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Title:
MANUFACTURE OF POLYMERIC SUGARS
Document Type and Number:
WIPO Patent Application WO/2017/064143
Kind Code:
A1
Abstract:
A method of manufacturing prebiotic oligossacharrides is disclosed which comprises the steps of: coagulating milk to create whey; separating the fat from the whey; removing minerals from the whey; retaining within the whey a substantial proportion of the protein originally present subsequent to the removal of minerals; and creating an enzyme reaction within the whey to convert lactose in the whey to prebiotic oligosaccharides.

Inventors:
HUNT, Simon (Claygate House Littleworth Road, Esher Surrey KT10 9PN, KT10 9PN, GB)
Application Number:
EP2016/074512
Publication Date:
April 20, 2017
Filing Date:
October 12, 2016
Export Citation:
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Assignee:
PROMOVITA INGREDIENTS LIMITED (Claygate House Littleworth Road, Esher Surrey KT10 9PN, KT10 9PN, GB)
International Classes:
C12P19/04; A23C21/02; C07H1/08; C07H3/06; C12P19/14
Domestic Patent References:
WO2004052900A12004-06-24
WO2000072692A12000-12-07
WO2006087391A12006-08-24
WO2015034356A12015-03-12
WO2002050089A12002-06-27
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Claims:
CLAIMS

1 . A method of manufacturing prebiotic oligossacharrides comprising the steps of:

coagulating milk to create whey;

separating the fat from the whey;

removing minerals from the whey;

retaining within the whey a substantial proportion of the protein originally present subsequent to the removal of minerals; and

creating an enzyme reaction within the whey to convert lactose in the whey to prebiotic oligosaccharides.

2. A method according to claim 1 comprising, prior to the creation of the enzyme reaction, the step of evaporating demineralised whey to a concentration of between 30 and 70% solids. 3. A method according to claim 2 wherein the step of evaporating the whey comprises the steps of reducing the pressure in an evaporation vessel and heating the whey.

4. A method according to claim 2 or claim 3 comprising the step, after evaporating and prior to creating the enzyme reaction, of cooling evaporated whey to a temperature of between 40 and 60°C.

5. A method according to any one of the preceding claims further comprising the step of stirring evaporated, cooled whey during the enzyme reaction. 6. A method according to any one of the preceding claims further comprising the steps, after creating an enzyme reaction, of concentrating and drying the product of the enzyme reaction.

7 A method according to claim 6 wherein the step of concentrating comprises the step of heating.

8. A method according to claim 6 or claim 7 wherein the step of drying comprises the step of spray drying. 9. A method according to any one of the preceding claims wherein the at least 6% w/w tein present subsequent to the removal of minerals.

10. A method according to any one of the preceding claims wherein the reaction temperature for the enzyme reaction conditions is 60°C or lower.

1 1 . A method according to any one of the preceding claims wherein the reaction temperature for the enzyme reaction conditions is 55°C.

12. A method according to any one of the preceding claims wherein the pH for the enzyme reaction conditions is less than 7. 13. A method according to any one of the preceding claims wherein the pH for the enzyme reaction conditions is 6.5 or lower.

14. A method according to any one of the preceding claims wherein the proportion of protein retained within the whey after demineralisation is at least 50% of the original protein.

15. A method according to any one of the preceding claims wherein the prebiotic oligosaccharides comprise galactooligosaccharide.

Description:
MANUFACTURE OF POLYMERIC SUGARS

BACKGROUND TO THE INVENTION

1 . FIELD OF THE INVENTION

The present invention relates to the manufacture of the class of sugars known as oligosaccharides. Examples of oligosaccharides are mano, fructo and galacto-oligosaccharide.

Prebiotics are non-digestible food ingredients that beneficially affect a host organism by stimulating the growth or activity of beneficial bacteria in the colon. Oligosaccharides, such as galacto-oligosaccharide are known to have prebiotic qualities. Because of the configuration of its glycosidic bonds, galacto-oligosaccharides (for example) largely resist hydrolysis by salivary and intestinal digestive enzymes. Consequently, they reach the colon or fermentative pat of the gastrointestinal tract in other monogastric animals virtually intact. There, they assist the promotion of health-promoting bacteria such as Bifidobacteria and Lactobacilli.

Generally speaking, oligosaccharides are derived from the enzyme catalysed transglycosylation reaction of lactose. The enzyme catalyses many sequential reactions in which for example, a galactose (gal) is transferred from lactose to the non-reducing end of an oligosaccharide (e.g. lactose) with the release of glucose. The galacto-oligosaccharides have the general form - (galactosyl)n-lactose and typically range in size from trisaccharides to octasaccharides. Structural complexity is introduced by the different intermolecular bonds GOS products contain a mixture of galacto-oligosaccharides, lactose, glucose and galactose.

2. DESCRIPTION OF RELATED ART

Thus, it is known to hydrolyse lactose into its monosaccharide components, glucose and galactose and, where that conversion is undertaken enzymatically, then to cause a substantial part of the galactose fraction of the conversion to undergo consecutive transgalactosylation reactions to create multiple monomeric galactose units, thereby to create oligosaccharides such as galactooligosaccharides (GOS). Refined lactose is used as a starting material for this kind of reaction, since it provides assured levels of purity. This can be of importance where the oligosaccharide produced is to be used as an ingredient in products such as infant formula, for example. One source of refined lactose by its extraction from whey, a by-product in the manufacture of cheese. That extraction process is not trivial. It involves at least one cycle of evaporation, crystallisation, washing, physical separation (e.g. by centrifuge), drying and milling. WO2006/087391 discloses an oligosaccharide mixture derived from animal milk, food products comprising said oligosaccharide mixture and a process for producing said oligosaccharide mixture. W)2004/052900 discloses a process for production of a carbohydrate composition from lactose. WO2015/034356 relates to the enzymatic preparation of galacto-oligosaccharides. WO2015/023281 discloses a method contacting yoghurt whey with an enzyme to form soluble fibre. WO02/50089 relates to improved methods of purification of sugars.

SUMMARY OF THE INVENTION

One aspect of the present invention provides an alternative manner of manufacture of prebiotic oligosaccharides. Prebiotic is used, herein, in relation to a substance having prebiotic characteristics for a monogastric animals. Embodiments of the present invention avoid the necessity to create pure or substantially pure lactose but instead uses de-mineralised whey as a starting material. A further aspect of the present invention lies in an appreciation of the fact that it is not necessary to remove substantial quantities of protein from a mixture which includes lactose in order to avoid browning when manufacturing oligosaccharides.

Yet a further aspect of the present invention lies in an appreciation of the fact that, in spite of a lower concentration of lactose within a mixture which incorporates, relatively speaking, substantial quantities of protein and therefore a correspondinigly lower concentration of lactose from which to create prebiotic oligosaccharides, the yield for the manufacture of prebiotic oligosaccharides using such a mixture is substantially the same. Yet a further aspect of the present invention provides for a product created by the manufacturing process of prebiotic oligosaccharides from de-mineralised whey including substantial quantities of protein. Such a products is advantageous for a number of reasons, since it readily contains substantial quantities of protein which is of significant nutritional benefit.

BRIEF DESCRIPTION OF DRAWINGS

Embodiments of the present invention will now be described, by way of example, and with reference to the accompanying drawings, in which:

Fig. 1 is a flow chart illustrating the process of creation of whey;

Fig. 2 is a flow chart illustrating an embodiment of the manufacture of galactooligosaccharide according to the present invention; and Figs. 3A to 3B are graphs illustrating the results of an experiment comparing reactions and yields of lactose containing substantial quantities of protein with relatively pure lactose.

DESCRIPTION OF PREFERRED EMBODIMENTS

Preferred embodiments of the invention include a method of treating whey which retains a substantial proportion of the protein originally present in the whey, to produce prebiotic oligosaccharide comprising the steps of: demineralising the whey by 70-95%; concentrating demineralised whey to a concentration of between 30 and 70 % solids by mass; and creating an enzyme reaction within the concentrated whey to convert lactose within the whey to oligosaccharide.

In a preferred embodiment the step of concentrating the whey comprises the steps of heating the whey under reduced pressure and subsequently cooling the whey to a temperature of between 40 and 60°C. In yet a further preferred embodiment the method further comprises the steps, subsequent to creating the enzyme reaction, of concentrating and drying the oligosaccharide. In one embodiment of such methods, concentrating and drying the oligosaccharide comprises the steps of heating and spray drying. Preferably, the method also comprises the step, prior to the step of creating an enzyme reaction, of removing starter bacteria from the whey. Preferably, the starter bacteria are removed from the whey by heating.

In various preferred embodiments of the present invention, including but not limited to those disclosed immediately above, the enzyme reaction from which the prebiotic oligosaccharides are created is performed by heating the mixture to a temperature of less than 60°C, more preferably a temperature of less than 57°C and more preferably a temperature of 55°C

Further preferred embodiments will now be disclosed below. It should be borne in mind that, in connection with the description of embodiments within the present description and specification as a whole, that modifications or variations to processes or reaction conditions are not limited in their application to the emobidment in connection with which they were first disclosed. To avoid needless repetition, therefore, unless expressly stated otherwise, each variation is capable of application to all other embodiments.

Lactating animals produce oligosaccharides in their milk. Human milk oligosaccharides are a very diverse group of oligosaccharides. More than 100 have been identified, which are based around lactose. The most common are: 2'-fucosyllactose, lacto-N-fucopentaose, lacto-N- (Neo)tetraose, 3'-fucosyllactose, 3'- and 6'-sialyllactose, lactodifucotetraose and sialyllacto-N- tetraose. The present invention relates to the creation of oligosaccharides having prebiotic characteristics in monogastric animals. More preferably the present invention relates to the creation of that group of oligosaccharides that are galactooligosaccharides (GOS).

Referring to Fig. 1 , the creation of a suitable form of whey from milk as a starting material is illustrated in very general terms. Milk which has been coagulated into curds and whey then undergoes a separation process 10, with the whey being drained off. That whey is then heat treated at step 12 to remove the starter bacteria organisms and demineralised at step 14. The demineralisation process, according to one embodiment involves removal of inorganic salts, together with some reduction in the content of organic ions, such as lactates and citrates.

The partial demineralization is mainly based on utilisation of cross-flow membranes in the nanometre (10-9 m) range. This type of filtration is called nanofiltration (NF), after this process further higher degree of desalination is conducted on electrodialysis and/or ion exchange. Typically the process will reduce the mineral content by 70-95%.. The reduction of the mineral content in the whey to this level is of significance. Oligosaccharides, such as GOS are used in infant formula, whose mineral composition is very accurately controlled. Levels of minerals within infant formula are required to be significantly below the levels which are present in untreated whey. This is one reason why refined lactose is used as a starting material for the production of oligosaccharides; because it has a very low mineral content.

Further information on the known aspects of the manufacture of whey and whey products can be found in "Lecturer's Handbook on whey and whey products, First Edition, by DR J.N. de Wit, published by European Whey Products Association and the contents of which are hereby incorporated herein by reference.

Notably, although demineralised, the whey which is then used for the subsequent process of the embodiments of the present invention is not de-proteinised (though some very small level of protein extraction may occur unintentionally as a consequence of the filtration process described above which removes the minerals). Thus, the whey used for the present embodiment contains substantially a full complement of the original protein present in the milk. Further preferred embodiments of the present invention use whey having a substantial proportion (as measured by reference to mass of protein as a proportion of the overall mass of the product) of the protein originally present in the milk, for example 50% or more of the original protein present; 60% or more of the original protein present; 70% of the original protein present; 80% of the original protein present; and 90% of the original protein present, respectively.

