In Vitro Analysis of Lactase Activity in Commercial Probiotics Containing Lactobacillus Species as a Possible Treatment for Lactose Intolerance

By Jess Breda

Sciences

Lactase

Abstract


Over half of the adult population has a reduced ability to digest lactose, yet treatments for lactose intolerance have varying successes. This research investigated the potential for using commercial probiotics as treatments for lactose intolerance by analyzing the in vitro lactase activity of various supplements containing differing compositions of Lactobacillus species. Lactase was first purified and analyzed from Escherichia coli via concentration and kinetic assays to establish parameters. Probiotic cultures were then grown aerobically in MRS and TPY medium under induced and uninduced conditions. Lactase activity was analyzed using a modified Miller ONPG assay. Probiotic analysis displayed that a supplement with a middle range of Lactobacillus diversity had the highest lactase activity. These findings suggest that probiotics are a possible treatment for lactose intolerance and further, more comprehensive research is warranted. 

Introduction

Lactase is commonly known for its importance in the LacZ Operon model or as a marker in molecular biology, but it has recently become of great interest to the food and pharmacological industries due to its association with lactose intolerance (LI).1 This condition is characterized by deficiency of lactase production in the intestines causing a buildup of non-absorbable lactose, and symptoms such as bloating and indigestion.2  It is estimated that 70% of adult humans have a reduced ability to digest lactose, and while diagnostic tools for LI have greatly developed in the last decade, treatments and their success vary.3,4

The most common treatment is to abstain from lactose consumption, but this can lead to nutritional deficits. Of the most concern is calcium deficiency, as diary tends to be the primary source of calcium in the diets of industrialized nations.5 Research has shown that calcium can help to remove carcinogens in the colon, lower blood pressure and improve bone health.5-7 For LI patients, unless calcium is properly supplemented from nondairy sources, reduced bone density and increased fracture risk can occur.8-10

An alternative strategy is enzyme replacement therapy via consuming exogenous lactase or adding lactase to dairy products pre-consumption.11 Overall, it has been shown that exogenous lactase administration has significant variability on LI symptoms and markers and this could be due to varying degrees of lactose consumption from one patient to another, or degradation of the enzyme in acidic gastric areas prior to intestine contact.12, 13

A second pharmaceutical treatment for LI is the administration of probiotics. Probiotics contain viable microorganism that are thought to benefit a host’s microflora community and have been shown to have a positive effect on treatment and prevention of other gastrointestinal disorders.14 Further, these microorganisms contain lactase and can aid in the digestion of lactose in the host.4

The efficacy of probiotics to break down lactose has been minimally researched in vitro, even though clinical studies have shown some success.15-18 Lactobacillus acidophilus is the most commonly studied probiotic in this field and it has shown in vitro lactase activity as well as decreased symptoms in those living with LI.16, 19 Additional Lactobacillus species such as L. rhamnosus, L. delbrueckii and L. bulgaricus have also shown potential for LI treatment, but in vitro analysis has yet to be performed.20

The goal of this experiment was to further the working knowledge of lactase activity in a variety of Lactobacillus strains to determine which may be the most effective in the treatment of lactose intolerance. It was hypothesized that the greater diversity of Lactobacilli, the more lactase activity the probiotic would have.

To achieve this goal, basal knowledge and experience was first needed in protein purification techniques and assays, so lactase was harvested, purified and analyzed from Escherichia coli (E. coli). Purification occurred by ammonium sulfate precipitation and affinity His-Tag column chromatography. Results were visualized through SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). Analysis was performed by calculating specific activity and performing kinetic activity assays under various conditions using absorbance spectroscopy. Additionally, molecular modeling software was used to examine the structure and properties of lactase. To test the hypothesis, three probiotic strains were grown in MRS and TPY broth and analyzed via a modified Miller ONPG (O-nitrophenyl-β-D galactopyranoside) assay.

Methods

Purification

An E.coli cell pellet was resuspended in 50 mL TE buffer (50 mM TRIS, 1 mM EDTA, pH 8.0) and homogenized in a BeadBeater™ blender (Biospec Products Inc., Bartlesville, OK) following the manufacture’s operating protocol with homogenization and rest occurring in one minute intervals for three cycles.21 The contents were filtered, washed with 20 mL TE buffer and centrifuged at 14,000 RPM for 15 minutes (JA 25.50 rotor, 4 °C). The supernatant was decanted and placed on ice. Ammonium sulfate precipitation occurred at 35% saturation and the salt was slowly added until completely dissolved. The solution equilibrated for 10 minutes and was then centrifuged as reported above. The pellet was resuspended in 25 mL Column Load buffer (50 mM Sodium Phosphate, 300 mM NaCl, pH 8.0) and this solution was placed in a dialysis membrane (3,500 MWCO, Thermo Fischer Scientific, Waltham, MA) and incubated in 1 L Column Load buffer for one week at 4 °C.

A pre-poured Ni-NTA column was equilibrated with 15 mL Column Load buffer, and the sample from dialysis was centrifuged at 5,000 RPM for 10 minutes (JA 25.50 rotor, 4 °C). This supernatant was loaded into the column, followed by a wash with 15 mL Column Load buffer. The column was eluted with 30 mL Elution buffer (50 mM Sodium Phosphate, 300 mM NaCl, 0.3 M Imidazole, pH 8.0). The elution fraction was placed in a dialysis membrane as mentioned above and incubated in 1 L 1X Reaction buffer (50 mM K2HPO4, pH 8.0) for one week at 4 °C.

