Escherichia coli inactivation on tenderloin beef medallions fried to different degrees of doneness

Food Control 106 (2019) 106683

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Escherichia coli inactivation on tenderloin beef medallions fried to different degrees of doneness

T

Clarissa Rech Peixotoa, Paulo Armendarisb, Andrius Grassic, Fabiani Andréa Walker Henglesa, Eduardo Cesar Tondoa,∗ a

Universidade Federal do Rio Grande do Sul (UFRGS), Food Science and Food Technology Institute, Food Microbiology and Food Control Laboratory – ICTA/UFRGS, Av. Bento Gonçalves, 9500, Porto Alegre, RS, 91501-970, Brazil b Rio Grande do Sul National Agriculture Laboratory (LANAGRO/RS), Estr. Ponta Grossa, 3036, Porto Alegre, RS, Brazil c Bascom do Brasil – School of the Gastronomy and Cooking, Rua dos Andradas, 1170, Porto Alegre, RS, Brazil

ARTICLE INFO

ABSTRACT

Keywords: Food safety Beef microbiota Gastronomic preparations MALDI-TOF/MS

Restaurants used to prepare tenderloin beef medallions using different frying parameters in order to satisfy their customers. However, some of those cooking methods are not in accordance with food safety recommendations, because they do not reach internal temperatures recognized as save, in countries such as US, Canada and Brazil. This study aimed to assess the microbial inactivation in tenderloin beef medallions fried to five pre-determined degrees of doneness. Two different frying techniques were investigated, allowing beef pieces to reach the following degrees of doneness: rare, medium-rare, medium, medium-well and well-done. Before thermal processing, tenderloin beef medallions were artificially contaminated with approximately 106 CFU/g of Escherichia coli and the survival of this microorganism and mesophilic total counts were measured using VRBA + MUG and BHI plates, respectively. Bacterial colonies grew on BHI plates were also analyzed by Matrix-Assisted Laser and TimeOf-Flight desorption/ionization mass spectrometry (MALDI-TOF/MS) in order to identify survivors. Results indicated that complete E. coli inactivation was only observed on tenderloin beefs subjected to well-done degree established by both techniques, given that Technique 1 recommended a final heating inside oven after frying. Without this final oven processing, which is not always done in restaurants, more than 2 log CFU/g of viable E. coli were detected. Modeling of the kinetic parameters by GInaFIT revealed a D value (time needed to reduce 90% of the bacterial population) of 1.67 min for Technique 1 and D = 3.81 min for Technique 2 and this difference was associated to the difference of temperatures of olive oil. MALDI-TOF analyses demonstrated high diversity in natural microbiota of tenderloin beef pieces and among the main identified survivor genera was Citrobacter, Enterobacter, Hafnia, Serratia, Raoultella, Bacillus, Lactobacillus, Micrococcus, Pseudomonas, Serratia and Staphylococcus. Pathogens such as Salmonella and Listeria monocytogenes were not found. Based on the results, we recommend the use of high microbiological quality beef for the preparation of tenderloin beef medallions and the validation of frying process considering specific conditions of restaurants, because different degrees of fried may result in different counts and types of bacterial survivors.

1. Introduction The current context of gastronomy, with renowned Chefs and starred restaurants, feeds a growing global market. Waiting times lasting for months to enjoy a meal at Michelin Guide1 starred

restaurants, the emphasis of specialized media outlets, such as culinary magazines, books, and TV shows, the increase in vocational education and in university programs in gastronomy - those are all factors responsible for a large share of tourism and business worldwide (Levine, Chaifetz, & Chapman, 2017).

Corresponding author. E-mail address: [email protected] (E.C. Tondo). 1 The Michelin Guide (or “Red Guide”) is a travel guide created by French tire manufacturer Michelin. Since 1900, it seeks to help those who travel by car. Since then, it broadened its scope to include restaurant suggestions and other services. Over the years, its scoring system (stars) gained credibility, especially given its sound criteria that have barely changed since it was implemented. Its inspectors assess each dish based on five criteria: quality of products employed, kitchen personality, cooking technique and flavor harmony, the price to quality relation, and consistency. Restaurants may receive up to 3 starts, but only 5% of restaurants analyzed receive one star. The respect and reputation of the Guide make it a legitimate gauge of the quality of a restaurant (Surlemont & Johnson, 2005). ∗

https://doi.org/10.1016/j.foodcont.2019.06.009 Received 11 March 2019; Received in revised form 4 June 2019; Accepted 5 June 2019 Available online 07 June 2019 0956-7135/ © 2019 Published by Elsevier Ltd.

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Table 1 Parameters for frying beef tenderloin medallions preparation based on the technique described by Le Cordon Bleu (Wright & Treuille, 2016) and by IGA (Zicarelli, 2017). DEGREES OF DONENESS

Technique 1: Le Cordon Bleu Controlled parameter: fry time

Technique 2: IGA Controlled parameter: Central Temperature a

Rare (MALPA) Medium-rare (APMAL) Medium (AOPO) Medium-well (APBEM) Well-done (BEMPA)

1 min (0.5 min each side) 2 min (1 min each side) 3 min (1.5 min each side) 4 min (2 min each side) 6 min (3 min each side) and 6–10 min inside oven at low heat

52–55 °C 55–60 °C 60–65 °C 65–69 °C 71–100 °C

a

The medallions were flipped when they reached half of the final temperature.

