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Microbial ecology of cereal fermentations
Trends in Food Science & Technology 16 (2005) 4–11
Review
Microbial ecology of cereal fermentations Walter P. Hammes*, Markus J. Brandt, Kerstin L. Francis, Julia Rosenheim, Michael F.H. Seitter and Stephanie A. Vogelmann
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Institut fu¨r Lebensmitteltechnologie, Fachgebiet Allgemeine Lebensmitteltechnologie und-Mikrobiologie, Universita¨t Hohenheim, Garbenstr. 28, 70599 Stuttgart, Germany (Tel.: C49 711 459 2305; fax: C49 711 459 4199; e-mail: [email protected]) Cereals are globally number one as food crops as well as substrates for fermentation. Well known products are beer, sake, spirits, malt vinegar, and baked goods made from doughs leavened by yeasts or sourdough. Fermentation processes are enabled by technological measures that act on the metabolically resting grains and direct ecological factors controlling the activity of lactic acid bacteria and yeasts. Fermentable substrates originate from endogenous or added hydrolytic enzyme activities. Examples of their management are malting, koji technology, addition of enzymes from external sources and sourdough, which stands on the origin of all fermentation. When sourdough is continuously propagated under the conditions applied in bakery practice, a stable association of only few species of lactic acid bacteria (LAB) and yeasts achieve dominance and ensure a controlled process. The variation of the ecological parameters acting on the microbial association such as the nature of cereal, temperature, size of inoculum, and length of propagation intervals leads in each case to a characteristic species association, thus explaining that altogether 46 LAB species and 13 yeast species have been identified as sourdough specific.
* Corresponding author. 0924-2244/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2004.02.010
Introduction When considering the multitude of foods made from cereals one has to recognise that their greater part has been subjected to fermentation processes taking place at least at one step of their generation. In general, fermentation is a process that proceeds under the influence of activities exerted by enzymes and/or microorganisms. In fact, both activities are important in cereal fermentation (Hammes & Ga¨nzle, 1998), but in the context of this treatise neither plain enzymatic processes are considered, nor those numerous fermentations in which cereals are mixed with substantial amounts of additional substrates, such as legumes or milk (e.g. soy sauce and miso in south-east Asia; idli and dosa in India; kishk in the Middle East (Campbell-Platt, 1987)). Like with any other fermentation process the understanding of the microbial ecology of cereal fermentations needs the knowledge of the fermentation substrates, i.e. the grains or seeds of the various cereal plants, as well as the products obtained thereof. This framework includes the characterisation of the microbial associations and ecological factors, which govern the fermentation process and arise from the nature of the cereal substrate. In cereal fermentations endogenous enzymes, bacteria, yeast and moulds play roles either singularly or in combination, and contribute to the creation of a great variety of products. In Table 1 the phylogenetic position and relationship between cereals are depicted. Included in this group of crops are species that belong to the family of Poaceae (formerly Gramineae). In addition so-named pseudo-cereals are of more or less importance in certain geographic regions. Their composition is similar to that of cereals especially with regard to the starch content. The knowledge of the phylogenetic relationship is not only important for breeders but also for selecting appropriate cereals for fabrication of foods for persons that suffer from food component intolerance, such as allergy or celiac disease. The bioactive compound responsible for the latter disease is exclusively found in the subfamily Poideae. Cereals have been, and still are, the most important food crop. Their cultivation dates back to 7000 B.C. for wheat and barley, 4500 B.C. for rice and maize, 4000 B.C. for millet and sorghum, 400 B.C. for rye, and 100 B.C. for oats. Triticale is the only cereal (since 1930) of our time (McGee, 1984). At present (FAO, 2002) the total global production of food crops amounts to roughly 3.6 billion tonnes, and 60% thereof are cereals. In developed countries up to 70% of
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Table 1. Phylogenetic relationship between cereals and pseudo-cereals
the cereal harvest is used as animal feed. The remaining part plus nearly all cereals in developing countries are used for human nutrition. The greater part thereof is subjected to fermentation and its volume surpasses by far that of all other fermented foods such as those made from milk (cheese and yoghurt), meat (fermented sausages), fish (fish sauce), soy (soy sauce), olives (fermented), or cabbage (sauerkraut). It is assumed that initially porridges were consumed that were made of pounded or ground grains, which were later baked, yielding still unfermented flat bread. These products have long been, and still are, in use. Porridges, more or less liquid, had also a tradition as fermented food in Europe (Fenton, 1974), and numerous examples can still be found outside of the western world (Nout & Rombouts, 2000). It is just a short way to boiling or baking these fermented products and, thus, already at the fourth millennium B.C. leavened bread was produced in Egypt. Remarkably, this first use of cereal fermentation for bread making was tightly related to alcohol production, as leftovers of gruels, porridge, dough, or bread after suspending in water will spontaneously undergo lactic acid and alcoholic fermentation. In such a way the history of beer production runs parallel of that of leavened bread. Aims of cereal fermentations A multitude of fermented products made from cereals have been created in the history of human nutrition. In their production, the fermentation steps aim to achieve the following: – Conditioning for wet milling by steeping of maize (Johnson, 2000) and wild rice (Oelke & Boedicker, 2000). – Affecting sensory properties (aroma, taste, colour, texture).
– Saccharification by use of koji (Yoshizawa, 1977) prior to alcoholic fermentation or producing sweetened rice (Wang & Hesseltine, 1970). – Preservation, which relies mainly on acidification and/or alcohol production (Hammes & Tichaczek, 1994). – Enhancing food safety by inhibition of pathogens, e.g. Burkholderia gladioli that had caused Bongkrek poisoning in products made from pre-soaked corn (Meng et al., 1988), Staphylococcus aureus and Bacillus cereus causing enterotoxicoses. – Improving the nutritive value by removing antinutritive compounds (e.g. phytate, enzyme inhibitors, polyphenols, tannins), and enhancing the bioavailability of components by, e.g. affecting physio-chemical properties of starch and associations of fibre constituents with vitamins, minerals or proteins (Chavan & Kadam, 1989). – Removal of undesired compounds such as mycotoxins (FAO, 1999; Nakazato et al., 1990; Nout, 1994), endogenous toxins, cyanogenic compounds, flatulence producing carbohydrates. – Reducing energy required for cooking. – Achieving the condition of bakeability as it is required for producing leavened rye bread. (Hammes & Ga¨nzle, 1998). In the course of the fermentation process the arising products are characterised by quite different properties and uses. They serve for example to the following: – Leavened baked goods obtained from sourdough and/or yeast leavened dough. – African lactic acid fermented gruels such as ogi obtained from cereals or fufu from cassava, which are further heat processed to obtain porridges and dumplings, respectively.
