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Biotechnology Advances 26 (2008) 22 – 34

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Research review paper

Amylolytic bacterial lactic acid fermentation — A review
Gopal Reddy ⁎, Md. Altaf 1 , B.J. Naveena 1 , M. Venkateshwar, E. Vijay Kumar
Department of Microbiology, Osmania University, Hyderabad-500 007, India
Received 29 June 2007; accepted 25 July 2007
Available online 31 July 2007

Abstract

Lactic acid, an enigmatic chemical has wide applications in food, pharmaceutical, leather, textile industries and as chemical feed
stock. Novel applications in synthesis of biodegradable plastics have increased the demand for lactic acid. Microbial fermentations
are preferred over chemical synthesis of lactic acid due to various factors. Refined sugars, though costly, are the choice substrates
for lactic acid production using Lactobacillus sps. Complex natural starchy raw materials used for production of lactic acid involve
pretreatment by gelatinization and liquefaction followed by enzymatic saccharification to glucose and subsequent conversion of
glucose to lactic acid by Lactobacillus fermentation. Direct conversion of starchy biomass to lactic acid by bacteria possessing both
amylolytic and lactic acid producing character will eliminate the two step process to make it economical. Very few amylolytic lactic
acid bacteria with high potential to produce lactic acid at high substrate concentrations are reported till date. In this view, a search
has been made for various amylolytic LAB involved in production of lactic acid and utilization of cheaply available renewable
agricultural starchy biomass. Lactobacillus amylophilus GV6 is an efficient and widely studied amylolytic lactic acid producing
bacteria capable of utilizing inexpensive carbon and nitrogen substrates with high lactic acid production efficiency. This is the first
review on amylolytic bacterial lactic acid fermentations till date.
© 2007 Elsevier Inc. All rights reserved.

Keywords: Amylolytic lactic acid bacteria; Lactic acid; Starch; Fermentation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2. Lactic acid and its importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3. Lactic acid bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4. Amylolytic lactic acid bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5. Amylolytic lactic acid fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
6. Substrates available for amylolytic lactic acid fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
7. Amylolytic enzymes in LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
8. Submerged fermentations involving amylolytic LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
9. Solid-state fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

⁎ Corresponding author. Tel.: +91 40 27682246/27090661.

E-mail addresses: gopalred@hotmail.com (G. Reddy), altafmicro_79@yahoo.com (M. Altaf).
1
Present address: Oklahoma University Cancer Institute, University of Oklahoma Health Sciences, Center, Oklahoma City, OK-73104, USA.

0734-9750/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2007.07.004
 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
1. Introduction
Lactic acid is one of the most important organic acids
produced by lactic acid bacteria (LAB), discovered by
Swedish scientist C. W. Scheele in 1780 from sour milk.
Lactic acid exists in two optically active stereo-isomers,
the L(+) and the D(−). Lactic acid has a wide range of
beneficial uses in the sectors relating to food preserva-
tion, flavor enhancement etc. Since elevated levels of
D(−) lactic acid is harmful to humans, L(+) lactic acid is
the preferred isomer in food and pharmaceutical
industries as humans have only L-lactate dehydrogenase
that metabolizes L(+) lactic acid (Akerberg et al., 1998;
Hofvendahl et al., 2000).
Currently, lactic acid is used in a wide variety of
specialized industrial applications where the functional
specialty of the molecule is desirable (Datta et al., 1995).
Leo Hepner of L. Hepner and Associates, a UK based
management consultancy for food ingredients and
biotechnology industries, rates worldwide consumption
of lactic acid at 130,000 to 150, 000 MT per year (Mirasol,
1999). In 1999, Hepner rated the demand for lactic acid to
grow continually at 5–8% annual clip. Its use as a raw
material for synthesis of biodegradable plastics was
identified in late 1940s and early 1950s (Vickroy, 1985).
Demand for lactic acid is expected to increase as rated by
different surveys due to its use in biodegradable plastics
and other large-scale industrial products. Yet the market is
limited by cost in competition with polystyrene as prices
for heat stable [L(+)] and higher grades of lactic acid are
more (Mirasol, 1999). If polylactides and lactate esters are
commercially successful, global demand will be around
14–19% (Chem systems reports, 2002; Jarvis, 2003). By
the end of year 2011, lactic acid global demand is
expected to shoot up to 200,000 MT world wide and
domestic demand for lactic acid and in India is expected to
touch 2000 tonnes from the present demand of 560 tonnes
(Ramesh, 2001). The current global production of lactic
acid is about 120,000 tonnes per year (Datta and Henry,
2006). New applications of L(+) lactic acid, such as a
monomer in biodegradable plastics or as an intermediate
in the synthesis of high-volume oxygenated chemicals,
have the potential to greatly expand the market for it.
Lactic acid can be manufactured either by chemical
synthesis or by microbial fermentations. Chemical synthe-
23
sis results in racemic DL-lactic acid whereas stereospecific
[L(+),D(−) and DL mixture] form is produced by fermen-
tation using specific microbial strain (Datta et al., 1993;
Litchfield, 1996). Lactic acid bacteria (LAB) can be
homofermentative or heterofermentative and can produce
either L(+) or D(−) or racemic mixture of lactic acid.
