Patent Application
20210070892
The present invention relates to gum arabic, methods of improving the properties of gum arabic and uses of gum arabic.
Acacia gum (gum arabic) is the oldest and best known of all the tree gum exudates. It is obtained from the stems and branches of Acacia trees which grow widely across Sub-Saharan Africa, from Mauritania, Senegal and Mali in the west, through Burkina Faso, Niger, northern parts of Nigeria and Chad to Sudan, Eritrea, Ethiopia and Somalia in the east, and northern parts of Uganda and Kenya. They form the so called “gum belt”. Gum arabic was introduced to Europe through various Arabian ports and came to be called “Gum Arabic” after its place of origin or port of export.
Acacia which is one of the most popular vegetations of the plant kingdom and is a cosmopolitan genus containing in excess of 1,350 species (Maslin et al., 2003). Acacia senegal and Acacia seyal remain the only commercially exploited species of the whole Acacia resource.
Acacia senegal belongs to the Vulgares series while A. seyal to the Gummiferae series. A. senegal is generally regarded as occurring as four varieties. These are: A. senegal (L.) Willd var. senegal (syn. A. verek Guill. & Perry); A. senegal (L.) Willd var. kerensis Schweinf; A. senegal (L.) Willd var. rostata Brenan; and A. senegal (L.) Willd var. leiorhachis Brenan (syn. A. circummarginata Chiov.).
Acacia seyal occurs as two varieties: A. seyal Del. var. seyal; and A. seyal Del. var. fistula (Schweinf) Oliv (Coppen, 1995). According to the Joint Expert Committee for Food Additives (JECFA) of FAO gum arabic is defined as a dried exudate obtained from the stems and branches of Acacia senegal (L.) Willdenow or Acacia seyal (fam. Leguminosae) (JECFA-FAO, 1998).
The systematic wounding of Acacia tree to induce gum exudation (Gummosis) is called tapping and is considered the standard practice applied for A. senegal. On the other hand, A. seyal is generally collected from natural exudation without tapping but there is some evidence that it is applied in certain location in Sudan. In certain gum sources the natural exudates were noted as being darker in colour compared to gums obtained by tapping (Anderson & Bridgeman, 1985).
Recently, Andres-Brull et al reported that tannin is contaminant from the Acacia bark and found to correlate with the exudates form (i.e. seep or noldules) (Andres-Brull et al., 2015). The highest concentration of tannin is associated with the exudate which has more contact with the bark during the process of gummosis. The gum exudates from seyal do not harden quickly and hence has acquired the name “friable”.
Gum arabic is made up of complex branched polysaccharides as well as some proteinaceous material which is integral to its structure. Upon hydrolysis of gum arabic: galactopyranose, arabinopyranose, arabinofuranose, rhamnopyranose, glucopyranosyl uronic acid and 4-O-methyl glucuropyranosyl uronic acid have been identified (Anderson & Stoddart, 1966; Anderson et al., 1967).
A. seyal consists of the same sugar residue as A. senegal, but has lower rhamnose and glucuronic acid contents and higher arabinose and 4-O-methyl glucuronic acid contents (Jurasek et al., 1995). A. seyal has lower nitrogen contents than A. senegal (Jurasek et al., 1993), and the specific optical rotation of the two species is different (Biswas & Phillips, 2003). A. seyal is proposed to be more highly branched and more compact in structure than A. senegal (Flindt et al., 2005; Hassan et al., 2005; Street & Anderson, 1983).
Street and Anderson (Street & Anderson, 1983) in a reinterpretation of the work of Churms et al. (Churms et al., 1983) suggested different structures of the polysaccharide for A. senegal and A. seyal gum. A. senegal is suggested to contain entirely of Type 1 repeat units, whereas A. seyal consists of small blocks of two or three modified Type 1 repeat units separated by significant blocks of Type 2 repeat units (Flindt et al., 2005). Recently (Nie, Wang, Cui, Wang, Xie & Phillips, 2013) elucidated the fine structure of Acacia seyal var. seyal and reported the presence of 13.6% galacturonic acid, not previously identified.
Studies have revealed the presence of three major components in gum arabic (A. senegal): a high molecular weight arabinogalactan-protein (AGP) gum, an arabinogalactan (AG) and a glycoprotein (GP), accounting for in the region of 10%, in the region of 90% and in the region of 1% of the total gum, respectively (Randall et al., Food Hydrocolloids, 1988, 2, 131; Randall et al., Food Hydrocolloids, 1989, 3, 65).
The different components are responsible for different functionalities of gum arabic. For example, the arabinogalactan-protein (AGP) component, in Acacia senegal, has been identified as an active emulsifier component, providing interfacial activity and stability during emulsification (Williams & Phillips, Handbook of Hydrocolloids, 2000, 155).
Underwood and Cheetham (Underwood & Cheetham, 1994) reported the fractionation of A. seyal using the same condition applied for Acacia senegal (Underwood et al., Journal of the Science of Food and Agriculture, 1994, 66, 217). As for the fraction of A. seyal that corresponded to AGP of A. senegal, there was only 2.4% of the total, and the protein content was also lower than that of A. senegal. Molecular weight fractions with differing emulsification properties were identified for gum talha (A. seyal) (Underwood & Cheetham, 1994). The three main components designated arabinogalactan protein (AGP), arabinogalactan (AG) and glycoprotein (GP) known to be present in Acacia senegal are also present in Acacia seyal. Subsequently, Siddig et al. reported that the protein in A. seyal is not mainly located in the high molecular weight component (AGP) as for A. senegal and indicated that at least two components are present in the high molecular weight fraction in A. seyal (Siddig et al., 2005).
Flindt et al. have reported about the fractions which were adsorbed on to oil droplets of A. seyal emulsions and showed that they were less efficient than Acacia senegal (Flindt, Al-Assaf, Phillips & Williams, 2005). For A. senegal it was mainly the proteinaceous material of the high molecular weight peak (AGP) which is adsorbed on to oil droplets. On the other hand, A. seyal showed a different behaviour. The high molecular weight peak of A. seyal is hardly adsorbed, and the proteinaceous material belonging to the second peak is mainly adsorbed. It is thought that the difference of this adsorbed component is related to the difference of the emulsion ability of A. senegal and A. seyal.
Senegal type gives high levels of functionality in formation and stability of emulsions and micro-encapsulation of flavours, and plays a very important role in the food and beverage industries. According to Coppen (1995) “Gum talha from Sudan (the local name for A. seyal) is intrinsically a poorer quality gum than hashab (the local name for A. senegal)—it has inferior emulsifying properties and even light-coloured samples of whole gum sometimes forms dark solutions in water due to the presence of tannins and other impurities. It is more friable than hashab”. Acacia seyal does not offer good emulsification performance, and is consumed directly as a confectionary in India and used in coatings, adhesives etc in other markets. The general consensus is that the emulsification ability of A. seyal is inferior compared with that of A. senegal (Fauconnier et al., 2000).
Acacia seyal showed considerably less surface activity compared to Acacia senegal both at air/liquid and liquid/liquid interface (Elmanan, Al-Assaf, Phillips & Williams, 2008). Acacia seyal was found to be more resistant to enzymatic degradation, since only 40% of the high molecular weight fraction can be digested again indicating the presence of two components. However, there is a conflicting report which showed that A. seyal sample that have the less protein content gave better emulsion stability than some A. senegal samples (Buffo et al., 2001). It should be noted, that although A. senegal is considered to be good emulsifier for oil in water emulsion but performance from various samples collected from different areas show considerable variation. Some of which can be considered as poor as the widely acknowledged emulsification performance of A. seyal (Al-Assaf et al., 2008).
It has been shown that A. seyal gums have larger variation especially commercial samples from different locations (Jurasek et al., 1995, A-Assaf et al., 2005).
The industry problems associated with the use of gum Arabic in general is that it has inconsistent performance and functionality. The inconsistency is partially due at least to the variation in the proportion of the three major components. Additionally, due to its variable sourcing from countries across the Sahelian belt of Africa, with different rainfall, soils and overall geography, the product has considerable inconsistency due to the natural variability. As a result, the raw material as delivered from primary producers often does not behave consistently in the various applications, (Williams, P. A. and Phillips, G. O., (2000) in Handbook of Hydrocolloids, Editors Williams, P. A. and Phillips, G. O pp 155-168, Woodhead, London and New York). Various approaches, therefore, have been used to try and eliminate the inconsistency as listed below.
There have been several modification procedures to improve the emulsification performance of gum Arabic in general and specifically Acacia seyal in some examples as given below.
In U.S. Pat. No. 6,841,644 (Phillips et al.) and US/GB 7,462,710 B2 (Al-Assaf) there are disclosed modification methods by radiation and heat treatment respectively to obtain gum arabic with one or more improved functionalities whereby the molecular weight and AGP content of the modified gum arabic were increased. In addition, it is known that excessive modification brings about negative effects including reduced solubility in water and degradation of the gum arabic.
EP-A-1 505 078 is directed to a process for modifying gum arabic by means of heating the gum arabic at a temperature higher than 40° C. in humid conditions, which enhances its emulsifying ability.
Similarly, EP-A-1 666 502 discloses a heat treatment of gum arabic, albeit under dry conditions, leading to an improvement of its emulsifying ability.
EP-A-1 734 056 relates to a process for the modification of gum arabic by dissolving gum arabic in water and then heat-treating the solution at a temperature below 60° C., thus improving its emulsification properties.
US-A-2005/124805 also discloses modified gum arabic (from Acacia senegal or Acacia seyal), which has improved emulsifying ability. The modified gum arabic is obtained by heating the gum arabic, in the solid state, at 110° C. for not less than 10 hours.
The most relevant patents, which report the modification of Acacia seyal, to this invention, are:
JP 2008-297359 (A) which describes the removal of tannin from gum arabic (Talha gum) solution originating from Acacia seyal, using a processing method that does not use hydrogen peroxide. The process relies on dissolving (Talha gum) originating from Acacia seyal in water from which dissolved oxygen has been removed. This aqueous solution is then processed with a synthetic adsorbent to absorb and remove tannin. In this way, tannin-free talha gum usable in food applications is obtained.
WO 02/069981 A1 describes a chemical process based on the reaction product of a hydrocolloid (such as Acacia seyal) with dicarboxlyic anhydrides specifically for the production of an emulsifier for oil-in-water emulsions.
WO 2013/091799 A1 describes a method for preparing modified gum arabic comprising treating gum arabic with an enzyme selected from the group of glycosidases at a concentration of 1 to 1000 units of enzyme per gram of gum arabic, a modified gum arabic obtainable by said method, an emulsion comprising the modified gum arabic and a beverage concentrate and ready-to-drink beverage comprising the emulsion.
In all above-mentioned patents, a physical or chemical treatment of gum arabic is used for achieving remarkably higher emulsifying abilities, expressed in the form of smaller oil droplet diameters in gum arabic stabilized oil-in-water emulsions.
It is one object of the invention to provide methods to prepare preparations of arabic gum from Acacia seyal preferably preparations having improved properties.
This object is solved in one embodiment by a method for preparing an improved gum arabic comprising the steps of
It was realized that the properties of gum arabic from Acacia seyal depend on the tannin content. The measurement of tannin is performed according to the method described in the examples.
Any gum arabic from Acacia seyal may be used, preferably one may use gum arabic of Acacia seyal var., Acacia seyal var. fistula and mixtures thereof.
Preferably, the tannin content is >750 ppm (w/w) or >1000 ppm (w/w) or >2000 ppm (w/w).
A further embodiment of the invention is composition of gum arabic from Acacia seyal having a tannin content >700 ppm (w/w). Preferably, the tannin content is >750 ppm (w/w) or >1000 ppm (w/w) or >2000 ppm (w/w).
The tannin content is measured according to the examples. As an alternative, selection may be made for gum arabic from Acacia seyal colour Gardner at 1% of 2.5 or greater, measured according to the examples.
Therefore, an embodiment of the invention is the method, wherein the gum has
i) a colour Gardner index of at least 2.5, more preferably 2.5-3.0 and even more preferably >3 at 1 wt % in water or
ii) a colour Gardner index of at least 15, more preferably 15-16 and even more preferably >16 at 20 wt % in water.
A further embodiment of the invention is a method for preparing an improved gum arabic comprising the steps of
It was further found, that gum arabic may be improved by increasing the content of phenols. Therefore, a further embodiment of the invention is a process for improving gum arabic comprising the steps of
This process is applicable to gum arabic of Acacia seyal, gum arabic of Acacia senegal, or a mixture thereof. Any Acacia senegal variety may be used, preferably A. senegal (L.) Willd var. senegal (syn. A. verek Guill. & Perry); A. senegal (L.) Willd var. kerensis Schweinf; A. senegal (L.) Willd var. rostata Brenan; and A. senegal (L.) Willd var. leiorhachis Brenan (syn. A. circummarginata Chiov.). The process is also applicable to other gum varieties such as Acacia polyyacantha var campylacantha, Acacia sieberana var. sieberana, Acacia Nilotica, Acacia mellifere and Acacia laeta.
In a simple process, bark may be prepared in powder form, combined with water and incubated. Incubation may be at elevated temperatures, e.g. between 20 and 80° C. It is possible to add the gum arabic after incubation or combine it with the water phase during incubation.
As a phenol source bark, polyphenols and gallic acid are especially preferred.
The bark should preferably contain polyphenols. Bark from Acacia seyal is a preferred bark. Other preferred phenol sources are those obtained from other Acacia gums varieties such as Acacia nilotica, Acacia tortilis as well as polyphenol extracts from olive and olive leaves.
In general, an addition of small amounts of a phenol source is sufficient. A preferred ratio (w/w) of gum arabic:phenol source is 100:1 to 100:5.
The product of the method is a further embodiment of the invention, i.e. a composition comprising
This process is applicable to gum arabic of Acacia seyal, gum arabic of Acacia senegal, or a mixture thereof.
As a phenol source bark, polyphenols and gallic acid are especially preferred.
The bark should preferably contain polyphenols. Bark from Acacia seyal is a preferred bark.
It has further been found, that the properties of gum arabic of Acacia seyal may be improved by adding gum arabic of Acacia senegal.
Therefore, an embodiment of the invention is a method for improving gum arabic of Acacia senegal comprising the step of:
A further embodiment is a composition comprising
The ratio of the amount of gum arabic of Acacia seyal to the amount of gum arabic of Acacia senegal may 5:95 to 95:5 (w/w). A preferred ratio is 20:80 to 80:20 (w/w) or 40:60 to 60:40 (w/w). A very preferred ratio is 50:50 (w/w).
Any of the compositions of the present invention may be used especially for making emulsion.
Therefore a further embodiment of the invention is an emulsion comprising water, a hydrophobic compound and a composition of the invention. Preferably, the emulsion is an O/W or W/O/W emulsion which contains at least one hydrophobic substance.
