hm, rnational lounzal o[ Ft~M Microbiology.'. 17 (1992127-35
27
© 1992 Elsevier Science Publishers B.V. All rights reserved 11168-16tl5/92/$115.1111 FOOD 011538
Ropy slime-producing lactic acid bacteria contamination at meat processing plants P i a M . M i i k e l i i t, H a n n u
J. K o r k e a l a
i a n d J o r m a J. L a i n e -"
t College of Veterinao' Medicine. Department of F~nxl and Em'iromm'ntal tlygiem: lh'L~'inki and " Fimzish Meat Re,~earc'h ('entre. tliimcenlimza. Finlaml
(Received 27 December 1991:accepted 15 July 19t12)
At three Finnish meat processing phmts the processing rtmms, meat trimmings and carcasses were examined for the presence of ropy slime-pn'n.lucinglactic acid bacteria. Bacterial isolates similar to ropy slime-producing lactobacilli strains able to produce ropy slime on meat pn~lucts, were recovered from the processing rooms and meat trimmings, indicating that these r,n)ms and raw materials are a ~mrce of contamimltion for ropy bacteria. The ability to produce ropy slime would appear to be a common characteristic of lactobacilli, since altogether Ill different ropy lactobacilli groups were i~)lated in this study. Key words: Contamination" Ropiness: Lactic acid bacteria
Introduction During the last few years the formation of ropy slime on vacuum-packed cooked meat products has been a common spoilage problem in the Finnish meat industry (Korkeala et al., 1988). This ropiness was shown by Korkeala ct al. (1988) to be caused by lactic acid bacteria. Three ropy slime-producing lactic acid bacteria groups have been isolated by Korkeala et ai. (1988) from slimy meat products; and one ropy slime-producing lactobaeilli group was isolated from raw pork meat by Miikelfi and Korkeala (1989). In experiments, all of these bacterial groups caused ropiness on vacuum-packed cooked sausages. Miikelii et ai. (1990) showed that the meat used as the raw material of meat products contained bacterial strains similar to the lactic acid bacteria isolated from spoiled coocked sausages, and the conclusion was drawn that raw meat was an important source of these spoilage bacteria at meat processing plants. At the plants, the bacteria were thought to be able to spread from meat to processing rooms via air, personnel and equipment. Cooked meat products ha~e been found by several workers (Kempton and Bobier, 1970; Moi et al., 1971; M~ikel~i and
Correspondence address: Pia MSkcl[i, Department of Food and Environmental Hygiene, College of
Veterinary Medicine, P.O. Box 6, SF-011581Helsinki. Finland.
Korkeala, 1987, Borchet al., 1988) to be recontaminated with lactic acid bacteria during handling after heat processing, and the processing rooms and equipment, from which lactic acid bacteria have been isolated (Kempton and Bobicr, 1970; Miikelii and Korkeala, 1987), were considered to form one source of this contamination. Minimizing contamination by unwanted bacteria is one of the most effective ways of preventing spoilage problems in the meat industry, and therefore it is important to know the contamination sources of those bacteria which are the cause of the particular spoilage problem. The purpose of this study was to determine the sources of ropy slime-producing lactic acid bacteria at meat processing phmts. For this reason, the raw meat used in processing and the processing rooms of three meat plants, were examined for the presence of such organisms.
Materials and Methods
Sanlpling in the prt~cessing romns of meat plants The processing rooms of three Finnish meat processing phmts were examined. All of these plants have had occasional ropiness problems with their vacuum-packed meat products during the last few years. For sampling at each plant a production line was chosen in which the spoilage problems had commonly occurred. At Plant ! the production line studied was that fi~r a sliced Bologna-type ct~ked sausage and sliced cooked ham; at Plant 2 the line studied was that of ct~ked grill sausages, which are an emulsion type of sausage stuffed in casing from pork intestine. At Plant 3 the production line studied was manufacturing skinless grill sausages. Air and surface samples were taken from the processing rooms in which the products were handled after cooking. The rt~ms studied were the area of cooking and water chilling cabinets, the peeling room, the chill rt~ms, the vacuum-packing rooms and the slicing rtr~m. At Plant ! the rt~ms were sampled three times, and at Plants 2 and 3 the rooms were sampled twice.
