Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2005
Volume 8
Issue 4
Topic:
Biotechnology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Żyła K. , Mika M. , Fortuna T. , Szymczyk B. , Czubak M. 2005. EFFECTS OF SESAME SEEDS GERMINATION ON PHYTATE DEGRADATION, IN VITRO NUTRIENT DIGESTIBILITY AND PLASMA LIPID PROFILE IN RATS, EJPAU 8(4), #53.
Available Online: http://www.ejpau.media.pl/volume8/issue4/art-53.html

EFFECTS OF SESAME SEEDS GERMINATION ON PHYTATE DEGRADATION, IN VITRO NUTRIENT DIGESTIBILITY AND PLASMA LIPID PROFILE IN RATS

Krzysztof Żyła1, Magdalena Mika1, Teresa Fortuna2, Beata Szymczyk3, Marta Czubak1
1 Department of Food Biotechnology, University of Agriculture in Cracow, Poland
2 Department of Analysis and Food Quality Evaluation, Agricultural University, Cracow, Poland
3 National Research Institute of Animal Production Department of Animal Nutrition and Feed Science in Aleksandrowice, Poland

 

ABSTRACT

Germination of sesame seeds was used to activate endogenous phytate-degrading enzymes that were expected to dephosphorylate phytate into lower myo-inositol phosphates. Effects of germination were related to the activity of endogenous phytase, chemical composition of seeds and in vitro digestibility of nutrients, as well as phosphorus and calcium metabolic balance and lipid composition in the plasma of rats. The activity of sesame phytase grew from 1.4 FTU/g in raw seeds to 5.0 FTU/g after 36 hours of germination at 30°C and pH 5.5, but only minor changes in the profile of myo-inositol phosphates in germinated seeds were observed. In spite of limited phytate hydrolysis, germination improved in vitro digestibility of phosphorus, calcium, magnesium and carbohydrates, but did not influence digestibility of protein. In rats, germination improved feed intake and feed efficiency (p<0.01), improved HDL/total cholesterol ratio (p<0.05) and lowered concentration of plasma triacyloglicerols p<0.05).

Key words: sesame seed, germination, phytate, phytase, in vitro digestibility, rat, serum lipids.

INTRODUCTION

Sesame (Sesamum indicum L.) being one of world’s most important oil seed crops offers good source of edible oil and nutritious food for humans. It is extensively used in baked goods and confectionery products, whereas sesame oil cake is used as valuable feed for farm animals [26, 14]. Since some backed goods contain as much as 50% of sesame seeds, they offer promising possibilities for their application in the production of functional foods. Sesame seeds are primarily known for their high lignans (sesamin and sesamolin) contents in oil that create excellent resistance to oxidative deterioration but they are also an abundant source of phytic acid (myo-inositol hexakisdihydrogen phosphate, IP6). Sesame lignans have been claimed to exert important physiological effects including antioxidant, anticarcinogen, blood pressure lowering and serum lipids lowering in experimental animals and humans [7, 23]. Similar claims have been made in relation to phytate and to lower myo-inositol phosphates – products of enzymatic phytate hydrolysis [16, 19]. Germination is a well known bioprocess that improves nutritional quality of oilseeds [4]. During germination, cereal grains and oilseeds synthesize the enzyme phytase (EC. 3.1.3.8), that dephosphorylates phytate to lower myo-inositol phosphates (IP1-5) releasing phosphate residues. Lower myo-inositol phosphates (mainly tri- and tetraphosphates, IP3+4) produced by the action of certain plant phytases may have important and unique physiological functions that relate mostly to phosphorus and calcium metabolism [17, 24]. Extracellular inositol 1,2,3,6-tetraphosphate have been shown to enhance calcium absorption in rats [20]. Furthermore, myo-inositol phosphates released from phytate by 6-phytase A have been evidenced to affect birds physiology differently than their counterparts produced by microbial 3-phytase A [29]. Although different effects of germination have been studied thoroughly in many oilseeds [6], there are no literature data about biosynthesis of endogenous phytase in sesame seeds during germination, and nothing is known about the impact of that process on the nutritional value of sesame seeds. From among enzymes that participate to the phosphorus metabolism in germinating sesame seeds inorganic pyrophosphatases have only been studied [5].

The objective of the study was to stimulate biosynthesis of phytate-degrading enzymes in sesame seeds by germination and to learn changes in seeds composition and in in vitro digestibilities of protein, carbohydrates, phosphorus, calcium and magnesium produced by that process. Yet another objective was to learn the influence of sesame seeds germination and defatting on growth performance and serum lipids composition in rats.

MATERIAL AND METHODS

Material
Seeds of sesame (Sesamum indicum L.) were purchased in a local healthy food store. Cellulase, xylanase, pepsin and pancreatin were purchased from Sigma Chemical Inc,. Pancreatin had an activity of 8 x United States Pharmacopeia (USP). Dialysis tubings had the molecular weight cut-off 12 kDa.

Sesame seeds germination
Sesame seeds were soaked in 0.2 % of potassium sorbate, placed on thin layers of wet cotton in the Petri dishes and germinated at different temperatures in laboratory incubators. Germination times were 0, 12, 24, 36, 48, 72 and 96 h whereas germination temperatures 25, 30, 35, 40, and 45°C. Samples of germinated seeds were washed from the sorbate solution, freeze dried and ground to pass a 1 mm screen and stored at a room temperature.

Sesame seeds defatting
Sesame seeds were defatted with hexane in a Soxhlet apparatus and dried under vacuum.

Phytase activity determinations
Phytase activity in seeds was determined by a procedure that involved 30 min extraction of a 0.25 g sample at 30°C and pH 4.0 with 100 units of ultrafiltrated (membrane cut-off 30 kDa) Aspergillus niger cellulase and xylanase preparations. The extract was centrifuged and mixed with 5 mM sodium phytate solution. Amounts of phosphate liberated after 60 minutes reaction at 40°C and pH 5.0 were determined colorimetrically. One unit of phytase activity (FTU) was defined as the amount of enzyme required to liberate 1µM of inorganic phosphorus under the conditions of the assay.

Determination of myo-inositol phosphates
Samples were defatted, mixed with 0.4M HClO4 and extracted with a shaker for 2 h. The extract was centrifuged (8000 g, 30 minutes), neutralized with 1M K2CO3 and put on a HyperSep NH2 SPE column. A fraction eluted from the column by 0.6 mL 1M HCl was used for HPLC analysis of inositol phosphates. Inositol phosphates (IP6-4) were determined by a modified Lehrfeld procedure [12] using ion-pair C18 reverse phase HPLC column and a refractive index detector.

In vitro determination of nutrients digestibility
An in vitro procedure designed to simulate digestion in the intestinal tract was used. Triplicate samples (0.4 ± 0.001 g) of ground sesame seeds were weighted into plastic syringes without Luer-lock tips. Samples were hydrated with 1.0 mL of water and an HCl solution so that a pH value of 2.0 was obtained. Then 6200 units of pepsin were added, and the contents of each tube were mixed, sealed with parafilm, vortexed, and incubated in a water bath at 37°C for 2 hours. At the end of this period, 0.5 mL of a NaHCO3 solution along with 2 mg of pancreatin and 25 mg of bile extract (0.6 mL) were added to reach pH 7.0. The slurry was transferred quantitatively to segments of dialysis tubings (cut-off 14 kDa) by means of syringe piston. Segments were placed in 250 mL Erlenmeyer flasks containing 40 mL of 0.1 M NaCl in a 0.05M imidazole buffer pH 7.0 and were incubated in a shaking water bath. The volumetric ratio between the digesta and dialysing medium was about 1:25. After 4 hours, samples of the dialysate were withdrawn for determination of inorganic phosphate, calcium, magnesium, protein and reducing sugars as detailed elsewhere [28].

