Development and Application of an Automated Raman Sensor for Bioprocess Monitoring: From the Laboratory to an Algae Production Platform.

Microalgae provide valuable bio-components with economic and environmental benefits. The monitoring of microalgal production is mostly performed using different sensors and analytical methods that, although very powerful, are limited to qualified users. This study proposes an automated Raman spectroscopy-based sensor for the online monitoring of microalgal production. For this purpose, an in situ system with a sampling station was made of a light-tight optical chamber connected to a Raman probe. Microalgal cultures were routed to this chamber by pipes connected to pumps and valves controlled and programmed by a computer. The developed approach was evaluated on Parachlorella kessleri under different culture conditions at a laboratory and an industrial algal platform. As a result, more than 4000 Raman spectra were generated and analysed by statistical methods. These spectra reflected the physiological state of the cells and demonstrate the ability of the developed sensor to monitor the physiology of microalgal cells and their intracellular molecules of interest in a complex production environment.

Fungal Sterol Analyses by Gas Chromatography-Mass Spectrometry Using Different Derivatives.

Sterols are the main components of the fungal membrane. Their study can be used to describe the chemical biodiversity among the strains and species or to work on antifungal treatment. Those molecules can be analyzed by gas chromatography coupled with mass spectrometry (GC-MS) as free molecules or after derivation as acetate or trimethylsilyl ether (TMSi). Here we describe how to extract sterols from fungal biomass according to its physiological form and the culture conditions (liquid and solid media). The different methodologies that can be used to obtain free sterols, acetate, and/or TMSi derivatives are presented. Identification keys using the fragmentation at 70 eV are also described.

Effects of light and nitrogen availability on photosynthetic efficiency and fatty acid content of three original benthic diatom strains.

Microalgal biotechnology has gained considerable importance in recent decades. Applications range from simple biomass production for food and animal feed to valuable products for fuel, pharmaceuticals, health, biomolecules and materials relevant to nanotechnology. There are few reports of the exploration of wider microalgae biodiversity in the literature on high value microalgal compounds, however, because it is believed that there is little to be gained in terms of biomass productivity by examining new strains. Still, without diversity, innovation in biotechnology applications is currently limited. Using microalgal diversity is a very promising way to match species and processes for a specific biotechnological application. In this context, three benthic marine diatom strains (Entomoneis paludosa NCC18.2, Nitzschia alexandrina NCC33, and Staurosira sp NCC182) were selected for their lipid production and growth capacities. Using PAM fluorometry and FTIR spectroscopy, this study investigated the impact of nitrogen repletion and depletion as well as light intensity (30, 100, and 400 µmol.photons.m-2.s-1) on their growth, photosynthetic performance and macromolecular content, with the aim of improving the quality of their lipid composition. Results suggest that under high light and nitrogen limitation, the photosynthetic machinery is negatively impacted, leading cells to reduce their growth and accumulate lipids and/or carbohydrates. However, increasing lipid content under stressful conditions does not increase the production of lipids of interest: PUFA, ARA and EPA production decreases. Culture conditions to optimize production of such fatty acids in these three original strains led to a balance between economic and ecophysiological constraints: low light and no nitrogen limitation led to better photosynthetic capacities associated with energy savings, and hence a more profitable approach.

Steroids from Marine-Derived Fungi: Evaluation of Antiproliferative and Antimicrobial Activities of Eburicol.

The most common sterol in fungi is ergosterol, which has frequently been investigated in human pathogenic fungal strains. This sterol, and others isolated from fungal strains, has also demonstrated cytotoxicity against cancer cell lines and antimicrobial activities. Marine fungi can produce high amounts of bioactive compounds. So, a screening was performed to study sterol composition using GC/MS in 19 marine fungal strains and ergosterol was always the major one. One strain, Clonostachys rosea MMS1090, was selected due to its high amount of eburicol and a one strain many compounds approach was performed on seven culture media to optimize its production. After purification and structural identification by NMR, eburicol was assessed against four cancer cell lines, MCF-7, MDA-MB-231, NSCLC-N6-L16 and A549, and seven human pathogenic bacteria Staphylococcus aureus, Bacillus sp., Bacillus cereus, Listeria ivanovii, Escherichia coli, Citrobacter freundii and Salmonella spp. The most significant activity was cytotoxicity against MCF-7 cells (2 µM). This is the first report of such an accumulation of eburicol in the marine fungal strain C. rosea confirming its potential in the production of bioactive lipids.

Carbon Orientation in the Diatom Phaeodactylum tricornutum: The Effects of Carbon Limitation and Photon Flux Density.

Diatoms adapt to changing environmental conditions in very efficient ways. Among the mechanisms that can be activated, the reorientation of carbon metabolism is crucial because it allows the storage of energy into energy-dense molecules, typically lipids. Beside their roles in physiology, lipids are commercially interesting compounds. Therefore studies dealing with this topic are relevant for both basic and applied science. Although the molecular mechanisms involved in the reorientation of carbon metabolism as a response to a deficiency in nutrients such as nitrogen or phosphorus has been partially elucidated, the impacts of carbon availability on the implementation of the reorientation mechanisms remain unclear. Indeed, it has not been determined if the same types of mechanisms are activated under carbon and other nutrient deficiencies or limitations. The first aim of this work was to get insights into the physiological, biological and molecular processes triggered by progressive carbon starvation in the model diatom Phaeodactylum tricornutum. The second aim was to investigate the effects of the growth light intensity on these processes. For such a purpose three different photon flux densities 30, 300, and 1000 µmol photons m-2 s-1 were used. The results presented here demonstrate that under carbon limitation, diatom cells still reorient carbon metabolism toward either phosphoenolpyruvate or pyruvate, which serves as a hub for the production of more complex molecules. The distribution of carbon atoms between the different pathways was partially affected by the growth photon flux density because low light (LL) provides conditions for the accumulation of chrysolaminarin, while medium light mostly stimulated lipid synthesis. A significant increase in the amount of proteins was observed under high light (HL).

Response of CO2-starved diatom Phaeodactylum tricornutum to light intensity transition.

In this study, we investigated the responses of Phaeodactylum tricornutum cells acclimated to 300 µmol m-2 s-1 photon flux density to an increase (1000 µmol m-2 s-1) or decrease (30 µmol m-2 s-1) in photon flux densities. The light shift occurred abruptly after 5 days of growth and the acclimation to new conditions was followed during the next 6 days at the physiological and molecular levels. The molecular data reflect a rearrangement of carbon metabolism towards the production of phosphoenolpyruvic acid (PEP) and/or pyruvate. These intermediates were used differently by the cell as a function of the photon flux density: under low light, photosynthesis was depressed while respiration was increased. Under high light, lipids and proteins accumulated. Of great interest, under high light, the genes coding for the synthesis of aromatic amino acids and phenolic compounds were upregulated suggesting that the shikimate pathway was activated.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

Exploitable Lipids and Fatty Acids in the Invasive Oyster Crassostrea gigas on the French Atlantic Coast.

Economic exploitation is one means to offset the cost of controlling invasive species, such as the introduced Pacific oyster (Crassostrea gigas Thunberg) on the French Atlantic coast. Total lipid and phospholipid (PL) fatty acids (FAs) and sterols were examined in an invasive population of C. gigas in Bourgneuf Bay, France, over four successive seasons, with a view to identify possible sources of exploitable substances. The total lipid level (% dry weight) varied from 7.1% (winter) to 8.6% (spring). Of this, PLs accounted for 28.1% (spring) to 50.4% (winter). Phosphatidylcholine was the dominant PL throughout the year (up to 74% of total PLs in winter). Plasmalogens were identified throughout the year as a series of eleven dimethylacetals (DMAs) with chain lengths between C16 and C20 (up to 14.5% of PL FAs + DMAs in winter). Thirty-seven FAs were identified in the PL FAs. Eicosapentaenoic acid (20:5n-3 EPA/7.53% to 14.5%) and docosahexaenoic acid (22:6n-3 DHA/5.51% to 9.5%) were the dominant polyunsaturated FAs in all seasons. Two non-methylene-interrupted dienoic (NMID) FAs were identified in all seasons: 7,13-docosadienoic and 7,15-docosadienoic acids, the latter being present at relatively high levels (up to 9.6% in winter). Twenty free sterols were identified, including cholesterol at 29.9% of the sterol mixture and about 33% of phytosterols. C. gigas tissues thus contained exploitable lipids for health benefits or as a potential source of high-quality commercial lecithin.

Lipid Composition, Fatty Acids and Sterols in the Seaweeds Ulva armoricana, and Solieria chordalis from Brittany (France): An Analysis from Nutritional, Chemotaxonomic, and Antiproliferative Activity Perspectives.

Lipids from the proliferative macroalgae Ulva armoricana (Chlorophyta) and Solieria chordalis (Rhodophyta) from Brittany, France, were investigated. The total content of lipids was 2.6% and 3.0% dry weight for U. armoricana and S. chordalis, respectively. The main fractions of S. chordalis were neutral lipids (37%) and glycolipids (38%), whereas U. armoricana contained mostly neutral lipids (55%). Polyunsaturated fatty acids (PUFA) represented 29% and 15% of the total lipids in U. armoricana and S. chordalis, respectively. In both studied algae, the phospholipids were composed of PUFA for 18%. In addition, PUFA were shown to represent 9% and 4.5% of glycolipids in U. armoricana and S. chordalis, respectively. The essential PUFA were 16:4n-3, 18:4n-3, 18:2n-3, 18:2n-6, and 22:6n-3 in U. armoricana, and 20:4n-6 and 20:5n-3 in S. chordalis. It is important to notice that six 2-hydroxy-, three 3-hydroxy-, and two monounsaturated hydroxy fatty acids were also identified and may provide a chemotaxonomic basis for algae. These seaweeds contained interesting compounds such as squalene, α-tocopherol, cholest-4-en-3-one and phytosterols. The antiproliferative effect was evaluated in vitro on human non-small-cell bronchopulmonary carcinoma line (NSCLC-N6) with an IC50 of 23 µg/mL for monogalactosyldiacylglycerols isolated from S. chordalis and 24 µg/mL for digalactosyldiacylglycerols from U. armoricana. These results confirm the potentialities of valorization of these two species in the fields of health, nutrition and chemotaxonomy.

The Marine-Derived Fungus Clonostachys rosea, Source of a Rare Conjugated 4-Me-6E,8E-hexadecadienoic Acid Reducing Viability of MCF-7 Breast Cancer Cells and Gene Expression of Lipogenic Enzymes.

A marine-derived strain of Clonostachys rosea isolated from sediments of the river Loire estuary (France) was investigated for its high lipid production. The fungal strain was grown on six different culture media to explore lipid production changes. An original branched conjugated fatty acid, mainly present in triglycerides and mostly produced when grown on DCA (23% of total fatty acid composition). It was identified as 4-Me-6E,8E-hexadecadienoic on the basis of spectroscopic analyses. This fatty acid reduced viability of MCF-7 breast cancer cells in a dose dependent manner (up to 63%) at physiological free fatty acid human plasma concentration (100 µM). Reduction of gene expression of two lipogenic enzymes, the acetyl CoA carboxylase (ACC) and the fatty acid synthase (FAS) was evaluated to explore the mechanisms of action of 4-Me-6E,8E-16:2 acid. At 50 µM, 50% and 35% of mRNA gene expression inhibition were observed for ACC and FAS, respectively.

Multiple beneficial lipids including lecithin detected in the edible invasive mollusk Crepidula fornicata from the French Northeastern Atlantic coast.

The invasive mollusk Crepidula fornicata, occurring in large amounts in bays along the French Northeastern Atlantic coasts, may have huge environmental effects in highly productive ecosystems where shellfish are exploited. The present study aims at determining the potential economic value of this marine species in terms of exploitable substances with high added value. Lipid content and phospholipid (PL) composition of this mollusk collected on the Bourgneuf Bay were studied through four seasons. Winter specimens contained the highest lipid levels (5.3% dry weight), including 69% of PLs. Phosphatidylcholine (PC) was the major PL class all year, accounting for 63.9% to 88.9% of total PLs. Consequently, the winter specimens were then investigated for PL fatty acids (FAs), and free sterols. Dimethylacetals (DMAs) were present (10.7% of PL FA + DMA mixture) revealing the occurrence of plasmalogens. More than forty FAs were identified, including 20:5n-3 (9.4%) and 22:6n-3 (7.3%) acids. Fourteen free sterols were present, including cholesterol at 31.3% of the sterol mixture and about 40% of phytosterols. These data on lipids of C. fornicata demonstrate their positive attributes for human nutrition and health. The PL mixture, rich in PC and polyunsaturated FAs, offers an interesting alternative source of high value-added marine lecithin.

