P1: SFK/UKS BLBS102-c31 P2: SFK BLBS102-Simpson March 21, 2012 14:0 Trim: 276mm X 219mm Printer Name: Yet to Come 31 Bakery and Cereal Products Defined cultures have also been marketed; however, the suitability of Type I and Type II cultures appears to outcompete the alternatives offered In addition to baker’s yeast, the collection of defined cultures comprise at least pure cultures of Lb brevis (heterofermentative) and Lb delbrueckii and Lb plantarum (homofermentative) The homofermentative cultures produce mainly lactic acid under anaerobic conditions, whereas the heterofermentative cultures will also produce acetic acid or ethanol and carbon dioxide By controlling, if possible, the contributions from these different cultures, the relative amounts of acetic and lactic acids may be regulated This important relationship: Lactic acid (Mole) Acetic acid (Mole) is called the fermentation quotient (FQ) Relatively mild acidity will have an FQ about 4–9, whereas a more strongly flavored rye bread, as produced in Germany, requires a much lower FQ, for example, 1.5–4.0 (Spicher 1983, Gobbetti and Corsetti 1997, Hammes and Găanzle 1998, Martinez-Anaya 2003, Stolz 2003) Sourdough Processes Several more or less traditional sourdough processes are practiced on a large scale in present-day bakery industry (Fig 31.5) One line comprises processes designed for baking with rye flour They may be the Type I processes mentioned above The Berliner short-sour process, the Detmolder Figure 31.5 The sourdough process 603 one-stage process, and the Lăonner one-stage process are typical for central and northern Europe In every case, the process is initiated by a starter culture, 2–20% (often, 9–10%), in a rye flour–water (close to 50:50 w/w) mixture that is incubated for 3–24 hours at 20–35◦ C, depending on the process When the resulting sourdough is ready, bread making starts with an “inoculation” of about 30% sourdough together with rye flour, wheat flour, baker’s yeast, and salt The final rye:wheat ratio is regularly 70:30 w/w Dough yield is adjusted to satisfy handling (e.g., pumping) and microbial nutrition requirements Another line of sourdough processes comprises those for baking with wheat only The San Francisco sourdough process is often mentioned in this connection; however, Italian wheat sourdough products, for example, Panettone, Colomba, and Pandoro, are also very important (Cauvain 1998, Gobbetti and Corsetti 1997, Spicher 1983, Wood 2000) The starter for San Francisco sourdough is ideally rebuilt every hours to maintain maximum activity However, a mature sponge may be kept refrigerated for days with acceptable performance For rebuilding the starter, sponge (40%) is mixed with high-gluten wheat flour (40%) and water (about 20%) to ferment at bakery temperature Final bread making requires a dough consisting of the ripe sourdough (9.2%), regular wheat flour (45.7%), water (44.2%), and salt (0.9%) Proofing for hours follows, during which the pH decreases from about 5.3 to about 3.9 A sourdough starter culture for San Francisco French bread production commonly contains Lb sanfranciscensis and Candida holmii P1: SFK/UKS BLBS102-c31 P2: SFK BLBS102-Simpson March 21, 2012 14:0 Trim: 276mm X 219mm 604 Printer Name: Yet to Come Part 5: Fruits, Vegetables, and Cereals Selection and Biochemistry of Microorganisms in Sourdough Sourdough breads based on rye or wheat flour or their mixtures enjoys a remarkable standing in many societies, either as established, traditional products or as “innovative” developments for more natural products and a wider choice of flavors Well-functioning and popular sourdough starters that have been maintained by simple rebuilding for decades, and the reestablishment of the “same” stable starter over and over again from a constant quality flour, are both expressions of stable ecological conditions The simplicity of the procedures may be somewhat deceiving with respect to the actual complexity of these biological systems In the following sections, metabolic events and biochemical aspects of sourdough fermentation will be discussed Attention will be drawn to some of the more clear points about the metabolic events and other biochemical facts The near future should bring us closer to a comprehensive understanding thus appears to be a near ideal partner for Lb sanfranciscensis (Gobbetti and Corsetti 1997, Gobbetti 1998, Wood 2000, Hammes and Găanzle 1998) Wheat and rye flour contain mainly maltose as a readily available carbohydrate, although rye flour has greater amylase activity and therefore has a greater potential for release of maltose Early work in the United States on Lb sanfranciscensis indicated that this organism would only ferment maltose (Kline and Sugihara 1971) However, strains isolated in Europe appeared more diversified, and some