REVIEW

Starch-modifying enzymes of lactic acidbacteria – structures, properties, and applications

Penka Petrova1, Kaloyan Petrov2 and Galina Stoyancheva1

1 Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria2 Institute of Chemical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria

In spite that lactic acid bacteria (LAB) are used for production of fermented foods and drinks

for millennia, their ability to grow using starch as a sole carbon source was noticed by the

scientists in the last 30 years. A number of amylolytic LAB (ALAB) strains were isolated and

several detailed investigations of biochemical and genetic basis of starch hydrolysis were

performed. The purpose of this review is to summarize for the first time the available data

about the starch-modifying enzymes in ALAB. The most important amylolytic representatives

of the genera Lactobacillus, Lactococcus, Streptococcus, Pediococcus, Carnobacterium,

and Weissella are described. Amino acid sequences, corresponding to ALAB amylase

enzymes are compared and some features of the gene expression are analyzed. The

possible application of ALAB strains for direct production of lactic acid from starch, as well

as their participation in food manufacturing is discussed.

Received: August 31, 2012

Revised: October 19, 2012

Accepted: October 22, 2012

Keywords:

Amylase / Lactic acid bacteria / Pullulanase / Starch

1 Introduction

Starch is the second carbohydrate, after cellulose that is

most abundant in terrestrial plants. As part of the food

since the dawn of civilization, starch can be defined as the

most valuable polysaccharide used bymankind. According

to FAO statistics (http://faostat.fao.org), the world total

agricultural production of the starch-rich plants maize, rice,

wheat, potatoes and cassava reached 2.72 billion tons in

2010, with the largest share of maize (0.8 billion tons).

Starch is harvested and used chemically or enzymatically

processed into a variety of different products such as

starch hydrolysates, glucose syrups, fructose, starch or

maltodextrin derivatives, and cyclodextrins [1]. It is also

used as a raw material with many industrial applications:

for production of polyols used as sweeteners, in the paper

industry, and as a cheap and abundant substrate for

variety of microbial fermentations.

The ability of lactic acid bacteria (LAB) to live in starchy

media without symbiotic partner’s help was noticed in

the last 30 years when two new species Lactobacillus

amylophilus and L. amylovorus were described [2, 3].

LAB strains with starch-degrading activities are very

rare: so far, representatives of only three LAB genera

(Lactobacillus, Lactococcus, and Streptococcus) are

reported to produce lactic acid directly from starch as a

carbon source. On the other hand, the complete genome

sequencing and annotations revealed the existence of

amylase genes in almost all LAB. The purpose of this

review is to summarize the available data about the

starch-modifying enzymes in LAB. The most important

amylolytic representatives of each genus are described

and their potential industrial importance is evaluated.

Amino acid sequences, corresponding to LAB amylase

enzymes are compared and analyzed.

Colour online: See the article online to view Fig. 1 in colour.

Correspondence: Dr. Penka Petrova, Institute of Microbiology,Bulgarian Academy of Sciences, 26, Acad. G. Bontchev str., 1113Sofia, BulgariaE-mail: pepipetrova@yahoo.comFax: þ359-2-8700109

Abbreviations: ALAB, amylolytic lactic acid bacteria; LA, lacticacid; LAB, lactic acid bacteria

DOI 10.1002/star.20120019234 Starch/Starke 2013, 65, 34–47

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2 Lactic acid bacteria (LAB) withstarch-modifying properties – sources,methods of isolation, and exploration

Fermented foods high in starch are a widespread menu of

the population in Asia, Africa, and South America. The

lactic acid fermentation is an approach to increase their

nutritional value and taste. It can also save energy used for

the preparation of foods of plant origin, cereals and bread.

ALAB are mainly distributed in directly fermented meals,

based onmanioc, sorghum, rice, millet, maize, cassava, or

taro [4–9]. Other sources of ALAB are the fermented cereal

beverages, such as maize pozol [10], non-alcoholic drink

bushera [11], sorghum beer [12], beer malt and beer wort

[13], and the probiotic drink boza [14, 15]. Thirdly, ALAB

could be found in the sour sourdoughs from sorghum,

maize (ogi and mawe), wheat or rye [16–20]; and also

in cooked rice-fish products as burong isda and plaa-som

[21, 22]. Other habitats of ALAB are the digestive tract of

animals, as well as plants and plant wastes [3, 4, 23–31].

The methods of isolation of ALAB include sampling

from the potential source, enrichment, isolation of single

strains, identification and assessment of their amylolytic

ability. For initial growth media, MRS or M17 broths var-

iants supplemented with soluble starch along with, or

instead of glucose are used [2–7, 9–26, 28–38]. The

optimal conditions for LAB growth are considered to be

temperature of 30–378C and medium pH 6.5–6.8.

In the majority of the studies, the LAB isolates are

characterized and identified by a polyphasic approach

based on phenotypic and genotypic methods, such

as carbohydrate fermentation patterns using API 50CH

[4, 10, 11, 25, 26], ribotyping [10], intergenic transcribed

spacers-PCR/restriction fragment length polymorphism

(ITS-PCR/RFLP) [12], pulsed-field gel electrophoresis

(PFGE) [12, 26], ARDRA, rep-PCR, RAPD-PCR [7, 12,

14, 20], multiplex-PCR [9], and 16S rRNA gene sequenc-

ing [5, 9–12, 15, 20, 26].

