WO2019202329A1

WO2019202329A1 - Microbiome modulated response to dietary nitrate

Info

Publication number: WO2019202329A1
Application number: PCT/GB2019/051106
Authority: WO
Inventors: Paul Graham Winyard; Anni VANHATALO; Andrew Jones; Mark van der GIEZEN
Original assignee: University Of Exeter
Priority date: 2018-04-20
Filing date: 2019-04-18
Publication date: 2019-10-24
Also published as: GB201806437D0

Abstract

There is provided a method for improving a response of an individual to a dose of dietary nitrate, comprising one or more of (a) reducing the prevalence of the total bacteria in the oral microbiome of the individual of the phylum Bacteroidetes, optionally of the genus Prevotella,optionally of the species Prevotella melaninogenica; (b) reducing the prevalence of the total bacteria in the oral microbiome of the individual of the phylum Firmicutes, optionally of the genus \/eillonella,optionally of the species Veillonella parvula;(c) increasing the prevalence of the total bacteria in the oral microbiome of the individual of the phylum Actinobacteria, optionally of the genus Rothia,optionally of the species Rothia mucilaginosa;(d) increasing the prevalence of the total bacteria in the oral microbiome of the individual of the species Fusobacterium nucleatum, optionally of the species Fusobacterium nucleatumsubsp.vincentii,optionally of the species Fusobacterium nucleatumsubsp.nucleatum;(e) increasing the prevalence of the total bacteria in the oral microbiome of the individual of the phylum Proteobacteria, optionally of the genus Neisseria,optionally of the species Neisseria meningitidis; (f) decreasing the ratio of prevalence in the individual's oral microbiome of total bacteria of the phylum Bacteroidetes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria; (g) decreasing the ratio of prevalence in the individual's oral microbiome of total bacteria of the phylum Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria; and/or (h) decreasing the ratio of prevalence in the individual's oral microbiome of total bacteria of the phyla Bacteroidetes plus Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phyla Proteobacteria plus Actinobacteria. The response to dietary nitrate is a change in the individual after an intake of the dose of dietary nitrate of one or more of a) systolic blood pressure; b) diastolic blood pressure; c) arterial stiffness; d) mean arterial pressure; and/or e) plasma nitrite concentration; and/or wherein the response is the ratio between plasma nitrite concentration and dietary nitrate dose per kg body mass of the individual ([N0 ₂ -]/N0 ₃ - dose).

Description

MICROBIOME MODULATED RESPONSE TO DIETARY NITRATE

FIELD OF THE INVENTION

The invention relates to the impact of the relative proportions of various bacteria within the oral microbiome of an individual on the ability of the individual to utilise dietary nitrate.

BACKGROUND

Inorganic nitrate (NO3 ) is a natural part of the human diet that is found in high concentrations in many vegetables. NCb itself is biologically inert and human cells are believed to lack NO3 - reductase capability. However, commensal bacteria in the oral cavity can use it as a terminal electron acceptor for ATP synthesis, reducing NO3 to nitrite (NO₂ ; which can be vasoactive in low-oxygen and low-pH conditions) and this NO₂ can be further reduced to the potent vasodilator, nitric oxide (NO) (Dejam et al. 2004). The NO3 - NO₂ -NO reduction pathway underpins the discovery that dietary NO3 supplementation through consumption of N03 salts (Larsen et al. 2006) or vegetable products such as beetroot juice (Kelly et al. 2013; Webb et al. 2008) reduces blood pressure (BP) in healthy young and old humans.

The importance of a functioning oral microbiome for the NO3 - NO₂ -NO reduction pathway is highlighted in cases where use of antibacterial mouthwash markedly blunts the increase in plasma and saliva NO2 concentrations and associated decrease in BP following ingestion of a standardised NO3 dose (Govoni et al. 2008; Kapil et al. 2013; McDonagh et al. 2015).

Epidemiological studies also indicate that dysbiosis of the oral microbial community is associated with poor cardiovascular health (Briskey et al. 2016). Conversely, a diet rich in vegetables, which contain high concentrations of inorganic NO3 , significantly protects against both coronary heart disease and stroke (Bondonno et al. 2017; Hung et al. 2004; Joshipura et al. 2009). Ageing has been associated with reduced salivary flow rate ('dry mouth') and altered oral bacterial colonisation (Percival et al. 1991), but it is not known whether the abundances of NO3 reducing oral bacteria decline with age. Dietary NO3 intake, and the abundance of N03 -reducing oral bacteria, therefore represent routes to lower blood pressure and maintain and improve cardiovascular health across the human lifespan.

It is possible that dietary NO3 as a prebiotic treatment might promote proliferation of NO3 - redudng bacteria. In a rodent model, a N03 -rich diet over 7 days increased abundance of oral bacteria ( Streptococcus and Haemophilus ) that contain NO3 reductase genes (Hyde et al. 2014a). In saliva samples of hypercholesterolaemic humans, 6 weeks of NO₃ supplementation with beetroot juice increased the abundance of Neisseria flavescens and Rothia mudiaginosa which are known NO3 reducers (Velmurugan et al. 2016). These studies indicate that increased dietary NO3 intake may alter the oral microbiome in a way which enhances an individual's ability to reduce ingested NO3 , resulting in greater plasma NO2 concentration and a greater reduction in systemic blood pressure. However, characterisation of potential changes in the oral microbiome of healthy young and old humans in response to NO3 supplementation is lacking.

SUMMARY OF THE INVENTION

A first aspect of the invention provides a method for improving a response of an individual to a dose of dietary nitrate, comprising decreasing the prevalence in the individual's oral microbiome of the total bacteria of the phylum Bacteroidetes. The method may comprise decreasing the prevalence in the individual's oral microbiome of the total bacteria of the genus Prevotella, optionally of the species Prevotella melaninogenica. The method according to the first aspect of the invention may be conducted in combination with any one or more of the second to eighth aspects of the invention.

The term "response of an individual to a dose of dietary nitrate", as used throughout the present application in relation to any aspect of the invention, indicates the change in one or more parameters measured before and after the individual has received a source of nitrate via enteric administration, typically by eating or drinking a source of nitrate. This may be a food such as a leafy green vegetables including brassicas such as rocket and broccoli, salad greens, beets, spinach and potato, as well as rhubarb; or may be a juice derived from or containing any of these. Alternatively, this may be a dietary supplement such as beetroot juice or any other nitrate-containing material such as a nitrate salt, for example an alkali metal nitrate solution, such as a potassium nitrate solution. The "dose of dietary nitrate" referred to herein is the amount of nitrate contained within the source of nitrate.

The parameters to be measured to determine the response to a dose of dietary nitrate, in relation to any aspect of the invention, may be selected from (but are not limited to) the following : a) systolic blood pressure; b) diastolic blood pressure c) arterial stiffness (for example measure by pulse wave velocity); d) mean arterial pressure; e) plasma nitrite concentration; and/or f) plasma nitrate concentration. The response may alternatively or additionally be measured as the ratio between plasma nitrite concentration and dietary nitrate dose per kg body mass of the individual ("[N0₂ ]/N0₃ dose").

The term "prevalence in the individual's oral microbiome of the total bacteria", as used throughout this specification in relation to any aspect of the invention, indicates the abundance (i.e., proportion or prevalence) of the phylum, order, genus or species of bacteria referred to, within the overall abundance of all types of bacteria which are detectable in an oral sample obtained from the individual, an "oral sample" being a sample of saliva or a mouth swab sample such as a tongue or a cheek swab sample. The types of bacteria present within such a sample may be determined by routine methods as described elsewhere herein, for example using a 16S rRNA gene amplification and detection technique. For example, a given genus of bacteria, such as Prevotella, may form 25% of the total bacteria within an oral sample obtained prior to the method according to the invention being carried out on the individual, rising to 30% of the total bacteria in an equivalent (i.e., obtained using the same method) oral sample obtained after the method has been carried out. In this hypothetical example, the prevalence in the individual's oral microbiome of Prevotella has increased. The prevalence of total bacteria of the genus Prevotella would include bacteria of all Prevotella species, whereas the prevalence of total bacteria of species Prevotella melaninogenica would include only bacteria of this species and not other species of Prevotella. Likewise, the prevalence of total bacteria of the phylum Bacteroidetes would include all bacteria of this phylum, not limited to Prevotella bacteria.

Prevotella is a genus of bacteria known to reduce nitrate to nitrite, so high abundances of these bacteria were expected in the art to be associated with improved responses to dietary nitrate supplementation. The inventors in fact found that a high abundance of these bacteria was associated with a less favourable response to dietary nitrate supplementation.

The prevalence of total bacteria of the phylum Bacteroidetes and/or the prevalence of the genus Prevotella and/or the prevalence of the species Prevotella melaninogenica may be decreased prior to intake by the individual of the dose of dietary nitrate. For example, this may be achieved by administration to the individual of an anti-Bacteroidetes and/or anti -Prevotella and/or anti -Prevotella melaninogenica compound, and/or by administration of a prebiotic or probiotic capable of reducing the prevalence in the individual's oral microbiome of total bacteria of the phylum Bacteroidetes and/or genus Prevotella and/or Prevotella

melaninogenica. Anti-Bacteroidetes and/or ant\-Prevotella and/or anti -Prevotella

melaninogenica compounds include compounds that kill or prevent or slow the growth of bacteria of the phylum Bacteroidetes and/or genus Prevotella and/or Prevotella melaninogenica. The compounds may act specifically on bacteria of the phylum Bacteroidetes and/or genus Prevotella and/or Prevotella melaninogenica, killing or preventing or slowing the growth of only those bacteria, or may also act on other bacteria. Where they act on other bacteria, the compounds preferably act more efficaciously on bacteria of the phylum

Bacteroidetes and/or genus Prevotella and/or Prevotella melaninogenica, that is to say the compounds kill or prevent or slow the growth of those bacteria more than other bacteria, especially other bacteria found in the oral cavity.

Alternatively or additionally, the prevalence of total bacteria of the genus Prevotella and/or the prevalence of the species Prevotella melaninogenica may be decreased concurrently with intake by the individual of the dose of dietary nitrate. This may be achieved by concurrent intake of the compound, prebiotic or probiotic referred to in the preceding paragraph, or may be achieved by the effect of consuming the dose of dietary nitrate, for example in the form of beetroot juice or a nitrate-containing material such as a potassium nitrate solution.

In the first aspect, the method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in plasma nitrite concentration and/or systolic blood pressure and/or pulse wave velocity in response to the dose of dietary nitrate in the individual, the method comprising decreasing the prevalence in the individual's oral microbiome, prior to intake by the individual of the dose of dietary nitrate, of the total bacteria of the genus Prevotella and/or of the species Prevotella melaninogenica.

The method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in plasma nitrite concentration and/or plasma nitrate concentration and/or [NO2Ί/NO3 dose in response to the dose of dietary nitrate in the individual, the method comprising decreasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the phylum Bacteroidetes.

The method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in plasma nitrite concentration and/or plasma nitrate concentration in response to the dose of dietary nitrate in the individual, the method comprising decreasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the genus Prevotella and/or of the species Prevotella melaninogenica.

According to the second aspect of the invention, there is provided a method for improving a response of an individual to a dose of dietary nitrate, comprising decreasing the prevalence in the individual's oral microbiome of the total bacteria of the phylum Firmicutes. The method may comprise decreasing the prevalence in the individual's oral microbiome of the total bacteria of the genus Veillonella, optionally of the species Veillonella parvula. The method according to the second aspect of the invention may be conducted in combination with any one or more of the first or third to eighth aspects of the invention.

Veillonella is a genus of bacteria known to reduce nitrate to nitrite, so high abundances of these bacteria were expected in the art to be associated with improved responses to dietary nitrate supplementation. The inventors in fact found that a high abundance of these bacteria was associated with a less favourable response to dietary nitrate supplementation.

The prevalence of total bacteria of the phylum Firmicutes and/or the prevalence of the genus Veillonella and/or the prevalence of the species Veillonella parvula may be decreased prior to intake by the individual of the dose of dietary nitrate. For example, this may be achieved by administration to the individual of an anti-Firmicutes and/or anti -Veillonella and/or anti- Veillonella parvula compound, and/or by administration of a prebiotic or probiotic capable of reducing the prevalence in the individual's oral microbiome of total bacteria of the phylum Firmicutes and/or genus Veillonella and/or Veillonella parvula. Anti-Firmicutes and/or anti- Veillonella and/or anti-Veillonella parvula compounds include compounds that kill or prevent or slow the growth of bacteria of the phylum Firmicutes and/or genus Veillonella and/or

Veillonella parvula. The compounds may act specifically on bacteria of the phylum Firmicutes and/or genus Veillonella and/or Veillonella parvula, killing or preventing or slowing the growth of only those bacteria, or may also act on other bacteria. Where they act on other bacteria, the compounds preferably act more efficaciously on bacteria of the phylum Firmicutes and/or genus Veillonella and/or Veillonella parvula, that is to say the compounds kill or prevent or slow the growth of those bacteria more than other bacteria, especially other bacteria found in the oral cavity.

Alternatively or additionally, the prevalence of total bacteria of the genus Veillonella and/or the prevalence of the species Veillonella parvula may be decreased concurrently with intake by the individual of the dose of dietary nitrate. This may be achieved by concurrent intake of the compound, prebiotic or probiotic referred to in the preceding paragraph, or may be achieved by the effect of consuming the dose of dietary nitrate, for example in the form of beetroot juice or a nitrate-containing material such as a potassium nitrate solution.

In the second aspect, the method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in plasma nitrite concentration and/or systolic blood pressure in response to the dose of dietary nitrate in the individual, the method comprising decreasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the phylum Firmicutes and/or the genus Veillonella and/or of the species Veillonella parvula. The method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in plasma nitrate concentration in response to the dose of dietary nitrate in the individual, the method comprising decreasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the genus Veillonella and/or of the species Veillonella parvula.

According to the third aspect of the invention, there is provided a method for improving a response of an individual to a dose of dietary nitrate, comprising increasing the prevalence in the individual's oral microbiome of the total bacteria of the phylum Actinobacteria, for example of the orders Actinomycetales and/or Micrococcales. The method may comprise increasing the prevalence in the individual's oral microbiome of the total bacteria of the genus Rothia, optionally of the species Rothia mucilaginosa. The method according to the third aspect of the invention may be conducted in combination with any one or more of the first, second or fourth to eighth aspects of the invention.

The prevalence of total bacteria of the phylum Actinobacteria and/or the prevalence of the genus Rothia and/or the prevalence of the species Rothia mucilaginosa may be increased prior to intake by the individual of the dose of dietary nitrate. For example, this may be achieved by administration to the individual of a prebiotic or probiotic capable of increasing the prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria and/or genus Rothia and/or Rothia mucilaginosa.

Alternatively or additionally, the prevalence of total bacteria of the genus Rothia and/or the prevalence of the species Rothia mucilaginosa may be increased concurrently with intake by the individual of the dose of dietary nitrate. This may be achieved by concurrent intake of the prebiotic or probiotic referred to in the preceding paragraph, or may be achieved by the effect of consuming the dose of dietary nitrate, for example in the form of beetroot juice or a nitrate- containing material such as a potassium nitrate solution.

In the third aspect, the method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in systolic blood pressure in response to the dose of dietary nitrate in the individual, the method comprising increasing the prevalence in the individual's oral microbiome, prior to intake by the individual of the dose of dietary nitrate, of the total bacteria of the phylum Actinobacteria, for example of the orders Actinomycetales and/or Micrococcales. The method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in plasma nitrite concentration and/or plasma nitrate concentration in response to the dose of dietary nitrate in the individual, the method comprising increasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the genus Rothia and/or of the species Rothia mucilaginosa.

According to the fourth aspect of the invention, there is provided a method for improving a response of an individual to a dose of dietary nitrate, comprising increasing the prevalence in the individual's oral microbiome of the total bacteria of the species Fusobacterium nudeatum. The method may comprise increasing the prevalence in the individual's oral microbiome of the total bacteria of the species Fusobacterium nudeatum subsp. vincentii and/or the species Fusobacterium nudeatum subsp. nudeatum. The method according to the fourth aspect of the invention may be conducted in combination with any one or more of the first to third, or fifth to eighth aspects of the invention.

The prevalence of total bacteria of the species Fusobacterium nudeatum subsp. vincentii may be increased prior to intake by the individual of the dose of dietary nitrate. For example, this may be achieved by administration to the individual of a prebiotic or probiotic capable of increasing the prevalence in the individual's oral microbiome of total bacteria of the species Fusobacterium nudeatum subsp. vincentii.

Alternatively or additionally, the prevalence of total bacteria of the species Fusobacterium nudeatum subsp. vincentii and/or the species Fusobacterium nudeatum subsp. nudeatum may be increased concurrently with intake by the individual of the dose of dietary nitrate. This may be achieved by concurrent intake of the prebiotic or probiotic referred to in the preceding paragraph, or may be achieved by the effect of consuming the dose of dietary nitrate, for example in the form of beetroot juice or a nitrate-containing material such as a potassium nitrate solution.

In the fourth aspect, the method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in [N0₂ ]/NC>3 dose and/or systolic blood pressure in response to the dose of dietary nitrate in the individual, the method comprising increasing the prevalence in the individual's oral microbiome, prior to intake by the individual of the dose of dietary nitrate, of the total bacteria of the species Fusobacterium nudeatum subsp. nudeatum.

The method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in [I\I02 ]/I\IC>3 dose and/or diastolic blood pressure and/or systolic blood pressure and/or mean arterial pressure and/or pulse wave velocity in response to the dose of dietary nitrate in the individual, the method comprising increasing the prevalence in the individual's oral microbiome, prior to intake by the individual of the dose of dietary nitrate, of the total bacteria of the species Fusobacterium nucleatum subsp. vincentii.

According to the fifth aspect of the invention, there is provided a method for improving a response of an individual to a dose of dietary nitrate, comprising increasing the prevalence in the individual's oral microbiome of the total bacteria of the phylum Proteobacteria. The method may comprise increasing the prevalence in the individual's oral microbiome of the total bacteria of the genus Neisseria, optionally of the species Neisseria meningitidis. The method according to the second aspect of the invention may be conducted in combination with any one or more of the first to fourth, or sixth to eighth aspects of the invention.

The prevalence of total bacteria of the phylum Proteobacteria and/or the prevalence of the genus Neisseria and/or the prevalence of the species Neisseria meningitidis may be increased prior to intake by the individual of the dose of dietary nitrate. For example, this may be achieved by administration to the individual of a prebiotic or probiotic capable of increasing the prevalence in the individual's oral microbiome of total bacteria of the phylum Proteobacteria and/or genus Neisseria and/or Neisseria meningitidis.

Alternatively or additionally, the prevalence of total bacteria of the phylum Proteobacteria and/or of the genus Neisseria and/or the prevalence of the species Neisseria meningitidis may be increased concurrently with intake by the individual of the dose of dietary nitrate. This may be achieved by concurrent intake of the prebiotic or probiotic referred to in the preceding paragraph, or may be achieved by the effect of consuming the dose of dietary nitrate, for example in the form of beetroot juice or a nitrate-containing material such as a potassium nitrate solution.

In the fifth aspect, the method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in plasma nitrite concentration and/or plasma nitrate concentration in response to the dose of dietary nitrate in the individual, the method comprising increasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the phylum Proteobacteria and/or the genus Neisseria and/or of the species Neisseria meningitidis. The method may be a method for improving a response of an individual to a dose of dietary nitrate, determined by an increased change in [IM0₂ ]/N03 dose in response to the dose of dietary nitrate in the individual, the method comprising increasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the phylum Proteobacteria.

According to the sixth aspect of the invention, there is provided a method for improving a response of an individual to a dose of dietary nitrate, comprising, concurrently with intake by the individual of the dose of dietary nitrate, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum

Bacteroidetes : prevalence in the individual's oral microbiome of total bacteria of the phylum Acti nobacteria.

The method according to the sixth aspect of the invention may be conducted in combination with any one or more of the first to fifth, seventh or eighth aspects of the invention. The method may comprise promoting a greater change in concentration of plasma nitrite and/or concentration of plasma nitrate and/or [NO₂Ί/NO3 dose in an individual in response to the dose of dietary nitrate.

According to the seventh aspect of the invention, there is provided a method for improving a response of an individual to a dose of dietary nitrate, comprising, concurrently with intake by the individual of the dose of dietary nitrate, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phylum

Actinobacteria.

The method according to the seventh aspect of the invention may be conducted in combination with any one or more of the first to sixth or eighth aspects of the invention. The method may comprise promoting a greater change in concentration of plasma nitrite and/or concentration of plasma nitrate in an individual in response to the dose of dietary nitrate.

According to the eighth aspect of the invention, there is provided a method for improving a response of an individual to a dose of dietary nitrate, comprising, concurrently with intake by the individual of the dose of dietary nitrate, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phyla Bacteroidetes plus Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phyla Proteobacteria plus Actinobacteria.

The method according to the eighth aspect of the invention may be conducted in combination with any one or more of the first to seventh aspects of the invention. The method may comprise promoting a greater change in concentration of plasma nitrite and/or concentration of plasma nitrate in an individual in response to the dose of dietary nitrate.

According to a ninth aspect of the invention, there is provided a method of predicting a response of an individual (a "subject individual") to dietary nitrate, comprising determining the prevalence in the individual's oral microbiome of the total bacteria of the genus Prevotella and/or of the total bacteria of the order Actinomycetales and/or bacteria of the species Fusobacterium nucleatum.

The inventors have found that individuals with a higher baseline abundance of the order Actinomycetales and/or a higher abundance of the species Fusobacterium nucleatum

(subspecies nucleatum or vincentii ) have improved responses to a dose of dietary nitrate, particularly as assessed by [IMO₂ j/NCb dose, change in systolic and/or diastolic blood pressure and/or change in mean arterial pressure. The inventors have found that individuals with a higher baseline abundance of the species Prevotella melaninogenica have poorer responses to a dose of dietary nitrate, particularly as assessed by change in plasma nitrite concentration, systolic blood pressure and pulse wave velocity.

