CN117778274A - Fermented lactobacillus mucilaginosus, edible additives, foods, medicines, feeds and applications and methods of the same - Google Patents

Abstract

The application discloses a fermented lactobacillus mucilaginosusLimosilactobacillus fermentum) WIS32 with the preservation number of CGMCC No.29244, and also discloses edible additives, medicines, foods and feeds containing the bacteria, and application and a method of the bacteria. The fermented lactobacillus mucilaginosus WIS32 has good biological activity under a certain acidic environment and can resist artificial gastric juice; also has good in vitro antioxidation capability, reduces inflammatory factors in serum, increases the content of brain-derived neurotrophic factors and 5-hydroxytryptamine, and can inhibit pathogenic bacteria; the application provides an edible additive, a medicine, food, feed and application and a method of the bacterium, which also have the beneficial effects respectively.

Description

Fermented lactobacillus mucilaginosus, edible additives, foods, medicines, feeds and applications and methods of the same

Technical Field

The application belongs to the technical field of microbiology, and particularly relates to lactobacillus mucilaginosus, edible additives, foods, medicines, feeds and application and a method of the lactobacillus mucilaginosus.

Background

Mental disorders refer to malfunctions that occur in thinking, emotion, behavior, or social aspects, resulting in individuals experiencing difficulty in daily life. This term is commonly used to describe a range of psychological diseases and symptoms associated with brain dysfunction, depression, bipolar disorder, obsessive-compulsive disorder being a typical mental disorder. Neurological disorders refer to a class of disorders that affect the function of the central nervous system (including the brain and spinal cord) or the peripheral nervous system (including nerve roots, plexuses, and nerve endings). These diseases may involve nerve cells, connections between neurons, neurotransmitter balance, and the like. Neurological diseases are closely related to the content of brain-derived neurotrophic factors.

For example, a great deal of research suggests that inflammatory response is closely related to pathophysiological mechanisms of depression, and when systemic infection, cancer or autoimmune diseases occur, immune signals caused by activation of immune systems can cause disease deterioration, so that emotion of susceptible people is influenced, and depression symptoms occur. Injection of Lipopolysaccharide (LPS) into the lateral ventricle causes hippocampal neuroinflammatory states, increases mRNA expression of pro-inflammatory cytokines in the brain, including IL1B, IL-6 and TNF-a, and induces depression-like behavior. At present, antidepressant drugs widely applied to clinical treatment, such as selective 5-hydroxytryptamine reuptake inhibitor (Selective Serotonin Reuptake Inhibitor, SSRI) have the defects of slow onset of action, inconsistent treatment effect, large side effect and the like.

Probiotics are a widely accepted dietary supplement by consumers. The probiotic bacteria are used for regulating intestinal flora and further improving the nerve function, and become a new means for treating depression. Certain bifidobacteria and lactobacilli are able to modulate neurological functions, e.gBifidobacterium longum R0175、Lactobacillus helveticus R0052, etc. Thus, finding a strain having improved nerve function is an important direction of research。

Disclosure of Invention

The embodiment of the application provides a lactobacillus mucilaginosus fermentation methodLimosilactobacillus fermentum) WIS32, which has good biological activity under certain acid environment and can resist artificial gastric juice; also has good in vitro antioxidant capacity, and can inhibit pathogenic bacteria, reduce inflammatory factors in serum, and increase brain-derived neurotrophic factor and 5-hydroxytryptamine content. The application also provides edible additives, foods, medicines, feeds and applications and methods of the bacteria, which have the beneficial effects respectively.

In a first aspect, embodiments of the present application provide a fermented lactobacillus mucilaginosusLimosilactobacillus fermentum) WIS32 with preservation number of CGMCC No.29244.

In a second aspect, embodiments of the present application provide a cell comprising lactobacillus mucilaginosus.

In a third aspect, embodiments of the present application provide an extract comprising lactobacillus mucilaginosus.

In a fourth aspect, embodiments of the present application provide a culture comprising lactobacillus mucilaginosus.

In a fifth aspect, embodiments of the present application provide a lysate comprising lactobacillus fermentum.

In a sixth aspect, embodiments of the present application provide an edible additive comprising:

lactobacillus mucilaginosus of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect; and edible excipient.

In a seventh aspect, embodiments of the present application provide a food product comprising lactobacillus fermentum of the first aspect or thallus of the second aspect or extract of the third aspect or culture of the fourth aspect or lysate of the fifth aspect or edible additive of the sixth aspect.

According to an embodiment of one aspect of the present application, the food product comprises 1×10 ³ ~1×10 ¹⁴ CFU/mL or 1X 10 ³ ~1×10 ¹⁴ CFU/g fermented mucilageAnd (3) bacillus.

In an eighth aspect, embodiments of the present application provide a feed comprising lactobacillus fermentum of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect or the edible additive of the sixth aspect.

According to an embodiment of an aspect of the present application, the feed comprises 1×10 ³ ~1×10 ¹⁴ CFU/mL or 1X 10 ³ ~1×10 ¹⁴ CFU/g fermented lactobacillus mucilaginosus.

In a ninth aspect, embodiments of the present application provide a medicament comprising lactobacillus fermentum of the first aspect or thallus of the second aspect or extract of the third aspect or culture of the fourth aspect or lysate of the fifth aspect; and

a pharmaceutically acceptable carrier.

According to an embodiment of one aspect of the present application, the medicament comprises 1×10 ³ ~1×10 ¹⁴ CFU/mL or 1X 10 ³ ~1×10 ¹⁴ CFU/g fermented lactobacillus mucilaginosus.

In a tenth aspect, embodiments of the present application provide the use of the lactobacillus fermentum of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect in the manufacture of a medicament for preventing or ameliorating a psychotic disorder or in the manufacture of a medicament for delaying or preventing or treating a disorder associated with free radicals, wherein the psychotic disorder comprises one or more of schizophrenia pedigree disorder, attention deficit hyperactivity disorder, anxiety disorder, affective disorder; the diseases related to free radicals are any one or more of arteriosclerosis, diabetes, cardiovascular diseases, rheumatic arthritis, senile eye diseases and enteritis.

In an eleventh aspect, the embodiments of the present application provide the use of the lactobacillus fermentum of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect in the manufacture of a medicament for preventing or ameliorating a neurological disorder, wherein the neurological disorder comprises one or more of a neurodegenerative disorder, schizophrenia; the neurodegenerative disease includes Alzheimer's disease.

In a twelfth aspect, embodiments of the present application provide a method of relaxing and/or mood easing a person, comprising:

providing one or more of lactobacillus mucilaginosus of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect or the edible additive of the sixth aspect;

mixing a carrier with one or more of lactobacillus mucilaginosus, and thalli thereof, and extracts thereof, and cultures thereof, and lysates thereof, to obtain a mixture;

allowing the subject in need of relaxation and/or mood relief to consume the mixture.

In a thirteenth aspect, embodiments of the present application provide a method for preventing or ameliorating a mental disorder, comprising:

providing one or more of lactobacillus mucilaginosus of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect or the edible additive of the sixth aspect;

the carrier and the fermented lactobacillus mucilaginosus are treatedLimosilactobacillus fermentum) Mixing one or more of WIS32, and dry products thereof, and extracts thereof, and cultures thereof, and lysates thereof, to obtain a mixture;

a subject in need of prevention or amelioration of a psychotic disorder comprising one or more of schizophrenic spectrum disorders, attention deficit hyperactivity disorder, anxiety disorders, affective disorders is fed a mixture.

In a fourteenth aspect, embodiments of the present application provide a method for delaying or preventing or treating a disease associated with free radicals, comprising:

providing one or more of lactobacillus mucilaginosus of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect or the edible additive of the sixth aspect;

mixing a carrier with one or more of the lactobacillus mucilaginosus, and the thalli thereof, and the extracts thereof, and the cultures thereof, and the lysates thereof, to obtain a mixture;

feeding the mixture to a subject in need of delay or prevention or treatment of a free radical related disease, wherein the free radical related disease is any one or more of arteriosclerosis, diabetes, cardiovascular disease, rheumatoid arthritis, senile eye disease, enteritis.

The embodiment of the application has at least the following beneficial effects:

the embodiment of the application provides lactobacillus mucilaginosusLimosilactobacillus fermentum) WIS32 is a gram positive, motionless, facultative anaerobic lactic acid bacterium. The research shows that the strain can be used as probiotics, and has the physiological functions of resisting pathogenic bacteria and virus infection, regulating human immunity and the like. More importantly, the composition has good biological activity under a certain acidic environment and can resist artificial gastric juice; the strain also has good in-vitro antioxidation capability, can inhibit pathogenic bacteria, reduce inflammatory factors in serum, increase the content of brain-derived neurotrophic factors and 5-hydroxytryptamine, expands the application range of the strain, and has great significance for deep excavation of the function of probiotics and development of probiotics with higher health care value. Because the fermented lactobacillus mucilaginosus has antidepressant potential, a new way and a new solution are opened up for alleviating depression by using a diet strategy.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows a colony characterization graph (A), a gram staining microscopy graph (B) and a Columbia haemolysis characterization graph (C) of strains of the examples of the present application on MRS medium;

FIG. 2 shows a growth curve of Lactobacillus fermentum according to an embodiment of the present application;

FIG. 3 shows statistical graphs of experimental results of improved LPS-induced depression mice behavior of lactobacillus fermentum of the examples of the present application;

FIG. 4 shows statistical graphs of the levels of hippocampal 5-hydroxytryptamine and brain-derived neurotrophic factors in depressed mice induced by LPS increase by Lactobacillus fermentum according to the examples of the present application;

figure 5 shows statistical graphs of inflammatory factor levels in hippocampus and serum of depressed mice induced by reduced LPS by lactobacillus fermentum of the examples of the present application.

Information on preservation of strains

The application provides fermented lactobacillus mucilaginosusLimosilactobacillus fermentumThe strain is named WIS32 and is preserved in China general microbiological culture Collection center (CGMCC) with the following addresses: the collection number is CGMCC No.29244, and the strain is received by the collection center on the 12 th year of 2023 and registered in a book on the 06 th year of 2023, and is detected as a survival strain on the 12 th year of 2023 by the collection center.

Detailed Description

In order to make the objects, technical solutions and advantageous technical effects of the present application clearer, the present application is described in detail below with reference to specific embodiments. It should be understood that the embodiments described in this specification are for purposes of illustration only and are not intended to limit the present application.

For simplicity, only a few numerical ranges are explicitly disclosed herein. However, any lower limit may be combined with any upper limit to form a range not explicitly recited; and any lower limit may be combined with any other lower limit to form a range not explicitly recited, and any upper limit may be combined with any other upper limit to form a range not explicitly recited. Furthermore, each point or individual value between the endpoints of the range is included within the range, although not explicitly recited. Thus, each point or individual value may be combined as a lower or upper limit on itself with any other point or individual value or with other lower or upper limit to form a range that is not explicitly recited.

In the description herein, unless otherwise indicated, "above" and "below" are intended to include the present number, and the meaning of "several" in "one or several" means two or more. The meaning of "plural" in "one or more" means two or more.

The above summary of the present application is not intended to describe each disclosed embodiment or every implementation of the present application. The following description more particularly exemplifies illustrative embodiments. Guidance is provided throughout this application by a series of embodiments, which may be used in various combinations. In the various examples, the list is merely a representative group and should not be construed as exhaustive.

In a first aspect, embodiments of the present application provide a fermented lactobacillus mucilaginosusLimosilactobacillus fermentum) WIS32 with preservation number of CGMCC No.29244.

The fermented lactobacillus mucilaginosus is preserved in China general microbiological culture collection center (CGMCC) for 12 and 06 days in 2023, and is classified and named as fermented lactobacillus mucilaginosus (CGMCC, no. 3 of North Xiyun, the Korean region of Beijing, china)Limosilactobacillus fermentum) The preservation number is CGMCC No.29244.

The study shows that the fermented lactobacillus mucilaginosus is a kind of gram-positive, motionless and facultative anaerobic lactobacillus. Many researches show that the fermented lactobacillus mucilaginosus is used as probiotics and has the physiological functions of resisting pathogenic bacteria and virus infection, regulating human immunity and the like. The fermented lactobacillus mucilaginosus WIS32 screened by the invention can effectively relieve depression symptoms, expands the application range of lactobacillus paracasei as probiotics, and has very important significance for deep excavation of the functions of the probiotics and development of the probiotics with higher health care value. Meanwhile, the antidepressant potential of the lactobacillus mucilaginosus is fermented, and a new way and a solution are opened up for alleviating depression by using a diet strategy.

The fermented lactobacillus mucilaginosus has good biological activity under a certain acidic environment and can resist artificial gastric juice; also has good in vitro antioxidation capability, can inhibit pathogenic bacteria, reduce inflammatory factors in serum, and increase BNDF and 5-hydroxytryptamine content.

In some alternative embodiments, the lactobacillus fermentum comprises the amino acid sequence set forth in SEQ ID NO:1, and a nucleotide sequence shown in 1.

In the examples herein, the sequence set forth as SEQ ID NO:1 may be a gene encoding a 16S rRNA sequence (16S rDNA).

In the embodiments of the present application, the culture medium and other culture conditions for culturing the lactobacillus fermentum of the first aspect may be used without particular limitation as long as they are generally used for culturing microorganisms of the genus lactobacillus.

In the embodiment of the present application, the culture temperature may be 25 ℃ to 48 ℃, and more specifically, 36 ℃ to 38 ℃, but the temperature is not limited thereto. The cultivation may be continued until a desired amount of the useful material is obtained, specifically, 1 to 100 hours, but the cultivation time is not limited thereto.

In a second aspect, embodiments of the present application provide a cell comprising lactobacillus mucilaginosus.

In any of the embodiments of the present application, the term "cell" may refer to a cell obtained by preparation of a strain of lactobacillus mucilaginosus or a culture thereof or the like by dry fermentation, or by centrifugal precipitation. The drying may be drying with hot air, or freeze drying. The lyophilization may be performed by taking a culture solution after culturing the above-mentioned fermented lactobacillus mucilaginosa or by subjecting the culture solution to lyophilization treatment.

In some embodiments, the bacterial cells are viable bacteria of lactobacillus mucilaginosus. In other embodiments, the bacterial cells are inactivated bacterial cells of lactobacillus mucilaginosus. For example, pasteurized inactivated bacteria. Dead bacteria may be included in the cells. In other embodiments, the lactobacillus fermentum in the cells is a mixture of live lactobacillus fermentum and inactivated lactobacillus fermentum.

In a third aspect, embodiments of the present application provide an extract comprising lactobacillus mucilaginosus.

As used herein, the term "extract of lactobacillus fermentum" may be a conceptual interchange of "strain extracts" and may refer to the material remaining after separation from the cell bodies of the strain. In particular, it may be obtained by lysing cells of the strain. The extract may include one or more water-soluble components of proteins, carbohydrates, nucleic acids, and fibers, but the components included therein are not limited thereto.

In a fourth aspect, embodiments of the present application provide a culture comprising lactobacillus mucilaginosus.

In any of the embodiments herein, the term "culture" may refer to the product resulting from culturing the strain of the invention. For example, the culture may be a medium containing byproducts that are produced by nutrient uptake and metabolism during cultivation of the strain in the medium. As another example, the culture may include all types of cultures formed by using the above-described culture medium. Such as a dilution or a concentrate of the medium, cells obtained by drying the medium, a crude purified product or a purified product of the medium, or a mixture thereof, or the like. The lactobacillus mucilaginosus culture of embodiments of the present application may or may not include lactobacillus mucilaginosus.

