Gelatin is an important hydrocolloid in food and biotechnological applications. There are two basic sources of gelatins; mammalian and fish-derived gelatins, but fish gelatins are less appreciated due to their weak gel properties. Hence, to reinforce the gel properties of fish gelatin, mixtures of gelatins are used. In this study, the mixtures of cold-water ocean fish scale (OFS) and pork skin (PS) gelatin were studied via rheological measurements, micro differential scanning calorimetry (DSC), and particle tracking at different cooling conditions and incubation times to understand the gelation mechanism and the network structure of the gels. The rheological and micro-DSC measurements provide a detailed understanding of the gelation mechanism of gelatin gels under different cooling conditions while particle tracking revealed the local physical properties of the gels. A single or two-step melting was demonstrated in the mixed gelatin gels depending on the cooling conditions suggesting that the aggregation of PS in the mixtures influences the physical and thermal properties of the gels. Under rapid cooling, co-aggregation of OFS and PS chains is considered to cause a single-step melting of the mixed gels. Under gradual cooling, conversely, the PS may form aggregates independently before the OFS. This is evidenced by a two-step decrease in moduli and two clear endothermic peaks in the micro-DSC measurement. Particle tracking further reveals information on the local physical properties of pure and mixed gelatin gels on reheating, indicating that the local structures of mixed gels resemble those of pure PS gels. This suggests that mixed OFS and PS gels may form a cooperative aggregation, thereby improving the physical and thermal properties of the mixed gelatin gels.

Alginates consist of distinct blocks with different physical properties. This short review focuses on research carried out in Trondheim related to the early discovery of the block structure, their isolation, their different chemical and physical properties, and how they recently are utilized in diblock polysaccharides to obtain new nanostructuring properties.

Fish gelatin is becoming a popular alternative to mammalian gelatins due to religious restrictions, cultural preferences, ecological, and ethical concerns. Warm water fish gelatins (WWFG), as opposed to gelatins from cold water fish species, have more similar physical properties, and hence represent an alternative, to mammalian gelatins albeit a lower sol/gel transition temperature and gel strength. In warm climates, WWFG gels may therefore exhibit reduced storage stability at temperatures above this transition temperature because of e.g., a more pronounced acid hydrolysis of the sol fraction. To improve the long-term storage stability of WWFG, gels were prepared with two different sugar alcohols, sorbitol and xylitol, and a non-reducing sugar, sucrose. The change in the sol/gel transition temperature, gelling and melting kinetics, and gel strength of the gels were analyzed using small amplitude oscillatory shear measurements. Short-term and long-term storage stability tests at ambient temperature, 30 °C and 40 °C indicated improved stability of gels with co-solutes without significant differences between the type of sugar alcohol or sucrose. The stability of the gels increased with increasing concentrations of sugar alcohols. The degree of hydrolysis of the gelatin in the gels were investigated using SEC-MALS analyses which supported the bulk rheology stability results. Using sucrose led to browning and high viscosity, which may pose challenges regarding the processability and industrial applications.

For gelatin-based confectionery, nutraceutical, and pharmaceutical products, storage stability is important for maintaining textural properties during transportation and throughout the shelf life of the final product. Exposure to elevated temperatures causes degradation of the gelatin molecule, which results in a reduction in the average molecular weight, further impacting its mechanical properties. In this study, the long-term stability of gelatin systems prepared with different gelatins (type B from bovine, type A from pig skin, or type A from fish skin) stored below and above the sol-gel transition temperature was compared. The effect of storage temperature, Bloom strength, and the inclusion of co-solutes (sugar alcohols or gum arabic) on storage stability was evaluated. The long-term stability was investigated using small amplitude oscillatory shear measurements and molecular weight analyses via size-exclusion chromatography coupled with online multiangle laser light scattering. The storage modulus of the gels and the average molecular weight of the gelatin indicated that incubation above or close to the equilibrium sol-gel transition temperature resulted in an increased degradation rate of gelatin. Type A gelatin gels exhibited better storage stability than type B gelatin gels. In addition, gels prepared with high Bloom strength gelatins exhibited improved storage stability compared to low Bloom strength gelatins. The addition of sugar alcohols increased stability, whereas gum arabic did not have a large impact on the long-term stability of the type B gelatin gel.

Gelatin-based pre-emulsified chewable gels have presented advantages over traditional tablets, bulk oils, hard and soft capsules for oral delivery. Ethical, ecological, and religious considerations have increased the demand for plant-based gelling agents which can be formulated into chewable emulsion gels. Plant-based polysaccharide emulsion gels prepared with agar and pectin were compared to gelatin emulsion gels regarding rheological, textural, and functional properties. The agar emulsion gel had higher gelling/melting temperatures (Tg: 40 °C, Tm: 90 °C) than the gelatin emulsion gel (Tg: 37 °C, Tm: 45 °C), whereas pectin emulsion gel had a more similar gelling/melting profile to the gelatin formulation (Tg: 38 °C, Tm: 54 °C). Texture analyses revealed that the agar emulsion gel had a harder and more brittle texture, whereas pectin emulsion gel had a softer texture than the gelatin emulsion gel. Pectin emulsion gels had the largest average droplet size (32 μm), followed by agar (13 μm) and gelatin emulsion gels (1 μm). The in vitro lipolysis experiments indicated that the polysaccharide emulsion gels were lipolyzed to a lower extent and had a slower initial lipolysis rate (agar: 2.8 μmol FFA/sec, pectin: 4.3 μmol FFA/sec), compared to the gelatin emulsion gel (24.9 μmol FFA/sec). The industrial potential and challenges of the polysaccharide emulsion gels were evaluated, and the results show that plant based pre-emulsified chewable gels can be manufactured for the oral delivery of nutraceuticals.

In this study, gels were successfully prepared at room temperature from mixtures of dry fractionated faba bean protein concentrate (FPC) and λ-carrageenan (λ-CGN), through acidification with glucono-δ-lactone (GDL). At neutral pH, the mixtures were shear thinning liquids, although the shear viscosity increased dramatically with λ-CGN addition. After adding GDL, the gelling kinetics were followed through small amplitude oscillatory rheology for 19 hours, at which point all gels had reached a gel modulus plateau. Elastic moduli for the prepared gels were in the range of 1500 – 4500 Pa, dependent on FPC:λ-CGN ratio and concentrations, and final pH (3.5 – 4). Rheological data further indicated the gels had properties typical of aggregated particle gels, e.g., low yield strains (∼1%). All gels showed some syneresis upon centrifugation (2000g), with the least amount of syneresis (15 – 20%) at the highest λ-CGN concentrations (1.5 – 2%). FPC is a good emulsifier, and gelled emulsions were successfully prepared. Inclusion of emulsion droplets had significant impact on the gel network, with ∼40% and ∼60% increased gel storage modulus at 20% and 30% oil, respectively. Preparing similar formulations using a more extensively processed commercial faba bean protein isolate was also attempted, but this resulted in poor gels with very high syneresis. This indicates that dry fractionation methods may be beneficial to preserve native protein functionality.

Collagen is the most abundant protein in mammals as it constitutes the main fraction of structural proteins in connective tissue. Gelatin, obtained by partial hydrolysis of collagen, is one of the most versatile biopolymers and has numerous applications in food, confectionery, pharmaceutical/medical, cosmetic, and technical products. This is also reflected by the approximately 450,000 metric tonnes of gelatin produced annually worldwide. Gelatin has been investigated and studied by scientists at least since the early 20th century but has been applied in foods even before this. The properties of the manufactured gelatins depend on the origin of the parent collagen and the severity of the extraction procedures. Today, gelatins are mainly produced from bovine and porcine sources, but gelatin may also be extracted from fish and poultry. This chapter focuses mainly on the manufacturing of mammalian gelatin, and the connection between the chemical compositions and the structure-function relationship of gelatins from mammalian sources as well as from cold and warmwater fish species.

Emulsions play an important role in the process of triglyceride (TG) digestion (lipolysis). Through emulsification, the oil-water interface is increased by orders of magnitude. This often leads to faster and more efficient lipolysis, which is potentially beneficial for the intestinal uptake of oils and lipophilic compounds. In this paper, we first examined the effect of emulsion droplet size on the in vitro lipolysis rate. Then an in vivo experiment was performed, to examine the plasma uptake kinetics of TGs and vitamin D3 (vitD3) over a 24 hours period after oral administration of the emulsions in rats. Basic corn oil emulsions loaded with vitD3 were prepared using polysorbate 80 as the emulsifier, with three different droplet sizes (D[3,2]): ∼3 μm (large), ∼1 μm (medium) and ∼0.3 μm (small). In vitro lipolysis experiments showed, as expected, that smaller droplets were lipolyzed more rapidly. However, the medium emulsion had by far the highest rate of lipolysis per surface area. This was attributed to bile salt limitation, polysorbate 80 lipolysis inhibition and TG digestion product accumulation. In vivo, the two smallest emulsions showed the highest uptake (Cmax and AUC) of vitD3 and TG, while the largest emulsion and bulk oil control showed lower values. However, only the (incremental) TG plasma values and kinetics displayed some statistically significant differences. These findings may have relevance for the formulation of functional foods/beverages or delivery units containing oils or lipophilic bioactives.

