CN111714581A

CN111714581A - Sleep-aiding enzyme and preparation method thereof

Info

Publication number: CN111714581A
Application number: CN202010712615.2A
Authority: CN
Inventors: 张国栋
Original assignee: Jiangxi Renren Health Industry Co ltd
Current assignee: Jiangxi Renren Health Industry Co ltd
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2020-09-29

Abstract

The invention relates to the field of functional beverages, and particularly relates to a sleep-aiding ferment and a preparation method thereof. The ferment comprises the following raw materials in parts by weight: 4-6 parts of lotus seed, 1-2 parts of spina date seed, 8-10 parts of arillus longan, 8-10 parts of lily, 8-10 parts of poria cocos, 2-4 parts of cinnamon, 1-2 parts of ginseng, 2.5-4.5 parts of noni pulp and 0.5-1 part of gamma-aminobutyric acid. The sleep-aiding ferment provided by the invention takes lotus seeds and cinnamon as monarch drugs, spina date seeds and arillus longan as ministerial drugs, lily, poria cocos and ginseng as adjuvant drugs, and gamma-aminobutyric acid and noni fruit pulp are added in an auxiliary manner, the compatibility of the drugs is limited according to the traditional Chinese medicine theory of monarch, ministerial, assistant and guide, and the sleep-aiding ferment has a good sleep-improving effect.

Description

Sleep-aiding enzyme and preparation method thereof

Technical Field

The invention relates to the field of functional beverages, and particularly relates to a sleep-aiding ferment and a preparation method thereof.

Background

Insomnia, i.e., sleep disorder, is a condition in which the excitation and inhibition functions of the cerebral cortex are disturbed due to excessive catecholamine secretion in the body, insufficient 5-hydroxytryptamine secretion, which is usually caused by physical, physiological, psychological, medicinal, artificial, nutritional deficiency, etc., thereby causing disturbance of slow-wave sleep and fast-wave sleep during sleep, which is usually manifested as difficulty in falling asleep or difficulty in maintaining sleep (easy-to-wake, early-wake and difficulty in falling asleep again).

According to WHO data, people WHO are nearly 1/4 all over the world are suffering from insomnia, and insomnia patients in developing countries account for about 30% -35% of the general population. According to the survey data of the Chinese medical society, the prevalence rate of sleep disorder in China is 42.7%, and about 3 hundred million middle-aged people suffer from sleep disorder. Insomnia is characterized by difficulty in falling asleep, difficulty in keeping sleep after falling asleep, or feeling of cachexia and poor spirit after waking up, and difficulty in entering work and study, so that insomnia has great adverse effect on normal life and work and study of people.

At present, a plurality of treatment methods for treating insomnia exist, wherein non-drug therapy includes cognitive behavior therapy, physical therapy and the like, drug therapy includes benzodiazepine hypnotic drug therapy, non-benzodiazepine hypnotic drug therapy, Chinese herbal medicine therapy and the like, at present, the most widely used drug therapy is still drug therapy, but the drug therapy has certain side effects, can generate dependence and has the risk of addiction, so that a method for treating insomnia with obvious effect, no side effect, safety and reliability needs to be researched.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defects that the insomnia treatment by drug therapy in the prior art has certain side effects, can generate dependence and has addiction risk, thereby providing the sleep-aiding ferment and the preparation method thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the sleep-aiding ferment comprises the following raw materials in parts by weight:

4-6 parts of lotus seed, 1-2 parts of spina date seed, 8-10 parts of arillus longan, 8-10 parts of lily, 8-10 parts of poria cocos, 2-4 parts of cinnamon, 1-2 parts of ginseng, 2.5-4.5 parts of noni pulp and 0.5-1 part of gamma-aminobutyric acid.

Further, the enzyme comprises the following raw materials in parts by weight:

6 parts of lotus seed, 1 part of spina date seed, 10 parts of arillus longan, 10 parts of lily, 10 parts of poria cocos, 2 parts of cinnamon, 1 part of ginseng, 3 parts of noni pulp and 1 part of gamma-aminobutyric acid.

Further, the ferment also comprises seasoning sugar, and the additive amount of the seasoning sugar accounts for 10% of the total mass of the ferment.

