GB1558538A - Oligosaccaride separation - Google Patents

Description

(54) OLIGOSACCHARIDE SEPARATION (71) We, THE BOARD OF TRUSTEES of the University of Alabama, a body corporate of the State of Alabama of Birmingham, Alabama, 35294, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - The present invention relates to a method for obtaining an oligosaccharide fraction. More particularly, it relates to a method for extract ing a suitable molecular weight range of oligo saccharides for use, for example, as a substrate for amylase assay.

Heretofore, many processes for preparing maltooligosaccharides have been used. Generally, these involve chromatographic separation by such techniques as partition column chromatography on cellulose, adsorption chromatography on charcoal and exclusion chromatography on polyacrylamide gels or dextran gels. (Whistler et al,JACS 77, (1955); Whelan et al, Biochem. J. 58, 569 (1954); Whistler et al, JACS 77, (19.55); French etal, JACS 71, 356 (1949); Trenel et al, J. Chrom. 42, 476 (1969); Whistler et al, JACKS 72, 677 (1950); Hugh eft al, J. Chem. Soc. 2511 (1949)). In all of these methods the presence of large amounts of glucose, maltose (G2) and maltotriose (G3) severly limits the amount of material that can be fractionated.

Other methods utilize a combination of a fermentation step followed by a chromatographic separation. For example, in U.S. Patent No. 3,788,910, a brewer's wort is fermented with a yeast to remove sugars of lower molecular weight than those desired. After the yeast is removed, the desired maltotriose/ maltotetraose fraction is collected by gel filtration chromatography.

Similarly, studies have shown that commercial baker's yeast is capable of com pletely removing glucose, maltose and maltotriose but not oligosaccharides of higher weight. Fractionation by column chromato graphy is required to remove the lower unwanted oligosaccharides such as G4 and G5.

However, this general method is not very amenable to commercial preparation of large quantities of oligosaccharide fractions of higher molecular weight, such as from about maltopentaose (G5) to about maltodecaose (Glo). Moreover, since fractionation by column chromatography is required, it is expensive and time consuming.

Because of this inability to prepare oligosaccharides in a commercially feasible and technically acceptable fashion, significant im provements in several analytical systems, which would be possible if such substrates were available, have been prevented. For example, a conventional system for amylase assay incor porates the following reactions:

1. Starch glycogen or F substrate amylase maltose + smaller maltodextrin J > oligosaccharides 2. Maltose maltase glucose 3. Glucose + ATP hexokinase glucose-6-phosphate + ADP 4. Glucose-6-phosphate + NAD glucose-6-phosphate dehydrogenase 6-phosphogluconolactone + NADH The rate of formation of NADH, once zeroorder kinetics is established for equation (4), is directly proportional to the amount of amylase present in the sample, NADH is monitored spectrophotometrically by its absorbance at 340 nm.

However, the use of polymeric oligo saccharides such as starch has the distinct dis advantage that a considerable lag is observed before the indicator reaction (Reaction 4) shows zeroorder kinetics. Another disadvantage is the relatively low sensitivity of the resultant amylase determination since the hydrolysis of large polymeric oligosaccharides produces many other smaller oligosaccharides rather than predominantly maltose, maltotriose or maltotetraose, which are readily hydrolyzed to glucose by the maltase in Reaction 2, as required. Thus, amylase can cleave several glycosidic bonds in these large polymeric oligosaccharides but on indication of amylase activity will be observed until a proper substrate for maltase activity is produced (Reaction 2). The use of substrates of smaller oligosaccharides which are not hydrolyzed by maltase such as maltopentaose, maltohexaose, maltoheptaose, maltooctaose, maltononaose or mixtures of these substrates would increase the sensitivity of the test as well as reduce the time required to reach zero-order kinetics of the indicator reaction (Reaction 4), thereby significantly improving the conventional assay.

Ideally, pure G5 would be the preffered substrate since only one G2 could be produced per glucosidic bond severance, but its cost of production would be commercially prohibitive. The use of the mixture described above, however, would give analytical values for amylase comparable to those obtained if pure G5 alone were used.

Accordingly, it is an object of this invention to provide a method for obtaining large quantities of G5 and higher oligosaccharides in relatively high purity.

It is another object of this invention to provide such a method which does not require a chromatographic step for fractional separation of oligosaccharides.

It is still another object of this invention to provide a method for obtaining oligosaccharides which are suitable as substrates in amylase assay systems.

It is further an object of this invention to provide a method for obtaining an oligo saccharide fraction containing substantially no maltooligosaccharides having a molecular weight of less than G5.

Briefly, these and other objects of this invention as will hereinafter be made clear have been attained by providing a method for obtaining an oligosaccharide fraction which comprises removing substantially all oligosaccharides below G5 from a sample containing a mixture of Gs and higher oligosaccharides with G4 and lower oligosaccharides by simultaneously treating the sample with a yeast and a maltase.

The present invention is based upon the discovery of a synergistic effect occurring when an oligosaccharide-containing solution is simultaneously treated with both a yeast and a maltase. When such a solution is fermented with the yeast alone, only G1-G3 oligosaccharides are removed. Similarly, when maltase is used alone, only G2 and G3 are significantly hydrolyzed. If the remaining oligosaccharides were used, for example, as an amylase assay substrate, G4 would typically be hydrolyzed by the maltase in the assay reagent thereby providing non-amylase-produced glucose for detection. However, when the yeast and the maltase are present simultaneously in the oligosaccharide-containing sample, maltotetraose (G4) is removed and the relative concentrations of Gg, G6 and G7 are increased such that the resulting oligosaccharide mixture forms an excellent substrate for an amylase assay. Most significantly, this result is achieved without the need for any fractional column chromatography for removing lower weight oligosaccharides. This is a completely unexpected result.

While the mechanism of the synergism is not fully understood, it is theorized that the maltase alone will not hydrolyze G4 present in the starting solution, such as corn syrup, because the presence of glucose in high concentrations in the syrup inhibits the maltase enzyme activity. Thus, an oligosaccharide substrate produced by the action of maltase alone and followed by removal or inactivitation of the maltase prior to treatment with yeast would yield a product containing large amounts of G4.

However, the simultaneous presence of a viable yeast and an active maltase results in the removal of glucose as it is formed which permits removal of G4 by maltase. Thus, the process can be carried out either by 1. fermenting the corn syrup with yeast alone followed by the addition of maltase; or 2. adding the maltase alone followed by the addition of yeast.

The critical and novel feature is the simultaneous presence of viable yeast and active maltase at one stage of the preparation. However, other techniques of removing glucose from the maltase-containing solutions can also be used to achieve G4 removal by the maltase.

The source of the oligosaccharide-containsample is not critical. Generally, natural extracts derived from enzymatic conversion or acid hydrolysis of starch-containing materials, such as grains or tubers such as potato starch, into the large sugars are suitable sources. These include corn syrup, brewer's wort, corn steep liquor, barley malt extract and the like. Corn syrup is preferred since it has a very high content (20-45 of oligosaccharides in the desired range from about G5 to about Glo.

Typically, corn syrup should have a degree of polymerization (D.P.) in the range of 8-40, preferably 10-15, since the oligosccharide contents are higher. An oligosaccharidecontaining solution suitable for use in this invention can be prepared by dissolving approximately 0.2 to 2.5 kg, preferably 1.9 2.1 kg, of corn syrup in 4.5 to 7.0 liters, preferably 5.5 to 6.0 liters, of H20.

The yeast used in this invention may be, in general, a species ofSaccharomyces, e.g.

S. cerevisiae, S. italicus or S. uvarum. Any strain of baker's yeast should be usable. A convenient source of such a yeast is commercial baker's yeast. The only requirement is that the yeast be able to ferment maltose and glucose and not be able to ferment Gg, G6, G7 etc. to any significant extend and, of course, that it be un contaminated with fungus. The amount of yeast required is an amount effective to ferment the maltose and glucose, preferably in 18 to 36 hours. Generally, from .0075 to 0.20 kg, preferably from 0.05 to .10 kg of yeast per 1.0 liter of oligosaccharide-containing solution should be added.

The maltases (a-glucosidase) suitable for use in the invention include those having a specificity such that maltotetraose (G4) is hydrolyzed to maltose or to maltotriose and glucose, and in addition that G5, G6, G7, G8 etc. and higher oligosaccharides are not significantly hydrolyzed. If the yeast strain used in the preparation is not able to ferment maltotriose, then the maltase must also be able to hydrolyze G3. Maltases obtained from Saccharomyces cerevisiae, and from Saccharomyces italicus, for example, are usable; while those from Saccharomyces uvarum or molds such as Aspergillus niger are not suitable. Particularly preferred is a specific maltase isolated from a strain of brewer's yeast (Hallstrain or Saccharomyces, ATCC 20,488). The maltase of this strain of Saccharomyces, suitable for use in this invention is that labelled "Peak II maltase".