Suitable hydrophobic compounds are vegetable oils, middle chain triglyceride (MCT) oils, and mixtures thereof. Further examples of hydrophobic substances include essential oil obtained from pant sources such as orange, lemon, lime, grapefruit; oleoresin obtained from plant sources such as pepper, cinnamon and ginger by the oleoresin process; oil based flavouring such as oil-based synthetic flavouring compounds and oil-based flavouring compositions, oil based colourants such as b-carotene, oil-soluble vitamins such as vitamins A, D, E and K; polyunsaturated fatty acids such as docosahexaenoic acid, eicosapentaenoic acid and linolenic acid; animal and vegetable fats and oils such as soybean oil, rapeseed oil, corn oil, plant sterol and fish oil; SAIB (sucrose acetate isobutyrate), ester gum (glycerol triabietate ester); processed food oils such as C6-C12 medium chain triglycerides and mixtures of any such edible oil materials.
The emulsions may be part of a food or a beverage, for examples soft drinks. Therefore, a further embodiment of the invention is a beverage or food comprising the emulsion of the invention. It can be also used in many fields including confectionary, health food, coating for tablets, gum jelly, emulsified flavours and paints.
The present invention will now be described in further details with reference to the following examples and Figures. These examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.
Samples and Test Methods
Authenticated samples of Acacia seyal (gum talha) obtained as a mixture of small nodules and broken gum pieces with varying colour were obtained from various locations in Sudan, from two seasons. In total 23 samples were used. Acacia senegal samples, in the lump gum form with various sizes, were also obtained from Sudan from various suppliers. Additionally, spray dried Acacia senegal were also used and were obtained from Norevo (Germany). Spray dried Acacia seyal subjected to bleaching by hydrogen peroxide was obtained from Dansa Food (Nigeria). The spray dried gums were used as supplied. The crude gum samples were kibbled using pestle and mortar in order to prepare a homogenous sample (in powder form) which was then used for all measurements listed below:
The practical problem is that Acacia seyal from various producing countries is supplied with different colour and thus properties. The main reason for this variation is believed due to (i) the varieties of this species, i.e. Acacia seyal var seyal and Acacia seyal var. Fistula. In a recent paper (Andres Brull 2015) it was shown how these varieties can be different even when they come from the same area and season. Variety fistula has a powdery bark, normally white or greenish-yellow, whereas var. seyal has a reddish bark. In the above paper it was stated “Why seyal obtained from various production areas shows such variation in terms of colour and shape is still not well understood. Currently, all A. seyal which originates from Sudan is sold as “seyal” (locally known as talha gum) irrespective of its taxonomic variety.” Additionally, the other reason why Acacia seyal is different and has not been possible to use in the industry is that different production areas within the same producing country use different methods for pre and post harvest treatments. One area tap Acacia tree as it is the normal procedure used for Acacia senegal whereas other areas simply collect the natural exudate without tapping. For this reason nodules with various colour are often the result since tannin is actually a containment which comes from the bark of Acacia seyal tree. It has already been known that when the tree is tapped the gum is lighter in colour since tapping essentially means manually wounding tree by removing the bark to induce gummosis.
Loss on Drying:
The % loss on drying was determined according to the JECFA method which is a measure of the moisture loss when the sample is heated for 5 hrs at 105° C. (Al-Assaf, S., et al. 2005);
see www.cybercolloids.net/library/jecfa/gum-arabic.
Optical Rotation:
The optical rotation was measured on 1 wt % solution (based on dry weight) prepared in distilled water and hydrated over night by tumble mixing. The solution was then filtered through 100 micron mesh.
Colour Gardner: The same solution was also used to determine the colour Gardner index as follows: A calibrated Lovibond Tintometer PFXi—195/1 colorimeter was used to determine colour Gardner of the Acacia seyal samples. Measurements were carried out using 10 mm path length cell on 1 wt % solutions prepared as described above. The Gardner colour scale is from 1 to 18 with 1 containing the least amount of colour and 18 with the maximum amount of colour. The Gardner is a one dimensional scale used to grade liquids such as varnishes, resins and oils.
Total Phenols:
Total phenols were determined using the modified Prussian Blue Assay as described in details in Andres-Brull 2015 and given below.
Tannin content is taken here to represents the “total phenols” and more accurately the “gallic acid equivalents” as gallic acid—99% in purity purchased from Sigma Aldrich—was used as the analytical standard for determining the hydrolysable tannins. 1 wt % solution was made according to the method described above was used to determine the tannin content in the respective samples.
