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Approaches for the Development of New Food Biopreservatives Obtained by Self-Microemulsifying Formulation of Raw Coconut Fat and Lipase

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Bulletin UASVM Agriculture, 67(2)/2010

Print ISSN 1843-5246; Electronic ISSN 1843-5386

Approaches for the Development of New Food Biopreservatives Obtained by Self-Microemulsifying Formulation of Raw Coconut Fat and Lipase

Georgiana PARFENE, Stefan DIMA, Vicentiu HORINCAR, Rodica DINICA, Gabriela BAHRIM

Dunarea de Jos University of Galati, Faculty of Food Science and Engeneering, Domneasca Street, No. 111, 800201, Galati Romania, e-mail: parfene_georgiana@yahoo.com

Abstract. Lipids hydrolysis in self-microemulsifying system with lipase increases the practical approaches of emulsions as food additives with biopreservation potential. This study aimed improved bioactive functions of raw coconut fat hydrolysates using self-microemulsifying oily formulation to intensify hydrolysis yield, stabilization of compounds of hydrolysis and control antimicrobial action by controlled release in model and food systems. For microemulsions characterization has been made pseudoternary phase diagrams and have highlighted reports of mixing A / W / S / CoS corresponding states of microemulsion. Microemulsions obtained are stable and provides good stabilization of oil, up to 70% w/w water. Through viscosimetric and conductometric analysis have highlighted the transition states of micro W/O, O/W bicontinuous structures. The antimicrobial activity of microemulsions was evaluated upon spoilage microorganisms as: Bacillus subtilis, Sarcina flava, Saccharomyces cerevisiae, Rhodothorula glutinis, Candida mycoderma, Aspergillus niger, Penicillium glaucum and Geotrichum candidum. It was observed that microemulsions have a higher antimicrobial potential, comparing with classical enzymatic hydrolisates, due to large surface of contact between enzyme and oil and also due to release a large amount of saturated fatty acids with antimicrobial activity.

Keywords: coconut oil, lipase, self-microemulsifying systems (SMES) .

INTRODUTION

Microemulsions are thermodinamically stable systems, transparent, with a low viscosity and are optically isotropic droplets having sizes ranging from 500 to 100 nm. They are prepared by mixing the lipophilic phase with the hydrophilic phase in the presence of surfactants and cosurfactants.Cosurfactants are generally short-chain saturated alcohols (C2- C6) which have the role of reducing surface tension, thereby leading to the formation of microemulsions with free decreasing energy (Moulik et al., 2000).

Generally, a low concentration of aqueous phase (less than 30%) form microemulsions type O/W and a low water content form microemulsions W/O. During the formation of mezophasic microemulsions the system run through different structures, transparent, formed by the association of amphiphilic surfactant molecules such as: direct and reverse micelles, lamellar phases, continuous phases until the cloudy emulsions. Highlighting the mixing range of different phases of components through which the system is done is based on pseudoternary phase diagrams (Ruckenstein et al., 2008). Due to its properties, microemulsions have found numerous applications in food and pharmaceutical industries. The

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microemulsions ensure solubility of active principles and increase their bioavailability (Flanagan et al., 2006; Garte et al., 2004).

Maintaining the stability of food chemistry and eliminating biological hazards induced by eating food contaminated with pathogens or toxins produced by microorganisms, requires the use of compounds having antimicrobial, microbiostatical and microbicidal activities. This research started raising considerable interest in finding new sources of antimicrobial agents as preservatives in food.

In recent years, there has been a considerable pressure by consumers to reduce or eliminate chemically synthesized additives in foods. Plants and plant products are a source of natural alternatives to improve the shelf-life and the safety of food (Lanciotti et al., 2004).

Oils obtained from plants that provide the main vegetable fat source have a higher content of compounds with antimicrobial potential and preservative role. The structural composition of fatty acids has been known to exert antimicrobial effects. An important part is played by saturated fatty acids (capric acid, caprinic acid, caprylic acid, lauric acid, lauric acid, myristic acid) and unsaturated fatty acids (palmitoleic acid, linoleic acid, linolenic aidul) of oil composition which has demonstrated high antimicrobial potential against pathogens (Agoramoorthy et al., 2007;Walters et al., 2004).

