adding new flavourzyme post

Author: CamDavidsonPilon

_posts/2021-06-04-flavourzyme-a-purified-enzyme-mixture-from-a-oryzae.md

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+---
+title: 'Flavourzyme: a purified enzyme mixture from A. oryzae'
+author: keith
+image: assets/images/flavourzyme/Cover.png
+---
+
+Flavourzyme™️ is a trademark name given to a mixture of refined proteolytic enzymes branded by Danish biotech company Novozymes A/S. It's produced by the submerged fermentation of our friend *A. oryzae* - in this post we'll look at what it is, and how it's made. 
+
+### What is Flavourzyme?
+
+The modern food processing industry uses proteolytic everywhere: from accelerating cheese aging, modifying breads, as a flavouring (any time you see "hydrolysed vegetable protein"), processed meats, in detergents... the list goes on and on. 
+
+Flavourzyme is a specific mixture of proteolytic enzymes extracted from an *Aspergillus oryzae* liquid culture. What sets it apart from other industrial proteolytic enzymes is that it contains a cocktail of both endo- and exo-peptidases that can efficiently convert proteins into something with more umami, making it useful for applications requiring the development of flavour [1].
+
+Why does this matter? If a protein is fully decomposed into amino acids, it tends to taste nice and savoury - even if certain amino acids have a slightly bitter taste. However, if a protein is only partially decomposed, certain peptides can be extremely bitter: for example, the free amino acids Leucine (Leu) and Phenylalanine (Phe) are slightly bitter (with a tasting threshold of 15-20mM), but their simple dipeptide forms, Leu-Leu and Leu-Phe, are **ten times** more bitter [2]. 
+
+![how endo and exo peptidases act on proteins](/assets/images/flavourzyme/Endo-Exo-Peptidases.png){:height="550" .center}
+*Simplified diagram of the action of endo- and exo-peptidases*
+
+This is why Flavourzyme works well in food applications requiring umami: it contains endopeptidases, which snip peptide bonds in the **middle** of a peptide chain, as well as exopeptidases, which snip peptide bonds at the **terminal ends** of a peptide chain.
+
+Working together in the correct proportions, you end up with an enzyme cocktail that yields plenty of amino acids without accumulating short, bitter peptides. 
+
+
+<div class="callout">
+💡 A quick sidenote on proteolytic enzymes: first of all, endopeptidases are typically very specific - they will cut only a specific bond at a specific pH (for example, Trypsin cuts ONLY after Arg OR Lys UNLESS followed by Pro). Secondly, every time an endopeptidase cuts, it frees up two new terminal ends for exopeptidases to cut. Exopeptidases that cut on the amino end are called aminopeptidases, and those that cut on the carboxy end are called carboxypeptidase. Exopeptidases can also cut at the terminal peptide bond, one or two bonds away from the terminal peptide bond, or right in the middle of a dipeptide. 
+</div>
+
+
+But wait - how do you make it? And how on earth do you grow *A. oryzae* in a liquid?
+
+### Growing *A. oryzae* in a liquid - upstream processing
+
+In bioprocess engineering, the process of cultivating a culture is called upstream processing (refining the resultant products is called downstream processing). For *A. oryzae*, the process can be a little more involved than growing unicellular organisms such as yeast or bacteria. 
+
+Information here is based off a paper from researchers at the University of Denmark, working at the Novozymes A/S Fermentation Pilot Plant, who wanted to develop a mathematical model for enzyme production with *A. oryzae* in submerged culture [3]. Even though they scrubbed some critical units and sig-figs from their research (for proprietary reasons), it was still enough to piece together details of the process. The process is similar to any other submerged, septic, aerated culture:
+
+![](/assets/images/flavourzyme/Bioreactor.png){:height="450" .center}
+*Process diagram of an aerated bioreactor*
+
+A seed train is propagated from a pure culture of A. oryzae and then added to an aerated, stirred bioreactor and allowed to grow. Once the culture reaches a certain point, the bioreactor is harvested to yield a crude mixture of enzymes, mycelia, and leftover substrate.
+
+There's a few quirks to this specific culture method, which applies to some other filamentous fungi fermentations:
+
+#### 1. Use a fed-batch configuration
+
+The bioreactor is primed with substrate and inoculum around 70% full, then additional substrate is slowly dosed in. This is done so that substrate is consumed at basically the same rate that it's fed into the bioreactor, which keeps glucose in the environment very low. As a result, enzyme yields are high and plenty of oxygen is available in the medium (the culture is substrate-limited rather than oxygen limited).
+
+#### 2. Filamentous fungi make everything viscous 
+
+Filamentous fungi don't normally grow in a submerged environment. The mycelia will aggregate to form pellets or clumps - which reduces substrate uptake and enzyme secretion. To complicate things, a bioreactor full of filamentous mycelia will make the mixture more and more viscous. This makes stirring harder, and also reduces the ability of air bubbles to mix and for oxygen to dissolve. By necessity, oxygen transfer drops as the mycelia grows. 
+
+#### 3. Use a pulsed-paused feeding mode 
+
+In addition to using a fed-batch setup, researchers have also found that pulsed-paused feeding, in which feeding of the substrate is turned on/off intermittently in 300s cycles, improves the fungi's morphology. The rationale is that under starvation considerations, fungal mycelium are known to autolyse (self-digest) older parts of the network to re-allocate resources to the growing tip. While this mechanism is yet to be studied in detail, it's been shown that you get better morphology and lower viscosity with pulsed-paused feeding, without sacrificing enzyme productivity [4]. 
+
+Once the upstream process is finished, you end up with a crude liquid containing fungi, enzymes, spent substrate, and all sorts of metabolites.
+
+### Refining the enzymes - downstream processing
+
+In *downstream processing*, we want to refine out the enzymes only. While I couldn't find any literature specifically on the downstream processing of flavourzyme, reading textbook on enzymes was enough to cover the basics [5]. Then, a producer of a Flavourzyme-like product on Alibaba just gave me their HACCP flow diagram, so that made things easy. Here's one example of how it can be done:
+
+#### 1. Filter Press 
+
+The first step is to filter out the mycelia, cell debris, any any other large solids. A *filter press* is used here with perlite, followed by diatomaceous earth, as a filtering aid. The solid matter is captured in the filter aid and removed as filter cake - leaving behind an enzyme mixture. 
+
+![](/assets/images/flavourzyme/Deadendfiltration.png)
+*Schematic of dead-end filtration with a filter aid - elements not to scale*
+
+Note that filtration is the first step because we are dealing with **extracellular** enzymes. If the product was an **intracellular** enzyme, this step would be preceded by a 'disruption' step to break apart cells. 
+
+#### 2. Ultrafiltration Concentration 
+
+At this point, our enzyme mixture is still at a fairly low concentration. Another type of filtration called *ultrafiltration* is used to remove water. By passing the liquid across a filter of the appropriate size (in this case, a filter rated around 10kDa), water will permeate through, leaving behind a more concentrated enzyme solution. This can be repeated until the desired concentration is achieved.
+
+![](/assets/images/flavourzyme/Ultrafiltration.png)
+*Schematic of cross-flow ultrafiltration - elements not to scale*
+
+#### 3. Final Steps
+
+I won't go into too much detail here, but the final mixture can either be dried by spray drying with the help of a carrier solid (typically sodium chloride), or packaged as a liquid after passing through a sterilizing filter and adding stabilizers and preservatives.
+
+### The Final Product
+
+If everything's gone well so far we should have a powerful enzyme mixture rated for 500 LAPU/g (one LAPU is the amount of enzyme which hydrolyzes 1 µmol of L-leucine-pnitroanilide per minute - this is one of many enzyme assays for proteolytic enzymes) and between 6% w/w of pure enzyme protein, depending on formulation [6]. By comparison, unrefined shoyu koji contains around 0.2% w/w protease [7].
+
+Novozymes A/S won't sell to non-commercial or non-institutional end-users. So naturally, I'd have to import an imitation product from Alibaba. The other question is, of course, can I brew this at home?
+
+### References
+
+1. Merz, M.; Eisele, T.; Berends, P.; Appel, D.; Rabe, S.; Blank, I.; Stressler, T.; Fischer, L. Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes. *Journal of Agricultural and Food Chemistry* **2015**, *63*. https://doi.org/10.1021/acs.jafc.5b01665.
+
+2. Maehashi, K.; Huang, L. Bitter Peptides and Bitter Taste Receptors. *Cell Mol Life Sci* **2009**, *66* (10), 1661–1671. https://doi.org/10.1007/s00018-009-8755-9. 
+
+3. Albaek, M. O.; Gernaey, K. V.; Hansen, M. S.; Stocks, S. M. Modeling Enzyme Production with Aspergillus Oryzae in Pilot Scale Vessels with Different Agitation, Aeration, and Agitator Types. *Biotechnol Bioeng* **2011**, *108* (8), 1828–1840. https://doi.org/10.1002/bit.23121.
+
+4. Bhargava, S.; Wenger, K. S.; Marten, M. R. Pulsed Feeding during Fed-Batch Aspergillus Oryzae Fermentation Leads to Improved Oxygen Mass Transfer. *Biotechnol Prog* **2003**, *19* (3), 1091–1094. https://doi.org/10.1021/bp025694p.
+
+5. *Enzymes in Industry: Production and Applications*, 3rd ed.; Aehle, W., Ed.; Wiley-VCH: Weinheim, 2007.
+
+6. Flavourzyme Product Data Sheet - Novozymes A/S. 
+
+7. *Koji Studies*, 6th ed.; Hideya, M.; The Brewing Society of Japan: Tokyo, 1986.