determination of preservatives in canned food products

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Jan 18, 1991 - standard deviation. SB -. Sodium Benzoate. SMBS-. Sodium Metabisulphite. USFDA-. United States Food and Drugs Authority. UV -. Ultraviolet.
DETERMINATION OF PRESERVATIVES IN CANNED FOOD PRODUCTS

ANTHONY MUTUNGA CHARLES

A RESEARCH PROJECT REPORT SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF BACHELOR OF SCIENCE DEGREE IN ANALYTICAL CHEMISTRY, JOMO KENYATTA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY

©2014

DECLARATION This research is my original work and has not been wholly or partially been presented to any other university or institution for award of a degree or any other certificate.

Signature………………………………………

Date.…………………………………..

ANTHONY MUTUNGA CHARLES SC 232-0774/2010

This project has been submitted with my approval as the official university supervisor.

Signature……………………………………….

Date………………………………………

DR. HARRISON WANYIKA Department of chemistry, JKUAT

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ACKNOWLEDGEMENT I acknowledge the relentless help of our God Almighty for his blessing and provision of good health before, during and after my research project. Special thanks to my Supervisor Dr. H.Wanyika for the professional advice and guidance on the whole process of my project work. Special thanks to our chief technician Mr. Mawili for his advice on various procedures and protocols. I also acknowledge Mr. Karanja from Food Science Department for his help in the use of HPLC machine; I also acknowledge the sincere help of Madam Jessica also from Food Science Department for going out of her way in helping me. Lastly my friends have really given me support and I thank them all.

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DEDICATION I would like to dedicate this work to my loving parent Madam Angeline Nduku for she offered immense support through counsel, encouragements and sincere prayers throughout the whole period in Campus. I also dedicate the work to my very dear friend and partner Faith Kalu for she offered a lot moral support. I finally offer special dedications to the BSc. Analytical class of 2014/2015 and the JKUAT fraternity.

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LIST OF ABBREVIATIONS CAN -

Acetonitrile,

ATSDR-

Agency for Toxic Substances and Disease Registry

CFR

Code of Federal Regulations

-

Conc. –

concentration

GRAS-

Generally Recognized as Safe

HPLC-

High Performance Liquid Chromatograph

KEBS-

Kenya Bureau of Standards

LC

-

Liquid Chromatography

PS

-

Potassium Sorbate

R



Regression coefficient,

S.D



standard deviation

SB

-

Sodium Benzoate

SMBS-

Sodium Metabisulphite

USFDA-

United States Food and Drugs Authority

UV

Ultraviolet

-

UV-VIS-

Ultraviolet-Visible

v

FSSI

-

CS-L/Nak –

Food Safety and Standard Authority of India Chili sauce Lyons brand from Nakumatt supermarket

CS-K/P

-

Chili sauce Ken brand from Pawa Supermarket

CS-P/U

-

Chili sauce Peptang brand from Uchumi supermarket

TS-K/P

-

Tomato sauce ken brand from Pawa supermarket

TS-Z/U

-

Tomato sauce Zesta brand from Uchumi supermarket

TS-P/U

-

Tomato sauce Peptang brand from Uchumi supermarket

KT-H/Ha

-

Ketchup sauce Heinz brand from happy supermarket

KT-AG/U

-

Ketchup sauce American Garden brand from Uchumi supermarket

Fr-Rh/Nak



Fruit cocktail, Rhodes Co. from Nakumatt supermarket

Pn-Rh/L

-

canned pineapple, Rhodes Co. from Leens supermarket

Pn-Del/T

-

canned pineapple, Delmonte. From Turkeys supermarket

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TABLE OF CONTENTS DECLARATION................................................................................................................................ii ACKNOWLEDGEMENT.................................................................................................................. iii DEDICATION .................................................................................................................................. iv LIST OF ABBREVIATIONS..............................................................................................................v TABLE OF CONTENTS .................................................................................................................. vii LIST OF FIGURES ........................................................................................................................... ix LIST OF CHROMATOGRAMS ........................................................................................................ ix LIST OF TABLES ............................................................................................................................. x LIST OF GRAPHS............................................................................................................................. x ABSTRACT ..................................................................................................................................... xi CHAPTER ONE ................................................................................................................................1 1.

INTRODUCTION AND LITERATURE REVIEW .......................................................................1 1.1

General introduction............................................................................................................1

1.2

Analytical methods ..............................................................................................................2

1.2.1

UV-Visible (UV-VIS) spectroscopy...............................................................................2

1.2.2

High performance liquid chromatography (HPLC)..........................................................3

1.3

Preservatives.......................................................................................................................5

1.3.1

Sodium benzoate and Benzoic acid ................................................................................5

1.3.2

Sorbic acid and potassium sorbate..................................................................................7

1.3.3

Sodium Metabisulphite ...............................................................................................10

1.4

Permissible limits of preservatives ......................................................................................11

1.5

Problem statement ............................................................................................................12

1.6

Justification .......................................................................................................................12

1.7

Hypothesis ........................................................................................................................12

1.8

Objectives .........................................................................................................................13 vii

1.8.1

Main objective............................................................................................................13

1.8.2

Specific objective .......................................................................................................13

CHAPTER TWO..............................................................................................................................14 2

MATERIALS AND METHODS................................................................................................14 2.1

Reagents and miscellaneous equipments ............................................................................14

2.2

Samples and Sample collection ...........................................................................................14

2.2.1

Sampling process........................................................................................................14

2.2.2

Sample treatment........................................................................................................15

2.2.3

Sample preparation .....................................................................................................15

2.3

Preparation of standard solutions .......................................................................................16

2.3.1

HPLC standards .........................................................................................................16

2.3.2

UV-VIS standards ......................................................................................................17

2.4

Instruments’ conditions for analysis of Benzoic acid.............................................................17

CHAPTER THREE ..........................................................................................................................18 3

4

RESULTS AND DISCUSSION .................................................................................................18 3.1

Spectra for some random samples ......................................................................................18

3.2

Concentration calculation...................................................................................................24

3.2.1

HPLC ........................................................................................................................24

3.2.2

UV-Visible .................................................................................................................26

3.3

Comparison of the methods ...............................................................................................29

3.4

CONCLUSION .....................................................................................................................32

3.5

RECOMMENDATIONS.........................................................................................................33

REFERENCES .........................................................................................................................34

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LIST OF FIGURES Figure 1: The UV-Visible spectrometer sketch diagram.........................................................................3 Figure 2: The HPLC chromatograph sketch diagram .............................................................................5 Figure 3: Molecular structure of benzoic acid .......................................................................................5 Figure 4: Molecular structure of sodium benzoate .................................................................................6 Figure 5: Molecular structure of sorbic acid..........................................................................................8 Figure 6: Molecular structure of potassium Sorbate...............................................................................8 Figure 7: Molecular structure of sodium metabisulphite ......................................................................10

LIST OF CHROMATOGRAMS Chromatogram 1: Chili sauce, Lyons from Nakumatt ..........................................................................18 Chromatogram 2: Chili sauce ken Pawa .............................................................................................19 Chromatogram 3: Chili sauce Peptang from Uchumi ...........................................................................19 Chromatogram 4:Tomato sauce Zesta from Uchumi............................................................................20 Chromatogram 5: Tomato sauce Ken from Pawa ................................................................................20 Chromatogram 6; crushed pineapple Rhodes Co. Ltd from Leens ........................................................21 Chromatogram 7: fruit cocktail Rhodes Co. Ltd ..................................................................................21 Chromatogram 8: ketchup Heinz from Happy Supermarket .................................................................21 Chromatogram 9: Ketchup American Garden from Uchumi ................................................................22 Chromatogram 10:sliced pineapples Delmonte from Tuskeys ..............................................................22 Chromatogram 11: 10ppm SB ...........................................................................................................23 Chromatogram 12: 40ppm SB ............................................................................................................23 Chromatogram 13: 80ppm SB ...........................................................................................................23

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LIST OF TABLES Table 1: Permissible limits of preservative content..............................................................................11 Table 2: Sample collection areas ........................................................................................................15 Table 3: the standards peak area information from HPLC analysis .......................................................24 Table 4: sample concentration calculated from the regression equation ................................................26 Table 5: Absorption values of the working standards for UV-Visible analysis.......................................27 Table 6:Sample concentration calculated from the regression equation .................................................28 Table 7: Concentration readings from the two methods of analysis used compared by paired T-test .......29

LIST OF GRAPHS Graph 1: standard curve of the HPLC working standard peak areas against concentrations ....................25 Graph 2: Standard curve of the UV-Visible working standard absorbance against concentrations ...........27 Graph 3: a graph showing comparisons between the two methods of analysis used ...............................30 Graph 4: Data comparison with the maximum allowed levels by various FDA bodies ...........................30

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ABSTRACT Food preservatives inhibit, stop or delay the growth of microorganisms or any deterioration of food due to microorganisms and are used alone or in conjunction with other substances to achieve shelf life prolongation of foods, [Food Safety and Standard Authority of India (FSSI), 2012] More than 3,000 additives as well as preservatives are available in the market and they are used as antioxidants and anti- microbial agents. Unfortunately some of the best food preservatives prove to be lethal to human health. The preservatives are strictly regulated because their overuse can cause health problems in humans. The concentration of Benzoates was determined in this study for canned food products (fruits, tomato sauce and ketchup) sampled in Kenyan market. Two techniques for quantitative determination of the said preservatives were employed. These are use of High Performance Liquid Chromatography and the results compared with UV-Visible Spectroscopy. After analytical treatment of the data, it was determined that the concentration of the data collected was as follows: all the brands exceeded the European Union maximum allowed concentration, but only one brand exceeded KEBS, USFDA, WHO and CFR maximum allowed limits, which is Tomato Sauce of brand Peptang from Uchumi supermarket and it had 1048.1527ppm by use of UV-VIS method. On average, the brands contained the following concentrations:- 858.449ppm, 874.539ppm, 640.269ppm, 1023.704ppm, 925.236ppm, 615.701ppm, 53.933ppm, 0.587ppm, 0.795ppm, 10.696ppn and 1.308ppm for CS-L/N (Chili Sauce, Lyons from Nakumatt Thika), CS-K/P (Chili Sauce, Ken from Pawa supermarket Juja), CS-P/U (Chili Sauce, Peptang from Uchumi supermarket Juja), TS-P/U (Tomato Sauce, Peptang from Uchumi supermarket Juja), TS-K/P xi

(Tomato Sauce, Ken from Pawa supermarket Juja), TS-Z/U (Tomato Sauce, Zesta from Uchumi Juja), KT-H/Ha (Ketchup, Heinz from Happy supermarket Juja), KT-AG/U (Ketchup, American Garden from Uchumi Juja), Fr-Rh/N (Canned Fruit cocktail, Rhodes Ltd from Nakumatt Thika), Pn-Del/T (Canned pineapples, Del-Monte Ltd from Tuskeys Thika) and Pn-Rh/L (Canned crushed Pineapples, Rhodes Ltd from Leens Supermarket Thika) respectively. The

significant

amount

of

the

preservatives

ranged

from 615.701±3.274ppm

to

1023.704±23.960ppm, when the average from the two methods was done. From the study done, it can be concluded that the food industries manufacturing canned food products adhere to the limiting amount of SB by the Kenyan FDA, KEBS. However, when compared to EU, none of the samples would pass the test and sell to the market. Also, the two methods used are not significantly different as seen by the paired T-test analysis. This is because they are both approved methods to analyze SB in food products. I would recommend a research to ascertain why the EU limits are so low, as this will give us an avenue to trade with the European Nations with these products.