Significant advantage over chemical synthesis is that
biological production can use cheap raw materials such as
whey, molasses, starch waste, beet, cane sugar and other
carbohydrate rich materials (Anuradha et al., 1999; Ritcher
and Berthold, 1998; Tsao et al., 1999; Vishnu et al., 1998,
2000). Raw material cost is one of the major factors in
economic production of lactic acid. The efficiency and
economics of the ultimate lactic acid fermentation is
however still a problem from many points of view and
media compositions play vital role in the improvement of
such a process. Research efforts are focused on looking for
new and effective nutritional source and new progressive
fermentation techniques enabling the achievement of both
high substrate conversion and high production yields (Sule
Bulut et al., 2004). Direct conversion of starch to lactic acid
by bacteria with both amylolytic and lactic acid producing
character will eliminate the two step process of sacchar-
ification followed by microbial fermentation to make it
economical.
Many reviews on lactic acid fermentation are
published, focus of this review is on amylolytic lactic
acid fermentation with emphasis to use starch or starchy
substrates and other low cost substrates to replace sugars
and costly nitrogenous materials.
2. Lactic acid and its importance
Lactic acid (C3H6O3) is present in almost every form
of organized life. Its most important function in animals
and humans is related to the supply of energy to muscle
tissues. This is a water soluble and highly hygroscopic
aliphatic acid and an enigmatic chemical. It is the first
biotechnologically produced multi-functional versatile
organic acid having wide range of applications. It is a
product of natural fermentation processes occurring in
buttermilk, cheese, beer, sourdough and many other
fermented foods. Litchfield (1996) has summarized
typical food applications for lactic acid and its salts. It is
non-volatile, odorless organic acid and is classified as
24 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
GRAS (Generally Recognized As Safe) for use as a
general purpose food additive by FDA in U.S.A. and
other regulatory agencies (Datta et al., 1995). The lactic
acid consumption market is dominated by the food and
beverage sector since 1982. Even today, lactic acid
market still exists for food and beverage industries.
More than 50% of lactic acid produced is used as
emulsifying agent in bakery products (Datta et al., 1993;
Litchfield, 1996). It is used as acidulant/flavoring/pH
buffering agent or inhibitor of bacterial spoilage in a
wide variety of processed foods, such as candy, breads
and bakery products, soft drinks, soups, sherbets, dairy
products, beer, jams and jellies, mayonnaise, and pro-
cessed eggs, often in conjunction with other acidulants.
Lactic acid or its salts are used in the disinfection and
packaging of carcasses, particularly those of poultry and
fish, where the addition of aqueous solutions during
processing increased shelf life and reduced microbial
spoilage (Datta et al., 1995; Naveena, 2004). The esters
of calcium and sodium salts of lactate with longer chain
fatty acids have been used as very good dough con-
ditioners and emulsifiers in bakery products. The water-
retaining capacity of lactic acid makes it suitable for use
as moisturizer in cosmetic formulations. Ethyl lactate is
the active ingredient in many anti-acne preparations.
The natural occurrence of lactic acid in human body
makes it very useful as an active ingredient in cosmetics
(Wee et al., 2006). Lactic acid has long been used in
pharmaceutical formulations, mainly in topical oint-
ments, lotions, and parenteral solutions. It also finds
applications in the preparation of biodegradable poly-
mers for medical uses such as surgical sutures, pros-
theses and controlled drug delivery systems (Wee et al.,
2006). The presence of two reactive functional groups
makes lactic acid the most potential feedstock monomer
for chemical conversions to potentially useful chemicals
such as propionic acid, acetic acid, acrylic acid etc.
(Dimerci et al., 1993). Technical-grade lactic acid is
extensively used in leather tanning industries as an
acidulant for deliming hides and in vegetable tanning.
Lactic acid is used as descaling agent, solvent, cleaning
agent, slow acid-releasing agent and humectants in a
variety of technical processes. Because of ever-increas-
ing amount of plastic wastes worldwide, considerable
research and development efforts have been devoted
towards making a single-use, biodegradable substitute
of conventional thermoplastics.
Biodegradable polymers are classified as a family of
polymers that will degrade completely – either into the
corresponding monomers or into products, which are
otherwise part of nature – through metabolic action of
living organisms. International organizations such as the
American Society for Testing of Materials (ASTM), the
Institute for Standards Research (ISR), the European
Standardization Committee (CEN), the International
Standardization Organization (ISO), the German Insti-
tute for Standardization (DIN), the Italian Standardiza-
tion Agency (UNI), and the Organic Reclamation and
Composting Association (ORCA), are all actively
involved in developing tests of biodegradability in
different environments and compostability. The demand
for lactic acid has been increasing considerably, owing
to the promising applications of its polymer, the
polylactic acid (PLA), as an environment-friendly
alternative to plastics derived from petrochemicals.
