Microbial and
Bioconversion Production of D-xylitol and Its Detection and Application
Abstract
D-Xylitol is found in low content as a natural
constituent of many fruits and vegetables. It is a five-carbon sugar polyol and
has been used as a food additive and sweetening agent to replace sucrose,
especially for non-insulin dependent diabetics. It has multiple beneficial
health effects, such as the prevention of dental caries, and acute otitis
media. In industry, it has been produced by chemical reduction of D-xylose
mainly from photosynthetic biomass hydrolysates. As an alternative method of
chemical reduction, biosynthesis of D-xylitol has been focused on the
metabolically engineered Saccharomyces cerevisiae and Candida strains.
In order to detect D-xylitol in the production processes, several detection
methods have been established, such as gas chromatography (GC)-based methods,
high performance liquid chromatography (HPLC)-based methods, LC-MS methods, and
capillary electrophoresis methods (CE). The advantages and disadvantages of
these methods are compared in this review.
Keywords: D-xylitol,
Bioconversion production, Detection methods, Saccharomyces cerevisiae, Candida.
Introduction
D-Xylitol is a five-carbon polyol (five-carbon sugar
alcohol), which has the capacity to form complexes with certain cations,
including Cu2+, Ca2+, and Fe2+ . It displaces
water molecules from these metal ions and the hydration layer of proteins. In
nature, D-xylitol is found in various fruits and vegetables, such as berries,
corn husks, oats, lettuces, cauliflowers, and mushrooms. It can be extracted
from birch, raspberries, plums and corn fiber and so on. The content of
D-xylitol in fruits and vegetables is usually low, and thus it is uneconomical
to extract large amounts of D-xylitol from such sources. In industrial scale
production, hemicellulose is utilized as the material to separate pure
D-xylose, which is subsequently reduced to D-xylitol.
D-Xylitol has attracted worldwide interest because of
its unique properties and huge potential. It has almost the same sweetness as
sucrose, but lower energy value than sucrose (2.4 cal/g vs. 4.0 cal/g), thus it
has been used as a sugar substitute in dietary foods, especially for
insulin-deficiency patients. Due to its anticariogenicity, tooth rehardening
and remineralization properties, D-xylitol has been widely applied in the
odontological industry. It could also prevent ear and upper respiratory
infections and benefit pregnant and nursing women. According to the current
price of D-xylitol of $4 - 5 kg-1, the global market is about $340
million year-1, and it will definitely grow bigger and bigger.
Production of D-xylitol and
screening of D-xylitol-producing microorganisms
D-xylitol is industrially produced by the chemical
reduction of D-xylose derived mainly from photosynthetic biomass hydrolysates.
Photosynthetic biomass is the most abundant renewable resources in the world,
consisting of cellulose, hemicellulose, lignin and a low quantity of pectin,
protein, extractives, and ash. Hemicellulose is the second most abundant
polysaccharide in nature, representing 19 - 34% of the photosynthetic biomass,
just next to the most abundant biopolymer: cellulose (34 - 50%). Hemicellulose,
a good resource for producing D-xylitol, is composed of D-glucose, D-galactose,
D-mannose, D-xylose, D-arabinose, and D-glucuronic acid with acetyl side
chains. In biomass materials, the three major components (cellulose,
hemicellulose and lignin) are strongly intermeshed and chemically bound by
non-covalent force or covalent cross-linkages. In order to obtain pure
fermentable sugar, D-xylose, from the complicated structure of biomass, it is
necessary to pre-treat those materials by chemical [or biological hydrolysis
methods. In 1970, the industrial scale chromatographic method was developed in
Finland, and pure D-xylose was separated from hemicellulose. Subsequently,
D-xylitol could be produced from D-xylose through catalytic hydrogenation, in
the presence of a nickel catalyst at high temperature (80 - 140oC)
and pressure (up to 50 atm). The conversion rate of xylan (a polymer of
D-xylose) to D-xylitol reached 50% - 60% .
Figure 1
Production of D-xylitol from photosynthetic biomass.
With the increase of interest in exploring more
environment-friendly and economical D-xylitol production methods, the
biosynthesis of D-xylitol using microorganisms recently became increasingly
popular. A considerable number of bacteria, fungi and yeasts can produce
D-xylitol. In Table, we list ten D-xylitol producing
microorganisms which have been used. Corynebacterium sp. produced 69
mg/ml of D-xylitol after 14 days of incubation. Enterobacter liquefaciens,
which was isolated from soil, could yield 33.3 mg/ml of D-xylitol when D-xylose
was used as a single carbon source. When grown in anaerobic conditions, Mycobacterium
smegmatis could transform 70% of D-xylulose to D-xylitol. Petromyces
albertensis yielded 39.8 g/L of D-xylitol, in the presence of 100 g/L of
D-xylose in the medium supplemented with 1% (v/v) methanol. Among the
microorganisms, yeasts were the preferred producer. In 1981, Barbosal et al.
screened 44 yeast strains, among which the best D-xylitol producers were Candida
guilliermondii and C. tropicalis. A mutant strain, C. tropicalis
HXP2, was reported to yield more than 90% D-xylitol from D-xylose . Guo et al.
(2006) tested five D-xylitol-producing strains from 274 strains. Two of them, C.
guilliermondii Xu280 and C. maltosa Xu316 had the highest ability to
consume D-xylose and produce D-xylitol in the batch fermentation with
micro-aerobic condition.
Suryadi et al. found that after 4 days of cultivation,
Hansenula polymorpha could produce 58 g/L of D-xylitol, using 125 g/L of
D-xylose in the medium. Sampaio et al. (2008) screened approximately 270 yeasts
for D-xylitol production using D-xylose as the sole carbon source. The best
producer was Debaryomyces hansenii UFV-170, which produced 5.84 g/L of
D-xylitol from 10 g/L D-xylose after 24h incubation.
Microbial conversion of
D-xylose to D-xylitol
In bacteria, the conversion of D-xylose to D-xylulose
is catalyzed by xylose isomerase in a single step. This xylose isomerase was
also detected in some yeasts and molds, such as C. boidinii, Malbranchea
pulchella, and Meurospora crassa. However, in the majority of yeasts
and fungi, the conversion of D-xylose to D-xylulose needs two steps, a
reduction step followed by an oxidation step. In these yeasts and fungi,
D-xylose was first reduced to D-xylitol by either NADH- or NADPH-dependent
xylose reductase (aldose redutase EC 1.1.1.21) (XR); the resulting D-xylitol was
either secreted or further oxidized to D-xylulose by NAD- or NADP-dependent
xylitol dehydrogenase (EC 1.1.1.9) (XDH). These two reactions were considered
to be the rate-limiting steps in D-xylose fermentation and D-xylitol
production. Some strains of yeast could metabolize D-xylulose to
xylulose-5-phosphate by xylulokinase (EC 2.7.1.17) (XK). Xylulose-5-phosphate
can subsequently enter the pentose phosphate pathway .
