KR800000570B1 - Method for extracting phenols and oligo saccharides from vegetables tissues - Google Patents

Abstract

Phenolic compds. and oligosaccharides were extd. from sunflower, cottonseed and soybean meals with solvent(n-BuOH and aq. HCl) in order to prep. protein concs. and isolates of increased parity. Defatted soybean meals were mixed with solvent for 15 min at 25≰C. A suspension was filtered through paper and the residue was reextn. 7 times. The product was dried 3 hr under N2 atm. This protein concn. can be processed to a light cream-colored protein isolate by extn. with ail. NaOH.

Description

Extraction of Fenol and Origosaccharides from Vegetable Tissues

The present invention relates to a method for coldening fenol and origosaccharides from vegetable tissues to obtain isolates and protein concentrates having low content of non-nutrient components and color source compounds.

It is known that penol and polyphene compounds in plants are widely distributed and are found in free form or in combination with protein and glutside components when present in different parts of plants, mainly seeds, leaves, stems and roots.

(See Loomis, WDand Battaile. J. Phytochemistry, 5, 423 (1966) Pierpoint, WS Biochem, J., 112 112, 609 (1969)). These natural substances are oxidized to quinone and then rapidly polymerized in alkaline environments. And react with proteins to form covalent and hydrogen bonds.

Oxidation of phenol to produce quinones occurs in the presence of oxygen or by several enzymes such as phenol oxidase, oxidase, and the like.

The use of proteins extracted from vegetables grown by humans is greatly influenced by the presence of penol components for the following economic and nutritional reasons. In other words

Covalent formation between the penol and several essential amino acids in the isolate reduces their nutritional value. Because new condensation compounds formed in this way cannot be digested by humans. Therefore, if the penol is not removed, the penol materials act as non-nutritional elements.

Rapid oxidation of these compounds causes the protein to decolorize with pH, making it unusable as food.

The presence of the phenol component reduces the ability of the proteins to be extracted due to internal interaction with the protein itself.

Several phenols, such as fennel and penol aldehydes present in cottonseed, are toxic.

Conventionally found methods for the removal of phenol compounds from vegetable powders do not allow for sufficient extraction to warrant the preparation of colorless extracts, without the use of {A.K. Smith or V.L. Johnsen, Ceral, Chem. 25, 399 (1948) and J.V. Formenta and E.P. Bruns Food Sci., 36, 490 (1971)} or because it causes some hepatic degeneration of the protein {F.J. Zobelt, Biochem. Biophys. Acta, 16, 520 (1955), S. Gaya Shudding, Carter and C.M. Matil Kood Technol., 24,242 (1970), F. W. Soulsky, C. M. McCreeley and F. S. Sorman, J. Food Sci., 37,253 (1972)}.

Undesired analogous compounds in vegetable powders are origosaccharides of fermentable properties (raffinose, starchose, verbacose, etc.) that cause bloating. Because of this, the products desired by humans are limited to the use of powders and protein concentrates.

The present invention relates to a method suitable for effective extraction under conditions that do not modify the ferol pigments and fermentable origosaccharides present in vegetables for protein. The chemical methods described herein utilize diffusion through membranes of cells and subcellular organisms in polar solvents of low molecular weight materials. The extraction process provided by the present invention provides a protein concentrate with high physiological value from which unwanted compounds (such as chlorogenic acid grass, raffinose, starch, etc.) are directly extracted, and have high purity and complete dissolution of the protein, from white to cream white. It is suitable for the preparation of protein isolates with up to color.

The present invention uses an organic polar solvent composed of an aqueous solution of an electrolyte having an acid selected from alcohols, ketones, esters or organic or inorganic acids and acid salts thereof.

The electrolyte weakens the internal action between the protein and the penol, while the weak acidic environment provided by the electrolyte increases the solubility of the penol. The extraction process is carried out in a powder solvent ratio of 1: 5-1: 240 and a pH range of 2.0-6.0 within a range from 4 ° C. to the temperature at which protein denaturation begins.

Among vegetable powders including penol and fermentable origosaccharides, sunflower seed powder, soybean powder and hwahwa powder are treated with acid solution in n-butanol.

[Sunflower Seed Powder]

Chlorogenic acid (3: 3′-cafeylquinic acid) corresponds to about 70% of the sunflower penol compound and is present in seeds in concentrations ranging from 1% -7%. M.A Sabil, F.W. J.Agr, Food Chem. 22 572, (1974), the remaining 30% of the seven known phenolic acids (iso-clogenic acid, kefeic acid p-coumaric acid, iso-ferulic acid, ferulic acid, cinnaphtran trans-cinnamic acid) And several unidentified compounds.

Sunflower seeds are protein isolates from powders used in human foods. Chlorogenic acids are oxidized to quinones in a weak alkaline environment used to use proteins to give extracts or protein isolates a color change from green to brown depending on pH. It is thoroughly disturbed by the existence of.

Soybean Powder

The non-nutritional component that causes swelling in soybean powder is the fermentable starchiose of Origosaccharide Raffinose (JJ Lakis, J. Food Sci., 35,634 (1970), which on average is 40 of water-soluble low molecular weight hydrocarbons. These compounds are present in the protein concentrate but are not in the isolate as they are removed during the protein extraction process.

[Cotton Seed Powder]

Principle limitations on the use of cotton protein as food include toxic polyphenol aldehydes, gosipol (1,1,6,6,7,7-hexahydroxy-5,5-biisopropyl-3,3′- Dimethyl-2,2-vinaphthare-8,8'-dicarboxyaldehyde) is due to association with the protein fraction. In all Gosipoli, part of Gosipol reacts with cottonseed proteins and some are found to be free and the toxic effects of Gosipol break down the use of proteins in food.

Several methods have been described for removing or deactivating gosipol (C. Bagarino J. Amer. Oil chemsoc., 38, 143 (1961): SM T. Mari and BJF Hudson J. Sci. Food Agric 26, 109 (1975) Some of these methods worked but they used only low-golf cotton seed powder.

In order to explain in detail the method of the present invention, several examples of the extraction of raffinose and starchose from chlorogenic acid and raffinose from cotton seed powder and from Gosipol and soybean powder from sunflower seed oil are listed for reference. In this example the following materials were used and the processes described below were applied.

[matter]

Chlorogenic acid and caffeic acid in pure form are from Fruka AG and Briggs SG, and sucrose, raffinose and stickynose in pure form are from Sigma Chemical Company.

The hydrochloric acid used was a melk agent, and n-butyl alcohol and n-hexane as a solvent were Karelbaze (Rea ttivo puro Erba Erba pure reagent) as RPE.

Gossipol-Vinelic acid is described by W. H. King and F. H. Turber, J. Am. Oil chem soc. , 30-70 (1953) prepared by extracting from the stripped cotton seed in accordance with the method of 98% purity.

[Way]

The macro-Kjeldahl method was used to measure nitrogen and protein nitrogen values were obtained by multiplying total nitrogen by 6.25.

Moisture, fat and fiber were measured according to standard procedures by A.O.A.C. (Association Official Analytical Chemist, 12th deition (1975)).

Quantification of chlorogenic acid in Example 1 was carried out according to the A.O.A.C. method 14,025, 11th edition (1971).

Chlorogenic acid, caffeic acid, sucrose and raffinose are derivatives in Examples 2 and 3, as measured by gas chromatography (M.A. Sabir, F.W.Soslsky and J.A.Kernan).

J. Agr Food Chem. 22 572 (1774)) Sucrose, Raffinose and Stachiose were determined in the same manner in Example 5. The analysis of organic and total testosterone is described in A.O.C.S. (Official and Tentatiue Methods of American oil Chemists Society) 3rd Edition (1972)

Ba. It was carried out according to the standard method by 8-85 and Ba-8-55.

[Gas Chromatography Method (glc)]

Preparation of Samples—Penol compounds and origosaccharides present in degreased powders and protein concentrates were extracted by refluxing with 80% aqueous methanol in a powder: solvent ratio of 1: 100 for 5 hours.

(KJ Microzac, Jr., CR Smith and IA Wolf J. Agr. Food chem. 18,27, (1970)) Methanol extract was evaporated to dryness at 40 ° C. under vacuum and then the dried residue was hydrochloric acid at pH 2.0. The solution was adjusted to pH 6.0 by adding dilute sodium hydroxide to the solution. This solution is evaporated to dryness at 40 ° C. under vacuum. The amount of phenol compound and origosaccharide extracted with anhydrous methanol at room temperature is extracted. The suspension is centrifuged and the supernatant is evaporated to dryness at room temperature in a nitrogen stream and the residue is then silylated by reacting with a known amount of TRI-SIL 'Z' at 60 ° C for 2 hours.

TRI-SIL is N-trimethylsilylimidazole from Pierce Chemical Company.

[Conditions of Gas Chromatography]

Gas chromatography analysis was performed using an HP 7620A gas chromatograph equipped with an automatic integrator HP 3380A.

Experimental conditions are as follows.

Glass column 1/8 inch x 6 feet

On stationary phase OV-1,3%, chromosolve WHP 80/100 mesh

Injector, temperature 300 ℃

Detector, temperature 300 ℃

150 ° C for 6 minutes, 150 ° C-260 ° C for 30 minutes, 260 ° C for 30 minutes

Carrier Gas Helium

Flow rate 35ml / min

Detector frame ionization

For soy flour, the analysis of origosaccharides is carried out using the following changes.

Column temperature 4 min 150 ° C., 6 ° C./min, 150 ° C.-250 ° C. for 10 min 250 ° C.,

250 ° C-320 ° C at 150 ° C / min 320 ° C-4,340 ° C at 10 ° C / min,

340 ℃ for 30 minutes

Injector temperature 350 ℃

Detector temperature 350 ℃

The 0-bifenol and origosaccharides of the extracts are identified based on the retention time of the corresponding pure compound. Quantitative analysis of several peaks was carried out by automatic integrator to recover known amounts of chlorogenic acid, caffeic acid, sucrose, raffinose and starchiose added to the extract in quantitative yield.

[Electrophoresis]

Electrophoretic analysis on 7.5% polyacrylamide gels is carried out in a vertical apparatus manufactured by Canalco Industries Co., Ltd., by using Tris-Glycine, pH 9.5, as a roundworm.

(B. Davis, Ann. N.Y. Acaol. Sci. 121,404 (1964))

[Production of Vegetable Green Powder]

The powder from which phenol and origosaccharides were extracted is manufactured by the following method. Completely peeled sunflower seeds, cotton seeds and soybeans were ground in an omni-mixing funnel in the presence of n-hexane at a ratio of 1: 2 at + 4 ° C. and then n-hexane with a volume ratio of 1: 10 It is degreased by stirring at 25 ° C. for 16 hours. Whatman No. 4 After removing the solvent by filtration under reduced pressure using a filter paper, the powder was dried for 1 hour at 25 ° C. in a nitrogen stream, and then the Great No. Crush up to 2. The chemical composition of the dried product is determined for moisture, protein, spleen and fiber using standard methods, and the content of penol and origosaccharides in sunflower seed powder and soybean powder is determined by gas chromatography.

[Extraction of Chlorogenic Acid from Sunflower Seed Oil (Example 1)]

The preparation of the solvent used in Example 1 for the extraction of chlorogenic acid from sunflower seed oil is carried out as follows.

1 L of -butyl alcohol is mixed with 1 L hydrochloric acid solution at 0.5 10 ^-2 N, pH 2.48 and allowed to stand overnight with occasional stirring in a separating funnel. The upper layer phase recovered after discarding the acidic aqueous phase is used as the solvent of the extraction step.

Sunflower seeds prepared by the method described above were screened with a size of 0.050 mm using a Pretty Analyset 3, screen machine, and then mixed with a solvent having a volume ratio of 1/30 and stirred at 30 ° C. for 30 minutes.

The turbidity is centrifuged at 5,000 rpm for 10 minutes at room temperature using a brushed RB-2 centrifuge, Laura SS-34.

After decanting the supernatant, the extraction is repeated several times (5-10 times) as a solvent. The number of extraction runs was determined by measuring the butanol extract at 328 mm, the maximum absorption of chlorogenic acid in the solvent, and the number of absorption of chlorogenic acid (Aimg / ml cm) at 328 mm was 51.3. After extraction with solvent, the solid phase is dried in nitrogen stream for 3 hours, and the residual content of chlorogenic acid is measured by A.D.A.C 14,025 method.

