CLIAMS:1. A prebiotic composition comprising:
(i) prebiotic components consisting of lactulose and pectin; and
(ii) additives
wherein the lactulose and pectin is in a range of ratio of 2:1 to 4:1.
2. The composition as claimed in claim 1, wherein lactulose and pectin is in a range of 2:1 to 3:1.
3. The composition as claimed in claim 1, further comprises probiotic microorganisms.
4. The composition as claimed in claim 3, wherein said probiotic microorganisms are present in the range of 106 cfu to 109 cfu.
5. The composition as claimed in claim 3, wherein said probiotic microorganisms are selected from Lactobacillus sp., Bacillus sp., Streptococcus sp., Bifidobacteria sp., Lactococci sp.
6. The composition as claimed in claim 5, wherein the said probiotic microorganisms are selected from Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus salivarious, Bacillus subtilis, Streptococcus thermophilus, Streptococcus salivarius, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium infantis and Lactococcus lactis.
7. The composition as claimed in claim 1, wherein said additives are selected from vitamins, minerals, proteins, lipids, carbohydrates, citric acid, fruit powder, flavors, herbs, beverage mix, dairy mix, seasonings, stabilizers, emulsifiers, actives, bases for formulations.
8. The composition as claimed in claim 1, wherein said composition is adapted to promote growth of probiotic microorganisms.
9. The composition as claimed in claim 1 or claim 3, wherein said composition is adapted to promote the production of short chain fatty acids.
10. The composition as claimed in claim 1 or claim 3, wherein said composition is adapted to increase the ratio of propionate to acetate content.
11. The composition as claimed in claim 1 or claim 3, wherein said composition enhances the production of vitamin B12 by the bacteria.
12. The composition as claimed in any of claims 1 to 7, wherein the said composition is in the form of tablets, powdered sachets, capsules or incorporated in food formats.
13. The composition as claimed in any of claims 1 to 7, wherein the said composition is adapted to be administered to mammals.
14. A synbiotic formulation comprising:
(i) prebiotic components consisting of lactulose and pectin;
(ii) probiotic microorganisms; and
(iii) additives
wherein the lactulose and pectin is in a range of ratio of 2:1 to 4:1. ,TagSPECI:Field of the invention
The present invention relates to synergistic mixtures of prebiotic components. More particularly, the invention relates to synergistic combination of prebiotic components which significantly stimulate the growth and production of bioactive metabolites by probiotic bacterium.
Background and prior art
The microflora of the human large intestine plays a crucial role in both human nutrition and health. The bacterial composition can be influenced and modulated by dietary intake. Carbohydrates which have passed through the stomach and small intestine undigested; are metabolized by the bacteria to produce metabolites like short-chain fatty acids (SCFAs), such as acetic acid, butyric acid, propionic acid etc. These SCFAs are subsequently absorbed in the gut and get released into the blood. By administering right type and right quantity of prebiotics one can beneficially influence the gut microflora and in turn can have positive health effects.
Prebiotics are defined as ‘a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or more bacteria in the colon, and thus improves host health’. Prebiotics are known to be selectively used by beneficial gut microbes (probiotic bacteria) as an energy source, thus delivering various health benefits (like gut health, metabolic health, immune health and vitamin absorption etc.). However usually, a high dose (10-20g/day) of prebiotic is required to get the desired beneficial effects.
A large amount of scientific endeavor has been put in by various scientists, globally, to understand the various mechanisms by which different prebiotics influence the beneficial gut microflora and can confer health benefits. Additionally, a few patents do exist on this subject as discussed below:
EP1685763 relates to synergistic mixtures of prebiotic carbohydrates, galacto-oligosaccharides (GOS, for example TOS) and polyfructose (for example inulin). The invention suggested the use of GOS and polyfructose combination for prevention of allergy, eczema or atopic diseases in bottle fed or partially bottle fed infants. The composition showed an improvement in fecal microbiota and levels of end products like SCFAs.