Referring now additionally to Fig. 2, demineralised whey containing full protein is first concentrated to between 30 to 70 % solids, measured by mass and preferably to between 45 - 65% solids. This process involves reducing the pressure within the evaporation vessel at step 20 to 160 - 320 hP. This reduces the boiling temperature which, therefore, ensures that the evaporation process does not burn any of the constituents of the demineralised whey. Heat is then applied at step 22 at 55 - 70 °C to create evaporation at step 24. These operations typically take place within a falling film evaporator, though this is not essential and other apparatus may be used.

Once the evaporated whey has been concentrated to the desired level, it is then cooled to within a temperature range of 45° - 65°C and, in a preferred embodiment, to a temperature of 58°C and stirred to remain homogeneous at step 26 and transferred to a reaction vessel at step 28. Suitable enzyme is then added at step 30 and the reactants are stirred. The preferred enzyme is beta-D-galactosidase galactohydrolase, the IUBMB number of which is (EC 3.2.1 .23). It is produced, for example, by selected microbial strains of Aspergillus oryzae, Bacillus circulans, Bifidobacterium sp., Escherichia coli, Kluyveromyces sp., Sporolomyces singularis. One preferred enzyme system involves the use of beta-galactosidase from Aspergillus oryzae. Another involves the sequential use of beta-galactosidase from Aspergillus oryzae and beta- galactosidase from Kluyveromyces lactis.

Further, in connection with preferred enzymes, it is noted that, based on sequence homology, beta-galactosidases have been classified as members of GH 1 , GH 2, GH 35 and GH 42 of the GH-A superfamily of glycoside hydrolase. Beta-Galactosidases like all glycoside hydrolases of the GH-A superfamily have an (alpha/beta) 8-barrel as catalytic domain. Two glutamic acid residues act as an acid/base catalyst and a nucleophile inside the barrel. · Aspergillus oryzae beta-galactosidase: belongs to GH 35 family;

Kluyveromyces lactis beta-galactosidase: whilst the beta-galactosidases from most eukaryotic organisms are grouped into GH 35, the enzymes from Kluyveromyces lactis and Kluyveromyces marxianus (sharing 99% identity) belong to GH 2 (along with the prokaryotic beta-galactosidases from Escherichia coli and Arthrobacter sp.

In broad and simple terms, the enzymatic reaction can be thought of as a first stage, were the lactose is initially hydrolised into glucose and galactose monomers; and then a second stage where those monomers are subsequently recombined to create oligosaccharide chains. A more accurate description is, necessarily, more complicated.

The preferred enzyme is known to catalyse the hydrolysis of terminal non-reducing beta-D- galactose residues in beta-D-galactosides (e.g. lactose, giving glucose and galactose). The enzyme catalysed formation of galacto-oligosaccharides (say) is a special case in which the acceptor for the galactose, bound to the enzyme during the hydrolysis of lactose, is lactose (or another carbohydrate) and not water. Therefore the beta-galactosidase enzyme is better described as a glycosyl (galactosyl) transferase that catalyses the hydrolysis of a beta-D- galactoside transferring the terminal non-reducing beta-D-galactose to a suitable acceptor: at low lactose concentrations water is a suitable acceptor and lactose is hydrolysed to glucose and galactose;

at high lactose concentrations lactose (or typically other galacto-oligosaccharides, or other carbohydrates) is a suitable acceptor and a galactosyl-lactose molecule is formed.

As noted above and implied from the enzyme-catalysed reactions, lactose and glucose are other reactants and are present in the GOS product. Thus, in addition there are the following reactions taking place:

the hydrolysis of lactose to galactose and glucose;

the hydrolysis of (galactosyl)n-lactose to (galactosyl)n and glucose (e.g. digalactans and glucose);

the conversion lactose to allo-lactose;

the possible conversion of lactose to lactulose and the hydrolysis of the latter to galactose and fructose.

