Phytochemical Content Of Coffee Substitute Beverage Biology Essay

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Being identified as one of the plants that can be used as food, feed, and fuel, sweet sorghum is a multipurpose crop. Its grains can be a staple food among people while its leaves can be forage for animals. High prices of of fossil- fuels resulted in the increased interest in the production and use of biofuel. Its stem produces high amount of sugar which is beneficial for fuel production. Sweet sorghum [Sorghum bicolor (L) Moench] is similar to grain sorghum with a sugar-rich stalk comparable to sugarcane. It has wide adaptability and grows rapidly. In recent years, there is an increasing interest in the utilization of sweet sorghum for ethanol production in India (Reddy, 2007).

Although about 75% of the world's sorghum crop is consumed by human, the use of sorghum as a whole is very limited in the Philippines, where rice and corn have been recognized as important energy sources. Efforts were already made towards developing recipes and products out of sorghum grains in order to increase its utilization among Filipinos. One of the popular beverages in the world is coffee. Even in the Philippines, coffee has been identified as one of the commonly consumed food item. However, coffee consumption is coupled with health issues concerning its caffeine content. With all the negative issues on coffee, potential products to substitute coffee in human's diet had surfaced in the market. Coffee substitutes are non-coffee products, usually without caffeine, that are used to imitate coffee.

Importance of the Study

Due to increase demand for fossil-fuel all over the world, a lot of studies focused for alternative source of fuel nowadays. Aside from jatropha, cassava, corn, coconut, soybeans, and sugarcane, sweet sorghum is identified as one of the promising crop for biofuel production. The stem of sweet sorghum produces high yield of syrup used in making biofuel. Due to its low production cost, propagation of sweet sorghum is being pushed globally. Sweet sorghum has been introduced in the Philippines in 2005 mainly for biofuel production. If the country will go full-scale in the implementation of 10% bio-ethanol-blended gasoline as mandated by Republic Act 9367, it can save approximately 565 million liters of gasoline per year. However, mass production of sweet sorghum poses threat on the food security as this will lessen the farmlands for Filipino food crops. In order to address this issue, full-scale production of sweet sorghum should target both energy and food security in the Philippines. Thus, if the sweet sorghum is the prime feedstock for ethanol production, its grains should be beneficial as food for Filipinos. However, Filipinos are not yet fully aware on the utilization of sweet sorghum as food, hence; many efforts are focused in developing recipes and products out of sorghum grains. The Pampanga State College already developed recipes from sweet sorghum which include porridge, burger, soups, native cakes, pastillas, suman, tupig, pop sorghum,biko, sapin sapin, and shanghai. This study wishes to help increasing the utilization of sweet sorghum, particularly the grains, by developing it into coffee-substitute. Coffee is one of the commonly consumed food items among Filipinos according to the 7th National Nutrition Survey (2008). Developing sweet sorghum-coffee substitute would not just help in increasing the utilization of grain sorghum among Filipinos, this study will also provide a healthful beverage which has phytochemicals, no caffeine, high in zinc, and cheaper coffee substitute option.

Objectives

In general, the objective of this study is to produce a healthful coffee made from sweet sorghum. Specifically, this study aims to:

standardize process in making sweet sorghum coffee-substitute;

identify appropriate sweet sorghum coffee-substitute powder to water ratio;

determine the acceptability and sensory characteristics of sweet sorghum coffee-substitute;

measure proximate composition (moisture, protein, fat, fiber and total ash), starch and amylose content, dietary fiber, fatty acid profile, amino acid profile, mineral content (iron and zinc), and photochemical (tannins, phenols, flavonoids, and saponins) present in sweet sorghum coffee-substitute;and

determine the effect of processing sweet sorghum into coffee in terms of its nutritional composition and antioxidant capacity;

Time and Place of the Study

The samples will be prepared and evaluated at the Institute of Human Nutrition and Food while the analysis of the nutritional and phytochemical content will be done the Institute of Plant Breeding (IPB), UP Los Baños. The study will commence on November 2012 and will be completed by March, 2013.

REVIEW OF RELATED LITERATURE

Sweet Sorghum

Characteristics

Sweet sorghum (Sorghum bicolor L. Moench) species belongs to the tribe Andropogoneae of the family Gramineae (Harlan and de Wet, 1972). It said to be the only crop that provides grain and stem that can be used for sugar, alcohol, syrup, jaggery, fodder, fuel, bedding, roofing, fencing, paper and chewing [Schaffert, R. E. 1992]. This crop can be grown all year round in tropics and in the sub-tropics. It is a major crop grown in the semi-arid and arid regions of Africa and Asia where it is used as a staple food (NRI, 1999). Sorghum is one among the few resilient crops that can adapt well to climate change conditions, particularly the increasing drought, soil salinity and high temperatures (ICRISAT,2012).

Sorghum plays an important role in crop rotation systems. Sweet sorghum is a C4 pathway crop. Among other particularities, C4 plants have a characteristic leaf anatomy, called "Kranz anatomy", which gives special separation between the photosynthetic CO2 fixation and the synthesis of assimilates - compounds produced by plants as a result of the photosynthesis and responsible for plant growth. This compartmentalization allows a higher solar radiation use and high photosynthetic efficiency of sorghum comparing to C3 crops, more common in temperate regions of the world (Intelligent Energy, 2011).

Structure

The roots of the sweet sorghum are adventitious and have numerous lateral roots (Dogget, 1988). Its stalk range from 1.5 m - 3.0 m tall and contain sweet juice (Martin et. Al, 1975). Sweet sorghum follows the same pattern of tillering as in the other sorghums. There is a single bud at each node and the lowest node buds may develop to form tillers and prop roots, while those on the upper nodes may produce branches (Dogget, 1988). The leaves of sweet sorghum differ from those of grain sorghum with a dull midrib due to the presence of juice in the air spaces of the pitting tissue (Martin, et al., 1975). It is caryopsis with a roughly rounded shape and differently coloured, depending on the variety. Sweet sorghum fruits are usually smaller than the grain sorghum ones. The weight of one thousand seeds is about 21g, varying between 16-28 g (Petrini et al., 1993).

The sorghum grain

The grain of the sweet sorghum is almost appears like the other cereals (Figure 1). Its major components are the pericarp (outer covering), the testa between pericarp and endosperm (which may or may not be present), the endosperm, and the embryo. The endosperm may be corneous (vitreous) or floury, and the testa may contain tannins which affect the nutritional quality of the grain. Tannins are high molecular weight phenolic compounds which are found in grains with a brown pericarp and pigmented testa. Certain tannins known as condensed tannins, form complexes with proteins and reduce their digestibility. They can also form complexes with the alimentary tract proteases, reducing the digestibility of the proteins in the grain. Despite this negative nutritional effect, high tannin varieties continue to be grown due to their bird and insect resistance, and higher malting potential than white grain varieties. In some traditional foods and beverages, the phenolics of red sorghum give a desired flavour and colour. The negative effects of tannins on nutritional value can partially be overcome by removal of the testa by mechanical dehulling, or by alkaline treatment at the village level (traditionally by using wood ash) (Chantereau and Nicou, 1994).

Figure 1. Structure of sorghum grain (after Sautier and O'Deye, 1989 as cited by NRI, 1999)

Varieties

Commercial varieties of Sorghum bicolor (L.) Moench are categorized into the

following agronomic types: grain sorghum, fiber sorghum, forage (or fodder) sorghum, broomcorn and sweet sorghum. Grain sorghums are generally grown in regions which are too dry or too hot for successful maize production. They are adapted to the drier climates due to several factors (Bennett et al. 1990)

A total of 242 cultivars have been released using germplasm and ICRISAT-bred lines and hybrid parents, over the years in all regions (Asia, Eastern and Southern Africa, West and Central Africa, and Latin America) (Figures 1-5). The number of cultivar releases was highest in Asia (75) including 35 in India, closely followed by Eastern and Southern Africa (74), Western and Central Africal (58) and Latin America (35) (ICRISAT,2012).

Nutritional Quality

 Sorghum has the potential for high levels of iron (more than 70 ppm) and zinc (more than 50 ppm) in the grain, and hence sorghum biofortification (genetic enhancement) of grain iron (Fe) and zinc (Zn) contents is targeted to complement other methods to reduce micronutrient malnutrition globally (ICRISAT, 2012)

However, nutrient from the grain are hardly absorbed by the body due to its high tannin content. In fact, sorghum is commonly called "coarse food "because of its amino acids imbalance and high tannin content (FAO, 1994).

Livestock feed manufacturers prefer to use grains from white sorghums or low tannin pigmented sorghums due to the effect of tannins on protein digestibility. Sorghum is therefore not a direct replacement for maize in a livestock ration. Sorghum has a lower energy density and protein digestibility compared to maize (Table 1) which is reflected in the price offered for sorghum (NRI, 1988).

Table 1. Comparative data on energy and protein levels of sorghum and maize (as feed)

Metabolisable energy for ruminants (MJ / kg)

Metabolisable energy for poultry (MJ / kg)

Protein content (%)

Lysine content (%)

Available lysine content (%)

Sorghum

12.4

13.7

11.0

0.27

0.19

Maize

12.1

14.2

9.0

0.27

0.22

Utilization as Food

As was tagged by Dr. William Dar, president of the International Cereal Research Institute for the Semi-Arid Tropics (ICRISAT), sweet sorghum is a "smart crop" as it can be used as both food and fuel. Aside from its use as food and fuel, it can also be used as feed/forage for poultry and livestock and its bagasse as fertilizer (de Leon, 2011).

In communities where sorghum is grown as a subsistence crop the main food products prepared include thin and thick porridges, fermented and unfermented breads, lactic and alcoholic beers and beverages, malted flours for brewing, malted porridge mixes and weaning foods. In Kenya and South Africa, there is a small but growing market for pearled sorghum as an alternative to rice. In India, proposals have been made for use of dehulled sorghum within feeding regimes for infants and children (Pushpama, 1987). Many countries have investigated the options for a composite wheat-sorghum flour but few have found commercial adoption. Sorghum does not contain the elastic protein, gluten, and thus the functional properties of sorghum for wheat-based bread and biscuit type products limits its inclusion level to a practical maximum of 10-15 percentage before changes in the structure of the product can be positively identified. Inclusion is also dependent upon availability of sorghum, appropriate varieties and the relative price of wheat and sorghum at the mill gate (NRI, 1999).

Many urban consumers consider sorghum to be a subsistence crop of low quality. This low social status for the grain constrains its desirability for inclusion in commercial products designed for urban consumers. In regions where the crop is not a staple, it may have low acceptability relative to maize due to its different organoleptic properties - unpleasant colour, aroma, mouthfeel, aftertaste and stomach-feel (NRI, 1999).

Africa has a tradition of making opaque beers by the use of sorghum as the source of malt and the adjunct, though for commercial brewing maize may often be the source of the adjunct. Opaque beer is a product of a lactic and alcoholic fermentation which is sold in a microbially active state, with a shelf life of only 5-7 days. The principles of the process whether by traditional or commercial methods are illustrated in Figure 4 (Daiber and Taylor, 1995).

In China, sweet sorghum is used as fresh fruit juice for children during autumn for many years. Also, sweet sorghum juice from the stems has been used as raw materials for liqour (50% alcohol), and bagasse used as raw material for fibreboard production in Honan (FAO, 1994). Furthermore, in China, strong liquors are made from the sorghum kernels like the world famous Maotai and Fen liquors. Sorghum grains produce starch, starch noodles and vinegar (NRI, 1999).

Most grain sorghum in China is used as food to make various breads, cakes, dumplings and noodles.

Production

It is a major crop grown in the semi-arid and arid regions of Africa and Asia where it is used as a staple food. In China, sorghum or kaoliang, is one of the earliest cultivated crops growing mainly in Northeast, Northwest and North China temperate zones.

The trade in sorghum is small compared with the major grains such as wheat, maize, barley and rice. The main importers of sorghum are Japan, Mexico, the former USSR (CIS) and Venezuela. Within most developing countries, the sorghum crop rarely reaches the market. It is grown for home consumption unless there is a bumper crop, or if cash is needed. The major producers of sorghum for domestic or foreign trade are the USA, Argentina and Australia. Most is used in livestock feed.

Regional Production

The sorghum area in Asia declined continuously from 23 million ha in the early 1970s to 9 million ha in 2009. The same situation happened in India in which there were more than 16 million ha in 1981, but has gradually decreased to 7.8 million ha in 2007-08 (still 20% of the world's sorghum area).  Despite the decrease in area over the years, production has been sustained at 7.3 million tons (2009) due mainly to adoption of improved varieties and hybrids. Sorghum grain yields in India have averaged 1170 kg/ha in the rainy season and 880 kg/ha in the postrainy season in recent years. On the other hand, area and production in Eastern and South Africa has increased significantly from the early 1970s to 2009, while there has been a marginal (18%) increase in productivity from 800 kg/ha to over 940 kg/ha during the same period. In West and Central Africa, the increase in sorghum area was more than two-fold from 1972 to 2008 (7.39 to 16.59 million ha), while production increased by almost four times during the same period. Sorghum area in Latin America increased from 4 million ha in the early 1970s to 5 million ha in the early 1980s followed by a decrease to the 4 million ha level of the early 1970s. In 2009 production increased to 11 million tons. Productivity increased by 55%, from 2000 kg/ha in the early 1970s to 3100 kg/ha in 2009.

