Title of Invention

A TEMPERATURE CONTROLLED IMMOBILIZED ENZYME BIOREACTOR

Abstract A bioreactor comprising of three concentric cylinders of different diameter for demarcating the liquid flow and enzyme cartridge, said cylinders comprising an outer water circulation cylinder (2) to maintain the reactor temperature from 5° - 95°C, a middle concentric cylinder (1) to hold the lactose hydrolyzed milk/product and is connected to a water delivery tank (14) and an innermost cylinder (4) is enzyme cartridge to hold the immobilized lactase in the form of a packed bed.
Full Text FIELD OF INVENTION
The invention relates to a temperature controlled immobilized enzyme bioreactor for lactose hydrolysis in milk and other lactose containing liquids.
PRIOR ART:
Enzymatic hydrolysis of lactose by (3-galactosidase is one of the important bioprocesses in food processing. The beneficial effects of lactose hydrolysis include assimilation of the resulting glucose and galactose as readily available energy sources prior to the fermentative metabolism by the gut micro flora. Hydrolysis of lactose is desirable to overcome the problem of its moderate solubility in concentrated milk products and to ensure its easy digestion as in the case lactose intolerant infants/and adults.
Furthermore, trans-galactosidase activity of P-galactosidase is widely used for the production of galacto-oligosaccharides and various glycol-conjugates used as pharmaceuticals. In this regard, thermostable and cold-tolerant p-galactosidase have attained great importance for lactose hydrolysis in milk at higher (>35° to 75°C) as well
as below ambient (from 5° to 15°C) temperature to reduce microbial contamination during the course of lactose hydrolysis in the bioreactors.
Application of soluble galactosidase on industrial scale is limited mainly due to the logistics involved in the use of enzyme preparations. Enzymes in soluble form cannot be reused and the hydrolysis reaction can only be a batch process. As a result design and fabrication of bioreactors is an imperative aspect to control feed stream (flow rate,) temperature, and sampling and effectively recover the immobilized biocatalyst as well as to ensure enzyme free product.
Immobilized enzymes and cells are preferred over their soluble forms to increase the overall efficiency of the hydrolytic process. Not may bioreactors have been reported either in the form of published papers or patents. Some of the reported bioreactor configuration are mostly known to be packed bed columns for lactose at fixed process temperature mostly ambient.
In immobilized packed bed bioreactors the contact between biocatalyst and substrate is known to be a mild mixing phenomenon due to the gravitational force. In a patent JP62044183 an immobilized lactase was packed in a column bioreactor and it was contacted with a stream of lactose containing liquid to affect the hydrolysis. In another patent WO 2004050822 a bioreactor was used by Ferreira et al to preserve the physical integrity and catalytic activity of the enzyme. Packed bed columns are simple to fabricate and they are used even for experimental purposes under laboratory conditions. Illanes et al (1998) have used a packed bed reactor with immobilized lactase. An ultra high temperature process employing a packed bed column using an outer thermostat column was explained by Petzelbauer et al (2002). Febbraio et al (1998) reported a non-isothermal bioreactor for using immobilized thermophilic p-galactosidase.
The process efficiency and thereby the cost benefit ratio of lactose hydrolysis can be improved using a continuous flow-bioreactor, packed with an immobilized p-galactosidase preparation. Immobilization of enzymes has been found to enhance their thermo stability above ambient temperatures. Immobilized enzymes and cells enable the optimum utilization of their activity in bioreactors.
The bioreactors which can hold immobilized enzyme/whole cell catalysts at high density and varying operational temperature play a major role in the development of effective bioprocess. Further more, bioreactor able to retain immobilized biocatalysts prepared in the form of granules, blocks, plain sheets or fibrous materials are in demand in the recent years. In view of this requirements a bioreactor has been invented to make the lactose hydrolysis a cost effective method by addressing several limitations of bioreactors available so for. The biocatalyst if reused for several times can contribute to improve the cost benefit ratio. Lactose hydrolysis if carried out either at 5° to 15° C or >35° C can drastically reduce the microbial contamination in the bioreactor. Hence, bioreactors which can operate continuously or in batch mode at 5°-15° C/or 35° -75° C are in important. Therefore, we intended to invent a bioreactor which is cost effective to fabricate, novel and simple to operate either in batch or continuous mode for the hydrolysis of lactose in milk or other lactose containing solutions.
In the past, different bioreactor configurations have been reported based on the immobilized enzyme preparation to be used. Bioreactors can provide long term mechanical and biochemical stability to immobilized enzyme preparations. Moreover, in the absence of a bioreactor the immobilized enzyme preparation need to be stirred in a vessel with milk for the hydrolysis of lactose.
OBJECTS OF THE INVENTION
The primary object of the invention is to propose an immobilized enzymes bioreactor
which is simple, cost effective and novel for lactose hydrolysis biocatalyst.
Another object of the invention is to propose an immobilized enzymes bioreactor
which maintains the temperature in the range of 5° to 95 °C
Still another object of the invention is to propose an immobilized enzymes bioreactor
which can efficiently retain the immobilized biocatalyst.
DESCRIPTION OF INVENTION
According to this invention there is provided a bioreactor comprising of three concentric cylinders of different diameter for demarcating the liquid flow and enzyme cartridge, said cylinders comprising an outer water circulation cylinder to maintain the reactor temperature from 5° -95°C, a middle concentric cylinder to hold the lactose hydrolyzed milk/product, and an innermost enzyme cartridge to hold the immobilized lactase in the form of a packed bed.
DESCRIPTION WITH REFERENCE TO DRAWINGS
Further objects and advantages of this invention will be more apparent from the ensuing
description when read in conjunction with the accompanying drawings and wherein:
Fig. 1. shows the bioreactor of this invention;
Referring to Fig.l the bioreactor for lactose hydrolysis comprises an outer cylindrical zinconium vessel (2) meant for water circulation maintainable at a temperature between 5° to 95°C, with its outer surface covered for insulation with 1.5-2.0 cm thick cylindrical zirconium mat. However, the immobilized enzyme is stable and functional between 50 and 75°C. The length and the diameter of the cylinder may, for example be 50 cm and 15 cm, respectively and can retain a liquid of 7.5 litres. A water jacket 3 is provided an inlet 18 and outlet. On the water jacket the water inlet at top may be arranged below 2.5cm and at the bottom the outlet is fixed at above 2.5 cm of the bottom surface. Water inlet 18 is connected to a hot water delivery tank 14 having a heater 12 a, a mixing blade 10 and a temperature indiacator 13 a. The middle cylinder (1) is tapered at the bottom and fitted with a filter mesh 5. The innermost cylinder (4) is an enzyme cartridge able to retain the immobilized enzyme. The outer surface of the enzyme
cartridge is perforated over a length of 26-30 cm from the bottom for treated feed stream (lactose hydrolyzed milk/solution) for collection within the middle cylinder. The upper lid (8) is threaded and it is fixed with a filter mesh inside the ring. It is also fitted with an inlet through a stainless steel T-joint, ball valve and nipple. The SS nipple in turn is connected to silicon tubing and connected to the feed tank 11. The feed tank has a heating element 12, a stirrer 10 and a temperature indicator 13, said tank 11 connected to feed inlet 9. The bioreactor is supported by a tripod stand 16. Feed tank 11 and hot water tank are each supported on stand 16. At the center of the stand the product out-let 6 is arranged by a T-joint, ball valve and nipple and connected to feed/storage/collection tanks 7, device further comprises a hot water/chilled water collecting tank for recirculation (17).






WE CLAM:
1. A bioreactor comprising of three concentric cylinders of different diameter for demarcating the liquid flow and enzyme cartridge, said cylinders comprising an outer water circulation cylinder (2) to maintain the reactor temperature from 5° - 95°C, a middle concentric cylinder (1) to hold the lactose hydrolyzed milk/product and is connected to a water delivery tank (14) and an innermost cylinder (4) is enzyme cartridge to hold the immobilized lactase in the form of a packed bed.
2. A bioreactor as claimed in claim 1, wherein the enzyme cartridge is provided with perforations to hold the Immobilized enzymes particles or sheets or pellets.
3. A bioreactor as claimed in claim 1, wherein the innermost cylinder (4) has an outlet for lactose hydrolyzed milk provided at the lower half of the cylinder.
4. A bioreactor as claimed in claim 1, wherein a top lid arranged with threads to the middle cylinder (1) and inner cylinder (4).
5. A bioreactor as claimed in claim 1, wherein top and bottom lids (8) are fitted with a mesh for filtration of the incoming and outgoing liquid streams.
6. A bioreactors as claimed in claim 1, comprising a stainless steel feed tank (11) having a heating element (12) to maintain the circulating water temperature in the outer jacket between 35° - 95° C.

7. A bioreactors as claimed in claim 1 comprising a stainless steel water
tank, (11) fitted with a heating element (12) to maintain the
circulating water temperature in outer jacket between 35 -95°C.
8. A bioreactor as claimed in claim 1 having a stainless steel water collection tank(ll) for recirculation of the hot/chilled water to conserve energy.

Documents:

2412-DEL-2005-Abstract-(08-08-2011).pdf

2412-DEL-2005-Abstract-281114.pdf

2412-del-2005-abstract.pdf

2412-DEL-2005-Claims-(08-08-2011).pdf

2412-DEL-2005-Claims-281114.pdf

2412-del-2005-claims.pdf

2412-DEL-2005-Correspondence Others-(08-08-2011).pdf

2412-del-2005-Correspondence Others-(10-10-2014).pdf

2412-DEL-2005-Correspondence Others-(24-06-2011).pdf

2412-DEL-2005-Correspondence-141114.pdf

2412-DEL-2005-Correspondence-281114.pdf

2412-del-2005-correspondence-others.pdf

2412-del-2005-correspondence-po.pdf

2412-del-2005-description (complete).pdf

2412-DEL-2005-Drawings-(08-08-2011).pdf

2412-del-2005-drawings.pdf

2412-del-2005-form-1.pdf

2412-del-2005-form-18.pdf

2412-del-2005-form-2.pdf

2412-DEL-2005-Form-3-(08-08-2011).pdf

2412-DEL-2005-Form-3-(14-11-2014).pdf

2412-DEL-2005-Form-5-(08-08-2011).pdf

2412-DEL-2005-GPA-(24-06-2011).pdf


Patent Number 264609
Indian Patent Application Number 2412/DEL/2005
PG Journal Number 03/2015
Publication Date 16-Jan-2015
Grant Date 09-Jan-2015
Date of Filing 08-Sep-2005
Name of Patentee DIRECTOR GENERAL, DEFENCE RESEARCH & DEVELOPMENT ORGANISATION
Applicant Address WEST BLOCK-VIII, WING 1, SECTOR 1, RK PURAM, NEW DELHI-110 066, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 AMARINDER SINGH BAWA WEST BLOCK-VIII, WING 1, SECTOR 1, RK PURAM, NEW DELHI-110 066, INDIA
2 KARNA VENKATA RAMANA WEST BLOCK-VIII, WING 1, SECTOR 1, RK PURAM, NEW DELHI-110 066, INDIA
3 ALAPPA RAMAKRISHNA WEST BLOCK-VIII, WING 1, SECTOR 1, RK PURAM, NEW DELHI-110 066, INDIA
PCT International Classification Number C12N,A61B 5/01,C12M,
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA