There is an increasing demand for alternative and sustainable protein sources, such as vegetables, insects and microorganisms, that can meet the nutritional and sensory pleasantness needs of consumers. This emergent interest for novel protein sources, allied with "green" and cost-effective processing technologies, such as high hydrostatic pressure, ohmic heating and pulsed electric fields, can be used as strategies to improve the consumption of proteins from sustainable sources without compromising food security. In addition to their nutritional value, these novel proteins present several technological-functional properties that can be used to create various protein systems in different scales (i.e., macro, micro and nano scale), which can be tailored for a specific application in innovative food products. However, in order for these novel protein sources to be broadly used in future food products, their fate in the human gastrointestinal tract (e.g., digestion and bioavailability) must be assessed, as well as their safety for consumers must be clearly demonstrated. In particular, these proteins may become novel allergens triggering adverse reactions and, therefore, a comprehensive allergenicity risk assessment is needed. This review presents an overview of the most promising alternative protein sources, their application in the production of innovative food systems, as well as their potential effects on human health. In addition, new insights on sustainable processing strategies are given.

High-level expression and secretion of heterologous proteins in yeast cause an increased energy demand, which may result in altered metabolic flux distributions. Moreover, recombinant protein overproduction often results in endoplasmic reticulum (ER) stress and oxidative stress, causing deviations from the optimal NAD(P)H regeneration balance. In this context, overexpression of genes encoding enzymes catalyzing endogenous NADPH-producing reactions, such as the oxidative branch of the pentose phosphate pathway, has been previously shown to improve protein production in Pichia pastoris (syn. Komagataella spp.). In this study, we evaluate the overexpression of the Saccharomyces cerevisiae POS5-encoded NADH kinase in a recombinant P. pastoris strain as an alternative approach to overcome such redox constraints. Specifically, POS5 was cooverexpressed in a strain secreting an antibody fragment, either by directing Pos5 to the cytosol or to the mitochondria. The physiology of the resulting strains was evaluated in continuous cultivations with glycerol or glucose as the sole carbon source, as well as under hypoxia (on glucose). Cytosolic targeting of Pos5 NADH kinase resulted in lower biomass-substrate yields but allowed for a 2-fold increase in product specific productivity. In contrast, Pos5 NADH kinase targeting to the mitochondria did not affect growth physiology and recombinant protein production significantly. Growth physiological parameters were in silico evaluated using the recent upgraded version (v3.0) of the P. pastoris consensus genome-scale metabolic model iMT1026, providing insights on the impact of POS5 overexpression on metabolic flux distributions. IMPORTANCE Recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of redox-affecting enzymes has been explored in other organisms, such as Saccharomyces cerevisiae, as a means to fine tune the cofactor regeneration balance in order to obtain higher protein titers. In the present work, this strategy is explored in P. pastoris. In particular, one NADH kinase enzyme from S. cerevisiae (Pos5) is used, either in the cytosol or in mitochondria of P. pastoris, and its impact on the production of a model protein (antibody fragment) is evaluated. A significant improvement in the production of the model protein is observed when the kinase is directed to the cytosol. These results are significant in the field of heterologous protein production in general and in particular in the development of improved metabolic engineering strategies for P. pastoris.

Viscosity-modified diet through thickeners is used as a strategy to circumvent swallowing problems by oropharyngeal dysphagia patients. Most commercial products present xanthan and starch in their formulations, but flaxseed gum (FG) is a potential thickener for liquid food that provides additional health benefits. FG was mixed either with modified starch (MS) and/or xanthan gum (XG), varying biopolymers' concentration according to a central composite rotational design in which rheological and colour properties in water were the evaluated responses. All formulations showed a shear time-independent and shear-thinning behaviour, mainly influenced by XG and MS concentrations. In oscillatory measurements, the formulations presented a prevailing elastic character attributed to MS and mainly to XG, which despite the lower concentration in which it was incorporated, exerted a similar influence on this rheological property. However, the increase of FG concentration was the most significant factor influencing viscosity, but also favoured an increase of both viscoelastic moduli mainly G'. Analysis of the microstructure disclosed different network structures as a result of biopolymers interactions, which was related to rheological behaviour giving insights to design new thickeners for dysphagia management. In addition, the amount of glucose released after in vitro digestion was evaluated and compared to a commercial MS-based thickener. Interestingly, the commercial formulation showed a glucose release significantly higher than the proposed FG/MS/XG-based formulations. These results open the opportunity to tailor the rheological characteristics of food systems by adding and combining natural ingredients, improving technological and nutritional properties.