In a fifth aspect, embodiments of the present application provide a lysate comprising lactobacillus fermentum.

In any of the embodiments herein, the term "lysate" may refer to the product produced by disrupting or lysing the strain or culture thereof. The lysate may include water-soluble ingredients or components that do not have water-soluble properties. For example, the lysate may be obtained by centrifuging the lysate, including its supernatant.

In any of the embodiments herein, cultures, lysates, supernatants thereof, and fractions thereof are also included within the scope of the present application. The method may further comprise the step of lysing the strain prior to or concurrent with the collecting step. Lysis of the strain may be carried out by methods commonly used in the art to which the invention pertains, such as lysis buffers, sonicators, heat treatment, french press (French press), and the like. In addition, the cleavage step may include a reaction of an enzyme such as a cell wall degrading enzyme, a nuclease, a nucleotide transferase, a protease, etc., but the cleavage step is not limited thereto.

According to the above examples, the cells, extracts, cultures and lysates containing the fermented lactobacillus mucilaginosus also have the effect of fermenting lactobacillus mucilaginosus, respectively.

In a sixth aspect, embodiments of the present application provide an edible additive comprising:

lactobacillus mucilaginosus of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect; and edible excipient.

The edible excipient may include a cell body or extract of the strain of the embodiments herein, and more specifically, according to the formulation to be applied, the preparation for use in a suitable formulation (e.g., food, feed, etc.) may be performed by mixing the strain extract with additives, but is not limited thereto.

The edible excipient of any of the above embodiments may further comprise a naturally occurring material or a non-naturally occurring material. Depending on the intended use of the formulation, the substance may include, but is not limited to, a pharmaceutically acceptable carrier, a dietetic acceptable carrier, a cosmetically acceptable carrier, and the like. The carrier may be appropriately selected by one skilled in the art based on the known contents. As one example, the edible excipient is milk powder.

The edible additive of any embodiment comprises one or more of lactobacillus fermentum, thallus thereof, extract thereof, culture thereof and lysate thereof, and has good biological activity under certain acidic environment respectively, and can resist artificial gastric juice; also has good in vitro antioxidation capability, can inhibit pathogenic bacteria, reduce inflammatory factors in serum, and increase BNDF and 5-hydroxytryptamine content.

In some embodiments, the number of bacteria of the lactobacillus mucilaginosus fermented in the edible additive is 1×10 ³ ~1×10 ¹⁴ CFU/mL or 1X 10 ³ ~1×10 ¹⁴ CFU/g, the bacteria can be viable bacteria number. Specifically, it may be 1×10 ³ CFU/mL（CFU/g）、1×10 ⁴ CFU/mL（CFU/g）、1×10 ⁵ CFU/mL（CFU/g）、1×10 ⁶ CFU/mL（CFU/g）、1×10 ⁷ CFU/mL（CFU/g）、1×10 ⁸ CFU/mL（CFU/g）、1×10 ⁹ CFU/mL（CFU/g）、1×10 ¹⁰ CFU/mL（CFU/g）、1×10 ¹¹ CFU/mL（CFU/g）、1×10 ¹² CFU/mL（CFU/g）、1×10 ¹³ CFU/mL（CFU/g）、1×10 ¹⁴ Any number or range of compositions in CFU/mL (CFU/g).

In some embodiments, the lactobacillus mucilaginosus in the edible additives described above is a viable bacterium. In other embodiments, the lactobacillus fermentum in the edible additive is an inactivated thallus of lactobacillus fermentum. In other embodiments, the lactobacillus fermentum in the edible additive is a mixture of live lactobacillus fermentum and inactivated lactobacillus fermentum.

The formulation of the above edible additive is not particularly limited, and may be, for example, powder, lozenge, tablet or capsule.

For example, the edible additive can be used as a raw material for preparing foods such as fermented milk, cheese, milk-containing beverages, solid beverages, milk powder, pressed candies, gel soft sweets, fermented fruit and vegetable juices, fermented bean products and the like.

In a seventh aspect, embodiments of the present application provide a food product comprising lactobacillus fermentum of the first aspect or thallus of the second aspect or extract of the third aspect or culture of the fourth aspect or lysate of the fifth aspect or edible additive of the sixth aspect.

The food of any of the above embodiments comprises one or more of lactobacillus mucilaginosus, and thallus, extract, culture and lysate thereof, which have good biological activity under certain acidic environment, respectively, and can resist artificial gastric juice; also has good in vitro antioxidation capability, can inhibit pathogenic bacteria, reduce inflammatory factors in serum, and increase BNDF and 5-hydroxytryptamine content.

In some embodiments, the number of lactobacillus mucilaginosus bacteria in the food product is 1×10 ³ ~1×10 ¹⁴ CFU/mL or 1X 10 ³ ~1×10 ¹⁴ CFU/g, the bacteria can be viable bacteria number. Specifically, it may be 1×10 ³ CFU/mL（CFU/g）、1×10 ⁴ CFU/mL（CFU/g）、1×10 ⁵ CFU/mL（CFU/g）、1×10 ⁶ CFU/mL（CFU/g）、1×10 ⁷ CFU/mL（CFU/g）、1×10 ⁸ CFU/mL（CFU/g）、1×10 ⁹ CFU/mL（CFU/g）、1×10 ¹⁰ CFU/mL（CFU/g）、1×10 ¹¹ CFU/mL（CFU/g）、1×10 ¹² CFU/mL（CFU/g）、1×10 ¹³ CFU/mL（CFU/g）、1×10 ¹⁴ Any number or range of compositions in CFU/mL (CFU/g).

The "food" of embodiments of the present application may include all types of functional foods, nutritional supplements, health foods, and food additives. For example, functional foods include biomedical foods well known in the art.

Food products of the above type may be prepared in various forms according to conventional methods known in the art.

The food can be prepared in the form of pill, powder, granule, infusion, tablet, capsule, liquid, etc. The food products to which the present invention may be added may include, for example, various foods such as rice, edible cereal powder (edible cereal flour), cereal soup, noodles, rice soup, instant rice, seasonings, lunch box, dried rice, bread, table sugar, rice cake, mixed sauces, spices, table salt, seasonings, seasoning powder, processed, frozen, dried and boiled fruits and vegetables, jellies, jams, candies, eggs, milk and other dairy products, edible fats and oils, coffee, cocoa and coffee substitutes, tapioca flour, cereal flour and cereal products, hand-pulled noodles, black winter noodles, crushed noodles, cold noodles, porridge, soup dishes, instant foods, frozen foods, steamed foods, other beverages, chewing gums, teas, vitamin complexes, health products, and the like, but the form of the food products is not limited thereto.

As ingredients of the food product that may be included in the embodiments of the present application, other ingredients, such as various herbal extracts, auxiliary food additives, or natural carbohydrates, may be included as in the general food product. In addition, the supplementary food additives may include supplementary food additives commonly used in the art, such as flavoring agents, coloring agents, fillers, stabilizers, and the like.

Examples of natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, and the like; and polysaccharides, for example, ordinary sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, erythritol, and the like. In addition to those described above, natural flavoring agents (e.g., rebaudioside A (rebaudioside A), glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used as flavoring agents. In addition, usual food additives for taste and nutrition supplement purposes, such as nucleic acids, amino acids, organic acids, etc., may be added.

In addition to those described above, the food products for use in the embodiments of the present application may include a number of nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and fillers (cheese, chocolate, etc.), pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation used in carbonated beverages, and the like. In addition, the food product may include pulp for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used singly or in combination.

The food product of the examples herein may be prepared by methods commonly used in the art, and may be prepared during preparation by adding raw materials and ingredients commonly added in the art. Furthermore, the food product may be made into any formulation without limitation, as long as the formulation is considered a food product.

Furthermore, while the food products of the embodiments of the present application may be used as health functional food products, the food products may be prepared in various forms of formulations.

