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. 2021 Jan 19;52(2):847–858. doi: 10.1007/s42770-021-00431-4

Listeria monocytogenes inhibition by lactic acid bacteria and coliforms in Brazilian fresh white cheese

Lina Casale Aragon-Alegro 1, Emília Maria França Lima 1, Gabriela Palcich 1, Tatiana Pacheco Nunes 1, Kátia Leani Oliveira de Souza 1, Cecília Geraldes Martins 1, Patrícia Kary Noda 1, Maria Teresa Destro 1,✉, Uelinton Manoel Pinto 1
PMCID: PMC8105483  PMID: 33462722

Abstract

Introduction

Minas fresh cheese (MFC), a Brazilian white cheese, is one of the most popular cheeses nationwide. Studies have shown that Listeria monocytogenes occurrence in this product is generally low, while high populations of coliforms can be found. This study aimed to evaluate the influence of coliforms in the behavior of L. monocytogenes in MFC.

Methods

Pasteurized milk was inoculated with L. monocytogenes and coliforms, and the acidification was made by lactic acid or by the addition of a starter culture. The cheeses of each production were divided into 3 groups and stored at 5 ºC, 12 ºC and cycles of 5 ºC followed by 25 ºC. In predetermined days, samples were taken and L. monocytogenes, coliforms and lactic acid bacteria populations were evaluated, besides the pH, water activity (aw), titratable acidity and NaCl concentration.

Results

The inhibition of L. monocytogenes in the presence of coliforms was observed (p < 0.05), except for those samples prepared with lactic acid and stored at temperature cycles. The values of pH and aw were not sufficiently low to cause inhibition; however, titratable acidity was higher in cheeses containing coliforms. In vitro tests containing lactic acid and L. monocytogenes showed that the bacterium is sensitive to concentration of lactic acid ≥ 0.3%, indicating that lactic acid produced by coliforms strongly influences the population of L. monocytogenes.

Conclusions

Thus, it can be concluded that coliforms negatively impact populations of L. monocytogenes in MFC. We strongly recommend that producers of MFC adopt good hygiene practices to not only avoid contamination with L. monocytogenes, but also coliforms.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-021-00431-4.

Keywords: Co-culture, Inhibition, Lactic acid, Minas fresh cheese, Storage temperature

Introduction

Listeria monocytogenes is a cause of concern to the food industry, especially to the dairy sector [1–4]. The ability of L. monocytogenes to survive and grow under refrigeration temperatures, in the presence of moderate salt concentrations, neutral pH, and high water activity makes it a dangerous hazard for dairy products [2, 5]. This microorganism has been associated with several foodborne outbreaks [6–10], and soft cheeses have been implicated in several of them [2, 3, 9, 11, 12]. In the USA, of 67 listeriosis outbreaks and 118 listeriosis deaths reported during 2010–2018, 4 outbreaks and 3 deaths were associated with soft cheeses [13]. Its occurrence in this type of food has been widely studied [3, 6, 14–17].

Minas fresh cheese (MFC) is the Brazilian representative of the Spanish soft cheese and is one of the most popular cheeses nationwide. It can be produced by direct acidification of pasteurized milk with lactic acid or by using lactic cultures for acidification, followed by enzymatic coagulation [18]. Its characteristics such as high water activity (> 0.97), pH above 5.0, low salt content (1.4–1.6%), and the absence of preservatives make this cheese an excellent medium for survival and growth of food spoilage organisms such as coliforms and pathogenic bacteria such as L. monocytogenes [5, 11, 14].

The MFC shelf-life estimated by manufacturers ranges from 21 to 30 days. However, Rocha et al. [19] observed that samples of MFC were unfit for consumption from the seventh day, and some cheeses were visibly deteriorated at 21 days. MFC has a short shelf-life, after which the product may present unpleasant flavor and odor; however, it may be kept in domestic refrigerators for longer periods of time. At home it can be subject to temperature abuse, since temperature control of the domestic refrigerator is not a popular habit, despite misuse by the consumers, in addition to abuse temperatures during transport and distribution. Fresh white cheeses made with raw milk and without inspection by regulatory agencies are also sold in open markets [20–22], sometimes with inadequate temperature control, as well as door to door and in ethnic fairs [16, 19].

Although these characteristics favor L. monocytogenes multiplication in this kind of cheese, its occurrence is relatively low in this food [15, 16]. On the other hand, many studies have found that the population of thermotolerant coliforms in Brazilian fresh white cheeses is usually high (> 106 NMP/g) [15, 16, 19], even though in disagreement with official standards [23, 24].

Apolinário et al. [15] evaluated 31 samples of MFC from Zona da Mata Mineira, Brazil. Of these, 77.4% presented nonconformity for total coliforms and 54.8% for thermotolerant coliforms, while L. monocytogenes was found in 9.6% of the samples. Similar results were observed by Vissoto et al. [16], when evaluating 30 samples of MFC purchased in grocery stores and free markets in São Paulo, Brazil. Ninety percent of the samples presented total coliform population above 1.1 × 103 NMP/g. On the other hand, Shigella spp., Salmonella spp., and L. monocytogenes were not detected in the study, suggesting that the absence of these pathogenic microorganisms may be due to unfavorable competition of these species in relation to the other bacteria present in the analyzed products.

Melo et al. [3] showed several studies performed in the last decade regarding the isolation of L. monocytogenes from dairy products in many countries. The results showed a significant variation in the occurrence of this pathogen in cheese products (0.2 to 46.0%) and cheese dairy environment (0 to 52.0%). In a study conducted in Brazil by Abrahão et al. [17] with 90 samples of different kinds of cheese, 11 (12.2%) were positive for Listeria sp., subdivided as L. monocytogenes in six samples (6.7%), and L. innocua in five samples (5.5%).

According to the Brazilian Institute of Geography and Statistics, the Brazilian cheese market represented 24.6% of the total of Brazilian dairy market in 2016, close to UHT milk (24.9%) [25]. The average cheese consumption corresponded to 2.17 kg/hab in 2009, rising to 5.4 kg/hab in 2017 [26]. Fresh cheese consumption is expected to increase according to the Brazilian Association of Cheese Industries (ABIQ), as the Association’s goal is to achieve a consumption of 7.5 kg/hab in 2020 [26]. The increased consumption of cheese highlights the importance of understanding the behavior of L. monocytogenes in these foods [11], and the ability of L. monocytogenes to associate with other microorganisms, such as Escherichia coli, is a possibility that should be further investigated [27].

We hypothesized that the high populations of coliforms in MFC may negatively influence the occurrence of L. monocytogenes. Thus, we evaluated the combined effect of the presence of coliform bacteria in Minas fresh cheeses associated with different production methods, storage time, and temperature on the population of L. monocytogenes.

Material and methods

Strains

Three L. monocytogenes strains were used in this study: ATCC 7644, 15E (isolated from chicken breast), and 30 (isolated from the environment of a chicken processing plant). Although 15E and 30 were not isolated from dairy products, they also belonged to products of animal origin, kept under refrigeration. The strains belong to the Food Microbiology Lab of the Faculty of Pharmaceutical Sciences, University of Sao Paulo, Brazil. The commercial starter culture was formulated with Lactobacillus lactis subsp. lactis and Lactobacillus lactis subsp. cremoris (Ezal MA011, Danisco) [28]. Coliforms were obtained from fresh white cheeses and isolated as described next.

Preparation of microbial cultures

L. monocytogenes

The three strains were grown separately in trypticase soy broth plus 0.6% yeast extract (TSB-YE, Oxoid Basingstoke, UK) and incubated at 37 °C for 24 h; the broths were mixed, centrifuged at 1050×g for 15 min at 4 °C, washed with 0.85% (w/v) saline solution (Merck, Darmstadt, Germany) aliquoted in 1-mL portions, and kept frozen at − 70 °C with 25% (v/v) glycerol. Each 1-mL portion contained approximately 107 CFU/mL.