Coffee and Coffee Substitutes

Coffee

Coffee is one of the world's most popular stimulants, consumed by about one-third of the world's population. Its total consumption as a beverage is second only to tea. The history of coffee as a beverage is an interesting and intriguing blend of political, financial, cultural and religious influences. Coffee was first known to be cultivated by the Arab colony at Harrar in the thirteenth century. In 1688, coffee took the place of beer as New York City's favorite breakfast drink. In the early 1900s, afternoon coffee was a standard occasion in Germany. Today coffee is grown, brewed and enjoyed worldwide (Jami, 2003).

 

Health Concerns against Coffee Consumption (Maam I still have to search

published literatures supporting these claims)

Coffee is a central nervous system stimulator that gives the adrenals a kick and causes production of the stress handling hormone adrenalin and the production of more cortisol resulting in short term benefits of heightened awareness / alertness and more energy, but long term may result in a crash after each consumption to lower levels of energy than previously thereby necessitating another cup and another cup, etc. Thus, it may be addictive and ultimately may result in adrenal exhaustion.

Even though coffee has never been conclusively linked to cancer, it does contain acknowledged carcinogens such as caffeine and other chemicals produced by the high heat of roasting such as creosote, pymdine, tars, and polycyclic aromatic hydrocarbons.

Caffeine interferes with adenosine, a brain chemical that normally has a calming effect.

Cortisol levels are raised which in turn results in constriction of the blood vessels, harder pumping of the heart and higher blood pressure.

The liver in fetuses and newborns cannot metabolize caffeine, so it remains in the body for up to four days stimulating the nervous system resulting in irritability and sleeplessness.

Coffee has been associated with low birth weight, birth defects, miscarriages, premature birth, inability to conceive, and sluggish sperm.

Many of the chemicals in coffee and decaf irritate the stomach lining causing an increase of stomach acid leading to digestive disorders.

Caffeine may cause problems with blood sugar control after meals for type 2 diabetics.

Coffee excites more rapid peristaltic movements of the intestines resulting in shorted transit times and less absorption of nutrients.

Coffee hampers the absorption of essential minerals and vitamins such as magnesium, zinc, iron, potassium, and B's.

Coffee-substitutes

With all the negative issues on coffee particularly due to its caffeine content, potential products to substitute coffee in human's diet had surfaced in the market. Coffee substitutes are non-coffee products, usually without caffeine, that are used to imitate coffee. The purpose of coffee substitutes can be used for medical, economic and religious reasons, or simply because coffee is not readily available. Roasted grain beverages are common substitutes for coffee. Coffee substitutes are sometimes used in preparing foods served to children or to people who avoid caffeine, or in the belief that they are healthier than coffee. For religious reasons, some members of the Church of Jesus Christ of Latter-day Saints, also known as Mormons, refrain from drinking coffee but may enjoy a substitute.

MATERIALS AND METHODS

Source of Raw Material

The sweet sorghum variety ICSV 700 from the IPB, UP Los Baños will be utilized in making the coffee-substitute grounds/powder.

Experimental Design

The sweet sorghum grains will be divided into parts, one will be well-milled (de-hulled then milled) while the other will only be de-hulled (Figure 1). Both the de-hulled and well-milled grains will be ground into grits (size around 12-14). The grits will then be roasted to a coffee-like ground. Moreover, the grits will be made into coffee-like powder using two different processes. The first process would subject the sweet sorghum grains to roasting before it will be ground. For the second process, the sweet sorghum grits will be ground first into powder before roasting.

ICSV 700

De-hulled

Well-milled

Coffee-like grounds

Grits

(size:12-14)

Grits

(size:12-14)

Coffee-like grounds

Coffee-like powder

Coffee-like powder

Roasted then powdered

Powdered then roasted

Roasted then powdered

Powdered then roasted

Sweet Sorghum Coffee-substitute Processing

Drying and Hulling

The sweet sorghum grains will be dried in a dehydrator until it reaches 10-12% moisture content. Moisture content will be determined using the digital grain moisture meter. After the grains have been dried, it will pass through the hulling machine to remove the husk. A part of the grains will be milled further after de-hulling that would represent the well-milled sample. Both the sample grains (de-hulled only and well-milled) will be initially ground into grits (size).

Roasting and Grinding

Process 1. Put the sweet sorghum grits in a square pan. Preheat the oven to 420oF. Roast the sorghum for 55 minutes and stir the grains every after 10 minutes. Set aside to cool and then grind to 80-100 mesh size powder using an electric coffee grinder.

Process 2. Grind the sweet sorghum grits up to 80-100 mesh size powder using an electric coffee grinder. Put the sweet sorghum powder in a square pan. Preheat the oven to 420oF. Roast the sorghum powder for 55 minutes and stir the grains every after 10 minutes. Set aside to cool.

Brewing

The sweet sorghum grounds/powder will be brewed in a saucepan. The proportion that will be used is 2 tablespoon of sweet sorghum grounds/powder per cup of water. The grounds/powder will be brew for six minutes maintaining the optimum temperature of 195-205°F. Afterwards, brewed sweet sorghum coffee will be poured into paper filter to strain out the floating particles.

Sensory Evaluation of Sweet Sorghum Coffee-substitute

Sensory evaluation will be carried out to determine the acceptability and sensory qualities of the Sweet Sorghum Coffee-substitute. A laboratory panel composed of thirty UPLB Faculty and Staff will evaluate the coffee-substitute. Sensory attributes of the sweet sorghum coffee substitute such as color, consistency, aroma, taste, and the general acceptability will be rated using a 7-point hedonic scale (Appendix D). The sample that would gain the highest acceptability will be subjected to chemical analyses.

Chemical Analyses

Proximate composition (moisture, protein, fat, fiber and total ash), starch and amylose content, dietary fiber, fatty acid profile, amino acid profile, zinc, and phytochemicals present in sweet sorghum coffee-substitute will be measured following the methods in Appendix B.

Statistical Analyses

RESULTS AND DISCUSSION

Table 1.

SUMMARY AND CONCLUSION

RECOMMENDATION

LITERATURE CITED

BENNETT, W.F., TUCKER, B.B. AND MAUNDER, A.B. (1990). Modern Grain Sorghum Production. First Edition. Iowa State University Press. Iowa, USA.

DAIBER, K.H. AND TAYLOR, J.R.N. (1995). Opaque beers. Sorghum and millets: chemistry and technology. Dendy, D., ed. American Association of Cereal Chemists.

DE LEON, DR. A. 2011. BAR Research and Development Digest: Sweet Sorghum-The search for the best cultivar for Western Visayas. Vol.13 (2). Date Accessed: August 9,2012. Available at:<http://www.bar.gov.ph/bardigest/2011/aprjun2011_sweetsorghum.asp>

DOGGET, H. (1988). Sorghum, 2nd ed. Longmans, Green and Co. Ltd: London.

FOOD AND AGRICULTURE OFFICE OF THE UNITED NATION (FAO-UN).1994. Integrated energy systems in China-The cold Northeastern region experience.

HARLAN, J.R. AND DE WET, J.M.J. (1972). A simplified classification of cultivated sorghum. CropScience.12:172-176.

INTELLIGENT ENERGY-EUROPEAN COMMISSION. (2011). Sweethanol- Intersectorial Manual:. Italy:Poligrafiche San Marco S.a.s.

INTERNATIONAL CEREAL RESEARCH INSTITUTE FOR THE SEMI-ARID TROPICS (ICRISAT). Sorghum (Sorghum bicolor (L.) Moench). Date Accessed: August 7, 2012. Available at <http://www.icrisat.org/crop-sorghum.htm>

Jami, X. (2003). Coffee. Journal Of Agricultural & Food Information, 5(3), 79-86. doi:10.1300/J108v05n03_07

MARTIN, JH., LEONARD, WH., AND STAMP, D.L. (1975). Principles of field crop production. 3rd ed. Collier McMillan International Edition, London.

NATURAL RESOURCES INSTITUTE (NRI).(1999). Sorghum: Post Harvest Operations. Food and Agriculture Office. Accessed August 3, 2012. Available at: < http://www.fao.org/fileadmin/user_upload/inpho/docs/Post_Harvest_Compendium_-_SORGHUM.pdf >

PETRINI C., BELLETTI A., AND SALAMINI F.(1993) "Breeding and growing sweet sorghum for fuel",Chapter 1. Morphology and Reproduction. Elsevier Science Publishers BN

PUSHPAMA, P. (1987). Supplementary foods in India. pp. 69-77. IDRC monograph. No 249e.

REDDY BVS., RAMESH, S., REDDY PS., KUMAR, AA., SHARMA, KK., CHETTY, SMK., AND AR PALANISWAMY.(2006). Sweet Sorghum: Food, Feed, Fodder, and Fue Crop. ICRISAT.Date accessed: August 10, 2012. Available at: www.icrisat.org

SAUTIER, D. AND O'DEYE, M. (1989). Mil, Mais, Sorgho - Techniques et alimentation au Sahel. pp. 171. Harmattan. Paris, France.

SORIANO, JR, H. M. S., DE JESUS, N. G. D., ZABALA, E. C., LORIA, R. D., COSIO, R. D., RAFAEL, R. R., SOLIS, L. G., PINEDA, E.B., PINEDA, L.M., AND Z.M. BATTAD. (2010). The Amazing Sweet Sorghum: Pampanga Agricultural College's Initiatives in Promoting and Commercializing its Utilization as Human Food, Animal Feed And Bio-Fuel. J. ISSAAS 16(1), 8-16.

THE PHILIPPINE STAR: Sweet sorghum - the next anti-diabetic sweetener. Issue date:May 20,2012Accessed dae: August 9, 2012. Available at:<http://www.philstar.com/Article.aspx?articleId=808691>

Zabala, E.C., Battad, Z.M, and de Jesus N.G.(2009). Sweet sorghum food products :a compendium.Magalang, Pampanga.PAC and BAR

APPENDICES

Appendix A. BUDGET REQUIREMENT

Activity

Chemical Analyses

Proximate analysis (moisture, protein, fat, fiber and total ash)

Starch Content

Amylose Content

Lysine Content

Tryptophan

Total Antioxidant Content

Amino acid profile

Fatty Acid Profile (2000/FA est. 3FA needed)

Tannin Content

Phenols

Zinc

Sensory Evaluation

Office supplies

Sensory evaluation Paraphernalia

Raw material (sweet sorghum grain)

TOTAL

APPENDIX B

NUTRITIONAL COMPOSITION

Analysis of Moisture

Moisture content refers to the amount of water present in the sample. Fresh samples such as leaves and tubers contain water as high as 80-90%. Dried samples such as flours contain water less than 10%. Samples dried under the sun generally have moisture content in the range of the atmospheric moisture which Is about 14%. Unless samples are hygroscopic after drying, as characterized by samples with high sugar content, they normally do not absorb as much water in the atmosphere to equilibrate with the atmosphere.

There are several ways of determining moisture content, although the most common is the oven-drying method. Toluene method is another way of doing moisture determination.

The method described here makes use of an air-forced draft oven and low temperature drying for a long period of time. This is done to preserve some of the components that may be destroyed when high temperature is employed. Sugars generally are caramelized in high temperature drying. Chlorophylls are also altered resulting in browning of leaves when dried at temperatures over 40ËšC.

Equipment and Supplies

Analytical balance

Moisture dish

Oven

Dessicator

Knife

Chopping board

Procedure

Chop the sample to smaller particles.

Weigh 10 gram sample in duplicate and place in a moisture dish.

Place the sample in an oven, set the oven temperature at 45ËšC.

Dry the samples overnight.

Remove the samples from the oven and place in a dessicator.

Equilibrate to room temperature.

Get the exact weight of the sample.

Calculation

Calculate percent moisture from the following equation:

% Moisture =

Analysis of crude fat (Ether-Soluble Fraction)

Crude fat is an estimation of the oil content and other pigments that are removed by organic solvents. The most common organic solvent used is diethyl ether or petroleum ether although many other organic solvents are applicable.

Equipment and Supplies

Analytical balance

Filter paper

Soxhlet extraction apparatus

Dessicator

Oven

Reagent

Petroleum ether

Procedure

Prepare filter paper thimbles by folding small sheets of filter paper and sealing off one end by a stapler

Get the exact weight of the paper thimble.

Place the approximately 1g of the dried ground sample.

Get the exact weight of the thimble and the sample.

Staple the other end of the thimble and place the thimble on a soxhlet fat extractor.

Put petroleum ether into the boiling flask and reflux the samples for 8 hours.

Remove the thimbles from the extractor and place in an oven for at least an hour at 45ËšC.

Equilibrate in the dessicator.

Remove the staple on one side of the thimble and get the exact weight of the thimble and defatted sample.

Calculation

Calculate the percent fats in the sample as follows:

% Fat =

Where:

Wt. of fats= (wt of thimble + sample before refluxing) - (wt. of thimble + sample after drying)

Wt. of sample = (wt. of sample + thimble) - (wt. of thimble)

Protein or total nitrogen analysis using Kjeldahl method

Protein is far the most common food and feed constituent that are always analyzed, it being used as a nutrition index, aside from moisture and fat content.

Crude protein or total nitrogen content is determined using the classical method developed by Kjeldahl in 1872; hence the method is known as Kjeldahl method. The method is highly sensitive although it was developed using large amounts of samples.

While several other methods follow later on, the advent of colorimetric and spectrometric methods are used in Kjeldahl digest, the reference for nitrogen analysis is still the traditional Kjeldahl method.

The method is divided into three parts: digestion with an inorganic acid, distillation of the nitrates into nitrogen and titration of the excess ammonia with standard acid.

Digestion is carried in the presence of heat and strong acid H2SO4. This general reaction takes place:

Protein + H2SO4 ----------------> (NH4)2SO4

In this reaction, nitrogen in protein is transformed into an inorganic salt.

Distillation with a strong base, like 40% NaOH, converts the ammonium salt into ammonia (NH3) according to the reaction:

(NH4)2SO4 + NaOH -----------> 2NH3

The ammonia is trapped in the distillation end with boric acid (H3BO3).

The excess ammonium is titrated against a standard acid, 0.020N HCl using a mixed indicator to a gray end point with pH 5.77.

NH3 + H3BO3 ------------------> NH4+ + 2H2BO3-

H2BO3- + HCl -----------------> H3BO3 + Cl-

Equipment and supplies

Analytical balance

Digestion flask, 30 mL

Micro digester

Micro distillation set-up

Titration apparatus

Erlenmeyer flask, 125 mL

Reagents

Sulfuric acid, concentrated

Sodium hydroxide, 40% w/v - Weigh 40.0 gmNaOH and dissolve and dilute 100mL with distilled water.

Boric acid, 4% w/v- Weigh 4.0 gm H3BOand dissolve and dilute to 100mL distilled water.

Mixed indicator- A mixture of 0.2% methyl red and 0.2% bromcresol green in a 1:5 proportion. Dissolve 100 mg methyl red indicator in 50 ml 95% ethanol. Add 500 mg brocresol green to the solution and dilute to a total volume of 300 ml with 95% ethanol.

Catalyst mixture- Mix 1 part selenium powder, 10 parts copper sulfate and 100 parts potassium sulfate thoroughly by pulverizing a porcelain mortar and pestle. Alternately, a prepared selenium catalyst mixture can be bought.

Hydrochloric acid, 0.020N standardized- Dilute 0.167 ml conc. HCL to 100ml with distilled water. Standardize against standard NaOH solution.

Procedure

C1. Digestion

Weigh 0.05g dry, defatted round sample and place in a 30 ml digestion flask.

Add 2 ml concentrated H2SO4 and about 0.20gm catalyst mixture.

Place in a microdigestor, adjust heat setting to high and digest for about 10 minutes or until the solution is clear.

Cool to room temperature.

C2. Distillation

Place the flask in an ice bath and add a small amount of distilled water.

Transfer the sample quantitatively into a steam distiller.

Wash flask at least at least three times with about 15ml distilled water.

Place a125ml Erlenmeyer flask on the receiver end of the distiller containing 10ml of 4% H3BO3 and three drops of mix indicator.

Immediately add 10ml of 40% NaOH into the distiller and close the inlet.

Distill the sample until about 50ml of the distillate is collected into the distiller.

C3. Calculation

Calculate %nitrogen as follows:

%Nitrogen =

Calculate %protein as follows:

%Protein = %Nitrogen x 5.95 (factor for rice)

Nitrogen Assay from KJELDAHL Digest by Colorimetry

Equipment and supplies

Analytical balance 5. Pipetor and dispensers

Spectronic 20 6.Kjeldahl flask, 30 ml

Volumetric flask 7. Microdigester

Test tubes

Reagents

Sulfuric acid, concentrated

Selenium catalyst

Solution A- dissolve 14.19g Na2HPO4, 4.0g NaOH, 50.8g, potassium tartarate in about 300 ml. dilute to 500 ml with distilled water.

Solution B - dissolve 50g NaOH and dilute to a final volume of 500ml with distilled water.

Working buffer - mix equal volumes of solutions A and B just before use

Salicylate nitroprussidereagent - dissolve 200g sodium nitroprusside and dilute to 1L with distilled water.

Hypochlorite solution, 2.5% - dilute 6.5ml of commercial bleach (5.25% NaOCl) to 25ml with distilled water.

Ammonium sulfate, analytical grade.

Procedure

Weigh 0.05g dried powdered sample ad place in a 00ml digestion flask

Add 0.50g selenium catalyst mixture and 2ml H2SO4

Digest in a microdigestor for 3 minutes or until solution is clear.

Cool and transfer quantitatively into an 18x100mm test tube.

Dilute to volume with distilled water and mix well (10 ml).

Take 0.01ml aliquot and place in an 18x150mm test tube.

Add 1.5ml working buffer.

Add 0.40ml salicylate-nitroprusside reagent and mix well.

Add 0.20ml hypochlorite for 30 minutes for color development.

Stand at room temperature for 30 minutes for color development.

Dilute to a final volume of 10 ml with distilled water and mix well.

Get absorbance readings at 650nm.

Run a blank to check for reagents used during assay.

Prepare a standard curve using ammonium sulfate to calculate nitrogen content in a sample.

Preparation of standard curve

Standard-ammonium sulfate, 100µN/ml - dry (NH4)2SO4 at 105˚C for 3 hours. Cool in a dessicator and weigh exactly 47.16mg. Digest salt as in sample. Dilute digest with distilled water to a final volume of 100ml.

Prepare the following tubes:

Tube #

Standard (ml)

Buffer (ml)

Nitroprusside (ml)

Hypochlorite (ml)

1

0

1.5

0.40

0.20

2

0.02

1.5

0.40

0.20

3

0.04

1.5

0.40

0.20

4

0.06

1.5

0.40

0.20

5

0.08

1.5

0.40

0.20

Do analyses as in sample.

Get absorbance readings at 650nm.

Plot the concentration of nitrogen against the absorbance and calculate the slope of lime.

From the slope of the line, calculate nitrogen content of the sample. Convert nitrogen into protein by multiplying %N with 5.95(rice).

Calculation

Use the following equation to calculate for nitrogen content:

% Nitrogen =

Where : slope =

Analysis of Crude Fiber

Crude fiber is that fraction of the sample that is not removed by either an acid or base hydrolysis. It is used as an index to determine the amount of the indigestible matter and approximates the amount of dietary fibers present in the food or feed. It is made up of structural carbohydrates - cellulose, hemicelluloses, lignin and silica, which are left after the proteins and non-structural carbohydrates like sugars and starches are subjected to successive acid and base hydrolysis.

Equipment and supplies

Analytical balance

Reflux apparatus

Suction filtration system

Oven

Dessicator

Furnace

Muslin cloth or equivalent

Sintered glass fiber

Gooch crucibles

Reagents

Sodium hydroxide, 2.5% w/v - dissolve 2.5gram NaOH in 100ml

Sulfuric acid, 2.5% v/v - add 2.5ml concentrated H2SO4 in distilled water to a total of 100ml.

Asbestos/Katsa

Procedure

Weigh 0.1g dry, defatted sample

Place in a 100mlBercillus beaker

Add 50ml 2.5% sulfuric acid.

Place in a reflux apparatus and reflux for 30 minutes.

Wash sample with hot distilled water until the washing with litmus paper.

Add 50ml 2.5% sodium hydroxide and reflux for another 30 minutes.

Wash with hot distilled water until the washing is neutral with litmus paper.

Place in an oven and dry overnight at 105ËšC.

Cool in a dessicator and get exact weight.

Ignite in a furnace overnight at 550ËšC.

Cool in a dessicator and get exact weight.

Calculation

Calculate % crude fiber from the following equation:

% Crude Fiber =

Where : wt of fiber = (wt. crucible + sample after ignition)

Total Ash Content Analysis

Total ash represents the mineral content of the sample. This is usually in the form of silicates and sulfates and other trace elements.

The method discussed here is a direct ignition of the sample in a furnace to remove all the organic components of the sample. The residue after ignition is the ash content of the sample.

Equipment and supplies

Analytical balance

Porcelain crucible

Furnace

Dessicator

Procedure

Weigh 100mg dry, ground samples.

Place in a 30ml porcelain crucible whose tared weight has been taken.

Place crucible with sample into a furnace and ignite overnight at 650ËšC.

Cool to a lower temperature and transfer into a dessicator.

Equilibrate to a lower temperature.

Get the exact weight.

Calculation

Calculate % total ash from the following equation:

%Total ash =

Where : wt. of ash = (wt. crucible + sample after ignition) - (wt. of crucible)

References: AOAC. 1980 Methods of Analysis. Washington: Pergamon Press

RODRIGUEZ F. AND MENDOZA E. 1997. Methods of analysis for screening crops of appropriate qualities, vol.2 (Institute of Plant Breeding, Analytical Services lab manual unpublished). IPB-UPLB. Pp6-7, 34-36, 58-63, 50-51, 92-93

Determination of Starch using Anthrone Reagent

Starch determination is carried on by the hydrolysis of starch to glucose with a strong acid under hot water bath. The breakdown of starch results to glucose and glucose concentration is measured using anthrone reagent. Starch is then calculated from glucose by multiplying the amount of glucose with a factor of 0.90. This factor is obtained from the loss in OH- as a result of polymerization.

Equipment and supplies

Spectrophotometer

Centrifuge

Test tube

Volumetric flask

Pipetor

Water bath

Vortex mixer

Analytical balance

Reagents

Sulfuric acid, 5% - dilute 50ml of concentrated H2SO4 with distilled water to 1000mL

Perchloric acid, 2.5% - prepare a 1:1 5%sulfuric acid:water solution

Anthronereagent - weigh 0.20gram anthrone and dissolve in 100ml of 95% H2SO4.

Ethanol, 95% - dilute 95 ml absolute ethanol to 100ml with distilled water.

Ethanol, 80% - dilute 80 ml absolute ethanol to 100ml with distilled water.

Determination of Starch Content

C1. Hydrolysis

Weigh 50 mg dried, ground, defatted sample into a 13x100 mm test tube.

Add 0.50 ml 80% ethanol.

Mix to wet the sample completely

Add 5ml 50% sulfuric acid and mix gently.

Incubate in boiling water bath for 2.5 hours.

Cool and mix well.

C2. Determination of starch content

Get 0.10 ml starch extract and place in an 8x150mm test tube.

Add 2.0 ml 2.5% sulfuric acid and mix well.

Place in an ice cold bath and add 2.0 ml anthrone reagent.

Mix well, cover the tubes with marbles and place tubes in a boiling water bath for 10 minutes.

Stop the reaction by placing the tubes in an ice cold bath.

Cool to room temperature.

Take absorbance reading at 630nm.

Preparation of Standard Curve

For starch analysis

Standard: Glucose, 100ug/ml - Weigh 10.0 mg glucose and dissolve and dilute 100 ml with 5.0% sulfuric acid.

Prepare the duplicate in the following tubes:

Tube

Glucose (ml)

H2SO4 (ml)

Blank (ml)

Anthrone (ml)

1

0.00

2.00

0.10

2.00

2

0.20

1.80

0.10

2.00

3

0.40

1.60

0.10

2.00

4

0.60

1.40

0.10

2.00

5

0.80

1.20

0.10

2.00

Perform the rest of the steps of the assay in sample.

Get absorbance reading at 630nm.

Plot absorbance against concentration of glucose and calculate the slope of the regression line.

Calculate from the slope of the line the amount of starch present in the sample.

Calculation

For all the analysis, use the following equation to calculate for the sugars and starch contents of the samples:

% glucose = x 100

Where:

Slope std = mg glucose/unit absorbance

Weight of sample = 50 mg/50 ml x ml= 0.10 mg

%starch = % glucose x 0.90

Calculate the weight of the sample based on the aliquot used during the analysis. Consider also the dilutions made.

References:

Shallenberger, RS and Birch, GG. 1975. Sugar Chemistry. AVI Publishing Company, Inc. Connecticut. PP. 17-46

Bottle, RT and Gilbert, GA. 1958.Use of alkaline reagents to determine the carbohydrate reducing groups.Analyst. 83: 4

Determination of Amylose

The method used in the assay is a simple base hydrolysis of the starch in corn to release the amylose and subsequently react the amylose with iodine-potassium iodide solution after neutralizing the hydrolysate with hydrochloric acid.

Equipment and supplies

Test tubes, 18x150mm

Analytical balance

Repipets and dispensers

Water bath

Vortex mixer

Spectronic 20

Reagents

Ethanol, 95% - dilute 95ml absolute ethanol to 100 ml with distilled water

Sodium hydroxide, 1.0N - Weigh 4g NaOH pellets and dissolve and dilute to 100 ml with distilled water.

Hydrochloric acid, 30% (w/v) - Measure 80ml conc. HCl and dilute to 100ml with distilled water.

Iodine-potassium iodide solution (0.2% I2 and 2.0% KI) - Weigh 2.0g KI and 0.20g I2 crystal. Dissolve KI in about 20 ml distilled water and then add I2 crystals. Dissolve crystals completely and dilute to 100 ml with distilled water.

Procedure

Weigh 50 mg dried, ground, defated sample in 18x150mm test tube.

Add 0.50 ml 95% ethanol and 9.5ml 1N NaOH.

Shake in vortex mixer and boil for 10 minutes in water bath.

Cool to room temperature.

Pipet 0.5ml aliquot into a 16x160mm test tube.

Add 0.20m iodine-potassium iodide solution and mix well.

Add 0.10 ml 30% HCl solution and mix well.

Make up to 5ml volume with distilled water and shake.

Let stand for 30 minutes.

Read absorbance of the solution at 570nm.

Use 0.5 ml ethanol and 10 ml 1N NaOH as blank. Get 0.5 ml aliquot and do color development as in sample.

Preparation of Standard Curve

Standard: Amylose, 2.5 mg amylose/ml - Weigh 25 mg amylose I 10 ml volumetric flask. Add 0.5 ml ethanol and 1N NaOH. Mix well. Dilute to volume with 1N NaOH.

Prepare in duplicate the following tubes:

Tube No.

Stock (ml)

Amylose (mg)

NaOH (ml)

1

0.00

0.00

0.40

2

0.10

0.25

0.30

3

0.20

0.50

0.20

4

0.30

0.75

0.10

5

0.40

1.00

0.00

Perform the rest of the steps of the assay sample.

Plot absorbance values against the amount of amylose and calculate the slope of the line in mg amylose/absorbance.

Calculation

Calculate amylose content of the samples using the following equation:

% amylose =

Where:

Slope std = mg amylose/absorbance

Weight of sample = 50 mg/ 10 ml x 0.50 ml = 2.5 mg

References:

Williams Vr, Wu WT, Tsai HY and Bates HG. 1958. Varietal differences in amylose content of rice starch. J. Agr. Food Chem. 6:47-48.

RODRIGUEZ F. AND MENDOZA E. 1997.Methods of analysis for screening crops of appropriate qualities, vol.2 (Institute of Plant Breeding, Analytical Services lab manual-unpublished).IPB-UPLB.pp6-7, 34-36, 58-63, 50-51, 92-93.

Analyzing Lysine Content in Cereals

Lysine is another limiting essential amino acid in cereals such as corn. Efforts to increase lysine content in corn are done by breeding for high lysine corn. In such program, monitoring the lysine content is important. To analyze for the amino acid profile only to check for the lysine content is a tedious work and is costly as well. This is colorimetric method offers a simple and inexpensive analysis of lysine for screening purposes. It is also accurate enough to detect small levels of the amino acid, and its result is comparable to the values obtained using an amino acid analyzer.

Equipment and supplies

Analytical balance

Test tube, 13 x 100 mm

Test tube, 15 x 160 mm

Vortex mixer

Water bath

Centrifuge

Repipets and dispensers

Spectronic

Reagents

Sodium carbonate buffer, pH 9.0

Sodium bicarbonate solution - weigh 500 mg NaHCO3 and dissolve in 90 ml distilled water.

Sodium carbonate solution - weigh 3.15gm Na2CO3 and dissolve in 50 ml distilled water.

Add B to A slowly until the pH is 9.0. Dilute to a final volume of 100 ml with distilled water.

Phosphate buffer, 0.03M, pH 7.4 - weigh 160 mg NaH2PO4 and 260 mg Na2HPO4 and dissolve in a about 80 ml of distilled water. Check pH and adjust when necessary. Dilute to a final volume of 100 ml with distilled water.

Papain solution, 4mg/ml - weigh 400 mg papain powder and dissolve and dilute to 100 ml with phosphate buffer.

Hydrochloric acid, 1.2N - Dilute 10 ml concentrated HCl to 100 ml with distilled water.

Ethyl acetate - analytical grade

2-chloro-3,5 dinitropyridine (DNP) solution, 30 mg/ml methanol - weigh 1.50 gm DNP and dissolve in 50 ml absolute methanol.

Cupric phosphate reagent

Cupric chloride solution - weigh 1.4 gm CuCl2.2H2O and dissolve in 50 ml distilled water.

Sodium phosphate solution - weigh 6.8 gm Na3PO4.12H2O and dissolve in 100 ml distilled water.

Borax solution, 0.05M, pH 9.2 - weigh 1.91 gm Na2B4O7.10H2O and dissolve in 70 ml water. Adjust pH to 9.0 with H3BO3 solution. Dilute to100 ml with distilled water.

Borate buffer, pH 9.0 - weigh 1.24 gm Na2B4O7.10H2O and dissolve in 70 ml distilled water. Adjust pH to 9.0 with H3BO3 solution. Dilute to a final volume of 100 ml with distilled water.

Add A to B slowly with stirring using a magnetic stirrer. Centrifuge at 3000 rpm. Collect precipitate.

Wash twice the precipitate with C and centrifuge at 3000 rpm.

Finally, disperse cupric phosphate precipitate in 40 ml of D. stir until particles are fine. Stir during use.

Procedure

Weigh 100 mg dried, defatted, powdered sample into a 13 x 100 mm test tube.

Add 3.0 ml papain solution.

Shake well using a vortex mixer.

Incubate for 16 hours at 65ËšC in an incubator.

Cool to room temperature.

Centrifuge at 3000 rpm for 5 minutes.

Collect hydrolysate.

Get 1.0 ml aliquot and place in a 13 x 100 mm test tube.

Add 0.50 ml carbonate buffer and .50 ml cupric phosphate suspension.

Stand for 5 minutes.

Centrifuge at 3000 rpm for 5 minutes.

Collect supernate.

Get 1.0 ml aliquot and add 0.10 ml DNP solution.

Stand for 2 hours with intermittent shaking.

Add 5.0 ml 1.2 N HCl and mix well.

Add 5.0 ml ethyl acetate and mix well.

Allow the two phases to settle and siphon off the acetate layer.

Repeat the washings two more times.

Read absorbance of aqeous at 390 nm.

Use papain treated as sample as blank to zero the instrument.

Preparation of Standard Curve

Standard - DL-Lysine, 400 ug/ml - weigh 20 mg standard in a 50 ml volumetric flask and dissolve and dilute to volume with carbonate buffer.

Prepare the following tubes:

Tube #

Stock (ml)

Try (ug)

Carbonate buffer (ml)

Phosphate buffer (ml)

1

0.10

40

1.00

0.50

2

0.20

80

0.90

0.50

3

0.30

120

0.80

0.50

4

0.40

160

0.70

0.50

5

0.50

200

0,60

0.50

Mix well and do the assay as in sample.

Read absorbance at 390 nm.

Plot concentration of lysine against absorbance and get the slope of the line.

Calculate from the slope of the line the concentration of lysine in the sample.

Calculation

Use the formula

%lys in sample =

Where:

Weight of sample = (100 mg/3) x 1 ml = 33.3 mg

%lys in protein =

Reference:

Tsai, CY, LW Hansel, and OE Nelson. 1972. A colorimetric method of screening maize seeds for lysine content. Cereal Chem. 49:572-579.

Analyzing Tryptophan Content in Cereals

Tryptophan is an essential amino acid that is found to be limiting in cereals such as corn. As a nutrient indicator and as an index in breeding purposes, the amount of tryptophan is important. To use the main amino acid profile however is expensive, tedious and time consuming for screening purposes. This colorimetric method offers a simple and inexpensive analysis of tryptophan for screening purposes. In accuracy however rivals that of the method using an amino acid analyzer.

Equipment and supplies

Analytical balance

Test tubes, 13 x 100 mm

Oven incubator

Vortex mixer

Water bath

Centrifuge

Repipets and dispensers

Spectronic 20

Reagents

Reagent A - Weigh 27 mg FeCl2.6H2O in 100 ml volumetric flask and dissolve in 50 µL distilled water. Dilute to volume with glacial acetic acid.

Reagent B - Dilute 83.3 ml concentrated sulfuric acid to 100 ml with distilled water.

Reagent C - Mix equal volumes of Reagents A and B. prepare one to two hours before use.

Sodium acetate buffer, 0.10N, pH 7.0 - Weigh 0.82 gm anhydrous NaAc and dissolve in about 50 ml distilled water. Adjust the pH to 7.0 with dilute acetic acid, and dilute to 100 ml with distilled water.

Papain solution, 4.0mg/ml - Dissolve 400 mg papain powder in 100 ml sodium acetate buffer, pH 7.0.

Procedure

Weigh 100 mg dried, defatted, powdered sample into a 13 x 10 mm test tube.

Add 3.0 ml papain solution.

Shake well using a vortex mixer.

Incubate for 16 hours at 65ËšC in an incubator.

Cool to room temperature.

Centrifuge at 3000 rpm for 5 minutes.

Collect hydrolysate.

Get 1.0 ml aliquot and place in a 13 x 100 mm test tube.

Add 4.0 ml reagent C.

Mix well and incubate at 65ËšC for 15 minutes.

Cool to room temperature.

Measure absorbance at 545 nm.

Use papain as blank to zero the instrument.

Preparation of standard curve

Standard - DL Tryptophan, 100 µg/ml - Weigh 10 mg tryptophan in a 100 ml volumetric flask and dissolve and dilute to volume with distilled water.

Prepare the following tubes in duplicate:

Tube #

Stock (ml)

Try (ug)

Papain (ml)

dH2O (ml)

Reagent C(ml)

1

0.05

5

0.80

0.20

4.00

2

0.0

10

0.80

015

4.00

3

0.15

15

0.80

0.10

4.00

4

0,20

20

0.80

0.05

4.00

5

025

25

0.80

0.00

4.00

Mix well and incubate as in sample.

Read absorbance at 545 nm.

Plot concentration of tryptophan against absorbance and get the slope of the line.

Calculate from the slope of the line the concentration of tryptophan in the sample.

Calculation:

Calculate concentration of tryptophan from the standard curve using the following equations:

% tryptophan =

Weight of sample = (100 mg/3) x ml = 33.3 mg

Use the slope from the regression line to calculate for concentration of tryptophan from the equation of the line:

y = mx + b

where:

y= absorbance reading

m= slope of the line

x= concentration

b = 0 at intercept

If percent transmittance was taken instead of absorbance, convert transmittance readings to absorbance using the equation:

Abs = 2 log (T), where T is the transmittance reading

To calculate for the amount of tryptophan in protein, use the following equation:

% try in protein =

Reference:

Opienska-Blauth, JM, M Charezinski and H Berbec. 1963. A new rapid method of determining tryptophan. Anal.Biochem.6:6976.

Enzymatic analysis of total dietary fibers in foods

Dietary fibers do not have a nutritive value although it is said to be of physiological importance like being a preventive factor against constipation, gallstone and other intestinal disorders related to digestion.

Reagents:

95% ethanol - w/v, technical grade

78% ethanol - 207 mml H2O was placed into a 1L volumetric flask. It has been mixed after it has been diluted to volume with 9% ethyl alcohol. Dilution and mixing was repeated if necessary. One volume water mixed with 4 volumes 95% ethyl alcohol will also give 78% ethyl alcohol final concentration.

Acetone - reagent grade

Phosphate buffer - 0.08M, pH 6.0 an amount of 1.40g sodium phosphate dibasic, anhydrous (Na2HPO4) or (1.753g dehydrate) and 9.68g sodium phosphate monobasic monohydrate (NaH2PO4) or (10.94g dehydrate) was dissolved in about 700 ml H2O. After diluting to 1L, pH was checked using a pH meter.

Termamyl (heat-stable alpha amylase) solution - No. 120L, Novo Laboratories, Inc. Wilton, CT 06897. The solution is stored in the refrigerator.

Protease - No. p-3910, Sigma Chemical Co. This was kept refrigerated.

Amyloglucosidase- No. A-9913, Sigma Chemical Co. Keep refrigerated.

Sodium hydroxide solution - 0.275N. about 11g NaOH ACS was dissolved in 700 ml H2Oin 1L volume flask and was then diluted to H2O.

Hydrochloric acid solution - 0.325M. Stock solution of known liter, e.g. 325 ml 1M HCl was diluted to 11 water.

Celite C-211 - acid washed, Fisher Scientific Co. or Sigma Corporation

Procedure

Weigh in duplicate 0.500g sample, made accurate to 0.1mg, into 100 ml tall-form beakers.

Add 25.0 ml pH 6.0 phosphate buffer.

Check pH and adjust to pH 6.0±0.2 if necessary.

Add 0.5 ml termamyl (heat stable alpha amylase) solution.

Cover the beaker with aluminum foil and lace in boiling water bath for 15 minutes.

Shake gently at 5 minutes interval.

Incubate in boiling water bath for 30 minutes at an internal temperature of 95-100ËšC.

Cool to room temperature.

Add 5 ml 0.275N NaOH solution to adjust pH to 7.5 + 0.2.

Add 2.50 mg protease by pipeting 0.05 ml of solution to each sample just before use.

Cover the beaker with aluminum foil and incubation for 30 minutes at 60ËšC with continuous agitation and then cool.

Add 5.0 ml 0.325 HCl solution.

Measure the pH and acid dropwise if necessary to a final pH of 4.0-4.6.

Add 0.15 ml amyloglucosidase, then covered with aluminum foil and incubated for 30 minutes at 60ËšC with continuous agitation.

Add 25 ml of 95% ethanol preheated at 60ËšC.

Allow precipitate formation at room temperature for 60 minutes.

Weigh the crucible containing celite to nearest 0.1 mg.

Wet to distribute the celite evenly in crucible by using stream of 785 ethanol from wash bottle. Note: suction was applied to draw celite into fritted glass at even mat.

Quantify the solution

Transfer the precipitate from enzyme digest to crucible.

Wash the residue successively with 3 20 ml portions of 78% ethanol and 2 10 ml portions of acetone.

Dry the crucible containing residue overnight in 70ËšC vacuum oven or 105ËšC air oven.

Cool in dessicator and weigh to nearest 0.1 mg.

Determine the weight of residue by subtracting the weight of crucible from the total weight.

Analyze residue from one sample of set duplicate for protein by Kjeldahl method, using n x 6.25 as conversion factor, except in cases where N content in protein is known.

Second residue sample of duplicate was incubated 5 hours at 525ËšC.

Cool in dessicator and weigh to nearest 0.1 mg.

Determine weight of ash by subtracting weight of crucible and celite from total weight.

Calculation:

Determination of blank:

B-blank, mg - wt. residue - PB - AB

Where:

Wt. of residue = average of residue weights (mg) for duplicate blank determination

PB and AB = weights (mg) of protein and ash, respectively in 1st and 2nd blank residues.

TDF % =

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