CA2758980A1 - Low glycaemic index maple product, methods and processes for producing same - Google Patents

Abstract

The present invention relates to a maple product comprising isomaltulose enzymatically converted from a maple starting product and having a low glycaemic index. The present invention also relates to a maple product comprising isomaltulose enzymatically converted from a maple starting product comprising sucrose wherein the enzymatically converted isomaltulose corresponds to about 20% to 98% of the sucrose present in the maple starting product prior to the enzymatic conversion. The present invention also relates to a process for obtaining an isomaltulose maple product comprising the step of enzymatically converting the sucrose present in a maple starting product into isomaltulose wherein at least 20% to 98% of the sucrose present in the maple starting product is converted into isomaltulose.

Description

LOW GLYCAEMIC INDEX MAPLE PRODUCT, METHODS AND PROCESSES FOR
PRODUCING SAME

The present invention relates to low glycaemic index maple products, methods and process for producing same. The present invention relates isomaltulose containing maple products, methods and process for producing same.
The sap of maple trees forms the basis of maple base products including maple syrup, maple sugar and maple confection products. Maple syrup is obtained by the concentration of maple sap. To produce maple syrup, the sap from maple tree is concentrated and the concentration can be achieved through simple boiling in an open kettle or using more advanced evaporation techniques such as vacuum pump evaporators.
The main sugar found in maple sap is sucrose. Sucrose is the organic compound commonly known as table sugar and sometimes called sucrose. Sucrose is a saccharide composed of glucose and fructose. Sucrose is easily assimilated and provokes a rapid raise in blood sugar upon ingestion. Sucrose is also associated with dental caries and tooth decay. It is well known that the rapidity with which sucrose raises blood glucose is a concern for people with hyperglycaemia or diabetes.
In view of the above disadvantages associated with sucrose, the food and beverage industry has been replacing sugar with artificial sweeteners such as sugar-alcohols or polyols and high intensity sweeteners. Sugar-alcohols or polyols do not have an effect on glycaemia and do not increase tooth decay. High intensity sweeteners are compounds with many times the sweetness of sucrose which can be used in a small amount to provide the desired sweetness without a significant increase in calories or otherwise affect the glycaemia.

2 Isomaltulose (also known as Palatinose, 6-0-a-D-glucopyranosyl-D-fructose) is a naturally occurring molecule that is found in honey, cane juice and molasses.
The physical and organoleptic properties of isomaltulose are similar to those of sucrose.
However, the sweetness intensity of isomaltulose is 45% lower than the sweetness power of sucrose. Isomaltulose does not cause tooth decay and also inhibits the formation of insoluble glucanes (Minami, T, Fujiwara, T, Ooshima, T, Nakajima, Y, Hamada, S, Interaction of structural isomers of sucrose in the reaction between sucrose and glucosyltransferases from mutans streptococci, Oral Microbial Immunol., 5, 1990, 189.; Ooshima, T, lzumitani, A, Minami, T, Fujiwara, T, Nakajima, Y, Hamada, S, Trehalose does not induce caries in rats infected with mutan streptococci, Caries Res., 25, 1991, 227). The level of insulin associated with isomaltulose intake is considerably reduced compared to the level of insulin associated with other sugars since monosaccharides are slowly released in the blood circulation. Isomaltulose is thus very low glycaemic with a glycaemic index (GI) of 32 as referenced in:

- (Holub I, Gostner A, Theis S, Nosek L, Kudlich T, Melcher R, Scheppach W :
Novel findings on the metabolic effects of the low glycaemic carbohydrate isomaltulose (Palatinosem"). British Journal of Nutrition (2010), 103, 1730-1737;

- http://www.i-dietetique.pro/?action=articles&id=7872;

- http://www. beneopa latinit.com/en/Food_Ing red ients/lsomaltulose/VVhat_is_lsomalt ulose/Palatinose-Brochure_EN_1.pdf; and - http://www.3actionshop.com/index.php?option=com_content&view=article&id=69&
Itemid=72&lang=en).

The use of isomaltulose in sport drinks or drinks intended for diabetics is therefore appropriate. Isomaltulose or palatinose can be safely used for human consumption as described in the article from Bar et al. (Lina, BAR, Jonker, D, Kozianowski, G, , =

3 e Isomaltulose (Palatinose ): a review of biological and toxicological studies, Foods Chem.

Toxicol., 40, 2002, 1375).

