The Art and Science of Food Alchemy: Innovations in Enzyme Applications from Cheesemaking to Brewing

Think about the last time you went to a party or a potluck; what did you eat and drink? Was there a cheese platter with a variety of milky and savory flavors? Maybe there was homemade sourdough bread, an apple pie with a crisscross lattice topping, or some cookies hot and fresh out of the oven. There might have even been commercial or homebrewed beer.

Enzymes are involved in making all of these delicious treats, and many more!

Traditionally, these enzymes come from the source ingredients that make the product. For instance, the enzymes used to make bread come from flour and yeast. However, additional enzymes are frequently added in commercial and artisanal applications to:

• make the production process more reproducible.
• provide new textures and flavors.

Adding extra recombinant enzymes can make the production process more reproducible. The amount of any given enzyme in flour, for example, can vary based on what type of flour you use. By adding a defined amount of recombinant proteins, you make the production process more consistent and reproducible time after time.

Adding additional enzymes can confer different textures and densities. Think about bread, cookies, and pie crust. They each have different densities and elasticities. Yet they all use flour and therefore flour derived enzymes.

However, adjusting the types and amounts of recombinant enzymes added can provide different physical properties to the dough that is amenable for making each distinct baked good.

Furthermore, solely relying on the enzymes in source ingredients limits the taste and mouthfeel of beer. Testing similar enzymes from different species allows brewers to explore different tastes and textures for their brews.

Article Table of Contents

Cheese making

Baking

Brewing

References

Cheese making

Humans have been making cheese for millennia. The earliest physical evidence of cheese comes from Egyptian tombs and is estimated to be four to five thousand years old.

However, cheese making likely occurred well before that as cheese is mentioned in the ancient Greek epic The Odyssey, which itself is estimated to be ten thousand years old.

One origin story for cheese is that a traveler was storing milk in a ruminant animal’s stomach. Ruminant animals are mammals such as cows and sheep that chew cud regurgitated from their rumen or first stomach.

According to this story, the desert heat and the rhythm of the camel’s stride converted milk into whey and cheese curds inside the ruminant stomach pouch.

This version of the cheese origin story is consistent with how cheese has been made in more recent times. Cheese makers used rennet, a complex enzyme mixture from the digestive juices of calves, to coagulate milk into cheese curds.

Figure 1. Top, chymosin (green) cuts milk casein protein hairs (purple) to form cheese curds (PDB: 1CZI). Bottom, hairless casein globules aggregate together forming cheese curds.

Nowadays, approximately 5% of cheese is still made using rennet whereas 95% of cheese is made using recombinant chymosin purified from genetically-modified bacteria (Yacoubou, 2008).

Recombinant chymosin is preferred by most cheese makers because it is cheaper, purer, more easily and reliably sourced, and converts milk into cheese more reproducibly than rennet.

There are a few artisanal cheese makers that continue to use rennet, particularly for aged cheddar and Parmesan cheeses, because they feel that recombinant chymosin does not recapitulate the full flavors that rennet brings out in these cheeses (Yacoubou, 2008).

Baking

If you are an experienced baker, or know one, then you know that developing dough is serious work! There are many different dough development techniques including kneading, fraisage, folding, mechanical, and passive. Distinct dough types - such as various breads, pie crust, and pasta noodles, for example – turn out best with particular developmental techniques and times.

You might be wondering, “why is this so complicated?!?” Essentially, it all comes down to preparing the right physical properties for the type of dough that you are making. Key to this process is breaking down carbohydrates and proteins in the ingredients to form dough with the appropriate stretchability and strength.

Ingredients such as flour and yeast provide all of the enzymes that are required for baking. For example, alpha- and beta-amylases from yeast break down long oligosaccharides, or starches, into simple sugar molecules (Figure 2) which slows down the rate at which the bread goes stale.

Figure 2. Alpha-amylase cuts carbohydrates into simple sugar molecules. Structure of alpha- amylase (purple) with bound glucose molecules (orange) (PDB: 1MXD).

In commercial baking, additional recombinant enzymes, besides those present in flour and yeast, are often added to:

• standardize the baking process.
• confer new properties or characteristics on the baked good.

