The roles and potential of lentil prebiotic carbohydrates in human and plant health

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors, Plants, People, Planet © New Phytologist Trust 1Plant and Environmental Sciences, 270 Poole Agricultural Center, Clemson University, Clemson, SC, USA 2Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN, USA 3Biodiversity and Crop Improvement Program, International Centre for Agricultural Research in the Dry Areas (ICARDA), Rabat-Institute, Rabat, Morocco

Prebiotic carbohydrates are specific colonic nutrients that act as biosynthetic precursors for human microbiota activity, which in turn leads to possible health benefits related to combating type II diabetes and obesity. In addition to human health benefits, prebiotic carbohydrates also benefit plant health by increasing leaf raffinose family oligosaccharides (RFOs) to enhance drought (Bartels & Sunkar, 2005), chilling (Nishizawa, Yabuta, & Shigeoka, 2008), and freezing tolerance (Pennycooke, Jones, & Stushnoff, 2003). Sugar alcohols (SAs) also increase chilling (Chiang, Stushnoff, McSay, Jones, & Bohnert, 2005), drought (Pujni, Chaudhary, & Rajam, 2007), and salinity tolerance in a range of plants (Zhifang & Loescher, 2003). These RFOs and SAs generally act as signaling compounds for both biotic and abiotic stresses (Valluru & Van den Ende, 2011). With climate conditions changing globally, future lentil production might be limited due to increased incidence of drought and higher temperatures. The significance of prebiotic carbohydrates to human and plant health means the type and concentration thereof in lentil are essential traits for nutrigenomic breeding efforts. Nutritionally improved lentil cultivars could help to combat global health problems, while simultaneously enhancing resilience to the effects of climate change (Muehlbauer et al., 2006).

| PREB IOTIC C ARBOHYDR ATE S
The definition of a prebiotic has evolved since its coining in 1995.
Complementary to the probiotic concept, Gibson and Roberfroid (1995) originally defined a prebiotic as a "non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria already resident in the colon." This definition was revised in 2004 to three criteria that restricted prebiotic foods to ingredients that are (a) resistant to mammalian digestion; (b) fermented by intestinal microflora; and (c) selectively stimulate the growth and/or activity of intestinal bacteria associated with health and well-being (Gibson, Probert, Van, Rastall, & Roberfroid, 2004 (Pineiro et al., 2008). The definition was critiqued by Gibson et al. (2010) for this latter omission and also for not adequately excluding antibiotics. Reaffirming selective fermentation and establishing "a niche," Gibson et al. (2010) Roberfroid, 2007). These prebiotic carbohydrates are associated with many human health benefits, because they promote satiety, lower high cholesterol, and regulate postprandial blood glucose levels (Beserra et al., 2015). Most naturally occurring prebiotic carbohydrates are found in fresh vegetables, legumes, and fruits at concentrations ranging from trace amounts in wheat, to moderate levels in onion and green bananas, to relatively high concentrations (35.7-47.6 g/100 g) in chicory root (Van Loo et al., 1999).
As a staple part of many diets, legumes, such as lentil and chickpea, provide an excellent source of prebiotic carbohydrates (Table 2).
Legumes tend to have higher concentrations of SA, RFO, fiber, and RS than prebiotic-rich fruits and vegetables, which tend to be higher in simple sugars and fructooligosaccharides (Table 2). For example, lentil and chickpea contain mean sorbitol concentrations of 0.66 and 0.52 g/100 g, respectively, compared to not detected and 1.09 g/100 g in onion and nectarine, respectively. With the exception of 0.23 g/100 g of raffinose in onion, nectarine and onion are void of detectable concentrations of RFO. Lentil and chickpea, however, have total RFO concentrations of 4.14 and 1.09 g/100 g, respectively.
Although all legumes have merit as prebiotic-rich foods, our focus here is lentil, which is one of the most studied cool-season food legumes.

| LENTIL PREB I OTI C C ARBOHYDR ATE S
Lentil contains a range of prebiotic carbohydrates including average concentrations of 4,071 mg of RFOs, 1,423 mg of SAs, 62 mg of FOSs, and 7,500 mg of RS per 100 g (Johnson et al., 2013). A recent study reported the prebiotic carbohydrate profile after removing protein and fat from lentil seeds (  (Johnson, Thavarajah, Thavarajah, Fenlason, et al., 2015).
Concentration of prebiotic carbohydrate can also vary by location and genotype, or by method of food processing (Johnson, Thavarajah, Thavarajah, Payne, et al., 2015;. Lentils are often cooked, cooled, and reheated before consumption, hence these processes are important consid-

