A RESEARCH NOTE

EFFECT OF COMMERCIAL PROCESSING STAGES OF

APPLE JUICE ON PATULIN, FUMARIC ACID AND
HYDROXYMETHYLFURFURAL 0 LEVELS
CETM KADAKAL'v3, NAS SEBAHATTIIV and ENDER SINAN POYRAZOGLU'

'Ankara University
Faculty of Agriculture
Department of Food Engineering
06110 Di&apr-AnRara, Turkey

2Pamukkale University
College of Engineering
Department of Food Engineering
20020 ~aml&Denizli,Turkey

Accepted for Publication May 15,2001

ABSTRACT

The effects of different stages of commercial apple juice production on the

patulin, fumaric acid and hydroxymethylfurfural (HMF) contents of apple juice
were investigated. Heat treatment and activated charcoal were effective for the
reduction of patulin. The average reduction ofpatulin were 13.4% and 22.9%
for heat treatment and activated charcoal, respectively. Statistical analysis
showed no significant differences (PcO.05) in the presence of furnark acid
between different treatments. Filtration and heat treatment caused an apparent
increase in the HMF content, while activated charcoal caused a decrease. The
average increments of HMF were 40.4% and 38.0% for separation and heat
treatment, respectively, while the average reduction of HMF was 23.7% for
activated charcoal. Spiking of samples with known amounts of patulin and
fumaric acid revealed respective recovery rates of 96.4% and 95.9%.

INTRODUCTION

Patulin, fumaric acid and hydroxymethylfurfural (HMF) are important

quality criteria in fruitjuices. Patulin (Chydroxy-4 H-furo (3,2 c)-pyran-2-(6H)
one], a mycotoxin produced by several species of Aspergillus. Penicillium and
ByssochZamys fungi, is often a contaminate in apples. Studies with fungi or

Correspondent author. TEL: 0312 3170550; E-mail:ckadakal@hotmail.com

Journal of Food Quality 25 (2002) 359-368. All Rights Reserved.

"Copyright 2002 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 359
360 C. KADAKAL, N. SEBAHATTIN and E.S. POYRAZOGLU

animals have demonstrated that patulin is mutagenic, carcinogenic and

teratogenic. Consequently, patulin is an important quality parameter in apple and
apple products for human health (Lai ef al. 2000). The World Health Organiza-
tion (WHO) and many countries such as Sweden, Belgium, Russia and Norway
have established 50 pg/L patulin as the upper allowed concentration in apple
juice (AJ) (Prieta ef al. 1992).
Patulin cannot be fully inactivated in AJ and apple juice concentrate (AJC),
with heat treatment and storage because of the stability of patulin in acidic
media. Lovett and Peeler (1973) demonstrated patulin to be resistant to thermal
destruction at a pH range of 3.5-5.5 when heated up to 125C. On the other hand
Wheeler ef al. (1987) have reported that high temperature-short time (HTST)
treatments (60, 80 and 9OC for 10 s) and batch pasteurization (9OC for 10 min)
significantly reduced the patulin level in pasteurized apple cider, but did not
completely destroy the patulin. Acar ef al. (1998) have reported 39% and 25%
average patulin reduction for conventional clarification and filtration, and
ultrafiltration, respectively.
Fumaric acid is one other quality criteria of AJ for the determination of
natural juice content and adulteration in AJ (Coppola and Stan 1986). The
presence of D-malic acid indicates adulteration due to DL-malic acid because D-
malic acid does not occur naturally. L-malic acid can be produced biotechnologi-
cally/enzymatically by adding water to fumaric acid in the presence of ftunarase
enzyme (Karadeniz and Ekgi 1997). Since natural L-malic acid is much more
expensive than synthetic malic acid, synthetic malic acid is used as an acidifier
in foods (Zyren and Elkins 1985). Fumaric acid is formed as a by-product in the
production of synthetic L-malic acid and the presence of high fumaric acid in AJ
( > 3 mg/L) indicates the addition of synthetic malic acid (Junge and Spadingen
1982). On the other hand, fumaric acid can be formed due to extreme
conditions, depectinization and evaporation in processing (Artik ef al. 2001).
Moreover, the importance of fumaric acid in AJ is not only due to the addition
of malic acid and to the production of fumaric acid in processing and storage,
but is also due to microbiological degradation of apples by Rhizopus sfolonifer
(Karadeniz and Ekgi 1997).
The presence of HMF is considered as an indication of quality deteriora-
tion. It is formed as a result of dehydration of ketopentoses, particularly in
acidic or high-temperature environments and a high level of HMF suggests that
thermal exposure has been excessive (When and Acar 1999). The Internation-
al Federation of Fruit Juice Processors (IFFJP) recommends a maximum
concentration of 5 mg/L HMF in fruit juices (IFFJP 1974).
The aim of this research was to determine the effect of different stages of
the AJ processing (enzymatic mash treatment, pressing, heat treatment,
depectinization and activated charcoal) on patulin, fumaric acid and HMF levels
of AJ.
 REDUCING PATULIN CONTENT OF APPLE JUICE 361

