Patent Application
20170303557
The present invention relates to a process for the purification of polyphenols from a starting material. In particular the present invention relates to a fraction enriched in polyphenols, in particular chlorogenic acids (CGA's) obtainable from a plant material, wherein the isomer balance of the enriched fraction is substantially maintained compared to the starting material.
An important property of beverages, such as coffee is persistent foam also referred to as “crema” and intense aroma which are considered important quality criteria. The volume, texture, finesse, color and stability of the crema are distinctive characteristics appealing to the consumer. Crema results from the extraction of surface active coffee components that coat and stabilize the gas bubbles created by blasting the tamped espresso coffee matrix with pressurized heated water.
Coffee aroma is also an important property because the intensity of the coffee aroma is the first sensoric experience that meets the consumer. When smelling coffee, the aroma can help you evaluate the coffee flavor and the brightness of the coffee. During roasting many aromatic compounds are formed or liberated from the coffee bean matrix and affect the experience (aroma and flavor) of the coffee. One of the most important precursor of aromatic compounds are chlorogenic acids, in particular caffeoyl quinic acid.
Thus, there has been an interest to isolate a polyphenol enriched fraction from many different kind of plant materials, e.g. berries, grapes, citrus, vegetables, cereals, herbs, tea, coffee, cocoa. In particular there has been an interest to isolate a polyphenol fraction enriched in chlorogenic acid compounds and which has an isomeric balance of the chlorogenic acid compounds that resemples the original plant material, in order to spike food and beverage products with e.g. aroma and flavor.
Furthermore, polyphenols have been reported to have antimicrobial, antiviral, antimutagenic, anticarcinogenic, antiproliferative and vasodilatory effects. Polyphenols may have antibacterial activity which may be useful in the combat of tooth decay caused by Streptococcus mutans. Polyphenols are listed as nootropics purported to improve mental functions such as cognition, memory, intelligence, motivation, attention and concentration.
However, strengthening of the rules for health claim in food legislation makes it compulsory to ensure a consistent level in the active ingredient as well as to refer to scientific evidence obtained on the manufactured products.
Thus, there is a need for a process for providing a polyphenol enriched fraction, especially comprising chlorogenic acids (CGA's) where the chlorogenic acid balance resembles the chlorogenic acid balance originally present in the plant material. The enriched fraction may subsequently be used to ensure a constant level in active ingredient of food product as well as in intervention studies to demonstrate the cause and effect relationship between the intake of polyphenols, such as from coffee, and a given claimed effect.
Thus, there is a need in the industry to provide a process for providing a fraction enriched in polyphenols, in particular chlorogenic acid, having the most authentic characteristics relative to the plant material it originates from which may be used to enhance the properties of a food product or a beverage.
Accordingly, the present invention describes a novel process for the purification of polyphenols where the polyphenolic balance is maintained or substantially maintained. Furthermore, the process of the present invention may result in a limited or no formation of derivatives formed during the process e.g. ethylesters of the polyphenols. Ethylesters are undesirable because they do not have the same properties as the native polyphenols naturally occurring in the plant material. In addition, ethylesters change the polyphenol balance of the enriched fraction relative to the original product.
Thus, one aspect of the invention relates to a process for providing a fraction enriched in polyphenols from a starting material, the process comprises the steps of:
Another aspect of the present invention relates to a fraction enriched in polyphenols obtainable from the process according to the present invention.
Yet another aspect of the present invention is to provide a food ingredient comprising the fraction enriched in polyphenols according to the present invention.
Still another aspect of the present invention is to provide a food product comprising the fraction enriched in polyphenols according to the invention and/or the food ingredient according to the present invention.
A further aspect relates to the use of the fraction enriched in polyphenols according to the present invention and/or the food ingredient according to the present invention as a precursor for generating aroma, flavor and/or foaming.
Yet another aspect relates to a pharmaceutical composition comprising the fraction enriched in polyphenols according to the present invention and a pharmaceutical acceptable carrier.
The present invention will now be described in more detail in the following.
The present invention describes a novel process for providing a fraction enriched in polyphenols using a chromatographic resin. The enriched fraction obtained from the present invention may provide improved properties, in particular aroma, flavour and foaming properties, since the isomer balance of the polyphenols (such as chlorogenic acid compounds) present in the enriched fraction is substantially maintained and resemble the isomer balance of the polyphenols (such as chlorogenic acid compounds) originally present in the plant material. Furthermore, the process for providing a fraction enriched in polyphenols according to the present invention also provides an enriched fraction with a high purity and/or good properties (product performance).
In an embodiment of the present invention the enriched fraction has a content of chlorogenic acid compounds of at least 40% (w/w), such as at least 50% (w/w), e.g. at least 60% (w/w), e.g. at least 70% (w/w).
The presented process may have several advantages compared to other purification processes. One advantage of the present invention may be that the isomeric balance of polyphenols, in particular, chlorogenic acid compounds, of the enriched fraction may be maintained. Another advantage may be that the process generates limited or no ethylesters of polyphenols. A further advantage of the present invention may be that a high level of purity (i.e. >60%) may be obtained.
Schematic details of the process according to the invention are displayed in
Thus, an aspect of the present invention relates to a process for providing a fraction enriched in polyphenols from a starting material, the process comprises the steps of:
This process was developed based on the chemical and physical properties of polyphenol compounds, for example polyphenol compounds as described in
Chlorogenic acids are low molecular weight compounds, globally below 600 g/mol. Chlorogenic acids bear one or several aromatic groups (i.e. phenolic moiety). Non-covalent aromatic interactions, also called π-π stacking interactions, can be established between compounds containing these aromatic moieties. This property is used to specifically adsorb chlorogenic acids to matrices bearing aromatic groups.
Chlorogenic acids bear a quinic acid group which confers acid-base properties (pKa=3.2). At pH below pKa, the chlorogenic acid is protonated. The molecule is uncharged and therefore it is rather hydrophobic. At pH above the pKa, the acid function is under a carboxylate form, which means that the entire molecule is more hydrophilic due to the charge. This property can be used to modulate the hydrophobicity of the molecule for purification purposes.
The starting material comprising the polyphenols may be of different origin. In an embodiment the starting material is a plant material. In yet an embodiment the plant material is selected from the group consisting of coffee, such as green coffee, caffeinated coffee, decaffeinated coffee and decaffeinated green coffee, malt, cocoa, tea, berries, grapes, vegetable, citrus, herbs and cereals. The starting material may initially be solubilized e.g. in boiling water. It may be advantageous to perform an initial precipitation step of the solubilized starting material before starting the above described purification process in order to remove high molecular weight (HMW) compounds like proteins, melanoidins and polypsaccharides e.g. arabinogalactans. Thus, in an embodiment the solubilized starting material obtained in step (i) may be subjected to precipitation e.g. with an alcohol (preferably ethanol) to provide a solid phase and a liquid phase, before the pH-adjustment in step (ii). In yet an embodiment the solid phase may be separated from the liquid phase by filtration, centrifugation and/or decantation, before the pH-adjustment in step (ii). Following the optional separation step the liquid phase may be subjected to an evaporation treatment to remove the alcohol used for precipitating HMW's from the liquid phase, before the pH-adjustment in step (ii).
The fraction enriched in polyphenols may be of different forms. Thus, in an embodiment the fraction enriched in polyphenols may be in liquid form or in dried form. In a further embodiment the fraction enriched in polyphenols is freeze dried.
Example 1 below discloses a particular example, where the solubilized starting material is precipitated with 80% alcohol. Thus, in an embodiment the alcohol is ethanol. In a further embodiment the alcohol such as ethanol has a concentration in the range 50-99% (w/w), such as 60-99% (w/w), such as 60-90% (w/w), such as 70-90% (w/w), such as 75-85% (w/w).
The process according to the present invention includes acidification of the starting material to below pH 3 before it is loaded on to the resin column. In an embodiment of the present invention the pH adjustment in step (ii) is an adjustment to below pH 2.5, preferably below pH 2, more preferably below pH 1.5, or even more preferably to about pH 1. In another embodiment the acid used is selected from the group consisting of strong acid e.g. hydrochloric acid, sulfuric acid and phosphoric acid. The reason for the relatively low pH is that pure water and/or use of higher pH-values may cause desorption of CGA's from the resin. On the other hand, under acidic condition, as chlorogenic acids remain in their protonated and hydrophobic form, interactions with the resin is maintained.
Example 2 presented below demonstrates the effect of lowering the pH to pH 1 before loading the starting material onto the chromatographic resin. After pH adjustment, and before loading the starting material onto the chromatographic resin, the material may be filtered to remove larger debris. The chromatographic resin may also be pre-conditioned with an acid before applying the starting material to be purified. Thus, in an embodiment the chromatographic resin is pre-conditioned with an acid at a pH below 3, preferably below pH 2, more preferably below pH 1.5, or even more preferably to about pH 1. In another embodiment the acid used for pre-conditioning the column is selected from the group consisting of hydrochloric acid, sulfuric acid and phosphoric acid. The exact concentration of the acid may vary. Thus, in an embodiment the concentration of the acid is in the range 0.001M-1M, preferably 0.001M-0.5M, or even more preferably in the range 0.05M-0.3M.
After loading the starting material onto the chromatographic resin it may be advantageous to rinse the chromatographic resin to remove unbound materials. Thus, in an embodiment the chromatographic resin may be treated with a rinsing solution before the polyphenols are desorbed. Preferably, the rinsing solution has a pH value below pH 3, preferably below pH 2, more preferably below pH 1.5, even more preferably about pH 1. In an embodiment the washing solution comprises an acid, such as hydrochloric acid, sulfuric acid or phosphoric acid. As the exact concentration of the acid may vary it is an embodiment of the present invention the concentration of the acid in the rinsing solution may be in the range 0.001M-1M, preferably 0.001M-0.5M, or even more preferably in the range 15 0.05M-0.3M.
Example 3 shows the effect of rinsing the column with 0.1M HCl, resulting in an increase in the purity of the final fraction from 50% to 66% compared to when no rinsing step is included. Thus, it may be concluded that rinsing the column before desorption may further improve the purity of the final fraction.
The ratio between the starting material and the chromatographic material may influence the amount of purified product. Overloading the column may cause the column to be saturated which may result in insufficient isolation of valuable polyphenolic compounds because a fraction of the solubilized starting material may not be adsorbed to the column. Thus, in an embodiment of the present invention the ratio between the starting material and the chromatographic material may be from 2:1 to 1:4 (on a weight:weight basis), such as from 1:1 to 1:3 (on a weight:weight basis), e.g. about 1:2 (on a weight:weight basis). In the present context the term “chromatographic resin” relates to the chromatographic media present in the chromatographic column and responsible for the separation of the starting material. In an embodiment of the present invention the chromatographic resin may be packed in a packed bed or in an expanded bed. Different types of chromatographic resins may be used. Thus in an embodiment of the present invention step (iii) may involve adsorption chromatography on a polymer resin and/or any other chromatographic media known to the skilled person.
For desorption, the interactions between the chromatographic resin and phenolic compounds of the polyphenols have to be weakened. Different parameters may influence the desorption step of the present invention.
The inventors of the present invention found that temperature may be a parameter that has a significant influence on the undesirable formation of ethylesters of the polyphenols in the presence of a hydroxyl component, e.g. ethanol.
Thus, in an embodiment of the present invention the temperature during desorption may be below 70° C., such as below 60° C., such as below 50° C., such as below 40° C. In another embodiment the temperature is in the range 10-40° C. such as in the range of 25-35° C., e.g. at about 30° C. Example 7 demonstrates the advantage of keeping the temperature down to avoid ethylester formation of the CGA's. Thus, it may generally be an advantage to keep the temperature as low as possible during every step of the process. However, it is always a tradeoff between the speed of the assay (e.g. during an evaporation and/or a desorption step) and avoiding the generation of ethylesters.
Another parameter that may influence the desorption process is the composition of the desorption eluent. The desorption eluent may comprise different constituents that may improve desorption of the polyphenols. In an embodiment of the present invention the desorption eluent may comprise a hydroxyl component, preferably the hydroxyl component is ethanol. Other alcohols or combination of alcohols may also be used as desorption eluent.
The inventors of the present invention also found that the pH of the eluate and the desorption eluent may have a significant influence on the undesirable formation of ethylesters of polyphenols during the process in the presence of a hydroxyl compound, e.g. ethanol. In an embodiment of the present invention the pH of the eluate and/or the desorption eluent may be above pH 3, such as above pH 3.5, preferably about pH 4, or even more preferably in the pH range 4-5. At high pH values, e.g. strong alkaline conditions like pH 12, the undesired formation of ethyldesters of chlorogenic acids may be reduced, but may cause other undesirable reactions of the polyphenols.
In yet an embodiment of the present invention the desorption eluent may comprise a hydroxyl component, preferably ethanol, in combination with an aqueous base, the aqueous base may preferably be KOH.
In an embodiment of the present invention the desorption eluent may comprise at least 20% of the hydroxyl component, e.g. at least 30% of the hydroxyl component, such as at least 40% of the hydroxyl component, such as at least 50% of the hydroxyl component, e.g. at least 60% of the hydroxyl component, such as at least 70% of the hydroxyl component, e.g. at least 80% of the hydroxyl component and at most 20% of the aqueous base, such as at least 90% of the hydroxyl component and at most 10% of the aqueous base, or a stepwise combination thereof.
Since the fraction enriched in polyphenols may comprise many different polyphenolic and chlorogenic acid compounds a stepwise elution may be used. Thus, in an embodiment of the present invention the desorption eluent may comprise
In a further embodiment of the present invention the desorption step may be a stepwise elution comprising the combination of two or more of the concentrations of hydroxyl components in the desorption eluent as mentioned in (i), (ii) or (iii), such as a stepwise combination of all three. It may also be advantageous to elute by adding a gradient of the concentration of the hydroxyl component during desorption, thereby obtaining an desorption eluent comprising different polyphenolic compounds. Thus, in an embodiment of the present invention the hydroxyl component may be provided as a gradient going from at the most 100% to minimum 1% during desorption, such as from at the most 90% to minimum 15%, such as from at the most 90% to minimum 70%, such as from at the most 60% to minimum 40%, such as from at the most 30% to minimum 10%. In the above embodiment the hydroxyl component is initially a high concentration, however, it may be advantageous to go from a low concentration to a highconcentration. Thus, in another embodiment of the present invention the hydroxyl component may be provided as a gradient going from at least 1% to at the most 100% during desorption, such as from at least 15% to at the most 90% such as from at least 10% to at the most 30%, such as from at least 40% to at the most 60%, such as from at least 70% to at the most 90%. In a further embodiment the desorption step is provided as a stepwise combination of any of the gradient ranges according to the invention. By having a stepwise elution where multiple eluates are collected (each with a different content of the different polyphenols,) it may subsequently be possible to mix two or more of the collected eluates to obtain a final product with an isomeric balance of polyphenols, in particular chlorogenic acids,which resembles the isomeric balance of polyphenols, in particular chlorogenic acid, present in the starting material more accurately. If only one eluate is collected, this may not be possible.
As mentioned previously, temperature influences the formation of ethylesters. Thus, in an embodiment of the present invention desorption may be performed at a temperature below 80° C., such as below 75° C., such as below 50° C., such as below 35° C., or such as in the range 20-30° C. However, since the speed of the desorption is faster at higher temperature, this is again a tradeoff
After desorption it may be an advantage to further adjust the pH of the eluate to avoid ethylester formations. Thus, in an embodiment the pH is adjusted after desorption to a pH in the range 3.5-6, such as 3.5-5, such as 3.5-4.5, such as 4-5, or such as around 4 or 5.
The eluate obtained comprises a hydroxyl component and in order to further enhance the concentration of the polyphenols in the eluate the hydroxyl component may be separated from the polyphenols.
The separation step after elution, and optional pH adjustment, may be performed in different ways. In an embodiment of the present invention the separation in step (vi) may performed by evaporation. Since temperature also influences ethylester formation the evaporation of the eluate is performed at a temperature below 80° C., such as below 75° C., below 60° C., such as in the range 40-60° C., or such as in the range 45-5° C., preferably around 50° C. However, since the speed of the desorption is much faster at elevated temperatures, this is again a tradeoff. After evaporation an evaporated phase comprising the hydroxyl component and a condensate phase comprising the polyphenols are provided.
Preferably the content of hydroxyl component present in the residual phase is less than 5% (w/w), more preferably less than 1% (w/w), even more preferably less than 0.1% (w/w), even more preferably less than 0.01% (w/w).
