hr.

Du;~J, Journal 5 (1995) 221m246

‘c 1995 Elsevier Science Limited
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Review Paper

Benzoic Acid as a Natural Compound in Cultured Dairy

Products and Cheese

R. Sieber, U. Biitikofer & J.O. Bosset

Federal Dairy Research Institute, CH-3097 Liebefeld-Bern, Switzerland

(Received 24 November 1993; revised version accepted 7 April 1994)

ABSTRACT

This review deals with the ‘state of the art’ of benzoic acid in cultured dairy products
and cheese. During fermentation, benzoic acid is produced from hippuric acid, a
component of milk naturally present ut concentrations of up to 50 mgjkg. In smear-
ripened cheese, however, higher benzoic acid concentrations have often been
measured on the surface or even inside such cheeses. A second metabolic pathway
has been proposed: during ripening, an additional quantity of benzoic acid originates
from phenylalanine degradation, with P-phenyl-propionic (hydrocinnamic) acid and
cinnamic acid as intermediate products. Acetophenone is a by-product of this
breakdown, which essentially occurs in the rind and the smear. The presence of these
intermediate products and their concentration gradient, the concentration of benzoic
acid and its formation during cheese ripening, and the simultaneous production of
ammoniu resulting from deamination support the validity of’ this second metabolic
pathway. A third way could be the auto-oxidation of benzaldehyde, produced by
certain strains of lactic acid bacteria. In addition to the transformation of hippuric
acid, these two sources (phenylalanine degradation, auto-oxidation of benzalde-
hyde) supply benzoic acid in cheese.

INTRODUCTION

Benzoic acid is widely used in the food industry as a preservative in acid

foods, under code number E210, owing to its antimicrobial activity against
various bacteria, yeasts and fungi involved in food poisoning and food spoi-
lage, such as Escherichia coli, Listeria monocytogenes, Aspergillus sp. and

227
228 R. Sieber, U. Btitikgfer, J.O. Basset

Penicillium sp. (Chipley, 1993). Benzoic acid occurs naturally in different foods,
such as fruits, vegetables, spices and nuts (Nagayama et al., 1983, 1986;
Heimhuber & Herrmann, 1990) and also in milk, especially dairy products, at
low concentration.
The objective of this paper is to collect together some of the results obtained by
the authors from previous investigations on benzoic acid in cultured dairy
products and cheese. The results have been supplemented by data from recently
published literature. A review on the occurrence of benzoic acid in various foods
has been published by Sieber et al. (1989).
The intake of benzoic acid differs in different countries. In the Netherlands,
the mean total daily intake of benzoic acid by 16-l&year-old male adolescents
was 34 mg and the maximum was 66 mg (van Dokkum et al., 1982). In
Finland, a comparable consumption of 40 mg benzoic acid per person and per
day was estimated (PenttiE et al., 1988). The daily intake of Japanese people
during the period 1976-8 1 was calculated to be 10.9 mg benzoic acid and
1.Ol mg of esters of p-hydroxybenzoic acid (Toyoda et al., 1983). The FAO/
WHO evaluated acceptable daily intake (ADI) is in the order of 5 mg benzoic
acid/kg body weight.
Benzoic acid is a colourless aromatic compound. Its acidic character
(pK = 4.19 at 25°C) is due to its carboxylic group. Its solubility in water and/or in
organic solvents depends primarily on its degree of protonation, i.e. on the pH of
the medium. Its main chemical and physical properties are summarized in
Table 1.
The first methods for the determination of benzoic acid in processed cheese
were described in the 1930s (Hostettler, 1933). For analytical purposes,
benzoic acid is usually extracted from foods by hot steam distillation after
acidification (stripping) or by liquid/liquid extraction (Brand1 & Binder, 1982).
A preliminary alkaline pre-concentration may be required. The liquid/liquid
extraction technique is more often used because it is more rapid than distilla-
tion. Benzoic acid can be quantified in foods by using different analytical
methods, such as polarography, (spectro)photometry, isotachophoresis, gas
chromatography with different detectors (FID, MSD), or high performance
liquid chromatography (HPLC) with UV detection (Table 2). A provisional

TABLE 1
Some Physical and Chemical Properties of Benzoic Acid*

Other names Phenylformic acid, benzenecarboxylic acid

Formula C6H,COOH
Molecular weight 122.1
In pure form Colourless, white needles, leaflets
Solubility 1.8, 2.7, 3.4, 22 g/L of water at 4”C, 18”C, room
temperature and 75”C, respectively
10-20 g/kg of fatty oils
Soluble in anhydrous ethanol
Melting point 122°C
*From Liick (1980) and Chipley (1993).
 Benzoic acid in cultured dairy products and cheese 229

TABLE 2
Principal Methods for Determination of Preservatives

Product Method Recovery Detection Com- First-name

(Xi limit pound author
(mglk or

Milk HPLC 95.2-98.7 B Wiench (1984)

Milk, yoghurt Polarography B Vogel (19656)
Milk, sour
milk HPLC >98 0.1 B. H Hatanaka (1986)
Milk, dairy
products GC/MS B Drawert (1978)
Liquid food HPLCjUV 955101.5 5 B, S, E Matsunaga (1985)
Yoghurt HPLCjUV 91.2 B, S Arimoto (1983)
Yoghurt HPLCjUV 93.3-96.7 0.2 B, S Ito (1983)
Yoghurt LC 7 1-74 20 B, S Puttemans (1985)
Yoghurt HPLC/UV 5 B, H, S Stijve (1984)
Yoghurt,
cheese HPLC/UV 98.5, 96.3 B, S, E Leuenberger (1979)
Quarg, cheese LC 95 1 R S Ktippers (1988)
Cheese GC S, B Stark (1976)
Cheese GC/MS 96.4 B Kurisaki (1973)
Cheese HPLC l-2 B Toppino (19876,
1990)
Dairy products uv 92175 B, S Raissouni (1991)
Dairy products HPLCjUV 988-100.8 1 B, S, E Gieger (1982)
Dairy products GC/MS 95.2 0.2 B Richardson (1981)
Dairy products HPLCjUV 3 B, S Btitikofer (1988)
Dairy products HPLCjUV 5 B, S Anonymous (1988a)
Food GC B, S Vogel (1965a)
Food LC 9(r-105 20 B, S Bui (1987)
Food (cheese) GC/MS 955102 0.1-0.25 B, S Kakemoto (1992)
Beverage, jam HPLC 988104 <l B, S Hannisdal (1992)
Fatty foods HPLCjUV B, S, E Aitzetmtiller (1984)
Food SP 96-103 B, S Zoneveld (1975)
Water HPLCjUV >95 0.1 B Dietz (1993)

(HP)LC(UV) = (high performance) liquid chromatography (with UV-detection),

GC(MS) = gas chromatography (with mass spectrometric detection), SP = spec-
trophotometry.
B = benzoic acid; H = hippuric acid; S = sorbic acid; E = ester of p-hydroxybenzoic acid.

IDF standard method is available (Anon, 1987; 1988a). A HPLC method has
been developed at the Swiss Dairy Research Institute to determine benzoic
acid alone or together with other preservatives such as p-hydroxybenzoic acid
methylester, -ethylester, -propylester, sorbic and hippuric acid and pimaricin
(Biitikofer et al., 1988).
230 R. Sieber, U. Btitikofer, J.O. Basset

OCCURRENCE OF BENZOIC ACID IN FERMENTED DAIRY

PRODUCTS

Cultured or fermented dairy products such as yoghurt, fruit yoghurt, sour milk
with bitidobacteria and kefir contain up to 50 mg/kg (0.41 mmol/kg) benzoic acid
with most mean values around 20 mg/kg (Tables 3 and 4) but milk contains only
a few mg/kg (Vogel & Deshusses, 19656; Nishimoto et al., 19680, 1969; Chandan
et al., 1977; Drawert & Leupold, 1978; Richardson & Gray, 1981; Sajko et al.,
1984; Hatanaka & Kaneda, 1986; Nagayama et al., 1986). According to Nishi-
moto et ul. (19686, 1969) lactic acid bacteria convert hippuric acid naturally
present in milk (Fleischmann, 1932; Karabinos & Dittiner, 1943; Kieffer et al.,
1964; Svensen, 1974; Marsili et al., 19X1; Wolfschoon-Pombo & Klostermeyer,
1981; Lavanchy & Steiger, 1984; Hatanaka & Kaneda, 1986) to benzoic acid so
that the latter could also be considered as a natural component of milk and milk
products (Fig. 1). Wolfschoon-Pombo & Klostermeyer (198 1) found a maximum
of 62.4 mg/kg (0.35 mmol/kg) of hippuric acid in evening milk, which could

TABLE 3
Benzoic Acid Content of Some Yoghurt and Fruit Yoghurt (mg/kg)

Product N x s A x First-named Author

Yoghurt 4 33.9 2747 Vogel (19653)

