CN115058468B

CN115058468B - Schizophyllan compound polysaccharide, ion-reduced transdermal toning lotion and preparation method thereof

Info

Publication number: CN115058468B
Application number: CN202210792418.5A
Authority: CN
Inventors: 郑暘; 周路
Original assignee: Ningbo Quanshui Biotechnology Co ltd
Current assignee: Ningbo Quanshui Biotechnology Co ltd
Priority date: 2022-07-07
Filing date: 2022-07-07
Publication date: 2023-04-28
Anticipated expiration: 2042-07-07
Also published as: CN115058468A

Abstract

The application discloses schizophyllan compound polysaccharide, ion reduction transdermal toning lotion and a preparation method thereof. The schizophyllan compound polysaccharide has broad-spectrum antibacterial performance in vitro. The cosmetic water has antibacterial and moisturizing effects, has no cytotoxicity, and has super redox activity and antioxidant effect. In addition, the cosmetic water can effectively permeate the cortex, clean hair follicles and pores, and is helpful for removing redundant oxygen free radicals of skin. Experiments show that the cosmetic water provided by the application has more moisture retention property, nutrient substances are easier to absorb, and the transdermal function is strong.

Description

Schizophyllan compound polysaccharide, ion-reduced transdermal toning lotion and preparation method thereof

Technical Field

The application relates to the technical field of ionic toning lotion, in particular to schizophyllan compound polysaccharide, ionic reduction transdermal toning lotion and a preparation method thereof.

Background

The existing cosmetics are various and classified according to the purpose of use: 1) Cleaning cosmetics; 2) A base cosmetic; 3) Cosmetic preparations; 4) Therapeutic cosmetics. The basic cosmetic comprises: (1) a mask; (2) toning lotion; (3) essence (4) honey; (5) a face cream; (6) hair cream, hair spray, etc. The cosmetic water is also called toner, moisturizing lotion and astringent. After the towel for washing face is used for wiping water, the cosmetic water is used in the preparation stage for absorbing nutrition, and has the functions of regulating skin and supplying water to horny layer. The astringent water not only has the function of shrinking, but also supplies water to the skin, and has the functions of regulating excessive sebum and controlling oil content. The classification can be as follows: clean-type toning lotion, moisturizing toning lotion and sun-proof toning lotion. The existing toning lotion contains chemical preservatives, and excessive chemical harmful substances can cause damage to skin; the existing cosmetic water has poor antibacterial property and often cannot be used for skin penetration and moisture preservation.

Disclosure of Invention

Therefore, the inventor creatively prepares a schizophyllan compound polysaccharide and uses the schizophyllan compound polysaccharide as a main raw material for preparing the toning lotion so as to solve the defects in the prior art.

In a first aspect, embodiments of the present application disclose a method for preparing a schizophyllan complex polysaccharide, comprising:

obtaining an activation solution of schizophyllum commune;

inoculating the activation solution into a working seed solution, and culturing on a shaking table to obtain the working seed solution, wherein the working seed solution comprises 15wt% of glucose, 0.8wt% of yeast powder, 0.2% of ammonium nitrate and K ₂ HPO ₄ 0.2％、MgSO ₄ 0.1 percent of Tween-80, 0.03v/v percent and 0.02 to 0.05v/v percent of aloe juice;

inoculating the working seed liquid into a fermentation culture medium, controlling ventilation and stirring speed at 28 ℃, and culturing to obtain a fermentation liquid, wherein the fermentation culture medium comprises 10wt% of glucose, 0.65wt% of yeast powder and 0.18% of ammonium nitrate, and K ₂ HPO ₄ 0.18％、MgSO ₄ 0.08%, tween-80 0.02v/v% and aloe juice 0.1-0.25 v/v%;

and collecting the fermentation liquor, and extracting and purifying to obtain a pure product of the schizophyllan compound polysaccharide.

In the embodiment of the application, the extracellular polysaccharide content in the working seed liquid is not lower than 40g/L, and the mycelium biomass is not lower than 90g/L.

In the embodiment of the application, the extracellular polysaccharide content in the fermentation broth is not lower than 27g/L, and the mycelium biomass is not lower than 55g/L.

In an embodiment of the present application, the step of extracting the fermentation broth includes:

adding 0.6v/m% active carbon into the fermentation broth, heating and stirring for 45min, filtering to obtain filtrate, adjusting pH=4, adding pepsin, preheating in a water bath kettle at 37 ℃ for 10min, oscillating and holding for 30min, then placing in a water bath kettle at 100 ℃ for enzyme deactivation for 10min, cooling to room temperature, adding 2.5 times volume of absolute ethyl alcohol, generating a large amount of white flocculent precipitate, shaking, standing at 4 ℃ overnight, centrifuging at 6000r/min for 20min, collecting precipitate, and freeze-drying to obtain crude schizophyllan compound polysaccharide.

In the embodiment of the application, dissolving the crude product by PBS with pH=6.8, then statically loading the solution into a chromatographic column filled with Sephadex G-200, eluting by using a sodium chloride solution with the concentration of 0.8%, collecting the solution step by step at the elution flow rate of 0.3mL/min, collecting 2mL of the solution by each tube, detecting the sugar content in the solution by using a phenol-sulfuric acid method, drawing an elution curve according to the sugar content concentration corresponding to the tube number, collecting the eluent corresponding to the elution peak, concentrating under reduced pressure, and freeze-drying to obtain the pure product of the schizophyllan compound polysaccharide.

In a second aspect, the embodiments of the present application disclose the schizophyllan compound polysaccharide prepared by the preparation method of the first aspect, wherein the schizophyllan compound polysaccharide has a structure formed by polymerizing schizophyllan and hyaluronic acid.

