Innovative Approaches to Camembert Cheese: Optimizing Prebiotics and Coagulation Conditions for Enhanced Quality and Nutrition

Abstract

The objective of this study is to investigate how different factors, such as lactic acid bacteria, prebiotics (flaxseed powder, watercress seed powder, okra mucilage), and coagulation temperature influence the final quality of curd by conducting three optimization experiments and implementing a structured experimental plan. In the first phase, milk coagulation was assessed at 45 °C with various combinations of lactic acid bacteria (probiotics) and prebiotics (powdered flaxseed and watercress). In the second investigation phase, the effects of lowered probiotic and prebiotic (powdered flaxseed and watercress) concentrations were examined at the coagulation temperature of 38 °C. We investigated the concentration of lactic acid bacteria at 3 mg/mL of milk and the effects of temperature and prebiotics (okra mucilage and flaxseed powder). We observed short milk clotting time (2 s) using the optimized mixture (0.18 mg of probiotics, 1.5 mg of flaxseed powder, and 1.147 mg of watercress powder) per 10 mL of milk. It contrasts with the classical coagulation way optimized at (5.9 and 9.5 s), which were generated at optimal temperatures of 45 and 45.7 °C, respectively. Our new mixture improves the fermentation process of camembert cheese at 38 °C. This cheese had a high flavonoid content, fewer lactic bacteria and molds, a homogeneous texture, and no outer crust, and exceptional sensory attributes such as a creamy and fluid paste. These attributes suggest its potential benefits as a dairy product for individuals with cardiovascular and gastrointestinal conditions.

1. Introduction

Functional foods and nutraceuticals are increasingly recognized for their role in promoting health and well-being. These include a broad range of products such as probiotics and prebiotics [1], which have been demonstrated to possess qualities that promote health and well-being. Probiotics are live microorganisms that, when consumed in adequate amounts, provide health benefits to the host. They are commonly found in fermented foods such as yogurt, kefir, sauerkraut, and kimchi.

Fuller defined probiotics as preparations containing live microorganisms are used as food additives [2]. Their viability and availability depend on various factors, including manufacturing conditions, storage, gastric acidity, and interactions with milk constituents.

Prebiotics, on the other hand, are non-digestible food ingredients that selectively stimulate the growth and activity of beneficial bacteria in the gut as defined by the International Scientific Association of Prebiotics and Prebiotics (ISAPP) in 2017.

Prebiotics are found in high-fiber foods such as fruits, vegetables, whole grains, and legumes. Together, these components form a critical part of the human diet and contribute to the maintenance of a healthy gut microbiota, which is essential for overall health.

Polysaccharides are common in conventional pharmacopeia [3] and are often used as prebiotics [4]. They can help prevent diarrhea and constipation by promoting regular bowel movements and maintaining a healthy gut microbiota. They also help lower blood cholesterol levels and intestinal pH, which can reduce the risk of cardiovascular disease and improve overall gut health.

Furthermore, they lower blood cholesterol and intestinal pH, reducing cardiovascular risk [5,6,7]. Additionally, prebiotics inhibit pathogenic bacteria in the gastrointestinal tract, stimulate mineral absorption, and help prevent cancer and obesity [8,9].

Prebiotic fibers, known for their gelling properties, are often added to foods in acceptable amounts to enhance their texture and sensory qualities [10]. Studies have shown that a mixture of lactic bacteria with prebiotics, such as cardon flowers, vine leaves, okra fruit mucilage powders, flaxseed, and watercress seed, reduce the milk coagulation time and enhance the nutritional and functional properties of the final product [11,12,13].

Flaxseed and watercress seed are rich sources of antioxidants, such as flavonoids and mucilage [13,14,15].

Mucilages are polysaccharide substances that swell in the presence of water and acquire a viscous consistency [16,17]. These substances are used as gelling agents as well as for thickening, emulsification, and stabilization [18].

Flaxseed mucilages have demonstrated benefits such as preventing intestinal irritation, reducing blood sugar and cholesterol levels, and moisturizing the skin. These properties make flaxseed a valuable ingredient in functional foods and nutraceutical products. Additionally, flaxseed mucilage can act as an artificial mucus, providing a protective barrier for the gastrointestinal tract and enhancing the overall health of the digestive system [18,19].

Moreover, watercress seed and flaxseed are high in polysaturated fats (66% and 41.57%, respectively), with linolenic acid (50.11% and 29.92%, respectively). These fats are essential to human health and can help prevent various illnesses, such as the respiratory diseases [20], hypertension, cardiovascular conditions, diabetes, and inflammatory disorders [21].