The coconut palm (Cocos nucifera) is generally belived to have originated in the jungle forests of central and east Africa. The major fatty acids in coconut oil are C12 (lauric acid) about 48%, C14 (myristic acid) about 18%, and C8 (caprylic acid) about 8% (Codex Alimentarius, 1999). Of the saturated fatty acids, lauric acid has the greatest antiviral activity (C-6) than caprylic acid (C-8), capric acid (C-10) or myristic acid (C-14). Approximately 50%

of the fatty acids in coconut oil are lauric acid. Lauric acid is known for its unique uses in the manufacture of soaps and cosmetics. More recently, lauric acid has been recognized for its unique properties in food use, which are related to its antiviral, antibacterial and antiprotozoal functions. Lauric acid is a medium-chain fatty acid which has an additional beneficial function of being formed into monolaurin in the human body. Monolaurin is the anti-viral, anti-bacterial and anti-protozoal monoglyceride used by the human body to destroy lipid- coated viruses such as HIV, herpes, cytomegalovirus, influenza, various pathogenic bacteria such as Listeria monocytogenes and Helicobacter pylori, and protozoa such as Giardia lamblia.

No other fatty acid is present at more than 10% and it is this heavy preponderance of lauric acid, which give coconut oil, it’s sharp melting properties, meaning hardness at room temperature which is combined with a low melting point (Pantzaris et al., 2000).

The aims of this study are: solubilisation of virgine coconut oil in order to increase the efficiency of hydrolysis of esters and fatty acids to enhance of the antimicrobial potential;

preparation of self-microemulsifying oily formulation and their physico-chemical characterization; comparative study between the antimicrobial activity of hidrolysed oil and microemulsions.

MATERIALS AND METHODS

Reagents and indicator microorganisms

Raw coconut fat was obtained from Cameroon, Africa.

The reagents tween 80, polysorbate(Merck), ethanol (Sigma)and glycerol (Merck) are used to obtain microemulsions.

The lipase (lyophilized enzyme) produced by a Yarowia lipolytica selected yeast strain was obtained from Microbial Biotechnology Reserch Centrer of Faculty of Biotechnology ,

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University of Agronomical Sciences and Veterinary Medicine, Bucharest, Romania. The lipase activity was measured as 4666.66 Units per gram of enzyme dry powder.

Lipase activity was measured by a titrimetric assay using emulsified coconut oil as the substrate. The assay was carried out at 37°C, during 60 minutes. The reaction was stopped by adding 3 ml acetone-ethanol 1:1 (vol. /vol.) and the amount of fatty acids was then titrated with NaOH 0.1N (Banu et al., 2008). For the acidity index evaluation a volume of 5 ml of sample was dissolved in a mixture of ethanol: ether (1:1 v / v). 2-3 drops phenolphthalein 0.1% in ethanol were added in the clear solution and titrated to turn red with 0.1N NaOH (Florea et al., 2009). One unit of lipase activity was defined as the amount of enzyme that released 1 µ equivalent of carboxyl groups of fatty acid under analysis conditions (temperature 370C, pH = 7.0, reaction time = 60 minutes).

As indicator microorganisms for evaluation of antimicrobial activity were used some bacterial and fungal strains belonging of the species: Bacillus subtilis, Sarcina flava, Saccharomyces cerevisae, Rhodothorula glutinis, Candida mycoderma, Aspergillus niger, Penicillium glaucum and Geotrichum candidum, wich were grown on specific agar broth media (malt extract agar -MEA, for fungi, plate count agar - PCA, for bacteria).

Classical oil hydrolysis and self-microemulsifying oily formulation

For classical hydolysis, a raw coconut fat (sample P1) was used with the next procedure: 10g raw coconut fat and 0.15g of enzyme in 100ml phosphate buffer solution at pH = 7.0, by stirring at 400 rot/min, at 35°C, during 48h.

Oil-water microemulsions were prepared based on the phase diagram layout and studied by combining various quantities of fat; enzyme and microemulsifying reagents (sample P2). The raw coconut fat and enzyme ratio was 80:1. Cosurfactant mass ratio for each sample was 5:1 (wt/wt). For preparation of microemulsions, surfactant percentage remained stable at 30% and the aqueous phase content varied from 0% to 90% every 5%

(wt/wt). Mixtures were formed by ultrasonic for 3 minutes at amplitude of 30% and a frequency of 20 kHz. All samples were kept at room temperature for 24 hours to establish steady state before the investigation.

Microemulsions properties evaluation

The viscosity of samples was measured at 25°C with Brookfield DVIII rheometer using a cone-plate of 60 mm diameter and 1 degree angle. Shear rate was 10-20 s-1. Electric conductivity and pH was measured at 25°C with C868 (Consort), with conductometric electrode SK10B type with a cell constant of 0.11 cm-1, and a pH-metric electrode SP10B type.

