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The study of the process for beer brewing and production, which includes the bioprocesses of the enzyme action of alpha amylase and fermentation of brewing yeast and how these can be improved are the components for this report. The many stepped process for beer brewing has been developed over hundreds of years since the very first production of alcohol. The enzyme alpha amylase breakdown the starches in the barley grain to simple sugars, producing wort which is fermented by yeast to produce the alcoholic beer beverage. Improvements to the bioprocesses of beer brewing would be the addition of enzymes to the mashing process, also the genetic modification of the activity sites of these enzymes to be more efficient. The genetic modification of the yeast strain used for brewing to be higher in fermentation yield of the sugars also would be a vast improvement.
The brewing and fermentation of beer is a many stepped process that has been developed over hundreds of years resulting in numerous different types of beer products, becoming an art form for some suppliers and a great commodity in various countries. The preparation of ingredients is always the first step, and although they may differ between beer types giving each their unique flavour, the essential ingredients and process does not change. As shown in process diagram 1, barley is the first essential ingredient that must go through a malting process. Malting is the conversion of barley grain into malt (Bruxelles 2008). The barley used must reach certain specifications before being malted where the quality is often determined by genetic selectivity for the best grains, these being barley with water content of 12-14%, a germination index minimum of 6.0, protein content of 9-11.5%, microorganisms and pesticides below a set level and so on (CRC 2002). Malting starts off with steeping the barely in water, in order to raise the moisture content of barley to approximately 45%, without over hydrating the grain which causes it to burst. It involves a balance of water then drying off with air then immersing in water multiple times to reach the desired result (Notts 2009). Towards the final stages of steeping, the beginnings of germination start to occur. Humid air is pushed beneath the barley causing the grain to produce enzymes that breakdown the cell wall structure of starch in the grain to produce sugars that will later be fermented. Before the germination process is completed by the grain, which is before a root system begins to form; the barley is finally kilned, which halts the germination process at this specific time (Notts 2009). The kiln halts all modifications to the barley and is very tightly temperature controlled to around 65Â°C. This step has high operating costs due to the energy it consumes, this therefore needs to be improved. After kilning is completed the product is now malt with a moisture content of approximately 4% (Notts 2009), and has a certain colour and flavour. Therefore differences to malt produce the many different types of beer.
Milling which takes place in an operating machine known as a declumer (Casey 2005) and is step 2 in process diagram 1. This process removes any roots that may have formed during malting, which becomes culm and can be sold as a by product as animal feed being high in proteins. By doing this the particle size of the malt is reduced which will improve efficiency and the economy of the brewing process (Goldammer 2009). The greater the malt is milled, the higher the surface area to volume ratio it becomes which therefore makes it easier for the sugars, which were broken down from the starch by enzymatic action, to be extracted. The milled malt (or grit as it is often also named) must be ground to a specified particle size of the next processes equipment. As an example, a lauter tun as seen in the process diagram; requires fine grit (Goldammer 2009).
Before the milled malt (grit) is used for brewing, the starch that was broken down into sugars in the barley has to be made soluble in water. This process is known as mashing and is the next step shown in process diagram 1. The grit is mixed with water in the mash mixer where it is then transfer into a lauter tun. In the lauter tun, the grit is mixed with the hot water at around 65Â°C, which is the optimum temperature for the enzyme alpha amylase to take action in liquefying the sugars from the starch where they are rinsed from the grit to produce syrup known as wort. This process is very tightly temperature controlled as the enzymes only act within a range of Â±5Â°C before they become denatured and no longer function (Casey 2005). The syrupy product, wort, is then transferred to a kettle for brewing.
