Food spoilage fungi. I!. Heat-resistant fungi

By Ailsa D. Hocking and J. I. Pitt CSIRO Division of Food Research, North Ryde, N.S.W., 2113

Spoilage problems caused by heat-resistant fungi have been reported frequently in Australia in the past few years. New developments in packaging technology, along with increased consumer demand for fruit juices and other fruit-based products, have led to a large increase in the marketed volume of these products. A trend towards pasteurized preservative-free juices has increased the risk of spoilage caused by heat-resistant fungi. The species capable of causing this type of spoilage are described in this paper.

Introduction particularly susceptible to contamination by It is probable that spoilage problems caused by Byssochlamys and other heat-resistant fungi. heat-resistant moulds have been with us since man began to preserve fruit by heat processing. The situation The problem was first recognized in canned In Australia, spoilage problems caused by strawberries in the early 1930s at the Campden Byssochlamys were first recognized in the early Fruit and Vegetable Preservation Station, 1960s, again in canned strawberries (Spurgin England. Olliver and Rendle (1934) showed 1964; Richardson 1965). In recent years, there that Byssochlamys fulva was responsible for the has been an increase in the volume and variety spoilage. They conducted extensive of heat-processed fruit-based products on the investigations on the incidence of B.fulva in market. New packaging technology, and an fruits, in sugar and water, and in packing increase in consumer demand, have seen a materials. Although wooden trays, baskets, huge increase in the marketed quantity of fruit glass bottles and jars were found to be juices and fruit-juice drinks. Preservation often contaminated with the fungus, the initial source has relied on either pasteurization or ultra high was shown to be the fruits coming in from the temperature (UHT) processing, rather than the fields and orchards. Strawberries and plums use of preservatives. Spoilage problems due to were infected more heavily than most other heat-resistant moulds have occurred in these fruits, but Olliver and Rendle (1934) also found products, and other products of a similar type, B.fulva on gooseberries, loganberries, such as fruit-gel baby foods. blackberries, black currants and apples. Since In Australia, in our experience, the raw all positive samples of stone fruits were taken materials most likely to be contaminated with from the ground, and strawberries, too, are heat-resistant fungal spores are passionfruit and easily contaminated with soil, Olliver and strawberries. Pineapple and mango juices and Rendle suggested that B.fulva may be a soil- pulps may also be a source of contamination, borne fungus. but orange and other citrus products have Hull (1939), investigating the sources of rarely been implicated. Passionfruit are infection by Byssochlamys, found the fungus on particularly susceptible to contamination leaves, fruits and straw from the strawberry because the fruit are usually harvested after fields, and also on mummified plums, they have fallen, and have thus been in contact raspberry refuse, and in the baskets used to with the soil. Strawberries may also come in collect fruit. He concluded that Byssochlamys contact with soil, and both strawberries and was a saprophytic rather than a parasitic pineapples may be contaminated with soil by fungus, and that the ascospores could survive in rain splash. orchard refuse over winter, to contaminate the The best way to ensure that heat-resistant following season's fruit. Although Byssochlamys moulds will not cause spoilage of susceptible did not actually grow in soil, the soil acted as an products is careful selection and handling of important reservoir for the ascospores, and raw materials, in conjunction with an effective fruits which came into contact with soil were screening procedure for heat-resistant fun a1 CSIRO FoodRes. Q. 44 (4f 73-82 73 spores. For example, passionfruit crops consist distributed over four 150 mm Petri dishes and of mostly smooth, sound fruit, together with a mixed with 1'12 strength potato dextrose agar. proportion which have wrinkled skins. The The Petri dishes are loosely sealed in a plastic smooth fruit will generally have a lower mould bag to prevent drying, and incubated at 30°C spore count than the wrinkled fruit, and are for up to 30 days. Plates are examined weekly much easier to decontaminate. Smooth fruit for growth. Most surviving moulds produce should be thoroughly washed, preferably in visible colonies within ten days, but incubation hypochlorite solution (approximately 100 ppm), for up to 30 days allows for the possible presence then rinsed before processing. Wrinkled fruit of badly heat-damaged spores, which may should be processed separately, and the juice or germinate very slowly. This long incubation pulp used in products such as refrigerated or time also allows most moulds to mature and frozen desserts, that will not be spoiled by heat- sporulate, aiding their identification. resistant moulds. The main problem associated with this dilution technique is the possibility of aerial Screening for heat-resistant fungi contamination of the plates with common A number of methods have been developed mould spores which will give false positive for the enumeration of heat-resistant fungal results. The appearance of green Penicillium spores. All include a heat treatment, ranging colonies, or colonies of common Aspergillus from 5 minutes at 75OC to 35 minutes at 80°C. species such as A. jlavus and A. niger, is a clear Most of these methods are outlined by Beuchat indication of contamination, as these fungi are and Rice (1979). One of these, developed not heat-resistant, and their spores will not specifically for screening fruit juices and survive the 75' to 80°C heat treatment. To concentrates by Murdock and Hatcher (1978), minimize this problem, plates should be poured has been adapted by Budnik (unpublished) to in clean, still air, or a laminar flow cabinet if enable the screening of a larger sample (100 ml) possible. If a product contains large numbers of of raw material. The method is described heat-resistant bacterial spores (e.g. Bacillus below, and outlined diagrammatically in Fig. 1. species), antibiotics can be added to the potato If the sample to be tested is greater than 35' dextrose agar. The addition of 100 mg of Brix, it should first be diluted l : 1 with 0.1% chloramphenicol per litre of medium will peptone or similar diluent. Two 50 m1 samples prevent the growth of these bacteria. are taken for examination. For passionfruit A more direct method can be used for juice, which normally has a pH of about 2.0, the screening fruit pulps and other semisolid pH should be adjusted to 3.4-3.6. The two materials which avoids the problems of aerial samples are heated in 200 X 30 mm test tubes contamination. Place approximately 30 m1 of in a closed water bath at 80°C for 30 minutes, pulp in a flat bottle such as a 100 m1 medicine then rapidly cooled. Each 50 m1 sample is then flat. Heat the bottle in the upright position for Fruit juice sample 2x50 ml samples Pasteurization in Samples rapidly (35" Brix or less) taken for water bath, cooled after examination 80°C for 30 min pasteurization

adjust pH 3.4-3.6- with 10% NaOH

10 ml of 1'/2 strength Dishes loosely sealed potato dextrose or malt extract agar / in plastic bag and incubated at 30°C for added riw 4 up to 30 days d- 10 (examine weekly) Each 50 ml lot distributed over 4x1 50 mm diam. Petri dishes Fig; 1. Procedure for detection and enumeration of heat-resistant mould spores.

74 30 minutes at 80°C and cool, as described moulds cause spoilage by growing as visible previously. The bottles of pulp can then be colonies on the surface of the product. incubated directly, without opening and without the addition of agar. They should be Heat resistance incubated flat, allowing as large a surface area Studies on the heat resistance of fungal as possible, at 30°C, for up to 30 days. Any spores have concentrated on Byssochlamys mould colonies which develop will need to be species, no doubt because of their dominant sub-cultured onto a suitable medium for role in the spoilage of heat-processed foods. identification. If suitable containers such as Moreover, ascospores of Byssochlamys species Roux bottles are available, larger samples can appear to be ambng the most heat-resistant be examined by this technique. fungal spores known. Many variables can affect heat resistance. Acceptable limits for heat-resistant moulds For Byssochlamys species, information on this

The acce~tablelevel of contamination- -- of a topic has been comprehensively reviewed by raw material with heat-resistant ascospores will Beuchat and Rice (1979). Heat resistance can depend very much on the end product. vary markedly from isolate to isolate (Bayne Considerations such as whether the raw and Michener 1979; Hatcher et al. 1979). material is a major or a minor ingredient, Factors such as IDH. ' water activitv.,, and the whether the final product will contain presence of preservatives also have an effect. preservative, and the heat process to which the Ascospores are more susceptible to heat if the product or the raw material will be subjected, pH is low (Bayne and Michener 1979), andlor if all must be taken into account. Practical preservatives such as SO2are present (King, experience has shown that, for passionfruit Michener and Ito 1969). On the other hand. juice, a contamination level of less than 2 spores high levels of sugar have a protective effect per 100 m1 should give a negligible spoilage rate (Beuchat and Toledo 1977). For Byssochlamys in most finished products. Contamination fulva, a D value between l and 12 minutes at levels of more than 2 spores per 100 m1 sample 90°C (Bayne and Michener 1979) and a z value are often unacceptable. However, for some of 6 to 7 minutes (King et al. 1969) are practical . . products, such as UHT processed fruit juice working values. blends which contain a high proportion of The heat resistance of Byssochlamys nivea is passionfruit juice and do not contain marginally lower than that of B. fulva (Put and preservative, an even lower level of Kruiswijk 1964; Beuchat and Rice 1979). contamination is required. One manufacturer The most heat resistant of other moulds of this type of product specifies that heat- isolated from heat-processed foods appears to be resistant mould spores should be absent from a the ascomycete Neosartoryafischeri, which is 100ml sample of passionfruit juice. more commonly known by the name of its conidial state, As~ergillwfischeri. Kavanagh et Types of spoilage al. (1963) reported that ascospores of an isolate In canned or bottled fruits affected by more recently identified as N.fischeri withstood Byssochlamys species, the first sign of fungal boiling in distilled water for 60 min. They spoilage is usually a slight softening of the fruit. reported that spore age, pH and sugar This progresses until total disintegration takes concentration affected heat resistance, but no place, due to the production of a powerful details of spore numbers heated or pectinase by the fungus (Hull 1939; Beuchat experimental procedures were given. McEvoy and Rice 1979). Off-odours, and a slightly sour and Stuart (1970) also heated ascospores of N. taste may develop, and there may be gas fischeri in distilled water: they reported 100% production. It is rare for species other than survival after 20 min at 80°C, and 0.002% Byssochlamys to be responsible for spoilage of survival after 5 min at 100°C. Splittstoesser canned fruits. and Splittstoesser (1977), however, reported Byssochlamys species are capable of growth at that the heat resistance of a Neosartorya isolate, extremely low oxygen tensions. In liquid probably N.fischeri, was comparable to that of products under these conditions, fermentation B.fulva. apparently occurs, with the production of C02. The production of gas then causes visible Taxonomy of heat-resistant fungi swelling and spoilage of the product. Even The basis for high heat resistance in these small amounts of oxygen in the headspace of a fungi is the production of a teleomorphic state, jar or bottle, or slow leakage of oxygen through that is, they form ascospores. Ascospores are a package such as a Tetra-Brik, can provide produced, generally in groups of eight, within a sufficient oxygen for these fungi to grow. In closed sac, the ascus @l. ascz]; ascospores are the solidified products, like fruit gels, heat-resistant prime characteristic of the class of fungi called Fig. 2. Different bodies in which ascospores may be formed: (a)cleistothecium (Neosamrya,Eupenicillium); (b) gymnothecium (Talaromyces);(c) unenclosed asci (Byssochlamys).