In this context, many restaurants adopt different cooking methods using techniques that excel at appealing to our senses. However, some of those methods are not in accordance to food safety recommendations or those set by legislation of different countries, such as US, Canada and Brazil. One of the most important factors to inactivate micro-organisms commonly present on raw material and ingredients is the controlling of temperature and time during thermal processing. Effective heat processing helps and may guarantee safety of food to be consumed, especially for highly perishable foods such as red meat. As expressed in its Safe Minimum Internal Temperature Chart (USDA, 2015), according to the Food Safety and Inspection Service, food as steaks, chops, and beef roasts must be prepared to a minimum internal temperature of at least 62.8 °C (145 °F), and left to rest for at least 3 min before serving. In Canada, doneness degrees indications are slightly different from those stablished in the United States. The Canadian Food Inspection Agency – CFIA – (Government of Canada, 2015) defines minimum internal temperature for beef at 63 °C (145 °F) for medium-rare degree; at least 71 °C (160 °F) for medium degree and 77 °C (170 °F) for well-done degree. In Brazil, federal legislation determines that all parts of a thermally processed food shall reach at least 70 °C, or be processed by other time and temperature parameters considered safe (Brasil, 2004). Even though temperatures around 70 °C are not difficult to reach when processing red meat, clients of high level gastronomy restaurants usually do not order well-done beef tenderloin medallions, based on undesirable sensorial characteristics. For example, in the study of Savell et al. (1995) consumers were asked the reason for choosing the degree of doneness of their preference. Those consumers who preferred meat under degree of doneness less than medium rare were interested on characteristics which could increase the sensory attributes of softness, juiciness and flavor of beef, but they were not interested on the safety of beef. On other hand, consumers who preferred the well-done point for beef were often motivated by emotional reasons, as blood aversion and food safety principles. Depending on the initial microbiological quality of tenderloin beefs, different quantities and types of survivors will be found, which may put in risk costumers. Therefore, this study aimed to assess the microbial inactivation on tenderloin beef medallions fried to five degrees of doneness (rare, medium-rare, medium, medium-well and well-done) as usually carried out in gastronomy restaurants.

6.0 ± 0.2 cm and height 2.5 ± 0.3 cm, weight 100.0 ± 5.0 g) were cut and the pH and Water Activity (aw) were measured. The pH was measured by potentiometric method (Kasvi – model K39–1014B) and the aw was measure using an Aqualab 3 TE – Decagon equipment. Tenderloin beef medallions were artificially contaminated as described below and subjected to two frying techniques (1 and 2). Technique 1 was described by the Le Cordon Bleu Culinary School (Wright & Treuille, 2016), while Technique 2 was preconized by Gastronomic Institute of the Americas (“Instituto Gastronômico das Américas”, IGA) (Zicarelli, 2017). The difference between both techniques is based on the control of final degree of doneness. Technique 1 defines each degree of doneness by controlling grilling time, on high fire temperature. For these techniques, well-done degree (BEMPA) instructions include heat treatment at an oven to complete cooking (time and temperature parameters are described below, in Table 1). Technique 2 instructs to control the degree of doneness by monitoring the central temperature of medallions. In this technique only the grilling process was carried out and this was done using low intensity fire in order to investigate the increase of internal temperature assuming that as the worst scenario found during grilling in restaurants. Medallions were prepared using a thick bottom skillet, 22 cm diameter, sprayed with 10 mL olive oil to grill each medallion to the desired degree of doneness. Olive oil was used only to avoid the medallion's surface burning. We tested five different degrees of doneness described by each technique. They were determined as follows: rare (MALPA), mediumrare (APMAL), medium (AOPO), medium-well (APBEM), and well-done (BEMPA). Process parameters of each degree are demonstrated in Table 1. We carried out 3 replicas per variable (technique and frying time), and 3 samples per each technique and frying time were analyzed. For both techniques, we monitored heat treatment time, temperature at the center of each medallion, surface temperature and oil temperature. Temperatures for each medallion were measured using a type K thermocouple, TENMARS model TM-747 DU, with four simultaneous measurement channels. The equipment was duly calibrated. Instructions for well-done degree (BEMPA), stablished by Technique 1, included cooking inside an oven after grilling was complete. In that case, medallions were put into a pre-heated oven at 180 °C for 9 min, and thermocouples were inserted at the beginning of the thermic process at the center and at the surface of the medallion, as described above.

2. Materials and methods 2.1. Intrinsic characteristics and medallion preparation

2.2. Inoculations and microbiological analyses

Chilled tenderloin (psoas major muscle) pieces were acquired at a butcher shop in the city of Porto Alegre/RS, Southern Brazil. After purchase, pieces were transported refrigerated (< 7 °C) inside thermal boxes to the Food Microbiology and Food Control Laboratory (ICTA/ UFRGS). Pieces were stored at refrigeration temperatures (4 ± 2 °C) before processing and analysis. A total of thirty tenderloin pieces (75.5 ± 0.1 cm3, diameter

An Escherichia coli pool was used to artificially contaminate beef tenderloin medallions. E. coli was chosen as an bacterial indicator in order to assess the effects of heat treatment on microbial inactivation in grilled tenderloin and because it can be found on beef (Forsythe, 2010; N. da Silva et al., 2017). The pool was composed by the following strains: E. coli CQ (isolated from a hot-dog sold in the streets of Porto 2

