KR100229948B1 - Extraction and Purification of Polysaccharide Sulfate from Off-White Peel - Google Patents

Abstract

The present invention enables the extraction and purification of sulfated polysaccharides from off-white shells such as sea squirts (buckwheat) in various fields to use sulfated polysaccharides such as feed preparations, pharmaceuticals, cosmetics, health drinks, etc. From this, it is possible to supply a cheaper effect, the present invention, by adding distilled water to the off-white shell powder and reacted for 48 hours by adding protease, centrifuged to take the supernatant and TCA (trichloroacetic acid) The solution was added, treated, and then centrifuged to remove the precipitate, ethanol was added to the supernatant, and left at -10 ° C. for 24 hours to precipitate enzyme digestibles, and the precipitated crude polysaccharide was dialyzed for 24 hours. After lyophilization or high temperature and high pressure treatment, the crude polysaccharide is extracted, and the extracted crude polysaccharide is phosphate buffered with Nacl. Eluted until no sugar was detected, and the eluate was fractionated and treated with alcohol, and the precipitate was retreated with Sephadex G-25 to obtain partially purified sulfated polysaccharide by alcohol treatment, dialysis and lyophilization. has exist.

Description

[Name of invention]

Extraction and Purification of Polysaccharide Sulfate from Off-White Peel

Detailed description of the invention

[Purpose of invention]

The present invention is to enable the extraction and purification of sulfated polysaccharides from off-white shells such as sea squirt (squirt) to various fields that want to use polysaccharide sulfates, such as feed preparations, pharmaceuticals, cosmetics, health drinks, and the like.

[Technical field to which the invention belongs and the prior art in that field]

There are many techniques for application to the industrial field using sulfated polysaccharides. In particular, the search for resources containing chondroitin sulfate has continued since the 1950s, and the physiological functions of chondroitin sulfate are contributing to the regulation of extracellular fluid capacity, water metabolism, ion transport and regulation of extracellular fluid, bone calcification, It is known that there is treatment of damaged tissues, smoothing joint tissues, preventing blood coagulation, maintaining the transparency of the cornea, preventing infections, health drinks, cosmetic effects, and anti-aging.

Chondroitin, a polysaccharide sulfate, is produced industrially from cartilage of shark fins, and some attempts have been made to produce sulfated polysaccharides by combining sulfur with sugars.

Techniques related to sulfate polysaccharides are disclosed in Patent Publication Nos. 97-009608, 97-010792, 96-000238, 96-003746, 96-040174, 95-024690, and 95-700918 No. 90-018129, and the like, and sulfated polysaccharides are widely used in foods, cosmetics, medicines, and the like.

However, if sulfate polysaccharides are extracted and used from high-quality natural resources such as shark fins and sea cucumbers as of now, it is difficult to continuously supply resources, and the production price is also very high, which may cause the unit price of products to rise.

[Technical problem to be achieved]

Therefore, in the present invention, without using high-quality natural resources such as shark fins and sea cucumbers, by using off-white shell including a large amount of discarded slugs, etc., it is possible to extract and use sulfated polysaccharides at low cost. The off-whites such as oolongs are produced in 100,000 tons per year, mainly in Tongyeong, Gyeongnam, and 50% of these are 50,000 tons discarded as shells, and the present invention is discarded as described above. By recycling the resources used, we will achieve the industrial effect of recycling the waste resources and supply of cheap sulfate polysaccharides.

[Configuration and Function of Invention]

The process of extracting and purifying the sulfated polysaccharide from the off-whites such as the larvae of the present invention is as follows.

Example 1

Extraction of Sulfate Polysaccharides

The sulfated polysaccharide was first extracted from the dried off-white bark and extracted again from the remaining residue.

First, the method by enzyme treatment was treated with the proteolytic enzymes alkalase, neutrase, mixture-2000.

That is, an appropriate amount of distilled water was added to a certain amount of off-white powder, and each enzyme was added thereto at a concentration, followed by reacting for 48 hours in a shaking constant temperature bath.

After the reaction, the supernatant was collected by centrifugation (3000 rpm, 15 min), and treated with 10% TCA (trichloroacetic acid) solution.

The precipitate was removed by centrifugation (3000 rpm, 15 min) again, ethanol was added to the supernatant by concentration, and left at -10 ° C for 24 hours to precipitate crude digestibles (enzyme digestibles).

Precipitated crude polysaccharide was lyophilized after dialysis for 24 hours.

