A method for extracting high-purity starch from microalgae and microalgae starch

Abstract

The present invention belongs to the technical field of polysaccharide extraction, and specifically relates to a method for extracting high-purity starch from microalgae and microalgae starch. A method for extracting high-purity starch from microalgae, including microalgae cultivation, collection and pretreatment, cell wall breaking, starch separation and purification and other steps. The present invention adopts a segmented cultivation strategy to promote starch accumulation, uses liquid nitrogen pretreatment to improve wall breaking efficiency, realizes efficient cell wall breaking by combining enzymolysis and ultrasonic crushing with multiple enzymes, and combines SDS washing and ultrafiltration technology for deep purification. The method of the present invention can be applied to a variety of microalgae, such as Spirulina, Chlorella, Chlamydomonas reinhardtii, etc., and the obtained starch purity is ≥98%, and the percentage of amylose is 40-75%. The method of the present invention is simple in process, environmentally friendly, and widely applicable, and the obtained high-purity, high-amylose content microalgae starch has broad application prospects in the fields of food, medicine, and chemical industry.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for extracting high-purity starch from microalgae and microalgae starch. Background Art

[0002] Starch is an important polysaccharide substance, which is widely used in food, medicine, chemical industry and other fields. Traditional starch extraction usually relies on terrestrial plants, such as corn, potato, etc. However, these methods not only have an impact on the environment, but are also limited by land resources. The ocean breeds a variety of algae that use starch as an energy storage substance. At present, the research on algae starch is mainly focused on brown algae starch, and there have been a few studies on red algae starch in the past 30 years. Starch is an important component of the cytoplasm of large red algae such as Gracilaria and Gracilaria. As an emerging biological resource, microalgae have rapid growth, efficient photosynthesis and high starch content, and are a potential source of starch.

[0003] However, the existing technology is difficult to extract starch from microalgae, mainly due to the following problems:

[0004] (1) Low purity: Existing extraction methods make it difficult to obtain high-purity microalgae starch, and the impurity content is high, which affects product quality;

[0005] (2) Complex process: Many extraction methods involve complex process flows, which increases production costs and difficulty;

[0006] (3) Low yield: The starch extraction efficiency of existing technologies is not high and it is difficult to meet the needs of large-scale production;

[0007] (4) Environmental impact: Some extraction methods use large amounts of organic solvents, which may have a negative impact on the environment;

[0008] (5) Temperature influence: Some extraction methods use high temperature treatment, which causes the microalgae starch to gelatinize during the processing and extraction process, significantly affecting the subsequent product quality.

[0009] Therefore, it is of great significance to develop an efficient, environmentally friendly method for extracting microalgae starch that can obtain high-purity starch. Summary of the invention

[0010] The purpose of the present invention is to provide a method for extracting high-purity starch from microalgae, which can simplify the process flow, improve the extraction purity and yield of starch, and obtain a starch product with a high amylose content.

[0011] To achieve the above object, the present invention adopts the following technical scheme: A method for extracting high-purity starch from microalgae, comprising the following steps:

[0012] (1) Cultivation of microalgae: selecting a microalgae species suitable for starch accumulation and culturing the microalgae; using a nitrogen-deficient medium to culture the microalgae after the microalgae enters the stationary phase; wherein the microalgae species is selected from any one of Spirulina, Chlamydomonas, Chlorella, Rhodophyta, Schizochytrium, and Dunaliella;

[0013] (2) Collection and pretreatment of microalgae: harvesting the microalgae 2-10 days after the microalgae are cultured under nitrogen deficiency; freezing the harvested microalgae with liquid nitrogen;

[0014] (3) Microalgae cell wall disruption: The microalgae frozen by liquid nitrogen are enzymatically hydrolyzed by a complex enzyme consisting of cellulose, pectinase and lytic enzyme, and then the cell wall is completely destroyed by ultrasonic disruption to release the starch in the cells; the amount of complex enzyme added per gram weight of freeze-dried microalgae is 1 U-4U of cellulase, 0.5 U-3U of pectinase, and 0.3 U-1.5U of lytic enzyme.

[0015] (4) Separation and purification of starch: The microalgae suspension after cell wall destruction is separated to obtain crude starch; the crude starch is dissolved in water and washed multiple times with SDS solution to remove impurities.

[0016] Preferably, in step (1), the nitrogen-deficient culture medium of Schizochytrium does not contain a phosphorus source.

[0017] Preferably, in step (1), the nitrogen-deficient culture medium of Chlamydomonas does not contain a sulfur source.

[0018] Preferably, in step (1), the concentration of sodium chloride in the nitrogen-deficient culture medium of Dunaliella salina is 3 M.

[0019] Preferably, in step (3), the complex enzyme of red hair algae also contains β-glucanase, and the added amount of β-glucanase is 0.3-0.6 U.

[0020] Preferably, in step (3), the complex enzyme of Schizochytrium sp. further comprises chitinase, and the added amount of chitinase is 0.4-0.8 U.

[0021] Preferably, in step (3), the Dunaliella salina salt complex enzyme further comprises alkaline protease, and the added amount of alkaline protease is 0.2-0.5 U.

[0022] Preferably, in step (4), the volume ratio of the SDS solution to the aqueous solution of the crude starch is 10:1, and the mass volume concentration of the SDS solution is 1%-16%.

[0023] The present invention further provides microalgae starch prepared by the method, wherein the total starch content in the microalgae starch is ≥98%, and the percentage of amylose is 40-75%.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) High purity: Through a multi-step purification process, microalgae starch with a purity of more than 98% is obtained, which is significantly better than existing technologies;

[0026] (2) High amylose content: By adopting a segmented cultivation strategy and precise process control, the amylose percentage obtained is 40-75%, which is higher than that of traditional crop starch;

[0027] (3) High efficiency: Through liquid nitrogen freezing pretreatment and multiple enzymes combined cell wall breaking, the cell wall breaking rate is as high as 95%, which greatly improves the starch extraction efficiency;

[0028] (4) Process simplification: The entire extraction process is relatively simple and easy to implement industrial production;

[0029] (5) Environmentally friendly: Water and ethanol are mainly used as solvents, which reduces the use of organic solvents and is more environmentally friendly;

[0030] (7) Diverse raw materials: Applicable to a variety of microalgae, such as Spirulina, Chlamydomonas, etc., with a wide range of raw material sources;

[0031] (8) Product diversification: By adjusting process parameters, starch products with different linear chain contents can be obtained to meet different application requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 1 is a graph showing the changes in the density and dry weight of microalgae during the culture process in the embodiment of the present invention; wherein: (a) the curve of the change in algae density; (b) the curve of the change in algae dry weight;

[0033] Figure 2 The change of starch content during the microalgae cultivation process in the embodiment of the present invention;

[0034] Figure 3 A particle size distribution diagram of microalgae starch provided in an embodiment of the present invention;

[0035] Figure 4 A transmission electron microscope image of the microalgae starch provided by an embodiment of the present invention;

[0036] Figure 5 A scanning electron microscope image of the microalgae starch provided in an embodiment of the present invention;

[0037] Figure 6 This is a comparison chart of the effects of different breaking methods provided in the embodiments of the present invention on the microalgae wall breaking rate. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0039] Example 1: This example provides a method for extracting high-purity starch from microalgae, and the specific steps are as follows:

[0040] (1) Microalgae culture: Spirulina platensis was selected as the experimental object. Spirulina was inoculated in a modified Zarrouk medium under the following culture conditions: temperature 28±1℃, light intensity 6000 lux, light-dark ratio 14:10h. After the logarithmic growth period (about 8 days), Figure 1 As shown in (a), the algae solution was collected by centrifugation and transferred to a medium without a nitrogen source for further cultivation for 7 days to promote starch accumulation. The dry weight and starch content of the microalgae reached the maximum value on the 7th day of cultivation, at which time the dry weight was 4.8 g / L. Figure 1 As shown in (b), the starch content is 56%. Figure 2 shown.

[0041] (2) Microalgae collection and pretreatment: Use a centrifuge (5000 rpm, 10 min) to collect algae. Wash the collected algae with deionized water three times and then freeze them in liquid nitrogen. Store the frozen algae in a -80℃ refrigerator for later use.

[0042] (3) Cell disruption: Take 10 g (dry weight) of quick-frozen algae and add 100 mL of pH 5.0 acetate buffer (50 mM). Add cellulase (2 U / g algae), pectinase (1 U / g algae) and lytic enzyme (0.5 U / g algae). Perform enzymatic hydrolysis at 37°C for 2 h. Subsequently, place the enzymatic hydrolyzate in an ultrasonic disruptor (power 400 W, working 5 s, rest 5 s), and the total treatment time is 10 min.

[0043] (4) Starch separation and purification: The algae solution was centrifuged (8000 rpm, 15 min) and the precipitate was collected. The precipitate was resuspended in 100 mL of deionized water, and 10 mL of 1% (w / v) SDS solution was added. The solution was treated in a 30°C water bath for 30 min. The SDS washing step was repeated 3 times. Finally, the washed sample was purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa and replaced with deionized water 5 times.

[0044] (5) Starch drying and storage: The purified starch suspension was freeze-dried (-50°C, 48 hours). The dried starch sample was sealed and stored in a desiccator. The final starch dry weight was 3.87 g, the percentage of amylose was 40%, and the starch purity was 98%. Figure 3 As shown, the particle size of the microalgae is 0.5-3 μm, and its transmission electron microscope and scanning electron microscope images are shown in Figure 4 , Figure 5 shown.

[0045] Example 2: This example provides a method for extracting high-purity starch from Chlorella, and the specific steps are as follows:

[0046] (1) Microalgae culture: Chlorella vulgaris was selected as the experimental object. Chlorella vulgaris was inoculated in BG11 culture medium under the following culture conditions: temperature 25±1℃, light intensity 5000 lux, light-dark ratio 12:12h. After the logarithmic growth period (about 5 days), the algae liquid was collected by centrifugation and transferred to a culture medium without nitrogen source for further culture for 7 days to promote starch accumulation and maximize the starch content in the microalgae.

[0047] (2) Microalgae collection and pretreatment: Use a centrifuge (4500 rpm, 15 min) to collect algae. Wash the collected algae with deionized water three times and then freeze them in liquid nitrogen. Store the frozen algae in a -80℃ refrigerator for later use.

[0048] (3) Cell wall disruption: Take 8 g (dry weight) of quick-frozen algae and add 80 mL of citric acid buffer (50 mM) at pH 5.5. Add cellulase (2.5 U / g algae), pectinase (1.5 U / g algae) and lytic enzyme (0.8 U / g algae). Perform enzymatic hydrolysis at 40°C for 2.5 hours. Subsequently, place the enzymatic hydrolyzate in an ultrasonic disruptor (power 450 W, working 8 s, rest 5 s), and the total treatment time is 12 min.

[0049] (4) Starch separation and purification: The crushed algae liquid was centrifuged (9000 rpm, 20 min) and the precipitate was collected. The precipitate was resuspended in 80 mL of deionized water, and 8 mL of 12% (w / v) SDS solution was added and treated in a 35°C water bath for 35 min. The SDS washing step was repeated 4 times. Finally, the washed sample was purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 50 kDa and replaced with deionized water 6 times.

[0050] (5) Starch drying and storage: The purified starch suspension was spray dried (inlet temperature 180°C, outlet temperature 85°C). The dried starch sample was sealed and stored in a brown glass bottle. The final starch dry weight was 3.87 g, the percentage of amylose was 36%, and the starch purity was 97%.

[0051] Example 3: This example provides a method for extracting high-purity starch from Chlamydomonas reinhardtii, and the specific steps are as follows:

[0052] (1) Microalgae culture: Chlamydomonas reinhardtii was selected as the experimental subject. Chlamydomonas reinhardtii was inoculated in TAP medium under the following culture conditions: temperature 23±1℃, light intensity 4500 lux, light-dark ratio 16:8h. After the logarithmic growth period (about 6 days), the algae liquid was collected by centrifugation and transferred to a culture medium without nitrogen and sulfur sources for further culture for 10 days to promote starch accumulation and maximize the starch content in the microalgae.

[0053] (2) Microalgae collection and pretreatment: Use a centrifuge (4000 rpm, 20 min) to collect algae. Wash the collected algae with deionized water three times and then freeze them in liquid nitrogen. Store the frozen algae in a -80℃ refrigerator for later use.

[0054] (3) Cell wall disruption: Take 6 g (dry weight) of quick-frozen algae and add 60 mL of phosphate buffer (50 mM) at pH 6.0. Add cellulase (3 U / g algae), pectinase (2 U / g algae) and lytic enzyme (1 U / g algae). Perform enzymatic hydrolysis at 35°C for 3 h. Subsequently, place the enzymatic hydrolyzate in an ultrasonic disruptor (power 500 W, working 10 s, rest 8 s), and the total treatment time is 15 min.

[0055] (4) Starch separation and purification: The crushed algae liquid was centrifuged (10,000 rpm, 25 min) and the precipitate was collected. The precipitate was resuspended in 60 mL of deionized water, and 6 mL of 5% (w / v) SDS solution was added and stirred at room temperature for 40 min. The SDS washing step was repeated 5 times. Finally, the washed sample was purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa and replaced with deionized water 7 times.

[0056] (5) Starch drying and storage: The purified starch suspension was freeze-dried (-60°C, 60 hours). The dried starch sample was sealed and stored under nitrogen conditions. The final starch dry weight was 3.06 g, the percentage of amylose was 75%, and the starch purity was 98%.

[0057] Example 4: This example provides a method for extracting high-purity starch from red hair algae, and the specific steps are as follows:

[0058] (1) Microalgae culture: Porphyridium cruentum was selected as the experimental object. The red algae was inoculated in f / 2 culture medium under the following culture conditions: temperature 22±1℃, light intensity 3500 lux, light-dark ratio 14:10h. After the logarithmic growth period (about 8 days), the algae liquid was collected by centrifugation and transferred to f / 2 culture medium without nitrogen source for further culture for 6 days to promote starch accumulation and maximize the starch content in the microalgae.

[0059] (2) Microalgae collection and pretreatment: Use a centrifuge (4200 rpm, 18 min) to collect algae. Wash the collected algae with 0.5M NaCl solution three times and then freeze them in liquid nitrogen. Store the frozen algae in a -80℃ refrigerator for later use.

[0060] (3) Cell wall disruption: Take 7 g (dry weight) of quick-frozen algae and add 70 mL of acetic acid buffer (50 mM) at pH 5.8. Add cellulase (2.8 U / g algae), pectinase (1.8 U / g algae), lytic enzyme (0.9 U / g algae) and β-glucanase (0.5 U / g algae). Perform enzymatic hydrolysis at 38°C for 2.8 hours. Subsequently, the enzymatic hydrolyzate was placed in an ultrasonic disruptor (power 480 W, working 9 s, rest 6 s), and the total processing time was 14 min.

[0061] (4) Starch separation and purification: The algae solution was centrifuged (9500 rpm, 22 min) and the precipitate was collected. The precipitate was resuspended in 70 mL of deionized water, and 7 mL of 8% (w / v) SDS solution was added and stirred at room temperature for 38 min. The SDS washing step was repeated 4 times. Finally, the washed sample was purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 40 kDa and replaced with deionized water 6 times.

[0062] (5) Starch drying and storage: The purified starch suspension was freeze-dried in a vacuum oven (-55°C, 56 hours). The dried starch sample was sealed and stored in a vacuum package. The final starch dry weight was 3.27 g, the percentage of amylose was 42%, and the starch purity was 98%.

[0063] Example 5: This example provides a method for extracting high-purity starch from Schizochytrium sp., and the specific steps are as follows:

[0064] (1) Microalgae culture: Haematococcus pluvialis was selected as the experimental subject. Haematococcus pluvialis was inoculated in OHM medium under the following culture conditions: temperature 24±1℃, light intensity 5500 lux, light-dark ratio 16:8h. After the logarithmic growth period (about 7 days), the algae liquid was collected by centrifugation and transferred to OHM medium without nitrogen and phosphorus sources for further culture for 12 days to promote starch accumulation and maximize the starch content in the microalgae.

[0065] (2) Microalgae collection and pretreatment: Use a centrifuge (3800 rpm, 25 min) to collect algae. Wash the collected algae with 0.9% NaCl solution three times and then freeze them in liquid nitrogen. Store the frozen algae in a -80℃ refrigerator for later use.

[0066] (3) Cell disruption: Take 9 g (dry weight) of quick-frozen algae and add 90 mL of citric acid buffer (60 mM) at pH 5.2. Add cellulase (3.2 U / g algae), pectinase (2.2 U / g algae), lytic enzyme (1.2 U / g algae) and chitinase (0.6 U / g algae). Perform enzymatic hydrolysis at 36°C for 3.2 hours. Subsequently, place the enzymatic hydrolyzate in an ultrasonic disruptor (power 520 W, working 12 s, rest 7 s), and the total processing time is 18 min.

[0067] (4) Starch separation and purification: The broken algae liquid was centrifuged (10500 rpm, 28 min) and the precipitate was collected. The precipitate was resuspended in 90 mL of deionized water, and 9 mL of 16% (w / v) SDS solution was added. The solution was treated in a 42°C water bath for 42 min. The SDS washing step was repeated 5 times. Finally, the washed sample was purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 20 kDa and replaced 8 times with deionized water.

[0068] (5) Starch drying and storage: The purified starch suspension was freeze-dried in vacuum (-55°C, 56 hours). The dried starch sample was sealed and stored in a vacuum package. The dried starch sample was sealed and stored in a nitrogen-filled aluminum foil bag. The final starch dry weight was 4.13 g, the percentage of amylose was 45%, and the starch purity was 98.5%.

[0069] Example 6: This example provides a method for extracting high-purity starch from Dunaliella salina, and the specific steps are as follows:

[0070] (1) Microalgae culture: Dunaliella salina was selected as the experimental subject. Dunaliella salina was inoculated in a modified Johnson's medium (2M NaCl) under the following culture conditions: temperature 26±1℃, light intensity 6500 lux, light-dark ratio 18:6h. After the logarithmic growth period (about 9 days), the algae liquid was collected by centrifugation and transferred to a high-salt medium (3M NaCl) without a nitrogen source for further culture for 8 days to promote starch accumulation and maximize the starch content in the microalgae.

[0071] (2) Microalgae collection and pretreatment: Use a centrifuge (3500 rpm, 30 min) to collect algae. Wash the collected algae with 2M NaCl solution three times and then freeze them in liquid nitrogen. Store the frozen algae in a -80℃ refrigerator for later use.

[0072] (3) Cell disruption: Take 5 g (dry weight) of quick-frozen algae and add 50 mL of HEPES buffer (70 mM, containing 1 M NaCl) at pH 6.2. Add cellulase (2.5 U / g algae), pectinase (1.5 U / g algae), lytic enzyme (0.8 U / g algae) and salt-alkaline protease (0.4 U / g algae). Perform enzymatic hydrolysis at 40°C for 2.5 hours. Subsequently, place the enzymatic hydrolyzate in an ultrasonic disruptor (power 450 W, working 8 s, rest 5 s), and the total treatment time is 12 min.

[0073] (4) Starch separation and purification: The algae solution was centrifuged (11000 rpm, 30 min) and the precipitate was collected. The precipitate was resuspended in 50 mL of deionized water, and 5 mL of 10% (w / v) SDS solution was added. The solution was treated in a 35°C water bath for 35 min. The SDS washing step was repeated 4 times. Finally, the washed sample was purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa and replaced with deionized water 7 times.

[0074] (5) Starch drying and storage: The purified starch suspension was dried (45°C, 48 hours). The dried starch sample was ground and sealed in a ziplock bag for storage. The final starch dry weight was 2.08 g, the percentage of amylose was 38%, and the starch purity was 98%.

