Tuesday, 16 June 2015
Monday, 15 June 2015
Sunday, 14 June 2015
Biotechnological Production of Alternate Sugars
1.
Introduction:
The use of low-calorie
sugar-free products tripled in the final two decades of the 20th century. In
the United States alone, more than 150 million people use these products
regularly. Even though hundreds of good-tasting low-calorie, sugar-free
products are now available, most light product consumers say they would like to
have additional low-calorie sugar-free products available. Of particular
interest are baked goods and desserts. A number of events that occurred in the
late 1990s are expected to facilitate providing additional good-tasting,
low-calorie, sugar-free products. The approval of acesulfame potassium for soft
drinks and aspartame and sucralose as general purpose sweeteners in the United
States and recognition by regulatory agencies around the world that polyols
have reduced caloric values compared with sucrose are examples. (A general
purpose sweetener may be used in accordance with good manufacturing practices
to sweeten any food when a standard of identity does not preclude its use.)
These events should expand the use of sweeteners alone and in combination as
well.
On the scientific
front, after more than 100 years of use, scientists around the world are
publicly acknowledging that saccharin is safe for humans. For example, in 1997,
a special International Agency for Research on Cancer (IARC) panel determined
the bladder tumors in male rats resulting from the ingestion of high doses of
sodium saccharin are not relevant to man. And, in late 1998, IARC downgraded
saccharin from a Category 2B substance, possible carcinogenic to humans, to
Category 3, not classifiable as to its carcinogenicity to humans. Agents for
which the evidence of carcinogenicity is inadequate in humans but sufficient in
experimental animals may be placed in Group 3 when strong evidence exists that
the mechanism of carcinogenicity in experimental animals does not operate in
humans. This is the first time IARC has considered mechanistic data, and the
IARC panel voted unanimously that saccharin could cause tumors in rats but that
this is not predictive of human cancer.
Unfortunately, not all
events surrounding sweeteners have been positive. Although some new
technologies have provided a means of supporting the safety of sweeteners, the
Internet has made disseminating negative information without accountability a
new art form. No adverse health effects related to aspartame have been
confirmed, but this has not stopped its critics. An extremely negative,
inaccurate article making absurd claims about aspartame began circulating on
the Internet in late 1998. The article asserts that aspartame is responsible
for any number of ailments, without supporting data and creating new challenges
for industry. Fortunately, many of the negative claims from this article and
other antiaspartame fanatics are so absurd that the sources are not considered
credible by many.
1.1.
The Ideal Sweetener:
The search for the
perfect sweetener continues, but it has long been recognized that the ideal
sweetener does not exist. Even sucrose, the ‘‘gold standard,’’ is not perfect
and is unsuitable for some pharmaceuticals and chewing gums.
Alternative sweeteners
(a) provide and expand food and beverage choices to control caloric,
carbohydrate, or specific sugar intake; (b) assist in weight maintenance or
reduction; (c) aid in the management of diabetes; (d) assist in the control of
dental caries; (e) enhance the usability of pharmaceuticals and cosmetics; (f)
provide sweetness in times of sugar shortage; and (g) assist in the
costeffective use of limited resources. The ideal sweetener should be at least
as sweet as sucrose, colorless, odorless, and noncariogenic. It should have a
clean, pleasant taste with immediate onset without lingering. The more a
sweetener tastes and functions like sucrose the greater the consumer
acceptability. If it can be processed much like sucrose with existing equipment,
the more desirable it is to industry.
The ideal sweetener
should be water soluble and stable in both acidic and basic conditions and over
a wide range of temperatures. Length of stability and consequently the
shelf-life of the final product are also important. The final food product
should taste much like the traditional one. A sweetener must be compatible with
a wide range of food ingredients because sweetness is but one component of
complex flavor systems. Safety is essential. The sweetener must be nontoxic and
metabolized normally or excreted unchanged, and studies verifying its safety
should be in the public domain.
To be successful, a
sweetener should be competitively priced with sucrose and other comparable
sweeteners. It should be easily produced, stored, and transported.
1.2.
Relative Sweetness:
Perceived sweetness is
subjective and depends on or can be modified by a number of factors. The
chemical and physical composition of the medium in which the sweetener is
dispersed has an impact on the taste and intensity. The concentration of the
sweetener, the temperature at which the product is consumed, pH, other
ingredients in the product, and the sensitivity of the taster are all
important. Again, sucrose is the usual standard. Intensity of the sweetness of
a given substance in relation to sucrose is made on a weight basis.
1.3.
The Multiple Ingredient Approach:
The advantages of the
multiple sweetener approach have long been known. A variety of approved
sweeteners are essential because no sweetener is perfect for all uses. With
several available, each sweetener can be used in the application(s) for which
it is best suited. Manufacturers also can overcome limitations of individual
sweeteners by using them in blends.
During the 1960s,
cyclamate and saccharin were blended together in a variety of popular diet soft
drinks and other products. This was really the first practical application of
the multiple sweetener approach. The primary advantage of this sweetener blend
was that saccharin (300 times sweeter than sucrose) boosted the sweetening
power of cyclamate (30 times sweeter), whereas cyclamate masked the aftertaste
that some people associate with saccharin. The two sweeteners when combined
have a synergistic effect—that is the sweetness of the combination is greater
than the sum of the individual parts. This is true for most sweetener blends.
Cyclamate was the major factor in launching the diet segment of the carbonated
beverage industry. By the time it was banned in the United States in 1970, the
products and trademarks had been well established.
Such a large market for
diet beverages provided a tremendous incentive to develop new sweeteners. After
cyclamate was taken off the market in 1970, saccharin was the only available
low-calorie alternative to sugar available in the United States for more than a
decade. But now with the availability of acesulfame potassium, aspartame,
sucralose, and saccharin, the multiple sweetener approach is a visible reality
in the United States. Fountain soft drinks generally contain a combination of
saccharin and aspartame and bottled drinks are available with combinations of
aspartame and acesulfame K, as well as sucralose and aspartame.
Triple blends, such as
acesulfame potassium, aspartame, and saccharin and aspartame, cyclamate, and
acesulfame potassium are being used in some parts of the world.
The polyols also are
important adjuncts to sugar-free product development. These sweeteners provide
the bulk of sugar but are generally less sweet than sucrose. The polyols, which
are reduced in calories, combine well (e.g., they are synergistic) with
low-calorie sweeteners, resulting in good-tasting reducedcalorie products that
are similar to their traditional counterparts. With the availability of fat
replacers and low-calorie bulking agents (e.g., polydextrose), not just a
multiple sweetener approach but a multiple ingredient approach to calorie
control is being used. In addition to the evidence that humans have an innate
desire for sweets, research indicates that the obese and those who once were
obese may have a greater preference than others for fatty liquids mixed with
sugar. Replacing the fat and the sugar is therefore important in the
development of products to assist in calorie control.
2.
Alternate Sweeteners and their Productions:
A)
High Intensity Sweeteners:
2.1.
Acesulfame K:
Acesulfame K is 200
times sweeter than sucrose
(table sugar), as sweet as aspartame,
about 2/3 as sweet as saccharin, and 1/3 as sweet as sucralose.
Like saccharin, it has a slightly bitter aftertaste,
especially at high concentrations. Kraft Foods
has patented the use of sodium ferulate
to mask acesulfame's aftertaste. Acesulfame K is often blended with other
sweeteners (usually sucralose or aspartame). These blends are reputed to give a
more sugar-like taste whereby each sweetener masks the other's aftertaste,
and/or exhibits a synergistic
effect by which the blend is sweeter than its components.
Unlike aspartame,
acesulfame K is stable under heat, even under moderately acidic or basic
conditions, allowing it to be used as a food additive
in baking, or in products that require a long shelf life. In carbonated
drinks, it is almost always used in conjunction with another sweetener, such as
aspartame or sucralose. It is also used as a sweetener in protein shakes and
pharmaceutical products, especially chewable and liquid medications, where it
can make the active ingredients more palatable.
2.1.1.
Production:
Biotechnological
production of Acesulfame K:
Not yet been identified.
2.2.
Cyclamate:
Sodium cyclamate (sweetener code 952) is an artificial sweetener. It is 30–50
times sweeter than sugar (depending on concentration; it is not a linear
relationship), making it the least potent of the commercially used artificial
sweeteners. Some people find it to have an unpleasant aftertaste,
but, in general, less so than saccharin
or acesulfame potassium. It is often
used with other artificial sweeteners, especially saccharin; the mixture of 10
parts cyclamate to 1 part saccharin is common and masks the off-tastes of both
sweeteners. It is less expensive than most sweeteners, including sucralose,
and is stable under heating.
2.2.1.
Production:
Cyclamates are produced
from cyclohexylamine (obtained by the reduction of aniline) by sulfonation.
Production of sodium and calcium cyclamates in western Europe (annual capacity,
4200 tonnes) in 1995 was estimated to be 4000 tonnes. Brazil (annual capacities
of 4000 and 2000 tonnes of sodium and calcium cyclamate, respectively) produced
over 2300 and 300 tonnes of sodium and calcium cyclamate, respectively, in
1993. There is no commercial production of cyclamates in Japan or the United
States. Information available in 1995 indicated that cyclamic acid was produced
in Japan, Spain, Taiwan and the United States, that sodium cyclamate was
produced in Argentina, Brazil, Germany, Indonesia, Japan, the Netherlands,
Romania, South Africa, Spain, Taiwan, Thailand and the United States and that
calcium cyclamate was produced in Argentina, Brazil, Japan, South Africa,
Taiwan and the United States (Chemical Infor- mation Services, 1995). Sodium
and calcium cyclamates are used as non-nutritive sweeteners; sodium cyclamate,
simply known as ‘cyclamate’, is the more common salt. Calcium cyclamate is used
in low-sodium and sodium-free products. World con- sumption of cyclamates in
1995 was estimated to be 15 000 tonnes. Western European con- sumption of
cyclamates was estimated to be 4000 tonnes. Canadian consumption of cyclamates
in 1995 was estimated to be 60 tonnes. Consumption of sodium cyclamate in China
in 1995 was estimated to be 4400 tonnes.
