Method for improving yield of agar in gracilaria lemaneiformis by using 1-hydroxyethylidene-1, 1-diphosphonic acid

Abstract

The invention provides a method for increasing the yield of agar in gracilaria lemaneiformis by using 1-hydroxyethylidene-1, 1-diphosphonic acid. The method comprises the following steps: firstly, pre-culturing the gracilaria lemaneiformis to enable the frond to adapt to a culture environment, then adding hydroxyethylidene diphosphonic acid to culture the gracilaria lemaneiformis, and when the concentration of the hydroxyethylidene diphosphonic acid is 3.75 mg / L, obviously improving the agar content of the gracilaria lemaneiformis by 25.22% compared with a control group; by means of the culture method, the agar content and the agar yield of the asparagus can be remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for increasing the yield of Gracilaria lemaneiformis agar by utilizing hydroxyethylidene diphosphonic acid. Background Art

[0002] Agar is a major component of the extracellular matrix of some marine red macroalgae. For these macroalgae, agar plays a crucial role in their adaptation to the marine environment, such as enhancing their strength, coping with strong currents and waves, and acting as a barrier against osmotic pressure and pathogens. Due to its unique and excellent physical and chemical properties, agar has a wide range of applications in the food industry, medicine, cosmetics, scientific research, and other fields. Globally, agar production has increased from 9,600 tons in 2009 to 15,000 tons in 2019. Despite this, agar continues to be in short supply.

[0003] However, agar cannot be synthesized artificially, and industrial-scale agar can only be extracted from members of the Geliaceae and Gracilariaceae families. In recent years, Gracilaria has become the main source of agar extraction, among which Gracilaria ( Gracilariopsis lemaneiformis ) is widely cultivated in China as a source of commercial agar. However, industrial production of Gracilaria lemaneiformis is limited by its low agar content. Therefore, finding an effective strategy to increase agar yield in Gracilaria lemaneiformis is crucial.

[0004] Existing methods for increasing alginate production in seaweed often involve modifying environmental factors, such as altering salt concentration (Hayashi et al., 2011), nitrogen concentration (Martinez and Buschmann, 1996), and light intensity and duration (Said et al., 2018). However, environmental conditions are extremely difficult to alter for cultivating Gracilaria lemaneiformis. Therefore, these methods are primarily used to study the biosynthesis pathway of Gracilaria lemaneiformis in laboratory conditions. Summary of the Invention

[0005] The invention provides a method for increasing the yield of Gracilaria lemaneiformis agar by utilizing hydroxyethylidene diphosphonic acid, thereby overcoming the deficiencies of the prior art.

[0006] The present invention provides a method for increasing the yield of agar-agar in Gracilaria lemaneiformis, which comprises adding hydroxyethylidene diphosphonic acid into the culture liquid of Gracilaria lemaneiformis; Furthermore, the added concentration of hydroxyethylidene diphosphonic acid is not higher than 30 mg / L; Furthermore, the added concentration of hydroxyethylidene diphosphonic acid is 3.75-15 mg / L; In more detail, the method comprises the following steps: 1) Pre-cultivation of Gracilaria: At a temperature of 23°C and a light intensity of 150 mmol / m 2 / s environment to cultivate Gracilaria; The culture medium used is seawater supplemented with Provasoli medium; 2) Cultivation of Gracilaria using hydroxyethylidene diphosphonic acid: Seawater supplemented with Pro medium was used as the basal medium, hydroxyethylidene diphosphonic acid was added, and pre-cultured Gracilaria was inoculated. Air was continuously introduced and the medium was kept at a temperature of 23°C and a light intensity of 150 mmol / m 2 / s environment.

[0007] The method of the present invention can significantly increase the agar content and yield of Gracilaria lemaneiformis, thereby making the agar production of Gracilaria lemaneiformis more efficient and economical. This provides a new technical approach for the industrialized production of agar by Gracilaria lemaneiformis and strongly supports the sustainable development of the agar industry. Furthermore, the technical advantage of the present invention is also reflected in its environmental friendliness. Since HEDP can be purified from the environment and waste discharge can be reduced during the production process, it conforms to the concept of modern green chemical industry.

[0008] In summary, the present invention is not only technologically innovative and advanced, but also exhibits significant economic and environmental advantages. It provides a new, efficient, economical, and environmentally friendly method for the industrialized production of Gracilaria agar, and is expected to promote the development of the entire agar industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0010] Figure 1 Schematic diagram of the structure of hydroxyethylidene diphosphonic acid used in the present invention.

[0011] Figure 2A 1 is a graph showing the effect of different concentrations of hydroxyethylidene diphosphonic acid on the biomass of Gracilaria lemaneiformis provided in an embodiment of the present invention; Figure 2B 3. This is a graph showing the effect of different concentrations of hydroxyethylidene diphosphonic acid on the content of agar in Gracilaria lemaneiformis provided in an embodiment of the present invention; Figure 3A 1 is a graph showing the effect of different concentrations of sodium silicate on the biomass of Gracilaria lemaneiformis provided in an embodiment of the present invention; Figure 3B 1 is a graph showing the effect of different concentrations of sodium silicate on the agar content of Gracilaria lemaneiformis provided in an embodiment of the present invention; Figure 4A 1 is a graph showing the effect of different concentrations of glycerol on the biomass of Gracilaria lemaneiformis provided in an embodiment of the present invention; Figure 4B 3. This is a graph showing the effect of different glycerol concentrations on the agar content of Gracilaria lemaneiformis provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0012] The applicant has found that adding HEDP during the cultivation of Gracilaria lemaneiformis can increase the agar content of Gracilaria lemaneiformis without losing biomass.

[0013] like Figure 1 As shown, the hydroxyethylidene diphosphonic acid (HEDP) used in this invention is an organophosphoric acid scale and corrosion inhibitor that forms stable complexes with various metal ions, such as iron, copper, and zinc, and can dissolve oxides on metal surfaces. The core structure of HEDP consists of a carbon atom (C) connected to two phosphonic acid groups (-PO3H2), also attached to a hydroxyl group (-OH). This structure makes HEDP stable, resistant to hydrolysis, and difficult to decompose under normal light and heat conditions. It also exhibits good resistance to acids, alkalis, and chlorine oxidation.

[0014] The method for improving Gracilaria agar using hydroxyethylidene diphosphonic acid provided by the present invention comprises the following steps: 1) Gracilaria Gracilariopsis lemaneiformis ) pre-culture: at a temperature of 23°C and a light intensity of 150 mmol / m 2 / s environment, using seawater added with Pro culture medium to cultivate Gracilaria, Gracilaria adapts to the culture environment, and enters the next stage after 2-3 days of culture.

[0015] 2) Cultivation of Gracilaria using hydroxyethylidene diphosphonic acid: Using seawater supplemented with Pro medium as the basal medium, add different concentrations of hydroxyethylidene diphosphonic acid, including 0 mg / L, 3.75 mg / L, 7.5 mg / L, 15 mg / L, and 30 mg / L. Weigh Gracilaria into a 2000 mL conical flask containing 1500 mL of medium, ensuring that the inoculum size is 2.0 g per flask. Continuously aerate the flask and keep it at 23°C under a light intensity of 150 mmol / m 2 / s environment, and the algae were collected after 13 days of culture.

