Adjusting the proportion of a substance by enzyme treatment

The proportion of a compound such as lactose in a solution or suspension such as milk using a soluble enzyme such as beta-galactosidase is carried out by removing matter larger than the enzyme in the solution or suspension by filtering with a first filter to produce a filtrate containing the compound, enzymatically converting the compound present in the filtrate, filtering off the enzyme with a second filter and recombining the resultant filtrate with the matter filtered off by the first filter.

The present invention relates to a process and apparatus for adjusting the 
proportion of a compound in a solution or suspension and more particularly 
to such a process and apparatus utilizing a soluble enzyme. 
Enzymes have found increasing use in industry for the purpose of promoting 
chemical reactions. For reasons of economy, and to avoid contamination of 
the final product, it is frequently necessary to recover the enzyme used 
in the process from the liquid in which the reaction has taken place. 
Conventionally two processes have been used to entrain the enzymes, the 
first is to insolubulise the enzyme by attachment to an insoluble support 
and the second is to retain the enzyme in a membrane diffusion bag reactor 
and to flow the liquid past the bag. The first method has the draw-back 
that insolubulised enzymes tend to lose activity quite rapidly with time; 
it has also been found that large areas of enzyme support, or long 
residence times, are required which adds to the cost of the process. The 
second method which depends upon diffusion of the reactants and the 
reaction products across a semipermeable membrane is slow and requires 
large areas of membrane and is thus unsuitable to commercial use. 
The present invention consists in a process for adjusting the proportion of 
a compound in a solution using a soluble enzyme having a molecular size 
greater than that of the compound, comprising the steps of filtering from 
the solution all matter having a size larger than the molecular size of 
the enzyme in a first filter, adding the enzyme to the first filtrate to 
achieve the desired enzymatic conversion of the said compound; filtering 
the enzyme from the solution in a second filter and adding the second 
filtrate to the matter filtered out by the first filter. 
The present invention further consists in apparatus for adjusting the 
proportion of a compound in a solution using a soluble enzyme having a 
molecular size greater than that of the said compound, comprising a first 
filter having a pore size not larger than the molecular size of the 
enzyme, a reaction vessel for the enzymatic conversion of the substance, a 
second filter having a pore size smaller than the molecular size of the 
enzyme and means to combine the filtrate from the second filter with 
matter filtered out by the first filter. 
The present invention has particular applicability to the hydrolysis of 
lactose in milk, however, it can also be used in numerous other systems 
such as the adjustment of the glucose content in egg albumen using glucose 
ozidase or the removal of limonin from citrus juices using limoninase. 
It is preferred that the enzymatic material is recycled from the second 
filter to the reactor in order to maintain a continuous flow of solution 
from the apparatus. The reactor may be of the plug flow type although 
other types of reactors, particularly continuous reactors such as back-mix 
reactors may be used. 
It will be appreciated that if the pore size of the two filters is the 
same, or if the pore size of the second filter is larger than the pore 
size of the first filter, the final composition of the liquid will be the 
same as its initial composition, with the exception of the enzymatic 
conversion of the substrate compound. This process can thus be used to 
effect the economic conversion of specific substances in a heterogeneous 
solution or suspension provided that the substance can be enzymatically 
converted by an enzyme having a larger molecular size than that of the 
substance. The process is of particular use where it is desired to remove 
from the solution a compound, other than the substrate compound, which 
would also be converted by the enzyme or which would deactivate the 
enzyme. It has been found that the removal of the larger particles or 
molecules from the solution prior to enzymatic conversion of the substrate 
compound increases the activity shown by the enzyme. 
If desired enzyme may be grafted onto a soluble substrate in order to 
increase its molecular size and hence the rate at which the reaction 
solution may be filtered. As used throughout this specification, the term 
"the molecular size of enzyme" is taken to mean molecular size of the 
enzyme itself together with any soluble substrate to which it may be 
grafted.

Apparatus for the adjustment of the lactose content of skim milk using 
.beta.-galactosidase comprises a feed tank 10, ultrafilters 11 and 12 and 
an enzyme reactor 13. 
Skim milk is fed intermittently into feed tank 10 through line 14 and is 
fed continuously out along line 15 through pump 16 to the first 
ultrafilter 11. 
Milk passing through ultrafilter 11 is separated into a filtrate consisting 
of a solution of molecules of a molecular size smaller than that of the 
enzyme and a concentrate containing larger molecular size molecules. In 
practice it was found convenient to operate the ultrafilter under 
conditions wherein 90% of the skim milk comprises the filtrate and 10% is 
separated off into the concentrate. 
The filtrate is passed along line 17 to the enzyme reactor 13 while the 
concentrate is passed directly through line 18 to an in-line mixer 19 for 
recombination with enzyme treated filtrate. 
In order to facilitate the starting up of the apparatus a recycle line 21 
and pump 22 are provided to recycle milk concentrate through the 
ultrafilter 11. 
Line 17 carries the filtrate from ultrafilter 11 to the enzyme reactor 13 
which is fitted with an agitator 23. The enzyme .beta.-glactosidase is 
added to the filtrate and converts the lactose in the milk filtrate to 
glucose and galactose. A conversion rate of 90% has been found to be 
obtainable with short residense times in the reactor. 
The reacted milk is carried by line 24 through pump 25 and heat exchanger 
26 to the second ultrafilter 12. The heat exchaner 26 is used to maintain 
the temperature in the enzyme reactor 13 at the temperature which 
corresponds to the maximum activity of the enzyme present in the reactor. 
Ultrafilter 12 separates the reacted milk into a filtrate containing the 
reacted milk constituents and a concentrate containing the enzyme. The 
filtrate is carried by line 27 to the in-line mixer 19 for recombination 
with the milk concentrate produced in the first ultrafilter 11. The enzyme 
concentrate from the second ultrafilter 12 is returned to the enzyme 13 
through line 28. 
The lactose adjusted milk contained 19% of its initial lactose content. The 
residual lactose content can be adjusted in the process by varying the 
ratio of separation achieved by the first filter, or by adjusting the 
degree of hydrolysis of the filtrate in the enzyme reactor. 
Milk having an adjusted lactose can be fed to persons who are unable to 
metabolise lactose; a condition common in large areas of Asia and the 
Middle East. The adjusted milk has considerable use in food processing 
e.g. lactose tends to precipitate out from frozen dairy products such as 
ice cream, the use of the milk treated according to this invention 
alleviates this problem as well as reducing the amount of sucrose which 
must be added to the product due to the greater sweetening power of 
glucose and galactose as compared with the original lactose. Baked goods 
such as buns and cakes have better browning properties when made with 
lactose adjusted milk. 
The apparatus described above can be used to carry out other processes 
according to the present invention. These examples are 
EXAMPLE 1 
Adjustment of the glucose content of egg albumen. 
Egg albumen normally contains about 0.5% of glucose. When egg albumen is 
dried to a low moisture content, the glucose will react with amino groups 
supplied by the major constituent of albumen, the proteins, to form dark 
coloured polymers which detract from the appearance, solubility and 
functional characteristics of albumen when used for cooking purposes. 
It is customary to remove the glucose from albumen prior to drying. This 
may be achieved by the present process by using glucose-oxidase as the 
enzyme, and filters that are of such a pore size so as to retain 
glucose-oxidase. In this case it is necessary to dilute the albumen with 
water prior to feeding to the first filter, as albumen viscosity is high 
and hampers flow through the system. 
EXAMPLE 2 
Removal of glucose from blood serum prior to drying 
Blood is fed to the first filter where red blood cells and molecules larger 
than glucose-oxidase are removed. The residual serum passes through the 
filter and the glucose contained in it is converted to gluconic acid by 
the glucose-oxidase enzyme. 
The reasons for removal of glucose from bood are similar to those given in 
Example 1, i.e. if dried blood is to be used as a feed, or as a 
microbiological media, it undergoes "browning reaction" if glucose and 
amino groups are allowed to interact to form coloured polymers. 
EXAMPLE 3 
Removal of the bitter principle from orange juice 
Orange juice that is bitter contains the material limonin which may be 
broken down and debittered by the enzyme limoninase. 
Orange juice is fed to the first filter where insoluble solids and soluble 
matter of molecular size greater than limoninase are removed. The enzyme 
limoninase operates on limonin between the two filters. It is not clear 
what constitutes the breakdown products of limonin, however it has been 
found that they are smaller than limonin and are non-bitter.