Combined efficiency of preloaded beta-galactosidase and ultra-filtration in the production of lactose-free milk products
Author: Bryn Werley
Lactose intolerance is a condition which results in the inability to digest lactose, a disaccharide naturally found in milk. Lactose intolerance is caused by a deficiency in beta-galactosidase (lactase) enzyme production. This results in a lack of lactose hydrolyzation during digestion. A lactase-deficient individual will experience gastrointestinal distress upon consumption of lactose. Lactose-free milk products are manufactured by preloading lactase into milk, stimulating the hydrolysis of lactose prior to consumption. However, commercial lactase has been shown to be less efficient than other forms of the beta-galactosidase enzyme. Therefore, the investigation of the combined efficiency of preloaded lactase and ultra-filtration in the production of lactose-free milk products is supported. In order to evaluate the combined efficiency of preloaded lactase and ultra-filtration, a preliminary high school study was designed. In this study, commercial unfiltered and ultra-filtered lactose-free milk samples were analyzed for glucose, a byproduct of the hydrolysis of lactose that indicates activity of the lactase enzyme. Glucose was detected in all lactose-free samples, filtered and unfiltered, indicating active hydrolysis of lactose by the lactase enzyme. Milk samples were further analyzed for lactose with high-performance liquid chromatography (HPLC). HPLC shows residual lactose in unfiltered lactose-free milk, indicating a deficiency of lactose removal in unfiltered milk products. Altogether, glucose detection and HPLC analysis show that preloading lactase degrades lactose in milk products. However, this study also indicates inadequate removal of residual lactose in unfiltered lactose-free milk products. Unfiltered lactose-free milk shows a lactose concentration 11.7 times greater than the legal limit for “lactose-free” product designation. Conversely, ultra-filtered lactose-free milk shows no presence of lactose, indicating increased efficiency of lactose removal when ultra-filtration is combined with the addition of lactase. This study supports future analysis of milk processing systems to ensure complete lactose removal for lactase-deficient consumers as well as accurate labeling of products.
Keywords: Lactose intolerance, lactose, beta-galactosidase, lactase, high-performance liquid chromatography, ultra-filtration, milk
Approximately 70% of the global population suffers from a reduced ability to digest lactose, the primary sugar in milk ((Ugidos-Rodriguez, S., Matallana-Gonzalez, M. C. & Sanchez-Mata, M. C. Lactose malabsorption and intolerance: a review. Food Funct. 2018.)). This condition is known as lactose intolerance ((Ugidos-Rodriguez, S., Matallana-Gonzalez, M. C. & Sanchez-Mata, M. C. Lactose malabsorption and intolerance: a review. Food Funct. 2018.)). Lactose intolerance is caused by low amounts of beta-galactosidase (lactase), the enzyme that breaks down lactose into its component sugars: glucose and galactose. [((Kretchmer, N. Lactose and Lactase. Scientific American. 1972.)), ((Ugidos-Rodriguez, S., Matallana-Gonzalez, M. C. & Sanchez-Mata, M. C. Lactose malabsorption and intolerance: a review. Food Funct. 2018.))]. The symptoms of lactose intolerance include abdominal pain, diarrhea, gas, nausea, and vomiting that occur after consuming dairy ((Suarez, F. L., Savaiano, D. A., & Levitt, M. D. A comparison of symptoms after the consumption of milk or lactose-hydrolyzed milk by people with self-reported severe lactose intolerance. New England Journal of Medicine. 1995.)). In order to prevent these symptoms, lactose-free products exist as alternatives for the lactose-intolerant population ((Adhikari, K., Dooley, L. M., Chambers IV, E., & Bhumiratana, N. Sensory characteristics of commercial lactose-free milks manufactured in the United States. LWT-Food Science and Technology. 2010.)). These products are required to contain no more than 0.01% lactose ((van Scheppingen, W. B., van Hilten, P. H., Vijverberg, M. P., & Duchateau, A. L. L., Selective and sensitive determination of lactose in low-lactose dairy products with HPAEC-PAD. 2017.)). Lactose-free milk products can be manufactured by preloading lactase into the milk, stimulating lactose hydrolysis prior to consumption ((Rosado, J. L., Solomons, N. W., Lisker, R., Bourges, H., Anrubio, G., Garcia, A. Perez-Briceño, R., Aizupuru, E. Enzyme replacement therapy for primary adult lactase deficiency: effective reduction of lactose malabsorption and milk intolerance by direct addition of beta-galactosidase to milk at mealtime. Gastroenterology. 1984.)). Lactose-free milk products can also be produced through combining the addition of lactase with ultra-filtration, a technique commonly used to remove sugars from fluids ((Limsawat, P. & Pruksasri, S. Separation of lactose from milk by ultrafiltration. Asian Journal of Food and Agro-Industry. 2010.)). However, commercial lactase has been shown to be less efficient than other forms of beta-galactosidase, which could result in the presence of residual lactose in lactose-free dairy products produced using commercial lactase ((Onwulata, C. I., Rao, D. R., & Vankineni, P. Relative efficiency of yogurt, sweet acidophilus milk, hydrolyzed-lactose milk, and a commercial lactase tablet in alleviating lactose maldigestion. The American Journal of Clinical Nutrition. 1989.)).
