NOVEL THERMOTOLERANT LACTOBACILLUS

Abstract

The present invention relates to a high temperature resistant Lactobacillus delbrueckii subsp. lactis DSM 32009 strain that can be used in the production of cheese where the curd is cooked at high temperature (>50° C.).

Claims

1-32. (canceled)

33. A process for producing cheese, comprising: (i) adding to milk a starter culture and an acidifying Lactobacillus strain to obtain a mixture, wherein the acidifying Lactobacillus strain is the Lactobacillus delbrueckii subsp. lactis strain deposited under Accession No. DSM 32009 with the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ); and (ii) heating the mixture to a temperature in the range of 45 to 65° C. or maintaining the mixture at a temperature in the range of 45 to 65° C., wherein the acidifying Lactobacillus delbrueckii subsp. lactis strain of step (i) is a thermotolerant strain, as determined by being able to acidify milk by at least 0.4 pH units in 2.5 hours at a temperature around 50° C. when inoculated with a 3% whey starter culture comprising Lactobacillus ssp. and Streptococcus thermophilus, wherein the process achieves acidification of the milk by at least 0.4 pH units in a shorter time than an identical process that does not use the acidifying Lactobacillus strain.

34. The process according to claim 33, further comprising adding to the milk one or more additional bacterial strains or cultures.

35. The process according to claim 34, wherein the one or more additional bacterial strains or cultures includes a bacterial strain or culture of genus Propionibacterium.

36. The process according to claim 33, wherein the starter culture comprises one or more bacterial strains other than the acidifying Lactobacillus strain, wherein the one or more bacterial strains are selected from Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus pentosaceus, Lactobacillus casei, Lactobacillus paracasei, Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus spp., Lactobacillus delbrueckii, Lactobacillus acidophilus and Propionic bacterium.

37. The process according to claim 36, wherein the starter culture comprises one or more other bacterial strains selected from Lactobacillus helveticus spp., Lactobacillus delbrueckii, Streptococcus thermophilus, and Lactobacillus casei.

38. The process according to claim 33, further comprising adding to the milk one or more coagulants.

39. The process according to claim 38, wherein the one or more coagulants is selected from bovine derived coagulants, microbial derived coagulants, camel derived coagulants, rennets, and chymosins.

40. The process according to claim 33, wherein, in step (i), the starter culture is added to the milk as a direct vat set (DVS) culture.

41. The process according to claim 33, wherein the starter culture is a whey starter culture.

42. The process according to claim 41, wherein, before being added to the milk, the whey starter culture has been subjected to physical conditions comprising a temperature drop from around 48° C. to around 39° C. in 9 hours, a constant temperature of around 39° C. for around 8 hours, and cooling and storing at around 16° C. until it is added to the milk.

43. The process according to claim 41, further comprising, prior to step (i), adding the acidifying Lactobacillus strain as a DVS culture to the whey starter culture.

44. The process according to claim 43, wherein the acidifying Lactobacillus strain is added as a DVS culture to the whey starter culture in an amount of at least 5×10.sup.10 CFU/500 liter whey.

45. The process according to claim 33, wherein, in step (i), the acidifying Lactobacillus strain is added directly to the milk.

46. The process according to claim 33, wherein, in step (i), the acidifying Lactobacillus strain is added to the milk in a composition that further comprises one or more excipients selected from polyhydroxy compounds, trehalose, and dimethyl sulfoxide (DMSO).

47. The process according to claim 33, wherein, in step (i), the starter culture and acidifying Lactobacillus strain are added to the milk in an amount sufficient to achieve acidification of the milk by at least 0.4 pH units in 2.5 hours at a temperature around 50° C.

48. The process according to claim 33, wherein the process comprises heating the mixture to a temperature in the range of 45 to 65° C.

49. The process according to claim 33, wherein the process comprises maintaining the mixture at a temperature in the range of 45 to 65° C.

50. An acidifying Lactobacillus delbrueckii subsp. lactis strain, wherein the strain is the acidifying Lactobacillus delbrueckii subsp. lactis strain deposited at the DSMZ under Accession No. DSM 32009, or a mutant or variant thereof able to acidify milk by at least 0.4 pH units in 2.5 hours at a temperature around 50° C. when inoculated with a 3% starter culture.

51. The acidifying Lactobacillus delbrueckii subsp. lactis strain of claim 50, wherein the strain is the acidifying Lactobacillus delbrueckii subsp. lactis strain deposited at the DSMZ under Accession No. DSM 32009.

52. A composition comprising the acidifying Lactobacillus delbrueckii subsp. lactis strain of claim 50 and one or more excipients.

Description

DRAWINGS

[0068] FIG. 1 depicts the temperature profile used for the generation of the whey starter culture containing the Lactobacillus delbrueckii subsp. lactis DSM 32009, cf. the example 1.

[0069] FIG. 2 depicts the evolution of the pH in the whey starter solution during the incubation step as described in FIG. 1.

[0070] FIG. 3 depicts the temperature profile used for the milk acidification using a whey starter containing the L. delbrueckii subsp. lactis.

[0071] FIG. 4 depicts the evolution of the pH in milk inoculated with a whey starter solution with and without the L. delbrueckii subsp. lactis.

EXPERIMENTAL

Example 1: Acidification of Milk by a Whey Starter Containing the Lactobacillus delbrueckii Subsp. Lactis DSM 32009 Strain when a Temperature Gradient with a High Temperature Cooking Step is Applied

[0072] a) Generation of Whey Starter Containing the Lactobacillus delbrueckii Subsp. Lactis DSM 32009 Strain.

[0073] Whey starter media consisted of a 7% sweet whey powder solution in water. After dissolution of the whey powder, the whey media was pasteurized for 20 minutes at 90° C. 200 ml of sweet whey media were inoculated with the whey starter culture comprising a mix of Lactobacillus spp and Streptococcus thermophilus with and without DSM 32009 The starter was incubated as described in FIG. 1. pH was measured continuously with pH probes connected to a datalogger as shown in FIG. 2. Temperature compensation was used to secure accurate pH measurement throughout the temperature gradient. The whey starter culture containing the Lactobacillus delbrueckii subsp. lactis DSM 32009 strain acidified to pH 3.5 in 16 hours.

[0074] FIG. 1 shows the temperature profile used for the generation of the whey starter culture. This temperature profile simulates the temperature profile used for the production of whey starter for manufacture of Grana Padano cheese. The temperature profile consists of: [0075] A temperature drop from 48° C. to 39° C. in 9 hours [0076] A constant temperature of 39° C. for 8 hours [0077] The whey starter is then cooled and maintained at 16° C. until it is used for milk acidification.

[0078] FIG. 2 shows the evolution of the pH in the whey starter solution during the incubation step as described in FIG. 1.

[0079] b) Acidification of Milk with a Whey Starter Containing the Lactobacillus delbrueckii Subsp. Lactis DSM 32009 Strain

[0080] Milk used for acidification experiment was prepared from 9.5% skim milk powder rehydrated in water and subsequently heat treated at 140° C./8 sec and 100° C./30 min. Milk was inoculated with 3% of the whey starter prepared as described in Example 1.a. The pH was measured continuously with pH probes connected to a datalogger as shown in FIG. 4. Temperature compensation was used to secure accurate pH measurement throughout the temperature gradient. Incubation temperature was as described in FIG. 3. During the time where the temperature is above 50° C., the whey starter culture containing the Lactobacillus delbrueckii subsp. lactis DSM 32009 strain decreased the pH of the milk from pH 6.30 to pH 5.80 (deltapH=0.50 pH units). The whey starter culture without the Lactobacillus delbrueckii subsp. lactis DSM 32009 strain decreased the pH of the milk from pH 6.40 to pH 6.20 (deltapH=0.2 pH units). Moreover, it is observed that the whey starter culture containing the Lactobacillus delbrueckii subsp. lactis DSM 32009 strain continues to lower the milk pH after the cooking step while the whey starter culture without the Lactobacillus delbrueckii subsp. lactis DSM 32009 strain has a longer lag phase with no or very little pH drop. As a comparison, the milk acidified with the whey starter culture containing the Lactobacillus delbrueckii subsp. lactis DSM 32009 strain reaches pH 5.0 in 10 hours while the milk acidified with the whey starter culture without the Lactobacillus delbrueckii subsp. lactis DSM 32009 strain reaches pH 5.0 in 12.5 hours.

[0081] FIG. 3 shows the temperature profile used for the milk acidification using a whey starter containing the Lactobacillus delbrueckii subsp. lactis DSM 32009 strain. This temperature profile simulates the temperature profile used for the manufacture of Grana Padano cheese. The temperature profile consists of: [0082] A constant temperature of 32° C. for 30 min [0083] A temperature increase to 54° C. and kept constant for 3 hours [0084] A linear temperature decrease to 30° C.

[0085] FIG. 4 shows the evolution of the pH in milk inoculated with a whey starter solution containing or not the Lactobacillus delbrueckii subsp. lactis DSM 32009 strain. Temperature gradient is as described in FIG. 3.

REFERENCES

[0086] Rosetti, L., Fornasari, M. E., Gatti, M., Lazzi, C., Neviani, E. and Giraffa, G. Grana Padano cheese whey starters: Microbial composition and strain distribution. International Journal of Food Microbiology 127 (2008) pp. 168-171.