**3.8. Analytical methods**

Cell growth was evaluated by reading the absorbance of culture medium at 600 nm using a Secoman spectrophotometer and numeration of total colony forming unit by 10-fold serial dilution of fermented broth and pour plating on MRS-starch agar (De Man Rogosa and Sharpe medium in which glucose has been replaced by soluble starch (Prolabo-Merck Eurolab, France)). In order to evaluate the capacity of microorganism to acidify the culture medium, the pH of the fermented broth was measured using an electronic pH meter (Mettler Seven S20, Japan)

Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 641

14BYA42, 20BBA60, 17BNG51, 23BYA21, 26BMB81) presented very high amylolytic power (≥15mm) and were qualified as amylase overproducing isolates. The amylolytic power was defined as the average diameter (mm) of starch hydrolysis halo (Fig.1.) provoked by a strain after its inoculation in micro-well on MRS-starch agar plate for 48 h incubation at optimum temperature of growth for three assays. The amylolytic power is an expression of the capacity of an isolate to degrade starch during the culture. Among the amylase overproducing isolates, two (04BBA19, 26BMB81) were aero-anaerobic non spore forming, gram positive and catalase negative bacteria; this characteristic is proper to lactic acid bacteria. Microscopic observation showed rod cells. Biochemical characteristics of these isolates were carried out using API 50 CH kit bioMerieux system, the results are summarized in Table 1, the isolates were tested for their possibility to ferment 50 carbohydrates, and this fermentation profile was use for their numerical identification. According to their biochemical profile, 04BBA19 and 26BMB81 were respectively identified as *Lactobacillus fermentum* and *Lactobacillus plantarum.* The strain 04BBA19 (*Lactobacillus fermentum*) presented a very high amylolytic power, as it was able to cause a starch hydrolysis halo of 45 ±1.5mm on MRS-starch agar plate after 48 h of incubation at 40 °C; consequently it was selected for further studies. The preliminary test of thermostability carried out on its crude extract amylase showed that it produce a very high thermostable enzyme and it was selected for an application on simultaneous production of thermostable

**Figure 1.** Plate assays for detection of amylase activity of lactic acid bacteria (04BBA19, 26BMB81) on MRS-starch agar plate medium. The diameter of hydrolysis halo was revealed by flooding the plates

with Iodine solution (0.1% I2+1% KI) after 48 h of culture at 40°C

amylase and lactic acid from starchy material.

The amylolytic power of Lactic acid bacteria was determined using the method of wells by inoculation of 10 μl of microbial strain in 4 mm depth micro-wells on the surface of MRSstarch agar plate. The starch hydrolysis halo was revealed after 48 h of incubation using iodine solution. The amylolytic power was defined as the average diameter (mm) of hydrolysis halo provoked by a strain after its inoculation in micro-well on MRS-starch agar plate for 48 h incubation at optimum temperature of growth for three assays

The activity of amylase both in crude and purified extracts was assayed by iodine method. In a typical run, 5 ml of 1% soluble starch solution and 2 ml of 0.1M phosphate buffer (pH 6.0) were mixed and maintained at a desired temperature for 10 min, then 0.5 ml of appropriately diluted enzyme solution was added. After 30 min the enzyme reaction was stopped by rapidly adding 1ml of 1M HCl into the reaction mixture. For the determination of residual starch, 1 ml of the reaction mixture was added to 2.4 ml of diluted iodine solution and its optical density was read at 620 nm using a spectrophotometer (Secoman). One unit of amylase activity (U) was defined as the amount of enzyme able to hydrolyse 1 g of soluble starch during 60 min under the experimental condition. The lactic acid was determined according to Kimberley and Taylor [51]. The nature of amylase (endo-acting or exo-acting) was determined according to Ceralpha method (Megazyme) which uses a blocked maltoheptaoside as substratre [57].

The affinity of the enzyme preparation from selected LAB toward raw cassava starch was studied by incubating 0.2 g of raw cassava flour with 1ml of the enzyme solution at 60 ◦C for 15 min. After centrifugation, the -amylase activity of the supernatant was measured and

the adsorption percentage was calculated as follows: 100 *A B Adsorption (%) <sup>A</sup>* , *A* is the

original -amylase activity and *B* is the -amylase activity in the supernatant after adsorption on raw potato starch granules.

For the determination of raw starch digestibility, raw cassava was used and the reaction mixture containing 100 U of -amylase preparation from the selected LAB and 100 mg of raw cassava starch in a final volume of 10ml dispensed in 100ml Erlenmeyer flasks were incubated in alternative water bath shaker at 60◦C and 150 oscillations per min. After a time interval of 6 h, the reducing sugars liberated in the reaction mixtures were determined by dinitrosalicyclic acid method [58].

Light microscopy was used for the examination of the effect of enzyme on raw starch granules using Olympus microscope BH-2.

### **4. Results and discussion**

### **4.1. Biochemical properties of amylolytic LAB isolated**

From the 28 samples of soil collected from different localities of Cameroon, 90 amylolytic isolates were screened but only 9 isolates (04BBA15, 04BBA19, 05BBA22, 05BBA23, 14BYA42, 20BBA60, 17BNG51, 23BYA21, 26BMB81) presented very high amylolytic power (≥15mm) and were qualified as amylase overproducing isolates. The amylolytic power was defined as the average diameter (mm) of starch hydrolysis halo (Fig.1.) provoked by a strain after its inoculation in micro-well on MRS-starch agar plate for 48 h incubation at optimum temperature of growth for three assays. The amylolytic power is an expression of the capacity of an isolate to degrade starch during the culture. Among the amylase overproducing isolates, two (04BBA19, 26BMB81) were aero-anaerobic non spore forming, gram positive and catalase negative bacteria; this characteristic is proper to lactic acid bacteria. Microscopic observation showed rod cells. Biochemical characteristics of these isolates were carried out using API 50 CH kit bioMerieux system, the results are summarized in Table 1, the isolates were tested for their possibility to ferment 50 carbohydrates, and this fermentation profile was use for their numerical identification. According to their biochemical profile, 04BBA19 and 26BMB81 were respectively identified as *Lactobacillus fermentum* and *Lactobacillus plantarum.* The strain 04BBA19 (*Lactobacillus fermentum*) presented a very high amylolytic power, as it was able to cause a starch hydrolysis halo of 45 ±1.5mm on MRS-starch agar plate after 48 h of incubation at 40 °C; consequently it was selected for further studies. The preliminary test of thermostability carried out on its crude extract amylase showed that it produce a very high thermostable enzyme and it was selected for an application on simultaneous production of thermostable amylase and lactic acid from starchy material.

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

(Mettler Seven S20, Japan)

medium, the pH of the fermented broth was measured using an electronic pH meter

The amylolytic power of Lactic acid bacteria was determined using the method of wells by inoculation of 10 μl of microbial strain in 4 mm depth micro-wells on the surface of MRSstarch agar plate. The starch hydrolysis halo was revealed after 48 h of incubation using iodine solution. The amylolytic power was defined as the average diameter (mm) of hydrolysis halo provoked by a strain after its inoculation in micro-well on MRS-starch agar

The activity of amylase both in crude and purified extracts was assayed by iodine method. In a typical run, 5 ml of 1% soluble starch solution and 2 ml of 0.1M phosphate buffer (pH 6.0) were mixed and maintained at a desired temperature for 10 min, then 0.5 ml of appropriately diluted enzyme solution was added. After 30 min the enzyme reaction was stopped by rapidly adding 1ml of 1M HCl into the reaction mixture. For the determination of residual starch, 1 ml of the reaction mixture was added to 2.4 ml of diluted iodine solution and its optical density was read at 620 nm using a spectrophotometer (Secoman). One unit of amylase activity (U) was defined as the amount of enzyme able to hydrolyse 1 g of soluble starch during 60 min under the experimental condition. The lactic acid was determined according to Kimberley and Taylor [51]. The nature of amylase (endo-acting or exo-acting) was determined according to Ceralpha method (Megazyme) which uses a blocked maltoheptaoside as substratre [57].

