A method for processing marine unicellular diatoms and processed products thereof

Abstract

The present invention discloses a method for processing marine unicellular diatoms and a processed product thereof, comprising the following steps: step A: preparing fresh algae mud using fresh marine unicellular diatoms, and obtaining the pickled algae mud after pickling; step B: dissolving the polysaccharide in the pickled algae mud by microwave heating using urea-alkaline solution to obtain a first solution, ultrafiltering the first solution and concentrating, and obtaining chitosan composite polysaccharide powder through vacuum freeze drying; step C: granulating the chitosan composite polysaccharide powder using an ethanol-glycerol mixture, and obtaining a processed product after drying; the processed product is a chitosan composite polysaccharide particle, including chitosan and its derivatives, hemicellulose and cellulose. The processing method provided by the present invention is simple, and raw materials are easy to breed and enrich, and the processed chitosan composite polysaccharide has moisture-retaining properties and can be widely used in aquaculture feed, biomedicine, environmental engineering, engineering materials or chemical and chemical fields.

Description

Technical Field

[0001] The present invention relates to the technical field of marine algae processing, in particular to a method for processing marine unicellular diatoms and processed products thereof, and particularly to C12N1 / 12 (unicellular algae; and culture medium thereof) and C08B37 / 08 (chitin polysaccharide; chondroitin sulfate; hyaluronic acid; and derivatives thereof) in the international patent classification numbers. Background Art

[0002] In the field of marine resource development and utilization, unicellular diatoms, as fundamental producers in marine ecosystems, possess significant ecological value and economic potential. Diatoms fix carbon dioxide through photosynthesis, contributing to primary productivity in the marine food chain. Their cellular metabolites are rich in active ingredients such as polysaccharides, fatty acids, and proteins, offering broad application prospects in biomaterials, aquaculture, and environmental governance.

[0003] The cell wall of single-celled diatoms is composed of a SiO2 skeleton and organic matter. While the silica skeleton is currently well-developed and utilized, the organic matter is often discarded during processing and has not been properly developed. The polysaccharides in this organic matter exist as intracellular and extracellular polysaccharides, exhibiting excellent water retention, film-forming properties, and biocompatibility. Research has shown that diatom polysaccharides can undergo a moisture absorption and release cycle through their pore structure at a relative humidity of 50%, achieving a 24-hour moisture absorption rate of 5.89% and a moisture release rate of 3.90%. This respirable property makes it an ideal candidate for a natural moisturizing material. Brunner E et al. used solid-state nuclear magnetic resonance (NMR) spectroscopy to identify chitin present inside and outside the cell walls of diatoms in samples treated with Thalassiosira pseudonana (Brunner E et al, Chitin-based organic networks: an integral part of cell wall biosilica in the diatom Thalassiosira pseudonana[J]. Angewandte Chemie International Edition, 2009, 48(51): 9724-9727). Shao Z et al. retrieved genes related to chitin metabolism in the genomes of Phaeodactylum tricornutum and Thalassiosirapseudonana, especially the chitin deacetylase (CDA) gene, and found increased chitin content in the cell walls of diatoms overexpressing the CDA gene, further supporting the view that diatoms contain chitin (Shao Z et al, Comparative characterization ofputative chitin deacetylases from Phaeodactylum tricornutum and Thalassiosirapseudonana highlights the potential for distinct chitin-based metabolicprocesses in diatoms[J]. New Phytologist, 2019, 221(4): 1890-1905).

[0004] Microwave-assisted extraction, also known as microwave extraction (MAE), is a novel extraction technology that utilizes microwave energy. Electromagnetic waves act directly on molecules, inducing dipole rotation and ion conduction, generating an internal heating effect. This promotes molecular vibration and friction, leading to the breakdown of chemical bonds and dissolution of the target product. However, existing technologies often use deep eutectic solvents (DES) combined with microwave-assisted extraction to extract polysaccharides. However, DES typically has a high viscosity, which can hinder heat transfer in the microwave field, leading to localized overheating and inactivation of the target product. Furthermore, if some DES components, such as choline chloride, are not fully recovered, they can accumulate in water or soil, causing environmental pollution.

[0005] Urea-alkaline solution can dissolve chitin to a certain extent under repeated freeze-thaw conditions. For example, patent document with publication number CN117510680A discloses a β-chitosan and its rapid extraction method and application. It extracts chitin through urea-alkaline solution combined with repeated freeze-thaw cycles. However, it is time-consuming, energy-intensive, and environmentally unfriendly, making it unsuitable for mass production or processing.

[0006] Currently, there is no polysaccharide extraction method that utilizes microwave-assisted extraction combined with urea-alkaline solution to process single-cell diatoms. Therefore, it is urgent to develop a new single-cell diatom processing method to break the limitations of traditional polysaccharide extraction technology, provide an innovative path for the efficient utilization and ecological protection of marine diatom resources, further optimize the particle preparation process, and explore the molecular modification of polysaccharide components to enhance the functionalization and industrialization of the product. Summary of the Invention

[0007] The purpose of the present invention is to utilize a polysaccharide extraction method combining microwave-assisted extraction with urea-alkaline solution to realize the processing of unicellular diatoms, so as to break the limitations of traditional polysaccharide extraction technology, provide an innovative path for the efficient utilization and ecological protection of marine diatom resources, further optimize the particle preparation process, explore the molecular modification of polysaccharide components, so as to enhance the functionalization and industrialization level of the product, so that it can be widely used in aquaculture feed, biomedicine, environmental engineering, engineering materials or chemical and chemical fields, in order to solve the technical problems:

[0008] A first aspect of the present invention provides a method for processing marine unicellular diatoms, comprising the following steps:

[0009] Step A: preparing fresh algae mud using fresh marine unicellular diatoms, and washing with acid to obtain acid-washed algae mud;

[0010] Step B: using a urea-strong base solution to dissolve the polysaccharide in the acid-washed algae mud by microwave heating to obtain a first solution, ultrafiltering and concentrating the first solution, and performing vacuum freeze-drying to obtain a chitosan composite polysaccharide powder;

[0011] Step C: Granulate the chitosan polysaccharide powder with an ethanol-glycerol mixture, and obtain a processed product after drying. The processed product is chitosan polysaccharide particles, which include chitosan and its derivatives, hemicellulose and cellulose.