Meanwhile, the food may further include flavoring agents, coloring agents, fillers, stabilizers, and flavoring agents, which may also be classified as food additives.

As used herein, the term "flavorant" may be a material added to enhance the flavor of a food product. The flavorant may be a material that imparts superior flavoring characteristics to the food.

The flavorants may be categorized according to taste components. In other words, the flavor can be classified into neutral flavor, beef flavor, chicken flavor, pork flavor, thick flavor, and the like, depending on the flavor.

The term "neutral flavor" refers to a flavor that maximizes "umami" while minimizing other flavors to produce a mild and clean flavor. For example, among oils, oils such as rapeseed oil or grape seed oil may be considered to have a neutral fragrance. The term "flavoring property" may mean having a sour, sweet, salty, bitter, or umami taste, but is not limited thereto.

In an eighth aspect, embodiments of the present application provide a feed comprising lactobacillus fermentum of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect or the edible additive of the sixth aspect.

The feed of any embodiment comprises one or more of lactobacillus mucilaginosus, thallus thereof, extract thereof, culture thereof and lysate thereof, has good biological activity under certain acidic environment and can resist artificial gastric juice; also has good in vitro antioxidation capability, can inhibit pathogenic bacteria, reduce inflammatory factors in serum, and increase BNDF and 5-hydroxytryptamine content.

The feed may include any one or more of a strain, a thallus of a strain, and an extract of a strain according to the present application, or may include a cell wall component obtained during preparation of an extract of a strain, and, for example, thallus of a strain (dry strain) and/or an extract of a strain (strain extract), but is not limited thereto.

The "feed" of the present disclosure is any suitable natural or artificial diet, meal alone, etc., or component of a meal alone, for consumption, ingestion, and digestion by an animal. The feed comprising the above strain according to the present application may be prepared in various feed forms known in the art, and in particular may comprise concentrated feed, roughage and/or special feed.

The type of feed is not particularly limited, and any feed commonly used in the art may be used. Non-limiting examples of feeds may include plant feeds such as grains, root plants, food processing byproducts, algae, fibers, pharmaceutical byproducts, oils, starches, cereals, cereal byproducts, and the like; and animal feeds such as proteins, minerals, fats and oils, single cell proteins, zooplankton, foods, and the like. These feeds may be used alone or in combination of two or more thereof.

In some embodiments, the number of lactobacillus mucilaginosus bacteria in the feed is 1×10 ³ ~1×10 ¹⁴ CFU/mL or 1X 10 ³ ~1×10 ¹⁴ CFU/g, the bacteria can be viable bacteria number. Specifically, it may be 1×10 ³ CFU/mL（CFU/g）、1×10 ⁴ CFU/mL（CFU/g）、1×10 ⁵ CFU/mL（CFU/g）、1×10 ⁶ CFU/mL（CFU/g）、1×10 ⁷ CFU/mL（CFU/g）、1×10 ⁸ CFU/mL（CFU/g）、1×10 ⁹ CFU/mL（CFU/g）、1×10 ¹⁰ CFU/mL（CFU/g）、1×10 ¹¹ CFU/mL（CFU/g）、1×10 ¹² CFU/mL（CFU/g）、1×10 ¹³ CFU/mL（CFU/g）、1×10 ¹⁴ Any number or range of compositions in CFU/mL (CFU/g).

In any of the above embodiments of the present application, such as in food or feed, a carrier may also be included. The carrier may be a naturally occurring or non-naturally occurring additive, but is not limited thereto.

In a ninth aspect, embodiments of the present application provide a medicament comprising lactobacillus fermentum of the first aspect or thallus of the second aspect or extract of the third aspect or culture of the fourth aspect or lysate of the fifth aspect; and

A pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be any one or more of excipients or diluents commonly used in the art, and pharmaceutically acceptable salts. Pharmaceutically acceptable carriers include, for example, carriers for oral or parenteral administration, and may additionally include pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier may be a solid carrier or a liquid carrier. The solid carrier comprises lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, the liquid carrier includes water, suitable oil, saline, aqueous dextrose, ethylene glycol, and the like, and stabilizers and preservatives may also be added. For example, suitable stabilizers may include sodium bisulphite, sodium sulphite or ascorbic acid may be antioxidants, and suitable preservatives include benzalkonium chloride, methyl or propyl p-hydroxybenzoate and chlorobutanol.

According to the embodiment of the application, the medicament uses the lactobacillus mucilaginosus, and the medicament of any embodiment has good biological activity under certain acid environment and can resist artificial gastric juice; also has good in vitro antioxidation capability, can inhibit pathogenic bacteria, reduce inflammatory factors in serum, and increase BNDF and 5-hydroxytryptamine content.

In some embodiments, the number of lactobacillus mucilaginosus bacteria in the food product is 1×10 ³ ~1×10 ¹⁴ CFU/mL or 1X 10 ³ ~1×10 ¹⁴ CFU/g, the bacteria can be viable bacteria number. Specifically, it may be 1×10 ³ CFU/mL（CFU/g）、1×10 ⁴ CFU/mL（CFU/g）、1×10 ⁵ CFU/mL（CFU/g）、1×10 ⁶ CFU/mL（CFU/g）、1×10 ⁷ CFU/mL（CFU/g）、1×10 ⁸ CFU/mL（CFU/g）、1×10 ⁹ CFU/mL（CFU/g）、1×10 ¹⁰ CFU/mL（CFU/g）、1×10 ¹¹ CFU/mL（CFU/g）、1×10 ¹² CFU/mL（CFU/g）、1×10 ¹³ CFU/mL（CFU/g）、1×10 ¹⁴ Any number or range of compositions in CFU/mL (CFU/g).

In a tenth aspect, embodiments of the present application provide the use of a lactobacillus fermentum of the first aspect or a thallus of the second aspect or an extract of the third aspect or a culture of the fourth aspect or a lysate of the fifth aspect in the manufacture of a medicament for preventing or ameliorating a psychotic disorder or delaying or preventing or treating a disorder associated with free radicals, the psychotic disorder comprising one or more of schizophrenia lineage disorder, attention deficit hyperactivity disorder, anxiety disorder, affective disorder; the diseases related to free radicals comprise any one or more of arteriosclerosis, diabetes, cardiovascular diseases, rheumatic arthritis, senile eye diseases and enteritis.

Mental disorder diseases may also broadly include: 1) Schizophrenic lineage disorders, such as schizophrenia; 2) Attention deficit hyperactivity disorder; 3) Anxiety disorders such as anxiety disorder, social anxiety disorder, obsessive compulsive disorder, post psychological trauma stress disorder, and the like; 4) Affective disorders such as depression, bipolar disorder, etc.

According to the embodiment of the application, the lactobacillus fermentum is used, and the lactobacillus fermentum has the functions of reducing inflammatory factors in serum and increasing the content of brain-derived neurotrophic factors and 5-hydroxytryptamine, so that mental disorder diseases can be prevented or improved or diseases related to free radicals can be delayed or prevented or treated. Such as improving depressed mood and/or anxiety disorders. The application can be applied to sub-healthy people or diseased people.

In an eleventh aspect, the embodiments of the present application provide the use of the lactobacillus fermentum of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect in the manufacture of a medicament for preventing or ameliorating a neurological disorder, wherein the neurological disorder comprises one or more of a neurodegenerative disorder, schizophrenia; the neurodegenerative disease includes Alzheimer's disease.

According to the embodiments of the present application, lactobacillus fermentum is used, which has the effects of reducing inflammatory factors in serum, increasing brain-derived neurotrophic factors and 5-hydroxytryptamine content, and thus can prevent or improve neurological diseases. For example to improve schizophrenia. The application can be applied to sub-healthy people or diseased people.

In a twelfth aspect, embodiments of the present application provide a method of relaxing and/or mood easing a person, comprising:

providing one or more of lactobacillus mucilaginosus of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect or the edible additive of the sixth aspect;

mixing a carrier with one or more of lactobacillus mucilaginosus, and thalli thereof, and extracts thereof, and cultures thereof, and lysates thereof, to obtain a mixture;

allowing the subject in need of relaxation and/or mood relief to consume the mixture.

According to the method, the lactobacillus mucilaginosus is used, so that the method has good biological activity under a certain acidic environment and can resist artificial gastric juice; also has good in vitro antioxidant capacity, can inhibit pathogenic bacteria, reduce inflammatory factors in serum, and relax and/or relieve emotion. The method can be applied to healthy people, and further, people without mental diseases.

In a thirteenth aspect, embodiments of the present application provide a method for preventing or ameliorating a mental disorder, comprising:

providing one or more of lactobacillus mucilaginosus of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect or the edible additive of the sixth aspect;

Mixing a carrier with one or more of lactobacillus mucilaginosus, dried products thereof, extracts thereof, cultures thereof, and lysates thereof to obtain a mixture;

a subject in need of prevention or amelioration of a psychotic disorder comprising one or more of schizophrenic spectrum disorders, attention deficit hyperactivity disorder, anxiety disorders, affective disorders is fed a mixture.

According to the method, the lactobacillus mucilaginosus is used, so that the method has good biological activity under a certain acidic environment and can resist artificial gastric juice; also has good in vitro antioxidant capacity, and can inhibit pathogenic bacteria, reduce inflammatory factors in serum, and prevent or improve depression and anxiety disorder. The method can be applied to sub-health people or sick people, and further, people with mental diseases.

In a fourteenth aspect, embodiments of the present application provide a method for delaying or preventing or treating a disease associated with free radicals, comprising:

providing one or more of lactobacillus mucilaginosus of the first aspect or the thallus of the second aspect or the extract of the third aspect or the culture of the fourth aspect or the lysate of the fifth aspect or the edible additive of the sixth aspect;

Mixing a carrier with one or more of the lactobacillus mucilaginosus, and the thalli thereof, and the extracts thereof, and the cultures thereof, and the lysates thereof, to obtain a mixture;

feeding the mixture to a subject in need of delay or prevention or treatment of a free radical related disease, wherein the free radical related disease is any one or more of arteriosclerosis, diabetes, cardiovascular disease, rheumatoid arthritis, senile eye disease, enteritis.

According to the method, the lactobacillus mucilaginosus is used, so that the method has good biological activity under a certain acidic environment and can resist artificial gastric juice; also has good in vitro antioxidant capacity, and can inhibit pathogenic bacteria, reduce inflammatory factors in serum, and prevent or improve depression and anxiety disorder. The method can be applied to sub-health people or sick people, and further, people with mental diseases.

In any of the above embodiments of the present application, the carrier may be a naturally occurring or non-naturally occurring additive, but is not limited thereto.

In any of the above embodiments of the present application, the carrier may be an edible excipient, which may be appropriately selected for use according to its intended use or form, and may be, for example, one or more selected from starch, glucose, cellulose, lactose, glycogen, D-mannitol, sorbitol, lactitol, maltodextrin, calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, dextrin, sodium alginate, methylcellulose, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose, propylene glycol, casein, calcium lactate, sodium starch (primogel), gum arabic, specifically one or more selected from starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, and maltodextrin, but the edible excipient is not limited thereto. The emulsifier may be a glyceride, a sorbitan ester, a monoglyceride, a diglyceride, a triglyceride, a sucrose ester, a sorbitan ester, a propylene glycol ester, a glycerin fatty acid ester, a sorbitan fatty acid ester, a propylene glycol fatty acid ester, a sucrose fatty acid ester, lecithin, or a mixture thereof, but the emulsifier is not limited thereto, and those known in the art may be suitably used.

In any of the above embodiments of the present application, the edible excipient may include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffering agent, a stabilizing agent, or an isotonic agent, but the edible excipient is not limited thereto.

Examples

The following examples more particularly describe the disclosure of the present application, which are intended as illustrative only, since numerous modifications and variations within the scope of the disclosure will be apparent to those skilled in the art. Unless otherwise indicated, all parts, percentages, and ratios reported in the examples below are by weight, and all reagents used in the examples are commercially available or were obtained synthetically according to conventional methods and can be used directly without further treatment, as well as the instruments used in the examples.

Example 1 isolation and identification and biological Properties of Lactobacillus mucilaginosus WIS32

And (3) screening out dominant strains from a plurality of strains separated from the healthy breast milk, and identifying the dominant strains.

16S DNA identification: the cultured bacterial liquid was treated with bacterial universal primer 27F: AGAGTTTGAT CCTGGCTCAG;1540R: AGGAGGTGATCCAGCCGCA, after colony PCR amplification, the nucleotide sequence of the gene (16S rDNA) encoding the 16S rRNA sequence is shown as SEQ ID NO: 1. Comparing the 16S DNA sequence of each strain with the gene sequences of all the determined microorganisms in the database, wherein the strain WIS32 and the lactobacillus fermentum are found Limosilactobacillus fermentum) Is encoded by 16S rRNAThe homology of the sequence genes is 99.93 percent, and the species is judged to belong to the fermentation lactobacillus mucilaginosus, and the species is named as a strain of fermentation lactobacillus mucilaginosusLimosilactobacillus fermentum）WIS32。

The screened lactobacillus mucilaginosus WIS32 is preserved in CGMCC for 12-06 days in 2023, the preservation number is CGMCC No.29244, and the lactobacillus mucilaginosus is classified and named as lactobacillus mucilaginosus @Limosilactobacillus fermentum）WIS32。

The strain WIS32 obtained by screening was streaked on MRS agar medium (available from Beijing land bridge biotechnology Co., ltd.) and anaerobically cultured at 37℃for 48 hours, and the colony morphology was as shown in FIG. 1A, and white colonies were grown in the MRS agar medium in the form of opaque, round, smooth and moist surface, regular edges and raised center. The optimal growth temperature was found to be 37℃and facultative anaerobic.

The strain was subjected to gram staining, and the result is shown in FIG. 1B, and the gram staining of the strain was typical positive, and the cells were observed under a microscope to be in a short rod shape, free of spores, and motionless.

The strain was streaked on Columbia blood agar medium (available from Qingdao sea Bo Biotechnology Co., ltd.) and cultured at 37℃for 48 hours, and as a result, as shown in FIG. 1, white opaque colonies were grown on Columbia blood agar without hemolysis.

The strain WIS02 is streaked on an MRS agar plate, colonies on the plate are picked into 2mL of physiological saline, shaking and mixing are carried out, a proper amount of bacterial liquid (v) is taken into 5mL of physiological saline, the bacterial liquid is measured and adjusted to 2 McMaillard turbidity, and then (2 v) is taken from the original bacterial liquid into 10mL of supporting culture medium of API (Mei Liai carbohydrate identification reagent strip API50 CH), and mixing is carried out uniformly. The strain was cultured using the API50 CHL system according to the instructions in the API50 CH carbohydrate identifier reagent (purchased from Mei Liai company), wherein the control group was cultured using the API50 CHL system without the strain added.

The results of the API50 CHL test of the strain Lactobacillus fermentum WIS32 of this example are presented in Table 1.

TABLE 1 API50 CHL results

Wherein, -indicates that the substance is not available, and +indicates that the substance is available.

From the results of Table 1, it was demonstrated that the strain can utilize L-arabinose, D-ribose, D-xylose, D-fructose, arbutin, D-sucrose, gluconate, potassium 5-ketogluconate as its energy supply source.

The bacterial strain obtained by screening is detected, the gene of the coding 16S rRNA sequence is shown as SEQ ID NO.1, and classification and comparison are carried out by utilizing a phylogenetic tree according to colony characteristics and nucleic acid sequences, and the bacterial strain is identified as lactobacillus mucilaginosus WIS32, belonging to a new microorganism variety.

EXAMPLE 2 cultivation of Lactobacillus mucilaginosus WIS32

After the fermentation of lactobacillus mucilaginosus WIS32 for the second generation, inoculating the fermentation lactobacillus mucilaginosus WIS32 into MRS culture medium according to the inoculum size of 1 percent, culturing the fermentation lactobacillus mucilaginosus in the MRS culture medium for 24 hours at the temperature of 37 ℃, sampling 200 mu L of the fermentation lactobacillus mucilaginosus every 1 hour, measuring the Optical Density (OD) value of the fermentation lactobacillus mucilaginosus at the wavelength of 600nm on an enzyme-labeled instrument, drawing a growth curve, and setting three repetitions.

As a result, as shown in FIG. 2, the Lactobacillus fermentum WIS32 grew rapidly from 3 hours into the logarithmic phase after the growth retardation period of 0-3 hours, and 9 hours ended the logarithmic phase into the stationary phase.

EXAMPLE 3 Lactobacillus mucilaginosus WIS32 tolerance to gastric juice intestinal juice experiment

1) Artificial gastric juice resistance experiment

Preparing artificial gastric juice: weighing NaCl 0.1 g, pepsin 0.175 g, adding water 50 mL, dissolving thoroughly, regulating pH to 2.5 with dilute hydrochloric acid, mixing, and filtering with 0.22 μm filter membrane under aseptic environment condition.

Glycerol-preserved Lactobacillus mucilaginosus WIS32 strain (1×10) ¹² CFU/mL), and derived from commercially available Lactobacillus fermentum LF-G89 (1X 10) ¹² CFU/mL) (wetting bioengineering (Shanghai) limited) was subjected to second-generation activation, and then inoculated into sterilized MRS liquid medium at an inoculum size of 1%, respectively, and subjected to anaerobic stationary culture at 37 ℃ for 16 h. Get 1 0 mL culture solution, 6000 g, centrifuging for 10 min, discarding supernatant, cleaning thallus for 2 times with sterile physiological saline, adding 3 mL artificial gastric juice to resuspend bacterial mud, mixing, fixing volume to 10 mL with artificial gastric juice, mixing uniformly, taking 100 μl, diluting step by step, counting with MRS plate, and determining initial viable count. After gastric juice is added, the mixture is subjected to anaerobic stationary culture at 37 ℃ for 3h, 100 mu L of the mixture is taken and diluted step by step, the mixture is counted by an MRS plate, and the viable count of the gastric juice treated 3h is measured.

Gastric juice resistance = (3 h viable count/initial viable count) ×100%.

Table 2 survival of wis32 in artificial gastric juice at ph=2.5 for 3h

The results show that after artificial gastric juice 3h at ph=2.5, the survival rate of both the strain of the invention (lactobacillus fermentum WIS 32) and the control strain (lactobacillus fermentum LF-G89) was lower than 10%, but the survival rate of the strain of the invention WIS32 was higher than that of the control strain LF-G89, as shown in table 2.

2) Experiment of artificial intestinal juice resistance

Preparing artificial intestinal juice: dissolving potassium dihydrogen phosphate 6.8 and g in 500-mL water, adjusting pH to 6.8 with 0.1 mol/L sodium hydroxide solution, dissolving pancreatin 10 g in water, mixing the two solutions, diluting with water to 1000 mL, mixing, and filtering with 0.22 μm filter membrane under aseptic condition.

Glycerol-preserved Lactobacillus mucilaginosus WIS32 (1×10) ¹² CFU/mL), and derived from commercially available Lactobacillus fermentum LF-G89 (1X 10) ¹² CFU/mL) was subjected to secondary activation, and inoculated into a sterilized MRS liquid medium at an inoculum size of 1%, respectively, and subjected to anaerobic stationary culture at 37 ℃ for 16 h. Taking 10 mL culture solution, centrifuging 6000 g for 10 min, discarding supernatant, cleaning thallus for 2 times with sterile physiological saline, adding 3 mL artificial intestinal juice to resuspend bacterial mud, mixing uniformly, fixing the volume to 10 mL with the artificial intestinal juice, mixing uniformly, taking 100 mu L, diluting step by step, and determining the initial viable count; after culturing at 37℃for 3h, 100. Mu.L was taken, diluted stepwise, counted on a plate, and the viable count of 3h in intestinal juice treatment was measured.

Intestinal juice resistance = (3 h viable count/initial viable count) ×100%

TABLE 3 survival of two Lactobacillus paracasei in artificial intestinal juice%

The results are shown in Table 3, and the fermented lactobacillus mucilaginosus WIS32 and the control strain LF-G89 show excellent artificial intestinal juice resistance after 3 hours of artificial intestinal juice, and the tolerance of the strain WIS32 of the invention is better.

Example 4 in vitro antioxidant capacity

Fermentation broth supernatant samples: the fermentation broth of lactobacillus mucilaginosus WIS32 and the control strain LF-G89 are subjected to three generations (50 mL, 5 percent and 36 ℃ for 24 hours) of bacterial liquid activation and are centrifuged, supernatant fluid is taken and filtered by a 0.22 microporous filter membrane to obtain a fermentation broth supernatant sample.

Bacterial extract samples: washing the obtained centrifuged thallus with pre-cooled PBS twice, and adjusting the thallus concentration to 10 ⁹ CFU/mL, 8mL of the bacterial liquid with the concentration adjusted is taken in a sterile 15mL centrifuge tube, and ultrasonic wall breaking is carried out. Centrifuging the cell wall-broken bacterial liquid, and filtering the supernatant with a 0.22 μm filter membrane to obtain a bacterial extract.

And respectively testing the free radical scavenging capacity, the hydroxyl free radical scavenging capacity and the total antioxidant capacity of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine free radical) by using the supernatant of the fermentation liquor and the bacterial extract according to the instruction of a kit.

DPPH radical scavenging ability was measured using a DPPH radical scavenging ability kit (available from Nanjing under the trade designation A153-1-1); the hydroxyl radical scavenging ability was measured using a hydroxyl radical scavenging ability test kit (microfabricated) (available from lei living beings under the trade designation YX-W-a 505); the total antioxidant capacity was measured using the total antioxidant capacity (T-AOC) assay kit (FRAP method) microplate method (commercially available from Nanjing under the designation A015-3-1).

As can be seen from Table 4, lactobacillus fermentum WIS32 has a certain DPPH radical scavenging ability and a certain OH radical scavenging ability, as well as a certain total antioxidant ability. The DPPH free radical scavenging ability is more excellent than that of the control strain LF-G89.

TABLE 4 antioxidant capacity

EXAMPLE 5 pathogenic bacteria inhibitory Capacity

The antibacterial activity of the strain was measured by oxford cup method. The pathogen indicator is 6 common pathogenic bacteria: escherichia coli ATCC25922, staphylococcus aureus ATCC25923, pseudomonas aeruginosa ATCC27853, enterococcus faecalis ATCC29212, shigella flexneri CICC 21534, enterobacter sakazakii CICC 21560. Inoculating 6 pathogenic bacteria into BHI liquid culture medium at 1%, culturing in 37 deg.C incubator for 16 hr, adding the culture solution into BHI agar culture medium maintained at about 50deg.C to make OD ₆₀₀ After mixing, add to agar plates, after the upper medium has condensed, place oxford cup with sterile forceps. And adding 200 mu L of supernatant of the test strain WIS32 and the control strain LF-G89 into an oxford cup, taking an MRS liquid culture medium which is not inoculated with bacteria after high-pressure sterilization as a blank control, placing the blank control in a refrigerator at 4 ℃ for diffusion for 24 hours, and standing at 37 ℃ for culturing for 24 hours. Observing the inhibition zone, and measuring the diameter of the inhibition zone by using a vernier caliper.

TABLE 5 inhibition of pathogenic bacteria by WIS32 fermentation supernatant

Note that: the values are the diameter of the inhibition zone, and the unit is mm. "-" means no bacteriostasis. Wherein, the diameter of oxford cup is 7.8mm.