Screening of microorganisms for
D-xylitol production.
|
Strians
|
Carbon Sources
|
Growth Conditions
|
Yield
|
References
|
|
Corynebacterium sp.
|
D-xylose
|
─
|
69 mg/ml
|
11
|
|
Enterobacter liquefaciens
|
D-xylose
|
─
|
33.3 mg/ml
|
12, 13
|
|
Mycobacterium smegmatis
|
D-xylulose, xylitol or D-mannitol
|
Anaerobic condition
|
0.7 g/g
|
14
|
|
Petromyces albertensis
|
D-xylose and methanol
|
Initial pH of 7.0
|
39.8 g/L
|
15
|
|
C. guilliermondii FTI-20037
|
D-xylose
|
Aerobic condition, 30~35℃
|
77.2 g/L
|
16
|
|
C. tropicalis HXP2
|
D-xylose
|
Aerobic condition, 30℃
|
0.96 g/g
|
17
|
|
C. guilliermondii Xu280
|
D-xylose
|
Micro-aerobic condition
|
0.63 g/g
|
18
|
|
C. maltosa Xu316
|
D-xylose
|
Micro-aerobic condition
|
0.43 g/g
|
18
|
|
Hansenula polymorpha
|
D-xylose and glycerol
|
pH of 8
|
0.52 g/g
|
19
|
|
Debaryomyces hansenii UFV-170
|
D-xylose
|
Micro-aerobic conditio
|
0.54 g/g
|
20
|
Figure 2
Metabolic pathway of D-xylitol in yeasts.
The wild type Saccharomyces cerevisiae was
known to be a non-xylose-fermenting yeast because of its lacking the D-xylose
metabolic pathway. Nevertheless, its GRAS status (generally recognized as safe)
and strong tolerance to inhibitors present in lignocellulose hydrolysates
attracted researchers' attention. They constructed the recombinant S.
cerevisiae strain, by introducing xylitol metabolism requisite genes into
it. In 1991, xylose reductase gene (XYL1) cloned from Pichia stititis
CBS 6054 was transformed into S. cerevisiae under the control of the
phosphoglycerate kinase (PGK) promoter. In this recombinant strain, the
conversion ratio of D-xylose to D-xylitol reached over 95%. Some other
researchers tried to use various strategies to express xylose reductase gene
derived from different strains. A xylose reductase gene from P. stipitis
was expressed in S. cerevisiae in 2000. The recombinant strain could
produce 0.95 g of D-xylitol from 1 g of D-xylose in the presence of glucose
used as a co-substrate for co-factors regeneration. Handumrongkul et al. (1998)
cloned a xylose reductase gene from C. guilliermondii ATCC 20118 and
expressed the gene under the control of an alcohol oxidase promoter (AOX1) in
methylotrophic yeast P. pastoris. The resulting strains were able to
utilize D-xylose and accumulate D-xylitol. In particular, when grown in aerobic
conditions, it could produce the maximum amount of D-xylitol (7.8 g/L). Chung
et al. (2002) reported that a xylose reductase gene was integrated into the
chromosome of S. cerevisiae by constructing two different vectors. The
recombinant strains could produce 0.90 g of D-xylitol per gram of D-xylose.
During the continuous D-xylose metabolic process, xylose reductase (XR)
activity was not the pivotal factor which controlled the conversion. However,
the deficiency of the key co-factor NAD(P)H broke the redox balance in the
cell, thus the production of D-xylitol could not be increased further in the
recombinant S. cerevisiae.
The effect of co-substrates on D-xylitol production by
S. cerevisiae was evaluated. Co-substrates were necessary for growth by
supplying the metabolic maintenance energy and generating the reduced
co-factors. It was proved that D-glucose, D-mannose and D-fructose had high
affinity with the transport system for D-xylose, and inhibited D-xylose
conversion by 99%, 77% and 78%, respectively, due to the competitive
relationship. However, D-maltose and D-galactose had their own special
transport systems. There was no inhibition of D-xylose metabolism with
D-maltose and 51% inhibition with D-galactose. More than 5 times higher
D-xylitol production was obtained in the presence of D-galactose than
D-glucose. The differences in D-xylitol yield observed with various
co-substrates were hypothesized to be due to the differences in redox
metabolism. The Candida yeasts were considered as better potential
candidates than the metabolically engineered S. cerevisiae, due to their
characteristics: natural D-xylose consumers and maintaining the reduction-oxidation
balance during D-xylitol accumulation. However, its application was limited in
food industry because of the opportunistic pathogenic nature of some Candida
spp. Reports about metabolic engineering methods in Candida strains are
rare: Using the Ura-blasting method, two copies of xyl2 gene which
encodes the xylitol dehydrogenase (XDH) in the diploid yeast C. tropicalis
were sequentially disrupted. The conversion of D-xylose to D-xylitol reached
98% when glycerol was utilized as a co-substrate. The C. parapsilosis
xyl1 gene encoding xylose reductase (XR) which is controlled by an alcohol
dehydrogenase promoter was introduced into C. tropicalis. This
recombinant yeast exhibited higher D-xylitol yield than the wild type strain.
In optimizing the D-xylitol-fermenting conditions, such as aeration,
temperature and pH, the oxygen availability was the key parameter for D-xylitol
production from D-xylose. In transient oxygen limited condition, a surplus of
NADH inhibited the activity of xylitol dehydrogenase, which resulted in
D-xylitol accumulation.
In general, the optimal temperature and pH for
D-xylitol-yielding yeasts was 30 - 37 oC and 4 - 6, respectively.
The applications of D-xylitol
As an effective and safe tooth-decay-preventive agent,
D-xylitol is used in chewing gums, mouth rinse and toothpaste ,Streptococcus
mutans is most notably associated with human dental decay, by attachment to
the acquired enamel pellicle and direct interaction with the salivary
components. The most important steps in the development of dental caries are
the adherence of S. mutans to tooth surfaces and the formation of dental
plaque. These bacteria are known to be agglutinated by the whole saliva. The
salivary agglutinating factor is a high-molecular-weight glycoprotein which
occurs optimally between pH 5.0 - 7.5. S. mutans possess the ability to
produce large amounts of intercellular polysaccharides from sucrose, which
could be converted to lactic acid after prolonged incubation and markedly
facilitate the colonization of S. Mutans. However, S. mutans
cannot utilize D-xylitol. After people take D-xylitol-containing products, the
lactic acid production from fermentation by these strains will be decreased.
Saliva with D-xylitol is more alkaline than that containing other sugar
products. When pH in the mouth rises above 7, calcium and phosphate salts in
saliva start to precipitate into the parts of enamel where they are lacking. At
the same time, D-xylitol increases the potential of saliva in buffering the
acid in plaque. D-Xylitol is able to reduce the ability of S. mutans to
adhere to plaque, making it more easily removed from the plaque. In 1989,
adults of mean age 22.5 years consumed 10.9 g of D-xylitol daily in chewing
gum. The plaque of the subjects decreased and showed an obvious ability to
resist pH drops induced by the sucrose rinse. In 1995, sixty 11 to 15 years old
children who wore fixed orthodontic appliances were given chewing gums
containing D-xylitol. The fresh and dry weight of the dental plaque, collected
at baseline and 28 days from incisors, canines and premolars from the area
between gingival margin and the bracket, reduced significantly by 43% to 47%.
The plaque and saliva levels of S. mutans were reduced by 13% to 33% in
children receiving D-xylitol gum. A 40-month double blind cohost study on the
relationship between the use of chewing gum and dental caries was performed
from 1989 - 1993 in Belize, Central America. The results showed that the D-xylitol-containing
gum was effective in reducing caries rates and the most effective agent was a
100% D-xylitol pellet gum. Makinen et al. (2001) reported the effect of a
2-month usage of saliva-stimulating pastils containing erythritol or D-xylitol.
In D-xylitol-group, the mean weight of total plaque
mass was reduced significantly; the plaque and salivary levels of S. mutans
and plaque levels of total streptococcus were reduced significantly as well.
Milgrom et al. suggested the effective dose of D-xylitol was between 6.44 g/day
and 10.32 g/day.
It was found that regular use of D-xylitol in chewing
gums or syrup prevented the incidence of acute otitis media (AOM) in children.