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Alegre, Brazil), E. coli DH5-α (laboratory strain), and E. coli ATCC 25922 and E. coli ATCC 8739, which belong to the bacterial cultures collection of the Food Microbiology and Food Control Laboratory (ICTA/UFRGS). Each E. coli strain was activated by inoculating an isolated colony into two 10 mL BHI tubes. Tubes were then incubated at 37 °C for 18–24 h. Subsequently, each tube of each culture was centrifuged (Hettich Mikro 120 centrifuge) at room temperature for 5 min at 14.000 rpm. The supernatant was discarded and the pellet was washed with 2 mL of 0.1% peptone water (Oxoid, Hampshire, England). This process was repeated twice. Cells of each tube were resuspended in 10 mL of 0.1% peptone water and the contents of both 10 mL tubes of each strain were added into a 1000 mL Beaker, which was added with 820 mL of 0.1% peptone water, completing 900 mL. Each medallion was put into this E. coli suspension for 2 min. The goal was to reach an initial inoculum of approximately 6–8 log CFU/g, as defined by the International Commission on Microbiological Specifications for Foods 8 (ICMSF, 2011). After artificial contamination, medallions were subjected to heat treatment, individually for each degree of doneness and separately for each technique investigated. Samples weighting 25 g were collected at each degree of doneness for each technique used. Those samples were diluted into 225 mL of 0.1% peptone water, generating a 10−1 dilution, which was then homogenized in Stomacher equipment (Stomacher 400, Seward London Clinical) for 5 min. Subsequently, decimal serial dilutions were carried out, according to the expected contamination for each degree of doneness. One-milliliter aliquots from the last three dilutions from each sample was plated on VRBA (Merck, Darmstadt, Germany) added of MUG (Oxoid, Hampshire, England) using the pour plate technique. At the same time, 1 mL aliquots were placed on nonselective BHI medium (Himedia, Mumbai, India) using the spread plate method. Petri dishes containing VRBA + MUG, as well as the dishes containing BHI were incubated at 37 °C/18/24 h. Colony-forming units (CFU) were counted after incubation. Colonies on VRBA + MUG plates were counted inside a 365 nm wavelength light chamber in order to identify E. coli (Davidson, Roth, & Gambrel-Lenarz, 2004). All bacterial counts were done at least three times in 3 replicas per variable (technique and frying time). Results were expressed in colonyforming units per gram of sample logarithms (log CFU/g ± Standard Deviation).

The software includes nine different mathematical models that were chosen by the user according to the results obtained in the investigation. The adequacy criterium is R2 closest to 1,0000. 2.4. Microbiota analysis Microbiota on beef tenderloin pieces was assessed before and after different grilling times by proteomic analysis through Matrix-Assisted Laser and Time-Of-Flight desorption/ionization mass spectrometry (MALDI-TOF/MS), model Autoflex Speed (Bruker Corporation, Bremen, Germany). The analyses were carried out in the National Agriculture Laboratory (LANAGRO/RS). MALDI-TOF/MS was performed in order to identify bacterial microbiota initially present on the raw beef, as well as to identify possible bacterial survivors after beef medallions processing. All isolated colonies obtained from the total mesophilic count method (Silva et al., 2017) performed on BHI plates were collected and inoculated on other BHI plates incubated at 37 °C for about 30–48 h. After grow, colonies were transported inside an isothermal container to be analyzed by MALDI-TOF/MS equipment at LANAGRO/RS. Samples were prepared through two protocols. The first one was directly loading colonies onto MALDI–TOF/MS equipment plates’ spots (Protocol 1). The second one was used to induce cell wall lysis (Protocol 2). Both Protocols followed the guidelines of the equipment manufacturer. To undertake MALDI-TOF/MS analyses under the direct loading protocol, colonies were transferred one by one through a sterilized inoculation loop directly onto the metallic plate reading spots in the MALDI-TOF/MS device. Colonies producing Identification scores below 2000 were processed by the cell wall breaking protocol (2). In those cases, colonies were transferred to Eppendorf tubes containing 300 μL deionized water. Subsequently, each sample was homogenized by pipetting and 900 μL of ethanol was added. The contents of the tubes were mixed, and the sample was centrifuged at a speed of ≥13,000 rpm for 2 min. The resulting ethanol-pellet complex was dried at room temperature for two to 3 min. Right after, formic acid 70% (25 μL) was added to the precipitate and mixed using a pipette or vortex. Pure acetonitrile (25 μL) was added to the pellet, mixed, and centrifuged for 2 min at a speed of ≥13,000 rpm, inducing cell lysis. After the procedure, 1 μL of the supernatant was loaded onto the metal plaque spots and left to dry in a flow cabinet. For both Protocols (1 or 2), 1 μL of matrix solution (α-cyano-4-hidroxycinnamic acid) was added over the dried material on metallic plate spots, and co-crystallization between the analyte and the matrix on site occurred. Drying happened under air flow, in a cabinet. Subsequently, the plate was put into the equipment and subjected to small laser pulses in a vacuum. Thus, the sample was ionized. According to the time-of-flight (TOF) from the analyte to the detector at the end of the vacuum tube, spectrum characteristics were generated (unique for each microorganism). This allowed microorganisms to be identified at species level, or in some cases genus level. Each analyte was loaded three times onto the plate's spots in order to confirm the results. Three spots in each metallic plate were loaded with calibration solution (Protein Standard 1, Bruker Daltonics) to validate all analytical runs. The spectrum was analyzed using the MALDI Biotyper Realtime Classification Wizard automation control, together with the Bruker Taxonomy Software MALDI Biotyper 3.1 (Bruker Daltonics) database.

2.3. Modeling of inactivation kinetic parameters Microbial inactivation of artificially contaminated beef tenderloin medallions was modeled using GInaFiT software, is a freeware add-in for Microsoft® Excel, aiming to bridge a gap between researchers who develop predictive modeling approaches and end users in the food industry or research groups that are disposable with the analysis of nonlinear regression (Geeraerd, Valdramidis, & Van Impe, 2005). The quality of models generated was assessed by the coefficient of determination (R2, Equation (1)). This coefficient calculates the general prediction measurement of the developed model and the root mean square error (RMSE, Equation (2)), which is a standard measurement of how adequate the model is in comparison with the data used.

R2 = 1

N˜ i=1

(

ˆ)

i i N˜ 2 i=1 i

Where N˜ is the number of points in the dataset; value; ŷi is the predicted value.

RMSE =

(µ n

µˆ)

(1) i

2.5. Data analysis

is the observed

For microbiology analysis purposes, data was stored in an Excel 2010 (Microsoft, USA) database and analyzed using the SPSS statistical package, version 21.0 (IBM, USA). To compare degrees of doneness and techniques, we used generalized estimating equations, Friedman tests and Bonferroni post-hoc tests. A significance level of 95% was used (P < 0.05).

(2)

Where n is the number of observations; μ is the observed value; µˆ is the predicted value. 3

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Conflicts of interest

7, pre-heated to its highest setting. Their results were similar to those found in this study regarding temperature at the center of the pieces versus time of exposure to heat treatment. According to those authors, temperature at the center reached 70 °C after 13 min of pan-frying.

Results obtained in this project did not generate any conflict of interest between collaborating entities i.e. LANAGRO and UFRGS. There was also no conflict of interest between collaborating authors.

3.3. Escherichia coli thermal inactivation

3. Results and discussion

Results of E. coli inactivation on the tenderloin beef medallions after each degree of doneness analyzed in the present study are shown in Table 4. Results indicated that E. coli total inactivation happened when Technique 1 was applied, reaching well-done degree (BEMPA) after the oven heating. Without this final oven processing 2.49 log CFU/g of E. coli still viable. The well-done degree (BEMPA) obtained by Technique 1 had a significant difference in mean counts when compared with other degrees of doneness from the same technique. From the degree of doneness APMAL, a 1 log CFU/g reduction was observed in E. coli counts. In Technique 2, the total E. coli inactivation occurred at the welldone degree (BEMPA) after subjecting medallions to grilling for 11 min and 35 s. The difference between the two techniques related to BEMPA degree of doneness can be explained because Technique 2 took almost 2-fold times to reach this degree of doneness, once it was determined when beef medallions reach internal temperatures higher than 71 °C. When Technique 2 was used, E. coli reductions of approximately 2 log CFU/g were observed at MALPA. For Technique 1, maximum reduction obtained was approximately 3.7 log CFU/g. As shown in Fig. 1, generated by GInaFit, the initial concentration of E. coli artificially inoculated onto the tenderloin beef medallions was 6.21 ± 0.09 log CFU/g. It was reduced to 2.49 ± 0.11 log CFU/g after 6 min of process on the skillet. Results generated from bacterial counts fitted to GInaFit models. Luchansky et al. (2011) assessed Shiga toxin-producing E. coli O157:H7 (ECOH) and O157:H7 (STEC) inactivation on the surfaces of five beef pieces. Microorganisms (6 log CFU/g) were artificially inoculated on the pieces and left under refrigeration for 30 min. Pieces were subsequently fractioned into units of the 2.54 cm in height which were then grilled using open fire. Results indicated that initial ECOH counts of 6.40 ± 0.22 CFU/g on raw beef were reduced to 5.19 ± 0.03 CFU/g after the central temperature of pieces reached 37.8 °C. Finally, bacterial numbers of 2.25 ± 0.59 CFU/g were observed when temperature at the center of pieces reached 71.1 °C. Initial STEC counts of 5.77 ± 0.19 CFU/g were reduced to 4.99 ± 0.32 CFU/ g when temperature in the center of pieces reached 37.8° and 2.81 ± 1.26 CFU/g when central temperature was 71.1 °C. Authors concluded that ECOH and STEC behaviors were similar and that the survival of those bacteria at temperatures exceeding 70 °C was due to non-uniform heating, as there were cold spots in the samples. These results are in agreement with our results, because E. coli survival patterns were similar and the survival of E. coli at degree

3.1. Intrinsic factors and initial microbial loads of beef tenderloin medallions Intrinsic factors of the raw beef medallions were measured. Results of water activity (aw) of 0.995 ± 0.004 (23.6 °C), pH 6.47 ± 0.02 (24.0 °C) and mesophilic total count of 5.38 ± 0.34 log CFU/g were obtained. Water activity and pH of tenderloin pieces used to produce medallions were in conformity with normal characteristics of red meat (Forsythe, 2010; ICMSF, 2011). Total mesophilic counts also were considered adequate, once counts agreed with results obtained by other studies carried out in Brazil (Becker & Kiel, 2016; J. B. da Silva et al., 2016) and abroad (Forsythe, 2010). 3.2. Thermal processing of tenderloin medallions according to two techniques (Le Cordon Bleu and IGA) For all the degrees of doneness, the oil temperatures used in the Technique 1 were higher than the oil temperatures used in Technique 2, presenting a difference greater than 58.0 °C. These differences were due to the initial intensity of fire (heat) used, which in Technique 1 was high fire (according to the stablished technique) and in Technique 2 was low fire. The results of this study identified the maximum core temperature reached at each degree of doneness for both techniques applied by insertion of thermocouples in each medallion. This is demonstrated in Table 3. In Technique 1, the highest central temperature reached was in the degrees of doneness BEMPA (fry + Oven), exceeding 78 °C in the center of the medallions. The other degrees of doneness, in this technique, reached a central temperature below 66 °C. These observations are justified by the short time of exposure to heat, as recommended by the technique, even when high fire was applied and the oil temperature was higher than that Technique 2. For Technique 2, the grilling process was sufficient to reach the central temperature above 75 °C, at the BEMPA point, as established by preparation instructions, which recommend to control the central temperature of beef pieces. In this case, the time of exposure to grilling heat was greater than 11 min, much higher than those used at Technique 1 (6 min). Lahou et al. (2015) studied beef filets prepared with butter on an electrical pan with a temperature control knob with settings from 1 to

Table 2 Olive oil temperature: average temperatures, standard deviation, median, minimum and maximum temperatures for both techniques analyzed, at each degree of doneness. MALPA = rare tenderloin, APMAL = medium-rare medallion; AOPO = medium medallion; APBEM = medium-well medallion; BEMPA = well-done medallion SD = standard deviation, SE = standard error. Temperatures in °C. Degree of Doneness

MALPA APMAL AOPO APBEM BEMPA

Technique 1

Technique 2

Difference

P

Average ± SD

Median

Minimum

Maximum

Average ± SD

Median

Minimum

Maximum

Average ± SE

Significance interval Inferior Superior

238.6 230.1 238.4 244.1 256.3

237.8 230.4 240.8 246.5 256.6

215.8 203.7 202.5 218.7 233.3

268.4 266.6 264.7 265.3 272.9

175.9 178.6 160.9 168.0 171.7

176.3 179.1 159.4 167.7 170.5

150.6 154.1 150.1 150.4 150.4

197.8 192.3 179,0 189.1 190.8

62.3 58.0 71.7 77.7 80.4

58.5 55.3 69.9 75.8 79.5

± ± ± ± ±

10.8 14.0 11.7 11.7 9.7

± ± ± ± ±

10.1 8.4 7.1 8.8 8.8

± ± ± ± ±

1.9 1.4 0.9 1.0 0.5

Significant differences (p < 0.05) were found between the average temperatures of oils used in Technique 1 and 2 (Table 2). 4

66.0 60.7 73.4 79.6 81.4

< 0,001 < 0,001 < 0,001 < 0,001 < 0,001

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Table 3 Processing time and maximum temperature at the center of beef tenderloin medallions (n = 9) subjected to different thermal processing stablished by two different gastronomic techniques. MALPA = rare medallion, APMAL = medium-rare medallion; AOPO = medium medallion; APBEM = medium-well medallion and BEMPA = well-done medallion. T. max. = maximum temperature.

Temperature and time parameters

Degrees of doneness MALPA

Max. process time T Max. at the center Max. process time T Max. at the center

APMAL

AOPO

APBEM

BEMPA

BEMPA BEMPA (Grill + (FryOven) + Oven)

6 min

15 min

Technique 1- Le Cordon Bleu 2 min 3 min 4 min

1 min o

o

11.5 C

o

19.3 C

Technique 2- IGA 6 min, 5 sec 7 min, 45 sec

5 min, 49 sec o

o

52.1 C

o

30.7 C

o

65.5 C

8 min, 17 sec

11 min, 35 sec

o

56.2 C

o

35.8 C

o

63.6 C

78.6 C

o

68.2 C

75.2 C

Table 4 Log E. coli counts/g after thermal processing of tenderloin medallions (n = 9) subjected to different degrees of doneness, stablished by two gastronomic techniques. MALPA = rare medallion, APMAL = medium-rare medallion; AOPO = medium medallion; APBEM = medium-well medallion; BEMPA = well-done medallion. BEMPA (grilled + oven) = well-done medallion subjected to grilling and oven. Results in log CFU/g. SD = standard deviation. n.d. = not detected.

Degrees of doneness Technique used

RAW BEEF

MALPA

APMAL

AOPO

APBEM

BEMPA

BEMPA (Fry ++Oven) (Grill Oven)

P

Average±SD Average±SD Average±SD Average±SD Average±SD Average±SD Average±SD Technique 1 Technique 2 P

a

6.21 ± 0.09 a

5.56 ± 0.50 0.002

ab

abc

ab

ab

5.39 ± 0.04 5.02 3.59 ± 0.14 <0.001

abc

± 0.10 4.53

2.99 ± 0.40 <0.001

ab

bc

± 0.14 3.76 ± 0.15

2.17 ± 0.13 <0.001

ab

1.74 ± 0.38 <0.001

c

2.49 ± 0.12 c

n.d. <0.001

n.d.

d

<0.001 0.011

Generalized estimating equations, Friedman tests, and Bonferroni post-hoc tests.

BEMPA, reached after using Technique 1 (without the use of oven), also can be explained by cold spots especially those located on the lateral of tenderloin beef medallions, which did not get in contact with skillet and olive oil. For the grilling technique which temperature at the center piece was greater than 78 °C, differences found between this study and the one carried out by Luchansky et al. (2011) may be related to the fact that the heat process applied by them was non-uniform. The heat

generated by the process of cooking inside oven (Technique 1) was uniform, being able to inactivate surviving E. coli population after grilling process. However, we highlight that at home kitchens and at many restaurants, this additional oven heating is not an usual practice and highly contaminated beef tenderloins medallions may still be contaminated by E. coli and other potential pathogens. McMinn et al. (2018) carried out a study to evaluate the thermal Fig. 1. Escherichia coli inactivation after tenderloin medallions (n = 9) were subjected to heat processing, according to degree of doneness, applying Technique 1, which prescribes processing time control. Generated by GInaFIT, points represent the average results from experiments and the line represents the predicted model. R2 = 0.9899 and RMSE = 0.1318. Time (in minutes) for each degree of doneness is as follows: 0,00 = CRU (raw beef); 1,00 = MALPA (rare medallion); 2,00 = APMAL (medium-rare medallion); 3,00 = AOPO (medium medallion); 4,00 = APBEM (medium-well medallion) and 6,00 = BEMPA (well-done medallion). Standard deviations were less than 0,15 log CFU/g for all processing times.

