The physicochemical and antimicrobial properties of cocoa pod husk (CPH) pectin intended as a versatile pharmaceutical excipient and nutraceutical were studied. Properties investigated include pH, moisture content, ash values, swelling index, viscosity, degree of esterification (DE), flow properties, SEM, FTIR, NMR, and elemental content. Antimicrobial screening and determination of MICs against test microorganisms were undertaken using agar diffusion and broth dilution methods, respectively. CPH pectin had a DE of 26.8% and exhibited good physicochemical properties. Pectin had good microbiological quality and exhibited pseudoplastic, shear thinning behaviour, and high swelling capacity in aqueous media. The DE, FTIR, and NMR results were similar to those of previous studies and supported highly acetylated low methoxy pectin. CPH pectin was found to be a rich source of minerals and has potential as a nutraceutical. Pectin showed dose-dependent moderate activity against gram positive and gram negative microorganisms but weak activity against
Cocoa or
The recovery of cocoa beans from the cocoa fruit generates large amounts of waste in the form of cocoa pod shells or cocoa pod husks (CPHs) estimated at 52–76% of the cocoa fruit [
An economical and environmentally friendly way of dealing with the CPH waste menace is to process them into pectins which are natural polymers containing linear chains of (1, 4)-linked
Various techniques and solvent systems such as water, citric acid, hydrochloric acid, and nitric acid have been employed in the recovery and extraction of pectins from CPH with varying levels of success, with respect to yield and quality of pectin extracted [
Although considerable research has been devoted to the development of pectin from commercial sources, such as apple pomace and citrus peel, with versatile functional properties in pharmaceutical applications, little attention has been paid to the pharmaceutical applications of CPH pectin. The objective of the present study was to evaluate the physicochemical properties, elemental composition, and antimicrobial properties of CPH extracted with water and citric acid. It is envisaged that results from this study would help in determining the suitability or otherwise of CPH pectin as a potential functional pharmaceutical excipient and nutraceutical.
Sodium hydroxide (UK), gelatin and lead acetate (France), ferric chloride (India), hydrochloric acid and Mayer’s reagent (England), and Dragendorff’s reagent and Marquis reagent (England) were purchased. Mannitol salt agar, MacConkey agar, Bismuth sulphite agar, Cetrimide agar, Sabouraud dextrose agar, nutrient agar, potato dextrose agar, and nutrient broth were obtained from Oxoid (England). Ciprofloxacin powder (batch number AV 4008, Maxheal Labs Pvt. Ltd., India), Amoksiklav powder (Amoxicillin + clavulanic acid) (Lot EN 2737, Lek Pharmaceuticals, Slovenia), and Nystatin (100,000 IU/drop, Egyptian Pharmaceutical Industries, Egypt) were used. All other chemicals used were of analytical grade.
Two typed cultures,
Ripe mature cocoa pods were harvested from
One milliliter of 2 N NaOH was added to 5 mL of 1 in 100 solutions of the CPH extract and was allowed to stand at room temperature for 15 minutes. The gel from the preceding test was acidified with 3 N HCl, shaken vigorously, and boiled [
The moisture content was determined by weighing 1 g of CPH pectin into each of three petri dishes and dried in an oven at 105°C to constant weight. The moisture content was determined as the ratio of the weight of moisture loss to weight of sample expressed as a percentage. The pH of 1% w/v solution of hot water soluble pectin and citric acid soluble pectin samples was determined with a calibrated pH meter. The total ash content and insoluble ash residue were determined according to the British Pharmacopoeia method [
In the determination of the bulk and tapped densities, 3 g of pectin powder was weighed into a 10 mL measuring cylinder and the volume occupied was noted. The sample was tapped till the powder was consolidated and the volume after tapping was noted. The bulk and tapped densities, as well as the Hausner ratio and compressibility index, were calculated as follows:
Specimens of hot water soluble pectin and citric acid soluble pectin were prepared for SEM analysis with a thin coating of colloidal carbon for electron conductivity. The morphological features of the samples were studied with a scanning electron microscope (Hitachi S3200N, Japan), using EDAX Genesis. All imaging was viewed under conventional high-vacuum mode and secondary electron scintillator detection mode.
A sample of hot water soluble pectin and citric acid soluble pectin was analysed for main functional groups using Bruker Alpha Fourier transform infrared spectrophotometer (Germany) operating on Platinum-ATR to obtain FTIR spectra at 400–4000 cm−1. Specimens of hot water soluble and citric acid soluble pectin were prepared for NMR analysis using a Varian 500 NMR spectrometer (USA). The 13C NMR spectra of the hot water soluble pectin and citric acid soluble pectin extracts in D2O were obtained at 25°C and 50°C, respectively. Chemical shifts were expressed in
Profiling of possible microbial contaminants from cocoa pectin was undertaken [
Four concentrations (1.25, 2.5, 5.0, and 10.0 mg/mL) of hot water soluble CPH pectin and standard antibacterial agents, Amoksiklav and ciprofloxacin, as well as the antifungal Nystatin, were used to assess their comparative antimicrobial activities by agar diffusion method [
The MIC of hot water soluble CPH pectin was determined using the broth dilution technique. Graded concentrations of pectin (0.125, 0.25, 0.5, 1.0, 2.0, 4.0., and 8.0 mg/mL) in nutrient broth and potato dextrose liquid medium were compared to those of Amoksiklav, ciprofloxacin, and Nystatin. A set of seven double strength nutrient broth tubes were arranged from a prepared stock solution of 50 mg/mL pectin test sample. Volumes of the stock solution required to produce the respective concentrations with the double strength nutrient broth were calculated and added aseptically to the broth by means of sterile syringes in a laminar flow chamber. The volumes of sterile distilled water required to make the broth tubes single strength were also calculated for and added to the broth tubes aseptically. Finally, 0.1 mL inoculum of a 24 h test microbial culture was inoculated into the broth to complete the procedure. Uniform mixing was ensured and the tubes were incubated at 37°C for 24 h. The tubes were observed for growth (turbidity) after the incubation period and MICs for pectin and the standard antimicrobial agents were determined.
The extraction yield obtained from CPHs was
Phytochemical screening of CPH yielded polyphenols such as tannins, alkaloids, and saponins. Phenolic compounds of cacao include catechins, epicatechins, anthocyanins, proanthocyanidins, phenolic acids, condensed tannins, other flavonoids, and some minor compounds [
Table
Some physicochemical properties of hot water soluble CPH pectin.
Parameter | Value |
---|---|
Yield on extraction (%) | 23.3 ± 2.00 |
10.5 ± 0.04 | |
Moisture content (%) | 0.19 ± 0.06 |
Ash value (%) | 1.0 |
pH (1% w/v @ 25°C) | 6.73 ± 0.06 |
3.43 ± 0.06 | |
Swelling index | |
0.1 M HCl | 357.3 ± 4.6 |
Phosphate buffer pH 6.8 | 274.7 ± 4.6 |
Distilled water | 360.0 ± 0.0 |
Degree of esterification (%) | 26.8 ± 2.5 |
Precompression properties | |
Bulk density (g/mL) | 1.881 |
Tapped density (g/mL) | 2.200 |
Hausner ratio | 1.17 |
Compressibility index (%) | 14.58 |
Angle of repose (°) | 37.97 |
The swelling characteristics of cocoa pectin in various media were investigated. The swelling index of cocoa pectin was 274.7 in 0.1 N HCl, 357.3 in phosphate buffer pH 6.8, and 360 in water. Cocoa pectin can swell to varying extents depending on the pH, ionic strength, and presence of salts in the medium. The swelling behaviour of CPH pectin shows that it can function as a binder or matrix agent in controlled release formulations. This is because swelling is an important mechanism in diffusion controlled release in drug delivery [
The precompression parameters of cocoa pectin powder studied were the angle of repose, bulk density, tapped density, Hausner ratio, and Carr’s compressibility. The ease of flow of powders is of paramount importance in tablets and capsules formulation as free flowing powders ensure reproducible filling of tablet dies and capsule dosators, thereby improving weight uniformity and consistency in physical properties. Hausner ratio is related to interparticle friction in a powder and values close to 1.2 are indicative of less cohesive and free flowing powder while values greater than 1.6 are powders which are cohesive and have poor flow properties. In terms of flowability, powders with compressibility index of 5–15% are regarded as excellent, 12–16% good, 18–21 fair, and >40% extremely poor. A high angle of repose is indicative of a cohesive powder, while a low angle of repose connotes a noncohesive powder. In general, powders with angles of repose >50° have unsatisfactory flow properties, whereas minimum angles close to 25° have very good flow properties [
The rheograms of 5% w/v hot water soluble cocoa pectin at 25°C and 33°C showed a non-Newtonian, pseudoplastic, shear thinning behaviour (Figure
Viscosity profiles of 5% w/v hot water soluble pectin.
The scanning electron micrographs of hot water soluble pectin and hot citric acid soluble pectin are shown in Figure
Scanning electron micrographs of (a) hot water soluble pectin (mag ×20) and (b) citric acid soluble pectin (mag ×20).
Figure
FTIR spectra of (a) citric acid soluble pectin and (b) hot water soluble pectin.
The 3C NMR spectrum of hot water pectin is shown in Figure
13C NMR spectrum of hot aqueous extract of pectin at 25°C in D2O.
Figure
13C NMR spectrum of citric acid soluble pectin at 50°C in D2O.
Results of the elemental analysis of hot water soluble CPH pectin are shown in Table
Elemental content of hot water soluble CPH pectin.
Type of element | Content (%) |
---|---|
Macroelements | |
Na | >0.038 |
Mg | 0.219 |
P | 0.096 |
S | 0.094 |
K | 2.269 |
Ca | 0.011 |
Fe | 0.024 |
Microelements | |
Cr | <0.0006 |
Co | <1.90 |
Ni | 3.70 |
Cu | 10.90 |
Zn | 8.30 |
Ga | 0.70 |
Mo | <0.9 |
Previous reports show that CPH flour also contained a variety of minerals. The qualitative components are similar to those reported in previous studies [
The wide range of macro- and microminerals found in cocoa pectin shows the potential of this natural polymer to provide medical or health benefits users. Cocoa pectin is therefore a potential plant-based nutraceutical. There is a growing interest in the use of plant-derived bioactive compounds in foods as “multifunctional food additives” due to their additional nutritional and therapeutic effects [
In general, microbial contaminants may be grouped into harmful, objectionable, and opportunistic organisms. Harmful organisms are toxins-producing, disease causing organisms such as
Microbial quality of hot water soluble CPH pectin.
Test protocol | Results | Inference |
---|---|---|
Total aerobic viable count of sample |
1.2 × 101 cfu/mL | Passed |
Test for |
None detected | Passed |
Test for |
None detected | Passed |
Test for |
None detected | Passed |
Test for |
None detected | Passed |
Test for |
None detected | Passed |
MCA = MacConkey agar; MSA = Mannitol salt agar; BSA = Bismuth sulphite agar; CA = Cetrimide agar; SDA = Sabouraud dextrose agar.
The antimicrobial activity of CPH pectin against selected microbial strains is presented in Table
Antimicrobial properties of hot water soluble CPH pectin and standard antimicrobial agents against test organisms.
Organisms | Mean zones of inhibition (mm) | |||
---|---|---|---|---|
10 mg/mL | 5 mg/mL | 2.5 mg/mL | 1.25 mg/mL | |
Gram negative bacteria | ||||
|
26.0 ± 0.5 | 25.0 ± 1.0 | 22.5 ± 0.5 | 20.0 ± 0.0 |
35.0 ± 0.0 |
32.0 ± 1.0 |
30.9 ± 0.1 |
29.0 ± 0.0 | |
37.0 ± 1.0 |
34.9 ± 0.9 |
30.0 ± 1.0 |
26.9 ± 0.9 | |
|
24.0 ± 0.5 | 23.2 ± 0.2 | 22.0 ± 0.0 | 19.4 ± 0.6 |
36.9 ± 0.1 |
35.0 ± 1.0 |
30.0 ± 1.0 |
27.8 ± 0.8 | |
38.9 ± 0.8 |
37.8 ± 0.8 |
32.0 ± 0.0 |
30.0 ± 0.0 | |
|
25.0 ± 1.0 | 23.2 ± 0.2 | 20.5 ± 0.5 | 18.0 ± 0.0 |
|
22.0 ± 0.0 | 20.3 ± 0.8 | 18.0 ± 0.0 | 16.0 ± 0.0 |
Gram positive bacteria | ||||
|
24.0 ± 0.0 | 22.5 ± 0.5 | 19.6 ± 0.6 | 17.0 ± 0.0 |
33.9 ± 0.1 |
27.3 ± 0.6 |
25.0 ± 0.0 |
22.0 ± 1.0 | |
36.0 ± 0.0 |
33.9 ± 0.9 |
29.9 ± 0.1 |
24.7 ± 0.6 | |
|
24.0 ± 0.0 | 22.5 ± 0.5 | 20.0 ± 0.5 | 17.0 ± 0.0 |
36.0 ± 0.0 |
30.9 ± 0.2 |
28.0 ± 1.0 |
25.0 ± 1.0 | |
38.0 ± 0.0 |
33.0 ± 1.0 |
24.9 ± 0.9 |
20.1 ± 0.9 | |
|
18.0 ± 0.5 | 16.0 ± 0.0 | 15.0 ± 0.0 | 12.7 ± 0.3 |
|
15.0 ± 0.0 | 13.0 ± 0.0 | 12.0 ± 0.0 | ND |
Fungus | ||||
|
18.0 ± 0.7 | 16.3 ± 0.4 | 15.0 ± 0.0 | ND |
20.3 ± 0.4 |
18.5 ± 0.0 |
17.0 ± 0.0 |
15.1 ± 0.1 |
Table
Minimum inhibitory concentrations (MICs) of CPH pectin and standard antimicrobial agents against test organisms.
Organisms | MIC (mg/mL) | |||
---|---|---|---|---|
CPH pectin | Amoksiklav | Ciprofloxacin | Nystatin | |
Gram negative bacteria | ||||
|
0.5–1.0 | ND | 0.125–0.250 | ND |
|
1.0–2.0 | ND | 0.500–1.000 | ND |
|
1.0–2.0 | ND | 0.250–0.500 | ND |
Gram positive bacteria | ||||
|
0.5–1.0 | 0.25–0.50 | ND | ND |
|
1.0–2.0 | 0.50–1.00 | ND | ND |
Fungus | ||||
|
2.0–4.0 | ND | ND | 0.5–1.0 |
ND = not determined.
The antimicrobial activity of CPH extract was assessed recently against
It can be concluded from the study that cocoa pectin has the requisite microbial quality and physicochemical parameters to be employed as a multifunctional excipient in the pharmaceutical, food, and allied industries. The elemental content analysis showed the presence of a broad range of micro- and macronutrients in cocoa pectin, making it a potentially useful health promotion polymer. Cocoa pectin showed moderate activity against selected gram positive and gram negative bacteria and could be useful as a preservation agent in pharmaceutical formulations and food products. The study has demonstrated the enormous potential of cocoa pectin as a pharmaceutical excipient, a nutraceutical agent, and an antibacterial agent.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors gratefully acknowledge the University of Ghana Office of Research, Innovation and Development (ORID) for providing a Faculty Development Grant to OAD in support of this study. Special thanks are also due to Dr. Jeremy Takrama of the Cocoa Research Institute of Ghana (CRIG), Tafo, Ghana, for his technical assistance.