WO2021048283A1

WO2021048283A1 - Method for promoting weight loss by administering a diet rich in arabinoxylan oligosaccharides

Info

Publication number: WO2021048283A1
Application number: PCT/EP2020/075337
Authority: WO
Inventors: Arne Astrup; Mads Fiil HJORTH; Lars Christensen
Original assignee: University Of Copenhagen
Priority date: 2019-09-10
Filing date: 2020-09-10
Publication date: 2021-03-18

Abstract

The present invention relates to a method for promoting weight loss in an individual by matching the gut microbiota in said individual with a specific diet. More specifically, the present invention relates to a method for promoting weight loss in an individual having a high P/B ratio (a high relative abundance of Prevotella spp./Bacteroides spp.) in its microbiota by providing a diet rich in arabinoxylan oligosaccharides (AXOS) to said individual. The disclosure further relates to a method for promoting weight maintenance and/or reducing appetite in said individual.

Description

Method for promoting weight loss by administering a diet rich in arabinoxylan oligosaccharides

Technical field

The present invention relates to methods for promoting weight loss in an individual by matching characteristics of the gut microbiota in said individual with a specific diet. More specifically, the present invention relates to methods for promoting weight loss in an individual having a high P/B ratio (a high relative abundance of Prevotella spp ./Bacteroides spp.) in its microbiota by providing a diet rich in arabinoxylan oligosaccharides (AXOS) to said individual. The disclosure further relates to methods for promoting weight maintenance and/or reducing appetite in said individual.

Background

As the prevalence of obesity has reached epidemic proportions globally over the past few decades the search for causes and approaches to treat obesity continues. Multiple dietary approaches have been tested on weight loss and weight management; however, the efficacy of a specific diet over another has not been established. The limited evidence for the most effective diet has given rise to conclude that there is no “one diet fits all”. Thus, the conventional view that different people will respond similarly to a specific diet might be too simplistic and instead it is more likely that the success of a diet might be predicted based on specific individual characteristics.

Identification of biomarkers in regard to diet-induced weight loss are being investigated. Although, many biomarkers have failed to be a robust marker, fasting plasma glucose (FPG), fasting insulin (FI) and gut microbial enterotypes have been identified as promising biomarkers. The dominant bacterial phyla in the gastrointestinal tract (GIT) are Firmicutes and Bacteroidetes. Bacteroidetes represents around 20 different genera including Bacteroides and Prevotella as the most dominant. The Prevotella and Bacteroides enterotypes are characterized by different functionalities, where the Prevotella enterotype seems to effectively degrade plant fibers and is consistently associated with a diet rich in carbohydrates and dietary fiber. On the other hand, the Bacteroides enterotype may represent a more diverse set of species which have proteolytic and saccharolytic potential, and is associated with a “Western diet” low in fiber and high in fat. Advances in personalized nutrition have found that metabolic responses to specific foods are dependent on the gut microbiota composition. This diet-gut microbiota dependency may also play a substantial role within obesity management, suggesting that the key to effective weight loss requires a match between diet and gut microbiota.

Summary

The inventors of the present disclosure have surprisingly identified that by matching a high P/B ratio (a high relative abundance of Prevotella spp. /Bacteroides spp.) in the microbiota of an individual with a diet rich in arabinoxylan oligosaccharides (AXOS), a weight loss is observed.

Advantages of using AXOS as a diet supplement include a pleasant sweet taste and a high solubility allowing for easy administration by e.g. mixing into food or beverages. Thus customer compliance for using AXOS as diet supplement is high.

Thus, one aspect of the present disclosure is a method for promoting weight loss in an individual in need thereof having a high P/B ratio in its microbiota, the method comprising administering to said individual a diet comprising arabinoxylan oligosaccharides (AXOS).

In a second aspect, the present disclosure provides a method for promoting weight maintenance in an individual in need thereof having a high P/B ratio in its microbiota, the method comprising administering to said individual a diet comprising AXOS.

In a further aspect, the present disclosure provides a method for reducing appetite in an individual in need thereof having a high P/B ratio in its microbiota, the method comprising administering to said individual a diet comprising AXOS.

Description of Drawings

Figure 1.

An outline of the study. The outline includes the measurements applied during the study. Abbreviations: AXOS: arabinoxylan-oligosaccharides PUFA: polyunsaturated fatty acids, Wk: week.

Figure 2. Histogram showing the stratification of participants by low and high Prevotella-to- Bacteroides ratio (n = 29). The cutoff of -0.8 was used to stratify participants into either the high or low Prevotella/Bacteroides group and was based on the baseline log- transformed relative abundance of Prevotella-to-Bacteroides ratio. <-0.8 represents the low Prevotella/Bacteroides group (n = 15) and >-0.8 represents the high Prevotella/Bacteroides group (n = 14).

Figure 3.

Correlation between baseline log Prevotella-to-Bacteroides ratio and body weight change after a 4-week supplement intervention with arabinoxylan-oligosaccharides or polyunsaturated fatty acids. The vertical dashed line indicates the cutoff between high and low Prevotella/Bacteroides groups. <-0.8 represents the low Prevotella/Bacteroides groups and >-0.8 represents the high Prevotella/Bacteroides groups. A: Pearson correlation coefficient (r= -0.51 , P = 0.055). B: Pearson correlation coefficient (r= 0.58, P = 0.030). Abbreviations: AXOS: arabinoxylan oligosaccharides, PUFA: polyunsaturated fatty acids.

Detailed description

Definitions

The term “P/B ratio”, as used herein, refers to the ratio of the relative abundance of Prevotella spp and Bacteroides spp in the microbiota of an individual. The P/B ratio refers to log10(Prevotelia spp. /Bacteroides spp.). The relative abundance of Prevotella spp and Bacteroides spp. may be determined using genera-specific quantitative PCR targeting the bacterial 16S ribosomal gene regions (Roager et al. 2014). A P/B ratio of 1 refers to a microbiota wherein the abundance of Prevotella spp. is 10 times higher relative to the Bacteroides spp.. A P/B ratio of -1 refer to a microbiota wherein the abundance of Bacteroides spp. is 10 times higher relative to the Prevotella spp.

The term “gene copy number” (also "copy number variants" or CNVs), as used herein, refers to the number of copies of a particular gene in the genotype of an individual. The term “human salivary amylase gene copy numbers (AMY1 CN)” refers to the number of copies of the gene encoding human salivary amylase in the genotype of an individual. The term “average degree of polymerization (avDP)”, as used herein, refers to the average number of monomeric units in a macromolecule, polymer or oligomer molecule, such as the average number of monomeric units in AXOS.

Method of treatment

The present disclosure relates to a method for promoting weight loss in an individual in need thereof having a high P/B ratio (a high relative abundance of Prevotella spp JBacteroides spp.) in its microbiota, the method comprising administering to said individual a diet comprising arabinoxylan oligosaccharides (AXOS).

The present disclosure further relates to a method for promoting weight maintenance in an individual in need thereof having a high P/B ratio in its microbiota, the method comprising administering to said individual a diet comprising AXOS.

The present disclosure also relates to a method for reducing appetite in an individual in need thereof having a high P/B ratio in its microbiota, the method comprising administering to said individual a diet comprising AXOS.

The method of promoting weight loss, the method of promoting weight maintenance, and the method of reducing appetite may be of therapeutic nature, such as for treatment of obese individuals. Alternatively, the methods may be of non-therapeutic nature, such as for promoting weight loss, weight maintenance or reducing appetite for cosmetic reasons.

Thus, in one embodiment, a diet comprising AXOS for use in a therapeutic method for promoting weight loss in an individual in need thereof having a high P/B ratio in its microbiota is provided.

In one embodiment, a diet comprising AXOS for use in a therapeutic method for promoting weight maintenance in an individual in need thereof having a high P/B ratio in its microbiota is provided.