Step four in beer production is brewing which occurs inside a piece of equipment called a kettle. Boiling in the kettle is predominantly the most energy intensive process in the entire brewery. Therefore many kettle types have been designed to try to combat this such as equipping the kettle with external heating jackets and internal heating systems, or to quicken the process, wort can be preheated using a plate heat exchanger (Goldammer 2009). As it was, wort used to be boiled in direct-fired kettles of copper due to coppers high heat conductivity, although as heat transfer only occurred at the base of the kettle equipment, this turned out to be inefficient, where the wort could burn. External heating jackets are now more common as well as internal heating systems, as they provide even heat transfer and as the wort flows through the kettle the turbulent flow of the fluid also improves heat transfer. Many kettles now implement external wort boilers, otherwise known as a calandria, which the wort is pumped through. These heat exchanger type systems give a greater surface area to which heat exchange can occur, giving effective heat transfer and therefore greater efficiency in energy consumption (Goldammer 2009). The boiling process within the kettle ensures that bacteria that have aggregated during the process so far are killed off, so as when the fermentation takes place, there is little to no competition for the fermentable sugars, which the enzymes produced during mashing, that the yeast cells will use to convert the sugars to alcohol. Also during the boiling hops are added, certain types at certain times, which also give rise to the flavour of the end product, and reason to differing varieties (Fuller 2007). The addition of hops also helps to preserve the solution thus far. The boiling times vary greatly depending on the flavour you are expecting to achieve, but the purpose is always the same. That is to concentrate and sterilize the wort and hops from bacteria by reaching a temperature of around 100Â°C. And also to boil at a temperature which now denatures the enzymes that have been present and served their purpose of breaking down the starches into simpler sugars (Stint 2008) as they are no longer needed. The sterilized wort is the filtered in a whirlpool separator (as seen on process diagram 1), which usually occurs within the kettle equipment as well, of many of the solids that have coagulated during boiling from the hops (Stint 2008).
Step 5 from the process diagram is the cooling of the filtered wort in a plate heat exchanger. The purpose of this step after boiling is to cool the wort to temperatures tolerable for yeast cells that are used in the fermentation process. The plate heat exchanger uses cooled water which lowers the temperature of the wort to approximately 20Â°C (Stint 2008). The plate heat exchangers are used due to their high heat transfer per unit volume and fine temperature control. Finally air enriched with oxygen is added to dissolve in this cooled wort, to aid in the growth of yeast cells when added (Stint 2008), as yeast optimum conditions require oxygen to respire aerobically, although when oxygen runs out it can also respire anaerobically where at this stage alcohol is still produced as desired.
The next step of the process, fermentation is the most important of the whole procedure. The cooled oxygen rich wort is added to a fermenter, whereby the yeast is also added and is stirred though the mixture to further oxygenate the yeast cells (Vaux 2009). Fermentation is the reaction that converts the simple sugars (glucose) into alcohol and carbon dioxide by the yeast cells, as follows:
The glucose has a higher concentration on the outside of the yeast cells initially then the inside, so enters the cell through simple diffusion whereby the process of glycolysis the glucose is converted into energy (ATP) to allow the yeast cells to reproduce and also produces pyruvates which later get converted in carbon dioxide and ethanol. The 10-step process is shown in process diagram 2.
The optimum conditions for this action to occur is at temperatures of 15-27Â°C, below these temperatures the activity of the yeast cells is greatly reduced and is very slow, above this the yeast cells cannot tolerate and begin to die off. Depending on the alcohol content of the beer product desired, certain yeast strains will be used that can tolerate an alcohol content of approximately 5% (Preedy 2009), as above this concentration the enzymes that the yeast cells use to undergo this reaction will no longer function. The initial oxygen rich environment which lasts depending on the amount of oxygen added from 24-48 hours is where the yeast cells are respiring aerobically and quickly multiply, doubling its population every 4 hours (Preedy 2009). At the point where oxygen is diminished, there is slower activity of the yeast cells and they no longer reproduce and concentrate solely on converting the sugar to alcohol (Preedy 2009). The temperature of the fermentation vessel must be carefully maintained, for optimum yeast cell activity, however as fermentation is an exothermic reaction that releases energy as heat, the vessel therefore must be cooled. For certain types of beer the temperature conditions vary, for example for ales a temperature of 20Â°C, for lagers approximate 10Â°C, each requiring their own yeast strain to work under these conditions and largely determine the quality and character of the beer flavour(Nice 2009), and will be left to ferment for approximately 2-6 weeks. A measure of when the fermentation process is completed is by the specific gravity of the mixture, to determine how much alcohol is present, thereby the alcohol content of the beer (Nice 2009). As well as producing alcohol the fermentation process also carbonates the beer. The fermenter has a small outlet pipe to release carbon dioxide though does not allow air to enter through, preventing contamination. When the specific gravity of the beer has reached a specified level, the CO2 pipe is sealed and the pressure of the vessel increases, due to continuing carbon dioxide production by the yeast cells, where it is then taken up by the beer, carbonating it at an equilibrium level, remaining at this pressure until being packaged and released by the consumer (Nice 2009). Towards the conclusion of the fermentation process the yeast has either settled to the bottom or floated to the top to be removed for reuse in the next batch, for example ales have yeast that float whereas lagers have yeasts that sediment to the bottom of the vessel (Fuller 2007). The yeast is used again and again until mutation causes the yeast to change the flavour of the beer, which therefore makes it unusable. The beer is finally cooled to approximately 0Â°C to sediment any remaining solids including yeast cells and proteins of out the solution, as alcohol freezes at below 0Â°C temperature this therefore remains in solution.
