CN111228316B - Composite probiotics for improving diabetes - Google Patents
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- CN111228316B CN111228316B CN202010135872.4A CN202010135872A CN111228316B CN 111228316 B CN111228316 B CN 111228316B CN 202010135872 A CN202010135872 A CN 202010135872A CN 111228316 B CN111228316 B CN 111228316B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses a composite probiotic for improving diabetes, which comprises the following components: lactobacillus casei CGMCC NO.4775, Lactobacillus rhamnosus CGMCC NO.3994 and other necessary auxiliary agents. More preferably, the fermentation medium of Lactobacillus casei CGMCC NO.4775 contains cochlearol E. The complex microbial inoculum can also comprise other probiotics, such as lactobacillus casei and lactobacillus paracasei. The compound microbial inoculum can be prepared into oral liquid, freeze-dried powder, capsules and the like, and is preferably used as the oral liquid. The compound microbial inoculum can also be used for preparing health-care food from fermented food.
Description
Technical Field
The invention relates to a composite probiotic for improving diabetes, belonging to the technical field of microorganisms.
Background
Probiotics have many advantageous functions in the human organism. The probiotic bacteria can effectively inhibit the growth of pathogenic bacteria, such as clostridium perfringens, escherichia coli, staphylococcus and the like, and prevent food poisoning. Probiotic microorganisms, such as lactobacilli, bifidobacteria, etc., enhance immune function, enhance the absorption of vitamins and mineral compounds, and stimulate the production of organic acids and amino acids. They may also produce a variety of enzymes such as lipases and coenzyme A, etc. Molecular and genetic studies have established that the beneficial effects of probiotics may be based on four mechanisms: antagonizing by producing antibacterial substances; ② compete with pathogens for adhesion to epithelial cells and nutrients; ③ immunological regulation of the host; and fourthly, inhibiting the generation of bacterial toxin. In view of the importance of probiotics to the health of the organism, it is one of the current research hotspots and is also widely used in clinical.
Type 2 diabetes (T2DM) is a group of metabolic diseases characterized by chronic elevated plasma glucose levels, the onset of which is mainly associated with insulin resistance and defects in islet beta cell function, obesity, a high calorie diet, and lifestyle. Recent researches show that the intestinal flora is closely related to T2DM, and a T2DM patient can generate intestinal flora imbalance and influence energy metabolism, inflammatory reaction and the like of a host; meanwhile, the regulation of intestinal flora can influence glucose metabolism, improve insulin resistance and improve insulin sensitivity.
There are two cases of diabetes during pregnancy, one is diabetes that has been diagnosed before pregnancy, and is called "diabetes combined with pregnancy"; the other is diabetes which is normal in glucose metabolism before pregnancy or has potential impaired glucose tolerance and appears or is diagnosed only in gestational period, and is also called Gestational Diabetes Mellitus (GDM). In the early and middle term of pregnancy, the demands of the fetus for nutrients increase with the increase of the gestational week, and the acquisition of glucose from the mother by the placenta is the main source of energy of the fetus. The plasma glucose level of pregnant women decreases with the progress of pregnancy, and fasting blood glucose decreases by about 10%. The reason is as follows: increased glucose uptake by the fetus from the mother; the renal plasma flow and the glomerular filtration rate are increased during pregnancy, but the reabsorption rate of renal tubules on sugar cannot be correspondingly increased, so that the sugar discharge of a part of pregnant women is increased; estrogens and progestogens increase the glucose utilization of the mother. Thus, the ability of a pregnant woman to clear glucose is enhanced during fasting compared to non-pregnant women. The fasting blood sugar of pregnant women is lower than that of non-pregnant women, which is the pathological basis that the pregnant women easily have hypoglycemia and ketoacidosis after long-time fasting. By the middle and late gestation period, the insulin resistance of pregnant women is increased, and the sensitivity of the pregnant women to insulin is reduced along with the increase of gestation period, such as placenta lactogen, estrogen, progesterone, cortisol and placenta insulinase. To maintain normal levels of glucose metabolism, the insulin requirement must be increased accordingly. For pregnant women with limited insulin secretion, the physiological change cannot be compensated in gestation period to increase blood sugar, so that the original diabetes is aggravated or GDM appears. Pregnancy is not easy to take medicine at will, while gestational diabetes is very harmful to both the mother and the fetus.
The invention aims to provide a composite probiotic for improving diabetes, which can effectively improve type 2 diabetes and gestational diabetes, and the main component of the composite probiotic is a strain which can be safely applied to food and is very safe.
Disclosure of Invention
The purpose of the present invention is to provide a composite probiotic for improving diabetes, which is effective in improving type 2 diabetes as well as gestational diabetes.
