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Effects of irradiation on the storage quality of fresh straw mushrooms (Volvariella volvacea)
Food Quality and Preference Food Quality and Preference 1989 1 O) 113-119~) Lougman Group UK Lid 1989 0950-3293189/013041131503.50 Receired 29 September Accepted 28 J.ne 1989
Linda Nayga-Mercado* and Estrella F. Alabastrot *Dean, College of Home Economics, Central Mindanao University, Mindanao, The Philippines tProfessor, Department of Food Science and Nutrition, University of the Philippines, Diliman, Quezon City, Luzon, The Philippines
Effects of irradiation on the storage quality of fresh straw mushrooms
( Volvariella volvacea)
Abstract The effects of ionizing radiation on certain properties of freshly-harvested Volvariella volvacea mushrooms were investigated at specified periods of storage at 22-25 *C and
85-95% r.h. Irradiation at 0.5 and 1.0 kGy delayed and inhibited veil opening and significantly suppressed the respiration rate, but did not affect the reducing sugar content. Irradiated mushrooms had lower polyphenol oxidase (PPO) activity, which decreased with storage time in contrast to an increasing activity in the control. Irradiation at 0.5 or 1.0 kGy significantly improved the colour, texture/ appearance and odour of fresh mushrooms. About 35% of the mushrooms irradiated at 0.5-1.0 kGy were without defects after storage for 5 d at ambient conditions of 2225°C, 85-95% r.h. All non-irradiated mushrooms were defective after 3 d.
Keywords: irradiation; mushrooms; sensory quality; shelf life
Introduction Cultured mushrooms are highly perishable. They can be kept in prime condition for only 1 d at 10 °C, 2 d at 4.4 °C or 5 d at 0 °C (Gill, Nicholas and Markakis, 1969). The obvious undesirable changes are opening of the cap, elongation of the stem, darkening and shrivelling. Such changes make mushrooms unattractive to the consumer and reduce their economic value. Irradiation is one possible method of pre-
servation which can be used by mushroom producers for wider and greater distribution of their produce. Gill et al. (1969) asserted that gamma irradiation retards the rate at which cultured mushrooms open and darken during storage. This study was conducted to specifically analyse the effect of low-dose ionizing radiation on the following properties of fresh Volvariella volvacea mushrooms: weight, veil condition, proportion of buttons without defects, respiration rate, reducing sugars content, PPO activity and sensory qualities.
Materials and methods Factorial design was used to determine the effect of two factors, namely, radiation dose and time of harvest (or trial date), on the postharvest quality of button straw mushrooms at any given storage time. Materials
The fresh mushrooms in the first flush stage (10-14 d old) were obtained, within 5-6 h of harvesting, ranging in Weight from 7 to 15g. A sample lot of 10kg was provided for each trial. Three trial runs were conducted, 4 - 6 wks apart. Low density polyethylene bags (100 film gauge), 17.8 c m x 30.5 cm in size, with 20 perforations (0.64cm dia.) to provide aeration, were used as storage containers. Before packing, mushrooms were culled to remove offsized, damaged, and partially opened buttons. About 65-75 buttons were placed inside each bag. Three packs were prepared for every irradiation dose.
114
Nayga-Mercado and Alabastro
One pack was assigned for the physical quality determinations and the other packs were used for the biochemical and sensory quality aspects of the study.
Irradiation The filled packages to be irradiated were transported to the irradiation facility in a container maintained at about 0 °C. The Gamma Cell 220 equipped with a Cobalt60 irradiation unit delivering a dose rate of 2.57-2.60 kGy/h, stationed at the Philippine Atomic Energy Commission (PAEC), provided the desired irradiation doses. Three irradiation treatments were used, namely: 0 k G y (control), 0.5 kGy and 1.0 kGy. After irradiation, the samples were stored in desiccators maintained for the duration of the experiment at 85-95% r.h. by saturated KCI solution and at 22-25 °C.
Determination of physical properties The mean weight per button and the number of closed and opened buttons without defects were noted daily starting 4 h after irradiation. The irradiated mushrooms were sampled for analysis until 136 h after irradiation, while the controls were stored for only 76 h, since by this time the non-irradiated mushrooms had completely deteriorated. A button was considered defective when it manifested one or more of the following symptoms: (a) physical damage or opening; (b) brownish black colour; (c) sliminess; (d) very strong earthy, ammoniacal odour.
Measurement of respiration rate About 30-50 buttons, averaging 335 g in weight, were withdrawn from the storage desiccator at the predetermined storage times. The samples from each treatment were placed in an air-tight desiccator with rubber fittings connected to an Orsat apparatus. The mushrooms were allowed to respire for 25-30 min. Replicate determinations of the respiration rate, expressed as mg COa iiberated/h/kg of samples, were conducted.
Determination of the PPO activity The micro methods of Halim and Montgomery (1978) and Tate et al. (1964) were applied to extract crude PPO and assay its activity. Triplicate runs were conducted, using 0.05 M acetate buffer (pH 5.6) as extracting solution. Catechol of concentration 0.01 M was used as substrate in the assay of PPO activity. The protein content of the enzyme preparation was analysed using the procedure of Lowry et al. (1951). Enzyme activity was expressed in absorbance units at 420 nm/ mg protein/g sample.
Evaluation of sensory qualities A total of 24-30 buttons was taken daily from each treatment, coded, and prepared for sensory evaluation by six selected panel members. Replicate judgements of colour, odour, texture and appearance, done in two sittings, were conducted by the panel daily, using descriptive scorecards, until the mushrooms were no longer acceptable. Scores were assigned after the panel evaluation for quantitative analysis of the descriptive qualities.
Statistical analysis of data Analysis of variance ( A N O V A ) was used to test the difference among means of each parameter at various storage times. Whenever A N O V A results indicated significant difference at 5% level of significance among trial and treatment means, Duncart's multiple range test was applied to determine which sample groups were diffe-
Food Quality and Preference (1989) 1 (3)
Results and discussion Physical changes Weight loss
Irradiation effects on weight loss are presented in Table 1. For each treatment, there were significant differences in the mean weights of the mushrooms between trials, which indicates that this physical property is affected by the time of culture. The gap between trial runs was 4 - 6 wks; the first was conducted in July, the second in August and the third in October. At all the sampling periods during storage, A N O V A results showed no significant differences in weights between treatments. A pattern of gradual weight decrease was observed for all samples except for the non-irradiated mushrooms in trial II, which showed a slight increase in weight during the first 28 h of storage followed by a decrease. At all storage times, the 1.0 kGy samples had lower mean weights compared to the 0.5 kGy samples, although the difference was not significant. Differing results of studies have been reported concerning the effects of irradiation on mushroom weight. Differences in storage conditions, such as r.h., airflow and packaging, may partially explain the discrepancies reported in the literature. The results of the present study agree with those of Skou et al. (1974), who observed a
Table 1 Weights (g) of fresh irradiated and unirradiated Voh,ariella voh,acea mushrooms stored at 22-25 °C and 85-95% r.h.
