Using Bacteria to Increase Organic Acids

Brett Steigerwaldt, Head Distiller, Lyon Rum

This project will be part of my Heriot-Watt MSc dissertation, under the supervision of Dr. Annie Hill. It will be performed at Windon Distilling Company, home of LYON RUM, in St. Michaels, MD, and will investigate the effects of co-inoculation of commercially available yeast and bacteria on the organoleptic properties of pot-distilled rum produced from blackstrap molasses and raw cane sugar. The research questions to be tested are: (1) can the intentional co-inoculation of yeast and bacteria into a rum fermentation produce rum with significantly more organoleptic characteristics than rum produced solely from yeast; (2) what effects each bacterium has on the organoleptic characteristics of the produced rum; (3) what effects each bacterium has on fermentation performance.

To answer these questions, five fermentation trials will be performed, each in triplicate, using the same blackstrap molasses and raw cane sugar source for all trials. The control trial will use a single chosen yeast strain, and each subsequent trial will use this yeast strain in concert with a single selected bacterial strain. Each trial will be double pot distilled and the aggregate hearts portion will be collected and stored in a non-reactive, glass container. Samples will be taken to BDAS Testing (www.bdastesting.com) in Lexington, KY, for GC-MS testing and sensory panel analysis. The results will be analyzed for statistical significance using Microsoft Excel and other statistics software.

It is expected that each bacterial strain will increase the amounts of organic acids present in the rum fermentation and therefore, increase the amounts of esters and higher alcohols present in the resulting distillate. Thus, yielding rum with significantly more organoleptic characteristics than rum produced solely from yeast. If this project is successful, it could serve as a model for other small-to-medium US-based rum producers, by presenting them with a commercial means to improve the organoleptic qualities of their rum with minimal additional process complexity, and lead to further product differentiation. It will also show the benefit of working directly with yeast suppliers to create custom bacteria solutions to customer requirements. This will encourage suppliers to create novel bacteria or yeast-bacteria products for the benefit of the larger rum industry.

The microbiology of rum and cachaça production

The microbiology of the rum production process has been studied since the late 1890s (Greg, 1895, Pairault, 1903, Allan, 1906, Ashby, 1909). However, even with over a century of research, our current understanding of many of the microbiological processes remains very limited, especially when compared to beer, wine or whisky (Green, 2015). This is a major concern for rum producers, as these microorganisms can impact rum quality at two places in the production process: raw materials and during fermentation (Fleet and Green, 2010).

Many studies have reported on the various microflora present in molasses and sugarcane juice, and found that the majority of species were Candida, Pichia, Saccharomyces, and Torulopsis (Hall et al., 1935, Shehata, 1960, Parfait and Sabin, 1975, Bonilla-Salinas et al., 1995, Todorov and Dicks, 2005). Others have reported on the microflora species associated from molasses-based rum fermentations, or sugarcane juice-based rhum agricole fermentations, and found a variety of Schizosaccharomyces, Bacillus, Clostridium, Propionibacterium, Lactobacillus, Saccharomyces, and Torulopsis species were present (Greg, 1895, Parfait and Sabin, 1975, Ganou-parfait et al., 1987, Fahrasmane et al., 1988, Ganou-Parfait et al., 1989, Fahrasmane and Ganou-Parfait, 1998). In Brazil, many studies reported on the microflora present in different cachaça production regions, including the isolated yeast strains, process microbiology, dynamics, and biochemistry (Schwan et al., 2001, Badotti et al., 2010, de Souza et al., 2012, Badotti et al., 2014, Portugal et al., 2016, Barbosa et al., 2016, Portugal et al., 2017). This has allowed others to explore the effects of these bacteria and yeasts, through deliberate use in, or inoculation of, a sugarcane byproduct-based fermentation.

Similar works and justification for this project

Bacteria and wild yeasts play a major role in the production of the high ester rums of Jamaica and the aromatic rhums of Martinique, through the addition of “dunder” to each fermentation or before distillation (I’Anson, 1971, Burglass, 2011, Green, 2015, Hill et al., 2017). Dunder is the stillage from previous runs, that has been stored and left to naturally ferment via a combination of wild yeasts and bacteria, which produce large amounts of esters and volatile fatty acids (Nicol, 2003). During distillation, the large amounts of acids present in the dunder enriched wash undergo esterification reactions with various alcohols, and yield a highly prized rum (Nicol, 2003). The use of dunder in a controlled fermentation and the characterization of its microbiology has recently been explored in Scotland, and was found to contain five strains of Lactobacillus, and to increase the levels of flavor active compounds of the final distillate (Hill et al., 2017). However, establishing a dunder program and shepherding the wild yeasts and bacteria that establish themselves takes time, and may be cost or space prohibitive for many small rum producers. Alternatively, the symbiotic use of yeast and bacteria or the mixed inoculation of multiple yeast strains in a rum fermentation offer viable means of heavy, aromatic rum production.

Arroyo explored the production of heavy, aromatic rums from pre-treated blackstrap molasses, using Schizosaccharomyces yeast in concert with a Clostridium saccharobutyricum, isolated from sugarcane bagasse, and found that a ratio of bacteria to yeast of 1:5 produced the best rum, as long as a fermentation temperature of 30°C, and a pH of 5.0 – 5.6 was maintained (Arroyo, 1945). Additionally, Arroyo developed a selection criteria for other bacterial candidates capable of producing heavy rums, with a principal concern being their ability to produce a range of acids capable of esterifying with alcohols produced by the yeast during fermentation (Arroyo, 1945). Nemoto further explored the symbiotic use of S. pombe and a C. butyricum strain for heavy rum production in Japan and found that both the symbiotic fermentation, and the addition of high acid wash (acidified by the bacterium) to a rum wash fermenting with the yeast, produced heavy rum (Nemoto, 1975). Lactobacillus and Propionibacterium species have also been found to positively impact the organoleptic properties of molasses-based rum by the production of various acids that can later esterify (Fahrasmane and Ganou-Parfait, 1998).

Finally, the use of non-Saccharomyces yeast to produce alcoholic beverages has been thoroughly reviewed (Varela, 2016). In Brazil, several studies examined their effects on cachaça production (Oliveira et al., 2005, Duarte et al., 2013, Castro Parente et al., 2015, Amorim et al., 2016). It was found that a mixed inoculation of yeast strains (Meyerozyma carabbica and S. cerevisiae) produced a cachaça with greater amounts of volatile compounds (esters, and higher alcohols) and preferable organoleptic properties, than cachaça produced from solely from S. cerevisiae (Duarte et al., 2013, Amorim et al., 2016).

There is growing interest by many rum producers in using novel strains of yeasts and bacteria to produce aromatic and flavorful rums. However, there is still a limited amount of published research on a controlled means of achieving this, without detrimentally affecting yield or rum quality. This project will explore a commercial method of producing rum in this manner.

Please note: I have submitted additional documents containing referenced tables, figures, and the detailed Gantt chart timeline to Erik Owens.

The project will take place at Windon Distilling Company in St. Michaels, MD, and be performed solely by the applicant. The distillery, like many small producers, does not have modern laboratory equipment that allow in depth analysis of fermentations (plating and culturing, cell counts, microscopy, etc.) and distillates (GC-MS and others). Therefore, if any significant testing is required, samples will be taken and sent off-site for laboratory analysis.

Experiment materials, methods, and timeline

The target completion date for project submission is August 2023.

Raw materials

The blackstrap molasses and raw cane sugar are both non-GMO products of the Lula-Westfield Sugar Factory of Paincourtville, Louisiana, USA.

Yeast and bacteria selection

Since Schizosaccharomyces strains are hard to source commercially and S. cerevisiae strains are ubiquitous across the industry, this project will use S. cerevisiae strains for all trials. Bacteria selection depends on which of the following project options are possible.

Option 1:

The author is in discussion with Dr. Pat Heist, chief science officer and co-founder of Ferm Solutions, a yeast and bacteria supplier in Danville, KY, regarding bacterial selection using what they have isolated from sites around the world. This will require the use of their yeast, and the author has selected FermPro 921, a popular whisky/rum yeast known for a robust congener profile. The bacteria and yeast will be added to each trial according to the direction of Dr. Heist. However, if this option is not possible, the second option will be performed.

Option 2:

This option will use Lallemand EC-1118, an efficient, predictable yeast, with neutral sensory impact on the resulting product, and commonly used by American rum producers. Commercially available strains of L. plantarum, L. helveticus, and Oenococcus oeni, from Lallemand and Fermentis will be used. The bacteria and yeast will be added to each trial according to manufacturer directions.

Methods

All trials will be performed in triplicate using the same container source of molasses (IBC tote) and raw cane sugar (Super Sack). Any trial that stalls or doesn’t complete satisfactorily, will be documented and repeated. All vessels and equipment will be cleaned and sanitized before use. Each trial will be performed in a 55-gallon, stainless steel drum with stainless steel lid. Yeast will be rehydrated according to manufacturer directions and pitched at 1 g/gal. The following measurements will be taken, twice per day: specific gravity, pH, temperature, and visual/sensory check of activity. Fermentation will be complete when there is no change in specific gravity for two days and no fermentation activity is visually present. However, fermentation length may change, depending on the activity of the bacteria. The experimental setup can be seen in Table 1, with each trial having an A, B, and C segment.

Fermentation and distillation

Fermentation volume will be 20 gallons and have the following composition: two gallons blackstrap molasses by weight, 20 lbs of raw cane sugar, and sufficient filtered municipal water to bring the total volume to 20 gallons, determined by weight. A marine/recreational vehicle filter will be used to remove any chlorine from the water. Fermentation is expected to take one week for each trial segment and three weeks for the entirety.

The first distillation will be performed in a 26-gallon stainless steel pot still, as seen in figure 1, heated with an internal electric element. The low wines will be collected into a 5-gallon glass carboy. The author expects to collect ≈ 4 gallons low wines per stripping run, depending on rum wash % ABV. The volume and %ABV will be recorded for each run.

The second distillation will be performed in a 3-gallon copper alembic pot still, as seen in figure 2, heated by an electric hot plate. The resulting distillate will be collected in series of 200 mL glass jars. The author will use sensory and an Anton Paar Snap 41 to determine heads, hearts, and tails. Estimates are: ≈1,000 mL for heads, ≈2,000-2,300 mL for hearts, ≈600 mL for tails. Once determined for the control, the same cuts will be made for all subsequent trials. The hearts for each run will be stored separately in a large glass jar and labeled with their volume and %ABV. Once the entire triplicate has been processed, the three jars of hearts will be combined into one large aggregate and labeled with the total volume and %ABV.

Analysis and conclusion

Samples will be taken from the aggregate hearts of each trial. These will be taken to BDAS Testing in Lexington, KY, for analysis via GC-MS, to determine compounds present and their concentrations, and will also include a sensory panel analysis. After testing and sensory analysis, the data will be analyzed for statistical significance using Microsoft Excel and other statistics software. The results will be discussed with my advisor to determine if any other testing is required before write-up can begin.  

Budget

$286.15 justification: Yeast and bacteria supplies.

$164.63 justification: Raw ingredients: blackstrap molasses, raw cane sugar.

$98.19 justification: Collection supplies: 200mL jars and 5-gallon carboy

$2,325 justification: Analysis and testing performed by BDAS Testing: 5 x CP09 distilled spirits comprehensive chemical profile ($305 each), 5 x CP11 taste panel evaluation ($160 each)

Request: $2,873.97.

During fermentation it is expected that each bacterial strain will increase the amounts of organic acids present in the wash. This acidification will affect the rate of pH drop and produce distinct aromatic differences from the control fermentation and will increase the amounts of esters and higher alcohols present in the resulting distillate. Thus, yielding rum with significantly more organoleptic characteristics than rum produced solely from yeast.

This project will significantly benefit small-to-medium US-based rum producers by presenting them with a commercial means to improve the organoleptic qualities of their rum with minimal additional process complexity, and lead to further product differentiation. It will also show the benefit of working directly with yeast suppliers to create custom bacteria solutions to customer requirements. This will encourage suppliers to create novel bacteria or yeast-bacteria products for the benefit of the larger rum industry.

ALLAN, C. 1906. The Manufacture of Jamaica Rum. West Indian Bulletin.

AMORIM, J. C., SCHWAN, R. F. & DUARTE, W. F. 2016. Sugar cane spirit (cachaça): Effects of mixed inoculum of yeasts on the sensory and chemical characteristics. Food Research International, 85, 76-83.

ARROYO, R. 1945. Production of Heavy Rums.

ASHBY, S. F. 1909. The study of fermentations in the manufacture of Jamaica rum. International Sugar Journal 11, p. 243-251, 300-307.

BADOTTI, F., BELLOCH, C., ROSA, C. A., BARRIO, E. & QUEROL, A. 2010. Physiological and molecular characterisation of Saccharomyces cerevisiae cachaça strains isolated from different geographic regions in Brazil. World Journal of Microbiology and Biotechnology, 26, 579-587.

BADOTTI, F., VILAÇA, S. T., ARIAS, A., ROSA, C. A. & BARRIO, E. 2014. Two interbreeding populations of Saccharomyces cerevisiae strains coexist in cachaça fermentations from Brazil. FEMS Yeast Research, 14, 289-301.

BARBOSA, E. A., SOUZA, M. T., DINIZ, R. H. S., GODOY-SANTOS, F., FARIA-OLIVEIRA, F., CORREA, L. F. M., ALVAREZ, F., COUTRIM, M. X., AFONSO, R. J. C. F., CASTRO, I. M. & BRANDÃO, R. L. 2016. Quality improvement and geographical indication of cachaça (Brazilian spirit) by using locally selected yeast strains. Journal of Applied Microbiology, 121, 1038-1051.

BONILLA-SALINAS, M., LAPPE, P., ULLOA, M., GARCIA-GARIBAY, M. & GÓMEZ-RUIZ, L. 1995. Isolation and identification of killer yeasts from sugar cane molasses. Letters in Applied Microbiology, 21, 115-116.

BURGLASS, A. J. 2011. Cane Spirits, Vegetable based Spirits and Aniseed Flavored Spirits. Handbook of Alcoholic Beverages.

CASTRO PARENTE, D., VIDAL, E. E., LEITE, F. C. B., DE BARROS PITA, W. & DE MORAIS JR, M. A. 2015. Production of sensory compounds by means of the yeast Dekkera bruxellensis in different nitrogen sources with the prospect of producing cachaça. Yeast, 32, 77-87.

CROSQ 2008. CARICOM Regional Standard for Rum: Specifications. Technical Report No. CRS 25: 2008. Belleville, St Michael, Barbados.

DE SOUZA, A. P. G., VICENTE, M. D. A., KLEIN, R. C., FIETTO, L. G., COUTRIM, M. X., DE CÁSSIA FRANCO AFONSO, R. J., ARAÚJO, L. D., DA SILVA, P. H. A., BOUILLET, L. É. M., CASTRO, I. M. & BRANDÃO, R. L. 2012. Strategies to select yeast starters cultures for production of flavor compounds in cachaça fermentations. Antonie van Leeuwenhoek, 101, 379-392.

DUARTE, W. F., AMORIM, J. C. & SCHWAN, R. F. 2013. The effects of co-culturing non-Saccharomyces yeasts with S. cerevisiae on the sugar cane spirit (cachaça) fermentation process. Antonie van Leeuwenhoek, 103, 175-194.

FAHRASMANE & GANOU-PARFAIT 1998. Microbial flora of rum fermentation media. Journal of Applied Microbiology, 84, 921-928.

FAHRASMANE, L., GANOU-PARFAIT, B. & PARFAIT, A. 1988. Research note: Yeast flora of Haitian rum distilleries. MIRCEN journal of applied microbiology and biotechnology, 4, 239-241.