In a third aspect, the embodiment of the application discloses an ionic reduction transdermal toning lotion, which is a deionized solution containing 5-12% of glycerol, 0.15-0.5% of sodium carboxymethyl cellulose, 0.5-2.5-4.5% of schizophyllan compound polysaccharide in the second aspect, 1.0-2.5% of polyoxyethylene, 0.05-0.1% of polybutylimidazole zinc acetate, 0.05-0.1% of polybutylimidazole iron acetate, 0.5-1.5% of polyethylene glycol, 0.05-0.2% of essence and 5-20% of ethanol according to mass concentration.

In the embodiment of the application, the toning lotion is a deionized solution containing 12% of glycerol, 0.5% of sodium carboxymethyl cellulose, 3.5% of schizophyllan compound polysaccharide provided in the embodiment 1, 0.08% of polybutylimidazole zinc acetate, 0.07% of polybutylimidazole iron acetate, 1.0% of polyethylene glycol, 0.05% of essence and 15% of ethanol according to mass concentration.

In a fourth aspect, the embodiment of the application discloses the preparation method of the first aspect or the application of the schizophyllan compound polysaccharide of the second aspect in preparation of cosmetics.

Compared with the prior art, the application has at least one of the following beneficial effects:

the schizophyllum commune compound polysaccharide is obtained by fermenting schizophyllum commune, adding glucose and aloe juice into a culture medium, separating and purifying, and carrying out structural analysis, and has a structure formed by polymerizing beta-pyrane glucan and hyaluronic acid, and has broad-spectrum antibacterial performance in vitro.

The embodiment of the application also prepares the ion reduction transdermal toning lotion by using the schizophyllan compound polysaccharide, the toning lotion not only has antibacterial and moisturizing properties, but also has no cytotoxicity, and the toning lotion also has super-strong redox activity and antioxidation function. In addition, the cosmetic water can effectively permeate the cortex, clean hair follicles and pores, and is helpful for removing redundant oxygen free radicals of skin. Experiments show that the cosmetic water provided by the application has more moisture retention property, nutrient substances are easier to absorb, and the transdermal function is strong.

Drawings

Fig. 1 is a GC diagram of a complete hydrolysate of pure schizophyllan compound polysaccharide provided in example 1 of the present application.

Fig. 2 is a GC diagram of a complete hydrolysate of pure schizophyllan compound polysaccharide provided in example 2 of the present application.

Fig. 3 is a GC diagram of a complete hydrolysate of pure schizophyllan compound polysaccharide as provided in comparative example 1 of the present application.

Fig. 4 is an infrared spectrum of a pure schizophyllan compound polysaccharide product provided in example 1 of the present application.

Fig. 5 is an infrared spectrum of a pure schizophyllan compound polysaccharide product provided in example 2 of the present application.

Fig. 6 is an infrared spectrum of a pure schizophyllan compound polysaccharide provided in comparative example 1 of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

Preparation of schizophyllan compound polysaccharide

1. Materials and methods

1. Bacterial strain

Schizophyllum commune (Schizophyllum commune Fr.) employed in the examples herein was purchased from testuo organism under the accession number TS303024.

2. Strain activation

After the freeze-dried tube of schizophyllum commune is thawed by sterile water, the schizophyllum commune is inoculated on a PDA solid culture medium for standing culture for 18 to 24 hours at room temperature, and then the schizophyllum commune is inoculated into a seed shake flask for culture, wherein the culture medium formula comprises the following components: 12wt% of glucose, 1wt% of yeast powder and K ₂ HPO ₄ 0.2wt％、MgSO ₄ 0.1wt% of the mixture was cultured at a shaking flask culture condition of 26℃and 180rpm for 5 days to obtain an activated liquid.

3. Working seed liquid

In example 1, the above-mentioned activated liquid was inoculated into a working seed culture liquid in an inoculum size of 12v/v% (volume percent), and cultured on a shaker at 180rpm for 96 hours to obtain a working seed liquid. Wherein the working seed culture medium comprises 15wt% of glucose, 0.8wt% of yeast powder and 0.2 wt% of ammonium nitrate, and K ₂ HPO ₄ 0.2％、MgSO ₄ 0.1%, tween-80 0.03v/v% and aloe vera juice (obtained by high-speed shearing fresh aloe vera and filtering the filtrate) 0.05v/v%.

In example 2, the above-mentioned activated liquid was inoculated into a working seed culture liquid in an inoculum size of 12v/v% (volume percent), and cultured on a shaker at 180rpm for 96 hours to obtain a working seed liquid. Wherein the working seed culture medium comprises 15wt% of glucose, 0.8wt% of yeast powder and 0.2 wt% of ammonium nitrate, and K ₂ HPO ₄ 0.2％、MgSO ₄ 0.1%, tween-80.03 v/v%, and aloe vera juice (obtained by shearing fresh aloe vera at high speed and filtering the filtrate) 0.02v/v%.

In comparative example 1, the above-mentioned activated liquid was inoculated into a working seed culture liquid in an inoculum size of 12v/v% (volume percent), and cultured on a shaker at 180rpm for 96 hours to obtain a working seed liquid. Wherein the working seed culture medium comprises 15wt% of glucose, 0.8wt% of yeast powder and 0.2 wt% of ammonium nitrate, and K ₂ HPO ₄ 0.2％、MgSO ₄ 0.1% Tween-80 0.03v/v%.

4. Preparation of fermentation broths

In example 1, a Biostat stirred tank controlled bioreactor (b.braun Biotech International company), an empty tank was sterilized at about 120 ℃ for about 40 minutes, and the bioreactor was transferred to a sterilized fermentation medium, and subjected to sterile culture observation with a liquid loading amount of 3L and an empty culture period of 1 d; the prepared working seed liquid is transferred into the seed liquid according to the inoculation amount of 12 percent, the ventilation and stirring rate are controlled at 28 ℃, the seed liquid is cultured for 144 hours, sampling analysis is carried out every 12 hours, and meanwhile, the seed liquid is filled with sterile water. Wherein the fermentation medium comprises 10wt% of glucose, 0.65wt% of yeast powder, 0.18% of ammonium nitrate and K ₂ HPO ₄ 0.18％、MgSO ₄ 0.08%, tween-80 0.02v/v% and aloe juice 0.1v/v%.

In example 2, a Biostat stirred tank control bioreactor was sterilized at about 120℃for about 40 minutes, transferred to a sterilized fermentation medium, filled with 3L of liquid, and cultured for 1d in an empty state, and subjected to sterile culture observation; the prepared working seed liquid is transferred into the seed liquid according to the inoculation amount of 12 percent, the ventilation and stirring rate are controlled at 28 ℃, the seed liquid is cultured for 144 hours, sampling analysis is carried out every 12 hours, and meanwhile, the seed liquid is filled with sterile water. Wherein the fermentation medium comprises 10wt% of glucose, 0.65wt% of yeast powder and ammonium nitrate0.18％，K ₂ HPO ₄ 0.18％、MgSO ₄ 0.08%, tween-80 0.02v/v% and aloe juice 0.25v/v%.

In comparative example 1, a Biostat stirred tank controlled bioreactor empty tank was sterilized at about 120℃for about 40 minutes, transferred to a sterilized fermentation medium, charged with 3L of liquid, and subjected to an empty culture for 1d, and subjected to a sterile culture observation; the prepared working seed liquid is transferred into the seed liquid according to the inoculation amount of 12 percent, the ventilation and stirring rate are controlled at 28 ℃, the seed liquid is cultured for 144 hours, sampling analysis is carried out every 12 hours, and meanwhile, the seed liquid is filled with sterile water. Wherein the fermentation medium comprises 10wt% of glucose, 0.65wt% of yeast powder, 0.18% of ammonium nitrate and K ₂ HPO ₄ 0.18％、MgSO ₄ 0.08% and Tween-80 0.02v/v%.

5. Determination of extracellular polysaccharide

Taking a proper amount of working seed liquid or fermentation liquid provided in examples 1-2 and comparative example 1 respectively, centrifuging at 5000rpm for 20min, adding twice volume of absolute ethyl alcohol into supernatant, fully stirring, standing for 6h, centrifuging at 5000rpm for polysaccharide precipitation, drying and weighing, and calculating to obtain the yield of extracellular polysaccharide in the working seed liquid or fermentation liquid, wherein the unit is g polysaccharide/L working seed liquid or fermentation liquid.

6. Determination of mycelium biomass of working seed or fermentation broth

Taking a proper amount of working seed liquid or fermentation liquid provided in examples 1-2 and comparative example 1 respectively, centrifuging at 5000rpm for 20min, separating supernatant from mycelium to obtain mycelium precipitate, drying to constant weight, and calculating mycelium biomass in g mycelium dry weight/L working seed liquid or fermentation liquid.

8. Separation of schizophyllan compound polysaccharide

(1) Extracting crude product

Taking 100mL of fermentation liquid provided in examples 1-2 and comparative example 1 respectively, adding 0.6v/m% active carbon, heating and stirring for 45min, filtering to obtain filtrate, adding 100mL of decolored fermentation liquid, adjusting pH=4, weighing 0.8g of pepsin (CAS No. 9001-75-6, microphone) into the filtrate, preheating for 10min in a 37 ℃ water bath kettle, oscillating and holding for 30min, putting the filtrate into the 100 ℃ water bath kettle for enzyme deactivation for 10min, cooling to room temperature, adding 2.5 times of absolute ethyl alcohol, generating a large amount of white flocculent precipitate, shaking uniformly, standing at 4 ℃ overnight, centrifuging for 20min at 6000r/min, collecting precipitate, freeze-drying to obtain crude schizophyllan compound polysaccharide.

(2) Pure product

Purifying the crude product by a column chromatography method, dissolving the crude product by PBS with pH of 6.8, loading the solution into a chromatographic column filled with Sephadex G-200, eluting by 0.8% sodium chloride solution with the elution flow rate of 0.3mL/min, collecting 2mL each tube, detecting the sugar content in each tube by a phenol-sulfuric acid method, drawing an elution curve according to the sugar content concentration corresponding to the tube number, collecting the eluent corresponding to the elution peak, concentrating under reduced pressure, and freeze-drying to obtain the pure product of the schizophyllan compound polysaccharide.

(3) Refining

In some embodiments, to obtain a pure product with higher purity, purification can be performed for multiple times by using a chromatographic column filled with Sephadex G-200, and the molecular weight of the pure product is measured by using a multi-angle laser light scattering instrument (specific detection method can refer to "preparation process optimization of Sparassis crispa low molecular weight polysaccharide and determination of its molecular weight [ J ], food industry science and technology, 2020, 4 th phase").

9. Gas chromatography detection of monosaccharide composition analysis

Accurately weighing 50.0mg of pure products provided in examples 1-2 and comparative example 1 respectively, sealing with 5mL of 1M sulfuric acid aqueous solution, placing in a 90 ℃ oven for hydrolysis for 10 hours, adjusting pH to 7.0 with 1M barium sulfate solution, centrifuging for 10 minutes at 4000r/min, concentrating the supernatant to dryness, adding 100.0mg of hydroxylamine hydrochloride and 5mL of pyridine, heating in a water bath at 90 ℃ for reaction for 10 minutes, taking out, cooling to room temperature, adding 5mL of acetic anhydride solution into the desired reaction solution, and placing in a water bath at 90 ℃ for reaction for 20 minutes to obtain the sugar nitrile acetyl ester derivative. Then, 10mg of each standard monosaccharide was accurately weighed, and the standard product of the sugar nitrile acetyl ester derivative was prepared by the same method, and was analyzed by gas chromatography. The chromatographic conditions are OV-225 capillary column, column temperature 240 deg.c, carrier gas of nitrogen gas in flow rate of 30ml/min, hydrogen gas flow rate of 48ml/min and attenuation of 6-7, sample and standard sample feeding amount of 2. Mu.L, and monosaccharide analysis.

