The chemical properties of prepared apple juice were determined as follow: pH = 4.08, total acidity = 0.28 g mallic acid/100 g, reducing sugar content = 12 g/100 g, total sugar content = 14.09 g/100 g, Brix = 15 and diazinon concentration < 10 µg/L. The results of effect of different parameters on chemical properties (pH, total acidity, reducing sugar and total sugar), diazinon content and microbiological properties (viability of probiotic bacteria) are discussed in the following.
3.1. Effect of probiotic strain’s type on diazinon reduction (first stage)
The effect of different probiotic strains i.e. L. rhamnosus, L. plantarum, L. acidophilus and L. casei) at a primary concentration of 9 log CFU/mL and presence of 1000 µg/L of spiked diazinon on chemical and microbiological properties of apple juice samples was shown in Table 1 after 48 h fermentation at 37°C.
Table 1
Chemical and microbial changes of apple juice samples in the first stage*
Treatment**
|
pH
|
Total acidity (g/100 g)
|
Reducing sugar content (g/100 g)
|
Total sugar content (g/100 g)
|
Viability of probiotic (CFU/mL)
|
Diazinon reduction (%)
|
B
|
4.06 ± 0.07a
|
0.28 ± 0.22a
|
11.86 ± 0.01a
|
13.80 ± 0.15a
|
-
|
-
|
B-1
|
4.06 ± 0.07 a
|
0.28 ± 0.23a
|
11.86 ± 0.01a
|
13.86 ± 0.15a
|
-
|
15.46a
|
A-9-1
|
3.94 ± 0.02bd
|
0.30 ± 0.28a
|
11.42 ± 0.03b
|
12.89 ± 0.18b
|
10.22 ± 0.03a
|
59.80b
|
A-9
|
3.86 ± 0.01c
|
0.44 ± 0.40b
|
10.64 ± 0.05c
|
12.06 ± 0.14c
|
10.83 ± 0.02b
|
-
|
R-9-1
|
3.96 ± 0.01d
|
0.29 ± 0.22a
|
11.61 ± 0.02d
|
13.07 ± 0.32d
|
10.20 ± 0.01a
|
56.97c
|
R-9
|
3.88 ± 0.01cb
|
0.43 ± 0.42b
|
11.02 ± 0.02e
|
12.12 ± 0.12c
|
10.80 ± 0.01b
|
-
|
C-9-1
|
3.98 ± 0.08d
|
0.29 ± 0.32a
|
11.61 ± 0.03d
|
13.29 ± 0.25e
|
10.17 ± 0.06a
|
56.77d
|
C-9
|
3.89 ± 0.01bc
|
0.42 ± 0.32b
|
11.07 ± 0.02e
|
12.50 ± 0.38f
|
10.79 ± 0.01b
|
-
|
P-9-1
|
3.99 ± 0.01bd
|
0.29 ± 0.23a
|
11.70 ± 0.04d
|
13.50 ± 0.13g
|
10.10 ± 0.05c
|
56.37e
|
P-9
|
3.90 ± 0.01c
|
0.41 ± 0.31b
|
11.22 ± 0.02f
|
12.70 ± 0.12h
|
10.67 ± 0.02d
|
-
|
*Each value in the table is the mean ± standard deviation (SD) of three trials. Different letters in each column indicate a statistically significant difference (P < 0.05). |
** B: control sample, 1: 1000 µg/L of spiked diazinon, A: L. acidophilus, R: L. rhamnosus, C: L. casei and P: L. plantarum, 9: Primary probiotic population (9 log CFU/mL). |
As presented in this table, the pH of all treatments decreased from 4.08 to 3.86–4.06 after 48 h fermentation. These decline were significant (p < 0.05) for all treatments except control samples (B and B-1) which contained no probiotic bacteria. The lowest pH content (3.86) was showed in samples containing L. acidophilus (A-9). The total acidity of all treatments increased significantly (p < 0.05) after 48 h fermentation from 0.28 to 0.29–0.44 g mallic acid/100 g apple juice with the exception of control samples without probiotic addition (B and B-1). The highest total acidity (0.44 g mallic acid/100 g) was showed in samples containing L. acidophilus (A-9). The reducing and total sugar content of all samples also decreased after fermentation. The lowest decline in sugar concentration was occurred in control samples (B and B-1), while the highest decline in reducing and total sugar content were observed in samples containing L. acidophilus (A-9) which determined 10.64 and 12.06 g/100 g apple juice, respectively.