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– Alcoholic drinks such as beer, sake and spirits. – Acid fermented drinks such as boza (Turkey), Berliner Weibe (Germany), kwass (Russia), or mahewu (South Africa). These processes occur commonly in combination with alcoholic fermentation. – Vinegar, obtained through a secondary aerobic fermentation of, e.g. alcoholic fermented rice (East Asia), or from beer without added hops (Europe). – Colorants such as Angkak obtained by fermenting rice grains with Monascus purpureus.
Basics of cereal fermentations The stored grains of cereals are metabolically in a resting state, which is primarily controlled by low water activity (aw%0.6, 14% moisture). In this state the constituents are not available for microorganisms, and the endogenous enzymes are inactive. Fermentation processes will be enabled under the influence of technological measures including addition of water, comminution by milling, and controlled management of microorganisms and enzyme activities. It is especially the addition of water that affects the ecological factors dramatically. After the water activity increases by water absorption, a reduction of the redox potential takes place by respiration, as well as a drop of pH by respiration and fermentation, whereupon substrates become available from (i) endogenous hydrolytic activities (e.g. amylolysis, proteolysis and lipolysis) and (ii) physiological activities of deliberately added or contaminating microorganisms. These events cause a continuous change of the ecological state in the cereal matrix, which is evident, e.g. in sourdough (Hammes & Ga¨nzle, 1998). Cereal fermentation processes are affected by characteristic variables, the control of which is the basis of all technological measures that are used to obtain the various products at a defined quality. These variables include the following (Hammes & Ga¨nzle, 1998): – Type of cereal determining the fermentable substrates, nutrients, growth factors, minerals, buffering capacity, and efficacy of growth inhibiting principles. – Water content. – Degree and moment of comminution of the grains, i.e. before or after soaking or fermentation. – Duration and temperature of fermentation. – Components added to the fermenting substrate, such as sugar, salt, hops and oxygen. – Source of amylolytic activities that are required to gain fermentable sugars from starch or even other polysaccharides. Among these variables, the type of cereal plays a key role. It affects the amount and quality of carbohydrates as primary fermentation substrates, nitrogen sources, growth factors such as vitamins, minerals, buffering capacity, and the efficacy of growth inhibitors. With regard to fermentable
carbohydrates, microorganisms are initially well supplied. The concentrations of free total sugars in cereal grains range between 0.5 and 3%. Therein sucrose is the major compound (Shelton & Lee, 2000), representing a percentage of O50%. Especially through the activities of b-amylase present in the endosperm, the maltose generation in dough proceeds efficiently after the addition of water to flour. The endogenous hydrolytic activities provide a more or less strong continuous further supply with free sugars. Similarly, peptides and amino acids become available through proteolytic activities. As shown by Prieto, Collar, and Benedito de Barber (1990), the content of total free amino acids increases by 64% in the course of 15 min mixing of an unfermented wheat dough. The mineral content of grains is generally sufficient for microbial growth but differs in the various fractions obtained after milling (Betschart, 1988). It is strongly decreased in the white flour and increased in the germ and bran fractions. For example, manganese as an important growth factor of LAB occurs in whole wheat, white flour, wheat germ, and wheat bran at concentrations of 4.6, 0.7, 13.7, and 6.4–11.9 mg/100 g, respectively. The minerals of the grain are not readily available for microorganisms as they are complexed with phytate. However, at pH values of !5.5 the endogenous grain phytase hydrolyses phytate and minerals are released from the complex. Therefore, a limitation in minerals may occur only at starting a spontaneous fermentation. In processes as exemplified by sourdough propagation, the addition of sourdough to the bread dough lowers the pH and, thus, ensures that phytase activity is sufficient and no need for physiological microbial activity exists (Fretzdorff & Bru¨mmer, 1993; Tangkongchitr, Seib, & Hoseney, 1982). The concentration of phytate in the various cereals ranges between 0.2 and 1.35%, and again is strongly increased in the bran fraction. As phytate develops a high buffering capacity, the degree of flour extraction affects the metabolic activity of LAB in substrates such as doughs. Therein the formation of titrable acids correlates with the phytate content. Inhibitors in cereals exert a selective effect on microbial growth. Known compounds are purothionins and complexing compounds that interfere with the hydrolytic activities of the organisms or the availability of growth factors (Wrigley & Bietz, 1988). Little is known to what extent these factors determine the development of a specific fermentation association, which can be shown to become established, for example, in sourdoughs prepared from different types or fractions of cereals (see below). The addition of water to cereals usually ensures optimum water activity for fermenting microorganisms. The ‘driest’ fermenting substrates are traditional sourdoughs, which are commonly adjusted to dough yields [(mass(water)C mass(flour))/mass(flour)!100] ranging between 160 and 220, corresponding to aw values of 0.965 and 0.980, respectively. Clearly, the lower value is already in
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the stress range for LAB, and optimum values are approached with increasing dough yields. Categories of fermentation technologies The management of hydrolytic activities needed to obtain the fermentable carbohydrates requires specific technologies, which have evolved in history and have different regional importance. Four basic technologies can be identified, namely (i) Malting, i.e. the management of endogenous activities. (ii) Koji technology, i.e. the use of physiological fungal activities. (iii) Use of hydrolytic activities originating from external enzyme sources, e.g. from fungi, bacteria, plants or human saliva. (iv) Dough (batter or gruel) fermentation.
Products from malt In Fig. 1, the scheme of production of beer and whisky from grains subjected to a malting process is shown. Whisky is one example of a great variety of spirits, which are obtained by distillation of a yeast-fermented wort (or mash). In addition to distillation, specific differences between whisky and beer production exist in the process design and in the fermentation events. Whereas in beer brewing wort is boiled and, thus, microorganisms are killed, for whisky production yeasts (Saccharomyces cerevisiae) are added to the unboiled wort. Therefore, the soluble hydrolytic activities are retained and contribute to a maximized ethanol yield. Furthermore, LAB grow spontaneously and acidify the wort during that ‘mixed’ fermentation step (van Beek & Priest, 2002). This practice is said to have
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a beneficial effect on whisky flavour. The LAB species involved are compiled in Fig. 1. A LAB-yeast mixed fermentation takes also place in certain types of acid beers, such as the Belgian Gueuze (Verachtert & Debourg, 1995) or Rodenbach-types or the Berliner Weibbier. Of more general importance is the controlled use of thermophilic lactobacilli to perform a biological acidification, which has technological advantages as it improves the sensory quality of beer with regard to flavour, colour and foam stability. One modern method is the acidification of an aliquot of wort. It is then used to lower the pH of the wort to 5.2 before boiling and/or that of the mash to pH 5.5–5.6. In the packaged beer microbial growth means spoilage and, therefore, contamination has to be prevented. In addition the hop added to mash before boiling exerts an antibacterial effect. Certain resistant organisms however exist and may cause spoilage. Among these LAB are of special importance (Back, 1994). Products from koji The use of the physiological hydrolytic activities of molds to achieve the saccharification of starch has evolved in East Asia and is known as koji technology. From there it spread also to the western world and is used for production of soy sauce and condiments on a cereal basis. The fermentation is a solid phase process and well-known products are sweet rice in China and rice wine. One example is the Japanese sake, whose production process is shown in Fig. 2. This scheme provides also an example for a process involving starch gelatinisation before fermentation. Steamed polished rice is used to prepare the inoculum, called tane koji. In Japanese industrial practice selected strains of Aspergillus
Fig. 1. Use of malt for production of beer and whisky. Modifications specific of the whiskey production are shown in the shadowed area and the process flow is indicated by dotted lines.
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Fig. 2. Application of koji in the production of sake (Yoshizawa & Ishikawa, 1989).
oryzae are propagated, whereas in other countries such as China, various indigenous Mucorales are in use. A seed mash, called moto, is then prepared by inoculation of steamed rice with tane koji. Upon diluting with water, sake yeast (S. cerevisiae) is added and lactic acid, or traditionally LAB, are used to achieve controlled acidification. Katagiri, Kitahara, and Fukami (1934) identified in moto Lactobacillus sakei, Lactobacillus plantarum, and Leuconostoc mesenteroides. In this environment the yeast is especially protected and is able to form ethanol up to a concentration of 20%. Moto is used to inoculate the main mash, called moromi, which ferments over three succeeding steps by feeding steamed rice as new substrate. The finally resulting sake may spoil and turns then in so called hiochi. From this spoiled product Kitahara, Kaneko, and Goto (1957) isolated Leuconostoc mesenteroides, Lactobacillus fermentum, Lactobacillus homohiochii, and, most abundant, Lactobacillus heterohiochii, which is identical with Lactobacillus fructivorans.
Products from use of hydrolytic enzymes originating from external sources The use of hydrolytic enzymes from external sources is common practice in industrial ethanol production (Senn & Pieper, 1996), and has also a tradition in brewing chicha, which is a type of South American beer produced by making use of diastase originating from human saliva (FAO, 1999). Products from fermented dough The concerted activity of hydrolytic activities of the grain and microorganisms (LAB and yeasts) is the origin of all cereal fermentations and is best represented by the traditional sourdough fermentation. A basic scheme of dough fermentation for production of baked goods is depicted in Fig. 3. The process requires comminuted grains or fractions thereof that have not been heat treated before and still possess the hydrolytic activities. By addition of water, contaminating microorganisms will become metabolically active, multiply and, at extended incubation, the most competitive organisms will inevitably dominate
Fig. 3. Basic scheme of dough fermentation for the production of baked goods.
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the process. These will always be LAB and yeasts. This type of processing is still the basic practice for preparing so-named predoughs. The only modification rests in addition of baker’s yeast when preparing the dough. The more often the fermented product is used as an inoculum for a succeeding fermentation run the greater becomes the adaptation of the microbial association to the process. Indeed, in continuously propagated doughs a few strains may prevail for years (Bo¨cker, Vogel, & Hammes, 1990). The continuous propagation results in so-called type I doughs, which fulfil all requirements needed to obtain baked goods such as bread when added in the appropriate ratio to the bread dough. They recieve the acidity from the metabolism of LAB, and LAB and yeasts together provide the gas formation capacity, development of flavour, and principles acting on texture of the crumb and the nutritional value. A disadvantage of this traditional sourdough propagation process is, that they are time and labour consuming (e.g. in rye bread 24 h, up to three propagation steps), and therefore, more convenient one-stage processes have been established. These ensure that all ingredients can be mixed together, and within a short time the baking condition of the dough is achieved. In these so-called type II doughs the leavening capacity needs, however, to be achieved by addition of baker’s yeast. The ecological conditions of the production of inocula differ greatly from those described for type I doughs and, therefore, usually species of different taxons are employed that are adapted to high acid tolerance and often affect the sensory profile of the baked goods recognisable for the gourmet. Microbial associations in sourdough The LAB species characteristic for the various types of sourdough are subject of the papers of Ehrmann and Vogel and De Vuyst and Neysens of this issue. We counted from published work a number of 46 LAB species, the majority of which belongs to the genus Lactobacillus (30 species). When all types of cereal fermentations are considered more than half of the 80 recognised Lactobacillus species (Hammes & Hertel, 2004) occur in association with cereal fermentation. Furthermore it is of interest that 15 Lactobacillus species known to occur in sourdough are also known as inhabitants of the intestines of humans and animals. For example L. acidophilus was found in humans, swine, poultry, cattle and horse and L. reuteri in the same organisms, as well as in mouse and hamster. Remarkably, no other habitat is known for Lactobacillus sanfranciscensis as the most characteristic sourdough LAB. As LAB in type I doughs are obligately heterofermentative they contribute decisively to gas production (Hammes & Ga¨nzle, 1998). Numerous yeasts have been isolated from sourdoughs (see Table 2) but only part of them can be considered to play a substantial role in fermentation processes. For example, non-fermenting species may be just ubiquitous contaminants although they may affect the flavour of the baked
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Table 2. Yeasts isolated from and adapted to doughs Species Candida boidinii Candida glabrata Candida humilis Candida parapsilosis Candida stellata Debaryomyces hansenii Dekkera bruxellensis Galactomyces geotrichum Issatchenkia orientalis Kluyveromyces marxianus Pichia anomala Pichia fermentans Pichia ohmeri Pichia subpelliculosa Pichia minuta var. minuta Saccharomyces bayanus Saccharomyces cerevisiae Saccharomyces exiguus Saccharomyces kluyveri Saccharomyces servazzi Saccharomycopsis fibuligera Saturnispora saitoi Torulaspora delbrueckii
Torulaspora pretoriensis
Synonyms Torulopsis glabrata Candida milleri Torulopsis stellata Torulopsis candida, Candida famata Brettanomyces custersii Geotrichum candidum Candida krusei Candida pelliculosa, Hansenula anomala Candida lambica Hansenula subpelliculosa Hansenula minuta Saccharomyces inusitatus Saccharomyces fructuum Torulopsis holmii, Candida holmii, Saccharomyces minor
Endomyces fibuliger Pichia saitoi Torulopsis colliculosa, Candida colliculosa, Saccharomyces rosei, Saccharomyces delbrueckii, Saccharomyces inconspicuous Saccharomyces pretoriensis
The fermenting sourdough specific species are printed in bold.
goods when occurring at high numbers. In Table 2 well adapted and frequently isolated sourdough specific species are printed in bold and synonyms are indicated. Yeasts in doughs may originate from the flour or the environment in the bakery. Truly adapted species are characteristic for type I doughs, and Candida humilis (originally described by Sugihara, Kline, and Miller (1971) as Candida holmii) is of superior importance therein. The predominant occurrence of other species depends strongly on the condition of dough propagation. It was shown by Meroth, Hammes, and Hertel (2003) and Meroth, Walter, Hertel, Brandt, and Hammes (2003) that, under otherwise identical conditions, at 25 8C and propagation cycles of 12 h (Batch A), C. humilis (together with L. sanfranciscensis and L. mindensis) becomes a dominant yeast in sourdough, whereas at 30 8C and 24 h cycles (Batch B) S. cerevisiae (together with Lactobacillus crispatus, Lactobacillus frumenti and Lactobcillus pontis) achieved dominance. Furthermore, at 40 8C (Batch C), a shift to the more thermophilic species Candida glabrata and Issatchenkia orientalis (but also S. cerevisiae) was observed. In addition to the effects on the ecological factors controlled by the process conditions, the nature of the cereals has a profound effect on the establishment of a characteristic fermentation association. We have propagated
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(unpublished results) according to the conditions described above (Batch B) doughs from seven cereals and three pseudocereals. It was observed that in each dough an association of LAB and yeasts developed, which was characteristic for a specific type of substrate with regard to the numbers of species as well as their composition. Conclusion Up to now we know little about the ecological factors and their interplay, which specifically affect the microbial associations in the fermentation of the various cereals. It can be expected that better-controlled processes and greater sensory variety or standardization of baked goods can be achieved when this knowledge will become available through research directed toward this field of microbial ecology. Acknowledgements We thank Drs M. Kicherer and C. Hertel for helpful discussions. References Back, W. (1994). Farbatlas und Handbuch der Getra¨nkebiologie. Teil 1: Kultivierung/Methoden Brauerei, Winzerei. Nu¨rnberg: Hans Carl Getra¨nkefachverlag. Betschart, A. A. (1988). Nutritional quality of wheat and wheat foods. V. Minerals. In Y. Pomeranz,3rd ed. Wheat chemistry and technology (Vol. II) (pp. 112–118). St. Paul, MN: American Association of Cereal Chemists, Inc. Bo¨cker, G., Vogel, R. F., & Hammes, W. P. (1990). Lactobacillus sanfrancisco als stabiles Element in einem Reinzucht-SauerteigPra¨parat. Getreide Mehl und Brot, 9, 269–274. Campbell-Platt, G. (1987). Fermented foods of the world—a dictionary and guide. Kent, England: Butterworths. Chavan, J. K., & Kadam, S. S. (1989). Nutritional improvement of cereals by fermentation. CRC Critical Reviews in Food Science and Nutrition, 28, 349–400. De Vuyst, L., Neysens, P. The sourdough microflora: Biodiversity and metabolic interactions. Trends in Food Science and Technology, this issue, doi: 10.1016/j.tifs.2004.02.012. Ehrman, M.A., Vogel, R.F. Molecular taxonomy and genetics of sourdough lactic acid bacteria. Trends in Food Science and Technology, this issue, doi: 10.1016/j.tifs.2004.06.004. FAO (2002): http://apps.fao.org FAO (1999). Fermented cereals. A global perspective (FAO agricultural services bulletin No. 138). http://www.fao.org/ docrep/x2184E/x2184E00.htm. Fenton, A. (1974). Sowens in Scotland. Folk-life, a Journal of Ethnological Studies, 12, 41–47. Fretzdorff, B., & Bru¨mmer, H. J. (1993). Reduction of phytic acid during breadmaking of whole meal breads. Cereal Chemistry, 69, 266–270. Hammes, W. P., & Ga¨nzle, M. G. (1998). Sourdough breads and related products. In B. J. B. Wood,2nd ed. Microbiology of fermented foods (Vol. 1) (pp. 199–216). London: Blackie Academic and Professional. Hammes, W. P., & Hertel, C. (2004). The genera Lactobacillus and CarnobacteriumDworkin, M. et al. The prokaryotes: An evolving electronic resource for the microbiological community,3rd ed. Springer, New Yorkhttp://link.springer-ny.com/link/service/ books/10125/ release 3.15.
