EP2890249A1 - Use of an intensive sweetener as food additive - Google Patents

Abstract

The invention concerns the use of a food additive containing at least one intense sweetener, orally and/or rectally administered, to modify flora and/or fermentation in the digestive tract of an animal. The food additive is used to increase the amount of fiber in a diet formula for the animal compared to a previous diet formula without modifying animal zootechnical performance and/or to decrease the amount of starch and/or simple carbohydrates in a diet formula for the animal compared to a previous diet formula without modifying animal zootechnical performance. For example, the animal is fed with a diet formula containing a decreased amount of carbohydrates minus fiber, said decreased amount corresponding to an amount of carbohydrates minus fiber inducing, without said food additive, a glucose uptake rate inferior to an optimal glucose uptake rate, especially inferior to 80% of the maximal glucose uptake rate for the animal.

Description

Title : Use of an intense sweetener as food additive

Introduction

The invention concerns the use of an intense sweetener orally or rectally as a food additive for an animal.

The basic philosophy behind Pancosma scientific approach is to create value in the animal nutrition field by incorporating recent scientific advances that will lead to consistent enhancement of zootechnical efficiency. Up to now, the most achieved example of Pancosma's scientific global approach is certainly XTRACT® for poultry. The first step was to understand the basic mechanism by which phytonutrients affect the immune response and microbial structure of the gut ecosystem. The next step was then to legitimize the product, which consisted of demonstrating that these effects are converted into an improvement in metabolizable energy and in net energy via a decrease in the maintenance energy requirement. This led to the "upper value for XTRACT®" suggesting an equivalence: "100 ppm of XTRACT® = 50 kcal/kg of ME". Finally, the third step was to measure efficacy of the additive and this was performed by compiling trials and carrying out a meta-analysis.

This illustrates the interest in basic research at Pancosma that materialized the scientific approach strategy. Indeed, it legitimizes the intellectual way of thinking that eventually led to the upper value system. In a way, it finalizes the whole strategy as it supports a tool that improves either growth of the animal and the return for our customer.

The effects of sweeteners are fascinating because it illustrates the technical dossier and supports unexplained but consistent improvements of growth efficiency with the sweetener (SUCRAM®). Glucagon-like peptide 2 (GLP-2) is undoubtedly the new frontier of nutrition and nutritional endocrinology of transition animals for the 21 st century.

Summary of the invention In this context, the invention concerns the use of a food additive containing at least one intense sweetener, orally and/or rectally administered, to modify flora and/or fermentation in the digestive tract of an animal.

Advantageously, the use of the invention comprises one or more of the following features:

- the food additive is used to increase the amount of fiber in a diet formula for the animal compared to a previous diet formula without modifying animal zootechnical performance;

- the sweetener is used to decrease the amount of starch and/or simple carbohydrates (for example glucose, lactose, saccharose, ...) in a diet formula for the animal compared to a previous diet formula without modifying animal zootechnical performance;

- wherein the animal is fed with a diet formula containing a decreased amount of carbohydrates minus fiber, said decreased amount corresponding to an amount of carbohydrates minus fiber inducing, without said food additive, a glucose uptake rate inferior to an optimal glucose uptake rate, especially inferior to 80% of the maximal glucose uptake rate for the animal;

- the animal being fed with a diet formula containing a determined level of digestible energy, the food additive is used to decrease the level of digestible energy of the diet formula compared to a previous diet formula without modifying animal zootechnical performance;

- the reduced digestible energy level of the diet formula corresponds to a digestible energy level inducing, without food additive, a glucose uptake rate inferior to an optimal glucose uptake rate, especially inferior to 80% of a maximal glucose uptake rate for the animal;

- the diet of the animal contains an amount of carbohydrates inducing a level of glucose uptake inferior to the optimal level of glucose uptake of the animal, especially inferior to a level of glucose uptake higher than 80% of the maximal glucose uptake rate for the animal, in order to improve animal performance;

- the diet of the animal is formulated with digestible energy levels inducing a level of glucose uptake inferior to the optimal level of glucose , especially inferior to a level of glucose uptake higher than 80% of the maximal glucose uptake rate for the animal, in order to improve animal performance;

- the animal is a monogastric animal;

- the animal is a ruminant;

- the animal is a pre-ruminant;

- the animal is a swine selected among the following categories: Piglet, Pig, Sow;

- the animal is selected among the following species: Chicken, Guinea fowl, Turkey, Quail, Fish;

- the animal is a bovine, a caprine or an ovine;

- the animal is a calf, a kid or a lamb;

- the animal is an equine;

- the animal is a rabbit;

- wherein the intense sweetener is selected among artificial sweeteners, sweeteners from natural origin and/or identical to nature;

- the sweetener is selected among saccharin and its salts, acesulfame-K, cyclamate, aspartam, stevioside or any other intense sweetener from natural origin such as stevia or a stevia extract;

- the food additive comprises a potentiator;

- the potentiator comprises at least one of the comprising compounds, glycyrrhizin, ammonium glycyrrhizinate, potassium glycyrrhizinate, sodium glycyrrhizinate, thaumatin, kokumi, neohesperidin dihydrochalcone (NHDC), the ribotides and sodium glutamate;

- the food additive comprises from 20% to 0% by weight of potentiator(s);

- the intense sweetener includes a mixture of NHDC and saccharin and/or stevia; - the intense sweetener is detected by a bacteria receptor membrane able to detect nutrients within its environment;

- it causes an increase of Lactobacilli populations;

- it causes an increase in the L. amylovorus population;

- it causes an increase in the population of Lactobacilli OUT 1946 population; - it causes a decrease in the Clostridia and/or Escherichia coli populations in the digestive tract;

- it causes an increase in lactate and/or lactate and/or lactic acid production; - it causes a reduction of propionate production;

- it acts on animal immunity.

Brief description of the figures

The invention will be better understood by the following specification in reference to the attached figures in which:

Figure 1 represents the caecal lactobacillus population abundance in several groups of piglets having different diets;

Figures 2 represents the relative proportions of different lactobacillus OTUs in the caecal lactobacillus population for several groups of piglets having different diets;

Figures 3A and 3B represent the microbial community structure in several groups of piglets having different diets;

Figure 4 represents the bacteroides population abundance and the Clostridia population abundance in several groups of piglets having different diets;

Figure 5 represents the caecal population abundance of the Lactobacilli in several groups of piglets having different diets;

Figure 6 represents the lactate concentrations in caecum in several groups of piglets having different diets;

Figure 7 represents lactic acid concentrations in the caecum in two groups of piglets having different diets;

Figure 8 represents some parameters of performances in several groups of piglets having different diets;

- Figure 9 represents the stool quality in several groups of piglets having different diets;

Figure 10 represents the rate of glucose update with increasing doses of glucose or lactose with ou without sucram;

Figure 1 1 represents the caecal microflora composition in several groups of piglets having different diets; Figure 12 represents the caecal microflora abundance of lactobacillus and caecal lactate concentration in several groups of piglets having different diets;

Figure 13 represents the microbial community structure of piglets in several groups of piglets having different diets;

Figure 14 represents the caecal lactobacillus population abundance in several groups of piglets having different diets;

Figure 15 represents the total lactobacillus populations in pigs fed the diets represented in figure 14 and the relative proportions of different lactobacilli OTU's;

Figure 16 represents the relative MCT1 mRNA expression in pig proximal colon in several groups of piglets having different diets;

Figure 17 represents performances of piglets in several groups of piglets having different diets;

Figure 18 represents the rate of glucose uptake with the different diets of figure 17;

Figure 19 represents the microbial community structure of piglets in several groups of piglets having different diets;

Figure 20 represents the caecal lactobacillus population abundance in several groups of piglets having different diets;

Figure 21 represents the lactate concentrations in the caecum of pigs fed differents diets;

Figure 22 represents the evolution of the rate of glucose uptake with the dietary amount of carbohydrates minus fibers;

Figure 23 represents the evolution of average daily gain with dietary digestible energy content;

Figure 24 represents glucose or sweet sensors connected with metabolic processes utilizing carbohydrates in mammalian and yeast, compared with lactobacillus.

MICROFLORA AND IMPACT ON PERFORMANCE MICROFLORA KNOWN TO BE IMPACTED BY NUTRIENTS NO INFORMATION THAT MICROFLORA COULD DE INFLUENCED BY SWEETENERS ?

The way to manipulate GLP-2 secretion should attract a lot of interest as GLP-2 is eminently protective for the gut structure. It inaugurates a relatively new way to consider gut health. Up to now, most interest in gut health was dedicated to research on directly controlling gut microbiota or on modulating the immune response. The new way of thinking consists of considering the host as a target for technologies and, in this case, GLP-2 is a candidate bioactive peptide. However, injecting or delivering GLP-2 via the diet is unlikely to be authorized in farm animals due to the obvious concern held by consumers regarding the use of drugs or synthetic compounds in the diet of the farm animals. Nonetheless, finding agonists that could be included in the diet and evoke the secretion of this bioactive compound remains very interesting. Thanks to the work performed at Pancosma, we now know that GLP-2 is released by the gut of piglets, dairy cows, and calves when these animals are fed SUCRAM®. The detailed effects of SUCRAM are established and mediated by enteroendocrine cells and their taste receptors. In addition to its influence on the gut architecture, GLP-2 also enhances sugar digestion and absorption.

At weaning, the industry is providing highly energetic compounds in order to improve animal growth. In order to provide this energy, different types of carbohydrates are used alone or in combinations. They can be splitted into different categories such as sugars (ex. :_glucose, lactose), hydrolysable carbohydrates (ex. starch in ground wheat), quickly fermentable fibers (ex. : soyhull) or slowly fermentable fibers (beet pulp).

MICROFLORA MODIFICATIONS AT WEANING

In reference to figure 1 , we used pyrosequencing which is a latest method for DNA sequencing. We were allowed to determine changes in bacterial diversity and community structure of piglet's large intestine when they were suckling (S), weaned and fed a control diet (D1 ), a diet containing SUCRAM® (D2) or a diet containing lactose (D7).

Our data show, (see previous figure) that in response to inclusion of SUCRAM® in piglets' feed, there is a significant increase in caecal (first part of the large intestine) lactobacillus population abundance (D2) compared to samples obtained from the caecal contents of piglets given the same diet without SUCRAM® (D1 ). Inclusion of lactose in the diet also resulted in significant increase in caecal lactobacillus population (D7). Figure 2 shows that enhancement of caecal lactobacillus population abundance, due to addition of SUCRAM® or lactose, is entirely due to increase abundance of OTU9641 , a pig specific lactobacillus species.

Abbreviations used: Sucking= 2 week old animals having sow's milk; HC=hydrolysable carbohydrate diet with no supplementation; HC+S= the same diet + Sucram; HC+Lac= the same diet+ lactose.

As known lactobacillus is important to enhance immunity, growth and fight the intestinal pathogens.

It is known that lactose - a substrate for lactic acid production by lactobacillus - enhances lactobacillus population. Lactose is absorbed by lactobacillus and used as the source of energy leading to propagation of this species. However saccharin is neither absorbed nor metabolised. We have measured the concentration of saccharin in caecal contents of piglets given SUCRAM® and it is equal to that included in the feed. We conclude that saccharin must be sensed by a membrane sensor (bacterial sweet sensor) activating a downstream pathway resulting in the enhancement in the level of transport protein responsible for the absorption of available nutrients such as propionate or butyrate, providing energy for growth and propagation of this species. This is the first ever indication of a bacterial sweet sensor. Based on this observation, the mode of administration of the sweetener could also be the rectum. Accordingly, an embodiment of the invention concerns the use of intense sweetener as a technology to target the gut ecosystem and increase the population of beneficial bacteria. An embodiment of the invention concerns also the fact that, for the first time, a bacterial sweet sensor has been identified and can be a target for manipulation. Under sweeteners one can understand products and/or molecules such as saccharin and its salts, acesulfame-K, cyclamate, aspartame, steviosides or any intense sweetener from natural origin such as stevia or stevia extract. They can be combined with some potentiators such as glycyrrhizin, ammonium and/or potassium glycyrrhizinate, thaumatin, kokumi, neohesperidin dihydrochalcone (NHDC), ribotides and sodium glutamate. The sweetener being preferably a mixture of NHDC and saccharin and/or stevia. The term sweetener is meant as any substance or compound, chemical or natural, having a sweet taste. In an embodiment of the invention, by "intense sweetener" is meant a sweetener with a sweetening power at least 20 times higher than sugar.

Method and material

Overall aims was to characterize the large intestinal microbiota of piglets weaned to diets containing either soluble digestible or fermentable carbohydrates, and piglets weaned to diets supplemented with natural or artificial sweeteners.

A total of 15 sets of 8 piglets (1 suckling; 14 weaned; total number of pigs = 120) fed different dietary formulations have been sacrificed and colonic contents and tissue samples have been collected from various regions of both the small and large intestine.

DNA has been extracted from caecal and faecal contents from several study groups of pigs:

[1]: 28 day old suckling pigs only fed on sow's milk.

[2]: 41 day old pigs weaned onto a hydrolysable carbohydrate diet (Diet 1 ) with principle component: Ground wheat (30% w/w). [3]: 41 day old pigs weaned onto a hydrolysable carbohydrate + SUCRAM® diet (Diet 2) with principle components: Ground wheat (30% w/w), NHDC + Saccharin (SUCRAM®).

[4]: 41 day old pigs weaned onto a fermentable carbohydrate diet (Diet 13) with principle components: Unmolassed sugar beet pulp (5% w/w), Inulin (0.75% w/w) & Wheat dextrin (3% w/w).

[5]: 41 d old pigs weaned onto Diet 1 (hydrolysable carbohydrate) with Stevia added to their drinking water (Diet 15).

Extracted DNA was used as template for the PCR amplification of 16S rRNA gene sequences using universal primers targeted to the V1 -V3 region of eubacterial 16S rDNA. Sequences generated were ~500bp. This sequence length will allow over 99% of sequences to be phylogenetically classified.

Each amplicon library consisted of 16 different samples from each study group (8 caecal samples / 8 faecal samples) identifiable by Muliplex Identifier (MID) tags included in each primer set. Amplicon libraries were sequenced on a GS-FLX Titanium sequencing platform producing an average of over 20,000 sequence reads per sample.

Bioinformatic analysis of sequences is ongoing. Sequences are being clustered and aligned using the Pyrosequencing Pipeline tool of the Ribosomal Database Project (RDP), using a similarity threshold of 97% to define each operational taxonomic unit (OTU). Taxonomic assignments are then made by directly comparing OTU's to reference RDP databases. Comparison of community structure and diversity between samples employs several statistical methods including UniFrac analysis. UniFrac analysis allows the creation of 3D plots that show spatial representation of community structure and profile by clustering samples that contain common OTU's and separating those that contain distinct OTU's.

Phylogenetic analysis and classification of OTU's allows identification of bacterial groups that are altered with dietary variation. Quantitative analysis of such alterations is possible by counting OTU's belonging to specific taxa and expressing as a percentage of the total number of OTU's. Also measured in colonic content samples from these 4 study groups of pigs are the concentrations of monocarboxylic acids (short chain fatty acids; SCFA), which are the metabolic products of micriobial fermentation of dietary carbohydrate. The proportions of SCFA produced by microbial fermentation can often be altered by dietary variation, and may be beneficial or deleterious to the host animal.

Results on microbial community structure and phylogenic classification

Control versus control + SUCRAM® versus control + fermentable fiber

UniFrac analysis has highlighted significant differences in microbial community structure, between suckling and weaned pigs; between weaned pigs fed diets containing different carbohydrate types; between weaned pigs fed identical diets with or without SUCRAM®®, as it appears on figures 3A and 3B.

UniFrac analysis also highlighted significant differences in microbial community structure between different regions of the colonic tract (caecal [right] v faecal [left] samples)

Sequence analysis and subsequent phylogenetic classification has identified the major microbial taxa present in the pig colonic tract and highlighted alterations in population abundance of certain microbial groups in response to dietary variation. Results show 3 major taxa dominate the pig colonic microbiota: Bacteroides, Clostridia and Lactobacilli. As it appears on figure 4, using results from caecal samples, the population abundance of Bacteroides tended to be lower in weaned pigs compared to suckling. In contrast, the population abundance of the Clostridia group tended to be higher in weaned pigs compared to suckling. The results are showing that a decrease of the population of Clostridia with SUCRAM®. As it appears on figure 5, the caecal population abundance of the Lactobacilli was very similar in 3 of the 4 study groups (suckling, hydrolysable carbohydrate & fermentable carbohydrate). However, in pigs fed the hydrolysable carbohydrate + SUCRAM® diet, the caecal population abundance of this group was up to 4-fold higher than in pigs fed the identical diet without SUCRAM®.

Enhancement of caecal Lactobacilli population is potentially very important due to the benefits that this group is believed to have on gut health. In addition, the abundance of Lactobacilli decreases dramatically from caecum to faeces in pigs fed the hydrolysable carbohydrate + SUCRAM® diet.

Using faeces only to assess microbiota would not reveal enhancement of

Lactobacilli in response to diet containing SUCRAM®. This is an important result because it points out that this result is not obvious.

Other results were very consistent with this observed increase of

Lactobacilli with SUCRAM®. For example, figure 6 presents that lactate concentrations were significantly higher in the caecum of suckling pigs compared to weaned ones. It is worth noting that analysis of lactic acid (the major metabolic product of

Lactobacilli) concentrations in the caecum showed an enhancement of lactic acid in pigs fed the hydrolysable carbohydrate + SUCRAM® diet compared to hydrolysable carbohydrate only, as represented on figure 7. This correlates with the observed increase of Lactobacilli in pigs fed the hydrolysable carbohydrate + SUCRAM® diet.

GUT PROTECTIVE EFFECTS OF SUCRAM® IN DIETS CONTAINING DIFFERENT CARBOHYDRATES TYPES OR LEVELS

In each trial, 28 days old Landrace X Large White crossbred suckling piglets (N = 4 of 2 piglets) were weaned onto proprietary formulation diets. RAW MATERIAL % ANALYSIS %

PORRIDGE OATS 17.00 OIL EE 8.44

STANDARD

MICRO GROUND 37.25 PROTEIN 22.1

WHEAT

MICRO GROUND 10.00 FIBER 2.9

CORN

POTATO PROTEIN 2.50 ASH 5.4

FFS EXTRUDED 7.50 DE PIG 16.7^*

WHITE FISH 20.00 T LYSINE 1 .65

L LYSINE 0.55 M+C 0.97

DL METHIONINE 0.25 THREO 1 .03

L THREONINE 0.20 TRYPT 0.25

SOYA OIL 2.40 STARCH 38.9

LIMESTONE 0.30 SUGAR 2.80

MONOCALCIUM PHOSPHATE 1 .00

SALT 0.50

WEANER TRIAL SUPPLEMENT 0.50

PIGLET FLAVOUR VANILLA 0.05

[1 ] Control diet; [2] As [1 ] + 150 ppm SUCRAM® C150;

[3] As [1 ] + 5% glucose; [4] As [3] + 150 ppm SUCRAM® C150;

[5] As [1 ] + 5% lactose; [6] As [5]+ 150 ppm SUCRAM® C150;

[7] As [1 ] + 5% beet pulp; [8] As [7] + 150 ppm SUCRAM® C150;

[9] As [1 ] + 5% soybean hulls; [10] As [9]+ 150 ppm SUCRAM® C150;

[1 1 ] As [1 ] + 5% beet pulp; [12] As [1 1 ] + 150 ppm SUCRAM® C150

Energy content was controlled by changing the inclusion of oil. Zootechnical parameters were measured daily during 13 days after weaning with BW, ADG, ADFI, ADWI and G : F. Stool grading was evaluated daily. SGLT1 and microflora composition were evaluated at the end of the experiment. Microbiota was characterized via sequence analysis of bacterial 16S rRNA genes using next-generation pyrosequencing technology. The 16S rRNA gene is ubiquitous in all bacteria; -1 ,500 nucleotides in length - long enough to provide sufficient information yet short enough to be easily sequenced. It contains both highly conserved and hypervariable regions at specific intervals. Microbial DNA was extracted from caecal contents of 41 d old piglets weaned onto each of the diets. 16S rRNA gene sequences (~500bp) encompassing the V1 -V3 region were amplified using PCR. Amplicons were sequenced on a GS-FLX Titanium 454 sequencing platform producing an average of over 15,000 sequence reads per sample. Bioinformatic analysis of sequence data sets allowed for spatial representations of the microbial community structure and profiles.

The data were analyzed using one treatment with Yij = μ + axBWO + ai + £ij, j = 4. Least Square Means corrected from the initial variability on BW0. Means separated by a Tukey test with P = 0.05. In a second analysis, the data were analyzed by a meta-analytical tool. The magnitude of the response to SUCRAM® was calculated in order to see if there was an interaction with dietary changes.

Results

Effect of SUCRAM® or 5% glucose or 5% lactose alone combination

In reference to figure 8, inclusion of SUCRAM® in the control diet increased feed intake and daily gain by a similar magnitude of +5.3% and +8.0%, respectively, leading to marginal changes in feed efficiency. Inclusions of either 5% glucose or lactose resulted in a highly deteriorated feed efficiency of -68% and -50%, respectively. Although the resulting gain to feed ratio was similar, the way to achieve this result was different: inclusion of 5% glucose did not affect feed intake but dramatically decreased daily gain (-64%). Conversely, inclusion of 5% lactose in the diet strongly increased feed intake (+20.9%) but decreased daily gain (-15.2%). On figure 8, "ADG" means average daily gain, "ADFI" means average daily feed intake and "G:F" represents the ratio between gain and feed intake. The quality of stools, as represented in figure 9,was degraded with the inclusion of both carbohydrates to 4.16 and 3.78 compared to 3.34 for the control diet. Finally, glucose absorption was not affected by the inclusion of neither glucose (253 pmol/s/mg) nor lactose (225 pmol/s/mg, value of the control diet: 234 pmol/s/mg).

When added on top of a 5% glucose or 5% lactose diet, SUCRAM® produced the same type of result on feed efficiency, with an increased gain to feed ratio compared with 5% glucose (0.24 vs. 0.12) or 5% lactose (0.35 vs. 0.19), that totally restored the efficiency of the control for lactose (0.35 vs. 0.38) but not for glucose (0.24 vs. 0.38). The way this improvement was achieved was different in the 2 diets. When added on top of 5% glucose, inclusion of SUCRAM® restored the daily gain to a level comparable with the control diet (1 19.8 g/d vs. 99.58 g/d), which was a dramatic increase compared to the 5% glucose diet (35.2 g/d). This was the consequence of a higher feed intake compared with the 5% glucose diet (498 g/d vs. 328 g/d). When added on top of 5% lactose, inclusion of SUCRAM® numerically depressed feed intake and weight gain but this was the result of a dramatic increase in feed efficiency.

