LACTOSE HYDROLYSIS AND ORGANIC ACIDS PRODUCTION IN YOGURT PREPARED WITH DIFFERENT ONSET TEMPERATURES OF ENZYMATIC ACTION AND FERMENTATION

The aim of this study was to evaluate lactose hydrolysis between two different initial temperatures of enzymatic and fermentation action to improve the fermentation period by checking the components formed and hydrolysis levels of lactose by HPLC. pH and titratable acidity analysis between treatments – higher initial temperature (HIT), 42 0C, and lower initial temperature (LIT), 30 0C – were different (P<0.05) during the fermentation process. HIT obtained lower pH and higher titratable acidity values as 4.6±0.04 and 0.73±0.01 g lactic acid.100 mL-1, respectively, against 4.82±0.01 and 0.64±0.01 g lactic acid.100 mL-1 from LIT. Lactose content was different (P<0.05) between treatments while fermenting; however, at the end of the fermentation, it reached 4.565±0.34 mg.mL1 and 4.398±0.18 mg.mL-1 for LIT and HIT (P>0,05), respectively. Glucose and galactose remained buoyant during the fermentation period, which indicates its production and consumption at the same time by the lactic acid bacteria involved. The lactic acid concentration in LIT was higher (P<0.05) (18.64±0.62 mg.mL-1) than in HIT (17.56±0.53 mg.mL-1) although citric and acetic acids content decreased throughout the process. In conclusion, the lactase enzyme contributed to reduce the lactose content without affecting the fermentation process. In addition, both treatments obtained lower values of lactose, which is sufficient for the consumption by those who have lactose malabsorption.


Introduction
Milk is considered a complete food: source of protein (3.2 w/w); carbohydrates, particularly lactose, (4.7 % w/w), fat (3.6 % w/w); minerals (0.6 w/w); and vitamins.Due to the essential nutrients and the high digestibility (bioavailability) of nutrients, milk and dairy products are considered highly nutritious food (1) .Lactose is the main carbohydrate in milk and is formed by one molecule of α-Dglucose and β-D-galactose.In cow's milk, lactose concentration is around 4.8%, which means 50 to 52% of total solids (2)(3) .However, this lactose percentage can be detrimental to subjects with impaired or completely absent intestinal lactase activity (lactose malabsorption) (4) .
Lactose malabsorption, also known as lactose non-persistent, is due to poor digestion of this carbohydrate (5) , causing gastrointestinal symptoms (lactose intolerance) (4) .Researchers have shown that more than 65% of the world's population -mostly adults -has a certain level of this disorder.Raithel (6) reported that lactose malabsorption has two different sources: a primary deficiency that includes physiological lactase reduction and autosomal recessive familial lactase deficiency; and a secondary deficiency featuring inflammatory intestinal diseases.Therefore, various technologies are employed for the development of reduced-lactose products, such as milk fermentation and enzymatic hydrolysis.
According to Codex Standards for Fermented Milk (7) , yogurt is a fermented milk product by homofermentative acid lactic bacteria.In fermentation process, milk lactose is fermented by specific acid lactic cultures as Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus culminated mainly lactic acid production at the end of the metabolism chain (8)(9) .Although there is a certain degree of lactose consumption by yogurt starter culture, a considerable amount of lactose remains intact in yogurt (3) .
Yogurt presents about 30% less lactose than milk; however, it is still considered relatively high for some lactose malabsorption levels (10) .Only the milk fermentation process may not be not sufficient for people who have some level of lactose intolerance; therefore, one of the solutions for such cases is to elaborate a dairy product with reduced lactose or even lactose free, as the ones obtained with BARROS, R.F. et al.
fermented milk added of β-galactosidase enzyme.Enzyme use in the manufacture of low-lactose dairy products is nowadays an usual practice (11) .
For these reasons, the hydrolysis conditions of milk lactose by the β-galactosidase enzyme action and the fermentation conditions by S. thermophilus and L. delbrueckii subsp.bulgaricus were compared between two treatments with different initial temperatures: low initial temperature (25 o C) and high initial temperature (40 o C).Therefore, the aim of this study was to evaluate lactose hydrolysis between two different initial temperatures of enzymatic and fermentation action.Components formation and hydrolysis levels of lactose were also checked, as well as whether the process obtained a low lactose concentration as required by humans with lactose malabsorption.

Materials and Methods
Commercial cow's whole UHT milk (Ninho ® , Nestle, São Paulo, Brazil), was used to elaborate the fermented milk.The milk was acquired in the retail market, transported and stored at room temperature.A whole milk sample was analyzed to indicate its physicochemical quality, determinate titratable acidity (Official Method 947.05), and pH (Official Method 973.41) (12) .The pH was determined by inserting a pH probe into milk (pH meter PG1800, CapLab, São Paulo, Brazil) after calibrating it with fresh pH 4.0 and 7.0 standard buffers.
To produce the yogurt, the dosage of 50 U of thermophilic lactic acid culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus strains YF L-812 CHR HANSEN ® ) to 500 L of milk was used, as suggested by the supplier, aiming at a cellular count of 10 11 CFU.g -1 of product The β-galactosidase enzyme used in this research to hydrolyze the lactose was Maxilact Lx 5000 (DSM Food Specialties, Delft, Netherlands), which is a refined lactase formulation derived from dairy yeast Kluyveromyces lactis.The dosage used was 0.45 mL of enzyme per liter of milk, as suggested by the supplier.The enzyme was added into milk and stirred at the start of fermentation in both treatments.The lactose hydrolysis and fermentation process were performed at an oven at 41 ± 1 °C during fermentation time.
Yogurt was made by applying the improved traditional method (Figure 1) adapted to laboratory scale (3) .It was prepared with whole milk, into two separated groups, using 3 L for each treatment.The first group, Low Initial Temperature (LIT), was prepared with milk at the temperature of 30 °C and the second one, High Initial Temperature (HIT), was prepared with milk at the temperature of 42 °C.In both groups a single-stage process took place with the simultaneous addition of βgalactosidase enzyme and lactic acid culture, using the dosages recommended by the respective manufacturers (as reported above).The fermentation was performed in an oven at 42 °C for 5 hours (300 minutes); thus, after the first hour, both temperatures were the same.The process was interrupted when the pH reached 4.7 ± 0.1.After that, the yogurt was cooled immediately in a refrigerator at 4 °C.
Lactose hydrolysis and organic acids production in yogurt prepared with different… During fermentation, the yogurt was analyzed every each hour to determinate pH and titratable acidity (12) .In addition, the yogurt was also analyzed to determinate carbohydrates and organics acids, which was extracted and carried out as following described.
The carbohydrates and organics acids extraction was carried out using a modification of the method described by González de Llano et al. (13) , performed in triplicate.Five milliliters of 45 mMol H2SO4 were added onto 1 mL of each sample and homogenized by vortexing for one minute.After that, all samples remained under agitation for one hour in a shaker table (TS -2000 A VDRL shaker, Biomixer ® ) and then they were centrifugated at 5500 g for 30 minutes at 4 °C.Finally, the supernatant was filtered through Whatman no. 1 filter paper (Sigma-Aldrich, St. Louis, MO, USA).
Filtered samples were injected (20 µL) in triplicate into an HPLC system consisted of a LC/20 AT pump integrated with CBM-20A and equipped with SPD-M20A diode array and refractive index RID-10A detectors (Shimadzu Corp., Tokyo, Japan).Carbohydrates and lactic acid separations were performed with an HPX-87H 300 x 7.8 mm Aminex cation-exchange column (Bio-Rad, Hercules, CA, USA), maintained at 60 ºC.The mobile phase used was 3 mM H2SO4 at isocratic flow rate at 0.5 mL.min -1 .Chromatograms from HPLC and compound quantification were obtained using the LC Solution software (Shimadzu Corp., Tokyo, Japan).Calibration curves were made from standard solutions prepared in Milli-Q water (Millipore, Billerica, MA, USA).The interest peaks were identified by comparing retention times of the standards with the samples.The concentration of each samples components was determined from the area of individual peaks.Carbohydrates identification was performed by refractive index detector while lactic acid by using a diode array detector model monitoring the absorbance at 210 nm.
One-way ANOVA with repeated measures of analytical methods was used to identify differences between pH values, titratable values, organic acids, and carbohydrates content over the yoghurt fermentation, as well as Tukey's test.Statistical significance was set at a 0.05 level of confidence.

