Cooked meat products made of coarsely ground pork: the main bacterial strains of bacterial flora, their heat resistance and effect on spoilage Esko Petäjä and Eero Puolanne Petäjä. E. & Puolanne, E. 1993. Cooked meat products made of coarsely ground pork: the main bacterial strains of bacterial flora, their heat resistance and effect on spoilage. Agric. Sei. Finl. 2: 413-421. (Dept. Food Technol., Meat Sect., FIN-00014 University ofHelsinki, Finland.) This study was conducted to investigate the bacterial flora of the surface layer and the core of meat products made of coarsely ground pork at the moment of spoilage when stored at 7°C or 4°C. The dominating strains were isolated, their heat resistance was studied in APT-broth, on APT-agar and incoarsely ground cured pork, and their growth after heating and effect on spoilage were followed in coarsely ground cured pork. The first signs of spoilage appeared in the surface layer of the products. The strains were coccoid lactic acid bacteria with counts ranging from 3,5 to 7.8 log efu (colony forming units)/g. They survived only accidentally after heating for 15 minutes at 72°C in APT-broth. The core of the products contained only coccoid lactic acid bacteria or only pseudomonads or both as the main bacterial strains. The counts ranged from 2.6 to 6.0 log efu/g. Most of the strains isolated from the core survived after heating for 30 minutes at 72°C in APT-broth in at least three tests out ofsix. The most noticeable result of the study was the occurence of heat-resistant pseudomonads in the core. It must be pointed out that all pseudomonads found survived after heating for 60 minutes at 72°C in APT-broth, and often after heating for 15 minutes at 72°C in coarsely ground cured pork (core 72°C). The efu number of the two most heat-resistant streptococcus strains decreased only I log unit over 15 minutes at 72°C in coarsely ground cured pork. The numbers of inoculated pseudomonads decreased but those of streptococci rose by a maximum of I log unit when the experimental porks were kept at 4°C after heating. This indicates that streptococci and pseudomonads probably do not constitute a serious spoilage factor in cooked meat products, but spoilage is generally effected by bacteria which have contaminated the surface layer of the products after heat treatment. Key words: keepability, spoilage, cooked meat, heat resistance Introduction The manufacture and consumption of food prod- ucts made of coarsely ground meat has grown over the last 10 years in Finland. These products some- times spoil quite quickly so that not only the surface layer but also the core is spoiled. There is not much information available on the keeping times or the microbial flora of these kinds of products, or about whether the bacteria which can survive heat treat- ment in the core spoil the product. These products can be assumed to resemble bologna-type cooked sausages and luncheon meat in many respects. In these products high bacterial counts may be found 413 Agric. Sei. Fint. 2 (1993) https://www.c-info.fi/en/info/?token=_T_NI_QAHDi8y59O.-Xdga9IkREcTZWNo4FiZGA.UZ8AexR5uwtWbQh2WnzViBMNhIA2KIENKCvs0Df8vvZDLsTCohvDIOoT2wNWXUwGE53aRxYx5S3qGRJswVM65xnL4qD1-hJIsy-18XqcWi8JeGZZXAIhsMp_1tKqoTrbiE-mBqRjEfYb9it9UajlMqssqDPe3SOizgLv2ISc7uN0vsjS3Ot6L4imIAfSPkVRslimRl3ulUC2ROLfOLbuNm3BYsLBoNnd5e0yzXYNo4bJQRUFxD5DFYjWWDOlbjyOTrur_6UNb5kuaN89HV6NgH4cTtU6kl2KDqkdmaJr although the products would not always have spoiled. Paradis and Stiles (1978) have found 60% ofcommercial bologna type sausages contain bacteria over 106 efu/g. According to Allen and Foster (1960) and Hill et al. (1976), 4-week-old bolognas contain over I07 bacteria/g. DuiTSCHA- ever (1977) reported over 106 bacteria/g in 50% of commercial luncheon meats and Hill et al. (1976) found that 71% of luncheon