10. Periodate oxidation-Smith degradation

In this experimental example, pure products of sodium periodate oxidized schizophyllan compound polysaccharide (refer to chemical structure and morphology analysis of Lily polysaccharide BHP-1 in Lanzhou [ J ] journal of Chinese experimental prescription, 2020, 8 th stage) are adopted to judge structural information such as type and connection form of glycosidic bond, branched chain positioning, number self and the like according to consumption of periodic acid and generation of formic acid. In this experimental example, smith degradation was performed on the basis of periodic acid oxidation to reduce periodic acid oxidation products to polyhydroxy compounds having good stability, such as glycerin, erythritol and non-oxidized monosaccharide residues, wherein the sugar bond types that can produce glycerin are (1- & gt), (1- & gt 6), (1- & gt 2) and (1- & gt 2, 6), and the glycosidic bonds that can produce erythritol are (1- & gt 4) and (1- & gt 4, 6). Thus, by identifying the hydrolysis product, the type of glycosidic bond and the branching location can be deduced.

11. Ultraviolet and infrared spectroscopic analysis

In this experimental example, pure schizophyllan compound polysaccharide provided in each of examples 1 to 2 and comparative example 1 was tableted with KBr at 4000 to 500cm ^-1 And infrared scanning is carried out in the interval.

2. Results

Table 1 shows the extracellular polysaccharide content and the mycelium biomass in the working seed solutions provided in examples 1 to 2 and comparative example 1, respectively, and table 2 shows the extracellular polysaccharide content and the mycelium biomass in the fermentation broths provided in examples 1 to 2 and comparative example 1, respectively, and in tables 1 and 2, multiple comparisons and significant difference marks were made for each column of data. As can be seen from tables 1 and 2, the working seed solutions and fermentation broths provided in examples 1-2 were significantly higher in extracellular polysaccharide content and mycelium biomass than comparative example 1.

TABLE 1 working seed fluids

TABLE 2 fermentation broths

Further, the molecular weight of the pure product was measured by a multi-angle laser light scattering instrument, and the pure product provided in example 1, example 2 and comparative example 1 had a relative molecular weight of 1.63×10, respectively ⁵ 、2.05×10 ⁵ And 5.62X10 ⁴ Da. Further, using rhamnose, xylose, mannose, glucose, galactose, D-glucuronic acid and N-acetylglucosamine as standard samples, the pure products provided in examples 1 to 2 and comparative example 1, respectively, were subjected to GC analysis after being derivatized with a glycolonitrile acetyl ester, and as a result, as shown in FIGS. 1 to 3, it was found that single peaks of glucose (Glu), glucuronic acid (GluA) and acetylglucosamine (GluNAc) were present in the GC patterns of the pure products provided in examples 1 to 2, whereas only single peaks of glucose (Glu) were present in the pure products provided in comparative example 1.

As shown in fig. 4 to 6, the results of the infrared spectroscopic analysis of schizophyllan compound polysaccharide show characteristic absorption peaks of general polysaccharide substances: 3545-3400 cm ^-1 The broad peak of (2) is the stretching vibration peak of polysaccharide hydroxyl and 1623cm ^-1 The left and right sides of the vibration peak are provided with C-O asymmetric stretching vibration peaks of 1138cm ^-1 The left and right have O-H angle-changing vibration peaks. 669cm ^-1 The absorption peak at the position is the characteristic peak of the pyran type beta-sugar ordinary bond; however, in the infrared spectra of the schizophyllan compound polysaccharide provided in examples 1-2, characteristic absorption peaks of-C=O and-C-N stretching vibration and N-H bending vibration are respectively at 1654cm-1 and 1560cm-1, which indicate that an acetamido structure exists, and two absorption peaks generated by coupling O=C-O-stretching vibration-OH bending vibration are respectively at 1412cm-1 and 1384cm-1, which indicate that a dissociated spindle group and polyhydroxy structure on uronic acid exist. It is thus shown that the schizophyllan complex polysaccharide provided in examples 1-2 is a complex polysaccharide formed by the polymerization of schizophyllan and hyaluronic acid, whereas comparative example 1 provides only schizophyllan.

Further, the present application also uses the periodic acid-Smith test to analyze the glycosidic bond in the schizophyllan compound polysaccharide provided in examples 1-2 and comparative example 1, respectively, and as a result, it is found that the ratio of the consumption of periodic acid to the amount of formic acid generated after the schizophyllan compound polysaccharide provided in examples 1-2 is oxidized by periodic acid is greater than 2, which indicates that the glucan bond types contained in the schizophyllan compound polysaccharide may include type I: 1- & gt, 1- & gt 2 or 1- & gt 6; type II: 1-4, 1-2, 6 or 1-4, 6 linkages; type III: 1→3,4,6, 1→2,3,6, 1→2,4 or 1→2, 46; in contrast, the schizophyllan compound polysaccharide provided in comparative example 1 was detected by gas chromatography using periodic acid oxidized Smith degradation product, and a large amount of glucose was detected, but no glucuronic acid and sugar amine were detected. The schizophyllan compound polysaccharide provided in examples 1-2 is a polysaccharide obtained by polymerizing schizophyllan and hyaluronic acid.

Antibacterial performance analysis of schizophyllan compound polysaccharide

1. Test strain

Staphylococcus aureus, RN4220 strain, beijing hua vietnamese organism; coli, cat No. B98084, minghuake; pseudomonas aeruginosa, cat No. B94631, minghuake; tubercle bacillus, cat No. CBS562.66, minghuake; candida albicans, cat No. BMZ134172, minghuake; streptococcus pneumoniae, cat No. B80924, minghuake; salmonella enteritidis, cat No. CICC21482, commodity No. e132161, medical fluid mall; lactobacillus pneumophilus, cat No. BMZ065915, minghuake; candida albicans, cat No. BMZ132601, minghuake; candida glabrata, cat No. B83111, minghuake.

2. Micro broth dilution method for detecting antibacterial property

The pure products provided in examples 1 to 2 and comparative example 1 were prepared with sterile water to give concentrations of 50mg/mL, 10mg/mL, 2mg/mL, 0.5mg/mL, 0.1mg/mL and 0.05mg/mL, respectively, as test solutions, and the Minimum Inhibitory Concentrations (MIC) of polysaccharide in the test solutions for the above-mentioned various strains were quantitatively determined by a trace broth dilution method. The specific method comprises the following steps:

the above bacterial strains were inoculated on LB solid medium and cultured upside down in an incubator at 37 ℃. After the colony grows out, a single colony is picked by an inoculating loop and transferred into LB liquid culture medium, shake culture is carried out at 37 ℃ until logarithmic phase, and the concentration of bacterial liquid is adjusted to 10 ⁶ CFU/mL. In a sterile environmentIn the following procedure, 90. Mu.L of MH broth was added to well 1 and 50. Mu.L of MH broth was added to wells 2 to 11 on a sterile 96-well plate. Then 10 mu L of the sample to be detected is added into the 1 st hole, 50 mu L of the sample is added into the 2 nd hole after fully mixing, the mixture is diluted by multiple ratio, 50 mu L of the sample is sucked out from the 10 th hole and discarded, 50 mu L of bacterial liquid (without the sample) is added into the 11 th hole to serve as a control, and 50 mu L of MH broth is added into the 12 th hole. After mixing, the mixture is placed at 37 ℃ and cultured for 6 to 8 hours at 150r/min, and the absorbance is measured at the wavelength of 600 nm. The MIC is calculated as the minimum mass concentration at which the haze level of the control well (11 th well) is lower by more than 50%.

2. Results

Table 3 shows that polysaccharides provided in examples 1 to 2 and comparative example 1, respectively, were used as test pieces, MIC antibacterial tests were performed, and in table 3, "x" indicates a significant difference (P < 0.05) from comparative example 1, and "-" indicates no antibacterial property was detected. As is clear from Table 3, the schizophyllan complex polysaccharides provided in examples 1-2 have bacteriostatic properties against gram-positive, gram-negative bacteria, and fungi of the type described in above 10, respectively, whereas the polysaccharides provided in comparative example 1 have no significant bacteriostatic effects against Streptococcus pneumoniae, salmonella enteritidis, corynebacterium pulmonarium, candida albicans, and Candida glabrata, and the MIC values of the schizophyllan complex polysaccharides provided in examples 1-2 are lower. Therefore, the schizophyllan compound polysaccharide provided by the embodiment of the application has wider antibacterial performance and stronger antibacterial performance.

TABLE 3 MIC of polysaccharide products (mg/mL)

Description of the embodiments	Example 1	Example 2	Comparative example 1
				Staphylococcus aureus	2.34±0.28*	2.58±0.42*	2.98±0.47
Coli bacterium	0.72±0.08*	0.63±0.05*	1.29±0.53
				Pseudomonas aeruginosa	1.68±0.34*	1.57±0.42*	7.83±1.25
Tubercle bacillus	12.41±2.34	13.01±1.89	13.82±2.92
				Candida albicans	4.65±0.74*	4.48±0.61*	12.08±1.36
Streptococcus pneumoniae	25.42±6.58	24.97±5.13	－
				Salmonella enteritidis	18.16±3.74	17.51±4.25	－
Bacillus pneumophilus	25.64±5.22	25.29±4.37	－
				Candida albicans	49.35±11.42	52.72±8.62	－
Candida glabrata (C.glabrata)	34.26±5.82	22.36±4.67	－

Toning lotion

1. Preparation of test article

For this reason, a lotion having not only antibacterial and moisturizing properties but also no cytotoxicity, and having superior redox activity and antioxidant function was prepared using the schizophyllan complex polysaccharide provided in examples 1 to 2 above. In addition, the cosmetic water can effectively permeate the cortex, clean hair follicles and pores, and is helpful for removing redundant oxygen free radicals of skin. Experiments show that the cosmetic water provided by the application has more moisture retention property, nutrient substances are easier to absorb, and the transdermal function is strong.

The embodiment of the application provides an ion reduction transdermal toning lotion which is a deionized solution containing 5-12% of glycerol, 0.15-0.5% of sodium carboxymethyl cellulose, 0.5-2.5-4.5% of schizophyllan compound polysaccharide, 1.0-2.5% of polyoxyethylene, 0.05-0.1% of polybutylimidazole zinc acetate, 0.05-0.1% of polybutylimidazole iron acetate, 0.5-1.5% of polyethylene glycol, 0.05-0.2% of essence and 5-20% of ethanol according to mass concentration. Wherein, the poly-butyl-imidazole-acetic-acid-iron and poly-butyl-imidazole-zinc are synthesized by the method disclosed in the 2021 and 3 rd phase by referring to the preparation of poly-ionic liquid containing carboxylic acid imidazole salts with different chain lengths and the biological performance [ J ] biomedical engineering research. Wherein, schizophyllan compound polysaccharide is provided by examples 1-2.

To further verify the above ion-reduced transdermal cosmetic water, the cosmetic water prepared with the schizophyllan complex polysaccharide provided in examples 1 to 2 and comparative example 1 was respectively provided in this experimental example, and the schizophyllan complex polysaccharide therein was additionally replaced with fruit acid as comparative example 2.

To this end, example 1 provides a toning lotion which is a deionized solution comprising, in mass concentration, 12% glycerin, 0.5% sodium carboxymethylcellulose, 3.5% schizophyllan complex polysaccharide provided in example 1, 0.08% zinc polybutylimidazole acetate, 0.07% iron polybutylimidazole acetate, 1.0% polyethylene glycol, 0.05% perfume, and 15% ethanol. Example 2 provides a toning lotion which is a deionized solution comprising, in mass concentration, 12% glycerol, 0.5% sodium carboxymethylcellulose, 3.5% schizophyllan complex polysaccharide provided in example 2, 0.08% polybutylimidazole zinc acetate, 0.07% polybutylimidazole iron acetate, 1.0% polyethylene glycol, 0.05% perfume and 15% ethanol. Comparative example 1 provides a toning lotion which is a deionized solution comprising, in mass concentration, 12% glycerin, 0.5% sodium carboxymethylcellulose, 3.5% schizophyllan polysaccharide as provided in comparative example 1, 0.08% zinc polybutylimidazole acetate, 0.07% iron polybutylimidazole acetate, 1.0% polyethylene glycol, 0.05% perfume, and 15% ethanol. Comparative example 2 provides a toning lotion which is a deionized solution comprising, in mass concentration, 12% glycerin, 0.5% sodium carboxymethylcellulose, 3.5% fruit acid, 0.08% zinc polybutylimidazole acetate, 0.07% iron polybutylimidazole acetate, 1.0% polyethylene glycol, 0.05% essence and 15% ethanol.

2. Antibacterial properties of test article

The test was performed as described above. The results are shown in table 4, "×" indicates a significant difference (P < 0.05) from both comparative example 1 and comparative example 2, and "-" indicates no antimicrobial activity was detected. As is clear from Table 4, the lotions provided in examples 1 to 2 have bacteriostatic properties against gram-positive, gram-negative bacteria, and fungi of the type 10 described above, respectively, while the lotions provided in comparative examples 1 and 2 have no remarkable bacteriostatic effects against Streptococcus pneumoniae, salmonella enteritidis, kerobacter pneumophila, candida albicans, and Candida glabrata, and the lotions provided in examples 1 to 2 have lower MIC values. Therefore, the toning lotion provided by the embodiment of the application has broader antibacterial performance and stronger antibacterial performance.

TABLE 4 MIC of lotion (mg/mL)

Description of the embodiments	Example 1	Example 2	Comparative example 1	Comparative example 2
					Staphylococcus aureus	5.36±1.25*	5.01±0.87*	8.92±1.14	28.32±4.62
Coli bacterium	6.82±0.74*	6.37±0.85*	12.05±1.42	35.74±3.27
					Pseudomonas aeruginosa	5.18±0.63*	4.89±0.38*	10.52±1.69	－
Tubercle bacillus	18.68±2.34*	17.82±2.25*	38.45±5.81	－
					Candida albicans	8.12±0.74*	7.85±0.56*	24.63±3.55	－
Streptococcus pneumoniae	38.21±6.22	36.82±5.82	－	－
					Salmonella enteritidis	35.82±4.41	36.07±6.15	－	－
Bacillus pneumophilus	33.14±7.04	34.73±6.12	－	－
					Candida albicans	78.35±15.14	76.97±12.51	－	－
Candida glabrata (C.glabrata)	63.52±11.37	61.73±10.54	－	－

3. Zeta potential test

The Zeta potential test was performed on the lotions provided in examples 1 to 2 and comparative examples 1 to 2, respectively, using a laser particle sizer (ZEN 3600,MalvernInstruments Ltd, uk). The results are shown in table 5, "×" indicates that there was a significant difference (P < 0.05) relative to the lotions provided in comparative examples 1 and 2. As is clear from Table 5, the lotions provided in examples 1 and 2 were lower in Zeta potential and higher in reducibility than the lotions provided in comparative examples 1 and 2.

TABLE 5

Description of the embodiments	Zeta potential (mV)
		Example 1	-253.4±3.5*
Example 2	-261.1±2.2*
		Comparative example 1	-62.3±1.8
Comparative example 2	-34.6±0.6

4. Hemolysis test

In this experimental example, fresh human serum was collected and centrifuged at 1500rpm for 15min, and human blood erythrocytes were collected and washed with PBS until the supernatant became clear and transparent. The obtained red blood cells were diluted to 2vol% with PBS, the lotions provided in examples 1 to 2 and comparative examples 1 to 2 were added in amounts of 20v/v%, respectively, and after co-culturing for 3 hours at 30℃the cultured cell solutions were centrifuged at 1500rpm for 10 minutes, 100. Mu.L of the supernatant was added to a 96-well plate and the OD576 value was measured by a microplate reader, and the hemolysis rates of the human serum red blood cells of the lotions provided in examples 1 to 2 and comparative examples 1 to 2 were sequentially calculated, and PBS groups and Triton reagent groups were used as negative controls and positive controls, respectively. Hemolysis = (OD experimental group-OD negative group)/(OD positive group-OD negative group) ×100%. The results are shown in Table 6. As is clear from Table 6, the lotions provided in examples 1 to 2 and comparative examples 1 to 2 each had a very low hemolysis rate, indicating excellent biocompatibility.

TABLE 6

Description of the embodiments	Hemolysis rate (%)
		Example 1	0.02±0.01
Example 2	0.01±0.002
		Comparative example 1	0.03±0.01
Comparative example 2	0.03±0.01
		Positive group	100

5. Toxicity test

TABLE 7

Description of the embodiments	Relative growth rate of cells (%)
		Example 1	104.7±2.5
Example 2	106.2±4.1
		Comparative example 1	101.5±1.8
Comparative example 2	102.1±2.4

In this experimental example, 100. Mu.L of the toning lotion provided in each of examples 1 to 2 and comparative examples 1 to 2 was added to a 96-well plate, and mixed with 100. Mu.L of a human skin fibroblast suspension (10 ⁵ cells/mL, cat No. CL-465h, wuhansaios) and at 37℃with 5% CO ₂ Incubation was carried out for 70h with 100 μl of 1-fold volume diluted PBS as a blank; and 100. Mu.L of PBS solution containing 5g/L MTT is added into the mixed solution, the culture is continued for 4 hours at 37 ℃, after the supernatant is removed, 0.75mL of DMSO is dripped into the supernatant and mixed uniformly, the OD value at 490nm is measured by using an enzyme-labeled instrument, and the relative growth rate of cells is calculated as the relative growth rate of the cells is calculated as the OD experimental group/the OD control group multiplied by 100%. As shown in Table 7, the relative growth rates of cells of the lotions provided in examples 1 to 2 and comparative examples 1 to 2 were all over 100%, indicating that the lotions provided were non-cytotoxic and have excellent biocompatibility.

Animal test

1. In vitro transdermal test

Healthy BALB/c mice (Beijing Hui Zhi and Source biotechnology Co., ltd.) were carefully shaved off hairs on the abdominal skin with scissors, sacrificed by neck breakage, and the abdominal skin was peeled off. Spreading the peeled skin on a glass plate, removing subcutaneous fat and adhesion, repeatedly washing with 0.9% sodium chloride solution, shearing to proper size, and maintaining skin integrity. The skin tissue is fixed between the upper and lower chambers of the diffusion cell, the horny layer is upward, the receiving liquid is 0.9% sodium chloride solution, the total transfer body of the cistanche phenylethanol or 1mL of total aqueous solution of the cistanche phenylethanol is added into the supply cell, and the cell opening is covered by a preservative film. Placing the transdermal absorption and diffusion device in a constant-temperature water bath at 37deg.C, stirring at 200rpm, timing, sampling 3mL at 1,2, 4,6, 8, 10, 12 and 24 hr respectively, simultaneously supplementing fresh receiving liquid with the same temperature and the same volume, detecting the content of related components in the sample solution, and calculating cumulative permeation according to the following formula

Wherein Cn is the nth smallWhen the concentration in the receiving tank is higher than the concentration in the receiving tank, V is the sampling volume, and V0 is the volume of the receiving tank; and (3) drawing a graph of Qn to t, performing linear regression, and solving the slope of a straight line to obtain the steady-state permeation rate Jss. />

In addition, the concentrations of sodium carboxymethylcellulose (CMC), schizophyllan complex polysaccharide (SPGHA), polybutylimidazole zinc acetate (PBMA-Zn) and polybutylimidazole iron acetate (PBMA-Fe) in the receiving cells were examined in this experimental example. Wherein, the concentration detection of sodium carboxymethyl cellulose is carried out by referring to the method of qualitative detection of sodium carboxymethyl cellulose in aquatic products [ J ] Guangzhou chemical industry, 2017, 3 rd phase, the detection of the content of schizophyllan compound polysaccharide is carried out by referring to the method of detecting yeast beta-glucan by Congo red method, the method of researching [ J ] food and fermentation industry, 2022, 9 th phase, the content detection of polybutyl imidazole zinc acetate and polybutyl imidazole iron acetate is carried out by referring to the method of solid phase extraction-high performance liquid chromatography for measuring byproducts 2-methylimidazole, 4-methylimidazole and 2-acetyl-4- (1, 2,3, 4-tetrahydroxybutyl) imidazole [ J ] analytical chemistry, 2015-05-15.

The transdermal results of the lotions provided in examples 1-2 and comparative examples 1-2, respectively, on the skin of mice are shown in table 8, which shows the cumulative permeation quantity Qn and steady state permeation rate Jss for 24 hours, "-" in table 8 indicates undetected, ".+ -. Indicates that both the mark of significant difference and the schizophyllan complex polysaccharide have higher cumulative permeation quantity and steady state permeation rate on the skin of mice in the skin tissue of mice as compared with comparative examples 1 and 2, while the polybutyl imidazole zinc acetate and polybutyl imidazole iron acetate have no significant permeation effect, so that the effects on other tissues and cells below the skin tissue can be reduced.

TABLE 8

2. Skin acute toxicity test and moisturizing test

Healthy BALB/c mice (Beijing Hui Zhi and Source biotechnology Co., ltd.) were set with acute toxicity groups, male and female halves, and the hair on the backs of the mice were removed with a dehairing knife. To be used forThe mice without skin injury and skin disease in the process of removing are taken as experimental objects, and the corresponding areas with the size of 2cm multiplied by 2cm are selected from the back for test according to the ratio of 5mL/cm respectively ² Two doses of the cosmetic water test pieces provided in examples 1 to 2 and comparative examples 1 to 2, respectively, were applied to this area. During the experiment, the mice were observed for skin poisoning and death by free diet once daily for two consecutive weeks.

Meanwhile, the mice of the oral group are respectively provided with male and female mice, after the mice are fasted and not forbidden for 12 hours before the oral acute toxicity test, the toning water provided in the examples 1-2 and the comparative examples 1-2 are respectively given as test products according to the weight of 5mL/Kg, the oral group is subjected to one-time gastric lavage administration, the oral group is fed after 2 hours of gastric lavage, the oral group is subjected to one-time daily continuous gastric lavage for two weeks, and the poisoning and death conditions of the mice are observed and recorded.

The skin-smeared mice were used as test groups, and a blank group and a positive group were additionally set. The blank group was applied with distilled water without any treatment, the positive control group was applied with the purchased cosmetic water product containing hyaluronic acid once daily, continuously applied for one week, and after the last administration for 6 hours, the skin moisture content was measured. The water content detection method comprises the following steps: immediately after killing the mice, shearing the middle smearing area of the mice, removing fat after annular skin taking, firstly weighing the wet weights of each group of dissected mice skin respectively, then spreading the mice skin on a glass plate smoothly, and drying the mice skin by a constant-temperature drying oven at the temperature of 80 ℃ for 12 hours; finally, weighing the weight of each group of dried mouse skin again, and calculating the change of the water content of the mouse skin before and after drying by a differential method. Skin moisture (%) = (wet weight-dry weight)/wet weight x 100%

The results show that through mice skin smearing or oral acute toxicity test, diet is normal, skin growth is good, obvious poisoning phenomenon does not appear after stomach irrigation, activity is reduced, but no death cases appear, and the fact that the toning lotion provided by the embodiment of the application has no acute toxic or side effect is shown. The skin moisture content is shown in table 9, which performs multiple comparisons and significance differential markers on the column data. As is clear from table 9, the lotions provided in examples 1 and 2 each had a better moisturizing effect.

TABLE 9

Description of the embodiments	Skin moisture content (%)
		Example 1	75.2±2.5a
Example 2	78.5±3.2a
		Comparative example 1	62.9±2.5b
Comparative example 2	62.6±2.2b
		Blank group	59.3±1.8c
Positive group	60.5±2.7c

In summary, in the embodiment of the application, schizophyllan is used for fermentation, glucose and aloe juice are added into a culture medium of the schizophyllan, and the schizophyllan compound polysaccharide is obtained through structural analysis, has a structure formed by polymerization of beta-pyran glucan and hyaluronic acid, and has broad-spectrum antibacterial performance in vitro.

The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Claims

1. A method for preparing schizophyllan compound polysaccharide, which comprises the following steps:

obtaining an activation solution of schizophyllum commune; wherein the schizophyllum commune is of academic nameSchizophyllum commune Fr.Purchased from testuo organism, cat No. TS303024;

inoculating the activation solution into a working seed solution, and culturing on a shaking table to obtain the working seed solution, wherein the working seed solution comprises 15wt% of glucose, 0.8wt% of yeast powder, 0.2% of ammonium nitrate and K ₂ HPO ₄ 0.2%、MgSO ₄ 0.1%, tween-80 0.03v/v% and aloe juice 0.02-0.05 v/v%;

inoculating the working seed liquid into a fermentation culture medium, controlling ventilation and stirring speed at 28 ℃, and culturing to obtain a fermentation liquid, wherein the fermentation culture medium comprises 10wt% of glucose, 0.65wt% of yeast powder and 0.18% of ammonium nitrate, and K ₂ HPO ₄ 0.18%、MgSO ₄ 0.08%, tween-80 0.02v/v% and aloe juice 0.1-0.25 v/v%;

2. The method according to claim 1, wherein the working seed liquid has an extracellular polysaccharide content of not less than 40g/L and a mycelium biomass of not less than 90g/L.

3. The process according to claim 2, wherein the fermentation broth has an extracellular polysaccharide content of not less than 27g/L and a mycelium biomass of not less than 55g/L.

4. The method of claim 1, wherein the step of extracting the fermentation broth comprises:

adding 0.6v/m% active carbon into the fermentation broth, heating and stirring for 45min, filtering to obtain filtrate, adjusting pH=4, adding pepsin, preheating in a water bath kettle at 37 ℃ for 10min, oscillating and holding for 30min, inactivating enzyme in a water bath kettle at 100 ℃ for 10min, cooling to room temperature, adding 2.5 times volume of absolute ethyl alcohol, generating a large amount of white flocculent precipitate, shaking, standing at 4 ℃ overnight, centrifuging for 20min at 6000r/min, collecting precipitate, and freeze-drying to obtain crude schizophyllan compound polysaccharide.

5. The preparation method of claim 4, wherein the crude product is dissolved by PBS with pH of 6.8 and then is statically loaded into a chromatographic column filled with Sephadex G-200, the chromatographic column is eluted by a sodium chloride solution with the concentration of 0.8%, the elution flow rate is 0.3mL/min, the concentration is collected step by step, 2mL is collected by each tube, the sugar content in each tube is detected by adopting a phenol-sulfuric acid method, an elution curve is drawn according to the sugar content concentration corresponding to the management number, the eluent corresponding to the elution peak is collected, the concentration is reduced, and the pure schizophyllan compound polysaccharide is obtained after freeze drying.

6. A schizophyllan compound polysaccharide prepared by the preparation method of any one of claims 1 to 5, wherein the schizophyllan compound polysaccharide has a structure formed by polymerization of schizophyllan and hyaluronic acid.

7. An ion-reduced transdermal toning lotion which comprises, by mass, 5-12% of glycerol, 0.15-0.5% of sodium carboxymethylcellulose, 0.5-4.5% of schizophyllan compound polysaccharide according to claim 6, 1.0-2.5% of polyoxyethylene, 0.05-0.1% of zinc polybutylimidazole acetate, 0.05-0.1% of iron polybutylimidazole acetate, 0.5-1.5% of polyethylene glycol, 0.05-0.2% of essence and 5-20% of ethanol.

8. The toning lotion according to claim 7, which is a deionized solution comprising, in mass concentration, 12% glycerol, 0.5% sodium carboxymethyl cellulose, 3.5% schizophyllan complex polysaccharide according to claim 6, 0.08% polybutylimidazole zinc acetate, 0.07% polybutylimidazole iron acetate, 1.0% polyethylene glycol, 0.05% essence, and 15% ethanol.

9. The method for preparing the schizophyllan compound polysaccharide according to any one of claims 1 to 5 or the application of the schizophyllan compound polysaccharide according to claim 6 in preparing cosmetics.

10. The use according to claim 9, wherein the cosmetic product is a toning lotion.