Chinese patent 201110325010 discloses a strain of Lactobacillus casei (Lactobacillus casei)2, which is preserved in China general microbiological culture Collection center (CGMCC) at 22 months and 4 months in 2011, with the preservation number of CGMCC No. 4775. The screening method of the lactobacillus casei 2 is to collect a fermented yak milk sample prepared by autonomous citizens in Tibetan of Gannan, and the sample is derived from traditional food. The strain has good fermentation performance under the condition of low temperature. Research and development teams of the company find that the lactobacillus casei strain has a good effect of improving diabetes, can be safely applied to improving gestational diabetes, and can be applied to health care and improvement of diabetes.
Chinese patent 2012105787207 discloses a strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus LC-STH-13), which is preserved in the China general microbiological culture Collection center (CGMCC), with the registration number as follows: CGMCC NO. 3994; classification name Lactobacillus rhamnous LC-STH-13, deposit date 2012, 7/8, address: xilu No. 1 Hospital, Beijing, Chaoyang, area. The strain has high lactic acid production concentration, can be planted and survived in intestinal tracts, enhances the function of gastrointestinal mucosa barrier, has the function of improving the gastrointestinal tracts, can inhibit the planting of harmful bacteria, can survive in a low-acid environment, has strong capacity of enduring the intestinal environments (such as gastric acid, bile and the like) and can be colonized in the intestinal tracts of human bodies. And the lactobacillus rhamnosus and the cell wall components thereof can promote peripheral blood mononuclear cells of a human body to secrete tumor necrosis factors, and have the function of improving the immunity of the organism. Research and development teams of the company find that the lactobacillus rhamnosus and lactobacillus casei are jointly used, so that the effect of improving diabetes of lactobacillus casei can be remarkably promoted.
Ganoderma lucidum belongs to a fungus Chinese medicament, is also a food with homology of medicine and food, has been applied for more than 2000 years in China, and is praised as 'Chinese immortal grass'. The Ganoderma lucidum is named as Ganoderma sinense, and Ganoderma sporum, and is named because its morphological stipe is different from Ganoderma sinense, and the Ganoderma lucidum is distributed in Yunnan, Fujian, Guangdong, Guangxi, hong Kong, etc. of China, and can also be found in other countries such as Indonesia, etc. The ganoderma lucidum is a very magical tonic, is rich in nutrition, is deeply loved by people, can enhance the human body resistance, prevent and resist cancer, protect the liver, detoxify, reduce hypertension, hyperlipidemia and hyperglycemia, improve the cardiovascular system, has good conditioning effect on long-term insomnia, neurasthenia, gastroenteropathy, diabetes, chronic pneumonia, bronchitis, asthma, kidney deficiency and the like of middle-aged and old people, and also has the effects of improving brain memory, preventing aging, maintaining beauty and prolonging life.
Qinpowengying et al isolated 2 new compounds from Ganoderma retrostematum ("two new compounds in Ganoderma retrostematum", Nat Prod Res Dev 2016, 28:821-824), one of which was named cochlearolE, with the side chain actually being a norsesquiterpene with 1 carbon atom loss. The structure is as follows:
research and development teams of the company find that cochlearol E alone does not have an obvious effect of improving diabetes, and has no obvious promotion effect when being directly used with lactobacillus casei CGMCC NO.4775 bacterial suspension, but if the cochlearol E is added into a culture medium of lactobacillus casei 2 to participate in fermentation, the cochlearol E can greatly promote the effect of improving the diabetes of CGMCC NO.4775 by possibly influencing physiological functions, extracellular secretion and the like of the CGMCC NO. 4775.
The technical problem to be solved by the invention can be realized by the following technical scheme.
A complex probiotic for ameliorating diabetes comprising:
lactobacillus casei CGMCC NO.4775, Lactobacillus rhamnosus CGMCC NO.3994 and other necessary auxiliary agents.
A method for preparing the composite probiotics.
More preferably, the fermentation medium of Lactobacillus casei CGMCC NO.4775 contains cochlearolE.
The complex microbial inoculum can also comprise other probiotics, such as lactobacillus casei and lactobacillus paracasei.
The compound microbial inoculum can be prepared into oral liquid, freeze-dried powder, capsules and the like, and is preferably used as the oral liquid.
The compound microbial inoculum can also be used for preparing health-care food from fermented food.
The food product is preferably a dairy product.
The research method of the invention comprises the following steps: model mice were selected for efficacy testing.
The invention has the advantages that:
(1) the lactobacillus casei selected by the invention is separated from the traditional cheese, is a probiotic bacterium with high safety for food, has good health care effect, is confirmed to be safe, and is a widely used strain with remarkable physiological activity.
(2) Ganoderma lucidum belongs to a fungus traditional Chinese medicine, is also a food with homology of medicine and food, has been known as 'Chinese immortal grass' after being applied for more than 2000 years in China, and a compound separated from the Ganoderma lucidum also has very high safety and potential other health-care effects.
