Journal of Cereal Science 14 (1991) 47-61

Glycemic Response to Starch in Pasta: a Study ofMechanisms of Limited Enzyme Availability

Y. GRANFELDT and I. BJORCK

University of Lund, Department of Applied Nutrition, Chemical Center, Box 124,S-221 00 Lund, Sweden

Received 25 June 1990

The aim of this work was to measure the availability of starch in pasta products andto study the mechanisms which affect the rate of digestion and absorption of starchin pasta. Healthy subjects were given test meals with an equivalent amount ofavailable carbohydrate from macaroni (25/75 % durum/Swedish wheat flour),spaghetti (100% durum wheat flour +monoglycerides), 'spaghetti porridge' (cookedspaghetti mixed in a food processor) and, for comparison, bread (made fromspaghetti ingredients) or mashed potatoes. Blood glucose levels were measured overa 3 h period and glycemic indices (GI) were calculated using 90 or 120 min areasunder the glucose curves. A new in vitro test was introduced to measure the rate ofstarch hydrolysis in products with a food form' as eaten'.

The three pasta products produced significantly lower peak blood glucose valuesand lower GI (90 min) than the corresponding bread. The spaghetti also displayed amore favourable late post-prandial glucose response, with a low but sustainedincrement above the fasting level in the late phase. Significant differences betweenbread and mashed potatoes were only detected in the late phase. The 'lente'properties of the pasta were assigned to a restricted enzymic availability due to a morecompact food texture. The importance of the food structure was further substantiatedby a significant increase in GI following mixing of the spaghetti product. The in vitromethod ranked the different wheat products similarly to the in vivo situation and isrecommended for prediction of the glycemic response to various food items.

Introduction

Quantitatively, starch is the most important carbohydrate in the human diet. Recentdietary recommendations prescribe an increased intake of starch and dietary fibre. Inview of current recommendations, it is particularly important to characterize thenutritional properties of starch in different foods. Studies in this area have documentedimportant differences in post-prandial glucose and hormonal responses to starchy foodsboth when eaten alone1- s and as a component in a mixed meal4-9.

Among cereal products, pasta appears to possess unique nutritional features in thatthe starch is slowly digested and absorbed in the small intestine1- s. A reduced rate ofstarch uptake is particularly beneficial in the dietary management of diabetes. However,'lente' food items, with slowly digested and absorbed starch, have also been suggested

Abbreviations used: GI = Glycemic Index; Pb = fasting blood glucose level; E % = per cent of energy fromtotal energy.

0733-5210/91/040047 + 15 $03.00/0 1991 Academic Press Limited

48 Y. GRANFELDT AND 1. BJORCK

as a preventive factor against development of diseases common in affluent societies e.g.hyperlipidemia, coronary heart disease, obesity and diabetes1o- 13 The mechanism of the'lente' features ofpasta is not fully elucidated. Moreover, the products investigated havebeen poorly characterized from a technological point of view. In Sweden, there has beena shift lately in the pasta industry towards use of higher temperatures during drying ofpasta. No data are available in the literature on the effect of this comparatively newtechnology on starch bioavailability. With respect to dietary guidelines, it is essential toknow whether or not results on pasta published in the literature can be generalized toall kinds of pasta.

The purposes of this study were to:1. Measure the availability of starch in two of the most common Swedish pasta

products.2. Study the mechanism for possible 'lente' feature of starch in pasta.Starch availability was evaluated by measurement of post-prandial glucose responses

and calculation of glycemic indices in healthy subjects. In addition, the rate of starchhydrolysis in products 'as eaten', was measured by using a new in vitro test.