[0012] Furthermore, chitin, also known as chitin and chitin, is a chitin polysaccharide.

[0013] Furthermore, step A includes: step A1: settling the algae liquid of fresh marine unicellular diatoms, washing with pure water at least once and centrifuging to obtain a precipitate; the precipitate is fresh algae mud; step A2: washing the fresh algae mud with hydrochloric acid with a concentration of 1 to 4 M to obtain acid-washed algae mud.

[0014] Preferably, after the fresh marine unicellular diatom algae liquid is settled, it is washed with pure water twice and centrifuged to obtain a precipitate; the precipitate is fresh algae mud.

[0015] Further, fresh marine unicellular diatoms include Central Bacillales or Pennate Bacillales; Central Bacillales include Cyclostriales, Rhizoschiales and Box-shaped Bacillales; Pennate Bacillales include Naviculales, Curculigoles, Diploschiales, Phaeoschiales, Aconomorphales and Brachyschiales; Cyclostriales include Cyclostriaceae and Thalassiosiraceae; Box-shaped Bacillales include Ceratocystaceae; Naviculales include Naviculaceae; Phaeoschiales includes Phaeoschiaceae.

[0016] Furthermore, the Cyclostriaceae includes Cyclostridium radiata and Cyclostridium crypticum; the Thalassiosirae family includes Thalassiosira pseudomicron and Thalassiosira wiseri; the Chaetoceros family includes Chaetoceros muelleri; the Naviculae family includes Navicula splendida; and the Phaeodactylaceae family includes Phaeodactylaceae triangularis.

[0017] Furthermore, step B includes:

[0018] Step B-1: Mixing a urea-strong base solution with an acid-washed algae mud to obtain an algae mud suspension; wherein the acid-washed algae mud and the urea-strong base solution are mixed at a W / V ratio of 1:1 to 1:10; the urea-strong base solution contains 80% to 95% water; and the ratio of urea to strong base is 1:(1-4);

[0019] Step B-2: using a microwave synthesis reactor to microwave-treat the algae mud suspension while heating it, and centrifuging it to obtain a first solution and an algae mud precipitate;

[0020] Step B-3: Using an ultrafiltration membrane to perform tangential flow ultrafiltration on the first solution to obtain a retentate, concentrating the retentate and then performing ultrafiltration again, repeating this process 2-3 times to increase the purity and obtain a concentrated solution;

[0021] Step B-4: freeze-drying the concentrate in a freeze dryer to obtain freeze-dried powder, which is then ground and sieved to obtain chitosan composite polysaccharide powder;

[0022] Step B does not include polar treatment, oxidation treatment and deacetylating treatment.

[0023] Furthermore, the base in step B-2 is potassium hydroxide or sodium hydroxide; the microwave treatment power in step B-2 is 200-800 W, the treatment temperature is 20-60° C., and the treatment time is 15-20 min; the retention capacity of the ultrafiltration membrane in step B-3 is 5000-20000 Da; the ultrafiltrate is centrifuged at a speed of 4000-10000 rpm for 5-20 min; the vacuum freeze-drying conditions in step B-4 are treatment at -80° C. for 24-32 h or at -60° C. for 32-52 h; and the mesh size is 100-400 mesh.

[0024] Furthermore, step C specifically comprises: spraying the chitosan polysaccharide powder on the surface with an ethanol-glycerol mixture, performing rotary granulation and mesh sieving, and obtaining chitosan polysaccharide particles after drying.

[0025] Furthermore, the ethanol-glycerol mixture includes anhydrous ethanol with a concentration by mass ratio of 30-90% and glycerol with a concentration by mass ratio of 1-10%; the mesh size is 20-80.

[0026] Furthermore, the drying treatment in step C is natural air drying or oven drying, and the oven drying conditions are 50-80° C. for 0.8-5.0 h.

[0027] Another aspect of the present invention provides a processed product, which is obtained by the above-mentioned marine unicellular diatom processing method; the processed product includes chitin.

[0028] Furthermore, the processed product further comprises one or more of chitosan derivatives, cellulose and hemicellulose.

[0029] Another aspect of the present invention provides an application of a processed product in aquaculture, biomedicine, environmental engineering, engineering materials or chemical engineering fields, wherein the processed product is a chitosan composite polysaccharide particle.

[0030] Further, applications in aquaculture include the use of feed additives;

[0031] Furthermore, applications in the biomedical field include applications in moist wound healing drugs; chitosan composite polysaccharide particles can be used alone, covered or bandaged with medical dressings, or used as an auxiliary drug; chitosan composite polysaccharide particles can be applied to the surface of the wound with a thickness of 0.5~1.0 mm; medical dressings include gauze, hydrocolloid dressings, alginate dressings, foam dressings and film dressings.

[0032] Furthermore, the chitosan composite polysaccharide particles have moisturizing properties, and the moisturizing properties of the chitosan composite polysaccharide particles are greater than 45% after 72 h.

[0033] Compared with the prior art, the marine unicellular diatom processing method and processed products provided by this application have the following beneficial effects:

[0034] (1) The raw materials required for the marine unicellular diatom processing method of the present application are easy to cultivate, extract, and enrich, and the operation is simple and convenient, which is a further development of marine resources.

[0035] (2) The processing method of marine unicellular diatoms in the present application abandons the traditional polar treatment, oxidation treatment and deacetylation treatment, so that the processed product is a chitin complex polysaccharide, including chitin and its derivatives and small molecular polysaccharides such as hemicellulose and / or cellulose, etc., which does not contain irritating chemicals and has mild ingredients.

[0036] (3) The dynamic wet environment is established by the process's own characteristics of water absorption, insolubility, and degradability. The process has a small molecular weight, short molecular chain, large specific surface area, and is easier to disperse. It can absorb trace moisture in the air through surface exposed groups such as hydroxyl (-OH) and acetylamino (-NHCOCH3) to achieve rapid moisture absorption. It releases some moisture at high humidity and directly binds to water molecules through hydrogen bonds, increasing local humidity in a short period of time. This allows the process to maintain a moisture retention of more than 45% after 72 hours. It can be widely used in aquaculture feed, biomedicine, environmental engineering, engineering materials, or chemical engineering. It also provides an innovative path for the efficient utilization and ecological protection of marine biomass. Future research can further optimize the particle preparation process and explore the molecular modification of polysaccharide components to improve the functionalization and industrialization level of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The above content and the following specific embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only examples of the technical solutions claimed.