Results
Milk physicochemical analyses results are presented in Table 1.Regarding hydrolysis and fermentation processes, the pH reached at the end of fermentation (4.82 ± 0.01 for LIT and 4.6 ± 0.04 for HIT) was considered a P< 0.05 among the groups (Table 2).Moreover, the titratable acidity presented a similar result with HIT (0.73 g lactic acid.mL - ), higher (P<0.05)than LIT (0.64 g lactic acid.mL - ), as seen in Table 3.
Lactose concentration started at 56.29±1.009mg.mL -1 .On the first four hours, HIT kept the lactose content P<0.05 lower than LIT.Nevertheless, at the end of 300 minutes, the concentrations reached were 4.565 ± 0.34 mg.mL -1 and 4.398 ± 0.18 mg.mL -1 of lactose for LIT and HIT (Table 4), respectively.Hence, with simultaneous fermentation process and lactose enzymatic hydrolysis, this compound decreased in both treatments at the end of fermentation (P>0.05).
In contrast, glucose and galactose concentration started at a very low value (0.16± 0.007 mg.mL -1 and 0.89±0.044mg.mL -1 , respectively) and increased during the first hour.Then, these compounds decreased slowly and slightly in both treatments.At the end of the process, its glucose and galactose concentrations were P<0.05.LIT ended the process with glucose and galactose concentration equal to 26.010 ± 1.13 mg.mL -1 and 21.72 ± 0.75 mg.mL -1 , respectively.Likewise, HIT obtained concentrations of 21.72 ± 0.75 mg.mL -1 of glucose and 14.74 ± 0.25 mg.mL -1 of galactose.

Discussion
Raw milk was analyzed and its result indicated good quality as expected.During fermentation, the initial pH of milk decreased, while titratable acidity increased in both treatments (LIT and HIT).These decrease and increase were evidently due the lactic acid production by lactose fermentation, as a result of microorganism metabolism.One of the lactic acid bacteria features are their capacity to ferment the mainly carbohydrate in milk and dairy products producing lactic acid as end product.The organic acids produced contribute not only to the flavor and aroma of fermented dairy products but also to their preservation.
Although a lower level of lactose results in a higher level of glucose and galactose, as it is seen in traditional yogurts (20) , the addition of the enzyme showed a significantly lower lactose content, however, galactose and glucose values were not very high.The lactose hydrolysis by enzymatic via is one of the most evaluated strategies to achieve yogurt with low lactose content (14) .The addition of β-galactosidase changes the carbohydrate pattern, which may affect the sugar consumption by the starter culture.Consequently, this fact could interfere with the production of derived compounds as well as the physicochemical and sensory characteristics of the products (11,15) .Furthermore, lactic acid culture cleaved the lactose into its monosaccharides and organic acids component, being lactic acid the predominant one, as previously reported (3,(16)(17)(18)(19) .
According to Mora et al. (21) and De Vin et al. (22) , most of the lactic acid bacteria are efficiently capable of using the glucose portion of lactose and releasing galactose into the medium.S. thermophilus is able to metabolize lactose.In addition, the majority of this strains are galactose negative (Gal-).However, some strains are galactose positive (Gal+), i.e., they are able to metabolize galactose (9) .This fact may explain the galactose behavior after 60 minutes fermentation.
In preheat treatment, the greater hydrolysis of lactose, observed at the beginning of fermentation, as compared to LIT treatment, is related to the increase in β-galactosidase activity.Whilst βgalactosidases are stable at temperatures between 20 and 37 °C, these enzymes are found to have optimum temperatures around 40 and 50 °C.In this condition, they retained 90-100% of their activity (23) .As reported by Chandan and O'Rell (24) , 40-45 °C is an optimum growth temperature for yogurt culture.It contributes to HIT quickly decreased pH and ripping.For the Maxilact ® manufacturer description, the enzyme used to hydrolyze the lactose is a compound extracted from a milk yeast; therefore, the optimum conditions for their activity are similar to the natural milk pH (6.6) and temperature (35-40 °C).

Conclusion
Our findings confirmed that both treatments obtained a final low lactose concentration although there was P>0.05 between the different initial temperatures (30 or 41 °C.Thus, the enzyme contributed to reduce lactose to a low-level providing for consumption by humans who have lactose malabsorption.The simultaneous enzymatic lactose hydrolysis and lactose-cleaving activity by yogurt cultures did not prevent the fermentation process, regardless of the initial temperature, including proper yogurt BARROS, R.F. et al.
fermentation and maintaining the physical and chemical characteristics.