The main distinctive feature of ALAB is their ability to

destroy the starch in MRS-starch agar plates that may be

observed when the plates are exposed to iodine vapors

[16]. The production of lactic acid (LA) from starch as a sole

carbon source is usually detected by the pH drop of the

liquid medium from pH 6.8 down to pH 3.7 [11, 15, 26],

followed by quantitative estimation of LA by gas chroma-

tography [3] or HPLC [4, 5, 15, 20], as well as by analysis of

carbohydrates, obtained in the course of the soluble starch

hydrolysis [4, 5, 36]. Then, the amylolytic activity of ALAB is

assayed by estimation of reducing sugars produced [4, 16,

32] or by measurement of iodine-complexing ability of

the residual starch [10, 15, 16, 20, 25]. A great deal of

the studies demonstrate amylase enzymes purification

by visualization of proteins by SDS–PAGE [4, 16, 23,

29, 33–37], and prove their properties by zymograms

[36] or enzyme’s N-terminal sequencing [23, 34]. The

molecular analyses of the responsible genes include

PCR amplification, sequencing and sequence comparison

[15, 20, 36, 38–40]. However, the investigations of ALAB

are so far focused mainly towards enhancement of starch

fermentation processes and only few strains were studied

for their amylolytic enzyme [41].

3 The spectrum of starch-modifyingenzymes, known to be produced by LAB

Pure starch consists predominantly of a-glucan in the form

of amylose and amylopectin. Amylose is a roughly linear

molecule containing �99% a-(1-4) and �1% a-(1-6)

bonds. Amylopectin is molecule with �95% a-(1-4) and

�5% a-(1-6) bonds. The number of repeated glucose

subunits in the amylose is usually in the range of 300–

3000, but can be many thousands. Amylopectin is highly

branched, being formed of 2000–200,000 glucose units.

Its inner-chains are formed of 20–24 glucose subunits.

Starches are defined as waxy when the ratio of amylose

to amylopectin is low (under 15%), normal when amylose

represents 16–35% and high-amylose when amylose

exceeds 36% [42]. Because of its widespread occurrence,

many enzymes for starch hydrolysis (glycosidases) or

modification (transglycosidases) are spread throughout

the whole biodiversity [43]. Basically there are four groups

of starch-converting enzymes: (i) endoamylases; (ii) exoa-

mylases; (iii) debranching enzymes; and (iv) transferases.

A number of these starch-converting enzymes belong to a

single family: the a-amylase family or GH13 family of

glycosyl hydrolases. This group of enzymes shares a

number of common characteristics such as a (b/a)8 barrel

structure (Fig. 1), the hydrolysis or formation of glycosidic

bonds in a-conformation, and a number of conserved

amino acid residues in the active site [1]. The KEGG

pathway of starch metabolism (http://www.genome.jp)

summarizes two possible catabolic directions of starch

utilization: (i) hydrolysis to dextrin and then to glucose,

Figure 1. Three-dimensional model of the amylase ofLactobacillus amylovorus (a) and amylopullulanaseof Lactobacillus paracasei B41 (b); http://swissmodel.expasy.org/.

Starch/Starke 2013, 65, 34–47 35

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or (ii) cleavage and conversion of the terminal glucose

residues to a-D-glucose-1-phosphate. Thus, the starch-

modifying enzymes in ALAB belong to a-amylases, malto-

genic amylases (MAases), amylopullulanases, pullula-

nases, neopullulanases, glycogen phosphorylases and

olygo-1,6-glucosidases.

4 Amylases and amylopullulanases of thegenus Lactobacillus

The lactobacilli group is that most efficiently utilizes starch

as a carbon and energy source among all ALAB. As it

is given away in Fig. 2, strains belonging to 11 from 96

distinct Lactobacillus species are reported to be able to

degrade starch [44].

Lactobacillus amylophilus was the first ALAB reported

in 1979 by Nakamura and Crowell. It is recognized as a

homofermentative lactic acid bacterium, which ferments

starch to L(þ)-lactic acid [2]. The following strains of the

species were described in the literature: JCM 1125 [45,

46], NCIB11546 [47], and NRRL B-4437 [33, 48].

L. amylovorus was isolated by Nakamura [3]. The type

strain is NRRL B-4540 (LMG9496). The amylases of

L. amylovorus and L. amylophilus were purified for the

first time by Pompeyo et al. [33] and later were investigated

by other authors [37, 38, 49]. In general, both species

possess very high amylase activity, as the maximum of

L. amylovorus was even higher than that of L. amylophilus

[33]. The enzymes are a-amylases with molecular masses

140 kDa for L. amylovorus and 105 kDa for L. amylophilus

amylase (Table 1). Of the six examined substrates, both

enzymes had the highest activity on soluble starch. Neither

of the enzymes hydrolyzed pullulan or cyclodextrins.

Various metal ions, such as 1 mM Ca2þ and Ba2þ, stimu-

lated L. amylophilus amylase activity and inhibited

L. amylovorus amylase activity.

Another strain, displaying extremely high starch-

degrading activity is GV6, which was initially identified

as L. amylophilus [50–58]. However, the sequence

comparison of 16S rRNA gene of GV6 (accession No.

