Abstract:
The present invention describes a process for preparing ultrapure (≧95%) galacto-oligosaccharides (GOS), starting from GOS at lower purities by using sequential microbiological purifications involving  Saccharomyces cerevisiae  and  Streptococcus thermophilus.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to the field of preparing high purity (≧95%) galacto-oligosaccharides (GOS), starting from lactose-derived syrups with low GOS content. 
       STATE OF THE ART 
       [0002]    Galacto-oligosaccharides (GOS) consist of a mixture of at least di-, tri-, tetra-, penta- and hexa-saccharides, in which glucose is the free reducing-end sugar and the other sugars in the chain are galactoses linked to each other and to glucose in varying ways depending on the enzyme used in the transglycosylation reaction starting from lactose. 
         [0003]    Currently, interest in GOS is steadily growing because recent research has demonstrated the efficiency of these oligosaccharides as prebiotics: in this sense they are a mixture of non-cariogenic non-digestible low-calorie oligosaccharides which stimulate the development of gastrointestinal microflora. 
         [0004]    A further benefit of GOS is their anti-adhesive activity: these oligosaccharides can directly inhibit infections caused by enteric pathogens such as  E. coli , acting as mimics of the binding sites of pathogens which generally attack gastrointestinal epithelial cells. 
         [0005]    Commercially available GOS are synthesized from lactose by using the transglycosylic activity of beta-galactosidases (lactases) isolated from various natural microorganisms (e.g.  Aspergillus oryzae, Penicillium expansum  and  Bacillus circulans ) or modified microorganisms. The GOS structure varies according to the enzyme source. The yield of GOS produced from natural enzymes is generally 20-45% and can be increased by employing recombinant or modified enzymes. The most widely commercially available GOS form contains the GOS at a concentration of about 50-60% by weight and also contains considerable amounts of glucose (by-product of the GOS formation reaction) and non-reacted lactose (starting material). This renders them unusable by people affected by diabetes mellitus or lactose intolerance. 
         [0006]    Methods are known in the literature for obtaining high purity GOS, namely those in which all the digestible sugars (lactose, glucose, galactose) are absent. Such methods involve the removal of glucose and lactose in GOS by chromatography, enzymatic oxidation or microbial fermentation. The aforesaid methods however lack large-scale applicability (in the case of chromatography) or present drawbacks. 
         [0007]    In Shoaf, K. et al.  Infect. Immun.  74 (12) 6920-6928, 2006, a mixture containing mono- and disaccharide-free GOS was made in the laboratory by preparative TLC (hence in quantities of a few milligrams). 
         [0008]    Splechtna, B. et al.  Enzyme and Microbial Technology  29 (6) 434-440, 2001 describes the removal of residual lactose by its selective oxidation with fungal cellobiose dehydrogenase into lactobionic acid. This enzyme is not easily available and works by coupling the lactose oxidation with reduction of 2,6-dichloro-indophenol, being present in catalytic concentrations. The oxidized redox mediator is continuously regenerated by the fungal laccase-catalyzed reduction of molecular oxygen into water. Ion exchange chromatographies were used to remove the lactobionic acid. 
         [0009]    Cheng, C.-C. et al.  Biotechnol. Lett.  28 793-797, 2006 describes the removal of residual lactose by fermentation with  Kluyveromyces marxianus : the process has good yields and leads to a high purity product devoid of glucose, galactose, lactose, but as well as lactose, all other disaccharides present in the GOS mixture prior to treatment with  K. marxianus  are also consumed by the microorganism. Li, Z. et al.  Process Biochemistry  2008, 43(8), 896-899 describes the removal of digestible sugars by fermentation with  Saccharomyces cerevisiae  or  Kluyveromyces lactis  immobilized on calcium alginate. The results are good when using  K. lactis , but unsatisfactory when using  S. cerevisiae.    
         [0010]    The drawback of using  Kluyveromyces marxianus  or  lactis  is that although both are publicly available to the public, they are not commonly employed in the food industry and are therefore not commercially available at low cost in ready-to-use form. 
         [0011]    There is an evident need for an alternative method for producing high purity GOS, which is also applicable on an industrial scale and in which all the digestible sugars (lactose, glucose, galactose) are either absent or present in small amounts, and which utilizes easily and widely available microorganisms 
       Definitions and Abbreviations 
       [0012]    GOS=Galacto-oligosaccharides 
       SUMMARY OF THE INVENTION 
       [0013]    The present invention overcomes the aforesaid problem by means of a process for preparing GOS of purity ≧95% in which the overall percentage of the digestible sugars lactose, glucose and galactose is ≦5%, starting from GOS mixtures of a lower purity, said process comprising a fermentation step with  Streptococcus thermophilus  and at least one fermentation step with  Saccharomyces cerevisiae,  where said purity is calculated by any analytical method able to distinguish and quantify GOS and said digestible sugars. 
         [0014]    The process enables said digestible sugars to be selectively removed by microbiological purification. 
         [0015]    Advantageously said process enables a GOS mixture to be obtained with low contents of the digestible sugars lactose, glucose and galactose, in which not all the disaccharides formed in the transglycosylation are removed by microorganisms. 
         [0016]    Advantageously the fermentation step with  S. thermophilus  is found to enable lactose to be selectively removed while preserving other disaccharides which have HPLC retention times very close to that of lactose. This distinctive preservation of the oligosaccharide, identifiable by HPLC with a peak immediately following that of lactose, makes the product obtained by the process different from others known in the art. 
         [0017]    The aforesaid process uses low cost commercially available microorganisms widely employed in the food industry; they can be used directly in lyophilized form (the form in which they are purchased) without having to activate them by preparing a pre-fermentation mixture. 
         [0018]    The aforesaid process can be easily applied on an industrial scale, i.e. on a multi-kg scale. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0019]      FIG. 1 —shows the HPLC trace of a starting GOS sample at 40% purity, of example 1, obtained from lactose by transglycosylation catalyzed by  B. circulans  lactase; 
           [0020]      FIG. 2 —shows the HPLC trace of a GOS sample, of example 1, after deglucosation with  Saccharomyces cerevisiae  according to the present invention; 
           [0021]      FIG. 3 —shows the HPLC trace of a GOS sample, of example 1, after delactosation with  Streptococcus thermophilus  according to the present invention; 
           [0022]      FIG. 4A —shows the HPLC trace of a GOS sample, of example 1, after degalactosation of the sample corresponding to  FIG. 3  with  Saccharomyces cerevisiae  according to the present invention after at least 20 hours of fermentation; 
           [0023]      FIG. 4B —shows the HPLC trace of a GOS sample, of example 1, after degalactosation of the sample corresponding to  FIG. 3  with  Saccharomyces cerevisiae  according to the present invention B: after at least 40 hours of fermentation; 
           [0024]      FIG. 5 —shows the HPLC trace of a GOS sample with a purity ≧95% obtained at completion, of example 1, according to the process of the present invention; 
           [0025]      FIG. 6 —shows the HPLC trace of a starting GOS sample, of example 2, namely Purimune™ (GO-P90) purchased from GTC Nutrition, Golden, Colo., USA; 
           [0026]      FIG. 7 —shows the HPLC trace of a GOS sample with a purity ≧95%, obtained at completion, of example 2, according to the process of the present invention; 
           [0027]      FIG. 8 —shows the HPLC trace of a GOS sample of example 4, obtained after fermentation with  Kluveromyces marxianus.    
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0028]    In the process of the present invention, any GOS mixture with a purity of &lt;95% can be used as the starting material, most conveniently GOS mixtures with a purity ≧40% being used in which the impurities consist essentially of the digestible sugars lactose, glucose and galactose. 
         [0029]    For the purposes of the present invention, to evaluate the purity of the GOS mixtures any HPLC method capable of distinguishing and quantifying GOS, lactose, galactose and glucose is preferably used. 
         [0030]    The term “purity” means the area percent by HPLC corresponding to the peaks of said GOS and digestible sugars. 
         [0031]    GOS mixtures at 40-60% purity (or higher) are available commercially (e.g. Oligomate 55, Vivinal GOS, Purimune, Cup oligo P) or can be prepared from lactose by transglycosylation catalyzed by a suitable lactase known for the purpose, such as that isolated from  Bacillus circulans . In particular, the process of the invention is convenient when starting from 40-60% GOS mixtures. 
         [0032]    Preferably the process of the invention is such that said fermentation step with  S. thermophilus  is followed by a fermentation step with  S. cerevisiae. S. thermophilus  hydrolyses lactose then consumes the glucose produced and accumulates galactose which is then eliminated by  S. cerevisiae . By means of the fermentation step with  S. thermophilus  the lactose content can be reduced to the desired levels, preferably &lt;5% by HPLC area percent, and more preferably &lt;3%. In case the starting GOS mixture has a glucose content &lt;5% by HPLC area percent, then said process comprises a fermentation step with  S. thermophilus  followed by a fermentation step with  S. cerevisiae. S. thermophilus  hydrolyses the lactose then consumes the small amount of glucose initially present and that which it produces during accumulation of galactose, which is then later eliminated by  S. cerevisiae.    
         [0033]    Thus, for example (see example 2), by starting from a commercially available GOS mixture at a purity of about approximately 80% in which glucose is present in a quantity of 2% by HPLC area percent, the lactose content can be reduced by direct fermentation with  S. thermophilus  to the desired levels i.e. &lt;5% by area percent relative to the sum of the area percents of the GOS. 
         [0034]    In case the GOS starting mixture has a glucose content 5% by HPLC area percent, the fermentation step with  S. thermophilus  is preceded by a fermentation step with  S. cerevisiae ; in this case the process therefore comprises the following 3 steps:
       (a) fermentation with  Saccharomyces cerevisiae;      (b) fermentation with  Streptococcus thermophilus;      (c) fermentation with  Saccaromyces cerevisiae.          
 
         [0038]    Thus, for example (see example 1), when starting from a GOS mixture with a purity of about 40% (see  FIG. 1 ) obtained by transglycosylation with  B. circulans , fermentation (a) with  S. cerevisiae  enables the glucose present in the starting GOS mixture to be almost completely removed (see  FIG. 2 .) 
         [0039]    Fermentation (b) allows lactose to be lowered to HPLC area percents of less than 3% (see  FIG. 3 ). 
         [0040]    It could be seen that when starting with GOS mixtures at 40-60% purity in which glucose is present at a purity of about 20%, if fermentation (b) with  S. thermophilus  is preceded by a deglucosation step such as fermentation (a) then the lactose can be lowered to below 3 area %, whereas direct fermentation with  S. thermophilus  even if prolonged for more than 40 hours, does not enable lactose to be lowered to the desired levels if undertaken without first effecting a deglucosation step (a) with  S. cerevisiae.    
         [0041]    Fermentation (c) with  S. cerevisiae  allows residual galactose which formed after fermentation with  S. thermophilus  to be almost completely removed (see  FIGS. 4A and 4B ). 
         [0042]    At the end of the process, the GOS mixture obtained has a purity of ≧95% in which glucose and galactose if not absent are present in negligible quantities which, together with lactose, nevertheless amount to ≦5% (see  FIG. 5 ). 
         [0043]    Fermentations with  S. cerevisiae  are preferably conducted at pH 6.5±0.5 at a temperature of 35±5° C. for at least 12 hours, using 40-15 g of dehydrated  S. cerevisiae  per kg of dry weight of starting GOS. The step (c) can even be conducted for time periods exceeding 40 hours and in such case, a substantial removal of the lactic acid, formed after fermentation (b) with  S. thermophilus , is actually achieved (see  FIG. 4B ). 
         [0044]    Fermentation with  S. thermophilus  is preferably undertaken at a temperature of 40 ±5° C., maintaining the pH at 6.0-6.5 by addition of a base, for at least 15 hours and using 1-0.4 g of dehydrated  S. thermophilus  per kg of dry weight of starting GOS. 
         [0045]    According to the process of the invention, after the fermentation steps the mixture is preferably subjected to further processing by means of ceramic ultrafiltration, nanofiltration, decolorization with carbon and ion-exchange resin deionization. 
         [0046]    According to the process of the invention, the fermentation steps (b) and (c) are preceded by pasteurization, preferably undertaken at 75±5° C. for at least 5 minutes. 
         [0047]    In effect, the product obtained at the end of the aforesaid process presents a distinctive GOS profile; during the fermentation step with  S. thermophilus , lactose is mainly removed, this being evident from the HPLC traces (compare  FIG. 2 . with  FIG. 3 , and  FIG. 6  with  FIG. 7 ) and it can also be noted that in the disaccharide region there remains a component with a retention time of about 13.6 minutes (indicated as GOS6 in the examples) immediately after the lactose retention time (about 12.9 minutes). Concomitant to the lactose removal, the disappearance of the component with a retention time of about 12.4 minutes (indicated as GOS5 in the examples) can be noted immediately preceding the lactose retention time. This preservation of the GOS profile is distinctive and is distinguishable from other GOS mixtures known in the state of the art such as those obtained by chromatographic separation or after purification with  Kluyveromyces  genus microorganisms (see example 4 and compare  FIG. 1  with  FIG. 8 ), these methods removing indiscriminately all disaccharides present in the GOS mixture. 
         [0048]    Therefore the present invention also relates to GOS mixtures obtained from the process i.e. GOS mixtures with a purity ≧95% in which galacto-disaccharides other than lactose are present in quantities ≧1% and the overall percentage of digestible sugars lactose, glucose and galactose is ≦5%. 
         [0049]    The aforesaid product, obtained by the process, can be employed for the known purposes including preparation of pharmaceutical compositions, baby formulas and food compositions, these being further aspects of the present invention. 
         [0050]    The present invention may be better understood in the light of the following working examples. 
       EXPERIMENTAL PART 
     HPLC Method 
       [0051]    This is a HPLC method using comparison with an external standard. A HPLC instrument is used with an isocratic pump, an autosampler, a Peltier oven for column temperature control, a refraction index detector and a Transgenomic column ICE-SEP ICE-ION 300, product code ICE-99-9850 equipped with an analogous precolumn. The analyses were carried out under the following working conditions: 
         [0000]    Column temperature: 40° C. 
       Injections: 20 μl 
       [0052]    Mobile phase: H 2 SO 4  0.015N
 
Flow rate: 0.4 ml/minute
 
Analysis time 36 minutes
 
       Integrator: Perkin Elmer Totalchrom Workstation 
       [0053]    Under the conditions indicated above the retention time of each product was about 12.5 minutes for lactose, 15.0 minutes for glucose, 16.2 minutes for galactose, 21.3 minutes for lactic acid and 22.2 minutes for glycerin (against the external standard). 
         [0054]    In the sample solutions, 6 peaks of GOS product were distinguished and identified as: 
         [0000]    GOS 1: peak at about 9.3 minutes
 
GOS 2: peak at about 9.7 minutes
 
GOS 3: peak at about 10.3 minutes
 
GOS 4: peak at about 11.3 minutes
 
GOS 5: peak at about 12.4 minutes
 
GOS 6: peak at about 13.6 minutes.
 
         [0055]    In the HPLC tabulation, the terms GOS1-6 simply mean the peaks attributed to GOS, irrespective of the number of saccharide units present therein. 
         [0056]    The integration system automatically calculated the contents by the formula: 
         [0000]      %= A   s   ×C   STD   ×V× 100/ A   STD   ×W   s   
         [0057]    In which: 
         [0000]    A C =Area of the peak in the sample solution
 
C STD  =Percentage sugar concentration in the standard solution
 
W s =Sample weight in grams
 
A STD =Area of the sugar peak in the standard solution
 
         [0058]    During process monitoring, the contents of the GOS peaks were not calculated against their own standards but by using the response factor of lactose or by evaluating its purity expressed as area/total area. 
         [0059]    Using the analytical method just described, the determination of lactose was invalidated by the possible overlap with other galacto-oligosaccharides. 
         [0060]    It is important to note the strong discrepancy found between the determination of lactose in commercial samples using the aforedescribed analytical method and the values provided on the certificates. 
         [0061]    For instance, the sample VIVINAL® GOS60 Batch no. 6297770 supplied by Friesland Foods Domo had a certificated lactose content on a dry matter basis of 17.5%, while by HPLC performed in our analytical laboratory the lactose content was 33.2%. 
         [0062]    A similar assessment in estimating lactose was found in the Purimune™ sample, Batch no. 20081217 in which the certificated overall purity of the GOS was 90.4%, whereas by HPLC performed in our analytical laboratory (attached) said purity was 83.0% with the purity of lactose being 13.6% by area percent. 
         [0063]    The determination of lactose was therefore overestimated by our analytical method, resulting in an exceeding assessment of the lactose concentration present in the final product. 
         [0064]    So as not to create ambiguities in the description, in the examples presented hereinafter and in the figures section, only the purity values obtained with the aforedescribed HPLC method are given, as are the values for the commercial products. 
       EXAMPLE 1 
     Example of Preparation Starting from Lactose 
       [0065]    80 kg of lactose monohydrate (0.22 kmols) were suspended in 120 l of water and heated to 70° C. under mild agitation until the lactose was completely dissolved. 
         [0066]    The solution was temperature controlled at 50° C. and the pH adjusted from 5.5 to 5.0 with 27 ml of 75% phosphoric acid. 
         [0067]    266 g of lactase from  Bacillus circulans  were added (1500 U/g). 
         [0068]    The reaction was monitored by HPLC and after 22 hours the formation of GOS and the appearance of glucose and galactose were detected with a consequent lowering of the lactose area percent purity to less than 40%. 
         [0069]    200 kg of a 40% crude GOS solution were obtained, the HPLC trace of which is shown in  FIG. 1  and presented in tabular form below: 
         [0000]                                                                                                  Peak   Delta RT   Time   Component   Conc.   Area   Response   Amount   Norm. Area       #   [%]   [min]   Name   %   [uV*sec]   Factor   [Norm. %]   [%]                                1       9.093   GOS 1   0.309071   154704   8.6372e+06   0.8   0.8       2   0.00   9.511   GOS 2   1.338322   669890   8.6372e+06   3.5   3.5       3       10.137   GOS3   4.122173   2063331   8.6372e+06   10.6   10.7       4   0.75   11.082   GOS4   8.925738   4467730   8.6372e+06   23.0   23.2       5   —   11.433       5.511e−95   31940    1.000e+100   1e−94   0.2       6       12.247   GOS5   0.414881   207666   8.6372e+06   1.1   1.1       7   0.22   12.666   Lattosio   15.240043   7628321   8.6372e+06   39.3   39.6       8       13.372   GOS6   0.471197   235855   8.6372e+06   1.2   1.2       9   0.05   15.131   Glucosio   7.519908   3601195   8.2635e+06   19.4   18.7       10   0.07   16.431   Galattosio   0.395477   190570   8.3150e+06   1.0   1.0       11   —   17.629       3.856e−96   2235    1.000e+100   1e−95   0.0       12   0.16   21.766   LCTH   0.003749   1173   5.3962e+06   0.0   0.0       13       22.328   Glicernia   0.015260   6177   6.9847e+06   0.0   0.0                       38.755819   19260786    2.000e+100   100.0   100.0                    
Step 1—Deglucosation with  S. cerevisiae  
 
         [0070]    The aforestated starting mixture was acidified to pH 3.0 with 250 ml of 75% phosphoric acid: under these pH conditions preservation at ambient temperature was possible. 
         [0071]    46 kg of 40% crude GOS solution (Batch no. 014491N9) were diluted with 140 l of water and temperature controlled at 37° C. After adjusting the solution pH from pH 2.8 to pH 7.0 with 600 ml of 24% ammonia, 200 g of lyophilized brewer&#39;s yeast were added. The deglucosation step, monitored by HPLC, was complete after 24 hours of vigorous agitation. 
         [0072]    The mixture was pasteurized at 70° C. for about 5 minutes then temperature controlled at 40° C. 
         [0073]      FIG. 2  shows the HPLC trace of the mixture after pasteurization, presented below in tabular form. 
         [0000]                                                                                                  Peak   Delta RT   Time   Component   Conc.   Area   Response   Amount   Norm. Area       #   [%]   [min]   Name   %   [uV*sec]   Factor   [Norm. %]   [%]                                1   2.62   9.275   GOS 1   0.095989   402093   8.7961e+06   1.2   1.2       2   0.16   9.694   GOS 2   0.358713   1502624   8.7961e+06   4.4   4.3       3       10.329   GOS3   1.074666   4501696   8.7961e+06   13.3   12.9       4   2.36   11.284   GOS4   2.277114   9538665   8.7961e+06   28.2   27.4       5   0.00   12.432   GOS5   0.105556   442165   8.7961e+06   1.3   1.3       6   0.31   12.882   Lattosio   3.842640   16096537   8.7961e+06   47.6   46.2       7   0.48   13.585   GOS6   0.127645   534696   8.7961e+06   1.6   1.5       8   4.14   16.059   Glucosio   0.004367   17492   8.4113e+06   0.1   0.1       9       16.697   Galattosio   0.082239   329174   8.4049e+06   1.0   0.9       10   —   17.882       3.713e−96   17680    1.000e+100   5e−95   0.1       11   —   18.925       1.129e−96   5378    1.000e+100   1e−95   0.0       12       21.431   LCTH   0.020144   51870   5.4069e+06   0.2   0.1       13       22.683   Glicernia   0.086023   280713   6.8522e+06   1.1   0.8       14   —   25.713       1.111e−95   52902    1.000e+100   1e−94   0.2       15   —   29.315       1.650e−96   7859    1.000e+100   2e−95   0.0       16   —   30.666       4.041e−96   19245    1.000e+100   5e−95   0.1       17   —   31.229       4.144e−96   19734    1.000e+100   5e−95   0.1       18   —   33.589       2.158e−94   1027592    1.000e+100   3e−93   2.9                       8.075096   34848113    7.000e+100   100.0   100.0                    
Step 2—Delactosation with  S. thermophilus  
 
         [0074]    2.5 g/l of yeast extract were added to the above mixture (resulting from step 1) and the pH was adjusted from pH 5.2 to pH 6.6 with 400 ml of 15% sodium hydroxide. The mixture was inoculated with 5 g of  Streptococcus thermophilus : fermentation proceeded at pH 6.4-6.5 by adding 15% sodium hydroxide by means of a pH-stat. The end of the reaction, monitored by HPLC, was reached after 26 hours when the lactose content in the mixture was less than 3 area %. 
         [0075]    The mixture was pasteurized at 70° C. for about 5 minutes then temperature controlled at 37° C. 
         [0076]      FIG. 3  shows the HPLC trace of the mixture after pasteurization, presented below in tabular form. 
         [0000]                                                                                                  Peak   Delta RT   Time   Component   Conc.   Area   Response   Amount   Norm. Area       #   [%]   [min]   Name   %   [uV*sec]   Factor   [Norm. %]   [%]                                1   1.42   9.257   GOS 1   0.089800   306895   8.8584e+06    1.3   1.4       2   0.14   9.692   GOS 2   0.296940   1014808   8.8584e+06    4.2   4.7       3       10.324   GOS3   0.792435   2708190   8.8584e+06    11.1    12.4       4   1.62   11.277   GOS4   1.907072   6517527   8.8584e+06    26.7    30.0       5   —   12.477       2.801e−96   10806   1.000e+100   4e−95   0.0       6       12.889   Lattosio   0.163174   557655   8.8584e+06    2.3   2.6       7   —   13.268       3.809e−95   146969   1.000e+100   5e−94   0.7       8   0.68   13.612   GOS6   0.114462   391180   8.8584e+06    1.6   1.8       9   —   14.374       1.105e−95   42639   1.000e+100   2e−94   0.2       10   4.21   16.051   Glucosio   0.007876   25635   8.4366e+06    0.1   0.1       11       16.701   Galattosio   1.324742   4362275   8.5353e+06    18.5    20.1       12   —   17.870       1.276e−95   49224   1.000e+100   2e−94   0.2       13   —   18.923       6.937e−95   267628   1.000e+100   1e−93   1.2       14   —   20.163       6.679e−97   2577   1.000e+100   9e−96   0.0       15   —   21.459   LCTH   1.524e−95   59474   1.000e+100   2e−94   0.3       16       22.127   Glicernia   2.375647   4931565   5.3807e+06    33.2    22.7       17   0.07   22.746       0.077039   203888   6.8599e+06    1.1   0.9       18   —   23.647       1.215e−95   46875   1.000e+100   2e−94   0.2       19   —   24.491       2.779e−97   1072   1.000e+100   4e−96   0.0       20   —   25.801       2.805e−95   108218   1.000e+100   4e−94   0.5                       7.149186   21755103   1.000e+101   100.0    100.0                    
Step 3—Decalactosation with  S. cerevisiae  
 
         [0077]    200 g of lyophilized brewer&#39;s yeast were added to the mixture resulting from step 2: after 20 hours of vigorous agitation the degalactosation step, monitored by HPLC, was complete. 
         [0078]    The reaction was stopped by adding 5 l of 50% sulphuric acid until pH 3.0 was reached. 
         [0079]      FIGS. 4A and 4B  show the HPLC trace of the mixture after degalactosation, presented below in tabular form. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Peak 
                 Delta RT 
                 Time 
                 Component 
                 Conc. 
                 Area 
                 Response 
                 Amount 
                 Norm. Area 
               
               
                 # 
                 [%] 
                 [min] 
                 Name 
                 % 
                 [uV*sec] 
                 Factor 
                 [Norm. %] 
                 [%] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 1.57 
                 9.270 
                 GOS 1 
                 0.097962 
                 358655 
                 8.7717e+06 
                 1.6 
                 1.8 
               
               
                 2 
                 0.18 
                 9.696 
                 GOS 2 
                 0.312481 
                 1144050 
                 8.7717e+06 
                 5.1 
                 5.8 
               
               
                 3 
                   
                 10.328 
                 GOS3 
                 0.827617 
                 3030055 
                 8.7717e+06 
                 13.5  
                 15.4 
               
               
                 4 
                 1.66 
                 11.282 
                 GOS4 
                 1.968015 
                 7205257 
                 8.7717e+06 
                 32.0  
                 36.7 
               
               
                 5 
                   
                 11.827 
                 GOS5 
                 0.018272 
                 66898 
                 8.7717e+06 
                 0.3 
                 0.3 
               
               
                 6 
                 — 
                 12.508 
                   
                 3.997e−96 
                 16683 
                  1.000e+100 
                 6e−95 
                 0.1 
               
               
                 7 
                   
                 12.894 
                 Lattosio 
                 0.157271 
                 575796 
                 8.7717e+06 
                 2.6 
                 2.9 
               
               
                 8 
                 — 
                 13.276 
                   
                 3.002e−95 
                 125300 
                  1.000e+100 
                 5e−94 
                 0.6 
               
               
                 9 
                 0.68 
                 13.613 
                 GOS6 
                 0.159856 
                 563296 
                 8.7717e+06 
                 2.5 
                 2.9 
               
               
                 10 
                 — 
                 14.381 
                   
                 1.490e−95 
                 62185 
                  1.000e+100 
                 2e−94 
                 0.3 
               
               
                 11 
                 4.96 
                 16.177 
                 Glucosio 
                 0.021663 
                 75309 
                 8.3290e+06 
                 0.4 
                 0.4 
               
               
                 12 
                 2.15 
                 17.117 
                 Galattosio 
                 0.001645 
                 5858 
                 8.5330e+06 
                 0.0 
                 0.0 
               
               
                 13 
                 — 
                 17.881 
                   
                 1.395e−95 
                 58207 
                  1.000e+100 
                 2e−94 
                 0.3 
               
               
                 14 
                 — 
                 18.936 
                   
                 6.927e−95 
                 289128 
                  1.000e+100 
                 1e−93 
                 1.5 
               
               
                 15 
                 — 
                 20.187 
                   
                 1.127e−96 
                 4705 
                  1.000e+100 
                 2e−95 
                 0.0 
               
               
                 16 
                 — 
                 21.466 
                   
                 2.285e−95 
                 95358 
                  1.000e+100 
                 4e−94 
                 0.5 
               
               
                 17 
                   
                 22.147 
                 LCTH 
                 2.528752 
                 5590606 
                 5.2968e+06 
                 41.1  
                 28.5 
               
               
                 18 
                 0.17 
                 22.776 
                 Glicernia 
                 0.061780 
                 174711 
                 6.7754e+06 
                 1.0 
                 0.9 
               
               
                 19 
                 — 
                 23.668 
                   
                 8.130e−96 
                 33932 
                  1.000e+100 
                 1e−94 
                 0.2 
               
               
                 20 
                 — 
                 24.907 
                   
                 8.769e−97 
                 3660 
                  1.000e+100 
                 1e−95 
                 0.0 
               
               
                 21 
                 — 
                 25.733 
                   
                 3.543e−95 
                 147894 
                  1.000e+100 
                 6e−94 
                 0.8 
               
               
                   
                   
                   
                   
                 6.149314 
                 19627543 
                  1.000e+101 
                 100.0  
                 100.0 
               
               
                   
               
             
          
         
       
     
         [0080]    The mixture was clarified by removing the cells by ultrafiltration. 
         [0081]    The low molecular weight fermentation by-products (lactic acid, glycerin etc.) were then removed by nanofiltration. 
         [0082]    The solution was then decolorized over carbon, desalinated with a pair of ion-exchange resins, namely a strong cationic (Amberlite C-200 H +  form, 3 l) and a weak anionic (IRA-96 free base form, 3 l), arranged in series. 
         [0083]    The demineralised solution was then microfiltered and concentrated under vacuum until a saccharometric concentration of 75° Brix was attained. 
         [0084]    9 kg of a GOS mixture were obtained with a purity ≧95%; the HPLC trace of the GOS mixture obtained at the end of the process is shown in  FIG. 5 , presented below in tabular form. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Peak 
                 Delta RT 
                 Time 
                 Component 
                 Conc. 
                 Area 
                 Response 
                 Amount 
                 Norm. Area 
               
               
                 # 
                 [%] 
                 [min] 
                 Name 
                 % 
                 [uV*sec] 
                 Factor 
                 [Norm. %] 
                 [%] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                   
                 9.228 
                 GOS1 
                 1.828791 
                 418901 
                 8.5581e+06 
                 2.7 
                 2.6 
               
               
                 2 
                   
                 9.648 
                 GOS2 
                 6.640325 
                 1521025 
                 8.5581e+06 
                 9.7 
                 9.6 
               
               
                 3 
                   
                 10.274 
                 GOS3 
                 17.010960 
                 3896510 
                 8.5581e+06 
                 24.8  
                 24.5 
               
               
                 4 
                 2.13 
                 11.222 
                 GOS4 
                 39.207543 
                 8980833 
                 8.5581e+06 
                 57.1  
                 56.6 
               
               
                 5 
                 — 
                 12.351 
                   
                 2.085e−94 
                 55794 
                  1.000e+100 
                 3e−94 
                 0.4 
               
               
                 6 
                 0.23 
                 12.819 
                 Lattosio 
                 2.769118 
                 634291 
                 8.5581e+06 
                 4.0 
                 4.0 
               
               
                 7 
                   
                 13.511 
                 GOS6 
                 1.233729 
                 282596 
                 8.5581e+06 
                 1.8 
                 1.8 
               
               
                 8 
                 — 
                 14.335 
                   
                 5.725e−95 
                 15322 
                  1.000e+100 
                 8e−95 
                 0.1 
               
               
                 9 
                 3.83 
                 15.869 
                 Glucosio 
                 0.031758 
                 7003 
                 8.2394e+06 
                 0.0 
                 0.0 
               
               
                 10 
                 — 
                 17.703 
                   
                 1.500e−95 
                 4014 
                  1.000e+100 
                 2e−95 
                 0.0 
               
               
                 11 
                 — 
                 18.822 
                   
                 2.397e−94 
                 64148 
                  1.000e+100 
                 3e−94 
                 0.4 
               
               
                   
                   
                   
                   
                 68.722223 
                 15880437 
                  4.000e+100 
                 100.0  
                 100.0 
               
               
                   
               
             
          
         
       
     
         [0085]    From the tabulated data and the attached chromatograms of the various steps, it can be seen that during step 2 (fermentation with  S. thermophilus ) the GOS5 peak disappears, while the GOS6 peak remains more or less unaltered. 
         [0086]    Table 1 presents a summary of the results of the various steps in example 1: 
         [0000]                                                                  TABLE 1               EX 1       GOS   LACTOSE   GLUCOSE   GALACTOSE                                START   % CONC   15.6   15.24   7.52   0.4           % PURITY   40.5   39.6   18.7   1           RELATIVE %   40.6   39.7   18.7   1       STEP 1    % CONC   4   3.83   0   0.09       (fermentation with   % PURITY   48.3   46.3   0   1         S. cerevisiae )   RELATIVE %   50.5   48.4   0   1       STEP 2    % CONC   3.13   0.18   0.01   1.29       (fermentation with   % PURITY   50   2.8   0.1   20.1         S. thermophilus )   RELATIVE %   68.5   3.8   0.1   27.5       STEP 3    % CONC   3.38   0.16   0.02   0       (fermentation with   % PURITY   62.9   2.9   0.4   0         S. cerevisiae )   RELATIVE %   95   4.4   0.6   0       FINAL PRODUCT   % CONC   66.2   2.78   0.03   n.d.           % PURITY   95.5   4   0   n.d.           RELATIVE %   96   4   0   n.d.           %                    
in which: % CONC=concentration in weight/weight %
 
% PURITY=area percent subtended by the HPLC peak RELATIVE %=purity of GOS/Σ purity of GOS, lactose, glucose and galactose
 
       EXAMPLE 2 
     Example of Preparation Starting from GOS at 83% (90% Commercial) 200 g of Purimune GOS 
       [0087]    (GO—P90) Batch no. 20081217 were solubilized in 1800 ml of water.  FIG. 6  shows the chromatogram and the solution HPLC is presented below in tabular form. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Peak 
                 Time 
                 Component 
                 Conc. 
                 Area 
                 Response 
                 Amount 
                 Area Norm. 
               
               
                 # 
                 [min] 
                 Name 
                 % 
                 [μN · s] 
                 factor 
                 norm. % 
                 [%] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 9.181 
                 GOS1 
                 0.494174 
                 209703 
                 8.3152e+06 
                 5.8 
                 5.8 
               
               
                 2 
                 9.562 
                 GOS2 
                 0.833452 
                 353676 
                 8.3152e+06 
                 9.8 
                 9.9 
               
               
                 3 
                 10.072 
                 GOS3 
                 2.055456 
                 872234 
                 8.3152e+06 
                 24.3 
                 24.3 
               
               
                 4 
                 10.951 
                 GOS4 
                 3.297243 
                 1399187 
                 8.3152e+06 
                 39.0 
                 39.0 
               
               
                 5 
                 12.044 
                 GOS5 
                 0.202695 
                 86014 
                 8.3152e+06 
                 2.4 
                 2.4 
               
               
                 6 
                 12.428 
                 Lattosio 
                 1.144282 
                 485577 
                 8.3152e+06 
                 13.5 
                 13.5 
               
               
                 7 
                 13.041 
                 GOS6 
                 0.142975 
                 60672 
                 8.3152e+06 
                 1.7 
                 1.7 
               
               
                 8 
                 14.735 
                 Glucosio 
                 0.155892 
                 62950 
                 7.9126e+06 
                 1.8 
                 1.8 
               
               
                 9 
                 15.993 
                 Galattosio 
                 0.136621 
                 64958 
                 7.8825e+06 
                 1.6 
                 1.5 
               
               
                 10 
                 28.904 
                   
                 7.133e−97 
                 364 
                  1.000e+100 
                 8e−96 
                 0.0 
               
               
                   
                   
                   
                 8.462789 
                 3585334 
                   
                 100.0 
                 100.0 
               
               
                   
               
             
          
         
       
     
         [0088]    The glucose present has an area percent of &lt;5% hence the procedure continued directly with fermentation by lactic acid bacteria culture. 
         [0089]    5 g/l of yeast extract were added and the pH adjusted from pH 4.9 to pH 6.4 with 0.3 ml of 15% sodium hydroxide. Following inoculation with 75 mg of  Streptococcus thermophilus , the fermentation proceeded at pH 6.3-6.5 by adding 15% sodium hydroxide by means of a pH-stat. The end of the reaction, monitored by HPLC, was reached after 15 hours when the lactose content of the mixture was less than 5% by area percent relative to the sum of the area percents of GOS. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Peak 
                 Time 
                 Component 
                 Conc. 
                 Area 
                 Response 
                 Amount 
                 Area Norm. 
               
               
                 # 
                 [min] 
                 Name 
                 % 
                 [μN · s] 
                 factor 
                 norm. % 
                 [%] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 9.223 
                 GOS1 
                 0.496176 
                 455733 
                 8.3252e+06 
                 6.0 
                 6.4 
               
               
                 2 
                 9.628 
                 GOS2 
                 0.886683 
                 816247 
                 8.3252e+06 
                 10.7 
                 11.4 
               
               
                 3 
                 10.143 
                 GOS3 
                 2.031244 
                 1865678 
                 8.3252e+06 
                 24.4 
                 26.1 
               
               
                 4 
                 11.041 
                 GOS4 
                 2.578800 
                 2368602 
                 8.3252e+06 
                 31.0 
                 33.1 
               
               
                 5 
                 12.513 
                 Lattosio 
                 0.282604 
                 259569 
                 8.3252e+06 
                 3.4 
                 3.6 
               
               
                 6 
                 13.124 
                 GOS6 
                 0.074653 
                 68605 
                 8.3252e+06 
                 0.9 
                 1.0 
               
               
                 7 
                 16.043 
                 Galattosio 
                 0.574881 
                 503523 
                 7.9890e+06 
                 6.9 
                 7.0 
               
               
                 8 
                 21.256 
                 LCTH 
                 1.393852 
                 810476 
                 5.2712e+06 
                 16.7 
                 11.3 
               
               
                   
                   
                   
                 8.320732 
                 7148433 
                   
                 100.0 
                 100.0 
               
               
                   
               
             
          
         
       
     
         [0090]    A summary of the results obtained are presented in table 2. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 EX 2 
                   
                 GOS 
                 LACTOSE 
                 GLUCOSE 
                 GALACTOSE 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 START 
                 % CONC 
                 7.03 
                 1.14 
                 0.16 
                 0.14 
               
               
                   
                 % PURITY 
                 83.1 
                 13.5 
                 1.8 
                 1.5 
               
               
                   
                 RELATIVE % 
                 83.2 
                 13.5 
                 1.8 
                 1.5 
               
               
                 STEP 1  
                 % CONC 
                 6.07 
                 0.28 
                 0 
                 1.39 
               
               
                 (fermentation with  
                 % PURITY 
                 78 
                 3.6 
                 0 
                 7 
               
               
                   S. thermophilus ) 
                 RELATIVE % 
                 88 
                 4 
                 0 
                 7.9 
               
               
                   
               
             
          
         
       
     
       Abbreviations: 
       [0091]    % CONC=concentration in weight/weight %
 
% PURITY=area percent subtended by the HPLC peak
 
RELATIVE %=purity of GOS/Σ purity of GOS, lactose, glucose and galactose
 
         [0092]    The experiment was stopped at this stage, but it was clear that the mixture could be subsequently degalactosated and purified in the manner already described in example 1. 
         [0093]    Direct fermentation with  Streptococcus thermophilus , thus avoiding deglucosation with  S. cerevisiae  which in this case was superfluous, resulted in the desired area ratio of GOS to lactose. Moreover, considering that the subsequent fermentation with  S. cerevisiae  was able to remove galactose, a GOS purity of 95.6% relative to the lactose alone can theoretically be calculated based on the area percent values reported in table 2. 
       EXAMPLE 3 
     Example of Preparation Starting from GOS at 43% (60% Commercial) 
       [0094]    427 g of Vivinal Gos60 Batch no. 6297770 were solubilized in 1600 ml of water: the HPLC data of the solution is presented below in tabular form: 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Peak 
                 Time 
                 Component 
                 Conc. 
                 Area 
                 Response 
                 Amount 
                 Area Norm. 
               
               
                 # 
                 [min] 
                 Name 
                 % 
                 [μV · s] 
                 factor 
                 norm. % 
                 [%] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 9.365 
                 GOS1 
                 0.132997 
                 117776 
                 8.4830e+06 
                 1.4 
                 1.4 
               
               
                 2 
                 9.710 
                 GOS2 
                 0.386673 
                 342420 
                 8.4830e+06 
                 4.0 
                 4.1 
               
               
                 3 
                 10.230 
                 GOS3 
                 0.978558 
                 866565 
                 8.4830e+06 
                 10.2 
                 10.3 
               
               
                 4 
                 11.086 
                 GOS4 
                 2.153239 
                 1906809 
                 8.4830e+06 
                 22.3 
                 22.7 
               
               
                 5 
                 12.198 
                 GOS5 
                 0.283797 
                 251318 
                 8.4830e+06 
                 2.9 
                 3.0 
               
               
                 6 
                 12.556 
                 Lattosio 
                 3.315665 
                 2936200 
                 8.4830e+06 
                 34.4 
                 34.9 
               
               
                 7 
                 13.186 
                 GOS6 
                 0.153348 
                 135798 
                 8.4830e+06 
                 1.6 
                 1.6 
               
               
                 8 
                 14.855 
                 Glucosio 
                 2.070889 
                 1719196 
                 7.9525e+06 
                 21.5 
                 20.4 
               
               
                 9 
                 16.122 
                 Galattosio 
                 0.160862 
                 137854 
                 8.2093e+06 
                 1.7 
                 1.6 
               
               
                   
                   
                   
                 9.636028 
                 8413937 
                   
                 100.0 
                 100.0 
               
               
                   
               
             
          
         
       
     
         [0095]    The procedure was carried out exactly as in example 1, with the first deglucosation step with  S. cerevisiae  followed by fermentation with  S. thermophilus ; at the end of this step (step 2 in Ex. 1) an HPLC was obtained and the result, being absolutely comparable to that of Example 1, is tabulated below: 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Peak 
                 Time 
                 Component 
                 Conc. 
                 Area 
                 Response 
                 Amount 
                 Area Norm. 
               
               
                 # 
                 [min] 
                 Name 
                 % 
                 [μV · s] 
                 factor 
                 norm. % 
                 [%] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 9.265 
                 GOS1 
                 0.103310 
                 93473 
                 8.4830e+06 
                 1.4 
                 1.6 
               
               
                 2 
                 9.641 
                 GOS2 
                 0.382881 
                 346426 
                 8.4830e+06 
                 5.3 
                 6.1 
               
               
                 3 
                 10.163 
                 GOS3 
                 0.908611 
                 822101 
                 8.4830e+06 
                 12.6 
                 14.5 
               
               
                 4 
                 11.025 
                 GOS4 
                 1.991562 
                 1801941 
                 8.4830e+06 
                 27.6 
                 31.7 
               
               
                 5 
                 12.104 
                   
                 4.315e−96 
                 12100 
                  1.000e+100 
                 7e−95 
                 0.2 
               
               
                 6 
                 12.498 
                 Lattosio 
                 0.167432 
                 151490 
                 8.4830e+06 
                 2.3 
                 2.7 
               
               
                 7 
                 13.124 
                 GOS6 
                 0.145504 
                 144421 
                 8.4830e+06 
                 2.0 
                 2.5 
               
               
                 8 
                 14.788 
                 Glucosio 
                 0.005412 
                 4590 
                 7.9525e+06 
                 0.1 
                 0.1 
               
               
                 9 
                 16.048 
                 Galattosio 
                 1.003410 
                 878581 
                 8.2093e+06 
                 13.9 
                 15.4 
               
               
                 10 
                 21.841 
                 LCTH 
                 2.387452 
                 1342275 
                 5.2712e+06 
                 33.1 
                 23.6 
               
               
                 11 
                 21.841 
                 GIOH 
                 0.125566 
                 89471 
                 6.6806e+06 
                 1.7 
                 1.6 
               
               
                   
                   
                   
                 7.221140 
                 5686869 
                   
                 100.0 
                 100.0 
               
               
                   
               
             
          
         
       
     
         [0096]    It can also be seen in this example that the GOS5 peak disappears during fermentation with  S. thermophilus , while the GOS6 peak remains more or less unaltered. 
         [0097]    As in example 2, this experiment was stopped at this stage and the mixture was degalactosated and purified in the manner already described in example 1. Moreover, considering that the second fermentation with  S. cerevisiae  was able to remove galactose, a GOS purity of 95.2% relative to the lactose alone can theoretically be calculated based on the area percent values reported in the table for the second HPLC of this example. 
       EXAMPLE 4 
     Example of Preparation Starting from GOS at 40% with  K. marxianus  (Cheng, C.-C.; et al.  Biotechnol. Lett.  28 793-797, 2006) 
       [0098]    The strain  K. marxianus  ATCC 56497 was cultured on a plate containing autoclave-sterilized YPD medium with agar for yeasts, and placed in an incubator at 30° C. for 48 hours. 
         [0099]    To prepare the pre-fermentation mixture, 100 ml of liquid YPD medium were prepared, autoclave-sterilized then inoculated with the colonies picked up from the previously prepared plate. The microorganisms were grown in a shaker at a temperature of 30° C. and shaken at 200 rpm for 24 hours. 
         [0100]    The test was carried out on the same starting solution as Example 1: 460 g of a 40% crude GOS solution (Batch no. 014491N9) diluted with 1.4 l of water. 
         [0101]    After adjusting the solution pH from pH 2.8 to pH 5.4 with 15% sodium hydroxide, it was temperature controlled at 30° C. 
         [0102]    The entire pre-fermentation mixture was used for the inoculation. The fermentation proceeded at pH 5.2-5.4 by adding 15% sodium hydroxide by means of a pH-stat. Sampling of the mixture was carried out after 48 hours and shows the reaction to have reached completion, as can be seen from the tabulated HPLC data (see also  FIG. 8 ) presented below: 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Peak 
                 Time 
                 Component 
                 Conc. 
                 Area 
                 Response 
                 Amount 
                 Area Norm. 
               
               
                 # 
                 [min] 
                 Name 
                 % 
                 [μV · s] 
                 factor 
                 norm. % 
                 [%] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 9.382 
                 GOS1 
                 0.070000 
                 29745 
                 8.5106e+06 
                 1.9 
                 1.5 
               
               
                 2 
                 9.700 
                 GOS2 
                 0.355858 
                 151213 
                 8.5106e+06 
                 9.7 
                 7.6 
               
               
                 3 
                 10.232 
                 GOS3 
                 0.944881 
                 401505 
                 8.5106e+06 
                 25.9 
                 20.0 
               
               
                 4 
                 11.065 
                 GOS4 
                 1.989309 
                 845311 
                 8.5106e+06 
                 54.5 
                 42.2 
               
               
                 5 
                 21.900 
                 GIOH 
                 0.289913 
                 96941 
                 6.6971e+06 
                 7.9 
                 4.8 
               
               
                 6 
                 33.135 
                   
                 9.570e−94 
                 477834 
                  1.000e+100 
                 3e−92 
                 23.9 
               
               
                   
                   
                   
                 3.649961 
                 2002549 
                   
                 100.0 
                 100.0 
               
               
                   
               
             
          
         
       
     
         [0103]    The reaction effectively resulted in the almost complete disappearance of lactose, but it should be noted from the attached chromatogram ( FIG. 8 ) that peaks attributable to the galacto-oligosaccharides are absent from the entire disaccharide region (between 12.2 min and 13.2 min).