Extraction, Purification, and Characterization of Olive (Olea europaea L., cv. Chemlal) Polyphenol Oxidase.

Among fruits susceptible to enzymatic browning, olive polyphenol oxidase (OePPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated OePPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. OePPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, OePPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six OePPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature OePPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).

Deciphering the Microbiota and Volatile Profiles of Algerian Smen, a Traditional Fermented Butter.

In Algeria, Smen is a fermented butter produced in households using empirical methods. Smen fermentation is driven by autochthonous microorganisms; it improves butter shelf-life and yields highly fragrant products used as ingredients in traditional dishes as well as in traditional medicine. The present study is aimed at investigating microbial diversity and dynamics during Algerian Smen fermentation using both culture-dependent and culture-independent approaches, as well as by monitoring volatile organic compound production. To reach this goal, fifteen Smen samples (final products) produced in households from different regions in Algeria were collected and analyzed. In addition, microbial and volatile compound dynamics at the different stages of Smen manufacturing were investigated for one Smen preparation. The results showed that Smen is a microbiologically safe product, as all hygiene and safety criteria were respected. The dominant microorganisms identified by both techniques were LAB and yeasts. Lactococcus spp. and Streptococcus thermophilus were the main bacterial species involved in spontaneous raw milk fermentation preceding butter-making, while lactobacilli and enterococci were the only bacteria found to be viable during Smen maturation. Regarding fungal diversity, yeast species were only recovered from two mature Smen samples by culturing, while different species (e.g., Geotrichum candidum, Moniliella sp.) were identified in all samples by the culture-independent approach. Using microbial analysis of a single batch, many of these were found viable during manufacturing. Concerning the volatile profiles, they were highly diverse and characterized by a high prevalence of short chain fatty acids, methylketones, and esters. Correlation analysis between microbial diversity and volatile profiles showed that several yeast (Moniliella sp., K. marxianus) and LAB (e.g., Lactococcus spp., S. thermophilus) species were strongly correlated with one or more volatile organic compound families, including several ethyl esters and methyl ketones that can be linked to pleasant, sweetly floral, fruity, buttery, and creamy odors. This study clearly identified key microorganisms involved in Smen fermentation and maturation that could be used in the future for better fermentation control and improvement of quality attributes.

Polyphenol oxidase and enzymatic browning in apricot (Prunus armeniaca L.): Effect on phenolic composition and deduction of main substrates.

In this study, we investigate the effect of enzymatic browning on the phenolic composition of apricot in vivo and in vitro. The in vitro browning was caused by the recombinant latent apricot polyphenol oxidase (L-PaPPO). Successful heterologous expression of PaPPO in Escherichia coli yielded substantial amounts of enzyme containing both copper ions in the catalytic active site. The expressed L-PaPPO was characterized with regard to its molecular mass (56531.3 Da), pH optimum (7.0), activation by SDS, and enzyme kinetics. LC-MS/MS was used to compare the phenolic profiles of brown and non-brown apricots. The browning reactions did significantly decrease total phenolics and antioxidant capacity (measured with DPPH and CUPRAC assays). Catechin, epicatechin, and B-type procyanidins were the individual phenolics most affected by browning, followed by chlorogenic and neochlorogenic acid. These phenolics are most likely the main endogenous substrates of L-PaPPO, as they were oxidized much faster than the other identified phenolics.

Effects of storage on the oxidative stability of acorn oils extracted from three different Quercus species.

BACKGROUND: Acorn fruit and its components and by-products are receiving renewed interest due to their nutritional and phytochemical features. In particular, the oil extracted from acorns is recognized for having high nutritional quality and for being rich in bioactive compounds. Despite the growing interest, few papers are available that consider the evolution of acorn-oil characteristics during storage. Our aim was to investigate the storage-related changes in acorn oils extracted from three Quercus species grown in Algeria (Q. ilex, Q. suber, and Q. coccifera) 180 days after production, with a focus on polar and volatile compounds, not yet investigated. Basic quality parameters, phenolic content, antioxidant activity and induction time were also monitored. RESULTS: The oxidation markers (peroxide value and UV absorptions) increased during storage, whereas antioxidants decreased. A distinctive volatile profile was observed at the time of production, which underwent changes during storage. Polar compounds increased, whereas induction time decreased. The oil extracted from Quercus suber L. was the most affected by storage time. CONCLUSION: Floral and fruity volatile compounds detected in the oils' headspace could explain the pleasant flavor of acorn oils reported by other authors. As with other vegetable oils, storage depletes both volatiles and antioxidants and produces oxidation compounds, such as oxidized triacylglycerols. However, the acorn oils that were studied were quite stable under storage in the dark at room temperature for 6 months. © 2020 Society of Chemical Industry.

Inhibition of apricot polyphenol oxidase by combinations of plant proteases and ascorbic acid.

The present research investigates the long term inhibition of enzymatic browning by inactivating the polyphenol oxidase (PPO) of apricot, using combinations of plant proteases and ascorbic acid (AA). The selected proteases were able to inactivate PPO at pH 4.5, with the degree of inactivation proportional to incubation time and protease concentration. Papain was the most effective protease, with 50 µg completely inactivating PPO in less than one hour. AA prevented browning reactions that occur before or during PPO inactivation by protease. The combinations of AA/proteases were highly effective in vitro, where 2 mM AA/500 µg proteases inhibited PPO activity completely over 24 h. The combination of AA/proteases was also effective in vivo, as treated apricot purees preserved their color (p < 0.0001, compared to untreated samples after 10 days of storage). The results demonstrate that AA/proteases combinations constitute a promising practical anti-browning method with feasible application in the food industry.

Effect of acorn flour on the physico-chemical and sensory properties of biscuits.

The aim of the work was to explore the feasibility of using acorn flour as a novel and healthy ingredient in biscuits. The physico-chemical characteristics of acorn flour obtained from three different Quercus species were compared. Acorns of Quercus coccifera L. were the most antioxidant and were therefore used for preparing biscuits at two levels of addition, 30 and 60 g 100 g-1 on wheat flour basis. The physico-chemical and sensory characteristics of the obtained biscuits were then assessed. Acorn-added biscuits showed significantly (p < 0.05) higher content of phenolics, antioxidant activity and oxidative stability than control biscuits, prepared without acorn flour. These features improved as the level of acorn flour increased. As for appearance, the acorn-added biscuits were darker, larger, more voluminous and more friable than control biscuits. Higher levels of fermentative alcohols and esters, as well as Maillard reaction volatile compounds (particularly furans), were observed in the acorn-added biscuits.

Antioxidant activity, tocopherols and polyphenols of acornoil obtained from Quercus species grown in Algeria.

Among the unusual vegetable fats, acorn oil has interesting nutritional and functional properties. Few studies are present in literature on acorn oil characterization and antioxidant activity evaluation while no studies are present regarding the analysis of the phenolic profile. The present study aims at investigating the content of hydrophilic and lipophilic phenolic compounds and pigments, as well as antioxidant properties and quality of the oil extracted from three Quercus species grown in Algeria. Oil yield, expressed as dry weight, was in the range 7.05-8.40%. Tocopherols contents were remarkable for the three species (539-676 mg kg-1), with (ß + γ)-tocopherols being the most abundant. Oils had also significant amounts of carotenoids (42-66 mg kg-1) and similar polyphenol patterns, but with a wide quantitative variability between species (195-436 mg kg-1). Twenty phenolic compounds were detected, 12 of which were tentatively identified. All of them were hydrolysable tannins derivatives (gallotannin or ellagitannin). Oils methanolic extracts had remarkable antioxidant activity; up to 3.34 and 3.79 µmol TE g-1 oil (DPPH and ABTS test, respectively). Quercus oil is a potential source of natural antioxidants, and it could be used as a new functional oil.

Optimization of Conditions for Extraction of Polyphenols and the Determination of the Impact of Cooking on Total Polyphenolic, Antioxidant, and Anticholinesterase Activities of Potato.

In this work we optimized the cooking and extraction conditions for obtaining high yields of total polyphenols from potato and studied the effect of three domestic methods of cooking on total phenols, antioxidant activity, and anticholinesterase activities. The optimization of the experiment was carried out by the experimental designs. The extraction of the polyphenols was carried out by maceration and ultrasonication. Determination of the polyphenols was performed by using the Folin-Ciocalteau reagent method. The antioxidant activity was evaluated by three methods: 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and CUPRAC(Cupric reducing antioxidant capacity), the anticholinesterase activity was evaluated by the method of Elmann. The optimum of total phenolic obtained was: 4.668 × 104, 1.406 × 104, 3357.009, 16,208.99 µg Gallic Acid Equivalent (GAE)/g of dry extract for crude potato, steamed potatoes, in boiling water, and by microwave, respectively. The three modes of cooking cause a decrease in the total polyphenol contents, antioxidant and anticholinesterase activities.

Purification and Characterization of Latent Polyphenol Oxidase from Apricot (Prunus armeniaca L.).

Polyphenol oxidase from apricot (Prunus armeniaca) (PaPPO) was purified in its latent form (L-PaPPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-PaPPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO4 and low concentrations (≤ 2 mM) of SDS. PaPPO has its pH and temperature optimum at pH 4.5 and 45 °C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (KM = 2.0 mM) and chlorogenic acid (KM = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-PaPPO). The mass of A-PaPPO was determined by mass spectrometry as 37 455.6 Da (Asp102 → Leu429). Both L-PaPPO and A-PaPPO were identified as polyphenol oxidase corresponding to the known PaPPO sequence (UniProt O81103 ) by means of peptide mass fingerprinting.

Olive mill wastewater microconstituents composition according to olive variety and extraction process.

Olive oil production yields a considerable amount of wastewater, a powerful pollutant that is currently discarded but could be considered as a potential source of valuable natural products due to its content in phenolic compounds and other natural antioxidants. The aim of this work was to explore the variability in olive mill wastewater composition from Algerian olive oil mills considering extraction processes (traditional discontinuous press vs 3-phases centrifugal system) and olive varieties (Azerraj, Sigoise, Chemlal). Whereas pH, dry or organic matter content didn't vary, there was a significant difference in ash content according to extraction process and olive variety. Carotenoid content was 2.2-fold higher with 3-phases than with press systems whereas tocopherol content was not significantly different. Among the phenolic compounds quantified, tyrosol was usually the most abundant whereas oleuropein concentrations were highly variable. Differences in phenolic compound concentrations were more pronounced between olive varieties than between processes.