The invention also provides a method for preparing the sleep-aiding ferment according to any one of the schemes, which comprises the following steps:

taking lotus seeds, spina date seeds, arillus longan, lily, poria cocos, cinnamon and ginseng according to a formula, mixing, adding boiling water for extraction, extracting twice, wherein the ratio of the adding amount of the boiling water to the total mass of the medicinal materials in each time is (5-15): 1, extracting for 1-2 hours each time, combining the two water extract solutions, and cooling to obtain a medicinal material water extract solution;

adding noni pulp into the water extract of the medicinal materials and performing fermentation treatment, wherein the final acidity of the fermentation liquid is more than or equal to 10g/L as a fermentation end point;

and (3) after the fermentation is finished, taking the supernatant, adding gamma-aminobutyric acid for blending, filtering and sterilizing to obtain the sleep-aiding enzyme.

Further, the fermentation comprises the step of sequentially carrying out lactobacillus fermentation, yeast fermentation and acetic acid bacteria fermentation on a mixed material formed by mixing the medicinal material water extract and the noni pulp.

Further, in the above-mentioned case,

the lactobacillus fermentation comprises the step of adding lactobacillus into the mixed material, and fermenting at 37-43 ℃ until the acidity of the mixed material is more than or equal to 2-5g/L to obtain a first fermentation solution; and/or the presence of a gas in the gas,

the yeast fermentation comprises the steps of adding yeast into the first fermentation liquid, and fermenting at 25-30 ℃ until the alcoholic strength of the first fermentation liquid is 0.5-1 degrees to obtain a second fermentation liquid; and/or the presence of a gas in the gas,

the acetic acid bacteria fermentation comprises the steps of adding acetic acid bacteria into the second fermentation liquid, wherein the volume of the second fermentation liquid per minute is as follows: and (3) fermenting under the environment of introducing air according to the aeration ratio of (0.07-0.1) when the air volume is 1 until the acidity of the fermentation liquor is more than or equal to 10 g/L.

Further, the addition amount of the lactic acid bacteria is 0.1-0.5 per mill of the total mass of the mixed material, the addition amount of the yeast is 0.5-1 per mill of the total mass of the first fermentation liquid, and the addition amount of the acetic acid bacteria is 0.5-1 per mill of the total mass of the second fermentation liquid.

Further, the preparation method also comprises the step of adding 20 parts of white granulated sugar and 10 parts of brown granulated sugar into the medicinal material water extract before fermentation.

Further, the blending step further comprises the step of adding a flavoring sugar to the supernatant.

Further, before the blending step, the method also comprises the step of sealing and aging the fermentation liquor after fermentation at normal temperature for 1-2 months.

The technical scheme of the invention has the following advantages:

1. according to the sleep-aiding enzyme provided by the invention, according to the record of pharmacopoeia and related Chinese medical data, lotus seeds are used for clearing heart fire and calming the heart, so that the heart fire can be lowered, cinnamon warms the kidney, and water and gas are dissolved, so that the heart can be nourished, therefore, the two medicines are used together, the heart and the kidney can be communicated, yin and yang can be harmonized, and yang can enter yin, so that the sleep can be achieved; the spina date seed mainly disturbs heart and does not sleep; arillus longan is a key herb for heart and spleen, and is mainly used for treating amnesia, severe palpitation and palpitation caused by mental and mental strain; lily has effects of tranquilizing mind, fixing gallbladder, benefiting mind, and nourishing five internal organs; poria helps to calm mind; ginseng is sweet and slightly cold in taste, and has the effects of nourishing five internal organs, calming the mind, calming soul and stopping palpitation; the lotus seed, the cinnamon, the spina date seed, the longan pulp, the lily, the tuckahoe and the ginseng can play a role in nourishing the heart and soothing the nerves; meanwhile, modern medicine shows that: gamma-aminobutyric acid is a strong nerve inhibitory amino acid and has the physiological effects of calming, hypnosis, resisting convulsion, reducing blood pressure and the like; noni pulp can promote melatonin secretion of pineal body and help sleep. The sleep-aiding ferment provided by the invention takes lotus seeds and cinnamon as monarch drugs, spina date seeds and arillus longan as ministerial drugs, lily, poria cocos and ginseng as adjuvant drugs, and gamma-aminobutyric acid and noni fruit pulp are added in an auxiliary manner, the compatibility of the drugs is limited according to the monarch, ministerial, assistant and guide pharmacology theory of traditional Chinese medicine, the sleep-aiding ferment has a good sleep-improving effect, the used traditional Chinese medicinal materials are medicinal and edible medicinal materials, and the gamma-aminobutyric acid is prepared from lactic acid bacteria, so that the product is high in edible safety, and free of side effects and dependence.

2. According to the method for preparing the sleep-aiding ferment, the ferment is prepared by extracting the traditional Chinese medicinal materials and then fermenting, the activity of the effective components in the traditional Chinese medicinal materials is enhanced by utilizing the conversion effect of microorganisms, and macromolecules in the materials can be converted into micromolecular substances, so that the absorption rate of a human body to the effective components in the ferment is improved, meanwhile, the product has special fermentation fragrance, the original smell and taste of the traditional Chinese medicinal materials are changed, and the acceptance of the public to the product is improved.

3. According to the method for preparing the sleep-aiding ferment, the lactic acid bacteria, the acetic acid bacteria and the saccharomycetes are sequentially fermented, on one hand, the fermentation end points of the three types of fermentation are just suitable for the survival of the microorganisms of the latter, so that the fermentation process can be accelerated to the maximum extent, and on the other hand, the microorganisms such as the lactic acid bacteria and the saccharomycetes can generate gamma-aminobutyric acid in the fermentation process, so that the amount of the gamma-aminobutyric acid which needs to be additionally added can be reduced, and the effect of saving raw materials is achieved.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

The noni pulp related to the invention is a new resource food approved by No. 9 bulletin of the Ministry of health in 2010, and is prepared by mixing noni and water according to a mass ratio of 1: 1, pulping.

In the invention, the main purpose of the white granulated sugar and the brown granulated sugar added before fermentation is to provide nutrient substances for microbial fermentation so as to improve the fermentation speed, and the main purpose of the seasoning sugar added in the blending step is to season the ferment, wherein the seasoning sugar can be honey and/or fructo-oligosaccharide, preferably honey and fructo-oligosaccharide.

Example 1

The embodiment relates to a sleep-aiding ferment which is prepared according to the following steps:

s1, extraction: mixing 6g of lotus seeds, 1g of spina date seeds, 10g of arillus longan, 10g of lily, 10g of poria cocos, 2g of cinnamon and 1g of ginseng, placing the mixture into an extraction container, adding 500g of boiling water for extraction for 2 hours to obtain a first water extract, separating the traditional Chinese medicinal materials, adding 500g of boiling water for second extraction for 1 hour to obtain a second water extract, and combining the first water extract and the second water extract to a constant volume of 1L to obtain the medicinal material water extract.

S2, fermentation: adding 4.5g of noni pulp, 20g of white granulated sugar and 10g of brown granulated sugar into the water extract of the medicinal material obtained in the step S1, mixing to obtain a mixed material, adding 0.4g of lactic acid bacteria into the mixed material, and fermenting at 43 ℃ until the acidity of the mixed material is 3g/L to obtain a first fermentation liquid;

continuously adding 0.5g of yeast into the first fermentation liquid, and fermenting at 28 ℃ until the alcoholic strength of the first fermentation liquid is 1 degree to obtain a second fermentation liquid;

continuously adding 0.5g of acetic acid bacteria into the second fermentation liquor, wherein the mass ratio of the second fermentation liquor per minute: fermenting under the environment of introducing air with the air ventilation ratio of 1:0.7 until the acidity of the fermentation liquid is 10g/L

S3, ageing: sealing the fermented material at room temperature and aging for 30 days

S4, blending: and taking the aged fermentation supernatant, and adding 1g of gamma-aminobutyric acid, 50g of honey and 50g of fructo-oligosaccharide into the supernatant.

S5, post-processing: and (5) filtering and sterilizing the material after the step S4 to obtain the sleep-aiding ferment.

Example 2

s1, extraction: mixing 4g of lotus seeds, 2g of spina date seeds, 8g of arillus longan, 8g of lily, 8g of poria cocos, 4g of cinnamon and 2g of ginseng, placing the mixture in an extraction container, adding 500g of boiling water for extraction for 2 hours to obtain a first water extract, separating the traditional Chinese medicinal materials, adding 500g of boiling water for secondary extraction for 1 hour to obtain a second water extract, combining the first water extract and the second water extract, and fixing the volume to 1L to obtain the medicinal material water extract.

S2, fermentation: adding 2.5g of noni pulp, 15g of white granulated sugar and 15g of brown granulated sugar into the water extract of the medicinal materials obtained in the step S1, mixing to obtain a mixed material, adding 0.2g of lactic acid bacteria into the mixed material, and fermenting at 43 ℃ until the acidity of the mixed material is 5g/L to obtain a first fermentation liquid;

continuously adding 0.8g of yeast into the first fermentation liquid, and fermenting at 25 ℃ until the alcoholic strength of the first fermentation liquid is 0.8 degrees to obtain a second fermentation liquid;

continuously adding 0.8g of acetic acid bacteria into the second fermentation liquor, wherein the mass ratio of the second fermentation liquor per minute: fermenting under the environment of introducing air with the air ventilation ratio of 1:0.7 until the acidity of the fermentation liquid is 10g/L

S3, ageing: sealing the fermented material at room temperature and aging for 60 days

S4, blending: collecting the aged fermentation supernatant, and adding 0.5g of gamma-aminobutyric acid, 20g of honey and 80g of fructo-oligosaccharide into the supernatant.

Example 3

s1, extraction: mixing 5g of lotus seeds, 1.5g of spina date seeds, 9g of arillus longan, 9g of lily, 9g of poria cocos, 3g of cinnamon and 1.5g of ginseng, placing the mixture into an extraction container, adding 500g of boiling water for extraction for 2 hours to obtain a first water extract, separating the traditional Chinese medicinal materials, adding 500g of boiling water for extraction for a second time, obtaining a second water extract, combining the first water extract and the second water extract, and fixing the volume to 1L to obtain the medicinal material water extract.

S2, fermentation: adding 3.5g of noni pulp, 10g of white granulated sugar and 20g of brown granulated sugar into the water extract of the medicinal materials obtained in the step S1, mixing to obtain a mixed material, adding 0.15g of lactic acid bacteria into the mixed material, and fermenting at 41 ℃ until the acidity of the mixed material is 4g/L to obtain a first fermentation liquid;

continuously adding 0.7g of yeast into the first fermentation liquid, and fermenting at 25 ℃ until the alcoholic strength of the first fermentation liquid is 0.9 degrees to obtain a second fermentation liquid;

continuously adding 0.7g of acetic acid bacteria into the second fermentation liquor, and mixing the second fermentation liquor: fermenting under the environment of introducing air with the air ventilation ratio of 1:0.7 until the acidity of the fermentation liquid is 10g/L

S3, ageing: sealing and aging the fermented material at room temperature for 45 days.

S4, blending: collecting the aged fermentation supernatant, and adding 0.75g of gamma-aminobutyric acid, 80g of honey and 20g of fructo-oligosaccharide into the supernatant.

Comparative example 1

The comparative example relates to a sleep-aiding enzyme, which is prepared according to the following steps:

mixing 6g of lotus seeds, 1g of spina date seeds, 10g of arillus longan, 10g of lily, 10g of poria cocos, 2g of cinnamon and 1g of ginseng, placing the mixture into an extraction container, adding 500g of boiling water for extraction for 2 hours to obtain a first water extract, separating the traditional Chinese medicinal materials, adding 500g of boiling water for secondary extraction for 1 hour to obtain a second water extract, combining the first water extract and the second water extract to a constant volume of 1L, and adding 1g of gamma-aminobutyric acid to obtain the sleep-aiding ferment.

Test examples

First, component analysis

1. Total phenol and total flavone contents

The contents of total phenols and total flavonoids in the ferments provided in examples 1-3 and comparative example 1 were analyzed using a spectrophotometer method, and the results are shown in table 1.

TABLE 1 Total phenol and Total flavone contents of examples and comparative examples

	Total phenols (mg/L)	Total Flavonoids (mg/L)
			Example 1	291	4.0
Example 2	250	5.1
			Example 3	265	4.3
Comparative example 1	237	4.1

2. Amino acid content

The contents of amino acids in the ferments provided in examples 1 to 3 and comparative example 1 were analyzed using an automatic amino acid analyzer, and the results are shown in table 2.

TABLE 2 amino acid contents (mg/L) of examples and comparative examples

3. Total acid and organic acid content

The total acid content of the ferments provided in examples 1-3 and comparative example 1 was analyzed by chemical titration, and the results are shown in table 3.

The contents of organic acids in the ferments provided in examples 1 to 3 and comparative example 1 were analyzed by liquid chromatography, and the results are shown in table 4.

TABLE 3 Total acid content of examples and comparative examples

	Example 1	Example 2	Example 3	Comparative example 1
					Total acid content (g/L)	16.7	16.58	18.07	1.33

TABLE 4 organic acid content of examples and comparative examples

Second, evaluation of function

1. Sleep test

(1) Test materials

The sleep-aiding ferment is obtained according to the preparation method of the example 1;

mouse male Kunming mouse, 18-22g

Distilled water

Experiment grouping of Anshen Bunao Jilin Aodongbai pharmaceutical industry Co., Ltd (2)

Mice were randomly divided into 4 groups of 10 mice each, and the administration method for each group was as follows:

negative control group: distilled water (gavage for 7 days, 0.2mL/day, gavage twice a day)

