Field

The invention relates to compositions and methods for the treatment of a gastrointestinal discomfort or a gastrointestinal disorder, such as but not limited to irritable bowel syndrome.

Background

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorderwith co- morbidities such as depression and anxiety. The prevalence of IBS is 10%-20% with incidence at 1-2% per year. Many IBS patients find that certain foods can provoke their symptoms, of these indigestible carbohydrates (i.e. fermentable oligosaccharides, disaccharides, monosaccharides and polyols [FODMAPs]) seem to be particularly IBS triggering. Low FODMAP diet is now recommended for IBS patients to help reduce symptoms. However, this diet is very restricted and has an adverse impact on the gut microbiota leading to a dysbiosis and additionally a low FODMAP diet may result in a negative effect on nutrient intake. An alternative to a restricted low FODMAP diet is the use of FODMAP digesting enzymes. These enzymes allegedly digest (part of) the FODMAPs and prevent triggering of IBS symptoms, however, the FODMAP digesting enzymes do not lead to full recovery from IBS symptoms. As will be detailed below, the enzyme dosages used in the currently available blends are very low and will not remove sufficient FODMAPS.

Dysbiosis in the gut microbiota is observed in IBS patients with low levels of bifidobacteria, and certain microbiota signatures have been associated with symptom severity. In addition, the gut microbiota profile in IBS patients shows high similarities with that of depressive patients, and depletion of species like Coprococcus and Dialister species has been linked to depression.

Human milk oligosaccharides (HMOs) are unique carbon structures and have structural homology to other glycan moieties found in the human body. HMOs structures are very different from the more plant derived FODMAP structures and cannot be compared. A clinical trial in healthy adults has shown that 2'-0-fucosyllactose (2'FL) and/or lacto-N-neotetraose (LNnT) specifically modulates the gut microbiota with an increase in bifidobacteria, and a recent clinical trial in IBS patients showed that these HMOs increase bifidobacteria without aggravating overall gastrointestinal symptoms in IBS patients. The same study also found that 2’FL and LNnT caused an increase in the level of the neurotransmitter GABA in plasma, and an increase in the abundance of Dialister in mucosal biopsies (Internal data).

Clinical trial data (Iribarren et al, 2020, Human milk oligosaccharide supplementation in irritable bowel syndrome patients: A parallel, randomized, double-blind, placebo-controlled study, Neurogastroenterology & Motility, volume 32, issue 10) show that HMOs have an effect on Bifidobacteria but no effect on IBS symptoms severity and overall gastrointestinal symptom severity. Figures

Figure 1 : The setup of the short-term colonic fermentation experiment

Figure 2: Impact of FODMAP; combination of FODMAP and HMO; combination of FODMAP and FODMAP-enzyme; and the combination of FODMAP, FODMAP-enzyme and HMO on absolute abundance of beneficial microbes, including Bifidobacterium (A), Faecalibacterium prausnitzii (B) and Firmicutes (C) in short-term colonic fermentation experiment.

Figure 3: Impact of FODMAP; combination of FODMAP and HMO; combination of FODMAP and FODMAP-enzyme; and the combination of FODMAP, FODMAP-enzyme and HMO on propionate (A), acetate (B) and total SOFA (C) concentrations in short-term colonic fermentation experiment.

Figure 4: Impact of FODMAP; combination of FODMAP and HMO; combination of FODMAP and FODMAP-enzyme; and the combination of FODMAP, FODMAP-enzyme and HMO on total gas production (A) and CO2 production (B).

Figure 5: Impact of FODMAP; combination of FODMAP and HMO; combination of FODMAP and FODMAP-enzyme; and the combination of FODMAP, FODMAP-enzyme and HMO on absolute abundance of beneficial microbes (A) and detrimental microbes (B) in medium-term colonic fermentation experiment.

Figure 6: Impact of FODMAP; combination of FODMAP and HMO; combination of FODMAP and FODMAP-enzyme; and the combination of FODMAP, FODMAP-enzyme and HMO on microbial diversity in medium-term colonic fermentation experiment.

Figure 7: HPAEC-PAD profile of fructans after treatment with either Quatrase or the Enzyme blend

Summary

The aim of the present invention is to provide a composition which can be used in the treatment of gastrointestinal discomfort and/or a gastrointestinal disorder. As a non-limiting example of a gastrointestinal disorder, irritable bowel syndrome (IBS) will be exemplified herein. However, the invention is also applicable to gastrointestinal discomfort and/or other gastrointestinal disorders such as but not limited to small intestine bacterial overgrowth (SIBO).

As will be shown herein within the experimental part, current enzyme blends on the market have a very low enzyme dose compared to the effective dose (for FODMAP’s) as identified by the inventors in the in vitro studies. Most available blends contain many other enzymes as well, that have no link to FODMAP’s. Single enzymes often have a higher dosage, but these only digest a single FODMAP. The current small amount of enzymes used does not remove all FODMAP’s, so problems relating to the presence of FODMAPs are not solved or only partly solved. Although higher amounts of enzymes could be added to remove all or more FODMAPS this results - as shown herein in the experimental part- in a lack of nutrients for the gut bacteria. The inventors show that adding HMO’s restores nutrients for only good bacteria, so the invention as described herein removes FODMAP’s and at the same time bacteria are selectively fed. The use of only enzymes provides instant relief but when used longer the overall improvements are limited due to detrimental effects on nutrition. The use of only HMO helps to feed the body’s own microbiome but this takes time meaning that people often do not persevere with a long term use.

As will be shown herein within the experimental part, the invention provides, amongst others, a composition which surprisingly is capable of reducing FODMAPs as well as restoring the human gut microbiome. By using a composition of the invention, a human individual suffering from gastrointestinal discomfort and/or a gastrointestinal disorder does not or hardly not need to follow a restricted diet such as a FODMAP reduced diet. The experimental part herein describes multiple surprising effects such as: the used enzyme mix is effectively degrading FODMAP and hence results in a lack of substrate for the gut microbiome which is for example reflected in the low level of short chain fatty acids (SCFAs) produced by gut bacteria preferably the dosage of the used enzymes is higher when compared to currently commercial enzyme mixtures

HMOs can effectively compensate for the effect caused by the enzymes which is reflected by an increase in SCFA and/or the combination of enzymes and HMOs have superior effect on feeding the microbiome as reflected by the highest concentration of propionate production and the highest number of beneficial microbes such as Firmicutes, Lachnospiraceae, Bifidobacteria, Faecalibacteria, Eubacterium ramulus, Clostridium boltae, Blautia obeum/wexlerae, Lachnospiraceae sp. the combination of enzymes and HMOs have superior effect on maintaining high microbial diversity compared to HMOs alone, and compared to the absence of HMOs and enzymes the combination of enzymes and HMOs decreases gas pressure and CO2

The invention provides a composition suitable for oral administration comprising at least one enzyme and at least one human milk oligosaccharide (HMO) a composition as claimed and/or described herein for use in the treatment of a gastrointestinal discomfort and/or gastrointestinal disorder, preferably irritable bowel syndrome (IBS) a method for alleviating symptoms associated with IBS comprising administering to a human individual in need thereof an effective amount of a composition as claimed and/or described herein a multipack for use in the treatment of a gastrointestinal discomfort or disorder, preferably IBS, the pack comprising at least 14 individually packed daily dosages of an effective amount of a composition as claimed and/or described herein Detailed description

In a first embodiment, the invention provides a composition suitable for oral administration, preferably oral administration to a human, comprising at least one enzyme and at least one human milk oligosaccharide (HMO). As used herein, the term “oral administration” comprises oral administration to a mammal. A preferred mammal is a human and hence this embodiment includes a composition suitable for oral administration to a human, comprising at least one enzyme and at least one HMO.

The term "oral administration" as used herein means any conventional form for the oral delivery of a composition to a patient or human that causes the deposition of the herein claimed and/or described composition in the gastrointestinal tract (including the stomach) of the patient. Accordingly, oral administration includes swallowing of a composition by the patient, enteral feeding through a naso-gastric tube, and the like.

As used herein, the term “at least one enzyme” refers to an enzyme which is capable of reducing the amount of at least one fermentable oligosaccharide, disaccharide, monosaccharide or polyol (FODMAP). Preferably, the “at least one enzyme” is a non-human enzyme, i.e. an enzyme which is not expressed in a human individual, i.e. the enzyme sequence of the “at least one enzyme” is different from the human enzyme sequence (if at all present). Examples of a suitable enzyme are alpha-galactosidase, lactase, invertase or xylose isomerase. Herein disclosed is a composition suitable for oral administration, preferably oral administration to a human, comprising at least one enzyme and at least one HMO, wherein said at least one enzyme is selected from alpha- galactosidase, beta-galactosidase, invertase and xylose isomerase. Yet another example of a suitable enzyme is inulinase.

Alpha-galactosidase

An alpha-galactosidase (EC3.2.1.22) is an enzyme which breaks down certain oligosaccharides, i.e. deals with the “O” and the “D” in FODMAP by breaking down melibiose, raffinose, stachyose, verbascose, and/or other galactans (also named galactooligosaccharides or GOS) which are for example found in beans, peas, cabbage, broccoli, some whole grains and some other vegetables. Most galactans in natural sources contain galactose units connected by a(1®3) or a(1®6) bonds. The human body does not produce alpha-galactosidase as a digestive enzyme. Commercial alpha-galactosidases are commonly obtained from fungi, like Aspergillus species , like Aspergillus niger or Aspergillus oryzae.

A commercial product comprising an alpha-galactosidase is Fibractase available from Disolut and BioCore Complete from Deerland Enzymes, Beano® Ultra from Beano.

Lactase Lactase or b-galactosidase (E.C: 3.2.1 .23) is an enzyme which catalyses the hydrolysis of lactose (a disaccharide) into its component monosaccharides glucose and galactose, i.e. a lactase deals with the “D” in FODMAP. Lactose is present in dairy products and more specifically in milk, skimmed milk, cream, yoghurt, ice-cream and other milk products.

Lactases have been described for and isolated from a large variety or organisms, including micro-organisms. Lactase is often an intracellular component of micro-organisms like Kluyveromyces and Bacillus. Kluyveromyces and especially K. fragilis, K. marxianus and K. lactis, and other yeasts such as those of the genera Candida, Torula and Torulopsis are a common source of yeast lactases, whereas B. coagulans or B circulans are well known sources for bacterial lactases. Several commercial lactase preparations, derived from these organisms are available such as Maxilact® (from K. lactis, produced by DSM, Delft, the Netherlands). All these lactases are so called neutral lactases since they have a pH optimum between pH=6 and pH=8. Several organisms such as Aspergillus niger and Aspergillus oryzae can produce extracellular lactase, and U.S. Pat. No. 5,736,374 describes an example of such lactase, produced by Aspergillus oryzae. The enzymatic properties of lactases like pH- and temperature optimum vary between species. In general, however, lactases that are excreted show a lower pH-optimum of pH=3.5 to pH=5.0 (acid lactases); intracellular lactases usually show a higher pH optimum in the region of pH=6.0 to pH=7.5 for neutral lactases, but exceptions on these general rules occur. Lactase that is present in a composition as disclosed herein is preferably an acidic lactase. A commercial product comprising an acid lactase is Tolerase L available from DSM Food-Specialties, the Netherlands and Disolact Lactase from Disolut, the Netherlands and Dairylytic® from Deerland Enzymes.

Invertase

Invertase is an enzyme which breaks down fructans, i.e. polymers of fructose, i.e. invertase deals with the “O” in FODMAP. Fructan, (also named fructooligosaccharides or FOS) such as inulin and levan, or small-chain FOS like 1-kestose, 6-kestose, neo-kestose, nystose, bifurcose, are present in several plants, such as cereals, for instance wheat, rye and barley and in onions, garlic, or artichokes. Most fructan in natural sources contain fructose units connected by b(2®1) or b(2®6) bonds. Fructan is considered to be beneficial to the health acting as a prebiotic, but at the same time fructan, such as fructan having a degree of polymerisation (DP) of DP3 to DP5 and being part of the Fermentable Oligo-, Di, Monosaccharides and Polyols (FODMAPs), are considered to contribute to symptoms in patients suffering from irritable bowel syndrome (IBS).

Invertase that is present in a composition as disclosed herein belongs to enzyme classification EC 3.2.1.26 and is also called beta-fructofuranosidase or beta-D-fructofuranoside fructohydrolase. Invertase may be derived from any suitable microorganism, such as fungi, yeast or bacteria, for instance Aspergillus sp, for instance A. fumigatus, A. niger, Fusarium oxysporum, or Saccharomyces cerevisiae. Invertase as disclosed herein may be derived from Saccharomyces cerevisiae. A commercial product comprising invertase EC 3.2.1.26 is Maxinvert® available from DSM Food-Specialties, the Netherlands and Quatrase® from Disolut. Xylose isomerase

Xylose isomerase (EC 5.3.1 .5) isomerizes fructose into glucose, and the reverse. Fructose in excess of glucose is regarded as the “M” in FODMAP. The human body does not produce xylose isomerase. Xylose isomerase may act in the small intestine by converting fructose into glucose, which can be taken up more efficiently by the body, and hence the excess of fructose will not reach the colon anymore. Commercial xylose isomerases are commonly obtained from Streptomyces species, like Streptomyces rubiginosus, Streptomyces griseofuscus, or Streptomyces olivochromogenes.

A commercial product comprising xylose isomerase is Lutomerase available from Disolut and Xylosolv or Fructosin from Stada, Germany.

"Human milk oligosaccharide" or "HMO" as used herein refers to a complex carbohydrate found in human breast milk (Urashima et al.: Milk Oligosaccharides. Nova Science Publisher (201 1); Chen Adv. Carbohydr. Chem. Biochem. 72, 1 13 (2015)). The HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more beta -N- acetyl-lactosaminyl and/or one or more beta - lacto-N-biosyl units, and which core structure can be substituted by an alpha L-fucopyranosyl and/or an alpha-N-acetyl-neuraminyl (sialyl) moiety. In this regard, the non-acidic (or neutral) HMOs are devoid of a sialyl residue, and the acidic HMOs have at least one sialyl residue in their structure. The non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto- N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH) and lacto- N-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N- fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP- III), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N- fucopentaose V (LNFP-V), lacto-N- difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para-lacto-N-hexaose I (FpLNH-l), fucosyl-para-lacto-N-neohexaose II (F- pLNnH II) and fucosyl-lacto-N-neohexaose (FLNnH). Examples of acidic HMOs include 3'- sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl- 3'-sialyllactose (FSL), LST a, fucosyl- LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl- LST c (FLST c), sialyl-LNH (SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N- neohexaose II (SLNH-II) and disialyl-lacto-N-tetraose (DSLNT).

The at least one HMO as present in a composition as disclosed herein is a neutral HMO which can be a fucosylated neutral HMO, or a non-fucosylated neutral HMO. In one embodiment, the neutral HMO is a mixture of neutral HMOs, even preferably a mixture comprising or consisting of a fucosylated and a non-fucosylated neutral HMO. A fucosylated neutral HMO may be selected from 2'-FL, 3-FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNDFH-I, LNDFH-II, LNDFH-III, FLNH-I, FLNH-II, FLNnH, FpLNH-l and F-pLNnH II, and a non-fucosylated neutral HMO may be selected from LNT, LNnT, LNH, LNnH, pLNH and pLNnH. Preferably, said fucosylated neutral HMO is selected from 2'-FL, 3-FL and DFL, and said non-fucosylated neutral HMO is selected from LNT and LNnT. In some preferred embodiments said fucosylated neutral HMO is 2’-FL. In another preferred embodiment, said non- fucosylated neutral HMO is LNnT.

The HMOs suitable for a composition as described herein can be isolated or enriched by well- known processes from milk(s) secreted by mammals including, but not limited to human, bovine, ovine, porcine, or caprine species. The HMOs can also be produced by well-known processes using microbial fermentation, enzymatic processes, chemical synthesis, or combinations of these technologies. As examples, using chemistry LNnT can be made as described in WO 2011/100980 and WO 2013/044928, LNT can be synthesized as described in WO 2012/155916 and WO 2013/044928, a mixture of LNT and LNnT can be made as described in WO 2013/091660, 2'-FL can be made as described in WO 2010/115934 and WO 2010/115935, 3-FL can be made as described in WO 2013/139344, 6'-SL and salts thereof can be made as described in WO 2010/100979, sialylated oligosaccharides can be made as described in WO 2012/113404 and mixtures of human milk oligosaccharides can be made as described in WO 2012/113405. As examples of enzymatic production, sialylated oligosaccharides can be made as described in WO 2012/007588, fucosylated oligosaccharides can be made as described in WO 2012/127410, and advantageously diversified blends of human milk oligosaccharides can be made as described in WO 2012/156897 and WO 2012/156898. With regard to biotechnological methods, WO 01/04341 and WO 2007/101862 describe how to make core human milk oligosaccharides optionally substituted by fucose or sialic acid using genetically modified E. coli.

Suitable HMOs can be obtained from DSM Glycom/DNP: GlyCare™ 2 Fucosyllactose & Difucosyllactose or GlyCare™ Lacto-N-neotetraoser or Holigos IBS Restore (2'-FL and LNnT) and Holigos Maintain (2'-FL).

Disclosed herein is a composition suitable for oral administration, preferably oral administration to a human, comprising at least one enzyme and at least one human milk oligosaccharide (HMO), wherein said at least one enzyme is alpha-galactosidase; wherein said at least one enzyme is lactase; wherein said at least one enzyme is invertase; wherein said at least one enzyme is xylose isomerase; wherein said at least one HMO is 2’-FL; wherein said at least one HMO is LNnT; wherein said at least one enzyme is alpha-galactosidase and said at least one HMO is 2’-FL; wherein said at least one enzyme is alpha-galactosidase and said at least one HMO is LNnT; wherein said at least one enzyme is lactase and said at least one HMO is 2’-FL; wherein said at least one enzyme is lactase and said at least one HMO is LNnT; wherein said at least one enzyme is invertase and said at least one HMO is 2’-FL; wherein said at least one enzyme is invertase and said at least one HMO is LNnT; wherein said at least one enzyme is xylose isomerase and said at least one HMO is 2’- FL; or wherein said at least one enzyme is xylose isomerase and said at least one HMO is LNnT.

Preferably, a composition as described herein comprises at least two different enzymes, more preferably at least 3 different enzymes and even more preferably at least 4 different enzymes. The term “different enzymes” refers to enzymes which act on different substrates and/or are taken from different enzyme classifications. Disclosed herein is a composition suitable for oral administration, preferably oral administration to a human, comprising at least one enzyme and at least one human milk oligosaccharide (HMO), which comprises at least 2 different enzymes; which comprises at least 3 different enzymes; or which comprises at least 4 different enzymes.

When more than one enzyme is used in a composition according to the invention, preferably at least one of them is a non-human enzyme, i.e. an enzyme which is not expressed in a human individual.

The different enzymes are preferably all capable of reducing the amount of at least one of fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FODMAP). Alternatively, at least one of the at least two, three or four different enzymes is capable of reducing the amount of at least one of fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FODMAP). Alternatively, at least two of the at least two, three or four different enzymes are capable of reducing the amount of at least one of fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FODMAP). Alternatively, at least three of the at least three or four different enzymes are capable of reducing the amount of at least one of fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FODMAP). Alternatively, at least four of the at least four different enzymes are capable of reducing the amount of at least one of fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FODMAP).

The composition may additionally (i.e. next to at least one enzyme which is capable of reducing the amount of at least one of fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FODMAP) include enzymes which are not capable of reducing the amount of at least one of fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FODMAP), these may for example be a lipase a protease an amylase an acid maltase a peptidase a diamine oxidase or histaminase a cellulase a xylanase a beta-glucanase a phytase a mixture of enzymes such as pancreatin which is a mixture of amylase, lipase and protease

A lipase assists in the breakdown of fats and lipids which plays an important role in the absorption of fat-soluble nutrients including vitamins A, D, E and K. A suitable lipase is a pancreatic lipase.

A protease assists in the breakdown of protein. The protease may be single type of protease or a mixture of acid, neutral and alkaline proteases which are effective through a wide range of pH levels, working through all portions of the digestive tract. A suitable protease is a prolyl- endoprotease DSM Tolerase G). Another suitable example is bromelain. Yet another example is papain.

An amylase allows digestion of carbohydrates during their transit through the gastrointestinal tract. The amylase hydrolyzes the starchy foods and liberates polysaccharides and maltose; the glucoamylase further hydrolyses polysaccharides producing glucose.An acid maltase hydrolyze the alpha 1 ,4 and 1 ,6 glucosidic bonds in starch, glycogen and other polysaccharides, and maltose.

A peptidase helps to digest protein into amino acids that are important building blocks in the human body. The amino acids are used to build muscles, metabolic enzymes, neurotransmitters and many other essential biochemicals. A suitable example of a peptidase is dipeptidyl peptidase IV (DPPIV), aminopeptidase (AP) or carboxypeptidase (CP).

A diamine oxidase (also called histaminase) assists in the digestion of histamine.

A cellulase assists in the breakdown of cellulose and some related polysaccharides. A suitable example is a hemicellulase.

A xylanase assists in the breakdown of xylans like hemicellulose.

A beta-glucanase assists in the breakdown of glucans.

A phytase is an enzyme that is capable of breaking down phytic acid/phytate (inositol hexaphosphate), by hydrolyzing phytic acid/phytate so as to release at least one phosphate group from phytate.

Also disclosed herein is a composition suitable for oral administration, preferably oral administration to a human, comprising at least one enzyme and at least one human milk oligosaccharide (HMO), which comprises at least 2 different enzymes and wherein said enzymes are alpha- galactosidase and lactase; which comprises at least 2 different enzymes and wherein said enzymes are alpha- galactosidase and invertase; which comprises at least 2 different enzymes and wherein said enzymes are alpha- galactosidase and xylose isomerase; which comprises at least 2 different enzymes and wherein said enzymes are lactase and invertase; which comprises at least 2 different enzymes and wherein said enzymes are lactase and xylose isomerase; which comprises at least 2 different enzymes and wherein said enzymes are invertase and xylose isomerase; which comprises at least 3 different enzymes and wherein said enzymes are alpha- galactosidase, lactase and invertase; which comprises at least 3 different enzymes and wherein said enzymes are alpha- galactosidase, lactase and xylose isomerase; which comprises at least 3 different enzymes and wherein said enzymes are alpha- galactosidase, invertase and xylose isomerase; which comprises at least 3 different enzymes and wherein said enzymes are lactase, invertase and xylose isomerase; or which comprises at least 4 different enzymes and wherein said enzymes are alpha- galactosidase, lactase, invertase and xylose isomerase.

Preferably, a composition as described herein comprises at least two different HMOs, more preferably at least 3 different HMOs and even more preferably at least 4 different HMOs. The term “different HMOs” refers to HMOs which have a different structure. Disclosed herein is a composition suitable for oral administration, preferably oral administration to a human, comprising at least one enzyme and at least one human milk oligosaccharide (HMO), which comprises at least 2 different HMOs; which comprises at least 3 different HMOs; or which comprises at least 4 different HMOs.

Also disclosed herein is a composition suitable for oral administration, preferably oral administration to a human, comprising at least one enzyme and at least one human milk oligosaccharide (HMO), which comprises at least 2 different HMOs and wherein said HMOs are 2’- FL and LNnT.

Further disclosed herein is a composition suitable for oral administration, preferably oral administration to a human, comprising at least one enzyme and at least one human milk oligosaccharide (HMO), which comprises at least 2 different enzymes and wherein said enzymes are alpha- galactosidase and lactase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT. which comprises at least 2 different enzymes and wherein said enzymes are alpha- galactosidase and invertase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT; which comprises at least 2 different enzymes and wherein said enzymes are alpha- galactosidase and xylose isomerase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT; which comprises at least 2 different enzymes and wherein said enzymes are lactase and invertase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT; which comprises at least 2 different enzymes and wherein said enzymes are lactase and xylose isomerase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT; which comprises at least 2 different enzymes and wherein said enzymes are invertase and xylose isomerase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT; which comprises at least 3 different enzymes and wherein said enzymes are alpha- galactosidase, lactase and invertase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT; which comprises at least 3 different enzymes and wherein said enzymes are alpha- galactosidase, lactase and xylose isomerase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT; which comprises at least 3 different enzymes and wherein said enzymes are alpha- galactosidase, invertase and xylose isomerase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT; which comprises at least 3 different enzymes and wherein said enzymes are lactase, invertase and xylose isomerase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT; which comprises at least 4 different enzymes and wherein said enzymes are alpha- galactosidase, lactase, invertase and xylose isomerase and wherein said composition further comprises at least two different HMOs, preferably said HMOs are 2’-FL and LNnT which comprises at least 2 different enzymes and wherein said enzymes are alpha- galactosidase and lactase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT. which comprises at least 2 different enzymes and wherein said enzymes are alpha- galactosidase and invertase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT; which comprises at least 2 different enzymes and wherein said enzymes are alpha- galactosidase and xylose isomerase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT; which comprises at least 2 different enzymes and wherein said enzymes are lactase and invertase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT; which comprises at least 2 different enzymes and wherein said enzymes are lactase and xylose isomerase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT; which comprises at least 2 different enzymes and wherein said enzymes are invertase and xylose isomerase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT; which comprises at least 3 different enzymes and wherein said enzymes are alpha- galactosidase, lactase and invertase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT; which comprises at least 3 different enzymes and wherein said enzymes are alpha- galactosidase, lactase and xylose isomerase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT; which comprises at least 3 different enzymes and wherein said enzymes are alpha- galactosidase, invertase and xylose isomerase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT; which comprises at least 3 different enzymes and wherein said enzymes are lactase, invertase and xylose isomerase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT; or which comprises at least 4 different enzymes and wherein said enzymes are alpha- galactosidase, lactase, invertase and xylose isomerase and wherein said composition further comprises at least three different HMOs, preferably said HMOs are 2’-FL, DFL and LNnT.

A composition as described herein is preferably a synthetic composition. The term "synthetic composition" is used herein to refer to a composition which is artificially prepared and preferably means a composition containing at least one compound that is produced ex vivo chemically and/or biologically, e.g. by means of chemical reaction, enzymatic reaction or recombinantly. In some embodiments a synthetic composition as described herein may be, but preferably is not, identical with a naturally occurring composition. The synthetic composition of the invention comprises at least one enzyme and at least one HMO. In some embodiments the synthetic composition may comprise one or more compounds or components other than enzymes and HMOs that may have an effect on bifidobacteria of a human subject microbiota in vivo, e.g. non-digestible oligosaccharides or prebiotics or probiotics. Also in some embodiments, the synthetic compositions may comprise one or more nutritionally or pharmaceutically active components which do not affect adversely the efficacy of the above mentioned compounds. Some non- limiting embodiments of a synthetic composition of the invention are also described below.

A composition as described herein is administered orally, e.g., as a tablet, capsule, pellet, powder or granules containing a predetermined concentration or a gel, paste, solution, suspension, emulsion, syrup, bolus, electuary, or slurry, in an aqueous or non-aqueous liquid. Orally administered compositions can include binders, lubricants, inert diluents, flavouring agents, and humectants. Orally administered compositions such as tablets can optionally be coated and can be formulated so as to provide sustained, delayed or controlled release of the mixture therein.

A composition as disclosed herein is a composition suitable for oral administration, preferably oral administration to a human, comprising at least one enzyme and at least one human milk oligosaccharide (HMO), wherein said composition is a liquid, a tablet, a powder, a granulate, a gummi, a gel, a capsule or any combination thereof.

The composition comprising at least one enzyme and at least HMO can be in a dosage form such as a capsule, tablet or sachet. For example, the composition can be in a tablet form comprising the at least one enzyme and the at least one HMO, and one or more additional components to aid formulation and administration, such as diluents, excipients, antioxidants, lubricants, colorants, binders, disintegrants, and the like.

