Influence of milk microbiota on Listeria monocytogenes survival during cheese ripening

Abstract This study aimed to compare the three strains of Listeria monocytogenes survival in raw milk cheese and pasteurized milk cheese and to suggest the effect of milk microbiota on survival. L. monocytogenes cell counts decreased in all cheese as ripening time increased, and the survival rate was different for the strains of L. monocytogenes. Furthermore, L. monocytogenes survived longer in raw milk cheese than in pasteurized milk cheese. The difference of bacterial survival in each cheese was independent of Aw or the Lactobacillus spp. populations in cheeses; there was no difference in Aw or Lactobacillus spp. populations in all cheeses. The richness of microbiota in raw milk was little higher than in pasteurized milk, and five phyla (Chloroflexi, Cyanobacteria, Deinococcus–Thermus, Lentisphaerae, and Verrucomicrobia) were present only in raw milk. Also, organic acid‐producing bacteria were presented more in pasteurized milk compared with raw milk; thus, the growth of L. monocytogenes was slower in pasteurized milk. In conclusion, differences in the microbial community of milk can affect the growth of L. monocytogenes. Making cheese using raw milk is a risk of L. monocytogenes infection; thus, efforts to prevent growth of L. monocytogenes such as the use of appropriate food additives are required.

coliforms (Van Kessel et al., 2004). In addition, Ombarak et al. (2018) reported that over 40% of raw milk cheese samples are contaminated with E. coli. Therefore, there is a genuine risk of food-borne illness associated with cheese (Baylis, 2009;De Buyser, Dufour, Maire, & Lafarge, 2001). The prevalence of L. monocytogenes in cheese reported and the detection rate ranges from 2.2% to 17% (Coroneo et al., 2016;Doménech et al., 2015;Iannetti et al., 2016;Kabuki et al., 2004). Listeriosis is a food-borne illness that can occur following consumption of cheese (Gaulin, Ramsay, & Bekal, 2012;McIntyre, Wilcott, & Naus, 2015). In northwest Switzerland, 10 cases of listeriosis were reported to be caused by the consumption of a soft cheese known as "tomme" and led to three deaths and septic abortion in two pregnant women (Bille et al., 2006).
Listeria monocytogenes is a zoonotic, gram-positive, facultative anaerobe (Nho, Abdelhamed, Reddy, Karsi, & Lawrence, 2015). This pathogen is the causative agent of human listeriosis (Allerberger & Wagner, 2010), for which the mortality rate is reported to be 20%-30% (World Health Organization, 2018). L. monocytogenes can survive in acidic and high salt environments and can tolerate low temperatures (Allerberger & Wagner, 2010;Lee et al., 2016). The ability of L. monocytogenes to survive in certain environments differs from strain to strain (Kale et al., 2017;Takahashi, Kuramoto, Miya, & Kimura, 2011). Kale et al. (2017) reported that among 104 L. monocytogenes strains, 13 strains could grow in a high salt environment (12.5% sodium chloride) and 22 strains showed tolerance to a low temperature (4°C). Cheese has a high salt content and a low pH and is ripened at a low temperature; nevertheless, it is possible that L. monocytogenes could survive in cheese, and its survival ability may vary among strains. Cheese can be made from raw milk or pasteurized milk, and the microbiota composition of raw milk and pasteurized milk may be different, which could affect the survival of L. monocytogenes.
Therefore, the objective of this study was to investigate the ability of different L. monocytogenes strains to survive in cheese made from raw or pasteurized milk and to reveal the effects of milk microbiota on the survival of L. monocytogenes.
The 0.1 ml portions of each culture were subcultured in 10 ml TSBYE and incubated at 30°C for 24 hr. Subcultures were centrifuged at 1,912 × g at 4°C for 15 min. Pellets were washed twice with phosphate-buffered saline (PBS; pH 7.4; 0.2 g KH 2 PO 4 , 1.5 g Na 2 HPO 4 , 8.0 g NaCl, and 0.2 g KCl in 1 L dH 2 O) and resuspended in PBS. Each suspension of L. monocytogenes was serially diluted with PBS to obtain 5-6 Log CFU/ml.

| Cheddar cheese preparation and inoculation
Three strains of L. monocytogenes were contaminated in raw milk and pasteurized milk, respectively, with a level of 3-4 Log CFU/ml. A total of six types of cheeses were prepared using two types of milk (raw milk and pasteurized milk) contaminated with three strains of L. (v/v); Mysecoren300FK, MAYSA, Istanbul, Turkey) was added to the milks, which were, then, left for 30-45 min to form curd. The curd was cut into cubes (2 × 2 cm) and stirred slowly. To remove the whey, the curd was slowly heated to 38°C via a 1°C increase every 5 min.
Cheddaring (the repeated process of cutting and stacking curd) was performed, and the whey was removed using a cotton cloth. NaCl (2% w/w) was added to the curd, which was mixed well before being placed into a cheese mold lined with cloth to form a block of cheese.
Curd was pressed at 25°C for 40-50 min and then re-pressed with 10 times the weight of the curd for 12 hr. The cheese was stored at 13-15°C for ripening. Cheese preparation was replicated twice.

| A w measurement in cheese
On the same days as the microbial analysis, the water activity (A w ) of the cheese was measured. Cheese samples were cut into small pieces and filled with a plastic shell up to 70%. The A w was then measured using an AQS-31-TC water activity meter (NAGY, Siedlerstrabe, Gaufelden, Germany).

| Comparison of microbiota between raw milk and pasteurized milk
To compare the effect of microbiota on the survival of L. monocytogenes, next-generation sequencing (NGS) analysis was performed to determine the microbiomes of the raw milk and pasteurized milk. DNA was extracted from the milk using a PowerSoil ® DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA), and a sequencing library was prepared by PCR using a Nextera XT Index Kit (Illumina, San Diego, CA, USA). Sequencing was performed using the Illumina MiSeq ® System (Illumina) to obtain raw data. A FASTQ file was created in the MiSeq Control Software (v2.2) and bcl2fastq (v1.8.4) using the raw data, and the PhiX sequence was removed by Burrows-Wheeler Aligner (Li & Durbin, 2009). Paired-end data were sorted using FLASH (v1.2.11) (Magoč & Salzberg, 2011). The data were then processed to remove sequencing errors, and CD-HIT-OTU was used to perform clustering with more than 97% sequence similarity to obtain operational taxonomic units (OTU) (Li, Fu, Niu, Wu, & Wooley, 2012). The taxonomic assignment was performed using the BLASTN program (v2.4.0) and the NCBI 16S Microbial reference database (Zhang, Schwartz, Wagner, & Miller, 2000).

| Statistical analysis
The experiment to obtain the microbial survival data was replicated twice with two samples in each replicate (n = 4). The data were analyzed using a mixed model procedure in SAS ® (version 9.4; SAS Institute Inc., USA). A pairwise t test at α = 0.05 was used for mean comparisons.

| RE SULTS AND D ISCUSS I ON
Listeria monocytogenes cell counts decreased gradually as ripening time increased in all raw milk cheese and pasteurized milk cheese.
Likewise, the initial A w (0.957-0.987) of the cheeses decreased to 0.708-0.801 during ripening. When the cell counts were significantly reduced for the first time compared to the initial cell counts (p < .05), it was judged as "the first significant reduction." The first significant reductions in L. monocytogenes cells in raw milk cheese were observed on days 90, 50, and 80 of ripening for strains SMFM-SI-1, SMFM-SI-6, and SMFM-CI-1, respectively (p < .05; Figure 1). The L. monocytogenes SMFM-SI-6 cell counts fell below the detection limit after 120 days of ripening, but this took 160 days for SMFM-SI-1 and SMFM-CI-1 in raw milk cheese (Figure 1). Otherwise, cell counts of three L. monocytogenes strains fell below the detection limit between days 120 and 130 in pasteurized milk cheese (Figure 1). As a result, the death rate of L. monocytogenes was slower in raw milk cheese than in pasteurized milk cheese during the ripening. We considered that these survival differences were related to differences in A w ; however, similar A w values were observed in both cheeses on day 160 of ripening (0.708-0.801). Thus, we thought that different F I G U R E 1 Cell counts of Listeria monocytogenes strains SMFM-SI-1, SMFM-SI-6, and SMFM-CI-1 in raw milk cheese (a) and pasteurized milk cheese (b) levels of Lactobacillus spp. between raw milk cheese and pasteurized milk cheese influenced L. monocytogenes survival ability; however, the concentration of Lactobacillus spp. in all cheeses was maintained at 6-9 Log CFU/g, with no difference between the two types of cheese. Next, we compared the microbiomes of raw milk and pasteurized milk using NGS. The values of Inverse Simpson were 0.98 in both milk samples; therefore, there was no difference in microbiota diversity between raw milk and pasteurized milk. However, raw milk exhibited higher richness (Chao1 value = 444) than pasteurized milk (Chao 1 value = 354), indicating that more microorganisms (specifically, the five phyla Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Lentisphaerae, and Verrucomicrobia) were present in raw milk (Table 1). In family level, 105 families in raw milk and 91 families in pasteurized milk were analyzed by NGS. Also, in genus level, 196 genus in raw milk and 162 genus in pasteurized milk were identified by NGS (Table 2). Based on these results, we speculated that the different compositions of these microbial communities may influence the longer survival of L. monocytogenes strains in raw milk cheese than in pasteurized milk cheese. Several organic acids (lactic acid, propionic acid, and acetic acid, etc.) had antimicrobial activity. In the genus level, the ratio of organic acid-producing [Proteobacteria phylum]) was higher in pasteurized milk (6.49%) compared with raw milk (4.81%). Acid production may have affected the survival of L. monocytogenes. Wemmenhove, van Valenberg, van Hooijdonk, Wells-Bennik, and Zwietering (2018) said that lactic acid was an inhibitor for growth of L. monocytogenes in Gouda cheese.
There was no Listeria spp. in both milk. Schvartzman et al. (2011) observed that L. monocytogenes grows in raw milk cheese, but its survival ability is weakened in pasteurized milk cheese. They also reported that the different composition of background microflora in raw milk and pasteurized milk could affect the fate of L. monocytogenes (Schvartzman et al., 2011). Although not clearly identified, microbial community differences may have affected L. monocytogenes survival. Additionally, it was revealed that several harmful bacteria such as Actinobaculum schaalii, Eubacterium moniliform, Flavonifractor plautii, Acinetobacter lwoffii, and Exiguobacterium aurantiacum existed only in raw milk (Bank, Jensen, Hansen, Søby, & Prag, 2010;Berger et al., 2018;Liang, Yin, Xu, & Chen, 2017;Pitt et al., 2007;Regalado, Martin, & Antony, 2009). F. plautii is gram-positive bacteria and can cause the acute cholecystitis (Berger et al., 2018). Also, E. moniliform and E. aurantiacum were bacteria isolated from patients with bacteremia (Liang et al., 2017;Pitt et al., 2007). Thus, it is considered that the possibility of outbreak is high when people intake raw milk cheese.
In conclusion, L. monocytogenes survival was observed to vary among strains. Furthermore, L. monocytogenes can survive longer in raw milk cheese than in pasteurized milk cheese during cheese ripening, and we believe that survival of L. monocytogenes is influenced by the differences of microbiota composition (i.e., organic acid-producing bacteria) between the raw milk and pasteurized milk. According to the results of this study, making cheese using raw milk as it cannot guarantee safety against listeriosis. Therefore, when making cheese using raw milk, various efforts will be required, such as adding appropriate food additives (e.g. lactic acid) that can control L. monocytogenes to inhibit the growth of L. monocytogenes and further prevent L. monocytogenes infection.