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Use of Probiotic Bacteria and Bacteriocins as an Alternative to Antibiotics in Aquaculture

Tengiliður

Viggó Marteinsson

Fagstjóri

viggo@matis.is

In addition to their use in human medicine, antimicrobials are also used in food animals and aquaculture, and their use can be categorized as therapeutic against bacterial infections. The use of antimicrobials in aquaculture may involve a broad environmental application that affects a wide variety of bacteria, promoting the spread of bacterial resistance genes. Probiotics and bacteriocins, antimicrobial peptides produced by some types of lactic acid bacteria (LAB), have been successfully tested in aquatic animals as alternatives to control bacterial infections. Supplementation might have beneficial impacts on the intestinal microbiota, immune response, development, and/or weight gain, without the issues associated with antibiotic use. Thus, probiotics and bacteriocins represent feasible alternatives to antibiotics. Here, we provide an update with respect to the relevance of aquaculture in the animal protein production sector, as well as the present and future challenges generated by outbreaks and antimicrobial resistance, while highlighting the potential role of probiotics and bacteriocins to address these challenges. In addition, we conducted data analysis using a simple linear regression model to determine whether a linear relationship exists between probiotic dose added to feed and three variables of interest selected, including specific growth rate, feed conversion ratio, and lysozyme activity.

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Protein Recovery of Tra Catfish (Pangasius hypophthalmus) Protein-Rich Side Streams by the pH-Shift Method

Tengiliður

Sigurjón Arason

Yfirverkfræðingur

sigurjon.arason@matis.is

Increasing protein demand has led to growing attention being given to the full utilization of proteins from side streams in industrial fish processing. In this study, proteins were recovered from three protein-rich side streams during Tra catfish (Pangasius hypophthalamus) processing (dark muscle; head-backbone; and abdominal cut-offs) by an optimized pH-shift process. Physicochemical characteristics of the resulting fish protein isolates (FPIs) were compared to industrial surimi from the same raw material batch. The pH had a significant influence on protein extraction, while extraction time and the ratio of the extraction solution to raw material had little effect on the protein and dry matter recoveries. Optimal protein extraction conditions were obtained at pH 12, a solvent to raw material ratio of 8, and an extraction duration of 150 min. The resulting FPI contained <10% of the fat and <15% of the ash of the raw material, while the FPI protein recovery was 83.0−88.9%, including a good amino acid profile. All FPIs had significantly higher protein content and lower lipid content than the surimi, indicating the high efficiency of using the pH-shift method to recover proteins from industrial Tra catfish side streams. The FPI made from abdominal cut-offs had high whiteness, increasing its potential for the development of a high-value product.

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Protein Characteristics and Bioactivity of Fish Protein Hydrolysates from Tra Catfish (Pangasius hypophthalmus) Side Stream Isolates

Tengiliður

Sigurjón Arason

Yfirverkfræðingur

sigurjon.arason@matis.is

Enzymatic hydrolysis is a novel method to recover highly potent bioactive fish protein hydrolysates (FPHs) from fish processing side-streams. The common way of producing FPHs directly from fish side-streams may be inappropriate due to the excess of lipids and pro-oxidants, especially in lipid-rich streams, as obtained from Tra catfish. This study aimed to optimise the hydrolysis conditions for a commercial enzyme (Alcalase® 2.4 L) (enzyme concentrate, temperature, and time) in FPH production from the fish protein isolate obtained from Tra catfish dark muscle (DM-FPI) using the pH-shift method. The degree of hydrolysis (DH), protein recovery (PR), and antioxidant properties, including DPPH radical scavenging activity (DPPH-RSA) and total reducing power capacity (TRPC), were measured to evaluate the effects of the hydrolysis conditions on the FPHs. Optimal hydrolysis was obtained at an enzyme/substrate protein ratio of 3% (v/w) and a hydrolysis temperature of 50 °C for 3 h. The FPHs obtained from different substrates, including DM-FPI, abdominal cut-off (ACO) FPI, and head and backbone blend (HBB) FPI, had similar DHs under these optimum conditions, ranging from 22.5% to 24.0%. However, the FPH obtained from abdominal cut-off isolate (ACO-FPH) showed the highest PR of 81.5 ± 4.3% and the highest antioxidant properties, with a DPPH-RSA of 86.1 ± 1.6% and a TRPC of 6.4 ± 0.4 equivalent mg vitamin C/g protein. The resulting FPHs present a natural source of antioxidants with great potential for food applications, especially the ACO-FPH. In addition, all FPHs had excellent amino acid profiles, indicating strong potential for their use as supplements. Tra catfish protein-rich side-streams can thus be processed into high-value bioactive FPHs using Alcalase for human consumption.

