Effects of organic acid and phosphates on the microbiological stability and on survival of salmonella typhimurium on turkey breast meat

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1 Effects of organic acid and phosphates on the microbiological stability and on survival of salmonella typhimurium on turkey breast meat A.H. DINÇER BAYSAL 1 * and A. ÜNLÜTÜRK 2 1 Department of Food Engineering, Faculty of Engineering, Izmir Institute of Technology, UrlaIzmir, Turkey 2 Department of Food Engineering, Faculty of Engineering, Ege University, Bornova Izmir, Turkey *handanbaysal@iyte.edu.tr Keywords: lactic acid; fumaric acid; trisodium phosphate; sodium tripolyphosphate; turkey breast meat; storage life; Salmonella; elimination Abstract The influence of dipping turkey breast meat in lactic acid (LA), fumaric acid (FA), lactic acid plus fumaric acid (LA+FA), trisodium phosphate (TSP) and sodium tripolyphosphate (STPP) solutions on aerobic mesophilic, psychrotrophic bacteria and yeast counts and also, the influence of dipping Salmonella inoculated turkey breast meat in LA, FA, TSP and STPP solutions on the elimination of S. typhimurium was assessed. 11.5% LA, 0.5% FA and 1% LA + 0.5% FA showed immediate inhibitory effects on aerobic mesophilic bacteria (0.30.6, 0.4 and 1.8 log unit reductions, respectively). 0.5% FA reduced psychrotrophic bacteria and yeast counts on fresh turkey breast fillets immediately by 1.1 and 0.4 log units, respectively. 5% TSP reduced aerobic mesophilic and psychrotrophic bacteria by 0.4 and 0.5 log units, respectively on fillets. 5% STPP reduced the yeast count on fillets immediately by 0.7 log unit. 1% LA, 0.5% FA and 1% LA + 0.5% FA reduced the psychrotrophic bacteria counts by 0.48, 0.23 and 1.27 log units after 8 days of storage, respectively in comparison to the untreated control samples. 1% LA + 0.5% FA treated breasts had a detectable discoloration and acid odour although there was no microbiological spoilage at 14 days of storage. Dipping fillets in both 1% LA and 0.5% FA extended the storage life up to 4 days, without adversely affecting the colour. Dipping fillets in 1% LA, 1.5% LA, 0.5% FA, 10% TSP and 10% STPP reduced the number of S. typhimurium by about 1.9, 2.7, 2.5, 3.5 and 3.2 log units, respectively. While there was only one Salmonella positive sample out of three samples 2 hours after dipping the fillets with an initial inoculum of 3.1 log CFU Salmonella per fillet in 1.5% LA solution, no Salmonella positive sample was observed in out of three samples dipped in 10% TSP solution. There were no Salmonella positive samples with an initial inoculum of 3.1 log CFU Salmonella per fillet after 2 and 24 hours of dipping in 10% TSP solution. There were no Salmonella positive fillets out of three samples dipped in 1.5% LA and 10% TSP solution with an initial inoculum of 2.6 log CFU Salmonella per fillet after 2 and 24 hours period. Introduction Contamination and growth of bacteria are a major problem to the poultry meat industry, even though good manufacturing practices are followed during processing. Therefore, numbers of microorganisms introduced on meat surfaces during slaughter should be reduced during production. The U.S. Department of Agriculture (USDA) has proposed that all slaughter establishments should apply at least one antimicrobial treatment or other approved intervention procedure to livestock and poultry carcasses (Nissen et al., 2001). Contamination of poultry products with pathogenic microorganisms is common. Among these microorganisms, Salmonella spp. and Campylobacter spp. are responsible for the largest proportion of poultry foodtransmitted incidents of human infection (Bean and Griffin, 1990; Bean et al., 1990; Tietjen and Fung, 1995; Tauxe, 1991). The U.S. Department of Agriculture (USDA) estimates that close to 40% of all raw poultry is contaminated with Salmonella (Rodriguez de Ledesma et al., 1996). In other countries, researchers determined the prevalence of Salmonella in poultry products ranged from 13.7 to 60%: 13.7% in Switzerland, 26.6% in Tokyo, 43% in Ohio, USA, 55% in Portugal, 66% in Thailand (Jerngklinchan et al., 1994; Uyttendaele et al., 1998). Turkey meat is frequently identified as a vehicle in outbreaks of salmonellosis. Consumers demand safe food 303

2 products and are unwilling to accept health risks that could be reduced by additional precautions being taken. The implementation of a HACCP system will force meat producers to study their production process and find, monitor and control the critical points. Control by endproduct decontamination is most attractive since it is applied just before the retailing of the carcasses or products and the loads of other pathogenic and spoilage bacteria also reduced. End product treatments that have been proposed for reducing or eliminating salmonellae from poultry carcasses in the processing plant include a variety of chemicals. Organic acids have been used as antimicrobial surface treatments to decrease pathogens, prevent the growth of spoilage microorganisms and extend the shelf life of fresh poultry meat. Organic acid dips or sprays such as acetic, lactic, citric and propionic acids either individually or combined, are beneficial in controlling undesired microorganisms on refrigerated meats (Bell et al., 1986; Hamby et al., 1987; Lillard et al., 1987; Marshall and Kim, 1996). Organic acids have been investigated because of their bactericidal activity and they are generally recognised as safe (GRAS) and therefore are utilised for preservation in many food applications (Izat et al., 1989; Dickson and Anderson, 1992; QuarteyPapafio et al., 1980). Phosphates have been used widely as texture modifiers, curing agents and to increase water holding capacity and more recently as surface decontaminating agents for meat products (Hwang and Beuchat, 1995; Slavik et al., 1994). Phosphates have broadspectrum antimicrobial activity and play an important role on the microbiological shelf life of meat products (Molins, 1991). USDA approved the use of trisodium phosphate (TSP) as a post chill processing aid to reduce bacteria on raw poultry carcasses. Although phosphates have antimicrobial effects, there are limited studies on the effects of phosphate type and concentration for use on refrigerated turkey meat to extend shelf life and to reduce or eliminate the Salmonella contamination. Nevertheless, conclusive evaluation of the efficacy of the organic acid or phosphate treatments has been hindered by the lack of reliable methodology for quantifying the levels of salmonellae on poultry carcasses or products before and after treatment. Since the level of natural salmonellae on poultry meat is unpredictable, it is necessary to evaluate decontamination treatments by using carcasses that have been inoculated with a known number of salmonellae. The purpose of the present study was to evaluate microbiological changes and microbiological shelf life of refrigerated turkey breast fillets treated with lactic acid, fumaric acid, their mixtures, trisodium phosphate and sodium tripolyphosphate. Also, this article presents, using plate count technique for rapidly and routinely enumerating salmonellae in conjunction with a nalidixic acid resistant (marker) strain of S. typhimurium, the efficacy of lactic acid, fumaric acid, trisodium phosphate and sodium tripolyphosphate dipping treatments for reducing and eliminating the Salmonella contamination of turkey breast fillets. Material and methods TURKEY BREAST FILLETS Turkey breast fillets were obtained from a local processing plant, PINAR Integrated Meat and Flour Industry Inc. Fillets were removed from the processing line just before packaging and immediately transported in ice bags to the University laboratories for the experiments. In two experiments, as indicated in the Result and Discussion text as the Experiment 2, refrigerated retail sliced breasts (fillets) were purchased from retail market. MICROORGANISM (BACTERIAL CULTURE) Salmonella typhimurium (CCM 835) was obtained from Ege University, Department of Biology and maintained on tryptone soy agar (TSA, Oxoid) slant at 4 C and was transferred every 3 months. S. typhimurium (CCM 835) was cultured in tryptone soy broth (TSB, Oxoid) at 37 C for 18 h before the experiments. TREATMENTS AND STORAGE Solutions of lactic acid (PURAC FCC80) at 1% and 1.5% (v/v), fumaric acid (Merck) at 0.5% (w/v), trisodium phosphate (Merck) at 5% (w/v) and sodium tripolyphosphate (Merck) at 5% (w/v) were prepared in tap water. The ph values of acid and phosphate solutions measured at the time of application with a ph meter were as follows: 1% LA (ph=2.72), 1.5% LA (ph=2.22), 0.5% FA (ph=2.70), 1% LA+0.5% FA (ph=2.14), 5% TSP (ph=12.68) and 5% STPP (ph=8.90). The administration of all the treatments and the sampling of the retail sliced breasts (fillets) were 304

3 conducted in a commercial poultry processing unit of a plant in İzmir. Fillets were dipped in 6 l solutions of 1%, 1.5% LA, 0.5% FA, 1% LA+0.5% FA, 5% TSP, 5% STPP for 1 min then drained on a sanitised tray for 10 min at 4 C. Fillets not treated with acids or phosphates were non treated control samples. After dipping, 3 fillets were packed in a polystyrene tray with a polyvinyl chloride cover films in an automatic machine and were transported on ice within 30 min to a storage room adjusted 4 ±1 C and 95% RH for storage. MICROBIOLOGICAL ANALYSES Immediately, after acid and phosphate treatment, and during the storage fillets were taken for analyses at defined intervals. 25 g sample was removed aseptically from ¼ of each fillet in the tray, placed in a stomacher bag and 225 ml of sterile 0.1% (w/v) peptone water (Oxoid L37) added. The sample was homogenised for 2 min in a stomacher (Seward Medical, Model 400), and a series of decimal dilutions were prepared. The enumeration of psychrotrophic microorganisms, total mesophilic aerobic microorganism, and yeast was carried out with the methods described by Anonymous (1989), APHA (1992); Gilliland et al. (1976); respectively. The results are presented as the means of duplicate plates of triplicate samples. ISOLATION OF NALIDIXIC ACID RESISTANT (NAR) S. TYPHIMURIUM STRAIN AND DETERMINATION OF COUNTING METHOD In order to isolate a nalidixic acid resistant (NAR) strain of S. typhimurium, 0.1 ml 18 h culture of S. typhimurium (CCM 835) was spread over the surface of the prepared and dried Brilliant Green Agar (BGA, Oxoid) plates containing 10 ppm NA and plates were incubated for 24 h at 37 C (Morrison & Fleet, 1985; Bilgili, S.F., personal communication). After incubation, typical Salmonella colonies were picked off the plates and confirmed with gram staining and biochemical tests. 18 h culture of the confirmed colonies were subcultured on to BGA containing 100 ppm nalidixic acid by streak method. Then the colonies, which developed on these plates after incubation at 37 C for 24 h, were presumed to be nalidixic acid resistant mutants and were subsequently confirmed by subculturing onto 100 ppm nalidixic acid containing Bismuth Sulphite Agar (BSA). Isolated mutants that had the same biochemical characteristics as the parent strain were reconfirmed as the NAR S. typhimurium (CCM 835). A modification of the procedure described by Morrison and Fleet (1985) was used as the counting method of NAR S. typhimurium. One fillet was placed into stomacher bag to which 50 ml of sterile buffered peptone water was added. After 2 min the rinse suspension was transferred to a sterile flask and incubated at 20 C for 2 h to resuscitate sublethally injured cells. After resuscitation, samples of suspension were then inoculated into three different media; in addition BSA supplemented with 100 ppm of nalidixic acid, Brilliant Green Agar, Plate Count Agar and Xylose Lysine Deoxycholate Agar (all supplemented with 100 ppm nalidixic acid) were also examined as counting media but were not selective enough to inhibit growth of non Salmonella species that were coming from turkey breast fillets. EXPERIMENT 1 Inoculation of turkey breast fillets with NAR S. typhimurium Each breast fillet surface was inoculated by spreading 0.1 ml of NAR S. typhimurium (CCM 835) across the fillet surface using a sterile, bent, glass rod and then allowed to set at room temperature for 20 min for cell attachment (to assure bacterial attachment prior to acid treatment). Then fillet samples were examined for numbers of the NAR S. typhimurium (marker organism) by plating on Bismuth Sulphite Agar containing 100 ppm nalidixic acid after resuscitation. Plates were incubated for 2448 h at 37 C before counting and recording the number of NAR S. typhimurium (CCM 835) per fillet. Treatments and storage Solutions of lactic acid (PURAC FCC80) at 1% and 1.5% (v/v), fumaric acid (Merck, ) at 0.5% (w/v), trisodium phosphate (Merck, ) at 5% and 10% (w/v), sodium tripolyphosphate (Merck, ) at 5% and 10% (w/v) were prepared in tap water. 1% LA, 1.5% LA and 0.5% FA solutions that determined effective in the reduction of microbiological load of turkey breast fillets, 305

4 without adversely affecting the colour, were used in the treatments (unpublished data). The ph values of acid and phosphate solutions are measured at the time of application with a ph meter as follows: 1% LA (ph=2.72), 1.5% LA (ph=2.22), 0.5% FA (ph=2.70), 5% TSP (ph=12.68), 10% TSP (ph=12.92), 5% STPP (ph=8.90) and 10% STPP (ph=9.07). Fillets were dipped in 6 l solutions of 1%, 1.5% LA, 0.5% FA, 5%, 10% TSP, 5% and 10% STPP for 1 min then drained on a sanitised tray for 10 min, at room temperature. Fillets not treated with acids or phosphates were untreated control samples. After dipping, 3 fillets were packed in a stomacher bag and were transported to a storage room adjusted to 4±1 C and 95% RH for storage. Enumeration of S. typhimurium (by Microbiological plating) After 2 h and 24 h following acid or phosphate treatment samples from each treatment were sampled to determine the NAR S. typhimurium (CCM 835) counts or prevalence. 25 g sample was removed aseptically from ¼ of each fillet in the tray, placed in a stomacher bag and 225 ml of sterile 0.1% (w/v) buffered peptone water (Oxoid L37) added. The sample was homogenised for 2 min in a stomacher (Seward Medical, Model 400), and a series of decimal dilutions were prepared. The enumeration of NAR S. typhimurium (CCM 835) was carried out with the plating on BSA supplemented with 100 ppm nalidixic acid after resuscitation. Plates were incubated for 2448 h at 37 C and the results are presented as the duplicate plates of triplicate samples. EXPERIMENT 2 In Experiment 2, the effects of organic acid and phosphate immersion treatments on prevalence (elimination) of S. typhimurium (CCM 835) on turkey breast fillets were evaluated with trials. After inoculation of turkey breast fillets with NAR S. typhimurium (CCM 835), fillets were treated with organic acid or phosphate solutions as described in Experiment 1 and then stored at 4±1 C and 95% RH. After 2 h and 24 h following treatment, samples from each treatment were analysed to determine the NAR S. typhimurium (CCM 835) counts and recovery of Salmonella (prevalence of Salmonella), by microbiological plating (as in Experiment 1) and traditional Salmonella detection method, respectively. Detection of S. typhimurium Salmonella were detected (observed) using the Turkish Standard (Anonymous, 1996). Statistical analyses Data were analysed employing completely randomised factorial design. Analyses of variance was conducted (STAT View) and mean differences were determined using Fisher s PLSD. The predetermined acceptable level of probability was 5% (P<0.05) for all comparisons. Results and discussion EFFECTS OF ORGANIC ACIDS AND PHOSPHATES ON AEROBIC MESOPHILIC MICROORGANISMS The effects of LA, FA and LA+FA, TSP and STPP treatments on the aerobic mesophilic microorganism count in the turkey breast fillets are shown in Table 1 and 2. There was a significant difference in aerobic mesophilic count between control, 1% LA, 0.5% FA and 1% LA+0.5% FA treated samples after 8 days of storage (P<0.05, Table 1). Significantly lower counts were observed in 1% LA+0.5% FA treated samples as compared to control after 8 days of storage ( P<0.05). But there were no significant difference in counts of 1% LA and 0.5% FA treatments after 8 days of storage ( P>0.05). The number of aerobic mesophilic microorganisms increased rapidly in the control from an initial population of 4.4 to 8.9 log cfu g 1 of turkey breast meat in 8 days where offodour was also detected. In 1% LA+0.5% FA treated samples after 14 days of storage the aerobic mesophilic count increased from 2.6 to 8.0 log cfu g 1 but there was no offodour. The treatment 1% LA+0.5 FA reached a maximum reduction by 2.2 log units after 6 days of storage in comparison to the untreated control samples. In the literature researchers recorded application of 1%, 2%, 3% LA caused decrease in total aerobic counts in the range of , and log units, respectively (Bautista et al., 1997; Izat et al., 1989; Podolak et al., 1996b; Sakhare et al., 1999; Xiong et al., 1998; Yang et al., 306

5 1998). Most researchers stated that the use of LA solutions at concentrations of 12% reduces the bacterial counts on poultry carcasses immediately after slaughter and during storage, without affecting organoleptic characteristics such as colour and flavour (Bolder, 1997). But, Bautista et al. (1997) reported that LA treatment at a concentration of greater than 1.24% caused discoloration on tissues of turkey carcasses and the same negative effect also has been observed with concentrations greater than 1.25% (Izat et al., 1989; Mulder et al., 1987). In our study no discoloration of the turkey breast fillets has been seen with dipping 1% LA solution. In summary, both lactic and fumaric acid seem to produce the desired initial microbial reduction, when mixtures of the two may possibly enhance the antimicrobial effects. The results obtained in this study are in agreement with the findings of others (Ariyapitipun et al., 1999; Izat et al., 1989; Podolak et al., 1996a,b). Table 1 Total aerobic mesophilic counts (log cfu g 1 ) of turkey breast meat treated with 1% lactic acid, 0.5% fumaric acid and combined acid solutions during storage at +4 C. 1,2. Aerobic mesophilic microorganism count (Day) C a 1% LA b 0.5% FA b 1% LA+0.5% FA c ± 0.7 a 3.8 ± 0.3 a 4.0 ± 0.4 a 2.6 ± 0.4 b ± 0.8 a 4.7 ± 0.2 ab 4.5 ± 0.3 b 3.8 ± 0.4 c ± 0.4 a 6.8 ± 0.2 b 7.7 ± 0.2 a 5.7 ± 0.2 c ± 0.2 a,2 8.5 ± 0.4 a 8.6 ± 0.4 a 7.1 ± 0.7 b ± 0.3 ab 8.9 ± 0.6 ab 8.4 ± 0.1 b ± 0.1 a 8.7 ± 0.1 a 9.0 ± 0.3 a ± 0.9 a,2 9.4 ± 0.1 a,2 8.0 ± 0.6 b 1 Each value in the table is the mean ± standard deviation of three samples. Different letters within the same period indicate significantly different means among treatments (P<0.05). C: untreated control, LA: dipped in lactic acid solution, FA: dipped in fumaric acid solution. 2 offodour was detected. Table 2 Total aerobic mesophilic counts (log cfu g 1 ) of turkey breast meat treated with 1.5% lactic acid, 5% trisodium phosphate and 5% sodium tripolyphosphate solutions during storage at +4 C. 1,2. Aerobic mesophilic microorganism count (Day) C ab 1.5% LA c 5% TSP b 5% STPP a ± 0.2 b 2.8 ± 0.2 b 2.7 ± 0.1 b 3.8 ± 0.4 a ± 0.4 ab 2.7 ± 0.2 c 3.7 ± 0.6 b 4.7 ± 0.3 a ± 0.3 a 5.1 ± 0.4 b 7.4 ± 0.3 a 7.3 ± 0.1 a ± 0.4 ab 7.1 ± 0.5 c 7.8 ± 0.3 b 8.4 ± 0.1 a ± 0.1 a 7.8 ± 0.5 b 8.7 ± 0.4 a 8.5 ± 0.2 a ± 0.2 a 8.9 ± 0.3 a 8.7 ± 0.4 a 8.7 ± 0.1 a ± 0.1 a,2 8.8 ± 0.3 a,2 8.7 ± 0.3 a,2 8.8 ± 0.1 a,2 1 Each value in the table is the mean ± standard deviation of three samples. Different letters within the same period indicate significantly different means among treatments (P<0.05). C: untreated control, LA: dipped in lactic acid solution, TSP: dipped in trisodium phosphate solution, STPP: dipped in sodium tripolyphosphate solution. 