ABSTRACT
The nutrient composition of yeast biofortified akamu, a cereal-based fermented food was determined using two different concentrations of inocula preparations at 5% and 10% glucose concentrations. While keeping the peptone and yeast extract constant at 2% and 1% respectively. These media were used to inoculate the akamu at different volumes of 2.5 ml, 5
ml and 7.5 ml and later incubated at room temperature (25oC) for 72 hours after which they
were analysed. The yeast used in this research was Pichia kudriavzevii. The result of the analysis showed that the pH of yeast biofortified akamu (YBA) reduced as the volume of the inocula increased for both concentrations of glucose while the titrable acidity increased in YBA as the volume of the inocula increased for both concentrations of glucose. The protein and reducing sugar contents of YBA increased significantly (p < 0.05) as the volume of the inocula increased for the two glucose concentrations. The carbohydrate and fibre contents of YBA was not significantly (p > 0.05) reduced while the fat and ash contents of YBA was not significantly altered. The increase and decrease in amino acids content of YBA at 5% and
10% glucose concentrations were not volume dependent. Relative to the unfortified akamu, the YBA at 10% glucose concentration yielded higher amounts of amino acids than YBA at
5% glucose concentration. Alanine, cysteine, phenylalanine, tyrosine and tryptophan contents increased as the volume of inocula increased for both media inoculated in YBA. In the case of proline, there was an inverse relation with inoculum volume. The vitamin contents of yeast biofortified akamu at 5% and 10% glucose concentrations showed no significant change in the amount in the YBA compared to the ordinary akamu except the amount of vitamin A that was significantly reduced (P < 0.05) at both glucose concentrations. In the mineral contents of YBA inoculated at 5% and 10% glucose concentrations, the calcium, copper, manganese and zinc contents increased at both concentrations though, the increase was not volume dependent. Magnesium and iron contents increased at both concentrations except in YBA inoculated with 2.5 ml at 5% glucose and sodium and potassium in YBA inoculated with 2.5 ml and 5 ml at 5% glucose concentration. There was a significant reduction (P < 0.05) in phosphorous content of YBA at all the three volumes at 5% glucose and also for 2.5 ml and 5 ml inocula at 10% glucose concentration. It is concluded from this study that yeast (Pichia kudriavzevii) can serve as a vehicle for biofortification and that YBA inoculated at 10% glucose concentration yielded more nutrients compared to that inoculated at 5% glucose concentration.
CHAPTER ONE INTRODUCTION
Good nutrition is essential for survival, health and reproduction (Frederick, 2010). The conventional strategies to combat nutrient deficiencies include dietary supplementations and food fortification programmes. This leads to introducing biofortified staple crops with increased nutrient content and can therefore have a very big impact, as the strategy relies on improving an already existing food supply. Biofortification represents one promising strategy to enhance the availability of vitamins and minerals for people whose diets are dominated by micronutrient-poor staple food crops. Biofortification help to alleviate certain forms of undernutrition. Biofortification techniques require the use of microorganisms such as micro- algae, bacteria and yeast to biofortify food (Ogbonna, 2013). In this research, yeast was used to biofortify akamu a cereal based fermented food. This is because of the enormous benefits the biofortified product possesses (Ogbonna, 2013).
Yeasts are used in the preparation of human foods and beverages, where besides having technological functions, confer different beneficial effects on human health and well-being. Among these, the most well known is the probiotic effect, which has been proven for Saccharomyces cerevisiae var. boulardii. This is the only yeast produced and used as a pharmaceutical product offering numerous valuable effects such as prevention and treatment of intestinal diseases and immunomodulatory effects. Since Saccharomyces cerevisiae var. boulardii is recognised as a member of the species Saccharomyces cerevisiae, it is most likely that other strains within Saccharomyces cerevisiae might also show probiotics properties. The other benefits of yeast include biodegradation of phytate, folate biofortification and detoxification of mycotoxins (Moslehi-Jenabian et al., 2010).
Yeasts have advantages over other micro organisms for biofortification because of their large size, making them easier to harvest, low nucleic acid content, high lysine content and ability to grow at lower hydrogen ion concentrations (pH). However, the most important advantage is their traditional use in fermentation, which makes them acceptable to the general public (Ogbonna, 2013).
Fermentation process serves as a means of providing a source of nourishment for large rural populations; fermentation enhances the nutrient content of foods (Egwim et al., 2013) through the synthesis of proteins, vitamins, and essential amino acids (Casanova et al., 2015; Ogbonnaya and Bernice, 2012). The traditional cereal based food that are consumed in West
Africa processed by the fermentation of maize, sorghum and millet are particularly important as weaning foods for infants and as dietary staples for adults. In terms of texture, the fermented cereal foods are either liquid (porridge or gruel), stiff gels (solid) or dry (fried or steam-cooked granulated products). The fermentation process is often carried out on small or household scales and are characterised by the use of simple, non-sterile equipment, random or natural innocula, unregulated conditions, sensory fluctuations, poor durability and unattractive packaging of the processed products (Annan et al., 2003).
There are four main types of fermentation processes: alcoholic, lactic acid, acetic acid and alkali fermentation. Alcoholic fermentation results in the production of ethanol (e.g. wines and beers) and yeasts are the predominant organisms. Lactic acid fermentation (e.g. fermented milks and cereals) is mainly carried out by lactic acid bacteria. Acetic acid produced by Acetobacter species convert alcohol to acetic acid in the presence of excess of Oxygen. Alkali fermentation often takes place during the fermentation of fish and seeds, popularly used as condiment (Blandino et al., 2003).
Research findings have brought to light the invaluable attributes of fermented food products. It is now known that the process of fermentation leads to the production of valuable products including flavour and aroma compounds; biomass proteins/amino acids; minerals; lipids; carbohydrates; vitamins and other products of the respiratory/ biosynthetic process such as lactic acid, ethanol, acetyaldehydes, pyruvic acid, some of which help in altering the pH of food to levels that do not favour growth of pathogenic microorganisms (Kalui et al., 2009). This in turn enhances food safety and increases food shelf life hence aiding in food preservation (Yasmine, 2002). The changes associated with the fermentation process are as a result of the action of enzymes produced by microorganisms.
1.1 Biofortification
Biofortification is the development of nutrient-dense staple crops using the best conventional breeding practices and modern biotechnology, without sacrificing agronomic performance and important consumer preferred traits. Fortification is done either to improve the taste and acceptability enjoyed by commercial (baby weaning) products like ceralac or for nutritional purpose. Local fortification for taste includes use of sugar, milk, chocolate, ‘kulikuli’ (ground nut cake), fried beans (i.e.beans cake), fruits and seeds/berry to enhance the sour taste. On a
laboratory scale, vanilla (Sanni et al., 2001) and other artificial flavours are used (Casanova
et al., 2015).
Maize-based ogi has been reported to have better protein score than sorghum based ogi when fortified with cowpea or soy bean (Nnam, 2000). Fortification has been reported to increase the content of protein from 1.4 % to 13 % in germinated and fortified preparation and increase lysine to more than 50% when cowpea is added (Egounlety et al., 2002). It is commonly recommended to add palm oil to the weaning gruel to improve the vitamin A content (Afolayan et al., 2009; Casanova et al., 2015).
Biofortification represents one promising strategy to enhance the availability of vitamins and minerals for people whose diets are dominated by micronutrients-poor staple food crops (Global Panel, 2015). Micronutrient biofortification is the most active area of research as a means of alleviating micronutrient malnutrition, particularly in the developing world (Frederick, 2010). In a research carried out by Hjortmo et al, (2008), on the impact of growth rate and chemical environments on folate levels in Saccharsomyces cerevisiae, a cereal based fermented porridge called togwa, it was found that folates increased about 23 folds during fermentations (in the cereal-based porridge togwa) compared to the unfermented raw material. The microbiology of many West African fermented cereal products including the maize products mawè, ogi and koko sour water (Hounhouigan et al., 1993; Oguntoyinbo et al., 2011; Adimpong et al., 2012), the sorghum products gowè, kunun-zaki and ogi-baba (Odunfa and Adeyele, 1985; Viera-Dalodé et al., 2007; Oguntoyinbo and Narbad, 2012) and the millet products arraw, ben-saalga and akamu (Totté et al., 2003; Songré-Ouattara et al.,
2008; Nwachukwu et al., 2010; Turpin et al., 2011) have been examined. The analysis of cereal foods in West Africa revealed that the natural fermentation process involves mixed cultures of lactic acid bacteria, yeasts, fungi and Bacillus species. Lactic acid bacteria species of the genera Lactobacillus, Leuconostoc, Weissella, Streptococcus and Pediococcus, as well as yeast species of the genera Saccharomyces, Candida and Kluyeromyces, have been identified as the dominant microorganisms in the fermentation process of cereal-based foods (Soro-Yao et al., 2014). The isolated lactic acid bacteria species have been used, often in combination with yeast species, as starter cultures in controlled laboratory trials. Overall, higher lactic acid production, rapid acidification, superior product shelf life, improved organoleptic properties and a greater degree of control over the fermentation process have been achieved with the use of starter cultures compared to traditional processes (i.e., without the use a selected starter culture). Although, many laboratory trials using lactic acid bacteria
starter cultures have been undertaken, reports on fermented cereal-based foods produced with the identified and selected strains are extremely scarce. However, a collaborative project between Belgian Cooperation and the Institute of Food Technology (ITA) in Dakar (Senegal) carried out to improve millet fermentation (Soro-Yao et al.,2014), led to the development of an improved processing of steam-cooked granulated products by small-scale urban processing units (Lardinois et al., 2003; Totté et al., 2003). The use of dried starter cultures could be of great benefit to the small-scale processing units in West Africa, considering the economic costs of and technical expertise required for production. A large majority of fermented cereal-based foods consumed in most West African countries are poor and disadvantaged, and as such, price, rather than food safety and quality, is the primary concern when purchasing food. In addition to economic considerations, the availability and the maintenance of the starter cultures could be suggested as another major factor; large quantities of starter cultures in active and pure forms are essential to the success of the fermentation stage of product manufacture. This can be achieved through the careful propagation of the inocula. Propagation of starter cultures is time consuming, laborious, requires skilled personnel and is prone to contamination, and necessitates significant investments in equipment (e.g. fermentors). Furthermore, contamination could occur during the process of culture production, which may result in poor growth of the LAB and defective products. Finally, dried starter cultures are easier to use and maintain, as they remain stable for up to two years (Brandt, 2014).
According to the research carried out by Jorge et al. (2009), zinc absorption was greater from biofortified wheat, than from typical wheat with lower zinc concentration, from the same quantities of each type of wheat flour consumed. While substantial amounts of zinc are lost with 80% extraction, absorption is still significantly higher than from the control. Because of the greater reduction in phytate with increased extraction at 80%, the quantity of zinc absorbed is similar to that from 95% wheat. Along with the model based predictions, this suggests that phytate intake must be considered in setting zinc target levels. There was a substantial increase in intake of bioavailable zinc from zinc‐biofortified wheat especially
from the higher extraction flour (95% versus 40% for the lower extraction flour). Under the scenario presented 300g wheat flour could provide about two thirds the physiological zinc requirements of adult women (Jorge et al., 2009). It has been shown that the control wheat used for the experiment had zinc concentrations at the lower end of the range. In addition, zinc content in the grain was affected by ecological condition.
1.2 Under Nutrition
Under nutrition is a common form of malnutrition caused by an inadequate diet, or diseases leading to an excessive loss of nutrients or an inability to absorb nutrients leading to wasting (thinness), stunting (shortness), and susceptibility to diseases. There are many complex underlying causes of under nutrition. They include lack of access, both physical and economic, to an adequate diet, often compounded by an associated higher burden of disease and lower ability to cope with disease. The long-term causes include poverty, government instability, lack of employment, lack of access to education and health services, and lack of national food security (Frederick, 2010).
Under nutrition is in various forms. It could be micronutrient malnutrition which is a deficiency in one or more vitamins and/or minerals. These micronutrients are required in the diet in small amounts but deficiencies could lead to serious health consequences. It could be the form of protein-energy malnutrition caused by an inadequate dietary intake of protein leading to stunting, wasting and kwashiorkor (a childhood condition in which lack of dietary protein is a significant factor) (Allen et al., 2006).
Micronutrient malnutrition is wide spread and affects all age groups, but young children and women of reproductive age tend to be among those most at risk of developing micronutrient deficiencies. Micronutrient malnutrition has adverse effects on human health, not all of which are clinically evident. Even moderate levels of deficiency can have serious detrimental effects on human function (Allen et al., 2006).
Protein-energy malnutrition is a major problem that frequently occurs during the important transitional phase of weaning in infants. It affects the physical and mental growth of many infants in developing countries. Protein energy malnutrition is a syndrome characterized by progressive onset and a series of symptoms and signs that encompass a continuum from clinically undetected manifestations to the full clinical picture of marasmus or kwashiorkor (Laminu et al.,2014).
1.3 Micronutrients
Micronutrients are those nutrients which the body need in minute quantity for growth and development and for the maintenance of healthy life. They include vitamins and minerals. Plants provide the bulk of human dietary calories and contain many essential nutrients
(Frederick, 2010). This provides a large number of potential targets for improving nutritional value.
1.3.1 Vitamins
Vitamins are organic nutrients which the body need in order to maintain functions such as immunity, metabolism, growth and physical well being. There is very little the body can do without a vitamin being needed. Vitamins are grouped in to two: fat soluble and water soluble. Fat soluble vitamins (include those vitamins) that are stored in fat cells when excess is present. They have toxicity levels associated with them as they are not got rid of by the body. They also need fat in order to be absorbed. Examples of fat soluble vitamins are: vitamin A, D, E and K.
Water soluble vitamins are not stored in the body. The body takes what it needs from food and then excretes what is not needed as waste. They also are easily destroyed by cooking and care should be taken when cooking vegetables. The examples of water soluble vitamins are the thiamin (vitamin B1), riboflavin (vitamin B2), niacin (Vitamin B3), pantothenic acid (vitamin B5), pyridoxine (Vitamin B6), folate or folic acid (vitamin B9 ), cobalamin (vitamin B12), biotin (vitamin B7) and vitamin C (Johnson, 2014; Adrienne, 2015).
1.3.1.1 Vitamin A
Vitamin A is an essential nutrient that is required in small amounts by humans for the normal functioning of the visual system, the maintenance of cell function for growth, epithelial cellular integrity, immune function and reproduction (Allen, 2001). Dietary requirements for vitamin A are normally provided as a mixture of pre-formed vitamin A (retinol), which is present in animal source foods, and provitamin A carotenoids, which are derived from foods of vegetable origin and which have to be converted into retinol by tissues such as the intestinal mucosa and the liver in order to be utilized by cells (Allen et al., 2006).
Aside from the clinical ocular signs; night blindness and xerophthalmia, symptoms of vitamin A deficiency are largely non-specific. Nevertheless, accumulated evidence suggests that vitamin A deficiency is an important determinant of child survival and safe motherhood. The non- specificity of symptoms, however, means that, in the absence of biochemical measures of vitamin A status, it is difficult to attribute non-ocular symptoms to vitamin A deficiency and it also complicates the definition of vitamin A deficiency. With these considerations in
mind, WHO has defined vitamin A deficiency as tissue concentrations of vitamin A low enough to have adverse health consequences, even if there is no evidence of clinical xerophthalmia. In more recent years, the term “vitamin A deficiency disorder” has been coined to reflect the diversity of adverse outcomes caused by vitamin A deficiency. The best sources of vitamin A are animal source foods, in particular, liver, eggs and dairy products, which contain vitamin A in the form of retinol, a form that can be readily used by the body (Allen et al., 2006).
1.3.1.2 The B Group Vitamins
The B group vitamins are a class of water-soluble vitamins that play important roles in cell metabolism. Though these vitamins share similar names, research shows that they are chemically distinct vitamins that often coexist in the same foods. In general, supplements containing all eight are referred to as a vitamin B complex. These water soluble vitamins are readily destroyed during cooking in water and by heat (even though niacin is stable to heat). More significantly, the milling and germinating of cereal grains removes almost all of the thiamine (B1), riboflavin (B2) and niacin (B3), which is the reason why restoration of these particular nutrients to wheat and corn flour has been widely practiced for the last 60 years (Allen et al., 2006).
1.3.1.2.1 Vitamin B1
Thiamine (vitamin B1) is a cofactor for several key enzymes involved in carbohydrate metabolism and is also directly involved in neural function. It is likely that thiamine deficiency, in its subclinical form, is a significant public health problem in many parts of the world (Allen et al., 2006). Severe deficiency causes beriberi, a disease that was once common among populations with a high carbohydrate intake, especially in the form of white rice.
1.3.1.2.2 Vitamin B2
Riboflavin (vitamin B2) is a precursor of various nucleotides, most notably flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which act as coenzymes in various metabolic pathways and in energy production. Riboflavin deficiency rarely occurs in isolation, and is frequently associated with deficiencies in one or more of the other B- complex vitamins (Allen et al., 2006).
1.3.1.2.3 Vitamin B3
Niacin (nicotinic acid or vitamin B3), as a functional group of coenzymes, nicotinamide and adenine dinucleotide (NAD) and its phosphate (NADP), is essential for oxidative processes. Deficiency results in pellagra and is associated with a heavily cereal-based diet that is low in bioavailable niacin, tryptophan (an amino acid) and other micronutrients needed for the synthesis of niacin and tryptophan. Niacin is unique among the vitamins in that at least part of the body’s requirement for it can be met through synthesis from an amino acid (tryptophan): the conversion of 60 mg tryptophan (via a niacin derivative) produces 1 mg of niacin (Allen et al., 2006).
1.3.1.2.4 Vitamin B6
Vitamin B6 is in fact a group of three naturally-occurring compounds: pyridoxine, pyridoxal and pyridoxamine. The different forms of vitamin B6 are phosphorylated and oxidized to generate pyridoxal 5-phosphate, which serves as a carbonyl-reactive coenzyme to various enzymes involved in the metabolism of amino acids. Vitamin B6 deficiency alone is relatively uncommon, but occurs most often in association with deficiencies of the other B vitamins (Allen et al., 2006).
1.3.1.2.5 Vitamin B9
Vitamin B9 (folate) plays a central role in the synthesis and methylation of nucleotides that intervene in cell multiplication and tissue growth. Its role in protein synthesis and metabolism is closely (inter) related to that of vitamin B12. The combination of severe folate deficiency and vitamin B12 deficiency can result in megaloblastic anaemia. Low intakes of folate are also associated with a higher risk of giving birth to infants with neural tube defects and possibly other birth defects, and with increased risk of cardiovascular diseases, cancer and impaired cognitive function in adults.
1.3.1.2.6 Vitamin B12
Vitamin B12 (cobalamin) is a cofactor in the synthesis of the essential amino acid, methionine. Its metabolic role is closely linked to that of folate .Vitamin B12 status is usually assessed by measuring its concentrations in plasma or serum. When serum vitamin B12 concentrations fall below 150 pmol/l, abnormalities in the function of some enzymes may occur with the risk, (at lower concentrations), of potentially irreversible poor memory and
cognitive function, impaired nerve condition and megaloblastic anaemia in individuals of all ages (Allen et al., 2006).
1.3.1.3 Vitamin C
Vitamin C is a redox system comprised of ascorbic acid and dehydroascorbic acid, and as such acts as an electron donor. Its main metabolic function is the maintenance of collagen formation. It is also an important antioxidant. Vitamin C deficiency (scurvy) is now marginally high. Vitamin C is widely available in foods of both plant and animal origin, but the best sources are fresh fruits and vegetables, and offal. Germinated grains and pulses also contain high levels of vitamin C (Allen et al., 2006).
1.3.2 Minerals
Dietary studies in developing countries have consistently shown that multiple micronutrient deficiencies, rather than single deficiencies, are common and that low dietary intake and poor bioavailability of micronutrients account for the high prevalence of these multiple deficiencies (Allen et al., 2006). The metabolic roles of minerals and the amounts of them in the body vary considerably (Walker, 2000). The adult recommended daily allowance (RDA) for iron is 10 mg/day for men and 15mg/day for women (Wardlaw, 1999; Walker, 2000). Iron deficiency has been reported to be extremely common in the developing world, with over
50% of the world’s population having some degree of iron deficiency status based on a wide variety of tests (Brune et al., 1989; Openheimer, 2000). This corresponds to studies by Elemo et al. (2010) on the iron status of premenopausal women in a Nigerian university. They reported that these women were at very high risk of nutrition anaemia. This could be attributed to their regular diet, socioeconomic status and consumption pattern. However, the presence of anti-nutrients such as phytate in food could reduce iron absorption and utilization in humans.
Calcium has been reported to be the most abundant mineral in the human body, with 99% of it contained in the skeleton where it exists as hydroxyapatite (Allen, 2001). In addition to its role in maintaining the rigidity and strength of the skeleton, calcium is involved in a large number of metabolic processes, including blood clotting, cell adhesion, muscle contraction, hormone and neurotransmitter release, glycogen metabolism, and cell proliferation and differentiation (Wardlaw, 1999). The adequate intake (AI) of calcium for adults is 1000-1200 mg/day and for adolescence, it is 1300 mg/day. Calcium deficiency is certainly a risk factor
for osteoporosis in later life (Allen, 2001). This makes supplementation very important. Although, no disease is currently associated with an inadequate phosphorus intake, its deficiency may contribute to bone loss in elderly women (Walker, 2000). Sodium is the major positive ion in extracellular fluid and a key factor in retaining body water. Food- labelling rules, the Daily Value for sodium is 2400 mg (Greely, 1997). High sodium content has been shown to contribute to hypertension in susceptible individuals, leading to increased calcium loss in urine (Wardlaw, 1999). Potassium plays a similar role with sodium in the biological system, but it is located in the intracellular fluid. Unlike sodium, it is associated with (lower rather than higher) blood pressure values (Wardlaw, 1999). Potassium RDA is (2000 mg/day). Deficiency of potassium leads to an irregular heartbeat, loss of appetite and muscle cramps.
Zinc is an essential component of a large number of enzymes, and plays a central role in cellular growth and differentiation in tissues that have a rapid differentiation and turnover, including those of the immune system and those in the growth of some stunted children, and on the prevalence of selected childhood diseases such as diarrhoea, especially in developing countries. However, the extent of zinc deficiency worldwide is not well documented. All population age groups are at risk of zinc deficiency, but infants and young children are probably the most vulnerable. Pregnant and lactating women are also (likely to be very) susceptible to zinc deficiency, and there is an urgent need for more information on the implications of low zinc status in those particular population groups (Walker, 2000; Allen et al., 2006).
1.4 Proteins and Amino acids
Proteins are the most abundant biological macromolecules, occurring in all cells and all parts of cells. They constitute the largest fraction (besides water) of a cell. Proteins also occur in great variety; thousands of different kinds, ranging in size from relatively small peptides to huge polymers with molecular weights in the millions, may be found in a single cell. Moreover, proteins exhibit enormous diversity of biological functions. Some proteins have catalytic activity and function as enzymes; others serve as structural elements, signal receptors, or transporters that carry specific substances into or out of cells. Proteins are the molecular instruments through which genetic information is expressed. Relatively simple monomeric subunits provide the key to the structure of the thousands different proteins. All
proteins, whether from the most ancient lines of bacteria or from the most complex forms of life, are constructed from the same ubiquitous set of 20 amino acids, covalently linked in characteristic linear sequences (Nelson and Cox, 2006). Because proteins are complex molecules, the body takes longer time to break them down. As a result, they are a much slower and longer-lasting source of energy than carbohydrates (Youdim, 2015).
Amino acids are organic compounds which contain both an amino group and a carboxyl group. They can be supplied by a combination of cereal grains (wheat, corn, rice etc.) and legumes (beans, peanuts, etc.). There are twenty (20) biologically active amino acids in humans. In nature, there are more than hundred 100 amino acids to be found. However, the human uses only twenty 20 of them. Since amino acids are the building blocks of vitamins,
20 different types of amino acids are quite a lot for the body to work with in many respects.
Amino acids can be placed in the category of either essential or non-essential.
Essential amino acids are those that are “essential” in the diet. In other words, our metabolism cannot create them. Therefore, we need to obtain them through foods containing these essential amino acids. Fortunately, protein-containing foods contain varying degrees of the essential amino acids. During times of starvation, the body relies on its own protein from sources that it normally shouldn’t have to utilize (muscle tissue). The essential amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine.
Non-essential amino acids can be produced from other amino acids and substances in the diet and metabolism. During times of need, metabolism can shift into producing the amino acids that it requires for synthesizing proteins essential to our survival. Examples of non-essential amino acids are alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, tyrosine (Youdim, 2015).
1.5 Fermentation
Fermentation is one of the oldest forms of food processing and preservation method in the world. Since very early times, humans have been exploiting yeasts and their metabolic products, mainly for baking and brewing. Nowadays, the products of modern yeast biotechnology form the backbone of many commercially important sectors, including foods, beverages, pharmaceuticals and industrial enzymes. Saccharomyces cerevisiae, which
according to EFSA (The European Food Safety Authority) has a QPS (Qualified Presumption of Safety) status (Blandino et al.,2003; Moslehi-Jenabian et al., 2010), is the most common yeast used in food fermentation where it has shown various technological properties. Yeasts do also play a significant role in the spontaneous fermentation of many indigenous food products. Several beneficial effects on human health and well-being have been reported (Moslehi-Jenabian et al., 2010) and there seems to be a need to understand the positive effects of yeasts, their mechanisms and employment. However, there are other reported effects such as enrichment of foods with prebiotics as fructo-oligosaccharides (Maugeri and Hernalsteens, 2007), lowering of serum cholesterol, anti oxidative properties, anti mutagenic and antitumour activities (Moslehi-Jenabia et al., 2010). In fermented foods, lactic acid bacteria have a long history of application because of their beneficial effects on nutritional, shelf-life and organoleptic characteristics of food. They cause acidification of the food through the production of organic acids, mainly lactic acid (Blandino et al., 2003; Omenu, 2011). The organic acids, bacte- riocins, aroma compounds and several enzymes production are highly important (Afolabi et al.,
2008). A food fermentation process with lactic acid bacteria is traditionally based on spontaneous fermentation. Lactobacilli exert strong antagonistic effects against many microorganisms, including food spoilage organisms and pathogens (Hartnett et al., 2002). They contribute to the preservation of foods by producing antimicrobial agents like bacteriocins which are considered as natural food preservatives. In akamu, lactic acid bacteria occur in large number and they confer qualities like extended shelf life, aroma and make the product safe for consumption. Akamu is a lactic acid fermented gruel or porridge traditionally made from maize, millet and sorghum. It contributes substantially to the daily diet of both rural and urban communities. It is especially used as weaning food for children and food for invalids because it is light and digests easily (Afolayan et al., 2010). It is locally prepared on small scale in homes or for commercial purposes, and its quality largely depends on the skills of the producers (as inherited over the years). The fermentation of akamu is spontaneous and mostly uncontrolled leading to products of variable quality. Akamu could be fortified with sugar, milk or chocolate to improve taste or sooth the sour taste (Ekwem, 2014).
Fermentation could lead to reduction of toxic products and has been reported to improve the bioavailability of minerals such as iron and zinc by significantly reducing the phytate compounds present in fermented cereals. Fermentation leads to production of acids and probable bacteriocins that prevent growth of microorganisms hence increasing shelf life of fermented products ( Kalui et al., 2009). This is a very valuable attribute especially in rural areas where advanced food preservation technologies such as refrigeration are not affordable and considering that people are beginning to appreciate more of naturally preserved than
chemically preserved foods. Fermented foods are associated with ‘good bacteria’ referred to as probiotics. Probiotics are beneficial bacteria in that they favourably alter the intestinal microflora balance, inhibit the growth of harmful bacteria, promote good digestion, boost immune function and increase resistance to infection (Kalui et al., 2010). People with flourishing intestinal colonies of beneficial bacteria are better equipped to fight the growth of disease causing bacteria. Examples of probiotics that have found application in probiotic products include some strains of Lactobacillus genera (L. plantarum, L. rhamnosus, L. acidophilus, L. reuteri, L. gasseri, and L. amylovorus); Bifidobacterium genera (B. adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, B. lactis, and B. longum); Enterococcus (E. faecalis, and E. faecium) (Hozapfel and Schillinger, 2002). Species of the genera Lactobacillus are the most widely studied for probiotic attributes (Kalui et al., 2010).
1.5.1 Fermentation Organisms
Changes occur during fermentation as a result of the activities of microorganisms: bacteria, yeast and moulds (Steinkraus, 2002). The most important group of microorganisms involved in the spontaneous or natural fermentation of foods are the lactic acid bacteria (Steinkraus,
2002; Jakobsen and Lei, 2004). Lactic acid bacteria are Gram positive, non-spore forming
catalase negative cocci or rods that are anaerobic, micro-aerophilic or aero-tolerant (Wood and Holzafel, 1995). These microorganisms produce lactic acid as the sole, major or an important product from the energy yielding fermentation of sugars (Wood and Holzapfel,
1995; Steinkraus, 2002, Egwin et al., 2013). They include the genus Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Bifidobacterium, some Enterococcus and some streptococcus (Wood and Holzafel, 1995; Steinkraus, 2002). Some yeast such as Saccharomyces and moulds including Penicillium, Aspergillus and Botrytis too produce lactic acid (Wood and Holzafel, 1995).
Lactic acid fermentations provide foods that have a variety of flavours, aromas, textures in addition to the foods being safe and having a long shelf-life. However, not all lactic acid bacteria are useful; some are involved in food spoilage due to action of proteinases and lipases that degrade proteins and lipids, respectively, producing by-products that cause off- flavours. Pediococcusdamnosus has been reported to produce off-flavour in beer while L. bifermentans and L. alimentarius are associated with spoilage of refrigerated and packaged foods. Some are pathogenic especially species belonging to the genera Streptococcus (which
cause throat infections) and Enterococcus (e.g. E. faecum causes urinary tract infections and abdominal septis).
Lactic acid bacteria have been claimed to have health benefits. These include antimicrobial effects against pathogenic bacteria, anti-tumour effects and protection against diarrhoea associated with antibiotics or food allergy. They have been reported to be acid and bile tolerant and hence are able to survive in the gastro intestinal tract (GIT). Lactic acid bacteria can therefore help enhance the gut bacterial population, estimated to be at a concentration of
5 x 1011 bacterial cells per gramme (Holzapfel and Schillinger, 2002). Gut flora are associated with health benefits to the host. As a result, these health enhancing bacteria have been termed ‘probiotics’. A lot of attention is being given to lactic acid bacteria fermented foods as potential sources of ‘probiotics’ that can enhance the balance of intestinal flora hence increasing the health benefits associated with them. Some examples of species that have been reported to possess probiotic attributes include L. plantarum, L. acidophilus, L.rhamnosus, L. reuteri and L. casei (Parves et al., 2006; Kalui et al., 2010).
1.6 Yeasts
Yeasts are a group of unicellular micro-organisms most of which belong to the fungi division of Ascomycota and Fungi imperfecti. Yeasts have been known to humans for thousands of years as they have been used in such fermentation processes as the production of alcoholic beverages and bread leavening. The industrial production and commercial use of yeasts started at the end of the 19th century after their identification and isolation by Pasteur. Today, the scientific knowledge and technology allow the isolation, construction and industrial production of yeast strains with specific properties to satisfy the demands of the baking and fermentation industry (beer, wine, cider and distillates). Food grade yeasts are also used as sources of high nutritional value proteins, enzymes and vitamins, with applications in the health food industry as nutritional supplements, as food additives, conditioners and flavouring agents, for the production of microbiology media, as well as livestock feeds. (Bekatorou et al., 2006) Yeasts are included in starter cultures, for the production of specific types of fermented foods like cheese, bread, sourdoughs, fermented meat and vegetable products, vinegar, etc. The significance of yeasts in food technology as well as in human nutrition, as alternative sources of protein to cover the demands in a world of low agricultural production and rapidly increasing population, makes the production of food grade yeasts
extremely important. A large part of the earth’s population is malnourished, due to poverty and inadequate distribution of food. Scientists are concerned whether the food supply can keep up with the pace of the world population increase, with the increasing demands for energy, the ratio of land are required for global food supply or production of bioenergy, the availability of raw materials, as well as the maintenance of wild biodiversity (Jespersen,
2003). Therefore, the production of microbial biomass for food consumption is a major
concern for the industry and the scientific community. The impressive advantages of microorganisms for single cell protein (SCP) production compared with conventional sources of protein (soybeans or meat) are well known. Microorganisms have high protein content and short growth times, leading to rapid biomass production, which can be continuous and is independent of the environmental conditions. The use of fungi, especially yeasts, for SCP production is more convenient, as they can be easily propagated using cheap raw materials and easily harvested due to their bigger cell sizes and flocculation abilities. Moreover, they contain lower amounts of nucleic acids than bacteria (Klimek et al., 2009).
1.6.1 Yeast metabolism
Yeasts are facultative anaerobes, and can grow with or without oxygen. In the presence of oxygen, they convert sugars to CO2, energy and biomass. In anaerobic conditions, as in alcoholic fermentation, yeasts do not grow efficiently, and sugars are converted to intermediate by-products such as ethanol, glycerol and CO2 (Bekatorou et al., 2006). Therefore, in yeast propagation, the supply of air is necessary for optimum biomass production. The main carbon and energy source for most yeast is glucose, which is converted via the glycolytic pathway to pyruvate and by the Krebs cycle to anabolites and energy in the form of ATP. Yeasts are classified according to their modes of further energy production from pyruvate: respiration and fermentation (Verstrepen et al., 2004). These processes are regulated by environmental factors, mainly glucose and oxygen concentrations. In respiration, pyruvate is decarboxylated in the mitochondrion to acetyl-CoA which is completely oxidized in the citric acid cycle to CO2, energy and intermediates to promote yeast growth. In anaerobic conditions, glucose is slowly utilized to produce the energy required just to keep the yeast cell alive. This process is called fermentation, in which the sugars are not completely oxidized to CO2 and ethanol. When the yeast cell is exposed to high glucose concentrations, catabolite repression occurs, during which gene expression and synthesis of respiratory enzymes are repressed, and fermentation prevails over respiration. In industrial practice, catabolite repression by glucose and sucrose, also known as Crabtree
effect, may lead to several problems, such as incomplete fermentation, development of off- flavours and undesirable by-products as well as loss of biomass yield and yeast vitality (Moslehi-Jenabian et al., 2010).
Yeasts can metabolize various carbon substrates but mainly utilize sugars such as glucose, sucrose and maltose. Sucrose is metabolized after hydrolysis (by extracellular enzyme invertase) into glucose and fructose. Maltose is transferred in the cell by maltose permease, and metabolized after hydrolysis into two molecules of glucose by maltase. Some yeast can utilize a number of unconventional carbon sources, such as biopolymers, pentoses, alcohols, polyols, hydrocarbons, fatty acids and organic acids, and this is of particular interest to food and environmental biotechnologists. For example, lactose can be utilized by yeasts that have the enzyme β-galactosidase. The yeasts of genera Kluyveromyces and Candida can grow e.g. in whey, yielding biomass under certain conditions, with applications in food production. Biopolymers like starch, cellulose, hemicellulose and pectin can be metabolized by some yeasts directly, or after hydrolysis by non-yeast enzymes. Some yeast species of Hansenula, Pichia, Candida and Torulopsis are also able to grow on methanol as sole energy and carbon source. The inability of yeasts to ferment certain sugars can be overcome by r-DNA technology, introducing genes of the corresponding enzymes from other species (Kumura et al., 2004). Finally, elements like N, P, S, Fe, Cu, Zn and Mn are essential to all yeasts and are usually added to the growth media. Most yeasts are capable of assimilating directly ammonium ions and urea, while very few species have the ability to utilize nitrates as nitrogen source. Phosphorus and sulphur are usually assimilated in the form of inorganic phosphates and sulphates, respectively.
1.6.2 Food Grade Yeasts
Various microorganisms are used for human consumption worldwide as single cell protein (SCP) or as components of traditional food starters, including algae (Spirulina, Chlorella, Laminaria, Rhodymenia, etc.), bacteria (Lactobacillus, Cellulomonas, Alcaligenes, etc.), fungi (Aspergillus, Penicillium, etc.) and yeasts (Saccharomyces, Candida, Kluyveromyces, Pichia and Torulopsis). The most common food grade yeast is Saccharomyces cerevisiae, also known as baker’s yeast, which is used worldwide for the production of bread and baking products. S. cerevisiae is the most widely used yeast species, whose selected strains are used in breweries, wineries and distilleries for the production of beer, wine, distillates and ethanol.
Baker’s yeast is produced utilizing molasses from sugar industry by-products as a raw material (Moslehi-Jenabian et al., 2010).
1.6.3 Pichia kudriavzevii (yeast)
Pichia kudriavzevii strain is used in biotechnology. It is unicellular with relatively high growth rates. It is relatively bigger than bacteria, making cell separation after cultivation easier and cheaper. Pichia kudriavzevii consist of 6.5–9.3 % of nitrogen, 40.6–58.0 % of proteins, 35.0–45.0 % of carbohydrates, 4.0–6.0 % of lipids, 5.0–7.5 % of minerals and various amounts of vitamins, depending on its conditions (Ogbonna, 2013).
1.6.4 Beneficial effects of yeast as probiotics
Probiotics are defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ (FAO/WHO, 2001). Probiotics may be consumed either as food components or as non-food preparations. There is a great interest in finding yeast strains with probiotic potential. Different yeast species such as Kluyveromyceslactis, Kluyveromyces marxianus, Kluyveromyces lodderae (Kumura et al.,
2004) have shown tolerance to passage through the gastrointestinal tract or inhibition of
enteropathogens. However, Saccharomyces boulardiiis the only yeast with clinical effects and the only yeast preparation with proven probiotic efficiency indouble-blind studies (Sazawal et al., 2006). S. boulardii, isolated from litchi fruit in Indochina by Henri Boulard in the1920s, is commonly used as a probiotic yeast especially in the pharmaceutical industry and in alyophilized form for prevention and treatment of diarrhoea. It is worthy to notice that contrary to e.g., probiotic strains of lactic acid bacteria, apparently there seems not to be different strains within S. cerevisiae var. boulardii. Based on the similarity in different molecular analyses, all isolates appear to originate from the one isolated from litchi fruit in Indochina by Henri Boulard (Moslehi-Jenabian et al., 2010).
1.6.4.1 Effects on Enteric Bacterial Pathogens
Several studies have shown that S. cerevisiae var. boulardii confers beneficial effects on various enteric pathogens, involving different mechanisms as: (i) prevention of bacterial adherence and translocation in the intestinal epithelial cells, (ii) production of factors that neutralize bacterial toxins and (iii) modulation of the host cell signalling pathway associated with pro-inflammatory response during bacterial infection. Prevention of bacterial adherence and translocation in the intestinal epithelial cells is due to the fact that the cell wall of S.
cerevisiae var. boulardii has the ability to bind enteropathogens. S. Cerevisiaevar. Boulardii cell wall has shown binding capacity to enter haemorrhagic Escherichia coli and Salmonella enterica serovar Typhimurium. Additionally, the yeast inhibits adherence of Clostridium difficile to Vero cells (derived from kidney epithelial cells). Pre-treatment of C. difficile or the Vero cells with S. cerevisiae var. boulardii or its cell wall particles results in lowering the adherence of bacteria to the Vero cells. Yeast cells or cell wall particles are able to modify the surface receptors involved in adhesion of C. difficile through a proteolytic activity and by sterichindrance. Administration of S. cerevisiae var. boulardii reduces adherence of enterotoxigenic E.coli to mesenteric lymph node in pigs intestine (Lessard et al., 2009 and (Moslehi-Jenabian et al., 2010). S. cerevisiae var. boulardii has also beneficial effect on Citrobacter rodentium-induced colitis in mice, and it is due to attenuating the adherence of C. rodentium to host epithelial cells, through reduction in EspB and Tir protein secretions, respectively a translocator and an effector protein implicated in the type III secretion system (TTSS) (Moslehi-Jenabian et al., 2010).
1.6.4.2 Anti-inflammatory Effects
Besides reducing inflammation during bacterial infection by interfering with the host cell signalling pathways, S. cerevisiae var. boulardii also stimulates the peroxisome proliferator- activated receptor gamma (PPAR-γ) expression in human colonocytes and reduces the response of human colon cells to pro-inflammatory cytokines. PPAR-γ is a nuclear receptor expressed by several cell types including intestinal epithelial cells, dendritic cells, T and B cells, and can act as a regulator of the inflammation. S. cerevisiae var. boulardii has been reported to modify the migratory behaviour of lymphocytes. This was observed in a mice model of inflammatory bowel disease (IBD), where inhibition of inflammation in the colon was detected in animals treated with S. cerevisiae var.boulardii. The inhibition was due to decrease in the production of IFN-γ and a modification of T cell distribution. There was a decrease in IFN-γ-producing CD4+ T cells within the colonic mucosa and an increase in IFN- γ-producing T cells in the mesenteric lymph nodes. In addition, S. cerevisiae var. boulardii supernatant modifies the capacity of endothelial cells to adhere to leucocytes, allowing better cell rolling and adhesion (Moslehi-Jenabian et al., 2010). In inflammatory bowel disease (IBD), production of high levels of nitricoxide (NO) and inducible nitric oxide synthase (iNOS) activity is associated with inflammatory effects. The inhibitory effect of S. cerevisiae var. boulardii on iNOS activity has been investigated by (Girard et al., 2005) in rats with castor oil-induced diarrhoea. Administration of yeast blocked the production of the citrulline
(a marker of NO production). The iNOS inhibition by S. cerevisiae var. boulardii may be beneficial in the treatment of diarrhoea and/or IBD associated with over production of NO.
1.6.4.3 Effects on Immune Response
There are several studies indicating the stimulation of the host cell immunity, both innate and adaptive immunity, by S. cerevisiae var. boulardii in response to pathogen infections. Oral administration of S. cerevisiae var. boulardii in healthy volunteers revealed several cellular and humoural changes in peripheral blood. This contributes to the activation of the reticulo endothelial and complement system, demonstrating the stimulation of the innate immune system by the yeast. Oral ingestion of S. cerevisiae var. boulardii stimulated secretion of immune factors, i.e., adaptive immunity. In a study by Buts et al. (1990), the level of secretory immunoglobulin A (sIgA) increased 57% in the duodenal fluid and the secretory component of immunoglobulins enhanced 69% in villus cells and 80% in crypt cells of rats treated with the high dose of yeast. Application of S. cerevisiae var. boulardii to mice treated with C. difficile toxin A caused a 1.8-fold increase in total sIgA levels and a 4.4-fold increase in specific antitoxin A (sIgA) levels. In another study, after intravenous administration of E. coli, germ-free mice mono-associated with S. cerevisiae var. boulardii showed higher clearance of the pathogen from the bloodstream compared to germ-free mice, which was correlated with earlier production of IFN-γ and IL-12 in the serum (Moslehi-Jenabian et al.,
2010).
1.6.5 Beneficial Effects of Yeasts on Bioavailability of Nutrients
1.6.5.1 Antinutrient Effects of Phytate
Phytic acid or phytate (myo-inositol hexakisphosphate, IP6) is the primary storage form of phosphorus in mature seeds of plants and it is particularly abundant in many cereal grains, oilseeds, legumes, flours and brans. Phytate has a strong chelating capacity and forms insoluble complexes with divalent minerals of nutritional importance such as iron, zinc, calcium and magnesium. Humans as well as monogastric animals like poultry and pigs, lack the required enzymes in the gastrointestinal tract for degradation and dephosphorylation of the phytate complex. Besides, lowering the bioavailability of divalent ions, phytate may have negative influence on the functional and nutritional properties of proteins such as digesting enzymes. In addition, lower inositol phosphates attained from degradation of phytate have a positive role in cancer prevention and treatment. Dephosphorylation of phytate is catalyzed by phytases (myo-inositol-hexakisphosphate6-phosphohydrolases). Characterized phytases
are nonspecific (phosphatise) enzymes, which release free inorganic phosphate (Pi) and inositol phosphate esters with a lower number of phosphate groups. Organisms such as plants and microorganisms extensively produce phytase (enzymes) and make the minerals and phosphorus present in the phytates available through a stepwise phytate hydrolysis (Moslehi- Jenabia et al., 2010).In food processing, degradation of phytate can be catalyzed either by endogenous enzymes, naturally present in cereals, or by microbial enzymes produced by e.g., yeasts or/and lactic acid bacteria naturally present in flour or added as starter cultures. Accordingly, improved adsorption of iron, zinc, magnesium and phosphorus can be achieved by degradation of phytate during food processing (Sandberg, 1991) or by degradation of phytate in the intestine (Sandberg et al., 1982).
1.6.5.2 Phytase Activity by Yeasts
Phytases are widespread in various microorganisms including filamentous fungi, Gram- positive and Gram-negative bacteria and yeasts. Among yeasts, Candida krusei (Issatchenkia orientalis), Schwanniomyces castellii, Debaryomyces castellii, Arxula adeninivorans, Pichia anomala, Pichia rhodanensis, Pichia spartinae, Cryptococcus laurentii, Rhodotorulagracilis, S. cerevisiae, Saccharomyces kluyveri, Torulaspora delbrueckii, Candida spp. and Kluyveromyces lactis have been identified as phytase producers. In a study by Olstorpe et al. (2009) on the ability of different yeast strains (122 strains from 61 species) to utilize phytic acid as sole phosphorus source, strains of A. adeninivorans and P. anomala showed the highest volumetric phytase activities. Production of phytase by S. cerevisiae has been investigated in different studies (Moslehi-Jenabian et al., 2010). The phytase activity of S. cerevisiae is partly due to the activity of the secretory acid phosphatases (SAPs), which are secreted by the cells to the growth media and are repressed by inorganic phosphate (Pi). However, the phytase activity of yeasts, e.g., during bread leavening, is relatively low (Turk et al., 1996; Moslehi-Jenabian et al., 2010). This could be due to the repression of the SAPs by Pi. Besides, repression of phytate-degrading enzymes is dependent on the pH and the medium composition. (Andlid et al., 2004) have shown that repression of phytate-degrading enzymes is weak in complex medium with pH 6.0 and high amount of phosphate. Regardless of Pi addition, the capacity to degrade phytase is highest at the pH far from the optimum pH for the SAPs, suggesting that pH has more effect on the expression of the enzyme than on the enzyme activity. S. cerevisiaeas a phytase carrier in the gastrointestinal tract and hydrolysis of phytate after digestion has also been investigated. In a study using a high-phytase producing recombinant yeast strain at simulated digestive conditions, a strong reduction of
phytate (up to 60%) in the early gastric phase was observed as compared to no degradation by wild-type strains (Andlid et al., 2004). The phytase activity during digestion was influenced by the type of yeast strain, cell density, and phytate concentration. However, degradation in the late gastric and early intestinal phases was insignificant, in spite of high phytate solubility, high resistance against proteolysis by pepsin, and high cell survival. This study also showed the importance of pH as a limiting factor for phytase expression and/or activity.
1.6.5.3 Application of Yeast Phytases in Foods
Yeasts or yeast phytases can be applied for pre-treatment of foods to reduce the phytate contents or they can be utilized as food supplement in order to hydrolyse the phytate after digestion. The phytase activity of yeast during bread making for reduction of phytate content of bread has been examined. It seems to be too low to significantly influence the iron absorption. Nevertheless, as explained earlier, during bread making, the content of phytic acid decreases. This is due to the action of phytases in the dough (cereal) and the activity of starter culture. Chaoui et al. (2003) have shown that phytase activity in sourdough bread is highest when using combinations of yeasts and lactic acid bacteria as starter culture. The same result was found by Lopez et al. (2003). Who showed that phytate contents in yeast and sourdough bread were lower than in reconstituted whole-wheat flour and that mineral bioavailability could be improved by bread making especially using both yeast and lactic acid bacteria. Therefore a high-phytase S. cerevisiae strain, may be suitable for the production of food grade phytase and for direct use in the food industry. Increasing the bioavailability of minerals is especially of importance in low-income countries. Therefore it is important to notice that apart from bread, reduction of phytates by yeast phytases has been observed in other plant-derived foods such as in ‘Icacina mannii paste’, a traditional food in Senegal, during fermentation with S. cerevisiae and in ‘Tarhana’, a traditional Turkish fermented food, using baker’s yeast as a phytase source (Moslehi-Jenabian et al., 2010).
1.6.6.0 Folate Biofortification by Yeasts
1.6.6.1 Importance of Folate in the Human Diet
Folates (vitamin B9) are essential cofactors in the biosynthesis of nucleotides and therefore crucial for cellular replication and growth (Hanson and Roje, 2001). Plants, yeasts and some bacterial species contain the folate biosynthesis pathway and produce natural folates, but mammals lack the ability to synthesize folate and they are therefore dependent on sufficient intake from the diet. During the last years, folates have drawn much attention due to the
various beneficial health effects (following an increased intake). The role of folate in the prevention of neural tube defects in the foetus has been established and sufficient folate intake may reduce the risk of cardio vascular disease, cancer (Bailey et al., 2003) and even Alzheimer’s disease (Wang, 2002). The recommended dietary intake (RDI) for the adult population is between 200–300 μg/day for males and between 170–300μg/day for females (according to the FAO/WHO in the USA and several European Countries). Insufficient folate levels result in prolonged cell division, which leads to megaloblastic anaemia (Moslehi- Jenabian et al., 2010).
