BACKGROUND
in vivo, lactase  is an enzyme secreted by intestinal villi that hydrolyses the disaccharide lactose into glucose and galactose  <cit>  and is essential for the digestion of bovine milk  <cit>  which contains an average of  <dig> % lactose.
deficiency for this gene  <cit>  leads to malabsorption of lactose and subsequent fermentation of lactose by the gut flora  <cit> .
lactose intolerance develops when afflicted individuals experience abdominal pain, diarrhea, bloating, flatulence, and other gastrointestinal symptoms following lactose consumption  <cit> .
avoidance of dairy products by lactose intolerant individuals often leads to insufficient calcium and vitamin d consumption and may cause adverse health outcomes, especially reduced bone mineral density and fractures  <cit> .
since lactose is a disaccharide composed of glucose and galactose, it can be hydrolyzed into these monosaccharides using either a β-glucosidase or β-galactosidase.
consumption of lactose-free milk, which can be produced by adding lactase enzyme  directly to milk, provides a means of maintaining good health while avoiding the symptoms of lactose intolerance.
the lactase enzymes most commonly used in the industry are β-galactosidases, which are generally inhibited as glucose concentration increases.
lactase can be synthesized from many sources including animals, plants, bacteria, fungi, and yeast  <cit> .
although bacterial sources can efficiently produce lactase, they are not considered safe for use in food production due the risk of microbial contamination  <cit> .
the lactase enzymes most widely used in industry are mesophilic enzymes from fungi  and yeast   <cit> .
fungal sources, with acidic ph-optima, are effective for hydrolyzing lactose in whey, while yeast sources, with neutral ph-optima, are more effective for hydrolyzing lactose in milk  <cit> .
although pasteurization conditions denature many enzymes, several thermostable lactase enzymes have been identified from a variety of sources  <cit> .
hyperthermophilic archeaeon pyrococcus furiosus harbors several hydrolytic enzyme activities; notably, it has a thermostable β-glucosidase  with a half-life of 85 h at 100°c.
this enzyme can also function as a β-galactosidase, but the β-galactosidase activity is  <dig> % of the β-glucosidase activity  <cit> .
although this enzyme has been cloned and functionally characterized in saccharomyces cerevisiae, expression in batch culture produced low yields  which are not suitable for industrial applications  <cit> .
still, this β-glucosidase serves as a model system to use for lactose hydrolysis since it displays extreme stability and high catalytic activity in the presence of lactose.
in the present study, we cloned the p. furiosus β-glucosidase gene  into the pgapzaa vector and electrotransformed it into the pichia pastoris x- <dig> strain for large-scale production of heterologous proteins by high-density cell culture.
the glyceraldehyde-3-phosphate dehydrogenase promoter  has been used for large-scale constitutive expression of many heterologous proteins and eliminates problems associated with using the traditional aox <dig> promoter such as the hazard and cost associated with the delivery and storage of large volumes of methanol  <cit> .
we then analyzed the lactose hydrolytic action of this β-glucosidase under pasteurization conditions in order to establish a new method for the production of low lactose pasteurized milk.
our system provides a method for high level production of “food grade” thermostable lactase enzyme that is appropriate for use in the milk industry during the process of pasteurization, eliminating the need to add enzyme following pasteurization and eliminating the risk of microbial contamination.
RESULTS
fed-batch fermentation
unlike the aox <dig> promoter which requires methanol to initiate gene expression, the gap promoter constitutively expresses genes in p. pastoris cells grown on many carbon sources  <cit> .
product formation appears to be growth associated in the gap promoter constitutive expression system, therefore, a higher cell density would help to achieve higher product concentration.
the fermentation was a fed-batch process carried out in 5 l fermenters.
the x- <dig> cells were initially grown on basal salts medium and fed with 50% glucose after 48 h. cell concentrations, total protein in supernatant, and enzyme activities during fermentation are shown in figure 1a.
the biomass accumulated at 120 h was 312 g/l, enzyme expression reached its peak with a yield of 740 mg/l, and the corresponding β-glucosidase activity was  <dig> 1u/ml.
we also tested the β-galactosidase activity of the enzyme and found it to be  <dig> u/ml, approximately 35% of the β-glucosidase activity.
a protein corresponding to recombinant β-glucosidase, with a molecular mass of approximately 120 kda, was detected upon performing sds-page analysis with coomassie brilliant blue staining .
this band corresponds to the dimer unit of the enzyme.
the native β-glucosidase is a tetramer, composed of four identical subunits, and each subunit has a molecular mass of 58 ± 2 kda.
the two monomer subunits dimerize by forming a disulfide bond and have a molecular mass of approximately 120 kda.
the enzyme dimers form a tetramer through non-covalent bonding, which is the functional native enzyme.
it has been previously demonstrated that boiling in sds is insufficient to denature the dimer which requires additional reagents, such as β-mercaptoethanol, to cleave the disulfide bonds.
purification of recombinant β-glucosidase
since the enzyme has a relatively low pi of  <dig>  and few secreted proteins are present in the supernatant, we used one-step weak anion exchange chromatography to isolate the target protein, thus making the recombinant production of the enzyme more favorable.
samples of supernatant before and after purification were run side by side using sds-page .
figure 2a shows the typical elution profile of chromatography.
this procedure resulted in  <dig>  fold purification and  <dig> % recovery from the supernatant .
pichia pastoris
enzyme characterization
the effects of temperature on enzyme activity and stability are shown in figure 3a.
optimum temperature was 100°c, and at 30°c the activity decreased to nearly 7% of the maximum activity.
β-glucosidase was stable and retained more than 80% of its maximum activity from 30°c to 120°c.
regarding ph, β-glucosidase presented optimum activity at ph  <dig>   and was nearly inactivated at extreme ph .
β-glucosidase retained more than 80% of its maximum activity from ph  <dig>  to  <dig>  when pre-incubated at 37°c for 1 hour.
the effect of metal cations on the β-glucosidase activity is shown in table  <dig>  and although cu2+ can reduce the relative activity to 78%, other metal cations have only negligible effects on the activity of the recombinant enzymes.
importantly, ca2+ did not significantly inhibit the activity of the enzyme.
effects of metal ions  on the activity of the recombinant β-glucosidase are shown.
data are presented as the means ± sd of triplicate determinations.
hydrolysis of lactose in milk under pasteurization conditions
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  and then analyzed by hplc  which showed that the lactose in milk can be hydrolyzed into glucose and galactose.
using  <dig> u/ml of recombinant β-glucosidase achieved 50% hydrolysis and increasing to  <dig> u/ml of recombinant enzyme resulted in greater than 90% hydrolysis.
we also tested the efficiency of hydrolysis of this enzyme  from 5–30 minutes  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  had minimal influence on the hydrolysis of lactose , 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.
discussion
voorhorst  <cit>  reported using e. coli to express the β-glucosidase gene.
the expressed product remained within the host cell and necessitates complicated purification methodologies.
moreover, e. coli is not considered safe for food applications.
smith  <cit>  reported expressing the β-glucosidase gene in s. cerevisiae, but the yield is very low at only 10 mg/l in batch culture.
the development of the s. cerevisiae expression system was limited by the lack of a strong promoter and being incapable of high-density cell culture.
recently, p. pastoris has been widely used as an efficient expression system, secreting very low levels of its own proteins.
the secreted heterologous protein is often virtually the only one protein in the culture medium.
thus, we cloned and expressed the β-glucosidase gene from p. furiosus into p. pastoris, and to our knowledge, this is the first use of p. pastoris to express this enzyme.
in the past, many researchers chose the methanol oxidase  promoter to construct expression vectors for the production of a variety of recombinant proteins in p. pastoris <cit> .
it is especially effective for gene expression of products that are toxic to the host.
however, methanol is a toxic substance, causes environment pollution, and is a fire hazard during production.
additionally, it is inconvenient to change from glucose or glycerol to methanol during fermentation.
therefore, we chose the constitutive gap promoter for the production of this recombinant enzyme.
this system is more desirable for large scale production because the hazard and cost associated with the storage and delivery of large volumes of methanol are eliminated.
the recombinant enzyme was cultivated in a 5 l fermentor, and after 120 h, we obtained 740 mg/l of β-glucosidase.
even when expression was not fully optimized, we achieved 700-fold higher levels than with e.coli expression and 70-fold greater than the s. cerevisiae system  <cit> .
in another 300 l fermentor, we obtained 7 g/l of recombinant β-glucosidase after 160 h, which is quite convenient to use for industrial production.
the ph optimum of recombinant enzyme lies slightly above the β-glucosidase from p. furiosus  and the temperature optimum  of the enzyme was similar with the β-glucosidase of p. furiosus .
