Plant Proteins

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Plant Proteins - How to Improve their Functional Properties with Enzyme Know-How www.biocatalysts.com Plant Proteins - How to Improve their Functional Properties with Enzyme Know-How Authors: Andrew Ellis, Technical and Compliance Director at Biocatalysts Ltd Carolyn Pritchard, Technical Marketing Manager at Biocatalysts Ltd

This white paper reviews the continued trend for incorporating plant protein into food products and how enzymes can improve their versatility and functionality including: flavour enhancement and solubility. With a summary of how soy, wheat, pea and rice hydrolysates are being utilised in food formulations to meet the market trends and growing health concerns and sustainability issues. Additionally we include a brief nod to what is coming next in the field of protein. Contents

03 Intoduction

04 Enzymes Unlock the Benefits

05 Established Plant Protein Hydrolysates

06 Plant Protein Hydrolysates - Conclusion and Outlook

07 References

www.biocatalysts.com 2 substrate concentration and enzyme dosage. Some of these modifications can be Introduction highly exquisite leading to highly valuable protein or protein-derived ingredients or Proteins are one of the essential components for growth and maintenance of components. In general, the greater the modification of the protein the higher the the human body, driving key functions to ensure good health. Traditionally (in the intrinsic (market) value of the protein; we describe in Figure 1 this as the “plant Western World), protein is consumed from animal derived sources such as meat, protein value chain”. eggs and dairy, but this is changing. The growing global population, consumer trends towards vegetarian and vegan foods and increasing concern for nutritional health are all drivers for exploitation of alternative sustainable sources of food Figure 1. Plant Protein Value Chain protein. A range of new solutions are required to meet the growing global demand for protein including algae, single cell protein, insects and new sustainable crops. While these novel protein sources are still being investigated, plant proteins such as soy, corn, wheat, pulses and lentils and more recently pea are being utilised in novel ways to introduce them into our diet. This drive to incorporate plant protein into everyday food stuff will begin to address the global needs.

Over the past decade, the increasing demand for plant-based protein has driven an explosive demand for “plant protein isolates”; these ingredients are incorporated into foodstuffs to enhance the protein content and quality. These protein isolates are extracted from their plant source, through processing to separate from other food macromolecules, namely carbohydrates and lipids. This separation can be achieved through one or more of the following processing methods: mechanical, chemical, thermal or enzymatic. When enzymes are used as a method for separation, this typically means enzymatic hydrolysis of the non-protein components, leaving the protein intact and easier to separate from the other components. For example, this has been demonstrated using different enzymes from Novozymes for extraction of oat protein using cellulase (Viscozyme® L) and rapeseed protein using pectinase (Pectinex® Ultra SP-L)1,2,3.

In addition to assisting protein extraction, enzymes can be used to make broad or very specific modifications to plant proteins (post isolation) to enhance their functional, nutritional and chemosensory properties. By far the most significant, in terms of volume of enzyme used for modifying food protein, are the proteases (peptidases) which act by breaking down (hydrolysing) the protein structure. In the process of hydrolysing these large molecules, functional amino groups and hydrophobic patches on the globular surface of the protein are exposed. This process also reduces the molecular weight of the resulting protein or peptides. The properties of the hydrolysed proteins (hydrolysates) are largely dependent on the type and specificity of enzyme used and the degree of hydrolysis (DH); the degree of hydrolysis being affected by the temperature, pH, process time

www.biocatalysts.com 3 Table 1. Some examples of commercial enzymes in the manufacture of plant protein Enzymes Unlock the Benefits hydrolysates16, 17, 18, 19. Endopeptidases (endoproteases) hydrolyse plant proteins by acting on ENZYME SUPPLIER TYPE SUBSTRATE EXAMPLE BENEFITS non-terminal amino acids (they tend to act away from the ends of the protein chain). There are numerous endopeptidases that have been applied to the Alcalase® Novozymes Alkaline protease Pea Broad spectrum hydrolysis of plant proteins including trypsin, bromelain, papain, subtilisin protease and fungal neutral endopeptidase. The hydrolytic effect and efficiency of Promod™ 24L Biocatalysts Ltd Casein protease Gluten Low DH with good each endopeptidase is determined by its intrinsic specificity. Commercial enzyme taste and solubility preparations can be highly specific. For example, porcine trypsin hydrolyses only Promod™ 950L Biocatalysts Ltd Microbial & Sulphite- Soya Low molecular weight at the carboxy terminal side of arginine or lysine4 in the protein substrate, whereas Free Alternative to peptides increase Papain solubility papain, containing four different cysteine endopeptidases exhibits broad specificity5. Glutaminase Amano Glutaminase Pea Improve solubility of Exopeptidases catalyse the cleavage of the terminal (last) or next-to-last peptide Amano 500 protein at low pH bond from the termini of a plant peptide or protein, releasing a single Tolerase® G DSM Proline specific Gluten Work at low pH amino acid or dipeptide. Examples of exopeptidases include dipeptidase and endopeptidase resistant to pepsin digestion leucine amino peptidase from Aspergillus oryzae. In most cases, for exopeptidases to work on their target peptide bond the intact globular protein requires pre- or COROLASE® AB Enzymes metalloprotease & Soya Broad spectrum 2TS serine protease protease parallel treatment with endopeptidases (and/or thermal, chemical or physical processing) to open up the globular structure and expose the termini.