Biotechnological
production of Cyclamate:
Not yet been
identified.
2.3.
Alitame, Aspertame and Neotame:
Alitame
is an artificial sweetener developed by Pfizer
in the early 1980s and currently marketed in some countries under the brand
name Aclame. Like aspartame, alitame is an aspartic acid-containing
dipeptide.
Most dipeptides are not sweet, but the unexpected discovery of aspartame in
1965 led to a search for similar compounds that shared its sweetness. Alitame
is one such second-generation dipeptide sweetener. Neotame,
developed by the owners of the NutraSweet brand, is another.
Alitame has several
distinct advantages over aspartame. It is about 2000 times sweeter than sucrose,
about 10 times sweeter than aspartame, and has no aftertaste.
Its half-life
under hot or acidic conditions is about twice as long as aspartame's, although
some other artificial sweeteners, including saccharin
and acesulfame potassium, are more
stable yet. Unlike aspartame, alitame does not contain phenylalanine,
and can therefore be used by people with phenylketonuria.
Aspartame
is an artificial, non-saccharide sweetener
used as a sugar substitute in some foods and
beverages. In the European Union, it is codified as E951.
Aspartame is a methyl
ester
of the aspartic acid/phenylalanine
dipeptide.
It was first sold under the brand name NutraSweet;
It was first synthesized in 1965 and the patent expired in 1992.
The safety of aspartame has been the subject
of several political and medical controversies, congressional hearings and Internet
hoaxes since its initial approval for use in food products by the U.S. Food and Drug Administration
(FDA) in 1981. A 2007 medical review on the subject concluded that "the
weight of existing scientific evidence indicates that aspartame is safe at
current levels of consumption as a non-nutritive sweetener". However,
because its breakdown products include phenylalanine,
aspartame must be avoided by people with the genetic condition phenylketonuria.
Neotame
is an artificial sweetener made by NutraSweet
that is between 7,000 and 13,000 times sweeter than sucrose
(table sugar). In the European Union, it is known by the E number
E961. It is moderately heat-stable, extremely potent, rapidly metabolized,
completely eliminated and does not appear to accumulate in the body.
The major metabolic
pathway is hydrolysis of the methyl
ester
by esterases
that are present throughout the body, which yields de-esterified neotame and methanol.
Because only trace amounts of neotame are needed to sweeten foods, the amount
of methanol derived from neotame is much lower than that found in common foods.
The product is
attractive to food manufacturers, as its use greatly lowers the cost of
production compared to using sugar or high fructose corn syrup (due to the
lower quantities needed to achieve the same sweetening), while also benefitting
the consumer by providing fewer "empty" sugar calories
and a lower impact on blood sugar.
2.3.1.
Production:
Biotechnological
Production:
All three above
Sweeteners are amino acid and peptide based sweeteners, so they can be produced
by formulation of individually produced Amino acids and peptides.
Production of amino
acids by biotechnology methods Biotechnological production processes have been
used for industrial production of amino acids for about 50 years now.
Microbiological
(biotechnology) methods for the industrial production of amino acids are of
three types: use of microbial enzymes or immobilized cells (enzymatic method),
semi-fermentation, and direct fermentation. Enzyme catalysis is well
established in the chemical industry. With the enzymatic method, an amino acid
precursor is converted to the target amino acid using one or two enzymes. This
method uses pure enzymes, rather than the enzyme systems of living
microorganisms, as in the fermentation methods. The enzymatic method allows the
conversion to a specific amino acid without microbial growth, thus eliminating
the long process from glucose. Industrial use of enzymes for production of
L-amino acids began about 40 years ago in Japan with the resolution of N-acetyl
D,L-amino acids by immobilized acylase. Significant production by enzymatic
catalysis is currently in place for alanine, aspartic acid, cysteine, cystine,
methionine, phenylalanine, serine, tryptophan and valine. For example,
L-aspartic acid is manufactured mainly by enzyme-catalyzed addition of ammonia
to fumaric acid and this is the preferred manufacturing method. Since only the
naturally occurring L-form is produced that way, resolution is not necessary,
unlike for chemically synthesized amino acid racemic mixtures. For the
production of L-methionine, enzymatic resolution with acylase from Aspergillus
oryzae in the enzyme membrane reactor is the method of choice. Several hundred
tons of L-methionine and L-valine are produced each year using this technology.
L-cysteine is manufactured by industrially used enzymatic process in which the
thiazoline derivative DL-2-amino- 2-thiazoline-4-carboxylic acid is converted
with the help of three enzymes (L-ATC hydrolase, S-carbamoyl-Lcysteine
hydrolase, and ATC racemase) from Pseudomonas thiazolinophilum.
According to
Leuchtenberger et al. enzyme catalysis is a particularly elegant and popular
method of producing D-amino acids and nonproteinogenic L-amino acids. The
enzymatic production of L-tryptophan from precursors involves a single reaction
step. It may be performed with isolated enzymes, either tryptophan synthase or
tryptophanase, or by a variety of microorganisms with these enzyme activities
such as E. coli. The enzymatic method has now been supplanted by a continuous
microbiological process in which the reacting solution passes over a fixed bed
of an immobilized microorganism. With semi-fermentation, a metabolic
intermediate in the amino acid biosynthesis or its precursor is converted to
the amino acid during fermentation. The fermentation method is being applied to
industrial production of most L-amino acids. This method utilizes the
phenomenon that microorganisms convert nutrients to various vital components
they need. With the fermentation method, raw materials such as syrups are added
to microorganism culture media, and the proliferating microorganisms are
allowed to produce amino acids. Enzymes play an important role in the
production of amino acids by fermentation. Consecutive reactions by 10 to 30
types of enzymes are involved in the process of fermentation, and various amino
acids are produced as a result of these reactions. The fermentation may take
place on a culture medium composed of grains, sugar, molasses, yeast, or other
biological material, e.g. petrochemicals, such as paraffin, and synthetic
nutrients such as ammonium chloride, ammonium nitrate, and potassium phosphate.
Extraction is achieved by physical and mechanical means, such as heat or
maceration, as well as by chemical methods such as petroleum solvents, ammonia,
strong acids, strong bases, and/or ion exchange. The final product is obtained
as crystalline powder. Once a suitable microorganism has been selected for the
fermentation method, it is necessary to enhance its potential in order to take
full advantage of the potential of the organism. Generally, microorganisms
produce the 20 kinds of amino acids only in the amounts they need. They have a
mechanism for regulating the quantities and qualities of enzymes to yield amino
acids only in the amounts necessary for themselves. Releasing this regulatory
mechanism allows manufacturing of the target amino acid in large amounts. The
yield of an amino acid depends on the quantities and qualities of the enzymes.
The yield increases if the enzymes involved in the production of the target
amino acid are present in large quantities under workable conditions, whereas,
if the enzymes are present in small quantities, it decreases. Strains are
improved using various techniques.
Improvements in
biotechnology production of amino acids:
The genome of the
production organism is altered at random, and more efficient mutants are identified
in a subsequent selection process. The mutants are usually characterized by
increased membrane permeability, regulation defects, or biosynthesis enzymes
with altered kinetic characteristics. Fundamental understanding of the
microbial amino acid metabolism is necessary for further increases in
productivity. Detailed analysis allows quantification of the flow of
metabolites in a fermentation process as a function of time (metabolic flux
analysis). In this way the addition of nutrients can be optimized, and yields
can be increased as a result. Metabolic flux analysis also makes it possible to
model the metabolism of a given production strain (metabolic modeling).
Another important
requirement for the targeted improvement of amino acid production strains is
knowledge about the genome of the microorganism concerned. The genome of C.
glutamicum has already been sequenced. Not only fermentation processes, but
also enzymatic processes are undergoing continual improvement, especially
concerning the identification of novel enzymes. Unlike the classical screening,
modern technologies access the genetic information directly. Specific
characteristics of known enzyme systems can also be improved through
incorporation of mutations into the genes of the biocatalyst. The optimized
biocatalysts can also be transferred into production strains to avoid bottle
necks in amino acid biosynthesis.
Direct
production of Aspertame by rDNA technology:
Nonribosomal peptide
synthesis is an alternative method for the production of pharmacologically and
biotechnologically important products. Many microorganisms produce these as
secondary metabolites by using nonribosomal peptide synthetases (NRPSs). The
assembly of nonribosomally synthesized peptides is determined by the sequence
of catalytically independent modules of the multifunctional NRPSs. Nonribosomal
Asp-Phe formation. Hybrid dimodular peptide synthetases were generated by
fusion of native gene fragments, to contain the domain sequence
A(Asp)-PCP-C-A(Phe)-PCP-Te, and used for in vitro production of the dipeptide
Asp-Phe.
Escherichia coli
BL21(kDE3)-gsp was transformed with the plasmids which was incorporated with
this Domain sequences. This E. coli strain carries the pREP4-gsp plasmid
supplying the gene gsp coding for the 4¢-Ppant transferase Gsp. The cells were
induced at D600 ¼ 0.6–0.7 with 0.2 mM isopropyl thio-b-D-galactoside and
incubated for an additional 1.5 h at 30
C before being harvested. Thus Aspertame will be directly produced by over expression of recombinant gene.
2.4.
Neohesperidin Dihydrochalcone:
The genus Citrus is
an important resource for natural products with direct benefits to humans
ranging from health-benefiting compounds to flavor and aroma compounds used by
the food, beverage, and cosmetics industries. Compounds of applied interest
include flavonoids belonging to the flavanone subgroup, which accumulate to
considerable levels in the leaves and fruit of citrus species where their
biological role in planta is not clear. The bitter flavanone neohesperidin,
abundant mainly in inedible citrus fruit species, is the substrate for the
commercial production of neohesperidin dihydrochalcone (NHDC), a seminatural
and generally regarded as safe lowcalorie sweetener with unique properties.
NHDC is approximately 1500-fold sweeter than sucrose, is stable under heat and
a wide pH range, and has also been documented to function as a bitterness
blocker and flavor enhancer with diverse uses in the food, beverage,
pharmaceutical, and animal feed industries. NHDC for human consumption is
usually combined with other sweeteners resulting in (i) a synergistic
sweetening effect allowing for lower overall concentrations of the sweetening
compounds and (ii) masking of sweetener aftertastes to derive a flavor
resembling sucrose. NHDC has been a commercial product for many years; however,
production is limited by the availability of the substrate neohesperidin, which
accumulates to significant levels only in inedible citrus species such as Citrus
aurantium (bitter orange) and other citrus hybrids that are especially
grown for this purpose. Neohesperidin has also been detected in Citrus aurantium
callus cultures; however, the growth rate of citrus callus/cell cultures is
extremely low, and therefore, this source does not have commercial potential.
NHDC may also be produced by chemical conversion of naringin (the dominant
bitter flavonoid in grapefruit peels) into neohesperidin, but the amount of
grapefruit peels is limiting. An isomeric flavanone, the rutinoside hesperidin,
is the predominant flavanone in orange peels (over 90%), as well as lemon and
mandarin peels, and is readily available in large quantities as a byproduct of
the vast orange juice industry. However, hesperidin (hesperetin-7-O-rutinoside)
could not be converted into neohesperidin (hesperetin-7-O-neohesperidoside)
by chemical or biological modification using the tools available up to now.
2.4.1.
Production:
Biotechnological
production:
Extraction of
Hesperidin from Orange Peels and Hydrolysis.
Hesperidin was
extracted from ripe orange (Citrus sinensis var. Shamouti) peels by a
modification of a published protocol as follows: Orange peels (100 g) were
pulverized in 500 mL of saturated Ca(OH)2 using a homogenizer (the
pH was measured to be _12). The homogenate was transferred to a bottle and
flushed with nitrogen before closing. The homogenate was heated to 60 °C for 1
h and left to cool slowly, and the pH was adjusted to _12 by the addition of
Ca(OH)2. Peel particles were filtered out of the homogenate using
gauze and filter paper. The homogenate was centrifuged at 20000g for 15
min, and the supernatant was collected and filtered through a nylon membrane
(0.45 ím). The pH of the now clear solution was lowered to 3.5 and
incubated at room temperature overnight to induce ring C closure and to
precipitate the hesperidin. The precipitate was collected by centrifugation at
20000g for 15 min, and the supernatant was discarded. The precipitate
was washed with 10 mL of acetone and dried into a powder.
For hydrolysis, 50 mg
of hesperidin was dissolved in 500 µL of DMSO that was then added to 25
mL of preheated (65 °C) 0.25% aqueous H2SO4 and immediately autoclaved for two
consecutive cycles at 121 °C for 25 min. Hydrolysis products were allowed to
precipitate overnight at room temperature, the supernatant was discarded, and
the precipitate was dried in a lyophilizer.
Process
for Conversion of Orange Peel Hesperidin into Neohesperidin:
Process to convert
hesperidin extracted from orange peels into neohesperidin using metabolic
engineering and biotransformation via three steps: (i) extraction of
hesperidin, (ii) full or partial hydrolysis of hesperidin sugar moieties, and
(iii) biotransformation of hesperidin hydrolysis products into neohesperidin. (i)
Various methods for the extraction of hesperidin from orange peels have been
described and are appropriate for industrial applications. (ii) Hesperidin can
be enzymatically hydrolyzed to hesperetin-7-O-glucoside (HG) and hesperetin
using hesperidinase (a mixture of R-L-rhamnosidase and â-D-glucosidase);
however, the solubility of hesperidin in aqueous solutions at temperatures compatible
with enzymatic activity is very low, making this approach somewhat of a
challenge for industrial applications.
Alternatively,
efficient hydrolysis of hesperidin into HG and hesperetin can be achieved using
dilute acid catalysis under high temperatures, which allows for efficient
solubilization of hesperidin in the aqueous solution. This process is simple
and is straightforward for application in the industry. (iii) A tobacco cell
suspension culture containing endogenous 7-Oglucosyltransferase activity was
transformed with the gene Cm1,2RhaT encoding a flavanone-7-O-glucoside-2-O-rhamnosyltransferase
and was shown to convert naringenin and naringenin-7-O-glucoside into
naringin. Because modifications on flavonoid ring B appear not to substantially
affect the affinity of the enzyme to the flavonoid substrate, it is expected
that HG is a substrate for the enzyme encoded by Cm 1,2RhaT. This
expectation is further supported by the fact that the 1,2RhaT enzyme purified
from pummelo leaves was active on both naringenin-7-O-glucoside and HG
substrates. Therefore, we propose to use cell suspension cultures transformed
with Cm 1,2RhaT for biotransformation of HG and hesperetin into
neohesperidin in one or two enzymatic steps, respectively.
Conversion of
neohesperidin into NHDC was first demonstrated by Horowitz and Gentili. This
simple and established procedure isomerizes neohesperidin into a chalcone under
alkaline conditions, and the reductive hydrogenation in the presence of a
catalyst completes the conversion to NHDC.
2.5.
Saccharin:
Saccharin
is an artificial sweetener. The basic
substance, benzoic sulfilimine, has effectively no food energy
and is much sweeter than sucrose,
but has a bitter or metallic aftertaste,
especially at high concentrations. It is used to sweeten products such as
drinks, candies, cookies, medicines, and toothpaste.
Saccharin is unstable
when heated but it does not react chemically with other food ingredients. As
such, it stores well. Blends of saccharin with other sweeteners are often used
to compensate for each sweetener's weaknesses and faults. A 10:1 cyclamate
: saccharin blend is common in countries where both these sweeteners are legal;
in this blend, each sweetener masks the other's off-taste. Saccharin is often
used together with aspartame in diet carbonated soft drinks, so that
some sweetness remains should the fountain syrup be stored beyond aspartame's
relatively short shelf-life. Saccharin is believed to be an important
discovery, especially for diabetics,
as it goes directly through the human digestive system
without being digested. Although saccharin has no food energy,
it may trigger the release of insulin
in humans and rats, presumably as a result of its taste, but this is not conclusive
as the same study states "No statistically significant changes in plasma
insulin were found."
2.5.1.
Production:
A number of companies
around the world manufacture saccharin. Most manufacturers use the basic
synthetic route described by Remsen and Fahlberg in which toluene is treated
with chlorosulfonic acid to produce ortho- and para-toluenesulfonyl chloride.
Subsequent treatment with ammonia forms the corresponding toluenesulfonamides.
ortho-Toluenesulfonamide is separated from the para-isomer (this separation is
alternatively performed on the sulfonyl chlorides), and
ortho-toluenesulfonamide is then oxidized to ortho-sulfamoylbenzoic acid, which
on heating is cyclized to saccharin. ortho-Toluenesulfonamide can occur as a
contaminant in saccharin produced by this process, but not in that produced by
the Maumee process, described below. The only producer in the United States
currently uses the Maumee process, in which saccharin is produced from purified
methyl anthranilate, a substance occurring naturally in grapes. In this
process, methyl anthranilate is first diazotized to form
2-carbomethoxybenzenediazonium chloride. Sulfonation followed by oxidation
yields 2-carbomethoxybenzenesulfonyl chloride. Amidation of this sulfonyl
chloride, followed by acidification, forms insoluble acid saccharin. Subsequent
addition of sodium hydroxide or calcium hydroxide produces the soluble sodium
or calcium salt.
China is the world’s
largest producer of saccharin, accounting for 30–40% of world production, with
an annual production of approximately 18 000 tonnes in recent years; its
exports amounted to approximately 8000 tonnes. In 1995, the United States
produced approximately 3400 tonnes of saccharin and its salts, and Japan
produced approximately 1900 tonnes. In the past, several western European
companies produced sodium saccharin; however, by 1995, western European
production had nearly ceased due to increasing imports of lower-priced
saccharin from Asia. Information available in 1995 indicated that saccharin was
produced in 20 countries, calcium saccharin was produced in five countries, and
sodium saccharin was produced in 22 countries.
Biotechnological
Production:
Not yet identified.
2.6.
Sucralose:
Sucralose
is an artificial sweetener. The majority
of ingested sucralose is not broken down by the body, so it is noncaloric. In
the European Union, it is also known
under the E number (additive code) E955. Sucralose
is approximately 320 to 1,000 times as sweet
as sucrose
(table sugar), twice as sweet as saccharin,
and three times as sweet as aspartame.
It is stable under heat and over a broad range of pH conditions.
Therefore, it can be used in baking or in products that require a longer shelf
life. The commercial success of sucralose-based products stems from its
favorable comparison to other low-calorie sweeteners in terms of taste,
stability, and safety.
2.6.1.
Production:
Sucralose is
manufactured by the selective chlorination of sucrose (table sugar), which
substitutes three of the hydroxyl groups with chlorine. This chlorination is
achieved by selective protection of the primary alcohol groups followed by
acetylation and then deprotection of the primary alcohol groups. Following an
induced acetyl migration on one of the hydroxyl groups, the partially
acetylated sugar is then chlorinated with a chlorinating agent such as
phosphorus oxychloride, followed by removal of the acetyl groups to give
sucralose.
Biotechnological
production:
This procedure involves
the chemical chlorination of raffinose to form a novel tetrachloroaffinose
intermediate (6,4′,1″,6″-tetrachloro-6,4′,1″,6″-tetradeoxygalactoraffinose;
TCR) followed by the enzymic hydrolysis of the α-1-6 glycosidic bond of TCR to
give sucralose and 6-chlorogalactose. Synthesis of raffinose was achieved from
saturated aqueous solutions of galactose and sucrose using a selected
α-galactosidase from Aspergillus niger. When raffinose is used as a
starting material for sucralose synthesis, this route has fewer steps than
either the preceeding method using glucose-6-acetate as an intermediate or the
complete chemical synthesis from sucrose.