These results were surprising because we would have expected positive results during the addition of glucose or lactose on the enhancement of growth performance. One Hypothesis could be that 5% glucose or lactose were not sufficient to enhance SGLT1 , thereby leading to a significant flow in unabsorbed glucose or lactose. These compounds would be fermented lower in the gut by the gut ecosystem; in weaning piglets, the gut ecosystem is known to be immature and easily disturbed.

In reference to figure 10, it was confirmed that the addition of 5% glucose or lactose does not upregulate SGLT1 , but this was accomplished with the addition of 10% of either carbohydrate. Furthermore, the addition of SUCRAM® alone or on top of each diet also raised SGLT1 .

Then, in reference to figure 1 1 , supplementation of the basal diet with 5% lactose resulted in a significant enhancement of the caecal lactobacillus population (expressed as a percentage of the total number of OTU's) from 8.3% to 23.8%, an increase of 286% (P < 0.01 ).

Furthermore, this increase was observed to be due to one particular OTU, designated lactobacillus OTU4228, which increased from 4.4% of the total microbiota in pigs weaned on to the basal diet to only 21 .0% of the total in pigs weaned on to a diet including 5% lactose. Moreover, measurements of lactic acid concentrations in caecal contents of piglets weaned on to the 5% lactose diet showed lactic acid to be present at a concentration of 15.2 mM, a 10-fold increase in caecal lactic acid concentrations in pigs weaned on to the basal diet only (1 .5 mM).

In reference to figure 12, a significant enhancement in the relative population size of caecal lactobacillus was similarly observed with inclusion of 150 ppm of SUCRAM® C150 to the basal diet. In these piglets, lactobacillus accounted for 18.0% of the total microbiota compared to 8.3% in piglets weaned on to the basal diet. This represents an increase of 217 %. Again, this increase was observed to be solely due to an increase in lactobacillus OTU4228 which comprised 15.3 % of the total microbiota in pigs weaned to the SUCRAM® containing diet. However, in contrast to that observed with addition of lactose, caecal lactic acid concentrations in response to SUCRAM® inclusion were increased by ~2-fold over caecal lactic acid in pigs weaned to the HC diet only (3.2 ±0.6 mM v 1 .5 ± 0.2 mM).

RESULTS ON MICROBIAL COMMUNITY STRUCTURE AND

PHYLOGENIC CLASSIFICATION (figure 13)

CONTROL versus CONTROL + SUCRAM® versus CONTROL + LACTOSE

UniFrac analysis of pyrosequencing data obtained from caecal samples of pigs weaned onto Diet 1 (HC), Diet 2 (HC + SUCRAM®) or Diet 7 (HC + Lactose) has revealed significant differences in microbial community profiles, especially between Diet 1 (unsupplemented) and Diets 2 & 7 (supplemented with SUCRAM® and lactose respectively):

In reference to figure 14, analysis of caecal microbiota of pigs fed Diet 7 (HC + Lactose) has revealed a dramatic increase in the population abundance of lactobacillus in comparison to pigs fed Diet 1 (HC), similar to that seen in pigs fed Diet 2 (HC + SUCRAM®). Lactobacillus abundance in pigs fed Diet 7 was 23.8% of the total microbiota (compared to 8.3% in pigs fed Diet 1 ). In reference to figure 15, detailed analysis of the lactobacillus populations in caecal contents of suckling pigs (S) and in weaned pigs fed Diet 1 (HC) show that the lactobacillus population is dominated by one particular strain (designated OTU9641 ; related to L. amylovorus) that accounts for over 70% of all lactobacilli in suckling pigs and over 50% in weaned pigs fed Diet 1 (HC). Furthermore, it is OTU9641 that is primarily responsible for the increase in total lactobacillus populations observed in response to SUCRAM® (Diet 2; HC+S) and also to lactose (Diet 7; HC+Lac), where OTU9641 represents over 80% of all lactobacilli in pigs fed these two diets.

Figure 15 shows the total lactobacillus populations in pigs fed these diets and also the relative proportions of different lactobacilli OTU's. OTU9641 is represented in blue. HC=Hydrolysable carbohydrate, S=SUCRAM®, Lac=lactose

GUT PROTECTIVE EFFECTS OF SUCRAM® IN DIFFERENT FIBER- CONTAINING DIETS

Inclusion of fermentable carbohydrate sources was postulated to cause an increase in SCFA concentrations due to increased fermentation. Soyhull and beet pulp were selected because they contain different types of fiber, with slowly or quickly fermented fibers, respectively. Analysis of the colonic SCFA transporter, monocarboxylate transporter 1 (MCT1 ), expressed on the luminal membrane of colonic enterocytes, revealed an upregulation in both mRNA and protein expression of MCT1 in the proximal colon (see figure 16). It was shown previously that MCT1 expression is subject to substrate- induced regulation. Therefore, the observed increase in MCT1 expression is indicative of increased SCFA production in response to the inclusion of fermentable carbohydrates. We did not observe this as an increase in SCFA steady-state levels due to the increased absorption of SCFA via MCT1 .

In reference to figure 17, the addition of soyhull did not affect feed intake but dramatically decreased weight gain (-23%), which was visible in the deteriorated feed conversion (1 1 .3 vs. 7.5). When beet pulp was added, feed intake was increased (+21 .8%) but weight gain was decreased (-29%), leading to a deteriorated feed conversion (53.7 vs.7.5).

For both dietary types, glucose uptake was dramatically reduced. When SUCRAM® was added on top of these 2 diets, these detrimental effects were totally counterbalanced (see figure 18).

RESULTS ON MICROBIAL COMMUNITY STRUCTURE AND PHYLOGENIC CLASSIFICATION

CONTROL versus CONTROL + LACTOSE versus CONTROL + FERMENTABLE FIBERS

Using UniFrac, we have analysed the caecal microbial community profiles of pigs weaned onto diets containing two different fermentable carbohydrate sources; soya hulls (Diet 1 1 ) and unmolassed sugar beet pulp (Diet 13) (both these diets also contained inulin and wheat dextrin). We have then compared these community profiles to profiles obtained from caecal samples of pigs fed Diet 7 (HC + Lactose), as both fermentable carbohydrate diets (1 1 & 13) also contain the same amount of lactose as Diet 7 (see figure 19).

One interesting aspect of the results obtained from caecal samples of pigs fed Diets 1 1 & 13 is that, even though these diets contain the same amount of lactose as Diet 7, we do not see the same enhancement in Lactobacillus population abundance (in comparison to Diet 1 ) observed with Diet 7, as it appears on figure 20. We have already reported increased lactate concentrations in the caecum of pigs fed Diet 2 (HC +SUCRAM®; 3.2mM) in comparison to pigs fed Diet 1 (HC; 1 .5mM), concomitant with an increased lactobacillus population (8.3% Diet 1 ; 20.5% Diet 2). We can now further report increased lactate concentrations in the caecum of pigs fed Diet 7 (HC + Lactose; 15.2mM) and Diet 15 (HC + Stevia; 8.8mM), as represented in figure 21 . The increase in lactate concentration seen in pigs fed Diet 7 is also concomitant with an increased lactobacillus population (see above). The analysis of microbiota for Diet 15 is still ongoing.

It is worth noting the difference in lactate concentration between Diet 2 (HC + SUCRAM) (3.2mM) and diet 7 (HC + Lactose) (15.2mM) despite similar lactobacillus population sizes (20.5% diet 2; 23.8% diet 7). This may due to the fact that lactose provides a metabolisable energy source for lactobacillus whereas SUCRAM® does not. This may also be true for Diet 15 (HC + Stevia) as Stevia is also metabolisable by colonic microbiota. It is also worth noting that the increase in lactate concentrations in caecal contents of pigs fed Diets 2, 7 & 15 are concomitant with decreases in the concentration of propionate.

Discussion

Taken together, these results support the use of SUCRAM® beyond simply enhancing feed intake and sweetening the diet. These new research insights demonstrate that SUCRAM® prompts two main molecular effects. The first one is the enhancement of SGLT1 via GLP-2, which corresponds with a host response; the second is the enhancement of the lactobacilli population, which represents a gut microbiota response. Interestingly, both molecular effects are protective and correlate very well with an improved zootechnical response when SUCRAM® was added to the diet. In contrast, a diet containing 5% lactose alone was not associated with improved zootechnical results. It could be speculated that the enhancement of favorable lactobacilli could be relatively detrimental to these weaning piglets whose gut ecosystem is still immature and whose gut mucosa undergoes major changes due to the weaning process. However, this may not explain the dramatic deterioration of zootechnical parameters. An additional hypothesis could be that by including fibers instead of starch, the gut of these piglets did not receive enough GLP-2 native agonists. This could limit gut maturation and exacerbate gut microbial proliferation. By affecting both the gut mucosal architecture and the gut ecosystem, SUCRAM® was able to provide a beneficial effect (via the gut ecosystem) while ensuring the protection of the epithelium in the case of over fermentation. Interestingly, SGLT1 was enhanced to a level comparable to the negative control. The decrease in SGLT1 with the addition of fiber, and the re-increase following the addition of SUCRAM®, correlates very well with the decrease and then recovery of zootechnical responses. This may suggest that upregulation of the GLP-2 pathway, which is sustaining the SGLT1 effect, is highly beneficial. GLP-2 is being secreted when there are nutrients in the gut but it may be possible that simple sugars are even more important and that they serve as a signal for gut maturation due to the presence of the energy fuel in the gut. By producing this effect, SUCRAM® may stimulate the GLP-2 pathway in piglets and promote gut maturity. Including fibers also decreased the GLP-2 pathway, which is detrimental as GLP-2 is the main factor controlling gut growth and gut health. It seems that there is a baseline of SGLT1 that promotes gut health.

These results also indicate that SGLT1 could be used as an indicator of gut maturity or quality. A level of 400 units of SGLT1 is considered good for mature mucosa of an adult pig. Then, a rough calculation could help to calculate the amount of SUCRAM® that could be added to diets for swine in order to optimize SGLT1 and, most importantly, GLP-2 which is sustaining it. More simply, taking the piglet as a model, equivalences between 150 ppm of SUCRAM® and dietary carbohydrates can also be calculated using a slope ratio assay technique (see figure 22). Another interesting outcome is shown in figure 23. Decreasing dietary digestible energy content in the control groups led to a numerically decrease average daily gain of the piglets. This correlated very well with a lower glucose absorption capacity which is an indicator of GLP-2 secretion. However, inclusion of SUCRAM® both enhanced the average daily gain, despite the DE decrease, and the glucose absorption capacity.

Lactobacilli are the predominant lactic acid bacteria found in pig intestine and constitute a major proportion of the entire intestinal microbiota. As such, they are of particular importance to the maintenance of gut health. The presence and activity of lactobacilli have a stimulatory effect on gut immunity and maturation, enhances immune protection, and reduces gastrointestinal inflammatory responses. Using PCR amplification of bacterial 16S rRNA gene sequences and subsequent pyrosequencing, we reported significant enhancements in the relative population abundance of lactobacilli in the caecal contents of piglets in response to dietary supplementation with either a natural sugar, lactose, or an artificial sweetener, saccharin. Although the response of lactobacillus OTU4228, in terms of increased population abundance, is similar in piglets weaned to diets supplemented with either lactose or SUCRAM®, the disparity between caecal lactic acid concentrations suggests that the underlying mechanisms are quite different. Increases in lactobacillus population abundance have previously been demonstrated in piglets fed diets supplemented with lactose, primarily due to the metabolism of lactose by lactobacilli. The highly fermentable nature of lactose is reflected in the large increase in lactic acid concentrations seen in the caecal contents of piglets weaned to the lactose containing diet (population abundance of lactobacilli increases ~3-fold; lactic acid increases ~10-fold). In contrast, the increase in lactic acid concentration measured in piglets weaned to SUCRAM® is in proportion to the increase in lactobacillus population abundance (both increase ~2-fold). This suggests that, unlike lactose, which is acting as a prebiotic by providing an additional substrate for the growth of lactobacilli and subsequent lactic acid production, saccharin is not a metabolizable energy source that can be fermented by lactobacillus populations to produce lactic acid. This is supported by HPLC measurements of saccharin concentrations showing no difference between caecal and rectal contents of piglets weaned to the HC + S diet (-300 ppm in each). These results indicate that saccharin remains intact during transit through the intestine and is not metabolized by the pig intestinal microbiota, in agreement with previous studies. The effects of artificial sweeteners on the gut microbiota have previously been studied in humans. The addition of maltitol, a sugar alcohol, to confectionary significantly enhanced the population abundance of both bifidobacteria and lactobacilli. It is notable, however, that maltitol is a fermentable substrate for these gut microbes, whereas saccharin is not.

In reference to figure 24, it has been shown that in the mammalian intestine, the sweet taste receptor, T1 R2-T1 R3, expressed in enteroendocrine cells, can detect the presence of sugars and SUCRAM® and initiate an intracellular signalling pathway leading to upregulation of the intestinal glucose transporter, SGLT1 . Likewise, yeasts, such as Saccharomyces cerevisiae, possess mutated glucose transporters (Snf3 and Rgt2) that act as transmembrane sweet sensors controlling the expression of hexose transporter proteins in the presence of glucose and other sugars. Lactobacilli, and many other enteric bacteria, express multiple sugar transport and metabolic systems that allow them to utilize a variety of carbohydrate substrates and adapt quickly to changes in nutrient availability. This versatility is of particular importance in an environment such as the gastrointestinal tract. The predominant sugar transport mechanism in these bacteria is the Phosphoenopyruvate:Carbohydrate Phosphotransferase system (PTS); with over 20 different PTS systems identified, each specific for only one or a few sugars. There are also multiple non-PTS sugar transport systems, such as non-PTS permeases and ABC transporters for various poly- and oligosaccharides. The vast majority of these systems are regulated in the presence of the specific substrate, or subject to catabolite repression/inducer exclusion in the presence of preferred substrates (eg. glucose)

Extracellular sensing is a key method employed by bacteria in order to respond to changes in their environment, such as alterations in pH, chemical composition, or nutrient availability. Many of these sensory responses are independent of transport or metabolism, but involve the binding of chemical ligands to membrane-spanning sensory proteins in order to initiate intracellular signalling. This can be observed in the phenomenon of chemotaxis, where bacteria can respond to environmental changes by moving up or down chemical or nutrient concentration gradients. One of the most important mechanisms for environmental nutrient sensing and signal transduction in enteric bacteria are hybrid two-component systems (HTCS), which are typically composed of a membrane-spanning sensor histidine kinase (HK) and an intracellular cytoplasmic response regulator (RR). Ligand activation of the HK sensory domain leads to a phosphoryl transfer cascade to the RR and initiation of intracellular signal transduction. Indeed, recent evidence has shown that transcription of genes responsible for utilization of diverse polysaccharides by enteric Bacteroides species can be directly activated by recognition of signature oligosaccharide ligands by specific HTCS, demonstrating that these systems play a key role in bacterial ability to sense and utilize polysaccharides in gut ecosystems.

The data presented here show that dietary supplementation with SUCRAM® can alter the gastrointestinal microbiota by positively influencing the population abundance of lactobacilli, commensal bacteria that are able to exert a beneficial effect on gut health, immunity and maturation. The prebiotic-like mechanism(s) underlying the increased abundance of caecal lactobacillus in response to dietary supplementation with SUCRAM® are presently unknown. However, the knowledge that SUCRAM® is not metabolized by the gut microbiota and remains structurally intact during transit through the gastrointestinal tract suggests the presence of an extracellular sweet sensor. Thus, we propose that lactobacillus OTU4228 may possess a cell membrane- associated receptor that can sense SUCRAM®, leading to the upregulation of sugar transport and metabolic systems. This will provide this strain of lactobacillus with a competitive advantage over other members of the pig intestinal microbiota, which is reflected in the increased population abundance observed when SUCRAM® is included in the diet. The identification and characterization of a SUCRAM® receptor in lactobacillus would be the first example of a cell membrane-associated bacterial sensor for an artificial sweetener, and will provide a novel and accessible target for nutritional strategies aimed at manipulating the commensal microbiota, helping to maintain the health of the gut particularly during the critical post-weaning period.

Conclusion

At Pancosma, our commitment is to include new scientific advances into technologies to create consistent value and return on investment for the animal feed industry. Reconsidering the use of intense artificial sweeteners as a GLP-2 stimulant technology is a good example. Indeed, basic research has unraveled the mechanism by which SUCRAM® triggers GLP-2 release and its consequences on the gut. Based on this demonstrated effect, the use of SUCRAM® could be reconsidered in growing animals as a way to secure gut health while affording the use of non-traditional feedstuffs. This is an original position that added value to SUCRAM, which derives all the benefits of understanding the mechanisms underlying technologies and justifies the basic research done at Pancosma. Without basic research, there would have been no way to understand or justify such a positioning, as artificial sweeteners are intended to make the diet sweeter. SUCRAM® is evolving into a gut protective key ingredients for the swine diet.

Accordingly, the invention concerns a particular use of food additive for animals, including the use for its prophylactic effect. It also concerns the food additive itself used for its prophylactic effect on the digestive tract of an animal.

In embodiments of the invention, the sweetener, or food additive, includes an intense sweetener. It may contain at least one of the compounds of the group consisting of saccharin, sodium saccharin, calcium saccharin, aspartame, acesulfame K, cyclamate and steviosides.

The sweetener (or additive) also contains a potentiator. The potentiator role is to extend the perception of sweetness and to hide the second taste or parasites of the sweetener (such as bitter or metallic tastes). It comprises at least one of the compounds from the group comprising glycyrrhizin, ammonium glycyrrhizinate, potassium glycyrrhizinate, sodium glycyrrhizinate, thaumatin, kokumi, neohesperidin dihydrochalcone, the ribotides and sodium glutamate.

The sweetener, or additive, may comprise from 80% to 100% by weight of sweetener(s) and from 20% to 0% by weight of potentiator(s).

The sweetener (or food additive) used as an example of illustration in the described experiments, is the product SUCRAM® C-150 of the Pancosma company. This product contains 80% by weight of sodium saccharin 10 and 20% by weight of potentiator(s).

The invention is not limited to a limited group of animals but also apply to the other mentioned animals.

Claims

Claims

1 . Use of a food additive containing at least one intense sweetener, orally and/or rectally administered, to modify flora and/or fermentation in the digestive tract of an animal.

2. Use according to claim 1 wherein, the food additive is used to increase the amount of fiber in a diet formula for the animal compared to a previous diet formula without modifying animal zootechnical performance.

3. Use according to claim 1 wherein the sweetener is used to decrease the amount of starch and/or simple carbohydrates in a diet formula for the animal compared to a previous diet formula without modifying animal zootechnical performance.

4. Use according to one of claims 1 to 3, wherein the animal is fed with a diet formula containing a decreased amount of carbohydrates minus fiber, said decreased amount corresponding to an amount of carbohydrates minus fiber inducing, without said food additive, a glucose uptake rate inferior to an optimal glucose uptake rate, especially inferior to 80% of the maximal glucose uptake rate for the animal.

5. Use according to claim 1 wherein, the animal being fed with a diet formula containing a determined level of digestible energy, the food additive is used to decrease the level of digestible energy of the diet formula compared to a previous diet formula without modifying animal zootechnical performance.

6. Use according to claim 5, wherein the reduced digestible energy level of the diet formula corresponds to a digestible energy level inducing, without food additive, a glucose uptake rate inferior to an optimal glucose uptake rate, especially inferior to 80% of a maximal glucose uptake rate for the animal.

7. Use according to claim 1 , wherein the diet of the animal contains an amount of carbohydrates inducing a level of glucose uptake inferior to the optimal level of glucose uptake of the animal, especially inferior to a level of glucose uptake higher than 80% of the maximal glucose uptake rate for the animal, in order to improve animal performance.

8. Use according to claim 1 , wherein the diet of the animal is formulated with digestible energy levels inducing a level of glucose uptake inferior to the optimal level of glucose , especially inferior to a level of glucose uptake higher than 80% of the maximal glucose uptake rate for the animal, in order to improve animal performance.

9. Use according to claim 1 to 8, wherein the animal is a monogastric animal.

10. Use according to claim 1 to 8, wherein the animal is a ruminant.

1 1 . Use according to claim 1 to 8, wherein the animal is a pre-ruminant.

12. Use according to claim 9, wherein the animal is a swine selected among the following categories:

a. Piglet,

b. Pig,

c. Sow.

13. Use according to claim 9, wherein the animal is selected among the following species:

a. Chicken,

b. Guinea fowl,

c. Turkey,

d. Quail,

e. Fish.

14. Use according to claim 10, wherein the animal is a bovine, a caprine or an ovine.

15. Use according to claim 1 1 , wherein the animal is a calf, a kid or a lamb.

16. Use according to one of claims 1 to 8, wherein the animal is an equine.

17. Use according to one of claims 1 to 8, wherein the animal is a rabbit.

18. Use according to one of claims 1 to 17, wherein the intense sweetener is selected among artificial sweeteners, sweeteners from natural origin and/or identical to nature.

19. Use according to any of claims 1 to 18, wherein the sweetener is selected among saccharin and its salts, acesulfame-K, cyclamate, aspartam, stevioside or any other intense sweetener from natural origin such as stevia or a stevia extract.

20. Use according to claim 19, wherein the food additive comprises a potentiator.

21 . Use according to claim 20, wherein the potentiator comprises at least one of the comprising compounds, glycyrrhizin, ammonium glycyrrhizinate, potassium glycyrrhizinate, sodium glycyrrhizinate, thaumatin, kokumi, neohesperidin dihydrochalcone (NHDC), the ribotides and sodium glutamate.

22. Use according to claim 21 , wherein the food additive comprises from 20% to 0% by weight of potentiator(s).

23. Use according to one of claims 1 to 22, wherein the intense sweetener includes a mixture of NHDC and saccharin and/or stevia.

24. Use according to one of claims 1 to 23, wherein the intense sweetener is detected by a bacteria receptor membrane able to detect nutrients within its environment.

25. Use according to one of claims 1 to 24, wherein it causes an increase of Lactobacilli populations.

26. Use according to one of claims 1 to 25, wherein it causes an increase in the L. amylovorus population.

27. Use according to one of claims 1 to 26, wherein it causes an increase in the population of Lactobacilli OUT 1946 population.

28. Use according to one of claims 1 to 27, wherein it causes a decrease in the Clostridia and/or Escherichia coli populations in the digestive tract.

29. Use according to one of claims 1 to 28, wherein it causes an increase in lactate and/or lactate and/or lactic acid production.

30. Use according to one of claims 1 to 29, wherein it causes a reduction of propionate production.

31 . Use according to one of claims 1 to 30, wherein it acts on animal immunity.