Commercial glucose analyses have been developed, and these devices have been validated for the analysis of milk ((Chaudhari, P. R. Evaluation of qualitative tests used for detection of adulterants in milk. 2015.)). High-performance liquid chromatography (HPLC) has also been shown to be useful for the analysis of sugars in dairy products ((Chávez-Servin, J. L., Castellote, A. I. & López-Sabater, M. C. Analysis of mono-and disaccharides in milk-based formulae by high-performance liquid chromatography with refractive index detection. Journal of Chromatography A. 2004.)). Combined, HPLC and a glucose analysis would be useful in determining the combined efficiency of lactase and ultra-filtration in reducing the lactose concentrations of lactose-free milk products. The objective of this study is to evaluate to what extent the efficiency of lactose removal is increased when ultra-filtration is combined with the use of preloaded lactase in the production of lactose-free milk products.
Glucose Analysis using Bayer Diastix
Milk samples were analyzed for glucose and lactose. Samples included 2% regular reduced-fat milk, 2% unfiltered lactose-free milk, and 2% ultra-filtered lactose-free milk.
In the first method of analysis, glucose concentrations of milk samples were measured as percentages with Bayer Diastix to detect glucose concentration. Measured glucose concentrations were compared to measurements of control solutions of glucose and lactose. The lactose control solution had a concentration of 4.6% (w/w), equal to that of an average milk sample. (Walstra, 1999) The glucose control solution had a concentration of 2.3% (w/w), equal to the theoretical amount of glucose in the unfiltered lactose-free sample assuming 100% lactose hydrolysis. Analysis of sample and control groups was performed in quintuplicate.
Sugar Analysis through HPLC
In the second method of analysis, lactose concentrations of milk samples were analyzed utilizing high-performance liquid chromatography, or HPLC. Standard curve development for lactose solutions with concentrations ranging from 0% (w/v) to 5% (w/v) confirmed test reliability. To develop this curve, lactose solutions of increasing concentrations were made dissolving 0.100, 0.200, 0.300, 0.400, 0.460, and 0.500 g of lactose in 10 mL of distilled water each. Milk samples were prepared for HPLC by removing fats and proteins from the samples using acetic acid and heat using standard laboratory procedures, which required dilution of the samples. (Pavia, D. L., Lampman, G. M., Kriz, G. S. & Engel, R. G. Introduction to organic laboratory techniques: a small scale approach. Cengage Learning. 2005.) The samples were then filtered through a syringe filter into microvials. Samples were subject to HPLC for 10 minutes using a Phenomenex Luna amine column, and chromatograms were generated and analyzed for lactose and glucose. Following HPLC analysis, calculations were performed to convert the measured lactose concentrations of the diluted milk samples to the concentrated values of the original milk samples.
Statistical analysis includes a one-way analysis of variance (ANOVA), significance p < 0.05, followed by a Tukey-Kramer test. Statistical calculations were performed in Microsoft Excel for Windows.
As indicated by the Bayer Diastix analysis, the concentration of glucose was greatest in the unfiltered lactose-free milk, which had a mean concentration of 1.0% (Table 1, Fig. 1). Glucose was also detected in the ultra-filtered lactose-free milk with a concentration of 0.5%, half of that of the unfiltered lactose-free milk (Table 1, Fig. 1). No glucose was detected in the regular reduced-fat milk (Table 1, Fig. 1).
Each box represents glucose concentration indicated by Bayer Diastix urinalysis reagent strips. Each sample was analyzed in quintuplicate. Average values represented graphically in Figure 1, statistical significance (p<0.05) in Tables 2 and 3.
(GCS= Glucose Control Solution, LCS= Lactose Control Solution, RM= 2% Reduced-Fat Milk, UnLFM= Unfiltered Lactose-free Milk, UlLFM= Ultra-filtered Lactose-free Milk)
Figure 1. Glucose Concentration (%(w/v)) Control Solutions vs. Milk Samples
Each bar in graph displays averages for each sample group. Individual concentrations shown in Table 1, statistical significance (p<0.05) in Tables 2 and 3. Error bars not shown for LCS, RM, UnLFM and UlLFM as standard deviation was calculated to be 0.
