Preparation method for low-temperature sol agar

Abstract

Disclosed in the present invention is a preparation method for a low-temperature sol agar, comprising the following steps: subjecting Gracilaria after microbial fermentation to an alkali treatment, cleaning, two-step pickling, cleaning, bleaching, sol boiling, dehydration, crushing, screw extrusion, microwave drying and the like to prepare the low-temperature sol agar. In the preparation method of the low-temperature sol agar according to the present invention, a microbial fermentation-assisted alkali method is used for extracting and modifying an agar product, and a thermal denaturation processing technology is further combined to prepare the low-temperature sol agar, which not only improves the application performance of the agar product as well as the commercial value of the product, but also reduces the usage amount of an alkali liquor in traditional technology while reducing the production cost, thereby providing environmental friendliness.

Description

A preparation method of low-temperature sol agar Technical Field

[0001] The present invention relates to the technical field of agar processing, in particular to a method for preparing low-temperature sol agar. Background Art

[0002] Agar is a functional polysaccharide extracted from red algae (primarily Gracilaria). It is a macromolecular polysaccharide formed by repeated crosslinking of β-D-galactose and α-3,6-endo-L-galactose. Agar is off-white or pale yellow, odorless, and has high gel strength, high transparency, high elasticity, and toughness. It is also hydrophilic and widely used in food processing, medicine, and bioengineering.

[0003] Currently, agar, a food additive, typically requires temperatures exceeding 95-100°C for more than 20 minutes to fully dissolve. This high temperature and time required for dissolution also contribute to poor dispersibility and diffusion of high-gel-strength agar, resulting in uneven dissolution. Furthermore, its ability to dissolve only at high temperatures makes agar unsuitable for use as a packaging material or compound for heat-sensitive food actives. These factors limit agar's application in food.

[0004] Cryosol agar is a high-value, deep-processed product that dissolves quickly at low temperatures by modifying agar raw material to break the hydrogen bonds within the agar chains. Because the double helix structure of agar is disrupted during processing, cryosol agar exhibits lower gel strength and poorer gel stability than agar. Cryosol agar also boasts high transparency, low viscosity, and high purity. The gelation temperature of cryosol agar is generally below 85°C, and the gelation time ranges from a few minutes to over ten minutes, making it easier to gel than agar.

[0005] At present, the preparation methods of low-temperature sol agar include physical, chemical and bio-enzymatic methods. The physical method is to destroy the hydrogen bonds between agar molecules, increase the solubility of agar in water, and reduce the sol temperature of agar by adding co-solvents or physical processing processes such as ball milling and spray drying. However, the effect of this physical modification is not sufficient and the improvement effect is not significant. The chemical method promotes low-temperature sol by adding chemical reagents to change the structure of agar, but its efficiency is low. At the same time, there is a lack of a process for removing chemical reagents, and the safety of the production process is difficult to control. Moreover, after adding chemical reagents, agar cannot be used in food. The bio-enzymatic method refers to the introduction of enzyme reagents to treat agar during the agar production process, which breaks the molecular bonds between agar and promotes its degradation. The cost is high, and the enzymes involved in the reaction cannot be reused, making mass production impossible.

[0006] In summary, there is a lack of a production process for low-temperature sol agar that simultaneously meets the requirements of low production cost, sufficient reaction, no introduction of new substances, simple operation, good safety, and the ability to achieve large-scale production.

[0007] Fermentation inhibits the growth of spoilage bacteria and common pathogens, improves the nutritional value of unfermented foods, and alters their color, flavor, and appearance. Using probiotics to ferment food ingredients can disrupt cell walls and promote the release of polysaccharides. Furthermore, the various enzymes produced by microorganisms during fermentation can biomodify polysaccharides, thereby unlocking new applications. Summary of the Invention

[0008] The present invention provides a method for preparing cryosol agar, which utilizes microbial fermentation-assisted alkaline extraction and modification of the agar product, and further combines it with a thermal denaturation process to prepare the cryosol agar. This method not only improves the application performance of the agar product and increases its commercial value, but also reduces production costs while reducing the amount of alkali solution used in traditional processes, thereby achieving environmental protection.

[0009] To achieve the above object, the present invention provides a method for preparing low-temperature sol agar, comprising the following steps:

[0010] S1. Microbial fermentation treatment of Gracilaria;

[0011] S2. treating the fermented Gracilaria obtained in step S1 with alkali and washing it to neutrality;

[0012] S3, acidifying the neutral fermented Gracilaria obtained in step S2 and washing it to neutrality;

[0013] S4, performing a second-step acidification on the neutral one-step acidified Gracilaria obtained in step S3 and washing it to neutrality;

[0014] S5, bleaching and washing the neutral two-step acidified Gracilaria obtained in step S4 until it is neutral;

[0015] S6. Bathing the neutrally bleached Gracilaria obtained in step S5 in a water bath, collecting the supernatant and cooling it to room temperature to obtain Gracilaria agar gel;

[0016] S7, cutting the Gracilaria agar gel into pieces, packaging them, freeze-dehydrating them, and drying them to a constant weight, and then crushing and sieving the dried Gracilaria agar;

[0017] S8. The agar powder obtained in step S7 is mixed with water, and the mixture is screw extruded, microwave dried, crushed and sieved to obtain low-temperature sol agar.

[0018] Preferably, the microbial fermentation in step S1 specifically comprises the following steps: weighing the washed, impurity-removed, and dried Gracilaria, adding the washed, impurity-removed, and dried Gracilaria to a glucose medium with a mass concentration of 1% to 5%, pasteurizing the mixture at 65°C for 30 minutes, and inoculating the mixture at 37°C under anaerobic conditions for fermentation after cooling; the solid-liquid ratio of Gracilaria to the glucose medium is 1:(15-80); the solid-liquid ratio of the inoculated microbial strain for fermentation is 1:(15-80), the inoculation amount is 1-10%, and the fermentation time is 6-36 hours.

[0019] Preferably, the microbial strain includes one of yeast, lactobacillus, acetobacillus, and bacillus.

[0020] Preferably, the specific steps of alkali treatment and cleaning in step S2 are:

[0021] S2-1: After the fermentation of Gracilaria is completed, drain the water, add 1% to 5% NaOH solution by mass, and keep it at 85 ºC for 2 to 8 hours;

[0022] S2-2. Filter and separate the fermented Gracilaria from the NaOH solution, and soak and wash the fermented Gracilaria with 20-90 times the volume of distilled water for 30-90 min, repeating several times until the pH of the fermented Gracilaria is 7.

[0023] Preferably, the specific steps of the acidification step in step S3 are:

[0024] S3-1. Drain the fermented Gracilaria that has been washed to neutrality and add distilled water at a solid-liquid ratio of 1:(15-80). Then add 0.043% concentrated sulfuric acid by volume, stir and mix thoroughly, and let it stand and soak for 30-90 minutes. Discard the acid washing solution.

[0025] S3-2. Add 20-90 times the volume of distilled water to the one-step acidified Gracilaria and soak and wash for 30-90 minutes. Repeat this process several times until the pH of the one-step acidified Gracilaria is 7.

[0026] Preferably, the specific steps of the two-step acidification and cleaning in step S4 are:

[0027] S4-1. Add 0.064% oxalic acid, 0.01267% EDTA-Na2, and distilled water at a material-to-liquid ratio of 1:(15-80) to the neutral one-step acidified Gracilaria, stir and mix, then let it stand and soak for 30-90 minutes, and discard the acid washing solution.

[0028] S4-2. Add 20-90 times the volume of distilled water to the two-step acidified Gracilaria and soak and wash for 30-90 minutes. Repeat this process several times until the pH of the two-step acidified Gracilaria is 7.

[0029] Preferably, the specific steps of bleaching and washing in step S5 are: adding a sodium hypochlorite solution with an available chlorine mass fraction of 0.04% to the neutral two-step acidified Gracilaria, stirring and mixing, and then standing for 30 to 90 minutes, discarding the bleaching solution, and washing the bleached Gracilaria with distilled water until the pH is 7; the solid-liquid ratio of the neutral two-step acidified Gracilaria to the sodium hypochlorite solution is 1:(15-80).

[0030] Preferably, the specific steps of the water bath in step S6 are: adding distilled water to the neutral bleached Gracilaria at a solid-liquid ratio of 1:(15-80), treating in a water bath at 100°C for 3-8 hours, and filtering to separate the algae residue and the supernatant.

[0031] Preferably, the specific steps of drying and crushing in step S7 are:

[0032] S7-1. Cut the Gracilaria agar gel into pieces and package them. Freeze for 24-48 hours, remove them, and thaw them at room temperature. Filter and separate the water. Place the Gracilaria agar, after removing most of the water, in an oven at 60-105°C for 12-48 hours.

[0033] S7-2: Grind the dried Gracilaria agar and pass it through a standard sieve of 80-200 mesh to obtain Gracilaria agar powder. Preferably, the moisture content of the agar powder after water addition in step S8 is 35%.

[0034] Therefore, the present invention adopts the preparation method of the above-mentioned low-temperature sol agar, which has the following technical effects:

[0035] (1) Microbial fermentation is used to assist the alkaline extraction and modification of agar products. Microbial fermentation can soften the cell wall and promote the outflow of pigments and colloids, which not only reduces the amount of alkali solution used in traditional processes but also degrades the internal structure of the colloid.

[0036] (2) Compared with the low-temperature sol agar extracted by the traditional alkaline process, the low-temperature sol agar prepared by the microbial fermentation-assisted alkaline process combined with the thermal denaturation processing technology of the present invention increased the yield from 7.57% to 10.02%, reduced the sol temperature from 80.93 ºC to 69.41 ºC, and reduced the gel strength from 676.5 g / cm2 to 379.3 g / cm2; the low-temperature sol agar extracted by this process can be widely used in heat-sensitive foods and special dietary foods, which improves the application range and economic value of low-temperature sol agar;

[0037] (3) This process not only improves the application performance of agar products and increases the commercial value of the products, but also reduces the amount of alkali used in traditional processes while reducing production costs, which is green and environmentally friendly.

[0038] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] To more clearly illustrate the embodiments of the present invention or technical solutions in the prior art, the following briefly describes the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are merely illustrative, and those skilled in the art can, without inventive effort, derive other implementation drawings from the provided drawings.

[0040] FIG1 shows the gel strength, yield, and sol temperature of the low-temperature sol agar prepared in Examples 1 to 3;

[0041] FIG2 shows the viscosity, turbidity and whiteness of the cryosol agar prepared in Examples 1 to 3;

[0042] FIG3 shows the sulfate and 3,6-galactose contents of the cryosol agar prepared in Examples 1 to 3. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0044] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0045] Example 1

[0046] The method for preparing low-temperature sol agar by fermentation-assisted alkaline method comprises the following steps:

[0047] S1. Accurately weigh 30 g of cleaned, dried Gracilaria officinalis and add 900 mL of 5% (w / v) glucose medium. Pasteurize at 65°C for 30 min. After cooling, inoculate with Lactobacillus for fermentation at 37°C under anaerobic conditions. The material-to-liquid ratio (w / v) is 1:30, the inoculum size (v / v) is 2%, and the fermentation time is 12 h.

[0048] After fermentation, drain the water, add 450 mL of 3% (w / v) NaOH solution, and heat at 85°C for 3 h. Separate the fermented Gracilaria from the alkali solution using a 200-mesh filter cloth. Soak and wash the fermented Gracilaria in 20 volumes of distilled water for 40 min. Repeat this process several times until the pH of the fermented Gracilaria reaches 7.

