Studies On Micro/Nano-particles Of Freshwater Clam(corbicula Fluminea Muller)Soup And Porcine Bone(sus Scrofa)Soup

Abundant amounts of nanoparticles(NPs)widely exist in the food and are often neglected till now.They are spontaneously assembled during the food processing,driven by the physical or chemical force presented.Their formations are the incidents of food processing or cooking rather than the results of subjective preparations.To distinguish between these NPs and other artificially synthesized NPs,which may also present in food in the form of food additives,we define these food-derived nanoparticles as ‘incidentally occurred nanoparticles'(io-NPs).As the fast-growing applications and rising concerns on the safety of long-term usage,nanomaterials have been in the spotlights of researches.Widely consumed as the concomitant compositions of food on daily and long-term basis,io-NPs may have conducted profound and inevitable physiological influences on human body and therefore affected the safety and health benefits of foods.In contrast to their vital importance,few studies have been performed on these families of NPs.This study set off to investigate the io-NPs from two daily foods,the freshwater clam(Corbicula fluminea Muller)soup and the porcine bone soup,by isolating io-NPs from the soups,characterizing their physical and chemical properties,and their biological activities in vitro and in vivo.(1)Demonstration of io-NPs in the soups.The freshwater clam soup contains 1.49×1013 nanoparticles per millilitre with hydrodynamic diameter(Dh)of 76±0.5 nm.The porcine bone soup contains 1.08×1010 nanoparticles per millilitre with hydrodynamic diameter(Dh)of 230±2 nm.(2)Using HPLC coupled with multiple angle lasers light scattering(MALLS)or dynamic light scattering(DLS)detector.Two spherical NPs,namely IEC-P1 and IEC-P2 io-NPs,derived from the freshwater clam soup were isolated and characterized,and were significantly different in average particle size and chargeability.The colloidal io-NPs were purified from porcine bone soup,namely SEC-colloidal.The SEC-colloidal particles were near spherical shape with hydrodynamic diameter of 275±2 nm.(3)Analysized the physical-chemical properties of these io-NPs,IEC-P1 and IEC-P2 io-NPs consisted of polysaccharides,lipids and proteins,carried six kinds of phytosterols occupying over 80% of total phytosterol content of the soup.Both subgroups of NPs were resistant to pH changes,but responsive to temperature and enzymatic hydrolysis,indicating possible applications in control release.The structure of the io-NPs was deduced as polysaccharides assembling the outer layer of particles with lipids bound to the surface,while proteins were hidden inside the particles.SEC-colloidal mainly consisted of lipids and proteins,i.e.type I collagen.They were stable in different temperatures but sensitive to changes in pH.(4)Characterization of toxicity and biological activities of io-NPs derived from the freshwater clam soup and porcine bone soup.(a)On L-02 hepatocytes,Tapilia fish and Mongolian gerbil(Meiiones unguiculataus Milme-Edwauds),the freshwater clam io-NPs exhibited preventive effects on non-alcoholic fatty liver disease.(b)On Mongolian gerbil(Meiiones unguiculataus Milme-Edwauds),the oral administration of the freshwater clam io-NPs was significantly more effective than the intragastric administration,indicating the mucosa of alimentary tract should be the major site where the biological effects of clam soup io-NPs took place.(c)No cytotoxicity of the two kinds of io-NPs was observed on Hep G2,L-02,Caco-2,MDCK and rat macrophages at all the NPs concentrations assayed.(d)The mucosal macrophages engulfed the io-NPs derived from freshwater clam soup and porcine bone soup.(e)The clam soup io-NPs showed no antioxidant activities determined with ABTS and FRAP assay,no intracellular antioxidant activities(CAA)on Hep G2,Caco-2 and MDCK cells.On the contrary,the bone soup colloidal io-NPs showed significant antioxidant activities determined with the same methods.But both kinds of io-NPs scavenged the reactive oxygen species in murine macrophages,restored the normal cellular membrane potential and mitochondrial respiratory rate,and maintained the uptake of neutral red,reporting the protective effects of io-NPs on macrophage's function.(f)The bone soup colloidal io-NPs exhibited protective effects on the epithelial cells of intestine,using Caco-2 cell monolayer measured with RTCA assay and Transwell assay.In summary,some conclusions can be drawn as below:Abundant amounts of io-NPs occurred and existed in the freshwater clam soup and porcine bone soup.They can be purified and remain stable in aqueous environment;carry biologically active ingredients,i.g.,phytosterols;resistant to temperature and pH changes.As no cytotoxicity was observed,which resulted from the cell survival rate,the direct interaction of io-NPs with mucosa macrophages and the influence on the cell membrane potential and mitochondrial superoxide content of mucosa macrophages,it implies the incidentally nanosizing during the food processing did not cause any negative effects on the safety of food.Two io-NPs exhibited different antioxidant activities or free radical scavenging activities,depending on the test methods or types of cells.The interaction between io-NPs and mucosal macrophages demonstrated the NPs could enrich in and directly affect the mucosa of alimentary tract.As the hepatoprotective effects of clam soup io-NPs have been demonstrated in vivo,it remains as an open question that how the clam soup NPs conduct the bioactivities on hepatocytes after ingestion by mucosal cells such as macrophages and enterocytes.A new mechanism,different from the targeted delivery of NPs,can be proposed as: the freshwater clam soup NPs reduces the oxidative stress allocated in the alimentary tract and eventually regulates the redox states and functions of remote organs,e.g.liver,via the recently revealed free radical signaling system.