AY330709) with that of L. amylophilus type strain

CIP102988T (accession No. HE573913) is only 97%.

GV6 possesses the highest similarity with L. amylovorus

(99% identity) thus suggesting the need of reclassification

of the strain (Fig. 2).

The starch-degrading enzyme of GV6 is extracellular

amylopullulanase (Mw 90 kDa) active towards soluble

starch, raw starch, amylose, glycogen, pullulan, with high-

est activity towards amylopectin.

In chronological order, L. acidophilus was the next

species, assigned to ALAB. In 1983, Champ et al. [28]

isolated two L. acidophilus strains – LEM 220 and LEM

207. They detected the presence of an intracellular amy-

lase, which production was more abundant in media con-

taining amylopectin or starch than in media containing

1

L. amylophilus GV6

L. amylovorus DSM 20531

L. acidophilus ATCC 4356T

L. amylolyticus DSM 11664T

L. amylophilus CIP 102988T

L. amylotrophicus NRRL B-4435

L. amylotrophicus LMG 11400T

L. fermentum NBRC 15885T

L. pentosus N3

L. plantarum Bom 816

L. paraplantarum DSM 10667T

P. acidilactici DSM 20284

P. pentosaceus DSM 20336T

L. manihotivorans 18010T

L. paracasei B41

Lc. lactis ssp. lactis B84

S. bovis JB1

Weissella confusa JCM 1093T

Figure 2. Phylogenetic tree of the ALAB based on full 16S rRNA gene sequences using the neighbor-joining method aftermultiple sequence alignment by ClustalW. In caseswhen 16S rRNA gene sequences of the amylolytic strains are not availablein the database, those of the type strains of the corresponding species were included in the comparison.

36 P. Petrova et al. Starch/Starke 2013, 65, 34–47

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glucose or maltose. Temperature and pH optima of LEM

220 amylase were 5.5 and 558C, and of LEM 207 – 6.4 and

408C, respectively. These two enzymes differed in their

products of starch hydrolysis. Lee et al. [25] isolated two

other amylolytic L. acidophilus strains A-4 and L23.

The enzyme activity of L. acidophilus L23 was associated

with the cell envelope and was inducible rather than con-

stitutive, whereas A4 possessed both cell-bond and extra-

cellular activity. However, these enzymes were not further

purified.

L. cellobiosus D-39 was isolated as a new ALAB

species in 1984 by Sen and Chakrabarty [59]. Later, it

was proposed that L. cellobiosus has to be reclassified

and, as a rule of priority of the first described species,

renamed as L. fermentum [60]. The amylase enzyme of

L. cellobiosus D-39 was purified and obtained in crystal

form. It was stable at neutral pH range, maximally active at

508C, and its molecular weight was 22.5 kDa. More than

a decade later, Agati et al. [16] isolated two intensively

starch-degrading L. fermentum strains OgiE1 and Mw2

and studied their amylase activities. It was found that

OgiE1 secreted extracellular amylase, while Mw2 pro-

duced predominantly cell-bond amylase. The distribution

of enzyme activity between extra- and intracellular frac-

tions and fermentation kinetics of OgiE1 indicates that the

extracellular a-amylase enzyme hydrolyzes the starch to

dextrins, which are then cleaved to maltose. Transferred

into the cell, maltose is hydrolyzed to glucose by the

intracellular a-glucosidase [24]. Further, several other

amylolytic L. fermentum strains were isolated [6, 9, 25,

26] and the presence of chromosomal genes a-amy and

dexC, encoding amylase and neopullulanase was demon-

strated [9].

L. plantarum is the most widespread ALAB species

in fermented foods [27]. L. plantarum A6 was isolated

by Giraud et al. [4] who reported the synthesis of extra-

cellular amylase enzyme. The enzyme was purified

and the capacity of the strain A6 to break down the

raw starch was demonstrated [35, 61]. Molecular charac-

terization of the amyA gene showed 30-end tandem

repeats that suggest a common evolutionary origin with

L. amylovorus amylase gene [49].

Olympia et al. [21] found a starch-hydrolyzing

L. plantarum strain L137. In difference to the other

Lactobacillus amylases, the enzyme of L137 is associated

with a 33 kb endogenous plasmid pLTK13. In 2008, Kim

et al. [62] cloned and sequenced the gene apuA, encoding

the amylopullulanase of L137. It consisted of an ORF of

6171 bp encoding a protein of 2056 amino acids, with Mw

216 kDa. The catalytic domain was located in themiddle of

Table 1. Starch-degrading enzymes produced by ALAB

Species Strain Gene Enzyme

MW

(kDa)

Amino

acids

Enzyme activity, pH and

temperature optimum Reference

L. amylophilus NRRL B-4437 amyA a-amylase 140 ND Extracellular, pH 5.5, 408C [33]

L. amylophilusa) GV6 ND Amylopullulanase 90 ND Extracellular [58]

L. amylovorus CIP 102989T amyA a-amylase 105 953 Extracellular, pH 5.0, 638C [33, 38, 49, 68]

L. acidophilus LEM 220 ND Amylase ND ND Intracellular, pH 6.4, 408C [28]

LEM 207 ND Amylase ND ND Intracellular, pH 5.5, 558C [28]