The dairy industry generates significant amounts of whey as a by-product of cheese production. Whey is considered a potent environmental pollutant due to its high chemical oxygen demand (COD) and biological oxygen demand (BOD), with COD ranging from 50 to 102 g/L and BOD from 37 to 60 g/L [22,23].

Fischer and Kleinschmidt [24] reported that 212.211 kilotons of whey are produced annually from 23.579 kilotons of cheese. Incorporating whey into functional foods and nutraceutical products could offer benefits and help reduce the environmental impact.

The processing temperature and the mix of lactic acid bacteria with prebiotics are key factors affecting the quality and safety of the final product in the dairy industry. Prebiotics can impact bacterial behavior during fermentation and interact with milk proteins, affecting texture, flavor, and the nutritional profile of the final product.

The temperature of fermentation also plays a crucial role in the speed of milk clotting and the overall quality of the dairy product. Optimizing these variables is essential for producing high-quality functional foods and nutraceutical products that provide maximum health benefits.

Three optimization studies were conducted using surface response methodology (SRM). The studies analyzed coagulation time, flavonoid content, and syneresis volume, identifying optimal conditions to maximize flavonoid content while minimizing coagulation time and syneresis volume.

The work’s novelty lies in its innovative approach to optimizing curd quality. The research integrates prebiotic materials—flaxseed powder, watercress seed powder, and okra mucilage—into the formulation process. These ingredients are selected for their unique nutritional properties and their ability to enhance the curd’s functional characteristics.

SRM could investigate how these prebiotics influence curd quality at varying temperatures. Gas chromatography was used for fatty acid profiling, while microbiological analysis ensured safety and quality standards.

2. Material and Methods

2.1. Plant Material

Flax (Linum usitatissimum) and watercress (Lepidium sativum) seeds were purchased from Rahma supermarket (Tizi-Ouzou). Following the cleaning and removal of excess moisture, the seeds were ground using an electric grinder to produce a 200-mesh powder. Okra fruit (Abelmuschus esculentus) was procured from the Ghardaia (Algeria).

2.2. Biological Material

Milk coagulation was initiated by a mixture of freeze-dried acidic lactic bacteria starter from Chr. Hansen in Horsholm, Denmark. To produce Camembert cheese, three specific strains of mold fungi from Chr. Hansen were employed: Penicillium candidum (PC Snow), which is crucial for both the development of the characteristic white, bloomy rind and the inhibition of undesirable microbial contamination; Penicillium candidum (PC SAM3), responsible for forming the delicate white surface thalli and contributing to the distinctive aroma of the cheese; and Geotrichum candidum (GEO 17), which enhances the cheese’s flavor, aroma, and final appearance, thereby ensuring the development of its unique texture and ripening characteristics.

2.3. Experimental Designs Methodology

Three optimization experiments were conducted using response surface methodology (RSM) to evaluate the effects of the various factors, such as the mixture of lactic acid bacteria and prebiotics (flaxseed powder, watercress seed powder, okra mucilage), and the milk coagulation temperature. Each experiment involved 18 tests with three replicates. This approach aimed at maximizing the curd’s total flavonoid content (TFC) while minimizing milk coagulation time and syneresis volume using a centered composite design (CCD) with four central points, as indicated by the response optimizer function of the Statgraphis Centurion 18 software.

In this study, SRM investigated factors including the mixture of lactic acid bacteria and prebiotics, as well as the temperature at which milk coagulation occurs.

The general form of the response surface model is expressed as follows:

$$Y=a_0+\sum _{i=1}^3{a}_i{X}_i+\sum _{i=1}^3{a}_i{X}_i^2+\sum _{i=1}^3\sum _{j=1}^3{a}_{ij}{X}_i{X}_j$$

(1)

where X_i (i = 1, 2, and 3) represent the independent variables, and a_i and a_ij are the coefficients for the linear, quadratic, and interaction terms, respectively. The variable Y denotes the predicted response.

The three optimization studies were as follows:

1. First Optimization Study: In this study, it was attempted to optimize the combination of lactic acid bacteria and prebiotics (flaxseed and watercress powders) at 45 °C to assess their impact on curd quality, including TFC and texture.

2. Second Optimization Study: In this study, how a lower temperature (38 °C) and reduced probiotic content affect coagulation process and curd quality was examined.

3. Third Optimization Study: In this study, the interaction between prebiotic composition and coagulation temperature was investigated, with a constant lactic bacteria concentration of 3 mg across all 18 tests.

