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Defatting dehulled hemp seeds is a crucial step prior to protein extraction. However, conventional methods rely on flammable solvents, posing significant health, safety, and environmental concerns. Additionally, hemp protein has poor extractability, challenging functionality, and flavor limitations, restricting its broader application in foods. Accordingly, a two-phase natural deep eutectic solvent (NADES)-assisted extraction was evaluated as a solvent-free alternative for co-extracting protein and oil from full-fat hemp flour. In comparison to the reference hemp protein isolate (R-HPI), produced from hexane-defatted flour following conventional alkaline extraction, NADES-extracted hemp protein isolate (N-HPI) had significantly higher protein extraction yield and purity. N-HPI exhibited enhanced surface charge, lower hydrophobicity, and thus higher solubility at an acidic pH compared to R-HPI. N-HPI had a higher abundance of edestin and lower levels of vicilin-like proteins, which contributed to superior gelation compared to R-HPI. N-HPI, compared to R-HPI, contained lower levels of lipid-derived off-flavor compounds, such as aldehydes, alcohols, and ketones. These findings highlighted, for the first time, the potential of a two-phase NADES-assisted extraction as a sustainable alternate and effective process for producing high-quality, functional hemp protein. The development of such a green process is an impetus for broadening the applications of hemp protein in food systems.

The effects of aqueous (AEP) and enzyme-assisted aqueous extraction processes (EAEP) on the biological and nutritional properties of green coffee extracts (protein and antioxidant-rich fraction) were investigated. All extracts exhibited high in vitro protein digestibility (>98%), regardless of the pH and use of enzymes during extraction, probably due to the low molecular weight of coffee proteins. Raising extraction pH from 7.0 to 9.0 resulted in extracts with lower concentrations of caffeine and some phenolic compounds such as chlorogenic and cinnamic acids, as well as catechin and epicatechin. This led to a reduction in the antioxidant activity of the extracts obtained at alkaline pH (AEP - pH 9.0). Overall, higher phenolic and caffeine extractability was achieved at neutral pH (AEP - pH 7.0), with no observed improvement in extraction yields when carbohydrases and/or proteases were employed. Coffee extracts generated by AEP at pH 7.0 exhibited the highest lipase inhibitory activity (66%), primarily attributed to their higher chlorogenic acid concentration. Conversely, EAEP extracts exhibited higher angiotensin-converting enzyme inhibition (up to 85%) compared to AEP extracts (68.5-74.3%). This strong inhibitory activity is likely related to the presence of both phenolic compounds (mainly chlorogenic acid) and smaller peptides. Nevertheless, all extracts exhibited low effectiveness for α-glucosidase inhibition (≤14%) and antimicrobial activity against S. aureus and E. coli . The current research underscores the feasibility of modulating the composition of green coffee extracts using sustainable and scalable AEP and EAEP, paving the way for developing tailored extracts with specific biological properties., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (© 2024 The Authors.)

Intermediate wheatgrass (IWG) is a promising perennial grain explored for mainstream food applications. This study investigated the effects of different germination temperatures (10, 15, and 20 °C) and durations (2, 4, and 6 days) on IWG's volatile and fatty acid (FA) profiles. A method using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was optimized through response surface design to extract the volatile compounds, achieving ideal extraction conditions at 60 °C for 55 min. Multiple headspace extraction (MHE) was used for volatile compound quantification. Fifty-eight compounds were identified and quantified in IWG flour, mainly alcohols, aldehydes, hydrocarbons, terpenes, esters, organic acids, and ketones. The main FAs found were linoleic acid (C18:2), oleic acid (C18:1), palmitic acid (C16:0), and linolenic acid (C18:3). Principal component analysis showed a direct correlation between volatile oxidation products and FA composition. Germination at 15 °C for 6 days led to a reduced presence of aldehydes and alcohols such as nonanal and 1-pentanol. Therefore, optimized germination was successful in reducing the presence of potential off-odor compounds. This study provides valuable insights into the effects of germination on IWG flour, showing a way for its broader use in food applications.

Lipids in human colostrum provide the majority of energy intake and essential fatty acids for developing infants. The fatty acid composition of human colostrum is highly variable and influenced by multiple factors. Human colostrum is a complex sample bringing challenges to fatty acid profiling. This work aimed to optimize the use of ionic liquid (IL) columns and flow-modulated comprehensive two-dimensional gas chromatography coupled to mass spectrometry (FM-GC×GC-MS) for fatty acid profiling in human colostrum. Derivatization strategies were optimized and the elution behavior of fatty acid methyl esters (FAME) on various 1 D column phases (Solgel-WAX, SLB-IL60i, SLB-IL76i, and SLB-IL111i). Derivatization with sodium methoxide yielded a satisfactory recovery rate (90%) at milder conditions and reduced time. The use of IL60 as the 1 D column provided superior separation, good peak shape, and better utilization of elution space. As a proof of concept, the developed method was applied to access the effects of the mode of neonatal delivery (vaginal vs. C-section) on the fatty acid profile of human colostrum samples. The integrated multidimensional gas chromatography strategy improved FAME detection and separation and can be a useful tool for accessing the effects of different factors on the fatty acid profiling of complex samples., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

