Method to improve Lactococcus preservation
09790510 · 2017-10-17
Assignee
Inventors
Cpc classification
C12N1/04
CHEMISTRY; METALLURGY
International classification
Abstract
Lactococcus lactis strains with improved preservation characteristics, and improved acid and bile salt tolerance are disclosed. More specifically, a L. lactis strain having a heterologous trehalose-6-phosphate synthase gene and/or a trehalose-6-phosphate phosphatase gene, results in an accumulation of trehalose in the cytoplasm and/or in the cytoplasmic membrane. A L. lactis strain, having an internal trehalose concentration of at least 10 mg per gram cells (w/w) is disclosed.
Claims
1. An isolated Lactococcus lactis strain comprising an internal trehalose concentration of at least 10 mg per gram wet weight of the L. lactis cells, wherein the strain expresses a heterologous trehalose-6-phosphate phosphatase gene, and wherein trehalose-6-phosphate phosphorylase activity is inactivated in the L. lactis cells.
2. The isolated Lactococcus lactis strain according to claim 1, further comprising a heterologous trehalose-6-phosphate synthase gene.
3. The isolated Lactococcus lactis strain according to claim 1, wherein said heterologous trehalose-6-phosphate phosphatase gene is situated on a self-replicating plasmid.
4. The isolated Lactococcus lactis strain according to claim 1, wherein said heterologous trehalose-6-phosphate phosphatase gene is integrated into the bacterial genome.
5. The isolated Lactococcus lactis strain according to claim 1, wherein said heterologous trehalose-6-phosphate phosphatase gene is operably linked to an inducible promoter.
6. The isolated Lactococcus lactis strain according to claim 1, wherein said heterologous trehalose-6-phosphate phosphatase gene is operably linked to a constitutive promoter.
7. The isolated Lactococcus lactis strain according to claim 1, wherein said heterologous trehalose-6-phosphate phosphatase gene is operably linked to an inducible Lactococcus lactis nisin promoter.
8. The isolated Lactococcus lactis strain according to claim 1, wherein said trehalose-6-phosphate phosphatase gene is under the control of a Lactococcus lactis P1 promoter.
9. The isolated Lactococcus lactis strain according to claim 1, wherein said heterologous trehalose-6-phosphate phosphatase gene is the Escherichia coli otsB gene.
10. The isolated Lactococcus lactis strain according to claim 2, wherein said heterologous trehalose-6-phosphate synthase gene is the Escherichia coli otsA gene.
11. The isolated Lactococcus lactis strain according to claim 1, comprising an internal trehalose concentration of 10-50 mg per gram wet weight of the L. lactis cells.
12. The isolated Lactococcus lactis strain according to claim 1, wherein said Lactococcus lactis strain has improved storage characteristics compared to a Lactococcus lactis strain comprising an internal trehalose concentration of less than 10 mg per gram wet weight of the L. lactis cells.
13. The isolated Lactococcus lactis strain according to claim 1, wherein said Lactococcus lactis strain has improved tolerance to freeze drying compared to a Lactococcus lactis strain comprising an internal trehalose concentration of less than 10 mg per gram wet weight of the L. lactis cells.
14. The isolated Lactococcus lactis strain according to claim 1, wherein said Lactococcus lactis strain has improved tolerance to acidic conditions compared to a Lactococcus lactis strain comprising an internal trehalose concentration of less than 10 mg per gram wet weight of the L. lactis cells.
15. The isolated Lactococcus lactis strain according to claim 14, wherein said Lactococcus lactis strain has improved tolerance to human gastric juice.
16. The isolated Lactococcus lactis strain according to claim 1, wherein said Lactococcus lactis strain has improved tolerance to bile salts compared to a Lactococcus lactis strain comprising an internal trehalose concentration of less than 10 mg per gram wet weight of the L. lactis cells.
17. The isolated Lactococcus lactis strain according to claim 1, wherein said Lactococcus lactis strain has an improved production capacity for producing a heterologous biologically active polypeptide per colony forming unit (cfu) compared to a Lactococcus lactis strain comprising an internal trehalose concentration of less than 10 mg per gram wet weight of the L. lactis cells.
18. The isolated Lactococcus lactis strain according to claim 1, wherein said internal trehalose concentration is measured using a method comprising converting said trehalose to glucose, and measuring said glucose.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1: Cloning of the Trehalose-Biosynthesis Genes from Escherichia coli onto the Lactococcal Expression Plasmid pNZ8048
(12) The DNA sequences encoding the trehalose-biosynthesis genes in Escherichia coli are retrieved from GenBank (Acc. Nr. X69160) (Kaasen et al. 1994).
(13) Escherichia coli strain DH5α (Woodcock et al., 1989) serves as the source of the trehalose biosynthesis genes, otsA and otsB, encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. Genomic DNA is purified from 10.sup.9 cells with the commercially available Qiagen DNeasy kit (Qiagen, Hilden, Germany) according to the supplier's protocol.
(14) The DNA sequence encompassing otsB-otsA is PCR-amplified with Vent® DNA polymerase (New England Biolabs, Beverly, Mass., USA) and the following primer sequences:
(15) forward primer: 5′-GCCCATGGGTGACAGAACCGTTAACCGAAACC-3′, in which GTG is the initiator codon of the otsB cistron and the CCATGG sequence is a NcoI restriction site;
(16) reverse primer: 5′-GCTCTAGACTACGCAAGCTTTGGAAAGGTAGC-3′, in which CTA is the complement of the TAG stop codon of the otsA cistron and the TCTAGA sequence is a XbaI restriction site.
(17) The amplified 2216 bp DNA fragment is digested with NcoI and XbaI, ligated into the NcoI-XbaI opened pNZ8048 vector (Ruyter et al., 1996) (
(18) Step 1.
(19) Forward primer: 5′-GGCACTCACCATGACAGAACCGTTAACC-3′
(20) Reverse primer: 5′-GCTCTAGACTACGCAAGCTTTGGAAAGGTAGC-3′, in which CTA is the complement of the TAG stop codon of the otsA cistron and the TCTAGA sequence is a XbaI restriction site.
(21) The amplified 2216 bp DNA fragment encompasses the otsB-otsA coding region.
(22) Step 2.
(23) Forward primer: 5′-GCGTCGACGGCAATAGTTACCCTTATTATCAAG-3′, in which GTCGAC coincides with the SalI restriction site in pNZEcTre0
(24) Reverse primer: 5′-GGTTAACGGTTCTGTCATGGTGAGTGCC-3′, in which CAT is the complement of the initiator codon preceding otsB.
(25) The amplified 1256 bp DNA fragment encompasses the chloramphenicol resistance gene, the P.sub.nis promoter and the nisA ribosome-binding site and ATG initiator codon fused to the coding region of otsB.
(26) Step 3.
(27) The 2216 bp DNA fragment from step1 and the 1256 bp DNA fragment from step 2 are mixed in equimolar amounts and subjected to PCR amplification with Vent® DNA polymerase, using 5′-GCGTCGACGGCAATAGTTACCCTTATTATCAAG-3′ and 5′-GCTCTAGACTACGCAAGCTTTGGAAAGGTAGC-3′ as forward and reverse primers, respectively. The amplified 3444 bp DNA fragment is digested with SalI and XbaI and ligated to a SalI-XbaI fragment, carrying the replicon of pNZ8048. Transformants are obtained in NZ9000 and their plasmids isolated as described above.
(28) A representative plasmid, whose structure can be identified by restriction enzyme analysis with SalI, XbaI, BglII, NcoI and combinations thereof, is designated pNZEcTre1 (
Example 2: Induction of the Cloned Trehalose-Biosynthesis Operon in L. lactis
(29) The strains L. lactis NZ9000 [pNZEcTre1] and L. lactis NZ9000 are grown as standing cultures at 30° C. overnight in M17 medium (Difco, Detroit, USA) supplemented with 0.5% glucose (=GM17 medium). The cultures are diluted 100-fold in fresh medium and incubated for another 3 hr at 30° C. The cells are collected by centrifugation and resuspended in the original volume of BM9G medium (M9 medium buffered at pH 8.5 and containing 0.5% glucose; Schotte et al., 2000). Nisin (Aplin&Barrett) is added to a final concentration of 0.4 μg/ml and the cultures are further incubated for up to 48 hr. At several time points samples are taken, the cells collected by centrifugation and lysed by addition of lysozyme (5 mg/ml) and mutanolysin (100 U/ml). SDS-PAGE reveals the nisin-dependent induction (Kuipers et al., 1998) of two additional protein bands in L. lactis NZ9000[pNZEcTre1]. Their molecular mass of 53.6 kDa and 29.1 kDa agree with the molecular mass of the E. coli trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB), respectively (
Example 3: Optimized Induction Protocol for Trehalose Biosynthesis in L. lactis
(30) Strain NZ9000[pNZEcTre1] is grown to saturation as standing culture at 30° C. in GM17Cm (=GM17 containing 5 μg chloramphenicol per ml) and diluted 3-fold into fresh medium containing 0.4 μg nisin/ml. Incubation is continued at 30° C. with shaking at 200 rpm for 8 hr. The growth rate of the culture is unaffected by the addition of nisin. Saturation is reached after 3 hr of incubation. Induction of OtsA and OtsB can be clearly identified by SDS-PAGE.
(31) Concentration of trehalose is determined by converting trehalose to glucose with trehalase (courtesy of J. Thevelein, Dept. of Molecular Microbiology, VIB-K.U.Leuven, Belgium), which is measured by a glucose assay protocol (Trinder, 1969).
(32) The cells are lysed with lysozyme and mutanolysin by incubation in 0.25 M Na.sub.2CO.sub.3 for one hr at 37 C and 20 min at 95 C. Cell debris is removed by centrifugation at 13,200 rpm. To one volume of supernatant, 0.5 volume of 1 M HAc and 0.5 volume of a buffer, consisting of 300 mM NaAc and 30 mM CaCl.sub.2 pH 5.5, are added. The mixture is incubated for 2 hr at 37 C in the presence of trehalase. Following centrifugation at 13,200 rpm, the supernatant is supplemented with Trinder reagent (glucose oxidase, phenol and 4-aminophenazone; Dialab, Austria) and incubated with shaking for 15 min at 30 C, after which the OD.sub.505 is automatically recorded in a 96-well VersaMax tunable microplate reader (Molecular Devices, USA)
(33) Experimental trehalose concentrations are read from a calibration curve, obtained with pure trehalose (Sigma-Aldrich Corp. St. Louis, USA), showing a linear correlation between the OD.sub.505 value and trehalose concentration up to 10 mM trehalose. The accumulation of trehalose in NZ9000[pNZEcTre1] nisin-induced as described above is shown in
Example 4: Freeze-Drying of L. lactis Cultures and Storage Conditions
(34) Strain NZ9000[pNZEcTre1] is grown to saturation as standing culture at 30° C. in GM17Cm and diluted 3-fold into fresh medium with or without nisin (0.4 μg/ml). Incubation is continued at 30° C. with shaking at 200 rpm for 3 hr. The cells are collected by centrifugation at 5000 rpm, resuspended in the original volume 10% (w/v) skim milk (Difco, Becton Dickinson) and kept on ice till ready for freeze-drying.
(35) All freeze-drying runs were performed in triplicate. A sample containing approximately 2 g of cells (wet weight) is filled in sterile vials (glass type 1, Gaash Packaging, Mollem, Belgium). The vials are covered with a lyophilisation stopper (V9032 FM 257/2 SAF1, Bromobutyl with magnesium silicate as filler, Helvoet Pharma, Alken, Belgium). The vials are loaded in the pre-cooled production chamber (−25° C.) of the freeze-dryer (Leybold GT4, Finn-aqua, Santasalo, Sohlberg, Germany) before freezing to −45° C. over a period of 105 min at 1000 mbar. The primary drying (12 hr) is performed at −15° C. and 0.8 to 1 mbar; the secondary drying (9 hr) at 10° C. and 0.1 to 0.2 mbar. After lyophilisation, the vials are closed under vacuum.
(36) The vials are stored at different conditions: (a) 8° C. and 10% relative humidity (RH), (b) 8° C. and 60% RH, (c) 20° C. and 10% RH.
(37) The water content of the freeze-dried culture is determined using a Mettler DL35 Karl Fisher titrator (Mettler-Toledo, Beersel, Belgium). The samples are stirred in the reaction medium for 60 s. Afterwards the water is titrated with Hydranal® Composite 5 (Riedel-de Haën, Seelze, Germany). The analysis is performed in triplicate.
Example 5: Viability of Freeze-Dried Samples of Induced and Non-Induced Cultures, Under Different Storage Conditions
(38) To determine the viability in the freeze-dried powder, 0.1 g powder is dissolved in 1 ml sterile water. Viability of the bacteria is determined by following the growth in a Bioscreen (Labsystems). To this end serial dilutions of the cultures are made, inoculated 1/100 in fresh GM17C and loaded in triplicate into the wells of the Bioscreen. OD.sub.600 values are automatically recorded at given intervals over a 21 hr period. The time necessary to reach an optical density at 600 nm (OD.sub.600) half way the minimum and maximum OD.sub.600 (50% time) is calculated based on the exponential growth phase. This 50% time is plotted against the natural logarithm of the viability and the equation of the standard curve is calculated. The viability of a freeze-dried sample is determined based on the standard curve of the starting culture and expressed as % of theoretical. The viability values of the samples as determined by this method corresponds very well with the results obtained by plating and colony counting.
(39) Table 1 summarizes the measured viability of induced (trehalose containing) or non-induced (trehalose free) NZ9000[pNZEcTre1] cultures, freeze-dried and stored under different conditions of temperature and relative humidity. Viability is expressed as percentage of the viability of the respective culture prior to the freeze-drying step.
Example 6: Internal Trehalose Accumulation Protects L. lactis Against Oxgall Challenge
(40) A saturated overnight culture of L. lactis NZ9000 [pNZEcTre1] was induced by nisin (0.4 μg/ml) for 3 hrs, at 30° C. and 200 rpm in a shacking water bath. The same culture, without nisin addition, was used as control. The saturated culture was centrifuged and resuspended in sterile double distilled water, with different fysiological concentrations of oxgall. The suspensions were incubated for a total of 4 hours, at 37° C., and samples were taken after 0, 0.5, 2 and 4 hours of incubation. The samples were plated on GM17Cm and the plates were incubated for 24 hours at 30° C. The results were expressed in colony forming units (cfu; table 2) or as percentage viable colonies, relative to the initial amount of cfu's (table 3). A graphical representation of the results after 0.5 hours of oxgall challenge is shown in
(41) The results are even more pronounced if the culture is freeze dried before applying the oxgall stress. A saturated overnight culture of L. lactis NZ9000 [pNZEcTre1] was induced by nisin for 3 hrs, at 30° C. and 200 rpm in a shacking water bath. The same culture, without nisin addition, was used as control. Both sets of culture was freeze dried, and after freeze drying, the powder was dissolved in sterile double distilled water and different fysiological concentrations of oxgall were added. The suspensions were incubated for a total of 4 hours, at 37° C., and samples were taken after 0, 0.5, 2 and 4 hours of incubation. The samples were plated on GM17Cm and the plates were incubated for 24 hours at 30° C. The results were expressed in colony forming units (cfu; table 4) or as percentage viable colonies, relative to the initial amount of cfu's (table 5).
(42) The trehalose accumulation in the nisin induced cultures was 58 mg/g ww of cells. Cells with internal trehalose accumulation do maintain their viability better, both after lyophilisation and upon oxgall challenge (Table 4 and 5).
Example 7: Internal Trehalose Accumulation Protects L. lactis Against High Acidity in the Medium
(43) A saturated overnight culture of L. lactis NZ9000 [pNZEcTre1] was induced by nisin (0.4 μg/ml) for 3 hrs, at 30° C. and 200 rpm in a shacking water bath. The same culture, without nisin addition, was used as control. Both sets of culture was freeze dried, and after freeze drying, the powder was dissolved in sterile double distilled water and different concentrations of human gastric juice were added (post-operative, pH 2.95)
(44) The suspensions were incubated for a total of 2 hours, at 37° C., and samples were taken after 0, 0.5, 1 and 2 hours of incubation. The samples were plated on GM17Cm and the plates were incubated for 24 hours at 30° C. The results were expressed in colony forming units (cfu; table 6) or as percentage viable colonies, relative to the initial amount of cfu's (table 7). A graphic representation of the relative results (% cfu after the treatment calculated on initial cfu) after 0.5 hour is given in
Example 8: Internal Trehalose Insures a Higher Productivity of a Prophylactic and/or Therapeutic Molecule after Freeze Drying
(45) Construction of pT1hIL10aPxA
(46) The build up of plasmid pT1hIL10aPxA is analogous to the plasmid, containing murine IL-10 (Schotte et al., 2000) It contains the hIL-10 gene fused to the usp45 secretion leader, preceded by the coliphage T7 gene 10 ribosome binding site and the P1 promoter. The sequence of the IL-10 gene is a synthetic one in which codon usage was adapted to the preferred codon usage in L lactis and in which the proline residue—the first amino acid of the mature protein in native human IL-10—was replaced by an alanine residue. The plasmid was transformed in L. lactis strain MG1363, according to Wells et al., 1993.
(47) Construction of pNZEcTre1-hIL10aPxA
(48) The plasmid pNZEcTre1-hIL10aPxA is obtained by PCR amplification with Vent® DNA polymerase (NEB) of the hIL-10 expression cassette from the plasmid pT1hIL10aPxA and the following primer sequences: 5′-GCACTAGTGAATTCGATTAAGTCATCTTACC-3′ and 5′-CGACTAGTTAGTTTCGTATCTTCATTGTCATGTAG-3′, in which ACTAGT is a SpeI restriction site. The amplified 796 bp DNA fragment is digested with SpeI, ligated into the XbaI opened pNZEcTre1 plasmid and transformed by electroporation into L. lactis strain NZ9000. Transformants are obtained as described by Wells et al (1993). The direction of the cloned hIL-10 expression cassette is sequence-verified (
(49) A saturated overnight culture of L. lactis NZ9000 [pNZEcTre1-hIL10aPxA] was induced with nisin (0.4 μg/ml), for 3 hours at 30° C., 200 rpm in an orbital shaker. The same culture without nisin was used as control, as well as a non-induced culture of L. lactis MG1363 [pT1hIL10aPxA]. The cultures were freeze dried as described in example 4. After freeze drying, the powder was redissolved in the original volume of 50 mM CO.sub.3.sup.2−, comprising 0.5% glucose. This solution was incubated at 37° C. Samples were taken after 0, 2, 4 and 6 hours, and the amount of hIL-10 was determined by an ELISA test (Maxisorp F96 plates (Nunc) were coated overnight with rat anti-human IL-10 antibody (BD). The plates were blocked with 0.1% casein solution for 2 hours. Serial dilutions of recombinant hIL-10 standard (BD) and supernatants were loaded on the plates. The bound hIL-10 was detected with biotinylated rat anti-human IL-10 (BD) and horseradish peroxidase coupled streptavidin (BD). The plates were developed with TMB substrate (BD). The reaction was stopped after 30 minutes with 1 M H.sub.2SO.sub.4. The absorbance was measured at 450 nm with 595 nm as reference wavelength.) as well as the cfu by plating. The results of the hIL-10 production in function of the culture volume and the number of cfu, after 8 hours of incubation are shown in
(50) Tables
(51) TABLE-US-00001 TABLE 1 Percentage survival of NZ9000[pNZEcTre1] following freeze-drying Storage condition Storage period Induced Non-induced directly after freeze-drying 96% 47% 8 C. - 10% RH 1 week.sup. 100% 49% 4 weeks 109% 37% 8 C. - 60% RH 1 week.sup. 103% 15% 4 weeks 86% 6% 20 C. - 10% RH 1 week.sup. 67% 19% 4 weeks 46% 8%
(52) TABLE-US-00002 TABLE 2 Effect of trehalose accumulation in nisin induced cultures on survival in different concentrations of oxgall, expressed as colony forming units after 0 h after 0.5 h after 2 h after 4 h cfu/ml −nisin +nisin −nisin +nisin −nisin +nisin −nisin +nisin before 2.68 × 10.sup.9 1.26 × 10.sup.9 2.68 × 10.sup.9 1.26 × 10.sup.9 2.68 × 10.sup.9 1.26 × 10.sup.9 2.68 × 10.sup.9 1.26 × 10.sup.9 A.C. after 3.16 × 10.sup.9 1.57 × 10.sup.9 3.16 × 10.sup.9 1.57 × 10.sup.9 3.16 × 10.sup.9 1.57 × 10.sup.9 3.16 × 10.sup.9 1.57 × 10.sup.9 A.C. 0% 3.16 × 10.sup.9 1.57 × 10.sup.9 3.44 × 10.sup.9 1.56 × 10.sup.9 3.29 × 10.sup.9 1.48 × 10.sup.9 2.84 × 10.sup.9 1.36 × 10.sup.9 oxgall 0.13% 3.38 × 10.sup.9 1.61 × 10.sup.9 1.12 × 10.sup.9 1.07 × 10.sup.9 2.87 × 10.sup.6 4.10 × 10.sup.7 6.00 × 10.sup.4 1.48 × 10.sup.7 oxgall 0.33% 3.43 × 10.sup.9 1.30 × 10.sup.9 1.12 × 10.sup.7 1.44 × 10.sup.7 3.00 × 10.sup.4 7.00 × 10.sup.4 <1.00 × 10.sup.3 <1.00 × 10.sup.3 oxgall 0.67% 3.28 × 10.sup.9 1.41 × 10.sup.9 3.51 × 10.sup.7 5.38 × 10.sup.7 5.06 × 10.sup.6 2.05 × 10.sup.5 <1.00 × 10.sup.3 <1.00 × 10.sup.3 oxgall
(53) TABLE-US-00003 TABLE 3 Effect of trehalose accumulation in nisin induced cultures on survival in different concentrations of oxgall, expressed as percentage of the initial concentration after 0 h after 0.5 h after 2 h after 4 h % −nisin +nisin −nisin +nisin −nisin +nisin −nisin +nisin before 100% 100% 100% 100% 100% 100% 100% 100% A.C. after 118% 124% 118% 124% 118% 124% 118% 124% A.C. 0% 118% 124% 128% 123% 123% 118% 106% 108% oxgall 0.13% 126% 128% 42% 85% 0% 3% 0% 1% oxgall 0.33% 128% 103% 0% 1% 0% 0% 0% 0% oxgall 0.67% 122% 112% 1% 4% 0% 0% 0% 0% oxgall
(54) TABLE-US-00004 TABLE 4 Effect of trehalose accumulation in nisin induced cultures on survival of freeze dried cultures in different concentrations of oxgall, expressed as colony forming units after 0 h after 0.5 h after 2 h after 4 h cfu/ml −nisin +nisin −nisin +nisin −nisin +nisin −nisin +nisin before lyo 3.33 × 10.sup.9 1.33 × 10.sup.9 3.33 × 10.sup.9 1.33 × 10.sup.9 3.33 × 10.sup.9 1.33 × 10.sup.9 3.33 × 10.sup.9 1.33 × 10.sup.9 after lyo 2.02 × 10.sup.9 1.28 × 10.sup.9 2.02 × 10.sup.9 1.28 × 10.sup.9 2.02 × 10.sup.9 1.28 × 10.sup.9 2.02 × 10.sup.9 1.28 × 10.sup.9 0% oxgall 2.02 × 10.sup.9 1.28 × 10.sup.9 2.16 × 10.sup.9 1.23 × 10.sup.9 1.72 × 10.sup.9 1.20 × 10.sup.9 1.73 × 10.sup.9 1.10 × 10.sup.9 0.13% oxgall 1.71 × 10.sup.9 1.13 × 10.sup.9 2.04 × 10.sup.9 1.09 × 10.sup.9 1.59 × 10.sup.9 1.21 × 10.sup.9 1.65 × 10.sup.9 1.07 × 10.sup.9 0.33% oxgall 1.93 × 10.sup.9 1.11 × 10.sup.9 1.54 × 10.sup.9 1.08 × 10.sup.9 1.31 × 10.sup.9 1.06 × 10.sup.9 9.15 × 10.sup.8 9.05 × 10.sup.8 0.67% oxgall 1.29 × 10.sup.9 1.07 × 10.sup.9 1.14 × 10.sup.9 1.11 × 10.sup.9 1.04 × 10.sup.9 7.53 × 10.sup.8 8.35 × 10.sup.8 8.41 × 10.sup.8
(55) TABLE-US-00005 TABLE 5 Effect of trehalose accumulation in nisin induced cultures on survival of freeze dried cultures in different concentrations of oxgall, expressed as percentage of the initial concentration after 0 h after 0.5 h after 2 h after 4 h % −nisin +nisin −nisin +nisin −nisin +nisin −nisin +nisin before lyo 100% 100% 100% 100% 100% 100% 100% 100% After Lyo 61% 96% 61% 96% 61% 96% 61% 96% 0% oxgall 61% 96% 65% 92% 52% 90% 52% 83% 0.13% oxgall 51% 85% 61% 82% 48% 91% 50% 80% 0.33% oxgall 58% 83% 46% 81% 39% 79% 27% 68% 0.67% oxgall 39% 81% 34% 84% 31% 57% 25% 63%
(56) TABLE-US-00006 TABLE 6 Effect of trehalose accumulation in nisin induced cultures on survival of freeze dried cultures in different concentrations of gastric juice, expressed as colony forming units after 0 h after 0.5 h after 1 h after 2 h cfu/ml −nisin +nisin −nisin +nisin −nisin +nisin −nisin +nisin before lyo 2.80 × 10.sup.9 1.20 × 10.sup.9 2.80 × 10.sup.9 1.20 × 10.sup.9 2.80 × 10.sup.9 1.20 × 10.sup.9 2.80 × 10.sup.9 1.20 × 10.sup.9 after lyo 2.05 × 10.sup.9 1.17 × 10.sup.9 2.05 × 10.sup.9 1.17 × 10.sup.9 2.05 × 10.sup.9 1.17 × 10.sup.9 2.05 × 10.sup.9 1.17 × 10.sup.9 0% gastric juice 2.05 × 10.sup.9 1.17 × 10.sup.9 1.82 × 10.sup.9 1.25 × 10.sup.9 1.75 × 10.sup.9 1.38 × 10.sup.9 1.86 × 10.sup.9 1.20 × 10.sup.9 25% gastric juice 2.06 × 10.sup.9 8.54 × 10.sup.8 1.55 × 10.sup.8 7.14 × 10.sup.8 1.03 × 10.sup.8 6.77 × 10.sup.7 6.07 × 10.sup.7 1.58 × 10.sup.7 50% gastric juice 1.63 × 10.sup.9 8.33 × 10.sup.8 1.06 × 10.sup.8 5.27 × 10.sup.8 5.81 × 10.sup.7 3.61 × 10.sup.7 3.13 × 10.sup.7 1.29 × 10.sup.7 75% gastric juice 2.10 × 10.sup.9 9.54 × 10.sup.8 1.26 × 10.sup.7 2.41 × 10.sup.7 9.00 × 10.sup.6 1.37 × 10.sup.7 4.81 × 10.sup.6 9.18 × 10.sup.6
(57) TABLE-US-00007 TABLE 7 Effect of trehalose accumulation in nisin induced cultures on survival of freeze dried cultures in different concentrations of gastric juice, expressed as percentage of the initial concentration. after 0 h after 0.5 h after 1 h after 2 h % −nisin +nisin −nisin +nisin −nisin +nisin −nisin +nisin Before lyo 100% 100% 100% 100% 100% 100% 100% 100% After lyo 73% 98% 73% 98% 73% 98% 73% 98% 0% gastric juice 73% 98% 65% 104% 62% 115% 67% 101% 25% gastric juice 74% 71% 6% 60% 4% 6% 2% 1% 50% gastric juice 58% 70% 4% 44% 2% 3% 1% 1% 75% gastric juice 75% 80% 0% 2% 0% 1% 0% 1%
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