METHOD FOR PREPARING CULTURES OF LACTIC ACID BACTERIA, PRODUCTS AND CULTURE MEDIA THEREFORE

Abstract

The present invention relates to microbial starter cultures. More specifically, a method for preparing a microbial culture such as a lactic acid bacteria (LAB) starter culture wherein at least one microbial strain such as a lactic acid bacteria and at least one hemoprotein is inoculated in a culture medium.

Claims

1: A method for obtaining a microbial culture, said method comprises the steps of: (i) culturing at least one microbial strain in a culture medium under aeration and obtaining a fermentate, and (ii) harvesting from the fermentate said microbial strain to obtain the microbial culture, wherein the culture medium comprises at least one hemoprotein.

2: The method according to claim 1, wherein the hemoprotein is a protein with native biological catalytical activity.

3: The method according to claim 2, wherein the hemoprotein is an enzyme.

4: The method according to claim 1, wherein the hemoprotein is a protein without native biological catalytical activity.

5: The method according to claim 1, wherein the oxygen consumption in the fermentate reaches 0.04 mol O.sub.2/L/h in less than 10 hours or less than 8 hours.

6: The method according to claim 1, wherein the hemoprotein is microbially produced.

7: The method according to claim 1, wherein the hemoprotein is produced by expression from the genus Aspergillus, Pichia, Bacillus, or Escherichia.

8: The method according to claim 7, wherein the hemoprotein is catalase or peroxidase.

9: The method according to claim 1, wherein the hemoprotein is added to a concentration of between about 0.1 g/kg fermentate and about 10 g/kg fermentate.

10: The method according to claim 1, said method further comprising: (iii) concentrating the microbial culture to obtain a concentrated microbial culture.

11: The method according to claim 10, said method further comprising: (iv) freezing or drying said microbial culture to obtain a frozen or dried microbial culture.

12: The method according to claim 11, said method further comprising: (v) packing said frozen microbial culture or the dried microbial culture obtained in step (iv).

13: The method according to claim 2, wherein the hemoprotein is inactivated hemoprotein.

14: The method according to claim 13, wherein the heat inactivated hemoprotein is heat inactivated.

15: The method according to claim 1, wherein the culture medium does not comprise a non-vegetarian compliant hemoprotein.

16: The method according to claim 1, wherein the hemoprotein is the enzyme catalase.

17: The method according to claim 1, wherein the culture medium further comprises a heat stabilizing compound selected from the group consisting of: polyols, sugars, biopolymers, amino acids, salt, polymers and non-ionic detergents.

18: The method according to claim 17, wherein the heat stabilizing compound is selected from the group consisting of: Sorbitol, Glycerol, Propylene glycol, Mannitol, Xylitol, Propanediol, Trehalose, Sucrose, Lactose, Maltose, Glucose, Levan (fructose homopolysaccharide), Dextrans, Dextran sulfate, Gelatins (type A and B), Hydroxyethyl starch, poly-L-glutamic acid, poly-L-lysine, Fucoidan, Pentosan polysulfate, Keratan sulfate, poly-Aspartate, poly-Glutamate, Hydroxyethylcellulose, Hydroxypropyl-p-Cyclodextrin, Glycine, L-Arginine hydrochloride, arginine, Proline, Lysine, Histidine, Aspartic acid, Glutamic acid, Acetate, Citrate, Sodium chloride, Phosphates, Ascorbate, poly(acrylic acid) randomly modified with n-octylamine and isopropylamine (A8-35), Polyethylene Glycols (PEG), Polyvinyl sulfate, Polysorbate 20, Polysorbate 80, Triton X-100, Pluronic F68, Pluronic F88, Pluoronic F-127, and Brij 35 (polyoxyethylene alkyl ether).

19: A culture obtained by the method according to claim 1.

20: A culture or a culture medium comprising at least one hemoprotein.

21: The culture or culture medium according to claim 20, wherein the hemoprotein is catalase.

22: The culture or culture medium according to claim 21, further comprising a heat stabilizing compound selected from the group consisting of: polyols, sugars, biopolymers, amino acids, salt, polymers and non-ionic detergents.

23: The culture or culture medium according to claim 22, wherein the heat stabilizing compound is selected from the group consisting of: Sorbitol, Glycerol, Propylene glycol, Mannitol, Xylitol, Propanediol, Trehalose, Sucrose, Lactose, Maltose, Glucose, Levan (fructose homopolysaccharide), Dextrans, Dextran sulfate, Gelatins (type A and B), Hydroxyethyl starch, poly-L-glutamic acid, poly-L-lysine, Fucoidan, Pentosan polysulfate, Keratan sulfate, poly-Aspartate, poly-Glutamate, Hydroxyethylcellulose, Hydroxypropyl-p-Cyclodextrin, Glycine, L-Arginine hydrochloride, arginine, Proline, Lysine, Histidine, Aspartic acid, Glutamic acid, Acetate, Citrate, Sodium chloride, Phosphates, Ascorbate, poly(acrylic acid) randomly modified with n-octylamine and isopropylamine (A8-35), Polyethylene Glycols (PEG), Polyvinyl sulfate, Polysorbate 20, Polysorbate 80, Triton X-100, Pluronic F68, Pluronic F88, Pluoronic F-127, and Brij 35 (polyoxyethylene alkyl ether).

24: A method of preparing a food product, feed product, a pharmaceutical product, a dairy flavor and a cheese flavoring product, said method comprising adding an effective amount of the culture according to claim 20, to a food, feed or pharmaceutical product starting material and keeping the inoculated culture under conditions where the at least one microbial strain is metabolically active.

25: A fermented food, feed or pharmaceutical product obtained by the method of claim 1.

26. (canceled)

27: A food product, feed product, a pharmaceutical product, a dairy flavor or a cheese flavoring product, comprising the culture according to claim 20.

Description

FIGURE LEGENDS

[0118] FIG. 1. A graph showing the oxygen transfer rate according to an embodiment of the invention.

EXAMPLES

Example 1: Fermentations in a Complex Fermentation Medium of Chr. Hansen A/S Performed with Catalase as Hemoprotein

[0119] A stock solution of catalase (Catazyme, purchased from NovoZymes) with a concentration of 24%-55% (w/w) is prepared in water.

[0120] The catalase solution is then treated at Ultra-High Temperatures (UHT) at 141 C. for 8-10 seconds, in order to sterilize the material and fully inactivate the enzyme.

[0121] The product Catazyme contains 9% w/w catalase and 42% w/w sorbitol. It was surprisingly found that sorbitol acts as a protein stabilizer. When UHT treating the Catazyme solution at concentrations lower than 24% w/w, the heme molecule is degraded at the high temperatures and is no longer able to support respiration of L. lactis. (data not shown).

[0122] Thus, sorbitol acts as a protein stabilizer and heme-protectant.

Culture

[0123] The present experiment was performed using the commercially available Lactococcus lactis culture deposited as DSM 24648 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by Chr. Hansen A/S, Horsholm, Denmark on 2011 Mar. 15.

Fermentation Medium

[0124] The fermentation medium used was a proprietary vegetarian friendly complex fermentation medium of Chr. Hansen A/S.

Negative Control

[0125] An anaerobic complex fermentation medium was used as medium for negative control. The medium was proprietary vegetarian friendly complex fermentation medium of Chr. Hansen A/S not including a heme source.

Positive Control

[0126] An aerobic complex fermentation medium was used as medium for positive control. The medium was proprietary vegetarian friendly complex fermentation medium of Chr. Hansen A/S including a non-vegetarian heme source.

[0127] The medium was sterilized by UHT-treatment (141 C. for 8-10 seconds). The finished medium had a pH of 6.5.

Fermentation Condition the Cultures

[0128] The fermentation was performed in a 2 L Lab scale fermentation tank with aeration at 30 C. using 1% (w/w) of the culture mentioned above as inoculum and one of the abovementioned hemoprotein Catazyme as heme source. For aerobic fermentation as a positive control, the same conditions as for the aerobic fermentation was applied with aeration in a proprietary vegetarian friendly complex fermentation medium of Chr. Hansen A/S including a non-vegetarian heme source. For anaerobic fermentation as a negative control, the same conditions as for the aerobic fermentation was applied but without aeration in a proprietary vegetarian friendly complex fermentation medium of Chr. Hansen A/S excluding heme source. The cultures were allowed to acidify to pH 6.0. The pH was subsequently maintained at 6.0 by controlled addition of 27% NH40H.

[0129] When no further base consumption was detected, the respective culture was cooled down to about 10 C.

[0130] Following cooling, the bacteria in the culture media were concentrated 6-18 times by centrifugation and subsequently frozen as pellets in liquid nitrogen at one atmosphere of pressure to produce a so-called frozen Direct Vat Set culture (F-DVS). The F-DVS pellets were stored at 50 C. until further analysis.

Process Parameters Evaluation

[0131] Lactococcus lactis changes metabolism profoundly when going from anaerobic to respiratory growth. Compared to anaerobic growth, biomass is approximately doubled, and acid production is reduced during respiratory growth. A key feature of respiratory growth is the reduction of dissolved oxygen (DO %) (FIG. 1). Compared to the Aerobic positive control, the respiratory fermentation process using Catazyme (hemoprotein, 9% w/w) showed similar dissolved oxygen (DO %)

[0132] As a conclusion, based on the process curves in FIG. 1, respiratory fermentation process using Catazyme as hemoprotein performed as good as the Aerobic positive control (FIG. 1, non-vegetarian heme source) to support respiratory growth.

Downstream Process Evaluation

[0133] After fermentation, a packed cell volume (PCV) test is done on the fermentate (centrifugation of the fermentate in special centrifuge tubes). This is the first indication of biomass. The PCV test show that fermentation with Catazyme resulted in about the same level of bacterial cells at PCV as the Aerobic positive control process whereas the Anaerobic negative control resulted in a lower level of PCV (data not shown).