The next stage of the process is maturation, which is for the sole purpose of improving the beer flavour. The beer is moved to a conditioning tank still under pressure, to evolve and mature in flavour. The beer becomes clearer as the final yeast and proteins particles coagulate at the lower temperature. These last sedimentary particles are filtered out through a filtration unit before packaging or pasteurization occurs. This stage is often named the secondary fermentation process, and can last between a few weeks to a few months where extra ingredients, such as honey and spices are added to concentrate the beer to a particular flavour and taste. A final filtration stage takes place using a sheet/pad filtration unit, followed by a small amount of CO2 being pumped through solution to give its last carbonation. The product is then packaged in bottles or kegs for consumer use.
This report will look at improvements for the fermentation bioprocess that occurs in the beer brewing process, where the limitations will also be developed, for the yeast cells undergoing glycolysis in producing alcohol. Also the enzymatic action that breakdown the starches in barley to provide the simple glucose sugars for the fermentation process will be researched for improvement and study, also being a bioprocess to the procedure.
The two main bioprocesses occurring during the whole beer brewing process are the enzyme action of alpha amylase to breakdown the starches in simple sugars for the yeast to perform its bioprocess of fermentation, that is glycolysis of the sugars which provides energy and produces alcohol.
The first bioprocess, the enzyme action of alpha amylase (structure shown in diagram 3), does not require the addition of enzymes to the barley, as these enzymes are already present in the barley grain. This process is exploiting the fact that the enzymes in the barley will breakdown the starches in simple sugars such as glucose naturally, as the grain wants to promote growth and germination. However, as discussed earlier, the germination process is halted before any radical growth can occur. Alpha amylase is a part of a group of enzymes known as hydrolases and is a hydrolytic enzyme that is they require water to break down biomacromolecules into their constituent parts, in this case starch to glucose. Alpha amylase breaks all bonds throughout the starch chain including branches in the presence of water. The optimum conditions for alpha amylase activity are temperatures of around 43Â°C, and a slightly acidic pH of 6.3, although it can function within a pH range of 5.8-7.5, with the best activators for increased activity of Ca2+ and Co2+ (McWethy and Hartman 1977) as shown in graph 1.
