Dose-related effects of early-life intake of sn-2 palmitate, a specific positionally distributed human milk fatty acid, on the composition and metabolism of the intestinal microbiota.

sn2 Palmitate in human milk plays an important role in the physiological health of infants by reducing mineral loss, improving stool hardness, and relieving constipation. Also, sn-2 palmitate modulates intestinal microbiota. However, it remains unclear whether the effects of sn-2 palmitate on infant gut microbiota are dose-dependent. In this study, we investigated the effects of low, medium, and high doses (600, 1,800, and 5,400 mg/kg body weight, respectively) of sn-2 palmitate on the structure, composition, and metabolic function of intestinal microbes in mice. Our results showed that high doses of sn-2 palmitate significantly modulated α- and ß-diversity of the intestinal microbiota. The relative abundance of Lachnospiraceae_NK4A136_group decreased with increasing doses of sn-2 palmitate. In contrast, the abundances of Bacteroidetes phylum, Bacteroides, uncultured_Lachnospiraceae, and uncultured_Muribaculaceae were positively correlated with sn-2 palmitate doses. The number of genes predicted encoding autophagy-yeast, phospholipase D signaling pathway, and pentose and glucuronate interconversion metabolic functions of intestinal microbiota increased with increasing doses of sn-2 palmitate. In addition, low and medium doses of sn-2 palmitate significantly upregulated the arginine and proline metabolic pathways, and high doses of sn-2 palmitate significantly increased purine metabolism. Our results revealed that the effects of sn-2 palmitate intake early in life on the composition and metabolism of the intestinal microbiota of mice showed dose-related differences. The study is expected to provide a scientific basis for the development of infant formulas.

Phytotoxic and antioxidant effects of dichloromethane fraction of Smilax Brasiliensis Sprengel.

Dichloromethane (DCM) fraction and sub-fractions obtained from Smilax brasiliensis leaves were examined in order to determine their phytotoxic and antioxidant effects. The dichloromethane fraction was submitted to a preparative layer chromatography leading to seven sub-fractions (DCM1-DCM7). Gas chromatography-mass spectrometry (GC-MS) was performed on the dichloromethane sub-fractions. The DCM sub-fractions presented phytotoxic potential; at a concentration of 125 µg per plate, DCM6 and DCM4 showed the strongest results on Lactuca sativa and Allium cepa, respectively. The DCM fraction and DCM4 sub-fraction were more effective than 2,6-di-tert-butyl-4-methylphenol (BHT) at scavenging the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Analysis by GC-MS showed the presence of methyl palmitate (33.05%) in DCM4 and methyl palmitate (17.29%) and methyl oleate (50.96%) in DCM6, suggesting that the activities exhibited by the sub-fractions may be attributed, at least partially, to these major compounds. These results indicate that the DCM sub-fractions of S. brasiliensis could be used as natural herbicides and antioxidants.

Evaluation of the bioaccessibility of carotenoid esters from Lycium barbarum L. in nano-emulsions: A kinetic approach.

Lycium barbarum L., known as goji berry, is a rich source of carotenoid esters, which are mainly composed of zeaxanthin dipalmitate (ZDP), lutein palmitate (LP), ß-cryptoxanthin palmitate (ß-CP), zeaxanthin palmitate (ZP), zeaxanthin myristate palmitate (ZMP), and zeaxanthin palmitate stearate (ZPS). Oil-in-water nano-emulsions containing carotenoid esters from L. barbarum L. with olive oil (ON) and soybean oil (SN) were prepared to investigate the liberation and bioaccessibility (BA) of in vitro digestion. The particle sizes of ON and SN were approximately 160 nm stabilized with sucrose esters and monoacylglyceride as emulsifiers. ON presented an equal liberation of each carotenoid ester as SN, except that LP had a high value. Incorporation of carotenoid esters into the micelle were evaluated using a fractional conversion model, containing two phases, namely, a rapid growth rate for the first phase, and then reaching a plateau for the second phase. The kinetic rate was related to the particle size, oil type and carotenoid ester nature. BA at plateau values for ZDP and ZPS were higher than that of the four other carotenoid esters in SN. Considering the great improvement of the liberation and BA, the excipient nano-emulsion prepared in this study is a good delivery system for carotenoid esters from goji berry.

Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis

Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p-nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p<0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well.

Expression of the Arabidopsis WRINKLED 1 transcription factor leads to higher accumulation of palmitate in soybean seed.

Soybean (Glycine max [L.] Merr.) is a commodity crop highly valued for its protein and oil content. The high percentage of polyunsaturated fatty acids in soybean oil results in low oxidative stability, which is a key parameter for usage in baking, high temperature frying applications, and affects shelf life of packaged products containing soybean oil. Introduction of a seed-specific expression cassette carrying the Arabidopsis transcription factor WRINKLED1 (AtWRI1) into soybean, led to seed oil with levels of palmitate up to approximately 20%. Stacking of the AtWRI1 transgenic allele with a transgenic locus harbouring the mangosteen steroyl-ACP thioesterase (GmFatA) resulted in oil with total saturates up to 30%. The creation of a triple stack in soybean, wherein the AtWRI1 and GmFatA alleles were combined with a FAD2-1 silencing allele led to the synthesis of an oil with 28% saturates and approximately 60% oleate. Constructs were then assembled that carry a dual FAD2-1 silencing element/GmFatA expression cassette, alone or combined with an AtWRI1 cassette. These plasmids are designated pPTN1289 and pPTN1301, respectively. Transgenic events carrying the T-DNA of pPTN1289 displayed an oil with stearate levels between 18% and 25%, and oleate in the upper 60%, with reduced palmitate (<5%). While soybean events harboring transgenic alleles of pPTN1301 had similar levels of stearic and oleate levels as that of the pPTRN1289 events, but with levels of palmitate closer to wild type. The modified fatty acid composition results in an oil with higher oxidative stability, and functionality attributes for end use in baking applications.

The genome and phenome of the green alga Chloroidium sp. UTEX 3007 reveal adaptive traits for desert acclimatization.

To investigate the phenomic and genomic traits that allow green algae to survive in deserts, we characterized a ubiquitous species, Chloroidium sp. UTEX 3007, which we isolated from multiple locations in the United Arab Emirates (UAE). Metabolomic analyses of Chloroidium sp. UTEX 3007 indicated that the alga accumulates a broad range of carbon sources, including several desiccation tolerance-promoting sugars and unusually large stores of palmitate. Growth assays revealed capacities to grow in salinities from zero to 60 g/L and to grow heterotrophically on >40 distinct carbon sources. Assembly and annotation of genomic reads yielded a 52.5 Mbp genome with 8153 functionally annotated genes. Comparison with other sequenced green algae revealed unique protein families involved in osmotic stress tolerance and saccharide metabolism that support phenomic studies. Our results reveal the robust and flexible biology utilized by a green alga to successfully inhabit a desert coastline.

Ultrastructural deposition forms and bioaccessibility of carotenoids and carotenoid esters from goji berries (Lycium barbarum L.).

Goji berries (Lycium barbarum L.) have been known to contain strikingly high levels of zeaxanthin, while the physical deposition form and bioaccessibility of the latter was yet unknown. In the present study, we associated ripening-induced modifications in the profile of carotenoids with fundamental changes of the deposition state of carotenoids in goji berries. Unripe fruit contained common chloroplast-specific carotenoids being protein-bound within chloroplastidal thylakoids. The subsequent ripening-induced transformation of chloroplasts to tubular chromoplasts was accompanied by an accumulation of up to 36mg/100g FW zeaxanthin dipalmitate and further minor xanthophyll esters, prevailing in a presumably liquid-crystalline state within the nano-scaled chromoplast tubules. The in vitro digestion unraveled the enhanced liberation and bioaccessibility of zeaxanthin from these tubular aggregates in goji berries as compared to protein-complexed lutein from spinach. Goji berries therefore might represent a more potent source of macular pigments than green leafy vegetables like spinach.

Enzymatic lipophilization of epicatechin with free fatty acids and its effect on antioxidative capacity in crude camellia seed oil.

BACKGROUND: Crude camellia seed oil is rich in free fatty acids, which must be removed to produce an oil of acceptable quality. In the present study, we reduced the free fatty acid content of crude camellia seed oil by lipophilization of epicatechin with these free fatty acids in the presence of Candida antarctica lipase B (Novozym 435), and this may enhance the oxidative stability of the oil at the same time. RESULTS: The acid value of crude camellia seed oil reduced from 3.7 to 2.5 mgKOH g-1 after lipophilization. Gas chomatography-mass spectrometry analysis revealed that epicatechin oleate and epicatechin palmitate were synthesized in the lipophilized oil. The peroxide, p-anisidine, and total oxidation values during heating of the lipophilized oil were much lower than that of the crude oil and commercially available camellia seed oil, suggesting that lipophilized epicatechin derivatives could help enhance the oxidative stability of edible oil. CONCLUSION: The enzymatic process to lipophilize epicatechin with the free fatty acids in crude camellia seed oil described in the present study could decrease the acid value to meet the quality standards for commercial camellia seed oil and, at the same time, obtain a new edible camellia seed oil product with good oxidative stability. © 2016 Society of Chemical Industry.

