Requeson is a type of traditional Mexican fresh cheese, not matured, without a crust, white, and spreadable, similar to Ricotta cheese. It is slightly salted and has a grainy texture (Ramírez-López and Vélez-Ruíz, Reference Ramírez-López and Vélez-Ruíz2012). It is made from pasteurised whey as its main ingredient, rich in lactose and whey proteins and low in fat, thereby utilising the proteins and other milk components (Álvarez-Martínez, Reference Álvarez-Martínez2012). This cheese is produced by coagulating whey proteins through the combination of an acid medium and heat application. The consumption of this traditional product is widespread in Mexico and is more frequent in rural areas (Ramírez-López and Vélez-Ruíz, Reference Ramírez-López and Vélez-Ruíz2012).
The yields of Requeson production are low, ranging between 2.5% and 4% (Masotti et al., Reference Masotti, Cattaneo, Stuknyte and De Noni2016). An alternative approach to increasing yields in the production of this type of product is the inclusion of exopolysaccharide (EPS) in the matrix through whey fermentation with lactic acid bacteria (LAB) or by adding it as an ingredient to the formulation. The EPS produced by LAB improves their sensory and techno-functional properties, such as viscosity and production yields, due to their high capacity for water retention (Hussain et al., Reference Hussain, Mahmood, Tabasum, Noreen, Ali, Iqbal and Zuber2017; Lynch et al., Reference Lynch, Coffey and Arendt2017). For this reason, its use in dairy products such as, yoghurt and cheese has been reported (Jiménez-Guzmán et al., Reference Jiménez-Guzmán, Flores-Nájera, Cruz-Guerrero and García-Garibay2009; Lluis-Arroyo et al., Reference Lluis-Arroyo, Flores-Nájera, Cruz-Guerrero, Gallardo-Escamilla, Lobato-Calleros, Jiménez-Gumán and García-Garibay2014).
Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 is well-suited for dairy fermentations due to its high EPS production, robust proteolytic system and efficient metabolism of lactose. The EPS produced by this strain improves the texture and water-holding capacity of dairy matrices, thereby enhancing both yield and structural consistency of whey-based cheeses. Furthermore, its generally recognised as safe (GRAS) status, along with its established role in yoghurt production, supports its potential application in clean-label, functional dairy products (Gentès et al., Reference Gentès, Turgeon and St-Gelais2016; Van Calsteren et al., Reference Van Calsteren, Gagnon, Nishimura and Makino2015).
In a related strain, L. delbrueckii subsp. bulgaricus OLL1073R-1, the EPS has been well characterised, exhibiting a weight-average molar mass of approximately 5.0 × 10⁶ Da. It is composed mainly of D-glucose and D-galactose in a molar ratio of 1:1.5. Unlike dextran, which is composed exclusively of glucose units, this EPS is a heteropolysaccharide (Van Calsteren et al., Reference Van Calsteren, Gagnon, Nishimura and Makino2015). Dextran, however, remains a common standard for EPS quantification due to its availability and structural similarity, facilitating reliable colourimetric estimations. Beyond its technological benefits, such as improving texture and reducing syneresis in dairy products, this biopolymer has also shown antioxidant activity in vitro (Durlu-Özkaya et al., Reference Durlu-Özkaya, Aslim and Taha Ozkaya2007; Tang et al., Reference Tang, Dong, Wang, Han, Rui, Chen, Jiang, Zhang, Wu and Li2017; Van Calsteren et al., Reference Van Calsteren, Gagnon, Nishimura and Makino2015). Therefore, the EPS from this species holds promise as a functional ingredient for developing novel dairy products, including traditional varieties such as Requeson. On the other hand, current consumption patterns have favoured the development of functional foods in the dairy industry, including those enhanced with probiotic cultures that offer immunological benefits. Most of these probiotics are added to liquid fermented dairy products, such as yoghurt; however, their use in cheese has been less studied. L. casei 431 is a probiotic LAB used in yoghurt production and fermentation (Kanmani et al., Reference Kanmani, Albarracin, Kobayashi, Iida, Komatsu, Kober, Ikeda-ohtsubo, Suda, Aso, Makino and Kano2018). It stands out from other probiotics since it helps decrease the incidence of respiratory and gastrointestinal diseases, strengthens the immune system and has been studied as an antimutagenic agent (Albano et al., Reference Albano, Morandi, Silvetti, Casiraghi, Manini and Brasca2018; Plessas et al., Reference Plessas, Nouska, Karapetsas, Kazakos, Alexopoulos, Mantzourani, Chondrou, Fournomiti, Galanis and Bezirtzoglou2017; Terpou et al., Reference Terpou, Bekatorou, Bosnea, Kanellaki, Ganatsios and Koutinas2018).
Material and methods
Microorganisms
A commercial culture of L. delbrueckii subsp. bulgaricus NCFB 2772 from the National Collection of Food Bacteria (Aberdeen, Scotland) produced EPS. Additionally, a commercial culture of L. paracasei subsp. paracasei, known as L. casei 431 from the CHR HANSEN laboratory (Horsholm, Denmark), was used as a probiotic bacterium.
Activation of starter culture
The activation process began with 1 mL of L. delbrueckii subsp. bulgaricus NCFB 2772 culture at a concentration of 107 CFU/mL, previously stored −21°C. This inoculum was transferred to 9 mL of sterile skimmed milk (Difco, France), which had been autoclaved at 121°C for 15 minutes. Then, it was incubated at 42°C for 24 hours. Subsequently, the entire 10 mL of fermented culture was transferred to a sterile flask containing 90 mL of sterile skimmed milk and was incubated at 37°C for 24 hours.
Sweet whey preparation
A total of 10 L of raw bovine milk was used to obtain the sweet whey required for the Requeson formulations evaluated in this study. The milk is sourced from Holstein cows raised in a cowshed located in Iztapalapa, Mexico City, and was first pasteurised at 65°C for 30 minutes. After cooling to 40°C, 2 mL of rennet (CHR HANSEN A/S, Denmark; 280 U/mL) was added and gently stirred to ensure uniform distribution of the coagulant. The mixture was then left undisturbed at 40°C until coagulation occurred (approximately 30 minutes). The curd was cut into ∼1 cm3 cubes, and the whey was separated by filtering through a sterile cheesecloth. The collected sweet whey was subsequently pasteurised (65°C for 30 minutes) and stored at 4°C until use.
Fermentation of sweet whey and EPS quantification
In the first phase of the study, sweet whey was fermented using L. delbrueckii subsp. bulgaricus NCFB 2772 at two incubation temperatures (37 and 42°C) for 48 hours. The sweet whey was inoculated with activated starter culture at 5% (v/v), and fermentations were carried out in triplicate.
For EPS quantification, 6 mL of fermented whey was mixed with 1 mL of 80% (w/v) trichloroacetic acid to precipitate proteins. The mixture was agitated for 30 seconds and allowed to stand at room temperature for 20 minutes. Subsequently, samples were centrifuged at 10,000 rpm and 4°C for 30 minutes. The supernatant, containing water-soluble EPS, was collected and mixed with an equal volume of absolute ethanol to precipitate the polysaccharides. After vortexing for 30 seconds, the mixture was kept at 4°C for 20 minutes.
The EPS concentration was then estimated by measuring turbidity at 720 nm using a UV-1800 spectrophotometer (Shimadzu, Japan). Absorbance values were interpolated from a standard calibration curve prepared with dextran (MW ∼9400; Sigma Aldrich, Germany) at concentrations ranging from 0 to 1 mg/mL. The use of dextran as a standard is justified by the compositional similarity, as EPS from L. delbrueckii subsp. bulgaricus predominantly contains glucose and galactose (Van Calsteren et al., Reference Van Calsteren, Gagnon, Nishimura and Makino2015).
Requeson production
Four different formulations of Requeson were prepared as follows: 1. Control: non-fermented sweet whey; 2. Whey + EPS: sweet whey fermented with L. delbrueckii subsp. bulgaricus NCFB 2772; 3. Whey + Probiotic: non-fermented sweet whey supplemented with L. casei 431 and 4. Whey + EPS + Probiotic: sweet whey fermented with L. delbrueckii subsp. bulgaricus NCFB 2772 and supplemented with L. casei 431. Each formulation was produced in triplicate.
For treatments involving fermentation (formulations 2 and 4), sweet whey was fermented as described previously. In the non-fermented treatments (formulations 1 and 3), the pH of the sweet whey was adjusted to 4.7 using 50% (w/v) citric acid.
All whey samples were subsequently heated at 90°C for 60 minutes, with temperature monitored every 10 minutes. Curd formation was promoted by rapid cooling to 4°C for 10 min. The resulting curd was collected using cheesecloth (0.5 mm pore size), and 1% (w/w) sodium chloride was added to each batch.
In probiotic-supplemented treatments (formulations 3 and 4), L. casei 431® was added at 5% (w/w), in accordance with supplier instructions, to reach a final concentration of 107 CFU/g. Finally, all Requeson samples were packaged in sterile polyethylene containers and stored at 4°C under refrigeration.
Physicochemical analysis
The pH of the fermented sweet whey was measured by direct immersion of a glass electrode. Moisture content was determined using the AOAC method 926.08 (AOAC, 2021), by drying 1 g of Requeson in pre-weighed aluminium capsules at 70°C in a drying oven until a constant weight was achieved. Fat content was assessed by solvent extraction using the AOAC method 920.39C (AOAC, 2021). Approximately 10 g of Requeson was treated with ammonia and ethanol, followed by successive extractions with diethyl ether and petroleum ether. The combined ether extracts were evaporated in a pre-weighed flask, and the residue was dried at 100°C until it reached a constant weight. Ash content was measured according to AOAC method 935.42 (AOAC, 2021). For this, 3 g of Requeson were weighed in porcelain crucibles and incinerated in a muffle furnace at 550°C for approximately 3 hours. The total protein content was determined using the Kjeldahl method. Initially, 0.15 g of Requeson was digested with sulfuric acid, followed by distillation with NaOH in 2 % (w/v) boric acid. Finally, the titration of the distilled product obtained was carried out with 0.1 N HCl. A nitrogen-to-protein conversion factor of 6.38 (AOAC, 2021) was used to convert the total nitrogen content into protein.
Texture analysis
The texture of the Requeson was measured using the texturometer model CT310K (Brookfield, United States), equipped with a 35 mm cylindrical probe. Each sample consisted of 18 g of Requeson, shaped into cubes measuring 40 mm in length, 30 mm in width and 15 mm in depth. The samples were wrapped in aluminium foil and allowed to rest at room temperature for 20 minutes before analysis. The samples were compressed up to 20% of their initial height at a speed of 2 mm/s and 30 mm from the surface. The test was performed in two cycles, where the following parameters were measured: hardness, cohesiveness, adhesiveness and elasticity.
Instrumental colour analysis
Colour measurements were performed using a portable colourimeter (Model NH310, Shenzhen ThreeNH Technology Co., Ltd., China), recording values in the CIELAB colour space: L* (luminosity), a* (chromaticity of green – a* to red + a*), b* (blue – b* to yellow + b*).
Shelf life of Requeson
Stability assessments were conducted on days 0, 7, 14 and 21. On each evaluation day, samples were vacuum-sealed in polyethylene bags at 0.8 bar and stored at 4°C. The following parameters were analysed: pH, total and faecal coliforms, moulds and yeasts (Secretaría de Salud, Reference de Salud2014), texture, and colour. Standard plate count assessed probiotic viability.
Consumer sensory evaluation
Sensory acceptability was assessed with 67 potential consumers aged 18–50. Each participant received 2 g portions of the four Requeson formulations and rated them on a 9-point hedonic scale (1 = dislike extremely; 9 = like extremely). The evaluation was conducted at the Universidad Autónoma Metropolitana, Unidad Iztapalapa.
Ethics statement
All participants provided informed consent prior to participation in the sensory test. This study is part of the divisional project “Obtaining bioactive compounds with an impact on health from lactic acid bacteria” (Obtención de compuestos bioactivos con incidencia en la salud a partir de bacterias ácido lácticas), approved by the Ethics Committee of UAM-Iztapalapa (Comisión Académica de Ética de la División de Ciencias Biológicas y de la Salud, UAM-Iztapalapa) under the ruling number 1913 (dictamen 1913).
Statistical analysis
All results were analysed using one-way analysis of variance (ANOVA), followed by a Tukey test for multiple comparisons with an α value of 0.05. Statistical computations were performed using Minitab 17 software (Pennsylvania, United States).
Results and discussion
EPS production in sweet whey fermentation
EPS production in cheese manufacturing enhances both yield and functional properties of the cheese. This study evaluated in situ EPS production via fermentation of sweet whey (initial pH 6.7) using L. delbrueckii subsp. bulgaricus NCFB 2772, which predominantly employs the Wzx/Wzy-dependent pathway for heteropolysaccharide biosynthesis (Schmid et al., Reference Schmid, Sieber and Rehm2015). During the initial phase, the production of EPS was studied in two temperature conditions: 37°C and 42°C. EPS values reached 1.126 ± 0.12 mgeq of dextran/mL. The high yield can be attributed to optimal activation of the biosynthetic pathway, wherein lactose hydrolysis provides glucose and galactose for glycosyltransferase-mediated assembly of repeating units. The subsequent polymerisation and export via the Wzx/Wzy pathway are favoured by the neutral pH and temperatures that maintain membrane fluidity (Nguyen et al., Reference Nguyen, Nguyen, Bui, Hong, Hoang and Nguyen2020; Schmid et al., Reference Schmid, Sieber and Rehm2015).
No statistically significant differences in EPS concentration were observed between the two fermentation temperatures (p = 0.241). In contrast, a previous study by this research group, using acid whey (pH 4.7) and the same bacterial strain, reported a markedly lower EPS concentration of 0.276 ± 0.04 mgeq dextran/mL (Carrero-Puentes et al., Reference Carrero-Puentes, Fuenmayor, Jiménez-Pérez, Guzmán-Rodríguez, Gómez-Ruiz, Rodríguez-Serrano, Alatorre-Santamaría, García-Garibay and Cruz-Guerrero2022). This supports the notion that sweet whey (pH 6.7) constitutes a more favourable substrate by optimising sugar transport and phosphorylation, enhancing glycosyltransferase activity and stabilising the undecaprenyl-phosphate carrier, all of which are critical components in the Wzx/Wzy-dependent mechanism (Kanmani et al., Reference Kanmani, Albarracin, Kobayashi, Iida, Komatsu, Kober, Ikeda-ohtsubo, Suda, Aso, Makino and Kano2018; Schmid et al., Reference Schmid, Sieber and Rehm2015).
Regarding oxygen availability, the present study was conducted under aerobic conditions. It yielded higher EPS concentrations than those reported by Briczinski and Roberts (Reference Briczinski and Roberts2002), who obtained 0.33 mg/mL after 28 hours of anaerobic fermentation of sweet whey using L. delbrueckii subsp. bulgaricus RR. Although anaerobic environments are often associated with enhanced EPS yields due to redirected carbon fluxes towards nucleotide sugar biosynthesis (De Vuyst and Degeest, Reference De Vuyst and Degeest1999), strain-specific metabolic responses may counter this trend. Under oxidative stress, some LAB may upregulate EPS secretion as a protective mechanism against reactive oxygen species or dehydration (Nguyen et al., Reference Nguyen, Nguyen, Bui, Hong, Hoang and Nguyen2020). In such cases, the greater energy availability under aerobic conditions may favour EPS biosynthesis, particularly when biomass accumulation is not a priority.
Furthermore, Nguyen et al. (Reference Nguyen, Nguyen, Bui, Hong, Hoang and Nguyen2020) noted that oxidative stress could enhance LAB growth in the presence of EPS, potentially promoting secretion in oxygen-sensitive strains. However, it is important to emphasise that these findings focused primarily on growth, not polysaccharide yield. Thus, while oxygen availability does not uniformly enhance EPS production, it can modulate stress responses and metabolic routing depending on strain-specific traits and environmental conditions. Finally, Domínguez-Soberanes (Reference Domínguez-Soberanes1997) reported lower EPS concentrations (0.130 mgeq dextran/mL) after 12 hours of sweet whey fermentation at 42°C using the same strain. The higher yields in the present study are likely due to the extended fermentation time.
Effects of EPS and probiotics on the yield and composition of Requeson
The incorporation of EPS into Requeson formulations significantly improved both yield and composition. As shown in Table 1, treatments containing EPS, either alone or combined with L. casei 431, achieved yields over 230% higher than the control. In contrast, the addition of the probiotic alone did not affect yield, highlighting the central role of EPS. These findings are consistent with those of Patlán-Velázquez et al. (Reference Patlan-Velázquez, González-Olivares, García-Garibay, Alatorre-Santamaría, Gómez-Ruiz, Rodríguez-Serrano and Cruz-Guerrero2024), who reported increased yield in Requeson-type cheese fermented with EPS-producing strains of the same L. delbrueckii subsp. Bulgaricus used in this work and Streptococcus thermophilus SY-102.
Yields, quantification of EPS and composition of types of Requeson
Different letters in the column indicate a significant difference (Tukey p < 0.05). EPS, exopolysaccharide. Probiotic: Lactobacillus casei 431.
