Two types of yogurt were manufactured. The first type was lactose added “plain” set yogurt. The second type was lactose added blueberry yogurt for the sensory study. Plain set-type yogurt was manufactured according to earlier published methods [21] [22] [23]. Briefly, the yogurt mixes contained skim milk and added lactose at 0%, 1%, 3% and 5% w/w. The yogurt mixes were preheated to 60˚C then homogenized in a two-stage homogenizer (Type: 300 DJP4 2PS, Gaulin, Manton-Gaulin MFG Co Inc., Everett, MA, USA) at 13.8 MPa for the first stage and 3.45 MPa for the second stage and later pasteurized at 85˚C for 30 minutes. Yogurt mixes were tempered to 40˚C and inoculated. Freshly thawed frozen yogurt starter culture concentrate of Streptococcus thermophilus (ST-M5) and Lactobacillus delbrueckii ssp. bulgaricus (LB-12) (CH-3, yogurt culture, Chr. Hansen’s Laboratory, Milwaukee, WI, USA) was added at 0.75 mL per 3.78 L of mix for each bacterial strain, 7.56 L of mix per treatment was used. After mixing, the yogurt mixes were poured into previously labeled 340 g plastic cups.

The inoculated mixes were incubated at 40˚C until pH reached 4.65 ± 0.1 to obtain a set-type yogurt and transferred to the cooler at 4˚C for refrigeration until further analyses. For the sensory evaluation blueberry flavored yogurt with the same lactose treatments (0%, 1%, 3% and 5% w/w added lactose) were manufactured. The manufacture process was the same with the exception that 15% w/w blueberry puree was incorporated after plain yogurt manufacture and refrigerated at 4˚C. Product manufacture was replicated 3 times.

Lactose concentration of yogurts was determined using the Lactose/ D-Galactose determination kit [24]. Briefly, 1 g of sample was weighed into a 100 mL volumetric flask. Then 60 mL of distilled water was added and flasks were incubated for 15 minutes at 70˚C. Samples were shaken from time to time. For clarification, 5 mL of Carrez-I-solution (3.60 g potassium hexacyanoferrate (II), 5 mL of Carrez-II-solution (7.20 g of zinc sulfate/100 mL) and 10 mL of NaOH (0.1 M) were added and mixed after each addition. The sample solution was adjusted to 20˚C - 25˚C and filled up to 100mL with distilled water, then filtered using a 12.5 cm Whatman® filter paper. The clear solution was used for the assay. Absorbance measurements were taken at a wavelength of 340 nm using an Evolution 100 spectrophotometer (Thermo Scientific, Waltham, Massachusetts). Lactose concentrations were measured every 7th day over the 35 days of storage for each of the 3 replications.

The pH of the yogurts was determined using the Oysters Series pH meter (Extech Instruments, Waltham, MA). The instrument was calibrated using commercial pH 4.00 and 7.00 buffers (Fisher Scientific, Fair Lawn, NJ) and instrument’s temperature was adjusted to the sample’s temperature of 8˚C ± 2˚C before reading. Two measurements were taken per replication for each of the 3 replications.

The titratable acidity (TA) was determined by weighing 9 g of yogurt to which 5 drops of phenolphthalein indicator solution were added. Samples were titrated with 0.1 N NaOH as until color changed to rose pink that persisted for 30 seconds.

Apparent viscosities were measured using a Brookfield DV II + viscometer (Brookfield Engineering Lab Inc., Stoughton, MA, USA) with a helipath stand at 10˚C ± 2˚C. A T bar B spindle was set to 10 rpm to obtain a torque between 10% - 90%. The T bar B spindle was inserted in the sample at a constant depth of 2 cm from the top level of the sample container. The helipath was set in downward motion to cut new circular layers at increasing depths of the sample. Sample’s container geometry was 4.55" top diameter, 3.25" bottom diameter and 2.45" height with 340 g capacity. The data were gathered using the Wingather® software (Brookfield Engineering Lab Inc., Stoughton, MA, USA). The viscosity measurements were continuous over 33 seconds required to collect one hundred data points averaged per sample per replication.

Syneresis was determined with the method described by [13] with slight modifications. The 300 mL of yogurt mix was poured into plastic cups prior to incubation during yogurt manufacture. The cups of set yogurt were kept at an angle of 45˚ and spontaneous whey was collected at the side of the cup with a pipette. Amount of whey in mL was measured at 22˚C. The yogurt gel was allowed to stand for 1 minute and any further surface whey was pipetted and total whey release in mL was measured.

Growths of Lactobacillus delbrueckii ssp. bulgaricus (LB-12) and Streptococcus thermophilus (ST-M5) were determined using the pour plate technique with serial dilutions of yogurt samples. Yogurts were sampled at days 1, 7, 14, 21, 28 and 35 of storage period. The yogurt in the cup was agitated and 1 g of yogurt was pipetted from the center of the cup into a sterile bottle containing 99 mL of sterile 0.1% peptone water (Difco, Detroit, MI, USA). Contents in bottle were agitated to prepare serial dilutions and plated on 5.2 modified pH Lactobacilli MRS agar for Lactobacillus bulgaricus LB-12 and Streptococcus thermophilus agar for Streptococcus thermophilus ST-M5. Pour plates were incubated anaerobically at 43˚C for 72 hours for Lactobacillus bulgaricus LB-12 [25] and aerobically at 37˚C for 24 hours for Streptococcus thermophilus ST-M5 [26] and colonies were counted.

The blueberry yogurt was tested for coliforms using petrifilms (3M®, St. Paul, MN) which contain violet red bile agar (VRBA). The procedure was performed by weighing 11 g of yogurt samples in 99 mL of sterile 0.1% peptone water (Difco, Detroit, MI, USA). Contents in bottle were agitated and serial dilutions were prepared. Aliquots of 1mL were taken from dilutions 10-1 and plated in duplicate for control and added lactose samples. Previously labeled petrifilms were incubated aerobically at 32˚C for 24 hours.

The sensory study was approved by the LSU Institutional Review Board with the IRB exemption number of HE13-6. Blueberry yogurt containing the four treatments (0%, 1%, 3% and 5% w/w added lactose) was poured into 2.5 oz. previously labeled shuffle cups. A 3-digits random number code was used to label the cups. Consumer acceptance study was performed with 100 consumers of yogurt. One cup per treatment, that is four cups in total, was given to each consumer along with the evaluation questionnaire which consisted of a 9-points rating scale (1 = Dislike extremely, 9 = Like extremely), with acceptability and purchase intent questions (yes/no). Panelists were asked to evaluate each yogurt sample for the following attributes: Appearance, Color, Aroma, Taste, Sourness, Sweetness, Thickness, Graininess and Overall like.

The Lactobacillus bulgaricus LB-12 and Streptococcus thermophilus ST-M5 counts were converted to log 10 scale before analyzing the data by SAS. Data were analyzed using Proc Mixed of the SAS® 9.3 program. Differences of Least Square Means were used to determine significant differences at P < 0.05 for main effects (lactose concentration and time) and interaction effects (lactose concentration*time). Sensory data are presented as mean ± standard deviation (SD) of the means. Significant differences between means were analyzed at α = 0.05 using Tukey adjustment to determine the best treatment. All experiments were replicated three times.

Lactose content of yogurts as influenced by lactose addition over storage of 35 days is shown in Figure 1. Treatment*day interaction effect was not significant

(P > 0.05) while treatment effect and day effect were significant (P < 0.05) (Table 1). Lactose content decreased for all treatments over time (Figure 1). Yogurts with lactose added at 1%, 3%, 5% stayed in that proportion higher than control throughout the 35 days of storage (Figure 1).

As expected, treatment containing 5% w/w added lactose had the highest lactose content (Table 2). The highest lactose content was observed at days 1 and 7 of storage period (Table 3). Also, as expected lactose content steadily decreases from day 21 onwards over the rest of the storage time (Table 3). Reference [27] found a reduction in lactose concentration of yogurt after storage period of 7 days. They attributed the loss of lactose to the fact that lactic acid bacteria not only produce lactic acid from lactose but also flavor compounds and polysaccharides during storage consuming more of this molecule.

^a-dLS means with the same letter does not indicate significant difference (P < 0.05).

^a-dLS means with the same letter does not indicate significant difference (P < 0.05).

The pH of yogurts as influenced by added lactose concentration over storage of 35 days is shown in Figure 2. Treatment*day interaction effect was not significant (P > 0.05) while treatment effect and day effect were significant (P < 0.05) (Table 1). The pH values decreased for all treatments at day 35 compared to day 1 (Figure 2). According to [28] a decrease in pH during storage is expected as a result of the metabolic activity of starter cultures. Yogurts containing 5% w/w added lactose had the lowest pH (Table 4). The more lactose present, the higher the production of lactic acid by the starter cultures. According to [29] at high pH most of the lactic acid is formed due to a growth-associated mechanism and the growth curve has a short stationary growth phase. On the contrary, at low pH most of the lactic acid produced is non-growth-associated [29]. The lowest pH values were obtained from day 14 onwards (Table 5).

The TA of yogurts as influenced by added lactose concentration over storage of

^a-bLS means with the same letter does not indicate significant difference (P < 0.05).

^a-cLS means with the same letter does not indicate significant difference (P < 0.05).

35 days is shown in Figure 3. Treatment*day interaction effect, treatment effect and day effect were significant (P < 0.05) (Table 1).

In general, yogurts containing 1% and 3% w/w lactose had higher TA at day 35 compared to days 1 and 7 (Table 6, Figure 3). Yogurts with 3% and 5% w/w added lactose had lower TA values compared to 0% and 1% w/w added lactose, except at day 7 for 3% w/w added lactose (Table 6). This phenomenon can be explained through the fact that whey separation caused by lactose hydrolysis leads to slow rate of acid production in yogurt [30]. As lactose is hydrolyzed by lactic acid bacteria the amount of lactic acid production increased. This behavior may be due to the availability of more quantity of easily fermentable sugar (glucose) which is required for the faster growth of starters [31] [32] [33]. Reference [34] reported that changes in titratable acidity do not necessarily have an effect on pH values.

The apparent viscosity of yogurts as influenced by lactose addition over storage of 35 days is shown in Figure 4. Treatment*day interaction effect and day effect were not significant (P > 0.05) while treatment effect was significant (P < 0.05) (Table 1). Yogurts containing 5% w/w added lactose had the lowest apparent viscosity values compared to 0%, 1% and 3% w/w added lactose (Table 7). This was because of the higher amount of whey released. Weaker body and texture of yogurt may be due to higher amount of whey separation which reduced the viscosity when lactose is hydrolyzed [35] [36] [37].

The syneresis of yogurts as influenced by lactose addition over storage of 35 days is shown in Figure 5. Treatment*day interaction effect, treatment effect and day effect were significant (P < 0.05) (Table 1). Yogurts containing 5% w/w added lactose had significantly the highest syneresis values compared to 0%, 1% and 3% w/w added lactose during storage period at day 7 and from day 21 onwards (Table 8). Reference [30] reported that as the degree of lactose hydrolysis in-

^ABCDEFGHLS means with the same letter does not indicate significant difference (P < 0.05).

^a-bLS means with the same letter does not indicate significant difference (P < 0.05).

^ABCDELS means with the same letter does not indicate significant difference (P < 0.05).

creased the amount of whey separation increased (P < 0.05).

There were no detectable coliforms in all samples.

Means for all tested attributes (appearance, color, aroma, taste, sourness, sweetness, thickness, graininess, and overall liking) are shown in Figure 8. The probabilities for fixed effect of sensory attributes are shown in Table 12. Samples containing 1% w/w added lactose had higher scores for thickness compared to 5% w/w added lactose, which might be explained by increased syneresis in added lactose samples. Control samples had lower scores for graininess compared to 5% w/w added lactose (Table 13), possibly due to difference in total solids content.

The overall liking scores indicated that samples containing added lactose were preferred over control (Table 13). Lactose addition did not appear to significantly (P > 0.05) impact appearance and color of yogurts (Table 12). Similar results were reported by [30] who studied lactose hydrolysis on enzymatically hydrolyzed yogurts. Aroma and taste are important sensory characteristics of yogurt [46]. Control had higher aroma scores compared to 1% and 3% w/w added lactose (Table 13). For taste, sourness and sweetness samples containing added lactose had higher scores than control (Table 13). Yogurt acceptability frequency values are shown in Figure 9(a). Added lactose yogurts had a greater acceptability compared to control yogurts. The consumer acceptability of yogurts increased as lactose addition increased. Yogurts containing 5% w/w added lactose led to the highest acceptability (88%) compared to control (70%). This is probably due to a better palatability given by the sweetness of lactose. According to [30] when lactose is hydrolyzed in yogurt it resulted in increased digestibility, sweeter

taste, and better mouth feel. Yogurt purchase intent frequency values are shown in Figure 9(b). Added lactose yogurts had greater purchase intent values compared to control yogurts. Purchase intent increased as lactose addition increased. Yogurts containing 5% w/w added lactose led to higher purchase intent (66%) compared to control (35%). Reference [47] reported that sweetness is one of the

^a-bMeans with the same letter does not indicate significant difference (P < 0.05).

main traits for flavored yogurts.