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Alfalfa Checkoff Watch: Enzyme-assisted protein isolation from alfalfa leaves

Youngmi Kim for Progressive Forage Published on 30 August 2019

Project Title: Enzyme-assisted Protein Isolation from Alfalfa Leaves
Industry Need: New Uses and Market Development
Principal: Youngmi Kim
Grantee: University of Wisconsin – River Falls
Duration: May 1, 2019 – April 30, 2020
Budget: $24,183

1. What (specifically) prompted this research project?

The technological advances and modernization in agriculture allowed agricultural output to increase faster than growth population in the past. But the world is now facing new threats and complex problems that demand innovative solutions: a rapidly changing global climate coupled with burgeoning energy and food demand. It is projected that by 2050 the world’s population will grow by 2.5 billion people.

The United Nations Food and Agricultural Organization (FAO) projects the global food and feed production needs to increase between 25 percent and 70 percent to meet the dietary demands of more than 10 billion people. On average, people will be wealthier than people today, which implies not only the increase of total calorie consumption but also increased demand for animal-based proteins. The production of animal-based protein requires considerably more resources than plant-based protein, such as land, water and fossil fuels, which are closely associated with climate change.

The research was prompted from the rather broad question I had in mind for a long time: “How can we produce food sustainably while meeting the growing demand and without causing any further harm to the environment?” There might be many approaches and solutions to this pressing challenge, but I have been particularly interested in alternative protein, especially plant-based protein.

One of the researches I have worked on while I was at Purdue University as a research engineer focused on increasing the value of distillers grains by extracting components (proteins, fibers, oils) and converting them into other value-added chemicals and materials. Distillers grains, which are a byproduct of the corn-to-ethanol process, contain high protein and typically are used as a protein supplement for livestock animals. The project provoked an idea about investigating an alternative plant source for human consumption.

While studies in the past have mainly focused on whey- or soy-based proteins, green leaf has been widely overlooked as a potential protein source for human consumption. Alfalfa leaves are one of the most important raw leaf protein sources due to the high crude protein content (260 grams per kilogram dry matter or 2,600 kilograms of protein per hectare) and balanced amino acid composition ratio, which is consistent with the FAO’s recommended adult amino acid profile. However, the application of alfalfa proteins for human consumption has been limited by undesirable sensory qualities such as color, taste and texture.

The main goal of my study is to use a combination of enzymes not only to increase extractability of proteins from alfalfa leaves but also to improve solubility and sensory properties of the extracted alfalfa proteins by hydrolyzing them into anti-oxidative peptides. Anti-oxidative peptides are low-molecular-weight polypeptides, comprising two to 20 amino acid residues, that exhibit various physiological functions. Anti-oxidative peptides produced from alfalfa leaves can be used as functional foods, nutraceuticals, dietary supplements and constituents of pharmaceuticals.

Our approach aims to develop an enzyme blend that can effectively loosen up and expose the cell wall matrices for enhancing the protein extractability in the subsequent steps at the lowest possible enzyme dose. Minimizing enzyme loading is an important requirement for reducing the cost of the overall processing, as the cost of enzymes is typically the main cost factor in bioprocessing.

2. What challenges have occurred and what progress has taken place since the research was initiated?

Since the research has officially started in May, 2019, we are currently at the early stage of the project, mainly focusing on harvesting fresh alfalfa leaves and preparing the samples for the study. In this study, we will extract proteins directly from fresh leaves rather than from dry leaves. While dried leaves are easier to transport and store than fresh leaves, protein extraction from dry leaves demand harsher chemicals or reaction conditions than for fresh leaves, which may negatively affect protein quality in terms of amino acid profile and digestibility.

Proteins are more labile compared to other components in plant cells. Some amino acids, such as lysine, cysteine and glycine are especially sensitive to extreme pH and high temperatures and can be irreversibly damaged during a process that involves such conditions. Thus, higher-quality protein concentrates can be obtained from fresh leaves than from dry biomass.

Also, the mild conditions applied for enzyme treatment are beneficial to preserve the quality of proteins extracted. The challenge has been to grind the fresh leaves to particles less than 1 millimeter while minimizing loss or damage to plant tissue components. Using liquid nitrogen during the grinding process allows us to preserve protein quality in the alfalfa leaves. While this might be challenging to apply in a large-scale system, the above-mentioned sample preparation process is necessary in the research-scale process to minimize external and internal variances in the feedstock materials and allows us to scientifically evaluate the impact of enzyme blends on protein yields and resulting amino acid profile of the extracted proteins.

Identifying an optimal blend of enzymes that maximizes protein extractability of alfalfa leaves is the critical part of this study. There are numerous commercial enzyme blends available that target plant cell wall carbohydrates for various industrial applications. However, it is often a challenge to find one specific product that contains all necessary enzymes that target different types and structures of carbohydrates found in plant materials.

Commercial cellulases, used for various applications such as for producing sugars for fuels or other bioproducts through fermentation, mainly contain cellulose-degrading enzymes and often lack other enzyme activities necessary to degrade xylan and pectin, the two other major components of plant cell wall matrix. The main challenge of this research will be formulating an optimal enzyme blend from the commercially available enzyme products that contain necessary enzyme activities to efficiently break down alfalfa leaves. We currently have identified and acquired five different enzymes that are to be investigated further and mixed together for alfalfa protein extraction.

3. When can producers expect to see the final results?

The research will finish by April 30, 2020. Most of the experiments will be completed by the end of this year, and the data analysis and report will be completed by April 2020.  end mark

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