MATERIALS AND METHODS

Materials: Date syrup (dips) was obtained from the local markets. The pasteurized cow milk was obtained from a dairy company on the same manufacturing day. The milk composition as shown on the packing was 3.1% fat and 8.5% non-fat solids. The probiotic bacteria namely Lactobacillus acidophilus La-5 and Bifidobacterium lactis Bb-12 were obtained from Christian Hansen/ Denemark.

Methods
Determination of acidity and pH values: Acidity and the pH of the fermented milk samples were determined according to the standard method (AOAC, 2000).

Count of probiotic bacteria: The counts of B. lactis and L. acidophilus were determined according to Adhikari et al. (2003) by using MRS agar medium (Oxoid, CM0361B). For bifidobacterial count, L.cysteine-HCl (Sigma chemical Co., St. Louis, Mo) was added at the rate of 0.5%, to decrease the redox potential of the medium. All plates were incubated anaerobically at 37°C for 48 h.

Experimental design: Cow milk was divided into five portions. The first portion was treated as control (0 syrup), while 5, 10, 15 and 20% dips were added to the second, third, fourth and fifth portions, respectively. Then, each portion was subdivided into two parts. The first part was inoculated with L. acidophilus while the other was inoculated with B. lactis. Both the parts were incubated at 37 and 40°C, respectively. Subsequent samples were taken after zero, 6, 12 and 24 h of incubation. The numbers of living bacteria, pH and the acidity for each treatment were determined during incubation

Data analysis: The data was analyzed by analysis of variance (ANOVA) and regression techniques according to SAS (2000).

RESULTS AND DISCUSSION

Effect of various concentrations of dips on the viable count of L. acidophilus in milk: The changes in the logarithm numbers of viable count of L. acidophilus in the presence of various concentrations of dips during incubation periods are given in Table 1.

After direct inoculation (zero time), the count of L. acidophilus ranged from 6.51-6.83 log cfu mL^-1 in all the treatments. The bacterial count in the control sample (0-Dips) increased significantly after 6 (6.90-7.94 log cfu mL^-1, 12 (6.82-7.94 log cfu mL^-1) and 24 h (6.04-8.34 log cfu mL^-1) of incubation as compared to zero time in different dips treatments. However, the rate of increase in the bacterial count was 1.35, 1.35 and 1.75 log cycle, respectively when compared to zero time. The viable count of L. acidophilus bacteria increased continuously with increasing the incubation times in all the treatments until 12 h. It was found that the log numbers of L. acidophilus for the treatment with 5% dips concentration increased significantly after 6, 12 and 24 h incubation as compared to zero time treatment. These increases were 0.62, 0.78, 0.71 log cycle for the different treatments, respectively. The increase in the count of L. acidophilus for the 5% dips was less than that found in the control sample at the same incubation time. This indicated that the addition of dips at 5% ratio showed a negative impact on the growth of L. acidophilus. Besides, there was no significant difference in the bacterial count between 5 and 15% addition of dips after 6 and 12 h incubation. The results showed significant differences in the bacterial counts when compared with zero time treatment. It was also observed that the numbers of bacterial counts showed decreasing trend with increasing the incubation period to 24 h. The results showed that the decrease in the bacterial count was more in 15% dips than 10% dips treatments.

Table 1:	Changes in the viable count of L. acidophilus in the presence of various concentrations of dips in milk
The upper different letters in each column indicates significant difference at p>0.05, The lower different letters in each row indicates significant difference at p>0.05

Furthermore, the bacterial count in 15% dips treatment after 24 h incubation was less than that recorded at zero time. The numbers of L. acidophilus in the 20% dips treatment recorded an increase of 0.35, 0.27 log cycle after 6 and 12 h of incubation, respectively. It was observed at the end of the incubation period that the count decreased by -0.51 log cycle as compared to zero time treatment. When comparing the results of L. acidophilus count for all the treatments after 24 h of incubation, there was a significant increase in count in the control treatment as compared to all other treatments followed by the treatment containing 5% dips. Moreover, there was no significant difference between 10 and 15% dips concentrations. The 20% dips treatment recorded the lowest count of these bacteria as compared to other treatments. In general, it can be concluded that the addition of dips may inhibit the growth and reproduction of L. acidophilus with increasing the incubation time. There was noticed an inverse relationship between the increase in the concentration of dips and the numbers of these bacteria. This could be due to the influence of the osmotic sugar (Jay et al., 2005) which caused the death of bacteria during incubation and the lack of resistance to the increased concentration of dips. Al Eid (2006) pointed out that dips contain 81% sugars which are subdivided into fructose (41%), glucose (39%) and sucrose (1%). Also, the presence of certain compounds in the dips have a toxic effect on the growth of these bacteria. Al-Jasass et al. (2010) found that the dips contain formic acid, acetic acid and propionic acid as 3.06, 2.38 and 0.68%, respectively. These Acids have a toxic effect on the yeast bread ″Saccharomyces cerevisia″.

Although, there was a reduction in the count of L. acidophilus at the end of incubation period but still the number is more than 10⁶ cfu mL^-1. This might explain the health benefits that can be obtained from the consumption of such product (Kurmann et al., 1992). These results are in agreement with the results of Curda and Plockova (1995), who reported about the addition of honey with and without heat treatment to milk with different concentration of fat (0, 1, 3, 5 and 10%). They found that the growth of L. acidophilus was inhibited in both types of honey at = 5% concentration.

Previous studies did not find any significant differences in the L. acidophilus count after 24 h of incubation between the control sample and the samples containing 5% sucrose and high fructose corn syrup or honey (Popa and Ustunol, 2011; Chick et al., 2001). This difference in the results with the present study can be explained by the presence of some organic acids in dips which has an inhibitory effect on the growth and survival of bacteria (Al-Jasass et al., 2010).

Effect of various concentrations of Dips on pH of milk inoculated with L. acidophilus: The pH values of fermented milk inoculated with L. acidophilus bacteria in the presence of various concentrations of dips during different incubation period are presented in Table 2. The pH values in all the treatments decreased significantly with increasing the storage time but without any significant difference from the control during different incubation periods i.e. 6, 12 and 24 h. The pH ranged from 6.15-6.35, 5.95-6.10, 5.05-5.20 and 4.90-5.10 after 0, 6, 12 and 24 h incubation, respectively in different dips treatments. These results are in agreement with the finding of Chick et al. (2001), who did not found any significant difference in pH values between L. acidophilus fermented milk without sweeteners and that containing 5% sucrose and high fructose corn syrup or honey. Curda and Plockova (1995) found that the L. acidophilus inhibition occurred at a concentration of = 5% honey. Overall in the present study, there was no significant difference in the pH values among all the treatments with different concentrations of dips.

Table 2:	Changes in pH values of milk inoculated with Lactobacillus acidophilus in the presence of various concentrations of dips during incubation periods
The upper different letters in each column indicates significant difference at p>0.05, The lower different letters in each row indicates significant difference at p>0.05

Table 3:	Changes in acidity of milk inoculated with Lactobacillus acidophilus in the presence of various concentrations of dips during incubation periods
The upper different letters in each column indicates significant difference at p>0.05, The lower different letters in each row indicates significant difference at p>0.05

Effect of Various Concentrations of Dips on the Acidity of Milk inoculated with L. acidophilus: The acidity of L. acidophilus fermented milk in presence of various concentrations of dips during incubation period is presented in Table 3. There was a slight increase in the acidity for all the treatments during the whole incubation period. The highest acidity was found in the control and 5% dips concentration treatment after 24 h of incubation. This increase could be due to the increase in the L. acidophilus count in these treatments than the others treatments (Table 1). Furthermore, the acidity of milk increased with increasing the dips proportion at zero time. The change in acidity ranged between 0.39-0.59, 0.47-0.61, 0.68-0.81 and 0.77-1.53 during 0, 6, 12 and 24 h incubation, respectively in different dips treatments. This increase in acidity might be due to the presence of organic acids in dips such as formic acid, acetic acid and propionic acids with relative proportions of 3.06, 2.38, 0.68%, respectively (Al-Jasass et al., 2010). In the present study, an inverse relationship was observed between the amount of dips concentrations and the acidity of milk after 24 h. incubation. Overall there was a clear relationship between the count of L. acidophilus and the acidity obtained as described in Table 1. The least acidity was found in the treatment containing 20% dips with 24 h incubation. Chick et al. (2001) found that the L. acidophilus produces lactic acid in milk containing sucrose, fructose and honey more than without their presence.

Table 4:	Changes in the viable count of Bifidobacterium lactis in the presence of various concentrations of dips in milk
The upper different letters in each column indicates significant difference at p>0.05, The lower different letters in each row indicates significant difference at p>0.05

Effect of various Concentrations of Dips on the viable count of Bifidobacterium lactis in milk: The changes in the logarithm numbers of Bifidobacterium lactis viable count in the presence of various concentrations of dips in milk during different incubation periods is given in Table 4. It was found that B. lactis followed different behaviors in their growth and reproduction in different treatments during different storage periods. The data indicate that the numbers of B. lactis was significantly high in the control treatment (without the addition of dips) after 12 and 24 h of incubation as compared to the rest of the treatments. The difference in the count of B. lactis was not significant between zero time and after 24 h of incubation. These results agree with those of Chick et al. (2001) who did not find significant difference between the numbers of B. lactis at zero and after 24 h of incubation using sucrose, fructose, honey and also without any sweetener. They further reported that the number of B. lactis decreased significantly (p>0.05) in all the treatments during the incubation period between 12 and 24 h. The number of bacteria ranged from 6.87-7.12, 6.88-7.17, 6.55-7.49 and 5.45-7.27 log₁₀ cfu mL^-1 after 0, 6, 12 and 24 h incubation in different dips treatments, respectively. This association was related to the effect of dips concentration. The results indicated that the number of B. lactis decreased with increasing concentration of dips. The lowest number of B. lactis was in the treatment with 20% dips concentration (5 and 45 cfu mL^-1). This directly reflects the inhibitory effect of dips towards the growth of bacteria B. lactis.

The other possible reason may be the presence of some compounds having toxic effect on the growth of bacteria as mentioned by Al-Jasass et al. (2010) or the osmotic effect as stated by Jay et al. (2005). Popa and Ustunol (2011) found that the number of B. bifidium rose by 0.28 log cycle in the presence of sucrose after 24 h of incubation. In the control sample (without sugar) the numbers rose by 0.36 log cycle indicating the inhibitory effect of sucrose on the growth and activity of the B. lactis. Though, there was a decline in the count of B. lactis in the end of incubation period yet the numbers exceeded to one million cells per mL of milk except that treatment containing 20% dips. This refers on the health effects of these bacteria (Kurmann et al., 1992).

Changes in pH values of fermented milk inoculated with Bifidobacterium lactis: The pH ranged from 6.00-6.30, 6.00-6.20, 5.90-6.20 and 5.55-6.10 after 0, 6, 12 and 24 h incubation in different dips treatments, respectively (Table 5). A gradual reduction in the pH values of all the treatments was observed with different concentration of dips (0, 5, 10, 15 and 20%).

Table 5:	Changes in pH values of milk inoculated with Bifidobacterium lactis in the presence of various concentrations of dips during incubation periods
The upper different letters in each column indicates significant difference at p>0.05, The lower different letters in each row indicates significant difference at p>0.05

Table 6:	Changes in acidity of milk inoculated with Bifidobacterium lactis in the presence of various concentrations of dips during incubation periods
The upper different letters in each column indicates significant difference at p>0.05, The lower different letters in each row indicates significant difference at p>0.05

However, this reduction in pH was significant (p>0.05) between the dips (10, 15, 20%) and the control treatment during different incubation periods (0, 6, 12 and 24 h). This reduction might be due to the presence of some organic acids in the dips as mentioned earlier by Al-Jasass et al. (2010) or the acids produced by the Bifidobacteria (Scardovi and Trovatelli, 1965). The results in Table 5 also showed significant differences in the pH values (p>0.05) between zero time and 24 h incubation for all the treatments. Chick et al. (2001) found that the pH of fermented milk decreased with B. bifidum in the presence of some sweeteners, but it was significant with 5% honey as compared to the control treatment (without sweetener).

Changes in acidity of fermented milk inoculated with Bifidobacterium lactis bacteria: The change acidity of fermented milk inoculated with Bifidobacterium lactis ranged from 0.42-0.62, 0.42-0.62, 0.45-0.67 and 0.48-0.69 after 0, 6, 12 and 24 incubation in different dips treatments, respectively (Table 6). The acidity of the fermented milk increased with increasing incubation time for all the treatments. It can be concluded from the results in Table 6 that the highest increase in pH value was in the treatment with 5% dips concentration. This increase in pH reached to 29.5% as compared to all other treatments. However, this increase in pH was less in 15% and 20% dips concentration treatments. The values for this increase was 7 and 11.7%, respectively for both these treatments. Moreover, the difference in acidity value was significant (p>0.05) among all the treatments in addition to the control at zero time incubation. The acidity values increased with increasing concentration of dips. The lowest value of acidity was in the control and the highest was in 20% dips treatments. This might be due to the reasons stated earlier by Al-Jasass et al. (2010), Scardovi and Trovatelli (1965) and Chick et al. (2001).

CONCLUSION

The study showed that high concentrations of dips have inhibitory effects on the growth and survival of probiotic bacteria (Lactobacillus acidophilus and Bifidobacterium lactis). However, the viable counts of bacteria were maintained high enough at the end of incubation period (more than 10⁶ cfu mL^-1) which is highly essential to achieve the health benefits of these probiotic bacteria. This conclusion holds true for all the treatments except the treatment with 20% concentration of dips in the presence of B. lactis after the 24 h incubation. The viable number of probiotic bacteria decreased to about 10⁵ cfu mL^-1. Therefore, the dips can safely be used as sweeteners for the fermented milk in the presence of the probiotic bacteria.