FOOD PRODUCT AND PROCESS FOR PREPARING IT

FIELD OF THE INVENTION

The invention relates to food products. More in particular, it relates to a carbohydrate containing food product having controlled or delayed energy release properties and to a process for preparing such product.

BACKGROUND TO THE INVENTION

According to World Health Organisation recommendations, the optimal diet to maintain health comprises at least 55% total energy from a variety of carbohydrate sources. Cereals with high starch content provide the main source of carbohydrates worldwide. Many other food products comprise starch, such as bread, pasta, and potatoes.

Starch is a homopolymer of glucose. It consists of essentially linear amylose molecules and highly branched amylopectin molecules. Starch can be rapidly converted to glucose in the intestinal tract. The glucose then enters the blood stream and provides the body with energy. In humans, starch degradation is initiated by the action of alpha-amylase in the saliva. The digestion of the remaining starch molecules is continued by the actions of pancreatic alpha-amylases . In general, pancreatic amylase is more important for digestion because food generally does not remain in the mouth long enough to be digested thoroughly by salivary amylase.

Hydrolysis of starch is completed in the duodenum and jejunum by the action of pancreatic amylase, which hydrolyses amylose to maltose and maltotriose and amylopectin to a mixture of maltose, isomaltose, and alpha-limit dextrins (three to five glucose units [alpha-1,4] and one glucose unit [alpha-1, 6] ) .

Final hydrolysis of these products is then carried out by the oligosaccharide-degrading enzymes amyloglucosidase (glucan 1,4- alpha-glucosidase) and isomaltase (oligo 1,6 glucosidase) . These enzymes that complete the hydrolysis of starch are anchored to the brush-border membrane.

Next to starch, many products contain high amounts of monosaccharides (e.g. glucose, fructose) and disaccharides (e.g. sucrose, maltose, lactose) and to a lesser extent sugar alcohols, oligo (tri-) saccharides and other polysaccharides. Disaccharides are too large to cross the mucosal cell membrane and must be hydrolysed for absorption to take place. Disaccharidases are located in the brush borders of the intestinal mucosa. Hydrolysis of most disaccharides and starches in the upper small intestine is rapid.

However, there is increasing evidence that a high intake of food products leading to a high glycaemic (blood glucose) response has a deleterious effect on type-2 diabetes and cardiovascular disease. Diets leading to a low glycaemic response appear to be useful in the management of the metabolic syndrome and of hyperlipidaemia. Lowering of cholesterol levels has also been observed in healthy subjects and there are also indications of improvements in fibrinolytic activity.

Differences in the post-prandial glucose profile may also be of physiological significance for satiety and weight maintenance. Data regarding the satiating capacity in relation to glycaemic features are, however, not consistent. Some studies have shown that a modest glucose rise will increase insulin sensitivity. This may induce stronger feelings of satiety and thereby a lower energy intake.

Much less information is present regarding the potential impact of post-prandial glycaemic level on cognitive function and

mental performance. There are some studies to support a relationship between glucose availability and changes in mood and/or mental function ( 'energy' , 'alertness' , 'concentration' , 'reduced irritability' , 'reduced fatigue' , 'vitality' ) . The optimal blood glucose curve has yet to be defined.

The concept of 'energy' is used widely in the food industry. However, most 'energy' claims are not scientifically substantiated and the underlying technology is mostly generic. Furthermore, the concept is very much restricted to cereals and biscuits. For other applications in which the water content is higher and heat is applied in the production process, this approach will not work. For example, when starch granules are heated in the presence of water, gelatinization occurs, which renders the starch molecules fully accessible to digestive enzymes, resulting in rapidly digestible starch. Depending on the process, part of the starch might also turn into indigestible starch without nutritional value.