the purified enzymes showed a remarkable thermostability, and even incubation at 100°c for 1 hour didn’t change the activity significantly.
the enzyme was not significantly stimulated or inhibited by divalent cations, as found for some glycosidases.
the ability of the recombinant β-glucosidase to hydrolyze lactose in milk under pasteurization conditions was tested and analyzed by hplc.
the lactose in the milk can be easily hydrolyzed into glucose and galactose.
the maximum amount of hydrolysis was 91% when using  <dig> u/ml of the recombinant β-glucosidase at 65°c for 30 min.
this is quite useful for industrial production of low lactose milk and milk powder under pasteurization conditions and the established industrial systems  would likely require less enzyme to achieve similar results.
in the past, the lactases most widely used in industry were obtained from aspergillus spp.
and kluyveromyces spp, but they are all mesophilic enzymes, which cannot be used before pasteurization, and adding the enzymes after pasteurization leads to a high risk of contamination.
moreover, the hydrolysis time of these enzymes is generally more than 24 hours  <cit> , which is too long for the production of pasteurized milk.
however, these problems are all solved by using the recombinant thermotolerant β-glucosidase.
using this enzyme before pasteurization decreases the risk of bacterial contamination, and incubating at 65°c for 30 min can effectively hydrolyze more than 90% of milk lactose.
in addition, we found the hydrolysis efficiency of the enzyme at 65°c for 20 minutes was similar to 65°c for 30 minutes.
different from many other glucosidases  <cit> , glucose has relatively small influence on this enzyme.
we added 5% glucose to the milk and found that the lactose hydrolysis efficiency only decreased slightly, highlighting another advantage of using this enzyme in low lactose milk production.
in conclusion, the enhanced stability of this recombinant thermostable 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 would be suitable for application in low-lactose milk production during milk pasteurization.
CONCLUSIONS
this report is the first on the cloning, expression, purification, and characterization of the β-glucosidase from p. furiosus in p. pastoris.
we achieved an enzyme yield of up to 740 mg/l using basal salts medium and purified the enzyme in one step anion exchange chromatography to near electrophoretic homogeneity.
in this study, we established a new method to produce low lactose milk using this enzyme under pasteurization conditions.
this method highlights the potential for low lactose milk production without additional steps required to add enzymes following pasteurization, and thus, decreases the risk of microbial contamination to a great extent.

 in vivo, lactase is an enzyme secreted by intestinal villi that hydrolyses the disaccharide lactose into glucose and galactose and is essential for the digestion of bovine milk which contains an average of % lactose. deficiency for this gene leads to malabsorption of lactose and subsequent fermentation of lactose by the gut flora. lactose intolerance develops when afflicted individuals experience abdominal pain, diarrhea, bloating, flatulence, and other gastrointestinal symptoms following lactose consumption. avoidance of dairy products by lactose intolerant individuals often leads to insufficient calcium and vitamin d consumption and may cause adverse health outcomes, especially reduced bone mineral density and fractures. since lactose is a disaccharide composed of glucose and galactose, it can be hydrolyzed into these monosaccharides using either a β-glucosidase or β-galactosidase. consumption of lactose-free milk, which can be produced by adding lactase enzyme directly to milk, provides a means of maintaining good health while avoiding the symptoms of lactose intolerance. the lactase enzymes most commonly used in the industry are β-galactosidases, which are generally inhibited as glucose concentration increases. lactase can be synthesized from many sources including animals, plants, bacteria, fungi, and yeast. although bacterial sources can efficiently produce lactase, they are not considered safe for use in food production due the risk of microbial contamination. the lactase enzymes most widely used in industry are mesophilic enzymes from fungi and yeast. fungal sources, with acidic ph-optima, are effective for hydrolyzing lactose in whey, while yeast sources, with neutral ph-optima, are more effective for hydrolyzing lactose in milk. although pasteurization conditions denature many enzymes, several thermostable lactase enzymes have been identified from a variety of sources. hyperthermophilic archeaeon pyrococcus furiosus harbors several hydrolytic enzyme activities; notably, it has a thermostable β-glucosidase with a half-life of 85 h at 100°c. this enzyme can also function as a β-galactosidase, but the β-galactosidase activity is % of the β-glucosidase activity. although this enzyme has been cloned and functionally characterized in saccharomyces cerevisiae, expression in batch culture produced low yields which are not suitable for industrial applications. still, this β-glucosidase serves as a model system to use for lactose hydrolysis since it displays extreme stability and high catalytic activity in the presence of lactose. in the present study, we cloned the p. furiosus β-glucosidase gene into the pgapzaa vector and electrotransformed it into the pichia pastoris x- strain for large-scale production of heterologous proteins by high-density cell culture. the glyceraldehyde-3-phosphate dehydrogenase promoter has been used for large-scale constitutive expression of many heterologous proteins and eliminates problems associated with using the traditional aox promoter such as the hazard and cost associated with the delivery and storage of large volumes of methanol. we then analyzed the lactose hydrolytic action of this β-glucosidase under pasteurization conditions in order to establish a new method for the production of low lactose pasteurized milk. our system provides a method for high level production of “food grade” thermostable lactase enzyme that is appropriate for use in the milk industry during the process of pasteurization, eliminating the need to add enzyme following pasteurization and eliminating the risk of microbial contamination.  fed-batch fermentation unlike the aox promoter which requires methanol to initiate gene expression, the gap promoter constitutively expresses genes in p. pastoris cells grown on many carbon sources. product formation appears to be growth associated in the gap promoter constitutive expression system, therefore, a higher cell density would help to achieve higher product concentration. the fermentation was a fed-batch process carried out in 5 l fermenters. the x- cells were initially grown on basal salts medium and fed with 50% glucose after 48 h. cell concentrations, total protein in supernatant, and enzyme activities during fermentation are shown in figure 1a. the biomass accumulated at 120 h was 312 g/l, enzyme expression reached its peak with a yield of 740 mg/l, and the corresponding β-glucosidase activity was 1u/ml. we also tested the β-galactosidase activity of the enzyme and found it to be u/ml, approximately 35% of the β-glucosidase activity. a protein corresponding to recombinant β-glucosidase, with a molecular mass of approximately 120 kda, was detected upon performing sds-page analysis with coomassie brilliant blue staining. this band corresponds to the dimer unit of the enzyme. the native β-glucosidase is a tetramer, composed of four identical subunits, and each subunit has a molecular mass of 58 ± 2 kda. the two monomer subunits dimerize by forming a disulfide bond and have a molecular mass of approximately 120 kda. the enzyme dimers form a tetramer through non-covalent bonding, which is the functional native enzyme. it has been previously demonstrated that boiling in sds is insufficient to denature the dimer which requires additional reagents, such as β-mercaptoethanol, to cleave the disulfide bonds. purification of recombinant β-glucosidase since the enzyme has a relatively low pi of and few secreted proteins are present in the supernatant, we used one-step weak anion exchange chromatography to isolate the target protein, thus making the recombinant production of the enzyme more favorable. samples of supernatant before and after purification were run side by side using sds-page. figure 2a shows the typical elution profile of chromatography. this procedure resulted in fold purification and % recovery from the supernatant. pichia pastoris enzyme characterization the effects of temperature on enzyme activity and stability are shown in figure 3a. optimum temperature was 100°c, and at 30°c the activity decreased to nearly 7% of the maximum activity. β-glucosidase was stable and retained more than 80% of its maximum activity from 30°c to 120°c. regarding ph, β-glucosidase presented optimum activity at ph  and was nearly inactivated at extreme ph. β-glucosidase retained more than 80% of its maximum activity from ph  to when pre-incubated at 37°c for 1 hour. the effect of metal cations on the β-glucosidase activity is shown in table  and although cu2+ can reduce the relative activity to 78%, other metal cations have only negligible effects on the activity of the recombinant enzymes. importantly, ca2+ did not significantly inhibit the activity of the enzyme. effects of metal ions on the activity of the recombinant β-glucosidase are shown. data are presented as the means ± sd of triplicate determinations. hydrolysis of lactose in milk under pasteurization conditions 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 and then analyzed by hplc which showed that the lactose in milk can be hydrolyzed into glucose and galactose. using u/ml of recombinant β-glucosidase achieved 50% hydrolysis and increasing to u/ml of recombinant enzyme resulted in greater than 90% hydrolysis. we also tested the efficiency of hydrolysis of this enzyme from 5–30 minutes 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 had minimal influence on the hydrolysis of lactose, 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. discussion voorhorst reported using e. coli to express the β-glucosidase gene. the expressed product remained within the host cell and necessitates complicated purification methodologies. moreover, e. coli is not considered safe for food applications. smith reported expressing the β-glucosidase gene in s. cerevisiae, but the yield is very low at only 10 mg/l in batch culture. the development of the s. cerevisiae expression system was limited by the lack of a strong promoter and being incapable of high-density cell culture. recently, p. pastoris has been widely used as an efficient expression