Ajatuksia pohdittavaksi.
Kun liian laktoosipitoisuuden ongelmasta on päästy, tulee sekundääriongelmaksi galaktoosin liian korkeat pitoisuudet käsitellyssä meijerituotteessa. Tässä artikkelissa pureudutaan tähän ongelmaan: miten vähentää galaktoosin liian korkeita määriä laktoosiredusoiduissa elintarvikkeissa? Tämä on harvan ongelmana, koska tietoisuus galaktoosin määrän noususta laktoosin fermentaation tuloksena ei ole mitenkään yleistietoa. Toisaalta ei myöskään ole vaatimusta galaktoosipitoisuuden ilmoittamisesta elintarvikkeessa. Kolmanneksi galaktoosin varoista on hämärät käsitykset. neljänneksi tavataan sanoa, että galaktoosi ei ole essentielli molekyyli, mitä pitäisi tarkemmin ilmoittaa taulukoissa, johtuu luonnollisesti siitä, että ruoassa yleensä on galaktoosia, eikä siitä etteikö se voisi olla essentielle. Se on kai niin tärkeä ja joka puolella, että se on "näkymätön". se ei maistu sokerina kuten sakkaroosi ja glukoosi heti kun niitä syödään, vaan se on "se jokin" karamellisoituneissa herkullisissa leivonnaisissa ja muissa hyvissä, joista ei voi luopua. Galaktoosin tarve kehossa on alitajuista astetta.
Täytyy vain toivoa että suoliston maitobakteerit ovat sitä sorttia jotka hajoittavat runsaan liiallisen galaktoosin tarpeeksi pitkälle lopputuotteiksi asti, joita ihminen voi haitatta saada tallennettua.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976262/
Abstract
Accumulation
of galactose in dairy products due to partial lactose fermentation by
lactic acid bacteria yields poor-quality products and precludes their
consumption by individuals suffering from galactosemia.
This study aimed
at extending our knowledge of galactose metabolism in Lactococcus lactis,
with the final goal of tailoring strains for enhanced galactose
consumption. We used directed genetically engineered strains to examine
galactose utilization in strain NZ9000 via the chromosomal Leloir
pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac
genes). Galactokinase (GalK), but not galactose permease (GalP), is
essential for growth on galactose. This finding led to the discovery of
an alternative route, comprising a galactose phosphotransferase system
(PTS) and a phosphatase, for galactose dissimilation in NZ9000.
Introduction of the Tag6P pathway in a galPMK mutant restored
the ability to metabolize galactose but did not sustain growth on this
sugar. The latter strain was used to prove that lacFE, encoding
the lactose PTS, is necessary for galactose metabolism, thus
implicating this transporter in galactose uptake. Both PTS transporters
have a low affinity for galactose, while GalP displays a high affinity
for the sugar. Furthermore, the GalP/Leloir route supported the highest
galactose consumption rate. To further increase this rate, we
overexpressed galPMKT, but this led to a substantial
accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate,
pointing to a bottleneck at the level of α-phosphoglucomutase.
Overexpression of a gene encoding α-phosphoglucomutase alone or in
combination with gal genes yielded strains with galactose
consumption rates enhanced up to 50% relative to that of NZ9000.
Approaches to further improve galactose metabolism are discussed.
Lactococcus lactis is a lactic acid bacterium widely used in
the dairy industry for the production of fermented milk products.
Because of its economic importance, L. lactis has been studied
extensively in the last 40 years. A small genome, a large set of genetic
tools, a wealth of physiological knowledge, and a relatively simple
metabolic potential render L. lactis an attractive model with which to implement metabolic engineering strategies (reviewed in references 21 and 57).
In the process of milk fermentation by L. lactis, lactose is
taken up and concomitantly
phosphorylated at the galactose moiety (C-6)
by the lactose-specific phosphoenolpyruvate (PEP)-dependent
phosphotransferase system (PTSLac), after which it is hydrolyzed to glucose and galactose 6-phosphate (Gal6P) (64).
The glucose moiety enters the glycolytic pathway upon phosphorylation
via glucokinase to glucose 6-phosphate (G6P), whereas Gal6P is
metabolized to triose phosphates via the d-tagatose
6-phosphate (Tag6P) pathway, encompassing the steps catalyzed by
galactose 6-phosphate isomerase (LacAB), Tag6P kinase (LacC), and
tagatose 1,6-bisphosphate aldolase (LacD) (Fig. (Fig.1).1). Curiously, during the metabolism of lactose by L. lactis, part of the Gal6P is dephosphorylated and excreted into the growth medium, while the glucose moiety is readily used (2, 7, 51, 56, 60).
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Schematic overview of the alternative routes for galactose uptake and further catabolism in L. lactis. Galactose can be imported by the non-PTS permease GalP and metabolized via the Leloir pathway (galMKTE) to α-G1P, which is converted to the glycolytic intermediate G6P by α-phosphoglucomutase (pgmH). Alternatively, galactose can be imported by PTSLac (lacFE) and further metabolized to triose phosphates by the Tag6P pathway (lacABCD).
Here, we propose a new uptake route consisting of galactose
translocation via the galactose PTS, followed by dephosphorylation of
the internalized Gal6P to galactose, which is further metabolized via
the Leloir pathway (highlighted in the gray box). galP, galactose permease; galM, galactose mutarotase; galK, galactokinase; galT, galactose 1-phosphate uridylyltransferase; galE, UDP-galactose-4-epimerase; pgmH, α-phosphoglucomutase; lacAB, galactose 6-phosphate isomerase; lacC, Tag6P kinase; lacD, tagatose 1,6-bisphosphate aldolase; lacFE, PTSLac; PTSGal, unidentified galactose PTS; Phosphatase; unidentified Gal6P-phosphatase; pgi, phosphoglucose isomerase; pfk, 6-phosphofructo-1-kinase; fba, fructose 1,6-bisphosphate aldolase; tpi,
triose phosphate isomerase; α-Gal1P, α-galactose 1-phosphate; α-G1P,
α-glucose 1-phosphate; UDP-gal, UDP-galactose; UDP-glc, UDP-glucose;
G6P, glucose 6-phosphate; Gal6P, galactose 6-phosphate; Tag6P, tagatose
6-phosphate; TBP, tagatose 1,6-bisphosphate; FBP, fructose
1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde
3-phosphate. The dotted arrow represents the conversions of GAP to
pyruvate via the glycolytic pathway. Steps essential to improve
galactose consumption are shown in black boxes.
As a result of incomplete lactose utilization, some fermented dairy
products contain significant residual amounts of galactose. The presence
of galactose has been associated with shoddier qualities of the
fermented product (6, 27, 43).
In particular, galactose is a major contributor to the browning that
occurs when dairy products (e.g., yogurt and mozzarella, Swiss, and
cheddar cheese) are cooked or heated in the manufacture of pizzas, sauce
preparation, or processed cheese. In addition, availability of residual
galactose may result in production of CO2 by
heterofermentative starters and, consequently, in textural defects such
as the development of slits and fractures in cheeses. Therefore, the
availability of starter strains with improved galactose utilization
capacity is desirable to develop higher-quality dairy products.
Additionally, strains with increased galactose metabolism could provide
galactose-free foods for individuals and, in particular, children
suffering from the rare disease galactosemia (36). To this end, a comprehensive understanding of galactose catabolism is essential.
Galactose metabolism in L. lactis was thoroughly studied in the
past and has been and still is the subject of some controversy. Indeed,
conflicting results regarding the type of PTS involved in galactose
uptake have been published. Some authors advocated that galactose is
exclusively transported via the plasmid-encoded PTSLac, whereas others proposed transport via a galactose-specific PTS (PTSGal) to the extreme of questioning the contribution of the PTSLac (17, 20, 50, 59). However, a gene encoding PTSGal has never been identified in L. lactis.
Independently of the nature of the PTS, it is generally accepted that
the resulting Gal6P is metabolized via the Tag6P pathway (lac operon) (Fig. (Fig.1).1).
On the other hand, galactose translocated via the highly specific
galactose permease (GalP) is metabolized via the Leloir pathway to
α-glucose 1-phosphate (α-G1P) through the sequential action of galactose
mutarotase (GalM), galactokinase (GalK), and galactose 1-phosphate
uridylyltransferase (GalT)/UDP-galactose-4-epimerase (GalE) (gal
operon). Entry in glycolysis is preceded by the α-phosphoglucomutase
(α-PGM)-catalyzed isomerization of α-G1P to G6P. The use of the Leloir
and/or the Tag6P pathway for galactose utilization is currently viewed
as being strain dependent (9, 16, 25, 32, 33, 58), but the relative efficacy in the degradation of the sugar has not been established.
The ultimate aim of this study was to engineer L. lactis for
improved galactose-fermenting capacity as a means to minimize the
galactose content in dairy products. To gain insight into galactose
catabolism via the Leloir (gal genes) and the Tag6P (lac genes) pathways, a series of L.
lactis subsp. cremoris NZ9000 isogenic gal and lac
mutants were constructed. Carbon 13 labeling experiments coupled with
nuclear magnetic resonance (NMR) spectroscopy were used to investigate
galactose metabolism in the gal and lac strains. The
data obtained revealed a novel route for galactose dissimilation and
provided clues to further enhance galactose utilization.
RESULTS
L. lactis NZ9000 can import galactose via more than one transport system.
L. lactis can use the Leloir pathway, the Tag6P pathway, or both pathways for galactose metabolism in a strain-dependent manner (58). Presumably, L. lactis subsp. cremoris NZ9000 (MG1363pepN::nisRK)
internalizes galactose by a secondary carrier symporter (GalP) and
further metabolizes the sugar exclusively via the Leloir pathway (25),
as this strain lacks the plasmid-linked genes encoding the Tag6P
pathway. To assess the potential of the Tag6P route for galactose
catabolism, a strategy was devised that consisted of introduction of
plasmid pMG820 (41) with genes lacABCDFEG, encoding the lactose PTS (lacFE), the Tag6P pathway enzymes (lacABCD), and a β-phosphogalactosidase (lacG) (64), in mutants in which galactose utilization via the Leloir pathway was prevented. Grossiord et al. (25) previously reported blockage of the Leloir pathway by inactivation of the galactose permease gene. In our study, galP was deleted in strain NZ9000 using a double-crossover recombination method. The extent of the deletion is shown in Fig. Fig.22 A. Unexpectedly, L. lactis NZ9000ΔgalP was still able to grow in a medium with galactose as the sole carbon source (Fig. (Fig.2B);2B);
growth was biphasic and characterized by an initial growth rate that
was 2.3-fold lower than a second growth rate, which was similar to that
of parent strain NZ9000 (0.38 ± 0.01 h−1). To exclude the possibility of residual GalP activity due to partial deletion only (Fig. (Fig.2A),2A),
a new out-of-frame deletion was made in which only the first four amino
acids of the original protein were left. The behavior of the resulting
strain was in all aspects similar to that of our original galP-deletion strain. Subsequently, a mutant strain of L. lactis NZ9000 was made in which, apart from galP, the downstream genes of the operon, galM (galactose mutarotase) and galK (galactose kinase), were also deleted (Fig. (Fig.2A).2A). Deletion of galPMK resulted in total loss of the capacity to grow in a medium with galactose as the sole source of carbon (Fig. (Fig.2B).2B). These data imply that L. lactis NZ9000 has an additional transport system with specificity for galactose.
L. lactis NZ9000 can catabolize galactose via the Tag6P pathway.
Introduction of pMG820 in L. lactis NZ9000ΔgalPMK
rendered a strain that could grow in CDM containing lactose (or
glucose, both at 1% [wt/vol]) but not when galactose (1% wt/vol) was the
sole carbon source (data not shown). However, resting cells of
lactose-grown cultures were able to convert [1-13C]galactose (20 mM) to a mixture of fermentation end products, including [3-13C]lactate, [2-13C]acetate, and [2-13C]ethanol, as determined by 1H NMR spectroscopy (Fig. (Fig.3).3). To determine which of the genes within the lac
operon were essential for galactose metabolism, the following
combinations of genes were cloned into pNZ8048 under the control of the
nisin promoter: lacABCD, lacFE, lacABCDFE, and lacABCDFEG. The various constructs were introduced into L. lactis NZ9000ΔgalPMK. Expression of the lac genes in the different constructs was confirmed by SDS-PAGE of cell extracts obtained from nisin-induced (1 μg liter−1) glucose-grown cultures (data not shown). Also, strain NZ9000ΔgalPMK(pLacABCDFEG) was able to grow on lactose (1%, wt/vol)-CDM when the inducer nisin (1 μg liter−1) was added at time 0 h (inoculation). Strain NZ9000ΔgalPMK(pLacABCDFE) showed moderate growth under the same conditions (Table (Table3),3), thus showing functional expression of the lac genes. Like NZ9000ΔgalPMK(pMG820),
none of the resulting strains was able to grow in CDM with galactose
(1%, wt/vol) as the carbon source, even though nisin was added at time 0
h. To determine the galactose-fermenting capacity of NZ9000ΔgalPMK and derivatives harboring the lac constructs, resting cell suspensions were incubated with [1-13C]galactose (20 mM), and the end products in the supernatants were examined by 1H NMR (Fig. (Fig.3).3). Strains NZ9000ΔgalPMK(pLacABCD) and NZ9000ΔgalPMK(pLacFE) and the negative control, NZ9000ΔgalPMK,
were unable to metabolize galactose, as indicated by the absence of
labeled end products in the supernatants. Lactate labeled on carbon 3
was detected in the supernatants of NZ9000ΔgalPMK(pLacABCDFE) and NZ9000ΔgalPMK(pLacABCDFEG), showing that, under the conditions tested (glucose-grown cells), both the Tag6P enzymes (lacABCD) and PTSLac (lacFE) are required for galactose consumption in L. lactis NZ9000ΔgalPMK.
.....
Future approaches, combining increased expression of catabolic genes,
namely, galactose permease and α-PGM, with engineering of the catabolite
control network could create L. lactis strains preferring
galactose over glucose and lactose, a desirable trait considering that
the concentration of lactose in milk fermentations is normally higher
than that of galactose (3). Thus, galactose scavengers would be ideal starters in the manufacture of galactose-free dairy products.
....
3. Alm, A. 1982. Effect of
fermentation on lactose, glucose, and galactose content in milk and
suitability of fermented milk products for lactose intolerant
individuals. J. Dairy Sci. 65:346-352. [PubMed]
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