1.6.6.2 Folate Production by Yeasts
S. cerevisiae is a rich dietary source of native folate and produces high levels of folate per weight. Besides the role as a biofortificant in fermented foods, high producing strains may be used as biocatalysts for biotechnological production of natural folates. The folate level can be considerably augmented in fermented foods using an appropriate yeast strain and by optimizing the growth phase and cultivation conditions for the selected strain. (Hjortmo et al.,
2008) have found that the growth medium and physiological state of cells are important factors in folate production. In synthetic growth medium, high growth rate subsequent to respiro-fermentative growth resulted in the highest specific folate content (folate per unit biomass). In complex media, the level of folate was much lower and less related to growth phase. The specific content of folate in yeast is not only species specific but also dependents on the yeast strain. In another study, Hjortmo et al. (2005) investigated the folate content and composition and the dominating forms of folate found in 44 different strains of yeasts belonging to 13 different yeast species cultivated in a synthetic medium at standard conditions. There was a large diversity in relative amounts of folate content among the studied yeasts. Tetrahydrofolate (H4folate) and 5-methyl-tetrahydrofolate (5-CH3-H4folate) were the dominating forms, which were varying extensively in relative amounts between different strains. Several strains showed a 2-fold or higher folate content as compared to the control strain, a commercial strain of Baker’s yeast. This indicates that by choosing an appropriate strain, the folate content in yeast fermented foods may be enhanced more than 2- fold. These scientists have shown that using a specific strain of S. cerevisiae cultured in defined medium and harvested in the respiro-fermentative phase of growth prior to dough preparation the folate content increased 3 to 5-fold (135–139 μg/100 g dry matter) in white wheat bread, (27–43 μg/100 g dry matter) (Moslehi-Jenabian et al., 2010).
1.6.6.3 Effect of Yeasts on Folate Biofortification of Food
Cereals, especially whole grain products, are the main supplier of folate in the diet. Yeasts are crucial effects on the folate contents of breads. Breads prepared with baking powder have very low folate contents, while addition of yeast results in higher folate content in bread. The variety of sourdough and baking processes obviously lead to great variation in folate content of breads. Total folate content increases considerably during sourdough fermentation due to the growth of yeasts. However, there would be some losses (about 25%) in the amount of folate following the baking (Kariluoto et al., 2004). Final folate content is dependent on the microflora and amylolytic activity of flour, starter cultures and baking conditions. Other microorganisms present in the sourdough like lactic acid bacteria may also influence the folate content. In a study, Kariluoto et al. (2006) investigated the ability of typical sourdough yeasts (S. cerevisiae, Candida milleri, and T. delbrueckii) and lactic acid bacteria to produce or consume folates during sourdough fermentation. Yeasts increased the folate contents of sterilised rye flour-water mixtures to about 3-fold after 19 hours, whereas lactobacilli not only did not produce folates but also decreased it to ultimately half amount. Although the lactobacilli consumed folates, their effect on folate contents in co-cultivations with yeasts was minimal. In beer, the amount of folate enhances due to synthesis by the yeast during the initial period of the fermentation. However, since yeast folate is intracellular, after cropping the yeast, folate will be eliminated from the beer and this is regardless the type of yeast. Some beer brands, which have a secondary fermentation step (often in the bottle), contain higher level of folate. Production of folate in kefir has also been investigated. Kefir is a fermented milk beverage that originated in Eastern Europe and regarded as a natural probiotic product, i.e., a health promoting product. It is produced by the fermentation of milk with kefir granules (grains) and contains different vitamins and minerals. Kefir granules have a varying and complex microbial composition including species of lactic acid bacteria (as the largest portion of microorganism), acetic acid bacteria, yeasts and mycelial fungi. Yeasts isolated from Kefir grains include Kluyveromyces marxianus, Saccharomyces exiguus, Candida lambica and C. krusei (I. orientalis). Kefir contains high folate content, which is produced by the yeast and not the lactic acid bacteria. Patring et al. (2006) also investigated the folate content of different yeast strains isolated from Russian kefir granules, belonging to different Saccharomyces and Candida species. Kefir yeast strains showed high folate-producing capacity. The most abundant folate forms were 5-CH3-H4folate (43–59%) and 5- formyltetrahydrofolate (5-HCO-H4folate, 23–38%), whereas H4folate occurred in a minor proportion (19–23%). By choosing yeast strains that produce a higher proportion of the most
stable folate forms such as 5-HCO-H4folate and 5-CH3-H4folate, it is possible to improve the stability of folates during fermentation and storage, and thus to increase the folate content in kefir products. Recently, the folate content of a traditionally fermented maize-based porridge, called togwa, consumed in rural areas in Tanzania has been investigated by Hjortmo et al. (2008). The yeasts strains belonged to C. krusei (I. orientalis), P. anomala, S. cerevisiae, K. marxianus and Candida glabrata. The major folate forms found during the fermentations were 5-CH3-H4folate and H4folate. The content of H4folate, per unit togwa, remained quite stable at a low level throughout the experiment for all strains, while the concentration of 5- CH3-H4folate was highly strain-and time-dependent. The highest folate concentration was found after 46 hours of fermentation with C. glabrata, corresponding to a 23-fold increase compared with unfermented togwa. As for degradation of phytate, selection of appropriate yeast strains as starter cultures in indigenous fermented foods appears to have high potential in especially developing countries where the vitamin intake generally is lower. Compared to lactic acid bacteria for example, yeast are much more robust and may therefore more easily be distributed as starter cultures.
1.6.7.0 Beneficial Effects of Yeasts on Detoxification of Mycotoxins
1.6.7.1 Prevention of Toxic Effects of Mycotoxins
Mycotoxins are secondary metabolites produced by fungi belonging mainly to the Aspergillus, Penicillium and Fusarium genera. Agricultural products, food and animal feeds can be contaminated by these toxins and lead to various diseases in humans and live stocks. Contamination of agricultural products by mycotoxins is a worldwide dilemma; however it is rigorous in tropical and subtropical regions (Moslehi-Jenabian et al., 2010). The most important mycotoxins are the aflatoxins, ochratoxins, fumonisins, deoxynivalenol (DON), zearalenone (ZEA) and trichothecenes. There are three general strategies in order to prevent the toxic effects of mycotoxins in foods: (i) prevention of mycotoxin contamination (ii) decontamination/detoxification of foods contaminated with mycotoxins and (iii) inhibition of absorption of consumed mycotoxin in the gastrointestinal tract. The ideal solution to reduce the health risk of mycotoxins is to prevent contamination of foods with them. Unfortunately, this cannot be completely avoided and sporadically mycotoxin contamination is reported in food products, especially in the developing world. Therefore, there is an increased focus on effective methods for detoxification of mycotoxins present in foods and also on the inhibition of mycotoxin absorption in the gastrointestinal tract. Various physical and chemical methods are available for the detoxification of food products contaminated with mycotoxins.
However, due to disadvantages of these methods, such as possible losses in the nutritional quality of treated commodities, limited efficacy, reduction of sensory quality and high cost of equipment, their application has been restricted (Kabak et al., 2006). An alternative strategy could be utilization of microorganisms capable of detoxifying mycotoxins in contaminated foods and feeds.
1.6.7.2 Biodegradation of Mycotoxins by Yeasts
Interests in biodegradation of mycotoxins have been increased significantly, since it is specific and environmentally friendly to reduce or eliminate the possible contaminations of mycotoxins in foods. Various microorganisms such as soil or water bacteria, fungi, and protozoa as well as specific enzymes isolated from microbial systems are able to some extent and with varied efficiency to degrade mycotoxins to less- or non-toxic products. Degradation of mycotoxins subsequent to yeast fermentation has been reported in different studies. Degradation of patulin during fermentation of apple juice by S. cerevisiae with E-ascladiol and Z-ascladiol as major metabolites and degradation of zearalenone by several yeast strains has been observed (Gard et al., 2005). However, degradation of zearalenone leads to conversion of that to α- and β-zearalenol, which are still toxic. Degradation of ochratoxin A, fumonisins B1 and B2 , deoxynivalenol and T-2 toxin (Gard et al., 2005) by S. cerevisiae has been reported. Two yeast strains, Phaffia rhodozyma and Xanthophyllomyces dendrorhous, have also been shown to have ochratoxin A (OTA) degrading activity by converting OTA to ochratoxin α possibly mediated by an enzyme related to carboxypeptidases (Moslehi- Jenabian et al., 2010).
