Title of Invention

A PROCESS FOR PREPARING LACTIC ACID WITH LACTOBACILLUS DELBRUECKII MTCC 5199

Abstract The present invention provides a process for preparing lactic acid comprising the steps of purging sterile air to the nutrient media; incubating Lactobacillus delbrukii MTCC 5199 at an effective concentration in a nutrient medium at an incubating pH sufficiently to allow production of lactic acid; adjusting the pH to 5 to 6.5; and adjusting the temperature to 45 to 55 degree C; adjusting the final pH to 7 to 9; and recovering lactic acid from the nutrient medium after 45 to 55 hrs. The incubating temperature is most preferably 52 degree C and the dissolved oxygen content is maintained between 0.55 ppm to 5.00 ppm. The process yields lactic acid salt about 97% with a conversion of about 91%. 2 2 DEC 2005
Full Text FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
& THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10, Rule 13]
LACTOBACILLUS DELBRUECKII
MTCC 5199 AND A METHOD OF PRODUCING LACTIC ACID;
THE GODAVARI SUGAR MILLS
LIMITED, A COMPANY
INCORPORATED UNDER THE COMPANIES ACT 1956 WHOSE ADDRESS IS FAZALBHOY BLDG., 45/47, MAHATMA GANDHI ROAD, FORT MUMBAI - 400 001 MAHARASHTRA, INDIA
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.


TECHNICAL FIELD
The present invention relates to a method of producing lactic acid using
Lactobacillus delbureckii.
BACKGROUND ART
Lactic acid has long been used as a food additive and in various chemical and
pharmaceutical applications.
The production of lactic acid is commonly carried out by fermentation of a strain of the bacterial genus Lactobacillus, and more particularly, for example, by the species Lactobacillus delbrueckii or Lactobacillus acidophilus.
US Patent 5,416,020 describes a Lactobacillus delbrueckii sub-species bulgaricus ATCC 55163, a strain of Lactobacillus delbureckii subspecies bulgaricus that can produce essentially stereoisometrically pure L- (+) lactic acid and a process for producing essentially stereoisometrically pure L- (+) lactic acid using the strain discloses. A method for producing a mutant strain of Lactobacillus that produces essentially stereoisometrically pure L- (+) lactic acid is also disclosed.
US Patent 5503750 describes a substantial continuous membrane separation process in which the known benefits of cell-recycle fermenter are unexpectedly enhanced because pH is controlled from 4.0 to 5.5 with a nitrogen-containing base. The ammonium lactate formed is not precipitated allowing continuous removal of lactic acid so as to maintain a concentration of lactic acid about 12% by weight in the broth. When combined with recycle of UF retnetate, operating this cell-recycle fermentor allows the build up of a cell population in the range from above 5 x 10 9

CFU/ml to 5 x 10 CFU/ml whicn population is much denser than one which can be produced with the specified pH control in a batch fermenter, after self-termination of the fermentation. This super-dense population of cells, in turn not only accelerates production of lactic acid but also minimizes contamination by foreign cells. UF retentate is recycled to produce a specified high fluid velocity when the effluent stream is flowed over UF membranes used in the UF modules. The high fluid velocity at relatively low pressure produces a scrubbing action over the membrane surfaces which are therefore not fouled over long periods of operation. In a batch fermentation with pH control, when the fermentation self-terminated the total lactic acid present is abotu10% by weight measured by HPLC) though free lactic acid is always A publication in Journal of Food Sci., Vol 68, No.4, 2003 entitled "Optimization of lactic acid production by Lactobacillus delbrueckii through response surface methodology" by S.J. Tellez-Luis, A.B. Moldes, J.L. Alonso, M. Vazquez. The article describes the optimization through response surface methodology of a low-cost medium based on corn steep liquor (CSL) for lactic acid production by Lactobacillus delbrueckii NRRL B 445. The effect of fermentation time was also considered. A maximum lactic acid concentration (93.4 g/l) was predicted using 15 g of CSL/L and 6g of yeast extract/L at a fermentation time of 80.1 hours. However, the maximum productivity (3.5 g/L/H) was predicted by using 15g of CSL/L, 6 g of yeast extract/L and 8.9 of peptone /L after 24 hours. The publication also suggests that from an economical perspective, better results obtained using 15g CSL/L alone and 24 hours achieving maximum economical productivity of 229.7 g of lactic acid per hour and considerable savings in nutrient.

