Hemp-based products as functional and sustainable ingredient for feed and food applications

The environmental impact of the food and feed sector and their direct competition are highly debated topics. This scenario is bound to get worse considering the increase of world population. For this, scientific research is investigating new matrices featured by both a high nutritional profile and a low ecological impact. Among them, hemp (Cannabis sativa L.) is an excellent candidate due to its important role in environmental health and its relevant nutritional and functional aspects, still little studied. In light of the above, the aim of this presentation will be to explore the use of hempseeds (HS) and hemp co-products (HCPs) (hulls, flowers, leaves and HS panel) as valuable resources for the food and feed sector. Specifically, hemp matrices were in vitro and ex vivo digested to investigate their antioxidant profile, phenolic content and the presence of bioactive peptides. In parallel, a HCP (mix of hulls, leaves and non-standardised HSs) was included in the diet of laying hens to evaluate the effect on health, animal performance and nutritional and functional profile of the eggs. Hempseeds in vitro digested showed a valuable peptidomic profile. Precisely, the MS/MS approach identified peptides with antioxidant, ace-inhibitory, anticancer, antibacterial, antifungal, antihypertensive and DPP-IV inhibitory activities. At the same time, HCP have proven to be matrices with an interesting nutritional and functional profile, but highly variable depending on the production processing. Although this represents a critical point, the inclusion of the HCP up to 9% in the diets of laying hens preserved animal health and increased the nutritional (e.g. lower saturated fatty acid and cholesterol content) and functional (e.g. higher content of vitamins and antioxidant molecules) profile of animal product. Although what has been presented supports the use of hemp-based products in the food and feed sector, future studies will be needed to ensure their increasing use

Benefits and risks associated with meat consumption during key life processes and in relation to the risk of chronic diseases

Red meat has been an important part of the diet throughout human evolution. Overall, when included as part of a healthy and varied diet, red meat can provide a rich source of bioavailable essential nutrients and high biological value protein. The present paper discusses the dietary role/impact of red and processed meat, with some reference to the relative effect of white meat, in a range of chronic conditions including iron-deficiency anaemia, cardiovascular diseases (CVD), cancer and dementia. The role of red meat in relation to key physiological conditions such as maintaining skeletal muscle and bone health and during pregnancy is also discussed. The inclusion of lean red meat in a healthy, varied diet may be beneficial during these critical conditions. There is however increasing evidence that red meat and especially processed meat are associated with increased risks of CVD, cancer and dementia whereas white meat is neutral or associated with a lower risk. There now seems little doubt that processed and unprocessed meat should have separate public dietary guidance

Relative bioefficacy of RRR-α-tocopherol versus all-rac-α-tocopherol in in vitro models

The aim of this study was to evaluate the in vitro relative bioefficacy of RRR-α-tocopherol (RRR- α-T) versus all-rac-α-tocopherol (all-rac-α-T) in counteracting the cytotoxic effect induced by H2O2 in Bovine Mammary Epithelium – University of Vermont (BME-UV1) and Madin-Darby Canine Kidney (MDCK) cells. The range of RRR- α-T and all-rac- α-T concentrations selected for the oxidative challenge experiments was 100µM - 1nM. To study the bioefficacy of RRR- α-T and all-rac- α-T, MTT and LDH tests were performed. Cells were pre-incubated for 3 h with  selected a-tocopherol concentrations and then exposed to increasing H2O2 concentrations ranging from 125 to 750µM for the following 24h. Concerning the cell viability, the pre-treatments with 100µM of RRR- α-T and 100µM all-rac-α-T were able to significantly (P<0.05) counteract the effect induced by 750 µM of H2O2 in BME-UV1. In MDCK the pre-treatment with 1nM of all-rac-α-T was able to significantly (P<0.05) reduce the effect of 125 and 150 mM H2O2. In MDCK cells, the pre-incubation with all-rac-α-T determines a significant reduction of the membrane damage, induced by 175 µM of H2O2. In conclusion, RRR-α-T and all-rac-α-T have shown the ability to counteract the oxidative effects of H2O2, however further investigation will help to better understand their specific mechanism of action in vitro.  

