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Biotechnology Advances 26 (2008) 22 – 34 www.elsevier.com/locate/biotechadv
Research review paper
Amylolytic bacterial lactic acid fermentation — A review Gopal Reddy ⁎, Md. Altaf 1 , B.J. Naveena 1 , M. Venkateshwar, E. Vijay Kumar Department of Microbiology, Osmania University, Hyderabad-500 007, India Received 29 June 2007; accepted 25 July 2007 Available online 31 July 2007
Abstract Lactic acid, an enigmatic chemical has wide applications in food, pharmaceutical, leather, textile industries and as chemical feed stock. Novel applications in synthesis of biodegradable plastics have increased the demand for lactic acid. Microbial fermentations are preferred over chemical synthesis of lactic acid due to various factors. Refined sugars, though costly, are the choice substrates for lactic acid production using Lactobacillus sps. Complex natural starchy raw materials used for production of lactic acid involve pretreatment by gelatinization and liquefaction followed by enzymatic saccharification to glucose and subsequent conversion of glucose to lactic acid by Lactobacillus fermentation. Direct conversion of starchy biomass to lactic acid by bacteria possessing both amylolytic and lactic acid producing character will eliminate the two step process to make it economical. Very few amylolytic lactic acid bacteria with high potential to produce lactic acid at high substrate concentrations are reported till date. In this view, a search has been made for various amylolytic LAB involved in production of lactic acid and utilization of cheaply available renewable agricultural starchy biomass. Lactobacillus amylophilus GV6 is an efficient and widely studied amylolytic lactic acid producing bacteria capable of utilizing inexpensive carbon and nitrogen substrates with high lactic acid production efficiency. This is the first review on amylolytic bacterial lactic acid fermentations till date. © 2007 Elsevier Inc. All rights reserved. Keywords: Amylolytic lactic acid bacteria; Lactic acid; Starch; Fermentation
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Lactic acid and its importance . . . . . . . . . . . . . . . . Lactic acid bacteria . . . . . . . . . . . . . . . . . . . . . Amylolytic lactic acid bacteria. . . . . . . . . . . . . . . . Amylolytic lactic acid fermentation . . . . . . . . . . . . . Substrates available for amylolytic lactic acid fermentation . Amylolytic enzymes in LAB . . . . . . . . . . . . . . . . Submerged fermentations involving amylolytic LAB . . . . Solid-state fermentation . . . . . . . . . . . . . . . . . . .
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⁎ Corresponding author. Tel.: +91 40 27682246/27090661. E-mail addresses:
[email protected] (G. Reddy),
[email protected] (M. Altaf). 1 Present address: Oklahoma University Cancer Institute, University of Oklahoma Health Sciences, Center, Oklahoma City, OK-73104, USA. 0734-9750/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2007.07.004
G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
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10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
1. Introduction Lactic acid is one of the most important organic acids produced by lactic acid bacteria (LAB), discovered by Swedish scientist C. W. Scheele in 1780 from sour milk. Lactic acid exists in two optically active stereo-isomers, the L(+) and the D(−). Lactic acid has a wide range of beneficial uses in the sectors relating to food preservation, flavor enhancement etc. Since elevated levels of D(−) lactic acid is harmful to humans, L(+) lactic acid is the preferred isomer in food and pharmaceutical industries as humans have only L-lactate dehydrogenase that metabolizes L(+) lactic acid (Akerberg et al., 1998; Hofvendahl et al., 2000). Currently, lactic acid is used in a wide variety of specialized industrial applications where the functional specialty of the molecule is desirable (Datta et al., 1995). Leo Hepner of L. Hepner and Associates, a UK based management consultancy for food ingredients and biotechnology industries, rates worldwide consumption of lactic acid at 130,000 to 150, 000 MT per year (Mirasol, 1999). In 1999, Hepner rated the demand for lactic acid to grow continually at 5–8% annual clip. Its use as a raw material for synthesis of biodegradable plastics was identified in late 1940s and early 1950s (Vickroy, 1985). Demand for lactic acid is expected to increase as rated by different surveys due to its use in biodegradable plastics and other large-scale industrial products. Yet the market is limited by cost in competition with polystyrene as prices for heat stable [L(+)] and higher grades of lactic acid are more (Mirasol, 1999). If polylactides and lactate esters are commercially successful, global demand will be around 14–19% (Chem systems reports, 2002; Jarvis, 2003). By the end of year 2011, lactic acid global demand is expected to shoot up to 200,000 MT world wide and domestic demand for lactic acid and in India is expected to touch 2000 tonnes from the present demand of 560 tonnes (Ramesh, 2001). The current global production of lactic acid is about 120,000 tonnes per year (Datta and Henry, 2006). New applications of L(+) lactic acid, such as a monomer in biodegradable plastics or as an intermediate in the synthesis of high-volume oxygenated chemicals, have the potential to greatly expand the market for it. Lactic acid can be manufactured either by chemical synthesis or by microbial fermentations. Chemical synthe-
sis results in racemic DL-lactic acid whereas stereospecific [L(+),D(−) and DL mixture] form is produced by fermentation using specific microbial strain (Datta et al., 1993; Litchfield, 1996). Lactic acid bacteria (LAB) can be homofermentative or heterofermentative and can produce either L(+) or D(−) or racemic mixture of lactic acid. Significant advantage over chemical synthesis is that biological production can use cheap raw materials such as whey, molasses, starch waste, beet, cane sugar and other carbohydrate rich materials (Anuradha et al., 1999; Ritcher and Berthold, 1998; Tsao et al., 1999; Vishnu et al., 1998, 2000). Raw material cost is one of the major factors in economic production of lactic acid. The efficiency and economics of the ultimate lactic acid fermentation is however still a problem from many points of view and media compositions play vital role in the improvement of such a process. Research efforts are focused on looking for new and effective nutritional source and new progressive fermentation techniques enabling the achievement of both high substrate conversion and high production yields (Sule Bulut et al., 2004). Direct conversion of starch to lactic acid by bacteria with both amylolytic and lactic acid producing character will eliminate the two step process of saccharification followed by microbial fermentation to make it economical. Many reviews on lactic acid fermentation are published, focus of this review is on amylolytic lactic acid fermentation with emphasis to use starch or starchy substrates and other low cost substrates to replace sugars and costly nitrogenous materials. 2. Lactic acid and its importance Lactic acid (C3H6O3) is present in almost every form of organized life. Its most important function in animals and humans is related to the supply of energy to muscle tissues. This is a water soluble and highly hygroscopic aliphatic acid and an enigmatic chemical. It is the first biotechnologically produced multi-functional versatile organic acid having wide range of applications. It is a product of natural fermentation processes occurring in buttermilk, cheese, beer, sourdough and many other fermented foods. Litchfield (1996) has summarized typical food applications for lactic acid and its salts. It is non-volatile, odorless organic acid and is classified as
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GRAS (Generally Recognized As Safe) for use as a general purpose food additive by FDA in U.S.A. and other regulatory agencies (Datta et al., 1995). The lactic acid consumption market is dominated by the food and beverage sector since 1982. Even today, lactic acid market still exists for food and beverage industries. More than 50% of lactic acid produced is used as emulsifying agent in bakery products (Datta et al., 1993; Litchfield, 1996). 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 (Datta et al., 1995; Naveena, 2004). 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 waterretaining 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 (Wee et al., 2006). 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 (Wee et al., 2006). The presence of two reactive functional groups makes lactic acid the most potential feedstock monomer for chemical conversions to potentially useful chemicals such as propionic acid, acetic acid, acrylic acid etc. (Dimerci et al., 1993). Technical-grade lactic acid is extensively used in leather tanning industries as an acidulant for deliming hides and in vegetable tanning. Lactic acid is used as descaling agent, solvent, cleaning agent, slow acid-releasing agent and humectants in a variety of technical processes. 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. International organizations such as the
American Society for Testing of Materials (ASTM), the Institute for Standards Research (ISR), the European Standardization Committee (CEN), the International Standardization Organization (ISO), the German Institute for Standardization (DIN), the Italian Standardization Agency (UNI), and the Organic Reclamation and Composting Association (ORCA), are all actively involved in developing tests of biodegradability in different environments and compostability. 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 (Senthuran et al., 1997). The lactic acid polymers, with tremendous advantages 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 (Wee et al., 2006).
3. Lactic acid bacteria Lactic acid bacteria (LAB) are a group of related bacteria that produce lactic acid as major metabolic product. LAB have the property of producing lactic acid from carbohydrates through fermentation. LAB have been used to ferment or culture foods for at least 4000 years. These organisms are heterotrophic and generally have complex nutritional requirements because they lack many biosynthetic capabilities. Most species have multiple requirements for amino acids and vitamins. Because of this, lactic acid bacteria are generally abundant only in communities where these
G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
requirements can be provided. Lactic acid bacteria are used in the food industry for several reasons. Their growth lowers both the carbohydrate content of the foods that they ferment, and the pH due to lactic acid production. It is this acidification process which is one of the most desirable effects of their growth. The pH may drop to as low as 4.0, low enough to inhibit the growth of most other microorganisms including the most common human pathogens, thus allowing these foods to prolong shelf life. LAB consist of bacterial genera within the phylum Firmicutes comprised of about 20 genera. The genera Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus and Weisella are the main members of the LAB (Axelsson, 2004; Davidson et al., 1995; Ercolini et al., 2001; Jay, 2000; Holzapfel et al., 2001; Stiles and Holzapfel, 1997). Lactobacillus is largest of these genera, comprising around 80 recognized species (Axelsson, 2004). The taxonomy of lactic acid bacteria has been based on the Gram reaction and the production of lactic acid from various fermentable carbohydrates. Lactobacilli vary in morphology from long, slender rods to short coccobacilli, which frequently form chains. Typical LAB are Gram-positive, nonsporing, catalase-negative, devoid of cytochromes, anaerobic but aerotolerant cocci or rods that are acidtolerant and produce lactic acid as the major end product during sugar fermentation (Axelsson, 2004). However, under certain conditions some LAB do not display all these characteristics. Thus, the most profound features of LAB are Gram positiveness and inability to synthesize porphyrin groups. The inability to synthesize porphyrin (e.g., heme) results in the LAB being devoid of catalase and cytochromes (without supplemented heme in the growth media). Therefore, the LAB do not possess an electron transport chain and rely on fermentation to generate energy (Axelsson, 2004). Since they do not use oxygen in their energy production, lactic acid bacteria grow under anaerobic conditions, but they can also grow in oxygen's presence. They are protected from oxygen by-products (e.g. H2O2) because they have peroxidases. These organisms are aerotolerant anaerobes. Because of the low energy yields, lactic acid bacteria often grow more slowly than microbes capable of respiration, and produce smaller colonies of 2–3 mm. Lactic acid bacteria can grow at temperatures from 5 to 45 °C and not surprisingly are tolerant to acidic conditions, with most strains able to grow at pH 4.4. The growth is optimum at pH 5.5–6.5 and the organisms have complex nutritional requirements for amino acids, peptides, nucleotide bases, vitamins, minerals, fatty
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acids and carbohydrates. The genus is divided into three groups based on fermentation patterns: • Homofermentative: produce more than 85% lactic acid from glucose. They ferment 1 mol of glucose to 2 mol of lactic acid, generating a net yield of 2 mol of ATP per molecule of glucose metabolized. Lactic acid is the major product of this fermentation (Fig. 1). • Heterofermentative: produce only 50% lactic acid. These ferment 1 mol of glucose to 1 mol of lactic acid, 1 mol of ethanol, and 1 mol of CO2. One mole of ATP is generated per mole of glucose, resulting in less growth per mole of glucose metabolized (Fig. 1). • Less well known heterofermentative species which produce DL-lactic acid, acetic acid and carbon dioxide. 4. Amylolytic lactic acid bacteria Amylolytic lactic acid bacteria (ALAB) have been reported from different tropical amylaceous fermented foods, prepared mainly from cassava and cereals (e.g., maize and sorghum). Strains of Lactobacillus plantarum have been isolated from African cassava-based fermented products (Nwankwo et al., 1989), and the new ALAB species Lactobacillus manihotivorans (MorlonGuyot et al., 1998) was isolated from cassava sour starch fermentations in Colombia. Olympia et al. (1995) characterized amylolytic strains of L. plantarum isolated from burong isda, a fermented food made from fish and rice in Philippines. Amylolytic strains of Lactobacillus fermentum were isolated for the first time from Benin maize sourdough (ogi and mawè) by Agati et al. (1998). Recently, Sanni et al. (2002) described amylolytic strains of L. plantarum and L. fermentum strains in various Nigerian traditional amylaceous fermented foods. The search for ALAB in fermented amylaceous foods has been justified by the high starch content of the raw material. Their role has yet to be elucidated since mono- and disaccharides, such as glucose and sucrose, which occur naturally in cereals and cassava, are readily available for lactic acid fermentation. The way the raw material is processed may determine the composition of the microbiota and, in particular, the occurrence of ALAB (Guyot et al., 2000). ALAB have repeatedly been isolated from traditional cereal or cassava-based fermented foods (Johansson et al., 1995; Morlon et al., 1998; Nwankwo et al., 1989; Olympia et al., 1995; Sanni et al., 2002). Due to the ability of their α-amylases to partially hydrolyze raw starch (Rodriguez-Sanoja et al., 2000), ALAB can ferment different types of amylaceous raw material, such as corn (Nakamura, 1981), potato (Chatterjee et al., 1997), or cassava
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Fig. 1. Metabolism of lactic acid bacteria.
5. Amylolytic lactic acid fermentation
sis 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 and many representative bacteria including Lactobacillus and Lactococcus species (Cheng et al., 1991; Zhang and Cheryan, 1994; Vishnu et al., 2002; Naveena et al., 2003, 2005a,b,c). Use of sugars is un-economical, still they are the choice substrates due to certain constraints such as
Conventional biotechnological production of lactic acid from starchy materials, for instance, requires pretreatment for gelatinisation and liquefaction, which 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 (Anuradha et al., 1999). This two step process involving consecutive enzymatic hydroly-
• Non-availability of potential amylolytic strains for lactic acid fermentation • Need to develop a potential strain for high yield efficiency of lactic acid • Inability of organisms for alternate substrate utilizations with high efficiencies • Inability of organisms to use abundantly available inexpensive crude agricultural renewable raw materials
(Giraud et al., 1994) and different starchy substrates (Vishnu et al., 2000, 2002; Naveena et al., 2003, 2005a, b,c). Amylolytic LAB utilize starchy biomass and convert into lactic acid in single step fermentation. Most of the amylolytic LAB are used in food fermentation. Amylolytic LAB (ALAB) are also 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.
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Fig. 2. Schematic representation of lactic acid production from starch as substrate.
In commercial scale, glucose addition is an expensive alternative. The use of a cheaper source of carbon, such as starch, the most abundantly available raw material on earth next to cellulose, in combination with amylolytic lactic acid bacteria may help to decrease the cost of the overall fermentation process. Use of raw starch or renewable easily available and cheap polysaccharide raw materials (complex organic sources) for lactic acid fermentation involves two step processes — saccharification followed by Lactobacillus fermentation. Amylolytic lactic acid bacteria can convert the starch directly into lactic acid (Fig. 2). 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 (Table 1). Single step Amylolytic Lactic acid fermentation amylolytic LAB Starch Y Lactic acid 6. Substrates available for amylolytic lactic acid fermentation Bioconversion of polysaccharide carbohydrate materials to lactic acid can be made much more effective by coupling the enzymatic hydrolysis of substrates and microbial fermentation of the derived glucose into a single step, which has been successfully employed for lactic acid production from raw starch materials. Historically, complex natural materials have been used in fermentation processes because they are much cheaper than pure substrates (Goel, 1994). Crop residues are annually renewable sources of energy. Approximately
3.5 billion tonnes of agricultural residues are produced per annum in the world (Pandey et al., 2001). 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 (Pandey et al., 2001). 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, (Vishnu et al., 2000, 2002; Altaf et al., 2005), L. amylophilus B4437 (Mercier et al., 1992), Lactobacillus amylovorus (Cheng et al., 1991; Zhang and Cheryan, 1991, 1994), Lactococcus lactis combined with Aspergillus awamorii (Kurusava et al., 1988) and Rhizopus arrhizus (Kristoficova et al., 1991). L. amylophilus NRRL B4437 (Nakamura and Crowell, 1979) L. amylovorus (Nakamura, 1981) 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 (Cheng et al., 1991; Zhang and Cheryan, 1994; Vishnu et al., 1998, 2000, 2002). 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 is reported that starch is used as substrate in two step
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Table 1 Amylolytic lactic acid producing bacteria so far reported Bacteria
Strain
Reference
L. manihotivorans L. manihotivorans
OND32T LMG18010T LMG 18011 Ogi E1 MW2 K9 ATCC33622 B-4542
