阅读说明：本技术 塔格糖和半乳糖糖浆 (Tagatose and galactose syrup ) 是由 贾瓦尼·斯波纳蒂 卢瓦纳·瓦尼奥利 雅格布·奇尼 西尔维亚·柏尔吉奥里尼 于 2019-10-10 设计创作，主要内容包括：本发明描述了作为主要组分的塔格糖和半乳糖连同少量的其它次要产物诸如甘油、低聚糖和其它糖的糖浆。(The present invention describes a syrup of tagatose and galactose as main components together with small amounts of other minor products such as glycerol, oligosaccharides and other sugars.)

1. a composition, comprising:

wherein the tagatose/galactose ratio is equal to 1.0-1.6, wherein% is by weight of the dry composition, said composition being in the form of a syrup having a concentration in the sugar amount of 58 ° -62 ° brix.

2. The composition according to claim 1, wherein the tagatose/galactose ratio is equal to 1.1-1.5.

3. The composition of any one of claims 1-2, having a brix concentration of 59 ° -61 ° brix.

4. The composition of any one of claims 1-3, having a pH of 3.0-3.5.

5. The composition according to any one of claims 1-4, comprising or consisting of:

6. a process for preparing the syrup of any one of claims 1-5, the process comprising:

i. subjecting lactose to enzymatic hydrolysis by lactase to obtain a mixture comprising glucose and galactose;

contacting the mixture comprising glucose and galactose with at least one food yeast to perform deglucosation and obtain a deglucosated mixture;

subjecting the deglucosated mixture to basic epimerization to perform an epimerization of galactose to tagatose and obtain an epimerized mixture;

contacting the epimerised mixture with at least one ion exchange resin to effect deionization and obtain a deionized mixture;

optionally subjecting the deionized mixture to nanofiltration to obtain a nanofiltration-filtered mixture;

optionally subjecting the deionized mixture, or optionally the nanofiltered mixture, to reverse osmosis to obtain a permeate retentate;

subjecting the deionized mixture, or optionally the nanofiltration mixture, or optionally the permeate retentate, to ceramic ultrafiltration to obtain an ultrafiltered mixture;

bringing the ultrafiltered mixture to a concentration of up to 58 ° -62 ° brix to obtain a syrup according to any one of claims 1-5.

7. The process according to claim 6, wherein the starting material is lactose monohydrate in crystalline form.

8. The process according to any one of claims 6-7, wherein the enzymatic hydrolysis (i) reaction of lactose is carried out at a temperature comprised between 5 ℃ and 60 ℃ and at a pH of 4.0-9.0, for a time comprised between 1 hour and 48 hours, preferably 20 hours, keeping the lactose solution recirculated on a column comprising immobilized lactase from Kluyveromyces lactis (K.Lactis), Kluyveromyces fragilis (K.fragilis), Aspergillus oryzae (A.oryzae), Aspergillus niger (A.niger), Escherichia coli (E.coli), Bacillus stearothermophilus (B.stearothermophilus) or Bacillus circulans (B.circulans).

9. Process according to any one of claims 6-8, wherein the de-saccharification is carried out under blowing air, kept at a temperature comprised between 25 ℃ and 40 ℃ at pH 4.0-9.0 under stirring for a period of at least 4 hours.

10. The process according to any one of claims 6-9, wherein the epimerization (iii) occurs by adding a calcium hydroxide molar ratio of 0.1-1.0 relative to the number of moles of galactose; said epimerisation (iii) is carried out at a temperature ranging from 0 ℃ to 30 ℃ with stirring for a time of at least 10 minutes, preferably 4 hours; wherein at the end of epimerization reaction (iii), the epimerization reaction (iii) is neutralized by adding 30% -50% sulfuric acid in water; after neutralization with the acid, the suspension is centrifuged to separate precipitated calcium sulfate and residual yeast from the deglucosation step.

11. Use of the composition according to any one of claims 1-5 in the preparation of functional foods, medical foods, sports drinks, fruit juices, yoghurts, food supplements and in the confectionery industry.

Technical Field

The present invention relates to the field of sweeteners and prebiotics.

Background

Tagatose is a hexose ketonic monosaccharide, an isomer of fructose. Tagatose is a rare sugar, and if a dairy product is subjected to heating, a small amount of tagatose can be found in the dairy product.

Despite having a sweetening power equal to 92% with respect to sucrose, tagatose offers a reduced caloric intake (38%), which is not cariogenic, and therefore it also finds application as a sweetener replacing ordinary table sugar (table sugar) in the preparation of baked products for the confectionery industry.

Tagatose has an antihyperglycemic effect because it achieves control of postprandial blood glucose levels by increasing the activity of glucokinase responsible for the transfer of glucose in glycogen. It also has an inhibitory effect on some enzymes in the intestinal tract involved in the degradation of carbohydrates, leading to their reduced absorption.

Studies have been conducted on the Effect of the reduction in Glycemic index (Glycemic index) caused by the intake of Tagatose (Mark Ensor et al, "Effect of Three Low-Doses of D-Tagatose on Glycemic control Over Six nutrients in Subjects with Milld Type 2Diabetes with Diet and excipient" J endocrine Diabetes Obes 2014.10 Months; 2(4): 1057).

Tagatose is therefore useful in the treatment of type 2diabetes, which has been clinically studied (clinicanttrials. gov, NCT00955747, first release: 8 months 10 days 2009).

However, the use of tagatose in crystalline form as a sweetener in foods, sports drinks, etc. and as a prebiotic is limited by the cost of the product, which is usually not very competitive with respect to other synthetic or extracted molecules.

Galactose is a simple sugar and is an epimer of glucose. It is produced in small amounts in humans, while most of it is introduced with the diet mainly by ingestion of milk and dairy products containing lactose disaccharide which is broken down into glucose and galactose by lactase.

Lactose is the most sugar present in the feeding of infants, who need to have available an efficient energy source as a growing organism, and furthermore, there is experimental evidence that Galactose produced by lactose is involved in the process of Myelin formation (Ravera S, Bartolucci M, Cazia D, Morelli a, Panfoli I, "Galactose and Hexose 6-posphatate Dehydrogenase supply the Myelin Metabolic Role", PARIPEX indiana journal of research 2015,4(9), pages 21-24).

With regard to The positive effect of galactose on The central nervous system for The treatment of degenerative diseases such as, for example, Alzheimer's disease, various studies have been carried out ("Therapeutic effect of oral interstitial treatment in rate model of systemic Alzheimer's" Alzheimer's & Dementia-The Journal of The Alzheimer's Association disease, 7 months 2014, volume 10, phase 4, supplementary page P464).

There is experimental evidence for the following: galactose and antioxidant intake may also be used to treat multiple sclerosis, especially in the early stages of the onset of the disease (Isabella Pandolfi, et al, "Missed evolution of demyelinating therapy with natural compounds: A case report", Medical Research instruments, Vol. 4, No. 1, 2016.4 months).

Clinical studies are underway for the use of galactose as a food supplement in the treatment of congenital glycosylation disorders (clinican trials. gov, NCT02955264, first release: 2016, 11, 4) and in the treatment of type 2diabetes (clinican trials. gov, NCT01776099, first release: 2013, 1, 25).

Many beneficial properties of oligosaccharides for health, such as galacto-oligosaccharides (galcto-oligosaccharides), are formed due to the action of lactase (β -galactosidase), which, in addition to having hydrolytic activity towards lactose, also has a synthetic effect of adding galactose units in variable amounts to lactose.

Galactooligosaccharides have a beneficial effect in promoting the growth of microorganisms in the intestinal tract, mainly bifidobacteria, and according to several studies they may inhibit the growth of potentially pathogenic microorganisms (Daniele Garrido et al, "inactivation of microbial microorganisms by Bifidobacterium subsp. Infantis isolates", Food Microbiol.2013, 33(2) 4/262-.

The invention aims to provide tagatose and galactose-based syrup and a preparation method thereof.

Summary of The Invention

The present invention solves the aforementioned problems by means of a composition comprising:

wherein the tagatose/galactose ratio is equal to 1.0-1.6, wherein% is by weight of the dry composition, said composition being in the form of a syrup having a concentration of sugars (saccharometric concentration) in the range of 58 ° to 62 ° Brix (brix).

Therefore, the object of the present invention is a syrup of tagatose and galactose as main components together with small amounts of other minor products such as glycerol, oligosaccharides and other sugars. The composition of the invention makes it possible to avoid undergoing crystallization of tagatose, which inevitably results in loss of product in the crystallization mother liquor, and allows to shorten the production time and to increase the productivity, benefiting the final cost.

The advantages of the compositions in the form of syrups are therefore evident, it being stressed however that one problem with sugar-containing syrups is that, depending on the purity and on the storage conditions, they tend to crystallize, but in the case of the compositions of the invention it has surprisingly been found that a ratio between tagatose and galactose equal to 1.0 to 1.6 prevents this from occurring, which has undoubted advantages from a commercial point of view and from the use of the product.

However, the composition of the invention, in addition to providing an intake of tagatose, allows other substances to be introduced into the diet, such as galactose and galactooligosaccharides, which, for the reasons mentioned, may act synergistically, which also provides a positive contribution to health. Due to the beneficial effects of tagatose and galactose, the syrup of the present invention can be used for preparing functional foods, medical foods, sports drinks, juices, yogurts, food supplements, and in the candy industry.

Moreover, the object of the present invention is a process for preparing the aforesaid syrup, comprising:

i. subjecting lactose to enzymatic hydrolysis by lactase to obtain a mixture comprising glucose and galactose;

contacting the mixture comprising glucose and galactose with at least one food yeast to perform deglucosation (deglucosation) and obtain a deglucosated mixture;

subjecting the deglucosated mixture to basic epimerization to perform an epimerization of galactose to tagatose and obtain an epimerized mixture;

contacting the epimerised mixture with at least one ion exchange resin to effect deionization and obtain a deionized mixture;

optionally subjecting the deionized mixture to nanofiltration to obtain a nanofiltration-filtered mixture;

optionally subjecting the deionized mixture, or optionally the nanofiltered mixture, to reverse osmosis to obtain a permeate retentate;

subjecting the deionized mixture, or optionally the nanofiltration mixture, or optionally the permeate retentate, to ceramic ultrafiltration to obtain an ultrafiltered mixture;

bringing the ultrafiltered mixture to a concentration of up to 58 ° -62 ° brix to obtain a syrup.

Brief Description of Drawings

Figure 1-example of HPLC chromatogram of a composition according to the invention on a sulfonic acid column (sulfonic column).

Figure 2-example of HPLC chromatogram of a composition according to the invention on an amine column.

Detailed Description

The syrup of the invention preferably has a tagatose/galactose ratio equal to 1.1-1.5, more preferably 1.2-1.4.

The syrup of the present invention preferably has a brix concentration of 59 ° to 61 ° brix.

The syrup of the present invention preferably has a pH of 3.0-3.5.

Preferably, the composition of the invention comprises:

according to the process of the present invention, the starting material is preferably lactose monohydrate in crystalline form. Alternatively, other sources of lactose may also be used, such as, for example, whey.

Enzymatic hydrolysis of lactose (i) is performed using commercial lactases from various sources; for example and preferably: lactase derived from kluyveromyces lactis (k.lactus), kluyveromyces fragilis (k.fragilis), aspergillus oryzae (a.oryzae), aspergillus niger (a.niger), escherichia coli (e.coli), bacillus stearothermophilus (b.stearothermophilus), bacillus circulans (b.circulans), more preferably an enzyme derived from aspergillus oryzae is used in the present invention.

Lactase can be used both in free form and in immobilized form on various types of solid supports, for example and preferably immobilized on synthetic resins, alginate beads, synthetic membranes or cotton fibers, preferably immobilized enzymes are used in the present invention on polystyrene synthetic resins, more preferably immobilized enzymes as described in patent application WO 2014006606.

The enzymatic hydrolysis (i) of lactose is carried out at a temperature comprised between 5 ℃ and 60 ℃, preferably at 52 ℃ and at a pH comprised between 4.0 and 9.0, preferably between 5.1 and 5.5, keeping the lactose solution recirculating on the column for a time comprised between 1 hour and 48 hours, preferably 20 hours.

According to the invention, the solution comprising lactose hydrolyzed to glucose and galactose is subjected to a deglucosation step (ii) by adding edible yeast, preferably lyophilized brewer's yeast (Scerevisiae), until a glucose concentration of 0.25% or less is obtained. The deglucosation is preferably carried out by keeping under stirring at a pH comprised between 25 ℃ and 40 ℃, preferably between 30 ℃ and 37 ℃, more preferably at 35 ℃ for a period of at least 4 hours, under blowing air, at a pH comprised between 4.0 and 9.0, preferably between 6.0 and 7.0.

Galactose present in the deglycated solution is converted to tagatose by epimerization (iii) via the addition of an alkaline substance, such as and preferably sodium hydroxide, potassium hydroxide or calcium hydroxide, more preferably calcium hydroxide is used according to the invention. The alkaline substance is preferably used in a molar ratio of 0.1 to 1.0, preferably 0.4 to 0.8, more preferably 0.6, moles per mole of galactose alkaline substance. The epimerization (iii) is preferably carried out at a temperature ranging from 0 ℃ to 30 ℃, preferably from 5 ℃ to 25 ℃, more preferably from 10 ℃ to 20 ℃, with stirring, for a time of at least 10 minutes, preferably 4 hours.

The epimerisation reaction (iii) is neutralized at the end by adding an acid preferably selected from the group consisting of hydrochloric acid, phosphoric acid, sulfuric acid, more preferably using 30% -50% sulfuric acid in water. After neutralization with acid, the suspension is preferably centrifuged to separate precipitated calcium sulfate and residual yeast from the deglucosation step.

The solution obtained after neutralization and centrifugation is deionized by passage over a pair of ion exchange resins, preferably strong cationic resins (such as, for example, Rohm and Haas Amberlite)^TM.200、Rohm and Haas Amberlite^TM.IR120、Rohm and Haas Amberlite^TM.FPC 23 and Dowex^TM Monosphere^TM88) Followed by a weakly anionic resin (such as, for example:Amberlite^TM FPA 55、Dowex^TM Monosphere^TM 66、Rohm and Haas Amberlite^TM.IRA 96、A120S andA109)。

the deionized mixture may then optionally but preferably be subjected to nanofiltration (v) in order to at least partially remove oligomeric components from the above dimers. The nanofiltration is preferably performed on spiral wound membranes (spiral wound membranes) selected for example from the group consisting of Dow Filmtec^TMNF270-4040, Koch Membrane System TFC-SR2 or the like recovering a permeate which is then optionally and preferably for at least partial removal of glycerol, subjected to a reverse osmosis treatment (vi) on a reverse osmosis Membrane, for example and preferably selected from the group consisting of Es^TMBW 30-4040.

According to the invention, the retentate obtained in the reverse osmosis step (vi) is clarified by ultrafiltration (vii) on a ceramic membrane, preferably with a cutoff of 300000Da, and the permeate is recovered and subjected to concentration (viii). After correcting the pH, preferably by adding an organic acid, to a value comprised between 3.0 and 3.5, the syrup is concentrated until a syrup having a saccharimetric concentration of 60 ± 2 ° brix is obtained. The organic acid is preferably selected from the group consisting of citric acid, lactic acid, acetic acid. Preferably, the organic acid used to correct the pH is citric acid, more preferably an aqueous solution of 40-60% by weight citric acid.

The invention will be better understood from the following examples of embodiments.

Experimental part

HPLC method:

perkin Elmer 200 series chromatograph with refractive index detector with thermostatic cell.

Analysis on sulfonic acid column: a Transgenomic ICE-SEP ION 300 column with a front column. The temperature is 45 ℃, the flow rate is 0.4ml/min, and the eluent is 0.015N sulfuric acid.

Analysis on amine column: thermo scientific Hypersil APS-2. The temperature was 40 ℃, the flow rate was 1.1ml/min, and the mobile phase was acetonitrile + sodium dihydrogen phosphate dihydrate 1.45 g/l.

Example 1: enzymatic hydrolysis of lactose and obtainment of a deglucosated solution on an industrial scale.

a) Preparation of immobilized enzyme on synthetic resin:

in a 10m chamber equipped with a thermostat (thermostat)³A steel jacketed reactor was charged with 750 liters of Purolite A120S resin. The resin was washed separately with three 750 liter aliquots of drinking water. 480 liters of 100mM sodium acetate solution at pH 5 and 55kg of 50% glutaraldehyde solution are added. The whole was kept under stirring at 25 ℃ for 30 hours, after which the resin was washed separately with three 1000 l aliquots of drinking water. 2000 liters of 100mM sodium acetate solution at pH 5 and 30kg of lactase from Aspergillus oryzae were added. The whole was kept at 25 ℃ for 65 hours with stirring. After this time had elapsed, the resin was washed separately with three 2000 l aliquots of drinking water.

b) Enzymatic hydrolysis of lactose:

2000kg of lactose monohydrate in crystalline form were dissolved at 10m³8000 litres of drinking water in a steel reactorThe steel reactor was equipped with stirring and a constant temperature jacket. The internal temperature of the reactor was brought to 53 ℃ and the pH was brought to 5.39 by adding 38% sulfuric acid.

The lactose solution was recirculated through a column containing 600 liters of resin (in which the lactase was immobilized as described in example 1a above) at a flow rate of 2400 liters/hour for 20 hours.

Analysis on sulfonic acid column:

galactose (HPLC)	9.023％
		Glucose (HPLC)	9.228％
Lactose (HPLC)	0.492％
		Glucose/galactose	1.02

After this time had elapsed, the solution containing glucose and galactose was transferred to 20m³In a steel jacketed reactor provided with a stirring and air blowing system. The temperature was brought to 35 ℃ and 12kg of lyophilized brewer's yeast and 100ml of antifoam (Silifood 1600) were added to the solution.

The whole was kept under agitation at 35 ± 2 ℃ for 10 hours under aeration. After this time had elapsed, the pH was returned to 6.8 by the addition of 9 liters of 30% sodium hydroxide. At the end of the pH correction, 10kg of freeze-dried brewer's yeast were introduced into the reactor and the fermentation was maintained under stirring with air blowing for a further 10 hours. After this time had elapsed, the pH was adjusted to 6.6 by adding 15 liters of 30% sodium hydroxide and 10kg of lyophilized Saccharomyces cerevisiae was introduced. After a further 15 hours had elapsed, the deglycated solution obtained was cooled to about 5 ℃.

Analysis on sulfonic acid column:

galactose (HPLC)	8.058％
		Glucose (HPLC)	0.013％
Lactose (HPLC)	0.491％
		Glucose/galactose	0.2

Example 2: starting from the deglycated solution of example 1, a laboratory scale epimerization was carried out at 40 ℃ with 50% moles of calcium hydroxide relative to the moles of galactose.

250g of the deglycated galactose solution prepared according to example 1 were introduced into a1 liter glass reactor thermostatted and provided with a stirring bar and combined with 4.16g of calcium hydroxide (ventilated lime), the whole being maintained at a temperature of 40 ℃ with stirring.

Samples were collected for HPLC analysis on a sulfonic acid column after the following times: 120min, 240min and 360 min.

The results are shown in the following table:

example 3: starting from the deglycated solution of example 1, a laboratory scale epimerization was carried out at 40 ℃ with 60% moles of calcium hydroxide relative to the moles of galactose.

250g of the deglycated galactose solution prepared according to example 1 were introduced into a1 liter glass reactor thermostatted and provided with a stirring bar and combined with 5.0g of calcium hydroxide (aerated lime), the whole being kept at a temperature of 40 ℃ with stirring.

Samples were collected for HPLC analysis on a sulfonic acid column after the following times: 120min, 240min and 360 min.

The results are shown in the following table:

example 4: starting from the deglycated solution of example 1, a laboratory scale epimerization was carried out at 30 ℃ with 50% moles of calcium hydroxide relative to the moles of galactose.

250g of the deglycated galactose solution prepared according to example 1 were introduced into a1 liter glass reactor thermostatted and provided with a stirring bar and combined with 4.16g of calcium hydroxide (aerated lime), the whole being kept at a temperature of 30 ℃ with stirring.

Samples were collected for HPLC analysis on a sulfonic acid column after the following times: 120min, 280min and 350 min.

The results are shown in the following table:

example 5: starting from the deglycated solution of example 1, a laboratory scale epimerization was carried out at 30 ℃ with 60% moles of calcium hydroxide relative to the moles of galactose.

250g of the deglycated galactose solution prepared according to example 1 were introduced into a1 liter glass reactor thermostatted and provided with a stirring bar and combined with 5.0g of calcium hydroxide (aerated lime), the whole being kept at a temperature of 30 ℃ with stirring.

Samples were collected for HPLC analysis on a sulfonic acid column after the following times: 120min, 280min and 350 min.

The results are shown in the following table:

example 6: starting from the deglycated solution of example 1, a laboratory scale epimerization was carried out at 25 ℃ with 50% moles of calcium hydroxide relative to the moles of galactose.

250g of the deglycated galactose solution prepared according to example 1 were introduced into a1 liter glass reactor thermostatted and provided with a stirring bar and combined with 4.16g of calcium hydroxide (ventilated lime), the whole being kept at a temperature of 25 ℃ with stirring.

Samples were collected for HPLC analysis on a sulfonic acid column after the following times: 120min, 280min, 350min, 470min, 590min, 710min

The results are shown in the following table:

example 7: starting from the deglycated solution of example 1, a laboratory scale epimerization was carried out at 25 ℃ with 60% moles of calcium hydroxide relative to the moles of galactose.

250g of the deglycated galactose solution prepared according to example 1 were introduced into a1 liter glass reactor thermostatted and provided with a stirring bar and combined with 5.0g of calcium hydroxide (aerated lime), the whole being kept at a temperature of 25 ℃ with stirring.

Samples were collected for HPLC analysis on a sulfonic acid column after the following times: 120min, 280min, 350min, 470min, 590min and 710 min.

The results are shown in the following table:

example 8: starting from the deglycated solution of example 1, a tagatose/galactose syrup was obtained on an industrial scale.

Basic epimerization:

9100kg of the deglycated galactose solution prepared according to example 1 was introduced into a 10m apparatus provided with a stirring and thermostatic jacket³In a steel reactor. 192.4kg of calcium hydroxide (the amount of calcium hydroxide represents a molar ratio of 60% with respect to galactose) were added to a 30% drinking water suspension. After the addition, the temperature was kept at 15 ± 5 ℃ for 4 hours with stirring. After this time has elapsed, the pH of the suspension is brought to 2.5 by maintaining the temperature below 45 ℃ by adding 590 liters of 38% sulfuric acid. The temperature was then lowered to 25 ℃ and the precipitated calcium sulphate was separated by 8 centrifugations at 350rpm for 25 minutes.

Analysis on sulfonic acid column:

deionization on ion exchange resin:

the solution obtained in the previous centrifugation step was placed on a pair of ion exchange resins (4000 liters of Amberlite, a strong cationic resin)^TMFPC 23 and 4000 liter weakly anionic typesResin Amberlite^TMFPA55) was deionized at a flow rate of 2000 l/h, and the eluted product was collected from the resin until the sugar concentration was 0.5 ℃ Brix or more and the conductivity was 50. mu.s/cm or less.

And (4) nanofiltration:

then using a membrane consisting of 12 membranes: (FILMTECH^TMNF 27040/40) at a pressure of about 30 bar, the deionized solution is subjected to a nanofiltration step.

Reverse osmosis:

using a membrane consisting of 12 membranes: (FILMTECH^TMBW30-400) was subjected to a reverse osmosis step at a pressure of about 10 bar, the retentate was concentrated up to a sugar concentration of 10 ° brix.

Ceramic ultrafiltration:

the concentrated solution is clarified by a tangential ultrafiltration step (volumetric ultrafiltration step) on a ceramic membrane with a cutoff of 300000Da, with an osmotic flow of 2000 l/h and a retentate recirculation of 9000 l/h. The retentate was concentrated until about 300 liters and washed 3 times with 150 liters of drinking water, respectively.

Concentration:

the clarified permeate from the previous ultrafiltration step was transferred to a 5000 liter steel reactor equipped with stirring, thermostatic jacket and condenser.

The pH was brought to 3.0 by adding 1.6 liters of 50% citric acid in water and the solution was concentrated under vacuum at a temperature of about 50 ℃ until a saccharimetric concentration of 60 ° brix.

As a result:

the syrup obtained at the end of the process was analyzed on both a sulfonic acid column and an amine column, which was used to determine the lactose content (see also the HPLC trace (HPLC trace) in fig. 1 and 2), and the results are represented in the following table: