CN114921363A - Composite probiotics for inhibiting fat accumulation and application thereof - Google Patents

Composite probiotics for inhibiting fat accumulation and application thereof Download PDF

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Publication number
CN114921363A
CN114921363A CN202210448054.9A CN202210448054A CN114921363A CN 114921363 A CN114921363 A CN 114921363A CN 202210448054 A CN202210448054 A CN 202210448054A CN 114921363 A CN114921363 A CN 114921363A
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freeze
fat
fat accumulation
dried powder
inhibiting
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方曙光
杨竹源
郭晓娟
汪欣
朱建国
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WeCare Probiotics Co Ltd
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WeCare Probiotics Co Ltd
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/385—Concentrates of non-alcoholic beverages
    • A23L2/39—Dry compositions
    • 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
    • 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
    • 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
    • A61K35/745—Bifidobacteria
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Abstract

The invention relates to a composite probiotic for inhibiting fat accumulation and application thereof, wherein the composite probiotic for inhibiting fat accumulation comprises bifidobacterium longum BL21 and lactobacillus rhamnosus LRa 05; the Bifidobacterium longum BL21 is Bifidobacterium longum (Bifidobacterium longum) BL21 strain, the preservation number is CGMCC No.10452, and the preservation date is 2015, 1 month and 27 days; the Lactobacillus rhamnosus LRa05 is a Lactobacillus rhamnosus LRa05 strain, the preservation number is CGMCC No.1.12734, and the preservation date is 2020, 7 and 20 days. Researches show that products taking BL21 strain and LRa05 strain as main functional components can obviously reduce TC, LDL and TG levels in serum and liver, improve HDL levels in serum and liver, reduce body fat, especially reduce perirenal fat or abdominal wall fat, and have good application prospects.

Description

Composite probiotics for inhibiting fat accumulation and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a composite probiotic for inhibiting fat accumulation and application thereof, in particular to a composite probiotic for inhibiting fat accumulation, a composite probiotic freeze-dried powder for inhibiting fat accumulation, fermented milk with fat-reducing and weight-losing effects, and a solid beverage with fat-reducing and weight-losing effects.
Background
The obesity brings about not only the change of body and the damage of immune function, but also can induce a plurality of chronic diseases, especially increase the incidence rate of a plurality of chronic diseases such as cardiovascular and cerebrovascular diseases, type II diabetes, cancer and the like, therefore, the influence mechanism of the obesity on the health needs to be fully mastered, the functional food with the functions of assisting weight losing and lipid lowering is developed, and the food has great economic and social values for people and social development.
Obesity is caused by excessive accumulation of body fat caused by family inheritance and environmental factors, excessive body energy intake and low consumption, so that balance of body energy inclines, intestinal flora disorder and other multiple factors. When the energy intake is far greater than the body's consumption of energy, it can lead to the accumulation of body fat, thus leading to obesity; metabolism and intestinal flora are destroyed, which causes the change of connexin in intestinal mucosa, and the permeability of intestinal tract to nutrient absorption is changed, which promotes the digestion and absorption of fat, and further causes the organism to form obesity and induce inflammation.
The intestinal flora is a main factor inducing obesity, and can regulate the digestion, absorption and metabolism of nutrients ingested by a human body, regulate the secretion of bile acid so as to regulate fat metabolism and influence the fat deposition level in blood and tissues, so that the balance of the intestinal flora is very important for the health of the human body. When the intestinal flora is disordered, the balance can be adjusted through the intake of a proper amount of probiotics, the environment in the intestinal tract of the organism is maintained in a balanced state, and the permeability of the intestinal mucosa for the absorption of nutrient substances is improved; meanwhile, healthy and stable intestinal flora and metabolites thereof influence obesity through the intestine-brain axis, and intestinal hormone secreted by the intestinal flora influences the central nervous system of a body, so that satiety is further increased, appetite is reduced, and energy metabolism of the body is further influenced, so that the effects of losing weight and reducing fat are realized; in addition, not only the thallus of the probiotics has excellent probiotic effect on the intestinal tract, but also the fermentation product and the enzyme system thereof formed by fermenting the probiotics can effectively reduce the body fat and the accumulation of the body fat. Therefore, the research on the probiotic strains with the functions of losing weight and reducing fat and the application of the probiotic strains in the aspects of food products, medicines and the like have great significance for obese people, preventing cardiovascular and cerebrovascular diseases and reducing the incidence rate of various chronic diseases.
CN109105902A discloses a composite probiotic preparation for losing weight, which comprises 30-50 parts of composite probiotic, 5-15 parts of dietary fiber, 1-3 parts of protein powder and 5-10 parts of plant extract according to the weight ratio; the composite probiotics comprise lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus bulgaricus and streptococcus thermophilus, and the plant extract comprises 30-35 parts of Chinese waxgourd peel, 9-12 parts of kudzu root, 30-35 parts of poria cocos and 25-30 parts of astragalus membranaceus. The formula improves the intestinal flora structure of obese people through scientific and reasonable combination, and effectively plays a weight reducing role.