The objective of this study was to use glycerol generated from the synthesis of biodiesel to study the oleaginous potential of wild yeasts. An initial selection was performed via a rapid and qualitative technique by staining with Sudan Black B. Initially 129 yeasts were present, from which 5 were selected and cultivated in liquid medium containing pure or raw glycerol. The yeast LEB-M3, isolated from the Pantanal, presented lipid content of 20.46% and 56.58% for cultivation in pure and raw glycerol, respectively. This strain was genotypically identified as Candida sp. The fatty acid profile showed predominance of oleic acid (C18:1), 57.35% for cultivation in pure glycerol, and in raw glycerol linoleic acid (C18:2) was predominant (46.0%). It was possible to select a yeast with high lipid concentrations 9.14 g/L and fatty acid profile similar to vegetable oils commonly used in the synthesis of biodiesel.

The main goals of the present study included the screening and identification of cellulase producing wild yeasts, isolated from samples collected from different Brazilian biomes. They were selected according to their capabilities of degrading carboxymethyl cellulose (CMC) and micro-crystalline cellulose (SERVACEL ®), as single carbon sources in solid medium. After the step of solid medium selection, yeast cells were grown in liquid medium containing cellulose (SERVACEL ®); in shake flasks at temperature of 30°C and 150 rpm agitation for 288 h. Three specific activities were evaluated: endoglucanase (CMCase), total activity (filter paper activity), and cellobiase. From a total of 390 strains of wild yeasts previously isolated, 16 strains performed cellulose hydrolysis, verified by the colorless halo in the solid medium. Among these 16 strains, 5 stood out as presenting higher levels of enzyme activity. The following step, screening in liquid medium, indicated only one strain as a potential producer of cellulases, named as AAJ6, for which the highest hydrolytic activity on carboxymethyl cellulose (0.33 U/ml) and filter paper (0.039 U/ml) was recorded. Afterwards, this wild yeast strain (AAJ6) was molecularly identified by sequencing the ITS1-5.8S-ITS2 and D1/D2 domains of the subunit (26 S) ribosomal DNA. Sequencing resulted in the identification of this strain as yeast-like fungus Acremonium strictum.

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The microbiological, chemical and sensory characteristics of licensed and unlicensed commercial Serrano cheese, a traditional product from the highlands of South Brazil, were studied. The average bacteria counts (log CFU/g) were 8.8, 3.4, 3.4, 5.1, and 8.8, for mesophilic bacteria, fecal coliforms, staphylococci, yeasts and moulds, and lactic acid bacteria (LAB), respectively. Differences in microbiological quality were detected between licensed and unlicensed cheeses. However, the presence of potential gastroentheric bacteria in both kind of cheeses is disturbing and point out the necessity of alternative manufacturing process and good manufacturing practices to minimize consumers’ health risks. The most prevalent LAB were <i>Lactobacillus</i> (91%), followed <i>Lactococcus</i> and <i>Enterococcus. Lactobacillus</i> isolates comprised <i>L. plantarum</i> (57.7%), <i>L. paracasei</i> (19.2%), <i>L. rhamnosus</i> (7.7%), <i>L. acidophilus</i> (7.7%), and 3.8% of both <i>L. curvatus</i> and <i>L. fermentum</i>. Non-significant physico-chemical differences were detected between licensed and unlicensed cheeses. Cheeses that received the highest sensorial scores exhibited the presence of both <i>L. plantarum</i> and <i>L. paracasei</i>, indicating that mixed populations of these species positively contributed to flavor development