Mainstream smoke was passed through a 92 mm glass fiber filter disc for particulate matter collection.To prepare the condensate samples,the respective filter pads were placed in a flask containing dimethyl sulfoxide and shaken on a wrist-action shaker for 20 min.Each condensate sample was standardized to a concentration of 30 mg total particulate matter per ml of DMSO.Cytotoxicity of the smoke condensates was determined using the lactate dehydrogenase assay and the XTT assay.The LDH assay was performed using a kit according to manufacturer’s instructions.Briefly,FE1 cells were grown in 12-well plates and exposed to 8 concentrations of 1.5–30 g/ml of MSC or 3–90 g/ml of TSC in serum free medium for 24 h r.After plates were centrifuged,an aliquot was transferred to flat-bottomed plates and the LDH Assay Mixture was added.Plates were covered with aluminum foil and incubated at room temperature for 20–30 min.1 N HCl was added and the absorbance was measured at 490 nm,with the background measured at 690 nm.The XTT assay was also performed using a kit according to manufacturer’s instructions.Briefly,FE1 cells were grown in 12-well plates and exposed to 8 concentrations of 1.5–30 g/ml of MSC or 3–90 g/ml of TSC in serum free media for 24 h.The XTT reagent was added and the plates were incubated for 2 h at 37 ◦C.The plates were mixed and the absorbance was measured at 450 nm.Absorbance at the reference wavelength of 690 nm was also read and subtracted from the 450 nm value.A reference design with arrays as blocks of size 2 was used to analyze the median signal intensities of the two-color micro-array data.The experiment included main effects of dose,time and dose-by-time interaction.Five biological replicates per condition were used for each of the eight conditions,for a total of 80 micro-arrays.Six MSC and four TSC “outlier” micro-arrays were removed based on quality control checks,cannabis vertical farming leaving a minimum of 3 replicates per group.

The background signal intensity for each array was estimated using the 1533xSLv1 negative controls present on each array.All pre-processing of the data was conducted using R.The data were normalized using the LOWESS normalization method in the R library “MAANOVA”.Differential expression between the control and exposed samples for each of the three dose levels at each of the two time points was tested using the MAANOVA library.The ANOVA model was fitted to include the main effects of dose and time,with a dose by time interaction term and the array as a blocking variable.The Fs statistic,a shrinkage estimator,was used for the gene-specific variance components,and the associated p-values for all the statistical tests were estimated using the permutation method.These p-values were then adjusted for multiple comparisons using the false discovery rate approach.The least squares mean,a function of the model parameters,was used to estimate the fold change for each pairwise comparison of the six pairwise comparisons of interest among the eight treatment-by-time groups.The micro-array data for this experiment has been submitted to the Gene Expression Omnibus repository and can be accessed under record number GSE44603.Visualization and analysis of significantly changing genes was performed using Gene Spring GX 7.3.Important pathways containing significantly expressed genes were identified using Ingenuity Pathway Analysis.Genes were assigned to functional categories using gene ontology in the Database for Annotation,Visualization and Integrated Discovery.BMD Express was used to calculate benchmark doses from gene expression data.Analyses were performed on genes that were identified as statistically significant by one-way ANOVA using four models: Hill,Power,Linear and 2◦ Polynomial.Models that described the data with the least complexity were selected.A nested chi-square test,with cutoff of 0.05,was first used to select among the linear and 2◦ polynomial model,followed by comparison of Akaike information criterion,which measured the relative goodness of fit of a statistical model,between nested models and the power model.The model with the lowest AIC was selected as the best fit.A maximum of 250 iterations and a confidence level of 0.95 were selected.For functional classifications and analyses,the resulting BMD datasets were mapped to KEGG pathways with promiscuous probes removed.BMDs that exceeded the highest exposure dose were removed from the analysis.

Eight nanograms of total RNA,from the same samples that were used for the micro-array study,were reverse transcribed to cDNA using an RT2 First Strand Kit.cDNA was mixed with the RT2 qPCR Master Mixes and aliquoted into 96-well plates containing primers for 84 pathway specific genes.Expression levels were evaluated using a CFX96 real-time Detection System.Relative gene expression was normalized to the Gapdh housekeeping gene,which remained unaffected under experimental conditions.Fold changes and statistical significance were calculated using the REST method for statistical significance.For the micro-array study,FE1 cells were exposed to 2.5,5 and10 g/ml of MSC and 25,50 and 90 g/ml of TSC.Exposed samples were compared to their matched controls,and genes were considered significantly differentially expressed if they had a fold change ≥2 with an FDR-adjusted p-value ≤0.05.A total of 1020 unique probe identifiers were significantly differentially expressed following exposure to MSC,and of these,979 were deemed “present”.Following exposure to TSC,557 probes were significantly differentially expressed and 527 were deemed “present”.Of these,356 were common to both MSC and TSC exposures.The number of significantly up- and down-regulated genes at each time point and concentration is shown in Table 1.Overall,there was an increase in the number of differentially expressed genes with increasing concentration of condensate,and there were more genes changing after the four hour recovery.At the highest concentration for both time points,cells exposed to MSC had a greater number of changing genes as compared to cells exposed to TSC.Gene expression was most altered for cells exposed to the highest concentration of MSC at the 6 + 4 h time point.Whether separated by dose or considered all together,Venn diagrams show considerable overlap in the genes that are significantly expressed at each time point following MSC or TSC exposure.Hierarchal clustering using all genes that were statistically significant revealed that the controls and the marijuana high concentration clustered independently from the rest of the samples.The remaining samples clustered first by concentration,then by condensate type,with the last branching resulting from time.When cells exposed to TSC and MSC were analyzed separately,samples clustered first by concentration and then by time point,suggesting that concentration has the largest overall effect on gene expression.For MSC,the high concentration samples were on the first main branch,followed by control,low and medium concentrations.

The results indicate that the expression profiles of the high concentration MSC exposed cells are quite distinct.For TSC,the controls branched separately from all the treatment groups.The top 10 genes with the largest overall fold changes are listed in Table 2.All of the top 10 genes were significantly up-regulated with the exception of low density lipoprotein receptor,which was down-regulated in MSC exposed cells.Of the top 10 changing genes,five genes were common to both MSC and TSC.The GO terms associated with these commongenes included multicellular organismal development,vasculogenesis,regulation of transcription,and regulation of inflammatory response.Ingenuity Pathway Analysis was used to define the pathways that were significantly altered following exposure to MSC or TSC.Fig.3 shows the overlap in all the significant pathways between the two condensate types.The top five most significantly altered pathways for cells treated with MSC or TSC are listed in Table 3.NRF2-Mediated Oxidative Stress Response was the most significant pathway for cells exposed to TSC at all concentrations and time points,with the exception of lowest concentration at time 6 + 4 h where LXR/RXR Activation was the most significant.For cells exposed to MSC,the most significantly altered pathways were Biosynthesis of Steroids,as well as NRF2-Mediated Oxidative Stress Response,Aminoacyl-tRNA Biosynthesis and HMGB1 Signaling.Some of the top five pathways were common to both the MSC and TSC including those related to oxidative stress and xenobiotic metabolism.However,inflammation pathways were more predominant for the MSC,whereas cell cycling and cancer signaling pathways were more predominant for the TSC.To further elucidate differences between the two smoke condensates,the genes that were uniquely expressed following TSC exposure or uniquely expressed following MSC exposure at the highest concentrations for the two separate time points were compared in IPA.The findings confirm the importance of inflammation and steroid biosynthesis pathways in MSC exposed cells and highlight the significance of apoptotic pathways particularly at the 6 h time point.For cells exposed to TSC,Mphase cell cycle pathways appear to be of particular importance.

Gene Ontology in the Database for Visualization,Annotation and Integrated Discovery was used to apply functional annotation to all the significantly differentially expressed genes for each condensate.The full results are shown in Supplementary Tables 1 and 2.For cells exposed to MSC,cannabis drying racks significant perturbations were associated with steroid/cholesterol/lipid biosynthesis,NOD like receptor signaling,tRNA aminoacylation,transcription regulation,unfolded protein response and DNA binding.Like MSC,cells exposed to TSC had significant perturbations in transcription regulation,unfolded protein response and DNA binding.In addition,perturbations in cell cycle,p53 signaling,oxidative stress,and cancer signaling were also noted in TSC exposed cells.Fig.5 shows the overlap of all the significantly affected on tologies between the two condensate types.Functional annotation clustering in DAVID was used to minimize redundancy in the GO terms.This analysis revealed 19 clusters with enrichment scores greater than 2 for MSC and 19 clusters for TSC.The top clusters for MSC relevant to toxicological processes include lipid/steroid biosynthesis,RNA processing,cellular response to unfolded protein,tRNA aminoacylation,and positive regulation of transcription.The top clusters for TSC relevant to toxicological processes include cellular response to unfolded protein,cell cycle,positive regulation of transcription,response to steroid hormone stimulus,and positive/negative regulation of apoptosis and cell death.To investigate early versus downstream effects,functional annotation was applied to significantly differentially expressed genes at the two separate time points.The results are shown in Supplementary Tables 5–8.For cells exposed to MSC at the 6 h time point,the analyses revealed 79 significant terms including those related to transcription activity,DNA binding,and steroid/cholesterol biosynthesis.Four KEGG pathways and 1 Biocarta pathway were also deemed significant at this time point.At the 6 + 4 h time point,76 significant terms were identified.These terms included unfolded protein response,and tRNA aminoacylation,as well as steroid/cholesterol biosynthesis which was found at the 6 h time point.Three KEGG pathways were significant at this time point including Steroid Biosynthesis,Terpenoid Backbone Biosynthesis,and Aminoacyl-tRNA Biosynthesis.Analyses of cells exposed to TSC at the 6 hr time point revealed 67 significant terms including those associated with oxidative stress,cell death,protein unfolding,transcription regulation,DNA binding and cell cycle.In addition,2 KEGG pathways were significant.At the 6 + 4 h time point,32 GO terms were identified as significant with oxidative stress being the only relevant toxicological endpoint.In addition,only one KEGG pathway was significant.

Overall for MSC,the DAVID analyses confirmed many of the significant pathways identified by IPA including steroid biosynthesis,tRNA aminoacylation,inflammation and apoptosis.In addition,the analyses highlighted transcription regulation,DNA binding and unfolded protein response as also significant.For TSC,the DAVID analyses confirmed the significance of IPA pathways related to oxidative stress and cell cycle.As with the MSC,the DAVID analyses also further highlighted the importance of transcription regulation,DNA binding and unfolded protein response,as well as cell death.Transcription regulation and DNA binding were significant terms common to both MSC and TSC at the 6 h time point,whereas no common terms existed for the two condensates at the 6 + 4 h time point.In our previous genotoxicity study we showed that MSC and TSC were both cytotoxic and genotoxic.However,quantitatively,MSC was more cytotoxic and mutagenic than TSC,and TSC appeared to induce chromosomal damage in a concentration-dependent manner whereas MSC did not.Our earlier chemical analyses of MSC and TSC noted that aside from the nicotine in tobacco and the cannabinoids in marijuana,the two smoke condensates contained mixtures of chemicals that were qualitatively similar though quantitatively different.The similarities in the chemical profiles and some of the toxicity findings suggested that the two smoke condensates might elicit somewhat comparable gene expression profiles.Hierarchal clustering of all the MSC and TSC exposed samples in the present study supported this notion and samples clustered first by concentration as opposed to smoke type.In addition,analysis of the top ten greatest gene expression changes relative to control revealed that half of the genes were common to both marijuana and tobacco.A number of previous studies have examined gene expression changes in pulmonary cells following exposure to tobacco smoke.Generally,these studies have shown that tobacco smoke stimulates xenobiotic metabolism,and that metabolized smoke constituents contribute to DNA damage.Following early insult,DNA damage leads to disruptions in the cell cycle such as arrest at the G2 checkpoint to allow time for response.Cellular response can include DNA repair,mutation induction through faulty repair or lack of repair,and programmed cell death of heavily damaged cells.

