GB2182936A - Method of producing a sugar syrup from sorghum - Google Patents

Abstract

A method of processing sorghum grain to produce a sugar syrup, and in particular, a method of producing a corn syrup substitute from sorghum grain comprising the steps of:- (a) partially hydrolysing, typically by use of heat stable alpha -amylase, the starch in the sorghum grain; (b) treating the partially hydrolysed starch with amyloglucosidase, alpha -amylase, beta -glucanase and cellulase enzymes whereby starch is hydrolysed to form sugars in a filterable aqueous medium; (c) filtering the aqueous medium; and (d) concentration of the aqueous medium to produce a sugar syrup.

Description

SPECIFICATION Method of producing a sugar syrup from sorghum The present invention relates to a method of processing sorghum grain to produce a sugar syrup, and in particular, a method of producing a corn syrup substitute from sorghum grain.

Sugar syrups, e.g. corn syrup, are well known staple products in the food industry and may be produced by acid hydrolysis of starch, e.g corn starch from maize grains. Sugar syrups find wide applicability in many areas of the food industry, for example in the production of beverages.

It is an object of the present invention to provide an economical process for the production of a sugar syrup from sorghum grain. It is also an object of the present invention to provide a processforthe production of a corn syrup equivalent sorghum grain.

The term "corn syrup equivalent" is used in this document to indicate a syrup which has the same dextrose equivalents as corn syrup produced by conventional processes from maize e.g. acid hydrolysis.

Previous attempts to process sorghum grain have encountered problems such as difficulty in extraction of starch from the grain and contamination of the products with insoluble or semi-soluble protein compounds which can cause hazing and sediment formation; both of which are particularly undesirable in the production of clear, bright beverages.

It is therefore an object of a preferred embodiment of the present invention to obviate or mitigatethese problems.

According to the present invention there is provided a processforthe production a sugarsyrupfrom sorghum grain comprising the steps of: (a) partially hydrolysing the starch in the sorghum grain; (b) treating the partially hydrolysed starch with amyloglucosidase, a-amylase, ss-glucanase and cellulose enzymes whereby starch is hydrolysed to form sugars in a filterable aqueous medium; (c) filtering the aqueous medium; and (d) concentration of the aqueous medium to produce a sugarsyrup.

Preferably, the partial hydrolysis in step (a) is effected by enzymatic means, typically athightemperature eg about90 C.

A particularly preferred enzyme for use in step (a) is a heat stable aamylase eg Hitempase (availa ble from Biocon U.K Ltd) Activity: (Heat stable) amylase Source: Bacillussuhtllis orbacillisllcheniformis ECN: 3.2.1.1 Units: 120,000 u/ml (as per method No 5) It is also preferred that the steps (a) to (d) are carried out under such conditions that any protein in the grain in coagulated to form an easily separable phase.

The preferred enzymes for use in step (b) are (all available from Biocon U.K. Ltd): Amylo 300 Activity: exo-1 ,4-a-D-glucosidase Source: Asperglllus awamorioraspergillus niger ECN: 3.2.1.3 Units: 1950 u/ml (as per method No.1) Biofiltrase Activity: cellulase Source: Aspergillusniger ECN: 3.2.1.4 Units: 2400 ulmI (as per method No 2) Bioglucanase 1600P Activity: endo-1 ,3(4)p-D-g lucanase Source: Penicillium emersonXi ECN: 3.2.1.6 Units: 1600 u/g (as per method No.3) Fun gal alpha amylase Activity: amylase Source:Aspergillus oryzae ECN: 3.2.1.1 Units: 40,000SKB (as per method No.4) Methods 1-5 are described in the appendix below.

The particular strain of sorghum used is selected according to the desired flavour of the sugar syrup.

Particularly preferred strains of sorghum are Fara-Fara which gives little flavour and Kaura which produces a syrup with a flavour similarto malt.

It will be appreciated that the various parameters such as time and temperatures may be adjusted according to the desired product.

The present invention will now be described in examples 1 and 2.

Example 1 This example illustrates a method of production of a 92 D.E. Syrup comprising the following steps 1 to 18: 1.Thesorghum grain is washed to cleanse the hull ofthe grain and remove any tannins and anyadverse flavours. Typically, the grain is washed with a cleansing agent such as dilute alkali or formaldehyde solution e.g. 0.2 w/v percent calcium hyroxide solution for 5 minutes.