B)
Polyols: (Bulk Sweeteners):
Second category of
sweeteners that can substitute for both the physical bulk and sweetness of
sugar includes the sugar alcohols (also called “polyols”) sorbitol, mannitol,
xylitol, isomalt, erythritol, lactitol, maltitol, hydrogenated starch
hydrolysates, and hydrogenated glucose syrups and often termed as “sugar
replacers” or “bulk sweeteners.” Other 2 sweeteners, namely, trehalose and
tagatose, although are actually sugars rather than sugar alcohols yet are
similar in function to the polyols. These sweeteners are being industrially
explored for their application as food ingredient in food products in which the
volume and texture of sugar, as well as its sweetness, are important, such as
sugar-free candies, cookies, and chewing gum. Many of these products are
marketed as “diabetic foods.” Some of these substances, such as sorbitol,
xylitol, and tagatose, also occur naturally in certain fruits or other food
while some are produced by yeast, fungi, and bacteria. Several health-promoting
effects have also been attributed to the addition of these ingredients and
hence addition/fortification to foods can lead to products with functional
benefits. These sweeteners can either be directly added to foods, or use of
these sweeteners-producing microorganisms can lead to natural foods containing
these sweeteners.
Total global sugar
alcohol production in the $10.92 billion global sweetener market has been
estimated to be at 836905 tons. The consumption of sugar alcohols and
high-intensity sugar is poised to rise as much as 15%. Sorbitol made up the
largest share of sugar alcohols (more than 54%), and tagatose is emerging as
another much sought after sweetener with a fast growth rate estimated at more
than 20% to 25% in near future. Other sugar alcohols including erythritol,
maltitol, and xylitol have also increased their share of this market.
Polyols and other bulk
sweeteners have a couple of potential advantages over sugar as food ingredients
:
Ø Do
not promote the development of dental caries (tooth decay) and hence, the USFDA
has authorized a health claim pertaining to dental caries on foods made with
polyols provided that these foods contain one or a combination of the approved
sugar alcohols, meet the criteria for “sugar free,” and do not lower plaque pH
in the mouth below 5.7 during consumption or up to 30 min afterward.
Ø Produce
a lower glycemic response than most sugars and starches do. Thus, their use may
be advantageous for people with diabetes.
Ø Have
low calorific values due to their poor digestion and poor absorption.
2.7. Mannitol:
Mannitol, a naturally
occurring polyol (sugar alcohol with 6 carbon atoms), is found in animals and
plants. It is present in small quantities in most fruits and vegetables.
Typically, it can be found in such plants as pumpkins, celery, onions, grasses,
olives, mistletoe, and lichens. Mannitol is also found in manna, the dried
exudate of the manna ash tree known as Fraxinus ornus. Manna is obtained
by heating the bark of the tree and it can contain up to 50% mannitol. Hence,
manna has been a commercial source of mannitol in Sicily, Italy. Marine algae,
especially brown algae, are also rich in mannitol. Furthermore, mannitol is
commonly found in the mycelium of various fungi and is present in fresh
mushrooms (about 1%). Also, some fungi and bacteria produce mannitol. Moreover,
small quantities of mannitol are found in wine. It is widely used in the food,
pharmaceutical, medicine, and chemical industries. It is only half as sweet as
sucrose. Mannitol also exhibits reduced caloric values compared to the
respective value of most sugars, for example, the calorific values of mannitol
and sucrose are 1.6 kcal/g and 4 kcal/g, respectively. The solubility of
mannitol in water is significantly lower than that of sorbitol and most of the
other sugar alcohols. At 14 °C, the solubility of mannitol in water is only
approximately 13% while at 25 °C the solubility of mannitol in water is
approximately 18%. Mannitol is sparingly soluble in organic solvents, like
ethanol and glycerol, and practically insoluble in ether, ketones, and
hydrocarbons. Mannitol forms white orthorhombic needles and the crystals have a
melting point of 165 to 168 °C. A cooling sensation occurs when mannitol
crystals dissolve in mouth. This effect is commercially used in chewing gums.
Crystalline mannitol exhibits a very low hygroscopicity and is chemically
inert. These properties make mannitol very useful in production of tablets and
granulated powders. Mannitol is considered safe for use in foods and it has a
food additive status. Mannitol is presently on the U.S. FDA GRAS-/INTERIM (generally
recognized as safe) list.
Technological
application
Mannitol is a valuable
nutritive sweetener because it is nontoxic, nonhygroscopic in its crystalline
form and has no teeth-decaying effects. At present, the main application for
mannitol in the food industry is as a sweetener in sugar-free chewing gums and
for dusting chewing gum sticks. In addition, mannitol is used as a bodying and
texturizing agent, anticaking agent, and humectant. Mannitol has been shown to
exhibit osmoprotecting effect during drying of LAB. Mannitol has the potential
to extend the shelf life of various foodstuffs. As mannitol is only partially
metabolized by humans and does not induce hyperglycemia, which makes it useful
for diabetics and for so-called light foods. Besides, mannitol is commercially
useful in making artificial resins and plasticizers.
Functional application
Mannitol belongs to a
group of drugs referred to as osmotic diuretics. In medicine, mannitol
(“Osmitrol”) is used to increase the formation of urine to prevent and treat
acute renal failure, and also in removal of toxic substances from the body.
Mannitol is also used to reduce both cerebral edema (increased brain water
content) and intraocular pressure. Furthermore, it is used to alter the
osmolarity of the glomerular filtrate in treating kidney failures. In the
pharmaceutical industry, mannitol is commonly used as a constituent in chewable
tablets and granulated powders. Furthermore, its sweet cool taste is used to
mask the unpleasant taste of many drugs.
Mannitol has also been
shown to have antioxidant effect by scavenging off free hydroxyl radicals. Kostopanagiotou
studied the effect of mannitol in the prevention of lipid peroxidation during
major liver resections performed during hepatic inflow occlusion. They
concluded that mannitol has an antioxidant activity, but were unable to confirm
a positive impact on the postoperative clinical course. Mendoza also found the
antioxidant effect of mannitol on hyaluronan depolymerization. Hyaluronan (HA)
was depolymerized by hydroxyl radicals generated from hydrogen peroxide and
cupric ions. About 26.51 mM of mannitol was needed to decrease the degradation
of HA by 50%.
2.7.1. Production:
Biotechnological
Production:
Mannitol is a 6-carbon
sugar alcohol that has been produced traditionally by chemical catalysis.
However, yeast, fungi, and especially LAB are also known to produce mannitol.
When compared fungal and bacterial processes for the production of mannitol,
the latter proved to be better. Biotechnology has brought advances to the
production in terms of substrate purity, process equipment requirements, and
safety. Enzymatic methods have improved the yields and the use of microbes has
brought versatility to the range of substrates that can be used in the
processes. Some of the microbial processes are already industrially feasible
and could be taken to further use.
Some homofermentative
LAB were found to produce small amounts of mannitol intracellularly, for
example, Streptococcus mutans, Lb. leichmanii, lactate
dehydrogenase-negative mutant of Lb. plantarum, and lactate
dehydrogenase deficient mutant of Lc. lactis.
The most promising
production strategy reported so far has been the utilization of nongrowing
cells of heterofermentative LAB for converting fructose to mannitol.
Heterofermentative LAB belonging to the genera Lactobacillus and Leuconnostoc
are the potent producer of mannitol. These bacteria contain the enzyme mannitol
dehydrogenase to convert fructose into mannitol. The LAB reported to produce
mannitol are Ln. pseudomesenteroides, Lactobacillus spp. B001, Lactobacillus
spp. KY-107, Lactobacillus spp. Y-107 and Leuconostoc spp. Y-002,
Ln. pseudomesenteroides ATCC 12291, Lb. sanfranciscensis, and
most recently Saha studied mannitol production by Lb. intermedius NRRL
B-3693 using inulin as a substrate. He reported that when fructose and inulin
mixture (3: 5, total 400 g/L) was used as substrate, the bacterium produced
227.9 ± 1.8 g/L of mannitol. He also used molasses and corn steep liquor as a
cheap substrate for mannitol production. Most studies on microbial mannitol
production are based on batch cultivations and commonly only moderate
production levels (yields or volumetric productivities) have been reported. The
yield can be improved by screening for more efficient production strains. To
improve the volumetric productivity of bioprocesses, the use of membrane
cell-recycle bioreactor (MCRB) systems has been suggested. Soetaert and
coauthors have studied extensively the production of mannitol with a
heterofermentative lactic acid bacterium (LAB), Ln. pseudomesenteroides
ATCC-12291. In fed–batch cultures an average volumetric mannitol productivity
of about 6.3 g/L/h and a mannitol yield from fructose of 94 mol% ([mole of
mannitol produced/mole of fructose used]× 100) were achieved. An improved
volumetric productivity (8.9 g/L/h) but a low yield (60 mol%) was reported for
cells immobilized to reticulated polyurethane foam. Ojamo suggested the
production of mannitol by high densities of immobilized Ln.
pseudomesenteroides cells. In this process, the average volumetric mannitol
productivity and mannitol yield from fructose were approximately 20 g/L/h and
85 mol%, respectively. Weymarn studied the production of mannitol by
heterofermentative LAB in a resting state. They developed a very efficient
mannitol production process by combining membrane cell-recycle technology and
high cell density batch cultures. They reported the volumetric mannitol
productivity (26.2 g/L/h) and mannitol yield 97 mol%. Using the same initial
biomass, a stable high-level production of mannitol was maintained for 14
successive bioconversion batches.
Helanto constructed
random mutants of Ln. pseudomesenteroides by chemical mutagenesis for
improving mannitol production of the mutant lacking the fructokinase activity.
