A composite probiotic and tranquilizing sleep-aiding fermentation composition, and a preparation method and application thereof

Abstract

The application provides a composite probiotic and a brain-calming and sleep-aiding fermentation composition as well as a preparation method and application thereof, relates to the technical field of microorganisms, and comprises plant lactobacillus KMHD-1 and lactobacillus gasseri KMBGI 03. The composite probiotic is used in combination, can enhance the fermentation efficacy, and can effectively improve the content of zizyphus jujuba mill. saponin, rare ginsenoside and polysaccharide in the product by using composite probiotic fermentation, and can effectively enhance the brain-calming and sleep-aiding efficacy of the product.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a compound probiotic and a sleep-aiding fermented composition, a preparation method and application thereof. BACKGROUND

[0002] In the modern fast-paced and stressful living environment, anxiety and insomnia symptoms are prevalent and on the rise, and severe anxiety and insomnia can cause serious physical and mental damage. Traditional Chinese medicine generally uses a mixture of various Chinese herbal medicines, decocting to prepare medicinal soup for taking, and some use refining methods to prepare tablets or capsules for taking, but the efficacy of traditional Chinese medicine generally cannot achieve rapid and obvious results, and long-term use is required, with a long course of treatment and no obvious effect. Western medicine treatment is fast and obvious, but generally has serious side effects and dependence, which is not suitable for patients with mild anxiety and insomnia, and is not worth the loss.

[0003] CN 117562958 A discloses a sophora fruit and pericarpium citri reticulatae composition for improving sleep and relieving anxiety and a preparation method thereof. The composition contains pericarpium citri reticulatae extract, sophora fruit extract, banana powder, golden needle vegetable powder, jujube seed powder, perilla powder, white kidney bean seed coat extract, modified lactobacillus reuteri, and modified animal bifidobacterium. The combination of the components treated in a special way has the effects of synergistically promoting sleep and improving anxiety, but the preparation requires a strict and complicated enzymolysis process, which is difficult to industrialize.

[0004] CN 116570662 A provides a sleep-aiding fermented composition, a preparation method and application thereof. The fermented composition comprises, by weight fraction, 80-160 parts of jujube seed, 1-20 parts of golden topped side ear, 20-60 parts of notoginseng flower, 1-10 parts of valerian, 0.3-1.5 parts of fermented bacteria powder, 5-30 parts of enzyme preparation, and 1-10 parts of acid-base regulator. The fermented composition is prepared by enzymolysis, extraction, and fermentation process. The combination of the components in the present application has a good effect on improving sleep quality, but the preparation requires a strict and complicated enzymolysis process, which is difficult to industrialize, and no sensory evaluation and human clinical verification are performed.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] One of the purposes of the present application is to provide a compound probiotic to at least solve one of the technical problems in the prior art. The compound probiotic comprises lactobacillus plantarum KMHD-1 and lactobacillus gasseri KMBGI 03, which are used in combination to enhance the fermentation efficacy.

[0007] A second objective of this invention is to provide an application of the aforementioned compound probiotics in the preparation of a calming and sleep-aiding fermented composition. By using the aforementioned compound probiotics for fermentation, a fermented composition with calming and sleep-aiding effects can be prepared.

[0008] A third objective of this invention is to provide a calming and sleep-aiding fermented composition. This composition includes a fermentation substrate and optional excipients. The composition is obtained by fermenting the substrate with the aforementioned compound probiotics. The fermentation substrate comprises jujube seed and ginseng. Using compound probiotics to ferment the substrate effectively increases the content of jujube seed saponins, rare ginseng saponins, and polysaccharides, thereby enhancing the product's calming and sleep-aiding effects.

[0009] The fourth objective of this invention is to provide a method for preparing a calming and sleep-aiding fermented composition. This method includes the following steps: sequentially mixing, extracting, and concentrating the raw material components of a fermentation substrate in the prescribed amounts to obtain a concentrated liquid; then mixing the seed liquid of a compound probiotic with the concentrated liquid and fermenting to obtain a fermented product. This preparation method can effectively increase the content of active ingredients in the product, and the process is simple, efficient, and feasible.

[0010] The fifth objective of this invention is to provide an application of the aforementioned calming and sleep-aiding fermented composition in the preparation of calming and sleep-aiding products.

[0011] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0012] In a first aspect, the present invention provides a compound probiotic, comprising: *Lactobacillus plantarum* KMHD-1 (… Lactiplantibacillus plantarum KMHD-1) and Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03);

[0013] The *Lactobacillus plantarum* KMHD-1 was deposited on August 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28269, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] The Lactobacillus gasseri KMBGI 03 was deposited on January 14, 2019, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 17177, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] In an optional embodiment, the weight ratio of *Lactobacillus plantarum* KMHD-1 to *Lactobacillus gasseri* KMBGI 03 is (0.5-1.5):1.

[0016] In a second aspect, the present application provides use of the composite probiotic in preparation of a sleep-aiding fermented composition.

[0017] In a third aspect, the present application provides a sleep-aiding fermented composition, comprising a fermentation substrate and optionally an auxiliary material.

[0018] The sleep-aiding fermented composition is prepared by fermenting the fermentation substrate with the composite probiotic, wherein the fermentation substrate comprises: Suanzaoren and Renshen.

[0019] In an optional embodiment, the fermentation substrate further comprises at least one of Fuling and Longyemr.

[0020] Preferably, the fermentation substrate comprises, by weight fraction: 12-15 parts of Renshen, 12-15 parts of Suanzaoren, 20-26 parts of Fuling, and 30-35 parts of Longyemr.

[0021] Preferably, the auxiliary material comprises at least one of blueberry concentrate, gamma-aminobutyric acid, casein peptide, and sweetener.

[0022] Preferably, the sweetener comprises erythritol and natural sweetener.

[0023] Preferably, the auxiliary material comprises, by weight fraction: 50-55 parts of blueberry concentrate, 12-16 parts of gamma-aminobutyric acid, 5-7 parts of casein peptide, 60-70 parts of erythritol, and 0.2-0.6 parts of natural sweetener.

[0024] In a fourth aspect, the present application provides a method for preparing the sleep-aiding fermented composition, comprising the following steps:

[0025] The raw material components of the fermentation substrate are sequentially mixed, extracted, and concentrated to obtain a concentrated solution, and then the composite probiotic is mixed with the concentrated solution to obtain a fermentation product.

[0026] In an optional embodiment, the extraction comprises primary extraction and secondary extraction.

[0027] Preferably, the primary extraction comprises sequentially adding water, primary boiling extraction, and primary filtration to the mixed components of the fermentation substrate to obtain a primary extraction solution.

[0028] Preferably, the amount of water added in the primary extraction is 10-20 times the weight of the raw material components of the fermentation substrate, and the time of the primary boiling extraction is 1-2 h.

[0029] Preferably, the secondary extraction comprises sequentially adding water, secondary boiling extraction, and secondary filtration to the components of the fermentation substrate after the primary extraction to obtain a secondary extraction solution.

[0030] Preferably, the amount of water added in the secondary extraction is 10-20 times the weight of the fermentation substrate raw material components, and the time of the secondary boiling extraction is 1-2 hours.

[0031] Preferably, the extraction further comprises mixing the primary extraction liquid and the secondary extraction liquid to obtain a total extraction liquid.

[0032] In an optional embodiment, the weight of the concentrated liquid is 4-7 times the weight of the fermentation substrate raw material components.

[0033] Preferably, the concentration temperature is 75-85 DEG C.

[0034] Preferably, after the concentration, the method further comprises sterilizing the concentrated liquid for the first time before fermentation.

[0035] Preferably, the first sterilization adopts the pasteurization method.

[0036] Preferably, the mixing of the compound probiotics and the concentrated liquid comprises mixing Lactobacillus plantarum KMHD-1 powder and Lactobacillus gasseri KMBGI 03 powder to obtain compound probiotic powder, and then dissolving the compound probiotics in water and inoculating into the concentrated liquid.

[0037] Preferably, the addition amount of the compound probiotic powder is 0.07-0.12% of the weight of the concentrated liquid.

[0038] Preferably, the fermentation time is 24-72 hours.

[0039] In an optional embodiment, the preparation method of the sleep-aiding and nerve-calming fermented composition further comprises mixing a formula amount of auxiliary materials and the fermentation product to obtain the sleep-aiding and nerve-calming fermented composition.

[0040] Preferably, the mixing process comprises dissolving casein peptides and sweeteners respectively, and then mixing to obtain a premix liquid, mixing gamma-aminobutyric acid and erythritol, and then adding the fermentation product, blueberry concentrated liquid and the premix liquid.

[0041] In a fifth aspect, the application provides a sleep-aiding and nerve-calming product prepared from the sleep-aiding and nerve-calming fermented composition.

[0042] Compared with the prior art, the application has the following beneficial effects:

[0043] The compound probiotics provided by the application comprise Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03, and in the compound probiotic fermentation process, the compound probiotics synergistically act to effectively increase the content of jujuboside, rare ginsenoside and polysaccharide in the product, and effectively enhance the sleep-aiding and nerve-calming effect of the product. DETAILED DESCRIPTION

[0044] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. In this application, the use of "or" means "and / or" unless specifically stated otherwise, e.g., "comprising A or B" means "comprising A or B or both". Also, the use of "comprising" or "including" or other forms for "comprise" or "including" is intended to be non- limiting.

[0045] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0046] The first aspect of the present application provides a composite probiotic, comprising: Lactobacillus plantarum KMHD-1 (Lactobacillus plantarum KMHD-1) Lactiplantibacillus plantarum and Lactobacillus gasseri KMBGI 03 (Lactobacillus gasseri KMBGI 03) Lactobacillus gasseri ;

[0047] The Lactobacillus plantarum KMHD-1 has been deposited with the China General Microbiological Culture Collection Center on August 28, 2023, with the accession number CGMCC No. 28269, and the address of the deposit is No. 3, Beichen West Road, Chaoyang District, Beijing;

[0048] The Lactobacillus gasseri KMBGI 03 has been deposited with the China General Microbiological Culture Collection Center on January 14, 2019, with the accession number CGMCC No. 17177, and the address of the deposit is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0049] The composite probiotic provided by the present application comprises Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03. During the fermentation process of the composite probiotic, the composite probiotic synergistically acts to effectively increase the content of zizyphus jujuba mill. saponins, rare ginsenosides and polysaccharides in the product, and can effectively enhance the efficacy of the product in soothing the nerves and helping sleep.

[0050] Further, the weight ratio of the Lactobacillus plantarum KMHD-1 and the Lactobacillus gasseri KMBGI 03 is (0.5-1.5):1, for example, it can be 0.5:1, 1:1, 1.5:1, etc.

[0051] The second aspect of the present application provides the use of the composite probiotic in the preparation of a soothing and sleep-aiding fermentation composition.

[0052] In the present application, the fermentation of the composite probiotics can prepare the fermentation composition with the effect of soothing the nerves and helping sleep.

[0053] The present application provides a soothing and sleep-aiding fermentation composition, which comprises a fermentation substrate and optional excipients.

[0054] The soothing and sleep-aiding fermentation composition is prepared by fermenting the fermentation substrate with the composite probiotics, and the components of the fermentation substrate comprise Suanzaoren and ginseng.

[0055] In the present application, the components of the fermentation substrate comprise Suanzaoren and ginseng, one of the main active ingredients in Suanzaoren is Suanzaoren saponin, which has a significant calming and soothing effect, and the fermentation of the composite probiotics can increase the content of Suanzaoren saponin; the main active ingredient in ginseng is ginsenoside, which can improve the body's tolerance and has an anti-fatigue effect, and the fermentation of the composite probiotics can promote the generation or release of ginsenoside in ginseng through specific enzymatic reactions; both Suanzaoren and ginseng contain rich polysaccharide components, which are further improved and activated through the action of the composite probiotics during the fermentation process, and these polysaccharides enhance the soothing and sleep-aiding effect of the product through various mechanisms such as antioxidant, immune regulation and anti-inflammatory effect.

[0056] Further, the components of the fermentation substrate further comprise at least one of Fuling and Longyanyanrou;

[0057] In the present application, the components of the fermentation substrate further comprise Fuling and Longyanyanrou, the effective components of Longyan include Longyan polysaccharide, and the main components of Fuling include Fuling polysaccharide, both Fuling and Longyanyanrou have a calming and anti-anxiety effect, and can also improve the body's immunity, the effective components of Fuling and Longyanyanrou are further activated during the fermentation process, and together enhance the soothing and sleep-aiding effect of the product, thereby providing better sleep quality and overall health status.

[0058] Preferably, the fermentation substrate comprises the following components by weight fraction: 12-15 parts of ginseng, 12-15 parts of Suanzaoren, 20-26 parts of Fuling and 30-35 parts of Longyanyanrou;

[0059] In the fermentation substrate, the addition amount of ginseng is 12 parts, 13 parts, 14 parts, 15 parts, etc.

[0060] In the fermentation substrate, the addition amount of Suanzaoren is 12 parts, 13 parts, 14 parts, 15 parts, etc.

[0061] In the fermentation substrate, the addition amount of Fuling is 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, etc.

[0062] The addition amount of the longan pulp in the fermentation substrate is 30 parts, 31 parts, 32 parts, 33 parts, 34 parts, 35 parts, etc.

[0063] Preferably, the auxiliary material comprises at least one of blueberry concentrate, gamma-aminobutyric acid, casein peptide and sweetener.

[0064] Preferably, the sweetener comprises erythritol and natural sweetener.

[0065] In the present application, the blueberry concentrate contains effective components such as anthocyanins, gamma-aminobutyric acid has a nerve regulation effect, casein peptide has a nerve system relaxation effect, improves sleep and regulates immunity, erythritol and natural sweetener are used to improve the taste of the product, and the natural sweetener comprises mogroside and steviol glycoside.

[0066] Preferably, the auxiliary material comprises the following components in parts by weight: 50-55 parts of blueberry concentrate, 12-16 parts of gamma-aminobutyric acid, 5-7 parts of casein peptide, 60-70 parts of erythritol and 0.2-0.6 parts of natural sweetener.

[0067] Preferably, the addition amount of the blueberry concentrate in the auxiliary material is 50 parts, 51 parts, 52 parts, 53 parts, 54 parts, 55 parts, etc.

[0068] Preferably, the addition amount of the gamma-aminobutyric acid in the auxiliary material is 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, etc.

[0069] Preferably, the addition amount of the casein peptide in the auxiliary material is 5 parts, 6 parts, 7 parts, etc.

[0070] Preferably, the addition amount of the erythritol in the auxiliary material is 60 parts, 61 parts, 62 parts, 63 parts, 64 parts, 65 parts, 66 parts, 67 parts, 68 parts, 69 parts, 70 parts, etc.

[0071] Preferably, the addition amount of the natural sweetener in the auxiliary material is 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, etc.

[0072] The fourth aspect of the present application provides a preparation method of the nerve-calming and sleep-aiding fermentation composition, and the preparation method comprises the following steps:

[0073] The raw material components of the formula amount of the fermentation substrate are sequentially mixed, extracted and concentrated to obtain a concentrated solution, and then the composite probiotics are mixed with the concentrated solution to obtain a fermentation product.

[0074] In the present application, the nerve-calming and sleep-aiding fermentation composition is prepared by the preparation method, which can effectively improve the content of effective components in the product, and the preparation method is simple, efficient and feasible.

[0075] Further, the extraction includes primary extraction and secondary extraction.

[0076] In the present application, the twice extraction is adopted to improve the extraction rate, avoid the waste of effective components in the product, ensure the complete extraction, and further improve the product quality.

[0077] Preferably, the primary extraction includes sequentially adding water, primary boiling extraction and primary filtration to the mixed components of the fermentation substrate to obtain the primary extraction liquid.

[0078] Preferably, the water adding amount of the primary extraction is 10-20 times of the weight of the fermentation substrate raw material components, for example, can be 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, etc., and the primary boiling extraction time is 1-2 h, for example, can be 1 h, 1.5 h, 2 h, etc.