FLEET, G. H. & GREEN, V. 2010. The microbiology and biotechnology of rum production. In: WALKER, G. M. & HUGHES, P. S. (eds.) Distilled spirits : new horizons : energy, environmental and enlightenment. Nottingham: Nottingham University Press.

GANOU-PARFAIT, B., FAHRASMANE, L., GALZY, P. & PARFAIT, A. 1989. Les bactéries aérobies des milieux fermentaires à base de jus de canne à sucre. Industries Alimentaires et Agricoles, 579-585.

GANOU-PARFAIT, B., FAHRASMANE, L. & PARFAIT, A. Bacillus spp in sugar cane fermentation media. 1987.

GREEN, V. 2015. The microbial ecology of a rum production process. PhD Dissertation, University of New South Wales.

GREG, P. H. 1895. The Jamaica Yeasts. Bulletin of the Botanical Department of Jamaica. Kingston.

HALL, H. H., L.H., J. & NELSON, E. K. 1935. Microorganisms causing fermentation flavors in cane sirups, especially Barbados “molasses”. Journal of Bacteriology, 33, p. 577-585.

HILL, A. E., SALVESEN, O. & RUSSELL, G. 2017. Deciphering dunder: an examination of rum fermentation. In: UNIVERSITY, H.-W. (ed.) Worldwide Distilled Spirits Conference Poster & Proceedings. Glasgow.

I’ANSON, J. A. P. 1971. Rum Manufacture. Process Biochemistry, p. 35-39.

LEHTONEN, M. & SOUMALAINEN, H. 1977. Rum. In: ROSE, A. H. (ed.) Economic Microbiology. London: Academic Press.

NEMOTO, S. 1975. Possibilities of utilization of butyric acid bacteria for rum making. Annales de technologie agricole, 24, 397-410.

NICOL, D. A. 2003. Rum. In: LEA, A. G. H. & PIGGOTT, J. R. (eds.) Fermented Beverage Production. Boston, MA: Springer US.

OLIVEIRA, E. S., CARDELLO, H. M. A. B., JERONIMO, E. M., SOUZA, E. L. R. & SERRA, G. E. 2005. The influence of different yeasts on the fermentation, composition and sensory quality of cachaça. World Journal of Microbiology and Biotechnology, 21, 707-715.

PAIRAULT, M. E. A. 1903. Le rhum et sa fabrication, Paris, Gauthier-Villars.

PARFAIT, A. & SABIN, G. 1975. Les fermentations traditionnelles de mélasse et de jus de canne aux Antilles françaises. Industries alimentaires et Agricoles, 27-34.

PORTUGAL, C. B., ALCARDE, A. R., BORTOLETTO, A. M. & DE SILVA, A. P. 2016. The role of spontaneous fermentation for the production of cachaça: a study of case. European Food Research and Technology, 242, 1587-1597.

PORTUGAL, C. B., DE SILVA, A. P., BORTOLETTO, A. M. & ALCARDE, A. R. 2017. How native yeasts may influence the chemical profile of the Brazilian spirit, cachaça? Food Research International, 91, 18-25.

SCHWAN, R. F., MENDONÇA, A. T., DA SILVA, J. J., JR., RODRIGUES, V. & WHEALS, A. E. 2001. Microbiology and physiology of Cachaça (Aguardente) fermentations. Antonie Van Leeuwenhoek, 79, 89-96.

SHEHATA, A. M. 1960. Yeasts isolated from sugar cane and its juice during the production of aguardente de cana. Appl Microbiol, 8, p. 73-5.

TODOROV, S. D. & DICKS, L. M. T. 2005. Lactobacillus plantarum isolated from molasses produces bacteriocins active against Gram-negative bacteria. Enzyme and Microbial Technology, 36, 318-326.

VARELA, C. 2016. The impact of non-Saccharomyces yeasts in the production of alcoholic beverages. Applied Microbiology and Biotechnology, 100, 9861-9874.