US2012/0121621 (WO2006/134409) relates to prebiotic composition comprising of one or more prebiotic components from a group including partially hydrolysed inulin, natural oligofructoses, fructo-oligosaccharides (FOS), lactulose, galactomannan and suitable hydrolysates thereof, indigestible polydestrose, indigestible dextrin and partial hydrolysates thereof, tran-galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), acemannan, lentinan or beta-glucan and partial hydrolysates thereof; gums, carob seed flour, oat bran, rice bran, polysaccharides P and K (PSP, PSK), tagatose etc.
WO2004089115 discloses synbiotic composition comprising Lactobacillus strain NCIMB 41114 (or an equivalent Lactobacillus strain) and one or more non-digestible oligosaccharides e.g. galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS), xylo-oligosaccharide (XOS), soybean-oligosaccharide (SOS), isomalto-oligosaccharide (IMO), gentio-oligosaccharide, fructan, inulin, lactulose, gluco-oligosaccharide and lactosucrose.
WO2011137249A1 discloses a prebiotic composition comprising one or more prebiotics { GOS, lactulose, raffinose, stachyose, lactosucrose, FOS (i.e. oligofructose or oligofructan), inulin, isomalto-oligosaccharide, xylo-oligosaccharide, paratinoseoligosaccharide, transgalactosylated oligosaccharides (i.e. transgalacto-oligosaccharides), transgalactosylate disaccharides, soybean oligosaccharides (i.e. soyoligosaccharides), gentiooligosaccharides, glucooligosaccharides, pecticoligosaccharides, palatinose polycondensates, difructose anhydride III, sorbitol, maltitol, lactitol, polyols, polydextrose, reduced paratinose, cellulose, β-glucose, β-galactose, β-fructose, verbascose, galactinol, and β-glucan, guar gum, pectin high, sodium alginate, and lambda carrageenan} for combating symptoms of lactose intolerance.
Dietary undigested carbohydrate fractions (prebiotics) are fermented by intestinal bacteria and produce various metabolites including short chain fatty acids. The major short chain fatty acids (1-6 carbons) produced post bacterial fermentation include acetic acid, propionic acid and butyric acid. The quality and quantity of short chain fatty acid (SCFA) products depends on the type and amount of carbohydrate substrates provided. Once formed in colon these short chain fatty acids are readily absorbed into the body, where they mediate different functions. Acetate is the major SCFA which when transported to liver was found to be involved in cholesterol synthesis (lipogenesis). However, propionate has been shown to inhibit cholesterol synthesis and stimulates lipolysis and gluconeogenesis. Therefore, substrates that can decrease the acetate: propionate ratio is suggested to reduce serum lipids and possibly cardiovascular disease risk (Wong JM, de Souza R, Kendall CW, Emam A, Jenkins DJ. (2006) Colonic health: fermentation and short chain fatty acids. J Clin Gastroenterol.; 40(3):235-43). Butyrate is the main energy source for colonic mucosa and has role in promoting cell differentiation, cell-cycle arrest and apoptosis. Thus Short chain fatty acids produced as the end products have implications in various functions and ratio of the different SCFAs is critical in exerting the desired effect.
As discussed above, various prior arts disclose various prebiotics and synbiotic compositions, but none of them really describes or suggests any specific prebiotic combinations in specific ratios delivering synergistic effect for higher health benefits.
Thus, there is a need to develop a synergistic prebiotic combination that can give higher bioactivity even at lower concentration.
Objects of the invention
Thus it is an object of the present invention to overcome the drawbacks of the prior arts.
It is another object of the present invention to provide a prebiotic composition comprising specific ratio of lactulose and pectin.
It is yet another object of the present invention to provide a prebiotic composition, which significantly stimulates the growth of probiotic bacterium.
It is a further object of the present invention to provide synergistic composition for metabolic health as the present prebiotic combination when combined with the bacterial strains e.g. Lactobacillus casei significantly modulated the short chain fatty acid profile from lipogenic (acetic acid) to lipolytic (propionic acid) side. The synbiotic formulation can also enhance the bioactive potential of the bacterial strains for potential applications in gut health, oral health, skin and nutritional health (vitamin production), cardiovascular health (exopolysaccharide and SCFA production), immune health (via immunogenic proteins) and neuronal health (tryptophan production).

Brief Description of the Accompanying Drawings
Figure 1a illustrates prebiotic potential of prebiotic components and combinations lactulose and pectin (L:P) in respective ratios with various bacterial strain.
Figure 1b illustrates prebiotic potential of prebiotic components and combinations; lactulose and pectin in a range of ratios (2:1 to 5:1).
Figure 2 illustrates (a) levels of different short chain fatty acid produced; (b) levels of acetic acid; and (c) relative proportions of acetic acid and propionic acid, in presence of Lactulose, Pectin and combination of both (LP) at different ratios.
Figure 3 illustrates the amount of vitamin B12 produced in presence of different prebiotic sources: Lactulose and Pectin individually and in combinations.
Detailed Description of the Invention
The present invention relates to a prebiotic composition comprising prebiotic components. The present invention provides a synergistic combination of prebiotic components which significantly stimulate the growth of probiotic bacterium.
Different prebiotic fibers carry different functional traits, thus, utilizing a synergistic combination ensures the delivery of more than collective effect in the same formulation. Therefore, an extensive scientific study has been carried out to understand and evaluate the functioning of various prebiotics (individually or in combination) and their interaction with the beneficial microbes (probiotics). For prebiotic combinations, effect of their relative content was also studied on their biological activity, so as to identify a synergistic composition to deliver higher level of health benefits by improving microflora and production of end products like short chain fatty acids (SCFA).
According to the present invention, prebiotic components when combined in specific ratios show synergistic activity by stimulating the growth of the beneficial microbes (Lactobacilli) and reducing the growth of pathogenic microbe (Salmonella). In vitro prebiotic potential of different components can be tested by evaluating their ability to stimulate the growth of probiotic microorganisms.
It is an embodiment of the present invention to provide a prebiotic composition consisting prebiotic components lactulose and pectin and additives. Suitable additives may be selected from vitamins, minerals, proteins, lipids, carbohydrates, citric acid, fruit powder, flavors, herbs, beverage mix, dairy mix, seasonings, stabilizers, emulsifiers, actives, bases for formulations.
The present invention provides a synergistic combination of the prebiotic components lactulose and pectin in a range of ratio i.e. 2:1 to 4:1. The present composition stimulates the growth of probiotic bacteria higher than the growth observed with individual prebiotics. The inventors have surprisingly found that among different ratios evaluated; only the specific ratios of lactulose and pectin in synergistic combination (i.e. in the range 2:1 to 4:1) showed synergistic effect on growth of probiotic bacteria.
The present invention further embodies that prebiotic composition when combined in specific ratios show synergistic activity by stimulating the growth of the beneficial microbes (Lactobacilli) and reducing the growth of pathogenic microbe (Salmonella typhi). Salmonella typhi MTCC734 was procured from microbial type culture collection (MTCC) bank, Institute of Microbial Technology (IMTECH), Chandigarh. The strain originally isolated in Russia, was used in the present experiment for evaluation of prebiotic potential of the prebiotic composition. In vitro prebiotic potential of different prebiotic components can be tested by evaluating their ability to stimulate the growth of probiotic microorganisms.
In the present study different prebiotic components (1% w/v) and their combinations (net concentration 1% w/v in media) were tested for their ability to stimulate the growth of selected probiotic strains (Lactobacillus sp). Prebiotic combination (lactulose: pectin, 2:1-4:1) significantly stimulated the growth of probiotic bacterium as compared to the individual component (prebiotic), in in- vitro culture model.
The present blend can be used for formulating synbiotic formulations with Lactobacillus strains the said bacterial strains used in the present invention for evaluation of prebiotic potential include Lactobacillus casei (ATCC SD5213) and Lactobacillus helveticus (ATCC 8018). American Type Culture Collection (ATCC) is an International Depository Authority (IDA) for the deposition of biological material for the purposes of patenting under the Budapest Treaty. The blend can improve the functionality of probiotic bacteria like secretion of various bioactives (vitamins, amino acids, proteins, short chain fatty acids).