Isomaltulose is obtained commercially from the enzymatic conversion of sucrose. More specifically, isomaltulose can be obtained from high concentrations sucrose solutions 5 using enzymes, free microorganisms, free immobilized cells or immobilized cells (Mcallister, M, Kelly, CT, Doyle, E, Fogarty, WM, The isomaltulose synthesising enzyme of Serratia plymuthica, Biotechnol. Lett., 12, 1990, 667; Nagai, Y, Sugitani, T, Tsuyuki, K, Characterization of a-glucosyltransferase from Pseudomonas mesoacidophila MX-45, Biosci. Biotech. Biochem., 58, 1994, 1789; Cheetham, PSJ, Garrett, C, Clark, J, 10 Isomaltulose production using immobilized cells, Biotechnol. Bioeng., 27, 1984, 471;

Shimizu, J, Suzuki, Y, Nakajima, Y, Method of producing an immobilized a-glycosyltransferase useful in the production of palatinose from sucrose, UK
Patent 2,082,591, 1982). The enzyme a-glucosyltransferase (sucrose isomerase; EC
5.4.99.11) converts sucrose into isomaltulose (a-D-glucosylpyranosy1-1,6-D-fructofuranose as 15 shown below) or trehalulose (a-D-glucosylpyranosy1-1,1-D-fructofuranose) along with the glucose and fructose as minor hydrolyses said products.

.11 , Lit

4 ____¨_____ .:
b.
...,, ,, .
:,:----1-1-111, õ (14 f , =

=.: -- 11,111 II I

il: , .1' II

4' i (1';' -1 =
= li !, =ili.ilf filo,.

it)-(-1)-:.111,,,iiiiono.1-i I - ')-titi,:...ctaii, =lovi 1-.1v,1011.=.fr.,,ei Strains such as Protaminobacter rubrum, Serratia plymuthica et Erwinia rhapontici can produce isomaltulose (75%-80%) and a small amount of trehalulose. It has also been reported that species from Klebsiella can produce from 60 to 70% isomaltulose while strains of Pseudomonas mesoacidophila and Agrobacterium radiobacetr can convert sucrose into trehalulose at a rate of about 90%.
In 1985 Mitsui Seito Company (Japon) reported using a bioreactor and the bacteria P.
rubrum for the commercial production of isomaltulose (Mitzutani, T, Preparation and use of palatinose oligosaccharides, New Food Ind., 33, 1999, 9-12). The conversion of sucrose into isomaltulose in the presence of Serratia plymuthica has also been reported (Vorlop, KD, Klein, J, Entrapment of microbial cells in chitosane in Mosbach K. editor, Methods in Enzymology, 135, 1987, 259; Krastanov, A, Yoshida, T, Production of palatinose using Serratia plymuthica cells immobilized in chitosane, J. Ind.
Microbiol.
Biotechnol., 30, 2003, 593) as well as techniques for immobilizing enzymes using calcium alginate and/or chitosane.
While the production of isomaltulose has been described, isomaltulose is generally obtained as a finished product which is then added to the desired beverage or food as a sugar substitute.
In view of the health concerns associated with sucrose, there is a need for food products which do not increase glycaemia and/or cause tooth decay. Maple products are complex products made from the concentration of maple sap. As sucrose concentration in maple products is high, the growth of strains converting sucrose into isomaltulose in maple products represents a challenge. Furthermore, conditions such as temperature, time and pH for growing said strains in maple products as well as conditions for obtaining an attractive concentration of isomaltulose are unknown. The production of maple products having a low glycaemic index having similar organoleptic and hygienic properties as regular maple products remains a challenge.
Recently, the company Natunola has issued a press release indicating that they have developed a maple syrup that contains isomaltulose. However, Natunola is silent about the characteristics, processes and/or methods for producing this modified maple syrup.

5 Contrary to other food products, low glycaemic index maple products can therefore not be made by simply adding isomaltulose that is produced by known methods. There is therefore a need for low glycaemic index maple products having organoleptic properties similar to sucrose based maple products. There is therefore a need for maple products having organoleptic properties similar to sucrose based maple products and having lower sucrose concentration.
The present invention provides a maple product comprising isomaltulose enzymatically converted from a maple starting product and sucrose in a ratio of 90:10 to 10:90.
The present invention also provides a maple product comprising isomaltulose enzymatically converted from a maple starting product wherein said maple product has a glycaemic index lower than 45.
The present invention also provides a maple product comprising isomaltulose enzymatically converted from a maple starting product comprising sucrose wherein the enzymatically converted isomaltulose corresponds to about 20% to 98% of the sucrose present in the maple starting product prior to the enzymatic conversion.
The present invention further provides a process for obtaining an isomaltulose maple product comprising the step of enzymatically converting the sucrose present in a maple starting product into isomaltulose wherein at least 20% to 98% of the sucrose present in the maple starting product is converted into isomaltulose.
The present invention provides a maple product obtained by the process as defined herein.
Brief description of the figures Fig. 1 represents the growth profile of strain #15 (1A) in the SPY media and the result of the Benedict test (16).

6 Fig. 2 represents the growth profile of strain #15 (2A) in the maple syrup media and the result of the Benedict test (26).

Fig. 3 represents the growth profile of strain #15 (A) in the maple sap media and the result of the Benedict test (2B).
Fig. 4 represents the growth kinetic and production of reducing sugars by strains #15 in maple syrup (4A) and maple sap (4C) along with the respective results of the Benedict test (4B and 40).
Fig. 5 represents the growth kinetic and production of reducing sugars (isomaltulose) by strain # 15 with maple syrup media at 70 g/L (5A), the conversion rate (5B) and the results of the Benedict test (5C).

Fig. 6: represents the growth kinetic and production of reducing sugars (isomaltulose) by strain #15 with maple syrup media at 100 gIL (6A), the conversion rate (6B) and the results of the Benedict test (6C).

Fig. 7: represents the growth kinetic and production of reducing sugars (isomaltulose) by strain #15 with maple syrup media at 440 g/L.
Fig. 8: represents the growth kinetic (8B) and production (8D) of reducing sugars (isomaltulose) by strain #15 with maple syrup media at 100 and 300 g/L and the Benedict results (8A, and 8C).
Fig. 9 represents the effect of sucrose concentration on the isomaltulose conversion.

Fig. 10 represents the effect of the enzyme concentration on the isomaltulose conversion (10B) and the results of the Benedict test (10A).

7 Fig. 11: represents the rate of conversion into isomaltulose and the enzymatic stability of the immobilized enzymes (1200 beads) in 30 ml of maple syrup (11A) and 60 ml of maple syrup (11B).
The present inventors have shown that low glycaemic index maple product can be obtained directly from maple syrup and/or maple sap and even from declassified maple syrups. The present inventors have shown that sucrose contained in maple syrup and/or maple sap can be converted into isomaltulose, a natural sugar substitute with reported benefit effects.
In one aspect the invention relates to a maple product having a lower glycaemic index and having the organoleptic properties of sucrose based maple products such as appearance, aroma, flavour and mouth-feel.
In one aspect, the maple product of the invention retains the flavour and essences (i.e. -the organoleptic properties) of the original sucrose based maple syrup.
In one aspect, low glycaemic maple product can be obtained using the process and methods of the invention without destroying or adversely affecting the flavour and organoleptic properties of original sucrose based maple product.
In one embodiment, the isomaltulose containing products are suitable for diabetics.
The glycaemic index is a measure of the effects of carbohydrates on blood sugar levels.
Carbohydrates that break down quickly during digestion and release glucose rapidly into the bloodstream have a high glycaemic index; carbohydrates that break down more slowly, releasing glucose more gradually into the bloodstream, have a low glycaemic index.
The glycaemic index of a food is defined as the area under the two hour blood glucose response curve (AUC) following the ingestion of a fixed portion of carbohydrate (usually

8 50 g). The AUC of the test food is divided by the AUC of the standard (either glucose or white bread, giving two different definitions) and multiplied by 100.
The term "isomaltulose" also known as Palatinosetm refers to the disaccharide:
6-0-a-D-glucopyranosyl-D-fructose.
The expression "maple starting product" refers to a concentrated or unconcentrated sap of the botanical genus Acer. In one aspect, the maple starting product contains 30 to 900 g/litre of sucrose, and for example; 30 g/l, 35 g/I, 70 g/I, 100 g/I, 200 WI, 280 g/I, 300 400 g/I, 440 gIl, 800 g/I, 850 g/I. The starting maple product may also contain organic acids such as malic acid, mineral such as potassium, calcium, zinc, manganese, amino acids and organic compounds such as vanillin, hydroxybutanone and propionaldehyde.
It is understood that the starting maple product may contain other sugars such as fructose and glucose.
The expression "enzymatically converting sucrose into isomaltulose" refers to the enzymatic conversion of the sucrose within the maple starting product into principally isomaltulose.
The conversion rate is calculated based on the conversion into isomaltulose of the sucrose present in the starting product. In one aspect, the process of the invention allows the enzymatic conversion of 20% to 98% of the sucrose present.
Methods for quantifying sucrose in a solution are known such HPLC and refractometer.
The identification and quantification of reducing and non-reducing sugar can be done using methods well known in the art, for example, the Benedict method and by thin layer chromatography (TLC). The quantification of existing sugars can also be conducted by incubating the solution to be tested with the invertase enzyme and quantifying the fructose obtained with HPLC (e.g. see example 1). It is well known in the art that invertase has no effect on isomaltulose.

