Ampicillin resistant texturizing lactic acid bacteria strains

Abstract

The present invention relates to mutants of lactic acid bacteria which are resistant to the antibiotic ampicillin and which were found to give an increased texture when grown in milk while maintaining the other growth properties of the parent strain. The present invention, furthermore, relates to compositions comprising such mutants, and to dairy products fermented with the lactic acid bacteria resistant to ampicillin.

Claims

1. A method for preparing a fermented milk product, comprising fermenting a milk substrate with a lactic acid bacteria strain selected from the Lactobacillus delbrueckii subsp. bulgaricus strain CHCC15466 that was deposited with the Deutsche Sammlung von Mikroorganismen and Zellkulturen under accession number DSM 25852, wherein strain CHCC15466 are resistant to ampicillin as determined by the amount of ampicillin that reduces the OD.sub.600 measured growth of the lactic acid bacteria strain, after 20 hours growth at 37 C. in a medium suitable for growth of the lactic acid bacteria strain, by 20% as compared to the OD.sub.600 measured growth in the medium without ampicillin being higher than 400 ng/ml, and exhibit a texturing property such that the efflux time of 28 ml of full fat cow milk containing 2% (w/v) skimmed milk powder, inoculated with at least 10.sup.4 CFU/ml of the strain CHCC15466 and acidified at 37 C. for 20 hours, from a polystyrene 25-ml pipette is at least 50 seconds.

2. The method of claim 1, wherein the fermented milk product is a yoghurt.

Description

EXAMPLES

(1) Materials:

(2) Media: For Streptococcus thermophilus, suitable media include the known M17 agar medium having the following composition:

(3) agar, 12.75 g/L

(4) ascorbic acid, 0.5 g/L

(5) casein peptone (tryptic), 2.5 g/L

(6) disodium -glycerophosphate pentahydrate, 19 g/L

(7) magnesium sulfate hydrate, 0.25 g/L

(8) meat extract, 5 g/L

(9) meat peptone (peptic), 2.5 g/L

(10) soyapeptone (papainic), 5 g/L

(11) yeast extract, 2.5 g/L

(12) and M17 broth medium with this composition:

(13) ascorbic acid, 0.5 g/L

(14) lactose, 5 g/L

(15) magnesium sulfate, 0.25 g/L

(16) meat extract, 5 g/L

(17) meat peptone (peptic), 2.5 g/L

(18) sodium glycerophosphate, 19 g/L

(19) soya peptone (papainic), 5 g/L

(20) tryptone, 2.5 g/L

(21) yeast extract, 2.5 g/L

(22) final pH 7.00.2 (25 C.)

(23) These media are normally used following the addition of 20 g/l of lactose (2% w/v).

(24) For Lactobacillus delbrueckii subsp. bulgaricus, suitable media include the known MRS medium.

(25) MRS agar medium has the following composition:

(26) TABLE-US-00001 Bacto Proteose Peptone no. 3 10 g/l Bacto Beef Extract 10 g/l Bacto Yeast Extract 5 g/l Dextrose 20 g/l Sorbitan Monooleate Complex 1 g/l Ammonium Citrate 2 g/l Sodium acetate 5 g/l Magnesium sulphate 0.1 g/l Manganese sulphate 0.05 g/l Potassium Phosphate Dibasis 2 g/l Bacto Agar 15 g/l

(27) and MRS broth medium has this composition:

(28) TABLE-US-00002 Bacto Proteose Peptone no. 3 10 g/l Bacto Beef Extract 10 g/l Bacto Yeast Extract 5 g/l Dextrose 20 g/l Sorbitan Monooleate Complex 1 g/l Ammonium Citrate 2 g/l Sodium acetate 5 g/l Magnesium sulphate 0.1 g/l Manganese sulphate 0.05 g/l Potassium Phosphate Dibasis 2 g/l final pH 6.5 0.2 (25 C.)

(29) As known to the skilled person, the M17 medium is a medium that is considered to be suitable for growth of Streptococcus thermophilus and the MRS medium is a medium that is considered to be suitable for growth of Lactobacillus delbrueckii subsp. bulgaricus.

(30) In the present context and as understood by the skilled person, the specific M17 and MRS media concentrate may be supplied from different suppliers and independently of the specific supplier one will (within standard measurement uncertainty) get the same herein relevant result of ampicillin resistance for a herein relevant strain of interest.

Example 1: Ampicillin Resistance Selection Assay

(31) The method is illustrated using a strain of Lactobacillus delbrueckii subsp. bulgaricus. To carry out the ampicillin resistance selection assay with Streptococcus thermophilus, M17 medium with lactose added to 2% w/v should be used instead of MRS medium.

(32) A Lactobacillus delbrueckii subsp. bulgaricus strain of interest is inoculated into 10 ml MRS broth medium and grown for at least 20 hours at 37 C. under anaerobic conditions. A cotton swab is dipped in the culture and used to streak out on the entire surface of a MRS agar plate (90 mm in diameter). An ampicillin E-test stick (0.016-256 g/ml) for Antimicrobial Susceptibility Testing (Biomerieux, cat. #501558) is placed on top of the agar, and the plate is incubated for not more than 24 hours at 37 C. under anaerobic conditions. The minimum inhibitory concentration (MIC) of ampicillin is the lowest concentration that will inhibit the visible growth of the plated strain and is read at the point where the elliptical zone of inhibition intersects the E-test strip.

(33) A Lactobacillus delbrueckii subsp. bulgaricus cell that has an increased resistance to ampicillin as discussed herein, is herein defined as a Lactobacillus delbrueckii subsp. bulgaricus cell, where the MIC value read is at least 1 increment higher as noted on the E-strip than for the mother strain.

(34) Cells that are capable of complying with this increased resistance to ampicillin criteria are herein defined as cells that are resistant to ampicillin in the ampicillin resistance assay of this Example 1.

(35) Conclusion:

(36) Based on the ampicillin resistance Selection assay of this Example 1for a specific strain of interest (e.g. one from a relevant commercial product)the skilled person can routinely test if this specific strain of interest has the herein relevant ampicillin resistance.

Example 2: Use of Ampicillin to Isolate Mutants of Lactobacillus delbrueckii subsp. bulgaricus with Improved Rheological Properties

(37) Strains:

(38) Lactobacillus delbrueckii subsp. bulgaricus CHCC13995

(39) Lactobacillus delbrueckii subsp. bulgaricus CHCC15466 (ampicillin resistant mutant of CHCC13995)

(40) Mutant Isolation:

(41) In order to isolate ampicillin resistant mutants of the Lactobacillus delbrueckii subsp. bulgaricus strain CHCC13995, cells derived from the growth of a single colony were inoculated anaerobically into 10 ml MRS broth containing one of the following amounts of ampicillin, 0 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml or 700 ng/ml, and grown for at least 20 hours at 37 C.

(42) After the 20 hours of growth OD.sub.600 is measured for all samples.

(43) Typically the concentration of ampicillin that reduces the OD.sub.600 measured anaerobic growth of Lactobacillus delbrueckii subsp. bulgaricus with at least 20% as compared to the growth in MRS medium without ampicillin (i.e. with 0 ng/ml ampicillin) is higher than 300 ng/ml. Cultures where the concentration of ampicillin was 500 ng/ml were diluted and plated on MRS agar plates with no ampicillin added and then incubated anaerobically for at least 20 hours at 37 C. Colonies were then picked and screened for anaerobic growth at 37 C. in microtiter plates in the presence of 500 ng/ml ampicillin. Typically, 25% of the resulting colonies were identified as fast growers in the presence of ampicillin. These were chosen for further study. The selected ampicillin resistant mutants were further purified and tested for their ability to grow in milk. During this work it was observed that some of the mutants produced considerably more texture than the parent strain under these conditions.

(44) Viscocity Screening:

(45) A rheological screening analysis was done by using a simple pipette viscosity test. In this test the ampicillin resistant mutants were pre-tested for their viscosity (before real rheology testing) by measuring the efflux time from a polystyrene 25-ml pipette (CELLSTAR) loaded with 28 ml of acidified milk:

(46) For each sample, full fat cow milk which was added 2% (w/v) skimmed milk powder where inoculated with an ampicillin resistant mutant in an amount of at least 10.sup.4 cells per ml of milk and left to acidify for 20 hours at 37 C.

(47) Before the pipetting test, the acidified milk was gently homogenized by stirring before loading into a polystyrene 25-ml pipette to the top (28 ml) and for each sample the efflux time from the pipette was measured 3 times. Table 1 lists the result of the pipette viscosity test on milk fermented at 37 C. for 20 hours by 21 ampicillin resistant mutants. The results show that most of these mutants result in a higher viscosity than mother strain. 6 out of the 21 ampicillin resistant mutants result in an efflux time which is double that of the mother strain and approximately 25% of the ampicillin resistant mutants result in an efflux time of at least 50 seconds.

(48) Especially mutant 18 (amp mut 18) have a high efflux time. This mutant derivative was designated CHCC15466 and was used for rheological test described in Example 3.

(49) TABLE-US-00003 TABLE 1 Efflux time (average of 3 measurements) measured in seconds from a polystyrene 25-ml pipette loaded with 28 ml of acidified milk Strain CHCC13995 mut1 mut2 mut3 mut4 mut5 mut6 mut7 mut8 mut9 Efflux 22 41 38 79.5 49 22 40.5 33.5 40 26.5 time (s) Strain mut10 mut11 mut12 mut13 mut14 mut15 mut16 mut17 mut18 mut19 Efflux 42 39 49 43 77 39 94 56 116 49 time (s) Strain mut20 mut21 Efflux 33 33.5 time (s)

(50) Conclusion

(51) The Lactobacillus delbrueckii subsp. bulgaricus ampicillin resistant mutants described herein may be incorporated into a culture, such as a starter culture, which produces a desirable high level of texture.

Example 3: Use of Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus Ampicillin Resistant Mutants for Preparation of a Fermented Milk Product

(52) Rheology:

(53) Rheology analyses were carried on a StressTech rheometer from ReoLogica Instruments AB, Sweden, following growth in full fat cow milk with 2% (w/v) added skimmed milk powder.

(54) The examples described above illustrate the improved texture of fermented milks fermented with selected ampicillin resistant mutants as single culture. In this example we will analyze the texture of fermented milks made as yoghurts i.e. with strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus used in combination. To document the improved texturizing effect of some of the mutants, yoghurts were made with combinations of one fixed wild type Streptococcus thermophilus strain CHCC4895 mixed with a Lactobacillus delbrueckii subsp bulgaricus wild type strain CHCC13995 and an ampicillin resistant strain CHCC15466, respectively. The yoghurt cultures were mixed at a ratio of 9:1 of Streptococcus thermophilus: Lactobacillus delbrueckii subsp bulgaricus and inoculated into full fat cow milk with 2% added skimmed milk powder and fermented at 40 C. to a final pH of 4.50. The following mixtures were analyzed after 5 days storage at 4 C. using the Stresstech rheometer. Ropiness is measured as described by Folkenberg et al. (2006. Int. Dairy J. 16(2); 111-118) The results are presented in Table 2.

(55) TABLE-US-00004 TABLE 2 Rheological measurements of yoghurts made with Streptococcus thermophilus strain CHCC4896 and either Lactobacillus delbrueckii subsp. bulgaricus strain CHCC13995 or annpiciliin resistant Lactobacillus delbrueckii subsp. bulgaricus strain CHCC15466. Shear stress at Gel stiffness at Ropiness Sample 300 1/s (Pa) 1 HZ (Pa) (Pa/Pa) CHCC4895 + CHCC13995 35.8 171 0.382 CHCC4895 + CHCC15466 35 193 0.422

(56) The results clearly show that the improved texturizing property of the ampicillin resistant mutant CHCC15466 as observed with the single cultures was preserved also in the presence of a strain of Streptococcus thermophilus.

(57) While the shear stress is only slightly affected, the gel stiffness and the ropiness are improved significantly where the mutant is used instead of the wild type strain of Lactobacillus delbrueckii subsp bulgaricus for the yoghurt fermentation.

(58) Conclusion

(59) The Lactobacillus delbrueckii subsp. bulgaricus ampicillin resistant mutant described herein may be incorporated into a culture, such as a starter culture, which produces a desirable high level of texture in a fermented milk product such as yoghurt.

DEPOSITS and EXPERT SOLUTION

(60) The strain of Streptococcus thermophilus CHCC4895 was deposited with Deutsche Sammlung von Mikrooganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany on 29 Mar. 2007 under the accession number DSM 19242.

(61) The strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC13995 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on 22 Sep. 2010 under the accession number DSM 24021.

(62) The strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC15466 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on 3 Apr. 2012 under the accession number DSM 25852

(63) The deposits have been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(64) The Applicant requests that a sample of the deposited microorganisms should be made available only to an expert approved by the Applicant.