**2.1. Amylolytic lactic acid bacteria**

Amylolytic lactic acid bacteria (ALAB) have been reported from different tropical starchy fermented foods, made especially from roots as cassava and sweet potato or grains as maize sorghum and rice. Strains of *Lactobacillus plantarum* have been isolated from African cassavabased fermented products [8], *L. plantarum* A6 (LMG 18053) have been isolated from retted cassava in the Congo [9] and *Lactobacillus manihotivorans* OND32 have been isolated from cassava sour starch fermentations in Colombia [10]. Amylolytic strains of *Lactobacillus fermentum* were isolated for the first time from Benin maize sourdough (ogi and mawè) by Agati et al. [11]. Sanni et al. [12] described amylolytic strains of *L. plantarum* and *L. fermentum* strains in various Nigerian traditional amylaceous fermented foods. ALAB are generally screened in fermented amylaceous foods. Owing to their relatively high starch content, starchy biomass appears as an important eco-niche for the screening and isolation of ALAB, which can be industrially applied to convert starch into mono- and disaccharides for lactic acid fermentation. The composition of the microbiota and in particular the occurrence of ALAB is determined by the way the raw material is processed [13]. Most ALAB isolated belong to the *Lactobacillus* genus, however few studies reported the existence of amylolytic activity in some strains of *Bifidobacterium* isolated from the human large intestinal tract [14, 15]. The distribution of amylolytic microorganisms in the human large intestinal tract has been investigated in various individuals of different ages using anaerobic cultures techniques. So far, twenty one amylolytic bifidobacteria have been isolated from adult faeces and tested for rice fermentation [16].

Owing to the ability of their α-amylases to partially hydrolyze raw starch, ALAB can ferment different types of amylaceous raw material, such as corn [17], potato [18], or cassava [19] and different starchy substrates [20, 21, 8]. Amylolytic LAB utilize starchy biomass and convert it into lactic acid in a single step fermentation. ALAB are mainly used in food fermentation, they are involved in cereal based fermented foods such as European sour rye bread, Asian salt bread, sour porridges, dumplings and non-alcoholic beverage production. Few of them are used for production of lactic acid in single step fermentation of starch [1]. The common method to produce lactic acid from starchy biomass involves the pretreatment for gelatinisation and hydrolysis (liquefaction and saccharification). The liquefaction of the starch is carried out at high temperatures of 90–130 °C for 15 min followed by enzymatic saccharification to glucose and subsequent conversion of glucose to lactic acid by fermentation [22, 1]. This two-step process involving consecutive enzymatic hydrolysis and fermentation makes it economically unattractive. The bioconversion of carbohydrate materials to lactic acid can be made much more effective by coupling the enzymatic hydrolysis of carbohydrate substrates and microbial fermentation of the derived glucose into a single step. This has been successfully employed for lactic acid production from raw starch materials with many representative bacteria including *Lactobacillus* and *Lactococcus*  species [23, 20, 24, 21].

Because at industrial scale, the use of glucose addition is an expensive alternative, there is interest in the use of a cheaper source of carbon, such as starch, the most abundantly available raw material on earth next to cellulose. This, in combination with amylolytic lactic acid bacteria may help to decrease the cost of the overall fermentation process. Amylolytic lactic acid bacteria can convert the starch directly into lactic acid. Development of production strains which ferment starch to lactic acid in a single step is necessary to make the process economical. Very few bacteria have been reported so far for direct fermentation of starch to lactic acid [1, 25, 26] Approximately 3.5 billion tonnes of agricultural residues are produced per annum in the world [27]. The use of a specific carbohydrate feedstock depends on its price, availability, and purity. Although agro-industrial residues are rich in carbohydrates, their utilization is limited [27]. Different food/agro-industrial products or residues form the cheaper alternatives to refined sugars as substrates for lactic acid production. Sucrose-containing materials such as molasses are commonly exploited raw materials for lactic acid production. Starch produced from various plant products is a potentially interesting raw material based on cost and availability. Laboratory-scale fermentations have been reported for lactic acid production from starch by *Lactobacillus amylophilus* GV6 [20], *L. amylophilus* B4437 [28], *Lactobacillus amylovorus* [29, 23*, Lactococcus lactis* combined with *Aspergillus awamorii* [30] and *Rhizopus arrhizus* [31]. *L. amylophilus* NRRL B4437 [32], *L. amylovorus* [17] and *L. amylophilus* GV6 are exceptions that have been described to actively ferment starch to lactic acid and this may lead to alternative process of industrial lactic acid production [23, 20]. To make the process cost effective in terms of substrate, various groups have worked on acid/enzyme hydrolysis of starchy substrates followed by *Lactobacillus* fermentation or simultaneous saccharification and fermentation by co-culture/mixed culture fermentations. It has been reported that starch is used as substrate in two steps [1].

### **2.2. Thermostable amylases**

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

the western region of Cameroon.

