Prebiotic composition and method for producing same

Abstract

The present invention relates to non-digestible prebiotic oligosaccharides that have been modified to incorporate one or more monosaccharide units and to have a higher sweetness value than unmodified oligosaccharides. Methods for the use and production of the non-digestible prebiotic oligosaccharides are also described. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] The present invention relates to sweet, non-digestible oligosaccharides that have particular application as functional food ingredients for foodstuffs and as incorporated ingredients in prepared food products, or that can be used alone to add sweetness. [Background technology]

[0002] Prebiotics, which are substrates selectively utilized by host microorganisms, such as lactobacilli or bifidobacteria, that confer health benefits, are finding ever-increasing application in the food sector. Prebiotics can be non-digestible food ingredients that are selectively metabolized by colonic bacteria, contributing to improved health. Thus, the use of prebiotics can promote beneficial changes in the indigenous gut bacterial environment, which can aid in the survival of probiotics. Prebiotics have a global effect on the gut bacterial population, distinct from many dietary fibers, such as pectin, cellulose, and xylan, which are not selectively metabolized in the gut. The criteria for classification as a prebiotic are that it must be resistant to gastric acidity, hydrolysis by mammalian enzymes, and absorption in the upper gastrointestinal tract, and must reach the colon in adequate amounts to be fermented by the gut microbiota and selectively stimulate the growth and / or activity of gut bacteria associated with health and well-being. Summary of the Invention [Problem to be solved by the invention]

[0003] It is an object of the present invention to provide non-digestible oligosaccharides with an enhanced sweetness value. In particular, it is an object of the present invention to provide non-digestible oligosaccharides with an enhanced sweetness value that are less bitter and / or have less undesirable aftertaste. It is a further object of the present invention to provide sweeter, naturally occurring fibers that are not digested in the human or animal intestine and therefore can be used as low-calorie functional ingredients that can improve microbiome diversity. [Means for solving the problem]

[0004] According to a first aspect of the present invention, there is provided a non-digestible oligosaccharide that has been modified to incorporate one or more monosaccharide units and to have a higher sweetness value than an unmodified oligosaccharide.

[0005] According to a related aspect of the present invention, synthetic non-digestible oligosaccharides are provided that have been modified to incorporate one or more monosaccharide units and to have a higher sweetness value than unmodified oligosaccharides.

[0006] In accordance with a further related aspect of the present invention, there is provided a prebiotic comprising non-digestible oligosaccharides modified to incorporate one or more monosaccharide units and to have a higher sweetness value than the unmodified prebiotic oligosaccharides.

[0007] According to still further related aspects of the present invention, there is provided a prebiotic composition comprising synthetic non-digestible prebiotic oligosaccharides modified to incorporate one or more monosaccharide units and to have a higher sweetness value than unmodified prebiotic oligosaccharides.

[0008] In a related aspect of the invention, the invention may provide non-digestible oligosaccharides that may or may not be prebiotic (may or may not be used as a prebiotic). [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a bar graph showing key attributes found to be significantly different between the samples of Example 1 for sweetness (bars represent mean values, error bars extend ± half LSD). [Figure 2] FIG. 2 is a bar graph showing key attributes found to be significantly different between the samples of Example 1 for off-taste (bars represent mean values, error bars extend ± half LSD). [Figure 3]FIG. 3 is a bar graph showing key attributes found to be significantly different between the samples of Example 1 for sweet aftertaste (bars represent mean values, error bars extend ± half LSD). [Figure 4] FIG. 4 is a plot showing the dose-response curve for the sweetness of the sucrose preparation used in Example 1. [Figure 5A] FIG. 5A shows the gas chromatography profile of the trimethylsilyl oxime of the disaccharide fraction of OPTI-GOS-Y tested in Example 1. [Figure 5B] FIG. 5B shows the gas chromatography profile of the trimethylsilyl oxime of the trisaccharide fraction of OPTI-GOS-Y tested in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0010] The terms "synthetic" and "synthesized" are intended to mean products that do not exist in nature or are not produced in nature. This term, of course, encompasses naturally occurring precursor compositions and "man-made" products using natural products, such as naturally occurring enzymes.

[0011] The oligosaccharides of all relevant aspects may be modified during synthesis. Preferably, the oligosaccharides are synthesized using a disaccharide precursor or an oligosaccharide precursor and a different monosaccharide.

[0012] The present inventors have surprisingly found that by incorporating different monosaccharides during the synthesis of non-digestible oligosaccharides, the different monosaccharides can improve the sweetness of the oligosaccharides, apparently without compromising other functional aspects of the oligosaccharides.

[0013] The terms "modify," "modified," or "modified" are intended to mean making partial or subtle changes to the overall structure of an oligosaccharide without substantially altering the functional aspects of the oligosaccharide. These terms are intended to encompass partial or subtle changes made during synthesis, but could also encompass changes made after synthesis.

[0014] The oligosaccharides of all relevant aspects may be selected from one or more of the following: inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), α-galactooligosaccharides, and β-glucooligosaccharides. Preferably, the oligosaccharides are selected from fructooligosaccharides (FOS) or galactooligosaccharides (GOS). Most preferably, the oligosaccharides are galactooligosaccharides (GOS).

[0015] In all relevant embodiments, the oligosaccharides are modified during synthesis using galactosidase or glycosidase in the presence of a different monosaccharide acceptor. The oligosaccharides may be fructosylated using β-galactosidase in the presence of a monosaccharide acceptor. Preferably, the monosaccharide acceptor is fructose and the oligosaccharide is modified by fructosylation.

[0016] It will be apparent to those skilled in the art that the β-galactosidase may be derived from a number of different species, however, it is preferred that the β-galactosidase is derived from Aspergillus. The species of the genus Aspergillus may be selected from one or more of the following: Aspergillus officinalis; Aspergillus aculeatus; Aspergillus awamori; Aspergillus carbonarius; Aspergillus cellulosae; Aspergillus oryzae; Aspergillus flavus; Aspergillus japonicas; Aspergillus nidulans; or Aspergillus niger.

[0017] The oligosaccharides may be used for several purposes, such as as a prebiotic. They may be incorporated into food products, food supplements, or calorie-restricted prepared foods. They may be used to replace some, most, or all of the bulk sugar content in food products, or may be used as sweeteners themselves. Advantageously, the inventors have found that the oligosaccharides have a clean flavor profile, a low glycemic index that may be classified as fiber, and help maintain gut microbiome diversity and healthy bacteria. The oligosaccharides may be in the form of granules or powder.

[0018] The term "foodstuff" is intended to mean any material that can be safely ingested by humans or animals, including, but not limited to, foods, beverages, cereals, bakery products, breaded and coated products (fried foods), dairy products, confectioneries, snack foods, and meal products. The term encompasses products that require cooking or reconstitution before being eaten. The term also encompasses any food / nutritional supplement or medicine (such as vitamin tablets or antibiotic liquids).

[0019] It will be apparent to those skilled in the art that the oligosaccharides may be incorporated into a product by blending or mixing the oligosaccharides with other ingredients, or the oligosaccharides may be used to coat a product.

[0020] The oligosaccharides may be present with or used in combination with native oligosaccharides of the same type. During synthesis, with very high yields, the modified oligosaccharides may account for up to about 99% of the oligosaccharides. The combination may include up to about 95% modified oligosaccharides relative to native oligosaccharides, or up to about 90% modified oligosaccharides relative to native oligosaccharides, or up to about 80% modified oligosaccharides relative to native oligosaccharides.

[0021] According to a second related aspect of the present invention, there is provided a combination of non-digestible oligosaccharides of the same type, the combination comprising modified and unmodified oligosaccharides, the modified oligosaccharides being modified to incorporate one or more monosaccharide units and to have a higher sweetness value than the unmodified oligosaccharides.

[0022] The modified oligosaccharides of the combination may include the non-digestible oligosaccharides described hereinbefore with reference to the first aspect.

[0023] The oligosaccharides may be for use as a prebiotic or for incorporation into foodstuffs, food supplements or calorie-restricted prepared food products.

[0024] According to a third related aspect of the present invention, there is provided a method for enhancing the sweetness of a non-digestible oligosaccharide, the method comprising modifying at least a portion of the oligosaccharide to incorporate one or more monosaccharide units.

[0025] In this method, the oligosaccharide is preferably modified to incorporate two or more monosaccharide units therein.

