Influence of Potassium Sorbate and Sodium Benzoate on Heat Inactivation of Aspergillus flavus, Penicillium puberulum and Geotrichum candidum

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1 45 Journal of Food Protetion, Vol. 44, No.6. Pages (June 1981) Copyright, International Assoiation of Milk, Food, and nvironmental Sanitarians Influene of Potassium Sorbate and Sodium Benzoate on Heat Inativation of Aspergillus flavus, Peniillium puberulum and Geotrihum andidum L. R. BUCHA T Department of Food Siene. University of Georgia Agriultural xperiment Station, xperiment. Georgia 3212 (Reeived for publiation September 29, 198) ABSTRACT xperiments were onduted to determine if two preservatives, potassium sorbate and sodium benzoate, had a synergisti effet with heat on inativation of onidia of Aspergillus flavus and Peniillium puberulum and vegetative ells of Geatrihum andidum. A seond objetive was to determine if heated onidia had inreased sensitivity to preservatives in a reovery medium. As the ph of heating menstrua was dereased from 7. to 2.5, the rates of inativation of molds were inreased. Conidia were not as adversely affeted by aid ph as were vegetative ells. At SO ppm, potassium sorbate "aused a signifiant (P"O.OS) inrease in the rate of thermal inativation of A. flavus and G. andidum; 1 ppm had a signifiant effet on P. puberulum. Sodium benzoate aused signifiant dereases in deimal redution times of A. flavus and P. puberulum when present at a onentration of SO ppm in heating media. Viable heated onidia of A. flavus and P. puberulum had inreased sensitivity to potassium sorbate and sodium benzoate, indiating heat injury. However, the relative effets of the two preservatives on olony formation in reovery agar were reversed from those noted in heating media, i.e.. at omparable onentrations, potassium sorbate was more effetive than was sodium benzoate for inhibiting olony formation. Numerous studies have been onduted to determine the effets of omposition, pretreatment and environmental onditions during and after treatment on survival of baterial spores at elevated temperatures. Comparatively few studies report the behavior of fungal spores and ells as affeted by these parameters. Available information does indiate, however, that several fators influene the tolerane of molds to heat. Doyle and Marth (5) reported that older onidia of Aspergillus flavus and Aspergillus parasitius were less tolerant to heat ompared to young onidia and that trends existed to suggest that onidia whih were produed by molds grown on a medium with a substantial amount of sugar were more heat tolerant than those formed when the amount of sugar was less. Irradiation of onidia of a non-toxigeni strain of A. jlavus has been shown to enhane sensitivity to heat (1). The nature of the heating medium may greatly affet the rates of inativation of molds. High levels of solutes suh as sodium hloride, surose, and gluose afford protetion against heat inativation of aspergilli (6); however, various genera of molds respond differently to solutes at elevated temperatures and some solutes may atually be lethal (3). Changes in sensitivity of molds as the ph of the heating medium is dereased from neutrality are dependent upon the buffering system as well as the type of aid used (6). Splittstoesser and Splittstoesser (13) demonstrated that the survival of asospores of an Aspergillus speies was unaffeted by various aids in the heating menstruum, whereas asospores of Byssohlamys.fulva were learly less tolerant to fumari, lati, suini and aeti aids ompared to mali, tartari and itri aids. Presene of sorbi aid has been reported to redue the heat resistane of Byssohlamys nivea (2) and Aspergillus niger (12) but not Peniillium thomii (12). The present study was designed to determine if two food preservatives, potassium sorbate and sodium benzoate, had a synergisti effet with heat on inativation of onidia of A. flavus and Peniillium pubernlum and vegetative ells of Geotrihum andidum at various ph values. A seond objetive was to determine if heated onidia had an inreased sensitivity to preservatives present in a reovery medium. Data obtained from these investigations would be helpful in reevaluating proess requirements for pasteurizing ertain aid foods. Organisms and ultural onditions MATRIALS AND MTHODS The three molds seleted for examination were A. flavus. P. puberulum and G. andidum. Organisms were ultured on a basal medium ontaining 3 g of yeast extrat. 3 g of malt extrat, 5 g of peptone, 1 g of gluose, and 1 liter of deionized water (YMPG, ph 5.5). Agar (2%) was added to YMPG broth before sterilizing at 121 C JOURNAL OF FOOD PROTCTION. VOL. 44. JUN 1981