Extraction of Fenol and Origosaccharides from Sunflower Seed Powder, Cotton Seed Powder, and Plum Powder (Examples 2, 3, 4, and 5)

The preparation of a solvent for extracting phenol and oligosaccharides from sunflower seed cotton seed and soybean powder is carried out as follows.

92 parts of n-butyl alcohol are mixed with 8 parts aqueous hydrochloric acid solution having a pH of 2.30. In this ratio the organic solvent is sufficiently mixed with the aqueous phase. The solvent obtained is added to the powder to be extracted in different ratios of powder to solvent and stirred for 15 minutes at different temperatures. The pH of the suspension is kept constant within the minimum solubility of the protein contained in the powder to be tested. The constant maintenance of pH is maintained by adding 6N hydrochloric acid or adding a pH 0.5 solution formed of 92 parts of n-butyl alcohol and 8 parts of hydrochloric acid to the stirred turbid solution. The turbidity is Whatman No. After filtration on the three filter papers, the extraction of the residue is repeated. The protein concentrate obtained after the extraction (2-8 times) is dried at least 3 hours in a stream of nitrogen.

The chemical composition of the different parts of the dried material is determined by the relevant moisture, protein, fat and fiber and the residual content of chlorogenic acid, caffeic acid, gosapol, sucrose, raffinose and starchiose on the concentrate is determined.

[Production of Protein Isolates]

The protein concentrate obtained in the above process is subjected to two acid extraction proteins. Firstly, a single-step non-selective extraction method in alkaline substitutes, and second, a low molecular weight water-soluble protein with high electrophoretic mobility and a high molecular weight protein dissolved in alkaline medium and having low electrophoretic mobility. Receive a two-step method of discernment. The second method yields two isolates of different composition and properties.

[Single step extraction]

A portion of the protein concentrate obtained from the extraction process is slurried in 15 parts of water and the pH is adjusted to 9.5 with 0.2 N sodium hydroxide (powder / solvent volume ratio of 1/15) and stirred at 25 ° C. for 30 minutes.

The slurry was centrifuged for 20 minutes at 17,000 rpm using a brushed RB-2-centrifuge with SS-34 rotor. Repeat the second extraction under the same conditions for the residue, combine the two supernatants and add 0.5N hydrochloric acid to the isoelectric branch to precipitate the protein. The precipitate is precipitated by centrifugation at 17,000 rpm for 10 minutes, washed with an acidic aqueous solution, placed in an aqueous medium, neutralized to pH 7.0, and lyophilized.

The Kjeldahl method determines the total nitrogen content for precipitates, supernatants and insoluble residues.

[2-step extraction]

1) A portion of the protein concentrate is treated with 15 parts of mole (1 weight / 15 volume powder / solvent ratio) at pH 6.5, stirred at 25 ° C. for 30 minutes, and the turbidity is centrifuged at 17,000 rpm for 20 minutes. For the residue, two combined supernatants obtained by performing a second extraction under the same conditions were coined to precipitate proteins. The precipitate is washed with acidic solution, slurried in water, neutralized to pH 7.0 and lyophilized.

2) Insoluble residue obtained from the first extraction step is dissolved in alkaline replacement at pH 9.5 to dissolve the protein and the single-extraction process is repeated under the same conditions.

More details have been described in the embodiments described in order to make the present invention easier to understand, but the embodiments are not intended to limit the present invention.

Example 1

Extraction of Chlorogenic Acid from Sunflower Seeds of Imeni Species

Chemical composition of sunflower seeds:

Moisture: 3.8% (on dry matter)

Protein (N. 6.25) 24.9%

Fat 58.7%

Chlorogenic Acid 1.9%

Impurity Fiber 2.4%

Non Nitrogen Extract 8.7%

Ash 3.4%

Sunflower seed powder produced by the above-described method has the following composition.