9 In one aspect the sucrose is enzymatically converted into isomaltulose with a microorganism or an enzyme.
In another embodiment, the step of enzymatically converting sucrose into isomaltulose is performed with a microorganism. The term "microorganism" refers to an organism that is unicellular or lives in a colony of cellular organisms such as bacteria, fungi, protist or archea. It is understood that the microorganism used in accordance with the present invention comprises an enzyme capable of converting sucrose into isomaltulose such as a-glucosyltransferase. Methods to identify the suitable microorganism are well known in the art such as standards enzymatic conversion tests. For example, a person skilled in the art would first evaluate the conversion capabilities of a microorganism in standard conditions. In a preferred embodiment, the microorganism is Serratia plymuthica.
In one embodiment, the step of converting sucrose into isomaltulose using microorganism is performed for a period of time sufficient for the enzyme to obtain the desired conversion of sucrose into isomaltulose. In one embodiment, the time required is about 30 minutes to about 80 hours and for example about 12, 24 or 48 hours.
The step of converting sucrose into isomaltulose using microorganism is performed at a temperature sufficient for the enzyme to obtain the desired conversion of sucrose into isomaltulose. In one aspect, the conversion is performed at a temperature of about 20 to about 30 C. The desired temperature can be obtained by known methods such as heating or cooling. In a preferred embodiment, the temperature is about 26 C.
In one embodiment, the present invention provides the use of an enzyme in order to convert the sucrose contained in maple syrup into isomaltulose.
In one aspect, the step of enzymatically converting sucrose into isomaltulose is performed with an enzyme. In a preferred embodiment, the enzyme a-glucosyltransferase (sucrose isomerase EC 5.4.99.11) is used to convert sucrose into isomaltulose. Sucrose isomerase and a-glucosyltransferase are used interchangeably.
Sucrose isomerase is known to produce at different concentration trehalulose (a-D-

10 glucosylpyranosy1-1,1-D-fructofuranose) as a by-product of the production of isomaltulose. Therefore the enzyme of the present invention may also convert sucrose into trehalulose. In another preferred embodiment, the enzyme a-glucosyltransferase is isolated from a microorganism such as Serratia plymuthica. Techniques for isolating enzyme from microorganism are well known such as centrifugation, sonication, filtration and homogenization. Sucrose isomerase can be purified and characterized as generally described in Appl. Biochem. Biotechnol (2011). 163:52-63. Sucrose isomerase can also be purified and characterized from microorganisms, including Etwinia rhapontici NCPPB
1578, Serratia plymuthica ATCC 15928, Pantoea dispersa UQ68J, Klebsiella sp., Enterobacter sp. FMB-1, Pseudomonas mesoacidophila MX-45 and also from a species of whitefly (e.g. Nagai, Y., Sugitani, T., & Tsuyuki, K. (1994). Bioscience, Biotechnology, and Biochemistry, 58(10)1789-1793; Zhang, D., Li, X., & Zhang, L. H. (2002).
Applied and Environmental Microbiology, 68(6):2676-2682; Wu, L., & Birch, R. G.
(2004).
Journal of Applied Microbiology, 97(1):93-103; Ravaud, S., Watzlawick, H., Haser, R., et at. (2006). Acta crystallographica section F structural biology and crystallization communications, 62(Pt1):74-76; Cha, J., Jung, J. H., Park, S. E. et al.
(2009). Journal of Applied Microbiology, 107(4):1119-30; Cheetham, P. S. J. (1984). The Biochemical Journal, 220, 213-220; Wu, L., & Birch, R. G. (2005). Applied and Environmental Microbiology, 71(3):1581-1590 and Veronese, T., & Perlot, P. (1999). Enzyme and Microbial Technology, 24, 263-269.).

Sucrose isomerase has been sequenced in different microorganisms. For example see D9MPF2 in uniprot (http://www.uniprot.org/uniprot/D9MPF2), AAK28735, AAP57083, AAP57084, AAP57085, ACF42098, ADJ56407, ADP12651, CAG74753, CA096700, CAX55502 and ADJ56407 in ebi (http://www.ebi.ac.uk). Moreover, methods for expressing isomaltulose into a microorganism are well known (Li S, Cai H, Qing Y, Ren B, Xu H, Zhu H, Yao J, Cloning and characterization of a sucrose isomerase from Erwinia rhapontici NX-5 for isomaltulose hyperproduction. Appl Biochem Biotechnol.
2011 Jan;163(1):52-63, Wu, L., & Birch, R. G. Characterization of the highly efficient

11 sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene. Applied and Environmental Microbiology, 2005 71(3):1581-1590 and Hyeon Cheol Lee, Jin Ha Kim, Sang Yong Kim, and Jung Kul Lee. Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from "Protaminobacter rubrum" (Applied and environmental microbiology, Aug. 2008, p. 5183-5194 Vol. 74, No. 16).
In a further embodiment, the enzyme can be immobilized on a support. In a further embodiment, the support is calcium alginate. The present inventors have found that conversion rate higher than 85% can be obtained using calcium alginate as a support and that the enzymatic activity can be maintained for many weeks.
In one aspect of the invention, the enzyme within the starting maple product is free. In this embodiment, the enzyme can be isolated from a microorganism as mentioned herein. In a preferred embodiment, the concentration of the enzyme within the maple starting product is about 20 mg/ml to about 300 mg/ml of maple starting product.
In another embodiment, the process further comprises the step of immobilizing the enzyme on a support prior to enzymatically converting the sucrose into isomaltulose.
Immobilized enzyme is economic as the enzyme can be reused. The term "immobilized the enzyme on a support" refers to the attachment of the enzyme to a support.
Techniques for immobilizing an enzyme on a support are known and include adsorption on glass or matrix, entrapment, cross-linkage molecule or spacer molecule. In a preferred embodiment, the support can comprise calcium alginate beads into which the beads are entrapped and for example, the support can comprise about 10 to 10 alginate beads and for example about 20, 200 or 800 beads and about 5 to 15 mg of enzyme/bead.
The step of converting sucrose into isomaltulose using free or immobilized enzyme is performed for a period of time sufficient for the enzyme to achieve the desired conversion of the sucrose into isomaltulose. In one embodiment, the time required is

12 about 10 minutes to about 50 hours and for example about 30 minutes, 1 hour, 1h30, 3, 4, 5, 6 or 17 hours. For example, the enzyme converts 87% of the sucrose into isomaltulose in a maple starting product comprising 850 g/litre of sucrose after 30 minutes when the enzyme is free and 90% of the sucrose is converted into isomaltulose in a starting maple product comprising 850 g/litre of sucrose after 17 hours when the enzyme is immobilized.
The step of converting sucrose into isomaltulose using free or immobilized enzyme is performed at a temperature sufficient for the enzyme to obtain the desired conversion of sucrose into isomaltulose. The conversion is performed at a temperature of about 20 to about 60 C. In a preferred embodiment, the temperature is about 37 C or about 45 C.
EXAMPLES
EXAMPLE 1: Production of isomaltulose via bacterial fermentation Strain selection Serratia plymuthica (#15) was cultivated in different media (in a synthetic media (SPY)), maple sap and maple syrup. Different fermentations were conducted at 26 C at rpm. The pH of the media was adjusted at pH 7 and microbiological control was conducted during the fermentation period. Bacterial growth was monitored by optic density at 600 nm (OD 600 nm) using a spectrophotometer. The conversion of sucrose in the SPY, maple sap and maple syrup media as well as the production of isomaltulose were monitored.
The results of Fig. 1 demonstrate that strain #15 can grow in SPY media (Fig 1A). The qualitative Benedict test (Fig. 1B) indicates the presence of reducing sugar such as isomaltulose.

13 Strain #15 grows in maple syrup media (Fig 2A). The Benedict test shows the presence of reducing sugar and for instant isomaltulose. As shown in Fig. 26, the coloration intensifies over time.

Similar results were obtained with maple sap media as shown in Figs 3A and 3B.

Method for quantifying isomaltulose with invertase combined with HPLC
(invertase-HPLC) It is well known that enzyme invertase hydrolyses sucrose into free glucose and fructose. It is also well known that this enzyme has no effect on isomaltulose. The presence or absence of isomaltulose can therefore be confirmed by treating a sample with invertase enzyme followed by HPLC analysis. Analysing a sample by HPLC
which does not comprise isomaltulose is characterized by the presence of peaks corresponding to glucose and fructose obtained from the hydrolysis of sucrose.

Conversion into isomaltulose Samples were analysed using qualitative test of Benedict (Figs 46 and 4D) followed by quantitative evaluation using invertase-HPLC method. The change of color in the Benedict test is indicative of the enzymatic conversion. Light coloration was obtained in the first sample which became increasingly darker indicating the conversion of non-reducing sucrose into reducing isomaltulose.

The results obtained indicate that after 24h in the above described condition, a conversion rate of 70% isomaltulose is obtained. This rate can increase up to 81% after an incubation period of 48h. It is important to note that the results obtained using the invertase-HPLC method correlate with the results obtained with the Benedict test.

The effect of sucrose concentration contained in maple syrup media on the growth of strain #15 and conversion rate into isomaltulose.

14 Fig. 5A shows the growth of strain #15 in maple syrup media containing 70 g/l of sucrose. The conversion of sucrose into isomaltulose is proportional with the growth rate of the strain. Fig. 5C confirms the conversion by the Benedict test and clearly indicates the presence of a reducing sugar after an incubation of 12h. The coloration becomes darker as the fermentation time increases. The results from the invertase-HPLC
method indicate the production of isomaltulose. The presence of peaks corresponding to glucose and fructose indicates the initial concentration of sucrose. Fig. 56 summarizes the HPLC
results of the samples following fermentation in maple syrup media at 70 g/l.
These quantitative results correlate the qualitative results of the Benedict test of Fig. 5C. A
conversion rate of 97% after 24h of fermentation is observed. This rate is maintained at 96% as indicated at Fig.5B.
Figs. 6A, 6B and 6C depict the results obtained using maple syrup media containing 100 g/l of sucrose. The inventors have observed that the isomaltulose conversion rate decreases from 64% (maple syrup 70 g/l) to 22% (maple syrup 100 g/l) after 12h of fermentation. After 24h of fermentation, a conversion rate of 89% using maple syrup media at 100 g/l of sucrose was observed (Fig. 6B).
The conversion rate is inversely proportional to the sucrose concentration of the starting media. Fig. 7 shows that following 3 days of fermentation, the conversion rate of sucrose into isomaltulose in maple syrup media containing 440 g/l of sucrose is 0%.
Results obtained with 1 litre fermentation The previous fermentations were conducted with volumes of 15 ml, 50 ml and 100 ml of different media. The following fermentations were studied: 1) 1 litre fermentation of maple syrup containing 100 g/l of sucrose; 2) 1 litre fermentation of maple syrup containing 300 g/I of sucrose. The fermentations were conducted using the experimental procedure described in this example. As can be shown in Fig. 8 (A to D), 98%
of sucrose contained in maple syrup 100 g/l and 52% of sucrose contained in maple syrup 300 g/l was converted after a period of 48h incubation.