Guyot and Morlon-Guyot (2001) Guyot et al. (2000) Ohkouchi and Inoue (2006) Calderon Santoyo et al. (2003), Agati et al. (1998) Agati et al. (1998) Sanni et al. (2002) Zhang and Cheryan (1991) Cheng et al. (1991) Nakamura (1981), Zhang and Cheryan (1991), Mercier et al. (1992), Litchfield (1996) Yumoto and Ikeda (1995) Mercier et al. (1992), Nakamura and Crowell (1979) Vishnu et al. (1998, 2000, 2002, 2006), Vishnu (2000), Naveena et al. (2003, 2004, 2005a,b,c), Altaf et al. (2005, 2006, 2007a,b), Gopal Reddy et al. (2004, 2006) Lee et al. (2001) Lee et al. (2001) Mette Hedegaard Thomsen et al. (2007), Giraud et al. (1991) Giraud et al. (1991) Krishnan et al. (1998) Junya Narita et al. (2004) Wang et al. (2005) Mette Hedegaard Thomsen et al. (2007) Chatterjee et al. (1997) Champ et al. (1983) Lindgren et al. (1984) Diaz-Ruiz et al. (2003) Bohak et al. (1998)
L. fermentum L. fermentum L. fermentum L. amylovorus L. amylovorus L. amylovorus L. amylophilus L. amylophilus L. amylophilus
L. acidophilus L. fermentum L. plantarum L. plantarum L. plantarum S. bovis Lactobacillus sp. Leuconostoc L. cellobiosus Lactobacillus strains Leuconostoc strains S. macedonicus L. amylolyticus
JCIM 1125 B 4437 GV6
L9 A6 LMG18053 NCIM 2084 148 TH165 St3-28 LEM 220, 207, 202
fermentation process of saccharification and Lactobacillus fermentation by enzyme/acid hydrolysis method which is relatively costly process (Vickroy, 1985; Datta et al., 1995; Yumoto and Ikeda, 1995; Litchfield, 1996; Xiaodong et al., 1997). Very few reports are available on isolation of amylolytic lactic acid bacteria for single step fermentation of inexpensive complex carbohydrates (starch) to lactic acid. Use of efficient amylolytic lactic acid producing bacteria will eliminate saccharification costs of substrate thereby reducing the production cost (Vickroy, 1985; Datta et al., 1995; Yumoto and Ikeda, 1995; Litchfield, 1996). In this direction we have reported single step lactic acid fermentation by an amylolytic bacterium L. amylophilus GV6 with high production efficiency (Vishnu et al., 1998, 2000, 2002; Naveena et al., 2003, 2004, 2005a,b, Altaf et al., 2005, 2006, 2007a,b). At high starch concentrations, lactic acid production is low with the known amylolytic organisms (Litchfield, 1996; Yumoto and Ikeda, 1995; Zhang and Cheryan, 1991; Mercier et al., 1992). Some agricultural by-products that are potential substrates for lactic acid production are cornstarch (Cheng et al., 1991; Hang, 1990), cassava starch (Yumoto and Ikeda, 1995),
lignocellulose/hemicellulose hydrolysates (Karel et al., 1997), cottonseed hulls, Jerusalem artichokes, corn cob, corn stalks (Vickroy, 1985), beet molasses (Goksungur and Guvenc, 1999; Kotzamanidis et al., 2002), wheat bran (Naveena et al., 2005a,b,c), rye flour (Raccach and Bamiro, 1997), sweet sorghum (Richter and Trager, 1994), sugarcane press mud (Xavier and Lonsane, 1994), cassava (Xiaodong et al., 1997; Rojan et al., 2005; John et al., 2006a,b), barley starch (Linko and Javanainen, 1996), cellulose (Venkatesh, 1997), carrot processing waste (Pandey et al., 2001), molasses spent wash (Sharma et al., 2003), corn fiber hydrolysates (Saha and Nakamura, 2003), and potato starch (Yumoto and Ikeda, 1995; Anuradha et al., 1999). 7. Amylolytic enzymes in LAB It is already mentioned that refined sugars or gelatinized starch are generally used for production of lactic acid by microbial fermentations. Many reports are available which emphasize on fungi producing enzymes to degrade raw starch (Bergmann et al., 1988; Hang, 1989a,b, 1990), but least work is done on isolation of
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galactosides (i.e. raffinose). Growth and amylase production of this organism were slightly higher with maltose than with starch. This might be explained by the fact that the efficiency of starch conversion was limited by the accumulation of limiting dextrins which were not further fermented, thus limiting growth and amylase synthesis (Calderon et al., 2001). Not many amylolytic lactic acid bacteria involved in production of lactic acid are studied for their amylolytic enzyme. 8. Submerged fermentations involving amylolytic LAB Fig. 3. Scanning Electron Microscope (SEM) photograph of unfermented wheat bran in SSF (with compact starch — cellulose fibers) (Naveena et al., 2005a,b,c).
amylolytic lactic acid bacterial strains (Figuerao et al., 1995; Morlon-Guyot et al., 1998). Some strains of Lactobacillus spp. produce extracellular amylase and ferment starch directly to lactic acid. Amylolytic activity of fermenting organism is a major characteristic for fermentation of starch to lactic acid. L. amylophilus GV6 was evaluated for its amylolytic activity by estimating the amount of extracellular amylolytic enzymes (amylase and pullulanase) production (Naveena, 2004; Vishnu et al., 2000, 2006). The amylase and pullulanase activities were 0.439 U/g/min and 0.18 U/g/min respectively in SSF with wheat bran (Naveena, 2004). Amylolytic enzyme having both amylase and pullulanase activities in L. amylophilus GV6 is a 90 KDa as protein characterized by Vishnu et al. (2006). The presence of both amylase and pullulanase (debranching enzyme) characteristics for the fermenting organism L. amylophilus GV6 is advantageous for efficient direct conversion of complex starchy substrates to lactic acid. This is evident from SEM photographs (Figs. 3 and 4) showing the hydrolysis of starch fibers in wheat bran to sugars which in turn are converted to L(+) lactic acid by L. amylophilus GV6 (Vishnu et al., 2000; Naveena et al., 2005c). Strain GV6 showed both amylase and pullulanase activities of 0.59 and 0.34 U/ml/min in submerged fermentation where maximum amylolytic activity was shown with amylopectin followed by soluble starch (Vishnu et al., 2006). The alpha amylase activity in fermentation of raw starch by Streptococcus bovis was (1.41 U/ml) higher than that from glucose (0.06 U/ml) (Junya Narita et al., 2004). The strain L. fermentum OGi E1 was able to grow and produce amylase from the main carbohydrates found in cereals (starch, maltose, glucose, sucrose, fructose) but also from other compound of cereals and legumes, α-
Soluble starchy substrates available in the form of agricultural wastes, soluble pure and crude starches are utilized in submerged fermentation. Among the various starches, cassava starch, sorghum starch and corn starch are the most abundant and relatively inexpensive raw materials. Amylolytic lactic acid bacterial fermentation has been receiving significant interest in recent past because of the cost effective nature of the starchy substrates. Soluble starch was utilized for production of lactic acid in studies by Yumoto and Ikeda (1995) and corn starch by Mercier et al. (1992). All the wild strains reported so far produced more than 90% lactic acid at low starch concentration, however at high starch concentrations the lactic acid yield was low (Yumoto and Ikeda, 1995; Nakamura and Crowell, 1979; Mercier et al., 1992). L. amylophilus GV6 was found to actively ferment various pure and crude starchy substrates at both low and high starch concentrations with more than 90% lactic acid yield efficiency in anaerobic submerged fermentation (Vishnu et al., 2000, 2002; Altaf et al., 2005, 2007a,b) (Table 2). Strain GV6 was found to utilize pure starches like soluble starch, corn starch and
Fig. 4. Scanning Electron Microscope photograph of fermented wheat bran with bacterial cells in SSF (showing the hydrolyzed starch in fibers) (Naveena et al., 2005a,b,c).
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Table 2 Fermentative production of L(+) lactic acid by amylolytic L. amylophilus GV6 Type of fermentation
Carbon source
Submerged
Soluble starch
Solid state Semi-solid state Submerged Solid state
Concentration of starch
2% 5% 9% Sorghum flour 6% 4.08% Cassava flour 6% 4.94% Wheat flour 6% 4.14% Rice flour 6% 4.68% Barley flour 6% 4.14% Wheat bran 54.2% Wheat bran 44.4% Starch 10% Corn flour 5% 3.7% Wheat bran 60%
Nitrogen source
Fermentation period (days)
LA % LA⁎ Reference
Peptone, YE Peptone, YE Peptone, YE Peptone, YE Peptone, YE Peptone, YE Peptone, YE Peptone, YE Peptone, YE Peptone, YE RL, YC RL, YC RL, YC
1 3 4 4 4 4 4 4 5 5 2 2.9 5
96
89 88 90 86 86 90 98 92 96 96
96 90 76 73 68 72 66 65 66 78 88 78.4 77.6
Vishnu (2000) Vishnu (2000) Vishnu (2000) Vishnu et al. (2002) Vishnu et al. (2002) Vishnu et al. (2002) Vishnu et al. (2002) Vishnu et al. (2002) Naveena et al. (2005b) Naveena (2004) Altaf et al. (2007a) Altaf et al. (2007b) Altaf et al. (2006)
RL — red lentil, YC — bakers yeast cells, YE — yeast extract, LA — lactic acid yield efficiency (g lactic acid produced/g substrate utilized), LA⁎ — lactic acid production efficiency (g lactic acid produced/g substrate taken).
