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Psychrotrophic, proteolytic and lipolytic properties of Enterobacteriaceae isolated from milk and dairy products
International Journal of Food Microbiology, 9 (1989) 79-83 Elsevier
79
JFM 00274
Short Communication
Psychrotrophic, proteolytic and lipolytic properties of Enterobacteriaceae isolated from milk and dairy products Delille Wessels 1 p . j . J o o s t e 2 a n d J . F . M o s t e r t 1 I Animal and Dairy Science Research Institute, Irene, Republic of South Africa and e Department of Food Science, University of the Orange Free State, Bloemfontein, Republic of South Africa Received 4 October 1988; accepted 14 March 1989)
Most of the Enterobacteriaceae strains (73 out of 75) isolated in a previous study (Wessels et al., 1988) were psychrotrophic on agar plates, with the exception of Enterobacter cloacae strains. The Enterobacteriaceae strains were largely non-proteolytic on milk agar m e d i u m although fimited n u m b e r s of E. cloacae, Serratia rubidaea and Klebsiella oxytoca strains were capable of proteolytic activity at 25 ° C. The E. cloacae and K. oxytoca strains positive at 25 o C were also proteolytic at 7 ° C. Most of the species tested were non-lipolytic on Victoria blue butterfat agar. The majority of Serratia marcescens and Klebsiella pneumoniae strains and a minority of E. cloacae and K. oxytoca strains, however, were lipolytic on this medium. Key words: Enterobacteriaceae, psychrotrophic, proteolytic and lipolytic properties; Dairy products
Introduction
A wealth of literature is available regarding known psychrotrophic organisms such as Pseudomonas, Flavobacterium and Alcaligenes spp. (Mikolajcik, 1979; Stead, 1986), but little is known about the occurrence and significance of psychrotrophic coliform bacteria and other Enterobacteriaceae in milk and dairy products. The bacterial population of milk produced under hygienic conditions normally contains less than 10% psychrotrophs; these organisms may, however, constitute up to 75% of the total population under unhygienic conditions (Cousin, 1982). Some psychrotrophic bacteria can produce heat-stable extra-cellular enzymes, and although the organisms are killed by pasteurisation temperature these enzymes may still cause spoilage (Cousin, 1982; Bigalke, 1985; Stead, 1986). Correspondence address: D. Wessels, Animal and Dairy Science Research Institute, Private Bag X2, 1675 Irene, Republic of South Africa. 0168-1605/89/$03.50 © 1989 Elsevier Science Publishers B.V.
80 A wide variety of genera and species of the family Enterobacteriaceae were isolated from milk and other dairy products in a previous study (Wessels et al., 1988). The species most often encountered, were Enterobacter cloacae, Klebsiella oxytoca, Citrobacter freundii and Enterobacter agglornerans. These organisms have also been found in dairy products by other workers (Cousin, 1982). The products most often involved in these studies included milk, cream, yoghurt, cottage cheese and ice-cream. It was also found that the organisms were responsible for defects such as bitter, fruity and 'dirty' flavours (Bigalke, 1985) as well as rancid and soapy flavours (Stead, 1986). It remains necessary, however, to determine the significance of species and strains of Enterobacteriaceae isolated from milk and dairy products in more detail. The purpose of this study, therefore, has been to examine members of the Enterobacteriaceae isolated from various dairy products by Wessels et al. (1988) with regard to their psychrotrophic, proteolytic and lipolytic properties in model systems. The model systems involved agar media containing skimmilk or butterfat and the possible significance of the strains in dairy products were inferred from these results.
Experimental procedure Seventy-five strains of species belonging to the family Enterobacteriaceae (Table I) and isolated previously from various dairy products (Wessels et al., 1988) were investigated with regard to their lipolytic and proteolytic activities, and psychrotrophic growth properties.
Lipolytic activity A sugar-free Nutrient Agar medium (pH 7.5) (Harrigan and McCance, 1976) with emulsified butterfat and Victoria blue B as indicator was used for the determination of lipolytic activity. 24 h cultures from Nutrient Agar (NA) slants were streaked onto pre-poured plates and incubated for 72 h at 30 o C. Bright blue colonies were regarded as lipase positive.
Proteolytic activity 24 h NA slant cultures of the bacterial strains tested were streaked onto pre-poured plates of the relevant medium. For monitoring proteolysis under psych° rotrophic conditions, 5 g of skimmilk powder and 1 g of Oxoid L l l agar were each suspended in a separate volume of 50 ml distilled water. After autoclaving separately the two volumes were cooled to 50 ° C, mixed and poured. The inoculated plates were incubated at 7 ° C (Mostert, 1981) and were examined daily for clear zones around the developing colonies. For determining proteolysis under mesophilic conditions Standard Plate Count (SPC) agar (Biolab C6) with 10% added skimmilk was used. The inoculated plates were incubated at 25 ° C for 48 h (Harrigan and
81
McCance, 1976) and subsequently flooded with a 10% acetic acid solution. Clear zones around the colonies after 1 min exposure were regarded as positive.
Psychrotrophic growth 24 h cultures were streaked onto SPC agar plates and incubated at 7 ° C for 10 days (Harrigan and McCance, 1976). Organisms that developed normal single colonies after 10 days were considered to be psychrotrophic.
Results and Discussion
The ability of coliform bacteria to grow over a wide temperature range is well known (Stead, 1986). Investigation of Enterobacteriaceae strains in the present study (Table I) has shown that all the species except certain strains of E. cloacae were capable of forming normal colonies on SPC agar at 7 ° C. These results are particularly important since all dairy products are stored under refrigeration for purposes of retarding microbiological deterioration and extending the shelf life. According to Cousin (1982) species of Enterobacter and Klebsiella are those members of the Enterobacteriaceae that occur most generally in refrigerated raw
TABL E I Psychrotrophic, proteolytic and lipolytic properties on agar plates of Enterobacteriaceae species isolated from milk and dairy products Species
No. strains
Enterobacter cloacae Klebsiella oxytoca Citrobacter freundii Enterobacter agglomerans Enterobacter zakazakii Serratia rubidaea Serratia marcescens Hafnia alvei Klebsiella pneumoniae Escherichia coli Klebsiella pneumoniae subsp, ozaenae Cedecea sp. Total
Percentage of strains exhibiting: Psychrotrophic
Proteolytic activity b
Lipolytic
growth a
7oC
25 o C
activity c
17 14 13 10 5 4 3 3 2 2
89 100 1O0 100 1O0 100 100 100 100 1O0
33 15 0 0 0 0 0 0 0 0
33 15 10 10 0 25 0 0 0 0
17 8 0 0 0 0 67 0 50 0
1 1 75
100 100
0 0
0 0
0 0
Normal colony development on SPC agar after 10 days at 7 ° C. b Clear zones on 10% milk agar at 7 o C after 10 days and at 25 ° C after 2 days. c Blue colonies on Victoria blue butterfat agar after 3 days at 30 o C.
82 milk. This has been confirmed in the study of Wessels et al. (1988) who found that E. cloacae (22.4%) and K. oxytoca (18.4%) were the dominant Enterobacteriaceae species in dairy products. The Enterobacteriaceae strains examined in the present study were generally non-proteolytic on agar medium. At an incubation temperature of 25 ° C (Table I) only 5 of the 12 species examined included strains that were at all capable of proteolytic activity. Of these 5 species ( E. cloacae, S. rubidaea, K. oxytoca, E. agglomerans and C. freundii) only a minority of strains (11 out of 54) were proteolytic at 25 o C. The strains of E. cloacae and K, oxytoca that were proteolytic at 25 ° C were also proteolytic at 7 ° C (Table I). Despite the low percentage of proteolytic strains amongst species of the family Enterobacteriaceae the activity of the proteolytic strains may prove to be significant. More than 40% of the proteolytic strains formed clear zones around the colonies on milkagar within 2 days at 7 o C. After 3 days, 50% of the strains had formed clear zones. These results show that the proteolysis of milk proteins in a model system can be brought about by these organisms under refrigeration conditions within 48 h and that proteolytic Enterobacteriaceae therefore may play a role in the spoilage of milk and dairy products. Such results have in fact been reported by Cousin (1982), who found that Enterobaeter and Klebsiella strains were actively involved in the proteolytic spoilage of milk. In an earlier study by Wessels et al. (1988) E. cloacae and K. oxytoca were virtually the only members of the family Enterobacteriaceae that were isolated from yoghurt and cottage cheese samples. Other workers (Sadovski et al., 1980) isolated Klebsiella and Enterobacter species from another fermented milk product, namely cultured buttermilk. When taints and off-flavours arose in the latter product after 5 days at 5 ° C these organisms were the predominant psychrotrophic bacterial types. The degree of lipase production by psychrotrophic bacteria generally varies from species to species and also depends on the storage conditions, temperature and p H (Cousin, 1982; Shelley et al., 1986). The present investigation (Table I) showed that the Enterobacteriaceae strains tested were not lipolytic as a rule. Seven of the 12 species tested in fact were non-lipolytic. Only 3 of the 17 E. cloacae and 1 of the 14 K. oxytoca strains were lipolytic. Although higher percentages of the S. marcescens and K. pneumoniae strains were lipolytic (Table I) the numbers of strains tested were too few to warrant any definite conclusions.
References Bigalke, D. (1985) Lipolytic and protcolytic microorganismsand their enzymes. Part 2. Dairy Food Sanit. 10, 388-389. Cousin, M.A. (1982) Presence and activity of psychrotrophic micro-organisms in milk and dairy products: A review. J. Food Protect. 45, 172-207. Harrigan, W.F. and McCance, M.E. (1976) Laboratory Methods in Food and Dairy Microbiology, Academic Press, London. Mikolajcik, E.M. (1979) Psychrotrophic bacteria and dairy product quality. 1. Major organisms involved and defects produced. Cult. Dairy Prod. J. 14, 6-10.
83 Mostert, J.F. (1981) The bacteriological spoilage of milk after treatment at ultra high temperatures. DSc (Agric)-thesis. University of Pretoria, Republic of South Africa. Stead, D. (1986) Microbial lipase: their characteristics, role in food spoilage and industrial uses. J. Dairy Res. 53, 481-505. Sadovski, A.J., Gordin, S. and Foreman, I. (1980) Psychrotrophic growth of microorganisms in a cultured milk product. J. Food Protect. 43, 765-768. Shelley, A.W., Deeth, H.C. and MacRae, I.C. (1986) Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk. J. Appl. Bacteriol. 61, 395-400. Wessels, D., Jooste, P.J. and Mostert, J.F. (1988) The occurrence and importance of Enterobacteriaceae isolates in milk and dairy products. S. Afr. J. Dairy Sci. 20, 23-28.
79
JFM 00274
Short Communication
Psychrotrophic, proteolytic and lipolytic properties of Enterobacteriaceae isolated from milk and dairy products Delille Wessels 1 p . j . J o o s t e 2 a n d J . F . M o s t e r t 1 I Animal and Dairy Science Research Institute, Irene, Republic of South Africa and e Department of Food Science, University of the Orange Free State, Bloemfontein, Republic of South Africa Received 4 October 1988; accepted 14 March 1989)
Most of the Enterobacteriaceae strains (73 out of 75) isolated in a previous study (Wessels et al., 1988) were psychrotrophic on agar plates, with the exception of Enterobacter cloacae strains. The Enterobacteriaceae strains were largely non-proteolytic on milk agar m e d i u m although fimited n u m b e r s of E. cloacae, Serratia rubidaea and Klebsiella oxytoca strains were capable of proteolytic activity at 25 ° C. The E. cloacae and K. oxytoca strains positive at 25 o C were also proteolytic at 7 ° C. Most of the species tested were non-lipolytic on Victoria blue butterfat agar. The majority of Serratia marcescens and Klebsiella pneumoniae strains and a minority of E. cloacae and K. oxytoca strains, however, were lipolytic on this medium. Key words: Enterobacteriaceae, psychrotrophic, proteolytic and lipolytic properties; Dairy products
Introduction
A wealth of literature is available regarding known psychrotrophic organisms such as Pseudomonas, Flavobacterium and Alcaligenes spp. (Mikolajcik, 1979; Stead, 1986), but little is known about the occurrence and significance of psychrotrophic coliform bacteria and other Enterobacteriaceae in milk and dairy products. The bacterial population of milk produced under hygienic conditions normally contains less than 10% psychrotrophs; these organisms may, however, constitute up to 75% of the total population under unhygienic conditions (Cousin, 1982). Some psychrotrophic bacteria can produce heat-stable extra-cellular enzymes, and although the organisms are killed by pasteurisation temperature these enzymes may still cause spoilage (Cousin, 1982; Bigalke, 1985; Stead, 1986). Correspondence address: D. Wessels, Animal and Dairy Science Research Institute, Private Bag X2, 1675 Irene, Republic of South Africa. 0168-1605/89/$03.50 © 1989 Elsevier Science Publishers B.V.
80 A wide variety of genera and species of the family Enterobacteriaceae were isolated from milk and other dairy products in a previous study (Wessels et al., 1988). The species most often encountered, were Enterobacter cloacae, Klebsiella oxytoca, Citrobacter freundii and Enterobacter agglornerans. These organisms have also been found in dairy products by other workers (Cousin, 1982). The products most often involved in these studies included milk, cream, yoghurt, cottage cheese and ice-cream. It was also found that the organisms were responsible for defects such as bitter, fruity and 'dirty' flavours (Bigalke, 1985) as well as rancid and soapy flavours (Stead, 1986). It remains necessary, however, to determine the significance of species and strains of Enterobacteriaceae isolated from milk and dairy products in more detail. The purpose of this study, therefore, has been to examine members of the Enterobacteriaceae isolated from various dairy products by Wessels et al. (1988) with regard to their psychrotrophic, proteolytic and lipolytic properties in model systems. The model systems involved agar media containing skimmilk or butterfat and the possible significance of the strains in dairy products were inferred from these results.
Experimental procedure Seventy-five strains of species belonging to the family Enterobacteriaceae (Table I) and isolated previously from various dairy products (Wessels et al., 1988) were investigated with regard to their lipolytic and proteolytic activities, and psychrotrophic growth properties.
Lipolytic activity A sugar-free Nutrient Agar medium (pH 7.5) (Harrigan and McCance, 1976) with emulsified butterfat and Victoria blue B as indicator was used for the determination of lipolytic activity. 24 h cultures from Nutrient Agar (NA) slants were streaked onto pre-poured plates and incubated for 72 h at 30 o C. Bright blue colonies were regarded as lipase positive.
Proteolytic activity 24 h NA slant cultures of the bacterial strains tested were streaked onto pre-poured plates of the relevant medium. For monitoring proteolysis under psych° rotrophic conditions, 5 g of skimmilk powder and 1 g of Oxoid L l l agar were each suspended in a separate volume of 50 ml distilled water. After autoclaving separately the two volumes were cooled to 50 ° C, mixed and poured. The inoculated plates were incubated at 7 ° C (Mostert, 1981) and were examined daily for clear zones around the developing colonies. For determining proteolysis under mesophilic conditions Standard Plate Count (SPC) agar (Biolab C6) with 10% added skimmilk was used. The inoculated plates were incubated at 25 ° C for 48 h (Harrigan and
81
McCance, 1976) and subsequently flooded with a 10% acetic acid solution. Clear zones around the colonies after 1 min exposure were regarded as positive.
Psychrotrophic growth 24 h cultures were streaked onto SPC agar plates and incubated at 7 ° C for 10 days (Harrigan and McCance, 1976). Organisms that developed normal single colonies after 10 days were considered to be psychrotrophic.
Results and Discussion
The ability of coliform bacteria to grow over a wide temperature range is well known (Stead, 1986). Investigation of Enterobacteriaceae strains in the present study (Table I) has shown that all the species except certain strains of E. cloacae were capable of forming normal colonies on SPC agar at 7 ° C. These results are particularly important since all dairy products are stored under refrigeration for purposes of retarding microbiological deterioration and extending the shelf life. According to Cousin (1982) species of Enterobacter and Klebsiella are those members of the Enterobacteriaceae that occur most generally in refrigerated raw
TABL E I Psychrotrophic, proteolytic and lipolytic properties on agar plates of Enterobacteriaceae species isolated from milk and dairy products Species
No. strains
Enterobacter cloacae Klebsiella oxytoca Citrobacter freundii Enterobacter agglomerans Enterobacter zakazakii Serratia rubidaea Serratia marcescens Hafnia alvei Klebsiella pneumoniae Escherichia coli Klebsiella pneumoniae subsp, ozaenae Cedecea sp. Total
Percentage of strains exhibiting: Psychrotrophic
Proteolytic activity b
Lipolytic
growth a
7oC
25 o C
activity c
17 14 13 10 5 4 3 3 2 2
89 100 1O0 100 1O0 100 100 100 100 1O0
33 15 0 0 0 0 0 0 0 0
33 15 10 10 0 25 0 0 0 0
17 8 0 0 0 0 67 0 50 0
1 1 75
100 100
0 0
0 0
0 0
Normal colony development on SPC agar after 10 days at 7 ° C. b Clear zones on 10% milk agar at 7 o C after 10 days and at 25 ° C after 2 days. c Blue colonies on Victoria blue butterfat agar after 3 days at 30 o C.