[0038] Figure 1 This is a schematic diagram of the chitosan composite polysaccharide sample in Example 1 of the present invention;

[0039] Figure 2This is a scanning electron microscope image of the chitosan complex polysaccharide in Example 1 of the present invention (scale bar: 10.0 μm);

[0040] Figure 3 This is the Fourier infrared spectrum of the chitosan composite polysaccharide in Example 1 of the present invention (the horizontal axis is the wavelength, the unit is (cm -1 ));

[0041] Figure 4 This is a schematic diagram of the chitosan composite polysaccharide sample in Example 2 of the present invention;

[0042] Figure 5 This is a scanning electron microscope image of the chitosan complex polysaccharide in Example 2 of the present invention (scale bar: 10.0 μm);

[0043] Figure 6 This is the Fourier infrared spectrum of the chitosan composite polysaccharide in Example 2 of the present invention (the horizontal axis is the wavelength, the unit is (cm -1 ));

[0044] Figure 7 This is a graph showing the water retention performance test results of the chitosan composite polysaccharide in Test Example 1 of the present invention (the horizontal axis is time, in units of (h), and the vertical axis is moisture retention, in units of (%)). DETAILED DESCRIPTION

[0045] The detailed features and advantages of the present invention are described in detail below in the specific embodiments, and the content is sufficient to enable any person skilled in the art to understand the technical content of the present invention and implement it accordingly. Based on the description, claims and drawings disclosed in this specification, those skilled in the art can easily understand the relevant purposes and advantages of the present invention.

[0046] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0047] To make the objectives, technical solutions, and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0048] Example 1

[0049] S1. Preparation of fresh algae mud. The specific steps are as follows:

[0050] (1) Let the algae solution stand: Take 30 L of Cyclotella cryptica (a living marine unicellular diatom belonging to the class Bacillariophyceae) at the end of the logarithmic growth period, which was independently isolated and cultured by the Marine Functional Materials and Protection Technology Innovation Center of Sanya Institute of Oceanology, Ocean University of China, and place it in a beaker. Let it stand at room temperature for 24 h to settle, and discard the supernatant to obtain the first precipitate.

[0051] In other specific embodiments, Cyclotella cryptica at the end of its logarithmic growth phase can be settled at room temperature for at least 14 hours. The rotational speed is adjusted to 4000-8000 rpm, and the corresponding centrifugation time is 5-20 minutes. The higher the rotational speed, the shorter the centrifugation time. The supernatant is discarded to obtain a first precipitate. Alternatively, an appropriate volume of Cyclotella cryptica can be used, such as 1-5 L, 5-10 L, or 10-20 L, depending on the specific experimental requirements.

[0052] (2) Pure water washing: Add 3 times the volume of pure water to the first precipitate for suspension, that is, the volume ratio of the first precipitate to pure water is 1:3, to obtain the first suspension; the first suspension is divided into 100 mL centrifuge tubes, centrifuged at 5000 rpm for 15 min, and the supernatant is discarded to obtain the second precipitate.

[0053] (3) Repeat the above step (2), wash with pure water again, and obtain the third precipitate after centrifugation, which is fresh algae mud.

[0054] In other embodiments, other volume multiples of pure water can be added, for example, 1-2 times. Alternatively, the speed can be adjusted to 4,000-10,000 rpm, corresponding to a centrifugation time of 5-20 minutes. Higher speeds result in shorter centrifugation times. If the suspension remains turbid after two washes with pure water, increase the number of washes.

[0055] S2, preparation of acid-washed algae mud, the specific steps are as follows:

[0056] (1) The fresh algae mud obtained above was mixed with 2 M hydrochloric acid in a ratio of 1:1, and stirred until the fresh algae mud was fluid and had no obvious large particles. The mixture was heated to 75°C and stirred for 2 h, then the heating was stopped and the mixture was allowed to cool for 24 h. The supernatant after the cooling treatment was removed to obtain the fourth precipitate.

[0057] (2) Add an equal volume of pure water to the fourth precipitate and stir for 1 h to thoroughly wash the fourth precipitate. Centrifuge at 4000 rpm for 20 min, discard the supernatant, and repeat this step twice. Finally, retain the fifth precipitate.

[0058] (3) Mix the fifth precipitate with 0.5 M sodium hydroxide solution, adjust the pH value of the mixture to neutral, and centrifuge at 4000 rpm for 20 min. Discard the supernatant to obtain the acid-washed algae mud of Hidden Cyclotella.

[0059] In other specific embodiments, the rotation speed can also be adjusted to 4000-10000 rpm, and the corresponding centrifugation time is 5-20 min. The higher the rotation speed, the shorter the centrifugation time, as long as the supernatant and the precipitate can be completely separated.

[0060] S3. Preparation of chitin composite polysaccharide powder, the specific steps are as follows:

[0061] (1) Preparation of urea-strong base solution: Prepare urea-strong base solution according to the ratio of urea to sodium hydroxide of 1:2, where urea accounts for 4% and sodium hydroxide accounts for 8%. That is, to prepare 100 mL of urea-strong base solution, take 4 g of urea (purchased from Aladdin Reagent (Shanghai) Co., Ltd.) and 8 g of potassium hydroxide (purchased from Sinopharm Chemical Reagent Co., Ltd.) into a beaker, add 88 mL of pure water, stir and dissolve, and obtain 100 mL of urea-strong base solution. Let it cool to room temperature before use.

[0062] In other specific embodiments, pure water accounts for 80-95% of the total amount of the urea-strong base solution; the strong base can be selected as a strong alkaline substance such as potassium hydroxide; the concentration mass ratio of urea can also be set to 1-10%, and the concentration mass ratio of the strong base can be set to 1-10%, wherein the ratio of urea to strong base is 1:(1-4).

[0063] (2) Dissolution of chitosan polysaccharides: 100 g of acid-washed algae mud of Cyclotella cryptica was taken, and 200 mL of urea-strong alkali solution cooled to room temperature was added. The mixture was stirred thoroughly to dissolve the chitosan polysaccharides in the acid-washed algae mud of Cyclotella cryptica in the urea-strong alkali solution. Since part of the algae mud could not be dissolved, an algae mud suspension was obtained.

[0064] In other specific embodiments, the addition amount may also be changed according to the ratio of fresh algae mud to urea-strong alkaline solution according to W / V=1:1~1:10.

[0065] (3) Microwave treatment: The algae mud suspension was placed in a microwave synthesis reactor (CEM Discover), with the microwave power set to 400 W and the temperature set to 50°C for 20 min. Stirring was performed during the microwave intervals to prevent local overheating and maintain the integrity of the polysaccharide structure.

[0066] (4) After microwave treatment, cool to room temperature and centrifuge at 5000 rpm for 20 min. Keep the upper layer, which is the first solution.

[0067] In other specific embodiments, a continuous microwave extraction system, an industrial microwave reactor, etc. can also be used to perform microwave heating on the algae mud suspension. The rotation speed can also be adjusted to 4000~10000 rpm, and the corresponding centrifugation time is 5~20 min. The higher the rotation speed, the shorter the centrifugation time, as long as the supernatant liquid and the precipitate can be completely separated.

[0068] (5) Ultrafiltration: The first solution was filtered through an ultrafiltration membrane with a molecular weight cutoff of 10,000 Dalton (purchased from Shanghai Mosu Scientific Instrument Co., Ltd.) by tangential flow filtration. The filtrate was discarded and the retentate was retained.

[0069] In other specific embodiments, an ultrafiltration membrane with a molecular weight cutoff of 5000 to 20000 Da can be used for filtration. The larger the molecular weight cutoff of the ultrafiltration membrane, the shorter the required filtration time, that is, the larger the molecular weight retained in the ultrafiltration membrane.

[0070] (6) Concentration: The intercepted liquid obtained above was concentrated using a hysteresis pump (Baoding Refu Fluid Technology Co., Ltd., BT3005 model) with the operating pressure set to 0.1 MPa and the temperature set to 4°C. The liquid was concentrated in a cycle to 1 / 5 of the original volume to obtain a primary concentrate.

[0071] In other embodiments, a pre-treatment step using a 0.45 μm membrane filter can be added before the concentration step to reduce clogging of the membrane pores by colloidal substances. During the concentration process, an operating pressure of 0.1-0.3 MPa and a temperature of 4-37°C can be selected. Each concentration cycle is concentrated to 1 / 5 of the original volume, and repeated 2-4 times to improve the purity of the final concentrate.

[0072] (7) The primary concentrated solution obtained in step (6) is again subjected to ultrafiltration in step (5) and concentration in step (6) to obtain a secondary concentrated solution, referred to as the concentrated solution.

[0073] (8) Freeze-drying: The above concentrate was placed in a freeze dryer (purchased from Ningbo Xinzhi Biotechnology Co., Ltd., SCIENTZ-18N / D model) for vacuum freeze drying at a temperature of -80°C for 24 hours to obtain freeze-dried powder, which was then ground and passed through a 200-mesh sieve to obtain chitosan composite polysaccharide powder. The chitosan composite polysaccharide powder is composed of a plurality of polysaccharides, including chitosan and its derivatives and other small molecule polysaccharides, and the other small molecule polysaccharides are hemicellulose and / or cellulose.

[0074] In other specific embodiments, the vacuum freeze-drying temperature can be set to -60~-80°C, and the vacuum freeze-drying time can be set to 24~52 h, that is, the lower the vacuum freeze-drying temperature, the shorter the time, and the ground freeze-dried powder can be sieved through 200~400 mesh to obtain a finer chitosan complex polysaccharide powder.

[0075] S4. Processing of chitin composite polysaccharide particles. The specific steps are as follows:

[0076] (1) Preparation of ethanol-glycerol complex: Anhydrous ethanol (purchased from Xilong Science Co., Ltd.) was prepared into an ethanol solution with a mass concentration of 90%, and glycerol with a mass concentration of 2% was added to form an ethanol-glycerol complex.

[0077] (2) Spread and level the chitosan composite polysaccharide powder obtained in step S3 (8), spray the ethanol-glycerol complex on its surface for rotary granulation, and sieve it using an 80-mesh sieve until the granulated particles can remain on the 80-mesh sieve.

[0078] (3) The particles retained on the 80-mesh sieve obtained by granulation in the above step S4 (2) were placed in an oven (purchased from Xi'an Zhicheng Electronics Co., Ltd., model ZXSD-B1270) at a temperature of 80°C and a drying time of 0.8 h, thereby obtaining chitin composite polysaccharide particles.

[0079] The results are as follows Figure 1 As shown, the chitin composite polysaccharide particle sample is light yellow granular.

[0080] In other specific embodiments, anhydrous ethanol can be prepared into an ethanol solution with a mass ratio of 30-90%, and glycerol (purchased from Aladdin Reagent (Shanghai) Co., Ltd.) with a mass ratio of 1-10% can be added and mixed to produce an ethanol-glycerol complex. Alternatively, sieving can be performed using a 60-80 mesh sieve until the granules obtained by granulation can be retained on the 60-80 mesh sieve. An oven temperature of 50-80°C and a drying time of 0.8-5.0 hours are selected. The higher the oven temperature, the shorter the drying time. Chitosan polysaccharide granules are obtained. These chitosan polysaccharide granules retain multiple polysaccharide complex components, namely, chitosan and its derivatives and other small molecule polysaccharides, the other small molecule polysaccharides being hemicellulose and / or cellulose.

[0081] S5. Detection of chitin composite polysaccharide particles. The specific steps are as follows:

[0082] (1) Take 100 μg of chitosan composite polysaccharide particles, use an ion sputtering device (referred to as a sputtering device, purchased from Beijing Zhongke Keji Co., Ltd., model SBC-12) to sputter-coat gold for 20 s, and place it under a scanning electron microscope (purchased from Hitachi, Ltd., model SU1000) for observation.

[0083] The results are as follows Figure 2 As shown, the chitosan composite polysaccharide particles have an obvious porous structure.

[0084] (2) Take 1 mg of chitosan composite polysaccharide particles and mix them evenly with 100 mg of potassium bromide (purchased from Sinopharm Chemical Reagent Co., Ltd.). After being fully ground and pressed into tablets, the particles were analyzed using a Fourier transform infrared spectrometer (purchased from BRUKER, USA, INVENIO-S model) in the wavelength range of 4000-400 cm -1 Testing is carried out at the site.

