Essence containing chlorella extract and preparation method thereof

Abstract

The invention provides essence containing a chlorella extract and a preparation method of the essence. A technology of the chlorella extract comprises the following steps: crushing raw materials; performing enzymolysis with cellulase and trypsin; bacillus natto and bacillus pumilus are mixed for primary fermentation; according to the chlorella extract disclosed by the invention, the bamboo leaf extract, the corn steep liquor and the cane sugar are added, the rhodotorula mucilaginosa is inoculated for secondary fermentation, then the chlorella extract is obtained, multi-strain mixed fermentation has a synergistic effect, and compared with single-strain fermentation, more natural products can be enriched in the fermentation process. In addition, the invention further provides essence prepared from the chlorella extract, glycerin, sodium hyaluronate, honey and water, and the essence has remarkable moisturizing, anti-oxidation and soothing effects.

Description

Technical Field

[0001] The invention belongs to the field of cosmetic preparation, and particularly relates to an essence containing chlorella extract and a preparation method thereof. Background Art

[0002] As a microalgae rich in protein, vitamins, minerals and bioactive substances, Chlorella has been widely studied and applied in the food, pharmaceutical and cosmetic industries. However, the traditional Chlorella extraction process has problems such as low efficiency, high cost and insufficient retention of active ingredients.

[0003] At present, the extraction methods of Chlorella mainly include physical methods and chemical methods. Physical methods such as mechanical crushing and ultrasonic crushing can maintain the active ingredients, but the extraction efficiency is low. Chemical methods such as organic solvent extraction, although the extraction efficiency is high, may introduce solvent residues and affect product safety. In addition, the existing extraction process often ignores the microbial fermentation link, which can significantly improve the release and conversion of active ingredients in Chlorella. Summary of the invention

[0004] The invention provides an essence containing chlorella extract and a preparation method thereof, which plays the role of skin anti-oxidation, moisturizing and soothing through natural ingredients extracted from plants.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for extracting a Chlorella extract, and the specific preparation steps are as follows:

[0007] S1: washing, removing impurities, drying to constant weight, breaking the cell wall and crushing the chlorella to obtain product A1;

[0008] S2: A1 is enzymatically hydrolyzed using cellulase and trypsin to obtain enzymatic hydrolysis product A2;

[0009] S3: Inoculate Bacillus natto and Bacillus pumilus into A2, ferment for 4-6 hours, and then sterilize and filter to obtain filtrate A3;

[0010] S4: adding bamboo leaf extract, corn steep liquor and sucrose to A3, stirring evenly, inoculating red yeast, fermenting for 3-4 hours, and then ultrasonically crushing and filtering to obtain filtrate A4;

[0011] S5: A4 is freeze-dried to obtain the final product A5.

[0012] The amount of cellulase and trypsin added in S2 is 1-3wt% of A1, the mass ratio of cellulase to trypsin is 1:0.4-0.7, and the enzymolysis is carried out for 30-60 minutes.

[0013] The added amount of Bacillus natto and Bacillus pumilus in S3 is 3-5wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.5-0.8; the fermentation starting temperature is 32-34°C, and the fermentation starting pH is 7±0.5.

[0014] The total amount of bamboo leaf extract, corn syrup and sucrose added in S4 is 10-15wt% of A3, and the mass ratio of bamboo leaf extract, corn syrup and sucrose is 1:0.7:0.1-0.3; the amount of red yeast added is 2-3wt% of A3; the fermentation starting temperature is 29±1°C, and the fermentation starting pH is 6.5±0.5.

[0015] In a second aspect, the present invention provides an essence composition, the specific components of which are as follows:

[0016] Chlorella extract 1-5wt%;

[0017] Glycerol 20-25wt%;

[0018] Sodium hyaluronate 10-12wt%;

[0019] Honey 1-5wt%;

[0020] The balance was made up with sterile deionized water.

[0021] In the present invention, three different bacteria are used to ferment Chlorella in a pioneering way. First, Bacillus natto and Bacillus pumilus are used for fermentation, and then bamboo leaf extract, corn syrup and sucrose are added to the fermentation product, and then red yeast is used for fermentation again, and then the final product is obtained. In the first fermentation (i.e., fermentation with Bacillus natto and Bacillus pumilus), the intervention of Bacillus pumilus can effectively enrich the products of Bacillus natto such as nattokinase, polymyxin and bacitracin; in the second fermentation (i.e., fermentation with red yeast), the red yeast is induced to selectively secrete astaxanthin, carotenoids and B vitamins by the preparation of the base raw materials, and the experiment found that the secretion of astaxanthin, carotenoids and B vitamins by red yeast can be effectively improved by adding a specific proportion of the base raw materials. The results show that the Chlorella extract obtained by the first fermentation and the second fermentation of Chlorella has significant antioxidant, moisturizing and soothing effects.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: the extract obtained by fermenting and extracting Chlorella with Bacillus natto, Bacillus pumilus and Rhodotorula glutinosus has significant antioxidant, moisturizing and soothing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1: The cumulative percentage of single-bacteria fermentation products to the cumulative percentage of mixed-bacteria fermentation products.

[0024] Among them, the secretion amount of each product of the mixed fermentation of Bacillus natto and Bacillus pumilus was used as the positive control, and the secretion amount of each product of the single bacteria fermentation was mapped as the percentage of the secretion amount of each product of the mixed fermentation of Bacillus natto and Bacillus pumilus; the fermentation experimental steps and the amount of each component were the same as in Example 1, and the detection of various secretions was carried out by conventional laboratory means or kits.

[0025] Figure 2 : The cumulative percentage of fermentation products without adding (corn syrup, bamboo leaf extract, sucrose) to the cumulative percentage of fermentation products with adding (corn syrup, bamboo leaf extract, sucrose).

[0026] Note: With the addition of (corn syrup, bamboo leaf extract, sucrose) as the positive control, the percentage of fermentation products without (corn syrup, bamboo leaf extract, sucrose) was mapped to the percentage of fermentation products with (corn syrup, bamboo leaf extract, sucrose); the fermentation experimental steps and the dosage of each component were the same as in Example 1; all kinds of secretions were detected by conventional laboratory methods or kits. DETAILED DESCRIPTION

[0027] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples, wherein the terms used in the examples are for describing specific embodiments and do not constitute limitations on the scope of protection of the present invention.

[0028] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0029] Some raw materials and their sources are as follows:

[0030] Bacillus natto strain: purchased from Ningbo Testo Biotechnology Co., Ltd., catalog number TS288161;

[0031] Bacillus pumilus strain: purchased from Ningbo Testo Biotechnology Co., Ltd., catalog number TS290749;

[0032] Rhodotorula glutinosus strain: purchased from Ningbo Testo Biotechnology Co., Ltd., catalog number TS324223;

[0033] Chlorella: The Chlorella is Xinjiang Tianshan Chlorella JY-QWWZ-1900386, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 7, 2021, with the deposit number CGMCC No.22396.

[0034] Bamboo leaf extract: purchased from Shaanxi Pinhong Biotechnology Co., Ltd.

[0035] The extraction process of Chlorella extract is as follows:

[0036] S1: washing, removing impurities, drying to constant weight, breaking the cell wall and crushing the chlorella to obtain product A1;

[0037] S2: A1 is enzymatically hydrolyzed using cellulase and trypsin to obtain enzymatic hydrolysis product A2;

[0038] S3: Inoculate Bacillus natto and Bacillus pumilus into A2, ferment for 4-6 hours, and then sterilize and filter to obtain filtrate A3;

[0039] S4: adding bamboo leaf extract, corn steep liquor and sucrose to A3, stirring evenly, inoculating red yeast, fermenting for 3-4 hours, and then ultrasonically crushing and filtering to obtain filtrate A4;

[0040] S5: A4 is freeze-dried to obtain the final product A5.

[0041] The amount of cellulase and trypsin added in S2 is 1-3wt% of A1, the mass ratio of cellulase to trypsin is 1:0.4-0.7, and the enzymolysis is carried out for 30-60 minutes.

[0042] The added amount of Bacillus natto and Bacillus pumilus in S3 is 3-5wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.5-0.8; the fermentation starting temperature is 32-34°C, and the fermentation starting pH is 7±0.5.

[0043] The total amount of bamboo leaf extract, corn syrup and sucrose added in S4 is 10-15wt% of A3, and the mass ratio of bamboo leaf extract, corn syrup and sucrose is 1:0.7:0.1-0.3; the amount of red yeast added is 2-3wt% of A3; the fermentation starting temperature is 29±1°C, and the fermentation starting pH is 6.5±0.5.

[0044] Example 1: Following the preparation methods S1-S5, the addition amount of S2 cellulase and trypsin was 2wt% of A1, the mass ratio of cellulase to trypsin was 1:0.5, and the enzymolysis was performed for 45min.

[0045] The addition amount of Bacillus natto and Bacillus pumilus in S3 is 4wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.6; the fermentation starting temperature is 33°C, and the fermentation starting pH is 7.

[0046] The total amount of bamboo leaf extract, corn syrup and sucrose added in S4 is 13wt% of A3, and the mass ratio of bamboo leaf extract, corn syrup and sucrose is 1:0.7:0.2; the amount of red yeast added is 3wt% of A3; the fermentation starting temperature is 30°C, and the fermentation starting pH is 6.5.

[0047] Example 2: Following the preparation methods S1-S5, the addition amount of S2 cellulase and trypsin was 1wt% of A1, the mass ratio of cellulase to trypsin was 1:0.4, and the enzymolysis was performed for 30 minutes.

[0048] The added amount of Bacillus natto and Bacillus pumilus in S3 is 3wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.8; the fermentation starting temperature is 32°C, and the fermentation starting pH is 6.5.

[0049] The total amount of bamboo leaf extract, corn syrup and sucrose added in S4 is 10wt% of A3, and the mass ratio of bamboo leaf extract, corn syrup and sucrose is 1:0.7:0.1; the amount of red yeast added is 2wt% of A3; the fermentation starting temperature is 31°C, and the fermentation starting pH is 6.0.

[0050] Example 3: Following the preparation methods S1-S5, the addition amount of S2 cellulase and trypsin is 3wt% of A1, the mass ratio of cellulase to trypsin is 1:0.7, and the enzymolysis is carried out for 60min.

[0051] The addition amount of Bacillus natto and Bacillus pumilus in S3 is 5wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.5; the fermentation starting temperature is 34°C, and the fermentation starting pH is 7.5.

[0052] The total amount of bamboo leaf extract, corn syrup and sucrose added in S4 is 15wt% of A3, and the mass ratio of bamboo leaf extract, corn syrup and sucrose is 1:0.7:0.3; the amount of red yeast added is 2wt% of A3; the fermentation starting temperature is 29°C, and the fermentation starting pH is 7.0.

[0053] Comparative Example 1: The difference from Example 1 is that only Bacillus natto is used in S3, and the rest is the same as Example 1.

[0054] Comparative Example 2: The difference from Example 1 is that only Bacillus pumilus is used in S3, and the rest is the same as Example 1.

[0055] Comparative Example 3: The difference from Example 1 is that step S3 is not included, and the rest is the same as Example 1.

[0056] Comparative Example 4: The difference from Example 1 is that different amounts of bamboo leaf extract, corn syrup and sucrose in a mass ratio of 1:2:1 are used in S4, and the rest is the same as Example 1.

[0057] Comparative Example 5: The difference from Example 1 is that bamboo leaf extract, corn syrup and sucrose are not added to S4, and the rest is the same as Example 1.

[0058] Comparative Example 6: The difference from Example 1 is that step S4 is not included, and the rest is the same as Example 1.

[0059] Comparative Example 7: The difference from Example 1 is that the order of steps S3 and S4 is changed.

