Biocatalysis: An Industrial Perspective

Nofima: Peptide Recovery and Commercialization by Enzymatic Hydrolysis of Marine Biomass

BIRTHE VANG^a, THEMIS ALTINTZOGLOU^a, INGRID MÅGE^b, SILESHI G. WUBSHET^b, NILS K. AFSETH^b AND RAGNHILD D. WHITAKER^*^a

17.1 Nofima: The Company

Nofima is a non-profit research organization that was established in 2008 as a merger between four different research institutes. Nofima is currently one of Europe’s largest institutes for applied research and development within the fields of aquaculture, fisheries and the food industry. The company is owned by the Norwegian government (56.8%), the Agricultural Food Research Foundation (33.2%) and Akvainvest Møre and Romsdal (10%). The company headquarters are located in Tromsø, and there are offices in Stavanger, Bergen, Ås and Sundalsøra. Nofima has 344 employees, of whom 203 are scientific personnel and the rest are employed in support functions within management, economy, communication and general infrastructure. The turnover in 2016 was about 550 million NOK and the company was engaged in over 600 different research projects with partners both domestically and internationally.¹

Nofima is divided into three sections: Fisheries Industries and Markets (FIM), Aquaculture (AKVA), and Food Science (FOOD), to describe competence and main working areas. However, scientific and support personnel work across divisions in order to provide the best solutions for the different customers. Nofima has a board of directors consisting of a combination of external leaders and Nofima employees. Nofima performs research in the area between academia and industry, which is usually referred to as the area of applied research. Through research, Nofima aims at results implementation to further develop environmentally, economically and socially sustainable food production. This goal can be achieved by delivering high quality research and solutions that create a competitive advantage and increase innovation and profitability for the customers. Customers include participants from the aquaculture industry, fisheries industry, land- and ocean based food and feed industry, cosmetic and pharmaceutical industry and public agencies.^1,2 The diversity of customers and research areas enables Nofima to have trans-sectorial knowledge and provide research and solutions in the entire value chain.

Nofima also runs and operates Biotep, a bio-test processing and production platform that opened in 2013. Biotep is one of the few facilities of its kind. It is designed to be a mini-factory where high technology companies can transfer promising research from the laboratory into advanced products on a larger scale. Companies can perform test productions based on their own processes and technology or in collaboration with Nofima. Smaller companies can also rent the facility to perform periodical or regular production. Biotep offers the option to test a production on a larger scale without the risk of large investments. From the test production, cost estimates can be made and a product prototype can be tested in the market. Biotep is used commercially, but it is also intended for use in research and for educational purposes. Each type of biomass requires an optimized process in order to extract and conserve all the desired components: water soluble, fat soluble and solids. Components produced are typically fish-, bone-, shell meal, purified oils, protein concentrate, small molecular components, bioactive peptides and ash. Commonly, processes are optimized on a laboratory scale and scaled up through a step-wise process to yield a small industrial scale. The processing plant is designed to receive most types of biomass, either pre-processed or raw/unprocessed including marine-, plant- and some animal-based. The plant is thus very flexible in order to allow each company to design their process to meet their needs exactly; however, this can lead to some loss in biomass due to longer pumping distances.

17.2 Hydrolysis of Marine Biomass

Marine biomass consists of various different kinds of fish, shellfish, marine plants and others. This chapter will focus on abundant and accessible marine biomass that is currently underutilized or used for low value products. Processing by-products from the fisheries industry is an example of this.

To access peptides from the proteinaceous material in biomass, peptides in the biomass need to be solubilized and made available. The biomass can in most cases be split into three fractions – an aqueous, a lipid and a solid phase. The chemical composition of the biomass will of course vary with species, but also depend on season, feed and what components of the fish are included, but usually a general distribution of 70–82% water can be expected, the remainder being proteins, fats and ash. On a dry matter basis, 40–70% of this will be protein, 1–50% fats and 3–4% ash. These are highly varying numbers, as already mentioned, and if heads, bones or shellfish are included, the ash component will be significantly increased.^3–5 Following this, all the components in the biomass can either be solubilized in the lipid or in the aqueous phase, or they can be considered insoluble and thus reside in the solid phase of the biomass. Lipids and lipophilic compounds can be recovered from the lipid phase, which is often considered the oil phase or the lighter phase. It has a lower density and will thus float on top of the aqueous phase. The aqueous phase will contain hydrophilic components such as peptides, some antioxidants and low molecular weight hydrophilic components.^6–10

17.2.1 Chemical Hydrolysis of Marine Biomass

To solubilize proteins in the biomass, chemical hydrolysis is a very common method. In a chemical extraction method, organic solvents, acids or alkali solutions can be employed. Solvent methods have been established and can result in high yield protein recovery; however, the use of organic solvents results in high costs and solvent trace residues in the product. Using organic solvents for protein extraction can also reduce the functionality of the proteins and cause an undesirable taste.^11,12 Organic solvent extraction methods are, due to the stated obstacles, not commercially viable from the type of biomass discussed in this chapter, and we will move on to acids or alkali extraction. In this approach, acid or alkali solutions are used to solubilize the proteins. The acidic or alkaline environment breaks down the proteins, causing unfolding and increased aqueous solubility of the smaller proteins and peptides.^{7,8,10,13–15} Acid hydrolysis, called ensilage or silage, is the most common process in the marine industry.^4,13 Apart from the acid hydrolysis, a combination of alkali and enzymatic digestion and extraction of proteins has demonstrated good protein solubility – here the pH is varied in order to solubilize the different types of proteins that are present in the biomass.¹⁵ Likewise alkaline hydrolysis has its drawbacks as it can reduce the nutritional value or properties of the biomass by causing total or partial destruction of the amino acids arginine, cysteine, lysine, threonine and tyrosine. In addition, formation of undesired toxic substances such as lysinoalanine, ornithinoalanine, lanthionine and β-amino-alanine can also be observed.^11,16–18 Due to these effects alkaline hydrolysis is less common in food and feed applications. In addition to acidic and alkali extraction of peptides from marine sources, techniques like ultrasound, microwaves, enzymes, supercritical fluid and pressurized liquid are techniques that are more novel and can be used together with traditional techniques to increase yield.^19,20

17.2.2 Enzymatic Hydrolysis of Marine Biomass

An alternative to chemical hydrolysis is enzymatic hydrolysis employing either autolytic and/or exogenous enzymes to disrupt tissues. In an enzyme-based hydrolysis process, enzymes cleave the peptide bond between two amino acids. The process is illustrated in Figure 17.1, where biomass that is raw, frozen or processed is exposed to one or more enzymes at predetermined times, temperatures and pH conditions. The goal of the enzymatic hydrolysis is to break down and enable separation of the components in the biomass. The enzymes can break down proteinaceous, lipid, carbohydrate or other components in the biomass. In this chapter the preparation and commercialization of peptides from marine biomass is the main focus and, thus, the enzymes generating peptides from proteins will be given our attention.

Figure 17.1 Schematic illustration of the process of biomass hydrolysis. The biomass is allowed to react with enzymes under predefined conditions. Upon reaching a defined end-point, the hydrolysis process is terminated through heating and thus inactivating the enzymes. The three phases are then separated and further treated and analyzed.

This kinds of enzymes can be grouped according to which part in the protein the bond is cleaved, e.g. on the C- or N-terminus or on the middle of the amino acids chain. The enzymes are also grouped according to which amino acids of the peptide bonds are cleaved. Some enzymes have a very narrow specificity, while others have a wider range of bonds. The rate of hydrolysis will depend on the biomass, as well as on the specificity of the enzyme, the enzyme and substrate concentration, temperature, pH and the duration of the reaction.²¹ The breakdown of the proteinaceous biomass can be measured using different parameters. The degree of hydrolysis (%DH) indicates to what extent the protein chain has been cleaved to shorter proteins, peptides and amino acids (see below).

Digestive endogenous enzymes are important in fish sauce production, where they hydrolyze proteins to peptide and free amino acids.⁸ However, as previously mentioned, marine biomass can include a large variety of species, and the composition of the heterogeneous raw material and the amount of endogenous enzyme can vary depending on among other things location, season and nutritional status. The raw material can also contain large amounts of enzyme inhibitors.^16,17 This variation makes predicting the outcome of a purely autolytic process (no enzymes added) challenging. Adding exogenous enzymes can provide control of the hydrolysis process to ensure certain physical-chemical, functional and or sensory properties (i.e. a tailor-made product). A tailor-made product can be achieved using predefined conditions and hydrolysis times, as well as optimized temperatures such that the degradation is well controlled and excessive degradation of the product, which can be experienced using chemical hydrolysis, can be avoided.^16,17

The three main fractions from a marine hydrolysis process (proteins, lipids and a mineral-rich sediment) form the basis for potential products. While products from the lipid fraction are already on the market, both within food and feed, the industry is struggling to take advantage of the tailor-making possibilities of the protein fraction to make products for the human consumption market. A vast amount of scientific work has shown the possibilities of using proteases to hydrolyze by-products from various sources into higher-value products directed towards many markets.^22,23 Among them, bioactive peptides for pharmaceuticals and/or nutraceuticals are the most lucrative. An extensive amount of work has been put into this particular field,^24,25 but only a few products have reached the market. Shrimp by-products have been commercialized in a peptide-based nutraceutical (see Section 17.8.1).^26,27 The use of hydrolysates in food and beverages, e.g. in sports drinks or in protein supplements for the elderly, are other examples of markets with high potential.^28,29 When the hydrolysates are employed as food ingredients, they need to meet requirements for a multitude of properties such as sensory attributes, digestibility, bioavailability and physical-chemical properties. The products also have to be produced with the same specifications from day-to-day regardless of the significant and inherent variation in the raw material at hand. These requirements result in a significant challenge, as there can be substantial amounts of uncontrollable variation in quality due to differences in composition (e.g. protein structure and amino acid composition due to variations in nutrient intake in the fish), oxidation state of oil and protein components, activity of spoilage enzymes, and microorganisms present in the material.

Another important consideration when employing enzymatic hydrolysis is that the enzymes require certain environmental conditions to have optimal activity. Temperature and pH values are two of the most important factors to consider when applying enzymes to a raw material. It is also important to use food grade enzymes if the products are intended for human use. The choice of which enzymes to use in a production process will be determined by a combination of efficacy, specificity and economics.¹⁷

In addition to the choice of enzyme, it is also important to regulate the termination of the hydrolysis at a given %DH in order to achieve desired functional properties like solubility, water-holding capacity, emulsifying and foaming-properties and sensory properties.³⁰ The degree of hydrolysis is an important measure of how the process of hydrolysis is progressing. There are several methods used to determine the degree of hydrolysis, the most common ones employ trinitrobenzenesulfonic acid (TNBS) or o-phthaldialdehyde (OPA), measure the trichloroacetic acid soluble nitrogen (SN-TCA), or perform titration with formol. All of these methods allow the determination of the amount of amine generated from the hydrolysis – and thereby the amount of peptide bonds cleaved – by colorimetric association or by precipitation. In addition, the pH-stat method, which consists of measuring the amount of protons released in the solution, serves to determine the degree of hydrolysis indirectly.^31,32 All these methods are difficult to perform on-line or at industrial real time, or can be difficult to reproduce, as they can give different results.^31,32 Therefore, research is being performed in this field to establish better and more industrially applicable methods for determining the degree of hydrolysis. The methods, their limitations and further research is described in more detail in Section 17.5.

Several different techniques including heat, pH alteration and high pressure can be employed to inactivate enzymes and terminate the hydrolysis process.^17,33 Heat is commonly used in large scale processing plants as it both inactivates and pasteurizes in a known fashion such that the product produced is safe for ingestion. After hydrolysis, ultrafiltration can be utilized to further separate the hydrolysates based on molecular size to obtain desirable peptides.¹⁷ In addition to high value products, other applications for protein hydrolysates like plant nutrients, fertilizers and animal feeds might be more feasible¹⁷ and economically viable.

17.3 Enzymes Used for Bioconversion

Enzymes are complex proteins synthesized by living cells and function as biological catalysts employed in a range of life processes including signaling, turnover and metabolic functions.⁷ Enzymes are substrate specific and it is therefore very important to choose enzymes based on the composition of the raw material as well as the desired outcome.

Enzymes are generally classified into six major classes according to the nature of the chemical reaction they catalyze, namely oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.^34–38 Most enzymes used by the food industry are hydrolases, including carbohydrases (glycosidases), proteases and lipases (esterase) that hydrolyze carbohydrates, proteins and lipids, respectively.^7,17 Hydrolysis of proteins involves cleavage of the peptide bonds resulting in breakdown of proteins to peptides and free amino acids (FAA). Protein hydrolysates therefore usually consist of a mix of peptides of different sizes as well as FAA. Both the size and the composition of the peptide chains determine the nutritional and functional characteristics of hydrolyzed proteins.²¹

Proteases can be divided into exopeptidases and endopeptidases based on their catalytic action. Exopeptidases cleave proteins and peptides stepwise from the N- or C- terminals,^17,35 while endopeptidases act on the polypeptide chain at specific peptide bonds.^17,38 A combination of endopeptidases and exopeptidases is sometimes used to achieve a higher degree of hydrolysis.¹⁷

Some plants and animals can be sources for enzymes, but in most cases enzymes are produced using microorganisms like fungi, bacteria and yeast.^39,40 Production of enzymes from microorganisms is easier to control, cheaper and more efficient than the production from animals and plants.³⁶ Microorganisms can be genetically engineered to produce large amounts of enzymes in a relatively short production time in inexpensive media. The culture medium must contain sufficient nutrients, and the environment needs to have suitable pH, temperature and amount of dissolved oxygen to facilitate growth.^37,39 Enzymes can be produced by continuous processes; however, batch production is more common. Often, liquid fermentation conditions are preferred, though solid substrates have been historically important, and are in some cases still employed.^8,39 Downstream processing, i.e. isolation and purification of the enzymes, is a key-step in the production. A typical process includes separation from solids, concentration to reduce the volume and purification to eliminate contaminants.³⁹ A wide range of techniques can be used to purify the enzymes, including crystallization, ultrafiltration and chromatographic methods.³⁷ The chosen method will depend on the intended use of the enzyme.³⁹

In recent years, the enzyme industry has grown and a large number of enzymes are now commercially available. According to the enzyme guide, there were more than 269 developers, producers and sellers of enzymes around the world in 2015.⁴¹ The applications are broad ranged including not only food and feed but also paper, brewing, winemaking, diagnostics, detergents, baking and many more.⁴¹

17.5 Functional Properties and Bioactivities of Hydrolyzed Marine Biomass

Detailed characterization of protein hydrolysates in terms of their chemical composition, sensory attributes, functional properties and bioactivities is an essential part of product development. A challenge when developing hydrolysis-based products from biomass is the complexity of the biomass, such that there is a multitude of different components in the mixture that affects the hydrolysis reaction. In addition, when using fisheries by-products, the composition of this heterogeneous biomass can vary from day to day, adding a further complexity to the production. This issue is addressed below. Much work has been done on demonstrating the functional properties and bioactivity of peptides derived from marine biomass.^{6,8,15,24,48,49} As mentioned in the classification of marine biomass, the functional screening will depend on the type of application. Most of the marine biomass that is classified as suitable for human consumption will go through the screening for bioactivity, in some cases category 3 or even category 2 biomass will be tested for bioactivity in those instances where a bioactivity can significantly enhance the biomass value. One example is animal feed where a bioactivity can significantly improve desired traits of the animal.⁸ In most cases, peptides will be recovered as a mix of peptides in a hydrolysate with a peptide size distribution. The size of the peptides will depend on the degree of hydrolysis of the proteins – the size will vary, but generally an enzymatic digestion that has been allowed to proceed for a longer time will contain smaller peptides.

A challenge of marine proteins is that they often can have an undesirable taste. The taste of the final product will affect its commercial value. Bitter taste is often associated with hydrophobic amino acids that are released, as well as other small molecular components and residual fats. De-bittering techniques have been developed, including solvent extraction, the use of activated carbon on matrices, chromatography techniques, addition of de-bittering components, or hydrolysis with exopeptidases. The research performed indicates that restricting the enzymatic digestion of the biomass to yield larger peptides will reduce undesired taste. However, this can again affect the bioavailability of the peptides.^21,28,29,50 Many of the de-bittering techniques are not easily implemented in large scale production; however, hydrolysate de-bittering with exopeptidases has shown promising results and is amenable to an industrial scale hydrolysis process.^11,20 The physicochemical properties such as water binding, emulsifying and foaming abilities are also important in order to obtain valuable products from the marine hydrolysates,^8,17 and are part of the deciding factors when evaluating the commercial value of the hydrolysate.

The bioactivity will of course affect the commercial potential of any given product. To determine bioactivity in the hydrolysate, screening is performed upon fractionation of the hydrolysate followed by in vitro bioassays to determine effects. Marine hydrolysates have been demonstrated to have several effects in humans (i.e. effects as functional foods and in the pharmaceutical area), including antioxidant and anticoagulant effects, and cardioprotective, antimicrobial, anti-diabetic and appetite suppressive effects, as well as improving nutritional status in people with nutritional needs. Antitumor, antiviral and neuroprotective effects have also been demonstrated. Upon initial screening, the hydrolysate is further fractionated and bioactivity effects can be further studied in several in vitro and in vivo assays, which have been extensively reviewed elsewhere.^{19,20,27,51,52} These processes are laborious and can usually only be applied in the developmental phase. A different and interesting approach is the use of multivariate analysis to relate the chemical fingerprint of a given hydrolysate with the corresponding sensory, functional and pharmacological properties. For example, van der Ven et al. developed Fourier-transform infrared spectroscopy (FTIR)-based multivariate regression models for the prediction of functional parameters (i.e. solubility, emulsification and foaming) and sensory properties (i.e. bitterness) of whey and casein hydrolysates.⁵³ Such an analytical and statistical approach is a beneficial alternative to measuring hydrolysate properties using the traditional biochemical assays especially in the scale up phase of the process. Overall, both the complexity of food processing by-products and a multistage action of catalytic enzymes poses significant challenges in creating the relevant biochemical understanding prior to product (i.e. protein hydrolysate) development. Knowledge of such biochemical parameters is mandatory, particularly in product development for higher-paying markets (i.e. for human consumption). In the processes described here, the biomass is utilized in the process such that protein, minerals and fats are not wasted. In other pharmaceutical processes, a bioactive peptide is identified from marine biomass; however, the compound is then synthesized chemically and used in drugs. In these pharmaceutical processes, the biomass is not utilized in the same way, such that proteins, minerals and fats in by-products are still wasted. Scale-up of laboratory developed processes and recent advances in the bioprocessing of marine biomass has allowed for more products that consume or use up the biomass to be developed; however, there are still challenges faced in the commercialization phase, and so the issue of scale up will be addressed next.

A lot of research is being carried out on the exploitation and valorization of low value marine biomass, especially remaining raw materials. Many of the high value products have been produced at lab scale, both within pharma, food and feed, but the number of project making the translation from lab scale to market is not very high. A few products have reached the market, but there is a need for research enabling the conversion of low value biomass into commercial products and at the same time enabling scale-up, marketing and consumer preferences. The demonstration phase is a critical phase of the development of sustainable products from under-utilized biomass. As mentioned above, by-products are complex and heterogeneous biomasses with a substantial, uncontrollable variation in quality due to differences in composition (e.g. protein structure and amino acid composition), oxidation state of oil and protein components, the amounts and activity of spoilage enzymes, and the microorganisms present in the material. It is therefore crucial to develop processing methods that can handle these variations, in order to obtain a stable end-product quality. The scale-up of a laboratory developed process can be challenging. Many factors can hamper the scale up. One challenge can be that the hydrolysis process on a large scale (2000 L plus) does not proceed as it does at laboratory scale. Another challenge can be that infrastructure that is readily available at laboratory scale can be either hard to access or not applicable to larger scales, or alternatively be very costly in larger scale. The demonstration phase is often challenging in terms of investments as the access to capital in this phase can be scare while the costs of scale-up of innovation and trial in the market are typically high.^54,55 Let us first focus on the scale-up of the process itself. Due to differences in heating, mixing, sheer flow and the formation of inhibitory products that were not significant at smaller scale, the speed and the result of the hydrolysis can differ from laboratory to larger scale. By scaling the process in a step-wise manner, going from smaller scale and increasing the volume 10× for each step, the issues can be more easily resolved, as the cause of the changes can be more easily identified. Another way of improving the scale-up process is to ensure proper monitoring of the process. Robust process design was introduced by the Japanese quality guru Taguchi around 1950, and has found use in biotechnology.⁵⁶ The method aims at finding process settings that minimize the effect of the so-called nuisance variables (e.g. raw material properties), and thereby obtaining a consistent product regardless of the raw material variation. Tailor-made hydrolysates do not only need to have the desired target functionality, but also satisfy a multitude of other requirements such as sensory and physiochemical properties. At the same time, it is important to maximize yield and maintain good quality of the oil and mineral fractions. The so-called desirability functions is a well-established method for multi-response optimization,⁵⁷ making it possible to set different weights on the responses.

Hydrolysis of biomass is commonly performed in large batch-based reactor tanks; however, an industrial improvement on production of peptide-based products from marine biomass is the introduction of continuous hydrolysis in large-scale production. In this setup, the hydrolysis is performed continually through smaller pipe-like constructions while being pushed from one end, the starting point, to the end-point of the hydrolysis. The process is monitored by recording viscosity in the biomass hydrolysate throughout the process, and the addition of monitoring-techniques as described below can improve the process additionally by slowing real-time adjustments of the processing settings and thus better targeting the desired end product quality. Parameters like temperature and pH can be modulated along the process, and the addition of different enzymes to sequentially break down components in the biomass can also lead to an improved product.

Earlier and on-going studies at Nofima show that characterization of both the hydrolysis process and the resulting hydrolysates using the classical wet chemistry approaches is a challenging task with a considerable risk of inaccurate or otherwise misleading results. Recent developments in analytical technologies open up several new possibilities in process monitoring as well as product and raw material characterization. In process monitoring, the degree of hydrolysis is an important parameter that can be measured using a variety of different methods including TNBS and OPA,^31,32 with the alternative approach based on titration of released protons (pH Stat) in the course of the hydrolysis.^32,58 Another valuable parameter used for characterization of protein hydrolysates is the molecular weight distributions (MWD).⁵⁹ Unlike DH%, MWD is a direct measure of the composition of the hydrolysate and relates to important quality parameters such as intestinal absorption, viscosity and bitterness. A major limitation of both MWD and DH% measurements is the lengthy analysis time, thus restricting their use as on-line monitoring tools in industrial setups. In this regard, the fast and non-invasive spectroscopic techniques are interesting process monitoring tools with a promising potential for industrial applications.⁶⁰ FTIR is also one of the techniques that potentially can be used to monitor the breakdown of proteins in hydrolysis reactions.^61,62 Thus, by using on-line or near real-time monitoring techniques one can significantly improve the scale-up of the hydrolysis process. As the biomass that is being processed is complex and can vary, the optimal parameters and the process can vary for each production. By allowing for continuous monitoring, the product produced can be more stable as it allows for small adjustments in process parameters in the large-scale production.

In addition to the process challenges and recent development in large scale production of peptide-based products from marine biomass, access to both infrastructure and capital can be a challenge. The demonstration phase has been described as the valley of death in product commercialization.⁵⁵ The access to demonstration plants can help alleviate the hurdle of the demonstration phase. Demonstration plants are flexible small scale production plants where companies or academic entities can test a process that has been developed at laboratory scale. A demonstration plant allows the company or academia to test out a process without having to make large investments in costly scale-up and production infrastructure. A product prototype can be made in this process and this product prototype can be tested in the market. The prototype assessment of the product in the market is an important part of product development as it will help evaluate whether the product can be sold in one or several markets as predicted in initial analyses, and that there is a customer segment willing to pay for the product. In addition to allowing for testing in the market, performing a prototype production will provide estimates of production costs and capital expenditures that will be associated with the product. As the demonstration plant will be built to be flexible and contain infrastructure that can be adapted to a number of processes, the cost figures will only be estimates. If the product is to be produced in a production plant designed specifically for the developed process, the production costs will likely be lower, and the yield will be higher. Figure 17.2 is a schematic illustration of the infrastructure available at Biotep, a demonstration plant that is owned and operated by Nofima. The different infrastructure is connected by pipes that can direct the biomass to the desired infrastructure. Different infrastructure is noted on the figure, and can also be viewed on the full 3D movie of the plant that is available (https://vimeo.com/174329346) for further explanation. Located outside of the area shown in Figure 17.2 are cooling and storage facilities for the different products. The plant is approved for food grade production, and according to EU regulations⁴⁴ all food and feed grade products have to be separated. The receiving room and the room with the reactor tanks (top left, Figure 17.2) are in what is called the unclean zone. All biomass is inactivated and pasteurized before being moved to the clean zone, which starts in the top-right room with the holding tanks and the filtration systems and continues further to the drying room and the storage and exit for the products.

Figure 17.2 Illustration of Biotep demonstration scale up plant. The different parts of the infrastructure are marked on the picture and an explanation is available on-line (https://vimeo.com/174329346) and at Biotep.no.

17.8 Case Examples

To better illustrate how marine biomass can be processed into commercial products, two examples are included here. These are from companies that are producing commercial products based on hydrolysis of marine by-products. The companies target very different market segments and are examples of the diverse possibilities obtainable by hydrolysis of marine by-products.

17.8.1 Marealis – Producing a Nutraceutical from Shrimp Peels

Marealis AS is a Norwegian marine biotechnology company focusing on the development of novel bioactive peptides from shrimp by-products. The Norwegian shrimp (Pandalus borealis) fisheries yields roughly 40% by-products, mainly the head and shells. Annually this amounts to 8000–10 000 tons of by-products containing approximately 8% protein. By applying enzymatic treatment, 70% of proteins can be recovered as a water soluble protein hydrolysate, while the solid fraction is suitable for production of chitin or chitosan.²⁷ The peptide fraction obtained from shrimp by-products hydrolysis has been shown to contain angiotensin I-converting enzyme (ACE) inhibitory peptides.^27,71 ACE indirectly increases blood pressure by causing blood vessels to constrict, and ACE inhibitors can therefore be useful agents to lower or prevent elevation of blood pressure.^27,72 Since 2008, Marealis has developed a patented process employing controlled enzymatic hydrolysis of shrimp by-products. The hydrolysate is refined to a concentrate, filtrated to isolate specific molecular weight compounds, dried and tableted. The product, Procardix®, is aimed at the nutraceutical market and production is performed at Biotep, which is described above. The product has undergone acute as well as long-term toxicological and efficacy studies in animal models, followed by a clinical pilot study, completed in 2012,⁷³ and a full scale clinical study conducted in Canada, Germany and the Czech Republic in 2014–2015.⁷⁴ According to Marealis AS, Procardix® will be ready for the market in 2017 and target adults with prehypertension (high normal blood pressure: 120–139/80–89 mm Hg).²⁶

17.8.2 Polybait AS – Producing Fishing Bait from Fisheries By-products

Polybait AS is a Norwegian company producing Kvalvik bait, a range of sustainable baits for anglers and commercial fisheries based on attractants released from hydrolyzed marine by-products. The increasing focus on improved utilization of marine resources and awareness concerning the use of fish for food, and not for feed or bait,⁷⁵ has prompted research on sustainable bait alternatives. In the North Atlantic fisheries, Atlantic cod (Gadus morhua L.) is one of the most important species, and longlining is a common fishing method. Longlining depends on bait to attract cod, and species like mackerel, herring and squid are often used as bait. It has been documented that hydrolysate made from shrimp and blue mussel have potential to replace natural bait for cod.⁷⁶

The bait is produced using hydrolyzed marine by-products combined with a specially designed swellable polymer to slowly release the attractants and specifically attract cod, halibut or salmon and trout depending on the by-products used in the production and the combination of hydrolysate and polymer. Artificial bait based on hydrolyzed marine by-products intended for commercial fisheries is currently in the final stage of testing and can prove very valuable for the industry. It is highly beneficial that these products can utilize feed-grade category 3 by-products that are typically used for lower value products or even discarded. In addition, artificial baits can decrease the weight of commercial fishing boats, which in turn reduces the cost of transport and CO₂ emissions.⁷⁶

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