potato starch and crude starches like sorghum flour, cassava flour, wheat flour, rice flour, barley flour, sweet potato flour, millet flour, jowar flour, tapioca flour, pearl millet flour, refined wheat flour (maida flour) and corn flour (Vishnu et al., 2002; Altaf et al., 2007b). Strain GV6 showed 89% lactic acid yield efficiency with soluble starch and sorghum flour, 85% with corn starch and potato starch, 86% with barley flour and rice flour, 88% with cassava flour and 90% with wheat flour respectively at high substrate concentrations of respective substrates (Vishnu et al., 2000, 2002; Gopal Reddy et al., 2006). L. amylophilus GV6 is the most widely studied amylolytic lactic acid bacterium due to its high lactic acid production ability even at higher substrate concentrations. Strain GV6 was also studied for its ability to utilize inexpensive nitrogenous materials with
starch as substrate and was found to produce more than 90% lactic acid yield (Altaf et al., 2005, 2007a,b) with good starch hydrolyzing ability (Figs. 5–7). S. bovis 148 was found to directly produce lactic acid from starch and maximum lactic acid concentration of 14.2 g/l was observed (Junya Narita et al., 2004). Batch fermentations on synthetic mixed sugar and starch medium with amylolytic lactic acid bacteria were studied by Mette Hedegaard Thomsen et al. (2007) where L .plantarum was found to actively ferment mixed carbohydrates (20 g/l) to produce 14.25 g/l lactic acid. Direct and effective lactic acid production by L. manihotivorans LMG18011 for simultaneous saccharification and fermentation using soluble starch and food wastes as substrates resulted in 19.5 g L(+)-lactic acid from 200 g food wastes (Ohkouchi and Inoue, 2006). L.
Fig. 5. Scanning Electron Microscope (SEM) photograph of pure soluble starch granules in MRS broth before sterilization.
Fig. 6. Scanning Electron Microscope (SEM) photograph of starch in MRS broth after sterilization (autoclaving).
G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
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2006). Lactobacillus cellobiosus produced lactic acid by direct fermentation of waste potato mash. Using a 5% (w/v) potato mash with 3% (w/v) CaCO3 to neutralise the lactic acid produced, 50% conversion of starch to lactic acid occurred in 48 h without any other media supplement (Chatterjee et al., 1997). Fermentative production of lactic acid directly from starch was studied in a batch fermentor using L. amylovorus, 96.2 g/l of lactic acid was produced from an initial liquefied starch concentration of 120 g/l starch in 20 h while 92.5 g/l of lactate was produced from the raw starch of the same concentration in 39 h (Zhang and Cheryan, 1991). Fig. 7. Scanning Electron Microscope (SEM) photograph of hydrolysis of starch by L. amylophilus GV6 in submerged fermentation.
9. Solid-state fermentation
plantarum produced lactate yield of 0.81 g/g substrate (Giraud et al., 1994) and L. amylophilus JCM 1125 produced 53.4 g/l using 100 g/l liquefied starch as reported by Yumoto and Ikeda (1995). LA production by L. plantarum NCIM 2084 was 72.9 g/l when provided with 100 g/l of liquefied starch (Krishnan et al., 1998). L. amylophilus NRRL B4437 produced 29 g/l lactic acid from 45 g/l of corn starch and L. amylovorus was used in conversion of 120 g/l liquefied starch to 92.5 g/l lactic acid in submerged fermentation (Zhang and Cheryan, 1991; Mercier et al., 1992). L. amylovorus utilized raw corn starch, rice starch and wheat starch medium to produce lactic acid with a productivity of 10.1, 7.9 and 7.8 g lactic acid/l respectively, but had lower productivities of 4.8 g/l and 4.2 g/l on cassava and potato starch in basal medium respectively. When peptone (1%) is added to basal medium with cassava starch as substrate, conversion rate increased from 43% to 70% (7.7 g lactic acid/l) (Xiaodong et al., 1997). A novel starch-degrading strain of Lactobacillus casei was constructed by genetically displaying α-amylase from the S. bovis strain 148 with a FLAG peptide tag (AmyAF) (Junya Narita et al., 2006). The lactic acid bacteria with AmyAF showed significantly elevated hydrolytic activity toward soluble starch. In fermentation using AmyAF-displaying L. casei cells, 50 g/l of soluble starch was reduced to 13.7 g/l, and 21.8 g/l of lactic acid was produced within 24 h. The yield in terms of gram lactic acid produced per gram of carbohydrate utilized was 0.60 g at 24 h. As AmyAF was immobilized, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h (Junya Narita et al.,
Solid-state fermentation (SSF) process is defined as “the growth of microorganisms (mainly fungi) on moist solid materials in the absence of free-flowing water” (Moo-Young et al., 1983; Pandey, 1992). Apparently, much work has been done on the production of industrial enzymes using SSF and good commercial success has been achieved. Moreover, till date there has been no report on production of lactic acid at high substrate concentrations in a single step through SSF using amylolytic bacterial strains except for L. amylophilus GV6. In SSF, the solid substrate not only supplies nutrients to the culture but also serves as an anchorage to the microbial cells. A study was made to develop a novel technology for L(+) lactic acid production by SSF using L. amylophilus GV6 culture for which wheat bran (a by-product of wheat milling industry) was selected as solid substrate and support (Naveena et al., 2003, 2004, 2005a,b,c; Altaf et al., 2006). Different brans like wheat bran, corn fiber, black gram bran, green gram bran, pigeon pea brans (different varieties) were used as substrates in SSF for lactic acid production by strain GV6 (Naveena et al., 2003). Of all the brans tested, L. amylophilus GV6 produced high lactic acid using starch present in wheat bran as support and substrate than other brans in SSF (Naveena et al., 2003, 2005a,b). The organism could produce 90.111% lactic acid yield which was comparable with that of submerged fermentation reported earlier for L. amylophilus GV6 (Naveena et al., 2003). The interaction of L. amylophilus GV6 with the wheat bran was observed using SEM. These observations (Figs. 3 and 4) explain the conversion of raw starch present in bran fibers to glucose, which in turn is converted to L(+) lactic acid by the organism. L. amylophilus GV6 was found to produce 36 g of lactic acid from high concentration of raw starch (54.4 g) present in 100 g of wheat bran after
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G. Reddy et al. / Biotechnology Advances 26 (2008) 22–34
optimization of fermentation parameters by RSM. (Naveena et al., 2005a,b,c). Substitution of peptone and yeast extract with low cost protein/nitrogen sources, red lentil flour and bakers yeast cells was studied for L(+) lactic acid production in SSF by L. amylophilus GV6 using wheat bran as support and substrate. The maximum lactic acid production of 46.3 g/100 g wheat bran having 60 g of starch was obtained at optimized conditions (Altaf et al., 2006). L. amylophilus GV6 showed 96% lactic acid yield efficiency (g lactic acid produced/g substrate utilized) and 77.6% lactic acid production efficiency (g lactic acid produced/ g substrate taken) in SSF (Altaf et al., 2005, 2006, 2007a,b). L. amylovorus NRRL B-4542 was utilized in production of lactic acid using deoiled groundnut cake as solid support with corn starch as substrate in solid-state fermentation (Nagarjun et al., 2005). 10. Conclusions Lactic acid fermentation has received extensive attention for a long time since its potential applications in various sectors in particular in foods and preparation of biodegradable plastics. Starchy biomass can become an attractive and alternative, cheap substrate replacing costly sugars for lactic acid fermentation. Only few amylolytic lactic acid bacteria are reported so far that could actively ferment starch to lactic acid in single step fermentation. Of all the amylolytic lactic acid fermenting bacteria, L. amylophilus GV6 was found to be potentially utilizing different starchy and nitrogenous substrates with high lactic acid production efficiency. Isolation and development of potential amylolytic organisms may lead to economical production of sterospecific lactic acid isomers. Acknowledgements The authors are grateful to CSIR, New Delhi, for providing fellowships to BJN and MV to carry out part of this work. References Agati VJP, Guyot J, Morlon-Guyot P, Talamond, Hounhouigan DJ. Isolation and characterization of new amylolytic strains of Lactobacillus fermentum from fermented maize doughs (mawe and ogi) from Benin. J Appl Microbiol 1998;85:512–20. Akerberg C, Hofvendahl K, Zacchi G, Hahn-Hagerdal B. Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis sp. lactis ATCC 19435 in whole wheat flour. Appl Microbiol Biotechnol 1998;49:682–90.
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