CN116059138A - Soxhlet Luo Jinxiao Chlorella with antioxidant activity and its application in cosmetics - Google Patents

Abstract

The invention discloses chlorella sorokinus with antioxidant activity and its application in cosmetics. The present invention isolates and identifies a strain of Chlorella sorokini, and its microorganism preservation number is: CCTCC M 20221450. The water extract of chlorella sorokinus provided by the invention is rich in active ingredients such as polysaccharides and growth factors, has a good scavenging effect on DPPH, and has outstanding antioxidation ability in vitro. After 100-fold dilution of the facial mask essence prepared with Sorokin chlorella water extract, the DPPH clearance rate exceeds 90%, with strong antioxidant capacity and good skin care effect. Not only does it contain the active substances of chlorella, but it also contains antioxidant and whitening plant natural active skin care ingredients such as vitamin C, niacinamide, arbutin, and rose hydrosol. The compounding of various ingredients works synergistically, and the skin is moisturized after use Delicate, elastic, good skin care effect, not only has outstanding anti-oxidation ability, but also has effects of radiation protection and skin repair.

Description

Chlorella sorokinensis with antioxidant activity and its application in cosmetics

technical field

The invention relates to chlorella (Chlorella), in particular to chlorella sorokiniana (Chlorella sorokiniana) with antioxidant activity and its application in cosmetics, belonging to the field of chlorella and its application.

Background technique

Chlorella is an ancient single-celled eukaryotic green algae, which originated about 500 million years ago and survived five mass extinctions on the earth. It has wide adaptability and good stress resistance. It has great development value.

Chlorella is rich in active ingredients such as growth factor (CGF) and polysaccharides. It has the functions of nourishing the skin, replenishing water and moisturizing, and can be applied to cosmetics. In particular, the unique CGF growth factor of chlorella, also known as "chlorella essence", is a water-soluble mixture, including proteins, amino acids, nucleotides, polysaccharides, vitamins, etc., with contents of 56.6%, 42.59%, 6.8%, 4.8%, 1.7%, etc., have strong antioxidant activity and can be applied to skin care products to promote growth, anti-aging, repair tissue, activate cells, etc., through hot water extraction, Active substances including growth factors can be obtained; however, the existing chlorella's antioxidant capacity is not significant enough, and the skin care effect applied to cosmetics is not good, and needs to be improved.

Therefore, screening out chlorella algae strains with higher antioxidant activity and applying them to the preparation of cosmetics with significant skin care effects will have important application value.

Contents of the invention

One of the objects of the present invention is to provide a Chlorella sorokiniana (Chlorellasorokiniana) strain with high antioxidant activity;

The second object of the present invention is to apply the described Chlorella sorogenus to the preparation of cosmetic and skin care products;

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention isolates and identifies a new Chlorella sorokiniana strain (Chlorella sorokiniana) BZ942 from a paddy field soil sample in Nanjiang County, Bazhong City, Sichuan Province. Its microbial preservation number is: CCTCC NO: M 20221450, and its classification name is: Chlorella sorokiniana BZ942, preserved in the China Center for Type Culture Collection, the preservation address is: Wuhan University, Wuhan, China, and the preservation time is: September 20, 2022.

The chlorella sorokinus strain BZ942 strain isolated by the present invention is green and spherical single cell under a microscope, has a protein nucleus, and the cell diameter is about 5 microns.

The nucleotide sequence of the ITS gene of the Chlorella sorokinensis strain BZ942 is SEQ ID No.1), after sequencing, through Blastn comparison, it is found that BZ942 is similar to the ITS gene of Chlorella sorokinii, and the consistency reaches 90.4 %; the nucleotide sequence of the 18s rRNA gene of the Chlorella sorokines algal strain BZ942 is shown in SEQ ID No.2, after Blastn comparison, it is found that BZ942 is close to the ribosomal RNA gene of Chlorella sorokines, Consistency reaches 97.9%; The nucleotide sequence of the COI gene (mitochondrial cytochrome C oxidase I gene) of the described Chlorella sorokini algal strain BZ942 is shown in SEQ ID No.3, after Blastn comparison, it is found that BZ942 The phylogenetic relationship with the mitochondrial COI gene of Chlorella sorokinensis is similar, with a consistency of 98.67%. Based on the COI gene, the phylogenetic tree analysis shows that BZ942 is aggregated in the Chlorella sorokinensis branch.

The invention provides a water extract of Chlorella sorokini BZ942, the preparation method of which comprises: (1) aseptically fermenting and culturing the Chlorella sorokini BZ942 to obtain the algae liquid; (2) centrifuging the algae liquid, and taking Precipitate and dry to obtain algae powder; (3) mix water and algae powder to obtain a mixed solution; (4) treat the mixed solution with ultrasound and then incubate at a high temperature of 80-100°C; (5) centrifuge, collect the supernatant, filter, Obtain the Sorokin chlorella water extract.

Preferably, in step (1), Chlorella sorokini BZ942 is cultured to obtain the algae liquid through aseptic fermentation of BG11 medium for 1-6 days; preferably, the centrifugal speed described in step (2) is 13000rpm/min, The centrifugation time is 10min; preferably, in step (3), water and algae powder are mixed according to the ratio of 40mL: 1g; preferably, in step (4), ultrasonic crushing with 50% power is used for 10min and then incubated at 95°C 30min; preferably, the centrifugation described in step (5) is 10000rpm/min centrifugation for 10min; the described filtration adopts 0.22μm membrane filtration.

The water extract of Chlorella sorokinensis BZ942 provided by the present invention is rich in active ingredients such as polysaccharides and growth factors, and has a good scavenging effect on DPPH, and the DPPH clearance rate of the chlorella water extract of 5 mg/mL reaches 70%. The anti-oxidation ability is outstanding, and the chlorella mask essence prepared by using it has excellent quality, strong anti-oxidation ability, and good skin care effect.

The present invention further provides a facial mask essence containing Chlorella sorogenus BZ942 water extract, which consists of Chlorella sorogenus water extract, squalane, vitamin C, nicotinamide, arbutin, rose hydrosol, glycerin, Propylene glycol, xanthan gum, sodium alginate, carbomer, triethanolamine, potassium sorbate and water; preferably, the percentages of each component are: 5% to 10% of Sorokin chlorella water extract, squalane 1%～6%, vitamin C 0.2%～0.6%, niacinamide 0.1%～0.5%, arbutin 0.3%～0.9%, rose hydrosol 2%～10%, glycerin 2%～6%, propylene glycol 2% ～6%, xanthan gum 0.01%～0.08%, sodium alginate 0.1%～0.5%, carbomer 0.1%～0.5%, triethanolamine 0.01%～0.05%, potassium sorbate 0.2%～0.8%, water 70% %～80%; preferably, the percentage of each component is: Sorokin Chlorella water extract 3%, squalane 3%, vitamin C 0.2%, nicotinamide 0.5%, arbutin 0.5%, rose hydrosol 5%, glycerin 5%, propylene glycol 2%, xanthan gum 0.05%, sodium alginate 0.3%, carbomer 0.2%, triethanolamine 0.05%, potassium sorbate 0.5%, water 79.7%.

The present invention further provides a method for preparing the mask essence containing Chlorella sorokinensis BZ942 water extract, comprising: (1) mixing xanthan gum, carbomer, sodium alginate and water at 85° C. to 90° C. Heat at ℃ and stir until there is no white solid; (2) Add glycerin, propylene glycol and triethanolamine and stir evenly, stop heating, and cool to below 45°C; 3) Add Sorokin chlorella water extract, potassium sorbate, squalane , vitamin C, niacinamide, arbutin and rose hydrosol are stirred evenly, emulsified by an emulsifier to obtain the primary product of the facial mask liquid, and left to stand for 2-3 days; 4) After the primary product of the facial mask liquid is sterilized by radiation, it can be packed in Form the finished product of chlorella mask essence, which can be used with mask paper.

The chlorella sorogenus BZ942 facial mask essence provided by the invention has a DPPH clearance rate of more than 90% after being diluted 100 times, has strong antioxidant capacity and good skin care effect. Its viscosity is moderate, it is translucent colloid, contains a touch of rose fragrance, mild and non-irritating, and has a significant antioxidant effect. It not only contains the active substance of chlorella BZ942, but also contains vitamin C, niacinamide, arbutin, rose pure Antioxidant and whitening plant natural active skin care ingredients such as dew, compounded with various ingredients, synergistically, after use, the skin is moist and delicate, elastic, and the skin care effect is good. It not only has outstanding anti-oxidation ability, but also has radiation protection, skin repair, etc. Efficacy, can be applied to sunscreen, hand cream, toner, body milk and other cosmetics.

Description of drawings

Figure 1 shows the culture solution of Chlorella sorokinus BZ942 and its observation under a microscope; Note: a represents the cultured Chlorella BZ942 liquid, and b represents the morphology of Chlorella BZ942 under the microscope objective lens 40x.

Fig. 2 shows the PCR method to amplify the ITS gene of Chlorella sorokinus BZ942 and identify its species.

Fig. 3 is the PCR method to amplify the 18s rRNA gene of Chlorella sorokinus BZ942, and identify its species.

Fig. 4 is a PCR method to amplify the COI gene of Chlorella sorokinus BZ942 and identify its species.

Fig. 5 is a phylogenetic tree diagram of Chlorella sorokines BZ942 strain.

Figure 6 shows the chlorella facial mask essence prepared by using the water extract of Chlorella sorogenus BZ942; Note: a represents the active substance prepared by using chlorella BZ942, and b represents the chlorella facial mask essence.

Fig. 7 is a flow chart of preparing facial mask essence by using Chlorella sorokinus BZ942.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Preliminary Example 1 Primer Design for Molecular Identification of Chlorella Species

The upstream and downstream primers were designed according to the ITS, 18s rRNA, and mitochondrial COI genes of Chlorella for molecular identification of Chlorella species. The sequences of gene amplification primers are as follows:

ITS-F: 5'-GGCAATAACAGGTCTGT-3'

ITS-R:5'-CCTCACGGTACTTGTTC-3'

18s rRNA-F: 5'-ATTGGAGGGCAAGTCTGGTG-3'

18s rRNA-R: 5'-TGATCCTTCTGCAGGTTCACCTACG-3'

COI-F: 5'-ATGTTGACTCGTTGGTTTTTATT-3'

COI-R: 5'-CAGAATAAGTGTTGGTAAAGAAT-3'.

Example 1 Separation, purification, screening and identification of Chlorella sorokinus BZ942

Select paddy field water samples collected from Nanjiang County, Bazhong City, Sichuan Province (106.86°E, 32.14°N) in the field, dilute the water samples 1000 times with sterile water, take 200ul diluted liquid, and evenly spread on 50mg /L kanamycin, 100mg/L cephalosporin on BG11 solid medium, cultured upside down in a light incubator (28°C, 12h light/12h dark), after culturing for about 5 days, observe whether green spots appear on the plate Monoclonal algae strains; after green monoclonal algae strains appear, use a sterile pipette tip to pick out a single clone, and mark on the BG11 medium containing 50mg/L kanamycin and 100mg/L cephalosporin antibiotics Then put it in the light incubator and continue to cultivate for about 5 days to observe whether there are monoclonal algae strains, and there are no bacteria on the medium. If there are still bacteria, continue to draw lines according to the above method to purify the algae strains; When the monoclonal algae strains are free of miscellaneous bacteria, select the monoclonal algae strains and place them in BG11 liquid medium containing 50mg/L kanamycin and 100mg/L cephalosporin, and shake them in a light shaking incubator (28°C , 12h light/12h dark, 200rpm/min), the algae liquid was obtained, which was named BZ942.

Take the algae liquid BZ942 and observe it under the optical microscope. Under the 40x magnification of the objective lens, it is observed that the shape of the algae strain is green, spherical, single-celled, contains 1 protein nucleus, and the cell diameter is about 5 microns. The shape is similar to Chlorella (Fig. 1 ). Take 0.5 mL of algae liquid BZ942 (OD680>0.5) in a 2 mL centrifuge tube, centrifuge at 10,000 rpm/min for 1 min, discard the culture liquid, add grinding steel balls, and grind for 6 min on a tissue grinder at a vibration frequency of 40 Hz, then add no 50 μL of bacterial water was centrifuged at 10,000 rpm/min for 1 min, and 1 μL of the supernatant was taken as a template. Chlorella ITS-F+ITS-R, 18srRNA-F+18s rRNA-R, COI-F+COI-R were used as primers for PCR amplification. Take 10 μL of the PCR product and observe it by gel imaging after electrophoresis; the remaining PCR product is sent for sequencing, and the sequencing primer is the corresponding ITS-F, 18s rRNA-F or COI-F. The sequencing results were processed through the reverse complement online tool (http://www.detaibio.com/sms2/rev_comp.htmL), and the reverse complement sequence was obtained, and the sequence was passed through the NCBI online tool BLAST (https://blast.ncbi .nlm.nih.gov/Blast.cgi) for sequence alignment to find homologous genes and analyze their species relationship.

PCR was used to amplify the ITS gene of microalgae (SEQ ID No.1), and after sequencing, Blastn comparison revealed that BZ942 was similar to the ITS gene of Chlorella sorokini, with a consistency of 90.4% (Figure 2).

PCR was used to amplify the microalgae 18s rRNA gene (SEQ ID No.2), and after sequencing, Blastn comparison revealed that BZ942 had a close relationship with the ribosomal RNA gene of Chlorella sorokini, with a consistency of 97.9% (Figure 3).

PCR was used to amplify the microalgae COI gene (SEQ ID No.3), and after sequencing, Blastn comparison revealed that BZ942 was closely related to the mitochondrial COI gene of Chlorella sorokinus, with a consistency of 98.67% (Figure 4). Based on the COI gene, the phylogenetic tree analysis was carried out, and it was found that BZ942 was gathered in the Sorokin Chlorella branch (Figure 5)

To sum up, in this example, a Chlorella sorokiniana strain (Chlorella sorokiniana) was identified in combination with microscope observation, gene sequencing and sequence analysis, named Chlorella sorokiniana BZ942, and was preserved in the China Center for Type Culture Collection with the preservation number CCTCC M 20221450.

The extraction of embodiment 2 Chlorella sorokinus active ingredient

Apply the chlorella BZ942 identified in Example 1, through BG11 medium for aseptic fermentation and culture for 3 days, to obtain the algae liquid, centrifuge in a centrifuge (13000rpm/min x 10min), and dry (120°C) with a spray dryer to obtain chlorella Algae powder of BZ942. Using algae powder as raw material and water as solvent, weigh 5g of algae powder and 200mL of water. After mixing evenly, it is ultrasonically crushed by a cell ultrasonic breaker at 50% power for 10 minutes, then heated in a water bath at 95°C for 30 minutes, and the algae liquid is centrifuged. Centrifuge at 10000rpm/min x 10min, collect the supernatant, and finally filter and sterilize through a 0.22μm filter membrane to obtain the water extract of Chlorella BZ942.

Example 3 Preparation of Chlorella Facial Mask Essence by Using Sorokin Chlorella Active Components

Using the chlorella BZ942 water extract prepared in Example 2, prepare 1000g of chlorella mask essence according to the following steps.

Step 1: In the clean workshop, weigh the following into a beaker: 0.5g xanthan gum, 2g carbomer, 3g sodium alginate, 797g water, heat at 85°C to 90°C and stir evenly until no white solid appears;

Step 2: Add 50g of glycerin, 20g of propylene glycol, and 0.5g of triethanolamine to the above beaker, stir evenly, stop heating, and cool to below 45°C;

In the third step, add 30g of chlorella water extract, 5g of potassium sorbate, 30g of squalane, 2g of vitamin C, 5g of nicotinamide, 5g of arbutin, and 50g of rose hydrosol, stir evenly, and emulsify through an emulsifier to obtain The first product of mask liquid, let it stand for 2-3 days;

The fourth step is to sterilize the primary product of the facial mask solution by radiation, and then pack it into a can to form a finished product of chlorella facial mask essence, which is used together with facial mask paper.

The prepared chlorella active water extract and chlorella mask essence are shown in Figure 6.

Test example 1 Antioxidant ability evaluation test of the chlorella mask essence prepared by Sorokin chlorella active ingredient

Get the facial mask essence 1mL prepared in Example 3, dilute 100 times with sterile water, take vitamin C as positive control, utilize DPPH free radical scavenging ability detection kit, measure the antioxidant capacity of chlorella facial mask essence.

Based on the QB/T 2872-2017 facial mask standard, the sensory and physical and chemical indicators of the facial mask were measured; 30 people were randomly selected to use the facial mask essence once a night for 1 consecutive week for evaluation.

The antioxidant evaluation results of Sorokin Chlorella BZ942 are shown in Table 1 and Table 2.

DPPH clearance rate of different samples in table 1

sample concentration DPPH clearance Sorokin Chlorella BZ942 water extract (embodiment 2) 5mg/mL 70% Sorokin chlorella mask essence finished product (embodiment 3) 100 times dilution 98% Vitamin C 0.1mg/mL 48%

Table 2 Sensory and Physicochemical Evaluation of Sorokin Chlorella Mask Essence

As can be seen from Table 1 and Figure 6a, the water extract of Chlorella sorokini BZ942 is a light green clear liquid (Figure 6a), which has a good DPPH free radical scavenging ability, wherein 5mg/mL of Chlorella sorokini BZ942 The DPPH clearance rate of the water extract is 70%, and the antioxidant effect is good.

As can be seen from Table 1, Table 2, and Figure 6, Chlorella sorogenus BZ942 is used to prepare the chlorella essence mask liquid, which is translucent colloidal, has a natural rose fragrance, is stable in properties, and has good The heat resistance and cold resistance of the mask essence were evaluated by a third party after use, and subjectively reflected that the mask essence was of excellent quality (Table 2, Figure 6b); the antioxidant capacity of the mask essence was evaluated, and it was found that after 100-fold dilution, it still had significant Antioxidant capacity, DPPH scavenging rate exceeded 90% (Table 1).

In conclusion, the facial mask essence prepared by Chlorella sorokinensis BZ942 has excellent comprehensive performance, good anti-oxidation effect, and good skin care effect.

Claims (10)

1. A strain of chlorella (Chlorella sorokiniana) of cable Luo Jinxiao, characterized by the microorganism deposit number: cctccc M20221450.

2. Use of the strain of chlorella so Luo Jinxiao of claim 1 in the preparation of a cosmetic skin care product; preferably, the cosmetic skin care product comprises a facial mask, a sun cream, a hand cream, a toner or body milk.

3. The use according to claim 2, wherein the cosmetic skin care product has antioxidant effect.

4. A water extract obtained by extracting the Chlorella strain of claim 1 with water as an extraction solvent.

5. Use of the aqueous extract of claim 4 in the preparation of a cosmetic skin care product; preferably, the cosmetic skin care product comprises a facial mask, a sun cream, a hand cream, a toner or body milk.

6. A method of preparing the aqueous extract of claim 4, comprising: (1) Culturing the chlorella strain of Luo Jinxiao in accordance with claim 1 by aseptic fermentation of a culture medium to obtain an algae liquid; (2) centrifuging the algae liquid, taking the precipitate and drying to obtain algae powder; (3) mixing water with algae powder to obtain a mixed solution; (4) Treating the mixed solution by ultrasonic treatment, and then incubating at a high temperature of 80-100 ℃; (5) Centrifuging, collecting supernatant, and filtering to obtain the Soxhlet Luo Jinxiao Chlorella water extract.

7. The method according to claim 6, wherein in the step (1), the chlorella strain of the cable Luo Jinxiao is cultured by the BG11 medium for 1-6 days to obtain an algae liquid; the centrifugation in the step (2) is that the centrifugal speed is 13000rpm/min and the centrifugation time is 10min; in the step (3), water and algae powder are mixed according to 40mL: mixing at a ratio of 1 g; in the step (4), 50% power ultrasonic crushing is adopted for 10min, and then high-temperature incubation is carried out for 30min at 95 ℃; the centrifugation in the step (5) is 10000rpm/min for 10min; the filtration is carried out by adopting a 0.22 mu m filter membrane.

8. A facial mask essence is characterized by comprising the water extract, squalane, vitamin C, nicotinamide, arbutin, rose hydrosol, glycerol, propylene glycol, xanthan gum, sodium alginate, carbomer, triethanolamine, potassium sorbate and water according to claim 4.

9. The facial mask essence according to claim 8, wherein the percentage of each component is: the water extract of claim 4, wherein the water extract comprises 5-10% of squalane 1-6%, 0.2-0.6% of vitamin C, 0.1-0.5% of nicotinamide, 0.3-0.9% of arbutin, 2-10% of rose hydrosol, 2-6% of glycerin, 2-6% of propylene glycol, 0.01-0.08% of xanthan gum, 0.1-0.5% of sodium alginate, 0.1-0.5% of carbomer, 0.01-0.05% of triethanolamine, 0.2-0.8% of potassium sorbate and 70-80% of water; preferably, the percentages of the components are: the aqueous extract of claim 4, wherein the aqueous extract comprises 3% of squalane, 3% of vitamin C, 0.2% of nicotinamide, 0.5% of arbutin, 5% of rose hydrosol, 5% of glycerin, 2% of propylene glycol, 0.05% of xanthan gum, 0.3% of sodium alginate, 0.2% of carbomer, 0.05% of triethanolamine, 0.5% of potassium sorbate and 79.7% of water.

10. A method of preparing the facial mask concentrate of claim 8 or 9, comprising: (1) Mixing xanthan gum, carbomer, sodium alginate and water, heating and stirring uniformly; (2) Adding glycerol, propylene glycol and triethanolamine, stirring, stopping heating, and cooling; 3) Adding the water extract, potassium sorbate, squalane, vitamin C, nicotinamide, arbutin and rose hydrosol according to claim 4, stirring uniformly, emulsifying by an emulsifying machine to obtain a mask liquid primary product, and standing; 4) Sterilizing and canning the primary product of the mask liquid to obtain a finished product of chlorella mask essence.

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