MSc Project > Results & Dicussion: Impact of Bacteria Pitch Rate (LF4)
This post is part of a series detailing the findings of my MSc research project which looked at the effects of different fermentation parameters on wort souring with Lactobacillus. If you haven’t already, take a look at the MSc project page for a full overview.
The effect of different LAB pitch rates on lactic fermentation performance was studied in LF4 by using a range of L. brevis WLP672 inoculum sizes in malt wort. The specific fermentation parameters used were outlined in Table 1.
The results indicated that all inoculation rates used were capable of reaching similarly low pH values (Fig. 11A). At 72 h the highest pH was with an inoculation rate of 2×106 CFU/ml (3.57 ± 0.03) while the lowest was with 4×107 CFU/ml (3.47 ± 0.01), both a mere pH 0.1 apart despite a twenty fold difference in the size of their initial bacterial populations. While the differences were quite small, the pH values for 2×106 CFU/ml fermentations were the highest at all time points compared with the 4×107 CFU/ml which were the lowest. This trend continued for TA and acid production, with 2×106 CFU/ml yielding the lowest values at both 48 and 72h. Meanwhile the 4×107 CFU/ml fermentations produced the highest values at both 48 and 72 h, reaching a maximum TA at 72 h of 5.11 ± 0.11 g/L of LA (Fig. 11B) and acid production of 3.92 ± 0.11 g/L of LA (Fig. 11C). Oddly at 24 h, despite having almost identical pH values, fermentations with an inoculation rate of 1×107 CFU/ml resulted in the lowest TA (2.96 ± 0.09 g/L of LA) while 2×107 CFU/ml resulted in the highest (3.47 ± 0.09 g/L of LA). By 48 h the TA values for these two inoculation rates had converged and by 72 h were only 0.02 g/L of LA apart.
Within the range tested here, the general trend from 48-72 h was that the larger the inoculation rate, the faster the pH drop and the lower it’s final value. Although the TA measurements were slightly more convoluted, ultimately at both 48 and 72 h, the highest inoculation rate fermentation produced the most organic acid. At 72 h this amounted to 0.79 g/L of LA or 25.3% more organic acid present in the 4×107 CFU/ml fermentations than the lowest average TA which was for the smallest 2×106 CFU/ml inoculation rate.
The fundamental outcome from this trial was that varying the L. brevis WLP672 pitch rate affected the fermentation performance and of particular interest was that using a greater inoculum concentration increased both the rate and degree of souring. As no literature information could be found investigating the relationship between Lactobacillus inoculum size in barley malt wort to changes in acid production it’s difficult to compare and evaluate these findings. Similar trends have however been reported for other substrates fermented with LAB. One such example was a study by Gebremariam et al. that found an increase in lactic acid production with cell concentration during lactic fermentation of teff malt wort by Lactobacillus amylolyticus.1
The cause of the irregular TA values at 24 h for the 1×107 CFU/ml and 2×107 CFU/ml fermentations, when compared to the trend at 48 h and 72 h, is not entirely clear. Considering the 1×107 CFU/ml results it’s worth emphasising again that this set of fermentations were performed as part of the Temperature (LF1) trials, at a different time and using a different batch of wort. It’s possible, as alluded to previously (see Wort pH discussion), that a degree of bacterial adaptation might had occurred in the stock culture in the time between experiments. There may also have been other variables that weren’t accounted for such as differences in wort composition. This could have been due to manufacturing variations inherent in the dry malt extract used or perhaps through experimental factors such as the autoclave cooldown time, which wasn’t controlled.
The other peculiar trait of the LF4 fermentations was that the apparent attenuations were unusually high for all fermentations at every time point (see AA % vs Acid Production). At 72 h the LF4 fermentations had an average AA of 6.0 ± 0.1 %, compared with a combined 72 h AA % for LF1-3 of 3.65 ± 0.9 %. The consistency of AA % values across the LF4 samples implied there was a factor that had affected all fermentations uniformly, such as might be expected with a change in the wort composition or bacterial physiology. The absence of yeast was also confirmed with a microscope as a precaution. While there were certainly some abnormalities in this trial it’s believed the overall trend is still relevant, even if comparing the absolute values with other fermentations in this study might be problematic. This is because the LF4 fermentations used the same wort and were inoculated with equivalent ratios of the same bacterial culture.
- Gebremariam, M. M., Hassani, A., Zarnkow, M., & Becker, T. (2015). Investigation of fermentation conditions for teff (Eragrostis tef) malt-wort by Lactobacillus amylolyticus. LWT-Food Sci. Technol., 61, 164-171. https://doi.org/10.1016/j.lwt.2014.11.008