As indicated before, the rate of digestion is the major determinant of glycaemia in the case of carbohydrate-rich (e.g. starchy) foods. To deliver a desired energy delivery profile for specific health and performance benefits, the carbohydrate delivery should be controlled. For most benefits a constant blood glucose level is beneficial, whereby the glucose release is slower, but sustained for longer. In that respect, Englyst et al. (Englyst KN, Englyst HN, Hudson GJ, Cole TJ, Cummings JH. Rapidly available glucose in foods: an in vitro measurement that reflects the glycaemic response. American Journal of Clinical Nutrition (1999) 69:448-54.) define slowly digested carbohydrates (or "slow carbs") as "carbohydrates that are likely to be completely digested in the small intestine but at a slower rate".

US-A-3 875 308 (Hayashibara Biochemical Laboratories) discloses food products comprising at least 0.05% pullulan and having a reduced caloric value. Pullulan is considered to be digestible only to an insignificant extent and it is added to the food products to replace starch.

US-A-2003/0232067 (Bryan Wolf) discloses the use of pullulan having a molecular weight between 50,000 and 500,000 daltons, as a slowly digested carbohydrate and its incorporation into food products, especially beverages and meal replacement products. However, a significant part of the pullulan seemed to be either resistant to degradation, or the digestion of pullulan occurred too slowly to achieve" complete hydrolysis at the end of the small intestine. This part of the pullulan was therefore not available as a short-term source for energy (e.g. available within 2 hours) and not available for characteristics associated with energy delivery (e.g. effect of glucose on satiety) .

Furthermore, pullulan reaching the colon undigested will be fermented by present micro-organisms and this might cause malabsorption and associated gastro-intestinal symptoms like flatulence.

From a product formulation perspective, the use of pullulan is limited to lower concentrations. Higher concentrations, effective for energy supply will lead to liquid food products having unacceptable high viscosity.

It is therefore an object of the invention to provide a carbohydrate containing food product having controlled energy release properties and which overcomes one or more of the above mentioned draw-backs. Surprisingly, it has now been found that the above-mentioned object can be achieved by the carbohydrate

containing food product according to the invention, comprising a pullulan having a number average molecular weight between about 1,000 and 45,000 daltons.

SUMMARY OF THE INVENTION

According to a first aspect, the invention provides a carbohydrate containing food product having controlled energy release properties comprising a pullulan having a number average molecular weight between about 1,000 and 45,000 daltons.

According to a second aspect, there is provided a process for preparing such a food product.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a food product having "controlled" energy release properties. There are now several ways to visualise and quantify the glycaemic effect of foods. The glycaemic index (GI) concept has been introduced to enable comparison of foods based on their glycaemic effect. It provides a standardised comparison for the 2 hour post-prandial glucose response of a carbohydrate with that of white bread or glucose .

Avoiding products that cause an immediate high blood sugar level will help to get a lower glucose response, but that can also be accomplished by "slow" carbohydrates. In that respect, one now speaks of rapidly available carbohydrates (RAC) or slowly available carbohydrates (SAC) . RAC is carbohydrate that is quickly hydrolysed, which results in high blood glucose concentrations, which are maintained for only a short time. SAC is defined as carbohydrate that is likely to be completely digested in the small intestine but at a slower rate, resulting

in lower blood glucose levels that are maintained for a longer time.

Resistant carbohydrate (RC) is the sum of carbohydrate and products of carbohydrate degradation that are not absorbed in the small intestine of healthy humans. RC therefore reaches the colon where it can be fermented by present micro-organisms and where it can play a role in the maintenance of human digestive health.

The determinants of post-prandial glucose excursions are numerous and include the amount and nature of the carbohydrates ingested, the rate of gastric emptying, the rates of intraluminal carbohydrate digestion and of intestinal glucose absorption, the entero-pancreatic hormonal response, and specific postabsorptive metabolic changes. Of these processes the rates of gastric emptying and digestion/ absorption are the most important ones. The rate of digestion is the major determinant of glycaemia in the case of carbohydrate-rich foods. Differences in glycaemic responses to dietary carbohydrate are directly related to the rate of carbohydrate digestion.