[0049] S3. Drain the fermented Gracilaria that has been washed to pH = 7 and add 450 mL of distilled water, then add 0.195 mL of concentrated sulfuric acid, stir and mix well, then let it stand for 30 minutes, discard the acid washing solution, add 20 times the volume of distilled water to the one-step acidified Gracilaria, soak and wash the one-step acidified Gracilaria for 30 minutes, and repeat several times until the pH of the one-step acidified Gracilaria is 7.

[0050] Add 0.288 g oxalic acid, 0.057 g EDTA-Na2 and 450 mL distilled water to the one-step acid-washed Gracilaria, stir and mix well, then soak for 30 min. Discard the acid-washing solution, and add 20 times the volume of distilled water to the second-step acid-washed Gracilaria and soak and wash for 30 min. Repeat several times until the pH of the second-step acid-washed Gracilaria is 7, completing the two-step acid-washing.

[0051] S4. Add 450 mL of sodium hypochlorite solution with an available chlorine mass fraction of 0.04% to the two-step acidified Gracilaria that has been washed to neutrality, stir and mix, and let it stand for 30 minutes. Discard the bleaching solution and wash the bleached Gracilaria with distilled water to pH = 7.

[0052] S5. Add 450 mL of distilled water to the washed Gracilaria bleached to neutrality, place in a constant temperature water bath at 100 ºC for 3 h, filter and separate the algal residue and supernatant, collect the supernatant and cool it to room temperature to form a gel.

[0053] S6. Cut the gelled Gracilaria agar into pieces and package them separately. Freeze them at -20°C for 24 hours, then take them out. Thaw them at room temperature and separate the water with a 200-mesh filter cloth. Place the agar, after most of the water has been removed, in an oven at 60°C for 12 hours until constant weight is reached.

[0054] S7. Use a grinder to grind the agar dried to constant weight and pass it through an 80-mesh standard sieve.

[0055] S8. The agar powder is mixed with water to a moisture content of 35%. The agar powder is screw extruded and microwave dried, and then crushed and passed through a 200-mesh sieve to obtain a low-temperature sol agar product (MLA).

[0056] Example 2

[0057] Low-temperature sol agar was prepared using a traditional alkaline process, including accurately weighing 30 g of Gracilaria truncatula, adding 450 mL of a 6% (w / v) NaOH solution, and incubating at 85°C for 3 hours to perform an alkali treatment. The remaining steps were the same as in Example 1, yielding a low-temperature sol agar product (HA).

[0058] Example 3

[0059] Low-temperature sol agar was prepared using a traditional alkaline process, including accurately weighing 30 g of Gracilaria truncatula, adding 450 mL of a 3% (w / v) NaOH solution, and incubating the mixture at 85°C for 3 hours to perform an alkali treatment. The remaining steps were the same as in Example 1, yielding a low-temperature sol agar product (LA).

[0060] Experimental testing

[0061] (1) The gel strength, yield, and gel temperature of the low-temperature sol agar products obtained in Examples 1 to 3 were tested. The results are shown in FIG1 . In FIG1 , a, b, and c represent significant differences (p < 0.05) between the values ​​of different samples. Indicators with one identical letter indicate no significant difference; indicators without identical letters indicate significant difference.

[0062] 1) Yield determination:

[0063] The percentage of low-temperature sol agar powder to the dry weight of Gracilaria is the product yield, and the yield calculation formula is as follows:

[0064] ;

[0065] Where: m1 is the mass of low-temperature sol agar powder / g; m2 is the mass of Gracilaria raw material after drying / g.

[0066] 2) Determination of gel strength:

[0067] Accurately weigh 1.5 g of cryosol agar powder and add it to 98.5 g of distilled water. Stir and mix thoroughly. Heat in a 100°C water bath to dissolve and replenish lost water. Once fully dissolved, pour into a 90 mm diameter glass dish and cool to solidify. Cover with plastic wrap and let stand at 20°C for 12 h.