CN113662996A discloses a probiotic composition with a weight-losing effect, which is prepared from the following raw materials in parts by weight: 6-7 parts of lactobacillus acidophilus, 4-5 parts of lactobacillus casei, 3-4 parts of lactobacillus plantarum, 1-2 parts of bifidobacterium lactis, 5-6 parts of lactobacillus rhamnosus, 2-3 parts of bacillus coagulans, 10-15 parts of stachyose, 20-25 parts of mannitol, 15-20 parts of isomaltooligosaccharide, 5-8 parts of ascorbic acid, 40-50 parts of resistant dextrin, 4-6 parts of noni extract and 50-60 parts of water. The probiotic composition has good acid and bile salt resistance and weight reducing effect.
However, the probiotic products with the effects of losing weight and reducing fat, particularly the effects of reducing fat of abdominal walls in the prior art are very limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a composite probiotic for inhibiting fat accumulation and application thereof, and particularly provides a composite probiotic for inhibiting fat accumulation, a composite probiotic freeze-dried powder for inhibiting fat accumulation, fermented milk with fat-reducing and weight-losing effects and a solid beverage with fat-reducing and weight-losing effects.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a composite probiotic for inhibiting fat accumulation, comprising bifidobacterium longum BL21 and lactobacillus rhamnosus LRa 05;
the Bifidobacterium longum BL21 is Bifidobacterium longum (Bifidobacterium longum) BL21 strain, the preservation number is CGMCC No.10452, and the preservation date is 2015, 1 month and 27 days;
the Lactobacillus rhamnosus LRa05 is Lactobacillus rhamnosus LRa05 strain, the preservation number is CGMCC No.1.12734, and the preservation date is 2020, 7 and 20 days.
The invention creatively discovers that the compounding of the Bifidobacterium longum (Bifidobacterium longum) BL21 strain and the Lactobacillus rhamnosus LRa05 strain has the obvious effect of reducing body fat, particularly the perirenal fat or abdominal wall fat, and creatively develops a new strategy for losing weight and reducing fat. Bifidobacterium longum and Lactobacillus rhamnosus belong to probiotics, are relatively healthy to human bodies, and are not easy to cause adverse reactions. Research shows that the product with BL21 strain and LRa05 strain as main effective components can obviously reduce the levels of TC (total cholesterol), LDL (low density lipoprotein cholesterol) and TG (triglyceride) in serum and liver, increase the levels of High Density Lipoprotein (HDL) in serum and liver, reduce body fat, especially reduce perirenal fat or abdominal wall fat, and has good application prospect.
Preferably, the ratio of the viable count of bifidobacterium longum BL21 to the viable count of lactobacillus rhamnosus LRa05 is (1-10):1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and the like, and other specific values in the numerical range can be selected, which is not described in detail herein.
Preferably, the complex probiotics that inhibit fat accumulation further comprise lactobacillus bulgaricus and/or streptococcus thermophilus; preferably a combination of Lactobacillus bulgaricus and Streptococcus thermophilus.
When the lactobacillus bulgaricus and/or streptococcus thermophilus which are probiotics are added to be compounded with the bifidobacterium longum BL21 and the lactobacillus rhamnosus LRa05, the lipid-lowering effect of the product can be further improved.
Preferably, the ratio of the viable count of the lactobacillus bulgaricus, the streptococcus thermophilus, the bifidobacterium longum BL21 and the lactobacillus rhamnosus LRa05 is (1-3): (2-4): (1-2): 1.
Wherein the specific point values in (1-3) can be selected from 1, 1.5, 2, 2.2, 2.5, 3, and the like; wherein the specific point values in (2-4) can be selected from 2, 2.5, 3, 3.5, 3.7, 4 and the like; wherein, the specific point value in (1-2) can be selected from 1, 1.2, 1.5, 1.8, 2, etc. Other specific values within the above range can be selected, and are not further described herein.
In a second aspect, the invention provides a composite probiotic freeze-dried powder for inhibiting fat accumulation, which comprises bifidobacterium longum BL21 freeze-dried powder and lactobacillus rhamnosus LRa05 freeze-dried powder.
Preferably, the composite probiotic freeze-dried powder for inhibiting fat accumulation further comprises lactobacillus bulgaricus freeze-dried powder and/or streptococcus thermophilus freeze-dried powder.
Preferably, the bifidobacterium longum BL21 freeze-dried powder, the lactobacillus rhamnosus LRa05 freeze-dried powder, the lactobacillus bulgaricus freeze-dried powder or the streptococcus thermophilus freeze-dried powder is prepared by a preparation method comprising the following steps:
centrifuging and washing the activated and expanded bacterial liquid to obtain bacterial sludge; mixing the bacterial sludge and the freeze-drying protective agent, and carrying out vacuum freeze-drying to obtain the microbial agent.
Preferably, the centrifugation is performed at 4000-7000rpm (e.g., 4000rpm, 5000rpm, 6000rpm, 7000rpm, etc.) for 10-25min (e.g., 10min, 12min, 15min, 20min, 25min, etc.); other specific values within the above range can be selected, and are not further described herein.
