Validation of an Analytical Method to Determine Melatonin and Compounds Related to <Emphasis Type="SmallCaps">l</Emphasis>-Tryptophan Metabolism Using UHPLC/HRMS

Melatonin is a bioactive compound that is present in wines because of the metabolism of l-tryptophan by yeasts. Even though the complete pathway of synthesis is not well elucidated, certain related indolic compounds might be involved in it. Consequently, their determination is a matter of interest. On one hand, their formation during fermentation might be related to a specific role for yeasts metabolism, not known so far. On the other hand, the synthesis by yeasts of bioactive compounds with putative health benefits for consumers, such as melatonin or serotonin, is a relevant matter. This paper aims to develop and validate an analytical method by ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC/HRMS) to monitor both melatonin and related indolic compounds, in order to decrease their detection limits and make it possible to assess their occurrence in culture medium and fermented products. In addition, the other objective is to evaluate the production of these compounds by a commercial Saccharomyces cerevisiae used to make white wines. Diminishing the limit of detection below 0.5 ng mL^?1 for all compounds under study is an achievement of this work. Furthermore, the strain under study (AROMA WHITE) has been found to synthesise melatonin and related compounds as serotonin. Additionally, the evolution of these compounds over time may contribute to understanding the role that they play in yeast metabolism.     

Determination of <Emphasis Type="SmallCaps">d</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis>-Amino Acids in Collagen from Pig and Cod Skins by UPLC Using Pre-column Fluorescent Derivatization

A simple and sensitive analytical method for the quantification of d,l-amino acids by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection is described. The reaction of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] with d,l-hydroxyproline (Hyp), glycine (Gly), d,l-aspartic acid (Asp), and d,l-proline (Pro) effectively proceeds at 55 °C for 20 min in the presence of 3% TEA to produce the corresponding fluorescent diastereomers (excitation at 460 nm, emission at 550 nm). The mixture was simultaneously separated within 20 min on an ACQUITY UPLCTM BEH C18 (1.7 μm, 100 mm?×?2.1 mm i.d.) by gradient elutions using water–acetonitrile containing 0.2% formic acid as the mobile phase. Peak resolution was in the range of 1.62 (d,l-Asp), 2.99 (d,l-Pro), and 6.74 (d,l-Hyp). A good linearity was achieved from the calibration curves (r²?>?0.9984) in the range of 1.0~100 pmol; the limit of detection (S/N?=?3) was 42.0–250 fmol, the inter-day and intra-day assay precisions were all less than 6.23%, and the mean recoveries (%) of the d,l-amino acids spiked in the collagen from pig and dried cod skins were 87.58–107.41%. The derivatives of the free d,l-amino acids in the collagen were successfully identified by the proposed procedure. To the best of our knowledge, for the first time, these three kinds of d-amino acids, which were d-Asp, d-Pro, and d-Hyp, were detected in the collagen samples.     

Improvement of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> by genome shuffling for the efficient production of arabitol from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

Arabitol is used in the food industry as a low-calorie sweetener. It is produced by yeasts during the biotransformation process of l-arabinose. Genome shuffling was performed in Candida parapsilosis DSM 70125, an efficient producer of arabitol, to obtain fusants with improved arabitol production ability. Four mutants from the parental library were used for the first round of genome shuffling. The best fusants, GSI-1 and GSI-10A, were subjected to a second round of genome shuffling. Finally, two fusants, GSII-3 and GSII-16, produced concentrations of arabitol that were 50% higher than that of the wild-type strain during selection culture. Under the optimal conditions established for C. parapsilosis, the two fusants produced 11.83 and 11.75 g/L of arabitol and were approximately 15–16% more efficient than the wild-type strain. Flow cytometry analysis showed that the ploidy of the new strains did not change.     

Lipase-catalyzed acylation of <Emphasis Type="SmallCaps">l</Emphasis>-carnitine with conjugated linoleic acid in [Bmim]PF<Subscript>6</Subscript> ionic liquid

Improving significantly the acylation reaction of l-carnitine with conjugated linoleic acid catalyzed by lipase needs to select an efficient and suitable reaction medium that is not only polar but also hydrophobic. [Bmim]PF₆ which is satisfied with the above two requirements was applied as the reaction medium. The optimal reaction conditions were: Novozyme 435 as the catalyst, 5:1 of molar ratio of fatty acid to l-carnitine, 40 g L⁻¹ lipase, 0.22 a_W of initial water activity, 125 g L⁻¹ molecular sieves (they were added within 0–3 h of reaction time), 60 °C of reaction temperature, 200 rpm of rotation speed, 32 h of reaction time. The overall conversion could reach 98.27%. The conversion in [Bmim]PF₆ was 2.13 and 1.56 times higher than that in acetonitrile and in solvent-free system, respectively. The lipase in the present work could be used eight times. [Bmim]PF₆ as the reaction medium was better than acetonitrile and solvent-free system.     