The best mutant showed fructokinase activity of only 10% of that of the
wild-type strain. The effect of the random mutation on mannitol production was
also studied in bioreactors under process conditions. The mutant strain grew
and consumed sugars faster than the parent strain. Less side products (acids,
carbon dioxide, and ethanol) were synthesized by the mutant cells and an
increased fraction of fructose was reduced to mannitol by mannitol
dehydrogenase (increased yield of mannitol from 74 to 86 mol%). The latest
developments in the field have dealt with the use of recombinant strains in
mannitol production.
In 2004, in response to
a petition filed by zuChem Inc. (Chicago, Ill., U.S.A.), the USFDA amended
additive regulations to permit the manufacture of mannitol by fermentation of
sugars such as fructose, glucose, or maltose by the action of the microorganism
Lb. intermedius (fermentum).
2.8. Tagatose:
Tagatose is an isomer
of fructose that occurs naturally in some dairy products. In comparison to
sugar it is metabolized differently, providing fewer calories and producing a
smaller glycemic response. In chemical terms, D-tagatose has melting
temperature, 134 °C; stable at pH 2 to 7; solubility, 58% (w/w) at 21 °C;
viscosity, 180 cP at 70% (w/w) at 20 °C. It is involved in browning reactions
during heat treatment and decomposes more readily than sucrose at high
temperatures. Tagatose is derived from lactose, the sugar found in milk. During
the production of tagatose, after enzyme hydrolysis of lactose the 2
monomers—galactose and glucose—are chromatographically separated. The galactose
fraction is then converted to tagatose under alkaline conditions. Tagatose is a
functional sweetener and is very similar in texture to sucrose (table sugar)
and is 92% as sweet, but with only 38% of the calories.
D-tagatose is a
malabsorbing sugar, as it is poorly absorbed in the small intestine. Its
unabsorbed fraction is completely fermented by the intestinal microflora in
intestine and the formed short-chain fatty acids are quickly absorbed and
metabolized. During fermentation, there is a relatively low-energy recovery,
and rather a certain amount of energy is lost due to increased biomass
excretion of the microflora. D-tagatose has a sucrose-like taste with no
cooling effect or aftertaste. It is similar to the polyols in having a low
caloric value and tooth-friendly property. However, it has no laxative effect
unlike polyols. Although similar to sucrose in taste, it does not contribute to
calorie production. D-tagatose has been found not to contribute to net energy
as revealed by a number of growth studies on rats and human clinical trials
show that subjects gradually and consistently lose weight at medically
desirable rates. As a result of these properties, D-tagatose is considered to
be a potential reduced-energy sweetener.
Technological
application
D-tagatose can be used
as a low-calorie sweetener (1.5 Kcal), as an intermediate for synthesis of
other optically active compounds, and as an additive in detergent, cosmetic, and
pharmaceutical formulation. Besides effecting enhancement of flavor, it
provides the natural taste and texture of sugar. According to Taylo who
evaluated “Physical properties and consumer liking of cookies prepared by
replacing sucrose with tagatose,” the latter appears to be suitable as a
partial replacer for sucrose in cookies based on similar dough properties,
cookie properties, and likeness scores. Using tagatose to replace sucrose in
foods would reduce the amount of metabolizable sugars in the diet as well as
provide the desirable prebiotic effect. On the commercial front, in 1996, Arla
Foods Ingredients in the United States licensed the exclusive worldwide rights
to produce and market tagatose for use in foods and beverages from Spherix.
This sweetener is being marketed and sold as an ingredient under the Gaio name
and used in chocolate products to be sold in New Zealand and Australia by Miada
Sports Nutrition of New Zealand since 2004.
Functional application
A number of health
benefits have been attributed to tagatose such as promotion of weight loss, no
glycemic effect, antiplaque, noncariogenic, antihalitosis, prebiotic, and
antibiofilm properties, organ transplants, improvement of pregnancy and fetal
development, treatment of obesity, reduction in symptoms associated with type 2
diabetes, hyperglycemia, anemia, and hemophilia.
Prebiotic effect
A small difference in chemical structure of tagatose compared to fructose gets
translated into a significant difference in the overall metabolism of the
sugar. The fructose carrier-mediated transport in the small intestine exhibits
little affinity for tagatose, and only approximately 20% of ingested tagatose
is absorbed in the small intestine. The absorbed part is metabolized in the
liver a la fructose. The major part of ingested tagatose is fermented in
the colon by the indigenous microflora resulting in the production of
short-chain fatty acids. In this respect, tagatose is a potential prebiotic.
Tagatose has been found to cause alteration in the composition of colonic
microflora in pigs as evidenced by changes in the proportions of the short-chain
fatty acids produced. This showed that the microflora favored by tagatose
consumption had the potential to produce large amounts of butyrate, which is
believed to be of importance for colonic health. Increased concentrations of
butyrate were also observed in portal vein blood from both adapted pigs fed
tagatose. The appearance of butyrate in the portal vein of unadapted pigs
showed that adaptation to tagatose took place within the 12 h of the
experimental period.
Similar trends were
observed in humans consuming 3 × 10 g tagatose per day for 2 wk. In vitro
fermentation of 1% tagatose with fecal samples from the human volunteers
revealed that the proportion of butyrate was higher when the volunteers were
adapted to tagatose (35 mol% after 4 h of incubation as compared to 25 mol% in
samples from unadapted ones). The human fecal microflora also adapted to
tagatose fermentation within the time span of the experiment (48 h).
Besides, tagatose
effected modification in microbial population density in the feces of the human
volunteers. Pathogenic bacteria (such as coliform bacteria) were reduced, and
specific beneficial bacteria (such as lactobacilli and lactic acid bacteria)
were increased. These results were in agreement with a study on evaluating pure
strains of intestinal bacteria as well as dairy starter cultures for their
ability to ferment tagatose. This study comprised isolates of normal (34
strains) or pathogenic (11 strains) human intestinal bacteria, 22 additional
intestinal isolates from healthy humans, and 107 dairy-type lactic acid
bacteria.
Tagatose was found only
to be fermented by a limited number of the intestinal bacteria and except for
one Clostridium species; the strains that were able to ferment tagatose
belonged to the lactic acid bacteria group, Lactobacillus and Enterococus
and pathogens failed to metabolize tagatose. The high frequency of tagatose
fermentation between intestinal Lactobacillus and Enterococus was
confirmed in the dairy-type lactobacilli and Enterococus species. None
of the Bifidobacterium tested was able to ferment tagatose. Lactobacilli
are important inhabitants of the intestinal tract of man and animals and they
are believed to exert positive effects on intestinal function and health. As
the viability of live bacteria in food products and during transit through the
gastrointestinal tract may be variable, an alternative approach is to stimulate
the growth of beneficial colonic bacteria by nondigestible food, the probiotic
concept. The stimulation of lactobacilli and the increase in butyrate
production in vitro and in vivo indicate that tagatose has
prebiotic properties that could find important applications in functional
foods.
Control of diabetes and
obesity Early human studies suggested tagatose as a
potential antidiabetic drug through its beneficial effects on postprandial
hyperglycemia and hyperinsulinaemia. The incidence of diabetes is increasing
worldwide representing a serious threat to public health. Glycemic control—that
is, maintaining the blood glucose levels as close to the normal range as
possible—has been shown to have positive effects against secondary
complications such as retinopathy (The Diabetes Control and Complications Trial
Research Group 1993. Intake of low glycemic foods, resulting in a lower blood
glucose relative to a similar intake (50 g) of digestible carbohydrates from
white bread or glucose, has been shown to improve glycemic control. In
addition, avoiding obesity and increasing intakes of food rich in dietary fiber
and low glycemic carbohydrate-containing foods have been advocated by WHO/FAO
(1998) as the best means of reducing the rapidly increasing rates of type 2
diabetes. Similar types of foods have also been suggested to have beneficial
effects against obesity and cardiovascular diseases. Lu carried out trials to
confirm the potential of tagatose branded (Naturlose®) for treating
type 2 diabetes, and showed promise for inducing weight loss and raising
high-density lipoprotein cholesterol, both important to the control of diabetes
and constituting benefits independent of the disease. No current therapies for
type 2 diabetes ensure these multiple health benefits. The predominant side
effects of tagatose are gastrointestinal disturbances associated with excessive
consumption, generally accommodated within 1- to 2-wk period. Under an
FDA-affirmed protocol, Spherix is currently conducting a phase-3 trial to
evaluate a placebo-subtracted treatment effect based on a decrease in HbA1c
levels and possible side effects, and contraindications.
Antioxidant activity
Tagatose has been observed to exhibit an antioxidant and a prebiotic, both
properties cited in the maintenance and promotion of health. Paterna studied
the antoioxidant properties of tagatose in cultured murine hepatocyte. They
investigated the effects of tagatose on both the generation of superoxide anion
radicals and the consequences of oxidative stress driven by prooxidant
compounds in intact cells and observed that the extent of nitrofurantoin (redox
cycling drug) (NFT) induced intracellular superoxide anion radical formation
was not altered by tagatose. Consequently, they concluded that tagatose is a
weak iron chelator, which can antagonize the iron-dependent toxic consequences
of intracellular oxidative stress in hepatocytes. The antioxidant properties of
tagatose may result from sequestering the redox-active iron, thereby protecting
more critical targets from the damaging potential of hydroxyl radical.
It has been discovered
that the addition of tagatose to an organ storage and preservative solution
might reduce reperfusion injury of the organ during surgery and/or following
removal of the organ from a subject. Tagatose exerts a dual effect that is
beneficial in preserving the organ and preventing reperfusion injury. First, it
is an aqueous-phase antioxidant. The mechanism of this protective effect
against oxidative cell injury is iron chelation, sequestering iron from
partitioning into membranes and promoting membrane lipid peroxidation. Second,
exposure of organ cells such as liver cells to tagatose massively decreases
ATP, which is beneficial in preventing apoptosis, as ATP is required for the
apoptotic process to be initiated.
2.8.1. Production:
Biotechnological
Production:
D-tagatose occurs
naturally in Sterculia setigera gum, and it is also found in small
quantities in various processed foods such as sterilized and powdered cow's
milk, hot cocoa, and a variety of cheeses, yogurts, and other dairy products.