Storage Time (hours after irradiation)
28
52
Analysis of reducing sugars Reducing sugars were extracted by blending 40 g of mushroom samples in deionized water, followed by centrifugation and filtration. The phenol-sulphuric acid micro technique (Joslyn, 1970) was used in determining the amount of reducing sugars in the filtrate. Replicate analyses were done for every treatment daily until the end of the storage period.
rent. The t-test was also applied on the mean values of selected parameters.
76
100
136
Irradiation dose (kGy) Trial
0
0.5
1.0
I II II1 I 1I III I II III I lI 1II l 11 III I II III
9.46 10.79 8.85 9.35 11.42 8.86 8.26 10.38 8.68 7.89 10.23 8.53
8.73 10.37 9.00 8.60 10.35 8.92 8.43 10.34 8.87 8.30 10.19 8.77 8.15 9.93 8.70 8.02 9.51 8.57
8.48 9.88 8.92 8.37 9.78 8.87 8.22 9.30 8.77 8.10 9.15 8.70 7.98 9.15 8.63 7.68 8.98 8.52
Effects of radiation on fresh straw mushrooms
small weight loss of less than 5% in irradiated fresh white and brown strains of mushrooms which were packed in PVC-foil bags and stored at 10 °C and 85-95% r.h. for 9-11 days; however, no significant differences among irradiation treatments, between 2.0-3.0 kGy, were noted. Campbell et al. (1968) found similar results and reported that the effects of irradiation at 1.0 kGy on weight loss in Agaricus campestris mushrooms stored for 4 d at 1 °C and 85% r.h. for one week were insignificant.
Cap opening Prior to irradiation, all mushroom buttons chosen for the study were closed. To induce approximate normality of percentages ranging from 0-100°/o, the arcsin (angular) transformation technique was applied to the data for opened buttons. A N O V A results of the arcsin transformed values indicate no significant differences between trials during the entire storage period for this physical attribute. At each sampling period, the averages of trials I, II and III were taken and the changes in the mean percentage of opened buttons during storage are shown in Fig. 1. Results of the t-test between treatment means indicate highly significant inhibition of cap opening in the 0.5 kGy treated mushrooms compared to the control; however, no significant difference was
noted between the 0.5 kGy and 1.0 kGy treatments in their effects on degree of veil opening of fresh mushrooms. These research results are in agreement with those of Bramlage and Lipton (1965) on Agaricus bisporus mushrooms; they noted that the degree of cap opening following storage for various times and temperatures was significantly less in irradiated samples at doses of 0.061.0 kGy. Gill et al. (1969) also found that 0.1 kGy appreciably reduced the normal increase in cap diameter, while doses of 1.0 kGy or more prevented lengthening of the stem of Agaricus bisporus mushrooms. Figure 1 further indicates that cap openings in the non-irradiated mushrooms occurred within the first 4 h of storage while for the irradiated samples, most of the openings took place after the 4th hour up to the 76th hour of storage.
Defective buttons While no significant differences among trials were noted, there were significant differences among treatments from the 4th up to the 76th hour. Duncan's multiple range test showed a significantly lower percentage of buttons without defects in the control compared to the 0.5 and 1.0 kGy treated materials in all storage times. Although irradiation at 0.5 kGy
115
produced lower percentage of buttons without defects than at 1.0 kGy, the difference was insignificant. A t the 76th hour, the control was 100% defective whereas 63% and 84% of the 0.5 and 1 . 0 k G y samples, respectively, were still without apparent defects. Results of t-test between means of the 0.5 and 1 . 0 k G y treated mushrooms for the entire storage period also showed insignificant difference between these two lots of samples. Graphs of mean percentage of defective buttons vs. storage time are shown in Fig. 2. The main defect of both irradiated and non-irradiated mushrooms in the first 28 h of storage was opened caps, but an opened cap, by itself, may not he sufficient to render the mushrooms unmarketable. At the 52nd h, slimy buttons comprised the major portion of the defective nonirradiated buttons. After 52 h, the nonirradiated mushrooms had completely deteriorated, manifesting very intense discoloration, whitish moulds, and very strong ammoniacal odour, in addition to the slime. In contrast, the major defect of the irradiated samples at 52 h was still opened caps. Slime formation in the irradiated materials was observed starting on the 100th hour. In some samples, irradiated and non-irradiated, it was difficult to differentiate mould growth from slime since moulds developed simultaneously with the slime.
40 13
35
30 U~ Z in
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z lld 0
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4 O Figure I
O KGY
I
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STORAGE TIME (HOURS) 0.,50 KGY O 1 .OO KGY
Mean percentage of opened buttons during post-irradiation storage at 22-25 °C, 85-95% r.h.
Food Quality and Preference (1989) 1 (3)
116
Nayga-Mercado and Alabastro
100 90 80
7O 60 50 40
-
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m s (HO~S) D Figure 2
OKGY
+
0.50 KGY
o
1.00 KGY
Mean percentage of mushrooms without defects during post-irradiation storage at 22-25 °C, 85-95% r.h.
Respiration rate
There was no significant variation in reSpiration rates among trials, at all the post-treatment sampling periods. Irradiation at 0.5 and 1.0 kGy produced a significant inhibition in respiration rates only on the 52nd hour of storage, towards the end of the normal shelf life of fresh mushrooms, but not at the earlier sampling periods. An obvious difference in the trend of respiration rate between the untreated and irradiated mushrooms can be seen in Fig. 3. The control mushrooms gave continuously increasing values of respiration rate until the end of their storage life, while irradiated samples showed a peak in the respiration rate on the 28th hour. A low respiratory rate is generally associated with a long shelf life. Thus, treatments which depress respiration rate are expected to result in the prolongation of shelf life. In this study, irradiated mushrooms, which had lower respiration rates compared to the control, starting at the 52nd hour of storage, also showed slower deterioration as evidenced by cap opening, discoloration and other defects. The results obtained in this study strongly support the finding of Stanek et al. (1977) that respiration is inhibited by irradiation treatment. Mercier & MacFood Quality and Preference (1989) 1 (3)
Queen (1965) found an increase in respiration 4 h after irradiation in all samples irradiated at 0.1-1.0 kGy, but, by one day after irradiation, the respiration of treated samples had dropped below that of control samples and continued to decrease up to 5 d after irradiation.
