108 ISSN 1120-1770 online, DOI 10.15586/ijfs.v33i2.1976 P U B L I C A T I O N S CODON Italian Journal of Food Science, 2021; 33 (2): 108–115 generally preserve their biological activity after exposure to heat (Jablonski and Bohach, 1997; Jørgensen et al., 2005). The presence of S. aureus in raw milk before pro- cessing is a concern because different physical and chem- ical production techniques are applied during processing and ripening of milk products to prevent growth of this pathogen and production of enterotoxins. Nevertheless, if one of these limiting factors fails, there is a risk of accu- mulation of staphylococcal enterotoxins (Jørgensen et al., 2005). Thus, it is important to control growth of S. aureus in raw milk and raw milk products. In order to ensure milk safety and prolong milk’s shelf life, while also improving its sensorial characteristics, the dairy industry is developing minimum processing techniques (Cava et al., 2007). It has been suggested P U B L I C A T I O N S CODON Anti-staphylococcal effect of cinnamaldehyde in milk Milijana Babic1†, Milica Glisic1†, Jasna Djordjevic1, Nemanja Zdravkovic2, Radoslava Savic-Radovanovic1, Milan Baltic1, Marija Boskovic Cabrol†1* 1Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, University of Belgrade, Bulevar Oslobodjenja, Belgrade, Serbia; 2Department of Bacteriology and Parasitology, Scientific Institute of Veterinary Medicine of Serbia, VojvodeToze, Belgrade, Serbia †These authors contributed equally to the work. *Corresponding Author: Marija Boskovic Cabrol, Research Associate, Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, University of Belgrade, Bulevar Oslobodjenja 18, 11000 Belgrade, Serbia. Email: marijaboskovic116@gmail.com Received: 28 October 2020; Accepted: 13 May 2021; Published: 12 June 2021 © 2021 Codon Publications OPEN ACCESS PAPER Abstract The survival of Staphylococcus aureus in inoculated (105 colony forming units [CFU]/mL) 3.2% and 0.5% fat ultra- high temperature-pasteurized milk samples containing 0%, 0.05%, or 0.1% cinnamaldehyde stored at 4°C or 10°C was evaluated within 15 days. S. aureus populations reached 7.92 (0.5% fat) and 7.95 (3.2% fat) log CFU/mL in con- trol milk samples stored at 10°C, while in milk sample stored at 4°C, S. aureus counts remained almost unchanged. At the end of the study, the number of this pathogen decreased by 1.52–4.04 log  CFU/mL in milk treated with cinnamaldehyde. The greatest anti-staphylococcal effect was achieved in low-fat milk at 10°C and treated with 0.1% cinnamaldehyde. Keywords: antibacterial activity, cinnamaldehyde, fat, milk safety, Staphylococcus aureus Introduction Milk and milk products, being highly nutritious foods, are excellent media for the growth of many spoilage and pathogenic microorganisms (Noël et al., 2016), includ- ing Staphylococcus aureus. This pathogen commonly exists in dairy production plants (Xing et al., 2016), and it is one of the most important causative infective agents of clinical and subclinical mastitis in dairy cattle (Nam et al., 2011; Basanisi et al., 2017). S. aureus presents an important public health burden since it is one of the major pathogens responsible for food intoxication (Jans et al., 2017). In spite of the fact that pasteurization kills S. aureus, it has little effect on thermostable enterotoxins, which mailto:marijaboskovic116@gmail.com Italian Journal of Food Science, 2021; 33 (2) 109 Anti-staphylococcal effect of cinnamaldehyde in milk were bought from a local supermarket. Cinnamaldehyde (CA) (98% purity) was purchased from Carl Roth, Germany and stored at 4°C prior to use. S. aureus was obtained from the American Type Culture Collection (ATCC 25923). Determination of minimum inhibitory concentration The minimum inhibitory concentration (MIC) of cin- namaldehyde was determined in a non-milk matrix using sterile U-bottom 96-well microplates. The bacterial inoc- ulum density was set to 0.5 on the McFarland scale, then further diluted 10 times in sterile saline; 5 μL of this sus- pension was inoculated into 0.1 mL of Cation-Adjusted Mueller–Hinton Broth (CAMHB; Becton, Dickinson and Company, Sparks, USA) to reach a final S. aureus ATCC 25923 inoculum of 5 × 104 colony forming units (CFU)/ well. Cinnamaldehyde was diluted in dimethyl sulfoxide (Serva, Heidelberg, Germany) and added to CAMHB in the levels of 2560–1.25 μg/mL by two-fold dilution in 96-well microtitre plates. After inoculation, plates were incubated for 24 h at 37°C. The MIC was the lowest concentration of cinnamaldehyde that did not show any visual growth of S. aureus after macroscopic evaluation, and it was expressed in μg/mL (Clinical and Laboratory Standards Institute [CLSI], 2006). The plates were pre- pared in triplicate. Sample preparation and storage conditions Milk containing 0.5% or 3.2% fat was analyzed for S. aureus to confirm the absence of this pathogen. Approximately 5 log CFU/mL of S. aureus was inoc- ulated into S. aureus-free milk containing 0.5% or 3.2% milk fat. The concentration of the inoculum was verified by the standard plate count method and determined as 5.55–5.60 log CFU/mL. To study the survival of S. aureus in milk, different concentrations of cinnamaldehyde (0.05% and 0.1%) were added to milk samples with 0.5% (reduced fat) and 3.2% (whole milk) milk fat, whereas controls were without cinnamaldehyde but were inoc- ulated with S. aureus. The selection of these concentra- tions of cinnamaldehyde was based on previous sensory evaluations (Babic et al., 2019). After addition of cin- namaldehyde, all milk samples were divided into halves and stored in sterile glass bottles at 4°C and 10°C for 15 days. This temperature of 10°C was selected as an abuse temperature. The milk samples are described in Table 1. Microbiological and pH analysis All milk samples were examined on storage days 0, 3, 6, 9, 12, and 15. For bacterial enumeration, 25 mL of milk that addition of plant extracts, including cinnamon, can enhance microbiological safety, and it positively affects the sensory attributes of processed dairy products and milk-based desserts such as rice pudding and vanilla cream pudding (Tayel et al., 2015; Lianou et al., 2018). When added to butter, cinnamon (3%) lowered microbial growth during storage and exhibited antioxidant activity, thus retarding the spoilage of butter by positively influ- encing its sensorial characteristics (Vidanagamage et al., 2016). Thus, cinnamon could be successfully incorpo- rated in butter as a natural preservative instead of syn- thetic preservatives. Cinnamon contains 85.3–90.5% cinnamaldehyde (Doyle and Stephens, 2019). Together with eugenol, isoeugenol, vanillin, and safrole, cinnamaldehyde is one of the best studied phenylpropenes (Nazzaro et al., 2013). Trans- cinnamaldehyde exhibits a wide range of beneficial effects, including antibacterial, antifungal, antioxidant, anti-inflammatory, anti-diabetic, neuroprotective, and antitumor (Masghati and Ghoreishi, 2018; Doyle and Stephens, 2019), while cis-cinnamaldehyde, the geomet- rical isomer of trans-cinnamaldehyde, exhibits antifun- gal properties (Doyle and Stephens, 2019). Essential oil (EO) of cinnamon has found application in food indus- try because of its various components, including cin- namaldehyde, a major ingredient of cinnamon bark oil (Masghati and Ghoreishi, 2018). Most essential oils and their components, including trans-cinnamaldehyde, are generally recognized as safe (GRAS) and accepted by consumers (Burt, 2004). Owing to their antibacterial and antioxidant properties, essential oils can be used as potential natural preservatives in different foods, including flavored drinks (Cava et al., 2007). Flavored milk has increased in popularity in recent years; never- theless, there are few data available in literature about the effect of adding essential oils directly to milk before cheese-making (Licon et al., 2020). The focus of the present study was to determine whether trans-cinnamaldehyde could be a potential natural anti- bacterial agent in milk, hence ultra-high temperature (UHT)-pasteurized milk was used as a matrix to elimi- nate any possible interactions with the microbiota nor- mally present in raw milk. The aims of the study were to: (1) evaluate the anti-staphylococcal effect of different concentrations of cinnamaldehyde (0.05% and 0.1%) on S. aureus in milk; and (2) determine the influence of dif- ferent fat contents (0.5% and 3.2% milk fat) and different storage temperatures on survival of S. aureus in milk. Materials and Methods Trans-cinnamaldehyde and S. aureus culture, UHT- pasteurized milk samples containing 0.5% and 3.2% fat 110 Italian Journal of Food Science, 2021; 33 (2) Babic M et al. Table 1. Experimental design. Medium Cinnamaldehyde Temperature Milk samples Milk containing 0.5% fat with S. aureus 0% 4°C 1. Milk containing 0.5% fat with S. aureus and without cinnamaldehyde stored at 4°C. 0.05% 2. Milk containing 0.5% fat with S. aureus and 0.05% cinnamaldehyde stored at 4°C. 0.1% 3. Milk containing 0.5% fat with S. aureus and 0.1% cinnamaldehyde stored at 4°C. 0% 10°C 4. Milk containing 0.5% fat with S. aureus and without cinnamaldehyde stored at 10°C. 0.05% 5. Milk containing 0.5% fat with S. aureus and 0.05% cinnamaldehyde stored at 10°C. 0.1% 6. Milk containing 0.5% fat with S. aureus and 0.1% cinnamaldehyde stored at 10°C. Milk containing 3.2% fat with S. aureus 0% 4°C 7. Milk containing 3.2% fat with S. aureus and without cinnamaldehyde stored at 4°C. 0.05% 8. Milk containing 3.2% fat with S. aureus and 0.05% cinnamaldehyde stored at 4°C. 0.1% 9. Milk containing 3.2% fat with S. aureus and 0.1% cinnamaldehyde stored at 4°C. 0% 10°C 10. Milk containing 3.2% fat with S. aureus and without cinnamaldehyde stored at 10°C. 0.05% 11. Milk containing 3.2% fat with S. aureus and 0.05% cinnamaldehyde stored at 10°C. 0.1% 12. Milk containing 3.2% fat with S. aureus and 0.1% cinnamaldehyde stored at 10°C. was transferred into a sterile Stomacher bag and 225 mL of Buffered Peptone Water (BPW; Merck, Germany) was added. The contents of each bag were homogenized in a Stomacher blender (Stomacher 400 Circulator, Seward, UK) for 2 min. Serial decimal dilutions were prepared and 0.1 mL of appropriately diluted suspension was plated on Baird Parker agar (Oxoid CM 275, Basingstoke, Hampshire, UK) with egg yolk tellurite emulsion (Oxoid CM 275, Basingstoke, Hampshire, UK) and incubated at 37°C for 24 h according to EN ISO 6888-1 (International Organization for Standardization [ISO], 1999). The num- ber of colonies was counted, and results were recorded as colony forming units per milliliter. The pH of milk samples was measured using a portable pH meter (Testo 205; Testo AG, Lenzkirch, Germany). The pH meter was calibrated with standard buffer solu- tions of pH 4.0 and 7.0 prior to use. Statistical analysis Six randomized milk samples from each group were analyzed on each examination day. Number of micro- organisms were transformed into logarithms (log) before statistical analysis. Statistical analysis of the results was conducted using the SPSS 20.0 software (IBM, Chicago, IL, USA). The S. aureus counts were expressed as mean ± standard deviation. A three-way ANOVA analysis was used to investigate factor effects (concentrations of cinnamaldehyde, temperature, and fat%) and interactions among them on log-transformed S. aureus counts. Statistical differences between exam- ined groups were determined by Tukey’s post hoc multiple comparisons test. P < 0.05 was considered statistically significant. Results and Discussion Anti-staphylococcal effect of cinnamaldehyde in milk during storage The MIC of cinnamaldehyde against S. aureus was 160 μg/mL, showing that cinnamaldehyde was able to inhibit growth of this pathogen at low concentra- tions in the non-milk matrix used. Alves et al. (2016) reported a cinnamaldehyde MIC of 100 µg/mL against S. aureus, in agreement with the result of the present study. Nevertheless, in spite of the good antibacterial effect in vitro, hydrophobic essential oil constituents are impaired by interactions with food matrix components, hence higher concentrations are needed to achieve the same antibacterial effect in food (Hyldgaard et al., 2012). Thus, in the present study, approximately 4- and 9-fold higher concentrations (0.05% and 0.1%) of cinnamalde- hyde than the obtained MIC were added to milk samples. Significant (P < 0.05) antibacterial activity against S. aureus was found in milk samples at the cinnamaldehyde concen- trations used (0.05% and 0.1%) when compared with the controls without cinnamaldehyde (Table 2). Initial S. aureus counts ranged from 5.55 to 5.60 log CFU/mL. On day 0, S. aureus counts were significantly (P < 0.05) higher in controls than in milk samples with cinnamaldehyde at 4°C and at 10°C, indicating the imme- diate antibacterial effect of cinnamaldehyde. Regardless of fat content, in control milk samples without cin- namaldehyde stored at 4°C, with the exception of a slight decrease observed on day 3, the S. aureus populations remained almost unchanged for 15 days compared with the initial populations in milk samples. Nevertheless, at 10°C, S. aureus counts increased to approximately 7.92 Italian Journal of Food Science, 2021; 33 (2) 111 Anti-staphylococcal effect of cinnamaldehyde in milk (3.2% milk fat with 0.05% cinnamaldehyde at 10°C), and 2.96  log  CFU/mL (3.2% milk fat with 0.1% cinnamalde- hyde at 10°C). The anti-staphylococcal effect of cinnamal- dehyde found in the present study was in agreement with previous reports. Alves et al. (2016) reported that growth of S. aureus was inhibited by the combination of nisin and cinnamaldehyde in pasteurized 3% fat milk stored at 4°C for 6 days. The mechanism of cinnamaldehyde’s antibacterial action is known and well described. The antibacterial activity of cinnamaldehyde is attributed to a free hydroxyl group (Nazzaro et al., 2013). Cui et al. (2016) reported that after treating S. aureus with cinnamon essential oil, cell mem- brane injury and leakage of intracellular material were observed. Loss of ATP and DNA were detected because of bacterial cell membrane damage. Some reports indi- cate that cinnamaldehyde inhibits the membrane-bound ATPase activity (Usta et al., 2003; Gill and Holley, 2004). Di Pasqua et al. (2006) found that trans-cinnamalde- hyde causes changes in the composition of fatty acid and large increase in the proportion of saturated fatty acids in membrane phospholipids. Shen et al. (2015) evalu- ated the effect of cinnamaldehyde on inner membrane permeability of S. aureus by measuring β-galactosidase activity. The authors found that β-galactosidase activity increased with increase in cinnamaldehyde concentra- tion, leading to the conclusion that effects on membranes are dose- dependent. In our previous pilot study (Babic log CFU/mL (0.5% milk fat) and 7.95 log CFU/mL (3.2% milk fat) by the end of storage (day 15) in milk samples without cinnamaldehyde. Growth of S. aureus is possible at temperatures above 8°C at optimum pH values