MSc Project > Results & Dicussion: Impact of Wort SG (LF2)
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 LF2 the effect of wort density or specific gravity (SG) on lactic fermentation was examined by fermenting worts of different density with L. brevis WLP672. The exact fermentation parameters were detailed in Table 1.
The effect of wort SG on lactic fermentation revealed a very similar overall pattern to the previous trial (LF1), where temperature was the independent variable. For all wort gravities the greatest pH drop and rise in organic acid production took place in the first 24 h. This was followed by more gentle pH decreases (Fig. 9A) and acid increases at 48 and 72 h (Fig. 9B). There was a clear correlation between increasing wort SG and both higher final pH and TA values. The 1.061 SG wort had the highest pH (3.65 ± 0.03) and TA (5.68 ± 0.12 g/L of LA) at 72 h (Fig. 9B). It did however also have the highest starting TA (1.66 ± 0.04 g/L of LA), which, when taken into consideration meant that the 1.049 SG wort fermentations produced slightly more acid at every time point (Fig. 9C). At 72 h the 1.049 SG wort had reached 4.22 ± 0.13 g/L of LA produced compared with 4.02 ± 0.12 g/L of LA for the 1.061 SG wort (Fig. 9C). Both the 1.031 SG and 1.043 SG wort fermentations achieved comparably low pH values after 72 h of 3.48 ± 0.01 and 3.48 ± 0.02 respectively (Fig. 9A), while the 1.031 SG resulted in the lowest yield of organic acids at all time points, reaching a maximum TA of 3.84 ± 0.24 g/L of LA at 72 h (Fig. 9C).
The results from LF2 showed that increasing wort SG translated into higher TA values but not necessarily a greater production of organic acids, as was the case with the 1.061 SG and 1.049 SG fermentations. Peyer et al. found a similar trend with L. brevis L1105 and L. brevis R2Δ, reporting that wort of increasing extract content (3, 6, 9 and 12% w/w) resulted in higher organic acid release.1 They also implicated a deficiency of essential nutrient(s) such as amino acids as a possible cause of the reduced acid production seen in the more dilute worts. No decline was however reported in acid production at higher extract contents. While it may be inconsequential, when comparing these results it’s worth noting that very different methods of acid detection were employed. Peyer et al. utilised HPLC to measure the concentrations of specific acids, whereas total acidity as used in this study, should be treated as an indicative determination of the total acid content.
LAB Nutrient Availability
LAB are known to be nutritionally demanding organisms, with organic nitrogen sources being particularly important due to the limited ability of many strains to synthesize their own amino acids.2 This also explains why many strains including L. brevis often show proteolytic behaviour.3 Fortunately all the main amino acids typically required by LAB are found in malt wort (L-glutamic acid, L-isoleucine, L-leucine and L-valine) and numerous studies have demonstrated the mediums ability to sustain LAB growth.4, 5, 6 Nsogning et al. however recently documented the exhaustion of a number of key amino acids including lysine, arginine and glutamic acid for various LAB during fermentation of 14°P (~ 1.057 SG) malt wort.7 They suggested that these deficiencies may have contributed to reduced LAB acid tolerance and cell growth.
Wort pH and buffering capacity
LAB are self inhibiting in a batch fermentation process due to the accumulation of acids and rapid lowering of pH. The mechanism of inhibition is largely attributed to the diffusion of organic acids across the bacterial membrane and particularly the undissociated forms of weak acids, which can later dissociate to release hydrogen ions (H+) inside a cell.8 This can lead to a lowering of the intracellular pH and disruption of normal cell functions, metabolism and enzyme activity, ultimately resulting in the complete inhibition of growth.9 A number of studies have concluded that lactic acid itself appears to be important in the inhibitory mechanism rather than just the effect of lowering the pH.10 It’s been suggested that the reason for this is the higher acid dissociation constant of lactic acid (PKa = 3.86) compared with many stronger acids, which significantly exacerbates the effect for LAB.11
The inhibition of LAB growth and acid production is related to both the abundance and relative proportion of the dissociated and undissociated forms of organic acids, which in turn is determined by the pH of the environment.12 As a consequence, the buffering capacity of the medium is an important factor to consider with respect to lactic fermentation. There are a number of buffering substances present in barley malt wort that enable the pH to resist change in response to small additions of acids or bases. It’s thought that a mixture of various buffers are responsible for the buffering capacity of wort and at the low pH of typical LAB fermentations the main buffering compounds of relevance are peptides, polypeptides and organic acids e.g. acetic, succinic and citric acid.13, 14 A high gravity wort will in general contain proportionally more nutrients and buffering compounds than an equivalent low gravity wort.15 It follows that as the wort gravity increased in LF2, the greater buffering capacity moderated the pH drop and delayed LAB self-inhibition. This in turn enabled more acid accumulation and a correspondingly higher final TA. The greater buffering capacity explains the consistently higher pH of the 1.061 SG fermentations despite simultaneously having the highest concentrations of organic acids (Fig. 9B). This hypothesis agrees with the findings of Peyer et al. who reported that for all LAB strains examined (Pediococcus acidilactici AB39, Lactobacillus amylovorus FST2.11 and Lactobacillus plantarum FST1.7), adding an external buffer to increase wort buffering capacity resulted in higher LA concentrations.16 A linear correlation (R2 = 0.990) was also found between the buffering capacity of the substrate and lactic acid released by L. plantarum FST1.7.
In a separate study Peyer et al. found a positive linear relationship (R2 = 0.996) between the growth of L. amylovorus FST2.11 and increasing wort extract up to 14% (w/w).17 Above extract values of 16% (w/w) growth was found to slow and eventually plateau between 18 and 20%. Narendranath et al. reported that in a liquid YPD medium, both growth and lactic acid production by LAB decreased linearly with increasing dissolved solids content.18 While not directly comparable to malt wort, it was suggested that osmotic stress of LAB resulted in the reduced growth observed at high dissolved solids concentrations. The effects of osmotic stress on LAB metabolism and physiology have been well documented with both increased external salt and sugar concentrations capable of inhibiting growth.19 As a wort density of 1.061 SG is approximately 15% (w/w), it seems probable, particularly with reference to the findings above that osmotic stress may have contributed to the lower observed acid production in fermentations using 1.061 SG wort when compared with 1.049 SG. This hints that osmotic stress may have became an increasingly limiting factor in the 1.061 SG wort fermentations and that it wasn’t offset by the potential benefits of a higher gravity wort, such as the greater buffering capacity or higher concentrations of essential nutrients.
The LF2 results demonstrated that the density of wort could have an effect on lactic fermentation performance. From the available research it seems plausible that a number of factors might have contributed to the observed fermentation behaviour, such as buffering capacity, essential nutrient concentration and osmotic stress.
- Peyer, L. C., Axel, C., Lynch, K. M., Zannini, E., Jacob, F., Arendt, E. K. (2016) Inhibition of Fusarium culmorum by carboxylic acids released from lactic acid bacteria in a barley malt substrate, Food Control, 69, 227-236. https://doi.org/10.1016/j.foodcont.2016.05.010
- Endo, A., Dicks, L. M. T. (2014) Physiology of the LAB, in Lactic acid bacteria biodiversity and taxonomy, (Holzapfel, W. H., Wood, B. J. B. Eds.) pp. 13–30, John Wiley & Sons, Ltd, Chichester, UK.
- Sasaki, M., Bosman, B. W., Tan, P. S. T. (1995) Comparison of proteolytic activities in various lactobacilli, J. Dairy Res., 62, 601- 610. https://doi.org/10.1017/S0022029900031332
- 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
- Patel, H. M., Pandiella, S. S., Wang, R. H., Webb, C. (2004) Influence of malt, wheat, and barley extracts on the bile tolerance of selected strains of lactobacilli, Food Microbiol., 21, 83-89. https://doi.org/10.1016/S0740-0020(03)00016-9
- 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
- Nsogning, S. D., Fischer, S., Becker, T. (2018) Investigating on the fermentation behavior of six lactic acid bacteria strains in barley malt wort reveals limitation in key amino acids and buffer capacity, Food Microbiol., 73, 245-253. https://doi.org/10.1016/j.fm.2018.01.010
- Piard, J. C., Desmazeaud M. (1991) Inhibiting factors produced by lactic acid bacteria. 1. Oxygen metabolites and catabolism end-products, Lait, 71, 525-541. https://doi.org/10.1051/lait:1991541
- Salmond, C. V., Kroll, R. G., Booth, I. R. (1984) The effect of food preservatives on pH homeostasis in Escherichia coli, J. Gen. Microbiol., 130, 2845-2850. https://doi.org/10.1099/00221287-130-11-2845
- Hongo, M., Nomura, Y., Iwahara, M. (1986) Novel method of lactic acid production by electrodialysis fermentation, Appl. Environ. Microbiol., 52, 314.
- Haynes, W. M. (2013) CRC handbook of chemistry and physics, 94th ed., CRC Press LLC, Boca Raton, FL.
- 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.
- Taylor, D. G. (1990) The importance of pH control during brewing, Tech. Q. Master Brew. Assoc. Am., 131-136.
- Li, H., Liu, F., Kang, L., Zheng, M. (2016) Study on the buffering capacity of wort, J. Inst. Brew., 122, 138-142. https://doi.org/10.1002/jib.286
- Bamforth, C. W. (2001) pH in brewing: An overview, Tech. Q. Master Brew. Assoc. Am., 38, 1-9.
- 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
- Peyer, L. C., Zarnkow, M., Jacob, F., De Schutter, D. P., Arendt, E. K. (2017) Sour brewing: Impact of Lactobacillus amylovorus FST2.11 on technological and quality attributes of acid beers, J. Am. Soc. Brew. Chem., 75, 207-216. http://dx.doi.org/10.1094/ASBCJ-2017-3861-01
- 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
- van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S., Maguin, E. (2002) Stress responses in lactic acid bacteria, Antonie van Leeuwenhoek, 82, 187-216. https://doi.org/10.1023/A:1020631532202