Brigham Young University Brigham Young University BYU ScholarsArchive BYU ScholarsArchive Theses and Dissertations 2010-08-09 Beta Galactosidose Activity of Commercial Lactase Samples in Beta Galactosidose Activity of Commercial Lactase Samples in Raw and Pasteurized Milk at Refrigerated Temperatures Raw and Pasteurized Milk at Refrigerated Temperatures Trenton W. Horner Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Food Science Commons, and the Nutrition Commons BYU ScholarsArchive Citation BYU ScholarsArchive Citation Horner, Trenton W., "Beta Galactosidose Activity of Commercial Lactase Samples in Raw and Pasteurized Milk at Refrigerated Temperatures" (2010). Theses and Dissertations. 2590. https://scholarsarchive.byu.edu/etd/2590 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected].
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Brigham Young University Brigham Young University
BYU ScholarsArchive BYU ScholarsArchive
Theses and Dissertations
2010-08-09
Beta Galactosidose Activity of Commercial Lactase Samples in Beta Galactosidose Activity of Commercial Lactase Samples in
Raw and Pasteurized Milk at Refrigerated Temperatures Raw and Pasteurized Milk at Refrigerated Temperatures
Trenton W. Horner Brigham Young University - Provo
Follow this and additional works at: https://scholarsarchive.byu.edu/etd
Part of the Food Science Commons, and the Nutrition Commons
BYU ScholarsArchive Citation BYU ScholarsArchive Citation Horner, Trenton W., "Beta Galactosidose Activity of Commercial Lactase Samples in Raw and Pasteurized Milk at Refrigerated Temperatures" (2010). Theses and Dissertations. 2590. https://scholarsarchive.byu.edu/etd/2590
This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected].
Department of Nutrition, Dietetics, and Food Science
Master of Science
Many consumers are unable to enjoy the benefits of milk, due to lactose-intolerance. Lactose-free milk is available, but at about 2 times the cost of regular milk or greater, it may be difficult for consumers to afford. The high cost of lactose-free milk is in part due to the added cost of the lactose hydrolysis process. Hydrolysis at refrigerated temperatures, possibly in the bulk tank or package, could increase the flexibility of the process, and potentially reduce the cost.
A rapid β-galactosidase assay was used to determine the relative activity of commercially
available lactase samples at different temperatures. Four enzymes exhibited low-temperature activity and were added to refrigerated raw and pasteurized milk at various concentrations and allowed to react for various lengths of time. The degree of lactose hydrolysis by each of the enzymes as a function of time and enzyme concentration was determined by HPLC.
The two most active enzymes, as determined by the β-galactosidase assay, hydrolyzed
over 98% of the lactose in 24 hours at 2⁰C using the supplier recommended dosage. The other two enzymes hydrolyzed over 95% of the lactose in 24 hours at two times the supplier recommended dosage at 2⁰C. Results were consistent in all milk types tested. The results show that it is feasible to hydrolyze lactose during refrigerated storage of milk using currently available enzymes.
Hydrolysis in chilled milk ........................................................................................................................... 6
High Performance Liquid Chromatography Analysis ................................................................................ 7
Data Analysis ............................................................................................................................................. 8
RESULTS AND DISCUSSION............................................................................................................................ 8
Relative activity of commercial enzymes .................................................................................................. 8
Lactase activity in chilled milk ................................................................................................................... 9
Effect of different milk types .................................................................................................................. 11
Applicability to the industry .................................................................................................................... 11
APPENDIX A ................................................................................................................................................. 23
Statement of the problem ...................................................................................................................... 24
Method to be followed ........................................................................................................................... 25
Justification of the problem .................................................................................................................... 25
Delimitations of the problem .................................................................................................................. 26
APPENDIX B ................................................................................................................................................. 27
TESTING RELATIVE ACTIVITY OF THE ENZYMES: THE BETA-GALACTOSIDASE ASSAY ................................ 27
Appendix B-3: Explanation of the how the β-Galactosidase assay works ............................................. 32
v
APPENDIX C ................................................................................................................................................. 33
HPLC ANALYSIS OF LACTOSE HYDROLYSIS .................................................................................................. 33
Appendix C-2: Results of HPLC Analysis ................................................................................................. 37
APPENDIX D ................................................................................................................................................. 44
STATISTICAL ANALYSIS OF HPLC DATA ........................................................................................................ 44
1
INTRODUCTION Milk is a nutrient-dense food and an important part of a healthy diet. Along with high-
quality protein, milk contains many vitamins and minerals, such as riboflavin and calcium (Choi,
2007). Milk is fortified with vitamin D and is one of the few commonly consumed food products
containing this vitamin (Mannion, 2006). Unfortunately, many consumers are unable to enjoy
the benefits of milk due to lactose-intolerance (Choi, 2007; Somkuti, 1997; Husain, 2010).
Lactose intolerance is caused by a deficiency of β-galactosidase (lactase) in the digestive
tract (Panesar, 2007). Lactose is a milk disaccharide composed of glucose and galactose bonded
with a β-1→4 linkage. Normally, lactose in milk is hydrolyzed by β-galactosidase located in the
mucosal cells of the small intestine. β-galactosidase hydrolyzes lactose into its respective
monosaccharides which are easily absorbed and metabolized (Gropper, 2005).
When lactose intolerant individuals consume milk, the undigested lactose passes into the
colon and increases the osmolarity of the digested food. This inhibits the absorption of water by
the colon and can cause diarrhea. Also, bacteria in the colon are able to digest the lactose, which
causes increased acidity and the production of gas and toxins (Choi, 2007). These unpleasant
symptoms may inhibit the consumption of milk by lactose-intolerant consumers (Messia, 2007).