Probiotic bacteria are able not only to survive but to use apple juice for their cell synthesis, as determined by a decrease in total and reducing sugar concentrations as well as pH value, and increase in acidity. Similar to previous studies concerning probiotic apple juice, probiotics may have consumed carbohydrates and formed small concentrations of organic acids thus lowering the pH and increasing the total acidity of the juice during fermentation and storage (Ding & Shah, 2008; Pimentel et al., 2015). It is worthy to note that different types of probiotic have different activity rate (Nematollahi et al., 2016). It is clear that L. acidophilus was observed to utilize the sugar and subsequently change pH and acidity at a faster rate than other strains.
The probiotic viability of samples containing different probiotic species increased significantly from 9 to 10.10-10.83 log CFU/mL during fermentation period. The highest and lowest probiotic viability were observed in samples containing L. acidophilus (A-9) and L. plantarum in the presence of added diazinon (P-9-1). It is worthy to mention that the probiotic viability in all samples with addition of diazinon was lower than samples without spiked diazinon. For example, the probiotic viability in A-9 and A-9-1 treatments were 10.83 and 10.22 log CFU/mL, respectively.
The diazinon reduction percent in control sample (B-1) was the lowest (15.46%), while the highest content was observed in samples containing L. acidophilus (A-9-1) which was 75.26 % which means the L. acidophilus decreased spiked diazinon as 59.80%. The diazinon reduction percent of other strains had low differences but significant (56.37–56.97 %). Yousefi et al. (2019) and Sarlak et al. (2016) also reported that L. acidophilus have the highest binding capacity to PAH and AFM1, respectively (Sarlak et al., 2017; Yousefi et al., 2019). Thus, L. acidophilus is the most effective probiotic strain in diazinon reduction in apple juice during fermentation and was chose for the next stage.
3.2. Effect of primary probiotic population on diazinon reduction (second stage)
In this stage, the effect of initial inoculated population (9 and 7 log CFU/mL) of selected probiotic strain from the previous stage (L. acidophilus) on diazinon reduction percent was evaluated. Table 2 shows the chemical and microbial changes of apple juice samples after 48 h fermentation at 37°C.
Table 2
Chemical and microbial changes of apple juice samples in the second stage*
Treatment**
|
pH
|
Total acidity (g/100 g)
|
Reducing sugar content (g/100 g)
|
Total sugar content (g/100 g)
|
Viability of probiotic (CFU/mL)
|
Diazinon reduction (%)
|
B
|
4.06 ± 0.01a
|
0.28 ± 0.32a
|
11.86 ± 0.02ab
|
13.82 ± 0.05a
|
-
|
-
|
B-1
|
4.06 ± 0.01a
|
0.28 ± 0.20a
|
11.93 ± 0.01a
|
13.82 ± 0.13a
|
-
|
15.53a
|
A-9-1
|
3.95 ± 0.01b
|
0.30 ± 0.30a
|
11.42 ± 0.08c
|
12.91 ± 0.19b
|
10.20 ± 0.01a
|
59.40b
|
A-9
|
3.86 ± 0.07c
|
0.43 ± 0.32b
|
10.53 ± 0.05d
|
11.98 ± 0.10c
|
10.83 ± 0.05b
|
-
|
A-7-1
|
3.98 ± 0.01b
|
0.29 ± 0.23a
|
11.80 ± 0.01b
|
13.71 ± 0.10d
|
7.35 ± 0.07c
|
44.40c
|
A-7
|
3.92 ± 0.01b
|
0.34 ± 0.32a
|
11.55 ± 0.03d
|
12.83 ± 0.10e
|
7.77 ± 0.04d
|
-
|
*Each value in the table is the mean ± standard deviation (SD) of three trials. Different letters in each column indicate a statistically significant difference (P < 0.05). |
** B: control sample, 1: 1000 µg/L of spiked diazinon, A: L. acidophilus, 9 or 7: Primary probiotic population (9 or 7 log CFU/mL). |
As shown in Table 2, the pH of all samples reduced after 48 h fermentation which this decline was significant (p < 0.05) for all samples except control samples (B and B-1). The lowest pH content (3.86) observed in samples containing 9 log CFU/mL of L. acidophilus (A-9). The total acidity of all samples increased from 0.28 to 0.29–0.43 g mallic acid/100 g apple juice except in control samples (B and B-1) which did not change during fermentation. The highest total acidity was also observed in samples containing 9 log CFU/mL of L. acidophilus (A-9). The reducing and total sugar content of all samples decreased to 10.53–11.86 and 11.98–13.82 g/100 g apple juice, respectively. The highest sugar consumption was also reported for samples containing 9 log CFU/mL of L. acidophilus (A-9).
