by Kristin Hals and Sofia Helena Lindahl, Research and Development Department, Borregaard AS


Ethoxyquin has, for decades, been widely used as an antioxidant in the feed sector, primarily in the marine industry.
This antioxidant inhibits the oxidation of highly unsaturated fatty acids in fishmeal and fish silage. Ethoxyquin has the unique property of being able to dissolve in both aqueous- and oil phases, depending on pH. However, there are concerns related to the use of this antioxidant (See Figure 1). In June 2017, the EU commission suspended the authorisation of ethoxyquin for all animal species and categories. Hence, there is a need to find an alternative solution.
A new alternative product containing the antioxidant propyl gallate has been developed by Borregaard. This product is optimised to ensure high quality, as well as stable product performance. The new combination of propyl gallate, lignosulphonic acid and formic acid provides a viable alternative for the market.

In June 2017, the EU commission suspended the authorisation of ethoxyquin (See Figure 1) for all animal species and categories [1]. Ethoxyquin has been the antioxidant of choice for decades, especially in the fish industry, in order to prevent rancidification of fats and oils during processing and storage.
Borregaard has many years of experience evaluating the efficiency of antioxidants. There are several different methods available on the market for testing antioxidant capacity [2-5] and the DPPH-assay [6] is one commonly used method. At Borregaard, an inhouse developed DPPH (2,2-diphenyl-1-picrylhydrazyl) based method – the BAU* method - is used, which will be described later in this text. 
*Borregaard Antioxidant Unit

Fish silage 
Fish silage contains fish, or parts of fish, combined with an additive for stabilising the silage during storage. Organic acids of different types are typically used as silage additives. Under the right conditions, temperature between 5 and 40 °C and at pH between 3.5 and 4.5, the fish mass will begin to decompose.
In this process, called autolysis, enzymes break down the muscles, and a liquid mass is formed, which is desirable given its ease of handling through pumps, piping, etc. (See Figure 2). Marine lipids contain high levels of long chained polyunsaturated fatty acids (PUFA). The PUFAs are readily oxidised by oxygen, which results in rancidification of fats and oils and consequently a decrease in product quality. Fishmeal and fish oil contain relatively high concentrations of PUFAs and are therefore especially prone to oxidation.
To prevent the oxidation of PUFAs in fishmeal and fish oil, the industry currently uses synthetic antioxidants like: ethoxyquin (E324), BHA (butylated hydroxy-anisole, E-320) and BHT (butylated hydroxy toluene, E321). In addition, natural antioxidants like tocopherols are also used. 

Screening of antioxidants
The list of approved antioxidants in feed are limited. In this study, several antioxidants, both synthetic and natural, have been evaluated. The antioxidant capacity was tested using the BAU method.
The BAU method is a spectrophotometric based method, using the stable free radical DPPH, to compare the antioxidant capacity of lignosulphonates and antioxidants. The change in absorbance of a solution containing DPPH and compound(s) of interest is measured.
Unreacted DPPH is violet, but after reaction, i.e. transfer of free radical to, for example, an antioxidant, the solution changes colour to yellow. The less antioxidant that is needed to quench the DPPH radical, the stronger is the antioxidant.
From the screening, BHA, propyl gallate and ascorbic acid showed the highest antioxidant capacities. Based on additional stability and solubility studies, propyl gallate (See Figure 3) was chosen for further testing.

Lignosulphonates and antioxidant capacities    
Lignin is a natural polymer. The word 'lignin' is derived from the Latin word 'lignum', meaning wood. Lignin is the binding element in wood and plays an important role in the transportation of water, metabolites and nutrients. It acts as an encrusting material and performs multiple functions that are essential for the life of the plant.
Lignin imparts rigidity to the wood cell walls and acts as a binder between the cell walls, creating a composite material that is outstandingly resistant to compression and bending. Lignin is one of the most abundant organic polymers on earth, exceeded only by cellulose.
Lignosulphonates are branched, water-soluble biopolymers produced from lignin. Natural polymers are understood as polymers which are the result of a polymerisation process that has taken place in nature, independently of the process with which they have been extracted. The monomeric units constituting the natural polymer of lignin can be seen in (See Figure 4).
Lignin-based products serve as additives in several industrial and commercial applications, and often replace petroleum-based products as a natural renewable solution. Building on inherent qualities, and further enhanced by chemical modification, our lignin-based products offer a unique and desirable set of functions for the chemical industry in areas such as: concrete admixtures, pesticide dispersants, battery expanders, oil well drilling chemicals, emulsions, ceramics, road binders, bypass protein and animal feed additives.
Lignin is known to have antioxidant properties. Especially, water-soluble lignosulphonates have shown synergistic effect in combination with antioxidants. Polyphenols are often used as antioxidants. Because of the phenolic molecular structure of lignosulphonates, associations to antioxidant effect can be attributed. In the literature, the antioxidant effect of lignosulphonates in various applications has been reported.
Lignosulphonates are available as lignosulphonic acid salts. Figure 5 gives an illustration of the lignosulphonic acid. The phenolic groups, and other easily oxidised structures in the lignosulphonic acid, can act as scavengers and stabilise reactive and potentially harmful free radicals.
In 2008, Borregaard filed a patent regarding use of lignosulphonic acid as a sacrificial agent for antioxidants. The patent describes that the antioxidants tested were less degraded in an organic acid solution if lignosulphonic acid was present, i.e., the lignosulphonic acid works as a sacrificial antioxidant.