The amount of lactose needed to cause symptoms can vary widely from person to person.
Some lactose-intolerant individuals are unable to consume any lactose without experiencing the
symptoms of lactose-intolerance while others are able to consume some milk without
experiencing symptoms or with only mild symptoms (Choi, 2007). Cultured dairy products such
as yogurt and cheese often have significantly lower levels of lactose and many lactose-intolerant
individuals are able to eat them without symptoms (Sieber, 1997). Lactose-free milk is another
option consumers have available (Somkuti, 1997).
2
Lactose-free milk is commercially prepared by allowing β-galactosidase enzymes (E.C.
3.2.1.23) to hydrolyze the lactose present, prior to packaging. This allows consumers to gain the
valuable nutrients and protein available in milk without unpleasant symptoms.
While enzyme-altered lactose-free milk is available, it is expensive, and many consumers
are unwilling or unable to pay for the extra cost. Lopez and Lopez (2009) report high-income
families are the main consumers of lactose-free milk. As household income rises, the purchase
and usage of lactose-free milk products also rises (Mintel, 2010). The higher cost of lactose-free
milk is due in part to the cost of the hydrolysis process used by manufacturers. It is hypothesized
that if enzymes were sufficiently active at the temperature of chilled milk, the hydrolysis reaction
could be carried out under refrigeration during transport or storage in trucks, tanks, or final
packaging. By doing this, the cost to manufacture lactose-free milk might be reduced.
The market for lactose-free milk has been growing for years. Jelen (2003) predicted
about a 20% per year increase in the market. This expected increase was due in part to the
increased awareness of lactose intolerance. The market for lactose-free milk is estimated at 30-
50 million consumers in the U.S. alone (Adhikari, 2009). Lactose intolerance is more common
in some ethnic groups than others (FDA, 2009). Among African-Americans and Native
Americans it is about 75%; among Asian populations it is about 90% (FDA, 2009). With such a
large potential market for the product, reducing the cost of lactose-free milk could significantly
increase its consumption.
Reduction of lactose in milk has been explored using many different methods.
Traditionally, soluble β-galactosidase enzymes are added to milk. Panesar and others (2007)
explored the use of immobilized enzymes. Novalin and others (2005) describe using an
innovative hollow-fiber ultra-filtration process with immobilized enzymes. Another study
3
described using ultra-filtration and concentration by evaporation, then adding the enzymes to the
concentrated permeate and later adding the hydrolysate back to the retentate (Chen and others,
2002).
Lactose-free milk that has been treated with β-galactosidase is sweeter than regular milk
due to glucose and galactose being sweeter than lactose, combining to be about 70% of the
sweetness of sucrose as compared to lactose, which is only 20% the sweetness of sucrose
(Novalin, 2005). This sweetness may be advantageous for certain products such as ice cream but
alters the flavor of milk used for drinking. Choi and others (2007) have shown that the resulting
sweetness can be removed by utilizing nanofiltration and reconstituting the milk. Harju (1989)
describes a chromatographic process to separate the lactose from the protein and other
components of milk, completely removing lactose without the use of enzymes and without any
residual sweetness. However, none of these or other methods reported in the literature have the
potential to significantly reduce the cost of producing lactose-free milk compared to traditional
methods.
β-galactosidase enzymes are commonly harvested from microorganisms such as fungus,
yeast, and bacteria; for example, Kluyveromyces lactis is a commonly used yeast for producing
β-galactosidase in the industry (Dabhole, 1998). Enzymes sourced from yeast or bacteria are
well suited for the hydrolysis of lactose in milk, often displaying an activity optimum near the
pH of milk (Chen, 2009). A fungal β-galactosidase is typically used in acidic dairy applications,
such as sour cream (Chen, 2009).
A number of bacterial β-galactosidase enzymes, having optimum hydrolytic activity at
temperatures lower than is normal for lactase enzymes, have been isolated (Fernandes, 2002;
Coker, 2006). Many lactases have optimum temperatures above 30⁰C, whereas cold-active
4
lactases can have temperature optimums of 15⁰C or below (Coker, 2006). Bacteria adapted to
colder climates can be found in soil and isolated for β-galactosidase production (Coker, 2003).
Cold-active β-galactosidase has also been isolated from psychrophilic yeasts, which may be
suitable for food use (Husain, 2010). Thus, there is a potential for developing lactase enzymes
with significantly increased activity at refrigerated temperatures.
The objective of this research was to determine if currently available lactase enzymes are
active enough in chilled milk to enable the hydrolysis process to be carried out during
refrigerated storage.
MATERIALS
Enzymes Five commercially available food-grade β-galactosidase enzymes were obtained from
four separate distributors. The enzymes examined in the initial screening included the following:
California, U.S.A.). For comparison purposes, reference samples made from untreated milk
were analyzed in the same manner for each run.
HPLC separation conditions were as follows: gradient mobile phase of 75% acetonitrile,
25% water; 30⁰C; 1ml/min flow rate; 20 µl injection volume; refractive index detector. Data
was quantified using external calibration based on a standard curve.
Data Analysis Data were analyzed using Statistical Analysis System software version 9.2 (SAS
Institute, Cary, NC). A mixed model analysis of variance procedure was used. The dependent
variable of the model tested for difference from 95% lactose hydrolysis (i.e. difference from
95%) with p<0.05. Analyses were also run at 98% and 99% lactose hydrolysis (difference from
98% or 99%). The independent variables included time, enzyme concentration, and the
time*concentration interaction. The statistical analysis is found in appendix D.
RESULTS AND DISCUSSION
Relative activity of commercial enzymes In initial activity comparison tests in buffer (β-galactosidase assay), DYL and VYL
showed steep increases with rising temperatures, while EYL, LYL, and VFL were less affected
by the temperature range tested (see figure 1). Of the five enzymes evaluated at manufacturers’
recommended levels, DYL and VYL showed greater activity than EYL, LYL, and VFL (see
figure 1). This may be due to the higher protein concentration of DYL and VYL compared to
the other enzyme samples. VFL, specified for use in acidic dairy products, had very little
enzyme activity compared to the other four enzymes due its pH optimum (4.0 to 5.0) not being
within the range of the pH of milk (6.6 to 6.8) (Validase Fungal Lactase Concentrate
9
Specification, Valley Research, South Bend Indiana, USA). VFL was not included in further
experimentation.
Lactase enzymes produced from Kluyveromyces lactis commonly have a temperature
optimum above room temperature. Schneider (1990) found that lactase derived from this
microorganism had an 85% to 95% increase in activity at 38⁰C compared to the activity between
4⁰C to 5⁰C. DYL and VYL showed about a fourfold increase in activity at ambient (~23⁰C)
compared to the activity at 4⁰C. EYL and LYL showed much more modest increases.
When the results of the β-galactosidase assay were adjusted for protein content of the
enzyme samples (activity per mass (ng) of protein), the activities of the enzyme preparations
were similar to the activities of the enzymes when measured per volume, except LYL (see figure
1). At refrigerated temperatures, LYL had the highest activity per unit of protein, yet it
performed more poorly than the other enzymes during the assay. This is likely due to the LYL
having the lowest concentration of protein among the enzyme preparations tested.
Lactase activity in chilled milk To determine lactose hydrolysis in milk for each enzyme, lactose content of pre-treatment
milk samples in each trial was compared to the lactose content after treatment. The galactose
and glucose peaks eluted at the same retention time during analysis and were inseparable without
a specialized column; this was consistent with the results of Sharma and others (2009). Thus,
percent hydrolysis was determined by disappearance of lactose.
The HPLC lactose peak area of each treated milk sample was divided by the area of the
respective un-treated milk sample to obtain a percentage of lactose remaining. That percentage
was then subtracted from 1 in order to determine percent hydrolysis.
10
Percent hydrolysis of lactose in refrigerated milk varied, depending on the enzyme
source, enzyme concentration, and reaction time. Performance of the enzymes correlated well
with the results of the β-galactosidase assay; the enzymes showing greatest activity in the β-
galactosidase assay also hydrolyzed the most lactose in milk.
Percent hydrolysis for each of the four enzymes over time at a series of concentrations is
reported in table 1. Statistics for these values are reported in table 2. In pasteurized whole milk,
DYL hydrolyzed, on average, 99% of the lactose present in 24 hours at the supplier’s
recommended dosage. VYL showed an average of 98% hydrolysis in 24 hours at the supplier’s
recommended dosage. EYL and LYL were able to hydrolyze 95% of the lactose on average in
24 hours, when added at 2 times the recommended dosage.
Lactose hydrolysis can be achieved with less enzyme if the reaction is allowed to proceed
for a longer time. DYL achieved 95% average lactose hydrolysis in 72 hours while using one-
fourth the recommended dosage in pasteurized whole milk and 99% hydrolysis under the same
conditions using one-half the recommended dosage. VYL achieved 99% hydrolysis in 72 hours
using one-half dosage. EYL and LYL both accomplished approximately 99% hydrolysis on
average in 72 hours by using the recommended dosage.
Statistics shown in table 2 indicate the target levels of hydrolysis were achieved under the
stated conditions. Actual hydrolysis levels were not different than specified target levels as
indicated by the p-values. Since the intent of the process was to achieve at least the target level
of hydrolysis, p-values greater than 0.05 are indicative of the target hydrolysis levels being met
under the conditions indicated.
The results show the four commercially available enzymes are capable of hydrolyzing
95% or more of the lactose in milk within 24 hours using the recommended dosage (DYL and
11
VYL) or double the recommended dosage (EYL and LYL). All four enzymes can also
hydrolyze at least 99% of the lactose in 72 hours using half the amount of enzyme used in the 24
hours assays. Hydrolyzing lactose during refrigerated storage will allow greater processing
flexibility and potentially reduce the costs of producing lactose-free milk.
Effect of different milk types The hydrolysis rate was not affected by fat content or processing. Hydrolysis in raw
whole milk, pasteurized skim milk, and pasteurized whole milk all proceeded at approximately
the same rate. For illustrative purposes, figure 2 shows the similarity of hydrolysis in different
milk substrates for LYL. All enzymes tested behaved similarly. The higher fat content of whole
milk did not impede enzyme performance. Enzyme concentration per volume was the same in
every milk type, even though the lactose content of whole milk is somewhat lower than skim
milk, due to the higher fat content. Despite the slight difference in lactose concentration between
skim and whole milk, the observed hydrolysis rates were similar across all three milk types.
Applicability to the industry Lactose hydrolysis during refrigerated storage could be applied several different ways to
produce lactose-free milk. Adding the enzyme after pasteurization and homogenization and
bottling or packaging the milk without further processing is one possibility. By the time the
consumer would purchase the milk, the lactose would be sufficiently hydrolyzed. The enzymes
would have to be sterile filtered before adding to milk post-pasteurization (personal
communication, Cody Huft, Utah Department of Agriculture).
Another possibility would be to add the enzyme to raw milk before the milk is processed
at the plant. The enzyme could be added in the farm bulk tanks or in the tanker trucks used for
transport, allowing sufficient time for the lactose to be hydrolyzed before pasteurization and
homogenization. Such a process would require the farms and/or trucks to have dedicated tanks
12
for the purpose of lactose-free milk production. The enzyme could also be added to the holding
tanks at the plant, and hydrolysis could be accomplished while the milk is waiting to be
processed.
The possibility of Maillard browning may be of concern when hydrolyzing the lactose
before pasteurization; however, this does not appear to be a problem with lactose-free milk
products currently on the market. Maillard browning occurs when reducing sugars are heated in
the presence of protein (Fennema, 2008). The amount of reducing carbohydrate groups is
doubled once lactose is hydrolyzed into glucose and galactose (Messia, 2007). Dattatreya and
others (2010) found that once glucose and galactose have accumulated in sweet whey protein,
the whey may be predisposed brown more readily. This may also be true of milk. Addition of
sterile β-galactosidase post-pasteurization would avoid this problem (Messia, 2007).
The possibility of browning may also be avoided by using additional methods of
processing, such as the nanofiltration method utilized by Choi and others (2007) to remove the
glucose and galactose prior to heating. However, Messia (2007) found lactose-free milk which is
ultra-pasteurized after hydrolysis and stored at refrigerated temperatures to be acceptable. This
is in agreement with our own assessment of lactose-free milks from the local market.
Cost Analysis Enzyme cost for each of the four enzymes, as determined at the time of this study, is
shown in table 3. The cost of the enzymes correlated well with the activity of each enzyme. As
the cost increased, the activity also increased. DYL, which hydrolyzed lactose the most
effectively at refrigerated temperatures, was the most expensive of the four enzymes with a
minimum volume pricing of $110 per kilogram. VYL, being the next most active enzyme, cost
$100 per kg. EYL cost $55 per kg and LYL cost $63 per kg. It is interesting to note that EYL
13
seems to be priced about the same as DYL on an activity basis, as EYL required twice as much
volume of the liquid preparation but only costs half as much as DYL.
By using a smaller dose and a longer time for lactose hydrolysis, enzyme cost for a liter
of lactose-free milk could be minimized. By using VYL at ½ the supplier recommended dosage
and allowing 72 hours for hydrolysis, cost could be as low as $0.05 per liter of milk. The price is
in addition to the normal processing costs of producing milk.
CONCLUSIONS Commercially available lactase enzymes are active enough in chilled milk to enable the
hydrolysis process to be carried out during refrigerated storage. With the proper
time/concentration combinations, >99% lactose hydrolysis can be achieved. Sweetness in
lactose-free milk made by this method is acceptable as compared to current products on the
market. By reacting the milk during cold storage, the hydrolysis process can be simplified with
greater flexibility, potentially reducing the cost of manufacturing lactose-free milk.
14
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Chen, C. S., C. K. Hsu, and B. H. Chiang. 2002. Optimization of the enzymic process for manufacturing low-lactose milk containing oligosaccharides. Process Biochemistry 38(5):801-808.
Chen, W., H. Chen, Y. Xia, J. Yang, J. Zhao, F. Tian, H. P. Zhang, and H. Zhang. 2009. Immobilization of recombinant thermostable beta-galactosidase from Bacillus stearothermophilus for lactose hydrolysis in milk. Journal of Dairy Science 92(2):491-498.
Choi, S. H., S. B. Lee, and H. R. Won. 2007. Development of lactose-hydrolyzed milk with low sweetness using nanofiltration. Asian-Australasian Journal of Animal Sciences 20(6):989-993.
Coker, J. A., P. P. Sheridan, J. Loveland-Curtze, K. R. Gutshall, A. J. Auman, and J. E. Brenchley. 2003. Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic Arthrobacter isolate. Journal of Bacteriology 185(18):5473-5482.
Coker, J. A. and J. E. Brenchley. 2006. Protein engineering of a cold-active beta-galactosidase from Arthrobacter sp SB to increase lactose hydrolysis reveals new sites affecting low temperature activity. Extremophiles 10(6):515-524.
Dabhole, M. P. and K. N. Joishy. 1998. beta-galactosidase from the yeast Kluyveromyces lactis. Journal of Scientific & Industrial Research 57(4):201-204.
Dattatreya, A., W. Lee, and S. A. Rankin. 2010. Short communication: Presence of galactose and glucose promotes browning of sweet whey powder. Journal of Dairy Science 93(6):2354-2357.
FDA.gov [internet]. Silver Spring, MD: U.S. Food and Drug Administration; 2009 [accessed 2010 Jun 17]. Available from: http://www.fda.gov/ForConsumers/ConsumerUpdates/ucm094550.htm
Fennema, O., S. Damodaran, and K. Parkin. 2008. Fennema’s Food Chemistry. 4th ed. Boca Raton, FL. CRC Press. 1144 p.
Fernandes, S., B. Geueke, O. Delgado, J. Coleman, and R. Hatti-Kaul. 2002. beta-galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis. Applied Microbiology and Biotechnology 58(3):313-321.
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Gropper, S.S., J.L. Smith, and J.L. Groff. 2005. Advanced nutrition and human metabolism. 4th ed. Belmont, CA. Thomson Wadsworth. 600 p.
Harju, M., inventor; Valio Meijerien Keskusosuusliike, assignee. 1989 Apr 11. Process for the specific separation of lactose from milk. U.S. patent 4,820,348.
Husain, Q. 2010. beta Galactosidases and their potential applications: a review. Critical Reviews in Biotechnology 30(1):41-62.
Jelen, P. and O. Tossavainen. 2003. Low lactose and lactose-free milk and dairy products - prospects, technologies and applications. Australian Journal of Dairy Technology 58(2):161-165.
Lopez, E. and R. A. Lopez. 2009. Demand for Differentiated Milk Products: Implications for Price Competition. Agribusiness 25(4):453-465.
Mannion, C. A., K. Gray-Donald, and K. G. Koski. 2006. Association of low intake of milk and vitamin D during pregnancy with decreased birth weight. Canadian Medical Association Journal 174(9):1273-1277.
Messia, M. C., T. Candigliota, and E. Marconi. 2007. Assessment of quality and technological characterization of lactose-hydrolyzed milk. Food Chemistry 104(3):910-917.
Mintel Oxygen. 2010. Milk – US – April 2010. London, UK: Mintel International Group Limited.
Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.].
Nielsen, S.S. 2003. Food Analysis. 3rd ed. New York, NY. Springer Science+Business Media, Inc. 557 p.
Novalin, S., W. Neuhaus, and K. D. Kulbe. 2005. A new innovative process to produce lactose-reduced skim milk. Journal of Biotechnology 119(2):212-218.
Panesar, R., P. S. Panesar, R. S. Singh, and M. B. Bera. 2007. Applicability of alginate entrapped yeast cells for the production of lactose-hydrolyzed milk. Journal of Food Process Engineering 30(4):472-484.
Schneider, R. E., E. Corona, F. Rosales, F. E. Schneider, O. Rodriguez, and O. Pineda. 1990. Effect of temperature on the lactose hydrolytic capacity of a lactse derived from Kluyveromyces-lactis. American Journal of Clinical Nutrition 51(2):197-201.
Sharma, R., Y. S. Rajput, Poonam, G. Dogra, and S. K. Tomar. 2009. Estimation of sugars in milk by HPLC and its application in detection of adulteration of milk with soymilk. International Journal of Dairy Technology 62(4):514-519.
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Sieber, R., M. Stransky, and M. de Vrese. 1997. Lactose intolerance and consumption of milk and dairy products. Zeitschrift Fur Ernahrungswissenschaft 36(4):375-393.
Somkuti, G. A. and V. H. Holsinger. 1997. Microbial technologies in the production of low-lactose dairy foods. Food Science and Technology International 3(3):163-169.
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Wehr, H.M., and J.F. Frank. 2004. Standard methods for the examination of dairy products. 17th ed. Washington DC: American Public Health Association. 570 p.
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Figure 1 – Comparison of β-galactosidase activity of five commercial lactases at different temperatures. Graph A shows the results expressed as change in optical density at 420 nm (ΔOD420) per minute per volume of enzyme based on the adjusted Miller assay. Graph B shows the ΔOD420 per minute per mass (ng) of protein for each enzyme. The mass of protein calculation adjusts for protein content of each enzyme solution and allows for comparison of the efficiency of each enzyme preparation.
0.0E+00
5.0E+05
1.0E+06
1.5E+06
2.0E+06
2.5E+06
3.0E+06
4⁰ 10⁰ 20⁰ ambient
ΔOD
420
per v
olum
e
Temperature (⁰C)
β-Galactosidase Assay Results
DYL
VYL
EYL
LYL
VFL
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
4⁰ 10⁰ 20⁰ ambient
ΔOD
420
per m
ass (
ng) p
rote
in
Temperature (⁰C)
Activity Adjusted for Protein Content
DYL
VYL
EYL
LYL
VFL
A
B
18
Figure 2 – Effects of milk type on enzyme performance of the LYL β-galactosidase at 2⁰C. Average percent lactose hydrolysis is shown. Hydrolysis rate is very similar for all enzymes in all milk types.
0.00%20.00%40.00%60.00%80.00%
100.00%120.00%
12 24 48 72
Perc
ent H
ydro
lysi
s
Hours
Pasteurized Skim Milk
0.5x
1.0x
1.5x
2.0x
0.00%20.00%40.00%60.00%80.00%
100.00%120.00%
12 24 48 72
Perc
ent H
ydro
lysi
s
Hours
Pasteurized Whole Milk
0.5x
1.0x
1.5x
2.0x
0.00%20.00%40.00%60.00%80.00%
100.00%120.00%
12 24 48 72
Perc
ent H
ydro
lysi
s
Hours
Raw Whole Milk
0.5x
1.0x
1.5x
2.0x
19
Table 1 – Time-dependent lactose hydrolysis (2⁰C) in pasteurized whole milk for four selected commercial enzymes at different concentrations. Concentrations represent the fraction of the suppliers’ recommended dosage used. The percentages listed in the table represent the average percent lactose hydrolysis for the time/concentration pairs.
Table 2 – Mixed model statistics for lactose hydrolysis by four commercial enzymes after 24 and 72 hours, in whole milk at 2⁰C. The concentrations are based on the suppliers’ recommended dosage. The statistics show the target levels of % lactose hydrolysis were achieved under the conditions indicated. A p-value greater than 0.05 indicates the mean % hydrolysis and the target % hydrolysis are not significantly different.
Table 3 – Cost analysis for lactose hydrolysis in refrigerated milk (2⁰C) using the four β-galactosidase enzymes evaluated in this study. Concentrations represent the fraction of the suppliers’ recommended dosage required to achieve at least >95% lactose hydrolysis in the time allotted. Costs are for enzyme only in addition to the normal costs of milk manufacture.
Minimum volume pricing of commercial enzymes per kg DYL $110.00 VYL $100.00 EYL $55.00 LYL $63.22
DEPARTMENT OF NUTRITION, DIETETICS AND FOOD SCIENCE THESIS OR DISSERTATION PROSPECTUS
MASTER CANDIDATES
Proposed Title: Beta-Galactosidase Activity of Commercial Lactase Samples in Raw and Pasteurized Milk at Refrigerated Temperatures.
Statement of the problem Milk is a nutrient-dense food and an important part of a healthy diet. Along with good amounts of high-quality protein, milk contains many vitamins and minerals, such as calcium, riboflavin, and phosphorus. Milk is fortified with vitamin D and is one of the few food products containing this vitamin. Unfortunately, many individuals are unable to consume milk due to lactose-intolerance and thus the benefits of milk are not readily available to them.
Lactose intolerance is caused by a deficiency of “lactase” (beta-galactosidase) in the digestive tract. Normally, lactose in milk is hydrolyzed while in the intestinal tract. The lactose in milk is broken down into glucose and galactose by the enzyme, and these monosaccharides are easily utilized by the digestive system. When lactose intolerant individuals consume milk, the undigested lactose passes into the colon and increases the osmolarity of digested food. This inhibits the absorption of water by the colon and can cause diarrhea. Also, the bacteria in the colon are able to digest the lactose which causes the acidity of the stools to increase and produces gas and toxins. These unpleasant symptoms further inhibit the consumption of milk by these individuals.
For those who are lactose-intolerant, lactose-reduced and lactose-free milk are available. These milk products have 75-100% of the lactose removed. Normally, a β-galactosidase is used during processing to break-down lactose into galactose and glucose which are easily digested. However, the process for making this milk is expensive, and can cause the cost to be two-to-four times the amount of normal milk. This higher price can be a problem for those who need the milk, making it difficult for them to obtain the benefits that milk can give.
If β-galactosidase enzymes are determined to have sufficient activity in the cold to accomplish hydrolysis in 24-72 hours, the reaction might be carried out during refrigeration storage and thereby eliminating the expensive in-plant process. This would result in a lower overall cost of producing lactose-reduced milk. The objective of this research was to determine if currently available lactase enzymes were active enough in chilled milk to enable the hydrolysis process to be carried out during refrigerated storage.
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Method to be followed There are two proposed methods for using potentially cold-active enzymes, outlined by the following:
-Process milk as normal, adding sterile cold-active enzymes as the milk is packaged. (Lactose hydrolysis would occur before being sold on store shelves)
-Add cold-active enzymes before milk arrives at the plant (Adding the enzymes at the farm, before shipping to the plant)
Commercially available β-galactosidase enzymes were obtained. The temperature-activity profiles of the enzymes were determined in milk at pH 6.8 using a rapid colorimetric assay. This consisted of adding the candidate enzymes to a prepared assay buffer and reacting for several minutes, recording the start time. The mixture produced a moderately yellow color, caused by the hydrolysis of O-nitrophenyl-β, D-galactopyranoside (present in the assay buffer). Once the yellow color was present, a stop buffer was added to halt the reaction and the stop time was recorded. The mixture was then centrifuged and moved to a cuvette for use in a spectrophotometer. The instrument was used to take a reading on the color of the mixture at OD 420nm, and the data gathered was used to calculate activity. This assay was performed at various temperatures to produce the temperature vs. activity profile for each enzyme.
Candidate enzymes with cold-activity were reacted with milk using various concentrations at 2°C for 12, 24, 48, and 72 hour periods. Lactose content was determined at the end of the reaction times by High Performance Liquid Chromatography (HPLC) to determine the extent of hydrolysis. The times represented possible reaction times during cold-storage.
For HPLC analysis, the enzymes were denatured and milk samples were prepared using the Lactose HPLC Method (Class B) 11(984.22), 50 (as referenced in Standard Methods for the Examination of Dairy Products (2004)). Milk proteins were precipitated by adding 0.9N sulfuric acid to each sample followed by centrifugation. Read-outs from the HPLC were checked against galactose, glucose, and lactose standards.
Justification of the problem The researched methods of reducing lactose in milk have the potential to save time and money. Over-all, the enzymes would take more time at the lower temperatures, but this would be time during shipping rather than time in the plant. The new methods require less employee time, less plant space, and less energy. Potentially, such processes would be safer as well, since the milk would stay at refrigeration temperatures during the entire process. It may also be possible to eliminate the second pasteurization step, further saving time and money.
Feasibility of research for cheaper product includes the following: -The market for lactose-reduced products is growing
-Jelen (2003) predicted about a 20% per year increase, due in part to increased awareness of lactose intolerance
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-Estimated market is about 50 million consumers (in U.S. alone)
Delimitations of the problem Issues with lactose-reduced milk include factors such as a sweeter taste (from glucose and galactose being sweeter than lactose) and potential off-flavors (potential for increased Maillard browning with glucose/galactose during pasteurization). This study looked at these issues and concluded that the milk produced by the researched methods was acceptable compared to products currently on the market.
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APPENDIX B
TESTING RELATIVE ACTIVITY OF THE ENZYMES: THE BETA-
GALACTOSIDASE ASSAY
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Notes about the entire analysis – Please read first:
This analysis is adapted from the Miller Assay (Miller 1972), which is a bacterial assay. In the bacterial assay, everything used must be sterile; however, sterility was not important for testing these β-galactosidase enzymes. Also, the purified enzymes were much more active than a broth of lactase producing bacteria. Thus, the enzymes were diluted (10-1 to 10-6) in order to achieve similar results to the bacterial assay.
Preparation for the analysis – β-galactosidase reaction Materials needed:
• 1.5 mL Microfuge tubes and rack for holding the tubes • Basal Buffer • Chloroform • Assay Buffer • Stop Buffer • 2 Mechanical pipettes, one with 1mL tips and one with 100µL tips • Stop watch or timer
1. Place 7 empty microfuge tubes in rack. The first 6 tubes are for each dilution (10-1 to 10-
6); the seventh tube is for making a blank. 2. Add 450µL of basal buffer to each tube. 3. Add 50µL of enzyme to the 450µL of basal buffer in the first microfuge tube (dilution 10-
1). Close the top of the tube and shake vigorously to mix well (at least 5 seconds of shaking).
4. To each tube in succession, create dilutions of 10-2 through 10-6 by adding 50µL of the previous dilution to the 450µL of basal buffer in the next tube and mix well (e.g. starting with the first tube, take 50µL from the first tube and put it into the 450µL of basal buffer in the second tube and mix).
5. For the blank, do not add any the enzyme to the tube. 6. Add 30µL of chloroform to each tube, including the blank. 7. Add 600µL of assay buffer to each dilution in order (including the blank). Close the top
of each tube and shake vigorously to mix well. Start the timer or record the start time once the assay buffer is added to the first tube.
8. When samples are sufficiently yellow (produces a spectrophotometer reading between 0.1 and 1.0 at 420nm; I suggest using dilution 10-4 or 10-5), add 600 micro-liters of stop buffer and mix well, stopping the timer or recording the stop time. The blank will not turn yellow.
Running the analysis – Spectrophotometer Materials needed:
• Samples and blank from the β-galactosidase reaction (I found the 10-5 dilution worked well)
• A Centrifuge and test-tubes that fit the centrifuge • Cuvettes for each sample and the blank
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• Mechanical pipette with 1mL tips
1. Transfer the assay solutions to test tubes for use in a table-top centrifuge in Dr. Dunn’s lab. Centrifuge the samples for at least a minute.
2. Transfer 1mL of each sample to cuvettes. Prepare the blank in the same manner. 3. Use the spectrophotometer in Dr. Dunn’s lab, set at 420nm and zeroed with the prepared
blank, to read the samples. Instructions for using the instrument are in appendix B-2. Record the readings for each sample.
4. Plug the data into the equation (adapted for a non-bacterial assay), where OD420 is the reading from the spectrophotometer:
Units per volume = [1000*OD420]/[volume(mL)*Time(min.)]
5. The units are useful for comparing enzyme activities. See figure 1 for the results of the assay.
6. The data were adjusted to show ΔOD420 per mass of protein of enzyme, or efficiency of the enzymes, as follows:
Units per mass = [OD420/(T)] * [Mass of protein(ng)] *1000
Where T = time in minutes and protein mass is calculated from the following:
Volume(ml)*(mg/ml of protein in the enzyme solution) * (dilution factor) * 10^6
Where volume is the amount of enzyme solution used in the assay (0.050 ml), dilution factor being the dilution used for each trial (either 10^-4 or 10^-5), and 10^6 to convert into nanograms.
• 500mL water • 4.3 g NA2HPO4 • 2.4 g NaH2PO4 • 0.75 g KCL • ~1.5mL 2N KOH • 1mL of 1M MgSO4_7H2O • 1 L autoclave bottle
Add the 500mL of water to the autoclave bottle. Add the NA2HPO4, NaH2PO4, and 0.75 g KCL and mix well. The KOH is then added as needed to bring the pH up to 7.0 (it is possible the solution will already be at pH 7.0 without adding KOH; in this case, don’t add the KOH). Autoclave the solution. When cool, add the MgSO4_7H2O. Autoclave again. Store in refrigerator to help prevent microbial growth after using. Stop Buffer Materials needed:
• 500mL water • 1 M Na2CO3 (Sodium carbonate) • 1 L autoclave bottle
Mix 53 g Na2CO3 with 500mL of water in the autoclave bottle. Autoclave (A precipitate may form). Refrigerate to help prevent microbial growth after using. Assay Buffer Materials needed:
• 15 ml basal buffer • 13.25 mg ONPG • 11 µl 10% SDS (make 10% SDS solution by adding 10 g SDS to 90 g water) • 45 µl β-mercaptoethanol (USE A VENT HOOD! It emits a strong, toxic sulfur smell) • 100 ml flask
Add the basal buffer to the flask. Add the SDS solution and the β-mercaptoethanol (when adding the β-mercaptoethanol, a vent hood must be used!). Add the ONPG, seal the flask, and mix everything well (ONPG does not dissolve easily; it may be necessary to warm the solution in order to get it to dissolve). Store in the refrigerator and keep it sealed while storing.
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Appendix B-2: Spectrophotometer Dr. Dunn lab Spectrophotometer (S-161 ESC)
Spectrophotometer model: Milton Roy Spectronic 1001 (Milton Roy Instrument Group)
1. Switch on the machine; wait for it to initialize the wavelength (WV) 2. Using the number pad, type in the desired wavelength (420) followed by pressing the “go
to λ” button. The machine will go to wavelength 420nm. 3. Open the samples’ compartment to the left of the display panel. Set the cuvettes in the
holders (be sure the clear side of the cuvettes correctly face the light source, which is right of the sample holders). Place the prepared blank in first. Be sure to close the compartment before taking any readings.
4. Zero the machine using the blank by pressing the “second function” button followed by the “zero ABS” button. The display will revert to zero.
5. Use the knob and arm attached to the sample holders to move the next sample into place. Record the data on the display panel, which should be between 0.1 and 1.0 for this assay.
6. Repeat the process for the remaining samples.
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Appendix B-3: Explanation of the how the β-Galactosidase assay works
Source: Switzer RL, Garrity LF. 1999. Experimental biochemistry, theory and exercises in fundamental methods. 3rd ed. New York: W H Freeman and Company. 453 p. When O-nitrophenyl-β, D-Galactopyranoside (ONPG) reacts with a β-galactosidase enzyme, galactose and O-Nitrophenol (ONP) are the products, and the solution turns a yellow color. ONP absorbs light at 420nm in alkaline conditions, allowing for measurement by a spectrophotometer. The gathered data can be put into the following equation (adapted from the original equation for bacterial assays):
Activity Units = [1000*OD420]/[volume(mL)*Time(min.)] OD420 is the reading from the spectrophotometer and time refers to the length of time in minutes that the ONPG was reacted with the β-galactosidase during the assay. Using the data from the Miller unit equation, the relative activity of the β-galactosidase enzymes can be compared to one another, allowing for selection of the most effective enzymes.
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APPENDIX C
HPLC ANALYSIS OF LACTOSE HYDROLYSIS
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Preparation for the analysis – Lactose hydrolysis
Notes about this section: The instructions below were made using enzyme #1. The method won’t change for the other enzymes other than the amount of enzyme used per 100 ml of milk based on supplier instructions. Materials needed:
• 100 ml milk • 100 µl β-galactosidase • 250 ml flask
1. Pour the milk into the flask and add the enzyme. Seal the flask and mix vigorously to
ensure thorough dispersion of the enzyme. 2. Place the flask in a refrigerator for the proper amount of hours according to the
experimental design. 3. Remove from refrigerator and HTST pasteurize the sample (161⁰C for 15 seconds) in
order to denature the enzyme. Cool the pasteurized sample in refrigerator to prepare for the next step.
Preparation for the analysis – Separation of milk proteins Materials needed:
• 100 ml volumetric flask • 10 g pasteurized milk sample from previous step • Analytical scale • Pasteur pipettes • 1 ml 0.9N sulfuric acid • Distilled-deionized water • 3 ml syringes with attached syringe filters, 0.45 µm • HPLC vials with caps • A capper to seal HPLC vials
1. Place the flask on the analytical balance and tare the weight. 2. Measure 10.0000 ± 0.0030 g milk into the flask. 3. Remove flask from balance, add 1 ml sulfuric acid to the milk and swirl. 4. Add ~50 ml of the water to the flask and swirl again. 5. Bring the mixture to volume (100 ml) with water and seal the flask. Shake vigorously for
20 seconds. 6. Let the mixture to stand for at least 5 minutes to allow good separation of the proteins
from the liquid fraction. A layer of white milk proteins will settle out on the bottom of the flask.
7. Use a Pasteur pipette to remove the liquid fraction of the milk and put the liquid into a syringe w/filter attached. Two pipettes of liquid should be plenty.
8. Use the syringe to filter the liquid into an HPLC vial. Fill the vial at least half-full and no more than ¾ full.
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9. Cap and seal the vial. Source: Wehr MH, Frank JF. 2004. Standard Methods for the examination of dairy products. 17th ed. Washington DC: American Public Health Association.
• 100mL volumetric flask • A separate flask/beaker/bottle for storage • Concentrated sulfuric acid (10M)
Add 9mL of 10M sulfuric acid to the flask. Bring up to volume with distilled-deionized water. Store the acid in a separate flask to enable easy access to the acid using Pasteur pipettes.
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Appendix C-2: Results of HPLC Analysis This appendix contains the raw data from the HPLC analysis of all the milk samples.
Each page contains one of the six trials from the three types of milk experimented upon, denoted by the label at the top of the page.
The tables on the top of each page contain the percent lactose hydrolysis of every sample (384 samples total). Underneath the tables are graphical summaries of the data in the tables. The legend on each graph shows the concentration of each enzyme.
Each table is organized with time (hours) at the top of each column and the concentration of each enzyme in the left column under the enzyme designation.
Statistics, calculated using SAS version 9.2, for the analysis of the HPLC data on hydrolysis of lactose are included in this appendix. The SAS code used is listed below. The data was originally pulled from an excel file; this data is found in appendix C-2. The statistical analysis was run based on a lactose hydrolysis of 95%, 98%, and 99%.
PROC IMPORT OUT= WORK.in DATAFILE= "C:\SAS\bioag\fsn\ogden\horner\lactase.xls" DBMS=EXCEL REPLACE; RANGE="Sheet1$"; GETNAMES=YES; MIXED=NO; SCANTEXT=YES; USEDATE=YES; SCANTIME=YES; run; *Enzyme Time Concentration Hydrolysis Milk Trial; title2 'difference from .95'; data good;set in; zeroed=.95-hydrolysis; run; proc sort data=good; by enzyme milk; proc mixed data=good; class time concentration; model zeroed=time concentration time*concentration; lsmeans time*concentration; by enzyme milk; run;