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Review
Microbial ecology of cereal fermentations Walter P. Hammes*, Markus J. Brandt, Kerstin L. Francis, Julia Rosenheim, Michael F.H. Seitter and Stephanie A. Vogelmann
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Institut fu¨r Lebensmitteltechnologie, Fachgebiet Allgemeine Lebensmitteltechnologie und-Mikrobiologie, Universita¨t Hohenheim, Garbenstr. 28, 70599 Stuttgart, Germany (Tel.: C49 711 459 2305; fax: C49 711 459 4199; e-mail: [email protected]) Cereals are globally number one as food crops as well as substrates for fermentation. Well known products are beer, sake, spirits, malt vinegar, and baked goods made from doughs leavened by yeasts or sourdough. Fermentation processes are enabled by technological measures that act on the metabolically resting grains and direct ecological factors controlling the activity of lactic acid bacteria and yeasts. Fermentable substrates originate from endogenous or added hydrolytic enzyme activities. Examples of their management are malting, koji technology, addition of enzymes from external sources and sourdough, which stands on the origin of all fermentation. When sourdough is continuously propagated under the conditions applied in bakery practice, a stable association of only few species of lactic acid bacteria (LAB) and yeasts achieve dominance and ensure a controlled process. The variation of the ecological parameters acting on the microbial association such as the nature of cereal, temperature, size of inoculum, and length of propagation intervals leads in each case to a characteristic species association, thus explaining that altogether 46 LAB species and 13 yeast species have been identified as sourdough specific.
* Corresponding author. 0924-2244/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2004.02.010
Introduction When considering the multitude of foods made from cereals one has to recognise that their greater part has been subjected to fermentation processes taking place at least at one step of their generation. In general, fermentation is a process that proceeds under the influence of activities exerted by enzymes and/or microorganisms. In fact, both activities are important in cereal fermentation (Hammes & Ga¨nzle, 1998), but in the context of this treatise neither plain enzymatic processes are considered, nor those numerous fermentations in which cereals are mixed with substantial amounts of additional substrates, such as legumes or milk (e.g. soy sauce and miso in south-east Asia; idli and dosa in India; kishk in the Middle East (Campbell-Platt, 1987)). Like with any other fermentation process the understanding of the microbial ecology of cereal fermentations needs the knowledge of the fermentation substrates, i.e. the grains or seeds of the various cereal plants, as well as the products obtained thereof. This framework includes the characterisation of the microbial associations and ecological factors, which govern the fermentation process and arise from the nature of the cereal substrate. In cereal fermentations endogenous enzymes, bacteria, yeast and moulds play roles either singularly or in combination, and contribute to the creation of a great variety of products. In Table 1 the phylogenetic position and relationship between cereals are depicted. Included in this group of crops are species that belong to the family of Poaceae (formerly Gramineae). In addition so-named pseudo-cereals are of more or less importance in certain geographic regions. Their composition is similar to that of cereals especially with regard to the starch content. The knowledge of the phylogenetic relationship is not only important for breeders but also for selecting appropriate cereals for fabrication of foods for persons that suffer from food component intolerance, such as allergy or celiac disease. The bioactive compound responsible for the latter disease is exclusively found in the subfamily Poideae. Cereals have been, and still are, the most important food crop. Their cultivation dates back to 7000 B.C. for wheat and barley, 4500 B.C. for rice and maize, 4000 B.C. for millet and sorghum, 400 B.C. for rye, and 100 B.C. for oats. Triticale is the only cereal (since 1930) of our time (McGee, 1984). At present (FAO, 2002) the total global production of food crops amounts to roughly 3.6 billion tonnes, and 60% thereof are cereals. In developed countries up to 70% of
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Table 1. Phylogenetic relationship between cereals and pseudo-cereals
the cereal harvest is used as animal feed. The remaining part plus nearly all cereals in developing countries are used for human nutrition. The greater part thereof is subjected to fermentation and its volume surpasses by far that of all other fermented foods such as those made from milk (cheese and yoghurt), meat (fermented sausages), fish (fish sauce), soy (soy sauce), olives (fermented), or cabbage (sauerkraut). It is assumed that initially porridges were consumed that were made of pounded or ground grains, which were later baked, yielding still unfermented flat bread. These products have long been, and still are, in use. Porridges, more or less liquid, had also a tradition as fermented food in Europe (Fenton, 1974), and numerous examples can still be found outside of the western world (Nout & Rombouts, 2000). It is just a short way to boiling or baking these fermented products and, thus, already at the fourth millennium B.C. leavened bread was produced in Egypt. Remarkably, this first use of cereal fermentation for bread making was tightly related to alcohol production, as leftovers of gruels, porridge, dough, or bread after suspending in water will spontaneously undergo lactic acid and alcoholic fermentation. In such a way the history of beer production runs parallel of that of leavened bread. Aims of cereal fermentations A multitude of fermented products made from cereals have been created in the history of human nutrition. In their production, the fermentation steps aim to achieve the following: – Conditioning for wet milling by steeping of maize (Johnson, 2000) and wild rice (Oelke & Boedicker, 2000). – Affecting sensory properties (aroma, taste, colour, texture).
– Saccharification by use of koji (Yoshizawa, 1977) prior to alcoholic fermentation or producing sweetened rice (Wang & Hesseltine, 1970). – Preservation, which relies mainly on acidification and/or alcohol production (Hammes & Tichaczek, 1994). – Enhancing food safety by inhibition of pathogens, e.g. Burkholderia gladioli that had caused Bongkrek poisoning in products made from pre-soaked corn (Meng et al., 1988), Staphylococcus aureus and Bacillus cereus causing enterotoxicoses. – Improving the nutritive value by removing antinutritive compounds (e.g. phytate, enzyme inhibitors, polyphenols, tannins), and enhancing the bioavailability of components by, e.g. affecting physio-chemical properties of starch and associations of fibre constituents with vitamins, minerals or proteins (Chavan & Kadam, 1989). – Removal of undesired compounds such as mycotoxins (FAO, 1999; Nakazato et al., 1990; Nout, 1994), endogenous toxins, cyanogenic compounds, flatulence producing carbohydrates. – Reducing energy required for cooking. – Achieving the condition of bakeability as it is required for producing leavened rye bread. (Hammes & Ga¨nzle, 1998). In the course of the fermentation process the arising products are characterised by quite different properties and uses. They serve for example to the following: – Leavened baked goods obtained from sourdough and/or yeast leavened dough. – African lactic acid fermented gruels such as ogi obtained from cereals or fufu from cassava, which are further heat processed to obtain porridges and dumplings, respectively.
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– Alcoholic drinks such as beer, sake and spirits. – Acid fermented drinks such as boza (Turkey), Berliner Weibe (Germany), kwass (Russia), or mahewu (South Africa). These processes occur commonly in combination with alcoholic fermentation. – Vinegar, obtained through a secondary aerobic fermentation of, e.g. alcoholic fermented rice (East Asia), or from beer without added hops (Europe). – Colorants such as Angkak obtained by fermenting rice grains with Monascus purpureus.
Basics of cereal fermentations The stored grains of cereals are metabolically in a resting state, which is primarily controlled by low water activity (aw%0.6, 14% moisture). In this state the constituents are not available for microorganisms, and the endogenous enzymes are inactive. Fermentation processes will be enabled under the influence of technological measures including addition of water, comminution by milling, and controlled management of microorganisms and enzyme activities. It is especially the addition of water that affects the ecological factors dramatically. After the water activity increases by water absorption, a reduction of the redox potential takes place by respiration, as well as a drop of pH by respiration and fermentation, whereupon substrates become available from (i) endogenous hydrolytic activities (e.g. amylolysis, proteolysis and lipolysis) and (ii) physiological activities of deliberately added or contaminating microorganisms. These events cause a continuous change of the ecological state in the cereal matrix, which is evident, e.g. in sourdough (Hammes & Ga¨nzle, 1998). Cereal fermentation processes are affected by characteristic variables, the control of which is the basis of all technological measures that are used to obtain the various products at a defined quality. These variables include the following (Hammes & Ga¨nzle, 1998): – Type of cereal determining the fermentable substrates, nutrients, growth factors, minerals, buffering capacity, and efficacy of growth inhibiting principles. – Water content. – Degree and moment of comminution of the grains, i.e. before or after soaking or fermentation. – Duration and temperature of fermentation. – Components added to the fermenting substrate, such as sugar, salt, hops and oxygen. – Source of amylolytic activities that are required to gain fermentable sugars from starch or even other polysaccharides. Among these variables, the type of cereal plays a key role. It affects the amount and quality of carbohydrates as primary fermentation substrates, nitrogen sources, growth factors such as vitamins, minerals, buffering capacity, and the efficacy of growth inhibitors. With regard to fermentable
carbohydrates, microorganisms are initially well supplied. The concentrations of free total sugars in cereal grains range between 0.5 and 3%. Therein sucrose is the major compound (Shelton & Lee, 2000), representing a percentage of O50%. Especially through the activities of b-amylase present in the endosperm, the maltose generation in dough proceeds efficiently after the addition of water to flour. The endogenous hydrolytic activities provide a more or less strong continuous further supply with free sugars. Similarly, peptides and amino acids become available through proteolytic activities. As shown by Prieto, Collar, and Benedito de Barber (1990), the content of total free amino acids increases by 64% in the course of 15 min mixing of an unfermented wheat dough. The mineral content of grains is generally sufficient for microbial growth but differs in the various fractions obtained after milling (Betschart, 1988). It is strongly decreased in the white flour and increased in the germ and bran fractions. For example, manganese as an important growth factor of LAB occurs in whole wheat, white flour, wheat germ, and wheat bran at concentrations of 4.6, 0.7, 13.7, and 6.4–11.9 mg/100 g, respectively. The minerals of the grain are not readily available for microorganisms as they are complexed with phytate. However, at pH values of !5.5 the endogenous grain phytase hydrolyses phytate and minerals are released from the complex. Therefore, a limitation in minerals may occur only at starting a spontaneous fermentation. In processes as exemplified by sourdough propagation, the addition of sourdough to the bread dough lowers the pH and, thus, ensures that phytase activity is sufficient and no need for physiological microbial activity exists (Fretzdorff & Bru¨mmer, 1993; Tangkongchitr, Seib, & Hoseney, 1982). The concentration of phytate in the various cereals ranges between 0.2 and 1.35%, and again is strongly increased in the bran fraction. As phytate develops a high buffering capacity, the degree of flour extraction affects the metabolic activity of LAB in substrates such as doughs. Therein the formation of titrable acids correlates with the phytate content. Inhibitors in cereals exert a selective effect on microbial growth. Known compounds are purothionins and complexing compounds that interfere with the hydrolytic activities of the organisms or the availability of growth factors (Wrigley & Bietz, 1988). Little is known to what extent these factors determine the development of a specific fermentation association, which can be shown to become established, for example, in sourdoughs prepared from different types or fractions of cereals (see below). The addition of water to cereals usually ensures optimum water activity for fermenting microorganisms. The ‘driest’ fermenting substrates are traditional sourdoughs, which are commonly adjusted to dough yields [(mass(water)C mass(flour))/mass(flour)!100] ranging between 160 and 220, corresponding to aw values of 0.965 and 0.980, respectively. Clearly, the lower value is already in
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the stress range for LAB, and optimum values are approached with increasing dough yields. Categories of fermentation technologies The management of hydrolytic activities needed to obtain the fermentable carbohydrates requires specific technologies, which have evolved in history and have different regional importance. Four basic technologies can be identified, namely (i) Malting, i.e. the management of endogenous activities. (ii) Koji technology, i.e. the use of physiological fungal activities. (iii) Use of hydrolytic activities originating from external enzyme sources, e.g. from fungi, bacteria, plants or human saliva. (iv) Dough (batter or gruel) fermentation.