Curation of the Mammalian Palmitoylome Indicates a Pivotal Role for Palmitoylation in Diseases and Disorders of the Nervous System and Cancers.

Palmitoylation involves the reversible posttranslational addition of palmitate to cysteines and promotes membrane binding and subcellular localization. Recent advancements in the detection and identification of palmitoylated proteins have led to multiple palmitoylation proteomics studies but these datasets are contained within large supplemental tables, making downstream analysis and data mining time-consuming and difficult. Consequently, we curated the data from 15 palmitoylation proteomics studies into one compendium containing 1,838 genes encoding palmitoylated proteins; representing approximately 10% of the genome. Enrichment analysis revealed highly significant enrichments for Gene Ontology biological processes, pathway maps, and process networks related to the nervous system. Strikingly, 41% of synaptic genes encode a palmitoylated protein in the compendium. The top disease associations included cancers and diseases and disorders of the nervous system, with Schizophrenia, HD, and pancreatic ductal carcinoma among the top five, suggesting that aberrant palmitoylation may play a pivotal role in the balance of cell death and survival. This compendium provides a much-needed resource for cell biologists and the palmitoylation field, providing new perspectives for cancer and neurodegeneration.

Efficient Separation and Analysis of Triacylglycerols: Quantitation of ß-Palmitate (OPO) in Oils and Infant Formulas.

A high-efficiency, convenient, and reliable method for the separation of structurally similar triacylglycerols is detailed and applied in the quantitative analysis of 1,3-dioleoyl-2-palmitoylglycerol (OPO) in infant formulas and OPO oils. OPO is an important lipid component in "humanized" infant formula. A fast preparative isolation of an OPO-containing fraction from the crude complex mixture, by nonaqueous reversed phase HPLC, followed by Ag(+)-HPLC with detection at 205 nm allowed fine separation and detection of the desired fraction. OPO was quantitated independently of its regioisomer 1,2-dioleoyl-3-palmitoylglycerol (OOP) and isomers of stearoyl-linoleoyl-palmitoyl glycerol that might be present in infant formulas. For samples with low OPO content, an evaporative light-scattering detector (ELSD) was more preferable than UV detection, with a calculated LOD of 0.1 µg of OPO injected and LOQ of 0.3 µg. The method, which showed high reproducibility (RSD < 5%), was suitable for both high OPO content oils and low OPO products such as unenriched infant formula. A number of possible interference issues were considered and dealt with.

Development of a quantitative GC-FID method for the determination of stearoyl-lactylates (E481/482) in foods.

Sodium and calcium salts of stearoyl-lactylates (SLs) are food emulsifiers especially used in bread and bakery products to improve texture. They should be used at the lowest level at which the desired technological effect is achieved in a specific food category and at amounts not exceeding the maximums set by European Commission Regulation No. 1129/2011. In order to be able to evaluate whether these emulsifiers are used correctly but also to evaluate whether the commercial additive formulations comply with legislation, a quantitative GC-FID method was developed. An internal standard (nonadecanoyl-1-lactylate) was synthesized in-house and pure ester standards were isolated from commercial additive formulations. The method showed a limit of detection of 0.04 and a limit of quantification of 0.12 mg esters ml⁻¹. The commercial additive formulations analysed proved to be complex mixtures of free lactic and fatty acids together with only 50-60% esters. Besides SLs important amounts of palmitoyl-lactylates were present. Different food matrices (with low- and high-fat contents) were spiked with commercial SL formulations and recoveries ranged between 85% and 109%. Determination of SLs in commercial foods (such as bakery and bread) indicated that pre-treatment with amylase was essential to determine accurately the SL content due to the interaction of SL with the amylose.

Characterization of electron ionization mass spectral (EIMS) fragmentation patterns of chloropropanol esters of palmitic acid using isotope labeling technique.

Chloropropanol (CP) esters are a class of thermally-induced toxicants that are mainly formed in refined edible oils. The structural diversity of these esters presents significant analytical challenges which have often been overcome through analysis of their corresponding free alcohols after a hydrolysis step. Mass spectrometry-based methodologies incorporating characteristic fragmentation patterns of particular isomers of CP esters greatly facilitates their identification. The electron ionization mass spectra (EIMS) of various isomers of synthetic and commercially available (13)C- and (2)H-labeled CP ester standards of palmitic (C16) and other short chain fatty acids (C3 to C10) were generated and analyzed using GC/MS. Short chain CP esters were synthesized by reacting their respective acid anhydrides with the corresponding 3-chloro- and 2-chloro- propanediols in addition to 1,3-dichloro- and 1,2-dichloropropanols. Five fragmentation pathways were identified. Four of the five pathways, such as α-cleavage, McLafferty rearrangement, α-H rearrangement and cyclic acyloxonium ion formation, were characteristic of CP mono- and diesters. The remaining pathway generating chloronium ion was found only in dichlorinated isomers. The proposed fragmentation pathways for the palmitic acid esters were confirmed through the use of (13)C- and (2)H-labeled CP ester standards of palmitic acid, and the generality of identified fragmentation patterns was confirmed through the identification of equivalent ions in the mass spectra of short chain fatty acids (C3 to C16). Characteristic ions that were identified in this study retaining the chlorine atom in their structures can be considered as potential markers for the presence of CP esters.

Reduced crying in term infants fed high beta-palmitate formula: a double-blind randomized clinical trial.

BACKGROUND: Beta-palmitate (sn-2 palmitate) mimics human milk fat, enabling easier digestion.Therefore, we hypothesized that infants consuming high beta-palmitate formula would have more frequent, softer stools and reduced crying compared to infants consuming low beta-palmitate formula. METHODS: Formula-fed infants were randomly assigned to receive either (1) formula with high beta-palmitate (HBP, n = 21) or (2) regular formula with a standard vegetable oil mix (LBP, n = 21). A matched group of breastfed infants served as a reference (BF, n = 21). Crying and stool characteristics data were recorded by the parents for 3 days before the 6- and 12-week visits. RESULTS: We found no significant differences in the stool frequency or consistency between the two formula groups. The percentage of crying infants in the LBP group was significantly higher than that in the HBP and BF groups during the evening at 6 weeks (88.2% vs. 56.3% and 55.6%, p < 0.05) and during the afternoon at 12 weeks (91.7% vs. 50.0% and 40%, p < 0.05). The infants fed HBP had significantly shorter crying durations when compared with infants fed LBP formula (14.90 ± 3.85 vs.63.96 ± 21.76 min/day, respectively; p = 0.047). CONCLUSIONS: Our study indicates that consumption of a high beta-palmitate formula affects infant crying patterns during the first weeks of life. Comparable to breastfeeding, it reduced crying duration and frequency, primarily during the afternoon and evening hours, thereby improving the well-being of formula-fed infants and their parents. TRIAL REGISTRATION: NCT00874068.Registration date March 31, 2009.

Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids.

The plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded ß,ß-carotenoid 3,3'-hydroxylase (CrtZ) and ß,ß-carotenoid 4,4'-ketolase (4,4'-oxygenase; CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Constructed transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. Berkeley and generate flowers and seeds. The germination ratio of the lettuce transformants (T0) (98.8%) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce (T1) leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2% of total carotenoids), astaxanthin monoester (18.2%), and the free forms of astaxanthin (10.0%) and the other ketocarotenoids (17.5%), which indicated that artificial ketocarotenoids corresponded to 94.9% of total carotenoids (230 µg/g fresh weight). Native carotenoids were there lactucaxanthin (3.8%) and lutein (1.3 %) only. This is the first report to structurally identify the astaxanthin esters biosynthesized in transgenic or transplastomic plants producing astaxanthin. The singlet oxygen-quenching activity of the total carotenoids extracted from the transplastomic leaves was similar to that of astaxanthin (mostly esterified) from the green algae Haematococcus pluvialis.

Mass spectrometry analysis of influenza virus reassortant clones does not reveal an influence of other viral proteins on S-acylation of hemagglutinin.

Hemagglutinin (HA) of influenza virus is S-acylated with stearate at a transmembrane cysteine and with palmitate at two cytoplasmic cysteines. The amount of stearate varies from 35 (in avian strains) to 12% (in human strains), although the acylation region exhibits only minor or even no amino acid differences between HAs. To address whether matrix proteins and neuraminidase affect stearoylation of HA, we used mass spectrometry to analyze laboratory reassortants containing avian virus HA and the internal proteins from a human virus. Only minor fluctuations in the amount of stearate were observed, implying that other viral proteins do not affect acylation of HA.

Effects of ethanol and long-chain ethyl ester concentrations on volatile partitioning in a whisky model system.

Ethanolic atmospheric pressure chemical ionization mass spectrometry (APCI-MS) was used to analyze the headspace concentrations of a test set of 14 whisky volatile compounds above a series of aqueous ethanolic solutions differing in alcohol content (5-40% ABV) and with regard to concentration of ethyl hexadecanoate (0-500 mg/L). The latter was selected to represent the long-chain ethyl esters found at various concentrations in new-make spirit. Headspace ion intensities were modeled against ethanol and ethyl hexadecanoate concentrations as factors. A separate model was prepared for each compound. Not surprisingly, ethanol content in the range of 5-40% ABV had a significant effect (P < 0.0001) on headspace volatile concentrations of all volatile compounds, whereas the ethyl hexadecanoate concentration had a selective effect of reducing headspace concentrations of the more hydrophobic compounds (log P > 2.5). This finding is discussed in terms of the "structuring" effects of ethyl hexadecanoate when present above critical micelle concentration, leading to the selective incorporation of hydrophobic volatile compounds into the interior of micelle-like structures. Data presented illustrate that dilution of whiskies to 23% ABV for "nosing" in the presence of long-chain ethyl esters is likely to change the balance of volatile compounds in the headspace and thus the perceived aroma character.

Analysis of lipids: metal oxide laser ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for lipid analysis; however, one of the drawbacks of this technique is matrix interference peaks at low masses. Metal oxide surfaces are described here for direct, matrix-free analysis of small (MW < 1000 Da) lipid compounds, without interferences in the resulting spectra from traditional matrix background peaks. Spectra from lipid standards produced protonated and sodiated molecular ions. More complex mixtures including vegetable oil shortening and lipid extracts from bacterial and algal sources provided similar results. Mechanistic insight into the mode of ionization from surface spectroscopy, negative ion mass spectrometry, and stable isotope studies is also presented. The metal oxide system is compared to other reported matrix-free systems.

Esterification of fatty acids using Candida antarctica lipase A in water-abundant systems.

The feasibility of using native lipase A from Candida antarctica (CAL-A) to esterify fatty acids with water-insoluble alcohols in the presence of excess water was investigated in stirred-tank reactors. For high reaction rates, a ratio of water:substrates of 0.6-1.4:1 (v/v) was required. CAL-A showed higher substrate selectivity for the esterification of saturated palmitic acid with branched-chain 2-ethyl-1-hexanol than for unsaturated oleic acid with linear alcohol (1-decanol). After 18 h at 70 °C in a 1.5 l bulk stirred-tank reactor, an 2-ethyl-1-hexyl palmitic acid ester was obtained near 100 % yield [molar ratio palmitic acid:2-ethyl-1-hexanol ~1:1.25, with 1.11 % (w/w) Novocor ADL (based on palmitic acid weight)].

Chemical composition and antifungal activity of Matricaria recutita flower essential oil against medically important dermatophytes and soil-borne pathogens.

OBJECTIVE: Fungal infections are potential public health threats all over the world. In the present study, effect of Matricaria recutita flower essential oil (EO) was evaluated against medically important dermatophytes and opportunistic saprophytes using microbioassay technique. MATERIALS AND METHODS: Flower essential oil (EO) of M. recutita prepared by hydrodistillation was analyzed by gas chromatography/mass spectrometry (GC/MS). The effect of plant EO on the growth of pathogenic dermatophytes and opportunistic saprophytes was assessed using microbioassay technique. In the bioassay, fungi were cultured in 6-well flat-bottom microplates in presence of various concentrations of plant EO (2.5-1000µg/mL) for 4-10days at 28°C. RESULTS: A total of 14 compounds were identified in the plant oil by GC/MS accounting for 97.5% of the oil composition. The main compound identified was chamazulene (61.3%) followed by isopropyl hexadecanoate (12.7%), trans-trans-farnesol (6.9%) and E-ß-farnesol (5.2%). Growth inhibition for the dermatophytes exposed to serial two-fold concentrations of plant EO (2.5 to 80µg/mL) was reported in the range of 3.24 to 68.15% for Microsporum gypseum, 24.48 to 100% for M. canis, 11.40 to 96.65% for Trichophyton mentagrophytes, 27.79 to 100% for T. rubrum and 45.73 to 100% for T. tonsurans. M. recutita EO inhibited the growth of opportunistic saprophytes by 3.98 to 64.29% for Aspergillus flavus, 6.38 to 93.62% for A. fumigatus, 3.52 to 89.45% for A. niger, 6.38 to 77.66% for Trichoderma harzianum and 17.41 to 89.41% for Fusarium oxysporum in serial two-fold concentrations of 15.62 to 1000µg/mL. CONCLUSION: Results of the present study indicate that M. recutita could be considered as a potential candidate for designing effective antifungal formulations suitable for treatment of dermatophytosis and other fungal infections.