[0060] Comparative Example 8: The steps S1-S3 in Example 1 were followed, wherein the strains added in S3 were Bacillus natto, Bacillus pumilus, and Rhodotorula glutinosus, and bamboo leaf extract, corn syrup, and sucrose were added before inoculating S3, and the added amounts were the same as in Example 1, and then the S3 fermentation product was sterilized, filtered, and freeze-dried to obtain the final product, Chlorella extract.

[0061] A serum composition, the specific mass percentage ratio of which is as follows:

[0062] Table 1

[0063]

[0064]

[0065] Note: The balance is sterile deionized water.

[0066] Safety Test

[0067] With reference to the 2015 Technical Specifications for Safety of Cosmetics, compositions 1-5 were evaluated for cosmetic irritation using a skin patch test on 50 randomly distributed subjects aged 16-65 years.

[0068] Test method: Put the test substance into the patch tester, the dosage is 0.020-0.025g, cover the patch tester with the test substance on the back or forearm of the subject with non-irritating cloth-based tape, and gently press it with the palm to make it evenly adhere to the skin surface for 24 hours. After removing the test patch tester, observe the skin reaction after the indentation disappears 30 minutes later. If the result is negative, observe again 24 hours and 48 hours after the patch test.

[0069] Evaluation criteria:

[0070] Grade 0: negative reaction;

[0071] Grade 1: Suspicious reaction, only slight erythema;

[0072] Grade 2: weak positive reaction, erythema, infiltration, edema, and papules may be present;

[0073] Grade 3: Strong positive reaction, erythema, infiltration, edema, papules, and the reaction may extend beyond the test area;

[0074] Grade 4: Very strong positive reaction, obvious erythema, severe infiltration, edema, confluent herpes, and the reaction exceeds the test area.

[0075] Test results: Skin reactions were negative in all subjects.

[0076] The above test results show that the composition provided by the present invention is mild and non-irritating to the skin and is safe to use.

[0077] DPPH detection

[0078] The antioxidant capacity of Examples 1-3 and Comparative Examples 1-8 was verified using DPPH free radicals.

[0079] Prepare 0.1mmol / L DPPH solution. Take Examples 1-3 and Comparative Examples 1-8 to prepare 10g / L sample solution, 1mg / mL positive control Vc solution and blank control solution. Take 0.1ml sample solution and 4.9ml DPPH solution respectively and place them in a dark room for reaction for 20 minutes. Then measure the absorbance at a wavelength of 517nm and calculate the clearance rate DVC%.

[0080]

[0081] Table 2

[0082] Experimental Group Clearance rate / % Example 1 87.42 Example 2 85.17 Example 3 86.23 Comparative Example 1 65.42 Comparative Example 2 41.52 Comparative Example 3 35.41 Comparative Example 4 74.23 Comparative Example 5 64.14 Comparative Example 6 42.25 Comparative Example 7 66.51 Low ratio 8 56.52 VC 100％

[0083] According to the results of Examples 1-3 in Table 2, the Chlorella extract provided by the embodiments of the present invention has a good antioxidant effect, especially the effect of Example 1 is better.

[0084] Comparing the results of Example 1 with Comparative Examples 1-3, it can be seen that Bacillus natto and Bacillus pumilus have significant synergistic effects.

[0085] By comparing the results of Example 1 with Comparative Examples 4-5, it can be seen that bamboo leaf extract, corn steep liquor and sucrose can effectively increase the secretion of astaxanthin, carotenoids and B vitamins by Rhodotorula glutinosae. Due to the addition of a specific proportion of bamboo leaf extract, corn steep liquor and sucrose, the antioxidant effect of Example 1 is significantly improved compared with Comparative Examples 4-5.

[0086] By comparing the results of Example 1 with Comparative Examples 3 and 6, it can be seen that the extract prepared by the secondary fermentation has a significant antioxidant effect compared with the extract prepared by the primary fermentation.

[0087] By comparing the results of Example 1 with Comparative Examples 7-8, it can be seen that the Chlorella extract obtained by mixed one-time fermentation has a worse antioxidant effect than the Chlorella extract obtained by the process of Example 1, indicating that the fermentation times and feeding sequence are specific and cannot be changed.

