Comparison Trial – By K. Dodds, Parallel 49 Brewing Company

Kelsey Dodds has worked as the laboratory manager at Parallel 49 Brewing since 2014. She is the on-staff beer scientist providing education, outreach and applied research to the company. Dodds provides much of the Quality Assurance/Quality Control testing procedures and manages the onsite yeast library of over 50 species. She obtained her Bachelor of Applied Science (BAS) degree from Mount Royal University in Calgary.

Introduction

The genus Lactobacillus currently contains over 180 species and encompasses a wide variety of organisms (Hammes W.P., 2006). Fortunately for breweries and homebrewers, the range of Lactobacilli encountered is fairly restricted. Especially when talking about strains that are purposely brought into a brewery as a positive, instead of the accidental and harmful spoilage bacteria. However there is some overlap between spoilage bacteria and desirable bacteria, and it all depends whether or not the strain was intended.

These desirable bacteria are supplied in one form or another at a commercial level for their ability to produce lactic acid. Knowing which species of this genus you intend on using can have a drastic effect on the final outcome of your brewing project.

Classification of the Genus

The large diversity in the Lactobacillus genus is widely due to the fact that the genus was originally classified by polyphyletic grouping in 1919 (Priest, 2003). A polyphyletic group is a set of organisms that have been classified together because they share a common characteristic, but do not share an immediate or common ancestor. There are some issues which this method of classification. Simply put, to polyphyleticly group Mammals; a mammal produces milk and has hair, therefore a coconut could be polyphyleticly classified as mammal… A coconut is not a mammal. We know it’s not a mammal. Which is why in many schools of taxonomy the existence of polyphyletic groups while classifying species are discouraged. But sometimes you just have to make do with what you have at the time and polyphyletic classification can be a good catch-all for “bulk” genus classification, even if some species (coconuts or Lactobacilli) end up finding loopholes.

Due to advancements in science, we do know that there are groups of Lactobacillus that have similar genetic traits (RNA grouping) as well as have shown to behave in a similar manner when exposed to comparable environments (phylogenic grouping). However genetic testing is time consuming and, at this time costly, so we do not have a complete genetic family tree of Lactobacillus genus. But we can fill in some gaps without having to fully sequence the fill chain of DNA. As expected, the two methods of classification do not perfectly differentiate the groups. Table 3 shows a detailed breakdown.

So naturally there are some flaws when talking about the Lactobacillus genus, and we will have to delay a perfect family tree until someone has the time and money to genetically sequence every single bacteria in the Lactobacillus genus. Luckily for us, many of the strains of Lactobacillus that are available commercially have been completely sequenced, so we have a very solid understanding of their fermentation and lactic production pathways. But it is worth noting that this genus is constantly getting updated and reclassified as more information comes out.

Figure 1 shows the two main metabolic pathways that a specie classified as a Lactobacillus can use to create both lactic acid (lactate) and/or ethanol. These fermentation pathways help differentiate the Lactobacillus genus into its RNA groups, as discussed before.

Obligatory homofermentation, where Lactobacillus only produces lactic acid.

Obligatory heterofermentative, where the Lactobacilli produce lactic acid, carbon dioxide and ethanol and possibly acetic acid.

Faculitativly heterofermentatiove create lactic acid, carbon dioxide and ethanol when there is an abundance of carbohydrates but can also switch to a homofermentative pathway when carbohydrates are scarce.

(Mehta, Kamal-Eldin, & Iwanski, 2012).

Knowing which classification of Lactobacilli you plan on working with greatly helps troubleshoot any fermentation issues that arise. Being aware of the RNA group also give you a good idea of what to expect from fermentation so you will be able to get the best performance out of your chosen Lactobacillus strain.

Lactobacilli Trial

During a period of research and development at the brewery, we chose to get some experience with as many commercially available Lactobacilli strains that we could get our hands on. As much as you can read books, websites and papers, sometimes seeing with your own eyes is the best. The desired outcome and objective of the project was to find a Lactobacillus specie that we were able to safely handle with consistent results, rapid and reliable pH drop, as well as produce positive sensory attributes.

Six (6) Lactobacillus strains were acquired to participate in the trials all of which ranged over the three RNA groups and all of which are available to a homebrewer. A breakdown of the six strains are provided in Table 1 along with a brief description of the strain as provided by the manufacturer.  

Table 1: Summary of Lactobacillus specie information from commercial supplier.

Code	Specie	Notes from website
WLP678	Lb. hilgardii	Low hop tolerance (>10 IBU), medium acidity
WLP677	Lb. delbruekii	Moderate Acidity
WLP673	Lb. buchneri	Low hop tolerance (>15 IBU), <5% Attenuation
WLP693	Lb. plantarum	High lactic acid production
WLP669	Lb. paracollinides	Good secondary fermenter to add acidity
WLP672	Lb. brevis	High lactic acid production

Procedure

Six 19L carboys were filled with a simple Pale Malt wort (11.4°P) that was less than 5.0 IBU, and were inoculated with a standard homebrewers pitch of each respective species.

Carboys were held in a warm room (20°C) while Plato and pH readings were taken daily for 10 days. A final reading on day 60 was also recorded along with sensory attributes of all carboys.

Observations and Results

As predicted, the initial 48 hours of the trial was fairly uneventful. pH and Plato readings remained steady, and there was little to no gas production. There were notable differences in the six trials over time, Figure 2 shows the change in pH over ten (10) days of all species. The two clear pH reduces being Lb. delbruekii and Lb. hilgardii. Figure 3 shows the Plato drop over time, also indicating that Lb. delbruekii was very active, while the other Lactobacillus species did not attenuate at all. Over the course of the 10 days very little gas production was observed in all but two of the carboys. The obligately heterofermentative species, (RNA Group 3’s, Lb. brevis and Lb. buchneri), has moderate gas production, as expected. It can be assumed that the facultative heterofermentative species, (RNA Group 2’s), used the homofermentative pathways, as there was plenty of carbohydrates in the wort, which would explain the lack of any major gas production.

The Lb. plantarum trial was abandoned on day 10 due to mold growth inside the carboy. This confirmed the inactivity of the bacteria in the trial, which is reflected in its lack of pH or Plato drop in carboy. It is believed the Lb. plantatum vial was well beyond its best before date. Therefore Lb. plantarums 60 day “final” readings are in fact, day 10 readings and no sensory attributes were recorded.

Figure 2: pH over time of six (6) Lactobacillus strains. Where the lines shows pH over time (0-10 days), and the bars show final pH at 60 days.

Figure 3: Plato over time of six (6) Lactobacillus strains. Where the line graph shows Plato over time (0-10 days), and the bars show final Plato at 60 days

As shown, the final 60 day readings were performed, and each carboys sensory attributes were described, which is summarized in Table 2.

Table 2: Summary of Lactobacillus Specie sensory attribute information.

Code	Specie	Tasting Notes
WLP678	Lb. hilgardii	Ripe,
WLP677	Lb. delbruekii	Clean, peach, apricot, tart
WLP673	Lb. duchneri	Honey, fruit, very sour
WLP693	Lb. plantarum	not tasted due to mold
WLP669	Lb. paracollinides	Apple, orange, grape, tannic, clean
WLP672	Lb. brevis	Apple, mango/pineapple, clean, sour

Conclusion

From the data presented, it is easy to see that all commercially available strains of Lactobacilli are not equal, and that reflects in the unique styles of sour beer that are being produced today. For our objectives; finding a Lactobacilli specie that provided consistent, rapid pH drops with positive sensory attributes, we chose to move forward with large scale testing of Lactobacillus delbrueckii. Which is now the base for most of our large scale souring operation. However we still do use other Lactobacillus species for some one-off sour beers or smaller projects, as Lb. delbrueckii can have a fairly one dimensional flavor. But Lb. delbrueckii has shown to be a rapid and consistent acidifier when handled properly which is precisely what we were looking for at the beginning of this trial.

Limit of scope

It is very important to note the limited scope of this project:

This was a 60 day trial completed with Lactobacillus as the primary fermenter, with no other secondary fermentation characteristics. Nor does this project cover the scope or sensory attributes of a Lactobacillus as a secondary fermenter or Lactobacillus specie used for kettle souring as both would greatly affect final flavors.

Also, if we were to redo this test, a final ABV on all carboys would be performed to see whether the facilitative heterofermenter actually used their heterofermentative pathways, creating alcohol and lactic acid, or their homofermentative pathway, just producing lactic.

Furthermore, to completely re-do the Lb. plantarum carboy as it obviously didn’t get a fair go at this trial.

Notes to the Homebrewer

We found that the descriptions provided online for all Lactobacillus species commercial source were true-to-target for the trials. We believe it is important to always try new things and improvements with some key take away points from this report.

While writing a sour beer recipe, be conscious about what final characteristics you want, and select a species of Lactobacillus that is right for the desired sensory attributes, exactly like when selecting a Saccharomyces strain.  As you can see from the data above, not all Lactobacilli are equal but all have their place in the brewery… so long as they are invited in.

Appendix A

Table 3: Some Lactobacillus species found in fermented beverages and related habitats and their allocation to taxonomic groups, (From; Priest, 2003)

Species	RNA Group	Polyphyletic Group	Common Habitats
Lb. delbrueckii	1	I	Grain, mashes, plant material, wine, cider
Lb. mali	2	I	Wine, cider
Lb. casei	2	II	Milk and milk products
Lb. collinoides	2	II	Cider
Lb. coryneformis	2	II	Fermented vegetables, silage, beer
Lb. curvatus	2	II	Milk, silage, sauerkraut
Lb. homohiochii	2	II	Wine
Lb. paracasei	2	II	Beer, whiskey fermentations
Lb. sakei	2	II	Wine, sake
Lb. brevis	3	III	Beer, wine, milk, sourdough
Lb. buchneri	3	III	Beer, wine, milk, cheese
Lb. fermentum	3	III	Fermented vegetables, whisky fermentations
Lb. fructivorans	2	III	Wine, vinegar preserves
Lb. hilgardii	2	III	Wine
Lb. lindneri	2	III	Beer
Lb. malefermentans	2	III	Brewery yeast
Lb. parabuchneri	2	III	Saliva, cheese, brewery yeast
Lb. plantarum	2	III	Milk, fermented vegetables

Works Cited

Hammes W.P., H. C. (2006). The Genera Lactobacillus and Carnobacterium. The Prokaryotes, 320-103.

Mehta, B. M., Kamal-Eldin, A., & Iwanski, R. Z. (2012). In Fermentation: Effects on Food Properties (pp. 76-77). Boca Raton: CRC Press.

Priest, F. G. (2003). Gram-positive brewery bacteria. In F. G. Priest, & I. Campbell, Brewing Microbiology (pp. 190-202). New York: Plenum Publishers.