ExperimentalProducts

The pasta products were obtained from Kungsomen AB, Jama, Sweden. Both products wereprocessed using 'high-temperature drying' following mixing/forming in a pasta extruder. Themacaroni were made from 25 % durum wheat flour (Canada Amber no. 1, Nord Mills AB, Malmo,Sweden) and 75 % Swedish wheat flour with extraction rates of approximately 73 and 76 %,respectively. The macaronies were very thin, with a product thickness of approximately 1 mm, toreduce boiling time (3 min). The spaghetti were made from 100 % durum wheat flour with additionof monoglycerides (0'6 %, flour basis). The monoglycerides were distilled from soya and palm oil(50/50 % w/w) and consisted mainly of C1B fatty-acids. (Monoglycerides are added commerciallyto improve the product quality of pasta intended for the catering industry). The product thicknesswas approximately 1'8 mm. One portion of each product was boiled in an excess of watercontaining approximately 01 % NaCI. The boiling time was (as recommended by the producer)3 min for macaronies and 12 min for spaghetti, to achieve the 'al dente' pasta texture.Immediately after cooking, the water was drained through a colander.

To evaluate the importance offood structure, the spaghetti was studied also after intense mixinginto a porridge. The' spaghetti porridge' was made from cooked and drained spaghetti, which wasmixed in a food processor for 15 +20 s with addition of 75 g water during the last mixing period.In addition, a bread was baked using the same ingredients as in the spaghetti.

Bread was made in a home baking machine, (panasonic SD-BT 2P, Matushiba Electric Trading,Osaka, Japan). Durum wheat flour (300 g) and monoglycerides (the same ingredients, in the sameproportion as used in spaghetti processing), water (200 g), salt (3 g) and dry yeast (3 g) were mixed,kneaded, fermented in four steps and baked in the machine. The whole procedure took 4 h. Afterbaking, the bread was allowed to cool to room temperature and was then frozen in slices. Beforeingestion it was thawed and the crust separated; only the crumb was used.

An alternative meal carbohydrate, mashed potato, was included as a reference material. Themashed potato was an industrially-produced flaked product (Bla Band, NOVIA Livsmedels-industrier AB, Kristianstad, Sweden). A portion corresponding to 50 g available carbohydrateswas swelled in 400 g of hot water instead of milk.

The starch contents of the products were (dry basis) 740 % (spaghetti), 788 % (macaroni),756 % (mashed potatoes) and 75'1 % (bread).

GLYCEMIC RESPONSE TO STARCH IN PASTA 49

Chemical analysis

Starch and low molecular weight carbohydrates. An amount ofsample (milled dry pasta and potato-flakes, and wet-homogenized bread) corresponding to 500 mg starch was suspended in distilledwater (15 ml) and incubated with a thermostable bacterial ex-amylase (100 J.lI; Termamyl 120 L,Novo A/S, Copenhagen, Denmark) in a boiling water bath for 15 min. The mixture was thendiluted to 50 ml with distilled water. To I ml of this suspension were added amyloglucosidase(50 J.lI; 14 U/mg, 10 mg/ml, Boehringer Mannheim, Germany) and 01 M Na-acetate buffer,pH 4'75 (1'95 ml). The mixture was incubated for 30 min at 60 ec, diluted to 100 ml with distilledwater and analysed for glucose with a glucose oxidase-peroxidase reagent14 A standard curve wasprepared using glucose and starch content was expressed in polymer weight. This method does notmeasure in vitro resistent starch, starch resistant in vitro unless solubilized in alkali. The contentof resistent starch in the products has been measured separately and was (dry basis) in bread,mashed potatoes, macaroni and spaghetti 03 %, 08 %, 0'4 % and 0'3 % respectively (to bepublished). Since the contents of resistent starch in these products are small, the total amount ofstarch are similar to the amount of available starch.

Free glucose in the spaghetti and macaroni was determined by dispersing milled products indistilled water. The sample was immediately boiled in a water bath for 5 min to inactivate starch-degrading enzymes. The content of free glucose was then analysed as above. In mashed potatoes,glucose, sucrose and fructose were analysed with an enzyme 'kit' (Boehringer Mannheim,Bromma, Sweden).

Dietary fibre. The total dietary fibre content in the pasta products was analysed gravimetricallyafter enzymic solubilization of protein and starch16 After a pre-incubation with Termamyl, theprotein was solubilized with pepsin. The final digestion of starch and protein was carried out byincubating the sample with pancreatin. The soluble dietary fibre components were precipitatedwith 95 % ethanol (4 vol.) and the total dietary fibre components were then recovered by filtration,using Celite as a filtering aid. All fibre values were corrected for remaining indigestible protein andash.

Protein. Protein content was determined by the Kjeldahl method using 5'70, 625 and 6'38 asconversion factor for wheat, mashed potatoes and cheese (see Test Meals below), respectively.

In vivo study

Subjects. Ten healthy volunteers participated in the study, five men and five women, with a meanage of 37 1'7 years. Their mean body mass indices were normal (22'6 08 kg/m2) and all hadnormal glucose tolerance.

The subjects consumed each of the five meals after an overnight fast on separate morningsapproximately one week apart. The meals were given between 8 and 8.40 a.m. and were eaten over15 min with zero time as the time eating commenced. Fingerprick blood samples were taken usingmini lancets (Clean Chemical Sweden AB, Borlange, Sweden) at -5, 15,30,45, 60, 90, 120 and180 min. Capillary blood (50 J.lI) was collected and mixed with 1 ml 0'025 M NaOH and 005 ml03 M ZnS04 to precipitate the proteins and the red blood cells. Glucose was then assayed withglucose-oxidase peroxidase reagent.

Test meals. The volunteers took test meals containing 50 g available carbohydrate, almostexclusively starch, from macaroni, spaghetti, 'spaghetti porridge', bread and mashed potatoes.The content of low molecular weight carbohydrates was very low. Mashed potatoes contained< 08 % and the pasta products < 0'2 % glucose, dry basis. To improve palatability, all the mealswere served with 100 g tomat%nion sauce. Grated cheese and/or butter were added to somemeals to get equal levels of protein and fat in all meals (Table I).

50 Y. GRANFELDT AND I. BJORCK

The tomat%nion sauce was prepared in one batch, divided in separate portions and frozen.Before ingestion it was thawed and cooked for some minutes.

A glass of water (150 g) was taken with the meal and a cup of coffee, tea or water (150 g) after.Only 75 g of water was served with the' spaghetti porridge' to standardize the amount of waterin all cereal based meals.

The contents of digestible carbohydrates, protein and fat were similar in the test meals: 530 gcarbohydrates (66E%); IO'5g protein (13E%), 7-8g fat (21 E%) and energy l369kJ. Thedietary fibre contents were 2 g in the cereal based meals and approached 5 g in the potato meal(dry basis). The wet weight of the meals (including a glass of water and a cup of coffee) was similar(560 30 g) except in the case of mashed potatoes (860 g), where it was necessary to add someextra water in order to get a palatable product.

The area under the glucose curve after each meal was determined in each subject. The glycemicindex (GI) was calculated as blood glucose area during 120 min following ingestion of the testmeal, as a percentage of the corresponding area with the bread made from spaghetti ingredients.For comparison, the GI was also calculated using the 90 min areas. Any area beneath the fastingblood glucose (Fb) level was ignored. The difference between the highest post prandial bloodglucose value minus Fb is referred to as 'peak above Fb'.

Three subjects produced higher blood glucose responses than the others. In these subjects thepeak above Fb with the reference bread was 17-60 % higher than the corresponding mean valuefor the entire group of 10 subjects. Their curves were printed out separately and are referred toas 'high responders'.

When the starch in the diet was slowly digested and absorbed in the small intestine it is, in thefollowing text, referred to as a 'lente' behaviour.

