Food Control 115 (2020) 107297

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Service temperature preservation approach for food safety: Microbiological T

evaluation of ready meals
Annalisa Riccia, Francesco Martellia, Roberta Razzanob,c, Davide Cassib,c, Camilla Lazzia,
Erasmo Neviania, Valentina Berninia,∗
a
Department of Food Science, University of Parma, Parco Area delle Scienze 49/A, 43124, Parma, Italy
b
Department of Mathematical, Physical and Computer Sciences, Parco Area delle Scienze 7/A, 43124, Parma, Italy
c
Future Cooking Lab, Parco Area delle Scienze 7/A, 43124, Parma, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: Microbial food contamination can take place in any step of food production from farms to factories and to retail,
Microbiological food contamination food services and storage, originating from different sources such as raw materials, operators and environmental
High temperature storage conditions of manufacturing plant. To avoid the range of temperature called “danger zone” (5°C–57 °C), oc-
Microbiological challenge test curring during food cooling, and to block microbial contamination post cooking, food service temperature
Food pathogens
preservation was evaluated in this study by an unconventional approach. This consists in the maintenance of
Thermal abuse
ready meals at temperatures above 60 °C, for long periods, and not just for less than 2 h as commonly used,
before their consumption. Applying this preservation method to different food preparations, the pathogenic
contamination was controlled. In particular, L. monocytogenes was absent in 25 g in all samples, and E. coli,
included O 157:H7, was under the detection threshold (1 Log CFU/g). Furthermore, different thermal abuses
were simulated and, overall, service temperature preservation maintained the microbiological contamination
under control. The microbial concentration did not increase in the most of the tested cases. Only S. aureus and B.
cereus were observed in concentration lower than 1.5 Log CFU/g and only after 24 h at room temperature some
critical situations were observed due to their presence near to 5 Log CFU/g. Microbiological challenge tests,
simulating food contaminations after preparation, were also performed to monitor microbial behaviour during
service temperature preservation. So, this work proposed a valid alternative to cold storage ensuring safety food
products stored with service temperature preservation.

1. Introduction “temperature danger zone”. For this reason, to well manage food after
cooking, a two-stage cooling method is commonly applied. The first
The increasing occurrence of foodborne illness caused by patho- step consists in cooling the product from 60 °C to 21 °C within 2 h,
genic microorganisms still represents a threat for consumers despite the instead, in the second step, the cooling occurs from 21 °C to 5 °C or
great attention to food safety and the continuous improvement in good below within 4 h (FDA, 2017). Among the just mentioned steps, the first
manufacturing practices made by food producers (Tajkarimia, is the most critical since in the temperature ranged from 51 °C to 21 °C
Ibrahima, & Cliver, 2010). Seventy percent of worldwide foodborne the microbial growth is generally faster than observed at lower tem-
illnesses is estimated to be related to food service establishments (Lund, peratures.
2015; Møller et al., 2015). In particular, the incorrected management of To avoid the microbial multiplication, the cooling speed is a critical
temperature, during heat treatment and food storage is the main cause point which is affected by many factors such as i) the initial food
of foodborne illnesses such as observed in U.K. (2005) and Australia temperature, ii) size and food density, iii) the materials/size of con-
(2010) (Giraudon et al., 2009; Liu et al., 2012). tainers used for its preparation (UME, 2018).
Food related microorganisms and, among them, pathogens, can In the USA, between 1998 and 2008, improper cooling practices
grow in a temperature range from 5 °C to 57 °C (U.S. Food Code, 2017). caused 504 outbreaks linked to restaurants and delis, showing how the
To ensure food safety, during processing steps after heat treatment, cooling of hot food is a critical point for foodborne illness. Considering
food should pass as quickly as possible through this range, called the complete process, not only the cooling steps but also the reheating is

∗
Corresponding author.
E-mail address: [email protected] (V. Bernini).