Preferably, the lyoprotectant includes any one or a combination of at least two of trehalose, sucrose, maltodextrin, or glycerol.
Preferably, the lyophilized powder after vacuum freeze-drying has a water content of less than 4% and a water activity of less than 0.1.
In a third aspect, the invention provides a composite probiotic for inhibiting fat accumulation as described in the first aspect or a composite probiotic freeze-dried powder for inhibiting fat accumulation as described in the second aspect, and the composite probiotic freeze-dried powder is used for preparing food, health-care products or medicines for reducing fat and losing weight.
Preferably, the food product comprises a solid food product, a liquid food product, a semi-solid food product.
Preferably, the dosage forms of the health care product and the medicament comprise any one of suspension, granules, capsules, powder, tablets, emulsion, solution, dripping pills and enemas.
Preferably, the medicament, health product or food further comprises any one of a filler, a wetting agent, a disintegrating agent, an adhesive, a protective agent and a prebiotic or a combination of at least two of the above.
In a fourth aspect, the invention provides a composite probiotic for inhibiting fat accumulation according to the first aspect or a composite probiotic freeze-dried powder for inhibiting fat accumulation according to the second aspect, and the composite probiotic freeze-dried powder is used for preparing a food, a health product or a medicine for inhibiting perirenal fat accumulation and abdominal wall fat accumulation.
The invention also creatively discovers that the composite probiotics can be used for specifically reducing perirenal fat accumulation and abdominal wall fat accumulation, and has important significance for people needing abdominal reduction.
In a fifth aspect, the invention provides fermented milk with lipid-lowering and weight-reducing effects, which is prepared from the following raw materials: raw milk, the composite probiotics for inhibiting fat accumulation, a sweetening agent and a stabilizing agent.
The fermented milk with the effects of reducing blood fat and weight is full in mouthfeel, free of essence and pigment, zero in sucrose, free of burden, high in protein and high in dietary fiber, capable of effectively promoting digestion and absorption of organisms, less in energy intake, and capable of meeting the requirements of contemporary people, particularly weight-losing people on low-sugar, healthy and nutritional diets.
Preferably, the preparation method comprises:
(1) preheating raw milk, mixing with sweetener and stabilizer, homogenizing, sterilizing, and cooling;
(2) inoculating the composite probiotics into the cooled raw milk, stirring and mixing uniformly, and fermenting to obtain the feed.
Preferably, the preheating is to 45-60 ℃, such as 45 ℃, 48 ℃, 50 ℃, 52 ℃, 55 ℃, 58 ℃, 60 ℃ and the like.
Preferably, the sweetener comprises sucralose and/or erythritol.
Preferably, the stabilizer comprises any one or a combination of at least two of polydextrose, whey protein powder, agar, pectin, diacetyl tartaric acid ester of mono-and diglycerides, or acetylated distarch phosphate.
The invention also creatively discovers that the combination of polydextrose, whey protein powder, agar, pectin, diacetyl tartaric acid ester of mono-and diglycerides and acetylated distarch phosphate ester of the fermented milk can be used as the stabilizer of the fermented milk to further improve the number of viable bacteria in the product, wherein the polydextrose, diacetyl tartaric acid ester of mono-and diglycerides and acetylated distarch phosphate ester of the fermented milk have a certain degree of synergistic interaction, after being eaten, the fermented milk can generate a probiotic effect on the intestinal tract of a human body, meet the requirements of mouth feel and nutrition of people, and simultaneously promote the digestion of fat in the body to the maximum extent, thereby helping people achieve the goal of losing weight.
Preferably, the homogenization is carried out at 55-65 ℃ (e.g., 55 ℃, 58 ℃, 60 ℃, 62 ℃, 65 ℃, etc.), 15-25MPa (e.g., 15MPa, 18MPa, 20MPa, 22MPa, 25MPa, etc.).
Preferably, the cooling is to 15-35 ℃, such as 15 ℃, 18 ℃, 20 ℃, 22 ℃, 25 ℃, 28 ℃, 30 ℃, 35 ℃ and the like.
Preferably, the complex probiotic is inoculated in an amount of 1-5%, such as 1%, 2%, 3%, 4%, 5%, etc.
Preferably, the fermentation is carried out at 37-41 ℃ (e.g., 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, etc.) with a fermentation acidity of 65-70 ° T (e.g., 65 ° T, 66 ° T, 67 ° T, 68 ° T, 69 ° T, 70 ° T, etc.) at the end of the fermentation.
Other specific point values within the above numerical range can be selected, and are not described in detail herein.
In a sixth aspect, the invention provides a solid beverage with lipid-lowering and weight-reducing effects, which is prepared from the following raw materials: the composite probiotic freeze-dried powder for inhibiting the fat accumulation, fructo-oligosaccharide, resistant dextrin, inulin and fruity powder of the second aspect.
