Microalgae protein digestibility: How to crack open the black box?

Microalgae are booming as a sustainable protein source for human nutrition and animal feed. Nevertheless, certain strains were reported to have robust cell walls limiting protein digestibility. There are several disruption approaches to break down the cell integrity and increase digestive enzyme accessibility. This review's intent is to discuss the digestibility of microalgae proteins in intact cells and after their disruption. In intact single cells, the extent of protein digestibility is chiefly related to cell wall structural properties (differing among strains) as well as digestion method and when added to food or feed protein digestibility changes depending on the matrix's composition. The degree of effectiveness of the disruption method varies among studies, and it is complicated to compare them due to variabilities in digestibility models, strains, disruption method/conditions and their consequent impact on the microalgae cell structure. More exhaustive studies are still required to fill knowledge gaps on the structure of microalgal cell walls and to find efficient and cost-effective disruption technologies to increase proteins availability without hindering their quality.

Innovative vs classical methods for drying heterotrophic Chlorella vulgaris: Impact on protein quality and sensory properties.

Drying is a necessary step in the microalgae production chain to reduce microbial load and oxidative degradation of the end product. Depending on the differences in applied temperature and treatment time, the process of drying can have a substantial impact on protein quality and aroma, important characteristics determining the incorporation potential in food products. In this study, we compared the drying of heterotrophic Chorella vulgaris with both innovative (agitated thin film drying (ATFD), pulse combustion drying (PCD) and solar drying (SolD)) and commonly used drying techniques (spray drying (SprD) and freeze drying (FD)). To evaluate the impact on protein quality, we evaluated techno-functional properties, in vitro digestibility (INFOGEST) as well as protein denaturation using differential scanning calorimetry (DSC). A sensory analysis was performed by a trained expert panel, combined with headspace solid-phase microextraction (HS-SPME) - gas chromatography-mass spectrometry (GC-MS) to determine volatile organic compounds (VOCs). ATFD was found to increase techno-functional properties such as gelling, water holding and solubility as well as in vitro protein digestibility. These observations could be related to induced cell disruption and protein denaturation by ATFD. Sensory analysis indicated an increased earthy off-flavor after ATFD. Interestingly, the high-temperature PCD led to an increase in cacao odor while low-temperature FD resulted in lower flavor, odors and VOCs. These results demonstrate that protein quality and sensorial properties of C. vulgaris can be steered through the type of drying, which could help in the selection of application-specific drying methods. Overall, this work could promote the incorporation of microalgal single cell proteins in different innovative food products.

Varying precipitation conditions allow directing the composition and physical properties of soy protein concentrates.

Soy protein concentrates (SPCs) are common food ingredients. They typically contain 65% (w/w) protein and ∼30% (w/w) carbohydrate. SPCs can be obtained with various protein precipitation conditions. A systematic study of the impact of these different protein precipitation protocols on the SPC protein composition and physical properties is still lacking. Here, SPCs were prepared via three different protocols, that is, isoelectric (pH 3.5-5.5), aqueous ethanol (50%-70% [v/v]), and Ca2+ ion (5-50 mM) based precipitations, and analyzed for (protein) composition, protein thermal properties, dispersibility, and water-holding capacity. SPCs precipitated at pH 5.5 or by adding 15 mM Ca2+ ions had a lower 7S/11S globulin ratio (∼0.40) than that (∼0.50) of all other SPC samples. Protein in SPCs obtained by isoelectric precipitation denatured at a significantly higher temperature than those in ethanol- or Ca2+ -precipitated SPCs. Precipitation with 50%-60% (v/v) ethanol resulted in pronounced denaturation of 2S albumin and 7S globulin fractions in SPCs. Additionally, increasing the precipitation pH from 3.5 to 5.5 and increasing the Ca2+ ion concentration from 15 to 50 mM caused a strong decrease of both the dispersibility of the protein in SPC and its water-holding capacity at pH 7.0. In conclusion, this study demonstrates that the SPC production process can be directed to obtain ingredients with versatile protein physicochemical properties toward potential food applications. PRACTICAL APPLICATION: This study demonstrates that applying different protein precipitation protocols allows obtaining SPCs that vary widely in (protein) composition and physical properties (such as protein dispersibility and water-holding capacity). These varying traits can greatly influence the suitability of SPCs as functional ingredients for specific applications, such as the production of food foams, emulsions, gels, and plant-based meat alternatives. The generated knowledge may allow targeted production of SPCs for specific applications.

Protein Quality and Protein Digestibility of Vegetable Creams Reformulated with Microalgae Inclusion.

Microalgae are considered a valuable source of proteins that are used to enhance the nutritional value of foods. In this study, a standard vegetable cream recipe was reformulated through the addition of single-cell ingredients from Arthrospira platensis (spirulina), Chlorella vulgaris, Tetraselmis chui, or Nannochloropsis oceanica at two levels of addition (1.5% and 3.0%). The impact of microalgae species and an addition level on the amino acid profile and protein in vitro digestibility of the vegetable creams was investigated. The addition of microalgae to vegetable creams improved the protein content and the amino acid nutritional profile of vegetable creams, whereas no significant differences were observed in protein digestibility, regardless of the species and level of addition, indicating a similar degree of protein digestibility in microalgae species despite differences in their protein content and amino acid profile. This study indicates that the incorporation of microalgae is a feasible strategy to increase the protein content and nutritional quality of foods.

A systematic study of the impact of the isoelectric precipitation process on the physical properties and protein composition of soy protein isolates.

The functional properties of soy protein isolates (SPIs), which are crucial for their successful use in food applications, depend on their protein physical properties and composition. Although the production process of SPIs is well-known and established industrial practice, fundamental knowledge on how the different isolation steps and varying isolation conditions influence these properties is lacking. Here, these characteristics were systematically investigated by assessing the impacts of the various steps of a conventional isoelectric precipitation based SPI production protocol. Protein denaturation and colloidal state were evaluated with differential scanning calorimetry and dynamic light scattering combined with (ultra)centrifugation, respectively. The protein composition (on protein subunit level) was assessed via size-exclusion chromatography. Hexane defatting was found not to cause protein denaturation. Alkaline extraction at pH values between 7.0 and 9.0 resulted in no differences in protein physical properties or composition. Subsequent acid precipitation at pH 5.5 resulted in SPIs with a lower 7S/11S ratio and higher protein solubility at neutral pH than when produced at pH 4.5 and 3.5. SPIs obtained at all evaluated precipitation pH values contained a considerable amount of aggregated protein structures. Spray-drying of SPI did not result in a higher degree of protein denaturation or in a loss of protein solubility compared to freeze-drying, but a smaller amount of soluble aggregates was observed in spray-dried SPIs. Hence, alterations in the isolation procedure can result in SPIs with moderately different physical properties and protein composition, which might lead to different functional properties and thus applicabilities in certain food systems.

Cultured meat and challenges ahead: A review on nutritional, technofunctional and sensorial properties, safety and legislation.

Cultured meat is introduced as a valuable traditional meat equivalent. However, before marketable end products are available, several hurdles need to be overcome. Among others, these issues comprise obtaining an optimal nutritional profile and approaching the texture, the colour and the unique flavour and taste of conventional meat. Furthermore, the impact of processing on these matters is also still subject of future research. Moreover, more profound knowledge on food-safety aspects, like microbial contamination, prions, possible genetically engineered starting material, etc., and ways to reduce such risks will determine the future success of cultured meat products. Undoubtedly, correct terminology and adequate definitions also require further attention, as these form the starting point of legislative/regulatory aspects. This review provides a state-of-the-art overview on nutritional, technofunctional and sensorial properties, and food-safety and legislative/regulatory aspects on cultured meat production. Additionally, the various challenges and future steps of these aspects of cultured meat are highlighted.

Drying Microalgae Using an Industrial Solar Dryer: A Biomass Quality Assessment.