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Changes in Protein and Non-Protein Nitrogen Compounds during Fishmeal Processing – Identification of Unoptimized Processing Steps

Tengiliður

Sigurjón Arason

Yfirverkfræðingur

sigurjon.arason@matis.is

Quality changes of protein and non-protein nitrogen compounds during industrial fishmeal processing of fatty pelagic species (mackerel/herring rest material blend, MHB) and lean fish (whole blue whiting, BW) were studied to identify processing steps that require optimization to allow production of products for human consumption. Samples from protein-rich processing streams throughout the fishmeal production were analyzed for proximate composition, salt soluble protein content (SSP), biogenic amines (BA), total volatile basic nitrogen (TVB-N), trimethylamine (TMA), and dimethylamine (DMA). Mass flows throughout processing were balanced based on the total mass and proximate composition data. The quality of the final fishmeal products was highly dependent on the fish species being processed, indicating that the processes require optimization towards each raw material. The chemical composition changed in each processing step, resulting in different properties in each stream. Most of the non-protein nitrogen compounds (including BA, TVB-N, TMA, and DMA) followed the liquid streams. However, the concentrate contributed less than 20% to the produced fishmeal quantity. Mixing of this stream into the fishmeal processing again, as currently carried out, should thus be avoided. Furthermore, the cooking, separating, and drying steps should be optimized to improve the water and lipid separation and avoid the formation of undesired nitrogen compounds to produce higher-value products intended for human consumption.

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The Arctic as a food-producing region. Chapter in Renewable Economies in the arctic 

Tengiliður

Ólafur Reykdal

Verkefnastjóri

olafur.reykdal@matis.is

This chapter provides a summary of food production in three regions of the Arctic. These include: the entirety of Iceland; Norway’s three northernmost counties—Nordland, Troms, and Finnmark; and northern Canada, including Yukon, Northwest Territories, Nunavut, Nunavik, and Labrador. In 2016, the Sustainable Development Working Group endorsed The Arctic as a Food Producing Region research project. The export of fish and fish products are by far the most important export items from Iceland and contribute significantly to the Icelandic economy. From 2012 to 2016, Norway exported fish products worth 339,207,335,000 Norwegian krone. Marine products accounted for 89% of northern Canada’s total food export. There are over 3,000 sheep farms in Iceland. These farms tend to be small and family owned. The sheep farming is as old as the human settlement of Iceland. The largest agricultural production systems in Northern Norway are based on meat production. The agricultural production in northern Norway is the northernmost active agricultural system in the world.

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Medium development and production of carotenoids and exopolysaccharides by the extremophile Rhodothermus marinus DSM16675 in glucose-based defined media  

Tengiliður

Þórdís Kristjánsdóttir

Verkefnastjóri

thordis@matis.is

Background

The marine thermophilic bacterium Rhodothermus marinus can degrade many polysaccharides which makes it interesting as a future cell factory. Progress using this bacterium has, however, been hampered by limited knowledge on media and conditions for biomass production, often resulting in low cell yields and low productivity, highlighting the need to develop conditions that allow studies of the microbe on molecular level. This study presents development of defined conditions that support growth, combined with evaluation of production of carotenoids and exopolysaccharides (EPSs) by R. marinus strain DSM 16675.

Results

Two defined media were initially prepared: one including a low addition of yeast extract (modified Wolfe’s medium) and one based on specific components (defined medium base, DMB) to which two amino acids (N and Q), were added. Cultivation trials of R. marinus DSM 16675 in shake flasks, resulted in maximum cell densities (OD620 nm) of 2.36 ± 0.057, cell dry weight (CDW) 1.2 ± 0.14 mg/L, total carotenoids 0.59 × 10–3 mg/L, and EPSs 1.72 ± 0.03 mg/L using 2 g/L glucose in DMB. In Wolfe’s medium (supplemented by 0.05 g/L yeast extract and 2.5 g/L glucose), maximum OD620 nm was 2.07 ± 0.05, CDW 1.05 ± 0.07 mg/L, total carotenoids 0.39 × 10–3 mg/L, and EPSs 1.74 ± 0.2 mg/L. Growth trials at 5 g/L glucose in these media either failed or resulted in incomplete substrate utilization. To improve reproducibility and increase substrate utilization, a screening of macroelements (e.g. phosphate) in DMB, was combined with use of trace elements and vitamins of the modified Wolfe’s medium. The resulting defined minimal R. marinus medium, (DRM), allowed reproducible cultivations to a final OD620nm of 6.6 ± 0.05, CDW 2.85 ± 0.07 mg/L, a maximum specific growth rate (µmax) of 0.26 h−1, total carotenoids 0.77 × 10–3 mg/L and EPSs 3.4 ± 0.17 mg/L in cultivations supplemented with up to 5 g/L glucose.

Conclusion

A minimal defined medium (DRM) was designed that resulted in reproducible growth and an almost doubled formation of both total carotenoids and EPSs. Such defined conditions, are necessary for systematic studies of metabolic pathways, to determine the specific requirements for growth and fully characterize metabolite production.

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Torula yeast in the diet of Atlantic salmon Salmo salarand the impact on growth performance and gut microbiome

Tengiliður

Birgir Örn Smárason

Fagstjóri

birgir@matis.is

Atlantic salmon aquaculture is expanding, and with it, the need to find suitable replacements for conventional protein sources used in formulated feeds. Torula yeast (Cyberlindnera jadinii), has been identified as a promising alternative protein for feed and can be sustainably cultivated on lignocellulosic biomasses. The present study investigated the impact of torula yeast on the growth performance and gut microbiome of freshwater Atlantic salmon. A marine protein base diet and a mixed marine and plant protein base diet were tested, where conventional proteins were replaced with increasing inclusion levels of torula yeast, (0%, 10%, 20%). This study demonstrated that 20% torula yeast can replace fish meal without alteration to growth performance while leading to potential benefits for the gut microbiome by increasing the presence of bacteria positively associated with the host. However, when torula yeast replaced plant meal in a mixed protein diet, results suggested that 10% inclusion of yeast produced the best growth performance results but at the 20% inclusion level of yeast, potentially negative changes were observed in the gut microbial community, such as a decrease in lactic acid bacteria. This study supports the continued investigation of torula yeast for Atlantic salmon as a partial replacement for conventional proteins.

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Feeding black soldier fly larvae (Hermetia illucens) reared on organic rest streams alters gut characteristics of Atlantic salmon (Salmo salar)

Tengiliður

Birgir Örn Smárason

Fagstjóri

birgir@matis.is

The Atlantic salmon (Salmo salar) aquaculture industry is growing, and with it, the need to source and optimise sustainable ingredients for aquafeeds. Black soldier fly (BSF) larvae (Hermetia illucens) have received increasing research attention since they are a good source of protein that can efficiently convert a wide range of low-value organic material into valuable resources. This study investigated the impact of three differently processed BSF meals, an untreated BSF diet (BSFC+), a dechitinated BSF diet (BSFC-) and a fermented BSF diet (BSFC+P+) at a 10% inclusion level replacing fish meal in a fish meal control (FM) and a marine and soy protein concentrate based control diet (SPC). Growth performance, gut microbiome and gut histology of salmon fry was assessed. The inclusion and processing methods of BSF showed no adverse impacts on either growth performance or gut histology. However, the gut microbiome of fish was significantly altered by both the protein source and the processing method of the BSF larvae. Fish fed BSFC+, had an increased diversity and evenness of the community compared with conventional protein sources alone, and compared with the other BSF processing methods. However, control diets had a greater presence of lactic acid bacteria and genera associated with faster growing hosts. Fish fed BSF had a high relative abundance of the genus, Exiguobacterium, a chitin-degrading bacterium and in BSFC+P+ fed fish this bacterium completely dominated the community, indicating the presence of dysbiosis. Future studies should determine, why Exiguobacterium has dominated the community for the BSFC+P+ diet, and if it provides a digestive function to the host and identify bacteria that are indicators of optimal host performance and resilience. The results confirmed that BSF is a promising fish meal replacement for salmon, and it demonstrated that BSFC+ has a potential prebiotic impact on the gut microbiome of Atlantic salmon.

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Lack of population genetic structure of lumpfish along the Norwegian coast: A reappraisal based on EST-STRs analyses

Tengiliður

Davíð Gíslason

Verkefnastjóri

davidg@matis.is

Lumpfish is now the single most important cleaner fish species to date and there is an extensive lumpfish translocation along the Norwegian coast. A reliable baseline information about the population genetic structure of lumpfish is a prerequisite for an optimal managing of the species to minimize possible genetic translocation and avoid possible hybridisation and introgression with local populations. The current study is a follow up of the study of Jónsdóttir et al. (2018) using expressed sequence tag-short tandem repeats (EST-STRs) markers. Samples (N = 291) were analysed from six sample locations along the Norwegian coastline from south to north, with additional 18 samples of first-generation (from wild fish) reared fish from a fish farm outside Tromsø (North Norway). Present findings show a lack of population differentiation among lumpfish sampling population along the Norwegian coast using EST-STRs, which is in accordance with the findings of Jónsdóttir et al. (2018) where genomic STRs (g-STRs) were analysed. Present findings indicate that should translocated lumpfish escape from salmon sea pens in Norway, this will probably have little impact on the genetic composition of the local lumpfish population.

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Impact of Putative Probiotics on Growth, Behavior, and the Gut Microbiome of Farmed Arctic Char (Salvelinus alpinus)

Tengiliður

Sigurlaug Skírnisdóttir

Verkefnastjóri

sigurlaug.skirnisdottir@matis.is

Beneficial bacteria promise to promote the health and productivity of farmed fish species. However, the impact on host physiology is largely strain-dependent, and studies on Arctic char (Salvelinus alpinus), a commercially farmed salmonid species, are lacking. In this study, 10 candidate probiotic strains were subjected to in vitro assays, small-scale growth trials, and behavioral analysis with juvenile Arctic char to examine the impact of probiotic supplementation on fish growth, behavior and the gut microbiome. Most strains showed high tolerance to gastric juice and fish bile acid, as well as high auto-aggregation activity, which are important probiotic characteristics. However, they neither markedly altered the core gut microbiome, which was dominated by three bacterial species, nor detectably colonized the gut environment after the 4-week probiotic treatment. Despite a lack of long-term colonization, the presence of the bacterial strains showed either beneficial or detrimental effects on the host through growth rate enhancement or reduction, as well as changes in fish motility under confinement. This study offers insights into the effect of bacterial strains on a salmonid host and highlights three strains, Carnobacterium divergensV41, Pediococcus acidilactici ASG16, and Lactiplantibacillus plantarum ISCAR-07436, for future research into growth promotion of salmonid fish through probiotic supplementation.

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