3-D printed meat alternatives based on pea and single cell proteins and hydrocolloids: Effect of paste formulation on process-induced fibre alignment and structural and textural properties.

Extrusion-based 3D food printing can be used as an alternative structuring technique to traditional extrusion processing for creating meat-like structures. This study focused on 3-D food printing to generate structures analogous to meat by using various combinations of texturized pea protein fibrils, microbial Single Cell Protein (SCP) and hydrocolloids locust bean gum and/or sodium alginate. Simple moulding was utilized as benchmarking to better understand the 3D printing-induced structural effects. To gain understanding of the interactions between proteins of different origin (plant and SCP) and with hydrocolloids, structural, textural and rheological properties were analysed. Oscillatory stress sweeps of all printing pastes revealed elastic-dominant rheological behaviour (G' 4000-6000 Pa) with a defined yield stress (25-60 Pa) explaining their printability and shape stability. X-ray microtomography of ion-crosslinked analogues showed a printing-induced preferential alignment of fibrils in the direction of nozzle movement, while moulding led to a random orientation. Textural characterization via bi-directional cutting tests demonstrated higher cutting force in transversal (FT) over longitudinal (FL) direction in 3D-printed samples and equal forces in moulded samples. The anisotropy index (AI = FT/FL) of printed samples ranged between 1.4 and 2.5, indicating anisotropic texture, and 0.8-1 for moulded samples indicating isotropic texture. This study demonstrated the applicability of paste-extrusion in generating anisotropic structures analogous to meat by process-induced fibril alignment. The results support further development of 3D food printing technology in design of sustainable meat alternatives resembling whole-muscle meat.

Production of bovine beta-lactoglobulin and hen egg ovalbumin by Trichoderma reesei using precision fermentation technology and testing of their techno-functional properties.

The food protein ingredient market is dominated by dairy and egg proteins. Both milk whey and egg proteins are challenging proteins to replace, e.g. with plant proteins, due to the unique structural features of the animal proteins that render them highly functional. Thus, to provide a non-animal source of these important proteins the fungal host Trichoderma reesei was utilized for the biotechnical production of recombinant hen ovalbumin (TrOVA) and bovine beta lactoglobulin (TrBLG). These food proteins were investigated using two different promoter systems to test the concept of effectively expressing them in a fungal host. Both proteins were successfully produced in 24 well plate and bioreactor scale. The production level of TrBLG and TrOVA were 1 g/L and 2 g/L, respectively. Both proteins were further purified and characterized, and their functional properties were tested. TrBLG and TrOVA secondary structures determined by circular dichroism corresponded to the proteins of bovine and hen. The T. reesei produced proteins were found to be N-glycosylated, mostly with Man 5. TrBLG had emulsification properties matching to corresponding bovine protein. TrOVA showed excellent foaming characteristics and heat-induced gelation, although the strength of the gel was somewhat lower than with hen ovalbumin, possibly due to the partial degradation of TrOVA or presence of other host proteins. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming. Industrial relevance: The food protein ingredient market is dominated by dairy (largely whey proteins) and egg proteins. Whey proteins are valuable and versatile food ingredients due to their functional and nutritional quality. They are largely used in meat and milk products, low fat products, bakery, confectionary, infant formulas and sports nutrition. Similarly, egg white protein ovalbumin is a highly functional protein ingredient that facilitates structure formation and high nutritional quality in most food products. Together they comprise 40-70% of the revenue in the animal protein ingredients market. Both whey and egg proteins are extremely challenging proteins to replace, e.g., by plant proteins due to their unique structural features that render them with high functionality. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming.

Tailoring sensory properties of plant cell cultures for food use.

The nutritional value of Rowan (Sorbus aucuparia L.) and Arctic bramble (Rubus arcticus L.) plant cell cultures in terms of protein and dietary fibre contents is very good, ∼ 18-22% and âˆ¼ 28-29% on dry matter basis, respectively. The aim of this study was to evaluate various processing methods and formulation to modulate sensory profiles of these plant cell cultures for food purposes. For fresh unprocessed plant cell cultures, treatment with sugar or sugar in combination with citric acid significantly improved the mouthfeel and flavour. The sugar and sugar + citric acid treated plant cell culture samples were perceived more moist, softer, less sandy and they had a less coarse mouthfeel when compared to untreated plant cell cultures. Freeze-drying produced sweet, intense, berry-like flavour and resulted in most promising sensory attributes for the studied plant cell cultures. When freeze-dried Rowan plant cell culture was further processed, the most balanced sweetness/sourness ratio was reached by using 9.5 % (w/w) sucrose and 0.1 % (w/w) citric acid or 4.8 % w/w fructose and 0.1 % w/w citric acid. We conclude that formulation and processing can greatly improve the performance of plant cell cultures for food use.

