Literature Review title:
The composition and relevant qualities of a wort that ensures it is suitable for pitching yeast.

INTRODUCTION

Brewing is one of the oldest known biotechnological processes, with the first records dating the process back to Mesopotamia then Egypt circa 6000BC, during the revolution of hunter-gatherers to farmers and breeders. Kas was the first grained fermented beverage brewed by Sumerians between 3000 to 2800 BCE (Karabín et al., 2017). A sweet and viscous wort is produced by allowing grain to germinate and dry, it is then crushed and mixed with hot water.
Fermentation is one of the most important steps in the production of alcohol, and it is greatly influenced by the wort composition. Wort can be defined as ‘an aqueous solution of extract made from grain, intended for fermentation by yeast into beer’ (Craft Beer & Brewing, 2018). Wort is produced during the mashing process, the lautering process then separates it from the grain husk material. The brew kettle collects the wort, and at the stage it is boiled with the hops. The fermentation process finally begins when yeast is added to the cooled wort. Yeast is the vital ingredient in the conversion of sugar to ethanol and carbon dioxide in any alcohol fermentation reaction, a phenomenon finally explained by Louis Pasteur in 1861 (Pasteur, 1876 cited in Karabín et al., 2017).

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Pitching can be defined as ‘the process of adding yeast to a wort to begin the fermentation reaction and produce beer’ (Craft Beer & Brewing, 2018b). Pitching rate plays a direct influence on the fermentation rate. The typical pitching rate for a normal gravity lager wort, with a Plato scale equaled to 12°P, is approximately 10-15×106 cells per ml. However, factors including yeast strain and desired fermentation characteristics all play a role in determining the exact concentration. Higher pitching rates result in short lag phase time periods, higher maximum fermentation rates, and shorter carbohydrate fermentation completion times (Hardwick, 1995).
Changes in the composition of a wort triggers different responses from the pitching yeast. The response of the yeast to changes an environment, in this case the wort, is vital to maintain the quality and consistency of the end-product; therefore, controlling raw ingredients and wort preparation are critical to ensure the wort is suitable for pitching yeast (He et. al, 2014). Before a wort is deemed suitable for pitching, several relevant factors related to its composition and quality must be perfected. The main components of a standard wort include carbohydrates, nitrogen compounds including proteins, salts and minerals, acids, phenols, hops, and lipids. However, the composition of the wort is dependent on several factors including the composition of the grain bill, the mashing process, the hops and the brewing water (Craft Beer & Brewing, 2018). The nutritional composition of wort is difficult to optimise due to the many complex chemical constituents of the medium. The objective of this literature review is to investigate the relevant factors influencing the composition and qualities of a wort to ensure it is suitable for pitching yeast.

CARBOHYDRATES AND SUGARS

The sugar profile of a wort is dependent on the raw ingredient used, with the most commonly used in the Western world being barley. Approximately 90-92% of the total solids in a standard brewer’s wort is made up of carbohydrates, with approximately 70-80% of these being fermentable. However, the final value of each individual wort is dependent on the enzymic power of the malt, yeast utilisation and the mashing method used (MacWilliam, 1968; He et al., 2014).  Chromatography techniques developed only a decade before MacWilliam’s paper was published, and microbiological techniques have been used to assess the carbohydrate content of a wort; however, these techniques express individual sugars as a percentage of the total carbohydrate content and fail to provide the actual concentrations of individual sugars present in the wort (Macwilliam, 1968). The sugar profile of a standard all-malt brewer’s wort mainly consists of 47% maltose, 15% maltotriose, 12% monosaccharides, 5% sucrose, while the remaining 25% is made up of the non-fermentable dextrin fraction (Craft Beer & Brewing, 2018).  However, it is important to note the exact percentage of each sugar differs due to batch-to-batch variation.
Following pitching, yeast utilise sugars present in the wort in a sequential order, with sucrose, which is present in the lowest concentration of only 5%, being depleted first. Followed by the monosaccharides – glucose and fructose. The most abundant sugars maltose and maltotriose, which represent almost 60% of the total sugar content of most standard malts, are only utilized after the depletion of monosaccharides due to the carbon catabolite repression of metabolic pathways involved in the uptake and utilization of alternative sugars (Gibson et al., 2008; He et. al, 2014). Lager and most industrial ale yeast strains fail to metabolise dextrins, which make up a quarter of the malt’s sugar profile, so they remain in the final beer product. Alpha-amylases are often added as exogenous enzymes to reduce the dextrin content; however, they result in an undesirable wort-like taste from the final product. The genetic modification of yeast strains to ferment the dextrin fraction is currently a vogue topic amongst consumers in the food and beverage industry (Karabín et al., 2017).
Adjuncts, a non-fermentable source of sugar, are also often added to the wort prior to pitching. Adjuncts do not serve as a source of essential nutrients. Examples of adjuncts used in Ireland’s temperate climate include wheat, rye and oats.

NITROGEN COMPOUNDS

Nitrogen compounds represent approximately 5% of the wort. Assimilable nitrogen or free amino nitrogen (FAN) are the nitrogenous compounds available for consumption by yeast, these can be defined as the sum of the individual amino acids, ammonium ions and small peptides in the wort. The relevant concentration of these components varies depending on several factors, such as the malting and mashing processes, adjuncts utilised, raw materials, and cereal to water ratio (He et al., 2014). Proteolytic enzymes are required in the wort to adjust the soluble nitrogen content.
Amino acids play a key role in: 1) the complex system regulating the biosynthesis of the flavour-active compounds formed by yeast, and 2) ensuring adequate growth of yeast, which has a direct effect on a healthy fermentation.  However, the presence of amino acids in wort is critical because they are a primary source of nitrogen for the yeast (Morebeer.com, 2018). The nitrogen compound concentration of wort is made up of approximately 40% polypeptides, 30% α-amino nitrogen, 20% proteins of a high molecular weight, and the remaining 10% is made up of purines and other nitrogen compounds. Table 1 provides a condensed summary of the concentration of nitrogen and amino acids present in wort (Ferreria and Guido, 2018). A healthy wort contains approximately 19 amino acids which are consumed at different stages throughout a fermentation. The use of adjuncts will result in a lower FAN concentration, a high FAN concentration and adequate aeration are important factors in ensuring a quick and clean fermentation process, as yeast pitched in this environment will rapidly move through the aerobic activity and produce strong cell walls by the consumption of internal glycerol and oxygen, resulting in the formation of sterols. (Morebeer.com, 2018).
Amino acids are grouped into three classes, a summary of this can be viewed on Table 2. Class I are referred to as the primary amino acids, their presence or absence in wort is insignificant because they are readily synthesised by yeast cells who use the products of normal sugar catabolism as their carbon source, a healthy fermentation consumes most primary amino acids during the first 20-hours following pitching. Class II are synthesised by their, their presence or absence in wort plays a role in the flavour of beer. Yeast are unable to utilise Class III; therefore, their presence in wort is essential (Morebeer.com, 2018).

LIPIDS

Lipids are a minor component of wort. The total concentration of crude lipids ranges from 5-7mg/100ml, accounting for 5-7% of the wort. The literature suggests the kilning temperature has a direct correlation on the fatty acid of the wort (MacWilliam, 1968). Lipids have a beneficial effect on the growth of yeast during fermentation, but play a negative impact on beer quality, affecting factors such as: 1) foam head stability, and 2) beer spoilage.
Effective removal of spent hops and hot break ensures a low concentration of lipids in the wort, at a value of approximately 3%, prior to pitching yeast, because even concentrations as low as this may result in considerable problems during fermentation (Anness and Reud, 1985).
 VITAMINS AND INORGANIC IONS
Yeast requires vitamins and a wide variety of inorganic ions for efficient proliferation and fermentation performance to occur. Wort is a rich source of vitamins and inorganic ions. The significance and importance of certain vitamins in wort is summarised in Table 3. The main function of these components is to ensure coenzymes and enzymes present in yeast function properly, inadequate concentrations of these components may result in malfunctions in the metabolic activities of yeast, causing problems during fermentation (He et al., 2014). A general rule of thumb suggests the concentration of arsenic, copper and iron in any wort should be kept as low as possible as studies demonstrate they have an adverse effect on the growth of yeast; however, small concentrations of iron (0.075ppm), copper (0.012ppm) and zinc (0.2ppm) are required to support yeast growth (MacWilliam, 1968).
Zinc plays a vital role in the production of ethanol and is a less common essential mineral naturally occurring in all worts. An insufficient zinc concentration will result in a delayed or sticking fermentation. An excessive zinc concentration will result in yeast autolysis, and the production of undesirable sulphur-like off-flavours (Murphy and Sons, n.d.).
Normal levels of nitrate have little effect on yeast. However, nitrate concentration up to 50ppm retard the growth of yeast, while concentrations in excess of 100ppm play an adverse effect on the growth of yeast (MacWilliam, 1968).

DISSOLVED OXYGEN AND AIR

A variation in the dissolved oxygen concentration of wort at the pitching stage will result in a variation in the flavours of beer (Murphy and Son, n.d). Oxygen is necessary for the growth and reproduction of yeast. Yeast require oxygen for the synthesis of sterols and fatty acids required for the expansion of their cell walls.  Several factors affect the oxygen requirement of yeast cells including: i) wort trub levels; ii) original gravity of the wort; and iii) the yeast strain used. For these reasons, some strains have a higher oxygen requirement compared to others, but breweries generally aim to use between 8 and 10ppm dissolved oxygen in a wort. The bare minimum required to ensure suitable conditions for pitching yeast is 5ppm (Brew Your Own, 2017). To allow for the adequate distribution of oxygen, the pressure at the injection point should be approximately 10 p.s.i with small gas bubbles (Muphy and Sons, n.d.).
Extremes in dissolved oxygen concentration of wort will have detrimental consequences. Low levels of dissolved oxygen will lead to insufficient yeast growth; therefore, the fermentation will halt before all the available sugar has been converted to alcohol and carbon dioxide. However, high levels of dissolved oxygen results in excessive yeast production. This will cause a long lag phase at the beginning of the fermentation, low beer pH, and a low original gravity determination (Murphys and Sons, n.d.).
If the appropriate fermentation temperature and good pitching yeast are used, but incomplete fermentation occurs, the literature suggests investigating the oxygenation technique employed until successful results are achieved. The concentration of dissolved oxygen and air varies greatly depending on whether the wort is cooled in an open or closed vessel, and its final temperature before pitching yeast (MacWilliam, 1968).  Failure to provide adequate wort aeration may lead to problems including: i) prolonged lag times prior to the beginning of fermentation, ii) incomplete fermentation, and iii) the production of excessive amounts of ester (Brew Your Own, 2017).

PITCHING YEAST

Following boiling, once the wort is sufficiently cooled to an optimum temperature, the yeast is ‘pitched’, and the fermentation process begins. Prior to pitching, the yeast are placed under microscopic examination to determine if enough viable cells are present. The viable cell count is used as a tool to estimate the density of cells, to ensure the correct amount of yeast is pitched. Following an in-depth analysis of several brewing blogs and websites, it suggests pitching 1 million yeast cells per °Plato, per ml of wort (Morebeer.com, 2018). To allow for the compensation of variations in yeast viability and composition, the pitching rate can be increased to a maximum of between 10-25% above the norm. The recommended temperature for pitching yeast lies within the range of 15-17°C (Murphy and Sons, n.d.).
There are two methods used to obtain efficient quantities of yeast for fermentation – 1) reusing the yeast from the slurry of a previous fermentation, or 2) adding a small amount of yeast from a commercial ‘yeast starter’ pack. When choosing suitable yeast for reusing, it is important to ensure the previous fermentation finished properly, and to never reuse yeast from a batch which did not fully ferment (Morebeer.com, 2018).

CONCLUSION

Following an in-depth analysis of the literature regarding this topic, yeast is introduced into an extremely complex environment consisting of simple sugars, dextrins, amino acids, peptides, proteins, vitamins, ions, and other constituents upon pitching. Wort is a highly sophisticated medium because it serves two functions: i) a growth medium for the development of new yeast cells; and ii) a fermentation medium to allow the conversion of sugar to ethanol and carbon dioxide (Stewart, Hill and Russell, 2013).  In conclusion, a good quality wort is essential to ensure a successful fermentation. Therefore, to ensure the pitching yeast will metabolise the medium nutrients in a predictable and reproducible fashion, it is essential to ensure the composition of the wort is constant.
REFERENCES

Anness, B. and Reud, R. (1985). LIPIDS IN WORT. Journal of the Institute of Brewing, 91(5), pp.313-317.

Bokulich, N. and Bamforth, C. (2013). The Microbiology of Malting and Brewing. Microbiology and Molecular Biology Reviews, 77(2), pp.157-172.

Ferreira, I. and Guido, L. (2018). Impact of Wort Amino Acids on Beer Flavour: A Review. Fermentation, 4(2), p.23.

Gibson, B., Boulton, C., Box, W., Graham, N., Lawrence, S., Linforth, R. and Smart, K. (2008). Carbohydrate utilization and the lager yeast transcriptome during brewery fermentation. Yeast, 25(8), pp.549-562.

Hardwick, W. (1995). Handbook of brewing. New York: M. Dekker.

He, Y., Dong, J., Yin, H., Zhao, Y., Chen, R., Wan, X., Chen, P., Hou, X., Liu, J. and Chen, L. (2014). Wort composition and its impact on the flavour-active higher alcohol and ester formation of beer – a review. Journal of the Institute of Brewing, 120(3), pp.157-163.

Hornsey, I. (1999). Brewing. Cambridge, U.K.: Royal Society of Chemistry.

MacWilliam, I. (1968). WORT COMPOSITION-A REVIEW. Journal of the Institute of Brewing, 74(1), pp.38-54.

Morebeer.com. (2018). Preparing Wort & Yeast for Fermentation | MoreBeer. [online] Available at: https://www.morebeer.com/articles/Wort_Yeast_Fermentation [Accessed 10 Nov. 2018].
Murphy and Son. (n.d.). Oxygen in Wort prior to Pitching – Murphy and Son. [online] Available at: https://www.murphyandson.co.uk/resources/technical-articles/oxygen-in-wort-prior-to-pitching/ [Accessed 19 Nov. 2018].