Application of Enzyme Biotechnology in Foods

The choice of appropriate proteases and pretreatment methods significantly influences the preparation of bioactive peptides. This study aimed to investigate the effects of different pretreatment methods on the hydrolytic performance of diverse proteases during the production of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides derived from wheat and their foaming and emulsion properties. Dry heating, aqueous heating, and ultrasound treatment were employed as pretreatments for the protein prior to the enzymatic hydrolysis of wheat gluten. FTIR analysis results indicated that all pretreatment methods altered the secondary structure of the protein; however, the effects of dry heating treatment on the secondary structure content were opposite to those of aqueous heating and ultrasound treatment. Nevertheless, all three methods enhanced the protein solubility and surface hydrophobicity. By using pretreated proteins as substrates, five different types of proteases were employed for DPP-IV inhibitory peptide production. The analysis of the DPP-IV inhibitory activity, degree of hydrolysis, and TCA-soluble peptide content revealed that the specific pretreatments had a promoting or inhibiting effect on DPP-IV inhibitory peptide production depending on the protease used. Furthermore, the pretreatment method and the selected type of protease collectively influenced the foaming and emulsifying properties of the prepared peptides. Full article

Consumers are more than ever in search of novel and exciting beer choices, and brewers are, therefore, continuously experimenting to adapt their product portfolio. One interesting way to naturally incorporate novel flavors and tastes is by using alternative adjuncts, but this is not always an easy and straightforward process. In this study, a 40% unmalted alternative adjunct (einkorn, emmer, spelt, khorasan, quinoa, amaranth, buckwheat, sorghum, teff, and tritordeum) or reference (barley malt, unmalted barley, and unmalted wheat) was added to 60% barley malt, after which three different laboratory mashing processes (Congress mash, Congress mash with pre-gelatinization of the adjunct, and Evans mash) were performed, and their behavior during mashing and the resulting wort characteristics were investigated in detail. Overall, the extraction process of all 10 unmalted alternative adjuncts was not complete for all three laboratory mashing processes, whereby Congress mashing resulted in the highest extract and fastest filtration, whereas Evans mashing resulted in the lowest extract and slowest filtration. Pre-gelatinization of the unmalted was generally only beneficial for adjuncts with high onset starch gelatinization temperatures. This process also inactivated endogenous enzymes in the unmalted adjuncts, which had an adverse effect on the mashing process. Full article

Bee pollen is one of the most valuable apitherapeutic products with high nutritional value. To obtain a higher diversity of compounds, higher bioactivity, and improve the release of nutrients from bee pollen, additional processing of the raw material may be applied: fermentation using microorganisms or hydrolysis using selective enzymes. This research aimed to determine the impact of enzymatic hydrolysis on the antioxidant and antibacterial activities of bee pollen. Bee pollen samples from Sweden, Spain, Netherlands, Italy, Poland, Denmark, Slovakia, Malta, and Lithuania were hydrolyzed using pure enzymes, including lipase, cellulase, protease, and amyloglucosidase, as well as enzyme mixtures such as Viscozyme^® L and Clara-diastase. Total phenolic content, total flavonoid content, and antioxidant activity were analyzed spectrophotometrically. Antibacterial activity against Staphylococcus aureus, Listeria monocytogenes, Staphylococcus enteritidis, and Salmonella typhimurium was evaluated using the agar well diffusion assay. Obtained results revealed a positive effect of enzymatic hydrolysis on biologically active compound content and activity: total phenolic content increased by 1.1 to 2.5 times, total flavonoid content by 1.1 to 3.0 times, radical scavenging activity by 1.1–3.5 times, and antibacterial activity by 1.1 to 3.3 times. K-means clustering analysis grouped samples into 5–9 clusters and was dependent on the measured characteristic used as an input—total phenolic compounds content, total flavonoid content, antioxidant activity, and antibacterial activity against four different bacteria. Chemometrics showed, that the enzyme used for the hydrolysis had a higher impact on clustering results than the geographical origin of the samples. Full article

Food-derived nattokinase has strong thrombolytic activity and few side effects. In the field of medicine, nattokinase has been developed as an adjuvant drug for the treatment of thrombosis, and nattokinase-rich beverages and health foods have also shown great potential in the field of food development. At present, the poor thermostability of nattokinase limits its industrial production and application. In this study, we used several thermostability-prediction algorithms to predict nattokinase from Bacillus mojavensis LY-06 (AprY), and screened two variants S33T and T174V with increased thermostability and fibrinolytic activity. The t_1/2 of S33T and T174V were 8.87-fold and 2.51-fold those of the wild type AprY, respectively, and their enzyme activities were also increased (1.17-fold and 1.28-fold, respectively). Although the thermostability of N218L was increased by 2.7 times, the fibrinolytic activity of N218L was only 73.3% of that of wild type AprY. The multiple-point mutation results showed that S33T-N218L and S33T-T174V-N218L variants lost their activity, and the T174V-N218L variant did not show any significant change in catalytic performance, while S33T-T174V increased its thermostability and activity by 21.3% and 24.8%, respectively. Although the S33T-T174V variant did not show the additive effect of thermostability, it combined the excellent transient thermostability of S33T with the better thrombolytic activity of T174V. Bioinformatics analysis showed that the overall structure of S33T and T174V variants tended to be stable, while the structure of S33T-T174V variant was more flexible. Local structure analysis showed that the increased rigidity of the active center region (positions 64–75) and the key loop region (positions 129–130, 155–163, 187–192, 237–241, and 268–270) determined the increased thermostability of all variants. In addition, the enhanced flexibility of S33T-T174V variant in the Ca1 binding region (positions 1–4, 75–82) and the peripheral region of the catalytic pocket (positions 210–216) may account for the inability to superpose its thermostability. We explored the effective strategy to enhance the thermostability of nattokinase, and the resulting variants have potential industrial production and application. Full article