By Federico Bernuzzi

The DIME study? What is it?

When we think of food, we tend to think of how many carbs, fats, and, perhaps even, protein a portion of food or meal contains. Rarely, do we consider the micronutrients, such as vitamins and minerals. But, what about phytochemicals? Phytochemicals, also referred to as plant bioactives, are entirely ignored, yet they can play a fundamental role in keeping us healthy.

Our gut is home to a community of complex microorganisms (mainly bacteria, along with viruses, archaea and fungi) collectively referred to as the gut microbiome. The gut microbiome can break down phytochemicals found in fruits and vegetables into compounds that have been shown to promote our health.

Hopefully, this has got you thinking, or wondering, which foods contain these phytochemicals. The answer can be complex, as different fruit and vegetables have various food bioactives. Polyphenols, sulphur metabolites, and carotenoids are the most characterised.

Polyphenols have long been recognised to have antioxidant properties, mopping up free radicals in the body (Pandey and Rizvi, 2009). They are found in a wide range of fruit and vegetables, such as apples, pears, berries, kale, onions, broccoli and chocolate. A group of polyphenols, known as anthocyanins, is what gives berries, grapes and strawberries their purple/blue colour.

Dietary intake of polyphenols has been shown to:

• Maintain cardiovascular health by blocking oxidation of LDL cholesterol (which can build in the arteries resulting in atherosclerotic plaques), supporting ventricular health, and reducing harmful platelet activity (Guo et al., 2014, Zhang et al., 2016, Guo et al., 2013).
• Several polyphenolic compounds, most notably anthocyanins,have been shown to protect beta cells of the pancreas from excess glucose and slow digestion of starch, leading to better glycaemic control (Xiao and Högger, 2015, Jakobek, 2015, Barrett et al., 2018).
• Have anti-obesogenic effects. Polyphenols, such as catechins (found in tea), resveratrol (present in red wine) and curcumin (what gives turmeric its bright orange colour), affect fat metabolism through both inhibition of genes involved in lipid biosynthesis and oxidation of lipids present in fat cells, and reducing inflammation (Wang et al., 2014, Zhang et al., 2015).

Dietary sulphur-metabolites, including sulfoxides and glucosinolates, are mainly present in allium (garlic and onions) and cruciferous vegetables (broccoli, cauliflower, cabbage, Brussel sprouts, kale and so on). Substantial epidemiological evidence from prospective cohort studies and retrospective case-control studies have shown that consumption of cruciferous vegetables is associated with a reduced risk of developing the following cancers:

• lung (London et al., 2000, Spitz et al., 2000, Zhao et al., 2001, Wang et al., 2004),
• stomach (Hansson et al., 1993, Wu et al., 2013),
• colorectal (Lin et al., 1998, Kim et al., 2014),
• breast (Nechuta et al., 2013, Lin et al., 2017),
• bladder (Abbaoui et al., 2018, Tang et al., 2010, Veeranki et al., 2015),
• prostate (Traka et al., 2014, Livingstone et al., 2019).

In addition, cruciferous-rich diets have been associated with reduced risk of cardiovascular and metabolic diseases such as type 2 diabetes (Aiso et al., 2014, Bahadoran et al., 2013, Kim et al., 2008, Murashima et al., 2004, Suido et al., 2002, Kurotani et al., 2013, Armah et al., 2015, Axelsson et al., 2017).

Glucosinolates are not bioactive, but rely on fermentation in the colon by gut microbiota to produce active compounds (e.g., isothiocyanates), and have been shown to modulate gut microbiota (Traka and Mithen, 2008, Kaczmarek et al., 2019, Kellingray et al., 2017).

Finally, carotenoids are pigments produced by photosynthetic organisms (these include, plants, algae and some bacteria) and are responsible for the red, yellow and orange hues of many fruits and vegetables. Some carotenoids are vitamin A precursors; others are required for good visual health, and most act as antioxidants in lipid-rich environments. Foods rich in carotenoids include apricots, melons, carrots, mangoes, and sweet potatoes. Carotenoid intake has been shown to alter gut microbiome composition and regulate gut immune function (Lyu et al., 2018, Schmidt et al., 2021).

How is the research at the Quadram helping us understand how phytochemicals affect the gut microbiome?

Research from the Food Databanks led by Dr Maria Traka is looking into the effects of phytochemicals in a pilot human study referred to as Dietary BIoactives and Microbiome DivErsity (DIME). This study, funded by the European Commission, is part of an EU-funded project called Food Nutrition Security (FNS) Cloud. FNS-Cloud, which aims to develop an infrastructure and services to make food, nutrition and security data more accessible for the community and user communities more confident in their exploitation of these resources.

DIME is a randomised 2×2 crossover study, where 20 healthy adults (18-65 years old) are placed on a high- or low-bioactive diet for two weeks, followed by a 4-week washout before ending the study in the opposite arm. The primary objectives of the study are to:

• Assess the effect of a high- vs low-bioactive diets on the gut microbiome’s composition and diversity and its impact on metabolic regulation.
• Test the feasibility of altering intakes of bioactives in participants’ diets in a research setting, while keeping other nutrient groups constant

The secondary objectives of the study are to assess the effects of high- versus low-bioactive diets on:

• Host metabolism, by looking at metabolites present in the urine (urine metabolomics). Metabolites which are substances made in the body from the break down food,drugs or chemicals, can be used as proxy to asses metabolic reactions present in human tissues
• Vascular health (measured through lipid profiles and markers of inflammation C-reactive protein)
• Sleep, and estimating post-prandial glycemic metabolism (through continuous glucose monitoring and blood sugar response to standardised meals at the end of each intervention period).

What are the types of data generated by DIME?

The answer is quite a few, as it is a complex study. Starting with the microbiome, participants in the study donate stool samples at five time points;

• Study visit one, baseline, which is the day before the start of the study,
• Study visit 2, end of the first arm,
• A mid-point study visit sample, which is some point during the 4-week washout,
• Study visit three, day before the start of the second arm
• Study visit 4, end of the second arm.

To understand the impact of a high- vs low-bioactive diets on gut microbiota composition and microbial diversity, metagenomics analysis will be carried out, which involves sequencing all bacteria DNA present in the stool samples. These are bioinformatically stitched together to gives us information about not only who they are but what they can do. By combining this information with metabolomics analysis in the same stool samples, we will also capture information about metabolic activities of the various bacteria.

Urine metabolomics will determine how metabolites produced by the gut microbiome affect the human host.

Data on anthropometric measurements, such as weight and BMI, blood pressure and blood lipid as well as inflammatory profiles, will help determine whether the diets impact blood markers of health. In addition, the study will also assess how a high- vs low-bioactive diets affect postprandial glucose concentrations, which are also an indication of metabolic health.

Blood glucose measurements will be captured during the study using continuous glucose monitors (CGM) while participants are eating meals, and when we give them a bolus of glucose (oral glucose tolerance test).

In this study, we are using a smartphone app, Libro (Nutritics Ltd) which makes it easier for participants to record their meals, by typing, barcode scanning, or voice recognition. This will allow us to measure how well the participants adhered to the dietary interventions and link their meals to post-prandial glucose concentrations.

Finally, the participants were asked to record their bowel habits in a daily stool questionnaire, assessing stool frequency and state, and gut transit time by consuming a meal of sweetcorn and noting its first appearance in their stools. Data on stress levels, anxiety, and mood have been recorded through validated questionnaires. Information about activity levels and sleeping patterns have also been collected through a Fitbit wearable device.

Data generated by this study will be used as a test case for developing a technical cloud-based solution for analysis and information exchange for future diet and microbiome studies, and contribute to better understanding the effects of complex diets on regulating gut microbiota and metabolism and their impact on health.

