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Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus.

Li B, Wang Z, Li S, Donelan W, Wang X, Cui T, Tang D - BMC Biotechnol. (2013)

Bottom Line: The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively.The enzyme activity was not significantly inhibited by Ca2+.We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, China. Taixing.Cui@uscmed.sc.edu.

ABSTRACT

Background: Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions.

Results: A thermostable β-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose.

Conclusions: The thermostablity of this recombinant β-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization.

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Efficiency of β-glucosidase amount on lactose hydrolysis in milk and analyzed by HPLC. A. Lactose hydrolysis was measured as a function of enzyme quantity (U/ml) at 65°C for 30 min using different concentrations of enzyme ranging from 1.7 U/ml to 498 U/ml as indicated. All data are presented as the mean ± SD of triplicate determinations. B. HPLC analysis of the reactions performed at 65°C for 30 min with 0 U/ml (Panel 1), 17 U/ml (Panel 2), 186 U/ml (Panel 3), or 498 U/ml (Panel 4) β-glucosidase enzyme. Lac = lactose, Glc = glucose, Gal = galactose.
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Figure 4: Efficiency of β-glucosidase amount on lactose hydrolysis in milk and analyzed by HPLC. A. Lactose hydrolysis was measured as a function of enzyme quantity (U/ml) at 65°C for 30 min using different concentrations of enzyme ranging from 1.7 U/ml to 498 U/ml as indicated. All data are presented as the mean ± SD of triplicate determinations. B. HPLC analysis of the reactions performed at 65°C for 30 min with 0 U/ml (Panel 1), 17 U/ml (Panel 2), 186 U/ml (Panel 3), or 498 U/ml (Panel 4) β-glucosidase enzyme. Lac = lactose, Glc = glucose, Gal = galactose.

Mentions: The ability of β-glucosidase to hydrolyze the lactose in milk at 65°C for 30 min was tested using different amounts of the recombinant enzyme (Figure 4A) and then analyzed by HPLC (Figure 4B) which showed that the lactose in milk can be hydrolyzed into glucose and galactose. Using 17 U/ml of recombinant β-glucosidase achieved 50% hydrolysis and increasing to 498 U/ml of recombinant enzyme resulted in greater than 90% hydrolysis. We also tested the efficiency of hydrolysis of this enzyme (311 U/ml) from 5–30 minutes (Figure 5A-B) and found that after 20 minutes, the amount of hydrolysis was similar to 30 minutes. Therefore, we have demonstrated that the enzyme activity was more than sufficient to remove lactose in milk under pasteurization conditions. The effects of glucose concentration on hydrolysis was also tested, and we found that even high concentrations of glucose (5%) had minimal influence on the hydrolysis of lactose (Figure 6A-B), demonstrating that the enzyme had a relatively good tolerance to glucose. These results show that this recombinant thermostable β-glucosidase is highly-effective at hydrolyzing lactose in milk under pasteurization conditions.


Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus.

Li B, Wang Z, Li S, Donelan W, Wang X, Cui T, Tang D - BMC Biotechnol. (2013)

Efficiency of β-glucosidase amount on lactose hydrolysis in milk and analyzed by HPLC. A. Lactose hydrolysis was measured as a function of enzyme quantity (U/ml) at 65°C for 30 min using different concentrations of enzyme ranging from 1.7 U/ml to 498 U/ml as indicated. All data are presented as the mean ± SD of triplicate determinations. B. HPLC analysis of the reactions performed at 65°C for 30 min with 0 U/ml (Panel 1), 17 U/ml (Panel 2), 186 U/ml (Panel 3), or 498 U/ml (Panel 4) β-glucosidase enzyme. Lac = lactose, Glc = glucose, Gal = galactose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016594&req=5

Figure 4: Efficiency of β-glucosidase amount on lactose hydrolysis in milk and analyzed by HPLC. A. Lactose hydrolysis was measured as a function of enzyme quantity (U/ml) at 65°C for 30 min using different concentrations of enzyme ranging from 1.7 U/ml to 498 U/ml as indicated. All data are presented as the mean ± SD of triplicate determinations. B. HPLC analysis of the reactions performed at 65°C for 30 min with 0 U/ml (Panel 1), 17 U/ml (Panel 2), 186 U/ml (Panel 3), or 498 U/ml (Panel 4) β-glucosidase enzyme. Lac = lactose, Glc = glucose, Gal = galactose.
Mentions: The ability of β-glucosidase to hydrolyze the lactose in milk at 65°C for 30 min was tested using different amounts of the recombinant enzyme (Figure 4A) and then analyzed by HPLC (Figure 4B) which showed that the lactose in milk can be hydrolyzed into glucose and galactose. Using 17 U/ml of recombinant β-glucosidase achieved 50% hydrolysis and increasing to 498 U/ml of recombinant enzyme resulted in greater than 90% hydrolysis. We also tested the efficiency of hydrolysis of this enzyme (311 U/ml) from 5–30 minutes (Figure 5A-B) and found that after 20 minutes, the amount of hydrolysis was similar to 30 minutes. Therefore, we have demonstrated that the enzyme activity was more than sufficient to remove lactose in milk under pasteurization conditions. The effects of glucose concentration on hydrolysis was also tested, and we found that even high concentrations of glucose (5%) had minimal influence on the hydrolysis of lactose (Figure 6A-B), demonstrating that the enzyme had a relatively good tolerance to glucose. These results show that this recombinant thermostable β-glucosidase is highly-effective at hydrolyzing lactose in milk under pasteurization conditions.

Bottom Line: The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively.The enzyme activity was not significantly inhibited by Ca2+.We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, China. Taixing.Cui@uscmed.sc.edu.

ABSTRACT

Background: Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions.

Results: A thermostable β-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose.

Conclusions: The thermostablity of this recombinant β-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization.

Show MeSH
Related in: MedlinePlus