Process for increasing the yield of Lactococcus lactis subsp, lactis and/or Lactococcus lactis subsp. cremoris bacteria cultures during aerobic fermentation

Abstract

Microbial starter cultures. More specifically, a method for preparing a microbial starter culture wherein the microorganism is inoculated in a culture medium comprising at least one 5 yield enhancing agent selected from the group consisting of a purine base, a pyrimidine base, a nucleoside and a nucleotide. Such microbial starter cultures are useful in the manufacturing of food, feed and pharmaceutical products.

Claims

1. A method for obtaining harvested Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp. cremoris bacteria, comprising: (i) culturing one or more of Lactococcus lactis subsp. lactis bacteria and Lactococcus lactis subsp. cremoris bacteria by aerating the culture in the presence of oxygen at conditions that allow the fermentation to proceed beyond an Optical Density measured at 600 nm (OD.sub.600) of 10, wherein said culture medium comprises: yeast extract; at least one porphyrin compound; and at least one yield-enhancing agent at a concentration that is from 1 mM to 70 mM at the initiation of the fermentation and that is sufficient to yield at least 1 M of said at least one yield-enhancing agent in the bacteria culture at the termination of the fermentation, wherein said at least one yield-enhancing agent is selected from the group consisting of inosine, inosinate (IMP), deoxyinosine, and deoxyinosinate (dIMP), wherein the OD.sub.600 is at least 10 at the termination of the fermentation; and (ii) harvesting said bacteria to obtain harvested Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp. cremoris bacteria.

2. The method according to claim 1, wherein said culture medium comprises at least two yield-enhancing agents.

3. The method according to claim 2, wherein two of said at least two yield-enhancing agents are inosine and IMP.

4. The method according to claim 1, wherein said culture medium initially comprises an amount of said at least one yield-enhancing agent selected from the group consisting of 1 to 60 mM, 1.3 to 60 mM, 1.5 to 50 mM, 2 to 40 mM, 2.5 to 30 mM, 3 to 20 mM, 3 to 15 mM, 4 to 10 mM, and about 7 mM.

5. The method according to claim 1, wherein the OD.sub.600 of the culture medium at the termination of fermentation is selected from the group consisting of OD.sub.600=10 to OD.sub.600=200, OD.sub.600=15 to OD.sub.600=100, and OD.sub.600=20 to OD.sub.600=80.

6. The method according to claim 1, wherein the Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp. cremoris bacteria are Lactococcus lactis subsp. cremoris bacteria.

7. The method according to claim 1, wherein the Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp. cremoris bacteria are Lactococcus lactis subsp. lactis bacteria.

8. The method according to claim 1, further comprising freezing said harvested Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp. cremoris bacteria to obtain frozen Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp. cremoris bacteria.

9. The method according to claim 8, further comprising sublimating water from the frozen Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp. cremoris bacteria to obtain freeze-dried Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp. cremoris bacteria.

10. The method according to claim 8, further comprising packing the frozen Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp. cremoris bacteria.

11. The method according to claim 8, wherein at least one cryoprotectant is added to the harvested Lactococcus lactis subsp. lactis bacteria and/or Lactococcus lactis subsp, cremoris bacteria prior to freezing.

12. The method according to claim 1, wherein the at least one yield-enhancing agent is selected from the group consisting of inosine and inosinate (IMP).

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIG. 1. Shows the yield of three S. thermophilus fermentations performed under anaerobic conditions. The graph shows biomass measured as the OD600 of non-concentrated samples of the fermentation medium as a function of time (hours) in the fermentor. Solid triangles indicate addition of 0.2% w/w IMP, solid spheres indicates addition of 0.2% w/w Inosine and solid squares indicates that no yield enhancing agents were added. 0.2% w/w inosine is approximately 7 mM inosine.

(2) FIG. 2. The level of various nucleo compounds and the Optical density during fermentation in a rich complex medium comprising relatively high amounts of purines. Briefly, a Lactococcus lactis (strain CHCC2862) was grown aerobically in the complex medium containing yeast extract and other complex components. The concentration in M (primary axis) and OD600 (secondary axis) is plotted against time. Abbreviations: G, guanine; A, adenine; Hx, hypoxanthine; X, xanthine; IR, inosine; GR, guanosine; GdR, deoxyguanosine; AR, adenosine.

(3) FIG. 3. The level of purine compounds during fermentation with 2 g/L of inosine added to the medium. Briefly, a Lactococcus lactis (strain CHCC2862) was grown aerobically in the same complex medium as used in FIG. 2 except that 2 g inosine pr. L medium was added. The concentration in M (primary axis) and OD600100 (secondary axis) is plotted against time. Note that the level of inosine is indicated by the secondary axis. Abbreviations: G, guanine; A, adenine; Hx, hypoxanthine; X, xanthine; IR, inosine; GR, guanosine; GdR, deoxyguanosine; AR, adenosine and IR, inosine (note different axis for inosine).

EXAMPLES

Example 1: Yield from Fermentations Performed in Three Different Types of Culture Media

(4) To illustrate the effect of adding extra purine containing compounds to an already enriched and optimized media three different types of industrial scale cultures were compared.

(5) All three types of cultures were performed under aeration and in a nutrient medium, in which at least one porphyrin compound is present or is added as described in international patent application WO 00/05342 (the EMIL procedure), and in all instances it was a so-called O-culture comprising Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. O-cultures are typically used to make cheese without holes (Cheddar, Cheshire, Feta). The particular culture is commercially available under the name R 604 from Chr. Hansen A/S, Hoersholm, Denmark (catalogue no. 200113).

(6) The three media are described in table 1

(7) TABLE-US-00001 TABLE 1 culture basic culture designation medium added yeast extract other additives Old EMIL BD-5-ex3* 1.7% w/w standard yeast extract** New EMIL BD-5-ex3* 1.7% w/w new yeast extract** Super EMIL BD-5-ex3* 1.7% w/w new 0.2% w/w IMP*** yeast extract** 0.2% w/w inosine.sup. *BD-5-ex3 is an optimized porphyrin-containing culture medium according to WO 00/05342 and WO 01/52668. **standard yeast extract and yeast new extract are two commercially available yeast extracts. ***IMP was: inosine-5-monophosphate (IMP) (Alsiano A/S, Birkeroed, DK). .sup.inosine was: inosine (Alsiano A/S, Birkeroed, DK).

(8) The culturing was performed in a 550 L or a 10,000 L industrial fermentation tank at 30 C. using 0.5% (w/w) of the culture mentioned above as inoculum. The fermentations were run under aerobic conditions as described in WO 00/05342. The cultures were allowed to acidify to pH 6.2. The pH was subsequently maintained at 6.2 by controlled addition of 13.4 N NH.sub.4OH.

(9) When no further base consumption was detected, the respective culture was cooled down to about 10 C.

(10) Following cooling, the bacteria in 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

(11) The yields of the fermentations were specified in two different ways. 1) by the obtained biomass measured as the Optical Density at 600 nm (OD.sub.600), 2) by kg of F-DVS culture with an acidification activity of 4.8-5.1 according to the according to Pearce test described in Example 2: Analytical Procedure QAm-043.

(12) The results are shown in table 2 below.

(13) TABLE-US-00002 TABLE 2 Yields of fermentations measured as OD.sub.600 and culture Biomass Yield in kg of F-DVS designation enhancing agent (OD.sub.600) pr 100 L medium* Old EMIL 32 5.56 New EMIL 45 8.33 Super EMIL 0.2% w/w IMP 76 16.67 0.2% w/w inosine *the acidification activity of the F-DVS is 4.8-5.1 according to the to Pearce test

(14) Conclusion:

(15) From these results it is clear that the addition of an enhancing agent composed of 0.2% w/w IMP and 0.2% w/w inosine, which were added to the culture medium before the start of the aerobic fermentation, results in increased yields.

Example 2: Analytical Procedure QAm-043, Acidification ActivityProgrammed Temperature Profile Chr. Hansen A/S (Denmark)

(16) Application

(17) This method is used for determination of acidification activity according to Pearce test. The Pearce test is included by the IDF standard (international dairy standard).

(18) Principle

(19) The acidification is performed according to a temperature profile reflecting the temperature course, which the culture will typically encounter when used in the dairy for production of a given dairy product.

(20) For Pearce test this is the cheese making temperature during the production of Cheddar.

(21) pH is measured at a fixed time.

(22) For cultures where rennet is not added during analysis, a continuous pH measurement may be applied.

(23) Analysing Parameters

(24) Analyzing parameters, which are product specific, are given in LIMS.

(25) Definition of temperature profile (for products where Pearce profile is not used).

(26) Control standard to be used.

(27) Type of pH measurement.

(28) Inoculation percents for sample and control standards.

(29) Dilution milk: 206.9 g cold (4 C.) LAB-milk (i.e. UHT-sterilized reconstituted skimmed milk (RSM) containing 9.5% (w/w) solid matter and heated at 99 C. for 30 minutes).

(30) Activity milk: 200 g cold (4 C.) low pasteurized whole milk 3.5% fat.

(31) Rennet: Naturn standard 190 diluted 1:40 with water.

(32) Apparatus and Reagents

(33) pH meter/pH meter for semi continuously pH measurement eks. Radiometer PHM92.

(34) pH electrode Radiometer PFC2401.

(35) Buffers: pH 7.000.01 and pH 4.010.01.

(36) Water bath with a thermostat programmed for heating according to a predetermined temperature profile 0.2 C.

(37) Temperature sensor.

(38) Balance, precision 0.01 g with minimum two decimals

(39) Watch.

(40) Magnetic stirrer.

(41) Magnets

(42) Beakers, 50 ml.

(43) Small plastic cups.

(44) Rotation apparatus.

(45) Procedure

(46) Preparation of analyze

(47) All bottles should be from the same batch i.e. with the same date.

(48) Water bath/s is/are tempered to the initial temperature of the temperature profile to be used.

(49) Bottles for dilution (=first weighing) and for activity (second weighing) are placed at 4 C. until just before use.

(50) Buffers pH 4.01 and pH 7.00 are placed in water bath at the specified measuring temperature 0.2 C. at least 30 min before calibration of pH meter.

(51) Preparation of samples before analysis.

(52) Frozen cultures:

(53) Frozen samples/control standards are before first weighing placed in a foam box with dry ice and are kept here till all weighings are done.

(54) Frozen cultures, which are thawn before use:

(55) For frozen products, where a whole carton is used, the product is thawn according to current instructions.

(56) After thawing the sample may be kept at 4 C. for max. 30 min, before use.

(57) Freeze dried cultures:

(58) Freeze dried samples and control standards are acclimatized at room temperature for at least 15 min before start of analysis.

(59) Provided that the sample are going to be used for retest the day after, it may be stored at +8 C.

(60) Inoculation procedure

(61) Weighing of product/control standard is carried out directly into the milk.

(62) The actual amount of inoculum (1st weighing) is entered with at least two decimals.

(63) Frozen and thawn products are turned carefully about 4 times, after which the bottle stands for approx. 50 sec.

(64) For freeze dried products the rotation apparatus must be used. It has to be driven with frequent speed for 5 minutes or till the product is completely soluted. This is controlled by leaving the bottle on the table for a moment and then checking the solution by looking in the bottom of the bottle.

(65) Note:

(66) If convenient for the working routine a cold, first weighing can stand at room temperature for max. 15 minutes before second weighing.

(67) 2nd weighing:

(68) The dilution bottle is turned before 2. weighing is carried out.

(69) The actual amount of inoculum (2nd weighing) is entered with at least 2 decimals.

(70) The activity bottle is turned and the inoculation procedure is repeated for samples/control standards.

(71) Activity bottles, which are inoculated from the same 1st weighing, are inoculated in succession.

(72) 2 ml rennet is added each bottle either before or after 2. weighing. After this the bottles are turned so the rennet been distributed.

(73) The bottles are subsequently incubated at one time, as described in 6.

(74) In the end 2 uninoculated milk bottles are placed in a water bath; one for measuring of the water bath temperature and one for measuring pH in the blind milk.

(75) Incubation

(76) Note: When more water baths are required, the control standard with corresponding samples MUST be incubated in the same water bath.

(77) All activity bottles are incubated at the same time in a pre-heated water bath at the defined starting temperature for the temperature profile.

(78) The temperature profile is started at the same time as the bottles are placed in the water bath.

(79) Hereafter the incubation temperature is controlled by a thermostat programmed for following a certain temperature profile. For Pearce test see table 3.

(80) The water level in the water bath should be min. 2 cm higher than the surface of the milk.

(81) TABLE-US-00003 TABLE 3 Temperature program in Pearce profile (following the IDF) Time, minutes Temperature, C. Variation 0 31.0 0.2 C. 50 31.0 0.2 C. 54 31.7 0.5 C. 58 32.2 0.5 C. 62 32.8 0.5 C. 66 33.3 0.5 C. 70 33.9 0.5 C. 73 34.4 0.5 C. 76 35.0 0.5 C. 79 35.6 0.5 C. 82 36.1 0.5 C. 85 36.7 0.5 C. 87.5 37.2 0.5 C. 90 37.8 0.2 C. 360 37.8 0.2 C.

(82) Calibration of pH Electrode

(83) Calibration is carried out at initial temperature according to current instructions regarding electrode calibration and maintenance.

(84) Measurement of pH

(85) After incubation the bottles are shaken well and pH is measured.

(86) The pH measurement is carried out in the bottle or in a sample, which is poured into a 50 ml beaker with magnet stirring.

(87) pH is entered with at least 2 decimals.

(88) Possible remarks on the measurement are entered.

(89) The measuring procedure is continued till all samples/control standards and the uninoculated milk are measured.

(90) Finally pH in buffers are measured and entered.

(91) Continuous pH measurement

(92) The pH values are sampled from the moment, the temperature profile is started. After the incubation is completed, the measured pH values in both buffers at initial temperature are registered.

Example 3: Yield of Lactococcus lactis Fermentations Performed at Standard Anaerobic High-OD Conditions

(93) To illustrate the effect of adding extra purine containing compounds to an already enriched and optimized media two different types of industrial scale fermentation were compared, one with and one without 0.3% w/w inosine added.

(94) Culture:

(95) The present experiment was performed using the commercially available R 604 culture, which is available from Chr. Hansen A/S, Hoersholm, Denmark (catalogue no. 200113).

(96) Fermentation Medium:

(97) The cultures were cultured in a medium having the following composition: Casein hydrolysate (Oxoid, Basingstoke, UK, Product Code L41), 30 g/l; Primatone RL (Quest, Naarden, The Netherlands, Product Code 5X59051), 30 g/l; soya peptone (Oxoid, Basingstoke, UK, Product Code L44), 30 g/l; yeast extract (Oxoid, Basingstoke, UK, Product Code L21), 15 g/l; MgSO4, 1.5 g/l; Na-ascorbate, 3 g/l; and lactose 50 g/l.

(98) The medium was sterilized by UHT-treatment (143 C. for 8 sec.). The finished medium had a pH of 6.5.

(99) Fermentation Condition the Cultures:

(100) The fermentation was performed in a 550 L industrial fermentation tank without aeration at 30 C. using 1% (w/w) of the culture mentioned above as inoculum. At high OD conditions the fermentation is essentially anaerobic. The cultures were allowed to acidify to pH 6.0. The pH was subsequently maintained at 6.0 by controlled addition of 13.4 N NH.sub.4OH.

(101) When no further base consumption was detected, the respective culture was cooled down to about 10 C.

(102) Following cooling, the bacteria in 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

(103) The yields of the fermentations were specified in two different ways: 1) by the obtained biomass measured as the Optical Density at 600 nm (OD600), or 2) by kg of F-DVS culture pr. 100 L fermentation medium wherein the F-DVS culture have an acidification activity of 4.8-5.1 according to the Pearce test described in Example 2: Analytical Procedure QAm-043.

(104) The results are shown in the table 4 below.

(105) TABLE-US-00004 TABLE 4 culture extra additive to Yield as Yield according to designation fermentation medium OD600 Pearce.sup. PP11145 nothing 25.1 4.95 PP11146 0.3% w/w inosine.sup.@ 25.2 4.98 .sup.@inosine was: inosine (Alsiano A/S, Birkeroed, DK). .sup.see example 2

(106) Conclusion:

(107) From these results it was clear that the addition of an enhancing agent composed of 0.3% w/w inosine, which were added to the anaerobic culture medium before the start of the fermentation, did not result in increased yields.

Example 4: Yield of Streptococcus thermophilus Fermentations Performed at Standard Anaerobic High-OD Conditions

(108) This experiment was performed to investigate the effect of adding extra purine containing compounds to an already enriched and optimized media to be used for anaerobic fermentation at high OD-conditions. In the present experiment three cultures of Streptococcus thermophilus were prepared, one with 0.2% w/w inosine added, one with 0.2% w/w IMP added and one culture where no extra purine containing compounds were added to the medium.

(109) Culture:

(110) The present experiment was performed using the commercially available Streptococcus thermophilus culture CHQ-18, which is available from Chr. Hansen A/S, Hoersholm, Denmark.

(111) Fermentation Medium:

(112) The cultures were cultured in a rich medium based on complex medium components, BioSpringer yeast extract 207, Arla skim milk powder (Milex 240), and lactose.

(113) The medium was sterilized by UHT-treatment (143 C. for 8 sec.). The finished medium had a pH of 6.0.

(114) Fermentation Condition of the Cultures:

(115) The fermentation was performed in a 3 L agitated fermentation tank at 40 C. using 0.1% (w/w) of the culture mentioned above as inoculum. The pH was maintained at 6.0 by addition of 13.4 N NH.sub.4OH. Anaerobic conditions were ensured by flushing with nitrogen in head space (1.5 l/min). Agitation was 300 rpm.

(116) The yields of the fermentations were specified by the obtained biomass measured as the Optical Density at 600 nm (OD600) of unconcentrated samples sampled during the fermentation.

(117) The result of the three fermentations is shown in FIG. 1 below.

(118) Conclusion:

(119) From these results it appears that the addition of an enhancing agent composed of either 0.2% w/w inosine or 0.2% w/w IMP did not result in increased yields.

Example 5: Use of Chemical Analysis for Detection of the Presence of Excess Nucleo Compounds in a Fermentation

(120) Even though lactic acid bacteria in general are prototrophic for purine and pyrimidines, and can thus synthesize these compounds, the cells utilize available exogenous purine and pyrimidine sources during fermentation. Specifically, all common purine nucleo compounds may be completely depleted already around OD 15 (see FIG. 2). Interestingly, the biomass accumulation continues to around OD 45 even though the purine compound is depleted. A similar result is found for the pyrimidine compounds (data not shown). This means that the fermentation broth (i.e. fermentate without cells) at the end of growth is devoid of purine and pyrimidine compounds.

(121) If on the other hand for example inosine is added in excess to the growth medium this compound, and/or the corresponding nucleo base hypoxanthine (present due to hydrolysis of the inosine), will be present in the fermentation broth at the end of growth (see FIG. 3). Such excess nucleo compounds may easily be detected in the fermentation broth.

(122) For the production of for example F-DVS the cells in the fermentate are concentrated several-fold. Although, the cells are present in a several-fold higher level in the F-DVS than in the fermentate substantial amounts of fermentation broth is still present in the F-DVS. This pure fermentation broth can be obtained through further concentrating the cells. Useful methods for isolating the broth may be through defrosting the F-DVS and then use a filter or by using a higher g-force during centrifugation than was used through production of the F-DVS.

(123) With just small amounts of pure broth available it can be tested if any nucleo compounds were present in the fermentate, and thus is such compounds had been added in excess to the fermentation medium. Such detection method may be conventional HPLC (see for example http://www.laubscherlabs.com/Presentation/YMC%20ODS-AQ.pdf) where the common nucleo bases and nucleosides (cytosine, cytidine, uracil, deoxycytidine, guanine, adenine, hypoxanthine, uridine, xanthine, thymine, inosine, guanosine, deoxyinosine, deoxyguanosine, xanthosine, thymidine, adenosine, and deoxyadenosine) can be conveniently detected. Other available methods may be used for the detection of the corresponding nucleotides. The presence of either of these compounds in the broth will strongly indicate that the fermentation has been performed according to the present invention.

Example 6: Use of Proteomics for Detection of the Presence of Excess Nucleo Compounds in a Fermentation

(124) During growth the cells require a continuous flow of purine and pyrimidine nucleotides for synthesis of RNA and DNA. These nucleotides can either be supplied exogenously from the medium (salvage) or be synthesized de novo from simpler compounds. For the de novo synthesis the specific de novo synthesis genes must be expressed. Conversely, the genes do not have to be expressed when an exogenous source is present.

(125) In the case of purine de novo synthesis around 10 gene-products are involved. It has previously been found that the purDEK operon involved in the purine de novo synthesis in L. lactis is about 35-fold regulated depending on the presence/absence of an exogenous purine source (Nilsson & Kilstrup 1998). Also, the presence/absence of several purine de novo synthesis proteins on 2D protein gels has been found to depend on the availability of exogenous purines (Gitton et al. 2005).

(126) To set up a specific method for detection of the presence/absence of exogenous purines in the medium we inoculated L. lactis subsp. lactis CHCC2862 into defined SA medium with 1% glucose. This medium is devoid of nucleo compounds (Jensen & Hammer 1993). Cultures were set up at 30 C. with and without 0.2% inosine and incubated overnight. Exponentially growing cells were then inoculated into fresh medium at an OD600 of around 0.1. At OD 0.8 (exponential growth) and in stationary phase cells were harvested and 2D protein gels were produced.

(127) In general around 3-400 protein spots were detectable on the gels with the pH range 4-7. For OD 0.8 there were less than 10 spots that were present from the culture without inosine, but absent (or very weak) from the culture with inosine. The presence/absence pattern of these spots indicates that the respective proteins are only present when there is no exogenous purine source. Four of the strongest spots present only on the gel produced from the culture without purines were subjected to in-gel digestion and mass spectrometry identification. The proteins were identified as: purH (bifunctional purine biosynthesis protein), purM (phosphoribosylaminoimidazole synthase), yphF (phosphoribosylformylglycinamidine synthase PurS), and fhs (formyltetrahydrofolate synthetase). The gene-products of the three genes purH, purM and yphF (purS) are all directly involved in the purine de novo biosynthesis, whereas fhs is involved in the formation of one-carbon units, which are used for purine de novo synthesis. For the cells in stationary phase, which is similar to the situation found in F-DVS, a similar absence/presence pattern was obtained. Overall, this shows that the presence of excess nucleo compounds in a medium can be detected using proteomics.

(128) Whereas the present examples illustrate the detection of yield enhancing purines a similar detection of the presence of an excess of pyrimidines can easily be set up. Thus proteomics can be used to provide very strong indications of a fermentation that has been performed according to the present invention.

(129) Materials & Methods

(130) The methods described below are based on standard methods published previously (Fey at al. 1998; Vido et al. 2004; Gitton et al. 2005).

(131) Preparation of cell free extract. Cells were harvested by centrifugation and washed twice in ice-cold 10 mM Tris-HCl, pH 7, 0.25 M sucrose. Cells were transferred to a 2 mL Eppendorf tube containing approx. 1.0 g glass beads (0.25-0.50 mm) and shaken in a mixer mill for two times 6 min. Subsequently, the extracts were centrifuged at 10,000 rpm for 5 min and 250-300 L supernatant was transferred to a new Eppendorf tube. The supernatant was centrifuged once more at 15,000 rpm for 5 min and all except 10-20 l from the bottom of the tube was transferred to yet a new Eppendorf tube. DTT was added from a 1 M stock solution to a final concentration of 10 mM and the lysate stored frozen at 20 C.

(132) 2D gel electrophoresis (isoelectric focusing and gel electrophoresis). For each gel between 75 and up to 300 g of protein was precipitated by a chloroform/methanol procedure and resuspended in 190 l rehydration buffer (8 M urea, 50 mM DTT, 4% CHAPS, 0.2% carrier ampholytes). First dimension was run on 11 cm IPG strips pH 4-7 and pH 4.7-5.9 from Bio-Rad with active rehydration for 12 hours followed by the standard program for 11 cm strips on a Protean IEF cell from Bio-Rad. For strips of pH 3.9-5.1, the proteins were cup loaded after the strips were rehydrated. After IEF electrophoresis the strips were either stored frozen at 20 C. or directly prepared for electrophoresis in the second dimension.

(133) The strips were equilibrated in SDS buffer prior to second dimension PAGE for 215 min, first in the presence of DTT, second in excess of IAA. Thereafter the strips were attached to 10-20% and 12.5% polyacrylamide gels (Criterion Tris-HCl from Bio-Rad) by agarose sealing, and the second dimension was run at 200V for one hour on a Criterion Dodeca Cell. The gels were stained in BioSafe Coomassie and scanned on a densitometer GS-800 from Bio-Rad.

(134) Identification of proteins. The identity of proteins in chosen spots was determined by in-gel digestion and analysis of peptide profile and amino acid content using mass spectrometry. The generated data were subsequently used for searching in public databases for proteins with similar properties (Alphalyse A/S, Odense, Denmark)

REFERENCES

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