[0026] In the method, the oligosaccharide may be selected from one or more of the following: inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), α-galactooligosaccharides, and β-glucooligosaccharides. Preferably, the oligosaccharide is selected from fructooligosaccharides (FOS) or galactooligosaccharides (GOS). Most preferably, the oligosaccharide is a galactooligosaccharide (GOS).

[0027] The oligosaccharides may be modified during synthesis using galactosidase or glycosidase in the presence of a monosaccharide acceptor, or may be fructosylated using β-galactosidase in the presence of a monosaccharide acceptor.

[0028] As described above, the β-galactosidase may be derived from the genus Aspergillus. The β-galactosidase may be derived from one or more of the following species of Aspergillus: Aspergillus officinalis, Aspergillus aculeatus, Aspergillus awamori, Aspergillus carbonarius, Aspergillus cellulosae, Aspergillus oryzae, Aspergillus flavus, Aspergillus japonicus, Aspergillus nidulans, or Aspergillus niger.

[0029] The monosaccharide acceptor may be fructose and the oligosaccharides modified by fructosylation, however, it will be apparent that a range of novel sweet, non-digestible oligosaccharides can be produced using different enzymes and monosaccharide acceptors.

[0030] In this method, modified oligosaccharides may be produced using the same type of unmodified oligosaccharides. During production, the yield of modified oligosaccharides may be about 99% or less. However, depending on the synthesis conditions, the yield may be lower, such as about 95% or less, about 90% or less, or about 80% or less.

[0031] All of the oligosaccharides described herein thus far have been shown to advantageously form sweet, natural, healthy fibers that are not digested in the human or animal intestine and can therefore be used as non-caloric or substantially non-caloric functional ingredients. These sweet fibers have been developed as potential bulk sugar substitutes, as products with a sweetness similar to sucrose but no or substantially no calories, while also improving microbiome diversity.

[0032] The oligosaccharides were surprisingly found to be significantly sweeter than all other samples with the advantage of being less off-taste (eg bitter, sour, musty, salty, etc.).

[0033] Through experiments, the inventors have shown that the prebiotic composition provides a natural, low-calorie, sweet, healthy fiber with gut microbiome functionality as a potential bulk sugar substitute in a wide range of foods.

[0034] According to a fourth aspect of the present invention, there is provided the use of the prebiotic composition hereinbefore described as a low- or non-calorie sweet prebiotic. The use may be as a bulk sugar substitute ingredient to replace all or part of the sugar or sucrose content of foodstuffs. It will be apparent to those skilled in the art that the composition may be incorporated into a range of foodstuffs, food supplements or calorie-restricted prepared food products.

[0035] According to a related fifth aspect of the present invention, there is provided a method for enhancing the sweetness of a non-digestible oligosaccharide, the method comprising modifying at least a portion of the oligosaccharide during synthesis to incorporate one or more monosaccharide units.

[0036] The method for producing modified non-digestible oligosaccharides may comprise mixing together a disaccharide precursor, a galactosidase or glycosidase, and a monosaccharide acceptor under suitable conditions that allow the enzymatic production of non-digestible oligosaccharides having different monosaccharide units.

[0037] In this production method, the non-digestible oligosaccharide precursor largely depends on which non-digestible oligosaccharide is to be produced. For example, if GOS is to be produced, the non-digestible oligosaccharide precursor may include lactose. The galactosidase or glycosidase selected also largely depends on the non-digestible oligosaccharide to be produced. For example, if GOS is to be produced, the galactosidase or glycosidase may be β-galactosidase. The monosaccharide acceptor may include several acceptors; for example, if the GOS to be produced is a fructosylated GOS, fructose may be selected. It will be apparent to those skilled in the art that other non-digestible oligosaccharide precursors, β-glucosidases, and other glycosidases, such as dextrose, may also be used. It will also be apparent that other donors, such as sucrose, raffinose, or lactulose, and other monosaccharide acceptors may be used to produce novel modified non-digestible oligosaccharides having a sweetness value greater than that of the same type of unmodified non-digestible oligosaccharide.

[0038] It will be apparent to one skilled in the art that some features of the oligosaccharides listed with respect to some aspects of the present invention may also be interchangeable with respect to the combinations and methods described.

[0039] Embodiments of the present invention will now be described, by way of example only. [Example]

[0040] Example 1 – Sensory profiling of oligosaccharides The purpose of these experiments was to determine the sweetness and any off-taste intensity of several oligosaccharides.