The integration of green technologies such as microwave- and enzyme-assisted extraction (MEAE) has been shown to improve the extraction efficiency of bioactive compounds while reducing processing time and costs. MEAE using tannase alone (MEAE-Tan), or in combination with cellulase and pectinase (MEAE-Tan-Cel-Pec), was optimized to produce enriched phenolic and antioxidant extracts from olive pomace. The individual and integrated impact of enzyme concentration, temperature, and pomace/water ratio were determined using a central composite rotatable design. Optimal extraction conditions for MEAE-Tan (60 °C, 15 min, 2.34% of enzyme ( w / w ), and 1:15 pomace/water ratio) and MEAE-Tan-Cel-Pec (46 °C, 15 min, 2% of enzymes ( w / w ), in the proportion of 1:1:1, and 1:20 pomace/water ratio) resulted in extracts containing 7110.6 and 2938.25 mg GAE/kg, respectively. The antioxidant activity of the extracts was correlated with phenolic acid release, which was enzyme-dependent, as determined with HPLC-DAD analysis. Enzyme selection had a significant impact on the phenolic profile of extracts, with tannase releasing high concentrations of chlorogenic acid and the combined use of enzymes releasing high concentrations of hydroxytyrosol and chlorogenic and ferulic acids. The novelty of this study relies on the integration and optimization of two green technologies (microwave- and enzyme-assisted extraction) to improve the extraction efficiency of bioactive phenolics from olive pomace while reducing processing time and costs. While these techniques have been evaluated isolated, the benefits of using both processing strategies simultaneously remain largely unexplored. This study demonstrates the effectiveness of the integration and processing optimization of two environmentally friendly technologies as a promising alternative to treat agro-industrial byproducts.

Identification of bioactive peptides is an increasingly important target for food chemists, particularly in consideration of the widespread application of proteolytic enzymes in food processing. Because the characterization of small peptides by LC-MS/MS is challenging, we optimized a dimethyl labeling technique to facilitate small peptide identification, using almond proteins as a model. The method was validated by comparing the MS/MS spectra of standards and almond-derived peptides in their nonderivatized and derivatized forms. Signal enhancement of a 1 ions was proved to effectively aid in the full-length sequencing of small peptides. We further validated this method using two industrially-relevant protein-rich extracts from almond flour: 1737 medium-sized peptides (5-39 amino acids) and 843 small peptides (2-4 amino acids) were identified. The use of an online bioactive peptide database, complemented by the existing literature, allowed the discovery of 208 small bioactive peptides, whereas for medium-sized peptides, only one was reported being bioactive., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Reverse-phase solid-phase extraction (SPE) is regularly used for separating and purifying food-derived oligosaccharides and peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, the diversity in physicochemical properties of peptides may prevent the complete separation of the two types of analytes. Peptides present in the oligosaccharide fraction not only interfere with glycomics analysis but also escape peptidomics analysis. This work evaluated different SPE approaches for improving LC-MS/MS analysis of both oligosaccharides and peptides through testing on peptide standards and a food sample of commercial interest (proteolyzed almond extract). Compared with conventional reverse-phase SPE, mixed-mode SPE (reverse-phase/strong cation exchange) was more effective in retaining small/hydrophilic peptides and capturing them in the high-organic fraction and thus allowed the identification of more oligosaccharides and dipeptides in the proteolyzed almond extract, with satisfactory MS/MS confirmation. Overall, mixed-mode SPE emerged as the ideal method for simultaneously improving the identification of food-derived oligosaccharides and small peptides using LC-MS/MS analysis.

The effects of enzymatic extraction strategies on extractability, bioconversion, and bioaccessibility of biologically active isoflavone aglycones, total phenolic content, and antioxidant activity of aqueous extracts from full-fat soy flour were evaluated. Protease, tannase, and cellulase enzymes were used individually or in combination. Except for the protease treatment, all enzymatic treatments increased the extraction of biologically active isoflavones (daidzein and genistein) compared with the control. The use of a mixture of protease, tannase, and cellulase resulted in increased extractability and/or bioconversion of aglycones from soy flour, indicating a synergistic effect amongst the enzymes. Daidzein and genistein concentrations increased from 29.0 to 158.2 μg/g and from 27.0 to 156.5 μg/g (compared to the control), respectively. Furthermore, enzymatic extraction followed by in vitro gastrointestinal digestion significantly increased the bioaccessibility of isoflavone aglycones, total phenolic content (by 22-45%), and antioxidant activity (by 15-22%) of the extracts. These results demonstrate that enzyme selection is an efficient strategy to maximize the extraction, bioconversion, and bioaccessibility of bioactive isoflavones from soy flour, which could contribute to health benefits associated with the consumption of soy-rich products., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The oxidation of polyunsaturated fatty acids produces bioactive primary oxidation products known as oxylipins. In many biological matrices, the majority of oxylipins are bound (i.e. esterified), and a relatively small proportion (<10%) exists in the free form. The present study tested whether this extends to bovine milk following method evaluation of various extraction and base hydrolysis protocols for measuring bound oxylipins. Free (unbound) oxylipins were also measured. Folch extraction followed by sodium carbonate hydrolysis in the presence of methanol containing 0.1% of acetic acid and 0.1% of butylated hydroxytoluene resulted in greater oxylipin concentrations and better surrogate standard recoveries compared to other methods that did not involve Folch extraction or the addition of methanol with hydrolysis base. Sodium hydroxide was better than sodium carbonate in hydrolyzing bound oxylipins under the same conditions. Milk analysis of oxylipins with mass-spectrometry following Folch extraction and sodium hydroxide hydrolysis revealed that 95% of oxylipins in bovine milk were esterified. Most of the detected oxylipins were derived from linoleic acid, which accounted for 92 and 88% of oxylipins in the free and esterified pools, respectively. These results demonstrate that the majority of bovine milk oxylipins are bound, and that linoleic-acid derived metabolites are the most abundant oxylipin species in free and bound lipid pools. Additional studies are needed to understand the role of different oxylipin pools in both calf and human nutrition. PRACTICAL APPLICATION: A method involving Folch lipid extraction and sodium hydroxide hydrolysis was validated for esterified oxylipin measurements in bovine milk. Application of the method revealed that the majority (∼95%) of oxylipins in bovine milk were bound. Linoleic-acid derived oxylipins were the most abundant species in both bound and free milk fractions (88-92%). The results highlight the presence of a new pool of oxidized lipids in milk, potentially involved in modifying its sensory and nutritional properties., (© 2021 Institute of Food Technologists®.)

Soybean is one of the most important commodities in the world, being applied in feed crops and food, pharmaceutical industries in different ways. Soy is rich in isoflavones that in aglycone forms have exhibited significant anti-obesity and anti-lipogenic effects. Obesity is a global problem as several diseases have been related to this worldwide epidemic. The aim of this work was to verify the effect of free and immobilized β-glucosidase, testing Lentikats, and sol-gel as carriers. Moreover, we wanted to examine if the different types of hydrolysis would generate extracts with distinct biological activity concerning lipid accumulation, PPAR-α regulation, and TNF-α, IL-6, and IL-10 concentrations using in vitro assays. Our results show that all formulations of β-glucosidase could hydrolyze soy isoflavones. Thus, after 24 h of incubation, daidzein content increased 2.6-, 10.8-, and 12.2-fold; and genistein content increased 11.7, 11.4, and 11.4 times with the use of free enzyme, Lentikats®, and sol-gel immobilized enzyme, respectively. Moreover, both methodologies for enzyme immobilization led to promising forms of biocatalysts for application in the production of soy extracts rich in isoflavones aglycones, which are expected to bring about health benefits. A mild lipogenic effect was observed for some concentrations of extracts, as well as a slight inhibition in PPAR-α expression, although no significant differences were noticeable in the cytokines TNF-α, IL-10, and IL-6 as compared with the control.

The almond cake is a protein-rich residue generated by the mechanical expression of the almond oil. The effects of the aqueous (AEP) and enzyme-assisted aqueous extraction processes (EAEP) on the biological properties of the almond cake protein were evaluated. Total phenolic content (TPC), antioxidant capacity, inhibitory effects against crucial enzymes related to metabolic syndrome, antimicrobial potential, and in vitro protein digestibility profile were assessed. EAEP provided the best results for antioxidant capacity by both ORAC (397.2 µmol TE per g) and ABTS (650.5 µmol TE per g) methods and also showed a high (~ 98%) potential for α-glucosidase inhibition. The AEP resulted in protein extracts with the highest lipase inhibition (~ 70%) in a dose-dependent way. Enzymatic kinetic analyses revealed that EAEP generated uncompetitive inhibitors against α-glucosidase, while EAEP, AEP, and HEX-AEP (used as control) generated the same kind of inhibitors against lipase. No protein extract was effective against any of the bacteria strains tested at antimicrobial assays. An in silico theoretical hydrolysis of amandin subunits corroborated with the results found for antioxidant capacity, enzyme inhibitory experiments, and antimicrobial activity. Digestibility results indicated that the digestive proteases used were efficient in hydrolyzing almond proteins, regardless of the extraction applied and that HEX-AEP presented the highest digestibility (85%). In summary, EAEP and AEP skim proteins have the potential to be used as a nutraceutical ingredient. The biological properties observed in these extracts could help mitigate the development of metabolic syndrome where EAEP and AEP skim proteins could be potentially used as a prophylactic therapy for diabetes and obesity, respectively.