meats stored for 3 weeks at 4 - 7°C contained over I06 bacteria/g. According to many scientists (Reuter 1969, Man- tel and Beck 1977, Daelman and Hoof 1975) lactobacilli are the biggest bacterial group in cooked meat products. However, Dowdell and Board (1968) and Gardner (1966, 1968) report that Microbacterium thermosphactum is the most important bacterial group causing spoilage in cooked sausage. Gardner (1966) also found that cooked sausage may contain gram-negative bac- teria, pseudomonads and achromobacteria, al- though gram-negative bacteria are generally as- sumed to die during the processing of cooked meat products (Hallenbach and Potter (1981) and Palumbo et al. (1974). The purpose of this study was to find out the main bacterial types in the surface layer and core of cooked meat products made of coarsely ground pork, their survival after heat treatment in APT- broth, on APT-agar, and in coarsely ground cured pork, as well as their growth after heating and their effect on spoilage during storage in coarsely ground cured pork. Various methods were used since heat resistance is known to vary in different media. Material and methods Product samples Seven samples of coarsely ground pork products were received directly from the manufacturers. The samples were 2-4 weeks old and they were kept at 4°C and 7°C up to the moment when the first signs of spoilage, smell and/or slime, were detected. Dif- ferent samples were investigated for the two tem- peratures. At the moment when the first signs of spoilage appeared the samples were examined mi- crobiologically. Microbiological examination of product samples The product was peeled and the surface layer sample was taken aseptically at 1-2 mm in thick- ness. The core sample was taken from the centre of product after cutting the product in two parts so that nothing came into contact with the sampling area. The following examinations were made: Total count of bacteria on plate count agar (Difco 0479, 2-4 d at 30°C), on APT-agar (Merck 10453, 3 d at 30°C) and on blood agar (Orion, Espoo, Finland, 2 d at 37°C). Because the bacteria to be counted may not grow in all media, three different agars for aerobic total plate count were used to ensure the growth of the dominating strains. Total count of anaerobic bacteria on SPS-agar (Merck 10235, 2 d at 37°C in anaerobic jar). Number of lactic acid bacteria on Rogosa-agar (Merck 5463,4 dat 30°C). Number of Brocholhrix thermosphacta on STAA-agar (Gardner 1966, 2 d at 22°C). Number of staphylococci and micrococci on Baird-Parker-agar (Lab 85 and XOB5, 2 d at 37°C). Number ofcoliforms on VRB-agar (Lab 31, 1 d at 37°C). The results were tested by variance analysis (Epistat program) in relation to storage temperature and surface layer and core. Isolation of the main bacterial types The surface layer and core of all product samples contained only a few strains in high numbers, and these were isolated. The main colonies were se- lected from the APT-agar dishes on which the samples had been plated. The dishes with the high- est dilution were used. The colonies to be isolated were transferred to APT-agar and incubated for 2 414 Agric. Sei. Fint. 2(1993) days at 30°C. One typical colony ofeach strain was removed to APT-agar for storing the culture. Survival of the isolated strains after heating on solid and in liquid medium Solid medium The survival of the strains after heating on a solid medium was studied with a Gradiplate WlO-tem- perature-gradient incubator (Biodata Oy, Helsinki, Finland) with which it is possible to achieve a stepless temperature gradient within a range of 0- 10°C. The operating principle is as follows: The bacteria are grown in cuvettes of 65*98*12 mm, into which the agar is poured. The limit temperature for growth or survival is determined by measuring the distance of the growth limit from the lower temperature sensor and by calculating the respect- ive temperature. The survival of the strains was investigated by growing them on APT-agar (Merck 10453) for 30 minutes at a temperature range of 50 - 70°C. The inoculation was carried out by rolling a drop (0.05 ml) of APT-broth culture onto the sur- face of the agar. Two strains were inoculated in the same cuvette. The 18-hour-old APT-broth culture was used for inoculation. Table 2 shows the ranges ofinoculatedcell count. Three determinations were made for each strain. Liquid medium The survival of the strains after heating in APT- broth at 72°C was investigated side by side with the experiments on solid medium. The 5 ml APT broth was heated in a test tube to 72°C and inoculated with 0.05 ml of 18-hour-old APT-broth culture. The ranges of the inoculated cell counts are pre- sented in Table 2. Different tubes were prepared for each heating period and temperature. The inocu- lated test tubes were heated for 15, 30, 60, 90 and 120 minutes. After heat treatment they were incu- bated at 30°C for 2 days and the growth (turbid broth) was recorded. Six experiments were made for each strain. Survival ofsome isolated Pseudomonas and Streptococcus strains after heating in coarsely ground cured pork The survival of four Pseudomonas strains (lb2, 4b, 6b, 7b2) and two Streptococcus strains (3bi, 7bi) was also examined in coarsely ground cured pork after heating at 72°C. One experimental batch con- tained 150 g pork, 0.67 g glucose and 15 g water. The following additives were used: NaCl (2%), NaNO2 (0.012%), phosphates (0.15% P205) and Na-ascorbate (0.04%). The bacterial inoculum was added as APT-broth culture (15 ml). The target number of colony forming units (efu) in the inocu- lum was 107/g meat. Control batches were made without bacterial inoculum. Instead, 15 ml of water was added to the control batch. The coarse grinding of the meat and mixing of additives and bacteria were done in a Moulinex mixer (Moulinex, France) to a particle size of 0.5-1 cm (diameter). Both experimental and control meat batches were packed in the 10ml glass tubes in which they were heated. Different tubes were prepared for each heating period. The pseudomonas tubes were heated in a water bath for 5,15 and 30 minutes after the temperature had been raised to 72°C. The re- spective heating periods for Streptococcus 3b 1 tubes were 0.5,2,5,and 15 minutes and for Strepto- coccus 7bi tubes 0.5, 2,5, 15, 30 and 60 minutes. After heating the tubes were cooled for 10 minutes at 10°C in a water bath and for 1 hour at 22°C. The following microbiological determinations were made for the non-heated and heated coarsely ground meats, as well as for the meats heated for 5 minutes and stored for 2-3 weeks at 6°C: total plate count on APT-agar, the number of pseudomonads on GSP-agar, staphylococci and micrococci on Baird-Parker-agar, lactic acid bacteria on Rogosa- agar, and Brochothrix thermosphacta on STAA- agar. The manufacturers of the media as well as the incubations were the same as those in the micro- biological studies of the meat product samples. The number of colony forming units of the in- oculums was determined on APT-agar by incubat- ing for 2 days at 30°C. The survival of the inocu- lums after heating in APT-broth at 72°C (0.05 ml in 5 ml APT-broth) was also tested side by side with 415 Agric. Sei. Fint. 2 (1993) Table 1. Bacterial flora of cooked meat products made of coarsely ground pork at the moment of spoilage. The bacterial numbers are presented as logarithms of colony forming units (cfu)/g. Storage temperature 4°C 7°C Surface layer Core Surface layer Core XRXR XRXR Total plate count -Plate count agar 7.0 A 4.9-7.8 5.58 5.2-5.9 6.0A8 4.2-7.5 3.6 C 2.6-4.7 -APTS-agar 7.3 A 4.9-8.5 5.38 4.6-5.7 6.2A8 4.6-7.7 4.3 C 3.0-6.6 -Blood agar 6.4 A 5.0-7-7 5.28 4.6-6.0 5.7A8 3.5-6.9 3.4 C 2.5-4.4 Total count of anaerobes 5.4 A 3.8-6.3 3.98 2.7-4.8 2.5A8 1.0-4.8 1.3 C 1.0T 2.7 Lactic acid bacteria 4.8 A 1.0-6.7 2.18 1.0-3.4 4.BAC 2.9-6.9 2.9A8C 1.0-4.6 Brochothrix thermosphacta < 2.0-6.6 < 2.0-4.2 4.3 2.7-5.3 <2.0-3.5 Staphylococci + micrococci 2.6 2.3-3.0 < 2.0-2.9 < 2.0-5.7 < 2.0-4.8 Coliforms 1 2 4b 6.7-7.8 5 4 3 4 2 7.0-7.2 6.0 1 1 2 6b 6.4-7-1 6 4 4 4 2 7.4-7.7 5.8 2 2 0 7b2 7.3-8.2 6 6 4 5 2 7.6-8.5 7.2 3.4 3.6 2 11 Number of samples containing over 100 cfu/g. Total number of samples = 3. Survival of isolated bacterial strains after heat treatment in APT-broth and on APT-agar Seven strains from the surface layers survived after heating for 15 minutes at 72°C in either one or two of the six tests conducted (Table 2). Only one sur- face layerstrain survived after heating on APT-agar at temperatures over 55°C. The highest survival temperatures of most surface layer strains on APT- agar varied around 50°C. Most of the strains isolated from the core of coarsely ground pork products survived after heat- ing for 30 minutes at 72°C in APT-broth in at least three of the six tests (Table 2). Two gram-positive cocci and one gram-negative rod survived after heating for 30 minutes at 72°C in all six tests. These three strains also survived after heating for 90 minutes in at least five of the tests. It is noteworthy that all gram-negative rods isolated from the core of coarsely ground pork products were heat-resistant to the extent that all of them survived after heating for 60 minutes at 72°C in APT-broth. The isolated strains survived heating on APT-agar at distinctly lower temperatures than after heating in APT- broth. Only three strains exhibited the highest sur- vival temperature of over 60°C in some of the experiments. Survival of four isolated Pseudomonas strains after heating at 72°C in coarsely ground cured pork Of the Pseudomonas strains isolated in this study from the core of the cooked products made of coarsely ground pork, strain 7b2 survived best after heating in coarsely ground cured pork (Table 3). The mean number of colony forming units de- creased from log 7.2/g to log 3.6/g during heating for 15 minutes. After heating for 30 minutes inocu- lated pseudomonads were found in two of the three tests over log 2.0 efu/g. After heating strains 1b2,4b and 6b in coarsely ground cured pork for 15 minutes each strain were found over log 2.0 efu/g in 1 - 2 tests of the three. The total plate counts of bacteria in the heat- treated coarsely ground experimental porks ranged between log 3.1 and 5.6 efu/g. The control porks (not inoculated) contained pseudomonads and staphylococci/micrococci over 2.0 efu/g only acci- dentally. They did not contain lactic acid bacteria over log 1.0 efu/g or Brochothrix thermosphacta over log 2.0 efu/g. The non-heated coarsely ground, cured pork did not exceed these values either. When the inoculated pork samples which had been heated for 5 minutes at 72°C were incubated 418 Agric. Sei. Finl. 2 (1993) Table 4. Bacterial flora of coarsely ground cured pork inoculated with four Pseudomonas strains after heating for 5 minutes at 72°C and incubating for 2 days at 30°C or 2-3 weeks at 4°C (3 test series). After heating 2 days at 30°C 2-3 weeks at 4°C Strains Strains Stains lb, 4b 6b 7b, lb, 4b 6b 7b, lb, 4b 6b 7b, Total plate count, APT-agar »X4.1 4.4 4.9 5.1 5.3 6.4 6.3 5.2 5.2 5.3 4.2 6.8 s 0.8 1.4 0.7 0.1 1.0 1.8 1.5 0.1 0.4 1.8 0.3 2.0 Pseudomonads, inoculated X 2.6 2> 1 2 3.4 2 2 0 1 0 0 0 1 s 0.9 1.3 Staphylococci + micrococci 212- 2222 2212 I actic acid bacteria " 0 0 0 0 0 0 0 0 0 0 0 0 II log cfu/g :i Number of" samples containing over 100 cfu/g. Total number of samples = 3. " Number of samples containing over 10 cfu/g. for two days at 30°C, the efu number of