[0041] Sample preparation The following commercial and experimental products were supplied to the Sensory Science Centre, Department of Food and Nutritional Sciences, University of Reading by Optibiotix Health Ltd, York, UK: GOS1; GOS2; GOS3; GOS4; OPTI-GOS-Y; GOS derived from lactulose; lactose and lactulose.

[0042] All samples underwent microbiological clearance testing.

[0043] All materials were stored at room temperature. Sucrose was purchased as white granulated sugar (Sainsbury's Plc, London, UK). Water was Harrogate Spa mineral water (Harrogate Water Brands, Harrogate, UK).

[0044] All oligosaccharides were prepared as 5% w / v solutions in mineral water. Weights were accurate to three decimal places, and samples were prepared in volumetric flasks. All samples were well dispersed and easily solubilized in water. Sucrose samples were prepared at 0.5%, 1.0%, 2.0%, and 2.6% w / v.

[0045] Sensory Profiling Methods A trained sensory panel from the Sensory Science Centre was recruited for the sensory profiling of the samples. There were 11 panelists with experience ranging from 6 months to 9 years. A QDA (Quantitative Descriptive Analysis) profiling approach was employed. The panel underwent retraining at the beginning of the sample set. This retraining focused on ensuring that the panelists could reliably score sweetness for the new concentrations of sucrose standard positions.

[0046] Scoring was performed in duplicate in a separate sensory evaluation booth using an arbitrary linear scale (scale of 0 to 100). However, to improve sweetness discrimination, four sucrose samples were used as standards. The mean values ​​for each of these samples, as agreed upon by the panel, are shown in Table 1 below.

[0047] [Table 1]

[0048] At the beginning of each scoring session, the panel tasted the four reference samples in order of increasing intensity to reacquaint themselves with the positioning of these levels of sweetness on the line scale. Reference samples (10 mL) were served in clear polystyrene cups (30 mL). The panel then cleansed their palates with warm filtered tap water and low-salt crackers (Carr's water crackers) before beginning the sample tasting session, and again between each sample scoring session.

[0049] Samples labeled with random three-letter codes were presented in a sequential monadic manner in an unbiased presentation order, with four samples per session. The room was air-conditioned and served at room temperature (23–24°C).

[0050] The panel used 16 characteristics to define the oligosaccharide samples, as shown in Table 2 below.

[0051] [Table 2]

[0052] Panelists were given 5 mL of each sample to taste. 5 mL was measured into a clear tasting cup (30 mL) with a plastic syringe. Panelists carefully sipped the sample, ensuring that the sample ran over their tongue before swallowing. Panelists sipped half of the sample into their mouths and scored the first six attributes, and the second half was used to score the following six attributes: after-effects were scored after a 30-second time delay.

[0053] Data analysis Data were analyzed using a mixed-model ANOVA, treating panelist as a random effect, sample as a fixed effect, and testing main effects for sample by assessor interaction. Multiple pairwise comparisons were performed using Fisher's LSD, and significance was declared at a 5% alpha risk (p ≤ 0.05). Data analysis was performed using Senpaq software (Qi Statistics, Reading, UK).

[0054] Sensory profiling results Of the 11 ranked attributes, eight differed significantly between samples (as shown in Table 2 above). The most substantial differences were in sweetness, off-taste intensity, and sweet aftertaste (see Figures 1-3). OPTI-GOS-Y (fructose-derived galactooligosaccharide, synthesized at CIAL (CSIC UAM) Madrid, 5% w / v) was significantly sweeter than all other samples. OPTI-GOS-Y had an average sweetness of 76, equivalent to a 2% sucrose (w / v) standard. The least sweet samples were lactulose-derived GOS1, GOS2, and GOS (synthesized by CIAL (CSIC UAM) Madrid).

[0055] The sample with the strongest off-taste was the lactulose-derived GOS, which was attributed to bitterness, cardboard / musty, sour / rancid, and metallic tastes, as shown in Table 2. The OPTI-GOS-Y sample had a significantly lower overall off-taste intensity, and the off-taste in this sample was attributed to different flavors; the only other attribute that was substantially and significantly higher was cotton candy taste.