Written by Federico Bernuzzi, PhD. Research Scientist, Food Databanks National Capability (FDNC)

Acknowlgements

The Food Nutrition Security Cloud (FNS-Cloud) has received funding from the European Union’s Horizon 2020 Research and Innovation programme (H2020-EU.3.2.2.3. – A sustainable and competitive agri-food industry) under Grant Agreement No. 863059 – www.fns-cloud.eu

References:

ABBAOUI, B., LUCAS, C. R., RIEDL, K. M., CLINTON, S. K. & MORTAZAVI, A. 2018. Cruciferous Vegetables, Isothiocyanates, and Bladder Cancer Prevention. Mol Nutr Food Res, 62, e1800079.

AISO, I., INOUE, H., SEIYAMA, Y. & KUWANO, T. 2014. Compared with the intake of commercial vegetable juice, the intake of fresh fruit and komatsuna (Brassica rapa L. var. perviridis) juice mixture reduces serum cholesterol in middle-aged men: a randomized controlled pilot study. Lipids Health Dis, 13, 102.

ARMAH, C. N., DERDEMEZIS, C., TRAKA, M. H., DAINTY, J. R., DOLEMAN, J. F., SAHA, S., LEUNG, W., POTTER, J. F., LOVEGROVE, J. A. & MITHEN, R. F. 2015. Diet rich in high glucoraphanin broccoli reduces plasma LDL cholesterol: Evidence from randomised controlled trials. Molecular nutrition & food research, 59, 918-926.

AXELSSON, A. S., TUBBS, E., MECHAM, B., CHACKO, S., NENONEN, H. A., TANG, Y., FAHEY, J. W., DERRY, J. M. J., WOLLHEIM, C. B., WIERUP, N., HAYMOND, M. W., FRIEND, S. H., MULDER, H. & ROSENGREN, A. H. 2017. Sulforaphane reduces hepatic glucose production and improves glucose control in patients with type 2 diabetes. Sci Transl Med, 9.

BAHADORAN, Z., MIRMIRAN, P. & AZIZI, F. 2013. Potential efficacy of broccoli sprouts as a unique supplement for management of type 2 diabetes and its complications. J Med Food, 16, 375-82.

BARRETT, A. H., FARHADI, N. F. & SMITH, T. J. 2018. Slowing starch digestion and inhibiting digestive enzyme activity using plant flavanols/tannins— A review of efficacy and mechanisms. LWT, 87, 394-399.

GUO, H., CHEN, Y., LIAO, L. & WU, W. 2013. Resveratrol protects HUVECs from oxidized-LDL induced oxidative damage by autophagy upregulation via the AMPK/SIRT1 pathway. Cardiovasc Drugs Ther, 27, 189-98.

GUO, R., LI, W., LIU, B., LI, S., ZHANG, B. & XU, Y. 2014. Resveratrol protects vascular smooth muscle cells against high glucose-induced oxidative stress and cell proliferation in vitro. Med Sci Monit Basic Res, 20, 82-92.

HANSSON, L. E., NYREN, O., BERGSTROM, R., WOLK, A., LINDGREN, A., BARON, J. & ADAMI, H. O. 1993. Diet and risk of gastric cancer. A population-based case-control study in Sweden. Int J Cancer, 55, 181-9.

JAKOBEK, L. 2015. Interactions of polyphenols with carbohydrates, lipids and proteins. Food Chem, 175, 556-67.

KACZMAREK, J. L., LIU, X., CHARRON, C. S., NOVOTNY, J. A., JEFFERY, E. H., SEIFRIED, H. E., ROSS, S. A., MILLER, M. J., SWANSON, K. S. & HOLSCHER, H. D. 2019. Broccoli consumption affects the human gastrointestinal microbiota. J Nutr Biochem, 63, 27-34.

KELLINGRAY, L., TAPP, H. S., SAHA, S., DOLEMAN, J. F., NARBAD, A. & MITHEN, R. F. 2017. Consumption of a diet rich in Brassica vegetables is associated with a reduced abundance of sulphate-reducing bacteria: A randomised crossover study. Mol Nutr Food Res, 61.

KIM, J. K., SHIN, D.-H., PARK, H. G. & SHIN, E.-C. 2014. Cruciferous vegetables, glutathione S-transferases, and implications of their interaction to colorectal cancer risk: A review. Journal of the Korean Society for Applied Biological Chemistry, 57, 511-517.

KIM, S. Y., YOON, S., KWON, S. M., PARK, K. S. & LEE-KIM, Y. C. 2008. Kale juice improves coronary artery disease risk factors in hypercholesterolemic men. Biomed Environ Sci, 21, 91-7.

KUROTANI, K., NANRI, A., GOTO, A., MIZOUE, T., NODA, M., KATO, M., INOUE, M. & TSUGANE, S. 2013. Vegetable and fruit intake and risk of type 2 diabetes: Japan Public Health Center-based Prospective Study. Br J Nutr, 109, 709-17.

LIN, H. J., PROBST-HENSCH, N. M., LOUIE, A. D., KAU, I. H., WITTE, J. S., INGLES, S. A., FRANKL, H. D., LEE, E. R. & HAILE, R. W. 1998. Glutathione transferase null genotype, broccoli, and lower prevalence of colorectal adenomas. Cancer Epidemiol Biomarkers Prev, 7, 647-52.

LIN, T., ZIRPOLI, G. R., MCCANN, S. E., MOYSICH, K. B., AMBROSONE, C. B. & TANG, L. 2017. Trends in Cruciferous Vegetable Consumption and Associations with Breast Cancer Risk: A Case-Control Study. Current developments in nutrition, 1, e000448-e000448.

LIVINGSTONE, T. L., BEASY, G., MILLS, R. D., PLUMB, J., NEEDS, P. W., MITHEN, R. & TRAKA, M. H. 2019. Plant Bioactives and the Prevention of Prostate Cancer: Evidence from Human Studies. Nutrients, 11, 2245.

LONDON, S. J., YUAN, J. M., CHUNG, F. L., GAO, Y. T., COETZEE, G. A., ROSS, R. K. & YU, M. C. 2000. Isothiocyanates, glutathione S-transferase M1 and T1 polymorphisms, and lung-cancer risk: a prospective study of men in Shanghai, China. Lancet, 356, 724-9.

LYU, Y., WU, L., WANG, F., SHEN, X. & LIN, D. 2018. Carotenoid supplementation and retinoic acid in immunoglobulin A regulation of the gut microbiota dysbiosis. Experimental biology and medicine (Maywood, N.J.), 243, 613-620.

MURASHIMA, M., WATANABE, S., ZHUO, X. G., UEHARA, M. & KURASHIGE, A. 2004. Phase 1 study of multiple biomarkers for metabolism and oxidative stress after one-week intake of broccoli sprouts. Biofactors, 22, 271-5.

NECHUTA, S., CAAN, B. J., CHEN, W. Y., KWAN, M. L., LU, W., CAI, H., POOLE, E. M., FLATT, S. W., ZHENG, W., PIERCE, J. P. & SHU, X. O. 2013. Postdiagnosis cruciferous vegetable consumption and breast cancer outcomes: a report from the After Breast Cancer Pooling Project. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 22, 1451-1456.

PANDEY, K. B. & RIZVI, S. I. 2009. Plant polyphenols as dietary antioxidants in human health and disease. Oxid Med Cell Longev, 2, 270-8.

SCHMIDT, K. M., HADDAD, E. N., SUGINO, K. Y., VEVANG, K. R., PETERSON, L. A., KORATKAR, R., GROSS, M. D., KERVER, J. M. & COMSTOCK, S. S. 2021. Dietary and plasma carotenoids are positively associated with alpha diversity in the fecal microbiota of pregnant women. J Food Sci, 86, 602-613.

SPITZ, M. R., DUPHORNE, C. M., DETRY, M. A., PILLOW, P. C., AMOS, C. I., LEI, L., DE ANDRADE, M., GU, X., HONG, W. K. & WU, X. 2000. Dietary intake of isothiocyanates: evidence of a joint effect with glutathione S-transferase polymorphisms in lung cancer risk. Cancer Epidemiol Biomarkers Prev, 9, 1017-20.

SUIDO, H., TANAKA, T., TABEI, T., TAKEUCHI, A., OKITA, M., KISHIMOTO, T., KASAYAMA, S. & HIGASHINO, K. 2002. A mixed green vegetable and fruit beverage decreased the serum level of low-density lipoprotein cholesterol in hypercholesterolemic patients. J Agric Food Chem, 50, 3346-50.

TANG, L., ZIRPOLI, G. R., GURU, K., MOYSICH, K. B., ZHANG, Y., AMBROSONE, C. B. & MCCANN, S. E. 2010. Intake of cruciferous vegetables modifies bladder cancer survival. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 19, 1806-1811.

TRAKA, M. & MITHEN, R. 2008. Glucosinolates, isothiocyanates and human health. Phytochemistry Reviews, 8, 269-282.

TRAKA, M. H., MELCHINI, A. & MITHEN, R. F. 2014. Sulforaphane and prostate cancer interception. Drug Discov Today, 19, 1488-92.

VEERANKI, O. L., BHATTACHARYA, A., TANG, L., MARSHALL, J. R. & ZHANG, Y. 2015. Cruciferous vegetables, isothiocyanates, and prevention of bladder cancer. Current pharmacology reports, 1, 272-282.

WANG, L. I., GIOVANNUCCI, E. L., HUNTER, D., NEUBERG, D., SU, L. & CHRISTIANI, D. C. 2004. Dietary intake of Cruciferous vegetables, Glutathione S-transferase (GST) polymorphisms and lung cancer risk in a Caucasian population. Cancer Causes & Control, 15, 977-985.

WANG, S., SUN, Z., DONG, S., LIU, Y. & LIU, Y. 2014. Molecular interactions between (-)-epigallocatechin gallate analogs and pancreatic lipase. PLoS One, 9, e111143.

WU, Q. J., YANG, Y., WANG, J., HAN, L. H. & XIANG, Y. B. 2013. Cruciferous vegetable consumption and gastric cancer risk: a meta-analysis of epidemiological studies. Cancer Sci, 104, 1067-73.

XIAO, J. B. & HÖGGER, P. 2015. Dietary polyphenols and type 2 diabetes: current insights and future perspectives. Curr Med Chem, 22, 23-38.

ZHANG, B., DENG, Z., RAMDATH, D. D., TANG, Y., CHEN, P. X., LIU, R., LIU, Q. & TSAO, R. 2015. Phenolic profiles of 20 Canadian lentil cultivars and their contribution to antioxidant activity and inhibitory effects on α-glucosidase and pancreatic lipase. Food Chem, 172, 862-72.

ZHANG, H., LIU, Q., LIN, J., WANG, Y., CHEN, S. & HOU, J. 2016. GW27-e0657 Resveratrol Protects against oxidized LDL-induced foam cells formation and apoptosis through inhibition of ER stress and downregulation of CD36. Journal of the American College of Cardiology, 68, C26-C26.

ZHAO, B., SEOW, A., LEE, E. J., POH, W. T., TEH, M., ENG, P., WANG, Y. T., TAN, W. C., YU, M. C. & LEE, H. P. 2001. Dietary isothiocyanates, glutathione S-transferase -M1, -T1 polymorphisms and lung cancer risk among Chinese women in Singapore. Cancer Epidemiol Biomarkers Prev, 10, 1063-7.