**1. Introduction**

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

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> Lactic acid bacteria (LAB) have diverse applications for both animals and humans. Food, pharmaceutical and chemical industries rely on these microorganisms to produce fermented beverage, foods and other important compounds of industrial interests. In recent years the industrial relevance of lactic acid bacteria is on an increasing trend because of the application of lactic acid as chemical for the production of biodegradable plastics [1]. Typical LAB are Gram-positive, non-sporing, catalase-negative, devoid of cytochromes, anaerobic but aerotolerant cocci or rods that are acid-tolerant and produce lactic acid as the major end product during sugar fermentation [2]. Although most LAB are unable to degrade starch because of the lack of the amylolytic activity, a few exhibit this activity and are qualified as amylolytic lactic acid bacteria (ALAB) which are able to decompose starchy material through the amylases production during the fermentation processes [3]. Regarding the importance and availability of starchy biomass in the world, amylases and lactic acid production from starch appear as two potential industrial applications of ALAB. Amylases play important role in degradation of starch and are produced in bulk from microorganisms and represent about 25 to 33% of the world enzyme market [4]. The spectrum of amylases application has widened in many fields, such as clinical, medical and analytical chemistry as well as in the textile, food, fermentation, paper, distillery and brewing industries [4]. The advantages of using thermostable amylases in industrial processes include the decreased risk of contamination, cost of external cooling and increased diffusion rate [4]. Several thermostable α-amylases have been purified from *Bacillus* sp. and the factors influencing their thermostability have been investigated [5]. However, no study has yet dealt with

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

Application of Amylolytic *Lactobacillus fermentum* 04BBA19 in

Fermentation for Simultaneous Production of Thermostable -Amylase and Lactic Acid 635

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* 

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

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

species [23, 20, 24, 21].

in two steps [1].

**2.2. Thermostable amylases** 

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 the western region of Cameroon.
