Universal Existence Of Fluorescent Carbon Dots In Commercial Beer And Assessment Of Their Potential Toxicity

Beer has plenty of nutritional ingredients,such as carbohydrates,vitamins and various amino acids.The saccharification and gelatinization processing technology of beer need to be heated,organic matter and small molecular substances are aggregated to form nano-scale substances.Small size effect and high specific surface area can be perceived as unique properties for nanoparticles,the research indicated that these properties have adverse effects on the body.Therefore,the toxicity of nanoparticles has been widely researched in recent years.However,the evaluation of the toxicity and property of fluorescent carbon dots?CDs?in beers is still not be reported,food safety has been a key issue to be solved urgently.This paper selected beers as raw material,we evaluated beers CDs potential toxicity in vivo and in vitro.Firstly,the presence of different beers CDs was studied.CDs were extracted from Snowbeer,Harbin,Wernesgruner Dark,FAXE,and YanJing,respectively.CDs were freely dispersed in the aqueous solution and the fractions of interest purified from the five beers were colorless or light yellow under visible light and exhibited strong blue fluorescence under excitation of ultraviolet light at 365 nm.CDs were spherical in shape and well dispersed.With a narrow size distribution in the range of 0.94-4.13 nm.Snowbeer and Harbin CDs have a strong aggregation and crystallization structure,HRTEM revealed a lattice spacing of 0.263 nm and 0.204 nm,respectively.The quantum yield of the CDs from five beers rangs from 1.42%to3.92%.The quantum yield of CDs from Snow beer?3.92%?and Harbin?2.64%?beer is relatively high.The CDs mainly contained C,N,and O elements,and hydroxyl,amino groups,and carboxylic groups exist in the surface of all the CDs from beer.Red shift phenomenon of emission wavelength will occur with the excitation wavelengh increasing.This could indicate that the fluorescent behavior might be attributed to different emitting centers present in the CDs.Through careful characterization of the CDs from beer,the particle size and elemental content were found to be distinctly different.It is likely that these variations are due,at least partly,to the differences in the raw materials and production processes for different brands of beers.As discussed above,the properties of CDs from beer were largely the same.Secondly,take the case of Snowbeer,cytotoxicity of CDs was evaluated by using MC3T3-E1 cells as a model after incubation with Snow beer CDs(1 mg mL^-1)for 4 h,the MC3T3-E1 cells emitted brilliant blue light excited by a 405 nm laser,while almost no fluorescence was observed from the cells without CDs treatment under the same conditions.The emission region was mainly located in the cytoplasm region,which suggested that the CDs were able to disperse onto both the cell membrane and the cytoplasm of the MC3T3-E1 cells.When the concentration of CDs was up to 4 mg mL^-1.The results showed that cell activity decreased remarkably.The total ratio of apoptosis and necrosis of snow beer CDs with the concentration of 4 mg/mL increased by 13.3%,it is reasonable to conjecture that Snowbeer CDs may lead to DNA damage and result in the G₂/M arrest of MC3T3-E1 cells.The results showed that cells have been effected on 4 mg mL^-1Snowbeer CDs,leading to apoptosis.Moreover,the main reason leading to apoptosis may be the intracellular production of ROS.Finally,take Snowbeer for example,in vivo toxicity of CDs was evaluated.When gastric juice was added to the mixture,20.2%of the fluorescent intensity was immediately quenched compared to the control groups,small intestinal juice was added,the fluorescence intensity slightly recovered.The fluorescence quenching decrease might be attributed to the decomposition or the inhibition of digestive juice.In vivo toxicity of CDs was evaluated by using BALB/c mice as a model.In order to get detailed information regarding their fate after uptake by human,biodistribution,and acute oral toxicity evaluations of CDs from beer were conducted.The distribution of CDs in mice was performed,the results demonstrated that CDs could accumulate in some important organs such as intestine,liver,and brain.CDs from Snow beer were excreted from the mice within 1 day.The acute toxicity of Snow beer CDs in mice was evaluated by determination of physiological parameters.The body weight and major parenchymatous organs/tissue?intestines,stomach,spleen,kidney,liver,heart,lung,and brain?of BALB/c mice were determined 24 h after oral administration.No significant differences?p>0.05?were observed between the control and experimental group.Additionally,neither death nor obvious clinical toxicity signs were observed within the 24 h evaluation,no notable change in the urea,creatinine,LDH,GPT,AST,and ALP levels between the tested and control groups was observed,indicating that Snow beer CDs cause no tissue damage in mice levels in mice were investigated 24 h after administration,which suggested that CDs from Snow beer exhibited no acute toxicity in mice at the tested dosage.These results may help to draw attention to the presence of CDs in commercialized foods and their potential health risk,which lay the foundation for follow-up studied.