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PROXIMATE ANALYSIS OF COMMERCIALLY AVAILABLE BISCUIT (MIXED CHIZ AND REGULAR BREAD STIX)

CATHERINE T. CHAN
School of Chemical Engineering, Chemistry, Biotechnology and Material Science, Mapua Institute of Technology Mapua Institute of Technology Muralla St., Intramuros, Manila Philippines chancath@hotmail.com

ABSTRACT In this fast-paced world that we are in, it is convenient to eat food products that are ready-to-eat. One of this ready-to-eat products that consumed by almost all people is biscuit. Essential amino acids and fatty acids, energy source (i.e. carbohydrates), and other nutrients needed are obtained from the diet. Since biscuit is in most peoples diet, proximate analysis of biscuit (bread stix) was determined. Two variety (regular and cheese) of bread stix were equally mixed and subjected to proximate analysis. Proximate analysis of this product includes moisture, crude fat, crude fiber, crude protein and ash. Specific mineral, iron, which is in ash content, was analyzed since the manufacturer claimed to have it in the product. Proximate analysis was then compared with the nutrition fact of the product. Apart from the proximate analysis, pH, total soluble solids and titrable acidity were determined. Keywords: biscuits, proximate analysis, iron, moisture, fiber, ash, fat, protein INTRODUCTION Biscuits are one of the most consumed food products in the market since it is ready to eat and at affordable price. We get our daily intake of nutrients, including vitamins, minerals, proteins, energy source, etc., to the food that we eat. Due to this, nutrition labeling of biscuit products is important since biscuits is included in most of peoples diet. For the nutrition labeling, proximate analysis of different constituents of the food product are being analyze. These constituents are moisture, fats, protein, ash, and particular minerals. Also in addition, pH, total soluble solids and titrable acidity is important factor for food quality. Moisture content in food products is important since it is an inexpensive ingredient of the product. It is an inexpensive filler in products thus, manufacturers used water to lower the product cost due to other ingredients which are expensive (i.e. flour). Water content of food can affect the shelf life and stability of food. But water activity is better indicator than moisture content. Microorganism, which contributes to the food perishable, can use the water for their growth.Protein is important in our diet. Protein is made up of chained amino acids (peptide). There are 20 amino acids but only 12 of this are able to synthesize by our body thus, the other 8 amino acids should be acquired from the diet. If there are essential amino acids that can be obtain in the diet, there are essential fatty acids which the body cannot synthesize. Due to this, fats are needed in the diet. Minerals are needed by our system for maintenance. There is a required daily intake of such and other food constituents approved by Food and Drug Administration (FDA) that is required by our system. Since biscuit is a ready to eat snack and consumed by most

people, most of required carbohydrate, proteins and other nutrients by the body can be acquired from it. In this paper, proximate analysis of equally mixed variety (regular and cheese) of Bread Stix was determined. Proximate analysis includes moisture, crude fats, crude fiber, crude protein and ash content. Iron content which is included in the ash was also analyzed. Apart from the proximate analysis, pH, total soluble solids and titrable acidity were determined. The proximate analysis was compared to the nutrition fact provided by the manufacturer. MATERIALS AND METHODS pH Determination. Biscuits (Bread Stix) were ground using mortar and pestle and 10 g was weighed in an analytical balance. This was homogenized with distilled water (10 mL). The pH was then determined using pH meter. This procedure was done in triplicate. Titrable Acidity Determination.Biscuits were ground using mortar and pestle. 5 g was weighed using analytical balance and placed into 100-mL Erlenmeyer flask. It was homogenized with distilled water (25 mL) making a 1:5 dilution. Homogenized sample was filtered Total Soluble Solids Determination.5 g of ground biscuit sample was weighed and homogenized with distilled water (20 mL). 2 drops of the homogenized sample was placed on the hand-held refractometer. The reading was recorder as oBx and the 1:4 dilution was accounted. This was done in triplicate. Moisture Determination. Porcelain crucible and filter paper was dried in drying oven and weighed in an analytical balance. Approximately 4 g of ground sample was placed at the center of the dried filter paper. This was folded and place inside the crucible. The crucible was subjected to overnight drying in oven drier. The oven had a temperature of 105oC during drying. After, the crucible with filter paper and sample was weighed to determine the moisture loss. This was done in triplicate. Crude Fat Determination.Weight of flask was determined.Approximately 1 g of ground sample was placed on a filter paper and then folded. The sample was placed into an extraction thimble. The thimble was placed inside the extractor. Ethyl ether (60 mL) was placed in weighed flask. The extractor was then connected to the flask and to the reflux condenser. The flask was placed in a steam bath for 6 hours. After, the solvents (ethyl ether) was removed and evaporated in a hot plate. The flask was dried in an oven with 100oC for 30 minutes, placed in a dessicator and weighed. Drying and weighing of the flask was repeated until the weight was constant or the change is too small. This was done in triplicate. Crude Fiber Determination.2 g of ground sample was weighed and placed in a 400-mL tall beaker. 1.25% sulfuric acid (200 mL) was added. The beaker was covered with watch glass and boiled for 30 minutes. Distilled water was added to make up for any loss in volume during boiling. Whatman No. 541 filter paper was fluted. It was placed in 250-mL beaker and dried at 105oC for one hour. The weight of the filter paper as determined after. The hot solution was filtered using ordinary filter paper and washed with distilled water. The residue was washed back

in the tall beaker with total of 100 mL hot distilled water. 2.5% NaOH solution (100 mL) was added. The solution was boiled for 30 minutes with addition of distilled water to make up for the loss volume. The solution was filtered through the weighed filter paper. The residue form the sides of the beaker was washed using hot distilled water. The residue together with the filter paper was transferred to the tared crucible. It was dried for 30 minutes in 105oC oven and weighed. The drying and weighing was repeated until the weight was constant or the change is very small. The filter paper and contents in tared crucible was incinerated in furnace overnight. The furnace was turned off and cooled to tolerable temperature. The crucible was transferred to a dessicator. After five minutes, The crucible was weighed. This was done in triplicate. For the computation of amount of crude fiber please refer to appendix. Total Ash Determination.Porcelain crucible was dried and weighed. Ground sample (10 g) was weighed and wrapped in a weighed filter paper (ashless). The crucible is then place in a muffle furnace and ignited at approximately 550oC overnight. The muffle furnace was turned off and waited to cool at tolerable temperature. The crucible was transferred to a dessicator carefully. After five minutes, the crucible was weighed and the amount of ash was determined by difference. This was done in triplicate. Crude Protein Determination.Ground sample (5.0g) was weighed and homogenized using water (30 mL). This made a 1:6 dilution factor. This was done in triplicates. The samples were subjected to UV/vis spectrophotometer at 280nm absorbance and 260nm absorbance. For the calculation for determination of amount of protein please refer to appendix. Mineral Analysis (Iron).Approximately 0.5 g of ground sample was placed in a 150-mL beaker. Distilled water (30 mL) and concentrated nitric acid (10 mL) was added. Hot plate was placed inside the hood. The beaker was slowly boiled on the hot plate until the volume was reduced to about half the original. Another concentrated nitric acid (10 mL) was added to the solution and heated until the volume was reduced to about 10-15 mL. The solution was filtered into 100-mL volumetric flask. The sides of the beaker was rinsed down and filtered then added to the solution in 100-mL volumetric flask and it was diluted to the mark with distilled water. 100 ppm of iron stock solution was made from ferric nitrate (solid). The stock solution was diluted to 0.1, 0.3, 0.5, 1.0 and 1.5 ppm for the standard solutions. The standard and the sample was then subjected to Atomic Absorption Spectrophotometer (AAS) at wavelength . This was done in triplicates.

RESULTS AND DISCUSSION Table 1: Proximate Analysis of Mixed Regular and Cheese Bread Stix (wet and dry basis)
Bread Stix Proximate Analysis (wet basis)
Moisture Content Crude Fat Crude Fiber Total Ash Iron Protein Carbohydrates Wet basis 4.54% 10.73% 4.13% 2.98% 8.18x10-3 % 0.68% 76.94% Dry basis --11.30% 4.34% 3.14% 8.60x10-3 % 0.71% 80.51%

Table 2: Average Nutrition Fact of Regular and Cheese Bread Stix


Nutrition Fact (per 30 g biscuit)
Total Fat Saturated Fat Sodium Total Carbohydrate Dietary Fiber Sugars Protein Iron Mass 5.0 g 4.5 g 65 mg or 0.065 g 22 g 1g 7g 2g 4% of 18 mg = 0.00072 g Percent 16.67% 15% 0.22% 73.33% 3.33% 23.33% 6.67% 0.0024%

Determination of pH, Titrable Acidity and Total Soluble Solids.

The pH and titrable acidity corresponds to acidity but the two are not synonymous. pHis the measure acidity or basicity (acid strength) while titrable acidity is the total acid present in the food. The pH of the biscuit is 6.71, approximately 7, means that it is in the neutral range. Since pH is at neutral, the titrable acidity or total acid should be very little. From the result, titrable acidity is 8.887x10-6% which is very low. Total soluble solid is measured in brix. 1 brix indicates that there is 1% sugar. From the paper of Omobuwajo, percent carbohydrate in biscuits made from wheat flour is 66.75%. carbohydrate is the main source of sugar in food products thus the total soluble solid of the bread stix should be near 66.75%. From the result above, the average total soluble solid is 46% which is much lower than 66.75% carbohydrate of biscuits analyze by Omobuwajo. This is due to the fact the readings from refractometer is subjective to analyst. Moisture Determination. Moisture is one of the important analysis made to determine food quality and also the most difficult to acquire an accurate data. From the nutrition fact of the two types of bread

stix(average), by difference (subtracting all components indicated from 30 g), the moisture content is 3.13%. From the result above, the moisture content determined is higher compared to the literature. This might due to the exposure of the crucible containing the sample during weighing after drying which gives a false high result. Crude Fat Determination. From the nutrition fact of the red and blue bread stix (refer to table 2), for every 30 g of bread stix there is 5g of total fat which is 16.67% (wet basis). In the paper of mamatet al., 10 semi-sweet biscuits were analyzed and their average total fat is 11.79%. Some of semi-sweet biscuits analyzed have about 14 to 15% total fat and the others have about 9% total fat. The percent total fat of the bread stix is near the value of other biscuits percent total fat. From the table above, the average percent fat is 10.16% which is lower than that of the literature (16.67%) this might be due to incomplete extraction. Since the reflux only run for 6 hours when it should be run for 8-16 hours. The solvent (ethyl ether) was removed in the thimble by transferring it to the flask but it should be left and let drip back to the flask (requires more time). Also, if other solvent was used, the result may vary. Crude Fiber Determination. Fiber is like a polysaccharide which contains chain of monosaccharide but humans are not able to digest fiber. It may be digested by the bacteria present in the large intestine. Even though it cannot be used as carbon source, it has a beneficial effect. Adequate consumption of fiber can minimized health problems such as cardiovascular diseases. From the nutrition fact, per 30 g of biscuit there will be 1 g of dietary fiber which is 3.33%. But for the proximate analysis done, crude fiber was determined. Dietary fiber of a food product is much larger compared to the crude fiber. Crude fiber is only one-seventh to one-half of dietary fiber. Therefore, percent crude fiber should be lower than percent of dietary fiber in Bread Stix biscuit. From the proximate analysis above (Table 1), percent crude fiber is 4.13% which is relatively high compared with Bread Stix dietary fiber, 3.33%. From the paper of Bose et al., proximate analysis of the controlled biscuit without husk (ordinary biscuit made of wheat flour) has 1.67% crude fiber. Comparing the result of 4.13%, it is really high for biscuit product. This inaccuracy may due to the analysis. During digestion, crude fiber from filter paper after acid hydrolysis should be washed and subjected to alkali digestion. But fiber in the filter paper was hard to separate from filter paper hence; there are some bits of filter paper still with the fiber. Due to this, there is a false high result. Total Ash Determination. Ash is the inorganic components or the minerals present in food (Nielsen, S. S. Food Analysis). From the nutrition fact of red and blue bread stix, the inorganic component is sodium and iron. The average sodium content of the two, per 30 g, is 65 mg and they have 4% of the required daily intake of iron (18 mg). Thus, the inorganic content is 65.72 mg or 0.06572 mg which is 0.22% of the food. From the paper of Omobuwajo, biscuits that made from wheat flour has 0.90% ash which is higher compared to 0.22% of bread stix (based from sodium and iron). From the result in table 6, the average of three trials for ash analysis content of bread

stix(average of red and blue) is 2.83% which is far higher compared to 0.22%. This may indicate that other minerals than sodium and iron is present like calcium, zinc, etc. And maybe theres a false high result since during the careful transfer of the crucible from the furnace to dessicator after ashing, the time it take for the transfer is too long due to small space and moisture may incorporate. Also ashing at 500-5250C results to false high result since there is less decomposition of carbonates. Crude Protein Determination.

For the protein analysis, Warburg-Christian assay was used. From the nutrition fact of the bread stix, for every 30 g there is 2 g of protein which is 6.67%. Again from the paper of Mamat et al., the average percent protein of ten semi-sweet biscuit is 6.45% which is similar to the protein content said in the nutrition fact of bread stix but from the result of the protein analysis using Warburg-Christian assay, only 0.62% of protein is present. The determined crude protein is too low compared to the literature. This might due to the fact the in Warburg-Christian assay, it quantify protein by the absorbance of the aromatic ring present in tryptophan and tyrosine. If there is few tryptophan and tyrosine in the protein present in bread stix, there will be a false low result. Mineral Analysis (Iron). Iron is one of minerals that are needed by the body. For example, in hemoglobin there is iron in the center of that protein. Iron is where oxygen binds. For this reason, blood carry oxygen throughout the body. In the nutrition fact provided by the manufacturer of Bread Stix, it is stated that both regular and cheese flavored have 4% of the percent daily value of Iron per 30 g or biscuit. FDA provides a list of the required daily intake of minerals, protein carbohydrates, etc. based on 2,000 calories daily value. 18 mg is the required daily intake or daily value for iron thus, 4% of it is 0.72 mg or 7.2x10-4 g. Per 30 g of bread stix there will be 0.0024% iron. From the result above, iron content is 0.00818% which is high compared to what the manufacturer stated. This must be due to

CONCLUSIONS Proximate analysis of food determines the major components of food which includes moisture, lipids (fats), fiber, ash, protein and carbohydrates. The components of food determine its quality and stability. Apart from the said food components; pH, total soluble solids and titrable acidity indicates food quality and stability. The result for proximate analysis (moisture, crude fat, crude fiber, crude protein, and specific mineral (iron) is not the same as with the manufacturer states in the nutrition fact. This inaccuracy is due to the following: for moisture, exposure to air before weighing of dried sample; for fiber, filter paper impurities gives a false high result; for protein, method use may contribute to error.

With the results of proximate analysis, even though it does not have the same value as stated in the nutrition fact (true value), Bread Stix can be one of the source of the required daily intake of protein, carbohydrates, iron. APPENDIX Determination of pH, Total Soluble Solids and Titrable Acidity Table 3: Determined pH and Total Soluble Solids of Bread Stix
Test
pH Total soluble solids

40

47

51

53.5

50

50

55

50

52

Table 4: Determined Titrable Acidity of Bread Stix


Test
% Titrable Acidity

Trial 1
7.2287x10-6

Trial 2
9.6383x10-6

Trial 3
9.6x10-6

Trial 4
8.4335x10-6

Trial 5
9.5620x10-6

Trial 6
8.9978x10-6

Ave. Std. Dev


8.9978x106 9.8148x107

      

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6.6711 0.0310 49.83 4.3445

Trial 1 6.65

Trial 2 6.66

Trial 3 6.66

Trial 4 6.65

Trial 5 6.63

Trial 6 6.66

Trial 7 6.70

Trial 8 6.71

Trial 9 6.72

Ave. Std. Dev

Table 5: Determined Moisture Content of Bread Stix


Trial 1
Wt. dried crucible w/ cover + filter paper + wet sample Wt. dried filter paper Wt. dried crucible w/ cover Wt. wet sample Wt. dried crucible w/ cover + filter paper + dried sample Wt. dried sample Loss in weight % Moisture Ave. % moisture Ave % moisture std. dev 42.22 g 1.14 g 36.98 g 4.10 g 42.03 g 3.91 g 0.19 g 4.63%

Trial 2
42.80 g 1.14 g 37.60 g 4.06 g 42.63 g 3.89 g 0.17 g 4.19%

Trial 3
41.69 g 1.15 g 36.35 g 4.01 g 41.45 g 3.77 g 0.24 g 5.99%

Trial 4
39.91g 1.11g 34.80g 4.09g 39.8g

Trial 5
39.35g 1.14g 34.10g 4.07g 39.05g

Trial 6
43.86g 1.15g 38.63g 4.08g 43.61

Trial 7
41.56g 1.13g 36.31 4.12g 41.37

Trial 8
40.32g 1.12g 35.12 4.08g 40.12

Trial 9
38.84g 1.15g 33.57 4.12g 38.64

3.91g 0.18g 3.91%

3.90g 3.91g 0.17g 0.17g 3.90% 3.91% 4.54% 4.54% 0.6770

3.93 0.19 g 4.61%

3.88 0.20 g 4.90%

3.92 0.20 g 4.85%

Wt. dried flask (Wo) Wt. sample (S) Wt. flask w/ fat (W) Wt. fat % Fat

        

Ave. % fat Ave. % fat

std. dev.

Table 6: Determined Crude Fat in Bread Stix


Trial 1
39.34 g 1.04 g 39.45 g 0.11 g 10.58 %

Trial 2
40.03 g 1.01 g 40.13 g 0.10 g 9.90%

Trial 3
38.40 g 1.00 g 38.50 g 0.10 g 9.00%

Trial 4
39.39g 1.04g 39.50g 0.11g 10.58%

Trial 5
40.03g 1.02g 40.15g 0.12g 11.76%

Trial 6
38.41g 1.03g 38.53g 0.12g 11.65%

Trial 7
40.02 g 1.01 g 40.12 g 0.10 g 9.90%

Trial 8
38.41 g 1.04 g 38.53 g 0.12 g 11.54%

Trial 9
39.15 g 1.03 g 39.27 0.12 g 11.65%

10.73% 10.73% 0.9890

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Table 7: Determined Crude Fiber, Dry and Wet Basis


Trial 1
Wt. fresh sample Wt. filter paper Wt. Crucible w/ cover Wt. residue + filter + crucible w/ cover Wt. residue Wt. ash Wt. dry sample % Crude fiber (dry basis) % Crude fiber (wet basis) Ave. % crude fiber (dry basis) Ave. % crude fiber std dev. (dry basis) Ave. % crude fiber (wet basis) Ave. % crude fiber std dev. (wet basis) 2.04 g 0.9 g 36.20 g 37.40 g 0.24 g 0.16 g 1.9392 g 4.13% 3.92%

Trial 2 Trial 3
2.01 g 0.9 g 35.17 g 36.29 g 0.22 g 0.14 g 1.9100 g 4.19% 3.98% 2.03 g 0.92 g 35.20 g 36.33 g 0.21 g 0.14 g 1.9297 g 3.63% 3.45%

Trial 4
2.00g 0.90g 33.67g 34.78g 0.20g 0.11g 1.915g 4.70% 4.45%

Trial 5
2.02g 0.90g 38.33g 39.44g 0.21g 0.12g 1.935g 4.65% 4.46% 4.34%

Trial 6
2.02g 0.89g 35.53g 36.61g 0.19g 0.11g 1.935g 4.13% 3.96%

Trial 7
2.003 g 0.93 g 35.93g 37.05g 0.19g 0.10g 1.907g 4.72% 4.49%

Trial 8
2.010 g 0.94 g 36.27g 37.40g 0.19g 0.11g 1.914g 4.18% 3.98%

Trial 9
2.00 g 0.89 g 34.75g 35.86g 0.22g 0.13g 1.904g 4.73% 4.50%

   

4.34% 0.3812 4.13% 4.13% 0.3637

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Table 8: Determined Total Ash of Bread Stix


Trial 1
Wt. sample before ashing Wt. crucible w/ cover Wt. filter paper Wt. sample after ashing % Ash Ave. % ash Ave. % ash std. dev 10.00 g 36.20 g 0.92 g 0.27 g 2.7%

Trial 2
10.00 g 36.40 g 0.91 g 0.32 g 3.2%

Trial 3
10.00 g 36.86 g 0.95 g 0.26 g 2.6%

Trial 4
10.04g 34.25g 0.96g 0.37g 3.69%

Trial 5
10.07g 34.01g

Trial 6
10.03g 38.63g

Trial 7
10.01g 35.42g 0.94g 0.22g 2.20%

Trial 8
10.03g 32.58g 0.93g 0.27g 2.69%

Trial 9
10.00g 46.55g 0.94g 0.33g 3.30%

0.96g 0.96g 0.39g 0.26g 3.87% 2.59% 2.98% 2.98% 0.5606

Wt. Sample Volume water Absorbance at 280 nm (A280) Absorbance at 260 nm (A260) Mass Protein % Protein Ave. % protein Ave % protein std. dev

   
  




 

Table 9: Determined Crude Protein in Bread Stix


Trial 1
5.00 g 30 mL 0.8507 0.3465 0.0317 g 0.63%

Trial 2
5.02 g 30 mL 0.8420 0.3719 0.0307 g 0.61%

Trial 3
5.01 g 30 mL 0.8547 0.3872 0.0309 g 0.62%

Trial 4
5.08g 32ml 0.8279 0.3751

Trial 5
5.05g 32ml 0.8107 0.3733

Trial 6
5.00g 32ml 0.7689 0.3218

Trial 7
5.00g 30 mL ----0.0416 g 0.83%

Trial 8
5.00g 30 mL ----0.0397 g 0.79%

Trial 9
5.00g 30 mL ----0.0385 g 0.77%

0.0319 0.0311 0.0303 g g g 0.63% 0.62% 0.61% 0.68% 0.68% 0.0900

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Table 10: Percent Iron Determined in Bread Stix


Trial 1
Wt. sample Volume nitric acid used Volume distilled water Volume sample (aliquot) Iron concentration 0.50 g 20 mL 30 mL 100 mL 0.4024 ppm 4.024x 10-5 g 0.0080 %

Trial 2
0.51 g 20 mL 30 mL 100 mL 0.3267 ppm 3.267x 10-5 g 0.0064 %

Trial 3
0.51 g 20 mL 30 mL 100 mL 0.4286 ppm

Trial Trial Trial 4 5 6


0.54g 0.51g 0.50g

Trial Trial 7 8
0.549 g 20ml 30ml 100m l 0.43p pm 4.30x 10-5 g 0.007 8% 0.531 g 20ml 30ml 100m l 0.5pp m 5.00x 10-5 g 0.009 4%

Trial 9
0.504 g 20ml 30ml 100m l 0.46p pm 4.60x 10-5 g 0.009 1%

Mass iron % Iron Ave. mass iron Ave. mass iron std. dev. Ave. % iron Ave. % iron std. dev.

20 ml 20 ml 20 ml 30 ml 30 ml 30 ml 100 100 100 ml ml ml 0.382 0.403 0.485 8 5 5 ppm ppm ppm 4.286x 3.828 4.035 4.855 10-5 g x10-5 x10-5 x10-5 g g g 0.0084 0.007 0.007 0.009 % 1% 9% 5% 4.2439x10-5 g 4.2439x10-5 5.36x10-6 g 8.18x10-3 % 8.18x10-3 1.04x10-3 %

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