Moisture: 8.8% (on dry matter)

Protein (N. 6.25) 64.6%

Fat less than 1.0

Chlorogenic Acid 4.8%

Impurity Fiber 3.9%

10 g of a powder containing 0.050 mm of particle size was mixed in a flask with 300 ml of solvent, stirred at 30 ° C. for 30 minutes, the mixture was centrifuged at 5,000 rpm at room temperature for 10 minutes, decanted and additionally added at 300 rpm. The extraction is repeated with fresh solvent. This treatment is carried out a total of eight times in succession (final ratio of powder / solvent of 1/240).

The chlorogenic acid content of each extract is measured by spectroscopic method at 328 mm for solvent blank as listed in Table 1.

TABLE 1

Eight extracted residues are dried in a nitrogen stream for 3 hours, and then chlorogenic acid is measured according to A.O.A.C 14,025 method. After 8 extractions, the content of chlorogenic acid is less than 0.2%.

Example 2

Preparation of Protein Concentrates and Disadvantages from Sunflower Seeds of Amiara Species with Chlorogenic Compounds and Fermentable Origosaccharides Removed

a) Preparation of protein concentrates free of C-bifenol and origosaccharides

The powder used has the following composition.

Moisture (in dry matter) 10.6%

Protein (N. 6.25) 58.7%

Less than 1.0% of fat

Chlorogenic Acid 1.56%

0.14% of caffeic acid

Sucrose 4.70%

Raffinose 3.32%

Impurity Fiber 4.2%

20 g of degreased sunflower seed powder is mixed in a flask with 400 ml of a solvent formed of 92 parts of n-butyl alcohol and 8 parts of aqueous hydrochloric acid solution and then extracted by stirring at 25 ° C. for 15 minutes. The initial pH of the turbidity is 6.2 and adjusted to 5.0 during extraction and this pH value is adjusted by addition of 0.5N hydrochloric acid. Whatman suspension

Filter under vacuum using three filter papers and repeat a total of eight extractions (final powder / solvent 1/160) on the solid residue. The product dried for 3 hours in a nitrogen stream has the following composition.

The product by its protein (72.9%) is defined as protein concentrate. This concentrate was removed from the origosaccharides and phenol components that reacted with the features formed during extraction of the white matter in alkaline medium. Electrophoretic analysis on polyacrylamide gels of proteins extracted from the concentrates indicate that they have the same electrophoretic morphology as those separated into sunflower seed powder.

b) Preparation of protein isolated mole by extraction in a single step.

10 g of protein concentrate is slurried in 150 ml of alkaline aqueous solution (1 weight / 15 volume powder / solvent ratio) at pH 9.5 and stirred at 25 ° C. for 30 minutes. The pH of the suspension is maintained at a constant value of 9.5 throughout the extraction process by the addition of dilute sodium hydroxide aqueous solution in small portions. The suspension is centrifuged at 17,000 ppm for 20 minutes and the residue is extracted again under the same conditions as above. The two protein solutions are combined and precipitated at pH 5.2 with 0.5 N hydrochloric acid. The precipitate was centrifuged at 17,000 rpm for 10 minutes, washed with main acidic acid of pH 5.2, slurried in water, neutralized to pH 7.0, and lyophilized. The protein content for isolates, supernatants and insoluble residues by a single step extraction method is reported in Table 11. The color of the bloated was bright creamy white.

c) Preparation of Protein Isolates by Two-Step Extraction

10 g of protein concentrate is added to 150 ml of an aqueous solution of pH 6.5 (1 weight / 15 volume powder / solvent ratio) and stirred at 25 ° C. for 30 minutes. The pH of the suspension is kept constant at 6.5 by addition of 0.02 N sodium hydroxide solution in small portions. The suspension is centrifuged at 17,000 rpm for 20 minutes and the residue is extracted again under the same conditions. Precipitate the two combined protein solutions with 0.5 N hydrochloric acid at pH 4.0, centrifuge at 17,000 rpm for 10 minutes, wash with acidic water at pH 4.0, neutralize to pH 7.0 with sludge in water, and freeze-dry.

The isolate of 1 is white.

2) Extraction of protein to insoluble residue obtained from the above process was effective in alkaline medium at pH 9.5 under the same conditions as described in the single-step protein extraction method.

The protein content for the isolates, supernatants and insoluble residues of the two-stage extraction process are listed in Table 2.

TABLE 2

Total nitrogen by 6.25

Example 3

Optimization of the Extraction Process of C-Bifenol and Fermentable Origosaccharides from Sunflower Seeds of Jomisei Species for Preparation of Protein Concentrates

The sunflower seed powder used had the same composition as reported in Example 2.

20 g of degreased powder is added to 100 ml of a solvent formed of 92 parts of n-butyl alcohol and 8 parts of aqueous hydrochloric acid solution, followed by extraction by stirring at 25 ° C. for 15 minutes. The initial pH of the suspension is 6.2 and the value of 5.0 is kept constant by small additions of 0.5 N hydrochloric acid during extraction. The suspension is vacuum filtered and eight extractions of the solid residue are carried out. (Final powder / solvent ratio of 1/40) The product to be dried for 3 hours in a nitrogen stream has the following composition.

Moisture (from dry matter) 10.8%

Protein (N. 6.25) 69.0%

Chlorogenic acid 0.05% or less

Caffeic acid 0.05% or less

Sucrose 1.38%

Rafinos 1.33%

4.6% impurities

The results indicate that the residual content of caffeic acid of chlorogenic acid is unchanged (below 0.05%) by varying the extraction ratio of powder / solvent by 1/160 (Example 1) -1/40, while adding sucrose and raffinose. Indicates that the output is reduced.

Example 4

Preparation of Protein Ponds with Low Gosipole Content from Stripped Cotton Seeds

The cotton seed powder used has the following composition.

Moisture (from dry matter) 10.0%

Protein (N. 6.25) 47.6%

Fat 1.0% or less

Yuri Goshipol 1.45%

Total Notice 1.93%

1.0% fiber

20 g of degreased cottonseed powder is added to a 40 ml solvent formed of 92 parts n-butyl alcohol and 8 parts aqueous hydrochloric acid solution in a flask, followed by extraction at 25 ° C. for 15 minutes. The initial pH of the suspension has a value of 6.1 and is kept constant at 4.0 by adding 0.5 N hydrochloric acid during extraction. The suspension is filtered and eight extractions of the solid residue are carried out under the conditions described above. The resulting protein concentrate was dried for 3 hours in a stream of nitrogen to have the following composition.

Moisture (from dry matter) 10.5%

Protein (N. 6.25) 66.5%

Less than 0.5% fat

Yuri Goshipol 0.07%

Total notice pole 0.344%

2.7% fiber

Treatment with n-butyl alcohol and aqueous hydrochloric acid solution yields protein concentrates (66.5% of protein) under undenatured conditions with low contents of free gosypol and total gosypol.

The method suggested by the present invention has the advantage of using cottonseed powder having a high goofol content, which is currently unsuitable for the production of protein concentrates or isolates.

Example 5

Preparation of Protein Concentrates with Low Content of Fermentable Origosaccharides from Stripped Soybeans of Adazole

Chemical composition of the powder 11.2%

Moisture (from dry matter) 53.8%

Protein (N. 6.25) 1.0 or less

Sucrose 8.24%

Lapis Sauce 0.95%

Starchios 4.70%

1.30% fiber

20 G of degreased soybean powder is added to a 400 ml solvent formed of 92 parts of n-butyl alcohol and 8 parts of hydrochloric acid solution in a flask, followed by extraction at 40 ° C. for 15 minutes. The initial pH of the suspension is maintained at a value of 4.5 by addition of small amounts of 0.5 N hydrochloric acid in the already 6.3 extract.

Suspension to Whatman No. Filter under reduced pressure using three filters and repeat the eight extractions to the solid residue. (1/160 final powder / solvent) The product, dried for 3 hours in a nitrogen stream, has the following composition.

Moisture (from dry matter) 9.3%

Protein (N. 6.25) 65.9%

Sucrose 0.41%

Papinos 0.83%

Starchios 2.82%

2.4% fiber

This product has a low content of fermentable origosaccharides, defined as protein concentrates with a protein content of 65.9%.

Claims (1)

Fenol, characterized in that the extraction of the vegetable protein from the powder, concentrate and isolate with a polar solvent consisting of a complete mixture of n-butyl alcohol and an aqueous solution of an electrolyte having an acid selected from inorganic organic or acid salts thereof. A method for extracting phenol and origosaccharides from plant protein substances, including origosaccharides.