Examples of suitable diluents, excipients, lubricants, colorants, binders, and disintegrants include, but not limited to polyethylene, polyvinyl chloride, ethyl cellulose, acrylate polymers and their copolymers, hydroxyethyl-cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose, polyhydroxyethyl methylacrylate (PHEMA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), or polyacrylamide (PA), carrageenan, sodium alginate, polycarbophil, polyacrylic acid, tragacanth, methyl cellulose, pectin, natural gums, xanthan gum, guar gum, karaya gum, hypromellose, magnesium stearate, microcrystalline cellulose, and colloidal silicon dioxide. Suitable antioxidants are vitamin A, carotenoids, vitamin C, vitamin E, selenium, flavonoids, polyphenols, lycopene, lutein, lignan, coenzyme Q10 ("CoQIO") and glutathione.

The unit dosage forms, especially those in sachet form, can also include various nutrients including macronutrients.

A composition as described herein can be a pharmaceutical composition. The pharmaceutical composition can contain a pharmaceutically acceptable carrier, e.g., phosphate buffered saline solution, mixtures of ethanol in water, water and emulsions such as an oil/water or water/oil emulsion, as well as various wetting agents or excipients. The pharmaceutical composition can also contain other materials that do not produce an adverse, allergic or otherwise unwanted reaction when administered to non-infants. The carriers and other materials can include solvents, dispersants, coatings, absorption promoting agents, controlled release agents, and one or more inert excipients, such as starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, and disintegrating agents. If desired, tablet dosages of the anti-infective compositions can be coated by standard aqueous or non-aqueous techniques.

Preferably, a composition as described herein is a dietary supplement or a medical food. For a dietary supplement, supervision by a physician is not necessary. For a medical food, supervision by a physician is typically required. A medical food is typically only given when a disease or disorder has been diagnosed.

Preferably, a composition according to the invention comprises an effective amount of said at least one enzyme and an effective amount of said at least HMO.

The term "effective amount" preferably means an amount of a composition that provides at least one enzyme and at least one HMO in a sufficient amount to render a desired treatment outcome in a(n) (human) individual or patient. An effective amount can be administered in one or more doses to a(n) (human) individual or the patient to achieve the desired treatment outcome.

Typically, the amount of HMO/HMOs is in the range from about 10 mg to about 20 g per day, in certain embodiments from about 10 mg to about 15 g per day, from about 100 mg to about 10 g per day, in certain embodiments from about 500 mg to about 10 g per day, in certain embodiments from about 1 g to about 7.5 g per day. An appropriate dose can be determined based on several factors, including, for example, body weight and/or condition, the severity of the of type discomfort/disorder, the inflammatory gastrointestinal condition or the enteropathogenic infection, being treated or prevented, other ailments and/or diseases, the incidence and/or severity of side effects and the manner of administration. Appropriate dose ranges may be determined by standard methods known to those skilled in the art. During an initial treatment phase, the dosing can be higher (for example 200 mg to 20 g per day, preferably 500 mg to 15 g per day, more preferably 1 g to 10 g per day, in certain embodiments 2.5 g to 7.5 g per day). During a maintenance phase, the dosing can be reduced (for example, 10 mg to 10 g per day, preferably 100 mg to 7.5 g per day, more preferably 500 mg to 5 g per day, in certain embodiments 1 g to 2.5 g per day). As disclosed herein within the experimental part, an appropriate range of HMO/HMOs dosage is 2 to 6 g per day, preferably 3 to 5 g per day and more preferably around 4 g per day.

The activity of an enzyme is expressed in units based on an enzymatic assay. Examples are described in the experimental part herein. Surprisingly, the enzymes need to be dosed at a higher level than what is described in prior art compositions. The mentioned dosages are per meal. Depending on the type of meal, i.e. , high or low on FODMAPs, the daily dosages need to be multiplied by for example a factor 2 (in case 2 FODMAP rich meal are consumed) or by a factor 3 (in case 3 high FODMAPs rich meals are used). Preferably, lactase is used in a concentration of at least 1000 ALU, more preferably at least 2500 ALU and most preferably at least 10000 ALU.

Preferably, alpha-galactosidase is used in a concentration of at least 600 GalU, more preferably at least 1200 GalU and most preferably at least 2400 GalU.

Preferably, invertase is used in a concentration of at least 500 SU and more preferably at least 900, 1800 or 2500 SU.

In a preferred embodiment, a composition of the invention comprises at least 3 enzymes and said at least 3 enzymes are lactase, alpha-galactosidase and invertase wherein said composition comprises at least 500 SU and more preferably at least 900, 1800 or 2500 SU invertase (dosage per meal). Even more preferred, said composition comprises at least 1000 ALU, more preferably at least 2500 ALU or at least 10000 ALU, most preferably between 5000 - 10000 ALU (lactase dosage per meal) and at least 600 GalU, more preferably at least 1200 GalU or at least 2400 GalU, most preferably between 600 - 1200 GalU (alpha-galactosidase dosage per meal).

A most preferred composition thus comprises at least 900, 1800 or 2500 SU invertase, 5000 - 10000 ALU lactase and 600 - 1200 GalU alpha-galactosidase (dosages per meal).

In another embodiment, the invention provides a composition as described herein for use in the treatment of a human individual suffering from a gastrointestinal discomfort or gastrointestinal disorder, preferably irritable bowel syndrome (IBS). Described herein is thus: a composition as described herein for use in the treatment of a human individual suffering from a gastrointestinal discomfort; or a composition as described herein for use in the treatment of a human individual suffering from a gastrointestinal disorder, preferably irritable bowel syndrome (IBS).

Also disclosed herein is a composition as described herein for use in the treatment of a human individual suffering from a gastrointestinal discomfort and wherein said human individual is not or hardly not on a FODMAP diet; or a composition as described herein for use in the treatment of a human individual suffering from a gastrointestinal disorder, preferably irritable bowel syndrome (IBS) and wherein said human individual is not or hardly not on a FODMAP diet.

Additionally disclosed herein is a method for alleviating symptoms associated with IBS comprising orally administering to a human individual in need thereof an effective amount of a composition as described herein. Such a method for alleviating symptoms associated with IBS may comprise additional steps, for example a step of identifying a human individual suffering from IBS symptoms. Preferably, such a human individual is not or hardly not on a FODMAP diet. Examples of “gastrointestinal discomfort” are “excessive wind”, diarrhoea, abdominal pain, bloating, constipation, nausea, vomiting, malnutrition, fatigue, weight loss, altered bowel movement patterns, or altered stool such as with blood in stool.

The terms “gastrointestinal disorder” and “gastrointestinal disease” are used interchangeably herein and refer to a disorder or disease which involves the gastrointestinal tract, namely the oesophagus, stomach, small intestine, large intestine and rectum.

IBS is an example of a gastrointestinal disorder/disease and the terms "irritable bowel syndrome" and "IBS" preferably mean a group of functional bowel disorders of humans, particularly adults, characterized by one or more chronic symptoms including abdominal pain, abdominal discomfort, abdominal bloating, fatigue, and changes in bowel movement patterns, such as patterns of loose or more frequent bowel movements, diarrhoea and constipation, typically in the absence of any apparent structural abnormality. There are at least three forms of IBS, depending on which symptom predominates: (1) diarrhoea- predominant (IBS-D); (2) constipation-predominant (IBS-C); and (3) IBS with alternating stool pattern (IBS-A or IBS-M). There are also various clinical subtypes of IBS, such as post-infectious IBS (IBS-PI).

Yet another example of a gastrointestinal disorder/disease is SIBO also referred to as BOS. The term "bacterial overgrowth" means bacterial overgrowth syndrome (BOS) (interchangeably termed small intestinal bacterial overgrowth (SIBO) or small bowel bacterial overgrowth syndrome (SBBOS)) relates to clinical manifestations that occur when the normally low number of bacteria that inhabit the stomach, duodenum, jejunum, and proximal ileum significantly increases or becomes overtaken by other pathogens. Bacterial overgrowth may be determined by a number of techniques, with the gold standard being an aspirate from the jejunum that grows in excess of 10 ⁵ bacteria per millilitre. Patients with bacterial overgrowth typically develop symptoms including nausea, bloating, vomiting, diarrhoea, malnutrition, weight loss and malabsorption. A composition as described herein may be combined - especially when used as a medical food- with the use of prescribed treatments such as antibiotics.

Most patients with gastrointestinal discomfort or gastrointestinal disorder (for example IBS) report worsening of symptoms after food ingestion. For example, foods rich in incompletely absorbed carbohydrates, fatty foods and high-caloric meals often cause issues. Dietary advice recommendations usually take the form of 'healthy eating' advice. The current recommendation is to modify intake of alcohol, caffeine, fat, spicy foods and gas- producing foods, to focus on meal size and number of meals per day and to assess the possibility of food intolerances, especially milk or lactose. Further, dietary fibre intake might be reduced if bloating is a predominant feature. These approaches are usually not fully satisfactory and do not lead to adequate symptom control. For this reason, many patients adopt a diet which is low in fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs). These carbohydrates can be incompletely absorbed in the small intestine and pass into the large intestine where they are fermented by intestinal bacteria, leading to gas production and bloating. Also, they may stimulate motility by causing a net flux of water into the lumen. Results from several clinical trials suggest that some patients with IBS have a favourable, short-term response to a low-FODMAP diet. However, even short-term use of the low-FODMAP diet has been associated with potentially unfavourable changes in intestinal microbiota composition. Also, the diet is difficult to comply with over the long term and proper application requires the gradual reintroduction of some sources of FODMAPs because many of these sources (e.g. fruits and vegetables) are important for healthy nutrition. Upon reintroduction of FODMAPs, the symptoms often return. Patients are therefore left with the choice of remaining on a difficult diet which is potentially unhealthy over the long term, or reintroducing foods which may trigger symptoms. A low-FODMAP diet has also a negative effect on the microbiome.