2.Thewashed grain is then milled (e.g. hammer mill or roll mill)to breakupthe grain to allowthe starch to be separated therefrom. The size ofthe particles achieved after milling is important so that a sufficient surface area for efficient extraction of starch can take place. In the present case, the grain is hammer milled until it passes through a 1.5 mm screen. It is undesirable to have too small a particle size as this can lead to problems in extraction ofthefinal product.

3. The milled grain (grist) is mashed in with liquorcomprising either water our a weaksugarsyrup. Inthe present case, the gristiliquor ratio is 1:3 an the mashing in is carried out at 6000. At this stage agitation ofthe mash begins, preferably by some low shear method of agitation (eg a low shear paddle mixer).

4. The mash is then treated to coagulate the tannin from the grain so that it may be separated from the syrup at a later stage. 200 ppm formaldehyde based on sorghum weight is added to coagulate the protein and the pH of the mash is adjusted to 6.5 and the Ca2+ ion concentration is adjusted to 150 ppm. This assists stabilization of the enzyme used later in this process.

5. The mash is then maintained at 600for 10 minutes to solubilize the starch. The temperature should be lessthan about70 Cto prevent gelling of the starch and dissolution ofthe coagulated protein.

6. Temperature stable amylase is then added to the mash to partially hydrolyse the starch. In the present case, HitempaseTlO (ex Biocon (U.K.) Ltd of Eardiston,Worcs, England) is used ata concentration of 0.1 percent based on grist weight.

7. The temperature of the mash is then raised to improve the hydrolysis. The temperature rise rate must be sufficiently low to maintain the protein in the coagulated state. The mash is heated at 10Cper minute to 7500.

8. Coagulation of the protein is then assisted by the addition of a protein coagulant, typically 40 ppm of potassium metabisulphite which generates SO2. The temperature is maintained at 750for 15 minutes.

9. The mash is then heated further to complete the partial hydrolysis of the starch and to achieve a pumpable liquid starch slurry. To this end, in this example the temperature is raised to 95"C at a rate of 10per minute and held at this temperature for 40 minutes.

10. The mash is then cooled and transferred to a mashing vessel. The temperature is reduced to 6000 and the transfer takes place via low shear pumps, again to maintain the protein in coagulated form.

11. The pH ofthe mash is adjusted again in preparation for the subsequent enzyme treatment. In this example, the pH is adjusted to 5.0 to 5.5 using HCI.

12. The amyloglucosidase, a-amylase, -glucanase and cellulase enzymes arethen added to the mash. The firsttwo mentioned enzymes effect hydrolysis of th starch to sugars and the amounts of enzymes used are dependant on the degree of hydrolysis required. The lasttwo enzymes mentioned are concerned with the process of extraction of the starch and sugars from the grist and the amount used is usually constant as it is desired to extract all of the starch from the remains of the grain. The amounts of enzyme used in the present case are asfollows: Amylo 300 0.15 percent Fungal amylase 0.05 percent Bioglucanase 1600P 0.05 percent Biofiltrase 0.05 percent The above four enzymes are obtainable from Biocon (UK) Ltd.

13. The mash is maintained at the required temperature until the required D.E. is achieved. In the present example,thetemperature is maintained at60 C,theAmylo 300 and the Fungal a-Amylase ceasing to function efficiently at temperatures above 63"C. Typically, the time of thins treatment is 12 hours.

14. The temperature is raised once the desired D.E. is achieved to denature the amyloglucosidase and fungal amylase. This temperature rise also serves to further coagulate protein.

15. The temperature is raised to 95"C and maintained for 20 minutes before cooling to 8000. The ss-gluconase and the cellulase continue the function in this step to release the sugars from the grain.

16. Protein coagulation is further assisted by the addition of more coagulating agent eg 30 ppm on mash weight of potassium metabisulphite.

17. The mash is then pumped to an extraction filter. Conveniently, a high pressure filter is used. In the present case, a Nordon HP Filter Press is used (available from Nordon et Cie, Nancy, France). Usually, the filter is purged two to three times with water, the last purge being typically recycled in step 3 above.

18. The resulting wort isthen concentrated, typically using a falling film evaporator giving upto 82 percent solids in solution.

Example2 For the production of a 42 D.E. syrup, the steps 1 to 18 of Example 1 are followed with thefollowing alteration in step 12. The amounts of enzyme used are asfollows: Amylo 300 0.025 percent Fungal ol-Amylase 0.025 percent Bioglucanase 1 600P 0.05 percent Biofiltrase 0.05 percent and the overall timefortreatmentin step 13 is about 1 hour.

It will be appreciated that the amounts of enzymes used, the duration of treatment and the activity ofthe enzymes used may be varied according to the conditions, desired product or desired treatmenttime.

The process is further illustrated on the accompanying flowchart. The figures 1 to 18 on this flowchart correspond with the respective steps 1 to 18 in Example 1.

Appendix Method No. 1 Estimation of amyloglucosidase activity (D.N.S method) Reagents 1. IMacetatebuffer pH 4.2 Prepare 1 M acetic acid and Im sodium acetate. To 50 ml of IM sodium acetate, add sufficient IM acetic acid to bring the pH down to 4.2.

2. Bufferedstarch solution (1 percent) Slurry 1 gram ofstarch (B.D.H. "Analar" grade water soluble) in a minimum ofcoldwaterand add to60 mls. of boiling water stirring continuously, taking care to rinse all the starch into boiling water. Boil for 10 minutes, while stirring. Cover and cool in ice bath. Place 5.6 mls of Acetate buffer in a 100ml volumetricflask, add the cooled starch and make up to 100 mis. with deionized water. Prepare starch solution daily.

3. D.N.S. Solution.

Add 1 gram of 3.5 Dinitro SalicyciicAcid and and 30 grams of Potassium Sodium TartrateTetrahydrate (Rochelle Salt) to 16 mis. of 10 percent w/v NaOH then add 50 mIs of deionized water. Warm to dissolve. When dissolved cool and add to 100 ml volumetric flask. Make up to markwith distilled water.

Apparatus Waterbath set at 40"C Stopwatch Spectrophotometer set at 540 mu Boiling water bath Cooling bath 1 m ml., 2ml., and 10 ml. pipettes 20 ml. testtubes Procedure Enzyme dilutions are prepared using deionised water. 1 ml. aliquots of substrate are placed in a series of 10 ml. test tubes. The tubes are then placed in water bath at 40 C. After 10 mins., 1 ml. of enzyme solution is added to each tube and reacted for exactly 10 mins. Stop the reaction with 2 mls. D.N.S. reagent in each tube.

The tubes are then placed in a boiling water bath for Exactly 10 mins. They are then cooled rapidly in cooling bath. Add 10 mls. deionized water to each. Set spectrophotometer at 540 mFand determine O.D. Read the amount of glucose produced from standard graph.

Standard graph The standard graph is obtained by preparing a range of glucose solutions (anhydrous analytic grade) from 0.1-1.5 mg/ml. 1 ml of the glucose solutions aretaken into test tubes then 1 ml. of deionized water is added.

Place test tubes in a water bath at 4000. After 10 mins., add 2 mls. of D.N.S. reagent in each tube, boil tubesfor 10 mins. Cool rapidly and add 10 mls deionized water. Determine the O.D. at 540 mu of each sample using a reagent blank to zero spectrophotometer. The reagent blank is prepared exactly the same as the glucose solutions, except that 1 ml. of distilled water is used instead of 1 ml. glucose solution. Plot O.D. V/S mg.

glucose/ml.

Calculation Glucose produced is obtained from graph.

Activity = mg of glucose x enzyme dilution 10 Method No.2 CMC Cellulase assay Principle: The assay is based on the production of glucose from a solution of OMO cellulose at 37"C over an hour atpH 4.6. The glucose formed is determined using the DNS method.

Reagents: 1. 1.6 percent OMO Cellulose (low viscosity, sigma). Carefully add 1.6 g of CMC cellulose to 90 ml deionized water while stirring vigorously. Allow to stirfor approximately 30 minutes then leave to stand overnight. Add 10 ml of 1 M acetate buffer pH 4.6, and stirfor 10 minutes. This solution will hold for 1 month at room temperature.

2. 1 M acetate buffer pH 4.6: Add 4.0g of sodium acetate to 30Or40 ml of deionized H2O. To this add 11.8 ml of glacial acetic acid. Adjustto 100 ml. 3. D.N.S. Solution prepare as indicated in method 1.

Procedure Incubate 1 ml samples of 1 .6percent CMC Cellulose art 3700 until equilibrated to this temperature. At zero time, add 1 ml of suitably diluted enzyme solution and react four 1 hour. Stop reaction with 2 ml DNS reagent.

Boil for 10 minutes, add 10 ml H2O and determine at 540 nm. Samples should be made up in triplicate and enzyme blanks should also be analysed. For the substrate blank, add 1 ml H2O instead of enzyme.

Calculations A standard curve is constructed on a range of glucose concentrations from 0.1 mg/ml to 1.0 mg/ml.

When OD's are obtained, the glucose formed is determined from the standard curve and the activity is expressed as below: mg Glucose x Dilution = zing per hour.

Method No.3 Beta glucanase estimation Principle: The enzyme is reacted with beta glucan substrate for 10 minutes and the reducing end groups determined colorimentrically by means of Dinitro Salicyclic Acid (D.N.S.) Reagents: Substrate 1.0 gram of pure barley beta glucan added slowly, with vigorous stirring, to deionized water (approx. 80 mls). Boil on hot plate for 5/10 minutes, cool, add 10 mls. of 1 M acetate buffer pH 5.00, make up to 100 mls.

Acetate buffer Add 79 ml of 1 M sodium acetate to 30 ml of 1 M acetic acid and adjust pH to 5.0 if necessary.

Dinitro salicyclic acid reagent To 1.0 gm of D.N.S. add to 16.0 mls of 10 percent sodium hydroxide w/v and 30 gms of potassium sodium tartratetetra hydrate and 50 mls of deionized water, warm to dissolve and bulkto 100 mls. after cooling. Store for5daysonlyat5 C.

Test procedure 1.0 ml of substrate pipetted into small test tube in a water bath art 5000, after 6 minutes add 1.0 ml of enzyme dilution, reactfor 10 mins exactly, then add 2.0 mix of D.N.S. reagent.

Perform blank determination by adding D.N.S. reagent, before the test solution, to the substrate. Place both tubes in a boiling water bath for 5 mins exactly, and cool, add 10 mls of distilled water.

Readthe colour of the test solution against the blank using 1.0cm cells at 540 m,u.

Standard maltose graph Construct a standard maltose graph using a solution containing between 0.2 and 1.0 mg. per ml maltoseto replace the enzyme solution in the above assay.

Calculation 1.0 unit of activity = 1.0 mg. of maltose equivalent produced per minute per gram of enzyme.

The OD 540 (Optical Density at 540 nm) obtained with test solution is converted to mg of maltose using standard curve and activity is calculated as follows: Activity = OD540 (converted to mg maltose) x enzyme dilution.

Method No.4 SKB Method for the determination of fun gal alpha amylase activity 1.0 Principle Alpha amylase (I.U.B. No 3.2.1.1.) breaks down the alpha (1.4) glycosidic linkages of buffered limitdextrin to yield maltose and small dextrins. The breakdown products are reacted with an iodine solution and the colour produced (changes from blue to red-brown as starch is broken down) is compared against a standard colour solution. For accuracy the dextrinization time should take place between 10 and 20 minutes.

2.0 Unitdefinition One SKB unit is the amount of alpha amylase which will dextrinize buffered limit dextrin (as prepared herein) atthe rate of 1 gr. per hourat30 C + 0.1 "C.

3.0 Equipment 3.1 Constanttemperature water bath regulated art3000 + 0.1"C.

3.2 Suitable visible range spectrophotometer 3.3 Whirlimixer 3.4 Timer 3.5 GradeAglassware 4.0 Reagents 4.1 iodine crystals Analargrade.

4.2 Potassiu m lodide anhydrous Analar g rade 4.3 Glacial Acetic Acid (Handle with Care) 4.4 Sodium acetate an hydrous Analar grade.

4.5 Soluble starch (Merck Lintner or equivalent) 4.6 B Amylase (Clodor UK) 4.7. Cobaltous chloride hexahydrate 4.8 Potassium dichromateAnalargrade.

4.9 Calcium chloride dihydrate Analar Grade.

5.0 Preparation ofreagents 5.1 Stock iodine solution: dissolve 5.50 go of iodine crystals and 11.0 gr. of potassium iodide in 150 ml of deionized water and make up to 250 mls. Store in darkness. Prepare fresh solution monthly.

5.2 Dilute iodine solution: dissolve 20 gr of potassium iodide in 400 ml of deionized water, add 2.0 ml of stock iodine solution and make up to 500 ml. This solution should be art 3000 when colour comparisons are made.

5.4 Buffered Limit Dextrin lor-amylodextrin) substrate.

Prepare suspension 10.0 gr (dryweight) soluble starch in cold water and pour slowly into approx.

350 ml of boiling water. Boil with stirring for 1 to 2 minutes, cool and add 25 ml of buffer solution and 250 mg of special P-amylase dissolved in a small amount of water.* Ensuretemperature of starch solution is 30"C or lower before addition of amylase. Make up to 500 ml with deionized water, add a few drops of toiuene, and store at 30"C for not less than 18 hours and not more than 72 hours before use.

*Ensure amylase is dissolved before addition to starch. This may take 10-20 minutes stirring on magnetic stirrer.

5.5 0.2percentcalcium chloride solution: dissolve 2.7 go of Calcium chloride dihydrate in 800 ml of deionized water and make upto 1000 ml.

5.6 Color standard: dissolve 25.0 gm of cobaltous chloride hexahydrate and 3.84 gm of potassium dichromate in 100 ml of 0.01 N, HCI. This colour is stable indefinitely if stored in a stoppered bottle.

6.0 Enzyme preparation Dilute enzyme in 0.2 percent Ca Cl2 using details listed below as a guide.

7.0 Controlofassay Include a suitable control with each assay run.

8.0 Procedure 8.1 Transfer 10.0 ml of buffered limit dextrin to a suitable test tube and incubate at 3000four 10 minutes.

8.2 Add 5.0 ml of suitably diluted enzyme (prewarmedfor3 min.) starttimeand mix.

8.3 Prepare a series oftesttubes each containing 5.0 ml of dilute iodine solution, 12-16 test tubes for each sample being assayed and place in a water bath art 3000 t 0.1 C 8.4 Zero the spectrnphotometerwkh deionized water and determine the optical density of the colour standard at 620 mm in a 1 cm light path cuvette.

8.5 10 minutes after 8.2 above and at definite time intervals, pipette a 1.0 ml sample from the starch -enzyme tube into 5.0 ml of the dilute iodine solution, vortex and read the optical density in a spectrophotometer at 620 nm.

8.6 As the colorofthe reaction approaches that ofthe colour standard, sample and react witch the dilute iodine every minute or half minute. When the O.D. of the sample reaches that of the colour standard, end point is reached.

8.7 In case two comparisons 30 seconds apart show that one is darker and the other is lighter than the colour standard, the end point is recorded to the nearest 0.25 of a minute. If the end point is reached in less than 10 minutes use a more dilute enzyme and vice versa. The time should fall between 10 and 20 minutes.

9.0 Calculation ofactivity 9,1 Powders 0.2 x 60 x D.F. = SKB units/ml.

Wxtxv where 0.2 = weight of starch (gm) in reaction mix 60 = minutes w = weightofsample t = dextrinizationtime D.F. = dilution factor v = volume of enzyme in reaction mix.

Reportto the nearest 1.0 unit.

9.2 Liquids 0.2 x 60 x S.G x D.F. = SKB units/ml.

wxtxv where S.G. = specific gravity at 20"C ia 0 Proceduralnoteslinformation 10.1 Determine dryweightof starch using oven method (4 hours at 1 20"C).

10.2 amylase is standardized to 2000"L and and to specifications set up byAACC's Malt Evaluation Committee. It is stable for up to 3 months when stored desiccated at 4 C.