Details of its properties and preparations may be found in our co-pending U.K. Patent Application No. 41256/77. The amount of maltase required is a catalytic amount, i.e., that sufficient for catalyzing the hydrolysis of oligosaccharides. Generally, from 300,000 to 2,000,000.00 preferably from 8no,000 to 1,200,000, units per liter of oligosaccharide solution and from .0075 to 0.20, preferably from 0.05 to 0.10 kg of yeast, should be added per liter. A unit of maltase activity is defined as the amount of enzyme giving rise to an absorbance change of 1.00/min in a 1.00 cm cell containing 1.0 ml of p-nitroplenyla-D-glucoside (0.20 mg/ml) in 0.10 M potassium phosphate buffer; 400 nm; 380 C, pH 6.80. The required purity is such that a- or amylase activity be absent from the maltase preparation.

The order of addition of the yeast and maltase is not at all critical. Either may be added first or both may be added together. In either of the first two cases, G2G3 only are hydrolyzed until the other component is added.

Thereafter, by synergistic action of the yeast and the maltase, G4 is hydrolyzed relatively rapidly and G5 only slowly. The time between addition of the components is also not critical.

The preferred order of addition is to add the yeast first and then to add the maltase later.

The preferred time of fermentation with yeast alone is 18-40 hours, preferably 34 to 38 hours.

There is no advantage in adding the maltase first, but it can be done if the total time of fermentation is increased by 36 to 48 hours.

Thus, a disadvantage of adding the maltase first and then the yeast hours later is that the total process is lengthened and the maltase enzyme is slowly inactivated, requiring a longer time of preparation to remove G4 or necessitating further addition of maltase after the yeast is added. However, with the preferred amounts of yeast and maltase given, the order of addition is not critical and suitable substrate can be obtained by either sequence. From the point at which the yeast and maltase are simultaneously present in the oligosaccharide-containing solution, generally from 36-72 hours are sufficient to remove malotetraose. Final contamination with glucose, maltase, maltotriose and maltotetraose is insignificant.

During the treatment with the yeast alone, the temperature - of the oligosaccharide-containing solution usually should be maintained at from 25 to 350 C, preferably from 29 to 320C, and the pH usually at from 6.0 to 7.5, preferably from 6.4 to 6.8. For maltase treatment alone the temperature usually should be from 25 to 320C, preferably from 28 to 310C, and the pH usually from 6.0 to 7.5, preferably from 6.4 to 6.8. During the simultaneous treatment with yeast and maltase the pH range usually is 6.0 to 7.2, preferably 6.4 to 6.8. The temperature range usually is 25 to 320C preferably 29-32"C.

The pH can be maintained by the addition of either KOH or NaOH pellets or solutions. In all cases, the yeast usually is slowly stirred to keep it in suspension throughout the treatment.

Vigorous stirring or aeration is to be avoided because denaturation of the maltase enzyme can result. From 0.2 to 0.4 g, preferably from 0.3 to 0.35 g, of sodium azide per liter of solution preferably should also be added to prevent bacterial growth. It is also preferred to add orthophosphoric acid in a form such as KH2PO4, which is preferred, K3PO4, NaH2 P04, Na21iPO4, (NH4)2HPO4, Na3PO4, or HPO4 to serve as a buffer to avoid rapid fluctuations in the pH and also to stimulate the yeast fermentation as is conventional. A suitable range of this additive is from 0.0 to 12 gm of KH2 PO4 per liter, preferably 6.0 to 7.0 gm per liter or equivalent amounts of the other forms.

After completion of the desired hydrolysis, generally after 72-120 hours of the simultaneous presence of the yeast'and the maltase, the fermentation mixture may be treated by any conventional method which is effective to denature the maltase and to remove the yeast. When maltase is not completely denatured, maltose and maltotriose accumulate in the solution of the oligosaccharide. A preferred method is as follows: 1. The pH of the mixture is adjusted to 2.0-4.0 with the addition of hydrochloric acid.