**2. Background and significance** 

**2.1. Amylolytic lactic acid bacteria** 

adult faeces and tested for rice fermentation [16].

thermostable amylase from lactic acid bacteria (LAB). The use of thermostable amylases from *Lactobacillus* is of advantage as they are generally non-pathogenic. On the other hand, the major end product of LAB fermentation, lactate, has applications as a preservative, acidulant and flavouring agent in the food industry, because of the tartness provided by

Thus a thermostable amylase producing lactic acid bacterium would be a potential candidate for food industries and especially for the making of high density gruel from starchy raw material as corn or wheat [7]. This would require a good knowledge of the conditions required to optimally produce amylase and lactic acid of good quality. The present study deals with the co-production of thermostable -amylase and lactic acid from a LAB, *Lactobacillus fermentum* 04BBA19, isolated from a starchy waste of a soil sample from

Amylolytic lactic acid bacteria (ALAB) have been reported from different tropical starchy fermented foods, made especially from roots as cassava and sweet potato or grains as maize sorghum and rice. Strains of *Lactobacillus plantarum* have been isolated from African cassavabased fermented products [8], *L. plantarum* A6 (LMG 18053) have been isolated from retted cassava in the Congo [9] and *Lactobacillus manihotivorans* OND32 have been isolated from cassava sour starch fermentations in Colombia [10]. Amylolytic strains of *Lactobacillus fermentum* were isolated for the first time from Benin maize sourdough (ogi and mawè) by Agati et al. [11]. Sanni et al. [12] described amylolytic strains of *L. plantarum* and *L. fermentum* strains in various Nigerian traditional amylaceous fermented foods. ALAB are generally screened in fermented amylaceous foods. Owing to their relatively high starch content, starchy biomass appears as an important eco-niche for the screening and isolation of ALAB, which can be industrially applied to convert starch into mono- and disaccharides for lactic acid fermentation. The composition of the microbiota and in particular the occurrence of ALAB is determined by the way the raw material is processed [13]. Most ALAB isolated belong to the *Lactobacillus* genus, however few studies reported the existence of amylolytic activity in some strains of *Bifidobacterium* isolated from the human large intestinal tract [14, 15]. The distribution of amylolytic microorganisms in the human large intestinal tract has been investigated in various individuals of different ages using anaerobic cultures techniques. So far, twenty one amylolytic bifidobacteria have been isolated from

Owing to the ability of their α-amylases to partially hydrolyze raw starch, ALAB can ferment different types of amylaceous raw material, such as corn [17], potato [18], or cassava [19] and different starchy substrates [20, 21, 8]. Amylolytic LAB utilize starchy biomass and convert it into lactic acid in a single step fermentation. ALAB are mainly used in food fermentation, they are involved in cereal based fermented foods such as European sour rye bread, Asian salt bread, sour porridges, dumplings and non-alcoholic beverage production.

lactate and also because lactate is generally regarded as safe (GRAS) [6].

Amylases are among the most important enzymes and are of great significance in presentday biotechnology. Although they can be derived from several sources, such as plants, animals and microorganisms; enzymes from microbial sources generally meet industrial 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 fermentation processes.

Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 637

body makes it very useful as an active ingredient in cosmetics [45]. Lactic acid has long been used in pharmaceutical formulations, mainly in topical ointments, lotions, and parenteral solutions. It also finds applications in the preparation of biodegradable polymers for medical uses such as surgical sutures, prostheses and controlled drug delivery systems [45]. Because of ever-increasing amount of plastic wastes worldwide, considerable research and development efforts have been devoted towards making a single-use, biodegradable substitute of conventional thermoplastics. Biodegradable polymers are classified as a family of polymers that will degrade completely – either into the corresponding monomers or into products, which are otherwise part of nature – through metabolic action of living organisms. The demand for lactic acid has been increasing considerably, owing to the promising applications of its polymer, the polylactic acid (PLA), as an environment-friendly alternative to plastics derived from petrochemicals. PLA has received considerable attention as the precursor for the synthesis of biodegradable plastic [46]. The lactic acid polymers have potentially large markets, as they many advantage like biodegradability, thermo plasticity, high strength etc., have potentially large markets. The substitution of existing synthetic polymers by biodegradable ones would also significantly alleviate waste disposal problems. As the physical properties of PLA depend on the isomeric composition of lactic acid, the production of optically pure lactic acid is essential for polymerization. L-Polylactic acid has a melting point of 175–178 °C and slow degradation time. L-Polylactide is a semicrystalline polymer exhibiting high tensile strength and low elongation with high modulus suitable for medical products in orthopedic fixation (pins, rods, ligaments etc.), cardiovascular applications (stents, grafts etc.), dental applications, intestinal applications, and sutures [45].

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

**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

transported to the laboratory and analyzed in the same week.

**3. Materials and methods** 

**3.1. Samples** 

### **2.3. Lactic acid**

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 body makes it very useful as an active ingredient in cosmetics [45]. Lactic acid has long been used in pharmaceutical formulations, mainly in topical ointments, lotions, and parenteral solutions. It also finds applications in the preparation of biodegradable polymers for medical uses such as surgical sutures, prostheses and controlled drug delivery systems [45]. Because of ever-increasing amount of plastic wastes worldwide, considerable research and development efforts have been devoted towards making a single-use, biodegradable substitute of conventional thermoplastics. Biodegradable polymers are classified as a family of polymers that will degrade completely – either into the corresponding monomers or into products, which are otherwise part of nature – through metabolic action of living organisms. The demand for lactic acid has been increasing considerably, owing to the promising applications of its polymer, the polylactic acid (PLA), as an environment-friendly alternative to plastics derived from petrochemicals. PLA has received considerable attention as the precursor for the synthesis of biodegradable plastic [46]. The lactic acid polymers have potentially large markets, as they many advantage like biodegradability, thermo plasticity, high strength etc., have potentially large markets. The substitution of existing synthetic polymers by biodegradable ones would also significantly alleviate waste disposal problems. As the physical properties of PLA depend on the isomeric composition of lactic acid, the production of optically pure lactic acid is essential for polymerization. L-Polylactic acid has a melting point of 175–178 °C and slow degradation time. L-Polylactide is a semicrystalline polymer exhibiting high tensile strength and low elongation with high modulus suitable for medical products in orthopedic fixation (pins, rods, ligaments etc.), cardiovascular applications (stents, grafts etc.), dental applications, intestinal applications, and sutures [45].