[0088] Zebrafish tail moisturizing test

[0089] The zebrafish epidermis has a certain osmotic pressure tolerance range, and water loss will occur if the tolerance range is exceeded. Using a high osmotic pressure solution to treat zebrafish will cause its tail to shrink and become smaller due to water loss. Therefore, an induced model of zebrafish hydration and moisturizing can be established to evaluate the effect of moisturizing products.

[0090] (1) Breeding: Zebrafish were paired at a male-female ratio of 2:1, and the eggs were collected and incubated overnight (28°C incubator).

[0091] (2) Experimental groups: The experiment set up a blank control group and an experimental group. The experimental groups included a model group, a positive group, and a sample group, with 8 2 dpf fry in each group.

[0092] (3) Solution preparation: Accurately weigh 1 mg of samples of Examples 1-3 and Comparative Examples 1-8 and prepare the sample solution to be tested, and use urea (0.05%) as a positive control for the experiment, adding 1 mg to each well. After the administration is completed, place the 6-well plate in a 28° C. incubator in the dark for 24 hours.

[0093]

[0094] Test results: as shown in Table 3 below.

[0095] Table 3

[0096] Experimental Group Zebrafish tail area reduction inhibition rate Positive group 1 blank 0 Example 1 0.89 Example 2 0.84 Example 3 0.83 Comparative Example 1 0.64 Comparative Example 2 0.31 Comparative Example 3 0.24 Comparative Example 4 0.63 Comparative Example 5 0.58 Comparative Example 6 0.42 Comparative Example 7 0.68 Low ratio 8 0.57

[0097] According to the results analysis in Table 3: It can be seen from the results of Examples 1-3 that the Chlorella extract provided by the examples of the present invention has a good antioxidant effect, especially the effect of Example 1 is better.

[0098] Comparing the results of Example 1 with Comparative Examples 1-3, it can be seen that Bacillus natto and Bacillus pumilus have significant synergistic effects.

[0099] By comparing the results of Example 1 with Comparative Examples 4-5, it can be seen that bamboo leaf extract, corn steep liquor and sucrose can effectively increase the secretion of astaxanthin, carotenoids and B vitamins by Rhodotorula glutinosae. Due to the addition of a specific proportion of bamboo leaf extract, corn steep liquor and sucrose, the antioxidant effect of Example 1 is significantly improved compared with Comparative Examples 4-5.

[0100] By comparing the results of Example 1 with Comparative Examples 3 and 6, it can be seen that the extract prepared by the secondary fermentation has a significant antioxidant effect compared with the extract prepared by the primary fermentation.

[0101] By comparing the results of Example 1 with Comparative Examples 7-8, it can be seen that the Chlorella extract obtained by mixed one-time fermentation has a worse antioxidant effect than the Chlorella extract obtained by the process of Example 1, indicating that the fermentation times and feeding sequence are specific and cannot be changed.

[0102] Human body 30s instant soothing effect test

[0103] Volunteer selection criteria: a) healthy volunteers aged 18 to 60 years old; b) subjects without skin lesions / dermatitis or other factors that affect measurement in the test area; c) those with sensitive and dry faces; d) those with positive lactic acid stinging test results.

[0104] The number of volunteers is guaranteed to be more than 30, and the subjects should mainly engage in indoor activities to avoid long-term exposure to light and high temperature or cold environments.

[0105] 140 volunteers aged 22-55 with sensitive facial skin (lactic acid stinging test score ≥ 3 points on both sides of the nose) and dry East Asian skin were selected. A sample group and a blank group (i.e., a model control group: no sample was applied) were set up, and the test areas were randomly distributed on the left and right faces of the volunteers. After using the composition 1-14 of the present invention, the redness map was collected by the skin facial image analyzer VISIA7, and the skin redness a* value was analyzed by Image-Pro Plus (IPP) analysis software to evaluate its moisturizing and soothing effects. The laboratory temperature is 21±1°C; the relative humidity is 50%±10%.