Yoghurt plain 1625 Chandan (1977)
Yoghurt 4 21-25 Gieger (1982)
Yoghurt 11 25 13-39 Van Renterghem (1982)
Yoghurt 2 13.0 12, 14 Hatanaka (1986)
Yoghurt natural 8 18.2 9-25 19.1 Sieber (1990a)
Yoghurt fat-free 53356 Chandan (1977)
Yoghurt skim milk 6 2430 Gieger (1982)
Yoghurt skim milk 4 25 13-34 Stijve (1984)
Yoghurt skimmed 2 19.3 13,25 Toppino (1990)
Yoghurt low fat 31-35 Chandan (1977)
Yoghurt 1% fat 32 14.8 3.4 Obentraut (1983)
Yoghurt 3.6% fat 40 14.3 4.0 Obentraut (1983)
Yoghurt cream 3 l&l7 Gieger (1982)
Yoghurt full cream 8 26 18-37 Stijve (1984)
Fruit yoghurt 18-26 Chandan (1977)
Fruit yoghurt 10 25 19-33 Van Renterghem (1982)
Fruit yoghurt 92 15.0 4.0 Obentraut (1983)
Fruit yoghurt 18 24.4 15539 23.0 Stijve (1984)
Fruit yoghurt 105 16.4 4.7 5-32 17.0 Sieber (1990a)
Fruit yoghurt 5 12.2 8816 12.2 Toppino (1990)
-
N = Number of samples.
5 = Mean.
s = Standard deviation.
A = Range.
X = Median.
 Benzoic acid in cultured dairy products and cheese 231

TABLE 4
Benzoic Acid Content of Some Other Fermented Dairy Products (mg/kg)

Product N x A % First-named Author

Bifidus sour milk 12.5 5-23 10.1 Sieber (1990a)

Cream, sour II-18 Chandan (1977)
Cream, sour, 10% fat lo-16 Gieger (1982)
Buttermilk 11-16 Chandan (1977)
Buttermilk lo-19 Gieger (1982)
Kefir 18-23 Gieger (1982)
Ketir 14.5 8-19 16.5 Sieber (1990a)
Whey 12.6 12-13 Toppino (1990)
Whey powder 50.0 23-15 Kopp (1992)

N, ,?, A, X: See footnotes to Table 3.

0 0 0
CoA Pi
OHM-----+
ATP AMP

Fig. 1. Stoichiometric (reversible) conversion of hippuric to benzoic acid (Pi = phosphate;

gly = glycine).

account for a maximum concentration of 0.35 mmol/kg of benzoic acid according

to a stoichiometrical conversion. According to Tschager et al. (1993) there was a
maximal hippuric acid content of 113 mg/L milk. In addition, Lactococcus luctis,
Escherichia coli and Pseudomonasfluorescens can synthetize benzoic acid in milk
(Sajko et al., 1984).

OCCURRENCE OF BENZOIC ACID IN CHEESE

Benzoic acid is also present in fresh cheeses, quargs and various cheeses
(Tables 5-7). Its concentration is normally below the above-mentioned, tolerated
threshold of 50 mg/kg, except for some cheese varieties for which data were
reported by Svensen (1974) Van Renterghem and Waes (1987) Mtiller (1990),
Sieber et al. (1990~) and Kopp (1992). In these publications, the mean and
median can be different. According to Toppino et al. (1990) higher concentra-
tions of benzoic acid were also detected in Provolone cheese samples, where an
additional amount of benzoyl peroxide, equivalent to 60 mg benzoic acid per kg,
was permitted (Toppino et al., 1987b); the concentration of benzoic acid
remained unchanged during ripening (Toppino et al., 1987~). Benzoic acid was
also found in lactic casein (13.3 mg/kg), in lactic casein whey (15.0 mg/kg), and in
lactic casein whey protein concentrate powder (50.4 mg/kg), but not in sulphuric
acid and rennet casein (Richardson & Gray, 1981).
Non-smear-ripened cheeses contain relatively low concentrations of benzoic
acid, comparable with those in fresh cultured dairy products. However, signifi-
cantly higher concentrations were found at the centre and especially in the rind of
232 R. Sieber, U. Btitikofer, J.O. Basset

TABLE 5
Benzoic Acid Content of Fresh Cheeses and Various Quargs (mg/kg)

Cheese N x s A x First-named Author

Fresh cheese 3 5-8 Grieger (1982)