system, secreting very low levels of its own proteins. the secreted heterologous protein is often virtually the only one protein in the culture medium. thus, we cloned and expressed the β-glucosidase gene from p. furiosus into p. pastoris, and to our knowledge, this is the first use of p. pastoris to express this enzyme. in the past, many researchers chose the methanol oxidase promoter to construct expression vectors for the production of a variety of recombinant proteins in p. pastoris. it is especially effective for gene expression of products that are toxic to the host. however, methanol is a toxic substance, causes environment pollution, and is a fire hazard during production. additionally, it is inconvenient to change from glucose or glycerol to methanol during fermentation. therefore, we chose the constitutive gap promoter for the production of this recombinant enzyme. this system is more desirable for large scale production because the hazard and cost associated with the storage and delivery of large volumes of methanol are eliminated. the recombinant enzyme was cultivated in a 5 l fermentor, and after 120 h, we obtained 740 mg/l of β-glucosidase. even when expression was not fully optimized, we achieved 700-fold higher levels than with e.coli expression and 70-fold greater than the s. cerevisiae system. in another 300 l fermentor, we obtained 7 g/l of recombinant β-glucosidase after 160 h, which is quite convenient to use for industrial production. the ph optimum of recombinant enzyme lies slightly above the β-glucosidase from p. furiosus and the temperature optimum of the enzyme was similar with the β-glucosidase of p. furiosus. the purified enzymes showed a remarkable thermostability, and even incubation at 100°c for 1 hour didn’t change the activity significantly. the enzyme was not significantly stimulated or inhibited by divalent cations, as found for some glycosidases. the ability of the recombinant β-glucosidase to hydrolyze lactose in milk under pasteurization conditions was tested and analyzed by hplc. the lactose in the milk can be easily hydrolyzed into glucose and galactose. the maximum amount of hydrolysis was 91% when using u/ml of the recombinant β-glucosidase at 65°c for 30 min. this is quite useful for industrial production of low lactose milk and milk powder under pasteurization conditions and the established industrial systems would likely require less enzyme to achieve similar results. in the past, the lactases most widely used in industry were obtained from aspergillus spp. and kluyveromyces spp, but they are all mesophilic enzymes, which cannot be used before pasteurization, and adding the enzymes after pasteurization leads to a high risk of contamination. moreover, the hydrolysis time of these enzymes is generally more than 24 hours, which is too long for the production of pasteurized milk. however, these problems are all solved by using the recombinant thermotolerant β-glucosidase. using this enzyme before pasteurization decreases the risk of bacterial contamination, and incubating at 65°c for 30 min can effectively hydrolyze more than 90% of milk lactose. in addition, we found the hydrolysis efficiency of the enzyme at 65°c for 20 minutes was similar to 65°c for 30 minutes. different from many other glucosidases, glucose has relatively small influence on this enzyme. we added 5% glucose to the milk and found that the lactose hydrolysis efficiency only decreased slightly, highlighting another advantage of using this enzyme in low lactose milk production. in conclusion, the enhanced stability of this recombinant thermostable 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 would be suitable for application in low-lactose milk production during milk pasteurization.  this report is the first on the cloning, expression, purification, and characterization of the β-glucosidase from p. furiosus in p. pastoris. we achieved an enzyme yield of up to 740 mg/l using basal salts medium and purified the enzyme in one step anion exchange chromatography to near electrophoretic homogeneity. in this study, we established a new method to produce low lactose milk using this enzyme under pasteurization conditions. this method highlights the potential for low lactose milk production without additional steps required to add enzymes following pasteurization, and thus, decreases the risk of microbial contamination to a great extent. 
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. 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. use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions. 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- the recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. 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. the optimum temperature and ph for this β-glucosidase activity was 100°c and ph , respectively. the enzyme activity was not significantly inhibited by ca2+. recombinant β-glucosidasethermostable enzymepichia pastorismilk lactose hydrolysispasteurization 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.
 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.  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- 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 , 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.  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. recombinant β-glucosidasethermostable enzymepichia pastorismilk lactose hydrolysispasteurization 