Impact of Enzymatic Hydrolysis and Microfluidization on the Techno-Functionality of Oat Bran in Suspension and Acid Milk Gel Models.

Oat bran is a nutritionally rich ingredient, but it is underutilized in semi-moist and liquid foods due to technological issues such as high viscosity and sliminess. The aim of this work was to improve the technological properties of oat bran concentrate (OBC) in high-moisture food applications by enzymatic and mechanical treatments. OBC was hydrolyzed with ß-glucanase (OBC-Hyd) and the water-soluble fraction (OBC-Sol) was separated. OBC, OBC-Hyd and OBC-Sol were further microfluidized at 5% dry matter content. Enzymatic treatment and microfluidization of OBC reduced the molecular weight (Mw) of ß-glucan from 2748 kDa to 893 and 350 kDa, respectively, as well as the average particle size of OBC (3.4 and 35 times, respectively). Both treatments increased the extractability of the soluble compounds from the OBC samples (up to 80%) and affected their water retention capacity. OBC in suspension had very high viscosity (969 mPa·s) when heated, which decreased after both enzyme and microfluidization treatments. The colloidal stability of the OBC in suspension was improved, especially after microfluidization. The addition of OBC samples to acid milk gels decreased syneresis, improved the water holding capacity and softened the texture. The changes in the suspension and gel characteristics were linked with reduced ß-glucan Mw and OBC particle size.

Techno-Economic Prospects and Desirability of 3D Food Printing: Perspectives of Industrial Experts, Researchers and Consumers.

3D food printing is an emerging food technology innovation that enables the personalization and on-demand production of edible products. While its academic and industrial relevance has increased over the past decade, the functional value of the technology remains largely unrealized on a commercial scale. This study aimed at updating the business outlook of 3D food printing so as to help entrepreneurs and researchers in the field to channel their research and development (R&D) activities. A three-phase mixed methods approach was utilized to gain perspectives of industrial experts, researchers, and potential consumers. Data were collected from two sets of interviews with experts, a survey with experts, and consumer focus group discussions. The results gave insights into key attributes and use cases for a 3D food printer system, including the techno-economic feasibility and consumer desirability of identified use cases. A business modelling workshop was then organized to translate these results into three refined value propositions for 3D food printing. Both the experts and consumers found personalized nutrition and convenience to be the most desirable aspects of 3D food printing. Accordingly, business models related to 3D printed snacks/meals in semi-public spaces such as fitness centers and hospitals were found to offer the highest business potential. While the technology might be mature enough at component level, the successful realization of such high-reward models however would require risk-taking during the developmental phase.

Structural and Textural Characteristics of 3D-Printed Protein- and Dietary Fibre-Rich Snacks Made of Milk Powder and Wholegrain Rye Flour.

This study addressed the potential of 3D printing as a processing technology for delivering personalized healthy eating solutions to consumers. Extrusion-based 3D printing was studied as a tool to produce protein- and dietary fibre-rich snack products from whole milk powder and wholegrain rye flour. Aqueous pastes were prepared from the raw materials at various ratios, grid-like samples printed from the pastes at ambient temperature and the printed samples post-processed by oven baking at 150 °C. Printing pastes were characterized by rheological measurements and the baked samples by X-ray micro tomography, texture measurements and sensory analysis. All formulations showed good printability and shape stability after printing. During baking, the milk powder-based samples expanded to a level that caused a total collapse of the printed multiple-layer samples. Shape retention during baking was greatly improved by adding rye flour to the milk formulation. Sensory evaluation revealed that the volume, glossiness, sweetness and saltiness of the baked samples increased with an increasing level of milk powder in the printing paste. A mixture of milk powder and rye flour shows great potential as a formulation for healthy snack products produced by extrusion-based 3D printing.

Modular protein architectures for pH-dependent interactions and switchable assembly of nanocellulose.