2.4. Assessment Methods

2.4.1. Extraction of Okra Fruit Mucilage

Mucilage extraction was performed according to the method described by Dick et al. [25]. Fresh okra fruits were thoroughly washed and cut into small pieces, then soaked in distilled water. Subsequently, they were mechanically agitated to promote the release of mucilage. Next, the mixture was homogenized using a high-speed blender to ensure maximum mucilage release. The resulting suspension was filtered through a fine mesh, and the filtrate was concentrated under reduced pressure to yield a viscous extract, which was then stored under refrigeration.

2.4.2. Total Flavonoid Content (TFC) Analysis

TFC was determined using a colorimetric assay at 430 nm [26]. A calibration curve was constructed using quercetin standards, and TFC was calculated using the regression equation Y = 0.0942X − 0.0387 with an R² value of 0.9982, expressed as milligrams of quercetin equivalent per gram of fresh matter (mg QE/g FM). Each sample was analyzed in triplicate, and statistical analysis was performed to validate the reproducibility of the results.

2.4.3. Method for Determining Clotting Milk Time

The method for determining milk coagulation time, measured until the acidity reached a pH of 4.5, involved using a chronometer.

2.4.4. Fatty Acid Profile Analysis

Fatty acid profiling was performed by gas chromatography using a CHROMPACK CP 9002 system (São Paulo, Brazil) following an ISO 5509 esterification method [27].

2.4.5. Microscopic Structure

Using a Carl Zeiss binocular photo microscope (Jena, Germany) attached to a camera and dyed with phenol red, the microscopic structures of both fresh curds generated using the optimal mixture (lactic bacteria and prebiotic-flaxseed powder and okra mucilage) and probiotic (lactic bacteria) were examined.

2.4.6. Camembert Cheese Fabrication

The stages in the fabrication of fermented Camembert cheese supplemented with prebiotics encompass the following steps:

1. The cow’s milk was pasteurized at 72 °C for 15 to 20 s, and kept at 38 °C for 15 min.

2. The optimized mixture of probiotics (0.18 mg/10 mL of milk) and prebiotics (1.5 mg of flaxseed powder and 1.147 mg of watercress seed powder) was added to pre-warmed cow’s milk, and the obtained mixture was inoculated with commercial fungal strains (Penicillium candidum (PC Snow), Penicillium candidum (PC SAM3), and Geotrichum candidum (GEO 17)) and incubated at 38 °C for 40 min.

3. Before being transferred to milk fermentation (20 L), presolubilized rennet was added to the inoculum after the mixture was incubated at 38 °C for 50 min to obtain a curd.

4. The curd was manually cut into small cubes using two trancheurs (horizontal and vertical), followed by mixing to remove the sour milk.

The plastic molds were filled with the filtered water and placed on shelves set on perforated aluminum plates that allow serum to drain.

5. After a complete molding, we moved the cheese to the evaporation chamber heated at 28 °C. It was left there for an hour before being turned over again. Two hours later, the cheese was turned over again, and then turned over a third time, this time resting on its first face until the following day.

6. The cheese balls were disassembled and arranged on grille trays a day after the harvest.

7. After being salted, the recovered cheeses were placed in the bleeding chamber at 12 °C for 48 h to remove any residual moisture.

8. The cheeses were placed in the first hollow to allow the aroma and white crust to develop, and then they were turned back so that the crust could continue to develop on the other side. After that, they were kept at room temperature (14 °C) for 12 days to continue the affinage process.

The same steps were followed to prepare the Camembert reference from ATTOUCHE industry, but no prebiotics were added.

2.4.7. Microbiological Analysis

This analysis followed Bourgeois and Malcoste’s [28] procedure to identify key microorganisms, including total coliforms, fecal coliforms, Staphylococcus aureus, mesophilic and thermophilic lactic acid bacteria, yeasts, and molds. Standard techniques such as selective media cultivation, incubation, and microscopic examination were employed for identification and quantification.

3. Results and Discussion

Table 1. Centered composite design (CCD) with process factors X_i and the responses Y_k of the curd milk (first optimization).

Run N°	Treatment Parameters (Independent Variables)			Responses (Dependent Variables)
	X1 (mg/10 mL of Milk)	X2 (g/10 mL of Milk)	X3 (g/10 mL of Milk)	Y1: tc (S)	Y2: TF (mg Quercetin/g FM)	Y3: SV (mL)
01	24.5 (1)	0.7 (−1)	1 (1)	60 ± 0	0.036 ± 0.05	0
02	16.5 (0)	1.5 (1.682)	0.7 (0)	30 ± 0	0.297 ± 0.02	0
03	16.5 (0)	0.5 (−1.682)	0.7 (0)	360 ± 60	0.598 ± 0.01	0
04	8.47 (−1)	1.3 (1)	1 (1)	60 ± 0	0.510 ± 0.05	0
05	8.47 (−1)	0.7 (−1)	0.4 (−1)	900 ± 0	0.268 ± 0.26	0
06	24.5 (1)	0.7 (−1)	0.4 (−1)	690 ± 42.42	0.508 ± 0.04	0
07	8.47 (−1)	0.7 (−1)	1 (1)	120 ± 0	0.428 ± 0.03	0
08	8.47 (−1)	1.3 (1)	0.4 (−1)	220 ± 34.64	0.147 ± 0.01	0
09	30 (1.682)	1 (0)	0.7 (0)	120 ± 0	0.226 ± 0.20	0
10	16.5 (0)	1 (0)	1.2 (1.682)	28 ± 3.46	0.393 ± 0.03	0
11	3 (−1.682)	1 (0)	0.7 (0)	105 ± 21.21	0.256 ± 0.01	0
12	24.5 (1)	1.3 (1)	1 (1)	30 ± 0	0.262 ± 0.02	0
13	24.5 (1)	1.3 (1)	0.4 (−1)	240 ± 0	0.223 ± 0.01	0
14	16.5 (0)	1 (0)	0.2 (−1.682)	910 ± 14.14	0	0
15	16.5 (0)	1 (0)	0.7 (0)	217.5 ± 3.53	0.158 ± 0.02	0
16	16.5 (0)	1 (0)	0.7 (0)	370 ± 28.284	0.199 ± 0.03	0
17	16.5 (0)	1 (0)	0.7 (0)	390 ± 42.426	0.055 ± 0.01	0
18	16.5 (0)	1 (0)	0.7 (0)	185.5 ± 2.121	0.319 ± 0.02	0

tc: time of coagulation (s); TF: total flavonoids (mg quercetin equivalent/g FM); SV: syneresis volume (mL); ${\mathrm{X}}_1$ = lactic bacteria [mg/10 mL of milk]; ${\mathrm{X}}_2$ = flaxseed powder [g/10 mL of milk]; ${\mathrm{X}}_3$ = watercress seeds [g/10 mL of milk].

,

Table 2. Centered composite design (CCD) with process factors X_i and the responses Y_k of the curd milk (second optimization).

Run N°	Treatment Parameters (Independent Variables)			Responses (Dependent Variables)
	X1 (mg/10 mL of Milk)	X2 (g/10 mL of Milk)	X3 (g/10 mL of Milk)	Y1: tc (S)	Y2: TF (mg Quercetin/g FM)	Y3: SV (mL)
01	0.1 (−1)	1.3 (1)	0.4 (−1)	300 ± 0.016	1.45 ± 0.028	0
02	0.25 (1)	0.7 (−1)	1.0 (1)	60 ± 0	1.1 ± 0	0
03	0.175 (0)	1.5 (1.682)	0.7 (0)	120 ± 0	1.85 ± 0.04	0
04	0.1 (−1)	0.7 (−1)	0.4 (−1)	139.8 ± 0.01	1.37 ± 0.04	0
05	0.05 (−1.682)	1.0 (0)	0.7 (0)	1020 ± 0.03	1.12 ± 0.01	0
06	0.30 (1.682)	1.0 (0)	0.7 (0)	240 ± 0.016	1.22 ± 0.05	0
07	0.25 (1)	1.3 (1)	1.0 (1)	120 ± 0	1.69 ± 0.01	0
08	0.25 (1)	0.7 (−1)	0.4 (−1)	1519.8 ± 0.01	2.21 ± 1.05	0
09	0.175 (0)	0.5 (−1.682)	0.7 (0)	820.2 ± 0.017	0.64 ± 0.05	0
10	0.25 (1)	1.3 (1)	0.4 (−1)	900 ± 0.016	0.89 ± 0.03	0
11	0.1 (−1)	0.7 (−1)	1.0 (1)	319.8 ± 0.007	0.95 ± 0.03	0
12	0.175 (0)	1.0 (0)	1.2 (1.682)	139.8 ± 0.001	1.19 ± 0.02	0
13	0.175 (0)	1.0 (0)	0.2 (−1.682)	1419.6 ± 0.02	0.51 ± 0.05	0
14	0.1 (−1)	1.3 (1)	1.0 (1)	100.2 ± 0.05	1.13 ± 0.02	0
15	0.175 (0)	1.0 (0)	0.7 (0)	280.2 ± 0.05	0.98 ± 0.02	0
16	0.175 (0)	1.0 (0)	0.7 (0)	280.2 ± 0.05	0.92 ± 0.01	0
17	0.175 (0)	1.0 (0)	0.7 (0)	300 ± 0	1.04 ± 0.05	0
18	0.175 (0)	1.0 (0)	0.7 (0)	160.2 ± 0.05	0.98 ± 0.01	0

tc: time of coagulation (s); TF: total flavonoids (mg quercetin equivalent/g FM); SV: syneresis volume (mL); ${\mathrm{X}}_1$ = lactic bacteria [mg/10 mL of milk]; ${\mathrm{X}}_2$ = flaxseed powder [g/10 mL of milk]; ${\mathrm{X}}_3$ = watercress seeds [g/10 mL of milk].

and

Table 3. Centered composite design (CCD) with process factors X_i and the responses Y_k for the curd milk (Third optimization).

Run N°	Treatment Parameters (Independent Variables)			Responses (Dependent Variables)
	X1 (mL/10 mL of Milk)	X2 (°C)	X3 (g/10 mL of Milk)	Y1: tc (S)	Y2: TF (mg Quercetin/g FM)	Y3: SV (mL)
01	2.75 (0)	42.85 (0)	0.85 (0)	31.333 ± 0.026	2.77 ± 0.03	1.066 ± 0.015
02	1.41 (−1)	41.16 (−1)	0.46 (−1)	26 ± 0.01	5.196 ± 0.03	1 ± 0.043
03	4.09 (1)	41.16 (−1)	0.46 (−1)	26.666 ± 0.015	4.453 ± 0.03	1.533 ± 0.025
04	1.41 (−1)	44.54 (1)	0.46 (−1)	20 ± 0.026	3.22 ± 0.03	1.4 ± 0.05
05	4.09 (1)	44.54 (1)	0.46 (−1)	24.333 ± 0.025	6.37 ± 0.036	1.066 ± 0.008
06	1.41 (−1)	41.16 (−1)	1.24 (1)	40.666 ± 0.014	3.496 ± 0.03	0.5 ± 0.02
07	4.09 (1)	41.16 (−1)	1.24 (1)	46.33 ± 0.037	12.63 ± 0.01	0.133 ± 0.05
08	1.41 (−1)	44.54 (1)	1.24 (1)	22.666 ± 0.041	0.3 ± 0.034	0.3 ± 0.034
09	4.09 (1)	44.54 (1)	1.24 (1)	17.333 ± 0.028	2.013 ± 0.90	0.3 ± 0.01
10	2.75 (0)	42.85 (0)	0.85 (0)	13 ± 0.05	1.704 ± 0.03	0.033 ± 0.057
11	2.75 (0)	42.85 (0)	0.85 (0)	40 ± 0	6.326 ± 0.02	0.833 ± 0.030
12	0.5 (−1.682)	42.85 (0)	0.85 (0)	30 ± 0	1.906 ± 0.05	0.133 ± 0.015
13	5 (1.682)	42.85 (0)	0.85 (0)	26.333 ± 0.015	2.73 ± 0.016	0.7 ± 0.043
14	2.75 (0)	40 (−1.682)	0.85 (0)	27 ± 0.024	5.303 ± 0.04	0.5 ± 0.036
15	2.75 (0)	45.7 (1.68)	0.85 (0)	13 ± 0.005	3.11 ± 0.037	0
16	2.75 (0)	42.85 (0)	0.2 (−1.682)	25.333 ± 0.05	3.146 ± 0.04	2.333 ± 0.007
17	2.75 (0)	42.85 (0)	1.5 (1.682)	33.333 ± 0.028	6.376 ± 0.03	0.366 ± 0.03
18	2.75 (0)	42.85 (0)	0.85 (0)	40 ± 0	3.143 ± 0.02	0.866 ± 0.05

tc: time of coagulation (s); TF: total flavonoids (mg quercetin equivalent/g FM); SV: syneresis volume (mL); ${\mathrm{X}}_1$ = volume of okra mucilage [mL/10 of milk]; ${\mathrm{X}}_2$ = temperature of coagulation [°C]; ${\mathrm{X}}_3$ = flaxseed powder [g/10 mL of milk].

show the responses from the optimization studies. They are as follows: Y₁: time of coagulation (tc) (s); Y₂: total flavonoids (TF) (mg quercetin equivalent/g FM), and Y₃: syneresis volume SV (mL).

3.1. First Optimization

The empirical models developed for predicting the milk clotting time (tc) (Y₁) and total flavonoids (Y₂) demonstrated high accuracy, with R² values of 94.49% and 76.69%, respectively. These high coefficients of determination indicate the robustness of the models in capturing the variability in the response variables.

These R² values indicate that the models account for a substantial portion of the variability observed in the residuals, confirming their effectiveness in predicting the outcomes.

Our study provides further insight by highlighting the interaction effects between flaxseed and watercress seed powders, which have not been as extensively explored in previous works.

Statistical analysis revealed that both flaxseed and watercress seed powders had a notable effect on coagulation time (p < 0.05). Specifically, the analysis identified a significant first-order negative linear effect for both types of prebiotics, with p-values less than 0.05. This suggests that increasing the concentration of either prebiotic resulted in a decrease in coagulation time (Figure 1a). This negative correlation highlights the efficiency of these prebiotics in accelerating the coagulation process. The rate of fermentation speed is related to the prebiotic effect as reported by Amirdivani and Baba [29].

In this study, a significant second-order quadratic interaction effect was found for the quantity of watercress seed powder (p < 0.05), indicating a notable interaction between the amounts of watercress seed powder and flaxseed powder.

The presence of lactic acid bacteria further influences this interaction, suggesting that the interactions between different prebiotics and probiotics are critical for understanding their combined effects on coagulation.

In our study, this was further enhanced by introducing the role of lactic acid bacteria, a crucial probiotic that has been shown to facilitate faster milk coagulation through enhanced lactose assimilation by improving lactic acid as reported by Walstra [30].

Lactic acid bacteria are classified as mesophilic microorganisms with an optimal growth temperature of 30 °C. Their maximum growth temperature is between 35 and 45 °C, and their minimum growth temperature may be around 0 °C [31].

The rate in milk coagulation speed is also associated with variations in the acidifying activity of different species of lactic acid bacteria, which translate into different coagulation speeds and lactic acid production. For instance, S. thermophilus produces less lactic acid at an optimal temperature of 45 °C but acidifies more quickly than Lactococci and Lactobacilli. On the other hand, the latter produce the highest amount of lactic acid [32,33].

Collaboration between these lactic species allows for a decrease in latency phase length, an increase in biomass production, and a rise in viscosity [34].

For TFC, the amount of probiotics (lactic acid bacteria) and prebiotics had a considerable impact on flavonoid bioavailability (Figure 1b). The quantity of flaxseed powder showed a significant second-order quadratic interaction effect (p < 0.05), implying that its effect on TFC is influenced by its interactions with other variables. Additionally, a significant interaction effect (p < 0.05) was observed between the amounts of probiotics and watercress seed powder. This interaction suggests that the relationship between prebiotics and probiotics is essential in modulating flavonoid availability, highlighting the importance of optimizing these components for desired curd properties.

These findings highlight the critical role of optimizing of prebiotic/probiotic ratios to improve bioactive compound retention in functional dairy products.

3.2. Second Optimization

The refined model for predicting the milk coagulation time (tc) (Y₁) showed significant regression (p < 0.05) and a satisfactory R² value of 70.96%, which indicates that the model accurately captures the variability in coagulation time.

However, in contrast to studies where factors such as temperature and pH were the primary influences on coagulation time, in our study, the significant contribution of watercress seed powder (p < 0.05) was emphasized, attributed to its mucilage content, which is known to enhance lactic acid bacteria growth at 38 °C as shown in the response surface graphs (Figure 2a). With their specific physical and chemical properties, mucilages facilitate the assimilation and distribution of lactic acid bacteria throughout the milk matrix, thereby accelerating the coagulation process.

This finding is corroborated by Guler-Aki et al. [35], who identified similar coagulation-enhancing properties in plant-derived mucilages.

In terms of TFC (Y₂), our model explained less variability with an R² value of 45.71%. This suggests that additional factors may need to be explored in future studies to improve the model’s predictive power.

Conversely, the models adjusted for TFC (Y₂) presented less favorable outcomes, with an R² value of 45.71%. Furthermore, the regression analysis was not statistically significant (p > 0.05), suggesting that the model did not adequately explain variations in flavonoid levels under test conditions.

According to Samuelsen et al. [36], earlier research has shown that environmental factors like extraction temperature affect the availability of bioactive compounds extracted from P. major leaves at 50 and 100 °C, which in turn affects the yields of lactic acid bacteria development, which range from 36% to 26%.

On the other hand, the quantities of lactic acid bacteria and the combined prebiotic powders (flaxseed and watercress seed) did not show a significant effect on the availability of TFC in the curd at 38 °C. These findings highlight that while prebiotics and probiotics concentrations influence coagulation time, they do not significantly affect flavonoid availability under the tested conditions.

3.3. Third Optimization

The model adapted for syneresis volume (SV) (Y₃) revealed a significant regression (p < 0.05) and an R² value of 82.93%.

This contrasts with the models for coagulation time (Y₁) and TFC (Y₂) (p > 0.05) and low R² values, ranging from 42.84% to 57.81%. The low R² values indicate that the models were not very effective in explaining the variability in coagulation time and flavonoid content.

A comprehensive analysis of the response surface graphs (Figure 3a–c) revealed several findings, as follows:

1: Coagulation Temperature: A significant negative first-order linear effect was observed (p < 0.05), showing that coagulation time decreases with increasing temperature. The data indicate that the coagulation temperature is the primary factor affecting milk clotting time

2: Syneresis Volume: A significant negative first-order linear effect (p < 0.05) was noted, indicating that increasing flaxseed powder amount results in reduced syneresis volume. This can be attributed to the high content of fibers, mucilage, and oil in flaxseed [13,37]. Fibers and mucilages form a network with calcium ions, which helps retain water within the curd. Additionally, the oil component helps form fine micelles that encapsulate lactic bacteria within the polymer gel matrix, enhancing the curd’s structural integrity and reducing water loss, as evidenced by microscopic examination (Figure 4a).

These findings reveal that the flaxseed-enriched curd is characterized by numerous small micelles containing lactic acid bacteria, uniformly distributed throughout the polymer gel network (Figure 4a). This distribution results in a curd with a light density and a soft, non-firm texture, devoid of visible pores. In contrast, curds made solely with lactic bacteria (probiotics) displayed a firmer texture with noticeable pores (Figure 4b), emphasizing the significant impact of flaxseed on the curd’s texture and structural properties. These observations are consistent with the research by Oliveira et al. [38], which demonstrated that fiber addition can significantly influence yogurt texture.

Moreover, studies have shown that incorporating polydextrose, inulin, and gluco-oligosaccharides (GOSs) into yogurt enhances elasticity, viscosity, and firmness [4].

These findings provide a comprehensive understanding of how different factors influence syneresis volume and curd texture.

3.4. Optimizations of Milk Coagulation Conditions by RSM

Table 4. Optimization results.

Optimization	Optimized Variables Values					Predicted Optimized Response Values			Desirability
	Lactic Bacteria (mg/10 mL of Milk)	Flax Seed Powder (g/10 mL of Milk)	Watercress Seed Powder (g/10 mL of Milk)	Volume of Okra Mucilage (mL/10 of Milk)	Temperature of Coagulation (°C)	Tc (s)	TF (mg Quercetin Equivalent/g FM)	SV (mL)
1	6.53	1.05	1.196	-	Fixed at 45	5.867	0.624	-	1
2	0.181	1.5	1.147	-	Fixed at 38	2	2.225	-	1
3	Fixed at 3	1.5	-	0.50	45.7	9.512	11.014	0.116	0.933

displays a summary of the experiments. Each experimental run varied in terms of concentrations of lactic bacteria, flaxseed powder, watercress seed powder, okra mucilage, and coagulation temperature. The optimized parameters include coagulation time, TFC, and syneresis volume. The desirability scores for each run indicate that Runs 1 and 2 achieved a perfect desirability score of 1.

The interaction between lactic bacteria, flaxseed, and watercress seed powders significantly influenced coagulation time. Table 4 highlights a short milk coagulation time of 2 s at 38 °C, for optimal amounts of probiotics (0.18 mg/10 mL of milk) and prebiotics (1.5 mg of flaxseed powder and 1.147 mg of watercress seed powder). These results stand in contrast to the extended coagulation times of 5.867 s and 9.512 s observed at 45 °C and 45.7 °C, respectively. This suggests that a lower temperature combined with specific prebiotic concentrations create a highly efficient coagulation environment.

These findings indicate that the interaction between prebiotic polysaccharides (flaxseed and watercress seed powders) and milk proteins had a strong quadratic effect which significantly accelerates coagulation time (Figure 1a). Mucilages help retain free water, thus reducing polarity and enhancing hydrophobic interactions. This results in the formation of micelles, characterized by a hydrophobic core and a hydrophilic exterior, which effectively retain lactic acid bacteria within the milk matrix.

The strong affinity of lactic acid bacteria for the polysaccharides in watercress seeds is evident. At 38 °C, these interactions are minimal, whereas higher temperatures slow down the acidification process, making these interactions more pronounced. This finding underscores the critical role of prebiotics in reducing milk clotting time, thereby improving processing efficiency.

Furthermore, Jiang et al. [39] emphasize the critical role of interaction effects between various components in improving system performance.

The interaction between the flaxseed and watercress seed powders also played a crucial role in increasing TFC, as illustrated by the response surface plot (Figure 1b).

The curd produced at 45.7 °C exhibited the highest TFC (11.014 mg QE/g FM), revealing the critical role of temperature in the release of phenolic compounds.

Higher temperatures generally enhance the release of these compounds, while the mobility, size, and conformation of polysaccharides (such as mucilages from prebiotics) affect the interaction between milk proteins and phenolic compounds. Stronger interactions lead to an increase in the availability of phenolic groups for binding to the forming gels, demonstrating a reversible interaction mechanism in an acidic environment.

In pH levels between 2 and 5, the binding of flavonoids to milk proteins remains relatively unaffected by the pH changes, suggesting that stronger ionic interactions enhance hydrophobic effects. Filamentous polysaccharides may exhibit relatively weak binding to phenolic compounds, due to their elongated structure. However, the hydrophobic cavities within the secondary structure of these polysaccharides create a significantly higher attraction for polyphenols [40]. Conversely, smaller polymers do not show notable interactions with phenolic compounds [41].

These results offer insight into how various factors influence the quality and properties of milk-based products, underscoring the importance of optimizing processing conditions.

3.5. Stability and Functionality of the Optimized Mix (Prebiotics and Probiotics)

To assess the viability and functionality of the optimized mixture of lactic acid bacteria and prebiotics at 38 °C derived from the second optimization, the curd was dried at the same temperature, then processed into powder, granules, and tablets (Figure 5a–c).

This mixture was used to produce Camembert cheese (fermented at 38 °C), which exhibited a creamy, fluid paste with a uniform texture and no outer crust (Figure 5d) in contrast to the firm crust, creamy, and smooth interior of the commercial Camembert from ATTOUCHE MILK INDUSTRY (Tizi-Ouzou, Algeria)(Figure 5e).

A comparative analysis between the two Camembert cheeses showed that the former exhibited lower counts of thermophilic lactic acid bacteria, yeasts, and molds compared to the commercial one (

Table 5. Microbiological analysis results expressed in CFU/g of cheese.

Researched Germs	Cheese Made with Optimized Curd	Camembert Cheese from ATTOUCHE MILK Industry	Standards (OJRA, 1998)
Total coliforms at 37 °C	Absent	Absent	100
Fecal coliforms at 44 °C	Absent	Absent	10
Mesophilic lactic acid bacteria at 30 °C	6.3 × 103	Absent	-
Thermophilic lactic acid bacteria at 44 °C	3.5 × 103	Non-significant	-
Staphylococcus aureus at 37 °C	Absent	Absent	Absent
Yeasts and Molds	>300	Non-significant	-

). This can be attributed to the prebiotic content (mucilages), rich in antioxidants (flavonoids) and antibacterial substances (fatty acids).

The optimized curd also exhibited higher TFC (4.1500 ± 0.015 mg QE/g FM) compared to the commercial cheese (1.8310 ± 0.046 mg QE/g FM). Swelam et al. [42] reported that incorporating additives derived from plants can increase bioactive compounds in dairy products. Furthermore, the optimized curd was rich in fatty acids, including palmitic acid (28%), oleic acid (21.2%), linoleic acid (1.70%), arachidic acid (0.91%), linolenic acid (0.23%), and pentadecanoic acid (0.87%), the latter being recognized for its notable anticarcinogenic properties [43].

According to Freitas and Malcata, [44], most soft Camembert cheeses exhibit high concentrations of fatty acids like C16:0 (palmitic acid) and C18:0 (stearic acid). Both analyzed cheeses showed neutral pH levels, with levels of 6.46 ± 0.05 and 7.4 ± 0.05, respectively.

The results are similar to those reported in other studies, which indicate that the incorporation of fennel extract in yoghurt leads to lower lactic acid bacteria [45,46,47].

Sensory assessment revealed that the Camembert cheese made from the optimized curd had a better taste, texture, and color, with a distinctive brownish hue. These enhanced sensory characteristics suggest that this Camembert not only meets but exceeds traditional standards, positioning itself as a potentially novel and nutritionally advantageous dairy product. It holds promise for individuals with specific health conditions, including gastrointestinal and cardiovascular diseases, due to its enhanced nutrient profile and beneficial bioactive compounds.

4. Conclusions

In this study, insights are provided into the optimization of curd quality through the evaluation of factors such as lactic acid bacteria, prebiotics, and coagulation temperature. The results showed that the combination of lactic acid bacteria with specific prebiotics (flaxseed powder and watercress seed powder) at an optimal temperature of 38 °C results in a short coagulation time. These results underscore the effectiveness of prebiotic and probiotic interactions in enhancing coagulation and curd quality. In conclusion, the optimization of curd production conditions and the resulting high-quality Camembert cheese suggest valuable applications in both the dairy industry and functional food sectors. Future research should explore broader implications, including the long-term health benefits.