Surface samples A sterile aluminium template was used to define an area of 25 cm 2, which was rubbed with a sterile, moistened cotton wool swab. In each sampling site an area of 10{I cm-" was sampled by use of the four swabs which were transferred to a bottle containing 10 ml of nutrient broth (Orion Diagnostica, Espoo, Finland). in the laboratory, the bottles were shaken manually for 3 min and preincubated at 30°C for 2 h to facilitate the repair of injured bacterial cells. The samples were then stirred and they and their 10-fold dilutions in 0.9% NaCI solution were plated onto MRS-S agar plates for the lactic acid bacteria count; MRS-S agar was prepared according to the method described by Korkeala and Lindroth (1987). MRS-medium (Oxoid, Basingstoke, England) was used as the base agar. The plates were incubated anaerobically at 20°C for 5 days in an anaerobic jar using an H , + CO, gas generating kit (Oxoid). All colonies on the plates were examined with a loop for the ability to rope (Korkeala et al., 1988).
For the i~lation of ropy slime producing lactic acid bacteria strains, an area of 100 cm 2 wlis sampled as above. The swabs were placed into l0 ml of MRS broth (Difco, Detroit, USA) and the bottles were shaken and preincuhated as above. The liquid sample was then added to IU0 ml of MRS broth containing 2 mg/ml NaNO: (Merck, Darmstad, Germany) and 0.25 p g / m l chloramphenicol (Carlo Erban, Milano, Italy). This modified MRS broth hits been hmnd to hc selective for the growth of ropy lactic acid bacteria strains (unpublished data). The broth wits incubated at 20°C. After 2 and 3 days of incubation the samples were plated onto 3 plates of MRS-S agar. These plates were incubated anaerobicaUy at 20°C for 5 dltys, after which the cohmies on the plates were examined for the ability to rope,
Air samples Air wits sampled using a n Andersen "l~vo-Stagc Vil,ble Particle Sizing Sampler (Andersen Samplers, Athmta, USA). Two petri dishes containing 20 ml of MRS-S agar were exposed to 0.849 m' of air. The plates were incubated as above. The ability of the cohmics to rope was then cxamincd. The "Positive hole" adjustment fi~r the air sltmples (Leopold, 1988) was used to correct the data. Meat trimmhags and carcass Smnl~les Pork and beef trimmings, and pork and beef carcasses, used as raw materials for meat products processed at Phmts I and 2, were studied. The meat trimmings contained 2.8% (w/w) sldt. A 25 g sample of meat trimmings wits mixed with 225 ml of 0.9% NaCI solution. The sample was preincubated hw 2 h at 30°C and then homogenized, diluted 10-fold in 0.9% NaCI solution, and plated onto MRS-S agar plates for the lactic acid bacteria count. For the isolation of slime-producing lactic acid bacteria strains, 10 ml of the 10-~dilution was added to 100 ml of the modified MRS broth described above. The MRS-S plates and the modified MRS broth were incubated and subcultured as alcove. Surfitce samples from pork carcasses were taken from cheek, mid-back, lateral abdomen and distal hind limb. Samples from beef carcasses were taken from lateral brisket, fore-rib, flank and medial round. Two tissue samples were removed from each site using sterile cork borers (diameter 2.52 cm), scalpels and tweezers. The combined surfilce area sampled from each carcass wits 40 cm 2. Sitmples from each carcass were placed in a sterile stomacher bag, to which 25 ml of 0.9% NaCI solution were added. The samples were preincubated fiw 2 h at 30°C and then homogenized in a stomacher (Seward Laboratory., London) h~r 2 rain. For the slime-producing lactic acid bacteria strains, 10 ml of this liquid was added to 100 ml of the modified MRS broth. For the lactic acid bacteria count the sample wlts diluted 10-fold and plated o n t o MRS-S plates. The modified MRS broth and the MRS-S plates were incubated and subcultured its above. Identification of ropy bacterial strabas if ropy colonies were detected, !-5 viscous colonies from each plate were isolated and grown on MRS at 20°C. The ropy isolates were examined for Gram reaction, general morphology and catalase production. The method described by
G i b s o n a n d A b d e l - M a l e k (1945) was used to test the ability to p r o d u c e gas from glucose. T h e A P i 50 C H L system was u s e d to f u r t h e r characterize the s t r a i n s ( A P ! System, M o n t a l i e u - V e r c i e u , F r a n c e ) .
Results
T h e lactic acid b a c t e r i a c o u n t s a n d the o c c u r r e n c e of ropy isolates in the surface s a m p l e s t a k e n from the r o o m s o f the m e a t processing p l a n t s are p r e s e n t e d in T a b l e !. Lactic acid b a c t e r i a w e r e f o u n d in all the r o o m s studied. R o p y bacterial isolates were r e c o v e r e d o n c e from the wall of the chill r o o m at P l a n t 2, o n c e from the wall of w a t e r chilling c a b i n e t of P l a n t 1 a n d o n c e from the roof of the slicing r o o m of P l a n t 1. R o p y isolates were isolated f r o m several s a m p l e s t a k e n from the surfaces of the slicing m a c h i n e at P l a n t 1 d u r i n g the first sampling. All the isolates
TABLE ! Lactic acid bacteria counts and occurrence of ropy isolates in surface samples taken from processing r ~ m s of three meat processing plants Sampling site Walls of water chilling cabinets Peeling area walls removing machine Chilling room walls
Plant I
Plant 2 LAB b
Ropy ~ No. LAB
Ropy No. LAB
9
27
© 1992 Elsevier Science Publishers B.V. All rights reserved 11168-16tl5/92/$115.1111 FOOD 011538
Ropy slime-producing lactic acid bacteria contamination at meat processing plants P i a M . M i i k e l i i t, H a n n u
J. K o r k e a l a
i a n d J o r m a J. L a i n e -"
t College of Veterinao' Medicine. Department of F~nxl and Em'iromm'ntal tlygiem: lh'L~'inki and " Fimzish Meat Re,~earc'h ('entre. tliimcenlimza. Finlaml
(Received 27 December 1991:accepted 15 July 19t12)
At three Finnish meat processing phmts the processing rtmms, meat trimmings and carcasses were examined for the presence of ropy slime-pn'n.lucinglactic acid bacteria. Bacterial isolates similar to ropy slime-producing lactobacilli strains able to produce ropy slime on meat pn~lucts, were recovered from the processing rooms and meat trimmings, indicating that these r,n)ms and raw materials are a ~mrce of contamimltion for ropy bacteria. The ability to produce ropy slime would appear to be a common characteristic of lactobacilli, since altogether Ill different ropy lactobacilli groups were i~)lated in this study. Key words: Contamination" Ropiness: Lactic acid bacteria
Introduction During the last few years the formation of ropy slime on vacuum-packed cooked meat products has been a common spoilage problem in the Finnish meat industry (Korkeala et al., 1988). This ropiness was shown by Korkeala ct al. (1988) to be caused by lactic acid bacteria. Three ropy slime-producing lactic acid bacteria groups have been isolated by Korkeala et ai. (1988) from slimy meat products; and one ropy slime-producing lactobaeilli group was isolated from raw pork meat by Miikelfi and Korkeala (1989). In experiments, all of these bacterial groups caused ropiness on vacuum-packed cooked sausages. Miikelii et ai. (1990) showed that the meat used as the raw material of meat products contained bacterial strains similar to the lactic acid bacteria isolated from spoiled coocked sausages, and the conclusion was drawn that raw meat was an important source of these spoilage bacteria at meat processing plants. At the plants, the bacteria were thought to be able to spread from meat to processing rooms via air, personnel and equipment. Cooked meat products ha~e been found by several workers (Kempton and Bobier, 1970; Moi et al., 1971; M~ikel~i and
Correspondence address: Pia MSkcl[i, Department of Food and Environmental Hygiene, College of
Veterinary Medicine, P.O. Box 6, SF-011581Helsinki. Finland.
Korkeala, 1987, Borchet al., 1988) to be recontaminated with lactic acid bacteria during handling after heat processing, and the processing rooms and equipment, from which lactic acid bacteria have been isolated (Kempton and Bobicr, 1970; Miikelii and Korkeala, 1987), were considered to form one source of this contamination. Minimizing contamination by unwanted bacteria is one of the most effective ways of preventing spoilage problems in the meat industry, and therefore it is important to know the contamination sources of those bacteria which are the cause of the particular spoilage problem. The purpose of this study was to determine the sources of ropy slime-producing lactic acid bacteria at meat processing phmts. For this reason, the raw meat used in processing and the processing rooms of three meat plants, were examined for the presence of such organisms.
Materials and Methods
Sanlpling in the prt~cessing romns of meat plants The processing rooms of three Finnish meat processing phmts were examined. All of these plants have had occasional ropiness problems with their vacuum-packed meat products during the last few years. For sampling at each plant a production line was chosen in which the spoilage problems had commonly occurred. At Plant ! the production line studied was that fi~r a sliced Bologna-type ct~ked sausage and sliced cooked ham; at Plant 2 the line studied was that of ct~ked grill sausages, which are an emulsion type of sausage stuffed in casing from pork intestine. At Plant 3 the production line studied was manufacturing skinless grill sausages. Air and surface samples were taken from the processing rooms in which the products were handled after cooking. The rt~ms studied were the area of cooking and water chilling cabinets, the peeling room, the chill rt~ms, the vacuum-packing rooms and the slicing rtr~m. At Plant ! the rt~ms were sampled three times, and at Plants 2 and 3 the rooms were sampled twice.
Surface samples A sterile aluminium template was used to define an area of 25 cm 2, which was rubbed with a sterile, moistened cotton wool swab. In each sampling site an area of 10{I cm-" was sampled by use of the four swabs which were transferred to a bottle containing 10 ml of nutrient broth (Orion Diagnostica, Espoo, Finland). in the laboratory, the bottles were shaken manually for 3 min and preincubated at 30°C for 2 h to facilitate the repair of injured bacterial cells. The samples were then stirred and they and their 10-fold dilutions in 0.9% NaCI solution were plated onto MRS-S agar plates for the lactic acid bacteria count; MRS-S agar was prepared according to the method described by Korkeala and Lindroth (1987). MRS-medium (Oxoid, Basingstoke, England) was used as the base agar. The plates were incubated anaerobically at 20°C for 5 days in an anaerobic jar using an H , + CO, gas generating kit (Oxoid). All colonies on the plates were examined with a loop for the ability to rope (Korkeala et al., 1988).
