- Email: [email protected]
Microbiological indicators for the assessment of performance in the hazard analysis and critical control points (HACCP) system in meat lasagna production
Available online at www.sciencedirect.com
Food Control 19 (2008) 764–771 www.elsevier.com/locate/foodcont
Microbiological indicators for the assessment of performance in the hazard analysis and critical control points (HACCP) system in meat lasagna production Elisabete Aparecida Martins *, Pedro Manuel Leal Germano Public Health School, University of Sa˜o Paulo, Avenida Doutor Arnaldo, 715, Sa˜o Paulo, SP, CEP: 01246-904, Brazil Received 10 September 2006; received in revised form 22 July 2007; accepted 3 August 2007
Abstract The HACCP system is being increasingly used to ensure food safety. This study investigated the validation of the control measures technique in order to establish performance indicators of this HACCP system in the manufacturing process of Lasagna Bolognese (meat lasagna). Samples were collected along the manufacturing process as a whole, before and after the CCPs. The following microorganism’s indicator (MIs) was assessed: total mesophile and faecal coliform counts. The same MIs were analyzed in the final product, as well as, the microbiological standards required by the current legislation. A significant reduction in the total mesophile count was observed after cooking (p < 0.001). After storage, there was a numerical, however non-significant change in the MI count. Faecal coliform counts were also significantly reduced (p < 0.001) after cooking. We were able to demonstrate that the HACCP system allowed us to meet the standards set by both, the company and the Brazilian regulations, proved by the reduction in the established indicators. 2007 Elsevier Ltd. All rights reserved. Keywords: HACCP; Validation; Control measure; Verification; Microbiological indicators
1. Introduction With globalization, food borne diseases have acquired a new dimension, as many food products are produced in one country to be imported and consumed in another. Sometimes, the country of origin of the product has precarious hygiene and sanitation conditions (Motarjemi & Ka¨ferstein, 1999). With the increased incidence of food borne diseases, the hazard analysis and critical control points (HACCP) system was developed as a method to ensure food safety. The HACCP system is being increasingly used by the food industry and regulatory agencies (Motarjemi, Ka¨ferstein, Moy, Miyagawa, & Miyasgishima, 1996). However, one of its least explored aspects is validation, not only
*
Corresponding author. Tel.: +55 11 3023 6037; fax: +55 11 3023 5316. E-mail address: [email protected] (E.A. Martins).
0956-7135/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2007.08.001
the validation of control measures, but also of the system itself. The validation and verification steps certify that the hazards have been identified and are being controlled, and whether the plan is being conducted as designed (Kvenberg & Schwalm, 2000; Mayes, 1999). The Codex Alimentarius Commission (2001) defines validation as the process to ensure that a certain group of measures can effectively control a specific hazard in a certain food. The validation of the measures requires a comparison between the actual and the expected results, as it is necessary to confirm the efficiency of the measures for controlling a specific hazard. Ideally, all procedures used to control a specific hazard should be validated by the validation of control measures, but there might be limitations to achieve this goal. In this case, some prioritization criteria may be considered: greater potential of human health risk or if there is no available historic data concerning the control of that hazard. Another aspect to be considered is if a control measure
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
is determinant to ensure the safety of the product; in this case, the validation can be focused on that measure. Focused validation implies that the control measure has a statistically higher contribution for the safety of the product. On the other hand, if there are multiple measures to control a certain hazard, and each of the measures has a similar influence, they will all be considered as having the same level of importance for validation (Codex Alimentarius Commission, 2001). The method used to validate a group of measures will depend on the nature of the hazard, on the product and on the type of control measures selected to control that hazard (Codex Alimentarius Commission, 2001). For example, considering the validation of a preventive measure related to the control of a microbiological hazard (pathogen), Kvenberg and Schwalm (2000); Swanson and Anderson (2000) do not advise testing for pathogens as a means of validation for that control measure, as the possibility of contamination of a product manufactured in a plant that uses the HACCP system is very remote. In this case, they suggest the use of quantifiable microorganism’s indicator (MIs) to validate the control measures. According to Scott and Moberg (1995), quoted by PAHO (Pan American Health Organization – Organizac¸a˜o Pan-Americana de Sau´de – OPAS, Brasil, 2001, p. 140), the MIs present in a food do not pose a direct threat to health, but they can be used as a signal of the presence of a potential threat. For Austin and Reynolds (2002), some indicators, such as enterobacteria, can demonstrate a possible presence of other pathogens in that environment and/or in the final product; the higher the MI count, the higher the possibility of pathogens in the product. Special attention should be paid also to the method chosen to assess the indicators. Thus, the method to be selected for validation must be approved and recognized (Austin & Reynolds, 2002). This means that it is necessary to define criteria for the validation of the control measures associated to the CCPs, and to determine the indicator organisms that will evaluate the microbiological performance of the HACCP system. With measurable controls, it is possible to verify if the product safety targets are being met. The present study was conducted to investigate the method for validation of control measures in order to establish the performance indicators of the system for the manufacturing process of the Lasagna Bolognese (meat lasagna). 2. Experiment 2.1. Type of study This experimental study was conducted in the manufacturing process, through industrial scale, of the product ‘‘Lasagna Bolognese’’ (or meat lasagna). The microbiological standard for this product is established by ANVISA (Ageˆncia Nacional de Vigilaˆncia Sanita´ria – Brazilian Sanitary Surveillance Agency – Brazil, 2001). It is made of special lasagna noodles, tomato meat sauce, Be´chamel
765
sauce, cooked ham and mozzarella cheese. The process includes the following steps: (a) preparation and cooking the tomato meat sauce and of the Be´chamel sauce, both sauces with pH more than 4.6; (b) assembling the lasagna by interlaying the noodles (pre-cooked and dehydrated lasagna pasta), the meat sauce, the ham and the mozzarella and finally by adding the Be´chamel sauce and grated parmesan cheese; (c) fitting the lid and sealing the package; (d) freezing the product; (e) passing the product through a metal detector; (f) final packaging, and (g) storage. The study involved the following stages: implementation of the HACCP system in the manufacturing process of the product Lasagna Bolognese; definition of the MIs and collection of samples for analysis; validation of the control measures associated with the critical control points (CCPs); validation of the HACCP system, and definition of the microbiological indicators for the assessment of the performance of the system. The implementation of the HACCP system in the manufacturing process of the Lasagna Bolognese was based on the steps described in Fig. 1. This figure also describes the preliminary stages and actions taken after the implementation of the system. Additionally, Table 1 presents a summary of the of HACCP Plan with the hazard identified, Clostridium perfringens, Escherichia coli and Bacillus cereus. Despite of the fact that Salmonella and Staphylococcus aureus are mention in the Brazilian Legislation for this kind of product (lasagna) they are not considered as hazards by the HACCP team. For Salmonella the literature shows that this bacteria is strongly associate with poultry and poultry products (FAO/WHO, 2000) and it is killed under pasteurization temperatures (Jay, 1996) and for S. aureus it is controlled through Good Manufacturing Practices. B. cereus and Clostridium perfringens vegetative cells are killed in cooking temperatures, more than 70 C. The most important control measure, for both, the to prevent the growing is to keep the product, after cooking, in temperature higher than 60 C (Germano & Germano, 2003b). These conditions, was observed in the process, in the cooking and storage steps. The CCPs and their respective steps in the process, the hazards that are controlled in each step and the associated control measures are identified. No chemical hazards were identified in the process. For the validation of the control measures associated with the CCPs, priority was given to those measures connected with the control of microbiological hazards as, according to Idexx (1998), mentioned by PAHO (2001, p. 128), they are the ones that represent a major risk to the wholesomeness of food. For Germano and Germano (2003a), the most important risks in food from animal origin are related to food poisonings. The microorganism’s indicators – (a) aerobic mesophiles and (b) faecal coliforms – were established based on references (Austin & Reynolds, 2002; Kvenberg & Schwalm,
766
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771 Documents produced Training of the HACCP team – main ideas and hazard analysis Preliminary actions to the implementation of the HACCP System
Training of the monitoring team – main ideas and monitoring procedures Establish criteria for checking the prerequisites for the implementation of the system are being met Develop the HACCP Plan by applying the seven principles
Evaluation of the training Record of training Monitoring operational procedure Evaluation of training Record of training Criteria Specification Ex.: minimum grades during the auditing of the prerequisite programs (GMP 1 and SSOP 2) Flow diagram of the process Description of the product and intended use HACCP Plan
A
Audit the implementation of the prerequisites and training program
Correct the criteria that are not being met
Criteria met? NO YES
A
Monitor the CCPs for one (1) month
Record of the monitoring Report of the record analysis
Are the Critical Limits being observed ? NO
YES
Validate the CCPs
Report of CCPs Validation
Have the CCPs been validated? NO YES
Validate HACCP System
Re-evaluate the HACCP Plan
Report of HACCP System Validation
Has the HACCP System been validated? NO
YES
1
End of process
Good Manufacturing Practices
2
Sanitation Standard Operating Procedures and Sanitation Standard Pre-operating Procedures
Fig. 1. Flow diagram of the implementation of the HACCP system.
2000; Scott and Moberg, 1995, cited in OPAS, 2001, p. 140; Swanson & Anderson, 2000). For the analysis of the indicators, five (5) samples of the product were collected each day during the processing
period, before and after each step related to each CCP, at one (1) hour intervals and during a period of five (5) days; thus, 25 samples were collected from each collecting point. Samples were also collected from the final product. The
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771 Table 1 Summary of the of HACCP plan with the hazard identified CCP #
Process step
Identified hazard
Control measure
1
Cooking of the tomato meat sauce
Survival of Clostridium perfringens and Survival of Escherichia coli
Minimum temperature of the sauce at the end of the process (80 C) Cooking time (3 min)
2
Storage of the tomato meat sauce
Forming spores of Clostridium perfringens
Minimum temperature of the sauce (75 C)
3
Cooking of the Be´chamel sauce
Survival of Bacillus cereus
Minimum temperature of the sauce at the end of the process (80 C) Cooking time (3 min)
4
Storage of the Be´chamel sauce
Forming spores of Bacillus cereus
Minimum temperature of the sauce (75 C)
5
Primary package
Presence of metal fragments
Metal detector
number of samples was based on the sampling plan of the International Commission on Microbiological Specification for Foods (ICMSF, 1974). The collection of samples during the processing steps was made as follows: aseptic collection of 500 g of the product being processed, before and after each CCP, and always from the same batch. The samples were placed in new plastic bags, tagged and identified, and were transported in a cooler with ice to the plant laboratory and stored under freezing temperature until tested. All samples were tested both for the selected indicators and according to the Brazilian legislation requirements. The samples from the final product were collected in duplicate; the first set of product samples underwent microbiological testing in the form that the product leaves the plant frozen, and the second set of samples was tested as prepared for consumption, after heating. The instructions for preparing the lasagna are printed on the preliminary package of the product. The product can be heated in a conventional oven or using a microwave oven. We chose to analyze the product heated in a microwave oven, as it is being used by an increasing number of consumers and it also has been a point of disagreement among researchers (Nunes, Germano, & Germano, 2003). The main point for the disagreement lies in the efficiency of the oven. Researchers have questioned the efficiency of microbiological count reduction with the use of the microwave oven, as the food is not equally heated, allowing for the formation of ‘‘cold spots’’ in the food, especially in the central portion of the serving. This fact emphasizes that food manufacturing should design microwaveable products to be safe prior to heating in microwave; once there is no consensus that microwave oven is a reliable kill step. The lasagna was prepared according to the instructions on the 650 g pack: 14 min under medium–high potency
767
(70%). The samples were heated in a domestic microwave oven, a Sanyo – 60 Hz and 120 W. After heating, temperature was measured in each sample in five different places: in each of the four corners of the pack and in the center. The methodology used for total mesophile count, faecal coliforms, sulfite-reducing Clostridium and B. cereus detection is described in the ‘‘Compendium of Methods for the Microbiological Examination of Foods’’ (Bennett & Belay, 2001; Kornacki & Johnson, 2001; Morton, 2001; Scott, Anderson, & Wang, 2001). The analysis for Salmonella spp. and S. aureus followed the criteria of the International Standard Organization, respectively ISO, ISO-6888-1 (1999), and ISO-6579 (2002). The obtained results, before and after each CCP, were evaluated according to the significant statistical difference. Along with the statistical analysis, the results were also evaluated according to sanitation and hygiene standards. Considering the MI counts after the CCPs and the established hygiene and sanitation standards, we formulated the following hypothesis: (a) cases with a significant statistical difference and with count reduction after the CCP were considered as a validated CCP, meaning that the hazard was under control; (b) cases without a significant statistical difference and with similar counts before and after the CCP, were considered validated, as the hazard was under control; (c) cases with significant statistical difference, and with higher counts after the CCP when compared to previous counts were considered a non-validated CCP, meaning that the hazard was not under control. The validation of the HACCP system was based on the ‘‘check-list’’ published by the World Health Organisation, (WHO, 1998) and performed according to technical and scientific analysis of: (a) all preliminary actions previous to the implementation of the HACCP system; (b) the implementation of the HACCP system; (c) traceability; (d) recall strategy. In order to establish the microbiological performance indicators of the system, an analysis of the following results was conducted: (a) MIs used during the process (validation of the control measures connected to the CCPs); (b) MIs in the final product; and (c) detection of microorganisms according to the microbiological standards defined for the product, allowing to establish the correlation of the performance of the product during the manufacturing process, at the CCPs, with the performance of the final product itself. From this data, the MIs for the process were established and assessed for one week. At the same time, the final product was analyzed according to the established microbiological standards, and the performance of the indicators was checked. The performance of the indicators was measured according to the following aspects: (1) The ability to maintain the characteristics observed during the validation of the control measures for the CCPs, considering the significance of the statistical difference between CCPs and the status of hazard
768
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
control. (2) The fulfillment of the microbiological guideline limit for total mesophile count. The following assumptions were made for this assessment: the chosen microbiological guiding limit for the total mesophile count was (m)104 to (M)106 CFU/g, considering the ICMSF (1974) classification for organisms that do not pose a direct threat to health. It must be noted, however, that the guideline was drawn for the final product, not being adequate for the analysis of the initial stages of food products processing. Thus, from the lower value established (m = 104) for the final product, a reduction of 1 (one) decimal exponent or 1 (one) logarithmic cycle was considered for each step of the manufacturing process. The following microbiological guiding limits were set for the total mesophile count: (a) assembly of the lasagna 104, (b) storage of sauces 103 and (c) end of cooking 102. (3) The ability to comply with the microbiological standards for faecal coliforms – a maximum of 102 CFU/g in the final product. The microbiological guiding limit was established for the manufacturing process based on the Brazilian legislation: maximum count of 5 · 101 CFU/g, storage – maximum count of 2 · 101 CFU/g, and cooking maximum count of 101 CFU/g. However, it should be stressed that the final product requires heating prior to consumption. 2.2. Data analysis The statistical analysis was conducted using the SPSS Incorporation Sigma Stat for Windows, version 2.0. In order to normalize the results, the absolute values of the microorganism’s indicator (MIs) counts were converted into base 10 logarithms. To compare different steps of the manufacturing process, variance analysis of repeated measurements was used; however, as the results were not parametric, the Friedman test was applied. Whenever the Friedman test revealed significant statistical difference (p < 0.05), the Duncan test was applied. The Wilcoxon test was used to compare the results after cooking with those obtained after storage. 3. Results The results are presented according to the statistical analysis used for comparing the MIs (total mesophile and faecal coliform counts), before cooking, after cooking and after storage – for the Be´chamel sauce and for the tomato meat sauce used in the Lasagna Bolognese. The figures are expressed as arithmetic mean ± standard deviation
from the mean, followed by the median value between parentheses. Initially, the results of the comparison between the different steps of the manufacturing process are shown – pre-cooking, after cooking, and after storage – for the tomato meat sauce and for the Be´chamel sauce. 3.1. Validation of control measures related to the CCPs The total mesophile count and the faecal coliforms count are presented in Table 2. After cooking, a significant reduction (p < 0.001) of the total mesophile count was observed in both sauces. In the meat sauce, no difference (p = 0.109) was observed between the total mesophile count after cooking and after storage. The same result was obtained for the Be´chamel sauce (p = 0.337). Thus, there was a considerable reduction in the total mesophile count resulting from the cooking step, without any significant increase during storage. The meat sauce samples showed the same faecal coliform count in the three steps that were assessed: <1.0 ± 0 (1.0) log10 CFU/g. As for the Be´chamel sauce, there was a significant reduction (p < 0.001) in the faecal coliforms count after the cooking step. There was no difference (p = 0.814) after cooking and after storage. Faecal coliform MIs had a similar performance in the cooking and storage steps to that observed in the total mesophile count; thus, a significant reduction of the MIs was detected after cooking, with maintenance of the microbiological conditions during storage. The comparative results of MIs in the frozen final product and in the heated one are presented in Section 3.2. 3.2. Further analysis – final product before and after preparation Both groups of MIs were also studied in the samples of the final product under two different conditions: frozen and after heating in a microwave oven. In order to assess the product after heating, the temperature of the lasagna was measured in 5 (five) different points of each product tray. In a 125 determinations, the lowest average temperature was measured in the center of the tray, 79.9 ± 14.2 C, and the highest was found in one of the corners, 86.3± 4.7 C. The frozen product had an average total mesophile count of 4.22 ± 0.79 (4.04) log10 CFU/g. After heating,
Table 2 Indicator organisms in the CCPs of the Lasagna Bolognese Total mesophile count (log10 CFU/g) Before cooking After cooking Storage
Faecal coliforms (log10 CFU/g)
Meat sauce
Be´chamel sauce
Meat sauce
Be´chamel sauce
3.58 ± 0.89 (3.34) 2.04 ± 0.12 (2.00) 2.19 ± 0.40 (2.00)
4.73 ± 0.92 (5.30) 1.69 ± 0.64 (1.60) 1.62 ± 0.83 (1.30)
<1.0 ± 0 (1.0) <1.0 ± 0 (1.0) <1.0 ± 0 (1.0)
1.50 ± 0.65 (1.0) 1.04 ± 0.20 <1.0 ± 0 (1.0)
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
the average was 2.72 ± 0.81 (2.61) log10 CFU/g. Thus, the process resulted in statistically significant difference (p < 0.001), with a reduction of 1.5 logarithmic cycle. There was no statistically significant difference in average faecal coliforms counts before and after heating, <1.0 log10 CFU/g. It must be noted that this group of MIs is part of the microbiological control standard of the product, with a maximum limit of 102 CFU/g (2 log10 CFU/g). Besides total mesophile count and faecal coliforms count, the MIs were also tested in the frozen final product, according to the requirements of the Brazilian regulation. All results (100%) were within the established limits, as described in Table 3. After the validation of the control measures associated with the CCPs, the two groups of MIs, total mesophile count and faecal coliforms, were also tested in the four (4) CCPs and in the final product during five days. The results are presented in Section 3.3. 3.3. Evaluation of the MIs – verification The results of the evaluation of the two groups of MIs, total mesophile count and faecal coliforms, in the CCPs – cooking and storage – are presented in Table 4. They were obtained from five different batches, assessed during a oneweek period. The microbiological guiding limit set for the cooking step was 102 CFU/g (2 log10 CFU/g), and for the storage step 103 CFU/g (3 log10 UFC/g). The average values found in this study were within the set range, but the deviation shows that there was lack of uniformity in the obtained results. The total mesophile count for the final product was 4.12 ± 0.47 (4.25), within the range limit set by the guideline: 104–106. As to faecal coliforms after cooking and after storage, the results were always equal to zero (0). Thus, all samples (100%) complied with the microbiological guideline limits set for storage – 2 · 101 CFU/g (1.30 log10 CFU/g) – and for cooking – 101 CFU/g (1 log10 CFU/g).
769
Table 4 Indicator: total mesophile count – verification of CCPs for the Lasagna Bolognese After cooking
Storage
Indicator: total mesophile count (log10 CFU/g) Meat sauce 1.76 ± 0.45 (1.9) Be´chamel sauce 1.76 ± 1.05 (1.3)
2.47 ± 0.66 (2.48) 2.07 ± 0.70 (2.00)
4. Discussion The validation is done on the control measures, most of the time based on the literature references. But in fact, is necessary to demonstrate, in a measurable way, that the control measures e.g. time and temperature of cookers can control the hazards. In this way, the use of MIs to validate the control measures established for the CCPs or even to validate the procedures for GMPs and SSOPs is highly recommended by many authors. Brashears, Dormedy, Mann, and Burson, (2002) used the total mesophile count, coliforms and generic E. coli counts to assess and to validate CCPs’ control measures related to temperature and time of exposure for beef and chicken. They also investigated the presence of Salmonella spp. and emphasized the importance of validation studies during the manufacturing process, to guide the control of hazards and to show that the process is properly conducted. Other authors (Gonzalez-Miret, Coello, Alonso, & Heredia, 2001) suggest the use of microbiological parameters such as total mesophile count, enterobacteria and Pseudomonas and S. aureus counts to validate CCPs at slaughter of poultry. They also highlight the importance of using statistical tools to analyze the data. The results from the total mesophile count, in the meat sauce and in the Be´chamel sauce, before and after cooking (CCP1 and CCP3), show that the parameters that were established in order to reduce the microbiological counts (p < 0.001) were effective, and the CCPs were validated. It was also verified that the MI counts during storage were maintained stable, without significant statistical
Table 3 Brazilian legislation (ANVISA/ RDC12/2001) standards and results from the final product Microorganism
Coliforms at 45 C/g Coagulase-positive Staphylococcus/g Bacillus cereus/g* Sulfite-reducing Clostridium at 46 C/g** Salmonella sp./25 g
Tolerance in a representative sample
Results from the frozen final product (CFU/g)
***
n
n
M
5 5 5 5 5
C 2 2 2 2 0
m 5 · 10 5 · 102 5 · 102 2 · 102 Absence
M 2
10 103 103 5 · 102 –
5 5 5 5 5
n = number of units to be randomly collected. c = maximum accepted number of samples with counts between limits m and M. m = upper limit point for the risk-free zone. M = limit point between acceptable and unacceptable products. Numbers above M are unacceptable. * Specific for grain or starch-based products. ** Specific for meat-based products. *** The results were the same for the 25 samples collected from the final products.
c 0 0 0 0 0
m 1
<10 <102 <102 <101 Absence
– – – – –
770
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
difference, allowing the maintenance of the microbiological status of the product. This fact characterizes the validation of the CCPs for the storage of the meat and Be´chamel sauces. As to faecal coliforms, a small difference (p < 0.001) was observed in the Be´chamel sauce after cooking. In all other assessments the result was <10 CFU/g. In the final product, the total mesophile count was 2.3 logarithmic cycles higher than the number found in the sauces. It should be noted that the Lasagna Bolognese includes many other ingredients that are added during the assembly step, and that the product is not subjected to any further thermal treatment in the production plant. The Brazilian legislation does not establish any limits for total mesophile counts for sensitive ingredients such as cooked ham and mozzarella cheese. There is, however, a 103 CFU/g limit for faecal coliforms. When these ingredients were received at the plant, besides the regular analysis of the faecal coliforms, an analysis of the total mesophile count was performed to determine the microbiological impact of such ingredients in the final product. The batches of mozzarella examined during the study had a maximum total mesophile count of 104 CFU/g and faecal coliforms of <10 CFU/g. The cooked ham had a total mesophile count of <10 CFU/g and faecal coliforms of <10 CFU/g. The mozzarella cheese was, probably, one of the contributors for the increase of the total mesophile count in the final product. Thus, the measurement of MIs in the final product, combined to the identification of CCPs in the manufacturing process, is an important means of ensuring that hygiene and sanitation standards of ingredients that are not submitted to further thermal treatment during the production of Lasagna Bolognese are met. Additionally, is necessary to evaluate the ingredients supplier’s quality assurance, such as GMP (Good Manufacturing Practices) and HACCP implemented in the process. The MIs are also important in ensuring the quality of the product along the steps that follow the storage of the sauces. Hansen and Knochel (1999) highlighted the importance of quantifying the MIs while assessing all steps of the manufacturing process, such as pasteurization, cooling and storage. The quantification of MIs is also important when there is a need to introduce changes in any of the process parameters. The analysis of the final product after heating adds information on the performance of the indicators all the way to the consumer. We observed that after the increase of 2.3 logarithmic cycles in the frozen final product, there was a reduction on the total mesophile count of 1.5 logarithmic cycles due to heating. It should be stressed, however, that there was an oscillation of the temperature of the product after heating, especially in the center of the lasagna tray (primary package), where the average temperature was 79.9 ± 14.2 C. Nunes et al. (2003) state that many researchers have proven that fewer colony counts are present if the product is heated
in a conventional oven or cooked in a conventional stove, if compared to using a microwave oven – 1 to 2 decimal exponents. Thus, if the lasagna is prepared in a conventional oven, there might be an even greater reduction in total mesophile counts. The faecal coliforms remained unaltered and the count was <1.0 log10 CFU/ g, before and after heating. The average total mesophile counts found during the one week evaluation of the MIs show that the microbiological guiding limits were being met. However, this data must be compared to the total mesophile count in the final product, so that a correlation between the results in the CCPs and in the final product can be established. There was no difference (p = 0.778) between the average total mesophile counts in the final product (4.12 ± 0.47) and the average counts in the final product obtained during the CCPs validation (4.22 ± 0.79), which means that the performance of the MIs has been maintained during the manufacturing process and, consequently, in the final product. With the results from the validation, it is possible to determine which MIs should be used for the verification, as well as, their limits, allowing the microbiological performance at the CCPs to be assessed, problems to be identified and dealt with preventively. At the same time, it is possible to search for pathogens that could become hazards in the final product. Thus, the evaluation of the indicators allows for the control of hazards at the CCPs and verification that specifications of the product and legal requirements as to the pathogen content are being met. Swanson and Anderson (2000) consider the use of microbiological tests in the validation and in the verification of CCPs as being essential. They also suggest the use of microbiological tests for the monitoring of the performance of processes such as cleaning and sanitation. These tests can signal trends and allow directed actions to be taken, preventing losses of process control. In turn, processes can also be improved by using the indicators preventively. 5. Conclusion Based on the arguments stated in the discussion, it can be concluded that the use of MIs combined with statistical analysis of the data confer a greater credibility to the validation and to the verification of control measures at the CCPs, aiming at ensuring food safety. The use of microbiological tests during the manufacturing process of the Lasagna Bolognese was essential to demonstrate that the validation and verification activities require standardized procedures, including frequency and expected microbiological results. These tests should be used not only to evaluate the microbiological limits established in the legislation for the final product, but also to guide the manufacturing process, enabling a proper evaluation and preventive actions. This way, the goal of guaranteeing food safety can be met.
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
References Austin, D., & Reynolds, E. (2002). Microbiological testing and validation of HACCP read set, validate. Available from: Accessed 03.06.03. Bennett, R. W., & Belay, N. (2001). Bacillus cereus. In F. P. Downes & K. Ito (Eds.), Compendium of methods for the microbiological examination of foods (4th ed., pp. 311–316). Washington: American Public Health Association (APHA). Brashears, M. M., Dormedy, E. S., Mann, J. E., & Burson, D. E. (2002). Validation and optimization of chilling and holding temperature parameters as critical control points in raw meat and poultry processing establishments. Diary, Food and Environmental Sanitation, 22(4), 246–251. Brasil. Ageˆncia Nacional de Vigilaˆncia Sanita´ria (ANVISA) (2001). Regulamento Te´cnico sobre Padro˜es Microbiolo´gicos para Alimentos. Resoluc¸a˜o-RDC 12, de 2 de janeiro de 2001. Dia´rio Oficial da Unia˜o, Sec¸a˜o 1 (pp. 45–53). Codex Alimentarius Commission (2001). Joint FAO/WHO Food Standard Programme. Codex Committee on Food Hygiene. Discussion Paper on Proposed Draft Guidelines for de Validation of Food Hygiene Control Measures. 34th Session. Bangkok, Thailand. Food and Agriculture Organization/World Health Organization (2000). Activities on risk assessment of microbiological hazards in food risk assessment: Salmonella spp. in broiler and eggs. Hazard Identification and Hazard Characterization of Salmonella in Broilers and Eggs. Rome, Italy. Germano, P. M. L., & Germano, M. I. S. (2003a). Qualidade das mate´rias-primas. In Higiene e Vigilaˆncia Sanita´ria de alimentos (pp. 15–32). Varela: Sa˜o Paulo. Germano, P. M. L., & Germano, M. I. S. (2003b). Qualidade das mate´rias-primas. In Higiene e Vigilaˆncia Sanita´ria de alimentos (pp. 220–234). Varela: Sa˜o Paulo. Gonzalez-Miret, M. L., Coello, M. T., Alonso, S., & Heredia, F. J. (2001). Validation of parameters in HACCP verification using univariate and multivariate statistics. Application to the final phases of poultry meat production. Food Control, 12, 261–268. Hansen, T. B., & Knochel, S. (1999). Quantitative considerations used in HACCP applied for a hot-fill production line. Food Control, 10, 149–159. International Commission on Microbiological Specifications for Foods (1974). Micro-organisms in Foods 2. Sampling for Microbiological Analysis: Principles and Specific Applications. Toronto: University of Toronto Press. International Standard Organization. ISO-6888-1 (1999) (E). Microbiology of food and animal feeding stuffs. Horizontal method for the
771
enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species). Part 1: Technique using Baird-Parker agar medium. International Standard Organization. ISO-6579 (2002) (E). Microbiology of food and animal feeding stuffs. Horizontal method for the detection of Salmonella spp. Jay, J. M. (1996). Modern food microbiology (5th ed.). New York, USA: Chapman & Hall, International Thompson Publishing, pp. 661. Kornacki, J. L., & Johnson, J. L. (2001). Enterobacteriaceae, coliforms and Escherichia coli as quality and safety indicators. In F. P. Downes & K. Ito (Eds.), Compendium of methods for the microbiological examination of foods (4th ed., pp. 69–82). Washington: American Public Health Association (APHA). Kvenberg, J. E., & Schwalm, D. J. (2000). Use of microbial data for hazard analysis and critical control point verification – Food and drug administration perspective. Journal of Food Protection, 63, 810–814. Mayes, T. (1999). How can the principles of validation and verification be applied to hazard analysis? Food Control, 10, 277–279. Morton, R. D. (2001). Aerobic plate count. In F. P. Downes & K. Ito (Eds.), Compendium of methods for the microbiological examination of foods (4th ed., pp. 63–67). Washington: American Public Health Association (APHA). Motarjemi, Y., & Ka¨ferstein, F. (1999). Food safety, hazard analysis and critical control point and the increase in food borne diseases: A paradox? Food Control, 10, 325–333. Motarjemi, Y., Ka¨ferstein, F., Moy, G., Miyagawa, S., & Miyasgishima, K. (1996). Importance of HACCP for public health and development. The role of the World Health Organization. Food Control, 7, 77–85. Nunes, I. A., Germano, P. M. L., & Germano, M. I. S. (2003). Forno de Microondas. In P. M. L. Germano & M. I. S. Germano (Eds.), . Higiene e Vigilaˆncia Sanita´ria de alimentos (pp. 443–453). Varela: Sa˜o Paulo. Organizac¸a˜o Pan-Americana da Sau´de (2001). HACCP: Instrumento Essencial para a Inocuidade de Alimentos. Buenos Aires: OPAS/ INPPAZ. Scott, V. N., Anderson, J. E., & Wang, G. (2001). Mesophilic anaerobic sporeformers. In F. P. Downes & K. Ito (Eds.), Compendium of methods for the microbiological examination of foods (4th ed., pp. 229–237). Washington: American Public Health Association (APHA). Swanson, K. M. J., & Anderson, J. E. (2000). Industry perspectives on the use of microbial data for hazard analysis and critical control point validation and verification. Journal of Food Protection, 63, 815–818. World Health Organization. Guidance on Regulatory Assessment of HACCP (1998). Report of a Joint FAO/WHO Consultation on the Role of Government Agencies in Assessing HACCP, Geneve.