Fresh cheese 82 26 - 68 Van Renterghem (1987)
Fresh cheese 5 8 l-17 3.0 Sieber (1990a)
Cottage cheese 4 9-18 Chandan (1977)
Cottage cheese 6 9 6 2-18 Takeba (1990)
Quarg, skim 4 17-29 Gieger (1982)
Quarg, skim 18 39.1 2.5 Obentraut (1983)
Quarg, skim 3 24.1 23-25 24.0 Sieber (1990a)
Q. 20% fat dry
matter 9 23.9 2.2 Obentraut (1983)
Q. 40% fat dry
matter 2 11, 13 Gieger (1982)
Q. 40% fat dry
matter 12 14.0 2.9 Obentraut (1983)
Quarg, cream 3 9.9 Cl3 11.O Sieber (1990a)
Quarg, sour milk 7 39.4 31-48 Anonymous (19886)
Quarg with fruit 11 16.0 lo-25 15.3 Sieber (1990a)
N, 3, s, A, 1: See footnotes to Table 3.

smear-ripened cheeses such as Biindner mountain cheese, Tilsit from pasteurised

milk, Vacherin fribourgeois and Vacherin Mont d’Or (Fig. 2).
By excluding any prohibited adulteration of the cheeses or addition of preser-
vatives to these cheeses, such high concentrations of benzoic acid cannot be
explained by a stoichiometric conversion of hippuric acid, which clearly indicates
the existence of at least one more metabolic pathway.

FORMATION AND DISTRIBUTION OF BENZOIC ACID DURING

MANUFACTURE AND RIPENING OF CHEESE, ESPECIALLY OF
SMEAR-RIPENED CHEESE

The formation and distribution of benzoic acid during the manufacture of cheese
was investigated by Chandan et al. (1977), Sieber et al. (1990b) and Kopp (1992).
During the manufacture of cottage cheese, Chandan et al. (1977) observed nearly
the same low concentration of benzoic acid in fermented skim milk and in the
cheese curd. Sieber et al. (1990b) and Kopp (1992) studied the formation and
distribution of benzoic acid during the manufacture of smear-ripened cheeses and
red smear-ripened cheese, respectively. In the study of Sieber et al. (19906), milk
was stored overnight at 3-7°C without or with supplementation with benzoic acid
(variants I and II, respectively) and also at room temperature after inoculation
with a mixed starter culture without or with added hippuric acid (variants III and
IV, respectively). The cheese was manufactured, and the behaviour and fate of
both acids were studied.
 Benzoic acid in cultured dairy products and cheese 233

TABLE 6
Benzoic Acid Content of Various Types of Cheese (mg/kg)

Cheese N x s A x First-named Author

Cheese 24 9.7 2241 6.1 Kurisaki (1973)

Cheese 11 trace-35 Chandan (1977)
Cheese - 70 &erstriim (1972)
Cheese 7 11.3 4-24 11.5 Nagayama (1986)
Cheese 3 17.7 12-22 Anonymous (19886)
Cheese 2 98, 118 Anonymous (1989)
Cheese 71 < l&200 Mtiller (1989)
Cheese 75 10.4 O-52 8.8 Toppino (1990)
Provolone’ 8 96.4 8-341 73 Toppino (1990)
Cheese 36 5.9 o-22 Takeba (1990)
Cheese 59 5.7 l-20 Takeba (1991)
Cheese 7 10.9 ~2-18 10.8 Kopp (1992)
Whey cheese 44 45.1 18-111 41.0 Svensen (1974)
Cheese non-smear-ripened
centre 16 58 O-20 3.5 Sieber (1990a)
rind 16 6.3 O-19 3.5 Sieber (1990a)
centre 5 16.5 12-25 12.9 Kopp (1992)
rind 4 15.9 5-41 9.1 Kopp (1992)
Cheese smear-ripened
centre 84 27.7 O-174 15.0 Sieber (1990~)
rind 84 618 O-622 18.5 Sieber (1990a)
centre 4 36.6 9-66 28.9 Kopp (1992)
rind 4 86.2 88285 56.4 Kopp (1992)

N, X, s, A, X: See footnotes to Table 3.

“Benzoyl peroxide declared on label.

TABLE 7
Benzoic Acid Content of Various Types of Processed Cheese (mg/kg)

Processed cheese N x S A First-named Author

Without ingredients
Austrian 22 9.4 4.3 417 Luf (1986)
Imported 24 9.2 4.4 5519 Luf (1986)
19 11.5 o-29 Sieber (1990a)
17 8 3 413 Takeba (1990)
10 6 (r21 Toppino (1990)
With ingredients
Austrian 11 10.1 4.4 7-22 Luf (1986)
Imported 8 13.1 4.9 8-24 Luf (1986)
10 11.8 O-20 Sieber (1990a)

N, 3, s, A: See footnotes to Table 3.