rang- ing between 6.0 and 7.0 (Valero et al., 2009). In all milk groups studied, the pH was within the optimal range (Figure 1) and enabled S. aureus to grow and survive at the utilized storage temperatures. In contrast, S. aureus counts decreased during 15 days’ storage in all milk samples with added cinnamalde- hyde. The decrease was less pronounced during the first 3 days of storage, and during this time no significant dif- ferences (P > 0.05) in S. aureus numbers were recorded between milk samples stored at 4°C and those stored at 10°C. From day 6 until the end of storage period (day 15), significantly greater S. aureus decrease (P < 0.05) was recorded in milk samples with added cinnamalde- hyde stored at 10°C than in comparable milk samples stored at 4°C. At the end of the study, in milk samples treated with cinnamaldehyde, S. aureus numbers had decreased by 1.61 log CFU/mL (0.5% milk fat with 0.05% cinnamaldehyde at 4°C), 2.45  log  CFU/mL (0.5% milk fat with 0.1% cinnamaldehyde at 4°C), 1.52  log  CFU/ mL (3.2% milk fat with 0.05% cinnamaldehyde at 4°C), 1.82  log  CFU/mL (3.2% milk fat with 0.1% cinnamalde- hyde at 4°C), 3.1  log  CFU/mL (0.5% milk fat with 0.05% cinnamaldehyde at 10°C), 4.04  log  CFU/mL (0.5% milk fat with 0.1% cinnamaldehyde at 10°C), 2.34 log CFU/mL Table 2. S. aureus counts (log CFU/mL) in milk with and without added cinnamaldehyde (CA), stored at 4°C and 10°C (mean ± SD), and the significance of interactions between cinnamaldehyde, storage temperature, and milk fat. CA concentration Temperature Fat Days 0 3 6 9 12 15 0% 4°C 0.5% 5.56 ± 0.06a 5.43 ± 0.10ac 5.46 ± 0.06a 5.48 ± 0.04a 5.57 ± 0.11a 5.56 ± 0.05a 3.2% 5.60 ± 0.07a 5.45 ± 0.09a 5.49 ± 0.10a 5.50 ± 0.06a 5.64 ± 0.08a 5.63 ± 0.06a 10°C 0.5% 5.55 ± 0.08a 6.98 ± 0.10b 7.21 ± 0.05b 7.49 ± 0.07b 7.47 ± 0.07b 7.92 ± 0.07b 3.2% 5.57 ± 0.09a 7.00 ± 0.16b 7.23 ± 0.15b 7.43 ± 0.07b 7.45 ± 0.08b 7.95 ± 0.10b 0.05% 4°C 0.5% 5.40 ± 0.06b 5.32 ± 0.07adce 5.15 ± 0.07c 4.62 ± 0.09c 4.44 ± 0.05c 3.95 ± 0.09c 3.2% 5.32 ± 0.07bc 5.23 ± 0.04def 5.11 ± 0.08c 4.89 ± 0.07d 4.28 ± 0.06d 4.08 ± 0.07c 10°C 0.5% 5.21 ± 0.05cd 5.26 ± 0.05cdef 4.51 ± 0.05d 3.81 ± 0.05e 3.04 ± 0.07e 2.45 ± 0.07d 3.2% 5.34 ± 0.07bc 5.30 ± 0.10adef 4.87 ± 0.08eg 4.00 ± 0.13f 3.90 ± 0.08f 3.23 ± 0.07e 0.1% 4°C 0.5% 5.28 ± 0.07bc 5.20 ± 0.08ef 4.94 ± 0.06g 4.46 ± 0.08g 4.10 ± 0.09g 3.11 ± 0.07e 3.2% 5.26 ± 0.06bc 5.18 ± 0.05ef 5.04 ± 0.08cg 4.56 ± 0.06cg 4.26 ± 0.08d 3.78 ± 0.07f 10°C 0.5% 5.11 ± 0.08d 5.14 ± 0.06f 4.08 ± 0.07f 3.62 ± 0.08h 2.51 ± 0.06h 1.51 ± 0.04g 3.2% 5.27 ± 0.06bc 5.25 ± 0.07ef 4.48 ± 0.08d 3.18 ± 0.09i 3.11 ± 0.07e 2.61 ± 0.10h Conc. CA × *Temp. NS ** ** ** ** ** Conc. CA × Fat% NS NS ** ** ** ** Conc. CA × Temp. × Fat% * NS ** ** ** ** a–iDifferent superscript letters in the same column, P < 0.05. NS: Not significant. *P < 0.05; **P < 0.001. 112 Italian Journal of Food Science, 2021; 33 (2) Babic M et al. 6.59 6.56 6.55 6.54 6.53 6.56 6.55 6.54 6.53 6.56 6.55 6.52 6.59 6.55 6.58 6.57 6.56 6.54 6.52 6.55 6.54 6.43 0% CA (0.5% fat milk) 0 6.3 6.35 6.4 6.45pH 6.5 6.55 6.6 3 6 Day 10ºC 9 12 15 0.1% CA (0.5% fat milk) 0.5% CA (3.2% fat milk) 0.5% CA (0.5% fat milk) 0% CA (3.2% fat milk) 0.1% CA (3.2% fat milk) 6.38 6.34 6.32 6.42 6.41 6.36 6.33 Figure 1. pH of milk stored at 10°C. et al., 2019), we found that the antibacterial effect of cin- namaldehyde was dependent on its concentration in 1.5% fat milk inoculated with 103 CFU/mL S. aureus stored at 4°C for 12 days. The same observation was made in the present study, showing significantly (P < 0.05) higher inhibition with 0.1% than 0.05% cinnamaldehyde used, but only for milk samples stored at the same temperature. It is supposed that essential oils are more effective when added at higher concentrations because after interactions with food matrix components (e.g. proteins and fats), more of the essential oil remains to interact with the bac- terial cells (Hyldgaard et al., 2012; Boskovic et al., 2017). One of the most important findings of the present study was the greater bacteriostatic effect of cinnamaldehyde at higher temperature. Significantly greater S. aureus decrease (P < 0.05) was recorded in milk samples with cinnamaldehyde stored at 10°C than in milk samples with same concentration of cinnamaldehyde stored at 4°C. With the expected exception of day 0, the interac- tions of storage temperature and cinnamaldehyde con- centration (P = 0.001; factorial ANOVA) on S. aureus counts (P = 0.207; factorial ANOVA) were statistically significant. At 4°C, 0.05% cinnamaldehyde decreased the number of S. aureus to 3.95 log CFU/mL in low-fat milk and to 4.08 log CFU/mL in whole milk, while at 10°C, this concentration of cinnamaldehyde decreased S. aureus counts to 2.45 log CFU/mL in low-fat milk and to 3.23 log CFU/mL in whole milk. When added at higher con- centration (0.1%), cinnamaldehyde reduced the initial S. aureus population to 3.11 log CFU/mL in low-fat milk and to 3.78 log CFU/mL in whole milk in samples stored at 4°C, while a significantly lower number (P < 0.05) of S. aureus was recorded in milk samples treated with the same concentration of cinnamaldehyde and stored at 10°C (1.51  log CFU/mL in low-fat milk and 2.61 log CFU/mL in whole milk). One possible explanation for this temperature-depen- dent antibacterial effect of cinnamaldehyde is that bacte- ria are metabolically more active at higher temperatures. Consequently, growth and death rates are higher at higher temperature (Smith-Palmer et al., 1998; Yuste and Fung, 2003; Guler and Seker, 2009). In addition, the lower growth rate of bacteria at lower temperatures can make them less susceptible to antimicrobials (Martinsen et al., 1992). Also, at lower temperatures, essential oils have lower diffusion rates, and this reduces the efficiency of their antibacterial activity (Wojtys and Jankowski, 2004; Leja et al., 2019). Even a small change in temperature causes significant changes in the efficiency of their action, which is why the doses of essential oils must be signifi- cantly higher at lower temperatures (Leja et al., 2019). These effects are in agreement with the results of present study. In milk samples with the same amount of fat, the anti-staphylococcal effect of 0.05% cinnamaldehyde was significantly (P < 0.05) more pronounced at 10°C than the effect of 0.1% cinnamaldehyde at 4°C (Table 2). In addition, Smith-Palmer et al. (1998) reported that the target site of cinnamon essential oil can change, and oil penetration to the interior of the cell can be reduced due to alterations in membranes at lower temperatures. Higher antibacterial Italian Journal of Food Science, 2021; 33 (2) 113 Anti-staphylococcal effect of cinnamaldehyde in milk However, when milk without cinnamaldehyde was stored at 10°C, decline in pH was observed regardless of the fat content. In fact, on day 3, the pH of milk without cin- namaldehyde stored at 10°C (Figure 1) decreased slightly compared with the initial pH values, but from day 6, sig- nificant (P < 0.05) pH declines were measured. The pH of this milk kept on decreasing throughout the 15 days’ storage, reaching pH values of 5.32 (0.5% milk fat) and 5.33 (3.2% milk fat). Growth of S. aureus in these milk samples (Table 2) matched decline in pH. Under aerobic conditions, S. aureus can ferment milk sugar and lac- tose, creating acids responsible for the storage-induced decline in milk pH (Medveďová and Valík, 2012). The pH of milk samples with cinnamaldehyde added and stored at 10 °C did not significantly differ between each other. Conclusion These results indicate that it could be possible to use cinnamaldehyde as a natural anti-staphylococcal agent in milk beverages. S. aureus numbers in milk were affected by cinnamaldehyde in a dose-dependent man- ner. Cinnamaldehyde showed a greater antibacterial effect against S. aureus in low-fat milk than in whole milk. Temperature had a strong effect on the anti-staphylo- coccal effect of cinnamaldehyde; hence, the lower concen- tration of cinnamaldehyde in milk stored at 10°C tended to have a better anti-staphylococcal effect than the higher concentration of cinnamaldehyde in milk stored at 4°C. Nevertheless, even if the results of our study are promising, and if flavored milk is becoming increasingly popular, fur- ther investigations are required to determine the antibacte- rial effectiveness of cinnamaldehyde in raw milk and dairy products and to conduct sensory analysis of final products. Acknowledgments The study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Contract numbers 451-03-68/2020-14/200143 and 451-03-9/2021-14/200143). Conflicts of Interest The authors have no conflicts of interest for this article. References Alves F.C., Barbosa L.N., Andrade B.F., Albano M., Furtado F.B., Pereira A.F.M., Rall W.L.M. and Júnior A.F. 2016. Inhibitory activities of the lantibioticnisin combined with pheno- lic compounds against Staphylococcus aureus and Listeria activities of essential oils at higher temperatures have been reported previously. Guler and Seker (2009) reported that the effect of cinnamon on Bacilluscereus  reductions in UHT-pasteurized milk during 28 days was significantly lower when milk samples were stored at 4°C than at 25°C. Yuste and Fung (2003) found that addition of 0.3% cinna- mon in apple juice was more effective against S. aureus during storage at higher temperatures. The initial con- tamination was lower (4.34–4.37 log CFU/mL) than in the present study, and counts decreased below detection lim- its in apple juice stored at 20°C after only 1 day, but it took 7 days of storage at 5°C to obtain the same results. In the present study, the greatest anti-staphylococcal effect was achieved in low-fat milk stored at 10°C and treated with 0.1% cinnamaldehyde, as S. aureus numbers were reduced by more than 4 log CFU/mL. Moreover, the effect of interaction between cinnamalde- hyde concentration and fat content in milk on S. aureus numbers was significant (P < 0.0001) from day 6 until the end of storage, while for the first 