2 offodour was detected. There was a significant difference in aerobic mesophilic microorganism count between 1.5% LA, 5% TSP and 5% STPP treated samples after 14 days of storage (P<0.05, Table 2). No significant difference (P>0.05) was also observed in counts between control and 5% STPP treated samples after 14 days of storage. Samples treated with the 1.5% LA and 5% TSP had significantly lower (P<0.05) counts than control after 14 days of storage. In control sample, the number of aerobic mesophilic microorganisms was 3.1 log units initially and offodour when this count reached to 9.2 log cfu g 1. The samples treated with 1.5% LA, 5% TSP and 5% STPP solutions had counts that were 8.8, 8.7 and

6 log cfu g 1, respectively, when offodour was noticeable at 14 th day of storage (Table 2). Results showed that dipping in 1.5% LA solution was more effective in decreasing microbial load than dipping 5% TSP and 5% STPP after storage for 10 days. In our study aerobic mesophilic counts of the samples were unaffected by dipping for 1 min in 5% STPP solution. In the literature there are conflicting results about the shelf life extension with different types of meat under different experiment conditions. Greer and Jones (1991) found that, although 1% LA sprays produced a 0.7 log units reduction in bacteria on beef carcasses, this effect was not extended the shelf life of fillets prepared from these carcasses. Similar results were obtained by Prasai et al. (1991). In our study dipping 1% LA+0.5% FA solution was found the most effective treatment among all applications for reduction of microbial load and the extension of shelf life (at least 6 days) of turkey breast fillets. Dipping fillets in both 1% LA and 0.5% FA extended the storage life up to 4 days, respectively, without adversely affecting the colour. When the results obtained in this study compared to data in the literature it was found dipping into 11.5% LA and 1% LA+ 0.5% FA solutions provided extension in the shelf life at the same level. Similar antimicrobial effect of FA at lower concentration (0.5%) than LA (1%) may have been related to its lower acid dissociation constant (pka) compared to those of lactic acid. The pka 1 and pka 2 of FA are 3.03 and 4.44, whereas the pka of lactic acid is 3.86 (BairdParker, 1980). The mechanisms of reductions in bacteria on poultry carcasses with the TSP treatment are still not fully understood. It has been suggested that the high ph and ionic strength of TSP solutions and the ability of these solutions to remove a thin layer of lipids (the detergent effect ) from skin are responsible for the bactericidal activity (Anonymous, 1994; Hwang and Beuchat, 1995; Rodriguez de Ledesma et al., 1996; Yang et al., 1998). In the literature researchers recorded immersion or spraying of % TSP for 17 s15 min caused immediate reduction in total aerobic counts in the range of log units (Bautista et al., 1997; Capita et al., 2000; Giese, 1993; Kim and Marshall, 1999; Marshall and Jindal, 1997; Morris et al., 1997; Vareltzis et al., 1997; Yang et al., 1998). The result obtained in this study was lower than the data in the literature and the reason for this, can be explained with the differences in treatment time, temperature and the material. This study supports the finding that using higher concentration of phosphate in poultry processing for improving shelf life should be successful in reducing bacterial contamination. EFFECTS OF ORGANIC ACIDS AND PHOSPHATES ON PSYCHROTROPHIC BACTERIA The effects of LA, FA and combined acid solutions on psychrotrophic bacteria in the turkey breast fillets are shown in Table 3. Table 3 Psychrotrophic bacteria counts (log cfu g 1 ) of turkey breast meats treated with 1% lactic acid, 0.5% fumaric acid and combined acid solutions during storage at +4 C. 1,2. Storage period Psychrotrophic bacteria counts (Day) C a 1% LA c 0.5% FA b 1% LA+0.5% FA d ± 0.1 a 4.0 ± 0.2 a 4.2 ± 0.5 a 3.1 ± 0.5 b ± 0.6 a 4.5 ± 0.5 b 4.8 ± 0.1 ab 3.9 ± 0.4 c ± 0.4 a 7.0 ± 0.3 b 7.9 ± 0.1 a 5.8 ± 0.2 c ± ± 0.3 a 8.6 ± 0.3 a 7.1 ± 0.7 b ± 0.3 a,2 8.7 ± 0.3 ab 8.9 ± 0.5 ab 8.4 ± 0.2 b , 2 Same as in Table ± 0.3 a 8.8 ± 0.1 a 8.6 ± 0.5 a 8.9 ± 0.5 a,2 9.3 ± 0.1 a,2 8.6 ± 0.5 b There was a significant difference in psychrotrophic bacteria count between control, 1% LA, 0.5% FA and 1% LA+0.5% FA treated samples after 10 days of storage (P<0.05). Similarly, significantly lower counts were observed in 1% LA+0.5% FA treated samples as compared to 1% LA and 0.5% FA treated samples after 14 days of (P<0.05). Generally, reduction was greater when the combinations of the acids are treated. In our study dipping turkey breast meat in 1% and 1.5% LA for 1 min reduced the psychrotroph counts by 0.2 and 0.6 log units reductions, respectively and the results obtained in this study from the standpoint of both immediate and delayed effect of LA on psychrotrophic counts are in parallel with the data ( log units reductions) presented in the literature (Hwang and Beuchat, 1995; Podolak et al., 1996a; Van der Marel et al., 1988). The results of the treatments 308

7 carried out with LA and phosphates are given in Table 4. There was a significant difference in psychrotrophic bacteria count between control, 1.5% LA, 5% TSP and 5% STPP treated samples after 14 days of storage (P<0.05, Table 4). 1.5% LA and 5% TSP treatments gave significantly lower psychrotroph counts than the control and 5% STPP treated samples after 14 days of storage ( P<0.05). In control the number of psychrotrophs increased 5.9 log units and reached 9.1log cfu g 1 when off odour was noticeable at 14 th day of storage. The samples treated with 1.5% LA, 5% TSP and 5% STPP solutions had psychrotroph bacteria counts that were become 8.9, 8.8 and 8.7 log cfu g 1, respectively, with initial counts of 2.6, 2.7 and 3.7 log cfu g 1. In literature there are studies made with different materials by the application of various concentrations of TSP and STPP solutions either dipping or spraying method. The results obtained in this study are lower than the data given in the literature about the reduction ( log units) in psychrotroph bacteria count (Hwang and Beuchat, 1995; Kim et al., 1996; Rodriguez de Ledesma et al., 1996) and this may be explained by the use of lower concentrations of phosphates, difference in time of application and the type (nature) of poultry meat. Table 4 Psychrotrophic bacteria counts (log cfu g 1 ) of turkey breast meat treated with 1.5% lactic acid, 5% trisodium phosphate and 5% sodium tripolyphosphate solutions during storage at +4 C. 1,2. Psychrotrophic bacteria counts (Day) C ab 1.5% LA c 5% TSP b 5% STPP a ± 0.2 ab 2.6 ± 0.1 b 2.7 ± 0.2 b 3.7 ± 0.8 a ± 0.7 a 2.7 ± 0.1 c 3.9 ± 0.6 b 5.0 ± 0.4 a ± 0.2 a 5.2 ± 0.6 b 7.2 ± 0.1 a 7.1 ± 0.1 a ± 0.5 ab 7.3 ± 0.6 c 7.8 ± 0.4 bc 8.5 ± 0.1 a ± 0.1 ab 8.1 ± 0.5 b 8.8 ± 0.1 a 9.2 ± 0.6 a ± 0.1 a 8.6 ± 0.2 ab 8.8 ± 0.4 a 8.1 ± 1.0 b ± 0.2 a,2 8.9 ± 0.2 a,2 8.8 ± 0.2 a,2 8.7 ± 0.1 a,2 1, 2 Same as in Table 2. EFFECTS OF ORGANIC ACIDS AND PHOSPHATES ON YEAST The effects of LA, FA and combined acid solutions on yeast in the turkey breast fillets are summarised in Table 5. There was a significant difference in yeast count between control, 1% LA, 0.5% FA and 1% LA+0.5% FA treated samples after 8 days of storage (P<0.05, Table 5). Samples treated with the 1% LA and 1% LA+0.5% FA had significantly lower (P<0.05) counts than control and the number of yeast in 1% LA+0.5% FA treated samples was significantly lower than that of the counts of 1% LA and 0.5% FA treated samples at day 8 (P<0.05). The number of yeast increased rapidly in the control from an initial population of 2.3 to 7.3 log cfu g 1 of turkey breast fillet in 8 days where that offodour was also detected. Table 5 Yeast counts (log cfu g 1 ) of turkey breast meat treated with 1% lactic acid, 0.5% fumaric acid and combined acid solutions during storage at +4 C. 1,2. Yeast Counts (Day) C a 1% LA b 0.5% FA ab 1% LA+0.5% FA c ± 0.4 ab 2.5 ± 0.2 a 2.3 ± 0.2 ab 1.9 ± 0.4 b ± 0.0 a 3.8 ± 0.5 a 3.9 ± 0.3 a 3.6 ± 0.4 a ± 0.0 a 5.2 ± 0.4 b 5.5 ± 0.4 b 4.7 ± 0.1 c ± 0.1 a,2 6.8 ± 0.1 b 6.3 ± 0.6 c 6.0 ± 0.3 c ± 0.1 a 6.9 ± 0.1 a 6.2 ± 0.2 b ± 0.1 a 6.9 ± 0.1 a 6.8 ± 0.2 a ± 0.4 b,2 7.6 ± 0.4 a,2 6.2 ± 0.2 c 1, 2 Same as in Table

8 The effects of LA, TSP and STPP treatments on yeast in the turkey breast fillets are summarised in Table 6. There was a significant difference in yeast count between control, 1.5% LA, 5% TSP and 5% STPP treated samples after 14 days of storage (P<0.05), but samples treated with the 1.5% LA and 5% TSP had significantly lower (P<0.05, Table 6) counts than control and 5% STPP treated samples. The number of yeast increased in the control from an initial population of 1.7 to 7.2 log cfu g 1 of turkey breast fillet in 14 days where offodour was also detected. Although many researchers have followed the effects of decontamination on poultry meat there are few published data (Sakhare et al., 1999; Woolthuis and Smulders, 1985) about the effects of organic acids on yeast count ( log units reductions). Results obtained in our study are in accordance with the data presented in the literature. Although there is an abundant of published data on poultry carcass decontamination no researchers have followed the effects of phosphates on yeast count. Table 6 Yeast