Food Control 19 (2008) 764–771 www.elsevier.com/locate/foodcont
Microbiological indicators for the assessment of performance in the hazard analysis and critical control points (HACCP) system in meat lasagna production Elisabete Aparecida Martins *, Pedro Manuel Leal Germano Public Health School, University of Sa˜o Paulo, Avenida Doutor Arnaldo, 715, Sa˜o Paulo, SP, CEP: 01246-904, Brazil Received 10 September 2006; received in revised form 22 July 2007; accepted 3 August 2007
Abstract The HACCP system is being increasingly used to ensure food safety. This study investigated the validation of the control measures technique in order to establish performance indicators of this HACCP system in the manufacturing process of Lasagna Bolognese (meat lasagna). Samples were collected along the manufacturing process as a whole, before and after the CCPs. The following microorganism’s indicator (MIs) was assessed: total mesophile and faecal coliform counts. The same MIs were analyzed in the final product, as well as, the microbiological standards required by the current legislation. A significant reduction in the total mesophile count was observed after cooking (p < 0.001). After storage, there was a numerical, however non-significant change in the MI count. Faecal coliform counts were also significantly reduced (p < 0.001) after cooking. We were able to demonstrate that the HACCP system allowed us to meet the standards set by both, the company and the Brazilian regulations, proved by the reduction in the established indicators. 2007 Elsevier Ltd. All rights reserved. Keywords: HACCP; Validation; Control measure; Verification; Microbiological indicators
1. Introduction With globalization, food borne diseases have acquired a new dimension, as many food products are produced in one country to be imported and consumed in another. Sometimes, the country of origin of the product has precarious hygiene and sanitation conditions (Motarjemi & Ka¨ferstein, 1999). With the increased incidence of food borne diseases, the hazard analysis and critical control points (HACCP) system was developed as a method to ensure food safety. The HACCP system is being increasingly used by the food industry and regulatory agencies (Motarjemi, Ka¨ferstein, Moy, Miyagawa, & Miyasgishima, 1996). However, one of its least explored aspects is validation, not only
*
Corresponding author. Tel.: +55 11 3023 6037; fax: +55 11 3023 5316. E-mail address: [email protected] (E.A. Martins).
0956-7135/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2007.08.001
the validation of control measures, but also of the system itself. The validation and verification steps certify that the hazards have been identified and are being controlled, and whether the plan is being conducted as designed (Kvenberg & Schwalm, 2000; Mayes, 1999). The Codex Alimentarius Commission (2001) defines validation as the process to ensure that a certain group of measures can effectively control a specific hazard in a certain food. The validation of the measures requires a comparison between the actual and the expected results, as it is necessary to confirm the efficiency of the measures for controlling a specific hazard. Ideally, all procedures used to control a specific hazard should be validated by the validation of control measures, but there might be limitations to achieve this goal. In this case, some prioritization criteria may be considered: greater potential of human health risk or if there is no available historic data concerning the control of that hazard. Another aspect to be considered is if a control measure
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
is determinant to ensure the safety of the product; in this case, the validation can be focused on that measure. Focused validation implies that the control measure has a statistically higher contribution for the safety of the product. On the other hand, if there are multiple measures to control a certain hazard, and each of the measures has a similar influence, they will all be considered as having the same level of importance for validation (Codex Alimentarius Commission, 2001). The method used to validate a group of measures will depend on the nature of the hazard, on the product and on the type of control measures selected to control that hazard (Codex Alimentarius Commission, 2001). For example, considering the validation of a preventive measure related to the control of a microbiological hazard (pathogen), Kvenberg and Schwalm (2000); Swanson and Anderson (2000) do not advise testing for pathogens as a means of validation for that control measure, as the possibility of contamination of a product manufactured in a plant that uses the HACCP system is very remote. In this case, they suggest the use of quantifiable microorganism’s indicator (MIs) to validate the control measures. According to Scott and Moberg (1995), quoted by PAHO (Pan American Health Organization – Organizac¸a˜o Pan-Americana de Sau´de – OPAS, Brasil, 2001, p. 140), the MIs present in a food do not pose a direct threat to health, but they can be used as a signal of the presence of a potential threat. For Austin and Reynolds (2002), some indicators, such as enterobacteria, can demonstrate a possible presence of other pathogens in that environment and/or in the final product; the higher the MI count, the higher the possibility of pathogens in the product. Special attention should be paid also to the method chosen to assess the indicators. Thus, the method to be selected for validation must be approved and recognized (Austin & Reynolds, 2002). This means that it is necessary to define criteria for the validation of the control measures associated to the CCPs, and to determine the indicator organisms that will evaluate the microbiological performance of the HACCP system. With measurable controls, it is possible to verify if the product safety targets are being met. The present study was conducted to investigate the method for validation of control measures in order to establish the performance indicators of the system for the manufacturing process of the Lasagna Bolognese (meat lasagna). 2. Experiment 2.1. Type of study This experimental study was conducted in the manufacturing process, through industrial scale, of the product ‘‘Lasagna Bolognese’’ (or meat lasagna). The microbiological standard for this product is established by ANVISA (Ageˆncia Nacional de Vigilaˆncia Sanita´ria – Brazilian Sanitary Surveillance Agency – Brazil, 2001). It is made of special lasagna noodles, tomato meat sauce, Be´chamel
765
sauce, cooked ham and mozzarella cheese. The process includes the following steps: (a) preparation and cooking the tomato meat sauce and of the Be´chamel sauce, both sauces with pH more than 4.6; (b) assembling the lasagna by interlaying the noodles (pre-cooked and dehydrated lasagna pasta), the meat sauce, the ham and the mozzarella and finally by adding the Be´chamel sauce and grated parmesan cheese; (c) fitting the lid and sealing the package; (d) freezing the product; (e) passing the product through a metal detector; (f) final packaging, and (g) storage. The study involved the following stages: implementation of the HACCP system in the manufacturing process of the product Lasagna Bolognese; definition of the MIs and collection of samples for analysis; validation of the control measures associated with the critical control points (CCPs); validation of the HACCP system, and definition of the microbiological indicators for the assessment of the performance of the system. The implementation of the HACCP system in the manufacturing process of the Lasagna Bolognese was based on the steps described in Fig. 1. This figure also describes the preliminary stages and actions taken after the implementation of the system. Additionally, Table 1 presents a summary of the of HACCP Plan with the hazard identified, Clostridium perfringens, Escherichia coli and Bacillus cereus. Despite of the fact that Salmonella and Staphylococcus aureus are mention in the Brazilian Legislation for this kind of product (lasagna) they are not considered as hazards by the HACCP team. For Salmonella the literature shows that this bacteria is strongly associate with poultry and poultry products (FAO/WHO, 2000) and it is killed under pasteurization temperatures (Jay, 1996) and for S. aureus it is controlled through Good Manufacturing Practices. B. cereus and Clostridium perfringens vegetative cells are killed in cooking temperatures, more than 70 C. The most important control measure, for both, the to prevent the growing is to keep the product, after cooking, in temperature higher than 60 C (Germano & Germano, 2003b). These conditions, was observed in the process, in the cooking and storage steps. The CCPs and their respective steps in the process, the hazards that are controlled in each step and the associated control measures are identified. No chemical hazards were identified in the process. For the validation of the control measures associated with the CCPs, priority was given to those measures connected with the control of microbiological hazards as, according to Idexx (1998), mentioned by PAHO (2001, p. 128), they are the ones that represent a major risk to the wholesomeness of food. For Germano and Germano (2003a), the most important risks in food from animal origin are related to food poisonings. The microorganism’s indicators – (a) aerobic mesophiles and (b) faecal coliforms – were established based on references (Austin & Reynolds, 2002; Kvenberg & Schwalm,
766
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771 Documents produced Training of the HACCP team – main ideas and hazard analysis Preliminary actions to the implementation of the HACCP System
Training of the monitoring team – main ideas and monitoring procedures Establish criteria for checking the prerequisites for the implementation of the system are being met Develop the HACCP Plan by applying the seven principles
Evaluation of the training Record of training Monitoring operational procedure Evaluation of training Record of training Criteria Specification Ex.: minimum grades during the auditing of the prerequisite programs (GMP 1 and SSOP 2) Flow diagram of the process Description of the product and intended use HACCP Plan
A
Audit the implementation of the prerequisites and training program
Correct the criteria that are not being met
Criteria met? NO YES
A
Monitor the CCPs for one (1) month
Record of the monitoring Report of the record analysis
Are the Critical Limits being observed ? NO
YES
Validate the CCPs
Report of CCPs Validation
Have the CCPs been validated? NO YES
Validate HACCP System
Re-evaluate the HACCP Plan
Report of HACCP System Validation
Has the HACCP System been validated? NO
YES
1
End of process
Good Manufacturing Practices
2
Sanitation Standard Operating Procedures and Sanitation Standard Pre-operating Procedures
Fig. 1. Flow diagram of the implementation of the HACCP system.
2000; Scott and Moberg, 1995, cited in OPAS, 2001, p. 140; Swanson & Anderson, 2000). For the analysis of the indicators, five (5) samples of the product were collected each day during the processing
period, before and after each step related to each CCP, at one (1) hour intervals and during a period of five (5) days; thus, 25 samples were collected from each collecting point. Samples were also collected from the final product. The
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771 Table 1 Summary of the of HACCP plan with the hazard identified CCP #
Process step
Identified hazard
Control measure
1
Cooking of the tomato meat sauce
Survival of Clostridium perfringens and Survival of Escherichia coli
Minimum temperature of the sauce at the end of the process (80 C) Cooking time (3 min)
2
Storage of the tomato meat sauce
Forming spores of Clostridium perfringens
Minimum temperature of the sauce (75 C)
3
Cooking of the Be´chamel sauce
Survival of Bacillus cereus
Minimum temperature of the sauce at the end of the process (80 C) Cooking time (3 min)
4
Storage of the Be´chamel sauce
Forming spores of Bacillus cereus
Minimum temperature of the sauce (75 C)
5
Primary package
Presence of metal fragments
Metal detector
number of samples was based on the sampling plan of the International Commission on Microbiological Specification for Foods (ICMSF, 1974). The collection of samples during the processing steps was made as follows: aseptic collection of 500 g of the product being processed, before and after each CCP, and always from the same batch. The samples were placed in new plastic bags, tagged and identified, and were transported in a cooler with ice to the plant laboratory and stored under freezing temperature until tested. All samples were tested both for the selected indicators and according to the Brazilian legislation requirements. The samples from the final product were collected in duplicate; the first set of product samples underwent microbiological testing in the form that the product leaves the plant frozen, and the second set of samples was tested as prepared for consumption, after heating. The instructions for preparing the lasagna are printed on the preliminary package of the product. The product can be heated in a conventional oven or using a microwave oven. We chose to analyze the product heated in a microwave oven, as it is being used by an increasing number of consumers and it also has been a point of disagreement among researchers (Nunes, Germano, & Germano, 2003). The main point for the disagreement lies in the efficiency of the oven. Researchers have questioned the efficiency of microbiological count reduction with the use of the microwave oven, as the food is not equally heated, allowing for the formation of ‘‘cold spots’’ in the food, especially in the central portion of the serving. This fact emphasizes that food manufacturing should design microwaveable products to be safe prior to heating in microwave; once there is no consensus that microwave oven is a reliable kill step. The lasagna was prepared according to the instructions on the 650 g pack: 14 min under medium–high potency
767
(70%). The samples were heated in a domestic microwave oven, a Sanyo – 60 Hz and 120 W. After heating, temperature was measured in each sample in five different places: in each of the four corners of the pack and in the center. The methodology used for total mesophile count, faecal coliforms, sulfite-reducing Clostridium and B. cereus detection is described in the ‘‘Compendium of Methods for the Microbiological Examination of Foods’’ (Bennett & Belay, 2001; Kornacki & Johnson, 2001; Morton, 2001; Scott, Anderson, & Wang, 2001). The analysis for Salmonella spp. and S. aureus followed the criteria of the International Standard Organization, respectively ISO, ISO-6888-1 (1999), and ISO-6579 (2002). The obtained results, before and after each CCP, were evaluated according to the significant statistical difference. Along with the statistical analysis, the results were also evaluated according to sanitation and hygiene standards. Considering the MI counts after the CCPs and the established hygiene and sanitation standards, we formulated the following hypothesis: (a) cases with a significant statistical difference and with count reduction after the CCP were considered as a validated CCP, meaning that the hazard was under control; (b) cases without a significant statistical difference and with similar counts before and after the CCP, were considered validated, as the hazard was under control; (c) cases with significant statistical difference, and with higher counts after the CCP when compared to previous counts were considered a non-validated CCP, meaning that the hazard was not under control. The validation of the HACCP system was based on the ‘‘check-list’’ published by the World Health Organisation, (WHO, 1998) and performed according to technical and scientific analysis of: (a) all preliminary actions previous to the implementation of the HACCP system; (b) the implementation of the HACCP system; (c) traceability; (d) recall strategy. In order to establish the microbiological performance indicators of the system, an analysis of the following results was conducted: (a) MIs used during the process (validation of the control measures connected to the CCPs); (b) MIs in the final product; and (c) detection of microorganisms according to the microbiological standards defined for the product, allowing to establish the correlation of the performance of the product during the manufacturing process, at the CCPs, with the performance of the final product itself. From this data, the MIs for the process were established and assessed for one week. At the same time, the final product was analyzed according to the established microbiological standards, and the performance of the indicators was checked. The performance of the indicators was measured according to the following aspects: (1) The ability to maintain the characteristics observed during the validation of the control measures for the CCPs, considering the significance of the statistical difference between CCPs and the status of hazard
768
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
control. (2) The fulfillment of the microbiological guideline limit for total mesophile count. The following assumptions were made for this assessment: the chosen microbiological guiding limit for the total mesophile count was (m)104 to (M)106 CFU/g, considering the ICMSF (1974) classification for organisms that do not pose a direct threat to health. It must be noted, however, that the guideline was drawn for the final product, not being adequate for the analysis of the initial stages of food products processing. Thus, from the lower value established (m = 104) for the final product, a reduction of 1 (one) decimal exponent or 1 (one) logarithmic cycle was considered for each step of the manufacturing process. The following microbiological guiding limits were set for the total mesophile count: (a) assembly of the lasagna 104, (b) storage of sauces 103 and (c) end of cooking 102. (3) The ability to comply with the microbiological standards for faecal coliforms – a maximum of 102 CFU/g in the final product. The microbiological guiding limit was established for the manufacturing process based on the Brazilian legislation: maximum count of 5 · 101 CFU/g, storage – maximum count of 2 · 101 CFU/g, and cooking maximum count of 101 CFU/g. However, it should be stressed that the final product requires heating prior to consumption. 2.2. Data analysis The statistical analysis was conducted using the SPSS Incorporation Sigma Stat for Windows, version 2.0. In order to normalize the results, the absolute values of the microorganism’s indicator (MIs) counts were converted into base 10 logarithms. To compare different steps of the manufacturing process, variance analysis of repeated measurements was used; however, as the results were not parametric, the Friedman test was applied. Whenever the Friedman test revealed significant statistical difference (p < 0.05), the Duncan test was applied. The Wilcoxon test was used to compare the results after cooking with those obtained after storage. 3. Results The results are presented according to the statistical analysis used for comparing the MIs (total mesophile and faecal coliform counts), before cooking, after cooking and after storage – for the Be´chamel sauce and for the tomato meat sauce used in the Lasagna Bolognese. The figures are expressed as arithmetic mean ± standard deviation
from the mean, followed by the median value between parentheses. Initially, the results of the comparison between the different steps of the manufacturing process are shown – pre-cooking, after cooking, and after storage – for the tomato meat sauce and for the Be´chamel sauce. 3.1. Validation of control measures related to the CCPs The total mesophile count and the faecal coliforms count are presented in Table 2. After cooking, a significant reduction (p < 0.001) of the total mesophile count was observed in both sauces. In the meat sauce, no difference (p = 0.109) was observed between the total mesophile count after cooking and after storage. The same result was obtained for the Be´chamel sauce (p = 0.337). Thus, there was a considerable reduction in the total mesophile count resulting from the cooking step, without any significant increase during storage. The meat sauce samples showed the same faecal coliform count in the three steps that were assessed: <1.0 ± 0 (1.0) log10 CFU/g. As for the Be´chamel sauce, there was a significant reduction (p < 0.001) in the faecal coliforms count after the cooking step. There was no difference (p = 0.814) after cooking and after storage. Faecal coliform MIs had a similar performance in the cooking and storage steps to that observed in the total mesophile count; thus, a significant reduction of the MIs was detected after cooking, with maintenance of the microbiological conditions during storage. The comparative results of MIs in the frozen final product and in the heated one are presented in Section 3.2. 3.2. Further analysis – final product before and after preparation Both groups of MIs were also studied in the samples of the final product under two different conditions: frozen and after heating in a microwave oven. In order to assess the product after heating, the temperature of the lasagna was measured in 5 (five) different points of each product tray. In a 125 determinations, the lowest average temperature was measured in the center of the tray, 79.9 ± 14.2 C, and the highest was found in one of the corners, 86.3± 4.7 C. The frozen product had an average total mesophile count of 4.22 ± 0.79 (4.04) log10 CFU/g. After heating,
Table 2 Indicator organisms in the CCPs of the Lasagna Bolognese Total mesophile count (log10 CFU/g) Before cooking After cooking Storage
Faecal coliforms (log10 CFU/g)
Meat sauce
Be´chamel sauce
Meat sauce
Be´chamel sauce
3.58 ± 0.89 (3.34) 2.04 ± 0.12 (2.00) 2.19 ± 0.40 (2.00)
4.73 ± 0.92 (5.30) 1.69 ± 0.64 (1.60) 1.62 ± 0.83 (1.30)
<1.0 ± 0 (1.0) <1.0 ± 0 (1.0) <1.0 ± 0 (1.0)
1.50 ± 0.65 (1.0) 1.04 ± 0.20 <1.0 ± 0 (1.0)
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
the average was 2.72 ± 0.81 (2.61) log10 CFU/g. Thus, the process resulted in statistically significant difference (p < 0.001), with a reduction of 1.5 logarithmic cycle. There was no statistically significant difference in average faecal coliforms counts before and after heating, <1.0 log10 CFU/g. It must be noted that this group of MIs is part of the microbiological control standard of the product, with a maximum limit of 102 CFU/g (2 log10 CFU/g). Besides total mesophile count and faecal coliforms count, the MIs were also tested in the frozen final product, according to the requirements of the Brazilian regulation. All results (100%) were within the established limits, as described in Table 3. After the validation of the control measures associated with the CCPs, the two groups of MIs, total mesophile count and faecal coliforms, were also tested in the four (4) CCPs and in the final product during five days. The results are presented in Section 3.3. 3.3. Evaluation of the MIs – verification The results of the evaluation of the two groups of MIs, total mesophile count and faecal coliforms, in the CCPs – cooking and storage – are presented in Table 4. They were obtained from five different batches, assessed during a oneweek period. The microbiological guiding limit set for the cooking step was 102 CFU/g (2 log10 CFU/g), and for the storage step 103 CFU/g (3 log10 UFC/g). The average values found in this study were within the set range, but the deviation shows that there was lack of uniformity in the obtained results. The total mesophile count for the final product was 4.12 ± 0.47 (4.25), within the range limit set by the guideline: 104–106. As to faecal coliforms after cooking and after storage, the results were always equal to zero (0). Thus, all samples (100%) complied with the microbiological guideline limits set for storage – 2 · 101 CFU/g (1.30 log10 CFU/g) – and for cooking – 101 CFU/g (1 log10 CFU/g).
769
Table 4 Indicator: total mesophile count – verification of CCPs for the Lasagna Bolognese After cooking
Storage
Indicator: total mesophile count (log10 CFU/g) Meat sauce 1.76 ± 0.45 (1.9) Be´chamel sauce 1.76 ± 1.05 (1.3)
2.47 ± 0.66 (2.48) 2.07 ± 0.70 (2.00)
4. Discussion The validation is done on the control measures, most of the time based on the literature references. But in fact, is necessary to demonstrate, in a measurable way, that the control measures e.g. time and temperature of cookers can control the hazards. In this way, the use of MIs to validate the control measures established for the CCPs or even to validate the procedures for GMPs and SSOPs is highly recommended by many authors. Brashears, Dormedy, Mann, and Burson, (2002) used the total mesophile count, coliforms and generic E. coli counts to assess and to validate CCPs’ control measures related to temperature and time of exposure for beef and chicken. They also investigated the presence of Salmonella spp. and emphasized the importance of validation studies during the manufacturing process, to guide the control of hazards and to show that the process is properly conducted. Other authors (Gonzalez-Miret, Coello, Alonso, & Heredia, 2001) suggest the use of microbiological parameters such as total mesophile count, enterobacteria and Pseudomonas and S. aureus counts to validate CCPs at slaughter of poultry. They also highlight the importance of using statistical tools to analyze the data. The results from the total mesophile count, in the meat sauce and in the Be´chamel sauce, before and after cooking (CCP1 and CCP3), show that the parameters that were established in order to reduce the microbiological counts (p < 0.001) were effective, and the CCPs were validated. It was also verified that the MI counts during storage were maintained stable, without significant statistical
Table 3 Brazilian legislation (ANVISA/ RDC12/2001) standards and results from the final product Microorganism
Coliforms at 45 C/g Coagulase-positive Staphylococcus/g Bacillus cereus/g* Sulfite-reducing Clostridium at 46 C/g** Salmonella sp./25 g
Tolerance in a representative sample
Results from the frozen final product (CFU/g)
***
n
n
M
5 5 5 5 5
C 2 2 2 2 0
m 5 · 10 5 · 102 5 · 102 2 · 102 Absence
M 2
10 103 103 5 · 102 –
5 5 5 5 5
n = number of units to be randomly collected. c = maximum accepted number of samples with counts between limits m and M. m = upper limit point for the risk-free zone. M = limit point between acceptable and unacceptable products. Numbers above M are unacceptable. * Specific for grain or starch-based products. ** Specific for meat-based products. *** The results were the same for the 25 samples collected from the final products.
