Quality of Lipids During Frozen Storage of Polychaete (Alitta virens) - a Low Trophic Marine Resource Relevant for Aquaculture Feed

ABSTRACT

Lipid quality during frozen storage of the low trophic marine resource polychaete (Alitta virens) was evaluated after blanching, vacuum packing, and different storage temperatures (−23°C vs −27°C). In non-blanched samples, free fatty acid (FAA) reached a level of 15–20% after 12-months of frozen storage. ¹H and³¹P nuclear magnetic resonance (NMR) analyses showed that the increase in FFA could be attributed to both hydrolysis of phospholipids and triacylglycerols. Vacuum packing prevented lipid oxidation. Blanching hindered lipid hydrolysis and is thereby recommended to preserve valuable phospholipids during long-term frozen storage of polychaete, a resource that has attracted growing interest for use in aquaculture.

GRAPHICAL ABSTRACT

KEYWORDS:

• Polychaete
• novel feed resources
• lipid hydrolysis
• lipid oxidation
• High resolution NMR

View correction statement:

Correction

Introduction

Fishmeal and fish oil are important components in feed for fish and marine crustaceans. Replacing these nutritional marine ingredients with feed ingredients of vegetable origin has allowed aquaculture to grow despite the limited supply of traditional forage fish. The composition of salmon feed has dramatically changed in the last decades, decreasing from 90% marine ingredients in 1990 to 25% in 2016 (Aas et al. 2019). However, marine oils are still vital components of aquaculture feed since several organisms require long-chain n-3 polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for optimal growth and healthy development (NRC 2011).

Cultivation of low trophic organisms, such as polychaetes, has been proposed as one means to recycle nutrients from side streams and at the same time produce omega-3 rich lipids and marine proteins that are in high demand both for feed and human applications (Brown et al. 2011; Kabeya et al. 2020; Marques et al. 2017; Wang et al. 2019). Polychaete is annelid worms that are part of the natural diet of marine fish and shrimps. Polychaete cultivation was initiated in the 1980’s (Olive 1999), and such worms are presently cultivated in both Europe and Asia but at relatively small scale and mainly sold in the premium feed market (Pombo et al. 2020) as freeze-dried (Chimsung 2014) or as live feed or bait. The ragworm Alitta virens is one of the species for which commercial cultivation has been established in Europe. Alitta virens is sold mainly as bait or for marine shrimp broodstock maturation diets (Chimsung 2014; Olive 1999). Recent research found that Alitta virens meal could replace fishmeal up to 40% in diets of European sea bass, without negative effects on growth or nutrient utilization while improving environmental performance (Monteiro et al. 2023).

Live polychaete worms are the preferred feed used for shrimp broodstocks, but the mechanisms responsible for the superior effect of live polychaetes are not known (Chimsung 2014; Meunpol et al. 2010). Their lipids, enzyme-pool, vitamins, carotenoids, pigments, chemo-attractant properties, and reproductive hormones are all possible reasons for the improved reproductive performance in shrimps (Chimsung 2014; Meunpol et al. 2010). The polychaete lipids, containing the LC PUFAs arachidonic acid (ARA, 20:4n-6), EPA (20:5n-3), and DHA (22:6n-3) (Wang et al. 2019), are particularly interesting for use in aquaculture feed (Olive 1999). Vertebrate and crustaceans have an absolute dietary requirement for these LC-PUFAs for many essential metabolic and physiological processes (NRC 2011; Tocher 2015). These fatty acids are required for successful production of high-quality crustacea and fish juveniles and also impact growth, health, and muscle quality of fish (Tocher 2015). Moreover, phospholipids (PLs) are essential components in feed for marine fish larvae (Tocher et al. 2008). However, the beneficial effect may vary due to the composition of the phospholipids, such as fatty acid composition and phospholipid classes (Li et al. 2018).

Marine raw materials are highly perishable; therefore, proper handling and preservation methods are needed to maintain valuable components during storage and further processing. High endogenous enzymatic activities may lead to degradation of both lipids and proteins soon after harvesting, and changes may also occur during frozen storage as reported for other low trophic marine species (Bergvik et al. 2012; Solgaard et al. 2007). In addition, biomass with high content of PUFAs is highly susceptible to lipid oxidation, (Mozuraityte et al. 2020; Wu et al. 2022) even though the biochemical composition of polychaete from different origins and cultivation practices has been reported (Bharath et al. 2021; Wang et al. 2019), there are few studies investigating the stability of the biomass during different storage conditions. In a previous study using earthworm meal in fish feed, the worms were blanched prior to processing (Sogbesan et al. 2007). Stabili et al. (2019) and Monteiro et al. (2023) used dried polychaete meal in fish feeding trials. For up-scaled and future use of polychaetes as an alternative aquafeed ingredient, the production, preservation, and processing should be cost and energy efficient while maintaining the quality of the raw material. To our knowledge, there are no studies on the effect of different storage or preservation conditions on important quality parameters of polychaete biomass.

The goal of the present study was to evaluate the lipid quality of polychaete (Alitta virens) biomass during different frozen storage conditions. The study included different packaging and frozen storage temperatures and the effect of thermal pre-treatment (blanching) prior to frozen storage. Quality was evaluated after 1-, 6-, and 12-months of frozen storage and compared with worms analyzed directly after snap freezing. Lipid quality was evaluated based on oxidative quality, amount of free fatty acids, fatty acid composition, and lipid profile with emphasis on phospholipids as evaluated by nuclear magnetic resonance (NMR) spectroscopy.

Material and methods

Raw material

Cultivated Alitta virens was procured from a commercial producer in The Netherlands (Topsy Baits). Parts of the harvested worms were transported live to the laboratory at SINTEF Ocean (Trondheim, Norway), while other parts of the same harvest were blast frozen at the plant before delivery to the same laboratory.

Live worms were rinsed to remove stones/substrate and transported to SINTEF in polystyrene boxes with ice in 1 L plastic bags with some seawater and air. After arrival, the live worms were stored at 4° until the experiment began the following day.

Storage procedures

After arrival at the laboratories, the live worms were washed in distilled water, and any remaining stones were removed. The biomass was divided into subsamples for the four following treatments:

1. Nitrogen: Samples were snap frozen in liquid nitrogen, and lipids were directly extracted as described below. This treatment served as control samples.

2. Frozen −23°C: Freezing at −23°C in a common freezer. Washed worms were divided into plastic bags of 100 g and stored frozen at −23°C in a common freezer.

3. Blanched −23°C: Blanching prior to freezing at −23°C in a common freezer. Worms were transferred to boiling water (1:2 on a weight basis), and the temperature was held at 70–80 degrees for 10 min prior to draining in a kitchen sieve. The experiments were performed with two replicate treatments. The solid biomass was kept on ice to reach room temperature and divided into subsamples of 100 g packed in plastic bags, before they were put in a common freezer at −23°C.

4. FrozenV −27°C: Vacuum packing prior to industrial freezing and storage at (−27°C). The worms were washed, packed in vacuum bags, and frozen at the industrial plant (blast freezing, −38°C). The samples were stored frozen 1 week at the plant and shipped frozen (at temperatures below <20°C) to the laboratories at SINTEF. After arrival, the vacuum packed worms were stored at −27°C until sampling at different storage times.

Temperature data loggers (type SL52T, Signatrol, UK) were used to monitor the temperature during the frozen storage. Samplings for quality analysis were performed after storage times of 1, 6, and 12 months; at each sampling, four replicate samples were analyzed.

Analysis of dry matter and ash

The dry matter content in the samples was determined gravimetrically after drying at 105°C until constant weight of samples was achieved (typically 24 h). Ash content was determined after heating dry samples at 590°C for 12 h. For each sampling time, four replicate samples were analyzed. The results are expressed as g/100 g dry weight.

Extraction of lipids and water-soluble components

Extraction by chloroform/methanol/water was performed as described by the procedure of Bligh and Dyer (1959) adjusted for a water content of 85%. The extraction was performed on 20 g of biomass, and the extractions were performed on n = 4 replicate samples for each treatment at each sampling time. Part of the methanol/water phase was sampled for analysis of polar 2-thiobarbituric acid reactive substances (TBARS). Parts of the chloroform phase were sampled for determination of lipid content and analysis of non-polar TBARS and peroxide values (PV). Samples for TBARS and PV were stored at −80°C until analysis. The remaining chloroform phase was evaporated under nitrogen, and the lipid extract was analyzed by fatty acid composition, content of free fatty acids, and phospholipid profiles analyzed by NMR spectroscopy (see further description below).

¹H and ³¹P NMR

¹H and ³¹P NMR spectra were recorded on a Bruker Avance 600 MHZ spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) at ambient temperature (25°C) using a BBO probe at a¹H frequency of 600.238 MHz and ³¹P NMR at frequency of 242.977 MHz. (at MR core facilities, NTNU, Trondheim).

A procedure with CsEDTA washing to remove cations in the sample prior to NMR analyses was used. The CsEDTA washing was performed according to Meneses and Glonek (1988) and Burri et al. (2016) with some small modifications. A CsEDTA solution was made by titrating a 0.2 M free acid (EDTA) by CsOH (CSOD, 1 M) to a pH of 7.0, at which point the EDTA was in solution. The lipid extract sample was added CDCL3 (0.5 mL), 0.5 mL MetOD containing triphenylphosphine (TPP) as internal standard (0.2 weight %), and 0.5 mL CsEDTA solution. The mixture was stirred 20 min, prior to centrifugation. The lower organic phase was transferred to the 5 mm NMR tubes. Acquisition parameters for ³¹P NMR were: pulse program zgig, time domain 251 904, spectral width 50.0 ppm, acquisition time 10.34 s, relaxation delay 2.0 s, number of scans 64, and dummy scans 4. The ³¹P NMR spectra were calibrated according to the TPP peak at −17.80 ppm. TPP has been reported with T1 value of ca 2 s in a similar solvent system (Monakhova et al. 2018). Identification of dominating phospholipid classes was done by analysis of internal standards and comparing chemical shifts with literature values on marine phospholipids (Burri et al. 2016; Meneses and Glonek 1988; Monakhova et al. 2018).

For¹H NMR, the following acquisition parameters were used: pulse program zg30, time domain 64k, spectral width 18.03 ppm, acquisition time 3.03 s, relaxation delay 2.0 s, number of scans 24, and dummy scans 4. Zero filling and exponential line broadening (0.30 Hz) was applied before Fourier transform.

The following equation was used for quantification of total phospholipid content from ³¹P NMR of the crude lipid extract.

$\mathrm{C}\left(\mathit{mg}/\mathrm{g}\right)={\mathrm{A}}_{\mathit{PL}}/{\mathrm{A}}_{\mathit{TPP}}{\ast }_{\mathit{\hspace{0.17em}}}\mathit{M}{W}_{\mathit{PLs}}/\mathit{M}{W}_{\mathit{TPP}\ast }{\mathrm{m}}_{\mathit{TPP}/}{\mathrm{m}}_{\mathit{sample}}$

where A is the area, MW is the molecular weight, m= the weight of sample (g) and TPP (mg). Since several of the new peaks arising in the ³¹P NMR spectra were not unambiguously identified, an average molecular weight of 790 g/moles for PLs was used in the calculations (e.g., as previously reported for PC of krill oil (Monakhova et al. 2018)).

³¹P NMR results were also reported as molar ratios of individual phospholipid species. In addition, as it was found that the amount of extracted fatty acids varied, the total PL content was also reported as weight % of total fatty acids. Quantification of the total fatty acids in the crude lipids was done from¹H NMR spectra as follows:

$\mathrm{C}\left(\mathrm{mg}/\mathrm{g}\right)={\mathrm{A}}_{\mathrm{FA}}/{\mathrm{A}}_{\mathrm{TPP}}\ast {\mathrm{n}}_{\mathrm{TPP}}/{\mathrm{n}}_{\mathrm{FA}\ast }\mathrm{M}{\mathrm{W}}_{\mathrm{FA}}/\mathrm{M}{\mathrm{w}}_{\mathrm{TPP}\ast }{\mathrm{m}}_{\mathrm{TPP}/}{\mathrm{m}}_{\mathrm{sample}}$

where A_FA is the area of methyl-group of FA, A_TPP the area of TPP, and n is the number of protons (n_FA = 3, while n_{TPP =} 15). An average molecular weight of 280 g/moles was used for fatty acids.

Free fatty acids

Free fatty acid content in the oils was analyzed according to Bernárdez et al. (2005). Isooctane was used as a solvent for lipids instead of cyclohexane. Standard curve prepared with an oleic acid standard (0–20 µmol) was used for calculation of the FFA content. The results are expressed as % FFA (weight %, as oleic acid equivalents) of crude lipid extract ± standard deviation of four parallels.

Fatty acid composition

The fatty acid (FA) profile of the extracted lipids in chloroform phase was determined using an Agilent Technologies 7890A (Agilent Technologies, Berlin, Germany) gas chromatography (GC) with a flame ionization detector (FID). The methylation step and GC-FID analysis were performed as described in detail by Kristinova et al. (13). An internal standard 21:0 methyl ester (purity 99%, Nu-Chek Prep Inc., Elysian, MN, USA) was added to the extracted sample prior to methylation. Fatty acid methyl esters were identified by the comparison of their retention times with those of a reference solution (Nu-Chek Prep Inc.) analyzed under identical gas chromatographic conditions. The results were expressed as g FA/g lipid crude lipids and the relative amount of each FA of total FA (weight %). Two replicates were run for each sample.

Statistical analysis

Statistical analysis and data processing were performed using Microsoft Excel 2013 and Minitab v20 (Minitab Inc., PA, USA). Results of the chemical analysis are reported as average values with standard deviation of at least n = 3 replicates.

One-way analysis of variance (ANOVA) was used to determine significant differences of selected components where the normal distribution and equal variance tests were fulfilled. Data were tested for normal distribution using the Anderson – Darling test and for equal variance by Levene´s test, prior to one-way ANOVA, using the Tukey method for comparison of means. The significance level was set to p < .05, and values marked with different superscript letters were significantly different. Also, individual values of replicates are given.

The unsupervised multivariate principal component analysis (PCA) (Wold et al. 1987) was performed to visualize differences and changes in the composition of phospholipids. The PCA was performed by the use of Minitab v.20 statistical software and employing the correlation matrix as calculated by the software (i.e., the standardized concentrations obtained by subtraction of the mean and divided by the standard deviation).

Results

Dry matter and ash content

Table 1 gives an overview of the gross chemical composition of polychaete biomass submitted to different treatments. The dry matter content of the polychaete varied between 12 and 19 g/100 g for the different treatments, but there were no significant changes during the frozen storage time for any of the treatments. The blanched samples had a higher dry matter content than the other samples (18.3 ± 1.2 g/100 g), indicating that these worms were drained more prior to frozen storage. Also, the worms frozen at the industrial facility (FrozenV −27°C) had a slightly higher dry matter content (15.3 ± 0.5 g/100 g) compared to the worms frozen at the laboratory (Nitrogen and Frozen −23°C with 11.7 ± 0.2 g/100 g and 12.7 ± 0.9 g/100 g, respectively). The ash content was similar in the control samples, and the non-blanched frozen stored samples, while there was a significant reduction in ash content in the blanched samples, from approximately 15 g/100 g dw in the control sample to 4.4 g/100 g dw in the blanched samples.

Table 1. Gross chemical composition of polychaete biomass submitted to different treatments. Dry matter is given as g/100 g, while ash and lipid content is given as g/100 g dry weight (dw) Significant differences (ANOVA p < .05) are given by different superscript letters.

Treatment	n	Dry matter (g/100 g)	St.dev	Ash (g/100 g dw)	St.dev	Lipid (g/100 g dw)	St.dev
Nitrogen	4	11.7^a	0.2	14.9^a	0.3	18.4a	0.7
Frozen-23°C	12	12.7^a	0.9	14.4^a	0.7	16.0b	1.3
Frozen-27°C	12	15.3^b	0.5	15.1^a	0.7	15.8b	0.6
Blanched-23°C	16	18.3 ^c	1.2	4.4 ^b	0.4	21.5c	0.7

Lipid content

The lipid content of the different treatments ranged between 2.2 and 2.5 g/100 g worm on a wet weight bases in the non-blanched samples, while the blanched samples had a higher lipid content (4.5 g/100 g wet weight), which can be attributed to the lower water content in blanched samples. Also, on a dry weight basis, the lipid content was highest in the blanched samples, with a lipid content of 21.5 g/100 g dry weight compared to 16–18 g/100 g dry weight in the other treatments.

Fatty acid composition

Supplementary Table S1 reports the total FA content and FA composition of the polychaete worms after different treatments and storage times. The total content of FA in the crude lipid extract did not show any clear differences due to treatment or storage time. On average, fatty acids accounted for 717 ± 56 mg/g crude oil. However, there was a large variation in total fatty acid extracted among replicates, especially for blanched −23°C samples after 1 month of storage. The content of the LC PUFAs DHA and EPA were 2.10 ± 0.04% and 8.3 ± 0.1% of total FA, respectively, in the control sample. A slight, but significant, decrease of both DHA and EPA was seen in non-vacuum-packed worms during the storage, which may be explained by the well-known oxidation of polyunsaturated fatty acids. After 12 months of storage, the DHA and EPA contents were reduced to ~1.74% and 7.6%, respectively, for the non-vacuum-packed samples (Frozen −23° and Blanched −23°C). For vacuum packed worms, the content of DHA and EPA was stable.

Free fatty acids

In samples without initial heat treatment, frozen storage at −23°C and −27°C led to a relatively fast increase in FFA values. FFA increased from 1.5 ± 0.2% in control samples to 4.8 ± 0.6% and 6.3 ± 0.4% after 1 month of storage for samples at −23°C and −27°C, respectively (reported as weight % of crude lipids). During further storage, the increase in FFA was faster at −23°C than at −27°C, and after 6 months, the FFA levels were 15.7 + 1.0% and 13.5 + 1.5%, respectively. After 12 months of storage, the amount of FFA was highest in samples stored at −23°C, with an FFA level of over 20%, while the −27°C stored samples had a content of 16.1 ± 0.6%. The amount of free fatty acids did not increase during frozen storage of the blanched samples (Figure 1).

Figure 1. Content of free fatty acid (weight % of crude lipids) in polychaete biomass during frozen storage up to 12 months at different conditions. ‘Nitrogen’ refers to live worms frozen with liquid nitrogen and extracted directly. ‘Frozen −23°C’ refers to worms frozen in plastic bags at − 23°C, while ‘FrozenV −27°C’ were vacuum packed prior to frozen storage at −27°C. ‘Blanched −23°C’ refers to blanched worms stored in plastic bags stored at −23°C. Results are reported as average- and individual values on four replicate extracts, and significant differences are denoted by different letters.

Total phospholipid content and profile of phospholipids

Results from ³¹P NMR showed that at the beginning of the experiment, phospholipids accounted for 26 ± 3 g/100 g (for nitrogen frozen samples) to 29 ± 5 g/100 g of crude lipid extract (blanched samples). There was a significant reduction in PL content after 12 months of storage for non-blanched samples (Frozen −23°C and FrozenV −27°C), where the PL content was reduced to similar levels, 21 ± 2 g/100 g of the lipid extract. For blanched samples, the results showed large variation between individual samples, as was also observed in the total fatty acids extracted. However, when normalizing the PL content to the sum of fatty acids from¹H NMR data on the same samples, the PL content showed less variation among parallels (Figure 2). The PL content was reduced from approximately 34 ± 3 weight % of total FA in the control to 26 ± 2% in non-blanched samples after 12 months of storage. In the blanched samples, the content of PL was not reduced after 12 months of storage (32.6 ± 0.6%). The reduction of PLs was lower than the formation of FFA (Figure 1), but from the¹H NMR spectra it was clear that also triacylglycerols were hydrolyzed to FFA during storage of non-blanched samples, as a reduction of intensities from peaks from triacylglycerols were seen (example from frozen samples after 1- and 12-months of storage shown in supplementary file B).

Figure 2. Content of total phospholipids from ³¹P NMR (weight % of total fatty acids) in polychaete biomass during frozen storage up to 12 months at different conditions. Results are reported as average- and individual values on four replicate extracts, and significant differences are denoted by different letters.

The ³¹P NMR analysis further showed that the profile of phospholipid changed during the frozen storage in samples not submitted to blanching as exemplified in Figure 3, whereas for the blanched samples, the PL composition did not change. The PCA analysis (Figure 4) illustrates this quite clearly, with control samples and blanched samples grouped closely, implying that the PL profile of these samples was similar.

Figure 3. ³¹P NMR profile of lipids extracted from polychaete biomass at the beginning of the experiment (nitrogen frozen), compared with profiles after 1-month frozen storage at −23°C and after 12 months of frozen storage at −27°C in vacuum pack. Significant differences are denoted by different letters.

Figure 4. Scores (a) and loadings plot (b) from principal component analysis (PCA). The PCA illustrates changes in phospholipid composition in polychaete biomass during frozen storage at different conditions. Labels refer to sampling month. The first two principal components explained 63% and 13% of the variance in the dataset, respectively.

The PCA analysis for the non-blanched samples shows that the relative amount of PC and LPC was decreasing, while the intensity of several new peaks was increasing during storage of non-blanched samples.

In Table 2, the relative content of different phospholipid species is given. In the control sample, PC accounted for 44.9 ± 0.8% (mole % of total PLs), followed by PE (20.5 ± 0.9%) and ePE/SPH (8.5 ± 0.7%). The PL species: ePC, 2LPC, LPC, PI, and APE accounted for ca 5% of the total PLs. The results showed a significant decrease in PC, 2LPC, and LPC in non-blanched samples (Frozen-23° and FrozenV −27°C), while the share of other PLs, such as LPE* at 0.51 ppm and PA* at 0.35 ppm, increased. Other PLs such as PI and PE seemed to be more stable during frozen storage. Among the new and unidentified (u*) peaks arising, those at −0.31 ppm (u1*) and −0.41 ppm (u0*) reached a level of about 3% (mole% of total PLs) after 12 months of frozen storage of non-blanched samples.

Table 2. Composition of phospholipids (mole % of total phospholipids) in polychaete biomass after different storage times for each phospholipid, values that do not share a letter are significantly different (ANOVA, p < .05).

Time (month)
	Group	0	1	12
PC	Nitrogen	44.9 ± 0.8^a
	Blanched −23°C	44.7 ± 0.8 ^a	45.2 ± 0.7^a	47.1 ± 0.6^a
	Frozen −23°C		41.3 ± 2.9^b	36.3 ± 1.8^c
	FrozenV −27°C		37.4 ± 0.7^c	35.5 ± 0.7^c
PE	Nitrogen	20.5 ± 0.9^c,d,e
	Blanched −23°C	21.6 ± 0.2 ^b,c,d	20.1 ± 0.8^d,e	18.9 ± 0.2^e
	Frozen −23°C		22.2 ± 0.6^b,c	21.9 ± 1.5^b,c,d
	FrozenV −27°C		23.1 ± 0.1^a,b	24.6 ± 0.5^a
ePE/SPH	Nitrogen	8.5 ± 0.7 ^a
	Blanched −23°C	8.2 ± 0.1^a,b	8.5 ± 0.8^a	8.0 ± 0.2^a,b,c
	Frozen −23°C		7.7 ± 0.4^c,d	6.8 ± 0.6^a,b,c,d
	FrozenV −27°C		7.0 ± 0.4^b,c,d	6.5 ± 0.3^d
ePC	Nitrogen	5.3 ± 0.3^a,b,c
	Blanched −23°C	4.9 ± 0.4^c	5.3 ± 0.1	5.1 ± 0.3^b,c
	Frozen −23°C		5.3 ± 0.3 ^a,b,c	5.6 ± 0.3 ^a,b
	FrozenV −27°C		5.2 ± 0.0^a,b,c	5.9 ± 0.1^a
2LPC	Nitrogen	4.7 ± 0.4 ^b,c
	Blanched −23°C	5.6 ± 0.2 ^a	5.7 ± 0.3^a	5.4 ± 0.3^a,b
	Frozen −23°C		3.0 ± 0.1^d	0.0 ± 0.0^f
	FrozenV −27°C		3.9 ± 0.3^c	1.3 ± 0.6^e
LPC	Nitrogen	4.7 ± 0.3 ^a
	Blanched −23°C	4.9 ± 0.4 ^a	5.3 ± 0.3^a	4.9 ± 0.2^a,b
	Frozen −23°C		3.7 ± 0.1^b	3.5 ± 0.5^b
	FrozenV −27°C		3.7 ± 0.4^b	3.4 ± 0.2^b
PI	Nitrogen	5.1 ± 0.1 ^c,d
	Blanched −23°C	4.5 ± 0.4 ^d,e	4.4 ± 0.1^e	5.0 ± 0.3^c,d,e
	Frozen −23°C		5.2 ± 0.2^c	6.9 ± 0.4^a
	FrozenV −27°C		5.6 ± 0.3^b,c	6.2 ± 0.3^b
APE	Nitrogen	4.5 ± 0.5
	Blanched −23°C	4.4 ± 0.3	4.3 ± 0.3	4.4 ± 0.3
	Frozen −23°C		4.3 ± 0.4	4.8 ± 0.4
	FrozenV −27°C		4.8 ± 0.4	4.8 ± 0.1
LPE*	Nitrogen	0.0 ± 0.0
	Blanched −23°C	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
	Frozen −23°C		0.7 ± 0.4^b	3.6 ± 0.9^a
	FrozenV −27°C		1.6 ± 0.1^b	3.8 ± 0.2^a
u0* (−0.41 ppm)	Nitrogen	0.0 ± 0.0
	Blanched −23°C	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
	Frozen −23°C		3.1 ± 2.2	3.5 ± 0.7
	FrozenV −27°C		0.0 ± 0.0	2.9 ± 0.4
u1* (− 0.31ppm)	Nitrogen	0.5 ± 0.6
	Blanched −23°C	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
	Frozen −23°C		1.3 ± 0.2	3.2 ± 0.6
	FrozenV −27°C		6.2 ± 0.6	3.2 ± 0.6
PA*	Nitrogen	0.0 ± 0.0
	Blanched −23°C	0.0 ± 0.0	0.3 ± 0.4	0.0 ± 0.0
	Frozen −23°C		0.3 ± 0.3	1.6 ± 0.4
	FrozenV −27°C		0.0 ± 0.0	1.4 ± 0.3

^{*Tentative assignments are labelled with *, u0 and u1 refers to unassigned peaks.}

Oxidative lipid quality

Primary oxidation products were evaluated by peroxide values (PV) in lipids extracted from polychaete biomass after the different storage times (Figure 5). PV in control sample was 9.8 ± 0.7 meq/kg oil, and no significant changes were seen during the first month of storage for any of the storage conditions. However, a significant increase was seen after 6 months of storage for samples that were not vacuum packed (Frozen −23°C and Blanched −23°C) with similar values of ca 18.2 ± 0.6 meq/kg oil at month 6. For the Frozen −23°C samples, PV reached 42.2 ± 6.6 after 12 months of storage. For blanched samples, the PV values decreased from 6 to 12 months of storage. For vacuum packed worms, no increase in PV was seen during the storage.

Figure 5. Peroxide values (PV, reported as meq/kg oil) of lipids extracted from polychaete biomass during frozen storage at different conditions. Results are reported as average- and individual values on four replicate extracts. Significant differences are denoted by different letters.

The development of secondary oxidation products was followed as changes in TBARS values of both the water/methanol phase (Figure 6a) and in the chloroform phase (Figure 6b) of the extraction. For vacuum packed worms (FrozenV −27°C), no significant increase in TBARS was seen during the storage for any of the two extracts. On the other hand, TBARS in chloroform phase increased significantly in Frozen −23°C stored samples (i.e., from control (0.3 ± 0.1 µmole MDA/100 g biomass) to month 6 (2.5 ± 0.2) and to month 12 (4.5 ± 0.3)). For the Blanched −23°C samples, a large variation in TBARS was seen between extracts of replicate treatments. Furthermore, while TBARS in chloroform phase was significantly higher at month 6 compared to earlier sampling times, the TBARS decreased afterwards. A similar trend was seen for TBARS in the methanol phase during storage of the different treatments. TBARS in the methanol phase was significantly higher for non-vacuum packed (Frozen −23°C and Blanched −23°C samples) samples at months 6 and 12, but there was a reduction from month 6 to month 12 in the Blanched −23°C samples.

Figure 6. Changes in TBARS values in (a) chloroform phase and (b) water/methanol phase of extracted polychaete biomass during storage at different conditions. Results are reported as average- and individual values on four replicate extracts. Significant differences are denoted by different letters.

TBARS levels for blanched samples were in the same range as worms frozen at −23°C in the same type of plastic bags (Figure 6a,b).

Discussion

Cultivated polychaetes have gained increasing interest for use in aquaculture, both as a feed ingredient with superior effect in broodstock diets but also as a novel and sustainable alternative to traditional fishmeal. Due to high activity of endogenous enzymes, high water content, and content of polyunsaturated fatty acids, marine raw materials are highly perishable, and proper handling and preservation methods are needed to ensure acceptable shelf life. Frozen storage is a commonly used method to hinder microbial and enzymatic changes in marine raw material, but degradation of components may take place during frozen storage (Bergvik et al. 2012; Standal et al. 2018). The goal of the present study was to evaluate the stability of polychaete biomass during different frozen storage conditions to increase knowledge on the stability of this cultivated organism that is of interest to the aquaculture feed industry.