The terms "microbiota", "microflora" and "microbiome" preferably mean a community of living microorganisms that typically inhabits a bodily organ or part. The most dominant members of the gastrointestinal microbiota include microorganisms of the phyla of Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Synergistetes, Verrucomicrobia, Fusobacteria, and Euryarchaeota; at genus level the microorganisms of Bacteroides, Faecalibacterium, Bifidobacterium, Roseburia, Alistipes, Collinsella, Blautia, Coprococcus, Ruminococcus, Eubacterium and Dorea; and at species level microorganisms of Bacteroides uniformis, Alistipes putredinis, Parabacteroides merdae, Ruminococcus bromii, Dorea longicatena, Bacteroides caccae, Bacteroides thetaiotaomicron, Eubacterium hallii, Ruminococcus torques, Faecalibacterium prausnitzii, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Bacteroides vulgatus and Roseburia intestinalis. In some instances, the gastrointestinal microbiota includes the mucosa- associated microbiota, which is located in or attached to the mucus layer covering the epithelium of the gastrointestinal tract, and luminal-associated microbiota, which is found in the lumen of the gastrointestinal tract.

As shown herein within the experimental part, a composition as disclosed herein has a positive effect on the microbiome. Also shown herein within the experimental part, a composition as disclosed herein is capable of decreasing gas pressure and/or CO2. Additionally, a human individual using a composition as disclosed herein does not or hardly not need to follow a low FODMAP diet (see for example https://www.monashfodmap.com/) The term “low FODMAP diet” is well accepted by the skilled person and involves a diet in which the consumption of fermentable oligosaccharides, disaccharides, monosaccharides and polyols are avoided as much as possible, i.e. are restricted. FODMAPs are part of a generally healthy diet that is rich in fruit, vegetables and legumes and hence following a low FODMAP diet has negative effect on nutrient intake. The phrase “hardly not need to follow a low FODMAP diet” refers to a diet in which typically only polyols are restricted or eliminated. Examples of a polyol (alternatively called sugar alcohols) are sorbitol, mannitol, xylitol and maltitol. These compounds are found in sugar-free chewing gum or sugar-free candy or other low-calorie or carbohydrate-free foods and also in mushrooms and stone fruits like prunes. A composition of the invention is preferably combined with a diet in which polyols are restricted or eliminated. "Treat" means to address a medical condition or disease with the objective of improving or stabilising an outcome in the person being treated. Treat includes the dietary or nutritional management of the medical condition or disease by addressing nutritional needs of the person being treated. "Treating" and "treatment" have grammatically corresponding meanings.

In yet another embodiment, the invention provides a multi-pack for use in the treatment of a human individual suffering from a gastrointestinal discomfort or disorder, preferably irritable bowel syndrome (IBS), the pack comprising at least 14 individually packed daily doses of an effective amount of a composition as described herein.

An individually packed daily dosage may be in different forms, for example: one sachet or stick pack which comprises any of the herein described compositions as powder or granulate and wherein the at least one enzyme and the at least one HMO are mixed together and are in one and the same compartment of said sachet or stick pack. A suitable example of such a sachet would be a sachet comprising 1 g 2’-FL, invertase (1800 SU), alpha-galactosidase (1500 GalU) and lactase (10,000 ALU) to be taken 3 times a day with every meal daily dosage. Preferably, the required 3 sachets are attached to each other and can be separated from each other.; one sachet or stick pack which comprises any of the herein described compositions as a powder or granulate and wherein the at least one enzyme and the at least one HMO are present in different and separated compartments of said sachet or stick pack; one sachet or stick pack which comprises any of the herein described compositions as a powder or granulate and wherein the at least one enzyme and the at least one HMO are present in different and separated compartments of said sachet or stick pack and wherein the amount of enzyme is divided over at least 2, preferably 3, different and separated compartments and wherein the amount of HMO is present in one different and separated compartment. A suitable example of such an arrangement is a first compartment or sachet which comprises 3 g 2'-FL and 3 separate compartments or sachets each comprising invertase (1800 SU), alpha-galactosidase (1500 GalU) and lactase (10,000 ALU). Alternatively, the compartment or sachet comprising the HMO also comprises a first dosage of the enzymes, i.e. a first compartment or sachet comprising 3 g 2'-FL and comprising invertase (1800 SU), alpha-galactosidase (1500 GalU) and lactase (10,000 ALU) and 2 separate compartments or sachets each comprising invertase (1800 SU), alpha-galactosidase (1500 GalU) and lactase (10,000 ALU). one sachet or stick pack which comprises any of the herein described compositions as powder or granulate and wherein the at least one enzyme and the at least one HMO are mixed together and divided over at least 2, preferably 3, different and separated compartments; one sachet or stick pack which comprises any of the herein described compositions as powder or granulate and wherein the at least one enzyme and part of the at least one HMO are mixed together and divided over at least 2, preferably 3, different and separated compartments and wherein the remainder of the HMO is present in a different and separated compartment; one capsule, gummi or gel which comprises any of the herein described compositions; at least two capsules, gummies or gels which comprise any of the herein described compositions and wherein the at least one enzyme is present in one capsule and the at least one HMO in another capsule; at least three capsules, gummies or gels which comprise any of the herein described compositions and wherein the at least one enzyme is divided over two of the three capsule and the at least one HMO is in the third capsule; or at least four capsules, gummies or gels which comprises any of the herein described compositions and wherein the at least one enzyme is divided over three of the four capsules and the at least one HMO is in the fourth capsule;

Alternatively, the invention provides a bottle with a drop dispenser for use in the treatment of a human individual suffering from a gastrointestinal discomfort or disorder, preferably irritable bowel syndrome (IBS), the bottle comprising any of the herein described compositions as a liquid wherein the at least one enzyme and the at least one HMO are mixed together.

Alternatively, the invention comprises a combination of a multi-pack wherein the individually packed daily dosage comprises one sachet or stick-pack comprising the at least one HMO and a bottle with a drop dispenser comprising the at least one enzyme as a liquid.

Alternatively, the invention comprises a combination of a sachet, multi-pack, capsule, gummy, gel or bottle with drop dispenser wherein the combination provides a composition as disclosed herein.

Irrespective of whether a composition of the invention is a liquid, a tablet, a powder, a granulate, a gummi, a gel or a capsule and irrespective of how the composition is packed (for example sachet, stick-pack or bottle with a drop dispenser, instructions for use are included to guide a human individual in how a composition as disclosed herein can best be used.

The features and definitions which are given above in the context of the method claims, equally apply to the use claims.

The present invention is further illustrated by the following non-limiting Examples. Materials and methods

Enzymes used in this study include:

• Maxinvert 200,000 MG (DSM) as source of invertase

• Tolerase L (DSM) as source of lactase

• Fibractase (Disolut) as source of alpha-galactosidase

• Pepsin (Chem-lab NV, Zedelgem, Belgium)

• Pancreatin (Sigma-Aldrich, Bornem, Belgium)

• Trypsin (Carl Roth, Karlsruhe, Germany)

• Chymotrypsin (Carl Roth, Karlsruhe, Germany)

HMOs used in this study include:

• 2’FL/DFL and LNnT (Glycom) in the ratio of 4:1 ; the mixture 2’FL/DFL comprises 80 wt% 2’F: and 15 wt% DFL, the remainder 5 wt% comprises other carbons structures

Description of enzyme units

Invertase activity was measured according to the Invertase Sumner Unit (SU) activity method described in the Food Chemical Codex (FCC 8, US Pharmacopeial Convention, Rockville MD). One SU is the quantity of enzyme which will convert 1 mg of sucrose to glucose and fructose in 5 minutes under the conditions of the assay (pH 4.5 and 20°C). The amount of monosaccharide produced by hydrolysis of the sucrose substrate is measured spectrophotometrically using a 3,5-Dinitrosalicylic Acid acid-phenol reagent correlated to a glucose standard.

Lactase activity was measured according to the Acid Lactase Unit (ALU) activity method described in the Food Chemical Codex (FCC 8, US Pharmacopeial Convention, Rockville MD). One ALU is defined as that quantity of enzyme that will liberate o-nitrophenol at a rate of 1 pmol/ minute under the conditions of the assay (pH 4.5 and 37°C). The hydrolysis of the o-nitrophenyl- b-D-galactopyranoside substrate is measured spectrophotometrically.

Alpha-galactosidase activity was measured according to the Alpha-galactosidase Unit (GalU) activity method described in the Food Chemical Codex (FCC 8, US Pharmacopeial Convention, Rockville MD). One GalU is defined as the quantity of the enzyme that will liberate p- nitrophenol at the rate of 1 pmol/ minute under the conditions of the assay (pH 5.5 and 37°C). The amount of p-nitrophenol liberated from the hydrolysis of the p-nitrophenyl-a-D-galactopyranoside substrate is measured spectrophotometrically.

Pepsin activity was measured as described in Mackie and Rigby (2015) InfoGest Consensus Method. In: The Impact of Food Bioactives on Health: in vitro and ex vivo models.

Eds: Verhoeckx et al. Cham (CH): Springer; 2015. Chapter 2 pp13-22, and references within.

One pepsin unit will produce a DA280 of 0.001 per minute at pH 2.0 and 37 °C, measured as TCA-soluble products using haemoglobin as a substrate. Pancreatin and trypsin activity was measured as TAME unit as described in Mackie and Rigby (2015) InfoGest Consensus Method. In: The Impact of Food Bioactives on Health: in vitro and ex vivo models. Eds: Verhoeckx et al. Cham (CH): Springer; 2015. Chapter 2 pp13-22, and references within. One unit hydrolyses 1 pmole of p -toluene-sulfonyl- L -arginine methyl ester (TAME) per minute at 25 °C, pH 8.1 , in the presence of 10 mM calcium ions.

Chymotrypsin was measured as N -Benzoyl- L -Tyrosine Ethyl Ester (BTEE) units as described in Mackie and Rigby (2015) InfoGest Consensus Method. In: The Impact of Food Bioactives on Health: in vitro and ex vivo models. Eds: Verhoeckx et al. Cham (CH): Springer; 2015. Chapter 2 pp13-22, and references within. One BTEE unit will hydrolyse 1.0 pmole of BTEE per minute at pH 7.8 at 25 °C.

The aim of the herein described examples was to evaluate the impact of the combination of FODMAP reducing enzymes and HMOs on FODMAP substrates in relation to interactions with the colonic microbiota of IBS donors.

Example 1: Upper GIT experiment

The typical experiment conducted by ProDigest (Gent, Belgium) to evaluate the digestion of food ingredients, makes use of an adapted SHIME® system

(https://www.prodiqest.eu/en/technoloqy/shime-and-m-shime ) representing the physiological conditions of stomach and small intestine within the same reactor over time. In order to mimic fed or fasted conditions, a specific gastric suspension is added to the reactor. After this, a standardized enzyme and bile liquid is added to simulate the small intestinal condition. Incubation conditions (pH profiles, incubation times) are optimized in order to resemble in vivo conditions in the different regions of the gastrointestinal tract for fasted or fed conditions.

To ensure that the simulation of the adult upper GIT underfed conditions is performed under the most representative conditions, the InfoGest consensus method was used with some adaptations (Mackie, A., and Rigby, N. (2015). InfoGest Consensus Method. In The Impact of Food Bioactives on Health’, Springer International Publishing, pp. 13-22).

The applied conditions are summarized below:

Gastric phase (fed state)

• First, the amount of test substrates was supplied to the reactors (Table 1). FOS was obtained from Actilight FOS 950P (Equisalud, Huarte, Spain), stachyose was obtained from Acros Organics through VWR, raffinose was obtained from Alfa Aesar through VWR, and lactose was obtained from VWR.

• Addition of gastric juice, which includes salts (NaCI and KCI; Chem-lab NV, Zedelgem, Belgium) and mucin (Carl Roth, Karlsruhe, Germany). The salt levels recommended by the consensus method (NaCI and KCI) were implemented, reaching approximately 50 mM and 7 mM respectively during gastric incubation. Mucin was added at 3 mg/ml_. • pH is adjusted to 5.5.

• Pepsin activity of 4000 U/mL is applied.

• Addition of phosphatidylcholine (Carl Roth, Karlsruhe, Germany) at a concentration of 0.17 mM.

• Samples were collected for HPAEC-PAD analysis (ST start samples)

• Enzymes invertase, a-galactosidase and b-galactosidase were added at 3 concentrations (Table 1)

• Incubation during 2h at 37°C, while mixing via stirring, with sigmoidal decrease of the pH profile from 5.5 to 2.0 by gradual addition of a 0.5M HCI solution.

• The gastric reactors had an initial volume of 57.1 ml and an end volume of 71.4 ml.

Small intestinal phase (fed state)

• Addition of pancreatic enzymes and bile salts.

• Regarding pancreatic enzymes both 5.6 TAME U/mL of a raw animal pancreatic extract (pancreatin), 15.4 TAME U/mL trypsin and 3.8 BTEE U/mL chymotrypsin was added.

• Regarding bile salts, 10mM bovine bile extract (BD, Erembodegem, Belgium) is supplemented.

• While mixing via stirring, the pH initially increases from 2.0 to 5.5 within a period of 5 minutes. After this initial phase, a linearly increasing pH from 5.5 to 7.0 was implemented during an incubation of 3h at 37°C. Increase in pH was due to the presence of the bicarbonate buffer in the pancreatic juice, and was controlled by the automatic addition of a 0.5M NaOH solution.

• After an additional 3h of incubation at pH 7.0, samples were collected for HPAEC-PAD analysis (SI end samples)

• The end volume of the small intestinal reactors was 100 ml.

HPAEC-PAD analysis

• Sample preparations

• First, samples were diluted in order to obtain a concentration between 0 and 200 pM for each of the sugars present. Dilutions were prepared in water, with a final 1 :1 dilution in acetonitrile to remove complex polymers from the samples.

• An internal standard (rhamnose) was included in the samples, reaching a final concentration of 25 pM.

• At the end, a centrifugation and filtration step was applied.

• Concentrations of the sugars were determined by HPAEC-PAD (Dionex, Sunnyvale, CA, USA) under highly alkaline conditions. A calibration curve of each sugar was included with concentrations ranging between 0-200 pM for each sugar. Table 1 : FODMAP substrates and enzyme concentration used in upper GIT experiment

By analysing the amount of sugars before and after upper GIT digestion, % digestion can be calculated as follow:

For Fructooligosaccharides (FOS), % digestion was calculated as ((fructose + glucose) at SI end - (fructose + glucose) at ST start)/total amount of FOS added;

For other test products, the % digestion can be calculated as 1- amount of intact test product at SI end/amount of intact test product at ST begin

Table 2: Digestion rates of test FODMAP enzymes (%)

Using the digestion rate (Table 2), we calculated the amount of substrates that will be tested in the short-term large intestine fermentation experiment, under the assumption that the monosaccharides are taken up by the small intestine, and are therefore excluded in the shortterm colonic experiment.

Example 2: Short-term colonic experiment

In short, four products (FODMAP 100% and pre-digested FODMAP, whether or not cosupplemented with HMOs) were fermented by the colonic microbiota of four IBS donors (Figure 1). Treatments were evaluated based on effects on production of saccharolytic fermentation products (SOFA), and on effects on specific members of the microbial community (targeted qPCR).

Donors and sample preparation

Preservation of fecal material

Fecal material was collected from four IBS-D donors. Fecal slurry were prepared by making a 1 :5 (mass:volume) mixture of a freshly collected fecal sample with anaerobic phosphate buffer (K2HPO4 8.8 g/L; KH2PO46.8 g/L; sodium thioglycolate 0.1 g/L; sodium dithionite 0.015 g/L). After homogenization (10 min, BagMixer 400, Interscience, Louvain-La-Neuve, Belgium) and removal of big particles via centrifugation (2 min, 500g), fecal slurry were mixed with an optimized cryoprotectant. The obtained suspensions were aliquoted, flash frozen and then preserved at -80°C (cryostock). Just before the experiment, fecal samples were defrosted and immediately added to the reactors.

Preparation of the cryostock from a single fecal suspension ensures that identical microbial communities are obtained in each aliquot, and thus that an identical inoculum is used throughout the different project phases. Moreover, preservation of aliquots ensures that the preserved samples undergo only one freeze-thawing cycle before introduction in a given incubation, as a new aliquot is used for each phase of the project. These actions ensure optimal reproducibility.

Short-term colonic incubations

The short-term screening assay consisted of a colonic incubation of a single dose of four test substrates (see also Table 3):

- 100% FODMAP (0.86 g/L FOS + 0.21 g/L stachyose + 0.21 g/L raffinose + 1 .71 g/L lactose)

100% FODMAP + HMOs (composition above + 2.4 g/L 2FL/DFL + 0.6 g/L lacto-N- triose)

Pre-digested FODMAP (0.05 g/L stachyose + 0.10 g/L raffinose + 0.11 g/L lactose)

Pre-digested FODMAP + HMOs (composition above + 2.4 g/L 2FL/DFL + 0.6 g/L lacto-N-triose)

At the start of the short-term colonic simulations aforementioned substrates were added to a sugar-depleted nutritional medium that contains basal nutrients of the colon consisting of K2HPO4 3.5 g/L; KH2PO4 10.9 g/L; NaHC03 2 g/L; yeast extract 2 g/L (OXOID, Pratteln, Switzerland); peptone 2 g/L (OXOID, Pratteln, Switzerland); mucin 1 g/L (Carl Roth, Karlsruhe, Germany); cysteine 0.5 g/L; Tween80 2 mL/L). 10% of a 7.5% fecal suspension from four IBS-D donors were added as sources of the colonic microbiota. A control condition, consisting of the sugar-depleted nutritional medium without test substrates, was included for each donor. The control condition serves as a reference, as it allows to assess the effects of fermentation of nutrients in the sugar- depleted nutritional medium on microbial activity and composition. Each condition was tested in single repetition. The experiment thus consisted of 20 independent incubations (Figure 1).

Incubations lasted 48h at a temperature of 37°C, under continuous shaking (90 rpm) and anaerobic conditions in fully independent reactors with sufficiently high volume to not only enable robust microbial fermentation, but also to allow collection of multiple samples over time. To achieve and maintain anaerobic conditions, the reactors were flushed with nitrogen and closed with air-tight caps. Sample collection enables assessment of metabolite production, which allows to understand the complex microbial interactions that are taking place.

Table 3: FODMAP substrates and enzyme concentration used in short-term colonic experiment

Analysis

Short chain fatty acids

Short chain fatty acids (SCFA) are an assessment of the microbial carbohydrate metabolism (acetate, propionate and butyrate) or protein metabolism (branched SCFA) and can be compared to typical fermentation patterns for normal Gl microbiota. Samples for SCFA analysis were analyzed after 0, 6, 24 and 48h of incubation under the simulated large intestinal conditions.

SCFA were extracted from the samples with diethyl ether, after the addition of 2-methyl hexanoic acid as an internal standard. Extracts were analysed using a GC-2014 gas chromatograph (Shimadzu, ‘s-Hertogenbosch, the Netherlands), equipped with a capillary fatty acid-free EC-1000Econo-Cap column (dimensions: 25 mm x 0.53 mm, film thickness 1.2 mM; Alltech, Laarne, Belgium), a flame ionization detector and a split injector. The injection volume was 1 mL and the temperature profile was set from 110 to 160°C, with a temperature increase of 6 °C min-1 . The carrier gas was nitrogen and the temperature of the injector and detector were 100 and 220°C, respectively. The production of unbranched and branched SCFA was calculated by summing the molar concentrations of acetate, propionate, butyrate, valerate and caproate, and summing isobutyrate, isovalerate and isocaproate molar concentrations, respectively. The total SCFA production was defined as the sum of unbranched and branched SCFA.

Microbial quantification by targeted qPCR Through careful selection of primers, qPCR allows the direct quantification of targeted taxonomic entities within complex microbial ecosystems. As this technique is independent of (the lack of) culturability of bacteria, data generated with this method offer a reliable insight in the effects of any treatment on the abundance of the taxonomic group(s) of interest. qPCR analysis was performed at Oh, 24h and 48h to assess treatment effects on Firmicutes, Bifidobacterium spp. and Faecalibacterium prausnitzii as described previously by Van den Abbeel et al (ACS Omega 2018, 3, 10, 12446-12456; https://doi.org/10.1021/acsomeqa.8b01360).

Results

The combination of FODMAP enzymes and HMOs increased number of beneficial microbe Bifidobacterium (Figure 2A)

^■ FODMAP alone (‘FODMAP’) and with HMO (‘FODMAP+HMO’) led to an increase in absolute abundance of beneficial microbe Bifidobacterium compared to the blank.

^■ The pre-digested FODMAP by FODMAP-enzyme mix (‘FODMAP+Enzyme’) reduced the number of Bifidobacterium, indicating that FODMAP substrates were degraded efficiently by the FODMAP-enzyme mix in the upper GIT experiment.

^■ Surprisingly, treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in highest number of Bifidobacterium compared to the blank, FODMAP alone, ‘FODMAP+HMO’, and ‘FODMAP+Enzyme’, despite that the total fermentable carbohydrate is lower than in the ‘FODMAP+HMO’ sample

The combination of FODMAP enzymes and HMOs increased number of beneficial microbe Faecalibacterium prausnitzii (Figure 2B)

^■ Surprisingly, treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in highest number of Faecalibacterium prausnitzii compared to the blank, FODMAP alone, ‘FODMAP+HMO’ and ‘FODMAP+Enzyme’, despite that the total fermentable carbohydrate is lower than in the ‘FODMAP+HMO’ sample

The combination of FODMAP enzymes and HMOs increased number of beneficial phylum Firmicutes (Figure 2B)

^■ Treatment with FODMAP+HMO and FODMAP+ enzyme resulted in the same level of Firmicutes compared to the blank.

^■ On the other hand, treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in an increase number of Firmicutes compared to the blank, ‘FODMAP+HMO’ and ‘FODMAP+Enzyme’. The combination of FODMAP enzymes and HMOs increased concentration of beneficial metabolite propionate (Figure 3A) Propionate is a major microbial fermentation metabolite in the human gut with several health benefits toward energy homeostasis. Propionate is thought to lower lipogenesis, serum cholesterol levels, and carcinogenesis in other tissues. Propionate may also decrease obesity by promoting the secretion of PYY and GLP-1 hormones from human colonic cells, inducing satiety and subsequently reducing energy intake and promoting weight loss. FODMAP alone (‘FODMAP’) and with HMO (‘FODMAP+HMO’) led to an increase in concentration of propionate compared to the blank. The pre-digested FODMAP by FODMAP-enzyme mix (‘FODMAP+Enzyme’) reduced the concentration of propionate to the blank level, indicating that FODMAP substrates were degraded efficiently by the FODMAP-enzyme mix. Surprisingly, treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in highest concentration of propionate compared to the blank, FODMAP alone, ‘FODMAP+HMO’, and ‘FODMAP+Enzyme’ despite that the total fermentable carbohydrate is lower than in the ‘FODMAP+HMO’ sample.

The combination of FODMAP enzymes and HMOs increased concentration of beneficial metabolite acetate (Figure 3B) Acetate is a primary metabolite produced from fermentation of prebiotic substrates. FODMAP alone (‘FODMAP’) and with HMO (‘FODMAP+HMO’) led to an increase in concentration of acetate compared to the blank. The pre-digested FODMAP by FODMAP-enzyme mix (‘FODMAP+Enzyme’) reduced the concentration of acetate to the blank level, indicating that FODMAP substrates were degraded efficiently by the FODMAP-enzyme mix. Treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in higher concentration of acetate compared to the blank, FODMAP alone, and ‘FODMAP+Enzyme’

The combination of FODMAP enzymes and HMOs increased concentration of total SCFA (Figure 3B) Total SCFA levels are reflective of the overall fermentation of test ingredients. FODMAP alone (‘FODMAP’) and with HMO (‘FODMAP+HMO’) led to an increase in concentration of total SCFA compared to the blank. The pre-digested FODMAP by FODMAP-enzyme mix (‘FODMAP+Enzyme’) reduced the concentration of total SCFA to the blank level, indicating that FODMAP substrates were degraded efficiently by the FODMAP-enzyme mix. Treatment with the combination of FODMAP-enzyme mix and HMO

(‘FODMAP+Enzyme+HMO’) resulted in higher concentration of total SCFA compared to the blank, FODMAP alone, and ‘FODMAP+Enzyme’.

Example 3: Medium-term SHIME experiment and short-term colonic simulation for gas analysis

Medium-term SHIME experiment

The typical reactor setup of the SHIME®, representing the gastrointestinal tract of the adult human, was described by Molly et al. (1993) Applied Microbiology and Biotechnology 39(2): 254-258. Inoculum preparation, retention time, pH, temperature settings and reactor feed composition were previously described by Possemiers et al. (2004) FEMS Microbiol Ecol. 49(3):495-507. Compared to the typical setup of the SHIME, the medium-term SHIME experiment used for this example included some adaptations. The stomach and small intestine were simulated subsequently in the same reactor vessel prior to addition of the small intestinal suspension in the colon region mimicking the transverse colon (pH 6.15-6.4; retention time = 20h; volume of 500 ml_).

The medium-term SHIME experiment consisted of two stages:

- Inoculation (day 0): On day 0, the colon reactors were inoculated with a faecal sample (conserved frozen prior to inoculation), and the microbiota was allowed to grow and colonize in the reactor for 1 day in order to support the maximum diversity of the gut microbiota originally present in the faecal inoculum. Briefly, a fecal sample was donated in a sampling box by an IBS donor (selected from Example 2). Immediately after sample collection, an Anaerogen bag was added and the box was immediately sealed. The powder in the Anaerogen bag immediately removed all oxygen from the sampling box. Subsequently, anaerobic PBS was added to the fecal sample and a fecal slurry was prepared by homogenization in a stomacher at a concentration of 40 g/100 ml_. The fecal slurry was briefly centrifuged to remove large particles. Afterwards, an equal volume of cryoprotectant solution, was added to the fecal supernatant in the anaerobic workstation to reach a concentration of 20 g/100 ml_. After homogenization, the cryoprotected fecal slurry was snap-frozen in liquid nitrogen and stored at -80°C. At the start of the SHIME experiment, 5% of preserved inoculum was added to the colonic reactors.

- Treatment period: During this 10-day period, the SHIME reactor was operated under nominal conditions (pH = 6.15-6.4, T = 37°C, Mix speed = 300 rpm, Volume = 500 mL) and fed 3x/day with a sugar-depleted SHIME nutritional medium ( 3g/l yeast extract; 1 g/L peptone; 3g/L mucin; 0.5 g/L L-cysteine), supplemented with the test products as described by Possemiers et al. (2004) FEMS Microbiol Ecol. 49(3):495-507. The medium-term SHIME consisted of the same test conditions as in example 2, with doses in the nutritional medium adjusted to reach similar concentrations in the colon vessels upon repeated intake:

- 100% FODMAP ( 2.38 g/L FOS + 0.6 g/L stachyose + 0.6 g/L raffinose + 4.76 g/L lactose in the nutritional medium)

- 100% FODMAP + HMOs (composition above + 6.67 g/L 2FL/DFL + 1 .67 g/L lacto-N- triose)

Pre-digested FODMAP (0.14 g/L stachyose + 0.29 g/L raffinose + 0.31 g/L lactose in the nutritional medium)

Pre-digested FODMAP + HMOs (composition above + 6.67 g/L 2FL/DFL + 1 .67 g/L lactose-N-triose)

On day 9, samples from each reactor of the medium-term SHIME experiment were taken for gas production analysis in a short-term colonic simulation.

Short-term colonic simulation for gas analysis

The short-term gas analysis colonic simulation consisted of the same test conditions as in example 2 (please see Table 3):

- 100% FODMAP (0.86 g/L FOS + 0.21 g/L stachyose + 0.21 g/L raffinose + 1 .71 g/L lactose)

100% FODMAP + HMOs (composition above + 2.4 g/L 2FL/DFL + 0.6 g/L lacto-N- triose)

Pre-digested FODMAP (0.05 g/L stachyose + 0.10 g/L raffinose + 0.11 g/L lactose)

Pre-digested FODMAP + HMOs (composition above + 2.4 g/L 2FL/DFL + 0.6 g/L lacto-N-triose)

At the start of the short-term colonic simulations aforementioned substrates were added to a sugar-depleted nutritional medium that contains basal nutrients of the colon consisting of K2HPO4 3.5 g/L; KH2PO4 10.9 g/L; NaHC03 2 g/L; yeast extract 2 g/L (OXOID, Pratteln, Switzerland); peptone 2 g/L (OXOID, Pratteln, Switzerland); mucin 1 g/L (Carl Roth, Karlsruhe, Germany); cysteine 0.5 g/L; Tween80 2 mL/L).