1.6.7.3 Mycotoxin Absorption by Yeasts
Inhibition of mycotoxin absorption in the gastrointestinal tract is another way to prevent the toxic effects of mycotoxins. There has been increased interest in the use of mycotoxin binding agents, e.g., yeasts and yeast-derived products, which can be added to the diet to bind mycotoxins. S. cerevisiae has the ability to bind mycotoxins as reviewed by Shetty and Jespersen (2006). The mechanism of detoxification by yeast is due to the adhesion of mycotoxins to cell-wall components. As for binding of pathogenic bacteria, mannan components of the cell wall play a major role in mycotoxin binding .In vitro efficacy of esterified glucomannan to bind aflatoxin B1, ochratoxin A and T-2toxin, when present alone or in combination, was assessed in toxin-contaminated feed. Esterified glucomannan showed significantly higher binding ability to aflatoxin B1 than to ochratoxin A and T-2toxin in a dose dependent manner (Moslehi-Jenabian et al, 2010). In a study by Aravind et al.,(2003) performed on broiler chicks to determine the efficacy of esterified glucomannan in counteracting the toxic effects of mycotoxins in naturally contaminated diet (aflatoxin, ochratoxin, zearalenone and T-2 toxin), it was observed that esterified glucomannan effectively improved the growth depression caused by mycotoxins. Strains of S.cerevisiae have been shown to bind ochratoxin A and zearalenone as well. Ochratoxin A and T-2 toxins also binds to glucomannan component of cell wall. However, zearalenone bind to β-d-glucans of yeast cell wall. It has been shown that yeast cell wall derived products efficiently adsorbed zearalenone (>70%) in an in vitro model that resembled the different pH conditions in the pig gastrointestinal tract, but they were not able to bind deoxynivalenol in a considerable percentage. In a study on mice, when dried yeast and yeast cell walls were added to the diet along with aflatoxin B1, a significant reduction in the toxicity was observed (Moslehi- Jenabian et al., 2010). Similarly, Madrigal-Santillán et al., (2006) described the potential of S. cerevisiae to improve weight gain and reduce genotoxicity of aflatoxin B1 in mice fed with contaminated corn. Even though several trials have been made for decontamination of animal feeds by yeast, very little has so far been done on decontamination of foods and beverages. Binding of mycotoxins to yeast has especially been investigated during winemaking. It has been shown that yeasts can bind to ochratoxin A and remove it from the white and red wine. Ochratoxin A removal from grape must was due to binding of the toxin to the yeast cell wall, and mannoproteins were involved in the mycotoxin absorption during winemaking. The implication of this finding could be very important in the winemaking of must contaminated with ochratoxin A. Oenological strains of Saccharomyces yeasts can also be used for the decontamination of ochratoxin A in synthetic and natural grape juice. Heat-treated cells
showed higher absorption (90% w/w) capacity compared to viable cells (35% w/w) showing the involvement of physical binding, and cell density played an important role in absorption efficiency. Dead yeasts do not pose any quality or safety problems and can be potentially used for detoxification of the grape juice (Moslehi-Jenabia et al., 2010). S. cerevisiae is one of the most important microorganisms involved in food fermentations in tropical countries with high level of mycotoxin contamination in the foods. Shetty et al. (2007) have investigated Aflatoxin B1 binding abilities of S. cerevisiae strains isolated from fermented maize dough (kenkey) and sorghum beer (pito), indigenous fermented foods from Ghana, West Africa. They showed that aflatoxin binding was strain specific and strains were found to bind10–40% equally and some of them more than 40% of the added aflatoxin B1 at standard conditions. The highest binding capacity was observed at the exponential growth phase with
53% binding of the total toxin and the binding reduced towards the stationary phase. Aflatoxin B1 binding increased in a dose dependent manner after addition of aflatoxin, regardless of the temperatures ranging from 20 to 37 °C, but was significantly reduced at 15
°C. Heat and acid treated cells showed higher binding capacity of up to78% binding of the total added toxin. Following this study unpublished results by Jespersen (2003) have shown the yeast-aflatoxin B1 complex to be stable during the passage of an in vitro gastrointestinal tract model indicating that the aflatoxin will not be absorbed in the gastrointestinal tract but excreted together with the yeast cells in the human faeces. Additionally, strains of S. cerevisiae isolated from indigenous fermented foods which are effective aflatoxin binders have been shown to be usable as starter cultures with additional capacities to decontaminate mycotoxins in fermented maize products.
1.7 Cereals (Maize)
Maize (Zea mays L., Poaceae) is the most important cereal in the world after wheat and rice with regard to cultivation areas and total production. The name maize is derived from the South American Indian Arawak-Carib word mahiz. It is also known as Indian corn or corn in
America. It was introduced into Nigeria probably in the 16th century by the Portuguese
(Osage and Offiong, 1998).
Maize is prepared and consumed in a multitude of ways which vary from region to region or from one ethnic group to the other. For instance, maize grains are prepared by boiling or roasting as paste (‘eko’), ‘abado’, and ‘elekute’ in Nigeria and ‘kenke’ in Ghana, or as popcorn which is eaten all over West Africa. Maize is an all-important crop which provides
an avenue for making various types of foods. It also has some medicinal values and serves as raw-materials for many industries. It is put to many uses like in akamu making and other fermented foods.
1.7.1 Nutrient Content of Maize
Cereal (maize) grains are considered to be one of the most important sources of dietary proteins, carbohydrates, vitamins, minerals and fibre for people all over the world. However, the nutritional quality of cereals and the sensorial properties of their products are sometimes inferior or poor in comparison with milk and milk products. The reasons for this are the lower protein content, the deficiency of certain essential amino acids (lysine), the low starch availability, the presence of determined anti nutrients (phytic acid, tannins and polyphenols) and the coarse nature of the grains (Blandino et al., 2003). Cereals as a main source of energy in diets for growing animals are supplemented with suitable sources of protein content in the diet and to balance the content with essential amino acids. A new source of protein has been introduced such as yeast which is very effective in optimizing the proper and harmonious development of the animal (Yabaya, 2011).
A number of methods have been employed with the aim of ameliorating the nutritional qualities of cereals. These include genetic improvement and amino acid supplementation with protein concentrates or other protein-rich sources such as grain legumes or defatted oil seed meals of cereals. Additionally, several processing technologies which include cooking, sprouting, milling and fermentation, have been put into practice to improve the nutritional properties of cereals, although probably the best one is fermentation (Blandino et al., 2003). The proteins, fatty matters and total soluble sugars contents of maize grain were respectively at 8.2%, 0.64 % and 0.64% %. Ash content of maize seeds was 1.85% and these values are significantly affected during the various stage of processing.
1.7.2 Akamu
Akamu is a porridge prepared from fermented maize, millet and sorghum. It is a popular breakfast cereal and infant weaning food in West Africa (Blandino et al.,2003). Akamu also called ogi, is a lactic acid fermented food made from maize, sorghum or millet it may may be fortified with legumes. Akamu is prepared by soaking clean maize grains in water for 2-3 days. The grains are washed and milled to a paste. The paste is sieved to smooth slurry which
is allowed to settle and the supernatant decanted. The slurry is mixed with hot water with stirring until it forms a gel which serves as food (Nwosu and Oyeka, 1998; Ogbonnaya and Bernice, 2012).
Approximately 20-50% of the nutrients available in the original cereal grains are lost through processing for akamu or ogi production, being the loss of aleurone layer and germ of grains during wet milling and wet sieving (Blandino et al.,2003), so the need for biofortification.
1.8 Aim and Specific Objectives of the Study
1.8.1 Aim of the study
This study was aimed at determining the nutrient compositions of yeast biofortified akamu, a cereal- based fermented food.
1.8.2 Specific Objectives of the Study
The specific objectives of this study were:
(To grow yeast at different glucose concentrations) to determine the effect of varying glucose amounts on nutrient availability in akamu at constant inocula of yeast.
To determine the effect of different yeast inocula volume at constant glucose level on the quality of akamu.
To compare the physicochemical composition of ordinary akamu to that of biofortified akamu.
To compare the amino acid contents in ordinary akamu to those of biofortified akamu
To compare the vitamin content in both ordinary akamu and biofortified akamu
To compare the minerals in both ordinary akamu and the biofortified akamu.
This material content is developed to serve as a GUIDE for students to conduct academic research
A1Project Hub Support Team Are Always (24/7) Online To Help You With Your Project
Chat Us on WhatsApp » 09063590000
DO YOU NEED CLARIFICATION? CALL OUR HELP DESK:
09063590000 (Country Code: +234)
YOU CAN REACH OUR SUPPORT TEAM VIA MAIL: [email protected]
09063590000 (Country Code: +234)