The improvement is mainly attributed to the high water-binding capacity of EPS and its interaction with whey proteins, which enhances moisture retention and protein aggregation. In the present study, EPS levels exceeded 200 mgeq dextran/g of Requeson, in agreement with prior reports. The resulting matrix exhibited significantly higher moisture and protein content (p < 0.05), likely due to reduced syneresis and improved structural cohesion. Similar effects have been described in other cheeses, where EPS reinforces the protein network, particularly under acidic conditions (pH < 5.0), by hydrophilic and electrostatic interactions.
Notably, fat content remained unaffected by EPS or probiotic addition, suggesting that the observed changes are specifically related to water and protein dynamics. Similar moisture-enhancing effects have been reported in Manchego and Panela cheeses (Lluis-Arroyo et al., Reference Lluis-Arroyo, Flores-Nájera, Cruz-Guerrero, Gallardo-Escamilla, Lobato-Calleros, Jiménez-Gumán and García-Garibay2014; Jiménez-Guzmán et al., Reference Jiménez-Guzmán, Flores-Nájera, Cruz-Guerrero and García-Garibay2009), supporting the role of EPS as a functional ingredient in dairy matrices.
Overall, EPS supplementation represents a promising strategy for whey valorisation, enhancing cheese yield and nutritional quality while reducing waste. Its reproducibility, compatibility with probiotic cultures and process scalability make it suitable for fresh cheese production.
In this study, the EPS concentration achieved 1.126 ± 0.12 mgeq dextran/mL using L. delbrueckii subsp. bulgaricus NCFB 2772 was consistent with previous observations. For instance, Patlan-Velázquez et al. (Reference Patlan-Velázquez, González-Olivares, García-Garibay, Alatorre-Santamaría, Gómez-Ruiz, Rodríguez-Serrano and Cruz-Guerrero2024) reported higher levels (up to 1.49 mg/mL) when this strain was cocultured with S. thermophilus SY-102, whereas lower concentrations were obtained in monocultures. Despite using a monoculture, our results confirm the robust EPS-producing capacity of this strain under the tested conditions. These outcomes are comparable to those of Wang et al. (Reference Wang, Song, Zhao, Xiao, Zhou and Han2019), who observed improved texture and antioxidant activity in low-fat Cheddar cheese supplemented with EPS from L. plantarum JLK0142. Interestingly, while their EPS-treated cheese showed reduced cohesiveness, our EPS-containing Requeson exhibited enhanced cohesiveness and firmness, differences likely attributed to cheese type and formulation. Finally, the use of in situ whey fermentation as a natural EPS delivery system provides a cost-effective and scalable alternative to EPS purification. This strategy aligns with the work of Li et al. (Reference Li, Ding, Chen, Shi and Wang2020), who demonstrated the feasibility of transforming cheese whey into value-added EPS using L. plantarum. Collectively, these results reinforce the technological potential and industrial relevance of the approach presented here.
Texture and colour analysis of Requeson
Instrumental texture analysis (Fig. 1) revealed that the highest values for hardness, elasticity and cohesiveness were observed in Requeson formulations with whey, EPS and L. casei 431, as well as in formulations containing whey and EPS alone. These results indicate that both EPS and the probiotic strain contribute to improving the cheese is rheological properties. These findings are consistent with Lluis-Arroyo et al. (Reference Lluis-Arroyo, Flores-Nájera, Cruz-Guerrero, Gallardo-Escamilla, Lobato-Calleros, Jiménez-Gumán and García-Garibay2014), who reported enhanced moisture and fat retention, yield, and cohesiveness in Manchego-type cheese supplemented with an EPS-producing S. thermophilus. In their study, EPS promoted a denser protein matrix, yielding a softer texture and improved water-holding capacity.
Texture profile analysis (TPA) of the different Requeson formulations.
Fundamental differences in cheese type and EPS characteristics may account for the contrasting textural outcomes observed between the two studies. Requesón is a fresh, high-moisture, whey-based cheese that does not undergo pressing or ripening, providing conditions in which EPS can effectively reinforce the protein–water network during curd formation. In contrast, Manchego-type cheese is subjected to pressing and maturation, resulting in a firmer, low-moisture matrix where EPS may promote water retention and lead to a softer texture. Moreover, structural and functional variations between the EPS produced by L. delbrueckii subsp. bulgaricus NCFB 2772 and S. thermophilus, as well as their distinct interactions with whey or casein-based protein matrices, likely contribute to the divergent effects on cheese texture.
Similarly, Patlán-Velázquez et al. (Reference Patlan-Velázquez, González-Olivares, García-Garibay, Alatorre-Santamaría, Gómez-Ruiz, Rodríguez-Serrano and Cruz-Guerrero2024) evaluated a Requeson-type cheese made from whey fermented with EPS-producing LAB. Using the same L. delbrueckii subsp. bulgaricus strain employed in the present study, co-cultured with S. thermophilus SY-102, and they observed increased EPS production and enhanced texture and sensory stability. Moreover, Buriti et al. (Reference Buriti, da Rocha, Assis and Saad2005) demonstrated that the inclusion of L. paracasei to Minas fresh cheese improved parameters such as hardness and cohesiveness, likely through protein–microbe interactions and microbial structuring of the matrix.
Conversely, the highest adhesiveness was recorded in the control Requeson. This inverse relationship between EPS content and adhesiveness agrees with Korish and Elhamid (Reference Korish and Elhamid2012), who reported that hydrocolloids like pectin and carboxymethylcellulose reduced adhesiveness and improved both rheological and sensory qualities in Karish cheese. In fresh cheeses, lower adhesiveness is typically associated with enhanced mouthfeel and higher consumer acceptance. It is worth noting that the L. casei 431 strain used in this study was added to the cheese matrix in accordance with the instructions provided by the supplier. Only the microbial biomass was incorporated, without any additional carriers or media. A treatment with the probiotic alone (without EPS) was included to evaluate the contribution of the microorganism independently. As no significant changes in yield or texture were observed in this treatment, the physical properties observed in the EPS-containing cheeses are likely attributable to the presence of the EPS. Regarding colour, CIELAB measurements (Fig. 2) revealed significantly lower L* values (p < 0.05) in EPS-containing samples, indicating decreased lightness. This pattern mirrors that described by Rubel et al. (Reference Rubel, Iraporda, Gallo, Manrique and Genovese2019) in Ricotta-type cheese enriched with hydrocolloids, where the reduction in L* was attributed to increased opacity and a denser microstructure, which reduced light reflection. No significant differences were found in the a* and b* coordinates (p > 0.05), indicating that the red and yellow tonalities remained stable across treatments. The observed positive b* values (yellow hue) are likely related to Maillard reaction intermediates generated during heat treatment, as also noted by Rubel et al. (Reference Rubel, Iraporda, Gallo, Manrique and Genovese2019).
Instrumental analysis of color for the formulations of Requeson: (A) luminosity (L*); (B) green–red chromaticity (a*); (C) blue–yellow chromaticity (b*). According to the Tukey test, results are expressed as mean ± standard deviation; values with different letters for the same attribute are significantly different (p < 0.05).
These results contrast with those reported by Ramírez-Rivas & Chávez-Martínez (Reference Ramírez-Rivas and Chávez-Martínez2017), who found no significant changes in Requeson colour following high-intensity ultrasound (HIU) treatment of whey. In contrast, EPS incorporation and HIU represent fundamentally different approaches, biochemical and physical, respectively. The comparison was made to emphasise their differing effects on a standard quality attribute: colour. Unlike HIU, which did not affect colour parameters, the addition of EPS in our study led to reduced L* values, likely due to microstructural densification and increased opacity. This highlights the impact of EPS on the optical properties of the matrix through compositional modification, rather than suggesting mechanistic equivalence between the two treatments.
Overall, the incorporation of EPS and L. casei 431 improved the rheological profile of Requeson, enhancing firmness, elasticity and cohesiveness while reducing adhesiveness. Although EPS addition led to a slight reduction in lightness, all colour attributes remained within acceptable sensory thresholds. These findings support the application of EPS and probiotic cultures as a viable strategy for enhancing texture and stability in fresh cheese, without compromising visual quality.
Shelf life of Requeson
The evolution of pH and viability of the probiotic strain L. casei 431 in Requeson samples, control, whey + L. casei 431 and whey + EPS + L. casei 431 were monitored over 21 days (Fig. 3). The pH remained stable throughout the storage period, showing no statistically significant changes (p > 0.05) between day 0 and day 21. Likewise, probiotic viability was maintained at functionally relevant concentrations for consumer health, in accordance with Al-Sheraji et al. (Reference Al-Sheraji, Ismail, Manap, Mustafa, Yusof and Hassan2013), with no significant reduction over time (p > 0.05).
pH and probiotic viability in Requeson during 21 days of refrigerated storage. According to the Tukey test, results are expressed as mean ± standard deviation; values with different letters for the same attribute are significantly different (p < 0.05).
Microbiological analyses confirmed that total counts of faecal coliforms, moulds and yeasts remained below the maximum thresholds established by Colombian (ICONTEC, 2012) and Mexican (Secretaría de Salud, Reference de Salud2014) food safety standards. These results indicate that the Requesón samples remained microbiologically stable and safe for consumption during the 21-day refrigeration period.
Sensory acceptability of Requeson
Figure 4 shows the results of the sensory acceptability test conducted with consumers, evaluating attributes such as flavour, colour, texture and overall perception. The formulations with the highest acceptance throughout the storage period were the control sample and the one produced with the probiotic strain (L. casei 431) in the absence of EPS. In contrast, formulations containing EPS consistently received lower scores, particularly for flavour and texture and were generally the least preferred by participants.
Sensory acceptability of different Requeson formulations. (I) control; (II) whey + Lactobacillus casei 431; (III) whey + EPS + Lactobacillus casei 431. Colour (C), flavour (F), texture (T) and general acceptability (G). The blue line corresponds to the results of day 0; the red dotted line corresponds to day 21. The scale is a 9-point hedonic structured scale, where 1 is the lowest acceptance level, and 9 is the highest.
A plausible explanation for the lower acceptability of EPS-containing samples is the increased hardness associated with their incorporation into the protein matrix (see Fig. 1). Although EPS are known to improve mouthfeel and viscosity in fermented products like yoghurt, their application in fresh cheeses such as Requeson, characterised by a soft and slightly granular texture, may have resulted in a firmer, gel-like consistency perceived as atypical or undesirable. This deviation from the expected sensory profile could have negatively influenced the perception of authenticity and palatability. Furthermore, interactions between EPS and casein micelles may have interfered with the release and perception of volatile flavour compounds, potentially reducing flavour intensity.
The sensory evaluation was conducted locally at Universidad Autónoma Metropolitana, Unidad Iztapalapa, with a sample of 67 untrained consumers aged 18–50 years, all habitual dairy consumers familiar with traditional Requeson. No formal stratification by demographic variables or consumption frequency was applied. Consequently, while the data offers valuable insight into localised consumer preferences, extrapolation to broader populations should be approached with caution.
Notably, although the control Requeson exhibited notable textural deterioration between day 0 and day 21, associated with a decline in consumer acceptance, the EPS- and probiotic-containing formulations maintained consistent textural properties over time. This suggests a stabilising effect of EPS during storage, even if it did not lead to improved sensory preference.
Future studies should explore strategies to optimise EPS concentration or molecular structure, for example, through fermentation parameters or strain selection, to better modulate its interaction with milk proteins and reproduce the expected texture of Requeson. Additionally, combining EPS with other texturising agents or adjusting moisture content may help counterbalance its mechanical effects, thereby improving overall sensory acceptability.
Conclusion
In this study, fermentation of sweet whey with L. delbrueckii subsp. bulgaricus NCFB 2772 at 42°C for 48 hours yielded 1.126 ± 0.12 mgeq dextran/mL of EPS. The incorporation of this EPS-rich whey into Requeson formulations resulted in a substantial increase in cheese yield (up to 230%) and improved textural attributes, specifically hardness, cohesiveness and elasticity, without negatively affecting protein content or microbiological safety. Additionally, the inclusion of L. casei 431 ensured the maintenance of probiotic viability above functional thresholds during 21 days of refrigerated storage.
Although samples containing EPS exhibited slightly lower sensory acceptability, particularly in terms of flavour and texture, they showed enhanced physicochemical and microbiological stability throughout shelf life. This suggests that in situ EPS production is a viable and cost-effective strategy for valorising sweet whey while enhancing the technological and functional quality of fresh cheeses.
Future research should focus on optimising EPS concentration, molecular characteristics, or fermentation parameters to minimise sensory drawbacks and expand the applicability of this approach in the development of functional dairy products with improved consumer acceptance.
Acknowledgements
This research work was supported by the Department of Biotechnology, Universidad Autónoma Metropolitana-Iztapalapa (Mexico) and the Institute of Food Science and Technology (ICTA), Universidad Nacional de Colombia in Bogota.
Competing interests
The authors declare no competing interests.
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