(3) The composite microbial inoculum has obvious effect of improving diabetes, and is suitable for vast type 2 diabetes patients and gestational diabetes patients.
Detailed Description
The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.
Specific examples of the present invention are described below.
Examples
1. Activating strains:
lactobacillus casei CGMCC NO. 4775: inoculating one-ring lactobacillus casei to MRS solid culture medium, streaking, and anaerobically culturing at 37 deg.C for 24 hr. Selecting single colony with better growth in the plate, inoculating the single colony into MRS liquid culture medium for activation, and performing anaerobic culture to obtain 1 x 108The cfu/ml lactobacillus casei CGMCC NO.4775 seed solution is adjusted to the pH value of 6.0 by using lactic acid.
Lactobacillus rhamnosus CGMCC NO. 3994: the culture method is the same as above.
2. Dispensing liquid
Preparing a cocalarol E solution: the mangrove fleshy ganoderma A solution with the concentration of 5mg/kg is prepared by water, Tween 80 (mass fraction of 1%) and mangrove fleshy ganoderma A.
3. Preparation of fermentation liquor
a. Lactobacillus casei CGMCC NO.4775 fermentation liquor: activating lactobacillus rhamnosus CGMCC No. 3698 seed solution in MRS liquid culture medium, anaerobically culturing for 24 hr, and regulating to 1 × 107cfu/ml, adjusting the pH value to 6.0 by lactic acid;
b. lactobacillus rhamnosus CGMCC NO. 3994: the same as a;
c. mix fermentation 1 (cochlearol E addition): activating lactobacillus casei seed solution in MRS liquid culture medium, adding cochlearol E solution with mass fraction of 10%, mixing, culturing for 24 hr under anaerobic condition, and adjusting to 1 × 10 with culture medium7cfu/ml, adjusting the pH value to 6.0 by lactic acid;
d. mixed fermentation broth 2 (lactobacillus rhamnosus added): activating lactobacillus casei seed solution and lactobacillus rhamnosus seed solution in MRS liquid culture medium simultaneously, anaerobically culturing for 24 hr, and regulating to 1 × 10 with culture medium7cfu/ml, lactic acid adjusted pH 6.0.
f. Mix fermentation broth 3 (lactobacillus rhamnosus + cochlearol E addition): activating lactobacillus casei seed solution and lactobacillus rhamnosus seed solution in MRS liquid culture medium simultaneously, adding cochlearol E solution with mass fraction of 10%, mixing, culturing for 24h under anaerobic condition, and regulating to 1 x 10 with culture medium7cfu/ml, lactic acid adjusted pH 6.0.
4. Mouse model preparation
KM mice, 15-20g, were selected and treated with female: breeding in 2: 1 cage. And (3) carrying out intraperitoneal injection of 1% STZ 80mg/kg on the 8 th day of pregnancy to construct a GDM mouse model, carrying out continuous injection for 3 days 1 time per day, and detecting the blood sugar, wherein if the blood sugar is more than or equal to 11mmol/L and lasts for 3 days, the modeling is successful. The 90 pregnant mice successfully modeled were randomly divided into a blank group, a control group, and an experimental group 1-4, each 10 mice, and there was no statistical difference between the mice in each group. The feed is normally fed in the first 1 day. Beginning on day 11, gavage was continued for 1 week, and the experimental protocol for each group was as follows:
blank group: intragastric administration of 0.9% NaCl 2 ml/d;
control group: intragastric administration of 60mg/kg/d pioglitazone;
lactobacillus casei treatment group: the lactobacillus casei CGMCC NO.4775 fermentation liquor (2ml/d) is used for intragastric administration;
lactobacillus rhamnosus treatment group: the lactobacillus rhamnosus CGMCC NO.3994 fermentation liquor (2ml/d) is used for intragastric administration;
cochlearol E treatment group: gavage cochlearol E (2 ml/d);
lactobacillus casei + cochlearol E treatment group: the lactobacillus casei CGMCC NO.4775 fermentation liquor (1ml/d) and cochlearol E solution (1ml/d) are perfused;
lactobacillus casei + lactobacillus rhamnosus treatment group: the lactobacillus casei CGMCC NO.4775 fermentation liquor (1ml/d) and the lactobacillus rhamnosus CGMCC NO.3994 fermentation liquor (1ml/d) are perfused;
cochlearol E + lactobacillus rhamnosus treatment group: gavage with 1ml/d of cochlearol E solution and 1ml/d of lactobacillus rhamnosus CGMCC NO.3994 fermentation liquor;
mixed fermentation broth 1 treatment group: mixed fermentation liquor (2ml/d) is used for intragastric administration;
mixed fermentation broth 2 treatment group: mixed fermentation liquor (2ml/d) is used for intragastric administration;
mixed fermentation broth 3 treatment group: the mixed fermentation liquor (2ml/d) is perfused into the stomach.