Protein engineering shows a wide range of possibilities for designing properties in novel materials. Following inspiration from natural systems we have studied how combinations or duplications of protein modules can be used to engineer their interactions and achieve functional properties. Here we used cellulose binding modules (CBM) coupled to spider silk N-terminal domains that dimerize in a pH-sensitive manner. We showed how the pH-sensitive switching into dimers affected cellulose binding affinity in relation to covalent coupling between CBMs. Finally, we showed how the pH-sensitive coupling could be used to assemble cellulose nanofibers in a dynamic pH-dependent way. The work shows how novel proteins can be designed by linking functional domains from widely different sources and thereby achieve new functions in the self-assembly of nanoscale materials.

Plant cells as food - A concept taking shape.

Plant cell cultures from cloudberry, lingonberry and stoneberry were studied in terms of their nutritional properties as food. Carbohydrate, lipid and protein composition, in vitro protein digestibility and sensory properties were investigated. Dietary fibre content varied between 21.2 and 36.7%, starch content between 0.3 and 1.3% and free sugar content between 17.6 and 33.6%. Glucose and fructose were the most abundant sugars. High protein contents between 13.7 and 18.9% were recorded and all samples had a balanced amino acid profile. In vitro protein digestion assay showed hydrolysis by digestive enzymes in fresh cells but only limited hydrolysis in freeze-dried samples. The lipid analysis indicated that the berry cells were rich sources of essential, polyunsaturated fatty acids. In sensory evaluation, all fresh berry cells showed fresh odour and flavour. Fresh cell cultures displayed a rather sandy, coarse mouthfeel, whereas freeze-dried cells melted quickly in the mouth. All in all the potential of plant cells as food was confirmed.

Structural properties and foaming of plant cell wall polysaccharide dispersions.

Water suspensions of cellulose nanofibres with xylan, xyloglucan and pectin were studied for foaming and structural properties as a new means for food structuring. The dispersions were analysed with rheological measurements, microscopy and optical coherence tomography. A combination of xylan with TEMPO-oxidized nanocellulose produced a mixture with well-dispersed air bubbles, while the addition of pectin improved the elastic modulus, hardness and toughness of the structures. A similar structure was observed with native nanocellulose, but the elastic modulus was not as high. Shear flow caused cellulose nanofibres to form plate-like flocs in the suspension that accumulated near bubble interfaces. This tendency could be affected by adding laccase to the dispersion, but the effect was opposite for native and TEMPO-oxidized nanocellulose. Nanocellulose type also influenced the interactions between nanofibers and other polysaccharides. For example, xyloglucan interacted strongly with TEMPO-oxidized nanocellulose (high storage modulus) but not with native nanocellulose.

Cross-linking of sodium caseinate-structured emulsion with transglutaminase alters postprandial metabolic and appetite responses in healthy young individuals.

The physico-chemical and interfacial properties of fat emulsions influence lipid digestion and may affect postprandial responses. The aim of the present study was to determine the effects of the modification of the interfacial layer of a fat emulsion by cross-linking on postprandial metabolic and appetite responses. A total of fifteen healthy individuals (26.5 (sem 6.9) years and BMI 21.9 (sem 2.0) kg/m2) participated in a cross-over design experiment in which they consumed two isoenergetic (1924 kJ (460 kcal)) and isovolumic (250 g) emulsions stabilised with either sodium caseinate (Cas) or transglutaminase-cross-linked sodium caseinate (Cas-TG) in a randomised order. Blood samples were collected from the individuals at baseline and for 6 h postprandially for the determination of serum TAG and plasma NEFA, cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), glucose and insulin responses. Appetite was assessed using visual analogue scales. Postprandial TAG and NEFA responses and gastric emptying (GE) rates were comparable between the emulsions. CCK increased more after the ingestion of Cas-TG than after the ingestion of Cas (P< 0.05), while GLP-1 responses did not differ between the two test emulsions. Glucose and insulin profiles were lower after consuming Cas-TG than after consuming Cas (P< 0.05). The overall insulin, glucose and CCK responses, expressed as areas above/under the curve, did not differ significantly between the Cas and Cas-TG meal conditions. Satiety ratings were reduced and hunger, desire to eat and thirst ratings increased more after the ingestion of Cas-TG than after the ingestion of Cas (P< 0.05). The present results suggest that even a subtle structural modification of the interfacial layer of a fat emulsion can alter the early postprandial profiles of glucose, insulin, CCK, appetite and satiety through decreased protein digestion without affecting significantly on GE or overall lipid digestion.

Versatile peroxidase as a valuable tool for generating new biomolecules by homogeneous and heterogeneous cross-linking.

The modification and generation of new biomolecules intended to give higher molecular-mass species for biotechnological purposes, can be achieved by enzymatic cross-linking. The versatile peroxidase (VP) from Pleurotus eryngii is a high redox-potential enzyme with oxidative activity on a wide variety of substrates. In this study, VP was successfully used to catalyze the polymerization of low molecular mass compounds, such as lignans and peptides, as well as larger macromolecules, such as protein and complex polysaccharides. Different analytical, spectroscopic, and rheological techniques were used to determine structural changes and/or variations of the physicochemical properties of the reaction products. The lignans secoisolariciresinol and hydroxymatairesinol were condensed by VP forming up to 8 unit polymers in the presence of organic co-solvents and Mn(2+). Moreover, 11 unit of the peptides YIGSR and VYV were homogeneously cross-linked. The heterogeneous cross-linking of one unit of the peptide YIGSR and several lignan units was also achieved. VP could also induce gelation of feruloylated arabinoxylan and the polymerization of ß-casein. These results demonstrate the efficacy of VP to catalyze homo- and hetero-condensation reactions, and reveal its potential exploitation for polymerizing different types of compounds.

Enzymatically structured emulsions in simulated gastrointestinal environment: impact on interfacial proteolysis and diffusion in intestinal mucus.

Fundamental knowledge of physicochemical interactions in the gastrointestinal environment is required in order to support rational designing of protein-stabilized colloidal food and pharmaceutical delivery systems with controlled behavior. In this paper, we report on the colloidal behavior of emulsions stabilized with the milk protein sodium caseinate (Na-Cas), and exposed to conditions simulating the human upper gastrointestinal tract. In particular, we looked at how the kinetics of proteolysis was affected by adsorption to an oil-water interface in emulsion and whether the proteolysis and the emulsion stability could be manipulated by enzymatic structuring of the interface. After cross-linking with the enzyme transglutaminase, the protein was digested with use of an in vitro model of gastro-duodenal proteolysis in the presence or absence of physiologically relevant surfactants (phosphatidylcholine, PC; bile salts, BS). Significant differences were found between the rates of digestion of Na-Cas cross-linked in emulsion (adsorbed protein) and in solution. In emulsion, the digestion of a population of polypeptides of M(r) ca. 50-100 kDa was significantly retarded through the gastric digestion. The persistent interfacial polypeptides maintained the original emulsion droplet size and prevented the system from phase separating. Rapid pepsinolysis of adsorbed, non-cross-linked Na-Cas and its displacement by PC led to emulsion destabilization. These results suggest that structuring of emulsions by enzymatic cross-linking of the interfacial protein may affect the phase behavior of emulsion in the stomach and the gastric digestion rate in vivo. Measurements of ζ-potential revealed that BS displaced the remaining protein from the oil droplets during the simulated duodenal phase of digestion. Diffusion of the postdigestion emulsion droplets through ex vivo porcine intestinal mucus was only significant in the presence of BS due to the high negative charge these biosurfactants imparted to the droplets. This implies that the electrostatic repulsion produced can prevent the droplets from being trapped by the mucus matrix and facilitate their transport across the small intestine mucosal barrier.

Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture.

Over the recent years, various materials have been introduced as potential 3D cell culture scaffolds. These include protein extracts, peptide amphiphiles, and synthetic polymers. Hydrogel scaffolds without human or animal borne components or added bioactive components are preferred from the immunological point of view. Here we demonstrate that native nanofibrillar cellulose (NFC) hydrogels derived from the abundant plant sources provide the desired functionalities. We show 1) rheological properties that allow formation of a 3D scaffold in-situ after facile injection, 2) cellular biocompatibility without added growth factors, 3) cellular polarization, and 4) differentiation of human hepatic cell lines HepaRG and HepG2. At high shear stress, the aqueous NFC has small viscosity that supports injectability, whereas at low shear stress conditions the material is converted to an elastic gel. Due to the inherent biocompatibility without any additives, we conclude that NFC generates a feasible and sustained microenvironment for 3D cell culture for potential applications, such as drug and chemical testing, tissue engineering, and cell therapy.

Crosslinking with transglutaminase does not change metabolic effects of sodium caseinate in model beverage in healthy young individuals.

BACKGROUND: Postprandial metabolic and appetitive responses of proteins are dependent on protein source and processing technique prior to ingestion. Studies on the postprandial effects of enzymatic crosslinking of milk proteins are sparse. Our aim was to study the effect of transglutaminase (TG)-induced crosslinking of sodium caseinate on postprandial metabolic and appetite responses. Whey protein was included as reference protein. METHODS: Thirteen healthy individuals (23.3 ± 1.1 y, BMI 21.7 ± 0.4 kg/m2) participated in a single-blind crossover design experiment in which the subjects consumed three different isovolumic (500 g) pourable beverages containing either sodium caseinate (Cas, 29 g), TG-treated sodium caseinate (Cas-TG, 29 g) or whey protein (Wh, 30 g) in a randomized order. Blood samples were collected at baseline and for 4 h postprandially for the determination of plasma glucose, insulin and amino acid (AA) concentrations. Gastric emptying (GE) was measured using the 13 C-breath test method. Appetite was assessed using visual analogue scales. RESULTS: All examined postprandial responses were comparable with Cas and Cas-TG. The protein type used in the beverages was reflected as differences in plasma AA concentrations between Wh and Cas, but there were no differences in plasma glucose or insulin responses. A tendency for faster GE rate after Wh was detected. Appetite ratings or subsequent energy intake did not differ among the protein beverages. CONCLUSIONS: Our results indicate that the metabolic responses of enzymatically crosslinked and native sodium caseinate in a liquid matrix are comparable, suggesting similar digestion and absorption rates and first pass metabolism despite the structural modification of Cas-TG.

Structure modification of a milk protein-based model food affects postprandial intestinal peptide release and fullness in healthy young men.

Physico-chemical and textural properties of foods in addition to their chemical composition modify postprandial metabolism and signals from the gastrointestinal tract. Enzymatic cross-linking of protein is a tool to modify food texture and structure without changing nutritional composition. We investigated the effects of structure modification of a milk protein-based model food and the type of milk protein used on postprandial hormonal, metabolic and appetitive responses. Healthy males (n 8) consumed an isoenergetic and isovolumic test product containing either whey protein (Wh, low-viscous liquid), casein (Cas, high-viscous liquid) or Cas protein cross-linked with transglutaminase (Cas-TG, rigid gel) in a randomised order. Blood samples were drawn for plasma glucose, insulin, cholecystokinin (CCK), glucagon-like peptide 1 and peptide YY analysis for 4 h. Appetite was assessed at concomitant time points. Cas and Wh were more potent in lowering postprandial glucose than Cas-TG during the first hour. Insulin concentrations peaked at 30 min, but the peaks were more pronounced for Cas and Wh than for Cas-TG. The increase in CCK was similar for Cas and Wh in the first 15 min, whereas for Cas-TG, the CCK release was significantly lower, but more sustained. The feeling of fullness was stronger after the consumption of Cas-TG than after the consumption of Cas and Wh. The present results suggest that food structure is more effective in modulating the postprandial responses than the type of dairy protein used. Modification of protein-based food structure could thus offer a possible tool for lowering postprandial glucose and insulin concentrations and enhancing postprandial fullness.

Combined enzymatic and high-pressure processing affect cell wall polysaccharides in berries.

The effect of high-pressure processing (HPP) on cell wall polysaccharides in berries was investigated. HPP decreased the degree of methyl esterification (DM), probably by activation of pectin methyl esterase (PME), and improved the extractability of pectins. When commercial enzyme mixtures were added to mashed berries, a synergistic effect was observed between treatment with commercial enzymes and HPP. Compared to treatment at atmospheric pressure, pectic polysaccharides were degraded to a larger extent when HPP was used. In contrast, hemicelluloses were hardly affected by the added enzymes when HPP was included, although they were degraded during similar treatment at atmospheric pressure. Additionally, the activity of rhamnose-releasing enzymes present in minor quantities might be enhanced after HPP, resulting in a decrease of rhamnose in the polymeric cell wall material. These results exploring the effect of HPP at representative conditions clearly point out the potential of HPP for polysaccharide modification.