In vitro starch hydrolysis

Enzymic hydrolysis of starch in heat-treated homogenized products. The samples (10 g of starch)were wet-homogenized in 0'5 M Na, K-phosphate buffer (pH 6,9), with an Ultra Turraxhomogenizer. The suspension was then diluted (I g of starch in 100 ml buffer) and incubated withporcine pancreatic ex-amylase (A 6255, Sigma Chemical Co, St Louis, U.S.A.). The ex-amylaseconcentration was 200 unitsjg starch (one unit liberates I mg of maltose from soluble starch in3 min at pH 69 and at 20C). Samples were taken after 5-60 min incubation at 37 C and analysedfor reducing sugar content16 In another experiment, the pH of the buffered suspensions wasadjusted to 1'5 followed by incubation with pepsin at 37C. After 60 min, the pH was re-adjustedto 6'9 and the mixture was incubated with ex-amylase as described above. Reference samples wereprepared and run under the same conditions, but using distilled water instead of pepsin solution.

Enzymic hydrolysis ofstarch in products' as eaten' (chewing test). An equivalent amount of starch(2 g) from macaroni, spaghetti, 'spaghetti porridge' and bread made from durum wheat flourwere used. The products were prepared in the same way as in the in vivo study but the other mealcomponents were excluded. Four healthy volunteers participated in the study. The samples werechewed 15 times, during approximately 15 s, and then spat out quantitatively in a 30 ml syringe.The subjects then rinsed their mouth with distilled water (10 ml) for 60 s and this rinsing water wastransferred quantitatively into the same syringe. The volume was then made up to 30 ml withdistilled water and the content were transfered to a dialysis bag. Spektra Por no 2. dialysis tubingswere used with a width of 45 mm and a mol. wt cut-off at 12-14000. The 22 cm tubes were cut toa length of 13 em. Each bag was incubated at 37C for 3 h in a beaker with distilled water(800 ml). The beaker was placed in a stirred water bath (number of revolutions were IOO/min).Every half hour, aliquots (1 ml) of the dialysate were removed for analysis of reducing power bythe 3,5-dinitro salicyclie (DNS) acid method17 . A standard curve was prepared using maltose. Theextent of hydrolysis was calculated as the proportion of starch degraded to maltose (per cent;maltose equivalents).

Each sample was chewed by each subject on three different occasions and the chewing tookplace at the same hour every day.

TABLE I. Composition of the test diets

Wet Available Dietaryweight carbohydrate Protein Fat& fibre Energy 0

MeaJs (g) (g) (g) (g) (g) (leI) r-'-

52 Y. GRANFELDT AND I. BJORCK

(a)

aab............

180

-'-,-9

160140

"b-" -" - ~ ~ ....~ ~ o''.- .. -..

b

12080 100Time (min)

a

604020

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: .7b ........ '~.":,, .:'./ ' .... ~,.. ': ..

J I C, "o,.c// ','....... -'9." .......:",

'\ bc . :.: .... ab'.. c

'B-- ~'~.,._ag_:-~__.:.: -' -c' - -. c~ ., -------~-~~----------ab ..... '. ac

.... :~ ~---~-"'. ab

3

25

2

." 158

:g....."iE

:ll 050u:::l

130

-0,5

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(b)

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" '0 "

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4

3,5

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Time (min)

FIGURE 1. (a) Mean incremental blood glucose responses obtained after ingestionof: --, bread, baked from spaghetti ingredients; -"-", mashed potatoes;-'-'-, macaroni; ----, spaghetti; and .. ...... 'spaghetti porridge'. For each time,means not sharing the same letter are significantly different (P < 0'05). (b) Meanincremental blood glucose' high responders' after ingestion of: --, bread, bakedfrom spaghetti ingredients; -"-", mashed potatoes; -'-'-. macaroni; ----,

spaghetti; and ......... , spaghetti porridge'.

