1. Introduction
The wine industry is a well-established sector in our society, producing significant amounts of waste, with only a small portion being utilized for fertilization, animal feed, or the production of other goods. A more sustainable approach could involve repurposing wine by-products to extract valuable functional ingredients, which could be used in the development of pharmaceutical and food products [1].
Enriching dairy-based foods with bioactive compounds is a technological strategy for developing products with functional appeal. Studies have demonstrated the potential of using winemaking by-products to enhance the functional and sensory properties of various food products [2,3]. In addition to incorporating bioactive ingredients, this strategy has strong environmental benefits and adds value to foods through the introduction of by-products [4].
Recent studies have reported that A2A2 milk can provide better digestibility when compared to milk containing the A1 variant of beta-casein. This is because A1 milk can release the peptide beta-casomorphin-7 (BCM-7), which is linked to digestive problems and other health issues in some individuals [5,6,7,8]. Although A2A2 milk is not a solution for cow’s milk protein allergy and/or lactose intolerance [9,10], it does not contain the BCM7 peptide, which is an allergenic agent for sensitive individuals [6,11]. Thus, dairy products made with A2A2 milk can be a valuable addition to the dairy market.
Petit Suisse cheese is a promising matrix for the addition of various ingredients, as it is a high-value product suitable for incorporating probiotics, prebiotics, and antioxidant compounds [12,13].
The recently reclassified lactobacillus [14] and bifidobacteria genera have been extensively studied for their probiotic potential [15,16,17,18,19]. The Bifidobacterium animalis subsp. lactis HN019 strain is known for its probiotic characteristics due to its high survival rate during passage through the gastrointestinal tract (GIT) [20,21] and its ability to adhere to the intestinal epithelium [18], as well as the various positive health effects it has demonstrated in clinical studies [22,23,24,25].
The development and use of novel ingredients aimed at promoting health and well-being have been the focus of several studies, enabling innovation in food products and catering to niche markets [26]. Therefore, the addition of probiotics, along with bioactive compounds, can be an interesting alternative for the manufacturing of dairy products made with A2A2 milk.
Milk naturally contains bioactive compounds, and A2A2 milk, in particular, has improved digestibility, enhancing its potential to provide health benefits to consumers of Petit Suisse within the context of cheese production [9,10,27].
In this context, the objectives of the present study were to develop and characterize Petit Suisse cheese produced with A2A2 milk and a probiotic culture and to evaluate the effects of adding a by-product from the winemaking industry. This study was divided into two stages: (a) the drying and characterization of the by-product; and (b) the production and characterization of the control Petit Suisse (without the addition of the by-product) and Petit Suisse with the addition of the by-product.
2. Materials and Methods
A by-product from the production of red wine and grape juice was used, which was donated by an agribusiness located in Rio Bonito do Iguaçu, PR (latitude: 25°28′14″ S, longitude: 52°32′41″ W, and altitude: 598 m); pasteurized A2A2 milk and A2A2 cream (Letti A2™) were donated by the company Letti A2; commercial coagulant Estrela (Christian Hansen, Valinhos, SP, Brazil), starter culture Streptococcus thermophilus (Christian Hansen, SP, Brazil), and probiotic culture Howaru™ Bifido Bifidobacterium animalis subsp. lactis HN019 (Dupont, Cotia, SP, Brazil) were used; and refined sugar (Camil Alimentos, São Paulo, SP, Brazil), calcium chloride, xanthan gum (Grupo Conexão Sul, Rio de Janeiro, RJ, Brazil), polypropylene plastic packaging (145 mL), and aluminum seals were purchased from a local market.
2.1. Winemaking By-Product
For the development of this research, a company provided approximately 20 kg of a frozen by-product and packaged it in rigid plastic packaging, divided into approximately 2 kg sections. The by-product was kept frozen until it reached the laboratory. The co-product is mainly composed of grape skins and pulp and may contain grape seeds in smaller quantities, but it did not contain grape stems. Each 2 kg portion was then subjected to thawing in refrigeration followed by another fractionation of 50 g of the by-product for each smaller plastic package. The packages containing 50 g of the by-product were frozen in an ultrafreezer at −18 °C. After 24 h, they were freeze-dried for 36 h. The freeze-drying yield was approximately 12%.
The freeze-dried by-product was characterized in triplicate for pH levels, titratable acidity, total soluble solids, moisture, ash, lipids, and protein contents according to the methodologies of the Association of Official Analytical Chemists [28]. The sample was also characterized for its water activity (AquaLab meter, Meter Group, Pullman, WA, USA); color parameters (Minolta Colorimeter, Konika Minolta, Kyoto, Japan) antioxidant activity using the iron reduction method (FRAP) [29], DPPH radical scavenging method [30], and ABTS radical scavenging method [31]; and total phenolic compounds [32].
The Extraction of the By-Product for the Determination of Antioxidant Activity
For the extraction, approximately 2.5 g of the by-product was vortexed for 3 min with 20 mL of 50% ethanol/water (v/v) and left to stand for one hour. The mixture was centrifuged, and the supernatant was stored. The resulting solid was then re-homogenized for 3 min with 20 mL of 70% acetone (v/v) in water and left to stand for 1 h. The mixture was centrifuged, the supernatant was combined with the one obtained from the first extraction, and distilled water was added until a volume of 50 mL was reached [29].
2.2. Manufacture of Petit Suisse
The Petit Suisse cheeses were made according to the methodology of Messias et al. [33] with adaptations (Figure 1). Initially, 0.5% (w/v) of Streptococcus thermophilus (starter) and Bifidobacterium animalis subsp. lactis HN019 cultures were separately activated in 11% (w/v) reconstituted milk powder, homogenized, and then incubated in an incubation chamber at 36 °C overnight.
The A2 milk was heated in a water bath, and the ingredients were then added according to the process outlined in Figure 1. After coagulation, the curd was cut, and the whey was drained. After draining, the resulting Quark cheese was weighed for the calculation of the yield and to determine the mass ratio for the Petit Suisse formulations. Next, xanthan gum (0.2%), A2 cream (14%), and sucrose (refined sugar, 10%) were added and homogenized in a planetary mixer at a rotational speed of 124 rpm until no lumps were visible and a smooth mass was obtained.
Two formulations were prepared from this mass: one control (F0) without the addition of the by-product and another with 10% (w/w) of the freeze-dried by-product (F1). The resulting products were packaged in individual polypropylene plastic containers (~100 g each), sealed with aluminum lids, and stored under refrigeration (5 ± 2 °C) until analyses. Therefore, for each 900 g batch of quark cheese, 100 g of the freeze-dried by-product was used. The quantity of highly freeze-dried by-products is important for obtaining the desired characteristics of Petit Suisse and can be acquired due to the high generation of this raw material in the wine industry.
2.3. Characterization and Shelf Life of Petit Suisse
The Petit Suisse cheeses were analyzed in triplicate the day after their production according to the methodologies of the Association of Official Analytical Chemists [28] for their moisture, ash, lipid, and protein contents. The total carbohydrate content was calculated using the difference method. Sampling at different analysis times during storage was conducted randomly.
For shelf life evaluation, the products (F0 and F1) were analyzed for pH levels [28], Aw [28], titratable acidity [34], total soluble solids (TSS) [35], instrumental color [36], syneresis [37], texture profile analysis (TPA) [38], antioxidant activity [29,31], phenolic compounds [32], and lactic acid bacteria and probiotic counts 1, 7, 14, 21, and 30 days after they were manufactured.
Syneresis was determined using 5.0 g of the sample, which was centrifuged at 8944× g for 15 min at 4 °C [37], and the result was expressed as a percentage of whey mass relative to the initial mass of the sample.
The instrumental texture analysis was carried out using a TA-XT2 texture analyzer (Stable Micro Systems, Surrey, UK) in quintuplicate, through a double compression test, using a 25 mm diameter aluminum cylinder and 1 cm compression, at a speed of 1 mm/s and a load of 0.05 N. The samples were removed from the refrigerator just before testing. The software Texture Expert for Windows—version 1.20 (Stable Micro Systems)—was used for data collection. The texture parameters analyzed were hardness, cohesiveness, gumminess, adhesiveness, and chewiness [38].
The viability of Streptococcus salivarius subsp. thermophilus and the probiotic bacteria was determined through serial dilutions in 0.1% (w/v) buffered peptone water (Oxoid, Cambridge, UK) on M17 agar at 42 °C for 24 h and selective MRS (de Man, Rogosa, and Sharpe) agar (MRS-ABC, with dicloxacillin, lithium chloride, and L-cysteine) at 36 °C for 72 h under anaerobic conditions (Anaerobac, Probac®, São Paulo, SP, Brazil), respectively [38,39,40].
2.4. Digestibility In Vitro
For the digestibility test, 5 g of each sample (F0 and F1) was subjected in triplicate to the oral, gastric, and enteric phases of simulated digestion based on the INFOGEST protocols [39,41]. The time for simulated digestibility study in each stage was as follows: 2 min in the oral phase, 2 h in the gastric phase, and 2 h in the intestinal phase. The reagents were prepared and combined in the indicated proportions for the preparation of simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF). Enzyme solutions were prepared immediately before the experiment by dissolving amylase, pepsin, and pancreatin, as well as bile, in the respective fluids. At the end of the gastric and enteric phases, the samples were collected and used for the determination of antioxidant activity and enumeration of the starter culture and probiotic bacteria [39]. The simulated in vitro digestion analyses were conducted at two distinct time points in triplicate. In the first stage, analysis was performed for Petit Suisse samples after 15 days of production. In the second stage, the Petit Suisse samples were 30 days old. This analysis was conducted at two different points, which made it possible to better understand the effect of the antioxidant compounds added in F1 and the viability of the probiotic microorganism added in both formulations.
2.5. Fatty Acid Profile
The fat contents of the Petit Suisse samples were determined using the Bligh–Dyer method, and 200 mg of the cold-extracted fat was analyzed for the fatty acid profile using gas chromatography (GC-MS QP2010 SE, Shimadzu, Kyoto, Japan), as described by Gonzalez et al. [42]. The analysis was performed in triplicate for the 3 independent productions after 15 days of storage to obtain sample results under conditions that were similar to those in the sensory analysis.
2.6. Scanning Electron Microscopy (SEM)
The Petit Suisse samples were fixed with 2.5% glutaraldehyde for 3 h and then dehydrated with an ethanol series (from 30% to 100%, increasing the concentration by 10% increments). Subsequently, the samples were sputter-coated with gold powder under high pressure (13 to 133 mPa or 10−4 to 10−3 Torr) using a sputter coater (BALTEC model SCD-050, Jundiaí, SP, Brazil). The images were evaluated using a scanning electron microscope (FEI-Phillips), model Quanta 200 (Thermo Fisher Scientific Brazil, São Paulo, SP, Brazil).
2.7. Sensory Evaluation and Purchase Intention Test
The sensory analysis was approved by the Research Ethics Committee involving Human Subjects (CEP/SH) of the State University of Londrina (UEL)—CAAE: 75079223.2.0000.5231, approval date: 13 November 2023. The analysis was conducted by untrained adult volunteers of both genders, according to their availability, their interest in Petit Suisse, and the frequency of their Petit Suisse consumption, as well as their interest in consuming A2 milk and grape-derived products. The Petit Suisse samples were subjected to microbiological safety evaluation, as established by Normative Instruction IN 161 [43], for the presence of coagulase-positive staphylococci, Escherichia coli, and the presence of Salmonella in 25 g according to AOAC methodologies [44] before the sensory evaluation.
The samples were evaluated using acceptance and purchase intention tests in individual booths under white light. The participants received 20 g of each Petit Suisse formulation at refrigeration temperature (5 to 7 °C) in plastic cups coded with three random digits, served randomly in balanced order (F0–F1 and F1–F0). The acceptance test was used to evaluate the following attributes: overall impression, color, flavor, and texture using a 9-point hedonic scale, with 1 corresponding to “disliked extremely” and 9 to “liked extremely” [45]. For the purchase intention test, a structured 5-point scale was used, with 1 corresponding to “definitely would buy” and 5 to “definitely would not buy” [46]. Finally, the participants stated whether they intended to purchase the products [47].
2.8. Statistical Analysis
The results were subjected to an analysis of variance (ANOVA) and Tukey’s test at a 5% significance level.
3. Results and Discussion
3.1. Winemaking By-Product Chemical Composition
The chemical composition of the freeze-dried by-product is shown in Table 1. Souza et al. [48] reported higher values of moisture, protein, lipids, and water activity in grape skin and similar values for ash, pH, and Brix when compared to those found in the present study.
The powdered by-product exhibited an intense violet color, with an L* (lightness) of 20.65, a* (red color intensity) of 10.12, b* (yellow color intensity) of 4.53, C* (chroma) of 110.89, and h* (hue) of 1.15. According to the CIELAB system, the value of chroma C* is the distance from the lightness axis (L*) and starts at zero in the center. The hue angle starts at the +a* axis and is expressed in degrees (e.g., 0° is +a*, or red, and 90° is +b, or yellow; 180 is −a* or green, and 270 is −b* or blue). Thus, it is evident that the by-product had low levels of lightness and tended to be red and yellow in color.
The results from the FRAP, DPPH, and ABTS assays used to assess antioxidant activity were 0.19, 0.18, and 0.14 mol trolox/kg, respectively, and the content of phenolic compounds was 55.7 mEq of gallic acid/kg. Guendez et al. [49] reported phenolic compound contents in grape seeds from 55 to 964 mg per 100 g, with an average of 380 mg per 100 g of dry mass. The differences found in phenolic compounds and antioxidant activity may be due to various factors, including the extraction method used, the grape species, and the harvest conditions [50]. Grape skin makes up 65% of the total grape pomace material and has been reported to contain a high concentration of phenolic compounds [51] According to the results in Table 1, the by-product showed relevant antioxidant activity and phenolic contents, reinforcing its potential for use in food products.
3.2. Characterization of Petit Suisse Formulations
The F0 and F1 formulations had ash contents below 2% and protein contents above 20% (Table 2). Thus, both formulations exhibited high protein contents, as the legislation recommends a minimum of 6% for Petit Suisse [52]. Both formulations had moisture values ranging from 46.0 to 54.5%, thus classifying them as “high-moisture cheeses” (>46%) [52], in accordance with the technical identity regulation of Petit Suisse cheese. The lipid contents were 14.03 and 15.06% for F0 and F1, respectively (Table 2), and the formulation F1 had a higher carbohydrate level, likely due to the characteristics of the by-product. The main source of added sugar in F0 was sucrose, while F1 contained sucrose and the natural sugar from the by-product. The use of the by-product, which is rich in carbohydrates, led to an increase in the carbohydrate and ash contents of F1, with values of 81.22 and 4.05%, respectively, accompanied by a consequent reduction in other parameters (Table 2).
3.3. Shelf Life of Petit Suisse Formulations
3.3.1. Physicochemical Characterization of Products
The physicochemical characterization (Table 3) of the formulations (F0 and F1) showed no changes throughout their shelf life (30 days) for the parameters of Aw, pH, and titratable acidity. However, there was a difference in total solids between the F0 and F1 formulations, probably due to the addition of the by-product, which led to a 10% increase in the total solids in F1. The addition of the by-product did not alter water activity due to the hygroscopic nature of the by-product, as there was an increase in its water absorption capacity despite the increase in its total solids, resulting in similar water activity values for F1 and F0. No differences were observed for pH or titratable acidity between the formulations and storage times.
Regarding the color parameters L*, a*, and b*, significant differences (p < 0.05) were observed between the F0 and F1 formulations, with no differences observed during their storage. This difference was due to the addition of the by-product, which gave the Petit Suisse a purple color due to the presence of anthocyanins in its composition [53]. For the color parameters C* and h*, there was no difference over time between the different formulations. For all color parameters, the products remained stable throughout their 30-day shelf life.
The results of the syneresis percentage were 0.03 and 0.01% for F0 and F1, respectively, with no difference between them over the five storage periods studied (p < 0.05). The low percentage of syneresis is possibly due to the use of xanthan gum (0.2%) in the formulations. The increase in syneresis during storage is generally associated with rearrangements of the casein network and is a common defect in fermented dairy products, thus affecting consumers’ acceptance. In this context, the adequate addition of gums and/or fibers can minimize or eliminate this phenomenon. Morales-Cortés et al. [12] studied symbiotic guava Petit Suisse containing inulin and reported a significant increase in syneresis 21 days after its manufacture.
3.3.2. Antioxidant Activity of Products
No differences (p < 0.05) were observed in the phenolic compounds or antioxidant activity of the F1 formulation throughout its shelf life, as determined using all of the assays (FRAP, DPPH, and ABTS) (Table 3). Therefore, the product could remain stable throughout a shelf life of up to 30 days. The control formulation (F0) showed no detectable values for antioxidant activity assessed using all assays, which could be due to the lower content of phenolic and antioxidant compounds naturally present in the samples, potentially being below the detection capacity of the methodologies used. These data reinforce the contribution of the by-product to the enrichment of bioactive compounds in the formulation.
