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Enzyme-aided production of lipid emulsifiers from side-streams of the food industry: rapeseed press cake and oat oil

Bouma, Renske LU (2017) KBTM01 20171
Biotechnology (MSc)
Abstract
The goal of this thesis project was to utilize side-streams of the food industry, namely rapeseed cake and oat oil, by enzyme-aided production of lipid emulsifiers.

The first objective was to extract lipids from the rapeseed press cake and increase protein yield, by adding an enzymatic step to the already existing protein extraction procedure developed by the food department of Lund University. A pre-treatment of the rapeseed press cake with Pectinex Ultra SP-L aimed to degrade the cell-wall matrix showed an increase in protein yield of approximately 10%. Lipids were not extracted as no separate oil layer was seen during any of the treatments. It is hypothesised that the lipids were hydrolysed during the procedure leading to the bitter... (More)
The goal of this thesis project was to utilize side-streams of the food industry, namely rapeseed cake and oat oil, by enzyme-aided production of lipid emulsifiers.

The first objective was to extract lipids from the rapeseed press cake and increase protein yield, by adding an enzymatic step to the already existing protein extraction procedure developed by the food department of Lund University. A pre-treatment of the rapeseed press cake with Pectinex Ultra SP-L aimed to degrade the cell-wall matrix showed an increase in protein yield of approximately 10%. Lipids were not extracted as no separate oil layer was seen during any of the treatments. It is hypothesised that the lipids were hydrolysed during the procedure leading to the bitter taste of the extract and solubilisation of the lipids. No starting material for modification of lipids was therefore obtained from rapeseed press cake.

The second objective was to determine the effect of enzymatic modification on the composition and emulsification properties of lipids. Two different oils were used: crude oat oil and polar lipid enriched oat oil (PL40). Firstly the unmodified oils were characterized. Column separation was performed, which successfully produced a neutral lipid, phospholipid and glycolipid fraction. Thin layer chromatography (TLC) showed that the neutral lipid fraction mainly contained di- and triglycerides and the phospholipid fraction contained phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and lyso-PC. Fatty acid profiles were determined by fatty acid methylation and gas chromatography. Main fatty acids in both oils were palmitic acid (16:0), oleic acid (18:1) and linoleic acid (18:2). Minor fatty acids were palmitoleic acid (16:1), stearic acid (18:0) linolenic acid (18:3), arachidic acid (20:0), eicosenoic acid (20:1) and avenoleic acid (18:2 15OH), of which the latter was only found in the glycolipid fraction. Differences in fatty acid profiles of the two oils could be explained by the profiles of the different fractions. Emulsion stability was assessed by a fine-tuned spectrophotometric method supplemented by visual observations and light microscopy. Stability was significantly increased by the presence of unmodified oils, especially by PL40 oil. Surprisingly, contact time between oat oil and water before mixing also significantly affected stability, which was ascribed to the formation of liquid crystals.
Modification of the lipids was performed by a lipase from Rhizopus arrhizus and hydrolysed lipids were initially extracted by adding chloroform and methanol. However, this method was inconsistent and three other extraction methods were tested. Addition of water, chloroform and methanol proofed most appropriate and was used for further experiments. Emulsion stabilizing ability increased as expected with incubation time for both crude oat oil and PL40 oil. Unexpectedly, the PL40 oil incubated without enzyme showed a higher stability than the hydrolysed oil. It is hypothesised that due to the hydrolysis more elaborate lipid structures are formed, which prevent coalescence but can cover less area than unmodified lipids, leading to bigger droplets and an increased creaming rate. From the results it is clear that the polar lipids in oat oil can be utilized as emulsifiers of which the properties can be changed by enzymatic hydrolysis.