D-Xylitol had the ability to reduce the growth of the major otopathogen of acute
otitis media, S. pneumonia, which caused 30% or more of such attacks,
and also suppressed Haemophilus influenza, another important pathogen
implicated in AOM. Kontiokari et al. reported that the exposure of either
epithelial cells or pneumococci or both to 5% D-xylitol reduced the adherence
of pneumococci. Some researchers implied that the inhibition of pneumonia
growth induced by D-xylitol was mediated via the fructose phosphotransferase
system. However the mechanism remains a matter for speculation.
In healthy humans, D-xylitol is metabolized to
glucose-6-phosphate through an insulin-independent pathway in the liver and red
blood cells. It is a very slow metabolism process from D-xylitol to D-glucose,
so in this way the blood glucose and the insulin concentration raise gently. In
insulin-deficiency conditions, D-xylitol could be used as a sugar substitute.
Adding the low energy content and the smaller thermogenic effects, D-xylitol
appears to be an attractive alternative for non-insulin dependent diabetics.
Detection methods of D-xylitol
The qualitative detection of D-xylitol in different
matrices is generally based on separation techniques coupled with
characterization methods, and its quantification requires a standard curve of
D-xylitol. The existing analytical methods for D-xylitol can be grouped into
four catagories: gas chromatography (GC)-based methods, high performance liquid
chromatography (HPLC)-based methods, LC-MS methods, and capillary
electrophoresis methods (CE). The analysis of low molecular weight
carbohydrates and sugar alcohols (including D-xylitol) by GC, HPLC and CE
methods was reviewed in 2004. In this section, analytical methods for the
determination of D-xylitol are briefly reviewed with emphasis on their recent
developments.
1. GC and GC-MS
GC is a powerful analytical tool for the determination
of volatile compounds in different matrices. Since D-xylitol is a polyol that
is non-volatile, direct analysis of D-xylitol with GC is not possible.
Therefore, GC analysis of D-xylitol requires pre-derivatization of the analyte,
and the commonly used derivatization methods include trimethylsilylation (TMS)
and acetylation . GC with flame ionization detection is one of the early
methods developed for the detection of D-xylitol and it is still a method of
choice for the determination of D-xylitol in a complex matrix. In more recent
years, the coupling of GC with mass spectrometry (GC-MS) has greatly enhanced
the capacity of this analytical method. GC-MS has high reproducibility, high
resolution and few matrix effects [and has become one of the commonly used
analytical approaches for the determination of D-xylitol. The hyphenation of MS
to GC has also increased the accuracy of GC methods in terms of structure
determination because MS methods are able to provide structural information
about the connectivity of atoms of the unknown molecule.
2. HPLC
Contrary to GC separation, HPLC does not require
complicated derivatization steps of the analytes to form volatile derivatives.
The HPLC method utilizes a suitable column for separation followed by specific
detection of individually separated compounds. Different types of columns have
been used for the separation of D-xylitol from carbohydrates and other sugar
polyols, e.g., amino-based carbohydrate column ,HPX-87H organic acid column,
TSK amide 80 column, and ion-exclusion column A variety of detection methods,
including UV detection, electrochemical detection, reflective index (RI)
detection, and evaporative light-scattering (ELS) detection are available for
HPLC methods. D-xylitol lacks chromophoric and fluorophoric moieties required
for UV and fluorescence detection. As a result, less sensitive HPLC detection
methods such as RI , ELS are more commonly used for the determination of
D-xylitol. Detection limits and sensitivity to interference depend on the type
of detector hyphenated to the HPLC separation. RI and ELS detection methods
typically provide detection limits in the range of 0.05 - 1.2 μg/injection.
Detection methods such as pulsed amperometric detection can also be efficiently
used for the detection of D-xylitol when coupled with ion chromatography, e.g.,
using Dionex column, and a strongly basic mobile phase (pH>12).
In the study to evaluate D-xylitol producing capacity
of several yeast strains isolated from different natural sources, authors first
used thin layer chromatography (TLC) to rapidly identify the best producers and
thereafter analyzed by HPLC with RI detection [71]. The TLC analysis was carried out
on a silica gel plate with ethyl acetate: 2-propanol: water (130:57:23) as the
developing solvent and stained with bromocresol green-boric acid. D-xylitol was
visualized as a yellow spot on a blue background on TLC plates. A strain of C.
tropicalis was found to be the most efficient D-xylitol producer in this
study. Recent development of column-switching techniques for chromatography
allows the coupling of different separation modes to resolve a wide range of
compounds in complicated samples. Cheng et al. reported a column-switching HPLC
technique by coupling H+ and Pb2+ ion-exclusion columns
to study enzyme hydrolysis components of waste cellulosic biomass. The
column-switching HPLC with RI detection was connected on-line to the
immobilized enzyme reactor for successive on-line desalting and simultaneous
analysis of carbohydrates in the hydrolysate of waste paper and waste tree
branch by incorporating the heart-cut and the elution-time-difference
techniques. D-xylitol was used as an internal standard in this study. Since RI
and ELS detection methods are of relatively low sensitivity, pre-column
derivatization of D-xylitol for more sensitive HPLC detection has been
investigated. Katayama et al. reported a simple and sensitive pre-column HPLC
method for the determination of sugar and sugar alcohols including D-xylitol in
the serum. The samples were first derivatized with benzoic acid in the presence
of condensing agents, 1-isopropyl-3-(3-dimethylaminopropyl) carbodiimide perchlorate
(IDC) and 4-piperidinopyridine at 80oC for 60 min. The benzoylated
derivatives were separated on a TSK amide 80 column and detected with a
fluorescence detector at λex 275 nm and λem 315 nm.
D-Xylitol was detected as its mono-benzoyl ester derivative and the detection
limit of D-xylitol was 10 ng/mL. Similarly, D-xylitol was determined among
other sugar alcohols after nitrobenzoylation by HPLC method with UV detection
(260 nm). In this case, HPLC was performed on a phenyl column.
3. LC-MS and LC-NMR
Liquid chromatography hyphenated to mass spectrometry
(LC-MS) has emerged as a popular and powerful tool for the determination of
compounds in sample mixtures. Initially, GC was the only separation method able
to be hyphenated to MS. However, the use of GC is restricted to a small set of
molecules, i.e., those that are volatile or could be derivatized. Compared to
GC-MS, sample pretreatment is usually simplified with LC-MS method by
eliminating the need of derivatization. The determination of D-xylitol in atmospheric
aerosols was carried out using LC-MS with positive ESI. Polymer-based amino
analytical columns were used to efficiently separate D-xylitol from eight other
monosaccharides and sugar polyols. Isocratic elution was carried out in a
mobile phase consisting of 20% of 10 mM NH4Ac aaqueous solution, 8%
of methanol, and 72% of water. The [M+NH4]+ ions were
found to be abundant and used for monitoring and quantification. Limit of
detection was 4.7 pmol/injection for D-xylitol. In a similar study, LC-MS with
negative ESI was employed to analyze sugars and sugar polyols in atmospheric
aerosols. Since sugars and sugar polyols lack highly acidic functional groups
in their structures, their ionization through deprotonation to produce [M-H]-
is not effective under either ESI or atmospheric pressure chemical ionization
(APCI) without derivatization. It was found that post-column addition of
chloroform in acetonitrile greatly enhanced ionization of these compounds by
forming chloride adduct ion in the negative mode ESI. The detection limit of
D-xylitol based on quantification of [M+35Cl]- adduct ion
was 0.016 μM. Proton NMR spectroscopy has become a routine tool for fast and
comprehensive characterization of complex mixtures; however, due to extensive
signal overlapping, the spectral complexity can be a serious hindrance when
mixtures are analyzed. The development of LC-NMR, with the coupling of an HPLC
step immediately prior to NMR measurement, allows compound separation and
analysis to be carried out quickly. LC-NMR was successfully applied for the
metabolic profiling of human amniotic fluid in which more than 30 compounds
including D-xylitol were identified.The NMR spectrometer was equipped with a 3
mm probe head (60 μL active volume) and coupled to an ION300 ion exchange
column with a mobile phase composed of 2.5 mM H2SO4 in
100% D2O. Although the LC-NMR method is a rather sophisticated
analytical procedure, it provides a higher degree of accuracy in structural
determination than other methods. This is because the NMR method provides
structural information about the atom connectivity as well as stereochemistry
of the unknown molecule.