c 0 0 0 0 0
m 1
<10 <102 <102 <101 Absence
– – – – –
770
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
difference, allowing the maintenance of the microbiological status of the product. This fact characterizes the validation of the CCPs for the storage of the meat and Be´chamel sauces. As to faecal coliforms, a small difference (p < 0.001) was observed in the Be´chamel sauce after cooking. In all other assessments the result was <10 CFU/g. In the final product, the total mesophile count was 2.3 logarithmic cycles higher than the number found in the sauces. It should be noted that the Lasagna Bolognese includes many other ingredients that are added during the assembly step, and that the product is not subjected to any further thermal treatment in the production plant. The Brazilian legislation does not establish any limits for total mesophile counts for sensitive ingredients such as cooked ham and mozzarella cheese. There is, however, a 103 CFU/g limit for faecal coliforms. When these ingredients were received at the plant, besides the regular analysis of the faecal coliforms, an analysis of the total mesophile count was performed to determine the microbiological impact of such ingredients in the final product. The batches of mozzarella examined during the study had a maximum total mesophile count of 104 CFU/g and faecal coliforms of <10 CFU/g. The cooked ham had a total mesophile count of <10 CFU/g and faecal coliforms of <10 CFU/g. The mozzarella cheese was, probably, one of the contributors for the increase of the total mesophile count in the final product. Thus, the measurement of MIs in the final product, combined to the identification of CCPs in the manufacturing process, is an important means of ensuring that hygiene and sanitation standards of ingredients that are not submitted to further thermal treatment during the production of Lasagna Bolognese are met. Additionally, is necessary to evaluate the ingredients supplier’s quality assurance, such as GMP (Good Manufacturing Practices) and HACCP implemented in the process. The MIs are also important in ensuring the quality of the product along the steps that follow the storage of the sauces. Hansen and Knochel (1999) highlighted the importance of quantifying the MIs while assessing all steps of the manufacturing process, such as pasteurization, cooling and storage. The quantification of MIs is also important when there is a need to introduce changes in any of the process parameters. The analysis of the final product after heating adds information on the performance of the indicators all the way to the consumer. We observed that after the increase of 2.3 logarithmic cycles in the frozen final product, there was a reduction on the total mesophile count of 1.5 logarithmic cycles due to heating. It should be stressed, however, that there was an oscillation of the temperature of the product after heating, especially in the center of the lasagna tray (primary package), where the average temperature was 79.9 ± 14.2 C. Nunes et al. (2003) state that many researchers have proven that fewer colony counts are present if the product is heated
in a conventional oven or cooked in a conventional stove, if compared to using a microwave oven – 1 to 2 decimal exponents. Thus, if the lasagna is prepared in a conventional oven, there might be an even greater reduction in total mesophile counts. The faecal coliforms remained unaltered and the count was <1.0 log10 CFU/ g, before and after heating. The average total mesophile counts found during the one week evaluation of the MIs show that the microbiological guiding limits were being met. However, this data must be compared to the total mesophile count in the final product, so that a correlation between the results in the CCPs and in the final product can be established. There was no difference (p = 0.778) between the average total mesophile counts in the final product (4.12 ± 0.47) and the average counts in the final product obtained during the CCPs validation (4.22 ± 0.79), which means that the performance of the MIs has been maintained during the manufacturing process and, consequently, in the final product. With the results from the validation, it is possible to determine which MIs should be used for the verification, as well as, their limits, allowing the microbiological performance at the CCPs to be assessed, problems to be identified and dealt with preventively. At the same time, it is possible to search for pathogens that could become hazards in the final product. Thus, the evaluation of the indicators allows for the control of hazards at the CCPs and verification that specifications of the product and legal requirements as to the pathogen content are being met. Swanson and Anderson (2000) consider the use of microbiological tests in the validation and in the verification of CCPs as being essential. They also suggest the use of microbiological tests for the monitoring of the performance of processes such as cleaning and sanitation. These tests can signal trends and allow directed actions to be taken, preventing losses of process control. In turn, processes can also be improved by using the indicators preventively. 5. Conclusion Based on the arguments stated in the discussion, it can be concluded that the use of MIs combined with statistical analysis of the data confer a greater credibility to the validation and to the verification of control measures at the CCPs, aiming at ensuring food safety. The use of microbiological tests during the manufacturing process of the Lasagna Bolognese was essential to demonstrate that the validation and verification activities require standardized procedures, including frequency and expected microbiological results. These tests should be used not only to evaluate the microbiological limits established in the legislation for the final product, but also to guide the manufacturing process, enabling a proper evaluation and preventive actions. This way, the goal of guaranteeing food safety can be met.
E.A. Martins, P.M.L. Germano / Food Control 19 (2008) 764–771
References Austin, D., & Reynolds, E. (2002). Microbiological testing and validation of HACCP read set, validate. Available from:
771
enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species). Part 1: Technique using Baird-Parker agar medium. International Standard Organization. ISO-6579 (2002) (E). Microbiology of food and animal feeding stuffs. Horizontal method for the detection of Salmonella spp. Jay, J. M. (1996). Modern food microbiology (5th ed.). New York, USA: Chapman & Hall, International Thompson Publishing, pp. 661. Kornacki, J. L., & Johnson, J. L. (2001). Enterobacteriaceae, coliforms and Escherichia coli as quality and safety indicators. In F. P. Downes & K. Ito (Eds.), Compendium of methods for the microbiological examination of foods (4th ed., pp. 69–82). Washington: American Public Health Association (APHA). Kvenberg, J. E., & Schwalm, D. J. (2000). Use of microbial data for hazard analysis and critical control point verification – Food and drug administration perspective. Journal of Food Protection, 63, 810–814. Mayes, T. (1999). How can the principles of validation and verification be applied to hazard analysis? Food Control, 10, 277–279. Morton, R. D. (2001). Aerobic plate count. In F. P. Downes & K. Ito (Eds.), Compendium of methods for the microbiological examination of foods (4th ed., pp. 63–67). Washington: American Public Health Association (APHA). Motarjemi, Y., & Ka¨ferstein, F. (1999). Food safety, hazard analysis and critical control point and the increase in food borne diseases: A paradox? Food Control, 10, 325–333. Motarjemi, Y., Ka¨ferstein, F., Moy, G., Miyagawa, S., & Miyasgishima, K. (1996). Importance of HACCP for public health and development. The role of the World Health Organization. Food Control, 7, 77–85. Nunes, I. A., Germano, P. M. L., & Germano, M. I. S. (2003). Forno de Microondas. In P. M. L. Germano & M. I. S. Germano (Eds.), . Higiene e Vigilaˆncia Sanita´ria de alimentos (pp. 443–453). Varela: Sa˜o Paulo. Organizac¸a˜o Pan-Americana da Sau´de (2001). HACCP: Instrumento Essencial para a Inocuidade de Alimentos. Buenos Aires: OPAS/ INPPAZ. Scott, V. N., Anderson, J. E., & Wang, G. (2001). Mesophilic anaerobic sporeformers. In F. P. Downes & K. Ito (Eds.), Compendium of methods for the microbiological examination of foods (4th ed., pp. 229–237). Washington: American Public Health Association (APHA). Swanson, K. M. J., & Anderson, J. E. (2000). Industry perspectives on the use of microbial data for hazard analysis and critical control point validation and verification. Journal of Food Protection, 63, 815–818. World Health Organization. Guidance on Regulatory Assessment of HACCP (1998). Report of a Joint FAO/WHO Consultation on the Role of Government Agencies in Assessing HACCP, Geneve.