10.3 This method conforms to AACC 22-01 reviewed 10-27-82.

10.4 Each lot of starch must be checked in parallel againstthe old lot of starch. Variations of more than j 3"L diastatic powder in the averages of a series of parallel tests indicate an unsuitable batch.

SKB fun gal a-am ylase Sam no. 01-20(see 6above) Activities Dilution Method of dilution 60,000 - 100,000 1/500,000 1/200 1/1001/25 30,000 - 60,000 1/250,000 1/250 1/1001/10 20,000 - 30,000 1/150,000 1/2001/150*1/15 12,000 - 20,000 1/100,000 1/2001/500 6,000 - 12,000 1/50,000 1/2001/250 3,000 - 6,000 1/25,000 1/2501/1000 2,400 - 4,800 1/20,000 1/200 1/100 1,200 - 2,400 1/10,000 1/2001/50 600 - 1,200 1/5,000 1/2001/25 300 - 600 1/2,500 1/2501/10 All powder samples should be stirred vigorously for 10 min. in 150 ml. of diluent before completing the initial dilution.

If the final dilution of a sample is cloudy, the stock must be filtered and diluted again as before. Mix each dilution thoroughly before proceeding with the next. Store dilutions on ice.

Method No.5 Determination ofbacterial a-A mylase 1.0 Principle amylase (lUB No.3.2.1.1.) breaks down the a(1.4) glycosidic linkages of buffered limit dextrin to yield maltose and smaller dextrins. The breakdown products are reacted with an iodine solution and the colour produced (changes from blue to a red brown as starch is broken down) is compared with a standard colour solution. For accuracy the dextrinization time should take place between 10 and 20 minutes.

2.0 Unit definition One bacterial a-amylase unit is the amount of enzyme which breaks down 5.830 mg of starch per hour at 4000 j 0.100 and pH 6.0 3.0 Equipment 3.1 Constant temperature water bath regulated at 40"C t 0.1 C 3.2 Suitable visible range spectrophotometer 3.3 Whirlmixer 3.4 Timer 3.5 GradeAglassware 4.0 Reagents 4.1 iodine crystals Analar grade 4.2 Potassium lodide anhydrous Analar grade 4.3 Glacial Acetic Acid (Handle with Care) 4.4 Sodium Acetate AnhydrousAnalar grade 4.5 Soluble starch Lintner 4.6 Calcium Chloride dihydrate Analargrade 4.7 Sodium Chloride Analar grade.

5.0 Preparation ofreagent 5.1 1 Molar acetate buffer pH 6.0: Prepare 1 moiar acetic acid (60.05 g in 1000 ml of deionized water) and 1 molarsodium acetate (82.03gm in 1000 mi). Mixthe 1 molar acetic acid with the 1 molarsodium acetate in a 1:40 ratio and check that the pH is 6.0.

5.2 Enzyme diluting solution: Dissolve 0.585 gm of sodium chloride and 2.22g calcium chloride dihydrate in 800 ml of deionized water. Add 20 ml of 1 molar acetate buffer, pH 6.0 and make upto 1.with deionized water.

5.3 Buffered starch solution: Slurry 1.39 gm of lintner starch (dry weight) in a small quantity of deionized water and add to 20 ml of boiling water. Boil for 1-2 minutes stirring continuously. Cool to room temperature and add 21 ml of 1 M acetate buffer and made up to 200 ml with deionized water. This is the enzyme substrate and should be made up daily.

5.4 Stock iodine Solution: Dissolve 5.3 gm of iodine crystals and 11.0 gms of potassium iodide in 150 ml of deionized water and make up to 250 mls. Store in darkness. Prepare fresh solution monthly.

5.5 Dilute iodine Solution: Dissolve 10 gms of potassium iodide in 200 ml of deionized water, add 1.0 ml of stock iodine solution and make upto 250 ml. Prepare fresh daily.

5.6 Standard colour solution: Dissolve 25.0 gm of cobaltous chloride hexahydrate and 3.84 gm of potassium dichromate in 100 ml of 0.01 N HCI. This colour is stable indefinitely if stored in a dark stoppered container.

6.0 Enzyme preparation Dilute enzyme in enzyme diluting solution using details below as a guideline.

7.0 Controlofassay Include a suitable control in each assay run 8.0 Procedure 8.1 Transfer 10 ml I of starch substrate to a suitable test tube and incubate at 40 Cfor a few minutes.

8.2 Add 5 ml of suitably diluted enzyme, (prewarmed for3 minutes) starttime and mixwell.

83 Prepare a series of testtubes each containing 5.0 ml of dilute iodine solution, 12-16 testtubesfor each sample being assayed.

8.4 Zero the Spectrophotometerwith deionized water and determine the optical density of the colour standard at 620 mm in a 1 cm light path cuvette.

8.5 10 minutes after8.2 above and at definite time intervals, pipette a 1.0 ml sample from the starch-enzyme tube into 5.0 ml of the dilute iodine solution vortex and read the optical density in a spectrophotomer at 620 nm.

8.6 Asthe colourof the reaction approachesthat ofthecolourstandard. sample and reactwith thedilute solution every minute or half minute. When the O.D ofthe sample reaches that of the colour standard, end point is reached.

8.7 In case two comparisons 30 seconds apart show that one is darker and the other is lighter than the colour standard,the end point is recorded to the nearest 0.25 minute. If the end point is reached in less than 10 minutes use a more dilute enzyme and vice versa. The time should fall between 10 and 20 minutes.

9.0 Calculation ofactivity 715 D.F. = B.A.A p/gm t xw xv Where: t = time of reaction in minutes v = enzyme solution added (mis) w = weight of sample in grams D.F = Dilution Factor 60 x lOx 6.95 =715 5.832 Where: 60 = lhourinminutes 10 = ml ofstarch solution 6.95 = mg starch in 1 ml solution 5.832 = mg starch hydrolysed per hour.

10,0 Proceduralnotes: 10.1 Determine dry weight of starch using oven method (4 hours at 1 20"C) 10.2 Each new lot of starch must be checked in parallel againstthe old batch. Variations exceeding + 5 percentforthe average determinations of each batch indicates an unsuitablestarch.

Bacterial a-amylase Sam no.01-30 (see 6above Activities dilution method of dilution 7,150 50 14,300 1/1000 1/200 1/5 13,300 - 28,600 1/2,000 1/200 1/10 17875 - 35,000 1/2,500 1/250 1/10 35,000 - 70,000 1/5,000 1/200 1/25 70,000 - 143,000 1/10,000 1/200 1/50 143,000 - 286,000 1/20,000 1/200 1/100 286,000 - 572,000 1/40,000 1/200 1/200 400,000 - 715,000 1/50,000 1/2001/250 715,000 - 1.43x10to6 1/100,000 1/200 1/1001/5 1.43 10to62.86 x 10to6 1/200,000 1/200 1/1001.10 2.86x 10to6 5.7 x 10to6 1/400,000 1/2001/2001/10 All powder samples should be stirred vigorouslyfor 10 min in 150 ml diluent before completing the initial dilution. Ifthefinal dilution is cloudy, the stock must befiltered and diluted again as before. Mix each dilution thoroughly before proceeding with the next. Store all dilutions on ice.

Claims (7)

1. A process for the production a sugarsyrupfrom sorghum grain comprising the steps of: (a) partially hydrolysing the starch in the sorghum grain; (b) treating the partially hydrolysed starch with amyloglucosidase, a-amylase,3-glucanase and cellulase enzymes whereby starch is hydrolysed to form sugars in a filterable aqueous medium; (c) removing any unwanted solids from said aqueous medium; and (d) concentration of the aqueous medium to produce a sugar syrup.

2. A process as claimed in claim 1 wherein, the partial hydrolysis in step (a) is effected by enzymatic means.

3. A process as claimed in claim 2, wherein the partial hydrolysis is carried out at high temperature.

4. A process as claimed in claim 2 or3wherein the enzymefor use in step (a) is a heatstableaamylase.

5. A process as claimed in any preceding claim wherein steps (a) to (d) are carried out under such conditions that any protein in the grain is coagulated to form an easily separable phase.

6. A process as claimed in any preceding claim wherein the sorghum grain used is of the strain Fara-Fara or Kaura.

7. Asugarsyrupwhen produced buy a process as defined in anyone of claims 1 - 6.