Coliforms

Three Minas fresh cheeses were acquired at retail in the city of Sao Paulo, Brazil. Portions of 50 g of each cheese were combined and homogenized in 2% (w/v) sodium citrate (Merck). Serial dilutions were poured-plated in violet red bile glucose agar (VRBGA, Oxoid) with an overlay of the same agar according to Kang and Fung [29] with modifications. The plates were incubated at 37 °C for 24 h, and coliform characteristic colonies were transferred to tubes containing brilliant green lactose bile broth (Oxoid) and Durham’s tube. The broths were incubated at 37 °C, and after 48 h, those that presented turbidity and production of gas were combined in Falcon tubes and centrifuged at 1050×g for 15 min at 4 °C. Cells were washed with saline solution, divided in 1-mL portions, and kept frozen at − 80 °C with 25% (v/v) glycerol. Each portion contained approximately 107 CFU/mL. Identification of the species present in this culture was done by transferring 1 mL of the culture to 150 mL of brain heart infusion broth (BHI, Oxoid) and incubating at 37 °C in a shaker (150 rpm—Innova 4000, New Brunswick Scientific, USA) for 3 h. Serial dilutions were streaked on McConkey agar (Oxoid) and VRBGA. After incubation for 24 h at 37 °C, 51 colonies were randomly chosen, streaked on BHI agar (Oxoid), and incubated at 37 °C for 24 h. These colonies were submitted to biochemical identification using EPM (Escola Paulista de Medicina) [30], MILi (Motility, Indole and Lysine) [31], and Simmons citrate (Oxoid) [32] agars. API 20E (bioMérieux, Marcy-l’Etoile, France) was also used according to manufacturer’s instructions.

Minas fresh cheese manufacture

Different formulations and storage conditions of MFC were prepared (Table 1). In two of them, the milk was acidified using lactic acid (Purac, Gorinchem, Netherlands), and in the others, lactic acid culture (Ezal MAO11 - Danisco, Copenhagen, Denmark) was used. One of each acidification type was inoculated with L. monocytogenes in order to have a population of 105 CFU/mL (high inoculum) and the other with a population lower than 101 CFU/mL of L. monocytogenes (low inoculum). Samples from each formulation were stored in the following conditions: 20 days at 5 °C (refrigeration temperature); 20 days at 12 °C (abuse temperature); and 8 days at 5 °C for 16 h followed by 25 °C for 8 h (to simulate open market conditions). The production of the cheeses was repeated three times on different days. Milk of the different formulations and productions were inoculated with L. monocytogenes and coliforms (LC). As controls, samples of milk inoculated with only coliforms (C) and only with L. monocytogenes (L) were used for cheese preparation (Table 1).

Table 1.

Experimental design of the Minas fresh cheese productions

Formulation Acidification* Contamination type** L. monocytogenes population (CFU/mL) Coliforms population (CFU/mL) Storage temperature
1 LA L 105 – 5 °C
LA C – 107
LA LC 105 107
2 LA L < 10 – 5 °C
LA C – 107
LA LC < 10 107
3 LA L 105 – 12 °C
LA C – 107
LA LC 105 107
4 LA L < 10 – 12 °C
LA C – 107
LA LC < 10 107
5 LA L 105 – 5 °C/16 h and 25 °C/8 h
LA C – 107
LA LC 105 107
6 LA L < 10 – 5 °C/16 h and 25 °C/8 h
LA C – 107
LA LC < 10 107
7 LAC L 105 – 5 °C
LAC C – 107
LAC LC 105 107
8 LAC L < 10 – 5 °C
LAC C – 107
LAC LC < 10 107
9 LAC L 105 – 12 °C
LAC C – 107
LAC LC 105 107
10 LAC L < 10 – 12 °C
LAC C – 107
LAC LC < 10 107
11 LAC L 105 – 5 °C/16 h and 25 °C/8 h
LAC C – 107
LAC LC 105 107
12 LAC L < 10 – 5 °C/16 h and 25 °C/8 h
LAC C – 107
LAC LC < 10 107
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*LA lactic acid; LAC lactic acid culture

**Cheese contaminated with only L. monocytogenes (L), with only coliforms (C), and with L. monocytogenes and coliforms (LC)

Each group of samples (LC, C, L) was produced with 2 L of pasteurized commercial whole milk (3% fat). For LC and C cheeses, 1 mL of the defrosted coliform aliquot was added to the milk, and the mixtures were incubated at 37 °C for 5.5 h. Milk that would be used for “L” cheeses was also incubated in the same conditions. Preliminary data indicated that this procedure was necessary to get coliforms population to 107 CFU/mL. After that, the pool of L. monocytogenes strains was added to the LC and L milk vats, and after 10 min at 37 °C, 85% lactic acid solution (0.25 mL/L) or lactic acid culture (109 CFU/mL) were added followed by calcium chloride (0.25 mL/L) (Merck). Commercial rennet (0.05 g/L) (HÁ-LA, Christian Hansen Ind. & Com. Ltda, Brazil) was incorporated, and the mixture kept at 37 °C until a firm curd was formed (approximately 40 min). The curd was gently cubed, and allowed to drain. After draining, 4 g/L of NaCl (Cisne, Refinaria Nacional de Sal S.A., Brazil) were added to the vat for salting and manually mixed. The curd was transferred to perforated cheese molds (500 g capacity) and kept for approximately 40 min for complete drainage. Cheese samples were cut into 25 g portions, packaged in polyethylene bags (Whirl-pak, Nasco), and stored at different temperatures, as shown in Table 1.

For titratable acidity determination and NaCl concentration, cheeses with no L. monocytogenes were prepared, as previously described.

All utensils used that were not autoclavable were sanitized with Dodigen 1% (v/v) (Royal Marck, Brazil) before being washed, and cheese whey was autoclaved before being disposed of.

Sampling

Samples were collected from milk (time zero), from fresh prepared cheeses and after 5, 10, 15, and 20 days of storage at 5 °C and 12 °C. Cheeses stored at temperature cycles (5 °C/16 h followed by 25 °C/8 h) were sampled after 2, 4, 6, and 8 days. Sampling for pH, water activity, and titratable acidity determinations were conducted in the same days. Salt concentration was evaluated on the last day of storage.

Physico-chemical analysis

Water activity (aw) at 25 °C was determined using the Novasina aw—Center instrument (Novasina AG, Switzerland). pH values were determined using pH-indicator strips (Special Indikator 4.0–7.0, Merck). Titratable acidity and salt concentration were estimated according to Brazilian official standard methods [33]. Salt concentration was determined by sodium chloride content, which was quantified from the sample ash, based on the reaction of silver nitrate with chlorides in the presence of potassium chromate as an indicator [33].

Microbiological analysis

Samples were homogenized with sterile phosphate buffer (Merck) (1:10) and decimal dilutions prepared. L. monocytogenes was enumerated by pour-plating 1 mL of the dilutions in Oxford agar (Oxoid) and incubating at 37 °C for 24 h. Black colonies were counted as L. monocytogenes and the population was estimated. For the enumeration of coliforms, 1 mL of each dilution was plated onto Petrifilm coliform count plates (3M, St Paul, USA) and incubated at 37 °C for 24 h [34]. Red colonies with gas were counted and the population was estimated. All samples were analyzed in duplicate and results expressed as mean of the counts.

Sensitivity of L. monocytogenes to coliforms, lactic acid bacteria and compounds produced by them

Antagonistic activity of coliforms and lactic acid bacteria against L. monocytogenes was evaluated using the agar diffusion technique according to Farias et al. [35] method. Three aliquots (5 μL each) of the coliforms culture (107 CFU/mL) or acid lactic bacteria (107 CFU/mL) were inoculated on TSB plus 1.5% agar plates. The plates were incubated at 37 °C for 24 h and then were covered with an overlay of TSB plus 0.75% agar containing each of the L. monocytogenes cultures (107 CFU/mL). After incubation at 37 °C for 24 h, the inhibition halo was measured using a ruler.

Sensitivity of L. monocytogenes to coliforms in broth

Five flasks containing 50 mL of TSB-YE were inoculated as follows: (1) 1 mL of coliforms culture (107 CFU/mL); (2) 1 mL of coliforms culture (107 CFU/mL) plus 1 mL of L. monocytogenes culture (107 CFU/mL); (3) 1 mL of coliforms culture (107 CFU/mL) plus 1 mL of L. monocytogenes culture (< 101 CFU/mL); (4) 1 mL of L. monocytogenes culture (107 CFU/mL); (5) 1 mL of L. monocytogenes culture (< 101 CFU/mL). The populations of coliforms and L. monocytogenes were estimated in the broths immediately after the addition of the bacteria and every hour for 10 h of incubation at 37 °C, and then, after 24 h. Aliquots were decimal pour-plated on Oxford Agar and plated on Petrifilm coliform count plates, and incubated at 37 °C. After 24 h, the colonies were counted and the populations estimated.

Sensitivity of L. monocytogenes to lactic acid

Ten flasks containing 50 mL of TSB-YE were prepared, and to each pair, different concentrations of lactic acid were added, in order to obtain final concentrations of 0.1, 0.3, 0.6, and 1.1%. Two flasks with no lactic acid were used as controls. To one set of flasks, aliquots of 1 mL of the L. monocytogenes culture (109 CFU/mL) were added, and to the other set of flasks, aliquots of 1 mL of the L. monocytogenes culture (101 CFU/mL) were added. The population of L. monocytogenes was immediately estimated (time zero) and after 6, 12, 24, and 32 h of incubation at 37 °C by serially diluting and pour-plating in TSA-YE, and incubating at 37 °C. After 24 h, the colonies were counted and the populations were estimated.

Statistical analysis

After a detailed exploratory data analysis, a mixed model (fixed and random effects) was used to obtain the mean comparisons between the different treatments [36]. Transformations in outcomes were applied when the residuals suppositions were violated. The PROC MIXED procedure of the software SAS 9.0 was used in the analysis. The mean comparisons were presented in tables containing the estimated values, standard error, and the p value.

Results and discussion

Water activity of the cheeses decreased slightly during storage under refrigeration, usually starting above 0.98 and finishing at around 0.97. They can be considered high aw values all throughout storage since they did not reach values lower than 0.975 for samples stored at 5 °C or 0.971 for the other conditions. The high aw observed throughout storage time is permissive for bacterial growth, since L. monocytogenes has the capacity of multiplying in aw as low as 0.92 [3, 11]. Similarly, small decreases in aw values of MFC produced with addition of lactic acid and lactic culture have been observed by Buriti et al. [37], during 21 days of storage at 5 °C.

The pH values obtained in cheeses prepared with lactic acid varied from 7.0 (in time zero) to 5.0 (last day) and from 7.0 (in time zero) to 4.7 (last day) when using lactic culture (Supplementary material). The decrease of the cheese pH was not low enough to be considered a limiting factor for the proliferation of the studied microorganisms. The optimal pH for the growth of most bacteria is near neutrality, and growth is decreased in values lower than 5.0 [38]. In this work, cheeses acidified with lactic acid presented higher pH values than the ones acidified with lactic culture (p < 0.05) (Supplementary material), which can be explained by the continuous production of acid by the lactic culture and, consequently, a decrease in the pH during the storage time [5]. Acidification with lactic acid is restricted to cheese manufacturing, with no additional pH decrease over time, as previously seen [38].

It was observed that cheeses stored at 5 °C generally had higher pH than those stored at 12 °C (p < 0.05). This pH reduction is caused by a higher growth rate of coliforms and lactic acid bacteria at abusive storage temperature (12 °C). When comparing the three kinds of cheese (L, LC, and C) maintained in the different storage temperatures, it was observed that cheeses L tend to present higher pH values, while lower pH values are found in cheeses containing coliforms. This can be due to lactose metabolism by coliforms that generates organic acids and causes the observed reduction in pH (Supplementary material).

In fact, this could be confirmed by the determination of titratable acidity. Cheeses produced with no coliforms (control cheeses) and stored at 5 °C presented less acidity, starting at 0.1 g/100 g on production day to a maximum of 0.3 g/100 g after 20 days of storage. Cheeses containing coliforms presented maximum acidity of 1.2 g/100 g after 20 days, when stored at the abuse temperature (12 °C), and 0.6 g/100 g, when stored at 5 °C. Cheeses inoculated with coliforms and stored at temperature cycles also showed higher titratable acidity, varying from 0.1 to 1.2 g/100 g, than those with no coliforms (0.1 to 0.6 g/100 g) and stored in the same condition (p < 0.05). In this study, of the 51 strains evaluated in the coliform suspension, 44 (86.3%) were identified as Escherichia coli, a strong lactose fermenter, besides Citrobacter diversus (5.9%), Citrobacter freundii (2.9%), Serratia liquefaciens (2%), and Klebsiella oxytoca (2%).

Sodium chloride addition to foods presents important functions such as modifying flavor, reducing water activity, and inhibiting the growth of undesirable bacteria. In MFC, the concentration of salt is around 1.2%, which is not sufficient to inhibit bacterial growth. The cheeses stored under temperature alternation regimen presented lower salt concentrations (0.10 to 0.30%), and those stored at 5 °C the highest values (0.28 to 0.43%) at the end of storage. This explains that loss of salt is directly proportional to storage temperature.

The behavior of coliforms in cheeses was similar in all conditions. The initial population of this group of microorganisms varied between 106 and 107 CFU/mL in the milk, and increased to 108–109 CFU/g by day 5 in the cheeses stored at 5 and 12 °C, and by day 2 for those stored under temperature alternation. This population remained constant until the end of storage. Significant differences (p < 0.05) were verified when comparing the populations of coliforms from cheeses produced with different combinations of acidification and storage. From cheeses storage at 5 and 12 °C, higher populations were found in those with abuse temperatures (12 °C), as expected. At various times when this difference was found (p < 0.05), the populations of coliforms were lower in cheeses acidified with lactic acid culture, indicating that lactic acid bacteria may have inhibited this group of microorganisms.

The increment in population of L. monocytogenes in Minas fresh cheese produced with high (105 CFU/mL) and low (< 101 CFU/mL) population of the pathogen is presented in Figs. 1 and 2, respectively. It can be observed that when L. monocytogenes is alone in cheeses stored at 5 °C (1A) or 12 °C (1B), the population is higher than when it is co-inoculated with coliforms (p < 0.05). Moreover, this increment was significantly higher in cheeses acidified with lactic acid than in those produced using lactic acid culture (p < 0.05). Cheeses containing higher population of L. monocytogenes and stored at 5 °C presented greater increment in the population of the pathogen, when compared to those stored at 12 °C (Fig. 1 a and b).

Fig. 1.

Fig. 1

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Increment in population of L. monocytogenes in minas fresh cheese produced with a high population of the pathogen inoculated alone or in co-cultured with coliforms. Cheeses were produced by acidification with lactic acid (full lines, LA) or lactic acid culture (dotted lines, LAC), stored at 5 °C (a), 12 °C (b), and 5 °C/16 h and 25 °C/8 h (c). Results represent the mean of three repetitions. Lm = L. monocytogenes

Fig. 2.

Fig. 2

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Increment in population of L. monocytogenes in minas fresh cheese produced with a low population of the pathogen inoculated alone or in co-cultured with coliforms. Cheeses were produced by acidification with lactic acid (full lines, LA) or lactic acid culture (dotted lines, LAC), stored at 5 °C (a), 12 °C (b), and 5 °C/16 h and 25 °C/8 h (c). Results represent the mean of three repetitions. Lm = L. monocytogenes

Many lactic acid bacteria (LAB) can inhibit pathogenic and spoilage microorganisms, besides exerting influence on the sensorial and physico-chemical characteristics of the cheeses [39, 40]. This inhibition can result from the production of natural antimicrobial agents, such as lactic acid, bacteriocins, and hydrogen peroxide, or due to competition [11, 41–43]. Recent studies indicated that lactic acid bacteria showed capacity to inhibit L. monocytogenes in cheeses [1, 14, 44]. The antimicrobial effect of LAB coupled with plant extract in packaging films against L. monocytogenes has also been investigated in fresh soft cheese [5].

An unexpected behavior of the pathogen was observed in cheeses prepared with a high population of L. monocytogenes and stored under temperature alternation regimen (Fig. 1c). It was verified that cheeses produced using lactic culture presented higher increment on populations of L. monocytogenes than those produced with lactic acid. A hypothesis to explain these findings is that the slow decrease in pH due to lactic acid bacteria metabolism could have triggered some stress response of L. monocytogenes, protecting the pathogen from the low pH [45, 46]. The pH values characteristic for soft cheese (4.5 to 6.5) and final NaCl content (2.3 to 3.5%) represent sublethal conditions for L. monocytogenes that can induce tolerance to more severe pH and salt conditions [3, 14, 44].

Listeria monocytogenes population increment is not affected by initial numbers of the pathogen. Figure 2 shows that when cheeses were produced only with low population of L. monocytogenes, a significant higher increment was also observed when compared to those produced with L. monocytogenes and coliforms, similarly to what has been described for initial high inoculum of L. monocytogenes.

Some reports could be found on the interactions between L. monocytogenes and other microorganisms in soft cheeses. Carlin et al. [46] evaluated the influence of the natural microbiota in the population of L. monocytogenes in minimally processed endive. The authors found that, similarly to what was observed in the present study, there is a competition between L. monocytogenes and the other microorganisms and that L. monocytogenes presented higher population increment when grown alone. Mirkovic et al. [14] revealed antibacterial potential of Lactococcus lactis subsp. lactis against L. monocytogenes and S. aureus in fresh soft cheese during 21 days at 4 °C. Trmcic et al. [47] detected the presence of coliforms and pathogenic bacteria in fresh cheeses, but no association was found between coliform and Listeria sp.

In the present study, in order to evaluate if the behavior observed in the cheeses was caused by antagonistic activities of coliforms and lactic acid bacteria, in vitro tests were carried out. According to Table 2, we observed that the largest zones of L. monocytogenes inhibition were promoted by coliforms or by compounds produced by them, indicating greater inhibition by this group of microorganisms. The strain ATCC 7644 was an exception, which was only inhibited by lactic acid bacteria.

Table 2.

Size of inhibition halo (mm) according to the method of Farias et al. (1992)

Indicator microorganism (L. monocytogenes) Inhibitor Size of inhibition zones (mm)
ATCC 7644 LAB 1.2
15 E LAB 1.2
30 LAB 1.2
ATCC 7644 coliform ND
15 E coliform 1.6
30 coliform 1.5
ATCC 7644 LAB_sup. 1.2
15 E LAB_sup. 1.3
30 LAB_sup. 1.3
ATCC 7644 coliform_sup. ND
15 E coliform_sup. 1.6
30 coliform_sup. 1.6
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LAB, lactic acid bacteria (Ezal MAO11—Danisco, Copenhagen, Denmark); LAB_sup, supernatant of the lactic acid bacteria culture; coliform_sup, supernatant of the coliform culture; ND, not detected

Interactions between the microorganisms were also evaluated in vitro by co-culturing high (105 CFU/mL) and low (< 101 CFU/mL) populations of L. monocytogenes and coliforms (107 CFU/mL) in TSB incubated at 37 °C (Fig. 3). In the first experiment (L. monocytogenes 105 CFU/mL), it was observed that the L. monocytogenes growth rate was slower when co-cultured with coliforms, comparing with L. monocytogenes alone, although the population increase of 3 logs was still observed in both cases (co-culture and L. monocytogenes alone), during 24 h. Experiments with low numbers of L. monocytogenes (< 101 CFU/mL) showed a stronger influence of coliforms on the pathogen’s growth rate. When L. monocytogenes was the only microorganism in the broth, there was a population increase of 8 logs, while in co-culture with coliforms, the pathogen population did not increase (Fig. 3).

Fig. 3.

Fig. 3

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Listeria monocytogenes and coliform population, isolated and in co-cultured in TSB broth, at 37 °C. Lm = L. monocytogenes

Concentrations of lactic acid similar to those found in the cheeses at the end of the shelf-life were used to simulate its influence on L. monocytogenes. The presence of at least 0.3% of lactic acid was efficient at inhibiting L. monocytogenes growth independent of the initial population (Fig. 4). It can be observed in Fig. 4b that 0.6 and 1.1% of lactic acid caused a reduction of 4 logs in the L. monocytogenes population after 24 h of incubation. This L. monocytogenes population reduction is higher than reported by others [48, 49]. However, in those studies, the matrix (beef or chicken) may have given some protection to the pathogen. The addition of 0.1% of lactic acid did not affect L. monocytogenes multiplication. This percentage was observed in the cheeses made by direct acidification, with no coliforms, and stored at 5 °C. This finding is troublesome because it indicates that cheeses prepared by direct acidification with high quality milk, and stored under good refrigeration, are more prone to allow L. monocytogenes multiplication. Thus, contamination with the pathogen is critical and preventive measures must be in place to avoid the presence of L. monocytogenes in the product. According to Engstrom et al. [50], L. monocytogenes inhibition via organic acids is attributed to the undissociated form of the acid, and predictive models confirmed antilisterial activity of several acids such as propionic, acetic, lactic, and citric acid in model soft cheese. The authors suggest that modifying the pH or the acidulant can significantly influence the growth of L. monocytogenes. Here, we have also demonstrated the influence of cheese making technology in L. monocytogenes behavior.

Fig. 4.

Fig. 4

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Behavior of L. monocytogenes in different concentrations of lactic acid in TSB, at 37 °C. Population of 101 CFU/mL (a) and population of 108–109 CFU/mL (b). Lm = L. monocytogenes

This work was developed in order to answer a question on the low occurrence of L. monocytogenes in Brazilian fresh cheeses. The objective is not to suggest the use of coliforms to inhibit the pathogen, but to understand the microbial interaction among L. monocytogenes and coliforms in this popular cheese consumed nationwide. For the control of L. monocytogenes and coliforms in food products, preventive measures and good production practices must be taken such as raw material of good microbiology quality, care for the handler’s health, pasteurization of milk, cleaning and sanitation of the production environment and utensils used, avoiding cross contamination in processing and post-processing, and cheese storage under refrigeration. The implementation and monitoring of programs such as hazards analysis and critical control points (HACCP) are highly encouraged.

Conclusions

There was a strong influence of coliforms on L. monocytogenes population, indicating that populations of L. monocytogenes in this Brazilian fresh cheese may be low due to high populations of contaminating coliforms. Despite these findings, the production of fresh cheeses with raw milk and/or in facilities lacking good hygiene practices is not recommended, in addition to being illegal. Only by following good hygiene and good manufacturing practices, together with a well-established and implemented HACCP program, will it be possible to produce safer fresh white cheeses.

Information obtained in this work regarding the influence of contaminating microbiota on L. monocytogenes multiplication capacity, as well as the information about the physicochemical properties of the cheese and technology used for its production, can be useful in elaboration of a risk assessment plan, which is a systematic way to estimate the severity of microbiological hazards and their potential to occur.

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Acknowledgments

The authors thank the São Paulo Research Foundation (FAPESP), National Council for Scientific and Technological Development (CNPq, Brazil), and Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) for financial support and scholarships. Purac do Brasil S.A. for providing the lactic acid and Rhodia S.A. (Danisco) for the lactic acid cultures and MSc Davi Casale Aragon for the statistical analysis.

Authors’ contributions

Conceptualization: [L.C. Aragon-Alegro, M.T. Destro]; Data curation: [L.C. Aragon-Alegro]; Formal Analysis [L.C. Aragon-Alegro, E.M.F. Lima]; Funding acquisition [M.T. Destro, U.M. Pinto]; Investigation [L.C. Aragon-Alegro, G. Palcich, T.P. Nunes, K.L.O. Souza, C.G. Martins, P.K. Noda]; Methodology [L.C. Aragon-Alegro; M.T. Destro]; Project administration [L.C. Aragon-Alegro, M.T. Destro]; Resources [M.T. Destro, U.M. Pinto]; Supervision [M.T. Destro]; Visualization: [E.M.F. Lima, U.M. Pinto]; Writing—original draft [L.C. Aragon-Alegro]; Writing—review and editing [E.M.F. Lima, U.M. Pinto].

Funding

The authors thank São Paulo Research Foundation (FAPESP) for financial support to: this research (04/05962–6, 04/10089–0) and to the Food Research Center – FoRC (2013/07914–8).

Data Availability

Not applicable.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Code availability

Not applicable

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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