D-tagatose can be produced from D-galactose by a chemical method using a
calcium catalyst, but the process has some disadvantages, such as complex
purification steps, chemical waste formation, and by-products formation. To
overcome these limitations, biological manufactures of D-tagatose using several
biocatalyst sources have been studied intensively in recent years. Among the
biocatalysts, L-arabinose isomerase catalyzes the conversion of D-galactose to
D-tagatose as well as the conversion of L-arabinose to L-ribulose, economically
feasible tagatose manufacturing process. L-arabinose isomerase has been of
interest for its potential application in galactose isomerization into tagatose
and among other microorganisms; LAB were recognized as source of this enzyme.
The LAB reported to contain L-arabinose isomerase are Lb. gayonii, Lb.
plantarum, and Bifidobacterium longum. Cheetham invented a process
to convert D-galactose to D-tagatose by using LAB. Further, Ibrahim have
patented an enzymatic isomerization process using arabinose isomerase
originating from a lactic acid bacterium. In a similar study, the L-arabinose
isomerase from Lb. plantarum SK-2 was purified to an apparent
homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa.
The optimum activity observed at 50 °C and pH 7.0 was found further to be
stimulated by Mn2+, Fe3+, Fe2+, and Ca2+
and inhibited by Cu2+, Ag+, Hg2+, and Pb2+.
D-galactose and L-arabinose as substrates were isomerized with high activity.
Using the purified L-arabinose isomerase, 390 mg tagatose could be converted
from 1000 mg galactose in 96 h, and this production corresponds to 39%
equilibrium.
The USFDA has granted
GRAS status and authorized a health claim pertaining to tooth decay on products
made with sugar alcohols to include tagatose as a substance eligible for the
health claim, even though tagatose is not a sugar alcohol. Besides, tagatose is
approved in Australia, New Zealand, Korea, and the European Union.
2.9. Sorbitol:
The most commonly used
polyol in the United States is sorbitol, which is the standard sweetener in
several sugar-free chewing gums and over-the-counter medicines. Sorbitol, also
referred to as D-glucitol, is naturally found in many fruits, for example,
berries, cherries, and apples and its worldwide production is estimated to be
higher than 500000 tons/year and continues to rise. Sorbitol is sweet tasting,
forms a viscous solution, stabilizes moisture, possesses bacterio-static
property and is generally chemically inert. These features and properties make
sorbitol an ideal and preferred ingredient in many products. It is freely
soluble in water and acetic acid, ethanol, and methanol. It is insoluble in
common organic solvent. Its melting point ranges from 93 to 98 °C. Sorbitol is
often used in modern cosmetics as a humectant and thickener. Sorbitol is used
as a cryo-protectant additive (mixed with sucrose and sodium polyphosphates) in
the manufacture of surimi, a highly refined, uncooked fish paste most commonly
produced from Alaska (or walleye) pollock (Theragra chalcogramma).
Sorbitol, together with other polyhydric alcohols such as glycerol, is one of
the ingredients in alkyl resins and rigid polyurethane foams manufacturing. In
tobacco industries, sorbitol may give mild effect in sniff, good humectant
agent, and is also to avoid acrolein formation which formed in burned
glycerine. In textile industries, sorbitol is used as a softener and color
stabilizer, and as a softener in leather industries.
Technological
application
This polyol has a
relative sweetness of around 60% vis-à-vis sucrose and displays a 20-fold
higher solubility in water than mannitol. Owing these properties, sorbitol is
widely used in a range of food products such as confectionery, chewing gum,
candy, desserts, ice cream, and diabetic foods. In these products, it imparts
sweetness and plays technological role such as a humectant, a texturizer, and a
softener. Besides, sorbitol is the starting material for the production of
pharmaceutical compounds such as sorbose and ascorbic acid. Several industrial
processes have been described for the production of sorbitol.
Functional application
In medication, it is
used as a laxative to treat occasional episodes of constipation. Vitamin C
(ascorbic acid) is mainly semi-synthesized from sorbitol by fermentation
process by Baccillus suboxydant. In oral or topical preparation, it is
used as humectant, sweetener, bodying and viscosity agent, vehicle,
anticaplocking, and texture improvement. In other case, sorbitol is useful to
promote the absorption of certain minerals such as Cs, Sr, F, and vitamins B12.
In high concentration, sorbitol is a stabilizer for unstable vitamins and
antibiotics. It is used as an excipient and intravenous osmotic diuretic in
pharmaceutical fields (PT. Sorini Corp., Tbk, Jawa Timur, Indonesia).
Sorbitol also has
antioxidant properties. Shih-Yung studied the differential effect of sorbitol
and polyethylene glycol on antioxidant enzymes in rice leaves. They reported
that sorbitol treatment had no effect on lipid peroxidation; however, there is
an increase in peroxidase, ascorbate peroxidase, and glutathione reductase
activities in rice leaves treated with sorbitol. Findings led them to suggest
that sorbitol treatment can upregulate antioxidant system in rice leaves.
2.9.1 Production:
Biotechnological
Production:
Sorbitol is claimed to
have important health-promoting effects. A recombinant strain of Lb. casei
was constructed, cells of which when pre-grown on lactose, were able to
synthesize sorbitol from glucose. Inactivation of the L-lactate dehydrogenase
gene led to an increase in sorbitol production. Lb. casei is a lactic
acid bacterium relevant as probiotic and used as a cheese starter culture. A
sorbitol-producing Lb. casei strain might therefore be of considerable
interest in the food industry.
Ladero reported the
capacity of Lb. plantarum, a lactic acid bacterium found in many
fermented food products and in the gastrointestinal tract of mammals, to
produce sorbitol from fructose-6-phosphate by reverting the sorbitol catabolic
pathway by over expressing sorbitol 6-P-dehydrogenase and the mutant strains
deficient for both L- and D-lactate dehydrogenase activities.
2.10. Trehalose
Trehalose, also known
as mycose, is a natural alpha-linked disaccharide formed by an α, α-1,
1-glucoside bond between 2 α-glucose units. In 1832, Wiggers discovered trehalose
in an ergot of rye and in 1859 Berthelot isolated it from trehala manna, a
substance made by weevils, and named it trehalose. Trehalose is found naturally
in insects, plants, fungi, and bacteria; the major natural dietary source is
mushrooms. It is implicated in anhydrobiosis—the ability of plants and animals
to withstand prolonged periods of desiccation. It has high water retention
capabilities and is used in food and cosmetics. The sugar forms a gel phase as
cells dehydrate, which prevents disruption of internal cell organelles by
effectively splinting them in position. Rehydration then allows normal cellular
activity to be resumed without the major, lethal damage that would normally
follow a dehydration/rehydration cycle. Trehalose has the added advantage of
being an antioxidant. Trehalose is a naturally occurring reducer of cell
stress, protecting these organisms from extremes in heat shock and osmotic
stress. It acts by altering or replacing the water shell that surrounds lipid
and protein macromolecules. It is thought that its flexible glycosidic bond
allows trehalose to conform to the irregular polar groups of marcromolecules.
In doing so, it is able to maintain the 3-dimensional structure of these
biologic molecules under stress, preserving biologic function. Trehalolipids
(trehalose linked at C-6 and C-6′ to mycolic acid) is produced from Rhodococcus
erythropolis and Arthrobacter spp. acts as biosurfactant.
Trehalose has been
accepted as a novel food ingredient under the GRAS terms in the United States
and European Union. Trehalose has also found commercial application as a food
ingredient. The uses for trehalose span a broad spectrum that cannot be found
in other sugars, the primary one being its use in the processing of foods.
Trehalose is used in a variety of processed foods such as dinners, western and
Japanese confectionery, bread, vegetables side dishes, animal-derived deli
foods, pouch-packed foods, frozen foods, and beverages, as well as foods for
lunches, eating out, or prepared at home. Technology for the production of
trehalose was developed in Japan, where enzyme-based processes convert wheat
and corn syrups to trehalose. It is also used as a protein-stabilizing agent in
research. It is particularly effective when combined with phosphate ions.
Trehalose has also been used in several biopharmaceutical monoclonal antibody
formulations: trastuzumab, marketed as Herceptin by Genentech, and ranibizumab,
marketed as Lucentis by Genentech and Novartis. Because of its
moisture-retaining capacity, it is used as a moisturizer in many basic
toiletries such as bath oils and hair growth tonics. Using trehalose's
properties to preserve tissue and protein to full advantage, it is used in
organ protection solutions for organ transplants. Other fields of use for
trehalose span a broad spectrum including fabrics that have deodorization
qualities, plant activation, antibacterial sheets, and nutrients for larvae.
Technological
application
It is anticariogenic,
synthesized by several microorganisms, only partially digested by humans, and
therefore considered a dietetic sugar. The protection of proteins against
denaturation under stress conditions is a well-known property of trehalose.
Trehalose is used in a wide range of products due to the multifaceted effects
of trehalose's, such as its inherently mild, sweet flavor; its preservative
properties that maintain the quality of the 3 main nutrients (carbohydrates,
proteins, fats); its powerful water-retention properties that preserve the
texture of foods by protecting them from drying out or freezing; and its
ability to suppress bitterness, stringency, harsh flavors, and the stench of
raw foods, meats, and packaged foods.
Functional application
Several recent studies
have revealed health-promoting attributes of trehalose. In rats, it was shown
to almost completely suppress dental caries, and in humans it reduced significantly
acidification in plaques. Furthermore, it has been demonstrated that trehalose
has important stabilizing effects on human proteins, preventing protein
aggregation as well as formation of pathological con formational forms. As an
extension of its natural capability to protect biological structures, trehalose
has been used for the preservation and protection of biologic materials. It
stabilizes bioactive-soluble proteins such as monoclonal antibodies and enzymes
for medical use. It stabilizes proteins for inhaled use. It has been successfully
used to preserve embryos, and cellular blood products. Cyopreservation of
transplant cells and tissue in the presence of trehalose has been shown to
increase viability and decrease host immune response.
As it inhibits lipid
and protein misfolding, trehalose has become an attractive molecule for study
in neurodegenerative disease characterized by protein misfolding and aggregate
pathology. Such diseases include Alzheimer's and Parkinson's disease, and the
less common triplet repeat diseases. Recent scientific publications describe
trehalose benefit in model systems that recapitulate aggregate pathology that
characterize Alzheimer's (AD), Huntington's (HD), and occulopharyngeal muscular
dystrophy (OPMD). Having beneficial properties, its production in food products
at the expense of other sugars is desirable.
Luo examined the
effects of trehalose on the activities of key antioxidant enzymes, including
superoxide dismutases (SODs), ascorbate catalases (CATs), and ascorbate
peroxidases (APX) from wheat (Triticum aestivum), and then measured the
ability of trehalose to scavenge hydrogen peroxide (H2O2)
and superoxide anions (O2-•). They indicated that trehalose
protected SOD activity slightly. However, it inhibited CAT and APX activities
under heat stress, with a little protection of CAT activity and trehalose
scavenged H2O2 and O2-• greatly in a
concentration-dependent manner. Their results suggest that trehalose plays a
direct role in eliminating H2O2 and O2-• in
wheat under heat stress. Kazuyuki studied the inhibitory effect of trehalose on
the autoxidation of unsaturated fatty acids (UFA) by water/ethanol system and reported
remarkable inhibition of formation of hydroperoxide from linoleic acid by
trehalose. They also observed that the inhibitory effect on the autoxidation
was dependent on the amount of trehalose. Similar to linoleic acid, the
formation of hydroperoxide from α-linolenic acid was inhibited by trehalose. On
the other hand, when the degradation of hydroperoxide was tested under the same
conditions of the autoxidation, it was found that the degradation was not
influenced by the presence of trehalose. Thus, it is clear that trehalose
inhibits the autoxidation of unsaturated fatty acids, but negligibly suppresses
the generation of volatile aldehydes from hydroperoxides. Trehalose depresses
the effect on the oxidation of UFA through the weak interaction with the double
bond(s).
2.10.1. Production:
Biotechnological
Production:
Trehalose is widespread
within the genus Propionibacterium. Trehalose accumulation in Propionibacterium,
for example, P. acidipropionici and P. freudenreichii subsp. shermanii
has also been observed to occur in response to stress conditions. In
particular, P. freudenreichii subsp. shermanii strain NIZO B365
accumulates trehalose to remarkable levels, and the trehalose content increases
considerably in response to osmotic, oxidative and acid stress (up to 40% [w/w]
of the cell protein). In this organism, trehalose results from the conversion
of glucose 6-P and ADP glucose via trehalose 6-P synthase to trehalose 6-P and
its subsequent dephosphorylation by trehalose 6-P phosphatase. Alternatively,
trehalose can be formed from maltose through the action of trehalose synthase.
While working on
isolates of Propionibacteria from Indian fermented milks for vitamin B12
production in our laboratory, we have also incidentally observed trehalose
production by these isolates as estimated by the HPLC method.
The safety of trehalose
is supported by animal and human studies and is marketed in the United States
as approved by USFDA since 2000. Trehalose is also approved in Japan, Korea, Taiwan,
and the United Kingdom.
2.11.
Erythritol:
Erythritol is a
biological sweetener with applications in food and pharmaceuticals. Not only
animal toxicological but also clinical studies have consistently demonstrated
its safety even when consumed on a daily basis in high amounts. Large-scale
production of erythritol uses fermentative processes with pure glucose, sucrose
and dextrose from chemically and enzymatically hydrolyzed wheat and corn
starches used as major carbon sources (Aoki et al. 1993; Marina et al. 1993).
Erythritol is produced by fermentation involving yeast-like fungi such as Trigonopsis
variabilis, Trichosporon sp., Torula sp., Candida magnoliae and Moniliella sp.
Leuconostoc oenos can also produce erythritol but only under anaerobic
conditions. A high initial concentration of glucose favors erythritol
production by osmophilic microorganisms. Generally, an increase in the initial
glucose concentration increases the production rate and yield in a batch
process if the microorganisms can tolerate a higher concentration of sugar and
a higher osmotic pressure. Erythritol has been produced commercially using a
mutant of Aureobasidium. The mutant produced erythritol at 1.8 g l-1 h-1 with a
44% yield in a medium containing 40% (w/v) glucose. Although erythritol
fermentation in synthetic media containing glucose or fructose has extensively
been studied, little information is available about the production of this
compound by Yarrowia lipolytica from glycerol. New uses of glycerol and new
microbial transformations to interesting products are of increasing importance.
What seems to be important for an economically competitive fermentation process
is the ability of Y. lipolytica to grow and to produce erythritol on renewable
products from industrial processes (such as raw glycerol discharged after
biodiesel manufacture), which are low-cost carbon substrates. A cost reduction
in erythritol production can be achieved by using less expensive substrates. We
have recently reported that an acetate-negative mutant of Y. lipolytica
Wratislavia K1 has the ability of simultaneously producing high amounts of
erythritol and citric acid in media containing glycerol, at pH 5.5 which is
optimal for citric acid biosynthesis by Y. lipolytica. In the current study, we
evaluate the dynamics and yield of erythritol production from raw glycerol by
the strain Wratislavia K1 at various pH, and thereafter investigate, in
fed-batch experiments, the effect of the optimal pH on the production of
erythritol by other strains belonging to the species Y. lipolytica.
2.11.1.
Production:
Biotechnological
Production:
Microorganisms
and media
The following strains
were used in this study: three acetate negative mutants of Yarrowia lipolytica:
Wratislavia AWG7, Wratislavia 1.31 and Wratislavia K1, one mutant of Y.
lipolytica 8661 UV1, and two wild strains of Y. lipolytica: A-101 and 1.22. All
were from Wroclaw University of Environmental and Life Sciences, Poland. The
growth medium for inoculum preparation contained: 50 g glycerol l-1, 3 g yeast
extract l-1, 3 gmalt extract l-1 and 5 gBactopepton l-1. Erythritol production
was conducted in a medium consisting of 150 g glycerol l-1, 3 g NH4Cl l-1, 1 g
MgSO47H2O l-1, 0.2 g KH2PO4 l-1, and 1 g yeast extract l-1. After 48 h of
cultivation, raw glycerol solution (86%w/v) was added (at a constant feeding
rate of 1.4 g h-1) until a total of 300 g glycerol l-1 was obtained.
Culture
conditions
A seed culture was
grown in a 300 ml flask (containing 50 ml of growth medium) on a shaker at 30C
for 3 days. An inoculum of 200 ml was introduced into the fermenter, which
contained 1.8 l of the production medium. All fed-batch cultures were performed
in a 5 l jar fermenter (Biostat B Plus, Sartorius, Germany) with a working
volume of 2 l at 30C for 7 days. The aeration rate was 0.6 l min-1. The stirrer
speed was adjusted to 800 rpm and the pH was maintained automatically at 3 by
the addition of NaOH (20% w/v). All the cultures were conducted in two
replications.
2.12.
Isomalt:
Isomalt is an
odourless, white, crystalline substance containing about 5 % water of
crystallisation. It tastes just as natural as sugar, is not sticky,
toothfriendly (it helps to prevent cavities and plaque), suitable for diabetics
and has only about half as many calories as sugar because it cannot be
completely metabolized. Isomalt is a bulk sweetener and can substitute sugar in
a 1:1 mass ratio. It should not be mistaken for intense sweeteners, which have
much greater sweetening power (between a hundred and a thousand times as high).
For this reason these intense sweeteners are only used in small quantities.
Because isomalt is similar to sucrose, it is particularly suitable for making
products such as candies, chewing gum, chocolate, compressed tablets or
lozenges, baked goods, baking mixtures, and pharmaceutical products using
conventional equipment. Isomalt’s nutritional and physiological benefits are
ideal for use in sugarfree, lowcalorie and diabetic products. It is
particularly suitable for food and pharmaceutical applications and also offers
many advantages for technical uses.
2.12.1. Production:
Biotechnological
Production:
The microorganism Leuconostoc
mesenteroides was found to produce an unknown disaccharide in1952. In 1957
Weidenhagen and Lorenz found independently that a bacterium they had isolated
from sugar beet raw juice effects a conversion of sucrose to an unknown
reducing disaccharide. The disaccharide was identified as isomaltulose and
given the trivial name palatinose. It was identical with the unknown
disaccharide Stodala, Koepsell, and Sharpe had isolated in 1952. The name
palatinose is derived from „palatinum“, the latin name of the German province
where Weidenhagen and Lorenz found the disaccharide. In 1958 Windisch
identified the isolated bacterium as Protaminobacter rubrum den Dooren de Jong.
Depending on culture conditions, P. rubrum produces a red pigment. The
bacterium is also able to degrade and modify amines. But its most remarkable
property is the transglycosylation of sucrose to palatinose. The first patent
for the production of palatinose was awarded to Süddeutsche Zucker AG,
Mannheim, Germany in 1957. In addition to P. rubrum the microorganisms Leuconostoc
mesenteroides, Serratia plymuthica, Serratia marcescens, Erwinia
carotovera, and Erwinia rhapontici have been reported to be capable
of transforming sucrose into palatinose. The enzymatic
transglycosylation of sucrose to palatinose effected by the
glycosyltransferase (sucrosemutase) is described with the molecules in
their steric representation in figure 2 for a better understanding. The
glucosyl group of the sucrose (alpha-Dglucopyranosyl- 1,2-beta-D-fructofuranoside)
changes from alpha(1->2) to alpha(1->6) position. The enzymatic
reaction results in the thermodynamically more stable palatinose
(alpha-glucopyranosyl-1,6-D-fructofuranose). Of all the microorganisms
reported, only the strain of P. rubrum originally isolated by
Weidenhagen and Lorenz in 1957 has been foreseen for industrial
scale use thus far.
