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Effect of ultra-high pressure on fruit juices contaminant yeasts
Trends in High Pressure Bioscience and Biotechnology R. Hayashi (editor) 9 2002 Elsevier Science B.V. All rights reserved.
511
E f f e c t o f u l t r a - h i g h p r e s s u r e on fruit j u i c e s c o n t a m i n a n t y e a s t s
A. Rosenthal ~'. B. MacKey b. A. Bird b Embrapa (Brazilian Company for Agricultural Research) - Food Technology Center, Av. das Americas 29501 - CEP 23.020-470, Rio de Janeiro-RJ, Brazil b Food Science and Technology Department. University of Reading. Whiteknights. Reading RG6 3AZ. UMted Kingdom
The pressure resistance of a number of yeasts associated with spoilage of fruit products
(Zygosaccharomyces bailii, Saccharono'ces cerevisiae, I'ichia anomola. ('andida magnoliae Rhodotorula ghainis) was examined. No especially pressure resistant strains were apparent. Pressure treatment at 300 MPa for 10 min reduced viable numbers of a cocktail of
Zygosaccharomyces bailii strains by 4 log units in orange, apple or pineapple juices. A smaller decrease occurred in tomato juice but. after storage for 24h at room temperature the level of reduction had increased to equal or exceeded that seen in the other juices.
(ca 20 ~
1. I N T R O D U C T I O N The increasing demand for products that maintain the quality of fresh foods has led to a search for new technologies of food preservation. These technologies aim mainly at replacing those that involve heat treatment, which can result in a decrease in the nutritional value and a change in the sensory characteristics of the products (1.2). In this sense, high presstire has been investigated in order to create products of high quality and microbiological stability. individually or in combination with moderate heat treatment (3-5). Unlike heat. high pressure affects only a few covalent bonds and therefore has little effect on ester molecules and other compounds responsible for tbod flavour and taste. Pressure also has negligible effects on vitamins. However pressure does cause denaturation or unfolding of proteins by disrupting hydrophobic interactions and causes phase changes in biological membranes which explain its efficiency in inactivating spoilage microorganisms and some enzymes (6,7). Furthermore, ultra-high pressure (UHP) can also disrupt cell wall and inner structures of microorganisms. In this sense, many intracellular organelles cells such as nucleus. mitochondria, endoplasmic reticulum and vacuoles from Saccharom3'ces cerevisae showed deformation and disorganization under UHP treatment (8). UItP induces physiological
512
imbalances due to the internal and external structural damages, which are responsible for killing the microorganisms (4). The effect of UHP was also associated to a permanent shrinkage of cell volume, which was associated to an irreversible mass transfer that occurs between the cell and pressure medium, due to the disruption or increase in membrane permeability. Adiabatic expansion of water was also reported to be associated with microorganisms' inactivation (4). In spite of being commercially used only recently, high-pressure technology has already originated a row of products in Japan, United States and Europe. Those are mainly low pH products, such as fruit juices, jams, jellies, fruit yogurts and salad sauces (9). However, many aspects of the technology still have to be clarified, mainly relating to the variation in pressure resistance of foodborne pathogenic and spoilage organisms, according to the type and composition of the product, and the microbial species and strain. This work aimed at evaluating the baroresistance of different spoilage yeasts, individually or in combination, inoculated in different fruit juices and in growth media, in order to evaluate the different factors that might influence process definition, and perspectives for future use of the technology.
2. MATERIALS AND METHODS 2.1.Preparation of fruit juices The skin and core of flesh pineapple fruits were removed and the flesh pulped in a sterile food processor. Pulp was centrifuged at 8 000 rpm for 20 min and the supernatant used as the juice. Orange juice was obtained by squeezing flesh oranges and centrifuging as above. Tomato and apple juices were purchased at a local supermarket (organic tomato juice containing 0.5% sea salt and pressed apple juice containing L-ascorbic acid). 2.2.Yeast cultures The following strains were obtained from the National Collection of Yeast Cultures (Institute of Food Research. Norwich UK): Zygosaccharomyces bailii (NCYC 1416, 1400, 1766, 2406, 1427, 1601), Saccharomyces cerevisiae (NCYC 505), Candida magnoliae (NCYC 765). Rhodotorula glutinis (NCYC 377). Pichia anomola (NCYC 2257). Zygosaccharomyces bailii is a common cause of spoilage of fruit products and Rhodotorula, Candida and Pichia species have been isolated from pressure-treated tropical fruits and derivatives. 2.3.Growth of organisms Strains were maintained frozen on glass beads at -70~ and on slopes of yeast malt (YM) agar at 5~ Cultures were prepared by inoculating 20 ml YM broth from a YM slope and incubating on a shaking platform for 6 days at 25~ Cells were harvested by centrifugation at 6000 rpm for 20 min and resuspended in one tenth the volume of fresh YM broth to give a concentration of between 10.7 to 10.8 cells per ml. Concentrated suspensions were mixed together to produce a 'cocktail" of strains, as indicated, or were used singly to inoculate fruit juice or medium prior to pressure treatment.
