Glucose-depleted liquid dairy milk, methods of producing the same and the use thereof to maintain health and to treat and prevent medical ailments

The present invention provides a glucose-depleted liquid dairy milk, methods of producing the same and the use thereof to assist in maintaining the health and well-being of a subject and in the treatment and prevention of medical ailments, specifically those associated with the over-consumption of glucose and/or inappropriate metabolism of glucose, e.g. metabolic syndrome, diabetes mellitus type II, obesity, dyslipidemia, insulin resistance, hypertension and liver steatosis.

The glucose-depleted liquid dairy milk of the invention may be prepared by treating liquid dairy milk with certain enzymes under pH conditions which do not cause coagulation of the milk, thereby resulting in a modified liquid dairy milk containing reduced amounts of free glucose and optionally reduced amounts of lactose, e.g. substantially or essentially no free glucose and optionally lactose, but an amount of gluconic acid corresponding to the amount by which the free glucose in the dairy milk is reduced, e.g. the amount of free glucose in the dairy milk prior to the enzyme treatment. The presence of gluconic acid in combination with reduced amounts, preferably the substantial or essential absence, of glucose and optionally lactose means the modified dairy milk of the invention has a surprisingly advantageous glycaemic profile and prebiotic properties and thus renders the modified dairy milk useful as part of a healthy diet in healthy subjects, and also surprisingly effective in treating subjects with or at risk of developing complex metabolic disorders associated with the over-consumption of glucose and/or inappropriate metabolism of glucose including metabolic syndrome, diabetes, obesity, dyslipidemia, insulin resistance, hypertension and liver steatosis, on account of its favourable insulin response and/or favourable effect on insulin sensitivity.

It is now well appreciated that a diet rich in simple sugars such as glucose can lead to health problems, in particular metabolic conditions including diabetes mellitus type II, metabolic syndrome and obesity. A well accepted measure expressing risk for the metabolic syndrome and diabetes and their sequelae, such as atherosclerosis, is the glycaemic response after ingestion of a meal or a food item and its "glycaemic index" and its "glycaemic load". It is also well established that the consumption of dairy milk, including the foodstuffs prepared therefrom, has certain health benefits, potentially including the prevention of osteoporosis and other bone disorders. However, dairy milk is also a rich source of glucose in the form of lactose, thus diminishing the overall benefit to health of milk and foodstuffs prepared therefrom. Consequently, it would potentially be advantageous to lower the glucose content of dairy milk thereby improving its glycaemic index, glycaemic load and the glycaemic and insulin responses thereto if this can be done without detrimentally affecting palatability and leaving the other components of the dairy milk substantially unaffected.

The consumption of dairy milk by certain human individuals and populations is also problematic because these subjects have difficulty in digesting lactose. These subjects are described as having lactose intolerance. This condition manifests in several undesirable symptoms such as abdominal bloating and cramps, flatulence, diarrhoea, nausea, rumbling stomach or vomiting upon consumption of lactose. The frequency of lactose intolerance in adults ranges from 5% in Northern European countries up to 100% in some African and Asian countries. It is known that the digestive problems of lactose intolerance can be avoided, or at least reduced, by the consumption of lactose-depleted dairy milk or products prepared therefrom. Lactose depletion may involve the treatment of dairy milk with lactase (which cleaves lactose into glucose and galactose), sometimes in combination with separation procedures to remove lactose, e.g. molecular sieve chromatography or ultrafiltration. When lactose is split into glucose and galactose the dairy product becomes enriched with free glucose as well as galactose. This results in an increased sweetness of such milks which has been perceived as excessive by some consumers and in a free glucose content (ca. 28g/l) which is higher than in regular milk (<1 g/l). This has a negative impact on the glycaemic index and glycaemic load of the treated milk and products prepared therefrom and the glycaemic and insulin responses thereto. The separation techniques result in the loss of other small molecules, including essential nutrients. Consequently, it would potentially be advantageous to lower the glucose content of lactase-treated dairy milk if this can be done without detrimentally affecting palatability and leaving the other components of the dairy milk substantially unaffected thereby allowing the use of lactose separation techniques to be avoided.

It has now been found however that liquid dairy milk can be glucose- depleted, e.g. rendered substantially or essentially devoid of glucose, by treatment with an enzyme that converts glucose into gluconic acid (glucose oxidase) and catalase under pH conditions which do not cause coagulation of the milk, optionally with a preceding step in which the dairy milk is treated with an enzyme which hydrolyses lactose to form glucose and galactose, and yet such products may retain sufficiently the taste, flavour and mouth-feel of the unmodified liquid dairy milk.

In addition it has been found that the gluconic acid content of a modified dairy milk in which substantially, e.g. essentially, all of the glucose has been converted to gluconic acid offers a dairy milk with a glycaemic response, a glycaemic index, a glycaemic load and an insulin response which are significantly lower than those of an untreated dairy milk, i.e. a more favourable glycaemic profile and lower available carbohydrate content, respectively, which in turn reduces the amount of dietary energy provided by the milk.

The difference between the insulin response to such a dairy milk of the invention as compared to the insulin response to an unmodified dairy milk may be more pronounced than that of the glucose response. Consuming a dairy milk of the invention may also increase insulin sensitivity.

Such a modified dairy milk is therefore surprisingly suited to the treatment and prevention of complex metabolic disorders associated with the over- consumption of glucose or the inappropriate metabolism of glucose including metabolic syndrome, diabetes, obesity, dyslipidemia, insulin resistance, hypertension and liver steatosis.

Thus, it can be seen that the present invention provides a modified dairy milk of superior nutritional value and therapeutic properties.

Therefore, in a one aspect the present invention provides a glucose- depleted liquid dairy milk, wherein said milk has a pH of about 4.5 to about 7.0 and comprises gluconic acid, e.g. at a concentration of at least about 0.1 g/l, and wherein the mass concentrations of one or more, e.g. any two or three, of Ca ²⁺ , K ⁺ , and Mg ²⁺ contained therein are increased by

(i) at least about 0.05 g/l for Ca ²⁺ ,

(ii) at least about 0.1 g/l for K ⁺ , and

(iii) at least about 0.01 g/l for Mg ²⁺ , as compared to the mass concentrations of Ca ²⁺ , K ⁺ , and Mg ²⁺ respectively present in an equivalent milk from the same mammal but which is not glucose- depleted.

By "dairy milk" it is meant the casein containing composition produced by the mammary glands of mammals for the nourishment of their offspring, including colostrum. In accordance with the invention it does not include vegetable, fruit or nut extracts resembling dairy milk in appearance. The term as used herein further includes processed forms of dairy milk, e.g. milk of any fat content (e.g. whole milk, partially skimmed milk or skimmed (fat free milk)), milk which has been treated enzymatically (e.g. with lactase) to deplete lactose through the conversion

(hydrolysis) of lactose to glucose and galactose, homogenised milk, pasteurised milk, heat treated milk (e.g. ultra heat treated milk, sterilised milk), filtered milk, flavoured milk, milk-based fruit smoothies, evaporated milk, condensed milk and reconstituted milk powder. The mammalian source is not limited and as such may be any domesticated ungulate, e.g. cow, goat, sheep, buffalo, yak, camel, horse and donkey, or human.

The proteins in the aqueous phase of a milk are not substantially coagulated and thus milk is a liquid composition which does not contain non-transient aggregates of milk proteins in the aqueous phase. Thus the liquid dairy milk of use in the invention is non-coagulated milk. More specifically, substantially, e.g.

essentially, all of the proteins, in particular the casein, in the aqueous phase of a milk are solubilised in the aqueous liquid phase, i.e. not coagulated. Expressed numerically less than 10%, e.g. less than 5%, 4%, 3%, 2%, 1 %, 0.5% or 0.1 % of the proteins, in particular the casein, in the aqueous phase of a milk are not solubilised therein. Expressed differently, greater than 90%, e.g. greater than 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% of the proteins, in particular the casein, in the aqueous phase of milk are solubilised therein.

In contrast, whey is the protein (whey protein, including beta-lactoglobulin, alpha-lactalbumin, serum albumin and immunoglobulin) containing liquid remaining after dairy milk is treated with rennet (specifically the chymosin contained therein) or an edible acid in order to effect the coagulation (or curdling) of milk (specifically the casein contained therein). Sweet whey is the co-product of rennet-coagulated milk and acid whey (also called sour whey) is the co-product of acid-coagulated milk. Sweet whey has a pH greater than or equal to about 5.6, acid whey has a pH less than or equal to about 5.1 . Dairy curds are the casein containing solid co- product of these processes.

A foodstuff prepared from dairy milk, dairy curd or whey is an edible product consisting essentially of compounds found in dairy milk, e.g. milk proteins (whey proteins and casein), lipids and sugars. Such products may be the result of microbial fermentation. As such the term specifically includes cheese, whey cheese, cream, butter, whey butter, yoghurt, creme fraiche, fromage frais, soured cream, kefir and buttermilk. Unless context dictates otherwise, the term "dairy product" is used herein as a simple and concise means to refer to dairy milk, dairy curd, whey, or a dairy foodstuff prepared therefrom. The term "glucose-depleted dairy product" is used herein analogously.

It is particularly preferred that the dairy milk is a milk which has been treated enzymatically (e.g. with lactase) to deplete lactose through the conversion

(hydrolysis) of lactose to glucose and galactose. Such enzymatically treated milk may contain at least trace amounts of the enzyme used to hydrolyse lactose into glucose and galactose (e.g. lactase) either in active, partially active or inactive form.

References to "an equivalent milk from the same mammal" are intended to convey that the milks under comparison come from the same mammalian animal and are essentially the same in terms of their processing, e.g. the examples provided above.

The dairy milk of use in the invention will contain free glucose (i.e. glucose molecules available for oxidation to gluconic acid). Such glucose may be present naturally or may arise from the enzymatic conversion of lactose into free glucose and galactose.

In other embodiments the dairy milk undergoing glucose depletion is not a dairy milk to which bioavailable glucose, in particular free glucose, has been added.

By "glucose-depleted" it is meant that a dairy milk has a reduced amount of bioavailable glucose compared to that of an equivalent milk from the same mammal but which is not glucose-depleted. More specifically, the liquid dairy milk of the invention is rendered "glucose-depleted" by the methods described herein.

Bioavailable glucose in dairy milk may be made up of "free glucose", i.e. glucose that is not covalently bound to another saccharide molecule, in other words glucose which is not part of a di-, oligo- or poly-saccharide, e.g. including but not limited to lactose and glycogen, and/or glucose which is that is covalently bound to another saccharide molecule, in particular in the form of lactose, and which may be used by an animal (in particular a human) as a source of energy. Preferably it is meant that a dairy milk has a reduced amount of free glucose and glucose that is covalently bound to another saccharide molecule, in particular in the form of lactose. More preferably, it is meant that a dairy milk has a reduced amount of glucose which is present in the form of lactose.

In these latter embodiments the term "glucose depleted" may alternatively be expressed more specifically as "glucose- and lactose-depleted" or "free glucose depleted and lactose depleted". Indeed, the mass concentrations of bioavailable glucose recited herein may be taken as a combined mass concentration of lactose and free glucose.

As such the invention provides a glucose-depleted and lactose-depleted liquid dairy milk, wherein said milk has a pH of about 4.5 to about 7.0 and comprises gluconic acid, e.g. at a concentration of at least about 0.1 g/l, and wherein the mass concentrations of one or more, e.g. any two or three, of Ca ²⁺ , K ⁺ , and Mg ²⁺ contained therein are increased by

(i) at least about 0.05 g/l for Ca ²⁺ ,

(ii) at least about 0.1 g/l for K ⁺ , and

(iii) at least about 0.01 g/l for Mg ²⁺ ,

as compared to the mass concentrations of Ca ²⁺ , K ⁺ , and Mg ²⁺ respectively present in an equivalent milk from the same mammal but which is not glucose- depleted.

More specifically the invention may be said to provide a free glucose- depleted and lactose-depleted liquid dairy milk, wherein said milk has a pH of about 4.5 to about 7.0 and comprises gluconic acid, e.g. at a concentration of at least about 0.1 g/l, and wherein the mass concentrations of one or more, e.g. any two or three, of Ca ²⁺ , K ⁺ , and Mg ²⁺ contained therein are increased by

(i) at least about 0.05 g/l for Ca ²⁺ ,

(ii) at least about 0.1 g/l for K ⁺ , and

(iii) at least about 0.01 g/l for Mg ²⁺ ,

as compared to the mass concentrations of Ca ²⁺ , K ⁺ , and Mg ²⁺ respectively present in an equivalent milk from the same mammal but which is not glucose- depleted.

The following discussion of the term "glucose depleted" and "bioavailable glucose" apply to these embodiments. More specifically, by "glucose depleted" it is meant that a dairy milk has a reduced amount of bioavailable glucose that results in a reduced glycaemic response in a subject as measured by the area under the curve (AUC) of a subject's blood glucose (preferably capillary blood glucose) levels over time, preferably over about 15mins, 30mins, 45 mins, 60mins, 75mins, 90mins, 105mins, 120mins, 150mins, 180mins, 210mins or 240mins immediately following

consumption of the glucose-depleted dairy milk, relative to an equivalent milk from the same mammal but which is not glucose-depleted. Preferably the AUC is calculated as the incremental AUC (iAUC), i.e. all area below the curve but above the fasting blood glucose concentration. Preferably the AUC, e.g. the iAUC, is calculated over about 120mins. The glycaemic response for each milk should be determined in the same way.

The glucose-depleted dairy milk of the invention preferably results in an area under the curve as defined above in response to its consumption that is no more than 75%, e.g. no more than about 70, 65, 60, 55, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 5 or 1 % of that of an equivalent milk from the same mammal but which is not glucose-depleted.

Expressed alternatively, "glucose-depleted" means that a dairy milk has a reduced amount of bioavailable glucose that results in the dairy milk having a reduced glycaemic load relative to that of an equivalent milk from the same mammal but which is not glucose-depleted. The glucose-depleted dairy milk preferably has a glycaemic load which is no more than 75%, e.g. no more than about 70, 65, 60, 55, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 5 or 1 %, of that of an equivalent milk from the same mammal but which is not glucose-depleted. The glycaemic load of each milk should be determined in the same way.

For the purposes of the invention, the glycaemic load of a foodstuff (e.g. a dairy milk) is calculated as the amount of available carbohydrate in a standard portion of the foodstuff multiplied by the glycaemic index (Gl) of the foodstuff divided by 100. For the dairy milk product of the invention a standard portion size may be taken as about 250ml or about 250g.

For the purposes of the invention the Gl of a foodstuff is defined as the iAUC of a blood glucose response curve over about 120mins after consumption of a 50 g available-carbohydrate portion of a foodstuff expressed as a percentage of that after 50 g oral glucose. For the purposes of the invention available carbohydrate is that fraction of carbohydrate that is absorbed across the

gastrointestinal tract and enters into intermediary metabolism. It does not include dietary fibre.

Expressed alternatively still, "glucose depleted" means that a dairy milk has a reduced amount of bioavailable glucose that results in the dairy milk having a reduced glycaemic index relative to that of an equivalent milk from the same mammal but which is not glucose-depleted. The glucose-depleted dairy milk of the invention preferably has a glycaemic index which is no more than 75%, e.g. no more than about 70, 65, 60, 55, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 5 or 1 %, of that of an equivalent milk from the same mammal but which is not glucose-depleted.

This may also be expressed as a dairy milk that has a lower mass concentration ratio of bioavailable glucose to non-saccharide soluble components than the corresponding ratio of an equivalent milk from the same mammal but which is not glucose-depleted. In these embodiments "lower" means a mass concentration ratio of bioavailable glucose to non-saccharide soluble components which is no more than 75%, e.g. no more than about 70, 65, 60, 55, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 5 or 1 % of that of an equivalent milk from the same mammal but which is not glucose-depleted.

Mass concentration is an indication of the mass of a first substance present in a defined mass or volume of a second substance. Mass concentration may therefore be expressed as grams per litre ("g/l"), grams per kilogram ("g/kg"), parts- per-million (ppm, i.e. mg of solute per litre of solvent); "% w/v"; "% w/w, "g/100ml"; or the like.

More specifically, "glucose-depleted" means that the mass concentrations of bioavailable glucose are no more than about 75% of the mass concentrations of bioavailable glucose typically present in an equivalent milk from the same mammal but which is not glucose-depleted, e.g. no more than about 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 % of the mass concentrations of bioavailable glucose typically present in an equivalent milk from the same mammal but which is not glucose-depleted.

More simply "glucose-depleted" means the mass concentrations of bioavailable glucose in the glucose-depleted dairy milk of the invention is reduced by at least about 25, 50, 60, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% as compared to the mass concentrations of bioavailable glucose in an equivalent milk from the same mammal but which is not glucose-depleted.

In certain embodiments the mass concentration of the bioavailable glucose in the glucose-depleted dairy milk of the invention is no more than about 20 g/l, e.g. no more than about 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 ,

0.5, 0.1 , 0.05, 0.01 g/l, when said dairy milk is adjusted in volume with water to give a gluconic acid concentration of about 0.1 g/l or the specific concentrations disclosed below (e.g. about 0.5, 1 , 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g/l).

The above mass concentration values may be considered to apply particularly in the context of dairy milk which has not undergone dilution or concentration or any kind other than removal of fats.

In other embodiments, "glucose-depleted" means that the dairy milk has been rendered substantially, e.g. essentially, devoid of bioavailable glucose, e.g. free glucose and lactose.

By "substantially devoid of bioavailable glucose" it is meant that the glucose- depleted dairy milk of the invention contains no more than about 5 g/l bioavailable glucose, e.g. no more than about 4, 3, 2, 1 , 0.5, 0.1 , 0.05, 0.01 g/l bioavailable glucose, when said dairy milk is adjusted in volume with water to give a gluconic acid concentration of about 0.1 g/l or the specific concentrations disclosed below.

By "essentially devoid of bioavailable glucose" it is meant that the glucose- depleted dairy milk of the invention contains a trace amount of bioavailable glucose (i.e. trace amounts of free glucose and lactose). This may also be expressed as essentially undetectable with standard analytical means, or at the limit of detection with such means. These measures preferably take place when said dairy milk is adjusted in volume with water to give a gluconic acid concentration of about 0.1 g/l or the specific concentrations disclosed below. Detection may be by any convenient means, e.g. the Reflectoquant™ system of Merck Millipore™ or by HPLC

(preferably with pulsed amperometric detection) as disclosed in the Examples or by high performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) (Perati, P., et al, Thermo Scientific Application Note 248, 20150. The above values for bioavailable glucose may be considered to apply particularly in the context of dairy milk which has not undergone dilution or concentration or any kind other than removal of fats.

The terms "free glucose-depleted" and "lactose-depleted" when used herein should be interpreted consistently with the foregoing mass concentrations.

In a still further alternative expression of the invention the (bioavailable) glucose-depleted dairy milk of the invention may be considered a sugar-depleted dairy milk wherein said milk has a reduced amount of free glucose and optionally a reduced amount of lactose. The specific measures of (bioavailable) glucose depletion recited above maybe recited under this alternative terminology.

In certain embodiments a glucose-depleted dairy milk of the invention may have an increased mass concentration ratio of other sugars, e.g. galactose or fructose, to non-saccharide soluble components than the corresponding ratios of an equivalent milk from the same mammal but which is not glucose-depleted.

In other embodiments the glucose-depleted dairy milk may have also been rendered devoid, or at least have a reduced content or be depleted, of other sugars, e.g. galactose or fructose, typically present in an equivalent milk from the same mammal but which is not glucose-depleted. The above embodiments relating to glucose can be applied mutatis mutandis in the context of the depletion of other sugars, e.g. galactose or fructose.

Therefore, in a further embodiment the present invention provides a glucose-depleted liquid dairy milk, wherein said milk has a pH of about 4.5 to about 7.0, is substantially, e.g. essentially, devoid of bioavailable glucose, and comprises gluconic acid, e.g. at a concentration of at least about 0.1 g/l, and wherein the mass concentrations of one or more, e.g. any two or three, of Ca ²⁺ , K ⁺ , and Mg ²⁺ contained therein are increased by

(iv) at least about 0.05 g/l for Ca ²⁺ ,

(v) at least about 0.1 g/l for K ⁺ , and

(vi) at least about 0.01 g/l for Mg ²⁺ ,

as compared to the mass concentrations of Ca ²⁺ , K ⁺ , and Mg ²⁺ respectively present in an equivalent milk from the same mammal but which is not glucose- depleted.

In preferred embodiments the glucose-depleted dairy milk has the same or substantially the same taste and flavour profile and mouth-feel as an equivalent milk from the same mammal but which is not glucose-depleted. In preferred embodiments the glucose-depleted dairy milk of the invention contains at least 0.1 g/l, e.g. at least about 0.5, 1 , 1 .5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 65, 70, 75 or 80 g/l gluconic acid.

In certain embodiments gluconic acid will typically be present in the glucose- depleted dairy milk at a mass concentration of about 0.1 to about 100 g/l, e.g. about any one of 0.5, 1 , 2.5, 5, 10, 15, 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 65, 70, 75, 80, 85, 90, or 95 to about 100g/l, preferably about any one of 2.5, 5, 10, 15, 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 65, 70, 75, 80 or 85 to about 90 g/l, more preferably about any one of 10, 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 65 or 70 to about 80 g/l, and still more about preferably about any one of 20, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 to about 75 g/l.

Any range which may be formed from any of the above recited mass concentrations is expressly contemplated.

In other embodiments gluconic acid will typically be present in the glucose- depleted dairy milk at a mass concentration of about 0.5 to about 70 g/l, e.g. about any one of 0.5, 1 , 1 .5, 2, 2.5, 3, 5, 10, 15, 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 65 g/l to about 70g/l, preferably about any one of 2.5, 3, 5, 10, 15, 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 or 60 to about 65 g/l, more preferably about any one of 10, 15, 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 or 56 to about 60 g/l, and still more preferably about any one of 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 or 56 to about 60 g/l.

Any range which may be formed from any of the above recited mass concentrations is expressly contemplated.

As used herein, the term "gluconic acid" is generic and represents all the equilibrium species of gluconic acid in an aqueous medium - e.g. lactone forms (e.g. D-gluconic acid δ-lactone and D-gluconic acid γ-lactone), gluconate salt forms and the acid form.

Gluconic acid content may be measured by any convenient means, e.g. via an appropriate enzymatic assay (e.g. as supplied by R-Biopharm™) or

chromatography based techniques. Such measurements should take into account all forms of gluconic acid, i.e. the free acid, salts thereof and the lactone forms thereof, e.g. D-gluconic acid δ-lactone and D-gluconic acid γ-lactone. As detailed later, because the method of producing the glucose-depleted dairy milk of the invention is based on the conversion of at least a portion of the bioavailable glucose present in a natural dairy milk to gluconic acid, the exact amount of gluconic acid present in these embodiments will be to an extent dictated by the amounts of bioavailable glucose (e.g. free glucose and glucose in lactose) converted to gluconic acid. Ultimately the amount of gluconic acid present in these embodiments will be dictated by the total bioavailable glucose (e.g. free glucose and lactose) content of the starting material.

In certain embodiments the glucose-depleted dairy milk of the invention has a pH of equal to or greater than about 4.5 and equal to or less than about 6.8, e.g. about 4.5 to about 6.5, about 4.5 to about 6.2, about 4.5 to about 6.0, about 4.5 to about 5.5, about 4.5 to about 5.0, about 4.5 to about 4.8, about 4.8 to about 7.0, about 4.8 to about 6.8, about 4.8 to about 6.5, about 4.8 to about 6.2, about 4.8 to about 6.0, about 4.5 to about 5.5, about 4.8 to about 5.0, about 5.0 to about 7.0, about 5.0 to about 6.8, about 5.0 to about 6.5, about 5.0 to about 6.2, about 5.0 to about 6.0, about 5.0 to about 5.5, about 6.0 to about 7.0, about 6.0 to about 6.8, about 6.0 to about 6.5, about 6.0 to about 6.2, about 6.2 to about 7.0, about 6.2 to about 6.8, about 6.2 to about 6.5, about 6.5 to about 7.0, about 6.5 to about 6.8, or about 6.8 to about 7.0

The above recited pH ranges must be maintained during the enzymatic treatment of the dairy milk otherwise the milk may coagulate and no longer be a liquid milk product of the invention. In accordance with the method of the invention pH is controlled by the addition of an oxide or a hydroxide of calcium, potassium and/or magnesium. As a result of this the glucose depleted dairy milk of the invention comprises mass concentrations of Ca ²⁺ , K ⁺ and/or Mg ²⁺ which are increased compared to the an equivalent milk from the same mammal but which is not glucose-depleted.

Without wishing to be bound by theory, it is believed that the

supplementation of the glucose-depleted dairy milk of the invention with the above described metal ions may cause a favourable shift in the amounts of gluconate- metal salts present in the glucose-depleted dairy milk and these salt profiles may promote the retention of the flavour and mouth-feel of the unmodified dairy milk. The skilled person would be easily able to determine workable and optimal combinations of metal ion concentrations for his particular dairy milk of interest in accordance with the invention, i.e. those that give rise to a glucose-depleted dairy milk that is highly palatable and which retains sufficiently the flavour and mouth-feel of an unmodified dairy milk. Routine techniques such as the use of trained sensory assessors can be employed in this regard.

In preferred embodiments the mass concentration of Ca ²⁺ in the glucose- depleted dairy milk of the invention may be increased by at least about 0.05 g/l, e.g. at least about 0.1 , 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 g/l Ca ²⁺ .

In preferred embodiments the mass concentration of K ⁺ in the glucose- depleted dairy milk of the invention may be increased by at least about 0.1 g/l, e.g. at least about 0.5, 1 , 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 1 1 , 1 1 .5, 12, 12.5, 13, 13.5, 14, 14.5 or 15 g/l K ⁺ .

In preferred embodiments the mass concentration of Mg ²⁺ in the glucose- depleted dairy milk of the invention may be increased by at least about 0.01 g/l, e.g. at least about 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 g/l Mg ²⁺ .

All ratios of Ca ²⁺ , K ⁺ and/or Mg ²⁺ mass concentrations increases which may be formed from the above values are expressly contemplated.

In certain embodiments the glucose-depleted dairy milk contains Ca ²⁺ and K ⁺ at the above described increased concentrations. In other embodiments the selected metal ions are Ca ²⁺ and Mg ²⁺ , Mg ²⁺ and K ⁺ , or Ca ²⁺ , K ⁺ and Mg ²⁺ all at the above described increased concentrations.

In certain embodiments the glucose-depleted dairy milk of the invention comprises one or more, e.g. any two or three, of Ca ²⁺ , K ⁺ and Mg ²⁺ at increased mass concentrations relative to an equivalent milk from the same mammal but which is not glucose-depleted and which, when said dairy milk is adjusted in volume with water to give a gluconic acid concentration of about 0.1 g/l, or the specific concentrations disclosed above, gives the above recited values for said ions.

The concentration of metal ions referred to herein are concentrations as may be determined by the physiochemical pressure digestion method described in EN 13805 (2013). Should alternative approaches be used to measure the concentration of metal ions in a dairy milk, an acid (e.g. nitric acid) digestion step, equivalent to that of EN 13805, must be incorporated immediately prior to analysis.

In these embodiments the mass concentration of Ca ²⁺ in the glucose- depleted dairy milk of the invention may be increased by about 0.05 to about 10 g/l, e.g. about any one of 0.05, 0.1 , 0.5, 1 , 1 .5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 to about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 9.5 g/l Ca ²⁺ , preferably about any one of 0.05, 0.1 , 0.5, 1 , 1 .5, 2, 2.5, 3, 3.5, or 4 to about any one of 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 g/l Ca ²⁺ , more preferably about any one of 0.05, 0.1 , 0.5, 1 , 1 .5, 2, 2.5 or 3, to about any one of 3.5, 4, 4.5, 5, 5.5 or 6 g/l Ca ²⁺ , and still more preferably about any one of 0.05, 0.1 , 0.5, 1 , 1 .5, 2 or 2.5 to about any one of 3, 3.5, 4, 4.5 or 5 g/l Ca ²⁺ . Any range which may be formed from any of the above recited mass concentrations is expressly contemplated.

In these embodiments the mass concentration of K ⁺ in the glucose-depleted dairy milk of the invention may be increased by about 0.1 to about 20 g/l, e.g. about any one of 0.1 , 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 to about any one of 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 g/l K ⁺ , preferably about any one of 0.1 , 0.5, 1 , 2, 3, 4, 5, 6, 7 or 8 to about any one of 9, 10, 1 1 , 12, 13, 14, 15 or 16 g/l K ⁺ , more preferably about any one of 0.1 , 0.5, 1 , 2, 3, 4, 5, or 6 to about any one of 7, 8, 9, 10 1 1 or 12 g/l K ⁺ , and still more preferably about any one of 0.1 , 0.5, 1 , 2, 3, 4, or 5 to about any one of 6, 7, 8, 9 or 10 g/l K ⁺ . Any range which may be formed from any of the above recited mass concentrations is expressly contemplated.

In these embodiments the mass concentration of Mg ²⁺ in the glucose- depleted dairy milk of the invention may be increased by about 0.01 to about 2 g/l, e.g. about any one of 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 to about any one of 1.1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1.7, 1 .8, 1 .9 or 2 g/l Mg ²⁺ , preferably about any one of 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 to about any one of 0.9, 1 , 1.1 , 1 .2, 1 .3, 1 .4, 1.5 or 1 .6 g/l Mg ²⁺ , more preferably about any one of

0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5 or 0.6 to about any one of 0.7, 0.8, 0.9, 1 1 .1 or 1 .2 g/l Mg ²⁺ , and still more preferably about any one of 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4 or 0.5, to about any one of 0.6, 0.7, 0.8, 0.9 or 1 g/l Mg ²⁺ . Any range which may be formed from any of the above recited mass concentrations is expressly

contemplated.

All ratios of increased Ca ²⁺ , K ⁺ and/or Mg ²⁺ mass concentrations which may be formed from the above values are expressly contemplated.

In other embodiments the modified dairy milk of the invention may be considered to have a Ca ²⁺ mass concentration which is relative to the mass concentration of Ca ²⁺ present naturally in an equivalent milk from the same mammal. In these embodiments a natural Ca ²⁺ mass concentration of 0 g/l to less than 0.5g/l in an equivalent milk from the same mammal will result in the dairy milk of the invention having at least about 0.5 g/l Ca ²⁺ . A natural Ca ²⁺ mass

concentration of 0.5 g/l to less than 1 g/l will result in the dairy milk of the invention having at least about 1 g/l Ca ²⁺ . A natural Ca ²⁺ mass concentration of 1 g/l to less than 1 .5 g/l will result in the dairy milk of the invention having at least about 1 .5 g/l Ca ²⁺ . A natural Ca ²⁺ mass concentration of 1 .5 g/l to less than 2 g/l will result in the dairy milk of the invention having at least about 2 g/l Ca ²⁺ . A natural Ca ²⁺ mass concentration of 2 g/l to less than 2.5 g/l will result in the dairy milk of the invention having at least about 2.5 g/l Ca ²⁺ . A natural Ca ²⁺ mass concentration of 2.5 g/l to less than 3 g/l will result in the dairy milk of the invention having at least about 3 g/l Ca ²⁺ . A natural Ca ²⁺ mass concentration of 3 g/l to less than 3.5 g/l will result in the dairy milk of the invention having at least about 3.5 g/l Ca ²⁺ . A natural Ca ²⁺ mass concentration of 3.5 g/l to less than 4 g/l will result in the dairy milk of the invention having at least about 4 g/l Ca ²⁺ .

In other embodiments the dairy milk of the invention may have a K ⁺ mass concentration which is relative to the mass concentration of K ⁺ present naturally in an equivalent milk from the same mammal. In these embodiments a natural K ⁺ mass concentration of 0 g/l to less than 1 g/l in an equivalent milk from the same mammal will result in the dairy milk of the invention having at least about 1 g/l K ⁺ . A natural K ⁺ mass concentration of 1 g/l to less than 1 .5 g/l will result in the dairy milk of the invention having at least about 1 .5 g/l K ⁺ . A natural K ⁺ mass concentration of 1 .5 g/l to less than 2 g/l will result in the dairy milk of the invention having at least about 2 g/l K ⁺ . A natural K ⁺ mass concentration of 2 g/l to less than 2.5 g/l will result in the dairy milk of the invention having at least about 2.5 g/l K ⁺ . A natural K ⁺ mass concentration of 2.5 g/l to less than 3 g/l will result in the dairy milk of the invention having at least about 3 g/l K ⁺ . A natural K ⁺ mass concentration of 3 g/l to less than 3.5 g/l will result in the dairy milk of the invention having at least about 3.5 g/l K ⁺ . A natural K ⁺ mass concentration of 3.5 g/l to less than 4 g/l will result in the dairy milk of the invention having at least about 4 g/l K ⁺ . A natural K ⁺ mass concentration of 4 g/l to less than 4.5 g/l will result in the dairy milk of the invention having at least about 4.5 g/l K ⁺ . A natural K ⁺ mass concentration of 4.5 g/l to less than 5 g/l will result in the dairy milk of the invention having at least about 5 g/l K ⁺ .

In other embodiments the glucose-depleted dairy milk of the invention may have a Mg ²⁺ mass concentration which is relative to the mass concentration of Mg ²⁺ present naturally in an equivalent milk from the same mammal. In these embodiments a natural Mg ⁺ mass concentration of 0 g/l to less than 0.1 g/l in an equivalent milk from the same mammal will result in the dairy milk of the invention having at least about 0.1 g/l Mg ²⁺ . A natural Mg ²⁺ mass concentration of 0.1 g/l to less than 0.15 g/l will result in the dairy milk of the invention having at least about 0.15 g/l Mg ²⁺ . A natural Mg ²⁺ mass concentration of 0.15 g/l to less than 0.2 g/l will result in the dairy milk of the invention having at least about 0.2 g/l Mg ²⁺ . A natural Mg ⁺ mass concentration of 0.2 g/l to less than 0.25 g/l will result in the dairy milk of the invention having at least about 0.25 g/l Mg ²⁺ . A natural Mg ²⁺ mass concentration of 0.25 g/l to less than 0.3 g/l will result in the dairy milk of the invention having at least about 0.3 g/l Mg ²⁺ . A natural Mg ²⁺ mass concentration of 0.3 g/l to less than 0.35 g/l will result in the dairy milk of the invention having at least about 0.35 g/l Mg ²⁺ . A natural Mg ²⁺ mass concentration of 0.35 g/l to less than 0.4 g/l will result in the dairy milk of the invention having at least about 0.4 g/l Mg ²⁺ . A natural Mg ²⁺ mass concentration of 0.4 g/l to less than 0.45 g/l will result in the dairy milk of the invention having at least about 0.45 g/l Mg ²⁺ . A natural Mg ²⁺ mass concentration of 0.45 g/l to less than 0.5 g/l will result in the dairy milk of the invention having at least about 0.5 g/l Mg ²⁺ .

The Ca ²⁺ , K ⁺ , and Mg ²⁺ mass concentrations present naturally in an equivalent milk from the same mammal can be easily determined as described herein. In addition, databases of food composition, e.g. the McCance and

Widdowson's Composition of Foods Integrated Dataset of Public Health England and the United States Department of Agriculture Agricultural Research Service National Nutrient Database for Standard Reference, the contents of which are incorporated in their entirety by reference, may be consulted.

In other embodiments the glucose-depleted dairy milk of the invention comprise may contain at least about 0.5 g/l Ca ²⁺ , e.g. at least about 0.6, 0.7, 0.8, 0.9, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 g/l Ca ²⁺ .

In preferred embodiments the glucose-depleted dairy milk of the invention may contain at least about 1 g/l K ⁺ , e.g. at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,

5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 1 1 , 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15 g/l K ⁺ .

In preferred embodiments the glucose-depleted dairy milk of the invention may contain at least about 0.1 g/l Mg ²⁺ , e.g. at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 g/l Mg ²⁺ .

All ratios of Ca ²⁺ , K ⁺ and/or Mg ²⁺ mass concentrations which may be formed from the above values are expressly contemplated.

In certain embodiments the glucose-depleted dairy milk of the invention contains Ca ²⁺ and K ⁺ at the above described concentrations. In other embodiments the selected metal ions are Ca ²⁺ and Mg ²⁺ , Mg ²⁺ and K ⁺ , or Ca ²⁺ , K ⁺ and Mg ²⁺ all at the above described concentrations.

In certain embodiments the glucose-depleted dairy milk of the invention comprises one or more, e.g. any two or three, of Ca ²⁺ , K ⁺ and Mg ²⁺ at mass concentrations which, when said dairy milk is adjusted in volume with water to give a gluconic acid concentration of about 0.1 g/l, or the specific concentrations disclosed above, gives the above recited values.

Any specific embodiments of individual elements of the invention described above apply mutatis mutandis to this aspect of the invention, in particular the specific values and ranges for glucose and/or gluconic acid mass concentration Thus, the present invention provides a glucose-depleted liquid dairy milk, wherein said milk has a pH of about 4.5 to about 7.0 and comprises gluconic acid, e.g. at a concentration of least about 0.1 g/l, is preferably substantially, e.g.

essentially, devoid of bioavailable glucose, and contains one or more of Ca ²⁺ , K ⁺ , and Mg ²⁺ at a mass concentration which, when said dairy milk is adjusted in volume with water to give a gluconic acid concentration of about 0.1 g/l or the specific concentrations described above, gives:

(i) a mass concentration of Ca ²⁺ of about 0.05 to about 10g/I

(ii) a mass concentration of K ⁺ of about 0.1 to about 20 g/l,

(iii) a mass concentration of Mg ²⁺ of about 0.01 to about 2 g/l.

The above values for bioavailable glucose may be considered to apply mutatis mutandis.

The above embodiments may be considered to apply particularly in the context of dairy milk which has not undergone dilution or concentration or any kind other than removal of fats.

The above disclosed ranges of gluconic acid, Ca ²⁺ , K ⁺ , and Mg ²⁺ mass concentrations apply mutatis mutandis to this aspect of the invention.

In a further embodiment there is provided a glucose-depleted liquid dairy milk, wherein at least 75%, 80%, 85%, 90%, 95% or essentially all of said dairy milk is cow's milk, wherein said milk has a pH of about 4.5 to about 7.0, comprises gluconic acid, e.g. at a concentration of least about 0.1 g/l, and one or more of

(i) at least about 1 .5 g/l Ca ²⁺ ,

(ii) at least about 2.0 g/l K ⁺ , and

(iii) at least about 0.2 g/l Mg ²⁺ . ln a further embodiment there is provided a glucose-depleted liquid dairy milk, wherein at least 75%, 80%, 85%, 90%, 95% or essentially all of said dairy milk is sheep's milk, wherein said milk has a pH of about 4.5 to about 7.0, comprises gluconic acid, e.g. at a concentration of least about 0.1 g/l, and one or more of (i) at least about 2.0 g/l Ca ²⁺ ,

(ii) at least about 1 .5 g/l K ⁺ , and

(iii) at least about 0.2 g/l Mg ²⁺ .

In a further embodiment there is provided a glucose-depleted dairy milk, wherein at least 75%, 80%, 85%, 90%, 95% or essentially all of said dairy milk is goat's milk, wherein said milk has a pH of about 4.5 to about 7.0, comprises gluconic acid, e.g. at a concentration of least about 0.1 g/l, and one or more of

(i) at least about 1 .5 g/l Ca ²⁺ ,

(ii) at least about 2.5 g/l K ⁺ , and

(iii) at least about 0.5 g/l Mg ²⁺ .

In a further embodiment there is provided a glucose-depleted dairy milk, wherein at least 75%, 80%, 85%, 90%, 95% or essentially all of said dairy milk is buffalo's milk, wherein said milk has a pH of about 4.5 to about 7.0, comprises gluconic acid, e.g. at a concentration of least about 0.1 g/l, and one or more of (i) at least about 2.0 g/l Ca ²⁺ ,

(ii) at least about 2.0 g/l K ⁺ , and

(iii) at least about 0.4 g/l Mg ²⁺ .

Any specific embodiments of individual elements of the invention described above apply mutatis mutandis to this aspect of the invention, in particular the specific values and ranges for gluconic acid, bioavailable glucose and increased Ca ²⁺ , K ⁺ and Mg ²⁺ mass concentrations. In certain embodiments the above specific dairy milk products of the invention comprise one or more, e.g. any two or three of Ca ²⁺ , K ⁺ and Mg ²⁺ , at mass concentrations which, when said dairy milk is adjusted in volume with water to give a gluconic acid concentration of about 0.1 g/l, or the specific concentrations disclosed above, gives the above recited values.

In other embodiments the glucose-depleted dairy milk of the present invention is also substantially, e.g. essentially, devoid of fructose, which term is to be interpreted as discussed above for glucose and lactose. This may be conveniently achieved by further treating the glucose-depleted dairy milk products of the present invention with an enzyme capable of converting fructose into a derivative form, preferably a derivative form with a lower calorific value and/or more favourable glycaemic profile. Such enzymes may include 5-D-fructose

dehydrogenase (e.g. as described in US 2009/0214620). Alternatively, fructose may be enzymatically converted to glucose prior to or concurrent with treatment with glucose oxidase. Such enzymes may be defined as glucose isomerases and include glucose-6-phosphate isomerase and D-xylose isomerase (e.g. as described in US 2009/031 1232). In these embodiments in may be necessary to supplement the glucose-depleted dairy milk of the present invention with a sweetening agent, e.g. an artificial sweetening agent that is not a sugar (e.g. stevia, sucralose and aspartame).

In certain embodiments the glucose-depleted dairy milk of the present invention does not contain detectable amounts of active glucose oxidase, and/or catalase, and/or lactase and/or 5-D-fructose dehydrogenase and/or a glucose isomerase (e.g. glucose-6-phosphate isomerase or D-xylose isomerase) or any other enzyme of use in accordance with the invention (e.g. those as described herein). Expressed numerically the glucose-depleted dairy milk of the present invention displays enzyme activities for the above enzymes of no more than 1 U/ml, e.g. no more than 0.1 U/ml, 0.05 U/ml or 0.01 U/ml. Nevertheless, such glucose- depleted dairy milk of the present invention may still comprise inactivated, e.g. denatured, forms of one or more of the above-mentioned enzymes, e.g. glucose oxidase, and/or catalase, and/or lactase. In other embodiments the glucose- depleted dairy milk contains active forms of one or more of the above-mentioned enzymes, e.g. glucose oxidase, and/or catalase, and/or lactase.

The glucose-depleted dairy milk of the present invention of the invention may be supplemented with other compounds to enhance the palatability of the milk, e.g. by enhancing taste, flavour and mouth-feel. Such compounds include, but are not limited to other metal ions (e.g. Na ⁺ , Fe ²⁺ , Fe ³⁺ ), vitamins (e.g. vitamins A, B, C, D, E, K and subtypes thereof), minerals (e.g. compounds containing phosphorous, sulphur, fluorine, chlorine, boron, chromium, cobalt, copper, iron, manganese, molybdenum, selenium, silicon, tin, vanadium and zinc), flavourings (natural and artificial), flavour enhancers (e.g. monosodium glutamate), preservatives, artificial sweeteners (e.g. stevia, sucralose and aspartame), polyphenols, organic acids (other than gluconic acid), acidity regulators and stabilisers. However, in other embodiments the glucose-depleted dairy milk of the present invention does not contain the above classes of additives in quantities greater than those found naturally in the unmodified dairy milk. ln preferred embodiments the glucose-depleted dairy milk of the present invention contains only the sodium present naturally in the unmodified dairy milk. Expressed numerically, in preferred embodiments the glucose-depleted dairy milk of the present in contains Na ⁺ at a mass concentration which, when said dairy milk is adjusted in volume with water to give a gluconic acid concentration of about 0.1 g/l, or the specific concentrations disclosed above, gives a mass concentration of Na ⁺ of no more than about 0.6 g/l, e.g. no more than about 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25 or 0.20 g/l.

The glucose-depleted dairy milk of the present invention may be a mixture of milk from different mammals. Said dairy milks may be blended once each has been rendered glucose-depleted or may be blended in their natural sugar-complete state and subsequently rendered glucose-depleted.

Preferably the glucose-depleted dairy milk of the present invention is a dairy milk of, or a mixture of dairy milks from, a mammal selected from cow, goat, sheep, buffalo, bison, yak, and camel.

As mentioned above, the glucose-depleted dairy milk of the present invention may be prepared through one or more enzyme treatments of liquid dairy milk under pH controlled conditions. Specifically, a treatment that converts glucose into gluconic acid via the action of a glucose oxidase in the presence of catalase with pH control using, at least in part, an oxide or hydroxide of one or more, preferably any two or three, of calcium, magnesium or potassium, and optionally a pretreatment with an enzyme which hydrolyses lactose to glucose and galactose (e.g. lactase).

The dairy milk undergoing the treatments of the invention may be in concentrated form or diluted form. In other embodiments the glucose-depleted dairy milk that has been prepared in accordance with the invention may undergo concentration or dilution. In still further embodiments, concentration and/or dilution steps may interspace the treatment steps of the invention.

Thus, in another aspect of the invention, there is provided a method for the preparation of a glucose-depleted liquid dairy milk, wherein said milk has a pH of about 4.5 to about 7.0 and comprises gluconic acid, said method comprising

(i) providing a liquid dairy milk containing free glucose and/or lactose and:

(a) contacting said milk with an enzyme which hydrolyses lactose to glucose and galactose, and (b) contacting the enzyme treated milk of step (a) with a glucose oxidase and a catalase,

wherein steps (a) and (b) may be performed simultaneously; or

(ii) providing a liquid dairy milk containing free glucose, optionally

wherein said milk is lactose-depleted, and:

(c) contacting said milk with a glucose oxidase and a catalase, wherein the pH of the milk is controlled at a pH of about 4.5 to about 7.0, at least in part, by the addition of an oxide and/or hydroxide of calcium, potassium and/or magnesium.

In these embodiments the final step recited therein is sufficient to result in the formation of a glucose-depleted liquid dairy milk of the invention. However, further processing steps may be included before or following or intervening the recited steps and the final product may still be considered a glucose-depleted liquid dairy milk of the invention to the extent such products are defined herein.

Preferably in these embodiments "depleted" means substantially, e.g.

essentially, devoid as defined herein.

The various specific characteristics of the glucose-depleted liquid dairy milk of the invention recited above apply mutatis mutandis to this aspect of the invention.

Preferably the enzyme which is used to hydrolyse lactose to glucose and galactose is a lactase (also referred to as lactase-phlorizin hydrolase, or LPH) or a beta-galactosidase. Lactase or beta-galactosidase enzymes of use in the invention may be or mammalian or fungal origin, e.g. a lactase from Kluyveromyces or Aspergillus or a lactase or a beta-galactosidase from lactic acid producing bacteria, e.g. lactobacilli.

The step of contacting with the glucose oxidase is performed for a time and with an amount of enzyme that, under the physical conditions used (e.g.

temperature, pressure and oxygen concentration), are sufficient to convert a sufficient amount of free glucose in the sample to gluconic acid to render the sample free glucose-depleted. Preferably, the step of contacting with the glucose oxidase is performed for a time and with an amount of enzyme that, under the physical conditions used, are sufficient to convert substantially, e.g. essentially, all of the free glucose in the sample to gluconic acid, i.e. to render the sample substantially, e.g. essentially, devoid of free glucose. Preferably the action of the glucose oxidase results in the requisite amounts of gluconic acid. The glucose oxidase will typically be used at a concentration 300-30000 U/l, more preferably 1000-1 OOOOU/I, most preferably at about 3000 U/l, e.g. 2500-3500 U/l and should be allowed to incubate with the sample for up to 48 hours, preferably 2-48, 2-36, 2-24, 2-18, 2-12 or 2-10 hours, most preferably 3-4 hours at a temperature of 5 to 30°C, e.g. 10 to 28°C, 16 to 24°C or about room temperature (20°C). Typically, this step of the methods of the invention will be conducted at atmospheric pressure, e.g. about 70 kPa to about 105 kPa.

Commercially available glucose oxidase preparations of note include Hydrase™ and BIO-CAT™ gluocose oxidase.

A by-product of the conversion of glucose to gluconic acid by glucose oxidase is hydrogen peroxide. Accordingly, a catalase is present during the glucose oxidase treatment step and in some embodiments a glucose oxidase treated sample may be further treated with catalase or other hydrogen peroxide degrading enzyme.

The pH of the liquid dairy milk during the method, in particular the glucose oxidase and catalase treatment step(s), is controlled, at least in part, by the addition of amounts of an oxide and/or hydroxide of calcium, potassium and/or magnesium appropriate to maintain a pH of about 4.5 to about 7.0, e.g. any of the pH ranges recited above in the context of the glucose depleted liquid dairy milk of the invention, thereby minimising the curdling effects of low pH on dairy milk (milk casein coagulates at pH 4.6 or below) and to replicate the organoleptic properties of the equivalent untreated milk. In preferred embodiments, pH is controlled, at least in part, with MgO, Mg(OH ₂ ), KOH and/or Ca(OH) ₂ (e.g. in solid form, for instance as a powder, granule or pellet, or in the form of a slurry, e.g. a slurry wherein the liquid part is an aliquot of the dairy milk undergoing treatment so that dilution of the milk is avoided) may be conveniently used.

The pH of the milk may also be controlled by any other convenient means, e.g. by the use of appropriate, acids, bases and/or buffers. It may be convenient to adjust pH prior to treatment of the sample with glucose oxidase as well as during the treatment itself, in which case the pH-adjusting agent(s) may be introduced in a plurality of applications. pH may be monitored by any convenient means, e.g. pH meter

The presence of oxygen is necessary for glucose oxidase to effectively convert glucose into gluconic acid. The treatment of certain dairy milks in accordance with the invention may therefore benefit from oxygen supplementation in order to ensure optimal enzyme activity and/or conversion of sufficient amounts of glucose to gluconic acid. Accordingly, in another preferred embodiment of the present invention, oxygenation is performed at least during the glucose oxidase treatment step. The oxygen may be supplied in the form of air, but pure oxygen (0 ₂ ) is preferable since the process of enzymatic conversion of glucose to gluconic acid tends to be faster when pure oxygen is supplied. On the other hand, the lipid and protein components of dairy milk are susceptible to oxidation and the consequent formation of undesired flavours and aromas. As such, the amount of 0 ₂ supplied should be carefully controlled and/or antioxidants may be used to minimise such oxidation. Suitable antioxidants include but are not limited to lipophilic anti- oxidative substances, e.g. tocopherol, or a combination of lipophilic and hydrophilic anti-oxidants, e.g. tocopherol and ascorbic acid.

Glucose oxidases typically tolerate a pH of 3-8, although optimal performance is typically at about pH 3-6.

The step of contacting with the enzyme which hydrolyses lactose to glucose and galactose is performed for a time and with an amount of enzyme that, under the physical conditions used (e.g. pH, temperature, pressure and oxygen concentration), are sufficient to hydrolyse a sufficient amount of lactose in the sample to glucose and galactose to render the sample lactose-depleted.

Preferably, the step of contacting with the enzyme which hydrolyses lactose to glucose and galactose is performed for a time and with an amount of enzyme that, under the physical conditions used, are sufficient to hydrolyse substantially, e.g. essentially, all of the lactose in the sample to glucose and galactose, i.e. to render the sample substantially, e.g. essentially, devoid of lactose.

In embodiments in which lactase is selected as the enzyme to hydrolyse lactose to glucose and galactose, the enzyme will typically be used at a

concentration of 500-50000 U/l, preferably 1000-10000 U/l, most preferably at about 5000 U/l, e.g. 4500-5500 U/l, or 3000 U/l, e.g. 2500-3500 U/l, and the lactase should be allowed to incubate with the sample for up to 48 hours, preferably 6-48, 6-36, 6-24 or 6-20 hours, most preferably 8-12 hours at a temperature of 5 to 30°C, e.g. 10 to 28°C, 16 to 24°C or about room temperature (20°C). Typically, this step of the methods of the invention will be conducted at atmospheric pressure, e.g. about 70 kPa to about 105 kPa. Typically this step of the methods of the invention will be conducted at the pHs described above. These conditions may also be applied generally in the context of the enzyme used to hydrolyse lactose to glucose and galactose.

The composition of dairy milk (in particular the milk proteins) is such that foaming may be a processing problem. As such the invention contemplates the use of antifoaming agents in the enzyme treatment steps. Anti-foaming agents suitable for use in the invention include silicone oils (e.g. polysiloxane), diatomaceous earth, surfactants (e.g. fatty acid esters, phospholipids, e.g. those found naturally in dairy milk), polyglycols (e.g. PEG) and other agents capable of destabilising foam. In other embodiment the reaction apparatus used may comprise a device or structure to control foaming, e.g. a device which breaks down foam or prevents foam formation (e.g. an ultrasound, thermal or electrical foam breaker or a rotating liquid spray nozzle) or a device which removes foam (e.g. a foam separation unit or structure) from the reaction mixture. Suitable devices and structures are disclosed in Atri, M. R., et al., 2010, Pak. J. Biotechnol., Vol 7(1 -2), 19-39, the contents of which are incorporated herein by reference.

Other sources of Ca ²⁺ , Mg ²⁺ and/or K ⁺ which may be used to supplement the glucose-depleted dairy milk of the invention and such sources are only restricted insofar as the sources must be compatible with food products, they do not affect the activities of any enzymes used after their introduction to the dairy milk and they do not have a detrimental effect on the advantageous properties of the glucose- depleted dairy milk of the invention, i.e. its palatability, its favourable glycaemic profile and prebiotic effects. The skilled person would have no trouble in selecting appropriate sources of Ca ²⁺ , Mg ²⁺ and/or K ⁺ . By way of example, suitable sources include but are not limited to salts (e.g. halide salts, including fluoride, chloride, bromide, iodide salts; organic salts, including acetate, citrate, glutamate), peroxides, sulphates, phosphates, nitrites, nitrates, bicarbonates and carbonates. Peroxides are of note as the inventors have found that these compounds, when present in the glucose-depleted milk of the invention, do not go on to form compounds which may detrimentally affect the flavour and mouth-feel of the dairy milk, and may in fact improve such properties. For such reasons bicarbonate and carbonate salts of Ca ²⁺ , Mg ²⁺ and/or K ⁺ should be used with some care and preferably will not be used. In certain embodiments calcium carbonate in particular is not used.

In other embodiments the glucose-depleted liquid dairy milk of the invention is also depleted of, e.g. rendered essentially devoid of, free fructose. This may be conveniently achieved by incorporating a step in which the dairy milk undergoing treatment is exposed to an enzyme capable of converting free fructose into a derivative form, preferably a derivative form with a lower calorific value and/or more favourable glycaemic profile. Such enzymes may include 5-D-fructose

dehydrogenase.

Alternatively, free fructose may be enzymatically converted to free glucose, e.g. prior to or concurrently with treatment with glucose oxidase. Such enzymes may be define as glucose isomerases and include glucose-6-phosphate isomerase and D-xylose isomerase.

In a further embodiment the starting material for the above described methods is a material that is fructose-depleted, e.g. substantially or essentially devoid of fructose.

Glucose, lactose, gluconic acid and, if required, fructose may be monitored in the methods of the invention by any of the numerous routine and convenient techniques available to the skilled person. By way of example, the free glucose and the lactose concentration in dairy samples may be measured using a rapid and simple reflectometric based kit (e.g. Reflectoquant from Merck) and free fructose and gluconic acid may be determined via an appropriate enzymatic assay (e.g. as supplied by R-Biopharm) .

Any or all of the enzymes used in the methods of the invention may be used in a form immobilised in or on a solid support, preferably a particulate solid support, e.g. a magnetic particulate solid support, or the internal surfaces of the reaction vessel. In this way recovery or retention of the enzyme(s) is convenient.

Preferably, lactase, glucose oxidase and/or catalase are used in a form immobilised in or on a solid support, preferably a solid support carrying both glucose oxidase and catalase immobilised therein or thereon is employed in the methods of the invention. The particulate solid support may be formed from alginate particles, resin particles, plastic particles or particles formed from other particle forming polymers. In other embodiments the enzymes are used in polymer or resin encapsulated form.

In certain embodiments the glucose-depleted liquid dairy milk of the invention does not contain detectable amounts of an active form of one or more the above-mentioned enzymes, e.g. glucose oxidase, and/or catalase, and/or lactase. This may be achieved by mechanical removal of the enzymes, e.g. by affinity chromatography or by collecting the enzyme linked solid support or removing the reaction mixture from the solid support if such supports are used. Alternatively or additionally the glucose-depleted liquid dairy milk of the invention may undergo heat treatment to inactivate the enzyme(s). Conveniently this may take the form of a pasteurisation process. Thus, in certain embodiments, the glucose-depleted liquid dairy milk of the invention may still comprise inactivated forms of one or more of the above-mentioned enzymes, e.g. a glucose oxidase, a catalase and/or an enzyme which hydrolyses lactose to glucose and galactose (e.g. lactase).

In other embodiments the glucose-depleted dairy milk contains active forms of one or more of the above-mentioned enzymes, e.g. glucose oxidase, and/or catalase, and/or lactase.

The starting materials for the above described methods may be provided in pasteurised, heat treated or microfiltered form.

In a further aspect of the invention there is provided a glucose-depleted liquid dairy milk obtained or obtainable from the methods disclosed herein. Such products may undergo pasteurisation, heat treatment and/or microfiltration to remove microorganisms. Such products may undergo further processing, e.g. ultrafiltration or molecular size chromatography, to remove gluconic acid and other sugars. Such products may be diluted with glucose-depleted and/or lactose- depleted dairy products which do not comprise gluconic acid.

The glucose-depleted liquid dairy milk of the invention may of course also be used in the preparation of other food products, e.g. ice creams, sauces, flavoured milks, milkshakes, smoothies, cakes, spreads, confectionary, dessert products, diabetic foodstuffs, low carbohydrate and low calorie products, or dietary supplements containing dairy milk.

The presence of gluconic acid in combination with reduced amounts of bioavailable glucose means the modified dairy milk of the invention has a glycaemic response, a glycaemic index, a glycaemic load and an insulin response which are significantly lower than those of an equivalent untreated dairy milk, i.e. a more favourable glycaemic profile and lower available carbohydrate content, respectively, which in turn reduces the amount of dietary energy provided by the product, and thus renders the modified dairy milk useful as part of a healthy diet in healthy subjects, and also surprisingly effective in treating subjects with or at risk of developing complex metabolic disorders associated with the over-consumption of glucose and/or inappropriate metabolism of glucose including metabolic syndrome, diabetes, obesity, dyslipidemia, insulin resistance, hypertension and liver steatosis. The potentially superior palatability of the glucose-depleted dairy milk of invention means that the product is a viable alternative to natural dairy milks that will see enthusiastic adoption and prolonged use by consumers, resulting in the above beneficial effects in consumers and patients.

The glucose-depleted liquid dairy milk of the invention preferably results in a reduced insulin response in a subject as measured by the area under the curve (AUC) of a subject's blood insulin (preferably venous blood insulin) levels over time, preferably over about 15mins, 30mins, 45 mins, 60mins, 75mins, 90mins, 105mins, 120mins, 150mins, 180mins, 210mins or 240mins immediately following

consumption of the glucose-depleted liquid dairy milk, relative to an equivalent milk from the same mammal but which is not glucose-depleted. Preferably the AUC is calculated as the incremental AUC (iAUC), i.e. all area below the curve but above the fasting blood insulin concentration. Preferably the AUC, e.g. the iAUC, is calculated over about 120mins. The insulin response of each product should be determined in the same way.

The glucose-depleted liquid dairy milk of the invention preferably results in an area under the blood insulin curve as defined above in response to its consumption that is no more than 75%, e.g. no more than about 70, 65, 60, 55, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 5 or 1 % of that of an equivalent milk from the same mammal but which is not glucose- depleted.

The glucose-depleted dairy milk of the invention preferably results in enhanced postprandial insulin sensitivity, e.g. as measured by ISI according to Belifore (ISI = 2/[AUC insulin x AUC glucose + 1]) relative to an equivalent milk from the same mammal but which is not glucose-depleted. The glucose-depleted dairy milk of the invention preferably results in postprandial insulin sensitivity that is at least 2 times, e.g. 3, 4, 5, 6, 7, 8, 9 or 10 times greater than that of an equivalent milk from the same mammal but which is not glucose-depleted.

Thus, in a further aspect the invention provides a method of assisting in maintaining the health and well-being of a subject or for maintaining or promoting health and well-being in a subject, said method comprising consuming a glucose- depleted liquid dairy milk of the invention. The use of the glucose-depleted liquid dairy milk of the invention in such methods is contemplated as is the use of the glucose-depleted liquid dairy milk of the invention in the manufacture of a nutraceutical or food-substitute for use in such methods. Complex metabolic conditions associated with the over-consumption of glucose and/or inappropriate metabolism of glucose, e.g. metabolic syndrome, diabetes mellitus type II, obesity, dyslipidemia, insulin resistance, hypertension and liver steatosis are well known, however successful treatment or prevention thereof has remained elusive. It has now surprisingly been found that simply reducing a subject's dietary intake of glucose does not effectively treat these conditions but that a more fruitful approach is to provide these subjects with a food-substitute having a more favourable glycaemic profile and/or insulin response and/or the ability to increase insulin sensitivity, specifically a glucose-depleted liquid dairy milk containing gluconic acid in amounts corresponding to amounts of bioavailable glucose removed from the milk, i.e. a glucose-depleted liquid dairy milk of the invention.