The results show that lactobacillus mucilaginosus WIS32 has a certain inhibition on enterobacter sakazakii and pseudomonas aeruginosa as shown in Table 5. Compared with LF-G89, the strain WIS32 has better inhibition effect on enterobacter sakazakii and pseudomonas aeruginosa and better antibacterial capability.

EXAMPLE 6 Lactobacillus mucilaginosus WIS32 prevention or amelioration of symptoms of depression and anxiety disorders

Preparation of lactobacillus stomach-lavage agent: after secondary activation, the lactobacillus mucilaginosus WIS32 preserved in the glycerol tube and the control strain LF-G89 are respectively inoculated into MRS liquid culture media according to the proportion of 2% (v/v), and are cultured for 24 hours at 37 ℃ to respectively obtain fermentation culture solutions. Centrifuging culture solution at 4deg.C and 6000g for 10min, collecting thallus, discarding supernatant, and re-suspending thallus with physiological saline to obtain 5×10 final concentrations ⁹ CFU/mL of fermented lactobacillus mucilaginosus WIS32 bacterial suspension and control strain LF-G89 bacterial suspension.

The bacterial suspension is used for lavage, wherein the volume of the lavage is 0.2 mL/day.

6.2 LPS-induced mouse depression model

50 male KM mice of 8 weeks old were selected and randomly divided into five groups after one week of acclimatization: blank, model, WIS32 test bacteria, control LF-G89, fluoxetine positive, each group contained 10 mice. The animals were grouped and treated as indicated in Table 6, with the blank intervention in normal saline. Behavioural observations were made on day 17.

TABLE 6 grouping and processing method for animal experiments

1.1 fermentation of Lactobacillus mucilaginosus WIS32 for preventing and improving depressive anxiety behavior in mice

1) Open field test

To evaluate symptoms of depression anxiety, open field analysis was performed on mice in the above-described rat model that caused depression. Open field analysis is based on behavioral observations of experimental animals after exposure to a large new space that cannot escape or evade, examining the level of depression and anxiety of experimental animals. The method comprises the steps of firstly placing a mouse in an open field laboratory 3h in advance, and placing the mouse in the open field laboratory box 1h in advance to adapt to the environment. The mice were placed in the center of a square open field box (100 cm ×100 cm ×40 cm) at the beginning of the formal experiment, and the time and activity trajectory of the mice in the center area of the open field box for the next 5min were photographed and recorded. After each mouse passes the open field experiment, the mouse is put back into the mouse cage, the excrement and urine in the open field box are removed, the bottom and the inner wall of the open field box are wiped clean by alcohol cotton balls, and the influence of breath on the motion trail of the next mouse is avoided. All mice were assayed for activity in open field boxes using supermaze+ software after testing. The total distance of activity of the mice in the open field box reflects their spontaneous activity. The anxiety level of the mice was evaluated by defining the central area as the central area in the central area of the bottom of the open field box, where the mice remained.

As a result of the open field analysis: there was a significant difference in center residence time between the blank and model group mine trials, indicating that LPS induced depressive behavior in mice. Compared with the model group, the WIS32 test group (in the WIS32 intake group of Lactobacillus fermentum) showed a significant increase in residence time in the central zone (panel A in FIG. 3), and the effect was superior to that of the control group (control group

LF-G89) while the WIS32 intake group was indistinguishable from the blank group treated with physiological saline. Indicating that after intake of WIS32, symptoms of LPS-induced depression and anxiety disorder are ameliorated.

2) Tail suspension experiment (Tail suspension test, TST):

one hour in advance, the mice in the above rat model causing depression were brought to a laboratory where the tail suspension experiment was performed, and the mice were taken out of the cage to perform the tail suspension experiment. The tail of the mouse is fixed by using a medical adhesive tape with the length of 12 cm and the width of 3 cm, so that the mouse is fixed at the fastest speed, and the unnecessary stress and stress state on the mouse should be avoided. The adhesive tape is 2-3 mm from the tip of the tail of the mouse. The other side of the tape was secured to the edge of the table such that the mouth of the hanging mouse was spaced from the floor 30 cm. The mice were tested for 6 min with tail suspension, and the total time of mice with tail suspension was 4 min after manual recording. In the test, mice were considered to be stationary when the forelimbs were moving, the hindlimbs were stationary, or the passive suspension was completely stationary, and can be considered to be in a disappointing state. The experiment is that the original environmental pressure of the mouse is forcefully changed by hanging the tail of the mouse, the mouse often shows the behavior that the mouse struggles to try to escape and the behavior that the mouse falls into the hopeless immobility because the mouse cannot escape, and the longer the struggle is abandoned to enter the hopeless immobility state, the depression-like behavior of the mouse is judged.

The strong analysis results of the tail suspension experiment are shown in a diagram B in FIG. 3: there was a significant difference in tail suspension time between the blank and model groups (x), indicating that LPS induced depressive-like behavior in mice. Compared with the model group, the tail suspension time in the lactobacillus mucilaginosus WIS32 ingestion group is obviously reduced, and the effect is superior to that of a positive drug fluoxetine group. The lactobacillus fermentum WIS32 proved to be able to significantly prevent and ameliorate depression and anxiety symptoms.

The Lipopolysaccharide (LPS) is a macromolecular complex existing in the outer membrane of bacteria. Lipopolysaccharide is a complex biological molecule consisting of two parts, lipid and polysaccharide, and the immune response of the human body to lipopolysaccharide usually includes an inflammatory response, and lipopolysaccharide can also cause excessive inflammatory response, leading to inflammatory diseases such as septic shock. Because of its strong immunostimulatory properties, lipopolysaccharide is also used as an immunological research tool in the laboratory for mimicking the immune response of infectious diseases.

3) Forced swimming test

The mice in the rat model causing depression are placed in a cylindrical glass jar with the height of 30cm, the diameter of 18cm, the water depth of 20 cm and the water temperature of 22-25 ℃ so as to ensure that the heads of the mice can not touch the water bottom when the heads of the mice are above the water surface. The mice swim for 6 min, the first 2min is the adaptation time, and the accumulated immobility time of the mice is 4 min after manual recording. After the experiment is completed, the mice are taken out from the basin, and are wiped by towels and dried; finally, the water in the basin is poured out to change the water into new water and the feces in the basin are cleaned, so that preparation is made for the next mouse to swim.

The analysis results of the forced swimming test are shown in a graph C in FIG. 3: there was a significant difference in tail-holding time between the blank and model groups (x), compared to the depressed model group, the forced swimming time of the lactobacillus mucilaginosus WIS32 intake group is obviously reduced, and the effect is better than that of the control bacteria group. Lactobacillus mucilaginosus WIS32 can ameliorate symptoms of depression and anxiety disorders caused by LPS.

1.2 fermentation of Lactobacillus mucilaginosus WIS32 to increase levels of hippocampal 5-hydroxytryptamine and brain-derived neurotrophic factor (BDNF) in depressed mice

Mice in the above rat model causing depression were euthanized for 18 days, and brain tissues of the mice were taken separately, and hippocampal tissues were isolated on ice. Fresh hippocampal tissue of a certain mass is taken respectively, physiological saline (equivalent to 1g of tissue and 9mL of homogenate physiological saline) is added, the tissue homogenate is carried out by using a tissue homogenate device, and the content of the hippocampal 5-hydroxytryptamine and BDNF is detected by using an ELISA kit.

The experimental results of the 5-hydroxytryptamine of the mouse hippocampus are shown in a graph A in fig. 4, wherein the content of the mouse hippocampus BDNF of the blank group, the model group, the WIS32 group, the control bacteria group and the fluoxetine group is respectively as follows: 261.57 + -54.14, 153.49 + -19.97, 209.66 + -35.31, 198.65 + -10.39, 251.40+ -49.44 ng/mg protein; the results indicate that administration of lactobacillus fermentum WIS32 significantly prevented and reversed the reduction of 5-hydroxytryptamine levels in hippocampal tissue caused by LPS compared to the depressed model group.