In order to assess the gas production and composition, the reactors were inoculated with 10% of the pre-adapted communities derived from the medium-term SHIME experiment on day 9. A control condition, consisting of the sugar-depleted nutritional medium without test substrates, was included. Each condition was tested in duplicate, thus leading to 10 independent incubations.

Incubations lasted 48h at a temperature of 37°C, under continuous shaking (90 rpm) and anaerobic conditions. To achieve and maintain anaerobic conditions, the reactors were flushed with nitrogen and closed with air-tight caps. Analysis

Gas production and composition

The short-term incubations were performed in closed incubation systems. This allows to evaluate the accumulation of gases in the headspace, which can be measured with a pressure meter (Hand-held pressure indicator CPH6200; Wika, Echt, The Netherlands). Gas composition was analysed with gas chromatography. The gas phase composition was analyzed with a Compact GC4.0 (Global Analyser Solutions, Breda, The Netherlands), equipped with a Molsieve 5A pre-column and Porabond Q column (CH4, 02, H2 and N2) and a Rt-Q-bond pre-column and column (C02, N20 and H2S). Concentrations of gases were determined by means of a thermal conductivity detector.

Gas pressure and composition were determined at Oh, 6h, 24h and 48h.

Quantitative Microbial Community Analysis by 16S rRNA Gene Sequencing and

Flow Cytometry

Next-generation 16S rRNA gene amplicon sequencing of the V3-V4 region was performed by LGC Genomics GmbH (Berlin, Germany) on samples from the medium-term SHIME experiment. Library preparation and sequencing were performed on an lllumina MiSeq platform with v3 chemistry. The 341 F (50-CCTACGGGNGGCWGCAG-30) and 785R (50- GACTACHVGGGTATCTAAKCC-30) primers were used as described by De Paepe et al. (2017) with the reverse primer being adapted to increase coverage. Quality control PCR was conducted using Taq DNA Polymerase with the Fermentas PCR Kit according to the manufacturers’ instructions (Thermo Fisher Scientific, Waltham, MA, USA). The DNA quality was verified by electrophoresis on a 2% (w/v) agarose gel for 30 min at 100 V. Bioinformatics analysis of amplicon data was performed as described by De Paepe et al. (2017).

The obtained high-resolution proportional phylogenetic information (i.e., proportional abundances (%)) was combined with an accurate quantification of total bacterial cells via flow cytometry to obtain quantitative data at phylum, family and species level. This was done by multiplying the proportional abundances with absolute cell numbers (cells/mL) obtained via flow cytometry. For flow cytometry analysis, 10-fold serial dilutions were prepared in Dulbecco’s phosphate-buffered Saline (DPBS) (Sigma-Aldrich, Bornem, Belgium) of all samples and stained with 0.01 mM SYT024 (Life Technologies Europe, Merelbeke, Belgium) for 15’ at 37°C in the dark. Samples were analyzed on a BD Facsverse (BDBiosciences, Erembodegem, Belgium) using the high-flow-rate setting and bacteria were separated from medium debris and signal noise by applying a threshold level of 200 on the SYTO channel.

Simpson diversity index was calculated as described by De Paepe et al. (2017).

Results

The combination of FODMAP enzymes and HMOs decrease gas pressure after 6 h incubation (Figure 4A) FODMAP alone (‘FODMAP’) and with HMO (‘FODMAP+HMO’) led to an increase in gas pressure compared to the blank. The pre-digested FODMAP by FODMAP-enzyme mix (‘FODMAP+Enzyme’) reduced gas pressure significantly, indicating that FODMAP substrates were degraded efficiently by the FODMAP-enzyme mix. Treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in lower gas pressure compared to FODMAP alone, and ‘FODMAP+HMO’. Especially the reduction in gas pressure in the ‘FODMAP+Enzyme+HMO’ sample, compared to the FODMAP sample, is surprising since the total fermentable sugar in the ‘FODMAP+Enzyme+HMO’ sample is even higher (see Table 3).

The combination of FODMAP enzymes and HMOs decrease C0 ₂ after 6 h incubation (Figure 4B) FODMAP alone (‘FODMAP’) and with HMO (‘FODMAP+HMO’) led to an increase in C0 ₂ percentage of the headspace inside the reactors compared to the blank. The pre-digested FODMAP by FODMAP-enzyme mix (‘FODMAP+Enzyme’) reduced the CO2 percentage to the blank level, indicating that FODMAP substrates were degraded efficiently by the FODMAP-enzyme mix.

^■ Treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in lower C0 ₂ percentage compared to FODMAP alone, and ‘FODMAP+HMO’.

The combination of FODMAP enzymes and HMOs increased absolute abundance of beneficial microbes and decreased absolute abundance of detrimental microbes (Figure 5) Treatment with ‘FODMAP’ and ‘FODMAP+Enzyme’ resulted in the same abundance of Firmicutes compared to the blank. On the other hand, treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in an increase abundance of Firmicutes compared to the blank, ‘FODMAP’, ‘FODMAP+HMO’ and ‘FODMAP+Enzyme’ (Figure 5A). This increase in abundance of the phylum Firmicutes corresponded to the increase in abundance of the family Lachnospiraceae, a family belongs to the phylum Firmicutes. Indeed, treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in an increase abundance of Lachnospiraceae compared to the blank, ‘FODMAP’, ‘FODMAP+HMO’ and ‘FODMAP+Enzyme’ (Figure 5A). This increase in abundance of the family Lachnospiraceae corresponded to the increase in abundance of several species, including Eubacterium ramulus, Clostridium boitae, Blautia obeum/wexlerae and Lachnospiraceae sp., which belong to the family Lachnospiraceae. Indeed, treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in a higher abundance of Eubacterium ramulus, Clostridium boitae, Blautia obeum/wexlerae and Lachnospiraceae sp. compared to the blank, ‘FODMAP’, ‘FODMAP+HMO’ and ‘FODMAP+Enzyme’ (Figure 5A). Treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in a decrease abundance of phylum Desulfobacteria compared to the blank, ‘FODMAP’, ‘FODMAP+HMO’ and ‘FODMAP+Enzyme’. This decrease in abundance of the phylum Desulfobacteria corresponded to the decrease in abundance of the family Desulfovibrionaceae, a family belongs to the phylum Desulfobacteria. Indeed, treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in a lower abundance of Desulfovibrionaceae compared to the blank, ‘FODMAP’, ‘FODMAP+HMO’ and ‘FODMAP+Enzyme’ (Figure 5B).

The combination of FODMAP enzymes and HMOs increased microbial diversity compared to FODMAP alone and FODMAP + HMO (Figure 6) Treatment with ‘FODMAP’ and ‘FODMAP+HMO’ resulted in lower microbial diversity compared to the blank. Treatment with the combination of FODMAP-enzyme mix and HMO (‘FODMAP+Enzyme+HMO’) resulted in a higher microbial diversity compared to the ‘FODMAP’ and ‘FODMAP+HMO’.

Example 4: Comparison of FODMAP digestion

The enzymes used in Example 1 were compared with the commercial product Quatrase Forte (Disolut). This enzyme blend contains Alpha galactosidase (1200 units), Xylose Isomerase (7500 Units), Lactase (10,000 FCC), Invertase (500 Units) per capsule according to the specifications of the supplier. Measurement of the activity of the enzymes in the Quatrase material using the ALU, GalU and SU assays described above confirmed the presence of similar amounts of the lactase, alpha-galactosidase and invertase in the Quatrase material, when compared to the numbers provided by Disolut. Xylose isomerase activity was undetectable in the Quatrase material.

This experiment was performed exactly as described in Example 1 , except that both the enzymes and the substrates were blended before addition to the gastric phase. Enzyme dosage was a blend of 2500 SU Maxinvert, 2400 GalU Fibractase and 10,000 ALU Tolerase L per 100 ml final volume. The Quatrase enzyme dosage (200 mg) was calculated from the recommended dosage given by the supplier (3 capsules per meal) and adjusted for the meal-size in the in-vitro experiment. This dosage of Quatrase contains 300 SU invertase, 750 GalU alpha-galactosidase and 6000 ALU lactase per 100 ml final volume The substrate addition to the experiment was 2 g FOS, 5 g Lactose, 0.5 g Stachyose and 0.5 g Raffinose per 100 ml final volume. After the gastric and small intestinal incubations, as described in Example 1 , the concentrations of the sugars were determined by HPAEC-PAD. The results shown in Table 4. The conversion of both lactose and raffinose with the enzyme blend was comparable to that of Quatrase. The amount of stachyose that was converted by the enzyme blend was however much higher than with Quatrase. The amount of free glucose and galactose was comparable between the two experiments, but the amount of released fructose was a factor 4 higher with the enzyme blend. A higher fructose release in this experiment indicates that the Enzyme blend as described here digests more fructans compared to the prior art Quatrase blend. This was confirmed by examining the conversion of fructan as shown in Figure 7. The Enzyme blend of this invention shows a clearly improved fructan digestion compared to Quatrase, while the lactose and raffinose digestion is comparable.

Table 4: HPAEC-PAD quantification of the sugar content (mg/100 ml) after enzyme incubation.