PLA has received considerable attention as the precur-
sor for the synthesis of biodegradable plastic (Senthuran
et al., 1997). The lactic acid polymers, with tremendous
advantages like biodegradability, thermo plasticity, high
strength etc., have potentially large markets. The
substitution of existing synthetic polymers by biode-
gradable ones would also significantly alleviate waste
disposal problems. As the physical properties of PLA
depend on the isomeric composition of lactic acid, the
production of optically pure lactic acid is essential for
polymerization. L-Polylactic acid has a melting point of
175–178 °C and slow degradation time. L-Polylactide is
a semicrystalline polymer exhibiting high tensile
strength and low elongation with high modulus suitable
for medical products in orthopedic fixation (pins, rods,
ligaments etc.), cardiovascular applications (stents,
grafts etc.), dental applications, intestinal applications,
and sutures (Wee et al., 2006).
3. Lactic acid bacteria
Lactic acid bacteria (LAB) are a group of related
bacteria that produce lactic acid as major metabolic
product. LAB have the property of producing lactic acid
from carbohydrates through fermentation. LAB have
been used to ferment or culture foods for at least
4000 years. These organisms are heterotrophic and
generally have complex nutritional requirements be-
cause they lack many biosynthetic capabilities. Most
species have multiple requirements for amino acids and
vitamins. Because of this, lactic acid bacteria are
generally abundant only in communities where these
 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
requirements can be provided. Lactic acid bacteria are
used in the food industry for several reasons. Their
growth lowers both the carbohydrate content of the
foods that they ferment, and the pH due to lactic acid
production. It is this acidification process which is one
of the most desirable effects of their growth. The pH
may drop to as low as 4.0, low enough to inhibit the
growth of most other microorganisms including the
most common human pathogens, thus allowing these
foods to prolong shelf life. LAB consist of bacterial
genera within the phylum Firmicutes comprised of
about 20 genera. The genera Lactococcus, Lactobacil-
lus, Streptococcus, Leuconostoc, Pediococcus, Aero-
coccus, Carnobacterium, Enterococcus, Oenococcus,
Tetragenococcus, Vagococcus and Weisella are the
main members of the LAB (Axelsson, 2004; Davidson
et al., 1995; Ercolini et al., 2001; Jay, 2000; Holzapfel et
al., 2001; Stiles and Holzapfel, 1997). Lactobacillus is
largest of these genera, comprising around 80 recog-
nized species (Axelsson, 2004). The taxonomy of lactic
acid bacteria has been based on the Gram reaction and
the production of lactic acid from various fermentable
carbohydrates. Lactobacilli vary in morphology from
long, slender rods to short coccobacilli, which frequent-
ly form chains. Typical LAB are Gram-positive,
nonsporing, catalase-negative, devoid of cytochromes,
anaerobic but aerotolerant cocci or rods that are acid-
tolerant and produce lactic acid as the major end product
during sugar fermentation (Axelsson, 2004). However,
under certain conditions some LAB do not display all
these characteristics. Thus, the most profound features
of LAB are Gram positiveness and inability to
synthesize porphyrin groups. The inability to synthesize
porphyrin (e.g., heme) results in the LAB being devoid
of catalase and cytochromes (without supplemented
heme in the growth media). Therefore, the LAB do not
possess an electron transport chain and rely on
fermentation to generate energy (Axelsson, 2004).
Since they do not use oxygen in their energy production,
lactic acid bacteria grow under anaerobic conditions, but
they can also grow in oxygen's presence. They are
protected from oxygen by-products (e.g. H2O2) because
they have peroxidases. These organisms are aerotolerant
anaerobes. Because of the low energy yields, lactic acid
bacteria often grow more slowly than microbes capable
of respiration, and produce smaller colonies of 2–3 mm.
Lactic acid bacteria can grow at temperatures from 5 to
45 °C and not surprisingly are tolerant to acidic
conditions, with most strains able to grow at pH 4.4.
The growth is optimum at pH 5.5–6.5 and the organisms
have complex nutritional requirements for amino acids,
peptides, nucleotide bases, vitamins, minerals, fatty
25
acids and carbohydrates. The genus is divided into three
groups based on fermentation patterns:
• Homofermentative: produce more than 85% lactic
acid from glucose. They ferment 1 mol of glucose to
2 mol of lactic acid, generating a net yield of 2 mol of
ATP per molecule of glucose metabolized. Lactic
acid is the major product of this fermentation (Fig. 1).
• Heterofermentative: produce only 50% lactic acid.
These ferment 1 mol of glucose to 1 mol of lactic
acid, 1 mol of ethanol, and 1 mol of CO2. One mole
of ATP is generated per mole of glucose, resulting in
less growth per mole of glucose metabolized (Fig. 1).
• Less well known heterofermentative species which
produce DL-lactic acid, acetic acid and carbon dioxide.
4. Amylolytic lactic acid bacteria
Amylolytic lactic acid bacteria (ALAB) have been
reported from different tropical amylaceous fermented
foods, prepared mainly from cassava and cereals (e.g.,
maize and sorghum). Strains of Lactobacillus plantarum
have been isolated from African cassava-based fermen-
ted products (Nwankwo et al., 1989), and the new
ALAB species Lactobacillus manihotivorans (Morlon-
Guyot et al., 1998) was isolated from cassava sour starch
fermentations in Colombia. Olympia et al. (1995)
characterized amylolytic strains of L. plantarum isolated
from burong isda, a fermented food made from fish and
rice in Philippines. Amylolytic strains of Lactobacillus
fermentum were isolated for the first time from Benin
maize sourdough (ogi and mawè) by Agati et al. (1998).
