FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION (Section 10 and Rule 13)
“SCALE-UP AND PRODUCTION OF LACTIPLANTIBACILLUS PLANTARUM NCIM5777”
TROPILITE FOODS PRIVATE LIMITED
DAVAR CAMPUS, TANSEN ROAD INDUSTRIES ESTATE, GWALIOR
MADHYA PRADESH – 474 002, INDIA
The following specification particularly describes the invention and the manner in which it is to be performed.

Field of the Invention
The invention relates to an optimized, economic process for high cell density cultivation of an isolated bacterial strain of Lactiplantibacillus plantarum having accession number NCIM5777, wherein the said strain being deposited at the National Collection of Industrial Microorganisms (NCIM), Pune, India, a national facility and microbial culture repository dedicated to isolation, preservation and distribution of authentic and industrially important microbial strains, and a member of World Federation for Culture Collections (WFCC/WDCM with Reg. No. 3).
Background of the Invention
Lactiplantibacillus plantarum (previously Lactobacillus plantarum) is a widespread member of the genus Lactiplantibacillus and commonly found in many fermented food products as well as anaerobic plant matter. Lactiplantibacillus plantarum is a kind of Lactic Acid Bacteria (LAB) which ferments arabinose, sucrose, glucose, fructose, galactose, maltose, dextran and the like, to generate lactic acids. In general, Lactiplantibacillus plantarum distributes widely in nature and is often isolated from dairy products (milk, cheese, and butter), kefir, fermented crops, kimchi, soil and the like.
Lactiplantibacillus plantarum has a feature to resist gastric acids and bile acids more strongly than other bacteria in a human body. Besides, it is reported as a beneficial bacterium to increase the secretion of cytokines in human immunocytes and decrease inflammation inducing factors, thereby helping to alleviate irritable bowel syndrome and improve atopic and allergic dermatitis. Lactiplantibacillus plantarum genome is also elucidated to generate an antibiotic substance referred to as lactolin that is effective to suppress herpes virus.
Lactiplantibacillus plantarum has significant antioxidant activities and helps to maintain intestinal permeability. It can be used as a probiotics microorganism and a component of health supplement. Lactiplantibacillus plantarum also maintains microflora balance and stabilization of digestive enzyme patterns in the gut.

Lactiplantibacillus plantarum NCIM5777 strain has been isolated from soil, and deposited at the National Collection of Industrial Microorganisms (NCIM), Pune, India. Lactiplantibacillus plantarum NCIM5777 has been identified as a potent probiotic culture which exhibits a good pH and temperature tolerance, stability with optimum pH 5 and 37°C. Thus, Lactiplantibacillus plantarum NCIM5777 has been selected for further investigations on process optimization of high cell density cultivation of probiotic culture.
Summary of the Invention
The present invention relates to an optimized, economic process for high cell density cultivation of an isolated bacterial strain of Lactiplantibacillus plantarum having accession number NCIM5777.
Accordingly, the present invention includes, but not limited to, one or more of the following embodiments:
[1] A process for the high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777, wherein the said process comprises, growing the Lactiplantibacillus plantarum NCIM5777 in a production medium under optimized conditions.
[2] The process according to [1], wherein for a maximum Lactiplantibacillus plantarum NCIM5777 cultivation, the parameters are optimized by one variable at a time approach.
[3] The process according to [2], wherein the parameters include one or more of agitation rate, dissolved oxygen (DO) or aeration rate.
[4] The process according to [1] to [3], wherein the said process comprises, growing the Lactiplantibacillus plantarum NCIM5777 in a fermentor having about 0.8 % of a carbon source, about 0.8 % of a nitrogen source.
[5] The process according to [4], wherein pH during cultivation is in the range of 3.0 to 12.0 and the temperature ranges from 10 to 45°C.
[6] The process according to [4] and [5], wherein the fermentor is 3000 litres fermentor containing production medium, which comprises:

