Conjugated Fatty Acids Research Articles

Conjugated fatty acids drive ferroptosis through chaperone-mediated autophagic degradation of GPX4 by targeting mitochondria

Conjugated fatty acids (CFAs) have been known for their anti-tumor activity. However, the mechanism of action remains unclear. Here, we identify CFAs as inducers of glutathione peroxidase 4 (GPX4) degradation through chaperone-mediated autophagy (CMA). CFAs, such as (10E,12Z)-octadecadienoic acid and α-eleostearic acid (ESA), induced GPX4 degradation, generation of mitochondrial reactive oxygen species (ROS) and lipid peroxides, and ultimately ferroptosis in cancer cell lines, including HT1080 and A549 cells, which were suppressed by either pharmacological blockade of CMA or genetic deletion of LAMP2A, a crucial molecule for CMA. Mitochondrial ROS were sufficient and necessary for CMA-dependent GPX4 degradation. Oral administration of an ESA-rich oil attenuated xenograft tumor growth of wild-type, but not that of LAMP2A-deficient HT1080 cells, accompanied by increased lipid peroxidation, GPX4 degradation and cell death. Our study establishes mitochondria as the key target of CFAs to trigger lipid peroxidation and GPX4 degradation, providing insight into ferroptosis-based cancer therapy.

Detailed lipid investigation of edible seaweeds by photochemical derivatization and untargeted lipidomics

Seaweeds are macrophytic algae that have been gaining interest as alternative healthy foods, renewable drug sources, and climate change mitigation agents. In terms of their nutritional value, seaweeds are renowned for their high content of biologically active polyunsaturated fatty acids. However, little is known about the regiochemistry—the geometry and position of carbon–carbon double bonds—of free and conjugated fatty acids in seaweeds. In the present work, a detailed characterization of the seaweed lipidome was achieved based on untargeted HRMS-based analysis and lipid derivatization with a photochemical aza-Paternò–Büchi reaction. A triple-data processing strategy was carried out to achieve high structural detail on the seaweed lipidome, i.e., (i) a first data processing workflow with all samples for aligning peak and statistical analysis that led to the definition of lipid sum compositions (e.g., phosphatidylglycerol (PG) 34:1), (ii) a second data processing workflow in which the samples of each seaweed were processed separately to annotate molecular lipids with known fatty acyl isomerism (e.g., PG 16:0_18:1), and (iii) the annotation of lipid regioisomers following MS/MS annotation of the lipid derivatives obtained following the aza-Paternò–Büchi reaction (e.g., PG 16:0_18:1 ω-9). Once the platform was set up, the lipid extracts from 8 seaweed species from different seaweed families were characterized, describing over 900 different lipid species, and information on the regiochemistry of carbon–carbon double bonds uncovered unknown peculiarities of seaweeds belonging to different families. The overall analytical approach helped to fill a gap in the knowledge of the nutritional composition of seaweeds.Graphical

Engineering Rhodosporidium toruloides for sustainable production of value-added punicic acid from glucose and wood residues

Rhodosporidium toruloides has emerged as a prominent candidate for producing single-cell oil from cost-effective feedstocks. In this study, the capability of R. toruloides to produce punicic acid (PuA), a representative plant unusual fatty acid, was investigated. The introduction of acyl lipid desaturase and conjugase (PgFADX) allowed R. toruloides to accumulate 3.7 % of total fatty acids as PuA. Delta-12 acyl lipid desaturase (PgFAD2) and diacylglycerol acyltransferase 2 were shown to benefit PuA production. The strain with PgFADX and PgFAD2 coexpression accumulated 12 % of its lipids as PuA from glucose, which translated into a PuA titer of 451.6 mg/L in shake flask condition. Utilizing wood hydrolysate as the feedstock, this strain produced 6.4 % PuA with a titer of 310 mg/L. Taken together, the results demonstrated that R. toruloides could serve as an ideal platform for the production of plant-derived high-value conjugated fatty acid using agricultural and forestry waste as feedstock.

First Proof of Concept of a Click Inverse Electron Demand Diels-Alder Reaction for Assigning the Regiochemistry of Carbon-Carbon Double Bonds in Untargeted Lipidomics.

Lipidomics by high-resolution mass spectrometry (HRMS) has become a prominent tool in clinical chemistry due to the proven connections between lipid dysregulation and the insurgence of pathologies. However, it is difficult to achieve structural characterization beyond the fatty acid level by HRMS, especially when it comes to the regiochemistry of carbon-carbon double bonds, which play a major role in determining the properties of cell membranes. Several approaches have been proposed for elucidating the regiochemistry of double bonds, such as derivatization before MS analysis by photochemical reactions, which have shown great potential for their versatility but have the unavoidable drawback of splitting the MS signal. Among other possible approaches for derivatizing electron-rich double bonds, the emerging inverse-electron-demand Diels-Alder (IEDDA) reaction with tetrazines stands out for its unmatchable kinetics and has found several applications in basic biology and protein imaging. In this study, a catalyst-free click IEDDA reaction was employed for the first time to pinpoint carbon-carbon double bonds in free and conjugated fatty acids. Fatty acid and glycerophospholipid regioisomers were analyzed alone and in combination, demonstrating that the IEDDA reaction had click character and allowed the obtention of diagnostic product ions following MS/MS fragmentation as well as the possibility of performing relative quantitation of lipid regioisomers. The IEDDA protocol was later employed in an untargeted lipidomics study on plasma samples of patients suffering from prostate cancer and benign prostatic conditions, confirming the applicability of the proposed reaction to complex matrices of clinical interest.

Biomolecular investigations into BBI reveal an enzymatic mechanism for PUFA isomerisation in bifidobacterium CFA bioconversion strains

Multiple species of Bifidobacterium exhibit the ability to bioconvert conjugated fatty acids (CFAs), which is considered an important pathway for these strains to promote host health. However, there has been limited progress in understanding the enzymatic mechanism of CFA bioconversion by bifidobacteria, despite the increasing number of studies identifying CFA-producing strains. The protein responsible for polyunsaturated fatty acid (PUFA) isomerization in B. breve CCFM683 has recently been discovered and named BBI, providing a starting point for exploring Bifidobacterium isomerases (BIs). This study presents the sequence classification of membrane-bound isomerases from four common Bifidobacterium species that produce CFA. Heterologous expression, purification, and enzymatic studies of the typical sequences revealed that all possess a single c9, t11 isomer as the product and share common features in terms of enzymatic properties and catalytic kinetics. Using molecular docking and alanine scanning, Lys84, Tyr198, Asn202, and Leu245 located in the binding pocket were identified as critical to the catalytic activity, a finding further confirmed by site-directed mutagenesis-based screening assays. Overall, these findings provide insightful knowledge concerning the molecular mechanisms of BIs. This will open up additional opportunities for the use of bifidobacteria and CFAs in probiotic foods and precision nutrition.

Edible Biopolymers from Marine Algae used as an Alternate Packaging material: A Review on their characteristics and properties

Food packaging is estimated to account for two-thirds of all plastic waste. As a result, it is crucial to discover alternative packaging materials that are both environmentally friendly and safe for human health. Marine algae are becoming more well-known and in demand as cutting-edge resources for producing biopolymers like proteins and polysaccharides. Because of their biocompatibility, biodegradability, and lack of toxicity, biopolymers have been suggested as potential sources for food packaging materials. Numerous research has thoroughly examined the extraction, separation, and use of marine biopolymers in the creation of sustainable packaging. Marine algae are also rich in protein and mineral content, they also have anticancer, anti-obesity, and hypolipidemic properties due to the presence of polyunsaturated conjugated fatty acids. The edible films enhance the shelf life of food by controlling moisture without changing the elements of food. The marine algae are collected either in the intertidal or subtidal areas and they will be dried for further process. The edible films are environmentally friendly. The edible film made from marine algae is a mixture of protein, polysaccharides, lipids, and resins. The factors which affect the properties of the edible film are the source of raw material, surface charge, hydrophobicity, polymer chain length, plasticizer type, proportion, and synthesis method. There are numerous research has been conducted to develop edible film using various matrix constituents. This review provides an overview of Marine algae, its process, and edible films, its characteristics, and factors affecting the film.

Biosynthesis of Fatty Acid Derivatives by Recombinant Yarrowia lipolytica Containing MsexD2 and MsexD3 Desaturase Genes from Manduca sexta.

