biotechnology and traditional fermented foods

13 B i otechnolo gy and Tr ad iti onal Fermented F o ods A . K . N AT H , A N U PA M A G U P TA , B H A N U N E O PA N Y, G I TA N J A L I V YA S , J A R U WA N M A N E E S R I , N I V E D I TA S H A R M A , N I S H A T H A K U R , O M K A L U AC H I , U L R I C H S C H I L L I N G E R , A N D V. K . J O S H I Contents

13.1 Introduction 13.2 Industrial Development of Processes for Fermented Foods 13.2.1 Raw Material Development 13.2.2 Starter Development 13.2.3 Process Development 13.2.4 Equipment 13.2.5 Finishing and Packaging of Product 13.3 Molecular Characterization of Microbial Diversity of Fermented Foods 13.3.1 Application of Molecular Methods in Microbial Diversity Studies 13.3.1.1 Cultivation-Independent Methods 13.3.1.2 Culture-Dependent Methods 13.3.2 Biofortification Through Fermentation and Concentration 13.3.3 Fermented Food Packaging Systems and Presentation 13.3.4 Production of Probiotic Functional Food 13.3.5 Alkaline Fermented Vegetable Proteins 13.3.6 Upgrading Fermented Cereal Products 13.4 Role of Killer Yeast in Indigenous Fermented Food 13.5 Role of Metagenomics and Metabolomics in Indigeneous Fermented Foods 13.5.1 Metabolomics 13.5.1.1 General Methodology for Metabolic Engineering 13.5.1.2 Tool Used in Metabolic Engineering 13.5.1.3 Detection of Metabolites 13.5.1.4 Bioinformatic Tools for Metabolomic Analysis 13.5.2 Metagenomics 13.5.2.1 Metagenomic Techniques 13.5.3 Role of Metabolomics and Metagenomics in Indigeneous Fermented Foods of South Asia 13.5.3.1 Fruit Based Products 13.6 Control of Toxins in Food and Animal Feed 13.6.1 Mycotoxins

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In d i g en o us F erm en t ed F o o d s o f S o u t h A sia

13.6.2 Patulin 789 13.6.2.1 Methods to Reduce Patulin 792 13.7 Summary and Future Advances 793 References 794 Q1

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13.1 Introduction

Food is the basic survival necessity for all human beings, and traditional methods of food processing were aimed at food preservation and economy of fuel (Nout et al., 2014), so food fermentation methods arose historically from the need for processing and preservation food (Law et al., 2011; Ravyts et al., 2012). More than anything else, man has been employing microbes for the preparation of fermented food products for thousands of years. All over the world, a wide range of fermented foods and beverages are produced, which contribute significantly to the diets of many people (Achi, 2005a,b). Fermentation and drying are the oldest methods of food preservation and processing. It gives food a variety of flavors, tastes, textures, sensory attributes, and nutritional and therapeutic values (Bhavbhuti and Kamal-Eldin, 2012). The availability of storable and hygienically safe food was a decisive prerequisite for the development of mankind and society (Prajapati and Nair, 2003). The skills of food fermentation are embedded in traditional knowledge systems among the native peoples of many areas of the world, and the knowledge is maintained and propagated orally. The art of fermentation practiced by the common man has continued, in spite of the scientific and technological revolution, but has largely remained confined to the rural and tribal areas due to (i) high cost or inaccessibility of the industry-made products in remote areas, (ii) the tastes of the people for the traditional fermented products, and (iii) their sociocultural linkages with such products (Chelule et al., 2010; Thakur et al., 2004). However, with the advent of microbiology, biochemistry, molecular biology, and biochemical engineering, the art of fermentation practiced by the common man has been improved and upgraded, which has led to the rise of fermentation industries, adding quality and expanding the range of products (Thakur et al., 2004). A wide range of literature testifies to the multifaceted existing importance of traditional fermented foods and beverages. Whether of plant or animal origin, they remained important components of diets in many parts of the world, including the South Asian countries (Kabak and Dobson, 2011). Almost in contrast, some local indigenous fermented foods lack appeal and have very restricted popularity, while modern consumers prefer imported and exotic food items due to their attractive form, long shelf-life, ease of transportation, and other forms of utility associated with such foods (Achi, 2005b; Marshall and Danilo, 2012). Thus, the enhanced awareness of consumers of health, and the consequent interest in functional foods to achieve a healthy lifestyle, has established a need for food products with versatile health-benefiting properties, which is being felt all-over the world. Research on spontaneously

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fermented indigenous foods, their probiotic potential, and predominant microorganisms, has led to the exploration and exploitation of this traditional technology, with the major aim of producing consistent, safe products with longer shelf-life, which, in addition to the normal functionality of foods, also impart health benefits to the consumers. The indigenous fermented food produced in South Asia have major functions like bio-preservation of perishables, bioenrichment of nutritional components, protective properties, increased bioavailability of minerals, production of antioxidants and omega-3-polyunsaturated fatty acids, therapeutic values, and immunological effects (Tamang, 2011, 2012). However, little exploration and exploitation of technologies used in their production has been made. Technologies are called traditional if they remain unaffected by modernization, have been commonly applied over a long period of time by the native inhabitants of a region, and constitute an important part of their cultural inheritance (Dirar, 1993). Many traditional fermented foods usually meet this criterion (Hicks, 2002). At the same time, it is essential that traditional knowledge should go together with developments in science and technology to find mutually beneficial outcomes, and many biotechnological innovations have greatly assisted in upgrading certain indigenous fermented foods to a commercial level. Several traditional fermentations, for example, Indonesian tempe and soy sauce, have been upgraded to high technological production systems and have been expanded on a global scale (becoming household products around the world and a multi-million dollar industry) due to a strong research tradition in fermented food technology (Achi, 2005a,b; Hicks, 1983). In a bid to enhance food availability and quality, and alleviate malnutrition in many developing countries, research in indigenous fermented foods has seen a marked improvement during the last few decades (Dirar, 1993; Parkouda et al., 2009; Steinkraus, 2004). For more information, excellent reviews on these aspects are available (Gadaga et al., 2013; Holzapfel, 2002; Olasupo et al., 2010; Tamang, 2010). Traditional fermented foods can be improved in a number of ways, as has been outlined earlier (Nout, 1985). Developments made in genetics, enzymology, recombinant Q2 technology, and fermentation technology have led to our understanding of several processes and methods that can be employed to improve traditional food fermentation technology. Research priorities should include four broad categories, such as improvement in the understanding of fermentation process, refinement of the process, increasing the utility of the process, and, finally, developing local capabilities (Anonymous, 2012). Since the microorganisms are the most important component of food fermen- Q2 tation, understanding their physiology, genetic capability, and the conditions for their optimum growth are of great significance. The genetic engineering of microorganisms for maximum output and the development of new products and processes is an essential priority in the improvement of traditional fermented foods (Harlander, 1992). Biotechnology has a long history of application in food production and processing (Joshi and Pandey, 1999). It represents both traditional processing techniques and the latest techniques based on molecular biology. These techniques open up a large

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number of possibilities for rapidly improving the quantity and quality of fermented foods, especially the traditional fermentation processes, and can assist in upgrading the technology and changing the traditional processes into controllable, predictable, and efficient ones (Moreno et al., 2010). Not only this, it is essential in designing a biotechnology process to know the nature of the biological system to be used, the substrate to be transformed, and the operating conditions to be applied, so as to maximize the metabolic activity of the microorganisms, increase the process yield, and ensure the quality of the final product (Achi, 2005a). Significant biotechnological innovations have been made in research to understand various facets of the production of indigenous fermented foods, the microbiology and biochemistry of the fermentation, and the protocols of production, to enhance their nutritional and overall food value (Achi, 1990, 1992, 2005a,b; Eka, 1980; Moreno et al., 2010; Odunfa, 1985b; Okafor, 1983). The safety and health of consumers must be protected, and efforts to control the levels of salt and toxins (including mycotoxins), and the occurrence of parasites and pathogenic microorganism need to be addressed (Nout et al., 2014). In this chapter, a focus is made on some economically important and well-known traditional fermented food products based on biotechnological-driven fermentations (Ravyts et al., 2012), some of which present interesting models for safe and functional traditional foods that can be upgraded. To this end, current knowledge about the production of fermented foods and applications of biotechnological knowledge, along with constraints and related issues, have been highlighted. 13.2 Industrial Development of Processes for Fermented Foods

The art of indigenous fermented food processing needs to be transformed into a very precise technology, and the steps of processing must also have provision for quality control and optimization, without losing their originality (Anonymous, 2012). Before any product is considered for production at industrial scale, several aspects are considered. Besides the economics of the process and modeling of the product, major emphasis is laid on the scientific angles of the process. The industrial development of indigenous fermented foods and beverages can be divided into four areas: raw material development, starter development, process development, and finished product development. Research and development in these areas can certainly improve the product in terms of better quality and efficiency of the process. 13.2.1 Raw Material Development

Raw materials can be tested to find the most suitable variety of food item and its availability for use as a substrate. For example, it is now generally accepted that sorghum cultivars with low tannin content give a better quality European-type beer (Achi, 2005a,b). The raw material should be free from antinutritional factors like trypsin inhibitors, saponins, etc., when cereals, legumes, fruits, milk, etc., are the starting

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materials for the fermented foods. The availability of a proper quality and quantity of raw material for industrial production of any product is a prerequisite. 13.2.2 Starter Development

All the indigenous fermented foods were originally made by fermentation by natural microorganisms, and the knowledge has been transferred from generation to generation. Isolation, selection, preservation, or collection, and starter-making of a highly-efficient microbial strain for use as inoculum has to be made before industrialized large-scale production of any product can take place (Daengsubha and Suwana-adth, 1985; Okafor, 1983, 1990). In general, the microorganisms selected should not be able to synthesize toxins or toxic metabolites, be thermotolerant and osmotolerant (Anonymous, 2012). Lactic acid bacteria (LAB) and yeasts are probably the most important groups of organisms in making fermented foods (see Chapter 3). Pure wine yeast in grape fermentation or “tempeh rhizopus” in tempeh will make the fermentation process more precise, controllable, and promising. The development of strains with better and more stable genetic properties is a major task before the microbiologists, as they may offer nutritional benefits and compatibility to multi-strain fermentations carried out at present under non-sterile conditions (Achi, 2005a,b; Nout, 1985). 13.2.3 Process Development

This aspect has been illustrated by citing an example of how a traditional fermented food has been modified for industrial production. It is the work on the South African sorghum beer, which is an alcoholic, effervescent, pinkish-brown beverage with a sour flavor, an opaque appearance and the consistency of a thin gruel. Its preparation follows a pattern similar to that of burukutu or pito fermentation. The main steps in the brewing are mashing, souring, boiling, conversion, straining, and alcoholic fermentation (Achi, 2005a,b; Hesseltine and Wang, 1979). In the traditional process, the malt is made by soaking sorghum grains in water for 8–24 h, draining, and then allowing the grains to sprout for 5–7 days. The malt is sun-dried and ground into a fine powder. The ground malt is made into thin slurry and boiled. A small amount of uncooked malt is added and left for one day, during which it undergoes natural lactic fermentation, where Lactobacillus is chiefly responsible. The mash is boiled and left for alcoholic fermentation to take place. More ground malt is added and after the fermentation on the 5th day, it is strained and is then ready to drink. The factory process is less complicated, though it still incorporates the lactic and alcoholic fermentation steps with the top fermenting yeast Saccharomyces cerevisiae (Achi, 2005a,b), and leads to better fermentation and better quality of beer. So, in selecting a modification and or improvement to the process, adequate attention must be paid to its usefulness.

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13.2.4 Equipment

This is the most vital component of the technology package for industrialization of the process, if the home scale methods, as employed in traditional fermented foods, are to be scaled up. To make such equipment is a challenge, but can be done, as number of advances in the engineering sciences have been made. At the same time, the fact that traditional fermentations are carried out vessels with unusual characteristics, such as barrels of wood, semi-porous clay (Anonymous, 2012), etc., means that the equipment should be designed accordingly. 13.2.5 Finishing and Packaging of Product

After the product is finished, it needs to bottled andpackaged for proper marketing. Packaging is an essential process in the industrial production of fermented products such as South Africa sorghum beer. In South Africa it is currently packaged in milk cartons, which are filled and sealed in just the same way as milk (Achi, 2005a,b; Hesseltine and Wang, 1979), and sold. Because the product is consumed in an active state of fermentation, so each carton is left with an opening, large enough to allow CO2 to escape, but small enough so that the corn fragments will sealed the hole if the carton is turned on its side (Achi, 2005a,b; Hesseltine, 1983a,b). Similar improvements in the process for wine and brandy making from grapes is illustrated by traditional technology and the modified industrial process, as shown in Figure 13.1 (Joshi, 1997; Joshi et al., 1990). Improvement in the production of wine and indigenous brandy from wild apricots in the Kinnaur district of Himachal Pradesh (a)

Black grape

(b)

Black grape Addition of additives: SO2, DAHP and pectinase

Crushing Natural fermentation Filtration Finishing Distillation

Bottling

Bottling

Wine

Brandy

Macerating Inoculation with S. cerevisiae var ellipsoideus On the skin fermentation for 72 h Skin removed Fermentation Filtration Maturation Wine Bottling

Fractional distillation Oak wood Distllation Brandy Bottling

Figure 13.1 Comparison of traditional and industrial methods of wine and brandy production: (a) traditional method, (b) industrial method.

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(India) has been made (Joshi et al., 1990). The improvement introduced includes addition of sulphur dioxide, diammonium hydrogen phosphate, pectinase, a specific culture of yeast for wine making, and fractional distillation to make the brandy. More examples of improvement will be discussed or cited in the subsequent sections of this text. 13.3 Molecular Characterization of Microbial Diversity of Fermented Foods

To improve the quality of fermented foods, microbial culture screening is an important activity in fermented food research. Better species with a higher enzyme activity are still desired. Advanced molecular techniques have been explored to screen and obtain a microorganism that possesses multifunctional biotechnological characteristics—like enzyme activities, bile tolerance, and antimicrobial activity—and to improve the aroma properties of the product. 13.3.1 Application of Molecular Methods in Microbial Diversity Studies

13.3.1.1 Cultivation-Independent Methods Traditionally, the microbial composi-

tion and ecology of fermented foods are explored by using microbiological methods based on plate culturing and subsequent biochemical identification of isolated strains (Rantsiou and Cocolin, 2008). But all microorganisms are not amenable to cultivation in the laboratory, and the artificial media support the growth of only a small fraction of the organisms present in an ecosystem (Carraro et al., 2011). Stressed, weakened, and sub-lethally injured cells often need specific culture conditions to recover and produce colonies. Species occurring in low numbers may be efficiently out-competed by numerically more abundant and faster growing microorganisms (Hugenholtz et al., 1998). Consequently, the isolated organisms may not be representative of the community and culture-based methods, and therefore cannot accurately capture the in situ diversity of complex food ecosystems. Thus, for biodiversity studies of complex microbial communities, it has become mandatory to use culture-independent techniques based on the direct analysis of DNA (or RNA) from food. Molecular techniques are the major tools for the analysis of microorganisms from food and other biological substances. The techniques provide ways to screen for a broad range of agents in a single test (Field and Wills, 1998). Its use in studies has allowed for both a more definitive analysis of the structure of the microbial community and the determination of the metabolic status of different components of microbial populations (Egert et al., 2005). Most of these molecular methods include amplification by PCR and separation of the DNA fragments by gel or capillary electrophoresis or hybridization to specific probes (Sensabaugh, 2009). At present, the molecular marker predominantly used as a target is the bacterial gene coding for 16S ribosomal RNA. Finger printing assays allow the simultaneous analysis of multiple

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samples without prior knowledge of their diversity. Molecular techniques not relying on culturing are cost-effective valuable tools to compare microbial diversities and to monitor the dynamic changes in composition of a microbial community, leading to better understanding and improved management of the microbial processes involved in food fermentation (Van Hoorde et al., 2008). An improvement of the microbial safety due to the fast detection of potential pathogens in the food product can also be a direct outcome of this application (Justé et al., 2008). During the last decades various DNA fingerprinting techniques, such as DGGE, ribotyping, ARDRA, and rep PCR have become available, and in the meantime polyphasic approaches combining classical biochemical analyzes and molecular techniques, such as 16S rRNA sequencing, are frequently used for identification of bacterial isolates from food fermentations. The greatest advantage of molecular methods is that they can be applied without the need for prior culturing of the microorganisms, and have proved extremely useful to establish biodiversity profiles of food-associated microbial communities. Our knowledge of the biodiversity of microorganisms involved in food fermentations is being extended considerably by the use of advanced tools like DNA microarray technology and quantitative PCR, and next-generation sequencing and bioinformatics analyzes that have become available during recent years, especially the introduction of second generation sequencing techniques, allowing parallelizing of the sequence process towards thousands or millions of sequences at once, has revolutionized gene sequencing methodology. Pyrosequencing provides the benefit of reduced labor time, lower reaction volumes, and extended number of sequence reads, as well as high throughput sampling (Quigley et al., 2011). Current techniques including genetic finger printing, gene sequencing, oligonucleotide probes, and specific primer selection, discriminate closely related bacteria with varying degree of success. In addition to 16S rRNA primers and RAPDderived PCR primers, there is a growing interest in exploiting intergenic sequences (ITS; notably the 16S–23S rRNA spacer region) as well as functional genes, such as heat-shock protein (hsp) genes, the recA gene, and the Idh gene (McCartny, 2002). A comparison of the advantages and disadvantages of PCR methods is given in Table 13.1. Wherever possible, throughout the text, specific examples have been cited to illustrate the specific method. Two methods of identification which can be used which are discussed here: (i) PCR-dependent methods, and (ii) PCR-independent methods. 13.3.1.1.1 PCR Dependent Methods The polymerase chain reaction (PCR) tech-

nique has gained acceptance as a powerful microbial tool (Mustapha and Lc, 2006). Protocols for bacterial typing using PCR techniques are becoming increasingly valuable (De Urraza et al., 2000). This can be discussed by taking an example of LAB, which have traditionally been classified on the basis of phenotypic properties, which

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Table 13.1 Advantages and Distavantages of PCR Based Molecular Methods ADVANTAGES • Provide ways to screen for a broad range of agents in a single test.

• Allows both a more definitive analysis of the structure of the microbial community and the determination of the metabolic status of different components of microbial populations • Is a valuable tool to compare microbial diversities and to monitor the dynamic changes in composition of a microbial community, leading to better understanding and improved management of the microbial processes involved in food fermentation. • Provides microbial safety due to the fast detection of potential pathogens in the food product.

DISADVANTAGES

SOURCE

• Selective extraction of nucleic acids as a result of differences in the levels of cell lysis efficiency and selective amplification of target genes. • Co-migration of different fragments may occur as different sequences may have identical electrophoretic mobility. • Different species may yield PCR-products which co-migrate in the DGGE/TGGE gel. • Two species Leuconostoc mesenteroides and Weissella paramesenteroides had the same melting position, thus can not have this application. • The microheterogeneity in rRNA encoding genes present in some species may result in multiple bands for a single species and subsequently, to an overestimation of community diversity.

Sekiguchi et al. (2001)

• The low sensitivity due to traditional gel staining, resulting in the loss of bands, representing less abundant community members and gels of complex communities may look smeared due to the large number of bands that could hamper the interpretation of the fingerprint. • An incomplete extension of the GC clamp during PCR amplification may result in artifactual double bands in DGGE analysis that may complicate the interpretation of the profiles.