ONPG Activity Assay

An activity assay was performed on collected fractions with ONPG in a SpectroVis® spectrophotometer (Vernier Software & Technology, Beaverton, OR). The recipe used was 50 μL 10X Reaction buffer, 450 μL deionized water (DI-H2O), 250 μL 3mM ONPG, and 50 μL protein fraction. Protein fraction volume was adjusted if absorbance value was > 1 and controlled for during data analysis. The reaction proceeded for 2 minutes at room temperature and was then stopped with 500 μL sodium phosphate (pH 12) before measuring the absorbance at 420 nm.

BCA Activity Assay

A BCA assay was performed on collected fractions and BSA (bovine serum albumin) standards that ranged from 0.0625 – 1.0 mg/mL. The recipe used was 100 μL BCA-Cupric Sulfate (50:1) and 5 μL protein sample. The sample was incubated at 37 °C for 30 minutes before absorbance was measured at 562 nm in the NanoDropTM spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

SDS-PAGE

SDS-PAGE was performed on collected fractions on a Bio-Rad mini-Protean II (Hercules, CA) as recommend by manufacturer’s protocol.22 Samples were combined with 2X SDS Loading buffer (1:1), incubated at 95 °C for 5 minutes and loaded into separate wells. The standard used was the broad range standards from New England Biolabs (Ipswich, MA). SDS-PAGE was performed in 1X SDS-PAGE buffer at 140 V for 1 hour. Staining occurred overnight in Coomaise blue, destaining occurred for 4 hours, and an image was taken once complete.

Qualitative Kinetic Assays

Kinetic assays were performed on purified lactase to determine the effect of environmental variables. The general recipe followed for each kinetic assay was: 100 μL 10X Reaction buffer, 870 μL DI-H2O, 500 μL 1X Reaction buffer with ONPG (3mM), and 130 μL EL fraction. The recipe was mixed well and measured in the SpectroVis® at 420 nm until a linear trend was well defined. If enzyme or substrate volume was altered in the recipe, DI-H2O volume was adjusted to keep final reaction volume at 1600 μL.

The kinetic assays tested the individual effect of four variables: substrate concentration, enzyme concentration, pH and temperature. Substrate concentration was varied from 0.047 to 0.938 mM. Enzyme concentration was varied from 4.9 x 10-4 to 2.42 x 10-3 mg/mL.  The pH of the reaction was varied from 3.5 to 11, and the temperature was varied from 21 to 72 °C. It should be noted that enzyme volume was reduced by one third in the temperature assay to prevent absorbance readings from being too high.
Quantitative Kinetic Assays

Additional kinetic assays were performed on purified lactase to determine Km and Vmax under normal conditions and in the presence of inhibitors.  The same procedure was followed as mentioned above for qualitative kinetic analysis with the following adjustments: substrate concentration variation from 0.049 to 1.69 mM, a second absorbance measurement was taken when trial was fully reacted to determine the concentration of product (ONP), and the addition of inhibitors. The two inhibitors tested were galactose (20% w/v, 220 μL added) and glucose (30% w/v, 300 μL added).

Molecular Modeling

Lactase was modeled in Swiss-PDB Viewer (version 4.1, Swiss Institute of Bioinformatics, Zürich, Switzerland). The files analyzed from the RCSB Protein Data Bank were: catalytically active (1JZ7) and catalytically inactive lactase (IJYN). The active site of each protein was analyzed along with the overall protein structure.

Probiotics

Three probiotic stains were tested from Vitamin Shoppe ®. The strains were: 10 Probiotic TM 13 Billion (PT), Probiotic Complex TM 4 Billion (PC) and Mega Acidophilus 2 Billion (PA). PT contained six Lactobacillus species, PC contained three species, which were all shared with PT, and PA only contained L. acidophilus, which was shared with PT and PC. See Supplemental Figures 1a-c for more information on each probiotic.

Cell Culture

This procedure was modified from Johnson et al.15 6.5 billion colony-forming units (CFUs) of PT, and 6 billion CFUs of PC and PA were added separately to 40 mL of MRS broth made per the manufacturer’s instructions.23 Growth media was incubated anaerobically for 28 hours at 37 °C. 2 mL of growth media was added to fresh MRS broth and centrifuged for 10 minutes, three times (2300 RPM, JA 25.50, 4 °C). The pellet was washed with 500 µL sterile Phosphate buffer (pH 6.3) and added to two conditions of TPY broth- induced with lactose (10 g/L) and uninduced with glucose (10 g/L). Media was incubated anaerobically for 12 hours at 37 °C, and absorbance was measured at 600 nm on the NanoDropTM to assess bacterial growth.

Probiotic ONPG Assay

This procedure was modified from Liu.24 20 µL of TPY culture was added to 80 µL of Permeabilization solution (100 mM Na2HPO4, 20 mM KCl, 2 mM MgSO4, 0.8 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, 5.4 µL/mL beta-mercaptoethanol). Samples and Substrate solution (60 mM Na2HPO4, 40 mM NaH2PO4, 1 mg/mL ONPG, 2.7 µL/mL beta-mercaptoethanol) were incubated at 30 °C for 30 minutes before Substrate solution was added (600 µL for first trial, 700 µL for second trial). Reactions occurred until color was close to that of Lysogeny broth, or 2 hours elapsed. The reaction was stopped with 700 μL sodium phosphate (pH 12). Stopped samples were microcentrifuged at 15,000 RPM for 10 minutes (FA-45-24-11 Rotor, 25 °C) and the supernatant absorbance was measured in the NanoDropTM at 420 nm. Activity was calculated using the formula:

1000*Abs 420Abs 600* volume* reaction time

Results

The activity of lactase in each collected faction was determined by reacting each sample with ONPG and measuring the absorbance. Lactase hydrolyzed ONPG into galactose and an ortho-nitro-phenyl group. The latter was a measure of lactase’s enzymatic activity as it was soluble, yellow and detectable in the SpectroVis®. The volume of protein fraction added was reduced if the absorbance was greater than 1, as it indicated the assay was saturated. For these reasons, activity was expressed relative to time collected and volume added. These results are displayed in Table 1 and show that the load fraction had the highest lactase activity, while the elution fraction had the lowest. This indicated an error was made during affinity chromatography and it will be discussed later.

Fraction

Absorbance

Volume (mL)

Activity ((Absorbance/min)/mL)

Cell Lysate (CL)

0.816

0.016

25.5

Ammonium Sulfate Supernatant (AS)

0.294

0.05

2.94

Load (LO)

0.641

0.008

40.1

Flow Through (FT)

0.851

0.05

8.51

Wash (WA)

0.302

0.016

9.44

Elution (EL)

0.147

0.05

1.47

Table 1. Collected absorbance data from the ONPG activity assay. It should be noted that reaction time for all fractions was two minutes.

Although the ONPG assay can determine lactase activity, this value is nearly impossible to compare across fractions without a measure of overall protein concentration in each sample. To determine this, a BCA assay was used. This assay reacts with peptide bonds and cysteine, tryptophan and tyrosine residues to produce a green-blue color that can be measured in the NanoDropTM.25 To determine fraction concentrations, a standard curve was generated using BSA protein standards and these results are displayed in Figure 1.

../Desktop/BCA.png

Figure 1. Standard curve produced from BSA concentration standards.

The BCA assay was then performed on the collected purification fractions to determine protein concentration. Using this information, specific activity was calculated as a measure of lactase purity. These results are displayed in Table 2 and suggest that the wash fraction had the highest specific activity, while the elution fraction had the lowest. Again, this indicates an error with the affinity column, which is further supported by a low purification factor of 0.328.

Fraction

Absorbance

Concentration (mg/mL)

Specific Activity ((Absorbance/min)/mg)

CL

0.067

0.085

301

AS

0.021

0.015

198

LO

0.038

0.041

985

FT

0.045

0.051

166

WA

0.013

0.003

3450

EL

0.021

0.015

98.8

Table 2. Collected absorbance data during BCA assay, calculated protein concentration and specific activity for each purification fraction.

Once specific activity was determined, it was necessary to visualize each fraction to determine if overall protein concentration was due to lactase or other contaminating proteins. This was performed by SDS-PAGE. The results from this assay are displayed in Figure 2 and they indicate high concentration of lactase in the cell lysate and load fractions. The elution, wash and flow through fractions display smaller amounts of lactase and the elution fraction has no contamination. The standard ladder did not develop, and this error will be further discussed.

Figure 2. Results of SDS-PAGE of collected purification fractions.

Once it was determined that purified lactase was obtained, it was of interest to see how its kinetic activity changed under various environmental conditions. The following experiments were performed on the purified lactase (EL fraction) to determine its quantitative kinetic properties. The environmental parameters adjusted were: concentration of ONPG, concentration of lactase, pH and temperature. The results for concentration of ONPG variation are displayed in Figure 3a. Results show an asymptotic trend similar to that found in Michaelis-Menten (M-M) kinetic models. Initially there is a linear trend between concentration of ONPG and reaction rate, but eventually a threshold is reached, and the consumption rate begins to plateau at higher concentrations.

Figure 3a. Effects of substrate concentration on reaction rate in purified lactase.

The next parameter adjusted was concentration of lactase. These results are displayed in Figure 3b and suggest a linear trend between lactase concentration and reaction rate. Again, this is similar to results found in MM kinetic models.

Figure 3b. Effects of enzyme concentration on reaction rate in purified lactase.

Following the changes in reactant concentrations, the pH of the 10X Reaction buffer in the general recipe was adjusted. These results are displayed in Figure 3c and suggest an optimal pH curve for the enzyme, and limited activity at high and low pH conditions.

Figure 3c. Effects of pH on reaction rate in purified lactase.

The final parameter adjusted was reaction temperature. These results are displayed in Figure 3d. Initially, there is a linear increase in reaction rate until 50 °C, then it begins to plateau before quickly dropping to 0 at 70 °C where the protein likely denatured.

Figure 3d. Effects of temperature on reaction rate in purified lactase.

Once it was understood how lactase activity responded to different environmental conditions, it was of interest to quantify its activity under normal conditions. This was done by varying substrate concentration, while measuring rate of product formation to determine the two M-M kinetic parameters: Km and Vmax. Vmax is a value of how efficient the enzyme is in the catalytic step, while Km is a value substrate binding quality and catalytic efficiency. Trials were performed by measuring Absorbance vs. Time at varying substrate concentrations and recording the slope as a measure of substrate consumption rate. The results from a trial are seen in Figure 4 along with a linear fit corresponding with a R2 value of 0.9995. All trials in the assay had similar coefficients of determination.

../Desktop/Abs%20V%20T.png

Figure 4. Data collected during qualitative kinetic assays. In this trail, ONPG concentration is 0.56250 mM with glucose inhibitor.  

For a more accurate measure of Km and Vmax, product formation rate was determined by measuring the absorbance twice for each sample: first to obtain initial velocity of substrate consumption (Figure 4) and second to obtain the absorbance value once the sample was fully reacted. Using these values, the extinction coefficient was calculated (ε = 4.9945 mM-1 cm-1) and rates were adjusted in terms of product formation.

In addition to quantitatively assessing lactase under normal conditions, it was also assessed in the presence of two inhibitors, galactose and glucose, to determine their effects on kinetics and type of inhibitor. The results from the three kinetic conditions are displayed in Table 3 and indicate reaction rate decreased in the presence of inhibitors at similar substrate concentrations.


Normal

Galactose

Glucose

[ONPG] (mM)

Δ [ONP]/t (mM/s)

Δ [ONP]/t (mM/s)

Δ [ONP]/t (mM/s)

0.04875

3.57 x 10-4

1.17 x 10-4

2.10 x 10-4

0.05625

3.60 x 10-4

1.30 x 10-4

2.12 x 10-4

0.069375

4.48 x 10-4

1.35 x 10-4

2.98 x 10-4

0.09375

5.64 x 10-4

2.03 x 10-4

3.76 x 10-4

0.140625

7.48 x 10-4

2.77 x 10-4

5.18 x 10-4

0.234375

9.66 x 10-4

4.15 x 10-4

6.09 x 10-4

0.5625

1.33 x 10-3

7.42 x 10-4

8.36 x 10-4

1.6875

1.44 x 10-3

1.13 x 10-3

9.59 x 10-4

Table 3. Reaction rate for each condition tested during the quantities kinetic analysis.

To calculate Km and Vmax, the results from table 3 were visualized in a kinetic plot. This was first done in a Lineweaver-Burke (LB) is displayed in Figure 5a.

../Desktop/LB%20Plot.pngFigure 5a. LB plot of the three conditions tested in the quantitative kinetic analysis.

Note: Km = -1/x-intercept, Vmax = 1/b and slope = Km/Vmax.

The Km and Vmax values were extrapolated from the plot and shown below in Table 4a. The results indicated that the normal and glucose conditions had similar Km values, while the galactose condition had an increased Km. Initially, this suggested that galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor, but the Vmax values neither supported this claim nor clarified inhibitor type.

Condition

Km (mM)

Vmax (mM/s)

Normal

0.2011

1.75 x 10-3

Galactose

0.5407

1.35 x 10-3

Glucose

0.2596

1.31 x 10-3

Table 4a. Kinetic parameter values extrapolated from the LB plot.

Issues often arise when using LB plots due to the long extrapolations required to calculate kinetic parameters, so an Eadie-Hoftsee (EH) plot was generated to find a more accurate value for Km. These results are displayed below in Figure 5b.

../Desktop/EH%20plot.png

Figure 5b. EH plot of the three conditions tested in the quantitative kinetic analysis.

Note: Km = -m and Vmax = b. 

The Km for each condition was determined from the EH plot, and then used to calculate Vmax with the LB slope. These results are displayed below in Table 4b and suggest that with regards to the normal condition, galactose is a competitive inhibitor due to an increased Km and similar Vmax while glucose is a noncompetitive inhibitor due to a similar Km and decreased Vmax. Additionally, the purified lactase appears to have reduced catalytic activity in comparison with other values in literature and this will be further discussed.

Condition

Km (mM)

Percent change (%)

Vmax (mM/s)

Percent Error (%)

Normal

0.1876

1.64 x 10-3

Galactose

0.6165

228.6

1.54 x 10-3

6.098

Glucose

0.1926

2.6

9.75 x 10-4

40.55

Table 4b. Kinetic parameter values calculated from the EH and LB plot.

Once basal understanding of bacterial lactase was achieved, probiotic analysis proceeded. Bacterial growth was assessed after 12 hours of incubation in TPY broth by measuring the absorbance at 600 nm. The goal was to perform activity analysis on bacteria during their exponential growth phase (absorbance 0.6 - 0.9), as the highest concentrations of lactase would be expressed then. These results are displayed below in Table 5 and suggest that the bacteria grew much faster than anticipated, exponential growth phase was missed, and the bacteria had entered stationary phase. This error will be further discussed. These results also display that significantly more growth occurred in the induced condition for PT and this will also be further discussed.


PT Absorbance

PC Absorbance

PA Absorbance

Uninduced

2.092

3.179

3.008

Induced

3.535

3.251

3.1396

Table 5. Absorbance values at 600 nm for each growth condition after 12 hours of incubation in TPY broth.

Even though exponential growth phase was missed, activity analysis was still performed using a modified ONPG assay. Activity accounted for bacterial growth, reaction time, total volume and substrate consumption. A second trial of the assay was performed because PT and PA uninduced conditions did not show adequate color change within the two-hour time frame, so their reaction time was not accurate, and additional Substrate solution was added to compensate. Results from both trials can be seen below in Table 6.