Reducing sugars
Any change in the reducing sugar content of mushrooms brought about by irradiation may affect flavour. Accordingly, this chemical constituent was monitored during storage, and the results of the analyses are given in Table 2. Analysis of variance showed insignificant differences in the reducing sugar content between treatments but showed significant differences to exist between trials. A general pattern of increasing reducing monosaccharides with time was observed. The results reported herein are compatible with those of Campbell et al. (1968) who found the reducing sugars content of Agaricus campestris mushrooms treated by 1.0 kGy to be slightly higher than that of non-irradiated mushrooms after storage for 4 d at 1 °C and 85% r.h., but the difference was not significant.
Polyphenol oxidase activity
Significant differences between trials were shown to exist only in the first sampling period (4 h after irradiation), but significant differences between treatment means were observed at all sampling periods. The activity of the PPO enzyme in the control was significantly higher than that of the PPO in the 0.5 and 1.0kGy treated mushrooms. However, no significant difference was observed between 0.5 and 1.0kGy treated samples in their PPO activity. The results in Fig. 4 also show a difference between irradiated and nonirradiated samples, in the effect of storage time on PPO activity. In the non-irradiated samples, enzyme activity increased during storage, while in the 0.5 and 1.0kGy treated samples the PPO activity decreased with storage time. The data in Fig. 4 further show that doses higher than 0.5 and 1.0 kGy are necessary to effect complete inactivation of PPO. Sensory qualities
As shown in Table 3, there was a significant variation in colour scores between trials at the 18th h but not at the subsequent sampling periods. The finding that
Effects of radiation on fresh straw m u s h r o o m s
117
5.4 5.2 6 4.8
£
I
4,6
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4.4 4.2 4 vo
3.8 3.G 3.4
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3.2
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2.8 2.6 2.4 2.2
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1 O0
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0 KGY
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136
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Respiration rate of mushrooms during post-irradiation storage at 22-25 °C, 85-95% r.h.
higher than that of the control, although the difference between the two irradiated samples was not significant. Irradiation dose (kGy) On the third day (76 h), the control had Storage time visibly darkened with an average colour .(hours after Trial 0 0.5 1.0 rating of almost 'brownish black'. The irradiation) irradiated mushrooms, on the other hand, 4 I 4.29 5.15 5.52 were still 'light brown'. II 9.10 9.34 9.54 The beneficial effect of irradiation on lII 9.19 9.32 9.95 coiour is consistent with findings on the 28 I 4.83 5.28 5.70 inhibition of PPO activity by irradiation II 10.22 10.50 9.52 treatment. At any storage time, nonIll 9.91 9.83 10.12 irradiated mushrooms had higher enzyme 52 I 6.22 5.74 6.78 activity than irradiated mushrooms and II 10.51 9.80 9.78 were judged to be more discoloured. III 10.18 9.87 10.57 There were no significant differences 76 I 6.70 7.09 between trials in terms of panel scores for II 9.82 9.82 texture/appearance of stored mushrooms. III 10.04 10.64 However, the data show that irradiation 100 I 8.74 9.86 treatment had a significant effect. At all II 9.85 9.96 sampling periods, the control had signifiIII 10.39 10.73 cantly lower scores than the 0.5 and 136 I 10.53 10.47 1.0 kGy irradiated batches. After 7 6 h II 10.40 11.70 storage, the control mushrooms appeared III 10.94 10.38 'very soft, definitely shrivelled', while the irradiated mushrooms showed only slight shrivelling. From the 18th to the 76th h, 0.5 and 1.0 kGy of ionizing radiation prothe date of culture affected colour scores significantly different from that of the duced indistinguishable effects on texture/ only in the initial hours of storage parallels non-irradiated sample, for all trials. appearance. that on PPO activity. After 58 and 76 h, the mean colour Just like the panel scores for colour, Analysis of variance showed that colour scores (averaged over the three trials) of significant differences in odour scores scores of the irradiated mushrooms were the irradiated samples were significantly. among trials were noted at the 18th h of Table 2 Reducing sugars (%) of mushrooms during post-irradiation storage at 22-25 *C, 85-95% r.h.
Food Quality and Preference (1989) 1 (3)
118
Nayga-Mercado and Alabastro
27 26 25 24 23 22 21 20 19
18 17 16 15 14 o
13 12
11 10
O Figure 4
I
I
I
I
I
I
4
28
52
76
1 O0
1 36
0 KGY
+
STORAGE TIME (HOURS) 0 . 5 0 KGY O 1 .OO KGY
Mean PPO activity of mushrooms during post-irradiation storage at 22-25 °C, 85-95% r.h.
storage. At this initial sampling period, the odour scores for the irradiated mushrooms were significantly higher than that of the control, in two out of the three trials. Subsequent samplings did not show significant variation among trials. At the 58th and 76th h, the mean odour scores (averaged over the three trials) of the irradiated mushrooms were significantly higher than that of the non-irradiated mushrooms, but the 0.5 and 1.0 kGy treated mushrooms were rated similarly by the panel. At the 76th h of storage, the odour of the nonirradiated mushrooms was judged as being 'strongly earthy and ammoniacal' while the irradiated mushrooms were assessed as having a 'just discernible earthy, ammoniacal smell'. Just as irradiation improved colour and texture quality during storage, a positive effect was likewise noted on the odour. The present results, which show that irradiation at 0.5 and 1.0 kGy definitely improved the sensory quality of stored straw mushrooms, corroborate the research findings of Mercier & MacQueen (1965), Staden (1966), Skou et al. (1974) and Gill et al. (1969) who all reported better colour and texture of mushrooms irradiated at doses of 2.0-5.0 kGy. The reports of Stanek et al. (1977) and Maxie et al. also stated that irradiated mushrooms Food Quality and Preference (1989) 1 (3)
kept the appearance, texture and flavour of fresh ones for a longer time than non-irradiated mushrooms.