4. Capillary electrophoresis
Capillary electrophoresis (CE) is a powerful
separation technique suitable for assaying a variety of analytes in relatively
complex matrices. The application of CE for the analysis of sugar polyols has
been demonstrated recently in a review. Since sugar polyols lack both a charge
and a strong UV chromophore, CE analysis may be carried out after derivatization.
At the same time, the analysis of underivatized sugar polyol by CE method has
been developed using high-alkaline pH to ionize the polyol making them suitable
for indirect UV detection in a buffer solution. A CE method has been reported
for the simultaneous analysis of underivatized acidic, neutral and amino sugars
and sugar alcohols with indirect UV detection. Separations are carried out on
fused silica capillaries with an electrolyte consisting of 20 mM
2,6-pyridinedicarboxylic acid and 0.5 mM cetyltrimethylammonium bromide which
is used to reverse the direction of electroosmotic flow. Optimun separation of
carbohydrates and sugar alcohols has been achieved at pH 12.1 with the minimum
detection level ranged from 23 - 71 μM for carbohydrates. Under these
conditions, D-xylitol has been determined along with other 18 monosaccharides
and sugar polyols. The method has been applied to the monosaccharide composion
analysis in fetuin as a model glycoprotein after acid hydrolysis. The
advantages of the described CE method include direct analysis of
monosaccharides and sugar alcohos without deriavatization and its high
separation capacity for acidic, neutral, and amino sugars and sugar alcohols
under a single electrophoretic condition. For the analysis of polyols by CE
method under less alkaline condition, in-situ derivatization of polyols
with boric acid can be employed. Boric acid [B(OH)3] reacts readily
with a diol forming borate diester complex, (RO)2BOH. The remaining
hydroxyl group on the boron atom of the complex is readily ionizable, rendering
the borate-complex able to migrate electrophoretically. By using on-column
complexation with borate and indirect UV detection, the separation and
determination of D-mannitol, D-sorbitol and D-xylitol in the form of anionic
borate-polyol complexes by CE method has been demonstrated. The separation is
carried out in a fused silica capillary with background electrolyte of 200 mM
borate buffer containing 10 mM 3-nitrobenzoate as the chromogenic co-ion.
Similarly, this boric acid chemistry has been utilized in capillary
isotachophoresis (ITP) for the determination of D-xylitol in multicomponent
pharmaceutical formulations and foods. Using conductivity detection, the
detection limit of D-xylitol by ITP method is 52 μM. Simplicity, accuracy, and
low cost of analyses make ITP an alternative procedure to other methods
described for the determination of D-xylitol.
5. Other methods
The advent of electrospray ionization has
significantly increased the speed of MS analysis of complex mixtures. Direct MS
method has also been used for the determination of carbohydrate compounds. In
order to gain detailed information about structures of unknown molecules,
Watkins et al. recently described an ESI-MS method equipped with Fourier
transform ion cyclotron resonance (FT-ICR), in which ion-molecule reactions
were employed for the characterization of polyols and polyol mixtures including
D-xylitol. Analytes introduced in the mass spectrometer were ionized by
positive mode ESI and then allowed to react with the neutral reagent
diethylmethoxyborane. Consecutive reactions of the hydroxyl groups of polyols
resulted in products which were separated by 68 mass units in the mass
spectrum, along with 30 mass shifts arising from intra-molecular derivatization
of the primary derivatized products. The generated data provided structural
information about the number of hydroxyl groups and their relative positions
present in the unknown molecule. Recently, Sreenath and Venkatesh reported an
indirect competitive immunoassay to detect and quantify D-xylitol in foods by
making use of affinity-purified heptan-specific anti-D-xylitol antibodies.
Reductively aminated D-xylitol-albumin conjugate has been used as the immunogen
to raise IgG and IgE antibodies specific for D-xylitol. The limit of detection
is 1 ng for D-xylitol with this immunoassay, and the linear range of the assay
for D-xylitol quantification is 5 - 400 ng. This indirect competitive ELISA may
serve as a sensitive analytical tool to detect and quantify nanogram amounts of
D-xylitol in various biological samples and natural/processed foods. Biosensor
technology is now widely used in the detection and control of specific
compounds in fermentation broths. Takamizawa et al. reported a D-xylitol
biosensor composed of the partially purified D-xylitol dehydrogenase from C.
tropicalis IFO 0618. When the biosensor was applied for the measurement of
D-xylitol in the flow injection system, optimal operation pH and temperature
were found to be 8.0 and 30 oC, respectively. The biosensor was
characterized to have high affinity for NAD+ and medium affinity for
D-xylitol, slow reaction time (15 min), and a narrow linear range of detection
for D-xylitol (up to 3 mM).
Conclusion and future
prospects
D-Xylitol is widely used in food, odontological and
pharmaceutical industry due to its significant benefits on human health.
Cost-effective production methods of D-xylitol are being explored. From
hemicelluloses, as a component of the widespread, substantive, and inexpensive
photosynthetic biomass, D-xylose could be separated and purified, and then
reduced to D-xylitol. Some researchers have focused on screening for efficient
D-xylitol-producing microbial strains for industry applications. Meanwhile, a
large number of recombinant strains have been obtained, having improved
D-xylitol yields. It is very common in yeasts to change the pathway of xylose
metabolism by introducing or enhancing the xylose reductase and/or inactivating
the D-xylitol dehydrogenase. In some genetically engineered Escherichia coli
strains, the operon containing a set of genes responsible for the D-xylitol
metabolism has been constructed in order to increase the productivity of
D-xylitol. Other microorganisms such as filamentous fungus, which has the same
D-xylose metabolic pathway as D-xylitol-producing yeasts, could be utilized in
future for metabolically engineering modification. There are a few analytical
methods available for the detection of D-xylitol. Some analytical methods do
not allow the direct determination of elemental connectivity and
stereochemistry of analytes; therefore the accurate assignment of the analyte's
structure may not be possible based on these data alone. In general,
spectroscopic methods such as NMR, MS and X-ray crystallography, are the main
analytical platforms which provide structural information about elemental
connectivity and stereochemistry of unknown compounds.
Mikroba dan Biokonversi Produksi D-xylitol Serta Deteksi dan Aplikasinya
Abstrak
D-Xylitol ditemukan dalam konten rendah sebagai konstituen alami dari banyak buah-buahan dan sayuran. Ini adalah lima karbon gula poliol dan telah digunakan sebagai aditif makanan dan agen pemanis untuk menggantikan sukrosa, terutama untuk penderita diabetes non-insulin dependen. Ini memiliki beberapa efek kesehatan yang bermanfaat, seperti pencegahan karies gigi, dan otitis media akut. Dalam industri, telah diproduksi oleh reduksi kimia dari D-xylose terutama dari hidrolisat biomassa fotosintetik. Sebagai metode alternatif dari pengurangan kimia, biosintesis D-xylitol telah difokuskan pada Saccharomyces cerevisiae metabolisme direkayasa dan strain Candida. Dalam rangka untuk mendeteksi D-xylitol dalam proses produksi, beberapa metode deteksi telah ditetapkan, seperti kromatografi gas (GC)-berdasarkan metode, kinerja kromatografi cairan yang tinggi (HPLC)-berdasarkan metode, LC-MS metode, dan metode elektroforesis kapiler (CE). Keuntungan dan kerugian dari metode ini dibandingkan dalam review ini.
