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Procedia Food Science 7 (2016) 17 – 20

9th International Conference on Predictive Modelling in Food

Contribution of Enterobacteriaceae to sensory characteristics in soft

cheeses made from raw milk
Magnus Westlinga*, Marie-Louise Danielsson-Thama, Jana Jassb, Asgeir Nilsena, Åsa
Öströma, Wilhelm Thama
a
Hospitality, Culinary Arts and Meal Science, Örebro University, Sörälgsvägen 1–2, 712 60 Grythyttan, Sweden
b
School of Science and Technology, Örebro University, Fakultetsgatan 1, 701 82 Örebro, Sweden

Abstract

Microbiological and sensory methods were used to analyze 22 soft cheeses, of which 19 were made from raw milk, one was
made from both raw and pasteurized milk and two were made from pasteurized milk. Moderate correlations (r-value 0.5–0.6 and
p-value <0.01–0.05) were found between the levels of Enterobacteriaceae 37 °C and the intensity of the sensory characteristics
“bitter”, “metallic”, “pungent”, “manure”
and “ammonia”. The present study indicates that it is possible to predict high levels of
Enterobacteriaceae in soft cheeses made from raw milk using only the human senses (odor and taste).
© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
([Link]
Peer-review under responsibility of Department of Food Science, Faculty of Food Engineering, University of Campinas.
Keywords: soft cheeses; pasteurization; sensory profile; Enterobacteriaceae; raw milk.

1. Introduction

Soft cheeses made from raw milk contribute a diversity of flavors that some consumers perceive positively [1]
and also associate with artisanal production [2]. However, several food-borne outbreaks have been traced back to
such cheeses [3, 4, 5]. Microbiological limits are used to determine if the food is satisfactory from a public health
perspective. The European Union (EU) has set limits for the number of coagulase-positive staphylococci per gram
of cheese for cheeses made from raw milk [6]. There are also limits for the number of Listeria monocytogenes – up
to 99 colony-forming units (CFU) per gram of food is generally accepted [6].
Cheeses made from raw milk have a higher flavor intensity than cheeses made from pasteurized milk [7, 8, 9].
Furthermore, numerous studies have shown that the production of volatile compounds varies considerably between
bacterial isolates and is strain dependent [10, 11, 12, 13]. Cheese made from raw milk is therefore particularly
complex due to the varying composition of microbes [14, 15]. Most of the studies that compare flavor compounds
and bacteria in cheese use chemical analytical instruments such as gas chromatography and mass spectrometry [12].
In contrast, our study is based on quantitative descriptive sensory methods. Sensory methods study the sensory
attributes of products, e.g. cheese, giving a profile consisting of basic taste, flavor and/or texture for the analyzed
product. This profiling is carried out by selected and trained assessors, which form a panel with the purpose of

* Corresponding author. Tel.: +46 (0)19-30 20 14; fax: +46 (0)591-144 43.
E-mail address: [Link]@[Link]

2211-601X © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
([Link]
Peer-review under responsibility of Department of Food Science, Faculty of Food Engineering, University of Campinas.
doi:10.1016/[Link].2016.02.075
18 Magnus Westling et al. / Procedia Food Science 7 (2016) 17 – 20

objectively evaluating the products [16].

The formation of flavor compounds due to microbial metabolism during ripening may be grouped into primary
and secondary metabolic pathways. The primary metabolic pathways include lipolysis, proteolysis and metabolism
of residual lactose, lactate and citrate [17]. Free fatty acids can be precursors to flavor compounds such as ketones,
acids, alcohols and esters [10]. These flavors are especially important in soft cheeses [18]. Peptides and amino acids
do not generally impact the flavor characteristics in cheese, other than the basic tastes such as bitter [18]. Further
conversion is required for flavor formation [19]. Metabolism of residual lactose and of lactate and citrate is a rapid
process during the early stages of ripening [17]. Further conversion can contribute to various flavor compounds such
as acetaldehyde, diacetyl, acetoin and acetate [19]. These flavors are especially important in yoghurt [18]. The
secondary metabolic pathways include metabolism of fatty acids and of amino acids [17]. Amino acids are degraded
through transamination reactions and various flavor compounds are then produced from one or two additional
reactions [20]. The flavor compounds resulting from this process have significant impact on the resulting flavor in
cheese [13]. For example, some acids can contribute to the sensory characteristics “rancid”, “putrid”
and “sour”,
which contribute to the perception of ripened cheese [20].
The aim of the present study was to test the hypothesis that it is possible to predict the number of
Enterobacteriaceae using human senses.