Proteose peptone (2.0%),
Sucrose (0.8%),
Sodium nitrate (0.8%),
Potassium dihydrogen phosphate (KH2PO4) (0.15%),
Magnesium sulphate (MgSO4) (0.08%),
Potassium chloride (KCl) (0.52 g/l),
Tween-80 (0.3%),
Zinc sulphate heptahydrate (ZnSO4.7H2O),
Ferrous sulphate heptahydrate (FeSO4.7H2O) and
Cupric nitrate trihydrate (Cu(NO3)2.3H2O) (traces).
[7] The process according to [1] to [6], wherein agitation rate is about 200 rpm, dissolved oxygen is about 20%, and aeration rate is about 0.5 vvm.
[8] The process according to [1] to [6], which the cultivation produces about 36.936 mg dcw/ml Lactiplantibacillus plantarum NCIM5777 using 20% dissolved oxygen (DO) saturation level.
[9] The process according to [1] to [6], wherein the cultivation produces 38.45 mg dcw/ml Lactiplantibacillus plantarum NCIM5777 at 200 rpm agitation rate.
[10] The process according to [1] to [6], wherein the cultivation produces 38.45 mg dcw/ml Lactiplantibacillus plantarum NCIM5777 at 0.5 vvm of aeration rate.
[11] The process according to [1] to [10], wherein the cultivation produces high cell density cultivation of bacterial strain Lactiplantibacillus plantarum NCIM5777 in about 22 hours of incubation period.
[12] The process according to [1] to [10], wherein the maximum yield is about 38.454 mg/ml in about 22 hours at 200 rpm (equivalent) agitation rate, 20% dissolved oxygen (DO) saturation and 0.5 vvm of aeration rate.

Brief Description of the Drawings
Figure 1: Lactiplantibacillus plantarum NCIM5777 production under uncontrolled conditions in a 3000 litres fermentor.
Figure 2: Lactiplantibacillus plantarum NCIM5777 production at 20% dissolved oxygen level in relation to time in a 3000 litres fermentor.
Figure 3: Lactiplantibacillus plantarum NCIM5777 production at 200 rpm (equivalent) in relation to time in a 3000 litres fermentor.
Figure 4: Lactiplantibacillus plantarum NCIM5777 production at different aeration rate (vvm) in a 3000 litres fermentor.
Detailed Description of the Invention
The present invention relates to an optimized, economic process for high cell density cultivation of an isolated bacterial strain of Lactiplantibacillus plantarum having accession number NCIM5777.
In one aspect, the present invention provides a process for the high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777, wherein the said process comprises, growing the Lactiplantibacillus plantarum NCIM5777 in a production medium under optimised conditions.
In another aspect, the present invention provides a process for the high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777, wherein the process is optimised by one variable at a time approach for a maximum Lactiplantibacillus plantarum NCIM5777 cultivation.
In one embodiment of this aspect, different process parameters are optimized by one variable at a time approach for a maximum Lactiplantibacillus plantarum NCIM5777 cultivation.
In another embodiment, the process parameters include one or more of agitation rate, dissolved oxygen (DO), aeration rate.

In particular, the present invention provides a process for the high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777, wherein the said process comprises, growing the Lactiplantibacillus plantarum NCIM5777 in a fermentor having about 0.8 % of a carbon source, about 0.8 % of a nitrogen source,
wherein pH during cultivation is in the range of 3.0 to 12.0 and the temperature ranges from 10 to 45°C,
wherein agitation rate is about 200 rpm (equivalent), dissolved oxygen is about 20%, and aeration rate is about 0.5 vvm.
In one embodiment of this aspect, the fermentor is a 3000 litres fermentor.
In the following section, aspects are described by way of examples to illustrate the process of the invention. However, these do not limit the scope of the present invention. Several variants of these examples would be evident to persons ordinarily skilled in the art.
In a first set of experiments, Lactiplantibacillus plantarum NCIM5777 production was scaled up and optimized in a 3000 litres fermentor using the OVT (one variable optimized at a time) optimized medium in shake flasks. The production medium containing Proteose peptone (2.0%), sucrose (0.8%), sodium nitrate (0.8%), Potassium dihydrogen phosphate (KH2PO4) (0.15%), Magnesium sulphate (MgSO4) (0.08%), Potassium chloride (KCl) (0.52 g/l), Tween-80 (0.3%), Zinc sulphate heptahydrate (ZnSO4.7H2O), Ferrous sulphate heptahydrate (FeSO4.7H2O) and Cupric nitrate trihydrate (Cu(NO3)2.3H2O) (traces), pH 5.0, was inoculated with 4.0% culture. Fermentation was initiated at 30°C, 150 rpm (revolutions per minute) and aeration of 0.5 vvm (The unit ‘vvm’ is used for bioreactor culture. The first ‘v’ stands for volume of air (e.g. litre); the second ‘v’ stands for per unit of medium (e.g. liter); ‘m’ stands for per unit of time (e.g. minute). For example, 2 vvm (l/l/m) means in 1 minute time there is 2 liter of air passing through 1 liter of medium). Foaming was controlled by the addition of sterilized silicon antifoam (10% v/v). Samples were withdrawn at regular intervals of 6 hours up to 30 hours.

Units Base Case (shake flasks) Scale up
Volume m3 0.005 2.7
Vessel Diameter mm 304.8 2419.2
Shaft Speed rpm 150 38
Reynold’s No. 37162 588363
Tip Speed m/s 0.96 1.91
The production was initially scaled up in a 3000 litres fermentor using 2500 litres optimized production medium. The conditions optimized under shake flask were identically translated to the fermentor. The dissolved oxygen (DO) and pH were monitored and recorded but not controlled. Foaming was controlled using 10% sterilized silicon (antifoaming) agent. The samples were withdrawn at periodic 2 hours intervals up to 30 hours and centrifuged at 10,000 rpm for 10 minutes at 4°C. The observations are presented in Figure 1.
The observations were presented in terms of biomass (mg dcw/ml), lactic acid (mg/ml), dissolved oxygen level (%) and residual sugar (%).
Figure 1 clearly shows that, under initial optimized conditions as optimized for shake flasks, only 31.12 mg dcw /ml of Lactiplantibacillus plantarum was produced in 24 hours of incubation. This is approximately 14.6% less as compared to biomass obtained in shake flasks (35.65 mg dcw/ml). The biomass production started in the fermentor after 8 hours of inoculation with gradual increase in titres up to 24 hours. The dissolved oxygen (DO) drop on the other hand was steep with dissolved oxygen (DO) levels reaching nearly zero by 20 hours. Therefore, there is a need to for optimizing biomass production.
The present inventors have found that optimization of dissolved oxygen (DO) level results in increased Lactiplantibacillus plantarum NCIM5777 production.

Limiting oxygen tension in the culture medium lowers the biomass production and extends the fermentation time. Thus, the effect of varying levels of dissolved oxygen (DO) was examined on biomass production. This was carried out in order to compare the cell mass production profiles under limiting and non limiting oxygen supply. Dissolved oxygen levels were maintained above 20% and 40% saturation after the dissolved oxygen (DO) fell below a critical level of 10 - 15% saturation. Dissolved oxygen (DO) level was maintained above a critical saturation level by using a computerized agitation-aeration cascade facility provided in the fermentor control unit. The observations on Lactiplantibacillus plantarum NCIM5777 production at two different saturation percent of dissolved oxygen (DO) (20% and 40%) are presented in Table 1 and Figure 2.

Incubation period (h) 20% DO saturation 40% DO saturation

DCW (mg/ml) LA mg/ml DCW (mg/ml) LA mg/ml
6 0.31 0.0 0..32 0.0
8 0.648 0.45 0.648 0.45
10 1.944 0.81 1.944 0.81
12 3.888 1.36 3.888 1.36
14 9.072 2.18 9.072 2.18
16 18.792 3.27 18.792 4.27
18 27.864 7.21 27.864 6.18
20 33.948 13.5 31.326 11.63
22 36.936 18.23 33.96 15.76
24 33.246 15.27 30.024 11.31

28 30.618 13.21 25.38 10.27
Table 1: Lactiplantibacillus plantarum NCIM5777 production at 20% and 40% dissolved oxygen (DO) levels in relation to time.
DCW: Dry Cell Weight
LA: Lactic Acid
Table 1 and Figure 2 clearly shows that, when dissolved oxygen (DO) level was controlled, the biomass production increased to 36.936 (18.6%) and 33.96 mg dcw /ml (9.1%) as against 31.12 mg dcw/ml (shown in Figure 1) obtained under initial unoptimized reactor conditions. Maximum biomass production was achieved in 22 hours as against 24 hours in shake flasks which resulted in a higher productivity of 1.67 mg dcw/ml/h when compared against 1.48mg dcw/ml/h obtained under shake flask condition. Further optimization was now carried out using 20% DO saturation level.
The present inventors have found that further to optimization of dissolved oxygen (DO) level, optimization of agitation rate results in increased Lactiplantibacillus plantarum NCIM5777 production.
The effect of different initial agitation rates were examined for Lactiplantibacillus plantarum NCIM5777 production in the 3000 litres fermentor. The Dissolved oxygen (DO) was maintained at 20% saturation level. Lactiplantibacillus plantarum NCIM5777 production was carried out using different initial agitation rates of 100, 150 and 200 rpm (equivalent). The production medium was inoculated with 4% inoculum and incubated at 30°C with an aeration rate of 0.5 vvm. Foaming was controlled using 10% sterilized silicon antifoaming agent. The observations in terms of biomass mg dcw/ml and lactic acid concentration are presented in the Table 2 and Figure 3.

Incubation period Agitation rate (rpm)

100 rpm (control) 150 rpm 200 rpm

(h) DCW (mg/ml) LA mg/ml DCW (mg/ml) LA mg/ml DCW (mg/ml) LA mg/ml
6 0.32 0.0 0.31 0.0 0.34 0.0
8 0.612 0.25 0.648 0.52 0.822 0.41
10 1.656 0.61 1.944 0.92 1.944 0.85
12 3.234 1.26 3.888 1.31 3.774 1.39
14 6.792 1.98 9.072 2.43 8.874 3.49
16 14.058 3.67 18.792 4.27 20.136 5.60
18 19.362 4.38 27.864 6.93 28.362 8.59
20 24.174 9.56 33.948 13.26 35.034 14.37
22 31.296 12.16 36.846 18.16 38.454 18.72
24 24.186 10.61 33.246 15.37 36.102 15.27
28 19.524 8.52 30.618 13.21 32.454 13.42
Table 2: Effect of different initial agitation rates on Lactiplantibacillus plantarum NCIM5777 production
DCW: Dry Cell Weight
LA: Lactic Acid
Table 2 and Figure 3 shows that on increasing the agitation rate to 200 rpm (equivalent), the biomass increased to 38.45 mg dcw/ml which is higher to the yield obtained at 150 rpm (control). A further decrease in agitation rate up to 100 rpm led to a decline in Lactiplantibacillus plantarum NCIM5777 production and yielded 31.296 mg/ml DCW in 22 hours of incubation.

The present inventors have found that further to optimization of dissolved oxygen (DO) level and optimization of agitation rate, optimization of aeration rate plays a role in Lactiplantibacillus plantarum NCIM5777 production.
The effect of varying levels of aeration rate (0.5, 0.75 and 1.0 vvm) were examined for Lactiplantibacillus plantarum NCIM5777 production up to 28 hours at a regular intervals of 2 hours with an optimal agitation rate of 200 rpm. Observations are presented in Table 3 and Figure 4.

Incubation period
(h) Aeration rate (vvm)

0.5 vvm (control) 0.75 vvm 1.0 vvm

DCW (mg/ml) LA mg/ml DCW (mg/ml) LA mg/ml DCW (mg/ml) LA mg/ml
6 0.34 0.0 0.0 0.0 0.0 0.0
8 0.822 0.45 0.792 0.40 1.176 0.37
10 1.944 0.93 2.016 0.67 2.532 0.85
12 3.774 1.29 5.052 1.17 4.41 1.32
14 8.874 3.52 9.792 2.33 9.762 2.39
16 20.136 5.72 19.578 3.49 18.456 4.06
18 28.362 8.42 26.712 6.75 28.944 6.45
20 35.034 14.29 31.446 10.32 31.974 12.61
22 38.454 18.57 35.226 14.76 35.256 15.28
24 36.102 15.26 32.022 13.29 26.532 13.04
28 32.454 13.58 24.132 11.47 21.144 12.42

Table 3: Effect of different aeration rate (vvm) on Lactiplantibacillus plantarum NCIM5777 production
Table 3 and Figure 4 shows that 0.5 vvm of aeration rate supported maximum biomass production. However, further increase in the aeration rate to 0.75 vvm and 1.0 vvm has resulted in lowering of biomass yield.
Thus, from the results, it can be inferred that optimized physiological parameters in 3000 litres fermentor are 200 rpm (equivalent) agitation rate, 20% dissolved oxygen (DO) saturation and 0.5 vvm of aeration rate resulted in the production (maximum yield) of about 38.454 mg/ml DCW of Lactiplantibacillus plantarum NCIM5777 in about 22 hours.
While the present invention has been described in terms of its specific aspects, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

We Claim:
(Claim 1) A process for the high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777, wherein the said process comprises, growing the Lactiplantibacillus plantarum NCIM5777 in a fermentor having about 0.8 % of a carbon source, about 0.8 % of a nitrogen source.
(Claim 2) The process according to claim 1, wherein pH during cultivation is in the range of 3.0 to 12.0 and the temperature ranges from 10 to 45°C.
(Claim 3) The process according to claim 1, wherein the fermentor is 3000 litres fermentor containing production medium, which comprises:
Proteose peptone (2.0%),
Sucrose (0.8%),
Sodium nitrate (0.8%),
Potassium dihydrogen phosphate (KH2PO4) (0.15%),
Magnesium sulphate (MgSO4) (0.08%),
Potassium chloride (KCl) (0.52 g/l),
Tween-80 (0.3%),
Zinc sulphate heptahydrate (ZnSO4.7H2O),
Ferrous sulphate heptahydrate (FeSO4.7H2O) and
Cupric nitrate trihydrate (Cu(NO3)2.3H2O) (traces).
(Claim 4) The process according to claims 1 to 3, wherein agitation rate is about 200 rpm, dissolved oxygen is about 20%, and aeration rate is about 0.5 vvm.
(Claim 5) The process according to claim 4, wherein the cultivation produces about 36.936 mg dcw/ml Lactiplantibacillus plantarum NCIM5777 using 20% dissolved oxygen (DO) saturation level.
(Claim 6) The process according to claim 4, wherein the cultivation produces 38.45 mg dcw/ml Lactiplantibacillus plantarum NCIM5777 at 200 rpm agitation rate.

(Claim 7) The process according to claim 4, wherein the cultivation produces 38.45 mg dcw/ml Lactiplantibacillus plantarum NCIM5777 at 0.5 vvm of aeration rate.
(Claim 8) The process according to any of the preceding claims, wherein the cultivation produces high cell density cultivation of bacterial strain Lactiplantibacillus plantarum NCIM5777 in about 22 hours of incubation period at 200 rpm agitation rate, 20% dissolved oxygen (DO) saturation and 0.5 vvm of aeration rate.
(Claim 9) A process for the high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777,
wherein the said process comprises, growing the Lactiplantibacillus plantarum NCIM5777 in a fermentor having about 0.8 % of a carbon source, about 0.8 % of a nitrogen source;
wherein pH during cultivation is in the range of 3.0 to 12.0 and the temperature ranges from 10 to 45°C;
wherein agitation rate is about 200 rpm, dissolved oxygen is about 20%, and aeration rate is about 0.5 vvm.
(Claim 10) A process for the high cell density cultivation of Lactiplantibacillus plantarum having accession number NCIM5777, substantially as described herein and in the appended drawings.