阅读说明：本技术 一种赤芝乳酸菌发酵提取物的制备及应用 (Preparation and application of ganoderma lucidum lactobacillus fermentation extract ) 是由 芦艳荣 方祥铭 王昌涛 于 2019-11-01 设计创作，主要内容包括：本发明公开了一种赤芝乳酸菌发酵提取物的制备方法,其包括如下步骤：将乳酸菌活化后制得的乳酸菌菌液接种在含有灭活赤芝发酵液和水的培养基中,混合得到初始体系,并对初始体系进行发酵培养,得到赤芝乳酸菌发酵提取物。该方法利用发酵工程技术从赤芝和乳酸菌发酵并提取活性物质,操作简单,且由于不添加任何香精、乳化剂、增稠剂、防腐剂等化学添加剂,提取物可直接作为化妆品使用,制得的赤芝乳酸菌发酵提取物对皮肤安全无伤害,具有更高的抗衰老、抗炎活性,同时具有良好的补水保湿效果。(The invention discloses a preparation method of a ganoderma lucidum lactobacillus fermentation extract, which comprises the following steps: inoculating lactobacillus bacteria solution obtained by activating lactobacillus in a culture medium containing inactivated Ganoderma lucidum fermentation liquid and water, mixing to obtain an initial system, and performing fermentation culture on the initial system to obtain Ganoderma lucidum lactobacillus fermentation extract. The method utilizes fermentation engineering technology to ferment and extract active substances from Ganoderma lucidum and lactobacillus, has simple operation, and can be directly used as cosmetics due to no addition of chemical additives such as essence, emulsifier, thickener, antiseptic, etc., and the prepared Ganoderma lucidum lactobacillus fermented extract has no harm to skin, has higher anti-aging and anti-inflammatory activity, and simultaneously has good moisturizing effect.)

1. A preparation method of Ganoderma lucidum lactobacillus fermentation extract comprises the following steps: inoculating lactobacillus bacteria liquid obtained after lactobacillus activation into a culture medium containing inactivated ganoderma lucidum fermentation liquid and water, mixing to obtain an initial system, and performing fermentation culture on the initial system to obtain the ganoderma lucidum lactobacillus fermentation extract.

2. The method according to claim 1, wherein the volume ratio of the lactobacillus solution, the inactivated ganoderma lucidum fermentation liquid and the water in the initial system is (5-15): (50-150): (200- & ltSUB- & gt 400), & lt 8-12 > -, preferably: (80-120): (250-350), more preferably 10: 100: 300.

3. the method according to claim 1 or 2, wherein the concentration of the lactic acid bacteria liquid is 10⁴To 10¹⁰CFU/mL, preferably 10⁵To 10⁹CFU/mL, more preferably 10⁶To 10⁸CFU/mL。

4. The production method according to any one of claims 1 to 3, wherein the temperature of the fermentation culture is 35 to 40 ℃, preferably 38 ℃, and the time of the fermentation culture is 30 to 38 hours, preferably 32 hours.

5. The preparation method according to any one of claims 1 to 4, wherein the lactic acid bacterium is a Lactobacillus, preferably Lactobacillus reuteri (Lactobacillus reuteri).

6. The preparation method according to any one of claims 1 to 5, wherein the preparation of the inactivated Ganoderma lucidum fermentation broth comprises picking Ganoderma lucidum mycelia to inoculate in potato dextrose broth, shake-culturing at 20 to 30 ℃, preferably 22 to 28 ℃, more preferably 25 ℃ for 30 to 60h, preferably 44 to 52h, more preferably 48 h; after grinding the pellet with a homogenizer, the pellet is inactivated at 100 to 130 ℃, preferably 110 to 120 ℃, more preferably 115 ℃ for 10 to 20min, preferably 13 to 17min, more preferably 15 min.

7. The method according to any one of claims 1 to 6, wherein the lactobacillus suspension is in the form of a culture solution or suspension, and the pH value of the culture solution or suspension is 6.0 to 8.0, preferably 7.0.

8. The preparation method according to any one of claims 1 to 7, further comprising the steps of sequentially sterilizing, centrifuging and taking a supernatant of the Ganoderma lucidum lactic acid bacteria fermentation extract after the fermentation culture.

9. The preparation method according to claim 8, wherein the sterilization temperature is 115 to 125 ℃, preferably 120 ℃, and the sterilization time is 20 to 30min, preferably 25 min; the centrifugation speed is 3800 to 4200r/min, preferably 4000r/min, the centrifugation time is 15 to 30min, preferably 20min, and the centrifugation radius is 8 to 10cm, preferably 9 cm.

10. A fermented extract of Ganoderma lucidum with lactic acid bacteria, which is prepared by the method according to any one of claims 1 to 9.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to preparation and application of a ganoderma lucidum lactobacillus fermentation extract.

Background

Ganoderma lucidum is a medicinal fungus of Ganoderma of Polyporaceae of Basidiomycetes, is one of main sources of traditional Chinese medicinal materials Ganoderma, has effects of invigorating qi and blood, tranquilizing mind, and invigorating spleen and stomach, and is mainly used for treating diseases such as asthenia, cardiopalmus, insomnia, dizziness, listlessness debilitation, chronic cough, asthma, coronary heart disease, tumor, etc.

Lactic acid bacteria are a generic term for a group of spore-free, gram-positive bacteria that can ferment and utilize sugars to produce large amounts of lactic acid, and are widely distributed in humans, animals, plants and fermented foods. Lactic acid bacteria are well known as a probiotic in a wide range of applications. The FAO and WHO identify probiotics as viable microorganisms that are beneficial to the body and promote the health of the body when dosed in sufficient amounts. Lactic acid bacteria are an important group of probiotics, and attract attention and research. Probiotics have now been widely used in a variety of fields, including promoting and maintaining human health, promoting the growth of agricultural animals, and the like. The probiotic bacteria include Bifidobacterium, Lactobacillus, Streptococcus, etc., and some of the strains have been listed as useful for food.

According to the excellent effects of the two microorganisms, the ganoderma lucidum and the lactic acid bacteria are subjected to compound fermentation. The product obtained by fermentation has a small molecular polypeptide product through the synergistic effect of the composite fermentation. The small molecular polypeptide is easier to be absorbed by skin, and has good anti-aging, whitening, moisturizing and hydrating effects.

Disclosure of Invention

The invention aims to provide a preparation method and application of a ganoderma lucidum lactobacillus fermentation extract. The method utilizes fermentation engineering technology to ferment and extract active substances from ganoderma lucidum and lactobacillus, has simple operation, can be directly used as cosmetics due to no addition of any chemical additives such as essence, emulsifier, thickener, preservative and the like, and the prepared ganoderma lucidum lactobacillus fermentation extract has the advantages of safety and no harm to skin, higher anti-aging and anti-inflammatory activity, and good moisturizing effect.

According to a first aspect of the present invention, there is provided a method for preparing a ganoderma lucidum lactic acid bacteria fermentation extract, comprising the steps of: inoculating lactobacillus bacteria solution obtained by activating lactobacillus in a culture medium containing inactivated Ganoderma lucidum fermentation liquid and water, mixing to obtain an initial system, and performing fermentation culture on the initial system to obtain Ganoderma lucidum lactobacillus fermentation extract.

According to a second aspect of the present invention, there is provided a ganoderma lucidum lactic acid bacteria fermented extract prepared by the above preparation method.

According to a third aspect of the present invention, there is provided a use of the above-mentioned fermented extract of ganoderma lucidum lactic acid bacteria for preparing a cosmetic having at least one function of 1) to 7) below:

1) inhibiting melanin generation;

2) skin whitening;

3) removing DPPH free radicals;

4) promoting collagen synthesis;

5) anti-aging;

6) increasing skin moisture content; and

7) and (5) moisturizing.

According to a fourth aspect of the present invention, there is provided the use of the above-mentioned fermented extract of ganoderma lucidum lactic acid bacteria as a cosmetic having at least one function of 1) to 7) below:

1) inhibiting melanin generation;

2) skin whitening;

3) removing DPPH free radicals;

4) promoting collagen synthesis;

5) anti-aging;

6) increasing skin moisture content; and

7) and (5) moisturizing.

The invention has the following beneficial effects:

(1) the preparation method provided by the invention adopts a fermentation engineering technology, and extracts and obtains the ganoderma lucidum lactobacillus fermentation extract by selecting proper strains and fermentation conditions. On one hand, active ingredients can be retained to the maximum extent, and compared with the traditional extraction method, the method avoids the introduction of organic solvents, and is safe and environment-friendly; on the other hand, no foreign substances such as enzyme and the like are required to be added, so that the production cost is saved, the production steps are simplified to the maximum extent, the industrial production can be realized by the fermentation technology, and the stability of the product quality can be fully ensured.

(2) The ganoderma lucidum lactobacillus fermentation extract provided by the invention contains indispensable micromolecular polypeptide components for healthy skin, does not contain any synthetic chemical component because no foreign substance is introduced in the preparation process, can be directly used as a finished product of a mask or essence or toner, is more natural than other products on the market, and does not cause any side effect on the skin.

(3) The molecular weight of the small molecular polypeptide in the ganoderma lucidum lactobacillus fermentation extract is 275.40Da, and accounts for more than 99% of the total amount, so that the small molecular polypeptide is easy to be fully absorbed by skin.

(4) The ganoderma lucidum lactobacillus fermentation extract provided by the invention has strong oxidation resistance, can remove free radicals, promote cell metabolism, enhance cell activity, promote collagen synthesis, improve the structure and function of an organism and improve the vitality of the organism, thereby delaying cell aging and having the anti-aging effect.

(5) The ganoderma lucidum lactobacillus fermentation extract provided by the invention can inhibit melanin synthesis, has an inhibition effect on melanin synthesis at a concentration of 5% better than that of 33mM arbutin, and has a good whitening effect.

(6) The ganoderma lucidum lactobacillus fermentation extract provided by the invention can obviously improve the water content of skin and has an excellent moisturizing effect.

Drawings

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

FIG. 1 is a HPLC chromatogram of the molecular weight distribution of active polypeptide of the fermented extract of Ganoderma lucidum lactic acid bacteria prepared according to one embodiment of the present invention.

FIG. 2 is a graph showing the relationship between the DPPH radical scavenging rate and the fermentation extract of Ganoderma lucidum and lactobacillus according to an embodiment of the present invention at different volume percentage concentrations.

FIG. 3 shows the effect of fermented extract of Ganoderma lucidum lactic acid bacteria on collagen synthesis by fibroblasts prepared according to an embodiment of the present invention.

FIG. 4 shows the effect of the fermented extract of Ganoderma lucidum lactic acid bacteria on the synthesis of melanin by B16 cells, prepared according to an embodiment of the present invention.

Detailed Description

In the following description, certain specific details are included to provide a thorough understanding of various disclosed embodiments. One skilled in the relevant art will recognize, however, that the embodiments may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth.

According to a first aspect of the present invention, there is provided a method for preparing a ganoderma lucidum lactic acid bacteria fermentation extract, comprising the steps of: inoculating lactobacillus bacteria solution obtained by activating lactobacillus in a culture medium containing inactivated Ganoderma lucidum fermentation liquid and water, mixing to obtain an initial system, and performing fermentation culture on the initial system to obtain Ganoderma lucidum lactobacillus fermentation extract.

In some specific embodiments, the volume ratio of the lactobacillus liquid, the inactivated ganoderma lucidum fermentation liquid and the water in the initial system is (5-15): (50-150): (200-400). In some preferred embodiments, the volume ratio of the lactobacillus liquid, the inactivated ganoderma lucidum fermentation liquid and the water in the initial system is (8-12): (80-120): (250-350). In certain more preferred embodiments, the volume ratio of the lactobacillus liquid, the inactivated ganoderma lucidum fermentation liquid and the water in the initial system is 10: 100: 300.

in some specific embodiments, the concentration of the lactobacillus liquid is 10⁴To 10¹⁰CFU/mL. In some preferred embodiments, the concentration of the lactobacillus liquid is 10⁵To 10⁹CFU/mL. In certain more preferred embodiments, the concentration of the lactic acid bacteria liquid is 10⁶To 10⁸CFU/mL。

In certain specific embodiments, the temperature of the fermentation culture is from 35 to 40 ℃. In certain preferred embodiments, the temperature of the fermentation culture is 38 ℃.

In certain specific embodiments, the time of the fermentation culture is 30 to 38 hours. In certain preferred embodiments, the time of the fermentation culture is 32 h.

In certain specific embodiments, the lactic acid bacteria are lactobacillus. In certain preferred embodiments, the lactic acid bacterium is Lactobacillus reuteri (Lactobacillus reuteri).

In certain embodiments, the preparation of the inactivated ganoderma lucidum fermentation broth comprises selecting ganoderma lucidum mycelia, inoculating into potato dextrose broth, and shake-culturing at 20-30 ℃ for 30-60 h; after grinding the pellet with a homogenizer, the pellet is inactivated at 100 to 130 ℃ for 10 to 20 min. In certain preferred embodiments, the preparation of the inactivated Ganoderma lucidum fermentation broth comprises selecting Ganoderma lucidum mycelia, inoculating into potato dextrose broth, and shake-culturing at 22-28 deg.C for 44-52 h; after grinding the pellet with a homogenizer, the pellet is inactivated at 110 to 120 ℃ for 13 to 17 min. In certain more preferred embodiments, the preparation of the inactivated Ganoderma lucidum fermentation broth comprises selecting Ganoderma lucidum mycelia to inoculate in potato dextrose broth, shake culturing at 25 ℃ for 48 h; after grinding the pellet with a homogenizer, the pellet was inactivated at 115 ℃ for 15 min.

In some embodiments, the lactic acid bacteria solution may be in the form of a culture solution or suspension having a pH of 6.0 to 8.0. In certain preferred embodiments, the pH of the culture broth or suspension is 7.0.

In some specific embodiments, the above preparation method further comprises the steps of sequentially sterilizing, centrifuging and collecting supernatant of the fermented extract of Ganoderma lucidum lactic acid bacteria after the fermentation culture.

In certain specific embodiments, the sterilization temperature is 115 to 125 ℃ and the sterilization time is 20 to 30 min. In certain preferred embodiments, the sterilization temperature is 120 ℃ and the sterilization time is 25 min.

In certain specific embodiments, the centrifugation speed is 3800 to 4200r/min, the centrifugation time is 15 to 30min, and the centrifugation radius is 8 to 10 cm. In certain preferred embodiments, the centrifugation speed is 4000r/min, the centrifugation time is 20min, and the centrifugation radius is 9 cm.

According to a second aspect of the present invention, there is provided a ganoderma lucidum lactic acid bacteria fermented extract prepared by the above preparation method.

According to a third aspect of the present invention, there is provided a use of the above-mentioned fermented extract of ganoderma lucidum lactic acid bacteria for preparing a cosmetic having at least one function of 1) to 7) below:

1) inhibiting melanin generation;

2) skin whitening;

3) removing DPPH free radicals;

4) promoting collagen synthesis;

5) anti-aging;

6) increasing skin moisture content; and

7) and (5) moisturizing.

According to a fourth aspect of the present invention, there is provided the use of the above-mentioned fermented extract of ganoderma lucidum lactic acid bacteria as a cosmetic having at least one function of 1) to 7) below:

1) inhibiting melanin generation;

2) skin whitening;

3) removing DPPH free radicals;

4) promoting collagen synthesis;

5) anti-aging;

6) increasing skin moisture content; and

7) and (5) moisturizing.

In certain specific embodiments, in the above applications, the cosmetic includes, but is not limited to: facial mask, essence or toner.

Hereinafter, the present invention will be explained in detail by the following examples in order to better understand various aspects of the present invention and advantages thereof. It should be understood, however, that the following examples are not limiting and are merely illustrative of certain embodiments of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The medium for activating Ganoderma lucidum in the following examples is composed of a solute and a solvent, the solvent is potato extract, and the concentration of the solute and its concentration in the liquid medium are 20g/L glucose.

The lactic acid bacteria activating liquid medium in the following examples consists of solute and solutionThe preparation comprises the following components in parts by weight, wherein the solvent is water, and the solute and the concentration thereof in a liquid culture medium are as follows: casein peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 5g/L, sodium acetate 5g/L, diammonium citrate 2g/L, Tween 801g/L, K₂HPO₄2 g/L，MgSO₄.7H₂O 0.2g/L，MnSO₄.H₂O 0.05g/L，CaCO₃20g/L, agar 15g/L, pH 6.8.

The Lactobacillus reuteri (Lactobacillus reuteri) bacteria in the following examples were obtained from the China center for Industrial culture Collection of microorganisms.

The Ganoderma lucidum in the following examples is derived from China general microbiological culture Collection center (CGMCC).