Challenge tests were done to determine the ability of the selected Lb. plantarum BFE 5092 and Lb.
fermentum BFE 6620 starter cultures to inhibit the growth of the pathogenic bacteria Salmonella Enteritidis S489 and Listeria monocytogenes SLCC 8210 serovar 1/2a. The fermentation of nightshade leaves was performed in 1000 ml beakers with 3 % sugar and 3 % salt solution (section 2.2.5). The experiment was performed in four batches, the first batch served as a control (no starters or pathogens were inoculated), the second batch was inoculated with the starter cultures Lb.
plantarum BFE 5092 and Lb. fermentum BFE 6620 at 107 cfu/ml, while in the third batch starter cultures were inoculated at 107 cfu/ml together with the 2 pathogenic bacteria Salmonella Enteritidis S489 and Listeria monocytogenes SLCC 8210 each at 103 cfu/ml and the final batch was inoculated with only pathogens each at 103 cfu/ml. The batches were fermented for 144 h at 25 oC.
98 The pH and microbial enumeration was performed during the entire fermentation as described in section 2.2.5.3.
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
0 h 24 h 48 h 72 h 144 h
pH values
Time
pH (uninoculated batch) pH (with starter cultures) pH (starter cultures with pathogens) pH (pathogens without starters)
Figure 3.23:Mean pH values from three nightshade fermentation uninoculated (blue line), inoculated only with starter cultures (Lb. plantarum BFE 5092 and Lb. fermentum BFE 6620 (red line) inoculated with starters cultures in
combination with pathogens (green line) and only pathogens without starters (purple line) during 144 h of fermentation in 1000 ml beakers at MRI, Karlsruhe, Germany. The standard deviation is indicated.
The fermentation of the all the batches had an initial pH of ca. 7.1 to 7.2. However, after 24 h of fermentation, the batches inoculated either with only the starter cultures, or inoculated with the starter cultures together with the pathogens, showed a sharp decrease of pH to below 3.9 (Fig.3.23).
After 24 h, these pH values decreased further slowly and remained stable after ca. 72 h until 144 h, reaching a final pH of 3.48 for the batches inoculated with starter culture and pathogens, or a final pH of 3.53 (Fig.3.23) for the batches inoculated with only the starter cultures. However, in both the control (natural fermentation without starters inoculated) and the batches inoculated only with the pathogens, there was only a relatively slow reduction in the pH. Nevertheless, in the batch inoculated with only the pathogens, the pH still decreased to pH 4.9 and 4.5 after 24 h and 48 h, respectively. For the control batches, which were not inoculated with either starter bacteria or pathogens, the pH decreased only to pH 6.4 after 24 h. As the fermentation progressed, the
99 pathogens inoculated batch reached a final pH of 4.1 after 144 h, while the control batch without inoculated bacteria reached a final pH of 4.8 (Fig.3.23).
3.6.1 Microbial enumeration during nightshade fermentation in challenge tests with foodborne pathogens
Microbial enumeration was carried out to study the progress of fermentation of nightshade and the growth of the different starter bacteria and pathogens during the fermentation using different selective culturing media and incubation conditions as described in section 2.2.5.3. The fermentation trials were conducted in triplicates on separate occasions from different nightshade harvests. The mean yeast/moulds and bacterial counts from control (natural fermentation) and starter cultures inoculated batches are shown in Fig.3.24.
0 1 2 3 4 5 6 7 8 9 10 11 12
0 h 24 h 48 h 72 h 144 h
Log10 cfu/ml
Time
LAB (With starters ) LAB (Without starters ) TAC (With starters) TAC (Without starters) TEC (with starters) TEC (without starters) Y+M (With starters) Y+M (Without starters)
Figure 3.24: The mean lactic acid bacteria (LAB) counts, total aerobic mesophilic plate counts (TAC), total enterobacterial counts (TEC) and yeast and moulds (Y+M) counts from triplicate nightshade fermentations uninoculated with starter cultures (control) and inoculated with Lb. plantarum BFE 5092 and Lb. fermentum BFE 6620 at MRI, Karlsruhe, Germany for 144 h in 1000 ml beakers. The standard deviation is indicated.
100 The results showed that at the beginning of the fermentation, the mean LAB count in the starter culture inoculated batches was approximately 107 cfu/ml. The count increased rapidly to about 109 cfu/ml after 24 h, and remained at this approximate level for up to 144 h (Fig.3.24). In the control batches without inoculated starter bacteria, the mean LAB count was about 101 cfu/ml after 0 h, and increased to approximately 103 cfu/ml after 24 h. It further increased to over 105 cfu/ml and remained in this range until 144 h. The mean total aerobic mesophilic colony counts on Std.I agar from the batches that were inoculated with starter cultures showed almost similar mean counts compared to the LAB counts (Fig.3.24), suggesting that the inoculated LAB starter cultures most likely constituted the highest number of growing bacteria on Std.I agar plates. However, in the uninoculated controls, the mean total aerobic count was about 101 cfu/ml after 0 h and increased to about 104 cfu/ml after 24 h and reached almost similar high levels of approximately 108 cfu/ml after 48 to 144 h fermentation as in the case with the starter inoculated batch (Fig.3.24). The mean counts of enterobacteria from the fermentation batch inoculated with starter culture were below 101 cfu/ml after 24 h and no bacteria were detected afterwards. In the uninoculated control batches, the mean enterobacterial count was less than 101 cfu/ml at 0 h and the count increased to approximately 102 cfu/ml after 24 h, after which it further increased to about 105 cfu/m after 48-72 h, before decreasing again to approximately 104 cfu/ml after 144 h (Fig.3.24). The mean yeast and moulds count were below 101 cfu/ml for all of the fermentation batches.
The mean bacterial counts from the batches with pathogens co-inoculated with starter cultures and control batches inoculated only with pathogens are shown in Figure 3.25. This experiment was performed to determine the role of selected LAB starter cultures to inhibit the growth of pathogenic bacteria such as L. monocytogenes and S. Enteritidis, thus ensuring the safety of the vegetables. The nightshade fermentation trials were co-inoculated with approximately 1 x 107 cfu/ml Lb. plantarum BFE 5092 and Lb. fermentum BFE 6620 and 1 x 103 cfu/ml Salmonella Enteritidis and L.
monocytogenes. The results in figure 3.25 show that at the beginning of the fermentation, the mean LAB count in the fermentation batches containing the starter culture which were co-inoculated with pathogens was approximately 1 x 107 cfu/ml. This mean rapidly increased to over 109 cfu/ml after 24 h and remained at this high level of ca. 109 cfu/ml up to 144 h of fermentation.
In the batch inoculated with pathogens only, the mean LAB count was much lower at about 102 cfu/ml after 0 h, and it increased to approximately 105 cfu/ml after 24 h and then remained in this range until 144 h of fermentation. The mean total aerobic mesophilic bacterial count on Std.I agar
101 from the fermentation batches inoculated with both starter cultures and the pathogens showed very similar mean counts when compared to the mean LAB counts (Fig.3.25), suggesting that the inoculated LAB starter cultures most likely were the highest number of bacteria growing on Std.I agar plates. However, in fermentation batches inoculated only with the pathogens, the mean total aerobic, mesophilic bacterial count was ca. 103 cfu/ml after 0 h and it increased to about 108 cfu/ml after 24 h, and remained at this high level cfu/ml for 48 to 144 h fermentation, and these counts were almost at similar levels as in the case with the fermentations inoculated with starter cultures together with pathogens (Fig.3.25).
0 1 2 3 4 5 6 7 8 9 10 11
0 h 24 h 48 h 72 h 144 h
Log10cfu/ml
Time
LAB (starters with pathogens) LAB (pathogens only) TAC (starters with pathogens) TAC (pathogens only) Salmonella (with starters) Salmonella (without starters) Listeria (with starters) Listeria (without starters) TEC (starters with pathogens) TEC (pathogens only) Y+M (starters with pathogens) Y+M (pathogens only)
Figure 3.25: Lactic acid bacteria (LAB) counts, total aerobic mesophilic colony counts (TAC), Salmonella counts, Listeria counts and total enterobacteria counts (TEC) from triplicate nightshade fermentation with pathogens and starter cultures Lb. plantarum BFE 5092 and Lb. fermentum BFE 6620 at MRI, Karlsruhe, Germany for 144 h in 1000 ml beakers. The standard deviation is indicated.
The results also showed that in the batch with starter cultures co-inoculated with pathogens, the mean number of Salmonella Enteritidis increased from approximately 103 cfu/ml after 0 h to about 106 cfu/ml after 24 h, after which it decreased by 4 logs to about 102 cfu/ml after 48 h. It further decreased to log 0.8 cfu/ml after 72 h. After 144 h, no viable cells could be detected either after
102 directly plating from nightshade fermentation brine or after enrichment (Fig.3.25). However, in the fermentation batch inoculated with pathogens only, the mean Salmonella Enteritidis count increased from 103 cfu/ml after 0 h to ca. 108 cfu/ml after 24 h, it then decreased to ca. 107 cfu/ml after 48 h it further reduced to ca. 105 cfu/ml after 72 h and reached about 103 after 144 h of fermentation (Fig.3.25).
The mean number of Listeria monocytogenes from fermentations co-inoculated with starter cultures and pathogens was approximately 103 cfu/ml after 0 h and remained at this level for up to 24 h of fermentation. After 48 h, no viable cells were detected from samples plated directly from nightshade fermentation brine, or after enrichment (Fig.3.25). However, the mean L.
monocytogenes count from fermentations only inoculated with pathogens increased from 103 cfu/ml after 0 h to ca. 108 cfu/ml after 24 h, remained at this high level up to 72 h, it then decreased to about 106 cfu/ml after 72 h, and reached ca. 104 cfu/ml after 144 h of fermentation (Fig.3.25). The mean enterobacterial count from fermentation with starter cultures co-inoculated with pathogens almost resembled the mean Salmonella Enteritidis count, suggesting that the majority of the bacteria on VRBD agar probably were Salmonella Enteritidis. However, the mean enterobacterial count from the batch inoculated with pathogens only increased from 103 cfu/ml after 0 h to 108 cfu/ml after 24 h, it further decreased by 1 log to 107 cfu/ml after 48 h then 105 cfu/ml after 72 h and reached 103 cfu/ml after 144 h fermentation (Fig.3.25).
3.7 Isolation of genomic DNA from fermentation brine and PCR amplification of 16S rRNA