Most carbohydrates are digested and 95% absorbed in the first part of the small intestine (duodenum and jejunum). In the first steps of digestion and absorption the physico-chemical characteristics of carbohydrates determine the rate and extent of these processes. An example is the fact that an α-1,4 bond is easier to digest than an α-1,6 bond.

As indicated above, slowly available carbohydrate or, specifically for glucose-polymers, slowly available glucose (SAG) is likely to be completely digested in the small intestine but at a slower rate, resulting in lower blood

glucose levels that are maintained for a longer time. On the other hand, rapidly available glucose (RAG) is glucose-polymers that are quickly hydrolysed, which results in high blood glucose concentrations, which are maintained for only a relatively short time.

Englyst et al. (1999, see above) used an in vitro test that correlates significantly to the in vivo glucose curves. The in vitro measurement of RAG and SAG could predict the glycaemic response measured in human studies. Englyst et al. defined RAG in the in vitro situation by the amount of carbohydrate hydrolysed to glucose after 20 min (called G20) . Also the amount hydrolysed was measured after 120 minutes (called G120) . The amount hydrolysed during these 120 minutes was considered to be available for absorption in the small intestine. Anything hydrolysed after the 120 min was considered not available for absorption and considered resistant. The amount of carbohydrates hydrolysed between 20 and 120 min (i.e. G120 - G20) was defined as SAG. In the ideal situation one would like to have a carbohydrate with a very low G20 and a very high G120, resulting in a high difference between G20 and G120. However, many efforts in industry to make certain products slowly digestible render them (partly) resistant. As such one wants to keep G120 as close as possible to the theoretical maximum (i.e. 100% of the total amount of theoretically available carbohydrate) .

In the present invention, we define "controlled energy release" as the release of carbohydrates represented by an in vitro hydrolysis (curve) , where G120- G20 is significantly higher than in a proper control that contains the same amount of available carbohydrate, while G120 is as high as possible, i.e. at least 50, 65, 80 or even 90% of the theoretical maximum.

By varying the relative amounts and by combining rapidly digestible carbohydrates (e.g. starch, glucose, sucrose) and pullulan with the above-mentioned properties, the release properties of energy in a food product can be controlled.

We found that one route to slow down energy delivery from carbohydrates in the human gut is the use of pullulan having a specific molecular weight. Pullulan is a fermentation product of the yeast Aerobasidium pullulans. In the food industry, pullulan is applied as a texturiser. It is a water-soluble, viscous polysaccharide consisting of three α-1,4 linked glucose molecules (one maltotriose-unit) that are repeatedly polymerised by an α-1,6 linkage on the terminal glucose, resulting in a stair-step structure (see Fig. 1) .

Pullulan has a molecular weight of 50,000 and 500,000 Dalton. It has the properties of a soluble fiber, i.e. it is resistant against enzymatic hydrolysis by intestinal enzymes and upon ingestion the major part of it reaches the colon undigested. The reason for the low degree of hydrolysis is that intestinal enzymes only have a limited capacity to hydrolyse the α-1,6 linkages of the polymer.

Wolf et al. (Am. Soc. For Nutritional Sciences 0022-3166/03) showed that pullulan, in comparison with rapidly digestible maltodextrins, maintained blood glucose levels for a longer time above the basal level, which supported the hypothesis that

pullulan was digested slowly. However, a significant part of the pullulan seemed to be either resistant to degradation, or the digestion of pullulan occurred too slowly to achieve complete hydrolysis at the end of the small intestine. This part of the pullulan was therefore unavailable as a short-term (e.g. available within 3 hours) energy source and unavailable for characteristics associated with energy delivery (e.g. effect of glucose on satiety) . The unavailability of pullulan was also confirmed in our own in vitro experiments as shown in the Example. More than two thirds of pullulan remained unhydrolysed after a 3 hour incubation.