There were some differences in dry matter content between worms due to the different treatments, and it is likely that this is due to differences in the extent of draining prior to frozen storage. All samples were within the commonly used quality limit of ash content in fishmeal for salmon feed (i.e., ash values of <14% of dry weight is recommended (Einarsson et al. 2019)). The drastic reduction in ash content after blanching (from approximately 15 g/100 g dw ash in the control sample to 4.4 g/100 g dw in the blanched samples) shows that blanching is an efficient means to reduce salt and mineral content and may be relevant for the use of these organisms for feed purposes. Blanching led to a reduction of minerals/salt and low molecular weight metabolites. After blanching, the liquid phase had a slight green and cloudy appearance, and it cannot be excluded that some leakage of lipidic components also took place, as observed in blanching of Calanus finmarchicus (Bergvik et al. 2012). Blanched samples, however, had a higher lipid content than the other treatments on a dry weight basis, which can be explained by the washing out of other components (such as salt/ash and other easily soluble components such as water-soluble proteins and osmolytes) leading to a higher relative content of the remaining constituents. The lipid content of 16–18% in non-blanched worms was in accordance with previous reported values for cultivated Alitta virens, but both the lipid content and composition are highly dependent on the diet used (Brown et al. 2011). The content of total fatty acids in the crude lipid extract did not show any significant changes according to treatment or storage time, but there were large variations among parallel extracts, especially for blanched samples, indicating that the extraction efficiency varied. It is well known that membrane lipids, such as phospholipids, are challenging to extract fully, even with the effective solvent system of chloroform and methanol (Bettjeman et al. 2018). The variation in both the total fatty acids, the content of phospholipids, and the oxidation quality parameters (PV and TBARS) was highest for blanched samples. A possible explanation for the larger variation for blanched samples is that there may have been a varying degree of washing out of lipids or changes in extractability due to the heating, and that the samples were therefore not as homogeneous as for the other treatments.

A relatively fast increase in FFA content was seen during frozen storage of polychaete biomass that had not been submitted to blanching. The content of free fatty acids (weight % of total lipids) increased from below 2% in the control to about 5% after 1-month frozen storage to nearly 13.5% and 15.5% after 6 months of storage in non-blanched samples. Lowered frozen storage temperature (−27°C vs −23°C) only slightly reduced the formation of FAA up to 6 months, but from 6 to 12 months, the FFA content was significantly lower in the −27°C group (16.1% vs 21.8%) vs the −23°C group. The increase in FFA was in similar range, as observed during frozen storage of intact low trophic organisms, such as different Calanus species (Bergvik et al. 2012; Ohman 1996) but remarkably higher than observed in frozen storage of other marine raw material, such as mackerel fillets (Standal et al. 2018). The traditional frozen storage temperatures used in the fishery industry today (−18°C to −27°C) may not be sufficient to prevent lipid hydrolysis in these novel feed raw materials. FFA levels in extracted lipids were stable in blanched worms (Figure 1), so introducing heat-treatment prior to frozen storage was effective for preventing activity of endogenous lipases/phospholipases.

This is the first study, as far as we know, reporting the detailed composition of the phospholipids in Alitta virens by ³¹P NMR. The composition differs from previous ³¹P NMR profiles reported for earthworm (Meneses and Glonek 1988) and krill oil (Monakhova and Diehl 2018). The PLs were composed of several different phospholipid species: phosphatidyl choline (PC) accounted for 44.9 ± 0.8%, followed by phosphatidyl ethanolamine (PE) (20.5 ± 0.9%) and ether ethanolamine/sphingomyelin ePE/SPH (8.5 ± 0.7%). The other PLs were ether PC (ePC), 2-lyso PC (2LPC), lyso-PC (LPC), phosphatidyl inositol (PI), and acylphosphatidylethanolamine (APE), accounting for 5% of the total PLs. Phospholipids are known to be valuable components in fish feed, and the current study shows that the phospholipid profile changes during frozen storage, which may be of relevance for the use of the worms for feed ingredients.

The³¹P and¹H NMR analysis showed that not only phospholipids but also triacylglycerols, were hydrolyzed during storage. Previous studies on frozen storage of the zooplankton Calanus finmarchicus found that the increase in FFA was mainly due to hydrolysis of phospholipids (Bergvik et al. 2012). The degree of hydrolysis of lipids is influenced by both the composition of the lipids and the activity of the endogenous enzymes, which have been shown to vary both due to internal and external factors such as season and prey (Sovik and Rustad 2005; Tande 1982), in addition to temperature, pH, salt/bile content, and other factors (Aaen et al. 1995; Bergvik et al. 2012; Burgaard and Jørgensen 2011; Kishimura 2012; Sovik and Rustad 2005; Tatiyaborworntham et al. 2022). The current study indicates that lipases and phospholipases were released from cells and were highly active during the frozen storage conditions of polychaete worms. This has previously been shown in fish muscle, although at higher temperatures (−18°C) than applied in the current study (−23 and −27°C). It has also been shown that fractions containing intestines, such as by-products after filleting, are labile towards lipolysis (Sovik and Rustad 2005).

PV is a commonly used indicator of early oxidation of oils. Proposed upper limits of PV varies according to type of oil and applications, but fresh marine oils typically have PV levels below 10 meq/kg oil (Ismail et al. 2016). To the best of our knowledge, there are no studies reporting the oxidative quality of lipids extracted from polychaete worms; but the current study shows that for fresh biomass, a PV of 10 meq/kg oil could be expected. As summarized by Bell and Koppe (2010), the quality guideline specification of marine oil used in aquaculture feed varies for oil from different species, with values ranging from 3 to 20 meq/kg (Bell and Koppe 2010). The worms stored at −27°C were packed in vacuum bags, and the results show that this was effective for preventing oxidation during frozen storage up to 12 months, with stable PV values (<10 meq/kg oil). The effect of vacuum packing to prevent rancidity is well-known (Domínguez et al. 2019). For the blanched samples, an increase in PV was seen from 0 to 6 months (from below <10 to 18.2 ± 0.6 meq/kg), indicating that oxidation took place but was followed by a reduction from 6 to 12 months of storage. This can be explained by the well-known degradation of peroxides into secondary oxidation products during later stages of oxidation. The results indicate that blanching did not decrease the formation of secondary oxidation products (measured by TBARS values), as the blanched samples stored in plastic bags had as high TBARS levels as the non-blanched samples stored at similar conditions. TBARS is a commonly used method to evaluate the content of carbonyl compounds formed by oxidation, primarily malondialdehyde.