of them would ferment up to eight different sugars (Hammes and Găanzle 1998) Utilization of maltose by Lb sanfranciscensis, Lb pontis, Lb reuteri, and Lb fermentum through phosphorolytic cleavage with maltose phosphorylase is energetically very favorable (Stolz et al 1993) and shows increased cell yield and excretion of glucose when maltose is available In these conditions the cells have very low levels of hexokinase Co-metabolism Carbohydrate Metabolism The development of a mixture (1:1) of rye flour or even wheat flour with water incubated some hours at 25–30◦ C will almost inevitably lead to a microbiological population consisting of lactic acid bacteria (LAB) and yeasts It may need rebuilding several times in order to stabilize it, but from then on the composition of the microflora may be constant for years, provided the composition of the flour and the conditions for growth are not changed much Representative LAB and yeasts have been presented in Table 31.2 These microorganisms have certain characteristics in common First, the selected LAB are very efficient maltose fermenters, a prime reason why they competed so well in the first place Several lactobacilli in sourdoughs, e.g Lb sanfranciscensis, Lb pontis, Lb reuteri, and Lb fermentum, harbor a key enzyme, maltose phosphorylase, which cleaves maltose (the phosphorolytic reaction) to glucose-1-phosphate and glucose Glucose-1-phosphate is metabolized heterofermentatively via the phosphogluconate pathway, while glucose is excreted into the growth medium Glucose repression has not been observed with these lactobacilli Most of the yeast species identified in sourdoughs are, per se, maltose negative, and will thus prefer to take up glucose when it is available Other microorganisms may experience glucose repression of the maltose enzymes, to the benefit of the sourdough lactobacilli Among the yeasts, S cerevisiae, which is maltose positive and transports maltose and hexoses very efficiently, cannot take up maltose due to glucose repression and will, as a consequence, be defeated from the sourdough flora S cerevisiae as baker’s yeast is, however, used at the bread-making stage, but as an addition in the recipe Additional yeast cells may also be necessary for fast and efficient CO2 production, because the yeasts are relatively sensitive to acids, particularly to acetic acid, which is excreted by the heterofermentative lactobacilli that often dominate the LAB flora of the sourdough Candida milleri (syn S exiguus, Torulopsis holmii) is common in sourdoughs for San Francisco French bread This yeast tolerates the acetic acid from heterolactic fermentation and thrives on glucose and sucrose in preference to maltose; it Lactobacilli in sourdough production are not only specialized for maltose fermentation they also exploit co-fermentations for optimized energy yield (Gobbetti and Corsetti 1997, Hammes and Găanzle 1998, Stolz et al 1995, Romano et al 1987) Lactobacillus sanfranciscensis, Lb pontis and Lb fermentum all have mannitol dehydrogenase Thus, fructose may be used as an electron acceptor for the reoxidation of NADH in maltose or glucose metabolism, and then acetylphosphate may react on acetate kinase to yield ATP and acetate (Axelsson 1993) The lactobacilli gain energetically and more acetic acid may contribute to the desirable taste and flavor of bread In practical terms addition of fructose is used to increase acetate in the products, that is, lower FQ (Spicher 1983) Comparable regulation of acetate production may be achieved by providing citrate, malate or oxygen as electron acceptors, resulting in products like succinate, glycerol and acetate (Gobbetti and Corsetti 1996, Condon 1987, Stolz et al 1993) Proteolysis and Amino Compounds In a sourdough, the flour contributes considerable amounts of amino acids and peptides; however, in order to satisfy nutritional requirements of growing LAB and provide sufficient amino compounds, precursors, for flavor development, some proteolytic action is necessary The LAB have been suspected as the main contributors of proteinase and peptidase activities for release of amino acids in sourdoughs (Spicher and Nierle 1984, Spicher and Nierle 1988, Gobbetti et al 1996), although the our enzymes may also have considerable input (Hammes and Găanzle 1998) In addition, lysis of microbial cells, particularly yeast cells, add to the pool of amino acids; a stimulant peptide containing aspartic acid, cysteine, glutamic acid, glycine, and lysine that appears in the autolytic process of C milleri has also been identified (Berg et al 1981) Lactobacillus sanfranciscensis has been found to have a regime of intracellular peptidases, endopeptidase, and proteinase, as well as a dipeptidase and proteinase in the cell envelope (Gobbetti et al 1996) Limited autolysis of P1: SFK/UKS