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Fresh cheese produced in Manabí province is an Ecuadorian artisan cheese with a moisture of 37% (Minga and Pérez, 2019), with good acceptance among consumers, especially in the province of Manabí (Ecuadorian coast). Previous studies have shown that fresh cheese processing does not fulfill Ecuadorian regulations due to the presence of pathogenic microorganisms such as Salmonella (Zambrano, 2014). Additionally, during the commercialization the cold chain is affected in some points, since the cheese is exhibited at 25 °C, which increases the microbiological load affecting the health of consumers (Lobacz, 2020). Salmonellosis cases in the province of Manabí, Ecuador are around 600 annually (Ministerio de Salud Pública, 2015-2018). The presence of Salmonella and other pathogenic microorganisms has been detected in cheeses from Mexico (Plumb et al., 2019) and Egypt (El-Baz et al., 2017) among others.
Several authors have studied the control of pathogenic microorganisms in different varieties of cheese such as the control of E. coli, S. enterica, B. cereus, S. scuiri and P. aeruginosa using lactic acid bacteria (LAB) (Al-Gamal et al., 2019); the inoculation of L. rhamnosus in semi-hard goat cheese to inhibit the growth of pathogenic bacteria (Rodrigues et al., 2015) and the use of L. acidophilus to control the development of mesophilic aerobic bacteria and Salmonella in fresh cheese (Santacruz and Castro, 2018).
Coating is a layer that works as barrier against moisture, gases, loss of aroma and flavors of food. At the same time, it can act as carriers of antimicrobial microorganisms or substances (Guimarães et al., 2018). In fact, coatings have been used in cheese preservation, reducing water loss and spoilage damage (Costa et al., 2018). Moreover, the effect of coatings on several types of cheeses have been studied previously such as Gouda (Göksen et al., 2020); Port Salut (Ollé et al., 2014); Kashar (Kavas et al., 2015) among others.
The present work is a complementary study of a previous work where the growth of mesophilic aerobic bacteria and Salmonella in fresh cheese could be inhibited using L. acidophilus. The effect of a cassava starch + L. acidophilus as a coating on fresh cheese was studied, evaluating its acidity, pH, weight loss, firmness and counting of mesophilic aerobic bacteria during 30 days of storage at 4 °C. Additionally, the difference of satisfaction among panelists was evaluated for coated and uncoated cheeses.
MATERIALS Y METHODS
Strains of L. acidophilus were obtained from Chr. Hansen A/S (Denmark). The fresh cheese was purchased in stores located in the center of the city of Manta, Ecuador.
Preparation of coated cheese
The fresh cheese was coated with a starch + L. acidophilus (SL) film. A Cassava starch solution 1% (w/v) was prepared according to Santacruz et al. (2015), with slight modifications. The starch solution was heated with constant stirring from room temperature (approximately 25 °C) to 90 °C, keeping this temperature for 5 min. Afterwards, the mixture was homogenized with an ultraturrax (Polytron, Switzerland) at 11,000 rpm for 4 min. Free L. acidophilus was added to the starch solution guaranteeing a starch coating with an absorbance between 0.08 and 0.1, measured at 625 nm (Medina et al., 2005). This absorbance guaranteed an equivalent of 1x108 CFU mL-1 of L. acidophilus of starch solution.
The fresh cheese was immersed in the previous solution for 5 min, dried at room temperature and finally transferred to plastic bags before storage for 30 days at 4 °C. Every day during storage, cheese was taken out from refrigerator and kept at room temperature for 8 h, then placed again into the refrigerator. This procedure allowed to simulate commercialization conditions.
Physical, chemical and microbiological analyses of fresh cheese
Cheese samples previously coated with a SL film were analyzed after 0, 10, 20 and 30 days of storage at 4 °C. Moisture content, titratable acidity, pH, and firmness of the cheese were examined. Moisture content was determined according to method 15.259 (AOAC, 1984), titratable acidity with method 16.276 (AOAC, 1984), pH was measured using a potentiometer (HANNA, Germany), firmness was measured as the maximum force (N) required to penetrate cheese cubes with a side length of 5 cm, using a texturometer (SHIMADZU EZ-XL, Japan). For the penetration test, a 2 mm diameter penetration probe was used at a speed of 20 mm s-1, with a penetration depth of 15 mm (Santacruz, 2021). The determination of mesophilic aerobic bacteria was carried out according to Castro et al., (2014). Uncoated cheese was used as a control sample for all analyzes.
Sensory analysis
Sensory analyses were done at 0 day of storage only, with cheese containing no Salmonella. This avoided health problems to panelists due to the growth of mesophilic aerobic bacteria in cheese during storage. Presence of Salmonella was analyzed by the Ecuadorian regulation for microbiological food control (INEN, 2015). For this stage, eight semi-trained panelists were involved in both tests (UPAEP, 2014). The panelists were previously chosen based on their ability to identify basic flavors (Sharif et al., 2017). First, a duo-trio test was carried out in two sessions, which aimed to determine if there was a difference between the sample coated with SL and the control sample. In this case, the number of correct responses was determined for a probability level of 5%. Afterwards, a satisfaction test was performed using a 5-point verbal hedonic scale (Wadhwani and McMahon, 2012), with a scale ranging from "I like it a lot" (5) to "I dislike it a lot" (1).
RESULTS AND DISCUSSION
The presence of L. acidophilus in cheese caused a lower pH and acidity compared to the control sample after 30 days of storage. Similar results were obtained by Mozuriene et al. (2016). The lower presence of mesophilic aerobic bacteria due to the inhibitory action of L. acidophilus may trigger a lower production of acidic metabolites and thus a lower acidity. Additionally, the increase of L. acidophilus may be responsible of a higher production of organic acids that act as buffer, controlling the increase of pH during storage. Regarding the weight loss, coated cheese showed a lower loss compared to control sample. The coating may act as a barrier against moisture loss.
Results of microbiological analyzes showed that control cheese had a higher quantity of mesophilic aerobic bacteria than the sample coated with L. acidophilus (P<0.05, Table 1). Santacruz and Castro (2018) showed that viability of L. acidophilus decreased along storage, however the viable cells inhibited the development of mesophilic aerobic (Mozuriene et al., 2016) and Salmonella. Coatings were able to control the release of the antimicrobial agents on the cheese surface improving the microbiological quality of the cheese (Santacruz and Castro, 2018; Krishnan et al., 2015) and Salmonella spp. in fresh cheese. Previous studies showed the inhibition of E. coli, L. monocytogenes and S. enteritidis by LAB (Winkowski et al., 1993; Lord, 2002). The presence of LAB could inhibit microorganisms through the production of bacteriocins (Aymerich et al., 2000).
Table 1 pH, acidity, weight loss, firmness and mesophilic aerobic bacteria counting in cheese stored at 4 °C during 30 days.
Sensory analysis
Results of the duo-trio test showed that the number of correct responses was 12 of 16, which showed a difference between the cheese samples coated with L. acidophilus compared to control sample (Wittig, 2001). Another aspect to evaluate was the sensorial acceptation by the panelists. The results showed that there was no difference in panelists satisfaction between coated and control sample (P<0.05). Both received a rating between "I neither like nor dislike" and "I like it moderately", with values of 3.63 and 3.50 for coated and control sample, respectively. Previous works showed similar results; in fact, LAB strains increased the acceptability and shelf-life of unripe curd cheese (Mozuriene et al., 2016). Coelho et al. (2014) found no significant differences in overall sensory evaluation of fresh cheese. Even after the addition of L. acidophilus, the semi-hard goat cheese showed better sensory scores in the acceptance test (Fernandes et al., 2012). The SL film could be used as an agent to control the growth of mesophilic aerobic bacteria in fresh cheese with no changes on the satisfaction among consumers.
CONCLUSIONS
The use of a cassava starch +L. acidophilus film inhibited the development of mesophilic aerobic in fresh cheese showing no difference in panelists satisfaction between coated and control sample. SL film-coated cheese could act as a barrier against moisture loss and may be a promising option to control mesophilic aerobic bacteria in fresh cheese during storage under refrigeration conditions with no changes in consumers satisfaction.