Effects of <Emphasis Type="SmallCaps">l</Emphasis>-lysine on thermal gelation properties of chicken breast actomyosin

The effects of l-lysine (l-Lys) on the water holding capacity (WHC) and texture of actomyosin (AM) gel and the possible mechanisms were investigated. l-Lys increased the WHC and hardness of the AM gel. These effects may be related to the even and continuous microstructure of the gel according to the scanning electron microscopy analysis. Furthermore, l-Lys increased the surface hydrophobic residues and the reactive sulfhydryl groups. l-Lys decreased the storage modulus at the first transition temperature but increased it at the second transition temperature and the third transition enthalpy. These results suggested that l-Lys varied the thermal behaviors and the microstructure of the AM gel by increasing the surface hydrophobicity and reactive sulfhydryl groups, ultimately contributing to the increased WHC and hardness. The changes in pH did not fully explain the results from the present study. The results were useful for understanding previous findings and may serve as a reference for the preparation of reduced-sodium and phosphate-free meat products.     

<Emphasis Type="SmallCaps">l</Emphasis>-Cysteine Functionalized Silica Gel as an Efficient Adsorbent for the Determination of Heavy Metals in Foods by ICP-MS

A novel l-cysteine functionalized silica gel adsorbent (SG-Cys) was successfully synthesized and characterized by Fourier transform infrared (FTIR) spectra and elemental analysis. An efficient method for simultaneous determination of heavy metals [V (V), Cr (VI), Cu (II), As (V), Cd (II), and Pb (II)] was developed by inductively coupled plasma-mass spectrometry (ICP-MS) coupled with pre-concentration with the prepared SG-Cys adsorbent. The experimental parameters, including the solution pH, sample flow rate, eluent volume, eluent type, and concentration, have been systematically optimized to obtain higher enrichment factors of target ions. Under the optimum conditions, the enrichment factors of V (V), Cr (VI), Cu (II), As (V), Cd (II), and Pb (II) reached 123, 100, 86, 106, 97, and 94, respectively, and the detection limits were as low as 1.8–3.7 ng L^?1. The proposed method has been applied to the determination of trace heavy metals in water, rice, wheat, corn, and tea samples, which exhibited a satisfactory recovery in the range of 90.6–106.0 %.     

Aqueous medium enzymatic preparation of <Emphasis Type="SmallCaps">l</Emphasis>-alpha glycerylphosphorylcholine optimized by response surface methodology

The ability of Rhizopus chinensis lipase (Thermo 4S-3) to catalyze the deacylation of l,2-diacyl-sn-glycero-3-phosphocholines (sn-1,2-PC) for l-alpha glycerylphosphorylcholine (l-α-GPC) preparation was investigated. Response surface methodology (RSM), based on a modified central composite rotatable design, was employed to examine the effects of substrate concentrations, temperature, enzyme loading, and dosage of CaCl₂ on the l-α-GPC yield. RSM analysis indicated good correlation between experimental and predicated values. The optimal condition was confirmed as follows: reaction time, 3.5 h; temperature, 43 °C; enzyme loading, 28.2 U mL⁻¹; substrate concentration, 51.5 mg mL⁻¹; and dosage of CaCl₂, 1.9 mg mL⁻¹. Under these conditions, the l-α-GPC yield increased by 96.8%, which was close to the amount predicted by the model.     

Content and distribution of vicine,convicine and <Emphasis Type="SmallCaps">l</Emphasis>-DOPA during germination and seedling growth of two <Emphasis Type="Italic">Vicia faba</Emphasis> L. varieties

This work examined the content and distribution of the pyrimidine glucosides vicine and convicine, and the nonproteic amino acid l-DOPA in cotyledons and embryo axes along the germination and seedling growth of Vicia faba L. vars. Alameda and Brocal. The behaviour of these compounds was similar in both varieties. Vicine and convicine, implicated in favism disease, slowly declined in cotyledons. In embryo axis, vicine levels were sharply reduced and convicine amount was slightly increased as assay progressed. Total pyrimidine glucosides remained unchanged in the whole plant during the study. l-DOPA only appeared in the embryo axis of V. faba seeds and the highest content was observed in Brocal variety at 6 DAI (days after imbibition). The information provided in this study could be valuable for a possible role of embryo axis, rich in l-DOPA, for the treatment of Parkinson’s disease and also as a choice ingredient for functional foods or as nutraceutical.     

Tyramine production of technological important strains of <Emphasis Type="Italic">Lactobacillus</Emphasis>, <Emphasis Type="Italic">Lactococcus</Emphasis> and <Emphasis Type="Italic">Streptococcus</Emphasis>

The aim of this paper was to study the biogenic amines (histamine, tyramine, putrescine, cadaverine, agmatine, spermine and spermidine) production of selected technological important lactic acid bacteria (strains of the genera Lactococcus, Lactobacillus and Streptococcus). Three methods (ion-exchange chromatography (IEC), PCR and cultivation method with pH indicator) were used. Within the 39 strains of lactic acid bacteria tested, the production of tyramine (formed by tyrosine decarboxylase) was detected in eight strains (3 strains of Lactococcus lactis subsp. lactis, three strains of Lactococcus lactis subsp. cremoris, 1 strain of Streptococcus thermophilus and 1 strain of Lactobacillus delbrueckii subsp. bulgaricus). The other tested biogenic amines were not detected. Cultivation in decarboxylation broth seems to be the least accurate method for the detection of biogenic amines due to enhanced risk of false-positive reactions. Therefore, in order to detect bacteria producing biogenic amines, the combination of PCR and chromatographic methods (e.g. IEC) can be recommended.     

Estrogenic effects of various extracts from <Emphasis Type="Italic">Chamdanggui</Emphasis> (<Emphasis Type="Italic">Angelica gigas</Emphasis> Nakai) and <Emphasis Type="Italic">sogdan</Emphasis> (<Emphasis Type="Italic">Phlomis umbrosa</Emphasis> Turcz)

The various extracts from chamdanggui (Angelica gigas Nakai) and sogdan (Phlomis umbrosa Turcz) were evaluated for estrogenic activity and characterized according to HPLC profile. Chamdanggui and sogdan were individually extracted with 4 solvents (hot water, 70% ethanol, n-butanol, and dichloromethane) of differing polarities. Estrogenic activity was determined by E-screen using an estrogen-dependent MCF-7 BUS cell. Although almost all extracts showed estrogenic effects in a concentrationdependent manner, the hot water extract from chamdanggui (250 μg/mL) had the higher effect (138%). Among 90 fractions using HPLC separation of the hot water extract from chamdanggui, fraction 21 and 28 produced the highest estrogenic effects of 178 and 163% at 10 μg/mL, respectively. The results imply that the hot water extract from chamdanggui could be useful as an alternative hormone replacement therapy.     

A Novel Technological Process of Extracting <Emphasis Type="SmallCaps">l</Emphasis>-Tyrosine with Low Fluorine Content from Defatted Antarctic Krill (<Emphasis Type="Italic">Euphausia superba</Emphasis>) By-product by Enzymatic Hydrolysis

Antarctic krill (Euphausia superba) offers a high potential for development of innovative food products due to its large reserves and satisfied nutritional values. Defatted krill powder by-product generates from the process of krill oil, which is rich in various types of amino acids. This study aimed at recovering tyrosine from defatted krill by-product through enzymatic hydrolysis. Defatted krill by-product was hydrolyzed with three types of protease under optimum conditions with different incubation time. Free tyrosine dissociated from protein through hydrolysis by trypsin and was detected in the protein hydrolysates. Content of tyrosine increased with the increasing of incubation time till 5 h. Tyrosine extract contained 6.43 mg/kg fluorine and over 95 % of tyrosine, which demonstrated its high purity and safety. Tyrosine extract showed rod-shaped microstructure through scanning electron microscope analysis. Crystalline structure and thermal properties of tyrosine extract agreed well with those of l-tyrosine standard. During this process, both tyrosine of high purity and bioactive peptide products were obtained.     

Thermal inactivation kinetics of quality-related enzymes in cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">botrytis</Emphasis><Emphasis Type="Bold">)</Emphasis>

Thermal inactivation of quality-related enzymes in both cauliflower crude enzyme extracts and fresh tissue samples was studied in temperature range 50–100 °C. For crude enzyme extracts, several parameters, reaction rate constants (k) and activation energy (E _a) as well as decimal reduction time (D) and (z) values, were used to characterize the thermal stability. The rates of inactivation were found to follow first-order inactivation kinetics. Activation energies varied between 101.18 and 208.42 kJ mol⁻¹ with z values of 10.59–24.09 °C. The examined kinetics indicated that lipoxygenase was the most heat resistant followed by peroxidase, polyphenol oxidase, pectin methyl esterase and ascorbic acid oxidase. Furthermore, the obtained results from the blanched fresh tissues indicated that inactivation of lipoxygenase secured disappearing of any other enzyme activities. Therefore, this study recommends using lipoxygenase as an indicator enzyme to optimize the thermal treatments of cauliflower products.     

