CN113995139A - Probiotic composition and application thereof - Google Patents
Probiotic composition and application thereof Download PDFInfo
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- CN113995139A CN113995139A CN202111262275.9A CN202111262275A CN113995139A CN 113995139 A CN113995139 A CN 113995139A CN 202111262275 A CN202111262275 A CN 202111262275A CN 113995139 A CN113995139 A CN 113995139A
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Images
Classifications
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- 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
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- 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
- A23L33/17—Amino acids, peptides or proteins
- A23L33/175—Amino acids
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- 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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- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/113—Acidophilus
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- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/121—Brevis
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- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
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- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The application belongs to the technical field of probiotics, and particularly relates to a probiotic composition and an application patent application thereof. The probiotic composition comprises: lactobacillus brevis, Lactobacillus paracasei, and Lactobacillus acidophilus. The probiotic composition can be used for relieving anxiety. In the application, the activity and the stability of the probiotics are taken as basic indexes, the actual function difference is combined, and a better probiotic combination is obtained by selecting and combining the existing probiotics. Preliminary experiment results show that the probiotic composition has good activity and stability, and the viable count can be further increased after the probiotic composition is further compounded with gamma-aminobutyric acid, so that the culture effect is improved. Through preliminary mouse behavior experiments, the probiotic composition also has a certain technical effect of relieving anxiety, so that the probiotic composition has the potential of being used as a probiotic dietary supplement for food or health care products.
Description
Technical Field
The application belongs to the technical field of probiotics, and particularly relates to a probiotic composition and an application patent application thereof.
Background
Probiotics are a general term for a group of active microorganisms beneficial to the host, usually residing mainly in the gut of the host, and exerting a beneficial effect on the life activities of the host by participating in the conversion and absorption of nutrients or by synthesizing related metabolites. Common probiotics mainly comprise yeast, probiotic bacillus, clostridium butyricum, lactobacillus, bifidobacterium, actinomycetes and the like.
Some studies have shown that intestinal microorganisms not only play an important role in the transformation of nutrients in the intestine, but also influence the relevant neuromodulation activities of humans and animals via the "gut-brain axis". That is, under the influence of external stimulation, intestinal microorganisms and intestinal cells generate various signal molecules, the signal molecules can further communicate with the central nervous system through vagus nerve activation, and the central nervous system generates corresponding stress signals for the intestinal tract and the organism after analyzing the signals, so as to guide the organism to respond to external pressure and stress.
Probiotic dietary supplements have developed rapidly in recent years as a stand-alone product because of the safety and good mouthfeel adaptability of probiotics. It should be noted that unlike traditional probiotic products, probiotic fermented products such as yoghurt are mainly concerned with taste and nutrition, the number of live probiotic bacteria is relatively low, and cold chain preservation is required. The probiotic acid yoghurt product has high viable count and high moisture, and also needs cold chain storage, and has a shelf life of about one week. The probiotic dietary supplement is used as a separate health-care and additive product, is usually formed by combining high-concentration probiotic freeze-dried powder and prebiotics, and is packaged in a tin foil strip bag isolated from air (filled with nitrogen gas), moisture and light in a powdery form to achieve the functions of protecting intestinal health, reducing anaphylactic reaction, improving the immunity level of an organism and the like.
As the active microbial strains have different functions of different probiotics, the proper combination of the probiotics and how to ensure the higher survival rate of the probiotics are important prerequisites for ensuring that the probiotics exert the corresponding 'probiotic' function. Researches have shown that the number of live bacteria of the probiotics is usually more than 100 hundred million/g to ensure certain probiotic effect. On the other hand, the shelf life of the probiotic dietary supplement product is usually 12 months, which is far beyond the common probiotic fermented food, so that the proper combination of the probiotic strains is the primary technical premise for better ensuring the functions and the shelf life of the probiotic dietary supplement product and delaying the reduction of the viable count of the probiotics.
Disclosure of Invention
Based on the consideration of probiotic activity and specific functional application, the application aims to provide a probiotic combination, which is beneficial to prolonging shelf life on the one hand and has certain beneficial functions in relieving anxiety and improving sleep on the other hand on the premise of ensuring that different probiotic combinations have better biological activity and stability.