Condition

Probiotic

Absorbance @ 600 nm

Absorbance @ 420 nm

Reaction Time (min)

Activity

(µL -1 min-1)

Uninduced

PT

2.092

0.183

120

5.21 x 10-4

0.192

56

1.09 x 10-3*

PC

3.179

0.236

32

1.66 x 10-3

0.355

6

1.24 x 10-2*

PA

3.008

0.085

120

1.68 x 10-4

0.112

74

3.35 x 10-4*

Induced

PT

3.535

0.43

16

5.43 x 10-3

0.88

6

2.77 x 10-2*

PC

3.251

0.517

16

7.10 x 10-3

1.103

4

5.65 x 10-2*

PA

3.193

0.361

32

2.52 x 10-3

0.426

6

1.48 x 10-2*

Table 6. Parameters used to calculate activity for each condition from trials 1 and 2. * indicates activity for trial 2 with additional 100 µL of Substrate solution. It should be noted that final reaction volume for trial 1 was 1400 µL and for trial 2 was 1500 µL.

Results from both trials suggested the induced condition had higher activity, which was anticipated as the presence of lactose should increase lactase expression. Further, PC had the highest activity, followed by PT and then PA. Results for each trail are visualized below in Figure 6 and provide conflicting support for the hypothesis in that the probiotic containing the most Lactobacillus species did not have the highest lactase activity, but neither did the probiotic containing the fewest. This will be further discussed.

Figure 6. Activity of the three probiotic supplements from both trials. It should be noted that trials have different y-axis scales due to additional substrate added in trial 2 increasing activity.

Discussion

The goal of this experiment was to analyze lactase activity in commercial probiotics containing varying amounts of Lactobacillus species to assess which combinations may be best for lactose intolerance treatment. It was hypothesized that the greater diversity of Lactobacilli, the more lactase activity the probiotic would have. Results showed that PT, which had three Lactobacillus species, had the highest lactase activity. These results conflicted with the hypothesis and suggest that a probitoic with a middle range of Lactobacillus diversity may be the best treatment for LI.  

Additional experiments were first performed to gain basal knowledge on purification and analysis of lactase from E. coli. Overall these experiments showed that pure, active lactase was obtained, although there was some protein lost. Kinetic assays displayed that lactase responded to environmental conditions as anticipated. Additionally, the measured Km and Vmax values suggested that the lactase obtain was less active than typically found in literature, and that galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor.

The ONPG assay results (Table 1) suggested that initial purification steps went well, but an issue occurred during column chromatography. This was indicated by high activity in the Load fraction (LO), higher than expected activity in intermediate Flow Through (FT) and Wash (WA) fractions (as the lactase should be bound to the Ni-NTA column via His-Tag during these collections) and low activity in the Elution (EL) fraction.

Although it was apparent some protein was lost in the column, the BCA assay performed displayed that there was protein present in the EL fraction (Figure 1, Table 2). The activity and concentration values were relatively small, which lead to the EL fraction having the lowest specific activity. SDS-PAGE was run to visualize protein type and relative amount in each fraction (Figure 2). It should be noted the marker did not develop due to degradation prior to the experiment. Results from this gel showed lactase presence in relatively similar, small amounts in the LO, WA and EL with no contamination in the EL fraction. This result confirmed that purification of lactase was successful.

Moving into additional analysis, the kinetic activity of the purified lactase influenced by different parameters was similar to that anticipated. The purified lactase displayed M-M properties with an asymptotic trend as concentration of ONPG was adjusted (Figure 3a). When modeled, lactase displayed an active sight conformationally primed to bind substrate, suggesting it was non-cooperative enzyme and should obey M-M kinetics. This is additionally supported by previous research.26 A linear trend was initially seen as increased concentrations of substrate allowed for faster consumption of substrate, but as enzyme active sites became increasingly saturated, the curve began to plateau as maximum velocity was approached.

Continuing to follow M-M kinetic properties, the purified lactase displayed a linear relationship between lactase concentration and ONPG consumption rate (Figure 3b). An assumption of M-M kinetics is the concentration of substrate greatly exceeds the concentration of enzyme in the system. In this assay, as the concentration of lactase increased, so did the rate of substrate consumption and a linear trend was observed (rather than an asymptotic trend) because the reaction rate was not limited by concentration of substrate or enzyme, allowing for a direct relationship between the two variables.

When the pH of the reaction environment was adjusted, the activity of lactase had a parabolic shape with the highest activity found between pH 6.5 to 9.5 (Figure 3c). Although larger, this range corresponds to the optimum pH of 6.8 to 7.4 of lactase from E. coli.27 The initial rate of ONPG consumption gradually increased from acidic values into the optimum range, but then quickly declined around a pH of 10, indicating lactase is more robust in acidic environments and denatures in basic environments. This was anticipated as lactase in many biological systems can commonly be found near acidic gastric areas, and in the ONPG activity assay, a solution with a pH of 12 was used to stop the reaction. Further, when looking at the molecular model, glutamate, histidine and tyrosine are located near the active site and likely involved in acid/base catalysis. The pKas of these residues are 4.07, 6.04, and 10.46, respectively, and when complied with the data suggest that lactase has optimum activity when glutamate is deprotonated, histidine is protonated and tyrosine is protonated (pH 6 - 10).28 Outside of this pH range, activity begins to decline due to the disruption of acid/base catalysis, as seen in Figure 3c.

The last environmental parameter tested was temperature, and a similar parabolic trend was found (Figure 3d). Initially, there was a linear increase in ONPG consumption rate as the temperature increased the kinetic energy of the system. The velocity then began to plateau between 50 and 65 °C as temperature was increasing activity, but some protein was beginning to denature. At 70 °C, activity plummeted and all protein was denatured.

The quantitative kinetic assays displayed that purified lactase had lower kinetic activity than anticipated (Table 4b). Reported Km values for lactase range from 0.161 to 0.950 mM in E. coli strains under various conditions, while the Km from this assay was 0.1876 mM.29 This suggests that the purified lactase had good binding. Reported Vmax values are slightly less standardized and documented in a variety of units, but overall it appears that the Vmax of the purified lactase (1.64 x 10-3 mM/s) was lower than other reported values. 30-32 This suggests that the purified lactase had reduced catalytic efficiency. Reduction in activity could be due to the conditions the kinetic assay was run in, as they did not match with literature conditions, or an error made during purification, storage and/or the kinetic procedure that damaged the protein. To clarify this error, this assay should be performed again matching a set of conditions from previous research.

Inhibitor assays displayed that galactose was a competitive inhibitor of lactase, while glucose was a non-competitive inhibitor (Figure 5a-b, Table 4b). The molecular model of active lactase displayed that the galactose portion of the lactose substrate bound deeper and made more contact with the active site of the enzyme than the glucose portion. This binding orientation allowed the enzyme to function normally and position lactose for catalysis via cleavage of its glyosidic bond. With the addition of competitive and noncompetitive inhibitors, the active site function was disrupted in two different ways.

When galactose was added into the system, it bound the active site of lactase, but there was no glyosidic bond to cleave and the reaction for that individual enzyme could not proceed. The Km increased due to reduced binding affinity of substrate (galactose is blocking ONPG from the active site), and the Vmax stayed constant as galactose did not affect the function of the enzyme substrate complex. These features of competitive inhibition were seen in the data (Table 4b) and previous research on galactose inhibition in lactase has produced similar results.26,33,34

When glucose was added to the system, it bound away from the active site of the enzyme or enzyme substrate complex, but changed the conformation of the active site in such a way that catalysis could not proceed for that individual enzyme. The Km stayed constant as glucose was not interfering with the binding of substrate and the Vmax decreased as rate of catalysis was reduced. These features of non-competitive inhibition were seen in the results (Table 4b) and depending on the source of lactase, previous research has suggested glucose has either no effect or is a non-competitive inhibitor of lactase.26,33,35,36

Transitioning to probiotic analysis, the probiotics grew much faster than the anticipated 14 – 16 hours to reach exponential phase (Table 5). This expedited growth could have been due to procedural discrepancies such as inoculation volumes or differing probiotic strains. Growth rates appeared similar between conditions apart from unindiuced PT having a much lower growth value. This was not anticipated as Lactobacillus strains tend to have enhanced growth in glucose, and suggests an error was made during inoculation or absorbance readings, or the combination of bacterial strains and supplemental ingredients in PT have abnormal growth in glucose.37  

Due to all conditions being in stationary phase and bacterial growth being controlled for when calculating activity, the experiment was continued and lactase activity was analyzed. Across trials, the induced condition had increased activity compared to the uninduced, which was anticipated as lactose, or more specifically one of its isomers allolactose, promotes lactase production (Table 6).38, 39 Additionally, both trials produced similar results regardless of reaction conditions; PC had the highest activity, followed by PT and then PA (Table 6, Figure 6).

It was hypothesized that PT would have the highest activity as it had the most Lactobacillus species, but this was not supported by the data, and there are multiple possibilities as to why this occurred. First, a limitation of this study was that total CFUs for each probiotic were known, but CFUs of individual bacterial species were not. Even though PT had a more diverse subset of Lactobacillus, the concentration of Lactobacillus species may have been lower than PC due to a larger presence other bacteria such as Bifidobacterium. Further, non-Lactobacillus bacteria would not have grown as well in the selected media, and measured lactase activity would have been suppressed. To control for this in the future, commercial probiotics with only Lactobacillus strains should be tested, or a nonspecific growth media should be used with a different research question.

In addition to bacterial strains, each commercial probiotic contained a different subset of capsule ingredients including compounds such as rice flour, silica or fructo-oligosaccharides. Some of these compounds are carbon sources that could influence growth, others were possible immobilizers of the enzyme, and most had unknown influence on lactase activity.40, 41 As their quantities and systemic effect were unaccounted for, these capsule ingredients could have influenced the results.

Another important lurking variable to discuss is that PA was a diary free supplement, while PC and PT were not. This means that PC and PT were already induced, and thereby were primed for higher lactase activity. This limits comparison across supplements due to inconsistent ingredients and induced growth. It would be of interest to investigate if the increase in lactase activity from lactose presence in these probiotics would compensate for the negative effects of a LI patient consuming lactose.

For future studies, this assay could be repeated in the presence of bile as performed by Johnson et al. to test how robust the probiotics are in acidic gastric environments.15 Additionally, after growth, lactase could be purified from each probiotic and further analyzed in a variety of kinetic assays as performed on the lactase from E. coli. Once there was sufficient in vitro evidence on which supplement has the highest activity in the appropriate environmental conditions, clinical in vivo analysis could assess the effects of the strains and validity of the in vitro results. It should be noted that all future studies should analyze probiotics with more comparable ingredients to increase control of the experiment and produce more conclusive results.

A significant error was made during column chromatography which lead to a loss of protein. There were many reasons as to why the lactase did not properly bind to the column such as micro cracks or channels in the column that allowed the sample to run through or diminished column resin binding capacity due use in the previous lab section. To obtain higher, more usable concentrations of lactase, column chromatography technique should be improved upon and a fresh column should be used eliminate the potential for the errors discussed above.

A second procedural error was made during the growth of the probiotic strains in TPY broth. Exponential growth phase, when lactase expression is highest, was missed. This meant activity was analyzed during stationary phase, which lowered activity values and made comparison to previous literature difficult. To avoid this error, growth in TPY should occur at a time when hourly samples can be taken to assess progress and analysis can begin at the appropriate stage.

Although some protein was lost, pure lactase was obtained in the EL fraction (Figure 2). Purified lactase activity was characterized under four different environmental conditions (Figures 3a-d) and kinetic parameters under normal conditions and in the presence of inhibitors were found (Figure 5b, Table 4b). The catalytic activity of the purified enzyme was reduced, and it was experimentally determined that galactose was a competitive inhibitor of lactase, while glucose was a noncompetitive inhibitor and these results are further supported by previous research. Probiotic analysis displayed that a supplement with a middle range of Lactobacillus diversity had the highest lactase activity, and this should be further verified by additional studies with increased controls (Table 6). In summation, this experiment provided in vitro support for probiotics as a possible treatment to lactose intolerance and further, more comprehensive research on the topic is warranted.

Supplemental Information

Supplemental Figure 1a. PA nutritional facts and information.

Supplemental Figure 1b. PC nutritional facts and information.

Supplemental Figure 1c. PT nutritional facts and information.

References

  • Juers DM, Matthews BW, Huber RE. Lac-Z Beta-galactosidase: Structure and function of an enzyme of historical and molecular biological importance. Protein Sci. 2012:21(12):1792-1807.
  • Bayless TM, Rothfeld B, Massa C, Wise L, Paige D, Bedine M. Lactose and milk intolerance: clinical implications. N Engl J Med. 2975:292(22):1156-1159.
  • Saqib S, Akram A, Halim SA, Tassaduq R. Sources of β-galactosidase and its applications in food industry. 3 Biotech. 2017:7(79).
  • Usai-Satta P, Scarpa M., Oppia F, Cabras F. Lactose malabsorption and intolerance: What should be the best clinical management? World J Gastrointest Pharmacol Ther. 2013:3(3):29-33.
  • U.S. Department of Health and Human ServicesBone Health and Osteoporosis: A Report of the Surgeon General. Rockville, MD: U.S. Department of Health and Human Services, Office of the Surgeon General, 2004.
  • Lipkin M, Newmak H. Calcium and prevention of color cancer. J Cell Biochem Suppl. 1995:22:65-73.
  • Appel LJ, Moore TJ, Obarzanek E, Vollmer WM, Svetkey LP, Sacks FM, Bray GA, Vogt TM, Culter JA, Windhauser MM, Lin PH, Karanja N. A clinical trial of the effects of dietary patterns on blood pressure. N Engl J Med. 1997:336(16):1117-24.
  • Honkanen R, Pukkinen P, Järvinen R, Kröger H, Lindstedk K, Tuppurainen M, Uusitupa M. Does lactose intolerance predispose to low bone density? A population-based study of perimenopausal Finnish women. Bone. 1996:19(1):23-28.
  • Honkanen R, Kröger H, Alhava E, Turpeinen P, Tuppurainen M, Saarikoski S. Lactose intolerance associated with fractures of weight-bearing bones in Finnish women aged 38-57 years. Bone. 1997:21(6):473-477.
  • Enattah N, Pekkarinen T, Välimäki MJ, Löyttyniemi E, Järvelä I. Genetically defined adult-type hypolactasia and self-reported lactose intolerance as risk factors of osteoporosis in Finnish postmenopausal women. Eur J Clin Nutr. 2005:59(10):1105-1111.
  • Montalto M, Nucera G, Santoro L, Curiglinao V, Vastola M, Covino M, . . . Gasbarrini G. Effect of exogenous beta-galactosidase in patients with lactose malabsorption and intolerance: a crossover double-blind placebo-controlled study. European Journal of Clinical Nutrition. 2005: 59:489-493.
  • Ibba I, Gilli A, Boi MF, Usai P. Effects of Exogenous Lactase Administration on Hydrogen Breath Excretion and Intestinal Symptoms in Patients Presenting Lactose Malabsorption and Intolerance. Biomed Res Int. 2014:680196.
  • Onwulata CI, Rao DR, Vankineni P. Relative efficiency of yogurt, sweet acidophilus milk, hydrolyzed-lactose milk, and a commercial lactase tablet in alleviating lactose maldigestion. Am J Clin Nutr. 1989:49(6):1233-1237.
  • Ritchie ML, Romanuk TN. A Meta-Analysis of Probiotic Efficacy for Gastrointestinal Diseases. PLOS. 2012:7(4):e34938.
  • Johnson DN, Aljaloud SO, Gyawali R, Ibrahim SA. Determining β-Galactosidase Activity of Commercially Available Probiotic Supplements. Journal of Nutrition & Food Sciences. 2016:5(4).
  • Selvarajan E, Mohanasrinivasan V. Kinetic studies on exploring lactose hydrolysis potential of β galactosidase extracted from Lactobacillus plantarum. J Food Sci Technol. 2015:52(10):6206-6217
  • Almedia CC, Lorena SL, Pavan CR, Akasaka HM, Mesquita MA. Beneficial effects of long-term consumption of a probiotic combination of Lactobacillus casei Shirota and Bifidobacterium breve Yakult may persist after suspension of therapy in lactose-intolerant patients. Nutr Clin Pract. 2012:27(2):247-251.
  • Shaukat A, Levitt MD, Taylor BC, Shamilyan TA, Kane RL, Wilt TJ. Systematic review: effective management strategies for lactose intolerance. Ann Intern Med. 2010:152(12):797-803.
  • Kim SH, Gillard SE. Lactobacillus acidophilus as a Dietary Adjunct for Milk to Aid Lactose Digestion in Humans. J Dairy Sci. 1983:66:959-966.
  • Adolfsson O, Meydani SN, Russell RM. Yogurt and Gut Function. Am J Clin Nutr. 2004:80(2):245-256.
  • BeadBeater Operating Instructions. Biospec Products. https://biospec.com/instructions/beadbeater. Accessed September 25, 2017.
  • Mini-PROTEAN Tetra Cell Instruction Manual. BioRad. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10007296D.pdf. Accessed September 25, 2017.
  • Remel MRS Broth. ThermoFisher Scientific. https://www.thermofisher.com/order/catalog/product/R454062. Accessed December 7, 2017.
  • Liu Lab. A Better Miler Assay. Pomona. http://research.pomona.edu/jane-liu/files/2012/08/Beta-Galactosidase-Assay-A-better-Miller-LIU-LAB.pdf. Publisehd August 26, 2014. Accessed December 3, 2017.
  • PierceTM BCA Protein Assay Kit. Thermo Scientific. https://tools.thermofisher.com/content/sfs/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf. September 25, 2017.
  • Lederberg J. The beta-D-galactosidase of Escherichia coli, strain K-12. J Bacteriol. 1950:60(4):381-392.
  • Beta-galactosidase from E. Coli Product Information. Sigma. https://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Sigma/Datasheet/7/g3153dat.pdf Accessed November 12, 2017.
  • Amino acids with Acidity Values. Keene. http://academics.keene.edu/rblatchly/Chem220/hand/npaa/aawpka.htm. Accessed November 16, 2017.
  • 3.2.1.23: beta-galactosidase. BRENDA. http://brenda-enzymes.org/all_enzymes.php?ecno=3.2.1.23&table=KM_Value#TAB. Accessed November 16, 2017.
  • Kinetic Analysis of Beta-Galactosidase activity using the PowerWaveTM and Gen5Tm Data analysis software. Held, P BioTek. https://www.biotek.com/resources/docs/B-Gal_Michaelis-Menten_App_Note.pdf. Accessed November 16, 2017.
  • Michaelis Menten Kinetics of Beta-Galactosidase with UV5Bio and LabX. Miller Toledo. https://www.mt.com/dam/Analytical/uv-vis/uv-glen/LAB_enzyme_1604_ANA_Academia.pdf. Accessed November 16, 2017.
  • 2-Nitropheny beta-D-galactopyranoside Product Information. Sigma. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/n1127pis.pdf. Accessed November 16, 2017.
  • Cavaille D, Combes D. Characterization of beta-galactosidase from Kluyveromyces lactis. Biotech and applied biochem. 1995.
  • Portaccio M, Stellato S, Rossi S, Bencivenga U, Mohy Eldin MS, Gaeta FS, Mita DG. Galactose compeietive inhibition of beta-galactosidase (aspergillus oryzae) immobilized on chitosan and nylon supports. Enzymes and Microbial Technology. 1998:23(1-2):101-106.
  • Guven RG, Kaplan A, Guven K, Matpan F, Dogru M. Effects of various inhibitors on beta-galactosidase purified from thermoacidophillic Alicyclobacillus acidocaldarius  subsp. Rittmannii isolated from Antarctica. Biotechnology and Bioprocess Engineering. 2011: 16(1):114-119.
  • Huber RE, Brockbank RL. Strong Inhibitory Effect of Furanoses and Sugar Lactones on beta-galactosidase of Escherichia coli. Biochemistry. 1987:26:1526-1531.
  • Srinivas D, Mital BK, Garg SK. Utilization of sugars by Lactobacillus acidophilus strains. International Journal of Food Microbiology. 1990:10:51-58.
  • Jobe A, Bourgrois S. Repressor-operon interaction. VI. The natural inducer of the the lac operon. J. Mol. Biology. 1972:69:397-408.
  • Burstein C, Cohn M, Kepes A, Monod J. Role of lactose and its metabolic products in the induction of the lactose operon in Escherichia coli. Biochem. Biophys. 1965:95:634-639.
  • Akcan N. High level production of extracellular β-galactosidase from Bacillus licheniformis ATCC 12759 in submerged fermentation. African Journal of Microbiology Research. 2011:5(26):4615-4621.
  • Bernal C, Sierra L, Mesa M. Design of β-galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance. Engineering in Life Sentences. 2014:14(1):85-94.

Jess

Jess Breda

Author Major

Neuroscience

Author Hometown

Marshfield, Massachusetts

About the Author

When she’s not in school, Jess spends most of her time outside climbing, skiing, and biking. Jess’ favorite aspect of this research is it’s applicability. Since lactose intolerance or maldigestion is quite prevalent, it is exciting for her to perform research that could positively contribute to the lives of others.

The most challenging part of this research was acquiring the methods to grow and analyze the enzyme of interest from commercial probiotics. This had only been done once before in literature, so contact was made with that lab and protocols were adjusted to fit this research question. This is not Jess’ first research project – she is currently working in the neuroscience lab with Westminster faculty.