https://doi.org/10.1016/j.foodcont.2020.107297
Received 17 February 2020; Received in revised form 2 April 2020; Accepted 3 April 2020
Available online 05 April 2020
0956-7135/ © 2020 Elsevier Ltd. All rights reserved.
A. Ricci, et al. Food Control 115 (2020) 107297

a critical point that should be monitored (Brown et al., 2012; UME, A, B and C were aimed to evaluate food safety implications in different
2018). Despite U.S. Food and Drug Administration (FDA) stated in 60 °C scenarios occurring during service temperature preservation, experi-
the critical limit for potentially hazardous hot foods, there are no stu- mental plan D consisted in two microbiological challenge test (MCT)
dies reporting the range of hot holding times used for food preservation. aimed to simulate pathogens behaviour in case of contamination
Vegetative cells of pathogenic bacteria will not survive to the re- (Fig. 1).
commended U.S. Food Code time/temperature combinations for
cooking. Although, food can be manipulated and inadvertent con- A) HS, CS, LS and R were considered as food models to evaluate
tamination may be introduced into cooked food from workers, custo- foodborne pathogens contamination during correct service tem-
mers or from unclean equipment (Greig, Todd, Bartleson, & Michaels, perature preservation at 70 °C. Microbiological analysis were per-
2007). To avoid this hazard, keeping food at a sufficiently high tem- formed at different times depending on the specific foods analysed.
perature could be an additional tool to prevent survival and growth of In particular, after 4, 7 and 13 days for HS and CS, 3 and 5 days for
post cooking bacterial contamination. LS while after 14 days for R (Fig. 1A).
In order to avoid the range of temperature called “danger zone”, B) WR, RV, RP, TS and MS were used as food models to evaluate the
occurring during food cooling, and to block post cooking contamina- contamination at the end of the preservation during which a thermal
tion, an unconventional approach, consisting in the maintenance of abuse has occurred. In particular, products were preserved at 62 °C
ready meals at temperatures above 60 °C for long periods, and not just for 5 days, then maintained at room temperature for 1 day, in order
for less than 2 h as commonly used before their consumption, is pro- to simulate thermal abuse, and finally bring again to 62 °C for 1 day
posed in this study. Several meals were prepared and different micro- (Fig. 1B). The microbiological analysis were carried out at the end of
biological food safety aspects were evaluated during service tempera- storage (seventh day).
ture preservation. In particular, the presence of the main foodborne C) BS and LS were used as food models to evaluate pathogens con-
pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus tamination during a not correct service temperature preservation.
aureus, Escherichia coli, E. coli O157:H7, Bacillus cereus and Clostridium Specifically, products were correctly maintained at 70 °C for 12 h,
spp., was monitored. Three different scenarios that may occur during then a thermal abuse at room temperature was simulated for 24 h.
the preservation process were considered: i) correct service temperature Samples were monitored after 12 h at 70 °C, then after 3 h, 6 h as
preservation at 70 °C; ii) thermal abuse occurring along service tem- well as 24 h of room temperature maintenance (Fig. 1C).
perature preservation; iii) thermal abuse interrupting service tempera- D) H and CLS were used as food models to simulate, through micro-
ture preservation. Moreover, microbiological challenge tests (MCT), biological challenge test, the behaviour of foodborne pathogens, L.
simulating food contaminations after preparation, were performed to monocytogenes and E. coli, eventually food contaminants after pre-
monitor microbial behaviour during service temperature preservation. paration. Non-pathogenic strains (Listeria innocua, LMG 11387T and
Lin6, and E. coli, K88 and K9) were used to ensure operator safety
2. Material and methods since the oven where contaminated samples were stored was si-
tuated outside the microbiological laboratory. All the strains were
2.1. Food models and service temperature preservation conditions selected from the Microbial Collection of Food Microbiology Unit,
Department of Food and Drug (University of Parma, Italy) excepted
The thirteen food models considered and the related ingredients LMG 11387T, purchased from BCCM (Belgian Co-ordinated
were reported in Table 1. They were cooked and provided by Future Collections of Microorganisms of Ghent University, Belgium). The
Cooking Lab (Parma, Italy), and, once cooked, stored in proper con- strains, stored at −80 °C with the addition of glycerol, were re-
tainers. For liquid or semiliquid foods, sterilized glass containers, with vitalized twice in Tryptic Soy Broth (TSB) (Oxoid Ltd., Basingstoke,
suitable geometry to reduce the exposed surface with the atmosphere, UK) (5% inoculum) and incubated at 37 °C for 16 h, in order to
were used. The surface was then covered with a layer of olive oil in reach a bacterial concentration of 8 Log CFU mL−1. Before samples
order to reduce the oxygen content. Sterile bags for packaging were artificial contamination, the strains belonging to the same species
used for solid foods. Service temperature preservation was set in ovens were mixed in equal volume. Two sets of MCT were set up for both
(Exever, Padova, Italy) at 70 °C for food rich in connective tissues and H and CLS to evaluate the behaviour of L. innocua and E. coli se-
legumes and at 62 °C for the remaining foods. parately. Food models were contaminated with high bacterial con-
centrations (approximatively 7 Log CFU g−1). Six hamburgers,
cooked and vacuum packed, were overall considered: 4 samples
2.2. Experimental design were contaminated and 2 were kept as negative control samples.
The artificial contamination was carried out under sterile conditions
Four experimental designs were overall designed: experimental plan

Table 1
Food models provided by Future Cooking Lab and their ingredients.
Food model ID Code Ingredients

Herb Soup HS Herbs, chickpeas, olive oil, dried mushrooms, tomato sauce, celery, carrot, onion, garlic, sage, salt, pepper
Chickpea Soup CS Chickpeas, pork sausage, raw ham, onion, tomato sauce, herbs, garlic, celery, parsley, salt, pepper
Legume Soup LS Red and white beans, chickpeas, bacon, onion, celery, carrot, tomato concentrate, olive oil, salt
Risotto with Gulasch R Rice, beef meat, onion, cream, Parmigiano Reggiano grated cheese, red wine, tomato concentrate, extra virgin olive oil, sweet and spicy
paprika, flour, salt
White Rice WR Rice, seed oil, salt
Rice with Vegetables RV Rice, peas, onion, carrot, olive oil, salt
Roasted Pork RP Pork loin, bacon, salamoia bolognese, apples, white wine, carrot, onion, celery, extra virgin oil, rosemary, salt
Tomato Sauce TS Tomato, extra virgin olive oil, onion, salt
Mushroom Sauce MS Portobello mushrooms, tomato concentrate, olive oil, garlic, parsley
Barley Soup BS Barley, soya, zucchini, celery, onion, carrot, laurel, olive oil
Lentil Soup LS Red lentil, tomato pulp, onion, carrot, garlic, olive oil, cumin seeds, curcuma, laurel, salt, pepper
Hamburger H Minced beef meat, onion, pepper, salt
Cereals and Legume Soup CLS Chickpeas, beans, wheat, celery, carrot, onion, olive oil, pepper, salt

2
A. Ricci, et al. Food Control 115 (2020) 107297

Fig. 1. Experimental plans to evaluate food safety implications in service temperature preservation: evolution of microbial contamination in different scenarios
occurring during preservation (A, B and C), and simulation by microbiological challenge test of pathogens behaviour in case of contamination (D).