For the i~lation of ropy slime producing lactic acid bacteria strains, an area of 100 cm 2 wlis sampled as above. The swabs were placed into l0 ml of MRS broth (Difco, Detroit, USA) and the bottles were shaken and preincuhated as above. The liquid sample was then added to IU0 ml of MRS broth containing 2 mg/ml NaNO: (Merck, Darmstad, Germany) and 0.25 p g / m l chloramphenicol (Carlo Erban, Milano, Italy). This modified MRS broth hits been hmnd to hc selective for the growth of ropy lactic acid bacteria strains (unpublished data). The broth wits incubated at 20°C. After 2 and 3 days of incubation the samples were plated onto 3 plates of MRS-S agar. These plates were incubated anaerobicaUy at 20°C for 5 dltys, after which the cohmies on the plates were examined for the ability to rope,
Air samples Air wits sampled using a n Andersen "l~vo-Stagc Vil,ble Particle Sizing Sampler (Andersen Samplers, Athmta, USA). Two petri dishes containing 20 ml of MRS-S agar were exposed to 0.849 m' of air. The plates were incubated as above. The ability of the cohmics to rope was then cxamincd. The "Positive hole" adjustment fi~r the air sltmples (Leopold, 1988) was used to correct the data. Meat trimmhags and carcass Smnl~les Pork and beef trimmings, and pork and beef carcasses, used as raw materials for meat products processed at Phmts I and 2, were studied. The meat trimmings contained 2.8% (w/w) sldt. A 25 g sample of meat trimmings wits mixed with 225 ml of 0.9% NaCI solution. The sample was preincubated hw 2 h at 30°C and then homogenized, diluted 10-fold in 0.9% NaCI solution, and plated onto MRS-S agar plates for the lactic acid bacteria count. For the isolation of slime-producing lactic acid bacteria strains, 10 ml of the 10-~dilution was added to 100 ml of the modified MRS broth described above. The MRS-S plates and the modified MRS broth were incubated and subcultured as alcove. Surfitce samples from pork carcasses were taken from cheek, mid-back, lateral abdomen and distal hind limb. Samples from beef carcasses were taken from lateral brisket, fore-rib, flank and medial round. Two tissue samples were removed from each site using sterile cork borers (diameter 2.52 cm), scalpels and tweezers. The combined surfilce area sampled from each carcass wits 40 cm 2. Sitmples from each carcass were placed in a sterile stomacher bag, to which 25 ml of 0.9% NaCI solution were added. The samples were preincubated fiw 2 h at 30°C and then homogenized in a stomacher (Seward Laboratory., London) h~r 2 rain. For the slime-producing lactic acid bacteria strains, 10 ml of this liquid was added to 100 ml of the modified MRS broth. For the lactic acid bacteria count the sample wlts diluted 10-fold and plated o n t o MRS-S plates. The modified MRS broth and the MRS-S plates were incubated and subcultured its above. Identification of ropy bacterial strabas if ropy colonies were detected, !-5 viscous colonies from each plate were isolated and grown on MRS at 20°C. The ropy isolates were examined for Gram reaction, general morphology and catalase production. The method described by
G i b s o n a n d A b d e l - M a l e k (1945) was used to test the ability to p r o d u c e gas from glucose. T h e A P i 50 C H L system was u s e d to f u r t h e r characterize the s t r a i n s ( A P ! System, M o n t a l i e u - V e r c i e u , F r a n c e ) .
Results
T h e lactic acid b a c t e r i a c o u n t s a n d the o c c u r r e n c e of ropy isolates in the surface s a m p l e s t a k e n from the r o o m s o f the m e a t processing p l a n t s are p r e s e n t e d in T a b l e !. Lactic acid b a c t e r i a w e r e f o u n d in all the r o o m s studied. R o p y bacterial isolates were r e c o v e r e d o n c e from the wall of the chill r o o m at P l a n t 2, o n c e from the wall of w a t e r chilling c a b i n e t of P l a n t 1 a n d o n c e from the roof of the slicing r o o m of P l a n t 1. R o p y isolates were isolated f r o m several s a m p l e s t a k e n from the surfaces of the slicing m a c h i n e at P l a n t 1 d u r i n g the first sampling. All the isolates
TABLE ! Lactic acid bacteria counts and occurrence of ropy isolates in surface samples taken from processing r ~ m s of three meat processing plants Sampling site Walls of water chilling cabinets Peeling area walls removing machine Chilling room walls
Plant I
Plant 2 LAB b
Ropy ~ No. LAB
Ropy No. LAB
9