Thus, in a further aspect the invention provides a method for the treatment or prevention of a disease or condition associated with the over-consumption of glucose and/or inappropriate metabolism of glucose, said method comprising administering a glucose-depleted liquid dairy milk of the invention to a subject on a calorie-controlled diet.

Expressed differently, the invention provides a glucose-depleted liquid dairy milk of the invention for use in the treatment or prevention of a disease or condition associated with the over-consumption of glucose and/or inappropriate metabolism of glucose in a subject on a calorie-controlled diet.

Expressed differently, the invention provides for the use of a glucose- depleted liquid dairy milk of the invention in the manufacture of a medicinal product for use in the treatment or prevention of a disease or condition associated with the over-consumption of glucose and/or inappropriate metabolism of glucose in a subject on a calorie-controlled diet.

A calorie-controlled diet is a diet which permits a subject to consume a defined number of calories per day, typically this will be a calorie-restricted diet that permits the subject to consume a number of calories per day that is fewer than the number the subject consumed before adopting the diet. This may be fewer than the number of calories recommended by the skilled practitioner for the average subject or a subject of equivalent body proportions. Preferably the diet will be sugar- controlled/sugar-restricted, in particular will be glucose-controlled/restricted, which terms should be interpreted as for calorie-controlled and calorie restricted. The disease or condition associated with the over-consumption of glucose and/or inappropriate metabolism of glucose may be selected from metabolic syndrome, diabetes mellitus type II, obesity, abdominal obesity, dyslipidaemia, insulin resistance, hyperinsulinemia, impaired glucose metabolism, hypertension, liver steatosis, steatohepatitis, hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDL levels, pancreatitis, neurodegenerative disease, retinopathy, nephropathy and neuropathy. Obesity, abdominal obesity, dyslipidaemia, insulin resistance, hyperinsulinemia, impaired glucose metabolism, hypertension, hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDL levels, neurodegenerative disease, retinopathy, nephropathy and neuropathy are of note.

Acidification of the intestinal milieu by gluconic acid and complex formation between gluconic acid and minerals such as calcium, magnesium, potassium, selenium, zinc and iron increases their solubility and bioavailability. This leads to increased absorption and retention of these minerals and consequently the contribution of these minerals to the on-going health and well-being of a subject is maximised. By way of example, calcium plays a role in blood coagulation, energy- yielding metabolism, muscle function, neurotransmission, digestive enzyme function, cell division and differentiation, development and maintenance of bones and teeth; potassium plays a role in muscular and neurological function and blood pressure; magnesium plays a role in the reduction of tiredness and fatigue, electrolyte balance, energy-yielding metabolism, neurotransmission, muscle contraction, protein synthesis, psychological function, maintenance of bones and teeth, cell division; selenium plays a role in spermatogenesis, maintenance of hair and nails, immune system function, thyroid function, protection of DNA, proteins and lipids from oxidative damage; zinc plays a role in DNA synthesis and cell division, carbohydrate and macronutrient metabolism, cognitive function, fertility and reproduction, maintenance of serum testosterone concentrations, vitamin A metabolism, protein synthesis, maintenance of bones, hair, nails and skin, immune system function, protection of DNA, proteins and lipids from oxidative damage, DNA synthesis and cell division, and iron plays a role in cognitive function, energy- yielding metabolism, formation of red blood cells and haemoglobin, oxygen transport, immune system function, reduction of tiredness and fatigue, cell division and cognitive development of children. The specific levels of Ca ²⁺ , K ⁺ and Mg ²⁺ in the glucose-depleted liquid dairy milk of the invention may further enhance these effects. ln particular acidification of the intestinal milieu by gluconic acid and complex formation between gluconic acid and minerals such as calcium, magnesium, potassium, selenium, zinc and iron leads to mineralisation of bone and reduction of blood pressure.

Thus, in a further aspect the invention provides a method for increasing the absorption and retention of dietary minerals or the mineralisation of bone, said method comprising administering a glucose-depleted liquid dairy milk of the invention to a subject.

Expressed differently, the invention provides a glucose-depleted liquid dairy milk of the invention for use in increasing the absorption and retention of dietary minerals or the mineralisation of bone in a subject.

Expressed differently, the invention provides for the use of a glucose- depleted liquid dairy milk of the invention in the manufacture of a medicinal product for use in increasing the absorption and retention of dietary minerals or the mineralisation of bone in a subject.

In turn the invention provides a method for treating perturbations, caused by insufficient absorbance or retention of dietary minerals, in blood coagulation, energy-yielding metabolism, muscle function, neurotransmission, digestive enzyme function, cell division and differentiation, development and maintenance of bones and teeth, blood pressure, the reduction of tiredness and fatigue, electrolyte balance, protein synthesis, psychological function, spermatogenesis, maintenance of hair and nails, immune system function, thyroid function, protection of DNA, proteins and lipids from oxidative damage, DNA synthesis, carbohydrate and macronutrient metabolism, cognitive function, fertility and reproduction,

maintenance of serum testosterone concentrations, vitamin A metabolism, formation of red blood cells and haemoglobin, oxygen transport, and cognitive development of children. In particular, increased mineralisation of bone will lead to the effective treatment and prevention of bone loss disorders including osteoporosis and arthritis.

Osmotic effects by non-absorbed gluconate as well as short chain fatty acids released from gluconate utilizing intestinal microorganisms accelerate gastrointestinal transit, soften stools and increase faecal volume. Hence conversion from sugars to gluconate provides foods with effects against constipation. The glucose-depleted liquid dairy milk of the invention may be used to treat constipation and slow Gl transit. "Treatment" when used in relation to the treatment of a medical condition in a subject in accordance with the invention is used broadly herein to include any therapeutic effect, i.e. any beneficial effect on the condition or in relation to the condition. Thus, not only included is eradication or elimination of the condition, or cure of the subject, but also any improvement in the condition. Thus included for example, is an improvement in any symptom or sign of the condition, or in any clinically accepted indicator of the condition (for example, an improvement in the metabolism of glucose). Treatment thus includes both curative and palliative therapy, e.g. of a pre-existing or diagnosed condition, i.e. a reactionary treatment.

"Prevention" as used herein refers to any prophylactic or preventative effect.

It thus includes delaying, limiting, reducing or preventing the condition or the onset of the condition, or one or more symptoms or indications thereof, for example relative to the condition or symptom or indication prior to the prophylactic treatment. Prophylaxis thus explicitly includes both absolute prevention of occurrence or development of the condition, or symptom or indication thereof, and any delay in the onset or development of the condition or symptom or indication, or reduction or limitation on the development or progression of the condition or symptom or indication.

Preferably the subject is a human, especially a human suffering from or at risk of developing a disease or conditions recited herein, in other words a human subject in need of the treatments disclosed herein.

The invention will be further described with reference to the following non- limiting Examples in which: Figure 1 shows measurements of glucose (diamonds) and gluconic acid

(squares) concentrations (mM) and input of 0 ₂ (litres) (circles) over the course of the reaction described in Example 1. EXAMPLES

Example 1 - Preparation of a glucose-depleted lactase-treated liquid dairy milk product

An example of a preferred process and glucose-depleted liquid dairy milk according to the present invention is exemplified by the following preparation of a glucose- depleted lactase-treated liquid cow's milk product.

Methods

80 liter of lactase treated semi-skimmed milk (fat content = 1.2%, pH 6.8) with a glucose content of ca. 28 g/l was used as the starting ingredient. To this was added 4 ml (50 ppm) of Foam-Clear EscaPro (KCC Basildon, UK) siloxane based antifoaming agent. The oxygen concentration was set at 5 mg/l and the pump speed was set at 15% (1.6 l/s). 13.3 g Hydrase (3000U/I based on glucose oxidase activity) was then added to the milk by first pre-blending it in an aliquot of milk. pH, temp 0 ₂ concentration and 0 ₂ flow from the input 0 ₂ source were monitored in real time over the course of the reaction (approximately 4 hrs). The pH of the reaction mixture was maintained at 5-6 following the initial drop from pH 6.8 upon formation of gluconic acid with batch additions of potassium hydroxide (total 250g added) and calcium hydroxide (total 100 g added). Samples were removed for analysis of glucose at-line (Reflectoquant, Merck) and for subsequent determinations of carbohydrates glucose, galactose, lactose, sucrose and gluconic acid. Gluconic acid was analysed via enzymatic assay (r-biopharm). Analysis of all other sugars was done using an HPLC with pulsed amperometric detection using a Thermo CarboPac PA-1 analytical column with alkaline eluent. Figure 1 shows measurements of glucose and gluconic acid concentrations and input of 0 ₂ (litres) over the course of the reaction described in Example 1.

Results

The final milk product was confirmed as palatable and acceptably similar to the flavour and aroma of unmodified (lactose-complete) semi-skimmed milk. The content of carbohydrates in the final milk product was as follows:

Glucose: <1 g/l

Galactose: 29 g/l

Sucrose: 0.5 g/l

Lactose: 2 g/l

Gluconic acid: 30 g/l

Final pH = 5