Two commercial enzymes β-galactosidase obtained from Bacillus licheniformis, activity 5500 BLU∙g⁻¹ NOLA Fit 5500 (Chr. Hansen, Denmark) and β-galactosidase obtained from Kluyveromyces lactis, activity 5000 NLU∙g⁻¹ Maxilact LGi 5000 (Sedim Cedex, France), were used in this study, according to information provided by the manufacturer.

The research was carried out in the temperature range 4˚C - 70˚C, the duration of the hydrolysis process 4.0 hours. Milk with a pH of 6.55, a 1.5% fat content and 4.75% lactose content were used for the lactose hydrolysis. 100 ml of milk was mixed with each enzyme (0.45%) and transferred to 250 ml Erlenmeyer flasks. The flasks were incubated in water baths at temperatures of 4˚C, 10˚C, 15˚C, 20˚C, 25˚C, 30˚C, 35˚C, 40˚C, 45˚C, 50˚C, 55˚C, 60˚C, 65˚C, 70˚C for 4 hours. The D-glucose and lactose content were determined in each sample. All analyzes were performed in triplicate.

Adjustment of the pH value was achieved by adding monobasic potassium phosphate buffer and sodium acetate buffer. The tested variable was the pH value (4.0; 4.5; 5.0; 5.5; 6.0 and 7.0). For the lactose hydrolysis, 100 ml of milk with a temperature of 4˚C - 6˚C were used according to the conditions described above. The D-glucose content and the lactose content were determined in each sample. All analyzes were performed in triplicate.

Research on determining the optimal amount of enzyme was performed at two regimes: 4˚C - 6˚C/30 hours and 38˚C - 40˚C/5 hours, pH milk in both cases being 6.55. The tested variables were temperature (4˚C - 6˚C and 38˚C - 40˚C), hydrolysis duration at 4˚C - 6˚C (0.5; 1; 2; 3; 4; 5 hours) and temperature 38˚C - 40˚C (3, 4, 5, 6, 12, 22, 24 and 30 hours). The lactose enzymatic hydrolysis processes were performed using concentrations of 5.5, 8.25 and 16.5 BLU/ml for β-galactosidase obtained from Bacillus licheniformis and 5.8, 15 NLU/ml for β-galactosidase obtained from Kluyveromyces lactis. These concentrations corresponded to 0.10%; 0.15% and 0.30% (w/v). For the lactose hydrolysis, 100 ml of milk were used according to the conditions described above. The D-glucose content and the lactose content were determined in each sample. All analyzes were performed in triplicate.

The concentration of free D-glucose, as well as the D-glucose component of lactose was determined by glucose spectrophotometric method using the lactose test kit (k-LOLAC, Megazyme). The method includes pre-treatment steps to clarify and deproteinate samples and also to remove the high levels of free D-glucose in the samples.

The determinations were performed according to the method for the measurement of lactose in low-lactose and lactose-free products under Standard Method Performance Requirement (SMPRVR) 2018.009 [17]. The absorbance reading of the samples was performed at wavelength at 340 nm using UV Vis Shimadzu UV-1900 spectrophotometer. The analyses were performed in triplicate.

The amount of D-glucose was determined based on the relationship:

D − g l = 0.1673 × F × Δ A D − g l u c o s e , g / L (1)

The amount of lactose was determined based on the relationship:

L = 0.3233 × F × Δ A l a c t o s e , g / L (2)

where: F-dilution factor;

Δ A D − g l u c o s e -substract the absorbance difference (A₂ − A₁) of the blank from the absorbance difference (A₂ − A₁) of the sample;

Δ A l a c t o s e -substract the absorbance difference (A₃ − A₂) of the blank from the absorbance difference (A₃ − A₂) of the sample.

The initial lactose concentration and the concentration of D-glucose released were used to calculate the hydrolysis efficiency.

E = D − G l p × M M l a c L i × M M g l × 100 , % (3)

where: D − G l p -glucose concentration of the sample, g/L;

M M l a c -molar mass of lactose, g/mol;

L i -initial lactose concentration, g/L;

M M g l -molar mass of glucose, g/mol.

The degree of milk sweetness at different lactose hydrolysis stages was determined by the relation 4, [18] :

S = D − G a × 65 + D − G l × 72 + L × 16 , units (4)

where: D − G a -galactose concentration of the sample, %;

D − G l -glucose concentration of the sample, %;

L-lactose concentration of the sample, %;

65, 75 and 16-sweetening power of galactose, glucose and lactose, respectively, units.

Acceptance of dairy products is mainly due to the food sensory characteristics. Lactose-free ultrapasteurized milk is characterized by a more intense boiling aroma and a sweeter taste compared to lactose-free ultrapasteurized milk [19] [20]. This could be an obstacle to the consumption of lactose-free dairy products by the lactose intolerant population. More industry efforts are needed to develop higher quality lactose-free products and to educate consumers on lactose-free dairy products [4]. Therefore, before developing such products, it is important to analyze the sensory characteristics of lactose-free milk compared to normal milk.

According to the literature, for the production of milk with low lactose content, it is sufficient to achieve a degree of lactose hydrolysis of 70% - 80%, which is an optimal correlation between lactose intolerance and obtaining dairy products with high sensory characteristics. A degree of lactose hydrolysis of more than 90% is required only for lactose-free dairy products [21]. The dependence of the sweet index on the lactose hydrolysis degree is represented in Figure 1.

With the increase of the lactose hydrolysis degree, the intensity of the sweet taste of milk also increases, which leads to the milk sensory characteristics modification as a whole. Enzymatic hydrolysis of lactose in milk under the action of the enzyme β-galactosidase to the monosaccharides glucose and galactose leads to the appearance of sweet taste in milk, with increasing hydration increases the intensity of milk sweet taste.

To determine the influence of lactose hydrolysis on the milk sensory properties, the enzyme product β-galactosidase from B. licheniformis was used. Hydrolysis of lactose in milk was performed at 4˚C - 6˚C for 12 hours until a

hydrolysis degree of more than 95% was provided. The milk samples taste was performed in a group of 5 evaluators. The taste of sweet, boiled, caramel and butter was appreciated (Table 1).

The results of the sensory analysis of milk according to the hydrolysis degree of lactose showed that the increase of the lactose hydrolysis degree by more than 30% leads to the appearance in milk of the weakly noticeable sweet sensation. The sweet sensation becomes intense at a lactose hydrolysis degree of 70%. And at a hydrolysis degree higher than 70%, the milk boiling taste intensifies and a caramel nuances appears. Similar results have been reported by other authors [22] [23]. Therefore, we can mention that the lactose hydrolysis has a significant influence on the sensory properties of milk, especially on taste.

The research was performed in the temperature range of 4˚C - 70˚C, the duration of the hydrolysis process 4.0 hours, the milk-raw material pH of 6.55. For lactose hydrolysis, 100 ml of milk with 1.5% fat content of and 4.75% lactose content were taken.

According to the data presented in Figure 2, the higher inactivation rate of β-galactosidase from K. lactis was directly proportional to the increase in temperature, especially from 40˚C ± 2˚C and β-galactosidase from B. licheniformis of 45˚C ± 2˚C which corresponds to the technical characteristics of the studied enzymes. Subsequent increase in temperature leads to decreased enzyme activity. Thus, at a temperature of 60˚C ± 2˚C the hydrolysis degree is approximately 50% for the β-galactosidase enzyme from B. licheniformis and 30% for the β-galactosidase enzyme from K. lactis. Complete inactivation is attested at a

temperature of 70˚C ± 2˚C for both types of enzymes. In general, the enzyme from K. lactis was sensitive to temperatures above 40˚C, with complete inactivation at 55˚C at all studied pH ranges. The thermal stability results of commercial β-galactosidase enzymes have shown that they are inactivated during pasteurization/sterilization of milk, so there is no residual enzymatic activity in the final product, which is an advantage for food regulation and labeling. These data are consistent with literature data on microbial β-galactosidase [22] [24] and with the technical characteristics of these enzymes.

The pH value was adjusted by adding monobasic potassium phosphate buffer and sodium acetate buffer (Table 2).

Commercial β-galactosidase from K. lactis and B. licheniformis showed optimal activities under different reaction conditions (Figure 3).

At a pH of 6.5 the lactose hydrolysis degree for the enzyme from K. lactis is maximum (77.6%), the decrease of the pH below the value of 6.5 leads to the inactivation of the enzyme, demonstrating sensitivity to low values of pH. For the enzyme from B. licheniformis the maximum degree of lactose hydrolysis (85.2%) was reached at pH values of 5.5 - 6.0, the decrease of the pH below the value of 5.5 leads to inactivation of the enzyme.

The degree of lactose hydrolysis is determined by the temperature, the hydrolysis duration and the enzyme amount.

Enzymatic hydrolysis of lactose can be done by incubating milk with lactase before pasteurization in cold conditions (familiar to the batch process for milk) or adding lactase together with the starter culture after pasteurization of milk (familiar for yogurt making) [7]. The batch process has a number of advantages, among which we can list: low temperatures prevent the growth of microbial, the enzyme is inactivated during pasteurization/sterilization of milk and milk is characterized by higher sensory properties compared to the continuous method of hydrolysis of lactose [7] [25].

If referring to the process of adding lactase together with the starter culture after pasteurization of milk, most yogurt producers opt for this, because predigestion seems to inhibit the activity of some yogurt cultures [7] [26].

Therefore, research on determining the optimal amount of enzyme product was performed at two regimes: 4˚C - 6˚C/30 hours and 38˚C - 40˚C/5 hours, the pH of milk in both cases being 6.55. For the lactose hydrolysis, 100 ml of milk with 1.5% fat content and 4.75% lactose content were taken. The milk was previously heated at 63˚C - 65˚C for 10 - 15 seconds.

Figure 4 shows that with the increase of the fermentation time and the amount of enzymes, the hydrolysis degree increases. For example, at an enzymes amount from B. licheniformis of 0.1% after 6 hours, only 35.02% of the initial amount of lactose was hydrolyzed, when the amount of enzymes was increased to 0.3% in the same period of time 95.25% of lactose was hydrolyzed. The use of a quantity of 0.15% in the cold lactose hydrolysis process (temperature 4˚C - 6˚C) is not justified, because the hydrolysis duration process increases up to 22 hours, the degree of hydrolysis reaching the value of 80.75%. When using an amount of 0.1% in general, an optimal value of the hydrolysis degree is not reached (minimum of 70% - 75%).

When using the enzyme from K. Lactis, the maximum value of the cold lactose hydrolysis is established after 12 hours at an enzymes amount of 0.3%.

Figure 5 shows the same legitimacy for lactose hydrolysis under the action of the β-galactosidase enzyme at temperatures of 38˚C - 40˚C and hydrolysis at a temperature of 4˚C - 6˚C, namely the increase of the hydrolysis degree with the increase of the enzymes amount and hydrolysis duration. Therefore, there is a significant increase in the rate of hydrolysis, when use of enzyme from B. licheniformis in the amount of 0.3%, an increased hydrolysis degree (over 90%) is already recorded after 2 hours of hydrolysis. For the enzyme from K. lactis, the optimal regime identified is temperature of 38˚C - 40˚C, the amount of enzyme 0.15% and the duration of hydrolysis 3 - 4 hours.

The pH of the milk subjected to the lactose enzymatic hydrolysis under the action of β-galactosidase at the regimes of 4˚C - 6˚C/30 hours and 38˚C - 40˚C/5 hours practically did not change. No sensory defects were identified in the lactose hydrolysis process, such as defects due to the breakdown of fats or proteins. Therefore, we can say that the level of microorganisms activity in milk during the lactose hydrolysis process is very low.