It is notable that, all of the above other than lactose, glucose, fructose and galactose are within the purview of the present invention, since they are prebiotic oligosaccharides. A further and entirely independent invention disclosed herein is the use of one or more of the enzymes disclosed above in the use of an enzymatic reaction to create prebiotic oligosaccharides from lactose (including any mixture containing lactose). Such a reaction can be used in conjunction with other reaction conditions set out herein, or separately since the use of the enzymes per se for such a category of reactions is considered independently inventive.

The reaction continues, typically between 2 and 24 hours. Preferably the reaction continues until in excess of 80% of the lactose has been converted; more preferably more than 85% and more preferably still more than 90% conversion to oligosaccharides and other reactants such as glucose and galactose (where galacto-oligosaccharides are defined as all reactant carbohydrates except lactose, glucose and galactose). One limiting factor on the capacity of the enzyme to create the requisite reaction is the extent of the minerals within the whey. This, therefore, is a further reason for and benefit of the demineralisation process which is performed to create demineralised whey as a starting material.

Once the requisite level of reaction has taken place, the hydrolysed whey is then dried to a powder. In the present embodiment, this involves first a further evaporation step 32 including heating, followed by a step 34 of spray drying.

Hydrolysing de-mineralised whey which retains a substantial level of protein and preferably substantially all of the original quantity of protein present in the milk has a number of advantages and benefits. First, it avoids the expenditure of energy and expense involved in the creation of refined lactose as a starting material. Second, it produces a hydrolysed oligosaccharide product which additionally includes a substantial degree of valuable protein. This is of significant potential benefit in connection with the use, for example, of oligosaccharide as an additive to animal feeds. This is because a significant barrier to the adoption of oligosaccharide in such feeds arises in connection with the cost of reformulating very precisely established proportions of food groups such as fats, protein and carbohydrate. By producing a product already inherently containing significant amounts of protein in addition to the oligosaccharide, it is anticipated that reformulation costs of using such a product will be reduced. Thirdly, pure oligosaccharide product from this reaction has proved difficult to dry. This is because it is highly hygroscopic. It therefore exhibits a tendency to absorb moisture, creating lumps and making it difficult to process. Problems arising from this characteristic have, historically be ameliorated by the addition of carriers whose function is to reduce the hygroscopicity. Hydrolising oligosaccharide from lactose within demineralised whey results in a product which inherently contains additional material which will serve a comparable function to that of the previously-used carriers; but in this instance the step of adding carriers is not necessary and the additional material has nutritional benefit.

It is notable that the prior art indicates explicitly that a de-proteinised source of lactose is required in order to avoid browning and prevent a maillard reaction. The present inventors have found that provided the temperature is kept within the limits set out and disclosed herein, and preferably in the region of 55°C browning is avoided or minimised to an insignificant degree. Further, the pH of the reaction conditions are preferably below a pH of 7 and more preferably at or below a pH of 6.5, these conditions avoiding or minimising the occurrence of any Maillard reaction.

Typically, by way of illustration by reference to industrial scale processes milk will contain the following elements in the following proportions: %

Milk

Total solids 12.6

Fat 4.1

Casein 2.6

Whey protein 0.7

Lactose 4.6

Lactic acid 0.12

Minerals 0.5

(the reference to Thus, by reference to the mass of total solids, milk will contain (0.7/12.6) x 100 = 5.56% by mass of whey protein. Unseparated whey (i.e. whey which has not had whey cream removed for use in making butter contains the following elements in the following proportions

Where whey cream is removed, separated whey contains the following elements in the following proportions:

Thus separated whey contains, measured by proportion of mass of total solids, around 10% of whey protein. The NPN component, this being non-protein nitrogen contains, inter alia, urea, amino acids which come from hydrolysed peptides and the casein-macropeptide (from casein and the action of chymosin). These elements of demineralised whey are relevant because they are peptide-derived and have the same nutritional benefits as protein. Accordingly they are often included in the proportion of protein present. In the definitions and claims of the present application, NPN is not included within the definition of protein unless explicitly stated otherwise.