Antimalarial activity of axidjiferosides, new ß-galactosylceramides from the African sponge Axinyssa djiferi.

The marine sponge, Axinyssa djiferi, collected on mangrove tree roots in Senegal, was investigated for glycolipids. A mixture containing new glycosphingolipids, named axidjiferoside-A, -B and -C, accounted for 0.07% of sponge biomass (dry weight) and for 2.16% of total lipids. It showed a significant antimalarial activity, with a 50% inhibitory concentration (IC50) of 0.53 ± 0.2 µM against a chloroquine-resistant strain of Plasmodium falciparum. They were identified as homologous ß-galactopyranosylceramides composed of 2-amino-(6E)-octadec-6-en-1,3,4-triol, and the major one, axidjiferoside-A (around 60%), contained 2-hydroxytetracosanoic acid. Cytotoxicity was studied in vitro on human cancer cell lines (multiple myeloma, colorectal adenocarcinoma, glioblastoma and two lung cancer NSCLC-N6 and A549). Results of this investigation showed that axidjiferosides are of interest, because they proved a good antiplasmodial activity, with only a low cytotoxicity against various human cell lines and no significant antitrypanosomal and antileishmanial activity. Thus, it seems that galactosylceramides with a ß anomeric configuration may be suitable in searching for new antimalarial drugs.

Non-methylene interrupted and hydroxy fatty acids in polar lipids of the alga Grateloupia turuturu over the four seasons.

Phospholipids (PL) and glycolipids (GL) FA in the edible Rhodophyta Grateloupia turuturu, from Brittany, France, were investigated over four seasons. The major lipid class was GL in all seasons (around 45 %). More than 80 FA occurred in polar lipids, with chains from C12 to C26, identified as methyl esters and N-acyl pyrrolidides by gas chromatography-mass spectrometry (GC-MS). PUFA occurred at up to 47.1 % (summer) in PL, and up to 43.6 % (summer) in GL. The major PUFA were 20:5n-3 (12.2 % in PL and 29.0 % in GL) and 20:4n-6 (25.6 % in PL and 10.4 % in GL). The unusual 18:3n-7 acid was identified in PL up to 2.2 %. Several minor unsaturated FA were identified in PL and are previously unreported in seaweeds, namely 14-tricosenoic, 15-tetracosenoic, 5,11-octadecadienoic and 5,9-nonadecadienoic. Also unprecedented in seaweeds, ten 2-hydroxy and three 3-hydroxy FA occurred mainly in PL, 13.9 % in spring with the 3-hydroxyhexadecanoic acid as the major one (8.1 % winter). Three n-9 monounsaturated 2-hydroxy FA occurred in PL. The 2-hydroxy-15-tetracosenoic acid was characterized as the dimethyl disulfide adduct of its methyl ester. The 2-hydroxy-16-pentacosenoic and 2-hydroxy-17-hexacosenoic acids were identified by comparison of mass spectra and GC mobilities with those of the 2-hydroxy-15-tetracosenoic acid, and of other homogeneous FA series. These rare n-9 monounsaturated 2-hydroxy FA are unprecedented in seaweeds.

Antiproliferative activity against human non-small cell lung cancer of two O-alkyl-diglycosylglycerols from the marine sponges Myrmekioderma dendyi and Trikentrion laeve.

Glycolipids of Myrmekioderma sponges contain Myrmekiosides, a new family of glycolipids with a unique structure of mono-O-alkyl-diglycosylglycerols. This report deals with the identification and biological activity of the new Myrmekioside E from Myrmekioderma dendyi. Its structure has been elucidated from spectroscopic data and chemical degradation studies. It contained a glycerol backbone linked to xylose and N-acetylglucosamine, and an alkyl long-chain with a terminal alcohol group. A related glycolipid, Trikentroside, known in the sponge Trikentrion laeve, was subjected to a comparative biological evaluation. Both glycolipids inhibit proliferation of two human non-small cell lung cancer cell lines (NSCLC-N6 and A549).

Phospholipid distribution and phospholipid fatty acids of the tropical tunicates Eudistoma sp. and Leptoclinides uniorbis.

Two tunicates, Eudistoma sp. and Leptoclinides uniorbis, collected from the tropical waters off Djibouti were investigated for lipids and phospholipid (PL) fatty acids. PL accounted for 38.2% of the total lipids in Eudistoma sp. and for 30.2% in L. uniorbis. PL classes were quantified by normal-phase high-performance liquid chromatography using an evaporative light-scattering detector and revealed essential differences. Eudistoma sp. contained mainly phosphatidylcholine (PC, 70.3% of total PL) and lysophosphatidylcholine (LPC, 11.9%) and was devoid of phosphatidylserine (PS), whereas the major PL of L. uniorbis was PS (59.1%) followed by PC (22.5%) and LPC (8.8%). Gas chromatography-mass spectrometry analyses of fatty acid (FA) derivatives revealed 38 FA in Eudistoma sp., and 35 FA in L. uniorbis, ranged from C(12) to C(24) chain lengths. Polyunsaturated FA accounted for 25.9% in Eudistoma sp. and for 32.3% in L. uniorbis. Interestingly, L. uniorbis contained a high percentage (16.7%) of the 20:5n-3 acid (8.9% in Eudistoma sp.) and the 18:4n-3 acid (4.1%). Significant levels of the 20:4n-6 acid were observed in both organisms (7.8 and 6.0% respectively). Eudistoma sp. contained the rare 20:3n-7 acid (2.3%) only recorded to date in hydrothermal vent animals. The cyclopropane dihydrosterculic acid was identified in both tunicates (0.7 and 0.5% respectively). These latter FA, together with some unusual branched saturated and monounsaturated FA, revealed the occurrence of associated bacteria in the tunicates. Another noticeable feature was a series of eight C(16) to C(18) aldehyde dimethylacetals revealing the presence of plasmalogens at 5.0% in Eudistoma sp. and 14.2% in L. uniorbis. The results of this study were compared with those previously published for other tunicates regarding mainly PL content and FA composition.

Cytotoxicity on human cancer cells of ophidiacerebrosides isolated from the African starfish Narcissia canariensis.

The starfish Narcissia canariensis harvested from the coasts off Dakar, Senegal, was investigated for glycolipids (GL). This report deals with the isolation, characterization and biological activity of a fraction F13-3 separated from the GL mixture and selected according to its ability to inhibit KB cell proliferation after 72 hours of treatment. Firstly, a GL mixture F13 was obtained that accounted for 1.36% of starfish biomass (dry weight) and 0.36% of total lipids. The fraction F13-3 obtained from F13 contained three homologous GL identified as peracetylated derivatives on the basis of chemical and spectroscopic evidence. These contained a ß-glucopyranoside as sugar head, a 9-methyl-branched 4,8,10-triunsaturated long-chain aminoalcohol as sphingoid base and amide-linked 2-hydroxy fatty acid chains. The majority (63%) had an amide-linked 2-hydroxydocosanoic acid chain and was identified as the ophidiacerebroside-C, firstly isolated from the starfish Ophidiaster ophidiamus. The minor components of F13-3 differed by one more or one less methylene group, and corresponded to ophidiacerebroside-B and -D. We found that F13-3 displayed an interesting cytotoxic activity over 24 hours on various adherent human cancerous cell lines (multiple myeloma, colorectal adenocarcinoma and glioblastoma multiforme) with an IC(50) of around 20 µM.

New 17-methyl-13-octadecenoic and 3,16-docosadienoic acids from the sponge Polymastia penicillus.

The phospholipid fatty acid composition of the North-East Atlantic sponge Polymastia penicillus (South Brittany, France) was investigated. Sixty fatty acids (FA) were identified as methyl esters (FAME) and N-acyl pyrrolidides (NAP) by gas chromatography-mass spectrometry (GC/MS), including eight Delta5,9 unsaturated FA and three long-chain 2-hydroxylated FA. The major phospholipid FA were palmitic (14.3% of the total FA mixture), vaccenic (12.7%), 15(Z)-docosenoic (13.4%) and 5(Z),9(Z)-hexacosadienoic (13.3%) acids. In addition to the iso- and anteiso-branched saturated FA, several unusual short-chain branched saturated FA were identified. In addition to the known Delta5,9 FA, and interestingly regarding their identification by GC-MS as N-acyl pyrrolidides, was the co-occurrence of unusual FA possessing a Delta3, Delta4 and Delta5 double bond such as iso-4-pentadecenoic, iso-5-heptadecenoic, anteiso-5-heptadecenoic and two new compounds, not hitherto found in nature, namely 17-methyl-13-octadecenoic (0.8%) and 3,16-docosadienoic (1.1%) acids.

Unusual sterolic mixture, and 24-isopropylcholesterol, from the sponge Ciocalypta sp. reduce cholesterol uptake and basolateral secretion in Caco-2 cells.

An unusual sterolic mixture (82.3% of 24-isopropylated sterols) and its major component, 24-isopropylcholesterol, isolated from a marine sponge, Ciocalypta sp. (Halichondriidae), reduce cholesterol uptake, basolateral secretion and ACAT-2 mRNA expression and increase the expression of ABCA1 mRNA in Caco-2 cells. The decreases of cholesterol uptake and secretion induced by 24-isopropylcholesterol alone were more than that of both the sterolic mixture and beta-sitosterol. These data add a new sterol, 24-isopropylcholesterol, to sterols that may reduce intestinal cholesterol absorption.

New trends in phospholipid class composition of marine sponges.

The exceptional ability of marine sponges to adapt to often drastic changes of their environments could be due to special structural features in cell membranes, including firstly phospholipids (PL). Thus, PL class composition was investigated in marine sponges (22 species from 19 genera to 15 families) originating from various locations (East Atlantic, North Atlantic, South-West Pacific, Mediterranean Sea, Red Sea, Arabian-Persian Gulf). The quantitative determination of PL class composition was obtained by high-performance thin-layer chromatography (HPTLC) with scanning densitometry of the different spots. Previous reports have shown phosphatidylethanolamine (PE) as the major PL class in marine sponges, followed by phosphatidylcholine (PC), while other papers described PC as a minor class and even lacking. This survey found PE as the major PL class in only two species, while PC was the major class in 13 species including a calcareous one. The great abundance of bacteria in some sponges was evidenced from the relatively high proportions of particular PL classes. Various PL distributions were observed even for the sponge species collected in the same area and belonging to the same genus. Thus, no clear rule on PL composition in marine sponges can be stated to date.

New Trichobrachins, 11-residue peptaibols from a marine strain of Trichoderma longibrachiatum.

A marine strain of Trichoderma longibrachiatum isolated from blue mussels (Mytilus edulis) was investigated for short peptaibol production. Various 11-residue peptaibols, obtained as microheterogenous mixtures after a chromatographic fractionation, were identified by positive mass spectrometry fragmentation (ESI-IT-MS(n), CID-MS(n) and GC/EI-MS). Thirty sequences were identified, which is the largest number of analogous sequences so far observed at once. Twenty-one sequences were new, and nine others corresponded to peptaibols already described. These peptaibols belonged to the same peptidic family based on the model Ac-Aib-xxx-xxx-xxx-Aib-Pro-xxx-xxx-Aib-Pro-xxol. They were named trichobrachin A when the residue in position 2 was an Asn, and trichobrachin C when it was a Gln. Major chromatographic sub-fractions, corresponding to purified peptaibols, were assayed for their cytotoxic activity. Trichobrachin A-IX and trichobrachin C exhibited the highest activities. There was an exponential relation between their relative hydrophobicity and their cytotoxicity on KB cells.

8-Amino-5-nitro-6-phenoxyquinolines: potential non-peptidic neuropeptide Y receptor ligands.

The synthesis and pharmacological evaluation of analogues of PD 160170, a neuropeptide Y1 (NPY) receptor antagonist are reported. Phamacomodulation of this 8-amino-5-nitro-6-phenylsulfonylquinoline was carried out by replacing the sulfone moiety by oxygen. The corresponding ethers 11-16 were obtained by nucleophilic substitution of 8-acetamido-6-chloro-5-nitroquinoline 4 with phenols, followed by acidic hydrolysis of the intermediary amides 5-10. The test compounds 11-16 exerted no appreciable Y1 activity and they were also inactive in terms of Y5 receptor binding; their IC50 values were >1 microM and 10 microM, respectively. The dramatic decrease in potency resulting from replacement of the sulfone function by an ether was confirmed by IP administration of 16 to ob/ob mice; after a 4-day administration, no decrease in food consumption or weight was observed.