GLYCEMIC RESPONSE TO STARCH IN PASTA 53

Chewing tests were also made using pre-incubation with pepsin prior to extended amylolysiswith added ct-amylase. In this case, the subjects spat the sample into a beaker with 5 001 water and05001 pepsin solution (IOOmg/ml, 2000 FIP-U/g, Merck, Darmstadt, Germany). The pH ofthe pepsin solution was pre-adjusted to 1'5 with 1-2 MHCl. They then rinsed their mouth with5 001 of distilled water and spat it out into the same beaker. The pH of the resulting slurry wasadjusted to 15 and incubated at 37C for half an hour, with gentle mixing 4 to 5 times duringincubation. The pH was then re-adjusted to 6'9 (with 1-2 MNaOH) before incubation with porcinepancreatic o:-amylase (A 6255, Sigma Chemical Co, St Louis, U.S.A.). The enzyme (2160 Sigmaunits) was dissolved in 10 001 distilled water and 1 001 of this solution was added to the beaker. Thesample was brought to volume (30 001) and transferred to the dialysis tubing. Reference sampleswere prepared and run under the same conditions, but using distilled water instead of pepsinsolution.

The degree of hydrolysis at different time points with the above methods was taken as a measureof rate of hydrolysis, and was used to evaluate mechanisms for possible differences in glycemicresponses.

Statistical evaluation

The results from the in vivo study are expressed as means.E.M. Significant differences werecalculated by Wilcoxon matched-pair signed-ranks test (each person is his own control). In thein vitro study, results are expressed as meant S.D. Significant differences were calculated withanalysis of variance, a two-way cross-classification and a one-way classification with Tukey's test.

Results

Post-prandial blood glucose responses

The mean incremental blood glucose responses during 180 min following ingestion ofthe different test meals are shown in Fig. l(a). Relevant parameters, such as the Fb,peak- and 180-min blood glucose values are listed in Table II together with' areas underthe curves during 120 min' and GI. Similar Fb values were recorded prior to all the testmeals and any differences in post-prandial behaviour can thus be assigned to differentproperties. All pasta products (spaghetti, macaroni and' spaghetti porridge ') producedsignificantly lower blood glucose responses than' spaghetti bread' or mashed potatoesin the early phase (45 and 60 min). This slower behaviour in the case of pasta productswas generally manifested also by lower peak glucose values (Table II). Only fewsignificant differences were noted between the pasta products in the early phase. Oneexception was that the glucose response to spaghetti was significantly lower (P < 0'01)compared with that to 'spaghetti porridge' at 60 min [Fig. l(a)].

In contrast to a lower initial phase, the spaghetti resulted in a higher post-prandiallevel of glucose in the late phase than for all the other products. The difference wasparticularly noteworthy when compared with mashed potato. However, the late glucoseresponse (180 min) to spaghetti was significantly higher (P < 0'01) also when comparedto 'spaghetti porridge' and macaroni. A significant difference was also noted between'spaghetti porridge' and the macaroni product at 120 min, the macaroni producing ahigher response [Fig. l(a)].

The mean incremental blood glucose values for the' high responders' are shown inFig. l(b). The pattern concerning both the early and late glucose responses was similar

TABLE II. Fasting blood glucose and post-prandial glucose characteristics (means.E.M.)

Fasting blood 180-min blood Area under curveglucose (Ph) Peak above Pb glucose value (120 min) Glycemic index

Meals (roM) (roM) (roM) (roM/min) Gl (120 min)

Macaroni 438007a 193O24b 011 O'08a 1025 16-4a,c,d 78'0 114a.b.c

Spaghetti 436 006a l-69020 0'470'1Q" 80'0 112c 60'56'4b'Spaghetti porridge' 44S004a 2'030'22c - 006 0-09 96-99'4d 73-4S6c

Bread baked from 4'330-17a 2-61 O-28a -0-01 0-08 137-6 16S,b looaspaghetti ingredients

Mashed potatoes 4'380-07" 257 0-33",b -0'2S0-llb 140-219'1b 100-976a

Values not sharing the same superscript letter in each column are significantly different (P < 0'05).

Glycemic indexGl (90 min)

705 101 b,C56-2S'3b73-8 4'7c

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