On the other hand, in order to extract the sulfated polysaccharides by the high temperature and high pressure treatment was processed by the high-temperature heating sterilizer at 125 ℃ (pressure: 1.5kg / cm ² ) by time.

In other words, take a certain amount of sample to the Erlenmeyer flask, add 15 times the amount of water to the Erlenmeyer flask, cover the inlet of the Erlenmeyer flask with aluminum foil, and process it in a high-pressure heat sterilizer. Crude polysaccharides (water solubles) were obtained.

Example 2

[Refining Sulfuric Acid Polysaccharides]

Purification of polysaccharide sulfate was detected by adding crude polysaccharides to DEAE-Cellulose (ø35mm × 25cm) parallelized with 20mM phosphate buffer solution (pH 7.0) and linearly increasing the buffer solution containing sodium chloride (NaCl) to 1.5M. Elution was not performed until.

6 ml of the eluate was collected by a fractionator, and the starch was measured. Each sample fraction was combined, dialyzed in distilled water for 24 hours, concentrated and freeze-dried.

The protein content of each fraction (4 fractions) by chromatography was measured at 280 mm with a spectrophotometer.

The ion exchanged sample was subjected to gel filtration.

That is, Sephadex G-100 swelled in a glass column (ø1.5 × 100 cm) was filled up to 75 cm, and 2-5% of the sample liquid was eluted and fractionated with respect to the total amount.

The eluate was 0.05M NaCl solution, and 6 ml of each fraction was collected, and the whole fraction was analyzed and the active fractions were collected by alcohol treatment. The precipitate was retreated with Sephadex G-25 for alcohol treatment, and lyophilized after dialysis. Sulfate polysaccharide was obtained.

(3) The usage example of the sulfated polysaccharide extracted from the off-white shell is as follows.

[Example 1]

end. summary

Most fish-based color improving agents use fat-soluble carotenoids, so it is very difficult to use them as they are.

That is, because it is in a liquid state, it must be mixed with a certain amount of powdered feed and re-made into a feed, and it will be difficult for fishermen to use directly.

Therefore, an excipient capable of adsorbing a carotenoid pigment for improving color is needed. In addition, these excipients must meet two conditions that should not be toxic while maintaining fish growth normally.

I. Measurement of Pigment Adsorption

The pigment adsorption was measured on the water-soluble Coomassie brilliant blue R-250 and Red-2, and the fat-soluble oolong-pigment pigment extract.

1.0 g of each sample was placed in a Erlenmeyer flask and prepared in each dye solution (Coomassie brilliant blue R-250 and Red-2 were dissolved in 70% ethanol, and fat-soluble squirrel shell pigment extract was dissolved in 99% ethanol). 10 ml was added and left for 1 hour to adsorb the dye, followed by volatilization of ethanol as a solvent in a hot air dryer.

The adsorbed sample was extracted three times with acetone and the absorbance (UV / Vis spectrophotometer) was measured at 450 mm.

The pigment content was calculated according to the conventional method by measuring the absorbance.

All. result

Blue-R-250, Red-2 and Rhubarb husks were examined for pigment adsorption, and the solvents were evaporated to adsorb the pigment extracts to polysaccharide sulfate. Blue-R-250 and Red-2 husks with 70% ethanol. The pigment extract was extracted and quantified three times with acetone. Compared with chitin and chitosan, which are known to have pigment adsorption capacity, as shown in Table 1, the pigment extract was inferior. Sterilizers and neutrase treated polysaccharides were 85.2% and 85.6%, respectively.

TABLE 1

[Example 2]

end. summary

In general, sulfate polysaccharides present in algae and vertebrates have an anti-blood coagulation effect, in addition to the anti-cancer and anti-mutagenic effect.

I. Anti-blood coagulation ability

Anticoagulant activity was measured by taking 4.5 ml of venous blood from normal people, mixing it with 0.5 ml of sodium cirtrate (3.8%) solution, and centrifuging at 3,000 rpm for 10 minutes to separate plasma.

The separated plasma was stored at -20 ° C and used for the experiment.

Activated partial thromboplastin time (APTT) was measured by adding 10 μl of the sample solution to 100 μl of plasma and warming it in a 37 ° C. constant temperature water bath for 1 minute.