Monitoring of GDM mice fasting plasma glucose (FBG)
Collecting tail venous blood of mice before administration (day 1 of pregnancy) and 3d (day 13 of pregnancy) and 7d (day 17 of pregnancy), centrifuging, separating serum, and detecting blood serum FBG with whole blood glucose meter and matched test paper. Specific results are shown in table 1.
6. Statistical treatment
Statistical analysis was performed using SPSS16.0 and the results of the data were expressed as mean ± standard deviation using the t test between groups.
TABLE 1 fasting plasma glucose (FBG) of mice of each group
Note: t test, l: p <0.05 (compared to blank); 2: p <0.05 (compared to control);
the result of fasting plasma glucose (FBG) of each group of mice shows that lactobacillus casei CGMCC NO.4775 has certain effect (P is less than 0.05) on fasting plasma glucose alone, the effect of the lactobacillus casei E and lactobacillus rhamnosus alone is not good, the effect of the lactobacillus rhamnosus + lactobacillus casei E treatment group, the effect of the lactobacillus casei + lactobacillus rhamnosus treatment group and the effect of the lactobacillus casei simple group are similar, and the mixed use is considered, and the promotion effect is not good. However, the blood sugar reducing effect obtained by adding the cochlearol E or the lactobacillus rhamnosus into the culture medium is good, the combined culture effect of the cochlearol E, the lactobacillus casei and the lactobacillus rhamnosus is better, and the effect of reducing the blood sugar of the lactobacillus casei can be promoted when the lactobacillus rhamnosus and the cochlearol E are added into the culture medium.
In conclusion, research and development teams of the company find that lactobacillus casei CGMCC NO.4775 has a certain effect on fasting blood glucose alone, lactobacillus casei E and lactobacillus rhamnosus do not have obvious effect on improving diabetes when used alone, and the lactobacillus casei CGMCC NO.4775 has no obvious promotion effect when directly used with lactobacillus casei CGMCC NO.4775 bacterial suspension, but if lactobacillus casei 2 culture medium is added with lactobacillus rhamnosus or lactobacillus casei E, the effect of reducing blood glucose can be promoted. Considering that the newly added substances in the culture medium participate in fermentation, the effect of CGMCC NO.4775 on improving diabetes can be greatly promoted by influencing the physiological function, extracellular secretion and the like of the lactobacillus casei CGMCC NO. 4775.
The description of the terms "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (2)
1. A method for preparing a composite probiotic for improving diabetes, characterized by the steps of:
(1) activating strains:
lactobacillus casei CGMCC NO. 4775: inoculating one-ring lactobacillus casei to MRS solid culture medium, streaking, and anaerobically culturing at 37 deg.C for 24 hr. Selecting single colony with better growth in the plate, inoculating the single colony into MRS liquid culture medium for activation, and performing anaerobic culture to obtain 1 x 108The cfu/ml lactobacillus casei CGMCC NO.4775 seed solution is adjusted to the pH value of 6.0 by using lactic acid.
Lactobacillus rhamnosus CGMCC NO. 3994: the culture method is the same as above;
(2) preparing liquid:
preparing a solution with the concentration of cochlearol E of 5 mg/kg;
(3) preparing fermentation liquor:
activating lactobacillus casei seed solution and lactobacillus rhamnosus seed solution in MRS liquid culture medium simultaneously, adding cochlearol E solution with mass fraction of 10%, mixing, culturing for 24h under anaerobic condition, and regulating to 1 x 10 with culture medium7cfu/ml, lactic acid adjusted pH 6.0.
2. A method for preparing probiotics for improving diabetes, characterized by the steps of:
(1) activating strains:
lactobacillus casei CGMCC NO. 4775: inoculating one-ring lactobacillus casei to MRS solid culture medium, streaking, and anaerobically culturing at 37 deg.C for 24 hr. Selecting single colony with better growth in the plate, inoculating the single colony into MRS liquid culture medium for activation, and performing anaerobic culture to obtain 1 x 108cfu/ml lactobacillus casei CGMCC NO.4775 seed solution, and adjusting the pH value to 6.0 by using lactic acid;
(2) preparing liquid:
preparing a solution with the concentration of cochlearol E of 5 mg/kg;
(3) preparing fermentation liquor:
activating lactobacillus casei seed solution in MRS liquid culture medium, adding cochlearol E solution with mass fraction of 10%, mixing, culturing for 24 hr under anaerobic condition, and adjusting to 1 × 10 with culture medium7cfu/ml, lactic acid adjusted pH 6.0.
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