In a seventh aspect, the present invention provides a preparation method of a solid beverage with lipid lowering and weight reducing effects, the preparation method comprising: the preparation method comprises the following steps of physically and uniformly mixing the preparation raw materials.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively discovers that the compounding of the Bifidobacterium longum (Bifidobacterium longum) BL21 strain and the Lactobacillus rhamnosus LRa05 strain has the obvious effect of reducing body fat, particularly the perirenal fat or abdominal wall fat, and creatively develops a new strategy for losing weight and reducing fat. Bifidobacterium longum and Lactobacillus rhamnosus belong to probiotics, are relatively healthy to human bodies, and are not easy to cause adverse reactions. Research shows that products taking BL21 strain and LRa05 strain as main functional components can obviously reduce the levels of TC (total cholesterol), LDL (low-density lipoprotein cholesterol) and TG (triglyceride) in serum and liver, improve the level of high-density lipoprotein (HDL) in serum and liver, reduce body fat, particularly reduce perirenal fat or abdominal wall fat, and have good application prospect.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
The following references to commercially available Bifidobacterium longum ATCC15707, commercially available Lactobacillus rhamnosus ATCC53103, commercially available Lactobacillus bulgaricus ATCC11842 and commercially available Streptococcus thermophilus ATCC 19258.
The preserved strain Bifidobacterium longum BL21 related in the following content is Bifidobacterium longum (Bifidobacterium longum) BL21 strain, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation time is 2015, 1 month and 27 days, the preservation number is CGMCC No.10452, and the address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The preserved strain LRa05 is Lactobacillus rhamnosus LRa05 strain, the preservation unit is China general microbiological culture Collection center of culture Collection of microorganisms, the preservation time is 2020, 7 and 20 days, the preservation number is CGMCC No.1.12734, and the address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The test animals referred to in the following are SPF grade male 8 week old KM mice.
The following related MRS solid culture media: weighing 10g of peptone, 10g of beef extract, 20g of glucose, 2g of sodium acetate, 5g of yeast powder, 2g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2.6g、MgSO 4 ·7H 2 O 0.1g、MnSO 4 0.05g of agar, 20g of agar and 0.5g of cysteine hydrochloride, dissolving by using deionized water, adding 1mL of Tween 80, diluting to a constant volume of 1L, sterilizing, cooling, and pouring into a sterilized culture dish for later use.
The following related MRS liquid culture media: weighing 10g of peptone, 10g of beef extract, 20g of glucose, 2g of sodium acetate, 5g of yeast powder, 2g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2.6g、MgSO 4 ·7H 2 O 0.1g、MnSO 4 0.05g and 0.5g of cysteine hydrochloride are dissolved by deionized water, 1mL of Tween 80 is added, the volume is adjusted to 1L, and the mixture is sterilized and cooled for standby.
Polydextrose as referred to below was purchased from jaboticaba food; the whey protein powder is a product which is purchased from Hebei Runzhou and has the model of WPC 35; pectin was purchased from Chengxi organisms; diacetyl tartaric acid esters of mono-and diglycerides are obtained from Derun food; acetylated distarch phosphate is a product of type CR3020 available from alzelese.
Example 1
Acid resistance test:
adjusting pH of MRS liquid culture medium to 2.0, 3.0 and 4.0 with 0.1mol/L hydrochloric acid, sterilizing at 121 deg.C for 20min, and cooling. Respectively inoculating activated bifidobacterium BL21 and lactobacillus rhamnosus LRa05 into MRS liquid culture media with the pH values of 2.0, 3.0 and 4.0 by the inoculation amount of 2%, culturing at 37 ℃, sampling at 0, 2, 3 and 4 hours respectively, measuring the viable count of the activated bifidobacterium BL21 and the activated lactobacillus rhamnosus LRa05 in different time periods by a plate counting method, and calculating the survival rate, wherein the calculated formula is that the viable count (%) acts for 2, 3 and 4 hours respectively/the initial viable count of 0 hour multiplied by 100 percent. The results are shown in table 1 (bifidobacterium BL21) and table 2 (lactobacillus rhamnosus LRa 05):
TABLE 1
Survival rate (%) 0h 2h 3h 4h
pH＝2 100 95.44 94.29 93.13
pH＝3 100 96.47 95.94 94.62
pH＝4 100 96.93 96.11 95.31
TABLE 2
Survival rate (%) 0h 2h 3h 4h
pH＝2 100 93.37 93.14 92.88
pH＝3 100 94.25 93.92 93.52
pH＝4 100 95.06 94.72 94.46
As can be seen from the data in tables 1 and 2: under the conditions of pH2.0 and pH3.0, the survival rate of the strain after 4 hours can still reach more than 92 percent, and under the condition of pH4.0, the survival rate of viable bacteria of the strain after 4 hours is more than 94 percent, which indicates that the bifidobacterium BL21 and the lactobacillus rhamnosus LRa05 have better gastric acid resistance.