As a significant part of the pullulan seemed to be either resistant to degradation, or the digestion of pullulan occurred too slowly to achieve complete hydrolysis at the end of the small intestine these undigested carbohydrates reach the colon where they can be fermented by present micro-organisms and produce various gases. In their study, Wolf et al. (2003, vide supra) did find an increase in malabsorption (represented by the use of a hydrogen breath test) and in the incidence and frequency of flatulence. This is generally found when relatively high amounts of dietary fibres are ingested.

The reason for the low degree of hydrolysis is that pancreatic enzymes lack the specificity to break down the α-1,6 linkages of the polymer. We found that a reduction in the chain length of pullulan - by breaking part of the α-1,6 bonds via pretreatment by pullulanase - was more readily hydrolysable.

In vitro tests showed that not only the pretreated pullulan was degraded by amyloglucosidases, but also that with the degree of pretreatment the rate and extent of hydrolysis could be controlled. At all conditions the rate of hydrolysis was significantly slower than that of soluble starch in the

presence of both amylase and amyloglucosidase, so that a slow release of energy was obtained. Furthermore, almost all material was digested within 2 to 3 hours, depending on the amount/time of pullulanase used. Based on the high amount of energy that pullulan contains in the form of the glucose monomers pre-treated pullulan could potentially be an interesting dietary "slow-release" energy source.

Generation of pullulan fragments of predetermined chain lengths can be achieved in several ways. First, process control is an effective way to control the chain length of microbially produced pullulan. Although the exact mechanism of pullulan production is not known factors like, culture selection, cultivation techniques and control of incubation conditions, like pH or substrate are known to have a significant effect on the chain length distribution of biosynthetic pullulan. For instance, it is known that the presence of fructose in the culture medium promotes the production of high molecular weight pullulan. Another important factor is the harvest time, since early in the initial phases of biosynthesis high molecular weight pullulan with a wide chain length distribution is produced, while more pullulan of medium chain length and narrow chain length distribution is produced in the stationary growth phase. Alternatively pullulan with a specific chain length can be obtained by fractionation of crude material with a wider chain length distribution. This may be achieved by e.g. size exclusion techniques. Thirdly pullulan fragments with a defined chain length distribution can be obtained via partial hydrolysis of pullulan. For instance, pullulan P-200 (commercially available from Hayashibara Biochemical Laboratories, Japan) can be treated with the enzyme pullulanase that selectively hydrolyses alpha 1-6 bonds of the molecule. By careful control of the incubation conditions fractions with

desired chain length ranging from about 1,000 to 100,000 daltons can be obtained.

The specific type of pullulan used according to the present invention has a number average molecular weight between about 1,000 and 45,000 daltons, preferably between about 5,000 and 40,000 daltons. By average molecular weight, we mean in the context of the present invention the number average molecular weight (M _n ), unless indicated otherwise. It is preferred that at least 70%, more preferably at least 90% of the pullulan molecules have a molecular weight of less than 45,000 Dalton.

Preferably, the pullulan is present in the food product in an amount of 0.005 to 35% by weight of the product, more preferably in an amount of 0.05 to 10% by weight of the product .

It is preferred that the food product according to the invention has a high moisture content. In particular, the water activity (Aw) is preferably at least 0.70.

Some examples of food products in accordance with the present invention (but not limiting to these) are: drinks / beverages, meal replacement products such as drinks, bars, powders, soups, dry soups, powdered soup concentrates, (fat) spreads, dressings, (whole) meals, desserts, sauces, sport drinks, fruit juices, snack foods, ready-to-eat and pre-packed meal products, ice creams and dried meal products. (Dry) soups are especially preferred.

The food products can be prepared by mixing the pullulan, in dry form or in the forms of an aqueous suspension, with the rest of the food product.

The food product of the present invention may optionally further comprise ingredients such as proteins, fats, salts, flavour components, colourants, emulsifiers, preservatives, acidifying agents and the like. The invention is illustrated in the following examples.

Example 1

Preparation of pullulan fragments and determination of molecular mass of fragments

A 2 % (w/w) solution of pullulan (PI20 from Hayashibara Company Ltd. Japan, MM = 200,000 Da) was made in 0.1 M sodium acetate pH 5.2 and heated for 5 minutes at 100 ⁰ C. After cooling to room temperature the solution was divided into portions and to each portion different amounts of pullulanase (Prozyme from Novozyme) were added. Incubation was allowed to proceed for 7 hours at room temperature. The reaction was stopped by heating for 5 minutes at 100 ⁰ C. The amount of reducing end groups liberated by the hydrolytic action of pullulanase was colourometrically determined by means of the dinitrosalicylic acid (DNSA) reaction using a maltotriose calibration curve. From these data the average chain length c/q the average molecular mass of fragments obtained was calculated (Table 1) .

Table 1. Average Molecular Mass of pullulan fragments after hydrolysis with pullulanase.

Example 2

Colourimetric quantification of reducing end groups

Reducing end groups were measured with a method described by Bernfeld (Bernfeld, P., 1955, Amylases, α and β, Methods in Enzymology, Vol. 1, Academic Press, NY, 149-158) . Ten grams of 3, 5-dinitrosalicylic acid (DNSA) was dissolved in 200 ml 2 M NaCl and 500 ml H ₂ O. Stirring and heating the suspension up to 60 ⁰ C promoted dissolving. After that, 300 g Rochelle salt (sodium potassium tartrate tetrahydrate) was added and the solution was adjusted to 1000 ml with H ₂ O. The DNSA solution was kept from light at room temperature. 500 μl of samples to be analysed was added to 500 μl DNSA solution and heated for about 5 minutes at 100 ⁰ C in a thermomixer (Eppendorf thermomixer comfort) . After that, tubes containing the mixtures were cooled under running tap water or on ice. Solutions were diluted properly with H ₂ O and the absorbances were measured at 540 nm (Shimadzu) . Standard concentrations of maltose (ranging from 0- 5 mg/ml) were prepared in 0.02 M phosphate buffer pH 6.9, containing 0.067 M NaCl. From the absorbances measured, maltose concentrations were calculated.

Example 3

Rate of hydrolysis and fragment size

Pullulan was treated with pullulanase (0.0, 0.1 and 1.0 U/ml) as described above. To 14 ml of the samples 1 ml of amyloglucosidase (from Aspergillus niger, Fluka 10115) was added and glucose release was monitored in time. From Figure 2 it is clear that the unhydrolysed pullulan hardly released any glucose upon amyloglucosidase treatment, whereas the partially hydrolysed fractions of pullulan gave a sustained release of glucose over the two hours of incubation.

Example 4

Enzymatic glucose quantification

The glucose concentration of samples was measured using an enzymatic kit (Enzytec) . The measurement was based on the following principle: hexokinase D-glucose + ATf ► Glucose-6-phosphate + δDP

Glucose-6-phosphate-dehydrogenase Glucose-6-phosphate + NADP ⁺ _► 6-PG + NADPH

+ H ⁺

The reaction was performed in 3 ml plastic cuvettes. To 1 ml of triethanolamine (TEA) buffer of pH 7.6, containing

approximately 80 mg NADP and 190 mg ATP, 100 μl of sample or standard glucose solution was added, followed by 1.9 ml H ₂ O. To the blank solution, 2 ml H ₂ O was added. Solutions were mixed and after approximately 3 minutes the absorbance was measured at 340 nm against water. Then, 20 μl of a hexokinase/glucose-6- phosphate dehydrogenase suspension (200 U / 100 U) in ammonium sulfate was added to the solutions and solutions were mixed. After 10-15 minutes the absorbance was measured again and measurements were repeated after 2 minutes to check if the reactions had stopped. The glucose concentration of the samples was calculated with the following formula:

c = (V x Mw x δA) / (ε x d x v x 1000) [g glucose/1 sample solution] c = (3.020 x 180.16 x δA) / (6.3 x 1 x 0.1 x 1000) = 0.8636 x

δA [ g glucose/1 sample solution]