MATERIALS AND METHODS

Materials
The material used in this study was apple juice from a commercial factory
(KONFRUT) Denizliflurkey. Ten lots of apple were obtained from local
growers (IspWTurkey) as commercial raw material and processed into apple
juice lot by lot. Each lot was approximately eight tons and included the mixed
varieties of Starking, Golden delicious and Amasya apples. In each lot, the
proportions of the Starking, Golden delicious and Amasya apples were
approximately equal. The dust activated charcoal, used in this study, was
provided from Erbliish Inc. (GeisenheimKermany). The dust activated charcoal
had a pH of 5.02, 88.43% moisture, 4.12% ash (dry matter), 1.98% water-
soluble matter, and 1600 m2/g total surface area.
Samples were taken for analysis of patulin, fumaric acid, HMF, pH and
brix contents from processing stages of the flow diagram shown in Fig. 1 and
were f r o m at -18C until analysis. Samples, obtained from the beginning of
enzymatic mash treatment and end of enzymatic mash treatment, were pressed
in a cloth bag and then analyzed. The other samples were directly analyzed. All
samples were diluted to 11.2 "Bx at the time of analysis.

Production of Apple Juice

Flow diagram of the production of AJ is given in Fig. 1. Sampling stages
and codes are also shown in Fig. 1. This production procedure, given in Fig.
1, is the method generally used in Turkey.

Methods
The determination of patulin in the samples was carried out by using a high
pressure liquid chromatography (HPLC) apparatus (Shimadzu), as suggested by
the International Standard Organisation (IS0 1993). Patulin in the crystalline
form was obtained from Sigma Chemical Co., St. Louis, MO. Patulin was
extracted from samples of the AJ with three times the equivalent volume of ethyl
acetate. The mobile phase employed was a 10% aqueous acetonitrile solution
with a flow rate of 1.0 mL/min. For the analysis, a C-18 (250X4.6 mm)
column, a column oven (Shimadzu, Model CTO-IOASVP), a photodiode array
detector (Shimadzu, Model SPD-M1OAVP-UV-VIS), a degasser (Shimadzu,
Model DGU 14A),a liquidchromatography pump (Shimadzu, Model LC-1OAT-
VP), and a Hewlett Packard 3396 Series I1 integrator were used.
The HPLC system, used in the fumaric acid analysis, was the same as for
the patulin analysis except for the column. An ODS 2 4.6X250 Cmm <column
was used in the fumaric acid analysis (Hunter er al. 1991). Standard fumaric
362 C.KADAKAL, N. SEBAHATTIN and E.S. POYRAZOGLU

1
Washing and Smashing

Mashing
L min)
(60+10
A1

c-.m
Pressing

&--
Separation

&A4
Heat treatment (90 C for 10 sec)

b
Cooling (until 45-50 C)
AS

Depectinization and Clarification

(Pectinex, arnilaz, gelatine and bentonite)

$-p A6
Activated Charcoal ( 2 @I)
L A 7
Filtration*

High temperature Short Time (HTST)* ( 1 10 C for 7-10 sec)

1
Filling into bottle

4
Sealing

Apple juice
A l . AZ, A3. A4. AS, M, A7; Sampling stagcs
*.Tlic clTcct of filtration and HTST werc not dctcmiined

FIG.1. FLOW DIAGRAM OF THE PRODUCTION OF APPLE JUICE

 REDUCING PATULIN CONTENT OF APPLE JUICE 363

acid was obtained from the Sigma Chemical Co. The AJ samples were vacuum-
filtered through 0.45 pm membranes (Millipore Co.). A 5 mL aliquot was
pipetted onto the Bio-Rex 5 resin bed and allowed to run through freely. The
organic acids were desorbed from the resin with 5 mL 10% (v/v) H,S04,
followed by addition of deionized water to a final volume of 25 mL. The mobile
phase employed was phosphate buffer (0.02 M KH,P04) at a flow rate of 1.O
mL/min.

Recovery of Patulin and Fumaric Acid

Apple juice samples containing known amounts of patulin and fumaric acid
were spiked with different levels of patulin (25, 50, 75, 100, 200 ppb) and
fumaric acid (0.2, 0.4, 0.6, 0.8, 1.0 ppm) respectively, to determine the
recovery of the extraction procedure. The average percentage recoveries of
patulin and fumaric acid were found to be 96.4% and 95.996, respectively, for
added levels. The levels of patulin and fumaric acid in AJ samples were
corrected for the average percent recoveries of 96.4% and 95.9%, respectively.
The analysis of hydroxymethylfurfural(HMF) (Sigma, H 9877) was carried
out by the spectrophotometric method at 550 nm described by the IFFJP (IFFJP
1964). Barbituric acid (Sigma, B 0625) and p-toluidine (Sigma, T 8766) were
used in the HMF analysis.
Moisture were detamined by drying to a fixed weight, ash amount was
determined by burning in a furnace (Electrornag, Model 3, temperature
adjustable, 1000-12OOC) at 500 f 25C, water soluble matter (brix) was
determined by using a digital refractometer (RFM Model 340) and pH was
measured with a pH meter (WTW,Model 537) (AOAC 1980). Total surface
area (m2/g)of activated charcoal was provided by the manufacturing company.

Statistical Analysis
Statistical analysis of the data was performed using the SAS system (SAS
1985). When analysis of variance (ANOVA) revealed a significant effect (P <
0.05, P <0.01), data means were compared with the least significant difference
(LSD)test.

RESULTS AND DISCUSSION

Changing of patulin values of the samples obtained from different stages

of AJ processing are shown in Fig. 2. Mean patulin, fumaric acid and HMF
amounts of the samples in different stages of apple juice processing are given
in Table 1. Statistical analysis of the data showed that there were significant
differences (P<0.05)in the patulin amounts of the AJ samples between the test
364 C. KADAKAL, N. SEBAHATTIN and E.S. POYRAZOGLU

TABLE 1.
MEAN VALUES OF PATULIN (ppb), FUMAFUC ACID (ppm) AND HMF (ppm) IN
DIFFERENT STAGES OF APPLE JUICE PROCESSING
Lot Numbef Patulin Fumaric Acid HMF
1 116.6 b* 0.80 d 0.80 a
2 69.8 cd 0.76 e 0.67 a
3 81.2 c 0.79 f 0.52 a
4 51.0 e 0.82 b 0.30 b
5 56.8 e 0.57 f 0.58 ab
6 145.7 a 0.98 a 0.29 b
7 64.4 de 0.96 a 0.78 a
8 113.2 b 0.78 c 1.10 a
9 122.0 b 0.69 g 0.4 a
10 52.7 e 0.97 a 0.6 a
Processing Stages
A1 95.0 a* 0.87 **
A2 93.7 a 0.85 Y **
A3 95.8 a 0.85 0.34 d
A4 96.5 a 0.87 Y 0.57 c
A5 83.6 ab 0.82 Y 0.92 a
A6 82.9 ab 0.74 Y 0.93 a
A7 63.9 b 0.68 0.71 b
Average 87.3 0.81 0.70

* Means with the same letter are not significantly different according to least significant
difference test (lW0.05).The data for the mean of two experiments with two replicates.
** Not determined
I
Patulin, fumaric acid and HMF values are the average of the processing stages evaluated for
each lot
Y Not significant
Al; Beginning of enzymatic mash treatment
End of enzymatic mash treatment
A3; After press
A4; Before heat treatment
A5; After heat treatment
A6; After depectinization
A7 ; Before Ntration
 REDUCING PATULIN CONTENT OF APPLE JUICE 365

treatments (Al-A7). Patulin amounts of all A1 coded samples were over 50 ppb.
WHO has established 50 ppb patulin concentration as the recommended
maximum concentration in kl. In general, the amounts of patulin did not change
in the stages of enzymatic mash treatment (60 f 10 min), pressing and
depectinization-clarification.When the patulin levels of A4 coded samples were
compared with the A5 coded samples, the decrement ranged between 0.66-
14.59%.A 14.59%decrement indicates that heat treatment in plant conditions
is also effective in reducing the patulin content. Decrement ranges between
20.80-47.4696 and 26.39-47.88%were determined in A7 coded samples, when
compared with the A1 and A5 coded samples, respectively, which was probably
due to the adsorption of patulin on the activated charcoal. But, the patulin
content was never reduced to the legal limit (50 ppb), suggested by the ISO,
even after all of the treatments were combined. Leggott ef al. (2000) have
reported a significant decrease in mean patulin concentration from 110 f 35
ng/mL to 75 f 18 ng/mL during the combined depectinization/chaal/ultra-
filtration stage. Past research has shown a 65.2%decrease in patulin content of
AJ with 5 min a treatment at a dust activated charcoal level of 3 g/L (Kadakal
and Nas 2000). The effect of filtration and high temperature short time (HTST)
were not determined in this study.

FIG. 2. CHANGING OF MEAN VALUES OF PATULIN (ppb), FUMARIC ACID (ppm) AND
HMF @pm) IN DIFFERENT STAGES OF APPLE JUICE PROCESSING
366 C. KADAKAL, N. SEBAHATTIN and E.S. WYRAZOGLU

Statistical analysis showed no significantdifferences (PC0.05) between the

results of fumaric acid by different treatments ( A l , A2, A3, A4, A5, A6, A7).
On the other hand, statistical analysis showed significant differences (PC0.05)
between the results for patulin. Analysis of variance for patulin, fumaric acid
and HMF on the AJ samples obtained from different stages of AJ processing are
given in Table 2.

TABLE 2.
ANALYSIS OF VARIANCE FOR PATULIN, FUMARIC ACID AND HMF ON THE APPLE
JUICE SAMPLES OBTAINED FROM DIFFERENT STAGES OF APPLE JUICE
PROCESSING
Patulin HMF Fumaric Acid
Source of
Variation

DF Fvalue Pvalue MSE DF F P MSE DF Fvalue P MSE

value value value

Sample(A) 9 60.7 <0.05 268.2 9 2.8 <0.05 0.08 9 244.1 c0.05 0.01

Treatments 6 2.3 <0.05 1236.8 4 27.7 <0.05 0.04 6 0.6 NS 0.18

(B)

AXB 69 25847.6 C0.05 0.10 49 4.4 C0.05 0.03 69 1691.9 <O.M 0.00

MSE: Mean square error

NS: Not significant
DF: Degree of freedom

At the initial analysis, HMF values of the A1 and A2 coded samples were
found 0 or near 0. So, the amount of HMF of the samples were not determined
in these stages. Statistical analysis showed significant differences ( P C 0.05)
between the results for HMF by treatments Al-A7). HMF values of the A5
coded samples were found higher than the A4 coded samples. However, HMF
values for all A7 coded samples decreased. Decrements ranging between 30.25-
5 1.1 1 % were determined for A7 coded samples when compared with the A4
coded samples. International Federation of Fruit Juice Producers has permitted
5 ppm HMF level for AJ (IFFJP 1974). The determined HMF values of all
samples were under 5 ppm. Our results indicate that treatment by heat processes
under plant conditions increases the HMF value considerably. Commercial
process stages of AJ did not cause significant changes in pH (from 3.91 to 4.0)
and brix values (from 13.8 to 14.6).
 REDUCING PATULIN CONTENT OF APPLE JUICE 367

CONCLUSIONS

When compared with other stages of AJ processing, activated charcoal is

more effective in reducing patulin level. It is clear from the results that patulin
at approximately 100 ppb could not be reduced under 50 ppb by the processing
methods evaluated. Apple juice, having approximately 100 ppb initial patulin
content, is not safety for consumption. For that reason, careful selection of raw
material is critical. Enzymatic mash treatment, pressing and depectinization-
clarification stages of AJ production had no significant effect on patulin
concentration. Our results affirmed that conventional processing steps have no
significant effect on the fumaric acid content of juice during commercial AJ
production. Only activated charcoal was effective in decreasing patulin. Heat
treatment under plant conditions increases the HMF content considerably.

ACKNOWLEDGMENT

We thank Hasan Gurgen (Director of KONFRUT Apple Juice Plant) for

his contribution to the fmancial support of this work.

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