fermentum wh 3906 freeze-dried powder are weighed.
Wherein, the preparation method of the bifidobacterium longum WHH2270 freeze-dried powder is the same as that of example 3, the preparation method of the bifidobacterium animalis WHH2276 freeze-dried powder is the same as that of example 4, the preparation method of the lactobacillus fermentum WHH2644 freeze-dried powder is the same as that of example 5, the preparation method of the lactobacillus fermentum WH3906 freeze-dried powder is the same as that of example 6, and the viable count of the bifidobacterium longum WHH2270 freeze-dried powder is 1 multiplied by 10 11 cfu/g, viable count of bifidobacterium animalis WHH2276 freeze-dried powder is 5 multiplied by 10 11 cfu/g, viable count of Lactobacillus fermentum WH2644 lyophilized powder 1×10 11 cfu/g, viable count of lactobacillus fermentum WHH3906 freeze-dried powder is 5 multiplied by 10 11 cfu/g。
(2) And uniformly mixing maltodextrin with the 4 probiotics freeze-dried powders, and preparing powder.
Example 8: probiotic and prebiotic composite preparation
Probiotic and prebiotic composite preparation with viable count of 5×10 10 cfu/g, consisting of the following components: the preparation method comprises the steps of bifidobacterium longum WH2270 freeze-dried powder, bifidobacterium animalis WH2276 freeze-dried powder, lactobacillus fermentum WH2644 freeze-dried powder, lactobacillus fermentum WH3906 freeze-dried powder, fructo-oligosaccharide and galacto-oligosaccharide. Wherein the mass percentage of the bifidobacterium longum WH2270 freeze-dried powder is 12.5%, the mass percentage of the bifidobacterium animalis WH2276 freeze-dried powder is 2.5%, the mass percentage of the lactobacillus fermentum WH2644 freeze-dried powder is 12.5%, the mass percentage of the lactobacillus fermentum WH3906 freeze-dried powder is 2.5%, the mass percentage of the fructo-oligosaccharide is 17.5%, and the mass percentage of the galacto-oligosaccharide is 52.5%.
The preparation method of the probiotic prebiotic composite preparation comprises the following steps:
(1) Weighing fructo-oligosaccharide, galacto-oligosaccharide and bifidobacterium longum WH2270 freeze-dried powder, bifidobacterium animalis WH2276 freeze-dried powder, lactobacillus fermentum WH2644 freeze-dried powder and lactobacillus fermentum WH3906 freeze-dried powder.
The preparation method of the bifidobacterium longum WHH2270 freeze-dried powder is the same as that of example 3. The preparation method of bifidobacterium animalis WH2276 freeze-dried powder is the same as in example 4. The preparation method of the lactobacillus fermentum WHH2644 freeze-dried powder is the same as that of example 5. The preparation method of the lactobacillus fermentum WH3906 freeze-dried powder is the same as that of example 6. Viable count of bifidobacterium longum WHH2270 freeze-dried powder is 1 multiplied by 10 11 cfu/g, viable count of bifidobacterium animalis WHH2276 freeze-dried powder is 5 multiplied by 10 11 cfu/g, viable count of Lactobacillus fermentum WH2644 lyophilized powder 1×10 11 cfu/g, viable count of lactobacillus fermentum WHH3906 freeze-dried powder is 5 multiplied by 10 11 cfu/g。
(2) Mixing fructo-oligosaccharide, galacto-oligosaccharide and the 4 probiotics freeze-dried powder uniformly, and preparing into powder.
Comparative example 1: probiotic composition formulation B
Probiotic composition preparation B with viable count of 5×10 10 cfu/g, differing from example 7 in the replacement of bifidobacterium longum WHH2270 with other commercial bifidobacterium longum, consisting of the following components: commercial bifidobacterium longum freeze-dried powder, bifidobacterium animalis WH2276 freeze-dried powder, lactobacillus fermentum WH2644 freeze-dried powder, lactobacillus fermentum WH3906 freeze-dried powder and maltodextrin.
The preparation of the probiotic composition formulation B was the same as in example 7.
Comparative example 2: probiotic composition formulation C
Probiotic composition preparation C with viable count of 5×10 10 cfu/g, differing from example 7 in the replacement of bifidobacterium animalis WHH2276 with other commercial bifidobacterium animalis, consisting of the following components: bifidobacterium longum WH2270 freeze-dried powder, commercial bifidobacterium freeze-dried powder, lactobacillus fermentum WH2644 freeze-dried powder, lactobacillus fermentum WH3906 freeze-dried powder and maltodextrin.
The preparation of the probiotic composition formulation C was the same as in example 7.
Comparative example 3: probiotic composition formulation D
Probiotic composition preparation D with viable count of 5×10 10 cfu/g, differs from example 7 in that other commercial Lactobacillus fermentum was used insteadLactobacillus fermentum WHH2644, consisting of the following components: bifidobacterium longum WHH2270 freeze-dried powder, bifidobacterium animalis WHH2276 freeze-dried powder, commercial lactobacillus fermentum freeze-dried powder, lactobacillus fermentum WHH3906 freeze-dried powder and maltodextrin.