Summary
and conclusions
The time at which the mushrooms were cultured affected only a few postharvest properties, notably the weight loss during storage and the reducing sugars content. The other properties, namely degree of cap opening, level of defective mushrooms, respiration rate, and texture/appearance as evaluated by a panel, were unaffected by date of harvest of the mushrooms. Irradiation did not result ,in a lower weight loss during storage, but did inhibit cap opening. Significantly higher percentages of buttons without defects were noted in the irradiated samples during the period from 4-76 h of storage. By this latter time, the non-irradiated mushrooms had completely deteriorated. Differences in the pattern of respiration rate during storage were shown by CO2 liberation rates increasing in the irradiated mushrooms up to 28 h and then decreasing abruptly, with the controls showing progressively increasing rates until the end of their shelf life. Irradiation effected a significant gradual
decrease in PPO enzyme activity with storage time, contrasted with an increasing activity with time in the control. This decrease in PPO activity is reflected in the results of the sensory evaluation, the irradiated samples having markedly better colour, texture/appearance and odour than the non-irradiated mushrooms. Irradiation at 0 . 5 k G y produced the same improvement in the storage quality of fresh mushrooms as 1.0 kGy irradiation treatment. This lower dose treatment has obvious advantages in terms of less energy cost and lower probability of occurrence0f" undesirable effects which have been observed at higher doses by many researchers. The results of the present study indicate that mushrooms, if irradiated, can be held at tropical ambient conditions (22-25 °C, 85-95% r.h.) for as long as 5 d after harvest, about 35% of the irradiated mushrooms being without apparent defects after a 5 d period without refrigeration. In contrast, the non-irradiated mushrooms were completely defective after 3 d. Acknowledgement
The authors gratefully acknowledge the assistance and cooperation of the Philippine Atomic Energy Commission.
Effects of r a d i a t i o n on fresh s t r a w m u s h r o o m s
Table 3 Panel scores for colour, odour and texture/appearance of mushrooms during postirradiation storage at 22-25 °C, 85-95% r.h. Storage time (h after irradiation)
Irradiation dose (kGy) Trial
0
0.5
1.0
It lit lilt i II III Meant I II Ill Meant
3.67 a 3.75" 4.25" 2.40 2.33 3.58 2.77 " 2.13 2.25 2.92 2.43"
4.75 h 4.92 b 5.00 b 4.50 4.25 4.67 4.47 h 4.5(I 4.33 4.08 4.30 h
4.83 h 5.00 h 5.00 b 4.70 4.58 4.50 4.59 b 4.88 4.50 4.17 4.52 b
It Ilt lilt I II Ill Meant I II III Meant
3.58 a 3.17" 4.00 ~ 2.30 2.17 3.42 2.63 u 2.00 1.58 2.58 2.05 ~
4.17 a 4.33 b 5.00 b 4.30 3.92 4.42 4.21 b 4.25 3.25 4.08 3.86 b
4.42" 4.67 b 4.67 b 4.70 4.50 4.58 4.59 b 4.13 3.58 4.08 3.93 b
I II III Meant I II 1II Meant I II Ill Meant
4.00 4.08 4.17 4.08~ 2.90 2.58 3.83 3.10 ~ 1.75 2.25 2.92 2.30 a
4.83 5.00 5.00 4.94 b 4.70 4.42 4.67 4.60 b 4.75 4.25 3.92 4.31 b
4.92 4.92 4.92 4.92 b 4.60 5.00 4.58 4.73 b 4.63 4.58 4.42 4.54 b
Colour 18
58
76
Odour 18
58
76
Texture~appearance 18
58
76
Colo,rscores: 1 black; 2 brownish black; 3 dark brown; 4 light brown; 5 very light brown, close to beige.
Odour scores: 1 very strongly earthy, ammoniacal smell; 2 strongly earthy, ammoniacal smell; 3 moderately earthy, ammoniacal smell; 4 just discernible earthy, ammoniacal smell; 5 natural aroma of mushroom. Texture~appearance scores: 1 disintegrated, appears rotten, mouldy, slimy and deteriorated; 2 very soft, definitely shrivelled; 3 moderately soft and shrivelled; 4 soft and slightly shrivelled; 5 firm yet tender; not shrivelled. t Values with the same superscripts are not significantly different at P -< 0.05.
119
References (1965) G a m m a radiation of vegetables to extend market life. Marketing Research Report No. 703, Agricultural Research Service, US Department of Agriculture: Washington, DC
BRAMLAGE, W J & LIPTON, W J
CAMPBELL, J D, STOTHERS, S, VAISEY, M & BERCK,
B (1968) Gamma radiation influence on the storage and nutrional quality of mushrooms,
Journal of Food Science, 33,540-542 (1969) Irradiation of cultured mushrooms, Food Technology, 23,385-388 HALIM, O H a MONTGOMERY, M W (1978) Polyphenol oxidase of d ' A n j o u pears (Pyrus communis L.), Journal of Food Science, 43, 603-606 JOSLYN, M A (ed.) (1970) Methods in Food Analysis, 2nd edn. Academic Press: New York GILL, W J, NICHOLAS, R C & MARKAKIS, P
LOWRY, O H, RASENBROUGH, N J, FARR, A L &
RANDAL, R (1951) Protein measurement with the folin-phenol reagent, Journal of Biological Chemistry, 193,265-275 MAXIE, E C, SOMMER, N F, & BROWN, D S (1967) Radiation technology in conjunction with post-harvest procedures as a means of extending the shelf life of fruits and vegetables. Report UCD-34P80-5, United States Department of Commerce: Washington DC MERCIER, R G & MACOUEEN, K F (1965) Gamma-
irradiation to Extend Post-harvest Life of Fruits and Vegetables. Horticultural Experiment Station and Products Laboratory, Vineland Station: Ontario, Canada s n o u , J P, BECH, K, & LUNDSTEN, K (1974) Effects of ionizing irradiation on mushrooms as influenced by physiological and environmental conditions. Radiation Botany, 14,287-299 STADEN, O L (1966) Food Irradiation. International Atomic Energy Agency; Vienna STANEK, M, URBAN, M, SMOTLACHA, M & HAKO-
VA, K (1972) Effect of ionizing radiation on the activity of proteases in mushroom fruit bodies, (in Czech, English summary) cited by Salkova, Z, Kubin, M, Stanek, M & Horacek, P (1977) Present Status of Food Irradiation in Czechoslovakia. International Symposium on Food Preservation by Irradiation: Wageningen, The Netherlands TATE, J N, LUK, R S & YORK, G K (1964) Polyphenol oxidase in Bartlett pears, Journal of Food Science, 29,829-836
Food Quality and Preference (1989) 1 (3)
Linda Nayga-Mercado* and Estrella F. Alabastrot *Dean, College of Home Economics, Central Mindanao University, Mindanao, The Philippines tProfessor, Department of Food Science and Nutrition, University of the Philippines, Diliman, Quezon City, Luzon, The Philippines
Effects of irradiation on the storage quality of fresh straw mushrooms
( Volvariella volvacea)
Abstract The effects of ionizing radiation on certain properties of freshly-harvested Volvariella volvacea mushrooms were investigated at specified periods of storage at 22-25 *C and
85-95% r.h. Irradiation at 0.5 and 1.0 kGy delayed and inhibited veil opening and significantly suppressed the respiration rate, but did not affect the reducing sugar content. Irradiated mushrooms had lower polyphenol oxidase (PPO) activity, which decreased with storage time in contrast to an increasing activity in the control. Irradiation at 0.5 or 1.0 kGy significantly improved the colour, texture/ appearance and odour of fresh mushrooms. About 35% of the mushrooms irradiated at 0.5-1.0 kGy were without defects after storage for 5 d at ambient conditions of 2225°C, 85-95% r.h. All non-irradiated mushrooms were defective after 3 d.