2. Materials and methods

This study included 19 raw milk cheeses, one cheese made from both raw milk and pasteurized milk and two
cheeses made from pasteurized milk. The raw milk cheeses (n = 19) and the cheese made from both pasteurized and
raw milk (n = 1) were purchased from a cheese store in Stockholm, Sweden. The cheeses made from pasteurized
milk (n = 2) were purchased from a local grocery store in Grythyttan, Sweden. The cheeses were stored in
refrigerators (+4 °C) at the School of Hospitality, Culinary Arts and Meal Sciences, Örebro University, until
analysis. All cheeses were analyzed before the best-before date according to the cheeses’ labels.
The cheeses (n = 22) were examined for aerobic microorganisms 30 °C, Enterobacteriaceae 37 °C,
Enterobacteriaceae 44 °C, enterococci, staphylococci, Bacillus cereus and Listeria monocytogenes using the plate
count method. The media used were tryptone glucose yeast extract agar, violet red bile glucose agar, Slanetz and
Bartley agar, Baird-Parker agar, BACARA agar and Ottaviani Agosti agar (bioMérieux, France). Furthermore,
four cheeses each contributed five typical Enterobacteriaceae 37 °C and 44 °C isolates, that is, a total of 10 isolates
from each cheese. The total of 40 isolates were identified using API 20 E strips (bioMérieux, France). The results
were recorded using the dedicated apiweb version 5.1 software ([Link] by bioMérieux,
France), which yielded a profile and an identification rate in %.
The sensory evaluation by Quantitative Descriptive Analysis (QDA) [16] was conducted by a sensory panel of
students (n = 14) at the School of Hospitality, Culinary Arts and Meal Sciences, Örebro University, who participated
voluntarily. The assessors had experience in sensory evaluation with lectures in physiology of the senses, perception
and sensory methods. The evaluation was carried out in a sensory laboratory conforming to ISO standards [21]. One
training session prior to the evaluation was carried out to calibrate the assessors. They discussed and agreed on
intensity levels of three different cheeses (two made from raw milk, one made from pasteurized milk). The sensory
characteristics used (n = 10) were based on previous studies [9, 22, 23] and included the basic tastes “sweet”, “sour”,
“salt”
and “bitter”
and the flavors “earthy”, “manure”, “mold”, “ammonia”, “metallic” and “pungent”. The intensity
was assessed using line scales with labeled end points ranging from low intensity (1) to high intensity (9). The
cheeses were evaluated in random order in replicates of one. Data collection was carried out using EyeQuestion
version 3.9.7 software (Logic8 BV, The Netherlands). Univariate statistical methods were used to analyze the data.
The analysis was performed using PanelCheck version 1.4.0 software ([Link] by Nofima,
Norway) for two-way ANOVA with samples and assessors as factors (two-way ANOVA, 1-rep) and for Principal
Component Analysis with sensory characteristics (mean values) for all products.
Data from plate counting and QDA were compiled using Excel 2010 version 14.0 (Microsoft Corporation, USA).
The statistical analysis was performed using Statistica version 12.0 software ([Link] by StatSoft, USA)
for correlation (r-value and p-value) and visualized by scatter plots, with the levels of bacteria and the intensity of
sensory characteristics as factors.
 Magnus Westling et al. / Procedia Food Science 7 (2016) 17 – 20 19

3. Results

Coagulase-positive staphylococci were detected in four of the 22 cheeses, Bacillus cereus was found in one of the
cheeses and L. monocytogenes was not detected in any of the soft cheeses. The number of aerobic microorganisms
cultured at 30 °C in the cheeses made from raw milk ranged from 6.5 to 9.5 colony-forming units (CFU log10) per
gram of cheese; the average value was 7.1 CFU log10. The number of Enterobacteriaceae cultured at 37 °C in all of
the cheeses made from raw milk ranged from 2.7 to 7.8 CFU log10; the average value was 7.1 CFU log10. The cheese
made from both pasteurized and raw milk harbored relatively low levels of Enterobacteriaceae at 37 °C, specifically
1.3 CFU log10. The levels of enterococci in all of the cheeses made from raw milk, except one, ranged from 4.7 to
7.1 CFU log10; the average value was 6.5 CFU log10.
All Enterobacteriaceae 37 °C isolates were identified as Hafnia alvei. All Enterobacteriaceae isolates at 44 °C in
three of the cheeses were identified as Escherichia coli, and four isolates in one cheese were identified as Klebsiella
pneumoniae. Three of the four cheeses harbored H. alvei at levels exceeding 7.0 log10 CFU per gram of cheese. One
of the four cheeses harbored E. coli at a level of 3.0 log10 CFU per gram of cheese.
All the descriptive words have a p-value of <0.001 except for the sensory characteristics “sweet”
and “earthy”,
which were excluded from further analysis. Moderate correlation (r-value 0.6 and p-value <0.01) was found
between the levels of Enterobacteriaceae at 37 °C and the intensity of the sensory characteristics “bitter”
and
“metallic”. Moderate correlation (r-value 0.5 and p-value <0.05) was also found between the levels of
Enterobacteriaceae at 37 °C and the intensity of the sensory characteristics “pungent”, “manure”
and “ammonia”.

4. Discussion

To our knowledge, there are no similar studies that suggest the use of a sensory method to predict high levels of
Enterobacteriaceae in soft cheeses. The bacterial family Enterobacteriaceae also includes environmental species, as
well as pathogenic bacteria. Therefore, Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs [6]
states that “Enterobacteriaceae can be used for routine monitoring, and if they are present testing of specific
pathogens can be started”. Furthermore, the same regulation states that “food business operators should have the
possibility to use analytical methods other than reference methods, in particular more rapid methods, as long as the
use of these alternative methods provides equivalent results”. According to Giammanco et al. [24], the quality of the
cheese milk, as well as the hygienic status during manufacturing, packaging and handling of cheese, can be assessed
by analyzing the amount of Enterobacteriaceae in cheese samples.
Different Enterobacteriaceae species have the ability to decarboxylate both lysine and ornithine, which leads to
the production of the biogenic amines cadaverine and putrescine [25, 26, 27, 28]. This was not a property of other
bacteria (enterococci, Lactobacillus and Leuconostoc) tested by Pircher et al. [28]. There are, however, substantial
intra-species differences in putrescine and cadaverine production [28]. Ardö
[29] states that cadaverine and
putrescine contribute to fecal and putrid off-flavors. These odors relate to the sensory characteristics “manure”
and
“ammonia”
respectively. In Camembert cheese, cadaverine has been reported as tasting distinctly bitter [30].

5. Conclusion

The present study indicates that it is possible to predict high levels of Enterobacteriaceae in soft cheeses made
from raw milk using the human senses (odor and taste) alone. However, it was not possible to identify the species
of Enterobacteriaceae in the tested soft cheeses made from raw milk using only the human senses (odor and taste).
Based on these results, it was not sufficient to use this as an alternative method to ensure hygienic quality. Further
studies with different combinations of Enterobacteriaceae species and mixtures would need to be evaluated to
ensure equivalent and standardized results.

Acknowledgement

Funding from Stadsveterinär Billströms stipendiefond is acknowledged.

20 Magnus Westling et al. / Procedia Food Science 7 (2016) 17 – 20

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