Antimicrobial activity of classical hydrolyzed raw coconut fat and of self- microemulsifying oily formulation

The disc diffusion method performed as by Brauer et al., 1996, was used for the determination of the antimicrobial activity for both samples. Were obtained suspensions indicating microorganisms by mixing in a test tube sterile saline with which is harvested from each pure culture of microorganisms tested. 1ml of suspension was assigned to each of the sterile Petri dish. Was added a layer of 5 mm thick, the culture medium (MEA, respectively PCA) thinned and cooled to 42°C, spreading through circular movements all over the Petri plate. 2 ml of microorganisms suspension was embedded in the culture medium, then rested three minutes for solidification to the medium. At the same time, were made sterilized filter paper discs of 1cm diameter, and after they have been wetted for 5 min in sample P1 and sample P2. Then wetted discs were placed on agar mediu in Petri dishes after they were

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incubated at 37°C for 48 h (for bacteria) and respectively 25°C for 4 days (for yeasts and molds) (Ekwenye et al., 2005).

RESULTS AND DISCUSSIONS

Making of pseudoternar phase diagram

To find the proper mixing ratio of components was needed to build a state of microemulsion pseudoternar phase diagrams. For this, oily phase is obtained by mixing the oil with alcohol in the mass ratio of 2:1. Oily phase is mixed with surfactant (Tween 80) in the mass reports 1:9, 2:8, 3:7, 4:6, 5:5, 7:3; 8:2, 9:1. Shake the mixture slightly and are classified by visual and microscopic analysis.

Fig.1. Pseudoternar phase diagram.

a) cosurfactant ethanol; b) cosurfactant 1-butanol

M-microemulsion; E- emulsion; 2Φ- two phase; L-surfactant micelles

Phase diagrams were used to determine the concentration of components to determine the appropriate field micro-emulsions. In preparation of microemulsions, cosurfactants help to reduce interfacial tension in addition to surfactants, favoring the formation of micro- emulsions. They adsorb at interface O/W and change the flexibility favoring the formation of micro-emulsions interfacial membrane.

After analyze of AB line (Fig. 1) it’s observe that the first 10 samples obtained from progressive water addition are homogenous, transparent, colored in yellow-orange. This sample corresponds to micro-emulsion phase. When the water content increase with up to 50% at minimum concentrations of surfactant, the samples begin to lose their transparency and it becomes more turbid, until the separation of the 2 phases. The zones with a higher surfactant concentration present a higher viscosity and a semitransparent appearance, which correspond to micellar structures of amphiphilic molecules. It is to be mentioned that the phase diagram is a small area of emulsion formation, with milky appearance, whose drops can be seen under a microscope.

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A B

Fig. 2. Aspect in self-microemulsifying system: A-emulsion; B-microemulsion

Viscosity

Viscosity depends very much on the micro-emulsion structure, the type and the shape of micellar aggregates, on the concentration and the interaction between dispersed particles.

Therefore viscosity measurements provide useful information about alternate phases and structural transformations in a micro-emulsion. All micro-emulsion samples analyzed showed a Newtonian flow. If adequate evidence is analyzed, viscosity noted AC is the line in the field of 0-30% water, viscosity values are relatively small and slightly increasing from 13.77 to 15.12 mPa·s while in the field of 30% -50% water, viscosity increases from 14.37 at 18.65 mPa·s and then decreases again 116.21 mPa·s.

These variations are due to structural changes in microemulsion. When the mass fraction of aqueous phase is small, the emulsion consists of scattered cells.

Electric conductivity

Absence of ions in the samples analyzed has led to very low values of electrical conductivity of micro-emulsions. This research has however revealed variations of electrical conductivity by increased water content. At water content less than 0-10% wt/wt conductivity values is about 12.15, following a slight increase to 15.76, corresponding to a water content of 35-40% wt/wt and then a more pronounced increase after this value (Fig. 3). These variations, which correspond to variations in viscosity, can be due to water micro-emulsions transition O/W, with low values of conductivity in mezophasic structures, which increase the conductivity of aqueous continuous phase.

Fig. 3. Variation of electrical conductivity with water content of microemulsion

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Antimicrobial activity of classical hydrolyzed raw coconut fat and of self- microemulsifying oily formulation

The antimicrobial activity evaluation shown a larger inhibition zone of self- microemulsifying oily formulation (sample P2) than classical hydrolyzed raw coconut fat (sample P1). This is because in microemulsion the area of contact between enzyme and fat promotes greater enzymatic hydrolysis that releases fatty acids with antimicrobial potential (Tab. 1).

Tab. 1 Antimicrobial activity of raw cocoanut fat hydrolysates in classical system and self-microemulsifying system

Indicator microorganisms Antimicrobial potential* of raw coconut fat hydrolysates obtaining by

Classical hydrolysis (sample P1)

Self-microemulsifying formulation (sample P2) Bacteria

Bacillus subtilis + ++

Sarcina flava + ++

Yeasts

Sacharomyces cerevisiae ++ ++

Rhodothorula glutinis ++ ++

Candida mycoderma +++ +++

Molds

Aspergillus niger + +++

Penicillum glaucum +++ +++

Geothricum candidum + +++

* +++ large inhibition zone; ++ medium inhibition zone; + small inhibition zone

It was observed that sample P2, comparing with P1, has a high antifungal activity against yeasts Sacharomyces cerevisiae, Rhodothorula glutinis and Candida mycoderma and molds Aspergillus niger, Penicillium glaucum and Geotrichum candidum respectively (Fig.4).

