DE2333184C3 - Process for the extraction of mucopolysaccharides from animal connective tissue - Google Patents

Description

Follow. The mucopolysaceharidsäurehaltige extract is obtained by animal connective tissue are extracted only with water at 105 to 15O ⁰ C and an elevated pressure and the aqueous phase is separated and recovered.

Examples of connective tissue are cartilage or skins of marine animals such as whales, fish, e.g. B. Sharks, or land-dwelling mammals such as cows, pigs, horses, sheep and goats. The cartilage from on the

Extraction and separation processes are obtained than by conventional methods. The fat phase and the Residue can be used as a source of oil and fat for various applications. Further 5 these materials can be obtained in a single step during the separation of the extract.

The obtained mucopolysaccharic acid-containing extract can be treated by the usual method become, however, much easier. In particular, the

Land-living mammals are particularly preferred. io extract of a known protease treatment and / or

The use of tracheal cartilage is particularly subjected to alkali treatment, and then the

to recommend. separated liquid phase. A precipitant, both

The extraction of the connective tissue can thereby, for example, lower aliphatic alcohols with 1 to

take place that the starting connective tissue together 4 carbon atoms, preferably ethanol, becomes the

with water in a pressurized extra-liquid phase to precipitate the mucopolysaccharide

tion zone, e.g. B. an autoclave, brought and added to acids. The precipitated Mucopolysaccha-

105 to 150 ^c C. The heating can be separated from acids and by any

by any method. For example, liquid-solid separation processes, e.g. B. through Zen

the extraction zone can either be obtained externally or by trifugal separation or filtration. the

inside or both outside and inside heated ao mucopolysaccharide acids can be known by

will. Also, the heating can be cleaned by introducing procedures, for example by

of superheated steam to the extraction zone and column chromatography. If necessary, the

Withdrawing through a pressure adjustment valve separated liquid phase after the above

leads to be. "Protease treatment and / or alkali treatment directly

The extraction temperature can be subjected to column chromatography within 25.

Range from 105 to 150 ^c C depending on the extraction The above-mentioned known protease treatment,

The time and type of connective tissue used may vary. Alkali treatment, separation measure and cleaning

It is preferably from 110 to 130 ^° C., in particular measures can be taken in various combinations

at about 120 ± 5 ° C. The extraction time can be carried out in ab-

dependence on such factors as the extraction lungs and measures repeated two or three times.

temperature or the type of original connective tissue.

can be varied and is usually in the range of Since the process of isolating mucopoly-

1 to 10 hours. saccharic acids from those obtained by the above process

The primary connective tissues are cut to the obtained aqueous extracts of connective tissues of the desired size and, as such, can be used as a known method. The complicated degreasing precedents of this process, with the exception of the follow-up, which are essentially not described following the previous process, are not described.
can be omitted according to the invention. The proteolytic degradation of the raw, mucopoly-

Since water is used as the extraction agent in the process of the saccharic acid-containing extract, the heat extraction process of any commercially available protease, for example papain, takes place at the temperature indicated above Pronase * (neutral protease, which is naturally pressurized by a micronizer, and it becomes a certain organism, e.g. Streptomyces, has been created) pressure state can be carried out automatically by the temperature and ficin.
dos steam set. The pH and temperature of the raw extract

If the extraction temperature is lower than 45 during proteolysis must be at the optimum

the lower limit of the above range, pH and optimal temperature of each enzyme, the connective tissues will only swell, and the
intended extraclion effect cannot be achieved
will. On the other hand, when the extraction temperature
higher than the upper limit of the specified range 50
heat decomposition or degradation easily occurs, what
is undesirable for the mucopolysaccharides. Therefore
the temperature range given above should be adhered to.

The obtained extract containing mucopolysaccharic acids can easily be converted into an insoluble residue, an extracted aqueous phase and a fat phase are separated. There is no particular limitation regarding the separation method. For example

the separation can be done by decanting. 60 exchangers of bridge-like dextran, containing

However, for technical processes filtration and as an active group diethylaminoethyl), DEAE cellulose, ECTEOLA cellulose and anion exchangers can be selected.