Contamination of commercially available <Emphasis Type="SmallCaps"> L-</Emphasis>tryptophan by related substances

In 1989 a new autoimmune disease, the eosinophilia myalgia syndrome (EMS), was traced back to the intake of L-tryptophan (Trp) of a single manufacturer, Showa Denko (SD). In an epidemiological study, six minor contaminants (10-1,000 mg/kg Trp) were related to the occurrence of EMS. In this study the pattern of contaminants in pharmaceutical- and feed-grade Trp was investigated in a market survey of 21 lots of Trp raw materials of six different manufacturers, as well as one bioconversion broth, and four tablets. For total Trp-related impurities all UV_{220 nm} detectable substance peaks separated from Trp by RP-HPLC were calculated as N -acetyl-tryptophan. In all samples 4-,5-, 6-, and 7-OH-Trp, 1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (THCC), 1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (MTHCC) and 1-(3-indolylmethyl)-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (IMTHCC) as well as 2-[2,3-dihydroxy-1-(3-indolyl)-propyl]- L- tryptophan (dhPIT) were determined, since the last four are known to be major contaminants in biotechnologically manufactured Trp. Additionally, four EMS-related substances were been analysed: 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid (PIC), 2-(3-indolylmethyl)- L- tryptophan (IMT), 1,1'-ethylidenebis-( L- tryptophan) (EBT) and 3-(phenylamino)alanine (PAA). EBT was only detectable in a single SD-Trp sample known to be EMS associated. Low amounts of PAA (20 mg/kg) were detected in feed-grade Trp of one manufacturer as well as in SD-Trp. IMT was found in small amounts in recent pharmaceutical-grade Trp (10-25 mg/kg Trp), but in higher amounts in feed-grade Trp samples (120-1,400 mg/kg) as well as in pharmaceutical-grade Trp and Trp tablets manufactured prior to 1989 (70-660 mg/kg). PIC is a primary oxidation product of Trp; thus we detected 4-50 mg/kg of the earlier-eluting PIC diastereomer in recent pharmaceutical-grade Trp and up to 2,500 mg/kg in feed-grade Trp. Non-EMS correlated THCC, MTHCC, IMTHCC, and dhPIT proved to be the main contaminants in amounts of <10-13,500 mg/kg.     

Production of biogenic amines by<Emphasis Type="Italic"> Morganella morganii</Emphasis>,<Emphasis Type="Italic"> Klebsiella pneumoniae</Emphasis> and<Emphasis Type="Italic"> Hafnia alvei</Emphasis> using a rapid HPLC method

The histidine decarboxylating activity and production of biogenic amines by Morganella morganii (NCIMB, 10466), Klebsiella pneumoniae (NCIMB, 673) and Hafnia alvei (NCIMB, 11999) were investigated using a rapid HPLC method. Derivatisation of the bacterial samples was carried out using benzoyl chloride. A gradient elution system was used for analysis with a mixture of acetonitrile and HPLC grade water. Bacterial strains not only produce histamine in histidine-enriched broth but also the other biogenic amines. The chromatographic results show that bacterial strains are also capable of producing spermine and spermidine in histidine-enriched broth. Bacterial ammonia production by all three strains was clearly detected since ammonia is generated during the degradation of histidine. The study demonstrates that the highest histamine production was obtained by Morganella morganii, followed by Klebsiella pneumoniae, and the lowest with the Hafnia alvei. Therefore, Morganella morganii and Klebsiella pneumoniae have strong histidine decarboxylase activity since they are prolific histamine-forming bacteria     

A New Multiplexed Real-Time PCR Assay to Detect <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, <Emphasis Type="Italic">C. coli</Emphasis>, <Emphasis Type="Italic">C. lari</Emphasis>, and <Emphasis Type="Italic">C. upsaliensis</Emphasis>

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 10² genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.     

Prevalence,genetic diversity,and antibiotic susceptibility of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. (<Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>) isolated from <Emphasis Type="Italic">Sunshik</Emphasis>, its ingredients and soils

The prevalence, genetic diversity, and antibiotic susceptibility of Cronobacter species (Enterobacter sakazakii) isolated from sunshik products, its ingredients, and root vegetable farm’s soils were investigated to analyze main reservoirs and contaminated sources of Cronobacter spp. Cronobacter spp. was isolated from 9 of 15 sunshik products, 26 of 72 its ingredients, and 2 of 39 soils. The root vegetables such as sweet potato and carrot showed higher contamination rate (70%) than the other sunshik ingredients. All isolates showed 929 bp band amplified from 16S rRNA and resistant to ampicillin, amoxicillin/clavulanic acid, and cefazolin. All isolates showed diverse random-amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) band patterns. However, 3 cases of RAPD banding patterns between clinical strains and isolates from sunshik products and root vegetables (yam, carrot) were related with similarities level of 80%. These studies indicated that root vegetables can be an important contamination source of Cronobacter spp. in sunshik products. Thus, the preparation of root vegetables for manufacturing sunshik products used as a weaning diet was handled with more care than the other sunshik ingredients.     

Starch,protein, glycoalkaloids,and <Emphasis Type="SmallCaps">l</Emphasis>-ascorbic acid content in tubers of genetically modified potato cv. Irga

Changes in contents of protein, starch, ash, l-ascorbic acid, and glycoalkaloids in tubers of 15 clones of cultivar Irga transformed with viral genome sequences in order to improve their resistance to a necrotic strain of potato virus Y (PVY^N) were investigated. The influence of modification type and year of cultivation on the contents of all chemical compounds examined for particular modified clones appeared to be irregular over the 3-year period of examination, and the clone of the best and stable content of chemical compounds was not found. Stable tendencies in the chemical compounds of tubers composed into groups and subgroups of clones representing the same modification type were not confirmed either. An essential stable decrease in glycoalkaloid content and an increase in starch content (compared with parental cultivar Irga) were found in almost all genetically modified clones. Glycoalkaloid content in green tubers of modified clones was usually (except three clones) higher than this for parental cultivar Irga. Correlation between glycoalkaloid content and tuber size for all, normal and green tubers of selected clones and parental cultivar Irga was examined as well.     

In vivo study of antiallergenicity of ethanol extracts from <Emphasis Type="Italic">Sargassum tenerrimum</Emphasis>, <Emphasis Type="Italic">Sargassum cervicorne</Emphasis> and <Emphasis Type="Italic">Sargassum graminifolium turn</Emphasis>

Food allergy has becoming the serious threat in the world for which the search of an effective anti-allergic drug is the demand of time. Keeping in view of the potentiality of seaweeds, the ethanol extracts from Sargassum tenerrimum (ST), Sargassum cervicorne (SC), and Sargassum graminifolium turn (SG) have been studied in vivo for its antiallergenicity through passive cutaneous anaphylaxis (PCA) and active cutaneous anaphylaxis (ACA) in female BALB/c mice. Intraperitoneal administration of these ethanol extracts inhibit mouse PCA and ACA in a dose-dependent manner using ovalbumin (OVA) and shrimp allergen as triggering agents to induce allergenicity over mice. The extract of ST containing phlorotannin has been found most active over the suppression of PCA triggered by OVA and shrimp with IC₅₀ values of 25.64 and 40.98 mg/kg, respectively and an efficacy comparable to that of an anti-allergic drug disodiumcromoglycate. Similarly, ST inhibits ACA triggered by ova and shrimp allergen in the mouse, with 50% suppression at 25.5 and 43.53 mg/kg, respectively. The results presented here show that these extracts are active on the studied models among which ethanol extract of ST was the most potent, leading toward the promising development of a new class of anti-allergic drugs.     

Molecular diversity among natural populations of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>/<Emphasis Type="Italic">paraplantarum</Emphasis> strains isolated from autochthonous dairy products

The diversity of 87 Lactobacillus paracasei and Lactobacillus plantarum/paraplantarum strains, previously identified from different autochthonous dairy products, was investigated by phenotypic and genotypic approaches. The increased resolution obtained using phenotypic and genotypic characterization allowed the level of strain heterogeneity detection to be widened. Phenotypic diversity was evaluated by studying biochemical characteristics of technological interest, including antimicrobial and proteinase activities, resistance to nisin, aggregation ability, production of exopolysaccharides, acetoin and diacetyl, citrate utilization, and antibiotic susceptibility. Genotypic diversity was generally evaluated by PCR amplification of repetitive bacterial DNA element fingerprinting using the (GTG)₅ primer [(GTG)₅-PCR]. Moreover, in cases where strains were not discriminated by (GTG)₅-PCR combined with phenotypic analysis, pulsed-field gel electrophoresis (PFGE) analysis was performed. The results indicate that L. plantarum/paraplantarum and L. paracasei natural isolates from artisanal dairy products are a gold mine in terms of diversity of strains and could be potentially interesting to dairy companies for the formulation of functional starter cultures in the production of innovative foods.