**4.4. Enzyme properties**

The amylase produced by *L. fermentum* 04BBA19 showed high affinity toward cassava raw starch granules with 80% adsorption and brought about 79% hydrolysis of 1% (w/v) suspension of raw cassava starch. On the other hand, the enzyme was able to hydrolyze blocked p-nitro phenyl methyl heptaoside, releasing a yellow compound (p-nitro phenol) with maximum absorption at 530 nm. This result was a proof that amylase from *L. fermentum* 04BBA19 is an endo acting amylase (-amylase), since the blocked p-nitro phenyl methyl heptaoside is known to be hydrolysed only by endo-acting amylases [38].

The enzyme exhibited maximum activity at 60-70°C and maintained 100% of its initial activity at 80°C for 30 min of heat treatment (Fig 5-a). When the enzyme was treated for the same time (30 min.) at 90°C and 100°C, the remaining activities were 90 and 87% respectively. These results showed the thermophilic character and very high thermostability of -amylase from *L. fermentum* 04BBA19. In general, most of lactic acid bacteria do not produce amylases. However, this property have been observed in some genera of lactic acid bacteria, especially in *L. plantarum* and *L. amylovorus* [37], *L. manihotivorans* [70, 13], *L. fermentum* OGI E1 [38]. But amylases produced by these strains are not thermostable. Traditionally high thermostable and thermophiles amylases are found in *Bacillus* and *Thermococcus* genera as: *B. amyloliquefaciens* [71]; *B. licheniformis* [72]; *B. stearothermophilus* [73]; *B. subtilis* and *T. aggreganes* [74], *T. profundus* [75], *Bacillus* sp PN5 [4], *B. cohnii* US147 [35], *Chromohalobacter* sp. TVSP 101 [76].

Fig. 6 shows the thermostability pattern of -amylase from *L. fermentum* 04BBA19 at 80°C, 90°C and 100°C when the time of heat treatment is beyond 30 min. Table 3 presents the thermal inactivation rate constant (ki) and half-life (T) at these temperatures. The half-life of this enzyme is higher than that of -amylase from *B. licheniformis*: 120 min at 70°C [76]. The thermal stability was considerably improved by addition of 0.1% (w/v) CaCl2.2H2O. Goyal et al*.* [53] obtained a half-life value of 3.5 h at 80°C with -amylase from *Bacillus* sp.I-3 in the presence of 0.1 % (w/v) calcium chloride, while under the same conditions; -amylase from *L. fermentum* 04BBA19 displayed a half-life of 6.1 h.

Due to its high thermostability, -amylase from *L. fermentum* 04BBA19 could be highly competitive in industrial bioconversion reactions, as compared to -amylase from *Bacillus*. In addition, this competitiveness is enhanced by the fact that lactobacilli, due to their nonpathogen character, are easily used in food industry [6].

The *L. fermentum* 04BBA19 -amylase is active and stable in pH range of 4.0 – 7.0 (Fig. 5-b), which is the pH range of many foods. In this respect, this amylase could be used in starch hydrolysis, brewing and baking.

**4.4. Enzyme properties** 

superscripts within columns are significantly different (p<0.05).

The basal medium contained soluble starch, 1% (w/v); yeast extract, 0.5 % (w/v); while the optimized medium contained all parameters without CuSO4.5H2O. The data shown are averages of triplicate assays with SD within 10% of mean value. For each group of parameters (Carbohydrate, Nitrogen, Mineral, Starchy sources, media), means with different

The amylase produced by *L. fermentum* 04BBA19 showed high affinity toward cassava raw starch granules with 80% adsorption and brought about 79% hydrolysis of 1% (w/v) suspension of raw cassava starch. On the other hand, the enzyme was able to hydrolyze blocked p-nitro phenyl methyl heptaoside, releasing a yellow compound (p-nitro phenol) with maximum absorption at 530 nm. This result was a proof that amylase from *L. fermentum* 04BBA19 is an endo acting amylase (-amylase), since the blocked p-nitro phenyl

The enzyme exhibited maximum activity at 60-70°C and maintained 100% of its initial activity at 80°C for 30 min of heat treatment (Fig 5-a). When the enzyme was treated for the same time (30 min.) at 90°C and 100°C, the remaining activities were 90 and 87% respectively. These results showed the thermophilic character and very high thermostability of -amylase from *L. fermentum* 04BBA19. In general, most of lactic acid bacteria do not produce amylases. However, this property have been observed in some genera of lactic acid bacteria, especially in *L. plantarum* and *L. amylovorus* [37], *L. manihotivorans* [70, 13], *L. fermentum* OGI E1 [38]. But amylases produced by these strains are not thermostable. Traditionally high thermostable and thermophiles amylases are found in *Bacillus* and *Thermococcus* genera as: *B. amyloliquefaciens* [71]; *B. licheniformis* [72]; *B. stearothermophilus* [73]; *B. subtilis* and *T. aggreganes* [74], *T. profundus*

Fig. 6 shows the thermostability pattern of -amylase from *L. fermentum* 04BBA19 at 80°C, 90°C and 100°C when the time of heat treatment is beyond 30 min. Table 3 presents the thermal inactivation rate constant (ki) and half-life (T) at these temperatures. The half-life of this enzyme is higher than that of -amylase from *B. licheniformis*: 120 min at 70°C [76]. The thermal stability was considerably improved by addition of 0.1% (w/v) CaCl2.2H2O. Goyal et al*.* [53] obtained a half-life value of 3.5 h at 80°C with -amylase from *Bacillus* sp.I-3 in the presence of 0.1 % (w/v) calcium chloride, while under the same conditions; -amylase from

Due to its high thermostability, -amylase from *L. fermentum* 04BBA19 could be highly competitive in industrial bioconversion reactions, as compared to -amylase from *Bacillus*. In addition, this competitiveness is enhanced by the fact that lactobacilli, due to their non-

The *L. fermentum* 04BBA19 -amylase is active and stable in pH range of 4.0 – 7.0 (Fig. 5-b), which is the pH range of many foods. In this respect, this amylase could be used in starch

methyl heptaoside is known to be hydrolysed only by endo-acting amylases [38].

[75], *Bacillus* sp PN5 [4], *B. cohnii* US147 [35], *Chromohalobacter* sp. TVSP 101 [76].

*L. fermentum* 04BBA19 displayed a half-life of 6.1 h.

pathogen character, are easily used in food industry [6].

hydrolysis, brewing and baking.

**Figure 5.** (a) Effect of temperature on activity () and stability () of -amylase from *L. fermentum*  04BBA19. (b) Effect of pH on activity () and stability () of -amylase from *L. fermentum* 04BBA19. The data shown are averages of triplicate assays within 10% of the mean value.

The metal salts generally act on activity of enzyme through their ions. The enzyme activity was highly improved by Ca2+, while Fe2+, Fe3+, Na+ and Mg2+ had less significant effect. On the contrary Cu2+ and EDTA acted as inhibitors (Fig. 7). The behaviour of the enzyme towards metal ions, particularly calcium, indicates its metalloenzyme nature, which is confirmed by the action of EDTA.

Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 651

this strain and its amylase are potential candidates for food industries (making of high density gruels, baking, brewing) and for the production of biodegradable plastic from

**Figure 7.** Effect of metal salts and EDTA on the activity of -amylase from *L. fermentum* 04BBA19. The

**Bacteria Strains References** 

data shown are averages of triplicate assays with SD within 10% of mean value.

*L. fermentum* 04BBA19 [47, 48] *L. manihotivorans* LMG18010T [69] *L. fermentum* Ogi E1 [11] *L. fermentum* MW2 [11] *L. fermentum* K9 [12] *L. acidophilus* L9 [78] *L. amylovorus* ATCC33622 [23] *L. amylovorus* B-4542 [29] *L. amylovorus* [17] *L. manihotivorans* OND32T [10, 13] *L. manihotivorans* [13] *L. manihotivorans* LMG 18011 [79] *L. acidophilus* [78]

starchy raw material.

**Figure 6.** Thermostability pattern of -amylase from *L. fermentum* 04BBA19, at 80, 90, 100°C without CaCl2.2H2O () and with 0.1% (w/v) CaCl2.2H2O (). The enzyme was pre-incubated at optimum pH, for 30, 60, 90, 120 and 180 min at temperatures (80, 90 and 100°C). The remaining activity was determined incubating the enzyme at optimum temperature, 60°C for 30 min. The data shown are averages of triplicate assays with SD within 10% of mean value.


**Table 3.** Inactivation rate constant (ki) and half-live (T) of amylase from *L. fermentum* 04BBA19 at 80, 90 and 100°C in the absence and the presence of 0.1% (w/v) CaCl2.2H2O.

The main ALAB that have been isolated for the past decade are summarized in Table 4. No study has dealt with the thermostability of their amylases, except the case reported by Aguilar et al. (2000) concerning the properties of the extracellular amylase produced by *L. manihotivorans* LMG 18010T. This strain produced an amylase with a moderate themostability exhibiting maximum activity at 55°C.

The strain *L. fermentum* 04BBA19 appears as the first ALAB producing highly thermostable amylase. The potential industrial application of this strain could be the bioconversion of inexpensive raw material as starch into lactic acid in single step process. On the other hand this strain and its amylase are potential candidates for food industries (making of high density gruels, baking, brewing) and for the production of biodegradable plastic from starchy raw material.

650 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

confirmed by the action of EDTA.

The metal salts generally act on activity of enzyme through their ions. The enzyme activity was highly improved by Ca2+, while Fe2+, Fe3+, Na+ and Mg2+ had less significant effect. On the contrary Cu2+ and EDTA acted as inhibitors (Fig. 7). The behaviour of the enzyme towards metal ions, particularly calcium, indicates its metalloenzyme nature, which is

**Figure 6.** Thermostability pattern of -amylase from *L. fermentum* 04BBA19, at 80, 90, 100°C without CaCl2.2H2O () and with 0.1% (w/v) CaCl2.2H2O (). The enzyme was pre-incubated at optimum pH,

CaCl2.2H2O (% w/v) ki (10-3.min-1) T (min) ki (10-3.min-1) T (min) ki .(10-3.min-1) T (min)

**Table 3.** Inactivation rate constant (ki) and half-live (T) of amylase from *L. fermentum* 04BBA19 at 80, 90

The main ALAB that have been isolated for the past decade are summarized in Table 4. No study has dealt with the thermostability of their amylases, except the case reported by Aguilar et al. (2000) concerning the properties of the extracellular amylase produced by *L. manihotivorans* LMG 18010T. This strain produced an amylase with a moderate

The strain *L. fermentum* 04BBA19 appears as the first ALAB producing highly thermostable amylase. The potential industrial application of this strain could be the bioconversion of inexpensive raw material as starch into lactic acid in single step process. On the other hand

0 3.4 204.0 5.6 123.8 7.9 87.7 0.1 1.9 364.8 2.6 266.6 3.8 182.4

80 °C 90°C 100°C

for 30, 60, 90, 120 and 180 min at temperatures (80, 90 and 100°C). The remaining activity was determined incubating the enzyme at optimum temperature, 60°C for 30 min. The data shown are

Temperatures

averages of triplicate assays with SD within 10% of mean value.

and 100°C in the absence and the presence of 0.1% (w/v) CaCl2.2H2O.

themostability exhibiting maximum activity at 55°C.

**Figure 7.** Effect of metal salts and EDTA on the activity of -amylase from *L. fermentum* 04BBA19. The data shown are averages of triplicate assays with SD within 10% of mean value.



Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 653

We gratefully acknowledge the assistance of the Ministry of Higher Education and Brewing society "Société Anonyme des Brasseries du Cameroun" (SABC) who supported this work

[1] Reddy G, Altaf M, Naveeana BJ, Ventkateshwar M., Vijay Kumar E (2008) Amylolytic

[2] Axelsson L (2004) Lactic acid bacteria: classification and physiology. In: Salminen S, von Wright A, Ouwehand A, editors. Lactic acid bacteria: microbiological and functional

[3] Asoodeh A, Chamani J, Lagziana M (2010) A novel thermostable, acidophilic -amylase from a new thermophilic "*Bacillus* sp. Ferdowsicous" isolated from Ferdows hot mineral spring in Iran: Purification and biochemical characterization. Inter. J. Biol.

[4] Saxena RK, Dutt K, Argawal L, Nayyar P (2007) A highly thermostable and alkaline

[5] Haki GD, Rakshit, SK (2003) Developments in industrially important thermostable

[6] Singh SK, Ahmed SU, Pandey A (2006) Metabolic engineering approaches for lactic acid

[7] Nguyen TT, Loiseau G, Icard-Vernière C, Rochette I, Trèche S, Guyot, JP (2007) Effect of fermentation by amylolytic lactic acid bacteria in process combinations, on characteristics of rice/soybean slurries: A new method for preparing high energy

[8] Songré-Ouattara, LT, Mouquet-Rivier C., Icard-Vernière C, Humblot C, Diawara B, Guyot JP (2008) Enzyme activities of lactic acid bacteria from a pearl millet fermented gruel (ben-saalga) of functional interest in nutrition. Int. J. of Food Microbiol. 128: 395–

[9] Giraud E, Lelong B, Raimbault M (1991) Influence of pH and initial lactate concentration on the growth of *Lactobacillus plantarum*. Appl Microbiol Biotechnol 36:

[10] Morlon-Guyot J, Guyot JP, Pot B, Jacobe de Haut I, Raimbault M (1998). *Lactobacillus manihotivorans* sp. Nov., a new starch-hydrolysing lactic acid bacterium isolated during

[11] Agati VJP, Guyot J, Morlon-Guyot P, Talamond, Hounhouigan DJ (1998). Isolation and characterization of new amylolytic strains of *Lactobacillus fermentum* from fermented

[12] Sanni A, Morlon-Guyot J, Guyot JP. New efficient amylase-producing strains of *Lactobacillus plantarum* and *L. fermentum* isolated from different Nigerian traditional

cassava sour starch fermentation. Int. J. Syst. Bacteriol. 48: 1101-1109.

maize doughs (mawe and ogi) from Benin. J Appl Microbiol 85: 512–20.

fermented foods. Int J Food Microbiol 2002;72:53–62.

density complementary foods for young children. Food Chem. 100: 623-631.

bacterial fermentation. A review. Biotechnology Advanced 26: 22-34.

amylase from a *Bacillus* sp PN5. Biores. Technol. 98: 260-265.

enzymes: a review. Biores. Technol. 89: 17-34.

production. Process Biochem. 41: 991-1000.

aspects. 3rd rev. and exp. ed.New York: Marcel Dekker, Inc.; 2004. p. 1-66.

**Acknowledgement** 

Macromol. 46: 289-297.

**6. References** 

400.

96–9.

through Research-Development Grant Programme.

**Table 4.** The main amylolytic lactic bacteria strains isolated during the past two decade