In conclusion it can be said that both rapeseed press cake and oat oil can be utilized to a greater extent by aid of enzymes. The fine-tuned methods reported in this thesis for enzymatic hydrolysis and assessing change in molecular structure and emulsification properties can be used as a starting point for future research. This could eventually lead to implementation in industry and an increased utilisation of the side-streams of the food industry. (Less)
Popular Abstract
During the manufacture of food, by-products are produced. These side-streams are not the focus of the production and are therefore often considered a waste. However, the streams frequently contain valuable components, which if utilized can increase the value of the crop while decreasing waste. Two by-products with potential for added value were investigated in this thesis, namely rapeseed press cake and oat oil.

Rapeseed press cake is the main side-stream of rapeseed oil production. In the year 2011/2012 the worldwide production of rapeseed oil was approximately 24 million ton and as a by-product 33.6 million ton of rapeseed press cake was produced. The cake still contains some oil, including useful phospholipids. These lipids are often... (More)
During the manufacture of food, by-products are produced. These side-streams are not the focus of the production and are therefore often considered a waste. However, the streams frequently contain valuable components, which if utilized can increase the value of the crop while decreasing waste. Two by-products with potential for added value were investigated in this thesis, namely rapeseed press cake and oat oil.

Rapeseed press cake is the main side-stream of rapeseed oil production. In the year 2011/2012 the worldwide production of rapeseed oil was approximately 24 million ton and as a by-product 33.6 million ton of rapeseed press cake was produced. The cake still contains some oil, including useful phospholipids. These lipids are often used in the food industry to stabilize emulsions, like mayonnaise. The rapeseed press cake also contains valuable proteins, with a composition favourable for human consumption. The food department of Lund University has developed a method to extract these proteins. The first objective of this thesis project was to increase the yield of this procedure and to extract the remaining oil. To do this, a step was added to the method in which an enzyme cut the rapeseed cell-wall material into small pieces so to release entrapped protein and oil. This worked partly. More proteins were released, approximately 10%, but no oil-layer was seen. It turned out that the lipids were also cut into pieces during the procedure, which made them disappear into the water. This discovery had an upside though, as the pieces of the lipids turned out to be responsible for the bitter taste of the protein extract. Knowing this, the taste can be improved by slightly adjusting the method.

Oat oil is the main side-stream of the production of oat fibres by the Swedish company SweOat. The fibres in oat, especially beta glucans, have recently gotten much attention for their health promoting abilities and the production is growing steadily. Oat oil contains many polar lipids, like the previously mentioned phospholipids and galactolipids. Polar lipids are molecules that like both water and oil and therefore prefer to be at the boundary between water and oil. In an emulsion oil is dispersed as small droplets in water, or the reverse, and much of this water-oil interface exists. As oil and water do not like each other, the droplets will normally quickly come together to minimize contact with the other liquid. But when polar lipids are present the oil and water are shielded from each other and the droplets can remain dispersed. The second objective of this thesis project was to investigate oat oil’s ability to stabilize an emulsion and how this ability changes when an enzyme cuts off a piece of the lipids. It turned out that adding oat oil to an emulsion does indeed increase the stability. This was especially the case for oat oil enriched in polar lipids, as you would expect. Surprisingly, stability was also increased when the oat oil was soaked in water for more than an hour before making the emulsion. During this time the oil changes from a yellow liquid to a white gel-like structure. This structure is called a liquid crystal; lipids are partly flexible, like a liquid, and partly well-regulated, like a crystal. These structures form a protective layer around the emulsion droplets, increasing stability. For the second part of the objective, pieces of the lipids were cut off by an enzyme. After fine-tuning the way to extract the lipids from the reaction mixture, it could be seen that the lipid’s ability to stabilize emulsions was indeed affected by the enzyme. The longer the enzyme was in contact with the lipids, the better the lipids could stabilize the emulsion.

In conclusion it can be said that both rapeseed press cake and oat oil can be utilized to a greater extent. The insights in this report can help to produce in the future protein extract with a higher yield as well as without a bitter taste, increasing the feasibility to produce for human consumption. Oat oil can be used as emulsifier of which the properties can be changed by enzymes. Using the methods of this research as a starting point, eventually commercialized production of a range of lipid emulsifiers might be possible, in this way increasing the utilization and value of this side-stream from the food industry. (Less)
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author
Bouma, Renske LU
supervisor
organization
course
KBTM01 20171
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Lipid emulsifiers, enzymes, side-streams food industry, rapeseed press cake, oat oil
language
English
id
8912125
date added to LUP
2017-06-19 16:00:19
date last changed
2017-06-19 16:00:19
@misc{8912125,
  abstract     = {The goal of this thesis project was to utilize side-streams of the food industry, namely rapeseed cake and oat oil, by enzyme-aided production of lipid emulsifiers. 

The first objective was to extract lipids from the rapeseed press cake and increase protein yield, by adding an enzymatic step to the already existing protein extraction procedure developed by the food department of Lund University. A pre-treatment of the rapeseed press cake with Pectinex Ultra SP-L aimed to degrade the cell-wall matrix showed an increase in protein yield of approximately 10%. Lipids were not extracted as no separate oil layer was seen during any of the treatments. It is hypothesised that the lipids were hydrolysed during the procedure leading to the bitter taste of the extract and solubilisation of the lipids. No starting material for modification of lipids was therefore obtained from rapeseed press cake. 

The second objective was to determine the effect of enzymatic modification on the composition and emulsification properties of lipids. Two different oils were used: crude oat oil and polar lipid enriched oat oil (PL40). Firstly the unmodified oils were characterized. Column separation was performed, which successfully produced a neutral lipid, phospholipid and glycolipid fraction. Thin layer chromatography (TLC) showed that the neutral lipid fraction mainly contained di- and triglycerides and the phospholipid fraction contained phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and lyso-PC. Fatty acid profiles were determined by fatty acid methylation and gas chromatography. Main fatty acids in both oils were palmitic acid (16:0), oleic acid (18:1) and linoleic acid (18:2). Minor fatty acids were palmitoleic acid (16:1), stearic acid (18:0) linolenic acid (18:3), arachidic acid (20:0), eicosenoic acid (20:1) and avenoleic acid (18:2 15OH), of which the latter was only found in the glycolipid fraction. Differences in fatty acid profiles of the two oils could be explained by the profiles of the different fractions. Emulsion stability was assessed by a fine-tuned spectrophotometric method supplemented by visual observations and light microscopy. Stability was significantly increased by the presence of unmodified oils, especially by PL40 oil. Surprisingly, contact time between oat oil and water before mixing also significantly affected stability, which was ascribed to the formation of liquid crystals. 
Modification of the lipids was performed by a lipase from Rhizopus arrhizus and hydrolysed lipids were initially extracted by adding chloroform and methanol. However, this method was inconsistent and three other extraction methods were tested. Addition of water, chloroform and methanol proofed most appropriate and was used for further experiments. Emulsion stabilizing ability increased as expected with incubation time for both crude oat oil and PL40 oil. Unexpectedly, the PL40 oil incubated without enzyme showed a higher stability than the hydrolysed oil. It is hypothesised that due to the hydrolysis more elaborate lipid structures are formed, which prevent coalescence but can cover less area than unmodified lipids, leading to bigger droplets and an increased creaming rate. From the results it is clear that the polar lipids in oat oil can be utilized as emulsifiers of which the properties can be changed by enzymatic hydrolysis.

In conclusion it can be said that both rapeseed press cake and oat oil can be utilized to a greater extent by aid of enzymes. The fine-tuned methods reported in this thesis for enzymatic hydrolysis and assessing change in molecular structure and emulsification properties can be used as a starting point for future research. This could eventually lead to implementation in industry and an increased utilisation of the side-streams of the food industry.},
  author       = {Bouma, Renske},
  keyword      = {Lipid emulsifiers,enzymes,side-streams food industry,rapeseed press cake,oat oil},
  language     = {eng},
  note         = {Student Paper},
  title        = {Enzyme-aided production of lipid emulsifiers from side-streams of the food industry: rapeseed press cake and oat oil},
  year         = {2017},
}