It is to be understood that pH adjustments may take place before separation (such as evaporation) of the hydroxyl component or after separation (such as evaporation) of the hydroxyl component. In addition, pH adjustment may take place both before and after separation of the hydroxyl component. Thus, in an embodiment of the present invention the pH of the desorption eluent may be adjusted to a pH in the range 3.5-6, such as 3.5-5, such as 3.5-4.5, such as 4-5, or such as around 4 or 5. In an embodiment of the present invention the separation step, such as evaporation takes place before separation, after separation or both before and after separation. After a separation step the desorption eluent may be present in an acidic solution in which the polyphenols may be present in their hydrophobic and predominantly insoluble form. In order to better solubilise polyphenols, a pH adjustment as described above may be an advantage.
In order to make the purification protocol industrial applicable, processing time may be important. Thus, in an embodiment of the present invention the processing time may be 36 hours or less, such as 30 hours or less, e.g. 25 hours or less, such as 20 hours or less, e.g. 18 hours or less, such as 15 hours or less, e.g. 12 hours or less, such as 10 hours or less, e.g. 5 hours or less. In the present context the processing time relates to the period from solubilization of the starting material until the eluate has been separated into a phase comprising the hydroxyl component and a residual phase comprising the polyphenols thereby obtaining the fraction enriched in polyphenols according to the invention.
The fraction enriched in polyphenols may be further purified. Thus, in an embodiment of the present invention the fraction enriched in polyphenols may be further purified by chromatography, ultrafiltration, nanofiltration, microfiltration, reversed osmosis or any combination thereof. The final product may also be freeze-dried.
Besides relating to a purification process, the present invention also relates to products obtainable by such process. Thus, another aspect of the present invention relates to a fraction enriched in polyphenols obtainable from the process according to the present invention. Such products may be very unique as they comprise a balance of polyphenols, in particular the balance of cholorogenic acids, that resembles the balance found originally in the starting material.
In an embodiment of the present invention the fraction enriched in polyphenols may have a content of polyphenols of at least 35% (w/w) on a dry matter basis, such as at least 45%, such as at least 55%, such as at least 65%, or such as at least 75%. Thus, in an embodiment of the present invention the fraction enriched in polyphenols may have a content of ethylester of polyphenols below 20% (w/w) on a drymatter basis and relative to the total content of polyphenols, such as below 15% (w/w), e.g. below 10% (w/w), such as below 5% (w/w), e.g. below 1 (w/w), such as in the range of 0.1-20% (w/w), such as in the range 0.1-10%, such as in the range 0.1-5%, such as in the range 0.1-4%, such as in the range 0.1-3%, such as in the range 0.1-2%, or such as in the range 0.1-1%. In a more specific embodiment of the present invention the fraction enriched in polyphenols may have a content of polyphenols of at least 35% (w/w) on a dry matter basis and below 20% (w/w) on a drymatter basis and relative to the total content of polyphenols, such as between 0.1-20% (w/w) ethylesters of polyphenols.
Thus, in an embodiment of the present invention the polyphenol may be a chlorogenic acid (CGA), caffeic acid or a combination thereof. In yet an embodiment of the present invention the chlorogenic acid (CGA) may be selected from the group consisting of 3-caffeoyl quinic acid (3CQA), 4-caffeoyl quinic acid (4CQA), 5-caffeoyl quinic acid (5CQA), 3,4-dicaffeoyl-quinic acid (3,4diCQA), 3,5-dicaffeoyl-quinic acid (3,5diCQA), 4,5-dicaffeoyl-quinic acid (4,5diCQA), 3-feruloyl quinic acid (3FQA), 4-feruloyl quinic acid (4FQA), 5-feruloyl quinic acid (SFQA), caffeic acid and any combination thereof.
In yet another embodiment of the present invention the fraction enriched in polyphenols may comprise 3-caffeoyl quinic acid (3CQA), 4-caffeoyl quinic acid (4CQA), 5-caffeoyl quinic acid (5CQA), 3,4-dicaffeoyl-quinic acid (3,4diCQA), 3,5-dicaffeoyl-quinic acid 3,5diCQA), 4,5-dicaffeoyl-quinic acid (4,5diCQA), 3-feruloyl quinic acid (3FQA), 4-feruloyl quinic acid (4FQA), 5-feruloyl quinic acid (SFQA) and caffeic acid.