[0085] The results are as follows Figure 3 As shown, at a wavelength of 1646 cm -1 、1539 cm -1 and 1308 cm -1 The peaks representing the stretching of amide I group, amide II group and amide III group of chitin appeared at the bottom, indicating that the chitin composite polysaccharide particles were successfully processed.

[0086] Example 2

[0087] S1. Preparation of fresh algae mud. The specific steps are as follows:

[0088] According to the method in step S1 of Example 1, fresh algae mud and acid-washed algae mud were prepared from Phaeodactylum tricornutum Bohlin (a living marine unicellular diatom belonging to the order Pennate Bacillariophyceae) at the end of the logarithmic growth phase, which was independently isolated and cultured by the Marine Functional Materials and Protection Technology Innovation Center of Sanya Institute of Oceanology, Ocean University of China, to obtain fresh algae mud of Phaeodactylum tricornutum.

[0089] S2, preparation of acid-washed algae mud, the specific steps are as follows:

[0090] The fresh algae mud of Phaeodactylum tricornutum was acid-washed according to the method of step S2 in Example 1 to obtain the acid-washed algae mud of Phaeodactylum tricornutum.

[0091] S3. Preparation of chitin composite polysaccharide powder, the specific steps are as follows:

[0092] (1) Preparation of urea-strong base solution: Prepare urea-strong base solution according to the ratio of urea to potassium hydroxide of 3:4, where urea accounts for 6% and potassium hydroxide accounts for 8%. That is, to prepare 100 mL of urea-strong base solution, take 6 g of urea (purchased from Aladdin Reagent (Shanghai) Co., Ltd.) and 8 g of potassium hydroxide (purchased from Sinopharm Chemical Reagent Co., Ltd.) into a beaker, add 86 mL of pure water, stir and dissolve, and obtain 100 mL of urea-strong base solution. Let it cool to room temperature before use.

[0093] In other specific embodiments, pure water accounts for 80-95% of the total amount of the urea-strong base solution; the strong base can be selected as a strong alkaline substance such as sodium hydroxide; the concentration mass ratio of urea can also be set to 1-10%; the concentration mass ratio of the strong base is set to 1-10%, wherein the ratio of urea to strong base is 1:(1-4).

[0094] (2) Dissolution of chitosan polysaccharides: 100 g of acid-washed algae mud of P. tricornutum was taken, and 100 mL of urea-strong alkali solution cooled to room temperature was added. The mixture was stirred thoroughly to dissolve the chitosan polysaccharides in the acid-washed algae mud of P. tricornutum in the urea-strong alkali solution. Since part of the algae mud could not be dissolved, an algae mud suspension was obtained.

[0095] In other specific embodiments, the addition amount may also be changed according to the ratio of fresh algae mud to urea-strong alkaline solution according to W / V=1:1~1:10.

[0096] (3) Microwave treatment: Place the algae mud suspension in a microwave synthesis reactor, set the microwave power to 600 W, the temperature to 60°C, and microwave for 15 min. Stirring during the microwave intervals prevents local overheating and maintains the integrity of the polysaccharide structure.

[0097] (4) After microwave treatment, cool to room temperature and centrifuge at 6000 rpm for 15 min. Retain the upper layer, which is the first solution.

[0098] In other specific embodiments, a continuous microwave extraction system, an industrial microwave reactor, etc. can also be used to perform microwave heating on the algae mud suspension. The rotation speed can also be adjusted to 4000~10000 rpm, and the corresponding centrifugation time is 5~20 min. The higher the rotation speed, the shorter the centrifugation time, as long as the supernatant and the precipitate can be completely separated.

[0099] (5) Ultrafiltration: The first solution is placed in an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the first solution is subjected to tangential flow filtration. The filtrate is discarded and the retentate is retained.

[0100] In other specific embodiments, an ultrafiltration membrane with a molecular weight cutoff of 5000 to 20000 Da can be used for filtration. The larger the molecular weight cutoff of the ultrafiltration membrane, the shorter the required filtration time, that is, the larger the molecular weight retained in the ultrafiltration membrane.

[0101] (6) Concentration: The above-obtained retentate is concentrated using a pump with an operating pressure of 0.2 MPa and a temperature of 37°C. The retentate is concentrated to 1 / 5 of the original volume in each cycle to obtain a concentrated solution. In other specific embodiments, a pretreatment step of 0.45 μm membrane filtration can be added before the concentration step to reduce the clogging of the membrane pores by colloidal substances. During the concentration process, the operating pressure can be selected from 0.1 to 0.3 MPa and the temperature can be selected from 4 to 37°C. The retentate is concentrated to 1 / 5 of the original volume in each cycle, and this is repeated 2 to 4 times to improve the purity of the final concentrated solution.

[0102] (7) The primary concentrated solution obtained in step (6) is again subjected to ultrafiltration in step (5) and concentration in step (6) to obtain a secondary concentrated solution, referred to as the concentrated solution.

[0103] (8) Freeze-drying: The concentrated solution obtained in this example was placed in a freeze dryer for vacuum freeze drying at a temperature of -60°C for 52 hours to obtain a freeze-dried powder, which was then ground and passed through a 100-mesh sieve to obtain a chitosan polysaccharide powder. The chitosan polysaccharide powder is composed of a plurality of polysaccharides, including chitosan and its derivatives and other small molecule polysaccharides, wherein the other small molecule polysaccharides are hemicellulose and / or cellulose.

[0104] In other specific embodiments, the vacuum freeze-drying temperature can be set to -60~-80°C, and the vacuum freeze-drying time can be set to 24~52 h, that is, the lower the vacuum freeze-drying temperature, the shorter the time. The ground freeze-dried powder is sieved through a 100~200 mesh sieve to obtain a finer chitin complex polysaccharide powder.

[0105] S4. Processing of chitin composite polysaccharide particles. The specific steps are as follows:

[0106] (1) Preparation of ethanol-glycerol complex: Anhydrous ethanol is prepared into an ethanol solution with a mass ratio of 60%, and glycerol with a mass ratio of 1% is added to form an ethanol-glycerol complex.

[0107] (2) The chitosan composite polysaccharide powder obtained in this example was spread out and flattened, and the ethanol-glycerol complex was sprayed on the surface of the powder, and then the powder was subjected to rotary granulation and sieved using a 40-mesh sieve until the granules obtained by granulation could be retained on the 40-mesh sieve.

[0108] (3) The particles retained on the 40-mesh sieve obtained by the granulation in this embodiment are placed in an oven at a temperature of 50°C and a drying time of 5.0 h, thereby obtaining powdered chitosan composite polysaccharide particles.

[0109] The results are as follows Figure 4 As shown, the chitin composite polysaccharide particle sample is light yellow granular.

[0110] In other specific embodiments, anhydrous ethanol can be prepared into an ethanol solution with a mass ratio of 30-90%, and glycerol (purchased from Aladdin Reagent (Shanghai) Co., Ltd.) with a mass ratio of 1-10% can be added and mixed to produce an ethanol-glycerol complex. Alternatively, sieving can be performed using a 20-60 mesh sieve until the granules obtained by granulation can be retained on the 20-60 mesh sieve. An oven temperature of 50-80°C and a drying time of 0.8-5.0 hours are selected. The higher the oven temperature, the shorter the drying time. Chitosan polysaccharide granules are obtained. These chitosan polysaccharide granules retain multiple polysaccharide complex components, namely, chitosan and its derivatives and other small molecule polysaccharides, the other small molecule polysaccharides being hemicellulose and / or cellulose.

[0111] S5. Detection of chitin composite polysaccharide particles. The specific steps are as follows:

[0112] (1) Take 100 μg of chitosan polysaccharide particles of this example, use ion sputtering equipment to sputter gold for 20 s, then observe the gold-plated chitosan polysaccharide particles under a scanning electron microscope. The results are as follows: Figure 5 As shown, the chitosan composite polysaccharide particles have an obvious porous structure.

[0113] (2) Take 1 mg of chitosan composite polysaccharide particles of this example, mix them with 100 mg of potassium bromide, grind them thoroughly and press them into tablets, and then use Fourier transform infrared spectrometer to analyze the particles in the wavelength range of 4000-400 cm -1 Testing is carried out at the site.

[0114] The results are as follows Figure 6 As shown, at a wavelength of 1700 cm -1 、1543 cm -1 、1299 cm -1 The peaks representing the stretching of amide I group, amide II group and amide III group of chitin appeared at the bottom, indicating that the chitin composite polysaccharide particles were successfully prepared.

[0115] Comparative Example 1

[0116] Compared with Example 1, the difference is that the ratio of potassium hydroxide to urea and pure water in the urea-strong alkali solution is different. The urea-strong alkali solution in Comparative Example 1 is configured according to the ratio of urea to potassium hydroxide to pure water of 2:4:94, that is, 100 mL of urea-strong alkali solution contains 2 g of urea, 4 g of potassium hydroxide and 94 mL of pure water, and chitin composite polysaccharide particles are obtained by processing.

[0117] Comparative Example 2

[0118] Compared with Example 2, the difference is that urea-strong alkali solution is not used to suspend the fresh algae mud, but 100 mL of pure water is directly used for suspension to obtain chitosan composite polysaccharide particles.

[0119] Test Example 1 Moisturizing Test

[0120] The chitosan composite polysaccharide particles processed in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 were tested for moisture retention. The specific steps are as follows:

[0121] Appropriate amounts of the chitosan composite polysaccharide particles obtained in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were weighed respectively, and each was configured into a suspension with a weight ratio of 1% (i.e., 1 g of the chitosan composite polysaccharide particles was added to 99 mL of pure water and mixed evenly to obtain a pure water suspension with a weight ratio of 1%), and were respectively labeled as Example 1 suspension, Example 2 suspension, Comparative Example 1 suspension, and Comparative Example 2 suspension.

[0122] The suspension of Example 1, the suspension of Example 2, the suspension of Comparative Example 1, and the suspension of Comparative Example 2 were respectively placed in a constant temperature and humidity environment, the constant temperature was 24°C, the constant humidity was 81% water content, and the suspensions were taken out and weighed at the 1st hour, 2nd hour, 4th hour, 8th hour, 12th hour, 24th hour, 36th hour, 48th hour, and 72nd hour, respectively. The experiment was repeated three times, and the moisture retention (Rh) of the suspension of Example 1, the suspension of Example 2, the suspension of Comparative Example 1, and the suspension of Comparative Example 2 was calculated according to the moisture retention (Rh) formula.

[0123] The moisturizing (Rh) formula is as follows: ;

[0124] Among them, H n It represents the mass of the sample at a certain moment during the experiment; H0 represents the mass of the initial sample.

[0125] Because the moisturizing data of the suspension in Comparative Example 2 was too low at 72 h, it was shown that the chitosan composite polysaccharide particles obtained in Comparative Example 2 were not suitable for wide application in aquaculture, biomedicine, environmental engineering, engineering materials or chemical engineering, and were therefore not included in the moisturizing statistical results.

[0126] The test results are as follows Figure 7 As shown in the results, the suspension of Example 1 can still maintain more than 50% of the moisture retention after 72 hours, and the suspension of Example 2 can still maintain more than 47% of the moisture retention after 72 hours, indicating that the chitosan composite polysaccharide particles obtained by the processing method in Example 1 and the chitosan composite polysaccharide particles obtained by the processing method in Example 2 both have good moisture retention properties.

[0127] The suspension of Example 1 had a moisture retention of 77.10% at 8 hours, 63.84% at 24 hours, 55.39% at 48 hours, and 50.16% at 72 hours. The suspension of Example 2 had a moisture retention of 75.93% at 8 hours, 62.92% at 24 hours, 53.53% at 48 hours, and 47.85% at 72 hours.

[0128] The moisturizing performance of the suspension in Comparative Example 1 was significantly lower than that of the suspensions in Example 1 and Example 2, with the moisturizing performance being less than 35% after 72 hours, indicating that the powdered chitosan composite polysaccharide particles obtained in Comparative Example 1 had poor moisturizing performance. The moisturizing performance of the suspension in Comparative Example 1 was 69.49% at 8 hours, 51.96% at 24 hours, 41.25% at 48 hours, and only 31.74% at 72 hours.

[0129] The results showed that reducing the ratio of urea to strong alkali in the urea-strong alkali solution, or not using the urea-strong alkali solution to suspend the algae mud, resulted in insufficient solubility of the chitin complex polysaccharide, and the resulting chitin complex polysaccharide granules also decreased in moisture retention, making them difficult to apply in other fields such as aquaculture feed, environmental engineering, food science, engineering materials, or chemical engineering. Increasing the ratio of urea to strong alkali in the urea-strong alkali solution within a certain range increased the content of chitin and other polysaccharides in the chitin complex polysaccharide granules, thereby improving the moisture retention of the single chitin complex polysaccharide granules.

[0130] Example 3

[0131] The results of Test Example 1 show that the chitosan composite polysaccharide particles prepared in Example 1 have the highest moisture retention. Therefore, Example 3 adopts the preparation method of Example 1, with the difference that Example 3 uses Thalassiosira pseudonana (a living marine unicellular diatom belonging to the Thalassiosiraceae family) for processing to obtain chitosan composite polysaccharide particles derived from Thalassiosira pseudonana.

[0132] Example 4

[0133] The results of Test Example 1 show that the chitosan composite polysaccharide particles prepared in Example 1 have the highest moisturizing properties. Therefore, Example 4 adopts the preparation method of Example 1, with the difference that Example 4 uses unicellular diatom Coscinodiscus lacustris (a living marine unicellular diatom belonging to the Coscinodiscus family) for processing to obtain chitosan composite polysaccharide particles derived from Coscinodiscus lacustris.

[0134] Test Example 2

[0135] The chitosan composite polysaccharide particles prepared in Example 3 and Example 4 were further subjected to a moisture retention (Rh) test using the same testing method as in Test Example 1. The results showed that the chitosan composite polysaccharide particles prepared in Example 3 and Example 4 had good moisture retention, and similar results to those obtained for the moisture retention of the chitosan composite polysaccharide particles prepared in Example 1 were obtained, i.e., the moisture retention was greater than 75% at 8 h, greater than 60% at 24 h, greater than 50% at 48 h, and greater than 45% at 72 h.

[0136] Test Example 3 Moisture absorption rate test

[0137] The chitin composite polysaccharide particles prepared in Examples 1, 2, 3 and 4 above and the homozygous β-chitosan prepared earlier by our research group (derived from the patent document with publication number CN117510680A) were used to test moisture absorption rates.

[0138] (1) Weigh 1 g of the chitin composite polysaccharide particles prepared in Examples 1, 2, 3 and 4 and the homozygous β-chitosan prepared in the early stage of this research group, respectively, and dry them under the same temperature and humidity conditions. Each sample was repeated three times, and the same number of weighing cups were used to dry the samples at the same time. The drying conditions were drying at 60°C for 4 h, and then naturally cooling and setting aside.

[0139] (2) The cooled chitin composite polysaccharide particles obtained in Example 1, Example 2, Example 3 and Example 4 and the homozygous β-chitosan prepared in the early stage of this research group were spread flat in a weighing cup and placed in a moisturizer respectively, and marked as the first group, the second group, the third group, the fourth group and the fifth group respectively; and took out and weighed them at the 1st minute, the 5th minute, the 10th minute, the 30th minute and the 60th minute respectively, or took out and weighed them at a certain time according to the actual situation. The experiment was repeated three times, and the moisture absorption rate of the first group, the second group and the third group of test samples was calculated according to the moisture absorption rate (W) formula.

[0140] The formula for moisture absorption (W) is as follows: ;

[0141] Where Wi represents the moisture absorption rate of the test sample in air at a certain humidity; mi represents the mass of the weighing cup and sample after being placed in the humidifier for i minutes (in g); ms represents the combined mass of the dry, empty weighing cup and the test sample (in g); and m0 represents the mass of the dry, empty weighing cup (in g). The average value of Wi calculated from several test results is the moisture absorption rate (W) of the test sample.

[0142] The results showed that the first group of chitin composite polysaccharide particles had the fastest moisture absorption rate, followed by the second, third, and fourth groups, and finally the fifth group of pure β-chitosan. It can be found that the moisture absorption rate of the chitin composite polysaccharide particles processed in this application is significantly greater than the moisture absorption rate of the pure β-chitosan prepared by the research team in the early stage.

[0143] In addition, chitosan composite polysaccharide particles can also be prepared using other unicellular diatoms according to the methods described in the above examples, and the technical effect of maintaining a moisture retention of more than 45% after 72 hours can also be achieved. Among them, the other unicellular diatoms are Central Bacillales or Pennate Bacillales; Central Bacillales includes Cyclostriales, Rhizoschiales, and Boxes; Pennate Bacillales includes Navicales, Curculales, Diploschiales, Phaeodactylales, Aconicales, and Brachyschiales. Cyclostriales includes Cyclostriaceae; Boxes include Chaetocerosaceae; Navicales includes Navicaceae; and Phaeodactylales includes Phaeodactylaceae. Chaetocerosaceae includes Chaetoceros muelleri; and Navicula includes Navicula elegans.

[0144] It can be concluded that: in this application, fresh algae mud is first prepared from unicellular diatoms, which is then acid-washed to obtain acid-washed algae mud. The chitin complex polysaccharide in the unicellular diatom is dissolved in a urea-strong alkaline solution by microwave heating, and ultrafiltration is performed to remove salt, urea and biological small molecule impurities. After concentration, the mixture is vacuum freeze-dried, ground and sieved to obtain chitin complex polysaccharide powder. The ethanol-glycerol complex obtained by mixing a 30~90% concentration mass ratio of ethanol solution and a 1~10% concentration mass ratio of glycerol is sprayed on its surface, rotary granulated, sieved and dried to obtain chitin complex polysaccharide particles, which retain a variety of polysaccharide complex components, such as chitin and its derivatives and small molecule polysaccharides such as hemicellulose and / or cellulose.

[0145] Compared with the prior art, the marine unicellular diatom processing method and processed products provided by this application have the following beneficial effects:

[0146] (1) The raw materials required for the marine unicellular diatom processing method of the present application are easy to cultivate, extract, and enrich, and the operation is simple and convenient, which is a further development of marine resources.

[0147] (2) The processing method of marine unicellular diatoms in the present application abandons the traditional polar treatment, oxidation treatment and deacetylation treatment, so that the processed product is a chitin complex polysaccharide, including chitin and its derivatives and small molecular polysaccharides such as hemicellulose and / or cellulose, etc., which does not contain irritating chemicals and has mild ingredients.

[0148] (3) The dynamic wet environment is established by the process's own characteristics of water absorption, insolubility, and degradability. The process has a small molecular weight, short molecular chain, large specific surface area, and is easier to disperse. It can absorb trace moisture in the air through surface exposed groups such as hydroxyl (-OH) and acetylamino (-NHCOCH3) to achieve rapid moisture absorption. It releases some moisture at high humidity and directly binds to water molecules through hydrogen bonds, increasing local humidity in a short period of time. This allows the process to maintain a moisture retention of more than 45% after 72 hours. It can be widely used in aquaculture feed, biomedicine, environmental engineering, engineering materials, or chemical engineering. It also provides an innovative path for the efficient utilization and ecological protection of marine biomass. Future research can further optimize the particle preparation process and explore the molecular modification of polysaccharide components to improve the functionalization and industrialization level of the product.

[0149] The terms and expressions used herein are for descriptive purposes only, and the present invention should not be limited to these terms and expressions. The use of these terms and expressions is not intended to exclude any equivalent features illustrated and described (or portions thereof), and it should be recognized that various modifications are also within the scope of the claims. Other modifications, variations, and substitutions are possible. Accordingly, the claims are intended to cover all such equivalents.

[0150] Similarly, it should be pointed out that although the present invention has been described with reference to the current specific embodiments, ordinary technicians in this technical field should realize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions can be made without departing from the spirit of the present invention. Therefore, as long as the changes and modifications to the above embodiments are within the scope of the essential spirit of the present invention, they will fall within the scope of the claims of the present invention.

Claims

1. A method for processing marine unicellular diatoms, characterized in that: The following steps are involved: Step A: preparing fresh algae mud using fresh marine unicellular diatoms, and washing with acid to obtain acid-washed algae mud; Step B: using a urea-strong base solution to dissolve the polysaccharide in the acid-washed algae mud by microwave heating to obtain a first solution, ultrafiltering the first solution and concentrating it, and vacuum freeze-drying it to obtain a chitosan composite polysaccharide powder; the urea-strong base solution contains 80-88% water; the ratio of urea to strong base is 1:1-4; the microwave dissolution is performed at a power of 200-800 W, a temperature of 20-60° C., and a time of 15-20 min; Step C: Granulating the chitosan polysaccharide powder with an ethanol-glycerol mixture, and obtaining a processed product after drying; the processed product is chitosan polysaccharide particles, which include chitosan and its derivatives, hemicellulose and cellulose.

2. The method for processing marine unicellular diatoms according to claim 1, characterized in that: The step A comprises: Step A1: sedimenting the algae liquid of the fresh marine unicellular diatom, washing it with pure water at least once, and centrifuging it to obtain a precipitate; the precipitate is the fresh algae mud; Step A2: washing the fresh algae mud with hydrochloric acid having a concentration of 1 to 4 M to obtain the acid-washed algae mud.

3. The method for processing marine unicellular diatoms according to claim 2, characterized in that: The fresh marine unicellular diatoms include the Central Diatoms or the Pennate Diatoms; The central diatoms include Cyclostriales, Rhizoschiales and Boxes; the feathered diatoms include Naviculales, Curculigoles, Diplodocrophorales and Phaeodactylales; the Cyclostriales include Cyclostriaceae and Thalassiosirae; the Boxes include Chaetocerosaceae; the Naviculales include Naviculaceae; the Phaeodactylales include Phaeodactylaceae.

4. The method for processing marine unicellular diatoms according to claim 1, characterized in that: The step B consists of step B-1, step B-2, step B-3 and step B-4: Step B-1: mixing the urea-strong alkali solution with the acid-washed algae mud to obtain an algae mud suspension; wherein the acid-washed algae mud and the urea-strong alkali solution are mixed at a W / V ratio of (1:1) to (1:10); The step B-2: subjecting the algae mud suspension to microwave treatment and centrifuging to obtain the first solution and algae mud precipitate; Step B-3: ultrafiltration of the first solution using an ultrafiltration membrane to obtain a retentate, centrifuging and concentrating the supernatant to obtain a concentrated solution; The step B-4: using a freeze dryer to vacuum freeze-dry the concentrated solution to obtain freeze-dried powder, grinding and sieving to obtain the chitosan composite polysaccharide powder.

5. The method for processing marine unicellular diatoms according to claim 4, characterized in that: The strong base in step B-1 is potassium hydroxide or sodium hydroxide; The ultrafiltration membrane in step B-3 has a cutoff of 5000-20000 Da; the centrifugation is performed at a speed of 4000-10000 rpm for 5-20 min; The vacuum freeze-drying conditions in step B-4 are: -80°C for 24-32 hours or -60°C for 32-52 hours; and the mesh size is 100-400 mesh.

6. The method for processing marine unicellular diatoms according to claim 1, characterized in that: The step C is specifically as follows: The chitosan composite polysaccharide powder is surface-sprayed with the ethanol-glycerol mixture, and rotary granulation and mesh sieving are performed, and the chitosan composite polysaccharide particles are obtained after drying.

7. The method for processing marine unicellular diatoms according to claim 6, characterized in that: The ethanol-glycerol mixture comprises anhydrous ethanol with a mass ratio of 30-90% and glycerol with a mass ratio of 1-10%; the mesh size of the sieve is 20-80 meshes.

8. The method for processing marine unicellular diatoms according to claim 1, characterized in that: The chitosan composite polysaccharide particles have moisturizing properties, and the moisturizing properties of the chitosan composite polysaccharide particles are greater than 45% after 72 hours; The test conditions for the moisture retention are a constant temperature and humidity environment, with the constant temperature being 24° C. and the constant humidity being 81%.

9. A chitosan composite polysaccharide particle, characterized in that: The chitosan composite polysaccharide particles are obtained by processing the marine unicellular diatoms according to any one of claims 1 to 8; The chitosan composite polysaccharide particles can be used alone, covered or bandaged with medical dressings, or used as auxiliary medicines.

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