**3. Materials and methods**

### **3.1. Samples**

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

fermentation processes.

**2.3. Lactic acid** 

demands. The spectrum of amylase application has widened in many other fields, such as clinical, medical and analytical chemistries, as well as their widespread application in starch saccharification and in the textile, food, brewing and distilling industries. Thermostability is one of the main features of many enzymes sold for bulk industrial usage. Thermostable αamylases are of interest because of their potential industrial applications. They have extensive commercial applications in starch liquefaction, brewing, sizing in textile industries, paper and detergent manufacturing processes. [33,34, 35]. The advantages of using thermostable amylases in industrial processes include the decreased risk of contamination and cost of external cooling, a better solubility of substrates, a lower viscosity allowing accelerated mixing and pumping [36]. Several thermostable α-amylase have been purified from *Bacillus sp*. and the factors influencing their thermostability have been investigated, but the thermostability of amylases from lactic acid bacteria have attracted very few scientific attention. *Lactobacillus amylovorus, Lactobacillus plantarum, Lactobacillus manihotivorans*, and *Lactobacillus fermentum* are some of the lactic acid bacteria exhibiting amylolytic activity which have been studied [37, 10, 38, 5, 39, 40]. However, most of αamylase from these bacteria presented weak thermostability compared to those of genus *Bacillus*. Owing to the important acidification of fermenting medium by most lactic acid bacteria, the production of thermostable amylase by a lactic acid bacterium under submerged or solid-state fermentation can help to reduce the risk of contamination caused by undesirable micro-organisms during the process [41, 42]. Another advantage is the nonpathogen character of the genus *Lactobacillus* that allows their utilization in food

Lactic acid a water soluble and highly hygroscopic aliphatic acid is present in humans, animals and microorganisms. It is the first biotechnologically produced multi-functional versatile organic acid having wide range of applications [1], namely as a preservative in many food products. It can be produce by LAB trough fermentation or synthetically from lactonitrile [43]. It is non-volatile, odorless organic acid and is classified as GRAS (Generally Recognized As Safe) for use as a general purpose food additive. The lactic acid consumption market is dominated by the food and beverage sector since 1982 [1]. More than 50% of lactic acid produced is used as emulsifying agent in bakery products [44]. 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 processed eggs, often in conjunction with other acidulants. Lactic acid or its salts are used in the disinfection and packaging of carcasses, particularly those of poultry and fish, where the addition of aqueous solutions during processing increased shelf life and reduced microbial spoilage. The esters of calcium and sodium salts of lactate with longer chain fatty acids have been used as very good dough conditioners 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

Twenty-eight samples of soils were collected from main geographic zones of Cameroon in four localities: (Ngaoundere, Yaounde, Bafoussam and Mbouda) at the factories where starchy wastes are frequently submitted to natural fermentation. Four kinds of factories were investigated: "gari" factories, corn and cassava mills, cassava plantation after harvesting and treatment of tubers and flour markets. At the site where degradation of starchy material was remarkable and visible, one to five grams of soils were collected and transferred to polyethylene aseptic bag, the factory age recorded; the samples were finally transported to the laboratory and analyzed in the same week.

### **3.2. Screening of thermostable amylases and lactic acid producing bacteria**

The starch degrading amylolytic lactic acid bacterial strains were isolated from different samples of soil. Amylolytic micro-organisms were firstly enriched by introduction of 1 g of soil sample in 100 ml Erlenmeyer flasks containing 50 ml of enrichment liquid medium, composed of (gram per litre): 5 g soluble starch, 5 g peptone, 5 g yeast extract 0.5 g MgSO4.7H2O, 0.01g FeSO4.7H2O, 0.01 g NaCl. Enrichment of thermostable amylases producing bacteria was carried out by heating Erlenmeyer flasks at 90°C for 5 min followed

by incubation in an alternative shaker at 37-40°C and speed of 150 oscillations per minute for 24 h. Amylases producing bacteria strains were screened on agar plate, containing (gram per liter): 10 g soluble starch, 5 g peptone, 5 g yeast extract, 0.5 g MgSO4.7H2O, 0.01g FeSO4.7H2O, 0.01 g NaCl, 15 g agar. Incubation at 37-40°C was carried out for 48 h, after which the plates were stained with lugol solution (Gram iodine solution: 0.1% I2 and 1% KI). The colonies with the largest halo forming zone were pre-selected and tested for Gram staining and catalase activity.

Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 639

The culture supernatant was supplemented with solid ammonium sulphate to 65% (w/v) final concentration, with mechanical stirring at 4°C. The suspension was retained for 1 h at 4 °C, and centrifuged at 8000 g for 30 min at the same temperature. The resultant supernatant was brought to 70 % w/v ammonium sulphate saturation at 4°C. 50-70% (w/v) ammonium sulphate precipitate was recovered, dissolved in 0.1 M phosphate buffer and dialysed using Spectra/PorR, VWR 2003 dialysis membrane overnight against the same buffer at 4°C and

The optimal temperature for amylase activity was determined by assaying activity between 30 and 100°C for 30 min in 50 mM phosphate buffer. Measurement of optimum pH for amylase activity was carried out under the assay conditions for pH range of 3.0-10.0, using 50mM of three buffer solutions: Tris-HCl (pH 3.0), Na2HPO4-Citrate (pH 4.0 – 6.0), and

The temperature stability was determined by incubating the partial purified enzyme solution in water bath for temperature range of 30-100°C for 30, 60, 90, 120, 180 min and then cooled with tap water. The remaining -amylase activity was measured and expressed as the percentage of the activity of untreated control taken as 100%. The first order inactivation rate constants, ki were calculated from the equation: lnA lnA k t 0 i , where A0 is the initial value

For the determination of pH stability, the enzyme was incubated in a water bath at 60 °C at varying pH value for 30 min. The residual activity was detected under the same conditions

The effect of metal salts and EDTA on amylase activity was determined by adding 0.05 to 0.1% (w/v) of metal salts (CaCl2.2H2O, MgSO4.7H2O, FeSO4.7H2O, NaCl, FeCl3, CuSO4.5H2O) and EDTA to the standard assay. The effect of metal salts and chelating agent on amylase activity were evaluated by pre-incubating the enzyme in the presence of effectors for 30 min at 60°C. The remaining amylase activity was determined and expressed as the percentage of

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

and expressed as the percentage of the activity of untreated control taken as 100%.

**3.5. Partial enzyme purification** 

used as partial purified enzyme solution.

Glycine-NaOH (pH 7.0-10.0).

**3.6. Effect of temperature and pH on activity and stability** 

of amylase activity and A the value of amylase activity after a time t (min).

**3.7. Effect of metal salts and chelating agent** 

the activity of untreated control taken as 100%.

**3.8. Analytical methods** 

Preliminary tests were carried out to determine the heat stability of the amylase of each isolate as we described previously [47, 48]. The gas production from glucose, growth at different temperature (10, 40, 45 °C) as well as the ability to grow in different concentration of NaCl was determined as described by Schillinger and Lucke [49] and Dykes et al [50]. The isolates which were Gram positive and catalase negative, non-motile and producing heat stable amylase and lactic acid were finally selected and identified using API 50 CH test kit (bioMerieux, France). The APILAB PLUS database identification was used to interpret the results.