Kata kunci: D-xylitol, biokonversi produksi, metode Deteksi, Saccharomyces cerevisiae, Candida.
Pengenalan
D-Xylitol adalah lima karbon poliol (lima karbon gula alkohol), yang memiliki kapasitas untuk membentuk kompleks dengan kation tertentu, termasuk Cu2 +, Ca2 +, dan Fe2 +. Ini menggantikan molekul air dari ion-ion logam dan hidrasi lapisan protein. Di alam, D-xylitol ditemukan dalam berbagai buah-buahan dan sayuran, seperti berry, sekam jagung, gandum, selada, kembang kol, dan jamur. Hal ini dapat diekstraksi dari birch, raspberry, plum dan serat jagung dan sebagainya. Isi dari D-xylitol dalam buah-buahan dan sayuran biasanya rendah, sehingga tidak ekonomis untuk mengekstrak sejumlah besar D-xylitol dari sumber tersebut. Dalam produksi skala industri, hemiselulosa digunakan sebagai bahan untuk memisahkan D-xylose murni, yang kemudian dikurangi ke D-xylitol. Produksi D-xylitol dan skrining D-xylitol mikroorganisme penghasil D-xylitol adalah industri yang dihasilkan oleh pengurangan kimia dari D-xylose terutama berasal dari biomassa hidrolisat fotosintetik. Fotosintetik biomassa adalah sumber daya terbarukan yang paling berlimpah di dunia, yang terdiri dari selulosa, hemiselulosa, lignin dan kuantitas rendah pektin, protein, ekstraktif, dan abu. Hemiselulosa adalah polisakarida kedua yang paling berlimpah di alam, yang mewakili 19-34% dari biomassa fotosintesis, persis di samping biopolimer yang paling berlimpah: selulosa (34 - 50%). Hemiselulosa, sumber yang baik untuk memproduksi D-xylitol, terdiri dari D-glukosa, D-galaktosa, D-mannose, D-xylose, D-arabinosa, dan D-glukuronat asam dengan rantai samping asetil. Dalam bahan biomassa, tiga komponen utama (selulosa, hemiselulosa dan lignin) sangat intermeshed dan kimia terikat oleh non-kovalen kekuatan atau kovalen lintas-hubungan. Dalam rangka untuk memperoleh gula difermentasi murni, D-xylose, dari struktur yang rumit dari biomassa, perlu untuk pra-memperlakukan bahan-bahan oleh kimia [atau metode hidrolisis biologis. Pada tahun 1970, metode kromatografi skala industri dikembangkan di Finlandia, dan D-xylose murni dipisahkan dari hemiselulosa. Selanjutnya, D-xylitol bisa diproduksi dari D-xylose melalui hidrogenasi katalitik, di hadapan sebuah katalis nikel pada suhu tinggi (80 - 140oC) dan tekanan (hingga 50 atm). Tingkat konversi xilan (polimer dari D-xylose) ke D-xylitol mencapai 50% - 60%.
Gambar 1
Produksi D-xylitol dari biomassa fotosintetik
D-Xylitol ditemukan dalam konten rendah sebagai konstituen alami dari banyak buah-buahan dan sayuran. Ini adalah lima karbon gula poliol dan telah digunakan sebagai aditif makanan dan agen pemanis untuk menggantikan sukrosa, terutama untuk penderita diabetes non-insulin dependen. Ini memiliki beberapa efek kesehatan yang bermanfaat, seperti pencegahan karies gigi, dan otitis media akut. Dalam industri, telah diproduksi oleh reduksi kimia dari D-xylose terutama dari hidrolisat biomassa fotosintetik. Sebagai metode alternatif dari pengurangan kimia, biosintesis D-xylitol telah difokuskan pada Saccharomyces cerevisiae metabolisme direkayasa dan strain Candida. Dalam rangka untuk mendeteksi D-xylitol dalam proses produksi, beberapa metode deteksi telah ditetapkan, seperti kromatografi gas (GC)-berdasarkan metode, kinerja kromatografi cairan yang tinggi (HPLC)-berdasarkan metode, LC-MS metode, dan metode elektroforesis kapiler (CE). Keuntungan dan kerugian dari metode ini dibandingkan dalam review ini.
Kata kunci: D-xylitol, biokonversi produksi, metode Deteksi, Saccharomyces cerevisiae, Candida.
Pengenalan
D-Xylitol adalah lima karbon poliol (lima karbon gula alkohol), yang memiliki kapasitas untuk membentuk kompleks dengan kation tertentu, termasuk Cu2 +, Ca2 +, dan Fe2 +. Ini menggantikan molekul air dari ion-ion logam dan hidrasi lapisan protein. Di alam, D-xylitol ditemukan dalam berbagai buah-buahan dan sayuran, seperti berry, sekam jagung, gandum, selada, kembang kol, dan jamur. Hal ini dapat diekstraksi dari birch, raspberry, plum dan serat jagung dan sebagainya. Isi dari D-xylitol dalam buah-buahan dan sayuran biasanya rendah, sehingga tidak ekonomis untuk mengekstrak sejumlah besar D-xylitol dari sumber tersebut. Dalam produksi skala industri, hemiselulosa digunakan sebagai bahan untuk memisahkan D-xylose murni, yang kemudian dikurangi ke D-xylitol. Produksi D-xylitol dan skrining D-xylitol mikroorganisme penghasil D-xylitol adalah industri yang dihasilkan oleh pengurangan kimia dari D-xylose terutama berasal dari biomassa hidrolisat fotosintetik. Fotosintetik biomassa adalah sumber daya terbarukan yang paling berlimpah di dunia, yang terdiri dari selulosa, hemiselulosa, lignin dan kuantitas rendah pektin, protein, ekstraktif, dan abu. Hemiselulosa adalah polisakarida kedua yang paling berlimpah di alam, yang mewakili 19-34% dari biomassa fotosintesis, persis di samping biopolimer yang paling berlimpah: selulosa (34 - 50%). Hemiselulosa, sumber yang baik untuk memproduksi D-xylitol, terdiri dari D-glukosa, D-galaktosa, D-mannose, D-xylose, D-arabinosa, dan D-glukuronat asam dengan rantai samping asetil. Dalam bahan biomassa, tiga komponen utama (selulosa, hemiselulosa dan lignin) sangat intermeshed dan kimia terikat oleh non-kovalen kekuatan atau kovalen lintas-hubungan. Dalam rangka untuk memperoleh gula difermentasi murni, D-xylose, dari struktur yang rumit dari biomassa, perlu untuk pra-memperlakukan bahan-bahan oleh kimia [atau metode hidrolisis biologis. Pada tahun 1970, metode kromatografi skala industri dikembangkan di Finlandia, dan D-xylose murni dipisahkan dari hemiselulosa. Selanjutnya, D-xylitol bisa diproduksi dari D-xylose melalui hidrogenasi katalitik, di hadapan sebuah katalis nikel pada suhu tinggi (80 - 140oC) dan tekanan (hingga 50 atm). Tingkat konversi xilan (polimer dari D-xylose) ke D-xylitol mencapai 50% - 60%.
Gambar 1
Produksi D-xylitol dari biomassa fotosintetik
. 
Dengan peningkatan minat dalam mengeksplorasi lebih ramah lingkungan dan ekonomis D-xylitol metode produksi, biosintesis D-xylitol menggunakan mikroorganisme baru-baru ini menjadi semakin populer. Sejumlah besar bakteri, jamur dan ragi dapat menghasilkan D-xylitol. Dalam Tabel, kami daftar sepuluh D-xylitol menghasilkan mikroorganisme yang telah digunakan. Corynebacterium sp. menghasilkan 69 mg / ml D-xylitol setelah 14 hari inkubasi. Enterobacter liquefaciens, yang diisolasi dari tanah, bisa menghasilkan 33,3 mg / ml D-xylitol bila D-xylose digunakan sebagai sumber karbon tunggal. Ketika tumbuh dalam kondisi anaerobik, Mycobacterium smegmatis bisa mengubah 70% dari D-xylulose ke D-xylitol. Petromyces albertensis menghasilkan 39,8 g / L D-xylitol, di hadapan 100 g / L D-xylose dalam medium ditambah dengan 1% (v / v) metanol. Di antara mikroorganisme, ragi adalah produser disukai. Pada tahun 1981, Barbosal dkk. disaring strain ragi 44, di antaranya D-xylitol terbaik produser Candida dan C. tropicalis guilliermondii. Sebuah strain mutan, C. tropicalis HXP2, dilaporkan untuk menghasilkan lebih dari 90% D-xylitol dari D-xylose. Guo et al. (2006) menguji lima D-xylitol-memproduksi strain dari 274 strain. Dua dari mereka, C. guilliermondii Xu280 dan C. maltosa Xu316 memiliki kemampuan tertinggi untuk mengkonsumsi D-xylose dan menghasilkan D-xylitol dalam fermentasi batch dengan mikro-kondisi aerobik. Suryadi et al. menemukan bahwa setelah 4 hari budidaya, Hansenula polymorpha bisa menghasilkan 58 g / L D-xylitol, menggunakan 125 g / L D-xylose dalam medium. Sampaio et al. (2008) diputar sekitar 270 ragi untuk D-xylitol produksi menggunakan D-xylose sebagai sumber karbon tunggal. Produser terbaik adalah Debaryomyces hansenii UFV-170, yang menghasilkan 5,84 g / L D-xylitol dari 10 g / L D-xylose setelah inkubasi 24 jam (Tabel 1).Pada tahun 1991, xylose reduktase gen (XYL1) kloning dari stititis Pichia CBS 6054 berubah menjadi S. cerevisiae bawah kendali phosphoglycerate kinase (PGK) promotor. Dalam strain rekombinan, rasio konversi D-xylose ke D-xylitol mencapai lebih dari 95%. Beberapa peneliti lainnya mencoba untuk menggunakan berbagai strategi untuk mengekspresikan gen xilosa reduktase berasal dari strain yang berbeda. A reduktase xilosa gen dari P. stipitis dinyatakan dalam S. cerevisiae pada tahun 2000. Strain rekombinan bisa menghasilkan 0,95 g D-xylitol dari 1 g D-xylose dalam kehadiran glukosa digunakan sebagai substrat untuk co-co-faktor regenerasi. Handumrongkul et al. (1998) kloning gen reduktase xilosa dari C. guilliermondii ATCC 20118 dan menyatakan gen di bawah kontrol promotor alkohol oksidase (AOX1) di P. pastoris methylotrophic ragi. Strain yang dihasilkan mampu memanfaatkan D-xylose dan menumpuk D-xylitol. Secara khusus, ketika tumbuh dalam kondisi aerobik, bisa menghasilkan jumlah maksimum D-xylitol (7,8 g / L). Chung et al. (2002) melaporkan bahwa sebuah gen reduktase xilosa telah diintegrasikan ke dalam kromosom S. cerevisiae dengan membangun dua vektor yang berbeda. Strain rekombinan bisa menghasilkan 0,90 g D-xylitol per gram D-xylose. Selama proses D-xylose metabolisme terus menerus, xylose reduktase (XR) aktivitas bukan faktor penting yang mengendalikan konversi. Namun, kekurangan dari co-faktor kunci NAD (P) H memecahkan keseimbangan redoks dalam sel, sehingga produksi D-xylitol tidak dapat ditingkatkan lebih lanjut dalam S. cerevisiae rekombinan. Efek co-substrat pada D-xylitol produksi oleh S. cerevisiae dievaluasi. Co-substrat yang diperlukan untuk pertumbuhan dengan memasok energi metabolisme dan menghasilkan pemeliharaan mengurangi co-faktor. Itu membuktikan bahwa D-glukosa, D-mannose dan D-fruktosa memiliki afinitas yang tinggi dengan sistem transportasi untuk D-xylose, dan menghambat D-xylose konversi dengan 99%, 77% dan 78%, masing-masing, karena hubungan kompetitif . Namun, D-maltosa dan D-galaktosa memiliki sistem transportasi khusus mereka sendiri. Tidak ada penghambatan D-xylose metabolisme dengan D-maltosa dan penghambatan 51% dengan D-galaktosa. Lebih dari 5 kali lebih tinggi D-xylitol produksi diperoleh di hadapan D-galaktosa dari D-glukosa. Perbedaan dalam D-xylitol menghasilkan diamati dengan berbagai co-substrat yang diduga disebabkan oleh perbedaan dalam metabolisme redoks. Ragi Candida dianggap sebagai kandidat potensial lebih baik daripada S. cerevisiae metabolik direkayasa, karena karakteristik mereka: D-xylose alami konsumen dan menjaga keseimbangan reduksi-oksidasi selama D-xylitol akumulasi. Namun, penerapannya terbatas dalam industri makanan karena sifat patogen oportunistik dari beberapa spp Candida. Laporan tentang metode rekayasa metabolik di strain Candida jarang terjadi: Menggunakan metode Ura-peledakan, dua salinan dari gen yang mengkode xyl2 dehidrogenase xylitol (XDH) dalam ragi diploid C. tropicalis secara berurutan terganggu. Konversi dari D-xylose ke D-xylitol mencapai 98% ketika gliserol dimanfaatkan sebagai substrat co-. C. parapsilosis xyl1 pengkodean gen xilosa reduktase (XR) yang dikendalikan oleh promotor alkohol dehidrogenase diperkenalkan ke C. tropicalis. Ragi rekombinan ini dipamerkan lebih tinggi D-xylitol hasil dari strain tipe liar. Dalam mengoptimalkan fermentasi-xylitol-D kondisi, seperti aerasi, suhu dan pH, ketersediaan oksigen adalah parameter kunci untuk D-xylitol produksi dari D-xylose. Dalam kondisi transien oksigen terbatas, surplus NADH menghambat aktivitas dehidrogenase xylitol, yang mengakibatkan akumulasi D-xylitol. Secara umum, suhu dan pH yang optimal untuk D-xylitol menghasilkan ragi adalah 30-37 oC dan 4 - 6, masing-masing.
Aplikasi D-xylitol
1. GC dan GC-MS
GC adalah alat analisis yang kuat untuk penentuan senyawa atsiri
dalam matriks yang berbeda. Karena D-xylitol adalah poliol yang non-volatile,
analisis langsung dari D-xylitol dengan GC tidak mungkin. Oleh karena itu,
analisis GC D-xylitol membutuhkan pra-derivatisasi analit, dan metode yang umum
digunakan termasuk derivatisasi trimethylsilylation (TMS) dan asetilasi. GC dengan
deteksi api ionisasi adalah salah satu metode awal yang dikembangkan untuk
mendeteksi D-xylitol dan masih merupakan metode pilihan untuk penentuan
D-xylitol dalam matriks yang kompleks.
Dalam beberapa tahun terakhir lebih, kopling GC dengan spektrometri massa (GC-MS) telah sangat meningkatkan kapasitas dari metode analitis. GC-MS telah reproduktifitas tinggi, resolusi tinggi dan efek matriks beberapa [dan telah menjadi salah satu pendekatan analitis yang umum digunakan untuk penentuan D-xylitol. Para hyphenation MS GC juga telah meningkatkan akurasi metode GC dalam hal penentuan struktur karena MS metode yang mampu memberikan informasi tentang struktur konektivitas atom molekul tidak diketahui.
Dalam beberapa tahun terakhir lebih, kopling GC dengan spektrometri massa (GC-MS) telah sangat meningkatkan kapasitas dari metode analitis. GC-MS telah reproduktifitas tinggi, resolusi tinggi dan efek matriks beberapa [dan telah menjadi salah satu pendekatan analitis yang umum digunakan untuk penentuan D-xylitol. Para hyphenation MS GC juga telah meningkatkan akurasi metode GC dalam hal penentuan struktur karena MS metode yang mampu memberikan informasi tentang struktur konektivitas atom molekul tidak diketahui.
2. HPLC
Bertentangan dengan
pemisahan GC, HPLC tidak memerlukan langkah-langkah derivatisasi rumit dari
analit untuk membentuk turunan yang mudah menguap. Metode HPLC menggunakan
kolom yang sesuai untuk pemisahan diikuti oleh deteksi spesifik senyawa
individual terpisah. Berbagai jenis kolom telah digunakan untuk pemisahan
D-xylitol dari karbohidrat dan poliol gula lainnya, misalnya, amino berbasis
kolom karbohidrat, HPX-87H kolom asam organik, TSK amida 80 kolom, dan
ion-kolom eksklusi Berbagai deteksi metode, termasuk deteksi UV, deteksi
elektrokimia, indeks reflektif (RI) deteksi, dan menguapkan cahaya hamburan
(ELS) deteksi yang tersedia untuk metode HPLC. D-xylitol memiliki gugus
kromoforik dan fluorophoric diperlukan untuk deteksi UV dan fluoresensi.
Akibatnya, metode deteksi kurang sensitif HPLC seperti RI, ELS lebih umum
digunakan untuk penentuan D-xylitol. Deteksi batas dan kepekaan terhadap
gangguan tergantung pada jenis detektor ditulis dgn tanda penghubung untuk
pemisahan HPLC. RI dan ELS metode deteksi biasanya memberikan batas deteksi
dalam kisaran 0,05-1,2 ug / injeksi. Deteksi metode seperti deteksi amperometri
berdenyut juga dapat digunakan secara efisien untuk deteksi D-xylitol ketika
digabungkan dengan ion kromatografi, misalnya, menggunakan kolom Dionex, dan
fase bergerak sangat dasar (pH> 12). Dalam studi untuk mengevaluasi
D-xylitol kapasitas memproduksi strain ragi yang diisolasi dari sumber alami
yang berbeda, penulis pertama kali digunakan kromatografi lapis tipis (KLT)
untuk secara cepat mengidentifikasi produsen terbaik dan selanjutnya dianalisis
dengan HPLC dengan deteksi RI [71]. Analisis KLT dilakukan pada piring silika
gel dengan etil asetat: 2-propanol: air (130:57:23) sebagai pelarut berkembang
dan diwarnai dengan Bromocresol hijau asam borat. D-xylitol adalah
divisualisasikan sebagai titik kuning pada latar belakang biru pada pelat KLT.
Sebuah strain C. tropicalis ditemukan menjadi produsen D-xylitol paling efisien
dalam penelitian ini.
Perkembangan terbaru dari kolom switching teknik untuk kromatografi memungkinkan kopling mode pemisahan yang berbeda untuk menyelesaikan berbagai senyawa dalam sampel yang rumit. Cheng et al. melaporkan teknik-switching kolom HPLC dengan kopling H + dan ion Pb2 + pengecualian kolom untuk mempelajari komponen enzim hidrolisis biomassa limbah selulosa.
Perkembangan terbaru dari kolom switching teknik untuk kromatografi memungkinkan kopling mode pemisahan yang berbeda untuk menyelesaikan berbagai senyawa dalam sampel yang rumit. Cheng et al. melaporkan teknik-switching kolom HPLC dengan kopling H + dan ion Pb2 + pengecualian kolom untuk mempelajari komponen enzim hidrolisis biomassa limbah selulosa.
3. LC-MS dan LC-NMR
Kromatografi cair ditulis dgn tanda penghubung untuk
spektrometri massa (LC-MS) telah muncul sebagai alat yang populer dan kuat
untuk penentuan senyawa dalam campuran sampel. Awalnya, GC adalah metode
pemisahan hanya mampu menjadi ditulis dgn tanda penghubung ke MS. Namun,
penggunaan GC dibatasi untuk satu set kecil dari molekul, yaitu, mereka yang
mudah menguap atau dapat diderivatisasi. Dibandingkan dengan GC-MS,
pretreatment sampel biasanya disederhanakan dengan LC-MS metode dengan
menghilangkan kebutuhan derivatisasi. Penentuan D-xylitol dalam aerosol
atmosfer dilakukan dengan menggunakan LC-ESI MS dengan positif. Berbasis
polimer kolom analitis amino digunakan untuk efisien memisahkan D-xylitol dari
delapan monosakarida lain dan poliol gula. Elusi isokratik dilakukan dalam fase
gerak yang terdiri dari 20% dari 10 solusi aaqueous mM NH4Ac, 8% dari metanol,
dan 72% air. Para [M + NH4] + ion ditemukan berlimpah dan digunakan untuk
pemantauan dan kuantifikasi. Batas deteksi adalah 4,7 pmol / injeksi untuk
D-xylitol. Dalam sebuah penelitian serupa, LC-MS dengan ESI negatif digunakan
untuk menganalisis gula dan polyol gula dalam aerosol atmosfer. Karena gula dan
poliol gula kurangnya kelompok fungsional yang sangat asam dalam struktur mereka,
ionisasi mereka melalui deprotonasi untuk menghasilkan [MH] - adalah tidak
efektif di bawah baik ESI atau tekanan ionisasi kimia atmosfer (APCI) tanpa
derivatisasi. Ditemukan bahwa pasca-kolom penambahan kloroform dalam
asetonitril sangat meningkatkan ionisasi senyawa ini dengan membentuk ion
klorida aduk dalam modus ESI negatif. Batas deteksi D-xylitol berdasarkan
kuantifikasi [M 35 Cl] - ion adisi adalah 0,016 pM.
Proton NMR spektroskopi telah menjadi alat rutin untuk karakterisasi cepat dan komprehensif campuran yang kompleks, namun, karena sinyal yang luas tumpang tindih, kompleksitas spektral dapat menjadi penghalang serius ketika campuran dianalisis. Pengembangan LC-NMR, dengan kopling langkah HPLC segera sebelum pengukuran NMR, memungkinkan senyawa pemisahan dan analisis yang akan dilakukan dengan cepat. LC-NMR telah berhasil diterapkan untuk profil metabolik cairan ketuban manusia di mana lebih dari 30 senyawa termasuk D-xylitol adalah NMR spektrometer identified.The dilengkapi dengan kepala Probe 3 mm (60 volume yang aktif uL) dan digabungkan ke ion ION300 pertukaran kolom dengan fase gerak 2,5 mM terdiri dari H2SO4 di D2O 100%.
Proton NMR spektroskopi telah menjadi alat rutin untuk karakterisasi cepat dan komprehensif campuran yang kompleks, namun, karena sinyal yang luas tumpang tindih, kompleksitas spektral dapat menjadi penghalang serius ketika campuran dianalisis. Pengembangan LC-NMR, dengan kopling langkah HPLC segera sebelum pengukuran NMR, memungkinkan senyawa pemisahan dan analisis yang akan dilakukan dengan cepat. LC-NMR telah berhasil diterapkan untuk profil metabolik cairan ketuban manusia di mana lebih dari 30 senyawa termasuk D-xylitol adalah NMR spektrometer identified.The dilengkapi dengan kepala Probe 3 mm (60 volume yang aktif uL) dan digabungkan ke ion ION300 pertukaran kolom dengan fase gerak 2,5 mM terdiri dari H2SO4 di D2O 100%.
4. Elektroforesis kapiler
Elektroforesis kapiler (CE) adalah teknik pemisahan yang kuat
cocok untuk pengujian berbagai analit dalam matriks relatif kompleks. Penerapan
CE untuk analisis gula poliol telah dibuktikan baru-baru ini di review. Karena
kurangnya poliol gula baik muatan dan kromofor UV yang kuat, CE analisis dapat
dilakukan setelah derivatisasi. Pada saat yang sama, analisis gula poliol
underivatized dengan metode CE telah dikembangkan menggunakan high-basa pH
untuk mengionisasi poliol membuat mereka cocok untuk deteksi UV tidak langsung
dalam larutan buffer. Sebuah metode CE telah dilaporkan untuk analisis simultan
dari underivatized asam, netral dan gula amino dan gula alkohol dengan deteksi
UV tidak langsung. Pemisahan dilakukan pada kapiler leburan silika dengan
elektrolit yang terdiri dari 20 mM 2,6-pyridinedicarboxylic asam dan 0,5 mM
cetyltrimethylammonium bromida yang digunakan untuk membalikkan arah aliran
electroosmotic.Untuk analisis poliol dengan metode CE bawah kondisi alkali
kurang, di-situ derivatisasi poliol dengan asam borat dapat digunakan Asam
borat [B (OH) 3] mudah bereaksi dengan kompleks borat diol membentuk diester,
(RO) 2BOH. Gugus hidroksil yang tersisa pada atom boron kompleks tersebut mudah
terionisasi, rendering borat-kompleks mampu bermigrasi elektroforesis. Dengan
menggunakan on-kolom kompleksasi dengan borat dan deteksi UV tidak langsung,
pemisahan dan penentuan D-manitol, sorbitol D-dan D-xylitol dalam bentuk
anionik borat-poliol kompleks dengan metode CE telah dibuktikan. Pemisahan ini
dilakukan dalam leburan silika kapiler dengan elektrolit latar belakang
penyangga borat 200 mM yang mengandung 10 mM 3-nitrobenzoate sebagai
chromogenic co-ion. Demikian pula, ini kimia asam borat telah digunakan di
isotachophoresis kapiler (ITP) untuk penentuan D-xylitol dalam formulasi
multikomponen farmasi dan makanan. Menggunakan deteksi konduktivitas, batas
deteksi D-xylitol dengan metode ITP adalah 52 pM. Kesederhanaan, akurasi, dan
biaya rendah membuat ITP analisis prosedur alternatif untuk metode lain yang
dijelaskan untuk penentuan D-xylitol.
5. Metode lain
Munculnya ionisasi elektrospray telah secara signifikan
meningkatkan kecepatan analisis MS dari campuran kompleks. Metode Direct MS juga telah digunakan untuk penentuan
senyawa karbohidrat. Dalam rangka untuk memperoleh informasi rinci
tentang struktur molekul yang tidak diketahui, Watkins et al. baru-baru ini menggambarkan sebuah metode ESI-MS
dilengkapi dengan Transformasi Fourier resonansi siklotron ion (FT-ICR), di
mana ion-molekul reaksi dipekerjakan untuk karakterisasi poliol dan campuran
poliol termasuk D-xylitol. Analit diperkenalkan pada spektrometer massa
terionisasi oleh ESI modus positif dan kemudian dibiarkan untuk bereaksi dengan
reagen diethylmethoxyborane netral. Reaksi
berturut-turut kelompok hidroksil dari poliol menghasilkan produk yang dipisahkan
oleh 68 unit massa pada spektrum massa, bersama dengan 30 shift massa yang
timbul dari intra-molekul derivatisasi produk diderivatisasi primer. Data yang
dihasilkan memberikan informasi tentang struktur jumlah gugus hidroksil dan
posisi relatif mereka hadir dalam molekul tidak diketahui. Baru-baru ini,
Sreenath dan Venkatesh melaporkan immunoassay kompetitif tidak langsung untuk
mendeteksi dan mengukur D-xylitol dalam makanan dengan memanfaatkan
afinitas-dimurnikan heptan-spesifik anti-D-xylitol antibodi. Reductively
aminated D-xylitol-albumin konjugat telah digunakan sebagai imunogen untuk
meningkatkan antibodi IgG dan IgE spesifik untuk D-xylitol. Batas deteksi adalah 1 ng untuk D-xylitol dengan
immunoassay ini, dan rentang linier dari assay untuk D-xylitol kuantifikasi
adalah 5 - 400 ng. Ini ELISA kompetitif tidak langsung dapat berfungsi sebagai
alat analisis sensitif untuk mendeteksi dan mengukur jumlah nanogram D-xylitol
dalam sampel biologis berbagai makanan alami / diproses. Teknologi biosensor
yang sekarang banyak digunakan dalam deteksi dan kontrol senyawa tertentu dalam
kaldu fermentasi. Takamizawa et al. melaporkan
biosensor D-xylitol terdiri dari dehidrogenase D-xylitol sebagian dimurnikan
dari C. tropicalis IFO 0618. Ketika biosensor diterapkan untuk pengukuran
D-xylitol dalam sistem injeksi aliran, operasi pH dan suhu yang optimal
ditemukan 8,0 dan 30 oC, masing-masing
Kesimpulan
D-Xilitol banyak digunakan dalam
makanan, industri odontological dan farmasi karena manfaat yang signifikan
terhadap kesehatan manusia. Metode produksi biaya-efektif dari D-xylitol sedang
dieksplorasi. Dari hemiselulosa, sebagai komponen dari biomassa fotosintesis
luas, substantif, dan murah, D-xylose dapat dipisahkan dan dimurnikan, dan
kemudian dikurangi ke D-xylitol. Beberapa peneliti telah berfokus pada skrining
untuk efisien D-xylitol-memproduksi strain mikroba untuk aplikasi industri.
Sementara itu, sejumlah besar strain rekombinan telah diperoleh, setelah
diperbaiki D-xylitol hasil. Hal ini sangat umum dalam ragi untuk mengubah jalur
metabolisme xylose dengan memperkenalkan atau meningkatkan reduktase xilosa dan
/ atau menonaktifkan dehidrogenase D-xylitol. Dalam beberapa strain Escherichia
coli rekayasa genetika, operon yang mengandung satu set gen yang bertanggung
jawab untuk metabolisme D-xylitol telah dibangun dalam rangka meningkatkan
produktivitas D-xylitol. Mikroorganisme lain nya seperti jamur filamentous,
yang memiliki jalur D-xylose sama metabolik sebagai D-xylitol-memproduksi ragi,
dapat dimanfaatkan dimasa depan untuk modifikasi rekayasa metabolik.