[0106] The experiment adopted the self-before and after control method and the blank group control method. The test period was 30s, and the skin image collection times were 2 times, 0s and 30s respectively. Set up the sample group (compositions 1-3) and the blank group (composition 9), and the test areas were randomly distributed on the left and right faces of the volunteers. Apply the sample at a dosage of 0.25g / half face. Select qualified subjects according to the requirements, and when the volunteers visit, sign the volunteer informed consent form with them, collect skin images and questionnaire data after using the product for 30s. When visiting, after washing the face with the designated cleanser, sit quietly in the test environment for about 20-30 minutes. After the meditation, start the test.

[0107] With the help of facial image analyzer VISIA 7 ( Company, USA), the instrument uses 3 different light sources to capture facial images and can intuitively observe changes in facial skin conditions.

[0108] The redness image a* value was analyzed using Image-Pro Plus software.

[0109] Skin redness a* value: The a* value represents the redness of the skin. An increase in the a* value indicates an increase in the vascular reactivity of the skin.

[0110] The data were analyzed using SPSS19.0 data analysis software.

[0111] Table 4

[0112]

[0113] From the analysis of the results in Table 4: Comparing compositions 1-3, the results show that the skin redness a* of compositions 1-3 is reduced, and composition 1 has the highest degree of reduction, indicating that the skin soothing effect of the composition prepared by the process of the present invention does not show a linear trend with its added amount.

[0114] Comparison of composition 1 and compositions 6-13 shows that the use of the Chlorella extract prepared by the present invention can achieve the maximum effect of the composition on reducing the a* value of skin redness, and the human efficacy experiment corroborates the previous experiment.

[0115] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present application.

Claims

1. An extraction process of Chlorella extract, It is characterized in that The extraction process of the chlorella extract is specifically as follows: S1: washing, drying and crushing the Chlorella vulgaris to obtain product A1; S2: A1 is enzymatically hydrolyzed using cellulase and trypsin to obtain enzymatic hydrolysis product A2; S3: Inoculate Bacillus natto and Bacillus pumilus into A2, ferment for 4-6 hours, and then sterilize and filter to obtain filtrate A3; S4: adding bamboo leaf extract, corn steep liquor and sucrose to A3, stirring evenly, inoculating red yeast, fermenting for 3-4 hours, and then crushing and filtering to obtain filtrate A4; S5: A4 is freeze-dried to obtain the final product A5.

2. The extraction process according to claim 1, It is characterized in that The Chlorella is Xinjiang Tianshan Chlorella JY-QWWZ-1900386, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 7, 2021, with the deposit number CGMCC No.22396.

3. According to the extraction process of claim 1, It is characterized in that The added amount of cellulase and trypsin in S2 is 1-3wt% of that in A1, the mass ratio of cellulase to trypsin is 1:0.4-0.7, and the enzymolysis time is 30-60min.

4. The extraction process according to claim 1, It is characterized in that The added amount of Bacillus natto and Bacillus pumilus in S3 is 3-5wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.5-0.

8.

5. The extraction process according to claim 1, It is characterized in that The total amount of bamboo leaf extract, corn steep liquor and sucrose added in S4 is 10-15wt% of A3, and the mass ratio of bamboo leaf extract, corn steep liquor and sucrose is 1:0.7:0.1-0.3; the amount of red yeast added is 2-3wt% of A3.

6. The extraction process according to claim 1, It is characterized in that The S3 fermentation starting temperature is 32-34°C, and the fermentation starting pH is 7±0.

5.

7. The extraction process according to claim 1, It is characterized in that The S4 fermentation starting temperature is 29±1° C., and the fermentation starting pH is 6.5±0.

5.

8. A chlorella extract, It is characterized in that The Chlorella extract is prepared by the Chlorella extract extraction process described in any one of claims 1-7.

9. A serum, It is characterized in that The invention comprises the Chlorella extract according to claim 8.

10. The essence according to claim 9, It is characterized in that The following components are included in mass percentage: Chlorella extract 1-5wt%; Glycerol 20-25wt%; Sodium hyaluronate 10-12wt%; Honey 1-5wt%; The balance was made up with sterile deionized water.

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