513
2.4.Pressure treatment Suspensions to be pressurized were divided into 1.5-ml portions and placed in polyethylene bags, which were heat-sealed. Samples were pressure treated in a 300 ml pressure vessel (Stanstead Mark II Enhanced mini-food lab. Stanstead Fluid Power, Stanstead, United Kingdom). The pressure transmitting fluid was ethanol: castor oil (80:20). The temperature increase in the pressurization fluid caused by adiabatic heating was monitored using a thermocouple and was about 25~ at 600 MPa and lasted about 30 sec. The samples were kept in ice before and after pressurization. All the pressure experiments were carried out in duplicate. 2.5.Counting of viable cells Samples were diluted in maximum recover5, diluent (Oxoid Ltd. Basingstoke UK) and appropriate dilutions were spread onto YM agar. Plates were incubated at 25~ and colonies counted after incubation for up to 4 days. All the counting procedure was done in duplicate.
3. RESULTS AND DISCUSSION 3.1.Inactivation of different spoilage yeasts In preliminary experiments the pressure resistance of several yeast species associated with spoilage of fruit juice was compared in broth. Suspensions of yeast in YM broth were pressurised at ambient temperature for 10 rain at 0.1 to 300 MPa (Figure 1). It is noteworthy that where there is no point corresponding to a certain combination of the parameters in any one of the figures, for any particularly yeast, it means that no viable cell was detected in the previous combination (point) considered.
Zygosaccharomvces bailii 1766 9
0
Zygosaccharomyces bailii 2406 Zygosaccharomyces bailii I400 ---96- Saccharomyces cerevisae
~-4
---qk-- Picchia anomala
O
('andida magnoliae
--1 -6
-8 0
100
200
300
Figure 1. Effect of pressure on spoilage yeasts inactivation (time of pressurisation = 10 rain)
514
Saccharomyces cerevisiae and Pichia anomola vcere more sensitive than the other spoilage yeasts (Zygosaccharomyces bailii, Candida magnoliae). Numbers of the more resistant species were reduced by 3-4 log units after 10 rain at 300 MPa. 3.2.Time course of yeast inactivation Differences in pressure resistance between strains of spoilage yeast were seen more clearly in time-course experiments at 250 MPa. The fast-growing pink yeast Rhodotorula glutinis was included in these experiments and its resistance matched that of the Z3'gosaccharomyces bailii strains (Figure 2).
0 -1 -2 0
Z3(gossacharomyces bailii 1766 -------- Zygossacharorro~ces bailii 1400 A Zygossacharo.o,ces bailii 0778 X Zygossacharomvces bailii 2406 ~ Saccharom3~ces cerevisae Picchia anomala I Candida magnoliae --!1-- Rhodotorula ghainis
-3
Z-4 ~-5 9
-6 -7 -8 -9 0
5
10
15
20
Time (min) Figure 2. Inactivation of yeasts with pressurisation time at 250 MPa
3.3.1nactivation in fruit juice The preliminary trials indicated that Z),gosaccharom3'ces hailii was relatively resistant to pressure so a cocktail consisting of 6 strains of Z3'gosaccharomyces hailii was tested in different fruit .juices (Figure 3). After 10 rain at 300 MPa viable number decreased by 4 log units in orange, apple and pineapple .juice. but by only 2.3 log units in tomato juice. However. after storing for 24h at ambient temperature, viable numbers in the tomato juice samples that had been treated at 250 or 300 MPa decreased by a further 3 log units, resulting in a net reduction of nearly 5 log units (Figure 4). No further reduction in count occurred in the other juices. The study has shown that is possible to greatly reduce the number of viable cells of the yeasts usually responsible for product spoilage. In this way. it may be possible to use the
515 technology as a non-conventional method for preserving fruit juices. Several factors such as pressure levels, product storage after the treatment and time of treatment affect the lethality of the process to yeasts, and the response to the treatment may greatly depend on the type of fruit juice, species and strain of yeast being considered. Zy,~osaccharomyces hailii was amongst the more resistant group of organisms but was not unusually resistant. A relatively' short treatment at 300 MPa was sufficient to reduce numbers in fruit juices by' 4 to 5 log units. Others have found that treatment at 300 MPa for 10 rain was sufficient to reduce numbers of Zygosaccharomyces bailii in citrate buffer by 7 log units (10). The differences between that work and ours may be due to differences in the suspending media or strains used. A very slight elevation of temperature to 45 ~ was found to increase inactivation substantially (10). In agreement with our results. ALEM,/~N et al. (4) obtained decimal reductions of 0.7 and 5.1 in pineapple juice inoculated with Saccharomyces cerevisae and treated under 270 MPa for 40 and 400 sec. respectively.
I
1
.
0 -!
-1
o
z z
z
-2
--- in apv,e
Ub 0
\ I
-2
z -.I,
-*- ~ ~
3
--~ Tomato
-4
---- Apple
-5 0
! 00
200
300
Pressure (MPa) Figure 3. Inactivation of Zygosaccharono'ces hailii in fruit,juices (time = 10 min)
0
100
200
300
(MPa)
F'igure 4. Inactivation of Zygosaccharomyces , o ,/? hailii after post-treatment storage(25 C _4 hr)
Of particular interest was the observation that the initial level of yeast inactivation in tomato was less than in the other juices but viable counts subsequently declined on storage. It is worth noting that the pH value of the tomato juice was 4.07. while the other juices presented values in the range between 3.34 to 3.67. Tomato and apple juices bought from commercial suppliers contained salt (0.5 %) and L-ascorbic acid, respectively. In the case of the tomato ,juice. the higher pH value and/or the presence of salt seelned to have contributed to a decrease in the number of cells after the storage period. Inactivation of spoilage yeasts ma\' be influenced by the product composition and processing conditions. For example reduced water activity has a protective effect whereas relativeh' small increases in temperature, up to 40~ increase inactivation ( 11 - 13).
516 Future studies related to processing and storing fruit juices may clarity such aspects and provide opportunities for enhancing the preservative effects of pressure.
4. CONCLUSION Tile study has shown that is possible to greatly reduce the number of viable cells of yeasts usually responsible for the fruit juices spoilage. In this wav. it is potentially viable to use the technology as a non-conventional method of fruit.juices preservation. Some factors such as pressure levels, product storage alter treatment and time of treatment are relevant to the process, and the response to the treatment may greatly' depend on the type of fruit .juice. specie and strain of the yeasts being considered. In particular the responses patterns greatly varied among the species and strains studied. what turns it difficult to generalise the results for all spoiling microorganisms.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
R.G. Earnshaw; J. Appleyard and R.M. Hurst., Intern. J. Food Microb, 28 (1995) 197. J.P.P.M. Smelt., Trends Food Sci. Technol.. 9 (1998) 152. A. Williams, Nut. & Food Sci., 1 (1994) 20. G.D. Alemfin; E.Y. Ting: S.C. Mordre: A.C.O. Hawes. M. Walker: D.F. Farkas and J.A. Torres., J. Food Sci., 61 (1996) 388. J.C. Cheftel, Food Sci. Yechnol. Int.. 1 (1995) 75. D. Knorr. Food Technol., 47 (1993) 156. T. Vardag: H. Dierkes and P. K6rner, Food Technol. Europe, 3 (1995) 106. S. Shimada; M. andou: N. Naito: N. Yamada: M. Osumi and R. Hayashi. Appl. Microbiol. Biotechnol., 40 (1993) 123. R.G. Earnshaw, Nut. & Food Sci., 2 (1996) 8. Y. P. Pandya, F. F jewett and D. Hoover, J. Food Protect. 58 (1995) 301. H. Ogawa. K. Fukuhisa, Y. Kubo and H. Fukumoto. Agric. Biol. Chem., 54 (1990) 1219. C. Chen and C. W. Tseng, Process Biochem., 32 (1997) 337. E. Palou, A. L6pez-Malo. G. V. Barbosa-Cfinovas. J. Welti-Chanes and B. G. Swanson, Letters in Applied Microbiology, 24 (1997) 417.
511
E f f e c t o f u l t r a - h i g h p r e s s u r e on fruit j u i c e s c o n t a m i n a n t y e a s t s
A. Rosenthal ~'. B. MacKey b. A. Bird b Embrapa (Brazilian Company for Agricultural Research) - Food Technology Center, Av. das Americas 29501 - CEP 23.020-470, Rio de Janeiro-RJ, Brazil b Food Science and Technology Department. University of Reading. Whiteknights. Reading RG6 3AZ. UMted Kingdom
The pressure resistance of a number of yeasts associated with spoilage of fruit products
(Zygosaccharomyces bailii, Saccharono'ces cerevisiae, I'ichia anomola. ('andida magnoliae Rhodotorula ghainis) was examined. No especially pressure resistant strains were apparent. Pressure treatment at 300 MPa for 10 min reduced viable numbers of a cocktail of
Zygosaccharomyces bailii strains by 4 log units in orange, apple or pineapple juices. A smaller decrease occurred in tomato juice but. after storage for 24h at room temperature the level of reduction had increased to equal or exceeded that seen in the other juices.
(ca 20 ~
1. I N T R O D U C T I O N The increasing demand for products that maintain the quality of fresh foods has led to a search for new technologies of food preservation. These technologies aim mainly at replacing those that involve heat treatment, which can result in a decrease in the nutritional value and a change in the sensory characteristics of the products (1.2). In this sense, high presstire has been investigated in order to create products of high quality and microbiological stability. individually or in combination with moderate heat treatment (3-5). Unlike heat. high pressure affects only a few covalent bonds and therefore has little effect on ester molecules and other compounds responsible for tbod flavour and taste. Pressure also has negligible effects on vitamins. However pressure does cause denaturation or unfolding of proteins by disrupting hydrophobic interactions and causes phase changes in biological membranes which explain its efficiency in inactivating spoilage microorganisms and some enzymes (6,7). Furthermore, ultra-high pressure (UHP) can also disrupt cell wall and inner structures of microorganisms. In this sense, many intracellular organelles cells such as nucleus. mitochondria, endoplasmic reticulum and vacuoles from Saccharom3'ces cerevisae showed deformation and disorganization under UHP treatment (8). UItP induces physiological
512
imbalances due to the internal and external structural damages, which are responsible for killing the microorganisms (4). The effect of UHP was also associated to a permanent shrinkage of cell volume, which was associated to an irreversible mass transfer that occurs between the cell and pressure medium, due to the disruption or increase in membrane permeability. Adiabatic expansion of water was also reported to be associated with microorganisms' inactivation (4). In spite of being commercially used only recently, high-pressure technology has already originated a row of products in Japan, United States and Europe. Those are mainly low pH products, such as fruit juices, jams, jellies, fruit yogurts and salad sauces (9). However, many aspects of the technology still have to be clarified, mainly relating to the variation in pressure resistance of foodborne pathogenic and spoilage organisms, according to the type and composition of the product, and the microbial species and strain. This work aimed at evaluating the baroresistance of different spoilage yeasts, individually or in combination, inoculated in different fruit juices and in growth media, in order to evaluate the different factors that might influence process definition, and perspectives for future use of the technology.
2. MATERIALS AND METHODS 2.1.Preparation of fruit juices The skin and core of flesh pineapple fruits were removed and the flesh pulped in a sterile food processor. Pulp was centrifuged at 8 000 rpm for 20 min and the supernatant used as the juice. Orange juice was obtained by squeezing flesh oranges and centrifuging as above. Tomato and apple juices were purchased at a local supermarket (organic tomato juice containing 0.5% sea salt and pressed apple juice containing L-ascorbic acid). 2.2.Yeast cultures The following strains were obtained from the National Collection of Yeast Cultures (Institute of Food Research. Norwich UK): Zygosaccharomyces bailii (NCYC 1416, 1400, 1766, 2406, 1427, 1601), Saccharomyces cerevisiae (NCYC 505), Candida magnoliae (NCYC 765). Rhodotorula glutinis (NCYC 377). Pichia anomola (NCYC 2257). Zygosaccharomyces bailii is a common cause of spoilage of fruit products and Rhodotorula, Candida and Pichia species have been isolated from pressure-treated tropical fruits and derivatives. 2.3.Growth of organisms Strains were maintained frozen on glass beads at -70~ and on slopes of yeast malt (YM) agar at 5~ Cultures were prepared by inoculating 20 ml YM broth from a YM slope and incubating on a shaking platform for 6 days at 25~ Cells were harvested by centrifugation at 6000 rpm for 20 min and resuspended in one tenth the volume of fresh YM broth to give a concentration of between 10.7 to 10.8 cells per ml. Concentrated suspensions were mixed together to produce a 'cocktail" of strains, as indicated, or were used singly to inoculate fruit juice or medium prior to pressure treatment.
513
2.4.Pressure treatment Suspensions to be pressurized were divided into 1.5-ml portions and placed in polyethylene bags, which were heat-sealed. Samples were pressure treated in a 300 ml pressure vessel (Stanstead Mark II Enhanced mini-food lab. Stanstead Fluid Power, Stanstead, United Kingdom). The pressure transmitting fluid was ethanol: castor oil (80:20). The temperature increase in the pressurization fluid caused by adiabatic heating was monitored using a thermocouple and was about 25~ at 600 MPa and lasted about 30 sec. The samples were kept in ice before and after pressurization. All the pressure experiments were carried out in duplicate. 2.5.Counting of viable cells Samples were diluted in maximum recover5, diluent (Oxoid Ltd. Basingstoke UK) and appropriate dilutions were spread onto YM agar. Plates were incubated at 25~ and colonies counted after incubation for up to 4 days. All the counting procedure was done in duplicate.
3. RESULTS AND DISCUSSION 3.1.Inactivation of different spoilage yeasts In preliminary experiments the pressure resistance of several yeast species associated with spoilage of fruit juice was compared in broth. Suspensions of yeast in YM broth were pressurised at ambient temperature for 10 rain at 0.1 to 300 MPa (Figure 1). It is noteworthy that where there is no point corresponding to a certain combination of the parameters in any one of the figures, for any particularly yeast, it means that no viable cell was detected in the previous combination (point) considered.
Zygosaccharomvces bailii 1766 9
0
Zygosaccharomyces bailii 2406 Zygosaccharomyces bailii I400 ---96- Saccharomyces cerevisae
~-4
---qk-- Picchia anomala
O
('andida magnoliae
--1 -6
-8 0
100
200
300
Figure 1. Effect of pressure on spoilage yeasts inactivation (time of pressurisation = 10 rain)
514
Saccharomyces cerevisiae and Pichia anomola vcere more sensitive than the other spoilage yeasts (Zygosaccharomyces bailii, Candida magnoliae). Numbers of the more resistant species were reduced by 3-4 log units after 10 rain at 300 MPa. 3.2.Time course of yeast inactivation Differences in pressure resistance between strains of spoilage yeast were seen more clearly in time-course experiments at 250 MPa. The fast-growing pink yeast Rhodotorula glutinis was included in these experiments and its resistance matched that of the Z3'gosaccharomyces bailii strains (Figure 2).
0 -1 -2 0
Z3(gossacharomyces bailii 1766 -------- Zygossacharorro~ces bailii 1400 A Zygossacharo.o,ces bailii 0778 X Zygossacharomvces bailii 2406 ~ Saccharom3~ces cerevisae Picchia anomala I Candida magnoliae --!1-- Rhodotorula ghainis
-3
Z-4 ~-5 9
-6 -7 -8 -9 0
5
10
15
20
Time (min) Figure 2. Inactivation of yeasts with pressurisation time at 250 MPa
3.3.1nactivation in fruit juice The preliminary trials indicated that Z),gosaccharom3'ces hailii was relatively resistant to pressure so a cocktail consisting of 6 strains of Z3'gosaccharomyces hailii was tested in different fruit .juices (Figure 3). After 10 rain at 300 MPa viable number decreased by 4 log units in orange, apple and pineapple .juice. but by only 2.3 log units in tomato juice. However. after storing for 24h at ambient temperature, viable numbers in the tomato juice samples that had been treated at 250 or 300 MPa decreased by a further 3 log units, resulting in a net reduction of nearly 5 log units (Figure 4). No further reduction in count occurred in the other juices. The study has shown that is possible to greatly reduce the number of viable cells of the yeasts usually responsible for product spoilage. In this way. it may be possible to use the
515 technology as a non-conventional method for preserving fruit juices. Several factors such as pressure levels, product storage after the treatment and time of treatment affect the lethality of the process to yeasts, and the response to the treatment may greatly depend on the type of fruit juice, species and strain of yeast being considered. Zy,~osaccharomyces hailii was amongst the more resistant group of organisms but was not unusually resistant. A relatively' short treatment at 300 MPa was sufficient to reduce numbers in fruit juices by' 4 to 5 log units. Others have found that treatment at 300 MPa for 10 rain was sufficient to reduce numbers of Zygosaccharomyces bailii in citrate buffer by 7 log units (10). The differences between that work and ours may be due to differences in the suspending media or strains used. A very slight elevation of temperature to 45 ~ was found to increase inactivation substantially (10). In agreement with our results. ALEM,/~N et al. (4) obtained decimal reductions of 0.7 and 5.1 in pineapple juice inoculated with Saccharomyces cerevisae and treated under 270 MPa for 40 and 400 sec. respectively.
I
1
.
0 -!
-1
o
z z
z
-2
--- in apv,e
Ub 0
\ I
-2
z -.I,
-*- ~ ~
3
--~ Tomato
-4
---- Apple
-5 0
! 00
200
300
Pressure (MPa) Figure 3. Inactivation of Zygosaccharono'ces hailii in fruit,juices (time = 10 min)
0
100
200
300
(MPa)
F'igure 4. Inactivation of Zygosaccharomyces , o ,/? hailii after post-treatment storage(25 C _4 hr)
Of particular interest was the observation that the initial level of yeast inactivation in tomato was less than in the other juices but viable counts subsequently declined on storage. It is worth noting that the pH value of the tomato juice was 4.07. while the other juices presented values in the range between 3.34 to 3.67. Tomato and apple juices bought from commercial suppliers contained salt (0.5 %) and L-ascorbic acid, respectively. In the case of the tomato ,juice. the higher pH value and/or the presence of salt seelned to have contributed to a decrease in the number of cells after the storage period. Inactivation of spoilage yeasts ma\' be influenced by the product composition and processing conditions. For example reduced water activity has a protective effect whereas relativeh' small increases in temperature, up to 40~ increase inactivation ( 11 - 13).
516 Future studies related to processing and storing fruit juices may clarity such aspects and provide opportunities for enhancing the preservative effects of pressure.
4. CONCLUSION Tile study has shown that is possible to greatly reduce the number of viable cells of yeasts usually responsible for the fruit juices spoilage. In this wav. it is potentially viable to use the technology as a non-conventional method of fruit.juices preservation. Some factors such as pressure levels, product storage alter treatment and time of treatment are relevant to the process, and the response to the treatment may greatly' depend on the type of fruit .juice. specie and strain of the yeasts being considered. In particular the responses patterns greatly varied among the species and strains studied. what turns it difficult to generalise the results for all spoiling microorganisms.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
R.G. Earnshaw; J. Appleyard and R.M. Hurst., Intern. J. Food Microb, 28 (1995) 197. J.P.P.M. Smelt., Trends Food Sci. Technol.. 9 (1998) 152. A. Williams, Nut. & Food Sci., 1 (1994) 20. G.D. Alemfin; E.Y. Ting: S.C. Mordre: A.C.O. Hawes. M. Walker: D.F. Farkas and J.A. Torres., J. Food Sci., 61 (1996) 388. J.C. Cheftel, Food Sci. Yechnol. Int.. 1 (1995) 75. D. Knorr. Food Technol., 47 (1993) 156. T. Vardag: H. Dierkes and P. K6rner, Food Technol. Europe, 3 (1995) 106. S. Shimada; M. andou: N. Naito: N. Yamada: M. Osumi and R. Hayashi. Appl. Microbiol. Biotechnol., 40 (1993) 123. R.G. Earnshaw, Nut. & Food Sci., 2 (1996) 8. Y. P. Pandya, F. F jewett and D. Hoover, J. Food Protect. 58 (1995) 301. H. Ogawa. K. Fukuhisa, Y. Kubo and H. Fukumoto. Agric. Biol. Chem., 54 (1990) 1219. C. Chen and C. W. Tseng, Process Biochem., 32 (1997) 337. E. Palou, A. L6pez-Malo. G. V. Barbosa-Cfinovas. J. Welti-Chanes and B. G. Swanson, Letters in Applied Microbiology, 24 (1997) 417.