under a microbiological hood by drilling each pack and injecting the chromogenic media (Biomerieux, Askim, Sweden). After samples dilu-
bacterial culture (1% v/w). After injection, the contaminated sam- tion they were spread on plate surface and incubated for 18–24 h at
ples were kept 1 h under laminar flow hood in order to facilitate the 37 °C. E.coli O157:H7 colonies turn to blue-green while E. coli colonies
attachment of contaminating cells. Subsequently the holes on the ones turn to brown-red.
packaging were closed with adhesive film. Two samples were ana- Regarding B. cereus the research was aimed to vegetative and spore
lysed just after contamination (T0) and two after service tempera- forms (for the last, before plate count, the samples were treated at 80 °C
ture preservation at 62 °C for 24 h (T24). For each sampling time, for 10 min) (Abdelmassih, Planchon, Anceau, & Mahillon, 2011). For
average values ± standard deviations were reported. their enumeration Brillance Bacillus cereus agar (Oxoid, Milan, Italy)
was used (Kabir, Hsieh, Simpson, Kerdahi, & Sulaiman, 2017). Food
Regarding CLS, 500 g of product were contaminated and then ali- products were diluted before the analysis, spread on the plate, in-
quoted. Analysis were carried out just after contamination and after 4 h, cubated at 37 °C for 24 h and the presumptive B. cereus were char-
8 h and 24 h of service temperature preservation at 70 °C. Two aliquots acterised by blue-green colonies. At least, for Clostridium spp. enu-
were considered for each sampling time and two not contaminated meration Reinforced Clostridial Medium (Oxoid, Milan, Italy) was
samples were analysed as negative controls. For each sampling time, employed (Barnes & Goldberg, 1962) as follows. Samples were diluted
average values ± standard deviations were reported. before the analysis, spread on the plate, incubated at 37 °C for 24 h and
colonies of presumptive clostridia were checked.
2.3. Microbiological analysis
3. Results
Detection and quantification of L. monocytogenes, S. aureus, E. coli, E.
coli O157:H7, B. cereus and Clostridium spp. was carried out at the For the evaluation of service temperature preservation efficacy, the
scheduled time established for each experimental plan (Fig. 1A, B and concentration of pathogens was checked in different meals. First of all
1C). Regarding L. monocytogenes a qualitative determination was also HS, CS, LP, R microbial contamination was estimated after different
performed to detect cells below the limit of quantitative analysis ac- days of storage at 70 °C (Table 2). Overall, during preservation the
cording to ISO 11290–1:2017, with some modification. Briefly, in a microbial concentration was taken under control. In particular, L.
primary enrichment step, 25 g of each sample were blended in 225 mL monocytogenes resulted absent in 25 g in all samples. E. coli, included O
of Half concentrated Fraser Broth (HFB) (Oxoid, Milan, Italy) and in- 157:H7, was under the detection threshold (1 Log CFU/g) while S.
cubated at 30 °C for 24 h. Subsequently, in a second enrichment step, aureus, B. cereus and Clostridium spp. were found in low concentration
0.1 mL of the first step culture was transferred into 10 mL Fraser Broth with few variations during service temperature preservation, main-
(FB) (Oxoid Milan, Italy) and incubated at 37 °C for 48 h for a sec- taining a satisfactory quality of meals according to the UK Health
ondary enrichment. Subcultures of both enrichments were plated on Protection Agency (2009). Secondly, the presence of pathogens was
Agar Listeria Acc. To Ottaviani & Agosti (ALOA) (Biolife Italiana, Milan, investigated as a consequence of thermal abuse, consisting in a main-
Italy) and incubated at 37 °C for 48 h to check the presence of typical L. tenance at room temperature for one day, occurred during service
monocytogenes colonies. Determination of Listeria innocua was per- temperature preservation of WR, RV, LS, TS, MS (Table 3). Also in this
formed applying the ISO 11290–2:2017. case L. monocytogenes was absent in 25 g of samples and E. coli, E. coli
For coagulase-positive staphylococci and S. aureus quantification O157:H7 and Clostridium spp. were under the detection threshold. Only
the Mannitol Salt Agar medium (MSA) (Oxoid, Milan, Italy) was used as S. aureus and B. cereus, this last in vegetative and spore forms, were
suggested by the American Public Health Association (1996). Briefly, observed in concentration around 1.5 Log CFU/g or lower, demon-
each product was accurately diluted, then it was spread on MSA and strating that, even in case of a thermal abuse during service tempera-
incubated for 24–48 h at 37 °C. Presumptive coagulase positive sta- ture preservation, this treatment is able to maintain the concentration
phylococci produce colonies with bright yellow zones whilst coagulase- of pathogens under control. Finally, to better clarify the potentiality of
negative staphylococci are surrounded by a red or purple zone. E. coli this preservation method, the microbial concentration was checked
and E. coli O157:H7 were presumptively detected in E.coli O157:H7 after 12 h of service temperature preservation followed by different

3
A. Ricci, et al. Food Control 115 (2020) 107297

Table 2
Microbial contamination during storage at 70 °C in herb soup (HS), chickpea soup (CS), legume soup (LS) and risotto (R). Microbial concentration was expressed as
Log CFU/g.
Microorganism HERB SOUP CHICKPEA SOUP LEGUME SOUP RISOTTO

4 Days 7 Days 13 Days 4 Days 7 Days 13 Days 3 Days 5 Days 14 Days

L. monocytogenes < 1a < 1a < 1a < 1a < 1a < 1a < 1a < 1a < 1a
Coagulase positive Staphylococci and S. aureus <1 1.24 ± 0.34 <1 <1 <1 <1 <1 0.85 ± 0.21 <1
E. coli <1 <1 <1 <1 <1 <1 <1 <1 <1
E. coli O157:H7 <1 <1 <1 <1 <1 <1 <1 <1 <1
B. cereus <1 1.0 ± 0.0 <1 <1 1.87 ± 0.13 <1 2.48 ± 0.05 2.52 ± 0.06 <1
Clostridium spp. <1 1.0 ± 0.0 <1 <1 <1 <1 <1 <1 <1

a
Absent in 25 g.

thermal abuses at room temperature (for 3, 6 and 24 h) in BS and LS improper food handling (Kirk et al., 2015). In addition, in the last
(Table 4). During correct service temperature preservation microbial decades, globalization of food supply has caused the rapid spread of
contamination was taken under control, maintaining satisfactory or pathogens causing foodborne diseases in different countries
borderline food qualities, according to the U.K. Health Protection (Coulombier & Takkinen, 2013). L. monocytogenes, E. coli, S. aureus, B.
Agency (2009). After 3 and 6 h of thermal abuse the microbiological cereus are among the microorganisms potentially dangerous that can be
situation did not change. However, after 1 day at room temperature found in food causing human diseases.
some critical situations, potentially injurious for health, were observed Listeriosis is a relative rare but potentially severe foodborne illness
especially for the presence of S. aureus and B. cereus (Table 4). with high hospitalization and mortality. In 2016, human cases of lis-
In order to better understand what could happen if a high con- teriosis were 2536 in EU, 9.3% higher compared to 2015 (EFSA &
tamination occurs, microbial challenge tests were carried out using L. ECDC, 2017). Number remains constant also in 2018 (2549 cases) with
innocua and E. coli and the results were reported in Table 5. H and CLS a fatality rate of 15,6% (EFSA & ECDC, 2019). Instead, in USA the
were analysed before the microbial inoculum revealing a concentration Centre for Disease Control and Prevention has estimated L. mono-
of L. innocua and E. coli < 1 and < 1 Log CFU/g, respectively. Then, H cytogenes responsible for 1600 illnesses and 260 deaths annually (CDC,
was separately inoculated in order to reach an initial concentration of 2020; Scallan et al., 2011). This pathogen can contaminate food both
7.45 ± 0.20 Log CFU/g of L. innocua and of 7.20 ± 0.30 Log CFU/g from the environment or via cross contamination. Among food, ready-
of E. coli. After 24 h at 62 °C L. innocua and E. coli have shown a con- to-eat products are especially risky, in particular those of meat origin, in
centration lower than 1 Log CFU/g, reducing their concentrations more which the highest level of non-compliance were usually observed
than 6 Log cycles. The same procedure was followed for CLS. The during the processing and the retail stages (EFSA & ECDC, 2017). The
contamination of the control samples was lower than 1 Log CFU/g for recommended practices for the conservation of these foods are related
both L. innocua and E. coli. L. innocua was inoculated in order to have an to the use of cold storage (FDA, 2017), however L. monocytogenes can
initial concentration of 7.44 ± 0.06 Log CFU/g. Its concentration was grow in a range from 0 °C to 45 °C (Health Protection Agency, 2009).
checked after 4, 8 and 24 h during storage revealing a concentration Moreover, it can produce biofilm also at low temperature (4–10 °C)
lower than 1 Log CFU/g and the absence in 25 g after 4 and 24 h. Si- making difficult its elimination from different surfaces (Di Bonaventura
milarly E. coli, inoculated in order to reach an initial concentration of et al., 2008) also involved in food conservation, increasing the like-
6.65 ± 0.01 Log CFU/g, showed a reduction higher than 5 Log CFU/g lihood of cross-contamination. So, cold storage, in some cases could not
(concentration < 1 Log CFU/g after 4, 8 and 24 h at 62 °C) (Table 5). guarantee the safety of food products after their preparation. In this
study, applying service temperature preservation, a huge reduction of L.
innocua concentration, a non-pathogenic surrogate of L. monocytogenes,
4. Discussion
was observed (> 5 Log CFU/g), guaranteeing the safety of the tested
products, over 24 h.
The increasing occurrence of foodborne illnesses caused by patho-
Differently from L. monocytogenes, E. coli is commonly found in the
genic microorganisms represents a threat for consumers being an im-
intestinal tract of humans and animals and it is used as faecal indicator
portant cause of morbidity and mortality both in developed and de-
to control the hygiene status of food items (Health Protection Agency,
veloping countries.,. Food contamination can take place in any step of
2009). However, some clones acquire specific virulence factors in-
production from farms to factories and to retail, food services and
volved in a wide spectrum of diseases (Kaper, Nataro, & Mobley, 2004).
storage. In particular most of them are related to food service estab-
Among them the verocytotoxin-producing E. coli and in particular the
lishments and to an incorrect temperature management, especially
serotype O157 are involved in foodborne infection (Health Protection
during cooling, storage and re-heating (Hedberg et al., 2006). Con-
Agency, 2009). Some foods, such as raw or undercooked beef and
tamination can originate from different sources such as water, tools,

Table 3
Microbial concentration after the occurrence of a thermal abuse (room temperature for one day) during six days of hot storage at 62 °C. Microbial concentration was
expressed as Log CFU/g.
Microorganism WHITE RICE RICE WITH VEGETABLES STRIATED LOIN TOMATO SAUCE MUSHROOM SAUCE

L. monocytogenes < 1a < 1a < 1a < 1a < 1a

Coagulase positive Staphylococci and S. aureus 0.35 ± 0.49 1.15 ± 0.21 1.24 ± 0.09 <1 <1
E. coli <1 <1 <1 <1 <1
E. coli O157:H7 <1 <1 <1 <1 <1
B. cereus (cells) <1 1.39 ± 0.12 1.48 ± 0.00 <1 <1
B. cereus (spores) 1.54 ± 0.09 <1 <1 0.85 ± 0.21 <1
Clostridium spp. <1 <1 <1 <1 <1

a
Absent in 25 g.

4
A. Ricci, et al. Food Control 115 (2020) 107297

Table 4
Microbial concentration after 12 h of storage at 70 °C followed by the occurrence of a thermal abuse (room temperature for one day). Microbial concentration was
expressed as Log CFU/g. The product quality grade was assigned according to the U.K. Health Protection Agency guidelines for assessing the microbiological safety.
Microorganism 70 °C Room temperature

12 Hours Quality 3 Hours Quality 6 Hours Quality 24 Hours Quality

BARLEY L. monocytogenes < 1a Satisfactory < 1a Satisfactory < 1a Satisfactory < 1a Satisfactory
SOUP Coagulase positive 2.64 ± 0.06 Borderline 1.45 ± 0.21 Borderline 1.87 ± 0.04 Borderline 5.23 ± 0.04 Potentially injurious
Staphylococci and S. aureus for health
E. coli <1 Satisfactory <1 Satisfactory <1 Satisfactory <1 Satisfactory
E. coli O157:H7 <1 Satisfactory <1 Satisfactory <1 Satisfactory <1 Satisfactory
B. cereus (vegetative cells) 3.71 ± 0.11 Borderline 3.64 ± 0.02 Borderline 4.82 ± 0.01 Borderline >5 Potentially injurious
for health
B. cereus (spores) <1 Satisfactory 1.24 ± 0.34 Satisfactory 1.00 ± 0.00 Satisfactory 2.02 ± 0.09 Satisfactory
Clostridium spp. <1 Satisfactory <1 Satisfactory <1 Satisfactory <1 Satisfactory

LENTIL L. monocytogenes < 1a Satisfactory < 1a Satisfactory < 1a Satisfactory < 1a Satisfactory
SOUP Coagulase positive 2.23 ± 0.04 Borderline 2.26 ± 0.17 Borderline 2.15 ± 0.15 Borderline 4.48 ± 0.00 Potentially injurious
Staphylococci and S. aureus for health
E. coli <1 Satisfactory <1 Satisfactory <1 Satisfactory <1 Satisfactory
E. coli O157:H7 <1 Satisfactory <1 Satisfactory <1 Satisfactory <1 Satisfactory
B. cereus (vegetative cells) 2.54 ± 0.09 Satisfactory 2.82 ± 0.07 Satisfactory 2.64 ± 0.03 Satisfactory >5 Potentially injurious
for health
B. cereus (spores) 1.30 ± 0.43 Satisfactory 1.30 ± 0.43 Satisfactory <1 Satisfactory <1 Satisfactory
Clostridium spp. <1 Satisfactory <1 Satisfactory <1 Satisfactory <1 Satisfactory

a
Absent in 25 g.

products thereof, raw milk and raw milk products, fresh produced and correct procedure (70 °C) its concentration was taken under control,
unpasteurised fruit/vegetable juices, can be contaminated by patho- and also during a thermal abuse (room temperature) for the first 6 h no
genic E. coli becoming a risk for human health (Commission regulation, microbial multiplication was observed.
2005; Karmali, Gannon, & Sargeant, 2010). Verocytotoxin-producing B. cereus, as S. aureus, is a toxin-producing bacteria involved in
strains were found resistant to cold temperature (8 °C), maintaining or foodborne illness. Foods most frequently associated with B. cereus in-
even increasing their concentration over approximately two weeks toxication include vegetables, rice, potatoes, grains, cereals, spices,
(Massa, Goffredo, Altieri, & Natola, 2002), and since the infectious dose meat, etc. (Dodd, Aldsworth, Stein, Cliver, & Riemann, 2017; Doona &
is estimated to be really low (Karmali, 2004) an efficient treatment for Feehery, 2007). This microorganism is ubiquitous, and for this reason it
food storage is necessary. The use of service temperature preservation, is difficult to find raw materials free from spores or vegetative cells.
proposed in this study, eliminated the most of E. coli present in samples However, to cause the disease high concentrations of bacteria (5 Log
(more than 5 Log CFU/g), already after 4 h of maintenance and could CFU/g) are necessary. Usually, B. cereus related illness are associated
be successfully applied for food preservation replacing cold storage. with food holding, in particular to condition allowing the microbial
Another pathogenic species which could led to human food-poi- growth, such as during not adequate refrigeration, slow cooling or
soning is S. aureus because of the production of enterotoxins during its storage below 60 °C (Dodd et al., 2017). Moreover same B. cereus strains
growth and proliferation. To prevent foodborne disease it is necessary can be psychrotolerant and grow at low temperature (8–10 °C) (Guérin,
to keep food refrigerated (temperature lower than 10 °C) or hot (above Dargaignaratz, Broussolle, Clavel, & Nguyen-the, 2016; Guerin,
45 °C) in order to avoid microorganisms multiplication necessary for Dargaignaratz, Clavel, Broussolle, & Nguyen-the, 2017) so, for these
toxin production (concentration of 5 Log CFU/g or higher). However, microorganisms, refrigerated storage could not be an effective obstacle
the time-temperature abuse and the nutritional composition of the to growth. To overcome this risk, the employment of service tempera-
contaminated food can be critical points for S. aureus multiplication ture preservation for long period evaluated in the present study could
(Labbé & García, 2013, pp. 26–44). So, operations requiring food be an efficient alternative to the cold storage. Nevertheless, some ar-
heating or cooling should be performed as rapidly as possible, passing ticles reported that high temperature can induce spores germination
quickly through the danger zone (from 5 °C to 57 °C), to avoid the (Hornstra, de Vries, Wells-Bennik, de Vos, & Abee, 2006; Soni, Oey,
possibility of bacterial growth (Brown et al., 2012; FDA, 2017). Silcock, & Bremer, 2018). However, from the data obtained in the
Nevertheless, to avoid this range of temperature, after cooking, it could present study, spores germination was controlled during hot storage
be hypothesised to maintain meals to a service temperature higher that also when a thermal abuse occurred. As far as the food models used in
57 °C to prevent microbial multiplication, the production of toxin and our study are concerned, also food flavour and structure were main-
the consequent possible food-poisoning. The service temperature pre- tained at high quality level.
servation evaluated in the present paper could be a valid alternative to
cold storage to avoid the multiplication of S. aureus. Indeed during the

Table 5
Behaviour of L. innocua and E. coli after 4 (T4), 8 (T8) and 24 (T24) hours of preservation at 62 °C, determined by Microbiological challenge test performed on
Hamburger (H) and Cereals and Legume Soup (CLS). Values expressed as Log CFU/g.
Food model Microorganism control sample T0 T4 T8 T24 Reduction

Hamburger (H) L. innocua <1 7.45 ± 0.20 ND ND <1 >6

E. coli <1 7.20 ± 0.30 ND ND <1 >6
Cereals and Legume Soup (CLS) L. innocua <1 7.44 ± 0.06 < 1a <1 < 1a >6
E. coli <1 6.65 ± 0.01 <1 <1 <1 >5

a
Absent in 25 g; ND: not determined.

5
A. Ricci, et al. Food Control 115 (2020) 107297

5. Conclusions foods. Blackwell.

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draft. Francesco Martelli: Investigation. Roberta Razzano: Resources. two standard and two chromogenic selective media for optimal growth and enu-
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Authors declare that no conflict of interests exists. All authors have (2015). World health organization estimates of the global and regional disease
approved the final article. burden of 22 foodborne bacterial, Protozoal, and viral diseases, 2010: A data
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