[0079] Preferably, the secondary extraction includes sequentially adding water, secondary boiling extraction and secondary filtration to the components of the fermentation substrate after the primary extraction to obtain the secondary extraction liquid.

[0080] In the present application, the silk cloth double-layer filtration is adopted when performing the primary filtration and the secondary filtration.

[0081] Preferably, the water adding amount of the secondary extraction is 10-20 times of the weight of the fermentation substrate raw material components, for example, can be 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, etc., and the secondary boiling extraction time is 1-2 h, for example, can be 1 h, 1.5 h, 2 h, etc.

[0082] Preferably, the extraction further includes mixing the primary extraction liquid and the secondary extraction liquid to obtain the total extraction liquid.

[0083] Further, the weight of the concentrated liquid is 4-7 times of the weight of the fermentation substrate raw material components, for example, can be 4 times, 5 times, 6 times, 7 times, etc.

[0084] Preferably, the concentration temperature is 75-85℃, for example, can be 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, etc.

[0085] In the present application, the double-effect external circulation evaporator is preferably adopted for heating and concentration, and the vacuum degree is set to -0.08--0.03Mpa.

[0086] Preferably, after the concentration, the concentrated liquid is subjected to the first sterilization before the fermentation, and the pasteurization method is preferably adopted.

[0087] In the present application, the concentrated liquid is poured into the fermentation tank without additional carbon and nitrogen sources, and then pasteurized and cooled to about 37℃.

[0088] Preferably, the mixing of the complex probiotics with the concentrated liquid comprises: mixing the Lactobacillus plantarum KMHD-1 powder and the Lactobacillus gasseri KMBGI 03 powder to obtain complex probiotic powder, and then dissolving the complex probiotics with water and inoculating into the concentrated liquid.

[0089] Preferably, the addition amount of the complex probiotic powder is 0.07-0.12% of the weight of the concentrated liquid.

[0090] Preferably, the fermentation time is 24-72 h, for example, it can be 24 h, 48 h, 72 h, etc.

[0091] In the present application, the fermentation substrate is fermented by using complex probiotics, and after stirring at 37℃ for 24-72 h, pasteurization is performed, and the precipitate is removed after overnight refrigeration and standing, to prepare a fermentation liquid for standby.

[0092] Further, the preparation method of the sleep-aiding fermented composition further comprises: mixing the formula amount of auxiliary materials and the fermentation product to obtain the sleep-aiding fermented composition.

[0093] Preferably, the mixing process comprises: separately dissolving the casein peptide and the natural sweetener, then mixing to obtain a premix, and mixing the gamma-aminobutyric acid and erythritol, and then adding the fermentation product, the blueberry concentrate and the premix.

[0094] In the present application, the casein peptide and the natural sweetener are separately dissolved with an appropriate amount of purified water to obtain a pre-solution; an appropriate amount of purified water is added to a pre-dissolution tank, stirring and steam heating are turned on, heating to 60-70℃, gamma-aminobutyric acid and erythritol are added, after stirring and dissolving, the fermentation concentrate, the blueberry concentrate and the premix are added, and constant stirring is performed until they are uniformly mixed. The weight is adjusted to 1 ton with purified water, and the mixture is uniformly mixed after heat preservation and stirring. The prepared liquid is filtered through a plate frame while hot, and then filled into containers. The filled semi-finished products are placed in a sterilization tray and a sterilization cabinet for moist heat sterilization. The sterilization conditions are: sterilization temperature 121℃, sterilization time 20 min.

[0095] The fifth aspect of the present application provides a use of the sleep-aiding fermented composition in the preparation of a sleep-aiding product.

[0096] In the present application, the homologous medicinal and edible herbs such as sour jujube kernel and ginseng are fermented by using complex probiotics, to prepare a sleep-aiding homologous medicinal and edible herb fermentation liquid and a sleep-aiding product, for example, a sleep-aiding beverage.

[0097] The application will be further described by examples. The materials in the examples are prepared according to the existing method or directly purchased from the market, unless otherwise specified.

[0098] Example 1

[0099] The present example provides a nerve-calming and sleep-aiding fermentation composition prepared by fermentation of composite probiotics (Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03), the components of which include a fermentation substrate and an auxiliary material;

[0100] wherein the weight ratio of Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03 is 1:1;

[0101] wherein the fermentation substrate includes the following components in parts by weight: 14 parts of ginseng, 14 parts of spina date seed, 23 parts of poria and 34 parts of longan meat;

[0102] wherein the auxiliary material includes the following components in parts by weight: 53 parts of blueberry concentrate, 14 parts of gamma-aminobutyric acid, 6 parts of casein peptide, 65 parts of erythritol, 0.2 parts of steviol glycoside and 0.2 parts of mogroside.

[0103] Example 2

[0104] The present example provides a nerve-calming and sleep-aiding fermentation composition prepared by fermentation of composite probiotics (Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03), the components of which include a fermentation substrate and an auxiliary material;

[0105] wherein the weight ratio of Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03 is 0.5:1;

[0106] wherein the fermentation substrate includes the following components in parts by weight: 12 parts of ginseng, 15 parts of spina date seed, 20 parts of poria and 35 parts of longan meat;

[0107] wherein the auxiliary material includes the following components in parts by weight: 50 parts of blueberry concentrate, 16 parts of gamma-aminobutyric acid, 5 parts of casein peptide, 70 parts of erythritol, 0.1 parts of steviol glycoside and 0.1 parts of mogroside.

