TWI729928B - Concentrate of phellinus linteus extract, method of manufacturing the same and use of the same for preparing composition of improving sleep - Google Patents

Abstract

The present invention relates to a concentrate of Phellinus linteus extract, a method of manufacturing the same and a use of the same in preparation of a composition for improving sleep. The self-screened Phellinus linteus is subjected to a multistage cultivating step, a drying step, an extracting step and a concentrating step to obtain the concentrate of Phellinus linteus extract, which can be applied as an effective ingredient of a composition for improving sleep.

Description

Phellinus igniarius extract concentrate, its manufacturing method and its composition for improving sleep Use of things

The present invention relates to a fungal mycelium and/or its derivatives, its manufacturing method and its use, in particular to a Phellinus linteus extract concentrate, its manufacturing method and its use for preparing sleep-improving The purpose of the composition.

Good sleep can make the body metabolize normally and relax the mind. If you cannot have a good sleep, it will affect all aspects of life, including physical, psychological, cognitive, social functions and quality of life. In terms of physiology, long-term insomnia may lead to obesity, cardiovascular system, endocrine system, malignant tumors, bronchial asthma, ulcer disease, diabetes, and sexual dysfunction. In terms of psychological, cognitive, and social functions, it affects mood, work performance and interpersonal relationships during the day. According to statistics from the World Health Organization, the global sleep disorder rate is 27%, and a survey by the Taiwan Society of Sleep Medicine found that at least 2 million people in Taiwan suffer from chronic insomnia, that is, 1 out of every 5 people have chronic insomnia. Symptoms of insomnia.

The drugs used to treat insomnia include Benzodiazepines (BZD), Non-Benzodiazepines (Non-BZD), Tricyclic Antidepressants (TCA), and melatonin. Melatonin Agonist drugs and antihistamine drugs. In addition to the side effects of the aforementioned drugs such as dizziness, headache, gastrointestinal discomfort or drowsiness, long-term use of benzodiazepines may cause dependence and withdrawal symptoms. Non-benzodiazepine drugs may cause side effects such as transient memory loss and sleepwalking. Tricyclic antidepressants also have withdrawal symptoms.

The Chinese name of Phellinus linteus is Phellinus linteus, which belongs to the Phellinus genus of the Hymenochaetaceae family. Phellinus igniarius grows on the trunks of plants of the genus Morus, also known as mulberry or Sangchen. Li Shizhen's "Compendium of Materia Medica" in the Ming Dynasty recorded that it was cold in nature and slightly bitter in taste, which could benefit the five internal organs, promote intestinal qi, detoxify qi, or stop bleeding. It was often used in Chinese medicine for diuresis, stomach strengthening or diarrhea. However, there are few studies on the effect of Phellinus sibiricum in improving sleep.

Therefore, one aspect of the present invention is to provide a Phellinus linteus extract concentrate, wherein the Phellinus linteus extract concentrate is derived from the Phellinus linteus deposited by the Bioresource Center (BCRC) of the Food Industry Development Research Institute, and The Phellinus linteus extract concentrate can be used as an effective ingredient for sleep-improving compositions, for example.

Another aspect of the present invention is to provide a method for producing a Phellinus igniarius extract concentrate, which includes a multi-stage cultivation step, a drying step, an extraction step, and a concentration step.

Another aspect of the present invention is to provide a use of the above-mentioned Phellinus igniarius extract concentrate for preparing a sleep-improving composition.

According to the above aspect of the present invention, a Phellinus linteus extract concentrate is proposed, wherein the Phellinus linteus extract concentrate was deposited at the Food Industry Development Institute, No. 331 Food Road, Hsinchu, Taiwan on July 18, 2019 Biological Resource Center (BCRC), deposit the strain numbered as BCRC 930210, and this Phellinus igniarius extract concentrate can be used as the active ingredient of sleep-improving composition.

According to another aspect of the present invention, a method for producing a Phellinus igniarius extract concentrate is provided, which includes subjecting the first mycelium of Phellinus igniarius to a multi-stage cultivation step to obtain a Phellinus igniarius fermented liquid. The multi-stage cultivation steps are as follows. First, a solid-state culture step is performed, which uses a solid-state medium to cultivate the first mycelium at 15° C. to 30° C. for 1 to 2 weeks to obtain the second mycelium. Phellinus sanghuang was deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan on July 18, 2019, with the deposit number of BCRC 930210.

Then, a liquid culture step is performed, which uses the first culture solution to cultivate the second mycelium at 15°C to 30°C for 3 to 14 days to obtain the third mycelium, wherein the pH value of the first culture solution is It is pH 2 to pH 6. Then, a fermented culture step is performed, which uses the second culture solution to cultivate the third mycelium at 15°C to 30°C for 3 to 21 days to obtain the Phellinus linteus fermented solution, The acid-base value of the second culture solution is from pH 2 to pH 6.

According to the above-mentioned embodiment of the present invention, the above fermentation fermentation step is carried out in a fermentation tank, and when the fermentation fermentation step is performed, it may optionally include introducing gas into the fermentation tank, and the gas system is selected from air, oxygen , Carbon dioxide, helium, or any combination of the above.

According to the above-mentioned embodiment of the present invention, the above-mentioned ferment culture step is at a ^{tank pressure of 0.5kg/cm 2} to 1.0kg/cm ² and 0.01 [volume of gas introduced/volume of fermented liquid/min, VVM] to 1.5VVM Of the ventilation rate.

According to the above-mentioned embodiment of the present invention, the above-mentioned fermented fermentation step further includes a drying step of the Phellinus igniarius fermented liquid to obtain the dried Phellinus igniarius fermented product.

According to the above-mentioned embodiment of the present invention, after the drying step, it further comprises a step of extracting the dried ferment of Phellinus linteus using a polar solvent to obtain the Phellinus igniarius extract.

According to the above-mentioned embodiment of the present invention, the above-mentioned polar solvent includes water and/or lower alcohol.

According to the above-mentioned embodiment of the present invention, after the extraction step, a step of concentrating the Phellinus igniarius extract is further included to obtain the Phellinus igniarius extract concentrate.

According to the above-mentioned aspect of the present invention, a use of the Phellinus igniarius extract concentrate as described above for preparing a sleep-improving composition is proposed, wherein the composition is an oral composition.

The Phellinus igniarius extract concentrate of the present invention is used to carry out multi-stage cultivation steps, drying steps, extraction steps and concentration steps on the self-selected Phellinus igniarius The obtained Phellinus linteus extract concentrate can be used as an effective ingredient for sleep-improving composition.

100: method

110: Provide the first mycelium of Phellinus igniarius

120: Carry out multi-stage cultivation steps

121: Perform a solid-state culture step on the first mycelium to obtain the second mycelium

123: Perform a liquid culture step on the second mycelium to obtain the third mycelium

125: Perform fermentation step for the third mycelium

130: Obtain the Phellinus igniarius fermented liquid, wherein the Phellinus igniarius fermented liquid contains the mycelium of Phellinus igniarius and/or its derivatives

301,303,305,307: Polyline

In order to make the above and other objectives, features, advantages and embodiments of the present invention more comprehensible, the detailed description of the accompanying drawings is as follows: [FIG. 1] A diagram showing the hyphae of Phellinus igniarius according to an embodiment of the present invention Flow chart of the manufacturing method of body and/or its derivatives.

[Figure 2] A diagram showing the evolutionary tree of 18s rRNA sequence alignment of Phellinus igniarius (BCRC 930210) and other conventional Phellinus igniarius.

[Fig. 3A] is a line chart showing non-rapid eye movement (NREM) sleep according to an embodiment of the present invention.

[Fig. 3B] is a line graph showing rapid eye movement (REM) sleep according to an embodiment of the present invention.

Based on the above, the present invention provides a Phellinus linteus extract concentrate, its manufacturing method and its use for preparing a sleep-improving composition.

The Phellinus linteus of the present invention belongs to the Phellinus genus of the Hymenochaetaceae family (Hymenochaetaceae), and is named Schizothorus linteus in Chinese. It was deposited in Hsinchu Foods, Taiwan on July 18, 2019 No. 331 Road, Food Industry Development Research Institute Biological Resource Center (BCRC), deposited the strain number BCRC 930210.

The present invention also provides a method for producing the mycelium of Phellinus igniarius and/or its derivatives, which includes subjecting the mycelium of Phellinus igniarius to a multi-stage cultivation step to obtain the mycelium of Phellinus igniarius and/or its derivatives.

Please refer to FIG. 1, which shows a flow chart of a method 100 for manufacturing the mycelium of Phellinus igniarius and/or its derivatives according to an embodiment of the present invention. First, as shown in step 110 of the method 100, the first mycelium of Phellinus igniarius is provided. The first mycelium system of Phellinus igniarius was deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan on July 18, 2019, with the strain number BCRC 930210 deposited.

Next, as shown in step 120, the first mycelium of Phellinus igniarius is subjected to a multi-stage culture step. Due to conditions such as nutritional components and environmental factors, it has a direct impact on the growth and differentiation of Phellinus igniarius. Through multi-stage culturing steps, the growth conditions of Phellinus igniarius at each stage can be adjusted and products with different components can be obtained.

In this embodiment, as shown in step 121, the multi-stage culturing step includes performing a solid-state culturing step on the first mycelium to obtain the second mycelium. The solid-state culture step is performed using a solid-state culture medium. The solid medium contains carbon sources, nitrogen sources and necessary nutrients that can provide the growth of the mycelium of Phellinus igniarius. The solid medium may be, for example, a potato dextrin medium (Potato Dextrose Agar, PDA).

In this implementation, the conditions for the solid-state culture step are to culture the first mycelium at 15°C to 30°C for 1 week to 2 weeks. If the temperature exceeds the aforementioned range, the growth of mycelium will be inhibited. If the culture time is less than 1 week, the mycelium has not fully grown. In addition, when the culture time reaches 2 weeks, the mycelium has been completely For growth, it does not need to be cultured for more than 2 weeks.

Next, as shown in step 123, a liquid culture step is performed on the second mycelium to obtain a third mycelium. The liquid culture step is performed using the first culture solution. The first culture medium contains 1% to 3% by weight of comprehensive carbon and nitrogen sources (e.g., cereals and/or beans), 1% to 4% by weight of sugars (e.g., monosaccharides and/or disaccharides), and 0.1% by weight % To 1% by weight of yeast extract, 0.1% to 1% by weight of egg whites, and 0.01% to 0.05% by weight of inorganic salts (such as phosphate and/or sulfate). It should be understood that the composition of the aforementioned first culture solution can be adjusted appropriately according to the needs of use.

The acid-base value of the above-mentioned first culture solution is from pH 2 to pH 6. If the pH value exceeds the aforementioned range, the mycelium will grow poorly.

In this embodiment, the culture condition of the liquid culture step is to culture the second mycelium at 15°C to 30°C for 3 to 14 days. If the temperature exceeds the aforementioned range, the growth of mycelium will be inhibited. If the culture time exceeds 14 days, it will not help or even inhibit the growth of mycelium.

In other embodiments, the rotation speed of the liquid culture step is 110 rpm to 130 rpm.

Then, as shown in step 125, a fermentation step is performed on the third mycelium. The fermentation step is carried out by using the second culture solution. The composition of the second culture medium can be the same as that of the aforementioned first culture medium, or the composition may be adjusted appropriately according to the needs of use. The acid-base value of the aforementioned second culture solution is from pH 2 to pH 6, and if the acid-base value exceeds the aforementioned range, the mycelium will grow poorly.

The condition of the fermentation step is to cultivate the third at 15°C to 30°C Mycelium lasts for 3 to 21 days. If the temperature exceeds the aforementioned range, the growth of mycelium will be inhibited. If the fermentation time is less than 3 days, the effective amount of the mycelium of Phellinus igniarius and/or its derivatives is insufficient. In addition, when the fermentation time exceeds 21 days, it will not help the growth of mycelium or even inhibit its growth.

The fermentation step is carried out in the fermentation tank. In one embodiment, during the fermentation step, gas is introduced into the fermentation tank, and the gas system is selected from a group consisting of any combination of air, oxygen, carbon dioxide, and helium. In one embodiment, the tank pressure is 0.5 kg/cm ² to 1.0 kg/cm ² . In one embodiment, the aeration rate is 0.01 (volume of gas introduced/volume of fermented liquid/minute, VVM) to 1.5VVM. In other embodiments, the rotation speed of the fermentation step is 50 rpm to 150 rpm.

Next, as shown in step 130 of method 100, through the above-mentioned multi-stage culturing steps, a Phellinus igniarius fermented liquid containing specific components can be obtained, wherein the Phellinus igniarius fermented liquid contains the mycelium of Phellinus linteus and/or its derivatives Things.

In one embodiment, after the fermentation step, the step of drying the Phellinus igniarius fermented liquid may optionally be included to obtain the dried Phellinus igniarius fermented product. Phellinus igniarius fermented liquid can be dried by conventional drying treatment methods, such as freeze drying, vacuum drying or spray drying.

In one embodiment, after the drying step, it may optionally include a step of extracting the dried fermented product of Phellinus igniarius using a polar solvent to obtain the Phellinus igniarius extract. In other embodiments, the polar solvent includes water and/or lower alcohol (for example, methanol, ethanol, propanol, isopropanol, etc.).

In one embodiment, after the extraction step, it may optionally include a step of concentrating the Phellinus igniarius extract to obtain the Phellinus igniarius extract concentrate. Phellinus igniarius extract The liquid can be concentrated by a conventional concentration method, such as reduced pressure concentration, evaporation concentration method, or membrane concentration method.

The present invention also provides a mycelium of Phellinus igniarius and/or its derivatives, which are prepared by the above-mentioned manufacturing method. The mycelia of Phellinus igniarius and/or its derivatives include, but are not limited to, Phellinus igniarius fermented liquid, dried Phellinus igniarius fermented product, Phellinus igniarius extract and/or Phellinus igniarius extract concentrate.

The present invention further proposes a use of Phellinus igniarius extract concentrate for preparing a composition for improving sleep. In one embodiment, the above-mentioned Phellinus igniarius extract concentrate is prepared by the above-mentioned manufacturing method. The composition is an oral composition, and the type thereof is not particularly limited. Any mycelium and/or its derivatives containing Phellinus igniarius are all It.

In one embodiment, the above-mentioned composition may be, for example, a food composition or a medical composition. In one embodiment, the above composition may optionally include food or pharmaceutically acceptable carriers, excipients, diluents, adjuvants, and/or additives, such as solvents, emulsifiers, suspending agents, and disintegrating agents. , Binders, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants and/or absorption retardants, etc.

The dosage form of the composition of the present invention is not particularly limited. In one embodiment, the dosage form of the composition can be, for example, but not limited to, aqueous solution, suspension, dispersion, emulsion (single-phase or multi-phase dispersion system, single- or multi-lamellar liposome), hydrogel, gel, solid lipid Nanoparticles, lozenges, granules, powders and/or capsules, etc.

The aforementioned food composition can be, for example, but not limited to, cereal products, fruit products, vegetable products, meat products, fish products, egg products, Dairy products, beverages, health foods, health foods, functional foods, nutritional supplements or special nutritional foods.

Several embodiments are used below to illustrate the application of the present invention, but they are not used to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various modifications and changes without departing from the spirit and scope of the present invention. Retouch.

Example 1: Preparation of the mycelium of Phellinus igniarius and/or its derivatives

(1) Morphological characteristics and genetic analysis of Sanghuang

The mycelium of Phellinus igniarius and/or its derivatives were deposited at the Biological Resource Center (BCRC), Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan on July 18, 2019, with the deposit number BCRC 930210.

The morphological characteristics of the above-mentioned Phellinus linteus are as follows. The hyphae of Phellinus igniarius are unbranched and have no diaphragm, with a diameter of 3 to 5 microns. The spores of Phellinus igniarius are nearly spherical and have a smooth surface. The long diameter of the spores is about 5 to 6 microns, and the short diameter is about 4 to 5 microns. The fruiting body of Phellinus igniarius has a hard woody texture, a sterile stalk, and a cap about 3 cm to 20 cm wide. The back is brown or dark brown, and the ventral surface is yellow.

Then, through genetic analysis, the differences between the above-mentioned Phellinus igniarius (BCRC 930210) and the same species of conventional bacteria were evaluated. First, the gDNA of the mycelium of Phellinus igniarius is extracted, and the gene sequence of 18s rRNA is obtained by polymerase chain reaction (PCR) and sequenced. The above-mentioned PCR method is well-known to those with ordinary knowledge in the technical field to which the present invention belongs, and can be adjusted arbitrarily according to actual needs, and will not be repeated here.

Next, compare the 18s rRNA sequence of Phellinus igniarius (BCRC 930210) [as shown in the sequence identification number (SEQ ID NO): 1] with the conventional Phellinus igniarius strain [for example, GeneBank No. KT862140 (South Korea), AY558629 ( (Costa Rica) and JQ860322 (U.S.)] using commercially available software, such as Molecular Evolutionary Genetics Analysis (MEGA) software, to analyze the 18s rRNA sequence and use Neighbor- Joining mode draws the evolution tree, as shown in Figure 2.

Figure 2 shows the evolution tree of the 18s rRNA sequence alignment of Phellinus sibiricum (BCRC 930210) and other conventional Phellinus sibiricum, in which the horizontal line represents the evolutionary change measured in the unit of gene diversity, and the scale of the horizontal line is marked on Bottom left. As shown in Figure 2, although the Phellinus sibiricum (BCRC 930210) and the conventional Phellinus sibiricum strain belong to the same species, they are separated from the conventional Phellinus sibiricum in evolutionary relationship and form a separate line. This result shows that from the perspective of genetic differences, Phellinus igniarius (BCRC 930210) and the conventional Phellinus igniarius do have significant differences, and they are new strains of Phellinus igniarius.

(2) Preparation of Phellinus igniarius mycelium and/or its derivatives

First, the above Phellinus igniarius (BCRC 930210) was inoculated on a potato dextrin medium (Potato Dextrose Agar, PDA) and cultured at 25°C for 7 days. Then, the scraped part of the mycelium of Phellinus igniarius was inoculated into the first culture medium (containing 1% by weight of comprehensive carbon and nitrogen sources, 1.5% by weight of sugars, 0.3% by weight of yeast extract, and 0.3% by weight of egg whites). And 0.05% by weight of inorganic salts), the culture step is performed for 7 days at 25° C., pH 5, and rotation speed of 120 rpm. The above-mentioned comprehensive carbon and nitrogen sources are valleys Types (wheat flour and/or bran flour) and/or beans (soya bean flour, mung bean flour, soy flour and/or cinnamon powder). The above-mentioned sugars are monosaccharides (glucose and/or fructose) and/or disaccharides (maltose and/or sucrose). The above-mentioned inorganic salts are phosphate (dipotassium hydrogen phosphate, potassium dihydrogen phosphate) and/or sulfate (magnesium sulfate and/or iron sulfate).

Next, take part of the mycelia of Phellinus igniarius in the first culture solution and inoculate it in a ferment tank containing the second culture solution (the same composition as the first culture solution) at 25°C, pH 5, and 0.5 kg/cm ² Fermentation was carried out for 14 days at a pressure of 1.0VVM, an air aeration rate of 1.0VVM and a stirring speed of 80rpm to obtain the Phellinus igniarius fermented liquid.

Take the Phellinus igniarius fermented liquid for freeze-drying to obtain the dried Phellinus igniarius fermented product. Then, 20 times the weight of ethanol was added to the dried Phellinus igniarius fermented material, and the extraction was carried out with ultrasonic shaking for 1 hour. After centrifugation, the supernatant was collected and concentrated under reduced pressure to obtain the Phellinus igniarius extract concentrate.

Example 2: Establish an animal model for analyzing sleep

Male rats of the Sprague-Dawley (SD) strain purchased from BioLASCO Taiwan Co., Ltd. (Taiwan) were selected and weighed 250 g to 300 g. The rats were kept at a temperature of 22±3°C, a humidity of 40% to 70%, 12 hours of light and 12 hours of dark cycle light, and sufficient feed and sterile reverse osmosis water were provided for the rats to eat freely. Animal experiments are conducted in accordance with the relevant regulations of the Laboratory Animal Care and Use Committee of the Research and Development Office of National Taiwan University.

Before the experiment, the rats were incised on the head with a sterile surgical blade, the soft tissues on the skull were scraped off and the bleeding was stopped with an electrocautery hemostatic device, and then electroencephalography (EEG) electrodes for recording were implanted in the skull And fixing screws. On the first day after surgery, plug the EEG electrode set into the brainwave recording cable, and connect the signal EEG electrode and the signal amplifier to the computer. The brainwaves and activities of rats were recorded using ICELUS software (Mark R.Opp, University of Michigan), and the baseline of the brainwaves was recorded on the 8th day after the operation, as the basic EEG. In addition, an infrared motion sensor is used to detect the activity of the rat.

The rats were randomly divided into 2 groups, 5 in each group, namely Phellinus igniarius group and control group, and recorded for 24-hour EEG and motion detection. Twenty minutes before the start of dark light, the rats were tube-fed with 150 mg/kg Phellinus igniarius extract concentrate (Phellinus igniarius group) or 0.1 mL of 5.5% ethanol (control group).

Example 3: The effect of Phellinus igniarius mycelium and/or its derivatives in improving sleep

The brain wave waveform uses every 12 seconds as a band (epoch) unit, using the fast Fourier transform (FFT) diagram provided by the ICELUS software to manually determine whether the mouse is in a non-rapid eye movement (non-rapid eye movement) according to the waveform. Eye movement (NREM) sleep or rapid eye movement (REM) sleep. Generally speaking, the frequency of NREM sleep waveform is low, and the amplitude is large and consistent. The REM sleep waveform has a higher frequency, a smaller amplitude and no activity signal.

Please refer to FIG. 3A, which shows a line graph of NREM sleep according to an embodiment of the present invention. The X axis represents time post-injection, in hours. The Y-axis represents the amount of NREM sleep in percentage. The statistical method in Figure 3A uses a paired sample t-test to analyze the percentages, figure number* It means that there is a statistically significant difference (p<0.05), and the same is true in Figure 3B below.

The result of Figure 3A shows that compared with the control group (broken line 303), the NREM sleep amount of the Phellinus igniarius group (broken line 301) increased from 30.3±4.0% to 51.3±3.3% from the 19th hour to the 24th hour. Therefore, Phellinus igniarius extract concentrate can significantly increase the amount of NREM sleep in rats.

Please also refer to FIG. 3B, which shows a line graph of REM sleep according to an embodiment of the present invention. The X axis represents time post-injection, in hours. The Y-axis represents the amount of REM sleep in percentage.

The results in Figure 3B show that compared with the control group (broken line 307), the REM sleep of the Phellinus igniarius group (broken line 305) increased from 12.3±1.8% to 23.3±2.2% from the 17th hour to the 21st hour. Therefore, Phellinus igniarius extract concentrate can significantly increase the amount of REM sleep in rats.

It can be seen from the above examples that the Phellinus linteus extract concentrate of the present invention, its manufacturing method and its use for preparing sleep-improving compositions have the advantage of cultivating self-selected Phellinus linteus using multi-stage cultivation steps. The Phellinus igniarius extract concentrate is obtained. A composition containing Phellinus igniarius extract concentrate as an active ingredient can be administered to a subject to improve their sleep.

It should be understood that although the Phellinus igniarius extract concentrate is used in the present invention to prove that the mycelium of Phellinus igniarius and/or its derivatives have the effect of improving sleep, those with ordinary knowledge in the field of the present invention should know that the use of Phellinus igniarius Mycelium, Phellinus igniarius mycelium-containing culture medium, Phellinus igniarius mycelium culture solution, Phellinus igniarius fermented liquid, dry fermented Phellinus igniarius and/or Phellinus igniarius extract can Produce a similar effect.

It should be added that although the present invention takes a specific manufacturing process, a specific analytical method and/or a specific instrument as an example to illustrate the mycelial extract concentrate of Phellinus linteus, its manufacturing method and its use For the preparation of sleep-improving compositions, anyone with ordinary knowledge in the technical field to which the present invention belongs can know that the present invention is not limited to this. Without departing from the spirit and scope of the present invention, the Phellinus linteus of the present invention The extract concentrate, its manufacturing method and its use for preparing sleep-improving compositions can also be carried out by other processes, other analytical methods or other instruments.

Although the present invention has been disclosed in several embodiments as above, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs can make various modifications without departing from the spirit and scope of the present invention. Modifications and modifications, therefore, the scope of protection of the present invention shall be subject to those defined by the attached patent application scope.

【Biological Material Deposit】

Phellinus linteus (Phellinus linteus) mycelium and/or its derivatives were deposited at the Biological Resource Center (BCRC), Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan on July 18, 2019. The deposit number is BCRC Strain of 930210.

100: method

110: Provide the first mycelium of Phellinus igniarius

120: Carry out multi-stage cultivation steps

121: Perform a solid-state culture step on the first mycelium to obtain the second mycelium

123: Perform a liquid culture step on the second mycelium to obtain the third mycelium

125: Perform fermentation step for the third mycelium

130: Obtain Phellinus igniarius fermented liquid, which contains the mycelium of Phellinus igniarius And/or its derivatives

Claims (6)

A Phellinus linteus extract concentrate, wherein the Phellinus linteus extract concentrate is derived from the Biological Resource Center (BCRC) of the Food Industry Development Research Institute at No. 331 Food Road, Hsinchu, Taiwan on July 18, 2019. The registration number is BCRC 930210 Phellinus igniarius, and the Phellinus igniarius extract concentrate is an effective ingredient of a composition for improving sleep. A method for manufacturing Phellinus igniarius extract concentrate comprises: performing a multi-stage cultivation step on a first mycelium of Phellinus igniarius to obtain a Phellinus igniarius fermented liquid, wherein the multi-stage cultivation step comprises: performing a solid-state culture The step is to use a solid medium to cultivate the first mycelium at 15°C to 30°C for 1 week to 2 weeks to obtain a second mycelium, wherein the Phellinus igniarius was deposited on July 18, 2019 The Bioresource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan, deposited Phellinus linteus with the number BCRC 930210; a liquid culture step was performed using a first culture medium at 15°C to 30°C Cultivate the second mycelium at ℃ for 3 to 14 days to obtain a third mycelium, wherein one of the first culture solutions has a pH value of pH 2 to pH 6; and a fermentation step is performed, It uses a second culture medium to cultivate the third mycelium at 15°C to 30°C for 3 to 21 days to obtain the Phellinus igniarius fermented liquid, wherein one of the second culture liquids has a pH value pH 2 to pH 6; Performing a drying step on the Phellinus igniarius fermented liquid to obtain a dried Phellinus igniarius fermented product; performing an extraction step on the dried Phellinus igniarius fermented product using a polar solvent to obtain a Phellinus igniarius extract; and The Phellinus igniarius extract is subjected to a concentration step to obtain the Phellinus igniarius extract concentrate. The method for manufacturing Phellinus linteus extract concentrate as described in item 2 of the scope of patent application, wherein the fermented fermentation step is carried out in a fermented tank, and when the fermented culture step is performed, the fermented ferment is further included. A gas is introduced into the tank, and the gas system is selected from a group consisting of air, oxygen, carbon dioxide, helium, or any combination of the foregoing. The method for manufacturing Phellinus linteus extract concentrate described in item 3 of the scope of patent application, wherein the fermentation step is at a ^{tank pressure of 0.5 kg/cm 2} to 1.0 kg/cm ² and 0.01 (volume of gas introduced/ The volume of fermented liquor/minute, VVM) is carried out at an aeration rate of 1.5VVM. According to the method for producing the Phellinus linteus extract concentrate described in item 2 of the scope of the patent application, the polar solvent contains water and/or lower alcohol. The use of a Phellinus igniarius extract concentrate for the preparation of a sleep-improving composition, wherein the Phellinus igniarius extract concentrate is derived from the Phellinus igniarius deposited with the BCRC on July 18, 2019 and the deposit number is BCRC 930210. The composition It is an oral composition, and the Phellinus igniarius extract concentrate is prepared by the manufacturing method described in any one of Claims 2 to 5.

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