5

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Fig. 2. E. coli inactivation on tenderloin medallions (n = 9) subjected to frying process, with degree of doneness monitored by temperature at the center (Technique 2 - IGA), generated by GInaFIT. Points represent the average results from experiments and the line represents the predicted model. R2 = 0.9888 and RMSE = 0.2229. Standard deviations were less than 0.45 log CFU/g for all processing times. Time 0 indicates raw beef and the numbers at the arrows indicate: 1 = MALPA (rare medallion); 2 = (APMAL) medium-rare medallion; 3 = AOPO (medium medallion); 4 = APBEM (medium-well medallion) and 5 = BEMPA (well-done medallion).

the kinetic parameters for the model were: kmax = 1.39 ± 0.06 min−1, N0 = 6.18 ± 0.09, with R2 = 0.9899 and RMSE = 0.1318.

resistance of E. coli, Salmonella and Listeria monocytogenes in different meats. The process was conducted in water-impermeable pouches, vacuum-packaged, and heated at 54.4, 60.0, 65.6, or 71.1 °C in a water bath. The results were compared to the guidelines of the USDA (United States Department of Agriculture) - FSIS (Food Safety and Inspection Service) for safe thermal processing, that requires a ≥6.5 log reduction of Salmonella spp. in RTE cooked beef, roast beef, and cooked corned beef. The results indicated that cooking temperatures of 60 °C or greater inactivate Salmonella and shiga toxin-producing E. coli, but a longer exposure time would be required to obtain a similar reduction of L. monocytogenes, ratifying the recommendations of the Institution. These results corroborate with our research, even though it is another form of thermal processing. Gill, Tamber, and Yang (2019) evaluated the relative response of generic Escherichia coli (GEC), Shiga toxin-producing E. coli (STEC) and Salmonella enterica when subjected to heat, alkaline or acid treatment. Focusing here GEC strains, this strains were obtained from beef carcasses and cuts from a small meat plant, after washing the carcass with cold water and strains obtained from a large meat industry, after multiple stages of decontamination: washing of carcass with 1.5% NaOH, spraying of carcasses with 5% of lactic acid and pasteurisation of carcasses with steam at > 90 °C. These strains were cultured in BHI and subjected to three different treatments: thermal (bath at 60 °C for 2min), lactic acid (pH 2.9) for 1 h at 4 °C or NaOH (pH 11.0), for 2 h at 4 °C. The results showed that the GEC reduction was 2.3–3.8 log CFU/ mL when subjected to heat treatment, a reduction of 0.7–2.2 log CFU/ mL for the alkaline treatment and 0, 7 to 1.2 log CFU/mL for acid treatment. The authors concluded that, in general, the model suggests that the strategies of heat decontamination or pH changes in meat plants are not associated with increased resistance among the E. coli species of these environments. This study confirms that an effective heat treatment reduces the contamination by generic E. coli, corroborating with the findings in our study. Heat is still the best way to reduce E. coli in this food matrix (meat). E. coli concentration data obtained at each degree of doneness through the grilling process were fed to GInaFiT software, which then simulated microbial reduction in the process. Through the software's linear regression model proposed by Bigelow and Esty (1920), we were able to assess the adequacy of experimental data by calculating their R2 (coefficient of determination) and RMSE (root mean square error). This simulation allowed us to obtain an equation (Equation (3)) and

log N = log N 0

kmax × t ln 10

(3)

Employing the obtained coefficients, the equation may be expressed as follows:

log N = 6.18

(4)

0.60 × t

Applying the technique prescribed by IGA (Technique 2) and with the microbiology data obtained, we observed that initial E. coli contamination was 5.56 ± 0.50 log CFU/g, however no E. coli was observed after the longest processing time (11 min and 35 s), as generated by GINAFIT and shown in Fig. 2. Results for Technique 2 were modeled in GInaFIT to predict kinetic parameters by describing the inactivation behavior for E. coli versus processing. Therefore, the best model (statistically assessed by R2 and RMSE) that fit the data was the one proposed by Geeraerd, Herremans, and Van Impe (2000). This model associates a linear regression to a “shoulder” or curve at the beginning of the bacterial decline process. The model provides the line presented in Fig. 2, which refers to the estimate of E. coli reduction, while the points refer to values obtained in the experiments. Kinetic parameters obtained by GInaFIT were Kmax = 1.22 ± 0.07 min−1, N0 = 5.53 ± 0.22 log CFU/g, with R2 = 0.9888 and RMSE = 0.2229, confirming the adequacy of the model to our data. Equation (5) describes the mathematical model for the inactivation curve in the experiment, while Equation (6) is the model employing the coefficients generated by the software. Therefore, the model confirmed the experimental data.

log N = log N 0 log N = 5.53

log e (kmax × SI ) kmax × t + kmax × SI ln 10 (1 + e 1) × e kmax × t

0.53 × t +

0.14 e1.22 × t

(5) (6)

Analyzing equations (4) and (6), we obtained a D value (time needed to reduce 90% of the bacterial population) of D = 1.67 min for Technique 1, and for Technique 2, D = 3.81. The difference in the D values is justified by the fact that in Technique 1 the control of the final degree of doneness was carried out through the process time, which was lower than the process times applied in the Technique 2, that define the 6

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Fig. 3. Microbiota of raw beef tenderloin medallions and of beef tenderloin medallions after heat treatment though frying, at different degrees of doneness. Highlighted Escherichia coli refers to artificial inoculation. Degrees of doneness: rare (MALPA), medium-rare (APMAL), medium (AOPO), medium-well (APBEM), and well-done (BEMPA).

control of the final degree of doneness by the central temperature control. Good quality meats generally have contaminations much lower than the inoculation goal of our experiments, i.e. 106 CFU/g. In general, raw beef from adequate suppliers have E. coli counts < 102 CFU/g on the surfaces (D. C. da Silva, Faria, Pereira, Duarte, & Nogueira, 2016). Therefore, assuming these counts in raw beef and the thermal reductions observed in our study, it would be necessary about 3 min for Technique 1 and 8 min for Technique 2 to eliminate 2 log CFU/g of E. coli on the beef medallions.

quadrants identified on Petri dishes containing BHI agar, which were incubated at 37 °C/24/36 h. Therefore, samples were analyzed though MALDI-TOF and the results are shown in Fig. 3. According to those results, we could verify the great diversity in natural viable microbiota present on raw beef. Among the main genera identified were Acinetobacter, Aeromonas, Alcaligenes, Carnobacterium, Lactobacillus, Micrococcus, and Pseudomonas. All those microorganisms are associated to chilled meat deterioration (Felipe, 2008), however we did not observe any organoleptic alteration in tenderloin pieces used in the experiments (results not shown). We further identified microorganisms that indicated fecal or environmental contamination, belonging to the Enterobacteriaceae family. We were able to identify the genera Citrobacter, Enterobacter, Hafnia, Serratia and Raoultella. In general, those micro-organisms cause deterioration in chilled meat. They are considered non-pathogenic, but may act as opportunistic pathogens (Barbosa, 2016), which confirms the need for adequate heat treatment before meat consumption and validation of the thermal processes, depending on the microbiological quality of beef.

3.4. Assessment of natural microbiota on beef tenderloin medallions We further assessed the natural microbiota on the beef tenderloin medallions before, during and after heat treatments. All colonies obtained from the total mesophilic counting technique (N. da Silva et al., 2017) in non-selective enrichment medium, BHI, at dilutions of 10−4 to 10−6 were collected. The sampled colonies were seeded into small 7

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Genera Bacillus and Staphylococcus were also identified on raw beef. Identified species presented in Fig. 3 are not pathogenic, but they may indicate raw material contamination during packaging, cooling, cooling maintenance and/or meat manipulation processes. MALDI-TOF/MS results demonstrated the presence of Acinetobacter jonhsonii in all degrees of doneness tested. Furthermore, bacteria of the genera Bacillus, Lactobacillus, Micrococcus, Pseudomonas, Serratia, Staphylococcus were also found at all degrees of doneness after heat treatment, including the longest processing time (well-done, BEMPA). These results indicated that those genera and species were resistant to heat treatment. Another possible explanation is due to the immersion inoculation method. This could result in a considerable penetration of the bacteria (including cells present on the surface of the raw material) into the meat. In this way, it is possible to explain, in part, the observed survival of some bacterial species known to be sensitive to heat (such as Gram-negative bacteria detected among survivors). This study did not identify pathogenic bacteria of the genus Salmonella or Listeria monocytogenes. This indicates that the analyzed meat pieces came from adequate suppliers. The number of bacterial genus identified after each degree of doneness were variable and this can be explained because different pieces of beef tenderloins were analyzed at each replica of experiments. These results have no intention to quantify or demonstrate the most resistant genus present on medallions, but only identify thermoduric microorganisms which resisted to the different processing times. A study conducted by Hilgarth, Behr, and Vogel (2017) also used MALDI–TOF/MS spectrometry analysis to monitor deteriorating microorganism growth and to differentiate psychrophilic and psychrotrophic microorganisms on beef packed under a modified atmosphere. Species found with most incidence during product shelf-life monitoring (21 days at 4–10 °C) were Hafnia alvei, Acinetobacter jonhsonii, Pseudomonas spp., Lactococcus sp. and Serratia liquefaciens. These results are similar to data found in our study. MALDI-TOF/MS results are in accordance with results found by microbiology analyses, since E. coli was identified at all the grilled degrees expect well-done degree (BEMPA).