Effect of Zinc Oxide and Zinc Chloride on Human and Swine Intestinal Epithelial Cell Lines

Zinc (Zn) salts are often used as nutritional additives in order to promote gut health. The aim of the present study was to assess the effect of two widely used additives in feedstuff, on the intestinal epithelium. In particular, the effect of zinc oxide (ZnO) and zinc chloride (ZnCl2) was investigated in human (INT-407) and porcine (IPI-2I) cell line models. The effect of Zn sources on IPI-21 and INT-407 cell lines was evaluated by a colorimetric viability test using an incubation period of 3 and 24 hours under serum-free conditions. INT407 and IPI-2I showed to be a suitable model of the intestine and a simple tool to investigate the role of Zn supplements. INT407 showed to be the most sensible model to Zn supplements considered, whereas IPI-2I were more resistant. The results of this study contribute to determine the role of zinc in human and swine intestinal epithelium. However, further in vivo experiments may be done to clarify the contribution of Zn supplements in gut health and to improve Zn supplementation in animal feed and in human formulations

Role of omega-3 polyunsaturated fatty acids, citrus pectin, and milk-derived exosomes on intestinal barrier integrity and immunity in animals

The gastrointestinal tract of livestock and poultry is prone to challenge by feedborne antigens, pathogens, and other stress factors in the farm environment. Excessive physiological inflammation and oxidative stress that arises firstly disrupts the intestinal epithelial barrier followed by other components of the gastrointestinal tract. In the present review, the interrelationship between intestinal barrier inflammation and oxidative stress that contributes to the pathogenesis of inflammatory bowel disease was described. Further, the role of naturally existing immunomodulatory nutrients such as the omega-3 polyunsaturated fatty acids, citrus pectin, and milk-derived exosomes in preventing intestinal barrier inflammation was discussed. Based on the existing evidence, the possible molecular mechanism of these bioactive nutrients in the intestinal barrier was outlined for application in animal diets

Anaemia in India and its prevalence and multifactorial aetiology: a narrative review

The prevalence of anaemia in India remains high in children, especially those in rural areas, and in women of childbearing age, and its impairment of neurological development can have serious lifelong effects. It is concerning that the most recent official data (2019–21) indicate an increased prevalence compared with 2015–16. There is also considerable variability in childhood anaemia between Indian states with socioeconomic factors, such as wealth and education contributing to the risk of anaemia among adolescent women and their children. Dietary iron deficiency is often regarded as the main contributor to anaemia but increasing evidence accumulated from the authors’ ongoing literature database coupled with recent literature research suggests that it has a multifactorial aetiology, some of which is not related to nutrition. This narrative review focused on these multifactorial issues, notably the contribution of vitamin B12/folate deficiency, which also has a high prevalence in India. It was also noted that the dietary intake of bioavailable iron remains an important contributor for reducing anaemia, and the role of millets as an improved iron source compared to traditional staple cereals is briefly discussed. The overall conclusion is that anaemia has a multifactorial aetiology requiring multifactorial assessment that must include assessment of vitamin B12 status

Exploring Plant-Based Proteins as an Alternative for Culture Media in Cellular Agriculture

The food sector is currently one of the most debated fields, particularly regarding its sustainabil-ity. While the livestock industry has actively worked to mitigate its environmental impact, scien-tific innovation must contribute further to address this challenge. In this context, cultured meat emerges as a revolutionary option, with the potential to overcome many ethical and environmen-tal issues associated with conventional meat production. However, the development of this inno-vative product faces significant challenges, chief among them being the formulation of culture media that are free of animal-derived components, economically sustainable, and compatible with cellular requirements. For this reason, the aim of this work was to evaluate protein extracts de-rived from hempseeds (HSs) as constituents of the culture medium for murine C2C12 muscle cells. Specifically, following alkaline extraction and isoelectric precipitation, protein isolates from HSs were tested at various concentrations (1:2 dilutions) on C2C12 cells. The results demonstrat-ed that all concentrations, except for the initial stock solution, effectively supported cell growth and proliferation for up to 48 h, as assessed using the Alamar Blue colorimetric assay. Among all the tested concentrations, 0.6 mg/mL was selected for further analysis and incorporated as a component of an edible bioink used for 3D bioprinting. The experiments showed that 0.6 mg/mL of HS protein extract was capable of sustaining cell viability in 3D structures for up to 7 days, as confirmed by the Live/Dead assay and confocal microscopy analysis. These findings indicate that HSs, characterized by high nutritional profile (94.6±0.12% of DM; 23.1±0.57% of crude protein; 27.9±0.75 of lipids), hold great potential as a sustainable, accessible, and alternative protein source for the formulation of culture media in the context of cellular agriculture. Further research is required to explore other plant-based matrices or food industry co-products that could reduce costs and optimize cell growth parameters. The integration of natural-origin ingredients with ad-vanced technologies such as bioprinting could pave the way for novel approaches to cultivated meat production, bringing this solution closer to commercial reality and aligning it with the prin-ciples of a circular economy

Effects of Euglena gracilis Supplementation on Growth Performance, Antioxidant Status, Meat Quality in Broiler Chickens

The current growth in global demand for animal products poses significant challenges in terms of environmental, economic and nutritional sustainability. The search for sustainable alternatives has become a priority. Innovative sources, such as microalgae, fungi and agro-industrial by-products, offer opportunities to improve the nutritional quality of feed, animal welfare and the resilience of livestock supply chains, contributing to a more circular and sustainable agriculture. The purpose of this project was to evaluate the performance, health status, and meat quality of broiler chickens that had been supplemented with Euglena gracilis. In particular, a 0.019% supplementation of Euglena gracilis was administered to 240 Ross 308 male broiler chickens for 42 days. Animals were housed in two identical rooms equipped with temperature and humidity control systems. Each room contained 120 chicks, divided into 12 boxes (10 chicks/pen), with 6 boxes allocated to each treatment group. The experimental groups are as follows: CTR (Control group) and TRT (Treatment group). At the end of the test, two animals per pen were slaughtered for analysis. Subsequently, performance, meat quality and antioxidant capacity of plasma and liver were evaluated. Key parameters evaluated for performance were Average Daily Gain (ADG), Average Daily Feed Intake (ADFI) and Feed Conversion Ratio (FCR). Meat quality parameters included color, pH, Water-Holding Capacity (WHC) and myoglobin content. Antioxidant capacity was assessed by T-AOC (Total Antioxidant Capacity) and Glutathione Reductase (GR) assays. Data (mean±SEM) were analyzed with one-way Anova using GraphPad Prism 9 (p<0.05). Performance in TRT with Euglena gracilis supplementation in the broiler diet showed no significant differences compared to the control group. The animal oxidative status in TRT resulted in an enhanced liver antioxidant capacity (6.64±3.34 U/ml) and plasma GR activity (5335.43±1072.78 nmol/min/ml) compared to the CTR (4.01±0.92 U/ml and 3824.74±695.86 nmol/min/ml, respectively). Meat quality parameters showed no significant differences in TRT. These preliminary findings emphasize the role of Euglena gracilis as a sustainable and functional feed ingredient in livestock production systems, addressing challenges related to animal health and stress resilience. Research supported by Agritech National Research Center and received funding from the European Union Next-GenerationEU (PNRR – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 – D.D. 1032 17/06/2022, CN00000022) grant

Functional characterisation of Euglena gracilis following growth medium enrichment

In recent years, microalgae, in particular Euglena gracilis, has been a candidate in the food/feed industry due to its high nutritional (high protein and lipid content) and functional properties due to its ability to produce vitamin C, E and paramylon, a high molecular weight β-1,3 glucan with immunomodulatory properties. However, the inclusion of E. gracilis in the diets of farm animals is little investigated, especially its antioxidant activity. Moreover, microalgae are known for their variability and adaptability in their composition and nutritional properties, mainly due to their cultivation’s conditions. For these reasons, the aim of the present work is to study the total phenolic content (TPC, Folin-Ciocalteu assay) and antioxidant activity (ABTS and FRAP assays) of E. gracilis grown under three different nutritional conditions. In particular, the algae growth media are characterised as follows: HEgM (yeast extract, soy peptones and casein broth); ETX (animal derived amino-acidic (AA) extract;) DOE-ETX (animal derived AA extract, MgSO4, KH2PO4, and a mixture of microelements). The samples undergo green chemical extraction and ex vivo digestion. For green extractions, 150 mg of Euglena HEgM, ETX, DOE-ETX were incubated for 1h, at room temperature, under shaking, with different concentrations of water:ethanol (W:E) (100:0, 75:25, 50:50, 25:75, 0:100). At the end of the incubation, the samples were centrifuged for 5 min at 4000 rpm and subjected to multiple extraction process (n=3). Considering ex vivo digestion, after collecting gastric and intestinal fluids from swine (50-110 d, n=24 at slaughter house), they were pooled and kept at 4oC until use. Then, 500 mg of Euglena HEgM, ETX, DOE-ETX were subjected to gastric digestion (39oC x 2h) in gastric fluid, and further exposed to intestinal fluid (39oC x 2h). Aliquots were taken at the beginning/end of gastric phase and at the end of the intestinal phase, to assess TPC and antioxidant activities. Data are expressed as mean±SEM. Considering green extraction, DOE-ETX showed a high TPC with statistically significant differences compared to ETX and HEgM, especially following the 25:75 W:E extraction, with values of 1104.4±68.2; 310.9±7.0 and 117.2±6.2 mg Tannic Acid Equivalent (TAE)/100g respectively (p<0.05). These values were also confirmed for ABTS (630.8±19.0; 41.8±7.0; 21.5±2.3 mg Trolox Equivalent (TE)/100g). Following ex vivo digestion, DOE-ETX showed good results for TPC (0h:501.0±12.0; 2h:584.9±6.7; 4h:465.2±37.4 mg TAE/100g). Although the functional profile was improved following medium enrichment, the production yields showed an inverse trend (0.67; 0.81; 1 g/l*day) for DOET-ETX, ETX and HEgM, respectively. These results confirmed the potential of E. gracilis as a valuable source of functional ingredients for feed application. Further investigations will be of paramount importance to optimise growth medium formulation to obtain high algae yield with improved functional characteristics