Often, synergistic effects can be sought through use of combinations of proteases 6,7 from different biological sources used sequentially or in parallel . Such synergism Figure 2. Enzymes ability to can reduce the processing time (producing improved properties faster; thereby unlock numerous innovative leading to cost reduction) and yield different or enhanced properties (relative to the and valuable benefits performance of enzymes derived from a single biological source). Some examples through hydrolysis of of commercial enzymes used for hydrolysing plant proteins are described in plant proteins. Table 1.

In order to access the required benefits from hydrolysis from any given plant protein, one must carefully select the most suitable peptidase(s) and the most suitable hydrolysis conditions such as pH, temperature and incubation time. The know-how, to inform such process and product development is beyond the scope of this review. Suffice it to say, that companies active in the development and manufacture of such hydrolysates often work closely with enzyme companies to realise one or more of the many benefits outlined in Figure 2.

www.biocatalysts.com 4 foaming and emulsification) enabling its utilisation as an ingredient in food, health Established Plant Protein Hydrolysates and sports nutrition products as well as in animal feed and cosmetic products.11

Soy Protein Hydrolysate The type of gluten hydrolysate produced is The first plant protein to be used specifically as very dependent on the type of protease used. an alternative to animal protein was soy. Soy has A bacterial neutral endopeptidase can achieve become an increasingly popular alternative a high DH and improve the solubility to >70% source of protein, especially for people total protein content, this results in a highly allergic to milk protein. Soybeans have great digestible protein source suitable for elderly potential for use in human food due to their or infant nutritional drinks and feeding young significant levels of high-quality protein and their animals. Due to the high content of glutamic acid unique functional and nutritional properties. present in gluten the use of an additional enzyme; Soy protein supplies all nine essential glutaminase during hydrolysis will enhance the amino acids and provides many functional flavour of wheat gluten hydrolysates producing benefits that have been widely embraced by the food industry. Soy an umami flavour. These hydrolysates can be used as flavour agents in products protein is most commonly used to supplement protein content in vegan meals, like dry soup mixes, stock cubes and seasoning mixes for snack foods and such as burgers and soups, in addition to aiding functions such as water sausages. There is also opportunity to use these properties in the aquafeed retention and adding a savoury flavour to vegan and vegetarian products. industry and pet food as palatents.

Historically, soy protein hydrolysates (SPH) were produced by chemical hydrolysis Pea Protein (acid HVP) as a cheap way to produce a product, but it had disadvantages. Soy Pea is becoming an increasingly popular substrate acid HVP had a high salt and monosodium glutamate (MSG) content and can be for manufacturers for producing plant-based carcinogenic. Using enzymes to hydrolyse soy (eHVP) meant it was a more natural protein ingredients. Pea protein has many benefits process, contained no carcinogens and the levels of salt and MSG could be for consumers, customers and farmers; as a great 8 controlled and reduced . eHVP is the earliest example of an enzyme source of protein that can be produced in an being used to produce hydrolysed plant protein. Improved knowledge of the environmentally friendly way, that provides a protein extraction of plant proteins to produce concentrates and isolates has also hydrolysate which is non-GMO, vegetarian/vegan, meant improved eHVP. Hydrolysis of isolates has allowed for improved low allergenicity and sustainable. Pea protein is solubility and taste providing better functional properties that can be exploited for food extracted from yellow pea and is promoted as ingredient applications. SPH have also been shown to have enhanced antioxidant an alternative to whey, animal-derived and other activity which could be used in a wide range of foods as a useful functional food plant-based proteins. Pea protein is rich in lysine, arginine and branched chain 9 ingredient. amino acids. Indeed, pea protein is said to be the richest source of arginine (about 8.7% on a protein weight basis)12. The pea market is predominantly made up of Gluten Hydrolysate protein isolates and concentrates. Pea protein isolates and concentrates are used in Wheat is one of the most commonly consumed cereal grains, its protein content supplementing additional protein values in meat products, cereals, bakery products, is usually between 7 to 20% with gluten accounting for around 80% of its total sports bars and drinks, and in meat analogue products. protein content.10 Gluten is an important co-product in the recovery of wheat starch in the wet processing of wheat flour. Gluten is a good nutrient source but it’s low Pea protein hydrolysates have been used in limited food categories over the dispersibility/solubility makes it very difficult to handle. Enzyme hydrolysis of wheat past five and a half years. The functional benefits of pea hydrolysates are gluten improves its solubility and other functional properties (water binding, foaming beginning to emerge and are slowly being introduced into new applications.