Subsequent to demineralisation, the following components are present in the following proportions:

The proportion of whey protein (again measured by mass) of the total solids therefore drops a little as a consequence of the demineralisation process to just under 10%. Embodiments of the present invention obtain the benefit from the inclusion of protein present in demineralised whey generally. The following proportions of such protein within the demineralised whey can be used:

≥ 50% of the original protein present - therefore approximately 0.3% or greater of the total solids

≥ 60% of the original protein present therefore approximately 0.37% or greater of the total solids

≥ 70% of the original protein present therefore approximately 0.43% or greater of the total solids

≥ 80% of the original protein present therefore approximately 0.49% or greater of the total solids

≥ 90% of the original protein present therefore approximately 0..55% or greater of the total solids An experiment was carried out to compare the yields of GOS created from de-mineralised whey powder within which a significant degree of protein was still present and a de-mineralised source of lactose from having comparatively little protein. Galacto-oligosaccharides are produced from lactose in an enzyme catalysed food-grade process. The enzyme used is β-galactosidase (EC 3.2.1 .23) also known as lactase and β-D- galactoside galactohydrolase.

Two sources of lactose used in the process were bovine milk-derived lactose. The first such source was food grade or refined lactose monohydrate powder. This is a pure source of lactose that contain little protein and ash (minerals).

The second source was de-mineralised whey powder, an example of which is demineralised whey powder 90% ("D90") which typically contains:

· ≥ 12.0 %(w/w) protein (6.38 x N);

≥ 81 .0 %(w/w) lactose;

< 1 .0 %(w/w) ash;

< 5.0 %(w/w) moisture. Experiment with D90 demineralised whey powder

Fonterra™ Demineralised Whey Powder 90%

Batch number: AQ6174

Lactose concentration 85.3 %(w/w) as lactose monohydrate

· Protein concentration 12.0 %(w/w)

Ash concentration 0.89 %(w/w)

Moisture concentration 1 .07 %(w/w)

Fat concentration 0.74 %(w/w) Experimental conditions

D90 concentration 55.0% (w/w solution)

Solvent water

Lactose concentration 44.6% (w/w solution)

Enzyme dose rate 16.9 (g/kg lactose)

· Reaction temperature 55 (°C)

Reaction pH 6.05 Results

Data are presented for a reaction time over 120 minutes. In this time the reaction for the production of galacto-oligosaccharides was complete giving a final product concentration of:

66.1 %(w/w dry solids) total galacto-oligosaccharides.

These results are shown in figures 3A and 3B.

Experiment with food grade lactose

Fonterra lactose monohydrate

Batch number 22797320

Lactose concentration 99.5 % (w/w) as lactose monohydrate

Protein concentration < 0.10 % (w/w)

· Ash concentration 0.30 % (w/w)

Moisture concentration 0.10 % (w/w)

Experimental conditions

Substrate concentration 55.4% (w/w solution)

Solvent water

Lactose concentration 51 .6% (w/w solution)

Enzyme dose rate 18.0 (g/kg lactose)

Reaction temperature55 (°C)

Reaction pH 6.50

Results

Data are presented for a reaction time over 180 minutes. In this time the reaction for the production of galacto-oligosaccharides was complete given a final product concentration of: · 67.6 %(w/w dry solids) total galacto-oligosaccharides, 66.0 %(w/w dry solids) after 120 minutes.

Given, compared to D90 demineralised whey powder, the lactose solution concentration was higher (conditions that suit a higher final total galacto-oligosaccharide concentration) - the results on the two materials is essentially the same. The results are shown in figures 3A to B.

The comparative results show that contrary to expectation, the two raw materials provided for this process has no significant effect on the kinetics or yield of prebiotic oligosaccharide production, even though the concentration of the key starting material, lactose, is significantly lower in one of those raw materials.