WO1994013743A1

WO1994013743A1 - Method for isolation of vegetable oleoresins producible by hexane extraction

Info

Publication number: WO1994013743A1
Application number: PCT/DK1993/000422
Authority: WO
Inventors: Klaus Pommer
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1992-12-17
Filing date: 1993-12-16
Publication date: 1994-06-23
Anticipated expiration: 1995-06-17

Abstract

The vegetable oleoresins are produced by extraction of vegetable starting products comprising the oleoresins with a mixture of water and an organic acid immiscible with water. In this manner the use of hexane or other environmentally dangerous solvents is avoided, and also, the yield of oleoresins is improved.

Description

METHOD FOR ISOLATION OF VEGETABLE OLEORESINS PRODUCIBLE BY HEXANE EXTRACTION

The invention comprises a method for isolation of vegetable oleoresins producible by hexane extraction. Examples of such vegetable oleoresins are plant pigments, such as the yellow pigment oleoresin from Tagetes erect , carotenoides from carrots and spices, such as pebber or paprika oleoresins. Reference is made to CRC Handbook of Food Additives, 2nd edition, Vol. II, 1980, page 324.

For decades these oleoresins have been isolated by extraction with hexane or other volatile organic solvents, which will later have to be evaporated in order to isolate the oleoresins. Also, for decades it has been a well established fact that this method is highly unsatisfactory, both from an environmental point of view and due to the explosion risk and fire hazard. Reference can be made to Kirk- Othmer, Encyclopedia of Chemical Technology, 3rd edition, Vol. 18, page 67, first paragraph after heading Vapor-Cloud Explosions.

Thus, it is the purpose of the invention to provide a method of the above indicated kind, in relation to which no extraction with volatile solvents or other environmentally dangerous processes are necessary, and in relation to which the yield of the wanted oleoresins is not less than the yield of the oleoresins if produced conventionally.

Surprisingly, according to the invention it has been found that the purpose of the invention can be fulfilled, if the extraction is carried out with a mixture of water and an organic acid immiscible with water under specified conditions. It is most surprising that the method according to the invention exhibits both the advantage that it does not use volatile solvents or other environmentally dangerous processes, and the advantage that the wanted oleoresins are produced in a yield not less than the yield of the oleoresins if produced conventionally. Actually it has been found that the yield of the oleoresins produced by means of the method according to the invention is of the order of magnitude 70%, whereas the yield of the oleoresins produced conventionally is of the order of magnitude 60%. In this specification with claims an organic acid immiscible with water is to be understood as an organic acid, of which the part thereof which exists as acid in an equilibrated mixture of water and organic acid is present in a concentration of less than 1 % in the water phase at a pH below 5.5. This means that organic acids with less than around 6 carbon atoms are not classified as organic acids immiscible with water in the sense of this invention. Also, it goes without saying that the organic acid immiscible with water should be fluid at reaction temperature (usually around 40-60°C), and should preferably exhibit relatively low viscosity. This means that organic acids with more than around 12 carbon atoms are not well suited for the invention.

Thus, the method according to the invention for isolation of vegetable oleoresins producible by hexane extraction is characterized by the fact that

1 ) vegetable starting products comprising the oleoresins are extracted with a mixture of water and an organic acid immiscible with water, at a pH below or equal to 5.5,

2) the thus produced organic acid phase from step 1 ) is separated from the thus produced aqueous phase from step 1),

3) the separated organic phase from step 2) is mixed with fresh water, whereafter pH is adjusted to a value above 8.0, whereby the extracted vegetable oleoresins are liberated, and

4) the thus liberated oleoresins from step 3) are separated from the alkaline phase from step 3),

It is to be understood that the vegetable starting products in relation to the method according to the invention preferably should be comminuted to a particle size of less than 5 mm, more preferable less than 2 mm.

The final products (i.e. the vegetable oleoresins) appear as the separated, "thus liberated oleoresins from step 3)" in step 4). This product can be further purified and/or dried, if necessary.

A preferred embodiment of the method according to the invention is characterized by the fact that the vegetable starting products are annato seeds, paprika, rhizome of Curcuma longa, Tagetes erecta, lucerne, carrots, tomatoes (all pigments), paprika, pepper, tumeric, ginger, cinnamon, cassia, nutmeg, mace, clove, pimento, or cardamon (all spices). These are the most common vegetable starting products. Also, waste of the above indicated vegetables are included as starting products in the method according to the invention, especially carrot waste. 5 A preferred embodiment of the method according to the invention is characterized by the fact that the organic acid immiscible with water is a straight chain, saturated fatty acid with a total number of C atoms between 6 and 12, inclusive, preferably between 8 and 10, inclusive. If the total number of C atoms in the organic acid is below 6, the smell of the organic acid is unagreeably strong, and

10 if the total number of C atoms in the organic acid is above 12, the tendency towards generation of an emulsion resulting in difficulties in regard to recovery of the wanted products is too large.

A preferred embodiment of the method according to the invention is characterized by the fact that during step 1) a cell wall degrading enzyme is added.

15 In this manner a larger yield is obtained, and it is easier to handle the mixture of step

1).

A preferred embodiment of the method according to the invention is characterized by the fact that the cell wall degrading enzyme is SPS-ase, which is preferably added in an amount corresponding to between 25 and 2500 SPS-ase

20 activity units/kg of dry vegetable starting products. The reference to "dry vegetable starting products" does not mean that the vegetable starting products used in the method according to the invention are dried. If the enzyme is used in an activity less than the above indicated minimum amount, the effect of the enzyme will be negligible during a reasonable time, and if the enzyme is used in an amount above the above

25 indicated maximum amount, the method will be commercially unattractive. The SPS- ase activity unit is defined in AF 201.

A preferred embodiment of the method according to the invention is characterized by the fact that the cell wall degrading enzyme is a pentosanase, which is preferably added in an amount corresponding to between 3600 and

30 360,000 pentosanase activity units/kg of dry vegetable starting products. If the enzyme is used in an activity less than the above indicated minimum amount, the effect of the enzyme will be negligible during a reasonable time, and if the enzyme is used in an amount above the above indicated maximum amount, the method will be commercially unattractive. The pentosanase activity unit PTU is defined in AF 284. A preferred embodiment of the method according to the invention is characterized by the fact that the cell wall degrading enzyme is a xylanase, which is preferably added in an amount corresponding to between 360 and 36,000 xylanase activity units/kg of dry vegetable starting products. If the enzyme is used in an activity less than the above indicated minimum amount, the effect of the enzyme will be negligible during a reasonable time, and if the enzyme is used in an amount above the above indicated maximum amount, the method will be commercially unattractive. The xylanase activity unit EXU is defined in AF 293.9.

A preferred embodiment of the method according to the invention is characterized by the fact that the cell wall degrading enzyme is a celiulase, which is preferably added in an amount corresponding to between 900 and 90,000 celiulase activity units/kg of dry vegetable starting products. If the enzyme is used in an activity less than the above indicated minimum amount, the effect of the enzyme will be negligible during a reasonable time, and if the enzyme is used in an amount above the above indicated maximum amount, the method will be commercially unattractive. The celiulase activity unit NCU is defined in AF 174.

A preferred embodiment of the method according to the invention is characterized by the fact that the cell wall degrading enzyme is a protease, which is preferably added in an amount corresponding to between 1.2 and 120 protease activity units/kg of dry vegetable starting products, preferably Neutrase^®. If the enzyme is used in an activity less than the above indicated minimum amount, the effect of the enzyme will be negligible during a reasonable time, and if the enzyme is used in an amount above the above indicated maximum amount, the method will be commercially unattractive. The protease activity unit AU is defined in AF 4.

A preferred embodiment of the method according to the invention is characterized by the fact that the cell wall degrading enzyme is galactomannase, which is preferably added in an amount corresponding to between 900 and 90,000 gammanase activity units/kg of dry vegetable starting products. If the enzyme is used in an activity less than the above indicated minimum amount, the effect of the enzyme will be negligible during a reasonable time, and if the enzyme is used in an amount above the above indicated maximum amount, the method will be commercially unattractive. The galactomannase activity unit KVHCU is defined in AF 156.

A preferred embodiment of the method according to the invention is characterized by the fact that the cell wall degrading enzyme is a pectinase, which is preferably added in an amount corresponding to between 7200 and 72,000 pectinase activity units/kg of dry vegetable starting products. If the enzyme is used in an activity less than the above indicated minimum amount, the effect of the enzyme will be negligible during a reasonable time, and if the enzyme is used in an amount above the above indicated maximum amount, the method will be commercially unattractive. The pectinase activity unit PSU is defined in AF 269.

A preferred embodiment of the method according to the invention is characterized by the fact that the cell wall degrading enzyme is a 0-glucanase, which is preferably added in an amount corresponding to between 60 and 6000 β- glucanase activity units/kg of dry vegetable starting products. If the enzyme is used in an activity less than the above indicated minimum amount, the effect of the enzyme will be negligible during a reasonable time, and if the enzyme is used in an amount above the above indicated maximum amount, the method will be commercially unattractive. The 0-glucanase activity unit FBU is defined in AF 70. It is to be understood that the invention can be carried out without any enzymes as auxiliary process aids or with one of the above enzymes as an auxiliary process aid or with any combination of two or more of the above enzymes as auxiliary process aids.

A preferred embodiment of the method according to the invention is characterized by the fact that the separation in step 2) is carried out by means of a three phase decanter centrifuge. This is a cheap and effective separation, which can be carried out in one single step.

A preferred embodiment of the method according to the invention is characterized by the fact that the separation in step 4) is carried out by centrifugation. This separation is rapid and effective. A preferred embodiment of the method according to the invention is characterized by the fact that the following two supplementary reaction steps are carried out after step 4):

5) the alkaline phase from step 4) is reacidified to a pH value of below 5 4.0, whereby an acid phase is formed, and

6) the acid phase from step 5) is separated and reused as the organic acid in step 1) for a following extraction.

In this manner a more economic process is provided, due to the recovery of the organic acid. o All the above AF publications are available on request from Novo Nordisk

A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark.

The following experiments were carried out in order to test the extraction efficiences of different organic acids inside the scope of the invention.

1 part of Tagetes erecta (silaged) and 2 parts of deionized water were 5 mixed and homogenized with Ultra Turrax type T 115 KT, and the pH was adjusted to 5.5 by potassium hydroxide. 4 x 450 g of the mixture was mixed with 4 x 45 g of an organic acid, as later indicated. The mixtures were heated to 50°C under thorough agitation.

The following organic acids were added: 0 a: Octanoic acid (C₈ acid) b: A mixture of octanoic acid and decanoic acid, (Edenor V 85 from Henkel) c: Oleic acid C_1β.„ (Edenor Ti 05 from Henkel) d: Rape fatty acids (Edenor NRa from Henkel)

The extraction was carried out for 60 minutes under thorough agitation. 5 Thereafter, the mixtures (4 x 495 g) were centrifuged in a Sorvall

Instruments RC-35 refrigerated centrifuge at 4,000 RPM for 5 minutes.

Only a part of the organic top phase was isolated in order to analyze the extraction efficiency of the various organic acids.

The content of lutein in the organic phase was determined by 0 spectrophotometric analysis at 474 nm, by means of petroleum ether and according to the following equation, reference being made to Handbook of U.S. Colorants, Third Edition, 1991, page 248 (recalculated to mg of lutein/g of organic phase).

LT OD,„ x dilution x 10 mg/g 2360 x sample weight

LT is the total amount of lutein, here the concentration of lutein in mg/g, the dilution is indicated in ml, and the sample weight is indicated in g. The factor 2360 is the absorptivity for lutein in petroleum ether, in L/cm.

Results:

150 g Tagetes erecta contains 162.8 mg Lutein. The total lutein extracted = LT x 45 mg The extraction efficiency = _ LT x 45 x 100%

162.8

It appears that the use of octanoic acid provides the best extraction efficiency.

EXAMPLE 1

1 part of Tagetes erecta (silaged) and 2 parts of deionized water were mixed and homogenized with Ultra Turrax type T 115 KT, and the pH was adjusted to 5.5 by potassium hydroxide. Three portions, each consisting of 450 g of the mixture, were mixed with 45 g of octanoic acid each. The mixtures were heated to 50°C under thorough agitation.

Immediately thereafter at time 0, the following was added to each of the three mixtures. 5 a: 0.75 g of water b: 0.75 g of SPS-ase corresponding to 22 SPS-ase activity units c: 0.75 g of the protease Neutrase^® corresponding to 0.375 AU

The extraction was carried out for 60 minutes under thorough agitation, and subsequently the mixtures were heated to 80°C and held at this temperature for 10 10 minutes in order to inactivate the enzymes.

Thereafter, the mixtures (3 x 495 g) were centrifuged in a Sorvall Instruments RC-35 refrigerated centrifuge at 4,000 RPM for 5 minutes.

In this example, only 25 g of the organic top phase (I) was isolated and mixed with 2 parts of deionized water. The pH was adjusted to pH 9.0 with 15 potassium hydroxide, and the whole mixture was centrifuged again at 4,000 RPM in a Heraeus Labofuge centrifuge type 2202 for 5 minutes. The top layer (oleoresin type) was collected and air dried (II). The water phase was reacidified to pH 4.0 with hydrochloric acid. The organic top layer (III) was isolated by centrifugation for 5 minutes in a Heraeus Labofuge centrifuge type 2202, and thus it can be reused in 0 future extractions.

The content of lutein in the organic phase was determined by spectrophotometric analysis at 474 nm, by means of petroleum ether and according to the following equation, reference being made to Handbook of U.S. Colorants, Third Edition, 1991 , page 248 (recalculated to mg of lutein/g of organic phase).:

25 LT = OP-*_" x dilution x 10 mg/g

2360 x sample weight

LT is the total amount of lutein, here the concentration of lutein in mg/g, the dilution is indicated in ml, and the sample weight is indicated in g. The factor 2360 is the absorptivity for lutein in petroleum ether, in L/cm. Results:

Yields:

150 g Tagetes erecta contains 162.8 mg of lutein. Total lutein, I = LT (I) x 45

Total lutein, II = LT (II) x weight (II) Total lutein, III = LT (III) x weight (III) Process yields:

Extraction yield Total lutein. I x 100% 162.8

Product yield Total lutein. ■U x 100% 162.8

Organic acid recovery = Total lutein. Ill _{χ 10}Q% 162.8

The use of SPS-ase provides better extraction yields than water, whereas the use of protease provides better product separation than water.

Product yield will be improved by additional extractions reusing the recovered organic acid.

Claims

1. Method for isolation of vegetable materials producible by hexane extraction, characterized by the fact that

4) the thus liberated oleoresins from step 3) are separated from the alkaline phase from step 3).

2. Method according to Claim 1 , characterized by the fact that the vegetable starting products are annato seeds, paprika, rhizome of Curcuma longa, Tagetes erecta, lucerne, carrots, tomatoes (all pigments), paprika, pepper, tumeric, ginger, cinnamon, cassia, nutmeg, mace, clove, pimento, or cardamon (all spices).

3. Method according to Claims 1 - 2, characterized by the fact that the organic acid immiscible with water is a straight chain, saturated fatty acid with a total number of C atoms between 6 and 12, inclusive, preferably between 8 and 10, inclusive.

4. Method according to Claims 1 - 3, characterized by the fact that during step 1) a cell wall degrading enzyme is added.

5. Method according to Claim 4, characterized by the fact that the cell wall degrading enzyme is SPS-ase, which is preferably added in an amount corresponding to between 25 and 2500 SPS-ase activity units/kg of dry vegetable starting products.

6. Method according to Claim 4, characterized by the fact that the cell wall degrading enzyme is a pentosanase, which is preferably added in an amount corresponding to between 3600 and 360,000 pentosanase activity units/kg of dry vegetable starting products.

7. Method according to Claim 4, characterized by the fact that the cell wall degrading enzyme is a xylanase, which is preferably added in an amount corresponding to between 360 and 36,000 xylanase activity units/kg of dry vegetable starting products.

8. Method according to Claim 4, characterized by the fact that the cell wall degrading enzyme is a celiulase, which is preferably added in an amount corresponding to between 900 and 90,000 celiulase activity units/kg of dry vegetable starting products.

9. Method according to Claim 4, characterized by the fact that the cell wall degrading enzyme is a protease, which is preferably added in an amount corresponding to between 1.2 and 120 protease activity units/kg of dry vegetable starting products, preferably Neutrase^®.

10. Method according to Claim 4, characterized by the fact that the cell wall degrading enzyme is galactomannase, which is preferably added in an amount corresponding to between 900 and 90,000 gammanase activity units/kg of dry vegetable starting products.

11. Method according to Claim 4, characterized by the fact that the cell wall degrading enzyme is a pectinase, which is preferably added in an amount corresponding to between 7200 and 72,000 pectinase activity units/kg of dry vegetable starting products.

12. Method according to Claim 4, characterized by the fact that the cell wall degrading enzyme is a 0-glucanase, which is preferably added in an amount corresponding to between 60 and 6000 3-glucanase activity units/kg of dry vegetable starting products.

13. Method according to Claims 1 - 12, characterized by the fact that the separation in step 2) is carried out by means of a three phase decanter centrifuge.

14. Method according to Claims 1 - 13, characterized by the fact that the separation in step 4) is carried out by centrifugation.

15. Method according to Claims 1 - 14, characterized by the fact that the following two supplementary reaction steps are carried out after step 4): 5) the alkaline phase from step 4) is reacidified to a pH value of below 4.0, whereby an acid phase is formed, and 6) the acid phase from step 5) is separated and reused as the organic acid in step 1) for a following extraction.