BLBS102-c31 P2: SFK BLBS102-Simpson March 21, 2012 14:0 Trim: 276mm X 219mm Printer Name: Yet to Come 31 Bakery and Cereal Products lactobacillus populations in sourdoughs may add to the repertoire of enzymes that will release amino acids from flour proteins, including those from proline-rich gluten in wheat Some of the enzymes have been purified for further characterization (Gobbetti et al 1996), and they express interesting activity levels at sourdough pH and temperatures The addition of exogenous microbial glucose oxidase, lipase, endoxylanase, α-amylase, or protease in the production of sourdough with 11 different LAB cultures showed positive effects on acidification rate and level for only three cultures, one Leuconostoc citreum, one Lactococcus lactis subsp lactis and one Lb hilgardii Lactobacillus hilgardii with lipase, endoxylanase or α-amylase showed increased production of acetic acid Lactobacillus hilgardii interacted with the different enzymes for higher stability and softening of doughs (Di Cagno et al 2003) Recent work with Lb sanfranciscensis, Lb brevis, and Lb alimentarius in model sourdough fermentations showed, by using two-dimensional electrophoresis, that 37–42 polypeptides had been hydrolyzed The polypeptides varied over wide ranges of pIs and molecular masses, and they originated from albumin, globulin, and gliadin, but not from glutenin Free amino acid concentrations increased, in particular those of proline and glutamic and aspartic acid Proteolysis by the lactobacilli had a positive effect on the softening of the dough A toxic peptide for celiac patients, A-gliadin fragment 31–43, was degraded by enzymes from lactobacilli The agglutination of human myelogenous leukemia–derived cells (K562) by toxic peptic-tryptic digest of gliadins was abolished by enzymes from lactobacilli (Di Cagno et al 2002) Volatile Compounds and Carbon Dioxide Both yeasts and LAB contribute to CO2 production in sourdough products, but the importance of the two varies In bread production with only the (natural) sourdough microflora, the input from LAB may even be decisive for leavening because the counts and kinds of yeast may not be optimal for gas production Relatively low temperature (e.g., 25◦ C) and low dough yield (e.g., 135) would select for LAB activities and less yeast metabolism More complete volatile profiles were obtained at higher temperatures (e.g., 30◦ C) and with a more fluid dough Of course, increasing the leavening time may give substantially richer volatile profiles (Gobbetti et al 1995) If baker’s yeast, S cerevisiae, is added to optimize and speed up the production process, the contribution from yeasts will dominate (Gobbetti 1998, Hammes and Găanzle 1998) Bread made with chemical acidification without fermentation starter failed in sensory analysis This indicates that fermentation with yeasts and LAB is important for good flavor, although high quality raw materials and proofing and baking are also decisive factors Flavor compounds distinguishing the different metabolic contributions in sourdough are as follows (Gobbetti 1998): r Yeast fermentation (alcoholic): 2-methyl-1-propanol, 2,3methyl-1-butanol r LAB homofermentative: diacetyl, other carbonyls r LAB heterofermentative: ethyl acetate, other alcohols and carbonyls 605 Antimicrobial Compounds from Sourdough LAB The primary antimicrobial compounds produced by sourdough LAB are lactic and acetic acid, diacetyl, hydrogen peroxide, carbon dioxide, and ethanol, and among these, the two organic acids continue to be the most important contributions for beneficial effects in fermentations Researchers in the field, of course, also consider and test possibilities that LAB may produce bacteriocins and other antimicrobials Thus antifungal compounds from Lb plantarum 21B have been identified, for example, phenyl lactic acid and 4-hydroxyphenyl lactic acid (Lavermicocca et al 2000) Caproic acid from Lb sanfranciscensis also has some antifungal activity (Corsetti et al 1998) A real broad-spectrum antimicrobial from Lb reuteri is reuterin (β-hydroxypropionic aldehyde), which comes as a monomer and a cyclic dimer (El-Ziney et al 2000) Reutericyclin, which was isolated from Lb reuteri LTH2584 after the screening of 65 lactobacilli, is a tetramic acid derivative Reutericyclin inhibited Gram-positive bacteria (e.g., Lactobacillus spp., Bacillus subtilis, B cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua), and it was bactericidal towards B subtilis, S aureus, and Lb sanfranciscensis The ability to produce reutericyclin was stable in sourdough fermentations over a period of several years Reutericyclin produced in sourdough was also active in the dough (Găanzle et al 2000; Hăoltzel et al 2000; Găanzle and Vogel 2003) A few bacteriocins or bacteriocin-like compounds have also been identified, isolated, and characterized (Messens and De Vuyst 2002) Bavaricin A from Lb sakei MI401 was selected by screening 335 LAB strains, including 58 positive strains (Larsen et al 1993) Bavaricin A (and Bavaricin MN from Lb sakei MN) have the N-terminal consensus motif of bacteriocin class IIA in common, comprise 41 and 42 amino acids, respectively, and have interesting sequence homologies and similar hydrophobic regions Bavaricin A inhibits Listeria strains and some other Grampositive bacteria but not Bacillus or Staphylococcus (Larsen et al 1993, Kaiser and Montville 1996) Plantaricin ST31 is produced by Lb plantarum ST; it contains 20 amino acids, and the activity spectrum includes several Gram-positive bacteria but not Listeria (Todorov et al 1999) A bacteriocin-like compound, BLIS C57 from Lb sanfranciscensis C57, was detected after screening 232 Lactobacillus isolates, including 52 strains expressing antimicrobial activity BLIS C57 inhibits Gram-positive bacteria including bacilli and Listeria strains (Corsetti et al 1996) TRADITIONAL FERMENTED CEREAL PRODUCTS Only two cereals, wheat and rye, contain gluten and are thereby suitable for the production of leavened bread, but many other food cereals are grown in the world On a global basis, a great proportion of cereals are consumed as spontaneously fermented products, in particular in Africa, Asia, and Latin America Most fermented cereals are dominated by lactic acid bacteria (LAB), and the microflora associated with the grains, flour, or any other ingredient, together with contamination from water, P1: SFK/UKS BLBS102-c31 P2: SFK BLBS102-Simpson 606 March 21, 2012 14:0 Trim: 276mm X 219mm Printer Name: Yet to Come Part 5: Fruits, Vegetables, and Cereals food-making equipment, and the producers themselves, represent the initial fermentation flora Malted flour is also an important source of microorganisms The changes that take place during the fermentation are due to both the metabolism of the microorganisms present and the activity of enzymes in the cereal, and these are in turn affected by the great variety of technologies that are used The technology may be simple, involving little more than a mixing of flour with water and allowing it to ferment, or it may be extremely complex and involve many steps with obscure roles Indigenous fermented foods are usually based upon raw materials that have a sustainable production in their country of origin and are therefore attracting increasing interest from researchers—both within pure and applied food science and also in anthropology These ancient technologies often have deep roots in the culture of a country, and there is increasing awareness of the importance of preserving these traditional foods Many products are not yet described in the literature, and knowledge of them is in danger of disappearing It is therefore necessary to document the technologies used and to identify the fermenting organisms and the metabolic changes that are essential for the characteristics of the product It is, however, often difficult to describe the sensory attributes of a product that is inherently variable In Africa, as much as 77% of the total caloric consumption is provided by cereals, of which rice, maize, sorghum, and millet are most important Cereals are also significant sources of protein Most of the cereal foods consumed in Africa are traditional fermented products and are very important both as weaning foods and as staple foods and beverages for adults In Asia, many products are based on rice, and maize is most widely utilized in Latin America (FAO 1999) Indigenous fermented cereals can be classified according to raw material, type of fermentation, technology used, product usage, or geographical location They can range from quite solid products such as baked flat breads to sour, sometimes mildly alcoholic, refreshing beverages Many factors have an influence on the characteristics of an indigenous product (Fig 31.6) The choice of raw material may be primarily influenced by price and availability rather than by preference For instance Togwa, a Tanzanian fermented beverage, may be made from maize in the inland areas of Morogoro and Iringa, but from sorghum in the coastal areas of Dar es Salaam and Zanzibar (Mugula 2001) Similarly, the Ethiopian product borde may be made from several different grains according to availability—sorghum, maize, millet, barley and also the Ethiopian cereal tef (Abegaz et al 2002) The use of different grains obviously affects the sensory characteristics of a product, and yet it may have the same name throughout the country Some fermented cereal products also contain other ingredients Idli is a leavened steamed cake made primarily from rice to which black gram dahl is added This not only improves the nutritional quality, but in addition, the black gram imparts a viscosity, apparently specific for this legume, which may aid air entrapment during fermentation and thereby lighten the texture of the product (Soni and Sandu 1990) However, on a broader basis, the addition of legumes such as soybean flour to fermented cereals Figure 31.6 Important factors determining the characteristics of spontaneously fermented cereal products has been suggested as an economically feasible way to generally improve the nutritional quality of cereal foods Some fermented cereal products are made using unmalted grain, with no extra addition of amylase, but they tend to either be very thick or of low nutritional density Malted flour is added to many indigenous fermented cereals, a traditional technology that has far-reaching effects on several product characteristics The addition of malt provides amylases (in particular, α-amylase) that hydrolyze the starch, sweeten the product, and also cause a considerable decrease in viscosity of the product after heat treatment The malting process, the germination of grain following steeping in water, is associated with colossal microbiological proliferation, and the organisms that develop during malting are a source of fermenting organisms Many Asian products, for example koji, a Japanese fermented cereal or soybean product, are first inoculated with a fungus, as a source of amylase, in order to liberate fermentable sugars from the cereal starch (Lotong 1998) Many fermented cereals are multipurpose A single product may be prepared in varying thicknesses and used as a fermented gruel for both adults and children, or it may be watered down and used as a fermented thirst-quenching beverage As Wood (1994) remarked, the latter type of product makes a meaningful contribution to nutrition; the potential of their replacement by cola-type beverages would result in a serious negative impact on the nutrition of people in developing countries The use of fermented cereals as weaning foods in developing countries raises several important issues Unfermented gruels deteriorate very rapidly in unhygienic conditions, especially if refrigeration is not available They then represent a significant source of foodborne infections that annually claim the lives of millions of young children (Adams 1998) Fermented malted cereal gruels have been shown on the whole to contain low numbers of pathogenic organisms since these are inhibited and killed by the low pH that rapidly develops in the product P1: SFK/UKS BLBS102-c31 P2: SFK BLBS102-Simpson March 21, 2012 14:0 Trim: 276mm X 219mm Printer Name: Yet to Come 31 Bakery and Cereal Products Fermented cereals are therefore usually regarded as safer than their unfermented counterparts (Nout and Motarjemi 1997) A weaning food made from unmalted cereals may be a cause of malnutrition because its thick viscosity limits the nutritional intake of a small child Addition of malted flour decreases the viscosity so that more food can be ingested If the fermentation flora includes yeasts in addition to LAB, a measurable reduction of carbohydrate will occur due to the production of CO2 and other volatile compounds (Muyanja 2001) Analysis of fermented cereal products therefore shows that the protein:carbohydrate ratio is improved during the fermentation, and this obviously has nutritional benefits Milling of cereals into flour is usually done prior to fermentation, but in some products, for example, borde (Ethiopia), wet milling is used This technique can be used when mechanical grain mills are not available and if the product is required to be smooth and without bits of suspended bran The starch is also liberated from the grain more thoroughly when slurried with water and sieved than if it has been previously dry milled (Abegaz 2002) A heat treatment step is found at some point in the production technology of most fermented cereal products and may involve boiling, steaming, or roasting The type of heating employed is likely to have an effect on the flavor of the product, certainly if the temperature attained is sufficient to promote Maillard reactions The heat also gelatinizes the starch, making it more susceptible to amylolytic enzymes, thus providing greater amounts of fermentable carbohydrates However, at the same time, most of the natural contaminating (and potentially fermenting) flora and cereal enzymes are destroyed Such products are also prone to contamination after the heat treatment step, and are thereby potentially unsafe should pathogenic organisms grow during the subsequent fermentation The traditional solution to this is to use “backslopping,” the addition of some of a previous batch of the product, and/or the addition of malted flour Regular backslopping results in a selection of acid-tolerant organisms and functions as an empirical starter culture Fermentation usually takes place at ambient temperatures, and this may cause seasonal variations in products due to selection of different microorganisms at different temperatures The duration of fermentation is largely a matter of personal choice, based on expected sensory attributes Heat treatment after fermentation makes for a safer product, but it has the disadvantage of change of taste or loss of volatile flavor and aroma compounds The Microflora of Spontaneously Fermented Cereals Spontaneously acid-fermented cereal products may contain a variety of microorganisms, but the flora in the final product is generally dominated by acid-tolerant LAB Yeasts are also invariably present in large numbers when the fermentation is prolonged A typical fermented cereal product contains approximately 109 and 107 cfu/g of product, of LAB and yeasts, respectively However, since yeast cells are considerably larger than bacteria cells, their metabolic contribution to product characteristics is likely to be just as important as that of the LAB 607 The buffer capacity of cereal slurries is low, and the pH therefore drops quickly as acid is produced Pathogenic organisms are inhibited by a fast acid production, so the addition of starter cultures, either as a pure culture or by “backslopping” promotes acid production and contributes to the safety of the fermented product (Nout et al 1989) The potential and the need for upgrading traditional fermentation technologies have initiated considerable research (Holzapfel 2002) In some recent studies of spontaneously fermented cereals, the LAB and yeasts have been isolated and identified as a first stage towards developing starter cultures for smallscale production of traditional fermented cereals Muyanja et al (2003) recorded that bushera, a traditional Ugandan fermented sorghum beverage that contains high numbers of LAB, was usually consumed by children after one day of fermentation as “sweet bushera.” After 2–4 days, the product became sour and alcoholic and was consumed by adults However, the sweet bushera showed very high counts of coliforms and had a reputation for causing diarrhea (Muyanja 2001) Clearly, the development of defined starter cultures would improve the safety of this and similar products Some recent examples of studies on the microbial flora of spontaneously fermented cereals are shown in Table 31.3 For each product, several different types of organisms have been isolated In other words, a specific product is not produced from fermentation by a specific organism or organisms Lb plantarum seems to be the most commonly isolated Lactobacillus species in fermented cereals In addition, heterofermentative LAB such as leuconostocs, Lb brevis, and Lb fermentum frequently occur Yeasts are always present in spontaneously fermented products, but few studies have characterized the predominating species However, Jespersen (2003) reported that S cerevisciae is the predominant yeast in many African fermented foods and beverages All spontaneously fermented products contain, or have contained, many different types of microorganisms These have grown in the product and will have metabolized some of the cereal components, thereby making a contribution (positive or negative) with their metabolites to the overall sensory characteristics of that product However, studies on spontaneously fermented products have focused on LAB and yeasts since these organisms are often associated with other, better known, fermented products and have a history of safe use in food Stanton (1998) proposed that the nature of the substrate (raw material) and the technology used to produce fermented foods are the predominating factors that determine the development of microorganisms and, thereby, the properties of a product Desirable Properties of the Fermenting Microflora The most important property of a starter culture for a fermented cereal is the ability to quickly produce copious amounts of lactic acid in order to achieve a rapid decline in pH and retard the growth of pathogens and other undesirable organisms Some workers (Sanni et al 2002) have sought amylolytic LAB strains, as this could remove the need for using the highly contaminated ... in sourdoughs (Spicher and Nierle 1 984 , Spicher and Nierle 1 988 , Gobbetti et al 1996), although the flour enzymes may also have considerable input (Hammes and Găanzle 19 98) In addition, lysis of... comprehensive understanding thus appears to be a near ideal partner for Lb sanfranciscensis (Gobbetti and Corsetti 1997, Gobbetti 19 98, Wood 2000, Hammes and Găanzle 19 98) Wheat and rye our contain... for optimized energy yield (Gobbetti and Corsetti 1997, Hammes and Găanzle 19 98, Stolz et al 1995, Romano et al 1 987 ) Lactobacillus sanfranciscensis, Lb pontis and Lb fermentum all have mannitol