L. cellobiosusb) D-39 ND a-amylase 22.5 ND Extracellular, pH 7.3, 508C [59]

L. fermentum Ogi E1 amyA a-amylase 103 980 Extracellular, pH 5.0, 408C [24, 37, 38]

L. plantarum A6 amyA a-amylase 95.5 913 Extracellular, pH 5.0, 608C [4, 37, 38]

L. plantarum L137 apuAc) Amylopullulanase 216 2056 Extracellular, pH 4.0, 358C [21, 62]

L. manihotivorans LMG 18010T amyAc) a-amylase 100 901 Extracellular, pH 5.5, 558C [37, 38, 64, 65]

L. paracasei B41 amy1 Amylopullulanase 67 592 Extracellular, pH 5.0, 458C [15]

Lc. lactis ssp. lactis B84 amyY a-amylase 58 524 Extracellular [20]

B84 amyL a-amylase 57 491 Intracellular, pH 5.4, 458C [20]

Lc. lactis ssp. lactis IBB500 amyAc) a-amylase 121 ND Extracellular, pH 4.5, 358C [69]

IBB500 pulAc) Pullulanase 74 ND Extracellular, pH 4.5, 458C [73]

S. bovis JB1 amyA a-amylase 70 742 Extracellular, pH 5.5, 508C [34]

JB1 amyB a-amylase 56 484 Intracellular, pH 6.8, 408C [76]

S. bovis 148 amyA a-amylase 77 703 Extracellular, pH 5.5, 508C [23, 72]

148 amyB a-amylase 57 484 Intracellular, pH 6.5, 408C [36]

ND, not determined.a) The strain GV6 most probably belongs to another species, here the original designation was kept.b) Later reclassified as L. fermentum.c) Plasmid-localized gene.

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the N-terminal region, where a six amino acid sequence is

repeated 39 times. Another three amino acid sequence

(Gln–Pro–Thr) is repeated 50 times in the C-terminal

region. The optimal conditions for soluble starch degra-

dation were 358C and pH 4.0. The enzyme hydrolyzes

starch, amylopectin, glycogen, pullulan, and to a small

extent amylose, and has no activity on dextran and cyclo-

dextrins. The major reaction products from soluble starch

were maltotriose, maltotetraose, and maltopentaose, but

no panose was detected, and maltotriose was the sole

product from pullulan [21].

L. plantarum Bom 816 and L. pentosus N3 with cell-

bond amylase activity were isolated by Petrova et al. [14].

The pH and temperature optima of the cell-bond amylases

for both strains were pH 5.5 and 458C. By analyzing the

starch-fermentation products it was demonstrated that

L. pentosus N3 produce succinic acid in addition to lactic

acid from starch. Twenty new amylolytic L. plantarum and

six L. paraplantarum strains were isolated by Turpin et al.

[9].

L. amylolyticus is an ALAB, isolated by Bohak et al.

[13]. They performed genotypic studies of 25 strains

isolated from beer malts and similar to L. amylovorus.

After investigation of DNA–DNA similarities and 16S

and 23S rRNA gene sequences these strains formed

new species and DSM 11664T was proposed as the type

strain.

L. amylotrophicus is a species closely related to

L. amylophilus. In 2006, Naser et al. [63] studied the first

isolated by Nakamura and Crowell [2] six strains of L.

amylophilus (NRRL B-4435, NRRL B-4436, LMG 6900T,

NRRL B-4438, NRRL B-4439, and NRRL B-4440) includ-

ing them in a multilocus sequence analysis. After this

reinvestigation, it was demonstrated that two strains,

NRRL B-4436 and NRRL B-4435, occupy a distinct taxo-

nomic position. Further genomic and phenotypic research

revealed that they represent a single novel species – L.

amylotrophicus sp. nov with type strain LMG 11400T

(NRRL B-4436T).

L. manihotivorans was developed as a new homolactic

ALAB species by Morlon-Guyot et al. [5]. The type

strain, LMG 18010T produces an extracellular a-amylase

that shares common characteristics with those of

L. plantarum A6 and L. amylovorus – high molecular

weight (100 kDa) [64], same temperature and pH

optima (558C, 5.5) [65]. Later, six new amylolytic

L. manihotivorans isolates were studied, as most of them

contain plasmids. Plasmid curing of the type strain

LMG 18010T (OND 32T) brought to loss of the starch

and raffinose fermentation ability, suggesting that the

gene amyA is plasmid-localized [66].

L. paracasei B41 was the most recently isolated ALAB

strain (2012) and is the first amylolytic representative of

L. casei group [15]. It produces extracellular amylopullu-

lanase, able to degrade soluble starch, and small amounts

of pullulan. The protein consists of 592 amino acids and

has Mw 67 kDa. Compared to the amylases of the closely

related species (L. casei, L. paracasei) B41 enzyme has

several amino acid substitutions, three of them in the

amylase catalytic domain. An inducible control at amy1

expression was proved.

