The cottage cheese was made following Kosikowski’s [25] method for short-setting cottage cheese, with some adaptations. Two systems were prepared, each with 1000 mL of skimmed milk. The process is divided into the following stages:

Partial-skim pasteurized milk was used (3.1 g protein, 0.6 g fat). The milk was warmed to 32.2°C and inoculated with or without a 1% (v/v) volume of a liquid culture of the probiotic lactic acid bacterium (L. acidophilus LA-5, standardized to ≈10⁹ CFU/mL, yielding an initial milk concentration of ≈10⁷ CFU/g). Calcium chloride was added in a proportion of 0.02% to improve coagulation properties. The mixture was incubated at 36°C until the pH reached approximately 5.5. Then 0.5% rennet (w/v) was added and incubated at 36°C for 1 hour, after which 20 mL/L of vinegar (5% acetic acid) was added. The grains were left to rest for 15 minutes to allow for initial syneresis and surface hardening.

The cooking process involved gradual heating, with the temperature rising every 5 minutes during the first half hour (to reach ≈42°C), then increasing more rapidly (≈ to 50°C). During this stage, gentle, steady stirring was maintained to avoid grain clumping and ensure even heat distribution. After scalding, the whey was drained, and the curd was washed three times with purified water at decreasing temperatures (29, 16, and 4.4°C). The cheese was then drained for 30 minutes before proceeding to the creaming step. The cream was prepared following the method described by Kosikowski [25], with the following composition: 56% skimmed milk, 40% cream, and 4% salt; 330 grams of prepared cream are added for every 1000 grams of curd.

The probiotic L. acidophilus LA-5 was obtained from a pharmaceutical preparation (Winthrop Pharmaceutical de México, S.A. de C.V., Edo. de México, Mexico). Inoculations were performed using a loop into previously prepared de Man Rogosa and Sharpe broth (Difco, BD, Sparks, MD), and the cultures were incubated for 48 hours at 35°C. For use in cottage cheese production, the incubated broth was centrifuged at 8000 × g for 10 minutes at 4°C (Marathon 21k/R, Fisher Scientific, Germany) and washed twice with phosphate buffer (pH 7.0). Wet cells were used on the same day for cheesemaking.

Water activity (a_w) was measured using an Aqualab Series 3TE instrument (Decagon Devices, Inc., Pullman, WA). Fat content was evaluated using the Mojonnier gravimetric method (AOAC 995.19) [26], and total crude protein content was determined by the micro-Kjeldahl method (AOAC 960.52A) [27]. Milk pH was measured by direct electrode immersion with a digital pH meter (UB-10, Denver Instrument, Bohemia, NY), previously calibrated with standard buffer solutions at pH 4.0 and 7.0. Milk titratable acidity stage was determined by titration with 0.1 N NaOH and expressed as a percentage of lactic acid. The tests were conducted in triplicate.

Physicochemical and optical evaluations of the cottage cheese samples were performed in triplicate. The moisture content was determined following the official AOAC gravimetric method 926.08 [28]. Fat content was determined by the Mojonnier method (995.19) [26], and total protein content (micro-Kjeldahl method) was determined according to AOAC Method 960.52A [27]. For pH monitoring initially and during refrigerated storage, measurements were taken by direct immersion of the calibrated electrode into the cheese matrix. Titratable acidity at the initial and during storage was determined by thoroughly homogenizing 10 g of cheese with 15 mL of distilled water, followed by titration with 0.1 N NaOH; results were expressed as a percentage of lactic acid. Water activity was measured using an Aqualab Series 3TE water activity meter (Decagon Devices, Inc., Pullman, WA). Color measurements were performed in triplicate using the Hunter scale parameters: Lh (lightness), ah (red-green), and bh (yellow-blue), with a Color Gard System/05 colorimeter (BYK-Gardner, Inc., Columbia, MD). The colorimeter was operated under illuminant D65 with a 10° standard observer and calibrated in reflectance mode before testing the samples. Based on the values of Lh and bh, the Hue (H) and Chroma (C) of the processed and commercial cottage cheeses were calculated, using the following equations:

To evaluate cheese behavior during storage, samples were packaged in opaque plastic containers, sealed with plastic film, and stored at 4°C for 24 days. Physicochemical and microbiological analyses were systematically conducted at designated storage intervals, with day 1 as the baseline, selected over immediate post-manufacturing testing to allow stabilization under cold storage conditions. The pH was measured every two days using the previously described pH meter. At the same intervals, 10 g of the cheese sample was aseptically collected, homogenized in 90 mL of sterile peptone water, and serially diluted (10-fold). Appropriate dilutions were plated on MRS agar (Difco, BD, Sparks, MD) using the pour plate method for probiotic enumeration after 24 h of incubation at 35°C.

To quantify the effect of acidification on probiotic survival during storage, an acid-viability sensitivity coefficient (AVSC) was calculated from probiotic counts and pH. It indicates the change in probiotic viability (log CFU/g) per unit pH change as:

where:

N = probiotic population (CFU/g)

∂(logN) = change in probiotic counts (log CFU g) during storage

∂(pH) = change in pH over the same period

The effects of incorporating probiotic microorganisms on sensory attributes were evaluated using two triangle tests and a hedonic test with a structured scale. The samples were evaluated by a panel of 30 untrained panelists who were regular consumers of cottage cheese. For the first triangle test, they were presented with two identical samples and one different sample, comparing commercial cheese and probiotic cottage cheese. In the second triangle test, they were presented with two identical samples and one different sample of the two cheeses elaborated in this work (with and without probiotics). In the hedonic test, the attributes evaluated were flavor, color, texture, and overall acceptability. For this test, the panelists were presented with samples of the produced cottage cheeses and the two commercial ones, along with a structured questionnaire, a 9-point hedonic scale (from 1 “I dislike it extremely” to 9 “I like it extremely”).

The sensory evaluation tests adhered to ethical principles for research involving human participants. The protocol was reviewed and approved by the Research and Ethics Committee on Sensory Evaluation of Foods of the doctoral program in Food Science at the Universidad de las Américas Puebla before testing. Each panelist received an Informed Consent form detailing the study’s purpose to evaluate the sensory properties of commercially produced cottage cheese, the ingredients, and allergens, and listing the ingredients used (cow’s milk, lactic cultures, enzymes, etc.). The form also stated the participant’s right to withdraw from the panel at any time without penalty. Collected data are handled anonymously and used solely for statistical and academic purposes. Only panelists who voluntarily signed the document participated in the tests.

The results obtained for each analyzed property and sensory attribute were analyzed with ANOVA and Tukey’s mean comparison tests (p < 0.05) using Minitab 20 software (Minitab LLC, PA, USA). For triangle difference sensory tests, the results were compared with the statistical tables of Roessler et al. at a significance level of p < 0.05. This analysis determines whether the panelists’ correct answers exceeded what would be expected by chance, thereby confirming significant differences between the cheese produced and the commercial references.

The physicochemical profile of the pasteurized, partially skimmed milk used for cottage cheese (Table 1) reflects typical raw material quality for acid-coagulated cheeses. The pH of 6.80 and low titratable acidity show minimal indigenous fermentation, typical of pasteurized milk [19]. Low acidity and near-neutral pH are essential for consistent acidification in cheese-making and probiotic growth, with high lactose availability [29,30].

The fat content aligns with partial skimming and supports the production of cottage cheeses [21]. High water activity was expected due to the milk’s high moisture content, which promotes microbial activity during processing [34,35]. The color parameters (Lh, ah, bh) describe a bright, slightly warm hue, typical of fresh milk with natural pigments like riboflavin and carotenoids [19]. The prepared cream dressing had a pH of 5.53 ± 0.02 and 11.2% fat, consistent with typical creaming mixtures used to adjust texture and mouthfeel in cottage cheese [36].

Adding cream significantly increased the yield of both cottage cheese types. For probiotic cheese, the yield went from 16.04 ± 1.12% without cream to 21.33 ± 1.37% with cream, while control cheese showed a similar pattern, rising from 15.54 ± 1.33% to 20.67 ± 1.25%. Overall, probiotic cheese consistently had slightly higher yields than the control cheese under both conditions. These results indicate that adding cream significantly increases cheese yield, likely due to improved retention of fat and total solids in the curd. Yields for cottage cheese were comparable to reported yields in the literature (~15-17%) [25,37]. Brito et al. [38] noted a practical yield of 16.62% for cottage cheese made with homogenized cream, which is also close to the yield values observed here. The similar yields of probiotic and control cheeses indicate that the addition of L. acidophilus LA-5 did not adversely affect curd formation or moisture retention.

