The AFM₁ standard was purchased from Sigma Chemical Company (St. Louis, MO, USA). Afla M1^TM HPLC was obtained from VICAM (Watertown, MA, USA). All solvents (HPLC grade) were purchased from Merck (Darmstadt, Germany). Water Millipore Milli-Q system was obtained from Millipore (Bedford, MA, USA).

In this study, two strains, Levilactobacillus brevis and Lactobacillus plantarum, were used to produce protease and lipase, respectively. These strains were isolated from some soft cheese and identified using the -50CHL API-20STREP identification system (BioMerieux) according to Bergey’s Manual of Systematic Bacteriology (2009) [17]. In another study, we performed the production, extraction, partial purification, and characterization of these enzymes [13].

Enzyme activity is affected by a number of factors, such as temperature, pH, and salt concentration. So, we determined the optimum pH according to the method proposed by Gomori (1955), as well as temperature, thermal stability, and the concentration of sodium chloride (NaCl) and some metal ions [18, 19].

We investigated the ability of protease and lipase to degrade AFM₁ at four different concentrations (50, 100, 150, and 200 U/ml for each enzyme) using phosphate-buffered saline (PBS) contaminated by AFM₁ at 10 µg/L. The enzyme was added to 100 ml of contaminated PBS and AFM₁, and the samples were incubated at 37 °C for 6, 12, and 18 h. AFM₁ was extracted and determined for each dose and incubation period. All treatments had three replicates for each incubation time and enzyme. After incubation, 5 ml of PBS was passed through the Afla M1^TM column, followed by washing with 20ml of distilled water. Then, AFM₁ was eluted with 1.5ml of acetonitrile, followed by 1.5ml of distilled water, and collected in a clean vial. According to AOAC (2005), the eluate was evaporated, and then AFM₁ was determined by HPLC as follows: 200 µL of hexane and 200 µL of trifluoroacetic acid were added to the dry residue in the vial, and the mixture was kept stable for 10 min at 40°C, and then evaporated to dryness. The residue was dissolved in 2 mL of water and acetonitrile (75 + 25) for injection by HPLC instrument with a quaternary pump fluorescence detector system set at 365 nm excitation and 435 nm emission wavelengths, with the mobile phase consisting of water: methanol: acetonitrile (66:17:17). The separation was carried out at 40 °C with a flow rate of 1 ml/min [21].

Cow’s milk (fat 3%) spiked with AFM₁ at 10µg/L was heated to 90 °C, held for 5 min, and then cooled to 45°C. This was followed by inoculation with 1.0% w/v of Streptococcus thermophilus and Lactobacillus bulgaricus. The samples were divided into eight groups as follows:

Group 1: Control sample of milk without AFM₁ and any enzyme.

Group 2: Positive sample of milk contaminated by AFM₁ without any enzyme.

Group 3: Milk contaminated by AFM₁ and treated with 0.3% of both lipase and protease.

Group 4: Milk contaminated by AFM₁ and treated with 0.6% of both lipase and protease.

Group 5: Milk contaminated by AFM₁ and treated with 0.9% of both lipase and protease.

Group 6: Milk free from AFM₁ and treated with 0.3% of both lipase and protease.

Group 7: Milk free from AFM₁ and treated with 0.6% of both lipase and protease.

Group 8: Milk free from AFM₁ and treated with 0.9% of both lipase and protease.

The infected mixtures were completely blended, transferred to 100 ml plastic cups, and incubated at 40°C until homogeneous coagulation was produced.

Groups 1, 6, 7, and 8 were subjected to chemical analysis and sensory evaluation at baseline when they were fresh, and 5, 10, and 15 days of storage at 5±2°C [22]. The percentage of AFM₁ degradation was calculated for groups 2, 3, 4, and 5 after storing the samples for two and five days as follows:

The percentage of degradation of

Where, C is the concentration of AFM₁ in the positive sample (group 2) and T is the concentration of AFM₁ in the treatment groups (groups 3, 4, and 5). AFM₁ was extracted according to the method proposed by Corassin et al. [23] and then determined by HPLC as mentioned earlier.

According to the procedures outlined in AOAC (2012), the contents of ash, fat, total nitrogen (TN), titratable acidity (TA), and moisture were all measured [24]. According to Less and Jago (1970), the yoghurt samples' acetaldehyde and diacetyl levels were assessed using a Shimadzu spectrophotometer (240-UV-Vis, Japan) [25]. A digital pH meter (HANNA, instrument, Italy) was used to measure the pH.

Using the scorecard proposed by Bodyfelt et al., yoghurt samples were assessed for their sensory qualities, including the appearance (10 points), body and texture (40 points), and flavor (50 points). The personnel at the NRC, Egypt's Dairy Science Department, judged the sample [26].

The SPSS version 18 (IBM Corp., NY) general linear model approach was employed for the statistical analysis. The Waller-Duncan k-ratio was used to determine the significance of the differences among the treatments. All statements of significance that depended on the probability of a P-value ≤0.05 were considered to be statistically significant. Values represent average standard deviations for triplicate experi-ments.

Fig. (1). The optimal pH values affecting on Protease and lipase enzymes activity.

Fig. (2). Effect of temperatures on purified Protease and lipase enzyme.

The results in Fig. (1) show the effect of different pH values from 3 to 10 on protease and lipase activity. The results show that protease retains its activity in the pH range of 5.0 to 8, while lipase retains its activity in the pH range of 5.5 to 9; the highest activity was recorded at pH 7 and 7.5 for protease and lipase, respectively. On the other hand, Fig. (2) shows the effect of different temperatures from 0 to 100 °C on purified protease and lipase activity. Protease and lipase enzyme activity increased as the temperature increased from 10 to 50°C. The results indicated the optimum activity at temperatures of 50°C and 30°C for protease and lipase, respectively. The activity decreased as the reaction temperature increased above 70°C due to the thermal denaturation of the protein.

Fig. (3). Thermal stability of the purified protease enzyme.

The protease and lipase enzyme activities were stable at temperatures from 30 to 40°C and 30 to 50°C, respectively, for 15–60 min of incubation, and then slightly decreased at 50 and 60 °C for protease and lipase, respectively. Incubation at 60 to 80°C caused a decrease in protease activity until it was completely absent at 90°C, as shown in Fig. (3), while lipase exhibited a sharp decrease in activity during incubation at 70 to 80°C at all times until the complete disappearance of enzyme activity at 90°C (Fig. 4).

Fig. (4). Thermal stability of the purified lipase enzyme.

Fig. (5). Effect of the different concentrations of NaCl on protease and lipase enzyme activity.

The concentration of sodium chloride in the solution limits the enzyme's ability to function. Depending on the concentration, salts can either activate or deactivate the enzyme through interactions in the enzyme's tertiary structure, which would be disrupted if the salt concentration is too high or too low [19]. So, this study evaluated the effects of different concentrations of NaCl (0–7%) on the activity of protease and lipase. Results showed a gradual reduction in protease activity when increasing the NaCl concentration. As shown in Fig. (5), increasing the concentration of NaCl with increasing protease activity (even 1% sodium chloride) caused the enzyme activity to decrease gradually with increasing concentration of sodium chloride, and then a sharp decline in the activity of the enzyme up to 7% has been observed. While lipase activity gradually reduced with increasing NaCl concentration.

Fig. (6). Some metal ions affect the lipase enzyme as inhibitors and activators.

Fig. (7). Some metal ions affect the protease enzyme as inhibitors and activators.

Under conventional assay conditions, chlorides and sulphates at concentrations of 1 and 5 mM each were used to examine the impact of monovalent and divalent ions on enzyme activity. Variable metal ions always have varied impacts on pure protease activity. Some metal ions and other materials were added at different concentrations of 1 and 5 mM, as shown in Fig. (7) indicating 1 and 5 mM Fe⁺², Cu⁺², and EDTA to act as inhibitors. Whereas MN⁺², Ba⁺², Ni⁺², Mg ⁺², Zn⁺² and CaCl₂ have been observed to be activators of the purified protease enzyme and in Fig. (6) indicating 1 and 5 mM Ba⁺², Fe⁺², Ni⁺² and CaCl₂ to act as activators. Whereas Mn⁺², EDTA, Mg⁺², Zn⁺² and Cu⁺² have been observed to be inhibitors of the purified lipase activity. Finally, the ability of an enzyme to tolerate different metal ions is essential for both its mechanisms of action and some of its industrial uses.

Concerning the structure and operation of enzymes, metal ions play a vital role. Enzyme activity may be altered by metal ions that modify enzyme conformation. They might act as cofactors in catalytic processes or create interactions with the side chains of amino acids in proteins, denaturing the structure of the enzyme and preventing it from doing its function [27].

The pH decreased on days 5 and 10, as shown in Table 3, but the subsequent drops were not statistically significant (P > 0.05) for the remainder of the storage period. All yoghurt samples underwent the same rate of pH lowering (p ≤ 0.05) during storage. Ozer et al. and Barros et al. reported that during fermentation, the initial pH of milk declined while titratable acidity rose in treatments [38, 39]. The pH of the control and each of the G6, G7, and G8 were the same (P ≤ 0.05). As a result, adding pure enzyme extract had no effect (P ≤ 0.05) on the fermentation process or the outcome. Changes in the acidity of yoghurt using cow’s milk treated with different concentrations from purified protease and lipase enzymes have been observed. All yoghurt samples with different concentrations of purified protease and lipase enzymes and starter culture exhibited obviously higher acidity values than those in control. According to Corrieu and Beal, the growth of lactic acid bacteria in milk causes yoghurt to undergo a number of favourable alterations. These modifications include the creation of several metabolites, like lactic acid, exopolysaccharides, and fragrance compounds, as well as adjustments to the product's texture and nutritional content [40]. The results showed that TN and moisture rose over the course of storage, but that there was no discernible difference (P ≤ 0.05) between the control and any of the treatments. Thus, neither the yoghurt's total protein content nor its moisture content was affected by the addition of purified protease and lipase enzyme extracts (P ≤ 0.05). The acquired results also showed that during the experiment, there was no observed rise in ash, and that there were substantial changes in fat between the control and all treatments.

According to the results shown in Table 4, there was no discernible change between the sensory characteristics’ scores (appearance, body, texture, and flavour) for all treatments at the fresh stage and after five days from storage compared to the control. During the storage time, there was a noticeable difference in the appearance, body, texture, and flavour between G8 and control, with the sensory attributes score in G8 being lower. Starch and pectin were found to significantly reduce the amount of aroma molecules in the headspace when present in yoghurt [43, 44]. Traditional yoghurt has a weak body (gelatinous body) and a sweet flavour, which may mask the pleasantly acidic flavour. It is manufactured with a 0.3% level of purified enzymes (G6). The sensory qualities were found to somewhat deteriorate during cold storage. Similar findings were obtained by Zhao and Routray and Mishra, who found that the amount of time yoghurt was stored had a negative impact on the flavour rating [45, 46]. As a result, sensory quality declined with time and it was found to be closely correlated with variations in the composition of scent components. These findings suggest that a viable starting point for the production of nutritional and therapeutic yoghurt could be the addition of 0.3% or 0.6% of purified enzymes.