strains lb2 and 4b rose, whereas the efu number of strains 6b and 7b2 decreased (Table 4). The total plate count for the porks rose to over log 6.0 efu/g. When the inoculated pork samples, which had been heated for 5 minutes at 72°C, were stored for 2 - 3 weeks at 6°C, the numbers of all inoculated strains decreased (Table 4). The total plate count rose distinctly only in the tests in which strain 7b2 was used. The incubation experiments both at 30°C and at 6°C indicate that even if pseudomonads survive after heat treatment, they are probably not a very serious factor causing spoilage in cooked meat pro- ducts made of coarsely ground meat or in cooked meat products in general. Survival oftwo most heat-resistant Streptococcus strains after heating at 72°C in coarsely ground cured pork The mean efu numbers of the porks before heating were 6.8 log efu/g (strain 3bi) and 7.6 log efu/g (strain 7b|) (Table 5). The decrease for both strains was about I log unit during heating for 2 minutes. The thermal death diminished when the heating was continued, and was about 2 log units for 3b| and 0.5 log unit for 7b i over the next 13 min. Coarsely ground experimental porks contained staphylococci and micrococci over 2.0 log efu/g only accidentally, while pseudomonads and Bro- chothrix thermosphacta never exeeded 2.0 log efu/g. When the heat-treated and cooled experi- mental coarsely ground cured experimental porks were stored at 6°C for 2-3 weeks the efu number of strain 3b 1 rose by 1 log unit and that of strain 7b i by 0.5 log unit. However, these bacteria probably do not constitute a spoilage problem because their efu number decreases by a few log units during cooking and rises only slightly during storage. Conclusion The core ofthe pork products were found to contain heat-resistant streptococci and pseudomonads as the main bacterial strains, but they were not the cause of spoilage of the products. The bacteria which contaminated the products after heat treat- ment caused the spoilage in the surface layer of the products. However, the reasons for eventual core spoilage were not found in this investigation. 419 Agric. Sei. Finl. 2 (1993) Table 5. Thermal death of Streptococcus strains 3b 1 and 7bl at 72°C in APT-broth (6 test series) and in coarsely ground cured pork (CCP) (4 test series). Heating Cfu/ml in APT-broth Cfu/g in CCP time, min 3bl 7bl 3bl 7bl X s X_ s X s X s 0 6.3 0.9 6.5 0.2 6.8 0.5 7.6 0.5 0.5 4.2 0.8 5.2 0.5 6.3 1.0 1 4.1 0.8 5.0 0.4 1.5 4.0 1.7 5.0 0.3 2 3.5 1.3 4.8 0.5 5.7 0.7 6.8 0.9 5 1.7 1.9 4.3 1.0 5.2 0.5 6.5 0.6 15 1.2 1.4 3.7 1.1 4.0 1.2 6.0 1.3 30 '> 4 2.1 0.9 5.5 1.1 60 1 2 5.3 1.4 120 0 I Cfu =colony forming unit X =mean of colony forming units s = standard deviation of mean 11 Number of samples containing colony forming units References Allen, J. R. & Foster, E. M. 1960. Spoilage of vacuum- packed sliced processed meats during refrigerated stor- age. Food Res. 25: 19-25. (Ref. Paradis & Stiles 1978.) Daelman, W. & Van Hoof, J. 1975. Einfluss des pH-Wertes, der Verwendung von Polyphosfat und der Lagerung auf die bakteriologiselle Beschaffenheit von Briihwurst und Briihwurstaufschnitt. Archiv fiir Lebensmittelhygienie 26:213-217. Dowdell, M. & Board, R. 1968. A microbiological survey of British fresh sausage. J. Appi. Bacteriol. 31; 378-396, Duitschaever, C. L. 1977. Bacteriological evaluation of some luncheon meats in Canadian retail market. J. Food Prot. 40: 382-384. Gardner, G. 1966. A selective medium for the enumeration of Microbacterium thermosphactum in meat and meat products. J. Appi. Bact. 29: 455-460. 1968. Effects of pasteurization or added sulphite on the microbiology of stored vacuum-packaged baconburgers. J. Appi. Bacteriol. 