One of the most interesting groups of fatty acid derivates is the group of conjugated fatty acids from which the most researched include: conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), which are associated with countless health benefits. Sex pheromone mixtures of some insect species, including tobacco horn-worm (Manduca sexta), are typical for the production of uncommon C16 long conjugated fatty acids with two and three conjugated double bonds, as opposed to C18 long CLA and CLNA. In this study, M. sexta desaturases MsexD2 and MsexD3 were expressed in multiple strains of Y. lipolytica with different genotypes. Experiments with the supplementation of fatty acid methyl esters into the medium resulted in the production of novel fatty acids. Using GCxGC-MS, 20 new fatty acids with two or three double bonds were identified. Fatty acids with conjugated or isolated double bonds, or a combination of both, were produced in trace amounts. The results of this study prove that Y. lipolytica is capable of synthesizing C16-conjugated fatty acids. Further genetic optimization of the Y. lipolytica genome and optimization of the fermentation process could lead to increased production of novel fatty acid derivatives with biotechnologically interesting properties.

Analysis of Triacylglyceride and Conjugated Fatty Acid Compositions in Seed Oils of some Plants of Cucurbitaceae Family in Vietnam

Conjugated fatty acids were known as potential sources for the pharmaceutical industry with beneficial bioactivities, including anti-cancer effects, anti-diabetes, anti-obesity, and anti-inflammatory properties. In the present work, characteristics of triglycerides and fatty acids of the seed oils of five plants (Momordica Cochinchinensis (Lour.) Spreng, MomordicaMomordica charantia, Thladiantha nudiflora Hemsl, and Trichosanthes cucumerina var. anguina) in Vietnam were analyzed by UV and FTIR spectroscopy, and reversed phase high performance liquid chromatography. Triacylglycerides compositions of the seed oils were determined using RP-HPLC with DAD detector connected with MS detector in series. The seed oils of M. Cochinchinensis, M. subangulata, and M. charantia are rich sources of conjugated octadecatrienoic acid with α-eleostearic acid (C18:39E,11E,13Z) as the base acid and β-eleostearic as the minor conjugated acid. In the seed of Thladiantha nudiflora (both α-eleostearic and punic acid (C18:39Z,11E,13Z) were synthesized with approximately equal proportions. In T. cucumerina var. anguina seed oil, punicic acid was synthesized as the main conjugated fatty acid. In addition, some oil parameters, such as oil yield and refractive index of these seed oils, were determined.

Production of conjugated fatty acids in probiotic-fermented walnut milk with the addition of lipase

Walnut milk is rich in linoleic acid (LA) and linolenic acid (LNA) in the form of triglycerides. Four LAB strains capable of converting LA and LNA could grow to 9.0 lg CFU/mL in walnut milk. However, no conjugated fatty acids were detected. Lipase was added to release LA and LNA and then LAB was inoculated to ferment walnut milk. However, conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) were only detected in walnut milk inoculated with Bifidobacterium breve CCFM683. When 60 U/mL lipase was added, the total amount of conjugated fatty acids reached 1.33 ± 0.01 mg/mL with conversion rates of 5.0% and 11.3% for CLA and CLNA, respectively. By monitoring the fermentation progress, the amount of conjugated fatty acids could reach a maximum of 2.30 ± 0.01 mg/mL at 20 h (p < 0.05). When 20 U/mL lipase was added, the amount of conjugated fatty acids in fermented walnut milk reached 1.58 mg/mL and conversion rates increased to 7.2% and 21.6% for CLA and CLNA, respectively. Thus, adding lipase can realize the conversion of CLA and CLNA in walnut milk and reducing the amount of lipase and hydrolysis time is beneficial to improve the conversion rates, but total amount is reduced.

Two unusual conjugated fatty acids, parinaric acid and α-eleostearic acid, are present in several Impatiens species, but not in congener Hydrocera triflora.

The online version contains supplementary material available at 10.1007/s12298-022-01194-4.

Bioavailability and Functionality of Conjugated Fatty Acids in Foods

Dietary conjugated fatty acids are associated with heart disease, obesity, diabetes, cancer and others; in addition, many functions of cell membranes are dependent on lipid composition and lipids ingested through the diet can modify this composition and their biochemical activity. Due to the discoveries of their chemical and physiological properties, there is a growing interest in conjugated fatty acids as well as in the properties of their isomers. Conjugated fatty acids (CFAs) are the general term to describe the positional and geometric isomers of polyunsaturated fatty acids with conjugated double bonds, which are separated by a single carbon-carbon bond rather than being separated by a methylene group (CH2). Theoretically, several isomers of conjugated fatty acids are possible, with multiple combinations of numerical, positional and geometric configurations of the conjugation of double bonds, which may present as conjugated linoleic and α-linolenic acids and may have different functionalities in the body.

Insights into gut microbiota metabolism of dietary lipids: the case of linoleic acid.

It has been recognized that, next to dietary fibre and proteins, gut microbiota can metabolize lipids producing bioactive metabolites. However, the metabolism of dietary lipids by human gut microbiota has been poorly explored so far. This study aimed to examine the change in lipids, particularly linoleic acid (LA), induced by the chemical form of lipids and the presence of the plant matrix. Short-chain fatty acid (SCFA) production was monitored to get an insight into microbial activity. Free LA, glyceryl trilinoleate and soybean oil as well as digested intact (DS) and broken (BS) soybean cells were subjected to in vitro fermentation using human faecal inoculums. Confocal microscopy was used to visualize the soybean cell integrity. Three LA metabolites, including two conjugated fatty acids (CLAs, 9z,11e and 9e,11e) and 12hydroxy, 9z C18:1, were identified and monitored. Free LA addition improved the LA metabolite production but reduced SCFA concentrations compared to trilinoleate and soybean oil. Breaking cell integrity had impacts on CLA, hydroxy C18:1 and SCFA production and free fatty acid release within the first 24 h of fermentation, but this effect vanished with time. In contrast, soybean oil only increased free LA release and hydroxy C18:1 production. The content of several FAs decreased during fermentation suggesting a substantial conversion in microbial metabolites. Besides, LA metabolites were also identified in the fermentation pellets suggesting the incorporation of microbial FA metabolites into bacterial cells. This study expands our understanding of microbial metabolism of dietary lipids with a special emphasis on the role of food- and diet-related factors.

Assessing the concentration of conjugated fatty acids within pomegranate seed oil using quantitative nuclear magnetic resonance (qNMR).

Pomegranate seed is rich in oil, and seed oil of pomegranate consists of conjugated fatty acids with different percentages. The current contribution covers how to determine percentages of different isomeric conjugated fatty acids. The percentages of these isomers are analysed by quantitative nuclear magnetic resonance (qNMR) using benzoic acid as an internal reference chemical with a well-defined amount. Linear mathematical equations are developed for the quantitative analysis of fatty acids found in pomegranate seed oil. The developed approach is utilised for the pomegranate seed oils prepared in the laboratory and tested for commercial samples. Among the oils derived at the laboratory, the Yeni Hicaz pomegranate cultivar seeds yielded the highest fraction of punicic acid. Among the acids, punicic acid was the one with the highest fraction, while linolenic acid was the one with the lowest percentage. These results are important in identifying pomegranate seed oils. Among the commercial samples tested with the current approach, only one of them showed similar content analysis as in the laboratory-derived oils.

Jacaranda oil administration improves serum biomarkers and bioavailability of bioactive conjugated fatty acids, and alters fatty acid profile of mice tissues.

Jacaric acid, a conjugated linolenic acid (CLNA) present in jacaranda oil (JO), is considered a potent anticarcinogenic agent. Several studies have focused on its biological effects, but the metabolism once consumed is not clear yet. The aim of this work was to evaluate the effects of two different daily doses of JO on serum parameters and fatty acid (FA) profile of mice tissues after 4 weeks of feeding. No significant changes on body weight gain, food intake, or tissue weight were determined after 0.7 or 2ml/kg of JO supplementation compared to control animals. Significantly lower blood low-density lipoproteins-cholesterol (20mg/dl) and glucose (~147-148mg/dl) levels were detected in both oil-treated groups compared to control (31.2 and 165mg/dl, respectively). Moreover, jacaric acid was partially converted into cis9, trans11 conjugated linoleic acid (CLA) and thus further incorporated into tissues. Liver evidenced the highest total conjugated fatty acid content (1.1%-2.2%), followed by epididymal (0.7%-1.9%) and mesenteric (1.4%-1.8%) fat. Lower saturated and higher unsaturated fatty acid content was detected in both oil-treated groups compared to control. Our results support the safety of JO and its potential application with a functional or nutraceutical propose, by increasing human CLNA consumption and further availability of CLA.

Improving the Production of Punicic Acid in Baker's Yeast by Engineering Genes in Acyl Channeling Processes and Adjusting Precursor Supply.

Punicic acid (PuA) is a high-value edible conjugated fatty acid with strong bioactivities and has important potential applications in nutraceutical, pharmaceutical, feeding, and oleochemical industries. Since the production of PuA is severely limited by the fact that its natural source (pomegranate seed oil) is not readily available on a large scale, there is considerable interest in understanding the biosynthesis and accumulation of this plant-based unusual fatty acid in transgenic microorganisms to support the rational design of biotechnological approaches for PuA production via fermentation. Here, we tested the effectiveness of genetic engineering and precursor supply in PuA production in the model yeast strain Saccharomyces cerevisiae. The results revealed that the combination of precursor feeding and co-expression of selected genes in acyl channeling processes created an effective "push-pull" approach to increase PuA content, which could prove valuable in future efforts to produce PuA in industrial yeast and other microorganisms via fermentation.

Nutritional evaluation, fatty acid analysis and anticancer properties investigation of seeds from kan-zaw, a potential diet oil and medicinal plant

Payenapara lleloneura Kurz. (Kan-zaw), an endemic medicinal plant only found in Tanintharyi Region of Myanmar, is widely used in the treatment of various cancer and different ailments. In the present research, the seeds were phytochemical investigated for their nutritional potential for their use as functional foods or novel diet oil resources. Nutritional evaluation showed that the seeds are rich in fats and carbohydrates (soluble sugars and starch). Fatty acid analyses showed that the seeds accumulate very rich α-eleostearic acid (α-ESA, 18:3Δ9cis,11trans,13trans), an important conjugated fatty acid, up to more than 70 ​% of total fatty acids. The seed oil derived from the Kan-zaw tree contains approximately 3.25 ​% β-eleostearic acid (18:3Δ9trans,11trans,13trans), an unusual conjugated fatty acid that imparts a potent anticancer application and industrially important drying qualities to Kan-zaw oil. Physicochemical properties of the Kan-zaw seeds were examined; petroleum ether (60–90 ​°C) extract of seed oils were also investigated for the saponification value, iodine value and estimation of acid value. Further, the present study investigated cytotoxic potential of ethanol, methanol, acetone, chloroform Kan-zaw seed extracts and commercial Kan-zaw oil against human cervical cancer cell line (HeLa). The Kan-zaw extracts and oil have shown significant anticancer activity on HeLa cells.

Oxysterols and lipidomic profile of myocardium of rats supplemented with pomegranate seed oil and/or bitter melon aqueous extract – Cardio-oncological animal model research

A close link between cardiovascular diseases and cancer results from sharing the same modifiable risk factors (e.g. nutritional) and cardiotoxicity of anti-cancerous therapies. It justifies cardio-oncological preliminary studies on dietary factors, especially on those of possible anti-carcinogenic or cardioprotective properties. The main purpose was to evaluate the effect of pomegranate seed oil (PSO) and/or bitter melon extract (BME) supplementation of the diet of female rats suffering from mammary tumors on lipidomic profile (expressed as fatty acids, conjugated fatty acids (CFA), malondialdehyde (MDA), cholesterol and oxysterols content) of cardiac tissue. Total lipidomic profile and intensity of lipid peroxidation in hearts of DMBA-treated Sprague-Dawley rats and their healthy equivalents, both obtaining diet supplementation, were evaluated with different chromatographic techniques coupled with appropriate detection systems (GC–MS, GC-TOFMS, Ag+-HPLC-DAD, UF-HPLC-DAD). Dietary modifications neither diminished breast cancer incidence nor exerted explicit cardio-protective influence, however, they diminished cholesterol content, i.a. because of inhibition of the endogenous conversion of squalene to cholesterol in cardiac tissue. CFA were incorporated into cardiac tissue to a lesser extent in the cancerous process. PSO and BME anti-oxidant properties in pathological condition were only slightly reflected in MDA levels but not in oxysterols formation. Obtained results indicate considerable changes in dietary supplements’ biological activity in pathological conditions and the need for clear distinction of drugs and dietary supplements, which is of utmost importance, especially for cancer survivors.

Measuring Conjugated Linoleic Acid (CLA) Production by Bifidobacteria.

The biological significance of conjugated fatty acids (CFAs) has been linked to positive health effects based on biomedical, in vitro, and clinical studies. Of note, conjugated linoleic acids (CLAs) are the most widely characterized fatty acids as geometric isomers cis-9,trans-11 and trans-10,cis-12CLA occur naturally in ruminant fats, dairy products, and hydrogenated oils. Concerning CLAs, it is known that bacterial biohydrogenation, a process whereby ruminal bacteria or starter cultures of lactic acid bacteria have the ability to synthesize CLA by altering the chemical structure of essential fatty acids via enzymatic mechanisms, produces a multitude of isomers with desirable properties. Bifidobacterium species are classed as food grade microorganisms and some of these strains harness molecular determinants that are responsible for the bioconversion of free fatty acids to CLAs. However, molecular mechanisms have yet to be fully elucidated. Reports pertaining to CLAs have been attributed to suppressing tumor growth, delaying the onset of diabetes mellitus and reducing body fat in obese individuals. Given the increased attention for their bioactive properties, we describe in this chapter the qualitative and quantitative methods used to identify and quantifyCLA isomers produced by bifidobacterial strains in supplemented broth media. These approaches enable rapid detection of potential CLA producing strains and accurate measurement of fatty acids in biological matrices.

Conjugated Fatty Acids in Muscle Food Products and Their Potential Health Benefits: A Review.

Conjugated fatty acids (CFAs) are a group of positional and geometric isomers of polyunsaturated fatty acids (PUFAs) with conjugated double bonds. There are several subgroups of CFAs including conjugated linoleic acids (CLAs), conjugated linolenic acids (CLNAs), conjugated eicosapentaenoic acids (CEPAs), and conjugated docosahexaenoic acids (CDHAs). CFAs, especially CLAs, have been studied in recent years both for their health benefits and factors that affect their level in muscle food products. CFAs have been reported in numerous studies as having antitumor, antiobesity, antidiabetes, anticardiovascular disease, and modulating immune system effects. These biological activies are involved in changes of lipid peroxidation and energy expenditure, as well as inhibitory effects on the hormone receptor, lipid metabolism, lipoprotein lipase activity, and adiponectin production. A large body of studies has revealed that the diet, processing, storage conditions, slaughter season, and age are common factors that affect CFA content in muscle food products, as detailed in this review. Recommendations are made regarding animal farming and meat product processing to obtain high CFA content meat products and to optimize the benefits of CFA for health promotion.

Microbial enrichment of blackcurrant press residue with conjugated linoleic and linolenic acids.

The aim of the study was to investigate the isomerization of linoleic (LA) and linolenic acids (LNAs) into their conjugated isomers by Propionibacterium freudenreichii DSM 20270 and utilize this feature for microbial enrichment of blackcurrant press residue (BCPR) with health-beneficial conjugated fatty acids. First, the ability of P. freudenreichii to isomerize 0·4mgml-1 of LA and LNA was studied in lactate growth medium. Free LA and α-LNA were efficiently converted into conjugated linoleic (CLA) and α-linolenic acid (α-CLNA), being the predominant isomers c9,t11-CLA and c9,t11,c15-CLNA, respectively. The bioconversion of α-LNA by P. freudenreichii was more efficient in terms of formation rate, yield and isomer-specificity. Thereafter, free LA and LNAs obtained from hydrolysed BCPR neutral lipids, by lipolytically active oat flour, were subjected to microbial isomerization in BCPR slurries. In 10% (w/v) slurries, a simultaneous enrichment in c9,t11-CLA and c9,t11,c15-CLNA of up to 0·51 and 0·29mgml-1 was observed from starting levels of 0·96mg LA ml-1 and 0·37mg α-LNA ml-1 respectively. This study shows that growing cultures of P. freudenreichii DSM 20270 are able to simultaneously enrich BCPR with health-beneficial conjugated isomers of LA and α-LNA. This study demonstrates that microbial isomerization technique can be utilized to enrich lipid-containing plant materials with bioactive compounds and thereby enable valorization of low value plant-based side streams from food industry into value-added food ingredients.