Is lysosomal acid lipase activity associated with the presence and severity of coronary artery disease?

BACKGROUND: Lipid metabolism is considerably complex and there can be many critical steps in atherogenesis. The association between lysosomal acid lipase (LAL) activity and coronary artery disease (CAD) has not been elucidated in detail. We aimed to evaluate the association between LAL activity with the presence and severity of CAD in patients who are seen in daily clinical practice. METHODS: Patients who underwent coronary angiography were divided into groups according to the angiography results. Syntax scores and Gensini scores were calculated. The LAL activity was measured from dried blood spots. RESULTS: Median LAL activity values were similar in all study groups (normal coronary arteries: 0.40 nmol/punch/h; non-obstructive CAD: 0.44 nmol/punch/h; obstructive chronic CAD: 0.40 nmol/punch/h; obstructive acute coronary syndrome: 0.48 nmol/punch/h) and there was no correlation between coronary atherosclerotic burden and LAL activity (correlation coefficients Syntax score and LAL: -0.032; Gensini score and LAL: -0.030). In addition, no relationship between serum lipid levels and LAL activity was detected. CONCLUSION: The presence of CAD and its severity is not associated with the LAL activity in patients encountered in daily clinical practice.

Differential lipase virulence in Malassezia furfur dimorphism isolated from pityriasis versicolor patients and healthy individuals.

BACKGROUND: Malassezia furfur is a member of the human skin microbiomes that can cause various skin diseases. Dimorphism plays a role as the yeast phase predominates during skin colonisation whereas mycelial forms are observed in the scales of patients with pityriasis versicolor (PV). However, due to their condition-dependence for growth, it is difficult to culture M. furfur and this is an additional challenge for studying the pathogenicity of this fungus. OBJECTIVE: To describe different media suitable for culturing Malassezia from the yeast phase into mycelial forms, with a particular focus on nutritional supplements and pH conditions. METHODS: Clinical M. furfur isolates from patients with PV and healthy individuals were used to investigate Malassezia dimorphism as well as the activity and expression of lipase enzymes. RESULTS: Our experimental media were significantly more likely to promote mycelial growth in strains from healthy individuals compared to those from patients with PV. Lipase activity was increased in the mycelial phase cells compared to yeast forms for all strains tested. Assessment of the relative transcriptional expression of lipase within M. furfur revealed that LIP-coding genes were upregulated in mycelium relative to yeast forms for the strains tested. However, the increases in LIP3, LIP5 and LIP6 gene expressions were significantly greater in strains from healthy individuals compared to those from patients with PV. CONCLUSION: Overall, this study validated effective growth conditions to study M. furfur virulence factors and demonstrated that lipase is associated with M. furfur dimorphism.

Monitoring polysorbate hydrolysis in therapeutic proteins using an ultrasensitive extraction-free fatty acid quantitation method.

Polysorbates (PSs) are surfactants commonly added to therapeutic protein drug product formulations to protect proteins from denaturation and aggregation during storage, transportation, and delivery. However, enzymatic hydrolysis of PSs has been recognized as the primary route of PS degradation in monoclonal antibody formulations, resulting in the release of free fatty acids that drive undesired particulate formation. Here, we present a rapid lipase activity assay with optimized incubation conditions for accurate quantitation of free fatty acids without a fatty acid extraction step. This assay can detect low levels of PS degradation (0.000024% PS20 degradation) within 1 day with minimal sample preparation. The levels of released free fatty acids were found to strongly correlate with the degree of PS20 degradation. The case study described herein suggests that this approach can detect low levels of PS20 degradation caused by sub-ppm lipase levels within 1 day, compared with the duration of 14 days needed for PS degradation assays based on two-dimensional liquid chromatography-charge aerosol detection.

Action of Mangifera indica Leaf Extract on Acne-Prone Skin through Sebum Harmonization and Targeting C. acnes.

(1) Background: Preclinical studies report that the ethanolic fraction from Mangifera indica leaves is a potential anti-acne agent. Nevertheless, the biological activity of Mangifera indica leaves has scarcely been investigated, and additional data are needed, especially in a clinical setting, for establishing the actual effectiveness of Mangifera indica extract as an active component of anti-acne therapy. (2) Methods: The evaluation of the biological activity of Mangifera indica extract was carried out through different experimental phases, which comprised in silico, in vitro, ex vivo and clinical evaluations. (3) Results: In silico and in vitro studies allowed us to identify the phytomarkers carrying the activity of seboregulation and acne management. Results showed that Mangifera indica extract reduced lipid production by 40% in sebocytes, and an improvement of the sebum quality was reported after the treatment in analyses performed on sebaceous glands from skin explants. The evaluation of the sebum quantity and quality using triglyceride/free fatty acid analysis conducted on Caucasian volunteers evidenced a strong improvement and a reduction of porphyrins expression. The C. acnes lipase activity from a severe acne phylotype was evaluated in the presence of Mangifera indica, and a reduction by 29% was reported. In addition, the analysis of the skin microbiota documented that Mangifera indica protected the microbiota equilibrium while the placebo induced dysbiosis. (4) Conclusions: Our results showed that Mangifera indica is microbiota friendly and efficient against lipase activity of C. acnes and supports a role for Mangifera indica in the therapeutic strategy for prevention and treatment of acne.

The Effect of Different Growth Stages of Black Chokeberry Fruits on Phytonutrients, Anti-Lipase Activity, and Antioxidant Capacity.

The present study investigated the nutrients, biologically-active compounds, as well as antioxidant and anti-lipase activities of chokeberry fruits across four different stages of development, from the unripe green to mature black forms. The highest content of total phenolics (12.30% dry weight (DW)), including proanthocyanidins (6.83% DW), phenolic acids (6.57% DW), flavanols (0.56% DW), flavonols (0.62% DW), and flavanones (0.10% DW), was observed in unripe fruits. The unripe green fruits were also characterized by the highest content of protein (2.02% DW), ash (4.05% DW), total fiber (39.43% DW), and chlorophylls (75.48 mg/100 g DW). Ripe black fruits were the richest source of total carotenoids (8.53 mg/100 g DW), total anthocyanins (2.64 g/100 g DW), and total sugars (33.84% DW). The phenolic compounds of green fruits were dominated by phenolic acids (above 83% of the total content), the semi-mature fruits by both phenolic acids and anthocyanins (90%), while the mature berries were dominated by anthocyanins (64%). Unripe fruits were the most effective inhibitor of pancreatic lipase in triolein emulsion, scavenger of 2,2'-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) radical cation, and reducer of ferric ion. Biological activities were mainly correlated with total proanthocyanidins and total phenolics. Considering their strong anti-lipase and antioxidant activities, unripe chokeberry fruits may have potential applications in nutraceuticals and functional foods.

Carboxylesterase 1d (Ces1d) does not contribute to cholesteryl ester hydrolysis in the liver.

The liver is the central organ regulating cholesterol synthesis, storage, transport, and elimination. Mouse carboxylesterase 1d (Ces1d) and its human ortholog CES1 have been described to possess lipase activity and play roles in hepatic triacylglycerol metabolism and VLDL assembly. It has been proposed that Ces1d/CES1 might also catalyze cholesteryl ester (CE) hydrolysis in the liver and thus be responsible for the hydrolysis of HDL-derived CE; this could contribute to the final step in the reverse cholesterol transport (RCT) pathway, wherein cholesterol is secreted from the liver into bile and feces, either directly or after conversion to water-soluble bile salts. However, the proposed function of Ces1d/CES1 as a CE hydrolase is controversial. In this study, we interrogated the role hepatic Ces1d plays in cholesterol homeostasis using liver-specific Ces1d-deficient mice. We rationalized that if Ces1d is a major hepatic CE hydrolase, its absence would (1) reduce in vivo RCT flux and (2) provoke liver CE accumulation after a high-cholesterol diet challenge. We found that liver-specific Ces1d-deficient mice did not show any difference in the flux of in vivo HDL-to-feces RCT nor did it cause additional liver CE accumulation after high-fat, high-cholesterol Western-type diet feeding. These findings challenge the importance of Ces1d as a major hepatic CE hydrolase.

In Silico Study, Physicochemical, and In Vitro Lipase Inhibitory Activity of α,ß-Amyrenone Inclusion Complexes with Cyclodextrins.

α,ß-amyrenone (ABAME) is a triterpene derivative with many biological activities; however, its potential pharmacological use is hindered by its low solubility in water. In this context, the present work aimed to develop inclusion complexes (ICs) of ABAME with γ- and ß-cyclodextrins (CD), which were systematically characterized through molecular modeling studies as well as FTIR, XRD, DSC, TGA, and SEM analyses. In vitro analyses of lipase activity were performed to evaluate possible anti-obesity properties. Molecular modeling studies indicated that the CD:ABAME ICs prepared at a 2:1 molar ratio would be more stable to the complexation process than those prepared at a 1:1 molar ratio. The physicochemical characterization showed strong evidence that corroborates with the in silico results, and the formation of ICs with CD was capable of inducing changes in ABAME physicochemical properties. ICs was shown to be a stronger inhibitor of lipase activity than Orlistat and to potentiate the inhibitory effects of ABAME on porcine pancreatic enzymes. In conclusion, a new pharmaceutical preparation with potentially improved physicochemical characteristics and inhibitory activity toward lipases was developed in this study, which could prove to be a promising ingredient for future formulations.

Rapid Identification of Mycoplasma bovis Strains from Bovine Bronchoalveolar Lavage Fluid with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry after Enrichment Procedure.

Mycoplasma bovis is a leading cause of pneumonia in modern calf rearing. Fast identification is essential to ensure appropriate antimicrobial therapy. Therefore, the objective of this study was to develop a protocol to identify M. bovis from bronchoalveolar lavage fluid (BALf) with matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS and to determine the diagnostic accuracy in comparison with other techniques. BALf was obtained from 104 cattle, and the presence of M. bovis was determined in the following three ways: (i) rapid identification of M. bovis with MALDI-TOF MS (RIMM) (BALf was enriched and after 24, 48, and 72 h of incubation and was analyzed using MALDI-TOF MS), (ii) triplex real-time PCR for M. bovis, Mycoplasma bovirhinis, and Mycoplasma dispar, and (iii) 10-day incubation on selective-indicative agar. The diagnostic accuracy of the three tests was determined with Bayesian latent class modeling (BLCM). After 24 h of enrichment, M. bovis was identified with MALDI-TOF MS in 3 out of 104 BALf samples. After 48 and 72 h of enrichment, 32/104 and 38/100 samples, respectively, were M. bovis positive. Lipase-positive Mycoplasma-like colonies were seen in 28 of 104 samples. Real-time PCR resulted in 28/104 positive and 12/104 doubtful results for M. bovis The BLCM showed a sensitivity (Se) and specificity (Sp) of 86.6% (95% credible interval [CI], 69.4% to 97.6%) and 86.4% (CI, 76.1 to 93.8) for RIMM. For real-time PCR, Se was 94.8% (CI, 89.9 to 97.9) and Sp was 88.9% (CI, 78.0 to 97.4). For selective-indicative agar, Se and Sp were 70.5% (CI, 52.1 to 87.1) and 93.9% (CI, 85.9 to 98.4), respectively. These results suggest that rapid identification of M. bovis with MALDI-TOF MS after an enrichment procedure is a promising test for routine diagnostics in veterinary laboratories.

Screening of solvents for favoring hydrolytic activity of Candida antarctica Lipase B.

Lipases are a group of enzymes of considerable significance in organic synthesis, among which Candida antarctica lipase B (CALB) is one of the most widely studied enzymes. The activity of the biocatalyst has been intensively characterized in many organic media, but this paper aimed to compare the effect of 20 different solvents on the activity of CALB in the hydrolysis of p-nitrophenyl laurate. Nonpolar, polar aprotic, and polar protic solvents were used for enzyme pretreatment and then entered the composition of mixed solvents reaction medium. An impact of solvents on solvation processes affecting the catalysis steps, protein denaturation, and changes of its conformation was discussed. Moreover the hydrolytic activity of CALB with partition coefficient (logP) of the solvent used was correlated. It was emphasized that the substrate solubility plays an important role in solvent selection. In the presence of hydrophobic solvents, hydration layer becomes more hydrophobic facilitating the substrate access to the enzyme surface. In turn, polar compounds are good solvents for organic substrates facilitating the penetration of the aqueous layer that surrounds the surface of the enzyme. Two variants proved to be favorable for ester hydrolysis reaction: isooctane or polar solvent such as acetone, tert -butyl methyl ether, tert-butanol or acetonitrile.

A fluorescence-based activity assay for immobilized lipases in non-native media.

A new method for the analysis of lipase activity in the immobilized state is developed. The fluorescence assay aims to quantify the potential of lipases for the application in organic solvents. As lipases are universally immobilized on polymeric carriers for the use in bioorganic synthesis, the assay includes an immobilization step on the walls of polymeric cuvettes. The activity of the immobilized lipase is probed by 4-methylumbelliferyl ester hydrolysis. The activity retention as a function of solvent concentration is used as a measure for the solvent resistance of the enzyme variant. The method is applied to two different lipases, Candida antarctica lipase B (CalB) and Bacillus subtilis lipase A (BSLA) in the presence of the solvents acetonitrile and ethanol. By comparison of the assay results with a commercial biocatalyst consisting of CalB on polymeric carrier (Novozyme 435) it is demonstrated that the assay allows a good prediction of the activity of the respective lipase as immobilisate on polymeric carriers. The assay surpasses the respective analysis in solution in terms of accuracy and precision.

Impact on the nutritional attributes of rice bran following various stabilization procedures.

Rice bran, a valuable byproduct of the rice milling process, has limitations in food industrial applications due to its instability during storage. This review summaries the methodology for stabilization and its impact on the nutritional properties of rice bran. A variety of treatments have been used and these include heat treatment, low-temperature storage, biological and chemical approaches and these will be discussed in terms of their ability to destroy/inhibit enzyme activity and improve storage performance of rice bran. More importantly, changes in the nutritional value of rice bran in terms of vitamins, polyphenols, tocopherols, flavonoids, free fatty acids caused by stabilization of rice bran will also be discussed. This review highlights the importance of appropriate design of processes for stabilization and controlling storage conditions to ensure quality of the rice bran and enhancing levels of phytochemicals in the bran for novel applications in functional foods.

Oroxylum indicum (L.) Kurz extract inhibits adipogenesis and lipase activity in vitro.

BACKGROUND: Oroxylum indicum (L.) Kurz (O. indicum) is found in Thailand. It has been used for the treatment of obesity. This study aimed to investigate the effects of an O. indicum extract (OIE) on the adipogenic and biomolecular change in 3T3-L1 adipocytes. METHODS: Initial studies examined the chemical components of OIE. The cell line 3T3-L1 was used to establish potential toxic effects of OIE during the differentiation of pre-adipocytes to adipocytes. The inhibitory effect of OIE on lipid accumulation in 3T3-L1 cells was investigated. Moreover, the impact of OIE on pancreatic lipase activity was determined. In further experiments, Fourier Transform Infrared (FTIR) was used to monitor and discriminate biomolecular changes caused by the potential anti-adipogenic effect of OIE on 3T3-L1 cells. RESULTS: Chemical screening methods indicated that OIE was composed of flavonoids, alkaloids, steroids, glycosides, and tannins. The percentage viability of 3T3-L1 cells was not significantly decreased after exposure to either 200 or 150 µg/mL of OIE for 2 and 10 days, respectively compared to control cells. The OIE exhibited a dose-dependent reduction of lipid accumulation compared to the control (p < 0.05). The extract also demonstrated a dose-dependent inhibitory effect upon lipase activity compared to the control. The inhibitory effect of the OIE on lipid accumulation in 3T3-L1 cells was also confirmed using FTIR microspectroscopy. The signal intensity and the integrated areas relating to lipids, lipid esters, nucleic acids, glycogen and carbohydrates of the OIE-treated 3T3-L1 adipocytes were significantly lower than the non-treated 3T3-L1 adipocytes (p < 0.05). Principal component analysis (PCA) indicated four distinct clusters for the FTIR spectra of 3T3-L1 adipocytes based on biomolecular changes (lipids, proteins, nucleic acids, and carbohydrates). This observation was confirmed using Unsupervised hierarchical cluster analysis (UHCA). CONCLUSIONS: These novel findings provide evidence that the OIE derived from the fruit pods of the plant is capable of inhibiting lipid and carbohydrate accumulation in adipocytes and also has the potential to inhibit an enzyme associated with fat absorption. The initial observations indicate that OIE may have important properties which in the future may be exploited for the management of the overweight or obese.

UHPLC/HR-ESI-MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti-lipase Activities' Evaluation.

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti-lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and ß-carotene-linoleic acid bleaching assays) while anti-lipase activity was evaluated using 4-methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR-ESI-MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N-caffeoyltyramine, lyciumide A, N-dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.

Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii.

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.

Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility.

Immobilization of lipase on porous monodisperse chitosan microspheres.

Porous monodisperse chitosan microspheres were synthesized for enzyme immobilization. The microspheres were prepared using microchannels and modified with glutaraldehyde. The microspheres had a mean diameter of 495 µm; the polydispersity indices were less than 0.08, and the specific surface area was between 121 and 173 m(2) /g. Candida sp. 1619 lipase was selected as a model lipase. Immobilization conditions such as enzyme loading, glutaraldehyde concentration, and immobilization time were optimized. The temperature, pH, and storage stability of the free and immobilized enzymes were also investigated. The immobilized enzyme had broad-ranging pH and temperature optima as compared with free enzyme. The storage stability of the immobilized enzyme was higher than that of the free enzyme.

Roles of tryptophan residue and disulfide bond in the variable lid region of oxidized polyvinyl alcohol hydrolase.

Oxidized polyvinyl alcohol hydrolase (OPH) catalyzes the cleavage of C-C bond in ß-diketone. It belongs to the α/ß-hydrolase family and contains a unique lid region that covers the active site. The lid is the most variable region when pOPH from Pseudomonas sp. VM15C and sOPH from Sphingopyxis sp. 113P3 are compared. The wild-type enzymes and the pOPH mutants W255A, W255Y and W255F were analyzed for lipase activity by using p-nitrophenyl (pNP) esters as the substrates. The wild-type enzymes showed increased Km and decreased kcat/Km with the acyl chain length, and the mutants showed reduced kcat/Km for pNP acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for pNP butyrate can be a result of product inhibition, as suggested by the complex crystal structures, in which butyric acid, DMSO or PEG occupied the same substrate-binding cleft. The mutant activity was retained with pNP caprylate and pNP laurate as the substrates, reflecting the amphipathic nature of the cleft. Moreover, the disulfide bond formation of Cys257/267 is important for the activity of pOPH, but it is not essential for sOPH, which has a shorter lid structure.

Solid Self-Nanoemulsifying Drug Delivery Systems of Furosemide: In Vivo Proof of Concept for Enhanced Predictable Therapeutic Response.

Liquid self-nano emulsifying drug delivery systems (SNEDDS) of furosemide (FSM) have been explored as a potential solution for enhancing solubility and permeability but are associated with rapid emulsification, spontaneous drug release, and poor in vivo correlation. To overcome the shortcoming, this study aimed to develop liquid and solid self-emulsifying drug delivery systems for FSM, compare formulation dynamics, continue in vivo therapeutic efficacy, and investigate the advantages of solidification. For this purpose, liquid SNEDDS (L-SEDDS-FSM) were formed using oleic acid as an oil, chremophore EL, Tween 80, Tween 20 as a surfactant, and PEG 400 as a co-surfactant containing 53 mg/mL FSM. At the same time, solid SNEDDS (S-SEDDS-FSM) was developed by adsorbing liquid SNEDDS onto microcrystalline cellulose in a 1:1 ratio. Both formulations were evaluated for size, zeta potential, lipase degradation, and drug release. Moreover, in vivo diuretic studies regarding urine volume were carried out in mice to investigate the therapeutic responses of liquid and solid SNEDDS formulations. After dilution, L-SEDDS-FSM showed a mean droplet size of 115 ± 4.5 nm, while S-SEDDS-FSM depicted 116 ± 2.6 nm and zeta potentials of -5.4 ± 0.55 and -6.22 ± 1.2, respectively. S-SEDDS-FSM showed 1.8-fold reduced degradation by lipase enzymes in comparison to L-SEDDS-FSM. S-SEDDS-FSM demonstrated a sustained drug release pattern, releasing 63% of the drug over 180 min, in contrast to L-SEDDS-FSM, exhibiting 90% spontaneous drug release within 30 min. L-SEDDS-FSM exhibited a rapid upsurge in urine output (1550 ± 56 µL) compared to S-SEDDS-FSM, showing gradual urine output (969 ± 29 µL) till the 4th h of the study, providing sustained urine output yet a predictable therapeutic response. The solidification of SNEDDS effectively addresses challenges associated with spontaneous drug release and precipitation observed in liquid SNEDDS, highlighting the potential benefits of solid SNEDDS in improving the therapeutic response of furosemide.

Exploration of the shelf-life difference between chilled beef and pork with similar initial levels of bacterial contamination.

This study compared the shelf-life of beef and pork longissimus lumborum muscles (loins) that had the same initial bacterial loads and were held under the same chilled storage conditions. To identify the underlying pathways, comparisons were conducted from the perspective of the spoilage indicators; protease/lipase activity, and the volatile organic compounds (VOC) generated over 28 d of chilled storage. The initial total viable microbial count (TVC) on Day 0 for both type of meat was 4.3 log10 CFU/g. It was found that the TVC of beef and pork did not differ throughout the total chilled storage period and both ultimately exceeded 7 log10 CFU/g after 28 d. Based on total volatile basic nitrogen (TVB-N) guidelines, pork was spoilt after 21 d of chilled storage and therefore 7 d earlier than beef. Changes in the concentration of VOC spoilage biomarkers, including 1-octen-3-ol, 1-octanol, nonanal, and others, confirmed that pork had a shorter shelf-life than beef. An important reason for the difference in shelf-life between the two types of meat was that pork had a higher protease activity, although the beef had higher levels of total lipase activity. These findings help us understand the differences in the spoilage process of raw meat from different species and explore specific measures to control the spoilage of beef or pork.

Lipase activity of recombinant KmYJR107Wp and KmLIP3p enzymes expressed in Saccharomyces cerevisiae BY4742 from Kluyveromyces marxianus L2029.

Lipases are used in many food, energy, and pharmaceutical processes. Thus, new systems have been sought to synthesize alternative lipases with potential biotechnological applications. Kluyveromyces marxianus is a yeast with recognized lipase activity; at least ten putative lipases/esterases in its genome have been detected, and two of them possess a signal peptide for extracellular secretion. The study of extracellular lipases becomes more relevant since they usually have higher activity rates than intracellular lipases and simpler purification mechanisms. For these reasons, this study aimed to characterize the production and lipase activity of the putative extracellular lipases of the K. marxianus L-2029 strain, encoded in the genes LIP3 and YJR107W. Both genes were heterologously expressed in Saccharomyces cerevisiae BY4742 (yeast strain without extracellular lipase activity) using a pYES2.1/V5-His-TOPO® plasmid. Herein, we show evidence that the strain transformed with the LIP3 gene did not show lipase activity during flask galactose induction. On the other hand, the strain transformed with the YJR107W gene showed a specific activity of 0.397 U/mg, with an optimum temperature of 37 °C and pH 6. For maximum cell production, glucose and yeast extract concentrations were evaluated by a 22 factorial design, followed by the validation of the best concentrations predicted by a statistical model; a 22 factorial design was also carried out to evaluate the concentration of the inducer galactose on the transformed strains, and the intracellular and extracellular lipase specific activities were quantified. Finally, the biomass and lipase production were determined for each strain, which was grown in a stirred tank bioreactor with a working volume of 1.5 L. The specific activities of the transformed strains obtained in the bioreactor were 1.36 U/mg for the LIP3 transformant and 1.25 U/mg for the YJR107W transformant, respectively.