[0016] The steps of extracting agar from the Gracilaria algae used in the present invention are as follows: Wash the Gracilaria algae twice with pure water, wipe the surface dry with absorbent paper, freeze at -20°C for two hours, and then freeze-dry in a freeze dryer. Grind the freeze-dried algae into a powder. Weigh 0.1 g of freeze-dried powder, add 4 ml of 2.5% NaOH solution, and heat in an 85°C water bath for 2 hours. Pass the treated material through a layer of 300-mesh gauze to remove the alkali solution and wash with distilled water three times. Collect the algae, place them in a beaker, add 10 mL of double-distilled water, adjust the pH to 6.5 with 0.1 mol / L HCl, pass the liquid through a layer of 300-mesh gauze, and wash the liquid three times with double-distilled water. Add 6 mL of distilled water to the treated algae and place in an autoclave at 120-125°C for 2 hours. Place the material through a layer of 300-mesh silk screen and filter by force. Place the collected supernatant into pre-weighed tin foil and freeze overnight at -20°C. Freeze the material in a freeze dryer. Weigh the extract and foil together and record the weight. Subtract the weight of the foil to obtain the dry weight of the agar.

[0017] The present invention is described in detail below with reference to the embodiments and accompanying drawings. Example 1:

[0018] The present embodiment provides a method for increasing the biomass and agar content of Gracilaria lemaneiformis at a concentration of 3.75 mg / L of hydroxyethylidene diphosphonic acid, and the specific steps are as follows: 1) Pre-cultivation of Gracilaria: Gracilaria lemaneiformis was cultured in seawater using Provasoli medium (hereinafter referred to as Pro medium) at a temperature of 23°C and a light intensity of 150 mmol / m 2 / s environment, the agaricus is adapted to the culture environment and enters the next stage after 2-3 days of culture.

[0019] 2) Cultivation of Gracilaria using hydroxyethylidene diphosphonic acid: To the seawater supplemented with Pro medium as the basal medium, hydroxyethylidene diphosphonic acid was added to a final concentration of 3.75 mg / L, and then the Gracilaria pre-cultured in step 1) was inoculated at an inoculum size of 2.0 g / bottle. Air was continuously aerated and the medium was kept at a temperature of 23°C and a light intensity of 150 mmol / m 2 / s environment and then culture for 10-15 days to detect the agar content.

[0020] Extraction of agar from the cells of Gracilaria lemaneiformis obtained in step (2): The final Gracilaria lemaneiformis algae obtained in step (2) was washed twice with pure water, and the surface moisture was wiped dry with absorbent paper. After freezing in a -20℃ refrigerator for two hours, it was placed in a freeze dryer for freeze drying. The freeze-dried algae was ground into powder. Weigh 0.1 g of freeze-dried powder, add 4 ml of 2.5% NaOH solution, and heat it in a constant temperature water bath at 85℃ for 2 h. The treated material was passed through a layer of 300 mesh gauze to remove the alkali solution, and washed with distilled water, and repeated 3 times. The algae were collected and placed in a beaker, 10 mL of double-distilled water was added, and the pH was adjusted to 6.5 with 0.1 mol / L HCl. The algae solution was passed through a layer of 300 mesh gauze and washed with double-distilled water 3 times. The treated algae were added with 6 mL of distilled water and placed in a pressure cooker at 120-125℃ for 2 h. The material was placed on a layer of 300 mesh sieve and squeezed and filtered. The collected supernatant was placed in pre-weighed tin foil and frozen at -20°C overnight. The above materials were freeze-dried in a freeze dryer. The extract and foil were weighed and recorded, and the dry weight of the agar was obtained by subtracting the weight of the foil. The biomass was 9.30 g, which was higher than the control group, but not significantly different. The agar content was 15.84% of the dry weight ( Figure 2A-2B ), an increase of 25.22% compared with the control group. Example 2:

[0021] Compared with Example 1, the added concentration of hydroxyethylidene diphosphonic acid was adjusted to 7.5 mg / L.

[0022] The final results showed that the biomass of Gracilaria lemaneiformis was 9.05 g, which was not significantly different from the control group; the agar content was 14.61% of the dry weight (see Figure 2A-2B ), an increase of 15.49% compared with the control group. Example 3:

[0023] Compared with Example 1, the added concentration of hydroxyethylidene diphosphonic acid was adjusted to 15 mg / L.

[0024] The final results showed that the biomass of Gracilaria lemaneiformis was 8.49 g, which was not significantly different from the control group; the agar content was 15.14% of the dry weight (see Figure 2A-2B ), an increase of 19.68% compared with the control group. Example 4:

[0025] In this example, the concentration of hydroxyethylidene diphosphonic acid is 30 mg / L The results of the culture showed that the biomass of Gracilaria lemaneiformis was 7.82, which was significantly lower than that of the control group; the agar content was 12.27% of the dry weight (see Figure 2A-2B ), which was 3.0% lower than that in the control group. Example 5:

[0026] This embodiment is to add sodium silicate to the culture medium to promote the effect of Gracilaria biomass and agar content, and the specific steps are as follows: 1) Pre-culture of Gracilaria lemaneiformis: same as in Example 1.

[0027] 2) Cultivating Gracilaria using Sodium Silicate: Seawater supplemented with Pro medium was used as the basal medium. Sodium silicate at different concentrations was added. Gracilaria was weighed and placed in a 2000 mL conical flask containing 1500 mL of medium. The inoculum size was 3.0 g / bottle. Air was continuously introduced and the flask was kept at 23°C and a light intensity of 150 mmol / m 2 / s environment.

[0028] The agar in the cells of Gracilaria obtained in step (2) was extracted. The results showed that the biomass of Gracilaria changed significantly, and lower concentrations of sodium silicate promoted the biomass; the highest agar content was 11.08% of the dry weight ( Figure 3A~Figure 3B ). Example 6:

[0029] In the embodiment of the present invention, glycerol is added to detect and analyze the biomass and agar content of Gracilaria lemaneiformis. The specific steps are as follows: 1) Pre-culture of Gracilaria lemaneiformis: same as in Example 1.

[0030] 2) Cultivation of Gracilaria using glycerol: Using seawater supplemented with Pro medium as the basal medium, add different concentrations of glycerol and weigh Gracilaria into a 2000 mL conical flask containing 1500 mL of medium, ensuring that the inoculum amount of Gracilaria is 3.0 g / flask. Continuously aerate the flask and keep it at 23°C and a light intensity of 150 mmol / m 2 / s environment.

[0031] Extraction of agar from Gracilaria cells obtained in step 2): The extraction steps for Gracilaria agar were the same as in Example 1. The final results showed that high concentrations of glycerol had a significant inhibitory effect on biomass, while other concentrations of glycerol had no significant effect on Gracilaria biomass; the agar content in the experimental group was not increased compared to the control group ( Figure 4A-4B ).

[0032] In summary, the present invention promotes the accumulation of agar in Gracilaria by exogenously adding hydroxyethylidene diphosphonic acid, and can significantly increase the agar content of Gracilaria at a concentration of 3.75 mg / L hydroxyethylidene diphosphonic acid.

Claims

1. A method for increasing the yield of Gracilaria lemaneiformis agar, characterized in that, The method comprises the steps of adding hydroxyethylidene diphosphonic acid into the culture liquid of Gracilaria lemaneiformis and then culturing the liquid.

2. The method according to claim 1, wherein The added concentration of the hydroxyethylidene diphosphonic acid is not higher than 30 mg / L.

3. The method according to claim 1, wherein The added concentration of the hydroxyethylidene diphosphonic acid is not higher than 15 mg / L.

4. The method according to claim 1, wherein The added concentration of the hydroxyethylidene diphosphonic acid is 3.75 mg / L.

5. The method according to claim 1, wherein The method comprises the steps of pre-culturing the Gracilaria lemaneiformis and then inoculating the Gracilaria lemaneiformis into a culture liquid added with hydroxyethylidene diphosphonic acid.

6. The method according to claim 5, wherein The pre-culture was carried out at a temperature of 23°C and a light intensity of 150 mmol / m 2 / s environment to cultivate Asparagus cochinchinensis.

7. The method according to claim 5, wherein The culture medium used in the pre-culture is seawater added with Provasoli culture medium.

8. The method according to claim 1, wherein The culture temperature is 23 ° C and the light intensity is 150 mmol / m 2 / s.

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