[0075] Example 7 In order to verify the beneficial effects of the method of the present invention, the following comparative experiments were conducted:

[0076] (1) Freeze-crushing method: Add 1L of distilled water to 10 g of dry weight of Spirulina to make a suspension, then quickly immerse it in liquid nitrogen for several minutes until the sample is completely frozen. After thawing the crushed sample, centrifuge (8000-12000rpm, 10-20 minutes) to separate cell fragments and starch. The cell crushing rate is 20%, and the purity of the extracted starch is 83%.

[0077] (2) Ultrasonic method: The method of Example 1 was followed, but the liquid nitrogen pretreatment and enzymatic hydrolysis steps were omitted. The cell disruption rate was 33%, and the purity of the extracted starch was 86%.

[0078] (3) Conventional enzymatic extraction (without liquid nitrogen pretreatment and ultrasonic assistance): The method of Example 1 was followed, but the liquid nitrogen pretreatment and ultrasonic disruption steps were omitted. The cell disruption rate was 80%, and the purity of the extracted starch was 90%.

[0079] (4) Freeze-crushing combined with enzymatic hydrolysis and ultrasonic method: The method of Example 1 was followed, the cell crushing rate was 98%, and the purity of the extracted starch was 98%.

[0080] The comparison results are as follows Figure 6 As shown, the method of the present invention is significantly superior to the traditional method in terms of microalgae cell wall breaking rate and starch purity, and obtains 40-75% of straight chain starch, which is higher than traditional crop starch.

Claims

1. A method for extracting high-purity starch from microalgae, characterized in that: The following steps are involved: (1) Cultivation of microalgae: selecting a microalgae species suitable for starch accumulation and culturing the microalgae; using a nitrogen-deficient medium to culture the microalgae after the microalgae enters the stationary phase; wherein the microalgae species is selected from any one of Spirulina, Chlamydomonas, Chlorella, Rhodophyta, Schizochytrium, and Dunaliella; (2) Collection and pretreatment of microalgae: harvesting the microalgae 2-10 days after the microalgae are cultured under nitrogen deficiency; freezing the harvested microalgae with liquid nitrogen; (3) Microalgae cell wall disruption: The microalgae frozen by liquid nitrogen are enzymatically hydrolyzed with a composite enzyme consisting of cellulose, pectinase and lytic enzyme, and then subjected to ultrasonic disruption to completely destroy the cell wall and release the starch in the cells; per gram weight of freeze-dried microalgae, the amount of composite enzyme added is 1 U-4U of cellulase, 0.5 U-3U of pectinase, and 0.3 U-1.5 U of lytic enzyme; the composite enzyme of red hair algae also contains β-glucanase, and the amount of β-glucanase added is 0.3-0.6 U; the composite enzyme of Schizochytrium also contains chitinase, and the amount of chitinase added is 0.4-0.8 U; the composite enzyme of Dunaliella salina also contains alkaline protease, and the amount of alkaline protease added is 0.2-0.5 U; (4) Separation and purification of starch: The suspension of microalgae after cell wall destruction is separated to obtain crude starch. The crude starch is dissolved in water and washed and ultrafiltered multiple times with SDS solution to remove impurities.

2. The method for extracting high-purity starch from microalgae according to claim 1, characterized in that: In step (1), the nitrogen-deficient culture medium for Schizochytrium does not contain a phosphorus source.

3. The method for extracting high-purity starch from microalgae according to claim 1, characterized in that: In step (1), the nitrogen-deficient culture medium for Chlamydomonas does not contain a sulfur source.

4. The method for extracting high-purity starch from microalgae according to claim 1, characterized in that: In step (1), the concentration of sodium chloride in the nitrogen-deficient culture medium of Dunaliella salina is 3 M.

5. The method for extracting high-purity starch from microalgae according to claim 1, characterized in that: In step (4), the volume ratio of the SDS solution to the aqueous solution of the crude starch is 10:1, and the mass volume concentration of the SDS solution is 1%-16%.

6. The method for extracting high-purity starch from microalgae according to claim 1, characterized in that: In step (4), ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 20-100 kDa, and deionized water is used for replacement 5-8 times.

7. A microalgae starch prepared by the method according to any one of claims 1 to 6, characterized in that: The total starch content of the microalgae starch is ≥98%, and the percentage of amylose is 40-75%.

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