500 μg/g gallic acid was prepared in distilled water. This was then serial diluted to obtain concentrations of 400, 300, 200, 100 and 50 μg/g used as standards. 0.10 mL of each sample or the standard was dispensed in a 30 ml universal. 3 mL of distilled water was added following by vortex mixing. Next, 1.00 mL of 0.016 M K3Fe(CN)6 followed by 1.00 mL of 0.02M FeCl3 were added and immediately mixed by vortex mixer. Exactly 15 minutes after adding the reagent to the sample 5.00 mL of stabiliser was added and vortex mixed. The stabiliser was prepared by mixing 10 mL of 85% H3PO4, 10 mL of 1 wt % gum arabic and 30 ml of distilled water. The gum arabic sample used was standard Acacia senegal var. senegal in the kibbled form obtained from Sudan. Solvent only blanks were also prepared by adding all reagents and 0.1 mL of solvent instead of seyal or gallic acid standards. The Absorbance was read at 700 nm in duplicate for all using Perkin Elmer Lambda 40 UV/Vis spectrophotometer. The error in measuring the tannin content was below 10% for all samples and the average was taken.
Molecular Weight:
The molecular weight parameters of gum arabic were determined by gel permeation chromatography coupled on line to laser light scattering, refractive index and UV detectors (GPC-MALLS). The use of a GPC-MALLS system first fractionates the material using gel permeation chromatography column (Superose 6 10/300GL), and then subsequently detects each fraction using: a protein detector (UV absorbance using Agilent 1100 series UV detector, Agilent Technologies, U.K.) operated at 214 nm or 280 nm), a multiangle laser light scattering detector (measured using DAWN EOS multiangle light scattering detector, Wyatt Technology Corporation, U.K., operated at 690 nm), and a concentration detector (refractive index, RI measured using Optilab refractometer, Wyatt Technology Corporation, U.K.). Aqueous NaCl solution (0.2 M) with 0.005% NaN3 filtered through 0.2 μm Millipore filter was adopted as an eluent and was delivered at a constant rate of 0.4 mL/min by a KNAUER HPLC pump K-501 (Kinesis, U.K.). The test material was prepared in the same solvent at a concentration of 2 mg/mL. It was injected into the GPC-MALLS system after being filtered through a 0.45 μm Nylon filter. Data was collected and analyzed by Astra 4.90.08 software. The system allows the molecular weight distribution of gum arabic to be measured and thus the molecular weight of whole gum as well as individual fractions can be determined together with its proportion compared to the total injected mass.
Emulsification:
The emulsification performance of the various Acacia seyal samples as well as Acacia senegal and mixes thereof were evaluated in a typical oil-in-water emulsion as described below. The method utilised medium chain triglycerides as the oil phase without using a weighting agent. Typical formulations were prepared as follows: 40 μm emulsions were made to contain 0.12 wt % citric acid (to adjust the pH), 0.13 wt % benzoic acid (as a preservative), Medium-chain triglyceride (MCT) oil was used as model oil at 20 wt % and gum arabic at 20 wt %. Gum Arabic mixes (seyal and senegal, kerensis, polyacantha etc) were made by mixing the solid gum which was used to make the stock solution at 30 wt % and from which dilution to 20 wt % was achieved using distilled water. Ingredients were initially mixed using a high shear homogeniser (Polytron PT-2100) at 26000 rpm for 3 minutes followed by two passes through a high-pressure homogeniser at 50 MPa (Nanovater, NV L, Yoshida, Japan). The emulsion droplet size and size distribution were measured using laser diffraction methods (Mastersizer, Malvern Instruments, UK) by fitting the data using the general purpose model. Values of 1.45 and 0.001 were used for MCT refractive index and absorption index respectively, and 1.33 and 0 for the dispersant (water) respectively. The emulsions were subjected to an accelerated stress testing by incubation at 60° C. The performance and stability of the emulsion were evaluated by measuring the initial droplet size immediately after preparing the emulsions and after storing them at ambient temperature and also at 60° C. (accelerated stress test) for 3 and 7 days. The results were expressed as volume-moment mean diameter (D4,3) since it is more sensitive to large particles. Additionally, the % of droplets greater than 1 μm or 2 μm or more were also reported since larger proportions of these droplets are mainly responsible for the development of ring formation which ultimately leads to emulsion failure.
Acacia seyal Sample (No. 5) was chosen to illustrate the example of making an oil in water emulsion with excellent emulsification and stability performance. The results are comparable to Acacia senegal and in some cases even better. The sample characteristics measured as outline above as follows:
% loss on drying 11.25%,
tannin (gallic acid equivalent) content 942 ppm,
Colour Gardner at 1% 3.3,
Colour Gardner at 20% 16.2,
pH 4.74 (measured at 20 wt % in water) and
Optical rotation of +50.
The molecular weight parameters measured by GPC-MALLS, the characteristics of which have been reported previously (Elmanan et al., 2008; Hassan et al., 2005) and summarized below.
The elution profile monitored by light scattering, refractive index and UV detectors is given respectively in
The refractive index detector (measure of concentration) also shows two peaks (
The sample was used to prepare the emulsion as described above.