As it is obvious the chemical changes of samples containing 9 log CFU/mL of L. acidophilus (A-9) was significantly higher than that in A-7 indicating higher activity of probiotics during fermentation in higher primary population count.
The viability of L. acidophilus increased more than one cycle in the samples containing 9 log CFU/mL and reached to 10.20 and 10.83 log CFU/mL after 48 h fermentation, while this increment was lower than one cycle in the samples containing 7 log CFU/mL and reached to 7.35 and 7.77 log CFU/mL. The diazinon reduction percent in samples containing 9 log CFU/mL of L. acidophilus was significantly higher than that in samples containing 7 log CFU/mL which were 59.40 and 44.40 %, respectively due to higher activity of L. acidophilus in higher primary population.
L. aciduphilus degraded diazinon by using it directly as a source of carbon, nitrogen and phosphorus, or by creating diazinon-degrading enzymes. Inoculum primary amount plat a significant role in pesticide biodegradation. Lower initial inoculum rate could lead to a small number of the probiotics contributing in chemical degradation (Zhou & Zhao, 2015). Similar result was also observed in the degradation of organophosphorus pesticides during fermentation of pickled Chinese cabbage (Lu et al., 2013), degradation of AFM1 in Doogh (Sarlak et al., 2017), in vitro removal of PAH (Yousefi et al., 2019) and AFM1 (Kabak & Var, 2008) from phosphate-buffer by probiotic bacteria. To this end the primary population rate of 9 log CFU/mL was selected for the next stage.
3.3. Effect of diazinon concentration spiked on its reduction (third stage)
In this stage, the effect of different concentrations of spiked diazinon (1000 and 5000 µg/L) on its reduction by 9 log CFU/mL of L. acidophilus in apple juice was evaluated during 48 h fermentation at 37°C. Table 3 shows the chemical and microbial changes of apple juice samples after 48 h fermentation. Similar to previous stages the pH, total and reducing sugar concentrations decreased, while the total acidity of all samples increased after fermentation.
Table 3
Chemical and microbial changes of apple juice samples in the third stage*
Treatment**
|
pH
|
Total acidity (g/100 g)
|
Reducing sugar content (g/100 g)
|
Total sugar content (g/100 g)
|
Viability of probiotic (CFU/mL)
|
Diazinon reduction (%)
|
B
|
4.06 ± 0.07a
|
0.28 ± 0.32a
|
11.86 ± 0.01a
|
13.86 ± 0.10a
|
-
|
-
|
B-1
|
4.06 ± 0.01a
|
0.28 ± 0.26a
|
11.93 ± 0.01b
|
13.86 ± 0.10a
|
-
|
17.50a
|
B-5
|
4.06 ± 0.01a
|
0.28 ± 0.32a
|
12.00 ± 0.01b
|
14.02 ± 0.15b
|
-
|
35.26b
|
A-9-1
|
3.94 ± 0.01b
|
0.30 ± 0.28a
|
11.42 ± 0.02c
|
12.93 ± 0.16c
|
10.20 ± 0.09a
|
57.53c
|
A-9
|
3.86 ± 0.09c
|
0.44 ± 0.60b
|
10.90 ± 0.02d
|
11.95 ± 0.22d
|
10.82 ± 0.01b
|
-
|
A-9-5
|
3.98 ± 0.06b
|
0.29 ± 0.39a
|
11.67 ± 0.02e
|
13.37 ± 0.30e
|
9.88 ± 0.04c
|
23.63d
|
*Each value in the table is the mean ± standard deviation (SD) of three trials. Different letters in each column indicate a statistically significant difference (P < 0.05) |
** B: control sample, 1 or 5: 1000 or 5000 µg/L of spiked diazinon, A: L. acidophilus, 9: Primary probiotic population (9 log CFU/mL). |
As depicted in Table 3, the presence of diazinon in apple juice samples containing probiotic bacteria (A-9-1 and A-9-5) led to decreasing in probiotic activity and viability, significantly (p < 0.05). For instance, the decline rate of pH, reducing and total sugar content in treatments containing L. acidophilus and 5000 µg/L of diazinon (A-9-5) was lower than that in samples containing 1000 µg/L of diazinon (A-9-1) and L. acidophilus without spiked diazinon (A-9). Furthermore, the viability of L. acidophilus in A-9, A-9-1 and A-9-5 was determined 10.82, 10.20 and 9.88 log CFU/mL, respectively which indicated that the presence of diazinon has negative effect on probiotic viability and subsequently their activity during fermentation. Thus, the ability of L. acidophilus to degrade the diazinon decreases with higher concentration of spiked diazinon. As shown in Table 3, the diazinon reduction percent by probiotic bacteria in samples containing 5000 µg/L of spiked diazinon (A-9-5) was lower than those containing 1000 µg/L of diazinon (A-9-1) which were 57.53 and 23.63 % which is in good agreement with Dordevic et al. (2013) (T. Đorđević et al., 2013 a) and Yousefi et al. (2019) (Yousefi et al., 2019) studies. In fact, the presence of diazinon could be effective in probiotic viability and activity (acid and other compounds’ production) which was also confirmed by Ayana et al. (2011) in a study done in yogurt in the presence of some pesticides (Ayana et al., 2011). Dordevic et al. (2013) also reported that pirimiphos-methyl (a pesticide) could just inhibit the growth of L. plantarom in concentrations higher than 5 mg/kg during wheat fermentation (T. Đorđević et al., 2013 a). In another study it is also reported that during the probiotic fermentation, bifenthrin reduction within wheat fortified with 500 µg/kg was 42%, although significantly lower in samples spiked with 2.5 µg/kg, maximum 18% (T. M. Đorđević et al., 2013 b). Thus, the spiked diazinon concentration of 1000 µg/L was selected for next stage.
3.4. Effect of alive and dead probiotic bacteria on diazinon reduction (fourth stage)
In this stage the effect of alive and dead L. acidophilus on diazinon degradation was compared during 48 h fermentation. Table 4 shows the chemical and microbial changes of apple juice samples after 48 h fermentation at 37°C.