Ascomycetes. In nearly all ascomycete genera, occasionally caused food spoilage (Williams asci are in turn enclosed. in large" numbers. et al. 1941). within larger bodies. In genera of interest here, In practical terms, recognition of these these bodies may have a solid, totally enclosed species of fungi relies on four principal factors; wall (a cleistothecium, Fig. 24 or be composed first, isolation from a heat screening process; of fine, interwoven hyphae (a gymnothecium, second, observation of colony growth and Fig. 2b). Only in Byssochlamys are asci borne appearance on standard media; third, singly and unenclosed (Fig. 2c). observation of cleistothecia, gymnothecia or As well as ascospores, ascomycetes generally unenclosed asci; fourth, microscopic produce an anamorphic state with asexual observation of conidial structures. The first of spores, that is they produce conidia (sing. these has been outlined above, the others are conidium). Conidia are not verv heat resistant. incorporated in the descriptions given below. and are usually readily destroyed by pasteurizing heat processes or the screening Isolation and culture of heat-resistant fungi techniques outlined above. The fungi of interest In general, heat-resistant fungi do not here produce conidial states characteristic of the require special media or cultural conditions. genera Aspergillus (Fig. 3a), Geosmithia (Fig. 3b), Sus~ectedheat-resistant colonies can be isolated Paecilomyces (Fig. 3c), and Penicillium (Fig. 3d). by picking spores or mycelium with a sterile needle from colonies which have grown on Common heat-resistant fungi heated product and transferring as a single It is probably true that most fungal point to a slant of suitable medium. Either ascospores possess relatively high heat Czapek yeast extract agar or malt extract agar resistance and could in theorv cause food is suitable (see formulae below). Transfer by spoilage. In practice only a few species have using a needle which has been flamed, and been encountered in our work or reported in the then cooled in agar, leaving a moist tip to which literature. Principal among these are spores will adhere. Incubate slants at 25' to Byssochlamys species: B. fulva, which is 30°C for a week or more, until fully grown. commonly associated with spoilage problems in Australia, and B. nivea, which appears to be Media much less common here than in Europe. Other Czapek yeast extract agar (CYA) has the fungi which less frequently cause spoilage in following composition: NaN03, 3.0 g; KH2P04, processed fruit products are Talaramycesflavus, 1.0 g; KC1, 0.5 g; MgS04.7H20, 0.5 g; T. bacillisporus and Neosartorya fischeri ( = FeS0+.5H20,0.01 g; yeast extract, 5.0 g; Aspergillusfischeri). Species from the genus sucrose, 30 g; agar, 15 g; water to 1 1. Malt Eupenjcillium also form heat-resistant extract agar (MEA) has the following

ascosDores.L, but do not commonlv cause composition: malt extract, 20 g; glucose, 20 g; spoilage. Some Penicillium species form hard bacteriological peptone, 1.0 g; agar, 20 g; sclerotia, really undeveloped cleistothecia distilled water to l 1. We have found light malt similar to those in Eupenicillium. These too extract sold for home brewing to be as effective appear to be highly heat resistant, and have for making media as malt extract sold Fig. 3. Anamorphic states produced by heat-resistant fungi: (a)Aspergillus anamorph of Neosartorya; (b) Geosmithia anarnorph of Talaromyces; (c)Paecilomyces anamorph of Byssochlamys; (d) Penicilliclm anamorph of Eupenicillium.

specifically for that purpose. slip, and gently blot away surplus stain. Detergent agar, optional for inoculating Petri Examine with 10 X, 40 X and 100 X objectives. dishes (see below), consists of one drop per Pieces of freshly sporing areas from the 100 m1 (0.05%) of a food grade detergent such as peripheries of colonies make the best wet polysorbitan 80 (Tween 80) in 0.2% agar. mounts for conidial structures; areas near the Dispense this medium in c. 0.25 m1 quantities centres of colonies are usually the first to in small vials. produce mature ascospores. All media should be sterilized by autoclaving The following key will assist in at 121°C for 15 min. identification.

Identification of isolates Key to common heat-resistant fungi To identify heat-resistant fungal isolates, 1. Asci produced in discrete bodies with totally proceed as follows. Inoculate each isolate onto enclosed walls (cleistothecia) 5 four Petri dishes, two each of CYA and MEA. Asci produced in bodies with walls of woven Inoculate each plate at three equally spaced hyphae (gymnothecia) or openly 2 points. Avoid chance inoculation of stray spores as far as possible, by one of two techniques. 2. Asci enclosed in gymnothecia (Talaromyces) 3 Either hold plates upside down and inoculate Asci produced openly; fine hyphae may be with a needle point of spores (wet needle), or present, but asci not enclosed(Byssoch1amys) 4 mix a needle point of spores with detergent agar 3. Colonies on MEA at 25' C exceeding in a small vial, and inoculate plates with a loop. 25 mm diam.; conidial state Penicillium Vials may be steamed or autoclaved and reused Talaromycesflavus several times before cleaning. Colonies on MEA at 25OC not exceeding Incubate plates for one week: one each of 25 mm diam.; conidial state Geosmithia MEA and CYA at 25OC or near, and the others Talaromyces bacillisporus at 30°C. 4. Colonies on CYA and MEA predominantly After incubation, examine plates by eye, buff or brown Byssochlamysfulva measuring colony diameters with a ruler, and Colonies on CYA and MEA persistently make wet mounts to examine small pieces of white or cream Byssochlamys niuea fungus under the compound microscope. 5. Colonies on CYA and MEA at 25OC To make wet mounts, cut a small piece of exceeding 50 mm diam. in 7 days; fungus from the colony using a chisel-pointed predominantly coloured white or cream wire or a steel sewing needle, and transfer to a Neosartoryafischeri clean slide, adding a single drop of 70% ethanol Colonies on CYA and MEA at 25OC to aid transfer and to wet the spores. After most otherwise Eupenicillium spp. of the ethanol has evaporated, add a single drop of 3 suitable stain, such as lactofuchsin (0.1% Genus Byssochlamys Westling acid fuchsin in pure lactic acid), add a cover Byssochlamys has the distinction of being Fig. 4. Byssochlamys species: (a)-(cl,(g) B. fulva; (d)-(f),(h); B. nivea. B. fulva: (a)colonies on CYA and MEA, 25OC. 7d.; (b)penicillus; (c)conidia; (g)ascospores. B. nivea: (d)colonies on CYA and MEA, 25" C, 7d.; (e)penicillus; (f) conidia; (h) ascospores. almost uniquely associated with food spoilage, ascomycetes envelop asci during development. and in particular with the spoilage of heat- Asci in Byssochlamys are borne in open clusters, processed acid foods. Apart from the original in association with, but not surrounded by, isolation and description of B. nivea from soil by unstructured wefts of fine, white hyphae. Westling in 1911, Byssochlamys has been In our experience, the temperature range for reported only rarely from sources other than observation of Byssochlamys asci and ascospores heat-processed foods. Its natural habitat in the laboratory is sometimes very narrow. appears to be soils, but the genus is mentioned Cultures need to be incubated at 30°C as some very seldom in lists of fungi from soils other isolates do not produce asci at 25OC or 37OC. than,those used for the cultivation of fruits. However, resumptive evidence of the presence Byssochlamys is an ascomycete genus of Byssochlamys can be made from plates at 25O characterized by the absence of cleistothecia, or 37OC if the isolate has come from heat- gymnothecia or other bodies which in most processed foods or raw materials. Byssochlamysfulva Olliver and G. Smith (Fig. 4, 10-14 days at 25OC and in 7-10 days at 30°C but a-c,g) rarely found at 37'C. Anamorph: Paecilomycesfulvus Stolk and Anamorphs of two kinds produced, Samson aleurioconidia and penicilli; aleurioconidia At 25OC, colonies on CYA and MEA at least borne singly, common at 30°C and 37OC, 60 mm diam., often covering the whole Petri spherical to pear-shaped, 7-10 pm diam.; dish, relatively sparse, low or somewhat irregular penicilli sparsely produced, and floccose; conidial production heavy, uniformly phialides sometimes borne solitarily from coloured olive brown; reverse in similar colours hyphae as well; phialides 12-20 pm long, or pale. At 30°C, colonies on CYA and MEA cylindrical then gradually tapering; conidia usually covering the whole Petri dish, low to ellipsoidal to pear-shaped, 3-6 pm long, smooth moderately deep, sparse, with moderate walled. conidial production, coloured brown, overlaid Distinguishing characteristics. - Byssochlamys by white hyphae from which asci produced; nivea is readily distinguished from B.fulva by its reverse olive brown to deep brown. persistently white to cream colonies. It differs Teleomorphic state single asci borne from, from other fungi by forming three but not enveloped by, wefts of contorted white characteristic types of reproductive structures: hyphae, best developed at 30°C, maturing in aleurioconidia, sparse penicilli, and solitary 7-12 days, occasionally formed at 25OC in fresh asci as well. isolates but maturing slowly if at all; asci Genus Eupenicil/ium Ludwig spherical to subspheroidal, 9-12 pm diam.; ascospores ellipsoidal, hyaline or straw Eupenicillium is characterized by the coloured, 5-7 pm long, smooth walled. production of macroscopic (100-500 pm diam.), Anamorphic state best observed at 25' C, smooth walled, often brightly coloured cleistothecia, in association with a Penicillium consisting of penicilli borne from surface hyphae or long, trailing, aerial hyphae; stipes anamorph. In many species cleistothecia 10-30 pm long; phialides of nonuniform become rock hard as they develop, and may appearance, flask-shaped or narrowing remain so for many weeks or months, finally gradually to the apices, 12-20 pm long; conidia maturing from the centre to yield numerous mostly cylindrical or barrel-shaped, narrow eight-spored asci. and 7-10 pm long, but sometimes longer, wider Most Eupenicillium species are soil fungi, and or ellipsoidal from particular phialides, smooth of little interest to the food microbiologist. walled. However, they do occur from time to time as survivors of heat processing. Williams et al. Distinguishing characteristics. - In culture at (1941) recorded that a new species, Penicillium 25OC, Byssochlamys fulva closely resembles the lapidosum (stone-like, an apt name) was causing common fungus Paecilomyces variotii. The spoilage of canned blueberries. It possessed simplest microscopic distinguishing feature is highly heat-resistant sclerotia (immature that P. variotii produces ellipsoidal not cleistothecia). This fungus was later shown to cylindrical conidia. At 30°C, B. fulva is produce a Eupenicillium state. Two points are distinguished by rapidly growing olive brown worth noting: first, it was the immature colonies with areas of fine white hyphae, in cleistothecium itself which was acting as the which asci are produced in open clusters. heat-resistant body, and second, most Eupenicillium species produce heat-resistant Byssochlamys nivea Westling (Fig. 4, d-f,h) ascospores. Fortunately, they rarely find their Anamorph: Paecilomyces niveus Stolk and way into heat-processed foods. Samson We and others (Anon. 1967) have isolated At 25OC, colonies on CYA 40-50 mm diam., Eupenicillium species as heat-resistant low and quite sparse, white to slightly grey; contaminants of fruit juices on several reverse pale to mid-brown. Colonies on MEA occasions. No particular species appears to be covering the whole Petri dish, low and sparse, significant, and growth of the fungus has white to creamish, with small knots of dense occurred in the product only rarely. As a cause hyphaq; reverse pale to brownish. At 30 'C on of food spoilage, Eupenicillium ascospores can be CYA, $olonies covering the whole Petri dish, safely ignored unless an unusual set of similar to those on MEA at 25OC, but often circumstances leads to excessive contamination more dense, enveloping distinct knots of dense of some raw material or product. hyphae. As an example of this genus, one species, Teleomorphic state similar to that of B. fulva Eupenicillium brefeldianum, is described below. exc$t for slightly smaller asci (8-11 pm diam.), This species was isolated from spoiled fruit and ascospores (4-6 pm diamJ, maturing in juice in South Africa (Anon. 1967). Eupenicilliurn brefeldianurn (B. Dodge) Stolk and strawberries; McEvoy and Stuart (1970) Scott reported that strawberry canneries in Ireland Anamorph: Penicillium dodgei Pitt experienced problems with N. fischeri in all but At 25OC, colonies on CYA 30-40 mm diam., one season between 1958 and 1968, despite radially furrowed, consisting of dense, velvety, increases in process severity. Splittstoesser and pale yellow to greyish orange mycelium; Splittstoesser (1977) studied a Neosartorya, probably N. fischeri, from a spoiled fruit drink. cleistothecia abundant, enveloped by the mycelium; conidia sparse; exudate limited to We have isolated this species quite frequently copious, clear; reverse pale to bright yellow, from passionfruit juice, and although it does not more usually ochre to umber. Colonies on appear to be as troublesome in finished product MEA 30-50 mm diam., low, plane and as Byssochlamys fulva, its presence on screening relatively sparse, otherwise similar to those on plates is an indication of potential problems. CYA but reverse usually yellow to yellow Neosartorya includes several species (see brown. At 30°C, colonies similar to those at Raper and Fennel1 1965, under Aspergillus 25OC, except reverse pale to bright orange. fumigatus group), but only N. fishceri is at all Cleistothecia buff to brown, 150-250 pm common. diam., relatively soft, maturing in 2 weelts at Neosaftoryafischeri (Wehmer) Malloch and Cain 25OC; ascospores ellipsoidal, 3.0-4.0 pm long, (Fig. 5) with roughened walls and a faint longitudinal Anamorph: Aspergillus fischeri Wehmer furrow. Conidiophores borne from aerial On CYA and MEA at 25 C, colonies hyphae, long and slender, 50-200 pm long, 50-65 mm or more in diam., of low and sparse smooth walled, bearing simple penicilli of to moderately deep and cottony white to cream phialides only; conidia subspheroidal to mycelium, surrounding abundant white ellipsoidal, 2.5-4.0 pm long. developing cleistothecia and overlaid by Distinguishing characteristics - Eupenicilliurn scattered, usually inconspicuous blue to green species are distinguished in general terms by conidial heads; reverse pale to yellow. At 30°C, relatively slowly growing, compact, brightly colonies covering the whole Petri dish, similar coloured colonies which produce hard, to those at 25OC, but often deeper and more spherical to ovoid cleistothecia in 1-2 weelts, and luxuriant. usually a sparsely-produced Penicillium Cleistothecia white, 300-400 pm diam., anamorphic state. maturing in 1-2 weeks at 25OC; ascospores ellipsoidal, overall 6-7 X 4-5 pm, ornamented Genus Neosartorya C. R. Benjamin with two prominent, sinuous, longitudinal A genus of soil fungi, Neosartorya is of interest ridges and usually with other irregular ridges here because of its highly heat-resistant as well. Anamorph Aspergillus, with sparse ascospores. It occurs from time to time in heat- conidiophores, 300-1000 pm long, terminating processed foods and has occasionally been in small swellings, 12-18 pm diam.; phialides reported as a cause of spoilage. Kavanagh et al. crowded, 5-7 pm long; conidia spheroidal, (1963) isolated it from spoiled canned 2.0-2.5 pm diam., with finely roughened walls.

Fig. 5. Neosartorya fischeri (a)colonies on CYA and MEA, 25OC, 7d.; (b)conidiophore. 80 Fig. 6. Talaromyces species: (a)-(c)T. flavus; (d)-(f) T. bacillisporus. T. flavus: (a) colonies on CYAand MEA, at 25"C, 7d.; (b)penicillus; (c)ascospores. T. bacillisporus: (d)colonies on CYAand MEA, 25OC, 7d; (e)penicillus; (f) ascospores

Distinguishing characteristics. - This is the only based products. No significant studies on the common heat-resistant fungus with an heat resistance of ascospores of T.jlauus or Aspergillus anamorph. Colonies grow rapidly other Talaromyces species appear to have been and are persistently white; cleistothecia are published, but it is significant that all known abundant and produce highly-ornamented heat-resistant isolates have possessed larger ascospores. than normal ascospores. The more common, smaller-spored isolates of T.jlauus presumably Genus Talaromyces C. R. Benjamin possess a much lower heat resistance. The name Talaromyces is derived from the A second species of interest here is T. Greek word for a basket, and aptly describes bacillisporus, a rare fungus with a Geosmithia the body in which this teleomorphic genus anamor~hwhich we have isolated on several produces its asci. Known as agymnothecium, this occasions in screening fruit juices for heat- body is composed of fine hyphae woven into a resistant fungi. more or less closed structure of indeterminate size (Fig. 2b). Talalamyces flauus (Klocker) Stolk and Samson Talaromyces is characterized by the (Fig. 6, a-c) production of yellow or white gymnothecia in Anamorph: Penicillium dangeardii Pitt association with an anamorphic state At 25OC, colonies on CYA 18-30 mm diam., characteristic of Penicillium, Paecilomyces or plane, low and quite sparse to moderately deep Geosmithia. It is a genus of about 25 species, and cottony; mycelium bright yellow, less mostly soil inhabiting. By far the most commonly buff or reddish brown, in most commonly encountered species is T.Jauus, and isolates concealing developing gymnothecia; we have isolated it as a heat-resistant species clear to reddish exudate present occasionally; quite frequently from fruit juices and fruit- reverse sometimes yellow, more usually orange, reddish or brown. Colonies on MEA 30-50 mm Distinguishing characteristics. - More rapid diam, generally similar to those on CYA but growth at 30°C than 25OC, dark green reverse gymnothecia more abundant; reverse usually colours and very narrow, cylindrical conidia dull orange or brown, but sometimes deep make Talaromyces bacillisporus a readily brown or deep red. At 30°C on CYA, colonies recognized species. 30-45 mm diam., generally similar to those at 25OC, but sometimes with white or brown References mycelium or overlaid with grey conidia or with Anon. (1967). Unusual heat resistance mould in applejuice. conspicuous red soluble pigment and reverse FoodInd. S. Afr. 19, 55-6. colour. At 30°C on MEA, similar to those at Bayne, H. G., and Michener, H. D. (1979). Heat resistance 25OC, usually producing abundant gymno- of Byssochlamys ascospores. Appl. Enuiron. Microbiol. 3;: thecia: reverse sometimes also red or olive. 449-53. Gymnothecia of tightly interwoven Beuchat, L. R., and Rice, S. L. (1979). Byssochlamys spp. mycelium, bright yellow, about 200-500 pm and their importance in processed fruits. Adu. Food Res. diam., closely packed, maturing within 2 25,237-88. weeks; ascospores yellow, ellipsoidal, 3.5-5.0 pm Beuchat, L. R., and Toledo, R. T (1977). Behaviour of Byssochlamys niuea ascospores in fruit syrups. Tram. Br. long, with spinose walls. Anamorph Penicillium, Mycol. Soc. 68, 65-71. with conidiophores borne from aerial hyphae, Hatcher, W. S., Weihe, J. L., Murdock, D. I., Folinazzo, stipes 20-80 pm long, bearing terminal J. E, Hill, E. C. and Albrigo, L. G. (1979). Growth biverticillate or less commonly monoverticillate requirements and thermal resistance of fungi belonging penicilli; phialides needle-shaped, 10-16 pm to the genus Byssochlamys. J. Food Sci. 44, 118-22. long; conidia ellipsoidal, 2.5-4.0 pm long, with Hull, R. (1939). Study ofByssochlamysfulua and control smooth to spinulose walls. measures in processed fruits. Ann. Appl. Biol. 26,800-22. Distinguishing characteristics. - Relatively Kavanagh, J., Larchet, N., and Stuart, M. (1963). rapidly growing, bright yellow colonies at both Occurrence of a heat-resistant species ofAspergillusin 25' and 30°C. and the Dresence of abundant canned strawberries. Nature(London) 198, 1322. yellow gymnothecia make Talaromycesjlauusa King, A. D., Jr, Michener, H. D., and Ito, I<. A. (1969). distinctive species. Control of Byssochlamys and related heat-resistant fungi in Talaromyces bacillisporus (Swift) C. R. Benjamin grape products. Appl. Microbiol. 18, 166-73. (Fig. 6, d-f) McEvoy, I. J., and Stuart, M. R. (1970). Temperature tolerance ofAspergi1lu.rfischeri var. glaber in canned Anamorph: Geosmithia swifiii Pitt strawberries. Irish J. A~ric.Res. 9, 59-67. At 25'~, colonies on CYA 18-25 mm diam., Murdock, D. I., and Hatcher, W. S. (1978). A simple plane, sparse, cottony; mycelium white to very method to screen fruit juices and concentrates for heat- pale yellow, surrounding abundant developing resistant mold. J. Food Prot. 41, 254-6. yellow gymnothecia; conidia sparse, greyish; Olliver, M,, and Rendle, T (1934). A new problem in fruit reverse characteristically very dark green, but preservation. Studies on Byssochlamysfulua and its effect occasionally pale or brown. Colonies on MEA on the tissues of processed fruit. J. Soc. Chem. Znd., 18-25 mm diam., similar to those on CYA London 53, 166-72. except reverse very dark greyish orange. At Put, H. M. C., and Kruiswijk, J. T (1964). Disintegration 30°C, colonies 20-40 mm diam., similar to and organoleptic deterioration of processed strawberries those at 25OC. caused by the mould Byssochlamys niuea. J. Appl. Bacteriol. Gymnothecia yellow, 80-150 pm diam., of 21 53-8. Raper, K. B., and Fennell, D. I. (1965). "The Genus fine, closely interwoven hyphae, maturing in 2 Aspergillus". (Williams and Wilkins: Baltimore). weeks at 30°C, but only sporadically after long Richardson, K. C. (1965). Incidence of Byssochlamysfulua in intervals at 25O or 37OC; ascospores spherical, Queensland-grown canned strawberries. Queensl.J. 3.5-4.5 pm diam., with spin~sewalls. Agric. Anim. Sci. 22, 347-50. Anamorph Geosmithia, with condiophores borne Splittstoesser,D. E, and Splittstoesser, C. M. (1977). from aerial hyphae, stipes 20-50 pm long, with Ascospores of Byssochlamysfulua compared with those of a thick, smooth to rough walls; penicilli heat-resistant Aspergillus, J. Food Sci. 42, 685-8. monoverticillate or biverticillate, with elements Spurgin, M. M. (1964). Suspected occurrence of rough walled; phialides needle-shaped, 10-12 Byssochlamysfulua in Queensland-grown canned pm long and occasionally up to 15 pm, often strawberries. Qeensl. J. Agric. Anim. Sci. 21, 247-50. with finely roughened walls; conidia mostly Williams, C. C., Cameron, E. J. and Williams, 0.B. (1941). cylindrikal and very narrow, 4.0-5.0 X 1.0-1.5 A facultatively anaerobic mold of unusual heat- pm, smooth walled. resistance. Food Res. 6, 69-73. " BI) Off-flavour in packaged foods*

By E. H. Ramshaw CSIRO Division of Food Research, Highett, Victoria, 3190

Introduction demonstrate that off-flavours can be avoided As one of the most important attributes that by a combination of awareness and careful determine the acceptance of a foodstuff, flavour practice. deserves more than a passing consideration because the absence of desirable flavour or the Techniques presence of an undesirable off-flavour may be As compounds responsible for flavour are the factor that determines the continued usually present at concentrations in the part per prosperity of any sector of the food industry. billion @pb) to part per million @pm) range and Chemicals are responsible for flavour when the amount of foodstuff available for analysis is they are present in a foodstuff above a limiting often restricted to a few grams, methods have concentration - the threshold concentration been developed for the analysis of small (TC) - and the intensity of the flavour can be amounts of material of the order of nanograms represented by the ratio of the actual to micrograms. Typically, an analysis includes concentration in the foodstuff to the threshold collection of a flavour extract by high vacuum concentration in that foodstuff - sometimes distillation, vacuum-steam-distillation or called an bdour unit' or 'flavour unit' headspace flushing; isolation of a flavour (Guadagni et al. 1966). The TC of a chemical essence from the aqueous phase by solvent will be different in various foodstuffs which extraction or acidlbase extraction; separation of have different proportions of fat, water and and identification of the individual components protein. It is necessary to remember that an off- by combined gas chromatography and mass *I) flavour, by definition, is a flavour that should spectrometry or gas chromatography with a not be present in a specified food, and a selective detector (e.g. electron capture for component which may be a desirable flavour in chlorophenols). The choice of the method of one matrix may be unacceptable and an off- analysis depends on the compound and the food flavour in a different one, or at a different matrix and is dictated by the flavour chemists' concentration. For example, 'contents of o-, rn- experience of similar taints and knowledge of andp-cresol form the main difference between the advantages and disadvantages of each whiskies produced from peated malt and other method for various foodstuffs. Thus, styrene whiskies' and 'it is possible that phenols may be collected from dairy products by contribute in a subliminal manner to the aroma vacuum distillation or by headspace flushing of Scotch Whisky' (Lehtonen 1982) but the whereas phenolic contaminants are removed occurrence of phenols or cresols in a dairy from dairy products more easily by vacuum- product at levels much above their TC causes steam-distillation using the apparatus shown in an unacceptable off-flavour defect. Fig. 1 (Forss and Holloway 1967). Typically, 2 1 This paper considers two types of off-flavour of distillate is collected in D by passing steam that have been observed many times in many through 0.5-1 1of an aqueous solution or slurry foodstuffs in Australia during the last twenty of the dairy product. Refluxing the distillate in years: styrene contamination and phenolic D through the vertical condenser at O°C allows contamination. The examples are used to the more volatile components to be taken off through capillary E and concentrated in 2-20 m1 *This paper is based on a talk presented to 'Horizon 90' the of water collected in trap G. Acidic and phenolic 16th Annual Convention of the Australian Institute of Food compounds remain with the less volatile or Science and Technology on 1 June 1983 in Melbourne. more hydrophilic components in D. The Readers should also refer to the paper 'Some flavours chlorophenols may be isolated by solvent which industry could well do without: Case studies of extraction of the aqueous phase at high pH to industrial problems' by Dr E B. Whitfield, which appeared remove non-acidic components followed by on pages 96-106 of the December 1983 issue of Food Research solvent extraction at low pH to collect the Quarterb. chlorophenols. Difficulties arise frequently CSIRO FoodRes. Q. 44 (4), 83-88 Fig. 1. Reduced pressure steam distillation of volatiles from dairy products and concentration of distillate. when compounds which play no part in the off- increase in the frequency of lymphocytic flavour are isolated at the same time as the chromosomal aberrations. Polystyrene is the flavour-active compounds due to their chemical second most frequently used polymer for similarities. Thus styrene collected by vacuum packaging foods and it may contain a residue of distillation or headspace flushing may be unpolymerized styrene monomer. At levels accompanied by large amounts of other below a toxicological threshold, styrene may be hydrocarbons, while phenolic compounds safe for human consumption but the isolated from dairy products by steam contamination will cause an off-flavour that distillation and acidlbase extraction will be will result in rejection of the foodstuff by the accompanied by large amounts of alkanoic consumer. In the sixties, styrene was a acids. Determination by gas chromatography troublesome contaminant that was isolated at with an electron capture detector can be the Dairy Research Laboratory (DRL) from complicated by the presence of large amounts of tins of shortbread biscuits (source: plastic compounds that show relatively high electron wrappers), fish fingers (source: transport capture response (e.g. plasticizers). containers) and butter (source: shipping As in all flavour research, it is prudent to containers). Characteristically, it was found in monitor the analysis by organoleptic evaluation the corners of the surface layer of the 56 lb of the isolated extract and the residue to ensure blocks of butter at higher levels than in the that the off-flavour has been removed from the centre of the butter block (CSIRO Division of food and collected unaltered. For final Dairy Research 1969). The off-flavour defect confirmation of the identity of the flavour, the caused rejection of the butter by the customer. pure compound is added to the uncontaminated Many studies have shown that styrene, foodstuff to confirm qualitatively the nature of present in packaging polymer as unreacted the off-flavour and quantitatively its occurrence residual monomer, migrates into the foodstuff above the TC. during storage. The strength of the off-flavour that develops depends on the concentration in Styrene contamination the packaging polymer, type of food matrix, Styrene has a characteristic unpleasant, time of contact and TC in the foodstuff. plasticdike chemical odour or taste and has Reported TC vary from 0.005 ppm in sour been blamed for several adverse health effects cream (Miltz et al. 1980) to about 5 ppm in (see, for example, Varner et al. 1983). butter (CSIRO Division of Dairy Research Occupational exposure to high concentrations 1969) and in one study varied from 0.2 ppm for of styrene vapour has caused eye, nose, throat tea through 0.5 ppm for yoghurt to 1.2 ppm for and skin irritation, toxic effects on the liver, whole milk (Jenne 1980). These results imply depression of the central nervous system and that the TC is higher in higher fat products making styrene defects less noticeable in high The potential for off-flavour occurrence is fat products. On the other hand, styrene increased greatly when they are chlorinated to migrates faster from the package polymer into form chlorophenols, which have a similar higher fat products. These two opposing medicinal flavour at a much lower TC. Further influences prevent accurate prediction of the reaction by methylation results in likelihood or strength of off-flavour chloroanisoles which are among the most development in any system except by potent flavouring compounds known but which experiment. A more useful approach is to limit are quite different qualitatively, having musty, the level of free styrene in the polymer: Rosli mouldy, earthy odours. Thus an acceptable and Marek (1977) concluded that the level of level of free phenols (i.e. non-tainting, below the styrene in dairy products rarely exceeded 0.05 TC) may be changed during processing (e.g. ppm if the level in the polymer was below 1000 chlorination by residues of in-plant sanitizers) ppm. In practice, empirical standard methods into a potent off-flavour, causing a severe of assessment have been devised to simulate problem. Similarly, chlorophenols may be storage and consumer reaction (British changed by fungal methylation during storage Standards Institution 1964, German into chloroanisoles resulting in a musty, mouldy Democratic Republic 1979, Standards off-flavour. Although the dairy industry has Association of Australia 1983). For example, to learnt to avoid direct contamination from assess the danger of the taste being transmitted causes such as phenols in the water supply, in the vapour phase to a fatty food of low water chlorophenols in teat-dips, phenols or content, a 10 dm2specimen of the packaging chlorophenols in boiler-water algicides, the material is placed in a closed glass container food industry has not learnt to recognize the with 50 g of grated milk chocolate. This is cause of musty, mouldy off-flavours as trace stored under ambient conditions for at least 48 amounts of chloroanisoles. Indeed, it is only in hours together with an identical test container recent times that sufficiently sensitive and of a reference material free from foreign tastes selective methods have been available to and aromas. The deviation of the experimental identify positively the chloroanisoles causing chocolate from that of the reference is musty taints in foodstuffs such as wine or milk determined by a panel of at least three assessors powder. using a scale from 0 (none) to 4 (pronounced). Such tests should become part of a code of Pentachlorophenol usage practice agreed on between package suppliers Many of the recent problems may be traced and food manufacturers and can reduce to the use of pentachlorophenol as a biocide. As greatly, if not eliminate, complaints of off- supplied, it contains up to 20% flavours due to packaging materials tetrachlorophenol with smaller amounts of (Goldenberg and Matheson 1975). trichlorophenol and hence, if methylated, may The causes and occurrence of styrene cause contamination with pentachloroanisole, contamination have become well-known to tetrachloroanisole and trichloroanisole. PCP* package and food manufacturers and the food is registered in the U.S.A. and the U.K. for use industry has learnt to avoid them. Polymers as a preservative, insecticide, herbicide, used in packaging now contain less residual microbiocide, fumigant, anti-mildew agent, styrene monomer and shipping containers are moth-proofing agent, paint preservative, anti- cured more completely. Food manufacturers fungal agent, and slimicide (Crosby 1981). can select packaging materials by using the Crosby (1981) suggests that the total world methods described in the standards referred to production of PCP rose to 5 X 107kg per above or specify that the packaging material annum with about 23 X 106kg per annum used must meet the relevant standard. Nevertheless, in the U.S.A. where 194 of the 468 wood- in an innovative world where new treatment plants used PCP (Cirelli 1978). manufacturers, new foodstuffs and new Similarly, most of the 5-6 X 106lb PCP per packaging ideas abound, constant vigilance is year used in Canada were applied to the necessary to avoid styrene off-flavours and the protection and preservation of wood products economic loss they cause. (Hoos 1978). In Japan, PCP has been an important herbicide and about 15 X 103tons Phenolic contamination were used on the rice crop in 1970 but usage has ~riephenols have a characteristic chemical, decreased because of its toxicity to fish medicinal, or antiseptic-like odour often (Kuwatsuka and Igarashi 1975). perceived more readily as a taste associated Although usage has dropped in the last few with a tingling sensation on the tongue. They are responsible only rarely for flavour defects in *In this paper PCP denotes the mixture of perlta-, tetra- foodstuffs as they have a relatively high TC. and trichlorophenols. years PCP does not degrade readily and Eventually, the dates when the external air had sufficient is present in the environment to cause a strong taint were compared with the factory concern because of such undesirable effects as records and this showed that complaints were toxicity to fish. Similarly, it is being recognized received only for biscuits that had been actually slowly that PCP can be recycled, for example in baked, processed or handled on those days, and PCP-treated wood or waste paper converted were unrelated to the type or age of the biscuit. into packages and animal hides, scraps or waste The contaminant, 6-chloro-o-cresol,caused a converted into animal foodstuffs. The residues clear, disinfectant-like off-flavour at a of PCP do not cause off-flavours but their concentration of only 0.0001 ppm. This report conversion into anisoles, with or without shows how difficult it is to determine the causes dechlorination, could produce the potent musty of off-flavours that occur intermittently unless off-flavours that cause much commercial good factory records and a representative heartache and significant loss of revenue - selection of 'library' samples are available. without a clear culprit on whom to pin the Chemical identification of the off-flavour commercial loss. component may be only one clue among many in the detective work necessary to pin-point the Occurrence of phenolic contamination cause or origin of a defect. In order to demonstrate the breadth of the The environment was blamed in a similar problem and draw attention to the need to manner when cheese was matured in a room control all facets of the production process, the whose floors had been treated with a phenol- following examples have been drawn from the based resin. The cheese developed a literature, as well as DRL experience. characteristic off-flavour and was found to contain up to 30 times the TC of phenol Raw materials (Toppino et al. 1977). Chicken meat and eggs developed It is interesting to note an experimental chloroanisole-type musty flavours as a result of study in which a piece of wood treated with using litter of wood shavings contaminated with preservative was placed in a closed vessel with PCP and in one study 32 broiler houses had 10 g of flour but out of physical contact (Kroyer litters with mean concentrations of 53 pprn et al. 1982). Over a period of 24 hours the flour tetrachlorophenol and 12 pprn picked up as much as a microgram of pentachlorophenol (Parr et al. 1974). Similar pentachlorophenol - a solid having a melting flavours were caused by feeding a proprietary point of 190°C, and a boiling point of 300°C, feed which included fleshings from hides but having a significant vapour pressure at treated with PCP (Scott et al. 1981). room temperature (Crosby 1981). Of special Gouda cheese manufactured in Holland importance to the food chemist is the fact that using rennet contaminated with lactobacilli the flour picked up more pentachlorophenol had a musty, stockyard flavour. This flavour than did aluminium oxide in similar was attributed to the production ofp-cresol by conditions. the lactobacilli (Badings et al. 1968). A similar defect in Australian Cheddar cheese was Manufacturing analysed at DRL and in both casesp-cresol Phenols or chlorophenols may contaminate a was isolated and was found to be above its TC pure product by accident during manufacture of 0.3 ppm. when contaminated ingredients are used, e.g. Mushrooms contained up to 0.2 pprn gelatin may contain PCP at pprn levels (Stijve pentachlorophenol after growth in wooden 1981). In one problem examined at DRL a large containers which were found to contain up to batch of product was ruined by the use of an 3400 pprn of pentachlorophenol (Meemken et ingredient packed in multiwall bags that had al. 1982). been standing on a pallet contaminated with phenol. Another cause of frequent problems in Environment the past has been the use of phenol compounds In a classic study of the cause of an off- as algicides or mould inhibitors in boiler water. flavour in biscuits, Goldenberg and Matheson A slight leak from boiler condensate or even the (1975) traced a disinfectant, antiseptic soapy direct use of live steam from such boiler water taste t~ airborne contamination from a has been responsible for medicinal off-flavours herbicide factory several miles distant. The in the product. case history showed more than 300 complaints had been received intermittently over 11 years. Packaging Baking tests did not implicate the raw It might be expected that the purest and ingredients, the water supply or contamination safest package, from a flavour viewpoint, would during manufacture and processing. be a glass jar but in one investigation the lids of preserving (Mason) jars were found to contain isolated from both the products mentioned up to 100 pg pentachlorophenol, presumably above. from biocide treatment. The preserved vegetables acquired up to 40 ppm Conclusions pentachlorophenol (Heikes and Griffitt 1980). This short presentation by no means Care is required with paper bags and cartons exhausts the list of known pathways of i since recycled paper may contain PCP and bags contamination. It demonstrates that there is the made from virgin paper were found to contain potential of a very serious problem, which could ! significant amounts of PCE The PCP was cost millions of dollars. Food manufacturers concentrated along the seam. It had been added must become aware of possible problems and to the glue as a mould inhibitor. While PCE as must appreciate the value of good factory l1 such, may not cause off-flavour in these records. Modern methods of analysis are very instances, there is circumstantial evidence powerful and a contaminant may be identified I: suggesting that it could be converted within days, but its identity may be of no value biologically to chloroanisole and cause musty if the history of the product, the ingredients or flavours. the packaging are not known. In these circumstances it may not be possible even to Presentation hazard a guess at the origin of the taint or to Many studies have emphasized the dangers make recommendations for its prevention, until of off-flavours from the solvents or printing inks it has occurred again. Whitfield (1983) in a associated with labelling and presenting the recent issue of this Journal has discussed some product. These off-flavours may be avoided similar off-flavours and recommended quite easily by prudent management and they precautions to avoid phenol, chlorophenol or should not cause problems. However, DRL has chloroanisole contamination. In summary, they examined icecream that had a medicinal demand that the food industry take positive I flavour caused by phenol. Phenol was present steps to ensure that such compounds are not in the glue used to stick the label onto a plastic added to foodstuffs during their processing, ice cream container and it migrated rapidly packaging and storage. A number of products through the plastic. have maximum limits for PCP and it may be necessary for the food industry to enforce Storage similar limits on raw materials from its Several occurrences of musty flavour in suppliers. products during storage andior transport have proved puzzling in recent years. In two References I instances examined at DRL products were Badings, H. T, Stadhouders, J., and Duin, H. van. (1968). 'I rejected for musty flavour on arrival at their Phenolic flavour in cheese.J. Dairy Sci. 51, 31-5. destinations while samples of the same British Standards Institution (1964). The assessment of production run, either held as 'library' samples odour from packaging materials used for foodstuffs. Brit or transported to different markets were found Stand. 3755: 1964. llpp. to be good quality and not musty. In both cases Cirelli, D. P (1978). Patterns of pentachlorophenol usage in 2,4,6-trichloroanisole could be isolated from the the United States of America - an overview. In musty-flavoured product but not from the Pentachlorophenol:chemistry, pharmacology and product of good quality. In the one case environmental toxicology, ed. K. R. Rao Plenum, NY, followed through, the package was found to be pp. 13-18. contaminated with PCP and the problem Crosby, D. G. (1981). Environmental chemistry of disappeared when packages not contaminated pentachlorophenol. PureAppl. Chem 53, 1051-80. with PCP were used. Coupled with the CSIRO Division of Dairy Research (1969). Examination of investigations of many workers into the taints caused by shipping containers. p 35. Annu. Rep. formation of chloroanisole from chlorophenol (CSIRO Melbourne). by fungal action, this suggests that the Forss, D. A., and Holloway, G. L. (1967). Recovery of chloroanisole has been formed during storage volatile compounds from butter oil. J Am. Oil Chem. Soc. andlor transport. 44, 572-5. It is difficult to prove the total chain of German Democratic Republic, Amt. fur Standardisier~n~, evide~ceunequivocally since the amounts of Messwesen und Warenprufung (1979). Evaluation of the chloroanisole are so small that it is difficult to influence on taste and smell of the product by packaging rule out alternative sources - probably materials, packages and auxiliary packaging materials. experiments with radioactive tracers would be DDR Stand. TGL 35703.4~~. necessary. Nevertheless, DRL has made some Goldenberg, N., and Matheson, H. R. (1975). 'Off- progress by showing that organisms capable of flavours' in foods, a summary of experience: 1948-74. metabolizing and existing on PCP could be Chem. Znd. (London), 551-7. Guadagni, D. G., Okano, S., Buttery, R. G., and Burr, H. threshold of styrene and the monomer migration from K.(1966). Correlation of sensory and gas-liquid polystyrene food packages. J. Food Proc. Pres. 4, 281-9. chromatographic measurements of apple volatiles. Food Parr. L. J., Gee, M. G., Land, D. G., Robinson, D., and Technol. 20, 518-21. Curtis, R. E (1974). Chlorophenols from wood Heikes, D. L., and Griffitt, K. R. (1980). Gas-liquid preservatives in broiler house litter.J. Sci. FoodAgric. 25, chromatographic determination of pentachlorophenol in 835-41. Mason jar lids and home-canned foods. J. Assoc. Off. Rosli, M,, and Marek, B. (1977). Ruckstande von Styrol- Anal. Chem. 63, 1125-2 Monomer in Polystyrol und in Milchprodukten. Mitt. Hoos, R. A. W. (1978). Patterns of pentachlorophenol usage Geb. Lebensm. Hyg. 68, 440-2 in Canada - an overview. In Pentachlorophenol: Scott,J. A., McLenaghan, C. C., and Lanthier, J. S. (1981). chemistry, pharmacology and environmental toxicology, Pentachlorophenolcontamination of a layer feed and ed. K. R. Rao. Plenum, NY, pp. 3-11. traceback to hide fleshings. Proc. 16th Annu. Workshop Jenne, H. (1980). Polystyrol und Polypropylen als Pestic. ResidueAnal. (West Can.), 25-32 (CA 96, 50791m). Spritzgussund Teifziehmaterial fur Molkereiprodukte. Standards Association of Australia (1983). Materials used Dtsch. Molk. Ztg 101, 1906-10. for the packaging of food and beverages - methods for Kroyer, G., Washuttl, J., Schorgrnayer, W, Steiner, I., and the assessment of odour and taint. Aust. Stand. 2609.1 - Winker, N. (1982). Modellversuche uber die 1983. 12pp. Kontamination von Lebensmitteln mit Bioziden St'Jve, T (1981). Determination of pentachlorophenol and fluchtigen Stoffen aus Holzschutzmitteln. Lebensm. Wiss. 2,3,4,6-tetrachlorophenolin edible gelatins. Dtsch. Technol. 15, 111-2. Lebensm. Rundrch. 77 249-53. Kuwatsuka, S., and Igarashi, M. (1975). Degradation of Toppino, F! M,, Nani, R., and Francani, R. (1977). PCP in soils. 11. The relationship between the Indagine su formaggio "Taleggio" inquinati da fenolo. degradation of PCP and the properties of soils, and the Riu. Latte 33, 6-8. identification of the degradation products of PCF! Soil Varner, S. L., Breder, C. V, and Fazio, T (1983). Sci. Plant Nutr. (Tokyo) 21, 405-14. Determination of styrene migration from food-contact Lehtonen, M. (1982). Phenols in whisky. Chromatographia polymers into margarine, using azeotropic distillation 16, 201-3. and headspace gas chromatography J. Assoc. OJ! Anal. Meemken, H. A., Furst, El, and Habersaat, K. (1982). Chem. 66, 1067-73. Nachweis und Bestimmung von Pentachlorphenol in Whitfield, E B. (1983). Some flavours which industry could Kulturchampignons. Dtsch. Lebensm. Rundsch. 78, 282-7. well do without: Case studies of industrial problems. Miltz, J., Elisha, C., and Mannheim, C. H. (1980). Sensory CSIRO Food Res. Q, 43, 96-106.

News from the Division

Review of the CSlRO Division of Food Research Dr K. A. Ferguson and Mr A. W Charles, It is the practice in CSIRO for Divisions to be Director and Secretary respectively of the 'reviewed' by an external committee in CSIRO Institute of Animal and Food anticipation of the completion of a Chief's term. Sciences, will be Chairman and Secretary of Accordingly the CSIRO Executive has decided the Committee. that the Division of Food Research will be The Review Committee's terms of reference are reviewed early in 1985, by a committee shown on page 96. comprising the following: Dr A. Skulberg, Director, Norwegian Food Research Institute, As, Norway - a food CSIRO has invited interested organizations technologist and ex-Member of Parliament and individuals to send comments relevant to Dr A. J. Bailey, Director, AFRC Meat the review to the Committee's Secretary; the Research Institute, Langford, UK - a Committee itself is likely to visit all of the biochemist and meat scientist Division's laboratories during March, 1985. Mr E Seale, Chief Chemist, Golden Circle Cannery, Brisbane - a food technologist and fruitand vegetable processor New llR Award Professor B. A. Stone, Foundation Professor The Australian National Committee of the and Chairman, Department of International Institute of Refrigeration has Biochemistry, La Trobe University, Victoria created an annual award to be called the James Mr E. W Barr, Chairman, Australian Dried R. Vickery Award for outstanding Fruits Corporation and a former senior achievements in the application of refrigeration executive with H. J. Heinz & Co. to the preservation of foods and food products. By K. C. Richardson CSlRO Division of Food Research, North Ryde, N.S.W., 2113

Many social and economic changes in answer these demands but they frequently Australia in the last few decades have mirrored require additives to do so. those occurring in other developed countries. These changes have led to an increasing role for Principles governing the use of food additives manufactured and processed foods in the diet. Australia uses as the basic element of its food With this increase in consumption of foods law the system of prohibition. In this system that have been partly prepared outside the anything that is not expressly authorized is home, there has been a growing interest at prohibited, and the Model Food Act and Model many levels in the nutritional value and safety Food Regulations developed by the National of foods, albeit accompanied by some unease Health and Medical Research Council (1983) about manufactured products. At times this state: 'Save where it is expressly permitted by unease develops into distrust of and even these regulations, the addition of a food additive hostility towards these foods and the industry . . . is prohibited'. The system involves the that produces them. Often, it seems that the preparation of lists of permitted food additives more remote that people are from food in order that approved substances may be production and preparation, the more added legally to specified foods. suspicious they are about how it is done. The classes of food additives described in the One aspect of this interest in and concern Model Food Regulations are shown in Table 1. about thefood supply has been concern over the h use of food additives. This is not to suggest that TABLE 1 i' the use of food additives or concern about them Classes of food additives described in the Model Food Regulations is new. Food additives, including some used today, have an ancient and, as critics are quick to point out, not always honourable history. If Class of additive Property of food influenced we include such substances as salt and vinegar, which are not included in the definition of Preservatives Shelf life preservatives in Australian food law, this Colourings Appearance history goes back still further. Flavourings Flavour It is true that with the growth in sales of Antioxidants Shelf life convenience foods, i.e. those that require little Artificial sweeteners Flavour, energy value or no preparation in the home, a greater range Vitamins and minerals Nutritive value of foods contains additives. Without certain Modifying agents classes of additives many convenience foods vegetable gums Texture, appearance could not be offered for sale in their present mineral salts Texture, appearance form and would never have appeared on the food acids Shelflife, flavour, texture market. emulsifiers Texture, appearance It is also evident that the introduction of open humectants Texture, shelf life date marking of foods has led to applications, thickeners Texture, appearance many of which have been approved, for use of preservatives and antioxidants in foods once For many years the Commonwealth and accepted as having a life of only a few days e.g. State Governments have cooperated in health cottage cheese, fruit yoghurt, and 'fresh' fruit matters, including food legislation, through the salad. Retailers and consumers now look for, National Health and Medical Research and demand, shelf life of some weelrs in these Council (NHMRC). The NHMRC relies on and similar products. Manufacturers can its Food Science and Technology Subcommittee 'This paper was presented to the 11th National Conference (FST) which reports to the Food Standards of the Australian Institute of Health Surveyors, Committee (FSC) for an evaluation of potential Launceston, Tasmania, 7-12 October, 1984. new additives and also for a continuing review CSZRO FoodRes. Q. 44 (4), 89-94 of the use of currently permitted additives. instances Australian authorities, in common with iv) the results of their own investigations. similar bodies in other countries, are guided by An essential role of the toxicologists on the the reports of the Joint FAOIWHO Expert FST is to identify poor data and unsound Committee on Food Additives UECFA). judgments, i.e. data derived from poorly Although they are independent of decisions constructed or executed experiments and taken in other countries on the use of food judgments based on such data. The banning in additives, in no sense are local authorities the U.S.A. in 1976 of the red food colour isolated from what is happening at a legislative amaranth is an illustration of a decision being or scientific level overseas. They must, taken on the basis of poorly designed and however, take into account patterns of food conducted studies (Fairweather and Swann production and usage which may be peculiar to 1981). The study on which the ban was based this country. was faulty in two important respects: (l) post- At the first meeting of JECFA in 1956 a mortems of dead rats were not carried out in number of principles were elaborated to be sufficient time to prevent serious autolysis, observed in evaluating any substance to be (2) at one stage in the study several rats were used as a food additive (FAOIWHO 1957). placed in the wrong cages thus producing These principles can be summarized briefly as: interchange. - of some treatment and control l) a technological need must be established for animals. the use of an additive A revised statistical analysis, which has been 2) the safety in use of the additive must be challenged, revealed that although there was no established. increase in total number of tumours in test Protection of the nutritive value of a foodstuff animals, the ratio of malignant to benign is inherent in the second principle. tumours did increase in female rats given diets Food additive legislation has been attacked containing 3% amaranth (the highest dose). Few on both scores. Critics argue that technological countries followed the American action yet this need has been inter~retedfar too widelv.,, particular trial is still used to suggest that particularly with regard to colours and Australian authorities were less than diligent in flavours. The same critics also argue that the not removing amaranth from the permitted results of animal feeding trials cannot be listings (Anon 1984). directly translated to consumption by humans, and that interactions between different dietary Establishing a technological need components are not studied adequately. When considering an application to use a Epidemiological studies are used to supplement food additive, it is usual for the FST to consider feeding trials where additives have been in use first the question of technological need. To for long periods in defined populations, e.g. establish a technological need, a petitioner has saccharin, but results of these studies are often to demonstrate that a satisfactory product equivocal. (Fairweather and Swann 1981.) cannot be presented to the consumer by the use of alternative technology. This term is used to Assessment of a food additive include physical means of processing, the use of With this background in mind, the FST new or more modern equipment or improved examines applications for the use of a food quality control. additive in a specified food. The application The use of potassium sorbate as a mav be for a new food additive not examined preservative in cottage cheese, already referred previously - these are now comparatively rare to as a recent change in regulations, can be except in the artificial sweetener field - or for used to illustrate some of these points. the use of an approved additive in a food in Potassium sorbate, at relatively low which it has not ~reviouslvbeen ~ermitted. concentrations. is an effective ~reservative e.g. potassium sorbate is widely used as a against mould growth. It is widely used preservative in fruit juices, cheese in flexible throughout the world for this purpose and it has packages and baked goods and its use has been allocated a relatively high acceptable daily recently been extended to cottage cheese. intake (ADI*) by JECFA because of its very low Members of the FST committee have to toxicity in animal studies. There is no evaluate immediate concern that the potential daily i) information supplied in support of a food intake of the additive will exceed the acceptable additive petition by the applicant daily intake. ii) the reports of JECFA iii) the scientific literature on relevant areas of *AD1 expressed as mglkg body weight is defined as the research particularly that published since amount of a chemical which might be ingested daily, even the latest JECFA report, and in many over alifetime, without appreciable risk to the consumer. An applicant who wished to use potassium The most important of these - and the one sorbate in cottage cheese made reference to a about which the others revolve - is that food number of features of modern food additives may not be used to deceive the manufacture and distribution that were consumer. When one studies the list of relevant to the request for the use of the standards for foods to which prescribed preservative in a product the name of which colourings may be added (National Health and indicates its humble origins. Medical Research Council 1983), it is clear that Manufacture of cottage cheese is now the majority of foods listed are such that a concentrated in a few large manufacturing serious question of deception does not arise, plants that supply one or more States. Direct e.g. with confectionery, cordials, dessert and deliveries from manufacturer to store are custard mixes, flavoured milk, ice cream and becoming uncommon as supermarket chains soft drinks. move to bulk warehousing. When the However, direct applications for the use of supermarket takes delivery of the product it colour (and flavour) in foods are rare indeed. requires a substantial proportion of the Most new products containing these additives nominated shelf life to be intact to suit its own find their way on to the market under the distribution and retailing practices. The Standard for Foods not Elsewhere consumer, not surprisingly, also has certain Standardized (National Health and Medical expectations about how long the product may Research Council 1983). This is a catch-all be kept before its 'use by' date expires. Standard designed to facilitate the marketing of Cottage cheese is therefore anything but new products without forcing manufacturers to 'cottage' cheese in a manufacturing sense and wait for an indefinite period before gaining to reach the urban shopper and consumer in specific approval to market each new product. consistently sound condition, some preservative Some which survive in the market place will is now necessary. The applicant made available ultimately have a standard described for them details of the process, including the quality but many more never achieve this status. control procedures used and the steps that had The Standard for Foods not Elsewhere been taken to minimize the initial level of Standardized permits the addition of permitted mould contamination. The nature of the colouring, flavouring and modifying agents. product precluded the use of a pasteurization A manufacturer seeking to use an additive step to inactivate contaminants introduced after outside these classes would have to seek specific the initial heat treatment of the ingredients. permission to do so. The option of nominating a lesser shelf life, Some of the objections to this broad brush for a product free from preservative, was also approach are overcome by the requirement to addressed. It was claimed that this would lead list all ingredients on the labels of packaged a) to the major distributors declining to foods. However, some foods are exempted from handle the product, this requirement, e.g. alcoholic beverages and orb) the eventual total rejection of the product most cheeses, and the listing of food additives by consumers because of its unreliable need not be specific. This means that class quality names such as 'colouring' and 'flavouring' may The application was duly approved and in some be used. This approach is clearly regarded as respects typifies the changes in food production unsatisfactory for some consumers. and marketing that have resulted in the While added colours are examined increased use of food additives. toxicologically in the same way as other less controversial food additives, flavours, in Colours and flavours general, are not. There is no list of permitted No comparable technologicaljustification flavours that may be added to food although in can be made for the use of colours and flavours. New South Wales, for example, there is a short This does not mean that to obtain permission list of prohibited flavours containing safrole to use a prescribed colouring in a food a and some related compounds (N.SN Pure Food manufacturer has to demonstrate only the Act No. 31, 1908 and Regulations Thereunder, safety in use of that colouring. Revised Issue 1978). When JECFA laid down its principles The food industry defends its use of flavours governing the use of food additives, it regarded and colours by arguing that, when used in a as redsonable the use of additives to make reasonable manner, they: sound food more attractive to the consumer. 1. restore desirable properties lost during However, in describing situations in which the processing use of a food additive was not justified, the 2. overcome undesirable properties resulting Committee set down a number of constraints from processing that are relevant to the use of food colourings. 3. overcome variations in properties to ensure 91 a consistent product alcohol in alcoholic beverages. However, it was I 4. introduce desirable sensory properties into subsequently demonstrated that, under certain W products that are not intrinsically appetizing conditions, the carcinogen urethane could be i (Coulson 1979). formed as a reaction product and permission to It is this fourth use listed above that has use the preservative in beverages was probably caused the strongest reaction to food withdrawn in 1974. With improvements in I colours. They have become recognized as an testing methodology, the possibility of other intrinsic part of many foods regarded as less current additives being removed from nutritionally desirable, e.g. snack foods. permitted listing cannot be ruled out. However, I Concern has been expressed by some groups since they have already met stringent (Commission of the European Communities requirements the possibility that, in practice, 1980) that the use of additives is permitting the they represent a hazard is extremely remote. manufacture of foods that encourage poor Of more concern to some people is the dietary habits. This would seem, however, to be problem posed by immediate adverse reaction a matter for education of the public rather than to a specific food component or additive. for regulatory control. Adverse reaction to food comDonents mav be a cause of chronic or recurrent symptoms and has Adverse reactions to food additives been reported to affect several systems, Cancer has always been a relatively common e.g. skin, gastrointestinal tract and upper disease and despite public concern there is no res~iratorvtract. Some of these reactions are sound evidence that its total incidence is true allergies, i.e. there is a demonstrable increasing. There is an overall decline in the immunological response. Many are more incidence of cancers associated with the accurately termed food sensitivity which implies alimentary tract in England and Wales there is an abnormal but reproducible reaction (Alderson 1980), the United States (Shubick to the ingestion of a specific food or compound 1980) and Australia (Rohan and McMichael but to which there is no immunological 1981). However, such trends conceal changes in response. Concern with food additives falls the death rate from individual cancers within mainly into this area of sensitivity. this group (Rohan and McMichael1981) and Recent reports (Baker, Collett and Allen epidemiological studies are being pursued in 1981; Hill 1982; MacGibbon 1983) g'ive some many countries ('Joossens and Geboers 1981; idea of the nature and extent of the problem McMichael1980) in relation to diet and cancer. and also estimates of the frequency of these A number of major dietary components (e.g. adverse reactions. Knowledge of these reactions fat and alcohol) have been examined and is not new (Hill 1982) but response at the associations have been found between some of regulatory level has probably been delayed for a them and certain types of cancer. At least one number of reasons: major study uoosens and Geboers 1981) has a reaction to food additives appears to be much also found an association between consumption less frequent than allergy to foods of traditional salted foods and gastric cancer. (MacGibbon 1983) Food contaminants of natural origin, particularly the aflatoxins, have been there is no good animal model in which to associated with potent carcinogenicity in man, study these reactions and human studies have in this case reinforcing the conclusions of many been restricted. laboratory studies. The contribution to the total it is only recently that the severity of some of cancer incidence of intentional food additives, these responses, particularly asthma with the possible exception of some component symptoms, have been adequately reported. of traditional salted foods, appears to be nil (Shubick 1980). Labelling These findings will, however, do little to It is now generally accepted that allay the fears of that section of the community comprehensive listing of ingredients on the that is influenced by the questionable public labels of packaged foods, which would include reporting of food safety matters. all additives as well as major components, is There have, of course, been a few instances desirable and necessary. Since early this where a;permitted additive has been century State food laws in Australia have determined to be a potential carcinogen and on required that foods to which colour, flavour or these occasions permission to use the additive preservative have been added must carry an has been immediately withdrawn. One which appropriate declaration, e.g. preservative comes to mind in Australia is the preservative added. In 1980 the NHMRC produced a model diethyl pyrocarbonate. This was thought to ingredient labelling standard which was break down completely to carbon dioxide and written into law by the individual States. However, as noted previously, this standard claim on the label. This claim takes the form of permitted the use of class names such as colour statements such as 'No preservatives' or 'No or antioxidant rather than requiring the artificial colour or flavour'. All too frequently specific name, e.g. tartrazine or sodium these claims appear on the labels of foods not metabisulphite. permitted to contain the nominated additives. This system is considered inadequate by By implication, products of other companies, those health professionals who have the operating under the same regulations, which do responsibiliG for compiling diets for people no make such a claim could contain these suffering" from food sensitivities. Consumer additives. The vast majority of canned and organizations also complain. frozen foods are not permitted to contain The food industry, however, has been against additives because there is simply no need for labels showing the specific name of food them. Their stability and palatability is ensured additives. Some objections by the industry have by the physical processing they receive. been based on practical grounds, notably that Even when a product is permitted to contain the formulation of a product will vary an additive, it is not unusual to find certain depending on availability and cost of brands of the product manufactured and ingredients. However, the main industry marketed without the additive. An example of objection has been that specific disclosure of this type of product is fruit juice. High additives on the label does not acknowledge the temperature short time processing ofjuice and essential chemical composition of all foods. filling the cooled juice aseptically into pre- This is seen as promoting undue concern about sterilized containers has eliminated the need for additives in the minds of consumers, preservative in this type of pack. However, the particularly as many chemicals used as equipment to execute this process is extremely additives occur naturally, and some of these e.g. expensive and it is not reasonable to require benzoates, are acknowledged as a cause of every processor ofjuice to avail himself of this adverse reations. Functional, added technology. Permission to use preservative in concentrations of these naturally occurring fruit juice, where required, continues. additives are usually higher than would However, for the manufacturer not using otherwise be present, however. preservative to imply, by negative labelling, Furthermore, a group such as the salicylates that his product is perhaps safer than the which in at least one study (Allen et al. 1984) opposition product damages the credibility of was the most common cause of adverse the entire food industry including, ultimately, reactions is widely distributed in plants and his own company. vegetables. The Food Standards Committee of the Conclusion National Health and Medical Research Providing an urbanized society with an Council in 1984 recommended that to hel~ adequate and safe food supply is not a simple overcome some of the problems posed, additives matter but the food processing industry has should be identified on the label of the responded well to the challenge. When packaged foods by either code numbers or individuals in such a society elect to follow specific names in addition to class names. It different lifestyles in which food preparation was suggested that the code numbers be based and consumption patterns vary considerably on the food additive numbering system the task becomes more complex. Food additives published.in directives of the European have come to play an important, but not Economic Community. dominant, role in this situation. If these recommendations are im~lemented in all States, it will permit consumers to identify and avoid foods containing additives to References which they believe they are sensitive. Charts Alderson, M. R. (1981). Nutrition and cancer: evidence listing food additives and their codes will, of from epidemiology Proc. Nutr. Soc. 40, 1-6 course, have to be freely available. While this Allen, D. H., Van Nunen, S., Loblay, R., Clarke, L., and system will not overcome identification Swain, A. (1984). In 'Allergy in Australia' (Eds. B. A. problems completely, it should certainly make Baldo and C. W. Wrigley) Spec. Suppl. Med. J. Aut. the task of health professionals simpler than it is Vol 141, No. 5 P S37-42. at the moment. Commission of the European Communities (1980).. . 'Food additives and the consumer.' (The author: Luxembourg..) Negative labelling Anon. (1984)). Your guide to food additives. Choice 25, 34-8. Unfortunately, one response to the criticism Baker, G. J., Collett, P, and Allen, D. H. (1981). to which the food industry has been subjected Bronchospasm induced by metabisulphite-containing over its use of additives has been the negative foods and drugs. Med. J. Aust. 2, 614-6. Coulson, J. (1979). The use of colours and flavours by the MacGibbon, B. (1983). Adverse reactions to food additives. 0 I food industry. Proc. Inst. FoodSci. Technol. 12, 159-70. Proc. Nutr. Soc. 42, 233-40. ! I Fairweather, E A., and Swann, C. A. (1981). Food additives McMichael, A. J. (1980). Diet and cancer: an I and cancer. PTOC.Nutr. SOC.40, 21-30. epidemiological perspective. Med. J. Aust. 2, 10-6,40. National Health and Medical Research Council. (1983). FAOlWHO Joint Expert Committee on Food Additives ! 'Model Food Legislation. ' (Australian Government (1957). 1st Rep. General principles governing the use of i Publishing Service: Canberra) l food additives. FAO Nutr. Meet. Rep. Ser. No. 15. Rohan, T., and McMichael, A. J. (1981). Alimentary tract Hill, A. (1982). Food additives, allergy and cancer. Proc. cancer mortality in Australia, 1908-1973.Med. J. Aust. 1, I Inst. FoodSci. Technol. 15, 45-50. 232-5. l Joossens, J. V, and Geboers, J. (1981). Nutrition and gastric Shubik, P. (1980). Food additives, contaminants and . ! cancer. Proc. Nutr. Soc. 40, 37-46. cancer. Preu. Med. 9, 197-201.

Meas~~ringthe, age of stored eggs

By M. B. Smith and Ly Nguyen CSIRO Division of Food Research, North Ryde, NSW, 2113

Although eggs are classed as a perishable food, there is often little attention given to their storage conditions in retail outlets. Unlike meat, fish and dairy products, which have had some form of preparation for sale, eggs are sold in their natural 'package' and their condition is only evident to the purchaser at the time of use.

Egg quality S-ovalbumin in eggs It is generally agreed that fresh eggs are more A unique solution to the problem of measuring attractive in appearance, have a better flavour, age is to make use of a built-in 'biological clock' and perform better in cakes and custards than in eggs, which was discovered in this laboratory eggs that have been kept for prolonged periods 20 years ago (Smith and Back 1962); the at room temperature. Most cooks also know reaction in which ovalbumin, the main protein that eggs deteriorate after laying and that their in egg white, is converted to a more heat-stable fresh qualities may be retained longer by form, S-ovalbumin. This reaction occurs when means of cool storage. However, the state of the pH of the egg white rises above 8.5, which it freshness of eggs at the time of purchase is normally does in about three days after laying, uncertain; it is not evident by inspection, and reach in^" a steadv value of 9.0 to 9.3. After this. any 'use by' date is based on unspecified the rate of conversion depends only on storage conditions. temperature, and the rates at different This state of uncertainty about the quality of temperatures may be compared by specifying the product begins with the packer and the times taken for half the ovalbumin to be wholesaler. 'Egg quality' may be assessed by converted to S-ovalbumin. These half-times are examining a reasonably large sample of shown in Table 1. broken-out eggs, e.g. by measuring Haugh The times at 20°C and 30°C are similar in units. However, quality may vary quite widely magnitude to the 'half-times' of acceptable even in fresh eggs, depending on the age and quality in eggs kept at different temperatures breeding of the hens (Shenstone 1967), so that (Coote et al. 1966). It was at one time considered these measurements will only indicate the that there was a connection between storage deterioration of a stored sample if the initial changes such as the thinning of the thick white quality is known. Even then it is not possible to and the weakening of the yolk membrane, and estimate the 'equivalent age' (see below) without the conversion to S-ovalbumin (Smith and Back taking into account other factors such as egg 1965). This theory has not been supported by size and water lost by evaporation. later investigations, and the only effect of CSIRO Food Res. Q 44 (4), 94-96 TABLE 1 affected by changes in water content, mucl1 I Rates of conversion of ovalbumin to more representative results are obtained than I I 8-ovalbumin in vifro by Haugh unit measurements. - This project was supported by a grant from Temperature Time for half convers~onat the Australian Poultry Research and Advisory I Committee. i ("c) pH9 0 pH 9.5 Analytical method 20 13 d 9 d Reagents 30 89 h 47h Phosphate buffer. - 13.7 g NaH2P04.2H20 40 27 h 11 h and 43.1 g Na2HP04in l 1. Dilute 20 times to 50 8.8 h 2.6 h give pH 7.5, ionic strength 0.05. Precipitatingsobition. - 0.5 M NaC1 plus 0.1 M l Na acetate adjusted to pH 4.7 with acetic acid. increased proportions of S-ovalbumin on the Biuret solution. - dissolve 1.8 g CuS04.5H20 culinary properties of eggs is now considered to and 7.2 g NaK tartrate. 4H20in 500 m1 water, be a loss of gelling or thickening properties add 36 g NaOH in 360 m1 H20,make up to 1 1. (Smith and Nguyen 1983). If we measure the proportions of ovalbumin Method

i

the calculation of reaction tim e the pH of the egg white rises to 9 (as carbon dioxide is lost through the shell). However, the half-times for the reaction (i.e. at -log 0.5) are 0.8 in reasonable accord with the half-times for the in vitro conversion at pH 9 shown in Table 1. For a practical unit of egg deterioration we 0.6 suggest that 'equivalent age at 20°C' be used, calculated, on the basis of 13 days half-life at pH 9, by means of the expression: 86-S Equivalent age at 20°C = - 43 log~days where S is the percent soluble determined by 0.2 the above method. References 0 0 5 10 15 20 25 30 Coote, G. G., Prater, A. R., Shenstone, E S., andvickery, J. (1966). A quality survey of shell egg marketing in Day5 Australia. CSIRO Diu. FoodPreseru. Tech. Pap. No. 32. Donovan J. W., and Mapes, C. J. (1976). A differential scanning calorimetric study of the conversion of ovalbumin to S-ovalbumin in eggs. J. Sci. FoodAzric. 27, Fig. 1. Solubility change in egg white proteins after heat 197-204. treatment versus storage time at two temperatures. Shenstone, E S. (1967). Market quality of eggs in Australia. A, Stored at 30°C. @ and 0,two storage experiments CSIRO Food Preseru. Q. 27, 86-94. at 20°C. Smith, M. B., and Back, J. E (1962). Modification of ovalbumin in stored eggs detected by heat denaturation. - log A against t should give a straight line with a slope k which will vary with temperature. Nature 193, 878-9. Smith, M. B., and Back, J. E (1965). Studies on ovalbumin. The results of storage experiments carried out 2. The formation and properties of S-ovalbumin, a more to test this method are shown in Fig. 1. The stable form of ovalbumin. Aust. J. Biol. Sci. 18, 365-77. straight-line relationship is followed up to about Smith, M. B., and Nguyen, Ly (1983). The gelling 30 days at 20' C and 15 days at 30°C, after properties of heat-denatured proteins: ovalbumin and its which the term (86 -S) becomes too small to natural heat-stable form, S-ovalbumin. Proc. Aust. measure accurately. There is also some Biochem. Soc. 15, 22. uncertainty about the zero time for the experiments, because the reaction is slow until

N~WSf r~m the Division (continued from page 88)

Review of the CSlRO Division of Food Research

The Review Committee's terms of reference are: research leadership provided by senior staff; (i) to assess the appropriateness of the (iv) to comment on any other matters peculiar Division's program objectives and its to the Division that may warrant special progress towards attaining them, the attention, as decided by the Executive or quality of the research and the Division's identified by the Committee; ordering of research priorities, and the (v) with due regard to the responsibilities and effectiveness of its efforts to transfer the activities of other groups in CSIRO and results to potential users; other organizations in Australia and (ii) to comment on the resources available to overseas, to comment on the future the Division (i.e. Appropriation1 direction and appropriate institutional contributory funding, staff, facilities, arrangements for those activities for which buildings), the effectiveness with which a continuing need is identified; this may these are utilized and any additional include comment on the continued opportunities for optimizing their use existence of the Division, and the through collaboration or sharing, either assessment and, if necessary, redefinition of internally or externally; the Division's role in contributing to the (iii) to review the management and internal research needs of relevant industry sectors1 organization of the Division, including the community interests appropriate to CSIRO.