Keywords: irradiation; mushrooms; sensory quality; shelf life
Introduction Cultured mushrooms are highly perishable. They can be kept in prime condition for only 1 d at 10 °C, 2 d at 4.4 °C or 5 d at 0 °C (Gill, Nicholas and Markakis, 1969). The obvious undesirable changes are opening of the cap, elongation of the stem, darkening and shrivelling. Such changes make mushrooms unattractive to the consumer and reduce their economic value. Irradiation is one possible method of pre-
servation which can be used by mushroom producers for wider and greater distribution of their produce. Gill et al. (1969) asserted that gamma irradiation retards the rate at which cultured mushrooms open and darken during storage. This study was conducted to specifically analyse the effect of low-dose ionizing radiation on the following properties of fresh Volvariella volvacea mushrooms: weight, veil condition, proportion of buttons without defects, respiration rate, reducing sugars content, PPO activity and sensory qualities.
Materials and methods Factorial design was used to determine the effect of two factors, namely, radiation dose and time of harvest (or trial date), on the postharvest quality of button straw mushrooms at any given storage time. Materials
The fresh mushrooms in the first flush stage (10-14 d old) were obtained, within 5-6 h of harvesting, ranging in Weight from 7 to 15g. A sample lot of 10kg was provided for each trial. Three trial runs were conducted, 4 - 6 wks apart. Low density polyethylene bags (100 film gauge), 17.8 c m x 30.5 cm in size, with 20 perforations (0.64cm dia.) to provide aeration, were used as storage containers. Before packing, mushrooms were culled to remove offsized, damaged, and partially opened buttons. About 65-75 buttons were placed inside each bag. Three packs were prepared for every irradiation dose.
114
Nayga-Mercado and Alabastro
One pack was assigned for the physical quality determinations and the other packs were used for the biochemical and sensory quality aspects of the study.
Irradiation The filled packages to be irradiated were transported to the irradiation facility in a container maintained at about 0 °C. The Gamma Cell 220 equipped with a Cobalt60 irradiation unit delivering a dose rate of 2.57-2.60 kGy/h, stationed at the Philippine Atomic Energy Commission (PAEC), provided the desired irradiation doses. Three irradiation treatments were used, namely: 0 k G y (control), 0.5 kGy and 1.0 kGy. After irradiation, the samples were stored in desiccators maintained for the duration of the experiment at 85-95% r.h. by saturated KCI solution and at 22-25 °C.
Determination of physical properties The mean weight per button and the number of closed and opened buttons without defects were noted daily starting 4 h after irradiation. The irradiated mushrooms were sampled for analysis until 136 h after irradiation, while the controls were stored for only 76 h, since by this time the non-irradiated mushrooms had completely deteriorated. A button was considered defective when it manifested one or more of the following symptoms: (a) physical damage or opening; (b) brownish black colour; (c) sliminess; (d) very strong earthy, ammoniacal odour.
Measurement of respiration rate About 30-50 buttons, averaging 335 g in weight, were withdrawn from the storage desiccator at the predetermined storage times. The samples from each treatment were placed in an air-tight desiccator with rubber fittings connected to an Orsat apparatus. The mushrooms were allowed to respire for 25-30 min. Replicate determinations of the respiration rate, expressed as mg COa iiberated/h/kg of samples, were conducted.
Determination of the PPO activity The micro methods of Halim and Montgomery (1978) and Tate et al. (1964) were applied to extract crude PPO and assay its activity. Triplicate runs were conducted, using 0.05 M acetate buffer (pH 5.6) as extracting solution. Catechol of concentration 0.01 M was used as substrate in the assay of PPO activity. The protein content of the enzyme preparation was analysed using the procedure of Lowry et al. (1951). Enzyme activity was expressed in absorbance units at 420 nm/ mg protein/g sample.
Evaluation of sensory qualities A total of 24-30 buttons was taken daily from each treatment, coded, and prepared for sensory evaluation by six selected panel members. Replicate judgements of colour, odour, texture and appearance, done in two sittings, were conducted by the panel daily, using descriptive scorecards, until the mushrooms were no longer acceptable. Scores were assigned after the panel evaluation for quantitative analysis of the descriptive qualities.
Statistical analysis of data Analysis of variance ( A N O V A ) was used to test the difference among means of each parameter at various storage times. Whenever A N O V A results indicated significant difference at 5% level of significance among trial and treatment means, Duncart's multiple range test was applied to determine which sample groups were diffe-
Food Quality and Preference (1989) 1 (3)
Results and discussion Physical changes Weight loss
Irradiation effects on weight loss are presented in Table 1. For each treatment, there were significant differences in the mean weights of the mushrooms between trials, which indicates that this physical property is affected by the time of culture. The gap between trial runs was 4 - 6 wks; the first was conducted in July, the second in August and the third in October. At all the sampling periods during storage, A N O V A results showed no significant differences in weights between treatments. A pattern of gradual weight decrease was observed for all samples except for the non-irradiated mushrooms in trial II, which showed a slight increase in weight during the first 28 h of storage followed by a decrease. At all storage times, the 1.0 kGy samples had lower mean weights compared to the 0.5 kGy samples, although the difference was not significant. Differing results of studies have been reported concerning the effects of irradiation on mushroom weight. Differences in storage conditions, such as r.h., airflow and packaging, may partially explain the discrepancies reported in the literature. The results of the present study agree with those of Skou et al. (1974), who observed a
Table 1 Weights (g) of fresh irradiated and unirradiated Voh,ariella voh,acea mushrooms stored at 22-25 °C and 85-95% r.h.
Storage Time (hours after irradiation)
28
52
Analysis of reducing sugars Reducing sugars were extracted by blending 40 g of mushroom samples in deionized water, followed by centrifugation and filtration. The phenol-sulphuric acid micro technique (Joslyn, 1970) was used in determining the amount of reducing sugars in the filtrate. Replicate analyses were done for every treatment daily until the end of the storage period.
rent. The t-test was also applied on the mean values of selected parameters.
76
100
136
Irradiation dose (kGy) Trial
0
0.5
1.0
I II II1 I 1I III I II III I lI 1II l 11 III I II III
9.46 10.79 8.85 9.35 11.42 8.86 8.26 10.38 8.68 7.89 10.23 8.53
8.73 10.37 9.00 8.60 10.35 8.92 8.43 10.34 8.87 8.30 10.19 8.77 8.15 9.93 8.70 8.02 9.51 8.57
8.48 9.88 8.92 8.37 9.78 8.87 8.22 9.30 8.77 8.10 9.15 8.70 7.98 9.15 8.63 7.68 8.98 8.52
Effects of radiation on fresh straw mushrooms
small weight loss of less than 5% in irradiated fresh white and brown strains of mushrooms which were packed in PVC-foil bags and stored at 10 °C and 85-95% r.h. for 9-11 days; however, no significant differences among irradiation treatments, between 2.0-3.0 kGy, were noted. Campbell et al. (1968) found similar results and reported that the effects of irradiation at 1.0 kGy on weight loss in Agaricus campestris mushrooms stored for 4 d at 1 °C and 85% r.h. for one week were insignificant.
Cap opening Prior to irradiation, all mushroom buttons chosen for the study were closed. To induce approximate normality of percentages ranging from 0-100°/o, the arcsin (angular) transformation technique was applied to the data for opened buttons. A N O V A results of the arcsin transformed values indicate no significant differences between trials during the entire storage period for this physical attribute. At each sampling period, the averages of trials I, II and III were taken and the changes in the mean percentage of opened buttons during storage are shown in Fig. 1. Results of the t-test between treatment means indicate highly significant inhibition of cap opening in the 0.5 kGy treated mushrooms compared to the control; however, no significant difference was
noted between the 0.5 kGy and 1.0 kGy treatments in their effects on degree of veil opening of fresh mushrooms. These research results are in agreement with those of Bramlage and Lipton (1965) on Agaricus bisporus mushrooms; they noted that the degree of cap opening following storage for various times and temperatures was significantly less in irradiated samples at doses of 0.061.0 kGy. Gill et al. (1969) also found that 0.1 kGy appreciably reduced the normal increase in cap diameter, while doses of 1.0 kGy or more prevented lengthening of the stem of Agaricus bisporus mushrooms. Figure 1 further indicates that cap openings in the non-irradiated mushrooms occurred within the first 4 h of storage while for the irradiated samples, most of the openings took place after the 4th hour up to the 76th hour of storage.
Defective buttons While no significant differences among trials were noted, there were significant differences among treatments from the 4th up to the 76th hour. Duncan's multiple range test showed a significantly lower percentage of buttons without defects in the control compared to the 0.5 and 1.0 kGy treated materials in all storage times. Although irradiation at 0.5 kGy
115
produced lower percentage of buttons without defects than at 1.0 kGy, the difference was insignificant. A t the 76th hour, the control was 100% defective whereas 63% and 84% of the 0.5 and 1 . 0 k G y samples, respectively, were still without apparent defects. Results of t-test between means of the 0.5 and 1 . 0 k G y treated mushrooms for the entire storage period also showed insignificant difference between these two lots of samples. Graphs of mean percentage of defective buttons vs. storage time are shown in Fig. 2. The main defect of both irradiated and non-irradiated mushrooms in the first 28 h of storage was opened caps, but an opened cap, by itself, may not he sufficient to render the mushrooms unmarketable. At the 52nd h, slimy buttons comprised the major portion of the defective nonirradiated buttons. After 52 h, the nonirradiated mushrooms had completely deteriorated, manifesting very intense discoloration, whitish moulds, and very strong ammoniacal odour, in addition to the slime. In contrast, the major defect of the irradiated samples at 52 h was still opened caps. Slime formation in the irradiated materials was observed starting on the 100th hour. In some samples, irradiated and non-irradiated, it was difficult to differentiate mould growth from slime since moulds developed simultaneously with the slime.
40 13
35
30 U~ Z in
B
2o
z lld 0
15
10
4 O Figure I
O KGY
I
I
l
!
28
52
76
100
+
13~
STORAGE TIME (HOURS) 0.,50 KGY O 1 .OO KGY
Mean percentage of opened buttons during post-irradiation storage at 22-25 °C, 85-95% r.h.
Food Quality and Preference (1989) 1 (3)
116
Nayga-Mercado and Alabastro
100 90 80
7O 60 50 40
-
30
-
20
-
10
-
T
0 4
I
i
T
!
28
52
76
100
|
136
m s (HO~S) D Figure 2
OKGY
+
0.50 KGY
o
1.00 KGY
Mean percentage of mushrooms without defects during post-irradiation storage at 22-25 °C, 85-95% r.h.
Respiration rate
There was no significant variation in reSpiration rates among trials, at all the post-treatment sampling periods. Irradiation at 0.5 and 1.0 kGy produced a significant inhibition in respiration rates only on the 52nd hour of storage, towards the end of the normal shelf life of fresh mushrooms, but not at the earlier sampling periods. An obvious difference in the trend of respiration rate between the untreated and irradiated mushrooms can be seen in Fig. 3. The control mushrooms gave continuously increasing values of respiration rate until the end of their storage life, while irradiated samples showed a peak in the respiration rate on the 28th hour. A low respiratory rate is generally associated with a long shelf life. Thus, treatments which depress respiration rate are expected to result in the prolongation of shelf life. In this study, irradiated mushrooms, which had lower respiration rates compared to the control, starting at the 52nd hour of storage, also showed slower deterioration as evidenced by cap opening, discoloration and other defects. The results obtained in this study strongly support the finding of Stanek et al. (1977) that respiration is inhibited by irradiation treatment. Mercier & MacFood Quality and Preference (1989) 1 (3)
Queen (1965) found an increase in respiration 4 h after irradiation in all samples irradiated at 0.1-1.0 kGy, but, by one day after irradiation, the respiration of treated samples had dropped below that of control samples and continued to decrease up to 5 d after irradiation.
Reducing sugars
Any change in the reducing sugar content of mushrooms brought about by irradiation may affect flavour. Accordingly, this chemical constituent was monitored during storage, and the results of the analyses are given in Table 2. Analysis of variance showed insignificant differences in the reducing sugar content between treatments but showed significant differences to exist between trials. A general pattern of increasing reducing monosaccharides with time was observed. The results reported herein are compatible with those of Campbell et al. (1968) who found the reducing sugars content of Agaricus campestris mushrooms treated by 1.0 kGy to be slightly higher than that of non-irradiated mushrooms after storage for 4 d at 1 °C and 85% r.h., but the difference was not significant.
Polyphenol oxidase activity
Significant differences between trials were shown to exist only in the first sampling period (4 h after irradiation), but significant differences between treatment means were observed at all sampling periods. The activity of the PPO enzyme in the control was significantly higher than that of the PPO in the 0.5 and 1.0kGy treated mushrooms. However, no significant difference was observed between 0.5 and 1.0kGy treated samples in their PPO activity. The results in Fig. 4 also show a difference between irradiated and nonirradiated samples, in the effect of storage time on PPO activity. In the non-irradiated samples, enzyme activity increased during storage, while in the 0.5 and 1.0kGy treated samples the PPO activity decreased with storage time. The data in Fig. 4 further show that doses higher than 0.5 and 1.0 kGy are necessary to effect complete inactivation of PPO. Sensory qualities
As shown in Table 3, there was a significant variation in colour scores between trials at the 18th h but not at the subsequent sampling periods. The finding that
Effects of radiation on fresh straw m u s h r o o m s
117
5.4 5.2 6 4.8
£
I
4,6
D
4.4 4.2 4 vo
3.8 3.G 3.4
Z
3.2
E
3
I[
2.8 2.6 2.4 2.2
I
I
!
I
I
4
28
52
76
1 O0
sxo~s O Figure 3
0 KGY
I-
~
0 . 5 0 KGY
136
04ou~) O
I .DO KGY
Respiration rate of mushrooms during post-irradiation storage at 22-25 °C, 85-95% r.h.
higher than that of the control, although the difference between the two irradiated samples was not significant. Irradiation dose (kGy) On the third day (76 h), the control had Storage time visibly darkened with an average colour .(hours after Trial 0 0.5 1.0 rating of almost 'brownish black'. The irradiation) irradiated mushrooms, on the other hand, 4 I 4.29 5.15 5.52 were still 'light brown'. II 9.10 9.34 9.54 The beneficial effect of irradiation on lII 9.19 9.32 9.95 coiour is consistent with findings on the 28 I 4.83 5.28 5.70 inhibition of PPO activity by irradiation II 10.22 10.50 9.52 treatment. At any storage time, nonIll 9.91 9.83 10.12 irradiated mushrooms had higher enzyme 52 I 6.22 5.74 6.78 activity than irradiated mushrooms and II 10.51 9.80 9.78 were judged to be more discoloured. III 10.18 9.87 10.57 There were no significant differences 76 I 6.70 7.09 between trials in terms of panel scores for II 9.82 9.82 texture/appearance of stored mushrooms. III 10.04 10.64 However, the data show that irradiation 100 I 8.74 9.86 treatment had a significant effect. At all II 9.85 9.96 sampling periods, the control had signifiIII 10.39 10.73 cantly lower scores than the 0.5 and 136 I 10.53 10.47 1.0 kGy irradiated batches. After 7 6 h II 10.40 11.70 storage, the control mushrooms appeared III 10.94 10.38 'very soft, definitely shrivelled', while the irradiated mushrooms showed only slight shrivelling. From the 18th to the 76th h, 0.5 and 1.0 kGy of ionizing radiation prothe date of culture affected colour scores significantly different from that of the duced indistinguishable effects on texture/ only in the initial hours of storage parallels non-irradiated sample, for all trials. appearance. that on PPO activity. After 58 and 76 h, the mean colour Just like the panel scores for colour, Analysis of variance showed that colour scores (averaged over the three trials) of significant differences in odour scores scores of the irradiated mushrooms were the irradiated samples were significantly. among trials were noted at the 18th h of Table 2 Reducing sugars (%) of mushrooms during post-irradiation storage at 22-25 *C, 85-95% r.h.
Food Quality and Preference (1989) 1 (3)
118
Nayga-Mercado and Alabastro
27 26 25 24 23 22 21 20 19
18 17 16 15 14 o
13 12
11 10
O Figure 4
I
I
I
I
I
I
4
28
52
76
1 O0
1 36
0 KGY
+
STORAGE TIME (HOURS) 0 . 5 0 KGY O 1 .OO KGY
Mean PPO activity of mushrooms during post-irradiation storage at 22-25 °C, 85-95% r.h.
storage. At this initial sampling period, the odour scores for the irradiated mushrooms were significantly higher than that of the control, in two out of the three trials. Subsequent samplings did not show significant variation among trials. At the 58th and 76th h, the mean odour scores (averaged over the three trials) of the irradiated mushrooms were significantly higher than that of the non-irradiated mushrooms, but the 0.5 and 1.0 kGy treated mushrooms were rated similarly by the panel. At the 76th h of storage, the odour of the nonirradiated mushrooms was judged as being 'strongly earthy and ammoniacal' while the irradiated mushrooms were assessed as having a 'just discernible earthy, ammoniacal smell'. Just as irradiation improved colour and texture quality during storage, a positive effect was likewise noted on the odour. The present results, which show that irradiation at 0.5 and 1.0 kGy definitely improved the sensory quality of stored straw mushrooms, corroborate the research findings of Mercier & MacQueen (1965), Staden (1966), Skou et al. (1974) and Gill et al. (1969) who all reported better colour and texture of mushrooms irradiated at doses of 2.0-5.0 kGy. The reports of Stanek et al. (1977) and Maxie et al. also stated that irradiated mushrooms Food Quality and Preference (1989) 1 (3)
kept the appearance, texture and flavour of fresh ones for a longer time than non-irradiated mushrooms.