In yet an embodiment of the present invention the enriched fraction has a content of each of 3-caffeoyl quinic acid (3CQA), 4-caffeoyl quinic acid (4CQA), 5-caffeoyl quinic acid (5CQA), 3,4-dicaffeoyl-quinic acid (3,4diCQA), 3,5-dicaffeoyl-quinic acid (3,5diCQA), 15 4,5-dicaffeoyl-quinic acid (4,5diCQA), 3-feruloyl quinic acid (3FQA), 4-feruloyl quinic acid (4FQA) and 5-feruloyl quinic acid (SFQA) that deviates at most by 30% (w/w) relative to the content present in the starting material, such as at most 25% (w/w); e.g. at most 20% (w/w), such as at most 15% (w/w); e.g. at most 10% (w/w).
In another embodiment of the present invention the chlorogenic acid (CGA) may comprise at least 50% (w/w) mono-caffeoyl quinic acid and/or at most 25% (w/w) di-caffeoyl quinic acid. In another similar embodiment the fraction enriched in polyphenols comprises at least 12% (w/w) on a dry matter basis of at least one of each of 3-caffeoyl quinic acid (3CQA), 4-caffeoyl quinic acid (4CQA) or 5-caffeoyl quinic acid (5CQA), such as at least two of said compounds, preferably all three of said compounds.
In yet an embodiment of the present invention the fraction enriched in polyphenols may comprise less than 12% (w/w) on a dry matter basis of at least one of each of 3,4-dicaffeoyl-quinic acid (3,4diCQA), 3,5-dicaffeoyl-quinic acid (3,5diCQA) or 4,5-dicaffeoyl-quinic acid (4,5diCQA) such as at least two of said compounds, preferably all three of said compounds.
In yet an even further embodiment of the present invention the fraction enriched in polyphenols may comprise less than 12% (w/w) on a dry matter basis of at least one of each of 3-feruloyl quinic acid (3FQA), 4-feruloyl quinic acid (4FQA) or 5-feruloyl quinic acid (5FQA), preferably all three.
The fraction enriched in polyphenols may be used directly as a food ingredient or form part of a food ingredient. Thus, a further aspect of the present invention relates to a food ingredient comprising the fraction enriched in polyphenols according to the present invention. In an embodiment of the present invention the food ingredient may comprise between 0.1-20% (w/w) ethylesters of polyphenols relative to the total amount of the polyphenols in the food ingredient, such as in the range 0.1-10%, such as in the range 0.1-5%, such as in the range 0.1-4%, such as in the range 0.1-3%, such as in the range 0.1-2%, or such as in the range 0.1-1%.
The fraction enriched in polyphenols or the food ingredient may be used in a food product. Thus, yet an aspect of the present invention relates to a food product comprising the fraction enriched in polyphenols and/or the food ingredient according to the present invention. In an embodiment the food product comprises between 0.1-20% (w/w) ethylesters of polyphenols relative to the total amount of the polyphenols in the food product, such as in the range 0.1-10%, such as in the range 0.1-5%, such as in the range 0.1-4%, such as in the range 0.1-3%, such as in the range 0.1-2%, or such as in the range 0.1-1%.
Polyphenols may be used for different purposes to improve a food product. Thus, an aspect of the present invention relates to the use of the fraction enriched in polyphenols according to the present invention and/or the food ingredient according to the present invention as a precursor for generating aroma, flavor and/or foaming.
In a further aspect the invention relates to a pharmaceutical composition comprising the fraction enriched in polyphenols according to the present invention and a pharmaceutical acceptable carrier. Since polyphenols may exhibit biological activity in vivo e.g. antioxidants, activator/inhibitor of enzyme and receptors they can be considered relevant components in pharmaceutical compositions.
It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.
The invention will now be described in further details in the following non-limiting examples.
Methods
Fractions
Green coffee fractions were manufactured from either plain or decaffeinated green Robusta beans.
Resin
The Amberlite FPX66 (CAS N° 9003-69-4) is a commercial food grade resin which chemical structure consists of a macro reticular aromatic polymer, namely styrene. As the styrene rings are not functionalised, the matrix is rather hydrophobic.
Purification Setup for Labscale Experiments
Purification Set-Up for Bench Scale
Chlorogenic Acids Analysis by HPLC-UV
Chlorogenic acid (CGA) analysis was performed by HPLC-UV with the following set up:
Concentrations, expressed in gram per 100 gram, of the other CQA are determined from that of the 5-CQA thanks to this formula:
LC-MS Analysis for Desorption Study and Fraction Composition
The coffee fractions were characterized by LC-UV-MS/MS as follows:
Green coffee extracts (i.e. caffeinated/decaffeinated) were prepared at 50% TC (dry matter content) in boiling water without any problems.
As untreated extract can lead to clogging of the column, the high molecular weight (HMW) compounds (i.e. proteins, melanoidins, arabinogalactans) of green coffee extract were eliminated by precipitating them in 80% ethanol (V/V). The precipitate was removed by filtration on a Buchner filter (porosity grade 1). Ethanol was further evaporated. Afterwards the concentration of CGAs was evaluated by HPLC-UV before and after treatment. The main results were:
In conclusion alcohols, such as ethanol, are particular useful in the process according to the invention.
The adsorption was first assessed with a coffee solution as obtained from example 1 as such. This revealed a poor adsorption rate. Next the coffee solution, as obtained from example 1, was acidified to pH 1 to reinforce the strength of the hydrophobic interactions between the CGA species and the aromatic polymer of the resin. Analysis of CGAs by HPLC-UV revealed that:
In conclusion an acidic environment improves the adsorption step of the present disclosed protocol.
To eliminate the compounds that were not retained but still present in the dead volume of the column, a rinse with water and 0.1M HCl was carried out. Wash water was checked for CGAs afterwards. As the main findings it was found that:
In conclusion a washing step increases the purity of the final product.
For CGAs desorption, the hydrophobic interactions between the resin and phenolic compounds had to be weakened. With a view to a food grade purified fraction, ethanol was chosen as organic solvent. KOH (10−4 M) was added in order to increase the pH of the desorption solution. This ethanol/KOH mixture (90/10 V/V) was used as desorption solvent and compared to pure ethanol or ethanol/water mixture. The findings were:
To ensure that the composition of CGA in the purified fraction remains the same as in the initial coffee it is crucial to know the desorption kinetics of the different isomers and for caffeinated samples that of caffeine as well. Therefore a series of fractions was collected upon desorption. As displayed in
To optimise the coffee/resin ratio, fraction collection at the output of the column during the adsorption phase was performed. Each fraction was analyzed by UV at 325 nm (characteristic wavelength of CGA). The optimal ratio corresponds to the volume of coffee solution at which CGA begin to elute again out of the column. From this volume of solution, the corresponding mass of coffee solids that pass through the column was calculated. The optimal coffee/resin ratio was determined to be approximately 0.55 g of coffee for 1 g of resin. However, as the content of chlorogenic acid may vary considerably from one type of coffee to another and within the same sort of coffee this ratio may change.
After having completed the purification protocol according to
The detailed composition of the enriched polyphenol fraction was further evaluated. Analysis of the CGA profile revealed the following results as shown in
In total the isomer balance can be considered as close to that of the initial extract.
After successive optimization of all purification steps a CGA fraction with a purity of ˜70% and preserved CGA balance could be obtained. The enrichment factor was globally around 2. The key steps of the process are:
From the findings of the labscale study in example 6, the optimized protocol as shown in
The results are:
The process conditions favoring the undesirable formation of ethylester from CGA were investigated. The kinetics of the degradation of CGA and respectively the formation of CGA esters were followed in model studies. During the process ethylesters of CGA may be formed. This is however, undesirable because it may significantly influence the CGA balance and the properties of the enriched fraction.
The scheme below displays the general reaction mechanism of an esterifaction resulting in the ethylester. The reaction between an acid and an alcohol is catalyzed by proton transfer making the reaction rate pH-dependent. As a second key parameter the temperature should influence the degradation rate as it is usually the case for chemical reactions. Both parameters were evaluated in order to determine the ideal conditions to avoid an ethylation of CGAs.
During the experiment 5-CQA was stored in the presence of ethanol (75% ethanolic solution) by varying the following parameters pH, temperature and time.
The process was conducted under the following conditions using 5-CQA esterification as the model.
For each trial 10 mg of 5-CQA were dissolved in 40 mL of a 75% ethanolic solution.
pH
The degradation of 5-CQA and the formation of the corresponding ethyl-ester were monitored by LC/MS-DAD.
In conclusion, in presence of ethanol the pH of the enriched fraction should be around 4, such as 4-5. Thus, a pH around 4-5 may be optimal for step (v) in the process according to the present invention as displayed in
Temperature
In a next step, the influence of the temperature was evaluated. To simulate the worst case scenario, pH 1 was chosen. Indeed, it corresponded to the most critical pH in the previous experiment and it currently reflects the conditions of the purification protocol. After 2 h the degradation of 5-CQA kept at three temperatures, namely ambient (RT), 50° C. and 80° C., was monitored by LC/MS-DAD (
In conclusion, in presence of ethanol, the temperature of the coffee extract and the enriched fraction should be lowered, in particular under acidic conditions.
Time
While pH and temperature are independent from the size of the purification setup, the residence time is clearly increased with up-scaling. Therefore the next study evaluated the esterification kinetics of 5-CQA into its ethylester at given pH and temperature. This study was performed at pH 1 for both room temperature and 50° C. (
In conclusion, the formation of ethylesters depends on pH and temperature of the desorption step.
From the findings of the labscale study in example 6, the optimized protocol (
5 batches were produced in order to assess the repeatability of the purification process.
A complete characterization of the decaffeinated fraction was performed and the results are displayed in
The detailed composition of CGA is displayed in
As already seen under lab scale conditions the CGA balance is maintained quite well (Example 6).
Moreover, the updated protocol (
In addition, the analysis of CGA and of their esters in the final product with HPLC-UV/MS revealed:
Thus, it is indeed possible to perform the process according to the present invention for decaffeinated compositions at a scale which is industrially applicable, without drastically affecting the polyphenols balance or generating large amounts of ethylesters.
For the caffeinated polyphenol enriched fraction, the updated protocol (
A complete characterization of the caffeinated fraction was performed and the results are available in
The detailed composition of CGA in the enriched caffeinated fraction is displayed in
For the caffeinated enriched fraction ten batches were analysed separately to evaluate the batch-to-batch performance.
An HPLC-UV/MS was also performed to control the content on CGA and their esters in the enriched caffeinated fraction. Data not shown.
Thus, it is indeed possible to perform the process according to the present invention for caffeinated compositions at a scale with is industrially applicable, without drastically affecting the polyphenols balance or generating large amounts of ethylesters.
Conclusions:
A foodgrade protocol to purify coffee polyphenols has been described in the previous examples, with adsorption of coffee polyphenols onto a hydrophobic resin. Desorption was performed under isocratic conditions using 90% aqueous ethanol with a polyphenol purity of 60-70% in the final enriched fraction.
With a view to produce more specific polyphenol products, the preparation of purified coffee polyphenol isomers was investigated. For this purpose, successive desorption steps with solvent of decreasing polarity (i.e. aqueous ethanol 20/80, 50/50, 80/20) were applied.
Desorption was performed in three successive steps with increasing ethanol ratio in the desorption mix. The protocol is displayed in
Step 1—desorption with ethanol/water/KOH 20/70/10 (v/v/v);
Step 2—desorption with ethanol/water/KOH 50/40/10 (v/v/v);
Step 3—desorption with ethanol/water/KOH 80/10/10 (v/v/v).
By applying a gradient for the desorption from the food grade resin three different enriched fractions were obtained. The chlorogenic acid composition of these enriched fractions was then assessed by HPLC-UV.
The main findings are:
This gradient application may be helpful in situations where it is desired to improve desorption of the various forms of chlorogenic acid compounds and enriched fractions may afterwards be combined to provide an isomeric balance of the CGA that resemples the isomeric balance of CGA originally in the coffee. The gradient desorption may also be useful in the case where a more specific composition is needed.
| Number | Date | Country | Kind |
|---|---|---|---|
| 14188365.2 | Oct 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/072978 | 10/6/2015 | WO | 00 |