www.biocatalysts.com 5 Due to its improved solubility pea protein hydrolysate is currently added in to vegan gummies to replace animal gelatin making confectionery a significant category Plant Protein Hydrolysates - for hydrolysates. Additionally, this functionality enables the pea protein hydrolysate to provide an excellent base for frozen products such as desserts and ice Conclusion and Outlook cream. More recently, leading innovators in this market are looking to utilise the improved solubility of pea protein hydrolysates in the production of beverages We have discussed in this paper the continued drive to replace animal protein 13 and shakes to supplement natural protein levels in the product . with plant protein, how plant proteins and their hydrolysates are providing fantastic benefits and functional properties that enhance food products. Rice Protein Hydrolysate Rice protein is frequently combined with pea protein powder, due to their We are seeing a pipeline of such products being released into the market by a complementary nutritional profiles. Rice protein is high in the sulphur containing significant number of western-based companies; how these plant-based products amino acids, cysteine and methionine, but low in lysine. Whereas pea protein are promoted for their health benefits and eco-friendly origins. Enzymes can is low in cysteine and methionine but high in provide high value benefits in the production of plant protein hydrolysates as well lysine. Therefore, combining the two plant as enhancing their functional properties that can be exploited to create nutritionally proteins offers a superior amino acid profile beneficial foods. that is comparable to dairy or egg proteins, but without the potential implications of allergies. Pea and rice are the current new kids on the block driving the novel inclusion of plant Additionally, rice protein has a higher NPU (Net protein hydrolysates in a growing range of products, but even soy and gluten are Protein Utilisation) and BV (Biological Value) than seeing some resurgence utilising the synergistic effects of novel enzyme to produce other vegetable proteins, which means highly specific protein modification, such as reducing the allergenicity of gluten. it is absorbed by the human body better than a pea protein or soy protein, for example. These It is certain the protein market will become more diverse in the coming years, superior benefits of rice protein make this vegetable the current trend towards high protein vegetable food ingredients will drive this protein a highly valuable and popular alternative to change. Only time will tell if alternative protein sources such as rapeseed, hemp and animal-derived protein.14 lupin will become as popular as soy and gluten or even pea and rice. The use of enzymes to further process these protein sources to produce higher functioning Rice protein hydrolysates are easily digestible, have good solubility in water and a hydrolysates is key to their adoption into the mainstream food ingredients market. bland flavour, they are gluten free and non-allergenic. These properties along with its Looking beyond plant protein, the next wave of innovative protein is already being amino acid profile being similar to milk make rice hydrolysates ideal as an investigated and developed from sources such as; insects, algae, single cell protein, ingredient in infant formula. A study by Reche 201016 demonstrated that 90% of poultry feathers and mesopelagic fish are all options being considered. You can infants allergic to cow’s milk could tolerate rice protein hydrolysates. It’s solubility be sure that enzymes will play a pivotal role in processing these protein sources to particularly at low pH’s lends itself to enriching juice-based beverages and its other produce functional foods. health-based benefits means that rice hydrolysates are used in sports nutrition and products designed for weight loss. Rice hydrolysates are also useful in applications Biocatalysts Ltd has over 35 years experience developing and manufacturing where a medium protein content is required in combination with a low level of enzymes to meet customer and market demands and trends. 25 years ago, we hydrolysis to enhance molecular weight distribution. developed a specific series of protease enzymes to hydrolyse soy to produce a hydrolysate and are currently providing proteases to produce pea protein hydrolysates. We are also involved in a number of projects specifically looking at developing existing and novel enzymes for existing and novel protein sources.

www.biocatalysts.com 6 Our enzyme knowledge enables us to manipulate the specific activity of enzymes to improve the functionality of a product, be it solubility, temperature stability, enhanced 512. Arla (2017) Research on the effect of papain coâextrusion on pea protein and enzymolysis antioxidant peptides. [online] http://europepmc.org/abstract/AGR/IND605872230 [Accessed 18th Feb. 2019] gelling properties, emulsifying properties, reduced allergenicity or digestibility. The 13. Zhou, Q. Feng, C. Liu, N. (2017) Protein beverages turn crystal-clear. [online] https://www.arla- vegetable protein sector is undergoing a transformation and we will be working foodsingredients.com/industries/sport-nutrition/cap-2---sport-beverages/ [Accessed 18th Feb. 2019] diligently to support our customers through the process. 14. Fang, Y. Chen, X. Luo, P. Pei, F. Kimatu, BM. Liu, K. Du, M. Qiu, W. Hu, Q. (2017) The Correla- tion Between In Vitro Antioxidant Activity and Immunomodulatory Activity of Enzymatic Hydrolysates from Selenium-Enriched Rice Protein. [online] https://onlinelibrary.wiley.com/doi/abs/10.1111/1750- If you would like to discuss how we can help with your enzymatic 3841.13595 [Accessed 18th Feb. 2019] hydrolysis, please contact us. Alternatively, sign up to our newsletter to 15. Reche, M. Pascual, C. Fiandor, A. Polanco, I. Rivero-Urgell, M. Chifre, R. Johnston, S. Martin-Este- receive future content like this. ban, M. (2010) The effect of a partially hydrolysed formula based on rice protein in the treatment of infants with cow’s milk protein allergy. [online] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904490/ [email protected] [Accessed 18th Feb. 2019] 16. Eriksen, S. Nielsen, PM. (1991) Method for production of pea protein hydrolyzate. US5520935A 17. Gray, N. (2014) Making the Most of Protein: DSM says Enzyme Toolbox will ‘Do More’ with Protein. [online] https://www.foodnavigator.com/Article/2014/05/15/Making-the-most-of-protein-DSM-says- Click to sign up to newsletter enzyme-toolbox-will-do-more-with-protein# [Accessed 18th Feb. 2019] 18. AB Enzymes (2019) COROLASE® 2TS. [online] https://www.abenzymes.com/en/your-industry/ References protein-processing-and-modification/protein-hydrolysis/corolase-2ts/ [Accessed 18th Feb. 2019] 1. Guan,X. Yao,H. (2008). Optimization of Viscozyme L-assisted extraction of oat bran protein using 19. Amano Enzyme (2018) Amano Enzyme Protein Glutaminase “Amano” 500. [online] https://www. response surface methodology. [online] Available at: https://www.sciencedirect.com/science/article/pii/ snackandbakery.com/articles/92093-amano-enzyme-protein-glutaminase-amano-500 [Accessed 18th S0308814607004591 [Accessed 18th Feb. 2019] Feb. 2019] 2. Zhang,SB. Wang,Z. Xu,SY. (2007). Optimization of the Aqueous Enzymatic Extraction of Rapeseed Oil and Protein Hydrolysates. [online] Available at: https://link.springer.com/article/10.1007/s11746- 006-1004-6 [Accessed 18th Feb. 2019] 3. VTT. (2016). Enzyme-aided recovery methods’ help in extracting protein from rapeseed press cake. [online] Available at: https://www.vttresearch.com/media/news/extracting-protein-from-rapeseed- press-cake [Accessed 18th Feb. 2019] 4. Kilcawley, K. N., Wilkinson, M. G. and Fox, P. F (2002) Determination of key enzyme activities in commercial peptidase and lipase preparations from microbial or animal sources. Enzyme and Microbial Technology, 31 (3), 310-320. 5. Amri, E. and Mambaya, F. (2012) Papain, a Plant Enzyme of Biological Importance: A Review. Ameri- can Journal of Biochemistry and Biotechnology, 8 (2), 99-104. 6. Yongjing, W. (2017) A method for producing a yeast extract. WO Patent 2017114403, application June 7, 2017. 7. Maynard, F., Salvatore, D. and Thevenier, A. (2017) Milk based protein hydrolysates and composi- tions thereof. WO Patent 2016156077, application date October 6, 2017. 8. Suzuki, H. Nakafuji, Y. Tamura, T. (2017) New Method To Produce Kokumi Seasoning from Pro- tein Hydrolysates Using Bacterial Enzymes. [online] Available at: http://europepmc.org/abstract/ MED/29111704 [Accessed 18th Feb. 2019] 9. Park, SY. Lee, JS. Baek, HH. Lee, HG. (2010) Purification and Characterization of Antioxidant Peptides from Soy Protein Hydrolysate. [online] Available at: http://europepmc.org/abstract/AGR/ IND44345101 [Accessed 18th Feb. 2019] 10. Zilic, S. Barac, M. Pesic, M. Dodig, D. Ignjatovic-Micic, D. (2011) Characterization of Proteins from Grain of Different Bread and Durum Wheat Genotypes. [online] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189758/ [Accessed 18th Feb. 2019] 11. Kong, X. Zhou, H. Qian, H. (2007) Enzymatic hydrolysis of wheat gluten by proteases and properties of the resulting hydrolysates. [online] https://www.researchgate.net/publication/223240216_En- zymatic_hydrolysis_of_wheat_gluten_by_proteases_and_properties_of_the_resulting_hydrolysates [Accessed 18th Feb. 2019]