Potential Pre Biotic Properties Of Fiber Biology Essay


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 (Kwak & Jukes, 2001). "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" (Diplock et al., 1999).

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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). 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 (Roberfroid, 2000b). The markets are flooded with a wide range of foods that claim to have functional food ingredients, like dietary fiber, lactic acid bacteria, vitamins, minerals, fish oils and plant extracts such as garlic, liquorices and celery, in them (Bogue & Ryan, 2000). Examples of food fortification include mineral fortification (calcium enriched milk), antioxidant fortification (Vitamins E and C), fiber fortification (high fiber cereal), live culture fortification (probiotics) and fat substitutes (low-fat chocolate) (Siro et al, 2008).

The global market share of functional foods is estimated to be $31 billion, United States being the largest market followed by Europe and Japan. Others report even higher market value (nearly $61 billion) (Kotilainen et al., 2006; Sloan, 2002). 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.

         Probiotics

Probiotic foods are fortified with naturally occurring live bacteria which, are also referred to as 'good bacteria' such as Lactobacillus and Bifidobacterium (Gibson, 2007). 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, year 2005 alone saw the launch of a staggering 379 product worldwide (Alzamora et al., 2005; Jones & Jew, 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 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 (Tuohy 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 (McNaught and MacFie, 2001).

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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 (Bird et al., 2002). 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 (Rountree, 2002).

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.

         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 such as inulin and fructo-oligosaccharides. At present, there were over 400 prebiotic food products such as cheese, yoghurts and breakfast cereals in the market while more than 20 companies are producing oligosaccharides and dietary 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). Inulin and fructo-oligosaccharides are frequently used in prebiotic foods as they resist digestion (Kolida and Gibson, 2007). Oligosaccharides in general may serve as prebiotic agents and also suppress potentially deleterious bacteria among the gastrointestinal microbiota (Roberfroid, 2007).

 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; Lo´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 fiber

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; 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, 2009).

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).

Identification of date fiber that show pre-biotic properties.

3. Methodology

3.1 Sample Preparation

Twenty varieties of dates (the most abundant and consumed in Saudi Arabia) were chosen for the study. The dates namely Manifi, Khodry, Sagiee, Khalas, Sukkari,Nabtat-ali, Barhi, Sabaka, Ruhodya, Sefri, Nabtat-seif, Maktomi, Wanana, Ruthana, Shaishee, Mabrom, Sari, Sullaj, Ruzeiz and Ajwa 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.

3.2 Total dietary fiber (TDF), insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) determination

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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 and to make the process economical we choose the cheapest one. Enzymatic- gravimetric method was used (AOAC, 991.43) (Lee et al., 1992) as follows:

         Preparation of test sample:

1-      Weigh duplicate 1g date flesh (dried by freeze dryer), into 600 ml beakers (W).

2-      Add 40 ml phosphate buffer to each beaker and stir on magnetic stirrer until test solution is completely dispersed.

3-      Add 50 µl -amylase solution and incubate in water bath at 95- 100oC with continuous agitation, 15 min. Then cool to 60oC.

4-      Add 100 µl protease solution to each beaker and incubate in water bath at 60oC, 30 min with continuous agitation.

5-      Adjust pH to 4.0- 4.7 by adding 1 M HCL solution at 60oC.

6-      Add 300µl amyloglucosidase solution with stirring. Then incubate in water bath at 60oC during 30 min with constant agitation.

         Determination of  total dietary fiber (TDF) and soluble dietary fiber (SDF):

7-      To each digested test solution add 225ml 95% ethanol and let precipitate from 1h at room temperature.

8-      Filter and transfer all remaining particles to fiber crucible with 95% ethanol. Using Fibertec system (cold extractor) with vacuum, wash residue twice with 95% ethanol.

9-       Dry crucible containing residue overnight in 105oC oven, then cool in desiccator 1h and weight residue (R).

  Use one duplicate to determine protein by Kjeldahl nitrogen determination (AOAC (960.52), 1995) (P).

  For ash analysis incinerate second duplicate 5h at 525oC. Then cool crucible in desiccators and weight (A).

         Determination of  insoluble dietary fiber (IDF):

10-   Wash digested test solution twice with 10ml water, then transfer to fiber crucible.

11-  Filter by using Fibertec system with vaccum, wash residue twice with ethanol 95%.

12-  Dry crucible containing residue overnight in 105oC oven, then cool in desiccators 1h and weight residue (R).

  Use one duplicate to determine protein by Kjeldahl nitrogen determination (AOAC (960.52), 1995) (P).

  For ash analysis incinerate second duplicate 5h at 525oC. Then cool crucible in desiccators and weight (A).

13-  Calculate DF% as follows:


Filtrate + water washing


Weigh solution

Add 4 vols 95% EtOH

Precipitate for 1 hour

Filter and dry residue



Insoluble Dietary

Fibre (IDF)



Soluble Dietary

Fibre (SDF)

Fig. 1: Soluble and insoluble dietary fibre determination procedures

Sample (1g) in 600mL beaker

Add 40 mL buffer

Add 50L -amylase

Water bath, 95 - 100oC, 35 min

Add 100L protease (no pH adjustment)

Water bath, 60oC, 30 min

Adjust pH to 4.1 - 4.8 and add 200L amyloglucosidase

Water bath 60oC, 30 min


Wash twice with 10mL water at 70oC

Determination of TDF, SDF and IDF was repeated twice to each type of dates (20 types).

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:

      The first used method to extract DF from the dates is for (Goňi et al., 2009) as follows:

1-      Weigh duplicate 0.3g date flesh (dried by freeze dryer), into 50 ml centrifuge tubes.

2-      Add 10 ml phosphate buffer pH 7.5 (0.1M) to each tube. And adjust pH to 1.5.

3-      Add 0.2 ml pepsin solution to each tube and incubate in water bath at 40oC, 1 h and adjust pH to 7.5.

4-      Add 1 ml pancreatin solution and incubate in water bath at 37oC during 6 h.

5-      Add 10 ml phosphate buffer pH 7.5 (0.1M) and adjust pH to 6.9.

6-       Add 1 ml -amylase solution and incubate in water bath at 37oC with continuous agitation, 16 h.

7-       Centrifugation of samples (15 min, 5000 rpm) and remove supernatants.

8-        Washing residues twice with 5 ml distilled water and dried overnight at 105oC, cool in desiccator and weigh to determine residue weight. This value is IDF.

9-       Add 10 ml sodium acetate buffer pH 4.75 to the supernatants (checking pH) follow by 0.1 ml amyloglucosidase solution and incubated at 60oC, 45 min, in water bath with constant shaking.

10-   Transfer the supernatants into dialysis tubes (12000-14000 m/ w) and dialyzed against water at 37oC, 48 h (water flow 7 L/h).

11-   Concentrate and dry the dialysis supernatants by freeze-dryer. This value is measured gravimetrically and it corresponds to SDF.


Pepsin (pH 1.5, 1h, 49oC)

Pancreatin (pH 7.5 6h, 37oC)

-amylase (pH 6.9, 16h, 37oC)




Amyloglucosidase, pH 4, 75, 45 min, 60oC

Dialysis (48h, water flow)

Dry (freeze dried)



Fig.2: Enzymatic gravimetric method procedures

                                            Dry (freeze dried)

         Non-enzymatic- Gravimetric Method (Fig. 3) (993.21 of AOAC) ( Li & Cardozo, 1994)

1-      Weigh duplicate. 5g date flesh (dried by freeze dryer) into 250 ml beakers.

2-      Add 25 ml water to each beaker with gently stirring until utterly wet, incubated at 37oC without stirring.

3-      Add 100 ml ethanol (95%) to each beaker and incubate 4 h at room temperature (25+ 2oC).