Products from malt In Fig. 1, the scheme of production of beer and whisky from grains subjected to a malting process is shown. Whisky is one example of a great variety of spirits, which are obtained by distillation of a yeast-fermented wort (or mash). In addition to distillation, specific differences between whisky and beer production exist in the process design and in the fermentation events. Whereas in beer brewing wort is boiled and, thus, microorganisms are killed, for whisky production yeasts (Saccharomyces cerevisiae) are added to the unboiled wort. Therefore, the soluble hydrolytic activities are retained and contribute to a maximized ethanol yield. Furthermore, LAB grow spontaneously and acidify the wort during that ‘mixed’ fermentation step (van Beek & Priest, 2002). This practice is said to have
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a beneficial effect on whisky flavour. The LAB species involved are compiled in Fig. 1. A LAB-yeast mixed fermentation takes also place in certain types of acid beers, such as the Belgian Gueuze (Verachtert & Debourg, 1995) or Rodenbach-types or the Berliner Weibbier. Of more general importance is the controlled use of thermophilic lactobacilli to perform a biological acidification, which has technological advantages as it improves the sensory quality of beer with regard to flavour, colour and foam stability. One modern method is the acidification of an aliquot of wort. It is then used to lower the pH of the wort to 5.2 before boiling and/or that of the mash to pH 5.5–5.6. In the packaged beer microbial growth means spoilage and, therefore, contamination has to be prevented. In addition the hop added to mash before boiling exerts an antibacterial effect. Certain resistant organisms however exist and may cause spoilage. Among these LAB are of special importance (Back, 1994). Products from koji The use of the physiological hydrolytic activities of molds to achieve the saccharification of starch has evolved in East Asia and is known as koji technology. From there it spread also to the western world and is used for production of soy sauce and condiments on a cereal basis. The fermentation is a solid phase process and well-known products are sweet rice in China and rice wine. One example is the Japanese sake, whose production process is shown in Fig. 2. This scheme provides also an example for a process involving starch gelatinisation before fermentation. Steamed polished rice is used to prepare the inoculum, called tane koji. In Japanese industrial practice selected strains of Aspergillus
Fig. 1. Use of malt for production of beer and whisky. Modifications specific of the whiskey production are shown in the shadowed area and the process flow is indicated by dotted lines.
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Fig. 2. Application of koji in the production of sake (Yoshizawa & Ishikawa, 1989).
oryzae are propagated, whereas in other countries such as China, various indigenous Mucorales are in use. A seed mash, called moto, is then prepared by inoculation of steamed rice with tane koji. Upon diluting with water, sake yeast (S. cerevisiae) is added and lactic acid, or traditionally LAB, are used to achieve controlled acidification. Katagiri, Kitahara, and Fukami (1934) identified in moto Lactobacillus sakei, Lactobacillus plantarum, and Leuconostoc mesenteroides. In this environment the yeast is especially protected and is able to form ethanol up to a concentration of 20%. Moto is used to inoculate the main mash, called moromi, which ferments over three succeeding steps by feeding steamed rice as new substrate. The finally resulting sake may spoil and turns then in so called hiochi. From this spoiled product Kitahara, Kaneko, and Goto (1957) isolated Leuconostoc mesenteroides, Lactobacillus fermentum, Lactobacillus homohiochii, and, most abundant, Lactobacillus heterohiochii, which is identical with Lactobacillus fructivorans.
Products from use of hydrolytic enzymes originating from external sources The use of hydrolytic enzymes from external sources is common practice in industrial ethanol production (Senn & Pieper, 1996), and has also a tradition in brewing chicha, which is a type of South American beer produced by making use of diastase originating from human saliva (FAO, 1999). Products from fermented dough The concerted activity of hydrolytic activities of the grain and microorganisms (LAB and yeasts) is the origin of all cereal fermentations and is best represented by the traditional sourdough fermentation. A basic scheme of dough fermentation for production of baked goods is depicted in Fig. 3. The process requires comminuted grains or fractions thereof that have not been heat treated before and still possess the hydrolytic activities. By addition of water, contaminating microorganisms will become metabolically active, multiply and, at extended incubation, the most competitive organisms will inevitably dominate
Fig. 3. Basic scheme of dough fermentation for the production of baked goods.
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the process. These will always be LAB and yeasts. This type of processing is still the basic practice for preparing so-named predoughs. The only modification rests in addition of baker’s yeast when preparing the dough. The more often the fermented product is used as an inoculum for a succeeding fermentation run the greater becomes the adaptation of the microbial association to the process. Indeed, in continuously propagated doughs a few strains may prevail for years (Bo¨cker, Vogel, & Hammes, 1990). The continuous propagation results in so-called type I doughs, which fulfil all requirements needed to obtain baked goods such as bread when added in the appropriate ratio to the bread dough. They recieve the acidity from the metabolism of LAB, and LAB and yeasts together provide the gas formation capacity, development of flavour, and principles acting on texture of the crumb and the nutritional value. A disadvantage of this traditional sourdough propagation process is, that they are time and labour consuming (e.g. in rye bread 24 h, up to three propagation steps), and therefore, more convenient one-stage processes have been established. These ensure that all ingredients can be mixed together, and within a short time the baking condition of the dough is achieved. In these so-called type II doughs the leavening capacity needs, however, to be achieved by addition of baker’s yeast. The ecological conditions of the production of inocula differ greatly from those described for type I doughs and, therefore, usually species of different taxons are employed that are adapted to high acid tolerance and often affect the sensory profile of the baked goods recognisable for the gourmet. Microbial associations in sourdough The LAB species characteristic for the various types of sourdough are subject of the papers of Ehrmann and Vogel and De Vuyst and Neysens of this issue. We counted from published work a number of 46 LAB species, the majority of which belongs to the genus Lactobacillus (30 species). When all types of cereal fermentations are considered more than half of the 80 recognised Lactobacillus species (Hammes & Hertel, 2004) occur in association with cereal fermentation. Furthermore it is of interest that 15 Lactobacillus species known to occur in sourdough are also known as inhabitants of the intestines of humans and animals. For example L. acidophilus was found in humans, swine, poultry, cattle and horse and L. reuteri in the same organisms, as well as in mouse and hamster. Remarkably, no other habitat is known for Lactobacillus sanfranciscensis as the most characteristic sourdough LAB. As LAB in type I doughs are obligately heterofermentative they contribute decisively to gas production (Hammes & Ga¨nzle, 1998). Numerous yeasts have been isolated from sourdoughs (see Table 2) but only part of them can be considered to play a substantial role in fermentation processes. For example, non-fermenting species may be just ubiquitous contaminants although they may affect the flavour of the baked
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Table 2. Yeasts isolated from and adapted to doughs Species Candida boidinii Candida glabrata Candida humilis Candida parapsilosis Candida stellata Debaryomyces hansenii Dekkera bruxellensis Galactomyces geotrichum Issatchenkia orientalis Kluyveromyces marxianus Pichia anomala Pichia fermentans Pichia ohmeri Pichia subpelliculosa Pichia minuta var. minuta Saccharomyces bayanus Saccharomyces cerevisiae Saccharomyces exiguus Saccharomyces kluyveri Saccharomyces servazzi Saccharomycopsis fibuligera Saturnispora saitoi Torulaspora delbrueckii
Torulaspora pretoriensis
Synonyms Torulopsis glabrata Candida milleri Torulopsis stellata Torulopsis candida, Candida famata Brettanomyces custersii Geotrichum candidum Candida krusei Candida pelliculosa, Hansenula anomala Candida lambica Hansenula subpelliculosa Hansenula minuta Saccharomyces inusitatus Saccharomyces fructuum Torulopsis holmii, Candida holmii, Saccharomyces minor
Endomyces fibuliger Pichia saitoi Torulopsis colliculosa, Candida colliculosa, Saccharomyces rosei, Saccharomyces delbrueckii, Saccharomyces inconspicuous Saccharomyces pretoriensis
The fermenting sourdough specific species are printed in bold.
goods when occurring at high numbers. In Table 2 well adapted and frequently isolated sourdough specific species are printed in bold and synonyms are indicated. Yeasts in doughs may originate from the flour or the environment in the bakery. Truly adapted species are characteristic for type I doughs, and Candida humilis (originally described by Sugihara, Kline, and Miller (1971) as Candida holmii) is of superior importance therein. The predominant occurrence of other species depends strongly on the condition of dough propagation. It was shown by Meroth, Hammes, and Hertel (2003) and Meroth, Walter, Hertel, Brandt, and Hammes (2003) that, under otherwise identical conditions, at 25 8C and propagation cycles of 12 h (Batch A), C. humilis (together with L. sanfranciscensis and L. mindensis) becomes a dominant yeast in sourdough, whereas at 30 8C and 24 h cycles (Batch B) S. cerevisiae (together with Lactobacillus crispatus, Lactobacillus frumenti and Lactobcillus pontis) achieved dominance. Furthermore, at 40 8C (Batch C), a shift to the more thermophilic species Candida glabrata and Issatchenkia orientalis (but also S. cerevisiae) was observed. In addition to the effects on the ecological factors controlled by the process conditions, the nature of the cereals has a profound effect on the establishment of a characteristic fermentation association. We have propagated
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(unpublished results) according to the conditions described above (Batch B) doughs from seven cereals and three pseudocereals. It was observed that in each dough an association of LAB and yeasts developed, which was characteristic for a specific type of substrate with regard to the numbers of species as well as their composition. Conclusion Up to now we know little about the ecological factors and their interplay, which specifically affect the microbial associations in the fermentation of the various cereals. It can be expected that better-controlled processes and greater sensory variety or standardization of baked goods can be achieved when this knowledge will become available through research directed toward this field of microbial ecology. Acknowledgements We thank Drs M. Kicherer and C. Hertel for helpful discussions. References Back, W. (1994). Farbatlas und Handbuch der Getra¨nkebiologie. Teil 1: Kultivierung/Methoden Brauerei, Winzerei. Nu¨rnberg: Hans Carl Getra¨nkefachverlag. Betschart, A. A. (1988). Nutritional quality of wheat and wheat foods. V. Minerals. In Y. Pomeranz,3rd ed. Wheat chemistry and technology (Vol. II) (pp. 112–118). St. Paul, MN: American Association of Cereal Chemists, Inc. Bo¨cker, G., Vogel, R. F., & Hammes, W. P. (1990). Lactobacillus sanfrancisco als stabiles Element in einem Reinzucht-SauerteigPra¨parat. Getreide Mehl und Brot, 9, 269–274. Campbell-Platt, G. (1987). Fermented foods of the world—a dictionary and guide. Kent, England: Butterworths. Chavan, J. K., & Kadam, S. S. (1989). Nutritional improvement of cereals by fermentation. CRC Critical Reviews in Food Science and Nutrition, 28, 349–400. De Vuyst, L., Neysens, P. The sourdough microflora: Biodiversity and metabolic interactions. Trends in Food Science and Technology, this issue, doi: 10.1016/j.tifs.2004.02.012. Ehrman, M.A., Vogel, R.F. Molecular taxonomy and genetics of sourdough lactic acid bacteria. Trends in Food Science and Technology, this issue, doi: 10.1016/j.tifs.2004.06.004. FAO (2002): http://apps.fao.org FAO (1999). Fermented cereals. A global perspective (FAO agricultural services bulletin No. 138). http://www.fao.org/ docrep/x2184E/x2184E00.htm. Fenton, A. (1974). Sowens in Scotland. Folk-life, a Journal of Ethnological Studies, 12, 41–47. Fretzdorff, B., & Bru¨mmer, H. J. (1993). Reduction of phytic acid during breadmaking of whole meal breads. Cereal Chemistry, 69, 266–270. Hammes, W. P., & Ga¨nzle, M. G. (1998). Sourdough breads and related products. In B. J. B. Wood,2nd ed. Microbiology of fermented foods (Vol. 1) (pp. 199–216). London: Blackie Academic and Professional. Hammes, W. P., & Hertel, C. (2004). The genera Lactobacillus and CarnobacteriumDworkin, M. et al. The prokaryotes: An evolving electronic resource for the microbiological community,3rd ed. Springer, New Yorkhttp://link.springer-ny.com/link/service/ books/10125/ release 3.15.
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