Positive control group: tranquilizing and brain nourishing liquid (Ten times diluted by sterile water, mixed well, ready to use, gavage for 7 days, 0.2mL/day, gavage twice a day)

Low dose group: sleep-aiding ferment (gavage for 7 days, 0.2mL/day, gavage twice a day)

High dose group: sleep-aiding ferment (gavage for 7 days, 0.4mL/day, gavage twice a day)

(3) Experimental methods

A. Direct sleep experiment

And (3) performing intragastric lavage on each group of mice respectively at one time according to a preset dose, and observing whether the animals sleep or not. The disappearance of the righting reflex is used as an index for sleeping. When the mouse is placed in the back lying position, the mouse can turn over to the right position immediately, if the mouse cannot turn over for more than 60s, the turning-over reflex is considered to disappear, and the mouse enters sleep. The recovery of the righting reflex is the awakening of the animal, the period from disappearance of the righting reflex to recovery is the sleeping time of the animal, the number of sleeping animals in 4 groups within 1h and the sleeping time are recorded, whether each tested object has the effect of directly inducing sleep is observed, and the experimental result is shown in a table 5.

B. Pentobarbital sodium subthreshold dose hypnosis experiment

After each group of mice are subjected to last gastric lavage for 30min, the maximum subthreshold hypnotic dose of the sodium pentobarbital is 30mg/kg.BW (the dose that 80% -90% of mice do not disappear after positive reflex turning), the abdominal cavities of the two groups of mice are injected, the number of animals falling asleep within 30min is recorded (the number of animals with positive reflex turning disappears is more than 60 s), and the experimental results are shown in Table 6.

C. Experiment of sleep latency of barbital sodium

After each group of mice had been subjected to the last gastric lavage for 30min, the mice were intraperitoneally injected with barbital sodium at a dose of 250mg/kg. BW, the time from the start of injection to the disappearance of the righting reflex was taken as the sleep latency, and the sleep latency of each group of mice was recorded, and the experimental results are shown in Table 7.

D. Experiment for prolonging sleep time of sodium pentobarbital

After each group of mice had been subjected to last gastric lavage for 30min, pentobarbital sodium was intraperitoneally injected at 50mg/kg. BW, disappearance of the righting reflex and recovery of the righting reflex were used as indexes, the period of time between the two was taken as sleep time, and the experimental results are shown in Table 8.

(4) Results of the experiment

TABLE 5 direct sleep test results

TABLE 6 hypnotic test results

Grouping	Number of experimental mice/mouse	Number of sleeping mice/mouse	Sleep rate/%)
				Negative control group	10	2	20
Positive control group	10	6	60
				Low dose group	10	5	50
High dose group	10	5	50

TABLE 7 sleep latency test results

Grouping	Number of experimental mice/mouse	Latency/s	Reduction/degree of
				Negative control group	10	390±92	-
Positive control group	10	271±46	30.51
				Low dose group	10	304±60	22.05
High dose group	10	309±73	20.77

TABLE 8 sleep time test results

(5) Conclusion of the experiment

Direct sleep experiment

In the direct sleep experiment, four groups of mice arranged in the experiment do not sleep, so that the positive control drug and the sleep-aiding ferment do not have direct hypnotic effect on the mice, the requirements of sleep-improving health-care food in a health-care food inspection and evaluation technical specification implementation manual are met, and the basic precondition that the subthreshold dose hypnosis experiment, the barbiturate sodium sleep latency experiment and the experiment for prolonging the sleep time of the barbiturate sodium are continuously carried out is met.

P. pentobarbital sodium subthreshold dose hypnosis experiment

The positive control drug and the sleep-aiding ferment in the high and low dose groups both show the effect of improving the sleep rate of mice. The positive medicine has the best effect; in the sleep-aiding enzyme group, the gastric lavage sample is suspected to have large acidity, and a mouse is stimulated by certain acidity after gastric lavage, so that the sleep-aiding effect of the sample is also hindered to a certain extent; the difference between the positive control group and the sleep-aiding enzyme group is not significant.

Experiment of sleep latency period of barbital sodium

By carrying out a barbital sodium sleep latency experiment on 4 groups of mice, a positive control group is found to be capable of obviously shortening the sleep latency of the mice. The sleep latency can be obviously shortened by the high and low dose group of sleep-aiding enzymes, but the range is inferior to that of a positive control group; the effect of the high dose of sleep-aiding enzymes was slightly lower than that of the low dose group. Similar to the above experiments, it is speculated that the higher acidity of the sleep-aiding enzymes stimulates the mice after gastric lavage, and the sleep-aiding effect is counteracted to a certain extent.

Experiment for prolonging sleep time of sodium pentobarbital

After high-dose sodium pentobarbital is injected into the abdominal cavity, the sleeping time of 4 groups of mice is counted, and the positive control drug group and high-dose sleep-aiding ferment group bacteria have the effect of improving the sleeping time of the mice by more than 100 percent and have extremely high statistical significance (p is less than 0.01); the improvement rate of the low dose of sleep-aiding ferment to the time of falling asleep is 66.85%, and the improvement rate also has statistical significance (p is less than 0.05). Clearly, the low dose of sleep-aid enzymes was not as effective in this test as the high dose group. It is speculated that after the mice are stimulated after stomach filling, the sleep-aiding enzymes play a good role in the falling asleep stage, and the mice show a positive correlation trend with the dosage.

2. Antioxidant capacity

2.1 Experimental procedure:

(1) accurately weigh 4mg of DPPH, dissolve with absolute ethanol to a constant volume of 100m l in a volumetric flask, and shake the solution evenly.

(2) Preparing different concentration gradients of sleep-aiding ferment sample solutions (ferment/diluted final volume: v/v): 1/200, 30/200, 60/200, 90/200, 120/200, 150/200, 170/200, 200/200.

(3) Adding 4.0ml of DPPH solution and the sleep-aiding ferment sample solution into a 10ml colorimetric tube in sequence, adding absolute ethyl alcohol to scale, immediately using a 1cm cuvette to measure absorbance (A) at the wavelength of 517nm after mixing, marking the absorbance as Ai, then measuring the absorbance after storing for 30min in a dark place at room temperature, marking as Aj, and taking an ethanol solution only added with DPPH as a control experiment, wherein the absorbance is Ac. The radical clearance (K) was calculated according to the following formula:

K(％)＝[1-(Ai-Aj)/Ac]×100％

2.2 Experimental results:

the scavenging capacity of the sleep-aiding ferment provided in the embodiment 1 on free radicals is investigated by DPPH and a spectrophotometer method, and the scavenging capacity of the free radicals can still reach 50% after the sleep-aiding ferment is diluted by about 4 times of the volume; corresponding to the vitamin C content of 95mg/100 g. The recommended basic dosage of vitamin C in China is 100 mg/day. Equivalent to drinking about 25mL of sleep-aiding ferment in one day, the recommended basic dose of vitamin C can be achieved.

3. Promoting intestinal peristalsis (relieving constipation)

A mouse is used as a test material, a medicine for moistening the intestines, namely trimebutine maleate tablets, is used as a reference, a mouse constipation model is prepared by using atropine sulfate, and the intestine-moistening and bowel-relaxing capability of sleep-aiding ferment is researched by using the ink propulsion rate in the intestinal tract of the mouse as an index.

(1) Test materials

Sleep-aiding ferment obtained by the preparation method of the example 1;

mouse C57BL/6J Male 7-8 week mouse (18-22g)

Distilled water

Trimebutine maleate tablet

(2) Experimental grouping

Mice were randomly divided into 5 groups of 10 mice each, and the administration method for each group was as follows:

CK (negative control group): distilled water (intragastric for 14 days 0.3mL/day)

MOD (model control group): distilled water (intragastric for 14 days 0.3mL/day)

PC (positive control group): 0.3mL of trimebutine maleate tablet solution (one tablet is dissolved in 250mL of water and completely dissolved. the tablet is used at present and is gavage for 14 days, 0.3mL/day)

B/LD (Low dose group): (sleep-aiding ferment, gavage for 14 days 0.3mL/day)

B/HD (high dose group): (sleep-aiding ferment, gavage for 14 days 0.6mL/day)

(3) Experimental method

S1, continuously gavage for 14d according to the grouping and the dosage, and fasting and not inhibiting water for 12h for each group of mice.

And S2, the PC group, the MOD group, the ferment LD group and the HD group are respectively given to atropine sulfate 100uL for intraperitoneal injection to inhibit small intestine peristalsis.

After S3, 30min, all groups were given 200uL of gavage ink. Preparing ink: accurately weighing 10g of Arabic gum, adding 80mL of water, boiling until the solution is transparent, weighing 5g of active carbon (powder) and adding the active carbon (powder) into the solution, boiling for three times, adding water to a constant volume of 100mL after the solution is cooled, storing in a refrigerator at 4 ℃, and shaking uniformly before use.

S4, after 25min, removing cervical vertebrae to kill animals, opening the abdominal cavity to separate mesentery, cutting the intestinal canal from the upper end to pylorus and from the lower end to ileocecal part, placing on a tray, slightly drawing the small intestine into a straight line, measuring the length of the intestinal canal to be the total length of the small intestine, measuring the ink propulsion length from the pylorus to the ink front edge, and calculating the ink propulsion rate.

Ink propelling rate/%, ink propelling length (cm)/total small intestine length (cm) × 100%

(4) And experimental results

Under experimental conditions, the high-dose sleep-aiding ferment (equivalent to 4 times of the daily recommended edible amount of an adult) can remarkably recover the peristalsis capability of the small intestine of a mouse, has stronger functions of relaxing bowels and moistening intestines, and has better effect than that of trimebutine maleate tablets.

4. Intestinal flora modulation

C57BL/6J male enteritis mice (18-22g) with 7-8 weeks are used as experimental materials, the species and the amount of microorganisms in intestinal tracts before and after the mice take enzymes are detected by a molecular biology method, and the strength of the sleep-aiding enzymes on the proliferation capacity of probiotics in the intestinal tracts of the mice is examined.

The experimental process comprises the following steps:

CK (negative control group): distilled water (intragastric for 4 weeks 0.3mL/day)

IBD (inflammatory bowel disease group): an IBD model with inflammatory enteritis is obtained by adopting drinking water containing 2.5% DSS to replace normal drinking water

PRO/IBD (enteritis, probiotic control): dissolving the Leli probiotics in 100mL of room-temperature distilled water for each bag, (gavage for 4 weeks, 0.3mL/day)

B/LD (Low dose group): (sleep-aiding ferment, gavage 4 weeks 0.3mL/day)

B/HD (high dose group): (sleep-aiding ferment, gavage 4 weeks 0.6mL/day)

B/LD/IBD (enteritis, sleep-aid enzyme low dose group): adopting drinking water containing 2.5% DSS to replace normal drinking water to obtain IBD model of inflammatory enteritis, and then irrigating stomach with sleep-assisting enzyme for 4 weeks, wherein the dosage of the model is 0.3mL/day)

B/HD/IBD (enteritis, sleep-aid enzyme high dose group): adopting drinking water containing 2.5% DSS to replace normal drinking water to obtain IBD model of inflammatory enteritis, and then irrigating stomach with sleep-assisting enzyme for 4 weeks, wherein the dosage of the model is 0.6mL/day)

Experiments were performed according to the above-mentioned group doses, animals were sacrificed by cervical dislocation after 4 weeks of continuous gavage, the cecum contents were stored at-80 ℃ and then subjected to colony sequencing analysis.

The steps of the flora sequencing analysis are as follows:

s1 extraction of total DNA of microbiome

Extracting the DNA of the mouse cecal content, detecting the extraction quality of the DNA by 0.8 percent agarose gel electrophoresis, and simultaneously quantifying the DNA by adopting an ultraviolet spectrophotometer.

S2 PCR amplification of target fragment

Usually, a target sequence such as microbial ribosomal RNA and the like capable of reflecting flora composition and diversity is taken as a target point, corresponding primers are designed according to a conserved region in the sequence, a sample-specific Barcode sequence is added, and then a rRNA gene variable region (single or continuous multiple) or a specific gene fragment is subjected to PCR amplification.

The PCR amplification adopts Q5 high-fidelity DNA polymerase of NEB company, and strictly controls the amplification cycle number, so that the amplification conditions of the same batch of samples are consistent while the cycle number is as low as possible.

S3, recovery and purification of amplification product

The PCR amplification product is detected by 2% agarose gel electrophoresis, and the target fragment is cut and recovered by adopting a gel recovery kit of AXYGEN company.

S4 fluorescence quantification of amplified product

Referring to the preliminary quantification result of electrophoresis, the PCR amplification recovery product is subjected to fluorescence quantification, the fluorescence reagent is Quant-iTPicoGreen dsDNA Assay Kit, and the quantification instrument is a Microplate reader (BioTek, FLx 800). And mixing the samples according to the corresponding proportion according to the fluorescent quantitative result and the sequencing quantity requirement of each sample.

S5 sequencing library preparation

Using Illumina MiSeq sequencing as an example, a sequencing library was prepared using TruSeq Nano DNA LT library Prep Kit from Illumina.

S5.1, firstly, carrying out sequence End Repair on the amplification product, cutting the protruding base at the 5 'End of the DNA sequence through End Repair Mix2 in the kit, adding a phosphate group and filling up the missing base at the 3' End;

s5.2, adding A base at the 3 'end of the DNA sequence to prevent the self-connection of the DNA fragment and ensure that the target sequence can be connected with a sequencing joint (the 3' end of the sequencing joint has a protruded T base);

s5.3, adding a sequencing joint containing a library-specific tag (namely an Index sequence) to the 5' end of the sequence to enable the DNA molecule to be fixed on the Flow Cell;

s5.4, adopting BECKMAN AMPure XP Beads, removing a joint self-connecting fragment through magnetic bead screening, and purifying a library system added with a joint;

s5.5, carrying out PCR amplification on the DNA fragment connected with the adaptor so as to enrich a sequencing library template, and purifying a library enrichment product again by adopting BECKMAN AMPure XP Beads;

s5.6, performing 2% agarose gel electrophoresis, and performing final fragment selection and purification on the library.

S6, processing the high-throughput sequencing

Paired-end sequencing of 2X 300bp was performed using a MiSeq sequencer, the corresponding Reagent being MiSeq Reagent Kit V3(600 cycles).

Due to the characteristic of short MiSeq sequencing read length, and in order to ensure the sequencing quality, the optimal sequencing length of the target fragment is recommended to be 200-450 bp.

Ribosomal RNA contains multiple conserved regions and highly variable regions, and generally we use the conserved regions to design primers to amplify single or multiple variable regions of the rRNA gene, followed by sequencing to analyze microbial diversity. Due to the restriction of MiSeq sequencing read length, and also to ensure sequencing quality, the insert range for optimal sequencing was 200-450 bp.

S7, result analysis

S7.1, firstly, carrying out primary screening on the original off-line data of high-throughput sequencing according to sequence quality;

s7.2, merging and dividing the obtained sequences, wherein the representative sequence of each OTU is used for classified position identification and phylogenetic analysis;

s7.3, evaluating the diversity level of each sample according to the abundance distribution of the OTU in different samples, and reflecting whether the sequencing depth reaches the standard or not through a sparse curve;

s7.4, analyzing specific compositions of each sample (group) at different classification levels (and testing whether the groups have statistical difference);

s7.5, further measuring the flora structure difference and species related to the difference among different samples (groups) through a plurality of multivariate statistical analysis tools;

s7.6, according to the sequencing result of the 16S rRNA gene, the flora metabolic function of each sample can be predicted.

The experimental results are as follows:

the sleep-aiding ferment can reduce the relative content of firmicutes, improve the content of bacteroides, improve the content of actinomycetes, increase the content of icotina, obviously improve the content of staphylococcus, obviously increase the content of bifidobacterium and obviously reduce the content of coprococcus. The results prove that the sleep-aiding ferment can improve the content of beneficial bacteria in the intestinal tract, reduce the content of harmful bacteria in the intestinal tract and has a good flora regulation effect.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. The sleep-aiding ferment is characterized by comprising the following raw materials in parts by weight:

2. The sleep-aiding ferment according to claim 1, comprising the following raw materials in parts by weight:

3. The sleep-aiding ferment according to claim 1 or 2, wherein the ferment further comprises a flavoring sugar, and the additive amount of the flavoring sugar is 10% of the total mass of the ferment.

4. A method of preparing a sleep-aid ferment according to any one of claims 1 to 3, comprising the steps of:

5. The method according to claim 4, wherein the fermentation comprises the step of sequentially performing lactic acid bacteria fermentation, yeast fermentation and acetic acid bacteria fermentation on a mixture of the aqueous extract of medicinal materials and the noni pulp.

6. The production method according to claim 5,

the lactobacillus fermentation comprises the step of adding lactobacillus into the mixed material, and fermenting at 37-43 ℃ until the acidity of the mixed material is more than or equal to 2-5g/L to obtain a first fermentation solution;

7. The preparation method of claim 6, wherein the addition amount of the lactic acid bacteria is 0.1-0.5% of the total mass of the mixture, the addition amount of the yeast is 0.5-1% of the total mass of the first fermentation solution, and the addition amount of the acetic acid bacteria is 0.5-1% of the total mass of the second fermentation solution.

8. The preparation method according to any one of claims 4 to 7, further comprising a step of adding 20 parts of white granulated sugar and 10 parts of brown granulated sugar to the aqueous extract of the medicinal material before fermentation.

9. The method of any one of claims 4-8, wherein the formulating step further comprises the step of adding a flavoring sugar to the supernatant.

10. The method according to any one of claims 4 to 9, further comprising a step of hermetically aging the fermentation broth after completion of the fermentation at room temperature for 1 to 2 months, before the blending step.