Several studies in the literature have reported the addition of fruits, such as acerola pulp, açai, grape, and currant, to Petit Suisse to enhance its functional properties, such as increasing the content of antioxidant compounds [13,54]. The inherent constituents of grapes have the potential to improve these properties in dairy products [53]. Thus, the use of waste from this industry can be an interesting alternative, as it combines health benefits and contributes to the use of a material that is underutilized in the food industry and can be reintroduced into this chain, with positive impacts on production and product characteristics.
3.3.3. Texture Profile Analysis (TPA) of Products
The texture properties (Table 3) remained constant from day 1 to day 30 of storage. This finding supports other analyses in this study, which demonstrated the product’s stability during 30 days of refrigerated storage. Similar results were found by Morales-Cortés et al. [12], who observed texture stability in symbiotic guava Petit Suisse over 21 days of storage. The parameters of hardness and adhesiveness showed a high level of variability, with less homogeneous data compared to the responses for cohesiveness and gumminess. Therefore, the latter parameters better represent the texture of the samples. The adhesiveness parameter is influenced by the fat content and increases with a higher fat content in the product [55]. No difference was observed for the proximate composition of the F0 and F1 formulations, which may have contributed to the maintenance of their texture, even after the incorporation of the by-product.
3.3.4. Lactic Acid Bacteria and Probiotic Counts
The addition of lactic acid bacteria (LAB) and microorganisms with probiotic potential and the maintenance of their viability in the food matrix are of interest because these bacteria can enhance food safety [18,24,56,57,58]. In addition, the presence of probiotics in high concentrations can enhance the functional properties of the cheese. Various authors have conducted in vivo studies on the health benefits of the HN019 strain [15,18,22,24,25].
The probiotic culture (Table 3) continued to be abundant (~eight log CFU/g) with no changes during storage for either formulation. The starter culture showed significant variations (p < 0.05) over time, with lower counts in the last two periods of analysis. According to the current legislation [59], Petit Suisse must have a total lactic acid bacteria count (CFU/g) above 106 according to International Standard Dairy Federation [60]. The high viability of the probiotic throughout the entire shelf life of the product is required for food to be characterized as having potential benefits [61], a characteristic observed in the Petit Suisse of the present study.
3.4. Simulated Digestibility In Vitro
Digestion in vitro aims to simulate the gastrointestinal conditions that the microorganisms are subjected to after the ingestion of the Petit Suisse samples, how they behave in terms of survival, and their possible effect on the human intestinal colon. The initial Streptococcus thermophilus counts were ~six log CFU/g, which dropped to five log CFU/g after the enteric phase for both F0 and F1, after 15 days of storage. After 30 days of storage, the count was three log CFU/g for both formulations (Table 4).
These results show that the HN019 strain exhibited a higher survival rate when compared to Streptococcus thermophilus during the passage through the gastrointestinal tract for both formulations. In the F0 formulation, the survival rates of Streptococcus thermophilus were 74.92 and 59.9% after 15 and 30 days of storage, respectively, while F1 showed survival rates of 83.25 and 66.67% for the same periods, respectively. For the probiotic bacteria, the survival rates were 83.25 and 81.97% after 15 and 30 days for F0 and 80.62 and 76.30% for F1, respectively.
This count of six log CFU/g in the assays performed after 15 and 30 days of storage demonstrated the good survival rate of the probiotic after the gastric and enteric phases for both formulations (Table 4). Thus, it is possible to affirm that the product has potential probiotic action. Although the digestive process creates adverse conditions for bacteria, survival under these conditions is a desirable characteristic of probiotic microorganisms [61]. In this regard, Bifidobacterium spp. has been reported as resistant to gastric conditions [62].
The probiotic bacteria studied are from a dairy-originated strain, with a high survival rate and well-established beneficial characteristics [15,18,24,57,58,63,64,65]. Thus, it is worth emphasizing that the Petit Suisse matrix in the present study provided favorable conditions for the high viability and survival rate of the HN019 strain throughout the product’s shelf life and after the simulation of gastrointestinal conditions.
The efficacy of probiotics is related to the presence of the necessary substrate for fermentation and the development of their metabolic functions in the intestine. In this context, the survival level of probiotic bacteria in fresh cheeses, such as Petit Suisse, is higher when compared to that of ripened cheeses [66]. This behavior is due to the higher moisture and water activity levels of Petit Suisse, as well as the lower salt contents, which interfere with probiotic multiplication [67].
Studies have shown that sucrose can protect probiotics, including B. lactis HN019, by increasing the number of intact cells after simulated digestion [68]. Some authors have reported that a higher fat content in ice cream and yogurt proved to be more effective in protecting and reducing exposure to acids and bile [69]. All this information can be extrapolated to Petit Suisse formulations due to their dairy base.
The maintenance of the high viability of the strain after digestion reinforces the product’s potential health benefits, such as in reducing oxidative stress; improving health and sensory properties; modulating the immune system and gut microbiota; reducing levels of obesity, glycemia, anxiety, and depression; and aiding in the treatment of food allergies [14,24,70,71,72].
Regarding the use of the by-product, no interference was observed in probiotic survival; in turn, there was an enrichment in antioxidant agents in Petit Suisse (Table 4). After 15 and 30 days of storage, no antioxidant activity was detected using the DPPH and ABTS assays in the simulated digestion phases for either formulation, except for the enteric phase of F0 after 30 days.
Conversely, a reduction in antioxidant activity during the passage through the simulated digestion stages was observed using the FRAP assay for both formulations during the periods studied, probably due to a matrix effect. In this sense, the ferric reducing antioxidant power (FRAP) method was used to detect the antioxidant activity of the Petit Suisse formulations at more stages and times of simulated digestion, followed by the DPPH and ABTS methods, respectively.
A total of 18 fatty acids were identified in both formulations (Table 5), including saturated fatty acids (SFAs: 4:0, 6:0, 8:0, 10:0, 12:0, 14:0, 15:0, 16:0, 18:0, 20:0, 21:0, 22:0, and 24:0), monounsaturated fatty acids (MUFAs: 14:1, 16:1, and 18:1), and polyunsaturated fatty acids (PUFAs: 18:2 and 18:3). Similar results for MUFAs and PUFAs were reported by Coutinho et al. [73] in a chocolate-flavored milk beverage. Among the saturated fatty acids, there was a significant increase (p < 0.05) in butyric acid (4:0), caproic acid (6:0), and caprylic acid (8:0) in the formulation with the addition of the by-product (F1). This behavior may be due to the production of short-chain fatty acids (SCFAs) by the probiotics during their development in the food matrix [74]. These SCFAs have various health benefits, including promoting gut health, improving the immune system, and potentially reducing inflammation [75]. Regarding MUFAs and PUFAs, the control formulation had a higher percentage of these fatty acids, probably due to the lower fat content of the by-product (0.96%).
3.5. Scanning Electron Microscopy (SEM) of the Products
Dairy-based products are well-known carriers of probiotic microorganisms, including the Petit Suisse cheese in the present study. Figure 2 presents the micrographs of the Petit Suisse formulations. The SEM images corroborate the bacterial count results (Table 4), showing the morphological structure of the two cultures used in the formulations. Both formulations showed very similar microstructures in both shape and abundance. Bifidobacterium sp. is a dominant genus among the diverse resistant microbiota in the human gastrointestinal tract [76]. These microorganisms are Gram-positive, anaerobic, non-sporulating, and appear as short, curved bacilli (Figure 2) and Y-shaped rods [77]. In turn, Streptococcus thermophilus is a coccus-shaped bacterium and a lactic acid producer, in addition to being Gram-positive, anaerobic, and non-sporulating.
3.6. Sensory Acceptance and Purchase Intention Tests
Both formulations met the microbiological safety criteria established by the current legislation [43]; thus, they were suitable for sensory analysis. There was no significant difference (p < 0.05) between the F0 and F1 formulations in terms of purchase intention, with 45.7% of the participants expressing an intention to buy F1 and 54.3% expressing an intention to buy F0. In a similar study, 82% of participants expressed an intention to buy guava Petit Suisse containing the probiotic B. animalis subsp. lactis BB-12 and inulin [12].
Concerning their overall impression and color attributes, higher scores were observed for the control formulation (F0), with 80 participants preferring F0 (57.14%) and 60 preferring F1 (42.86%). However, there was no significant difference in the respondents’ purchase intentions towards the samples.
On the evaluation forms, the participants reported that both samples had a strong milk flavor, and some considered both samples to be very sweet but tasty. Although no difference in instrumental texture was identified, the assessors found F0 to have a more pleasant texture when compared to F1. Occasionally, some small particles were felt in the mouth, which may have been mistakenly attributed to a lack of homogeneity.
Some participants also pointed out that the color of F1 was excessively purple, causing them to hesitate before tasting it. However, 30% of the participants experienced nostalgic memories, recalling the flavors of açai, plum, natural grape juice, and sweet wine. On the other hand, some participants commented that the grape flavor could be more pronounced. The contributions of the by-product to the flavor characteristics of dairy products primarily include a sour taste and unique ortho- and retro-olfactory sensations, such as grassy, wine-like, fruity, citrus, cereal, nutty, roasted, and spicy notes [78].
The formulation with the incorporation of the by-product was evaluated for the following attributes: overall impression, flavor, and texture. There were no significant differences when compared to the control. However, some participants reported that the intense purple color caused them to feel strange. Despite the differences between the samples for some parameters, there was no difference in purchase intention between the formulations (Table 6).
Lyophilization is a high-cost process, and Petit Suisse would likely have a high price due to the use of this technology. Therefore, the market niche for this product would be aimed at high-income consumers and/or those who do not prioritize price over the potential health benefits that can be obtained through regular consumption.
4. Conclusions
The by-products generated in the winemaking industry have significant potential as ingredients in the production of Petit Suisse cheese using A2A2 milk and probiotic cultures. This study demonstrated that the addition of the by-product enhanced the cheese’s functional properties, including its antioxidant activity and probiotic viability, while benefiting from the improved digestibility associated with A2A2 milk. Moreover, the addition of the by-product to the fresh cheese proved to be an effective approach concerning technological processing, microbiological safety, and nutritional value. This not only makes it a viable alternative for the dairy industry but also highlights its potential for use in other sectors of the production chain. The introduction of the by-product into the food production chain offers considerable economic and environmental advantages, underscoring the importance of recognizing and leveraging these benefits. The 10% concentration was chosen for the study based on sensory characteristics of color, flavor, aroma, and texture that are typical of Petit Suisse and/or reminiscent of grapes. However, this presents an opportunity for future studies to investigate ways to optimize this concentration and also explore alternatives for obtaining a co-product with an appropriate particle size so as to not negatively impact the characteristic texture of Petit Suisse cheese.
Conceptualization, C.M.S.C.W., L.C.B., G.N.C. and R.K.Y.; methodology, C.M.S.C.W., M.M.G., B.G.V., L.T., G.A.F., J.d.O.L., R.M.N.B., A.E.C.A., P.D.d.S.d.S. and O.O.S.; investigation, C.M.S.C.W.; writing—original draft preparation, C.M.S.C.W.; writing—review and editing, L.C.B., G.N.C. and R.K.Y.; visualization, C.M.S.C.W.; supervision, L.C.B., G.N.C. and R.K.Y.; project administration, R.K.Y. All authors have read and agreed to the published version of the manuscript.
This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee involving Human Subjects (CEP/SH) of the State University of Londrina (UEL) (protocol code CAAE: 75079223.2.0000.523; and 13 November 2023).
Not applicable.
The original data presented in the study are openly available in Open Science Framework (OSF) at DOI 10.17605/OSF.IO/PJC83.
The authors are thankful for the contributions of the Microscopy and Microanalysis laboratory (LMEM) at the State University of Londrina, Terra Telli, and Leite Letti.
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 2. Scanning electron micrographs of the products. (A) F0 at 8000× magnification; (B) = F0 at 15,000× magnification; (C) = F1 at 8000× magnification; (D) = F1 at 15,000× magnification.
Characterization of freeze-dried by-product.
| Parameter | Results (Mean ± SD) |
|---|---|
| Ash (%, w/w) | 4.05 ± 0.10 |
| Moisture (%, w/w) | 11.92 ± 0.65 |
| Protein (%, w/w) | 1.86 ± 0.04 |
| Fat (%, w/w) | 0.96 ± 0.02 |
| Total carbohydrates (%, w/w) | 81.22 ± 0.60 |
| Acidity (mol tartaric acid·kg−1) | 0.043 ± 0.08 |
| Aw | 0.40 ± 0.01 |
| TSS (°Brix) | 2.37 ± 0.12 |
| pH | 3.41 ± 0.02 |
| L* | 20.65 ± 0.11 |
| a* | 10.12 ± 0.37 |
| b* | 4.53 ± 0.14 |
| C* | 110.89 ± 0.33 |
| h* (°) | 1.15 ± 0.02 |
| Fe RC (mol trolox/kg) | 0.19 ± 0.01 |
| DPPH SC (mol trolox/kg) | 0.18 ± 0.01 |
| ABTS SC (mol trolox/kg) | 0.14 ± 0.01 |
| Phenolic compounds (mEq. kg−1) | 55.7 ± 1.30 |
Aw = water activity; L* = luminosity; a* and b* = chromaticity coordinates; C* = chroma; and h* = hue angle. Assay results of antioxidant activity and phenolic compounds of freeze-dried by-product on wet basis; Fe RC = ferric reduction capacity; DPPH SC = DPPH radical scavenging capacity; ABTS SC = ABTS radical scavenging capacity. Results expressed as mean of triplicates ± standard deviation (SD).
Proximate composition of formulations at storage time 1.
| Sample | % Ash | % Moisture | % Protein | % Fat | % Carbohydrates |
|---|---|---|---|---|---|
| F0 (control) | 1.14 ± 0.02 b | 41.49 ± 0.26 a | 22.69 ± 0.16 a | 14.03 ± 0.06 b | 20.640 ± 0.120 b |
| F1 (by-product) | 1.33 ± 0.01 a | 40.51 ± 0.25 b | 20.36 ± 0.21 b | 15.06 ± 0.06 a | 22.717 ± 0.111 a |
Results are expressed as mean ± standard deviation. Equal letters in same column do not differ according to Tukey’s test (p < 0.05).
Physicochemical parameters, antioxidant activity, texture, and LAB counts of formulations.
| F0 | F1 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| T1 | T2 | T3 | T4 | T5 | T1 | T2 | T3 | T4 | T5 | |
| Aw | 0.97 ± 0.02 a | 0.98 ± 0.01 a | 0.98 ± 0.03 a | 0.98 ± 0.01 a | 0.95 ± 0.04 a | 0.96 ± 0.01 a | 0.95 ± 0.01 a | 0.95 ± 0.01 a | 0.95 ± 0.01 a | 0.95 ± 0.01 a |
| TSS (°Brix) | 14.7 ± 0.2 a | 14.7 ± 0.01 a | 14.7 ± 0.01 a | 14.8 ± 0.1 a | 15.0 ± 0.1 a | 33.1 ± 0.9 b | 33.1 ± 0.2 b | 33.2 ± 0.03 b | 33.3 ± 0.03 b | 33.0 ± 0.6 b |
| pH | 5.84 ± 0.01 a | 5.89 ± 0.01 a | 5.84 ± 0.01 a | 5.81 ± 0.01 a | 5.79 ± 0.01 a | 5.70 ± 0.01 a | 5.72 ± 0.02 a | 5.70 ± 0.01 a | 5.74 ± 0.01 a | 5.69 ± 0.01 a |
| Titratable acidity (mEq. lactic acid·kg−1) | 66.67 ± 0.03 a | 56.66 ± 0.05 a | 64.44 ± 0.01 a | 76.67 ± 0.02 a | 73.33 ± 0.04 a | 82.22 ± 0.06 a | 73.33 ± 0.03 a | 69.99 ± 0.04 a | 77.77 ± 0.02 a | 82.22 ± 0.03 a |
| L* | 85.73 ± 0.94 a | 88.09 ± 0.46 a | 89.60 ± 0.15 a | 88.83 ± 0.39 a | 88.60 ± 0.39 a | 42.76 ± 0.47 b | 45.98 ± 0.39 b | 47.85 ± 0.12 b | 49.16 ± 1.16 b | 47.58 ± 0.76 b |
| a* | −2.02 ± 0.09 a | −1.96 ± 0.05 a | −1.87 ± 0.32 a | −2.55 ± 0.14 a | −1.74 ± 0.89 a | 8.93 ± 0.33 b | 9.26 ± 0.39 b | 10.56 ± 0.05 b | 10.47 ± 0.29 b | 10.07 ± 0.76 b |
| b* | 8.66 ± 0.52 a | 9.74 ± 0.42 a | 10.34 ± 0.13 a | 10.66 ± 0.15 a | 10.91 ± 0.18 a | −2.59 ± 0.09 b | −2.47 ± 0.15 b | −2.21 ± 0.07 b | −1.97 ± 0.33 b | −2.16 ± 0.02 b |
| C* | 8.91 ± 0.50 a | 9.94 ± 0.42 a | 9.81 ± 0.38 a | 10.90 ± 0.31 a | 11.13 ± 0.18 a | 9.32 ± 0.31 a | 9.61 ± 0.39 a | 10.84 ± 0.05 a | 10.68 ± 0.25 a | 10.32 ± 0.75 a |
| h* | −0.23 ± 0.02 a | −0.19 ± 0.01 a | −0.30 ± 0.02 a | −0.20 ± 0.01 a | −0.15 ± 0.08 a | −1.29 ± 0.01 a | −1.30 ± 0.01 a | −1.37 ± 0.01 a | −1.38 ± 0.03 a | −1.36 ± 0.01 a |
| Fe RC (mol trolox/kg ± SD) | ND | ND | ND | ND | ND | 0.04 ± 0.02 a | 0.05 ± 0.02 a | 0.05 ± 0.03 a | 0.09 ± 0.03 a | 0.06 ± 0.02 a |
| DPPH SC (mol trolox/kg ± SD) | ND | ND | ND | ND | ND | 0.55 ± 0.03 a | 0.48 ± 0.01 a | 0.39 ± 0.06 a | 0.28 ± 0.05 a | 0.26 ± 0.02 a |
| ABTS SC (mol trolox/kg ± SD) | ND | ND | ND | ND | ND | 0.24 ± 0.01 a | 0.28 ± 0.08 a | 0.31 ± 0.13 a | 0.51 ± 0.12 a | 0.30 ± 0.09 a |
| Phenolic compounds (mEq. gallic acid·kg−1 ± SD) | ND | ND | ND | ND | ND | 20.9 ± 0.1 a | 26.7 ± 0.01 a | 30.5 ± 0.5 a | 47.4 ± 0.5 a | 34.9 ± 0.1 a |
| Hardness (N) | 726 ± 145.37 a | 520 ± 62.46 a | 436 ± 68.60 a | 588 ± 141.32 a | 508 ± 61.54 a | 902 ± 85.84 a | 841 ± 75.50 a | 614 ± 91.70 a | 910 ± 272.33 a | 711 ± 102.37 a |
| Adhesiveness (J) | −821 ± 344.08 a | −641 ± 182.75 a | −557 ± 93.07 a | −591 ± 150.06 a | −608 ± 213.45 a | −1245 ± 312.55 a | −927 ± 203.35 a | −776 ± 253.70 a | −773 ± 184.71 a | −872 ± 308.35 a |
| Cohesiveness | 0.58 ± 0.09 a | 0.66 ± 0.06 a | 0.65 ± 0.05 a | 0.60 ± 0.06 a | 0,64 ± 0.07 a | 0.60 ± 0.03 a | 0.63 ± 0.07 a | 0.61 ± 0.11 a | 0.56 ± 0.06 a | 0.65 ± 0.04 a |
| Gumminess | 423 ± 89.76 a | 345 ± 20.28 a | 281 ± 28.93 a | 345 ± 76.25 a | 305 ± 46.09 a | 514 ± 51.77 a | 525 ± 85.04 a | 374 ± 89.72 a | 465 ± 164.67 a | 452 ± 75.77 a |
| Streptococcus thermophilus (CFU·g−1) | 6.71 ± 0.03 a | 6.69 ± 0.02 a | 6.69 ± 0.05 a | 6.43 ± 0.04 b | 6.31 ± 0.03 b | 6.17 ± 0.03 a | 6.11 ± 0.04 a | 6.11 ± 0.03 a | 5.89 ± 0.02 b | 5.45 ± 0.02 b |
| Bifidobacterium lactis HN019®(CFU·g−1) | 8.60 ± 0.06 a | 8.58 ± 0.06 a | 8.59 ± 0.04 a | 8.60 ± 0.04 a | 7.98 ± 0.05 b | 8.96 ± 0.03 a | 8.95 ± 0.04 a | 8.91 ± 0.02 a | 8.74 ± 0.02 a | 8.58 ± 0.03 a |
ND—not detected; Fe RC = ferric reduction capacity; DPPH SC = DPPH radical scavenging capacity; ABTS SC = ABTS radical scavenging capacity; LAB—lactic acid bacteria; CFU—colony-forming units. S. thermophilus was cultivated in M17 medium; Bifidobacterium lactis HN019 was cultivated in selective MRS medium. F0 = control formulation; F1 = formulation made with 10% by-product; T1 = analysis on 1st day of manufacture; T2 = 7th day; T3 = 14th day; T4 = 21st day; T5 = 30th day. Assay results of antioxidant activity and phenolic compounds for freeze-dried by-product on wet basis. Results are expressed as mean of triplicates ± standard deviation. Equal letters in same column do not differ according to Tukey’s test (p < 0.05).
Antioxidant capacity and viability of LAB and probiotic bacteria in product after simulated digestion in vitro after 15 and 30 days of storage.
| Antioxidant Capacity | LAB Counts | ||||||
|---|---|---|---|---|---|---|---|
| Sample | Phase | Fe RC (mol Trolox/kg) ± DP | DPPH SC (mol Trolox/kg) ± DP | ABTS SC (mol Trolox/kg) ± DP | Streptococcus (log CFU/g) ± SD | Bifidobacterium (log CFU/g) ± SD | |
| Digestion after 15 days of storage | F0 | Before digestion | 11,375 ± 1077 b | ND | ND | 6.74 ± 0.03 a | 8.39 ± 0.06 a |
| Gastric | 656 ± 170 a | ND | ND | 3.96 ± 0.02 a | 6.94 ± 0.02 a | ||
| Enteric | ND | ND | ND | 5.05 ± 0.04 a | 6.99 ± 0.10 a | ||
| F1 | Before digestion | 29,913 ± 1538 b | 1075 ± 90 b | 1613 ± 20 a | 6.03 ± 0.03 a | 8.67 ± 0.07 a | |
| Gastric | 5490 ± 188 b | 78 ± 16 b | 4 ± 3 a | 3.69 ± 0.16 a | 6.54 ± 0.01 a | ||
| Enteric | 682 ± 95 a | 246 ± 5 a | ND | 5.02 ± 0.20 a | 6.99 ± 0.10 a | ||
| Digestion after 30 days of storage | F0 | Before digestion | 15,978 ± 727 a | ND | ND | 6.11 ± 0.11 a | 8.60 ± 0.02 a |
| Gastric | 2422 ± 71 a | ND | ND | 3.66 ± 0.08 b | 7.29 ± 0.07 a | ||
| Enteric | ND | 27 ± 8 | ND | 3.66 ± 0.08 b | 7.05 ± 0.05 a | ||
| F1 | Before digestion | 43,033 ± 966 a | 1813 ± 114 b | 1649 ± 104 a | 5.22 ± 0.02 a | 7.88 ± 0.01 a | |
| Gastric | 5489 ± 280 b | 117 ± 22 b | 60 ± 35 a | 3.48 ± 0.04 b | 6.62 ± 0.10 a | ||
| Enteric | 4836 ± 63 b | 363 ± 11 a | ND | 3.48 ± 0.04 b | 6.01 ± 0.10 a | ||
ND = not detected by the method used; F0 = control formulation; F1 = formulation made with 10% by-product. Fe RC = ferric reducing capacity; DPPH SC = DPPH radical scavenging capacity; ABTS SC = ABTS radical scavenging capacity; LAB—lactic acid bacteria; CFU—colony-forming units. Results expressed as mean of triplicates ± standard deviation. Equal letters in same column do not differ according to Tukey’s test (p < 0.05).
Fatty acid profile of formulations (F0 and F1).
| Fatty Acid | Samples | ||
|---|---|---|---|
| Nomenclature | Cn:m | F0 | F1 |
| Butyric acid | 04:0 | 0.63 ± 0.03 a | 2.48 ± 0.31 b |
| Caproic acid | 06:0 | 0.26 ± 0.02 a | 0.76 ± 0.03 b |
| Caprylic acid | 08:0 | 0.43 ± 0.03 b | 0.54 ± 0.01 a |
| Capric acid | 10:0 | 0.12 ± 0.03 a | 0.12 ± 0.01 a |
| Lauric acid | 12:0 | 3.47 ± 0.12 a | 2.31 ± 0.23 b |
| Myristic acid | 14:0 | 12.11 ± 0.33 a | 15.15 ± 0.53 b |
| Myristoleic acid | 14:1 | 0.36 ± 0.03 b | 0.50 ± 0.02 a |
| Pentadecanoic acid | 15:0 | 1.06 ± 0.01 b | 1.26 ± 0.02 a |
| Palmitic acid | 16:0 | 34.66 ± 0.09 b | 35.92 ± 0.28 a |
| Palmotoleic acid | 16:1 | 1.27 ± 0.01 b | 1.32 ± 0.01 a |
| Stearic acid | 18:0 | 13.59 ± 0.22 a | 11.74 ± 0.15 b |
| Oleic acid | 18:1 | 26.21 ± 0.38 a | 23.12 ± 0.24 b |
| Linoleic acid | 18:2 | 4.54 ± 0.01 a | 4.07 ± 0.05 b |
| Alpha linolenic acid | 18:3 | 0.40 ± 0.02 a | 0.07 ± 0.01 b |
| Arachidic acid | 20:0 | 0.42 ± 0.03 a | 0.36 ± 0.03 b |
| Heneicosanoic acid | 21:0 | 0.14 ± 0.01 a | 0.12 ± 0.01 b |
| Behenic acid | 22:0 | 0.21 ± 0.01 a | 0.10 ± 0.01 b |
| Lignoceric acid | 24:0 | 0.04 ± 0.01 a | 0.05 ± 0.01 a |
F0 = control formulation; F1 = formulation with 10% by-product; Cn:m = n is number of carbons, and m is degree of unsaturation. Results are expressed as mean ± standard deviation. Equal letters in same column do not differ according to Tukey’s test (p < 0.05).
Respondents’ sensory acceptance of and purchase intention towards Petit Suisse made with addition of by-product and control.
| Overall Impression | Color | Flavor | Texture | Purchase Intention | |
|---|---|---|---|---|---|
| F0 | 7.4 ± 3.1 a | 7.8 ± 1.1 a | 6.9 ± 2.0 a | 7.6 ± 1.5 a | 2.5 ± 1.2 a |
| F1 | 6.7 ± 1.8 a | 6.2 ± 2.0 a | 6.7 ± 2.1 a | 7.4± 1.6 a | 2.7 ± 1.2 a |
Results are expressed as mean ± standard deviation. Data are averages from 140 repetitions and relate to structured scales from 1 to 9 for acceptance parameters. Purchase intention ranged from 1 to 5. Equal letters in same column do not differ according to Tukey’s test (p < 0.05).
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; Gomes, Márcia Miss 1 ; Bárbara Geremia Vicenzi 2
; Fabiano, Giovanna Alexandre 3
; de Oliveira Lopes, Jean 3
; Patrícia Daniele da Silva dos Santos 4 ; Tormen, Luciano 1 ; Oscar Oliveira Santos 4 ; Rosangela Maria Neves Bezerra 5 ; Adriane Elisabete Costa Antunes 3
; Bertan, Larissa Canhadas 1
; Giselle Nobre Costa 2 ; Ricardo Key Yamazaki 1
1 Food Science and Technology, Federal University South Border, BR 158, km 405, Laranjeiras do Sul 85319-899, PR, Brazil;
2 Food Science and Technology, State University of Londrina, PR-445, km 380, Londrina 86057-970, PR, Brazil;
3 Dairy, Probiotics and Prebiotics Laboratory (LLPP), College of Applied Sciences (FCA), University of Campinas, R. Pedro Zaccaria, 1300, Limeira 13484-350, SP, Brazil;
4 Postgraduate Program in Chemistry, State University of Maringa, Ave Colombo, 5790, Zona 7, Maringa 87020-900, SP, Brazil;
5 Laboratory of Functional Properties in Foods (LAPFAL), College of Applied Sciences (FCA), University of Campinas, R. Pedro Zaccaria, 1300, Limeira 13484-350, PR, Brazil;