The probiotic composition formulation D was prepared in the same manner as in example 7.
Comparative example 4: probiotic composition formulation E
Probiotic composition formulation E with viable count of 5×10 10 cfu/g, differing from example 7 in the replacement of lactobacillus fermentum WHH3906 with other commercial lactobacillus fermentum, consisting of the following components: bifidobacterium longum WHH2270 freeze-dried powder, bifidobacterium animalis WHH2276 freeze-dried powder, lactobacillus fermentum WHH2644 freeze-dried powder, commercial lactobacillus fermentum freeze-dried powder and maltodextrin.
The preparation of the probiotic composition formulation E was the same as in example 7.
Example 9: evaluation of hypoglycemic efficacy of formulations
The 5dpf wild type AB strain zebra fish was selected and divided into 13 groups: example 3 experimental group, example 4 experimental group, example 5 experimental group, example 6 experimental group, example 7 experimental group, example 8 experimental group, comparative example 1 experimental group, comparative example 2 experimental group, comparative example 3 experimental group, comparative example 4 experimental group, positive control pioglitazone hydrochloride 20.0 μg/mL, and a normal control group and a model control group were simultaneously set, each group of 30 zebra fish.
The samples were each given water-soluble at a concentration of 2mg/ml. Except for the normal control group, the rest experimental groups are water-soluble in the daytime and are given high-fat feed (yolk powder) and water-soluble in the evening and are given high sugar (glucose), so that a zebra fish high-sugar high-fat model is established. The samples were treated with high-fat feed for 15 hours (7.5 hours per day), glucose for 33 hours (16.5 hours per day), and at 28℃for 48 hours, and the blood glucose level in the zebra fish was analyzed by collecting data using a blood glucose meter, and the blood glucose-lowering effect of the samples was evaluated by the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS26.0 software, p <0.05 indicated that the differences were statistically significant.
The results of the experiments are shown in table 3, and the hypoglycemic effect of the probiotic composition preparation of example 7 is significantly better than that of the single strain preparation (examples 3-6), which shows that after the bifidobacterium longum WHH2270 is compounded with other three strains, the hypoglycemic capability of the bifidobacterium longum WHH2270 can be further enhanced, and the data of example 7 is compared with the data of comparative examples 1-4, and the data of example 7 is optimal, and the reason for analysis is that:
the replacement of bifidobacterium longum WHH2270 with other commercial bifidobacterium longum in comparative example 1 demonstrated that the other commercial bifidobacterium longum was not as effective as the bifidobacterium longum WHH2270 of the present invention.
Comparative examples 2-4 replacement of lactobacillus fermentum WHH2644 and lactobacillus fermentum WHH3906 with other commercial bifidobacterium animalis, commercial lactobacillus fermentum, respectively, resulted in less hypoglycemic capacity than example 7, demonstrating that only the four specific strains of the invention were optimal after compounding.
Example 7 the hypoglycemic ability of example 7 is inferior to example 8, demonstrating that the introduction of prebiotics can have a synergistic effect with the combination of strains, further enhancing the hypoglycemic ability of the strains.
TABLE 3 blood sugar measurement results for each group of zebra fish
| Group of | Blood glucose level of zebra fish (mmol/L) |
| Normal control group | 0.90±0.039 *** |
| Model control group | 3.31±0.134 |
| Pioglitazone hydrochloride | 2.62±0.090 *** |
| Example 3 (bifidobacterium longum WHH 2270) | 2.78±0.138 ** |
| Example 4 (bifidobacterium animalis WHH 2276) | 3.14±0.237 |
| Example 5 (Lactobacillus fermentum WH2644) | 2.88±0.114 ** |
| Example 6 (Lactobacillus fermentum WH3906) | 2.99±0.099 |
| Example 7 (probiotic composition formulation A) | 1.97±0.104 *** |
| Example 8 (Probiotics prebiotics Complex formulation) | 1.73±0.068 *** |
| Comparative example 1 (probiotic composition formulation B, replacement of WHH2270 with commercial bifidobacterium longum) | 2.43±0.189 *** |
| Comparative example 2 (probiotic composition formulation C, replacement of WHH2276 with bifidobacterium in a commercial animal) | 2.31±0.177 *** |
| Comparative example 3 (probiotic composition formulation D, substitution of WHH2644 with commercial lactobacillus fermentum) | 2.35±0.183 *** |
| Comparative example 4 (probiotic composition formulation E, substitution of commercial Lactobacillus fermentum for WH 3906) | 2.29±0.161 *** |
In comparison with the control group of the model, * p<0.05， ** p<0.01， *** p<0.001。
the raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
Claims (10)
1. A probiotic composition with hypoglycemic effect, characterized in that: comprises bifidobacterium longum WH2270, bifidobacterium animalis WH2276, lactobacillus fermentum A and lactobacillus fermentum B;
the microorganism classification of the bifidobacterium longum WHH2270 is named bifidobacterium longum subspecies longumBifidobacterium longum subsp. LongumThe method comprises the steps of carrying out a first treatment on the surface of the Has been deposited at the microorganism strain collection of Guangdong province at 10.18 of 2022, with the deposit address of Hirschner No. 100 in Guangzhou City and the deposit number of GDMCC No:62900;
the microorganism classification of the bifidobacterium animalis WHH2276 is named as bifidobacterium animalis subspecies lactisBifidobacterium animalis subsp. lactisThe method comprises the steps of carrying out a first treatment on the surface of the Has been deposited at the microorganism strain collection of Guangdong province at 10.18 of 2022, with the deposit address of Hirschner No. 100 in Guangzhou City and the deposit number of GDMCC No:62901.
2. the probiotic composition according to claim 1, characterized in that:
the saidLactobacillus fermentum A is Lactobacillus fermentum WH2644, and the microorganism classification is namedLimosilactobacillus fermentumThe strain has been deposited in China general microbiological culture Collection center, with a deposit address: the preservation number of the Beijing city Chaoyang area North Chenxi way No.1 and No. 3 is CGMCC No.16754;
the lactobacillus fermentum B is lactobacillus fermentum WH3906, and the microorganism classification is named asLimosilactobacillus fermentumThe strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and the preservation address is as follows: no.1 and No. 3 of North Chen Xili No.1 in the Chaoyang area of Beijing city are preserved with the number of CGMCC No.19472.
3. A probiotic composition according to claim 1 or 2, characterized in that: the bifidobacterium longum WH2270, the bifidobacterium animalis WH2276, the lactobacillus fermentum A and the lactobacillus fermentum B exist in the form of freeze-dried powder.
4. A probiotic composition according to claim 3, characterized in that: the bifidobacterium longum WH2270 freeze-dried powder is 10-15 parts by weight, the bifidobacterium animalis WH2276 freeze-dried powder is 1-4 parts by weight, the lactobacillus fermentum A freeze-dried powder is 10-15 parts by weight and the lactobacillus fermentum B freeze-dried powder is 1-4 parts by weight.
5. A probiotic and prebiotic composite preparation with blood glucose reducing effect, which is characterized in that: comprises bifidobacterium longum WH2270 freeze-dried powder, bifidobacterium animalis WH2276 freeze-dried powder, lactobacillus fermentum A freeze-dried powder, lactobacillus fermentum B freeze-dried powder, fructo-oligosaccharide and galacto-oligosaccharide;
the microorganism classification of the bifidobacterium longum WHH2270 is named bifidobacterium longum subspecies longumBifidobacterium longum subsp. LongumThe method comprises the steps of carrying out a first treatment on the surface of the Has been deposited at the microorganism strain collection of Guangdong province at 10.18 of 2022, with the deposit address of Hirschner No. 100 in Guangzhou City and the deposit number of GDMCC No:62900;
the microorganism classification of the bifidobacterium animalis WHH2276 is named as bifidobacterium animalis subspecies lactisBifidobacterium animalis subsp. lactisThe method comprises the steps of carrying out a first treatment on the surface of the Has been deposited at the microorganism strain collection of Guangdong province at 10.18 of 2022, with the deposit address of Hirschner No. 100 in Guangzhou City and the deposit number of GDMCC No:62901.
6. the probiotic prebiotic composite formulation of claim 1 wherein:
the lactobacillus fermentum A is lactobacillus fermentum WH2644, and the microorganism classification is named asLimosilactobacillus fermentumThe strain has been deposited in China general microbiological culture Collection center, with a deposit address: the preservation number of the Beijing city Chaoyang area North Chenxi way No.1 and No. 3 is CGMCC No.16754;
the lactobacillus fermentum B is lactobacillus fermentum WH3906, and the microorganism classification is named asLimosilactobacillus fermentumThe strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and the preservation address is as follows: no.1 and No. 3 of North Chen Xili No.1 in the Chaoyang area of Beijing city are preserved with the number of CGMCC No.19472.
7. The probiotic prebiotic composite formulation of claim 6 wherein: the bifidobacterium longum WH2270 freeze-dried powder comprises, by weight, 10-15 parts of bifidobacterium longum WH2270 freeze-dried powder, 1-4 parts of bifidobacterium animalis WH2276 freeze-dried powder, 10-15 parts of lactobacillus fermentum A freeze-dried powder, 1-4 parts of lactobacillus fermentum B freeze-dried powder, 10-20 parts of fructo-oligosaccharides and 50-55 parts of galacto-oligosaccharides.
8. Use of a probiotic composition according to one of claims 1 to 4 or a probiotic prebiotic composite formulation according to one of claims 5 to 7 for the preparation of a product having a prophylactic and/or therapeutic effect on diabetes.
9. The use according to claim 8, wherein: the product is tablet, capsule, powder, pill, granule or solution.
10. The use according to claim 8 or 9, wherein: the product is a medicament.
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