Recently, Sanni et al. (2002) described amylolytic
strains of L. plantarum and L. fermentum strains in
various Nigerian traditional amylaceous fermented
foods. The search for ALAB in fermented amylaceous
foods has been justified by the high starch content of the
raw material. Their role has yet to be elucidated since
mono- and disaccharides, such as glucose and sucrose,
which occur naturally in cereals and cassava, are readily
available for lactic acid fermentation. The way the raw
material is processed may determine the composition of
the microbiota and, in particular, the occurrence of
ALAB (Guyot et al., 2000). ALAB have repeatedly
been isolated from traditional cereal or cassava-based
fermented foods (Johansson et al., 1995; Morlon et al.,
1998; Nwankwo et al., 1989; Olympia et al., 1995;
Sanni et al., 2002). Due to the ability of their α-amylases
to partially hydrolyze raw starch (Rodriguez-Sanoja et
al., 2000), ALAB can ferment different types of
amylaceous raw material, such as corn (Nakamura,
1981), potato (Chatterjee et al., 1997), or cassava
26 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
Fig. 1. Metabolism of lactic acid bacteria.
(Giraud et al., 1994) and different starchy substrates
(Vishnu et al., 2000, 2002; Naveena et al., 2003, 2005a,
b,c). Amylolytic LAB utilize starchy biomass and
convert into lactic acid in single step fermentation.
Most of the amylolytic LAB are used in food
fermentation. Amylolytic LAB (ALAB) are also
involved in cereal based fermented foods such as
European sour rye bread, Asian salt bread, sour
porridges, dumplings and non-alcoholic beverage pro-
duction. Few of them are used for production of lactic
acid in single step fermentation of starch.
5. Amylolytic lactic acid fermentation
Conventional biotechnological production of lactic
acid from starchy materials, for instance, requires
pretreatment for gelatinisation and liquefaction, which
is carried out at high temperatures of 90–130 °C for
15 min followed by enzymatic saccharification to
glucose and subsequent conversion of glucose to lactic
acid by fermentation (Anuradha et al., 1999). This two
step process involving consecutive enzymatic hydroly-
sis and fermentation makes it economically unattractive.
The bioconversion of carbohydrate materials to lactic
acid can be made much more effective by coupling the
enzymatic hydrolysis of carbohydrate substrates and
microbial fermentation of the derived glucose into a
single step. This has been successfully employed for
lactic acid production from raw starch materials and
many representative bacteria including Lactobacillus
and Lactococcus species (Cheng et al., 1991; Zhang and
Cheryan, 1994; Vishnu et al., 2002; Naveena et al.,
2003, 2005a,b,c).
Use of sugars is un-economical, still they are the
choice substrates due to certain constraints such as
• Non-availability of potential amylolytic strains for
lactic acid fermentation
• Need to develop a potential strain for high yield
efficiency of lactic acid
• Inability of organisms for alternate substrate utiliza-
tions with high efficiencies
• Inability of organisms to use abundantly available in-
expensive crude agricultural renewable raw materials
 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
Fig. 2. Schematic representation of lactic acid production from starch as substrate.
In commercial scale, glucose addition is an expensive
alternative. The use of a cheaper source of carbon, such
as starch, the most abundantly available raw material on
earth next to cellulose, in combination with amylolytic
lactic acid bacteria may help to decrease the cost of the
overall fermentation process. Use of raw starch or
renewable easily available and cheap polysaccharide
raw materials (complex organic sources) for lactic acid
fermentation involves two step processes — sacchari-
fication followed by Lactobacillus fermentation.
Amylolytic lactic acid bacteria can convert the starch
directly into lactic acid (Fig. 2). Development of
production strains which ferment starch to lactic acid
in a single step is necessary to make the process
economical. Very few bacteria have been reported so far
for direct fermentation of starch to lactic acid (Table 1).
Single step Amylolytic Lactic acid fermentation
amylolytic LAB
Starch Y Lactic acid
6. Substrates available for amylolytic lactic acid
fermentation
Bioconversion of polysaccharide carbohydrate mate-
rials to lactic acid can be made much more effective by
coupling the enzymatic hydrolysis of substrates and
microbial fermentation of the derived glucose into a
single step, which has been successfully employed for
lactic acid production from raw starch materials.
Historically, complex natural materials have been used
in fermentation processes because they are much cheaper
than pure substrates (Goel, 1994). Crop residues are
annually renewable sources of energy. Approximately
27
3.5 billion tonnes of agricultural residues are produced
per annum in the world (Pandey et al., 2001). The use of
a specific carbohydrate feedstock depends on its price,
availability, and purity. Although agro-industrial resi-
dues are rich in carbohydrates, their utilization is limited
(Pandey et al., 2001). Different food/agro-industrial
products or residues form the cheaper alternatives to
refined sugars as substrates for lactic acid production.
Sucrose-containing materials such as molasses are com-
monly exploited raw materials for lactic acid production.
Starch produced from various plant products is a po-
tentially interesting raw material based on cost and
availability. Laboratory-scale fermentations have been
reported for lactic acid production from starch by Lac-
tobacillus amylophilus GV6, (Vishnu et al., 2000, 2002;
Altaf et al., 2005), L. amylophilus B4437 (Mercier et al.,
1992), Lactobacillus amylovorus (Cheng et al., 1991;
Zhang and Cheryan, 1991, 1994), Lactococcus lactis
combined with Aspergillus awamorii (Kurusava et al.,
1988) and Rhizopus arrhizus (Kristoficova et al., 1991).
L. amylophilus NRRL B4437 (Nakamura and Crowell,
1979) L. amylovorus (Nakamura, 1981) and L. amylo-
philus GV6 are exceptions that have been described to
actively ferment starch to lactic acid and this may lead to
alternative process of industrial lactic acid production
(Cheng et al., 1991; Zhang and Cheryan, 1994; Vishnu
et al., 1998, 2000, 2002).
To make the process cost effective in terms of
substrate, various groups have worked on acid/enzyme
hydrolysis of starchy substrates followed by Lactoba-
cillus fermentation or simultaneous saccharification and
fermentation by co-culture/mixed culture fermentations.
It is reported that starch is used as substrate in two step
28 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34

Table 1
Amylolytic lactic acid producing bacteria so far reported
Bacteria Strain Reference
L. manihotivorans OND32T Guyot and Morlon-Guyot (2001)
L. manihotivorans LMG18010T Guyot et al. (2000)
LMG 18011 Ohkouchi and Inoue (2006)
L. fermentum Ogi E1 Calderon Santoyo et al. (2003), Agati et al. (1998)
L. fermentum MW2 Agati et al. (1998)
L. fermentum K9 Sanni et al. (2002)
L. amylovorus ATCC33622 Zhang and Cheryan (1991)
L. amylovorus B-4542 Cheng et al. (1991)
L. amylovorus Nakamura (1981), Zhang and Cheryan (1991),
Mercier et al. (1992), Litchfield (1996)
L. amylophilus JCIM 1125 Yumoto and Ikeda (1995)
L. amylophilus B 4437 Mercier et al. (1992), Nakamura and Crowell (1979)
L. amylophilus GV6 Vishnu et al. (1998, 2000, 2002, 2006), Vishnu (2000),
Naveena et al. (2003, 2004, 2005a,b,c),
Altaf et al. (2005, 2006, 2007a,b),
Gopal Reddy et al. (2004, 2006)
L. acidophilus Lee et al. (2001)
L. fermentum L9 Lee et al. (2001)
L. plantarum A6 Mette Hedegaard Thomsen et al. (2007), Giraud et al. (1991)
L. plantarum LMG18053 Giraud et al. (1991)
L. plantarum NCIM 2084 Krishnan et al. (1998)
S. bovis 148 Junya Narita et al. (2004)
Lactobacillus sp. TH165 Wang et al. (2005)
Leuconostoc St3-28 Mette Hedegaard Thomsen et al. (2007)
L. cellobiosus Chatterjee et al. (1997)
Lactobacillus strains LEM 220, 207, 202 Champ et al. (1983)
Leuconostoc strains Lindgren et al. (1984)
S. macedonicus Diaz-Ruiz et al. (2003)
L. amylolyticus Bohak et al. (1998)

fermentation process of saccharification and Lactoba- lignocellulose/hemicellulose hydrolysates (Karel et al.,

cillus fermentation by enzyme/acid hydrolysis method
which is relatively costly process (Vickroy, 1985; Datta
et al., 1995; Yumoto and Ikeda, 1995; Litchfield, 1996;
Xiaodong et al., 1997). Very few reports are available on
isolation of amylolytic lactic acid bacteria for single step
fermentation of inexpensive complex carbohydrates
(starch) to lactic acid. Use of efficient amylolytic lactic
acid producing bacteria will eliminate saccharification
costs of substrate thereby reducing the production cost
(Vickroy, 1985; Datta et al., 1995; Yumoto and Ikeda,
1995; Litchfield, 1996). In this direction we have
reported single step lactic acid fermentation by an
amylolytic bacterium L. amylophilus GV6 with high
production efficiency (Vishnu et al., 1998, 2000, 2002;
Naveena et al., 2003, 2004, 2005a,b, Altaf et al., 2005,
2006, 2007a,b). At high starch concentrations, lactic
acid production is low with the known amylolytic
organisms (Litchfield, 1996; Yumoto and Ikeda, 1995;
Zhang and Cheryan, 1991; Mercier et al., 1992). Some
agricultural by-products that are potential substrates for
lactic acid production are cornstarch (Cheng et al., 1991;
Hang, 1990), cassava starch (Yumoto and Ikeda, 1995),
1997), cottonseed hulls, Jerusalem artichokes, corn cob,
corn stalks (Vickroy, 1985), beet molasses (Goksungur
and Guvenc, 1999; Kotzamanidis et al., 2002), wheat
bran (Naveena et al., 2005a,b,c), rye flour (Raccach and
Bamiro, 1997), sweet sorghum (Richter and Trager,
1994), sugarcane press mud (Xavier and Lonsane,
1994), cassava (Xiaodong et al., 1997; Rojan et al.,
2005; John et al., 2006a,b), barley starch (Linko and
Javanainen, 1996), cellulose (Venkatesh, 1997), carrot
processing waste (Pandey et al., 2001), molasses spent
wash (Sharma et al., 2003), corn fiber hydrolysates
(Saha and Nakamura, 2003), and potato starch (Yumoto
and Ikeda, 1995; Anuradha et al., 1999).
7. Amylolytic enzymes in LAB
It is already mentioned that refined sugars or
gelatinized starch are generally used for production of
lactic acid by microbial fermentations. Many reports are
available which emphasize on fungi producing enzymes
to degrade raw starch (Bergmann et al., 1988; Hang,
1989a,b, 1990), but least work is done on isolation of
 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
Fig. 3. Scanning Electron Microscope (SEM) photograph of
unfermented wheat bran in SSF (with compact starch — cellulose
fibers) (Naveena et al., 2005a,b,c).
amylolytic lactic acid bacterial strains (Figuerao et al.,
1995; Morlon-Guyot et al., 1998). Some strains of
Lactobacillus spp. produce extracellular amylase and
ferment starch directly to lactic acid. Amylolytic
activity of fermenting organism is a major characteristic
for fermentation of starch to lactic acid. L. amylophilus
GV6 was evaluated for its amylolytic activity by esti-
mating the amount of extracellular amylolytic enzymes
(amylase and pullulanase) production (Naveena, 2004;
Vishnu et al., 2000, 2006). The amylase and pullulanase
activities were 0.439 U/g/min and 0.18 U/g/min res-
pectively in SSF with wheat bran (Naveena, 2004).
Amylolytic enzyme having both amylase and pull-
ulanase activities in L. amylophilus GV6 is a 90 KDa as
protein characterized by Vishnu et al. (2006). The
presence of both amylase and pullulanase (debranching
enzyme) characteristics for the fermenting organism L.
amylophilus GV6 is advantageous for efficient direct
conversion of complex starchy substrates to lactic acid.
This is evident from SEM photographs (Figs. 3 and 4)
showing the hydrolysis of starch fibers in wheat bran to
sugars which in turn are converted to L(+) lactic acid by
L. amylophilus GV6 (Vishnu et al., 2000; Naveena
et al., 2005c). Strain GV6 showed both amylase and
pullulanase activities of 0.59 and 0.34 U/ml/min in
submerged fermentation where maximum amylolytic
activity was shown with amylopectin followed by
soluble starch (Vishnu et al., 2006). The alpha amylase
activity in fermentation of raw starch by Streptococcus
bovis was (1.41 U/ml) higher than that from glucose
(0.06 U/ml) (Junya Narita et al., 2004). The strain L.
fermentum OGi E1 was able to grow and produce
amylase from the main carbohydrates found in cereals
(starch, maltose, glucose, sucrose, fructose) but also
from other compound of cereals and legumes, α-
29
galactosides (i.e. raffinose). Growth and amylase
production of this organism were slightly higher with
maltose than with starch. This might be explained by
the fact that the efficiency of starch conversion was
limited by the accumulation of limiting dextrins which
were not further fermented, thus limiting growth and
amylase synthesis (Calderon et al., 2001). Not many
amylolytic lactic acid bacteria involved in production of
lactic acid are studied for their amylolytic enzyme.
8. Submerged fermentations involving amylolytic
LAB
Soluble starchy substrates available in the form of
agricultural wastes, soluble pure and crude starches are
utilized in submerged fermentation. Among the various
starches, cassava starch, sorghum starch and corn starch
are the most abundant and relatively inexpensive raw
materials. Amylolytic lactic acid bacterial fermentation
has been receiving significant interest in recent past
because of the cost effective nature of the starchy
substrates. Soluble starch was utilized for production of
lactic acid in studies by Yumoto and Ikeda (1995) and
corn starch by Mercier et al. (1992). All the wild strains
reported so far produced more than 90% lactic acid at
low starch concentration, however at high starch
concentrations the lactic acid yield was low (Yumoto
and Ikeda, 1995; Nakamura and Crowell, 1979; Mercier
et al., 1992). L. amylophilus GV6 was found to actively
ferment various pure and crude starchy substrates at
both low and high starch concentrations with more than
90% lactic acid yield efficiency in anaerobic submerged
fermentation (Vishnu et al., 2000, 2002; Altaf et al.,
2005, 2007a,b) (Table 2). Strain GV6 was found to
utilize pure starches like soluble starch, corn starch and
Fig. 4. Scanning Electron Microscope photograph of fermented wheat
bran with bacterial cells in SSF (showing the hydrolyzed starch in
fibers) (Naveena et al., 2005a,b,c).
30 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34

Table 2
Fermentative production of L(+) lactic acid by amylolytic L. amylophilus GV6
Type of Carbon source Concentration of Nitrogen Fermentation period LA % LA⁎ Reference
fermentation starch source (days)
Submerged Soluble starch 2% Peptone, YE 1 96 96 Vishnu (2000)
5% Peptone, YE 3 90 Vishnu (2000)
9% Peptone, YE 4 76 Vishnu (2000)
Sorghum flour 6% 4.08% Peptone, YE 4 89 73 Vishnu et al. (2002)
Cassava flour 6% 4.94% Peptone, YE 4 88 68 Vishnu et al. (2002)
Wheat flour 6% 4.14% Peptone, YE 4 90 72 Vishnu et al. (2002)
Rice flour 6% 4.68% Peptone, YE 4 86 66 Vishnu et al. (2002)
Barley flour 6% 4.14% Peptone, YE 4 86 65 Vishnu et al. (2002)
Solid state Wheat bran 54.2% Peptone, YE 5 90 66 Naveena et al. (2005b)
Semi-solid state Wheat bran 44.4% Peptone, YE 5 98 78 Naveena (2004)
Submerged Starch 10% RL, YC 2 92 88 Altaf et al. (2007a)
Corn flour 5% 3.7% RL, YC 2.9 96 78.4 Altaf et al. (2007b)
Solid state Wheat bran 60% RL, YC 5 96 77.6 Altaf et al. (2006)
RL — red lentil, YC — bakers yeast cells, YE — yeast extract, LA — lactic acid yield efficiency (g lactic acid produced/g substrate utilized), LA⁎ —
lactic acid production efficiency (g lactic acid produced/g substrate taken).

potato starch and crude starches like sorghum flour,
cassava flour, wheat flour, rice flour, barley flour, sweet
potato flour, millet flour, jowar flour, tapioca flour, pearl
millet flour, refined wheat flour (maida flour) and corn
flour (Vishnu et al., 2002; Altaf et al., 2007b). Strain
GV6 showed 89% lactic acid yield efficiency with
soluble starch and sorghum flour, 85% with corn starch
and potato starch, 86% with barley flour and rice flour,
88% with cassava flour and 90% with wheat flour
respectively at high substrate concentrations of respec-
tive substrates (Vishnu et al., 2000, 2002; Gopal Reddy
et al., 2006). L. amylophilus GV6 is the most widely
studied amylolytic lactic acid bacterium due to its high
lactic acid production ability even at higher substrate
concentrations. Strain GV6 was also studied for its
ability to utilize inexpensive nitrogenous materials with
starch as substrate and was found to produce more than
90% lactic acid yield (Altaf et al., 2005, 2007a,b) with
good starch hydrolyzing ability (Figs. 5–7).
S. bovis 148 was found to directly produce lactic acid
from starch and maximum lactic acid concentration of
14.2 g/l was observed (Junya Narita et al., 2004). Batch
fermentations on synthetic mixed sugar and starch
medium with amylolytic lactic acid bacteria were
studied by Mette Hedegaard Thomsen et al. (2007)
where L .plantarum was found to actively ferment
mixed carbohydrates (20 g/l) to produce 14.25 g/l lactic
acid. Direct and effective lactic acid production by L.
manihotivorans LMG18011 for simultaneous sacchar-
ification and fermentation using soluble starch and food
wastes as substrates resulted in 19.5 g L(+)-lactic acid
from 200 g food wastes (Ohkouchi and Inoue, 2006). L.

Fig. 5. Scanning Electron Microscope (SEM) photograph of pure Fig. 6. Scanning Electron Microscope (SEM) photograph of starch in
soluble starch granules in MRS broth before sterilization. MRS broth after sterilization (autoclaving).
 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
Fig. 7. Scanning Electron Microscope (SEM) photograph of hydrolysis
of starch by L. amylophilus GV6 in submerged fermentation.
plantarum produced lactate yield of 0.81 g/g substrate
(Giraud et al., 1994) and L. amylophilus JCM 1125
produced 53.4 g/l using 100 g/l liquefied starch as
reported by Yumoto and Ikeda (1995). LA production
by L. plantarum NCIM 2084 was 72.9 g/l when
provided with 100 g/l of liquefied starch (Krishnan
et al., 1998). L. amylophilus NRRL B4437 produced
29 g/l lactic acid from 45 g/l of corn starch and L.
amylovorus was used in conversion of 120 g/l liquefied
starch to 92.5 g/l lactic acid in submerged fermenta-
tion (Zhang and Cheryan, 1991; Mercier et al., 1992). L.
amylovorus utilized raw corn starch, rice starch and
wheat starch medium to produce lactic acid with a
productivity of 10.1, 7.9 and 7.8 g lactic acid/l res-
pectively, but had lower productivities of 4.8 g/l and
4.2 g/l on cassava and potato starch in basal medium
respectively. When peptone (1%) is added to basal
medium with cassava starch as substrate, conversion
rate increased from 43% to 70% (7.7 g lactic acid/l)
(Xiaodong et al., 1997). A novel starch-degrading strain
of Lactobacillus casei was constructed by genetically
displaying α-amylase from the S. bovis strain 148 with a
FLAG peptide tag (AmyAF) (Junya Narita et al., 2006).
The lactic acid bacteria with AmyAF showed signifi-
cantly elevated hydrolytic activity toward soluble starch.
In fermentation using AmyAF-displaying L. casei cells,
50 g/l of soluble starch was reduced to 13.7 g/l, and
21.8 g/l of lactic acid was produced within 24 h. The
yield in terms of gram lactic acid produced per gram of
carbohydrate utilized was 0.60 g at 24 h. As AmyAF
was immobilized, cells were recovered after fermenta-
tion and used repeatedly. During repeated utilization of
cells, the lactic acid yield was improved to 0.81 g per g
of carbohydrate consumed at 72 h (Junya Narita et al.,
31
2006). Lactobacillus cellobiosus produced lactic acid
by direct fermentation of waste potato mash. Using a 5%
(w/v) potato mash with 3% (w/v) CaCO3 to neutralise
the lactic acid produced, 50% conversion of starch to
lactic acid occurred in 48 h without any other media
supplement (Chatterjee et al., 1997). Fermentative
production of lactic acid directly from starch was
studied in a batch fermentor using L. amylovorus,
96.2 g/l of lactic acid was produced from an initial
liquefied starch concentration of 120 g/l starch in 20 h
while 92.5 g/l of lactate was produced from the raw
starch of the same concentration in 39 h (Zhang and
Cheryan, 1991).
9. Solid-state fermentation
Solid-state fermentation (SSF) process is defined as
“the growth of microorganisms (mainly fungi) on moist
solid materials in the absence of free-flowing water”
(Moo-Young et al., 1983; Pandey, 1992). Apparently,
much work has been done on the production of
industrial enzymes using SSF and good commercial
success has been achieved. Moreover, till date there has
been no report on production of lactic acid at high
substrate concentrations in a single step through SSF
using amylolytic bacterial strains except for L. amylo-
philus GV6. In SSF, the solid substrate not only supplies
nutrients to the culture but also serves as an anchorage to
the microbial cells. A study was made to develop a
novel technology for L(+) lactic acid production by SSF
using L. amylophilus GV6 culture for which wheat bran
(a by-product of wheat milling industry) was selected as
solid substrate and support (Naveena et al., 2003, 2004,
2005a,b,c; Altaf et al., 2006).
Different brans like wheat bran, corn fiber, black
gram bran, green gram bran, pigeon pea brans (different
varieties) were used as substrates in SSF for lactic acid
production by strain GV6 (Naveena et al., 2003). Of all
the brans tested, L. amylophilus GV6 produced high
lactic acid using starch present in wheat bran as support
and substrate than other brans in SSF (Naveena et al.,
2003, 2005a,b). The organism could produce 90.111%
lactic acid yield which was comparable with that of
submerged fermentation reported earlier for L. amylo-
philus GV6 (Naveena et al., 2003). The interaction of L.
amylophilus GV6 with the wheat bran was observed
using SEM. These observations (Figs. 3 and 4) explain
the conversion of raw starch present in bran fibers to
glucose, which in turn is converted to L(+) lactic acid by
the organism. L. amylophilus GV6 was found to pro-
duce 36 g of lactic acid from high concentration of raw
starch (54.4 g) present in 100 g of wheat bran after
32 G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
optimization of fermentation parameters by RSM.
(Naveena et al., 2005a,b,c). Substitution of peptone
and yeast extract with low cost protein/nitrogen sources,
red lentil flour and bakers yeast cells was studied for
L(+) lactic acid production in SSF by L. amylophilus
GV6 using wheat bran as support and substrate. The
maximum lactic acid production of 46.3 g/100 g wheat
bran having 60 g of starch was obtained at optimized
conditions (Altaf et al., 2006). L. amylophilus GV6
showed 96% lactic acid yield efficiency (g lactic acid
produced/g substrate utilized) and 77.6% lactic acid
production efficiency (g lactic acid produced/ g sub-
strate taken) in SSF (Altaf et al., 2005, 2006, 2007a,b).
L. amylovorus NRRL B-4542 was utilized in produc-
tion of lactic acid using deoiled groundnut cake as solid
support with corn starch as substrate in solid-state
fermentation (Nagarjun et al., 2005).
10. Conclusions
Lactic acid fermentation has received extensive
attention for a long time since its potential applications
in various sectors in particular in foods and preparation
of biodegradable plastics. Starchy biomass can become
an attractive and alternative, cheap substrate replacing
costly sugars for lactic acid fermentation. Only few
amylolytic lactic acid bacteria are reported so far that
could actively ferment starch to lactic acid in single step
fermentation. Of all the amylolytic lactic acid ferment-
ing bacteria, L. amylophilus GV6 was found to be po-
tentially utilizing different starchy and nitrogenous
substrates with high lactic acid production efficiency.
Isolation and development of potential amylolytic or-
ganisms may lead to economical production of ster-
ospecific lactic acid isomers.
Acknowledgements
The authors are grateful to CSIR, New Delhi, for
providing fellowships to BJN and MV to carry out part
of this work.
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