[0056] Although the sucrose standards were not scored blindly, it is useful to compare the mean sweetness values ​​of the samples to the sweetness values ​​of the four sucrose standards. Figure 4 shows the dose-response curves for the sweetness of the sucrose standards. Within the range of concentrations used, the relationship was linear, with the linear regression equation being: Sweetness = 15.5 (sucrose) - 22.5 (R 2 =0.99)

[0057] Using this regression equation, the sweetness values ​​of the samples were converted to equivalent sucrose (ES) concentrations. This was done for the mean sweetness score and the value ± standard deviation. To calculate the relative sweetness (RS) value, the following equation was used: RS = 1 / (5 / ES) × 100

[0058] The values ​​of ES and RS are given in Table 3 below.

[0059] [Table 3]

[0060] The 5% oligosaccharides were all equivalent (on average) to 0.9% to 2% sucrose, leading to relative sweetness values ​​of 18 to 40. These values ​​were not in perfect agreement with the literature available for lactose (literature RS 15, calculated RS 18 to 38). However, it should be noted that the sensory panel was scoring only 0.5 mL samples.

[0061] In conclusion, significant differences in sweetness were found for the oligosaccharides tested in this study. All oligosaccharides were prepared at 5% w / v in mineral water and rated for sweetness according to a defined scale anchored by 0.5%, 1.0%, 2.0%, and 2.6% w / v sucrose standards. The sweetness of the 5% w / v oligosaccharide samples ranged from 0.9 to 2% sucrose. The fructose-derived galactooligosaccharides were significantly sweeter than all other samples and exhibited lower levels of all scored off-flavors except cotton candy.

[0062] Optimization of OPTI-GOS-Y (a blend of GOS and fructosylated GOS) This experiment aimed to investigate the potential yield and preferred enzymes for producing a mixture of GOS (approximately 30%) and fructosylated GOS (approximately 5%) as a prebiotic. The three commercially available enzymes investigated were β-galactosidases from a range of microbial species, including Aspergillus. The substrates used were a range of ratios of lactose and fructose. Purification was performed using yeast fermentation, and the drying process involved freeze-drying and rotary evaporation.

[0063] FIG. 5A shows the disaccharide fraction and FIG. 5B shows the trisaccharide fraction.

[0064] The best results were obtained with β-galactosidase from Aspergillus sp., which showed a higher yield. This is believed to be the first report of fructosylated GOS as a prebiotic sweetener. The sweetness value is higher than that of other commercially available GOS, which has a sweetness value of 75 compared to sucrose (100).

[0065] Effect of OPTI-GOS on metabolic activity The effect of Opti-GOS (1% w / v) on the metabolic activity of the human gut microbiome was investigated in pH- and temperature-controlled batch cultures. The effect of opti-GOS on the concentration of organic acids was compared with short-chain fructooligosaccharides (FUJIFILM Wako Chemicals, Germany) and a carbohydrate negative control. Galactooligosaccharides produced by the activity of the same enzymes used to synthesize Opti-GOS were also tested.

[0066] Freshly defecated fecal samples were obtained from five healthy adults without gastrointestinal disorders who had not taken antibiotics for 6 months prior to the study and had not taken prebiotics and / or probiotics for 6 weeks prior to the study.

[0067] A sterile fermenter (20 mL working volume, Soham Scientific, Ely, UK) was filled with 2 g L of peptone water (Oxoid, Basingstoke, UK). -1 ; Yeast extract (Oxoid, Basingstoke, UK) 2gL -1 ;NaCl 0.1gL -1 ;K2HPO40.04gL -1 ;KH2PO4 0.04gL -1 ;MgSO4.7H2O 0.01gL -1 ;CaCl2.6H2O 0.01gL -1 ;NaHCO3 2gL -1 ;Hemin 0.05gL -1 ;Cysteine.HCl 0.5gL -1 ;Bile salts 0.5gL -1Anaerobic conditions were established and maintained by filling the fermentation vessels with pre-reduced (deoxygenated) sterile basal medium consisting of 10 μL of ethanol, 10 μL of vitamin K1, and 2 mL of Tween 80 (Sigma-Aldrich) and injecting oxygen-free N2. Agitation was performed using a magnetic stirrer. The carbohydrate to be tested (1% w / v) was added to the designated vessel and immediately inoculated with fecal slurry from a single donor (10% v / v prepared in anaerobic phosphate-buffered saline). All tests on a single donor were performed in parallel. The fermentation temperature was maintained at 37°C by a circulating water bath. An automated pH controller (Fermac 260; Electrolab, UK) maintained the broth pH within the range of 6.7–6.9 by adding 0.5 M NaOH and 0.5 M HCl as needed. Fermentations were run for 24 h, and samples were withdrawn at 0, 5, 10, and 24 h for organic acid analysis.