2. The mixture is heated rapidly to 70-750C (the total time to reach 70"C is not critical approximately 20 minutes is convenient), and cooled to from 0 to 250C.

3. A filter aid is added (e.g., the material available under the Trade Mark Celite) and the mixture filtered to remove the yeast and the denatured protein in conventional fashion.

The clear pale yellow filtrate which results may be lyophilized and used directly as a substrate in amylase assay. However, it is preferred to remove phosphates and other salts and to clarify the solution by passage of the filtrate through an adequate amount of conventional mixed-bed ion-exchange resin, such as Amberlite (Trade Mark) MB-3 or HN-High Capacity Hose Nipple Cartridge D8901 (Barnstead Company), and then to lyophilize the effluent to remove the enthanol produced during fermentation.

Yields are approximately 700-900 grams of substrate for a typical 2 kg sample of corn syrup. This represents an extracting yield of about 35455'o for G6 and higher weight oligosaccharides.

Of course, additional treatments are not excluded by the method of this invention, For example, the oligosaccharide-containing sample may be pretreated with a conventional ethanol solution for removal of unwanted larger oligosaccharides and dextrins. Alternatively, this ethanol treatment may be effected after removal of the yeast and the denatured maltase.

Other conventional pretreatments can also be performed such as dialysis to remove some of the lower molecular weight oligosaccharides.

Moreover, although a significant feature of this invention is the elimination of the need for a fractionating chromatography step, such an additional treatment is not precluded. The effects of such a chromatographic separation can be both beneficial and detrimental. In view of the time, complexity and, most importantly, the cost involved in utilization of such an extra step on an industrial scale, further separation on a column is strongly contraindicated. In any event, such a step is clearly not required, but when desired is fully compatible with the present process.

In the Examples, the materials and analytical techniques used were as follows.

Detection of Oligosaccharides by TLC: The procedures used were slight modifications of the published procedures by Mansfield, in Quantitative Thin Layer Chromatography, edited by Touchstone, J.C., John Wiley and Sons, New York, 1973, pp. 79-93. Plates were routinely scanned with a Schoeffel Spectrodensitometer. Relative concentrations of oligosaccharides were calculated from the peak height of the various oligosaccharide spots on the densitometer scan.

Yeast: Commercially available baker's yeast.

Corn Syrup: That used in all studies was a sample of #1033 corn syrup obtained from Corn Products International.

Maltase: The maltase was prepared from the Hall strain of yeast according to our copending U.K. Patent Application No. 41256/77.

Determination of Blank Rate and Sensitivity of Substrate in Amylase Assay: The measurement of the blank rate and sensitivity of the substrate in the amylase assay system was determined after the addition of suitable amounts of substrate dissolved in H2 O (final concentration of oligosaccharide was 3.0 mg/ml) to a lyophilized reagent reconstituted with H2 0. The final concentration of components in the assay cuvette (1.00 ml final volume; light path 1.00cm) were as follows: Potassium phosphate buffer, pH 6.8 0.10 M NaC1 0.05 M Na2ATP.3H20 2.0 mg/ml NAD 1.0 mg/ml Magnesium acetate 0.01 M Hexokinase 3.0 IU/ml Glucose-6-P dehydrogenase 4.0 IU/ml Maltase 191 Units ml Measurements were made at 340 nm, 38"C unless otherwise indicated, using a Gilford Recording Spectrophotometer (Model 2000).

The sensitivity of the substrate in the assay was determined by measuring the rate of absorbance change after the addition of 10 ul of Moni Trol (Trade Mark) IIX (Lot #xPT-540). The rate was determined after a 7 minute preincubation to ensure complete removal of the endogenous glucose in the Moni-Trol preparation.

Conductivity Measurements: The conductivity of solutions was determined with a Markson digital Conductivity Meter with automatic temperature compensation. (Model Electromark).

EXAMPLE I Demonstration of the Synergistic Action of a Combination of Maltase and Yeast in the Obtaining of Oligosaccharides Suitable for Use in an Amylase Assay.

It is well known from prior art that fermentation of oligosaccharide containing solutions with yeast removes G1, G2, and G3 from the solution, and that larger oligosaccharides are fermented poorly if at all. These observations were confirmed using baker's yeast in these investigations. However, as shown in Table I, the simultaneous presence of baker's yeast and maltase in the oligosaccharide-containing solution gave the completely unexpected result that in addition to fermentation of G1, G2 and G3, G4 is removed by fermentation and is decreased to an acceptable level for amylase assays. Also there was a relative increase in the content of Gg, G6 and G7 which is also desirable.

It is also clearly evident from Table I that maltase alone will not significantly hydrolyze G4 in the absence of yeast.

Table I Treatment of corn syrup solutions with either maltase alone, yeast alone, or a combination of maltase and yeast.

Additional treatment of corn Relative peak heights of oligo syrup solution* saccharides after TLC separation** G1 G2 G3 G4 G5 G6 G7 G8-Gn Yeast alone *** 0 0 0 30 20 15 8 100 Maltase alone *** 112 0 0 48 60 48 39 100 Yeast and maltase 0 0 0 2 42 37 29 100 in combination * Flasks containing 54 gm corn syrup, 150 ml of H20, 100 mg sodium azide, and 2 gm KH2PO4 were adjusted to pH 7.2 with KOH. Where indicated, 20 gm of baker's yeast or 153,000 units of maltase were added. The pH was maintained between 6.0 and 7.2 throughout the incubation at 300C by the addition of KOH.

** The contents of the flask were examined for oligosaccharides after six days at 300 C. Relative peak heights before treatment of the corn syrup were 63, 52,49,31,22,9.2,4.6, and 100 for G1, G2 .. . G8 n, respectively.

*** Comparative tests not in accordance with the present invention.

EXAMPLE II Large Scale Preparation of Amylase Substrate 2.0 kg of corn syrup was dissolved in a total of 6 liters of H2 0. After the addition of 40 gm of KH2PO4, the pH was adjusted to 7.2 with KOH, and 2 gm of sodium azide were added.

After bringing the temperature to 300 C, 454 gm of baker's yeast was added. Fermentation was allowed to proceed for 36 hours at which time complete removal of G1, G2, and G3 was observed. The pH of the mixture was readjusted to pH 7.0 with KOH and 2 x 106 units of Peak I maltase and 4 x 106 units of Peak II maltase activity were added. (Peak I and Peak II maltase are fully defined in our copending U.K. Patent Application No.

41256/77. It was subsequently determined that Peak I maltase activity was not effective in producing the synergistic effect described.

However, the presence of Peak I maltase activity has no deleterious effect in the preparation). Fermentation was allowed to proceed for an additional 120 hours during which the pH was adjusted daily to 7.0 with KOH. The yeast was occasionally resuspended by gentle mixing.

Approximately 120 hours after the addition of maltase, the fermentation was terminated by the addition of HC1 to the mixture to lower the pH to 4.0. The mixture was heated to 700C during a 20 min. interval and chilled to room temperature.

The mixture was filtered after the addition of 15 gm/liter of Celite. Filtration yielded a clear, pale yellow solution. The solution was desalted by passage through Barnstead Company mixed bed deionizer cartridges (Catalog number D 8901). The eluate from the ion exchanger had a pH of 6.0, a conductivity of 1.65 micromhos, and was colorless and nearly odorless.

The preparation was lyophilized, yielding 785 grams of material.

EXAMPLE III Demonstration of the Suitability of the Oligosaceharde Sample Prepared in Example II for use as an Amylase Substrate The suitability of the oligosaccharide prepared in Example II for use as an amylase substrate was demonstrated by measurement of: 1. The blank rate in the absence of added amylase (Table II).

2. The response (sensitivity) of the assay system upon addition of a control serum sample containing amylase activity (Table II).

3. The correlation of the response of an amylase assay system employing the oligosaccharide fraction with the response of a standard method for the determination of amylase (Table III).

Table II Evaluation of the suitability of the oligosaccharide fraction prepared in Example II as a substrate for the assay of amylase with respect to blank rate and sensitivity.

Blank Sensitivity Rate Average 2.78 689 Number of determinations 12 12 Standard Deviation 0.24 1.84 Coefficient of Variations (%) * The blank rate and sensitivity are expressed as the change in milliabsorbance per minute.

** The Sensitivity of detection of amylase activity was measured upon the addition of 10 ul of control serum (Moni-Trol IIX).