If we want to improve on this process, one method could be to genetically modify the activity site on the alpha amylase enzyme to make it faster and more efficient in its reaction rate. This can be done using a few methods. As this enzyme is found in barley grains naturally, a possible method is to genetically modify the barley to produce these specific modified and improved enzymes. This is quite complicated to do and most likely unreliable as to the inserting of this genetic material to ensure it reaches each and every plant that grows barley, throwing up a time consuming problem. Another method would be to add these modified alpha amylase enzymes to the beer brewing process when the activity is occurring at the steeping and mashing stage. They would first need to be grown and collected from an appropriate microorganism, such as bacteria (i.e. a Bacillus or E. coli), that have been genetically modified in themselves to produce the new improved alpha amylase enzyme (Vehmaanperä et al 1987). Currently the process is not reusing the enzymes. If these genetically modified alpha amylases were added, it would be beneficial to reuse them for the next batch to reduce material cost, improve reaction efficiency and extract those naturally occurring in barley. The use of Bacillus strains is common in the production of enzymes, especially alpha amylases, so using this bacterium would be a very appropriate choice to cultivate our required enzyme product in high yields (Vehmaanperä et al 1987). A recombinant DNA plasmid when inserted into a Bacillus strain was found to be highly reliable and stable (Vehmaanperä et al 1987). The best medium to grow these strains of Bacillus for alpha amylase production was a mixture of 6% (w/v) hydrolysed starch and 4% (w/v) corn step liquor (Vehmaanperä et al 1987) by fermentation, with optimum growth during stationary phase. Due to the high stability of the strain, scale up of the process would also be possible and reliable (Vehmaanperä et al 1987). The activity of the enzyme was found to occur over 48 hours, therefore the time between growth and cultivation and use would be in this time range. All clones of the strain of Bacillus showed characteristic that the recombinant plasmid for modified alpha amylase was retained in growth and replication (Vehmaanperä et al 1987). This would therefore prove further that the production of this enzyme at an industrial scale is possible.
The Fermentation process occurring in brewing beer is by a specified yeast strain for brewing, that undergoes glycolysis to produce energy and also alcohol from the simple glucose sugars broken down from the starch in barley by alpha amylase. Similar to the genetic modification of the enzyme, alpha amylase, a yeast mutant with near perfect fermentation of the sugars to be fermented for the beer brewing process could be made. An improvement for these needs to be found that will have a high increase in sugar fermentation whilst keeping the same characteristics and same conditions required for growth, which would also shorten the maturing stage of the brewing process. What is known is that "â€¦brewing yeasts are unable to utilize the oligosaccharides and maltotriose contained in wort efficientlyâ€¦" (Liu 2008), so a yeast that could perform this function efficiently and quickly would be of great use and produce a much higher yield in beer product. The main reason common yeasts do not ferment the maltotriose in wort is that yeast prefers to take up glucose sugars before maltose or maltotriose and will only take these up until glucose is completely used up, where it is then a slow process. However of the sugars, maltotriose is the second most common in wort and to ferment these would be greatly beneficial (Liu 2008). Therefore a major improvement to the fermentation process in beer brewing would be to create the recombinant yeast strains which has the material to be able to ferment the glucose sugars as well as the maltose and maltotriose sugars which would not only improve efficiency but improve revenue for the company.
As yeast performs glycolysis on these sugars which forms energy and alcohol as another product, the maltose and maltotriose must first be converted to the glucose subunit before being used in glycolysis. The yeast cell has the ability to take up these sugars in their larger saccharide state, although this process is slow and rate limiting to the reaction (Liu 2008). By further genetic modification it would be possible for the yeast cells to breakdown these maltotriose and maltose sugars, or possibly by using enzymes capable of this, and once done the fermentation of these sugars is possible. Using this mutant strain of yeast, it showed an increase of 40.5% fermentation in maltotriose alone, which would increase brewing revenue by an increase in product (Liu 2008). Most importantly it also showed that the quality of the beer product was not affected by these new properties and conditions, still keeping its characteristic flavour and taste. An overall increase of 13.4 % was seen in wort fermentation and a considerably shorter maturation period by almost half, this also would lower operating costs for fermentation (Liu 2008). A strain suitable for industrial beer production is what is essentially needed.
The bioprocesses involved in the brewing of beer are the enzyme action of alpha amylase in the breakdown of starches in the barley grain to produce simple sugars such as glucose, and also the fermentation of yeast strains from wort to produce the alcohol in the beverage. Improvements to these processes that would not only improve efficiency but also yield of product and therefore revenue for the brewing company, are the genetic modification of the alpha amylase in improving the activity site for the enzyme to be more efficient, and lastly mutangenizing the yeast strain to break down more sugars in the wort and improve fermentation capacity as well as yield.