31: 462-478. Hallenbach, C. H. & Potter, N. N. 1981. Effects of nitrite and sorbate on bacterial populations in frankfurters and thuringer cervelat. J.Food Prot. 44: 341-346. Hill, W. M., Reaume, J. & Wilcox, J. C. 1976. Total plate count and sensory evaluation as measures of luncheon meat shelf life. J. Milk Food Techn. 39: 759-762. Korkeala, H. & Lindroth, S. 1987. Differences in micro- biological growth in the surface layer and at the centre of vacuum-packed cooked ring sausage. Int. J.Food Micro- bi 01.4: 105-110. —, Suortti, T. & Mäkelä, P. 1988. Ropy slime formation in vacuum-packed cooked meat products caused by homo- fermentative lactobacilli and a Leuconostoc species. Int. J. Food Microbiol. 7: 339-347. Mantel, T, & Beck, G. 1977. Zur microbiologischen Situa- tion vorverpackter Briihwurst. Fleischwirtsch. 57: 245- 247. Palumbo, S. A., Huhtanen, C. N. & Smith, J.-L. 1974. Microbiology of frankfurter’s process: Salmonella and natural aerobic flora. Appi. Microbiol. 27: 724-732. Paradis, D. C. & Stiles, M. E. 1978. A study of microbial quality of vacuum packaged sliced bologna. J. Food Prot. 10: 811-815. Reuter, G. 1969. Untersuchungen zur Mikroflora von Vor- verpackten Aufgeschnittenen Briih- und Kochwursten. Proc. 15th Eur. Meet. Meat Res. Workers, Helsinki, p. 124-134. Manuscript received April 1993 Esko Petäjä Eero Puolanne Department of Food Technology Meat Section FIN-00014 University of Helsinki, Finland 2420 Agric. Sei. Fin!. 2 (1993) SELOSTUS Keitettyjen karkeahienonnetusta lihasta valmistettujen lihavalmisteiden bakteerillooran hallisi'vai bakteerikannat, niiden lämpöresistenssi ja vaikutus pilaantumiseen Esko Petäjä ja Eero Puolanne Helsingin yliopisto Keitettyjen karkeahienonnetusta lihasta valmistettujen liha- valmisteiden pintaosan ja sisäosan bakteeriflooraa tutkittiin pilaantumishetkellä, kun valmisteita oli säilytetty 7°C tai 4°C:ssa. Flooraa hallitsevat bakteerikannat eristettiin ja niiden lämpöresistenssiä tutkittiin APT-liemessä, APT-agarilla ja karkeahienonnetussa suolatussa sianlihassa. Valmisteiden pintaosan bakteeriflooraa hallitsevat kannat olivat kokkimaisia maitohappobakteereja lukumäärien vaih- dellessa välillä 3,5-7,8 log pmyfpesäkkeen muodostava yksik- kö)/g. Valmisteiden sisäosan bakteerifloora sisälsi valtaorga- nismeina kokkimaisia maitohappobakteereja, kokkimaisia maitohappobakteereja japseudomonadeja tai pseudomonade- ja.Kaksi lämpöresisteintä maitohappobakteeria olivatstrepto- kokkeja. Sisäosan bakteerien kokonaislukumäärät vaihtelivat välillä 2,5-6,0 log pmy/g. Valmisteiden pintaosasta eristetytbakteerikannat säilyivät lisääntymiskykyisinä vain satunnaisesti, kun niitä lämpökäsi- teltiin 15 min 72°C:ssa APT-liemessä. Useimmat valmistei- den sisäosasta eristetyistä kannoista säilyivät lisääntymisky- kyisinä vähintään kolmessa kokeessa kuudesta, kun niitä läm- pökäsiteltiin 30 min 72°C:ssa APT-liemessä. On merkille pantavaa, ettäkaikki sisäosasta eristetyt pseudomonadikannat säilyivät lisääntymiskykyisinä, kun niitä lämpökäsiteltiin 60 min 72°C:ssa APT-liemessä. ja usein, kun niitä lämpökäsitel- tiin karkeahienonnetussa sianlihassa 5 min 72°C:ssa. Inoku- loitujen pseudomonadien lukumäärä laski, kun lämpökäsitel- tyjä koelihoja säilytettiin 4°C:ssa. Siten ne eivät todennäköi- sesti ole vakava pilaantumistekijä karkeahienonnetusta lihasta valmistetuissa lihavalmisteissa tai keitetyissä lihavalmisteissa yleensä. Lämpöresisteimpien streptokokkien lukumäärä (pmy) laski APT-liemessä jyrkemmin kuin karkeahienonnetussa sianlihassa, jossa lasku oli noin yksi log-yksikkö 15 minuutis- sa. Kun lämpökäsiteltyjä karkeahienonnettuja ja suolattuja sianlihoja säilytettiin 4 viikkoa 4°C:ssa, streptokokkien luku- määrä nousi enintään yhden log-yksikön. Kuitenkin myös- kään streptokokit eivät aiheuttane pilaantumista, koska niiden lukumäärä laskee lämpökäsittelyn aikana selvästi ja nousee kylmävarastoinnin aikana vain vähän. 421 Agric. Sei. Finl. 2 (1993)