2 HAT INACTIVATION OF MOLDS 451 for 15 min, pouring into petri dishes, and allowing to dry at 22 C overnight. Suspensions (.2 ml) of onidia of A. ftavus and P. pubernlum in.1 M potassium phosphate buffer (ph 7.) ontaining.1 o/o Tween 8 (phosphate-tween buffer) were spread on YMPG agar plates followed by inubation for 2 days at 25 C (8o/o relative humidity). The medium used to ulture G. andidum was YMPG broth; 1 ml was dispensed into 25-m! rlenmeyer flasks, sterilized, ooled and inoulated from stok ultures. The mold was prepared for testing by suessively transferring 2-day-old ultures whih had been inubated at 3 C while being ontinuously agitated (15 rpm) on a rotary shaker. Proedure for harvesting spores and ells Conidia from A. ftavus and P. pubernlum were suspended in phosphate-tween buffer by gently rubbing sporulated myelial mats with a sterile glass rod. Conidial suspensions were removed from plates by pi petting and then filtered through sterile glass wool. After washing and filtering two additional times, stok suspensions in phosphate Tween buffer were held at 4 C for a maximum of 12 (P, pubernlum) or 27 (A. flavus) days before subjeting to test onditions. Vegetative ells (46- to 48-h-old) of G. andidum ultured in YMPG broth were diluted 1-fold in.1 M potassium phosphate buffer (ph 7.) before testing for tolerane to heat. Proedure for testing tolerane to heat Sterile phosphate-tween buffer (1 ml in 25-m1 rlenmeyer flasks) was adjusted to ph values ranging from 2.5 to 7. in.5-unit inrements. For tests designeo to evaluate the effets of preservatives on heat inativation. 1-ml quantities of sterile.5, 1., 5. or 1% solutions of potassium sorbate (Monsanto Industrial Chemials Co., St. Louis, MOl and sodium benzoate (Pftzer In., New York, NY) were added to phosphate-tween buffer before adjustment of ph to give final onentrations of SO, 1, 5 and 1 ppm. Heat tolerane of onidia from A. ftavus and P. pubernlum was tested in phosphate-tween buffer, whereas vegetative ells of G. andidum were tested by heating in YMPG broth adjusted at ph 2.5 to 7. and ontaining 5 and 1 ppm of preservatives. The effets of SO and 1 ppm of preservatives were tested only in the ph range of2.sto 4.5. Menstrua (1 ml) in whih mold propagules were to be heated were adjusted to various temperatures, depending upon relative heat resistane (3). in a water bath shaker. Inoula (1 mi) of onidia or vegetative ells (a. 1 7 per ml) were transferred to menstrua and heated for times up to 8 min. At seleted times throughout the period of heat treatment, ponions were withdrawn, immediately transferred to 9- or 99-ml blanks of phosphate-tween buffer (A. flavus and P. pubernlum) or phosphate buffer laking Tween 8 (G. andidum), serially diluted, and plated in YMPG agar (ph 5.5), using a pour-plate tehnique. Colonies were ounted after 3 to S days of inubation at 3 C. Deimal redution times (time required for 9% redution in viable ells) were alulated for eah mold subjeted to eah set of environmental onditions during heat treatment. Sensitivity of heated ells to preservatives in reovery medium Unheated and heated onidia of A. jlavus [2 min in phosphate Tween buffer (ph 7.), 52 CJ and P. pubernlum (2 min, 49 C) were plated on YMPG agar (ph 4.5) ontaining onentrations of potassium sorbate and sodium benzoate ranging to 1, ppm. Colonies appearing within a 21-day period of inubation at 3 C were ounted. Statistial analyses Data presented represent means of a minimum of two repliations done in dupliate. The multiple range test as desribed by Dunan (7} was used to test for statistially signifiant differenes (P.,;O.OS). RSl'LTS A:'\D DISCt:SSIO:'\ Results from studies to determine the effets of ph and preservatives on inativation of molds are presented in Fig. 1. Of the three speies tested, onidia of P. puberulum were the most heat sensitive at ph 7.. As the ph of heating medium was dereased, the rates of inativation of test molds were inreased, i.e., the D-values (time to redue the viable populations by 9%) were dereased. Conidia of P. puberulum and A. flavus appeared not to be as adversely affeted by aid ph as were vegetative ells of G. andidum. - -Q)... - :J '" Q) ::: CJ Q) P. puberu/um, 49 A. f/avus, 52 G. andi dum, ph Figure 1. ffets of ph and preservatives on rates of heat inativation of molds. Symbols:, no preservative present in heating medium; lj., 5 ppm potassium sorbate;. 1 ppm potassium sorbate; A., 5 ppm sodium benzoate; 1 ppm sodium benzoate. JOURNAL OF FOOD PROTCTION. VOL. 44. JUN 1981

452 BUCHAT Supplementation of heating media with 5 ppm of potassium sorbate or sodium benzoate resulted in marked dereases in D-values of all test molds. ffets on P. puberolum and A.

3 452 BUCHAT Supplementation of heating media with 5 ppm of potassium sorbate or sodium benzoate resulted in marked dereases in D-values of all test molds. ffets on P. puberolum and A. Jlavus were most pronouned at ph values below 5., where the preservatives are in undissoiated forms. Compared to dissoiated forms, the undissoiated forms of both preservatives are also known to be more detrimental to growth of molds. At 1 ppm, the synergisti effet.s of preservatives and heat on inativation of these test molds were somewhat greater than at 5 ppm. Potassium sorbate, at the same onentration as sodium benzoate, was less detrimental. A synergisti effet of ombined heat and radiation treatments on inativation of onidia of two strains of Aspergillus was demonstrated by Padwal-Desai et al. (9). They reported that a heat-radiation sequene was more effetive than a radiation-heat sequene. Noting the marked effets of 5 and 1 ppm of preservatives on heat inativation of molds, experiments were then onduted to determine the effets of 1-fold dereases in onentration. Results are summarized in Table 1. Observations on the general detrimental effets of low ph on survival onfirm earlier data. Vegetative ells of G. andidum appear to be more adversely affeted by high hydrogen ion onentration than were onidia of P. puberolum and A. flavus. At the same ph as ontrols, 5 ppm of potassium sorb ate did not ause a signifiant (P ~.5) derease in D-value of P. pubero [um. Signifiant dereases were noted, however, at ph 2.5 and 3. when the heating medium ontained 1 ppm of potassium sorbate. The presene of 5 ppm (ph 2.5 and 3.) and 1 ppm (all ph values tested) of sodium benzoate aused a signifiant inrease in the rate of thermal inativation of P. puberolum ompared to ontrols. Preservatives at 5 and 1 ppm were more effetive against A. flavus than against P. puberolum. xept for potassium sorbate at 5 ppm, ph 4.5, ompared to respetive ontrols, 5 ppm of either potassium sorbate or sodium benzoate aused a signifiant derease in D-values of A. flavus. It is more diffiult tg disern the effets of potassium sorbate on thermal inativation of G. andidum due to the independent adverse effets of low ph per se. However, at 5 and 1 ppm, the preservative aused signifiant dereases in D-values at ph 3. to 4.5. The effet of low onentrations of sodium benzoate on G. andidum was not tested. Although inreased sensitivity of spores of molds to heat at redued ph values has also been demonstrated by other researhers (5), the degree of sensitivity at any given ph may also depend upon the aid used to ahieve that ph as well as other onstituents in the heating medium (5, 12, 13). Hene, the effets of low ph observed in the present study may have been different if, for example, an organi aid instead of HCl had been used to adjust the hydrogen ion onentration of the heating medium. It is important to onsider these parameters when attempting to predit the effets of dereasing the ph of an individual food on the time/temperature requirements neessary to redue the viable mold populations in that food. In a previous study (4), we reported that a somewhat more heat-tolerant strain of A. flavus had inreased sensitivity to as low as 25 ppm of potassium sorbate and 15 ppm of sodium benzoate when reovered on potato dextrose agar (ph 3.5) ontaining these preservatives. Results from the present study indiate potassium sorbate inhibits olony formation by unheated and heated onidia of A. flavus when present in YMPG TABL 1. ffets ofpresf{rvatives on D values of molds heated in media adjusted to ph 2.5 to 4.5. Deimal redution times (min) at various ph values a --,.--- Organism Preservative Conentration (ppm) P. puberulum Control 28.7 efgh 29.6 def 29.5def 3.8 bde 32.8 a Potassium sorbate so 29.3 defg 29.defgh 29.4 defg 3.6 de 32.6 ab j 24.9 i 28.8defgh 3.1 def 3L4ab Sodium benzoate so 2.7k 23.8 ij 28.6fgh 3.4 def 3.9abd 1 9.n 12.8m gh 27.4 h A.flavus Control 4.8 be 41.7 ab 43.2ab 44.4 a 44.7 a Potassium sorbate so 2.8h 27.5f 36.6d 4.8 be 43.2ab hi 25.9fg 3.9de ab Sodium benzoate so 15.2jk 24.1 g 33.2e 34.2de 34.9de k 17.6 ij 2.9h G. andidum Control 6.7 gf 23.b 28.7 a 3.8a 31.2a Potassium sorbate so 6.2gf 6.7 gf 12.5e 15.8d g 5.5g 9.f 13.8de 14. de aconsidering eah organism separately, D-values not followed by the same letter are signifiantly different (P~.5). JOURNAL OF FOOD PROTCTION. VOL.44,JUN 1981

4 HATINACTIVATION OF MOLDS 453 reovery agar (ph 4.5) at ;;:,:25 ppm (Fig. 2). The inhibitory effet is pronouned at 7,5 ppm and total at 1, ppm. Differenes in ounts between the nu~b~r of olonies formed by unheated and heated omdta progressively inreased as the onentration of potassium sorbate in the reovery medium was inreased, indiating that a portion of onidia were injured (stressed) as a result of heat treatment and had inreased sensitivity to the preservative. Sodium benzoate, at 1, ppm, was only slightly inhibitory to olony development by A. jlavus, although inreased sensitivity due to heat treatment was noted at onentrations ;;;,: 2,5 ppm. Potassium sorbate.._ (I).. +- :;;)..... ' _j Potassium sorbate.._ (I).. +- :;;)..... :2 ' _j Conentration of Preservative (ppm) Figure 2. ffets of potassium sorbate and sodium benzoate in YMPG reovery agar (ph 4.5) on olony formation by A. flavus. Solid bars represent olony ounts from unheated suspensions of onidia; striped bars represent olony ounts from suspensions of onidia heated 2 min at 52 C. Results from experiments to determine the effets of preservatives on olony formation by unheated and heated onidia of P. puberulum are presented in Fig. 3. Growth of this mold was onsiderably more affeted by potassium sorbate than was growth of A. jlavus. Marked redution in olony development was noted when unheated onidia were plated on YMPG agar ontaining 2,5 ppm of potassium sorbate; 5, ppm ompletely inhibited olony development. Heated onidia were extremely sensitive to potassium sorbate at onentrations of 75 and 1, ppm, and failed to form olonies in YMPG agar ontaining 2,5 ppm. These observations represent a lassial demonstration of injury of onidia as a result of heat treatment. The sensitivity of onidia of P. puberulum to potassium sorbate, whether subjeted to heat or not, was somewhat surprising, sine the mold had been isolated from a heese ontaining a high onentration of the preservative (8). It is possible that the organism lost its Conentration of Preservative (ppm) Figure 3. ffets of potassium sorbate and sodium benzoate in YMPG reovery agar (ph 4.5) on olony formation by P. puberulum. Solid bars represent olony ounts from unheated suspensions of onidia; striped bars represent olony ounts from suspensions of onidia heated 2 min. at 49 C. tolerane to potassium sorbate upon repeated transfer and storage on laboratory media not ontaining the preservative or that sensitivity was enhaned when YMPG reovery agar was used as a growth medium. Sodium benzoate, at onentrations ranging to 1, ppm, had little if any effet on olony formation by unheated onidia of P. puberulum (Fig. 3). Heat treatment may have aused inreased sensitivity to sodium benzoate at a onentration of 1, ppm in YMPG reovery agar. Results serve to lengthen the list of molds demonstrated to undergo injury upon exposure to heat. Although most reports dealing with this phenomenon have been onerned with speies of Aspergillus (1, 4, 1, 12), it appears that others are also suseptible to sublethal debilitating effets of heat. The synergisti effets of potassium sorbate and sodium benzoate with heat against inativation of onidia were noted when levels of the preservatives were onsiderably lower than those whih aused inhibitory effets on olony formation in YMPG agar. Furthermore, at omparable onentrations during heat treatment, sodium benzoate was more detrimental to onidia than was potassium sorbate. However, observations onerning the effets of the two preservatives on olony formation by onidia of A. flavus and P. puberulum were reversed, i.e., at omparable onentrations, potassium sorbate was onsiderably more inhibitory than was sodium benzoate. It is diffiult to draw onlusions from the data on the relative effetiveness of these preservatives in heating versus reovery environments due to differenes in other omponents in the JOURNAL OF FOOD PROTCTION, VOL. 44, JUN 1981

5 454 BUCHAT suspending/reovery media. However, data do suggest that the mehanism of ation of potassium sorbate and sodium benzoate to synergistially ontribute to heat inativation may be different from that assoiated with preventing germination and/or outgrowth of onidia. Also, data suggest that the mehanism of ation of the two preservatives may differ. In addition to reports indiating that potassium sorbate (sorbi aid) inhibits ertain enzyme ativities in mirobial ells (14. 15, 16), the hemial has also been shown to ause a derease in the ATP ontent of onidia of A. parasitius (11). In the present study, the inreased sensitivity of molds to low onentrations of preservatives during heat treatment may be a result of inreased permeability due to expansion of the ell wall and ytoplasmi membrane at elevated temperature. That is, the preservatives might more easily enter ells as the temperature is inreased simply due to a weakening of the physial barriers inherent in the ell wall and membrane. Tween 8 may also have influened the suseptibility of onidia to thermal inativation. ase of transport or entry of sorbi and benzoi aids into ells of molds at various temperatures may be influened by differenes in their strutural onfiguration, thus also ontributing to the relative effetiveness of the two preservatives at various temperatures. ACKNOWLDGMNTS I am grateful to B. V. Nail for tehnial assistane and to. H. Marth for supplying the ulture of P. puberulum. RFRNCS 1. Adams, G. H., and Z. J. Ordal ffets of thermal stress and redued water ativity on onidia of Aspergillus parasitius. J. Food Si. 41: Beuhat, L. R ffetiveness of various food preservatives in ontrolling outgrowth of Byssohlamys nivea asospores. Myopa- thologia 59: Beuhat, L. R Combined effets of solutes and food preservatives on rates of inativation of and olony formation by heated spores and vegetative ells of molds. Appl. nviron. Mirobiol. (in press). 4. Beuhat, L. R., and W. K. Jones ffets of food preservatives and antioxidants on olony formation by heated onidia of Aspergillus flavus. Ata Aliment. Aad. Si. Hung. 7: Doyle, M. P., and. H. Marth Thermal inativation of onidia from Aspergillus jlavus and Aspergillus parasitius. I. ffets of moist heat, age of onidia, and sporulation medium. 1. Milk Food Tehno!. 38: Doyle. M. P., and. H. Marth Thermal inativation of onidia from Aspergillus flavus and Aspergillus parasitius. II. ffets of ph and buffers, gluose, surose, and sodium hloride. J. Milk Food Tehno!. 38: Dunan, D Multiple range and multiple F tests. Biometris 11: Marth.. H University of Wisonsin-Madison. Personal ommuniation. 9. Padwal-Desai, S. R., A. S. Ghanekar, and A. Sreenivasan Studies on Aspergillus.flavus. I. Fators influening radiation resistane of non-germinating onidia. nviron. xptl. Bot. 16: Padwal-Desai, S. R., A. S. Ghanekar, and A. Sreenivasan Studies on Aspergillus flavus. II. Responses of germinating onidia to single and ombined treatments of gamma radiation and heat. nviron. xptl. Bot. 16: Przybylski, K. S.. and L B. Bullerman Influene of sorbi aid on viability and ATP ontent of onidia of Aspergillus parasitius. J. Food Si. 45: , Shibasaki, I.. and T. Tsuhido nhaning effet of hemials on the thermal injury of miroorganisms. Ata Aliment. A ad. Si. Hung. 2: Splittstoesser, D. F., and C. M. Splittstoesser Asospores of Byssohlamys fulva ompared with those of heat resistant Aspergillus. J. Food Si. 44: Troller, J. A Catalase inhibition as a possible mehanism of fungistati ation of sorbi aid. Can. J. Mirobiol. 11: Whitaker, J. R Inhibition of sulfhydryl enzymes with sorbi aid. Food Res. 24: York, G. K., and R. H. Vaughn Mehanisms in the inhibition of miroorganisms by sorbi aid. J. Baterio!. 88: Doyle, on't.fromp. 488 in pigs. Ned.J. Vet. Si.1: Tiehan, W., and R. L. Vogt Waterborne ampylobater gastroenteritis. Morbid. Mortal. Week. Rep. 17: Tribe, G. W., and A. Frank Campylobater in monkeys. Vet. Re. 16: Tribe, G. W., P. S. MaKenzie, and M. P. Fleming Inidene of thermophili Campylobater speies in newly imported simian primates with enteritis. Vet. Re.15: Tritz, G. J., and J.. Ogg Physial and hemial onditions induing the ooid form of ovine Vibrio fetus. Am. J. Vet. Res. 18: Truszynski, M Further studies on the etiology of swine dysentery. Rozn. Nauk. Roln. Seria. 68: Tuker, J. C., and G. W. Roberts tad xperimentalvibriosis in sheep. J. Am. Vet. Med. Asso.129: Veron, M., and R. Chatelain Taxonomi study of the genus Campylobater Sebald and Veron and designation of the neotype strain for the type speies, Campylobater fetus (Smith and Taylor) Sebald and Veron. Int. J. Sys. Bateriol. 13: Vesely, D., S. Maintyre, and K. R. Ratzan Bilateral deep brahial vein thrombophlebitis due to Vibrio fetus. Arh. lnteru. Med. 135: Vinzent, R., J. Dumas, and N. Piard Septiemi grave au ours de Ia grossesse, due a un vibrion. Avortmentonseutif. Bull. Aad. Nat. Med. 131: Vogt, R. L., H.. Sours, T. J. Barrett, R. A. Feldman, and R. J. Dikinson Campylobater enteritis assoiated with water, Vermont, June, Abstr. Ann. Mtg. Am. So. Mirobial. p Willis, M. D., and W. J. Austin Human Vibrio fetus infetion. Am. J. Dis. Child. 111: Wilson, N. A., M. I. Blaser, L. B. Reller, and W. L. Wang.198. Isolation of Campylobater fetus subsp. jejuni from migratory waterfowl. Abstr. Ann. Mtg. Am. So. Mirobiol. p Yanagisawa, S Large outbreak of Campylobater enteritis among shoolhildren. Lanet 2:153. JOURNAL OF FOOD PROTCTION. VOL. 44. JUN 1981

EFFECT OF DIFFERENT METHODS OF PRESERVATION ON THE QUALITY OF CATTLE AND GOAT MEAT. Abstract

Bang. J. Anim. Si. 2009, 38(1&2) : 86 91 ISSN 0003-3588 EFFECT OF DIFFERENT METHODS OF PRESERVATION ON THE QUALITY OF CATTLE AND GOAT MEAT S. Bin. Faisal, S. Akhter 1 and M. M. Hossain Abstrat The study