=

15 Table 1 below summarizes the results of the growth of strain #15 and the conversion obtained in maple syrup.

Table I:
Fermentation Maple Maple Maple Maple Maple Maple Maple Maple /Conversion syrup syrup syrup syrup syrup syrup syrup syrup rate 70g/I 100g/1 140g/I 200g/1 280g/I 440g/I 100g/1 300g/I
100m1 100m1 100m1 100m1 100m1 100m1 1 I 1 I
Growth good good good good average Very good average low After 12h 61% 22% 15% 6% 1% 0%
After 24h 97% 89% 87% 88% 41% 0%
After 48h 96% 92% 92% 93% 85% 0% 98% 52%

Qualitative Benedict method as well as quantitative invertase-HPLC method is used to confirm the presence of isomaltulose.

EXAMPLE 2: Production of isomaltulose using free sucrose isomerase Fig. 9 clearly indicates that free enzyme is capable of converting sucrose into isomaltulose in concentration ranging from 100 to 800 g/I (Sucrose-100 to Sucrose-800).
The conversion rate in a 200 g/I solution is 82% at 37 C for 1h and the conversion rate in a solution comprising 800 g/I of sucrose is about 60%. Conversion rates ranging from 30 to 49% were obtained from half diluted maple syrup media (440 g/l) and original maple syrup at 850 WI tested in similar conditions. Increasing the enzyme concentration from 200 to 800 pl or 60 to 240 mg/ml, afforded a conversion rate of 77% with the original maple syrup concentrated at 850 g/I and 83% with the maple syrup media concentrated at 440 g/I (see Fig. 10B).

After 30 minutes the conversion rate in maple syrup concentrated at 440 g/I is 91% and is 87% in maple syrup concentrated at 850 g/I. Increasing the reaction time to minutes afforded a conversion rate of 82% (maple syrup concentrated at 440 g/1) and of 84% (maple syrup concentrated at 850 WI). Without being bound to any theory, the

16 inventors believe that this reduction may be caused by a retro-inhibition or a reactional reversing.

Table ll below summarizes the enzymatic activity observed at 37 C for lh relative to the enzyme concentration and substrate.

Table II:

Enzyme amount 60 60 240 mg/I
Concentration Sucrose solution g/I Maple syrup Maple syrup of substrate 100 200 400 800 440 g/I 850 g/I 440 g/I 850 g/I

% of conversion 76% 82% 69% 60% 49% 30% 83% 77%

EXAMPLE 3: Production of isomaltulose using an immobilized enzyme The results shown in Table 3 below summarize the different parameters used in the production of isomaltulose with sucrose isomerase in calcium alginate.

Tableau Ill: Immobilization using calcium alginate beads at 37 C for 17h beads 200 beads 800 beads Post-production 1 week 3 weeks 6 weeks time Volume of maple syrup 1 ml 4 ml 4 ml 8 ml 20 ml 20 ml % of conversion 50% 89% 87% 76% 90% 69%
into isomaltulose

17 One month and an half following post-production, the temperature effect (37, 45 and 50 C) was observed in relation to the incubation time (1h30, 3h and 0/N=17h).
A
conversion rate of 90% at 45 C was obtained following an incubation period of 17h.
The results in Fig. 11A indicate that for a volume of maple syrup of 30 ml, a conversion rate of 80% was observed after 6h of incubation. 74% of conversion rate was observed after 9 weeks and 53% after 12 weeks. Fig. 11B indicates that even if the amount of culture medium is doubled, similar results are obtained.
The inventors have also observed that increasing the number of alginate beads and the reaction volume (5000 beads and maple syrup concentrated at 440g/I, 500m1) yielded a conversion rate of 88% after 17h of incubation and yielded a conversion rate of 92%
after 36h of incubation.

Materials and Methods A) Bacterial Strain The following strain was used:
Serratia plymuthica; (ATCC 15928) renamed strain #15;
Culture media Conservation media Strain #15 was stored and maintained in a solid TSA gelose (Quelab #QB-39-5106) with 40 g/I of distilled water. After dissolution, pH was adjusted at 7, the media was sterilized by autoclaving (121 C / 15 min) cooled in a bain-marie to 45 C and distributed under sterile conditions, into sterile Petri boxes. The strain was kept at 4 C and maintained by regular re-seedings.

