237

Journal of Food Protection, Vol. 47, No. 3, Pages 237-241 (March 1984) Copyright*, International Association of Milk, Food and Environmental Sanitarians

Effects of Sorbate, Benzoate, and Temperature on Growth and Patulin Production by Byssochlamys nivea in Grape Juice

J. O. ROLAND, L. R. BEUCHAT*, R. E. WORTHINGTON and H. L. HITCHCOCK Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/3/237/1656172/0362-028x-47_3_237.pdf by guest on 30 September 2021 Department of Food Science, University ofGeorgia Agricultural Experiment Station, Experiment, Georgia 30212

(Received for publication September 19,1983)

ABSTRACT MATERIALS AND METHODS

The influence of potassium sorbate, sodium benzoate, sulfur Biomass study dioxide (S02) and temperature on biomass and patulin produc­ Organism. Byssochlamys nivea Westling strain NRRL-2615 was used tion by Byssochlamys nivea in grape juice was investigated. throughout the study. This strain was demonstrated in previous studies Growth of B. nivea was monitored over a 25-d incubation to produce high quantities of patulin on Czapek-Dox broth (15). The period at 21, 30 and 37°C. Approximately 2,500 mg (dry was maintained at 4°C on unacidified potato dextrose agar (PDA, weight) of biomass per 100 ml of juice was obtained in controls pH 5.5) (Difco, Detroit, MI). Cultures (8-10 d old) grown at 30°C on at 30 and 37°C; significantly lower amounts were observed at PDA were harvested by flooding plates with sterile 0.1 M potassium phosphate buffer (pH 7.0) containing 1% Tween 80. Conidiospores 21°C. Based on concentration, S02 had the most significant ef­ were suspended in buffer by gently rubbing a sterile glass rod over fect on reducing biomass production followed by potassium sor­ the surface of mycelial mat. The suspension was filtered through sterile bate and sodium benzoate, respectively. Patulin was produced glass wool and diluted with buffer to give an absorbance of 1.18 on in the highest concentrations (10 mg/100 ml) at 21°C after 20 d a spectrophotometer Bausch and Lomb Spectronic 20 at a setting of of incubation. Production was less at 30 and 37°C, with a fairly 620 nm; Bausch and Lomb Analytical System Division, Westbury, rapid decrease after reaching a maximum concentration. As in NY). The diluted suspension gave a viable population of 2.0-2.5 x 104 the biomass study, S02 had the most significant influence on colony forming units (CFU)/ml. All cultures were prepared just before inhibiting patulin production followed by potassium sorbate and inoculation to test media. sodium benzoate. Substrate. Welch's Concord Grape Juice (Welch Foods, Inc., Westfield, NY) was evaluated as a culture medium for supporting biomass production by B. nivea. The juice contained no added sugar, preservatives or artificial colors and flavors; it did contain added ascor­ The genus Byssochlamys is comprised of two species, bic acid and had been pasteurized. One-hundred milliliter quantities of B. nivea and B. fulva (9). Both species have been iden­ juice were aseptically dispensed into sterile 250-ml Erlenmeyer flasks. tified as spoilage organisms in processed fruit products Stock solutions of potassium sorbate (Monsanto Company, St. Louis, due to outgrowth of heat resistant ascospores after ther­ MO) and sodium benzoate (Pfizer Chemical Division, Pfizer Inc., New mal processing (5,12,14). York, NY) were prepared by adding 2.00 g to 100 ml of deionized water. An aqueous stock solution of sodium metabisulfite (pyrosulfite) The capacity of various fungi to produce toxic metabo­ (Mallinckrodt Chemical Works, St. Louis, MO) was prepared to give lites is well established but the potential role these toxic an equivalent of 2.0 g of S02 in solution (4). Stock solutions of all compounds play in disease processes is not well under­ test preservatives were filter-sterilized by passing through a 0.22-u, Mil- stood. Byssochlamys species produce several , lipore filter. Several concentrations of potassium sorbate (0, 50, 75, including byssochlamic acid (13), byssotoxin A (8), and 100, and 150 n-g/ml), sodium benzoate (0, 200, 300, 400 and 500 n-g/ ml) and S0 (0, 25, 50, 75 and 100 n-g/ml) were tested for their effects patulin (4-hydroxy-4H-furo[3,2c]-pyran-2-[6H]-one) (6). 2 on growth of B. nivea in the juice. The preservative-supplemented juice Patulin has been investigated extensively (18,20,21); it is was inoculated at a rate of 1.0 ml of the B. nivea suspension per 100 highly toxic and has been shown to be carcinogenic to ml. laboratory animals (2). Strains of B. nivea have demon­ The grape juice was incubated in a static condition at 21, 30 and 37°C strated a greater potential to produce patulin than have for various periods ranging to 25 d. Samples were taken at specified times strains of B. fulva (15). and analyzed for biomass production, pH and soluble solids. Mats were separated from duplicate samples of juice by passing the flask Although it is known that B. nivea can produce patulin contents through a tared Whatman No. 4 filter paper using a vacuum. The in several types of fruit juices (75), little is known of mycelial mat was washed several times with distilled water to remove any its ability to produce this in the presence of remaining juice residue. The filter pads were then placed into a forced air convection oven and dried at 60°C for 24 h. Biomass production was deter­ potassium sorbate, sodium benzoate or S02 at various mined by reweighing the dried mycelial mat and pads and calculating the temperatures. The study reported here was designed to biomass weight by difference. determine the effects of these food preservatives on the The pH of the juice was monitored over the 25-d incubation period. The rate of growth and patulin production by B. nivea in soluble solids content of the juice was determined using a Bausch and Lomb grape juice incubated at various temperatures. Refractometer (Bausch and Lomb Analytical System Division, Westbury,