The technical solution of the present application is detailed as follows.
A probiotic composition, the probiotic composition comprising: lactobacillus brevis, Lactobacillus paracasei, Lactobacillus acidophilus;
preferably, the probiotic composition further comprises gamma-aminobutyric acid, inulin or theanine for synergistically enhancing the activity and stability of the mixed probiotic;
selecting Lactobacillus brevis with a deposit number of CICC 6239;
selecting cheese bacillus paracasei with the deposit number of CICC 25051;
the Lactobacillus acidophilus is Lactobacillus acidophilus CICC 6093.
In the probiotic composition, the viable count of the three probiotics is not less than 150 hundred million/g respectively, and the dosage of the gamma-aminobutyric acid is preferably 100 mg/g.
A probiotic dietary supplement prepared using the probiotic composition for the relief of anxiety; the probiotic dietary supplement is in a solid powder form, and usually comprises prebiotic components (such as inulin, fructo-oligosaccharide and the like) besides the probiotic powder composition, and in terms of mass percentage, in an actual product, the proportion of the probiotic powder is usually about 10% -50%, the addition amount of the prebiotic is usually about 50% -80%, and the balance is a small amount of auxiliary material components for keeping fresh and keeping dry.
In the early experimental research process, based on the needs of the food field, the inventor preliminarily screens more than 20 strains of lactobacillus from the existing preservation organization, and further rescreens the strains according to the performance needs of in vitro stability, gastric acid environment, bile acid salt tolerance and the like. Meanwhile, a plurality of bacterial strains which have the effects of preventing and slowing down anxiety and depression caused by pressure are screened out by combining with a mouse model for stimulating acute stress response by external pressure. Furthermore, a better probiotic combination is obtained by selecting and combining the existing probiotics by taking the activity and stability of the probiotics as basic indexes and combining the actual function difference. On the other hand, it has been shown that gamma aminobutyric acid (also called GABA), an amino acid present in the human brain, is an important inhibitory neurotransmitter in the nervous system. Therefore, in order to further improve the probiotic effect of the probiotic composition on relieving mental stress and improving sleep, the inventor further compounds the probiotic composition with gamma-aminobutyric acid. The preliminary experiment result shows that the compound has a beneficial effect on further improving the viable count.
In general, the probiotic composition has certain probiotic effects on relieving anxiety and improving sleep on the premise of ensuring good activity and stability of the probiotic composition through compounding and combination of related probiotics and compounding with GABA, so that the probiotic composition also has certain application potential in preparation of health care products.
Drawings
FIG. 1 shows the cell counts in different combinations;
FIG. 2 is an elevated plus maze experimental scenario setup;
FIG. 3 is the time for different treatment groups of mice to enter the middle zone in the open field experiment;
FIG. 4 shows the number of times mice in different treatment groups entered the central area in the open field experiment;
FIG. 5 is the ratio of open arm residence time to total time for different treatment groups of mice in an elevated plus maze experiment;
FIG. 6 is the ratio of the number of open arms entered to the total number of arms entered for the mice of different treatment groups in the elevated plus maze experiment.
Detailed Description
The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.
And (3) microbial strains:
lactobacillus brevis with a deposit number CICC 6239, and the prior art is mainly used for producing pickle, lactic acid and flavor substances; belong to publicly available strains;
the cheese bacillus paracasei Lactcasei with the accession number of CICC 25051 is Lactcaseibacillus paracasei, and the prior art is mainly used for vinegar brewing and acid production; belong to publicly available strains;
lactobacillus acidophilus with a accession number CICC 6093, which is mainly used for the production of probiotic health food in the prior art; belong to publicly available strains.
Example 1
On the basis of early-stage preliminary experiments, based on the screening and use differences of strains used in fermented food processing, combined with strain activity and stability indexes, and combined with in-vitro stability and gastric acid environment tolerance performance (in an artificial gastric juice in-vitro simulation experiment, survival rate reaches more than 85% under the conditions of 37 ℃ and pH2.0 is taken as a screening index), the inventor finally selects lactobacillus brevis, cheese lactobacillus paracasei and lactobacillus acidophilus as probiotic combinations.
On the other hand, based on the existing GABA (gamma-aminobutyric acid) research, in view of the mutual feedback relationship between the GABA (gamma-aminobutyric acid) as a metabolite and the activity of the strain and in view of the probiotic effect on improving sleep and the like, for the probiotic combination designed by the application, the inventor introduces a proper amount of GABA (gamma-aminobutyric acid) into a culture system to examine the actual influence of GABA (gamma-aminobutyric acid) on the activity of the strain.
It should be noted that in the experimental design, GABA (gamma-aminobutyric acid) is introduced into the culture system, and meanwhile, based on the prior art data, commonly used probiotic substances such as maltitol, theanine, emblic leafflower fruit extract, inulin and the like are synchronously selected as controls (the related substances are all common commercial products) to investigate the influence of different materials on the activity of the probiotic combination. For these material choices, the main factors considered are: inulin is a common prebiotic and can be used as a carbon source for growth of lactic acid bacteria and the like; theanine as plant extract has strong antioxidant function, and can resist oxygen free radical generated by stress and protect probiotic activity.
The experimental process comprises the following steps:
respectively selecting freeze-dried probiotic powder (purchased from CICC) of 0.3 g of each of the following strains, and uniformly mixing (the number of viable bacteria is 500 hundred million/g): lactobacillus brevis CICC 6239; lactobacillus paracasei Lactcasei CICC 25051 and Lactobacillus acidophilus Acidophilus CICC 6093.
0.1 g of prebiotics (maltitol, GABA, theanine, emblic leafflower fruit extract or inulin) is added into the 0.9 g of probiotic mixture respectively to obtain 1g of experimental finished product which can be used as a probiotic dietary supplement (the total number of viable bacteria in 1g of finished product is 450 hundred million/g).
The probiotic dietary supplement is tested to be naturally placed for 7 days under the room temperature condition of constant temperature and humidity (the temperature is 24 ℃, and the humidity is 60%) (the shelf life of the probiotic dietary supplement is obviously shortened compared with that of a normal probiotic dietary supplement because a sealing storage mode is not adopted, namely, the test is accelerated to investigate the influence of different prebiotic components on the viable count).
After 7 days, the number of the remaining viable bacteria is detected according to the national standard lactobacillus detection method (GB 4789.35-2016 lactobacillus test).
The results are shown in FIG. 1 (control group is pure bacterial powder without other components). It can be seen that the number of viable bacteria was reduced to some extent during the storage period compared to the original number of viable bacteria (450 hundred million/gram), but when mixed with different prebiotics, the number of viable bacteria was reduced to a different extent. Wherein, when the inulin is compounded and mixed with the inulin, the retention degree of viable count is the highest, and theanine and GABA are the second. Therefore, from this experimental result, inulin, theanine and GABA are apparently better formulated compositions in terms of maintaining the activity of the cells with respect to the specific combination of strains of the present application.
Example 2
As mentioned above, in view of the potential health-care use of GABA, the inventors further investigated the use of GABA in combination with probiotics, and the specific experimental summary is as follows.
(one) GABA Effect on probiotic Combined Activity
Referring to the above operation, 0.3 g of each of the following strains of freeze-dried probiotic powder was selected and mixed uniformly (the number of viable bacteria was 500 hundred million/g): lactobacillus brevis CICC 6239; lactobacillus paracasei Lactcasei CICC 25051 and Lactobacillus acidophilus Acidophilus CICC 6093.
And then 0.1 g of GABA (namely the number of live bacteria of the three probiotics in the finished product is 150 hundred million/g respectively, and the total number of bacteria is equal to 450 hundred million/g) is added into the 0.9 g of probiotic mixture to obtain 1g of experimental finished product which can be used as a probiotic dietary supplement. The group treated without the addition of gamma-aminobutyric acid was used as a control.
According to the method for counting the viable lactobacillus by the national standard method, compared with the contrast without adding gamma-aminobutyric acid, the viable count is respectively increased by 26.3 percent, 51.5 percent and 37.6 percent. That is, the experimental result shows that the addition of the gamma-aminobutyric acid has a better synergistic enhancement effect on the maintenance of the activity of the mixed probiotics bacteria count.
(II) anxiety-relieving Effect
To accurately evaluate the application effect of the probiotic composition in the aspect of relieving anxiety, the inventor further evaluates the relevant effect through a mouse open field experiment and an elevated plus maze experiment. The specific experimental procedures are briefly described as follows.
In each experiment, 50 male ICR mice of SPF class 6 weeks old were randomly divided into 5 groups of 10 animals per group according to body weight, i.e.: blank group, probiotic + GABA water solution group (total viable bacteria number is 450 hundred million in each g of water solution, GABA100 mg), probiotic group (viable bacteria number is 450 hundred million in each g of probiotic water solution), GABA group (100 mg/g) and positive control group (diazepam water solution, dosage is calculated according to 3mg/kg.
Performing intragastric administration for each treatment group for 1 time every morning, wherein the intragastric administration amount is 20 mL/kg.bw; the blank group is filled with sterile normal saline every day, the probiotic and GABA water solution group is filled with probiotic and GABA water solution, and the other three groups are filled with probiotic solution, GABA solution and diazepam water solution respectively; the stomach is drenched for 30 consecutive days.
In the open field experiment process, the activity change of the mouse in the central area of the open field is used as an index for evaluating the anxiety state. It is to be explained that anxiolytic treatment does not by itself increase the mice's exploration for open field but they reduce the inhibitory effect of anxiety on exploration behavior, and thus an increase in the mice's trajectory of activity in the central open field may indicate that the mice are relieved of anxiety.
The elevated plus maze is an experimental device (structure shown in fig. 2) having a pair of open arms and a pair of closed arms, and during the experiment of the elevated plus maze, rodents tend to move in the closed arms due to darkness but move in the open arms due to curiosity and exploration. In the face of a novel stimulus, the animals produce both exploratory impulses and fear, which create conflicting exploratory and avoidant behaviors and thus create an anxious mind. The anxiolytic can obviously increase the times and time for entering the open arm. That is, the effect of the application of the anxiolytic is evaluated by the number of times and the time the animal enters the open arm.
The results of the open field experiment are shown in fig. 3 and 4. The statistical results show that: compared with a probiotic group or a GABA group, the probiotic and GABA water solution group can better increase the residence time of the mouse in the central area and increase the frequency of entering the central area, namely, the probiotics and the GABA have a certain synergistic effect, so that the anxiety relieving effect can be further improved; even if compared with the positive control group, the probiotic + GABA water solution group is slightly lower than the positive control group, but the product provided by the application still has better technical value by considering the characteristics of safety, no toxic or side effect and the like of the product provided by the application.
The results of the elevated plus maze experiment are shown in fig. 5 and 6. Statistical results show that compared with a single probiotic group or a GABA group, GABA and probiotics show a certain synergistic effect, when the GABA and the probiotics are used in a compounding mode, the times of entering the open arms of mice can be further increased, the staying time of the mice in the open arms can be prolonged, and the effect is equivalent to that of a positive group. This further demonstrates the technical effect of the products provided by the present application in terms of anxiety.
Claims (5)
1. A probiotic composition, characterized in that it comprises: lactobacillus brevis, Lactobacillus paracasei, Lactobacillus acidophilus.
2. The probiotic composition according to claim 1, characterized in that it contains gamma-aminobutyric acid, inulin or theanine for synergistically enhancing the activity and stability of the mixed probiotic.
3. The probiotic composition according to claim 1, wherein the Lactobacillus brevis is selected from Lactobacillus brevis with deposit number CICC 6239;
selecting cheese bacillus paracasei with the deposit number of CICC 25051;
the Lactobacillus acidophilus is Lactobacillus acidophilus CICC 6093.
4. The probiotic composition according to claim 1, wherein the number of viable bacteria of the three probiotics is not less than 150 hundred million/g, and the amount of the gamma-aminobutyric acid is 100 mg/g.
5. Use of a probiotic composition according to claim 1 in the preparation of a probiotic dietary supplement.
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