Microalgae are considered a promising resource of proteins, lipids, carbohydrates, and other functional biomolecules for food and feed markets. Competitive drying solutions are required to meet future demands for high-quality algal biomass while ensuring proper preservation at reduced costs. Since often used drying methods, such as freeze or spray drying, are energy and time consuming, more sustainable processes remain to be developed. This study tested an indirect and hybrid solar dryer as an alternative to conventional freeze drying of industrially produced Tetraselmis chui and Nannochloropsis oceanica wet paste. The effects of the drying method on biomass quality parameters, including biochemical profiles, functional properties, and microbial safety, were assessed. No significant differences were found between the applied drying technologies for total proteins, carbohydrates, lipids, and fatty acid profiles. On the other hand, some pigments showed significant differences, displaying up to 44.5% higher contents in freeze-dried samples. Minor differences were also registered in the mineral profiles (<10%). Analyses of microbial safety and functional properties of the solar-dried biomass appear adequate for food and feed products. In conclusion, industrial solar drying is a sustainable technology with a high potential to preserve high-quality microalgal biomass for various markets at expected lower costs.

Development of GLM regression models to predict the consumer acceptability of cooked ham based on analytical parameters.

Due to concerns about meat quality, health, sustainability and animal welfare, the typical Belgian meat products such as cooked ham are being threatened by a negative reputation. To address these concerns, an objective quality assessment tool was developed that could predict the consumer acceptability for a range of sensorial descriptors based on analytical parameters. A total of 28 commercial cooked hams were evaluated by a sensorial panel of consumers while simultaneously, a broad range of analytical tests were conducted on the same hams. Per sensorial descriptor, the analytical results and consumer acceptability for all cooked hams were processed by Generalized Linear Modelling (GLM). This holistic approach makes it possible to predict the consumer acceptability of a sensorial descriptor with great reliability and robustness by only using objective analytical parameters. An efficient R&D tool was developed to optimize the sensorial and analytical quality of the cooked ham that meets consumer demands.

Exploring the microbiological quality and safety of dry-aged beef: A cross-sectional study of loin surfaces during ripening and dry-aged beef steaks from commercial meat companies in Belgium.

A cross-sectional survey was undertaken in Belgian beef producing companies to study the current practices and the microbiological load of dry-aged loins (during production) and trimmed steaks (final product). In each company, the temperature and relative humidity of the ripening chamber were measured, and two loins (each in a different stage of the ripening process) were sampled. From the surface of each loin, a lean meat and adipose tissue sample was analysed separately, and different groups of bacteria, yeasts and moulds were enumerated. The average relative humidity in the ripening chambers was 72 ± 13% and the temperature ranged between 0.0 °C and 5.9 °C. During the drying process, most of the lean meat and adipose tissue samples showed high numbers of total psychrotrophic aerobic bacteria, Pseudomonas spp., psychrotrophic lactic acid bacteria, and yeasts, but the variation between loins was high. The microbiological load on freshly cut dry-aged steaks was generally lower than on loin surfaces, but both psychrotrophic aerobic and anaerobic bacteria were present inside several steaks. The water activity inside dry-aged beef steaks was high (aw ≥ 0.98), which could allow growth of psychrotrophic pathogens, though more in-depth studies are necessary to determine potential growth during the storage of (trimmed) steaks or even inside loins during the dry-aging process.

Apple phenolics as inhibitors of the carbonylation pathway during in vitro metal-catalyzed oxidation of myofibrillar proteins.

The effect of apple phenolics on the oxidative damage caused to myofibrillar proteins by an in vitro metal-catalyzed oxidation system was investigated. Three pure phenolic compounds (chlorogenic acid, (-)-epicatechin and phloridzin) and an apple peel extract were added to myofibrillar proteins in three concentrations (50, 100 and 200µM), and a blank treatment was included as a control. All suspensions were subjected to Fe(3+)/H2O2 oxidation at 37°C during 10days, and protein oxidation was evaluated as carbonylation (α-amino adipic and γ-glutamic semialdehydes) and Schiff base cross-links. Significant inhibition by apple phenolics was found as compared to the control treatment, with (-)-epicatechin being the most efficient antioxidant and phloridzin showing the weakest antioxidant effect. The higher concentrations of apple extract showed effective antioxidant activity against protein oxidation in myofibrillar proteins, emphasizing the potential of apple by-products as natural inhibitors of protein oxidation in meat products.

Ascorbate and Apple Phenolics Affect Protein Oxidation in Emulsion-Type Sausages during Storage and in Vitro Digestion.

The effect of sodium ascorbate and apple phenolics on the oxidative stability of emulsion-type sausages during storage and digestion was investigated. Emulsion-type sausages containing 0.05% sodium ascorbate or 3% freeze-dried apple pomace were subjected to chilled illuminated storage and subsequent in vitro digestion. Lipid oxidation was assessed as TBARS, and protein oxidation was evaluated as thiol oxidation, total carbonyls, and γ-glutamic and α-amino adipic semialdehyde. Proteolysis was measured after digestion to evaluate protein digestibility. The results suggest the presence of protein-ascorbate and protein-phenol interactions, which may decrease protein digestibility and may interfere with spectrophotometric methods for measuring oxidation.

Protein oxidation and proteolysis during storage and in vitro digestion of pork and beef patties.

The effect of protein oxidation on proteolysis during meat digestion was investigated following storage and subsequent in vitro digestion of beef and pork patties. Protein oxidation was evaluated as thiol oxidation, total carbonylation, and specific carbonylation (α-amino adipic and γ-glutamic semialdehyde). Furthermore, 4-hydroxyphenylalanine, a hydroxylation product of phenylalanine, was identified and quantified as a new protein oxidation marker. After 7days of chilled illuminated storage (4°C), significant oxidative modifications were quantified and the oxidative degradation was continued during in vitro digestion. The observed effects were more abundant in beef patties. Protein oxidation before digestion resulted in impaired proteolysis during digestion.

Evaluation and validation of the use of a molecularly imprinted polymer coupled to LC-MS for benzylpenicillin determination in meat samples.

This article describes a full analytical method for the clean-up and detection of benzylpenicillin in chicken and beef meat samples using a previously developed molecularly imprinted polymer in a solid-phase extraction cleanup step followed by analysis using LC-MS/MS. The method was validated based on the criteria and the requirements of the European Commission Decision 2002/657/EC. This validation revealed method performance characteristics that meet all the criteria in the Decision with limits of detection for chicken and beef meat samples of 6.2 and 14.4µg/kg, respectively, 8 and 3.5 times lower than the MRL. This method holds strong potential when a specific cleanup of benzylpenicillin is required, because with slight modifications it is also applicable in a milk matrix [1].

Protein thiols undergo reversible and irreversible oxidation during chill storage of ground beef as detected by 4,4'-dithiodipyridine.

Quantification of protein thiols and disulfides in ground beef during storage under high-oxygen atmosphere at 4 °C was performed by thiol detection using 4,4'-dithiodipyridine (4-DPS) before and after disulfide reduction using sodium borohydride. Two independent storage trials were performed, and in trial 1, only reversible thiol oxidation was observed (thiol loss was 30%). In trial 2, irreversible thiol oxidation occurred during the first days of storage, while further loss of thiols was caused by reversible disulfide formation (thiol loss was 33%, of which ca. half was lost because of irreversible oxidation). The results were compared to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cross-linked myosin heavy chain formed by disulfide bonding. Both methods confirmed increasing disulfide formation because of thiol oxidation in meat during storage, but the 4-DPS method showed higher disulfide percentages than the SDS-PAGE method (22.2 ± 0.3% and 8.5 ± 1.2%, respectively). The 4-DPS assay provides an accurate method to evaluate the thiol-disulfide redox state in meat.

Development and evaluation of a molecularly imprinted polymer for the detection and cleanup of benzylpenicillin in milk.

A molecularly imprinted polymer (MIP) was designed for benzylpenicillin via suspension polymerization. The specific absorption of benzylpenicillin to the MIP, applied in a molecularly imprinted solid-phase extraction (MISPE), was compared to the nonspecific binding using a NIP (nonimprinted polymer without a target molecule) in a non-molecularly imprinted solid-phase extraction. This validation was performed successfully in acetonitrile solutions and milk extracts spiked with benzylpenicillin. Significant differences in absorption were observed. In acetonitrile, the recoveries using MISPE (90-95%) were a fraction higher than those in milk extracts (70-80%). The validation revealed the limit of detection and the limit of quantitation for the MISPE application in milk samples to be 0.51 and 1.02 µg/kg, respectively. In addition, comparing the results of the analysis of positive milk samples using MISPE with those using a classic sample preparation step showed a Pearson correlation of 0.989. Finally, cross reactivity tests using other antibiotics showed a certain cross reactivity, but non-ß-lactams were barely bound.