METHOD FOR SEPARATING BIOMASS FROM A SOLUTION COMPRISING BIOMASS AND AT LEAST ONE OLIGOSACCARIDE

Abstract

The present invention relates to a method for separating biomass from a solution comprising biomass and at least one oligosaccharide.comprising providing the solution comprising biomass and oligosaccharides.lowering the pH value of the solution below 7 by adding at least one acid to the solution comprising biomass and the at least one oligosaccharide. adding an adsorbing agent to the solution comprising biomass and oligosaccharides. and carrying out first membrane filtration so as to separate the biomass from the solution comprising the at least one oligosaccharide.

Claims

1.-15. (canceled)

16. A method for separating biomass from a solution comprising biomass and at least one oligosaccharide, comprising: providing the solution comprising biomass and oligosaccharides; setting the pH value of the solution below 7 by adding at least one acid to the solution comprising biomass and the at least one oligosaccharide; adding at least one adsorbing agent to the solution comprising biomass and oligosaccharides; and carrying out a first membrane filtration, so as to separate the biomass from the solution comprising at least one oligosaccharide.

17. The method according to claim 16, wherein the pH value of the solution is lowered to a pH value in the range of 3.0 to 5.5.

18. The method according to claim 16, wherein said at least one acid is an acid selected from the group consisting of H.sub.2SO.sub.4, H.sub.3PO.sub.4, HCl, HNO.sub.3 and CH.sub.3CO.sub.2H.

19. The method according to claim 16, wherein said adsorbing agent is added in an amount in the range of 0.5% to 3% by weight.

20. The method according to claim 16, wherein said adsorbing agent is added as a powder having a particle size distribution with a diameter d50 in the range of 2 μm to 25 μm.

21. The method according to claim 20 wherein said adsorbing agent is added as a suspension of the powder in water.

22. The method according to claim 16, wherein said first membrane filtration is carried out as cross-flow microfiltration or cross-flow ultrafiltration.

23. The method according to claim 22, wherein said cross-flow microfiltration or cross-flow ultrafiltration includes a cross-flow speed in the range of 0.5 m/s to 6.0 m/s.

24. The method according to claim 22, wherein said cross-flow speed is equal to or below 3 m/s.

25. The method according to claim 16, wherein said first membrane filtration is carried out at a temperature of the solution in the range of 8° C. to 55° C.

26. The method according to claim 16, wherein said first membrane filtration is carried out by means of a ceramic microfiltration or ultrafiltration membrane having a pore size in the range of 20 nm to 800 nm, or wherein said first membrane filtration is carried out by means of a polymeric microfiltration membrane or polymeric ultrafiltration membrane having a cut-off in the range of 10 kDa to 200 nm.

27. The method according to claim 16, further comprising carrying out a second membrane filtration with the solution comprising oligosaccharides obtained by the first membrane filtration.

28. The method according to claim 27, wherein said second membrane filtration is an ultrafiltration and is carried out by means of an ultrafiltration membrane having a cut-off in the range of 1.5 kDa to 10 kDa.

29. The method according to claim 27, wherein said second membrane filtration is carried out at a temperature of the solution being in the range of 5° C. to 15° C.

30. The method according to claim 16, wherein said at least one oligosaccharide comprises human milk oligosaccharide.

31. The method according to claim 16, wherein the adsorbing agent comprises active carbon.

32. The method according to claim 16, wherein the pH value of the solution is lowered to a range from 4.0 to 4.5.

33. The method according to claim 16, wherein the adsorbing agent is added in an amount in the range of 1.0% to 2.0% by weight and the adsorbing is added as a powder having a particle size distribution with a diameter d50 in the range of 3 μm to 7 μm.

34. The method according to claim 16, wherein the first membrane filtration comprises microfiltration or ultrafiltration and is carried out at a temperature of the solution in the range of 30° C. to 40° C.

35. The method according to claim 16, wherein the at least one oligosaccharide comprises at least one of 2′-fucosyllactose, 6′-sialyllactose and Lacto-N-tetraose.

Description

FIGURES

[0162] FIG. 1 shows a block diagram of a method for separating biomass from a solution comprising biomass and at least one oligosaccharide according to the present invention.

EXAMPLES

[0163] The method according to the present invention will be described in further detail below. Whatsoever, the Examples shall not be construed as limiting the scope of the invention.

