Next Level of p-Phenylene Diisothiocyanate-based Covalent Immobilization of β-d-Galactosidase: technical optimization an application

Next Level of p-Phenylene Diisothiocyanate-based Covalent Immobilization of β-d-Galactosidase: technical optimization an application | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Next Level of p-Phenylene Diisothiocyanate-based Covalent Immobilization of β-d-Galactosidase: technical optimization an application Tabea L. Boehme, Bernadette Straub, Ursula Eschenhagen, Magnus S. Schmidt This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7637179/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this study, a continuous lactose hydrolysis process in a fixed-bed reactor was developed using β-galactosidase covalently immobilized on resin beads via 1,4-phenylenediisothiocyanate (PDC) as linker. Process conditions, including temperature, enzyme loading, perfusion speed, and repeated perfusion of the same substrate solution were systematically varied. The highest glucose yields were obtained at 55°C, with increased yields observed at low perfusion speeds, high enzyme loadings, and upon repeated perfusions. Under optimized cycle perfusion over 72 h, final lactose conversion reached approximately 90% at 37°C and 80% at 22°C. A hydrolysis process in a fixed-bed reactor was successfully established, although further optimization is required. covalent enzyme immobilization fixed-bed reactor β-D-galactosidase hydrolysis lactose Aminomethylated polystyrene resin phenylenediisothiocyanate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 INTRODUCTION Due to its wide spread prevalence in the global population, lactose intolerance gains a central importance in nutrition, rising interest in production of lactose free alternatives for the food industry. An economically efficient way of producing lactose free dairy products uses the hydrolysis properties of the enzyme β-Galactosidase ( 1 , 2 ). Currently, all industrially applicated processes for Lactase free dairy products use soluble Lactase that is added to the product ( 3 ). Hence, the Lactase enzyme can only be used for a single process, leading to higher production costs ( 4 ). This leads to higher market costs of the product, lowering the acceptance on the customer side ( 5 ). Immobilizing Lactase covalently can bypass that limitation, reducing production costs and create a more sustainable way of producing lactose free nutrition ( 6 ). In addition, immobilizing enzymes offers further advantages, such as a higher operational stability, as well as the absence in the final product leading to a lower risk of Maillard reactions ( 7 , 5 ). An initial study on covalently immobilized Lactase using p-phenylenediisothiocyanate as linker was already published and builds the basis of this paper ( 8 ). Unfortunately, immobilizing β-Galactosidase is also accompanied by several disadvantages including risk of microbial contamination and lower enzyme activity ( 9 ). The latter expresses the need of technical improvement, to achieve a complete conversion of lactose. In this study, a continuous hydrolysis process was established and optimized. For this, different parameters, such as temperature, outflow speed, number of perfusion cycles and amount of Lactase were examined, to establish improved process conditions. The resulting conditions were then implemented in a cycle perfusion, lasting several days, to examine the best possible degree of hydrolysis under these conditions. 2 MATERIALS AND METHODS The following section will give an overview of the used materials and methods to implement and optimize hydrolysis of lactose by β-Galactosidase. 2.1 Materials β-Galactosidase (from A. oryzae ( 10 )) was purchased from sanotact GmbH (Münster, Germany) in the form of Lactase tablets. Lactose monohydrate was purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Enzytec™ Liquid D-Glucose Test-Kits were obtained from R-Biopharm AG (Darmstadt, Germany). All solvents and other chemicals were of analytical grade. Two glass columns, one with a heating jacket and one without, were purchased from Th. Geyer GmbH & Co. KG (Renningen, Germany). 2.2 Solutions and buffer for the column perfusion experiments Preparation of phosphate buffer saline (PBS buffer) 11.8 mM, pH 7.3 Sodium dihydrogen phosphate dihydrate (0.365 g), disodium hydrogen phosphate dihydrate (1.37 g), sodium chloride (8.75 g), magnesium chloride heptahydrate (0.02 g) and calcium chloride dihydrate (0.013 g) were dissolved in deionized water. 1 ml of manganese (II) chloride stock solution (159.0 mM) was added and the pH was adjusted to 7.3 using 0.1 M hydrogen chloride. The prepared solution was filled up to 1 liter. Preparation of phosphate buffer saline with Tween® 20 and 20% (v/v) DSMO (PBS-T 20% (v/v) DMSO) 11,8 mM, pH 7,3 400 µl of Tween® 20, 800 ml of PBS buffer (pH 7.3) and 200 ml of DMSO were mixed. Preparation of lactose solution (4.7 g/L) 13.0 mM 2.093 g of 3-(N-morpholino)propanesulfonic acid (MOPS) was dissolved in 1 liter of deionized water and 4.95 g of lactose monohydrate was added to the 1 liter of MOPS buffer. 2.3 Methods Immobilized Lactase was inserted into a glass column, to build a fixed bed reactor. Different experiments were conducted to optimize the degradation degree of lactose during perfusion through the reactor. Immobilization of β-Galactosidase The immobilization procedure and efficiency data of the Lactase used in this study have been published previously (Boehme et al., ( 11 )). The approaches and reactors were screened before their selection for usage. An overview of the used immobilized reactor for each experiment can be seen in Table 1 . Experimental design The first column perfusion system, established by Straub, transported the lactose solution from a glass bottle into the column using a pressure system that operated at 0.2 to 0.4 bar. The efflux speed was adjusted by a stopcock integrated in the column. The heatable column was tempered via a thermostat (Thermomix ME 852 112/3, B.Braun Melsungen AG, Melsungen, Germany), pumping heated water through the column jacket. Using the system from Straub as basis, a peristaltic pump (Reglo, Ismatec® – Cole-Parmer GmbH, Wertheim, Germany) was attached to the end of the column, to adjust the efflux speed more precisely (Fig. 1 ). A cycle system was established, transporting the efflux back into the initial glass bottle, to enable repeated column perfusion (Fig. 2 ). Perfusion experiments In initial perfusion experiments conducted by Straub, a lactose solution (4.7 g/l) was passed through the column under varying conditions. Four experiments were performed in which temperature, flow rate, and Lactase loading were varied, and the same lactose solution was collected and perfused repeatedly. The exact perfusion conditions can be seen in Table 1 . Table 1 Perfusion experiments by Bernadette Straub and their parameter settings. Experiment Temperature Perfusion speed Utilized immobilized Lactase Duration Sampeling frequency Temperature 55°C 0.375–0.9 ml/min Approach Straub: Column 30 min 10 min Repeated perfusion 37°C 0.27–1.129 ml/min Approach Straub: Column 3x 30 min 30 min Various flow rates 37°C 0.312–1.282 ml/min Approach Straub: Column 2h 10 min Lactase loading 37°C 0.2–0.875 ml/min Approach Straub: Column + R4, R6, R12 + R13 4x 10 min 10 min Similar experiments were conducted using the pump controlled system set-up. All experiments were conducted at 22°C and 37°C. If not further specified, a basic pump speed of 0.5 ml/min was set. In the experiment examining temperature variation, a single run perfusion was repeated at 22°C, 37°C and 55°C. Samples were collected for 10 minutes over 60 minutes. Lactase from all reactors of Approach 1 and 2 were used to conduct the perfusion. The Perfusion speed was increased in another experiment from 0.5 ml/min to 4.5 ml/min and lastly to 9 ml/min. All three perfusions were conducted using the cycle system with a 100 ml lactose solution. During the perfusion at 37°C, one sample was taken every hour for a total of 6 hours, whereas at 22°C, samples were collected only in triplicate after 6 hours. Again, all reactors of Approach 1 and 2 were selected for perfusion. The increased Lactase amount experiment was conducted by adding reactor after reactor into the, at the beginning empty, column. For each added reactor, a single run perfusion was conducted for 30 minutes, collecting three samples in total over 10 minutes. The perfusion at 22°C used the Lactase from reactor 5, 6, 7, 8, 9 and 10 of Approach 4 in the respective order, while the one at 37°C was conducted using reactor 1, 2, 5, 6, 7 and 8 of Approach 3, again in the respective order. Lastly, a long time cycle perfusion experiment was carried out for 72 hours, using the same Lactase as in the Lactase increase experiment and was done for both temperatures respectively. 100 ml lactose was initially added to the cycle system and approximately 0.5 ml was taken for each sample, drawn each day at 7:30, 13:30 and 19:30 o’clock. All samples were analysed using the Enzytec™ Liquid D-Glucose Test-Kits and a UV-VIS Spectrometer (UVmini-1240, Shimadzu Corporation, Kyoto, Japan), following the manufacturer's instructions, with the exception, that all volumes were reduced by half. Lactose conversion was evaluated based on the glucose yield, defined as the amount of glucose produced relative to its maximum theoretical amount (Eq. I). \(\:\text{G}\text{l}\text{u}\text{c}\text{o}\text{s}\text{e}\:\text{y}\text{i}\text{e}\text{l}\text{d}\:\left(\text{%}\right)=\frac{{\text{C}}_{\text{m}\text{a}\text{x}.,\:\:\text{a}\text{c}\text{t}\text{u}\text{a}\text{l}}}{{\text{C}}_{\text{m}\text{a}\text{x}.,\:\text{t}\text{h}\text{e}\text{o}\text{r}\text{e}\text{t}\text{i}\text{c}\text{a}\text{l}}}\text{*}100\:\text{%}\) I 3 RESULTS The following passages show the results of the initial perfusion experiments performed by Staub and the following experiments using advanced set ups. 3.1 Initial column perfusion experiments by Straub performed under variable conditions The results from the initial perfusion experiments without a pump, performed by Straub, are seen in Table 2 to Table 5 . The results of the experiment with the perfusions at different speeds is shown in Table 2 . The perfusion over 125 minutes showed a speed range from 0.025 ml/min up to 1.650 ml/min. At the lowest speed, a yield of 42% was achieved. The highest yield of 52% was obtained at speed 0.35 ml/min, while another sample at the same speed only achieved 37%. At the highest speed, the lowest glucose yield of 13% was reached. In the perfusion over 60 minutes, a speed range from 0.275 ml/min to 0.675 ml/min was recorded. The lowest perfusion speed showed also the lowest yield with 22%, while the highest speed showed a yield of 29%. The highest yield was reached at the second lowest speed of 0.3 ml/min. Throughout both perfusions, the same yield was achieved by samples with different perfusion speeds. Table 2 Glucose yields, and Glucose concentration obtained at different perfusion speeds in two approaches. Fraction Perfusion speed (ml/min) Glucose (g/l) Glucose yield (%) Fraction Perfusion speed (ml/min) Glucose (g/l) Glucose yield (%) Perfusion 1a: Different perfusion speeds, 37°C Perfusion 1b: Different perfusion speeds, 37°C A 1.05 0.451 18 a 0.275 0.532 22 B 0.9 0.652 26 b 0.3 0.807 33 C 0.925 0.565 23 c 0.6 0.766 31 D 1.375 0.413 17 d 0.525 0.714 29 E 1.65 0.312 13 e 0.575 0.711 29 F 1.1 0.425 17 f 0.675 0.72 29 G 1.5 0.388 16 H 0.675 0.519 21 I 0.5 0.624 25 J 0.35 0.926 37 K 0.35 1.282 52 L 0.025 1.048 42 Repeating the perfusion of the same Lactase solution three times as displayed in Table 3 , showed a severe yield increase. The perfusion with 29 ml showed an yield increase from 21% over 39% to 46%. It presents a much higher increase of 18% in the first repetition at a lower perfusion speed of 0.675 ml/min when compared to the second repetition at 0.833 ml/min increasing by 7%. The second approach with a 15 ml Lactase solution showed overall lower yields, increasing from 11% up to 18% and finally 26%. This time, the second perfusion, at a speed of 1.3 ml/min, showed slightly higher increase compared to the first repetition at a speed of 2.6 ml/min. Table 3 Glucose concentration and yield with corresponding perfusion speeds for the same lactose solution, after repeated perfusions. Fraction Perfusion speed (ml/min) Glucose (g/l) Glucose yield (%) Fraction Perfusion speed (ml/min) Glucose (g/l) Glucose yield (%) Perfusion 2a: Repeated Perfusion of the same Lactase solution (29 ml), 37°C Perfusion 2b: Repeated Perfusion of the same Lactase solution (15 ml), 37°C 1.1 0.967 0.509 21 1.2 1.875 0.27 11 2.1 0.675 0.958 39 2.2 2.8 0.435 18 3.1 0.833 1.129 46 3.2 1.3 0.648 26 The perfusion experiment at 55°C (Table 4 ) showed glucose yields between 26% and 38% with an average of approximately 30% and standard deviation of 6.7%. In comparison to values with a similar perfusion speed at 37°C, only little or no increase was observed. Increasing the amount of immobilized Lactase to 150% (Table 5 ) showed an average yield of approximately 29% with a standard deviation of 10.8%. Both increase and decrease could be observed, when compared to samples with similar speeds, though certain variations of the speed between the approaches existed. Table 4 Glucose concentration and yield with corresponding perfusion speeds from a perfusion at 55°C. Fraction Perfusion speed (ml/min) Glucose (g/l) Glucose yield (%) Perfusion 3: Perfusion at a higher temperature, 55°C one 0.9 0.648 26 two 0.425 0.662 27 three 0.375 0.952 38 Table 5 Glucose concentration and yield with corresponding perfusion speeds for the perfusion containing higher amount of immobilized Lactase. Fraction Perfusion speed (ml/min) Glucose (g/l) Glucose yield (%) Perfusion 4: Increased amount of immobilized Lactase (150%), 37°C Blank I 0.875 0.381 15 II 0.2 0.67 27 III 0.15 1.02 41 IV 0.7 0.776 31 3.2 Column perfusion at 22°C, 37°C and 55°C The glucose yield, obtained from perfusions at three different temperatures, is presented in Fig. 3 . At 22°C, the lowest yield of 3% was recorded. Raising the temperature to 37°C resulted in a 25% yield. The highest yield of 31.8% was achieved at 55°C, showing an approximately tenfold increase compared to the yield at 22°C. 3.3 Perfusion cycle with different pump speeds for 6 hours at 22°C and 37°C The results obtained from the cycle run perfusions after six hours with three different pump speeds at 22°C as well as 37°C are shown in Fig. 4 . The highest yields at each temperature were achieved at the lowest pump speed of 0.5 ml/min, with 21.6% at 22°C and 30.1% at 37°C. At both temperatures, similar yields were obtained at pump speeds of 4.5 ml/min and 9 ml/min. The 22°C perfusion resulted in a slightly higher yield of 17.9% at 9 ml/min compared to 17.3% at 4.5 ml/min. At 37°C, a slightly lower yield of 25.3% was observed at a speed of 9 ml/min compared to a 25.9% yield at 4.5 ml/min. Across all pump speeds, the yields at 22°C were approximately 8% lower than the ones at 37°C. Figure 5 shows the hourly development of the glucose yield for the six-hour perfusion at 37°C, previously shown in Fig. 4 . Across all speeds, a linear increase in yield was observed. After one hour, the highest yield of 17.1% was observed at a speed of 0.5 ml/min. Yields of 4.5 ml/min and 9 ml/min were lower, at 6.0% and 5.4%, respectively. Over the time of 6 hours, the yields of speed 4.5 ml/min and 9 ml/min increased at a faster rate than at a speed of 0.5 ml/min, leading the final yields described above. 3.4 Column perfusion with different amounts of immobilized Lactase Column perfusions with an increasing amount of immobilized Lactase were performed at 22°C and 37°C. Figure 6 shows the amount of immobilized Lactase in the column for each perfusion and its corresponding yield. In both perfusions, the yield increased steadily. At 22°C, the initial yield started at 3.5% with 2.9 g immobilized Lactase and ended at a total of 17.9% and 18.1 g immobilized Lactase. At 37°C, the same initial amount of immobilized Lactase, as at 22°C, resulted in a glucose yield of 11.1%, corresponding to an approximately threefold increase. The yield increased to 36.1% with 17.4 g of Lactase, reaching a higher final value while having a lower enzyme loading, compared to the final yield at 22°C. 3.5 Perfusion cycle for 72 hours at 22°C and 37°C The results of the 72 hour cycle run perfusions at 22°C and 37°C are shown in Fig. 8. During the first 6 hours, glucose yields increased rapidly at each temperatures, leading to a yield of 30.9% and 46.3% at 22°C and 37°C, respectively. At 22°C, the yield continued to rise steadily until hour 42, reaching approximately 80%, after which it stabilized. At 37°C, 80% yield was reached by hour 24 and increased further to approximately 90% at hour 48, after which the yield also stabilized. 