| LENTIL PREB I OTI C C ARBOHYDR ATE S AND G UT HE ALTH
The human gastrointestinal tract, with a surface area of over 300 m 2 , hosts more than 100 trillion microorganisms (Savage, 1977). These microbes, collectively termed "the microbiome", comprise 10 times more cells than human cells and over 100 times more genetic information than the human genome (Bäckhed, Ley, Sonnenburg, Peterson, & Gordon, 2005). The microbiome is a dynamic ecosystem, with a myriad of interactions between microbes and human tissues that change throughout the course of human growth and development.
Increasingly, the microbiome is recognized as an extra-human organ, capable of protecting the host from invading pathogens, stimulating the immune system, increasing the availability of nutrients, stimulating bowel motility, and improving lipid levels in the body (Holzapfel & Schillinger, 2002). However, gut microbiota are also involved with a host of disease processes, including obesity, diabetes, infections, inflammatory bowel disease, cancer, and many others (Lynch & Pedersen, 2016). Primary determinants of microbiota composition and function include age, environment, genetic factors, diet, health status, and medical interventions, such as the use of antimicrobial agents (Lozupone, Stombaugh, Gordon, Jansson, & Knight, 2012).
The concept of modulating the gut microbiome's composition and function through diet, primarily through prebiotics, has gained enormous attention (Bindels et al., 2015). Prebiotics are fermented by hindgut microflora into active metabolites-short-chain fatty acids,  Original data obtained from Johnson, Thavarajah, Thavarajah, Fenlason, et al. (2015). We are now discovering the importance of the microbiome in early childhood growth and development. Moderate acute malnutrition in Bangladeshi children has been related to premature microbiota composition (Subramanian et al., 2014). Supplementation with gut microbial flora from healthy children and with foods rich in several prebiotic ingredients alleviated acute malnutrition with an associated normalization of age-appropriate hindgut microflora (Gehrig et al., 2019). Moreover, an altered gut microbiome has also been implicated in autism spectrum disorder, although this interaction is not yet thoroughly understood (Li, Hu, Ou, & Xia, 2019). Prospective studies with prebiotics in autistic children, when combined with exclusion of a dietary component, have revealed modest improvements in behavioral symptoms; however, randomized controlled trials have not been able to demonstrate these effects (Ng et al., 2019). These discoveries highlight opportunities for further research toward how novel dietary approaches can improve early childhood growth and development. As lentils provide significant levels of prebiotic carbohydrate, we propose they are an ideal food source for increasing prebiotic carbohydrates in people's diets and for imparting the health benefits these may provide. Indeed, the results from a recent study in rats further support the notion that a lentil-rich diet may have significant health benefits because of the superior nutritional value of its prebiotic carbohydrates and the concomitant increase in the activity of hindgut bacteria (Siva, Johnson, et al., 2018).

TA B L E 3 Prebiotic carbohydrate concentrations vary by growing location
Specifically, rats fed on a lentil diet had a significantly lower mean body weight (443 ± 47 g/rat) than those fed on control (511 ± 51 g/ rat) or corn (502 ± 38 g/rat) diets; in addition, mean percent body fat and triglyceride concentration were lower and lean body mass was higher in rats fed on the lentil diet. Moreover, the fecal abundance of Actinobacteria and Bacteroidetes (beneficial bacteria) was significantly higher and the abundance of Firmicutes (pathogenic bacteria) was lower in rats fed the lentil diet versus the control diet.
When considering the impact of diet on the microbiome and chronic disease, we recommend a diet with satisfactory levels of prebiotics. Legumes, such as lentil, are a rich and affordable source of prebiotic carbohydrates with 100 g of lentil providing 12 g of prebiotic carbohydrates (Siva et al., 2019). This recommendation is especially applicable to countries where legumes are often neglected in people's diets. Creativity in processing methods and marketing approaches, such as the recent advance of plant-based burgers, could help to popularize lentil and other legumes in countries where they are not generally widely consumed.

| PREB IOTIC C ARBOHYDR ATE S AND PL ANT HE ALTH
As would be expected due to their high concentrations in lentil seed, aids of abiotic stress tolerance, namely temperature, drought, and salinity stress.
Raffinose family oligosaccharides and SAs are primary photosynthetic products and carbon transport molecules in many higher plants. Labeled 14 CO 2 studies have revealed that the primary soluble carbon products synthesized through photosynthesis in higher plants are sucrose (ubiquitous), RFOs, and SAs (Loescher & Everard, 2000). The orders of plants that utilize RFOs as a photosynthetic product and storage molecule include Lamiales, Cucurbitales, Cornales, and some Celastrales (Sengupta & Majumder, 2015). Ajuga reptans L. is the premier example of this type of plant, which uses stachyose as its primary carbon transport molecule. To store carbon, it synthesizes RFO of higher degrees of polymerization (DP), which become trapped for storage purposes (Bachman, Matile, & Keller, 1994). Lentil is not known to synthesize RFOs in leaves as a primary photosynthetic product, and, consequently, also does not transport carbon via RFOs (Obendorf & Gorecki, 2012). Instead, sucrose and SAs function as the transport molecules to the seed during seed filling. RFOs are formed in maturing lentil seeds at high concentrations (Obendorf & Gorecki, 2012). Likewise, for SAs, Grant and ap Rees (1981) showed that approximately 70% of fixed carbon in apple leaves was made into sucrose and sorbitol. Similarly, Loescher, Tyson, Everard, Redgwell, and Bieleski (1992) found that 80%-90% of the fixed carbon was transformed into mannitol and sucrose in celery. Similar patterns of SA accumulation have been shown in lilac and apricot (Loescher & Everard, 2000). Although sucrose is the primary photosynthetic product and carbon transport molecule in legumes, SAs may also function passively in this capacity, being found in both the leaf and seed (Amede, Schubert, & Stahr, 2011;Johnson et al., 2013).
Raffinose family oligosaccharides and SAs also serve as a carbon store. As noted, some plants (i.e., A. reptans) store RFOs in their leaves by increasing DP. RFOs are primarily known for their accumulation in seeds during late development (Sengupta & Majumder, 2015) and are especially prevalent in legumes (Obendorf & Gorecki, 2012). RFOs protect the embryo during desiccation. During germination, RFOs are rapidly hydrolyzed by α-galactosidases but do not appear to be necessary for germination (Peterbauer & Richter, 2001). The use of SAs as a carbon store is largely dependent on tissue type, developmental stage, and environment. For example, apple leaves contain 0.9% sorbitol (dry weight) in June but 4.8% in late July (Loescher & Everard, 2000). Physiologically mature lentil seeds contain significant concentrations of both sorbitol and mannitol (Johnson et al., 2013).
Lastly, RFOs and SAs aid plants experiencing abiotic stress.
During abiotic stress, several compounds accumulate, including RFOs and SAs. These compounds aid the plant in survival through these extreme conditions by balancing osmotic pressures and have, therefore, been called "osmoprotectants" (Bohnert & Jensen, 1996).
RFOs and SAs substitute for water as compatible solutes; they may provide a medium for enzyme function and protect enzymes from free radicals and consequent denaturing (Smirnoff & Cumbes, 1989).
Biochemical synthesis pathways have been elucidated for both RFOs and SAs and are detailed separately below (Figure 3).
Understanding these pathways will help to identify and exploit molecular and genetic markers that can be used in lentil breeding programs. RFOs represent a series of increasing DP formed through the addition of galactose monomers to sucrose via 1,6-α glycosidic linkage, building raffinose (DP3), stachyose (DP4), and verbascose (DP5). Higher DP (DP15 or greater) exist in some plants, such as lupin seeds (Kannan, Sharma, Gangola, Sari, & Chibbar, 2018), but are not detected in lentil. The primary RFO biosynthesis pathway uses galactinol as the galactosyl donor. Galactinol is formed via galactinol synthase from UDP-galactose and L-myo-inositol ( Figure 3).
Raffinose synthase binds the galactosyl from galactinol to a sucrose molecule to form raffinose. Stachyose synthase binds galactosyl to raffinose to form stachyose. In addition, verbascose synthase binds galactosyl to stachyose to form verbascose. RFO synthesis takes place primarily in the cytosol. A secondary RFO biosynthesis pathway exists in A. reptans (Bachmann et al., 1994). This pathway is independent of galactinol, using a galactosyltransferase enzyme to transfer a galactosyl unit from one RFO to another to create higher DP oligosaccharides (Sengupta & Majumder, 2015).
The most abundant and well-studied SAs in higher plants are sorbitol (glucitol) and mannitol. Both have reduced forms of hexose sugars (glucose and mannose) and share similar pathways (Figure 3).

| B REED ING APPROACHE S FOR LENTIL PREB IOTIC C ARBOHYDR ATE S
Due to lentil's excellent overall nutritional makeup, it has already been targeted for biofortification (Kumar, Sen, Kumar, Gupta, & Singh, 2016). However, efforts have primarily been directed toward combatting micronutrient deficiency or "hidden hunger" (Kumar et al., 2016).

| CON CLUS ION
Lentil is a rich source of prebiotic carbohydrates including SAs, RFOs, FOSs, and other polysaccharides such as cellulose, hemicellulose, and amylose. In addition to the human nutritional benefits, prebiotic carbohydrates have a significant influence on plant health, a feature that will significantly benefit the breeding of pulse crops for climate resilience. Consequently, lentil prebiotic carbohydrates are an important breeding target, requiring further characterization and evaluation of germplasm. Phenotyping diverse lentil mapping populations could identify future genetic markers associated with high levels of prebiotic carbohydrates and thus significantly accelerate nutritional breeding for different growing environments and consumer preference (Varshney et al., 2013). These genetic markers could then be used to screen locally grown varieties as well as to develop new cultivars with special consumer requirements; for example, breeder-friendly genetic markers can be used to develop new varieties with moderate RFOs and increased levels of FOSs and RS to reduce flatulence in populations sensitive to RFOs. Globally, the development and selection of lentil genotypes with enhanced levels of prebiotic carbohydrates could not only provide significant health benefits to society, but could also provide economic benefits through improved crop sustainability and production.