Earlier processes using viable, free microorganisms have the
disadvantage of higher product purification costs and lower yield. More
cost-effective are techniques using immobilized non-viable cells. This is in
agreement with many other authors who prefer the use of immobilization
techniques for other processes. The process starts with the propagation of the
cells. P. rubrum can be cultivated either on thick juice supplemented with corn
steep liquor or on molasses with additional nitrogen and phosphate e. g.
(NH4)2HPO4. Molasses is a byproduct of the sugar industry. The growth medium is
adjusted to a concentration of 5 % total solids and pH of 7.2. Sterilization of
the medium is, of course, necessary.
In 1990, a large scale plant for production of isomalt went on stream
in Offstein/ Rhineland-Palatinate. Current isomalt production is about 35,000
tons yearly. Hydrogenation of palatinose solutions can be done easily under
moderate temperature and pressure conditions and at neutral pH or under mildly
alkaline conditions. Any of the usual hydrogenation catalysts is suitable for
carrying out the reaction. Quite suitable is Raney-Nickel in pelletized form in
a fixed bed reactor. Theoretically the reduction of palatinose should yield an
equimolar mixture of 1-O-alpha-Dglucopyranosyl- D-mannitol (1,1-GPM) and
6-Oalpha- D-glucopyranosyl-D-sorbitol (1,6-GPS) but depending on the conditions
of hydrogenation the rate of each component can vary between 43-57 %.
2.13.
Xylitol:
Xylitol is a
five-carbon sugar alcohol that is naturally found in some fruits and
vegetables. The most significant application of xylitol is its use as an ideal
sweetener for diabetic patients. Other potential uses of xylitol are: as an
anticariogenic agent in toothpaste formulations, as thin coatings on vitamin
tablets, in chewing gum, soft drinks, mouthwashes, beverages, and in bakery
products. Xylitol has received global demand mainly due to its
insulin-independent metabolism, anticariogenecity, high sweetening power, and
pharmacological properties. Xylitol is currently approved for usage in foods,
pharmaceuticals, and oral health products in more than 50 countries. Xylitol
can be produced either by chemical hydrogenation of pure xylose or by
biotechnological processes. Currently, xylitol is industrially produced by
chemical reduction of pure xylose in the presence of a nickel catalyst at high temperature
and pressure. The yield of xylitol is about 50–60% of the xylan fraction and
the resultant product is very expensive due to extensive purification
procedures. The chemical process is laborious, and cost- and energy-intensive.
In view of alternatives to the conventional process, two biotechnological
approaches seem promising: the fermentation process and the enzymatic approach.
These biotechnological processes are highly attractive alternatives that are
able to produce a high-quality and cost-effective product. The fermentation
process uses bacteria, fungi, and yeast for xylitol production from xylose or
hemicellulosic hydrolysate. Yeasts are considered as the best xylitol producers
among the microorganisms investigated. In the fermentation process, the yield
of xylitol obtainable from xylose is in a range of 65–85% of the theoretical
value (Nigam and Singh, 1995). The application of the fermentation process on
an industrial-scale is time-consuming because of some preparatory activities
such as sterilization and regular inoculum development. An advantage of the
fermentation process over chemical procedures is its lower cost due to the
non-necessity of xylose purification. However, the fermentation method has not
yet been able to accumulate the advantages of the chemical process. Xylitol
production from xylose using enzyme technology can be an attractive alternative
to both fermentation and chemical processes. Compared to the fermentation
process, the enzymatic approach employing xylose reductase (XR) for xylitol
synthesis is expected to obtain a substantial increase in productivity. There
are scarce reports on the enzymatic conversion of synthetic xylose to xylitol
using XR. The conversion of D-xylose to xylitol is more than 95% by the NADH-dependent
XR from yeast. Despite a wide range of applications, the use of xylitol as
sweetener is limited due to its high price. This has inspired researchers to
work toward the development of improved technologies to lower the production
costs. In this field, the enzymatic approach to xylitol production from xylose
present in the lignocellulosic biomass may provide an alternative for the
chemical process. This review attempts to describe the current literature on
the processes involving xylitol production, taking into account the chemical
and biotechnological processes, microorganisms involved and tries to identify
ways to improve enzymatic xylitol production so that it can compete with the
chemical process.
2.13.1. Production:
Chemical Production:
Xylitol is manufactured
industrially by reducing pure xylose, obtained from hardwood or hemicellulosic
hydrolysate in the presence of a Raney nickel catalyst. The chemical synthesis
of xylitol starts with the extraction of xylose from hemicellulose by
acid-catalyzed hydrolysis. After color removal and purification, xylose-rich
hemicellulosic hydrolysate can be employed for xylitol production through
hydrogenation of xylose at 80–140°C and hydrogen pressures up to 50 atmospheres
in the presence of metal catalysts (Raney nickel). The xylitol solution formed
requires further purification by chromatography, and then concentration and
crystallization of the product to obtain pure xylitol. The xylitol yield is
only about 50–60% of the xylan fraction and thus the xylitol production process
is expensive due to the extensive separation and purification stages.
Biotechnological
Production:
Fermentation
process
The fermentation
process uses bacteria, fungi, and yeast for xylitol production from commercial
pure xylose or hemicellulosic hydrolysate. The production of xylitol using
bacteria and fungi has been studied to a lesser extent compared to that using
yeast strains. A few bacteria, such as Enterobacter liquefaciens, Corynebacteriurn
sp., and Gluconobacter oxydans, have been reported to produce
xylitol. There are very few studies regarding xylitol production from D-xylose
using filamentous fungi. Yeasts are considered as the best xylitol producers
among the microorganisms. As a result, yeasts have been studied extensively in
the last few decades by several researchers. Forty-four yeast strains from the
five genera were screened by Barbosa for their ability to convert D-xylose to
xylitol. Candida guilliermondii and C. tropicalis were found to
be the best xylitol producers and these yeasts produced 77.2 g l-1 xylitol from
104 g l-1 xylose using high cell densities and a defined medium under aerobic
conditions. The fermentation conditions were optimized by da Silva and Afschar
(1994) during continuous cultivation of Candida tropicalis for xylitol
production. C. tropicalis produced xylitol at a yield of 77–80% of
theoretical value (0.91 g g-1) in a medium containing 100 g l-1 D-xylose. The
screening of different xylose-assimilating yeast has confirmed that the best
xylitol producers belong to the genus Candida. In the fermentation
process using yeast, the yield of xylitol obtainable from D-xylose is in a
range of 65–85% of the theoretical value. The production of xylitol through the
fermentation process is limited by certain factors, such as precise control of
culture conditions, expensive nutrients, huge water consumption, and the type
of process. Thus, the application of the fermentation process on an industrial
level is timeconsuming, being associated with some preparatory activities such
as sterilization and regular inoculum development involving input of energy,
labor, and time, leading to decreased productivity. The advantage of the
fermentation process over chemical procedures is its lower cost due to the
non-necessity of extensive xylose purification. The fermentative xylitol
production has been studied as an alternative, but its viability is dependent
on the optimization of the various fermentation variables such as nutritional
composition (substrate, nitrogen source, and micronutrients), the culture and process
conditions, as well as the genetic nature of the microorganisms.
Enzymatic
process
The production of
xylitol from xylose by using enzyme technology is an alternative and promising
approach. The enzymatic conversion of D-xylose into xylitol using xylose
reductase (XR) of Candida pelliculosa coupled with the
oxidoreductase system of Methanobacterium sp. has been reported by Kitpreechavanich.
The authors observed that the xylose was stoichiometrically converted to
xylitol with an equivalent consumption of NADPH and that an almost quantitative
conversion of xylose to xylitol was achieved using a NADP+-toxylose ratio of
over 1:30, whereas the coenzyme was successfully regenerated and retained using
a membrane reactor. About 90% conversion of xylose to xylitol could be achieved
at 35°C and pH 7.5 after a 24 h reaction period. Nidetzky optimized the
production of xylitol from xylose by XR from Candida tenuis coupled with
glucose dehydrogenase from Bacillus cereus for regenerating the NADH in
an enzyme reactor. In this system, the substrate was converted at
concentrations of 300 g l-1 xylose, with a 96% yield and xylitol productivity
of 3.33 g l-1 h-1. Neuhauser et al. (1998) reported on the C. tenuis XR-mediated
NADH-dependent xylose reduction coupled with formate dehydrogenase (FDH) from C.
boidinii for the byproduct-free recycling of NADH used in a pH-controlled
enzyme reactor. In this process, a fed-batch conversion of 0.5 M xylose to
xylitol using yeast XR yielded productivities of 2.8 g l-1 h-1. To optimize the
performance of the XRcatalyzed reactions for xylitol synthesis, the effect of
several process variables on productivity needs to be studied: pH, temperature,
initial substrate, and coenzyme concentration.
Limitations:
Despite the yield of
microbiological conversion of xylose to xylitol could be increased by 65–85%
using different production methods, the chemical process would still be very
competitive in terms of industrial scale manufacture. The synthesis of xylitol
from xylose using XR from yeast is an attractive alternative to chemical and
microbial processes. The application of XR constitutes an alternative of
economic interest regarding both the chemical reduction of pure xylose and the
fermentation of xylose present in the hemicellulosic hydrolysate. During
enzymatic production, all experiments were performed using commercial
xylose-containing medium. It is certainly still necessary to study the
enzymatic conversion of xylose in the lignocellulosic biomass to xylitol and to
optimize the reaction conditions. The production of xylitol through the
chemical process is expensive due to difficult separation and purification
steps. On the other hand, the fermentation process on an industrialscale is not
feasible due to reduced productivity. Hence, it is important to explore
alternative methods for the effective production of xylitol using XR enzyme.
The enzymatic approach might be able to overcome the disadvantages of the
chemical process that is largely being used at present and also the
fermentation process that is under investigation.
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