[0108] Example 3

[0109] The present example provides a nerve-calming and sleep-aiding fermentation composition prepared by fermentation of composite probiotics (Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03), the components of which include a fermentation substrate and an auxiliary material;

[0110] The weight ratio of Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03 is 1.5:1.

[0111] The fermentation substrate includes, by weight fraction, 15 parts of ginseng, 12 parts of spina date, 26 parts of poria cocos and 30 parts of longan meat.

[0112] The auxiliary material includes, by weight fraction, 55 parts of blueberry concentrate juice, 12 parts of gamma-aminobutyric acid, 7 parts of casein peptide, 60 parts of erythritol, 0.3 parts of steviol glycoside and 0.3 parts of mogroside.

[0113] Embodiments 4-6

[0114] Embodiments 4-6 provide a preparation method of the nerve-calming sleep-aiding fermented composition, including the following steps:

[0115] Step 1: Extraction, according to the formula amount of the fermentation substrate components in embodiments 1-3, respectively, the formula amount of the fermentation substrate components is put into a multifunctional extraction tank, 15 times the amount of purified water is added for one-time extraction, boiling extraction is performed for 1.5 h, and then double-layer filtering is performed with silk cloth; 15 times the amount of purified water is added for two-time extraction, boiling extraction is performed for 1.5 h, and then double-layer filtering is performed with silk cloth, and the two-time filtrates are combined to obtain a total extraction liquid.

[0116] Step 2: Concentration, the total extraction liquid is pumped into a double-effect external circulation evaporator, heating and concentration are started, the vacuum degree is set to -0.08--0.03 Mpa, the temperature is 80℃, concentration is performed, and the concentration is performed to 6 times the weight of the raw material; the concentrated liquid is the fermentation substrate.

[0117] Step 3: Embodiments 4-6 are fermented according to the formula amount of the bacteria powder ratio in embodiments 1-3, respectively, the concentrated liquid is poured into a fermentation tank, and no additional carbon and nitrogen sources are added. Then, pasteurization is performed, and the temperature is reduced to about 37℃. Lactobacillus plantarum KMHD-1 powder and Lactobacillus gasseri KMBGI 03 powder are mixed, the addition amount of the compound probiotic bacteria powder is 0.1% of the weight of the concentrated liquid, then the compound probiotic bacteria powder is fully dissolved with normal-temperature sterile purified water (wherein, the use amount of the sterile purified water is 7-10 times the weight of the compound probiotic bacteria powder, which can achieve the purpose of fully dissolving the compound probiotic bacteria powder), and stirring is uniformly performed. After 48 h of stirring fermentation at 37℃, pasteurization is performed, the precipitate is removed after cold storage and overnight standing, and the product is prepared.

[0118] Step 4: According to the formula amount of the auxiliary components in Example 1-3, respectively, the casein peptide and natural sweetener were fully dissolved in an appropriate amount of purified water to obtain a pre-solution; an appropriate amount of purified water was added to the pre-solution tank, and stirring and steam heating were turned on and heated to 60-70℃, then γ-aminobutyric acid and erythritol were added, and after stirring and dissolving, fermentation concentrate, blueberry concentrate and premix were added, and stirred constantly until uniform. The weight was adjusted to 1 ton with purified water, and stirred and mixed uniformly. The prepared liquid was filtered through a plate and frame while hot, and then filled into bottles. The filled semi-finished products were placed in a sterilization tray and a sterilization cabinet for moist heat sterilization at 121℃ for 20 min.

[0119] Example 7

[0120] The present example provides a preparation method of a nerve-calming and sleep-aiding fermentation composition, comprising the following steps:

[0121] Step 1: Extraction. According to the formula amount of the fermentation substrate components in Example 1, the formula amount of the fermentation substrate components was put into a multifunctional extraction tank, 10 times the amount of purified water was added for one-time extraction, and boiled for 2 h, then double-layer filtered with silk cloth; 20 times the amount of purified water was added for two-time extraction, and boiled for 1 h, then double-layer filtered with silk cloth, and the filtrates were combined to obtain the total extract.

[0122] Step 2: Concentration. The total extract was pumped into a double-effect external circulation evaporator, and heating and concentration were started, the vacuum degree was set to -0.08--0.03 Mpa, and the temperature was 85℃, and the concentration was carried out, and the concentration was carried out until the weight of the raw material was 4 times, and the concentrated solution was the fermentation substrate.

[0123] Step 3: Fermentation. The concentrated solution was poured into a fermentation tank without additional carbon and nitrogen sources. Then pasteurization was carried out, and the temperature was reduced to about 37℃. Plant lactobacillus KMHD-1 powder and georgi lactobacillus KMBGI 03 powder were mixed, and the addition amount of the compound probiotic powder was 0.12% of the weight of the concentrated solution. Then the compound probiotic powder was fully dissolved in normal temperature sterile purified water (the amount of sterile purified water was 7-10 times the weight of the compound probiotic powder, which could fully dissolve the compound probiotic powder), and stirred uniformly. After stirring and fermentation at 37℃ for 72 h, pasteurization was carried out, and the precipitate was removed after cold storage overnight for standby.

[0124] Step 4: According to the formula amount of the auxiliary components in Example 1, the step 4 of Example 7 was consistent with the step 4 of Example 4.