The method may further comprise comparing the prevalence in the individual's oral microbiome of the total bacteria of the genus Prevotella and/or of the total bacteria of the order Actinomycetales and/or bacteria of the species Fusobacterium nucleatum to the average prevalence of the same genus, phylum or species in a control group of individuals. The term "control group" is as is well understood in the art, meaning a group of individuals with similar characteristics to the subject individual. For example, the subject individual may share one or more characteristics with the individuals in the control group, the characteristics optionally selected from, for example (but not limited to), age, BMI (Body Mass Index), gender, geographical area of residence, ethnicity, diet or disease state (i.e. , the presence or absence of one or more diseases, such as a cardiovascular disease).

In the method, if the prevalence of the total bacteria of the genus Prevotella in the individual is higher than the average prevalence, the individual is predicted to have a lower response to dietary nitrate (for example, a smaller change in plasma nitrite concentration, systolic blood pressure and/or pulse wave velocity).

If the prevalence of the total bacteria of the order Actinomycetales and/or bacteria of the species Fusobacterium nucleatum in the individual is lower than the average prevalence, the individual is predicted to have a lower response to dietary nitrate (for example, a smaller change in systolic blood pressure). A tenth aspect of the invention provides a microbiome-altering composition for use in a method for improving a response of an individual to dietary nitrate, the microbiome-altering composition being capable of one or more of: a) reducing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Bacteroidetes, optionally of the genus Prevotella, optionally of the species Prevotella melaninogenica; b) reducing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Firmicutes, optionally of the genus Veiilonella, optionally of the species Veillonella parvula ; c) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Actinobacteria, optionally of the orders Actinomycetales and/or Micrococcales, optionally of the genus Rothia, optionally of the species Rothia mucilagmosa,- d) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the species Fusobacterium nucleatum, optionally of the species Fusobacterium nucleatum subsp. vincentii, optionally of the species Fusobacterium nucleatum subsp. nucleatum,· e) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Proteobacteria, optionally of the genus Neisseria, optionally of the species Neisseria meningitidis,· f) when administered to the individual, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum

Bacteroidetes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria; g) when administered to the individual, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phylum

Actinobacteria; h) when administered to the individual, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phyla

Bacteroidetes plus Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phyla Proteobacteria plus Actinobacteria.

The method according to the tenth aspect may be or form part of a method for the treatment or amelioration of a disease in an individual, the disease involving endothelial dysfunction, ischaemia, hypoxia-reperfusion events, metabolic syndrome, hypertension or inflammation. The microbiome-altering composition, and compounds useful in any other aspect of the invention, when capable of decreasing the prevalence of a phylum, genus and/or species, may be, for example, an antibiotic or a pre- or pro-biotic composition capable of decreasing the prevalence of bacteria in the phylum, genus and/or species. The microbiome-altering composition, and compounds useful in any other aspect of the invention, when capable of increasing the prevalence of a phylum, genus and/or species, may be a pre- or pro-biotic or other composition capable of increasing the prevalence of bacteria in the phylum, genus and/or species. In some embodiments, the microbiome-altering composition may be a nitrate- containing composition such as a nitrate solution (such as an alkali metal nitrate solution, optionally a potassium nitrate solution), or a beetroot juice.

An eleventh aspect of the invention provides a composition for use in a method for the treatment or amelioration of a disease in an individual, the disease involving endothelial dysfunction, ischaemia, hypoxia-reperfusion events, metabolic syndrome, hypertension or inflammation, the individual previously having been administered a microbiome-altering composition defined as in accordance with the tenth aspect of the invention.

A twelfth aspect of the invention provides a method for the treatment or amelioration of a disease in an individual, the disease involving endothelial dysfunction, ischaemia, hypoxia- reperfusion events, metabolic syndrome, hypertension or inflammation, the individual previously having been administered a microbiome-altering composition to improve a response of the individual to dietary nitrate, the microbiome-altering composition being defined as in accordance with the tenth aspect of the invention.

A thirteenth aspect of the invention provides a database comprising: a) a prevalence figure of a bacterial phylum, order, genus or species in the oral

microbiome in an individual; b) an indication of a response of the individual after administration to the human

individual of a dose of dietary nitrate. The phylum may be selected from Bacteroidetes, Firmicutes, Acti nobacteria, Proteobacteria or Firmicutes. The order may be Actinomycetales or Micrococcales. The genus may be selected from Prevotella, Veillonella, Rothia or Neisseria. The species may be selected from Prevoteila melaninogenica , Veillonella parvula, Rothia muciiaginosa, Fusobacterium nucleatum (optionally Fusobacterium nucleatum subsp. vincentii or Fusobacterium nucleatum subsp. nucleatum ), or Neisseria meningitidis.

The database may comprise the information of (a) and (b), for more than one individual, each more than one individual sharing at least one characteristic with all of the other individuals.

The database may comprise: i) a Prevotella prevalence figure of Prevotella genus bacteria in the oral microbiome in an individual; ii) an Actinomycetales prevalence figure of Actinomycetales order bacteria in the oral microbiome in the individual and/or a Fusobacterium nucleatum prevalence figure of Fusobacterium nucleatum species bacteria in the oral microbiome in the individual; iii) an indication of a response of the individual after administration to the human

individual of a dose of dietary nitrate.

The database may comprise the information of (i), (ii) and (iii) for more than one individual, each more than one individual sharing at least one characteristic with all of the other individuals.

In the database, the characteristic may optionally be selected from age, BMI, gender, geographical area of residence, ethnicity, diet or disease state. The "response of the individual" may be a change in the individual, after an intake of the dietary nitrate, of one or more of: systolic blood pressure, diastolic blood pressure, arterial stiffness (for example, measured by pulse wave velocity), mean arterial pressure, plasma nitrite concentration, and/or plasma nitrate concentration, or the response may be measured as the ratio between plasma nitrite concentration and dietary nitrate dose per kg body mass of the individual ([N0₂ ]/N0₃ dose).

A fourteenth aspect of the invention provides a computer-readable medium comprising a database according to the thirteenth aspect of the invention. A fifteenth aspect of the invention provides a device, optionally a computer, comprising a database according to the thirteenth aspect of the invention, or linked to a computer-readable medium according to the fourteenth aspect of the invention. A sixteenth aspect of the invention provides a method for obtaining a database according to the thirteenth aspect of the invention, the method comprising : a) determining the prevalence of total bacteria of a phylum, order, genus or species in the oral microbiome in an individual to provide the phylum, order, genus or species prevalence figure; and b) determining in the individual a first measurement of a parameter selected from one or more of systolic blood pressure, diastolic blood pressure, mean arterial pressure, arterial stiffness, mean arterial pressure, concentration of plasma nitrite and/or concentration of plasma nitrate; c) subsequently administering to the individual a dose of dietary nitrate; d) subsequently determining in the individual a second measurement of the parameter determined as the first measurement in (b); e) comparing the first and second measurements from (b) and (d) to provide a parameter change figure; and f) recording as a record in the database the microbiome/response information, which is the prevalence from (a) linked to the parameter change figure from (e).

The method may be conducted on more than one individual and the collective

microbiome/response information recorded in the database.

A seventeenth aspect of the invention provides a nitrate-containing dietary supplement for use in modulating an individual's microbiome, wherein modulation of the microbiome comprises one or more of the steps of: a) reducing the prevalence of the total bacteria in the oral microbiome of the

individual of the phylum Bacteroidetes, optionally of the genus Prevotella, optionally of the species Prevotella melaninogenica; b) reducing the prevalence, when administered to the individual, of the total

bacteria in the oral microbiome of the individual of the phylum Firmicutes, optionally of the genus Veillonella, optionally of the species Veillonella parvula ; c) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Actinobacteria, optionally of the genus Rothia, optionally of the species Rothia mucilaginosa ; d) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the species Fusobacterium nudeatum, optionally of the species Fusobacterium nudeatum subsp. vincentii, optionally of the species Fusobacterium nudeatum subsp. nudeatum; e) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Proteobacteria, optionally of the genus Neisseria, optionally of the species Neisseria meningitidis; f) decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Bacteroidetes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria; g) decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria; h) decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phyla Bacteroidetes plus Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phyla Proteobacteria plus Actinobacteria.

An eighteenth aspect of the invention provides a nitrate-containing dietary supplement for use in increasing an individual's oral nitrate reduction capacity.

As in other aspects of the invention, the nitrate-containing dietary supplement of the seventeenth and eighteenth aspects may comprise or consist of beetroot juice. It may further or alternatively comprise a nitrate salt, comprising for example inorganic nitrate (NO3 ), or potassium nitrate (KNCb), or sodium nitrate (NaNCh). In one embodiment, the dietary supplement is or comprises an alkali metal nitrate solution, particularly a potassium nitrate solution. The dietary supplement may also comprise or be for use in combination with compounds belonging in the following groups: polyphenols, and/or betacyanins, and/or phenolic acids, and/or flavonoids. Specific compounds belonging in these groups may include: Betanin-3-Oglucoside, betanidine, gallic acid, chlorogenic acid, caffeic acid, ferulic acid, ascorbic acid, rutinoside-3-O-quercetin, glucoside-3-O-quercetin, myrycetin, luteolin, quercetin, or kaempferol.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to" and do not exclude other components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers or characteristics described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose. BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described, by way of example only, with reference to Figures 1-8 which are as follows:

Beetroot Juice Supplementation Study

Figure 1: Participants underwent 10-day supplementation periods with nitrate (~12.4 mmol/d), in the form of beetroot juice supplement, and placebo in a balanced cross-over design. Screening, protocol familiarisation and Salivary Flow Rate Questionnaires (SFR-Q) were completed at baseline. Measurements of plasma nitrite ( NO2 ) and nitrate ( NO3^')

concentrations, blood pressure (BP) of the brachial artery, arterial stiffness as carotid-femoral pulse wave velocity (PWV), and the collection of saliva samples for microbiome analysis were undertaken on days 8, 9 and 10 of each supplementation period.

Figure 2: Plasma [ NO3^"] (panel A) and [ NO2 ] (panel B) were significantly greater after nitrate supplementation (white bars) compared to placebo (black bars) (n = 18). The change (D) in plasma [NO3 ] between nitrate and placebo conditions was similar in young (n=9) and old participants (n=9) (panel C), but D[N0₂ ^·] was significantly greater in the old compared to young participants. Error bars indicate standard deviations and black squares (panels C and D) indicate means for young and old participants. * P<0.05.

Figure 3: Mean arterial pressure (MAP; panel A), systolic blood pressure (SBP; panel D), diastolic blood pressure (DBP; panel G) and pulse wave velocity (PWV; panel J) were not different between placebo and nitrate conditions across all participants (n=18). The old participants (n=9) showed greater reductions (D) in MAP, SBP and DBP between placebo and nitrate conditions than the young participants (n=9; panels B, E and H), as well as a greater increase in PWV (young n=9, old n=8; panel K). DMAR, ASBP and ADBP inversely correlated with the change in plasma [NO2 ] relative to nitrate dose (D[N0₂ ]/N03 dose) (panels C, F and I) and APWV was positively correlated with D[Nq₂ ]/Nq3^~ dose (panel L). *P<0.05.

