Degree of Hydrolysis Affects the Techno-Functional Properties of Lesser Mealworm Protein Hydrolysates

Protein hydrolysates from lesser mealworm (Alphitobius diaperinus, LM) were obtained by enzymatic hydrolysis with protease from Bacillus licheniformis. A preliminary test performed for five hours of hydrolysis generated an insect protein hydrolysate with 15% of degree of hydrolysis (DH), optimum solubility property and oil holding capacity, but emulsifying and foaming ability were completely impaired. In order to investigate the potential implication of DH on techno-functional properties, a set of protein hydrolysates with a different DH was obtained by sub-sampling at different time points during three hours of enzymatic hydrolysis process. An increase in DH% had positive effects on the solubility property and oil holding ability, while a reduced emulsifying ability was observed up to five hours of hydrolysis. These results demonstrated that the enzymatic hydrolysis, if performed under controlled conditions and not for a long period, represents a valid method to extract high quality protein from insects with tailored techno-functionality, in order to produce tailored ingredients for feed and food purpose

Effectiveness of enzymatic hydrolysis for reducing the allergenic potential of legume by-products

The interest in agri-food residues and their valorization has grown considerably, and many of them are today considered to be valuable, under-exploited sources of different compounds and notably proteins. Despite the beneficial properties of legumes by-products, there are also some emerging risks to consider, including their potential allergenicity. In this work the immunoreactivity of chickpea, pea, and white bean by-products was assessed, and whether the production of enzymatic hydrolysates can be an effective strategy to reduce this allergenic potential. The results presented clearly indicate that the efficiency of this strategy is strongly related to the enzyme used and the food matrix. All legume by-products showed immunoreactivity towards serum of legume-allergic patients. Hydrolysates from alcalase did not show residual immunoreactivity for chickpea and green pea, whereas hydrolysates from papain still presented some immunoreactivity. However, for white beans, the presence of antinutritional factors prevented a complete hydrolysis, yielding a residual immunoreactivity even after enzymatic hydrolysis with alcalase

A RELAÇÃO DO AGENTE COMUNITÁRIO DE SAÚDE COM A EQUIPE DE SAÚDE DA FAMÍLIA E USUÁRIOS: UM ESTUDO EM UM MUNICÍPIO DA 13ª COORDENADORIA REGIONAL DE SAÚDE

O estudo teve por objetivo analisar a relação do Agente Comunitário de Saúde (ACS) com a equipe da unidade de saúde da família, com o enfermeiro coordenador e supervisor das atividades do ACS, assim como a percepção deste frente à receptividade dos usuários. Trata-se de um estudo quantitativo e representa um recorte da pesquisa: “O cotidiano e as perspectivas profissionais dos Agentes Comunitários de Saúde do Rio Grande do Sul”, desenvolvida pelo Grupo de Estudos e Pesquisa em Saúde (GEPS), da Universidade de Santa Cruz do Sul, RS. Participaram do estudo 51 ACS de um município com 10 unidades de Estratégia de Saúde da Família, pertencentes à 13ª Coordenadoria Regional de Saúde. Os dados foram coletados por meio da aplicação de questionário com questões fechadas. A análise dos dados foi realizada por meio da estatística descritiva com software SPSS 15.0. Identificou-se a existência de relação positiva entre os ACS, enfermeiros e usuários, pois as respostas indicam a satisfação deste trabalhador com os demais profissionais e comunidade onde está inserido. Isso demonstra a possibilidade de articulação das equipes de saúde com os usuários do sistema através de representantes comunitários

Insect-based protein ingredients: extraction, molecular characterization, techno-functionality and safety aspects

Recentemente, a causa dell'aumento della popolazione mondiale e del consumo pro-capite di carne, gli insetti sono stati esplorati come fonte alternativa e sostenibile di proteine animali. In questa tesi di dottorato, due diverse specie di insetti, lesser mealworm (Alphitobius diaperinus) e black soldier fly (Hermetia illucens), sono state studiate come fonte di proteine da poter inserire nel settore alimentare e mangimistico. Entrambi gli insetti hanno dimostrato di essere una valida fonte di proteine, non solo per la loro quantità, ma anche per l'alto profilo nutrizionale, ricco di aminoacidi essenziali. Al fine di integrare le formulazioni di mangimi e alimenti con ingredienti proteici a base di insetti, diversi protocolli di estrazione sono stati esplorati e confrontati. Oltre ai metodi chimici, basati sull'estrazione alcalina e sul frazionamento di Osborne, l'estrazione enzimatica assistita ha dimostrato di essere altrettanto promettente. L’utilizzo di enzimi proteolitici ha dimostrato di essere un valido metodo per preservare la qualità delle proteine e migliorarne la digeribilità. Inoltre, l'idrolisi enzimatica può essere modulata per raggiungere le proprietà tecnologiche desiderate, in base all’uso finale del prodotto. La qualità delle proteine degli insetti può essere danneggiata dalla presenza di enzimi endogeni. In particolare, l’imbrunimento enzimatico nelle frazioni proteiche è risultato essere responsabile di implicazioni a livello chimico e nutrizionale. Nella presente tesi, il blanching, utilizzato come metodo di uccisione degli insetti, è stato identificato come un valido trattamento per inattivare gli enzimi e preservare la qualità delle proteine. Al fine di valutare l'inclusione di ingredienti a base di proteine di insetti nella catena alimentare, è stata anche effettuata una valutazione della sicurezza, concentrandosi principalmente sulla presenza di micotossine e sul potenziale allergenico. Entrambe le specie di insetti hanno dimostrato di rappresentare un rischio allergenico per i futuri consumatori. In particolare, è stata identificata una reattività crociata tra proteine dell'insetto e allergeni noti da crostacei, a causa della stretta omologia tra specie tassonomicamente correlate. Attraverso un approccio proteomico, la tropomiosina è stata identificata come allergene prevalente in entrambi gli insetti e la sua immunoreattività è stata confermata con un siero prelevato da un paziente allergico ai crostacei. L'estrazione enzimatica, precedentemente menzionata, è stata dimostrata essere un valido strumento biotecnologico utilizzabile per ridurre l'immunoreattività delle proteine degli insetti. Oltre agli allergeni, è stata studiata anche la potenziale bioaccumulazione delle micotossine. Infatti, uno dei principali utilizzi degli insetti è correlato alla loro capacità di crescere su un diverso spettro di substrati e sottoprodotti che potrebbero essere potenzialmente contaminati. In questo progetto di tesi, nonostante gli insetti fossero stati coltivati su substrati contaminati da micotossine, non è stata dimostrata la loro capacità di bioaccumulare micotossine. Al contrario è stata identificata la loro capacità di espellerle e / o metabolizzarle in forme non ancora note. In conclusione, i risultati ottenuti in questa tesi di dottorato supportano pienamente gli insetti commestibili come promettente fonte alternativa e sostenibile di proteine, che potrebbero essere introdotte sia nella catena alimentare che in quella mangimistica al fine di soddisfare la futura domanda di proteine di alta qualità.In the recent years, due to the increasing in world population as well as rising in meat consumption, edible insects have been explored as a sustainable alternative source of animal proteins. In the present PhD thesis two different insect species, lesser mealworm (Alphitobius diaperinus) and black soldier fly (Hermetia illucens), have been deeply explored for their potential to provide high quality and safe proteins to the feed and food sector. Both insects have been demonstrated to be a valid source of proteic material, not only for their amount, but also for the high nutritional profile, rich in essential amino acids. In order to supplement feed and food formulations with insect-based protein ingredients, different extraction protocols have been explored and compared. Besides chemical methods, based on alkali extraction and Osborne fractionation, the enzymatic assisted extraction has been demonstrated to be very promising. As a matter of fact, the use of proteases has been demonstrated to preserve the protein quality and improve digestibility. Further to that, the enzymatic hydrolysis can be tailored in order to reach the desired techno-functionalities, based on the potential end use. Insect protein quality can be hampered by endogenous enzymes. In particular they trigger enzymatic browning in insect protein fractions, with implication also on their chemical and nutritional quality. In the present thesis, blanching treatment, used as killing method for insects, has been identified as valid way to inactivate enzymes and preserve protein quality. In order to evaluate the inclusion of insect protein-based ingredients in the food chain, a safety assessment has also been performed, mostly focusing on mycotoxin presence and allergen potential. Both lesser mealworm and black soldier fly have been determined to pose an allergenic risk for future consumers. In particular, a cross-reactivity between insect proteins and known crustacean allergens has been identified, due to the close homology between taxonomically related species. Through a shotgun proteomics approach, tropomyosin has been identified as prevalent allergen in both insects and its immunoreactivity confirmed with a serum from a patient allergic to crustaceans. The previously mentioned enzymatic assisted extraction has been found to be a valid biotechnological tool in order to reduce the insect protein immunoreactivity. Besides allergens, also the potential bioaccumulation of mycotoxins in insect body was studied. Indeed, one of the main potentials in using insects is their ability to grow on a different spectrum of substrates, and potentially contaminated by-products from the agri-food chain can be used to feed them. In the present thesis, insects were grown on mycotoxin contaminated substrates were demonstrated not to bioaccumulate mycotoxins into their body, but rather to excrete and/or metabolize them in not yet known forms. In conclusion, the results obtained in this PhD thesis fully support edible insects as a promising and sustainable alternative source of proteins which could be introduced in both feed and food chain in order to meet the future high-quality protein demand

Reduction of Mycotoxigenic Fungi Growth and Their Mycotoxin Production by Bacillus subtilis QST 713

The use of chemical pesticides to control the occurrence of mycotoxigenic fungi in crops has led to environmental and human health issues, driving the agriculture sector to a more sustainable system. Biocontrol agents such as Bacillus strains and their antimicrobial metabolites have been proposed as alternatives to chemical pesticides. In the present work, a broth obtained from a commercial product containing Bacillus subtilis QST 713 was tested for its ability to inhibit the growth of mycotoxigenic fungi as well as reduce their mycotoxin production. Mass spectrometry analysis of Bacillus subtilis broth allowed to detect the presence of 14 different lipopeptides, belonging to the iturin, fengycin, and surfactin families, already known for their antifungal properties. Bacillus subtilis broth demonstrated to be a useful tool to inhibit the growth of some of the most important mycotoxigenic fungi such as Aspergillus flavus, Fusarium verticillioides, Fusarium graminearum, Aspergillus carbonarius, and Alternaria alternata. In addition, cell-free Bacillus subtilis broth provided the most promising results against the growth of Fusarium graminearum and Alternaria alternata, where the radial growth was reduced up to 86% with respect to the untreated test. With regard to the mycotoxin reduction, raw Bacillus subtilis broth completely inhibited the production of aflatoxin B1, deoxynivalenol, zearalenone, and tenuazonic acid. Cell-free broth provided promising inhibitory properties toward all of the target mycotoxins, even if the results were less promising than the corresponding raw broth. In conclusion, this work showed that a commercial Bacillus subtilis, characterized by the presence of different lipopeptides, was able to reduce the growth of the main mycotoxigenic fungi and inhibit the production of related mycotoxins

Bioactive properties of fermented donkey milk, before and after in vitro simulated gastrointestinal digestion

Indigenous strains of lactic acid bacteria previously isolated from raw donkey milk samples were used to ferment donkey milk. Each sample was subjected to an in vitro simulated gastrointestinal digestion (SGID) and analysis of the digesta by HPLC-LTQ-Orbitrap XL was performed in order to obtain a comprehensive peptide profile of fermented donkey milk. Functional properties such as ACE-inhibitory, antimicrobial and antioxidant activities of the resulting fermented donkey milks, as well as characterization of the bioactive peptides produced by an in vitro SGID, were assayed. All bioactivities were found to be high in fermented milk and a further significant increase was observed after SGID. The Enterococcus faecium DM33 fermented milk exhibited the strongest antioxidant and the highest antimicrobial activities. The highest ACE-inhibitory activity was observed in milk fermented with Lactobacillus casei DM214. These findings will contribute to the development of a new functional dairy drink with anti-hypertensive, antimicrobial and/or antioxidant activities

Effectiveness of enzymatic hydrolysis for reducing the allergenic potential of legume by-products

The interest in agri-food residues and their valorization has grown considerably, and many of them are today considered to be valuable, under-exploited sources of different compounds and notably proteins. Despite the beneficial properties of legumes by-products, there are also some emerging risks to consider, including their potential allergenicity. In this work the immunoreactivity of chickpea, pea, and white bean by-products was assessed, and whether the production of enzymatic hydrolysates can be an effective strategy to reduce this allergenic potential. The results presented clearly indicate that the efficiency of this strategy is strongly related to the enzyme used and the food matrix. All legume by-products showed immunoreactivity towards serum of legume-allergic patients. Hydrolysates from alcalase did not show residual immunoreactivity for chickpea and green pea, whereas hydrolysates from papain still presented some immunoreactivity. However, for white beans, the presence of antinutritional factors prevented a complete hydrolysis, yielding a residual immunoreactivity even after enzymatic hydrolysis with alcalase

Enhancement of agri-food by-products: green extractions of bioactive molecules with fungicidal action against mycotoxigenic fungi and their mycotoxins

Introduction: Today, alternative strategies based on the use of bioactive compounds have been proposed to reduce mycotoxin contamination and limit the use of chemical fungicides. Methods: In the present work, several by-products collected from the agrifood chain (i.e., red and white grape marc, red grapevine leaves, grape seeds and stalks, pear, apple, green beans, tomato, and spent hops) were subjected to green extraction protocols (i.e., steam distillation, Ultrasound-Assisted, and Naviglio® extraction) to obtain extracts rich in polyphenols and terpenes. Each extract was assessed in vitro for its ability to inhibit the development of the main mycotoxigenic species and related mycotoxins. Results and Discussion: Aspergillus flavus and A. carbonarius were significantly reduced by pear (from −45 to −47%) and grape marc (from −21 to −51%) extracts, while F. graminearum was shown to be highly influenced by grape stalk, pear, and grape marc extracts (−24% on average). On the contrary, F. verticillioides was inhibited only by pear (−18%) and to a very low and negligible extent by apple (−1%) and green beans (−3%). Regarding the reduction of mycotoxins, the extracts were able to inhibit OTA from 2 to 57%, AFB1 from 5 to 75%, and DON from 14 to 72%. The highest percentages of reduction were obtained against FBs (from 11 to 94%), ZEN (from 17 to 100%), and Alternaria toxins (from 7 to 96%). In conclusion, this work provided promising results for the production of bioactive extracts obtained from agri-food by-products, which could be exploited as potential biofungicides against the development of mycotoxigenic fungi and related mycotoxins