The affinity of the enzyme preparation from selected LAB toward raw cassava starch was studied by incubating 0.2 g of raw cassava flour with 1ml of the enzyme solution at 60 ◦C for 15 min. After centrifugation, the -amylase activity of the supernatant was measured and

original -amylase activity and *B* is the -amylase activity in the supernatant after

For the determination of raw starch digestibility, raw cassava was used and the reaction mixture containing 100 U of -amylase preparation from the selected LAB and 100 mg of raw cassava starch in a final volume of 10ml dispensed in 100ml Erlenmeyer flasks were incubated in alternative water bath shaker at 60◦C and 150 oscillations per min. After a time interval of 6 h, the reducing sugars liberated in the reaction mixtures were determined by

Light microscopy was used for the examination of the effect of enzyme on raw starch

From the 28 samples of soil collected from different localities of Cameroon, 90 amylolytic isolates were screened but only 9 isolates (04BBA15, 04BBA19, 05BBA22, 05BBA23,

, *A* is the

the adsorption percentage was calculated as follows: 100 *A B Adsorption (%) <sup>A</sup>*

adsorption on raw potato starch granules.

dinitrosalicyclic acid method [58].

**4. Results and discussion** 

granules using Olympus microscope BH-2.

**4.1. Biochemical properties of amylolytic LAB isolated** 

plate for 48 h incubation at optimum temperature of growth for three assays

**Figure 1.** Plate assays for detection of amylase activity of lactic acid bacteria (04BBA19, 26BMB81) on MRS-starch agar plate medium. The diameter of hydrolysis halo was revealed by flooding the plates with Iodine solution (0.1% I2+1% KI) after 48 h of culture at 40°C


Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 643

**Figure 2.** Time course of growth (), pH (), -amylase () and lactic acid () production by *L. fermentum* 04BBA19 in 1% (w/v) soluble starch medium at 40°C, pH 6.0. The data shown are averages of

The study of cell growth and amylase production as a function of temperature (Fig. 3a) showed that *L. fermentum* 04BBA19 exhibited maximal growth and amylase activity at 45°C, confirming thus the strong relationships between cell growth and amylase production. On the other hand the maximum value of lactic acid was produced at the same temperature. Many other investigators reported that maximum amylase production occurred at the optimum growth temperature [56, 53]. These results are contrary to the findings of Chandra et al.[57] who studied the growth and amylase production of *Bacillus licheniformis* CUM 305. They have observed that this microorganism grew very well at 30°C, but did not produce amylase at that temperature. In addition, Saito and Yamamoto [58] found -amylase production at 50°C and cell growth at a temperature lower than 45°C for another strain of *B.* 

The amylase and lactic acid production by *L. fermentum* 04BBA19 was influenced significantly by initial pH of culture broth (Fig. 3b). Maximum amylase and lactic acid production was achieved for pH range of 4.0-6.5. These results could be explained by the fact that pH generally act by inducing morphological change in microorganism which

Amylase production is known to be induced by a variety of carbohydrate, nitrogen compounds and minerals [60, 61]. In order to achieve high enzyme yield, efforts are made to develop a suitable medium for proper growth and maximum secretion of enzyme, using an

triplicates assays within 10% of the mean value.

*licheniformis*.

facilitate enzyme production [59].

**4.3. Optimisation of amylase and lactic acid production** 

adequate combination of carbohydrates, nitrogen and minerals [53, 62].

+, positive reaction; -negative reaction, nc, non -conclusive

**Table 1.** Biochemical characteristics of amylases overproducing *Lactobacillus* isolated from soils

### **4.2. Amylase and lactic acid production**

In the presence of starch as carbon source at 40°C, *L. fermentum* 04BBA19 strain grew, exhibited amylolytic activity and produced lactic acid in the culture medium. The amylase production pattern in *L. fermentum* 04BBA19 (Fig.2) indicates that the induction of amylase took place during the lag phase (after 10 h of incubation) in the presence of starch. The level of amylase production increased significantly during the exponential phase of growth. Lactic acid production became visible around 15 h after incubation and also increased considerably during the exponential phase of growth. Cell growth, amylase and lactic acid production reached maxima values at the same time (40 h of fermentation). The values of those maxima were 1.1x109 cfu/ml, 107.30.5 U/ml, 8.70.5g/l for cell growth, amylase activity, and lactic acid production respectively. Such coincidence shows that amylase production by *L. fermentum* 04BBA19 was tightly linked to cell growth. These results are in agreement with the report of Goyal et al. [53], Liu and Xu [54] on the relationship between pattern of cell growth and amylase production. The decline of cell growth and amylase production after the peak occurred around 50 h of incubation and could be attributed to the rise of lactic acid concentration in fermented broth [6] or to the rise of protease levels [55]. The acidification was also expressed by the decrease of initial pH of culture broth (Fig. 2). The initial pH of culture broth declined significantly and reached a value of 3.0 around 50 h of incubation and then remained constant.

number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Test

Strains cod

Test

Strains cod

Heterofermentative

Amygdalin

Arbutin

Aesculin

Salicin

Cellobiose

+, positive reaction; -negative reaction, nc, non -conclusive

**4.2. Amylase and lactic acid production** 

Maltose

Lactose

Melibiose

Sucrose

Trehalose

Inulin

Gaz production

Optimum temperature of

growth

Growth at 10°C

Dextran

Ammonia from Arginine

Nitrate reduction

Glycerol

Erythritol

D-arabinose

L-arabinose

number 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Melezitose

04BBA19 - - nc - - + + + + - - - + + - - - - - - - - - - + - -

26BMB81 + + + + + + + + + + nc + + + + - + + - - - - - - + - -

**Table 1.** Biochemical characteristics of amylases overproducing *Lactobacillus* isolated from soils

In the presence of starch as carbon source at 40°C, *L. fermentum* 04BBA19 strain grew, exhibited amylolytic activity and produced lactic acid in the culture medium. The amylase production pattern in *L. fermentum* 04BBA19 (Fig.2) indicates that the induction of amylase took place during the lag phase (after 10 h of incubation) in the presence of starch. The level of amylase production increased significantly during the exponential phase of growth. Lactic acid production became visible around 15 h after incubation and also increased considerably during the exponential phase of growth. Cell growth, amylase and lactic acid production reached maxima values at the same time (40 h of fermentation). The values of those maxima were 1.1x109 cfu/ml, 107.30.5 U/ml, 8.70.5g/l for cell growth, amylase activity, and lactic acid production respectively. Such coincidence shows that amylase production by *L. fermentum* 04BBA19 was tightly linked to cell growth. These results are in agreement with the report of Goyal et al. [53], Liu and Xu [54] on the relationship between pattern of cell growth and amylase production. The decline of cell growth and amylase production after the peak occurred around 50 h of incubation and could be attributed to the rise of lactic acid concentration in fermented broth [6] or to the rise of protease levels [55]. The acidification was also expressed by the decrease of initial pH of culture broth (Fig. 2). The initial pH of culture broth declined significantly and reached a value of 3.0 around 50 h of incubation and then remained constant.

Raffinose

Starch

Glycogen

Xylitol

ß Gentiobiose

D-turanose

D-lyxose

D-tagatose

D-fucose

L-fucose

D-arabitol

L-arabitol

Gluconate

2-gh-Gluconate

5-Keto-Gluconate

Ribose

D-xylose

04BBA19 + + 45°C - - + - - - - - + - - - - + + + + - - - nc - - - - - 26BMB81 - + 40°C - - - - - - - + + - - - - + + + + - - - - + + - + +

L-xylose

Adonitol

ß methyl-D-Xyloside

Galactose

Glucose

Fructose

Mannose

Sorbose

Rhamnose

Dulcitol

Inositol

Mannitol

Sorbitol

α-Methyl-D-mannoside

α-Methyl-D-glucoside

Identification species (API 50CHL)

*Lactobacillus fermentum* 

*Lactobacillus plantarum* 

N-Acetyl-Glucosamine

**Figure 2.** Time course of growth (), pH (), -amylase () and lactic acid () production by *L. fermentum* 04BBA19 in 1% (w/v) soluble starch medium at 40°C, pH 6.0. The data shown are averages of triplicates assays within 10% of the mean value.

The study of cell growth and amylase production as a function of temperature (Fig. 3a) showed that *L. fermentum* 04BBA19 exhibited maximal growth and amylase activity at 45°C, confirming thus the strong relationships between cell growth and amylase production. On the other hand the maximum value of lactic acid was produced at the same temperature. Many other investigators reported that maximum amylase production occurred at the optimum growth temperature [56, 53]. These results are contrary to the findings of Chandra et al.[57] who studied the growth and amylase production of *Bacillus licheniformis* CUM 305. They have observed that this microorganism grew very well at 30°C, but did not produce amylase at that temperature. In addition, Saito and Yamamoto [58] found -amylase production at 50°C and cell growth at a temperature lower than 45°C for another strain of *B. licheniformis*.

The amylase and lactic acid production by *L. fermentum* 04BBA19 was influenced significantly by initial pH of culture broth (Fig. 3b). Maximum amylase and lactic acid production was achieved for pH range of 4.0-6.5. These results could be explained by the fact that pH generally act by inducing morphological change in microorganism which facilitate enzyme production [59].

### **4.3. Optimisation of amylase and lactic acid production**

Amylase production is known to be induced by a variety of carbohydrate, nitrogen compounds and minerals [60, 61]. In order to achieve high enzyme yield, efforts are made to develop a suitable medium for proper growth and maximum secretion of enzyme, using an adequate combination of carbohydrates, nitrogen and minerals [53, 62].

Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 645

enzyme yield was 67.10.5 U/ml. These results are in agreement with the reports of Cherry et al. [63], Saxena et al. [4] who reported maximum amylase production when starch was used as carbohydrate source. In the presence of glucose and fructose, amylase production was almost nil; and that was a proof that glucose and fructose repressed amylase synthesis by *L. fermentum* 04BBA19. This observation is in agreement with the reports of Theodoro and Martin [64] showing that synthesis of carbohydrate degrading enzymes in some microbial species leads to catabolic repression by substrate such as glucose and fructose. Similar results were observed by Halsetine et al. [65] for the production of amylase by the hyperthemophilic archeon *Sulfolobus solfataricus.* According to them, glucose prevented -amylase gene expression and not only secretion of performed enzyme. Since amylase yield is higher with amylose (92.3 U/ml) as carbohydrate source than with amylopectin (50.1 U/ml), the *L. fermentum* 04BBA19 amylase is more efficient for hydrolysis of alpha-1,4 linkages than those of alpha-1,6. The amylase production increased with the soluble starch concentration (Fig. 4), reaching a maximum (180.5 ± 0.3 U/ml) at the concentration range of 8-16 % (w/v). These optimum starch concentrations for amylase production by *L. fermentum* 04BBA19 are higher than that observed for amylase production in *Bacillus* sp. PN5 reported by Saxena et al. [4]. This microorganism presented an optimum soluble starch concentration of 0.6% (w/v) for amylase production. The lactic acid production also increased with the soluble starch concentration, the optimum starch concentration for lactic acid production was achieved

at the same range of concentration for amylase production.