Therefore, it is known that generally, large amounts of lactic acid can be produced by large-scale industrial bacterially-conducted fermentation processes, particularly using carbohydrates, such as dextrose, as the feed stock, along with suitable mineral and amino acid based nutrients. Typically, such productions occur at broth temperatures of at least 45° C, usually around 48° C.
However, in lactic acid fermentation the end-product inhibition by the lactic acid can be a major problem to efficient fermentation. Bacterial strains generally employed in lactate fermentations may be inhibited by low pH in addition to lactate concentration. To overcome this problem, industrial lactate fermentation processes are typically run at a higher pH, at least about 5.0 and often at or above 6.0. This results in the production of a lactate product, which is essentially present in the form of a salt. However, high concentrations of certain salts, e.g., sodium cations, may have an inhibitory effect on fermentation and may influence the efficiency of the fermentation.
The further problems associated with respect to lactic acid production include, downstream isolation and production involving the isolated lactic acid or lactic acid derived product and the yield of the end product.
SUMMARY OF INVENTION
An object of the present invention is to provide a process for producing lactic acid using Lactobacillus delbrueckii MTCC 5199 strain.
Another object of the present invention is to provide a process for producing lactic acid using Lactobacillus delbrueckii MTCC 5199 strain with a good yield.

Yet another object of the present invention is to provide a simple process for producing lactic acid using Lactobacillus delbrueckii MTCC 5199 strain.
According to an aspect of the present invention a process for preparing lactic acid comprising the steps of purging sterile oxygen to the nutrient media; incubating Lactobacillus delbruecki MTCC 5199 at an effective concentration in a nutrient medium at an incubating pH sufficiently to allow production of lactic acid; adjusting the pH to 5 to 6.5; and adjusting the temperature to 45 to 55 degree C; adjusting the final pH to 7 to 9; and recovering lactic acid from the nutrient medium after 45 to 60 hrs. The incubating temperature is most preferably 52 degree C and the dissolved oxygen content is maintained between 0.55 ppm to 5.00 ppm.
The process yields lactic acid of 97% with a conversion of about 91%.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows growth curve of lactobacillus delbrueckii MTCC 5199 in the medium. Figure 2 shows the agarose gel electrophoresis pattern of Lactobacillus delbrueckii MTCC 5199 identification.
DESCRIPTION OF INVENTION
According to broadest aspect of the invention, a strain of Lactobacillus delbrueckii MTCC 5199 is provided. Lactobacillus delbrueckii MTCC 5199, a strain of Lactobacillus delbrueckii is isolated from soil and was deposited at IMTECH. Lactobacillus delbrueckii MTCC 5199 is a Gram Positive, facultative anaerobe, which

is non-motile and non-spore forming, rod shaped. The cell size is about 0.5-0.8 x
2.0-9.0 mm.
Lactobacillus delbrueckii MTCC 5199 is acid tolerant and possess strictly
fermentative metabolism with lactic acid as the major metabolic end product L lactic
acid.
According to another aspect of the present invention a process for preparing Lactic acid using Lactobacillus delbrueckii MTCC 5199 is provided. According to an embodiment of the present invention, the process of preparing lactic acid using Lactobacillus delbrueckii MTCC 5199 includes the steps where oxygen is purged into the fermenter containing the media to maintain dissolved oxygen content of about 0.55 ppm to about 5.00 ppm. To improve the productivity of lactic acid, during fermentation fermenter broth is saturated with sterile air since the strain is micro-aerophilic in nature.
Characteristic of Lactobacillus delbureckii MTCC 5199
The micro-organism Lactobacillus delbureckii MTCC 5199 works in both the
conditions i.e. in aerobic and in anaerobic condition. According to an aspect of the
present invention, cultivation of micro-organism is done in anaerobic condition and
during the production stage of the fermentation media sterile air is saturated
before inoculation of culture which maintains aerobic condition.
Cultivation media for Lactobacillus delbrueckii MTCC 5199
Peptone 10gm
Yeast Extract 0.5gm
Beef Extract 0.5gm

Magnesium sulfate 0.1 gm
Urea 0.1 gm
Di-ammonium phosphate 0.1gm
Distilled water 100ml
The pH is maintain between 5.5-6.5. and temperature between 45-55 degree C.
Aerobic Fermentation Process
Raw material Molasses is hydrolysed at temperature between 90-95 degree C
for 1-1.5 hour. After hydrolysis neutralize acid by addition of calcium carbonate
and when temperature reaches between 60-65 degree C, nutrient media is
added and aeration is done for 1 hr to maintain the dissolved oxygen level
from 0.1 to 2ppm. The 12 hr old culture is transferred to 100 ml media and
fermented for 45-60 hours to produce lactic acid.
Anaerobic Fermentation process
Raw material Molasses is hydrolysed at temperature between 90-95 degree C
for 1-1.5 hour. After hydrolysis neutralize acid by addition of calcium carbonate
and when temperature reaches between 60-65 degree C, nutrient media is
added and temperature is maintained between 45-55 degree C and pH between
5.5-6.5.
During the fermentation the carbon dioxide is produced which maintain the
anaerobic condition in fermenter. The 12 hr old culture is transferred to 100 ml
media and fermented for 45-60 hours to produce lactic acid.
The rate of fermentation is faster in previously aerated fermenter than
anaerobic fermenter. The productivity of lactic acid without aeration is 0.5 gm/lit/hour
and after aeration the productivity is 2gm / L / hour.

The Lactobacillus delbrueckii MTCC 5199 grown was transferred to the fermenter. Preferably, the initial sugar concentration in the fermenter was maintained to at about 8 to 12 % w/v. The inoculum volume was about 8 to 12 % of fermenter volume. The pH of the broth was maintained in the range of 5.5 to 6.5 by addition of a base known to a person skilled in the art. Preferably, the base may be ammonium bicarbonate, calcium carbonate, sodium bicarbonate, sodium carbonate and the like. The temperature of the fermenter was maintained to about 45 to 55 degree C, preferably at 52 degree C. After 55 hours of conversion, the pH of the fermented broth is adjusted to about 7 to 9, preferably 8.5. The biomass is removed by conventional centrifugation technique. The salt of lactic acid was recovered from the fermenter by methods known to a person skilled in the art.
The conversion for carbohydrate using the process of the present invention is given below:

Time (hrs) Reducing Sugar (Gm/100ml)
0 9.25
24 4.64
36 2.63
48 1.8
60 0.85
The percentage yield of salts of lactic acid produced by the inventive process using Lactobacillus delbrueckii MTCC 5199 strain is 97% with the percentage conversion of around 91%.

According to an embodiment of the present invention, the Lactobacillus delbrueckii MTCC 5199 strain was grown in the media containing peptone, yeast extract, beef extract, magnesium sulfate, urea, diammonium phosphate, and distilled water.
The process of producing lactic acid according to the present invention using Lactobacillus delbrueckii MTCC 5199 strain produces 97% yield with 91% conversion that is substantially higher than the percentage yield of the strains of the prior art.

Lactobacillus delbrueckii strain % Yield % Glucose % Conversion
NRRL B-445 87 4.5 74
ATCC 53197 85 1.7 88
MTCC 5199 97 9.25 91
The following examples illustrate the invention, however does not limit the scope of
the invention.
Example 1
Isolation of Lactobacillus delbrueckii MTCC 5199
Isolated from distillery waste of distillery at Sameerwadi Karnataka. 1 gm of
distillery waste was taken and 10 ml sterile water was added and heated at 60
degree C on water bath. 1 ml of the aliquot is taken and added to the media
containing:
Peptone 10gm
Yeast Extract Q.5gm
Beef Extract 0.5gm

Magnesium sulfate 0.1 gm
Urea 0.1 gm
Di-ammonium phosphate 0.1gm
Distilled water 100ml
and heated to 48degree C for 24 hrs. Then it was streaked on above media
containing 2% agar and then incubated for 12 hour at maintaining the
tempreture between 50-52 degree C .The growth obtained was then
characterized as gram positive bacteria by known methods such as gram staining.
Example 2
Identification of microorganism: The microorganism was identified at Agarkhar
Institute , Pune as Lactobacillus delbrueckii. The agarose gel electrophoresis
pattern of the microorganism of the present invention as provided by Agarkhar
Institute, Pune is shown in Figure 2.
Example 3
Lactobacillus delbrueckii MTCC 5199 was grown in the following media:
Peptone 10gm
Yeast Extract 5gm
Beef Extract 5gm
Magnesium sulfate 1gm
Urea 1 gm
Di-ammonium phosphate 1gm
Distilled water 100ml.

The pH of the broth was maintained at 5.6 at temperature of 50 degree C. The 12 hr old culture was transferred to the 100 ml media in 250 ml conical flask. One liter media is prepared using 6 part hydrolysed molasses and 5 part calcium carbonate and nutrient media as described above. The temperature is maintained 50 degree C. The fermenter is saturated with sterile air for 1 hr to maintain the dissolved oxygen level from 0.55-5.00 to increase the yield of lactic acid then the fermenter is inoculated with 10% v/v of inoculum, and pH is maintained 5.4.After complete reduction of fermentable sugar add calcium oxide to raise the pH in the range between 9-10, heat the contents to 90 degree C for 1 hour The broth is then transferred to intermediate storage vessel for centrifugation to remove the biomass and calcium salts . Filtrate is then concentrated in evaporator and lactic acid is purified by crystallization. After 45 hours the fermenter broth contain 80 g/l of lactic acid. Example 4
Lactobacillus delbrueckii MTCC 5199 was grown in the media as in example 3. The pH of the broth was maintained at 5.4 at temperature of 52 degree C. The 12 hr old culture was transferred to the 100 ml media in 250 ml conical flask. One liter media is prepared using 6 part hydrolysed molasses and 5 part calcium carbonate and nutrient media as described above. The temperature is maintained at 52 degree C.The fermenter is saturated with sterile air for 1 hr to maintained the dissolved oxygen level 0.55-5.00 to increase the yield of lactic acid then the fermenter is inoculated with 10% v/v of inoculum, and pH is maintained at 5.4. After complete reduction of fermentable sugar add calcium carbonate to raise the pH to 9-10, heat the contents to 90 degree C the broth is then transferred to intermediate storage vessel for further centrifuge to remove

the biomass and calcium salts . Filtrate is then concentrated in evaporator and
lactic acid is purified by crystallization. After 45 hours the fermented broth
contain 80 g/l of lactic acid.
Example 5
Lactobacillus delbrueckii MTCC 5199 was grown as in example 3.
The pH of the broth was maintained at 5.4 at temperature of 54 degree C. The 12 hr
old culture was transferred to the 100 ml media in 250 ml conical flask.
One liter media is prepared using 6 part hydroiysed molasses and 5 part
calcium carbonate and nutrient media as described above. The temperature is
maintained at 52 degree C.The fermenter is saturated with sterile air for 1 hr
to maintained the dissolved oxygen level 0.55-5.00 inside the fermenter to
increase the yield of lactic acid then the fermenter is inoculated with10%v/vof
inoculum and pH is maintained 5.4 , After complete reduction in fermentable
sugar add calcium oxide to raise the pH to 9-10, heat the contents to 90 degree
C the broth is then transferred to intermediate storage vessel for further
centrifuge to remove the biomass and calcium salts . Filtrate is then
concentrated in evaporator and lactic acid is purified by crystallization. After 48
hours the fermenter broth contain 80 g/l of lactic acid.
Example 6
Lactobacillus delbrueckii MTCC 5199 was grown in the media as given example 3.
The pH of the broth was maintained at 5.4 at temperature of 48 degree C. The 12 hr
old culture was transferred to the 100 ml media in 250 ml conical flask.
One liter media is prepared using 6 part hydroiysed molasses and 5 part calcium
carbonate and nutrient media as described above. The temperature is

maintained between 52 degree C.The fermenter is saturated with sterile air for 1 hr to maintained the dissolved oxygen level 0.55-5.00 to increase the yield of lactic acid then the fermenter is inoculated with 10% v/v of inoculum, and pH is maintained at 5.6. After complete reduction of fermentable sugar add calcium oxide to raise the pH to 9-10, heat the contents to 90 degree C for 1 hour.The broth is then transferred to intermediate storage vessel for further centrifuge to remove the biomass and calcium salts . Filtrate is then concentrated in evaporator and lactic acid is purified by crystallization. After 50 hours the fermenter broth contain 80 g/l of lactic acid.
Example 7
The lactobacillus was grown as in example 3. The initial sugar content was maintained to about 10% w/v. The fermenter was saturated with sterile air and then the fermenter was inoculated with around 10% v/v of inoculum. The pH of the broth was maintained in the range of 5.5 to 6.5 with ammonium bicarbonate. The temperature of the fermenter was maintained between 45 to 55 degree C. After 55 hours, the conversion was 90% and selectivity was found to be 94%. Example 8
This example shows large scale production of lactic acid using Lactobacillus delbrueckii strain MTCC 5199. The organism was cultured in 500ml of an aqueous medium containing 50g hydrolyzed molasses, 0.5 g yeast extract, 0.5 g K2HP04 and 0.01 g MnSo4 for 12 hours at 45 to 55 degree C. The culture is transferred to a 10L fermenter containing a culture medium containing 200 g/L molasses having a lactose content of 50%, 3 g/L yeast extract and 0.02 g/L manganese sulfate. The fermenter broth is maintained between 45 and 55 degree C and pH at 5.4 controlled

with ammonium bicarbonate for 45 hours at which time, about 80g/L lactic acid is produced.
The fermenter broth is then transferred to a 200 L fermenter containing the same nutrient medium used in the 10L fermenter and also maintained at 43 degree C and pH 5.4. After 36 hours the lactic acid content of the fermenter broth reached about 35 g/L. the fermenter broth is transferred to a 6,000 L fermenter that containing the same nutrient media used for the 10L fermenter. The 6,000 L fermenter is maintained at temperature 45 to 48 degree C and pH 5.4 to 6.5 for about 60 hours at which time the fermenter broth contains about 80 g/l lactic acid.

WE CLAIM
1. A process for preparing lactic acid comprising:
(a) Purging sterile air to the nutrient media;
(b) Incubating Lactobacillus delbruckii MTCC 5199 at an effective concentration in a nutrient medium at an incubating pH sufficiently to allow production of lactic acid;
(c) Adjusting the pH to 5 to 6.5; and
(d) Adjusting the temperature to 45 to 55 degree C;
(e) Adjusting the final pH to 7 to 9; and
(f) Recovering lactic acid from the nutrient medium after 45 to 55 hrs.

2. The process for preparing lactic acid as claimed in claim 1 wherein the incubating temperature is 52 degree C.
3. The process of preparing lactic acid as claimed in claim 1 wherein the dissolved oxygen content is maintained between 0.55 ppm to 5.00 ppm.
4. A process for preparing lactic acid as claimed in claim 1 wherein the nutrient medium is Peptone 1%w/v; Yeast Extract 0.5%w/v; Beef Extract 0.5%w/v; Magnesium sulfate 0.1%w/v; Urea 0.1%w/v; Di-ammonium phosphate 0.1%w/v; and Distilled water 100ml.
5. A process for preparing lactic acid as claimed in claim 1 wherein the nutrient medium is hydrolyzed at an elevated temperature for at least 1 hour before incubating Lactobacillus delbruckii MTCC 5199.
6. A process for producing lactic acid as claimed in claim 1 wherein the process comprises adding base to the fermenter.

7. A process for producing lactic acid as claimed in claim 6 wherein the base includes calcium carbonate, ammonium bicarbonate, ammonia, sodium bicarbonate, sodium carbonate.
8. Lactic acid produced from the process as claimed in claims 1 to 7.
Dated this 22nd day of December 2004
FOR GODAVARI SUGAR MILLS LTD BY THEIR AGENT
(UMA BASKARAN) KRISHNA & SAURASTRI
To,
The Controller of Patents
The Patent Office
Mumbai

Documents:

1020-mum-2004-abstract(22-12-2005).doc

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1020-MUM-2004-ABSTRACT(GRANTED)-(7-2-2012).pdf

1020-MUM-2004-CANCELLED PAGES(20-12-2011).pdf

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1020-MUM-2004-CORRESPONDENCE(IPO)-(7-2-2012).pdf

1020-MUM-2004-DESCRIPTION COMPLETE(22-12-2005).pdf

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1020-mum-2004-form 2(complete)-(22-12-2004).doc

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1020-MUM-2004-FORM 5(22-12-2005).pdf

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1020-mum-2004-general power of attorney(22-9-2004).pdf

1020-MUM-2004-GENERAL POWER OF ATTORNEY(9-3-2010).pdf

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1020-MUM-2004-REPLY TO EXAMINATION REPORT(20-12-2011).pdf

abstract1.jpg


Patent Number 250922
Indian Patent Application Number 1020/MUM/2004
PG Journal Number 06/2012
Publication Date 10-Feb-2012
Grant Date 07-Feb-2012
Date of Filing 22-Dec-2004
Name of Patentee GODAVARI BIOREFINERIES LTD.
Applicant Address SOMAIY BHAVAN, 45/47, MAHATMA GANDHI ROAD, FORT,MUMBAI-400 001, MAHARASHTRA,INDIA
Inventors:
# Inventor's Name Inventor's Address
1 ANIL AMUNDKAR A-4, AMBIKA DHAM SOCIETY, NEW AYRE ROAD, DOMBIVALI(E), DIST. THANE-421201
2 SANGEETA SRIVASTAVA 1001, BHASKARA, TIFR COLONY, COLABA, MUMAI-400005.
PCT International Classification Number C12P7/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA