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1.
A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori +) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal
region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori + vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from
a homologous background. Single-cross-over integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on
medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions.
In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5–15) × 10 −2 copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations
of non-selective growth. These results show that the single-cross-over integration system described here represents a simple
procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.
Received: 23 September 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997 相似文献
2.
Various strains of coryneform bacteria, Micrococcaceae and commercial starters of Lactococcus lactis and Leuconostoc were compared for their aptitude to form S-methyl thioesters. Resting cells were incubated with methanethiol alone at pH 7 and in conjunction with a mixture of straight,
branched and hydroxy short-chain fatty acids up to C 6 at pH 7 and 5. Results showed that all the strains synthesized at least S-methyl thioacetate, with strains that were low and high producers in each group. This is the only thioester formed in small
amount by Leuconostoc. Brevibacterium linens (six strains) and Micrococcaceae (five strains) were able to form branched-chain thioesters especially from their intracellular
fatty acids at neutral pH, and straight-chain thioesters mostly from exogenous fatty acids at acid pH. Coryneform bacteria
other than B. linens (four strains) and L. lactis (four starters) synthesized thioesters up to S-methyl thiobutyrate from endogenous or exogenous fatty acids but not branched-chain ones, except for one starter which formed
a very little thioisovalerate. Some particular effects of pH and added fatty acids revealed differences between species or
strains in their specific enzymatic systems.
Received: 7 April 1997 / Received revision: 5 June 1997 / Accepted: 7 June 1997 相似文献
3.
The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification
kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly.
Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains.
Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in
the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains.
At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show
that citrate metabolism slightly stimulates the growth of lactococci in milk.
Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997 相似文献
4.
β-Galactosidases from Lactobacillus delbruekii subsp. bulgaricus 20056, Lb. casei 20094, Lactococcus lactis subsp. lactis 7962, Streptococcus thermophilus TS2, Pediococcus pentosaceus PE39 and Bifidobacterium bifidum 1901 were partially purified. The rate of hydrolysis of lactose given by the predominant β-galactosidase activity from each
of the bacteria studied was in all cases enhanced by Mg 2+, while the effect of K + and Na + differed from strain to strain. The β-galactosidases from all strains also catalysed transgalactosylation reactions. The
types of oligosaccharides produced appeared to be very similar in each case, but the rates of their production differed. All
the β-galactosidases were also capable of hydrolysing galactosyl-lactose although, unlike the other bacteria studied, Lb. delbruekii subsp. bulgaricus 20056 and Lc. lactis subsp. lactis 7962 were unable to utilise galactosyl-lactose as a carbon source for growth.
Received: 4 October 1995/Received revision: 5 March 1996/Accepted 11 March 1996 相似文献
5.
Twenty strains of Streptococcus bovis grew more slowly on lactose (1.21 ± 0.12 h −1) than on glucose (1.67 ± 0.12 h −1), and repeated transfers or prolonged growth in continuous culture (more than 200 generations each) did not enhance the growth
rate on lactose. Lactose transport activity was poorly correlated with growth rate, and slow growth could not be explained
by the ATP production rate (catabolic rate). Batch cultures growing on lactose always had less␣intracellular fructose 1,6-bisphosphate
(Fru1,6 P
2) than cells growing on glucose (6.6 mM compared to 16.7 mM), and this difference could be explained by the pathway of carbon
metabolism. Glucose and the glucose moiety of lactose were metabolized by the Embden-Meyerhoff-Parnas (EMP) pathway, but the
galactose moiety of lactose was catabolized by the tagatose pathway, a scheme that by-passed Fru1,6 P
2. A mutant capable of co-metabolizing lactose and glucose grew more rapidly when glucose was added, even though the total
rate of hexose fermentation did not change. Wild-type S. bovis grew rapidly with galactose and melibiose, but these galactose-containing sugars were activated by galactokinase and catabolized
via EMP. On the basis of these results, rapid glycolytic flux through the EMP pathway is needed for the rapid growth (more
than 1.2 h −1) of S.␣bovis.
Received: 3 June 1997 / Received revision: 10 September 1997 / Accepted: 6 January 1998 相似文献
7.
Xanthomonas campestris pv. translucens IFO13599 could produce xanthan gum (18.5 mg/100 mg, lactose) with lactose as the growth substrate in spite of a low level
of β-galactosidase. This productivity corresponded to one-fifth that with glucose. This strain could also produce ice-nucleating
material having an ice-nucleating temperature, T
50, of −2.8 °C with xanthan gum in the culture broth. We found that this strain produced both materials in whey medium from
which the insoluble components had been removed. The production of xanthan with ice-nucleating material reached a maximum
after cultivation for 168 h under optimum conditions. Furthermore, the xanthan obtained had a low viscosity because of its
variant structure revealed, by TLC and HPLC analyses, to be lacking pyruvic acid. Furthermore, we concluded that this mixture
had considerable potential as a regeneratic agent, when compared to other regeneratic agents such as carboxymethylcellulose.
Received: 29 August 1997 / Received revision: 17 November 1997 / Accepted: 18 November 1997 相似文献
8.
Various strains of Pediococcus genus were successfully transformed by electroporation using the broad host-range plasmid pSA3 and the lactococcal Rep22
based-replicon pUCB304. Failure to transform Tetragenococcus strains by electroporation have led to use conjugation as an alternative plasmid DNA transfer mechanism. Intergeneric matings
conducted with the broad host-range conjugative plasmid pVA797 from Lactococcus lactis subsp. lactis SL2/797A to Tetragenococcus halophilus and Pediococcus pentosaceus strains have shown that Tetragenococcus strains are not impervious to plasmid DNA transfer. Plasmid and metabolic profiles of wild and mutant strains of Pediococcus pentosaceus NCDO990 have shown that many metabolic traits including lactose utilization are plasmid linked. 相似文献
9.
It is critical that an inexpensive electron- donor/carbon-source be found for selenium bioremedia-tion using the selenate-respiring
bacterium, Thauera selenatis. Since acetate is a preferred substrate for growth of this organism, a method was developed for fermenting the lactose in
whey to large amounts of acetate. Indigenous whey microorganisms fermented the whey lactose in this manner when grown in continuous
culture at a very slow dilution rate ( D = 0.05 h −1). The successful use of the fermented whey lactose as the carbon-source/electron-donor feed for a laboratory-scale selenium-bioremediation
reactor system, inoculated with T. selenatis, treating selenium-contaminated drainage water was also demonstrated. Selenium oxyanions and nitrate were reduced by 98%.
Received: 30 October 1998 / Received revision: 26 January 1999 / Accepted: 5 February 1999 相似文献
10.
A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well
as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions
were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products
and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present.
The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to
180 g l −1 glucose and 89 g l −1 lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition
due to lactic acid.
Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998 相似文献
11.
Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures
(up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied
by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation
of lactate dehydrogenase and pyruvate formate-lyase.
Received: 14 December 1998 / Received revision: 12 January 1999 / Accepted: 22 January 1999 相似文献
12.
Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease
domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular
presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal
sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments
including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin
produced was shown to be biologically active.
Received: 2 December 1996 / Received revision: 17 March 1997 / Accepted: 27 April 1997 相似文献
13.
The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l −1 exopolysaccharides with glucose and 30 mg l −1 with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced
on glucose showed the presence of two fractions with relative molecular masses ( M
r) of 1.7 × 10 6 and 4 × 10 4 in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M
r of 4 × 10 4. The high- M
r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose
and rhamnose in the molar ratio of 5:1:1, whereas the low- M
r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide
fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose,
1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass
fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production
of the high- M
r fractions appeared to be dependent on the carbohydrate source, whereas the low- M
r fractions were produced more continuously.
Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997 相似文献
14.
An improved strain of Pseudomonas sp. ATCC 31461 ( Pseudomonas elodea), capable of producing broth viscosities of 11 000 and 4700 mPa s (cP) when grown in enriched whey permeate and enriched
sweet whey broths respectively, was isolated. The isolation was by serial transfers of the parent on lactose-rich and sweet
whey broths. Maximum viscosities and biopolymer production were observed in 25% (v/v) whey concentration. In whey concentrations
of 50% (v/v) or greater, residual glucose was detected in the broth and biopolymer production was low. This strain is capable
of totally utilising the lactose in up to 50% (v/v) whey in 64 h. Enzyme activities suggest that the transport of lactose
in P. elodea is by the permease system as opposed to the phosphotransferase system. The location of β-galactosidase is mainly intracellular.
The improved strain is able to utilise lactose better than the parent and produce 1.6 times more intracellular β-galactosidase
activity compared to the parent.
Received: 3 May 1996 / Received revision: 8 August 1996 / Accepted: 10 August 1996 相似文献
15.
Recombinant Lactococcus lactis MG1363/pMG36e-lacZ exhibiting high β-galactosidase activities were constructed by us in the previous study. However, erythromycin
resistance present in these recombinants restricted their practical application in food preparation. This study was conducted
to delete the gene coding for erythromycin resistance present in recombinant L. lactis, as a result of which these bacteria express food-grade β-galactosidase. In this study, the recombinant plasmid pMG36e-lacZ
was digested with restriction enzymes BclI and HpaI and the food-grade plasmid FGZW was rebuilt. FGZW was transformed into Escherichia coli JM109 and L. lactis MG1363. Erythromycin resistance, enzyme activity determination, gene sequencing and SDS-PAGE analysis indicated that these
new recombinant bacteria lost erythromycin resistance and its relevant gene but still expressed β-galactosidase activities,
although a decrease in the expression of β-galactosidase of these new strains was observed. The β-galactosidase food-grade
expression system was successfully constructed and it could provide a new solution for the management of lactose intolerance.
These results might promote the usage of gene-modified microorganisms and related technology in the food sector, which has
the highest priority for food safety. 相似文献
16.
An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones
could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 10 5 transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000
concentration and the wetness of selective plates were investigated.
Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997 相似文献
17.
Lactose conversion by lactic acid bacteria is of high industrial relevance and consistent starter culture quality is of outmost importance. We observed that Lactococcus lactis using the high-affinity lactose-phosphotransferase system excreted galactose towards the end of the lactose consumption phase. The excreted galactose was re-consumed after lactose depletion. The lacS gene, known to encode a lactose permease with affinity for galactose, a putative galactose–lactose antiporter, was upregulated under the conditions studied. When transferring cells from anaerobic to respiration-permissive conditions, lactose-assimilating strains exhibited a long and non-reproducible lag phase. Through systematic preculture experiments, the presence of galactose in the precultures was correlated to short and reproducible lag phases in respiration-permissive main cultivations. For starter culture production, the presence of galactose during propagation of dairy strains can provide a physiological marker for short culture lag phase in lactose-grown cultures. 相似文献
18.
Of three β-galactosidases from Aspergillus oryzae, Kluyveromyces lactis and Bacillus sp., used for the production of low-content galacto- oligosaccharides (GOS) from lactose, the latter produced the highest
yield of trisaccharides and tetrasaccharides. GOS production was enhanced by mixing β-galactosidase glucose oxidase. The low-content
GOS syrups, produced either by β-galactosidase alone or by the mixed enzyme system, were subjected to the fermentation by
Kluyveromyces marxianus, whereby glucose, galactose, lactose and other disaccharides were depleted, resulting in up to 97% and 98% on a dry weight
basis of high-content GOS with the yields of 31% and 32%, respectively.
An erratum to this article can be found at 相似文献
19.
The effects of Lactobacillus-GG-fermented oat bran product on the microbiota and its metabolic activity in the human gut were investigated, using a simulator
of the human intestinal microbial ecosystem (SHIME), by analysing the bacterial population, short-chain fatty acids and gas
production. In addition, the effects of fermented oat bran supernatant and supernatant samples from reactors 4, 5 and 6 (large
intestine) on the growth of Escherichia coli IHE 13047, Enterococcus faecalis VTT E-93203, Lactobacillus rhamnosus VTT E-94522 ( Lactobacillus GG) and Lactococcus lactis subsp. lactis VTT E-90414 were monitored to ascertain possible stimulatory/inhibitory effects by an in vitro turbidometric method. Our
experiments showed that Lactobacillus GG colonized the SHIME reactor and this colonization could be maintained for several weeks without extra supplementation.
Oat bran feeding also favoured the growth of bifidobacteria and caused an increase in the production of acetic, propionic
and butyric acid as well as CH 4 and CO 2. However, the effects of oat bran, either on bacterial populations or on their metabolic activity, were not directly dose-dependent.
In turbidometric measurements, the supernatant of fermented oat bran exerted an inhibitory effect of Lactobacillus GG, but stimulated the growth of enterococci.
Received: 19 January 1998 / Received revision: 6 April 1998 / Accepted: 13 April 1998 相似文献
20.
When Lactic Acid Bacterial cultures were frozen at −20°C for 24 h, the cell viability decreased drastically, but when they
were cold shocked at 10°C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp. lactis strains (25–37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp. cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2. When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed
cold shock cryotolerance. Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested. The PCR product from Lactococcus lactis ssp. lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csp's. Use
of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied
and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested.
Received: 18 October 1996 / Accepted: 28 January 1997 相似文献
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