Characterization of low-lipase oats

Abstract

Cereals constitute a large portion of the nutritional intake in the world and are an essential part of a sustainable future. The common oat (Avena Sativa L.) has a unique composition of nutrients, including many lipids. One problem with this is the inability to be stored during a longer period due to the high lipase activity which releases bitter free fatty acids (FFA). Heat treatment is the most common way of prolonging the shelf life, but an alternative is to develop low-lipase oats.
In this study, previously identified low-lipase oat lines were to be further characterized. To measure the lipase activity two different assays were used. One uses 4-methylumbeliferyl heptanoate (MUH) and the other uses a pH-stat-based method. MUH is a synthetic substrate previously used for screening lipase activity but in this work, no conclusive data could be obtained. This is thought to be the consequence of too old raw material.
The principle behind the pH-stat method is that the release of FFAs causes a decrease in pH which is measured and automatically neutralized by titration of a dilute base. Due to not using a synthetic substrate, this method has the potential to give values closer to reality.
The characterization also included the qualification and quantification of the total lipids and the fatty acids in the oat lines. Additionally, a 14-day storage assay was done to study the actual release of FFA. The extracted lipids were separated using thin-layer chromatography, followed by methylation and analysis of the fatty acid methyl esters using gas chromatography.
In conclusion, the identified low-lipase lines were insufficient to replace heat treatment, and the pH-stat method needs to be further studied.

Popular Abstract

Oat is an important cereal crop. In recent years, interest in oat as food has increased, partly because several health benefits of oat consumption have been discovered. Oat is the cereal with the highest fat content and is a good source of energy and unsaturated fatty acids. However, the amount of lipids in oat lead to a greater risk of developing rancidity and unappealing flavors and odors. This is mainly caused by the breakdown of lipids to free fatty acids by enzymes called lipases. Today's most common procedure to tackle this problem is to heat-treat the oat before storage, which will inactivate the enzymes. On the other hand, this is not energy- and time efficient and causes additional problems like degradation of nutrients, for example vitamins.
Another possible way of dealing with this issue is to develop oats without these lipases. Oats with lower lipase activity have been identified in populations with induced random mutations. In this work, these oats were studied in terms of lipase activity, using a molecule called 4-Methylumbelliferyl heptanoate (MUH), and fat content, using GC, to obtain a more extensive understanding of them, if they differ from ordinary oat, and what more must be done in the future to deliver a better product.
The most interesting result found in the thesis is the lack of results. No measurements of the enzyme activity showed significant differences between the various variants and the original oat. This is believed to be mainly due to the age of the raw material, although it was shown that the lipase in the different kinds of oat was still active.
Further studies of the fat content and the release of free fatty acids after a 14-day storage assay showed that the percentage of the free fatty acids increase in relation to the total fat content in the genetically modified oat and the ordinary oat. As with the enzyme activity measurements, no significant differences between the mutated oat and the original oat could be identified.
An alternative method to determine the lipase activity was also explored, using an instrument called a pH-stat. The use of unnatural molecules to measure the lipase activity results in some problems so if a method without those could be used, maybe the results would be closer to reality. The principle behind using this method is the decrease in pH caused by the release of free fatty acids. With the addition of base, the drop in pH is neutralized and recorded to calculate the lipase activity.
Taking all into account, it was determined that the identified oat variants with lower lipase activity are still not sufficiently low to be an alternative to heat treatment. Additionally, further optimization of the pH-stat method is needed to be used to measure the lipase activity in oat.

• author: Bro, Svante
• supervisor: Sofia Marmon
• organization: Pure and Applied Biochemistry; Computational Chemistry
• course: KBKM05 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: oat, lipase activity, lipid profile, total lipids, fatty acids, biotechnology, gas chromatography, ph-stat, biochemistry, applied biochemistry
• language: English
• id: 9125378
• date added to LUP: 2023-07-06 12:09:31
• date last changed: 2023-07-06 12:09:31