Justé et al. (2008) Anonymous (2007)

Ampe et al. (1999)

Anonymous (2007)

Rantsiou and Cocolin (2006, 2208)

involves physiological parameters and sugar fermentation patterns (Gerrs, 2001; Vandamme et al., 1996). However, these tests are difficult to interpret and the techniques are also time-consuming and laborious (Mohammed et al., 2009). Moreover the results of molecular-based approaches support many of the findings derived from culture-based methods of the presence of high proportions of LAB (Cabral, 2010). Among these methods, random amplified polymorphic DNA (RAPD-PCR) have proven to be very effective to enumerate specie and strain differentiation in food fermentations (Mohammed et al., 2009; Quiberoni et al., 1998). On the other hand, identification of LAB in traditional fermented foods by the use of molecular tools also offers the possibility of enhancing identification of bacterial composition in complex food fermentations. At the same time, molecular methods can complement biochemical species identification of isolated colonies (Aymerich et al., 2003). According to

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Arihara et al. (1993), based on the use of molecular tools strategies it may be possible to identify and then, combine the most desirable properties to construct LAB strains for probiotic application and starter culture development (Quiberoni et al., 1998). Hence, the development of PCR-based methods has opened new possibilities for rapid and specific identification of LAB. 13.3.1.1.1.1 Multiplex PCR Multiplex PCR (MPCR) methodology is based on the combination of several primer sets with different specificities in a single PCR reaction. It is the fastest culture-independent approach for simultaneous strainspecific detection of multiple microorganisms in complex matrices (Settanni and Corsetti, 2007). A pentaplex PCR assay was developed which allowed the simultaneous detection of 5 LAB species by direct DNA extraction from whey cultures for Grana Padano cheese (Cremonesi et al., 2011; Fornasari et al., 2006). Multiplex PCR approaches were also successfully applied to monitor the role of S. cerevisiae in wine fermentations (Hurtado et al., 2010; López et al., 2003) and, in combination with PCR-DGGE, to differentiate Lactobacillus species from sourdough (Settanni et al., 2006). Additionally, multiplex RAPD has been employed successfully for the differentiation of LAB isolated from the Gl tract and the identification of probiotic strains (McCartney, 2002). As with other DNA based methods, MPCR does not provide any information on the viability of the present organisms. MPCR assays targeting the mRNA (multiplex RT-PCR) could be an instrument to detect viable organisms. Such techniques, if applied to indigenous fermented foods, can give interesting and actionable information. 13.3.1.1.1.2 Quantitative PCR (qPCR) Real-time quantitative PCR (qPCR)

Q2

is a method of choice for the culture-independent detection and quantification of microorganisms. The exponential increase of amplicons can be monitored at every cycle (in real time) using a fluorescent reporter, and the increase in fluorescence is plotted against the cycle number to generate the amplification curve (Postollec et al., 2011). Combined with reverse transcription (RT), qPCR can also estimate transcript amounts, and this reversed transcription-qPCR can be used to study population dynamics and activity through quantification of gene expression during food fermentation (Postollec et al., 2011). In food microbiology, the technique is predominantly being used to detect pathogens and for the detection and quantification of microorganisms participating in food fermentations, such as LAB species in fermented milk products (Falentin et al., 2010; Furet et al., 2004; Masco et al., 2007). Real-time PCR assays have also been developed for the quantitative detection and quantification of Lactobacillus sakei and Leuconostoc mesenteroides in meat products (Elizaquivel et al., 2007; Martin et al., 2006) and for the enumeration of yeasts in fermented olives (Giraffa and Domenico, 2012; Tofalo et al., 2012). Application of this test can provide better insight into the microbiology of indigenous fermented foods.

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13.3.1.1.1.3 Denaturing Gradient Gel Electrophoresis and Temperature Gradient Gel Electrophoresis (DGGE/TGGE) Denaturing gradient gel electrophoresis

(DGGE) and temperature gradient gel electrophoresis (TGGE) are fingerprinting techniques based on the electrophoretic separation of DNA fragments according to their denaturing profiles. Currently, they are the most frequently used culture-independent methods to evaluate the microbial diversity of natural environments and to monitor community changes according to environmental variations (Giraffa, 2004). DGGE uses denaturing chemicals such as urea and formamide in a polyacrylamide gel, whereas TGGE is based on a linear temperature gradient (Lynch et al., 2004). The most commonly employed target for PCR prior to DGGE is phylogenetically informative ribosomal DNA, characterized by conserved and variable regions within the gene. The 16S rDNA V3 region is most frequently used for amplification. The rpoB gene, coding for the ß-subunit of the RNA polymerase, was also used as a target for PCR-DGGE (Rantsiou et al., 2004; Rantisou and Cocolin, 2006). PCR Q2 amplification of DNA extracted from mixed microbial communities with primers specific for 16S rRNA or rpoB gene fragments results in a mixture of PCR products which have about the same length but are different in base compositions (Nisiotou et al., 2014). These DNA molecules are subjected to increasing concentrations of the Q2 denaturing agents, and sequence differences bring about differences in their melting behaviors. Partial melting creates branched molecules with a decreased migration through the gel (Ercolini, 2004). Based on this principle, a sample containing a mixture of many different microorganisms, all with different melting domains, will result in many bands on the gel. The complexity of the profiles reflects the bacterial diversity of the sample. To prevent the complete dissociation of the double-stranded DNA, a 30–40 bp GC-rich sequence is usually attached to the 5′-end of one of the primers (Boutte, 2006; Sheffield et al., 1989). This clamp is very stable and holds the strands partially together. One of the major advantages of the DGGE/TGGE methods is that they allow simultaneous analysis of multiple samples and the use of universal primers, which permits the analysis of microbial communities without prior knowledge of the species present in the sample (Anonymous, 2007). The individual bands can be excised from the gel and identified by sequencing, although the small size of the PCR products may not always provide sufficient information for an unequivocal classification (Manzano et al., 2002; Ovreas, 2000). On the other hand, the DGGE/TGGE techniques suffer from the inherent bias of PCR-based molecular methods. DGGE and TGGE analyzes have been used as finger printing techniques for the identification of bacteria isolated from foods and, subsequently, more frequently for analysis of the bacterial communities in foods and in monitoring differences in the populations during fermentation or between different samples (Cocolin et al., 2000; Dawen and Tao, 2011; Jiang et al., 2010; Tsuchiya et al., 1994). Applications of DGGE and TGGE analysis for finger printing in the identification of bacteria are given in Table 13.2.

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Table 13.2 Application of DGGE and TGGE Analysis for Finger Printing in Identification of Bacteria PARTICULAR • Developed PCR-TGGE methods for identification of lactic acid bacteria in beer and Lactobacillus species isolated from Italian sausages, respectively used. • TGGE analysis was used to identify different genera present in dairy products and set up a data base for lactic acid bacteria and other dairy microorganisms with low G+C content and showed that it can be applied in complex liquid and solid dairy ecosystems. • DGGE and TGGE approaches have been used—mostly in combination with other molecular and culturedependent methods—to evaluate the microbial diversity of different fermented foods including pozol, a fermented maize dough. • Various cheeses from Italy, Spain, Belgium and Denmark kefir grains from different Asiatic countries nukadoko, fermented rice bran from Japan, coffee and West African cereal foods. • Studies focused to monitor the population dynamics during the fermentation process included Italian sausages artisanal cheeses from different countries, cassava various types of cereals, berries, rice vinegar, kimchi, cocoa beans and table olives. • The bacterial community changes during the malolactic fermentation of Spanish wine during the production of a fermented crucian carp with rice, called funazushi and a Philippine fermented mustard, called burong mustasa were also investigated using DGGE profiles.

SOURCE Ogier et al. (2002) Fontana et al. (2005)

Ampe et al. (1999) Guyot (2012) Björkroth and Holzapfel (2006)

Coppola et al. (2001), Ercolini et al. (2001), Randazzo et al. (2002), Flórez and Mayo (2006), Van Hoorde et al. (2008), Masoud et al. (2011), Chen et al. (2008), Jianzhong et al. (2009), Nakayama et al. (2007), Vilela et al. (2010), Oguntoyinbo et al. (2011). Cocolin et al. (2000), Dolci et al. (2010), Fuka et al. (2010), Randazzo et al. (2010), Miambi et al. (2003), Meroth et al. (2003), Weckx et al. (2010), Madoroba et al. (2011), Pulido et al. (2005), Haruta et al. (2006), Chang et al. (2008), Lefeber et al. (2011), Abriouel et al. (2011). Ruiz et al. (2010), Fujii et al. (2011), Larcia et al. (2011)

Most of these studies highlight the fact that a combination of both culture dependent methods and DGGE or TGGE approaches is essential for revealing microbial diversity and dynamics during fermentation (Madoroba et al., 2011). 13.3.1.1.1.4 Single-Strand-Conformation Polymorphism (SSCP) SSCP is a

molecular technique similar to DGGE/TGGE, as it is also based on the electrophoretic separation of PCR products of similar length (Ndoye et al., 2011; Thakur et al., 2004). It was originally developed in mutation research, mainly to detect novel polymorphisms and mutations in human genes (Spiegelman et al., 2005). Following denaturation, single-stranded DNA fragments are separated on a non-denaturing polyacrylamide gel or by capillary electrophoresis. Under denaturing conditions, single-stranded DNA fragments will adopt stable secondary structures according to their nucleotide sequence and their physico-chemical environment (Schwieger and Tebbe, 1998). Based on the migration of these secondary structures in the gel, PCR products of similar size can be separated and visualized (Justé et al., 2008). In contrast to

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DGGE/TGGE, no GC clamp primers are required (Peters et al., 2000). Caution should, however, be exercised when identifying bacterial populations using SSCP peak analyzes, due to the possible co-migration of different sequences (Delbès et al., 2007). This limitation can partly be overcome by the use of different sets of primers targeting various variable regions (V2, V3 of 16S rRNA) or specific groups of bacteria (Delbès et al., 2007). Another major limitation of SSCP analysis is the high rate of reannealing of DNA strands after an initial denaturation during electrophoresis. However, this problem can be minimized by the use of a 5′-phosphorylated primer allowing selective removal of the corresponding phosphorylated strand through digestion with lambda exonuclease (Schwieger and Tebbe, 1998). It has also been reported that several stable conformations out of one single DNA fragment may co-exist and result in multiple bands on the gel (Justé et al., 2008). SSCP analysis was applied in combination with clone library sequencing to investigate the dynamics of the complex microbial community of traditional French cheeses (Chamkha et al., 2008; Delbès et al., 2007; Duthoit et al., 2003), to study the microbial populations present in brines of Tunisian olives (Chamkha et al., 2008), and in Japanese traditional fermented foods made from fish and vegetables (An et al., 2011). The diversity of soft red-smear cheese populations was also investigated using SSCP analysis (Feurer et al., 2004). 13.3.1.1.1.5 Terminal Restriction Fragment Length Polymorphism (T-RFLP) The technique called terminal restriction fragment length polymorphism (T-RFLP) analysis combines selective PCR amplification of target genes with restriction enzyme digestion, high resolution electrophoresis, and fluorescent detection (McEniry et al., 2008; Rademaker et al., 2006; Slishi et al., 2006). Small subunit rRNA genes from total community DNA are amplified using primers designed to be non-discriminating, amplifying nearly all 16 SrDNAs, or selective, targeting specific domains or groups (Lord et al., 2002; Marsh et al., 2000). One of the two primers is fluorescently labeled at the 5′-end and the PCR is followed by digestion of the amplification products with one or more restriction endonucleases that usually have four base-pair recognition sites (Schütte et al., 2008). Only the fluorescently labeled terminal restriction fragments are detected on the sequencing gel, and their size can precisely be determined by using an automated DNA sequencer (Liu et al., 1997). The T-RFLP pattern is a distinct fingerprint of the microbial community, and the obtained TRFs can be compared to the sequence database of the Ribosomal Database Project (Cole et al., 2005), allowing tentative identification of primer-restriction enzyme combinations. Multiple restriction enzymes are typically used to increase the resolution, specificity, and reliability of the T-RFLP analysis (Osborne et al., 2006). The major advantages of the T-RFLP analysis are the high resolution of the capillary electrophoresis technology, its rapidity, and the possibility to screen and compare a large number of communities to investigate the response of the population structure to intrinsic and extrinsic factors (Nieminen et al., 2011). It is a valuable method

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for comparison of complex microbial communities when high through-put and high sensitivity are required, without a need for direct sequence information (Kopecký et al., 2009; Nocker et al., 2007). One of the major drawbacks of this technique is its dependence on restriction efficiency. Incomplete or non-specific restriction may occur, leading in an increased number of fragments and, therefore, to an overestimation of the microbial diversity of the sample (Justé et al., 2008; Zhang, 2010). To avoid this problem, the amplified product from a well-characterized isolate may be used as an internal standard, which allows checking of the restriction efficiency. Additional secondary T-RFs were observed to occur in T-RFLP analysis at high frequency and were called “pseudo-T-RFs” (Blaž, 2006; Egert and Friedrich, 2003). As a result, the microbial diversity may be overestimated because of the larger number of peaks in T-RFLP profiles. However, complete elimination of the pseudo-T-RFs can be achieved by the digestion of amplicons with a single-strand-specific nuclease prior to T-RFLP analysis (Egert and Friedrich, 2003). The number of PCR cycles should also be minimized, because the formation of pseudoterminal restriction fragments increases linearly with the cycle number (Egert and Friedrich, 2003). To improve the resolution and sensitivity of T-RFLP, another approach, called fluorophore ribosomal DNA restriction typing (f-DRT) has been developed (Wang et al., 2011), based on the end-labelling of all restriction fragments from a single enzyme digestion with a fluorescent dye, using high through-put capillary electrophoresis for detection of these fragments (Wang et al., 2011). The technique has been used to study the surface microflora of smear ripened Tilsit cheeses (Rademaker et al., 2005) and to monitor population dynamics during yoghurt and hard cheese fermentation (Rademaker et al., 2006). 13.3.1.1.1.6 Amplifed rDNA Restriction Analysis (ARDR A) Amplified ribo-

somal DNA restriction analysis (ARDRA), also known as restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes is a fingerprinting technique that has been successfully applied to bacterial identification at species level. However, it is also valuable and reliable for ecological studies and for comparison of microbial communities (Giraffa and Neviani, 2001). ARDRA is based on the digestion of PCRamplified ribosomal community DNA using appropriate restriction endonucleases, followed by gel electrophoretic separation of the generated restriction fragments. Multiple restriction endonucleases usually have to be used either separately or in combination. In contrast to T-RFLP, all fragments are visualized on the gel, increasing the resolution of this technique (Justé et al., 2008). On the other hand, the more complex patterns make the comparison and interpretation more difficult. A single species can produce 4–6 restriction fragments using a 4 bp cutting enzyme (Nocker et al., 2007). This, together with the limited staining sensitivity of DNA binding dyes, results in a suppression of bands from less abundant members of the microbial community (Nocker et al., 2007). In general, ARDRA is considered to be a useful technique for detecting structural changes in relatively simple microbial populations,

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but is not a method of choice to measure diversity of complex communities (Nocker et al., 2007; Sklarz et al., 2011). 13.3.1.1.1.7 Automated Ribosomal Intergenic Spacer Analysis (ARISA) Automated

ribosomal intergenic spacer analysis (ARISA) is based on the amplification of the intergenic region between the 16S and 23S ribosomal genes. In the case of molds and yeasts, the internal region (ITS) between the small subunit and the large subunit, including the 5.8S rRNA gene, is amplified (Filteau et al., 2011). The intergenic spacer region is more heterogeneous both in length and nucleotide sequence than the flanking ribosomal genes (Justé et al., 2008). Both types of variations make the intergenic spacer regions suitable for subtyping bacterial strains and closely related species in cases where the fingerprinting of ribosomal gene sequences does not provide sufficient resolution (Nocker et al., 2007). A fluorescent primer is used in the amplification of the ribosomal intergenic spacer region, and the PCR products are analyzed by an automated capillary electrophoretic system that produces an electropherogram, the peaks of which correspond to discrete DNA fragments (Cardinale et al., 2004). Potential problems associated with ARISA are the preferential amplification of shorter templates (Fisher and Triplett, 1999) and the fact that often more than one signal is generated by a single organism because of the IGS length variation within a single genome (Justé et al., 2008). As for DGGE/TGGE and T-RFLP analysis, ARISA has mainly been applied in ecological studies, and only a few applications in food matrices have been reported (Arteau et al., 2010). A multiplex automated ribosomal intergenic spacer analysis (MARISA) method has been developed and used to simultaneously analyze the bacterial and fungal microbiota composition of maple sap (Filteau et al., 2011). 13.3.1.1.1.8 Pyrosequence-Based rRNA Prof iling Pyrosequence-based rRNA

profiling is a new strategy allowing high through-put and in-depth monitoring of microbial communities. Up till now, it has mainly been applied in ecological studies of soil, the deep sea, and the human intestinal tract. The method is based on the detection of pyrophosphate released during nucleotide incorporation (Margulies et al., 2005). Pyrosequence-based rRNA profiling involves the amplification of variable regions of the 16S rRNA gene V1–V9 using primers that target adjacent conserved regions, followed by direct sequencing of individual PCR products (Liu et al., 2007). It monitors the luminescence of sequencing reactions in pico-liter plates (Nakayama, 2010). A barcode-tag sequence strategy can be used to analyze amplicons from different samples in one batch. Massive parallel sequencing means that more than 300,000 sequences can be determined simultaneously (Humblot and Guyot, 2009), and thus the costs associated with sequencing can be dramatically reduced. The second advantage is that cloning of the samples is not required, thus avoiding any problems associated with this step.

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Pearl millet slurries were the first fermented foods analyzed by this technique (Humblot and Guyot, 2009). Then, barcoded pyrosequencing was used to evaluate the archeal and bacterial diversities of seven types of fermented seafoods (Roh et al., 2010) and to study the microbial communities of various fermented foods, including Danish raw cheeses (Masoud et al., 2011), a fermented rice bran mash, called nukadoko (Sakamoto et al., 2011), narezushi, a traditional fermented food from Japan (Koyanagi et al., 2011), kimchi (Park et al., 2012), and traditional Korean alcoholic beverages such as makgeolli (Jung et al., 2012). 13.3.1.1.1.9 Cloning and Sequencing Individual members of the bacterial community may be identified at species level by cloning and subsequent sequencing of PCR-amplified sequences. 16S rRNA gene based libraries are used very frequently. A major drawback of the use of clone libraries is the high number of clones to be analyzed to detect rare organisms against the background of a few dominant species (Nocker et al., 2007). Therefore, it is common to construct clone libraries in parallel to fingerprinting techniques such as DGGE, or clone libraries initially are screened by restriction digestion yielding different restriction types which then, can be sequenced (Justé et al., 2008). For example, Kim and Chun (2005) used clone libraries in combination with ARDRA to investigate the microbial structure of kimchi samples, and a combination of 16S rRNA gene clone libraries and real-time quantitative PCR was applied to study the structure and dynamics of raw milk microbiota and changes in the microbial community during Montasio cheese ripening (Carraro et al., 2011; Rasolfo et al., 2010).

13.3.1.1.2 PCR Independent methods 13.3.1.1.2.1 DNA Array Technology The DNA microarray (microchip) tech-

nology allows parallel analysis of hundreds or thousands of genes in a single assay. Diagnostic nucleic acid sequences, referred to as probes, are immobilized on a miniaturized support (usually a glass slide) and are hybridized with the homologous labeled target amplicons, which can then be detected (Justé et al., 2007). Since hybridization signals are proportional to the quantity of target DNA, microarrays can also quantify genes in DNA samples (Cho and Tiedje, 2002). The DNA microarray technology enables rapid and simultaneous identification of hundreds of different organisms in one assay. So far, DNA arrays primarily have been developed to detect pathogens in food and clinical samples and have been less used to identify microorganisms participating in food fermentations. There is one report on the application of an oligonucleotide array in the analysis of raw milk bacterial communities (Giannino et al., 2009). A genome-probing microarray (GPM) was developed by Bae et al. (2005) which showed increased specificity as compared to oligonucleotide arrays. Using 149 microbial genomes as probes deposited on a glass slide, they were able to quantitatively analyze about 100 diverse LAB species involved in kimchi fermentation (Bae et al., 2005).

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Most of the currently applied methods, however, do not deliver information on the level of physiological activity and possible metabolic capabilities of the microorganisms, so the techniques targeting the mRNA will gain more importance in the future. In order to understand the role of different organisms in fermentation, for instance, their contribution to the aroma profile, it will be necessary to study gene expression by the means of transcriptomics and proteomics. A good example is the recent study of Nam et al. (2009) showing that metatranscriptome analysis with genome-probing microarrays can be used to determine the role and contribution of the different LAB species to kimchi fermentation. To monitor microbial dynamics during kimchi fermentation, another approach using environmental mRNAs (metascriptome) in addition to the metagenome analysis has also been applied (Nam et al., 2009). 13.3.1.1.2.2 Fluorescent in situ Hybridization (FISH) Fluorescent in situ hybridization (FISH) with 16S rRNA gene probes is another method of cultivation-independent detection and identification of microorganisms in a food matrix. It combines the simplicity of microscopic observation and the specificity of DNA hybridization (Justé et al., 2008; Machado et al., 2013). Following fixation and permeabilization, microbial cells are identified in situ within the original food sample by visualization of intercellular probe rRNA target hybrids (Ludwig, 2007). Cell fluorescence conferred by the probe attached fluorochromes is detected by epifluorescent microscopy or flow cytometry. It is a rapid method, allowing completion of the whole procedure within a few hours (Justé et al., 2008). Besides identification, the FISH technique provides information about the distribution of the microorganisms in the food sample and enables their enumeration and visualization of cell morphology. But the technique suffers from some system inherent pitfalls, such as the difficulty of achieving permeabilization of all cells in diverse communities, and the need for an extensive knowledge of the composition of the bacterial community to be able to construct suitable probes. Moreover, fluorescent in situ hybridization is less sensitive than PCR-based methods (Justé et al., 2008). Nevertheless, the FISH technique has been used for the rapid detection of LAB in wine (Sohier and Lonvaud-Funel, 1998), to analyze the surface microflora of Gruyère Swiss cheese (Kollöffel et al., 1999) and to study the spatial distribution of different microbial species in the Stilton cheese matrix (Ercolini et al., 2003). It has been demonstrated that Lactobacillus acetotolerans is a dominant bacterium in ripening of nukadoko (Nakayama et al., 2007), and it has been used in combination with flow cytometry to quantify the RNA content of yeast cells during alcoholic fermentation (Andorra et al., 2011).

13.3.1.2 Culture-Dependent Methods Molecular approaches may also be applied fol-

lowing common culture-dependent isolation and preliminary phenotypic characterization of strains. Several DNA-based techniques are available for genotyping of isolates, and the most commonly employed targets are the 16S and 23S rRNA

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encoding genes (Quigley et al., 2011). They involve 16S rRNA target oligonucleotide probes and the direct sequencing of the 16S rRNA gene, as well as many fingerprinting techniques, such as RAPD-PCR, rep PCR, PCR RFLP, ribo-typing, ARDRA, PFGE, and species-specific PCR. Basically, the pattern methods rely on the detection of DNA polymorphisms between species and strains, and differ in their discriminatory power, reproducibility, ease interpretation, and standardization. Several commonly applied techniques, including RFLP, ARDRA, ribotyping, and PFGE, make use of restriction fragment polymorphisms. Restriction fragment length polymorphism (RFLP) means that genomic DNA is digested using appropriate restriction endonucleases, and the generated fragments are electrophoretically separated (Sánchez and Sanz, 2011). Ribotyping is a variation of the conventional RFLP analysis using Southern hybridization of the DNA fragments with probes targeting rDNA and resulting in a less complex pattern to evaluate. Moreover, an automated riboprinter can be used. Amongst others, LAB from a Spanish blood sausage (Santos et al., 2005) and a Portuguese cheese (Kongo et al., 2007) were identified using ribotyping. ARDRA combines restriction fragment polymorphism with the application of site-specific PCR amplification, and has been used as a molecular tool in numerous studies, mostly in combination with 16S rDNA sequencing (Liu et al., 2007). It was shown to be useful in the identification of LAB from grape must and wine (Rodas et al., 2003), for the molecular characterization of enterococci from an African fermented sorghum product (Yousif et al., 2005), and for the classification of Bacillus isolates from cocoa fermentation (Quattara et al., 2011). Although 16S rRNA gene-based PCR-RFLP assays were preferentially used, rpo B gene sequences are increasingly being applied (Marty et al., 2012). Pulse field gel electrophoresis (PFGE) allows the separation of large fragments resulting from digestion of genomic DNA with rare-cutting enzymes. It is more discriminatory than most other techniques and, therefore, is suitable for strain typing (Sánchez and Sanz, 2011). PFGE was used for the differentiation of Staphylococcus strains isolated from fermented sausages (Corbière Morot-Bizot et al., 2006) and in combination with RAPD for characterization of strains involved in the malolactic fermentation of wine (Ruiz et al., 2008). Frequently used, easy-to-perform, PCR based techniques are RAPD-PCR, rep PCR, and species-specific PCR. RAPD (randomly amplified polymorphic DNA)PCR is a popular fingerprint method for intra- and inter-specific differentiation of LAB and other microorganisms involved in food fermentations. It uses short arbitrary primers targeting multiple sites on the genome and low-stringency conditions to randomly amplify DNA fragments which are then separated on a gel to give a fingerprint pattern specific for each strain (Quigley et al., 2011). It is frequently used for screening and a first grouping of isolates, and generally is followed by a more detailed molecular characterization (such as 16S rRNA gene sequencing) of typical representatives of the groups (Rebecchi et al., 1998). Another powerful typing method is rep (repetitive extragenic palindromic) PCR based on amplification of repetitive bacterial DNA

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elements. In contrast to RAPD-PCR, it uses primers such as (GTG)5 against known universal sequences in the bacterial genome (Singh et al., 2009). RAPD and rep PCR have both been used for identification of LAB from fermented vegetables and bamboo shoots produced in North East India (Tamang et al., 2005, 2008) and from fermented cassava (Kostinek et al., 2005). Rep PCR-fingerprinting using the (GTG)5 primer has also been successfully applied for the identification of acetic acid bacteria from fermented cocoa beans (De Vuyst et al., 2008) and LAB from raw milk Gouda-type cheese (Van Hoorde et al., 2008). RAPD-PCR analysis has been shown to be useful for the differentiation of the lactobacilli most commonly found in meat products (Andrighetto et al., 2001), and for the strain-specific identification of Leuconostoc mesenteroides used as starter culture in sauerkraut fermentation (Plengvidhya et al., 2004). Finally, species-specific primers are suitable tools in mono- or multiplex PCR assays, allowing a rapid and easy identification of species that are expected to be detectable in the food sample. For example, a total of 230 LAB isolates from French wheat sourdough were identified by PCR amplification using different species-specific primer sets (Robert et al., 2009). 13.3.2 Biofortification Through Fermentation and Concentration

Biofortification of food is an integrated approach to reducing malnutrition. The concept of in situ fortification by bacterial fermentation provides the basis to enhance the nutritional value of food products and their commercial value. In recent years, a number of biotechnological processes have been explored to perform a more economical and sustainable enhancement of the nutritional quality of bland traditional food (Capozzi et al., 2012). By this approach, food-grade biotechnological strategy for the production of protein-enriched yam flour has been reported (Achi, 1991, 1999; Achi and Akubor, 2000; Capozzi et al., 2012). Yam flour (Elubo) is an important processed yam product which is reconstituted by stirring in boiling water to form a paste (amala) and eaten with flavored sauces (Ige and Akintunde, 1981). Fermentation of yams to produce flour has been found to improve both product quality as well as to remove inherent coloration problems associated with the acceptability of the processed product (Achi, 1999; Ray and Sivakumar, 2009). Yam slices have been successfully fermented with Lactobacillus species isolated from the fermenting medium. Fermentation was found to give a product with a lighter color and a better quality product with enhanced consumer acceptability (Achi, 1992). The moisture, protein, and fat contents of the fermented flour are in the range of 7.0%–7.6%, 2.0%–3.5%, and 0.3%–0.4%, respectively, depending upon the varieties (Achi, 1999; Akingbala et al., 1995). Similarly, pretreated soy flour was used to replace 10, 20, 30, and 40% of fermented yam flour as a protein supplement. Higher concentrations of soy flour above 40% level, however, reduced the acceptability of the cooked paste (Achi, 1999; Achi and Akubor, 2000; Ray and Sivakumar, 2009). Such types of approaches can be applied very conveniently to similar products in the South Asian countries.

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13.3.3 Fermented Food Packaging Systems and Presentation

Q2

Packaging is an essential process in the production of many food products in industrialized countries. The principal roles of food packaging are to protect food products from outside influences and damage, to contain the food, prevent physico-chemical degradation, retain the beneficial effects of processing, extend shelf-life, maintain or increase the quality and safety of food, prevent microbial spoilage, and provide consumers with ingredient and nutritional information (Anonymous, 2011; Coles, 2003; Joshi and Pandey, 1999; Kailasapathy, 2004; Marsh and Bugushu, 2007; Verma and Joshi, 2000). As traditional fermented food production increases, new issues regarding problems of safety, spoilage, and sensory properties occur. The emergence of new package requirements for fermented foods is as a result of the continuous migration of people from their place of origin, population growth, and the desire to export the traditional fermented food to other cultures (Marsh and Bugushu, 2007). Fermented foods need special packaging. The required packaging protection depends on the stability and fragility of the food, the desired shelf-life of the food package, and the distribution chain. Good package integrity is also required to protect against loss of hermetic condition and microbial penetration (Kailasapathy, 2010; Lee et al., 2008). Several of the traditional packaging/presentation materials are now gradually being replaced by modern synthetic materials, such as plastics, paper, and cans (Teniola, 2009). The traditional presentation and packaging of some traditional fermented foods is shown in Figure 13.2. Fabricated improved fermentation trays constructed with maximum emphasis on maintenance, not only enhance sanitation and reliability for the process, but also involve minimum capital investment and operating cost (Ouoba, 2010; Sanni, 1993). Apart from the obvious effects of improving the shelf-life of the product, packaging helps to increase product popularity and general acceptability. 13.3.4 Production of Probiotic Functional Food

The primary role of diet is to provide sufficient nutrients to meet metabolic requirements, while giving the consumer a feeling of satisfaction and well-being (See Chapter 6 of this text for more information). In addition, some food components exert beneficial effects beyond basic nutrition, leading to the concept of functional foods and nutraceuticals (Anonymous, 2010; Ribiero, 2000). A growing public awareness of diet-related health issues, and mounting evidence regarding the health benefits of probiotics, have increased consumer demand for probiotic foods. “Let food be thy medicine and medicine be thy food ”, the age-old quote by Hippocrates, is certainly a widely-held opinion today (Vasiljevic and Shah, 2001). Some foods and their food components modulate various physiological functions and may play detrimental or beneficial roles in some diseases (Anonymous, 2010; Koletzko et al., 1998). Fermented foods are also functional probiotic foods, and are of great

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Figure 13.2 Example of some traditional packaging and presentation materials and modern packaging materials: (a) Agidi made ready for sale, (b) cooked ready-to-eat fufu on sale, (c) plastic package or paper cartons for processed dry fufu, gari, or elubo flour for sale, and (d) paper package/carton for fufu, gari, and elubo for sale. (Adapted from Teniola, O.D. 2009. Role of Packaging and Product Presentation in the Acceptability, Quality Improvement and Safety of the Traditional African Fermented Foods. Federal Institute of Industrial Research, Oshodi (FIIRO), Lagos, Nigeria.)

significance. Probiotics are defined as live microorganisms that exert health beneficial effects to the host when administered in sufficient quantities (Quigley et al., 2013). A food can be made functional by applying any technological or biotechnological means to increase the concentration, add, remove, or modify a particular component, as well as to improve its bioavailability, provided that the component has been demonstrated to have a functional effect, such as a probiotic effect (Das et al., 2012; Roberfroid, 1998). Many traditional fermented cereal food products have been found to contain components with potential health benefits (Anonymous, 2010). Some of the major categories of cereal based functional foods contain live microorganisms and may produce or release potential health promoting compounds in the substrate medium (Nwachukwu et al., 2010). In addition to these foods, new foods are being developed to enhance or incorporate these beneficial components for their health benefits or desirable physiological effects (Anonymous, 2010). There is a considerable amount of work being done on cereal containing foods fermented with complex indigenous cultures (Blandino et al., 2003). Studies using fermented cereals as delivery vehicles for potentially probiotic LAB for the production of probiotic foods have been reported (Angelov et al., 2006; Charalampopoulos et al., 2003; Kedia et al., 2008 Rathore et al., 2012). Cereal grains Q2 such as barley and oats are good substrates for LAB growth, which has led to the commercialization of cereal-based probiotic products (Salovaara, 2004). Strains of the

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Lactobacillus genus, such as Lactobacillus acidophilus, L. reuteri, and L. plantarum constitute significant proportion of cultures used in probiotic products (Charalampopoulos et al., 2003; Ravyts et al., 2012). Functional foods produced through traditional fermentation processes thus have potential to provide food sources with increased nutritional value to South Asia countries. For more details, see Chapter 6 on the health aspects of indigenous fermented foods. 13.3.5 Alkaline Fermented Vegetable Proteins

Q2

Q2

A very important class of fermented products is the indigenous alkaline-fermented food condiments (Sarkar and Nout, 2014). Alkaline fermentation is the process in which the pH of the substrate increases to alkaline values, maybe as high as pH 9 (Amadi et al., 1999; Omafuvbe et al., 2000; Sarkar and Nout, 1885; Sarkar and Tamang, 1995). Fermented vegetable proteins made up of legumes and oilseeds are generally used as soup flavoring condiments (Olasupo et al., 2010). The cooked forms of dietery proteins are eaten as meals, and are commonly used in fermented form as condiments to enhance the flavors of foods (Achi, 1991; Aidoo, 1986; Oniofiok et al., 1996). Apart from increasing the shelf-life and a reducing the anti-nutritional factors (Achi and Okereka, 1999; Barimalaa et al., 1989; Reddy and Pierson, 1999), fermentation markedly improves the digestibility, nutritive value, and flavors of the raw seeds. Excellent reviews of traditional fermented food condiments have been published earlier (Achi 2005b; Odunfa, 1985b; Olasupo, 2006; Parkouda et al., 2009). The methods employed in the manufacture of fermented condiments differ from one region to another because these processes are based on traditional systems. Various studies have been carried out to upgrade the traditional process and make it less labor intensive (Achi, 2005b; Odunfa et al., 2006). 13.3.6 Upgrading Fermented Cereal Products

Ogi is a fermented cereal gruel processed from maize, although sorghum or millet is also employed as the substrate for fermentation (Blandino et al., 2003). It is an example of traditional fermented food, which has been upgraded to a semi-industrial scale (Achi, 2005a). 13.4 Role of Killer Yeast in Indigenous Fermented Food

This is another application of biotechnology in indigenous fermented foods, where killer yeast is used (Cocolin and Comi, 2011). Killer yeast is the yeast which is capable of killing susceptible cells by producing toxic proteins or glycoproteins (so-called killer toxins or killer proteins). The phenomenon in yeast cells was first reported by Bevan and Makower (1963). S. cerevisiae can kill sensitive strains of the same species, though it has been found in the other strains also, such as Hansenula sp., Pichia sp., Candida

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sp., Willopsis sp., Torulopsis sp., Tricosporon sp., Kloeckers sp., and Kluyveromyces phaffi (Comitini et al., 2004a,b; Izgu et al., 2006). Killer yeasts have been isolated from various sources like fruit, mushrooms, decaying plants, soil, the skin of poultry, and fermented products (Cocolin and Comi, 2011). Killer toxins have been classified into different types, as K1, K2 and K3. They are usually active and stable at pH 4–5 and temperatures of 20–25°C (Bendova, 1986; Heard and Fleet, 1987; Michalcakova and Repova, 1992), and cause membrane permeability changes in sensitive cells (Kagan, 1983). In some cases, they can inhibit DNA synthesis, such as in S. cerevisiae (Izgu and Altinbay, 1997; Schmitt et al., 1989) or stop cell division (Stark et al., 1990). Q2 Clearly visible inhibition zones surrounding the killer toxin-producing yeast can be observed, and the killer phenomenon can be confirmed with purified killer toxins, which implies a unique form of bioaction. The inhibition zone produced by the activity of killer yeast is shown in Figure 13.3. The killer yeast toxin can be useful as a tool for the development of yeast-based biocontrol in several biotechnological applications (Marquina et al., 2002; Selvakumar et al., 2006; Silva da et al., 2008; Walker et al., 1995). In the food industry, killer characteristics can be conferred to yeast strains. The use of killer yeasts as starter cultures can protect the product against spoilage yeasts. They have also been considered useful in biological control of undesirable yeasts in the preservation of food (Izgu et al., 2004; Lowes et al., 2000; Petering et al., 1991). Therefore, killer yeast has the ability to control spoilage in the preservation of food. For example, C. tropicalis shows inhibitory effects against S. cerevisiae in contaminated starter cultures of the Turkish bakery industry (Izgu et al., 1997; Santos et al., 2004) and Kluyveromyces phaffii DBVPG 6076 killer toxin has been found effective against apiculate wine yeast

Figure 13.3 Inhibition zones of sensitive yeast strains surrounding the growth of killer yeast strains in YPD agar medium with methylene blue.

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from grape samples (Ciani and Fatichenti, 2001). Knowledge and understanding of each killer yeast/toxin is of importance in determining its possible use, such as in solving fermentation problems during commercial biomass production of baker’s yeast (Silva et al., 2008). It has been found that K3 killer toxin gene can be transferred into S. cerevisiae BSP 1 to construct a baking strain resistant to killer toxin producing C. tropicalis contamination by the protoplast fusion technique. The new S. cerevisiae BSP 1 (K3) can inhibit the growth of C. tropicalis in a pH of 3.5–5.0 and at a temperature of 20–30°C (Izgu et al., 2004). The phenomenon of killer yeast appears to be widely distributed within many yeast genera and such yeasts can be isolated from a great variety of food fermentation processes. Isolation of killer yeast from fermented vegetables (pak-sien, bamboo shoot, sator, and cabbage) has also been made and identified as Candida krusei (Hernández et al., 2008). It shows the killer activity against sensitive reference strains on YPD agar plate supplemented with 0.003% methylene blue (Figure 13.3). The killer yeast showed activity against food pathogenic bacteria (Escherichia coli TISTR 887, Salmonella typhimurium TISTR 292, Staphylococcus aureus TISTR 118, and Bacillus cereus TISTR 868) (Waema et al., 2009). Debaryomyces hansenii, Klyveromyce marxianus, Pichia guilliermondii, and S. cerevisiae have been isolated from seasoned green table olives, which had the broadest spectra of action against yeasts that cause spoilage (Hernandez et al., 2008). The effect of pH and salt on killer activity has been studied by testing the killer strains against a killer-sensitive strain. These killer yeasts show their killer activity at pH 8.5 in the presence of 5% and 8% NaCl, but a high concentration (10% NaCl) decreased it (Hernandez et al., 2008). Toxins produced at any salt concentration exhibited the highest activity against sensitive strains in the detection medium. Salt has been found to have induced the formation of ion-permeable channels, killing cells by disruption of the plasma membrane function and cellular ionic balance (Santos and Marquina, 2004; Sitva, 2008). Toxins at high salt levels can also have biotechnological applications, such as preservation of high-salt food products. Moreover, the killer activity is different at different temperature, pH, and salt level of the brine, depending upon the strains (Table 13.3) (Silva et al., 2008). Table 13.3 Optimum Temperature and pH for the Activity of Killer Toxins of Yeast Strains KILLER STRAIN Saccharomyces cerevisiae Kluyvermyces phaffii Saccharomyces cerevisiae Candida tropicalis Pichia membranifaciens Pichia anomala

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TEMPERATURE (°C)

pH

REFERENCE

15.0–25.0

3.5–4.5

Heard and Fleet (1987)

24.0 20.0

4.0 4.3–4.4

Ciani and Fatichenti (2001) Lebionka et al. (2002)

20.0–30.0 25.0

4.0 3.0–4.8

Izgu et al. (2004) Santos and Marquina (2004)

4.0–37.0

4.5

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Biopreservation is considered to improve food safety without changing the sensory quality of a product. In dairy products, there is a risk of contamination by Listeria monocytogenes, so killer yeast strains were isolated, screened, and tested against bacteria from different sources, but mainly from dairy products. Some strains showed an inhibitory potential against the growth of pathogenic bacteria (Gorgis, 2006). Candida intermedia was able to reduce the L. monocytogenes cell count by 4 log cycles, whereas Kluyveromyces marxianus suppressed the growth of L. monocytogenes by 3 log unit cycles (Gorges et al., 2006). Williopsis saturnus could inhibit spoilage by yeasts in cheese. It is ideal as a biopreservative and may replace chemical or antibiotics-based preservatives (Goerges et al., 2006; Liu and Tso, 2009). In brief, killer yeast and its toxins have potential for application in indigenous foods, mostly as biopreservatives. 13.5 Role of Metagenomics and Metabolomics in Indigeneous Fermented Foods

Fermented foods, a part of an important food ecosystem, harness microbial diversity and functional microbiota in the environment (Tamang, 2001). Filamentous molds, yeasts, and bacteria constitute the microbiota in indigenous fermented foods and beverages, which are present in the ingredients and are selected through adaptation to the substrate. For more details, see Chapter 3. New biotechnological methods are now applied to increase the capacity of microorganisms in terms of yield, longer time of preservation, detection of bioactive substances, etc. Metabolomics and metagenomics are new emerging fields of biotechnology that play an important role in indigenous fermented foods. These technologies interact with the metabolic pathways of microorganisms and, thus, lead to beneficial products. Metabolomics and metagenomics are derived constitutively from the suffix “OMICS” (OM)—the progenitor of all terminology including proteomics, genomics, transcriptomics, etc., with the ability to simultaneously measure the level of the several gene products and metabolites. It is thus possible to gain greater understanding of a cell. So meta- Q3 bolic engineering has become a tool for the directed evolution of microorganisms (Noronha et al., 2000). 13.5.1 Metabolomics

Metabolomics is the study of metabolome in a cell at different developmental stages under different conditions. It refers to the complete set of metabolites, including hormones, secondary metabolites, etc., found within a biological system. When this technology involves biotechnology for the improvement of the cellular activities of microorganisms, via the use of recombinant DNA technology, then it is called metabolic engineering, as illustrated in Figure 13.4. It refers to the systematic modification of metabolic pathways for the purpose of obtaining a desired product or trait (Norouha et al., 2010).

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Microorganism

Metabolic pathways

Improved by suppressing or enhancing particular pathways which leads to beneficial products

Figure 13.4 Steps in metabolic engineering.

13.5.1.1 General Methodology for Metabolic Engineering

Metabolic engineering involves different steps such as: • Identify the target phenotype or trait. • Increase the frequency of occurrence of genes that may confer the phenotype. a) Increase the mutation frequency in producing cells by mutagen treatment (UV, x-ray, chemical mutagen) (Classical method). b) Introduce additional gene (that may already exist or be absent in the host cell). c) Introduce a genetic element to inactivate the gene by random insertion of extra sequences. d) Introduce the gene that causes insertional inactivation of harmful alleles e) Suppress the harmful genes by inserting homologous gene sequences. 13.5.1.2 Tool Used in Metabolic Engineering To design microorganisms for hyperproduction of a metabolite, a detailed analysis of the metabolic process is needed, which in turn requires in-depth knowledge of the various metabolities, gene products, and the regulatory system involved (Noronha et al., 2030). These include functional genomics, metabolic profiling, metabolic analysis, and metabolic control analysis (Kopka et al., 2004; Trethewey, 2004). 13.5.1.2.1 Separation of Metabolites To acquire the technology of various metabo-

lites, different analytical tools are employed such as: Q4

• GC can separate various components, but only volatile biomolecules can be analyzed by this technique. • HPLC (high performance liquid chromatography): Compared to GC this gives low resolution, but a wide range of analytes can potentially be measured. • Capillary electrophoresis (CE): For charged analytes, it is the most widely used electrophoretic technique and gives higher theoretical efficiency than HPLC. 13.5.1.3 Detection of Metabolites The most common methods for identification of the

abundance of metabolites present in an organism include nuclear magnetic resonance (NMR) and mass spectrometry. NMR based metabolomics were developed in the

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laboratory of Jeremy Nicholson at Birkbeck, University of London. NMR provides a great deal of information for every biomolecule, thus allowing identification and quantification of the differences in the metabolites. Gas chromatography mass spectroscopy (GC–MS) and liquid chromatography mass spectroscopy (LC–MS) are able to detect several hundred chemicals, including sugars, sugar alcohols, organic acids, amino acids, fatty acids, etc. 13.5.1.4 Bioinformatic Tools for Metabolomic Analysis The tools employed for the analy-

sis of metabolomics include correlation optimized warping (COW) and chrompare. After detecting the metabolites present in a particular organism, whether in plants or microorganisms, metabolic engineering can be undertaken to yield the desired product.

13.5.2 Metagenomics

Metagenomics can be defined as the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, by-passing the need for isolation and laboratpry cultivation of individual species (Anonymous, 2005). The term was first used by Jo Handelsman, Jon Clardy, Robert M. Goodman, and others, and first appeared in publication in 1998. 13.5.2.1 Metagenomic Techniques Some of the techniques used in metagenomics

include:

• Sampling from habitat (microorganisms can be isolated from any environmental areas, such as the sea, mud, marine depopsits, etc) • Filtering particles typically by size with filter membrane • Lysis of cell wall followed by DNA extraction. Cell wall is ruptured with the help of calcium chloride or other extraction buffer to extract DNA • Cloning of DNA into vectors and construction of libraries in bacterial artificial chromosome (BAC) vectors. (BACs are used because they have cloning capacity of 100–300 kb) Sequencing of clones by the following methods: a) Shotgun sequencing methods: These provide information on which environment the microorganism will better adapt to, and what metabolic processes are possible in that community. b) High through-put sequencing: This uses massive parallel pyrosequencing methods to generate shorter DNA fragments, approx 400 bp, than Shotgum sequencing methods. This become advantageous as it does not require cloning of the DNA before sequencing, removing one of the main bases in environment sampling.

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• Assembly of sequence into contigs, which is a series of clones that contain overlapping pieces of DNA covering a specific region in a chromosome. • Application of bioinformatics software to detect the sequences. 13.5.3 Role of Metabolomics and Metagenomics in Indigeneous Fermented Foods of South Asia

This aspect will be illustrated by taking a few examples of foods, including those of South Asia. Cheese: Cheese is a generic term for a diverse group of milk-based products, and is produced in wide-ranging flavors, textures, and forms, and has proteins and fats derived from milk. It is produced by the milk protein casein. Typically, the milk is acidified and the addition of the enzyme rennet causes coagulation. The solids are separated and pressed into the final form. Most of the time the enzymes are produced by fermentation of the fungus Mucor miehei. Metabolic profiling technologies with GC and time-of-flight mass spectrometry (TOF-MS) have analyzed the low molecular weight components, including amino acids, fatty acids, amines, organic acids, and saccharides, in hard and semi-hard cheese. The compounds that play an important role in constructing each sensory prediction model include 12 amino acids and lactose for “Rich flavor” and 4-aminobutyric acid, ornithine, succinic acid, lactic acid, proline, and lactose for “Sour flavor” (Ochi et al., 2012). The metabolomics-based component profiling not only focused on hydrophilic low molecular weight components, but was also able to predict the sensory characteristics related to ripening (Ochi et al., 2012). Metabolic profiling of the microorganism Lactococcus lactis (used in cheese fermentation) under different conditions can be obtained by GC-MS and HS-MS. Such techniques are used to study different metabolites produced by the bacteria and manipulate the metabolic pathways for better yield (Azizan et al., 2012). Metagenomic analysis can be done using massively parallel DNA sequencing to characterize the microbial communities living in cheese. In addition to its economical importance, cheese production is also a concern for public health, with thousands of people becoming sick every year after eating improperly manufactured cheese. Description of the microbial populations and their temporal variations in cheese is thus essential. At the Genomic Medicine Institute, Cleveland Clinic, Cleveland, DNA was extracted from cheese at four time points: immediately after insemination (day 1), at the day of sale (day 30), at the expiration date (day 90) and at day 180. Then, short fragments of 16S and 18S ribosomal RNA genes were amplified using universal primers, and obtained 100,000 sequence reads from each sample. The analysis showed that, starting from an environment relatively free of microorganisms before being inseminated with a few bacterial species, the microbial diversity increased dramatically to include multiple species belonging to several orders of bacteria and fungi. The microbial composition at the different points of time reflected the changes in the environment (e.g., pH, nutrient content, and presence of oxygen)

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and the interactions between the different microorganisms. The potential of this high through-put sequencing (metagenomical technique) to study the complex microbial interactions in a changing model ecosystem is illustrated by this example. Yogurt: This is a dairy product produced by bacterial fermentation of milk. The bacteria employed to make yoghurt are known as “yoghurt cultures”. Fermentation of lactose by these bacteria produces lactic acid, which acts on milk protein to give yoghurt its texture. It is made by the addition of a culture of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus bacteria, besides other lactobacilli and bifidobacteria that are sometimes added during or after yoghurt culture (Anonymous, Wikipedia). Biochemical impacts in yoghurt produced by the addition of probiotic bacteria (Lactobacillus rhamnosus GG, Lactobacillus plantarum WCFS1, or Bifidobacterium animalis Bb12), especially in metabolite production via NMR spectroscopy and he GC–MS or LC–MS techniques, can be used to illustrate the simultaneous growth, viability, and metabolite production of starter cultures and probiotic bacteria in yoghurt fermentation. 13.5.3.1 Fruit Based Products Wines: Wine is an alcoholic beverage made from fer-

mented grapes or other fruits. Wines made from fruits besides grapes are usually named after the fruit from which they are produced (e.g., pomegranate wine, apple wine, and elderberry wine), and are generically called fruit wines (Joshi and Pandey, 1999). NMR is successfully being used to characterize wine and find an association of wine metabolites with environmental and fermentative factors in vineyard and wine-making. The metabolomics and matagenomics thus help in the detection of various bioactive substances present in the fermented foods. Metagenomic approaches present a fascinating opportunity to identify uncultured microorganisms and to understand their biodiversity, function, interactions, and evolution in different environments (Park et al., 2011). Similarly, GC-MS and HS analysis of the metabolites produced by L. lactis in response to temperature and agitation contribute to the understanding of metabolic changes during environmental stresses (Azizan et al., 2012). In a nutshell, the application of new “omics” technologies to unravel the interactions in mixed starter cultures will enable the rational development of new and more effective starter culture systems and increase understanding of the microbiota present in the food (Ivey et al., 2012).