Stability of propyl gallate in organic acids 
A stability study of propyl gallate in formic acid (85%), and formic acid (85%) containing 20 percent (w/w) lignosulphonic acid was performed. Ethoxyquin was included as a reference.
The inclusion level of antioxidants in the acids was 0.35 percent (w/w). 25-litre containers were stored outside during summer and early fall. The stability of the antioxidants was tested regularly by measuring the level of the antioxidants in the samples. The measurements were done using high pressure liquid chromatography in combination with UV detection (HPLC-UV).
The stability data of propyl gallate is presented in Figure 6a. The first measurements showed a remaining value of 58 percent of the added propyl gallate in formic acid. In the solution containing lignosulphonic acid, the corresponding value was 90 percent (See Figure 6a). After 75 days, the corresponding values were 48 percent in formic acid and 63 percent in the solution containing lignosulphonic acid.
The results clearly show that addition of lignosulphonic acid to the solution stabilises and protects propyl gallate better than the solution containing only formic acid. In Figure 6b the same stabilising effect of lignosulphonic acid is demonstrated for ethoxyquin. 

Measuring degree of rancidity
As a control of the performance of the new antioxidant in fish silage, rancidity tests were performed. Measuring oxidation/degree of rancidity involves testing for primary and secondary degradation products. The most common way is to measure the peroxide value (PV), i.e. measuring the primary oxidation products (mainly hydroperoxides), and to measure the anisidine value (AV), i.e. measuring the secondary oxidation products.
The secondary stage of oxidation occurs when hydroperoxides decompose to form carbonyls and other compounds like aldehydes. The latter will give the oil a rancid smell and is measured by AV.
It is, therefore, important to measure both PV and AV and evaluate the two parameters together. This is commonly done by calculation of total oxidation value, TOTOX, which gives an overall picture of the quality of the oil; TOTOX = PV*2 + AV. 

Fish silage trial in lab scale
Propyl gallate and lignosulphonic acid blends were used to prepare fish silage. Salmon was chopped and minced in a food processor in the laboratory. Different acid solutions were prepared containing:

  • 0.35 percent propyl gallate in formic acid 85 percent + lignosulphonic acid 
  • 0.70 percent propyl gallate in formic acid 85 percent + lignosulphonic acid
  • 0.35 percent ethoxyquin in formic acid 85 percent + lignosulphonic acid

Note: The solutions above all contained 80 percent w/w formic acid 85 percent and ~ 20 percent w/w lignosulphonic acid.
The minced fish was mixed with the different acid solutions and stored in two-litre containers in a water bath at 23°C. The acid solutions were added to the minced fish to reach the desired pH of 3.5-3.6. Parallel samples were made.
Extracted oil samples from the minced fish were collected at different time intervals over 11 weeks. The oil samples were analysed for rancidity products, expressed as peroxide- and anisidine values.  Acidity in the fish silage was controlled to ensure pH<4.

Stability of the fish silage
The TOTOX values can be seen in Figure 7. Each TOTOX value is calculated from the measured PV and AV in the samples.  After six weeks, the silage stabilised with propyl gallate and lignosulphonic acid having the same TOTOX level as the silage stabilised with ethoxyquin.
However, after 11 weeks, the silage stabilised with propyl gallate and lignosulphonic acid has lower TOTOX values than the silage containing ethoxyquin. There were no significant differences between the two dosages of propyl gallate tested.

By combining the data from the storage and fish stability trials it is clear that propyl gallate can replace ethoxyquin as antioxidant in silage additives. 
Borregaard has developed a new silage additive containing formic acid/lignosulphonic acid and propyl gallate for the fish by-product industry. This product offers the following benefits:

  • Ethoxyquin-free solution
  • Reduced degradation of the antioxidant
  • Longer shelf life of the silage additive
  • Stable and high-quality fish silage  

The work described was funded by the BioBased Industries Joint Undertaking under the Horizon 2020 European Union Funding for Research and Innovation programs within the BioForEver project (BIObased products from FORestry via Economically Viable European Routes, grant agreement No. 720710).

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