In one embodiment, a diet comprising AXOS for use in a therapeutic method for reducing appetite in an individual in need thereof having a high P/B ratio in its microbiota is provided. In one embodiment, a non-therapeutic method for promoting weight loss in an individual in need thereof having a high P/B ratio in its microbiota is provided, the method comprising administering to said individual a diet comprising AXOS.

In one embodiment, a non-therapeutic method for promoting weight maintenance in an individual in need thereof having a high P/B ratio in its microbiota is provided, the method comprising administering to said individual a diet comprising AXOS.

In one embodiment, a non-therapeutic method for reducing appetite in an individual in need thereof having a high P/B ratio in its microbiota is provided, the method comprising administering to said individual a diet comprising AXOS.

In one embodiment, the diet comprising AXOS of the present disclosure comprises a supplement of AXOS, such as a dietary supplement of AXOS, for example a food supplement or a beverage supplement of AXOS.

Thus, in one embodiment, a supplement comprising AXOS for use in a therapeutic method for promoting weight loss in an individual in need thereof having a high P/B ratio in its microbiota is provided.

In one embodiment, a supplement comprising AXOS for use in a therapeutic method for promoting weight maintenance in an individual in need thereof having a high P/B ratio in its microbiota is provided.

In one embodiment, a supplement comprising AXOS for use in a therapeutic method for reducing appetite in an individual in need thereof having a high P/B ratio in its microbiota is provided.

In one embodiment, a non-therapeutic method for promoting weight loss in an individual in need thereof having a high P/B ratio in its microbiota is provided, the method comprising administering to said individual a supplement comprising AXOS. In one embodiment, a non-therapeutic method for promoting weight maintenance in an individual in need thereof having a high P/B ratio in its microbiota is provided, the method comprising administering to said individual a supplement comprising AXOS.

In one embodiment, a non-therapeutic method for reducing appetite in an individual in need thereof having a high P/B ratio in its microbiota is provided, the method comprising administering to said individual a supplement comprising AXOS.

In one embodiment, the amount of AXOS administered to said individual is at least 8 g/d AXOS.

Thus, in one embodiment, a method for promoting weight loss in an individual in need thereof having a high P/B ratio (a high relative abundance of Prevotella spp JBacteroides spp.) in its microbiota is provided, the method comprising administering to said individual a diet comprising at least 8 g/d arabinoxylan oligosaccharides (AXOS).

In one embodiment, a method for promoting weight maintenance in an individual in need thereof having a high P/B ratio in its microbiota is provided, the method comprising administering to said individual a diet comprising at least 8 g/d AXOS.

In one embodiment, a method for reducing appetite in an individual in need thereof having a high P/B ratio in its microbiota is provided, the method comprising administering to said individual a diet comprising at least 8 g/d AXOS.

In one embodiment, the methods of the present disclosure comprise the steps of: a) obtaining a biological sample from said individual; b) determining the P/B ratio in said sample; and c) administering to said individual a diet comprising AXOS to said individual.

In one embodiment, the methods are performed for individuals having a high P/B ratio, such as for example when the P/B ratio determined in step b) is equal to or greater than 0 8

Thus, in one embodiment, the methods of the present disclosure comprise the steps of: a) obtaining a biological sample from said individual; b) determining the P/B ratio in said sample; and c) administering to said individual a diet comprising AXOS to said individual if the P/B ratio determined in step b) is high, such as for example equal to or greater than -0.8.

In one embodiment, the method further comprises a step of determining human salivary amylase gene copy numbers (AMY1 CN) in the biological sample.

In one embodiment, a method is provided wherein the diet comprising AXOS is provided to an individual having a low AMY1 CN.

In one embodiment, a low AMY1 CN is an AMY1 CN equal to or lower than 6.5.

Thus, in one embodiment, the methods of the present disclosure comprise the steps of: a) obtaining a biological sample from said individual; b) determining the P/B ratio and AMY1 CN in said sample; and c) administering to said individual a diet comprising AXOS to said individual if the P/B ratio determined in step b) is high, such as for example equal to or greater than -0.8 and the AMY1 CN determined in step b) is low, such as for example equal to or lower than 6.5..

In one embodiment, the biological sample is a fecal sample, an intestinal sample, a saliva sample or a blood sample, such as a fecal sample or an intestinal sample. In a preferred embodiment, the biological sample is a fecal sample.

P/B ratio

The methods of the present invention relates to administration of a diet comprising AXOS to an individual having a high P/B ratio. As defined above, the term P/B ratio as used herein refers to log10(Prevotella spp./Bacteroides spp.).

In one embodiment, the P/B ratio is the P/B ratio as found in the microbiota of said individual.

In one embodiment, a high P/B ratio is a P/B ratio equal to or greater than -0.8, such as equal to or greater than -0.6, for example equal to or greater than -0.5, for example equal to or greater than -0.45, such as equal to or greater than -0.4, for example equal to or greater than 0, such as equal to or greater than 0.5, for example equal to or greater than 0.8.

In one embodiment, a high P/B ratio is a P/B ratio equal to or greater than -0.8. In a separate embodiment, a high P/B ratio is a P/B ratio equal to or greater than -0,4. In one embodiment, a high P/B ratio is a P/B ratio equal to or greater than -0.45. In yet another embodiment, a high P/B ratio is a P/B ratio equal to or greater than 0.5.

In one embodiment, a low P/B ratio is a P/B ratio lower than -0.8.

The relative abundance of Prevotella spp. may be increased by administering a probiotic comprising Prevotella spp. to said individual, thus increasing the P/B ratio. In one embodiment, the relative abundance of Prevotella spp. is increased by administering a probiotic comprising Prevotella spp. to said individual prior to performing the methods of the present disclosure.

AXOS as supplement

The diet of the present disclosure comprises AXOS.

The AXOS may be obtained from grain extract from one or more cereal grains, such as from one or more of rye, wheat, barley, oats, sorghum, rice, maize, millet, psyllium, flaxseed, pangola grass, bamboo shoot and rye grass.

In one embodiment, the AXOS is obtained from grain extracts from wheat, maize, barley, rye, oat, rice or sorghum. In a preferred embodiment, the AXOS is obtained from grain extracts from wheat, or maize, such as from grain extracts from wheat.

Alternatively, the AXOS may be obtained from bran extract from one or more cereal grains, such as from one or more of rye, wheat, barley, oats, sorghum, maize, millet, psyllium, flaxseed, pangola grass, bamboo shoot and rye grass.

In one embodiment, the AXOS is obtained from bran extracts from wheat, maize, barley, rye, oat, rice or sorghum. In a preferred embodiment, the AXOS is obtained from bran extracts from wheat or maize, such as from bran extracts from wheat. In one embodiment, the diet comprising AXOS is wheat bran extract.

The grain extracts and/or bran extracts may be further processed in order to increase the percentage amount of AXOS in the product.

AXOS is an oligosaccharide comprising arabinose and xylose monomer units. The ratio of arabinose and xylose units may vary. In one embodiment, the AXOS of the present methods has an arabinose to xylose ratio in the range of 0.1 -0.8, such as in the range of 0.1 -0.7, for example in the range of 0.1 -0.6, such as in the range of 0.1 -0.5, for example in the range of 0.1-0.4, such as in the range of 0.1-0.3, for example in the range of 0.15- 0.3, such as in the range of 0.17-0.25. In a preferred embodiment, the arabinose to xylose ratio is in the range of 0.15-0.3.

The degree of polymerization of the AXOS of the present invention may vary. In one embodiment, the AXOS of the present methods has an average degree of polymerization in the range of 2-20, for example in the range of 2-19, such as in the range of 2-18, for example in the range of 2-17, such as in the range of 2-16, for example in the range of 2-15, such as in the range of 2-14, for example in the range of 2-12, such as in the range of 2-10, such as in the range of 3-9, for example in the range of 4-8, such as in the range of 4-7, for example in the range of 4-6, such as 5. In a preferred embodiment, the degree of polymerization is in the range of 4-6.