Table 4
Chemical and microbial changes of apple juice samples in the fourth stage*
Treatment**
|
pH
|
Total acidity (g/100 g)
|
Reducing sugar content (g/100 g)
|
Total sugar content (g/100 g)
|
Viability of probiotic (CFU/mL)
|
Diazinon reduction (%)
|
B
|
4.06 ± 0.01a
|
0.28 ± 0.26a
|
11.86 ± 0.06a
|
13.82 ± 0.06a
|
-
|
-
|
B-1
|
4.06 ± 0.01a
|
0.28 ± 0.25a
|
12.13 ± 0.06b
|
13.86 ± 0.06a
|
-
|
16.26a
|
A-9-1
|
3.94 ± 0.01b
|
0.30 ± 0.35ab
|
11.36 ± 0.05c
|
12.89 ± 0.05b
|
10.20 ± 0.05a
|
57.97b
|
A-9
|
3.86 ± 0.01c
|
0.44 ± 0.37c
|
10.96 ± 0.02d
|
12.13 ± 0.02c
|
10.83 ± 0.01b
|
-
|
A-9D-1
|
3.99 ± 0.01b
|
0.29 ± 0.42ab
|
11.67 ± 0.02e
|
13.29 ± 0.02d
|
-
|
27.74c
|
A-9D
|
3.93 ± 0.02b
|
0.35 ± 0.26b
|
11.61 ± 0.02e
|
13.57 ± 0.02e
|
-
|
-
|
*Each value in the table is the mean ± standard deviation (SD) of three trials. Different letters in each column indicate a statistically significant difference (P < 0.05). |
** B: control sample, 1: 1000 µg/L of spiked diazinon, A: L. acidophilus, 9: Primary probiotic population (9 log CFU/mL), D: dead probiotic bacteria (killed by heat). |
As depicted in Table 4, the apple juice treatments including heat-killed L. acidophilus (A-9D and A-9D-1) had slower chemical changes than samples containing alive probiotic bacteria i.e. A-9 and A-9-1, after 48 h fermentation. Ding and Shah (2008) reported that the dead probiotic bacteria could also release enzymes for hydrolyzing sugars in the fruit juice, consequently increasing the acidity and decreasing the pH and sugar content although it is lower than live cells (Ding & Shah, 2008) which is in good consistence with our study. Microbiological analysis of A-9 and A-9D-1 samples indicated the absence of any viable probiotic bacteria (data not shown). These results show that bacterial viability is a prerequisite for diazinon removal via enzyme production. However, the reduction percent in diazinon concentration by dead probiotic bacteria (27.74 %) revealed that probiotic bacteria could also reduce diazinon via binding capacity to this pesticide.
Generally, pesticide degradation is done either via degradation or absorption mechanisms. Several groups of microbial enzymes, including carboxylesterases, phosphatases, phosphotriesterases, organophosphorus hydrolases, may facilitate degradation of organophosphate pesticides like diazinon via the hydrolysis of phosphoric acid esters. Both acid and alkaline phosphatases may degrade organophosphate pesticides by hydrolyzing the C-O-P linkage of a wide variety of phosphate esters (Mohammadi et al., 2020; Y.-H. Zhang et al., 2014). Regarding to higher diazinon reduction percent in A-9-1 samples (57.97 %) than that in A-9D-1 samples (27.74 %), it could be concluded that 27.74 % and 30.23 % of diazinon reduction percent may be attributed to absorption and degradation mechanisms, respectively.
3.5. Effect of fermentation times on diazinon reduction (fifth stage)
Fermentation is a promising biological process, and decrease in pesticide residues through this procedure has been continuously investigated in several food commodities and buffer systems (Bo et al., 2011; T. Đorđević et al., 2013 a; T. M. Đorđević et al., 2013 b; KUMRAL et al., 2020). Some microorganisms are capable of utilizing pesticides as a carbon and phosphorus source and it has been observed that enzymes isolated from them are able to detoxify diazinon. They may also decrease diazinon through binding pesticides via agents existed in their cell wall (Cho et al., 2009; T. Đorđević et al., 2013 a; T. M. Đorđević et al., 2013 b; Mohammadi et al., 2020; Wang et al., 2016).
In this stage the effect of different fermentation times on diazinon reduction percent was investigated. Table 5 shows the chemical and microbial changes of apple juice samples during 72 h fermentation at 37°C with 24 h intervals.
Table 5
Chemical and microbial changes of apple juice samples in the fifth stage*
Parameter
|
Treatment
|
Fermentation times (h)
|
24
|
48
|
72
|
pH
|
B
|
4.08 ± 0.08aA
|
4.06 ± 0.01aA
|
4.05 ± 0.01aA
|
|
B-1
|
4.07 ± 0.08aA
|
4.06 ± 0.01aA
|
4.06 ± 0.01aA
|
|
A-9-1
|
4.02 ± 0.08abA
|
3.94 ± 0.02bB
|
3.90 ± 0.02cB
|
|
A-9
|
3.97 ± 0.02bA
|
3.86 ± 0.08cB
|
3.84 ± 0.01cB
|
Total acidity (g/100 g)
|
B
|
0.28 ± 0.20aA
|
0.28 ± 0.46aA
|
0.28 ± 0.32aA
|
|
B-1
|
0.28 ± 0.25aA
|
0.28 ± 0.26aA
|
0.29 ± 0.38aA
|
|
A-9-1
|
0.29 ± 0.27abA
|
0.30 ± 0.38aA
|
0.31 ± 0.32aA
|
|
A-9
|
0.35 ± 0.32bA
|
0.44 ± 0.50bB
|
0.45 ± 0.32bC
|
Reducing sugar content (g/100 g)
|
B
|
11.86 ± 0.01aA
|
11.86 ± 0.02aA
|
11.80 ± 0.01aA
|
|
B-1
|
12.00 ± 0.05aA
|
11.93 ± 0.03aA
|
11.86 ± 0.02aB
|
|
A-9-1
|
11.67 ± 0.02bA
|
11.36 ± 0.02bB
|
11.25 ± 0.02bC
|
|
A-9
|
11.36 ± 0.02cA
|
10.64 ± 0.20cB
|
9.47 ± 0.10cC
|
Total sugar content (g/100 g)
|
B
|
13.82 ± 0.05aA
|
13.75 ± 0.02aA
|
13.64 ± 0.05aB
|
|
B-1
|
13.98 ± 0.07bA
|
13.82 ± 0.15aB
|
13.75 ± 0.02aB
|
|
A-9-1
|
13.43 ± 0.07cA
|
12.91 ± 0.06bB
|
12.80 ± 0.05bC
|
|
A-9
|
12.87 ± 0.07dA
|
11.98 ± 0.30bB
|
11.44 ± 0.18cC
|
Viability of probiotic (CFU/mL)
|
B
|
-
|
-
|
-
|
|
B-1
|
-
|
-
|
-
|
|
A-9-1
|
9.44 ± 0.09aA
|
10.21 ± 0.06aB
|
10.23 ± 0.05aB
|
|
A-9
|
9.82 ± 0.08bA
|
10.82 ± 0.02bB
|
10.85 ± 0.04bB
|
Diazinon reduction (%)
|
B
|
-
|
-
|
-
|
|
B-1
|
5.16aA
|
15.36aB
|
20.13aC
|
|
A-9-1
|
22.57bA
|
58.70bB
|
64.37bC
|
|
A-9
|
-
|
-
|
-
|
*Each value in the table is the mean ± standard deviation (SD) of three trials. Different lower and upper letters in each column and row, respectively indicate a statistically significant difference (P < 0.05). |
** B: control sample, 1: 1000 µg/L of spiked diazinon, A: L. acidophilus, 9: Primary probiotic population (9 log CFU/mL). |
As shown in Table 5, the pH, reducing sugar content and total sugar content decreased with increasing fermentation time while, probiotic viability and diazinon reduction percent increased.
The pH value of samples containing L. acidophilus (A-9) and samples containing L. acidophilus and 1000 µg/L spiked diazinon (A-9-1) decreased from 4.08 to 3.84 and 3.90, respectively which are significantly (p < 0.05) lower than control samples (B and B-1) in all fermentation times. The total acidity of A-9 and A-9-1 treatments increased from 0.28 g mallic acid/100 g apple juice to 0.45 and 0.31, respectively which are significantly (p < 0.05) higher than control samples (B and B-1) in all fermentation times. The reducing sugar content of A-9 and A-9-1 treatments decreased from 12 g /100 g apple juice to 9.47 and 11.25, respectively which are significantly (p < 0.05) lower than control samples (B and B-1) in all fermentation times. The total sugar content of A-9 and A-9-1 treatments also decreased from 14.09 g /100 g apple juice to 11.44 and 12.80, respectively which are significantly (p < 0.05) lower than control samples (B and B-1) in all fermentation times. In the case of probiotic viability, it is observed that the viability of L. acidophilus in A-9 and A-9-1 treatments increased from 9 log CFU/mL to 10.85 and 10.23 log CFU/mL, respectively. The diazinon reduction percent by L. acidophilus was calculated 22.57, 58.70 and 64.37 % after 24, 48 and 72 h fermentation, respectively.
As it could be detected in Table 5, the majority of noted changes observed in probiotic population, pH, total acidity, sugar consumption and diazinon reduction percent, happened in the period between hours 24 to 48 of the fermentation. Further extension of the fermentation process (from 48 to 72 h) the changes were slower but significant (p < 0.05) except in the case of pH value. It is obvious that with increasing fermentation time, diazinon reduction percent occurred by L. acidophilus increased significantly (p < 0.05). Thus, 72 h fermentation time was chose for the final stage.
In a similar study, Cho et al. (2009) reported that with increasing fermentation time for kimchi production, the degradation content of organophosphorus insecticide chlorpyrifos increased significantly. They reported that lactic acid bacteria degraded 83.3% of chlorpyrifos until 72 h and degraded it completely by 216 h (Cho et al., 2009).
3.6. Effect of refrigerated storage on diazinon reduction (sixth stage)
In the final stage, the effect of 28 days of cold storage on diazinon reduction percent of fermented apple juice samples was investigated. Table 6 shows the chemical and microbial changes of fermented apple juice samples during 28 days of cold storage at 4°C with 7 days intervals.
Table 6
Chemical and microbial changes of apple juice samples in the sixth stage*
Parameter
|
Treatment**
|
Refrigerated storage times (day)
|
0
|
7
|
14
|
21
|
28
|
pH
|
B
|
4.05 ± 0.01aA
|
4.05 ± 0.01aA
|
4.04 ± 0.01aA
|
4.03 ± 0.01aA
|
4.02 ± 0.02aA
|
|
B-1
|
4.06 ± 0.01aA
|
4.05 ± 0.01aA
|
4.06 ± 0.01aA
|
4.03 ± 0.01aA
|
4.03 ± 0.02aA
|
|
A-9-1
|
3.91 ± 0.02bA
|
3.90 ± 0.02bA
|
3.87 ± 0.01bA
|
3.76 ± 0.01bB
|
3.62 ± 0.04bC
|
|
A-9
|
3.88 ± 0.07bA
|
3.84 ± 0.03bAB
|
3.80 ± 0.09bB
|
3.70 ± 0.08bC
|
3.64 ± 0.01bC
|
Total acidity (g/100 g)
|
B
|
0.28 ± 0.28aA
|
0.29 ± 0.39aA
|
0.29 ± 0.38aA
|
0.29 ± 0.39aA
|
0.20 ± 0.38aA
|
|
B-1
|
0.29 ± .0.32aA
|
0.29 ± 0.18aA
|
0.29 ± 0.25aA
|
0.30 ± 0.38aA
|
0.31 ± 0.30aA
|
|
A-9-1
|
0.31 ± 0.14aA
|
0.36 ± 0.32aAB
|
0.42 ± 0.28aB
|
0.53 ± 0.38aC
|
0.63 ± 0.25aD
|
|
A-9
|
0.45 ± 0.38bA
|
0.50 ± 0.25bAB
|
0.56 ± 0.38bBC
|
0.59 ± 0.38bC
|
0.66 ± 0.52bD
|
Reducing sugar content (g/100 g)
|
B
|
12.00 ± 0.03aA
|
11.93 ± 0.02aB
|
11.93 ± 0.01aB
|
12.00 ± 0.01aB
|
12.00 ± 0.02aB
|
|
B-1
|
11.86 ± 0.02aA
|
11.8 ± 0.01bA
|
11.73 ± 0.01bB
|
11.86 ± 0.05aB
|
11.86 ± 0.02aB
|
|
A-9-1
|
11.25 ± 0.02bA
|
11.13 ± 0.01cB
|
11.07 ± 0.02cBC
|
11.02 ± 0.02bC
|
10.43 ± 0.15bC
|
|
A-9
|
9.51 ± 0.13cA
|
9.27 ± 0.14dB
|
9.15 ± 0.11dC
|
8.88 ± 0.05cD
|
8.47 ± 0.18cD
|
Total sugar content (g/100 g)
|
B
|
13.71 ± 0.10aA
|
13.6 ± 0.10aA
|
13.64 ± 0.15aA
|
13.86 ± 0.07aB
|
14.16 ± 0.10aC
|
|
B-1
|
13.82 ± 0.05aA
|
13.71 ± 0.10aABC
|
13.64 ± 0.05aB
|
13.80 ± 0.15aC
|
14.16 ± 0.10aD
|
|
A-9-1
|
12.70 ± 0.03bA
|
12.65 ± 0.02bB
|
12.63 ± 0.05aB
|
12.55 ± 0.02bBC
|
12.50 ± 0.02bC
|
|
A-9
|
11.42 ± 0.05cA
|
11.36 ± 0.11cAB
|
11.33 ± 0.02bB
|
11.30 ± 0.01cB
|
11.14 ± 0.25cC
|
Viability of probiotic (CFU/mL)
|
B
|
-
|
-
|
-
|
-
|
-
|
|
B-1
|
-
|
-
|
-
|
-
|
-
|
|
A-9-1
|
10.23 ± 0.04aA
|
9.35 ± 0.01aB
|
9.25 ± 0.02aC
|
8.27 ± 0.01aD
|
7.13 ± 0.02aE
|
|
A-9
|
10.85 ± 0.02bA
|
9.96 ± 0.06bB
|
9.67 ± 0.05bC
|
8.92 ± 0.02bD
|
7.88 ± 0.06bE
|
Diazinon reduction (%)
|
B
|
-
|
-
|
-
|
-
|
-
|
|
B-1
|
19.66aA
|
38.40aB
|
44.96aC
|
49.86aD
|
58.73aE
|
|
A-9-1
|
64.97bA
|
49.60bB
|
47.64bC
|
45.04bD
|
41.27bE
|
|
A-9
|
-
|
-
|
-
|
-
|
-
|
*Each value in the table is the mean ± standard deviation (SD) of three trials. Different lower and upper letters in each column and row, respectively indicate a statistically significant difference (P < 0.05). |
** B: control sample, 1: 1000 µg/L of spiked diazinon, A: L. acidophilus, 9: Primary probiotic population (9 log CFU/mL). |
As depicted in Table 6 the pH of probiotic apple juice samples (A-9-1 and A-9) decreased significantly during storage period from 3.91 and 3.88 to 3.62 and 3.64, respectively. However, the total acidity of A-9- and A-9 treatments increased significantly from 0.31 and 0.45 to 0.63 and 0.66 g mallic acid/100 g apple juice, respectively which is in good agreement with previous works (Ding & Shah, 2008; Pimentel et al., 2015). The reducing and total sugar concentration of probiotic apple juice samples (A-9 and A-9-1) showed a slight significant decline (p < 0.05) indicating sugar consumption for their growth and activity. Fortunately, the probiotic viability of apple juice samples decreased around 3 logarithmic cycle probably due to low temperature inappropriate for growth of L. acidophilus as well as post acidification of this strain. It is well-known fruit and vegetable juices are proper matrixes for delivery of probiotics because of their level of sugar, vitamins, antioxidant and minerals (Zoghi et al., 2017). Our results showed that, the probiotic viability of A-9 and A-9-1 samples decreased from 10.85 and 10.23 to 7.88 and 7.13 log CFU/mL, respectively. When it comes to diazinon concentration, this value for control sample (B-1) and probiotic samples (A-9-1) declined from 803 to 412 µg/L and 153 to 0 µg/L, respectively at the end of storage time. It is worthy to note that 64.97, 49.60, 47.64, 45.04 and 41.27 % of diazinon reduction was related to probiotic effect via degradation or binding mechanism at day 0, 7, 14, 21 and 28 of cold storage, respectively. Results of this section are in good accordance with Zhoghi et al. (2017) study who investigated the effects of 6 weeks cold storage on patulin reduction by probiotic bacteria (Zoghi et al., 2017).