It is difficult to establish acceptable quality limits of TBARS, as the values obtained may vary between studies both with respect to the composition of the sample (Mozuraityte et al. 2017), the extract analyzed (Mozuraityte et al. 2020; Sajib and Undeland 2020), and also the protocol used for analysis (Mozuraityte et al. 2017). As summarized by Van’t Land et al. (2017), even though there are no specific quality limits for TBARS in feed raw materials, the oxidation quality may influence the feed acceptability. In the present study, both water/methanol soluble TBARS and chloroform soluble TBARS were measured. The results followed the same trend for both extracts. The TBARS values in the methanol/water fraction were slightly higher than in the lipid/chloroform fraction. It has been proposed that as oxidation progresses, there is relatively higher content of ‘free’ TBARS (methanol/water soluble) compared to bound TBARS (chloroform soluble) (Mozuraityte et al. 2020). During further oxidation, tertiary oxidation products may be formed such as volatile short chain acids, leading to reduced TBARS values (Van’t Land et al. 2017). The latter may be an explanation for the apparent reduction in TBARS in Blanched −23°C samples from 6 to 12 months. The large variation in TBARS values of replicate blanched samples may be due to different extraction efficacy as discussed above and also that the main carbonyl compound contributing to the TBARS values, i.e., MDA (malondialdehyde), could react with protein, peptides, or amino acids (Adams et al. 2011). It has also been shown that TBARS may be hydrolyzed at higher temperatures (Sajib and Undeland 2020). However, in the present study, the heating was performed before the oxidation took place. The results on FA composition show that the blanched −23°C group had a significant decrease in the LC PUFAs DHA and EPA, which may be explained by the relatively high oxidative status and thereby degradation of polyunsaturated fatty acids. On the other hand, vacuum packed worms stored frozen at −27°C, showed very low lipid oxidation as measured by TBARS, in line with the PV results, demonstrating the importance of preventing oxygen access to retain high oxidative quality of raw material lipids containing LC PUFAs. It is well established that DHA and EPA levels and lipid oxidation quality are important quality parameters of fish oil used in aquaculture (Bell and Koppe 2010). However, to the authors’ knowledge, studies assessing feed performance due to varying lipid oxidation status in oil or meal are lacking in the literature.

Recently, the effect of lipolysis on lipid oxidation has been reviewed (Tatiyaborworntham et al. 2022). Results in the literature are contradictory regarding whether products from lipolysis function as pro-oxidants or antioxidants. The present study does not imply that there are any large effects on oxidation parameters due to the hydrolysis of lipids, as the oxidation status is quite similar for both the Blanched −23°C samples and the Frozen −23°C (non-blanched), and the latter had much higher FFA content at later storage times.

Conclusion

The results obtained in this study show that both high endogenous lipase/phospholipase activity and lipid oxidation are challenges for maintaining lipid quality during frozen storage of polychaete biomass. In particular, phospholipids are valuable components in aquaculture feed, and the study demonstrates that to prevent the formation of free fatty acids, frozen storage time should be limited, or endogenous enzymes should be inactivated before storage. To also prevent lipid oxidation during frozen storage, polychaete biomass should be stored vacuum packed. Blanching is one method for hindering enzymatic activity in biomass with high endogenous activity and is also beneficial for salt/ash reduction; however, some loss of lipids, water-soluble proteins, and osmolytes are anticipated. The preservation approach selected may thereby differ depending on factors such as expected storage duration and the specific quality parameters considered most crucial to retain. Given the limited understanding to which extent lipid hydrolysis and oxidation influence the performance of feed ingredients, it is important to consider such quality factors and preferably take effective measures to hinder such degradation in marine low trophic raw materials.

Acknowledgments

This study was supported by The Research Council of Norway under Grant (number 280836, Cultivation of Polychaeta as raw material for feed) and also through the ERA-NET BlueBio COFUND (Grant ID 68 and number 311701, Secondary bio-production of low trophic organisms utilizing side streams from the Blue and Green sectors to produce novel feed ingredients for European aquaculture). The NMR analysis was performed at the MR Core Facility, Norwegian University of Science and Technology (NTNU) partly supported by the RCN through the Norwegian NMR Platform (NNP, 226244/F50). The authors are grateful to Topsy Baits for providing the live and frozen Alitta virens used in the study. The authors wish to thank Merethe Selnes for performing laboratory analysis.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10498850.2024.2316581.

Correction Statement

This article was originally published with errors, which have now been corrected in the online version. Please see Correction (http://doi.org/10.1080/10498850.2024.2338983)