JOURNAL OF FOOD PROTECTION, VOL. 47, MARCH 1984 238 ROLAND, BEUCHAT, WORTHINGTON AND HITCHCOCK

NY). Readings were corrected to 20°C and expressed as percentage soluble bited until day 20 of the incubation period. At the initial con­ solids. centration of 150 jxg of potassium sorbate/ml, growth was completely inhibited over the 25-d incubation period. Patulin study Substrate. Aqueous suspensions of B. nivea were prepared as described Biomass production at 30 and 37°C was very similar. At above. Conidial suspensions (2.0-2.5 x 104 CFU/ml) were added to grape both temperatures, growth was not observed at 50 and 75 (xg juice at a rate of 1.0 ml per 100 ml of juice. of potassium sorbate/ml until day 8, and at 100 and 150 (xg/ Potassium sorbate (0, 50 and 100 u.g/ml), sodum benzoate (0, 200 and ml, growth was delayed up to 17 d. Although at 30 and 37°C, 400 u.g/ml) and S02 (0, 25 and 50 fig/ml) were evaluated for their effects 50 and 75 (xg of potassium sorbate/ml did repress growth of on production of patulin by B. nivea. The preservative-supplemented juices B. nivea up to 20 d, the amount of biomass produced at the were prepared, inoculated and incubated as described previously. Duplicate samples of grape juice incubated at 21, 30 and 37°C were analyzed at 3-d end of the incubation period (25 d) was significantly differ­ intervals up to 20 d. ent than that obtained in the control juice. Biomass produc­ Extraction. At the end of each incubation period, the mycelial mat was tion at these concentrations was approximately 2000 mg/100 separated from the juice by vacuum filtration using Whatman No. 4 filter ml of grape juice after 25 d. Biomass production at 21CC ac­ paper. Fifty milliliters of juice were extracted three times with equal vol­ cumulated to approximately 750 mg/100 ml. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/3/237/1656172/0362-028x-47_3_237.pdf by guest on 30 September 2021 umes of HPLC-grade ethyl actate in a 250-ml separatory funnel. The com­ bined extracts were dried over approximately 20 g of anhydrous Na2S04 for Biomass production by B. nivea in the presence of 20 min. Lumps initially formed were broken up using a glass rod. The ex­ sodium benzoate is illustrated in Fig. 2. Substantially tract was decanted into a 500-ml round bottom flask and the Na2S04 was higher concentrations of sodium benzoate were required washed with two 25-ml portions of ethyl acetate. The extracts were com­ to retard growth as compared with that of potassium sor­ bined and evaporated to approximately 25 ml using a rotary flash evaporator. bate. B. nivea tolerated up to 400 (xg of sodium ben- A Teflon disc was placed in the bottom of a 12-cc Monoject syringe zoate/ml at 30°C while growth was completely inhibited (Brunswick Co., St. Louis, MO) and a slurry of 10 g of silica gel (60-200 at 21 and 37°C. Although not indicated on this figure, mesh) in ethyl acetate was added to the syringe. The was drained 500 (xg of sodium benzoate/ml was tested. At this con­ to the top of the absorbent and the sample was loaded onto the column. The centration, no growth was observed at the temperatures patulin was eluted from the column with 150 ml of ethyl acetate. The extract tested. Sodium benzoate repressed growth of B. nivea to was concentrated to approximately 5 ml, transferred to a