Faculty of Applied Sciences

B.Sc.(Hons) Food Science and Nutrition

Wong Vern Mein

1000821184
Supervisor: Ms. Pui
 • The types of drink made from fruit can be
separated into:
– Ready-to-drink
– Concentrate

• Preservation

(Murano 2003; Practical Action 2006)

Types Description

Fruit Juice Pure expressed juice

Fruit Nectar Sugar, water and sweetener added, 25 to 50%

juice, pulpy or clear
Fruit Squash, Fruit Normally contain at least 25% w/v of the juice
Cordial, Fruit Syrup mixed with syrup
Fruit Drink Contains at least 5% w/v of fruit juice

Fruit Juice Drink Contains at least 35% w/v fruit juice, water and
glucose or high fructose glucose syrup are added

(FAO Corporate Document Repository 2001; Legal Research Board

2010; Practical Action 2006)
 How to prolong the shelf-life?
• Preservative
– Any substance that, when added to food, is
capable of inhibiting, retarding, or arresting
the process of decomposition, fermentation,
or acidification of such food but shall not
include herb, spice, vinegar or wood smoke.
(Food Regulations 1985)

(Legal Research Board 2010)

 Benzoic acid (E200)
• White crystalline solid (scales or
needles)
• Salt: sodium (E 211)

Sorbic acid (E200)

• Colourless needles or white free flowing
powder
• Salt: potassium (E 202)

(Pylypiw Jr. and Grether 2000; Mota et al. 2003; Dong and Wang 2006;
FAO 2010; Food Standards Agency 2010)
• Benzoic acid and sodium benzoate
– Preserve freshness
– Inhibit yeasts, molds, and bacterial growth.

• Sorbic acid and potassium sorbate

– Inhibit yeasts, molds, and bacterial growth.

(Dong and Wang 2006; FAO 2010; Food Standards Agency 2010; Mota et
al. 2003; Pylypiw Jr. and Grether 2000)
 FAO/WHO Food Standards
Food Regulations 1985
Preservative
Preservative (Max permitted proportion
in mg/kg)
(Max permitted proportion in
mg/kg) Food
Food C7H6O2 (or C6H8O2 (or
C7H6O2 (or C6H8O2 (or benzoates) sorbates)
benzoates sorbates as
as C7H6O2) C 6 H 8 O2 ) Fruit Juice,& Fruit
Nectar
Fruit-based (Concentrated or 1000 1000
Beverages 800 800 Direct
(Concentrated) Consumption)
Fruit cordial, syrup,
Fruit-based
squash, drink, fruit
Beverages
350 350 juice
(Direct 600 -
drink(Concentrated
Consumption)
or Direct
Consumption)
(Legal Research Board 2010) (FAO and WHO 2010)
 Benzoic acid
•May cause
– Slightly irritating to the skin and irritating to the eyes.
– Pseudoallergy
– The yield of benzene

Sorbic Acid
•May cause:
– severe damage to the tissues of the eyes, skin and
respiratory tract
– Hydroxy free radicals and superoxide anions formation

(Adams 1997; Gardner and Lawrence 1993; WHO 2000; Yang 1984)
Types of fruit- Preservatives Method References
based beverages

Concentrated orange Benzoic acid, GC Dong and Wang

juice and concentrated sorbic acid 2006
plum syrup

Orange juice, apple Benzoic acid, HPLC Mota et.al 2003

juice, pineapple juice sorbic acid

Apple juice, grape Sodium benzoate, HPLC Pylypiw and

juice, cranberry juice potassium sorbate Grether 2000
Types of fruit- Preservatives Method References
based beverages

Orange juice, apple Sorbic acid Capillary zone Tang and Wu

juice, orange drink electrophoresis 2005
concentrate
Grape juice, guava Benzoate ion Potentiometric Pezza et.al
juice, passionfruit sensor 2001
juice, gooseberry immobilized in a
syrup graphite matrix
Lemon juice Benzoate ion Ion Dionex
Chromatography Corporation
2004
 HPLC
• High-performance liquid chromatography
(HPLC) is a form of liquid chromatography
to separate compounds that are dissolved
in solution.
To determine the levels of benzoic acid and sorbic acid
in fruit-based beverages found in the market.

To determine whether the amount of

benzoic acid and sorbic acid
added by the fruit-based beverages manufacturers
complies with
Food Regulations 1985 (Regulation 20)
And
FAO/WHO Food Standards.
Samples (purchased from local supermarket)
• Fruit juices
• Fruit nectars
• Fruit cordials
• Fruit drinks
• Fruit juice drinks
 Chemicals and Reagents
• Ammonium acetate
• Ultra-pure water
• Acetic acid (99.8%)
• HPLC-grade methanol
• Benzoic acid (99.5%)
• Sorbic acid (99%)

Filter
• 0.45-µm nylon membrane filter
(Saad et. al 2005)
 Standard Preparation
Preparation of Stock Solution

Dissolves with

10 mL methanol
0.1 g of benzoic
acid/sorbic acid

Top up to 100mL
using ultra pure
water
 *Stock Ultra-pure Total Concentration
Standard
solution,1 water (mL) volume (mg/L)
solution
g/L (mL) (mL)
1 0.2 9.8 10 20
2 0.4 9.6 10 40
3 0.6 9.4 10 60
4 0.8 9.2 10 80
5 1.0 9.0 10 100
6 1.2 8.8 10 120

*Stock solution: Benzoic acid / sorbic acid

 Mobile Phase Preparation

Dissolved + acetic acid Adjust

the pH
3.8 g of to 4.4
ammonium
1L of water
acetate
+

Methanol-acetate
buffer (pH 4.4) Produce
(35:65, v/v)
Methanol
(Saad et. al 2005)
 Sample Preparation

Centrifugation for 10
mins at 5000 rpm

Filtered
RTD
Dilute with mobile
phase to 1:10 (v/v)

Concentrated
Dilute with mobile 0.45-µm nylon
phase to 1:100 (v/v) membrane filter
(Mota et al. 2003; Pylypiw Jr. and Grether 2000)
 Chromatographic Conditions
Column: reversed phase C18, 5 µm (150 x 4.6 mm)
Column temperature: Room temperature
Injection volume: 20 µL
Flow rate: 1.4mL/min, isocratic elution
Mobile phase: methanol-acetate buffer pH 4.4 (35:65, v/v)
Detector: UV-vis spectrophotometer at 254 nm

(Saad et. al 2005)

• The levels of benzoic acid and sorbic acid
in fruit-based beverages are determined.
• The levels of benzoic acid and sorbic acid
in some fruit-based beverages are
conformed to the legal prescriptions of
both Food Regulations 1985 and
FAO/WHO Standards.
• Some of these beverages are complied
with FAO/WHO Standards only.
Adams, J.B., 1997. Food additive-additive interactions involving sulphur dioxide and ascorbic and
nitrous acids: a review. Food Chemistry, 59 (3), 401-409.

Canadean Liquid Intelligence, 2008. Beverage Categories Definitions [Online]. Available from:
<http://www.liquidforecasts.com/CanadeanDefinitions.pdf> [Accessed 24 February 2011].

Dong, C.Z., and Wang, W.F., 2006. Headspace solid-phase microextraction applied to the
simultaneous determination of sorbic and benzoic acids in beverages. Analytica Chimica Acta, 562,
23-29.

Dionex Corporation, 2004. Determination of benzoate in liquid food products by reagent-free ion
chromatography.

FAO, 2010. Food Additive Index [Online]. Available from: <http://www.fao.org/ag/agn/jecfa-

additives/index.html> [Accessed 19 February 2011].

FAO Corporate Document Repository, 2001. Defining Juices [Online]. Available from:
<http://www.fao.org/Docrep/005/Y2515e/y2515e03.htm#TopOfPage> [Accessed 23 February 2011].

FAO and WHO, 2010. GSFA Provisions for Food Category 14.1.2.1 (Fruit juice) [Online]. Available
from: <http://www.codexalimentarius.net/gsfaonline/foods/details.html?d-3586470-o=2&id=239&d-
3586470-s=2> [Accessed 19 February 2011].
Food Standards Agency, 2010. Current EU approved additives and their E Numbers [Online].
Available from: <http://www.food.gov.uk/safereating/chemsafe/additivesbranch/enumberlist>
[Accessed 19 February 2011].

Gardner, L.K., and Lawrence, G.D., 1993. Benzene production from decarboxylation of benzoic
acid in the presence of ascorbic acid and a transition-metal catalyst. J. Agric. Food Chem., 41,
693-695.

Legal Research Board, 2010. Food Act 1983 (Act 281) & Regulations. Malaysia: International
Law Book Services, pg. 82-83, 165-167, 241.

Lozano, V.A., Camina, J.M., Boeris, M.S., and Marchevsky, E.J., 2007. Simultaneous
determination of sorbic and benzoic acids in commercial juices using the PLS-2 multivariate
calibration method and validation by high performance liquid chromatography. Talanta,73,
282-286.

Mota, F.J.M., Ferreira, I.M.P.L.V.O., Cunha, S.C., Beatriz, M., and Oliveira, P.P., 2003.
Optimisation of extraction procedures for analysis of benzoic acid and sorbic acids in
foodstuffs. Food Chemistry, 82, 469-473.

Murano, P.S., 2003. Understanding Food Science and Technology. Belmont:

Wadsworth/Thomson Learning, pg. 267.

Nielsen, S.S., 2010. Food Analysis. 4th Ed. New York: Springer, pg. 232-234.
Perth District Health Unit, 2007. Fruit Juice and Fruit Drinks, What is the Difference? [Online].
Available from: <http://www.pdhu.on.ca/pdf/fruit.pdf> [Accessed 23 February 2011].

Pezza, L., Santini, A.O., Pezza, H.R., Melios, C.B., Ferreira, V.J.F., and Nasser, A.L.M., 2001.
Benzoate ion determination in beverages by using a potentiometric sensor immobilized in a
graphite matrix. Analytica Chimica Acta, 433, 281-288.

Practical Action, Schumacher Centre for Technology & Development, 2006. Fruit Juice
Processing [Online]. Available from:
<http://practicalaction.org/practicalanswers/product_info.php?products_id=94> [Accessed 23
February 2011].

Pylypiw Jr., H.M., and Grether, M.T., 2000. Rapid high-performance liquid chromatography
method for the analysis of sodium benzoate and potassium sorbate in foods. Journal of
Chromatography A, 883, 299-304.

Saad, B., Bari, M.F., Saleh, M.I., Ahmad, K., and Mohd. Talib, M.K., 2005. Simultaneous
determination of preservatives (benzoic acid, sorbic acid, methyparaben and propylparaben) in
foodstuffs using high-performance liquid chromatography. Journal of Chromatography A,
1073, 393-397.

Tang, Y.J., and Wu, M.j., 2005. A quick method for the simultaneous determination of ascorbic
acid and sorbic acid in fruit juices by capillary zone electrophoresis. Talanta, 65, 794-798.
WHO, 2000. Concise International Chemical Assessment Document 26- Benzoic Acid and
Sodium Benzoate.
Yang, S.F., 1984. Reactions of oxidation intermediates of sulphite species with some cellular
components of plants. Food Chem., 15, 113-124.