The AGP component of gum arabic (Acacia senegal) is responsible for the emulsification effectiveness of oil in water emulsions (Randall et al., Food Hydrocolloids, 1988, 2, 131; Randall et al., Food Hydrocolloids, 1989, 3, 65); (Dickinson, E. (2003)). The AGP is believed to coat the oil droplets and prevents them from re-associating. As a result, such an emulsion is stable over a long time scale. Higher AGP content usually leads to even more stable emulsions and therefore makes a gum more valuable. High AGP content is also considered to be associated with other functionalities such as water binding and flavour binding. Increasing the amount of the AGP component in a gum alone provides added value to gum arabic product. Acacia seyal on the other hand, can form good emulsion initially but the stability of the emulsion has always been the problem. Since the makeup of the first peak is different in seyal the stability of the emulsion usually deteriorate following acceleration. Without being bound to a particular theory this increase in stability is considered as a direct contribution of tannin. Specifically, the better stability is considered as a consequence of tannin contribution to the molecular association present in the first peak. Tannin is known to bind to protein and has also been reported to precipitate protein.
Good and stable emulsion can be obtained as further demonstrated below on a range of samples of Acacia seyal.
The results given in
In this example, we have used three samples which gave poor emulsification performance and stability to demonstrate how tannin (more specifically hydrolysable tannin) can improve the molecular weight properties and emulsification.
These samples were No. 18, No. 19 and No. 20 with tannin content of 373, 287 and 227 respectively. The samples were selected based on showing the least amount tannin in the 20 samples shown in
To this solution 1 μm of ground bark (as given above) was added to increase the total tannin content to 700, 614 and 554 ppm for samples 18, 19 and 20 respectively.
(Note: This was calculated as follows: 1% of solid bark (i.e. dissolving 5 μm in 500 ml water) gives 131 ppm of tannin. A. seyal stock solution was prepared at 30 wt % in water to which 1 μm of solid bark was added to the total volume 40 μm. The concentration of bark is then 2.5%. Each 1% solid bark gives 131 ppm tannin as shown above. Therefore
for sample 18 (131×2.5)+273 ppm (original tannin present=700 ppm total tannin in gum after adding the bark.
for sample 19 (131×2.5)+273 ppm (original tannin present=614 ppm total tannin in gum after adding the bark.
Sample 20 (131×2.5)+227 ppm (original tannin present=554 ppm total tannin in gum after adding the bark.
The mixture was heated for 3 hrs at 60° C. then left to tumble mix over night. The solution was then filtered using 100 micron mesh as described above. Dilution to 2 mg/mL in 0.2M NaCl was made from the control and the sample with added tannin. The samples were then injected into the GPC-MALLS system and the molecular weight parameters are given in Table 1 below.
Alternatively, the same method was applied using another source of polyphenol: namely an extract from Olive leaf (N-O-02.02 Olexelo (solution), supplied by N-Zyme, Germany). The total polyphenol for this extract was determined according to the method described already and a value of 3042 ppm was obtained. 0.5 ml of Olexelo was added to 3 g of 17.6 wt % solution of Acacia seyal (Sample No. 23) and the solution was treated the same way by incubation at 60° C. for 3 hours and subsequently diluted 2 mg/mL in 0.2M NaCl for GPC-MALLS measurements. The results are also given in Table 1.
Upon the addition of tannin all samples showed an increase in the molecular weight and accompanied by an increase in the Rg value, albeit to a different extent. The results show that the increase in the molecular weight parameters is directly related to the formation of high molecular weight fraction. These changes are clearly shown in
Without being bound to theory we attribute this change to simply association of gum arabic molecules throughout the distribution by tannin. The addition of tannin results in molecular association whereas almost all molecules are affected. Consequently, there is a clear increase in the first peak area and a reduction in the second peak (AG+GP fractions). It is interesting to note that in the total volume area (19-24 mL) the response is almost identical for control and that of added tannin which confirms that the only the soluble tannin is released from the bark during dispersion in water.
The emulsification performance of the samples in the presence and absence of added tannin is compared is
In this example we have selected Acacia seyal sample which has been subjected to bleaching, using hydrogen peroxide, to further demonstrate the effect of adding bark as a source of soluble tannin to Acacia seyal. First the molecular weight parameters before and after the addition of bark were determined using GPC-MALLS techniques as described above and the results are shown in Table 2 below.
The results given in Table 2 show that the weight average molecular weight for the whole gum is almost doubled upon the addition of bark and accompanied by an increase in the Rg (root mean square radius of gyration) value from 25 to 54 nm. While the proportion of the first peak is almost the same it is most significant to note the molecular weight increases from 1.9×106 to 5.5×106 g/mol and also an increase in its Rg value from 32 to 58 nm. The parameters for the second peak (i.e. AG and GP fraction) remain largely unchanged with the exception of the slight increase in the molecular weight. These changes are clearly shown in the comparison of the elution profile monitored by refractive index, light scattering (detector 90°) and UV detectors for bleached seyal before and after the addition of bark as shown in
Similarly, the emulsification performance of bleached Acacia seyal before and after the addition of Acacia seyal bark was evaluated. Emulsion at 20% gum:20% MCT oil prepared according to the standard method described above. The emulsions were then evaluated by measuring the droplet size distribution for fresh emulsion and after acceleration for 3 and 7 days at 60° C. and shown in
In the previous examples we demonstrated how Acacia seyal bark addition to Acacia seyal (gum talha) as well as to bleached Acacia seyal can enhance the emulsification performance and stability. The addition of bark is mainly as a source of soluble tannin (hydrolysable tannin) which is source of polyphenolic compounds. In this example we have used Acacia senegal which is widely known not to contain tannin. Here, initially we repeated the same procedure applied on Acacia seyal samples whereby 1 μm of bark was added the solid material during the dissolution process. In the second part, we have used pure gallic acid (99%) obtained from Sigma as a source of a soluble phenolic compound. The purpose of using gallic acid is to demonstrate that under the conditions used, i.e. adding the bark to gum arabic, only the soluble tannin (polyphenol) is released into the aqueous solution which is responsible for the molecular association between gum arabic fractions. The gum sample selected in this example originated from Sudan and was supplied in the lump gum form (Acacia senegal, sample No. 1). The sample was kibbled using pestle and mortar and subsequently was made into a fine powder. 0.04 g of gallic acid was added to 13 g of gum arabic and made up to 40 g with distilled water. The final concentration of gum arabic (based on dry weight) was 30 wt % containing 1000 ppm gallic acid. The solution was left to dissolve overnight by tumble mixing on a roller mixer. The control sample was prepared in similar way without the addition of gallic acid. After dissolution an appropriate weight of the respective solution was made up to 10 g with GPC solvent (0.2M NaCl) for determination of molecular weight. Emulsions were prepared as per usual method at 20% gum arabic and 20% MCT containing 0.13% benzoic acid and 0.12 citric acid. Then high shear mixing using the polytron and subsequently two passes at 50 MPa using the high pressure homogemiser. The weight average molecular weight for the whole gum and Rg values are given in Table 3 below for control and the sample added bark as well as with the addition of gallic acid.
The results give average molecular weight for the control sample of 4.0×105 g/mol with Rg value of 28 nm. This value is considered slightly below average A senegal and indicates that is a fresh sample since the % loss on drying was 13.7%. Viscosity of 98 cps (measured using the Brookfield viscometer, 100 rpm, spindle 02) at 20% with pH value of 4.4 which are typical values of Acacia senegal in the crude form. The elution profile monitored by light scattering, refractive index and UV detectors also showed typical Acacia senegal whereby the presence of three fractions: namely AGP, AG and GP can be identified. The emulsification performance was investigated and the droplet size distribution is shown in
The improved emulsification is mainly due to increasing the molecular weight of the whole gum from 4×105 at least by 2.5 folds compared to the starting material. The most striking changes is that following the addition of gallic acid an aggregate peak was formed at the start of the elution volume. The elution profile of the sample with added bark is almost identical to that with added gallic acid (
The results given in
Further to the example above we have also used other Acacia senegal samples with poor emulsification performance. These samples were in the spray dried form and were obtained from various suppliers. The molecular weight parameters including the aggregate peak are tabulated in Table 4 below before and after the addition of bark. In both cases the molecular weight increases as a result of increasing the proportion of the high molecular weight fraction termed AGP. There is also an increase in the Rg value for both gum following the addition of bark. This increase again similar to what we found in A. seyal and is accompanied by a decrease in the molecular weight of the second peak (AG+GP fractions). The reduction again attributed to the association of molecules following the addition of bark. Again, the addition of bark is purely as a source of soluble tannin which is released into the solution thus providing polyphenolic compounds required for the association process.
acacia seyal bark as source of soluble tannin.
We have therefore also examined the effect of bark addition on the emulsification performance and stability. Both samples were chosen with poor emulsification properties as determined by the increase in D4,3 values and % greater than 1 micron droplets following acceleration at 60° C. for 3 and 7 days. Both samples give an initial smaller droplet of 0.48 micron with % greater than 1 micron of 3.9 and 6.4% respectively. Upon acceleration at 60° C. for 7 days the proportion of droplets greater than 1 micron increases to >40% (
An example is shown here to demonstrate that oil in water emulsion can be made by mixing Acacia senegal and Acacia seyal. Acacia senegal (Senegal No. 4) obtained from Sudan in the lump gum form was processed by kibbling and subsequently made into powder using pestle and mortar. The Acacia senegal sample was used to make mixes with three Acacia seyal samples (Samples No. 21, 22 and 23 also processed similarly into the powder form) at 50:50% in the dry state by mixing equal weight of each sample to give Mix 1, Mix 2 and Mix 3 respectively. The samples were then left to tumble mix to ensure a homogenous mixture. First the % loss on drying, optical rotation, pH and colour Gardner index were determined using the methods described above. Values for the % loss on drying for the mixes are almost an average of the sum of the two respective components. The optical rotation obtained for Acacia senegal (−30) and Acacia seyal samples are typical values and in agreement with those reported previously (Hassan et al, 2005).
Upon mixing senegal and seyal the optical rotation changes as a function of the sugar components present. Also the pH values for all samples and mixes are similar to those reported in previous studies and within the range of 4.1-4.8. The major difference between Senegal and seyal samples are the L* value (a measure of the solution transparency) and colour Gardner, measured at 1 and 20% solution in distilled water. The colour Gardner shows an increase with increasing concentration for both senegal, seyal and mixes samples. At higher concentrations the Gardner values enter the redder end of the scale (numbers 9-18). L* decreases with increasing concentration due to the increase in darkness of the solution. For Acacia senegal at 1 wt % a value of 96 (almost fully transparent) is obtained compared to 80 at 20 wt %. On the other hand, for Acacia seyal samples the value ˜40 at 20 wt % compared to ˜95 at 1 wt % solution. This difference between Senegal and seyal is mainly caused by the presence of tannin in Seyal. Mixing Senegal and seyal resulted in lighter colour and more transparent solutions for the three mixes. Repeated measurements on the same solution or fresh solution gave similar values with good reproducibility which indicates the mixture is fully compatible and no phase separation takes place.
Table 6 below gives the molecular weight parameters of Acacia senegal and seyal samples together with the mixes made thereof. The molecular weight of Acacia senegal for the whole gum is 5.5×105 and the proportion of the arabinogalactan protein fraction (AGP) at 10.85% with Mw of 2.1×106 g/mol. These values are typical of standard gum Arabic in the crude form as reported previously (A-Assaf et al, 2005).
The molecular weight parameters for Acacia seyal samples for the whole gum were 11.9, 10.6 and 11.6×105 g/mol for samples 21, 22 and 23 respectively as shown in Table 6 below. The molecular weight for the 1 t peak were 3.6, 3.0 and 3.2 with a proportion of 15.2, 11.9 and 15.0 and Rg values from 26-37 nm. The molecular weight for the second was 8×105 g/mol. These are typical Acacia seyal molecular weight parameters as reported previously (Hassan et al, 2005).
The molecular weight for mixes prepared at 50:50 weight percentage were also determined using the method applied for the control samples and the results shown in Table 6. Acacia seyal and Senegal mixes gave average molecular weight parameters in terms of the weight average molecular weight for the whole gum and for the second peak (typically associated with the arabinogalactan AG and glycoprotein GP fractions). There is a reduction in the proportion of the first peak area compared to both Senegal and seyal samples but comparable molecular weight to Acacia seyal samples.
The emulsification performance and stability were evaluated using the methods outlined above and the results shown in
In this example, a typical recipe for cloud emulsion made on a large scale and further to the previous examples given with MCT oil shown in
Acacia seyal (No. 23) is used together with Acacia senegal variety senegal (Sample No. 5, MW 6.57×105), 11.1% AGP content). The dissolution method to prepare 1000 Kg of 20 wt % of Acacia seyal or 15 wt % Acacia seyal with 5 wt % Acacia senegal was carried out as follows: Appropriate weight (see Table 7 below) of gum arabic was added to deionised water at 10-15° C. and stirred using IKA-RW20 digital with 900 rpm, for 8 hours. Subsequently citric acid (at 46.5%) was added to the solution while stirring for 5 min. The solution was then pasteurised at 80° C./10 min. The viscosity, measured by Brookfield viscometer was 43.4 mPa·s for Acacia seyal alone and 40.1 mPa·s for Acacia seyal at 15 wt % mixed with 5 wt % Acacia senegal.
Table 8 gives the details of preparing the emulsion described as follows. For preparing of the oil phase ester gum was dissolved in orange Oil using IKA-RW20 digital mixer operated at 400 rpm, for 3 hours. Water phase was prepared by mixing of gum solution with citric acid, potassium sorbate and cold deionized water by using IKA-RW20 digital mixer at 400 rpm, for 10 min. The pre-emulsion was prepared by adding the oil phase to the water phase using Utra Turrax T50 basic high shear mixer, for 5 min operated at 10 000 rpm. The pre-emulsion was homogenized 3 times with 250/50 bar with high pressure homogeniser (Gaulin APV Typ LAB 60/60-TBS).
Acacia seyal (sample No. 23)
Acacia Senegal kibbled (Sample No. 5)
The droplet size distribution for fresh and stored (14 days at ambient temperature) emulsions made using Acacia seyal alone and mixture (15 wt % Acacia seyal and 5 wt % Acacia senegal) results are shown in
The results shown in
The colour Gardner index of the samples was measured and is shown in
All references cited herein are incorporated by reference to the full extent to which the incorporation is not inconsistent with the express teachings herein.
Relationships between the series Vulgares (Acacia senegal) and Gummiferae (Acacia seyal), Food Hydrocolloids, 9, 17-34
| Number | Date | Country | Kind |
|---|---|---|---|
| 15179164.7 | Jul 2015 | EP | regional |
This application is continuation of U.S. patent application Ser. No. 15/747,390, filed Jan. 24, 2018, which is a National Stage Application under 35 U.S.C. § 371 of International Application No. PCT/EP2016/068135, filed Jul. 29, 2016, which claims priority benefit from European Patent Application No. 15179164.7, filed on Jul. 30, 2015, the entire content of which is incorporated herein by reference. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15747390 | Jan 2018 | US |
| Child | 17039388 | US |