18 The strain was also kept in the freezer at -20 C and -70 C in a liquid synthetic SPY-glycerol having the following composition:
g/I of Peptone 4 g/I of yeast extract 5 40 g/I of sucrose Glycerol (final concentration of 10%) Synthetic medium for culture (SPY) The following SPY medium was used:
- Peptone (10 g/1) 10 - Yeast extract (4 g/I) - Sucrose (40 g/1) pH adjusted at 7 and media autoclaved and distributed in Erlenmeyer.
Fermentation medium (maple products) Studies were conducted with maple sap having a sucrose concentration between 3 and 10% and of grade A (from Citadelle).
Growth conditions Fermentations in different media were conducted in Erlenmeyer, seeded from isolated colonies and put in rotating incubator. Incubation was conducted at 26 C with an agitation of 250 rpm and samples were taken on a regular basis. Bacterial growth was determined using a spectrophotometer (optic density at 600 nm (OD 600nm)). PH
was also monitored during the fermentation process with a pH-meter.
Treatment of bacterial production Samples prepared for chemical analysis were centrifuged at 500 rpm, at 5 C for 5 to 10 minutes. The pellet was kept in glass tubes at 4 C or was frozen.

19 Preparation of samples for the enzymatic determination At the end of each fermentation period, the culture broth was centrifuged at 4000 rpm at C for 30 minutes in order to collect the cells. The pellet was rinsed twice with sterile water and centrifuged 30 minutes at 4000 rpm at 4 C. The pellet was weighed, 5 solubilised in 5 ml of phosphate buffer pH 6.0 and kept in a freezer until needed.

B) Determination of the enzymatic activity of sucrose isomerase (SI) The enzymatic extract was prepared as follows:
- from fresh or frozen biomass (from step A);
- sonication (Branson sonifier 150) for 15 seconds, 5 times at power 5;
- centrifugation for 1h at 4000 rpm at 4 C;
- addition of 5 ml of buffer (pH 6.0) to the pellet (humid crude extract).

pH 6.0 Phosphate Buffer A = Monobasic sodium phosphate solution at 0,2M;
B = Dibasic phosphate sodium solution at 0,2M;
87,7 ml of solution A + 12,3 ml of solution B, topped at 200 ml with water and pH was adjusted at 6,0. The buffer was autoclaved and kept at room temperature.

Following the enzymatic extraction, the enzymatic reaction was conducted in the following reaction media:
Substrate (sucrose, maple sap or maple syrup) 100 pl (variable concentration);

Enzymatic extract 200 p1(60 mg/ml humid crude extract); and Phosphate buffer pH 6,0, 200 pl.

The reactional volume was 500 pl and the reaction time was lh at 37 C. The enzymatic reaction was stopped by placing the solution in boiling water for 10 minutes.
The results from the enzymatic reaction were analysed by the Benedict method and HPLC.
Prior to the injection of HPLC, the samples were diluted and filtered.

20 C) Immobilization of sucrose isomerase (SI) The following reactants were used:
Sodium alginate 2 % (w/v) CaCI22% (w/v) Glutaraldehyde 0,06%
Sonicated enzymatic extract (from step B) and alginate were mixed (1:2) and transferred with gentle stirring into a syringe over a becher containing a CaCl2 solution.
The beads are formed by adding the enzymatic-alginate mix to the CaCl2 solution one drop at a time. The beads are placed in the refrigerator for at least 1 hour or over night.
The resulting beads are rinsed with water, dried and placed into a becher containing 0,06% glutaraldehyde and kept for 20 minutes at room temperature.
The beads are rinsed with phosphate buffer pH 6,0, dried and counted.

D) Sugar analysis Glucose, fructose, sucrose and isomaltulose were analyzed and quantified by qualitative and quantitative methods as follows:
Benedict method (Fehlinq Liquor);
0.5 ml of Benedict agent (Fisher #S79914-2) and a drop of NaOH was added to a 0,5 ml sample in a glass tube and the latter was placed into boiling water for 5 minutes;
Non-reducing sugars yield a blue coloration and reducing sugars change the coloration of the mixture to red/brown.
High pressure liquid chromatography Shimadzu Refraction index RID-10A
Pump Column over CTO-10AS vp Auto-injector SiL-9A

21 Protocols Rezex column: RHM-Monosaccharide H+ (300x7.80mm) of Phenomenex Mobile phase: 0,01N H2SO4 Rate: 0,5 ml/min Maximum pressure: 45bar Temperature of the column: 80 C
Guard column Cation H (#00H-0132K0) of Phenomenex"
Column Aminex: HPX=87H 300 x 7,8mm, BIO RAD"
Mobile phase: 0,01N H2SO4 Rate: 0,5 ml/min Maximum pressure: 65bar Temperature of the column: 65 C
Guard column Cation H+ (#125-0129), BIO RAD
Thin layer chromatography (TLC) 3 pl of each sample from the reaction medium was deposited on TLC silica K5F
(Silicycle") and developed twice with ethylacetate/isopropanol/water (1:7:2) and pulverized with 0,3% (w/v) N-ethylenediamine (1-naphthyl) and 5% (v/v) H2SO4 in methanol and developed at 110 C for 10 minutes.

Claims (38)

What is claimed:

1. A maple product comprising isomaltulose enzymatically converted from a maple starting product and sucrose in a ratio of 90:10 to 10:90.

2. A maple product comprising isomaltulose enzymatically converted from a maple starting product wherein said maple product has a glycaemic index lower than 45.

3. A maple product comprising isomaltulose enzymatically converted from a maple starting product comprising sucrose wherein the enzymatically converted isomaltulose corresponds to about 20% to 98% of the sucrose present in the maple starting product prior to the enzymatic conversion.

4. The maple product according to any one of claims 1 to 3 wherein the maple product is maple sap or maple syrup.

5. The maple product of claim 4 wherein the maple syrup comprises at least 200 g/l of isomaltulose.

6. The maple product of claim 4 wherein the maple syrup comprises at least 400 g/I
of isomaltulose.

7. The maple product of claim 4 wherein the maple syrup comprises at least 500 g/I
of isomaltulose.

8. The maple product of claim 4 wherein the maple syrup comprises at least 800 g/I
of isomaltulose.

9. A process for obtaining an isomaltulose maple product comprising the step of enzymatically converting the sucrose present in a maple starting product into isomaltulose wherein at least 20% to 98% of the sucrose present in the maple starting product is converted into isomaltulose.

10. The process of claim 9, wherein the conversion step is conducted with an enzyme.

11. The process of claim 10, wherein the enzyme is from Serratia plymuthica.

12. The process of claim 10, wherein the enzyme is .alpha.-glucosyltransferase.

13. The process of any one of claims 10 to 12, wherein the concentration of the enzyme is at least 20 to 300 mg/ml.

14. The process of any one of claims 10 to 13, further comprising the step of immobilizing the enzyme on a support, prior to enzymatically convert the sucrose into isomaltulose.

15. The process of claim 14, wherein the support comprises glass, matrix, cross-linkage molecule, spacer molecule or calcium alginate beads.

16. The process of claim 15, wherein the support comprises alginate beads.

17. The process of claim 16, wherein the support comprises at least 10 to 10 alginate beads.

18. The process of claim 17, wherein the support comprises at least 20, 200 or alginate beads.

19. The process of any one of claims 14 to 18, wherein the alginate beads comprise at least 5 to 15 mg of enzyme/bead

20. The process of any one of claims 9 to 19, wherein the concentration of sucrose presents in the maple starting product is from about 50 to about 900 g/litre.

21. The process of claim 20, wherein the concentration of sucrose present in the maple starting product is about 100, 200, 400, 440, 800 or 850 g/litre.

22. The process of any one of claims 9 to 21, wherein the step of enzymatically converting sucrose into isomaltulose lasts about 10 minutes to about 50 hours.

23. The process of claim 22, wherein the step of enzymatically converting sucrose into isomaltulose lasts about 30 minutes, 1, 1.5, 3, 4, 5, 6, or 17 hours.

24. The process of any one of claims 9 to 23, wherein the step of enzymatically converting sucrose into isomaltulose is at a temperature of about 20 to 60°C.

25. The process of claim 24, wherein the step of enzymatically converting sucrose into isomaltulose is at a temperature of about 37 or about 45°C.

26. The process of claim 9, wherein the sucrose is enzymatically converted into isomaltulose with a microorganism.

27. The process of claim 26, wherein the microorganism is Serratia plymuthica.

28. The process of claim 26 or 27, wherein the step of enzymatically converting sucrose into isomaltulose lasts about 30 minutes to about 80 hours.

29. The process of claim 28, wherein the step of enzymatically converting sucrose into isomaltulose lasts about 12, 24 or 48 hours

30. The process of any one of claims 26 to 29, wherein the step of enzymatically converting sucrose into isomaltulose is at a temperature of about 20 to 30°C.

31. The process of claim 30, wherein the temperature is about 26°C.

32. The process of any one of claims 26 to 31, wherein the sucrose concentration presents in the maple starting product is about 30 to 400 g/litre.

33. The process of claim 32, wherein the sucrose concentration present in the maple starting product is about 35, 70, 100, 200, 280 or 300 g/litre.

34. The process of any one of claims 9 to 33, wherein the maple starting product is maple syrup or maple sap.

35. The process of any one of claims 9 to 33, wherein the maple product is maple syrup or maple sap.

36 The process of claim 35, wherein the maple syrup or maple sap is evaporated to form maple butter, maple candy, maple sugar or maple toffee.

37. A maple product obtained by the process as defined in any one of claims 9 to 36.

38. The maple product of claim 37, wherein the maple product is maple syrup, maple sap, maple butter, maple candy, maple sugar or maple toffee.