[0068] Place the plate to be tested on the left tray of the tray balance and 2 Fix the plunger just in contact with the gel surface. Place the beaker on the right side of the tray balance and slowly pour distilled water into the beaker continuously without interruption. Stop pouring water immediately after the gel surface breaks. Record the weight of the beaker and distilled water at this time. The ratio of the weight of the beaker and distilled water to the cross-sectional area of ​​the plunger is the gel strength of the product:

[0069] ;

[0070] Where: M is the total weight of distilled water and beaker / g; S is the cross-sectional area of ​​the plunger / cm2.

[0071] 3) Determination of sol temperature:

[0072] Prepare a 1.5% cryosol agar solution and pour 10.0 mL into three 18 x 180 mm test tubes. Cool overnight at room temperature. Once a gel has formed, place a small glass bead on the surface of each gel and seal with a rubber stopper. Place the test tubes in a 30°C water bath and increase the temperature at a rate of 1°C / min. Record the temperature at the moment the glass bead falls.

[0073] Result Analysis

[0074] The yields of low-temperature sol agar (HA, LA, MLA) prepared by high alkali, low alkali, and fermentation low alkali processes were 7.57%, 8.08%, and 10.02%, respectively (p < 0.05).

[0075] Alkali treatment softens cell walls and promotes the release of mucilage and pigment. High alkali concentrations severely damage cell walls, promoting mucilage release. However, the subsequent water washing and neutralization process results in significant mucilage loss, leaving less mucilage within the algae, resulting in the lowest yield. Low-alkali solutions do not damage the algae cell walls as severely as high-alkali solutions, but due to reduced losses during washing and neutralization, mucilage can remain within the algae, resulting in a higher LA yield than HA. MLA yield is the highest because fermentation and low-alkali solutions damage the algae cell walls, promoting mucilage release. However, low-alkali treatment is easier to wash and neutralize than high-alkali treatment, resulting in less mucilage loss and the highest yield.

[0076] The gel strengths of HA, LA, and MLA were 676.5 g / cm2, 548.0 g / cm2, and 379.3 g / cm2, respectively (p < 0.05).

[0077] Alkali concentration, time, and temperature are the main factors affecting agar gel strength. HA prepared under high alkaline conditions has higher gel strength than LA and FLA prepared under low alkaline conditions. Fermentation degrades the internal structure of the colloid, resulting in MLA having the lowest gel strength.

[0078] The sol temperatures of HA, LA, and MLA were 80.93°C, 77.05°C, and 69.41°C, respectively. Sol temperature is related to the molecular structure and molecular weight of agar, and the density of the molecular structure is related to gel strength. The trends in sol temperature for HA, LA, and MLA were consistent with those in gel strength.

[0079] Low-temperature sol agar was prepared by fermentation and low-alkali process, with the yield, gel strength and sol temperature being 10.02%, 379.3 g / cm2 and 69.41 ºC, respectively. Compared with the low-temperature sol agar prepared under high-alkali conditions (7.57%, 676.5 g / cm2 and 80.93 ºC), not only the yield was improved and the sol temperature was reduced, but also 50% of the alkali consumption was saved.

[0080] (2) The viscosity, turbidity, and whiteness of the cryosol agar products obtained in Examples 1 to 3 are shown in FIG2 . a, b, and c represent significant differences between the values ​​of the different samples (p < 0.05). Indicators with one identical letter indicate no significant difference; indicators with no identical letters indicate significant difference.

[0081] 1) Turbidity determination:

[0082] Prepare 100 mL of 1.5% low-temperature sol agar solution by mass, heat in a 100°C water bath until fully dissolved, replenish the lost water, pour into a cuvette at 85°C, place in a turbidimeter, and record the reading after it stabilizes.

[0083] 2) Determination of viscosity:

[0084] Referring to GB1886.169-2016, weigh 4.5 g of cryosol agar powder and add approximately 300 mL of deionized water. Stir and mix thoroughly. Dissolve in a 100°C waterbath and replace any lost water. Once fully dissolved, cool to 77°C and place in a 75°C thermostat. Heat the viscometer's pendulum and protective sleeve in water to 75°C, dry them, and then install them in the viscometer. Viscosity is measured using an NDJ-9S digital rotational viscometer in mP·s.

[0085] 3) Determination of whiteness:

[0086] The low-temperature sol agar powder was passed through an 80-mesh standard sieve and the whiteness was measured using a whiteness meter.

[0087] Result Analysis

[0088] Testing revealed that the viscosities of HA, LA, and MLA were 9.97 mP.s, 8.53 mP.s, and 7.83 mP.s, respectively. HA has the highest viscosity due to its denser molecular structure. The turbidity of HA, LA, and MLA were 23.23 NTU, 25.40 NTU, and 24.07 NTU, respectively, and the whiteness was 52.20%, 46.20%, and 46.80%. Both turbidity and whiteness are indicators of agar color. Alkali treatment can remove pigments from Gracilaria and improve agar quality. HA has lower turbidity and higher whiteness than LA and MLA, indicating that high alkaline conditions facilitate pigment removal. The turbidity and whiteness of MLA are intermediate between those of HA and LA, indicating that fermentation softens the cell walls and also removes some pigment.

[0089] (3) The sulfate and 3,6-galactose contents of the cryosol agar obtained in Examples 1 to 3 were determined. The results are shown in FIG3 . a, b, and c represent significant differences between the values ​​of the different samples (p < 0.05). Indicators with one identical letter indicate no significant difference; indicators without identical letters indicate significant difference.

[0090] 1) Determination of sulfate content:

[0091] Preparation of 1% Tween-BaCl2 solution: Weigh 1 g of Tween 20 and 1 g of BaCl2 respectively, add 98 g of water, mix well, and filter to obtain a clear solution for later use.

[0092] Preparation of K2SO4 standard solution: Weigh 0.1088 g (accurate to 0.0001 g) of K2SO4 that has been dried to constant weight at 105 °C, dilute to 100 mL with 1.0 mol / L hydrochloric acid, and store for later use.

[0093] Digestion of cryosol agar: Weigh 0.05 g of sample into a 25 mL colorimetric tube and add 25 mL of 1 mol / L hydrochloric acid. Digest in a water bath at 100°C for 5 h. After digestion, cool the digestion solution to room temperature and dilute to 25 mL. Decolorize with activated charcoal and filter to obtain a clear digestion solution for later use.

[0094] Drawing of standard working curve: Dilute K2SO4 standard solution 5 times, take 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of dilution solution into test tubes respectively, and make up to 1.0 mL with 1.0 mol / L hydrochloric acid. Take 1 mL of sample solution and add 3 mL of Tween-BaCl2 The solution was mixed by oscillation, allowed to stand for 10 min, and the absorbance was measured at a wavelength of 360 nm to obtain a standard curve of absorbance of different concentrations of sulfate ion.

[0095] Determination of sulfate content: Take 1 mL of digestion solution, add 3 mL of Tween-BaCl2 solution, shake and mix, let it stand for 10 minutes, measure the absorbance at a wavelength of 360 nm, and calculate the sulfate content of the sample using the standard working curve.

[0096] 2) Determination of 3,6-galactose:

[0097] Preparation of standard solution: Dry fructose to constant weight, accurately weigh 1 mg, dilute to 10 mL volumetric flask with ultrapure water to prepare a 100 μg / mL standard solution.

[0098] Preparation of resorcinol stock solution: Dissolve 150 mg of resorcinol in distilled water, dilute to volume in a 100 mL brown volumetric flask, store at 4 ºC, and use within one month.

[0099] To prepare acetal stock solution: Pipette 84 μL of acetal into 9.916 mL of distilled water, mix thoroughly, and store in a brown bottle at 4°C. Use within one month. Before preparing the colorimetric reagent, pipette 1 mL and dilute to 25 mL. Use the same day.

[0100] Preparation of working solution (resorcinol-acetal reagent): Slowly add 100 mL of concentrated hydrochloric acid to 9 mL of resorcinol stock solution, then add 1 mL of diluted acetal stock solution. This solution must be prepared on the same day, protected from light, and used within 3 hours.

[0101] Prepare a standard curve: Add 0.02 mL, 0.04 mL, 0.08 mL, 0.12 mL, 0.16 mL, and 0.24 mL of 100 μg / mL fructose solution to test tubes, respectively. Make up to 0.4 mL with distilled water, and prepare a 0.4 mL blank. Incubate on ice for 5 minutes at 175 rpm. Add 2 mL of the newly prepared cold working solution to each test tube while still on ice. Incubate in an 80°C water bath for 15 minutes, then incubate on ice for 1.5 minutes at 175 rpm. Measure absorbance at 554 nm. Plot a standard curve using standard concentration as the horizontal axis and absorbance as the vertical axis.

[0102] Sample determination: Prepare a 30 μg / mL solution of the sample in distilled water. Pipette 400 μL and perform the same procedure as above. Measure the absorbance and substitute it into the standard curve to determine the 3,6-galactose content in the sample.

[0103] Result Analysis

[0104] The sulfate content of HA, LA, and MLA is 3.20%, 4.59%, and 4.58%, respectively, and the 3,6-galactose content is 33.06%, 28.04%, and 32.06%. Alkali treatment can increase the gel strength of agar, converting sulfate groups into 3,6-galactose. Agar gel strength is inversely proportional to sulfate content and directly proportional to 3,6-galactose content.

[0105] The sulfate content in HA prepared under high alkaline conditions was lower than that in LA and MLA prepared under low alkaline conditions. The difference in sulfate content between LA and MLA was not significant, indicating that the solubility of the alkali solution affects the sulfate content in cryosol agar. The difference in 3,6-ether galactose between MLA and HA was not significant, and the higher galactose content in MLA than in LA was due to the combined effects of the low alkaline solution and fermentation.

[0106] Therefore, the present invention adopts the above-mentioned low-temperature sol agar preparation method, adopts microbial fermentation-assisted alkali method to extract and modify the agar product, and further combines it with a thermal denaturation processing technology to prepare the low-temperature sol agar, which not only improves the application performance of the agar product and increases the commercial value of the product, but also reduces the use of alkali solution in traditional processes while reducing production costs, which is green and environmentally friendly.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing low temperature sol agar, characterized in that, The following steps are involved: S1. treating Gracilaria with microbial fermentation; S2, treating the fermented Gracilaria obtained in step S1 with alkali and washing it to neutrality; S3, acidifying the neutral fermented Gracilaria obtained in step S2 and washing it to neutrality; S4, performing a second-step acidification on the neutral one-step acidified Gracilaria obtained in step S3 and washing it to neutrality; S5, bleaching and washing the neutral two-step acidified Gracilaria obtained in step S4 to neutrality; S6, placing the neutrally bleached Gracilaria obtained in step S5 in a water bath, collecting the supernatant and cooling it at room temperature to obtain Gracilaria agar gel; S7, cutting the Gracilaria agar gel into pieces, packaging them, freezing and dehydrating them, and drying them to a constant weight, and crushing and sieving the dried Gracilaria agar; S8, the agar powder obtained in step S7 is mixed with water, and after screw extrusion, microwave drying, crushing and screening, low-temperature sol agar is obtained.

2. The method for preparing low temperature sol agar according to claim 1, wherein The specific steps of microbial fermentation in step S1 are: Weigh the washed, impurity-free and dried Gracilaria, add it to a glucose medium with a mass concentration of 1% to 5%, pasteurize it at 65°C for 30 minutes, and then inoculate microbial strains for fermentation at 37°C under anaerobic conditions after cooling; the solid-liquid ratio of Gracilaria to glucose medium is 1:(15-80); The microbial strains include one of yeast, lactobacillus, acetobacillus, and bacillus; the material-liquid ratio of the inoculated microbial strains for fermentation is 1:(15-80), the inoculation amount is 1-10%, and the fermentation time is 6-36 hours.

3. The method for preparing low temperature sol agar according to claim 1, characterized in that: The specific steps of alkali treatment and cleaning in step S2 are: S2-1, after the fermentation of Gracilaria, drain the water, add 1%~5% NaOH solution by mass, and keep it at 85 ºC for 2~8h; S2-2, filter and separate the fermented Gracilaria from the NaOH solution, and soak and wash the fermented Gracilaria with 20-90 times the volume of distilled water for 30-90 minutes, repeating the process several times until the pH value of the fermented Gracilaria is 7.

4. The method for preparing low temperature sol agar according to claim 1, characterized in that: The specific steps of the one-step acidification in step S3 are: S3-1, drain the water from the fermented Gracilaria that has been washed to neutrality and add distilled water at a solid-liquid ratio of 1: (15-80), then add 0.043% concentrated sulfuric acid by volume, stir and mix well, and then let it stand and soak for 30-90 minutes, and discard the pickling solution; S3-2. Add 20-90 times the volume of distilled water to the one-step acidified Gracilaria and soak and wash for 30-90 minutes. Repeat several times until the pH of the one-step acidified Gracilaria is 7.

5. The method for preparing low temperature sol agar according to claim 1, characterized in that: The specific steps of the two-step acidification and washing in step S4 are: S4-1, add 0.064% oxalic acid, 0.01267% EDTA-Na2 and distilled water with a solid-liquid ratio of 1:(15-80) to the neutral one-step acidified Gracilaria, stir and mix well, let stand and soak for 30-90 minutes, and discard the acid washing solution; S4-2. Add 20-90 times the volume of distilled water to the two-step acidified Gracilaria and soak and wash for 30-90 minutes. Repeat several times until the pH of the two-step acidified Gracilaria is 7.

6. The method for preparing low temperature sol agar according to claim 1, characterized in that: The specific steps of bleaching and washing in step S5 are: adding a sodium hypochlorite solution with an effective chlorine mass fraction of 0.04% to the neutral two-step acidified Gracilaria, stirring and mixing, and then standing for 30 to 90 minutes, discarding the bleaching solution, and washing the bleached Gracilaria with distilled water until the pH is 7; the solid-liquid ratio of the neutral two-step acidified Gracilaria to the sodium hypochlorite solution is 1: (15 to 80).

7. The method for preparing low temperature sol agar according to claim 1, characterized in that: The specific steps of the water bath in step S6 are: adding distilled water to the neutral bleached Gracilaria at a solid-liquid ratio of 1: (15-80), treating in a water bath at 100 ºC for 3-8 hours, and filtering and separating the algae residue and the supernatant.

8. The method for preparing low temperature sol agar according to claim 1, characterized in that: The specific steps of drying and crushing in step S7 are: S7-1, cut the Gracilaria agar gel into pieces, pack them, freeze them for 24-48 hours, take them out, thaw them at room temperature, filter and separate the water, and place the Gracilaria agar with most of the water removed in an oven at 60-105ºC for 12-48 hours; S7-2, crush the dried Gracilaria agar, and pass it through a standard sieve of 80-200 meshes to obtain Gracilaria agar powder.

9. The method for preparing low temperature sol agar according to claim 1, characterized in that: The moisture content of the agar powder after mixing with water in step S8 is 35%.

10. A low temperature sol agar prepared by the preparation method according to any one of claims 1 to 9.

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