[0068] Organic acid (OA) concentrations were determined by gas chromatography with a flame ionization detector (GC-FID) using 2-ethylbutyric acid as an internal standard, based on the method described by Richardson et al. (1989). A gas chromatograph analyzer (Agilent / HP 6890) equipped with a flame ionization detector (FID) and an HP-5MS column (30 m x 0.25 mm) with a 0.25 μm coating (crosslinked (5%-phenyl)-methylpolysiloxane, Hewlett-Packard, UK) was used for SCFA measurements. Helium was used as the carrier gas at a flow rate of 1.7 mL / min (head pressure 133 KPa). The initial oven temperature was set to 63 °C, followed by a 15 °C / min temperature ramp to 190 °C, at which the oven was held for 3 min. A split ratio of 100:1 was used. The appearance of OA in the chromatogram was confirmed based on the retention time of each commercial OA standard (lactic acid, acetic acid, propionic acid and butyric acid) (Sigma-Aldrich, UK).

[0069] Opti-GOS was found to mediate significantly higher levels of acetic acid compared to the baseline from 5 hours of fermentation. Acetic acid concentrations were significantly higher than the negative control at every time point and were comparable to those of the prebiotic positive control and GOS (as shown in Table 4 below).

[0070] Propionic acid concentrations were significantly higher than the negative control at 10 and 24 h of fermentation and were similar to levels observed with GOS and the positive control.

[0071] Similarly, butyric acid increased from 5 h of fermentation onwards and was significantly higher compared to the negative control, showing a pattern similar to that of the prebiotic control and GOS.

[0072] Lactic acid significantly increased at 5 and 10 hours of fermentation, at levels significantly higher than those of the negative control, following a pattern similar to that of the prebiotic control and GOS. Lactic acid is a fermentation intermediate that is rapidly utilized through cross-uptake by other members of the gut microbiome. Lactic acid accumulates in the broth when the production rate is greater than the utilization rate, a characteristic of the rapid gut microbiome fermentation rate observed during the glycolysis of oligosaccharides. This indicates that opti-GOS is rapidly fermented, following the well-established behavior of other prebiotic oligosaccharides, such as FOS and GOS.

[0073] Overall, the metabolic activity of the gut microbiome during fermentation of opti-GOS was similar to that of the prebiotic positive control (FOS) and GOS in terms of both fermentation kinetics and specific organic acid production. Opti-GOS behaved similarly to commercially available prebiotics, and its effects on the metabolic activity of the human gut microbiome are characteristic of oligosaccharide glycolysis. All of them significantly increased not only acetate but also propionate and butyrate, organic acids that play important roles in cholesterol production, appetite regulation, tight junction integrity, and immune regulation.

[0074] [Table 4]

[0075] The above embodiments are not intended to limit the scope of protection provided by the claims, but rather to describe examples of how the present invention may be practiced.

Claims

1. 1. A method for enhancing the sweetness of non-digestible oligosaccharides, comprising modifying at least a portion of the oligosaccharides during synthesis to incorporate one or more fructose units, wherein the oligosaccharides are galactooligosaccharides (GOS), and the oligosaccharides are modified using β-galactosidase in the presence of fructose.

2. The method of claim 1, wherein the β-galactosidase is derived from the genus Aspergillus.

3. 3. The method of claim 1 or claim 2, wherein the percentage of oligosaccharides modified to incorporate one or more monosaccharide units is 95% or less.

4. 4. The method of claim 3, wherein the percentage of oligosaccharides modified to incorporate one or more monosaccharide units is 90% or less.

5. 5. The method of claim 4, wherein the percentage of oligosaccharides modified to incorporate one or more monosaccharide units is 80% or less.

6. 3. The method of claim 2, wherein the Aspergillus is selected from one or more of the following species: Aspergillus officinalis; Aspergillus aculeatus; Aspergillus awamori; Aspergillus carbonarius; Aspergillus cellulosae; Aspergillus oryzae; Aspergillus flavus; Aspergillus japonicus; Aspergillus nidulans; or Aspergillus niger.

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