3 days no interaction was observed (day 0, P = 0.423; day 3, P = 0.370; factorial ANOVA). At the end of storage, significant differences (P < 0.05) between the S. aureus counts in whole milk (3.2%) and low-fat milk (0.5%) were found for the same concentrations of cinnamaldehyde. However, no signifi- cant differences (P > 0.05) in S. aureus counts were found between milk samples stored at the same temperatures without cinnamaldehyde, regardless of content of milk fat. Thus, cinnamaldehyde was more effective in inhibiting the pathogen in low-fat milk than in high-fat milk, which is also consistent with literature. Cava-Roda et al. (2012) found significant differences between whole milk (3.9% fat) and skimmed milk (0.3% fat) for inhibiting Escherichia coli O157:H7 and Listeria monocytogenes using the same concentration of vanillin (which also belongs to a group of phenylpropenes, as does cinnamaldehyde). The authors also reported that there was no effect of content of milk fat on pathogen numbers in control milk samples without vanillin. Therefore, the antibacterial effects of essential oils and other antimicrobial agents are likely to decrease or even limited by the amount of fat in the matrix (Liu and Yang, 2012; Boskovic et al., 2017, 2019). It has been suggested that fats may form a protective layer around bacterial cells and absorb essential oils, leaving the water phase free of antimicrobial agents, and therefore lower- ing the antibacterial activity (Tassou et al., 1995; Smith- Palmer et al., 2001; Perricone et al., 2015). pH of milk during storage The initial pH of milk samples ranged from 6.54 to 6.59. Cinnamaldehyde initially caused the milk pH to drop very slightly (from 6.59 to around 6.55). Milk pH did not change significantly during storage at 4°C. 114 Italian Journal of Food Science, 2021; 33 (2) Babic M et al. bacteria in animal feeds and human foods. J. Agric. Food Chem. 65(48):10406–10423. https://doi.org/10.1021/acs.jafc.7b04344 Gill A.O. and Holley R.A. 2004. Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of euge- nol against L. monocytogenes and Lactobacillus sakei. Appl. Environ. Microbiol. 70(10):5750–5755. https://doi.org/10.1128/ AEM.70.10.5750-5755.2004 Guler S. and Seker M. 2009. The effect of cinnamon and guar gum on Bacillus cereus population in milk. J. Food Process. Preserv. 33(3):415–426. https://doi.org/10.1111/j.1745-4549.2009.00417.x Hyldgaard M., Mygind T. and Meyer R.L. 2012. Essential oils in food preservation: mode of action, synergies, and interactions with food matrix components. Front. Microbiol. 3:12. https://doi. org/10.3389/fmicb.2012.00012 International Organization for Standardization (ISO). 1999. ISO Standard No. 6888-1: Microbiology of food and animal feeding stuffs—horizontal method for the enumeration of coagulase- positive staphylococci (Staphylococcus aureus and other species) part 1: Technique using Baird-Parker agar medium. International Organization for Standardization, Geneva, Switzerland. Jablonski L.M. and Bohach G.A. 1997. Staphylococcus aureus. In Doyle M.P., Beuchat L.R. and Montville T.J. (ed.). Food microbi- ology fundamentals and frontiers. ASM Press, Washington DC. 353–375. Jans C., Merz A., Johler S., Younan M., Tanner S.A., Kaindi D.W.M., et al. 2017. East and west African milk products are reservoirs for human and livestock-associated Staphylococcus aureus. Food Microbiol. 65:64–73. https://doi.org/10.1016/j.fm.2017.01.017 Jørgensen H.J., Mørk T. and Rørvik L.M. 2005. The occurrence of Staphylococcus aureus on a farm with small-scale production of raw milk cheese. J. Dairy Sci. 88(11):3810–3817. https://doi. org/10.3168/jds.S0022-0302(05)73066-6 Leja K., Szudera-Kończal K., Świtała E., Juzwa W., KowalczewskiP.Ł. and Czaczyk K. 2019. The influence of selected plant essen- tial oils on morphological and physiological characteristics in Pseudomonas orientalis. Foods. 8(7):277. https://doi.org/ 10.3390/foods8070277 Lianou A., Moschonas G., Nychas G.J.E. and Panagou, E.Z. 2018. Growth of Listeria monocytogenes in pasteurized vanilla cream pudding as affected by storage temperature and the presence of cinnamon extract. Food Res. Int. 106:1114–1122. https://doi. org/10.1016/j.foodres.2017.11.027 Licon C.C., Moro A., Librán C.M., Molina A.M., Zalacain A., Berruga M.I., et al. 2020. Volatile transference and antimicrobial activity of cheeses made with ewes’ milk fortified with essential oils. Foods. 9(1):35. https://doi.org/10.3390/foods9010035 Liu T.T. and Yang T.S. 2012. Antimicrobial impact of the compo- nents of essential oil of Litseacubeba from Taiwan and antimi- crobial activity of the oil in food systems. Int. J. Food Microbiol. 156(1):68–75. https://doi.org/10.1016/j.ijfoodmicro.2012.03.005 Martinsen B., Oppegaard H., Wichstrøm R. and Myhr E.G.I.L. 1992. Temperature-dependent in vitro antimicrobial activity of four 4-quinolones and oxytetracycline against bacteria pathogenic to fish. Antimicrob Agents Chemother. 36(8):1738–1743. https:// doi.org/10.1128/AAC.36.8.1738 monocytogenes in cow milk. J. Dairy Sci. 99(3):1831–1836. https://doi.org/10.3168/jds.2015-10025 Babic M., Glisic M., Zdravkovic N., Djordjevic J., Velebit B., Ledina T., et al. 2019. Inhibition of Staphylococcus aureus by cinnamaldehyde and its effect on sensory properties of milk. IOP Conf Ser Earth Environ Sci. 333:012042. https://doi. org/10.1088/1755-1315/333/1/012042 Basanisi M.G., La Bella G., Nobili G., Franconieri I., and La Salandra  G. 2017. Genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk and dairy products in South Italy. Food Microbiol. 62:141–146. https:// doi.org/10.1016/j.fm.2016.10.020 Boskovic M., Djordjevic J., Glisic M., Ciric J., Janjic J., Zdravkovic N., et al. 2019. The effect of oregano (Origanumvulgare) essential oil on four Salmonella serovars and shelf life of refrigerated pork meat packaged under vacuum and modified atmosphere. J. Food Process. Preserv. 44(1):e14311. https://doi.org/10.1111/ jfpp.14311 Boskovic M., Djordjevic J., Ivanovic J., Janjic J., Zdravkovic N., Glisic M., et al. 2017. Inhibition of Salmonella by thyme essen- tial oil and its effect on microbiological and sensory prop- erties of minced pork meat packaged under vacuum and modified atmosphere. Int. J. Food Microbiol. 258:58–67. https:// doi.org/10.1016/j.ijfoodmicro.2017.07.011 Burt S. 2004. Essential oils: their antibacterial properties and potential applications in foods-a review. Int. J. Food Microbiol. 94(3):223– 253. https://doi.org/10.1016/j.ijfoodmicro.2004.03.022 Cava R., Nowak E., Taboada A. and Marin-Iniesta F. 2007. Antimicrobial activity of clove and cinnamon essential oils against Listeria monocytogenes in pasteurized milk. J. Food Prot. 70(12):2757–2763. https://doi.org/10.4315/0362-028X- 70.12.2757 Cava-Roda R.M., Taboada-Rodríguez A., Valverde-Franco M.T. and Marín-Iniesta F. 2012. Antimicrobial activity of vanillin and mixtures with cinnamon and clove essential oils in controlling Listeria monocytogenes and Escherichia coli O157: H7 in milk. Food Bioproc. Tech. 5(6):2120–2131. https://doi.org/10.1007/ s11947-010-0484-4 Clinical and Laboratory Standards Institute (CLSI). 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard, 7th ed. CLSI Publication M7-A7. Clinical and Laboratory Standards Institute, Wayne PA. Cui H.Y., Zhou H., Lin L., Zhao C.T., Zhang X.J., Xiao Z.H., et  al. 2016. Antibacterial activity and mechanism of cinnamon essen- tial oil and its application in milk. J. Anim. Plant. Sci. 26:532–541. Di Pasqua R., Hoskins N., Betts G. and Mauriello G. 2006. Changes in membrane fatty acids composition of microbial cells induced by addiction of thymol, carvacrol, limonene, cinnamalde- hyde, and eugenol in the growing media. J. Agric. Food Chem. 54(7):2745–2749. https://doi.org/10.1021/jf052722l Doyle A.A. and Stephens J.C. 2019. A review of cinnamaldehyde and its derivatives as antibacterial agents. Fitoterapia.139:104405. https://doi.org/10.1016/j.fitote.2019.104405 Friedman M. 2017. Chemistry, antimicrobial mechanisms, and antibiotic activities of cinnamaldehyde against pathogenic https://doi.org/10.1021/acs.jafc.7b04344� https://doi.org/10.1128/AEM.70.10.5750-5755.2004� https://doi.org/10.1128/AEM.70.10.5750-5755.2004� https://doi.org/10.1111/j.1745-4549.2009.00417.x� https://doi.org/10.3389/fmicb.2012.00012� https://doi.org/10.3389/fmicb.2012.00012� https://doi.org/10.1016/j.fm.2017.01.017� https://doi.org/10.3168/jds.S0022-0302(05)73066-6� https://doi.org/10.3168/jds.S0022-0302(05)73066-6� https://doi.org/10.3390/foods8070277� https://doi.org/10.3390/foods8070277� https://doi.org/10.1016/j.foodres.2017.11.027� https://doi.org/10.1016/j.foodres.2017.11.027� https://doi.org/10.3390/foods9010035� https://doi.org/10.1016/j.ijfoodmicro.2012.03.005� https://doi.org/10.1128/AAC.36.8.1738� https://doi.org/10.1128/AAC.36.8.1738� https://doi.org/10.3168/jds.2015-10025� https://doi.org/10.1088/1755-1315/333/1/012042� https://doi.org/10.1088/1755-1315/333/1/012042� https://doi.org/10.1016/j.fm.2016.10.020� https://doi.org/10.1016/j.fm.2016.10.020� https://doi.org/10.1111/jfpp.14311� https://doi.org/10.1111/jfpp.14311� https://doi.org/10.1016/j.ijfoodmicro.2017.07.011� https://doi.org/10.1016/j.ijfoodmicro.2017.07.011� https://doi.org/10.1016/j.ijfoodmicro.2004.03.022� https://doi.org/10.4315/0362-028X-70.12.2757� https://doi.org/10.4315/0362-028X-70.12.2757� https://doi.org/10.1007/s11947-010-0484-4� https://doi.org/10.1007/s11947-010-0484-4� https://doi.org/10.1021/jf052722l� https://doi.org/10.1016/j.fitote.2019.104405� Italian Journal of Food Science, 2021; 33 (2) 115 Anti-staphylococcal effect of cinnamaldehyde in milk Tassou C.C., Drosinos E.H. and Nychas G.J.E. 1995. Effects of essential oil from mint (Menthapiperita) on Salmonella enter- itidis and Listeria monocytogenes in model food systems at 4°C and 10°C. J. Appl. Microbiol. 78(6):593–600. https://doi. org/10.1111/j.1365-2672.1995.tb03104.x Tayel A.A., Hussein H., Sorour N.M. and El-Tras W.F. 2015. Foodborne pathogens prevention and sensory attri- butes enhancement in processed cheese via flavoring with plant extracts. J. Food Sci. 80(12):2886–2891. https://doi. org/10.1111/1750-3841.13138 Usta J., Kreydiyyeh S., Barnabe P., Bou-Moughlabay Y. and Nakkash- Chmaisse H. 2003. Comparative study on the effect of cinna- mon and clove extracts and their main components on different types of ATPases. Hum. Exp.Toxicol. 22(7):355–362. https://doi. org/10.1191/0960327103ht379oa Valero A., Pérez-Rodríguez F., Carrasco E., Fuentes-Alventosa J.M., García-Gimeno R.M. and Zurera G. 2009. Modelling the growth boundaries of Staphylococcus aureus: effect of temperature, pH and water activity. Int. J. Food Microbiol. 133(1–3):186–194. https://doi.org/10.1016/j.ijfoodmicro.2009.05.023 Vidanagamage S.A., Pathiraje P.M.H.D. and Perera O.D.A.N. 2016. Effects of cinnamon (Cinnamomumverum) extract on func- tional properties of butter. Procedia Food Sci. 6:136–142. https://doi.org/10.1016/j.profoo.2016.02.033 Wojtys A. and Jankowski T. 2004. The effect of temperature on the permeation rate of some selected essential oils into baker’s yeast cells. Żywność Nauka Technologia Jakość, 11(3):77–86. Xing X., Zhang Y., Wu Q., Wang X., Ge W. and Wu C. 2016. Prevalence and characterization of Staphylococcus aureus iso- lated from goat milk powder processing plants. Food Control. 59:644–650. https://doi.org/10.1016/j.foodcont.2015.06.042 Yuste J. and Fung D.Y.C. 2003. Evaluation of Salmonella typh- imurium, Yersinia enterocolitica and Staphylococcus aureus counts in apple juice with cinnamon, by conventional media and thin agar layer method. Food Microbiol. 20(3):365–370. https:// doi.org/10.1016/S0740-0020(02)00130-2 Masghati S. and Ghoreishi S.M. 2018. Supercritical CO2 extraction of cinnamaldehyde and eugenol from cinnamon bark: optimi- zation of operating conditions via response surface methodol- ogy. J Supercrit Fluids. 140:62–71. https://doi.org/10.1016/j. supflu.2018.06.002 Medveďová A. and Valík Ľ. 2012. Staphylococcus aureus: character- isation and quantitative growth description in milk and artisanal raw milk cheese production. In: Eissa A.A. (ed.) Structure and Function of Food Engineering. Books on Demand, Intech Open, London, pp. 71–101. https://doi.org/10.5772/48175 Nam H.M., Lee A.L., Jung S.C., Kim M.N., Jang G.C., Wee S.H. and Lim S.K. 2011. Antimicrobial susceptibility of Staphylococcus aureus and characterization of methicillin-resistant Staphylococcus aureus isolated from bovine mastitis in Korea. Foodborne Pathog. Dis. 8(2):231–238. https://doi.org/10.1089/ fpd.2010.0661 Nazzaro F., Fratianni F., De Martino L., Coppola R. and De Feo V. 2013. Effect of essential oils on pathogenic bacteria. Pharmaceuticals. 6(12):1451–1474. https://doi.org/10.3390/ph6121451 Noël T.S., Kifouli A., Boniface Y., Edwige D.A., Farid B.M. and Fatiou T. 2016. Antimicrobial and physico-chemical effects of essential oils on fermented milk during preservation. J. Appl. Biosci. 99:9467–9475. https://doi.org/10.4314/jab.v99i1.12 Perricone M., Arace E., Corbo M.R., Sinigaglia M. and Bevilacqua A. 2015. Bioactivity of essential oils: a review on their interac- tion with food components. Front. Microbiol. 6:76. https://doi. org/10.3389/fmicb.2015.00076 Shen S., Zhang T., Yuan Y., Lin S., Xu J. and Ye H. 2015. Effects of cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane. Food Control. 47:196–202. https://doi.org/10.1016/j. foodcont.2014.07.003 Smith-Palmer A., Stewart J. and Fyfe L. 1998. Antimicrobial prop- erties of plant essential oils and essences against five important food-borne pathogens. Lett. Appl. Microbiol. 26(2):118–122. https://doi.org/10.1046/j.1472-765X.1998.00303.x Smith-Palmer A., Stewart J. and Fyfe L. 2001. The potential appli- cation of plant essential oils as natural food preservatives in soft cheese. Food Microbiol. 18(4):463–470. https://doi.org/10.1006/ fmic.2001.0415 https://doi.org/10.1111/j.1365-2672.1995.tb03104.x� https://doi.org/10.1111/j.1365-2672.1995.tb03104.x� https://doi.org/10.1111/1750-3841.13138� https://doi.org/10.1111/1750-3841.13138� https://doi.org/10.1191/0960327103ht379oa https://doi.org/10.1191/0960327103ht379oa https://doi.org/10.1016/j.ijfoodmicro.2009.05.023� https://doi.org/10.1016/j.profoo.2016.02.033� https://doi.org/10.1016/j.foodcont.2015.06.042� https://doi.org/10.1016/S0740-0020(02)00130-2 https://doi.org/10.1016/S0740-0020(02)00130-2 https://doi.org/10.1016/j.supflu.2018.06.002� https://doi.org/10.1016/j.supflu.2018.06.002� https://doi.org/10.5772/48175� https://doi.org/10.1089/fpd.2010.0661� https://doi.org/10.1089/fpd.2010.0661� https://doi.org/10.3390/ph6121451� https://doi.org/10.4314/jab.v99i1.12� https://doi.org/10.3389/fmicb.2015.00076� https://doi.org/10.3389/fmicb.2015.00076� https://doi.org/10.1016/j.foodcont.2014.07.003� https://doi.org/10.1016/j.foodcont.2014.07.003� https://doi.org/10.1046/j.1472-765X.1998.00303.x� https://doi.org/10.1006/fmic.2001.0415� https://doi.org/10.1006/fmic.2001.0415� _Hlk41220087 _Hlk38042043 bbib10 bbib12 _Hlk38142306 bbb0120 bBIB20 _GoBack