With the increase in production,advanced and standardized processing technologies,including drying,extraction and purification,are required to ensure the quality and safety of these products for food applications,as well as the processing efficiency and sustainability of hemp.Some terpenes,on the other hand,are considered generally regarded-as-safe substances,which are permitted to be added and have been used in foods.However,the incorporation of hemp terpenes in foods and preservation of terpenes during processing is still challenging,requiring further research.In recent years,many studies have been conducted to identify the functionalities and pharmaceutical values of hemp CBD and terpenes,as well as extraction technologies of CBD.However,there is currently not a comprehensive review that discusses the potential of hemp CBD and terpenes as future functional food ingredients and evaluates the available and potential processing technologies for hemp.Therefore,this article reviews the legal regulations,challenges in incorporating hemp CBD and terpenes in foods and potential solutions,as well as current research status and future prospects in processing technologies of hemp biomass.Through this review,we expect to draw a clearer image for food manufacturers and researchers on the potential of applying hemp CBD and terpenes as future functional food ingredients and provide information for standardizing and improving the processing technologies of hemp for efficient production of high quality and food-safe products.Future research needs are also identified in this article.In the hemp plant,cannabinoids mainly reside in the flowers and leaves.More specifically,they are primarily stored in the glandular and non-glandular epidermal appendages called trichomes,which are transparent glandular ‘hairs’ on the surface of inflorescence and leaves.

The cannabinoids are chemically bounded with the cell walls,secretory vesicles and fibrillar material in the secretory cavity of the disc cell.In this section,the physical properties and chemistry of CBD,cannabis growing system health benefits,potentials and associated challenges as future functional food ingredients are discussed.Chemically,cannabinoids are a type of molecules that contain terpenoids and alicyclic units.Currently,more than 120 types of cannabinoids have been found in hemp plants.Fig.3 shows the biosynthetic pathway of cannabinoids in hemp plant.The basic reaction in the cannabinoids synthesis involves the alkylation of the phenolic moiety of olivetolic acid with the terpenoid component of geranyl pyrophosphate ,which produces cannabigerolic acid ,the common precursor of all major cannabinoids in the hemp.CBGA can then convert into other cannabinoids such as tetrahydrocannabinolic acid ,cannabichromenic acid and cannabidiolic acid ,etc.,through the enzymatic catalytic reactions by THCA-synthase,CBCA-synthase and CBDA-synthase.It is worthy of mentioning that only the acidic cannabinoids exist naturally in hemp plants.According to their molecular structures,cannabinoids are non-polar,and thus have low solubility in aqueous solution.The boiling points of cannabinoids ranged from 157 °C to 220 °C,which is not volatile.However,CBD is thermally unstable and very sensitive to photolytic reaction and oxidation.The acidic form cannabinoids are not considered to be pharmacologically active since they do not affect the endocannabinoid system of human body in the same way as the neutral forms.Therefore,a decarboxylation process is usually needed to convert the CBDA to CBD,which involves the removal of a carboxyl group from the acidic molecule and the release of a CO2 under high temperature.The reaction mechanisms of the decarboxylation process are illustrated in Fig.3B.Recently,research has proven the functionalities of CBD,which include pain relief,reduction of anxiety/distress,nausea and promotion of relaxation.

In addition,since CBD is not psychoactive,it does not result in addictive effect as caused by THC.At present,there is an increasing prevalence of lifestyle induced chronic diseases,and consumers are pursuing healthier diets,seeking more functional foods reinforced with functional ingredients.Currently,the global health and wellness market is at $1.5 trillion,and is projected to increase annually at 5%–10%.In 2019,the global functional food market was $250 million,and was projected to $440 billion by 2022.Therefore,there is a high potential in this rising trend to add hemp CBD in foods or beverages as future functional food ingredients.However,there are several challenges that are limiting the development of CBD-containing foods,which involves legal regulations,consumer awareness,and technological deficiencies.Legal regulations and policies are one of the most important factors in directing the developments of markets and products.Currently,regulations on the hemp CBD varies country by country,and CBD is in the ‘grey zone’,of which the legality is not clearly defined.In the United States,the 2018 Farm Bill removed industrial hemp from the definition of marijuana,which allowed more research to be done on hemp.However,it is still prohibited in federal level to add CBD to any foods,beverages or nutrient supplements.FDA has not waived it from the illegal ingredients list due to safety concerns of the non-approved CBD products and the fact that CBD was first studied as a drug.In fact,in 2018,FDA approved the first and by far the only medicine containing CBD for the treatment of seizures associated with two rare and severe forms of epilepsy,Lennox-Gastaut syndrome and Dravet syndrome.Even though it is still not approved in federal level in the US,some food products are already in the markets in some states as they deregulated the CBD,such as tea,coffee and chocolate,etc..In Canada,cannabis is nationally legalized,but phyto-cannabinoids such as CBD is in the Prescription Drug List and cannot be legally sold in self-care natural health products or cosmetics.

Canadian Health Food Association has recently released a report,which emphasized the potential positive economic impacts of permitting CBD in NHPs,foods and beverages for the economic recovery.In Europe Union ,CBD was catalogued into ‘novel foods’ in January 2019,and the EU has required extensive testing and authorization of CBD products from food safety authorities.In addition,different countries have different standards in legalizing the THC level in the products.China was projected to be largest producer and consumer of hemp CBD,given the long cultivating traditional and production area,however,recently a national level regulation forbids any use of CBD in foods or cosmetics.The strict regulations have a significant impact on the development of the hemp industry,and the major food manufacturers are conservative in entering the CBD market.In fact,due to these strict regulations and the influence of pandemic on the global logistics,the hemp market and product development process have been significantly impacted.The hemp biomass and CBD are oversupplied in the market,and the price of CBD has hit the rock bottom in the first half of year 2021.The enthusiasm of farmers in cultivating hemp is cooling down.However,we envision that this ‘downturn’ could be temporary.With more research works done and more scientific evidence published proving the health benefits and safety of CBD,the regulations shall be more relaxed and standardized,which will be a strong booster for both the CBD-food market and in return benefit more related research to be conducted.In most cultures,the words ‘hemp’ and ‘cannabis’ are associated with ‘drugs’ and ‘illegal’,and thus are repelled.The word ‘CBD’ is still unfamiliar to most people in the world,not to mention its health benefits.Therefore,to promote the use of CBD as functional food ingredients,educating the consumers about the health benefits,safety,and its distinction from ‘drug’ is as important as the relaxing of legal regulations.At the same time,it is critical to find the right market position and target consumer groups.Currently,regular CBD consumers are buying CBD to obtain a functionality or health benefit,and usually taking it as a dietary supplement/medicine,which only represent a niche market.It is important to identify the types of foods that are compatible with CBD.Incorporating CBD into staple foods and beverages may potentially expand the consumer base,which is currently challenging and require more extensive research.The consumer needs for the CBD reinforced foods are another important factor to be considered in the product developments.For example,an afternoon pick-me-up ‘CBD-containing coffee’ or a sports drink need to be fast-responding,meanwhile it should not cause any adverse side effect to the human health.

Additionally,the food products need to be shelf-stable and maintain the activity of CBD under normal storage conditions,particularly given the fact that CBD is not a very stable substance.Besides the regulation and consumers aspects,there are currently some technological challenges that need to be resolved to add hemp CBD in foods and beverages.The types of products that can be reinforced with CBD are dependent on the physical and chemical properties of CBD.As discussed above,CBD is a highly non-polar molecule,meaning it has low aqueous solubility.Therefore,it is difficult to incorporate it into most aqueous-based food and beverage systems.Besides,recent studies have shown that the bio-accessibility of CBD is relatively poor in animal and human bodies,which may be due to the low cell penetration rate.In addition,CBD has relatively poor stability,which is affected by various factors such as temperature,humidity,pH,oxygen,light,and the solvent types,etc..Its instability suggests that CBD is vulnerable to deteriorate when it is inappropriately added to food systems or exposed under stress conditions.The oral ingestion of cannabinoids usually results in long lasting and slow effects,hydroponic rack system which is not desirable for the fast-responding needs,and is identified as an area of improvement.Currently,studies on the solubility and stability in various food and beverage systems are not well reported.The studies on the bioaccessbility of CBD in human body are also scarce.More extensive research in these aspects is urgently needed to address these challenges.The outcomes will provide scientific evidence to support the feasibility of adding CBD into foods and further prove their health benefits.Although the most of CBD-containing foods are considered illegal at present,the future can still be bright and optimistic.The 2018 Farm Bill of the U.S.opened the gate of industrial hemp to industries and researchers.Following that,more and more capital investments are putting into the hemp industry and scientific research.According to statistics,the global market size of hemp was $5.73 billion,and was expected to grow to $27.72 billion by 2028.This allows more universities and research institutions to conduct necessary research,which in return will be beneficial for the development of CBD-containing foods.Although current regulations prohibit the use of CBD as food ingredients,we envision that some regulations will ease up in the near future.This will depend on more scientific studies to be conducted and evidence that proves the health benefits and determines any side effects of CBD to human beings.In fact,United Nations has recently removed cannabis from its most rigid drug control list,the ‘Schedule IV of the 1961 Single Convention on Narcotic Drugs’,which was a huge win for the hemp industry.More organized and standardized regulations all over the world will also be helpful for the development of CBD-containing food products.In technological aspects,some studies have been conducted to improve the solubility,stability and bio-accessbility of CBD in medicine or food systems.Encapsulation,emulsification and microfluidization are being used to incorporate various bio-active compounds into food and beverage systems in the industry and offer potential solutions to the development of CBD-containing foods.

Micro-encapsulation is one possibility to improve the solubility and reduce the oxidative susceptibility of sensitive bio-active compounds,where the hydrophobic molecule is encapsulated and dispersed in aqueous phase.Feng et al.applied sesame oil as a delivery system for CBD in medicines and revealed that adding of medium-chain triglyceride improved the solubility of CBD and superior bio-availability.Lange and Schilderink found that micro-encapsulated CBD had higher CBD bio-accessibility compared to CBD-isolate without micro-encapsulation.Mozaffari et al.discovered that the CBD bio-accessbility was improved with the incorporation into a food system made of olive oil and baby food,which might be attributed to the improved efficiency of micelle forming from hydrolyzed lipids.Nanotechnology has already been applied in drug delivery systems and can be another promising solution.Durán-Lobato et al.developed cannabinoid-loaded lipid nanoparticles through solvent-emulsion evaporation and improved the stability of the products.With more relaxed regulations and more scientific research being conducted,it is envisioned that the technological challenges in the development of CBD edibles may be overcome in the future.Terpenes are another type of bio-active compounds and secondary metabolites found in hemp,which are gaining increasing interest in recent years.There are currently more than 120 terpenes already identified from hemp.The most distinct characteristic of terpenes is the smell,which depicts the unique aromatic characteristics of hemp plant and is used by the plant to repel and defense against herbivores,attract pollinators,and inhibit the microbial growth.Depending on the variety of hemp,the typical terpene contents in hemp range from 0.125% to 0.278% weight in leaves and 1.283% to 2.141% in the inflorescence on a dry basis.Although hemp is abundant in terpenes,it is not the only source.In fact,terpenes are found in lots of fruits,vegetables and herbs.Chemically,terpenes are hydrocarbons consisting of small isoprene units linked to one another to form chains.The biosynthetic pathway of terpenes is shown in Fig.4.Interestingly,terpenes and cannabinoids share a common precursor,which is the GPP,a 10-carbon molecule.As a result,the contents of terpenes and cannabinoids in the hemp are usually positively correlated.Monoterpenes are composed of two isoprenes such as pinene,limonene,myrcene etc..Sesquiterpenes consist of 3 isoprene units,such as humulene,farnesol and caryophyllene.More complex terpene molecules include triterpenes that are made of 6 isoprene units,and polyterpenes.As shown in Fig.4A,the monoterpenes and sesquiterpenes are the major terpene species found in the hemp.

The influence of the treatments with Na2CO3 and BTCA on the shives SHI-C and SHI-BTCA microstructure is clearly visible on the nano-CT scans.The removal of a part of the hemicelluloses in the case of sample SHI-C led to the partial deconstruction of the woody fibers cell wall,and general disorganization of the cells within the tissue,which is directly observed through the misorientation and waviness of the woody rays.The fiber walls were also strongly affected by grafting ,the woody rays degraded,the vessels distorted,and the tissues got fully disorganized as shown in Fig.3c.Indeed,the cellulose compartment seems the most affected,with a strong decrease,whereas the lignin content was increased,reflecting an enrichment effect.One can note that the pectin content is also higher in the BTCA sample,suggesting a selective chemical targeting of the treatment on cellulose polymers Fig.3.also shows EDX spectra of SHI-W,SHI-C,and SHI-BTCA samples before copper adsorption,highlighting the presence of sodium on the surface of hemp shives after Na2CO3 treatment and BTCA grafting.Fig.4 presents the X-ray nano-CT images and EDX spectra of the raw and modified hemp shives after copper adsorption.No significant effect of the exposition to copper on the shives’ microstructure can be detected but a major change in the EDX spectra can be observed.The data point out the presence of peaks that were not observed before the shives immersion in the copper salt solution,flood and drain tray which was assigned to copper.It is worthy to remind that EDX is a technique for surface characterization with limited penetration in matter thickness.

To better identify the spatial localization of the copper in the whole shives’ volume,a density-based segmentation of the 3D computed nanotomography images was done.The obtained images are proposed in Fig.5.The figures 5 b and d show the typical microstructure of hemp shives where the vessels are isolated or grouped by two or three,rarely by more,and then they deform one another.The vessels have a quite thin cell wall and a diameter of approximately 50 to 150 μm.They are surrounded by relatively thick-walled woody fibers with diameters of only a few μm and an irregularly polygonal section with a rounded cavity.It is evident that copper is adsorbed on all the free surfaces of the SHI-WCu sample,meaning shives external surface but also on the internal surface of the cells’ lumen.The situation is something different in the case of SHI-CCu shives where it is possible to observe that the copper is not only located on the internal surface of the cell wall bordering the lumen but also in the wall itself.This is attributed to the partial removal of the hemicelluloses in the cell wall induced by the treatment with sodium carbonate and by the micro- and nano-porosities created in the cell wall in which the aqueous solution can diffuse.Furthermore,the copper is massively adsorbed on the internal surface of the vessels’ wall bordering the lumen of the sample SHIBTCACu.The BTCA presence allowed more copper to be absorbed in these areas when compared to raw shives,which is in accordance with the results presented in Fig.1.The detailed analysis of EDX and nano-CT results showed that these two techniques were complementary in revealing the mechanism of copper adsorption on materials.In the EDX spectra ,we can observe an important decrease in the sodium peak and its replacement by copper cations,suggesting the presence of interaction by ion-exchange.In addition,some copper agglomerates are also observed in the internal part of the wall of some of the vessels,suggesting micro-precipitation during the adsorption onto the SHI-BTCA sample.So,the results point out that,even if the two treatments performed similarly in terms of copper removal,the spatial localization of copper and the adsorption mechanisms were significantly different.

FTIR and Raman spectroscopy were further used for the characterization of the shives before and after copper adsorption.The main IR and Raman absorption bands and their assignments according to the literature are summarized in Table 2 and Table 3,respectively.These bands were generally attributed to the three main shives components: cellulose,hemicelluloses,and lignin.The comparison of SHI-W and SHI-C samples shows an important difference in their IR spectra.Namely,the bands at 1730 cm− 1 and 1250 cm− 1 assigned to the stretching of unconjugated C=O groups present in hemicelluloses disappeared after the activation with sodium carbonate.This is in agreement with the data of the chemical composition and is also observed after the partial hemicellulose removal from jute fibers using alkaline treatment.When the FTIR spectrum of the SHI-BTCA sample was observed,the bands in the region 1500-1800 cm− 1 in particular those of the carboxyl and carboxylate groups ,are more intense than in the case of the two other samples which is ascribed to the esterification reaction.In addition,the comparison of SHI-W and SHI-BTCA samples also shows an important increase of the band at 1730 cm− 1 and a decrease of the band at 1250 cm− 1 corresponding to the elimination of hemicelluloses enhancing the lignin content in the material.The interpretation of Raman data indicated that the SHI-W and SHI-C spectra were similar with little differences in the intensity of the bands.On contrary,the Raman spectrum of the SHI-BTCA sample showed an important change in the intensity of the ester band at 1743 cm− 1.The comparison between the FTIR and Raman spectra before and after copper adsorption for the studied samples is shown in Fig.6.After copper adsorption,no changes were observed in the IR spectra of SHIWCu and SHI-CCu,while the spectrum of SHI-BTCACu showed an important change at 1590 cm− 1.Indeed,this band underwent a profound change in both intensity and width,and shifted to 1610 cm− 1.This data reflected the implication of the carboxylate groups on the copper removal through complexation and/or ion-exchange.By comparing the samples’ Raman spectra,we observed that the alkaline activation resulted in the disappearance of the ester band at 1743 cm− 1.The addition of copper contributed to obtain samples with a very high sensitivity to burning,resulting in spectra with enhanced background noise.

This is the reason why we had to work at 1% laser to avoid such noisy phenomena.However,it was difficult to explain the difference in the ratio between polysaccharides and lignin.In this case,we observed more lignin signals in samples containing copper.SHIBTCA gave very fluorescent samples,around 60,000 counts in comparison to 10,000 counts or less for SHI-C.For this sample,we also worked with a 1% laser both to avoid the detector’s ’ saturation and to burn the copper containing sample.The Raman spectrum for SHI-BTCACu also showed the involvement of the ester group in the removal of copper ions,confirming the presence of various interactions between them.Finally,the Raman spectra of samples after copper adsorption also revealed two bands at 1420 cm− 1 corresponding to carboxylate group and at 885 cm− 1 ,in agreement with IR data.Fig.7 compares the XPS wide-spectra of the SHI-W,SHI-C,and SHIBTCA samples before and after copper adsorption.The spectra obtained before copper adsorption are very similar showing the presence of oxygen and carbon C1s at their surfaces.Nevertheless,hydroponic tables canada the sample SHI-BTCA has an additional peak corresponding to the presence of sodium localized near the surface at 1072 eV.The presence of this element is due to the conversion of the carboxylic groups into a carboxylate by immersing the BTCA treated shives in a sodium hydrogen carbonate solution.The 1s level of carbon allows access to the different oxygen functions.The decomposition of level 1s of carbon is significantly different for sample SHI-BTCA with a strong decrease in C-O / C-OH bonds in favour of carboxylic functions.These results are in agreement with the data of the chemical composition.After copper adsorption,Fig.7 clearly indicates the disappearance of the sodium signal in the SHI-BTCA spectrum,due to the ion-exchange with copper cations.Fig.8 shows the main and satellite peaks of Cu 2p3/2 and Cu 2p1/2 after copper adsorption onto three studied samples,while Table 4 reports atomic concentrations of detected elements before and after adsorption.All three samples showed XPS peaks around 933 eV and 953 eV characteristic for Cu 2p3/2 and Cu 2p1/2,respectively.

The XPS analysis of the Cu 2p peaks showed that this element was not in the same oxidation state in all studied samples.Indeed,the detection of satellites on the 2p level of copper for the SHI-BTCACu sample makes it possible to deduce also the presence of copper at the oxidation number for 60%.These satellites are associated with the peak at 934.66 eV of the 2p3/2 level which corresponds to copper in the form of Cu2 [33].In the case of SHI-CCu and SHI-WCu,the detected copper was only in the I oxidation state.Indeed,there are no satellites,the 2p3/2 level showed only one component at about 933 eV.The measurement of the kinetic energy of the Auger transition of copper L3M45M45 at approximately 913.7 eV allowed us to calculate a modified Auger parameter value at 1846.7 eV close to the species Cu2O.This shows that the reactions involved in the removal of copper were different for each hemp sample.In order to know the rate of adsorbed copper on the surface,the atomic percentage of copper was calculated from the area of the Cu 2p peaks.SHI-BTCACu sample had the highest ratio Cu/C = 8.3,followed by SHI-CCu which ratio is 1.5.SHI-WCu sample had a very low Cu/C ratio of 0.4.These results agree with those obtained with other characterization techniques,notably on the fact that copper is well absorbed at the surface for SHI-BTCACu,which gives it this green colour.The less important Cu/C ratio for SHI-CCu confirms that,in this case,the mechanism of copper adsorption is different from those of SHI-BTCACu.Namely,the main interaction between copper and sodium carbonate activated shives is diffusion inside the particles.XANES spectroscopy provides quantitative insight into the oxidation states of copper present in materials.The XANES spectra of SHI-CCu and SHI-BTACCu display the same shape with a pre-edge at 8977.5 eV,the edge at 8991 eV,the maximum of the white line at 8996.5 eV,and a broad oscillation centered on 9052 eV.The absence of peak around 8983 eV,as found in Cu2O,shows that Cu was not detected and that Cu oxidation did not change during the Cu adsorption,it was kept as Cu.CuSO4,5H2O spectrum shows specific features such as a shoulder at 8990.0 eV and an oscillation at 9008.0 eV that are not present in hemp samples’ spectra.Moreover,the white line of CuSO4,5H2O spectra is slightly shifted toward lower energies and is more symmetric compared to that in hemp samples’ spectra.

These differences indicate that after adsorption onto hemp,Cu did not correspond to the crystalline form of CuSO4,5H2O.Furthermore,the hemp samples’ spectra are quite different from those of CuO and Cu2,corresponding to other Cu-bearing precipitates.The spectra of hemp samples are similar to the ones of Cu2+ measured in liquid phase; CuSO4 but also Cu2,another Cu complex,both when recorded in a liquid phase.The results obtained by XANES and those obtained by XPS or both Cu and Cu seems to differ.However,they are compatible and complementary as XANES at Cu K-edge sounded the bulk sample whereas XPS sounded the first atomic layers on the surface of the sample.XANES spectroscopy evidenced that most of the Cu atoms are present in Cu form in both SHI-CCu and SHI-BTCACu samples and XPS spectroscopy indicated that the external surfaces of SHI-WCu and SHI-CCu are coated by Cu2O and that the external surface of SHIBTCACu is coated by a mixture of Cu2O and Cu2,explaining this characteristic blue color.An increased interest in the acceptance of novel foods,that is,foods to which a consumer has not been previously exposed ,is borne of an increasing global population that should reach nine billion by the year 2050.Globalisation has resulted in increased exposure of the world’s population to foods from other cultures,and increased multicultural culinary experiences have altered normative perceptions for many.The increasing global population presents a challenge to food security as many traditional food production techniques will become environmentally unsustainable at the levels required to meet world food demands.Hemp foods might suitably address many food security issues.The hemp plant,Cannabis sativa,from which hemp foods are produced is environmentally sustainable due to a reported low water need and natural pest resistance ,can be economically lucrative with high yields and shorter growth cycles compared with many traditional crops ,and has many nutritional benefits.Nutritional benefits of hemp include being high in levels of plant protein,high in dietary fibre,a rich source of Omega 3 and 6,and contains all of the amino acids essential to human life.

Another benefit of hemp seeds is that they contain proteins that are rich in several essential amino acids including arginine and the sulfur-rich amino acids methionine and cysteine.Hemp contains other known beneficial and nutraceutical compounds but also some antinutrients including phytate and trypsin inhibitor that can impact on nutrient uptake especially of minerals and amino acids.However rat model studies confirm the high bio-availability of hemp proteins suggesting that the anti-nutrients may not be a major concern.A disadvantage of one of the earliest seed cultivars,FINOLA,is that the seed is about 50% smaller than other seed varieties.Comparisons between different lines are usually based on thousand seed weight.A recent comparison of 33 lines showed TSW ranged from approximately 7.5 g–23 g,with FINOLA at around 12 g,which is at the lower end of the expected range of 12–15 g for FINOLA.Seed size is not the only important consideration for seed varieties.A higher proportion of heart to hull tissue ,here called heart %,is desirable since it is the heart that attracts a higher value margin.A study of five hemp cultivars in Romania showed that heart % varied from 59.0% to 69.5%.However,current data on the heart % trait is limiting with very few published studies.Hemp hearts,also known as dehulled seed have more protein and more “digestible” fiber than whole seed and seed meal ,based on analysis of a “typical” variety.Interpretation of fiber data in food products can be difficult,due to the use of different terminology and methods.Scientific papers often report both neutral detergent fiber and acid detergent fiber ,where NDF includes cellulose,‘hemicellulose’,and lignin whereas ADF only includes cellulose and lignin,thus providing a measure of non-fermentable fiber.One disadvantage of NDF and ADF is that they underestimate the amount of total fiber,as the methods may not recover all of particular components such as pectins and gums,which can be some of the healthier fermentable fibers.However,this information is useful because it provides an indirect way to determine lignin content,trimming tray for weed and high levels of lignin can negatively affect palatability.

The nature of the non-cellulosic polysaccharides that contribute to dietary fiber in hemp has not yet been studied.One review suggests that hemp seed contain 25% starch,however the two cited references report soluble fiber and not starch.Since “detailed chemical characterization of dietary fiber is crucial to explain its effect on health” ,there is a clear knowledge gap of the nature of the polysaccharides in hemp lines grown for food.Different cell wall polysaccharides have remarkably different functional properties and these in turn influence fermentability,nutrient bio-availability and composition of gut microbes.The role of complex carbohydrates in microbiome diversity and human health is becoming clearer,and many positive effects relate to the fermentation products or short chain fatty acids such as butyrate,acetate and lactate.It has long been established that plant polysaccharides can differ in length of backbone,number and distribution of side branches and the form of each monosaccharide and pyranosyl and the linkages between them.Even within the same general class the structures can be markedly different.For example,heteroxylans from different species of Plantago have different proportions of unsubstituted xylan and linkages along the xylan backbone.It is now understood that microbes exist in cooperative metabolic networks where selected bacterial species initiate degradation and other species continue fermenting the partially degraded polysaccharides,thus supporting microbial biodiversity and a healthy colon.To fill the knowledge gap on the nature of hemp polysaccharides,we analyzed the composition of 20 different industrial hemp varieties and breeding lines that are currently available in Australia.We report analysis of 1000 seed weight,heart %,% nitrogen ,lipid profiles,lignin,phytate,and a detailed analysis of the carbohydrates in hemp heart and hull fractions including cellulose,starch,monosaccharide composition of complex carbohydrates,and soluble sugars.In addition,we use specific antibodies to investigate the distribution of plant polysaccharides in hull and heart fractions,thus providing critical information on the distribution of non-cellulosic polysaccharides in hemp seed.One hundred seed were counted out and weighed,then multiplied by 10 to provide 1000 seed weight.A single replicate of 100 seed was selected for further analysis.Hull and heart tissues were separated using a fine spatula and the separated fractions weighed.

Hull fractions were ground in a retsch mill for 30 s,and the softer heart fractions were ground in liquid nitrogen and air dried prior to analysis.These ground fractions were used for quantification of cellulose and soluble sugars and monosaccharide analysis ,lignin and starch.This research is the first detailed study of complex carbohydrates in hemp seed using a combination of chemical analysis and immunolabelling of tissue sections from a selected set of germplasm mainly sourced from Ecofibre’s seed bank.The focus of this study was on end-user/health traits where diversity of dietary complex carbohydrates is positively correlated with microbiome diversity and improved human health outcomes.The new findings on carbohydrate composition are combined with analysis of heart %,1000 seed weight,lipid composition,total protein,lignin and phytate,thus providing a foundation for selection of plant lines with improved human health attributes for expanding the hemp industry in Australia and internationally.Coefficient of variation % is a useful parameter for determining the characteristics that vary most between lines.Since the batches of seed used in this study were sourced from different regions of Australia,it is not possible to tell if the source of variation is from genotype or environment or an interaction of both.However,CV information is still useful for prioritising hemp lines for future field trials,and traits for subsequent analysis,and for identifying traits with a strong genetic component suitable for targeting in breeding programs.In the following discussion,we make the assumption that traits with a high CV have a strong genetic component,but obviously this will need to be tested in the future.There is some support for this assumption based on a hemp field trial from Canada that evaluated 11 cultivars over two years in seven different environments.Analysis of field trial data revealed that most of the traits evaluated had statistically significant genetic ,environment and G x E components,including seed protein,seed yield,plant height,biomass yield,biomass cellulose and biomass hemicellulose.

However,for the 11 varieties studied there was not a significant genetic component for biomass lignin and seed oil,but both had significant G x E components,and seed oil also had a significant environmental component.In our study,heart %,seed lignin and total lipids had a CV of <10% indicating low variability,whereas,many traits had intermediate CV ,including crystalline cellulose,hull monosaccharides ,non-resistant starch,phytate,the essential omega-3 fatty acid ALA.A few traits had high CV ,including 1000 seed weight,GLA,soluble sucrose and soluble raffinose.This information can be used to select a smaller number of appropriate lines for statistical validation of each desired trait,and used by breeders to select lines for future field trials to determine the extent of the genetic and environmental components of each trait.There is high variation in 1000 seed weight.Thousand seed weight provides an approximation of seed size,as generally the bigger the seed,the heavier it will be,although variation in the composition and ratio of heart % means that this correlation may not always hold.Furthermore,seed size does not usually correlate with overall seed yield which is key for profitability,since yield is heavily influenced by inflorescence architecture and shattering resistance.Higher heart % provides an opportunity to increase profitability,through increases in higher value hemp heart products.The heart % trait has not been widely reported for hemp,with only one published study to our knowledge,where heart % varied from 59.0% to 69.5%.Twelve of the 20 lines analyzed in this study had heart % greater than 59%.This does not guarantee that these lines are high yielding,profitable lines for current use,but rather they are potential lines for breeding,if the heart % trait has a strong genetic component.For other nuts,such as Macadamia,high heart % is called kernel recovery,and is known to be influenced by the genotype of the pollen donor.Kernel recovery in Macadamia is also sensitive to mild drought stress during nut development.To our knowledge there have not been any studies in C.sativa on what factors affect kernel recovery ,but we hypothesize that pollen donor and/or stress could be important,and should be explored in future to determine genotype by environment effects on this trait.Hemp hulls contain crystalline cellulose as the major polysaccharide ,with xylan as the next most abundant polysaccharide.The identification of xylan as a major complex carbohydrate in hemp hulls is based on monosaccharide analysis and immunolocalization analysis which demonstrates the polysaccharide is present as an unsubstituted xylan and not arabinoxylan.

The variability in hull xylose content is of significant interest to the food industry for adding value to agricultural wastes.Xylan is a source for the production of xylan oligosaccharides for prebiotics,which are non-digestible food ingredients that promote growth of bacteria in the colon and can improve health.Xylan oligosaccharides have been produced from peanut shells.The two hemp lines with the highest levels of xylose in their hulls,Frog-1 and Yunma-1 ,are likely to have higher levels of xylan than peanut shells,assuming that all the xylose is hemp is found in the xylan as suggested by immunolocalization.Hemp hulls and hearts are low in soluble sugars,with hemp hearts generally containing more soluble sugars than the hull fraction.The new data reported here is mostly consistent with a previous study that showed that sucrose was the most abundant soluble sugar.The major difference between the earlier study and this study is that they did not detect any raffinose in whole hemp seeds,whereas here raffinose was found in all 20 heart samples.This difference is puzzling since the 2018 study was able to detect raffinose in other food samples,weed trimming tray however it is not of great importance given the low levels <0.5% w/w observed in the 20 lines analyzed here.An important finding from the current study is that starch is not a major component of hemp seed,based on the trace levels of glucose in the polysaccharide fraction and <2% total starch in the six lines chosen for starch quantification.This contrasts with information in a recent review where the authors state that hemp seeds contain 25% starch.The results presented here show that the dietary fiber is not starch but predominantly xylan and pectin in hulls,with a small amount of xyloglucan and pectin in hemp hearts.This is important because it suggests that hemp will have a low glycemic index and contains a range of complex carbohydrates that could be fermented in the gut to varying degrees.This could be readily tested in future by including hemp as a protein source in human diets,because SCFAs,the products of gut fermentation,are readily detected in stools making them convenient biomarkers.Other studies adding whole seeds to human diet plans show that increases in SCFAs can be achieved in relatively short time frames.

The fate of the different cell wall polysaccharides is an area of active research,for example,xyloglucans from cranberry promote the growth of SCFA producing strains of Bifidobacterium longum.Furthermore a phenol-free xyloglucan derived oligosaccharide fraction from cranberry hulls reduces biofilm formation by strains of E.coli that cause urinary tract infections.Pectins are also substrates for gut bacteria,and although present in small amounts in hemp,they should contribute to microbiome diversity,especially as their fine-structure may be different to commonly consumed pectins such as citrus pectin.The fine-structure of the hemp carbohydrates remains to be determined,and xylan would be the obvious first target as it is the major noncellulosic polysaccharide in hemp.The hull should not be overlooked as a food source.It can be milled for example and used as a high-fiber ingredient,or better still,more emphasis could be placed on developing food products that use the whole seed.In addition to providing the best human health outcomes,use of whole seeds may have the benefit of reducing manufacturing costs and waste products generated through the dehulling process.Whole seed products would also contain lignin ,which can be perceived as a negative component.However,rather than being an inert compound,lignin is now thought to contain beneficial antioxidant activity.The mean value of lignin in hemp hulls observed in this current study was 17.6  1.0 ,which is higher than a prior study that reported a mean value of 11.2.However,the values are comparable given that our study used hemp hulls,not whole seed.The other major components of hemp hearts are proteins and lipids.The study conducted by House et al.,used a rat bio-assay to calculate protein digestibility-corrected amino acid score measurements.Importantly they showed that hemp protein had PDCAAS value equal to or greater than certain grains,nuts,and some pulses.specifically whole hemp seeds have a higher PDCAAS value than almonds and whole wheat,and hemp hearts have a higher score than lentils,pinto beans and rolled oats.