Summary
and conclusions
The time at which the mushrooms were cultured affected only a few postharvest properties, notably the weight loss during storage and the reducing sugars content. The other properties, namely degree of cap opening, level of defective mushrooms, respiration rate, and texture/appearance as evaluated by a panel, were unaffected by date of harvest of the mushrooms. Irradiation did not result ,in a lower weight loss during storage, but did inhibit cap opening. Significantly higher percentages of buttons without defects were noted in the irradiated samples during the period from 4-76 h of storage. By this latter time, the non-irradiated mushrooms had completely deteriorated. Differences in the pattern of respiration rate during storage were shown by CO2 liberation rates increasing in the irradiated mushrooms up to 28 h and then decreasing abruptly, with the controls showing progressively increasing rates until the end of their shelf life. Irradiation effected a significant gradual
decrease in PPO enzyme activity with storage time, contrasted with an increasing activity with time in the control. This decrease in PPO activity is reflected in the results of the sensory evaluation, the irradiated samples having markedly better colour, texture/appearance and odour than the non-irradiated mushrooms. Irradiation at 0 . 5 k G y produced the same improvement in the storage quality of fresh mushrooms as 1.0 kGy irradiation treatment. This lower dose treatment has obvious advantages in terms of less energy cost and lower probability of occurrence0f" undesirable effects which have been observed at higher doses by many researchers. The results of the present study indicate that mushrooms, if irradiated, can be held at tropical ambient conditions (22-25 °C, 85-95% r.h.) for as long as 5 d after harvest, about 35% of the irradiated mushrooms being without apparent defects after a 5 d period without refrigeration. In contrast, the non-irradiated mushrooms were completely defective after 3 d. Acknowledgement
The authors gratefully acknowledge the assistance and cooperation of the Philippine Atomic Energy Commission.
Effects of r a d i a t i o n on fresh s t r a w m u s h r o o m s
Table 3 Panel scores for colour, odour and texture/appearance of mushrooms during postirradiation storage at 22-25 °C, 85-95% r.h. Storage time (h after irradiation)
Irradiation dose (kGy) Trial
0
0.5
1.0
It lit lilt i II III Meant I II Ill Meant
3.67 a 3.75" 4.25" 2.40 2.33 3.58 2.77 " 2.13 2.25 2.92 2.43"
4.75 h 4.92 b 5.00 b 4.50 4.25 4.67 4.47 h 4.5(I 4.33 4.08 4.30 h
4.83 h 5.00 h 5.00 b 4.70 4.58 4.50 4.59 b 4.88 4.50 4.17 4.52 b
It Ilt lilt I II Ill Meant I II III Meant
3.58 a 3.17" 4.00 ~ 2.30 2.17 3.42 2.63 u 2.00 1.58 2.58 2.05 ~
4.17 a 4.33 b 5.00 b 4.30 3.92 4.42 4.21 b 4.25 3.25 4.08 3.86 b
4.42" 4.67 b 4.67 b 4.70 4.50 4.58 4.59 b 4.13 3.58 4.08 3.93 b
I II III Meant I II 1II Meant I II Ill Meant
4.00 4.08 4.17 4.08~ 2.90 2.58 3.83 3.10 ~ 1.75 2.25 2.92 2.30 a
4.83 5.00 5.00 4.94 b 4.70 4.42 4.67 4.60 b 4.75 4.25 3.92 4.31 b
4.92 4.92 4.92 4.92 b 4.60 5.00 4.58 4.73 b 4.63 4.58 4.42 4.54 b
Colour 18
58
76
Odour 18
58
76
Texture~appearance 18
58
76
Colo,rscores: 1 black; 2 brownish black; 3 dark brown; 4 light brown; 5 very light brown, close to beige.
Odour scores: 1 very strongly earthy, ammoniacal smell; 2 strongly earthy, ammoniacal smell; 3 moderately earthy, ammoniacal smell; 4 just discernible earthy, ammoniacal smell; 5 natural aroma of mushroom. Texture~appearance scores: 1 disintegrated, appears rotten, mouldy, slimy and deteriorated; 2 very soft, definitely shrivelled; 3 moderately soft and shrivelled; 4 soft and slightly shrivelled; 5 firm yet tender; not shrivelled. t Values with the same superscripts are not significantly different at P -< 0.05.
119
References (1965) G a m m a radiation of vegetables to extend market life. Marketing Research Report No. 703, Agricultural Research Service, US Department of Agriculture: Washington, DC
BRAMLAGE, W J & LIPTON, W J
CAMPBELL, J D, STOTHERS, S, VAISEY, M & BERCK,
B (1968) Gamma radiation influence on the storage and nutrional quality of mushrooms,
Journal of Food Science, 33,540-542 (1969) Irradiation of cultured mushrooms, Food Technology, 23,385-388 HALIM, O H a MONTGOMERY, M W (1978) Polyphenol oxidase of d ' A n j o u pears (Pyrus communis L.), Journal of Food Science, 43, 603-606 JOSLYN, M A (ed.) (1970) Methods in Food Analysis, 2nd edn. Academic Press: New York GILL, W J, NICHOLAS, R C & MARKAKIS, P
LOWRY, O H, RASENBROUGH, N J, FARR, A L &
RANDAL, R (1951) Protein measurement with the folin-phenol reagent, Journal of Biological Chemistry, 193,265-275 MAXIE, E C, SOMMER, N F, & BROWN, D S (1967) Radiation technology in conjunction with post-harvest procedures as a means of extending the shelf life of fruits and vegetables. Report UCD-34P80-5, United States Department of Commerce: Washington DC MERCIER, R G & MACOUEEN, K F (1965) Gamma-
irradiation to Extend Post-harvest Life of Fruits and Vegetables. Horticultural Experiment Station and Products Laboratory, Vineland Station: Ontario, Canada s n o u , J P, BECH, K, & LUNDSTEN, K (1974) Effects of ionizing irradiation on mushrooms as influenced by physiological and environmental conditions. Radiation Botany, 14,287-299 STADEN, O L (1966) Food Irradiation. International Atomic Energy Agency; Vienna STANEK, M, URBAN, M, SMOTLACHA, M & HAKO-
VA, K (1972) Effect of ionizing radiation on the activity of proteases in mushroom fruit bodies, (in Czech, English summary) cited by Salkova, Z, Kubin, M, Stanek, M & Horacek, P (1977) Present Status of Food Irradiation in Czechoslovakia. International Symposium on Food Preservation by Irradiation: Wageningen, The Netherlands TATE, J N, LUK, R S & YORK, G K (1964) Polyphenol oxidase in Bartlett pears, Journal of Food Science, 29,829-836
Food Quality and Preference (1989) 1 (3)