From the initial absorbance change found upon the addition ef substrate to the test reagent, G1--Gq contamination of the substrate was extremely low. From the data presented in Table II it is concluded that the substrate prepared by the procedure of Example II produces acceptable blank rates and acceptable sensitivities Table III Correlation of the response of an amylase assay system employing the oligosaccharide fraction prepared in Example II with the response of the Caraway iodomestric procedure for the determination of amylase activity*.

Number of paired determinations 52 Correlation Coefficient 0.998 Correlation equation ** Y = 0.292 X + 30.7 * The activity of amylase was determined by the standard method of Caraway (McNair, RD. in Standard Methods of Clinical Chemistry, edited by MacDonald, R.P.

Academic Press, New York, 1970, pp.

183-188).

** Y = Caraway amylase units; X = IU/liter of amylase determined kinetically by the described analytical method using the oligosaccharide fraction.

WHAT WE CLAIM IS: 1. A method for obtaining an oligosaccharide fraction having substantially no G4 or lower oligosaccharide content which comprises adding to a source solution containing at least G4 and higher oligosaccharides both a yeast and a maltase such that both are present simultaneously and subsequently separating said yeast and maltase from said solution.

2. A method as claimed in Claim 1, wherein said source solution is first treated with said yeast or said maltase alone.

3. A method as claimed in Claim 1 or Claim 2, wherein said yeast is baker's yeast or brewer's yeast.

4. A method as claimed in any one of the preceding Claims wherein said yeast is a species of Saccharomyces cerevisiae, Saccharomyces italicus or Saccharomyces uvarum.

5. A method as claimed in any one of the preceding Claims, wherein said maltase hydrolyzes malto-tetraose but not higher malto-oligosaccharides.

6. A method as claimed in any one of the preceeding Claims, wherein said maltase is derived from Saccharomyces cerevisiae or from Saccharomyces italicus.

7. A method as claimed in any one of the preceding Claims, wherein the maltase is the Peak II maltase enzyme (as hereinbefore defined) derived from a strain of Saccharomyces ATCC 20488.

8. A method as claimed in any one of the preceding Claims, wherein said source solution is a natural extract derived from the enzymatic or acid hydrolysis of starch-containing materials.

9. A method as claimed in any one of the preceding Claims, wherein said source solution is corn syrup.

10. A method as claimed in any one of the preceding Claims, wherein from 0.0075 to 0.20 kg of yeast and from 3 x 105 to 2 x 106 units of maltase activity per liter of said source solution is used.

11. A method as claimed in any one of the preceding Claims, wherein the temperature is maintained at from 20 to 350C and the pH at from 6.0 to 7.5, and wherein said source solution is simultaneously treated with said yeast and said maltase for from 36 to 164 hours 12. A method as claimed in any one of the preceding Claims, wherein after said treatment said maltase is denatured and said maltase and yeast are removed from said source solution.

13. A method as claimed in Claim 12 wherein said maltase and yeast are removed by adjusting the pH of the solution to from 2.0 to 4.0 rapidly heating it to from 70 to 750 C, cooling to from 0 to 250 and thereafter filtering the solution to remove said yeast and denatured maltase.

14. A method as claimed in Claim 12 or Claim 13, wherein after said removal o

Claims (25)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   Table II it is concluded that the substrate prepared by the procedure of Example II produces acceptable blank rates and acceptable sensitivities Table III Correlation of the response of an amylase assay system employing the oligosaccharide fraction prepared in Example II with the response of the Caraway iodomestric procedure for the determination of amylase activity*.

   Number of paired determinations 52 Correlation Coefficient 0.998 Correlation equation ** Y = 0.292 X + 30.7 * The activity of amylase was determined by the standard method of Caraway (McNair, RD. in Standard Methods of Clinical Chemistry, edited by MacDonald, R.P.

   Academic Press, New York, 1970, pp.

   183-188).

   ** Y = Caraway amylase units; X = IU/liter of amylase determined kinetically by the described analytical method using the oligosaccharide fraction.

   WHAT WE CLAIM IS: 1. A method for obtaining an oligosaccharide fraction having substantially no G4 or lower oligosaccharide content which comprises adding to a source solution containing at least G4 and higher oligosaccharides both a yeast and a maltase such that both are present simultaneously and subsequently separating said yeast and maltase from said solution.
2. 2. A method as claimed in Claim 1, wherein said source solution is first treated with said yeast or said maltase alone.
3. 3. A method as claimed in Claim 1 or Claim 2, wherein said yeast is baker's yeast or brewer's yeast.
4. 4. A method as claimed in any one of the preceding Claims wherein said yeast is a species of Saccharomyces cerevisiae, Saccharomyces italicus or Saccharomyces uvarum.
5. 5. A method as claimed in any one of the preceding Claims, wherein said maltase hydrolyzes malto-tetraose but not higher malto-oligosaccharides.
6. 6. A method as claimed in any one of the preceeding Claims, wherein said maltase is derived from Saccharomyces cerevisiae or from Saccharomyces italicus.
7. 7. A method as claimed in any one of the preceding Claims, wherein the maltase is the Peak II maltase enzyme (as hereinbefore defined) derived from a strain of Saccharomyces ATCC 20488.
8. 8. A method as claimed in any one of the preceding Claims, wherein said source solution is a natural extract derived from the enzymatic or acid hydrolysis of starch-containing materials.
9. 9. A method as claimed in any one of the preceding Claims, wherein said source solution is corn syrup.
10. 10. A method as claimed in any one of the preceding Claims, wherein from 0.0075 to 0.20 kg of yeast and from 3 x 105 to 2 x 106 units of maltase activity per liter of said source solution is used.
11. 11. A method as claimed in any one of the preceding Claims, wherein the temperature is maintained at from 20 to 350C and the pH at from 6.0 to 7.5, and wherein said source solution is simultaneously treated with said yeast and said maltase for from 36 to 164 hours
12. 12. A method as claimed in any one of the preceding Claims, wherein after said treatment said maltase is denatured and said maltase and yeast are removed from said source solution.
13. 13. A method as claimed in Claim 12 wherein said maltase and yeast are removed by adjusting the pH of the solution to from 2.0 to 4.0 rapidly heating it to from 70 to 750 C, cooling to from 0 to 250 and thereafter filtering the solution to remove said yeast and denatured maltase.
14. 14. A method as claimed in Claim 12 or Claim 13, wherein after said removal of yeast and maltase, said solution is further purified by treatment with a mixed-bed ionexchange resin and lyophilization.
15. 15. A method for obtaining a G5 and higher oligosaccharide fraction, which comprises: a. fermenting a solution containing from 0.05 to 0.40 kg of corn syrup per liter of H2O, from 0.20 to 0.40 gm of sodium azide per liter of solution, and from 0 to about 15 gm of KH2 PO4 per liter of solution, said solution being continually maintained at a pH of from 6.0 to 7.5 by the addition of NaOH or KOH and at a temperature of from 20 to 350C, with from 0.0075 to 0.20 kg of baker's yeast per liter of solution for a period of from 18 to 36 hours, and adding from 3 x 105 to 2 x 106 units of maltase per liter of said solution for a period of from 36 to 164 hours; b. adjusting the pH of the solution to a value from 2.0 to 4.0, rapidly heating it to a temperature of from 70 to 750C, and cooling it to from 0 to 250C; c. filtering said solution to remove denatured maltase and yeast; and d. passing said solution through a mixed-bed ion-exchange resin, and lyophilizing the effluent.
16. 16. An oligosaccharide fraction whenever obtained by a method as claimed in any one of the preceding Claims.
17. 17. An amylase assay method, in which an oligosaccharide fraction as claimed in Claim 16 is used as a substrate for the amylase and the amount of amylase degredation products produced from the substrate is determined.
18. 18. An amylase assay substrate produced by the process of Claim 14.
19. 19. A method for removing oligasaccharides in the range of G1 -G4 from a solution containing the same and higher oligosaccharides which comprises simultaneously adding to said solution both baker's yeast and the Peak II maltase enzyme derived from a strain ofSaccharomyces ATCC 20,488 and subsequently separating said

    yeast and maltase from the solution.
20. 20. A method of hydrolysing a G4 oligosaccharide which comprises contacting a solution thereof with a viable yeast in the presence of an active maltase.
21. 21. A method as claimed in Claim 1 and substantially as hereinbefore described.
22. 22. A method as claimed in Claim 15 and substantially as hereinbefore described.
23. 23. A method as claimed in Claim 17 and substantially as hereinbefore described.
24. 24. A method as claimed in Claim 19 and substantially as hereinbefore described.
25. 25. A method as claimed in Claim 20 and substantially as hereinbefore described.