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CHAPTER ONE 1. INTRODUCTION AND LITERATURE REVIEW 1.1

General introduction

With the advent of modern technology, methods of preserving food have changed significantly. Human beings have always found out ways to preserve food, right from their existence. In ancient times, the need was limited. It was easy for our ancestors to store food by drying it under the sun. But today, due to fast paced modern life, due to globalization and easy transport facilities, shelf life of various types of food is increased by employing various techniques. Introduction of new technology and discovery of new preservatives have completely changed the concept of marketing and food safety, (FSSI, 2012) Preservatives may be categorized as class 1 and class 2 preservatives. Class one are those which have no maximum allowed levels, such as common salt, sugar, glucose, dextrose, honey, etc. class 2 on the other hand are those which are regulated by drug and food administration agencies in the world, (FSSI, 2012). These preservatives may have a long term affect on the consumers’ health, hence a need to control their usage. Examples of these harmful food preservatives in food include; benzoic acid and its salt derivatives, sorbic acid and its derivatives, metabisulphites, nitrites and nitrates of potassium and sodium, Nisin, propionates of calcium or sodium, lactic acid and its salts, methyl or propyl parahydroxy benzoates sodium diacetate, etc., (FSSI, 2012) They can cause different allergies and conditions such as hyperactivity and attention deficit disorder in some people who are sensitive to specific chemicals. Some reactions can be very adverse, including the following among others; nausea and diarrhea, gastrointestinal discomforts and ulcers, tumors and bladder cancers, birth defects, genetic changes, etc.

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1.2

Analytical methods

Most of the preservatives are organic compounds providing a clear platform for their determination. Several methods for molecular determination are used, among them are; Headspace Concentration Techniques, capillary electrophoresis, voltametric techniques, HPLC, UV-Visible spectroscopy and Capillary Gas Chromatography, (McNeal et al, 1993). Simple analytical techniques like UVVisible spectroscopy and High Performance Liquid Chromatography were the major technique used for their determination. (Tfouni et al, 2002)

1.2.1

UV-Visible (UV-VIS) s pectroscopy

UV-VIS spectroscopy is an essential technique in quantification of compounds that absorb radiation either in the UV or in the visible region of electromagnetic spectrum. The UV-VIS spectrophotometer consists of a source of radiation, collimators, sample compartments, detector and the data system. A sample is placed in a sample cell made of quartz or glass that is placed in the path of radiation from the source. The UV or visible radiation causes electronic excitation of molecules that have UV chromophores and thus attenuating the incident radiation such that only a percentage of it is transmitted to the detector. The detector (usually a photomultiplier tube) converts radiant energy to an electrical signal and is recorded either as transmittance or a s absorbance by the data system, (McMahon 2007). This is principled on Beer-Lamberts law; A

cl log

Io It

log

1 T

log T , Where A= absorbance; T= Transmittance; C= Concentration in

molarity; l= path length in cm, ε = the molar absorbance coefficient in Lmole -1 cm-3 . Io and It are incident and transmitted UV-Visible light intensities respectively. To obtain quantitative data, a calibration curve transmittance/ absorbance against concentration is plotted and the sample concentration can thus be obtained from Beer’s la w. The schematic diagram for the instrument is shown below. 2

Figure 1: The UV-Visible spectrometer sketch diagram

1.2.2

High performance liquid chromatography (HPLC)

This is an analytical technique that involves separation and quantification of samples. All chromatographic systems contain: A stationary phase, a mobile phase and Sample molecules (mixture for separation). Movement of molecules is determined by the Impelling force of mobile phase which carries with it molecules for which it has affinity - favored by solubility (LC) Separation of sample components is achieved by interaction of sample molecules with the stationary phase. The analyte is carried through a stationary phase in a packed column by a liquid mobile phase at high pressure, during which time the components separate from each other on the column, (Gagliardi et al, 1997).

1.2.2.1 Instrumentation Polar HPLC columns and relatively non polar mobile phases used constitute normal phase mode of HPLC. Non polar columns and polar solvents constitute reversed phase HPLC. Often, a gradient of solvent composition passing through the column is used to separate mixtures, e.g. a water/methanol gradient. Else, isocratic elution is used, (McMahon 2007). A HPLC instrument consists:-

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a) Source The mobile phase and pump comprise the source. The mobile phase is chosen such that it has the optimal eluting power for the HPLC mode being used, low viscosity, high purity and stability, and such that it is compatible with the detection system. Solvents usually need to be degassed prior to use either by filtering under vacuum, use of an ultrasonic bath or degassing online. The most commonly used pumps are constant flow pumps. b) Sample injection Samples can be injected via a sample loop using a valve and the valve is then switched to deliver the sample plug onto the analytical column. The volumes injected are usually in the range 25-35 μL depending on the size of the loop and under certain conditions if required. The commonly used valve is a six port valve, (Gagliardi et al, 1997). c) Columns Efficient analytical columns must have a homogeneous stationary phase of small particle size. The use of narrower and shorter columns reduces the flow rate of the mobile phase and, hence, the overall amounts of solvent and sample consumed. It also reduces the time spent in the column by the analytes. Decrease in particle size with length creates efficiency and resolution. Connections between injector, column and detector should be of low volume and the inside diameters of components should be of similar size. Temperature control may be required if the separation is sensitive to changes in temperature. The choice of stationary phase depends on the mode of HPLC being used, size and polarity differences between analytes. d) Detectors They include refractive index detector (RID), UV absorbance detector, conductivity detector, light scattering detector, fluorescence detector, amperometric detector, MS detector and combination detector. The most commonly used detectors are the RID and the UV absorbance detector. UV absorbance detectors are easy to use non-destructive, insensitive to temperature and gradient 4

changes, selective, relatively sensitive and can be used with gradient elution. They are generally suitable for detecting of the majority of samples due to the large number of compounds that absorb in this wavelength, (McMahon 2007). Beer–Lambert Law is used for quantification.

Samples

Read out (chart)

Figure 2: The HPLC chromatograph sketch diagram

1.3 1.3.1

Preservatives Sodium benzoate and Benzoic acid

Benzoic acid also known as: Dracylic acid, Benzene formic acid, 65-85-0, benzene carboxylic acid, phenyl formic acid, Carboxybenzene, Benzenemethanoic acid is a white, crystalline organic compound belonging to the family of carboxylic acids, widely used as a food preservative and in the manufacture of various cosmetics, dyes, plastics, and insect repellents.

Figure 3: Molecular structure of benzoic acid It is soluble in ethanol and very slightly soluble in benzene and acetone 5

Sodium benzoate C7 H5 O2 Na is sodium salt of benzoic acid with molecular weight is 144.11g and a melting point above 300 °C. In its refined form it is a white, odorless or nearly odorless solid with a sweetish, astringent taste together with its aqueous solutions. It is very soluble in water (550–630 g/L at 20 °C) and is hygroscopic at a relative humidity above 50%. Its pH is about 7.5 at a concentration of 10 g/Lwater. It is soluble in ethanol, methanol, and ethylene glycol, mixtures with specific ratios of water and ethyl alcohol and is insoluble in ethyl ether. Dry sodium benzoate is electrically charged by friction and forms an explosive mixture when its dust is dispersed in air (Maki et al, 1985). SB can be produced naturally in various fruits like prunes, cinnamon, and cloves (Baldwin et al, 1995). It is also formed by most vertebrates during metabolism. Although undissociated benzoic acid is the more effective antimicrobial agent for preservation purposes, SB is used preferably, as it is about 200 times more soluble than benzoic acid (CFR, 1999). Abo ut 0.1% is usually sufficient to preserve a product that has been properly prepared and adjusted to pH 4.5 or below. A disadvantage is the off- flavor they may impart to foods (Chipley, 1983)

Figure 4: Molecular structure of sodium benzoate 1.3.1.1 Preparation Sodium benzoate is produced by the neutralization of benzoic acid with sodium hydroxide or by adding benzoic acid to a hot concentrated solution of sodium carbonate until effervescence ceases. The solution is then evaporated, cooled and allowed to crystallize or evaporate to dryness, and then granulated. Other methods can also be used for industrial production In all cases, the benzoic acid is further refined to produce sodium benzoate e.g. by dissolving the acid in a sodium hydroxide solution in a chemical reaction yielding Sodium Benzoate plus water. The crystals are isolated by evaporating off the water. (Srour, 1998)

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1.3.1.2 Uses SB can be used for food preservation, treating urea cycle disorders, [Bathsaw et al, 1981] and also as a fuel in fireworks in the whistle mixture, a powder that emits a whistling noise when compressed into a tube and ignited, (Scholz et al, 1991), (Srour, 1998). 1.3.1.3 Health effects Exposure to an overdose of both SB and benzoic acid has been shown to cause urticaria, asthma, rhinitis, or anaphylactic shock through oral, dermal, or inhalation of the said chemicals, (Maibach et al, 1975), (Clemmensen et al, 1982) and (Batshaw et al, 1981). Atopic individuals also demonstrated reactions to oral and dermal challenge with benzoic acid or Sodium Benzoate (Srour, 1989). Sodium Benzoate can trigger allergic reactions e.g. hyperactivity in children with attentiondeficit hyperactivity disorder. It forms little amounts of benzene in presence of ascorbic acid (Gagliardi et al, 1997) Benzene is a chemical that has been linked to increased risk of leukemia and other blood cancers (ATSDR, 2007). However, sodium benzoate has been GRAS (Generally Recognized as Safe), (Jones, 1992). The maximum allowed intake per day by the WHO is 5mg/kg body weight. (FAO/WHO, 1999)

1.3.2

Sorbic acid and potassium sorbate

Sorbic acid, or 2, 4-hexadienoic acid, is a natural organic compound used as a food preservative with the chemical formula C 6 H8 O2 . It is a colourless solid that is slightly soluble in water and sublimes readily. It was first isolated from the unripe berries of the rowan tree (Sorbus aucuparia), hence its name. It is a straight chain unsaturated fatty acid with a molecular weight of 112.13g. Sorbic acid is commercially produced as a powder or granules; it has a characteristic acrid odor and acid taste. The carboxyl (~COOH) group in sorbic acid is very reactive and can form salts with

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calcium, sodium, and potassium. The potassium salt of sorbic acid is commercially available as a powder or granules. Its molecular weight is 150.22 and it is very soluble in water.