**Figure 4.** Effect of starch concentration on -amylase () and lactic acid production ( ) by *L.* 

*fermentum* 04BBA19. The data shown are averages of triplicate assays with SD within 10% of mean value

Among the various gelatinized starchy sources tested, corn and sorghum flour were found to be the most suitable for -amylase and lactic acid production by *L. fermentum* 04BBA19

**Figure 3.** (a) Effect of temperature on microbial growth (), -amylase () and lactic acid ( ) production. (b) Effect of initial pH of culture broth on -amylase () and lactic acid ( ) production. The data shown are averages of triplicate assays within 10% of the mean value.

From the use of different carbohydrate sources in the present study, soluble starch proved to be the best inducer of amylase production (Table 1). In the presence of soluble starch at concentration of 1% (w/v), the enzyme yield reached 107.01.2 U/ml after 48 hours of fermentation, while in the presence of raw cassava starch at the same concentration, the enzyme yield was 67.10.5 U/ml. These results are in agreement with the reports of Cherry et al. [63], Saxena et al. [4] who reported maximum amylase production when starch was used as carbohydrate source. In the presence of glucose and fructose, amylase production was almost nil; and that was a proof that glucose and fructose repressed amylase synthesis by *L. fermentum* 04BBA19. This observation is in agreement with the reports of Theodoro and Martin [64] showing that synthesis of carbohydrate degrading enzymes in some microbial species leads to catabolic repression by substrate such as glucose and fructose. Similar results were observed by Halsetine et al. [65] for the production of amylase by the hyperthemophilic archeon *Sulfolobus solfataricus.* According to them, glucose prevented -amylase gene expression and not only secretion of performed enzyme. Since amylase yield is higher with amylose (92.3 U/ml) as carbohydrate source than with amylopectin (50.1 U/ml), the *L. fermentum* 04BBA19 amylase is more efficient for hydrolysis of alpha-1,4 linkages than those of alpha-1,6. The amylase production increased with the soluble starch concentration (Fig. 4), reaching a maximum (180.5 ± 0.3 U/ml) at the concentration range of 8-16 % (w/v). These optimum starch concentrations for amylase production by *L. fermentum* 04BBA19 are higher than that observed for amylase production in *Bacillus* sp. PN5 reported by Saxena et al. [4]. This microorganism presented an optimum soluble starch concentration of 0.6% (w/v) for amylase production. The lactic acid production also increased with the soluble starch concentration, the optimum starch concentration for lactic acid production was achieved at the same range of concentration for amylase production.

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

**Figure 3.** (a) Effect of temperature on microbial growth (), -amylase () and lactic acid ( ) production. (b) Effect of initial pH of culture broth on -amylase () and lactic acid ( ) production.

From the use of different carbohydrate sources in the present study, soluble starch proved to be the best inducer of amylase production (Table 1). In the presence of soluble starch at concentration of 1% (w/v), the enzyme yield reached 107.01.2 U/ml after 48 hours of fermentation, while in the presence of raw cassava starch at the same concentration, the

The data shown are averages of triplicate assays within 10% of the mean value.

**Figure 4.** Effect of starch concentration on -amylase () and lactic acid production ( ) by *L. fermentum* 04BBA19. The data shown are averages of triplicate assays with SD within 10% of mean value

Among the various gelatinized starchy sources tested, corn and sorghum flour were found to be the most suitable for -amylase and lactic acid production by *L. fermentum* 04BBA19 while for the raw starchy sources tested, potato starch was most suitable (Table 2). On the other hand the level of lactic acid was more important when corn and sorghum flours were used. The good production of -amylase and lactic acid when these starchy flours are used is based on their composition; they also contain proteins and vitamins which are required by lactic acid bacteria for their growth, enzymes and acids production [66].

Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 647

Parameters Enzyme yield (U/ml) Lactic acid(g/l)

Glucose 0.10.0d\* 14.30.5a Fructose 0.20.0d 12.10.5b Maltose 0.10.0d 12.80.4b Amylose 92.30.1b 12.20.1b Amylopectin 50.10.5c 10.30.5c Soluble starch 107.30.5a 8.70.5d

Yeast Extract 107.3±0.5b 8.70.5b Beef extract 92.4±0.5c 7.3±0.2b Peptone 88.31.7d 7.10.3b Tryptone 75.3±0.5e 6.5±0.5b Soya bean meal 397.30.4a 29.20.4a Ammonium sulphate 95.41.5c 7.2.0.8b Urea 76.30.3e 5.30.6c

CaCl2. 2H2O 412.10.6a 33.20.1a MgSO4. 7H2O 315.10.4b 31.20.5a FeSO4. 7H2O 237.30.7c 20.20.4b NaCl 315.20.9b 22.10.6b CuSO4.5H2O 12.20.6e 3.20.3d

Tween-40 209.50.1b 27.30.4b Tween-80 215.10.3a 35.20.3a

Corn flour 303.50.2a 36.30.6a Cassava flour 182.30.4c 24.20.8d Sorghum flour 305.80.7a 35.20.1a Rice flour 187.30.8c 30.10.5b Tapioca flour 237.40.6b 27.20.7c

Cassava starch 67.10.5c 21.30.4a Potato starch 87.20.5a 23.40.1a Cocoyam starch 78.60.2b 22.70.4a

Basal medium 107.50.3 8.70.5b Optimized medium 732.30.4 53.20.4a

**Table 2.** Effect of different parameters on -amylase and lactic acid production by *L. fermentum*

04BBA19 in submerged state fermentation at 45 °C and initial pH 6.5.

Carbohydrate sources (1% w/v)

Nitrogen sources (1.5% w/v)

Minerals (0.1% w/v)

Surfactants (1.5% w/v)

Raw starchy sources

Media

Gelatinized starchy sources (1 %w/v)

Among nitrogen sources used in the present study, soya bean meal and yeast extract showed significant effect on -amylase and lactic acid production. Soya bean meal, rich in protein is a potential nutrient for lactic acid fermentation. Similar results were obtained by several authors. Goyal et al. [53] reported that soybean meal presented a positive effect and was the best nitrogen source for raw starch digesting thermostable -amylase production by the *Bacillus* sp I-3 strain. The yeast extract was also reported to be a potential nutrient for lactic acid fermentation, since it contains vitamins, amino acids [66]. Though all nitrogen sources are positively influencing enzyme production by *L. fermentum* 04BBA19, an inverse behaviour has been observed with other bacterial strains, for instance, Tanyildizi et al. [67] reported zero effect of yeast extract on amylase production by *Bacillus* sp.

All metal salts tested in this study increased amylase and lactic acid production by *L. fermentum* 04BBA19, except CuSO4.5H2O that acted as inhibitor. The inhibition of amylase production by CuSO4.5H2O was also reported by Wu et al. [68] for the *Bacillus* sp CRP strain. Copper ion acted as poisonous compound for this strain and consequently inhibited amylase synthesis. The effect of CaCl2.2H2O was the most important, and was in agreement with the observation of Gangadharan et al. [61] who described the rise of amylase production by *B. amyloliquefaciens* when CaCl2.2H2O was supplemented to the culture medium. The supplementation of metal ions has been reported to provide good growth and also influence higher enzyme production. Most -amylases are metalloenzymes and in most cases, Ca2+ ions are required for maintaining the spatial conformation of the enzyme, thus play an important role in enzyme stability [61].

From the surfactants tested in this study, Tween-80 appeared to be the best surfactant sources for amylase production by *L. fermentum* 04BBA19. Similar results were obtained by Reddy et al. [69]. These authors reported that the supplementation of culture medium with Tween-80 resulted in a marked increase in the yields of thermostable -amylase and pullullanase by *C. thermosulfurogenes* SV2, and that the stimulation of enzyme production was greater when the surfactants were added after 18 h of incubation of culture. Beside stimulation, the surfactants caused and increased secretion of the enzymes into extracellular fluid [59].

From various environmental factors tested for -amylase and lactic acid production by *L.*  fermentum 04BBA19, it has been observed that all factors that increase amylase synthesis also positively affect lactic acid production. The optimization of the basal medium by supplementation of all carbohydrate, nitrogen, mineral and surfactant sources (excepted CuSO4.5H2O4) in culture medium resulted to a significant improvement of enzyme and lactic acid yield. In the optimized medium, amylase activity and lactic acid content reached 732.4±0.4 U/ml and 53.2±0.4 g/l respectively.

play an important role in enzyme stability [61].

732.4±0.4 U/ml and 53.2±0.4 g/l respectively.

fluid [59].

while for the raw starchy sources tested, potato starch was most suitable (Table 2). On the other hand the level of lactic acid was more important when corn and sorghum flours were used. The good production of -amylase and lactic acid when these starchy flours are used is based on their composition; they also contain proteins and vitamins which are required by

Among nitrogen sources used in the present study, soya bean meal and yeast extract showed significant effect on -amylase and lactic acid production. Soya bean meal, rich in protein is a potential nutrient for lactic acid fermentation. Similar results were obtained by several authors. Goyal et al. [53] reported that soybean meal presented a positive effect and was the best nitrogen source for raw starch digesting thermostable -amylase production by the *Bacillus* sp I-3 strain. The yeast extract was also reported to be a potential nutrient for lactic acid fermentation, since it contains vitamins, amino acids [66]. Though all nitrogen sources are positively influencing enzyme production by *L. fermentum* 04BBA19, an inverse behaviour has been observed with other bacterial strains, for instance, Tanyildizi et al. [67]

All metal salts tested in this study increased amylase and lactic acid production by *L. fermentum* 04BBA19, except CuSO4.5H2O that acted as inhibitor. The inhibition of amylase production by CuSO4.5H2O was also reported by Wu et al. [68] for the *Bacillus* sp CRP strain. Copper ion acted as poisonous compound for this strain and consequently inhibited amylase synthesis. The effect of CaCl2.2H2O was the most important, and was in agreement with the observation of Gangadharan et al. [61] who described the rise of amylase production by *B. amyloliquefaciens* when CaCl2.2H2O was supplemented to the culture medium. The supplementation of metal ions has been reported to provide good growth and also influence higher enzyme production. Most -amylases are metalloenzymes and in most cases, Ca2+ ions are required for maintaining the spatial conformation of the enzyme, thus

From the surfactants tested in this study, Tween-80 appeared to be the best surfactant sources for amylase production by *L. fermentum* 04BBA19. Similar results were obtained by Reddy et al. [69]. These authors reported that the supplementation of culture medium with Tween-80 resulted in a marked increase in the yields of thermostable -amylase and pullullanase by *C. thermosulfurogenes* SV2, and that the stimulation of enzyme production was greater when the surfactants were added after 18 h of incubation of culture. Beside stimulation, the surfactants caused and increased secretion of the enzymes into extracellular

From various environmental factors tested for -amylase and lactic acid production by *L.*  fermentum 04BBA19, it has been observed that all factors that increase amylase synthesis also positively affect lactic acid production. The optimization of the basal medium by supplementation of all carbohydrate, nitrogen, mineral and surfactant sources (excepted CuSO4.5H2O4) in culture medium resulted to a significant improvement of enzyme and lactic acid yield. In the optimized medium, amylase activity and lactic acid content reached

lactic acid bacteria for their growth, enzymes and acids production [66].

reported zero effect of yeast extract on amylase production by *Bacillus* sp.


**Table 2.** Effect of different parameters on -amylase and lactic acid production by *L. fermentum* 04BBA19 in submerged state fermentation at 45 °C and initial pH 6.5.

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

Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 649

**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.
