Customer demands in the field of food production have changed considerably over last two decades. More and more consumers believe that food contributes directly to their health. Food not only satisfies hunger but also provides essential nutrients for a healthy mind and body. Today consumers want more out of the foods and that is where 'functional foods' play an important role.
The term ''functional food'' was coined and first used in mid 1980's in Japan. The term was used to categorize food products fortified with special ingredients (trace elements and vitamins) believed to promote physiological well being and help prevent diseases (Hardy, 2000; Kwak & Jukes, 2001a; Stanton, Ross, Fitzgerald, & Van Sinderen, 2005). "A food can be regarded as 'functional' if it is satisfactorily demonstrated to affect beneficially one or more target functions in the body, beyond adequate nutritional effects in a way which is relevant to either an improved state of health and well-being and/or reduction of risk of disease. Functional foods must remain foods and they must demonstrate their effects in amounts, which can normally be expected to be consumed in the diet. They are not pills or capsules, but part of a normal food pattern" (EUFIC 2003).
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Functional foods are believed to improve the general well being and lower the risk of nutrition based illness (e.g. cholesterol-lowering products). They may also be effective in curing some diseases (Siro et al, 2008). The increasing demand of functional foods can be attributed to the ever increasing cost of healthcare, increase in average life expectancy, and more and more consumers' opting for healthy lifestyle choices (Kotilainen, et al., 2006; Roberfroid, 2000a, 2000b). Functional foods may prove to be an effective way of creating awareness about the significance between diet and health and also facilitating extensive savings in the health care sector (Bogue & Ryan, 2000). The markets are flooded with a wide range of foods that claim to have functional food ingredients, like dietary fibre, lactic acid bacteria, vitamins, minerals, fish oils and plant extracts such as garlic, liquorice and celery, in them (Niness, 1999). Examples of food fortification include mineral fortification (calcium enriched milk), antioxidant fortification (Vitamins E and C), fibre fortification (high fibre cereal), live culture fortification (probiotics) and fat substitutes (low-fat chocolate) (Bogue & Ryan, 2000; Siro et al, 2008).
The global market share of functional foods is estimated to be $33 billion (Hilliam, 2000b). Experts like Sloan (2000, 2002) believe it to be $47.6 billion with United States being the largest market followed by Europe and Japan. Others report even higher market value (nearly $61 billion) (Benkouider, 2004). Functional foods have been mainly used in the dairy, confectionery, soft-drinks, and baby-food products. The most prominent types of functional food products can be categorized into either probiotics or prebiotics.
Probiotic foods are fortified with naturally occurring live bacteria which, are also refrred to as 'good bacteria' like Lactobacillus sp., Lactococcus sp., and Bifidobacterium sp., (Gokool 2006). According to the Food and Agriculture Organization and World Health Organization, probiotics can be defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (FAO/WHO 2001). The functional food markets in Japan and Europe are mainly dominated by products thought to improve and maintain a healthy digestive system (Alzamora et al., 2005; Jones & Jew, 2007; Saarela, LaÂ¨hteenmaÂ¨ ki, Crittenden, Salminen, & Mattila-Sandholm, 2002). Year 2005 alone saw the launch of a staggering 379 product worldwide (Ouwehand, 2007).
Probiotics can mostly be found in dairy products such as yogurt, fermented and unfermented milk and some juices and soy beverages. They are either already present in the foods or added during preparation. Adding live microbial additions to appropriate food vehicles is the basis of what is now recognized as the probiotic concept (Gibson, 2007). Dairy products such as yoghurts and cheese provide excellent conditions for probiotic bacterial growth. Lactobacillus and Bifidobacterium sp. are the most studied and widely employed bacteria within the probiotic field. They are regular members of the intestinal microbiota and have a long tradition of safe application within the food industry (Ventura et al., 2009).
Probiotic foods have been investigated for their usefulness against a range of gastrointestinal diseases and disorders such as lactose intolerance and diarrhoea (Kieran et al., 2003; Salminen and Gueimonde, 2004). Other therapeutic benefits attributed to probiotic microorganisms include reduction of hypercholesterolemia, protection against cancer and prevention or treatment of peptic ulcer disease (Goyal and Gandhi, 2008). "There is only limited scientific evidence to suggest that healthy people will benefit from a regular intake of probiotics, however many people report feeling better after taking them" (Gokool 2006).