Figure 5: Molecular structure of sorbic acid Potassium Sorbate also known as 2, 4-Hexadienoic acid potassium salt, Trans 2, 4-hexadienoic acid with molecular formula: C 6 H7 KO2 and molecular Weight: 150.21. It is one kind of nonsaturated fatty acid compounds, and can be absorbed by human body rapidly, then decomposed into CO 2 and H2 O, moreover no remaining in body (Jones, 1992). It can restrain effectively the activity of mould, yeast and aerophile bacteria. It also restrains growth and reproduction of the pernicious microorganism as pseudomonas, staphylococcus salmonella action. Meanwhile, it cannot restrain useful micro-organism growth as anaerobic spore-bearing bacilli, acidophil therefore lengthen food store period preserve food original flavor. The preservative efficiency of PS is 5-10 times SB (Ferreira et al, 2000). It forms colorless crystals of a white crystalline powder which are freely soluble in water and sparingly soluble in ethanol. It is unstable and if not sealed well, it will be oxidized into colored ones and is hygroscopic when exposed to air for long (Decnop-Weever et al, 1997).

Figure 6: Molecular structure of potassium Sorbate 1.3.2.1 Production Potassium sorbate is produced industrially by neutralizing sorbic acid with potassium hydroxide. The precursor sorbic acid is produced in a two-step process via the condensation of croton aldehyde and ketene.

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1.3.2.2 Antimicrobial Activity The antimicrobial action of sorbic acid is primarily against yeasts and molds. Its action against bacteria appears to be selective. At concentrations used in wine it does not seem to prevent spoilage from either acetic or lactic acid bacteria. Moulds and wine related yeasts inhibited by sorbic acid include species of genera Brettanomyces, Candida, Hansenula, Pichia, Saccharomyces, Torulaspora, and Zygosaccharomyces. (Lueck, Erich. 1980) 1.3.2.3 Uses a. It has been the preferable food preservative because of its excellent preservatives and high safety for various foods like cheese, wine, yogurt, dried meats, apple cider and baked goods among many more. (Han et al, 1998). b. Also known as "wine stabilizer", potassium sorbate produces sorbic acid when added to wine. It serves two purposes. When active fermentation has ceased and the wine is racked for the final time after clearing, potassium sorbate will render any surviving yeast incapable of multiplying. Yeast living at that moment can continue fermenting any residual sugar into CO2 and alcohol, but when they die no new yeast will be present to cause future fermentation. When a wine is sweetened before bottling, potassium sorbate is used to prevent re- fermentation when used in conjunction with potassium metabisulfite. 1.3.2.4 Health effect Potassium sorbate is a skin, eye and respiratory irritant. Although some research implies it has a long term safety record, in vitro studies have shown that it is both geno-toxic and mutagenic to human blood cells. Potassium sorbate is found to be toxic to human DNA in peripheral blood lymphocytes, and hence found that it negatively affects immunity. It is often used with ascorbic acid and iron salts as they increase its effectiveness but this tends to form mutagenic compounds that damage DNA molecules, (Melnick et al., 1954b).

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1.3.3

Sodium Metabis ulphite

Figure 7: Molecular structure of sodium metabisulphite Sodium metabisulphite (SMBS) is an inorganic compound of chemical formula Na 2 S2O 5 . The substance is sometimes referred to as disodium (metabisulfite). SMBS is a white to slightly yellowish crystalline powder with sulfur dioxide odor; readily soluble in water. The anion is a hybrid of dithionite (S2 O 4 2-) and dithionate (S2 O62-). The anion consists of an SO 2 group linked to an SO 3 group, with the negative charge more localized on the SO 3 end. (Carter et al, 2004) 7 1.3.3.1 Chemical properties When mixed with water, SMBS releases sulfur dioxide (SO 2 ), a pungent, unpleasant smelling gas that can also cause breathing difficulties in some people. For this reason, SMBS has fallen from common use in recent times, with agents such as hydrogen peroxide becoming more popular for effective and odorless sterilization of equipment (Smith, 1985). Released sulfur dioxide however makes the water a strong reducing agent. SMBS releases sulfur dioxide in contact with strong acids: Na2 S2 O5 + 2 HCl → 2 NaCl + H2 O + 2 SO 2 On heating to high temperature, it releases sulfur dioxide, leaving sodium oxide behind, (Carter et al, 2004). Na2 S2 O5 → Na2 O + 2 SO 2 1.3.3.2 Uses a) It is used as a disinfectant, antioxidant, a preservative (Allen, 1985) and as a sanitization and cleaning agent in home-brewing and winemaking to sanitize equipment as an

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alternative of sulphur dioxide and as a cleaning agent for potable water reverse osmosis membranes in desalination systems. (Smith, 1987). b) In some tablets, such as paracetamol it is used as an excipient and it can be added to a blood smear in a test for sickle cell anemia. The substance causes defunct cells to sickle hence confirming disease (Jamieson et al, 1985). 1.3.3.3

Health effects

It may cause allergic reactions in those who are sensitive to sulfites, including respiratory reactions in asthmatics, anaphylaxis and other allergic reactions in sensitive individuals (Jamieson et al 1985. SMBS and potassium metabisulfite are the primary ingredients in Campden tablets, used for wine and beer making. The acceptable daily intake is up to 0.7 mg per kg of body weight. SMBS has no side effects; it is oxidized in the liver to harmless sulfate and excreted in urine 1.4

Permissible limits of preservatives

The table below contains the allowed limits of individual preservative in the non-carbonated beverages. A concentration that is higher than the permissible limit is termed as contamination and its accumulation in tissues lead to toxification. Table 1: Permissible limits of preservative content Preservative

Permissible limits in ppm

Preservatives code

KEBS

USFDA,WHO, CFR

EU

numbers*

Sodium benzoate

1000

1000

500

E210-E213

Potassium sorbate

1000

1000

500

E200-E203

(WHO, 1993), (CFR, 1999) and Food Standards Agency (2013); www.food.gov.uk *the numbers are used on labels as they take up less space and avoid possible confusion with additives with similar names 11

1.5

Proble m statement

With the technological advancement and industrialization, people have become so busy in their endeavors to accomplish much in a short term. This has led to development of more advanced food preservatives, which will make food to last the longest time possible. The main explanation is that time is limited and any chance we have we better go to the superma rket and do shopping to last us for a month or two. The industrialists also tend to use high concentration of these preservatives, which increase the food shelf life.

1.6

Justification

The use of high concentration of preservatives leads to health complication. There has been increasing cases of cancers, tumors and complicated ailment from the population across the world. This can be attributed to the living standards; and in this case, food preservatives and additives as one cause. Moreover, most of the research work sites a person’s lifestyle which include the food we eat, the perfumes and cosmetics we wear/use, the chemicals we use etc. This has in turn led to deaths and high cost of medication, (CFR, 1993). There is therefore, a need to control the use of these preservatives to the minimum safe levels in chemically preserved food products and ingredients, (DHHS, 1991). This has necessitated the determination of the levels of preservatives in commonly consumed foodstuff.

1.7

Hypothesis

The preservatives in canned food products sold in Kenya do not exceed the permitted amount by Kenya Bureau of Standards (KEBS), USFDA, WHO, CFR and EU.

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Objectives

1.8

1.8.1

Main objective

To determine the amount of food preservatives; (Sodium Benzoate) in canned food products (fruits, tomato sauce and ketchup) sampled in Kenyan market. 1.8.2

Specific objective

a) To sample various canned food products from various Kenyan markets. b) To quantify the amount of Sodium Benzoate in canned food products sold in Kenya. c) To compare the preservative level with stipulated maximum allowed limits, both by the World Health Organization (WHO) and the Kenya Bureau of Standards.

13

CHAPTER TWO

2

MATERIALS AND METHODS

2.1

Reagents and miscellaneous equipments

All the standards that were used were of analytical grade and they included the following: HPLCgrade acetonitrile (ACN), Potassium dihydrogen orthophosphate, concentrated orthophosphoric acid, Diethyl ether, Ammonium hydroxide, Hydrochloric acid, Saturated NaCl solution, Analytical grade Sodium Benzoate and 0.45 µm nylon filter obtained from Sigma-Aldrich outlets in Kenya, Kobian Kenya Limited. Other lab-ware In order to achieve the set objectives, the following equipments and apparatus were used. i.

Shimadzu 10A-VP HPLC for determination of concentrations

ii.

UV-VIS spectrophotometer for determination of concentrations

iii.

A pH meter, Syringe, sample vials, micro-syringe and micro- filters

iv.

Analytical balance

v.

50ml, 250ml and 1000ml volumetric flasks, measuring cylinder, 10ml and 25ml pipette and beakers

2.2 2.2.1

Samples and Sample collection Sampling process

The samples for the study were obtained from the major Kenyan supermarkets, Uchumi, Tuskeys and Nakumatt supermarkets and major local supermarkets in Juja and Thika area. This is because the four supermarkets quantitatively and qualitatively represented the shopping areas for most of the Kenyan population. Several brands were acquired for the same product from different companies. The following table shows the samples that were obtained for analysis. A total of eleven samples were collected. 14

Table 2: Sample collection areas Sample Name

Sample Brand

Place of Purchase

Area of Purchase

Canned Fruit Cocktail

Rhodes Limited

Nakumatt

Thika

Canned Sliced Pineapples

Delmonte

Uchumi

Juja

Crushed Pineapple

Rhodes Limited

Leens Supermarket

Thika

Ketchup

American Garden

Uchumi

Juja

Ketchup

Heinz

Tuskeys

Thika

Chili Sauce

Peptang

Uchumi

Juja

Chili Sauce

Lyons

Nakumatt

Thika

Chili Sauce

Ken

Pawa

Juja

Tomato Sauce

Ken

Pawa Supermarket

Juja

Tomato Sauce

Peptang

Uchumi

Juja

Tomato Sauce

Zesta

Uchumi

Juja

2.2.2

Sample treatment

The purchased samples were stored in a cool dry place, of temperatures below 4o C awaiting analysis plastic sample containers. 2.2.3 2.2.3.1

Sample preparation HPLC analysis of benzoic acid

Refers to the procedures of preparing or treating the sample and make it ready for analysis. For the fruit products, about 25g of sample was measured, mashed in a blender and put into a beaker and then 50ml of distilled water added and mixed thoroughly. The solution was then filtered and 20ml of the filtrate was centrifuged at 10000 revolutions per minute for 10 minutes.10ml of the 15

supernatant liquid was diluted to 50ml with potassium dihydrogen orthophosphate buffer, microfiltered and then 35µL injected into HPLC, (AOAC, 1990). For tomato sauce and ketchup, the method involved using ultrapure water for extraction. 25.000g of the homogenized sample was weighed to the nearest 1mg and put into a beaker. 50ml of distilled water was added and the flask placed in an ultrasonic bath at 20o C for 20minutes. The solution was then diluted to 50ml mark with potassium dihydrogen orthophosphate buffer. The test solution was filtered through a membrane filter, 0.45µm, before injection, (V. Nour et al, 2009) 2.2.3.2

UV-VIS analysis of benzoic acid

25.000g of the sample was blended and diluted to 50ml with saturated NaCl, then filtered through Whatman 42 filter paper, acidified with 2ml of 0.001M HCl, mixed thoroughly and then transferred to separating funnel. The prepared solution was then extracted with 30ml, 20ml and 10ml of diethyl ether and shaken well to break emulsions. The aqueous phase was then drained and discarded. The combined ether extracts was washed with 20ml, 15ml, and 10ml portions of 0.001M NH4 OH and the ether layer then discarded. Combined NH4 OH extracts was neutralized with 0.0001M HCl with 1ml HCl in excess. The acidified solution was extracted with 30ml, 15ml and 10ml of ether and the absorbance of micro- filtered combined ether extracts determined directly with UV-VIS Spectrophotometer at 272nm with diethyl ether as the reference solution (AOAC, 1990), (Pylypiw et al, 2000), (Gagliardi et al, 1997), (Smith, 1987).

2.3 2.3.1

Preparation of standard solutions HPLC standards

0.100g of sodium benzoate standards was weighed accurately and transferred to 100ml volumetric flask, dissolved in water by shaking and made up to the volume to yield 1000ppm sodium benzoate solution. Serial dilution using the equation C1 V1 =C2 V2 was done using phosphate buffer to top up to the mark to 50ml to yield 10ppm, 20ppm, 40ppm, 60ppm, 80ppm and 100ppm respectively. 16

These working standards were micro filtered and injected into HPLC and run at 230nm. A calibration graph of peak area against concentration was plotted and the regression equation obtained, (AOAC, 1990). 2.3.2 UV-VIS standards 1000ppm stock solution of benzoic acid was prepared by accurately weighing 0.2500g of sodium benzoate and dissolving it in diethyl ether. The solution was then transferred to 250ml volumetric flask and made up to the mark. Serial dilution using the equation C1 V1 =C2 V2 was done using diethyl ether to top up to the mark to 50ml to yield 10ppm, 20ppm, 40ppm, 60ppm, 80ppm and 100ppm respectively. The absorbance of standards were then spectrophotometrically determined at 272nm and then a calibration curve plotted. Regression equation was used to calculate the concentration of the samples, (AOAC, 1990), (Andersen Kenneth et al, 2006).

2.4 i.

Instruments’ conditions for analysis of Benzoic acid Instrument: Shimadzu HPLC

Detection: UV-VIS, 230nm Flow rate: 1ml/min Mobile phase: Acetonitrile: phosphate buffer (0.05M, pH=2.5) in the ratio 30:70 Column: Supelco C18 , 4.6x250mm, 5µm particle size. Injection volume: 30 µl, Column temperature: 25o C Diluent: distilled water and Phosphate buffer ii.

Mobile phase preparation

0.05M Potassium dihydrogen orthophosphate buffer was prepared by dissolving 6.8045g of potassium dihydrogen orthophosphate in 1000ml of distilled water and adjusting its pH with concentrated orthophosphoric acid to 2.5. 300ml of ACN was mixed with 700ml of the buffer filtered and degassed and used as the mobile phase for HPLC analysis.

17

CHAPTER THREE

3 3.1

RESULTS AND DISCUSSION Spectra for some random samples

The spectra for the standards contained many insignificant peaks with areas less than 500. These can be attributed to the instrumental noise and chemical interferences in the measurement of benzoic acid. However, there was a dominant peak at retention time of 9.3minutes. Since the standards were for SB, this was attributed to SB signal. For the samples, there were various peaks observed at different retention times with a dominant peak as 9.3 min, which is the analyte’s signal. The other small peaks can be attributed to the various food additives that are added to chili and tomato sauce, ketchup and even the canned fruit products. Such additives include artificial food colours, sugars, or even natural food coloring as well as flavonoids. The retention time of Sodium Benzoate using the instrumental conditions described earlier was found to be 9.3 minutes for both the samples and the standards. The following are the various chromatograms

Chromatogram 1: Chili sauce, Lyons from Nakumatt 18

Chromatogram 2: Chili sauce ken Pawa

Chromatogram 3: Chili sauce Peptang from Uchumi

19

Chromatogram 4: Tomato sauce Zesta from Uchumi

Chromatogram 5: Tomato sauce Ken from Pawa

20

Chromatogram 6; crushed pineapple Rhodes Co. Ltd from Leens

Chromatogram 7: fruit cocktail Rhodes Co. Ltd

Chromatogram 8: ketchup Heinz from Happy Supermarket 21

Chromatogram 9: Ketchup American Garden from Uchumi

Chromatogram 10: sliced pineapples Delmonte from Tuskeys

22

Chromatogram 11: 10ppm SB

Chromatogram 12: 40ppm SB

Chromatogram 13: 80ppm SB 23

3.2

Concentration calculation

3.2.1 HPLC When the standards were run in the HPLC columns, various readings were obtained including the peak heights and peak areas. Peak areas were used to do the calibration since they completely represent the amount of analyte in the sample, especially where chro matograms suffered broadening. Peak area for the working standards were used for the calibration graph and the following data was obtained. Table 3: the standards peak area information from HPLC analysis Standards concentration for SB

peak area

00

0.0000

10

927410

20

1684259

40

3107954

60

4776492

80

6104680

100

7558894

A calibration curve was drawn from the data obtained in the above table, (peak area against concentrations of the samples). From this graph, a linear curve was obtained with linear correlation coefficient of R 2 =0.998. This showed that there was a strong positive relationship between the peak areas of the standards and the samples with their concentrations, which was taken advantage of to calculate unknown concentration of SB in the samples analyzed. The following is a plot of the said peak areas against concentrations of the standards.

24

standard curve for SB determination by HPLC 9000000

y = 75041x + 12813 R² = 0.998

8000000 7000000

Peak areas

6000000 5000000 4000000 3000000 2000000 1000000 0

0

20

40

60

80

100

120

Concentrations ppm Graph 1: standard curve of the HPLC working standard peak areas against concentrations Using the drawn graph, the concentrations of the samples were calculated as follows A

Calibration equation → 75041x + 12813

then,

X X

75041x 12813 (A 12813 ) 75041 conc(ppm)

Using the above equation, the concentration of the samples were calculated and tabulated. The following

are

the

sample

peak

information

25

from

the

HPLC

analysis

method.

