Biochemical characteristics of amylases overproducing
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 . 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 . 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 . 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 . 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 . The advantages of using thermostable amylases in industrial processes include the decreased risk of contamination, cost of external cooling and increased diffusion rate . Several thermostable α-amylases have been purified from
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 . 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 α-amylase and lactic acid from a LAB,
2. Background and significance
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
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 , potato , or cassava  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 . 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
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 . 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 . 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
2.2. Thermostable amylases
Amylases are among the most important enzymes and are of great significance in present-day 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 α-amylases 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 . Several thermostable α-amylase have been purified from
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 , namely as a preservative in many food products. It can be produce by LAB trough fermentation or synthetically from lactonitrile . 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 . More than 50% of lactic acid produced is used as emulsifying agent in bakery products . 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 . 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 . 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 . 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 .
3. Materials and methods
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.
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  and Dykes et al . 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.
3.3. Microbial growth, amylase and lactic acid production
In order to study microbial growth, amylase and lactic acid production, the microorganism was propagated at 40°C for 70 h in 50 ml of a basal medium containing: soluble starch, 1% (w/v); yeast extract, 0.5 % (w/v) placed in 100 ml Erlenmeyer flask with shaking at 150 oscillations per minute in an alternative shaker (Kotterman, Germany). The initial pH of the medium was adjusted to 6.5 using 0.1 M HCl. After removal of cells by centrifugation (8000xg, 30 min, 4°C) in centrifugator (Heraeus, Germany), the supernatant was considered as the crude enzyme solution and was also used for lactic acid evaluation.
3.4. Optimisation of raw starch degrading thermostable amylase and lactic acid production
The amylase and lactic acid production was optimized by studying the effect of cultural and environmental variables (carbohydrate and nitrogen sources, metal salts and surfactants) individually and simultaneously. The effect of carbohydrate sources was studied by replacing soluble starch in basal medium with different sugars, gelatinized and raw natural crude starch sources (glucose, fructose, maltose, amylose, amylopectine, cassava, corn, rice tapioca, and sorghum flours at final concentration of 1% (w/v)). Nitrogen sources were tested by replacing yeast extract with various nitrogen sources (peptone, tryptone, beef extract, soyabean meal, ammonium sulphate, and urea at final concentration of 1.5% (w/v)). The effect of metal salts was studied by adding individually various metal salts (CaCl2.2H2O, MgSO4.7H2O, FeSO4.7H2O, FeCl3, NaCl at concentration of 0.1% (w/v)). Similarly the effect of surfactants was studied by supplementing the culture medium with Tween 80 and Tween 40 at concentration of 1.5% (v/v).
All media containing gelatinized starch sources were autoclaved at 121 °C for 20 min, while for the media containing raw starch flour, starch powder was sterilized by washing in ethanol and added to sterile nutrient broth.
3.5. Partial enzyme purification
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 used as partial purified enzyme solution.
3.6. Effect of temperature and pH on activity and stability
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 Glycine-NaOH (pH 7.0-10.0).
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 α-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:, where A0 is the initial value of amylase activity and A the value of amylase activity after a time t (min).
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 and expressed as the percentage of the activity of untreated control taken as 100%.
3.7. Effect of metal salts and chelating agent
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 the activity of untreated control taken as 100%.
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)
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 MRS-starch 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 . The nature of amylase (endo-acting or exo-acting) was determined according to Ceralpha method (Megazyme) which uses a blocked maltoheptaoside as substratre .
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 α-amylase activity of the supernatant was measured and the adsorption percentage was calculated as follows: ,
For the determination of raw starch digestibility, raw cassava was used and the reaction mixture containing 100 U of α-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 .
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
|Strains cod||Heterofermentative||Gaz production||Optimum temperature of growth||Growth at 10°C||Dextran||Ammonia from Arginine||Nitrate reduction||Glycerol||Erythritol||D-arabinose||L-arabinose||Ribose||D-xylose||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||N-Acetyl-Glucosamine|
|Strains cod||Amygdalin||Arbutin||Aesculin||Salicin||Cellobiose||Maltose||Lactose||Melibiose||Sucrose||Trehalose||Inulin||Melezitose||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||Identification species (API 50CHL)|
4.2. Amylase and lactic acid production
In the presence of starch as carbon source at 40°C,
The study of cell growth and amylase production as a function of temperature (Fig. 3a) showed that
The amylase and lactic acid production by
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].
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 107.0±1.2 U/ml after 48 hours of fermentation, while in the presence of raw cassava starch at the same concentration, the enzyme yield was 67.1±0.5 U/ml. These results are in agreement with the reports of Cherry et al. , Saxena et al.  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
Among the various gelatinized starchy sources tested, corn and sorghum flour were found to be the most suitable for α-amylase and lactic acid production by
Among nitrogen sources used in the present study, soya bean meal and yeast extract showed significant effect on α-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.  reported that soybean meal presented a positive effect and was the best nitrogen source for raw starch digesting thermostable α-amylase production by the
All metal salts tested in this study increased amylase and lactic acid production by
From the surfactants tested in this study, Tween-80 appeared to be the best surfactant sources for amylase production by
From various environmental factors tested for α-amylase and lactic acid production by
|Parameters||Enzyme yield (U/ml)||Lactic acid(g/l)|
|Carbohydrate sources (1% w/v)|
|Nitrogen sources (1.5% w/v)|
|Soya bean meal||397.3±0.4a||29.2±0.4a|
|Minerals (0.1% w/v)|
|Surfactants (1.5% w/v)|
|Gelatinized starchy sources (1 %w/v)|
|Raw starchy sources|
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).
4.4. Enzyme properties
The amylase produced by
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 α-amylase from
Fig. 6 shows the thermostability pattern of α-amylase from
Due to its high thermostability, α-amylase from
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.
|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)|
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
|B 4437||[28, 32]|
On the other hand the fact that thermostable amylase and lactic acid production can be combined in single fermentation step would not only provide a way to make gruels with high energy density, but also improve its safety, since lactic acid bacteria fermentation is an efficient way to inhibit food-borne pathogens.
Owing to the importance of this finding, further studies will focus on the development of an accurate method for preparing high energy density complementary food using local starchy sources and the L. fermentum 04BBA19 strain.
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 through Research-Development Grant Programme.