[0125] Example 8

[0126] The present example provides a preparation method of a nerve-calming and sleep-aiding fermentation composition, comprising the following steps:

[0127] Step 1: Extraction, according to the formula amount of the fermentation substrate component in Example 1, the formula amount of the fermentation substrate component is put into a multifunctional extraction tank, 20 times the amount of purified water is added for one extraction, boiling extraction for 1 h, then double-layer filtering with silk cloth; 10 times the amount of purified water is added for two extractions, boiling extraction for 2 h, then double-layer filtering with silk cloth, and the filtrates of the two extractions are combined to obtain the total extraction liquid.

[0128] Step 2: Concentration, the total extraction liquid is pumped into a double-effect external circulation evaporator, and heating and concentration are started, the vacuum degree is set to -0.08-0.03 Mpa, and the temperature is 75℃, and the concentration is performed, and the concentration is performed until the weight of the raw material is 7 times, and the concentrated liquid is the fermentation substrate.

[0129] Step 3: Fermentation, the concentrated liquid is poured into a fermentation tank without additional carbon and nitrogen sources. Then pasteurization is performed, and the temperature is reduced to about 37℃. Plantarum KMHD-1 bacteria powder and Lactobacillus gasseri KMBGI 03 bacteria powder are mixed, and the addition amount of the compound probiotic bacteria powder is 0.07% of the weight of the concentrated liquid. Then the compound probiotic bacteria powder is fully dissolved in normal temperature sterile purified water (wherein the amount of sterile purified water used is 7-10 times the weight of the compound probiotic bacteria powder, which can achieve the purpose of fully dissolving the compound probiotic bacteria powder), and stirring is uniform. After stirring fermentation at 37℃ for 24 h, pasteurization is performed, and the precipitate is removed after cold storage overnight for standby.

[0130] Step 4: According to the formula amount of the auxiliary component in Example 1, the step 4 of Example 8 is consistent with the step 4 of Example 4.

[0131] Comparative Example 1

[0132] This comparative example provides a sleep-aiding and nerve-calming fermented composition, which is different from Example 1 in that the sleep-aiding and nerve-calming fermented composition is prepared by fermenting a fermentation substrate with Lactobacillus plantarum KMHD-1, and the rest is consistent with Example 1.

[0133] Comparative Example 2

[0134] This comparative example provides a sleep-aiding and nerve-calming fermented composition, which is different from Example 1 in that the sleep-aiding and nerve-calming fermented composition is prepared by fermenting a fermentation substrate with Lactobacillus gasseri KMBGI 03, and the rest is consistent with Example 1.

[0135] Comparative Example 3

[0136] This comparative example provides a sleep-aiding and nerve-calming fermented composition, which is different from Example 1 in that the sleep-aiding and nerve-calming fermented composition is prepared by fermenting a fermentation substrate with a compound probiotic bacteria (commercial bacteria Lactobacillus plantarum and commercial bacteria Lactobacillus gasseri), and the rest is consistent with Example 1.

[0137] Comparative Example 4

[0138] This comparative example provides a calming and sleep-aiding fermented composition, which differs from Example 1 in that the calming and sleep-aiding fermented composition is prepared by fermenting a fermentation substrate with a compound probiotic (Lactobacillus plantarum KMHD-1 and commercial Lactobacillus gasseri), while the rest is the same as Example 1.

[0139] Comparative Example 5

[0140] This comparative example provides a calming and sleep-aiding fermented composition, which differs from Example 1 in that the calming and sleep-aiding fermented composition is prepared by fermenting a fermentation substrate with a compound probiotic (commercial bacteria Lactobacillus plantarum and Lactobacillus gasseri KMBGI 03), while the rest is the same as Example 1.

[0141] Comparative Examples 6-10

[0142] Comparative Examples 6-10 provide a method for preparing a calming and sleep-aiding fermented composition, and the calming and sleep-aiding fermented compositions of Comparative Examples 1-5 are prepared using the preparation method of Example 4.

[0143] Test Example 1. Detection of Active Ingredients.

[0144] (1) Detection method:

[0145] Ginsenosides were isolated using high performance liquid chromatography (HPLC): acetonitrile containing 0.1% formic acid was used as mobile phase B, and water containing 0.1% formic acid was used as mobile phase C, with gradient elution. The chromatographic column was an ACQUITY UPLC BEH Shield RP18 (2.1 mm × 100 mm, 1.7 μm); the flow rate was 0.4 mL / min; and the column temperature was 30℃.

[0146] Jujube seed saponins were determined by high performance liquid chromatography: the chromatographic column was Ultimate AQ-C18 (4.6 mm × 250 mm, 5 μm), the mobile phase was acetonitrile-water (33:77), the flow rate was 1.0 mL·min-1, and the column temperature was 30℃.

[0147] Polysaccharide determination was performed using the phenol-sulfuric acid colorimetric method: Accurately measure 0.0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of glucose standard solution into 20 mL stoppered test tubes, and bring the volume to 1.0 mL with water. Add 1.0 mL of 5% phenol solution, then quickly add 5.0 mL of sulfuric acid, and let stand for 10 minutes. Ensure the reaction solution is thoroughly mixed, and react in a 30°C water bath for 20 minutes. Measure the absorbance at 490 nm. Plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis. The standard curve is: y = 0.008x + 0.0885, R0 2 =0.9901.