Example 1

[0164] A fermentation broth as a complex solution comprising biomass and at least one oligosaccharide has been prepared by standard methods in the amount of 2.4 kg. The pH value thereof has been lowered to 4±0.1 by means of adding 92 g 10% sulfuric acid. Thereafter, 98 g of a 30% suspension of active carbon Carbopal Gn-P (Donau Carbon GmbH, Gwinnerstraβe 27-33, 60388 Frankfurt am Main, Germany), which is food safe, has been added and stirred for 20 min.

[0165] The thus prepared solution has been supplied to the process apparatus, a semi-automatic MF lab unit from Sartorius AG, Otto-Brenner-Str. 20, 37079 Goettingen, Germany, modified for the purpose, and heated to 37° C. in a circulating manner with closed permeate. For separation purposes, the process apparatus included a mono channel element (from Atech Innovations GmbH, Gladbeck, Germany) having an outer diameter of 10 mm, an inner diameter of 6 mm, a length of 1.2 m and a membrane made of Al.sub.2O.sub.3 having a pore size of 50 nm. As soon as the circulation of the solution is running and the solution comprises the target temperature of 37° C., the discharging of the permeate has been started and the control of the trans membrane pressure has been activated.

[0166] After terminating of the inventive method, the process apparatus has been stopped, the concentrate has been disposed and the process apparatus has been cleaned. Cleaning has been carried out by means of 0.5% to 1% NaOH at a temperature of 50° C. to 80° C., wherein the NaOH has been subsequently removed by purging.

[0167] In a preferred embodiment, the first membrane filtration of the inventive methods includes three steps as will be explained in further detail below. The first step includes a first diafiltration having a factor of 0.5 (amount of diafiltration water=starting amount of fermentation broth×diafiltration factor). During diafiltration, the amount of water added is identical to the amount of permeate discharged. The first step is a continuing step and the volume in the feed vessel is thus kept constant. The second step includes concentrating of the fermentation broth with the factor 2 by stopping the feed of diafiltration water and the level will decrease down to the target value (target value=volume or mass at the beginning of the fermentation broth/concentrating factor).

[0168] Subsequently, the third step includes a second diafiltration. The permeates collected during these three steps are typically combined to form the permeate referred to in the tables below. By means of these three steps a lower dilution of the product within the permeate and an increased yield of ≥95% are realized. By increasing the factor of the second diafiltration, the yield may even be increased.

[0169] The following analytical methods have been carried out. [0170] HPLC for the determination of the product, i.e. human milk oligosaccharides, and secondary components [0171] Drying balances for measuring the dry content [0172] APHA for measuring the color using standard methods, for example DIN EN ISO 6271 [0173] Bradford protein assay for measuring the concentration of protein.

[0174] Some experiments have been made with different fermentation broths as these may not be stored over a longer period of time. In order to be able to determine whether the method correctly works and provides the announced advantages, experiments have been made: [0175] without adjustment of pH value and without adding active carbon, [0176] without adjustment of pH value and with adding active carbon, [0177] after adjustment of pH value and without adding active carbon, [0178] after adjustment of pH value and with adding active carbon, [0179] after adding active carbon and then adjustment of pH value.

[0180] Hereinafter, the following abbreviations are used: [0181] AC=Active Carbon [0182] UF=Ultrafiltration [0183] DF=Diafiltration factor (ratio: amount of water and start volume) [0184] CF=Concentration factor (ratio between start volume and final volume) [0185] DP=Pressure drop along the module (p.sub.feed−p.sub.retentate) [0186] Flux=Permeate flow rate per m.sup.2 and hour (l/m.sup.2 h) [0187] Cross-flow velocity=linear speed of the suspension in membrane channels (m/s) [0188] Membrane load=amount of permeate produced by 1 m.sup.2 of membrane area (m.sup.3/m.sup.2)

[0189] Further, regarding the liquid separation, the following symbols and explanations are used.

TABLE-US-00001 Symbol Meaning Unit Definition Letters CF Concentration factor — m.sub.R,t=0/m.sub.R DF Diafiltration factor — m.sub.P/m.sub.R,t=0 J Flux LMH = L m.sup.−2 h.sup.−1 m Mass kg p Pressure bar R Retention — 1 − C.sub.permeate/C.sub.retentate TMP Trans-membrane bar (p.sub.feed + p.sub.retentate)/ pressure 2 − p.sub.permeate

[0190] Still further, in the following tables, the term “Series” refers to the respective experimental number.

[0191] Table 1 shows the membrane performance depending on the pH value and active carbon. Different batches of fermentation broth originating from fermentations with varying parameters resulting in a solution with differing color components and different oligosaccharide and disaccharide compositions of the solution demonstrate the broad applicability of the methods of the invention.

TABLE-US-00002 TABLE 1 AC Flux cross ads. [kg/ TMP DP Temp. flow Series Batch pH [%] [h] m.sup.2h] [bar] [bar] [°C.] [m/s] A1 Batch 1 7.0 15 1.3 1.2 39.5 4.0 5.0 9 1.3 1.4 39.5 4.0 4.0 25 1.4 1.5 39.5 3.9 3.5 21 1.3 1.4 39.5 3.9 A2 Batch 2 7.0 0.0 0.0 12 1.3 1.1 39.4 4.0 1.0 0.3 8 1.5 1.3 39.5 3.9 4.0 0.0 0.0 15 1.2 1.4 39.5 4.0 1.0 0.3 77 1.2 1.3 41.0 3.7 A3 Batch 3 4.0 1.0 24 85 1.3 1.4 38.9 3.4 2.0 24 75 1.6 1.7 38.8 3.8 1.0 0.3 47 1.2 1.2 39.3 3.0 2.0 0.3 37 1.3 1.3 39.4 3.0 1.0 3 53 1.2 1.0 39.4 2.9 The abbreviation “ads. [h]” is the time after addition of the adsorbing agent to the solution before the start of the first membrane filtration in hours.

[0192] Series A 1 was done in the absence of any adsorbing agent yet at different pH values. Series A 2 was done at pH 7.0 and 4.0 and with or without active carbon. Series A3 was done at pH 4 and varying amounts of active carbon and differing cross-flow speeds as indicated. For these three series, only a first diafiltration step with DF=1 and a concentrating step with CF=2 were performed and then the first membrane filtration was stopped and the resulting solutions and remainder of the starting solutions analysed and results compared.

[0193] The following results are derivable from table 1:

[0194] The membrane performance has its maximum at a pH value of 4 at a cross-flow speed of 4 m/s. The membrane performance is reduced at a pH value of 7 with presence of 1% active carbon. whereas the membrane performance is enhanced at a pH value of 4 and with presence of 1% active carbon with a cross-flow speed of 4 m/s by a factor of approximately 4. An increase of the adsorption time after adding active carbon from 0.3 hours to 24 hours provides only a negligible enhancement of the membrane performance. An increase of the added amount of active carbon from 1% to 2% lowers the membrane performance. A reduction of the cross-flow speed from 4 m/s to 3 m/s reduces the membrane performance but the same is still higher than without presence of active carbon. A reduction of the cross-flow speed significantly reduces the electric power consumption and also reduces the risk of membrane abrasion.

[0195] Table 2 shows the analytical results depending on the pH value and active carbon of Series A1. DC is the abbreviation for dry content. OD for the optical density. Feed denotes the solution comprising biomass and oligosaccharides and disaccharide. Permeate is the resulting solution after first membrane filtration, concentrate the remainder of the feed.

TABLE-US-00003 TABLE 2 H.sub.2SO.sub.4-10% DC 3.2-Di-Fl 2FL Lactose Protein Series Batch pH [g/kg] Sample [%] OD [g/l] [g/l] [g/l] [g/l] APHA A1 with 50 nm Batch 1 7.0 0 Feed 15.9 160 1.14 47.21 9.31 4.51 Al2O3 membrane Permeate 5.9 0.84 29.13 5.68 1.46 8116 Concentrate 19.0 307 0.78 24.7 1.23 5.81 5.0 26.3 Feed 16.1 242 1.14 47.21 9.31 4.51 Permeate 5.69 0.78 28.88 5.67 0.32 7952 Concentrate 18.9 237 5.74 4.0 41.7 Feed 15.9 160 1.14 47.21 9.31 1.31 Permeate 5.9 0.77 29.21 5.61 0.19 7854 Concentrate 19 307 0.7 24.51 4.66 1.38 3.5 53.8 Feed 15.9 151 1.14 47.21 9.31 4.51 Permeate 5.91 0.82 28.99 5.62 0.14 7814 Concentrate 17.7 293 0.74 24.83 0.53

[0196] The following results are derivable from table 2:

[0197] A variation of the pH value has no influence on the color value of the permeate. Lower APHA values at lower pH values are the result of a minor dilution of the fermentation broth by 10% sulfuric acid. The concentration of protein is significantly reduced at lower pH value. The pH value of the fermentation broth has no significant influence on the oligosaccharides 3.2-Di-fucosyllactose (3.2-Di-Fl) and 2′Fucosyllactose (2FL) or the disaccharide lactose.

[0198] Table 3 shows the analytical results depending on the pH value and active carbon of Series A2. DC is the abbreviation for dry content. OD for the optical density.

TABLE-US-00004 TABLE 3 DC 3.2-Di-Fl 2FL 2F-Lactulose Lactose Protein Series Batch pH Sample [%] APHA OD [g/l] [g/l] [g/l] [g/l] [g/l] A2 with 50 nm Batch 2 7.0 Feed 17.8 138 3.43 62.07 0.6 4.28 0.478 Al2O3 membrane Permeate 7.61 4196 1.99 34.54 0.43 2.54 0.124 Concentrate 18.5 136 1.882 7.0 + 1% AC Feed 18.3 119 3.29 62.22 0.33 3.93 0.964 Permeate 7.9 1467 2.14 37.69 0.26 2.25 0.073 Concentrate 17.3 237 1.89 31.39 0.54 0.12 1.41 4.0 Feed 17.3 150 2.83 54.98 0.59 0.89 0.76 Permeate 8.2 4784 1.90 35.00 0.37 2.57 0.019 Concentrate 17.7 434 0.026 4.0 + 1% AC Feed 16.8 151 2.83 55.52 0.34 3.65 0.760 Permeate 7.8 781 1.73 33.66 0.27 2.26 0.019 Concentrate 18.3 293 1.61 29.14 0.33 2.38 0.026

[0199] The following results are derivable from table 3:

[0200] Adding 1% active carbon to the fermentation broth reduces the color value of the permeate. At a pH value of 7. 1% active carbon reduces the color value at approximately 65%. At a pH value of 4.1% active carbon reduces the color value at approximately 84%. Thus. the color value is below the upper end of 1000 and a further decolorization is not necessary. Adding active carbon at a pH value of 7 reduces the concentration of protein within the permeate at approximately 40%. whereas no effect in this respect by adding active carbon can be derived at a pH value of 4 over the pH effect on protein concentration. Nevertheless. the concentration of protein within the permeate at a pH value of 4 and with adding 1% active carbon is smaller by a factor of 4 if compared to the concentration of protein within the permeate at a pH value of 7 and with adding of 1% active carbon. Adding active carbon has no significant influence on the concentration of the oligosaccharides 3.2-Di-fucosyllactose (3.2-Di-Fl). 2′Fucosyllactulose (2F-Lactulose) and 2′Fucosyllactose (2FL). within the permeate at both pH values. Thus. it can be derived that these components do not adhere to the active carbon in significant amounts. The disaccharide lactose shows in this experiment a small reduction in concentration when active carbon is used. yet the beneficial effect of lowered pH and active carbon allow for the application of the inventive method for this disaccharide.

[0201] Table 4 shows the analytical results depending on the pH value and active carbon of Series A3. DC is the abbreviation for dry content. OD for the optical density.

TABLE-US-00005 AC DC 3.2-Di-Fl 2FL 2F-Lactulose Lactose Protein Series Batch [%] [h] Sample [%] APHA OD [g/l] [g/l] [g/l] [g/l] [g/l] A3 Batch 3,. pH = 4 0 0 Feed 16.5 218 1.60 43.40 0.16 1.28 0.050 Permeate 7.3 4815 0.97 26.32 0.10 0.81 0.019 Concentrate 15.9 370 0.026 1 24 Feed 16.5 229 1.54 41.73 0.15 1.23 0.050 Permeate 7.3 772 1.00 26.90 1.2 0.001 Concentrate 20.4 1.06 23.79 0.1  0.79 0.001 2 24 Feed 16.7 1.46 37.35 na 1.47 0.050 Permeate 6.9 247 1.01 25.78 na 1.16 0.001 Concentrate 21.7 1.03 24.03 0.13 0.81 0.001 1 0.3 Feed 16.5 1.69 42.43 na 1.37 0.050 Permeate 7.1 795 1.09 27.50 na 0.94 0.002 Concentrate 19.2 0.97 24.7 na 0.89 0.000 2 0.3 Feed 17.2 251 1.54 39.41 na 1.37 0.050 Permeate 7.1 320 1.11 27.70 na 0.93 0.000 Concentrate 21.6 1.01 25.44 na 0.86 0.000 1 3 Feed 17.0 1.65 42.93 na 1.40 0.050 Permeate 7.4 700 1.04 26.53 na 1.03 0.002 Concentrate 18.6 0.94 24.07 na 0.85 0.001

[0202] The following results are derivable from table 4:

[0203] Adding active carbon reduces the concentration of protein within the permeate at 95% if compared to the experiment without adding active carbon. An increase of the added amount of active carbon from 1% to 2% has no significant or detectable influence on the concentration of protein. Adding active carbon significantly reduces APHA. The reduction is approximately 85% with adding 1% active carbon and is 93% to 95% with adding 2% active carbon. a Longer adsorption time before filtration has no significant or detectable influence on APHA or the concentration of protein. Neither a prolonged adsorption time nor an increase in the amount of active carbon used had a strong effect on the oligosaccharides 3.2-Di-fucosyllactose (3.2-Di-Fl). 2′Fucosyllactulose (2F-Lactulose) and 2′Fucosyllactose (2FL) or the disaccharide lactose in the permeate.

[0204] Additional tests showed that these good results could be further improved when a second diafiltration step was used.

[0205] In another experiment (Series A4) the effects of adding the active carbon before or after the pH lowering of the solution comprising biomass (also called feed) were tested. In this experiment, only the first diafiltration step was used with a DF of 3, the first membrane filtration was stopped and the permeate and the concentrated solution comprising the biomass were analysed and the two treatments compared.

[0206] Table 5 shows the analytical results depending on the pH value and active carbon of Series A4.

TABLE-US-00006 TABLE 5 DC 3.2-Di-Fl 2FL 2F-Lactulose Lactose Protein Series Batch pH Sample [%] APHA OD [g/l] [g/l] [g/l] [g/l] [g/l] A4 Batch 4 4.0 then Feed 17.3 146 1.61 50.79 0.20 1.11 0.133 1% AC Permeate. 5.9 1386 1.15 30.91 0.71 0.01 Concentrate 15.9 0.70 16.88 0.10 0.22 0.079 1% AC Feed 16 8 146 1 62 51.47 0.16 0.75 0.137 then Permeate 6.0 1555 1.13 29.06 0.12 0.51 0.031 pH 4 Concentrate 14.4 0.58 14.24 0.04 0.21 0.610

[0207] The following results are derivable from table 5:

[0208] If the pH value is adjusted to 4 first and then adding active carbon reduction of protein and color is better at approximately 15% than if the active carbon is added first and then the pH value is adjusted. No strong effects on the measured oligosaccharides were observed. A slightly stronger retention of the disaccharide lactose was seen when the pH value was lowered first. Empty cells indicate that a value was not determined.

[0209] A more stable performance of the membrane (Flux. TMP. DP) was obtained when the pH value was adjusted to 4 first and then the active carbon suspension was added than in an opposite way.

Example 2

[0210] Hereinafter only differences from Example 1 will be described and identical parameters are not repeated.

[0211] A fermentation broth as a complex solution comprising biomass and at least one oligosaccharide has been prepared. The pH value thereof has been lowered to 4±0.1 by means of adding 38 g 20% sulfuric acid per kg fermentation broth. Further. 1% active carbon powder has been added. The separation was carried out with a hydrophilic 50 kDa polyethersulfone (PES) membrane (NADIR® UH050 P. MICRODYN-NADIR GmbH. Kasteler Straβe 45. 65203 Wiesbaden. Germany).

[0212] Table 6 shows performance data of a biomass separation as a function of adsorption time at pH=4 with 1% active carbon powder and cross-flow speeds for different fermentation broths. “Pe” denote the permeate amount.

TABLE-US-00007 TABLE 6 AC Flux cross ads. Pe [kg/ TMP DP Temp. flow Batch pH [%] [h] [kg] m.sup.2h] [bar] [bar] [° C.] [m/s] Batch 4 4. 0 1.0 0.3 3.77 23 1.51 2.03 39.5 1.2 1.0 0.3 4.02 21 1.67 2.58 39.3 1.1 Batch 5 4. 0 1.0 1.0 9.00 45 1.90 2.46 37.6 1.5 Batch 6 1.0 1.0 6.83 35 1.94 2.12 39.0 1.7 Batch 7 4.0 1.0 1.0 4.47 12 1.2 1.18 38.2 1.0

[0213] The following results are derivable from table 6:

[0214] Using different starting solutions, pH4.4, 1% active carbon and low cross-flow speeds were tested and resulted in good membrane performance. Longer incubation periods of the adsorbing agent in the starting solution were not required.

[0215] Comparing the results from tables 2, 3 and 6, it can be concluded that at pH=4 and with 1% active carbon the membrane performance was higher by a factor 4 to 10 in comparison with performance measured with fermentation broth without active carbon and at pH value of 7 and that the reduction of the cross-flow speed from 1.5 m/s to 1.1 m/s results in significant flux reduction but the performance is still higher by a factor 2 in comparison to the trials at a pH value of 7 with a cross-flow speed of 1.5 m/s.

[0216] In a further experiment the batches of fermentation broth shown in table 6 were used as starting solution experiments for further tests. Batch 4 was used to test the two variants of first setting pH to 4.0 and then adding 1% w/w active carbon, or first a adding the same percentage of carbon and afterwards setting the pH to 4.0, followed by the first membrane filtration. As observed before, the permeate of the membrane filtration showed 3 to 4 times more protein when the pH was set after the addition of active carbon compared to adding the active carbon to a solution already at pH 4.0. Also, the APHA values of the permeate were higher when active carbon was added before the pH adjustment. Both treatments had similar effects on the oligosaccharides and disaccharides measured, these were largely recovered in the permeate and losses in the retentate were in the order of 10% to 20%, in some cases up to 30%.

[0217] Tests with batches 5, 6 and 7 and setting pH to 4.0 and then adding 1% w/w active carbon, confirmed the permeate to contain as little as 5 to 15% of the protein amount of the starting solution. APHA values of the permeate below 700 (batch 7) and below 400 (batches 5 and 6) were achieved. With respect to the oligosaccharides and disaccharides, again the large majority was recovered in the permeate, with the retentate containing amounts similar to those observed for batch 4.

[0218] Additional observations were that oligosaccharide and lactose concentration in fermentation broth may vary significantly. yet the inventive methods can be applied with similar results on the oligosaccharides and lactose nonetheless; and a lower color number in the permeate as a trend correlates with a lower the protein concentration in the permeate.

[0219] In addition, several batches of fermentation broths produced with standard methods comprising 6′-sialyllactose or Lacto-N-tetraose, have been tested in the inventive methods. The results when the pH was lowered first, and then active carbon was added were comparable and often even better than those shown above. For example, fermentation broths comprising Lacto-N-tetraose starting with a high concentration of color components resulting in APHA values of 7000 or more in the feed, gave permeates after the first membrane filtration with an APHA value of below 1000, but typically below 300 and even as low as below 100. The protein concentration was lowered from typically around 3 g/l to less than 0.01 g/l. The vast majority, typically above 95%, of the Lacto-N-tetraose originally found in the fermentation broth was present in the combined permeate. Similarly, for other oligosaccharides present and also for the disaccharide lactose most was present in the combined permeate and only minor amounts found in the retentate at the end of the first membrane filtration. The applied DF values were below 3.

[0220] Also, fermentation broths comprising 6′-sialyllactose with APHA values of around 7000, after said first membrane filtration resulted in permeates with an APHA value of below 300 and even as low as below 70. The protein concentration was lowered by a factor of 10 or more compared to the starting value in the fermentation broth, at DF values below 3. The vast majority, typically above 90% of the 6′-sialyllactose originally found in the fermentation broth was present in the combined permeate. Similarly, for other oligosaccharides present and also for the disaccharide lactose most was present in the combined permeate and only minor amounts found in the retentate at the end of the first membrane filtration.

[0221] It was also found that performing the methods with a pH of below 5.5 improved flux in the first membrane filtration compared to higher pH values (cross-flow speed 3.5 m/s, temperature 30° C.; DF=3). This improved even further when the pH value of the solution comprising the biomass and the 6′-sialyllactose was pH 4.2. Compared to pH 6.3, the flux more than doubled when pH 5.2 was used and tripled when the pH value was pH 4.2.

CITED LITERATURE

[0222] WO 2015/032412 [0223] EP 2 379 708 [0224] CN 100 549 019 [0225] EP 2 896 628 [0226] U.S. Pat. No. 9,944,965