Statistical analysis revealed significant differences between the glucose concentrations of the regular reduced-fat milk and the unfiltered lactose-free milk, the regular reduced-fat milk and the ultra-filtered lactose-free milk, and between the unfiltered lactose-free milk and the ultra-filtered lactose free milk (Table 2, 3).
Table 2. One-way ANOVA for Glucose Concentration (%(w/v)) Control Solutions vs. Milk Samples
Fcalc and Fcrit values shown in the graph indicate a significant difference, which was located using the Tukey-Kramer test in Table 3.
Table 3. Tukey-Kramer Test for Glucose Concentration (%(w/v)) Control Solutions vs. Milk Samples
Tukey-Kramer test used to locate significance between groups indicated by the one-way ANOVA in Table 2. Values indicating significance (>4.232) are shown in red. (p<0.05)
Analysis through HPLC was calibrated using the data and standard curve presented in Table 4 and Figures 2 and 3.
Figure 2. Calibration Chromatogram.
This figure shows the HPLC results of the standard lactose solutions. The integrated peak areas are shown in Table 4. (Blue = 0.0% Lactose, Red = 1.0% Lactose, Green = 2.0% Lactose, Pink = 3.0% Lactose, Yellow = 4.0% Lactose, Purple = 5.0% Lactose)
Table 4. Standard Solution Area Peak Areas
This table shows the areas of the peaks from Figure 2. An additional peak area was found using a 4.6% solution. These values were used to create the standard curve shown in Figure 3. (LCSS = Lactose Concentration of Standard Solution)
Figure 3. Lactose Standard Curve.
This standard curve was created from the values in Table 4. The displayed trendline was used to convert the peak values from Figure 4 to the concentrations displayed in Table 5.
HPLC analysis indicated the presence of lactose in the regular reduced-fat milk and the unfiltered lactose-free milk (Table 5, Fig. 4, 5, 6). No lactose was detected in the ultra-filtered lactose-free milk (Table 5, Fig. 4, 5, 6).
Figure 4. RM, UnLFM, UlLFM Overlay.
This chromatogram shows the results from the RM, UnLFM, and UlLFM. The peaks shown at 8.581 and 8.336 minutes indicate lactose. The peaks shown at 5.652 and 5.631 minutes indicate glucose. (Pink = RM, Green = UnLFM, Blue = UlLFM)
The equation y = 1658.9x was used to calculate the dilute concentration of each sample when y was equal to the lactose peak area and x was equal to the dilute concentration. The equation C1V1=C2V2 was used to convert the dilute lactose concentration to the original lactose concentration. Values are shown graphically in Figure 5. (LCDS = Lactose Concentration of Diluted Sample, LCCS = Lactose Concentration of Concentrated (Original) Sample)
Figure 5. Lactose Concentrations in RM, UnLFM, and UlLFM
This graph represents the values of the lactose concentrations of each milk sample when diluted and concentrated. Calculations are explained in Table 5.
Table 6. Comparisons of Lactose Content of Milk Samples
This table shows mathematical comparisons of the lactose concentrations of the lactose-free milks in comparison to the concentrations the RM sample and the legal definition for lactose-free milk as ratios. (LDLF = Legal Definition of Lactose-Free)
Figure 6. UnLFM and UlLFM Overlay. This chromatogram represents the results of the HPLC analysis of both lactose-free samples. The green peak at 8.336 indicates the presence of lactose in UnLFM. The peak blue peak at 8.194 represents baseline noise, not lactose in UlLFM. (Green = UnLFM, Blue = UlLFM)
Figure 7. RM, UnLFM, UlLFM, 0.5% Sugar Overlay
This overlay presents the analysis of each milk sample as well as a 0.5% solution of fructose, glucose, and sucrose (blue). The multiplicity of the blue, yellow, and green lines at 5.652 minutes confirms the presence of glucose in lactose-free milk samples. The peaks in the blue line at 4.749 minutes and 7.410 minutes represent fructose and sucrose, respectively. (Pink = RM, Yellow = UnLFM, Green = UlLFM, Blue = 0.5% Sugar Mix)
Based on statistical analysis, it was deduced that lactase is effective at hydrolyzing lactose in commercial milk products. Figures 4 and 5 show that regular reduced-fat milk had the highest lactose concentration of all samples. This was expected as no lactase had been added to this sample to hydrolyze the lactose. The unfiltered lactose-free milk also contained a detectable amount of lactose. However, as shown in Figures 4 and 5, the concentration of lactose in the unfiltered lactose-free milk was much smaller than the concentration of lactose in the regular reduced-fat milk. The ultra-filtered lactose-free milk did not contain any detectable lactose, supporting the hypothesis regarding increased efficiency of lactose removal when ultra-filtration is combined with preloading of lactase (Table 5, Fig. 6).
One consideration of sample error in this research may be found in the varying facilities of origin for milk samples. The milk-producing cows may have been different breeds, in different stages of lactation, on different feeds, or under different levels of stress. The aforementioned variables could have an effect on the amount of lactose in the samples, thereby altering glucose levels following the addition of the lactase enzyme and the amount of lactose that would be necessary to remove. This would have affected the analyses performed using Bayer Diastix and HPLC.
Analysis using Bayer Diastix evidenced the efficacy of lactase in catalyzing the hydrolysis of lactose. HPLC analysis further confirmed the efficacy of lactase. Chromatogram analysis shows that lactase alone is not 100% efficient as the lactose concentration of the unfiltered lactose-free milk was 11.7 times greater than the legal requirement for a “lactose-free” product (Table 6). However, chromatogram analysis also reveals that ultra-filtration is effective at removing residual lactose as no lactose was detected in the ultra-filtered lactose-free milk (Table 5). Further analysis using HPLC is supported in order to evaluate the concentrations of other unfiltered lactose-free milks as well as to better support the conclusions drawn here.
Ultimately, the lactose concentrations of lactose-free milks are much lower than the lactose concentration of regular reduced-fat milk. However, when lactase alone is used to reduce lactose concentrations in milks, the lactose concentrations of these products may exceed the legal limit for “lactose-free” product designation. The combination of lactase and ultra-filtration, however, has been demonstrated as more effective than the use of lactase alone in reducing lactose concentrations of milk products.
I thank Dr. Thomas Betts of Kutztown University for allowing the use of the analytical technology at the Boehm Science Center. I also thank Adelle L. Schade and Christina Ruoss for their assistance in the preparation of the manuscript and John Siefert for his role in overseeing this study. I additionally acknowledge the Conrad Weiser Science Research Institute for its role in providing the opportunity to complete this research.
Adhikari, Koushik, Dooley, Lauren M, Chambers IV, Edgar, & Bhumiratana, Natnicha. (2010). Sensory characteristics of commercial lactose-free milks manufactured in the United States. LWT-Food Science and Technology, 43(1), 113-118. (https://www.sciencedirect.com/science/article/pii/S0023643809001893)
Chaudhari, PRIYANKABEN RAMESHBHAI. (2015). Evaluation of qualitative tests used for detection of adulterants in milk. AAU, Anand,
Chávez-Servin, Jorge L, Castellote, Ana I, & López-Sabater, M Carmen. (2004). Analysis of mono-and disaccharides in milk-based formulae by high-performance liquid chromatography with refractive index detection. Journal of Chromatography A, 1043(2), 211-215.
Kretchmer, Norman. (1972). Lactose and lactase. Scientific American, 227(4), 70-79.
Limsawat, Pongsathon, & Pruksasri, Suwattana. (2010). Separation of lactose from milk by ultrafiltration. Asian Journal of Food and Agro-Industry, 3(2), 236-243.
Onwulata, C. I., Rao, D. R., & Vankineni, P. (1989). Relative efficiency of yogurt, sweet acidophilus milk, hydrolyzed-lactose milk, and a commercial lactase tablet in alleviating lactose maldigestion. The American Journal of Clinical Nutrition, 49(6), 1233-1237. doi:10.1093/ajcn/49.6.1233
Pavia, Donald L, Lampman, Gary M, Kriz, George S, & Engel, Randall G. (2005). Introduction to organic laboratory techniques: a small scale approach: Cengage Learning.
Rosado, Jorge L, Solomons, Noel W, Lisker, Ruben, Bourges, Hector, Anrubio, Guadalupe, García, Antonieta, . . . Aizupuru, Edna. (1984). Enzyme replacement therapy for primary adult lactase deficiency: effective reduction of lactose malabsorption and milk intolerance by direct addition of beta-galactosidase to milk at mealtime. Gastroenterology, 87(5), 1072-1082.
Suarez, Fabrizis L, Savaiano, Dennis A, & Levitt, Michael D. (1995). A comparison of symptoms after the consumption of milk or lactose-hydrolyzed milk by people with self-reported severe lactose intolerance. New England Journal of Medicine, 333(1), 1-4.
Ugidos-Rodriguez, S., Matallana-Gonzalez, M. C., & Sanchez-Mata, M. C. (2018). Lactose malabsorption and intolerance: a review. Food Funct, 9(8), 4056-4068. doi:10.1039/c8fo00555a
van Scheppingen, Wibo B, van Hilten, Piet H, Vijverberg, Marieke P, & Duchateau, Alexander LL. (2017). Selective and sensitive determination of lactose in low-lactose dairy products with HPAEC-PAD. Journal of Chromatography B, 1060, 395-399.
Walstra, Pieter. (1999). Dairy technology: principles of milk properties and processes: CRC Press. (https://www.cabdirect.org/cabdirect/abstract/19990403119)