Gelatin-based chewable emulsions can be a convenient vehicle for oral delivery of oils or lipid soluble bioactive components. Gelatin-stabilized emulsions do, however, rapidly flocculate and gradually coalesce in gastric fluids. This destabilization is caused by the combined action of pepsin and mucin and is most significant at pH 3, followed by pH 2, then pH 4. Through in vitro lipolysis experiments it is shown that this destabilization leads to a decrease in emulsion lipolysis rate after incubation in simulated gastric fluids (SGFs). In this paper a potential solution to this gastric instability is suggested: inclusion of 1 wt% sodium-kappa-carrageenan (kappa-CGN) of intermediate M-w into the gelatin emulsions. The kappa-CGN used has minimal impact on gelatin gelled emulsion properties, preserving the soft elastic gelatin texture. When these gelled emulsions disintegrate in gastric fluids, electrostatic interactions between the gelatin and kappa-CGN occur. While these interactions lead to heavy flocculation, they also protect the gelatin from pepsin action, providing full stability against emulsion coalescence during at least 2 h in SGF at pH 2-4. When the pH is neutralized upon mixing with intestinal fluids, the emulsion fully deflocculates and the rate of in vitro lipolysis is not affected by gastric residence time. Practical applications: Avoiding gastric coalescence in gelatin emulsions may lead to more reliable oral delivery of lipids or lipophilic components in gelatin-based chewable supplements or functional foods. Keeping the emulsion droplet size stable and small until reaching the intestine may lead to more rapid and efficient intestinal lipolysis, potentially advantageous in regards to bioavailability of slowly digested oils (e.g., omega-3 concentrate) or for people suffering from impaired lipid digestion. These findings may also be applicable to emulsion systems stabilized by other proteins.

Three alginates with fundamentally different block structures, poly-M, poly-G, and poly-MG, have been investigated upon ionic crosslinking with chitosan oligosaccharides (CHOS), using circular dichroism (CD), rheology, and computer simulations, supporting the previously proposed gelling principle of poly-M forming zipper-like junction zones with chitosan (match in charge distance along the two polyelectrolytes) and revealing a unique high gel strength poly-MG chitosan gelling system. CD spectroscopy revealed an increased chiroptical activity exclusively for the poly-M chitosan gelling system, indicative of induced conformational changes and higher ordered structures. Rheological measurement revealed gel strengths (G ' < 900 Pa) for poly-MG (1%) CHOS (0.3%) hydrogels, magnitudes of order greater than displayed by its poly-M analogue. Furthermore, the ionically crosslinked poly-MG chitosan hydrogel increased in gel strength upon the addition of salt (G ' < 1600 at 50 mM NaCl), suggesting a stabilization of the junction zones through hydrophobic interactions and/or a phase separation. Molecular dynamics simulations have been used to further investigate these findings, comparing interaction energies, charge distances and chain alignments. These alginates are displaying high gel strengths, are known to be fully biocompatible and have revealed a broad range of tolerance to salt concentrations present in biological systems, proving high relevance for biomedical applications.

Dehulling of peas and faba beans prior to milling and air classification was evaluated, namely its impact on physical properties, chemical composition and techno-functional properties of the fractions. Dry fractionation protocols for protein enrichment from whole and dehulled peas and faba beans were optimized and large-scale batches were produced. Fine fractions with protein contents of 44.0 and 46.2% dm from whole and dehulled peas and between 60.0 and 60.9% dm from whole and dehulled faba beans were obtained, respectively. A maximum protein recovery of 71.3% and 49.2% was obtained for peas and faba beans, respectively. Dehulling enabled a lighter colour of faba bean fractions and improved the starch enrichment in the coarse fractions from peas and faba beans. The total non-starch polysaccharides were significantly reduced in the coarse fractions when dehulling was conducted. Dehulling did not significantly improve the techno-functional properties of fine and coarse fractions.

Complex polysaccharides are widespread within the animal kingdom. At the molecular level, however, the importance of such components have largely been considered inferior compared to nucleic acids and proteins. This in spite of the fact that carbohydrates are well known to govern important biological processes like cell-cell and cell-ECM interactions. There are probably several reasons for this, out of which the huge challenges of characterizing and synthesizing complex carbohydrates play an important role. Secondly, the fact that complex carbohydrates are not primary gene products but rather the result of post polymerization processes like e.g. glycosylation of proteins adds to the intricacy to the science of Glycobiology. As of today, heparin and heparin analogues for the treatment of DVT represent some of the very few examples of carbohydrate, or carbohydrate-inspired pharmaceutical products on the market. This review will address the complexity of carbohydrates, but also that due to this complexity carbohydrates exhibit a huge potential as carriers of information. Finally, some examples of pharmaceutically active oligosaccharides of marine origin will be presented.

Raw materials for the production of gelatin may either be pre-treated with acid or alkali to hydrolyze interand intramolecular bonds between and within the collagen mother molecules. The resulting gelatins are denominated type A or type B, respectively, and the main difference being a conversion of the acid amides (Asn and Gln) to their acid analogues (Asp and Glu) after alkaline pretreatment leading to a lowering of the pI. Gelatins also possess amphiphilic properties and will therefore be able to stabilize O/W emulsions. In this study, gelled O/W emulsions with varying oil contents have been prepared with the two different types of gelatins. Small strain oscillatory measurements combined with temperature sweeps were used for the determination of the gelling kinetics. The acid pretreated gelatins exhibited a steeper increase in modulus as function of increased oil contents as well as increased setting and melting temperatures. This behavior is suggested to stem from a hydrogen bond mediated flocculation of the oil droplets. Additionally, gelled emulsions were made applying a cold-water fish gelatin sample, which typically show inferior physical properties compared to their mammalian counterparts. At high contents of oil inclusion, these suboptimal properties were improved. Within certain limitations, these results suggest that the properties of gelatinbased solid emulsions can be manufactured to meet specific demands. INTRODUCTION Chewable oral formulation is gathering an increased popularity; particularly in the neutraceutical segment but also as pharmaceutical formulations1. The main reason for this is linked to different dysphagia challenges, especially amongst children and the elderly, which makes it challenging to swallow traditional oral formulations such as tablets and capsules. Gelatins are especially preferred for such chewable formulations because of their long texture and melt-in-mouth properties. These properties are, however, exclusive to gelatins from mammalian sources as e.g. gelatin extracted from cold-water fish species has considerable lower setting and melting temperatures2. Another beneficial feature of applying proteins such as gelatins is that they are in general considerably more surface active compared to e.g. polysaccharides. In turn, this means that they can be exploited to stabilise emulsions without the need for additional emulsifiers. One example of this is that gelatin based chewable formulations are now made with a very high payload of long chain omega-3 fatty acids (DHA/EPA)3. Gelatins are categorized according to their gelling potential (“Bloom strength”) as well as to the pre-treatment of the raw Alkaline or Acidic Pretreated Gelatins; Effect on Rheological Properties of Gelatin-based Chewable Solid Emulsions K.I. Draget1 and M.N. Hattrem2 1Dept. of Biotechnology and Food Science, NTNU, Trondheim, Norway 2Vitux AS, Oslo, Norway ANNUAL TRANSACTIONS OF THE NORDIC RHEOLOGY SOCIETY, VOL. 26, 2018

Previous studies have demonstrated that oligoguluronate (guluronate block extracted from alginate, GB) was an efficient modulator of the gelation and gelling properties of macromolecular alginate in the presence of calcium. Here we report totally different modulatory effects of the oligomer when used to modify the gelation of low methoxyl pectin (LMP). GB was found to promote the gelation of LMP in the range of R ([Ca]/[guluronate + galacturonate]) < 0.25 and could make non-gelling systems gellable. This is significantly different from the case of alginate where no gelation could be induced at all. In the range of 0.25 < R < 0.60, the addition of GB was found to inhibit the gelation of LMP, whereas it had a negligible effect on the gelation of alginate as long as a fixed R was considered. In the range of R > 0.60, GB was found to promote the gelation of LMP again, which is similar to the case of alginate. The results were in consistence with microstructural observations by AFM. The different modulatory effects of GB were thought to arise from the different gelation mechanisms of LMP and alginate, that is, a progressive dotting growth of LMP dimers vs. a critical zippering growth of alginate dimers during Ca-induced crosslinking. The mechanism of GB modulating the gelation of LMP was proposed and compared to that for alginate.

Nanoparticulate drug delivery systems (nDDS) offer a variety of options when it comes to routes of administration. One possible path is crossing mucosal barriers, such as in the airways and in the GI tract, for systemic distribution or local treatment. The main challenge with this administration route is that the size and surface properties of the nanoparticles, as opposed to small molecular drugs, very often results in mucosal capture, immobilization and removal, which in turn results in a very low bioavailability. Strategies to overcome this challenge do exist, like surface ‘stealth’ modification with PEG. Here we review an alternative or supplemental strategy, co-association of mucus modulating agents with the nDDS to improve bioavailability, where the nDDS may be surface modified or unmodified. This contribution presents some examples on how possible co-association systems may be achieved, using currently marketed mucolytic drugs, alternative formulations or novel agents.

Abstract The use of oligosaccharides as active pharmaceutical ingredients lags behind that of other biological molecules such as proteins and nucleic acids. However, there is now a growing understanding of the complex biological functions of saccharide structures. This has resulted in an increased interest in exploring the pharmaceutical applications of both oligosaccharides and glycoconjugates. Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late stage disease and are often unable to tolerate drug combination regimens due to the associated toxicity. Here we report a novel oligosaccharide drug candidate, RiXOVA (G-blocks). The term “G-blocks” refers to highly defined and specialized short oligomers comprising α-L-guluronate residues derived from alginates extracted from brown seaweed. The G-block oligomers are well tolerated at i.v./i.p. doses >100mg/kg in mice. These oligomers have previously demonstrated the ability to alter matrices of biological macromolecules including mucins, giving rise to the hypothesis that they may have similar effects on the dense desmoplastic tissue within tumours. We here describe the anti-tumour properties of RiXOVA in different PDAC mouse models. Initial efficacy studies were performed in a xenograft model using the CAPAN-2 cells. RiXOVA was found to be non-toxic and non-immunogenic (I.P, 25mg/kg Q3D10 ) in mice. Relative to the start of dosing, the tumour growth in RiXOVA treated mice was 160% +/-30 (n=10) compared to 240% +/- 61 (n=10, p value=0.001) in the vehicle treated mice. Further testing was carried out in a Genetically Engineered Mouse-Derived Allograft (GEDA). The GEDA model recapitulates the dense desmoplastic stroma of the original donor (KPC mouse), unlike cell line-based allografts. KPC (LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre) mouse tumour fragments (2 donors) were implanted subcutaneously in the flank of recipient LSL-Trp53R172H; Pdx1-cre (PC), immunocompetent mice. Relative to the start of dosing, the tumour growth in the RiXOVA treated group (25 mg/kg TIW) was 260% +/- 160 (n=10), compared to 540% +/- 210 (n=8) for the Vehicle (p = 0.001) and 130 % +/- 57 (n=9) for the combination of RiXOVA + gemcitabine (100 mg/kg BIW p <0.001) compared to the vehicle. Ex vivo analysis of tumour tissue revealed changes in the expression and distribution of ECM proteins. Collectively, these results indicate RiXOVA to be a non-toxic oligosaccharide, that acts at the level of the tumour microenviroment to inhibit tumour growth. Whilst these results are in a preliminary stage they indicate G-block oligomers may represent a novel treatment modality in PDAC. Citation Format: Shalini V. Rao, Tonje S. Steigedal, Aarthi Gopinathan, Synnøve N. Magnussen, Sabina S. Strand, Duncan Jodrell, Fran Richards, Kurt I. Draget, Catherine T. Nordgård. Antitumor effect of an oligosaccharide API in a genetically engineered mouse-derived allograft (GEDA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 627.

Objective: Hard tablets and capsules for oral drug delivery cause problems for people experiencing dysphagia. This work describes the formulation and properties of a gelatin based, self-preserved, and soft chewable tablet as an alternative and novel drug delivery format.Materials and methods: Gelatin (8.8-10% in 24.7-29% water) constituted the matrix of the soft, semi-solid tablets. Three different pharmaceuticals (Ibuprofen 10%, Acetaminophen 15%, and Meloxicam 1.5%) were tested in this formulation. Microbial stability was controlled by lowering the water activity with a mixture of sorbitol and xylitol (45.6-55%). Rheological properties were tested applying small strain oscillation measurements. Taste masking of ibuprofen soft-chew tablets was achieved by keeping the ibuprofen insoluble at pH 4.5 and keeping the processing temperature below the crystalline-to-amorphous transition temperature.Results: Soft-chew formulations showed good stability for all three pharmaceuticals (up to 24 months), and the ibuprofen containing formulation exhibited comparable dissolution to a standard oral tablet as well as good microbial stability. The rheological properties of the ibuprofen/gelatin formulation had the fingerprint of a true gelatin gel, albeit higher moduli, and melting temperature.Conclusions: The results suggest that easy-to-swallow and well taste-masked soft chewable tablet formulations with extended shelf life are within reach for several active pharmaceutical ingredients (APIs).