13.6 Control of Toxins in Food and Animal Feed 13.6.1 Mycotoxins

Food is liable to be contaminated with toxins like mycotoxins, putulin, etc., if proper measures are not taken. A number of fungi are known to produce to mycotoxins

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Table 13.4 Examples of the Reported Effects of Fermentation on Mycotoxins in Raw Materials

TOXIN

RAW MATERIAL/ PRODUCT

TYPE OF FERMENTATION

NATURALLY CONTAMINATED/ SPIKED

EXTENT OF REDUCTION

Aflatoxin

Maize/kenkey

Lactic acid

None

Aflatoxin

Sorghum/ogi

Lactic acid

Natural with B1

12%–16%

Aflatoxin

Wheat/bread

Yeast (dough)

Spiked with B1

19%

Aflatoxin

Milk/yogurt

Lactic acid

Natural with M1

None

Aflatoxin

Milk/kefir Milk/yoghurt

Lactic acid Lactic acid

Decreased None

Aflatoxin

Melon seed/ogiri

Bacillus sp.

Aflatoxin

Peanut press cake/ pure mold cultures

Aflatoxin

Maize/ogi Sorghum/ogi Pure culture isolates from kenkey

Neurospora sitophila and Rhizopus oligosporus Lactic fermentation Lactic acid bacteria

As above spiked with B1, B2, G1, G2 and M1 Natural with B1 and G1 Not Stated

Alternariol and alternaniol mono-methyl ether

REFERENCE Jesperson et al. (1994) Dada and Muller (1983) (http:// www.karlsruherernaehrungstage. de/papers/Part1. pdf) El Banna and Scott (1983), Westby et al. (1997) Wiseman and Marth (1983) Blanco et al. (1993)

Complete removal after 4 days 50% and 70% respectively

Ogunsanwo et al. (1989) Steinkraus (1983), Westby et al. (1997)

Natural with B1

Greater with 70%

Spiked laboratory media

Reduction greater than 50% by all tested strains

Adegoke et al. (1994) Holzapfel (1995)

(Table 13.4). Formation of mycotoxins in food depends on a number of physical and biological factors, as reviewed earlier (Sharma, 1999; Sharma et al., 1980; Westby et al., 1997). The physical factors include moisture in the product, relative humidity, mechanical damage to seeds, temperature, and time of storage. The factors that determine formation of mycotoxins are the microenvironment of the food or its atmosphere, the nature of substrate, the mineral nutrition, the presence of inhibitory factors, and the treatment, if any. The biological factors that predispose the commodity to the attack of mycotoxin-producing fungi include plant stress, invertebrate vectors, fungal infection, load and strain of the fungus, varietal differences, and microbial competition. Naturally, if the raw material is contaminated with mycotoxin producing fungi, the product made out of such a crop is most likely to contain mycotoxins. Indigenous fermented foods also involve fermentation, thus, microorganisms and, hence, any contamination with undesirable microflora, can also lead to the formation of mycotoxins, even if the crop does not contain such toxins itself.

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The preferred method of controlling the occurence of mycotoxins in these foods is to prevent their formation, either in the field or during storage (Coker, 1995). However, this is not always feasible, particularly when mycotoxins are produced by field fungi or during storage in uncontrolled hot and humid environments. Efforts are being made to develop modern biotechnological approaches to prevent pre-harvest crop infection for the control of mycotoxin producing fungi, including the development of transgenic plants by cloning for genetic resistance to the offending fungus and the cloning of genes coding for known antifungal proteins and their tissue specific expression. The use of competitive microbes and natural inhibitors of fungi can also be developed as biological control agents for the prevention of pre-harvest crop infections. Of course, development of these strategies needs through understanding of the genes involved in plant disease resistance and those responsible for controlling mycotoxin production in the fungi. The cloning of mycotoxin genes could also obviate the need to isolate and study the enzymes of the pathway, while allowing understanding the effect of regulation on gene expression. Sequencing of mycotoxin genes can throw light on the evolutionary trends in mycotoxins production. Fungi with deleted mycotoxin genes could also be developed as ideal biocontrol agents. Cloning and characterization of mycotoxin genes could, therefore, help in the development of mycotoxin-resistant transgenic plants. The understanding of plant disease resistance genes can help in the control of plant infections and in the replacement of harmful chemicals through interspecific transfer. Postharvest infection of agricultural produce could be prevented through the elimination of mold spores using antifungal agents, both physical and chemical, and the development of storage with controlled conditions of atmosphere, humidity, and temperature (Sharma et al., 1980; Zaika and Buchanan, 1987). A number of compounds Q2 have been found to affect the biosynthesis or bioregulation of aflatoxin. Many of these studies could help in hazard analysis and in identifying the critical points for preventing mycotoxins formation in foods during processing and preservation. It is also possible to develop seeds resistant to fungal infection during storage through tissue-specific expression of fungal inhibitor genes in seeds using recombinant DNA technology. Unlike bacterial protein toxins, which can be destroyed at high temperatures, mycotoxins require the most drastic conditions for their destruction. But mycotoxins such as aflatoxin have been shown to be destructible by physical methods such as autoclaving and exposure to ultraviolet radiation, or by chemical methods. But extreme basic conditions, such as treatment with ammonia under pressure, as well as by biological methods such as enzymes and microbes, are also effective, but are known to affect the nutritional and sensory attributes of food as well, so these methods have limited application. Other simpler methods for the elimination of mycotoxins are the use of hand and machine sorting for the removal of infected seeds. It is essential to know that a decontamination process should essentially leave no toxic mutagenic or carcinogenic residue, while retaining the nutritive value and the acceptability of the product. It should also not significantly alter the technological properties of food, and must be technically and economically feasible (Bhatnagar et al., 1995).

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Strategies to eliminate reformed toxins in a commodity are rather cumbersome and less reassuring (Park et al., 1988). These include sorting of the infected material and general detoxification of a commodity using ammonia. The conventional techniques for the prevention of pre-harvest infection include breeding for resistance to evolve germ plasm or elite varieties of crop which can resist the infecting fungus, besides the use of fungicides and insecticides. There have been a number of reports of the effects of fermentation on the aflatoxin content of contaminated raw materials (Table 13.4) (Westby et al., 1997). Nout (1994) summarized the effects of fermentation on Aflatoxin B1. Fungi involved in food fermentations, such as Rhizopus oryzae (R. arrhizus) and Rhizopus oligosporus, are able to reduce the cyclopentanon moiety which results in aflatoxicol A, which is a reversible reaction. However, under some conditions (e.g., in the presence of organic acids) aflatoxicol A is irreversibly converted into the stereoisomer aflatoxicol B, which is about 18 times less toxic than aflatoxin B1. In the conditions of a lactic fermentation (pH 4.0), aflatoxin B1 is readily converted to aflatoxin B2 , which is also less toxic. These reactions do reduce toxicity, but cannot provide complete detoxification (Nout, 1994). Complete detoxification is only achieved when the lactone ring is broken, which corresponds to a loss in fluorescence at 366 nm, which has been used as a screening tool for this toxin. Bol and Smith (1989) used the technique to identify certain Rhizopus spp. that were able to degrade greater than 85% of aflatoxin B1 present into non-fluorescent substances. The toxicity of these substances, however, is unknown. Rhizopus strains with the ability to degrade aflatoxin B1 have also been reported (Kanittha, 1990; Westby et al., 1997). Along with four major aflatoxins, several of its metabolites (M1#M2, M4, B2a, G^, etc.) have been identified and characterized. Aflatoxin B1 (AFB) present in animal feed has been found to be excreted as aflatoxin M (AFM) in milk, which withstands processing steps like chilling, separation, pasteurization, and boiling. It gets concentrated in concentrated/dried dairy products, like khoa, chhana, condensed milks, dried milks, infant formula, etc. Unfortunately, there is no feasible method that can completely degrade the aflatoxin in milk and milk products. Some chemical methods, like treatment with hydrogen peroxide, sulphates, bisulphates, etc., and physical methods, like adsorption on particulate materials, treatment with UV light, etc., have been tried. The ability of certain strains to degrade aflatoxin offers the potential to develop defined starter cultures for this purpose. Notwithstanding this, fermentation cannot be relied upon as a means of detoxifying raw materials contaminated with aflatoxins, although, at the same time, the potential contribution of fermentation to the safety of some products should also not be ignored. Aflatoxin content was found to be decreased during the fermentation of milk (Blanco et al., 1993; Van Egmond et al., 1977; Wiseman and Marth, 1983). Dahi was prepared using Lactobacillus acidophilus (CJ), Streptococcus thermophilus (C2), and Streptococcus lactis plus Streptococcus cremoris (C3) as starter culture. The aflatoxin M1 was determined in milk and experimental dahi samples (Table 13.5).

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Table 13.5 Effect of Fermentation on Aflatoxin M1 Content During Preparation and Storage of Dahi STARTER STORAGE (H)

AF M1

REDUCTION

AF M1

REDUCTION

AFM4

REDUCTION

Milk 0 h (d*N) 24 48 72 96 120 144

3.00 2.10 2.10 2.12 2.15 2.18 1.96 1.89

0.00 30.00 20.87 30.00 29.86 27.33 37.10 34.66

3.00 2.12 1.96 1.72 1.79 1.84 1.67 1.64

0.00 29.33 34.67 42.67 40.33 44.33 81.33 45.33

340 1.27 1.61 1.46 1.96 1.92 1.70 1.56

0% S746 6347 464 – 0% 40.33 49.33

Source: From Wiseman, D.W. and Marth, E.E. 1983. J Food Prot 46: 115–118. With permission.

It can be seen that dahi samples contained lower aflatoxin M than the milk from which it was prepared. The maximum reduction of aflatoxin M1 was obtained using a combination of Streptococcus lactis plus Streptococcus cremoris starter culture (CJ). When the dahi samples were stored at 5 ± 1°C for 144 h, the aflaxtoxin M (AFM) content increased up to 72 h in the case of the dahi prepared by cultures C2 and C3, and upto 96 h in the case of culture C. The decrease in AFM, content in dahi during later stages may be due to degradation of the toxin by microbial enzymes (Bhatnagar et al., 1995; Zaika and Buchanan, 1987), Recently Govartfs et al. (2002) and Deveci (2007) studied the distribution and stability of AFM during the production and storage of yoghurt, and showed that, following fermentation, AFM was significantly lowered (P