The recommendation of 400–600 mg/d for flavan-3-ols to improve cardiometabolic health is based on beneficial effects observed across a range of disease biomarkers and endpoints. This recommendation is higher than the recent health claim of 200 mg/d for cocoa-flavanols by EFSA . The main reason for this discrepancy is that the EFSA health claim is only based on vasodilation as an endpoint and no other cardiometabolic disease markers. Regarding upper intake limits for flavan-3-ols, risk assessments of green tea catechins by EFSA concluded that no adverse effects are expected for intakes <800 mg/d . It must be acknowledged that challenges were encountered in establishing this guideline, such as limitations from lack of homogeneity in protocols. For example, studies included in the Raman et al. systematic review/meta-analysis reported large discrepancies in quality as well as lack of consensus in population description, duration of supplementation, form of bioactive/food/extract, and statistical methods . Implementing methodological consensus in executing and describing randomized clinical trials would allow for more rigorous assessment of study findings for comparison and pooling of data. Other limitations include the following: inclusion of more men than women in randomized clinical trials, different biomarkers used to assess prevention and development of cardiometabolic disease, and heterogeneity in dosages examined along with metabolism and assessment of circulating concentrations, which was not routinely evaluated. Additionally, it should be noted that cohort studies often relied on self-reported dietary intake, often at one time point, to assess benefit, which could contribute to information bias compromising internal validity; furthermore, package of blueberries the estimates of flavan-3-ol exposure were calculated from different food composition databases, which could preclude precise comparability.

Although FFQ data can clearly differentiate between extremes of intake, this assessment method does not account for the extensive inter individual metabolism that these compounds undergo after ingestion, which could impact effectiveness. As such, future studies should integrate biomarker, genetic, and dietary assessment methods to assess the effect of flavan-3-ols and their metabolites on cardiometabolic health. Considering a lack of homogeneity among studies, several research considerations would improve the generalizability of results from randomized clinical trials. For example, dose-dependent trials are warranted to assess minimal and maximal dose effects along with identifying potential negative effects from higher doses. Additional repository databases should be developed not only to report studies, but also to archive raw data and results to allow future ancillary analyses. This would allow for comparison and merging of results, thus increasing the total sample size, thereby increasing statistical power. Further, standardization in biomarkers of intake and exposure to flavan-3-ols is warranted. For example, γ -valerolactones, a flavan-3-ol metabolite formed by the colonic microbiome, can be used as markers of chronic flavan-3-ol intake . Future research should also include more diverse populations to assess inter individual variability for optimizing dietary recommendations and food product development, especially for specific population subgroups. Further, although this guideline was developed from research on the general adult population, additional research evaluating flavan-3-ol intake earlier in the lifespan is warranted because dietary habits adopted earlier in life can contribute to the magnitude of effect of flavan-3-ols on cardiometabolic health. In conclusion, when quality evidence is available to make an evidence-based intake guideline, such a recommendation can inform multiple stakeholders including clinicians, policymakers, public health entities, and consumers.

Evidence gaps identified in the review process can inform scientists, thereby guiding future randomized clinical trials. In summary, upon review of data from human studies reporting effects of foods rich in flavon-3-ols, the Expert Panel found moderate evidence supporting cardiometabolic protection resulting from flavan-3-ol intake in the range of 400–600 mg/d. It should be noted that the beneficial effects were observed across a range of disease biomarkers and endpoints; furthermore, this is a food-based guideline and not a recommendation for flavan-3-ol supplements.Horticulture likely originated 20,000years ago. There are over 100 species of horticultural crops, consisting of diverse fruits, vegetables, and tubers, many of which are of high economic value with enormous production volume worldwide. The amounts of fruits, vegetables, and tubers produced in 2018 were 868, 1089, and 832 million tons respectively , and the increased demand from a growing, and affluent global population, is predicted to drive further expansion of horticultural output . Horticultural crops not only provide basic calories , but also, are among the most crucial sources of fiber, organic acids, micro- and macro minerals, vitamins, and antioxidants in human diets. Healthy attributes, and a wide range of tastes, textures, and flavors make horticultural crops attractive. Starch is critical to human society given its versatile uses. Starch is the dominant energy source in the human diet, providing over 50% of our daily caloric needs. In the food industry, starch is widely used as a thickener, stabilizer, lipid replacer, defoaming agent, gelling agent, emulsifier, and dietary fiber, and in the pharmaceutical industry, starch is used as an excipient for drug delivery. In addition to these diverse uses, starch is an excellent renewable material for making ethanol biofuels and degradable ‘bioplastic’ products. Starch is almost ubiquitous in higher plants, including horticultural crops, in ways that may or may not be noticed.

For instance, potato, sweet potato, yam, and cassava are starchy, but spinach, lettuce, and ripe tomatoes, berries, and citrus are not, yet starch is likely to be important to the growth, development and fitness of all of these crops, as they are in better studied models. The widely accepted view is that starch accumulates either in a transitory state, or for long-term storage starch. Transitory starch follows a diurnal pattern: it is synthesized and accumulated directly from the products of photosynthesis in the leaf and in the stem during the daytime, and is then degraded into sugars as an energy source for the following night. In comparison, storage starch is defined as that located in perennating organs such as seeds, grain, embryos and tubers, where it provides sustenance for the next generation during germination and sprouting in sexual and asexual propagated crops, respectively. A third class of starch: ‘transitory-storage starch’ has been proposed. It describes starch that is accumulated and degraded during development in the storage organ. Transitory-storage starch is a feature of many species including horticultural crops of economic value such as tomato, banana, kiwi, strawberry, nectarine, and apple fruit. Starch accumulates as semi-crystalline, water insoluble granules that vary in diameter from 1 to 100μm depending on species. Starch is organized into two glucan polymers: amylose and amylopectin. Amylose and amylopectin consist primarily of linear chains of glucoses joined by α-1,4-glycosidic bonds. In amylopectin, the α-1,4-glucan chains are branched more frequently through α-1,6-glycosidic bonds, compared to amylose. The branching of the amylopectin chains is such that chains of different lengths are produced: short, medium and long chains, and the frequency with which each fraction occurs influences starch functionality. Side chains of amylopectin form clusters around branching points, and two adjacent chains make up a double helix. These physical features of amylopectin polymers leads to a semi-crystalline granule; amylose with a randomly coiled conformation, fills the matrices within the granule. Amylopectin and amylose account for around 25 and 75% of the starch in major heterotrophic storage organs, respectively , while the starch in leaf tissues is approximately 5 – 10% amylose. The core starch biosynthetic enzymes include ADP-glucose pyrophosphorylases , starch synthases , starch branching enzymes , and de-branching enzymes , of which there are many isoforms. In brief, AGPases initiate the first step of starch biosynthesis by catalyzing the formation of ADP-glucose. SSs elongate the glucan chains in amylose and amylopectin; SBEs branch the glucan chains, while the DBEs shorten and modify the starch chains which enable a higher-order semicrystalline structure to form. SBEs, the focus of this review, hydrolyze α-1,4-linked glucan chains, square plant pots and attach the newly-created ‘free’ chain to another glucan chain within the starch granule, via an α-1,6-linkage. Through this action, SBEs largely determine the proportion of the relatively unbranched amylose to the highly-branched amylopectin. Two major classes of SBEs are biofunctionally known: SBE1 and SBE2 , and they vary in terms of their substrate selectivity , whereas the function of SBE3 awaits verification across a broader set of species. SBE1 preferentially branches ‘amylose-like’ long glucan chains as the substrate, while SBE2 prefers a more branched substrate. The action of both forms further increases the number of branch points in starch polymers.

SBEs are the key players in the regulation of the amylose-to-amylopectin proportion in plants. However, their functions in many harvested horticultural crops have been under-investigated, although evidence points to the importance of starch in determining the postharvest quality of these crops. We aimed to develop a better understanding of the role of SBEs in fruits, tubers, and leafy greens in physiological processes by exploring SBE sequence relationships, expression, and starch phenotypes in diverse crops.SBEs have three classes of isozymes including two functional SBE classes and one putative class 3 SBE . SBE1 isoforms appeared earlier than SBE2 and SBE3 in the viridiplantae, but plant SBE1 and SBE2 are more homologous to each other, than to SBE3. SBEs have been identified and relatively well-characterized in cereal crops, tubers, and Arabidopsis thaliana over the last two decades,but, as mentioned, little attention has been paid to the diverse group of species that are classified as horticultural crops. Within each class of SBE, the cereals grouped together, while most non-cereals formed another cluster . This pattern is due to the divergence of monocots from dicots around 200 million years ago . In contrast to the presence of ‘a’ and ‘b’ sub-isoforms of SBE2 in cereal crops, horticultural plant species generally have one SBE2 isoform. It was also observed that not all species have a known or predicted class 3 isoform. The SBE sequences contained within diverse organs, i.e., fruits, tubers, roots, and leafy vegetables , clustered together based on their respective plant families. The class 1 SBE is absent in Arabidopsis thaliana, and so it was not surprising that this SBE class is not present in the Brassicaceae. However, the class 1 SBE is also absent in apple , and European olive , but these species all have two class 2 SBE isoforms . In addition, banana contains at least four types of SBE2, and transcripts corresponding to these SBE2s have been identified, indicating that they are expressed.Starch Branching Enzymes belong to the α-amylase family of enzymes, specifically the glycoside hydrolase family 13 superfamily, with multiple isoforms encoded by different genes . The overall structure of the SBE polypeptide is highly conserved: all SBEs possess a central α-amylase catalytic domain , and an NH2- terminus, and a carboxyl- terminus. The SBE NH2-terminus contains two conserved domains: a chloroplast transit peptide for plastid-targeting, and a CBM48 domain for binding to starch. The C-terminus contains the residues that determine substrate preference and catalytic activity. The central region of the enzyme contains the “A” catalytic domain, that is made up of 8–barrels. Notably, the class 3 SBE may not directly participate in starch biosynthesis in Arabidopsis, but it has a demonstrated function in mediating cesium toxicity of photosynthesis. However, the role of SBE3 is unlikely to be conserved. In potato, StSBE3 has a unique coiled-coil motif which is absent in the AtSBE3 polypeptide . Notably, the CBM48 domain is also deficient in AtSBE3 . It is possible that the StSBE3 may interact and complex with other starch biosynthetic enzymes through its coiled-coil domain, in a similar way to the SS4-PTST2 interaction in Arabidopsis, the GBSS-PTST1 interaction in rice or the SBEcontaining protein complexes in cereal endosperm, rendering an assistant function in starch biosynthesis. This species-specific mode of action of SBE3 may reveal a novel function of SBEs generally. Indeed, although all SBEs are predicted to form complexes with starch phosphorylases , the starch synthases and isoamylase , interactions with other proteins show differences depending on the species and SBE isoform.Four conserved regions critical for catalysis, named Regions 1-4 , are found within the catalytic A-domain . Regions 1-3 are directly involved in catalysis, while Region 4 is involved in direct substrate binding. SBE1 and 2 have largely invariant residues, but the residues in the SBE3 isoform of many species have substitutions at these sites. Post-transcriptional phosphorylation of the SBE-protein complexes formed with other starch biosynthetic enzymes has been found in cereal crops and in cassava, while experimental evidence of this regulation in the majority of horticultural crops is absent. SBE1 and SBE3 have fewer possible phosphorylation amino acid sites than SBE2 . Overall, the distinctive domain features of the SBE3 predicted protein, and the implifications for functionality may complicate current views of SBE function, but these features may also provide an opportunity to deepen our mechanistic understanding of starch biosynthesis and regulation.