Administration

The methods of the present disclosure involve administering a diet comprising AXOS to an individual in need thereof.

In one embodiment, the diet comprising AXOS is administered daily for at least 2 consecutive weeks, such as at least 3 consecutive weeks, for example at least 4 consecutive weeks, such as at least 5 consecutive weeks, for example at least 6 consecutive weeks, such as at least 7 consecutive weeks, for example at least 8 consecutive weeks, such as at least 10 consecutive weeks, for example at least 12 consecutive weeks, such as at least 14 consecutive weeks. The diet of the present disclosure may comprise at least 8 g/day of AXOS, such as at least 9 g/d, for example at least 10 g/d, such as at least 11 g/d of AXOS.

In one embodiment, the diet comprising AXOS is administered in one daily dose, such as two daily doses, for example in three daily doses, such as four or more daily doses.

In one embodiment, the diet comprising AXOS is administered in one daily does, in two daily doses, in three daily doses, or in four or more daily doses.

Thus, in one embodiment, the diet comprising AXOS is administered in at least one daily dose, such as at least two daily doses, for example in at least three daily doses, such as at least four or more daily doses.

In one embodiment, the diet comprising AXOS of the present disclosure comprises a supplement of AXOS, such as a dietary supplement of AXOS. Thus, in one embodiment, the AXOS is administered in the form of a supplement, such as a beverage supplement and/or a food supplement. The supplement may be in the form of for example biscuits, food bars, crackers, powder and/or a bread.

In one embodiment, the supplement is in the form of a powder. Said powder may be obtained from a grain extract and/or bran extract and may be further purified in order to increase the percentage content of AXOS in said powder.

In one embodiment, AXOS is administered in a capsule, such as an enterocoated capsule. Enterocoated capsules are known in the art and allow for protection of the content in the gastric environment until release of the content in the intestines, such as in the small intestine or in the colon. Such administration form may allow for a lower dosage of AXOS necessary to provide the observed effect.

Thus, in one embodiment, the diet of the present disclosure may comprise AXOS administered in enterocoated capsules in an amount in the range of 1-10 g/day, such as in the range of 1-8 g/day, for example in the range of 1-6 g/day, such as in the range of 2-4 g/day. In one embodiment, the beverage supplement and/or food supplement comprises in the range of 5-100% AXOS, for example in the range of 10-100% AXOS, such as for example in the range of 20-100% AXOS, for example in the range of 30-100% AXOS, such as in the range of 40-100% AXOS, for example in the range of 50-100% AXOS, such as in the range of 60-100% AXOS, for example in the range of 70-100% AXOS, such as in the range of 80-100% AXOS, for example in the range of 90-100% AXOS.

In one embodiment, the beverage supplement and/or food supplement is in powder form and comprises in the range of 50-100% AXOS, such as in the range of 60-100% AXOS, for example in the range of 70-100% AXOS, such as in the range of 80-100% AXOS, for example in the range of 90-100% AXOS.

In one embodiment, the beverage supplement and/or food supplement is in powder form and may be added to a beverage and/or a food preparation prior to consumption.

In one embodiment, the beverage supplement and/or food supplement comprises in the range of 6-10 kJ/g, such as in the range of 7-9 kJ/g.

In one embodiment, the beverage supplement and/or food supplement comprises in the range of 0.5-1 g of total dietary fibers/g, such as in the range of 0.6-0.9 g of total dietary fibers/g, for example in the range of 0.6-0.8 g of dietary fibers/g.

In one embodiment, the beverage supplement and/or food supplement comprises in the range of 0.05-0.5 g of carbohydrate/g, such as in the range of 0.1 -0.4 g of total carbohydrate/g, for example in the range of 0.1 -0.3 g of carbohydrate/g.

In one embodiment, the beverage supplement and/or food supplement comprises in the range of 0.4-1 g of AXOS/g, for example in the range of 0.5-1 g of AXOS/g, such as in the range of 0.6-0.9 g of AXOS/g, for example in the range of 0.6-0.8 g of AXOS/g.

Individual

The methods of the present disclosure relates to treatment of obesity, maintenance of weight and/or reduction of appetite in an individual in need thereof.

In one embodiment, the individual is overweight or obese. In one embodiment, the individual has a body mass index of more than 23 kg/m², for example more than 25 kg/m², such as more than 30 kg/m2, for example more than 35 kg/m², such as more than 40 kg/m².

In one embodiment, the individual has one or more of the following clinical characteristics: a) body mass index of 23-40 kg/m², b) waist circumference of ³80 cm for women or of ³94 cm for men, c) triglycerides of ³1.7 mmol/L, d) high density lipoprotein of <1.29 mmol/L for women or of <1.03 mmol/L for men, e) fasting plasma glucose (FPG) of ³5.6 mmol/L, f) high blood pressure (BP) , such as systolic BP of ³130 mmHg or diastolic BP of ³85 mmHg, g) Haemoglobin concentration of ³7 mmol/L.

Result obtained The methods of the present disclosure relates to treatment of obesity, maintenance of weight and/or reduction of appetite in an individual in need thereof.

In one embodiment, the methods of the present disclosure are methods for promoting weight loss. In one embodiment, the weight loss is primarily fat loss.

In one embodiment, the methods of the present disclosure are methods for promoting weight maintenance.

In one embodiment, the methods of the present disclosure are methods for reducing appetite.

AXOS is a soluble fiber and because of its complex chemical structure it can be fermented both in the small intestine, and in the proximal and distal part of the colon. However, where AXOS is fermented might depend on the average degree of polymerization (avDP), meaning that AXOS with a higher avDP is fermented in the distal colon where AXOS with a low avDP may be fermented already in the small instestine and proximal part of the colon (Sanchez et al. 2009). Due to the small average degree of polymerization of the AXOS of the present disclosure, it is likely fermented higher in the gastrointestinal tract, compared to dietary fibers having a higher average degree of polymerization. This may, through microbial metabolites (e.g. short-chain fatty acids (SOFA)), affect L-cells in the small intestine and GLP-1 production. This could be (one of) the mechanism the weight loss provided by AXOS in an individual having a high P/B- ratio. Thus in one embodiment, the weight loss provided by the methods of the present disclosure may be obtained through an increase in SOFA metabolites, such as acetate, propionate or butyrate.

A potential role of SCFAs in appetite regulation and energy intake has been demonstrated (Chambers et al. 2015). Thus, in one embodiment, the reduction in appetite as provided in the methods of the present disclosure may be a result of an increase in SOFA metabolites, such as acetate, propionate or butyrate.

Items

1. A method for promoting weight loss in an individual in need thereof having a high P/B ratio (a high relative abundance of Prevotella spp./Bacteroides spp.) in its microbiota, the method comprising administering to said individual a diet comprising arabinoxylan oligosaccharides (AXOS).

2. A method for promoting weight maintenance in an individual in need thereof having a high P/B ratio in its microbiota, the method comprising administering to said individual a diet comprising AXOS.

3. A method for reducing appetite in an individual in need thereof having a high P/B ratio in its microbiota, the method comprising administering to said individual a diet comprising AXOS.

4. A method for promoting weight loss in an individual in need thereof comprising: a) obtaining a biological sample from said individual; b) determining the P/B ratio in said sample; and c) administering to said individual a diet comprising AXOS to said individual if the P/B ratio determined in step b) is high. 5. A method for promoting weight maintenance in an individual in need thereof comprising: a) obtaining a biological sample from said individual; b) determining the P/B ratio in said sample; and c) administering to said individual a diet comprising AXOS to said individual if the P/B ratio determined in step b) is high.

6. A method for reducing appetite in an individual in need thereof comprising: a) obtaining a biological sample from said individual; b) determining the P/B ratio in said sample; and c) administering to said individual a diet comprising AXOS to said individual if the P/B ratio determined in step b) is high. 7. The method according to any one of the preceding items, wherein the diet comprises at least 8 g/day, such as at least 9 g/d, for example at least 10 g/d, such as at least 11 g/d of AXOS, preferably, the diet comprises at least 8 g/d of AXOS.

8. The method according to any one of the preceding items, wherein a high P/B ratio is a P/B ratio equal to or greater than -0.8, such as equal to or greater than -0.6, for example equal to or greater than -0.5, such as equal to or greater than - 0.4, for example equal to or greater than 0, such as equal to or greater than 0.5, for example equal to or greater than 0.8.

9. The method according to any one of the preceding items, wherein a high P/B ratio is a P/B ratio equal to or greater than -0.8.

10. The method according to any one of the preceding items, wherein a high P/B ratio is a P/B ratio equal to or greater than -0.4.

11. The method according to any one of the preceding items, wherein a low P/B ratio is a P/B ratio lower than 0.5. 12. The method according to any one of the preceding items, wherein the diet comprising AXOS is administered daily for at least 4 consecutive weeks, such as at least 8 consecutive weeks, for example at least 10 consecutive weeks, such as at least 14 consecutive weeks.

13. The method according to any one of the preceding items, wherein the at least 8 g/d AXOS are administered in one daily dose, such as two daily doses, such as in three or more daily doses.

14. The method according to any one of the preceding items, wherein the AXOS is obtained from grain extract from one or more cereal grains, such as from one or more of rye, wheat, barley, oats, sorghum, rice, maize, millet, psyllium, flaxseed, pangola grass, bamboo shoot and rye grass.

15. The method according to any one of the preceding items, wherein the AXOS is obtained from bran extract from one or more cereal grains, such as from one or more of rye, wheat, barley, oats, sorghum, maize, millet, psyllium, flaxseed, pangola grass, bamboo shoot and rye grass.

16. The method according to any one of the preceding items, wherein the AXOS has a arabinose to xylose ratio in the range of 0.1 -0.4, such as in the range of 0.1- 0.3, for example in the range of 0.15-0.3, such as in the range of 0.17-0.25.

17. The method according to any one of the preceding items, wherein the AXOS has an average degree of polymerization in the range of 2-10, such as in the range of 3-9, for example in the range of 4-8, such as in the range of 4-7, for example in the range of 4-6, such as 5.

18. The method according to any one of the preceding items, wherein the at least 8 g/d AXOS is administered in the form of a supplement, such as a beverage supplement and/or a food supplement.

19. The method according to any one of the preceding items, wherein the at least 8 g/d AXOS is administered in a capsule, such as an enterocoated capsule. 20. The method according to any one of the preceding items, wherein the diet of the present disclosure comprises AXOS administered in enterocoated capsules in an amount in the range of 1-10 g/day, such as in the range of 1-8 g/day, for example in the range of 1-6 g/day, such as in the range of 2-4 g/day.

21. The method according to any one of the preceding items, wherein the beverage supplement and/or food supplement comprises in the range of 6-10 kJ/g, such as in the range of 7-9 kJ/g. 22. The method according to any one of the preceding items, wherein the beverage supplement and/or food supplement comprises in the range of 0.5-1 g of total dietary fibers/g, such as in the range of 0.6-0.9 g of total dietary fibers/g, for example in the range of 0.6-0.8 g of dietary fibers/g. 23. The method according to any one of the preceding items, wherein the beverage supplement and/or food supplement comprises in the range of 0.05-0.5 g of total carbohydrate/g, such as in the range of 0.1 -0.4 g of total carbohydrate/g, for example in the range of 0.1 -0.3 g of carbohydrate/g. 24. The method according to any one of the preceding items, wherein the beverage supplement and/or food supplement comprises in the range of 0.5-1 g of AXOS/g, such as in the range of 0.6-0.9 g of AXOS/g, for example in the range of 0.6-0.8 g of AXOS/g. 25. The method according to any one of the preceding items, wherein the at least 8 g/d AXOS is in powder form and is added to a beverage and/or a food preparation prior to consumption.

26. The method according to any one of the preceding items, wherein the food supplement is provided in the form of biscuits, food bars, crackers, powder, bread and/or a beverage.

27. The method according to any one of the preceding items, wherein the weight loss is primarily fat loss. 28. The method according to any one of the preceding items, wherein the biological sample is a fecal sample, an intestinal sample, a saliva sample, a blood sample, such as a fecal sample or an intestinal sample, preferably a fecal sample.

29. The method according to any one of the preceding items, wherein the individual is overweight or obese.

30. The method according to any one of the preceding items, wherein the individual has a body mass index of more than 23 kg/m², for example more than 25 kg/m², such as more than 30 kg/m², for example more than 35 kg/m², such as more than 40 kg/m².

31. The method according to any one of the preceding items, wherein the individual has one or more of the following clinical characteristics: a) body mass index of 23-40 kg/m², b) waist circumference of ³80 cm for women or of ³94 cm for men, c) triglycerides of ³1.7 mmol/L, d) high density lipoprotein of <1.29 mmol/L for women or of <1.03 mmol/L for men, e) fasting plasma glucose (FPG) of ³5.6 mmol/L, f) high blood pressure (BP) , such as systolic BP of ³130 mmHg or diastolic BP of ³85 mmHg, g) Haemoglobin concentration of ³7 mmol/L.

32. The method according to any one of the preceding items, further comprising determining human salivary amylase gene copy numbers (AMY1 CN) in the biological sample.

33. The method according to any one of the preceding items, wherein the individual has a low AMY1 CN.

34. The method according to any one of the preceding items, wherein a low AMY1 CN is an AMY1 CN equal to or lower than 6.5. 35. The method according to any one of the preceding items, wherein the relative abundance of Prevotella spp. is increased by administering a probiotic comprising Prevotella spp. to the patient.

Examples

Objectives and hypotheses

The primary objective was to investigate the potential differences in 4-week body weight change after a 4-week intervention with a dietary supplement of Arabinoxylan- oligosaccharides (AXOS) compared to control (Poly-Unsaturated Fatty Acids (PUFA) as a function of baseline P/B ratio. The hyphothesis was that participants with a high Prevotella/Bacteriodes-ratio ( P/B ratio) lose more body weight than participants with a low P/B ratio on an AXOS supplement compared to a control (PUFA) supplement.

Method Study design

The study included two diet periods (AXOS and polyunsaturated fatty acids (PUFA)) with a duration of 4 weeks separated by a 4-week washout period (3x4 weeks) (Figure 1). Participants were instructed to maintain their body weight during the two diet periods. During the study, data collection was conducted on 4 clinical investigation days (CID): baseline (week 0), after the first intervention period (week 4), after the washout period (week 8), and after the second intervention period (week 12). To ensure body weight maintenance, the study participants had two consultations with a dietician (week 2 and 10), where body weight (non-fasting) and diet were evaluated. Furthermore, the dietician contacted the participants by phone in the remaining weeks (week 1 , 3, 9, 11). Before each CID the participants were instructed to consume a standardized dinner in the evening and to be fasted for a minimum of 8 hours. The present analyses focuses on the first period of the cross-over design as no response was observed to the AXOS diet in the second period, due to a potential carry-over effect from the PUFA intervention in period 1. The study was registered at clinicaltrial.gov (NCT02215343) and approved by the Danish Ethical Committee. All study procedures were carried out in accordance with the Helsinki Declaration and the Danish Protection Agency. Written informed consent was obtained before study start.

Randomization and blinding Participants were randomly allocated to one of the sequences of the two supplements (AXOS

AXOS) using a computer-generated randomization list conducted at randomization.com. Owing to the nature of the study supplements, blinding of participants and staff was not possible.

Study participants Eligibility criteria

Eligibility criteria were non-smoking men and women between 18-60 years with a BMI of 25-40 kg/m². Furthermore, participants should have a WC ³94 cm for men and ³80 cm for women and in addition it was required that they should have at least one criteria for metabolic syndrome; elevated FPG (³5.6 mmol/L), elevated triglycerides (TG) (>1.7 mmol/L), lowered high density lipoprotein (HDL) (men: <1.03 mmol/L, women: <1.29 mmol/L), or elevated blood pressure (BP) (systolic BP ³130 mmHg or diastolic BP ³85 mmHg). Exclusion criteria included use of antibiotics three months before and during the study, medication for dyslipidemia, T2DM, or hypertension. Participants were not allowed to consume dietary supplements with pre- and/or probiotics, fiber or fish oil for six weeks before study start. Furthermore, participants were excluded if they, two months before study start, have had a body weight change of ±3 kg.

A more detailed description of the complete inclusion and exclusion criteria can be found below:

Inclusion criteria:

• Men and women between 18-60 years

• Body mass index of 25-40 kg/m2

• Waist circumference of ³80 cm for women and ³94 cm for men

• At least one criteria for metabolic syndrome:

^■ Elevated triglycerides (³1.7 mmol/L)

^■ Reduced high density lipoprotein (women: <1.29 mmol/L, men: <1.03 mmol/L)

^■ Elevated FPG (³5.6 mmol/L) or raised blood pressure (systolic BP ³130 mmHg or diastolic BP ³85 mmHg)

• Haemoglobin concentration ³7 mmol/L

• Non-pregnant, non-lactating and not planning pregnancy during the study Exclusion criteria:

• Use of antibiotics three months before and during the study

• Medication for dyslipidemia, type-2 diabetes or elevated blood pressure

• Dietary supplements with pro- and/or prebiotics, fiber or fish oil six weeks before study start

• Special dietary habits as vegetarian and vegan

• Food allergy for the components of the study supplements (e.g. wheat, milk)

• Smoking

• Body weight change of ±3 kg two months before study start.

• Elite athletes or individuals with intensive physical training (>10 hours of strenuous physical activity per week)

• Blood donation one month before study start

• Gastrointestinal and liver disorders

• Chronic inflammatory disorders (excluding obesity)

• Psychiatric disorders including depression requiring treatment

• Surgical treatment of obesity and abdominal surgery

• Individuals who were unable to adhere to the procedures required by the study protocol

Intervention

The dietary supplements evaluated were wheat bran extract, rich in AXOS (10.4 g/d AXOS) and PUFA (3.6 g/d n-3 PUFA). For the AXOS intervention the goal was to reach a high-fiber diet consisting of approximately 30 g of fiber/day, of which 10 g was obtained from the AXOS supplementation. AXOS was provided as powder (5 g/d) and biscuits (4 per day). The powder was instructed to be consumed in the morning and in the evening and should be dissolved in water. A detailed nutritional description of the AXOS supplementation can be found in Table 1. The remaining fiber intake (~20 g) was achieved through the participants’ habitual diet which was ensured by consultation with the dietician. For the PUFA intervention, the goal was to reach approximately 10 energy percentage (E%) PUFA/day whereas the participants were guided to decrease their intake of saturated fatty acids. PUFA was provided as fish oil capsules (1.32 g/d of docosahexaenoic acid (DHA) and 1.86 g/d of eicosapentaenoic acid (EPA)). Three capsules should be consumed in the morning and 3 capsules in the evening. Participants on both interventions were instructed to maintain their body weight throughout the study which was controlled by weekly contact with the dietician. Table 1. Characterization of WBE and AXOS supplements consumed during the AXOS intervention

WBE Powder Crackers Biscuits Total daily (1 OOg) (5g (per (per intake

WBE) piece) piece)

Energy (kJ) 812 41 125 162 655

Protein (g) 0.7 0.04 0.6 0.5 2.3

Fat (g) 0 0.0 0.7 1.8 5.0

Carbohydrates (g) 19.7 1.0 4.8 4.8 21.2

Sugar (g) 3.8 0.2 0.7 1.7 5.2

Total dietary fibre 72.0 3.6 1.0 1.0 11.2

(9)

WBE (g) 100 5 1.3 1.2 15.0

AXOS (g) 69 3.5 0.9 0.8 10.4

Abbreviations: WBE, wheat bran extract; AXOS, arabinoxylan oligosaccharides.

Clinical examinations

Anthropometric measurements Body composition including height, body weight, and WC were evaluated using standard operational procedures. The anthropometric measurements were conducted in fasting state and all participants had to void their bladder before start. Body weight was measured using a calibrated digital scale (Lindells, Malmo, Sweden) to the nearest 0.1 kg with the participants wearing underwear, and height was measured without shoes to the nearest 0.5 cm using a wall-mounted stadiometer (Hultafors). BMI was calculated as weight in kilograms divided by height in meters squared (kg/m²). WC was measured twice using a non-elastic tape on the skin between the lowest rib and iliac crest after exhalation to the nearest 0.5 cm. The average of the two measurements was calculated. Blood samples

At the first CID (after an overnight fast), venous blood samples were drawn. The blood samples were analyzed for glucose, insulin, LDL, HDL, and TG. Plasma glucose samples were collected in fluoride tubes and directly stored on ice and centrifuged. For insulin, LDL, HDL, and TG, the samples were collected in serum tubes and stored for 20 minutes at room temperature for coagulation. Before processing, all blood samples were centrifuged at 2500 x g for 10 min at 4 °C and stored at -80 °C. Chemiluminescent immunometric assay was used to measure insulin on IMMULITE 200 INSULIN Analyzer (Siemens Healthcare Diagnostics Products Ltd., UK). Glucose was analyzed on the Pentra 400 Analyzer (HORIBA ABX, Montpellier, France) using enzymatic hexokinase method and immunoturbidimetric method. For quantification of insulin resistance (IR) and beta-cell function the Homeostasis model assessment (HOMA) was used based on measurements from glucose concentrations and FI. HOMA-IR was calculated as: ( insulin (mΐu/mL) x glucose (mmol/L))/22.5. LDL, HDL, and TG were analyzed on an auto analyzer platform DIMENSION VISTA® (Siemens Healthcare Diagnostics Inc., USA) and measured by enzymatic methods.

Fecal samples

To assess the gut microbiota composition fecal samples were analyzed using 16S rRNA gene sequencing. Participants collected a fecal sample in the morning prior to the first CID, kept it cold and delivered it to the department within three hours. Each fecal sample was weighted, and a small portion of the sample was brought to the EasySampler® kit for the collection of stool (GP Medical Devices, Denmark) and kept at -80 °C. To extract fecal deoxyribonucleic acid (DNA) the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) was used. Bead beating was conducted in 2 mL microcentrifuge tubes with 0.1 mm diameter glass beads, 1 mL InhibitEX buffer and -200 mg feces and carried out in a Mini-Bead Beater apparatus (BioSpec Products, Bartlesville, USA) including two cycles of shaking in 1 minute and incubation on ice in between cycles. Fecal DNA was measured using UV methods (Nanodrop, Thermo Scientific, Wilmington, USA). A portion of each sample was prepared at 20ng/pL with the use of nuclease free water for polymerase chain reaction (PCR). Amplification of the V3-V4 hypervariable regions of the 16S rRNA gene were done using 20 ng DNA (1 L diluted portion of feces) and furthermore 25 PCR cycles containing the following steps: 95°C for 20 sec., 55°C for 20 sec., and 72°C for 20 sec. During PCR, Phusion High-Fidelity Taq Polymerase (Thermo Scientific, 219 Wilmington, USA) and the 6-mer barcoded primers, S-D-Bact-0341-b-S- 17 (CCTACGGGNGGCWGCAG) and S-D-Bact-0785-a-A-21 (GACTACHVGGGTAT-

CTAATCC) were used, which target a broad range of bacterial 16S rRNA genes. Purification of dual barcoded PCR products which consisted of ~380bp were done from triplicate reactions using lllustra GFX DNA and Gel Band Purification Kit (GE Healthcare, Little Chalfont, UK) and furthermore quantified with Qubit 3.0 together with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). By mixing equimolar amounts of amplicon DNA (100 ng per sample) and sequencing it in an lllumina MiSeq platform alongside 2x300 PE configuration (Eurofins Genomics GmbH, Ebersberg, Germany), fecal samples were multiplexed. De-multiplexing of samples was conducted using sequence information from the DNA barcodes. After the removal of barcodes/primers, the sequences were prepared for removal of chimera using Uchime algorithm and the 16S rRNA database SILVA. The Ribosomal Database Project (RDP) classifier v2.12. was used for taxonomic assessment.

For quantification of fecal SCFAs (acetate, propionate, and butyrate), the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was applied based on the 3-nitrophenylhydrazone derivatization/reaction.

Dietary records and compliance

A 3-day dietary records were completed before study start and after the intervention and reviewed by a dietician. Instruction in how to complete a dietary record was provided to all participants. Energy content, macronutrients, and micronutrients were calculated based on the mean intake from the 3-day dietary records. All dietary records were calculated by dieticians using the Danish Food Database (Dankost Pro, Dankost APS, Denmark).

Compliance was measured from a food diary where participants were instructed to record their daily intake of the provided supplement/intervention product. Compliance was assessed on numbers of days during a week where the participants failed to consume 100% of the products. Compliance was then assessed as either very good, good, bad or very bad, defined as missing products of <4, >4 to <8, >8 to <12, and >12 days during the 4-week intervention, respectively. If information about consumption of the supplement was missing, compliance was downregulated one level (e.g. from very good to good).

Stratification of enterotypes

To stratify participants into high P/B or low P/B groups a histogram were created including the frequency of baseline relative abundance of the log-transformed P/B ratio. Furthermore, the log-transformed P/B ratio was tested for bimodal distribution using the dip test for unimodality ( diptest R package). Since no bimodal distribution was found in any of the studies as previously observed, a decision to use the median value of the log- transformed P/B ratio in both studies was made. This cutoff has been used previously to stratify participants into high and low P/B groups.

Statistical analyses

Baseline characteristics are presented as mean ± standard deviation (SD), median (interquartile range) or proportions (%). Differences between the four P/B groups across the intervention and control group were tested using a two-way ANOVA or Pearson's chi-squared test. Correlations between body weight change and the log-transformed baseline P/B ratio were analyzed using Pearson’s correlation coefficient for each treatment. Differences in body weight change, WC, metabolic markers, and gut microbiota markers from baseline within and between the two P/B groups in the intervention and the control group were analyzed by means of a linear mixed model (LMM). A similar model was used for analyzing the change in energy intake and macronutrients. An available-case approach was used for all outcomes. The LMM included a three-way interaction between treatment (AXOS vs. PUFA) x time x P/B group. Difference between the intervention and control group were analyzed by pairwise comparison of the estimated mean difference in change between P/B groups. Additionally, a LMM including an interaction term between P/B ratio as a continues biomarker and a treatment-time combination were fitted for body weight. Predictions were estimated as contrasts of intercepts and slopes of baseline P/B ratio. Based on the specific intercepts and slopes, delta models were fitted for a range between the upper and lower P/B ratio giving a minimum treatment effect. The mentioned LMMs included age, gender, and baseline BMI as fixed effects, and subject as random effect. Finally, a LMM with a three-way interaction between treatment x time x P/B group was carried out using the cross-over design for one of the sequences (AXOS to PUFA) adjusted for age and gender as fixed effects and subject and within-period subject-specific random effects. In general, model checking was validated by residuals and quantile-quantile probability plots. The results are reported as mean change from baseline with 95% confidence interval (Cl). The significance level was set at P < 0.05. All statistical analyses were carried out with the use of R statistical software, version 3.5.1 (R Foundation).

Results

Thirty participants were included in the study. In period one, a total of 29 participants completed the 4-week dietary intervention with AXOS (n = 15) and PUFA (n = 14). One participant was excluded before study start due to antibiotic treatment. The cutoff to stratify participants into high and low P/B groups was based on the median value of the baseline log-transformed P/B ratio (Figure 2) and resulted in four equal sized groups (n = 7 to 8). No differences were found in baseline characteristics between all groups within and across interventions, with exception of a higher relative abundance of Prevotella and a lower relative abundance of Bacteroides in the high P/B groups compared to the low P/B groups (P < 0.001) (Table 2). No additional differences in baseline characteristics were found between participants with high- and low P/B ratio independent of the intervention (Table 3).

Table 2. Baseline characteristics of the study participants in the AXOS and PUFA intervention stratified by high and low P/B ratio

AXOS PUFA

High P/B (n=7) Low P/B (n=8) High P/B (n=7) Low P/B (n=7) Age (years) 47 ± 8 44 ± 11 41 ± 16 43 ± 11

Gender (%

71/29 87/13 72/28 57/43 female/male) Body weight

87.1 ± 8.1 90.5 ± 15.4 84.2 ± 11.8 89.8 ± 18.9

(kg)

BMI (kg/m²) 29 ± 3 30 ± 3 29 ± 3 32 ± 3 WC (cm) 96.7 ± 7.4 96.8 ± 9.1 92.9 ± 6.6 99.3 ± 11.9 Glucose

5.61 ± 0.27 5.53 ± 0.46¹ 5.42 ± 0.60 5.35 ± 0.27 (mmol/L)

Insulin (pmol/L) 34 (25; 50) 61 (33; 72) 31 (24; 60) 20 (16; 41)

1.49 (1.05; 2.47 (1.40; 1.37 (0.93; 0.83 (0.65;

HOMA-IR 2.02) 3.06)¹ 2.45) 2.06)

1.11 (1.05; 1.13 (0.75; 1.19 (0.90; 1.33 (0.99;

TG (mmol/L) 1.33) 1.33)¹ 1.38) 1.49)

LDL (mmol/L) 3.21 ± 1.22 2.77 ± 0.70¹ 2.80 ± 0.83 3.47 ± 0.62 HDL (mmol/L) 1.50 ± 0.52 1.53 ± 0.34¹ 1.42 ± 0.49 1.17 ± 0.16 0.086 (0.009; 0.016 (0.009; 0.032 (0.030; 0.016 (0.014;

Prevotella (RA) 0.221)^ab 0.026)^ac 0.062)^cd 0.019)^bd

Bacteroides 0.097 (0.045; 0.250 (0.184; 0.073 (0.056; 0.344 (0.195

(RA) 0.272)^ade 0.354)^abc 0.088)^cef ;0.411)^bdf 0.00560 0.00420 0.00550 0.00390

Bifidobacterium

(0.00375; (0.00358; (0.00410; (0.00345;

(RA)

0.00830) 0.00970) 0.01840) 0.00565)

Total SCFA

261 ± 111² 327 ± 170³ 333 ± 167³ 350 ± 113

(mGTIqI/g)

Acetate

150 ± 68² 187 ± 97³ 184 ± 97³ 190 ± 67

(mGhoI/g)

Butyrate

51 (43; 60)² 70 (45; 76) 3 67 (51; 90) 3 84 (49; 99)

(m ol/g)

Propionate

53 (44; 55)² 60 (44; 83)³ 63 (44; 78) 3 76 (67; 81)

(m ol/g)

Energy (kJ/d) 8770 ± 3570 8580 ± 1690 8670 ± 3130 9380 ± 3080

Carbohydrates

42.7 ± 6.3 48.0 ± 5.8 46.4 ± 4.4 45.7 ± 10.1

(E%)

Protein (E%) 17.4 ± 4.9 17.1 ± 4.4 18.4 ± 4.7 17.7 ± 4.7 Fat (E%) 37.7 ± 5.6 33.3 ± 4.0 34.0 ± 5.0 35.3 ± 6.9

PUFA (g/d) 16.9 ± 8.1 14.0 ± 3.9 14.1 ± 4.6 14.1 ± 5.6 Total dietary 21.3 (16.8; 23.5 (15.4; 22.7 (17.2; 21.5 (18.3; fiber (g/d) 28.1) 32.5) 30.9) 25.3)

Data are shown as mean ± SD, median (interquartile range) or proportions (%). Difference between the groups was evaluated using a two-way ANOVA. Prevotella, Bacteroides, Bifidobacterium, propionate, butyrate, and total dietary fiber were log- transformed before analysis. Same letters within a row (a,b,c,d,e,f) indicate significant differences ( P < 0.05). All fecal short-chain fatty acids are shown as mGhoI/g in dry weight. Abbreviations: AXOS: arabinoxylan-oligosaccharides, PUFA: polyunsaturated fatty acids, P/B: Prevotella-to-Bacteroides, BMI: body mass index, WC: waist circumference, HOMA-IR: homeostatic model assessment-insulin resistance, TG: triglycerides, LDL: low-density lipoprotein, HDL: high-density lipoprotein, SCFA: short-chain fatty acid, RA: relative abundance, E%: energy percentage. ¹n=7, ²n=

Results of the changes from baseline to week 4 in dietary intake from the 3-day dietary records are shown in Table 4. A difference in intake of dietary fiber (g/d) and fat (E%) was seen between the high P/B group in the AXOS intervention and high P/B group in the PUFA intervention (P < 0.05). Energy (kJ/d) and the other macronutrients did not show a difference in change between groups (Table 4). Self-reported compliance showed that everyone had “very good compliance” except up to one participant within each group that had “good compliance”.

Table 3. Baseline characteristics of the study participants stratified by high and low P/B ratio

High P/B (n=14) Low P/B (n=15)

Age (years) 44 ± 13 44 ± 11

Gender (% female/male) 71/29 73/27 Body weight (kg) 85.7 ± 9.84 90.2 ± 16.5

BMI (kg/m²) 29 ± 3 31 ± 3

WC (cm) 94.8 ± 7.0 98.0 ± 10.2

Glucose (mmol/L) 5.51 ± 0.46 5.44 ± 0.37²

Insulin (pmol/L) 51.0 (41.0; 88.0) 48 (32.5; 76.0) HOMA-IR 1.43 (1.00; 2.08) 1.98 (0.71 ; 2.82)² TG (mmol/L) 1.15 (0.94; 1.40) 1.24 (0.82; 1.45)²

LDL (mmol/L) 3.00 ± 1.02 3.11 ± 0.73²

HDL (mmol/L) 1.47 ± 0.48 1.34 ± 0.31²

Prevotella (RA) 0.0402 (0.0297; 0.121) ^a 0.0157 (0.0142; 0.0176) ^a Bacteroides (RA) 0.0770 (0.0583; 0.121) ^a 0.282 (0.023; 0.346) ^a 0.00390 (0.00345;

Bifidobacterium (RA) 0.00555 (0.00398; 0.0132)

0.00585)

Total SCFA (mGhoI/g) 300 ± 143¹ 339 ± 136³ Acetate (mGhoI/g) 169 ± 83 189 ± 79 Butyrate (mGhoI/g) 52 (50; 77) ¹ 73 (45; 90)³ Propionate (mGhoI/g) 53 (43; 77) ¹ 72 (59; 84)³ Energy (kJ) 8720 ± 3220 8960 ± 2380 Carbohydrates (E%) 44.6 ± 5.6 46.9 ± 7.8 Protein (E%) 17.9 ± 4.7 17.4 ± 4.4

Fat (E%) 35.9 ± 5.6 34.2 ± 5.4

Total dietary fibre (g) 22.0 (16.8; 28.6) 23.0 (16.8; 27.6)

Data are presented as mean ± SD, median (interquartile range) or proportions (%). Difference between the groups were tested using one-way ANOVA (some variables were log-transformed before analysis) or Pearson’s chi-squared test. Same letters within a row (a) indicate significant differences (P < 0.05). All fecal SCFAs are presented as mGhoI/g in dry weight. Abbre-viations: P/B: Prevotella-to-Bacteroides, BMI: Body mass index, WC: waist circumference, HOMA-IR: homeostatic model assessment-insulin resistance, TG: triglycerides, LDL: low-density lipoprotein, HDL: high-density lipoprotein, RA: relative abundance, E%: energy percentage. ¹ n=11 ,

Table 4. Changes in dietary intake from baseline to week 4 for the study participants in the AXOS and PUFA intervention stratified by high and low P/B ratio

AXOS PUFA

High P/B Low P/B High P/B Low P/B

(n=7) (n=8) (n=7) (n=7)

D Energy (kJ/d) 161 ± 1453 454 ± 1359 1174 ±1453 -1566 ± 1453

D Carbohydrates -4.9 ± 3.6 -0.4 ± 3.6

3.3 ± 3.6 1.6 ± 3.4 (E%)

D Protein (E%) 1.1 ± 2.4 -8.8 ± 2.2 -5.7 ± 2.4 7.1 ± 2.4

D Fat (E%) -4.9 ± 2.6^a 0.1 ± 2.5 5.0 ± 2.6^a -0.7 ± 2.6

D PUFA (g) -2.8 ± 3.2 -1.7 ± 3.0 5.2 ± 3.2 0.6 ± 3.2

D Total dietary fiber -7.0 ± 5.6^a -3.0 ± 5.6

10.6 ± 5.6^a 7.7 ± 5.2

(g)

Data are shown as mean ± SEM. Difference between the groups was tested using a two-way ANOVA. Same letters within a row (a, b) indicate significant differences (P < 0.05). The presented total dietary fiber and polyunsaturated fatty acids include intervention supplements. Abbreviations: AXOS: arabinoxylan-oligosaccharides, PUFA: polyunsaturated fatty acids, P/B: Prevotella-to-Bacteroides, E%: energy percentage.

Difference in body weight change between P/B groups In period one, participants in the AXOS intervention in the high P/B group had an insignificant weight loss of 0.14 kg (95% Cl: -0.67; 0.38, P= 0.59) whereas participants in the low P/B group had a weight gain of 0.65 kg (95% Cl: 0.16; 1.14, P=0.009). Consequently, a difference of -0.79 kg was found between groups (95% Cl: -1.51; -0.08, P= 0.030). The participants in the PUFA intervention in the high P/B group had an insignificant weight gain of 0.41 kg (95% Cl: -0.11; 0.94, P=0.12) whereas participants in the low P/B group had an insignificant weight loss of 0.20 kg (95% Cl: -0.72; 0.32, P=0.45). Consequently, no difference was found between groups (0.61 kg, 95% Cl: - 0.13; 1.35, P=0.10). Nevertheless, when comparing these intervention differences in body weight change between the high- and low P/B groups a difference of -1.41 kg (95% Cl: -2.44; -0.38, P=0.007) was observed. When analyzing the cross-over for the sequence AXOS to PUFA, a difference of -1.85 kg (95% Cl: -3.13; -0.57, P= 0.004) in body weight was found when comparing intervention differences between P/B groups.

Consistent with these results a borderline negative correlation between P/B ratio and body weight change in the AXOS intervention (r =-0.51, P= 0.055) was identified, whereas a positive correlation was seen in the PUFA intervention (r=0.58, P= 0.03)

(Figure 3).

As an extension of using the stratified approach an additional analysis using baseline P/B ratio continuously was conducted. A linear trend in body weight change between the supplements was found across the spectrum of log(Prevotella/Bacteroides) (slope = - 1.26 kg/unit (95% Cl: -2.09; -0.43, P = 0.003)). Notably, participants with a P/B ratio < - 0.97 implied an average weight gain of 0.61-1.27 kg whereas a P/B ratio > 0.44 predicted an average weight loss of 1.17-1.75 kg (all P < 0.05) when choosing AXOS over PUFA (Table 5).

Table 5. Difference in body weight change (kg) between the two intervention diets (AXOS - PUFA) according to the participants’ baseline P/B ratio (n=29)

P/B ratio Aweight (kg) 95% Cl

1.27* 0.45; 2.10

1.15* 0.38; 1.92

^L3 1.02* 0.30; 1.74 2 0.90* 0.22; 1.57

0.77* 0.14; 1.40

^Gd 0.64* 0.04; 1.25

^09 0.52* -0.07; 1.10

^08 039 -0.18; 0.97 -0.7 0.27 -0.31; 0.85

-0.6 0.14 -0.46; 0.74

-0.5 0.01 -0.61; 0.64

-0.4 -0.11 -0.77; 0.55

-0.3 -0.24 -0.94; 0.46

-0.2 -0.36 -1.12; 0.39

-0.1 -0.49 -1.30; 0.32

0.0 -0.62 -1.49; 0.25

0.1 -0.74 -1.68; 0.19

0.2 -0.87 -1.87; 0.13

0.3 -1.00 -2.06; 0.08

0.4 -1.12 -2.26; 0.02

0.5 -1.25* -2.46; -0.03

0.6 -1.37* -2.66; -0.09

0.7 -1.50* -2.86; -0.14

0.8 -1.62* -3.06; -0.19

0.9 -1.75* -3.27; -0.24

Estimated body weight change in kg between the AXOS and the PUFA intervention and 95% confidence interval was conducted using the predictions from the linear mixed model fitted to body weight and Prevotella-to-Bacteroides ratio and diet interactions including age, gender and baseline body mass index as fixed effects and subject as random effects. * Indicates a P-value < 0.05. Abbreviation: P/B, Prevotella- to-Bacteroides.

Conclusion

Intake of AXOS for 4 weeks in a weight maintenance study resulted in a significant body weight change when comparing intervention differences (AXOS and PUFA) between the high- and low P/B groups. Interestingly, these results were further confirmed using baseline P/B ratio continuously as a predictor for body weight change instead of the stratified approach. Here we were able to predict an individualized treatment effect of both the AXOS and PUFA supplements. We found that an average weight loss between 1.17-1.75 kg could be predicted for participants with a baseline P/B ratio > 0.44 whereas a baseline P/B ratio of < -0.97 could predict an average weight gain of 0.61-1.27 kg when choosing AXOS over PUFA. Overall findings suggest that intake of AXOS positively affect weight loss in participants with a high P/B ratio but not participants with a low P/B ratio when compared to a control supplement.

References Sanchez Jl, Marzorati M, Grootaert C, Baran M, Craeyveld VV, Courtin CM, et al. Arabinoxylan-oligosaccharides (AXOS) affect the protein/carbohydrate fermentation balance and microbial population dynamics of the Simulator of Human Intestinal Microbial Ecosystem. Microb Biotechnol. 2009;2(1): 101— 13. Roager HM, Licht TR, Poulsen SK, Larsen TM, Bahl Ml. Microbial enterotypes, inferred by the prevotella-to-bacteroides ratio, remained stable during a 6-month randomized controlled diet intervention with the new nordic diet. Appl Environ Microbiol. 2014 Feb;80(3):1142-9.

Chambers ES, Viardot A, Psichas A, Morrison DJ, Murphy KG, Zac-Varghese SEK, et al. Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults. Gut. 2015;64(11): 1744- 54.

Claims

1. A diet comprising at least 8 g/d arabinoxylan oligosaccharides (AXOS) for use in a therapeutic method for promoting weight loss in an individual in need thereof having a P/B ratio (Iog10 (Prevotella spp./Bacteroides spp.)) equal to or greater than -0.8 in its microbiota.

2. A diet comprising at least 8 g/d AXOS for use in a therapeutic method for promoting weight maintenance in an individual in need thereof having a P/B ratio equal to or greater than -0.8 in its microbiota.

3. A diet comprising at least 8 g/d AXOS for use in a therapeutic method for reducing appetite in an individual in need thereof having a P/B ratio equal to or greater than -0.8 in its microbiota.

4. A non-therapeutic method for promoting weight loss in an individual in need thereof having a P/B ratio equal to or greater than -0.8 in its microbiota, the method comprising administering to said individual a diet comprising at least 8 g/d arabinoxylan oligosaccharides (AXOS).

5. A non-therapeutic method for promoting weight maintenance in an individual in need thereof having a P/B ratio equal to or greater than -0.8 in its microbiota, the method comprising administering to said individual a diet comprising at least 8 g/d AXOS.

6. A non-therapeutic method for reducing appetite in an individual in need thereof having a P/B ratio equal to or greater than -0.8 in its microbiota, the method comprising administering to said individual a diet comprising at least 8 g/d AXOS.

7. The method according to any one of the preceding claims, wherein the P/B ratio is equal to or greater than -0.4, such as equal to or greater than 0.5.

8. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the method comprises the steps of: a) obtaining a biological sample from said individual; b) determining the P/B ratio in said sample; and c) administering to said individual a diet comprising at least 8 g/d AXOS to said individual if the P/B ratio determined in step b)is equal to or greater than -0.8.

9. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the diet comprising at least 8 g/d AXOS is administered daily for at least 4 consecutive weeks, such as at least 8 consecutive weeks, for example at least 10 consecutive weeks, such as at least 14 consecutive weeks.

10. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the at least 8 g/d AXOS are administered in one daily dose, in two daily doses, or in three or more daily doses.

11. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the AXOS is obtained from bran extract from one or more cereal grains, such as from one or more of rye, wheat, barley, oats, sorghum, maize, millet, psyllium, flaxseed, pangola grass, bamboo shoot and rye grass.

12. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the AXOS has a arabinose to xylose ratio in the range of 0.1 -0.8, such as in the range of 0.1 -0.6, for example in the range of 0.1 -0.4, such as in the range of 0.1- 0.3, for example in the range of 0.15-0.3, such as in the range of 0.17-0.25.

13. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the AXOS has an average degree of polymerization in the range of 2-20, for example in the range of 2-18, such as in the range of 2-15, for example in the range of 2-12, such as in the range of 2-10, such as in the range of 3-9, for example in the range of 4-8, such as in the range of 4-7, for example in the range of 4-6, such as 5.

14. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the at least 8 g/d AXOS is administered in the form of a supplement, such as a beverage supplement and/or a food supplement.

15. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to claim 14, wherein the supplement comprises in the range of 0.4-1 g of AXOS/g, for example in the range of 0.5-1 g of AXOS/g, such as in the range of 0.6-0.9 g of AXOS/g, for example in the range of 0.6-0.8 g of AXOS/g.

16. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the at least 8 g/d AXOS is in powder form and is added to a beverage and/or a food preparation prior to consumption.

17. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the at least 8 g/d AXOS is administered in a capsule, such as in an enterocoated capsule.

18. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of claims 8 to 17, wherein the biological sample is a fecal sample or an intestinal sample.

19. The diet comprising at least 8 g/d AXOS for use in a therapeutic method or the non-therapeutic method according to any one of the preceding claims, wherein the individual has a body mass index of more than 23 kg/m², such as more than 25 kg/m², such as more than 30 kg/m², for example more than 35 kg/m², such as more than 40 kg/m².