100 μl actin was added thereto, followed by further warming for 2 minutes in a 37 ° C. constant temperature water bath. At the time of 2 minutes, 100 μl of 20 mM CaCl ₂ heated to 37 ° C. was added thereto, and the coagulation time was measured by pressing a stopwatch.

The control sample was diluted with heparin to measure the anticoagulant time.

Prothrombin time (PT) was measured in 200 µl of thromboplastin pre-warmed in a constant temperature water bath at 37 ° C for at least 1 minute, and a mixture of plasma and sample solution warmed at 37 ° C for at least 1 minute (plasma: sample solution = 10: 1) 100 μl was added and the stopwatch was pressed to measure the solidification time.

All. Angiotesin I Converting Enzyme (ACE) Inhibitory Activity

ACE inhibition was tested in two ways.

One method was pre-reacted at 37 ° C. for 5 minutes after adding 200 μl of ACE coenzyme solution and 200 μl of 0.1M sodium borate buffer (pH 8.3) to 50 μl of sample solution of Cushman and Cheung. 100 μl of Hippuryl-His-Leu solution (25 mg / 2.5 ml sodium borate buffer) was added thereto as a substrate, and the reaction was again performed at 37 ° C. for 30 minutes. Then, 500 μl of 1N HCl was added to stop the reaction. Distilled water was used, and the control was added 500 μl of 1N HCl, followed by 200 μl of ACE coenzyme solution).

1.5 ml of ethyl acetate was added thereto, followed by voltex for 15 seconds, followed by centrifugation at 3,000 rpm for 5 minutes to obtain 1 ml of supernatant.

After completely drying it, 3 ml of distilled water was added to dissolve it, and the absorbance was measured at 228 mm.

As another method, the experiment was performed using TNBS (trinitrobenzene sulfonate).

la. result

Table 2 shows the results of measuring the activated partial thromboplastin time (APTT) and prothrombin time (PT) at a constant concentration (100 µg / ml) to examine the anticoagulant activity of polysaccharide sulfate.

APTT inhibitory effect was observed in all samples, but the PT value was constant at about 14.5 sec.

Especially, F ₄ (fraction 4) obtained by DEAE-Celluse ion exchange chromatography was the most effective with APTT of 75 seconds, F ₂ of Sephadex G-100 was obtained by 70 seconds and DEAE-Celluse ion exchange chromatography. Crude polysaccharides extracted with F ₃ and autoclave were 66 and 65 seconds, respectively.

The anticoagulant activity of seaweed polysaccharide fucoidan increased with higher sulfate content, which is in good agreement with the results of this experiment.

TABLE 2

Table 3 looks at the ACE inhibitory effect of the extracted polysaccharide sulfate.

All samples had ACE inhibitory effects, and the degree of inhibition was also different in the same sample according to the adopted experimental method.

TNBS method showed higher value than Cushman and Cheung's method. Especially, DEAE-Cellulose ion exchange activity fractions showed better ACE detoxification effect, with fraction 4 being the most effective at 35.7%, and neutrase-treated polysaccharides had relatively low inhibition. Showed.

TABLE 3

[Example 3]

end. summary

In order to take advantage of the excellent pigment adsorption capacity of sulfated polysaccharides, rainbow trout was selected from salmon and fish whose red color influences product value.

In other words, the optimum amount of pigment extracts were adsorbed on the sulfated polysaccharide, and it was added to the feed for rainbow trout breeding experiments.

I. Experimental fish and breeding experiment

First of all, 500g of rainbow trout with an average weight of 4g preliminarily reared for 4 weeks was used, and 20 animals were received in each experimental group, and each experimental group was duplicated. The breeding experiment was conducted at the outdoor breeding ground at Gyeongsangnam-do Inland Water Development Test Center in Milyang-si, Gyeongsangnam-do for 8 weeks from June 3 to July 29, 1996. The breeding device is a flowing breeding device that continuously supplies groundwater. Fish droppings were allowed to flow out of the cage immediately.

The size of the breeding tank was a circular FRP tank with a diameter of 1.7m and a depth of 0.9m (2 tons). At this time, the main water was 20ℓ / min, the dissolved oxygen was around 8ppm, and the water temperature was 16.0 ~ 18.5 ℃ throughout the entire breeding period. (Average 17.3 ° C). Feeding amount was 1.5% of body weight three times a day, seven days a week.

Fish meal was used as white fish meal from the United States, and the amount of pigment source and astaxanthin content was optimally added.