4 DISCUSSION This study investigated the implementation and optimization of a continuous lactose hydrolysis process using p-phenylenediisothiocyanate-based covalent immobilization of β-D-galactosidase. The efficiency of the immobilization procedure has been characterized in detail previously ( 11 ). This data provided the basis for the present implementation of a continuous hydrolysis process. During initial perfusion experiments by Bernadette Straub, flow rates varied continuously due to manual adjustment using the stopcock. While this variability was useful for identifying the effect of the flow rate on the glucose yield, it led to heterogeneous conditions during sampling. This complicates data interpretation, particularly because the results from the glucose test kits were not always unambiguous. The subsequent implementation of a pump controlled flow successfully minimized these fluctuations, leading to a more consistent perfusion and better comparability within multiple samples. Nevertheless, due to the variety of samples with different speeds, a trend towards higher lactose conversion at lower perfusion speeds could be observed, suggesting a long time of contact to increase lactose conversation. This was also identified in a study from Klein et Al. ( 12 ), showing that an increase of the outflow of lactose and whey solution from 2.6 ml/min to 3.4 ml/min resulted in a reduction of the hydrolysis degree in both solutions from over 90 % to 86% in latose solution and 80 % in why solution. Perfusion at the reported temperature optimum of 55°C for β-Galactosidase, derived from the fungi Aspergillus oryzae , showed the highest level of lactose, though at 37°C, glucose levels were still at a sufficient level ( 9 , 13 ). Contradicting this, the initial experiment by Straub at 55°C displayed no enhanced yields compared to the samples at 37°C with similar speeds. This maybe attributed to the heterogeneous conditions, mentioned above, due to varying perfusion speeds within the samples. In all experiments done additionally at 22°C, highly lower glucose levels were observed in comparison to the tempered perfusions at 37°C, signalling insufficient lactose conversion for the use in industrial processes. Overall, this indicates, that temperature plays a crucial role in the optimization of the process. Since microbial contamination is a big problem in β-Galactosidase immobilization, this has to be addressed further ( 9 ). Recent literature suggests, that milk hydrolysis should be performed at low temperatures, to reduce microbial growth, contradicting the use of the Aspergillus oryzae β-Galactosidase ( 14 , 9 ). However, another approach to reduce microbial contamination suggested by recent studies is the use of thermostable β-Galactosidases that endure the pasteurization temperature of 65°C ( 15 , 16 ). A Study by Farag et. Al. showed, that Aspergillus oryzae β-Galactosidase maintains over 50 % of its enzyme activity at 70 °, indicating the possible use of the enzyme at pasteurization temperatures, to reduce the risk of a microbial contamination ( 17 ). Another aspect of operating at high temperatures is the need for a high thermal stability. Fortunately, Lactase of Aspergillus oryzae is known for its high thermostability, that is improved by immobilization ( 18 , 19 ). The initial study of an increasing amount of immobilized Lactase by Straub showed no clear increase in glucose yields, though a comparison is complicated due to the absence of similar conditions. An improved experimental set-up using the pump controlled system, presented a linear correlation between the lactose amount and glucose yield. This suggests, that the amount of immobilized Lactase can be further increased to lead to higher glucose yields. Though, due to the limited availability of immobilized Lactase and its production costs, the increase in amount of immobilized Lactase is restricted. Furthermore, an increase in yield is highly affected by the immobilized enzyme efficiency. Since single run perfusions showed yields far from 100%, a circular perfusion set-up was implemented, mimicking a possible industrial process at a small scale. The repeated perfusions of the same lactose solution by Straub showed a clear increase in yield, suggesting that the more cycles the Lactase undergoes, the higher the yield should be, while the time of contact also needs to be sufficient. Hence, a compromise between the number of cycles and time of contact hat to be examined by variation of the perfusion speed. Results after 6 hours showed the highest lactose conversion in the perfusion with the slowest perfusion speed, indicating the higher importance of contact time over numbers of cycles. Contradicting this, the development of the glucose yield over the 6 hours displays a faster increase at higher speeds, suggesting that the duration plays a crucial role in determining the optimal perfusion speed. Long term cycle perfusion, at optimal conditions showed a stagnation in lactose conversion at 90% after 48 hours, although 80% was already achieved after 24 hours. The same trend at lower levels has been observed in the 22°C perfusion, supporting the validity of the curve. The stagnation below a 100% yield might be addressed to the inhibitory effect of the hydrolysis product galactose on β-Galactosidase ( 9 ). This limitation can also be observed in another immobilization approach using the entrapment method, achieving only 87 % f hydrolysis degree after 36 hours ( 14 ). Another immobilization approach, using alginate immobilized Aspergillus oryzae β-Galactosidase, also shows limited degree of hydrolysis at 70.9 % but as only carried out for 12 hours and as a batch process ( 20 ). At this point it needs to be highlighted, that the mentioned approaches were carried out using skim milk, while the experiments of this study used a lactose solution containing a 10 times lower concentration than milk. Therefore, qualitative comprehension of the approaches is impossible. Nevertheless, this highlights the fact, that further improvement in yield is inevitable for industrial processes. The optimal perfusion speed could be examined at longer cycle perfusion durations for optimal balance of cycle number and contact time. Furthermore, the reduction of the possibility for microbial contamination should be addressed by testing the effectivity of the immobilized ß-Galactosidase at pasteurization temperatures, or investigating the use of low temperature operating ß-Galactosidase. However, a first approach of a continuous hydrolysis process using immobilized Lactase in a fixed bed reactor was successfully established. Declarations Supporting Information. The following files are available free of charge. Raw data perfusion experiments Bernadette Straub (XLSX) Raw data perfusion experiments Tabea Boehme including long term cycle perfusion experiments (XLSX) Author Contributions Tabea Boehme: Writing - Original Draft, Data Curation, Investigation. Bernadette Straub: Data Curation, Investigation. Ursula Eschenhagen: Resources, Methodology. Magnus S. Schmidt: Conceptualization, Supervision, Writing – review & editing. No funds, grants, or other support was received. Acknowledgement We would like to thank the ReAching program of the faculty Health, Medical and Life Sciences, Furtwangen University, for support. Author information Tabea L. Boehme, EMail: [email protected] Bernadette Straub: EMail: [email protected] Ursula Eschenhagen, Email: [email protected] Magnus S. Schmidt, Email: [email protected] Credit Photograph courtesy of Tabea L. Boehme. Copyright 2025 Tabea L. Boehme. Images are free domain. References Kalathinathan P, Sain A, Pulicherla K, Kodiveri Muthukaliannan G (2023) A Review on the Various Sources of β-Galactosidase and Its Lactose Hydrolysis Property. Curr Microbiol 80(4):122 Matthews BW (2005) The structure of E. coli beta-galactosidase. 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ACS Omega 8(30):27585–27596 Dekker P (2019) Enzymes Exogenous to Milk in Dairy Technology: β-d-Galactosidase. Reference Module in Food Science. Elsevier sanotact GmbH Sanotact lactase 24 000 6h depot (40 lactase tablets) Lactose tablets with depot effect for lactose intolerance & milk intolerance immediate effect & 6h long-term depot 24 000 depot 40 tablets [cited 2025 Aug 27]. Available from: URL: https://www.gosupps.com/sanotact-lactase-24-000-6h-depot-40-lactase-tablets-lactose-tablets-with-depot-effect-for-lactose-intolerance-milk-intolerance-immediate-effect-6h-long-term-depot-24-000-depot-40-tablets.html ? Boehme T, Straub B, Eschenhagen U, Schmidt M (2025) Dataset on the analysis of β-galactosidase immobilization efficiency on AMP resin in syringe and column reactors Klein MP, Fallavena LP, Da Schöffer JN, Ayub MAZ, Rodrigues RC, Ninow JL et al (2013) High stability of immobilized β-D-galactosidase for lactose hydrolysis and galactooligosaccharides synthesis. 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Enzym Microb Technol 34(2):85–93 Hirohara H, Yamamoto H, Kawano E, Nagase T (1982) Continuous Hydrolysis of Lactose in Skim Milk and Acid Whey by Immobilized Lactase of Aspergillus Oryzae. In: Chibata I, Fukui S, Wingard LB (eds) Enzyme Engineering. Springer US, Boston, MA, pp 295–297 Bayramoglu G, Cimen AG, Arica MY (2023) Immobilisation of β-galactosidase onto double layered hydrophilic polymer coated magnetic nanoparticles: Preparation, characterisation and lactose hydrolysis. Int Dairy J 138:105545 Katrolia P, Liu X, Li G, Kopparapu NK (2019) Enhanced Properties and Lactose Hydrolysis Efficiencies of Food-Grade β-Galactosidases Immobilized on Various Supports: a Comparative Approach. Appl Biochem Biotechnol 188(2):410–423 Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7637179","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516350951,"identity":"5d5938b1-15ab-4690-a5cd-390dd3922f4b","order_by":0,"name":"Tabea L. 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Schmidt","email":"data:image/png;base64,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","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Magnus","middleName":"S.","lastName":"Schmidt","suffix":""}],"badges":[],"createdAt":"2025-09-17 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07:05:07","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82472,"visible":true,"origin":"","legend":"","description":"","filename":"rs76371790structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/920c9fc9e843d20cdee00d01.xml"},{"id":91818914,"identity":"c2e2101d-58f4-4280-9086-305de476dddb","added_by":"auto","created_at":"2025-09-22 07:05:39","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92516,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/0c7035ef630ad3b28954ce59.html"},{"id":91818671,"identity":"e7f5d8fc-6f52-49ac-8184-431a72037c1c","added_by":"auto","created_at":"2025-09-22 07:05:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":409089,"visible":true,"origin":"","legend":"\u003cp\u003eSet-up of the singe run system.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/9a3d9c4645bee35f365ee89e.png"},{"id":91819012,"identity":"ff388d43-c09a-4118-966c-5da13148d03b","added_by":"auto","created_at":"2025-09-22 07:05:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":405707,"visible":true,"origin":"","legend":"\u003cp\u003eSet-up of the cycle run system.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/cd1874cce30f007495624439.png"},{"id":91818886,"identity":"192ea51e-3b5b-48df-bab0-6fdb8b7fc1da","added_by":"auto","created_at":"2025-09-22 07:05:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":26946,"visible":true,"origin":"","legend":"\u003cp\u003eGlucose yields of different column perfusions at 22 °C, 37°C and 55 °C.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/f7aec5951656d86037c94743.png"},{"id":91818906,"identity":"78ce7a97-af1e-474f-960e-23ab7f7abfe0","added_by":"auto","created_at":"2025-09-22 07:05:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33084,"visible":true,"origin":"","legend":"\u003cp\u003eGlucose yield of cycle run perfusions at 22 °C and 37 °C with different pump speeds.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/e64ef3149d77a0a6353154fc.png"},{"id":91818871,"identity":"b7a97d60-ca3a-4ef9-ac95-08905d2ff40d","added_by":"auto","created_at":"2025-09-22 07:05:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":75462,"visible":true,"origin":"","legend":"\u003cp\u003eHourly development of the glucose yield of three 6 hour cycle run perfusions with different pump speeds at 37 °C.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/fbb727df8ff6bf0380406b70.png"},{"id":91818813,"identity":"cb267333-c5d9-472d-8b03-4a8261abf9d8","added_by":"auto","created_at":"2025-09-22 07:05:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":110922,"visible":true,"origin":"","legend":"\u003cp\u003ePerfusion with increased amount of immobilized lactase at 22 °C and 37 °C.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/72d59ae58326b93e4fc9796b.png"},{"id":91818868,"identity":"6888fc95-60f6-4f0b-86e1-325ad9e58191","added_by":"auto","created_at":"2025-09-22 07:05:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":105322,"visible":true,"origin":"","legend":"\u003cp\u003eGlucose yields of cycle run perfusion for 72 hours at 22 °C and 37 °C.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/7f092b4048468eebb8c6f478.png"},{"id":91820209,"identity":"04230fb9-0ffa-4f29-8ef6-bc810021f28a","added_by":"auto","created_at":"2025-09-22 07:09:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2456995,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7637179/v1/173ad160-42a1-4f16-9596-fca5b5467f7e.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eNext Level of p-Phenylene Diisothiocyanate-based Covalent Immobilization of β-d-Galactosidase: technical optimization an application\u003c/p\u003e","fulltext":[{"header":"1 INTRODUCTION","content":"\u003cp\u003eDue to its wide spread prevalence in the global population, lactose intolerance gains a central importance in nutrition, rising interest in production of lactose free alternatives for the food industry. An economically efficient way of producing lactose free dairy products uses the hydrolysis properties of the enzyme β-Galactosidase (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Currently, all industrially applicated processes for Lactase free dairy products use soluble Lactase that is added to the product (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Hence, the Lactase enzyme can only be used for a single process, leading to higher production costs (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). This leads to higher market costs of the product, lowering the acceptance on the customer side (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Immobilizing Lactase covalently can bypass that limitation, reducing production costs and create a more sustainable way of producing lactose free nutrition (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In addition, immobilizing enzymes offers further advantages, such as a higher operational stability, as well as the absence in the final product leading to a lower risk of Maillard reactions (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). An initial study on covalently immobilized Lactase using p-phenylenediisothiocyanate as linker was already published and builds the basis of this paper (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Unfortunately, immobilizing β-Galactosidase is also accompanied by several disadvantages including risk of microbial contamination and lower enzyme activity (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The latter expresses the need of technical improvement, to achieve a complete conversion of lactose. In this study, a continuous hydrolysis process was established and optimized. For this, different parameters, such as temperature, outflow speed, number of perfusion cycles and amount of Lactase were examined, to establish improved process conditions. The resulting conditions were then implemented in a cycle perfusion, lasting several days, to examine the best possible degree of hydrolysis under these conditions.\u003c/p\u003e"},{"header":"2 MATERIALS AND METHODS","content":"\u003cp\u003eThe following section will give an overview of the used materials and methods to implement and optimize hydrolysis of lactose by β-Galactosidase.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eβ-Galactosidase (from A. oryzae (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)) was purchased from sanotact GmbH (M\u0026uuml;nster, Germany) in the form of Lactase tablets. Lactose monohydrate was purchased from Carl Roth GmbH \u0026amp; Co. KG (Karlsruhe, Germany). Enzytec\u0026trade; Liquid D-Glucose Test-Kits were obtained from R-Biopharm AG (Darmstadt, Germany). All solvents and other chemicals were of analytical grade. Two glass columns, one with a heating jacket and one without, were purchased from Th. Geyer GmbH \u0026amp; Co. KG (Renningen, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Solutions and buffer for the column perfusion experiments\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003ePreparation of phosphate buffer saline (PBS buffer)\u003c/strong\u003e\u003cp\u003e11.8 mM, pH 7.3\u003c/p\u003e\u003c/p\u003e\u003cp\u003eSodium dihydrogen phosphate dihydrate (0.365 g), disodium hydrogen phosphate dihydrate (1.37 g), sodium chloride (8.75 g), magnesium chloride heptahydrate (0.02 g) and calcium chloride dihydrate (0.013 g) were dissolved in deionized water. 