Improving Nutritional Properties and Extractability of Pea Proteins for Human Consumption via Fungal Bioprocessing

Yellow peas are a rich source of protein and phytochemicals. Peas are compatible with many diets restricted by sensitivities, allergies, or personal choice. As a result, peas are gaining popularity in food markets, despite the challenges they present. The biggest challenges for pea proteins in food markets are high content of fiber, starch, and saponins. These components cause processing challenges and create undesirable textures and bitter flavors. Pea proteins are typically extracted with alkaline extraction or air classification methods which result in 80% and 50% protein products respectively. With low protein purity, these products are not competitive with other, more established, plant-based proteins like soy. The objectives of this study are to (1) Determine the effect of fungal fermentation on the protein, saponin, phenolic, and dietary fiber content of dry processed proteins (DPP), (2) Describe the effects fungal fermentation has on the saponin, protein, starch, and phenolic composition of dehulled peas (DHP), (3) Understand how fermentation, drying method, and antimicrobial techniques affect alkaline extraction and isoelectric precipitation from DPP and DHP. In DPP and DHP, fermentation has potential to improve composition. Fermentation increased protein, total saponins, and total phenolic content for both substrates. During fermentation, total dietary fiber decreased in DPP, and starch content decreased in DHP. With high fiber- and starch- contamination in DPP and DHP respectively, these results indicate that fermentation may reduce these unwanted components. In addition, increased phenolic- and saponin content may provide increased antioxidant activity and health benefits. Extraction of both DPP and DHP indicate that chemical antimicrobials are superior to autoclaving in terms of protein yield, extraction yield and protein content of isolates. Drying method does not significantly impact protein extraction. Fermentation increased solubility but resulted in unrecoverable protein during isoelectric precipitation. Methods such as ultrafiltration may be a superior method for protein recovery. These results demonstrate that fungal fermentation has potential to broaden the application of pea proteins in food markets. Fungal fermentation improves composition of DPP and DHP in regard to protein and starch as well as non-nutritive components like saponins, phenolics and fiber. Fermentation also may produce a more soluble protein isolate

Advance in Recovery and Application of Bioactive Compounds from Seafood

Due to the increased focus on circular bioeconomies, the full utilization of marine biomasses, including side streams from the seafood processing industry, as well as the utilization of hitherto unexploited biomasses, such as star fish, mussels, seaweed, and microalgae, are receiving increased attention from both academia and the industry. These marine biomasses contain a wide array of bioactive compounds with beneficial and/or functional health properties, which can be exploited for applications in food, feed, dietary supplements, or pharma. New technologies are being developed for the recovery and preservation of bioactive compounds from these resources. Technologies for preserving perishable bioactive compounds are particularly important during the storage of seafood side streams before extraction as well as during the extraction, concentration, purification, and storage of the extracted compounds. Advanced application refers to new applications of the bioactive compounds in, for example, food products or new technologies for the incorporation of these bioactive compounds in food, feed, dietary supplements, or drugs

Soybean and Nutrition

Worldwide, soybean seed proteins represent a major source of amino acids for human and animal nutrition. Soybean seeds are an important and economical source of protein in the diet of many developed and developing countries. Soy is a complete protein and soy-foods are rich in vitamins and minerals. Soybean protein provides all the essential amino acids in the amounts needed for human health. Recent research suggests that soy may also lower risk of prostate, colon and breast cancers as well as osteoporosis and other bone health problems and alleviate hot flashes associated with menopause. This volume is expected to be useful for student, researchers and public who are interested in soybean

Plantebasert fiskefôr : overføringspotensialet til mykotoksiner og fytoøstrogener fra fôr til fisk og konsekvenser for fiskehelse og mattrygghet

The introduction of novel feed ingredients has brought tremendous changes in the field of aquaculture. The shift to non-marine resources was necessary because of rising prices for fish meal and fish oil and their decreasing availability. At the same time, the total global as well as the per-capita fish consumption has increased, which has caused an enormous expansion of the fish farming industry. The introduction of suitable alternatives for the fish-derived feed components was therefore a pre-requisite for the growth of aquaculture. Novel feed ingredients are currently mostly plant-based, including vegetable oils and protein concentrates from different legumes or wheat gluten. The change in aquafeeds towards “green” ingredients may have negative effects on fish health and product quality, which in turn can lead to economic losses. The main concerns are connected to the presence of plant-borne contaminants including endogenous anti-nutritional factors (ANF). However, new feed processing techniques methods have considerably reduced their occurrence, and sensitive detection methods allow controlling compliance with maximum levels that have been implemented by food and feed safety authorities. Nevertheless, some ANF such as natural toxins, phytoestrogens and allergenic peptides are rather resistant to heat and digestion and have the potential to be carried over into the food chain. The biological activities of these substances in fish is little known and requires more detailed investigation. This thesis addresses the impact of “green” aquafeeds on fish health and food safety, focussing on mycotoxins and phytoestrogens that are typically present in plant-based ingredients used for fish diets. The plant ingredients used in this study were wheat gluten (WG), soybean protein concentrate (SPC) and pea protein concentrate (PPC), which were fed to zebrafish (Danio rerio) and Atlantic salmon (Salmo salar L.) with the aim to investigate possible effects on the fish and the transmissibility potentials of target ANF into edible parts in connection with food safety. The project objectives were formulated to address these questions by analysing different aspects of this complex issue. All experiments performed in the course of the project were based on samples from initially conducted feeding trials in zebrafish and salmon using different custom-made diets produced with definite levels of the three selected plant protein preparations. Control feed was based on fish meal (FM) as the only source of protein, whereas the test diets contained WG, SPC or PPC at levels of 15% or 30%, replacing the FM. Salmon were exposed to five feed types, i.e. FM, WG15, WG30, SPC15 and SPC30, while zebrafish were additionally exposed to PPC15 and PPC30. The first analytical project activities were performed with the aim of identifying the carry-over potentials of 25 mycotoxins and phytoestrogens that are frequently occurring in the selected plant protein sources. A quantitative multi-analyte liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) method for the simultaneous determination of the target substances in feed and fish matrices was developed and validated. Sample preparation was optimised for each matrix by selecting suitable extraction solvents. The analysis of the method performance characteristics showed high specificities for all analytes with sufficient to excellent sensitivities in the different matrices. Linear calibration curves were generated either by using matching stable isotope-labelled derivates or similar-structure homologues as internal standards, or by using matrix-matched external standards. The method precision and accuracy data were in the range suggested in international validation guidelines for quantitative instrumental analysis. Applying the 25-in-1 method to the analysis of feed and fish samples from the feeding trials showed only low levels of the mycotoxin enniatin B in WG-containing diets and of the isoflavones daidzein (DAI), genistein (GEN) and glycitein (GLY) and their respective glucoside forms in SPC- and PPC-based diets, demonstrating the effectiveness of maximum level recommendations and modern feed processing technologies in the Norwegian aquaculture industry. Consequently, carry-over into fish muscle was not observed, confirming that fillets from plant-fed salmon were safe for human consumption. The second objective in this project was to understand the metabolic fate of major soybean isoflavones in exposed salmon. Products from the hepatic biotransformation of DAI, GEN and GLY in salmon had not been previously identified, but knowledge about possibly harmful metabolites is essential for the assessment of potential risks. Due to the lack of suitable metabolism models for salmon, liver microsomes and primary hepatocytes were prepared in-house for applications in in vitro metabolism assays. The salmon liver fractions were characterised with respect to Phase I cytochrome P450 (CYP) and Phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates. Two new methods, one for the simultaneous measurement of five CYP activities and one for five UGT activities, were developed using LC-Triple-Quadrupole tandem mass spectrometry (TripleQ-MS/MS) for the specific detection of the substrates and their metabolites. The methods were optimised in assays with commercially available human liver microsomes with known enzyme activities and then used for the salmon liver preparations, under consideration that the available probe substrates were specific for human CYP and UGT enzymes. Subsequently, DAI, GEN and GLY were metabolised by using the salmon liver microsomes and primary hepatocytes and metabolites were analysed by LC-HRMS/MS. The isoflavones were effectively eliminated by UGT to metabolites that were preliminarily identified as the 7-O-glucuronides based on the measurement of exact masses, fragmentation patterns, and retention times. In contrast, the production of oxidative metabolites was insignificant. Only small amounts of four mono-hydroxylated DAI metabolites were detectable, when the incubations were upscaled. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soybean-containing aquafeeds are unlikely. The third and fourth project activities were committed to the identification of potential nutrigenomic effects from plant protein-based diets on fish health using transcriptomic analysis of fast muscle, liver and intestine of exposed zebrafish and salmon. Microarray hybridisation analysis was conducted in liver and intestine of salmon fed with FM, WG, and SPC containing diets. Gene expression data were processed and analysed with Nofima’s bioinformatics software STARS. In addition, growth performance parameters and apparent digestibilities were recorded, and typical enzyme biomarkers for liver health were analysed in serum. The gene expression profiles in the different salmon tissues showed that several metabolic pathways were the least affected by the SPC15 diet and the most affected by the WG30 diet. The majority of the changes in gene expression patterns occurred in the intestine, in particular in genes related to lipid metabolism and transport, sterol metabolism, immunity and tissue structure and integrity. This study confirmed thus results of previous research on effects of SPC in salmon; however, the observations with regard to WG30 feed implied that the salmon were undergoing nutritional stress and showed symptoms similar to those of gluten sensitivity in humans. In zebrafish, global transcriptome changes were analysed in fast muscle after exposure to FM, SPC30 or WG30 diets using RNA-seq technology. The analysis was extended to on-growing salmon by testing selected differentially expressed genes in the zebrafish model using salmon paralogue-specific qPCR assays. Global gene expression changes in the muscle of zebrafish fed with plant diets were moderate, with the highest changes observed in fish fed with the SPC diets, whereas no changes were found for the PPC diets, when compared to the FM-control group. Differentially expressed genes in the SPC and WG feeding groups had important functions in regulating muscle growth, maintaining muscle structure and function, and muscle tissue homeostasis. Most of those genes and their paralogues were similarly affected in salmon fed with the same diets, with some species-specific regulation. In conclusion, the work presented in this thesis shows that the inclusion of plant-based ingredients into aquafeeds is not of concern for consumers of salmon products regarding potential health risks from the carry-over of mycotoxins and phytoestrogens, but that a high percentage of WG or SPC in the diet can cause adverse effects in fish.Introduksjonen av nye ingredienser i fôr har medført store forandringer innen fiskeoppdrett. Overgangen til ikke-marine ressurser har vært nødvendig i forhold til stigende priser og redusert tilgang på fiskemel og fiskeolje. Samtidig har etterspørselen etter fisk økt på både globalt og individuelt nivå, noe som har ført til sterk vekst innen fiskeoppdrett. Innføringen av passende alternativer til fiskebaserte ingredienser i fôret har vært og fortsetter å være grunnleggende for den sterke veksten innen denne sektoren. De fleste nye ingredienser til fiskefôr baserer seg på planter, for eksempel vegetabilske oljer og proteinkonsentrater fra forskjellige erteblomstplanter og hvetegluten. Overgangen til «grønne» ingredienser i fiskefôr kan ha negative konsekvenser for fiskens helse, og dermed sluttproduktets kvalitet, noe som kan igjen kan føre til økonomiske tap. Hovedbekymringene knytter seg til kontaminanter som forekommer i planter, blant annet endogene ernæringsmotvirkende faktorer (ANF). Likevel har nye fôrprossesseringsteknikker redusert forekomsten av disse, i tillegg til at presise målingsmetoder gjør at nivåene holder seg innenfor begrensningene som er satt av myndighetene. Til tross for dette viser enkelte ANF, blant annet naturlige toksiner, fytoøstrogener og peptider med allergipotensiale, motstandsdyktighet mot varmebehandling og fiskens fordøyelse, og kan muligens bli overført i næringskjeden. De biologiske virkningene til disse stoffene i fisk er lite kjent, og må forskes nøyere på. Denne avhandlingen omtaler virkningen av «grønt» fiskefôr på fiskens helse og matsikkerhet og fokuserer på mykotoksiner og fytoøstrogener som til vanlig forekommer i plantebaserte fôringredienser for fiskenæring. I denne studien ble det brukt hvetegluten (WG), soyaproteinkonsentrat (SPC) og erteproteinkonsentrat (PPC), som ble fôret til sebrafisk (Danio rerio) og laks (Salmo salar L.) med målet å undersøke mulige virkninger på fisken og overføringspotensialet av utpekte ANF i henhold til mattrygghet. Prosjektets mål ble utledet for å ta for seg problemstillingen ved å se på de forskjellige aspektene av det sammensatte emnet. Alle eksperimentene som ble utført i løpet av prosjektet er basert på fôringsforsøk på laks og sebrafisk, som ble utført i begynnelsen av studien, ved hjelp av skreddersydde fôrtyper, med fastsatte nivåer av de tre utvalgte preparatene av planteproteiner. Kontrollfôret var basert på fiskemel (FM) som eneste proteinkilde, imens fôrtypene som ble testet inneholdt WG, SPC eller PPC i 15% og 30% konsentrasjon, som erstatning for FM. Laksen ble eksponert for fem fôrtyper: FM, WG15, WG30, SPC15 og SPC30, imens sebrafiskene i tillegg ble eksponert for PPC15 og PPC30. De første analysene i prosjektet ble foretatt med mål om å identifisere overføringspotensialet til 25 ulike mykotoksiner og fytoøstrogener som ofte opptrer i de nevnte planteproteinene. En kvantitativ væskekromatografisk høyoppløsende massespektrometrisk metode (LC-HRMS/MS) som samtidig analyserer og måler de utvalgte analyttene i fiskefôr og fiskeprøver ved å velge ut egnede løsemidler. Metoden viste høy spesifisitet for alle analytter med tilstrekkelig til fremragende følsomhet i forskjellige matriser. Lineære kalibreringskurver ble laget ved å enten benytte stabile isotopmerkede derivater eller homologer med liknende struktur som interne standarder, eller ved å benytte eksterne standarder i sammensvarende matriser. Metodens resultater for presisjon og nøyaktighet var innenfor internasjonalt gitte retningslinjer for kvantitativ instrumentell analyse. Ved å anvende 25-i-én-metoden til å analysere fôr og fiskeprøver fra fôringseksperimentene viste kun lave nivåer av mykotoksinet enniatin B i fôr som inneholder WG, i tillegg til lave nivåer av isoflavonene daidzein (DAI), genistein (GEN) og glycitein (GLY) og deres hendholdsvise glukoside former i SPC- og PPC-baserte fôrsammensetninger. Dette demonstrerer effektiviteten til de satte grenseverdiene samt moderne fôrprosseseringsteknologi i norsk fiskeoppdrett. Det fører til at det ikke ble funnet spor av overført ANF i fiskenes muskelvev, som bekrefter at laks fôret med plantebasert fôr er trygt for mennesker å spise. Det andre målet med prosjektet var å undersøke og forstå den metabolske nedbrytningsprosessen til soya-isoflavoner i laks. Produkter fra den hepatiske biotransformasjonen til DAI, GEN og GLY i laks hadde ikke blitt identifisert tidligere, men kunnskap om potensielt skadelige metabolitter er avgjørende for å kartlegge mulige risikoer. På grunn av manglende metabolske nedbrytningsmodeller for laks, ble levermikrosomer og primære hepatocytter laget på laboratoriet for å brukes i in vitro metabolisme-assayer. Lakseleverfraksjonene ble karakterisert i henhold til fase I cytokrom P450 (CYP) og fase II uridin-difosfat-glukoronosyltransferase (UGT)- enzymaktiviteter ved å bruke spesifikke prøvesubstrater. To nye metoder, hvorav en for samtidig måling av fem CYP-aktiviteter og en for fem UGT-aktiviteter, ble utviklet ved å bruke LC-trippel-kvadrupol-massespektrometri (TripleQ-MS/MS) for å spesifikt kartlegge substratene og deres metabolitter. Metodene ble optimalisert i assayer med kommersielt tilgjengelige mikrosomer fra menneskelever med kjente enzymaktiviteter, og deretter brukt for lakseleverpreparatene, med forbehold at de tilgjengelige prøvesubstratene var spesifikke for menneskelige CYP og UGT-enzymer. I følge ble DAI, GEN og GLY metabolisert ved å bruke lakselevermikrosomer og primære hepatocytter, og metabolitter analysert med LC-HRMS/MS. Isoflavonene ble effektivt eliminert av UGT til metabolitter som foreløpig ble identifisert som 7-O-glukuronider basert på målinger av eksakte masser, fragmenteringsmønstre og retensjonstider. Til forskjell var produksjonen av oksidative metabolitter ubetydelig. Kun små mengder med fire mono-hydroksilerte DAI-metabolitter var målbare når inkubasjonene ble oppskalert. Disse funnene indikerer at bioakkumulering av fytoøstrogener i oppdrettslaks fra soyaproteiner i fôret er usannsynlig og derfor medfører liten risiko for forbrukere. Aktivitetene i den tredje og fjerde delen av prosjektet knyttet seg rundt kartlegging av potensielle nutrigenomiske effekter på fiskens helse som følge av et kosthold som baserer seg på planteproteiner, ved hjelp av transkriptomisk analyse av muskler, lever og tarm i eksponert laks og sebrafisk. En microarray hybridiseringsanalyse ble utført i leveren og tarmen til laks fôret med FM, WG og SPC-holdig fôr. Genekspresjonsdataene ble prosessert og analysert ved hjelp av Nofimas bioinformasjonsdatavare STARS. I tillegg ble andre variabler som tilvekst og apparent fordøyelighet, samt typiske enzymatiske biomarkører for leverhelse analysert i serum. Profilene til det genetiske uttrykket i de ulike vevsprøvene av laks, viste at vekst og biosynteseveier ble minst påvirket av fôret som inneholdt SPC15, og mest påvirket av det som inneholdt WG30. Flertallet av forandringene av uttrykkelsen til genmønstre forekom i tarmen, særlig i gener tilknyttet transport av lipoproteiner, sterolmetabolisme, immunitet, samt vevsstruktur og integritet. Studien bekrefter dermed resultatene av tidligere forskning på effektene SPC har i laks. Likevel viser observasjonene i forhold til WG30-fôret at laksen gjennomgikk ernæringsbetinget stress og viste symptomer som liknet glutensensitivitet i mennesker. I sebrafisk ble forandringer i hele transkriptomet analysert i muskel etter av fiskene var eksponert med FM, SPC30 eller WG30 i fôr ved hjelp av RNA-seq-teknologi. Analysen ble utvidet til laks i vekstfasen ved å teste gener som viste forskjeller i uttrykk i sebrafiskmodellen, ved å bruke paralog-spesifikke qPCR assayer for laks. Forandringer i det helhetlige genetiske uttrykket i musklene til sebrafisk som ble fôret med plantebasert fôr var moderate. De tydeligste forandringene ble funnet i fisk med SPC-holdig fôr, imens ingen forandringer ble funnet i sammenheng med PPC-holdig fôr, sammenlignet med FM-kontrollgruppen. Forskjellig uttrykte gener hadde viktige funksjoner i regulering av muskelvekst, opprettholdelse av muskelstruktur og funksjon samt homeostase i muskelvevet. De fleste av disse genene og paralogene ble påvirket på liknende måte i laks fôret med samme typer fôr, under forbehold av noe spesiesspesifikk regulering. Konklusjonen til arbeidet som er presentert i denne avhandlingen er at bruk av plantebaserte ingredienser i fiskefôr ikke utgjør grunn til bekymring for forbrukere av lakseprodukter i forhold til potensielle helserisikoer fra overføringen av mykotoksiner og fytoøstrogener. Likevel kan høye andeler av WG og SPC i fôret ha skadelige virkninger på fisken

Phytochemical Omics in Medicinal Plants

Medicinal plants are used to treat diseases and provide health benefits, and their applications are increasing around the world. A huge array of phytochemicals have been identified from medicinal plants, belonging to carotenoids, flavonoids, lignans, and phenolic acids, and so on, with a wide range of biological activities. In order to explore our knowledge of phytochemicals with the assistance of modern molecular tools and high-throughput technologies, this book collects recent innovative original research and review articles on subtopics of mechanistic insights into bioactivities, treatment of diseases, profiling, extraction and identification, and biotechnology

Food Processing and Its Impact on Phenolic and other Bioactive Constituents in Food

It is our pleasure to present this Special Issue of Molecules entitled “Food Processing and Its Impact on Phenolic and other Bioactive Constituents in Food”. Bioactive compounds including phenolic ingredients have long been used as important constituents of a healthy diet. As a result, consumer awareness about the important role of high-quality products rich in bioactive compounds—especially phenolic compounds—in human nutrition, health, and prevention against diseases has increased. Additionally, methods for food processing, regardless of the technology used, have a huge impact on the quality of the final products. Therefore, the big challenges for scientists lie in the monitoring of changes during food processing and the optimization of technology to achieve the minimal degradation of nutrients (including phenolic compounds). Thus, for this Special Issue, I encourage you to read some interesting papers aimed at bringing the latest scientific news, insights, and advances in the field of food processing and its impact on bioactive constituents in food, especially phenolic compounds. The information presented will certainly arouse considerable interest among a large group of our readers from different disciplines and research fields

Recent Advances and Future Trends in Fermented and Functional Foods

Health and wellness are among the core segments of quickly-changing consumer goods, with ever-increasing health consciousness among consumers around the globe. Functional foods and beverages, formulated from natural ingredients with targeted physiological functions, are at the heart of research and development in the food industry. The application of modern biotechnology methods in the food and agricultural industry is expected to alleviate hunger today and help avoid mass starvation in the future. Modern food biotechnology has in recent years been transforming existing methods of food production and preparation far beyond the traditional scope. Currently, at the global level, food biotechnological research has focused on traditional process optimization (starter culture development, enzymology, fermentation), food safety and quality, nutritional quality improvement, functional foods, and food preservation (improving shelf life). The fermentation of substrates considered for human consumption has been applied for centuries as a process that enhances shelf life, sensory properties, and nutritional value. Special emphasis has also been given to newly growing concepts, such as functional foods and probiotics. The application of biotechnology in the food sciences has led to an increase in food production and has enhanced the quality and safety of food