Figure 4: The proportions of five main phyla of oral bacteria identified in the saliva samples following placebo (PL) and NO3^' supplementation (BR). ^Difference between PL and BR (P<0.05).

Figure 5: Overall salivary microbiome composition illustrated by non-metric multidimensional scaling (NMDS) analysis. The salivary microbiome composition was different between nitrate (BR) and placebo (PL) conditions (P<0.05; panel A) but not between young and old participants (P>0.05; panel B).

Figure 6: The Shannon diversity index indicated no statistically significant difference in species diversity between nitrate (BR) and placebo (PL) conditions. Potassium Nitrate Supplementation Study

Figure 7: Oral nitrate reduction capacity at pre-supplementation baseline and following 5 days of KNO3 supplementation (n-7). The dashed lines indicate individual responses, the solid line indicates the group mean, and the error bars indicate standard deviation.

Figure 8: The overall tongue microbiome composition prior to KNO3 supplementation

(baseline; solid line, black symbols) and after 5 days of supplementation (dashed line, white symbols) illustrated by non-metric multidimensional scaling (NMDS) analysis.

EXAMPLES

In the present study, the inventors used 16S rRNA gene sequencing to investigate whether abundances of NCb -reducing bacteria on the surface of the human tongue modulate an individual's response to NO3 supplementation in young (18-22 years) and old (70-79 years) normotensive adults. It was hypothesised that at baseline, abundances of known NO3 - reducing bacteria (including Neisseria, Prevotella, Rothia, Veillonella and Actinomycetales) would be greater in young compared to old participants, and that high abundances of these bacteria at baseline would be associated with higher plasma NO2 concentrations, and greater changes in blood pressure and arterial stiffness in response to NO3 supplementation.

Secondly, the inventors investigated whether 10 days of regular dietary NO3 ingestion altered the oral microbiome compared with placebo supplementation. It was hypothesised that oral microbiomes would be different between placebo and IMO3 conditions, and specifically that the relative abundances of bacteria capable of NO3 reduction would be greater after NO3 compared to placebo supplementation.

1. METHODS - BEETROOT JUICE SUPPLEMENTATION STUDY

Ethical approval

The study was approved by the institutional Ethics Committee (Sport and Health Sciences, University of Exeter) and conducted in accordance with the code of the ethical principles of the World Medical Association ( Declaration of Helsinki) . All participants gave their written, informed consent before the commencement of the study, once the experimental procedures, associated risks, and potential benefits of participation had been explained.

Subjects

Nine old adults including six females (mean ± SD, age 75 ± 3 yrs, age range 70-79 yrs, height 162 ± 6 cm, body mass 61.8 ± 14.0 kg) and three males (age 73 ± 5 yrs, age range 70-78 yrs, height 172 ± 4 cm, body mass 77.7 ± 11.6 kg) and nine young adults including five females (age 20 ± 1 yrs, age range 19-22 yrs, height 168 ± 7 cm, body mass 67.9 ± 10.3 kg) and four males (age 20 ± 2 yrs, age range 18-22 yrs, height 180 ± 4 cm, body mass 73.4 ± 12.9 kg) volunteered to participate in this study (Table 1). The nine old adults represented a subsample of a larger cohort tested for a Dunhill Medical Trust funded project (R269/1112) from which the microbiome of the tongue and saliva were retrospectively analysed.

Participants were screened prior to participation to ensure suitability for the study. All participants were ostensibly healthy and were not taking medication or dietary supplements. None of the participants were tobacco smokers and all reported having no oral diseases.

Participants were instructed to arrive at the laboratory in a rested and fully hydrated state, at least 3 h postprandial, and to avoid strenuous physical exertion in the 24 h preceding each laboratory visit. Participants were also asked to refrain from caffeine and alcohol intake 6 and 24 h before each test, respectively. All tests were performed at approximately the same time of day (± 2 h) for each participant.

Experimental design

Prior to commencing dietary supplementation, participants visited the laboratory for health screening and familiarisation to test protocols. Participants completed a salivary flow rate questionnaire (SFR-Q; Fox et al. 1987). The SFR-Q included eleven questions asking the participant to rate the frequency of various symptoms of low salivary flow rate on a scale of 1 ('never') to 5 ('very often'). Participants then underwent two 10-day dietary supplementation periods with NO3 and placebo in a randomised, double-blind, cross-over design (Figure 1) . On days 8, 9 and 10 of each supplementation period, participants returned to the laboratory to perform physical and cognitive function tests. Upon the first arrival at the laboratory and on each subsequent visit venous blood samples were collected for the measurement of plasma [NO2 ] and [NO3 ] and resting BP was measured. The mean values of three measurements of [NO2 ], [NO3 ] and BP were used for further analyses. Saliva samples were also collected on each visit and three samples were pooled for analysis of the salivary microbiome. Arterial stiffness was assessed on one occasion (day 8, 9 or 10) using radial-femoral pulse wave velocity (PWV).

Supplementation

The supplements were NO3 -rich concentrated beetroot juice (BR) (2 x 70 ml-d ¹, each 70 ml containing _™6.2 mmol NO3 ; Beet It, James White Drinks, Ipswich, UK) and NO3 -depleted concentrated beetroot juice placebo (PL) (2 x 70 ml-d ¹, each 70 ml containing ~0.01 mmol NO3 ; Beet It, James White Drinks, Ipswich, UK). The PL was indistinguishable from the BR supplement in appearance, taste and smell . Participants were instructed to consume one 70-ml beverage in the morning and one in the afternoon. On testing days, participants were asked to ingest one 70-ml beverage in the morning and one 2.5 h prior to their laboratory visit. A washout period of at least three days and up to 47 days separated the supplementation periods. Participants were instructed to maintain their normal daily activities, food intake and oral hygiene regime throughout the study. However, participants were instructed to refrain from using antibacterial mouthwash during the study period. Participants were advised that supplementation may cause beeturia (red urine) and red stools temporarily, but that such side effects were harmless.

Oral bacteria

Oral swabs of the tongue dorsum were collected at baseline. Saliva samples (~ 1 ml_) were collected by expectoration, without stimulation, over a period of 5 min on three occasions following PL and BR supplementation periods. Oral swab and saliva samples were stored at -80°C until analysis. Genomic DNA was isolated from tongue swabs using a Gentra Puregene Buccal Cell Kit (Qiagen, Germantown, MD), and from saliva samples following the methods of Goode et al. (2014). Double-stranded DNA concentration was fluorometrically quantified (Qubit 3.0 high-sensitivity fluorescence detection, ThermoFisher Scientific, Waltham, MA). Library preparation employed a NEXTflex 16S V1-V3 Amplicon-Seq Kit (Bioo Scientific, Austin, USA) . The 16S V1-V3 rDNA region was amplified using 5ng of dsDNA and subjected to 8 thermal cycles of 30 s at 98°, 30 s at 60° and 30 s at 72° with primers A and B (Table SI). Following AMPure^® XP bead cleanup (Becton Dickinson, Franklin Lakes, NJ), a subsequent PCR with indexing primers to identify individual samples, containing Illumina flow cell binding sites, was performed. Paired-end 300 metagenomics next generation sequence analysis was performed on the MiSeq Illumina platform (Illumina, San Diego, CA) using v3 MiSeq reagents. Nucleotide sequence data in FASTQ format was trimmed using Trim-Galore! (Krueger F. Trim-Galore!, accessible at www.bioinformatics.babraham .ac.uk/projects/trim_galore/), and processed by the Kraken Taxonomic Sequence Classification System (Woods and Salzberg 2014). Variations in the V1-V3 regions enabled taxonomic identification which was visualised using Krona bioinformatics pie charts (Ondov et al. 2011).

Plasma [NO3^'] and [NO2·]

Blood samples for determination of plasma [NO2 ] and [NO3 ] were collected from an antecubital vein into lithium heparin tubes and centrifuged for 8 min at 3000 g and 4 °C within 2 min of collection. Plasma was extracted and samples stored at -80°C for later determination of [NO3 ] and [NO2 ] using a modified chemiluminescence technique as previously described (Kelly et al. 2013) .

Blood pressure and arterial stiffness

Blood pressure of the brachial artery was measured following 10 min of seated rest in a quiet room using an automated sphygmomanometer (Dinamap Pro, GE Medical Systems, Tampa, USA). A total of four measurements were taken, with the mean of the final three

measurements recorded. The mean of the systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) measurements made over three laboratory visits in each condition were calculated for each individual and used for subsequent analyses. Arterial stiffness was estimated via pulse-wave velocity (PWV) (Complior SP; Alam Medical, Vincennes, Paris,

France). Electrodes were placed on the carotid, femoral, and radial arteries, and the pulse transit time was calculated and recorded. A mean of three measurements was calculated and used for subsequent analyses. The position of each electrode was measured in relation to the nearest bony landmark to enable precise reproduction of the position of the electrodes in each condition.

Statistical analyses

The Kraken raw data output of phylogenetic data were analysed using R-script (R Development Core Team 2008), SPSS V20 and Microsoft Excel. Non-metric multidimensional scaling (NMDS) was used to assess the level of microbiome similarity between young and old, and PL and BR conditions using non-parametric relationships, and analysed using ADONIS (Vegan R

Software). Differences between PL and BR conditions were assessed using paired t-tests with Bonferroni-Hoechberg correction on bacteria that made up >0.01% of bacteria (R statistical software). The Shannon-Wiener diversity index (H') was used to explore differences in diversity (Vegan R Software). Paired-samples t-tests were used to assess differences between BR and PL conditions in plasma [NO2 ] and [NO3 ], BP and arterial stiffness. Relationships between plasma NO biomarkers, oral microbiome and physiological responses to

supplementation were assessed using Pearson's correlation coefficients. Statistical significance was accepted when P<0.05 and statistical trend was defined as P O.10. Data were expressed as mean ± SD.

2. RESULTS - BEETROOT JUICE SUPPLEMENTATION STUDY

The young and old participants were similar in terms of body mass and BMI (Table 1). The young participants had a greater mean score in SFR-Q than the old participants (Table 1), indicative of more frequent self-reported symptoms of low salivary flow rate. The young participants reported greater frequency of sensations associated with dry mouth (old 1.6 ± 1.0, young 2.7 ± 0.5; P<0.05) and having difficulty eating dry foods (old 1.1 ± 0.3, young 1.8 ± 0.4; P<0.05).

NO biomarkers, blood pressure and arterial stiffness

The NO3 dose relative to body mass was not different between young and old participants, but the latter had a greater increase in plasma [NO2 ] in response to BR supplementation (Table 1). Plasma [NO3 ] and [NO2 ] were significantly higher following BR supplementation compared with PL (all P<0.05; Figure 2, panels A and B). BR supplementation increased plasma [NO₃ ] relative to PL by a similar amount in old (1509 ± 744%) and young participants (1481 ±

909%) (Figure 2C), but the increase in [NO2 ] was greater in old (648 ± 477%) compared to young participants (365 ± 249%, P<0.05; Figure 2D). There were no differences between young and old participants in plasma [NO3 ] in PL (old : 28 ± 12 mM, young : 28 ± 14 mM ; P>0.05) or BR conditions (old : 379 ± 55 pM, young : 366 ± 101 pM ; P>0.05). Plasma [NO2 ] tended to be greater in the old in PL (old : 173 ± 97 nM, young : 104 ± 63 nM ; P=0.09) and was significantly greater in the old than young participants in the BR condition (old : 1029 ± 393 nM, young : 380 ± 175 nM; P<0.05).

MAP, SPB, DBP and PWV were not different between PL and BR conditions across all participants (n= 18, P>0.05; Figure 3, panels A, D, G and 3) . In old participants (n=9), SBP and MAP were significantly lower following BR supplementation compared with PL (Figure 3, panels B and E). Changes (D) in MAP, SBP, and DBP between PL and BR conditions inversely correlated with the change in plasma [IMO2 ] relative to NO3 dose per kg body mass (D[I\I02 ]/N0₃ dose; Figure 3, panels C, F and I). BR supplementation did not significantly alter radial- femoral PWV (Figure 3, panels J and K) across all participants, but APWV between BR and PL in the old participants (increase of 4.3 ± 10.9 m-s ¹; n=9) was different (P<0.05) from APWV in the young participants (decrease of -6.8 ± 9.8 rrvs ¹; n=9). APWV positively correlated with D[Nq₂ ]/Nq3 dose (Figure 3L). Absolute plasma [NO2 ] or [NO3 ] measured in the PL condition were not correlated with D[Nq2 ], DϋBR, ASBP, DMAR or D PWV. One old male participant did not wish to undertake PWV measurement and therefore all PWV data are derived from 17 participants.

Tongue microbiome at baseline

Relative abundances of the five main oral bacterial phyla in tongue swab samples collected at baseline were Bacteroidetes 32 ± 9%, Fusobacteriales 27 ± 11%, Proteobacteria 20 ± 7%, Firmicutes 17 ± 5% and Acti nobacteria 1 ± 1 %. The most abundant bacterial species found on the tongue were Fusobacterium nucleatum subsp. nudeatum (16 ± 8%), Prevotella melaninogenica (14 ± 7 %), Campylobacter condsus (13 ± 8%), Leptorichia buccal is, (7 ±

6%) Veillonella parvula (5 ± 3%), Prevotella intermedia (4 ± 2%), Fusobacterium nucleatum subsp. vincentii (3 ± 3%) and Neisseria meningitidis (3 ± 3%). Correlations between relative abundances of selected taxonomic units of tongue bacteria and physiological responses to BR supplementation are shown in Table 2. The greatest decreases in BP were associated with high abundances of Actinomycetales and Fusobacterium nucleatum subsp. vincentii at baseline, whereas a high relative abundance of Prevotella melaninogenica was associated with a greater mean score in SFR-Q, and smaller changes in plasma [NO2 ], SBP and PWV in response to BR supplementation (Table 2) . Saliva microbiome after PL and BR supplementation

Relative abundances of the main phyla of oral bacteria differed between PL and BR conditions (Figure 4). Relative abundance of Proteobacteria was greater and Bacteroidetes was lower following BR compared with PL (P<0.05), while abundances of Firmicutes and Fusobacteria tended to be lower following BR supplementation compared with PL (P O.lO). NMDS plots revealed that oral microbial communities differed significantly between PL and BR

supplemented conditions (Figure 5A), but there were no differences between young and old participants (Figure 5B). A Shannon diversity index revealed no significant differences in species diversity between PL and BR conditions (Figure 6). Overall, 52 taxonomic units were significantly different between BR and PL conditions. Table 4 illustrates statistically significant differences at genera and species levels between PL and BR. The trend for reduction in the relative abundance of Firmicutes following BR was primarily due to a decrease in Veillonella (-65%), including a 65% decrease in Veillonella parvula species (both P<0.05), while the order Lactobacilliales and genus Streptococcus were not affected by BR (P>0.05). Within the phylum Bacteroidetes, BR resulted in a reduction in the genus Prevotella (-60%), and specifically P. melaninogenica (-67%) compared to PL (both P<0.05). The increase in Proteobacteria after BR stemmed from an increase in the order Neisseriales (+348%), containing the genus Neisseria (+351%) and N. meningitidis (+439%) (all P<0.05), while there were no statistically significant changes in the genera Campylobacter or Haemophilus. Proportions of the phylum Actinobacteria were not significantly different between PL and BR, but there was an increase in the genus Rothia ( + 127%, P<0.05) and R. mucilaginosa (+234%, P<0.05) after BR supplementation relative to PL. There was insufficient microbial DNA in a saliva sample of one young male participant and therefore the saliva microbiome data were for 17 participants.

Correlations across PL and BR conditions showed that plasma [NCb ] and [NO2 ] positively correlated with relative abundances of Rothia and Neisseria and inversely correlated with Prevotella and Veillonella (Table 3). PWV positively correlated with relative abundance of Rothia and R. mucilaginosa (Table 3).

Phylum level correlations of oral bacteria with age, and indices of NO bioavailability and vascular function across placebo and NO3 supplemented conditions are shown in Table 5. Age was inversely correlated with the relative abundance of Actinobacteria (r=-0.48; P<0.05), and positively correlated with the relative abundance of Bacteroidetes (r=0.61; P<0.05) and the Bacteroidetes:Actinobacteria ratio (r=0.63; P<0.05). Plasma [NO3 ] was inversely correlated with the abundance of Bacteroidetes (r=-0.43; P<0.05), the ratio of

Bacteroidetes:Actinobacteria ratio (r=-0.39; P< 0.05), and the ratio of (Bacteroidetes + Firmicutes) : (Proteobacteria + Actinobacteria) (r=-0.39; P<0.05), and positively correlated with the abundance of Proteobacteria (r=0.58; PcO.Ol) . Plasma [NCh ] was positively correlated with the relative abundance of Proteobacteria (r= 0.62; PcO.Ol), and inversely correlated with the abundance of Firmicutes (r=-0.41 ; Pc0.05), and the ratio of (Bacteroidetes + Firmicutes): (Proteobacteria + Actinobacteria) (r=-0.35; Pc0.05) . D[Nq2 ]/Nq3 dose was positively correlated with relative abundance of Bacteroidetes (r=0.49; P O.Ol) and the

Bacteroidetes:Actinobacteria ratio (r=0.39; P<0.05). Among the indices of vascular function, the systolic blood pressure (SBP) was inversely correlated with the relative abundance of Firmicutes (r=-0.34; P<0.05) and the mean arterial pressure (MAP) was positively correlated with the Bacteroidetes:Actinobacteria ratio (r=0.35; P<0.05) . Furthermore, the pulse wave velocity (PWV) was positively correlated with the relative abundance of Actinobacteria (r=0.47; PcO.Ol).

(continued on page 28)

Table 1. Participant characteristics, nitrate (NO3 ) dose and plasma [NO2 ] responsiveness to supplementation, and saliva flow rate questionnaire (SFR-Q) results. The age groups were similar in terms of body mass and BMI. NO3 dose was similar in both groups but older participants had a greater increase than the young in plasma [NO2 ] in response to supplementation. The young reported feeling more frequent symptoms of low salivary flow rate than the older participants.

OLD

Sex Age Body BMI N0₃- dose A[N0₂-]/N0₃- SFR-Q (yrs) mass (kg/m²) (mmol/kg/d) dose mean

(kg) (nM/mmol/kg/d) score

10 F 22 71.5 29.4 0.17 803 1.9

11 F 19 60.8 22.9 0.20 2202 2.5

12 F 19 64.5 22.6 0.19 880 1.6

13 F 20 69.7 24.4 0.18 1604 2.7

14 F 19 85.2 28.5 0.15 999 1.5

15 M 18 55.7 18.0 0.22 2371 1.5

16 M 22 81.2 23.7 0.15 3254 1.6

17 M 19 72.3 22.3 0.17 1127 2.1

18 M 19 84.4 26.0 0.15 70 2.1

Mean 19.7 * 71.7 24.2 0.18 1479 * 2.0 *

SD 1.4 10.4 3.5 0.03 977 0.4

Mean 46.8 69.4 24.3 0.18 2857 1.7

SD 28.1 12.6 4.0 0.03 2079 0.5

F, female; M, male; BMI, body mass index; D[N02 ]/N0₃ dose, change in plasma [NOz ] relative to dose of NCU ingested per kg body mass; SFR-Q, salivary flow rate questionnaire; . ^Different from OLD, P<0.05. Table 2. Correlation coefficients for relationships between selected taxonomic units of tongue microbiome at baseline and changes in plasma [N02-], blood pressure and arterial stiffness in response to N03- supplementation.

D= change between placebo and NO3 ; [NO2Ί/NO3 dose = plasma [nitrite] relative to nitrate dose per kg body mass ingested; DBP=diastolic blood pressure; SBP= systolic blood pressure; MAP= mean arterial pressure; PWV= pulse wave velocity; SFR-Q= salivary flow rate questionnaire; **P<0.01, *P<0.05, #P< 0.10.

27

Table 3. Correlation coefficients (r) for relationships between relative abundances of selected taxonomic units of saliva microbiome (% of total bacteria) and plasma [N03-] and [N02-] ; diastolic (DBP), systolic (SBP) and mean arterial (MAP) blood pressure; and pulse wave velocity (PWV) across placebo and nitrate conditions. Microbiome data were not available for one young male subject and PWV data were not available for one older male subject, such that [N03-], [N02-] and BP correlations are for n=34 and PWV correlations are for n=32.

DBP=diastolic blood pressure; SBP= systolic blood pressure; MAP= mean arterial pressure; PWV= pulse wave velocity; **P<0.01, *P<0.05, #P<0.10. Table 4. The genera and species of salivary bacteria that comprised >0.01% of all bacteria and showed significant differences (P<0.05) between placebo (PL) and nitrate (BR) conditions.

Table 5. Correlation coefficients among the main phyla of oral bacteria and age, and indices of NO bioavailability, and vascular and cognitive function across placebo and N03- supplemented conditions. Saliva microbiome data were not available for one young male participant and PWV data were not available for one old male participant, such that [N03-], [N02-] and BP correlations are for n=34 and PWV correlations are for n=32. **P<0.01, * P<0.05, # P O. lO.

Bac:Act = ratio between relative abundances of Bacteroidetes and Actinobacteria

FinAct = ratio between relative abundances of Firmicutes and Actinobacteria

(B+F) : (P+A) = ratio between sum of relative abundances of Bacteroidetes and Firmicutes and sum of relative abundances of Proteobacteria and Actinobacteria

10 [N03 ]= plasma nitrate concentration; [N02^~] = plasma nitrite concentration; D[N02-]/N03- dose = change in plasma [nitrite]

relative to nitrate dose per kg body mass ingested; DBP=diastolic blood pressure; SBP= systolic blood pressure; MAP= mean arterial pressure; PWV= pulse wave velocity; EE VO2 = end-exercise oxygen uptake during moderate-intensity exercise; VO2 Amp = amplitude of the oxygen uptake response between baseline and end-exercise for moderate-intensity exercise.

3. DISCUSSION - BEETROOT JUICE SUPPLEMENTATION STUDY

We used an in vivo experimental model and bacterial 16S rRNA gene sequencing to examine relationships between the oral microbiome and physiological indices of NO bioavailability in humans and changes in these variables following NO3 supplementation. The principal finding of this study was that dietary NO3 supplementation altered the salivary microbiome in young (~20 yrs) and old (~74 yrs) normotensive humans, such that it increased relative abundances of some bacteria capable of N03 reduction ( Rothia and Neisseria ) while reducing the abundances of other NO3 reducers ( Prevotella and Veillonella). NO3 supplementation increased NO bioavailability in all participants, as indicated by plasma concentrations of NO3 and NO₂ , and reduced systemic blood pressure in the old, but not young, participants. Across placebo and NO3^· supplemented conditions, high abundances of Rothia and Neisseria and low abundances of Prevotella and Veillonella were associated with high NO bioavailability. The current findings indicate that the oral microbial community was malleable to change with increased dietary intake of inorganic NO3 , and, importantly, that the oral microbiome was related to indices of NO homeostasis and vascular health in vivo.

Relationships between tongue microbiome at baseline and responsiveness to NO3 supplementation

It has been proposed that the oral microbiome modulates the magnitude of plasma [NO2 ] increase, and changes in associated physiological indices, in response to NO3 supplementation (Bryan et al. 2017; Hyde et al. 2014ab; Koch et al. 2016). We found that individuals who had high proportions of Prevotella melaninogenica and Campylobacter concisus at baseline were less responsive to NO3 supplementation, i.e. had smaller increases in plasma [NCte^~] and smaller or no reductions in BP, than those individuals who had low abundances of P.

melaninogenica and C. concisus. In addition, a high mean SFR-Q score at baseline, which indicated more frequent self-reported symptoms of dry mouth, was associated with greater abundance of Prevotella and a smaller increase in plasma [NO2 ] in response to NO3 supplementation. Campylobacter concisus is believed to express dissimilatory NO3 reduction to NH₃ and its main physiological function Is N0₂ reduction (Simon & Klotz 2013), while P.

melaninogenica has been shown to encode NO2 , but not NO3 , reductase genes (Hyde et al. 2014b). It may, therefore, be speculated that during subsequent NO3 supplementation both C. concisus and P. melaninogenica , which were dominant species in tongue swab samples at baseline, may have acted as net consumers of N0₂ in the oral cavity. In contrast, high abundances of Actinomycetales and Fusobacterium nucleatum subspecies at baseline were associated with greater increases in plasma [N0₂ ] and greater reductions in blood pressure in response to NO3 supplementation (Table 2). Actinomycetales are generally obligate anaerobes, including several species such as Actinomyces odontolyticus and Actinomyces naeslundii that have been identified as effective NCb reducers (Doel et al. 2005). Fusobacterium nucleatum can reduce NO2 , but does not possess N03 -reductase genes.

However, F. nucleatum provides 'scaffolding' in biofilms, enabling microbial attachments (Kolenbrander et al. 2002), and it is possible that these bacteria may have facilitated attachment and proliferation of key NO3 reducing bacteria during subsequent dietary interventions.

Changes in saliva microbiome after NO3 supplementation

We showed that, relative to placebo, NO3 supplementation significantly increased relative abundances of the previously identified NO3 reducers Neisseria (Hyde et al. 2014b) and Rothia (Doel et al. 2005) in saliva. This was consistent with a report of a significant increase in N. flavescens and a trend for an increase in R. mucilaginosa in saliva samples of

hypercholesterolaemic patients after 6 weeks of NO3 supplementation (Velmurugan et al. 2015). A novel finding in the present study was that a high abundance of the facultative anaerobe R. mucilaginosa was associated with faster pulse wave velocity, indicative of lower arterial stiffness. These data suggest that high relative abundances of bacteria belonging to Neisseria and Rothia were related to high NO bioavailability and may promote vascular health.

We also found that compared to placebo, NO3 supplementation decreased relative abundances of the obligate anaerobic bacteria Prevotella and Veillonella in saliva. This finding appears to contradict studies that have used in vitro approaches to identify key oral NO3 reducing taxa. Hyde et al. (2014b) categorised biofilms prepared from tongue swab samples of six healthy humans as best, intermediate and worst NO3 reducers and found greater abundances of both Prevotella and Veillonella in the best versus worst O3^· reducing biofilms. Using tongue swab samples from ten healthy humans, which were incubated on solid medium under aerobic and anaerobic conditions and analysed by 16S rDNA sequencing, Doel et al. (2005) concluded that Veillonella were the most prevalent oral NO3 reducers and were major contributors to net NOS production. Intricate metabolic interactions among the oral microbiota might mean that increased O3· availability in the oral cavity may not facilitate the growth of all bacteria capable of NO3 reduction. It is not directly apparent why NO3 supplementation resulted in a decline in Veillonella. One factor that may have contributed to proliferation of some taxa and inhibition of others is the oral pH, which is a powerful modulator of the oral microbial community. Beetroot juice supplementation has been shown to increase oral pH from 7.0 to 7.5 (Hohensinn et al. 2016), and notably, a pH of 8 is optimal for NO3 reductase activity (van Maanen et al. 1996). Monitoring of salivary pH alongside alterations in the oral microbiome during NO3 supplementation, should be undertaken in future studies to address the possible effects of pH. Given that many oral bacteria with ability to reduce IMO3 are also capable of downstream metabolism of the produced NO2 , it is important to differentiate between NCb reducing bacteria in general and NO2 accumulating bacteria specifically. NO3 reducing oral bacteria have been identified in vitro from human samples, including Veillonella, Actinomyces , Rothia, Staphylococcus and Propionibacterium (Dole et al. 2005). More recently Hyde et al. (2014b) added Neisseria, Haemophilus parainfluenzae, Prevotalla (including P. melaninogenica ) and Granu/icatel/a to the list of candidate species for most potent contributors to oral NCh production. We showed that some of the oral bacteria that have been proposed as key NCh accumulators on the basis of in vitro experiments, such as Veillonella and Prevotelia, do not thrive under high NO3 availability in vivo. Indeed, we found no significant changes in relative abundances of Actinomyces, Staphylococcus, Propionibacterium, Granulicatella or Haemophilus after NO3 supplementation. In order to contribute significantly to the amount of NCh that is swallowed from the oral cavity, the bacteria need to reduce NO3 at a faster rate than they reduce NO₂ , or not undertake downstream metabolism of NCh at all. Overall, to maximise NO bioavailability from a N0₃ -rich diet, the composition of the oral microbial community needs to be such that it contains a greater quantity of net NO2 accumulators than net NO2 consumers. Further research is needed to establish whether the observed microbiome changes following chronic NO₃ supplementation in the present cohort, are replicated in different populations and whether such changes are associated with an increased capacity for acute NCh reduction in the oral cavity.

The present data suggest that the chronic (10-day) NCb supplementation serves to change the relative abundance of a few, but not all, NO3 reducing taxa and that these changes are correlated with beneficial changes in NO bioavailability and indices of cardiovascular health. It should be noted that elevated NO bioavailability may have further beneficial effects on aspects of healthy ageing, including maintenance of a strong immune response. A recent study showed that tongue microbiomes that had high abundances of Prevotelia and Veillonella species were associated with elevated risks of all-cause mortality and mortality from pneumonia in frail elderly nursing home residents (Kageyama et al. 2017). NCh supplementation in older people, which reduces the relative abundances of Prevotelia and Veillonella, may therefore have potential to enhance the NO-mediated immune response in this high-risk population.

Differences in [NO₂ ] and blood pressure responses between young and older subjects

Previous studies have shown appreciable inter-individual variability in the plasma [N0₂ ] and blood pressure responses to NO3 ingestion in both young and older populations (e.g. Casey et al. 2015; Jaija et al. 2014; Kelly et al. 2013). Since the human vascular response to NO3 supplementation is dependent on efficient bacterial reduction of NO3 to NO2 , it is possible that at least some of this variability is linked to differences between individuals in the oral microbiota and therefore oral NO3 reducing capacity. We found that despite ingesting the same dose of NO3 the old adults showed a greater plasma [NO2 ] increase than the young adults, and also exhibited a reduction in blood pressure which was absent in the young participants. NMDS analysis revealed no overall differences in the salivary microbiomes between young and old participants, or following placebo or NO3 supplementation, and there were no differences in tongue microbiomes at baseline. The greater responsiveness to supplementation in the old compared to young participants was surprising, given that ageing is typically associated with reduced salivary flow rate and altered oral bacterial colonisation (Percival et al. 1991). We did not measure salivary flow rate but the young adults reported a greater frequency of dry mouth symptoms than the old adults. Whether possible differences in salivary flow rate and/or NO3 uptake into the enterosalivary circulation via sialin 2N03 /H⁺ transporters (Qu et al. 2016) contribute to inter-individual variability in responsiveness to NO3 supplementation irrespective of age warrants further investigation with large cross-sectional cohorts across the human lifespan.

Physical exercise and diet are emerging as powerful modulators of the gut microbiota (Barton et al. 2017) and it is intuitive that the microbiota in the oral cavity, the uppermost section of the alimentary canal, may also vary according to age, diet, and with physical activity levels. There is evidence to suggest that the gut microbiome of healthy older people may be remarkably similar to that of young adults (Bian et al. 2017), and that age-related alterations in the gut microbiome may be related to advancing frailty and development of disease (Biagi et al . 2010). Whether the maintenance of a 'young' gut microbiome into older age is a cause or consequence of healthy ageing is unknown. Our data suggest that the oral microbiome of individuals who have reached their 8^th decade of life without chronic disease is

indistinguishable from oral microbiome of young adults. The inclusion criteria in the present study meant that the enrolled old participants were in exceptionally good health for their age and therefore not representative of an ageing population with poor cardiovascular and/or metabolic health, who may be less responsive to NO3 supplementation due to impairments in NO bioactivity (Siervo et al . 2015). Indeed, we found that there was no difference in blood pressure between young and old participants following NO3 supplementation. Longitudinal studies are necessary to identify possible changes that may occur in the oral microbiome, and NO bioavailability, at the onset of chronic disease.

4. CONCLUSIONS - BEETROOT SUPPLEMENTATION STUDY

Imbalances in the oral microbial community and poor dental health have been associated with reduced cardiovascular and metabolic health. We showed that ageing, per se, in the absence of chronic disease, does not impair an individual's ability to reduce dietary NO3 and increase plasma [NO2 ] in response to NO3 supplementation. Using 16S rRNA gene sequencing of oral bacteria in an in vivo experimental model, we showed that high abundances of oral bacteria belonging to genera Prevotella and Veillonella were likely detrimental, while high abundances of the genera Rothia and Neisseria were likely beneficial for the maintenance of NO homeostasis and associated indices of cardiovascular health. The symbiotic relationship between the oral microbiome and its human host is a fast evolving field of research with significant implications for development of prebiotic and probiotic interventions to improve cardiovascular and metabolic health. Our results identify dietary NO3 as a modulator of the oral N0₂ -producing microbiome in healthy humans and highlight the potential of oral microbiota-targeted therapies for ameliorating conditions hallmarked by low NO bioavailability.

5. METHODS - POTASSIUM NITRATE SUPPLEMENTATION STUDY

Ethical approval

The study was approved by the institutional Ethics Committee (Sport and Health Sciences, University of Exeter) and conducted in accordance with the code of the ethical principles of the World Medical Association (Declaration of Helsinki). All participants gave their written, informed consent before the commencement of the study, once the experimental procedures, associated risks, and potential benefits of participation had been explained.

Subjects

Ten healthy young adults volunteered to take part in the study (6 men, 4 women; age mean ± SD 29.5 ± 6.4 years, body mass 70.2 ± 11.6 kg). All participants had good oral health as assessed by the World Health Organization Oral Health Questionnaire for Adults.

Measurements

Oral swabs of the tongue dorsum were collected at baseline prior to dietary supplementation and blood pressure of the brachial artery was measured following 10 min of seated rest in a quiet room using an automated sphygmomanometer (Dinamap Pro, GE Medical Systems, Tampa, USA). A total of four measurements were taken, with the mean of the final three measurements recorded.

Participants ingested 8 mmol/d of food grade KNO3. The dose was ingested in two equal portions of 4 mmol diluted in 50 ml of water, one in the morning and one in the evening. Further oral swabs were collected on day 5 of the supplementation and a final swab was taken 72 h after supplement cessation. Venous blood samples were collected at baseline, at the end of supplementation period and 72 h after cessation of supplementation. Samples were immediately centrifuged and supernatant stored at -80°C until later analysis for plasma nitrate and nitrite concentrations.

Oral nitrate reductase activity was assessed at baseline and after five days of KN03 supplementation by using a 2-min mouth-hold test of a solution containing 1 mM of KN03, and by measuring the ratio of nitrite to total nitro-compounds (N02-/N0x) in saliva. Saliva samples (~ 1 mL) were collected by expectoration, without stimulation, over a period of 5 min.

Plasma was deproteinised 1 :3 in ice-cold ethanol, vortexed then incubated on ice for 30 minutes, then centrifuged for 10 minutes at 14 000 x G. Saliva samples were diluted 1: 200 in ddH20. For determination of nitrite concentration, samples were injected into a glass purge vessel containing glacial acetic acid and sodium iodide solution at 50°C. These conditions reduce nitrite to nitric oxide, which is carried in inert nitrogen to a Sievers 280i nitric oxide analyser, whereupon it reacts with ozone to form nitrogen dioxide, emitting light in the process. Filtered and detected on a photomultiplier tube, the resultant area under the peak in mV signal is calculated as nitrite molarity using standards of known value. Subsequently, total NOX is reduced to NO in Vanadium (III) Chloride in 1M HCI at 95°C. The nitrate concentration is determined by deducting the nitrite value from NOX.

Oral bacteria

Oral swab samples were stored at -80°C until analysis. Genomic DNA was isolated from tongue swabs using a Gentra Puregene Buccal Cell Kit (Qiagen, Germantown, MD), and from saliva samples following the methods of Goode et al. (2014). Double-stranded DNA concentration was fluorometrically quantified (Qubit 3.0 high-sensitivity fluorescence detection,

ThermoFisher Scientific, Waltham, MA). Library preparation employed a NEXTflex 16S V1-V3 Amplicon-Seq Kit (Bioo Scientific, Austin, USA). The 16S V1-V3 rDNA region was amplified using 5ng of dsDNA and subjected to 8 thermal cycles of 30 s at 98°, 30 s at 60° and 30 s at 72° with primers A and B. Following AMPure^® XP bead cleanup (Becton Dickinson, Franklin Lakes, NJ), a subsequent PCR with indexing primers to identify individual samples, containing Illumina flow cell binding sites, was performed.

The samples were sequenced using paired-end 300 base pair (bp) MiSeq Illumina platform (Illumina, San Diego, CA) using v3 MiSeq reagents. For each sample, the nucleotide sequence data in FASTQ format was trimmed using Trim-Galore! (Krueger F. Trim-Galore!, accessible at www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Quality trimming was performed by removing low-quality bases from the 3' read ends. The adapter sequences were

subsequently removed from the 3' end (the first 13 base pairs). Trim-Galore! paired-end validation was performed to remove short sequences once the trimming was complete, where the minimum specified length was 20 bp. The bacterial taxonomies and abundance were assigned using Kraken standard build, which uses the genomes in Refseq and NCBI taxonomic information (Kraken manual, accessible at;

www.ccb.jhu.edu/software/kraken/MANUAL.html#kraken-databases). The paired read sequences were classified and processed by the Kraken Taxonomic Sequence Classification System (Woods and Salzberg 2014). Variations in the V1-V3 regions enabled NCBI taxonomic identification, and kraken-translate was used to translate the NCBI Identifiers to taxonomy identifiers. A Kraken report was generated for each sample, which was visualised using Krona bioinformatics pie charts (Ondov et al. 2011).

Statistical analyses

The Kraken raw data output of phylogenetic data were analysed using R-script (R Development Core Team 2008), SPSS V20 and Microsoft Excel. Non-metric multidimensional scaling (NMDS) was used to assess the level of microbiome similarity between baseline, end of

supplementation, and 72 h post supplementation time points using non-parametric

relationships, and analysed using ADONIS (Vegan R Software). Specific differences in taxonomic units between the same time points were assessed using paired samples LSD t- tests (R statistical software). Statistical significance was defined as P<0.05 and statistical trend was defined as P O.10. Data were expressed as mean ± SD.

6. RESULTS - POTASSIUM NITRATE SUPPLEMENTATION STUDY

Oral nitrate reduction capacity was successfully assessed in 7 subjects. Nitrate reduction capacity increased from pre-supplementation baseline to 5 days of KNO3 supplementation in six out of seven subjects (Figure 7).

The NMDS plot illustrates that the overall compositions of the tongue microbiome were distinctly clustered at baseline and after five days of KNO3 supplementation, but this shift did not reach statistical significance (P= 0.026; Figure 8). Among the main phyla of oral bacteria, the relative abundance of Proteobacteria increased in response to the KNO3 supplementation (P<0.05) and there was a trend towards a decrease in Firmicutes (PcO.10) (Table 6), while Actinobacteria, Fusobacteria, and Bacteroidetes were not significantly altered by

supplementation. There were no significant differences in any of the five phyla between baseline and the samples collected 72 h post supplementation.

Genus and species level comparison between pre-supplementation baseline and end of the 5- day KNO₃ supplementation revealed a significant decrease in genus Veillonella and V. parvula and a significant increase in genus Neisseria, including N. meningitidis (P< 0.05 for all; Table 7). There were also trends towards a significant decrease in genus Prevotella (including P. me!aninogenica ) and an increase in genus Rothia (including R. mucilaginosa ) (P O.lO for all; Table 7).

7. DISCUSSION & CONCLUSIONS - POTASSIUM NITRATE SUPPLEMENTATION STUDY

The principal novel finding of this pilot study was that dietary nitrate supplementation results in similar alterations in oral microbiome irrespective of whether nitrate is ingested in the form of a natural food product (nitrate-rich beetroot juice) or a pure nitrate salt (KNO3). In the present study using KNO3 (8 mmol/d for 5 days) we were able to replicate 19 of the 32 differences observed at the genus and species level in our previous study where we used beetroot juice, as described. The nitrate-induced differences that were common between our two studies included genera Prevotella, Veillone!la, Neisseria , Biautia, Megasphaera,

Atopobium, Sanguibacter, Rothia and Spirochaeta , as well as species Prevotella

metaninogenica, Neisseria meningitidis, Veillonella parvula, Ruminococcus torques,

Megasphaera elsdenii, Clostridium difficile, Atopobium parvulum, Sanguibacter keddieii, Rothia mucilaginosa and Spirochaeta africana. Consistent with our previous study, we showed that KNO3 supplementation significantly increased the relative abundance of the phylum

Proteobacteria and tended to decrease Firmicutes. We also found that changes in the microbial community achieved by KNO3 supplementation returned to baseline 72 h after cessation of supplementation. This finding has important implications for the design of future studies that use a cross-over design for dietary interventions. Future work is warranted to explore the dose-response relationship between dietary nitrate and associated alterations in the oral microbiome (particularly relating to Veillonella, Prevotella, Neisseria and Rothia) and to establish key relationships between the oral microbiome and increased oral nitrate reduction capacity.

Table 6. Relative abundances of the five main phyla of oral bacteria at baseline and following five days of potassium nitrate supplementation. % change indicates the relative difference between baseline and day 5 of KNCb. * Significant difference P<0.05; # Trend towards a significant difference P O.lO.

Table 7. Relative abundances of selected genera and species that made up >0.01% of all oral bacteria, and that showed a significant difference (P<0.05) or a trend towards a significant difference (P<0.10), between baseline and five days of KNO3 supplementation. % change indicates the relative difference between baseline and day 5 of KNO3. Bold font indicates those taxonomic units which showed differences consistent with those reported above.

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Claims

1. A method for improving a response of an individual to a dose of dietary nitrate, comprising decreasing the prevalence in the individual's oral microbiome of the total bacteria of the phylum Bacteroidetes.

2. The method according to claim 1 comprising decreasing the prevalence in the individual's oral microbiome of the total bacteria of the genus Prevotella, optionally comprising decreasing the prevalence in the individual's oral microbiome of the total bacteria of the species Prevotella melaninogenica.

3. The method according to any preceding claim comprising decreasing the prevalence in the individual's oral microbiome of the total bacteria of the phylum Firmicutes and/or of the genus Veillonella, for example decreasing the prevalence in the individual's oral microbiome of the total bacteria of the species Veillonella parvula.

4. The method according to any preceding claim wherein the prevalence of total bacteria of the phylum Bacteroidetes and/or Firmicutes and/or the prevalence of the genus Prevotella and/or Veillonella and/or the prevalence of the species Prevotella melaninogenica and/or Veillonella parvula is decreased prior to intake by the individual of the dose of dietary nitrate.

5. The method according to any preceding claim comprising increasing the prevalence in the individual's oral microbiome prior to intake by the individual of the dose of dietary nitrate, of the total bacteria of the phylum Actinobacteria, optionally of the orders Actinomycetales and/or Micrococcales, and/or comprising increasing the prevalence in the individual's oral microbiome, prior to intake by the individual of the dose of dietary nitrate, of the total bacteria of the species Fusobacterium nucleatum subsp. vlncentii.

6. The method according to any of claims 1-3 wherein the prevalence of total bacteria of the phylum Bacteroidetes and/or Firmicutes and/or the prevalence of the genus Prevotella and/or Veillonella and/or the prevalence of the species Prevotella melaninogenica and/or Veillonella parvula is decreased concurrently with intake by the individual of the dose of dietary nitrate.

7. The method according to any preceding claim comprising : a) increasing the prevalence in the individual's oral microbiome of the total bacteria of the genus Rothia, for example of the species Rothia mucilaginosa,· and/or b) comprising increasing the prevalence in the individual's oral microbiome of the total bacteria of the genus Neisseria, for example of the species Neisseria meningitidis,· optionally wherein the prevalence of total bacteria of the genus Rothia and/or Neisseria and/or of the species Rothia mucilaginosa and/or Neisseria meningitidis is increased concurrently with intake by the individual of dietary nitrate

8. The method according to any preceding claim wherein the response to dietary nitrate is a change in the individual, after an intake of the dose of dietary nitrate, of one or more of: a) systolic blood pressure; b) diastolic blood pressure c) arterial stiffness; d) mean arterial pressure; and/or e) plasma nitrite concentration; and/or wherein the response is the ratio between plasma nitrite concentration and dietary nitrate dose per kg body mass of the individual ([NO₂Ί/NO₃ dose).

9. The method according to any preceding claim comprising :

(a) promoting a greater change in concentration of plasma nitrite and/or systolic blood pressure (SPB) and/or pulse wave velocity (PWV) in an individual in response to a dose of dietary nitrate, the method comprising decreasing the prevalence in the individual's oral microbiome, prior to intake by the individual of the dose of dietary nitrate, of the total bacteria of the species Prevotella meianinogenica,· and/or

(b) promoting a greater reduction in diastolic blood pressure (DPB) and/or systolic blood pressure (SPB) and/or mean arterial pressure (MAP) in an individual in response to a dose of dietary nitrate, the method comprising increasing the prevalence in the individual's oral microbiome, prior to intake by the individual of the dose of dietary nitrate, of the total bacteria of the phylum Actinobacteria, for example of the orders Actinomycetales and/or Micrococcales, and/or of the species Fusobacterium nucleatum subsp. vincentii and/or Fusobacterium nucleatum subsp. nucleatum ; and/or (c) promoting a greater change in concentration of plasma nitrite and/or plasma nitrate in an individual in response to a dose of dietary nitrate, the method comprising decreasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the phylum Bacteroidetes and/or Firmicutes; and/or

(d) promoting a greater change in concentration of plasma nitrite and/or concentration of plasma nitrate and/or [NCh ]/NC>3 dose in an individual in response to a dose of dietary nitrate, the method comprising, concurrently with intake by the individual of the dose of dietary nitrate, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Bacteroidetes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria; and/or

(e) promoting a greater change in concentration of plasma nitrite and/or concentration of plasma nitrate in an individual in response to a dose of dietary nitrate, the method comprising, concurrently with intake by the individual of the dose of dietary nitrate, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria; and/or

(f) promoting a greater change in concentration of plasma nitrite and/or concentration of plasma nitrate in an individual in response to a dose of dietary nitrate, the method comprising decreasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the genus Prevotella and/or Veillonella and/or of the species Prevotella melaninogenica and/or Veillonella parvula,· and/or

(g) promoting a greater change in concentration of plasma nitrite and/or plasma nitrate in an individual in response to a dose of dietary nitrate, the method comprising increasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the phylum Proteobacteria and/or Actinobacteria; and/or (h) promoting a greater change in concentration of plasma nitrite and/or plasma nitrate in an individual in response to a dose of dietary nitrate, the method comprising increasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the genera Rothia and/or Neisseria, for example of the species Rothia mucilaginosa and/or Neisseria meningitidis,· and/or

(i) promoting a greater change in pulse wave velocity (PWV) in an individual in

response to a dose of dietary nitrate, the method comprising increasing the prevalence in the individual's oral microbiome, concurrently with intake by the individual of the dose of dietary nitrate, of the total bacteria of the genus Rothia and/or species Rothia mucilaginosa,· and/or

(j) promoting a greater change in concentration of plasma nitrite and/or concentration of plasma nitrate in an individual in response to a dose of dietary nitrate, the method comprising, concurrently with intake by the individual of the dose of dietary nitrate, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phyla

10. A method of predicting a response of an individual to a dose of dietary nitrate, comprising determining the prevalence in the individual's oral microbiome of the total bacteria of the genus Prevotella and/or of the total bacteria of the order Actinomycetales and/or bacteria of the species Fusobacterium nucleatum, optionally, further comprising comparing the prevalence in the individual's oral microbiome of the total bacteria of the genus Prevotella and/or of the total bacteria of the order Actinomycetales and/or bacteria of the species Fusobacterium nucleatum to the average prevalence of the same genus, phylum or species in a control group of individuals.

11. A method according to claim 10 wherein the response to the dose of dietary nitrate is a change in the individual, after an intake of the dose of dietary nitrate, of one or more of:

(a) systolic blood pressure;

(b) diastolic blood pressure;

(c) arterial stiffness; (d) mean arterial pressure;

(e) plasma nitrite concentration; and/or

(f) plasma nitrate concentration; and/or wherein the response is the ratio between plasma nitrite concentration and dietary nitrate dose per kg body mass of the individual ([Nq2 ]/Nq3^~ dose).

12. A microbiome-altering composition for use in a method of improving a response of an

individual to a dose of dietary nitrate, the microbiome-altering composition being capable of one or more of:

(a) reducing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Bacteroidetes, optionally of the genus Prevotella, optionally of the species Prevotella melaninogenica;

(b) reducing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Firmicutes, optionally of the genus Veillonella, optionally of the species Veillonella parvula ;

(c) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Actinobacteria, optionally of the genus Rothia, optionally of the species Rothia mucilaginosa ;

(d) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the species Fusobacterium nucleatum, optionally of the species Fusobacterium nucleatum subsp. vincentii, optionally of the species Fusobacterium nucleatum subsp. nucleatum,·

(e) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Proteobacteria, optionally of the genus Neisseria, optionally of the species Neisseria meningitidis,·

(f) when administered to the individual, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Bacteroidetes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria;

(g) when administered to the individual, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria;

(h) when administered to the individual, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phyla

Bacteroidetes plus Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phyla Proteobacteria plus Actinobacteria; optionally wherein the method is or forms part of a method for the treatment or amelioration of a disease in an individual, the disease involving endothelial dysfunction, ischaemia, hypoxia-reperfusion events, metabolic syndrome, hypertension or inflammation.

13. A method for the treatment or amelioration of a disease in an individual comprising

administering a nitrate-containing composition to the individual, the disease involving endothelial dysfunction, ischaemia, hypoxia-reperfusion events, metabolic syndrome, hypertension or inflammation, the individual previously having been administered a microbiome-altering composition capable of one or more of:

(a) reducing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Bacteroidetes, optionally of the genus Prevotella, optionally of the species Prevotella me!aninogenica · (b) reducing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Firmicutes, optionally of the genus Veillonella, optionally of the species Veillonella parvula ;

(c) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Actinobacteria, optionally of the genus Rothia, optionally of the species Rothia muciiaginosa,·

(d) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the species Fusobacterium nudeatum, optionally of the species Fusobacterium nudeatum subsp. vincentii, optionally of the species Fusobacterium nudeatum subsp. nudeatum ; (e) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Proteobacteria, optionally of the genus Neisseria, optionally of the species Neisseria meningitidis ;

(f) when administered to the individual, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum

Bacteroidetes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria;

(h) when administered to the individual, decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phyla Bacteroidetes plus Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phyla Proteobacteria plus Actinobacteria.

14. A database comprising:

(a) a prevalence figure of a bacterial phylum, genus or species in the oral microbiome in an individual;

(b) an indication of a response of the individual after administration to the human

individual of a dose of dietary nitrate; optionally wherein he database comprises the information of (a) and (b), for more than one individual, each more than one individual sharing at least one characteristic with all of the other individuals.

15. A database according to claim 14 comprising: (a) a Prevotella prevalence figure of Prevotella genus bacteria in the oral microbiome in an individual; (b) an Actinomycetales prevalence figure of Actinomycetales order bacteria in the oral microbiome in the individual and/or a Fusobacterium nucleatum prevalence figure of Fusobacterium nucleatum species bacteria in the oral microbiome in the individual;

(c) an indication of a response of the individual after administration to the human

individual of a dose of dietary nitrate.

16. The database according to claim 15 comprising the information of (a), (b) and (c) for more than one individual, each more than one individual sharing at least one characteristic with all of the other individuals.

17. The database according to claim 14 or 15 wherein the characteristic Is selected from age, BMI, gender, geographical area of residence, ethnicity, diet or disease state.

18. The database according to any of claims 14-17 wherein the response is a change in the individual, after an intake of the dietary nitrate, of one or more of: a) systolic blood pressure; b) diastolic blood pressure c) arterial stiffness; d) mean arterial pressure; e) plasma nitrite concentration; and/or f) plasma nitrate concentration; and/or wherein the response is the ratio between plasma nitrite concentration and dietary nitrate dose per kg body mass of the individual ([NO2Ί/NO3 dose).

19. A computer-readable medium comprising a database according to any of claims 14-18

20. A computer comprising a database according to any of claims 14-18, or linked to a

computer-readable medium according to claim 19

21. A method for obtaining a database according to claim 14 comprising : a) determining the prevalence of total bacteria of a phylum, genus or species in the oral microbiome in an individual to provide the phylum, genus or species prevalence figure; and b) determining in the individual a first measurement of a parameter selected from one or more of systolic blood pressure, diastolic blood pressure, mean arterial pressure, arterial stiffness, mean arterial pressure, concentration of plasma nitrite and/or concentration of plasma nitrate; c) subsequently administering to the individual a dose of dietary nitrate; d) subsequently determining in the individual a second measurement of the parameter determined as the first measurement in (b) ; e) comparing the first and second measurements from (b) and (d) to provide a parameter change figure; and f) recording as a record in the database the microbiome/response information, which is the prevalence from (a) linked to the parameter change figure from (e).

22. A method for obtaining a database according to claim 14 comprising conducting the

method according to claim 21 on more than one individual and recording in the database the collective microbiome/response information.

23. A nitrate-containing dietary supplement for use in modulating an individual's microbiome, wherein modulation of the microbiome comprises one or more of the steps of: a) reducing the prevalence of the total bacteria in the oral microbiome of the

individual of the phylum Bacteroidetes, optionally of the genus Prevotella , optionally of the species Prevotella melaninogenlca; b) reducing the prevalence, when administered to the individual, of the total

bacteria in the oral microbiome of the individual of the phylum Firmicutes, optionally of the genus Veillonella, optionally of the species Veillonella parvula ; c) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Actinobacteria, optionally of the genus Rothia, optionally of the species Rothia mucilaginosa,· d) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the species Fusobacterium nucleatum, optionally of the species Fusobacterium nucleatum subsp, vincentii, optionally of the species Fusobacterium nucleatum subsp. nucleatum ; e) increasing the prevalence, when administered to the individual, of the total bacteria in the oral microbiome of the individual of the phylum Proteobacteria, optionally of the genus Neisseria, optionally of the species Neisseria meningitidis,- f) decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Bacteroidetes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria; g) decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phylum Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phylum Actinobacteria; h) decreasing the ratio: prevalence in the individual's oral microbiome of total bacteria of the phyla Bacteroidetes plus Firmicutes : prevalence in the individual's oral microbiome of total bacteria of the phyla Proteobacteria plus Actinobacteria.

24. A nitrate-containing dietary supplement for use in increasing an individual's oral nitrate reduction capacity.

25. The nitrate-containing dietary supplement of claim 23 or claim 24, wherein the supplement comprises or consists of beetroot juice.

26. The nitrate-containing dietary supplement of any of claims 23 to 25, wherein the

supplement comprises a nitrate salt, particularly an alkali metal nitrate solution, inorganic nitrate, potassium nitrate or sodium nitrate.