The expression “prebiotic” as used herein means non-digestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial to host (human).
The expression “probiotic” used herein refers to live microorganisms administered in adequate amounts that confer a beneficial health effect to the host as per FAO/WHO, 2002.
The expression “synbiotic formulation” as used herein is defined as a formulation composed of combination of probiotic bacteria and prebiotic fibers (prebiotic components).
The prebiotic composition according to the present invention can be prepared by mixing 2-4 g of lactulose powder with 1 g of pectin powder using mortar pestle (lactulose to pectin ratio is 2: 1 to 4:1).
The synbiotic formulation according to the present invention can be prepared by preparing a premix containing probiotic bacteria (106 - 109 cfu per serve) and prebiotic fibers/components (3-10g) and then incorporating the premix with the additives of the formulation. Alternatively, the prebiotic fibers/components are first added to the formulation additives and post processing (thermal or pressure), the probiotic bacteria (106 - 109 cfu per serve) are added to the formulation.
The composition according to the present invention can be prepared in several forms that include powder, tablets and capsules or incorporated in food formats (nutritional bars, candies, bruit beverages, snacks, bakery products and the like).
The present invention is described by way of non-limiting, illustrative examples.
Example 1
Preparation of prebiotic composition:
Prebiotic composition is prepared by mixing lactulose powder and pectin powder in different ratios (1:1, 2:1, 1:2, 3:1, 4:1, 5:1) using mortar pestle.
Batch culture model for bacterial growth (prebiotic potential):
Prebiotic activity of the individual prebiotics and combinations was estimated using batch culture model using two probiotic microorganisms (lactobacillus strains: Lactobacillus casei; Lactobacillus helveticus) and one pathogenic micro-organism (Salmonella typhi). Inoculation media was prepared without glucose (peptone, 10g/L; Beef extract, 8g/L; Yeast extract, 4g/L; Dipotassium hydrogen phosphate, 2g/L; Sodium acetate, 5g/L; Triammonium citrate, 2g/L; Magnesium sulphate, 0.2g/L; Manganese sulphate, 0.05g/L).
Prebiotic fibers/components and combinations were used in net concentration of 1mg/ml. For preparation of the combination, the prebiotics were blended in different ratios, however were used in the same concentration in which the net concentration of the prebiotics were used in 1mg/ml. Inoculation media (100µl) containing 1mg/ml of carbohydrate source (glucose and prebiotic components: lactulose and pectin) was incubated with 10µl of test bacteria (probiotic and pathogen) in the concentration of 106 cfu/ml and incubated at 37°C shaking incubator. Bacterial growth was observed by taking absorbance at 600nm.
Growth of bacterial strains was measured by taking absorbance at 600nm. The unit here means absorbance at 600nm. Thus, prebiotic index was calculated as change in absorbance at 600nm in the test carbohydrate source in 24 hr per unit change in absorbance (A600nm) in media containing glucose as the carbohydrate source.

Results:
Lactulose and pectin when combined in the ratios of 2:1 showed a synergistic effect towards stimulating the growth of probiotic strains Lactobacillus casei and Lactobacillus helveticus. It has been found that the effect is lesser when they were mixed in the ratios of 1:1 or 1:2 (Figure 1a). However, the synergistic prebiotic potential of lactulose and pectin combination was found to be extended over the range of concentration i.e. 2:1 to 4:1, with the highest effect observed for the ratio 2:1(Figure 1b).

Example 2
Effect of prebiotic combination on short chain fatty acid production:
To evaluate the pattern of short chain fatty acid production by Lactobacillus casei bacteria were cultivated in presence of lactulose and pectin, as prebiotic components, (individuals or combinations in 1% net concentration) incorporated into the bacterial fermentation media containing peptone, 10g/L; Beef extract, 8g/L; Yeast extract, 4g/L; Dipotassium hydrogen phosphate, 2g/L; Sodium acetate, 5g/L; Triammonium citrate, 2g/L; Magnesium sulphate, 0.2g/L; Manganese sulphate, 0.05g/L. Bacterial inoculum (1% of 108 cfu/ml of Lactobacillus casei) was added into the fermentation media and allowed to grow anaerobically at 37 oC. After 24 hr of incubation the culture medium was removed by centrifugation at 6000rpm for 10 min and acidified to pH:2 (using 1N HCl) and was then used for analysis of short chain fatty acids by direct injection method using gas chromatography.

Results:
As mentioned earlier short chain fatty acids are produced as end products of the fermentation using lactulose and pectin prebiotic components used alone or in combination. Figure 2(a) depicts the levels of different short chain fatty acids (SCFA) produced by the fermentation of prebiotic components and their combinations. There is a decrease in the content of lipogenic acetic acid production by the use of lactulose and pectin in ratios 2:1 to 4:1 (figure 2 b), which is further compensated by upregulation of other functional fatty acids like propionic and butyric acid (figure 2a). As discussed earlier acetic acid is known for its effects on lipogenesis (lipid producing) and propionic acid is considered as lipolytic (lipid degrading). Thus, the relative proportion of acetic acid to propionic acid can help in predicting the lipogenic/lipolytic potential of the formulation. It was observed the use of prebiotic combination of lactulose and pectin in a ratios 2:1 to 4:1 significantly enhanced the proportion of propionic acid relative to acetic acid (figure 2c), thus enhancing the lipolytic potential of the formulation.
Example 3
Effect of prebiotic combination on vitamin B12 production:
To evaluate the effect of prebiotic combination on production of vitamin B12 by Lactobacillus casei; bacteria were cultivated in the presence of prebiotic components: lactulose and pectin (individuals or combinations in 1% net concentration). These components were incorporated into the bacterial fermentation media containing peptone, 10g/L; Beef extract, 8g/L; Yeast extract, 4g/L; Dipotassium hydrogen phosphate, 2g/L; Sodium acetate, 5g/L; Triammonium citrate, 2g/L; Magnesium sulphate, 0.2g/L; Manganese sulphate, 0.05g/L. Bacterial inoculum (1% of 108 cfu/ml of Lactobacillus casei) was added into the fermentation media and allowed to grow anaerobically at 37oC. After 24 hr of incubation the culture medium was removed by centrifugation at 6000rpm for 10 min and then analyzed for vitamin B12 content by high performance liquid chromatography (HPLC) method.

Results:
It was observed that the combination of prebiotics lactulose and pectin in the ratio 2:1 to 4:1 synergistically enhanced the production of vitamin B12 in the media (Figure 3) compared to the individual component.
Example 4

Preparation of Synbiotic Formulations in various food/edible formats
Buttermilk/ Dahi: Milk (200ml) mixed with 3 g of prebiotic combination (lactulose and pectin, 2:1 to 4:1) and then inoculated with 1% inoculum of probiotic bacteria (108 cfu/ml) and Dahi forming bacteria (1%) and kept overnight (6-8 hr) at 30˚C for setting.
Fruit beverage mix: Probiotic bacteria (108 cfu) mixed with prebiotic combination (3 g) and then added to additives (20 g) like citric acid, fruit powder, flavors and stabilizers.
Tablets: A mixture can be prepared by combining probiotic bacteria (109 cfu, 10mg), prebiotic combination (3g), binder, filler and glidant to form a slug. And then the slug can be cut and then broken subsequently to compressed tablets
Capsules: The ingredients can be delivered in the capsule with gelatinous envelope enclosing the probiotic bacteria (108 cfu, 10mg), prebiotic fibers/components (1g), preservatives and surfactants.
Sachets: Probiotic bacteria (109cfu), prebiotic fibers/components (3-4 g), mixed with additives like surfactants, flavors and bulking agents and can be packaged in air-tight sachets.