[0148] (2) Test samples: The calming and sleep-aiding fermented compositions prepared in Examples 4-8 and Comparative Examples 6-10 were tested using the same method, and the results are shown in Table 1 below. Among them, 12 rare ginsenosides were tested: Rh1, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, 20(S)-PPT, 20(R)-PPT, CK, Rk1, Rg5, 20(S)-Rh2, 20(R)-Rh2, 20(S)-PPD.

[0149] Table 1

[0150]

[0151] As shown in Table 1, the sleep-aiding products prepared in the embodiments of the present invention all contain abundant rare ginsenosides, jujube seed saponins, and polysaccharides. The higher content of rare ginsenosides in Comparative Example 6 compared to Comparative Example 8 indicates that the fermentation and conversion of saponins by the self-screened bacteria *Lactobacillus plantarum* is significantly effective. Compared with Comparative Examples 6 and 7, Comparative Examples 9 and 10, which used a mixture of self-screened bacteria and commercial bacteria for fermentation, showed that the content of rare ginsenosides, jujube seed saponins, and polysaccharides was increased to varying degrees, indicating that mixed-bacterial fermentation is more effective than single-bacterial fermentation. The content of rare saponins, jujube seed saponins, and polysaccharides in the product of Example 4 was significantly higher than that in Comparative Examples 8, 9, and 10. This shows that the specific interaction produced by the fermentation of the two self-screened strains affected the conversion process of rare ginsenosides. In addition, the mixed fermentation of the two self-screened strains also significantly increased the content of jujube seed saponins and polysaccharides, indicating that the sleep-aiding effect of mixed fermentation of the two self-screened strains was more significant.

[0152] Test Example 2. Population Trial Evaluation.

[0153] (1) Testing method: In accordance with the "Guiding Principles for Clinical Research of New Traditional Chinese Medicine Drugs for the Treatment of Insomnia", during the recruitment of research subjects, those who simultaneously meet both the primary and secondary symptoms are diagnosed with insomnia and included in the study. The primary symptoms are: restlessness and insomnia, or light sleep and easy awakening, or difficulty falling back asleep after waking up; the secondary symptoms are: five-center heat, night sweats, dry mouth and throat, palpitations, and soreness and weakness of the lower back and knees.

[0154] The subjects were divided into four groups of 30 each. After completing a Traditional Chinese Medicine (TCM) symptom scoring form, the subjects received oral treatment with the product of this invention. The primary and secondary symptoms of the four groups were scored before and after treatment to obtain symptom scores. Each primary symptom was scored as 0, 2, 4, or 6 points according to a scale of none (mild, moderate, severe); each secondary symptom was scored as 0, 1, 2, or 3 points according to the same scale. The scores for each symptom were then summed to obtain the symptom score. The clinical efficacy of the subjects was then analyzed based on the symptom scores. According to the "Guiding Principles for Clinical Research of New Traditional Chinese Medicine Drugs," the clinical efficacy was divided into the following four levels:

[0155] Cure: Symptom score reduced by ≥95%, and TCM clinical symptoms and signs completely relieved or disappeared;

[0156] Significant effect: Symptom score reduced by ≥70%, and clinical symptoms and signs in traditional Chinese medicine showed significant improvement;

[0157] Effective: Symptom scores decreased by ≥30%, and both clinical symptoms and signs in Traditional Chinese Medicine showed signs of improvement;

[0158] Ineffective: Symptom score reduction of less than or equal to 30%, no improvement in TCM clinical symptoms and signs, or even signs of worsening.

[0159] The number of subjects who recovered, showed significant improvement, showed improvement, and showed no improvement were determined. The total number of effective cases and the overall effective rate were calculated and recorded in Table 2 below. The total number of effective cases = number of recovered cases + number of cases with significant improvement + number of cases with improvement; the overall effective rate = (number of recovered cases + number of cases with significant improvement + number of cases with improvement) / total number of cases × 100%.

[0160] (2) Test samples: The calming and sleep-aiding fermented compositions prepared in Example 4 and Comparative Examples 6-10 were tested using the same method, and the results are shown in Table 2 below.

[0161] Table 2. Clinical efficacy analysis table

[0162]

[0163] As shown in Table 2, after one month of continuous use of the above six products, the symptoms of the subjects were relieved to varying degrees. Symptom scores were calculated, and the effective rate of improving insomnia was 90% for subjects using the product of Example 4, 76.7% for subjects using Comparative Example 6, 73.3% for subjects using Comparative Example 7, 76.7% for subjects using Comparative Example 8, 83.3% for subjects using Comparative Example 9, and 80.8% for subjects using Comparative Example 10. This demonstrates that the product prepared in Example 4 of this invention has significant therapeutic effects on subjects with insomnia.

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nerve-calming sleep-aiding fermented composition, characterized by, The nerve-calming sleep-aiding fermentation composition comprises a fermentation substrate and an auxiliary material; The nerve-calming sleep-aiding fermentation composition is prepared by fermenting a fermentation substrate with compound probiotics, and the fermentation substrate comprises, by weight fraction, 12-15 parts of ginseng, 12-15 parts of spina date seed, 20-26 parts of poria cocos, and 30-35 parts of longan arillus; The complex probiotic includes: Lactobacillus plantarum (L. Lactiplantibacillus plantarum ) KMHD-1 and Lactobacillus gasseri (L. Lactobacillus gasseri ) KMBGI 03; The Lactiplantibacillus plantarum KMHD-1 has been preserved in the China General Microbiological Culture Collection Center on August 28, 2023, with a preservation number of CGMCC No.28269 and a preservation address of No.3, Beichen West Road, Chaoyang District, Beijing; The Lactobacillus gasseri KMBGI 03 has been preserved in the China General Microbiological Culture Collection Center on January 14, 2019, with a preservation number of CGMCC No.17177 and a preservation address of No.3, Beichen West Road, Chaoyang District, Beijing; The weight ratio of the Lactiplantibacillus plantarum KMHD-1 to the Lactobacillus gasseri KMBGI 03 is (0.5-1.5):

1.