A comprehensive study of the genetic basis of ALAB

enzymes was presented by Turpin et al. [9]. A collection

of 152 ALAB from ben-saalga and the metagenomes

of millet fermented foods were screened for the

presence of six genes encoding the enzymes involved

in starch degradation and its products entry into the

glycolytic pathway. The distribution of these genes among

the isolates was as follows: 79% for agl (a-glucosidase),

76% for glgP (glycogen phosphorylase, 75% for a-amy

(a-amylase), 66% for malP (maltose phosphorylase),

54% for dexC (neopullulanase), and 19% for malL

(olygo-1,6-glucosidase). Only 8% of the isolates tested

were positive for all the six genes, while 3% were negative

for all of them.

The recent full genomes annotations of the genus

Lactobacillus amassed new information about LAB amy-

lase genes. A comparison between the deduced amino

acid sequences of amyA gene orthologs in nine strains is

shown at Fig. 3. The strains are representatives of various

Lactobacillus species. Despite that amyA presents in the

genomes, none of these strains expressed it in vivo.

Interestingly, amy genes that are expressed, differ sub-

stantially in their sequences from these of the relative

species [15].

AmyA genes of L. amylovorus, L. plantarum A6, and

L. manihotivorans share 98% identity and are completely

different from amyA found in other lactobacilli [37]. A

comparison of their amino acid sequences is shown at

Fig. 4. The enzymes are organized in two functional

domains: catalytic (amino acids 1–474) and starch-binding

domain (SBD, amino acids 475 to 953) [40]. The catalytic

domain contains all conserved regions for GH13 family

[67]. Peculiar feature of these enzymes is the large SBD of

almost 500 amino acids. It consists of tandem repeat units

of 91 amino acids each: four repeats in L. manihotivorans

and L. plantarum A6 and five repeats in L. amylovorus.

The role of SBD is the adsorption onto raw starch

granules. Thus, it promotes attachment to the substrate

and increases its concentration at the active site of

the enzyme, which allows microorganisms to degrade

non-soluble starch [39, 40]. Therefore, Lactobacillus

amylases that lack SBD would be unable to degrade the

raw starch.

Important property of extracellular amylases is the

existence of a signal peptide that ensures the transport

of the polypeptide out of the cell before enzyme’s matu-

ration. It contains predominantly hydrophobic amino acids

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(28 in L. paracasei, 36 in L. amylovorus, and L. plantarum

A6), and a typical cleavage site AQA [15, 68].

The analysis of amy1 promoters revealed the presence

of cis-acting sequence termed catabolite-responsive

element (CRE). It is involved in the binding of the global

regulatory factor catabolite control protein (CcpA). CcpA

binds to the promoter at low glucose levels and activates

the expression of regulated genes of starch metabolism in

Gram-positive bacteria. Since the high glucose concen-

trations repressed amy1 in L. paracasei and L. amylovorus,

it was deduced that the most probable mechanism of

expression control is at transcriptional level [15].

5 Starch-modifying enzymes of the lacticacid cocci

5.1 Amylases and pullulanases of the genusLactococcus

Lactococcus lactis is widespread in fermented foods; it

can be isolated from fermented cereal beverages, sour-

doughs, and cooked fish products [12, 17–19, 22]. In spite

of the starch abundance in these inhabitant niches, only

several Lactococcus isolates were found to be amylolytic

[10, 11, 20, 69].

Figure 3. Amino acid sequences comparison of deduced amylases of Lactobacillus strains. Designations: ‘‘L_pl’’ –L. plantarum; ‘‘L_rham’’ – L. rhamnosus; ‘‘L_cas’’ – L. casei; ‘‘L_sali’’ – L. salivarius; ‘‘L_acid’’ – L. acidophilus; ‘‘L_kefiran’’ –L. kefiranofaciens. The regions conservative for all strains are black-highlighted. The alignment was done with the programGeneDoc 2.7.0.

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Figure 4. Amino acid sequences comparison of extracellular amylases of Lactobacillus plantarum A6, L. manihotivorans andL. amylovorus. The identical amino acids are designated with asterisks; the signal peptide – with bold, italics; the cleavage site –with arrows. Catalytic aspartates (D) and glutamate (E) are bold, underlined. The repeated sequences that form the starch-binding domains are underlined. For the alignment the ClustalW program was used (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

40 P. Petrova et al. Starch/Starke 2013, 65, 34–47

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Observing the fully sequenced genomes of lactococci,

there are four genes that are potentially involved in starch

utilization: amyY, amyL, glgP, and apu encoding extra-

cellular and cytoplasmic a-amylases; glycogen phos-

phorylase and amylopullulanase [70, 71]. All four genes

were detected in the genome of the first amylolytic Lc.

lactis subsp. lactis B84 [20]. Reverse transcription PCR

experiments showed that both genes, encoding a-amy-

lases (amyL and amyY) were expressed into mRNAs,

whereas apu and glgP were not. The cytoplasmic amylase

proved to be the key enzyme, involved in the starch

hydrolysis with maximum activity at 458C and pH 5.4

(Table 1). It has been proven that although bearing the

same genes, other Lactococcus strains IL1403 and DSM

20481 were unable to produce amylase enzymes [20, 72].

The strain Lc. lactis B84 is plasmid-free and the genes

encoding amylolytic enzymes have chromosomal location

[20], as it was also demonstrated for the majority of the

genes encoding a-amylases in Lactobacillus [47, 49, 68].

In contrast, other authors described plasmid localization of

pullulanase and a-amylase of Lc. lactis IBB500 [31, 69].

Plasmid with size 30 kb contained both pulA and amyA

genes that was proved by plasmid curing experiments [69].

The extracellular a-amylase from Lc. lactis IBB500 had

molecular mass of 121 kDa and showed an optimum

activity at a lower pH (4.5) and temperature (358C) in

difference to the amylases described in other studies

[69]. The extracellular pullulanase type I (73.9 kDa) was

also purified from a cell-free culture supernatant of Lc.

lactis IBB500 [73]. Pullulanase activity was increased by

addition of Co2þ and completely inhibited by Hg2þ. The

enzyme was specifically directed toward a-1,6 glycosidic

linkages of pullulan giving maltotriose units; from starch it

produced a mixture of maltose and maltotriose.

5.2 Amylases of lactic acid Streptococcus spp

Analysis of the full genome sequencing of Streptococcus

thermophilus revealed that its capacity to degrade starch is

severely reduced. The genome contains a complete amy-

lase gene (amyL) and two pseudogenes (cdexT and

cpulA) encoding remnants of a dextranase and a pullula-

nase [74]. According to Goh et al. [75], S. thermophilus

strain LMD-9 is unable to ferment starch, and the intra-

cellular alpha-amylase, encoded by amyL remains inactive

[75].

In contrast, the amylases of S. bovis strains were

essential for the rapid cell growth on starchy substrates.

Two different strains of S. bovis were found to degrade

starch – JB1 and 148 [23, 34]. Satoh et al. [23] cloned in

E. coli the genes, encoding extracellular and intracellular

a-amylases of S. bovis 148 – amyA and amyB (Table 1).

The intracellular a-amylase of S. bovis 148 hydrolyzed

soluble starch to a large amount of maltotriose and a small

amount of maltose, whereas the extracellular a-amylase

completely hydrolyzed soluble starch to maltose and

glucose. The extracellular a-amylase was able to hydro-

lyze raw starch, but the intracellular enzyme was not.

The deduced amino acid sequence of the intracellular

a-amylase corresponds to a 484-residue protein with a

calculated Mw 57 kDa, whereas the extracellular enzyme

consisted of 703 residues and has Mw 77 kDa. Highly

hydrophobic 39-amino acid sequence served as a signal

peptide for transport and maturation of the extracellular

enzyme [72].

Freer purified the extracellular enzyme of S. bovis JB1

[34]; a bit later Whitehead and Cotta [76] identified the

intracellular amylase enzyme of JB1 and cloned the

responsible gene in E. coli. Comparing S. bovis a-amy-

lases, the N terminus of the S. bovis 148 extracellular

amylase is almost identical to that of the S. bovis JB1.

The homology between intracellular a-amylases was

83.2% [34, 36].

Two other Streptococcus strains were also reported

to display low amylolytic activity – S. bovis 25124 and

S. macedonicus [10]. However, the enzymes interacting

with starch have not been discussed.

5.3 Starch-modifying enzymes of the generaPediococcus, Enterococcus, andOenococcus

Numbers of ALAB, belonging to these three genera were

isolated from fermented traditional starch-containing foods

and drinks. Of 152 ALAB, isolated by Turpin et al. [9], as

P. pentosaceus 26% of the strains were identified, and

10% – as P. acidilactici. This study demonstrated that

pediococci contain three genes, responsible for the

starch degradation: a-amy, encoding a-amylase, dexC –

neopullulanase, and malL – oligo-1,6-glucosidase. The

last enzyme is known as capable of hydrolysis of (1-6)-

alpha-D-glucosidic linkages with preference to short-chain

substrates.

Amylolytic representatives of the genus Enterococcus

were found in maize pozol – E. sulfureus [10]. E. faecium

was isolated from African starchy beverages bushera and

kisra [11], but there was no evidence for its ability to

convert starch.

With regard to amylolytic Oenococcus strains, none

have been isolated.

5.4 Starch-modifying enzymes of the generaLeuconostoc, Carnobacterium, andWeissella

These three LAB genera are related and belong to the

family of Leuconostocaceae. Two distinct starch-utilizing

representatives of Leuconostoc were found in Ugandan

Starch/Starke 2013, 65, 34–47 41

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bushera – Leuc. mesenteroides ssp. mesenteroides

and Leuc. mesenteroides ssp. dextranicum [11]. Leuc.

mesenteroides was also isolated from Nigerian gari [26],

and from ben-saalga [9]. The latter study (as well as

the KEGG database) showed that the type strain Leuc.

mesenteroides ssp. mesenteroides ATCC8293 lacks

genes, encoding amylases and pullulanases. It synthes-

izes only oligo-1,6-glucosidase, able to degrade short

chains of dextrins.

The analysis of the complete genome of Carnobacterium

sp. 17-4 showed the presence of all essential genes, encod-

ing starch-degrading enzymes: a-amylase, glycogen phos-

phorylase, and oligo-1,6-glucosidase [77]. However, this

genus is not involved in cereal fermentations [27].

Weissella confusa (formerly Lactobacillus confusus)

has been isolated from sugar cane, carrot juice, milk,

fermented cereals, and beverages [78]. Amylolytic

Weissella confusa strain was isolated from bushera [11].

The species is widespread also in the French sourdoughs

and recently the full genome sequence of Weissella

confusa LBAE C39-2 was completed [79]. The genome

contains genes, encoding neopullulanase (588 amino

acids), amylopullulanase (560 amino acids), and oligo-

1,6-glucosidase (560 amino acids).

6 Evolution of the amylolytic enzymesfamily in LAB

The results of the reconstruction of Lactobacillales

genomes suggest that their common ancestor had at least

2100–2200 genes. Hence, during the process of evolution,

this order of Gram-positive bacteria have lost 600–1200

genes (25–30%) and gained at least 100 genes after the

divergence from the bacilli ancestor [80]. Niche-specific

adaptation has played a central role in the evolution of LAB.

In the species with larger genomes, such as L. plantarum

and L. casei, the loss of ancestral genes was counter-

balanced by the emergence of many new genes via dupli-

cation and horizontal gene transfer [81].

The analysis of the orthologs, involved in starch

hydrolysis by the genus Lactobacillus reveals that the

amylase or pullulanase genes usually present in the

genome, but the majority of them are not expressed

due to mutation damages in the promoter, in the amylase

catalytic domain, or in the sequence encoding the signal

peptide [15]. With long-term propagation in dairy habitats

that do not engage the amylolytic enzymes, the respon-

sible genes were converted to pseudo-genes since

they are not subjected to the selective pressure of the

environment. The same phenomenon was observed in

S. thermophilus – the strain LMD-9 harbors a large number

of pseudogenes (13% of ORFeome), indicating that it has

also undergone major reductive evolution, with the loss of

carbohydrate metabolic genes found in their streptococcal

counterparts [75]. According to other authors, the large

number of pseudo-genes may be an indication for

possible niche shift of ancestor strain from plant to dairy

environment [74].

Many genes important for the growth of LAB have been

found on mobile elements such as plasmids or transpo-

sons. The analysis of the phylogenetic relationships

strongly supports the hypothesis of a horizontal gene

transfer between the remotely related bacteria. Wasko

et al. [69] noted the possibility of horizontal gene transfer

between Ralstonia and Lactococcus, supported by the

strong homology between the amino acid sequences of

the a-amylases of these two species. Doman-Pytka et al.

[31] and Kim et al. [62] affirmed that the plasmid DNA

represents a gene cassette, which is vital for the adap-

tation of lactococci or lactobacilli to the plant environment.

7 Heterologous expression ofstarch-modifying enzymes of ALAB

The initial attempts of heterologous expression of genes of

ALAB were aimed at LAB strains improvement for appli-

cation in lactic acid production from starch. An example is

the development of amylolytic L. plantarum by cloning and

chromosomal integration of L. amylovorus a-amylase

gene [82]. Narita et al. [83] constructed a novel starch-

degrading strain of L. casei by genetically displayed

a-amylase from S. bovis 148 on the cell surface. Aiming

to produce branched oligosaccharides, Cho et al.

employed the gene for the enzyme maltogenic amylase

(MAase) from L. gasseri. The gene was successfully

expressed in Lc. lactis as a host [84].

In other cases, the genes were cloned with the purpose

to investigate their function or to obtain larger amount of

the desired enzyme. Thus, the expression of genes amyA

and amyB of S. bovis 148 and JB1 in E. coli was achieved

[23, 34, 76]. The cloning of S. bovis amyA in L. lactis

IL1403, L. delbrueckii and S. thermophilus launched the

development of broad host vectors for LAB [72].

The expression of complete and truncated forms of

L. amylovorus amylase in L. plantarum enabled the

analysis of the starch-binding domain [39].

8 Applications of ALAB

The desired application of ALAB is at first place for pro-

duction of lactic acid (LA) from starch because of the

profitability of this substrate. Discovered by Swedish

scientist C.W. Scheele (1780) in sour milk, lactic acid

was initially considered as a milk component. In 1857,

Pasteur revealed that it was a fermentation metabolite

42 P. Petrova et al. Starch/Starke 2013, 65, 34–47

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[85]. Lactic acid is a valuable chemical with a wide variety

of applications in food, pharmaceutical, textile, and cos-

metic industry. Latter, lactic acid was given extra attention

because of its use as a monomer for biodegradable poly-

mers [86]. There are two optically active isomers of LA:

L(þ) and D(�) forms. While the first is human metabolized

and preferable for production LA form, the second is con-

sidered to be harmful to humans in high levels. Thus, the

D(�)-isomer has limited application in food and pharma-

ceutical industries [41]. On the other hand, the optical

purity is very important for the monomer feedstock in

LA-based polymers manufacturing [87].

Lactic acid can be produced commercially both by

chemical syntheses and microbial fermentation. By chemi-

cal synthesis, LA is obtained in racemic DL mixture,

whereas microbial fermentation can purvey the needed

stereo-isomer [88]. Another advantage of the biological

production is the possibility of using cheap raw materials

as a feedstock. Since the low substrate costs provide

economically feasible LA production, the attention shifted

on conversion of cellulosic and starchy materials [89, 90].

However this requires additional steps of hydrolysis prior to

fermentation, which makes the process uneconomic. The

starch conversion occurs in several steps: (i) liquefaction to

dextrines by enzymes or acids at high temperatures; (ii)

enzymatic saccharification to glucose (iii) glucose fermen-

tation to lactic acid. The ALAB ability of direct conversion of

starch to lactic acid, unifying the steps of saccharification

and fermentation in a single process, make it economically

attractive. Although the most ALAB, producing lactic

acid by direct conversion of starch are from the genus

Lactobacillus – strains of the species L. amylophilus,

L. amylovorus, L. fermentum, L. manihotivorans,

L. paracasei, L. pentosus, and L. plantarum, there are also

reports for strains of Lc. lactis [20] and S. bovis [91].

Very few ALAB strains are capable to convert starch

at high substrate concentrations (Table 2). The strains

L. amylophilus GV6 [92], L. amylovorus ATCC 33620

Table 2. Lactic acid productivity of ALAB after fermentation of various starch-containing substrates

ALAB species

L(þ) LA/total

LA ratio

Fermentation

mode Substrate

Initial

starch

(g/L)

LAa)

(g/L)

Duration

(h)

Yield (g LA/g

utilized starch) pH control Reference

L. amylophilus GV6 L(þ)b) Batch Soluble starch 100 75.7 96 0.89 6.5 [92]

Batch Corn starch 100 39.2 120 0.78 6.5 [92]

Batch Potato starch 100 38.1 144 0.81 6.5 [92]

SSSFc) Wheat bran 44.4 35 130 0.78 6.5 [56]

SSFc) Wheat bran 54 36 120 0.66 6.5 [54]

L. amylophilus

JCM 1125

92.5% Batch Soluble starch 50 30 150 0.6 6.8 [46]

Batch Soluble starch 100 53.4 >400 0.53 6.8 [46]

L. amylovorus

ATCC 33620

DLb) Batch Soluble starch 120 96.2 20 0.80 Uncontrolled [93]

Batch Raw cassava starch 10 7.7 36 0.77 5.5 [96]

L. fermentum

Ogi E1

40–43.5% Batch Soluble potato

starch

�18 �8 30 0.48d) 6.0 [24]

L. manihotivorans

LMG 18010T99% Batch Soluble starch 17.5 12.6 12 0.67d) 6.0 [32]

L. paracasei B41 92.5% Batch Soluble starch 40 37.3 48 0.93 5.0 [15]

L. pentosus N3 ND Batch Soluble potato starch 30 5.5 96 0.34 Uncontrolled [14]

L. plantarum A6 36% Batch Raw cassava starch 45 41 72 0.91 6.0 [61]

Batch Dry-heated starch 88 80 144 0.91 6.0 [61]

L. plantarum

Bom 816

ND Batch Soluble potato

starch

30 9.5 96 0.69 Uncontrolled [14]

Lc. lactis ssp.

lactis B84

95.8% Batch Soluble potato

starch

20 5.5 150 0.28 6.0 [20]

S. bovis 148 95.6% Batch Raw corn starch 20 14.7 48 0.88d) 6.0 [91]

LA, lactic acid; SSSF, semi-solid state fermentation, SSF, solid state fermentation, ND, not determined.a) The obtained LA is represented as an average amount indicated in the corresponding reference.b) % of L(þ) LA form was not presented by the authors.c) The initial starch concentration is estimated as grams in 100 g substrate; LA is presented as grams, after extraction.d) Yield as g lactic acid per g total sugars consumed.

Starch/Starke 2013, 65, 34–47 43

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[93], and L. plantarum A6 [61] produced promising

quantities of lactic acid (8–10% w/v) from soluble starch.

However, these three strains yielded significantly lower

amounts of LA (<4% w/v) from raw starchy substrates.

Moreover, only L. amylophilus GV6 produces the desired

L(þ)-form from raw materials in high amounts. In spite of

all, the process of lactic acid production by direct conver-

sion of starchy materials using ALAB is feasible and

preferable to the use of conventional ones.

Lactic acid fermentation is also a good approach for

maintaining the nutritional value of cereals and leads to a

general improvement in the durability, consistency, taste,

and aroma of the finished product. That is why the second

practical application of ALAB is in the manufacturing of

various fermented foods and beverages. Some of them are

used as colorants, spices, drinks, snacks, or light food,

while others are a main meal in many areas of the world

[27]. Depending on the species and strain composition of

ALAB starters, they may be applied for development of

probiotic fermented meals and beverages [9, 14, 94] and

for hipo-allergic children foods [8, 95]. ALAB producing

extracellular amylases are also applied as sourdough

starters, especially for the original in taste and aroma

bakery products, containing rye and corn flour [41]. This

technology improves the bread characteristics and

extends the period of bread’s life by suppression of unde-

sired microflora.

9 Conclusions

During the last decades, a lot of new data concerning the

presence of genes, encoding starch-modifying enzymes in

ALAB were garnered. Part of the genes was found as a

result of complete genome sequencing annotations; hence

their structures and the regulation of their expression will

be clarified in the future. The ALAB strains have promising

applications for direct conversion of starch into LA and

in production of fermented starch-containing foods and

beverages.

The authors thank to the National Scientific Fund,

Republic of Bulgaria, as this work was financially sup-

ported by the Research grant DMU 03/45.

The authors have declared no conflict of interest.

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