82 milk. This has been confirmed in the study of Wessels et al. (1988) who found that E. cloacae (22.4%) and K. oxytoca (18.4%) were the dominant Enterobacteriaceae species in dairy products. The Enterobacteriaceae strains examined in the present study were generally non-proteolytic on agar medium. At an incubation temperature of 25 ° C (Table I) only 5 of the 12 species examined included strains that were at all capable of proteolytic activity. Of these 5 species ( E. cloacae, S. rubidaea, K. oxytoca, E. agglomerans and C. freundii) only a minority of strains (11 out of 54) were proteolytic at 25 o C. The strains of E. cloacae and K, oxytoca that were proteolytic at 25 ° C were also proteolytic at 7 ° C (Table I). Despite the low percentage of proteolytic strains amongst species of the family Enterobacteriaceae the activity of the proteolytic strains may prove to be significant. More than 40% of the proteolytic strains formed clear zones around the colonies on milkagar within 2 days at 7 o C. After 3 days, 50% of the strains had formed clear zones. These results show that the proteolysis of milk proteins in a model system can be brought about by these organisms under refrigeration conditions within 48 h and that proteolytic Enterobacteriaceae therefore may play a role in the spoilage of milk and dairy products. Such results have in fact been reported by Cousin (1982), who found that Enterobaeter and Klebsiella strains were actively involved in the proteolytic spoilage of milk. In an earlier study by Wessels et al. (1988) E. cloacae and K. oxytoca were virtually the only members of the family Enterobacteriaceae that were isolated from yoghurt and cottage cheese samples. Other workers (Sadovski et al., 1980) isolated Klebsiella and Enterobacter species from another fermented milk product, namely cultured buttermilk. When taints and off-flavours arose in the latter product after 5 days at 5 ° C these organisms were the predominant psychrotrophic bacterial types. The degree of lipase production by psychrotrophic bacteria generally varies from species to species and also depends on the storage conditions, temperature and p H (Cousin, 1982; Shelley et al., 1986). The present investigation (Table I) showed that the Enterobacteriaceae strains tested were not lipolytic as a rule. Seven of the 12 species tested in fact were non-lipolytic. Only 3 of the 17 E. cloacae and 1 of the 14 K. oxytoca strains were lipolytic. Although higher percentages of the S. marcescens and K. pneumoniae strains were lipolytic (Table I) the numbers of strains tested were too few to warrant any definite conclusions.
References Bigalke, D. (1985) Lipolytic and protcolytic microorganismsand their enzymes. Part 2. Dairy Food Sanit. 10, 388-389. Cousin, M.A. (1982) Presence and activity of psychrotrophic micro-organisms in milk and dairy products: A review. J. Food Protect. 45, 172-207. Harrigan, W.F. and McCance, M.E. (1976) Laboratory Methods in Food and Dairy Microbiology, Academic Press, London. Mikolajcik, E.M. (1979) Psychrotrophic bacteria and dairy product quality. 1. Major organisms involved and defects produced. Cult. Dairy Prod. J. 14, 6-10.
83 Mostert, J.F. (1981) The bacteriological spoilage of milk after treatment at ultra high temperatures. DSc (Agric)-thesis. University of Pretoria, Republic of South Africa. Stead, D. (1986) Microbial lipase: their characteristics, role in food spoilage and industrial uses. J. Dairy Res. 53, 481-505. Sadovski, A.J., Gordin, S. and Foreman, I. (1980) Psychrotrophic growth of microorganisms in a cultured milk product. J. Food Protect. 43, 765-768. Shelley, A.W., Deeth, H.C. and MacRae, I.C. (1986) Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk. J. Appl. Bacteriol. 61, 395-400. Wessels, D., Jooste, P.J. and Mostert, J.F. (1988) The occurrence and importance of Enterobacteriaceae isolates in milk and dairy products. S. Afr. J. Dairy Sci. 20, 23-28.