result in great differences in microbial lethality of the grilling process and, therefore, the surviving microbiota. Natural microbiota demonstrated to be able to survive to different degrees of doneness on tenderloin medallions, demonstrating that even well-done beef tenderloins still containing viable bacterial contamination. Acknowledgements We would like to thank the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES) for the research grant and LANAGRO for allowing the use of MALDI-TOF/MS equipment. References Barbosa, T. A. (2016). Epidemiologia da colonização e infecção microbiana em Unidade de terapia intensiva neonatal: Abordagem clínica e molecular. Universidade estadual paulista (UNESP). February 29 . Retrieved from https://repositorio.unesp.br/handle/11449/ 136351. Becker, A. K., & Kiel, G. (2016). Análise microbiológica de carne bovina in natura comercializada em supermercados de Cascavel - PR. Revista Thêma et Scientia, 1(2), 149–155. Retrieved from http://www.themaetscientia.fag.edu.br/index.php/RTES/ article/view/41. Bigelow, W. D., & Esty, J. R. (1920). The thermal death point in relation to typical thermophylic organisms. Journal of Infectious Diseases, 27(602). Brasil Resolução RDC no 216, de 15 de setembro de 2004 - Dispõe sobre Regulamento Técnico de Boas Práticas para Serviços de Alimentação (2004). Brasil: D.O.U. - diário Oficial da União; Poder Executivo, de 16 de setembro de 2004. Retrieved from http:// portal.anvisa.gov.br/documents/33916/388704/RESOLU%25C3%2587%25C3% 2583O-RDC%2BN%2B216%2BDE%2B15%2BDE%2BSETEMBRO%2BDE%2B2004. pdf/23701496-925d-4d4d-99aa-9d479b316c4b. Davidson, P. M., Roth, L. A., & Gambrel-Lenarz, S. A. (2004). Chapter 7 coliform and other indicator bacteria. In J. Bruhn (Ed.). Standard methods for the examination of dairy productsAmerican Public Health Associationhttps://doi.org/10.2105/ 9780875530024ch07. Felipe, L. M. (2008). Associação de bactérias da família Enterobacteriaceae E Clostridium estertheticum com a deterioração“ blown pack ” em cortes cárneos embalados à vácuo. Universidade Estadual Paulista. Forsythe, S. J. (2010). The microbiology of safe food (2nd ed.). West Sussex: Blackwell Publishing Ltd. Geeraerd, A. H., Herremans, C. H., & Van Impe, J. F. (2000). Structural model requirements to describe microbial inactivation during a mild heat treatment. International Journal of Food Microbiology, 59(3), 185–209. Geeraerd, A. H., Valdramidis, V. P., & Van Impe, J. F. (2005). GInaFiT, a freeware tool to assess non-log-linear microbial survivor curves. International Journal of Food Microbiology, 102(1), 95–105. https://doi.org/10.1016/j.ijfoodmicro.2004.11.038. Gill, A., Tamber, S., & Yang, X. (2019). Relative response of populations of Escherichia coli and Salmonella enterica to exposure to thermal, alkaline and acidic treatments. International Journal of Food Microbiology, 293, 94–101. Government of Canada (2015). Safe internal cooking temperatures Chart (Food safety General food safety tips). Canadá: Health Canada - CFIA - Canadian Food Inspection Agency. Retrieved from https://www.canada.ca/en/health-canada/services/generalfood-safety-tips/safe-internal-cooking-temperatures-chart.html. Hilgarth, M., Behr, J., & Vogel, R. F. (2017). Monitoring of spoilage‐associated microbiota on modified atmosphere packaged beef and differentiation of psychrophilic and psychrotrophic strains. Journal of Applied Microbiology. https://doi.org/10.1111/jam. 13669. ICMSF (2011). Microorganisms in foods 8 : Use of data for assessing process control and product acceptance. ICMSF - international commission on microbiological Specifications for foods. New York: Springer-Verlag New York Inc Chapter 2 and 8;. Lahou, E., Wang, X., De Boeck, E., Verguldt, E., Geeraerd, A., Devlieghere, F., et al. (2015). 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4. Conclusions Grilling techniques tested in this study demonstrated differences regarding E. coli inactivation. Results indicated that controlling the temperature at the center of beef pieces (Technique 2) was more effective to inactivate E. coli than controlling processing time (Technique 1). We observed greater E. coli reduction through grilling when the Technique 2 was used, as the heat treatment lasts longer, and the center of the medallions reached temperatures higher than 70 °C. Using the oven heating to complete the BEMPA treatment as prescribed by Technique 1, E. coli inactivation also was complete, however many restaurants do not used this further heat treatment. Neither technique completely reduced E. coli contamination at degrees of doneness below well-done degree, highlighting the necessity of using tenderloin beef pieces from adequate suppliers, especially when clients ask for other degrees of doneness different from well-done. Aiming to comply with some legal requirements in various countries (Brasil, 2004; Government of Canada, 2015; USDA, 2015) in order to be considered safe, internal parts of beef and other foods shall reach at least 70 °C, or be subject to other time and temperatures patterns proved as safe. Tenderloin beef medallions tested in this study did not reached these temperatures, except the well-done ones, which could suggest they were not safe, especially if expressively contaminated beef are processed. Based on this, restaurants should use good quality beef and validate their own thermal processes considering the quality of their suppliers and receipts used. Kinetic parameters results indicate a direct correlation between E. coli survival and processing time to grilling the medallions. D values indicated that apparently differences in processing temperatures may 8

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C.R. Peixoto, et al. 2C+San+Antonio%2C+Texas&btnG=. Silva, D. C. da, Faria, P. B., Pereira, A. de A., Duarte, W. F., & Nogueira, T. M. (2016a). Parâmetros microbiológicos em diferentes cortes de carne bovina resfriada. Higiene Alimentar, 30(260/261), 116–120. Retrieved from http://docs.bvsalud.org/biblioref/ 2016/11/2789/260-261-sitecompressed-116-120.pdf. Silva, N. da, Junqueira, V. C. A., Silveira, N. F. de A., Taniwaki, M. H., Gomes, R. A. R., & Okazaki, M. M. (2017). Manual de Métodos de Análise Microbiológica de Alimentos e Água (5a ed.). São Paulo: Blucher. Silva, J. B. da, Prazeres, A. R.dos, Oliveira, A. C., do, S. de, Dantas, V. V., Barros, M. C. de S., et al. (2016b). Avaliação higiênico-sanitária de estabelecimentos comerciais e análise de micro-organismos indicadores em amostras de carne bovina (coxão mole) in natura comercializadas em mercados públicos. 0 Revista Do Instituto Adolfo Lutz, 75, 01–07. Retrieved from http://revistas.bvs-vet.org.br/rialutz/article/view/37122.

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