Title of Invention

A PROCESS FOR GROWING PROBIOTIC LACTIC ACID BACTERIA AS A SOURCE OF BETA-GALACTOSIDASE

Abstract This invention relates to a process for preparation of biofilm, amendable to permeabilization chemicals like ethanol or acetone; does not swell by read sorption of liquids and detach from the immobilized matrix even after incubation at a temperature between 5-80°C and which is to be used after permeabilization, for immobilization using agar, chitosan and cellulose sheet; provided stability to intracellular and cell found p-galactosidase from oxidation and thermal denaturation over longer duration of reaction at high temperature (after as high as 60-75°C), consisting of ß-galactosidase producing lactic acid bacteria, wherein Soypeptone 2.5-3 gm, Yeast extract 5-6gm, lactose 15-17.5 gm, Sodium acetate 5-6 gm, magnesium sulphate 0.1-0.2 gm, dipotassium hydrogen phosphate 3-4 gm, protein hydrolysate 100-125 ml dissolved in 1000 ml distilled water, pH adjusted to 5-8-6.5, sterilized, cooled, 10-15 ml of 96 hr old lactobacillus sp. Inoculum added, incubated at 30-35°C in a temperature controlled incubator for a period of 96-120 hrs, the bacterial biofilm removed by decanting; the growth medium used as inoculum for fresh hatches in the ratio of this broth: fresh medium around 1:3, wherein the biofilm weighing 5-5.5 gm was suspended in 100 ml of phosphate buffer pH 6.5 (0.1-0.05 M), centrifuged at 10000-12000 rpm for 15-20 minutes each time, then suspended in 50 ml of phosphate buffer to which added 50 ml of cold ethanol and the mixture allowed to stand at 30-35°C for 60-90 mins, centrifuged at 10000-12000 rpm for 15-20 mins.
Full Text
Field of Invention:
This invention relates to a process for the growth of lactic acid bacteria in the form of biofilm having fi-Galactosidase activity to be used as a biocatalyst for the hydrolysis of lactose in milk and other solutions and for probiotic use.
Prior Art;
Lactic acid bacteria are known to play an important role in the production of fermented dairy products owing to their technological, nutritional and health benefits. (3-galactosidase is an important hydrolytic enzyme that breaks down lactose into monomeric glucose and galactose with major implications in food technology, particularly for the preparation of low lactose milk. The enzyme can be useful in the utilization of whey resulting from cheese manufacturing industry as well as environmental protection by disposing lactose-containing wastewaters effectively into other by-products of some economical importance. The enzyme is commonly found in lactic and bacteria as well as amongst various other microorganisms comprising of yeast and fungi. Application of soluble (3-galactosidases on industrial scale is limited mainly because of the logistics involved, as enzymes in this form can not the reused and the sugar hydrolysis can be a mere batch process.
Lactic acid bacteria have been the subject of interest for the production of thermo-stable p-galactosidases activity at relatively high temperatures (above 60°C) and pH in the range of 6.0-7.5. Biomass production of lactic acid bacteria is an important step towards the development of whole-cell biocatalysts for the lactose hydrolysis. However, biomass yield of lactic

acid bacteria highly varies from batch to batch, due to rampant phase infection during active growth phase of these bacteria, often in complex media such as deMan Rogosa Sharpe (MRs) broth. The bacterial cell lysis is a major limitation in the production of large quantities of lactic acid bacteria for use as inoculum in the dairy industry or probiotic cultures as well as a enzyme source. Bacterial lysis can cause a substantial increase in viscosity of the growth medium. In addition lactic acid bacteria produce copious amounts of negatively charged exopoly-saccharides, making it tedious to sediment the bacteria even at relatively high centrifugal force.
In order to improve biomass production of the lactic acid bacteria and for
easy recovery of the cellmass in the form of a thick biofilm a process has
been developed and reported in the present invention. Here a process for
the preparation of biofilm consisting of lactic acid bacteria has been
described, wherein lactose is added to the extent of 15.0-20.0 g/litre to
the media for the induction of p-galactosidases, in place of glucose in
conventional media. The biofilm formation is attributed to reduced
infection by bacteriophages and the ensuing cell lysis, combined with
lower production of polysaccharides. The micro-aerophillic conditions
prevailing at the surface of growth medium also contribute to the biofilm
formation. ,
Objects of the invention;
1. The object of the invention is to improve biomass production of the lactic acid bacteria and develop a process for easy recovery of the cell mass in the form of a thick biofilm.

Another objective of the invention is to develop a process for producing a natural ammo acid and peptide source substituting the use of conventional media ingredients.
The other objective of the present invention is to propose a process wherein a mixed microbial consortium is used for the preparation of nitrogen source and biofilm inducing ingredient in the lactic acid bacteria.
Another objective is to propose a process using a medium to provide microaerophilic conditions at the surface of the liquid medium.
Yet more objective of the process is to reuse the culture broth as a source of nutrients for the production of second batch of biofilm with the addition of depleted nutrients i.e. adding hydrolyzed/fermented protein component.
Further objective of the invention is to reduce the polysaccharide production in the broth.
Yet another objective of the invention is to enhance the lyophilization process of biomass by way of reduced polysaccharide production.
Another objective is to augment the process of biofilm formation using an increased volume of the inoculum (200-300 ml/litre).
Yet another objection is to enhance the cell recovery by centrifugation or filtration.

Another objective of the present invention is to retain the extra cellular (3-galactosidase within the biofilm.
Another objective of this invention is to propose a process wherein the biofilm contents provided stability to the intracellular p-galactosidase against oxidative damage and thermal denaturation over longer periods of reaction at high temperatures (as high as 60-70°C).
One more objective of the invention is to obtain lactic acid bacteria suitable for permeabilization and immobilization using agar, chitosan and cellulose sheet as matrices.
Another objective of the invention is to develop a process wherein, the bacterial biomass after lyophilization did not swell by re-absorption of liquid and detach from the immobilized matrix even at higher reaction temperatures.
Further objective of the invention is to develop a process wherein, the immobilized whole-cell biocatalyst could be prepared by drying the immobilized catalyst at room temperature without lyophilization.
Yet another objective is to propose a process for the growth of lactic acid bacteria for use as probiotics in reproducible quantities.
Still another objective of this invention is to minimize the production of waste water by reusing the spent broth after the removal of biofilm as inoculum to start bacterial growth in another batch of liquid medium.

The other objective of the invention is to determine media ingredients suitable to scale up the lactic acid bacterial growth process for the development of cost effective bioprocess.
Statement of Invention
According to this invention there is provided a process for the production of thermo stable whole cell biocatalyst lactic acid bacteria in the form of biofilm with endogenous fJ-galactosidases for the hydrolysis of lactose in milk and other lactose containing solutions.
DETAILED DESCRIPTION OF THE INVENTION
Preparation of growth medium and growth of lactic acid bacteria
The (3-galactosidases producing strains of lactic acid bacteria have been isolated earlier at Defence Food Research Laboratory, Mysore, from fermented milk products, at 30-35°C using MRS broth and lactose as inducer instead of glucose. Soy peptone, yeast extract, lactose, sodium acetate magnesium sulphate and dipotassium hydrogen phosphate were obtained from commercial sources. However, the protein hydrolysate was prepared in the laboratory using a mixed microbial consortium.
The protein hydrolysate was prepared by adding 125-150 grams of casein to 1 litre of distilled water. The protein was dissolved in the distilled water by heating and adjusting the pH to 6.5-7.5 using 0.1 M sodium hydroxide solution. After cooling to room temperature (25-30°C) 10 ml of

24 hour inoculum was added and incubated for 24-36 hours at 35°C. The resulting solution was used as protein hydrolystate for the growth and biofilm formation by lactic acid bacteria. The remaining solution could be stored at 4-5°C till further experimentation.
The bacterial growth medium comprised of the following ingredients in the ranges (per litre) mentioned against them:
Soy peptone 2.5-3.0g
Protein hydrolysate 100.0ml-125ml
Yeast extract 5.0-6.0g
Lactose 15.0-17.5g
Sodium acetate 5.0-6.0g
K2HPO4 3.0-4.0g
MgSO4 0.10-0.20g
Firstly the ingredients were dissolved in 500 ml of distilled water and the final volume was made up to 1000 ml in a one litre volumetric flask. The pH of the solution was adjusted to 5.8-6.5 using 0.1M HCI. Around 250 ml each of the medium was distributed into 500 ml conical flasks and sterilized at 15 psi for 20 min. After cooling the conical flasks, 10-15 ml of 96 hr old Lactobacillus sp inoculum was added to each flask and incubated at 30-35°C in a temperature controlled incubator, for a period of96-120hrs.

Biofilm harvesting from growth medium and its permeasbilization
The bacterial biofilm was removed by gently decanting the clear bottom portion of the growth medium into another sterile conical flask of same volume. The biofilm removed, growth medium can also used as inoculum for fresh batches of growth medium, wherein, the ratio of this broth and fresh growth medium was optimized to be around 1:3. The biofilm remaining in the first flask was decanted into a breaker in a laminar flow. The residual moisture remaining with the biofilm was removed by spinning it in a cold centrifuge at 10000-12000 rpm for 15-20 minutes. The biofilm weighing 5.0-5.5g was suspended in the centrifuge bottle (250 ml volume) in 100ml of phosphate buffer pH 6.5 (0.1-0.05M), washed 2-3 times with the same buffer and centrifuged in a cold centrifuge at 10000-12000 rpm for 15-20 minutes each time. Again the biofilm was suspended in 50 ml of phosphate buffer in a centrifuge bottle and added 50ml of cold ethanol for permeabilization. The ethaol/buffer and biofilm mixture was allowed to stand at 30-35°C for 60-90 min. after incubation the mixture was centrifuged at 10000-12000 rpm for 15-20 min and the sedimented biofilm was further washed with the same phosphate buffer 2-3 times to remove the residual ethanol. The biofilm containing the endogenous and cell bound 3-galactosidases was preserved at 5-6°C till its use for immobilization.

B-Galactosidases activity of the permaeabilized biofilm
Enzyme activity in the biofilm was measure using 5% lactose solution in phosphate buffer pH 6.5 (0.05M). To 10 mg of permeablized biofilm 2 ml of 5% lactose solution was added and incubated at 70°C for 30 min in a temperature controlled incubator. Nearly 50ul of the hydrolyzed lactose solution was added to 2.0 ml Ependorf tube containing 2.0 ml glucose oxidase reagent. The colour development was allowed for 10-15 min. and optical density measured at 505nm using a UV-Vis Shimadzu spectrophotometer. The amount of glucose released as a result of lactose hydrolysis was obtained from a standard glucose curve.
Example 1
The specified quantities of the media ingredients were dissolved in 500ml of distilled water and the final volume was made upto 1000ml in a 1 litre flask. The pH of the solution was adjusted to 5.8-6.5 using 0.1 M HCI. Around 250ml of the medium was distributed into 500ml conical flask and sterilized at 15 psi for 20 min. After cooling the conical flask 50-75 ml of inoculum in the form of broth (biofilm removed) was added. The flasks were incubated statically for a period of 96-120 hrs at 30-35°C in a temperature-controlled incubator. A 1-2 mm thick biofilm was noticed 5-7 g. The biofilm was removed by gently decanting the broth into a sterile 500ml flask. The broth was stored, for further use an inoculum containing a diffused mass of lactic acid bacteria. The biomass after permeabilization with solvents in phosphate buffer pH 6.5, was used for immobilization, while for probiotic use the biomass can be used as such after lypholization.

Example 2
The specified quantities of the media ingredients were dissolved in 500ml of distilled water and the final volume was made upto 1000ml in a 1 litre flask. The pH of the solution was adjusted to 5.8-6.5 using 0.1 M HCI. Around 1000ml of the medium was distributed into 2000ml conical flask and sterilized at 15 psi for 20 min. After cooling the conial flask 200-300 ml of inoculum in the form of broth (biofilm removed) was added. The flasks were incubated statically for a period of 96-120 hrs at 30-35°C in a temperature-controlled incubator. A 1-2 mm thick biofilm was noticed 10-15g. The biofilm was removed by gently decanting the broth into a sterile 500ml flask. The broth was stored, for further use as inoculum containing a diffused mass of lactic acid bacteria. The biomass after permeabilization with solvents in phosphate buffer pH 6.5, was used for immobilization, while for probiotic use the biomass can be used as such after lypholization.
The above examples are merely illustrative of the invention and are not be construed as limiting thereof.






WE CLAIM:
1. A process for preparation of biofilm, amendable to permeabilization chemicals like ethanol or acetone; does not swell by read sorption of liquids and detach from the immobilized matrix even after incubation at a temperature between 5-80°C and which is to be used after permeabilization, for immobilization using agar, chitosan and cellulose sheet; provided stability to intracellular and cell found p-galactosidase from oxidation and thermal denaturation over longer duration of reaction at high temperature (after as high as 60-75°C), consisting of p-galactosidase producing lactic acid bacteria, wherein Soypeptone 2.5-3 gm, Yeast extract 5-6gm, lactose 15-17.5 gm, Sodium acetate 5-6 gm, magnesium sulphate 0.1-0.2 gm, dipotassium hydrogen phosphate 3-4 gm, protein hydrolysate 100-125 ml dissolved in 1000 ml distilled water, pH adjusted to 5-8-6.5, sterilized, cooled, 10-15 ml of 96 hr old lactobacillus sp. Inoculum added, incubated at 30-35°C in a temperature controlled incubator for a period of 96-120 hrs, the bacterial biofilm removed by decanting; the growth medium used as inoculum for fresh hatches in the ratio of this broth: fresh medium around 1:3, wherein the biofilm weighing 5-5.5 gm was suspended in 100 ml of phosphate buffer pH 6.5 (0.1-0.05 M), centrifuged at 10000-12000 rpm for 15-20 minutes each time, then suspended in 50 ml of phosphate buffer to which added 50 ml of cold ethanol and the mixture allowed to stand at 30-35°C for 60-90 mins, centrifuged at 10000-12000 rpm for 15-20 mins.
2. A process as claimed in claim 1, wherein microaerophilic conditions prevail at the surface of growth medium through protection of bacteria by protein layer to contribute to the biofilm formation and also reduce the viral infection.
3. A process as claimed in claim 1, wherein the exopolysaccharide production of lactic acid bacteria is being inhibited substantially which removes the slime so that the cell recovery is enhanced by centrifugation or filtration.


4. A process as claimed in claim 1, wherein protein hydrolysate is
prepared by adding 125-150 gm of casein to 1 litre of water,
dissolved by heating and adjusting pH to 6.5-7.5, cooling to 25-
30°C, 10 ml of 24 hour old inoculum from fermented milk added
and incubated for 24-36 hrs at 35°C.
5. A process as claimed in claim 1, wherein the growth of lactic acid
bacteria is enhanced by adding atleast 20-30% inoculum to the
growth media.
6. A process as claimed in claim 1, wherein the extra cellular p-
galactosidase is retained within the biofilm by non specific
adsorption to the particulate matter and entrapment.


Documents:

2078-del-2006-Abstract-(17-02-2011).pdf

2078-del-2006-abstract.pdf

2078-del-2006-Claims-(17-02-2011).pdf

2078-del-2006-claims.pdf

2078-DEL-2006-Correspondence-Others-(07-03-2011).pdf

2078-del-2006-Correspondence-Others-(17-02-2011).pdf

2078-del-2006-correspondence-others-1.pdf

2078-del-2006-correspondence-others.pdf

2078-del-2006-description (complete).pdf

2078-del-2006-form-1.pdf

2078-del-2006-form-18.pdf

2078-del-2006-Form-2-(17-02-2011).pdf

2078-del-2006-form-2.pdf

2078-del-2006-Form-3-(17-02-2011).pdf

2078-del-2006-Form-5-(17-02-2011).pdf

2078-del-2006-gpa.pdf

2078-del-2006-Petition 137-(17-02-2011).pdf


Patent Number 247904
Indian Patent Application Number 2078/DEL/2006
PG Journal Number 22/2011
Publication Date 03-Jun-2011
Grant Date 01-Jun-2011
Date of Filing 21-Sep-2006
Name of Patentee DIRECTOR GENERAL,MINISTRY OF DEFENCE, DEFENCE RESEARSH & DEVELOPMENT ORGANIZATION
Applicant Address DIRECTOEATE OF EXTRAMUNAL RES &IPR GROUP IIIRD FLOOR B WING ROOM NOS. 348 & 349, DRDO BHAWAN, RAJAJI MARG DHQPO, NEW DEHLI-110011
Inventors:
# Inventor's Name Inventor's Address
1 KARNA VENKATA RAMANA, DEFENCE FOOD RESEARCH LABORTRY, SIDDARTHANAGAR, MYSORE-570011
2 AJAY PAL DEFENCE FOOD RESEARCH LABORTRY, SIDDARTHANAGAR, MYSORE-570011
3 MAHANTESH MALLIKARJUN PATIL DEFENCE FOOD RESEARCH LABORTRY, SIDDARTHANAGAR, MYSORE-570011
4 AMARINDER SINGH BAWA DEFENCE FOOD RESEARCH LABORTRY, SIDDARTHANAGAR, MYSORE-570011
PCT International Classification Number A61K35/74
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA