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.