1 ml of manganese (II) chloride stock solution (159.0 mM) was added and the pH was adjusted to 7.3 using 0.1 M hydrogen chloride. The prepared solution was filled up to 1 liter.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePreparation of phosphate buffer saline with Tween\u0026reg; 20 and 20% (v/v) DSMO (PBS-T 20% (v/v) DMSO)\u003c/strong\u003e\u003cp\u003e11,8 mM, pH 7,3\u003c/p\u003e\u003c/p\u003e\u003cp\u003e400 \u0026micro;l of Tween\u0026reg; 20, 800 ml of PBS buffer (pH 7.3) and 200 ml of DMSO were mixed.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePreparation of lactose solution (4.7 g/L)\u003c/strong\u003e\u003cp\u003e13.0 mM\u003c/p\u003e\u003c/p\u003e\u003cp\u003e2.093 g of 3-(N-morpholino)propanesulfonic acid (MOPS) was dissolved in 1 liter of deionized water and 4.95 g of lactose monohydrate was added to the 1 liter of MOPS buffer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Methods\u003c/h2\u003e\u003cp\u003eImmobilized Lactase was inserted into a glass column, to build a fixed bed reactor. Different experiments were conducted to optimize the degradation degree of lactose during perfusion through the reactor.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmobilization of β-Galactosidase\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe immobilization procedure and efficiency data of the Lactase used in this study have been published previously (Boehme et al., (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)). The approaches and reactors were screened before their selection for usage. An overview of the used immobilized reactor for each experiment can be seen in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe first column perfusion system, established by Straub, transported the lactose solution from a glass bottle into the column using a pressure system that operated at 0.2 to 0.4 bar. The efflux speed was adjusted by a stopcock integrated in the column. The heatable column was tempered via a thermostat (Thermomix ME 852 112/3, B.Braun Melsungen AG, Melsungen, Germany), pumping heated water through the column jacket.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUsing the system from Straub as basis, a peristaltic pump (Reglo, Ismatec\u0026reg; \u0026ndash; Cole-Parmer GmbH, Wertheim, Germany) was attached to the end of the column, to adjust the efflux speed more precisely (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A cycle system was established, transporting the efflux back into the initial glass bottle, to enable repeated column perfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePerfusion experiments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn initial perfusion experiments conducted by Straub, a lactose solution (4.7 g/l) was passed through the column under varying conditions. Four experiments were performed in which temperature, flow rate, and Lactase loading were varied, and the same lactose solution was collected and perfused repeatedly. The exact perfusion conditions can be seen in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePerfusion experiments by Bernadette Straub and their parameter settings.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperiment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemperature\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePerfusion speed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUtilized immobilized Lactase\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDuration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSampeling frequency\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTemperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.375\u0026ndash;0.9 ml/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eApproach Straub: Column\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRepeated perfusion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.27\u0026ndash;1.129 ml/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eApproach Straub: Column\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3x 30 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e30 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVarious flow rates\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.312\u0026ndash;1.282 ml/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eApproach Straub: Column\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLactase loading\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.2\u0026ndash;0.875 ml/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eApproach Straub: Column\u0026thinsp;+\u0026thinsp;R4, R6, R12\u0026thinsp;+\u0026thinsp;R13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4x 10 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSimilar experiments were conducted using the pump controlled system set-up. All experiments were conducted at 22\u0026deg;C and 37\u0026deg;C. If not further specified, a basic pump speed of 0.5 ml/min was set.\u003c/p\u003e\u003cp\u003eIn the experiment examining temperature variation, a single run perfusion was repeated at 22\u0026deg;C, 37\u0026deg;C and 55\u0026deg;C. Samples were collected for 10 minutes over 60 minutes. Lactase from all reactors of Approach 1 and 2 were used to conduct the perfusion.\u003c/p\u003e\u003cp\u003eThe Perfusion speed was increased in another experiment from 0.5 ml/min to 4.5 ml/min and lastly to 9 ml/min. All three perfusions were conducted using the cycle system with a 100 ml lactose solution. During the perfusion at 37\u0026deg;C, one sample was taken every hour for a total of 6 hours, whereas at 22\u0026deg;C, samples were collected only in triplicate after 6 hours. Again, all reactors of Approach 1 and 2 were selected for perfusion.\u003c/p\u003e\u003cp\u003eThe increased Lactase amount experiment was conducted by adding reactor after reactor into the, at the beginning empty, column. For each added reactor, a single run perfusion was conducted for 30 minutes, collecting three samples in total over 10 minutes. The perfusion at 22\u0026deg;C used the Lactase from reactor 5, 6, 7, 8, 9 and 10 of Approach 4 in the respective order, while the one at 37\u0026deg;C was conducted using reactor 1, 2, 5, 6, 7 and 8 of Approach 3, again in the respective order.\u003c/p\u003e\u003cp\u003eLastly, a long time cycle perfusion experiment was carried out for 72 hours, using the same Lactase as in the Lactase increase experiment and was done for both temperatures respectively. 100 ml lactose was initially added to the cycle system and approximately 0.5 ml was taken for each sample, drawn each day at 7:30, 13:30 and 19:30 o\u0026rsquo;clock.\u003c/p\u003e\u003cp\u003eAll samples were analysed using the Enzytec\u0026trade; Liquid D-Glucose Test-Kits and a UV-VIS Spectrometer (UVmini-1240, Shimadzu Corporation, Kyoto, Japan), following the manufacturer's instructions, with the exception, that all volumes were reduced by half. Lactose conversion was evaluated based on the glucose yield, defined as the amount of glucose produced relative to its maximum theoretical amount (Eq. I).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{G}\\text{l}\\text{u}\\text{c}\\text{o}\\text{s}\\text{e}\\:\\text{y}\\text{i}\\text{e}\\text{l}\\text{d}\\:\\left(\\text{%}\\right)=\\frac{{\\text{C}}_{\\text{m}\\text{a}\\text{x}.,\\:\\:\\text{a}\\text{c}\\text{t}\\text{u}\\text{a}\\text{l}}}{{\\text{C}}_{\\text{m}\\text{a}\\text{x}.,\\:\\text{t}\\text{h}\\text{e}\\text{o}\\text{r}\\text{e}\\text{t}\\text{i}\\text{c}\\text{a}\\text{l}}}\\text{*}100\\:\\text{%}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cp\u003eThe following passages show the results of the initial perfusion experiments performed by Staub and the following experiments using advanced set ups.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Initial column perfusion experiments by Straub performed under variable conditions\u003c/h2\u003e\u003cp\u003eThe results from the initial perfusion experiments without a pump, performed by Straub, are seen in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e to Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe results of the experiment with the perfusions at different speeds is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The perfusion over 125 minutes showed a speed range from 0.025 ml/min up to 1.650 ml/min. At the lowest speed, a yield of 42% was achieved. The highest yield of 52% was obtained at speed 0.35 ml/min, while another sample at the same speed only achieved 37%. At the highest speed, the lowest glucose yield of 13% was reached. In the perfusion over 60 minutes, a speed range from 0.275 ml/min to 0.675 ml/min was recorded. The lowest perfusion speed showed also the lowest yield with 22%, while the highest speed showed a yield of 29%. The highest yield was reached at the second lowest speed of 0.3 ml/min. Throughout both perfusions, the same yield was achieved by samples with different perfusion speeds.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGlucose yields, and Glucose concentration obtained at different perfusion speeds in two approaches.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFraction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePerfusion speed (ml/min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlucose (g/l)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGlucose yield (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFraction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003ePerfusion speed (ml/min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eGlucose (g/l)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u003cp\u003eGlucose yield (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003ePerfusion 1a: Different perfusion speeds, 37\u0026deg;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c10\" namest=\"c5\"\u003e\u003cp\u003ePerfusion 1b: Different perfusion speeds, 37\u0026deg;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.451\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.275\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.532\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.652\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.807\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.925\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.565\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.766\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.375\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.413\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.525\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.714\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.312\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ee\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.575\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.711\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.425\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ef\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.675\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.388\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.675\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.519\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.624\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.926\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.282\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.048\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eRepeating the perfusion of the same Lactase solution three times as displayed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, showed a severe yield increase. The perfusion with 29 ml showed an yield increase from 21% over 39% to 46%. It presents a much higher increase of 18% in the first repetition at a lower perfusion speed of 0.675 ml/min when compared to the second repetition at 0.833 ml/min increasing by 7%. The second approach with a 15 ml Lactase solution showed overall lower yields, increasing from 11% up to 18% and finally 26%. This time, the second perfusion, at a speed of 1.3 ml/min, showed slightly higher increase compared to the first repetition at a speed of 2.6 ml/min.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGlucose concentration and yield with corresponding perfusion speeds for the same lactose solution, after repeated perfusions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFraction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePerfusion speed (ml/min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlucose (g/l)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGlucose yield (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFraction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePerfusion speed (ml/min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eGlucose (g/l)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eGlucose yield (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003ePerfusion 2a: Repeated Perfusion of the same Lactase solution (29 ml), 37\u0026deg;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e\u003cp\u003ePerfusion 2b: Repeated Perfusion of the same Lactase solution (15 ml), 37\u0026deg;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.967\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.509\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.875\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.675\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.958\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.435\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.833\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.648\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe perfusion experiment at 55\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) showed glucose yields between 26% and 38% with an average of approximately 30% and standard deviation of 6.7%. In comparison to values with a similar perfusion speed at 37\u0026deg;C, only little or no increase was observed.\u003c/p\u003e\u003cp\u003eIncreasing the amount of immobilized Lactase to 150% (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed an average yield of approximately 29% with a standard deviation of 10.8%. Both increase and decrease could be observed, when compared to samples with similar speeds, though certain variations of the speed between the approaches existed.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGlucose concentration and yield with corresponding perfusion speeds from a perfusion at 55\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFraction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePerfusion speed (ml/min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlucose (g/l)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGlucose yield (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003ePerfusion 3: Perfusion at a higher temperature, 55\u0026deg;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.648\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etwo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.425\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.662\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ethree\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.375\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.952\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGlucose concentration and yield with corresponding perfusion speeds for the perfusion containing higher amount of immobilized Lactase.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFraction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePerfusion speed (ml/min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlucose (g/l)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGlucose yield (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003ePerfusion 4: Increased amount of immobilized Lactase (150%), 37\u0026deg;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlank\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.875\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.381\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Column perfusion at 22\u0026deg;C, 37\u0026deg;C and 55\u0026deg;C\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe glucose yield, obtained from perfusions at three different temperatures, is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. At 22\u0026deg;C, the lowest yield of 3% was recorded. Raising the temperature to 37\u0026deg;C resulted in a 25% yield. The highest yield of 31.8% was achieved at 55\u0026deg;C, showing an approximately tenfold increase compared to the yield at 22\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Perfusion cycle with different pump speeds for 6 hours at 22\u0026deg;C and 37\u0026deg;C\u003c/h2\u003e\u003cp\u003eThe results obtained from the cycle run perfusions after six hours with three different pump speeds at 22\u0026deg;C as well as 37\u0026deg;C are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The highest yields at each temperature were achieved at the lowest pump speed of 0.5 ml/min, with 21.6% at 22\u0026deg;C and 30.1% at 37\u0026deg;C. At both temperatures, similar yields were obtained at pump speeds of 4.5 ml/min and 9 ml/min. The 22\u0026deg;C perfusion resulted in a slightly higher yield of 17.9% at 9 ml/min compared to 17.3% at 4.5 ml/min. At 37\u0026deg;C, a slightly lower yield of 25.3% was observed at a speed of 9 ml/min compared to a 25.9% yield at 4.5 ml/min. Across all pump speeds, the yields at 22\u0026deg;C were approximately 8% lower than the ones at 37\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the hourly development of the glucose yield for the six-hour perfusion at 37\u0026deg;C, previously shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Across all speeds, a linear increase in yield was observed. After one hour, the highest yield of 17.1% was observed at a speed of 0.5 ml/min. Yields of 4.5 ml/min and 9 ml/min were lower, at 6.0% and 5.4%, respectively. Over the time of 6 hours, the yields of speed 4.5 ml/min and 9 ml/min increased at a faster rate than at a speed of 0.5 ml/min, leading the final yields described above.\u003c/p\u003e\u003ch2\u003e3.4 Column perfusion with different amounts of immobilized Lactase\u003c/b\u003e\u003c/p\u003e\u003c/h2\u003e\u003cp\u003eColumn perfusions with an increasing amount of immobilized Lactase were performed at 22\u0026deg;C and 37\u0026deg;C. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the amount of immobilized Lactase in the column for each perfusion and its corresponding yield. In both perfusions, the yield increased steadily. At 22\u0026deg;C, the initial yield started at 3.5% with 2.9 g immobilized Lactase and ended at a total of 17.9% and 18.1 g immobilized Lactase. At 37\u0026deg;C, the same initial amount of immobilized Lactase, as at 22\u0026deg;C, resulted in a glucose yield of 11.1%, corresponding to an approximately threefold increase. The yield increased to 36.1% with 17.4 g of Lactase, reaching a higher final value while having a lower enzyme loading, compared to the final yield at 22\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Perfusion cycle for 72 hours at 22\u0026deg;C and 37\u0026deg;C\u003c/h2\u003e\u003cp\u003eThe results of the 72 hour cycle run perfusions at 22\u0026deg;C and 37\u0026deg;C are shown in Fig.\u0026nbsp;8. During the first 6 hours, glucose yields increased rapidly at each temperatures, leading to a yield of 30.9% and 46.3% at 22\u0026deg;C and 37\u0026deg;C, respectively. At 22\u0026deg;C, the yield continued to rise steadily until hour 42, reaching approximately 80%, after which it stabilized. At 37\u0026deg;C, 80% yield was reached by hour 24 and increased further to approximately 90% at hour 48, after which the yield also stabilized.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 DISCUSSION","content":"\u003cp\u003eThis study investigated the implementation and optimization of a continuous lactose hydrolysis process using p-phenylenediisothiocyanate-based covalent immobilization of β-D-galactosidase. The efficiency of the immobilization procedure has been characterized in detail previously (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This data provided the basis for the present implementation of a continuous hydrolysis process. During initial perfusion experiments by Bernadette Straub, flow rates varied continuously due to manual adjustment using the stopcock. While this variability was useful for identifying the effect of the flow rate on the glucose yield, it led to heterogeneous conditions during sampling. This complicates data interpretation, particularly because the results from the glucose test kits were not always unambiguous. The subsequent implementation of a pump controlled flow successfully minimized these fluctuations, leading to a more consistent perfusion and better comparability within multiple samples. Nevertheless, due to the variety of samples with different speeds, a trend towards higher lactose conversion at lower perfusion speeds could be observed, suggesting a long time of contact to increase lactose conversation. This was also identified in a study from Klein et Al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), showing that an increase of the outflow of lactose and whey solution from 2.6 ml/min to 3.4 ml/min resulted in a reduction of the hydrolysis degree in both solutions from over 90 % to 86% in latose solution and 80 % in why solution. Perfusion at the reported temperature optimum of 55\u0026deg;C for β-Galactosidase, derived from the fungi \u003cem\u003eAspergillus oryzae\u003c/em\u003e, showed the highest level of lactose, though at 37\u0026deg;C, glucose levels were still at a sufficient level (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Contradicting this, the initial experiment by Straub at 55\u0026deg;C displayed no enhanced yields compared to the samples at 37\u0026deg;C with similar speeds. This maybe attributed to the heterogeneous conditions, mentioned above, due to varying perfusion speeds within the samples. In all experiments done additionally at 22\u0026deg;C, highly lower glucose levels were observed in comparison to the tempered perfusions at 37\u0026deg;C, signalling insufficient lactose conversion for the use in industrial processes. Overall, this indicates, that temperature plays a crucial role in the optimization of the process. Since microbial contamination is a big problem in β-Galactosidase immobilization, this has to be addressed further (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Recent literature suggests, that milk hydrolysis should be performed at low temperatures, to reduce microbial growth, contradicting the use of the \u003cem\u003eAspergillus oryzae\u003c/em\u003e β-Galactosidase (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, another approach to reduce microbial contamination suggested by recent studies is the use of thermostable β-Galactosidases that endure the pasteurization temperature of 65\u0026deg;C (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). A Study by Farag et. Al. showed, that \u003cem\u003eAspergillus oryzae\u003c/em\u003e β-Galactosidase maintains over 50 % of its enzyme activity at 70 \u0026deg;, indicating the possible use of the enzyme at pasteurization temperatures, to reduce the risk of a microbial contamination (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Another aspect of operating at high temperatures is the need for a high thermal stability. Fortunately, Lactase of \u003cem\u003eAspergillus oryzae\u003c/em\u003e is known for its high thermostability, that is improved by immobilization (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe initial study of an increasing amount of immobilized Lactase by Straub showed no clear increase in glucose yields, though a comparison is complicated due to the absence of similar conditions. An improved experimental set-up using the pump controlled system, presented a linear correlation between the lactose amount and glucose yield. This suggests, that the amount of immobilized Lactase can be further increased to lead to higher glucose yields. Though, due to the limited availability of immobilized Lactase and its production costs, the increase in amount of immobilized Lactase is restricted. Furthermore, an increase in yield is highly affected by the immobilized enzyme efficiency.\u003c/p\u003e\u003cp\u003eSince single run perfusions showed yields far from 100%, a circular perfusion set-up was implemented, mimicking a possible industrial process at a small scale. The repeated perfusions of the same lactose solution by Straub showed a clear increase in yield, suggesting that the more cycles the Lactase undergoes, the higher the yield should be, while the time of contact also needs to be sufficient. Hence, a compromise between the number of cycles and time of contact hat to be examined by variation of the perfusion speed. Results after 6 hours showed the highest lactose conversion in the perfusion with the slowest perfusion speed, indicating the higher importance of contact time over numbers of cycles. Contradicting this, the development of the glucose yield over the 6 hours displays a faster increase at higher speeds, suggesting that the duration plays a crucial role in determining the optimal perfusion speed.\u003c/p\u003e\u003cp\u003eLong term cycle perfusion, at optimal conditions showed a stagnation in lactose conversion at 90% after 48 hours, although 80% was already achieved after 24 hours. The same trend at lower levels has been observed in the 22\u0026deg;C perfusion, supporting the validity of the curve. The stagnation below a 100% yield might be addressed to the inhibitory effect of the hydrolysis product galactose on β-Galactosidase (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This limitation can also be observed in another immobilization approach using the entrapment method, achieving only 87 % f hydrolysis degree after 36 hours (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Another immobilization approach, using alginate immobilized \u003cem\u003eAspergillus oryzae\u003c/em\u003e β-Galactosidase, also shows limited degree of hydrolysis at 70.9 % but as only carried out for 12 hours and as a batch process (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). At this point it needs to be highlighted, that the mentioned approaches were carried out using skim milk, while the experiments of this study used a lactose solution containing a 10 times lower concentration than milk. Therefore, qualitative comprehension of the approaches is impossible. Nevertheless, this highlights the fact, that further improvement in yield is inevitable for industrial processes. The optimal perfusion speed could be examined at longer cycle perfusion durations for optimal balance of cycle number and contact time. Furthermore, the reduction of the possibility for microbial contamination should be addressed by testing the effectivity of the immobilized \u0026szlig;-Galactosidase at pasteurization temperatures, or investigating the use of low temperature operating \u0026szlig;-Galactosidase. However, a first approach of a continuous hydrolysis process using immobilized Lactase in a fixed bed reactor was successfully established.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupporting Information.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following files are available free of charge. Raw data perfusion experiments Bernadette Straub (XLSX) Raw data perfusion experiments Tabea Boehme including long term cycle perfusion experiments (XLSX)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTabea Boehme: Writing - Original Draft, Data Curation, Investigation. Bernadette Straub: Data Curation, Investigation. Ursula Eschenhagen: Resources, Methodology. Magnus S. Schmidt: Conceptualization, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eNo funds, grants, or other support was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the ReAching program of the faculty Health, Medical and Life Sciences, Furtwangen University, for support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor information\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTabea L. Boehme, EMail: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBernadette Straub: EMail:\u0026nbsp;\u003c/em\u003e\u003cem\[email protected]\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Ursula Eschenhagen, Email: [email protected]\u003c/p\u003e\n\u003cp\u003eMagnus S. Schmidt, Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCredit\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhotograph courtesy of Tabea L. Boehme. Copyright 2025 Tabea L. Boehme. Images are free domain.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKalathinathan P, Sain A, Pulicherla K, Kodiveri Muthukaliannan G (2023) A Review on the Various Sources of β-Galactosidase and Its Lactose Hydrolysis Property. Curr Microbiol 80(4):122\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatthews BW (2005) The structure of E. coli beta-galactosidase. C R Biol 328(6):549\u0026ndash;556\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDekker PJT, Koenders D, Bruins MJ (2019) Lactose-Free Dairy Products: Market Developments, Production, Nutrition and Health Benefits. Nutrients ; 11(3)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBernal C, Urrutia P, Illanes A, Wilson L (2013) Hierarchical meso-macroporous silica grafted with glyoxyl groups: opportunities for covalent immobilization of enzymes. N Biotechnol 30(5):500\u0026ndash;506\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRizzo PV, Harwood WS, Drake MA (2020) Consumer desires and perceptions of lactose-free milk. J Dairy Sci 103(8):6950\u0026ndash;6966\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVeum L, Hanefeld U (2006) Carrier enabled catalytic reaction cascades. Chem Commun (Camb) ; (8):825\u0026ndash;831\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDamin BIS, Kovalski FC, Fischer J, Piccin JS, Dettmer A (2021) Challenges and perspectives of the β-galactosidase enzyme. Appl Microbiol Biotechnol 105(13):5281\u0026ndash;5298\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDayi DI, Eschenhagen U, Seidinger H, Schneider H, Schmidt MS (2023) p-Phenylene Diisothiocyanate-Based Covalent Immobilization of β-d-Galactosidase and Determination of Enzyme Activity by Cleavage of X-Gal and ONPG on Solid Support. ACS Omega 8(30):27585\u0026ndash;27596\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDekker P (2019) Enzymes Exogenous to Milk in Dairy Technology: β-d-Galactosidase. Reference Module in Food Science. Elsevier\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003esanotact GmbH Sanotact lactase 24 000 6h depot (40 lactase tablets) Lactose tablets with depot effect for lactose intolerance \u0026amp; milk intolerance immediate effect \u0026amp; 6h long-term depot 24 000 depot 40 tablets [cited 2025 Aug 27]. 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Springer US, Boston, MA, pp 295\u0026ndash;297\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBayramoglu G, Cimen AG, Arica MY (2023) Immobilisation of β-galactosidase onto double layered hydrophilic polymer coated magnetic nanoparticles: Preparation, characterisation and lactose hydrolysis. Int Dairy J 138:105545\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatrolia P, Liu X, Li G, Kopparapu NK (2019) Enhanced Properties and Lactose Hydrolysis Efficiencies of Food-Grade β-Galactosidases Immobilized on Various Supports: a Comparative Approach. Appl Biochem Biotechnol 188(2):410\u0026ndash;423\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"covalent enzyme immobilization, fixed-bed reactor, β-D-galactosidase, hydrolysis, lactose, Aminomethylated polystyrene resin, phenylenediisothiocyanate","lastPublishedDoi":"10.21203/rs.3.rs-7637179/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7637179/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, a continuous lactose hydrolysis process in a fixed-bed reactor was developed using β-galactosidase covalently immobilized on resin beads via 1,4-phenylenediisothiocyanate (PDC) as linker. Process conditions, including temperature, enzyme loading, perfusion speed, and repeated perfusion of the same substrate solution were systematically varied. The highest glucose yields were obtained at 55\u0026deg;C, with increased yields observed at low perfusion speeds, high enzyme loadings, and upon repeated perfusions. Under optimized cycle perfusion over 72 h, final lactose conversion reached approximately 90% at 37\u0026deg;C and 80% at 22\u0026deg;C. A hydrolysis process in a fixed-bed reactor was successfully established, although further optimization is required.\u003c/p\u003e","manuscriptTitle":"Next Level of p-Phenylene Diisothiocyanate-based Covalent Immobilization of β-d-Galactosidase: technical optimization an application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 06:53:37","doi":"10.21203/rs.3.rs-7637179/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1cb7cb65-d0da-4588-9cdf-057beec33015","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T06:53:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-22 06:53:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7637179","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7637179","identity":"rs-7637179","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}