fermentation

Article
Innovative Approaches to Camembert Cheese: Optimizing
Prebiotics and Coagulation Conditions for Enhanced Quality
and Nutrition
Adiba Benahmed Djilali 1,2, *, Mohammed Said Metahri 1 , Lynda Lakabi 1 , Hichem Tahraoui 3 ,
Abdelouahab Benseddik 4 , Colette Besombes 5 and Karim Allaf 5

1 Faculty of Biological and Agricultural Sciences, Mouloud Mammeri University of Tizi-Ouzou,

Tizi Ouzou 15000, Algeria; [email protected] (M.S.M.); [email protected] (L.L.)
2 Research Unit Laboratory, Materials, Processes & Environment (UR-MPE), M’Hamed Bougara University of
Boumerdes, Boumerdes 35000, Algeria
3 Laboratory of Biomaterials and Transport Phenomena (LBMPT), Nouveau Pôle Urbain, Medea University,
Medea 26000, Algeria
4 Unité de Recherche Appliquée en Energies Renouvelables, URAER, Centre de Développement des Energies
Renouvelables, CDER, Ghardaia 47133, Algeria; [email protected]
5 Laboratory of Engineering Science for Environment (LaSIE) UMRER7356 CNRS, La Rochelle University,
17000 La Rochelle, France; [email protected] (C.B.); [email protected] (K.A.)
* Correspondence: [email protected]

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
Citation: Benahmed Djilali, A.; the concentration of lactic acid bacteria at 3 mg/mL of milk and the effects of temperature and
Metahri, M.S.; Lakabi, L.; Tahraoui, H.; prebiotics (okra mucilage and flaxseed powder). We observed short milk clotting time (2 s) using
Benseddik, A.; Besombes, C.; Allaf, K. the optimized mixture (0.18 mg of probiotics, 1.5 mg of flaxseed powder, and 1.147 mg of watercress
Innovative Approaches to Camembert powder) per 10 mL of milk. It contrasts with the classical coagulation way optimized at (5.9 and
Cheese: Optimizing Prebiotics and
9.5 s), which were generated at optimal temperatures of 45 and 45.7 ◦ C, respectively. Our new mixture
Coagulation Conditions for Enhanced
improves the fermentation process of camembert cheese at 38 ◦ C. This cheese had a high flavonoid
Quality and Nutrition. Fermentation
content, fewer lactic bacteria and molds, a homogeneous texture, and no outer crust, and exceptional
2024, 10, 524. https://doi.org/
sensory attributes such as a creamy and fluid paste. These attributes suggest its potential benefits as
10.3390/fermentation10100524
a dairy product for individuals with cardiovascular and gastrointestinal conditions.
Academic Editor: Alexander Dimitrov
Kroumov Keywords: prebiotics; probiotics; milk coagulation optimization; nutraceutical Camembert; quality
Received: 29 August 2024
Revised: 23 September 2024
Accepted: 29 September 2024
Published: 15 October 2024 1. Introduction
Functional foods and nutraceuticals are increasingly recognized for their role in pro-
moting 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
Copyright: © 2024 by the authors.
and well-being. Probiotics are live microorganisms that, when consumed in adequate
Licensee MDPI, Basel, Switzerland.
This article is an open access article
amounts, provide health benefits to the host. They are commonly found in fermented foods
distributed under the terms and
such as yogurt, kefir, sauerkraut, and kimchi.
conditions of the Creative Commons Fuller defined probiotics as preparations containing live microorganisms are used as
Attribution (CC BY) license (https:// food additives [2]. Their viability and availability depend on various factors, including
creativecommons.org/licenses/by/ manufacturing conditions, storage, gastric acidity, and interactions with milk constituents.
4.0/).

Fermentation 2024, 10, 524. https://doi.org/10.3390/fermentation10100524 https://www.mdpi.com/journal/fermentation

Fermentation 2024, 10, 524 2 of 16

Prebiotics, on the other hand, are non-digestible food ingredients that selectively stim-
ulate 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 [5–7]. 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 accept-
able 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 [11–13].
Flaxseed and watercress seed are rich sources of antioxidants, such as flavonoids and
mucilage [13–15].
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. Addi-
tionally, 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 coagula-
tion 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
Fermentation 2024, 10, 524 3 of 16

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 tempera-
tures. 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:

3 3 3 3
Y = a0 + ∑ ai Xi + ∑ ai Xi2 + ∑ ∑ aij Xi Xj (1)
i =1 i =1 i =1 j =1

where Xi (i = 1, 2, and 3) represent the independent variables, and ai and aij 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 combi-
nation 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 com-
position and coagulation temperature was investigated, with a constant lactic bacteria
concentration of 3 mg across all 18 tests.
Fermentation 2024, 10, 524 4 of 16

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 ana-
lyzed 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.
Fermentation 2024, 10, 524 5 of 16

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 mi-
croorganisms, 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

Tables 1–3 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).

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

Treatment Parameters (Independent Variables) Responses (Dependent Variables)

Run N◦ X1 X2 X3
Y2 : TF
(mg/10 mL of (g/10 mL of (g/10 mL of Y1 : tc (S) Y3 : SV (mL)
(mg Quercetin/g FM)
Milk) Milk) Milk)
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);
X1 = lactic bacteria [mg/10 mL of milk]; X2 = flaxseed powder [g/10 mL of milk]; X3 = watercress seeds [g/10 mL
of milk].
Fermentation 2024, 10, 524 6 of 16

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

Treatment Parameters (Independent Variables) Responses (Dependent Variables)

Run N◦ X1 X2 X3 Y2 : TF
(mg/10 mL of (g/10 mL of (g/10 mL of Y1 : tc (S) (mg Quercetin/ Y3 : SV (mL)
Milk) Milk) Milk) g FM)
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);
X1 = lactic bacteria [mg/10 mL of milk]; X2 = flaxseed powder [g/10 mL of milk]; X3 = watercress seeds [g/10 mL
of milk].

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

Treatment Parameters (Independent Variables) Responses (Dependent Variables)

Run N◦ X1 X3 Y2 : TF
(mL/10 mL of X2 (◦ C) (g/10 mL of Y1 : tc (S) (mg Quercetin/g Y3 : SV (mL)
Milk) Milk) FM)
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
Fermentation 2024, 10, 524 7 of 16

Table 3. Cont.

Treatment Parameters (Independent Variables) Responses (Dependent Variables)

Run N◦ X1 X3 Y2 : TF
(mL/10 mL of X2 (◦ C) (g/10 mL of Y1 : tc (S) (mg Quercetin/g Y3 : SV (mL)
Milk) Milk) FM)
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);
X1 = volume of okra mucilage [mL/10 of milk]; X2 = temperature of coagulation [◦ C]; X3 = flaxseed powder
[g/10 mL of milk].

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 vari-
ability 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].
Fermentation 2024, 10, 524 8 of 16

Contours of Estimated Response Surface Contours of Estimated Response Surface

Watercress seed=0.7 Watercress seed=0.7

1.5 Time of coagulation 1.5 0.48 Total Flavonoids

0.0 0.0 0.42
0.36 0.0
0.0 100.0 100.0 0.06
1.3 200.0 1.3 0.12
300.0 0.18
Flax seed powder

Flax seed powder

200.0 400.0 0.24
1.1 500.0 1.1 0.3
0.24
600.0 0.3
0.36
300.0 700.0 0.18
0.42
0.9 800.0 0.9 0.48
900.0 0.54
1000.0 0.6
0.7 400.0 1100.0 0.7
1200.0 0.36

0.42
0.5 0.5 0.54 0.48
0.54
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Lactic bacteria Lactic bacteria

(a) (b)
Figure 1.
Figure 1. Effect of the
Effect of the mix
mix (probiotic and prebiotic)
(probiotic and prebiotic) on
on the
the response
response variables:
variables: (a):
(a): coagulation
coagulation time;
time;
(b): total flavonoids (response surface of the First Optimization).
(b): total flavonoids (response surface of the First Optimization).

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
Contours of Estimated
speedsResponse Surface
and lactic acid production. For instance, S. thermophilus
Contours of Estimated Response Surface produces less lactic acid at
Watercress seeds=0.7
Watercress seeds=0.7
an optimal temperature of 45 ◦ C but acidifies more quickly than Lactococci and Lactobacilli.
1.5 On the other hand, Timethe
of coagulation
latter produce the highest amount of lactic acid [32,33].
0.0 1.5 Total Flavonoids
2.4
700.0 1.6
0.0 2.2
Collaboration 100.0 between these lactic species allows for a decrease in latency 0.2
phase length,
2.0
1.8 1.4
1.3 200.0 1.6
an increase in biomass 300.0 production, 1.3 and a rise in viscosity [34]. 0.4 1.2
Flaxseed powder

200.0
400.0 0.6
Flaxseed powder

1.1 For TFC, the amount 500.0 of probiotics (lactic acid bacteria) and prebiotics 0.8 had a consid-
600.0 1.1 1.0
600.0 erable impact on flavonoid 700.0
bioavailability (Figure 1b). The quantity 1.2of flaxseed powder
300.0
0.9 showed a significant 800.0 second-order quadratic interaction effect (p < 0.05), 1.4 implying that
900.0 0.9 1.6
its effect on TFC is1000.0 influenced by its interactions with other variables. 1.8 Additionally, a
0.7 400.0 1100.0
600.0
2.0
significant interaction 1200.0 effect (p < 0.05)
700.0
800.0 0.7 was observed between the amounts 2.2 of probiotics and
1300.0 900.0 2.4 1.6
0.5 700.0 500.0 watercress seed powder. 1400.0 This interaction
1000.0 suggests that the relationship 2.6
between prebiotics 1.0 1.8
0 0.05 0.1 0.15
and 0.2 0.25
probiotics 0.3 1500.0
is essential in 0.5
modulating flavonoid availability, 2.8
highlighting the importance 2.0

Lactic bacteria 1600.0 3.0

of optimizing these1700.0 components for 0desired 0.05 0.1
curd 0.15
properties.
Lactic bacteria
0.2 0.25 0.3
1800.0
These findings highlight the critical role of optimizing of prebiotic/probiotic ratios to
improve (a) bioactive compound retention in functional dairy (b) products.
Figure 2. Effect of the mix (probiotic and prebiotic) on the response variables: (a): time of coagula-
3.2.
tion;Second Optimization
(b): total flavonoids (response surface of the 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.

Fermentation 2024, 10, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/fermentation

 (a) (b)
Figure 1. Effect of the mix (probiotic and prebiotic) on the response variables: (a): coagulation time;
(b): total flavonoids (response surface of the First Optimization).
Fermentation 2024, 10, 524 9 of 16

Contours of Estimated Response Surface

Contours of Estimated Response Surface
Watercress seeds=0.7
Watercress seeds=0.7

1.5 Time of coagulation

0.0 1.5 2.4 Total Flavonoids
700.0 1.6
100.0
2.2
2.0 0.0
1.4
1.3 200.0
1.8
1.6 0.2
300.0 1.3 1.2
0.4
Flaxseed powder

200.0
400.0 0.6

Flaxseed powder
1.1 500.0 0.8
600.0 1.1 1.0
600.0
300.0
700.0 1.2
0.9 800.0 1.4
900.0 0.9 1.6
1000.0 1.8
0.7 400.0 600.0
1100.0 2.0
700.0
800.0 1200.0 0.7 2.2
900.0 1300.0 1.6 2.4
700.0 500.0 1000.0
0.5 1400.0 1.0 1.8 2.6
0 0.05 0.1 0.15 0.2 0.25 0.3 1500.0 0.5 2.0 2.8
Lactic bacteria 1600.0 3.0
0 0.05 0.1 0.15 0.2 0.25 0.3
1700.0
1800.0 Lactic bacteria

(a) (b)
Figure
Figure2. Effect ofofthe
2. Effect themix (probioticand
mix (probiotic and prebiotic)
prebiotic) onresponse
on the the response variables:
variables: (a): time(a): time of coagula-
of coagulation;
tion;(b):
(b): total flavonoids (response surface of the second optimization).
total flavonoids (response surface of the second optimization).

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 probi-
otics concentrations influence coagulation time, they do not significantly affect flavonoid
availability under the tested conditions.

3.3. Third Optimization

Fermentation 2024, 10, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/fermentation
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:
Fermentation 2024, 10, x FOR PEER REVIEW 2 of 2
Fermentation 2024, 10, 524 10 of 16

Contours of Estimated Response Surface Contours of Estimated Response Surface

Flaxseeds powder=0.85 Flaxseeds powder=0.85

46 5.0 Coagulation time 46 9.0 Total Flavonoids

10.0 0.0 7.5 0.0
15.0
5.0 1.5
Temperature of coagulation

Temperature of coagulation
6.0 1.5
45 45
20.0
10.0 4.5
3.0
25.0 15.0 4.5
44 20.0 44 3.0 6.0
25.0 7.5
30.0
30.0 9.0
43 43
35.0 10.5
40.0 3.0 12.0
42 45.0 42 13.5
4.5
50.0 15.0
35.0 1.5 6.0
41 41
7.5
30.0 9.0
10.5
40 40
0 1 2 3 4 5 0 1 2 3 4 5
Volume of okra mucilage
Volume of okra mucilage
(a) (b)
Contours of Estimated Response Surface
Flaxseeds powder=0.85

46 -5.55112E-17
Syneresis Volume
-5.55112E-17
-0.3
-5.55112E-17
Temperature of coagulation

45
0.3
0.3
0.6
44 0.9
1.2
1.5
43
0.6 1.8
2.1
42 2.4
2.7
41
-5.55112E-17

40
0 1 2 3 4 5
Volume of okra mucilage

(c)
Figure 3.
Figure 3. Effect
Effectofofthe
themix
mix(probiotic
(probioticand
andprebiotic) and
prebiotic) andthethe
temperature
temperatureof coagulation on on
of coagulation the the
re-
sponse variables: (a): time of coagulation; (b): total flavonoids; (c): syneresis volume (response
response variables: (a): time of coagulation; (b): total flavonoids; (c): syneresis volume (response
surface of the third optimization).
surface of the third optimization).

1: Coagulation Temperature: A significant negative first-order linear effect was ob-

served (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 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
Fermentation 2024, 10, 524 and a soft, non-firm texture, devoid of visible pores. In contrast, curds made solely 11with
of 16
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
These findings
properties. provide a comprehensive
These observations understanding
are consistent with the researchof by
how different
Oliveira factors
et al. [38],
influence syneresis volume
which demonstrated andaddition
that fiber curd texture.
can significantly influence yogurt texture.

Figure 4. Microscopical structures of curd: (a): curd prepared with the mix (lactic bacteria, flaxseed
powder, and okra mucilage) (200X scaled image); (b): curd prepared with lactic bacteria (Scale 250×
scaled image).

3.4. Optimizations of Milk Coagulation Conditions by RSM

Table 4 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 coagu-
lation time, TFC, and syneresis volume. The desirability scores for each run indicate that
Runs 1 and 2 achieved a perfect desirability score of 1.

Table 4. Optimization results.

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

The interaction between lactic bacteria, flaxseed, and watercress seed powders sig-
nificantly 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.
Fermentation 2024, 10, 524 12 of 16

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), reveal-
ing the critical role of temperature in the release of phenolic compounds.
Fermentation 2024, 10, x FOR PEER REVIEW 12 of
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
tionsin
lead to an increase lead
thetoavailability
an increaseofinphenolic
the availability
groupsofforphenolic
bindinggroups
to thefor bindinggels,
forming to the formin
gels, demonstrating a reversible interaction mechanism
demonstrating a reversible interaction mechanism in an acidic environment. in an acidic environment.
In pH levels
In pH levels between between
2 and 2 and 5, of
5, the binding theflavonoids
binding ofto
flavonoids to milk
milk proteins proteins remai
remains
relatively unaffected by the pH changes, suggesting that stronger
relatively unaffected by the pH changes, suggesting that stronger ionic interactions enhance ionic interactions e
hance hydrophobic effects. Filamentous polysaccharides
hydrophobic effects. Filamentous polysaccharides may exhibit relatively weak bindingmay exhibit relatively we
binding to phenolic compounds, due to their elongated structure. However, the hydr
to phenolic compounds, due to their elongated structure. However, the hydrophobic
phobic cavities within the secondary structure of these polysaccharides create a signi
cavities within the secondary structure of these polysaccharides create a significantly
cantly higher attraction for polyphenols [40]. Conversely, smaller polymers do not sho
higher attraction for polyphenols [40]. Conversely, smaller polymers do not show notable
notable interactions with phenolic compounds [41].
interactions with phenolic compounds [41].
These results offer insight into how various factors influence the quality and pro
These results offer insight into how various factors influence the quality and properties
erties of milk-based products, underscoring the importance of optimizing processin
of milk-based products, underscoring the importance of optimizing processing conditions.
conditions.
3.5. Stability and Functionality of the Optimized Mix (Prebiotics and Probiotics)
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
To assess the viability and functionality of the optimized mixture of lactic acid ba
and prebiotics at 38 ◦ C derived from the second optimization, the curd was dried at the
teria and prebiotics at 38 °C derived from the second optimization, the curd was dried
same temperature, then processed into powder, granules, and tablets (Figure 5a–c).
the same temperature, then processed into powder, granules, and tablets (Figure 5a–c).

Figure
Figure 5. Different 5. Different
forms obtained forms
fromobtained from the curd
the optimized optimized curd at
obtained 38 ◦ C: at
obtained 38 powder;
(a): °C: (a): powder; (
granules; (c): tablets; (d): nutraceutical cheese; (e): commercial Camembert
(b): granules; (c): tablets; (d): nutraceutical cheese; (e): commercial Camembert from ATTOUCHE from ATTOUCHE M
dairy industry.
Milk dairy industry.

This mixture wasThisused

mixture was usedCamembert
to produce to produce cheese
Camembert cheese at
(fermented (fermented at 38 °C), whi
38 ◦ C), which
exhibited a creamy, fluid paste with a uniform texture and no outer crust (Figure 5d)
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 Camembe
from ATTOUCHE MILK INDUSTRY (Tizi-Ouzou, Algeria)(Figure 5e).
A comparative analysis between the two Camembert cheeses showed that the fo
mer exhibited lower counts of thermophilic lactic acid bacteria, yeasts, and molds com
pared to the commercial one (Table 5). This can be attributed to the prebiotic conte
(mucilages), rich in antioxidants (flavonoids) and antibacterial substances (fatty acids).
Fermentation 2024, 10, 524 13 of 16

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

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

Cheese Made with Camembert Cheese from Standards

Researched Germs
Optimized Curd ATTOUCHE MILK Industry (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 -

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 concen-
trations 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 [45–47].
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 gas-
trointestinal 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 opti-
mization 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.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/fermentation10100524/s1. Table S1. Central Composite
Design (CCD) independent variables and their levels (First optimization). Table S2. CCD independent
variables and their levels (Second optimization). Table S3. CCD independent variables and their
levels (Third optimization). Table S4. Second-order model regression coefficients and their meanings,
Fermentation 2024, 10, 524 14 of 16

with Y1 , Y2 and Y3 models used to understand the relationships between the studied factors and the
observed responses.
Author Contributions: Conceptualization, A.B.D.; Methodology, A.B.D.; Software, A.B.; Validation,
A.B.D., M.S.M. and K.A.; Formal analysis, A.B.D. and C.B.; Investigation, A.B.D.; Resources, M.S.M.;
Writing—original draft, A.B.D.; Writing—review & editing, A.B.D. and H.T.; Visualization, L.L.;
M.S.M., L.L., H.T., A.B. and K.A.; Funding acquisition, M.S.M. All authors have read and agreed to
the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Material, further inquiries can be directed to the corresponding author.
Acknowledgments: Grants received from the Laboratory of Physico-chemical and Microbiology of
Mouloud Mammeri of Tizi-Ouzou; the Laboratory of Engineering Science for Environment (LaSIE),
La Rochelle University, France; the National High School of Chemistry of Rennes, Scientific Research
National Center, Rennes University, France; and the Research Unit Laboratory, Materials, Processes
Environment (UR-MPE), M’Hamed Bougara University of Boumerdes (Algeria), have supported this
work. We would like to express our gratitude to ATTOUCHE Milk dairy industry, for providing us
with all the resources necessary to produce our cheese.
Conflicts of Interest: The authors declare no conflicts of interest.

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