Investigating the Relationship Between pH, Acid Production and Apparent Attenuation During Lactic Fermentation

MSc Project > Results & Dicussion: pH, TA & Attenuation

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.

Summary of lactic fermentation behaviour

During the fermentation trials LF1-4, conducted with an isolated L. brevis WLP672 strain, the largest drop in pH and increase in Total Acidity (TA) was almost always seen during the first 24 h. This corresponded to the growth phase, obseved while monitoring the bacterial growth behaviour. Considering as well that in general for most variables tested, the pH drop and acid production increase were more moderate between 24 – 72 h, it seems hightly likely that the stationary phase was reached at around 24 h for this strain. Peyer et al. likewise found that all four LAB strains used in their study, including L. brevis R2∆, had reached peak cell populations after 24 h in malt wort, whereas Nsogning et al. reported that L. brevis Lb.986 reached a maximum cell concentration at 48 h in wort produced from DME.1, 2

Both these studies also documented the ability of some LAB strains to continue to accumulate organic acids during the stationary and even death phases. In this work an average of 44 ± 14 % of the total acid production (measured at 72 h) took place between 24-72 h in LF1-4 fermentations, compared with 18 ± 8 % between 48-72 h. If the stationary phase for L. brevis WLP672 was reached after approximately 24 h then a sizeable proportion of the acid production occured after the growth phase. This LAB characteristic appears to be strain dependent with the rate of accumulation generally reduced after the exponential growth phase. In a study of Lactobacillus plantarum fermentation in cucumber juice, Passos et al. concluded that the amount of acid produced following the growth phase was strongly affected by the medium composition and reported a maximum of 51 % of lactic acid production occuring after growth had ceased.3

pH and Total Acidity

For L. brevis WLP672 the average 72 h pH for fermentation trials LF1-4 was 3.51 ± 0.09 while the lowest recorded value was pH 3.40 ± 0.01 for pH 4.84 wort (see Wort pH). These findings are similar to those of Peyer et al. who reported that both L. brevis R2∆ and L. brevis L1105 were able to grow to pH 3.57 and pH 3.55 after 48 h in 6% (w/w) malt wort.4

Fig. 12. Change in pH as a function of TA during lactic fermentation by L. brevis WLP672 for all variables examined in LF1-4. Fermentation durations of 24 h (), 48 h (), and 72 h (). Each point is the mean of independent fermentations performed in triplicate.

The drop in pH during lactic acid fermentation was consistent with the accumulation of organic acids as measured by TA (Fig. 12). Regression analysis revealed that TA had a statistically significant (p< 0.0001) linear relationship with pH (R2 = 0.49), when treating all the LF1-4 results as a single data set. It’s nonetheless clear that a given pH could have a wide range of TA values (Fig. 12). The combined mean pH after 48 h for all LF1-4 fermentation samples was pH 3.66 ± 0.13 with an average TA of 4.16 ± 0.62 g/L of LA, while at 72 h the mean pH was pH 3.51 ± 0.09 with a corresponding TA of 4.82 ± 0.67 g/L of LA. The large standard deviations for TA illustrate the greater dispersion of acid values for comparatively small pH ranges. Given the number of different fermentation parameters represented by these values, variability was to be expected. The point however, is that pH measurement during lactic fermentation of malt wort is a relatively poor indicator of organic acid concentration. That said, for all the variables examined in this study, over the 72 h time period considered, a longer duration of lactic fermentation resulted in a lower pH and higher TA (Fig. 12).

Apparent attenuation and organic acid production

Fig. 13. Change in AA % as a function of acid production during lactic fermentation by L. brevis WLP672. All time points from 24-72 h are included for fermentation variables of temperature LF1 (), wort SG LF2 (), wort pH LF3 () and bacterial pitch rate LF4 (). Each point is the mean of independent fermentations performed triplicate.

The amount of acid produced by LAB during lactic fermentation was found to increase almost linearly with the AA % for fermentations with variables of temperature LF1 (R2 = 0.95) and wort pH LF3 (R2 = 0.93). A weaker relationship was found with varying wort SG LF2(R2 = 0.64) and bacterial pitch rate LF4 (R2 = 0.71). As discussed previously, the change in AA % for the bacterial pitch rate LF4 fermentations was unusually large relative to the other fermentations. Therefore, excluding the LF4 results, a reasonably strong correlation was found between the combined AA % and acid production for LF1-3 (R2 = 0.80). LAB utilise sugars in wort for growth and metabolism and as organic acids, particularly lactic acid, are the main products of fermentation, it follows that as sugar consumption increases so to will the acid yield.

References

  1. Charalampopoulos, D., Pandiella, S. S., Webb, C. (2002) Growth studies of potentially probiotic lactic acid bacteria in cereal based substrates, J. Appl. Microbiol., 92, 851-859. https://doi.org/10.1046/j.1365-2672.2002.01592.x
  2. Peyer, L. C., Zannini, E., Jacob, F., Arendt, E. K. (2015) Growth study, metabolite development, and organoleptic profile of a maltbased substrate fermented by lactic acid bacteria, J. Am. Soc. Brew. Chem., 73, 303-313. https://doi.org/10.1094/ASBCJ-2015-0811-01
  3. Passos, F. V., Fleming, H. P., Ollis, D. F., Felder, R. M., McFeeters, R. F. (1994) Kinetics and modeling of lactic acid production by Lactobacillus plantarum, Appl. Environ. Microbiol., 60, 2627-2636
  4. Peyer, L. (2017) Lactic acid bacteria fermentation of wort as a tool to add functionality in malting, brewing and novel beverages, School of food and nutritional sciences, University College Cork, Cork, Ireland.

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