counts (log cfu g 1 ) of turkey breast meats treated with 1.5% lactic acid, 5% trisodium phosphate and 5% sodium tripolyphosphate solutions during storage at +4 C. 1,2. Yeast Counts (Day) K a 1.5% LA b 5% TSP b 5% STPP a ± 0.2 ab 1.6 ± 0.1 ab 1.8 ± 0.5 a 1.0 ± 0.3 b ± 0.6 a 1.8 ± 0.6 b 1.8 ± 0.2 b 2.6 ± 0.4 a ± 0.3 a 3.8 ± 0.7 b 5.2 ± 0.2 a 5.0 ± 0.1 a ± 0.5 b 5.4 ± 0.9 c 5.5 ± 0.1 c 7.0 ± 0.1 a ± 0.3 ab 6.5 ± 0.7 b 6.7 ± 0.3 b 7.5 ± 0.5 a ± 0.1 a 7.2 ± 0.6 ab 6.8 ± 0.1 b 6.9 ± 0.4 b ± 0.1 a,2 7.2 ± 0.2 a,2 7.0 ± 0.2 a,2 7.1 ± 0.0 a,2 1, 2 Same as in Table 2. EFFECT OF ORGANIC ACID IMMERSION TREATMENTS ON S. TYPHIMURIUM COUNTS OF TURKEY BREAST FILLETS The effectiveness of dipping of NAR Salmonella typhimurium CCM 835 inoculated turkey breast fillets at a level of 7.7 log CFU per fillet in 1%LA, 1.5% LA and 0.5% FA solutions on the reduction of Salmonella are shown in Table 7. There was a significant difference in S. typhimurium counts after 2 hours between control, 1% LA, 1.5% LA and 0.5% FA treated samples ( P<0.05, Table 7). Significantly lower S. typhimurium counts were observed in 1% LA, 1.5% LA and 0.5% FA treated samples as compared to control (P<0.05). After 2 h dipping treatment the S. typhimurium counts of control, 1% LA, 1.5% LA and 0.5% FA treated turkey breast fillets were 7.8, 5.9, 5.1 and 5.3 log CFU per fillet, showing 1.9, 2.7 and 2.5 log unit reductions respectively (Table 7). Table 7 S. typhimurium counts of turkey breast meats treated with 1%, 1.5% lactic acid and 0.5% fumaric acid solutions during storage at +4 C. 1 (Inoculum:7.7 log CFU per fillet). (Hour) S. typhimurium count (log CFU per fillet) C a 1% LA b 1.5% LA c 0.5% FA b ± 0.5 a 5.9 ± 0.5 b 5.1 ± 0.4 c 5.3 ± 0.7 bc ± 0.2 a 7.2 ± 0.3 a 6.1 ± 0.4 b 7.1 ± 0.5 a 1 Each value in the table is the mean ± standard deviation of three samples. Different letters within the same row are significantly different (P>0.05). C: Control, LA: dipped in lactic acid solution, FA: dipped in fumaric acid solution. After 24 h of storage at +4 C the number of S. typhimurium increased rapidly in the samples treated with each of the acid solutions (Table 7). There was no change (increase or reduction) in S. typhimurium count of control with an initial count of 7.8 log CFU per fillet after 24 h at +4 C. In 1% LA and 0.5% FA treated samples the number of S. typhimurium reached 7.2 and 7.1 log CFU per fillet after 24 h +4 C indicating 1.3 and 1.8 log unit increases, respectively. But this count remained at 6.1 log unit level in the samples treated with 1.5% LA indicating a 1.7 log unit lower count than those of 310

9 control samples, after 24 h at +4C (Table 7). The results obtained in this study from the standpoint of immediate effect ( log units reductions) of LA on S. typhimurium counts of turkey breast fillets are in parallel with the data presented in the literature (Xiong et al., 1998). Although there is no published data on the effect of FA on S. typhimurium the results obtained in this study showed that FA treatment as poultry decontaminating treatment is as effective as LA treatment. EFFECT OF PHOSPHATE IMMERSION TREATMENTS ON S. TYPHIMURIUM COUNTS OF TURKEY BREAST FILLETS There were no significant difference (P>0.05) in S. typhimurium count between control and 5% STPP treated samples, but significantly lower counts were observed in 5%, 10% TSP and 10% STPP treated samples as compared to control after 2 h of dipping treatment (Table 8). S. typhimurium counts of control and 5%, 10%TSP and 5%, 10% STPP treated turkey breast fillets were 7.2, 5.8, 3.7, 7.2 and 4.0 log CFU per fillet, respectively (Table 8). After 2 h dipping treatment Salmonella counts of 5%, 10% TSP and 10% STPP treated samples were 1.4, 3.5 and 3.2 log unit lower than those of control samples increased 0.8 log units and reached 8.0 log CFU per fillet. In 10% TSP and 10% STPP treated samples the number of S. typhimurium reached to a level of 6.3 and 7.8 log CFU per fillet indicating 2.6 and 3.8 log unit increases, respectively. These results indicate that storage period was effective on S. typhimurium counts and the most effective treatment in lowering the Salmonella count was dipping in 10% TSP. Table 8 S. typhimurium counts of turkey breast meats treated with 5%, 10% trisodium phosphate and 5%, 10% sodium tripolyphosphate solutions during storage at +4 C. 1 (Inoculum:7.6 log CFU per fillet). S. typhimurium count (log CFU per fillet) (Hour) C a 5% TSP b 10% TSP d 5% STPP a 10% STPP c ± 0.3 a 5.8 ± 0.2 b 3.7 ± 0.0 c 7.2 ± 0.4 a 4.0 ± 0.5 c ± 0.0 a 7.0 ± 0.2 b 6.3 ± 0.1 c 7.6 ± 0.2 a 7.8 ± 0.2 a 1 Each value in the table is the mean standard deviation of three samples. Different letters within the same row are significantly different (P>0.05). C: Control, LA: dipped in lactic acid solution, FA: dipped in fumaric acid solution, TSP: dipped in trisodium phosphate solution, STPP: dipped in sodium tripolyphosphate solution. Results obtained in this study indicate that phosphate solutions caused somewhat similar or slightly higher reductions on S. typhimurium than the reductions reported (1.0 to 2.2 log units) (Hwang and Beuchat, 1995; Kim et al., 1994; Lillard, 1994; Rodriguez de Ledesma et al., 1996; Slavik et al., 1994; Xiong et al., 1998; Yang et al., 1998). The inconsistencies could be attributed to a number of variables including different types of meat, method of phosphate application (dipping or spraying), different experimental conditions (temperaturetime), different S. typhimurium strain (initial number) and different counting and isolating methods. EFFECTS OF ORGANIC ACID AND PHOSPHATE IMMERSION ON THE INCIDENCE OF S. TYPHIMURIUM ON TURKEY BREAST FILLETS The results of the trials in order to determine the effect of lactic acid and trisodium phosphate solutions on the elimination of inoculated S. typhimurium on turkey breast fillets were presented in Table 9 and

10 Table 9 Effects of 1.5% LA and 10% TSP solutions on NAR S.typhimurium on turkey breast fillets. Low inoculum a High inoculum b (Hour) C c 1.5% LA 10% TSP C c 1.5% LA 10% TSP log CFU/fillet Qualitative/fillet d log CFU/fillet Qualitative/fillet d ± 0.1 1/3 0/3 3.9 ± 0.1 3/3 3/ ± 0.1 3/3 0/3 3.8 ± 0.1 3/3 3/3 a 3.1 log CFU/fillet 4.1 log CFU/fillet c Each value in the table is the mean standard deviation of three samples. C: Control, LA: dipped in lactic acid solution, TSP: dipped in trisodium phosphate solution. d Number of Salmonella positive sample of three samples. Table 10 Effects of 1.5% LA and 10% TSP solutions on incidence of NAR S. typhimurium on turkey breast fillets. (Hour) S. typhimurium /fillet a Low inoculum b High inoculum c C 1.5% LA 10% TSP C 1.5% LA 10% TSP 2 3/3 0/3 0/3 3/3 3/3 0/3 24 3/3 0/3 0/3 3/3 3/3 0/3 a Number of Salmonella positive sample of three samples. C: Control, LA: dipped in lactic acid solution, TSP: dipped in trisodium phosphate solution. b 2.6 log CFU/fillet c 3.6 log CFU/fillet While there was only one Salmonella positive sample out of three fillets with an initial inoculum of 3.1 log CFU S. typhimurium per fillet dipped in 1.5% LA solution, no Salmonella positive sample was observed in out of three samples dipped in 10% TSP solution. 2 hours after dipping. All three samples were Salmonella positive 2 hours after dipping the fillets with an initial inoculum of 4.1 log CFU S. typhimurium per fillet in 1.5% LA and 10% TSP solution. The only Salmonella negative samples were the 1.5% LA dipped samples with an initial inoculum of 3.1 log CFU Salmonella per fillet after 24 hours storage at +4 C. Table 10 shows the results of the LA and TSP dipping on the elimination of Salmonella at two different levels (2.6 and 3.6 log CFU per fillet) of initial inoculum slightly lower than the former experiment. All the samples dipped in 1.5% LA and 10% TSP with an initial inoculum level of 2.6 log CFU per fillet, and dipped in 10% TSP with an initial inoculum level of 3.6 log CFU per fillet were found to be Salmonella negative after 2 and 24 hours storage at +4 C (Table 10). The data presented in table 3 and 4 clearly indicates that dipping turkey breast fillets in 10% TSP solution for 1 min eliminates S. typhimurium inoculated at a level of approximately 10 3 CFU per fillet. This finding is in good accordance with the results presented in table 2, which cause similar immediate reductions (3.5 log unit) in the initial level of inoculated S. typhimurium when turkey breast fillets were dipped in 10% TSP solution for 1 minute. Similarly, Lillard (1994) reported that there were no Salmonella positive rinsing liquid and skin homogenate samples after dipping 10 2 CFU nalidixic acid resistant S. typhimurium inoculated 9 chicken carcasses in 15 min 10% TSP solution (ph= ). Salmonella are mainly killed or injured because of chemical interactions between extremely high ph TSP and cell wall elements. The effects of dipping in organic acids (LA, FA) and phosphates (TSP, STPP) on Salmonella were different. This is reasonable since the two treatments have different mechanisms of action. The TSP probably has a detergent effect, making clumps of the cells split and loosen from the skin, while the bactericidal effects of LA are due to the ability of the undissociated acid to penetrate the bacterial cell membrane and acid effect. Apart from the present investigation, there have been no known published studies on the susceptibility of S. typhimurium to fumaric and lactic acid, and phosphates (TSP, STPP) in turkey meat. The bactericidal activities of LA and FA were of a similar magnitude. The most effective treatment among the four treatment used in this study to eliminate S. typhimurium from turkey breast fillets was shown to be 10% TSP dipping. 312

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