Both naturally occurring and added recombinant enzymes modify the material properties of dough and change gas retention, which impacts the density and texture of the resulting baked goods.

For instance, proteases are used to reduce gluten elasticity and dough shrinkage when making cookies and other baked goods. Extra proteases are added in the mass production of cookies to form cookies with very reproducible shapes and sizes.

The type and amount of enzymes added will depend on what type of product is being made. Proteases, mentioned above, help produce the same size cookies over and over. However, this would not be a good additive for pie crust, for example, where the elasticity and stretchability of the dough plays an important functional role in shaping the crust around the fillings.

Therefore, by tailoring the type and amount of enzymes added, and varying how long they are allowed to function, bakers can sample a range of different textures and physical properties required for different types of baked goods.

Brewing

Like cheese, beer and other fermented drinks are part of our culture and social experiences, and the art of producing these drinks includes some significant science!

Three main types of proteins are involved in brewing:

• amylases which convert starch into fermentable sugars.
• proteases which cut proteins into a nitrogen source for yeast.
• beta-glucanases which degrade the endosperm cell wall.

Traditionally these enzymes are supplied through the barley or other grains used in brewing. The initial brewing steps vary temperature to sequentially activate proteases, beta-glucanases, and beta-amylases and alpha-amylases, in that order.

However, additional external proteins are sometimes added to the brewing process to provide additional functionalities, and to sample different taste and mouthfeel profiles.

Isinglass, which consists mainly of collagen protein purified from fish swim bladders, clarifies beer by removing yeast and molecular particles.

Brewers Clarex^® is a protease that cleaves proteins after proline residues to prevent chill haze in beers and is used to produce gluten-free beers.

Chill haze is when molecules in the beer aggregate together when the beer is stored at cold temperatures near freezing. Although chill haze does not change the taste of beer, it causes a cloudy visual appearance that is undesirable for many types of beers.

Alpha-amylases from different organisms have different activities at various pH values and temperatures. So, brewers often experiment with adding different alpha-amylases to see how the exogenous proteins change the taste and feel of various types of beer.

Table 1. Proteins involved in cheesemaking, baking, and brewing.

While cheese, baked goods, and beer are three common examples where enzymes play an important role in their production, there are many others including:

• fermenting cocoa beans in chocolate production.
• tenderizing meat.
• enhancing meat substitute flavors.
• fruit juice clarification.

The next time you are enjoying a slice of cheese, a warm baked good, or a refreshing cold beer, you can think about how enzymes, both naturally occurring and added recombinant versions, help produce these delicious treats!

References

Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., & Bourne, P. E. (2000). The Protein Data Bank. Nucleic acids research, 28(1), 235–242. https://doi.org/10.1093/nar/28.1.235

Berman, H., Henrick, K., & Nakamura, H. (2003). Announcing the worldwide Protein Data Bank. Nature structural biology, 10(12), 980. https://doi.org/10.1038/nsb1203-980

Carpenter, D. (2015). Fun with Enzymes. doi: https://beerandbrewing.com/fun-with-enzymes/

Cheng Z, Xian L, Chen D, Lu J, Wei Y, Du L, Wang Q, Chen Y, Lu B, Bi D, Zhang Z, Huang R. Development of an Innovative Process for High-Temperature Fruit Juice Extraction Using a Novel Thermophilic Endo-Polygalacturonase From Penicillium oxalicum. Front Microbiol. 2020 Jun 12;11:1200. doi: 10.3389/fmicb.2020.01200. PMID: 32595621; PMCID: PMC7303257.

Gilliland, G. L., Winborne, E. L., Nachman, J., & Wlodawer, A. (1990). The three-dimensional structure of recombinant bovine chymosin at 2.3 A resolution. Proteins, 8(1), 82–101. https://doi.org/10.1002/prot.340080110

Groves, M. R., Dhanaraj, V., Badasso, M., Nugent, P., Pitts, J. E., Hoover, D. J., & Blundell, T. L. (1998). A 2.3 A resolution structure of chymosin complexed with a reduced bond inhibitor shows that the active site beta-hairpin flap is rearranged when compared with the native crystal structure. Protein engineering, 11(10), 833–840. https://doi.org/10.1093/protein/11.10.833

Hickman, D. S., T.J.; Miles, C.A.; Bailey, A.J.; de Mari, M.; Koopmans, M. (2000). Isinglass/collagen: denaturation and functionality. J Biotechnol., 79(3), 245-257.