2. The preparation method of the nerve-calming sleep-aiding fermentation composition according to claim 1, characterized by, The preparation method comprises the following steps: The raw material components of the formula amount of the fermentation substrate are sequentially mixed, extracted, and concentrated to obtain a concentrated solution, and then the compound probiotics are mixed with the concentrated solution to obtain a fermentation product; The mixing of the compound probiotics with the concentrated solution comprises: mixing Lactiplantibacillus plantarum KMHD-1 powder and Lactobacillus gasseri KMBGI 03 powder to obtain compound probiotic powder, and then dissolving the compound probiotics in water and inoculating into the concentrated solution; The preparation method of the nerve-calming sleep-aiding fermentation composition further comprises: mixing the auxiliary material and the fermentation product to obtain the nerve-calming sleep-aiding fermentation composition; The nerve-calming sleep-aiding fermentation composition is prepared by fermenting a fermentation substrate with compound probiotics, and the fermentation substrate comprises, by weight fraction, 12-15 parts of ginseng, 12-15 parts of spina date seed, 20-26 parts of poria cocos, and 30-35 parts of longan arillus; The complex probiotic includes: Lactobacillus plantarum (L. Lactiplantibacillus plantarum ) KMHD-1 and Lactobacillus gasseri (L. Lactobacillus gasseri ) KMBGI 03; The Lactiplantibacillus plantarum KMHD-1 has been preserved in the China General Microbiological Culture Collection Center on August 28, 2023, with a preservation number of CGMCC No.28269 and a preservation address of No.3, Beichen West Road, Chaoyang District, Beijing; The Lactobacillus gasseri KMBGI 03 has been preserved in the China General Microbiological Culture Collection Center on January 14, 2019, with a preservation number of CGMCC No.17177 and a preservation address of No.3, Beichen West Road, Chaoyang District, Beijing; The weight ratio of the Lactiplantibacillus plantarum KMHD-1 to the Lactobacillus gasseri KMBGI 03 is (0.5-1.5):

1. The auxiliary material comprises at least one of blueberry concentrate, gamma-aminobutyric acid, casein peptide, and sweetener.

3. The method of preparing the nerve-calming sleep-aiding fermentation composition according to claim 2, wherein the fermentation is performed at a temperature of 30 to 40°C for 1 to 10 days. The sweetener comprises erythritol and natural sweetener.

4. The preparation method of the nerve-calming sleep-aiding fermentation composition according to claim 2, characterized by, The auxiliary material comprises the following components in parts by weight: blueberry concentrate 50-55 parts, gamma-aminobutyric acid 12-16 parts, casein peptide 5-7 parts, erythritol 60-70 parts, and natural sweetener 0.2-0.6 parts.

5. The preparation method according to claim 2, characterized in that, The extraction comprises primary extraction and secondary extraction.

6. The production method according to claim 5, characterized by, The primary extraction comprises sequentially adding water, primary boiling extraction, and primary filtration to the mixed components of the fermentation substrate, to obtain a primary extraction liquid.

7. The production method according to claim 6, characterized by, The water added in the primary extraction is 10-20 times the weight of the fermentation substrate raw material components, and the primary boiling extraction is performed for 1-2 h.

8. The preparation method according to claim 6, characterized in that, The secondary extraction comprises sequentially adding water, secondary boiling extraction, and secondary filtration to the components of the fermentation substrate after the primary extraction, to obtain a secondary extraction liquid.

9. The production method according to claim 8, characterized by, The water added in the secondary extraction is 10-20 times the weight of the fermentation substrate raw material components, and the secondary boiling extraction is performed for 1-2 h.

10. The preparation method according to claim 8, characterized in that, The extraction further comprises mixing the primary extraction liquid and the secondary extraction liquid to obtain a total extraction liquid.

11. The method of claim 2, wherein, The weight of the concentrated liquid is 4-7 times the weight of the fermentation substrate raw material components.

12. The method of claim 2, wherein, The concentration temperature is 75-85℃.

13. The preparation method according to claim 2, characterized in that, After the concentration, the concentrated liquid is subjected to first sterilization before fermentation.

14. The method of claim 13, wherein, The first sterilization is performed by pasteurization.

15. The preparation method according to claim 2, characterized in that, The added amount of the compound probiotic powder is 0.07-0.12% of the weight of the concentrated liquid.

16. The method of claim 2, wherein The fermentation time is 24-72 h.

17. The preparation method according to claim 2, characterized in that, The mixing process comprises dissolving the casein peptide and the natural sweetener respectively, then mixing to obtain a premix, and mixing the gamma-aminobutyric acid and the erythritol, then adding the fermentation product, the blueberry concentrate, and the premix.

18. Use of the nerve-calming and sleep-aiding fermentation composition according to claim 1 or the nerve-calming and sleep-aiding fermentation composition prepared by the preparation method of any one of claims 2-17 in the preparation of a nerve-calming and sleep-aiding product.

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