Impact of Wort pH (LF3) on Lactobacillus Fermentation Performance

MSc Project > Results & Dicussion: Impact of Wort pH (LF3)

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.

In LF3 the effect of pH on lactic fermentation was investigated by adjusting the pH of the base wort using lactic acid, prior to inoculation with L. brevis WLP672. The specific fermentation parameters used were outlined in Table 1.

Fig. 10. Change in pH (A), TA (g/L of LA) (B) and acid production (g/L of LA) (C) during lactic fermentation (LF3) by L. brevis WLP672 at 30°C in 1.042 SG worts with pH 3.76 (), pH 4.25 () and pH 4.84 (). The 30°C, 1.043 SG data from Temperature (LF1), pH 5.03 () is included for comparison. Error bars represent SD.

Results

Despite the large initial pH range (pH 3.76-5.03), within 24 h the difference between values was just pH 0.29 and by 72 h all fermentations had converged below pH 3.50, within a small range of pH 3.40-3.48 (Fig. 10A). The pH 4.84 wort attained the lowest pH at 72 h (3.40 ± 0.01) in addition to reaching the highest TA (5.50 ± 0.33 g/L of LA). Factoring in the different starting acidities (Fig. 10C), at 72 h the pH 4.84 wort still yielded the highest acid production (4.45 ± 0.33 g/L of LA), however at 48 h it had both the lowest TA and acid production. The results for pH 4.84 wort appeared to be quite unusual, especially considering the comparably large standard deviations in TA at both 48 and 72 h of 0.37 and 0.33 respectively (Fig. 10B). To further compound this irregularity the acid production (Fig. 10C) after 72 h in pH 3.76, pH 4.25 and pH 5.03 worts fell within a narrow range of 0.23 g/L of LA, with the pH 5.03 wort achieving a fractionally greater amount (3.67 ± 0.16 g/L of LA) than the pH 3.76 wort (3.62 ± 0.12 g/L of LA).

In contrast to many of the other lactic fermentations in this study, the increase in acid production in LF3 during the first 24 h was not as conspicuous. While the first 24 h still gave rise to the largest TA increases, for both pH 4.25 and pH 3.76 worts the difference from 0-24 h compared with 24-48 h was very slight. This trend was most pronounced for the pH 3.76 wort which showed an almost linear increase in TA from 1.55 ± 0.02 g/L to 3.17 ± 0.03 g/L of LA after 24 h to 4.58 ± 0.05 g/L of LA after 48 h.

Discussion

The results from LF3 demonstrated that regardless of the starting pH of the wort, within the range tested here, very similar pH values were reached after 72 h fermentations. The same trend was also largely true for acid production, aside from some peculiar results in pH 4.84 wort fermentations. Considering the importance of both pH and organic acid concentration in LAB inhibition (see Wort SG discussion) it was anticipated that a more acidic starting environment would ultimately hamper acid production. It was therefore quite remarkable that the acid production from the lowest pH 3.76 wort fermentations was comparable to the higher pH worts after 72 h. Narendranath et al. found that for four different Lactobacillus strains, lowering the starting pH (from pH 5.5 to 4.0) of the medium in all cases resulted in a reduced growth rate and lower lactic acid production.1 Peyer et al. also reported similar results for three LAB strains with growth dropping abruptly at pH 3.9 in malt wort by 63%, 81% and 94% when compared with growth in pH 5.9 wort.2

It’s worth noting that L. brevis is one of the leading beer spoilage microorganisms and thus able to grow well in typical beer pH of approximately pH 3.8-4.7.3, 4 Some LAB have also demonstrated a tolerance of extremely low pH conditions, with Ramos et al. documenting seven L. brevis strains that were found to grow after 3 h on pH 2.0 media.5 It’s feasible that the L. brevis WLP672 strain has a high intrinsic pH tolerance but it could also have developed greater resistance as a consequence of being initially propagated and stored in malt wort prior to inoculation. Another factor may have been that compared with the significant separation of initial wort pHs, the actual difference between the organic acid concentrations was not substantial. The starting TA of the pH 3.76 wort was 1.55 ± 0.03 g/L of LA, only 0.51 g/L of LA on average greater than the pH 4.84 wort, which had the lowest (1.04 ± 0.01 g/L of LA).

Based on the increasingly linear acid production as the starting wort pH was reduced, it’s probable that a degree of bacterial growth and/or acid production inhibition was taking place. As growth was not measured during these fermentations and it has been well documented that LAB acid production can continue under conditions that inhibit growth, it’s not possible without further work to quantify the effect.6 Despite the relatively small differences in starting wort TA, as lactic acid has a Pka = 3.86 both the pH 4.25 and pH 3.76 worts would have considerably higher concentrations of the undissociated acid form at the beginning of fermentation, known to inhibit LAB growth. Therefore the effect that lactic acid might have had on the growth and acid production response cannot be ignored and had an alternative acid been used for pH adjustment the outcome might have been different. It was nonetheless evident that the initial wort pH and organic acid concentration were not sufficient, even in the pH 3.76 wort, to severely affect L. brevis WLP672 acid production. For this strain the threshold for significant inhibition must therefore exist at a lower pH and/or higher acid concentration.

Considering the similarities between the pH 4.84 and pH 5.03 worts it was anticipated that the fermentation results would be close. Both worts had comparable starting organic acid concentrations, a small difference in pH and for the first 24 h, almost identical pH and TA fermentation profiles. It’s not clear why there was then such a marked deviation and variability in the measured TA for the pH 4.84 wort fermentations at 48 h and 72 h. It should be emphasised that the worts were nevertheless different, the pH 5.03 wort having a slightly higher SG and being produced and used for LF1 lactic fermentations at a different time. Yeast contamination was also not suspected as the average AA of 4.276 ± 0.94 % after 72 h for the pH 4.84 wort fermentations was comparable with other findings (see Problems with Yeast Contamination).

References

  1. Narendranath, N. V., Power, R. (2005) Relationship between pH and medium dissolved solids in terms of growth and metabolism of lactobacilli and Saccharomyces cerevisiae during ethanol production, Appl. Environ. Microbiol., 71, 2239-2243. https://doi.org/10.1128/AEM.71.5.2239-2243.2005
  2. Peyer, L. C., Bellut, K., Lynch, K. M., Zarnkow, M., Jacob, F., De Schutter, D. P., Arendt, E. K. (2017) Impact of buffering capacity on the acidification of wort by brewing relevant lactic acid bacteria, J. Inst. Brew., 123, 497-505. https://doi.org/10.1002/jib.447
  3. Suzuki, K. (2011) 125th Anniversary review: Microbiological instability of beer caused by spoilage bacteria, J. Inst. Brew., 117, 131-155. https://doi.org/10.1002/j.2050-0416.2011.tb00454.x
  4. Garofalo, C., Osimani, A., Milanović, V., Taccari, M., Aquilanti, L., Clementi, F. (2015) The occurrence of beer spoilage lactic acid bacteria in craft beer production, J. Food Sci., 80, M2845-M2852. https://doi.org/10.1111/1750-3841.13112
  5. Ramos, C. L., Thorsen, L., Schwan, R. F., Jespersen, L. (2013) Strain-specific probiotics properties of Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus brevis isolates from brazilian food products., Food Microbiol., 36, 22-29. https://doi.org/10.1016/j.fm.2013.03.010
  6. Passos, F. V., Fleming, H. P., Ollis, D. F., Hassan, H. M., Felder, R. M. (1993) Modeling the specific growth rate of Lactobacillus plantarum in cucumber extract, Appl. Microbiol. Biotechnol., 40, 143-150.

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