An unclear inhibition zone presented by P1 sample, was probably due to impurities contained in raw coconut fat which has not been no treatment beforehand. This was not noticed when P2 sample was examined, probably due to the content of surfactant and cosurfactant used in the preparation of microemulsions.

A B C

Fig. 4. Antifungal activity of raw coconut fat hydrolysates in classical system (P1) and self- microemulsifying system (P2).

Indicator strains: A) Rhodotorula glutinis; B) Penicillium glaucum; C) Aspergillus niger

In the future the extending and increasing of the antimicrobial activity of microemulsions against spoilage microorganisms and pathogens will be target by optimization of self-microemulsifying conditions.

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CONCLUSIONS

The raw coconut fat hydrolysates could be potential food biopreservatives. Making the continuous hydrolysis by self-microemulsifying oily formulation do it more efficient antimicrobial product.

The pseudoternary phase diagrams have been realized and have highlighted reports of mixing A / W / S / CoS corresponding states of micro-emulsion. Microemulsions obtained are stable and provides good stabilization of oil for up to 70% w/w water. For viscosimetric and conductometric analysis, the transition states of micro W/O, O/W bicontinuous structures have been highlighted.

It was shown that raw coconut fat microemulsions has a higher acidity than classical hydrolyzated coconut fat which confirm that in microemulsion it was obtained a high content of fatty acids, because in the microemulsion the large contact area between enzyme, with high antimicrobial potential against bacteria, yeasts and molds.

Acknowledgments. The authors are grateful to support provided by prof. dr. Stefana Jurcoane from lipase and by dr. Aboubakar to bring the raw coconut fat from Cameroun. A gratefully acknowledge from the financial support of BioAliment Research Platform and Grant no.POSDRU/88/1.1/S/61445,Project ID:61445/2009 in carrying out this research work.

REFERENCES

1. Moulik, S. P. and B. K. Paul. (2000). Structure, dynamic and transport properties of microemulsions. Adv. Drug. Deliv.Rev. 45. 89-121.

2. Ruckenstein, E., J. Chi and T. Farad. (2008). Stability of microemulsions, J. Chem. Soc. 271.

1690 – 1707.

3. Flanagan, J. and H. Singh. (2006). Microemulsions: A Pontential Delivery System for Bioactive in Food. Food Science and Nutrition. 46. 221-237.

4. Garti, N., S. Zakharia, R. Elfakess and S. Ezrahi. (2008). Solubilisation of water – insoluble nutraceuticals in nonionic microemulsions for water/based use. Progr. Colloid Polyim. Sci. 126:184 – 189.

5. Belletti N, M. Ndagijimana, C. Sisto, M. E. Guerzoni, R. Lanciotti and F. Gardini. (2004).

Evaluation of the antimicrobial activity of citrus essences on Saccharomyces cerevisiae. J. Agricult.

Food Chem. 52: 6932-6938.

6. Agoramoorthy, G., M. Chandrasekaran, V. Venkatesalu and M. J. Hsu. (2007). Antibacterial and antifungal activities of fatty acids methyl esters of the blind-your-eye mangrove from India.

Brazilian Journal of Microbiology. 38. 739–742.

7. Walters, D., L. Raynor, A. Mitchell, R. Walker and K. Walker. (2004). Antifungal activities of four fatty acids against plant pathogenic fungi. Mycopathologia. 157. 87–90.

8. Codex Alimentarius Commission (1999). Report of the Sixteenth Session of the Codex Committee on Fats and Oils. Alinorom 99/17: Appendix II. Rome, Italy.

9. Pantzaris, T. P. (2000). Pocketbook of palm Oil Uses. Fifth edition. MPOB. Bangi p.102-109.

10. Banu A., O. Radovici, M. Marcu, T. Spataru and S. Jurcoane (2008). Electrochemical synthesis and characterization of a polipyrrole/lipase composite film. Roumanian Biotechnological Letters. 13(1). 3551-3556.

11. Florea T., B. Furdui, R. Dinica and R. Cretu. (2009). Chimie organica, Sinteza si analiza functional. Ed. Academica. Galati .

12. Brauer, A. W., M. M. Shervis and N. Truck. (1996). Antibiotic Susceptibility Testing by a Standardized Sample Disc Method. J. of Chemical Pathology. 45. 493-496.

13.Ekwenye, U. N. and C. A. Ijeomah. (2005.), Antimicrobial effects of palm kernel oil and palm oil, KMITL Sci. J. Vol. 5 No. 2.

References

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