4-      Collect residue under vacuum. Wash residue twice with 20 ml ethanol (95%), twice.

5-      Dry residue overnight at 105oC. Then cool it 2 h in desiccators and weight.

Sample 500 mg

wet with 25 ml H2O


- Incubator, 90 min at 37oC

- Add 100ml 95% ethanol,

let 1h at (25±2oC)

Washing with

2 x 20ml 95% ethanol

Fig. 3: Non-enzymatic gravimetric method procedures

                                Residue              Filtration                          Filter


Drying 2h at 105oC, weight

         Extraction by hot water:

 The third used method to extract DF from dates is for (Elleuch et al., 2008) as follow:

1-      Add 600 ml hot water to 30g dates fleshes (100oC for 5 min).

2-      Dietary fibers recover by centrifugation (8500 rpm, 15 min).

3-      Washing the residue with 300 ml water (40oC) and Centrifugation (This operation repeat 5 times)

4-      The residues obtained were freeze-dried to give the DF concentrates.

Dates flesh

Hot water extraction

(600 ml H2O, 30g of fleshes)

100oC, 5 min


8500 rpm, 15 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 fiber

Repeated extraction twice by used three different methods on one chose sample. After employing three different methods for extraction of dietary fibre, total carbohydrate and TDF was determined in the samples.

         Total carbohydrate determination (Masuko, et al, 2005):

1-      Prepare 2mg/ ml solution of glucose (standard) and sample in water.

2-      Dilute to 80µg/µl (10µl stock + 900µl water).

3-      Aliquot into wells a range from 0 - 50µl (standard and sample) and make up to 50µl.

4-      Add 150µl of concentrated sulphuric acid.

5-      Immediately add 30µl of 5% phenol.

6-      Incubate for 5 min at 90oC in static water bath.

7-      Cool to room temperature for 5 min.

8-      Measure on microtitre plate reader to read the absorption at 490 nm.

9-      Repeat the measurement twice.

Prepare sample and standard (2mg / ml)

Dilute to 80 µg/ µl

Aliquot into wells a range from 0 - 40 mg (standard and sample) and make up to 50 ml

Add 150 ml of concentrated sulphuric acid

Immediately add 30 ml of 5% phenol

Incubate for 5 min at 90oC in static water

Cool at room temperature for 5 min

Measure on microtitre plate reader at 490 nm)

Total Carbohydrate

Figure 5: Determination of total carbohydrate

         TDF determination(AOAC, 991.43) (Lee et al., 1992):

See (Fig.1)

3.4. Prebiotic properties of date fiber extracted by water extract method:

PREBIO 7®, a probiotic mixture, contains 7 types of bacteria - Lactobacillus Acidophilus, Lactobacillus casei, Streptococcus Thermophilus, Bifidobacteriumbifidum, Lactobacillus bulgaricus, Bifidobacterium Longum and Lactobacillus Lactis. PREBIO 7®, 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 (glucose free) as control.

         50 ml MRS broth (with glucose) as control.

         50 ml MRS broth (glucose free) with date fiber (1 g/50 ml). and 0.5 mg/50ml).

         50 ml MRS broth (glucose free) with date fiber (0.5 g/50ml).

This experiment was carried out once. In this experiment, growth curves of duplicate samples were measured. The turbidimetry assay were done at 600 nm and values expressed as OD, this was repeated twice for each sample. The measurements were used to determine the growth of bacteria in the media.

4. Results

4.1 Total dietary fiber (TDF), insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) determination

TDF ranged from 6.73+ 0.23g\100g in Barhi dates to 11.73+ 0.27g\100g in Khodry dates, SDF ranged from 0.92+ 0.04g\100g in Sullaj dates to 3.22+ 0.23 g\100g in Nabtat- ali dates, while IDF were ranged from 4.56+ 0.28g\100g in Barhi dates to 9.69+ 0.23g\100g in Maktomi dates. Table 1 shows that Khodary dates contain the highest percent of TDF (11.73+ 0.27) so that DF of Khodry will use in this study.