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The probiotic bacteria have been implicated as playing important roles in various physiological processes; they are involved in digestive processes and produce fatty acids and vitamins for use in the body. These bacteria also serve as a protective barrier within the digestive tract and strengthen the immune system thereby preventing infection by pathogenic bacteria (Ouwehand, 2007). A potential probiotic culture must be of human origin, maintain high acid and bile stability and should adhere to mucosal surfaces apart from being edible and safe for clinical (Ronka et al, 2003; Roberfroid, 2000).
Probiotic bacteria produce acids which, are thought to improve health. They also act as bacteriocins by competing with pathogens for substrate and binding sites and help stimulate the immune system (Vanderpool et al, 2008). Most of probiotics bacteria need an energy source for growth in the intestinal tract which, could be seen as a disadvantage of probiotics. Improvements over this disadvantage lead to introduction of what is called prebiotics.
Gibson and Roberfroid (1995) defined prebiotic foods as "a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health". The Food and Agricultural Organization defines prebiotic food as "any non-viable food component that confers a health benefit on the host associated with modulation of the microbiota" (FAO, 2007). Prebiotics foods are considered to improve health by stimulating the growth of beneficial gut flora while inhibiting the growth of pathogenic bacteria (Tuohy et al., 2001).
The prebiotic food mainly comprises of indigestible carbohydrates like inulin and fructo-
oligosaccharides. At present, there were over 400 prebiotic food products like cheese, yoghurts and breakfast cereals in the market while more than 20 companies are producing oligosaccharides and fibres used as prebiotics. The prebiotics food market is growing rapidly and this dramatic growth spurt can in part be explained by the increase in diversity of food products containing prebiotics (Wang, 2009).
Natural sources of prebiotics include leeks, chicory, asparagus, bananas, artichokes, garlic, onion, wheat, soybean and oats (Murphy, 2001; Voragen, 1998). Inulin and fructo-oligosaccharides are frequently used in prebiotic foods as they resist digestion (Cummings et al., 2001). Oligosaccharides in general may serve as prebiotic agents and also suppress potentially deleterious bacteria among the gastrointestinal microbiota (Kajiwara et al., 2002).
A lot of research on potential prebiotic properties of certain food such as, chicory, artichokes, soybean and oats (Rupérez, 2006; Van de Wiele et al., 2004; LoÂ´pez-Molina et al., 2005) has been carried out but the study about potential prebiotic properties of dates fiber however, remain unreported. Therefore, this study aims to characterize dates fiber and explore its potential as a prebiotic agent.
Dates are a rich source of dietary fiber (Myhara et al., 1999; Al-Farsi et al., 2007; Elleuch et al., 2008). The fiber content depends on the variety and ripening stage of the dates. It can range from 4.4 to 11.4% (Spiller, 1993; El-Zoghbi, 1994; Al-Hooti et al., 1995; Al-Shahib and Marshall, 2002). A serving of five to six dates can provide up to 14% of the recommended daily intake of dietary fiber (Spiller, 1993).
The Kingdom of Saudi Arabia (KSA) is the fourth largest producer of dates. The dates, one of the most important fruit crops in KSA, are also processed to produce date syrup. Date fiber (DF), a by-product of date syrup extraction, may contain up to 51.57% total dietary fiber (Hashim, 2008).
2. Project Objectives
Determination of dietary fiber (DF) in 20 varieties of dates at different stages of maturity.
Finding most economic and efficient method or procedure for date fiber extraction.
Evaluation of various oligosaccharides in date fiber by High Performance Liquid Chromatography (HPLC).
Defining the prebiotic properties of date fiber.
3.1 Sample Preparation
Eight varieties of dates (the most abundant and consumed in Saudi Arabia) were chosen for the study. The dates namely Manifi, Khadari, Sagiee, Khalas, Sukari, Nabtat ali, Barhi and Reshwidya, were obtained from date's farm in Riyadh (Almohamadia farm and factory of dates).
After removing seeds, the date flesh was washed with tap water. The samples were freeze dried to remove water. The dried samples were stored in bottles for further analysis.
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3.2 Total dietary fiber (TDF), insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) determination
Fiber content was determined in all the mentioned types of dates (Fig. 1). This was done to select the variety with highest percentage of fiber. To make the process economical enzymatic- gravimetric method, the cheapest extraction method, was used (AOAC, 991.43).
Filtrate + water washing
Add 4 vols 95% EtOH at 60oC
Use a portion of EtOH to rinse
filtering flask and beaker
Precipitate for 1 hour
Filter and dry residue
Sample (1g) in duplicate in 600mL beaker
Add 400 mL TRIS buffer, 0.05M each, pH 6.2 at 24oC
Add 50ïL ï¡-amylase
Water bath, 95 - 100oC, 35 min
Add 100ïL protease (no pH adjustment)
Water bath, 60oC, 30 min
Add 5mL 0.56 N HCl to pH 4.1 - 4.8 and add 200ïL amyloglucosidase
(Leave beakers in 60oC water bath until pH checking/adjusting step.)
Water bath 60oC, 30 min
Wash twice with 10mL water at 70oC
(Use water to rinse beaker before washing residue)
Fig. 1: Soluble and insoluble dietary fibre determination procedures
3.3 Extraction of Dietary Fiber (DF)
Three methods were employed to extract DF from the dates. This was done to select the most efficient and cost-effective extraction process.
Enzymatic method (Fig. 2) (GoÅˆi et al., 2009).
Pepsin (pH 1/5. 1h, 49oC)
Pancreatin (pH 7.5 6h, 37oC)
ï¡-amylase (pH 6.9, 16h, 37oC)
Amyloglucosidase, pH 4, 75, 45 min, 60oC
Dialysis (48h, water flow)
Dry (freeze dried)
Fig. 2: Soluble and insoluble dietary fibre determination procedures
Dry (freeze dried)
Non-enzymatic- Gravimetric Method(Fig. 3) (993.21 of AOAC)
Sample (0.1 mg)
Duplicate 500 mg
freeze dried, ground
wet with 25 ml H2O
- Sonicate or gently stir
- Incubator 90 min at 37oC
- Add 100ml 95% ethanol,
let 1h at (25Â±2oC)
2 x 20ml 78% ethanol
2 x 10 ml 85% ethanol
1 x 10ml acetone
Drying 1h > 105oC
Residue Filtration Filter
Fig. 3: Non-enzymatic gravimetric method procedures
Extraction by hot water (Fig. 4) (Elleuch, M. et al., 2008).
Hot water extraction
(600 ml H2O2, 30g of fleshes)
100oC, 5 min
6500 g, 10 min, 25oC
Washing the residue with water
(300 ml, 40oC and Centrifugation
(This operation was repeated 5 times)
Dry (freeze dried)
Dietary fibre concentrates
Fig. 4: Process of elaboration of date dietary fibre concentrates
After employing three different methods for extraction of dietary fibre , total carbohydrate (Masuko, et al, 2005) and total DF was determined in the samples.
3.4. Prebiotic properties of date fiber
PREBIO 7Â®, a probiotic mixture, contains 7 types of bacteria - Lactobacillus Acidophilus, Lactobacillus casei, Streptococcus Thermophilus, Bifidobacterium bifidum, Lactobacillus bulgaricus, Bifidobacterium Longum and Lactobacillus Lactis. PREBIO 7Â®, used in this study, is a nutritional supplement found in a capsule form.
The probiotic mixture was cultured in de Man Rogosa Sharpe (MRS) broth and incubated at 28 °C overnight and at 37 °C afterwards. The bacterial growth was expressed as optical density (OD) obtained from absorbance at 600 nm. Observations were made at 0 hour and then every 2 hours over 24 hours. 1 ml from the culture was added to 50 ml MRS broth (without glucose) and MRS broth (without glucose) with date fiber. The turbidimetry assay were done at 600 nm and values expressed as OD. The measurements were used to determine the growth of bacteria in the media.
4.1 Total dietary fiber (TDF), insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) determination
TDF ranged from 6.89g\100g in Barhi dates to 11.92g\100g in Khadari dates, SDF ranged from 2.14g\100g in Barhi dates to 3.08 g\100g in Nabtat- ali dates, while IDF were ranged from 4.75g\100g in Barhi dates to 8.90g\100g in Sukari dates
Table 1.Fibre content of eight date varieties
4.2 Determination of dietary fiber
Table 2 compares total carbohydrates and total dietary fiber present in date samples extracted by three different methods.
Non-enzymatic- Gravimetric Method
Water extract method
Table 2: Total Carbohydrates and total dietary fiber in test samples: methods comparison
4.3 Prebiotic properties of date fiber
Fig5: Growth pattern of probiotic mixture with dates fiber
5. Ongoing Experiments
Evaluation of various types of oligosaccharides present in date fiber by HPLC.
Defining the prebiotic properties of date fiber.