The developing solution for chromatography

removal of the residue and the fat phase can be obtained from aqueous solutions of sodium chloride,

will. Thus, a mucopolysaccharide acid lithium chloride and sodium acetate can be chosen,

containing extract from the connective tissues of Examples of the mucopolysaccharides are Chon-

Animals by much simpler and less time-consuming droitin, chondroitin sulfate, hyaluronic acid, heparin,

are maintained, for example the ranges of the pH value from 4.0 to 9.0 and the temperature from 20 to 60 ⁰ C are normally used.

The period of proteolytic degradation can be 6 to 24 hours.

The alkali treatment can be carried out with an aqueous solution of sodium hydroxide or potassium hydroxide be performed.

Although the alkali treatment of the crude extract can be carried out at room temperature, any temperature can be applied from 0 to 5O ⁰ C.

The carrier for use in column chromatography can be made from DEAE Sephadex® (anion

Centrifugal separation appropriate. Most commonly, centrifugal separation is used, and the aqueous solution can be easily

Heparitin sulfate and keratosulfate. The method of the invention is particularly useful for the production of Chondroitin sulfate and / or hyaluronic acid are preferred. The following examples illustrate the invention.

example 1 and Comparative Examples 1, 2, 3 and 4

In an autoclave, 1 kg of crushed tracheal cartilage of an ox and 1 liter of water were placed and held at 121 ° C for 4 hours. The treated liquid was immediately subjected to centrifugal separation subject. To 21 of the obtained mucopolysaccharidic acid-containing extract became Pronase® (trade name for a protein-degrading enzyme. 200,000 units ^ added and the mixture was kept at 50 ° C. for 3 hours and then to obtain a liquid phase subjected to centrifugal separation. Sodium hydroxide became this liquid phase to an extent of 1 η was added, and the mixture was kept at room temperature for 1 hour. Then became 2.5 times the volume of the mixture of ethyl alcohol was added. The obtained precipitate became dissolved in water and neutralized. Again, twice the volume of ethyl alcohol was added. the The resulting precipitate was dried, and 20 g of chondroitin sulfate having an S content was obtained of 5.8%.

The quality of the chondroitin sulfate is given in Table I, expressed as the specific viscosity (measured by an Ostwald viscometer at 30 ^° C. using a 1% solution of the chondroitin in isotonic sodium chloride solution), as the S content (percent by weight) and as the N- Content (percent by weight).

For comparison, chondroitin sulfate was used in the same manner as in Example 1 except for manufactured that the mucopolysaccharidic acid-containing extract by the usual alkali extraction method (Comparative Example 1) and the enzyme decomposition extraction method (Comparative Example 2) was obtained. The quality of the chondroitin obtained is also shown in Table 1.

Table I.

tries • Extraction process Chopping second ent Extraction Quality of Chendroitin Sulphate S content N content Material of first design ningsvor- greasy conditions specific greasy gear occurrence viscosity (Ge occurrence (Weight weight percent) percent)

Example 1 ox - no yes no

trachea- (unnecessary) (unnecessary)

cartilage

Ochsen- yes yes yes

equal air tubes (emergency (emergency

example 1 cartilage agile) agile)

Ochsen- yes (not- yes yes (not-

same lift tubes manoeuvrable) manoeuvrable)

example 2 cartilage

Extraction only 0.48
with water
121 ⁰ C during
4 hours in one
Autoclave

Extraction with 0.21
aqueous 5N NaOH
at 6O ⁰ C during
1.5 hours

Extraction at
55 ° C during
4 hours under
use
of protease
(1500 units
per gram of
Raw material)

0.50

5.80 2.87

5.80 3.10

5.75 2.97

From the results obtained in Table I above, it can be seen that the alkali extraction method in Comparative Example 1 brings about a non-negligible decomposition by alkali and the quality of the chondroitin obtained with a significantly reduced specific viscosity bad is. A comparison of Example 1 with Comparative Example 2 shows that the qualities of the obtained Chondroitin sulfates are practically the same and behave well like natural substances that however, the enzyme decomposition extraction method requires two defatting processes that are complicated and complex are time consuming and also have the disadvantage of using a large amount of protease. When the degreasing process was omitted as in Example 1, the enzyme decomposition extraction was carried out insufficient and the fats in the extract made subsequent treatment extremely difficult. Chondroitin Sulphate was irrelevant with Loss separated during the process, but with removal of solidified fats. The received The product was discolored to a high degree and technically completely unusable.

For comparison, the above procedure was repeated with the exception that the extraction operation was carried out at 16O ⁰ C (Comparative Example 3). As in Comparative Example 1, there was an insignificant decrease in specific viscosity and the product was poor in quality.

For further comparison, the procedure of Example 1 was repeated except that the Material in a steam bath at atmospheric pressure

was steamed instead of using the autoclave (Comparative Example 4). A useful one The extract containing mucopolysaccharic acid could not be obtained.

Table II Yield (be at quality of the chondroitin N-Gc-
stop 2.87 pulled on 1 kg sulfate 3.38 raw ox
air tube
cartilage) speci
fishes
Viscose S-Ge
stop (Weight percent) activity 5.80 2.91 (B) 4.25 20th 0.48 example 1 6.2 0.28 5.78 Comparison example 3 3.7 0.48 Comparison example 4 game 2

In an autoclave 1.8 kg crushed skin were introduced from blue shark and 101 water and heated to 126 ⁰ C. The contents were then kept in this state for 4 hours. The treated liquid was immediately subjected to centrifugal separation. To 121 of the obtained extract containing the mucopolysaccharide acid was added 0.2η-sodium hydroxide, and the mixture was allowed to stand at room temperature overnight. Then, ethanol was added in an amount twice the volume of the mixture to precipitate mucopolysaccharic acids. The precipitate was collected by centrifugal separation and dissolved in water. The pH of the solution was adjusted to neutral with acetic acid. Pronase® was then added to the solution (360,000 units) and the same enzyme treatment as in Example 1 was carried out, followed by centrifugal separation to obtain a liquid phase. The liquid phase was allowed to stand, and the supernatant liquid obtained was passed through a Schiller column (S. Schüler el al; J. Biol. Chem., 236, 983 [1961]). The fractions adsorbed on the column were eluted with sodium chloride and separated. In one drain, hyaluronic acid with a sodium chloride concentration up to 0.5 M. Heparitin sulfate was formed in a drain with a sodium chloride concentration above 0.5 M and up to 1.25 M, and chondroitin sulfate was formed in a drain with a sodium chloride concentration above 1.0 Get 25 sts and up to 1.5 sts. These fractions were dried and 18 g of hyaluronic acid and 6.5 g of chondroitin sulfate were obtained. The obtained chondroitin sulfate is a mixture of B-type and C-type in a ratio of 20:80 as determined by the enzyme analysis method. The obtained heparitin sulfate was in an amount of 150 mg and its S content was 6.8%.

Example 3

10 kg of minced pig skin became 10 kg of water and the mixture was held at 115 ° C for 1 hour and 30 minutes. The treated Liquid was subjected to centrifugal separation. The extract obtained was in the same As in Example 1, subjected to an alkali treatment and then to an enzyme treatment. He was fractionated and purified using a Schiller column, yielding 12 g of hyaluronic acid and gave 10 g of chondroitin sulfate. The chondroitin sulfate was B-type and had one S content of 6.3%.

Example 4

101 water was added to crushed blue shark cartilage, and the mixture was left for 4 hours held at 120 ° C. The treated liquid was subjected to centrifugal separation. To 8.5 1 des 0.5N sodium hydroxide was added to the extract obtained, and the mixture was left overnight stand at room temperature, which is then followed by the same enzyme treatment as in Example 1. Then, ethyl alcohol in an amount twice the volume of the mixture was added to make mucopolysaccharic acids to fail. The obtained precipitate was dissolved in water and neutralized. Ethyl alcohol was again added and the resulting precipitate was precipitated to obtain 210 g Dried chondroitin sulfate with an S content of 6.5%.

According to the method of the invention, mucopolysaccharides can be obtained in good yields within a short period of time Periods without their decomposition can be extracted by choosing the temperature and time so that they do not affect the properties of the mucopolysaccharide acids as natural products.

509686/3

Claims (2)

extremely long periods of time are required to extract muco claims: pclysaccharide acids. The enzyme decomposition method has the disadvantage 1. Method of obtaining mucopoly- that the action of the protease, if not that in the saccharide acids from animal connective tissue through 5 connective tissues contained oils and fats in sufficient extraction with water, subsequent protease- to the extent to be removed, is hindered. Hence are treatment and / or alkali treatment of the ex- usually two or more degreasing processes and separation of the mucopolysaccharide required. The disadvantage also requires acids from the aqueous phase, which characterizes the process of enzymatic decomposition itself, that one the extraction io graced scheme. at a temperature of 105 to 15O ⁰ C below It has now been found that a Mucopoly- increased pressure. liquid containing saccharic acids in advantageous 2. The method of claim 1, characterized marked manner from animal connective tissues only characterized in that the extraction temperature to be extracted at a temperature-using water is carried out of 110 to 130 ⁰ C. 15 can and that the complicated degreasing process, which was essential in the usual method is omitted can be, and the extract in one single stage from the thinned connective tissues can be obtained. There was thus a method 20 for the separation and recovery of Mucopcly- The invention relates to a process for the recovery of saccharic acids on an industrial scale Mucopolysaccharidic acids from animal binding, which wraps all the difficulties of the state tissue. technology, such as the decomposition of the mucopoly- There are already processes of separation and saccharic acids that add complexity to the process Obtaining mucopolysaccharide acids from connective tissue or the excessive length of the extraction time known from animals by a mucopoly-sided. Liquid containing saccharic acids from the binding agent. An object of the invention is thus the tissues is extracted, the extract obtained is treated with the development of a process for obtaining high protease and / or alkali to remove the protein-pure mucopolysaccharide acids from animal binding impurities and then the 3 ° tissues in high yields and with economical remaining liquid of the precipitation an advantage whereby the various disadvantages of the prior art precipitant and / or column chromatography are eliminated,
is subjected. So far, the extraction of a Mucopolysaccharidic acid-containing liquid from deep down for the extraction of mucopolysaccharidic acids Roman connective tissue by an alkali extraction 35 from animal connective tissue by extraction with method using an aqueous alkali water, subsequent protease treatment and / or solution, by a neutral salt extraction method, alkali treatment of the extract and separation of the using an aqueous solution of a neu mucopolysaccharide acid from the aqueous phase central salt, can be dissolved by an enzyme degradation method by performing the extraction at Use of protease or a combination 40 at a temperature of 105 to 150 ° C under pressure these methods. performs. These known methods are in view of the extract obtained can be obtained even by mere the decomposition of mucopolysaccharidic acids which are easily and effectively allowed to stand into one The complexity of the process and / or the length of the solid residue phase, which is insoluble in water the extraction time disadvantageous and have been found to contain 45 proteins, one oil and one fat phase and one technical procedures as unsuitable. The aqueous phase containing the alkali mucopolysaccharic acids extraction method requires two or more decoupling. This makes the technical greasing processes - To prevent the extremely simple and effective implementation of the procedure, It was known that before the extraction of Soap and get into the extract in a dissolved state, animal connective tissues for the production of mucopolysaccharidic acids and the complexity of obtaining the polysaccharidic acids, the starting connective tissue through the desired mucopolysaccharidic acids and the loss of boiling or steam to facilitate the subsequent extraction of mucopolysaccharidic acids during the process to be softened. It could be prevented, however. Furthermore, it is not known in these extraction processes that the extraction of the connective tissue is required using careful process control, using water alone at above 105 ^° C. in order to decompose the desired mucopoly and under pressure. It is known to avoid saccharic acids, and even if careful control of the above pretreatment is exercised in a steam bath, but this is particularly the case for non-negligible amounts of muco-swelling of animal connective tissues such as shark polysaccharic acids . This method also requires skin, and facilitates a subsequent extraction, an additional measure of neutralization of the alkaline substance contained in the extract. extract containing polysaccharic acids from deep According to the neutral salt extraction method, the ric connective tissues can be preserved with good effectiveness The decomposition of mucopolysaccharide acids can be avoided. be, however, there is the disadvantage that in the process of the invention the ab- an aqueous solution of a neutral salt dissolved separation and recovery of amount of mucopolysaccharide protein contamination increases. Furthermore, acids from the extract by known means