3.03 +


2.74 +


2.52 +


2.76 +


3.22 +


2.18 +


2.70 +


2.76 +


3.22 +




7.13 +


8.7 +


6.86 +


5.35 +


8.78 +


6.55 +


4.56 +


6.04 +


6.64 +


7.27 +




9.91 +


11.73 +


9.59 +


7.87 +


11.54 +


9.76 +


6.73 +


8.74 +


9.14 +


9.99 +














1.45 +


2.00 +


1.05 +


1.17 +


0.41 +


1.40 +


1.94 +


0.92 +


0.79 +


3.16 +




9.69 +


5.84 +


6.01 +


6.66 +


7.45 +


8.25 +


7.81 +


7.67 +


7.11 +


5.93 +




11.13 +


7.84 +


7.07 +


7.83 +


7.87 +


9.65 +


9.75 +


8.58 +


7.895 +


9.09 +


Table 1: TDF, SDF and IDF content of twenty dates varieties

4.2 Determination of dietary fiber extracted by three different methods:

Table 2 compares total carbohydrates and total dietary fiber present in date samples extracted by three different methods. Where it shows the high concentration of fiber in water extracted samples.

Total Carbohydrates%


Enzymatic method

53.44+ 0.95

7.065+ 0.56

Non-enzymatic- Gravimetric Method

46.62+ 0.86

9.88+ 0.83

Water extract method

22.78+ 0.33

84.94+ 0.41

Table 2: Total Carbohydrates and total dietary fiber in test samples extracted by three different methods: methods comparison

4.3 Prebiotic properties of date fiber extracted by water extract method:

Table (3) shows the results of probiotic bacteria growth with date fiber, in which it shows significant growth for probiotic bacteria with date fiber in both concentration comparison with control samples.

Time (hrs)

MRS (control)

MRS (glucose free)


MRS (glucose free)+ date fiber (0.5 g/ 50 ml)

MRS (glucose free)+ date fiber (1g/ 50 ml)


0.19+ 0.00

0.15+ 0.01

0.99+ 0.01

0.99+ 0.01


0.19+ 0.01

0.23+ 0.00

1.72+ 0.00

1.95+ 0.00


0.21+ 0.01

0.26+ 0.01

1.8+ 0.00

1.97+ 0.00


0.21+ 0.01

0.25+ 0.00

1.82+ 0.02

2.18+ 0.01


0.21+ 0.01

0.25+ 0.00

1.85+ 0.00

2.26+ 0.01


0.21+  0.00

0.27+ 0.01

1.87+ 0.00

2.30+ 0.01


0.22+ 0.01

0.23+ 0.00

1.67+ 0.01

2.07+ 0.02


0.24+ 0.01

0.25+ 0.00

1.77+ 0.01

1.88+ 0.01


0.26+ 0.00

0.27+ 0.00

1.80+ 0.01

1.98+ 0.01


0.27+ 0.00

0.29+ 0.00

1.82+ 0.00

2.07+ 0.01


0.27+ 0.00

0.27+ 0.00

1.82+ 0.01

1.97+ 0.01


0.37+ 0.00

0.27+ 0.01

1.82+ 0.00

1.97+ 0.0


0.39+ 0.01

0.27+ 0.01

1.78+ 0.01

2.08+ 0.00


Table 3: Growth of probiotic mixture with dates fiber extracted by water extract method

Fig.5: Growth pattern of probiotic mixture with dates fiber

5. Conclusions

    date variety with highest fibre

    extraction method with highest yield of fibre

    prebiotics test: preliminary results (only one experiment) indicate positive prebiotic properties of date fibre.

6. Ongoing Experiments

         Evaluation of various types of oligosaccharides present in date fiber by HPLC.

         Identification of date fiber as prebiotic in presence of beneficial and harmful strains selected of bacteria.