234 R. Sieber, U. Btitikofer, J.O. Bosset

0 1GO 1700 300 400

centre content [mg/kgl

Fig. 2: Occurrence of benzo:c acid in various smear-ripened cheeses. (From Sieber, R.,
Btitikofer, U., Baumann, H. & Bosset, J.O. (1990) ijber das Vorkommen der BenzoesHure
in Sauermilchprodukten und klse. Mitt. Geb. Lebensm. Hyg., 81, 484-93. Reprinted by
permission.)

No benzoic acid was found before cheese manufacture (Table 8) but

hippuric acid remained at a practically constant concentration. The distri-
bution determined during cheese manufacture was as follows: 9% in curd
and 91% in whey for benzoic acid and 7% in curd and 93% in whey for
hippuric acid. Benzoic acid was transferred from milk into the cheese in the
same way as hippuric acid (variant II). Less than 10% of the benzoic acid
added to the milk was found in the curd. During cheese ripening, additional
benzoic acid also appeared in this variant, lying significantly outside the
expected balance (Table 8).

Formation of benzoic acid from bippuric acid during cheese manufacture

Nishimoto et al. (1968b, 1969) showed that lactic acid bacteria convert the indi-
genous hippuric acid in milk to benzoic acid. Considering that during the first
hours of Swiss cheese manufacture, only the streptococci, and then, a few hours
later, the lactobacilli, grow, i.e. during pressing of the cheese loaf (Steffen, 1971)
it could be assumed that hippuric acid is converted to benzoic acid by the latter
rather than by the former organisms.
The data in Table 8 confirm the stoichiometric conversion of hippuric acid
(Sieber et al., 19906). No difference of metabolism could be found between
variants I and IV. In both variants, no biogenesis of benzoic acid was observed
 TABLE 8
Balance of Hippuric and Benzoic Acid (mmol) under Different Cheese Manufacturing and Ripening Conditions*

Step/Product Variant I+ Variant II+ Variant III+ Variant IV + b

8
Benzoic Hippuric + Benzoic Hippuric + Benzoic Hippuric + Benzoic Hippuric •t :i;.
acid benzoic acid benzoic acid benzoic acid benzoic 8
acids acids acids acids C
%’
Milk 0 6.1 63.9 10.1 0 I.1 0.3 50.1 P
Vat milk 0 6.1 64.4 11.3 0 6.1 0.4 50.4 2
T
Vat milk + rennet + mixed Z
starter culture 0.6 I.4 62.4 69.0 0 6.1 0 50.4 %
Curd/whey o/o.5 I.2 5.1/56.1 61.1 O/O 6.5 o/1.1 49.3 k?’
After pressing: cheese/whey 0.410 I.2 3.1/0.3 66.3 0.410 6.6 2+/o. 1 49.1 ‘tr
a
Cheese 1-month-old” 0.5 I.4 3.5 66.1 0.6 6.1 2.8 49.1
B
Cheese 3-month-old” 0.5 I.4 6.1 69.3 6.1 12.9 9.6 55.9 2

*From Sieber, R., Biitikofer, U., Baumann, E. & Bosset, J.O. (1990). iiber die Benzoes%urebildung und -verteilung wshrend der Herstellung S
Q
und Reifung von geschmierten KBsen. Mitt. Geb. Lebensm. Hyg., 81, 722-30. Reprinted by permission. S
+Variant I: stored at 3-7°C overnight. 2
3
Variant II: as I with a total supplementation of 49.2 mmol sodium benzoate.
Variant III: stored at room temperature overnight, after inoculation with a mixed starter culture.
Variant IV: as III, with a total supplementation of 33.6 mmol sodium hippurate.
“The balance also included the portion of whey lost by the preparation and pressing of the curd.
236 R. Sieber, U. BQtikofer, J.O. Bosset

either in milk or in vat (Kessi) milk or even in curd. In variant I, milk with added
rennet and mixed starter culture already contained a low concentration of
benzoic acid (Table 8). In variant III, benzoic acid was present in cheese only
after pressing. Variants I and III contained no more hippuric acid after this
manufacturing step.
To determine whether an initially higher concentration of hippuric acid could
enhance the formation of benzoic acid by lactic acid bacteria, milk was inoculated
with a mixed starter culture, enriched with hippuric acid, and then ripened over-
night at room temperature (variant IV). Such a procedure led to the production
of only minute amounts of benzoic acid in the stored milk. This experiment
showed that most of the hippuric acid drained off and was lost with whey and
that less than 10% of the hippuric acid (indigenous or added) present was carried
over into the fresh curd.

TABLE 9
Concentration (mg/kg) of Benzoic, Hydrocinnamic, and Cinnamic Acid in l- and 3-
month-old Cheese after Different Storage Conditions of the Vat Milk With and Without
Supplementation of Hippuric and Benzoic Acid*

l-month-old cheese 3-month-old cheese

Centre Rind Rind Centre Rind Rind

(flat side) (hoop side) (fat side) (hoop side)

Variant I+
Cinnamic acid nd 7 nd nd 12 nd
Hydrocinnamic acid nd 54 45 41 202 53
Benzoic acid 9 39 10 9 126 39

Variant II+
Cinnamic acid nd 7 nd nd 13 nd
Hydrocinnamic acid nd 122 13 29 226 48
Benzoic acid 62 160 4 119 172 62

Variant III+
Cinnamic acid nd 28 nd nd 20 nd
Hydrocinnamic acid nd 97 10 41 323 46
Benzoic acid 10 144 18 114 212 55

Variant IV+
Cinnamic acid nd 8 nd nd 19 nd
Hydrocinnamic acid nd 95 8 69 292 54
Benzoic acid 46 133 44 159 247 99

*From Sieber, R., Btitikofer, U., Baumann, E. & Bosset, J.O. (1990). Uber die Benzoe-
saurebildung und -verteilung wahrend der Herstellung und Reifung von geschmierten
K&en. Mitt. Geb. Lebensm. Hyg., 81, 722-30. Reprinted by permission.
+For variants I-IV, see footnotes to Table 8.
nd = not detected (below the detection limit).
 Benzoic acid in cultured dairy products and cheese 231

Formation of benzoic acid in smear-ripened cheeses

During cheese ripening (1 and 3 months), the formation of benzoic acid was
demonstrated by Sieber et al. (1990b). In spite of a very low residual concen-
tration of hippuric acid (due to its conversion to benzoic acid) in cheese just
after pressing, a significant increase in the concentration of benzoic acid was
found in 3-month-old cheese in variant III, showing a non-stoichiometric
balance between these acids (Table 8). In variant I, the concentration of benzoic
acid remained practically constant in the centre of the loaf but increased parti-
cularly in the rind of the flat sides and, to a lesser extent, in the rind on the
hoop side (Table 9). In short, a stoichiometric transformation of the total
amount of hippuric acid available in milk to benzoic acid cannot account for
the high concentration of this substance in the outer zone of these cheese loaves.
This observation leads to the hypothesis that one or more natural pathways
exist for its formation.

BREAKDOWN OF PHENYLALANINE AS AN ALTERNATIVE PATH-

WAY FOR THE BIOGENESIS OF BENZOIC ACID IN SMEAR-RIPENED
CHEESES

Degradation of phenylalanine as a possible metabolic pathway for the formation of

benzoic acid

According to Leuthardt (1977), another metabolic pathway can explain the

biogenesis of benzoic acid from phenylalanine, as well as the simultaneous
production of acetophenone (Bosset & Liardon, 1984, 1985) and ammonia (Blanc
et al., 1983). According to Leuthardt (1977), acetophenone and benzoic acid are
formed from phenylvaleric acid with the intermediate product, phenylpropionic
acid, which can also be generated through the deamination of phenylalanine
(Fig. 3). The degradation of phenylalanine to benzoic acid was also demonstrated in
cell-free extracts of Sporobolomyces ro.seus (Moore et al., 1968) and in intact cells of
Rhodotorula rubra (Uchiyama et al., 1969); cinnamic acid was an intermediate.

Evidence of metabolites of phenylalanine degradation in cheeses

According to Teuber (1987) benzoic acid can be formed at up to 200 mg/kg

(1.64mmol/kg) in Harzer cheese by fermentation of aromatic amino acids. Kuri-
saki et al. (1973) showed that, in yeast-ripened cheese, benzoic acid might be
produced from phenylalanine but not from tyrosine. The yield from the conver-
sion of phenylalanine amounted to 0.06%, which was determined by using
radioactively labelled phenylalanine in curd and benzoic acid in ripened cheese.
Using GC/MS analysis, Kuzdzal-Savoie et al. (1971, 1973) identified P-phenyl-
propionic acid (hydrocinnamic acid), phenylacetic acid and benzoic acid in
Munster and Livarot cheese. Simonart and Mayaudon (1956) Jarczynski and
Kiermeier (1955) and Kiermeier and Jarczynski (1960) found p-hydroxybenzoic
acid in cheese. According to Schormiiller et al. (1954) this substance can be an
intermediate product from the degradation of tyrosine during cheese ripening.
Tyrosine itself is not converted into benzoic acid (Kurisaki et al., 1973).
238 R. Sieber, U. Btitikofer, J.O. Basset

The following metabolites (Fig. 3) were identified by GC/MS or even quanti-

tied by HPLC by studying the ripening of smear-ripened cheeses (Swiss Gruyere
and Vacherin fribourgeois):
phenylalanine by ion exchange chromatography (Lavanchy & Btihlmann,
1983):
/II-phenylpropionic acid by HPLC (Bosset et al., 1990; Sieber et al., 1990b) and
GC/MS (Bosset et al., 1993);
cinnamic acid by HPLC (Bosset et al., 1990; Sieber et al., 1990b);
acetophenone by GC/MS (Bosset & Liardon, 1984, 1985); and
benzoic acid by HPLC (Bosset et al., 1990; Sieber et al., 1990b) and by GC/MS
(Bosset et al., 1993).

phenylalanine phenylvaleric acid

kphenyl-propionic acid
OH (hydrocinnamic acid)

OH cinnamic acid

OH 0

R-phenyl-R-hydroxypropionic acid
OH

OH R-phenyl-R-cetopropionic acid

benzolc acid acetophenone

Fig. 3. Breakdown of phenylalanine as an alternative pathway for the biogenesis of

benzoic acid.
 Benzoic acid in cultured dairy products und cheese 239

These metabolites were found almost exclusively in the rind, including smear
(Bosset & Liardon, 1984, 1985; Bosset et al., 1993). Their concentrations
increased markedly (Bosset & Liardon, 1984; Bosset et al., 1993) during matura-
tion and an intensive emission of ammonia was observed (Blanc et al., 1983). The
relatively low concentration of benzoic acid measured in the middle and centre of
most cheese loaves could be ascribed to the relatively slow diffusion of this
substance from the rind to the centre. Similar concentration gradients were
observed for many other smear-produced polar compounds such as pyrazines,
aldehydes, and methyl ketones (Bosset & Liardon, 1984, 1985; Liardon et al.,
1982).
A further observation could support the validity of this theory: loaves of
smear-ripened Swiss Gruyere cheese ripened in a gas-tight container specially
designed for the determination of respiratory quotients under a controlled
anaerobic atmosphere (Bosset et al., 1982) showed a much lower tendency to
become mouldy than loaves of smearless Swiss Emmental cheese stored under
similar conditions (Fliickiger, 1983, personal communication). This can be
interpreted as a consequence of the biogenesis of a sufficient amount of
benzoic acid from phenylalanine by yeasts on the smear-ripened cheese. It is
known that this acid can inhibit the growth of bacteria, yeasts and moulds
(Chipley, 1993).
Free phenylalanine and other amino acids are present at relatively high
cdncentrations in different ripened cheeses (Lavanchy & Biihlmann, 1983;
Lavanchy & Sieber, 1993). Assuming that free or even peptide-bound phenylala-
nine of cheese could be stoichiometrically converted to benzoic acid, concentra-
tions of several g/kg of benzoic acid could be expected. Owing to the more
intensive degradation of protein and peptides by the smear, the available
concentration of this (free) amino acid should be higher in the rind than in the
centre of cheese. It is also known that benzoic acid can be degraded anaerobically
(Londry & Fedorak, 1992).

Sample benzoic acid content hydrocinnamic acid content

[mg/kg cheese] [mg/kg cheese]
1. centre 0 0
2. centre hoop site 9 25
3 rind, upper site 10 24
4. rind lower site 24 33
5. rind, hoop site 81 230

Fig. 4. Distribution of benzoic and hydrocinnamic acid in Vacherin fribourgeois cheeses.

(From Basset, J.O., Biitikofer, U. & Sieber, R. (1990). Phenylalaninabbau-ein weiterer
Weg zur natiirlichen Bildung der BenzoesZure in geschmierten K&en. Schweiz. Milch-
wirtsch. Forsch., 19, 46-50. Reprinted by permission.)
240 R. Sieber. U. Biitikofer, J.O. Basset

TABLE 10
Concentration of Benzoic and Hydrocinnamic Acid (mg/kg) in Vacherin Fribourgeois
from Different Manufacturers*

Manufacturer 1st investigation 2nd investigation

Centre Rind Centre Rind Hoop side

Benzoic Benzoic Benzoic Benzoic Benzoic Hydro-

acid acid acid acid acid cinnamic acid

A 61 161 nd nd nd nd
B 89141” 204145’ 131 359 229 168
C 22 0 137 366 454 313
D 16 26 5 7 3 nd
E 57 200 13 17 8 nd
F 45 94 12 11 22 nd
G nd nd 5 7 15 nd
H 72/l 15” 2721205’ 35 118 123 114
I 29 143 16 140 140 484
K 61 304 7 21 43 nd

*From Bosset, J.O., Btitikofer, U. & Sieber, R. (1990). Phenylalaninabbau-ein weiterer

Weg zur natiirlichen Bildung der Benzoesgure in geschmierten Kasen. Schweiz. Milch-
wirtsch. Forsch., 19, 4650. Reprinted by permission.
nd = not detected.
“Two samples within 3 weeks.

Model studies with Vacherin fribourgeois

Owing to the high concentrations of benzoic acid in many smear-ripened cheese

varieties, Vacherin fribourgeois was chosen as a model for further studies, which
should provide more information on the occurrence of various substances
formed during the degradation of phenylalanine (Bosset et al., 1990). The
different zones investigated showed a variable concentration of benzoic acid
(Fig. 4). The highest content was in the rind on the hoop side of cheese, which
thus supported an independent observation by Marchon and Deillon (1990,
personal communication). Simultaneously, a high concentration of hydro-
cinnamic acid was measured in the same region. Other samples of Vacherin
fribourgeois containing high concentrations of benzoic acid had at the same
time a high content of hydrocinnamic acid in the rind of the hoop side
(Table 10).
This surprising distribution of benzoic and hydrocinnamic acids can, however,
be explained. Only the rind on the hoop side is exposed all the time to aerobic
conditions. The formation of benzoic acid needs such conditions. In comparison,
the lower concentrations in the bottom and upper flat side are subjected to
different ripening and washing conditions: these sides rest (in close contact)
alternately on the wood and are therefore exposed to aerobic conditions for only
half of the ripening period. Moreover, the surfaces of the cheese loaves are peri-
 Benzoic acid in cultured dairy products and cheese 241

odically washed and therefore subjected to mechanical rubbing, which is certainly

more intensive on the flat surfaces than on the curved hoop side.
Both hydrocinnamic and cinnamic acids are metabolites of phenylalanine
degradation (Bosset et al., 1990). In all variants (I-IV) discussed above, the
concentration of hydrocinnamic acid in the rind of the flat surfaces as well as in
the hoop side of the l- and 3-month-ripe cheese loaves was comparable with that
of benzoic acid. Cinnamic acid was found at lower concentrations practically
only in the rind of the flat surfaces (Table 9).

FURTHER POSSIBLE SOURCES OF BENZOIC ACID IN SMEAR-

RIPENED CHEESES

A study in progress on the origin and content of volatile flavour components in

cultured dairy products revealed the occurrence of relatively high concentrations
of benzaldehyde in many of them (Imhof et al., 1994, 1995). This substance seems
to be produced by certain strains of lactic acid bacteria. Benzaldehyde has been
characterized as a product of phenylacetic acid oxidation by Penicillium chryso-
genum (Hockenhull et al., 1952). Benzaldehyde is known to be subjected to auto-
oxidation in the presence of air (Bosset et al., 1982), a condition that is fulfilled in
the smear-ripened rind. This reaction therefore explains the presence of benzoic
acid in the outer zone of cheese loaves.
Another possible mechanism for the formation of benzoic acid could be the
anaerobic metabolism of phenol (Londry & Fedorak, 1992) which was found at
low concentrations and almost exclusively in the outer zone of Gruyere, but also
in other cheeses (Bosset et al., 1993).

CONCLUSION

Benzoic acid is a natural constituent of fermented dairy products, particularly

cheese. It is produced from hippuric acid, another naturally occurring compound
in dairy products, according to a stoichiometric reaction (1:l). The high concen-
trations of benzoic acid found in some smear-ripened cheeses can be explained by
the breakdown of phenylalanine during ripening as well as, probably, to a certain
extent by the auto-oxidation of benzaldehyde, a constituent frequently identified
in cultured milk products produced with lactic acid bacteria.
The variable concentration of benzoic acid in the bottom and upper sides, as
well as in the hoop side, of smear-ripened cheese loaves, depends on many
factors, such as the composition of the microbiological flora, the frequency and
kind of cheese curing and ripening conditions in the cellar (temperature, humid-
ity, duration, etc.). The species of microorganisms able to break down phenyla-
lanine to benzoic acid have not yet been identified: lactobacilli, rather than
streptococci, seem to be involved. In addition to its role by generating cheese
flavour compounds, the smear seems to act as a natural preservative against yeast
and mould contamination on the surface of smear-ripened cheese loaves. This
natural protection could be due to the age-old practical knowledge (empiricism!)
of cheesemakers, whose critical observation on traditional cheese manufacture
242 R. Sieber, U. Btitikqfer, J.O. Bosset

could have anticipated in this field the newest developments and knowledge of
modern food science and technology!

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