5-hydroxytryptamine is a neurotransmitter, also known as serotonin, which is primarily found in the central nervous system and in the intestinal tract, and the 5-hydroxytryptamine system is associated with a number of mental disorders such as depression, anxiety, etc.

The results of the mice hippocampal BNDF experiments are shown in a graph B in FIG. 4, wherein the content of the mouse hippocampal brain-derived neurotrophic factors of a blank group, a model group, a WIS32 group, a control bacterium group and a fluoxetine group are respectively as follows: 1.32+ -0.36, 0.82+ -0.22, 1.24+ -0.16, 0.99+ -0.23, 1.17+ -0.31 ng/mg protein; the results show that administration of lactobacillus fermentum WIS32 can significantly prevent and reverse the reduction of brain-derived neurotrophic factor levels in hippocampal tissue caused by LPS. Wherein, the improvement degree of the fermented lactobacillus mucilaginosus WIS32 on the brain-derived neurotrophic factor in the hippocampus is superior to that of the control bacteria and the positive medicine, and the preparation shows good depression prevention and treatment potential.

Brain-derived neurotrophic factor is a neurotrophic factor that plays a key role in the survival, development and maintenance of function of nerve cells. Brain-derived neurotrophic factors are associated with a variety of neurological diseases such as neurodegenerative diseases, depression, and the like. Reduced brain-derived neurotrophic factor levels may be associated with the development and progression of these diseases.

Thus, by this experiment, it was shown that lactobacillus fermentum WIS32 is related to the content of 5-hydroxytryptamine and brain-derived neurotrophic factor, and that lactobacillus fermentum WIS32 has an association with improving neurological and mental health disorders.

1.3 fermentation of Lactobacillus mucilaginosus WIS32 to reduce inflammatory factors in the hippocampus and serum of depressed mice

Mice were euthanized at 17 days, and blood was collected from each of the mice and centrifuged at 1500g for 15min to obtain serum. Mouse brain tissue was isolated and hippocampal tissue was isolated on ice. The ELISA kit is used for detecting inflammatory factors in serum and hippocampal tissues respectively: TNF-alpha, IL-1 beta and IL-6.

Compared with the model group, the administration of the fermented lactobacillus viscosus WIS32 can remarkably prevent and reverse the rise of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in hippocampal tissues caused by LPS, the improvement degree is superior to that of a control bacterial group, and the potential of reducing the sea Ma Yanzheng factor in a LPS-induced depression model is shown.

The results of the serum inflammatory factors of the mice are shown in fig. 5D, 5E and 5F, and compared with a model group, the serum inflammatory factors TNF-alpha, IL-1 beta and IL-6 in serum caused by LPS can be obviously prevented and reversed by taking the fermented lactobacillus mucilaginosus WIS32, the improvement degree is superior to that of a control bacterial group, and the potential of reducing the serum inflammatory factors in a depression model induced by LPS is shown to be good.

Example 7 detection of Lactobacillus mucilaginosus WIS32 by fermentation

Preparing probiotic freeze-dried powder: fermenting the strain of WIS32 as probiotics, centrifuging to collect thalli, washing twice, adding 10% of skimmed milk powder as a protective agent according to mass fraction, performing vacuum freeze drying to prepare WIS32 probiotic freeze-dried powder, and preparing 2 g/bag of probiotic freeze-dried powder containing 600 hundred million fermented lactobacillus mucilaginosus WIS32 by using composite powder (20% of skimmed milk powder and 80% of maltodextrin).

Placebo: 20% nonfat milk powder +80% maltodextrin was mixed into a placebo bar pack of 2 g/bag.

WIS32 pre-examination anxiety relief effect detection

Recruiting subjects: 20 college student volunteers, healthy at 17-25 years, without mental illness, signed informed consent.

The intervention method comprises the following steps: at 8 weeks prior to the exam, 20 college student volunteers were recruited and tested in 2 groups at random, one group taking 2g of probiotic lyophilized powder per bag per day (300 million WIS32 fermented lactobacillus mucilaginous bacteria + skimmed milk powder + maltodextrin) and the other group taking 2g of placebo per bag per day (skimmed milk powder + maltodextrin) for the end of the end-of-period exam.

The evaluation method comprises the following steps: on the day before the examination, volunteers filled in SAS anxiety self-evaluation and SDS depression self-evaluation, and the symptoms of anxiety, fear, hypodynamia, sleep, stress and the like of the last week before the examination were faithfully reported, and were statistically analyzed by SPSS. The results are shown in Table 7.

TABLE 7 test of effects of improving anxiety and depression in students before examination

Note that: compared to placebo group,: p <0.05; * *: p <0.01

From table 7 above, the SAS anxiety total score and SDS depression total score were significantly reduced in the group taking the WIS32 probiotic solid beverage compared with the placebo group, demonstrating the effect of relieving anxiety and depression disorders caused by stress of the functional probiotic products prepared by the formulation provided by the invention.

It should also be noted that the exemplary embodiments mentioned in this application describe some methods or systems based on a series of steps or devices. However, the present application is not limited to the order of the above-described steps, that is, the steps may be performed in the order mentioned in the embodiments, may be different from the order in the embodiments, or several steps may be performed simultaneously.

While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the invention. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (10)

1. Fermented lactobacillus mucilaginosusLimosilactobacillus fermentum) WIS32 with preservation number of CGMCC No.29244.

2. A thallus, extract, culture or lysate comprising the fermented lactobacillus mucilaginosus of claim 1.

3. An edible additive comprising:

the lactobacillus mucilaginosus of claim 1 or the thallus, the extract, the culture, or the lysate of claim 2; and

edible excipient.

4. A food product comprising the fermented lactobacillus mucilaginosus of claim 1, one or more of its thalli, extract, culture, lysate or the edible additive of claim 2.

5. A feed comprising one or more of the lactobacillus fermentum of claim 1 or the thallus of claim 2, the extract, the culture or the lysate or the edible additive of claim 2.

6. A medicament comprising one or more of the lactobacillus mucilaginosus of claim 1, and a thallus, an extract, a culture and a lysate thereof; and

a pharmaceutically acceptable carrier.

7. A food additive according to claim 3 or a food according to claim 4 or a feed according to claim 5 or a medicament according to claim 6, Comprising 1X 10 ³ ~1×10 ¹⁴ CFU/mL or 1X 10 ³ ~1×10 ¹⁴ CFU/g fermented lactobacillus mucilaginosus.

8. Use of one or more of the lactobacillus mucilaginosus of claim 1 or the thallus, the extract, the culture or the lysate of claim 2 for the manufacture of a medicament for preventing or ameliorating a disease of a psychotic disorder or for the manufacture of a medicament for delaying or preventing or treating a disease associated with free radicals, wherein the psychotic disorder is one or more of a schizophrenic lineage disorder, attention deficit hyperactivity disorder, anxiety disorder, affective disorder; the diseases related to the free radicals are any one or more of arteriosclerosis, diabetes, cardiovascular diseases, rheumatic arthritis, senile eye diseases and enteritis.

9. Use of one or more of the lactobacillus mucilaginosus of claim 1 or the thallus, the extract, the culture or the lysate of claim 2 in the manufacture of a medicament for preventing or ameliorating a neurological disorder, wherein the neurological disorder is one or more of neurodegenerative diseases, schizophrenia; the neurodegenerative disease is Alzheimer's disease.

10. A method of relaxing and/or mood relieving a person comprising:

providing one or more of the lactobacillus mucilaginosus of claim 1 or the thallus, the extract, the culture, or the lysate of claim 2;

mixing a carrier with one or more of the lactobacillus mucilaginosus, and the thalli thereof, and the extracts thereof, and the cultures thereof, and the lysates thereof, to obtain a mixture;

allowing a subject in need of relaxation and/or mood relief to consume the mixture.