Table 4: sample concentration calculated from the regression equation samples

peak areas

concentration

CS-L/N

6408702.0000

852.3193

CS-K/P

6435827.0000

855.9340

CS-P/U

4808597.0000

639.0885

TS-P/U

7511327.0000

999.2556

TS-K/P

6869155.0000

913.6795

TS-Z/U

4608023.0000

612.3599

KT-H/Ha

427336.0000

55.2395

KT-AG/U

18891.0000

0.8100

Fr-Rh/N

3979.0000

-1.1772

Pn-Del/T

110493.0000

13.0169

Pn-Rh/L

329.0000

-1.6636

3.2.2 UV-Visible Absorption of analytes by UV-Visible spectroscopy is governed by presence of colour or chromophores in the analyte. In a similar way, Benzoic acid has a conjugated system of pielectrons; hence it was possible to utilize this method to study its concentration. Beers’ law came handy in this study. The prepared standards were run in the machine with blank as diethyl ether, which was the solvent used for extraction. The absorbance values for benzoic acid were measure in triplets and alongside are the absorption values for the working standards.

26

Table 5: Absorption values of the working standards for UV-Visible analysis

concentration

Average absorption values (Abs)

0

0

10

0.574

20

1.241

40

2.209

60

3.483

80

4.209

100

5.684

Similarly, a calibration graph was drawn using the data obtained above as follows

standard curve for SB determination by UV-Visible spectroscopy 6

y = 0.055x + 0.039 R² = 0.995

Absorption (Abs)

5 4 3 2 1 0 0

20

40

60

80

100

120

concentration (ppm)

Graph 2: Standard curve of the UV-Visible working standard absorbance against concentrations 27

Here a linear curve was obtained with linear correlation coefficient of R2 =0.995. This showed that there was a strong positive relationship between the absorption of the standards and the samples with their concentrations, which was taken advantage of to calculate the unknown concentration of SB in the samples analyzed as it is the case with HPLC. Again, using the drawn graph, the concentrations of the samples can be calculated as follows A

Calibration equation → 0.055X + 0.039

X

then,

X

0.055 x 0.039 ( A 0.039 ) 0.055 conc( ppm)

Using the above equation, the concentration of the samples were calculated and tabulated as shown in the following table Table 6: Sample concentration calculated from the regression equation Sample

Read1

Read1 Read1

average

Conc.1

Conc.2

Conc.3

Average

CS-L/N

4.9079

4.9196

4.9153

4.9143

863.4588

865.5172

864.7607

864.5789

CS-K/P

5.0784

5.1232

5.0283

5.0766

893.4553

901.3371

884.6411

893.1445

CS-P/U

3.6651

3.5945

3.6784

3.6460

644.8100

632.3892

647.1499

641.4497

TS-P/U

6.4930

5.6946

5.6855

5.9577

1142.3293

1001.8649

1000.2639

1048.1527

TS-K/P

5.3506

5.3029

5.3207

5.3247

941.3441

932.9521

936.0837

936.7933

TS-Z/U

3.5134

3.5109

3.5316

3.5186

618.1210

617.6812

621.3230

619.0418

KT-H/Ha

0.3567

0.2677

0.2730

0.2991

62.7551

47.0971

48.0296

52.6273

KT-AG/U

0.0008

0.0052

0.0002

0.0021

0.1407

0.9148

0.0352

0.3636

Fr-Rh/N

0.0076

0.0056

0.0139

0.0090

1.3371

0.9852

2.4455

1.5893

Pn-Del/T

0.0655

0.0058

0.0715

0.0476

11.5236

1.0204

12.5792

8.3744

Pn-Rh/L

0.0198

0.0121

0.0127

0.0149

3.4835

2.1288

2.2343

2.6155

28

There are variations in the above calculated results when they were compared to the HPLC method results. This is strongly attributed to the crude sample preparation techniques involving solvent extraction and use of volatile solvent, diethyl ether in UV-VIS method. However, the trend of SB concentrations in the sample is followed both in HPLC and UV-Visible spectroscopy. 3.3

Comparison of the methods

The two methods used were compared and checked if there was a significance difference between them. The following were the results. A paired T-test was done and at 95% confidence level, the Tcal was found to be less than the Tcrit ; i.e. Tcal< Tcrit. This statistically implies that the two methods are not significantly different at 95% confidence level. This was simply because the two methods are established as the acceptable analysis method for SB preservative in food products. The table below shows how the conclusion was arrived at. Table 7: Concentration readings from the two methods of analysis used compared by paired T-test

samples

HPLC method

UV-VIS method

×d

×d-µ

(×d-µ)2

CS-L/N CS-K/P CS-P/U TS-P/U TS-K/P TS-Z/U KT-H/Ha KT-AG/U Fr-Rh/N Pn-Del/T Pn-Rh/L

852.319 855.934 639.089 999.256 913.680 612.360 55.240 0.810 0.000 13.017 0.000

864.579 893.145 641.450 1048.153 936.793 619.042 52.627 0.364 1.589 8.374 2.616

-12.260 -37.211 -2.361 -48.897 -23.114 -6.682 2.612 0.446 -1.589 4.643 -2.616

-0.712 -25.663 9.187 -37.349 -11.566 4.866 14.160 11.994 9.959 16.190 8.932

0.506 658.565 84.397 1394.957 133.768 23.679 200.511 143.865 99.175 262.132 79.789

µ= 11.54798182 S.D=308.1343952

Σ(×d-µ)2=3081.343952 Tcal (1.3036)