WO2009004517A1

WO2009004517A1 - Process

Info

Publication number: WO2009004517A1
Application number: PCT/IB2008/052376
Authority: WO
Inventors: Olinda Canhoto; Patrick Dunphy; Jan Verhoeven
Original assignee: Firmenich Sa
Priority date: 2007-07-02
Filing date: 2008-06-17
Publication date: 2009-01-08

Abstract

The invention provides a process for the treatment of orris roots comprising steps of blanching and incubation. Processed orris root containing high levels of irones is obtained much more rapidly than is possible using conventional methods.

Description

PROCESS

This invention relates to a process for ripening orris roots, to roots thus ripened, to a process for extracting valuable components from such roots, and to the extracts thus obtained.

Background

Orris roots grown in Tuscany, Italy, in generally decreasing production are regarded as being of best quality and most expensive. Lower qualities and prices originate from China and Morocco. Orris roots are stored from two up to four years before being treated to recover the irone fraction.

To isolate the irone fraction the rhizomes after storage for 2-4 years (moisture content 8- 13%) are finely milled to a powdered state and steam distilled to yield an Orris butter enriched in irones and myristic acid. The butter may be applied as such or further concentrated with respect to irones by crystallisation at chill temperatures from ethanol as solvent. The liquor from this crystallisation process is high in irones.

The principal irones found are c/s-α-irone (I) and γ-irone (II) and they are derived principally from iris germanica and pallida.

(I) (II)

This prolonged processing time is required for the formation of irones which are absent from the freshly harvested roots, but are produced during this storage period. Since Orris roots are expensive the storage period of 3 years is often neglected and thus Orris roots often come onto on the market having a significantly lower level of irones. As a result of this, it is often necessary to add irone enriched extracts to primary extracts to keep the irone content at the required levels. This makes the final products even more expensive. On average the overall irone yield from different Orris sources is ca. 0.03% on a final tissue basis. FR2620702 describes a procedure whereby lipophilic extracts of fresh rhizomes are oxidised via permanganate either in the presence or absence of a phase transfer catalyst. The product was an odorant composition rich in irones.

The method, although yielding an irone rich fraction, the resulting product can not be labelled natural extract anymore.

US4963480 describes an improved process for the preparation of gamma-irone enriched product by bioconversion comprising treatment of Iris rhizome substrate. These are selected from the group of Iris rhizomes, Iris rhizome parts, Iris rhizome extracts, Iris rhizome extraction wastes, plant cell cultures of Iris rhizome and mixtures thereof with selected bacteria.

US5100790 describes a process for obtaining irone by an enzymatic route. The precursors extracted from fresh Iris rhizome are subjected to the action of peroxidising enzymatic compositions comprising oxygen and a lipoxidase (LOX) or hydrogen peroxide and a peroxidase. Lipid extracts of fresh rhizome were treated in this way to obtain irones in yield that are at least three times higher than those of the standard process.

US6338861 describes the use of "unicellularised" orris root material or homogenised orris root incubated at 50⁰C for 27 days. This produces total irones, α- and γ-irone, for homogenised tissue, of 1690 mg/ Kg dry weight of tissue. Traditional, 3 years storage of sliced tissue yielded 2720 mg/ Kg dry weight of tissue.

The present invention seeks to address the problems of the prior art.

Description

According to a first embodiment, there is provided a process for the treatment of orris root, comprising the steps of: i) blanching orris root; ii) optionally comminuting the orris root of step i); iii) incubating the orris root of step i or ii to obtain processed orris root.

According to a second embodiment, there is provided processed orris root obtainable by a process of the invention. According to a third embodiment, there is provided orris butter obtainable by a process of the invention.

According to a fourth embodiment, there is provided orris absolute obtainable by the process of the invention.

Brief description of the Figures

Figure 1 is a graph; Figure 2 is a graph; Figure 3 is a graph; Figure 4 is two graphs; Figure 5 is a graph.

Detailed description

Washing

Optionally, and preferably, harvested orris root is washed to remove soil and other contaminants.

Blanching

A step in the process according to the present invention is to blanch orris root. The blanching step is preferably conducted at a sufficient temperature and for a sufficient length of time to kill fungal organisms and other pathogens detrimental to the subsequent steps, but not for sufficient time to negatively affect the enzyme systems that are essential to the incubation (fermentation) of the root.

Preferably, blanching is conducted in water. Preferably, blanching is conducted at between 40 and 100 ⁰C. More preferably, blanching is conducted at between 50 and 80 ⁰C. Still more preferably, blanching is conducted at between 60 and 70 ⁰C. Still more preferably, blanching is conducted at between 60 and 65 ⁰C.

In a particularly preferred embodiment, the blanching step is conducted at between 60 and 65 ⁰C for between one and three minutes. Cooling

Subsequent to the blanching step, the blanched orris root is preferably cooled. The cooling may be achieved by any suitable means. For example the cooling may be achieved by allowing the root to rest at ambient temperature in air. Preferably, the root is cooled by plunging into water. This may be preferably performed immediately after the blanching step. Preferably, the water is at or below ambient temperature. Still more preferably, the water is between 12 and 15 ⁰C.

Comminuting

After the blanching and optional cooling step, the roots are optionally comminuted. Comminuting serves to disturb the cell structure of the plant material, causing the wound response to be initiated. During this wound response, enzymes are released that catalyse the conversion of compounds present in the roots to the compounds responsible for the characteristic qualities of orris in terms of flavour and aroma.

It will be appreciated that the term "comminuting" or "comminuted" or "comminute" includes any manner for breaking one or more orris roots into smaller fragments. This term includes for example chopping. Any suitable apparatus may be used for comminuting the roots. A bowl chopper has been found to be particularly convenient.

Any degree of comminuting is acceptable provided that it serves to elicit the wound response. Preferably, the roots are comminuted or chopped into longitudinal pieces (i.e. comminuted or chopped perpendicular to the length of the root). More preferably, the roots are comminuted into pieces wherein the average size is between 0.1 and 5 cm in length. Still more preferably, the roots are comminuted into pieces wherein the average size is between 0.3 and 2cm in length.

Most preferably, the roots are comminuted into pieces wherein the average size is between 0.5 and 1 cm in length. Surprisingly, it has been found that when the roots are comminuted into pieces of this size, optimal results are achieved in subsequent steps. Without wishing to be limited by any such theory, it is proposed that root pieces of this size have the optimum surface area exposed to air/the atmosphere. Finer comminuting or chopping tends to result in a pulp through which air cannot circulate, while larger pieces have a lower surface area to weight ratio. Additionally, small pieces tend to exude liquor containing enzymes and flavour component precursors, which it is desirable to retain. It has been found, however, that such comminution is unnecessary, and in fact if intact orris root is employed in the incubation step, this leads to improved content of desired irones. Thus, according to a preferred embodiment, the comminution step does not take place.

Incubation

Following optional comminuting, the roots (intact or in pieces) are incubated. By "incubation" it is meant that the process by which the compounds characteristic of orris are allowed to develop. This step is also referred to as fermentation, although microbes are not believed to be involved.

The progress of the incubation process is preferably monitored so that the content of the desired aroma components is optimised. In particular, it has been found advantageous to monitor the amount of irones and myristic acid. High performance liquid chromatography (HPLC) is useful in this regard for analysis of iridals (irone precursors); gas chromatography-mass spectrometry (GC-MS) is also useful for analysis of irones and other components.

By monitoring the levels of irones and/or myristic acid, one skilled in the art will be able to judge when the incubation process is complete, and so conclude it.

In a particularly preferred embodiment, the roots are incubated at about 30⁰C and 70 % relative humidity.

Preferably, a moving tray or bed may be used. For instance, the bed may be a conveyor belt. The use of such a moving bed or tray is advantageous in that it allows the incubation step to be carried out in a continuous fashion under controlled conditions of temperature and humidity. Alternatively, drum incubation may be employed, whereby the comminuted roots are constantly rotated to achieve maximum aeration and uniformity.

Preferably, the temperature of the roots is maintained between defined maxima and minima. Optionally, the temperature is allowed to vary between the maxima and minima during the course of the incubation. However, preferably, incubation is conducted at one fixed temperature (within the limits of operational practicality, it being understood that a certain degree of fluctuation is unavoidable. Preferably, the temperature of incubation is maintained at 30 to 70⁰C. More preferably, the temperature is maintained at between 40 and 60 ⁰C. Still more preferably, the temperature is maintained at between 45 and 55 ⁰C. Still more preferably, the temperature is maintained at between 48 and 52 ⁰C. Most preferably, the temperature is maintained at about 50 ⁰C.

Preferably, the incubation is conducted at a relative humidity of greater than 50 %. More preferably, the incubation is conducted at a relative humidity of greater than 70 %. More preferably, the incubation is conducted at a relative humidity of about 75 %.

Preferably, the incubation is conducted at a relative humidity of between 50 and 100 %. More preferably, the relative humidity is 60 to 80 %. Most preferably, the relative humidity is 65 to 75 %.

The relative humidity can be controlled by any means that will be well known to those skilled in the art. It has been found preferable to maintain the humidity of the atmosphere by periodic misting of the roots with water.

Optionally, the layer of roots is mixed during the incubation process, for instance by incubation in a rotating drum. The mixing may be achieved by any suitable means, as will be apparent to one skilled in the art.

Preferably, incubation is conducted under aerobic conditions.

Surprisingly, it has been found unnecessary to conduct the incubation step in the presence of external sources of enzyme. Thus, according to a preferred embodiment, the incubation step is conducted in the absence of an external source of enzyme.

As used herein, the term "external source of enzyme" refers to enzymes not originating in the natural orris root.

Conversion to Orris Extract

The isolation of the final product is conveniently carried out by the conventional methods used in the art for the isolation of the volatiles from plants or for the production of fragrances and flavours, i.e., steam distillation, extraction or the Likens-Nickerson technique involving simultaneous steam distillation and extraction using any suitable volatile organic solvent, preferably methylene chloride, diethyl ether, hexane, and the like, and mixtures thereof.

The root may, by methods known per se in the art, be processed into any desirable form of orris; for example orris oil, orris extract, orris absolute, resinoid orris, orris concrete and orris resin.

Examples

Example 1

Intact tissue incubation

Fresh Orris rhizome, ca. 25Og, was washed with water then blanched in water at 60-65⁰C for 2 min. This material was incubated at 50 ⁰C and relative humidity of ca. 74 % for 20 days. The rhizome was comminuted and irones were extracted with acetone/diethyl ether and concentration of the ether extract, lrone concentration was

718 mg/kg dry weight of rhizome. This compared with an average value for traditionally processed Orris rhizome of 530 mg/kg dry weight. Myristic acid (14:0) contents were 1430 and 2136 mg/kg for incubated and traditionally processed rhizome respectively.

Example 2

Comminuted tissue incubation

Fresh Orris rhizome, ca. 25Og, were blanched as above. This material was comminuted to a particle size of ca. 3-5 mm, then incubated at 50⁰C and relative humidity of ca. 74 % for 10 days. Irones recovered by extraction were 364mg/kg. Myristic acid (14:0) levels were 720 and 2136 mg/kg for incubated and traditional processed material respectively.

Example 3

Incubation studies with microorganisms and enzymes

Incubation of Orris rhizomes with two fungal species

Microbial species: Trametes versicolor (Cranfield University, UK); Lepista irina (CBS 551-87, Centraalbureau Voor Schimmelcutures, Holland). Culture media: Malt extract agar (Merk), malt extract broth (Oxoid) and standard nutrient medium (S2).

Microbial growth

Actively growing cultures were incubated at 25⁰C and sub-cultured every 7-day into malt extract agar medium plates. Five plugs of agar were used to inoculate 150 ml of liquid media. Liquid cultures were incubated in a rotary shaker-incubator at 25⁰C for 10 days.

Fresh Orris rhizomes were chopped and autoclaved for 30 min at 121⁰C for sterilisation. Approximately 8 g were introduced in 250 ml flasks together with 100 ml of malt extract broth. The sterile mixture was then inoculated with 2 ml of liquid culture from either T. versicolor or L. irina. Each treatment was incubated at 25⁰C for 7 days.

Results

There was no significant difference between samples incubated with each microorganism and the control sample after 7 days of incubation.

Incubation with extracellular enzymes from L. irina

Fresh Orris rhizomes were chopped and autoclaved for 30 min at 121⁰C for sterilisation. Approximately 8 g of sterile chopped rhizomes were introduced in 250 ml flasks together with 100 ml of filtered media from actively growing L irina liquid cultures. A control samples was also prepared using 8 g of sterile chopped rhizomes and 100 ml of malt extract broth. Samples were incubated in a rotary shaker-incubator at 25⁰C for 5 and 12 days.

Results

Figure 1 shows that after 12 days incubation with extracellular enzymes from L. irina, the total irone content obtained was 124.85 mg Kg^'1 dry weight.

Incubation with Lipoxidase

Fresh Orris rhizomes were finely chopped with a blender, and approximately 100g were homogenised and extracted with methanol (see extraction method). After filtration of the solid residue, the extract was concentrated until dryness with a rotary vacuum evaporator. The extract was then diluted in 20 ml of dichloromethane (DCM) and transferred into a conical flask. The solvent was evaporated in a hot water bath and the final weight of the extract was determined.

Approximately 5g of extract were mixed with 49 ml of Sodium borate (pH 9.2, 0.01 M) buffer solution, 0.125g of Tween 80 (Sigma), 3 mg of linoleic acid and 1 ml of a lipoxidase (Type I-B from soybean, 131 ,000 units/ mg solid, Sigma) solution (0.44mg/ml in buffer). The solution was stirred at 500 rpm for 2Oh at room temperature. After incubation, the enzymatic solution was steam striped using the Liker-Nickerson technique and distilled for 1.5 h. The extract was diluted in 200ml of HPLC water and the volatiles collected in 70 ml of ether. The ether fraction was dried with NaSO₄ and filtered into a Kudema-Danish vessel. The liquid-liquid extraction was carried out at 39⁰C and the solvent concentrated up to a final volume of 4 ml. The extract was analysed and quantified in duplicate by GC-MS, using Eugenol as an internal standard.

Results

Figure 2 shows the results in duplicate obtained after 20 h incubation with lipoxidase. The averaged total irone content obtained from fresh orris rhizome extract was 46.01 mg Kg^'1 dry weight.

Example 4

Results from studies on O₂ dependency, enzymatic inhibition and time/temperature optimization

O₂ dependency and enzymatic inhibition

Studies performed to investigate the effect of oxygen and enzymatic activity, were carried out with fresh Orris rhizomes. Steaming was carried out in a pressurized system for 20 minutes at 121⁰C. For the anaerobic treatment, blanched Orris rhizomes were placed in a 3.5 L capacity anaerobic incubator (Oxoid, UK), the lid was sealed and the O₂ scavenging device was placed in the incubator. Incubation conditions for both studies were: 5O⁰C and 70% RH. Results

The results shown in figure 3 indicate that the production of irones was significantly affected by the lack of oxygen. Steamed rhizomes had a higher increase in irone content from 0 to 8 days, yet at 16 days the concentration or irones remained almost identical.

Time and temperature optimization

Experiments were carried out for the determination of the optimum temperature and incubation time. Based on previous results, comminuted and intact tissues reach their maximum irone content at different stages. For comminuted rhizomes the material was sampled after 6, 8 and 10 days; intact rhizomes were samples after 14, 16 and 20 days.

These sampling points were based on previous results, for comminuted rhizomes the irone content after 16 days was lower then that of 8 days incubation; for intact rhizomes the maximum irone content had been found after 16 days incubation. Both treatments were incubated at 50°C/70% RH. The effect of temperature was determined using intact rhizomes, incubated for 16 days at 40, 50 and 6O⁰C and 70% RH.

Results

Figure 4 shows the total irone content obtained for both comminuted and intact tissue over time. Maximum levels were obtained after 10 days for comminuted rhizomes and after 20 days for intact tissue.

The effect of temperature on the production of irones after 16 days incubation at 40, 50 and 6O⁰C is shown in figure 5. Results indicate that the optimum incubation temperature is 5O⁰C, and the lowest levels of irone content were found at 4O⁰C. Results may suggest the participation of enzyme(s) in the reaction.

Claims

1. A process for the treatment of orris root, comprising the steps of: i) blanching orris root; ii) optionally comminuting the orris root of step i); iii) incubating the orris root of step i or ii to obtain processed orris root.

2. The process according to claim 1 , wherein the blanching step i) takes place in water between 60 and 65°C.

3. The process according to claim 1 or 2, wherein the orris root is blanched in step i) for between one and three minutes.

4. The process according to any preceding claim wherein step ii is present.

5. The process according to claim 4 wherein the orris root is comminuted into pieces of 3 - 5 mm average size.

6. The process according to any preceding claim wherein the incubation step iii is conducted at a temperature of between 30 and 70 ⁰C.

7. The process according to any preceding claim wherein the incubation step iii is conducted at a temperature of between 40 and 60 ⁰C.

8. The process according to any preceding claim wherein the incubation step iii is conducted at a temperature of between 45 and 55 ⁰C.

9. The process according to any preceding claim wherein the incubation step iii is conducted at a temperature of about 50 ⁰C.

10. The process according to any preceding claim wherein the incubation step is conducted for at least 5 days.

11. The process according to any preceding claim wherein the incubation step is conducted for at least 10 days.

12. The process according to any preceding claim wherein the incubation step is conducted for between 5 and 50 days.

13. A process according to any preceding claim wherein the process is conducted at a relative humidity of greater than 50 %.

14. A process according to any preceding claim wherein the process is conducted at a relative humidity of greater than 70 %.

15. A process according to any preceding claim wherein the process is conducted at a relative humidity of about 75 %.

16. A process according to any preceding claim conducted in the absence of an external source of enzymes.

17. A process according to any one of claims 1 to 16 comprising a further step of converting the processed orris root into orris butter.

18. A process according to any one of claims 1 to 16 comprising the further step of converting the processed orris root into orris absolute.

19. Processed orris root obtainable by a process of any one of claims 1 to 16.

20. Orris butter obtainable by the process of claim 17.

21. Orris absolute obtainable by the process of claim 18.

22. A process substantially as described herein with reference to the examples.