Employing Response Surface Methodology for Investigating the Impact of Nigella sativa L. Oil on Physicochemical and Organoleptic Changes of Gouda Cheese upon Accelerated Storage

The primary goal of many producers was to preserve the quality of Gouda during maturation storage. The concentration of Nigella sativa L. oil and ripening time were two main variables in the present research that were improved using the Response Surface approach (RSM). The optimal conditions for ripening Gouda cheese were 0.29 (v/v) SFNSO concentration and 2.8 months at 14°C. It was determined that the RSM models were the best methodology for maintaining the selected Gouda cheese. The optimal Gouda cheeses: pH, fat content, moisture content, total protein, water activity (aw), ADV, and overall acceptability were found to be 5.9, 30.67%, 38%, 23.8%, 0.9131, 4.3, and 8.4, respectively. Additionally, there was a sizable overlap between the experimental and anticipated outcomes, which supported the accuracy and dependability of the proposed approach. The use of supercritical fluid Nigella sativa L. oil SFNSO on Gouda cheese improved and sustained the stability of Gouda cheese over storage time while higher black cumin concentrations had a negative effect on the sensory properties, resulting in dark appearance and bitter taste. Finally, it is necessary to produce excellent Gouda cheese on a practical scale under ideal RSM conditions.


Introduction
A variety of diverse and intricate processes, including biochemical and microbiological alterations brought on by the metabolism of primary and adjunct cultures, go into the ripening of cheeses [1].As Gouda cheese can age for anywhere between one and twenty months depending on its qualities and grading criteria [2].Black cumin, also known as Nigella sativa L., has been used extensively for medicinal and culinary uses throughout history.Because they contain bioactive substances with antibacterial, antioxidant, antifungal, and anti-inflammatory effects, seeds are extremely beneficial [3].The intricate microbiological and physicochemical processes that occur in food matrices have an impact on Nigella sativa's ability to fight microbes.The majority of investigations concentrate on experimental food matrices injected with starter cultures (lactic acid bacteria, or LAB), as well as with pathogenic bacteria cultures, at varied doses, to assess the antimicrobial impact of Nigella sativa [4].Proteolysis, which mediates the breakdown of casein into peptides and amino acids, is essential for cheese ripening [5,6].Free fatty acids (FFA) are produced as a by-product of lipid hydrolysis, which may both directly contribute to the flavor of cheese and act as substrates for subsequent reactions that result in extremely flavorful catabolicend products [7].According to the hypothesis, the flavor of cheese is made up of a harmony of flavors produced by a variety of chemicals, which must be present at specific concentrations and in the right harmony to produce a flavor typical of a particular variety.However, for some cheese kinds, a particular class of ingredient is acknowledged as being the primary flavorenhancing component [8].There are currently a number of techniques used to extract plant oils, including supercritical fluid extraction (SFE), which has several advantages over the traditional techniques used in the production of oil [9].Thymoquinone (TQ) is a valuable component of the crude extracts of NSO that maintains antioxidant/anti-inflammatory activity, according to [10].Recent years have seen a substantial increase in the use of Response Surface Methodology (RSM), which is suited and efficient for analyzing systems with a large number of independent factors that have the ability to affect the final responses [11].The main advantage of RSM is that it significantly reduces the number of trials necessary to assess how the multiple factors affect interactions and responses [12].It is frequently expected that treated cheese will be a consistent product with a much extended storage duration.However, even uncontaminated items lose their great quality after a few months when left at room temperature.The goal of the current investigation was to ascertain how SFNSO affected the physicochemical and organoleptic properties of Gouda cheese.The results of the most recent study could be used to enhance novel Gouda cheese that is rich in bioactive compounds and has a long shelf life.

Preparation of Gouda Cheese
The method outlined by [13,14], was used to create gouda cheese.Based on the factorial design guidelines listed in Table 1.From fresh cow's milk, thirteen sets of Gouda cheese were created.According to [15], measured amounts of Nigella sativa L. oil (NSO) generated by supercritical fluid extraction (SFNSO) at a concentration of 0-0.6% (v/v) were combined with the curd.At 14°C, all cheese testing and analysis was done on days 0 through 3 of maturation.
Table 1.Experimental design of Gouda cheese from the central composite design (CCD).

Cheese Samples for Analysis
Each cheese item (10 cm 10 cm) was divided into smaller pieces (2 cm 2 cm) for analysis.Grated samples were obtained from representative samples.

Proximate Analysis
According to the AOAC method, the cheese samples, including the control Gouda cheese, were examined in triplicate for moisture content using oven drying at 102°C to obtain a constant weight [16].A digital pH meter was used to measure the pH of cheese samples (Mettler -Toledo, GmbH, 8603 Schwarzenbach, Switzerland).The British Standards Institution (Standard, 1969) used the butyrometer method to determine the crude fat level.Total nitrogen was measured using the micro-Kjedahl method, and protein content was calculated by multiplying the result by 6.38 (Kelrich) [17].Aqua Lab's water activity analyzer was used to measure the water activity (a w ) of cheese samples (Washington, USA).

Determination of Nitrogen Contents in Cheese
The approach outlined by [18], was used to fractionate cheese samples and commercial Gouda cheese nitrogen.The amount of protein in the cheese was calculated by multiplying the total nitrogen content, which was represented as a percentage of weight, by 6.38.(Kelrich).

Determination of Acid Degree Value (ADV) in Cheese
With a small modification, the method used by the Bureau of Dairy Industry [19], is used to determine the acid degree value.

Overall Acceptability Evaluation
According to [17] and with only minor process alterations, organoleptic (flavor and taste) and rheological (hardness, springiness, cohesiveness, gumminess, and chewiness) study of the Gouda cheeses during the maturing period was carried out.It was rated from 1 to 9, with 9 signifying "very like" and 1 signifying "extremely detest."Twenty trained Arabian panelists who were familiar with Gouda cheese participated in the study.Additionally, panelists were provided unlimited amounts of water and unsalted crackers throughout the examination to help them clear their palates between samples.Every session's sample analysis was conducted in an arbitrary order that took the cheese's age into account.All panelists' values for each sample were averaged.The sensory evaluation was carried out twice.

Aroma Profile
A single, high quartz, uncoated surface acoustic wave (SAW) resonator electronic nose was used to measure the scent profiles of industrial and the new ideal Gouda cheeses that had been aged for a month at 14°C.With the aid of an ultrafast GC known as the zNose 7100 analyser, the fragrance present in the cheeses was evaluated in accordance with the method outlined by [20].(Electronic Sensor Technology Co., Newbury Park, CA, USA).The zNose chromatogram and VaporprintsTM software displayed the fragrance component profiles obtained from the analysis (polar plots).

Statistical Data Analysis
Using MINITAB statistical software, version 16, samples were selected at random, and data was tabulated and subjected to analysis of variance (ANOVA) and MANOVA (PROC GLM) (MINITAB Inc., state college, PA and USA).If the absolute F-ratio increases, the outcome variables are more significant (p≤ 0.05) [21].The mean SD of separate trials was used to represent all measurements, which were all carried out in triplicate.

Chemical Changes in Cheese Composition During Storage
The experiment's proximate analysis of the cheeses at one day after manufacture revealed no significant variance (p>0.05)across all the manufacturing iterations of Gouda cheese.The experiment's initial pH value for mature cheeses is similar to that discovered by [22].The moisture content is in line with what [17].In addition, processed Gouda's fat and total protein levels are comparable to those discovered by [23].The findings of this study showed that the processed Gouda cheeses' main composition is comparable to that of the cheeses examined by other scientists

pH Value (Y2)
Table 3 shows the evaluation of changes in the pH variance composition for the Gouda cheese during the ripening phase.The results shown in Table 2 show that the basic effects of ripening time (x2) and the quadratic effects of the SFNSO concentration (x12) and ripening time (x2 2 ) with the interaction of the SFNSO concentration and ripening time (X1X2) significantly (p≤0.05)influenced the response of the pH (Y2) in the studied Gouda cheese acceleration.The perfect Gouda cheese is mostly impacted by ripening time, which is the factor that is most significant (p≤0.05).Figure 1A illustrates how the interactions between SFNSO concentration (x1) and ripening time (x2) resulted in a consistent increase in pH during ripening.

Fat Value (Y3)
Table 3 shows that the main effect of SFNSO concentration and maturation time, as well as the quadratic impacts of maturation time, as well as the interactions between SFNSO concentration and maturation time, had a significant impact on the Fat degree (p≤ 0.05).The primary effect of SFNSO concentration, which had the strongest effects (p≤0.05) on fat (Y3).
Figure 1B demonstrates the effect of lipase on increasing the fat content of Gouda cheese through periods of ripening in addition to the effect of ripening time due to loss of water over the course of the ripening process, showing that the value of fat increased significantly from 27.15 to 32.89 when the concentration of SFNSO was increased from 0.1 to 0.6.

Moisture Content (Y4)
It's interesting to note that, as shown in Table 3, the main influence of ripening time (x1) and the quadratic impact of ripening time both had a notable (p≤0.05)impact on the value of moisture content (x2 2 ).The value of moisture in Gouda cheese is significantly affected by ripening time (x1) over the course of the cheese's maturity.As a result, there was no discernible difference between the concentration of SFNSO and the moisture content.

Total Protein (Y5)
The main effects of SFNSO concentration (x1) and ripening time (x2), as well as the quadratic effect concentration of SFNSO (x1 2 ) and interaction of SFNSO concentration with time of ripening, have a significant (p≤0.05)impact on the total protein content of Gouda cheese during the maturation period, as shown in Table 3.The most important factor in this study's analysis of the total amount of protein was the x1 2 (p≤0.05).The impact of changing the SFNSO concentration from 0.1 to 0.6% (22.15 to 24.89%) on boosting the total protein content of Gouda cheese can be shown in Figure 1C, which also shows that this change has the greatest effect on the response.

Water Activity (a w ) (Y6)
In cheese, water activity (a w ) has a significant impact on the product's stability, quality, and safety [24].a w , which is related to the food's enzymatic and microbiological stability, can be used to determine the chemical potential of water [25].
According to Table 3, the independent factors and their interaction significantly (p0.05) impacted the water activity (a w ) value of Gouda cheese.Figure 1D shows that the SFNSO oil and maturation time had the greatest (p≤0.05)influence on the water activity of Gouda cheese during the maturation period.

Acid Degree Value (ADV) (Y7)
ADV determination is used to calculate the rate of lipolysis.This number represents the amount of FFA dissolved in certain fat and is related to the sensory appeal of the finished goods [26].Table 3 shows the changes in ADV that occurred while the cheese was being stored.The predominant effect of independent variables and their interaction resulted in ADV being noticeable (p≤value) in all batches of cheese during the ripening process.Table 3's results show that in the studied Gouda cheese, the main effects of SFNSO concentration (x1) and ripening time (x2), the quadratic effect of SFNSO concentration (x1 2 ) and ripening time (x2 2 ), as well as the interaction of SFNSO concentration and ripening time (x1x2), all significantly (p≤0.05)affected the response of the ADV.Additionally, it was found that the ADV (Y7) value was highly influenced by the main effect of SFNO oil, ripening period, and quadratic effect of the SFNSO concentration.
As demonstrated in Figure 1E, the rise in the acidity of Gouda cheese during the maturing period was substantially higher and represents ongoing hydrolysis of the lipid content.

Overall Acceptability Evaluation (Y1)
The acceptability, selection, and consumption of any edible product depend heavily on the three basic sensory qualities of flavor and rheology [27].The taste of cheeses like Cheddar and Gouda is another important consideration for consumers [28].Table 3 shows that the main effect of SFNSO concentration and ripening time, the quadratic effects of SFNSO concentration and ripening time, and the interactions of SFNSO concentration and ripening time all significantly (p≤0.05)influenced the overall acceptability (Y1) during the maturation period.The previous outcomes showed that the interaction impact of concentration of SFNSO concentration and ripening was responsible for the most significant (p≤0.05)effect on overall acceptance (Y1) of the finest Gouda cheese.The response surface plots used to optimize Gouda cheese's general appeal are displayed in Figure 1F.The main effect of Concentration of SFNSO and ripening time, respectively.X1 2 and X2 2 : The quadratic effect of Concentration of SFNSO and ripening time, respectively.X1X2: The interaction effect of Concentration of SFNSO and ripening time, respectively.a significant effect (p-value ≤0.05).

Optimization and Validity Conditions
To ensure that the final response surface models were adequate, the experimental data and fitted values were evaluated.The results must closely match the experimental data and the anticipated values and not show any evidence of a significant difference (p>0.05).The RSM approach is the most effective methodology for the recovery of desired Gouda cheese, as demonstrated in Figure 2 where it is shown that the respective values of the response variables acquired from observations were closer to those estimated in the modelled equations.The optimum characteristics of the Gouda cheese scheme were identified in this investigation.SFNSO concentration and ripening time measurements of 0.29% and 2.8 months, respectively, were used to validate the final model.The overall acceptability, pH, fat, moisture content, total protein, water activity (aw), and ADV of optimal Gouda cheese were found to be 8.4, 5.9, 30.7%, 38%, 23%, 0.9131, and 4.3, respectively, using this model, under the optimized conditions.

Aroma Profile
One of the most important elements influencing the sensory qualities and quality of cheese is its scent.Electronic noses are used to evaluate the sum of volatile substances by infusing them collectively into a mass spectrometer rather than to evaluate individual components after chromatographic division [29].Each fragrance compound's contact duration with the sensor is calculated, and a system software may calculate the substance's retention time and amount [30].
In this study, an electronic nose was used to analyze the qualitative evaluation of the scent profile of cheese made from 0.29% SFNSO during a 2,8-month period.Based on time of retention and polar plots, the results are displayed as chromatograms (Ultrafast GC) (VaporprintTM).Figure 3 shows the results of the chromatograms (Ultrafast GC).The aroma of cheese is thought to result from the equilibrium of several volatile components, which when taken individually do not represent the whole odor [31].However, due to interactions with other compounds and within the matrix itself, the flavor of a certain substance in food may differ from that discovered by analytical instruments [28].
Within the assessment time of 0-20 s, 8 compounds (a-g, reflecting various retention durations) were recorded.Five of these were common components in cheese across the board.Based on the volatile components discovered in the cheese, these results showed that the electronic nose device was successful in detecting the ripening stage.This result confirms the electronic nose's potential for identifying scent components in cheese during its maturation period [32].

Discussion
It is said that the synthesis of non-acidic decomposition products, lactic acid from lactate, and the release of alkaline products breakdown and dissociated amino acids are to blame for the pH rising throughout the maturation stage [8].In contrast to other treatments, the pH of the Gouda cheese was steadily raised by using concentration 0.29 percent of SFNSO.When SFNSO concentration was present at 0.29% (v/v) and 2.8 month ripening time, significant pH (5.9) of perfect Gouda cheeses was discovered.Also, The chemical composition of fat and its percentage in the dry matter of Gouda cheese made from modified mesophilic lactic bacteria and probiotic culture, according to [33], was noticeably altered over the cheese's maturing time.Literature frequently refers to patterns that show an increase in fat content as fruit ripens and links these trends to moisture loss, a decline in non-fat dry matter, the breakdown of protein, and whey loss [4].Gouda cheese's decreased moisture content throughout the ripening process may have been caused by biochemical changes and the growth of lactic acid, which caused the curd to constrict and ended the aqueous stage of the cheese.In the ripening process, it also caused moisture loss [13].Additionally, it led to agreement with other researchers who studied various Gouda cheese production and enhancement methods, including as [17] and [34].Moreover, According to a study by of [33], adding lactobacilli (24.6-29.4%)and freeze-shocked mesophilic lactic starter bacteria to Gouda cheese during processing boosted the total protein content.According to [13], adding heat-treated catalyst raised the average content of total nitrogen in low-fat Gouda cheeses from 29.45 to 29.76%, compared to about 26% in full-fat cheeses during the ripening process.
Our findings concur with those of [35], who claimed that Nigella sativa enhanced the sensory evaluations and proteolysis of raw sheep's milk cheese.However, some research suggest that utilizing Nigella sativa seeds rather of cold-pressed oil reduces sensory qualities of cheeses, leading to darker shades of color and an unpleasant bitter flavor, as well as a considerable reduction in dry matter and fat content [36].
To identify the microbial content of treated Gouda cheese, water activity was determinate.The water activity of cheese is typically in the range of 0.70-1.00even though most varieties have activity of water above 0•90, which is close to that of the experimental Gouda cheese, despite the significant impact of the two primary factors and the interaction of oil with time which decreased the value of a w [37].In addition, the impact of biological enzyme reactions and cheese ripening time on the matrix can be used to explain the decrease in water activity.
Higher molecular weight substances have fewer molecules overall than lower molecular weight substances, which has a larger a w -reducing impact.Additionally, lowering aw is affected by increasing salt in moisture percentage during ripening [38].Water's activity therefore has a positive relationship with moisture and a negative relationship with salt [39].According to [40], water activity (a w ) decreased in all Gouda cheeses with an increase in S/M percentage over the course of ripening periods for 60 days, it was 0.93 for new Gouda cheese at zero duration and decreased to 0.51 in 60 days.
If the SFNSO oil is changed, the value of ADV increases.This is due to the fact that when the cheese ripens, the fat in the cheese degrades more quickly, releasing various short-chain fatty acid constituents such methyl ketones, free fatty acids, and aldehydes [41].
The yellowness and dryness of the appearance attributes dramatically increased throughout sensory evaluation.Properties of the aroma, flavor, and taste, such as butyric, fruity, and musty, tended to rise. Figure 1a's results clarify that the interactions between the SFNSO concentration and ripening time (0.29% SFNSO and 2.8 months) increased the overall acceptability value (overall acceptability = 8.4) in comparison to the other cheese samples while maintaining the cheese's features.This may be because SFNSO concentration at this point just affects texture and fragrance profile properties, not its acceptance features, without causing any defects.Several Previous studies [42][43][44], have demonstrated the antimicrobial effects of N. sativa seeds against various pathogenic microbes, which have approved the stability of the characteristics of ideal Gouda cheese for almost 3 months.It's interesting to note that the acceptability of the current sample decreases when SFNSO concentration rises, which may be explained by SFNSO's effect on Lactic acid bacteria (LAB).These findings contradict earlier research on the impact of Nigella sativa L. oil on LAB.SFNSO's antimicrobial action revealed to be selective because it had little to no impact on the helpful LAB [3,45].These results support [46], finding that the total aerobic mesophilic bacteria, lactic acid bacteria, and coliform bacteria counts decreased during storage after the addition of essential oil.Finally, the various flavoring ingredients must be in proper balance.It should be emphasized, nonetheless, that an excessive production of sensory active components through the breakdown of fatty acids and amino acids may have a negative impact on the sensory qualities [1,47].The addition of supercritical fluid oil (SFO) of Nigella sativa L. concentration (0.29%) to Gouda cheese preserved significantly the cheese's quality characteristics throughout storage, which depended on the flavor enhancer.Significant differences were found between treated Gouda cheese and control findings in terms of physicochemical composition.Gouda cheese that had been treated showed selective antimicrobial activity because there was no discernible impact on LAB, which is very advantageous because LAB is crucial for maintaining the balance of the microbiota during the experiment.The experimental food matrix's sensory attributes were greatly enhanced by NSSO.The enrichment of the food matrix with NSSO has no effect on the dynamics that pH and titratable acidity follow and is thought to be advantageous for pathogen suppression.This study demonstrates that even when used to intricate, dynamic food matrices, NSO is still an effective antibacterial treatment.Pathogens are selectively inhibited through interaction with the intricate microbial environment of the Gouda matrix, with the beneficial lactic acid microbes remaining unaffected.These findings imply IOP Publishing doi:10.1088/1755-1315/1259/1/01206510 that NSO maintains an effective antibacterial activity even when added to natural food matrices, making it a potent natural antibiotic candidate for application as a food dietary supplement.

Conclusion
The addition of black cumin improved the cheese's volatiles both qualitatively and quantitatively while also significantly impacting the sensory scores and proteolysis of the cheese during maturing time.Despite all the promising findings of this study, more research is necessary to determine how SFNSO affects the microbial content of maturing Gouda cheese and on storage time.Finally, it is necessary to produce excellent Gouda cheese on a practical scale under ideal RSM conditions.

Figure 1 .
Figure 1.Response surface plots of the central composite design for the interaction effects of (x1) Concentration of SFNSO (x2) Ripening time on physicochemical properties of Gouda cheese throughout ripening.

Table 2 .
Fitting of the RSM ModelsTable2lists the expected regression coefficient values for the RSM models along with the related R 2 values.Regression coefficients, R 2 , and probability value of the Response Surface Models.

Table 3 .
The significance of each independent variable effect indicated by using F -ratio and p-value in the final models.