Example 2
Bile salt tolerance test:
adjusting the pH value of the MRS liquid culture medium to 8.0, taking the MRS liquid culture medium without bile salt as a control, respectively adding the bovine bile salt with different concentrations into the culture medium to ensure that the mass concentrations are 0.03, 0.2, 0.3 and 0.5g/100mL, sterilizing at 121 ℃ for 20min, and cooling for later use. Respectively inoculating activated bifidobacterium BL21 and lactobacillus rhamnosus LRa05 into the treated culture medium in an inoculation amount of 2%, culturing at the constant temperature of 37 ℃, sampling for 0, 2, 3 and 4 hours respectively, measuring the viable count by using a plate counting method, and calculating to obtain the survival rate, wherein the calculation formula is as follows: the survival rate (%) is the number of viable cells exposed to the cells for different periods of time/initial number of viable cells × 100%. The results are shown in table 3 (bifidobacterium BL21) and table 4 (lactobacillus rhamnosus LRa 05):
TABLE 3
Survival rate (%) 0h 2h 3h 4h
0.03% bile salt 100 100.67 100.93 101.12
0.2% bile salt 100 99.61 98.94 98.53
0.3% bile salt 100 99.36 98.49 97.62
0.5% bile salt 100 98.52 97.53 96.27
TABLE 4
Survival rate (%) 0h 2h 3h 4h
0.03% bile salt 100 100.32 100.51 101.01
0.2% bile salt 100 99.57 98.74 98.46
0.3% bile salt 100 99.23 98.50 97.71
0.5% bile salt 100 98.33 97.76 96.42
From the above table data: in the process of entering the intestinal tract of a human body, gastric acid produced in the stomach of the human body and bile salt produced by secretion in the small intestine of the human body are main factors influencing the activity of bacteria in the intestinal tract. Generally, the food stays for about 2-4 hours after entering the stomach, while the pH of the gastric acid produced in the stomach of a human is usually maintained between 2.5 and 3.5; the content of bile and salt in human small intestine is generally 0.03% -0.3%, and the residence time of food in small intestine is about 1-4 h. The test results show that the bifidobacteria BL21 and the lactobacillus rhamnosus LRa05 have excellent gastric acid resistance and cholate resistance, and can be excellently adhered and fixedly planted in the intestinal tract of a human body, so that fat metabolism in the intestinal tract is accelerated, enterotoxin generation is reduced, and a probiotic effect is generated on the human body.
Example 3
Animal experiments:
(1) feeding and grouping experimental animals:
56 healthy adult SPF-grade male KM mice were selected, and after 2 weeks of adaptive feeding, the mice were randomly assigned to a control group, a model group, an intervention group a, an intervention group B, an intervention group C, and an intervention group D, 8 mice each. Wherein the control group comprises common feed and normal saline; the high fat model group is high fat feed and normal saline; the probiotic dry preparation group comprises a high-fat feed and probiotic physiological saline bacterial suspension, a group A single intragastric Bifidobacterium longum BL21 bacterial suspension, a group B single intragastric Lactobacillus rhamnosus LRa05 bacterial suspension, a group C mixed bacterial suspension of BL21 and LRa05 in intragastric mass ratio of 1:1, and a group D mixed bacterial suspension of ATCC15707 and ATCC53103 in intragastric mass ratio of 1: 1.
The dose of the gavage normal saline of each mouse is 0.25mL/d, and the dose of the composite probiotics gavage is adjusted along with the weight change of the mouse, which is 10 9 CFU/kg d, the breeding cycle was 8 weeks, and the change of body weight, perirenal fat factor, abdominal wall fat factor, blood lipid, and lipid index in liver in 7 groups of mice under different intervention conditions were analyzed by comparison.
All test mice were fed freely during the experiment and gavaged 1 time per day. During the gavage period, the weight of the mice is weighed every day, and the gavage amount is adjusted in time according to the weight change of each experimental mouse. In the course of all the experiments on mice, the ambient temperature (22 + -2 ℃) and humidity (55% + -10%) were controlled, the light-dark period was 12 hours, and the padding was replaced once a day. The test period was 8 weeks, and the mice were fasted for 12 hours before sacrifice, and the pre-sacrifice body weight was recorded for each group. After slaughter, the organs and fat in the abdominal and thoracic cavities of the mice were quickly removed, weighed and recorded.
(2) Preparing a mouse lipid detection sample:
(2.1) adopting an orbital blood collection method to collect a blood sample of the mouse, and standing the mouse at 25 ℃ for a period of time. Centrifuging at 4 deg.C under 3000rpm for 10min, sucking upper layer serum, and placing separated serum in EP tube, and refrigerating at-80 deg.C for use.
(2.2) diluting the liver with normal saline by 10 times according to the proportion of 1:9, fully grinding and uniformly mixing to prepare liver homogenate, fully grinding and mixing, and preparing the liver homogenate by low-temperature high-speed centrifugation. The supernatant was placed in an EP tube and stored in a refrigerator at-80 ℃.
(3) Index detection results and analysis:
(3.1) mouse blood samples collected from orbital bleeding were tested according to the instructions on the TC, TG, LDL and HDL kits. The results are shown in table 5:
TABLE 5
Group of TC level Level of TG LDL levels HDL levels
Control group 1.5±0.43 0.69±0.41 1.23±0.22 1.47±0.18
High fat model group 3.35±0.33 0.76±0.25 2.24±0.21 0.83±0.17
Intervention group A 2.06±0.24 0.63±0.22 1.71±0.26 1.11±0.37
Intervention group B 1.83±0.21 0.66±0.27 1.63±0.30 1.02±0.43
Intervention group C 1.54±0.17 0.43±0.38 1.13±0.18 1.13±0.31
Intervention group D 1.91±0.32 0.65±0.26 1.7±0.34 1.05±0.28
From the data in table 5 it can be seen that: compared with a control group, the serum TC, LDL and TG levels of the mice with high-fat diet are obviously increased, which shows that obesity caused by high-fat diet can cause the change of serum indexes in vivo; after intervention of the composite probiotics, the levels of all indexes in the serum of the mouse are obviously reduced, and experimental results show that the composite probiotics have obvious relieving effect on all index changes in the serum caused by fat accumulation. A great deal of research shows that the action mechanism of the probiotic bacteria is probably related to the activity of the high bile salt hydrolase of the probiotic bacteria; can increase High Density Lipoprotein (HDL) level. The HDL can remove excessive cholesterol in atherosclerotic plaques and prevent the cholesterol from being deposited on blood vessel walls, so that the incidence rate of atherosclerosis and other related cardiovascular diseases is effectively reduced, and the intervention of the compound probiotics can effectively inhibit fat accumulation and further relieve dyslipidemia induced by high-fat diet.
And (3.2) detecting the prepared liver homogenate supernate by adopting a TC, TG and LDL kit according to the instruction of the operation steps on the kit. The results are shown in Table 6:
TABLE 6
Group of TC level TG level LDL levels
Control group 1.17±0.25 0.67±0.34 0.97±0.12
High fat model group 1.96±0.13 0.93±0.26 2.03±0.19
Intervention group A 1.50±0.24 0.81±0.33 1.62±0.23
Intervention group B 1.32±0.17 0.64±0.22 1.58±0.21
Intervention group C 1.15±0.32 0.58±0.18 1.11±0.27
Intervention group D 1.30±0.27 0.71±0.31 1.35±0.25
From the data in table 6, it can be seen that: the level of lipid indicators in the liver of the mice tested on the high-fat diet was generally higher than in the control group and the probiotic intervention group. Under the condition of high-fat diet, after the dry prognosis of the compound probiotics, the lipid index level in the liver of the mouse is obviously lower than that of a high-fat diet group, which shows that the compound probiotics has obvious relieving effect on liver injury caused by fat accumulation in the body of the mouse caused by the high-fat diet.
(3.3) the body weight, perirenal fat factor (calculated as mouse perirenal fat mass/mouse body weight), and abdominal wall fat factor (calculated as mouse abdominal wall fat mass/mouse body weight) of each group of mice after 8 weeks of breeding were changed as shown in Table 7:
TABLE 7
Figure BDA0003616204840000131
Figure BDA0003616204840000141
From the data in table 7: after 8 weeks of feeding, the weight of the mice is obviously increased compared with the initial weight of the mice, wherein the weight of the mice in a high-fat model group is most obviously increased, and the weight of the mice in a composite probiotic dry prognosis group is obviously lower than that of the mice in a high-fat group. Compared with a normal control group, the perirenal fat coefficient and the abdominal wall fat coefficient of the mouse are obviously improved under the condition of high-fat diet, and the perirenal fat coefficient and the abdominal wall fat coefficient of the mouse are obviously reduced after the composite probiotics are dried, and the result shows that the composite probiotics can obviously reduce the weight increase of the mouse caused by high-fat diet and effectively inhibit the risk of obesity caused by the increase of abdominal fat caused by high-fat diet.
Application example 1
Preparation of the sucrose-free flavored fermented milk:
(1) selecting raw milk which meets the international standard requirements and does not contain antibiotics;
(2) preheating 929.55g of prepared raw milk to 55 ℃, adding 0.1g of sucralose, 14.0g of erythritol, 50g of polydextrose, 5.0g of whey protein powder, 0.6g of agar, 0.4g of pectin, 0.15g of diacetyl tartaric acid monoglyceride and 0.2g of acetylated distarch phosphate, stirring for dissolving, heating to 60 ℃, homogenizing under 20MPa, sterilizing at 100 ℃ for 10min, and cooling to 20 ℃;
(3) inoculating a starter (prepared by compounding BL21, LRa05, ATCC11842 and ATCC19258 with the viable bacteria ratio of 1:1:2: 3) into the product obtained in the step (2) according to the inoculation amount of 2 percent, uniformly stirring, fermenting for about 8 hours at the temperature of 41 ℃, and stopping fermentation when the texture is changed from liquid state to solid curd and the fermentation acidity is 70 DEG T;
(4) and (4) cooling the product obtained in the step (3) to 20 ℃, stirring, demulsifying, carrying out aseptic filling, carrying out overnight refrigeration at 4 ℃ after filling, and then aging to obtain the sucrose-free flavored fermented milk.
Application example 2
Preparation of the sucrose-free flavored fermented milk:
the preparation method is different from the application example 1 only in that in the step (2), ' 0.15g of diacetyl tartaric acid ester of mono-and-diglycerides and 0.2g of acetylated distarch phosphate ester are replaced by ' 0.35g of diacetyl tartaric acid ester of mono-and-diglycerides ', and other conditions are kept unchanged.
Application example 3
Preparation of the sucrose-free flavored fermented milk:
the preparation method is different from the application example 1 only in that "0.15 g of diacetyl tartaric acid ester of mono-and diglycerides, 0.2g of acetylated distarch phosphate ester" is replaced by "0.35 g of acetylated distarch phosphate ester" in the step (2), and other conditions are kept unchanged.
The acid status and viable cell count of the products obtained in application examples 1 to 3 were measured by follow-up measurement, and the results are shown in tables 8 to 10:
TABLE 8
Total viable count (CFU/mL) 0d 7d 21d
Application example 1 7.76×10 8 7.53×10 8 7.40×10 8
Application example 2 7.31×10 8 5.11×10 8 4.01×10 8
Application example 3 7.89×10 8 6.52×10 8 4.03×10 8
TABLE 9
BL21 viable cell count (CFU/mL) 0d 7d 21d
Application example 1 4.85×10 7 4.24×10 7 3.87×10 7
Application example 2 4.78×10 7 2.74×10 7 1.33×10 7
Application example 3 4.33×10 7 2.63×10 7 1.64×10 7
Watch 10
LRa05 viable count (CFU/mL) 0d 7d 21d
Application example 1 5.76×10 7 5.24×10 7 4.51×10 7
Application example 2 5.31×10 7 4.04×10 7 2.77×10 7
Application example 3 5.96×10 7 4.37×10 7 3.12×10 7
From the above data, it can be seen that: within 21d of shelf life, the acid condition of the fermented milk without the sucrose flavor is good, the mouthfeel of the fermented milk is not obviously influenced, the viable count of bifidobacterium and lactobacillus rhamnosus is high, and the viable count can be obviously influenced by the composition formula of the stabilizer. The fermented milk without the sucrose flavor is full in mouthfeel, free of essence and pigment, zero in sucrose and burden, high in protein and dietary fiber, capable of effectively promoting digestion and absorption of organisms, less in energy intake, and capable of meeting requirements of contemporary people, particularly weight-losing people on low-sugar, healthy and nutritional diets, and high in viable count of bifidobacteria, and capable of generating a probiotic effect on human intestinal tracts after being eaten, and promoting digestion of fat in vivo while meeting requirements of taste and nutrition of people, so that people are helped to achieve the weight-losing goal.
Application example 5
Preparing the probiotic solid beverage:
(1) BL21 strain and LRa05 strain were cultured at 37 ℃ for 18 hours, inoculated in an amount of 3% of MRS liquid medium, and activated for 3 consecutive generations. Centrifuging the activated and expanded seed liquid at 6500r/min for 20min, washing with sterile water for 2 times, centrifuging, and removing supernatant to obtain bacterial sludge.
(2) Adding a freeze-drying protective agent into the bacterial sludge obtained in the step (1) to obtain 10 10 CFU/mL of bacterial suspension, wherein the lyoprotectant is prepared by: adding 10-25% trehalose, 1-5% sucrose, 10-15% maltodextrin, and 0.1-0.5% glycerol into the water solution, sterilizing at 121 deg.C for 20min, and making into lyophilized protectant. Placing the mixture at-40 deg.C for prefreezing for 3h, placing at-15 deg.C for sublimation for 15h, placing at 25 deg.C for sublimation for 2h, and placing under vacuum for 2h to obtain BL21 lyophilized powder and LRa05 lyophilized powder with water content less than 4% and water activity less than 0.1.
(3) The probiotic solid beverage comprises the following formula:
(3.1) preparing the blueberry-flavored solid beverage: uniformly mixing BL21 lyophilized powder 10%, LRa05 lyophilized powder 10%, Lactobacillus acidophilus lyophilized powder 10%, fructo-oligosaccharide 10%, resistant dextrin 30%, inulin 10% and blueberry fruit powder 20%.
(3.2) preparing the cranberry flavor solid beverage: uniformly mixing 10% of BL21 freeze-dried powder, 10% of LRa05 freeze-dried powder, 10% of lactobacillus acidophilus freeze-dried powder, 10% of fructo-oligosaccharide, 30% of resistant dextrin, 10% of inulin and 20% of cranberry powder.
(4) The product specification is as follows: the probiotic solid beverage is in the form of 2 g/bar and 10 bars/box.
The applicant states that the present invention is illustrated by the above examples, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims (10)

1. A composite probiotic for inhibiting fat accumulation, which is characterized by comprising bifidobacterium longum BL21 and lactobacillus rhamnosus LRa 05;
the Bifidobacterium longum BL21 is Bifidobacterium longum (Bifidobacterium longum) BL21 strain, the preservation number is CGMCC No.10452, and the preservation date is 2015, 1 month and 27 days;
the Lactobacillus rhamnosus LRa05 is Lactobacillus rhamnosus LRa05 strain, the preservation number is CGMCC No.1.12734, and the preservation date is 2020, 7 and 20 days.
2. The composite probiotic bacteria for inhibiting fat accumulation according to claim 1, wherein the ratio of viable count of Bifidobacterium longum BL21 to that of Lactobacillus rhamnosus LRa05 is (1-10) to 1.
3. The fat accumulation inhibiting complex probiotic according to claim 1 or 2, characterized in that it further comprises lactobacillus bulgaricus and/or streptococcus thermophilus; preferably lactobacillus bulgaricus and streptococcus thermophilus;
preferably, the ratio of the viable count of the lactobacillus bulgaricus, the streptococcus thermophilus, the bifidobacterium longum BL21 and the lactobacillus rhamnosus LRa05 is (1-3): (2-4): (1-2): 1.
4. The composite probiotic freeze-dried powder for inhibiting the fat accumulation is characterized by comprising bifidobacterium longum BL21 freeze-dried powder and lactobacillus rhamnosus LRa05 freeze-dried powder.
5. The fat accumulation inhibiting composite probiotic freeze-dried powder of claim 4, further comprising Lactobacillus bulgaricus freeze-dried powder and/or Streptococcus thermophilus freeze-dried powder;
preferably, the bifidobacterium longum BL21 freeze-dried powder, the lactobacillus rhamnosus LRa05 freeze-dried powder, the lactobacillus bulgaricus freeze-dried powder or the streptococcus thermophilus freeze-dried powder is prepared by a preparation method comprising the following steps:
centrifuging and washing the activated and expanded bacterium liquid to obtain bacterium mud; mixing the bacterial sludge with a freeze-drying protective agent, and carrying out vacuum freeze-drying to obtain the bacterial sludge;
preferably, the centrifugation is performed at 4000-7000rpm for 10-25 min;
preferably, the lyoprotectant comprises any one or a combination of at least two of trehalose, sucrose, maltodextrin, or glycerol;
preferably, the lyophilized powder after vacuum freeze drying has a water content of less than 4% and a water activity of less than 0.1.
6. Use of the composite probiotics for inhibiting fat accumulation according to any one of claims 1 to 3 or the freeze-dried powder of composite probiotics for inhibiting fat accumulation according to any one of claims 4 to 5 in the preparation of food, health product or medicine for reducing fat and losing weight.
7. Use of the composite probiotics for inhibiting fat accumulation according to any one of claims 1 to 3 or the freeze-dried powder of composite probiotics for inhibiting fat accumulation according to any one of claims 4 to 5 in the preparation of food, health-care product or medicine for inhibiting perirenal fat accumulation and abdominal wall fat accumulation.
8. The fermented milk with the effects of reducing fat and losing weight is characterized by being prepared from the following raw materials: raw milk, the composite probiotic for inhibiting fat accumulation according to any one of claims 1 to 3, a sweetener and a stabilizer.
9. The method for producing fermented milk with lipid-lowering and weight-reducing effects according to claim 8, comprising:
preheating raw milk, mixing with sweetener and stabilizer, homogenizing, sterilizing, and cooling;
inoculating the composite probiotics into the cooled raw milk, stirring and uniformly mixing, and fermenting to obtain the feed additive;
preferably, the preheating is to 45-60 ℃;
preferably, the sweetener comprises sucralose and/or erythritol;
preferably, the stabilizer comprises any one or a combination of at least two of polydextrose, whey protein powder, agar, pectin, diacetyl tartaric acid ester of mono-and diglycerides or acetylated distarch phosphate;
preferably, the homogenization is carried out at 55-65 ℃ and 15-25 MPa;
preferably, the cooling is to 15-35 ℃;
preferably, the inoculation amount of the composite probiotics is 1-5%;
preferably, the fermentation is carried out at 37-41 ℃ with fermentation acidity of 65-70 ° T at the end of the fermentation.
10. The solid beverage with the effects of reducing blood fat and losing weight is characterized in that the solid beverage with the effects of reducing blood fat and losing weight is prepared from the following raw materials: composite probiotic freeze-dried powder for inhibiting fat accumulation, fructo-oligosaccharide, resistant dextrin, inulin and fruity powder as claimed in any one of claims 4 to 5.
CN202210448054.9A 2022-04-26 2022-04-26 Composite probiotics for inhibiting fat accumulation and application thereof Pending CN114921363A (en)

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CN112210507A (en) * 2020-08-10 2021-01-12 江苏微康生物科技有限公司 Shigella-antagonistic lactobacillus rhamnosus LRa05, screening method and application thereof
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CN117821343A (en) * 2024-03-05 2024-04-05 微康益生菌(苏州)股份有限公司 Composite probiotics for regulating blood glucose metabolism and application thereof

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