As shown in Table 2, the two commercial cottage cheeses (brands A and B) were characterized for comparison; both products exhibited high water activity (aw ≈ 0.98), consistent with their elevated moisture content and those of typical fresh cheeses [34,35,39,40]. The pH values (5.01-5.27) complied with the Mexican standard NMX-F-092-1970 and fell within the expected range for cottage cheese (4.7-5.3), while titratable acidity (1.08-1.13% lactic acid) corresponded to values reported for acid-coagulated fresh cheeses and dairy matrices [12,19]. Moisture content in commercial cheeses (~80.5%) slightly exceeded the regulatory limit but was consistent with the high-moisture nature of this cheese variety [25]. Fat content in both brands met the Codex Alimentarius [41] requirement of at least 4% for creamed cottage cheese. Protein levels (10.6-11.1%) were lower than both the label claims and the reference values in NMX-F-092-1970. This variation has been observed in other studies of commercial fresh cheeses and is likely due to differences in formulation and the amount of dairy solids included [39]. Cottage cheese’s complex, biphasic structure can cause sampling issues and lead to an underestimation of crude protein. The discrepancy in protein content arises from industrial formulations and the analytical testing of moisture-rich, segmented dairy matrices.

Experimental cottage cheeses, produced without probiotic culture (control) and with L. acidophilus LA-5, showed physicochemical characteristics comparable to commercial products (Table 2). Both formulations complied with moisture specifications (<80%), in agreement with literature values for cottage cheese [25,37,38]. The absence of significant differences (p > 0.05) in moisture content between formulations is consistent with previous findings indicating that probiotic incorporation does not markedly affect water retention in fresh cheeses [8]. Protein contents (≈10.6-11.0%) were similar to those of commercial samples.

Fat content in the experimental cheeses was close to the Codex STAN 273-1968 minimum value; probiotic cheese (4.10%) met the requirement, whereas control cheese was marginally below (3.94%). Regarding acidification, the probiotic cheese exhibited higher (p < 0.05) titratable acidity (1.54 ± 0.12% lactic acid) and a lower (p < 0.05) pH (4.93) than the control (0.96 ± 0.07% lactic acid; pH 5.62), reflecting the metabolic activity of L. acidophilus LA-5. This behavior aligns with previous studies showing that probiotic lactobacilli increase lactic acid production in fresh cheeses, thereby enhancing product stability, flavor development, and probiotic viability [12,42,43]. Although the probiotic cheese presented a pH slightly below the minimum established by NMX-F-092-1970, it remained within the typical range reported for cottage cheese and probiotic fresh cheeses [40,44].

Color analysis of the cheeses (Table 2) revealed subtle but statistically significant variations among the evaluated cottage cheeses (Control, Probiotic, Brand A, and Brand B). Cottage cheese is a fresh dairy product characterized by high lightness and low chroma (C), resulting in a fresh appearance [37]. The statistical color analysis showed no significant differences (p > 0.05) between control and probiotic cheeses, indicating that the incorporation of L. acidophilus LA-5 did not affect visual attributes. This is consistent with previous reports indicating that the addition of probiotics generally preserves the color characteristics of fresh cheeses, an important factor for consumer acceptance [42,45]. Commercial samples had the highest Lh values (Table 2) compared with the control and probiotic batches, suggesting they use processes such as high-pressure homogenization, thickening agents, and natural hydrocolloids. These enhance light scattering from casein micelles and fat globules, boosting opacity and whiteness [46]. The ah parameter was negative (-1.9 to -2.83), indicating a shift towards the green spectrum typical of dairy products. The bh coordinate was positive, showing a yellow undertone. The hue angle was stable (102.78° to 104.40°), with only minimal changes upon adding probiotics (103.53° vs. 102.78°). This suggests probiotic fortification does not significantly alter the cheese’s color, maintaining consumer-acceptable attributes [37,46]. Overall, the results demonstrate that incorporating probiotic cultures can be achieved without major alterations to the physicochemical properties of cottage cheese, supporting its potential as a suitable carrier for probiotic delivery.

In the present study, probiotic viability was monitored during refrigerated storage through periodic microbial enumeration and pH measurements (Figure 1). The initial inoculation level of L. acidophilus LA-5 (~10⁷ CFU/g) increased rapidly to approximately 10¹⁰ CFU/g within the first day, indicating active adaptation to the cheese matrix during preparation steps before creaming and ensuring a high functional starting threshold from the first day of storage.

Viable counts remained stable until approximately day 15, then gradually declined, yet consistently exceeded 10⁹ CFU/g throughout the 24-day storage period (Figure 1). These values are significantly above the minimum levels recommended for probiotic efficacy, which are generally established at ≥10⁷ CFU/g at the time of consumption [2,47,48] and are consistent with earlier intake guidelines of 10⁸-10⁹ CFU/day [49]. The observed reduction in viability (~1.25 log CFU/g) followed a linear trend, consistent with previous reports of probiotic stability in cheese matrices [43,44]. Cuffia et al. [43] reported that L. acidophilus and Bifidobacterium animalis maintained populations above therapeutic levels in probiotic cheeses during refrigerated storage. Similarly, Hernández-Figueroa et al. [44] observed sustained viability of L. acidophilus LA-5 (>10⁸ CFU/g) in probiotic cream cheese during 30 days of storage. This stability has been attributed to the protective properties of cheese, including its protein-rich structure and buffering capacity [6,42].

Previous studies have demonstrated the potential of cottage cheese as a probiotic delivery system. For instance, Jeon et al. [50] reported improved stability of Lactobacillus plantarum Lb41 in cottage cheese compared to control samples during refrigerated storage, supporting its suitability as a probiotic carrier. The incorporation of different probiotic strains and adjunct ingredients has also been widely explored. Lactobacillus paracasei subsp. paracasei LBC81 has shown probiotic potential in cottage cheese systems, with the addition of plant-based flours not significantly affecting viability, although storage time influenced microbial stability [51]. In this context, post-acidification, commonly observed in fermented dairy products, has been associated with the continued metabolic activity of LAB during refrigeration, resulting in decreased pH and increased titratable acidity.

Some studies have evaluated strategies to enhance probiotic viability in cottage cheese. Araújo et al. [52] demonstrated that inulin supplementation did not adversely affect Lactobacillus delbrueckii viability, while Rostami et al. [53] reported increased Lacticaseibacillus casei populations in cottage cheeses enriched with black cumin seeds. Ahmed et al. [54] showed that incorporating Aloe vera gel and probiotic microcapsules into cottage cheese improved probiotic stability and maintained sensory quality over 28 days of storage, with minimal loss of viability (~1 log CFU/g). Likewise, Saeed et al. [13] observed that encapsulation of L. acidophilus and L. casei in alginate-carrageenan matrices significantly enhanced survival in cottage cheese compared to free cells and improved sensory attributes. Obando et al. [15] reported that probiotic populations in cottage cheese, including L. acidophilus LA-5, remained above 10⁶ CFU/g during refrigerated storage, with minimal impact on key sensory properties.

Acidification kinetics showed a gradual decline in pH during storage (Figure 2), with an overall decrease of about 0.79 units, along with an increase in titratable acidity from 0.32% to 0.46% lactic acid. The control cheese without L. acidophilus maintained greater pH stability during storage, while the probiotic cheese experienced steady acidification starting around day 6, with pH dropping from 4.8 to 4.04 by day 24. This pattern further supports the role of probiotic metabolic activity in driving acidification dynamics. Post-acidification is commonly observed in probiotic dairy products due to the continued fermentation of residual lactose during storage [42,55,56]. Similar pH reductions have been reported in lactobacilli-containing probiotic ricotta and fresh cheeses, where metabolic activity persists at low temperatures but at reduced rates [23,24,57].

A strong positive correlation between pH and probiotic viability (Figure 3) was found, indicating that acidification influenced microbial survival, although the decline was not sufficient to compromise functionality. The acid-viability sensitivity coefficient (AVSC = 1.58) indicates moderate probiotic sensitivity to pH changes, as limited acidification (<1 pH unit) maintained high counts during storage. Compared with probiotic strains in fluid systems such as fermented milk drinks or yogurt [58,59,60], which often have AVSC > 2.50, indicating greater sensitivity; lower AVSC indicates greater tolerance, such as in other cheeses with dense structures that exhibit low AVSC due to physical shielding [6,8]. The intermediate AVSC of 1.58 indicates cottage cheese offers a protective, balanced microenvironment. These results confirm that the cheese matrix supports probiotic stability and microbial delivery [6,42].

Lactic acid bacteria activity decreases pH and increases titratable acidity during refrigerated storage, a phenomenon known as post-acidification, which can cause acid stress and affect microbial viability [61,62]. However, in cottage cheese, this impact is reduced due to its composition: it maintains a pH above 4.0 and contains protein and mineral salts that help buffer the effects of acidity [8]. Despite increased acidity, probiotic counts stay above 10⁹ CFU/g, ensuring potential benefits. The results confirm that the cottage cheese matrix can protect microorganisms through its network of protein micelles, which can trap probiotic cells and reduce their exposure to oxygen and oxidative stress [7,8]. The cream can form a film that acts as a barrier against proton diffusion and osmotic shock, and the protein concentration and moderate pH also act as buffers. This type of matrix (solid or semi-solid) offers greater protection than liquid matrices [7].

The sensory properties of the cottage cheeses produced (probiotic and control cheese) were assessed using two complementary methodologies: a triangular discrimination test and a nine-point hedonic evaluation of flavor, color, texture, and overall acceptability. This combined approach enables both the detection of perceptible differences and the quantification of consumer preference [63]. The triangular test revealed that only 20% of panelists correctly identified the different samples when comparing probiotic cheeses with a control one. According to standard statistical tables, this proportion is insufficient to establish a significant difference at the 95% confidence level, indicating that the incorporation of L. acidophilus LA-5 did not produce perceptible sensory changes relative to the control formulation. In contrast, when probiotic cheese was compared with a commercial reference, 85% of panelists correctly identified the different sample, demonstrating a significant difference (p < 0.05). These findings suggest that while probiotic incorporation did not substantially modify the sensory profile of the experimental cottage cheese, differences between laboratory-produced and commercial products were perceptible, likely due to variations in formulation, processing conditions, or ingredient standardization.

These results are consistent with previous studies reporting minimal sensory impact from incorporating probiotics into fresh cheeses. For example, Abadía-García et al. [12] found no significant sensory differences between probiotic and control cottage cheese, while Felicio et al. [64] reported that probiotic Minas cheese maintained comparable sensory characteristics to conventional formulations. Similar observations have been reported in other fresh cheese systems, where probiotic cultures did not adversely affect sensory perception [42,65].

A more detailed evaluation using a nine-point hedonic scale (1 = “dislike very much”; 9 = “like very much”) showed (Table 3) that all samples achieved favorable acceptance, with mean scores ranging from 6 to 8 and clustering around 7 (“like moderately”).

These values indicate positive consumer perception across all formulations. For flavor, the highest scores were observed for the A-brand commercial cheese and the probiotic cheese, with the latter averaging 7.35. The B-brand cheese obtained the lowest flavor score, although all samples remained within the acceptable range and were not significantly different (p > 0.05). Color scores were slightly higher for commercial cheeses, likely reflecting their more standardized appearance. However, experimental cheeses showed comparable values (p > 0.05), with control (7.55) and probiotic (7.10) remaining within the same acceptance level. Texture evaluation followed a similar trend, with the A-brand cheese receiving the highest score, followed by the probiotic (7.15) and control (6.80) cheeses. Despite minor differences, all values indicated satisfactory texture perception.

Overall acceptability indicated that the control cheese achieved a score comparable to the A-brand product (7.45), whereas the probiotic formulation showed a slightly lower score (7.20). Statistical analysis indicated no significant differences (p > 0.05) among samples for most attributes, supporting the sensory equivalence of the probiotic formulation. These preliminary results, from a panel of 30 untrained consumers, suggest that including L. acidophilus LA-5 likely doesn’t affect quality, but larger sensory studies are needed to confirm market acceptance. The observed sensory acceptability aligns with previous findings on probiotic cheese products. Hernández-Figueroa et al. [44] reported high consumer acceptance of probiotic cream cheese containing L. acidophilus LA-5, with no significant differences compared to conventional products. Likewise, Granato et al. [66] and Karimi et al. [6] highlighted that cheese matrices are particularly suitable for probiotic incorporation due to their mild flavor and buffering capacity, which help maintain sensory quality. More recent studies have also emphasized that fresh cheeses can effectively deliver probiotics without compromising sensory attributes, even during storage [8,67].

Probiotic foods should deliver at least 10⁷ CFU/g or per serving [2,68]. In this study, L. acidophilus LA-5 (>10⁹ CFU/g) stayed viable during storage, ensuring a relevant dose. This supports health benefits and confirms cottage cheese as an effective probiotic carrier. Probiotic cottage cheese links nutrition and health, offering an alternative to supplements. Adding L. acidophilus LA-5 boosts its benefits without affecting taste, making fresh cheeses effective probiotic carriers.