The effects of industrial heat treatments of raw bovine milk subjected to Batch Pasteurization (BP), High Temperature Short Time (HTST) and Ultra High Temperature (UHT) on the formation of primary (hydroperoxide content and oxylipins) and secondary lipid oxidation products (thiobarbituric acid reactive species -TBARS) were evaluated. Total fatty acid content, percent of free fatty acids (FFA), and total antioxidant capacity (TAC) were also measured. Except for a 30% reduction in capric acid (C10:0) after UHT compared to BP, no significant differences in total fatty acid concentrations were detected amongst the heat treatments. Compared to raw bovine milk, no statistically significant effects of heat treatment were observed on percent FFA (0.29-0.31%), hydroperoxide concentration (0.0558-0.0624 mmol L -1 ), and TBARS values (13.4-18.9 µg MDA kg -1 ). HTST and UHT led to significant reductions (50-65%) in linoleic and alpha-linolenic acid oxidized metabolites compared with raw milk and batch pasteurized milk. Compared to raw milk (2943.7 μmol of TEAC L -1 ), TAC was significantly reduced by all heat treatments (2245 - 2393 μmol of TEAC L -1 ), although no statistically significant differences were observed amongst the treatments. The results demonstrate that heat processing reduces milk oxylipin content and antioxidant capacity and that oxylipin and TAC measurements provide a new sensitive approach to assess the impact of milk processing on lipid oxidation. The nutritional, shelf life and sensory implications of reduced oxylipins in HTST and UHT processed bovine milk merit further investigation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study describes the production of native l-asparaginases by submerged fermentation from Aspergillus strains and provides the biochemical characterization, kinetic and thermodynamic parameters of the three ones that stood out for high l-asparaginase production. For comparison, the commercial fungal l-asparaginase was also studied. Both commercial and l-asparaginase from Aspergillus oryzae CCT 3940 showed optimum activity and stability in the pH range from 5 to 8 and the asparaginase from Aspergillus niger LBA 02 was stable in a more alkaline pH range. About the kinetic parameters, the denaturation constant increased with the heating temperature for all l-asparaginases, indicating that the l-asparaginase activity decreased at higher temperatures, especially above 60 °C. Moreover, l-asparaginase from A. oryzae CCT 3940 remained stable after 60 min at 50 °C. None of the l-asparaginases were inhibited by high NaCl concentrations, which are highly desirable for food industry application. The catalytic activities of all the l-asparaginases were enhanced by the presence of Mn 2+ and inhibited by p-chloromercuribenzoate and iodoacetamide. The l-asparaginase from the Aspergillus strains and the commercial enzyme had similar K m when l-asparagine was used as substrate. None of the l-asparaginases, except the l-asparaginase from A. niger LBA 02, could hydrolyze the substrate l-glutamine, which is of interest for medical proposes, since the glutaminase activity is usually related to adverse reaction during the leukemia treatment. This study showed that these new three non-recombinant l-asparaginases studied have potential application in the food and pharmaceutical industries, especially due to their good thermostability., Competing Interests: Conflict of interestAll authors declare that they have no conflict of interest.

Microextractions have become an attractive class of techniques for metabolomics. The most popular technique is solid-phase microextraction that revolutionized the field of modern sample preparation in the early nineties. Ever since this milestone, microextractions have taken on many principles and formats comprising droplets, fibers, membranes, needles, and blades. Sampling devices may be customized to impart exhaustive or equilibrium-based characteristics to the extraction method. Equilibrium-based approaches may rely on additional methods for calibration, such as diffusion-based or on-fiber kinetic calibration to improve bioanalysis. In addition, microextraction-based methods may enable minimally invasive sampling protocols and measure the average free concentration of analytes in heterogeneous multiphasic biological systems. On-fiber derivatization has evidenced new opportunities for targeted and untargeted analysis in metabolomics. All these advantages have highlighted the potential of microextraction techniques for in vivo and on-site sampling and sample preparation, while many opportunities are still available for laboratory protocols. In this review, we outline and discuss some of the most recent applications using microextractions techniques for comprehensive two-dimensional gas chromatography-based metabolomics, including potential research opportunities., (Copyright © 2018 Elsevier B.V. All rights reserved.)