In vivo experiment on rats
The experiment was conducted following the Guidelines for Animal Care and Treatment set by the Council of European Communities and the protocols were approved by the local Animal Ethics Commission. Growing male albino Wistar rats, at 6 wk of age, weighing 100 ± 1.5 g were housed in stainless steel cages, maintained at 25°C, with 12 h light:dark cycle and unlimited access to feed and water. After adaptation period, the animals were randomly divided into four groups of five and fed different diets for five weeks. The control group (CON) was fed an egg white-sucrose diet, whereas in other groups egg white was partially substituted for by sesame seeds (SES), germinated sesame seeds (GER), or defatted sesame seeds (DEF). Detailed composition of experimental diets is given in Table 1.

Table 1. Ingredient composition and proximate analyses of experimental diets (g/kg) fed to rats

Item

CON

SEZ

GER

DEF

Sesame seeds

Sesame seeds germinated

Sesame seeds defatted

Egg white

Sucrose

Cellulose

Sunflower oil

Mineral premix

Vitamin premix

DL-methionine

Corn starch

Proximate composition (g/kg):

dry mass

protein

fat

fiber

ash

-

-

-

200

200

40

135

40

20

3

362

 

948

192

132

37

43

300

-

-

115

200

20

-

40

20

3

302

 

951

188

134

38

56

-

300

-

115

200

20

23

40

20

3

294

 

948

190

135

38

55

-

-

300

50

200

15

121

40

20

3

266

 

953

189

132

36

67

Four experimental diets were fed: CON, the control egg white-based diet; SEZ- a diet with untreated sesame sees, GER – with germinated sesame seeds, DEF- with defatted sesame seeds
Mineral premix supplied per kg of diet: CaCO3 12.4 g; KH2PO4 13.2 g; Ca(H2PO4)2 3 g; MgSO4.7H2O 4.2 g; NaCl 6.7g; MnSO4 4H2O 0.21 g; ZnSO4 7H2O 0.025 g; FeSO4 7 H2O 0.13 g; KJ 0.032 g; CuSO4 5 H2O 0.098 g.
Vitamin premix supplied per kg of diet: retinyl acetate 4375 IU; cholecalciferol 1750 IU; thiamin mononitrate 17.5 mg; riboflavin 35 mg; pyridoxine 35 mg; cyanocabalamin 33 µg; biotin 0.8 mg; p-aminobenzoic acid 10 mg; nicotinic acid 100 mg; calcium pantothenate 35 mg 2.926 mg of Ca); dl-α-tocopheryl acetate 8.7 mg; choline HCl 1 g.

Performance parameters of rats were recorded for the experimental period from week 3 to 5. At the completion of the experiment rats were deprived of food overnight, anesthetized intraperitoneally with sodium tiophenal (Biochemie, Viena, 25 mg/100 g body weight) and killed by withdrawing blood from heart. Blood samples were centrifuged (4000 g, 10 min) and the serum samples were analysed using commercially available test kits for total cholesterol (TC), HDL-cholesterol (HDL), and triacylglicerols (TG). Test kits were from Olympus Diagnostica GmbH, Hamburg, Germany. The concentration of total lipids in serum was determined enzymatically using standard kits (Cormay) on Beckman DU640 spectrophotometr.

Chemical analyses
Basic chemical analyses were performed using a Tecator autoanalyser. Inorganic phosphorus content was assayed using the procedure of Kessel [8].

Statistical analyses
Data were subjected to one-way analysis of variance using Statgraphics Plus for Windows 4.0 statistical package. Mean differences were determined using Fisher’s least significant difference test. Statistical significance was accepted at p < 0.05.

RESULTS AND DISCUSSION

Effects of temperature and time of germination on phytase activity in sesame seeds
The temperature optimal for phytase biosynthesis during sesame seeds germination at pH 5.5 was 30°C. The value is close to 31.9°C found by Baptista de Carlvalho et al. [1] as optimal germination temperature for a black variety of sesame. Wanasundara et al. [25] reported a maximum lipase biosynthesis after 4 days of germination Sesamum indicum L seeds. Similarly, the activities of acid and alkaline inorganic pyrophosphatases in sesame cotyledons were growing up to day 4 of germination [5]. In our study, in the course of the first 36 hours of sesame seeds germination the activity of endogenous phytase increased 3.4 fold and then slightly declined (Table 2).

Table 2. Phytase activity (FTU/g) in sesame seeds at different times and temperatures of germination

Time (h); Temperature = 30 °C

Temperature (°C); Time = 36 h

 

Mean

Std Error

 

Mean

Std Error

0

1.45

0.05

25

3.66

0.08

12

1.92

0.04

30

4.89

0.05

24

4.21

0.11

35

4.67

0.07

36

4.99

0.09

40

3.01

0.09

48

4.25

0.08

45

0.87

0.10

60

4.30

0.12

     
Values are means of five determinations

Quite different kinetic of phytase biosynthesis was reported by Muzquiz with co-workers [15] who observed a constant increase in the enzyme activity during 120 hours of germination in the seeds of lupine. Similarly, Laboure et al. [11] (1993) reported the increase of phytase activity in maize seedlings during the first 7 days of germination. Maximum phytase activity in lentils, broad beans and runner beans was found after 8 days of germination whereas in chick peas, after 6 days [10]. In the study presented here, phytase in a crude extract of sesame seeds was found to have pH 5.0 and temperature 40°C optimal for enzymatic activity (data not shown). These values are quite common for phytases from legumes and oilseeds [9]. A phytate-degrading enzyme, purified from germinated 4-day-old faba bean seedlings showed maximum of enzymatic activity at pH 5.0 and temperature 51°C [27]. In the seedlings of maize the pH and temperature optima of the purified enzyme were found to be 4.8 and 55°C , respectively. It is quite possible therefore, that the crude sesame seeds extract contained different phytate-degrading enzymes, similarly to wheat, barley, soybean or lupine [3].

Effects of germination and defatting on the chemical composition and on in vitro digestibility of nutrients in sesame seeds
In order to learn nutritional effects that might be related to the oil fraction of sesame seeds, a part of seeds was subjected to defatting. As compared to sesame seeds used in another study [26], the seeds employed in the work presented here had lower fat content (47.2 vs. 52.3 %) and higher protein content (27.4 vs. 21.0 %). Defatted seeds of sesame had the composition similar to commercially processed sesame seed meal (sesame seed residue after oil extraction, [14]), but were much higher in protein. As a result of sesame seeds germination, the fat and carbohydrates content decreased from 47 to 40 %, and from 6.05 to 2.03 %, respectively, but protein content was not changed significantly (27 vs. 28 %; Table 3).

Table 3. Changes in chemical composition, and in in vitro digestibilities of nutrients in sesame seeds subjected to germination or defatting

Sesame seeds

 

raw

germinated

Defatted

 

raw

germinated

defatted

Ash (%)

4.74

5.02

9.24

IP6 (µM/g )

38.20

36.37

70.29

Protein (%)

27.38

27.99

50.38

IP5 (µM/g)

3.11

2.69

5.72

Fiber (%)

7.11

7.23

9.03

IP4 (µM/g)

n.d.

0.46

n.d.

Fat (%)

47.20

39.85*

5.02

P dial (mg/g)

0.225

0.528*

0.2708

Carbohydrates (%)

6.05

2.03*

26.32

Ca dial (mg/g)

0.253

0.736*

0.198

P total (mg/g)

7.507

7.544

8.548

Mg dial (mg/g)

0.996

1.297*

1.3538

P inorganic (mg/g)

0.589

1.367*

1.404

Protein dial (mg/g)

86.13

87.81

124.06

Ca (mg/g)

10.67

10.03

22.25

Sugars dial (mg/g)

6.01

51.31*

12.33

Mg (mg/g)

3.695

3.699

5.715

       
Values are means of five determinations.
n.d. – not detected
* denotes significant difference between means of raw and germinated seeds at p<0,05; t-test. Data for defatted seeds were not included in the analysis

Total contents of phosphorus, calcium and magnesium remained unchanged, and the concentration of inorganic phosphorus in seeds increased from 0.6 to 1.4 mg/g. During germination the concentration of IP6 decreased only slightly (from 38.2 to 36.4 μM/g), the decrease in the concentration of IP5 was not significant, and minor amounts of IP4 were produced. Although germination of sesame seeds activated endogenous phytase more than three fold, this created only minor changes in the profile of myo-inositol phosphates. This observation is in agreement with findings of Muzquiz and co-workers [15], who reported similar phenomenon in germinated lupines. Contradictory results were provided by Silva and Trugo [22], who observed decreasing concentrations of phytate during 8 days germination of lupines. The hydrolysis of phytate by enzymes activated during germination was observed also in germinating soya beans [2] and rapeseed [13]. An effective mean of reducing phytic acid content in sesame seeds was suggested by Mukhopadhyay and Ray [14], who reported a substantial decrease in phytic acid content after bacterial fermentation with Lactobacillus acidophilus. We found that only at a high temperature (120°C), in acidic pH, after prolonged reaction time, sesame phytate can be in substantial quantities converted into lower myo-inositol phosphates (data not shown). A practical application of such a procedure is doubtful, however. In the in vitro digestion model, germination of sesame seeds improved digestibility of phosphorus, calcium, magnesium and carbohydrates, but did not influence digestibility of protein. As a result of defatting, the content of fat in seeds was decreased from 47.2 to 5.0 %. This produced a subsequent increase in other seeds constituents ranging from 14% for total phosphorus to 435% for carbohydrates. Germination considerably decreased the concentration of carbohydrates and fat in sesame seeds. The same was reported for germinating soybean [2], but in contrast to sesame, the germination of soybean produced also significant changes in dietary fiber and protein fractions. Protein content declined during the first day of rapeseed germination and thereafter increased [13]. Apparently there is not a single pattern for biochemical changes that take place during germination of different oilseeds.

Effects of germination and defatting of sesame seeds on growth performance of rats
There were significant effects of experimental diets on rats’ growth performance (Table 4). Animals fed the control egg white–sucrose based diet had superior feed intakes, daily weight gains, final body weights and feed efficiencies among all the rats tested. Partial substitution of egg white for different forms of sesame seeds substantially depressed growth parameters in rats. Feed intakes were the lowest in animals fed defatted or non-defatted seeds of sesame. Germination of sesame resulted in improved feed intakes, and better feed efficiencies than in animals fed non-germinated seeds. Similar observations were made by Mukhopadhyay and Ray [14] in fish fed sesame meal fermented by lactic acid bacteria. Among animals fed different forms of sesame, rats receiving defatted seeds had the best feed efficiencies, and, consequently, final body weights. It may be concluded that the growth suppressing factors were associated with the fat fraction of sesame seeds and these factors were partially removed during seed germination. These must have been biologically active compounds other than phytates.

Table 4. Growth performance of rats fed different experimental diets (means ± SEM)

Parameter

Diet

CON

SEZ

GER

DEF

Feed intake (g/rat/day)

Body weight gain (g/day)

Final body weight(g)

Feed efficiency (g/g gain)

17.00 ± 0.20 Aa

4.09 ± 0.11A

237.00 ± 1.41A

4.16 ± 0.12A

15.64 ± 0.26Cc

1.01 ± 0.09 C

135.50 ± 1.22C

14.22 ± 1.77D

16.18 ± 0.19Bb

1.24 ± 0.09C

142.83 ± 1.48C

13.04 ± 0.27C

15.73 ± 0.15BCc

1.82 ± 0.11B

163.17 ± 1.49B

8.64 ± 0.19B

CON – an egg white-sucrose diet, in other diets egg white was partially substituted for by sesame seeds (SES), germinated sesame seeds (GER), or defatted sesame seeds (DEF).
A, B, C, D – Means within rows with no common superscript capital letter differ significantly at p<0.01; a, b, c – Means within rows with no common superscript letter differ significantly at p<0.05

Effects of germination and defatting of sesame seeds on serum lipids profile in rats
In comparison to the control egg white-based diet, the rats receiving different forms of sesame seeds had higher levels of total cholesterol in the serum (Table 5).

Table 5. Effects of experimental diets on lipids profile in serum in rats (means ± SEM)

Lipid fraction

Diet

CON

SEZ

GER

DEF

TC (mg/dl)

HDL (mg/dl)

(HDL/TC)

TG (mg/d1)

Total lipids(mg/dl)

81.20 ± 0.92 a

67.38 ± 0.89 a

0.829 ± 0.05 Aa

63.68 ± 0.95 Aa

391.17 ± 1.84 a

88.97 ± 0.95 b

74.63 ± 1.00 b

0.838 ± 0.03 Aa

53.17 ± 0.78 Bb

435 ± 2.58 b

85.25 ± 0.58 ab

72.85 ± 0.62 ab

0.855 ± 0.05 Ac

46.58 ± 0.53 Bc

430 ± 2.23 b

88.08 ± 0.87 b

69.83 ± 0.79 ab

0.792 ± 0.06 Bb

47.15 ± 1.00 Bc

411.16 ± 1.64 ab

CON - an egg white-sucrose diet, in other diets egg white was partially substituted for by sesame seeds (SES), germinated sesame seeds (GER), or defatted sesame seeds (DEF). Detailed composition of experimental diets is given in Table 1.
A, B, C, D – Means within rows with no common superscript capital letter differ significantly at p<0.01; a, b, c – Means within columns with no common superscript letter differ significantly at p<0.05

The unexpected effect, observed also for the total lipids concentrations, was partially reduced by sesame seeds germination. Moreover, germination of sesame seeds resulted also in the highest HDL to TC ratios and in the lowest concentrations of triacylogycerols in sera of animals receiving diets with germinated seeds of sesame. On the other hand, the highest concentrations of HDL-cholesterol were found in animals fed the non germinated seeds of sesame. Substituting endogenous sesame oils with sunflower oil did not affect total cholesterol concentrations in blood, but resulted in lower HDL-cholesterol concentrations and the lowest HDL to TC ratios among all experimental animals. The substitution, however, lowered the concentrations of triacylglycerols in sera to levels which did not differ from those observed in rats fed the germinated seeds of sesame. Sirato-Yasumoto and co-workers [23] found that sesame seeds significantly decrease levels of triacylglycerols and total cholesterol in sera of rats, and attributed the effect to the high concentrations of sesame lignans. The findings of our work confirm the effect only in relation to triacylglycerols. Germination of sesame seeds however, additionally modulates plasma lipid profile in rats. The effects cannot be attributed to lower myo-inositol phosphates, since these compounds were not accumulated during germination in substantial quantities. Apart from phytate, many different biologically active compounds like lectins, protease inhibitors, α-amylase inhibitors, polyphenols are known to be present in oilseeds. Shyu and co-workers [21] cloned and characterised cystatin – a cysteine protease inhibitor from maturing sesame seeds that was effectively degraded in the course of germination. Similarly, Sandberg [18] reports germination to be an effective mean of reducing polyphenol contents and increasing bioavailability of minerals in legumes. Nutritionally favourable changes that may be attained by biotechnological modifications of antinutritional factors in legume and oilseeds have been reviewed by Hajos and Osagie [4].

Germination of sesame seeds, although ineffective in reducing their phytate content, decrease fat and carbohydrate concentrations, increase bioavailability of phosphorus, calcium and magnesium in vitro, and, in consequence, favourably alters lipid metabolism in rats enhancing HDL to TC ratio and lowering triacylogycerols concentration in blood plasma.

ACKNOWLEDGEMENTS

Authors would like to acknowledge financial support from the Polish Research Committee (grant Nb. AR 73/31/PBZ/021/P06/99).

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Krzysztof Żyła
Department of Food Biotechnology,
University of Agriculture in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
phone/fax: (+4812) 662 4794
email: kzyla@ar.krakow.pl

Magdalena Mika
Department of Food Biotechnology,
University of Agriculture in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland

Teresa Fortuna
Department of Analysis and Food Quality Evaluation,
Agricultural University, Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
email: rrfortun@cyf-kr.edu.pl

Beata Szymczyk
National Research Institute of Animal Production
Department of Animal Nutrition and Feed Science in Aleksandrowice, Poland
32-083 Balice, Poland

Marta Czubak
Department of Food Biotechnology,
University of Agriculture in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland

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