amylolytic bacterial lactic acid fermentation — a review.pdf

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Research review paper

Amylolytic bacterial lactic acid fermentation A review

Gopal Reddy , Md. Altaf1 , B.J. Naveena 1, M. Venkateshwar, E. Vijay Kumar

Department of Microbiology, O smania 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 usingLactobacillussps. 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 Lactobacillusfermentation. 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

Available online at www.sciencedirect.com

Biotechnology Advances 26 (2008) 2234

www.elsevier.com/locate/biotechadv

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

E-mail addresses: [email protected](G. Reddy), [email protected](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
mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.biotechadv.2007.07.004http://dx.doi.org/10.1016/j.biotechadv.2007.07.004mailto:[email protected]:[email protected]


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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 isthe 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 acidat 130,000 to150, 000 MTper year (Mirasol,

1999). In 1999, Hepner rated the demand for lactic acid to

grow continually at 58% annual clip. Its use as a rawmaterial 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

1419% (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 anddomestic demand for lactic acid andin 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-

sis results in racemic DL-lactic acid whereas stereospecific

[L(+),D() and DLmixture] 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; Ritcherand 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 animalsand 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

23G. Reddy et al. / Biotechnology Advances 26 (2008) 2234


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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 acidmarket 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 duringprocessing 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 findsapplications 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 avariety 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, highstrength 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

175178 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 relatedbacteria 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

24 G. Reddy et al. / Biotechnology Advances 26 (2008) 2234


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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 oneof 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 etal., 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 allthese 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) becausethey 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 23 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 5.56.5 and the organisms

have complex nutritional requirements for amino acids,

peptides, nucleotide bases, vitamins, minerals, fatty

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 to2 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

produceDL-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 ofLactobacillus 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 ofL. plantarumisolated

from burong isda, a fermented food made from fish and

rice in Philippines. Amylolytic strains ofLactobacillusfermentum were isolated for the first time from Benin

maize sourdough (ogi and maw) byAgati 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 ofthe 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-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



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(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 90130 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

andLactococcusspecies (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 thechoice 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

Fig. 1. Metabolism of lactic acid bacteria.



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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 byLactobacillusfermentation.

Amylolytic lactic acid bacteria can convert the starchdirectly 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

StarchYamylolytic LAB

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

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 amylophilusGV6, (Vishnu et al., 2000, 2002;

Altaf et al., 2005),L. amylophilusB4437 (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) andRhizopus 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 toactively 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-

cillusfermentation or simultaneous saccharification and

fermentation by co-culture/mixed culture fermentations.

It is reported that starch is used as substrate in two step

Fig. 2. Schematic representation of lactic acid production from starch as substrate.



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fermentation process of saccharification and Lactoba-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 anamylolytic 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),

lignocellulose/hemicellulose hydrolysates (Karel et al.,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 (Yumotoand 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

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)



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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 inL. amylophilusGV6 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 organismL.

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 insubmerged 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, -

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 whichwere 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 starchare 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 byYumoto and Ikeda (1995)and

corn starch byMercier 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. amylophilusGV6 was found to actively

ferment various pure and crude starchy substrates atboth 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. 3. Scanning Electron Microscope (SEM) photograph of

unfermented wheat bran in SSF (with compact starch cellulose

fibers) (Naveena et al., 2005a,b,c).

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).



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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 widelystudied 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. 57).

S. bovis148 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.

Table 2

Fermentative production ofL(+) lactic acid by amylolyticL. amylophilusGV6

Type of

fermentation

Carbon source Concentration of

starch

Nitrogen

source

Fermentation period

(days)

LA % LA Reference

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, YCbakers yeast cells, YE yeast extract, LA lactic acid yieldefficiency (g lactic acid produced/g substrate utilized), LA

lactic acid production efficiency (g lactic acid produced/g substrate taken).

Fig. 5. Scanning Electron Microscope (SEM) photograph of pure

soluble starch granules in MRS broth before sterilization.

Fig. 6. Scanning Electron Microscope (SEM) photograph of starch in

MRS broth after sterilization (autoclaving).



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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.

amylovoruswas 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 andwheat 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

ofLactobacillus casei was constructed by genetically

displaying -amylase from theS. bovisstrain 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-displayingL. 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.,

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 mediasupplement (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 asthe 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 forL. amylo-

philusGV6. 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 forL(+) lactic acid production by SSFusingL. amylophilusGV6 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-

philusGV6 (Naveena et al., 2003). The interaction ofL.

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

Fig. 7. Scanning Electron Microscope (SEM) photograph of hydrolysis

of starch byL. amylophilusGV6 in submerged fermentation.



11/13

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. amylophilusGV6 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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