vial and evaporated to dryness under a stream of nitrogen by immersing in a hot-water bath. The some extent to all temperatures. Maximum biomass pro­ resulting residue was immediately dissolved in 350 ml of ethyl acetate with duction occurred at 30 and 37°C, reaching approximately the aid of a Vortex mixer. Samples not immediately quantitated were stored 2000 mg/100 ml of grape juice, whereas only 500 mg at-18°C. of biomass was produced at 21°C. Quantitation. Thin-layer chromatography was carried out on 20 x 20 cm, 250 fx, K5 silica gel plates (Whatman Chemical Separation Division, Clif­ Growth of B. nivea was not significantly retarded in ton, NJ). The plates were scored to produce 1-cm columns, 15cm in height. the presence of up 25-75 (xg of S02/ml at 30 and 37°C Samples were spotted in 1-u.l portions of juice extracts, 1 cm from the bot­ (Fig. 3). Growth at these concentrations was very similar tom of the plate. The plates were developed in an equilibrated glass tank to that of the control, thus indicating that there was little with toluene:ethyl acetate:90% formic acid (5:4:1, v/v/v) and allowed to air to no inhibitory effect demonstrated at these levels. dry at room temperature. Plates were lightly sprayed with 4% phenylhyd- razine-hydrochloride and heated at 110°C for 2-3 min. Patulin appears as Maximum biomass production (2300-2500 mg/100 ml) in ayellow spot under visible light with an Rf value of 0.60. grape juice occurred at 30 and 37°C, regardless of the Patulin was quantitated by measuring the intensity of the yellow spots using a photodensitometer model 520-A (Photovolt Corporation, New York, NY) and a Strip Chart Recorder Model SR-255B (Heath Company, Potassium Sorbate (|ig ml): Benton Harbor, MI). A standard curve was established by dissolving a patu­ o o 0 lin standard (Sigma Chemical, St. Louis, MO) in ethyl acetate to give a con­ centration of 2.0 mg/ml. Various amounts of standard solution (0.2 to 20.0 100 |xg of patulin) were spotted and developed as described above. Values ob­ 150 tained for the samples were compared to the standard curve to give the equi­ valent u.g of patulin in the sample. Calculations were made using a formula described by Scott (7 7). To determine the percentage recovery of patulin from grape juice, an aqueous solution of patulin standard (5 mg/ml) was added to the juice to give concentrations ranging from 50 to 700 (ig/ml. The spiked juice was allowed to stand overnight before analysis. Duplicate samples of juice contaimng various known levels of patulin were analyzed and the percentage recovery was calculated. The extraction procedure pro­ duced an average percentage recovery of 80%. This figure was used to calculate the amount of patulin actually in the grape juice samples. RESULTS AND DISCUSSION Biomass Figure 1 illustrates biomass production by B. nivea in grape juice containing potassium sorbate at concentrations 10 15 20 25 O 5 10 15 20 25 ranging from 0 to 150 (xg/ml. Biomass production was se­ Time (days) verely repressed at 21°C; growth was not observed at 0 and Figure 1. Production of biomass by B. nivea grown in grape juice 50 (xg/ml until days 10 and 15 of incubation, respectively. containing various levels of potassium sorbate (0-150 \iglml) at 21, Growth at 75 and 100 (xg of potassium sorbate/ml was inhi­ 30and3TC.

JOURNAL OF FOOD PROTECTION, VOL. 47, MARCH 1984 ANTIFUNGAL AGENTS AFFECT BYSSOCHLAMYS 239

fulva occurred at 25 and 30°C. Incubation at 18 and 38CC Sodium Benzoate (ug/ml): resulted in a lower biomass production. At 38°C there o — „ 0 •—. 200 were definite signs of autolysis after 11 d of incubation. u a 300 Olliver and Rendle (10) found the optimum temperature — 400 for growth of B. fulva to be between 30 and 37°C. The results obtained in the present study tend to confirm the data reported in earlier studies. Although B. nivea will grow at cooler temperatures, e.g., 21CC, its maximum growth occurs at temperatures between 30 and 37CC. Olliver and Rendle (10) observed that B. fulva toler­ ated up to 50 (Jig of S02/ml in saline solution and plum syrup. King et al. (7) reported that growth of B. fulva was completely inhibited in grape juice containing 500

u.g of potassium sorbate/ml or 1000 u,g of sodium ben- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/3/237/1656172/0362-028x-47_3_237.pdf by guest on 30 September 2021 zoate/ml. Beuchat (1) reported that B. nivea was inhibited by 400 u,g of potassium sorbate/ml, but tolerated up to 1000 u,g of sodium benzoate/ml when cultured in grape juice at 30°C. 10 15 20 25 0 5 10 15 20 25 On the basis of concentration, S0 had the most signif­ Time (days) 2 icant effect on retarding the rate of biomass production Figure 2. Production ofbiomass by B. nivea grown in grape juice by B. nivea followed by potassium sorbate and sodium containing various levels of sodium benzoate (0-400 [i.glml) at 21, benzoate, respectively. 30and3TC. pH The pH of grape juice was monitored over the 25-d incuba­ tion period. The average pH values of the control samples 2TC Sulfur Dioxide (ug/ml): of grape juice are presented in Fig. 4. At 30 and 37°C, there 2000 „ o 0 .—. 25 was an increase in pH over the incubation period. Although u—c 50 the rise in pH was not great, from 3.3 to approximately 4.0, 1500 L „ 75 . . 100 data indicate a definite change occurred. At 21°C, the pH of 1000 J/' the grape juice decreased slightly. It is not fully understood why this occurred, but the possibility of production of secon­ />/; 500 dary metabolites such as byssochlamic acid or other organic

0 „ __a.^^^ acids may have been responsible for this decrease. Changes in pH correspond to the extent of biomass ac­ cumulation, regardless of the type or amount of preserva­ tive in the juice. Apparently B. nivea is primarily using sugars in the juices as an energy source and not utilizing organic acids which would cause greater changes in pH.

15 20 25 0 5 10 15 20 25 Time (days)

Figure 3. Production ofbiomass by B. nivea grown in grape juice containing various levels ofS02 (0-100 \Lglml) at21, 30 and37°C. presence of S02 at concentrations up to 75 (Jig/ml. Biomass production was repressed to some extent in the presence of 100 jjLg of S02/mI at 30 and 37°C. At this concentration, growth was delayed up to 17 d at 21 and 30°C. Biomass production by B. nivea was favored at 30 and 37CC. There were insignificant differences in the amount of growth that occurred in control juice at these tempera­ 10 15 20 25 0 5 10 15 20 25 tures. Hull (5) and King et al. (7) reported that 35°C Time (days) was the optimal temperature for growth of B. fulva. Rice Figure 4. Changes inpH in control samples of grape juice contami- et al. (15) reported optimum biomass production for B. natedwith B. nivea over a 25-d incubation period.

JOURNAL OF FOOD PROTECTION, VOL. 47, MARCH 1984 240 ROLAND, BEUCHAT, WORTHINGTON AND HITCHCOCK

B. nivea may be producing small amounts of ammonia which would explain the slight rise in pH. Rice et al. Potassium Sorbate (ug/ml): o o 0 (14) was observed only a small increase in pH in canned •—. 50 peaches contaminated with B. fulva. .— 100

Soluble solids Figure 5 illustrates the change in soluble solids in grape juice over the duration of the incubation period. A substantial decrease in soluble solids was observed during this study. The initial soluble solids content of grape juice was 15.8% but after 25 d at 30 and 37°C, B. nivea caused a decrease to approximately 11.3 to 11.8%, respectively. The decrease in soluble solids can be attributed to the use of sucrose and other

sugars by B. nivea during growth and ascospore production. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/3/237/1656172/0362-028x-47_3_237.pdf by guest on 30 September 2021 Patulin Patulin production in grape juice supplemented with 50 16 20 0 4 8 12 16 20 Time (days) and 100 jxg of potassium sorbate/ml is illustrated in Fig. 6. At 37°C, 50 and 100 jxg of potassium sorbate/ml completely Figure 6. Patulin production by B. nivea grown in grape juice con­ inhibited patulin production by B. nivea over a 20-d incuba­ taining various levels of potassium sorbate (0-100 ^glml) at 21, 30 tion period. Patulin production was reduced at 37°C with the and3TC. control juice reaching a maximum of approximately 3 mg/ 100 ml after 9 d. Again, after attaining maximum patulin Patulin production of B. nivea in the presence of 200 and concentration, a fairly rapid decrease was observed. Sommer 400 |ig of sodium benzoate/ml was observed at all tempera­ et al. (19) suggested that the decrease in patulin content in tures tested (Fig. 7). After 20 d of incubation, patulin con­ laboratory media may be due to metabolic destruction. For­ tent at 30 and 37°C was nondetectable. At 21°C, in juice con­ rester and Gaucher (3) reported the m-hydroxybenzyl al­ taining 200 and 400 jig of sodium benzoate/ml, patulin ac­ cohol dehydrogenase, a key in the biosynthesis of cumulated to 6 and 5 mg/100 ml, respectively. patulin, was highly reversible. Patulin has been reported to Patulin accumulation in the presence of 25 jxg of S02/ml gradually decrease in concentration in canned grape juice was significantly delayed up to 20 d at 21°C and effectively stored at 22°C (16). inhibited over the incubation period by 50 jig of S02/ml Patulin production was observed in grape juice containing (Fig. 8). Patulin accumulation at 30 and 37°C was severely 50 and 100 jig of potassium sorbate/ml at 21 and 30°C. Patu­ repressed by S02. The patulin content in preservative-sup­ lin production was reduced at 30°C in the preservative-sup­ plemented grape juice at 30 and 37°C never accumulated plemented juice, whereas at 21°C, 50 (xg of potassium sor­ above 0.5 mg/100 ml and had virtually disappeared from the bate/ml had very little affect. juice by the end of the test period. Wilson (21) reported that in the presence of free sulfhydryl groups patulin was unsta­ ble. Pohland and Allen (11) showed that when patulin was

Sodium Benzoate (ug/ml): o o 0 .—. 200 ._. 400

12 16 20 0 4 12 16 20 10 15 20 25 0 5 10 15 20 25 Time (days) Time (days) Figure 7. Patulin production by B. nivea grown in grape juice con­ Figure 5. Changes in soluble solids in control samples of grape taining various levels of sodium benzoate (0-400 \i,glml) 20, 30 and juice contaminated with B. nivea over a25-d incubation period. 3TC.

JOURNAL OF FOOD PROTECTION. VOL. 47, MARCH 1984 ANTIFUNGAL AGENTS AFFECTBYSSOCHLAMYS 241

2. Dickens, F., and H. E. H. Jones. 1961. Carcinogenic activity of a 21°C Sulfur Dioxide (ug/ml): series of reactive lactones and related substances. Brit. J. Cancer 15 o—o 0 • 15:85-92. .—. 25 3. Forrester, P. I., and G. M. Gaucher. 1972. M-hydroxybenzyl alcohol .—. 50 10 ^ dehydrogenase from Penicillium urticae. Biochemistry 11:1108-1114. 4. Green, L. F. 1976. Sulphur dioxide and food preservatives - a review. 5 y^. Food Chem. 1:103-124. / 5. Hull, R. 1939. Study of Byssochlamys fulva and control measures in 0 processed fruits. Ann. Appl. Biol. 26:800-822. 6. Karow, E. O., and J. W. Foster. 1944. An substance from 30°C species of Gymoascus and Penicillium. Science 99:265-266.

Patuli n 15 7. King, A. D., H. D. Michener, and K. A. Ito. 1969. Control of Bys­ sochlamys and related heat-resistant fungi in grape products. Appl. 10 Microbiol. 18:166-173. 8. Kramer, R. K., N. D. Davis, and U. L. Diener. 1976. Byssotoxin A, -° 5 —-— a secondary metabolite of Byssochlamys fulva. Appl. Environ. Micro­ /^ biol. 31:249-253. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/3/237/1656172/0362-028x-47_3_237.pdf by guest on 30 September 2021 0 / =t= . 9. Olliver, M., and G. Smith. 1933. Byssochlamys fulva sp. nov. J. Bot. o 2 =. 8 12 16 20 0 4 8 12 16 20 Lond. 71:196-197. Time (days) 10. Olliver, M., and T. Rendle. 1934. A new problem in fruit preservation. Studies on Byssochlamys fulva and its effect on the tissues of processed Figure 8. Patulin production by B. nivea grown in grape juice con­ fruit. J. Soc. Chem. Ind., London 53:166-172. taining various levels ofS02 (0-50 \ig/ml) at 21,30 and37°C. 11. Pohland, A. E., andR. Allen. 1970. Mycotoxins: Analysis and chemi­ cal confirmation of patulin in grains. J. Assoc. Offic. Anal. Chem. 53:686-687. added to a commercial apple juice (8 mg of patulin/L), it was 12. Put, H. M. C, and J. Th. Kruiswijk. 1964. Disintegration and or­ completely stable for at least 10 d, but they also stated that ganoleptic deterioration of processed strawberries caused by mould it disappeared rapidly from apple juice containing low con­ Byssochlamys nivea. J. Appl. Bacteriol. 27:53-58. centrations of S02. In the present study, patulin may have 13. Raistrick, H., and G. Smith. 1933. Studies in the biochemistry of been produced at higher levels than detected, since the microorganisms. 35. The metabolic products of Byssochlamys fulva Olliver and Smith. Biochem. J. 27:1814-1819. mycotoxin may have been rapidly inactivated by S02, thus reducing amounts detectable at each time of analysis. 14. Rice, S. L., L. R. Beuchat, and E. K. Heaton. 1977. Changes in the composition and texture of canned peach halves infected with In summary, S02 had the most significant inhibitory effect Byssochlamys fulva. J. Food Sci. 42:1562-1565. on patulin production by B. nivea in grape juice. Based on 15. Rice, S. L., L. R. Beuchat, and R. E. Worthington. 1977. Patulin concentration, potassium sorbate was the next most effective production by Byssochlamys spp. in fruit juices. Appl. Environ. inhibitor of patulin production followed by sodium benzoate. Microbiol. 34:791-796. Biomass was produced at 21 °C to a lesser extent as com­ 16. Scott, P. M., and E. Somers. 1968. Stability of patulin and penicillic acid in fruitjuices and flour. J. Agric. Food Chem. 16:483-485. pared to that at 30 and 37°C, while patulin production was 17. Scott, P. M. 1974. Collaborative study of a chromatographic method much greater at 21°C. There appeared to be no direct re­ for determination of patulin in apple juice. J. Assoc. Off. Anal. Chem. lationship between biomass and patulin production in grape 57:621-625. juice. 18. Singh, J. 1967. Patulin. pp. 621-630. In D. Gottieb andD. V. Shaw. (ed.) . Springer-Verlag, New York. 19. Sommer, N. F., J. R. Buchanan, andR. J. Fortlage. 1974. Production of patulin by Penicillin expansum. Appl. Microbiol. 28:589-593. REFERENCES 20. Stott, W. T., and L. B. Bullerman. 1975. Patulin: A mycotoxin of po­ tential concern in foods. J. Milk Food Technol. 38:695-705. 1. Beuchat, L. R. 1976. Effectiveness of various preservatives in control­ 21. Wilson, D. M. 1976. Patulin and penicillic acid. pp. 90-109. In J. V. ling the outgrowth of Byssochlamys nivea ascospores. Mycopathologia Rodricks (ed.), Mycotoxins and other fungi related food problems. 59:375-378. Am. Chem. Soc, Washington, DC.

JOURNAL OF FOOD PROTECTION, VOL. 47, MARCH 1984