Preparation of dry formulations of dairy probiotics

Abstract

The invention relates to a method for the preparation of dry formulations of dairy probiotic bacteria using a combination of different polymers. The said formulations having increased viability during processing, storage and upon transfers to the rumen of the dairy animals. Further said formulations having high tolerance to lactate and acetate in the rumen of dairy animals.

Claims

1. A method for the preparation of dry formulation of anaerobic probiotic bacteria comprising steps of: (a) providing a culture of Megasphaera elsdenii; (b) subjecting said culture to centrifugation to separate bacteria as a wet cake; (c) mixing said wet cake with a solution of carboxymethylcellulose, pectin, and glycerol to form a lyoslurry; (d) lyophilizing said lyoslurry to encapsulate the Megasphaera elsdenii cells in carboxymethylcellulose, pectin, and glycerol and form a lyopowder comprising particles of the encapsulated Megasphaera elsdenii cells; and (e) packaging said lyopowder in an air-tight container.

2. The method of claim 1, wherein said solution comprises carboxymethlycellulose about 1% by weight.

3. The method of claim 1, wherein said solution comprises pectin about 1% by weight.

4. The method of claim 1, wherein said solution comprises glycerol about 2.5% by weight.

5. The method of claim 1, wherein said lyophilisation is performed at about −50° C. for about 30 to about 36 hours.

6. The method of claim 1, wherein said lyopowder is stable for about 90 days when stored in the air-tight container.

7. The method of claim 1, wherein said lyopowder is suitable for use as a component of animal feed.

8. The method of claim 1, wherein providing the Megasphaera elsdenii culture includes culturing in deMan-Rogosa-Sharpe liquid medium.

9. The method of claim 1, wherein the solution has about 1% by weight carboxymethlycellulose, about 1% by weight pectin, and about 2.5% by weight glycerol.

10. The method of claim 9, wherein the solution is mixed with the wet cake at a ratio of 1:1 under anaerobic conditions.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-D provide details of the SEM analyses of Example 8. A & C show the encapsulating agent as a powder and B & D show the encapsulating agent enclosing the bacterial cells forming particles of definite size and shape.

(2) FIGS. 2A-D provide the details the FT-IR analyses of Example 9. FIG. 2A is the spectrum of pectin. FIG. 2B is the spectrum of carboxymethylcellulose (CMC). FIG. 2C is the spectrum of CMC+pectin mixture. FIG. 2D is the spectrum of CMC+pectin+glycerol mixture enclosing the bacterial cells.

DETAILED DESCRIPTION

(3) A strain of Megasphaera elsdenii is used as a probiotic culture for further development of dry solid powder formulations. Megasphaera elsdenii is selected on the basis of its tolerance to lactic acid and acetic acid condition and its hydrophobicity. Said culture can attaches well to ruminal walls and preferentially utilizes lactate over other sugars.

(4) In one of the embodiment of present invention, Megasphaera elsdenii is cultured in sodium lactate medium in static incubation conditions at about 37° C. for about 24 hours using degassed stoppered bottles. The culture is routinely transferred and stored at about −80° C. in 20% (vol/vol) glycerol stocks between transfers. Said sodium lactate medium is a complex medium with suspended solid in it. For the purpose of the formulation development deMan,-Rogosa-Sharpe (MRS) medium is used, which is simpler and clear growth medium and easy to handle, scale up and for enumeration of microorganisms. The total viable count in MRS medium is about 1.1×10.sup.9 CFU/ml in static incubation condition at about 37° C. for about 24 hours using degassed stoppered bottles. Said media is selected further for fermentation and for probiotic formulation development. Said Megasphaera elsdenii culture is inoculated in degassed MRS broth bottles and incubated in static condition at about 37° C. for about 24 hours. Further said inoculum is inoculated in MRS broth and incubated at static condition for about 24 hours at about 37° C. Further, said broth is subjected to centrifugation with feed rate of about 500 ml/min to separate the wet cake. Next, the wet cake is immediately removed aseptically in LAF unit (Laminar Air Flow) and transferred to an anaerobic chamber. Further about 1% carboxymethylcellulose (CMC) and about 1% of pectin; in combination with 2.5% glycerol is used as an encapsulation agent. Next, encapsulation material and 70% glycerol are sterilized at about 121° C. for about 20 minutes. Further said wet cake, encapsulating chemicals (1% pectin+1% CMC) and 2.5% glycerol is mixed properly in anaerobic chamber to prepare a lyoslurry by mixing. Then said prepared lyoslurry is poured in trays and subjected to freezing at about −80° C. for about 2 hours. Post freezing, the frozen lyoslurry is subjected to lyophilisation at about −50° C. for about 28 to about 36 hours to prepare lyopowder. The lyopowder obtained is analyzed for TVC and solids contents. Said broth, wet cake, lyoslurry and lyopowder are serially diluted inanaerobic chamber using sterile degassed saline Tween 80 solution and plated over sterile MRS agar plates. Said plates are incubated at about 37° C. for about 48 to about 72 hours under anaerobic condition. The total solid and TVC values for broth, wet cake, lyopowder and for lyoslurry are enlisted in TABLE 1.

(5) TABLE-US-00001 TABLE 1 TVC of Megasphaera elsdenii culture at different process stages. Sr. TS TVC No. Sample (% w/w) (CFU/gm) 1 Harvest/Broth 04.04 5 × 10.sup.8 2 Wet cake 23.57 .sup. 1 × 10.sup.11 3 Lyoslurry 12.75 1 × 10.sup.9 4 Lyopowder 96.63 1 × 10.sup.7

(6) In another embodiment of the invention, said lyopowder formulations are packed in air-tight containers and stored at room temperature at about 50% relative humidity. The moisture contents of lyopowder formulations are not more than 5% by weight. During storage after specified time periods the TVC analyses are carried out to check the viability of bacteria in the formulations.

(7) In another embodiment of the invention, during the production of dry formulations, Megasphaera elsdenii culture survived to oxygenated condition for a short period like centrifugation or serial dilution as checked by measurement of total viable counts. This is important feature as survival of the bacteria in aerobic conditions is necessary for creation of said dry formulations.

(8) In yet another embodiment of present invention, others polymers like hydroxy methyl propyl cellulose, gum acacia, kappa carrageenan, maltodextrin are also tested alone or in combination with or without glycerol as a capsulating agents for rumen bacteria for the preparation of the dry formulations.

(9) Embodiments provided above give wider utility of the invention without any limitations as to the variations that may be appreciated by the person skilled in the art. A non-limiting summary of various embodiments is given above, which demonstrate the advantages and novel aspects of the process disclosed herein.

Advantages

(10) 1. Due to use of deMan-Rogosa-Sharpe (MRS) medium for fermentation, the operational expenses are substantially reduced relative to conventional processes. 2. Megasphaera elsdenii has hydrophobicity properties which helps them for better attachment in rumen. 3. Megasphaera elsdenii has tolerance to lactic acid and acetic acid conditions. Said bacteria can tolerate pH up to 3 and it can also grow well at about 7 pH. 4. Said encapsulation agents used for M. elsdenii have a low porosity and which helps in low exposure to unfavourable environmental conditions. 5. The encapsulated dry formulations have increased bacterial viability during processing and transfer to the rumen. 6. The probiotic dry product is more stable and much easier for handling and transport. 7. Due to the powdery nature of probiotic product, the dosage requirements are very low.

(11) Examples provided below give wider utility of the invention without any limitations as to the variations that may be appreciated by the person skilled in the art. A non-limiting summary of various experimental results is given in the examples, which demonstrate the advantageous and novel aspects of the preparation of dry formulations of Megasphaera elsdenii using combination of polymers and glycerol.

Example 1

(12) A strain of Megasphaera elsdenii (ATCC 25940) was used as a probiotic culture for the development of dry solid powder formulations. The recommended media for Megasphaera elsdenii was sodium lactate media. Said culture was grown in sodium lactate medium in static incubation condition at about 37° C. for about 24 hours using degassed stoppered bottles. The culture was routinely transferred and stored at about −80° C. in about 20% glycerol by volume between transfers. The total viable count in sodium lactate medium is about 4.0×10.sup.7.Math.CFU/ml. Said sodium lactate medium was with suspended solids in it which made it non feasible and it was not useful for scale up and for cell counting by TVC method. Several different media were screened in which deMan-Rogosa-Sharpe (MRS) liquid medium was simpler and clear growth medium and it was easy to handle, to scale up and to count microbial cells. The total viable count in MRS medium was about 1.1×10.sup.9 CFU/ml in static incubation conditions at about 37° C. for about 24 hours using degassed stopper bottles. So it was selected further for fermentation and for probiotic formulation development.

Example 2: Oxygen Tolerance of M. elsdenii During Handling for the Preparation of Dry Formulations

(13) About 10 ml culture having initial count of about 4.0×10.sup.7 CFU/ml was used for the experimentation. About 1 ml culture each was exposed to three different conditions and TVCs were calculated to check the tolerance levels. In one test the culture was serially diluted in laminar air flow [aerobic condition] and then incubated in a glove box for about 37° C. for about 24 hrs and at the end TVC was counted. In second test the culture was serially diluted in anaerobic chamber and then incubated in a glove box for about 37° C. for about 24 hrs and at the end TVC was counted. In third test the culture was centrifuged, wet cake collected and serially diluted [TVC counted in aerobic conditions]; then incubated [anaerobic conditions] in a glove box for about 37° C. for about 24 hrs and at the end TVC was counted. Said three different conditions and its count are enlisted in TABLE 2:

(14) TABLE-US-00002 TABLE 2 Serial Dilution And Incubation TVC Plating Condition condition (CFU/ml) Aerobic Anaerobic 6.0 × 10.sup.5 Anaerobic 4.0 × 10.sup.7 Centrifugation, wet cake 3.0 × 10.sup.6 serial dilution and plating

Example 3

(15) The Megasphaera elsdenii culture was inoculated in degassed MRS broth bottles and incubated in static condition at about 37° C. for about 24 hours to form a starting culture. Next this starting culture was inoculated in 1.5 l MRS broth and incubated at static condition for about 24 hours at about 37° C. Then said broth was subjected to centrifugation to separate the biomass. Then a pectin solution [an encapsulation agent] having about 1% or 2% concentration were sterilized at about 121° C. for about 20 min. The biomass of Megasphaera elsdenii obtained after centrifugation was mixed with sterile pectin solution in 1:1 proportion under the anaerobic condition. Said mixture was mixed well using vortex mixer at low speed to avoid shear disruptions. Said prepared lyoslurry was subjected to freezing at about −80° C. for 2 hours. Post freezing, the frozen lyoslurry was subjected to lyophilisation at about −50° C. about 28 to about 36 hours to prepare the lyopowder. Said lyopowder and lyoslurry were serially diluted in anaerobic chamber using sterile degassed saline Tween 80 solution and plated over sterile MRS agar plates. Said plates were incubated at about 37° C. for about 48 to about 72 hours. Total viable counts analyses of said lyopowder and lyoslurry were carried out in anaerobic chamber to check the encapsulation potential of polymers and loss of viability of probiotic cells after lyophilisation. The total viable counts before lyophilisation (lyoslurry) and after lyophilisation (lyopowder) are given in TABLE 3.

(16) TABLE-US-00003 TABLE 3 Sr. Lyoslurry TVC Lyopowder TVC No. Material (cfu/ml) (cfu/g) 1 Control 6.4 × 10.sup.9 0 2 1% pectin 6.0 × 10.sup.6 3 2% pectin 6.0 × 10.sup.6

Example 4

(17) The Megasphaera elsdenii culture was inoculated in degassed MRS broth bottles and incubated in static condition at about 37° C. for about 24 hours to form a starting culture. Next this starting culture was inoculated in 1.5 l MRS broth and incubated at static condition for about 24 hours at about 37° C. Then said broth was subjected to centrifugation to separate the biomass. Then a carboxymethylcellulose (CMC), solution [an encapsulation agent] having about 1% or 2% concentration were sterilized at about 121° C. for about 20 min. The biomass of Megasphaera elsdenii obtained after centrifugation was mixed with sterile the CMC solution in 1:1 proportion under the anaerobic condition. Said mixture was mixed well using vortex mixer at low speed to avoid shear disruptions. Said prepared lyoslurry was subjected to freezing at about −80° C. for 2 hours. Post freezing, the frozen lyoslurry was subjected to lyophilisation at about −50° C. about 28 to about 36 hours to prepare the lyopowder. Said lyopowder and lyoslurry were serially diluted in anaerobic chamber using sterile degassed saline Tween 80 solution and plated over sterile MRS agar plates. Said plates were incubated at about 37° C. for about 48 to about 72 hours. Total viable counts analyses of said lyopowder and lyoslurry were carried out in anaerobic chamber to check the encapsulation potential of polymers and loss of viability of probiotic cells after lyophilisation. The total viable counts before lyophilisation (lyoslurry) and after lyophilisation (lyopowder) are given in TABLE 4.

(18) TABLE-US-00004 TABLE 4 Sr. Lyoslurry TVC Lyopowder TVC No. Material (cfu/ml) (cfu/g) 1 Control 7.0 × 10.sup.9 0 2 1% CMC 6.0 × 10.sup.6 3 2% CMC 2.0 × 10.sup.6

Example 5

(19) The Megasphaera elsdenii culture was inoculated in degassed MRS broth bottles and incubated in static condition at about 37° C. for about 24 hours to form a starting culture. Next this starting culture was inoculated in 1.5 l MRS broth and incubated at static condition for about 24 hours at about 37° C. Then said broth was subjected to centrifugation to separate the biomass. Then a solution of 1% carboxymethylcellulose (CMC) and 1% pectin [an encapsulation agent] was sterilized at about 121° C. for about 20 min. The biomass of Megasphaera elsdenii obtained after centrifugation was mixed with sterile the CMC+pectin solution in 1:1 proportion under the anaerobic condition. Said mixture was mixed well using vortex mixer at low speed to avoid shear disruptions. Said prepared lyoslurry was subjected to freezing at about −80° C. for 2 hours. Post freezing, the frozen lyoslurry was subjected to lyophilisation at about −50° C. about 28 to about 36 hours to prepare the lyopowder. Said lyopowder and lyoslurry were serially diluted in anaerobic chamber using sterile degassed saline Tween 80 solution and plated over sterile MRS agar plates. Said plates were incubated at about 37° C. for about 48 to about 72 hours. Total viable counts analyses of said lyopowder and lyoslurry were carried out in anaerobic chamber to check the encapsulation potential of polymers and loss of viability of probiotic cells after lyophilisation. The total viable counts before lyophilisation (lyoslurry) and after lyophilisation (lyopowder) were given in TABLE 5.

(20) TABLE-US-00005 TABLE 5 Sr. Lyoslurry TVC Lyopowder TVC No. Material (cfu/ml) (cfu/g) 1 Control 2.0 × 10.sup.9 0 2 1% CMC + 1% Pectin 2.0 × 10.sup.3

Example 6

(21) Next about 2.5% glycerol was included at the time of preparation of the dry formulation of M. elsdenii. The glycerol being moisture retaining agent in the encapsulation agent increased the TVC of the dry formulation significantly as shown in TABLE 6.

(22) TABLE-US-00006 TABLE 6 Lyopowder TVC Encapsulation Agent (cfu/g) 1% CMC + 1% Pectin 2.0 × 10.sup.3 1% CMC + 1% Pectin + 2.5% 2.0 × 10.sup.6 Glycerol

Example 7: Shelf Life Study of M. elsdenii Lyophilized Powder Formulations

(23) To estimate the shelf life periods for the dry formulations of M. elsdenii, they were prepared according to the EXAMPLE 6 and subjected to storage at STP conditions for 30, 60 or 90 days. After the storage periods, the TVC was counted for the each sample and is listed in TABLE 7.

(24) TABLE-US-00007 TABLE 7 Time for storage Shelf life TVC (days) (cfu/g) 0 4.0 × 10.sup.5 30 3.2 × 10.sup.4 60 3.0 × 10.sup.4 90 1.0 × 10.sup.4

Example 8: Analysis of Encapsulated Cells by Scanning Electron Microscopy

(25) To establish the morphology of the particles of the dry formulations of M. elsdenii the encapsulating agent [1% CMC+1% pectin+2.5% glycerol] as such and with the bacterial cells enclosed were analysed under a scanning electron microscope. FIG. 1 provides the details of the SEM analyses. FIG. 1 A & C show the encapsulating agent as such as a powdery matter. FIG. 1 B & D show the encapsulating agent enclosing the bacterial cells forming particles of definite size and shape.

Example 9: Infrared Spectral Analyses of Encapsulated Formulations

(26) To establish the spectral properties of the particles of the dry formulations of M. elsdenii the encapsulating agent components as such and the mixture as such and the mixture with the bacterial cells enclosed were analysed by FT-IR spectrometry. FIG. 2 provides the details the FT-IR analysis. FIG. 2 A is the spectrum of pectin as such. FIG. 2 B is the spectrum of CMC as such. FIG. 2 C is the spectrum of CMC+pectin mixture. FIG. 2 D is the spectrum of CMC+pectin+glycerol mixture enclosing the bacterial cells. The IR spectra were recorded at the wavelength between 4000 cm.sup.−1 to 500 cm.sup.−1 with spectral resolution of 4 cm.sup.−1. The pectin has a sharp absorption at about 1000 cm−1 and CMC has a sharp absorption at 1000 cm.sup.−1 and at 1600 cm.sup.−1. After mixing of both pectin and CMC with glycerol, absorption was not changed at these major frequencies and after cell encapsulation, it exhibited absorptions at about 1000 cm.sup.−1 and at 1600 cm.sup.−1 and a new absorption at about 1500 cm.sup.−1, which is remarkable for the encapsulated formulations of the bacteria herein in disclosed.

(27) Embodiments provided above give wider utility of the invention without any limitations as to the variations that may be appreciated by the person skilled in the art. A non-limiting summary of various embodiments is given in the examples and tables, which demonstrate the advantageous and novel aspects of the process disclosed herein.