Fabrication of Microalgae Oil Vesicles for Drug Delivery Applications.

In this study, it is demonstrated that natural microalgae oils, which contain fatty acid components including docosahexaenoic acid (DHA), could be directly applied to fabricate vesicular structures in aqueous phase through a forced formation process. The microalgae oil vesicles had initial average diameters of 170- 230 nm with negative charges apparently caused by dissociation of the fatty acid components. The vesicles possessed excellent stability with lifetimes for at least 450 days. The formation of the vesicular structures with hydrophilic cores/regions was confirmed by the transmission electron microscopy (TEM) image and successful encapsulation of a hydrophilic material. For encapsulation of a hydrophobic material, lutein, the vesicle size was increased probably due to the insertion of lutein into the hydrophobic vesicular bilayer structures. The analysis of Fourier transform infrared (FTIR) spectroscopy suggested that the vesicular bilayer fluidity was decreased by encapsulating lutein. However, the lutein-encapsulating microalgae oil vesicles still possessed high stability and the vesicular structures could maintain intact even at an environmental temperature up to 60℃. Applicability of the microalgae oil vesicles as drug delivery carriers was also demonstrated by successful encapsulation of curcumin. However, when the loaded curcumin was increased to a certain amount, physical stability of the microalgae oil vesicles was significantly reduced. This is probably because the vesicular structures with only limited spaces for accommodating hydrophobic materials were strongly affected by encapsulating a large amount of curcumin. It is interesting to note that by adding egg L-α-phosphatidylcholine, the curcumin encapsulation-induced instability of the microalgae oil vesicles could be alleviated. The results indicated that vesicular structures could be fabricated from microalgae oils and the microalgae oil vesicles were capable of encapsulating hydrophilic or hydrophobic materials for drug delivery applications. The findings lay a background for further dosage form development of nutritional supplements encapsulated by natural microalgae oils.

Enhanced production of bioactive compounds from marine microalgae Tetraselmis tetrathele under salinity and light stresses: A two-stage cultivation strategy.

This study leveraged the salinity and light intensity stresses during the stationary phase for enhancing the pigment contents and antioxidant capacity of Tetraselmis tetrathele. The highest pigments content was obtained in cultures under salinity stress (40 g L-1) illuminated using fluorescent light. Furthermore, the best inhibitory concentration (IC50) for scavenging the 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals was found as 79.53 µg mL-1 in ethanol extract and cultures under red LED light stress (300 µmol m-2 s-1). The highest antioxidant capacity in a ferric-reducing antioxidant power (FRAP) assay (1,778.6 µM Fe+2) was found in ethanol extract and cultures under salinity stress illuminated using fluorescent light. Maximum scavenging of the 2.2-diphenyl-1-picrylhydrazyl (DPPH) radical was found in ethyl acetate extracts under light and salinity stresses. These results indicated that abiotic stresses could enhance the pigment and antioxidant components of T. tetrathele, which are value-added compounds in the pharmaceutical, cosmetic, and food industries.

Emerging green cell disruption techniques to obtain valuable compounds from macro and microalgae: a review.

In the modern era, macro-microalgae attract a strong interest across scientific disciplines, owing to the wide application of these cost-effective valuable bioresources in food, fuel, nutraceuticals, and pharmaceuticals etc. The practice of eco-friendly extraction techniques has led scientists to create alternative processes to the conventional methods, to enhance the extraction of the key valuable compounds from macro-microalgae. This review narrates the possible use of novel cell disruption techniques, including use of ionic liquid, deep eutectic solvent, surfactant, switchable solvents, high voltage electrical discharge, explosive decompression, compressional-puffing, plasma, and ozonation, which can enable the recovery of value added substances from macro-microalgae, complying with the principles of green chemistry and sustainability. The above-mentioned innovative techniques are reviewed with respect to their working principles, benefits, and possible applications for macro-microalgae bioactive compound recovery and biofuel. The benefits of these techniques compared to conventional extraction methods include shorter extraction time, improved yield, and reduced cost. Furthermore, various combinations of these innovative technologies are used for the extraction of thermolabile bioactive compounds. The challenges and prospects of the innovative extraction processes for the forthcoming improvement of environmentally and cost-effective macro-microalgal biorefineries are also explained in this review.

Effects of Piperonyl Butoxide on the Accumulation of Lipid and the Transcript Levels of DtMFPα in Dunaliella tertiolecta.

As one of the sources of biodiesel, microalgae are expected to solve petroleum shortage. In this study, different concentrations of piperonyl butoxide were added to the culture medium to investigate their effects on the growth, pigment content, lipid accumulation, and content of carotenoids in Dunaliella tertiolecta. The results showed that piperonyl butoxide addition significantly decreased the biomass, chlorophyll content, and total carotenoid content but hugely increased the lipid accumulation. With the treatment of 150 ppm piperonyl butoxide combined with 8000 Lux light intensity, the final lipid accumulation and single-cell lipid content were further increased by 21.79 and 76.42% compared to those of the control, respectively. The lipid accumulation in D. tertiolecta is probably related to the increased expression of DtMFPα in D. tertiolecta under the action of piperonyl butoxide. The phylogenetic trees of D. tertiolecta and other oil-rich plants were constructed by multiple sequence alignment of DtMFPα, demonstrating their evolutionary relationship, and the tertiary structure of DtMFPα was predicted. In conclusion, piperonyl butoxide has a significant effect on lipid accumulation in D. tertiolecta, which provides valuable insights into chemical inducers to enhance biodiesel production in microalgae to solve the problem of diesel shortage.

Astaxanthin and eicosapentaenoic acid production by S4, a new mutant strain of Nannochloropsis gaditana.

BACKGROUND: Astaxanthin is a ketocarotenoid with high antioxidant power used in different fields as healthcare, food/feed supplementation and as pigmenting agent in aquaculture. Primary producers of astaxanthin are some species of microalgae, unicellular photosynthetic organisms, as Haematococcus lacustris. Astaxanthin production by cultivation of Haematococcus lacustris is costly due to low biomass productivity, high risk of contamination and the requirement of downstream extraction processes, causing an extremely high price on the market. Some microalgae species are also primary producers of omega-3 fatty acids, essential nutrients for humans, being related to cardiovascular wellness, and required for visual and cognitive development. One of the main well-known producers of omega-3 fatty eicosapentaenoic acid (EPA) is the marine microalga Nannochloropsis gaditana (named also Microchloropsis gaditana): this species has been already approved by the Food and Drug Administration (FDA) for human consumption and it is characterized by a fast grow phenotype. RESULTS: Here we obtained by chemical mutagenesis a Nannochloropsis gaditana mutant strain, called S4, characterized by increased carotenoid to chlorophyll ratio. S4 strain showed improved photosynthetic activity, increased lipid productivity and increased ketocarotenoids accumulation, producing not only canthaxanthin but also astaxanthin, usually found only in traces in the WT strain. Ketocarotenoids produced in S4 strain were extractible in different organic solvents, with the highest efficiency observed upon microwaves pre-treatment followed by methanol extraction. By cultivation of S4 strain at different irradiances it was possible to produce up to 1.3 and 5.2 mgL-1 day-1 of ketocarotenoids and EPA respectively, in a single cultivation phase, even in absence of stressing conditions. Genome sequencing of S4 strain allowed to identify 199 single nucleotide polymorphisms (SNP): among the mutated genes, mutations in a carotenoid oxygenase gene and in a glutamate synthase gene could explain the different carotenoids content and the lower chlorophylls content, respectively. CONCLUSIONS: By chemical mutagenesis and selection of strain with increased carotenoids to chlorophyll ratio it was possible to isolate a new Nannochloropsis gaditana strain, called S4 strain, characterized by increased lipids and ketocarotenoids accumulation. S4 strain can thus be considered as novel platform for ketocarotenoids and EPA production for different industrial applications.

Osteogenic activities of four calcium-chelating microalgae peptides.

BACKGROUND: Adequate calcium intake is necessary to prevent osteoporosis, which poses significant public health challenges. The natural bioactive peptide calcium chelates have been regarded as superior calcium supplements. Microalgae peptides are regarded as potential candidates for protection from bone loss in osteoporosis. This study aimed to prepare microalgae calcium-chelating peptides from four microalgae proteins and assess their osteogenic activities in osteoporosis-like zebrafish. RESULTS: After in vitro gastrointestinal digestion, 4.42% Chlorella pyrenoidosa protein, 2.74% Nannochloropsis oceanica protein, 6.07% Arthospira platensis protein and 10.47% Dunaliella salina protein were retained. The calcium-chelating capacities of four microalgae protein hydrolysates (MPHs) ranged from 14.10 ± 7.16% to 34.11 ± 9.34%. CaCl2 addition increased the maximum absorption peaks, absorption intensities and particle sizes of MPHs. Calcium-chelating MPHs showed stronger osteogenic activities than MPHs in the osteoporosis-like zebrafish model, with significantly increased mineralized tissue area and integrated optical density. CONCLUSION: Microalgae proteins have favorable digestibilities. Among the four MPHs, Nannochloropsis oceanica protein hydrolysates showed the highest calcium-chelating capacity, which might be due to its high degree of hydrolysis after in vitro digestion and high content of Ser, Tyr, Thr, Asp and Glu. The absorption intensities and particle sizes of MPHs both increased after calcium addition. MPH treatment could reverse dexamethasone-induced osteoporosis of zebrafish, and MPHs-Ca chelates showed higher osteogenic activities in osteoporosis-like phenotype zebrafish. © 2022 Society of Chemical Industry.

Innovative method to grow the probiotic Lactobacillus reuteri in the omega3-rich microalga Isochrysis galbana.

Microalgae are natural sources of valuable bioactive compounds, such as polyunsaturated fatty acids (PUFAs), that show antioxidant, anti-inflammatory, anticancer and antimicrobial activities. The marine microalga Isochrysis galbana (I. galbana) is extremely rich in ω3 PUFAs, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Probiotics are currently suggested as adjuvant therapy in the management of diseases associated with gut dysbiosis. The Lactobacillus reuteri (L. reuteri), one of the most widely used probiotics, has been shown to produce multiple beneficial effects on host health. The present study aimed to present an innovative method for growing the probiotic L. reuteri in the raw seaweed extracts from I. galbana as an alternative to the conventional medium, under conditions of oxygen deprivation (anaerobiosis). As a result, the microalga I. galbana was shown for the first time to be an excellent culture medium for growing L. reuteri. Furthermore, the gas-chromatography mass-spectrometry analysis showed that the microalga-derived ω3 PUFAs were still available after the fermentation by L. reuteri. Accordingly, the fermented compound (FC), obtained from the growth of L. reuteri in I. galbana in anaerobiosis, was able to significantly reduce the adhesiveness and invasiveness of the harmful adherent-invasive Escherichia coli to intestinal epithelial cells, due to a cooperative effect between L. reuteri and microalgae-released ω3 PUFAs. These findings open new perspectives in the use of unicellular microalgae as growth medium for probiotics and in the production of biofunctional compounds.

Multiproduct biorefinery from marine thraustochytrids towards a circular bioeconomy.

Microalgal biotechnology research continues to expand due to largely unexplored marine environments and growing consumer interest in healthy products. Thraustochytrids, which are marine oleaginous protists, are known for their production of bioactives with significant applications in nutraceuticals, pharmaceuticals, and aquaculture. A wide range of high-value biochemicals, such as nutritional supplements (omega-3 fatty acids), squalene, exopolysaccharides (EPSs), enzymes, aquaculture feed, and biodiesel and pigment compounds, have been investigated. We discuss thraustochytrids as potential feedstocks to produce various bioactive compounds and advocate developing a biorefinery to offset production costs. We anticipate that future advances in cell manufacturing, lipidomic analysis, and nanotechnology-guided lipid extraction would facilitate large-scale cost-competitive production through these microbes.

Freeze-dried Nannochloropsis oceanica biomass protects eicosapentaenoic acid (EPA) from metabolization in the rumen of lambs.

Eicosapentaenoic acid (EPA) from freeze-dried biomass of Nannochloropsis oceanica microalgae resists ruminal biohydrogenation in vitro, but in vivo demonstration is needed. Therefore, the present study was designed to test the rumen protective effects of N. oceanica in lambs. Twenty-eight lambs were assigned to one of four diets: Control (C); and C diets supplemented with: 1.2% Nannochloropsis sp. oil (O); 12.3% spray-dried N. oceanica (SD); or 9.2% N. oceanica (FD), to achieve 3 g EPA /kg dry matter. Lambs were slaughtered after 3 weeks and digestive contents and ruminal wall samples were collected. EPA concentration in the rumen of lambs fed FD was about 50% higher than lambs fed SD or O diets. Nevertheless, the high levels of EPA in cecum and faeces of animals fed N. oceanica biomass, independently of the drying method, suggests that EPA was not completely released and absorbed in the small intestine. Furthermore, supplementation with EPA sources also affected the ruminal biohydrogenation of C18 fatty acids, mitigating the shift from the t10 biohydrogenation pathways to the t11 pathways compared to the Control diet. Overall, our results demonstrate that FD N. oceanica biomass is a natural rumen-protected source of EPA to ruminants.

Haematococcus pluvialis as a Potential Source of Astaxanthin with Diverse Applications in Industrial Sectors: Current Research and Future Directions.

Haematococcus pluvialis, a green microalga, appears to be a rich source of valuable bioactive compounds, such as astaxanthin, carotenoids, proteins, lutein, and fatty acids (FAs). Astaxanthin has a variety of health benefits and is used in the nutraceutical and pharmaceutical industries. Astaxanthin, for example, preserves the redox state and functional integrity of mitochondria and shows advantages despite a low dietary intake. Because of its antioxidant capacity, astaxanthin has recently piqued the interest of researchers due to its potential pharmacological effects, which include anti-diabetic, anti-inflammatory, and antioxidant activities, as well as neuro-, cardiovascular-, ocular, and skin-protective properties. Astaxanthin is a popular nutritional ingredient and a significant component in animal and aquaculture feed. Extensive studies over the last two decades have established the mechanism by which persistent oxidative stress leads to chronic inflammation, which then mediates the majority of serious diseases. This mini-review provides an overview of contemporary research that makes use of the astaxanthin pigment. This mini-review provides insight into the potential of H. pluvialis as a potent antioxidant in the industry, as well as the broad range of applications for astaxanthin molecules as a potent antioxidant in the industrial sector.

An integration study of microalgae bioactive retention: From microalgae biomass to microalgae bioactives nanoparticle.

Microalgae metabolites include biologically active compounds with therapeutic effects such as anticancer, anti-inflammatory and immunomodulation effects. One of the most recent focuses is on utilizing microalgae lipid-based biologically active compounds in food applications. However, most microalgae biological active compounds in their natural forms have common drawbacks like low solubility, low physicochemical stability and strong susceptibility to degradation, which significantly limits their application in foods, therefore, it is important to find solutions to retain their functional properties. In the present work, a comprehensive review on multi-product biorefinery was carried out from upstream processing stage to downstream processing stage, and identify critical processes and factors that impact bioactive material acquisition and retention. Furthermore, since nanoencapsulation technology emerges as an effective solution for microalgae nutraceutical product's retention, this work also focus on the nanoparticle perspective and comprehensively reviews the current nanoencapsulation solutions of the microalgae bioactive extract products. The aim is to depict advances in the formulations of microalage bioactive nanoparticles and provide a critical analysis of the reported nanoparticle formation. Overall, through the investigation of microalgae from biomass to bioactive nanoparticles, we aim to facilitate microalgae nutraceuticals incorporation as high value-added ingredients in more functional food that can improve human health.

Microalgal Lipid Extracts Have Potential to Modulate the Inflammatory Response: A Critical Review.

Noncommunicable diseases (NCD) and age-associated diseases (AAD) are some of the gravest health concerns worldwide, accounting for up to 70% of total deaths globally. NCD and AAD, such as diabetes, obesity, cardiovascular disease, and cancer, are associated with low-grade chronic inflammation and poor dietary habits. Modulation of the inflammatory status through dietary components is a very appellative approach to fight these diseases and is supported by increasing evidence of natural and dietary components with strong anti-inflammatory activities. The consumption of bioactive lipids has a positive impact on preventing chronic inflammation and consequently NCD and AAD. Thus, new sources of bioactive lipids have been sought out. Microalgae are rich sources of bioactive lipids such as omega-6 and -3 polyunsaturated fatty acids (PUFA) and polar lipids with associated anti-inflammatory activity. PUFAs are enzymatically and non-enzymatically catalyzed to oxylipins and have a significant role in anti and pro-resolving inflammatory responses. Therefore, a large and rapidly growing body of research has been conducted in vivo and in vitro, investigating the potential anti-inflammatory activities of microalgae lipids. This review sought to summarize and critically analyze recent evidence of the anti-inflammatory potential of microalgae lipids and their possible use to prevent or mitigate chronic inflammation.

Safety evaluation of odd-chain fatty acid algal oil.

In the food industry, most fatty acid-rich oils are primarily composed of saturated even-chain fatty acids. However, saturated odd-chain fatty acids are potentially a beneficial alternative to other saturated fatty acid-containing oils. In this communication, we examine the safety of odd-chain fatty acid (OCFA) algal oil, a microalgal-sourced oil composed primarily of the saturated odd-chain fatty acids pentadecanoic acid and heptadecanoic acid. OCFA algal oil was assessed for toxicity in a 14-day palatability study and comprehensive 13-week dietary study at inclusion levels of 5%, 10%, and 15% in the diet, utilizing a DHA-rich algal oil as a comparator control. No adverse effects attributed to the consumption of OCFA algal oil were observed in either study. Therefore, we report a No Observable Adverse Effect Level (NOAEL) of 150,000 ppm (15% in the diet), equivalent to an OCFA algal oil intake of 7553.9 and 8387.7 mg/kg bw/day for male and female rats, respectively. The genotoxic potential of OCFA algal oil was also examined in an in vitro bacterial reverse mutation assay and in vivo mammalian bone marrow chromosome aberration test. OCFA algal oil was non-mutagenic in Salmonella typhimurium and Escherichia coli test strains and did not exhibit clastogenicity in vivo.

Variability in Macro- and Micronutrients of 15 Commercially Available Microalgae Powders.

The nutrient composition of 15 commercially available microalgae powders of Arthrospira platensis, Chlorella pyrenoidosa and vulgaris, Dunaliella salina, Haematococcus pluvialis, Tetraselmis chuii, and Aphanizomenon flos-aquae was analyzed. The Dunaliella salina powders were characterized by a high content of carbohydrates, saturated fatty acids (SFAs), omega-6-polyunsaturated fatty acids (n6-PUFAs), heavy metals, and α-tocopherol, whereas the protein amounts, essential amino acids (EAAs), omega-3-PUFAs (n3-PUFAs), vitamins, and minerals were low. In the powder of Haematococcus pluvialis, ten times higher amounts of carotenoids compared to all other analyzed powders were determined, yet it was low in vitamins D and E, protein, and EAAs, and the n6/n3-PUFAs ratio was comparably high. Vitamin B12, quantified as cobalamin, was below 0.02 mg/100 g dry weight (d.w.) in all studied powders. Based on our analysis, microalgae such as Aphanizomenon and Chlorella may contribute to an adequate intake of critical nutrients such as protein with a high content of EAAs, dietary fibers, n3-PUFAs, Ca, Fe, Mg, and Zn, as well as vitamin D and E. Yet, the nutritional value of Aphanizomenon flos-aquae was slightly decreased by high contents of SFAs. The present data show that microalgae are rich in valuable nutrients, but the macro- and micronutrient profiles differ strongly between and within species.

Spirulina Microalgae and Brain Health: A Scoping Review of Experimental and Clinical Evidence.

Spirulina microalgae contain a plethora of nutrient and non-nutrient molecules providing brain health benefits. Numerous in vivo evidence has provided support for the brain health potential of spirulina, highlighting antioxidant, anti-inflammatory, and neuroprotective mechanisms. Preliminary clinical studies have also suggested that spirulina can help to reduce mental fatigue, protect the vascular wall of brain vessels from endothelial damage and regulate internal pressure, thus contributing to the prevention and/or mitigating of cerebrovascular conditions. Furthermore, the use of spirulina in malnourished children appears to ameliorate motor, language, and cognitive skills, suggesting a reinforcing role in developmental mechanisms. Evidence of the central effect of spirulina on appetite regulation has also been shown. This review aims to understand the applicative potential of spirulina microalgae in the prevention and mitigation of brain disorders, highlighting the nutritional value of this "superfood", and providing the current knowledge on relevant molecular mechanisms in the brain associated with its dietary introduction.

Nutraceuticals and Bio-inspired Materials from Microalgae and their Future Perspectives.

Nutraceuticals are food or component of food that do not only promote health but also help in recovering and combating health disorders. Algae are microorganisms that are used as supplements used in treating health disorders. They are rich in essential fatty acids, antioxidant pigments, and other micronutrients. These algae are gaining importance as functional components in the green synthesis of metal nanoparticles and applications in fabrics incorporated antimicrobial agents and pharmaceuticals. The present review focus on the distinctive algal components that are beneficial in biomedical applications. It also focuses on the research techniques to enrich the macronutrients and micronutrients by altering growth conditions and susceptible nutritional factors. A diagram model for a systematic review is utilized for this search. The research is conducted through the following databases: PubMed, Web of Science, Scopus, and Science Direct. Results: Here in this review, current reviewers put forward the importance of microalgae and other algae as alternative marine nutrient sources of dietary supplements for human consumption. In this context, extrinsic and intrinsic environmental parameter manipulative studies by eminent research groups to enhance the nutrient composition of these marine creatures are focused on in this study. Some costeffective approach-based techniques for industrial output have also been manifested. The role of algae as bio-inspired material for the production of biosynthetic metal nanoparticles, water-soluble polymers, bioplastic, antimicrobials, antifouling agents has been incurred as research interests in the past decades. In spite of being so impressive as nutraceuticals and bio-inspired material components, research gaps still exist. The purpose of the manuscript is to cover such gaps and show a new paradigm of biomedical applications.

Investigating the action of the microalgal pigment marennine on Vibrio splendidus by in vivo2H and 31P solid-state NMR.

This work investigates the potential probiotic effect of marennine - a natural pigment produced by the diatom Haslea ostrearia - on Vibrio splendidus. These marine bacteria are often considered a threat for aquaculture; therefore, chemical antibiotics can be required to reduce bacterial outbreaks. In vivo2H solid-state NMR was used to probe the effects of marennine on the bacterial membrane in the exponential and stationary phases. Comparisons were made with polymyxin B (PxB) - an antibiotic used in aquaculture and known to interact with Gram(-) bacteria membranes. We also investigated the effect of marennine using 31P solid-state NMR on model membranes. Our results show that marennine has little effect on phospholipid headgroups dynamics, but reduces the acyl chain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus membranes through different mechanisms. While PxB would alter the bacterial outer and inner membranes, marennine would act through a membrane stiffening mechanism, without affecting the bilayer integrity. Our study proposes this microalgal pigment, which is harmless for humans, as a potential treatment against vibriosis.

Can algae contribute to the war with Covid-19?

The world at large is facing a new threat with the emergence of the Coronavirus Disease 2019 (COVID-19) pandemic. Though imperceptible by the naked eye, the medical, sociological and economical implications caused by this newly discovered virus have been and will continue to be a great impediment to our lives. This health threat has already caused over two million deaths worldwide in the span of a year and its mortality rate is projected to continue rising. In this review, the potential of algae in combating the spread of COVID-19 is investigated since algal compounds have been tested against viruses and algal anti-inflammatory compounds have the potential to treat the severe symptoms of COVID-19. The possible utilization of algae in producing value-added products such as serological test kits, vaccines, and supplements that would either mitigate or hinder the continued health risks caused by the virus is prominent. Many of the characteristics in algae can provide insights on the development of microalgae to fight against SARS-CoV-2 or other viruses and contribute in manufacturing various green and high-value products.

Microalgal extract from thermotolerant Coelastrella sp. F50 retards the liver tumor progression by targeting hepatic cancer stem cells.

Microalgae extracts have shown antitumor activities. However, the antitumor mechanism of them is not yet completely clear, especially the effect on cancer stem cells (CSCs). This study aimed to elucidate the antitumor activity and mechanism of microalgal extract from thermotolerant Coelastrella sp. F50 (F50) in hepatocellular carcinoma (HCC). Oncogenic behaviors were analyzed using cell proliferation, colony formation, invasion, sphere formation, and side population cells (SPCs) assays in HCC cells after F50 treatment. The molecular mechanism was further studied by quantitative real-time PCR, immunoblot, and immunofluorescence analyses. The chemopreventive efficacy of F50 was evaluated in rat orthotopic hepatoma, and the hepatic pathologies were investigated by immunohistochemical, immunoblot, and immunofluorescence analyses. F50 specifically suppressed hepatic CSCs (tumor spheres, drug efflux, CD133/ABCG2 CSCs markers) with no cytotoxicity in vitro. In the animal experiments, prophylactic F50 administration significantly attenuated tumor progression and improved liver function in HCC-bearing rats. In the mechanistic analysis, F50 potentially inhibited cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2 ) axis in HCC cells and rat hepatoma, and exogenous PGE2 restored CSCs properties in F50-treated HCC cells. In summary, F50 extract inhibits hepatic CSCs by COX-2/PGE2 downregulation and may facilitate a novel phytotherapy for HCC prevention.

Occurrence of Antibiotic Resistance Genes in Hermetia illucens Larvae Fed Coffee Silverskin Enriched with Schizochytrium limacinum or Isochrysis galbana Microalgae.

Hermetia illucens larvae are among the most promising insects for use as food or feed ingredients due to their ability to convert organic waste into biomass with high-quality proteins. In this novel food or feed source, the absence of antibiotic-resistant bacteria and their antibiotic resistance (AR) genes, which could be horizontally transferred to animal or human pathogens through the food chain, must be guaranteed. This study was conducted to enhance the extremely scarce knowledge on the occurrence of AR genes conferring resistance to the main classes of antibiotics in a rearing chain of H. illucens larvae and how they were affected by rearing substrates based on coffee silverskin supplemented with increasing percentages of Schizochytrium limacinum or Isochrysis galbana microalgae. Overall, the PCR and nested PCR assays showed a high prevalence of tetracycline resistance genes. No significant effect of rearing substrates on the distribution of the AR genes in the H. illucens larvae was observed. In contrast, the frass samples were characterized by a significant accumulation of AR genes, and this phenomenon was particularly evident for the samples collected after rearing H. illucens larvae on substrates supplemented with high percentages (>20%) of I. galbana. The latter finding indicates potential safety concerns in reusing frass in agriculture.