Kao, L., Krstenansky, J., Mendell, J., Rammohan, K. W., & Gruenstein, E. (1988). Immunological identification of a high molecular weight protein as a candidate for the product of the Duchenne muscular dystrophy gene. Proceedings of the National Academy of Sciences of the United States of America, 85(12), 4491–4495. https://doi.org/10.1073/pnas.85.12.4491

Kay, L. (2016). Brewers Clarex^TM: The Gluten Enzyme. Best Gluten Free Beers.https://bestglutenfreebeers.com/the-gluten-eating-...

Landegren, U., & Hammond, M. (2021). Cancer diagnostics based on plasma protein biomarkers: hard times but great expectations. Mol Oncol, 15(6), 1715-1726. doi:10.1002/1878-0261.12809

Lima, C. O. C., De Castro, G. M., Solar, R., Vaz, A. B. M., Lobo, F., Pereira, G., Rodrigues, C., Vandenberghe, L., Martins Pinto, L. R., da Costa, A. M., Koblitz, M. G. B., Benevides, R. G., Azevedo, V., Uetanabaro, A. P. T., Soccol, C. R., & Góes-Neto, A. (2022). Unraveling potential enzymes and their functional role in fine cocoa beans fermentation using temporal shotgun metagenomics. Frontiers in microbiology, 13, 994524. https://doi.org/10.3389/fmicb.2022.994524

Linden, A., Mayans, O., Meyer-Klaucke, W., Antranikian, G., & Wilmanns, M. (2003). Differential regulation of a hyperthermophilic alpha-amylase with a novel (Ca,Zn) two-metal center by zinc. The Journal of biological chemistry, 278(11), 9875–9884. https://doi.org/10.1074/jbc.M211339200

Liu, X., Wu, Y., Guan, R., Jia, G., Ma, Y., & Zhang, Y. (2021). Advances in research on calf rennet substitutes and their effects on cheese quality. Food Res Int, 149, 110704. doi:10.1016/j.foodres.2021.110704

Miguel, A. S. M., da Costa Figueiredo, E.V., Lobo, B.W.P., Dellamora-Ortiz, G.M. (2013). Enzymes in Bakery: Current and Future Trends. In Food Industry.

Mohd Azmi SI, Kumar P, Sharma N, Sazili AQ, Lee SJ, Ismail-Fitry MR. Application of Plant Proteases in Meat Tenderization: Recent Trends and Future Prospects. Foods. 2023 Mar 21;12(6):1336. doi: 10.3390/foods12061336. PMID: 36981262; PMCID: PMC10047955.

Palmer, D. S., Christensen, A. U., Sørensen, J., Celik, L., Qvist, K. B., & Schiøtt, B. (2010). Bovine chymosin: a computational study of recognition and binding of bovine kappa-casein. Biochemistry, 49(11), 2563–2573. https://doi.org/10.1021/bi902193u

Phillip, M. (2021, September 29). Why kneading isn’t always the best way to develop bread dough. King Arthur Baking. https://www.kingarthurbaking.com/blog/2021/09/29/w...

The PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger, LLC.

Thesseling, F. A., Bircham, P. W., Mertens, S., Voordeckers, K., & Verstrepen, K. J. (2019). A Hands-On Guide to Brewing and Analyzing Beer in the Laboratory. Current protocols in microbiology, 54(1), e91. https://doi.org/10.1002/cpmc.91

Yacoubou, J. (2008). An Update on Rennet. Vegetarian J, 3. https://www.vrg.org/journal/vj2008issue3/2008_issu...

Zhao, D., Huang, L., Li, H., Ren, Y., Cao, J., Zhang, T., & Liu, X. (2022). Ingredients and Process Affect the Structural Quality of Recombinant Plant-Based Meat Alternatives and Their Components. Foods (Basel, Switzerland), 11(15), 2202. https://doi.org/10.3390/foods11152202