All. Feed preparation

The oolong pigment extract was dissolved in a certain amount of ethanol and added about 1.6g to 6g of sulfated polysaccharide (Fig. 1), followed by uniform mixing for 30 minutes using a mixer to prepare pellet feed.

Carophyll Pink is an imported synthetic synthetic pigment that has a 64 ppm astaxanthin content in the feed.

The prepared experimental feed was used for the experiment while packing in a small amount of vinyl packed with nitrogen and stored in a refrigerator at -20 ° C.

la. Measurement of Pigment Content

In the pigment content measurement, the experimental fish were filled with ice and transported to the laboratory, and the tails and gills were removed and separated into meat and epidermis.

Meat was put in a certain amount into a grinder and acetone was crushed with a solvent, the epidermis was soaked in acetone and left overnight.

Extraction was repeated three times each and it was concentrated to an ether layer and concentrated.

The total carotenoid content was calculated as the absorption coefficient E ^1% _1cm = 2,400 using benzene as the solvent at the maximum absorbance of 480mm.

Isolation and identification of astaxanthin fractions in meat and epidermis were performed by high performance liquid chromatography.

Absorbance was analyzed at 476 mm with an uv / visible spectrophotometer while solvent was flowing with hexane / acetone (86:16) at a rate of 0.8 ml / min.

The retention time of the standard astaxanthin was identified.

hemp. result

Tables 4 and 5 show the changes in the pigment content of the experimental group to which the sulfated polysaccharide, pigment extract, and carophyll pink, etc., extracted from the bark powder.

The pigment contents in meat and skin of pre-incubated rainbow trout were 0.0157 and 0.2073 mg / 100g, respectively.

After four weeks, the rainbow trout meat pigment content was 0.0652mg / 100g, pink and pigment extracts were 0.1156 and 0.1263mg / 100g, respectively.

After 8 weeks of breeding, the control group was 0.0896mg / 100g, but the pigment extraction group was 0.2841mg / 100g.

Pink and crude polysaccharides added 0.2840, 0.2224 and 0.2137mg / 100g, respectively, indicating that pigmentation with sulfated polysaccharides was normal.

After 4 weeks, the pigment content of rainbow trout skin was 0.2363mg / 100g and 0.5734mg / 100g extract, which was slightly higher than pink or crude polysaccharide. After 8 weeks, the control group showed almost no change, but the highest value of the extract added was 0.9023mg / 100g, and the pink was 0.8816mg / 100g and the crude polysaccharide added 0.8625 and 0.8437mg / 100g, respectively, indicating that the pigmentation was smooth in the skin. appear.

Astaxanthin was the most prevalent form of astaxanthin diester, monoester, and free in the rainbow trout meat and skin.

However, after 4 weeks, the morphology was diester> free> monoester.

Changes in meat ratio were highest in diester (70-75%), free (13-18%) and monoester (7-9%), except for control.

However, the shells were 77-83% diester, 6-9% monoester and 5-10% free. In the control group, unidentified substance was found to be 14.04%, indicating that metabolism varies depending on the feeding source.

TABLE 4

TABLE 5

[Effects of the Invention]

As described above, the present invention enables the extraction of sulfated polysaccharides from off-white shells including the larvae, and enables the extraction, purification and use of sulfated polysaccharides as an industrial advantage and low cost of recycling waste materials. The invention is an excellent invention that can be obtained inexpensive sulfate polysaccharide extraction and use thereof as described above to obtain many economic benefits and effects in the field of use of various sulfate polysaccharides, such as feed preparation, pharmaceuticals, cosmetics, health drinks, and the like.

Claims (1)

Distilled water was added to the off-white shell powder, followed by reaction with proteolytic enzymes, followed by centrifugation to obtain a supernatant, followed by treatment with TCA (trichloroacetic acid) solution, followed by centrifugation to remove the precipitate and ethanol to the supernatant. Thereafter, precipitated crude polysaccharides (enzyme digestibles) and the precipitated crude polysaccharides are lyophilized after 24 hours of dialysis or extracted at high temperature and high pressure to extract crude polysaccharides, and the extracted crude polysaccharides are phosphate buffer containing sodium chloride (Nacl). Eluted until no sugar was detected, and the eluate was eluted, fractionated, and alcohol-treated. The precipitate was retreated with Sephadex G-25 to obtain purified sulfate polysaccharide by lyophilization after alcohol treatment and dialysis. Extraction and purification of polysaccharide sulfate from off-white shell.