Identification of bromelain subfamily proteases encoded in the pineapple genome.

Papain (aka C1A) family proteases, including bromelain enzymes, are widespread across the plant kingdom and play critical regulatory functions in protein turnover during development. The proteolytic activity exhibited by papain family proteases has led to their increased usage for a wide range of cosmetic, therapeutic, and medicinal purposes. Bromelain enzymes, or bromelains in short, are members of the papain family that are specific to the bromeliad plant family. The only major commercial extraction source of bromelain is pineapple. The importance of C1A family and bromelain subfamily proteases in pineapple development and their increasing economic importance led several researchers to utilize available genomic resources to identify protease-encoding genes in the pineapple genome. To date, studies are lacking in screening bromelain genes for targeted use in applied science studies. In addition, the bromelain genes coding for the enzymes present in commercially available bromelain products have not been identified and their evolutionary origin has remained unclear. Here, using the newly developed MD2 v2 pineapple genome, we aimed to identify bromelain-encoding genes and elucidate their evolutionary origin. Orthologous and phylogenetic analyses of all papain-family proteases encoded in the pineapple genome revealed a single orthogroup (189) and phylogenetic clade (XIII) containing the bromelain subfamily. Duplication mode and synteny analyses provided insight into the origin and expansion of the bromelain subfamily in pineapple. Proteomic analysis identified four bromelain enzymes present in two commercially available bromelain products derived from pineapple stem, corresponding to products of four putative bromelain genes. Gene expression analysis using publicly available transcriptome data showed that 31 papain-family genes identified in this study were up-regulated in specific tissues, including stem, fruit, and floral tissues. Some of these genes had higher expression in earlier developmental stages of different tissues. Similar expression patterns were identified by RT-qPCR analysis with leaf, stem, and fruit. Our results provide a strong foundation for future applicable studies on bromelain, such as transgenic approaches to increase bromelain content in pineapple, development of bromelain-producing bioreactors, and studies that aim to determine the medicinal and/or therapeutic viability of individual bromelain enzymes.

Freeze-Dried Watermelon Supplementation Has Modest Effects on Bone and Lipid Parameters of Ovariectomized Mice.

This study investigated the effects of two doses of freeze-dried watermelon (WM) on bone and lipid parameters in ovariectomized (OVX) mice, a model of post-menopausal osteoporosis. Three-month-old C57BL/6 female mice (n=46) were sham-operated (SHAM) or OVX and randomly assigned to the control or WM diets for 12 weeks: SHAM-control, OVX-control, OVX+1%, or 10% (wt/wt) freeze-dried WM. All diets were isocaloric and isonitrogenous, and had the same calcium and phosphorus concentrations. Freeze-dried WM supplementation was not able to prevent the decrease in whole body, tibial, and lumbar bone mineral density due to estrogen deficiency. Micro-computed tomography analyses showed that WM was also not able to modulate changes in tibial trabecular and cortical bone microarchitecture due to ovariectomy. However, the lumbar trabecular micro-architecture analyses revealed that the WM-10% group had a similar connectivity density, trabecular number, trabecular separation, and structure model index as the SHAM group. Supplementation with 10% WM reduced plasma cholesterol and total liver lipids to the level of the SHAM group but was still similar to that of the OVX-control group. Supplementation with 10% WM increased liver catalase (CAT) mRNA levels but had no effects on mRNA levels of glutathione peroxidase (GPX) and the pro-inflammatory cytokine interleukin-6. There were no differences in plasma activity of the antioxidant enzymes GPX and CAT between all treatment groups. Our findings demonstrate some positive effects of watermelon for modulating lipids and attenuating lumbar vertebral bone loss arising from ovarian hormone deficiency.

Daily watermelon consumption decreases plasma sVCAM-1 levels in overweight and obese postmenopausal women.

Postmenopausal status is associated with an increase in total and abdominal body fat as well as increased incidence of insulin resistance and cardiovascular disease. The purpose of this study was to determine if watermelon supplementation affects select systemic markers of atherosclerosis and measures of insulin resistance in overweight and obese postmenopausal women. We hypothesized that overweight and obese postmenopausal women consuming 100% watermelon puree daily for 6 weeks would have improved levels of select systemic markers connected with cardiovascular disease without changing markers of insulin resistance. To test this hypothesis, overweight and obese postmenopausal women were recruited to participate in this study. Participants were randomly assigned to either the control group (no intervention) or the watermelon puree group (WM) for 6 weeks. Plasma concentration of markers connected with atherosclerosis and glycemic control were measured pre- and poststudy. A significant 6% decrease in soluble vascular cell adhesion molecule-1 occurred pre- to poststudy in WM, P = .003. The pattern of change in fasting blood glucose (P = .633), insulin (P = .158), and homeostatic model assessment-estimated insulin resistance (P = .174) did not differ between groups. Pre- to poststudy increases were measured in the fasting plasma concentration of l-arginine (8%, P = .005), cis-lycopene (32%, P = .003), and trans-lycopene (42%, P = .003) in WM. We conclude that 6 weeks of watermelon supplementation improved soluble vascular cell adhesion molecule-1 levels, a marker connected to atherogenesis, independent of changes in body composition or glycemic control.

Mango Supplementation Modulates Gut Microbial Dysbiosis and Short-Chain Fatty Acid Production Independent of Body Weight Reduction in C57BL/6 Mice Fed a High-Fat Diet.

BACKGROUND: High-fat (HF) diet-induced obesity is associated with changes in the gut microbiota. Fiber and other bioactive compounds in plant-based foods are suggested to prevent gut dysbiosis brought on by HF feeding. Mango is high in fiber and has been reported to have anti-obesogenic, hypoglycemic, and immunomodulatory properties. OBJECTIVES: We investigated the effects of freeze-dried mango pulp combined with an HF diet on the cecal microbial population and its relation to body composition, lipids, glucose parameters, short-chain fatty acid (SCFA) production, and gut inflammatory markers in a mouse model of diet-induced obesity. METHODS: Six-wk-old male C57BL/6 mice were randomly assigned to 1 of 4 dietary treatment groups: control (AIN-93M, 10% fat kcal), HF (60% fat kcal), and HF + 1% or 10% mango (HF+1%M or HF+10%M, wt:wt) for 12 wk. The cecal microbial population was assessed by use of 16S rDNA sequencing. Body composition, plasma glucose and lipids, cecal and fecal SCFAs, and mRNA abundance of inflammatory markers in the ileum and colonic lamina propria were assessed. RESULTS: Compared with the control group, HF feeding significantly reduced (P < 0.05) 1 operational taxonomic unit (OTU) of the genus Bifidobacteria (64-fold) and 5 OTUs of the genus Akkermansia (≥16-fold). This reduction was prevented in the HF+10%M group, members of which had 10% higher final body weight compared with the HF group (P = 0.01) and similar fasting blood glucose concentrations (P = 0.24). The HF+10%M group had 135% (P = 0.004) and 133% (P < 0.0001) greater fecal acetic and n-butyric acids concentrations than the HF group, suggesting greater microbial fermentation. Furthermore, a 59% greater colonic interleukin 10 (Il10) gene expression was observed in the HF+10%M group than in the HF group (P = 0.048), indicating modulation of gut inflammation. The HF+1%M group generally did not differ from the HF group. CONCLUSIONS: The addition of mango to an HF diet modulated the gut microbiota and production of SCFAs in C57BL/6 mice; these changes may improve gut tolerance to the insult of an HF diet.

Effects of watermelon supplementation on aortic blood pressure and wave reflection in individuals with prehypertension: a pilot study.

BACKGROUND: Oral L-citrulline is efficiently converted to L-arginine, the precursor for endothelial nitric oxide (NO) synthesis. Oral L-arginine supplementation reduces brachial blood pressure (BP). We evaluated the effects of watermelon supplementation on aortic BP and arterial function in individuals with prehypertension. METHODS: Heart rate (HR), brachial systolic BP (bSBP), brachial pulse pressure (bPP), aortic SBP (aSBP), aortic PP (aPP), augmentation index (AIx), AIx adjusted for HR of 75 beats/min (AIx@75), amplitude of the first (P1) and second (P2) systolic peaks, reflection time (Tr), and carotid-femoral pulse wave velocity (PWV) were evaluated in the supine position in nine subjects (four men/five women, age 54 ± 3 years) with prehypertension (134/77 ± 5/3 mm Hg). Subjects were randomly assigned to 6 weeks of watermelon supplementation (L-citrulline/L arginine, 2.7 g/1.3 g/day) or placebo followed by a 4-week washout period and then crossover. RESULTS: There was a significant treatment effect (change in the value of watermelon minus placebo from baseline to 6 weeks) on bPP (-8 ± 3 mm Hg, P < 0.05), aSBP (-7 ± 2 mm Hg, P < 0.05), aPP (-6 ± 2 mm Hg, P < 0.01), AIx (-6 ± 3%, P < 0.05), AIx@75 (-4 ± 2%, P < 0.05), and P2 (-2 ± 1 mm Hg, P < 0.05). There was no significant treatment effect (P > 0.05) on bSBP, brachial diastolic BP (DBP), aortic DBP, Tr, P1, HR, and carotid-femoral PWV. CONCLUSIONS: This pilot study shows that watermelon supplementation improves aortic hemodynamics through a decrease in the amplitude of the reflected wave in individuals with prehypertension.

Dietary supplementation with watermelon pomace juice enhances arginine availability and ameliorates the metabolic syndrome in Zucker diabetic fatty rats.

Watermelon is rich in L-citrulline, an effective precursor of L-arginine. This study was conducted to determine whether dietary supplementation with watermelon pomace juice could ameliorate the metabolic syndrome in the Zucker diabetic fatty (ZDF) rat, an animal model of noninsulin-dependent diabetes mellitus. Nine-week-old ZDF rats were assigned randomly to receive drinking water containing 0% (control) or 0.2% L-arginine (as 0.24% L-arginine-HCl), 63% watermelon pomace juice, 0.01% lycopene, or 0.05% citrus pectin (n = 6 per treatment). At the end of the 4-wk supplementation period, blood samples, aortic rings, and hearts were obtained for biochemical and physiological analyses. Feed or energy intakes did not differ among the 5 groups of rats. However, dietary supplementation with watermelon pomace juice or L-arginine increased serum concentrations of arginine; reduced fat accretion; lowered serum concentrations of glucose, free fatty acids, homocysteine, and dimethylarginines; enhanced GTP cyclohydrolase-I activity and tetrahydrobiopterin concentrations in the heart; and improved acetylcholine-induced vascular relaxation. Compared with the control, dietary supplementation with lycopene or citrus pectin did not affect any measured parameter. These results provide the first evidence to our knowledge for a beneficial effect of watermelon pomace juice as a functional food for increasing arginine availability, reducing serum concentrations of cardiovascular risk factors, improving glycemic control, and ameliorating vascular dysfunction in obese animals with type-II diabetes.