AFS – Advances in Food Sciences Continuation of CMTL founded by F. Drawert

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2 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

CONTENTS

ORIGINAL PAPERS

DEVELOPING FUNCTIONAL FOODS USING RED PALM OLEIN. 138 I. PAN BREAD AND SUGAR-SNAP COOKIES S. N. Al-Hooti, J. S. Sidhu, J. M. Al-Saqer, H. A. Al-Amiri, A. Al-Othman, I. B. Mansour, and M. Johari

THERMAL RESISTANCE OF Bacillus coagulans 144 IN PHOSPHATE BUFFER AND TOMATO JUICE A. H. Dinçer and A. Ünlütürk

A COMPARATIVE STUDY OF THE MICROBIOLOGICAL QUALITY AND 148 CONSUMER ACCEPTABILITY OF SOYCHEESE AND MILK-BASED CHEESE O. O. Aboaba and M. S. Adeleye

MÖGLICHKEITEN UND GRENZEN DES EINSATZES VON GASSENSOR-ARRAYS 154 ZUR QUALITÄTSBEURTEILUNG VON LEBENSMITTELN S. Nitz und D. Hanrieder

SHORT COMMUNICATIONS

CONSTITUENTS OF ESSENTIAL OIL OF 170 Echinophora tenuifolia L. subsp. sibthorpiana (Guss.) Tutin. H. Ç. Özen and Z. Toker

FATTY ACID COMPOSITIONS OF Hypericum triquetrifolium TURRA 173 H. Ç. Özen and M. Başhan

PRESS RELEASE

Fraunhofer Institute for Process Engineering and Packaging IVV: 175 Conference on Packaging Food and Pharmaceuticals – Science for Compliance (May 27 and 28, 2003, Erding – GERMANY)

INDEX 176

SUBJECT INDEX for Advances in Food Sciences 2002 177

AUTHOR INDEX for Advances in Food Sciences 2002 179

137 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

DEVELOPING FUNCTIONAL FOODS USING RED PALM OLEIN. I. PAN BREAD AND SUGAR-SNAP COOKIES

Suad N. Al-Hooti, Jiwan S. Sidhu, Jameela M. Al-Saqer, Hanan A. Al-Amiri, Amani Al-Othman,1 Isa B. Mansour,2 and Minal Johari2

Biotechnology Dept. 1Systems Development Dept., Kuwait Institute for Scientific Research, Safat, Kuwait, 2Malaysian Palm Oil Board, Malaysia

SUMMARY

Red palm olein (RPOL) is a specially prepared vege- level in the blood responds to increasing dietary intake. table oil rich in two important antioxidants, β-carotenes The carotenoid profile of palm oil is rich in α-carotenes and vitamin E. Two functional foods, pan bread and (30 to 40%) and β-carotenes (50 to 60%). Apart from sugar-snap cookies, were prepared by replacing normal their functions as precursors of vitamin A [6], these caro- bakery shortening with red palm shortening (RPS) and tenoids can quench singlet oxygen and serve as antioxi- RPOL. Replacement of up to 100% RPS and RPOL in dants in tissues, particularly under conditions of low oxy- sugar-snap cookies had no adverse effect on their quality gen tension. They can stimulate the immune response and when compared with control bakery shortening. However, protect against some types of induced cancers [7]. Palm 100% replacement with RPOL in the bread formulations oil is also the largest potential source of vitamin E, which had a negative effect on the baking characteristics of test has been advocated for the prevention of heart attacks [8]. breads, when compared with control breads. A sensory The vitamin E content in palm oil is unique, represented evaluation of these functional foods revealed that all of mainly as tocotrienols (70%) rather than as tocopherols the products were well-accepted by the consumers. The (30%). α-Tocopherol and γ-tocotrienol account for the research data presented in this paper lends strong support major portions of the total tocopherols and tocotrienols. to the view that good quality, acceptable bread and sugar- Natural tocopherol, especially α-tocopherol, is a superior snap cookies, rich in natural antioxidant vitamins (vitamin radical chain-breaking antioxidant compared with synthetic E and β-carotene) can be prepared successfully using ones. The presence of this natural vitamin E in palm oil RPOL and RPS, thus providing healthy choices of nutri- ensures a longer shelf-life for palm-based food products. tionally superior functional foods to the consumers. Recent research shows a significant and adverse ef- fect of trans fatty acids (TFA) on LDL cholesterol levels, with each 1% increase in dietary energy from TFA lead- KEYWORDS: Functional foods, pan bread, cookies, vitamin E, ing to a 0.028-mmpl/L increase in LDL [9]. Main sources β-carotenes, nutritive value, sensory quality, red palm olein. of TFA in the human diet are deep-fried foods, spreads, margarine, cookies, cakes and bread. Palm oil needs very little or no hydrogenation and is being used extensively in the manufacture of hardened fats like vanaspati in India,

Nepal, Pakistan and the Middle East [1-2]. One advantage INTRODUCTION of palm olein is that it can be used without hydrogenation, thereby, excluding the possible adverse effects of dietary Crude palm oil is the richest natural plant source of trans fatty acids. carotenoids, containing between 500 and 700 ppm carote- noids [1-2]. Refined palm oil contains no carotenoids as Currently, there is a growing belief that healthy eating is they are removed or destroyed during processing. Red a better way to manage illness than medication. The cross- palm olein (RPOL), a product of palm oil, is refined by a over effect from dietary supplements to foods is increasing mild, specially designed process so as to retain most of the demand for fortified, functional and medical foods [10]. the natural carotenes [3-5]. RPOL is edible oil and is the There is an immense interest in the nutritional benefits of richest natural plant source of carotenoids as well as vi- vitamin E due to its preventive and therapeutic potentials tamin E. During digestion in the human alimentary canal, against free-radical-mediated degenerative diseases such as β-carotene is absorbed through the intestinal wall and its arteriosclerosis and certain types of cancer [11].

138 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

Considering the importance of β-carotenes and vitamin sodium bicarbonate, 2.75 g of ammonium bicarbonate, 7 g E, this study was carried out to utilize the nutritionally supe- of nonfat dry milk, 2.25 g of dextrose and 1.25 g of SSL. rior RPOL and RPS in the development of two important Initial trials were conducted by replacing bakery shorten- functional foods, viz., pan bread and cookies. The results of ing (Wesson, USA) with RPOL or RPS at 0, 25, 50, 75, the initial laboratory studies using RPS and RPOL in bread and 100% levels. and sugar-snap cookies are presented in this paper. Physical Measurements for Bread A proofed dough was weighed just before loading MATERIALS AND METHODS into the baking oven and then immediately after removal from the oven at the end of baking. Weight loss in baked Raw Materials bread was divided by the proofed dough weight and mul- tiplied by 100, and then expressed as the percentage bak- RPS and RPOL were provided free of charge by the ing loss. The specific loaf volume of the bread was deter- Carotino Company of Malaysia. White flour, whole- mined by the rapeseed displacement method [12]. wheat flour, and fine bran were obtained from the Kuwait

Flour Mills and Bakeries Co., Kuwait. Fine granulated Physical Measurements for Cookies sugar, common salt, bakery shortening (Wesson brand, USA), instant dry yeast and nonfat dry milk were pro- The mean diameter of a cookie was measured by cured from the local market. Diacetyl tartaric acid esters placing six cookies side by side, measuring their length, of monoglycerides (DATEM) and sodium stearoyl-2- then rotating each cookie 90° and measuring their total lactylate (SSL) emulsifiers were procured from the length again, and then taking the average of the two read- American Ingredients Co., Kansas City, USA. All other ings. The six cookies were then stacked one on top of the chemicals were of analytical grade. other, the height was measured, and the average thickness of one cookie was calculated. The cookie spread ratio was Pan Bread Formulation then calculated by dividing the average diameter by the For bread-making, whole-wheat flour, fine bran (20% average height of the cookie. replacement of white flour), and unbleached straight-grade Sensory Analysis flour with 72% extraction were used in various trials. For bread-making, the optimized straight-dough bread-making The baked bread and cookie samples were cooled to o method (Method 10-10B) of the American Association of room temperature (22±1 C) and subjected to sensory analy- Cereal Chemists [12] was used. The bread was baked at sis on a nine-point hedonic scale for crumb color, texture, 215°C for 24 min. The basic bread dough formulation flavor and overall acceptability, using a semi-trained panel contained 200 g of flour, 12 g of sucrose, 3 g of salt, 6 g of of 12 judges from among KISR’s employees [13]. Each shortening, 8 g of nonfat dry milk, 3.5 g of instant dry panelist was served a control sample along with test sam- yeast, 0.7 g of malt flour and 75 ppm of ascorbic acid. ples, and was asked to assign scores on a nine-point he- The amounts of flour and bran used in these trials were donic scale for crumb color, texture, flavor and overall calculated on a 14% moisture content basis. Two loaves acceptability. A sensory score of 5 or above was rated as were made per baking test, and each baking test was rep- acceptable, and a score below 5, was considered unaccept- licated two times. Initial trials were conducted for replace- able. These data were analyzed statistically for analysis of ment of bakery shortening (Wesson, USA) with RPOL or variance, and the average values are reported. RPS at 0, 25, 50, 75, and 100% levels. Trial breads were made with whole-wheat flour, white flour (72% extraction), Statistical Analysis or white flour and fine bran (80:20 ratios). In these tri- All the sensory data obtained were analyzed statisti- als, the corresponding control breads were formulated cally for analysis of variance, and the mean values were from whole-wheat flour, white flour, and white flour evaluated for statistical significance (p = 0.05) using with 20% bran (brown bread). The control breads in all Duncan's New Multiple Range Test (SAS Program, Win- trials contained only Wesson shortening and no RPS or dows Version 6.08), and inferences were reported at the RPOL. Different combinations of RPS and RPOL, i.e., appropriate places. Significance was accepted at the p = 0:100%, 25:75%, 50:50% and 100:0% were also tried. 0.05 level. For the baking characteristics, the mean values After the initial trials, 0.5% DATEM was used in the of the results are reported. optimized trials.

Sugar-Snap Cookies Formulations RESULTS AND DISCUSSION The cookies were made as per standard Method 10-50D of the American Association of Cereal Chemists [12]. The Three types of bread (whole-wheat flour bread, white cookies were baked at 205°C for 10 min. The basic bread and brown bread) and sugar-snap cookies made in cookie dough formulation contained 225 g of white flour, the laboratory were evaluated for their baking characteris- 135 g of sucrose, 2.25 g of salt, 70 g of shortening, 1 g of tics, and sensory quality, and the results are presented here.

139 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

Optimized Pan Bread Formulations 100% in white bread samples resulted in a decreased 3 3 After the preliminary trials, a number of pan bread specific loaf volume (3.7 cm /g) compared with 4.5 cm /g formulations were developed. Whole-wheat flour, for the control (Table 2). straight-grade flour (white flour with 72% extraction), or The baking characteristics of brown bread (made from white flour with 20% bran (as a replacement for flour for white flour and 20% bran) formulated with 100% RPS or making brown bread) were used for producing control 100% RPOL as well as the control bread samples are bread (with Wesson brand control shortening) and test presented in Table 3. The results indicate that 20% bran bread samples (with RPS and/or RPOL). bread samples with 100% RPS had a higher specific loaf volume of 4.3 cm3/g than those made with 100% RPOL As can be seen from the data in Table 1, increasing the of 3.3 cm3/g, when compared with whole wheat control level of RPS in whole-wheat bread formulations produced bread (4.0 cm3/g), white control bread (4.7 cm3/g), and comparable percentages of baking loss and specific loaf 20% bran control bread (4.3 cm3/g). This shows clearly volumes with those of the whole-wheat control bread, that in comparison with RPS, the RPOL performed poorly whereas increasing the level of RPOL resulted in decreased during bread-making. specific loaf volume compared with the whole-wheat con- trol bread. The lowest specific loaf volume (2.7 cm3/g) was The results of utilizing different combinations of RPS recorded for bread samples with 100% RPOL. and RPOL at 100% replacement of bakery shortening and use of DATEM emulsifier in the formulation of bread sam- Bread samples formulated with white flour had ples made from whole-wheat bread are presented in Table 4. higher baking loss values (Table 2) than the whole-wheat The results indicate that utilizing RPOL at the 100% level bread (Table 1). The specific loaf volume values for all resulted in a lower specific loaf volume (2.6 cm3/g) com- white bread samples, including the control, were higher than pared with the control whole-wheat bread (3.6 cm3/g). The those for whole-wheat bread at all levels of RPS or RPOL specific loaf volume of test breads increased with increas- replacement (Tables 1 and 2). Increasing levels of RPS up ing levels of RPS, and reached 3.3 cm3/g at 100% RPS, to 100% in white bread samples had no effect on the spe- which is comparable with that of the control whole-wheat cific loaf volume (4.1 to 4.3 cm3/g), which was compara- bread. Utilization of DATEM in the formulation resulted ble to that of control white bread samples (4.1 cm3/g), in further slight improvement in specific loaf volume with whereas increasing the replacement levels of RPOL to all combinations of RPS and RPOL.

TABLE 1 Effect of replacing bakery shortening (Wesson, USA) with red palm shortening (RPS) or red palm olein (RPOL) on the baking characteristics of whole-wheat bread.

Replacement Level Baking Loss (%) Specific Loaf Volume (cm3/g) (%) Mean ± SD Mean ± SD RPS RPOL RPS RPOL 0 (Control) 11.5±1.5 11.9±1.0 3.5±0.0 3.2±0.4 25 11.9±0.3 11.1±1.4 3.4±0.1 3.3±0.3 50 11.4±0.8 11.7±0.3 3.4±0.0 3.3±0.5 75 11.3±0.9 12.7±1.1 3.3±0.1 3.2±0.1 100 11.9±0.9 12.0±0.8 3.4±0.0 2.7±0.3 SD = Standard deviation

TABLE 2 Effect of replacing bakery shortening (Wesson, USA) with red palm shortening (RPS) or red palm olein (RPOL) on the baking characteristics of white bread.

Replacement Baking Loss (%) Specific Loaf Volume (cm3/g) Level (%) Mean ± SD Mean ± SD RPS RPOL RPS RPOL 0 (Control) 12.6±0.5 14.3±0.9 4.1±0.3 4.5±0.0 25 12.5±0.3 13.9±0.7 4.1±0.3 4.4±0.2 50 14.0±0.2 14.1±0.9 4.2±0.1 4.4±0.2 75 14.4±0.9 13.7±0.8 4.2±0.1 4.3±0.2 100 14.1±1.5 11.0±0.9 4.3±0.4 3.7±0.5 SD = Standard deviation

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TABLE 3 Effect of replacing bakery shortening (Wesson, USA) with red palm shortening (RPS) or red palm olein (RPOL) on the baking characteristics of brown bread made with white flour and 20% fine bran.

Replacement Level (%) Baking Loss (%) Specific Loaf Volume, cm3/g Mean ± SD Mean ± SD 0 (Control Whole-Wheat Bread) 14.1±1.3 4.0±0.2 0 (Control White Flour) 15.6±0.5 4.7±0.0 0 (Control Brown Bread , i.e., 14.7±0.7 4.3±0.0 White Flour + 20 % Bran) 100% (RPS Brown Bread) 12.8±0.7 4.3±0.0 100% (RPOL Brown Bread) 12.7±0.8 3.3±0.2 SD = Standard deviation

TABLE 4 Effect of using different combinations of red palm shortening (RPS) and red palm olein (RPOL), and diacetyl tartaric acid esters of monogycerides (DATEM) emulsifier on the baking characteristics of whole-wheat pan bread.

RPS:RPOL Ratio Baking Loss (%) Specific Loaf Volume (cm3/g) Mean ± SD Mean ± SD Without DATEM With DATEM Without DATEM With DATEM 100% Wesson (Control) 11.2±0.1 12.8±0.7 3.6±0.1 3.5±0.0 0:100 11.0±0.8 11.1±1.3 2.6±0.1 2.8±0.1 25:75 11.1±0.7 11.8±0.5 3.0±0.2 3.1±0.1 50:50 10.7±0.3 11.3±0.4 3.0±0.4 3.5±0.0 75:25 12.1±1.0 12.1±1.5 3.3±0.1 3.4±0.1 100:0 11.8±0.3 12.7±0.5 3.3±0.2 3.6±0.0 SD = Standard deviation

TABLE 5 Baking characteristics of optimized formulations of pan bread using diacetyl tartaric acid esters of monoglycerides (DATEM) emulsifier.

Replacement Level Baking Loss (%) Specific Loaf Volume (cm3/g) (%) Mean ± SD Mean ± SD Whole-Wheat White Brown Bread Whole-Wheat White Brown Bread Bread Bread Bread Bread 0 (Control) 11.8±0.0 15.2±1.4 14.7±0.7 3.5±0.1 4.1±0.0 4.3±0.0 100% RPS 12.4±0.6 13.0±0.6 12.3±0.4 3.4±0.3 4.1±0.1 3.7±0.2 50:50 RPS:RPOL 10.5±1.9 14.3±0.5 12.5±0.7 3.2±0.2 4.2±0.2 3.7±0.2 RPS = Red palm shortening; RPOL = Red palm olein, SD = Standard deviation

The baking characteristics of the optimized whole- were slightly lower than that of white bread. The value for wheat, white, and 20% bran test bread samples containing 100% RPS were 3.4 cm3/g, and for equal proportions of DATEM and optimized levels of RPS or RPS:RPOL RPS and RPOL, it was 3.2 cm3/g; both of which were combinations are presented in Table 5. The results indi- comparable to the 3.5 cm3/g value for whole-wheat con- cate that the percentage of baking loss values for white trol bread. The specific loaf volume values (3.7 cm3/g) for bread were higher (15.3 to 14.3%) than those for whole- 20% bran test bread using 100% RPS or equal proportions wheat bread (11.8 to 10.5%) and 20% bran bread (12.3 to of RPS and RPOL were slightly higher than that of the 12.5%). The specific loaf volumes of white bread using whole-wheat control bread (3.5 cm3/g). From these re- 100% RPS or equal proportions of RPS and RPOL were sults, it can be concluded that the RPS at all levels of almost equal to that of the control, with the values being replacement of control shortening and 50:50 combination 4.1, 4.2, and 4.1 cm3/g, respectively. The specific loaf of RPS:RPOL gave the best performance in producing volume values for whole-wheat bread and 20% bran bread antioxidant-rich bread.

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SUGAR-SNAP COOKIES RPOL reduced the texture score significantly over that of control bread as well as over that for bread made with The results on the spread ratio of sugar-snap cookies RPS. The flavor of the whole-wheat bread samples was are presented in Table 6. The results indicate that as the not affected to any significant degree. In overall accept- level of RPS or RPOL was increased in the formulations ability, all of the test whole-wheat bread samples were of cookies, the spread ratios of the cookies gradually acceptable to the consumers. increased; cookies with 100% replacement by RPS and In the case of white bread, although the control sample RPOL had the highest spread ratio values. This indicates gave a significantly higher crumb color score than the test that the utilization of RPS and RPOL, even at the 100% samples, the texture and flavor scores were not affected to level in the formulation, of cookies does not pose any any significant level with the addition of either RPS or technological problem, when compared with the control RPOL. Also, none of the sensory attributes were affected shortening. Evidently, RPS and RPOL are quite suitable adversely by the addition of RPS or RPOL in brown bread replacements for white shortening, making these RPOL samples made with white flour and 20% fine bran. As to cookies nutritionally superior in terms of carotenoid and the overall effect of RPS and RPOL in optimized test bread vitamin E contents. samples, it can be concluded from the sensory evaluation data that acceptable quality and antioxidant-rich (vitamin E TABLE 6 and β-carotenes) whole-wheat bread, white bread and Effect of replacement of bakery shortening with red palm shortening (RPS) or red palm olein (RPOL) brown bread can be produced using RPS or RPOL in the on the spread ratio of sugar-snap cookies. formulation. To maximize the nutritional benefits of vita- min E and β-carotenes, and at the same time, achieve a Spread Ratio (Diameter/Height) better loaf volume, a 50:50 combination of RPS and RPOL Replacement Level Mean ± SD should be used in bread-making. (%) RPS RPOL 0 (Control) 6.7±0.0 7.0±0.5 Cookies: The sugar-snap cookies were also evaluated for sensory attributes like color, texture, flavor and overall 25 7.5±0.4 7.5±0.7 acceptability on a nine-point hedonic scale, and the results 50 7.4±0.7 7.7±0.0 are presented in Table 8. Cookies made with RPOL alone 75 7.7±1.0 7.8±0.3 gave the highest sensory scores, though these differences 100 8.2±0.3 8.0±0.2 were not statistically significant. The panelists did not SD = Standard deviation perceive any difference in the sensory scores among the cookie samples made with the control shortening, RPS or RPOL. As cookies have much higher fat contents than SENSORY ANALYSIS bread, if made with RPS or RPOL, these would serve as very good carriers of vitamin E and β-carotenes, thus pro- Bread: All eight optimized bread samples prepared in viding health benefits to the consumers. RPOL being richer the laboratory were evaluated for sensory quality on a in these antioxidant vitamins than RPS, holds greater prom- nine-point hedonic scale, and the results are summarized ise for producing such nutritious cookies. The effect of in Table 7. The crumb color was not affected to any adding RPS and RPOL on the chemical composition, in- significant degree with the addition of RPS or RPOL in strumental texture and objective color of these baked prod- whole-wheat bread formulations, but the addition of ucts will be presented in separate publications.

TABLE 7 - Average sensory scores of optimized pan bread samples using red palm olein (RPOL) and red palm shortening (RPS).

Sample Description Color Texture Flavor Overall Acceptability a b d e Whole-Wheat Bread with Control Shortening 6.1 p 6.5 q 6.9 s 6.5 wy a bc d e Whole-Wheat Bread with RPS 5.5 p 5.9 qr 6.7 st 6.1 wxy a c d e Whole-Wheat Bread with RPS + RPOL (1:1) 5.3 p 5.1 r 6.3 t 6.0 x f j k m White Bread with Control Shortening 7.4 n 6.4 q 6.8 s 6.7 w fh j k m White Bread with RPS 6.4 np 6.6 q 6.7 s 6.7 wy h j k m White Bread with RPS + RPOL (1:1) 6.3 p 6.8 q 6.6 s 6.5 wy a b d e Brown Bread with RPS 5.8 p 5.9 qr 6.0 st 5.7 wxy a b d e Brown Bread with RPS + RPOL (1:1) 5.9 p 5.9 qr 5.9 st 5.5 xy For comparison of samples in each group (i.e., whole-wheat bread, white bread, and brown bread), values with same superscripts in a single column do not differ significantly (p = 0.05). For comparison of all eight bread samples together, values with the same subscripts in a single column do not differ significantly (p = 0.05). Brown bread was made using white flour and 20% replacement with fine wheat bran.

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TABLE 8 Average sensory scores of optimized sugar-snap cookie samples made with red palm olein (RPOL and red palm shortening (RPS).

Sample Description Color Texture Flavor Overall Acceptability Control Shortening 6.9a 6.1b 6.6c 6.6d RPS 6.1a 6.5b 5.5c 6.1d RPOL 6.3a 6.4b 6.5c 6.7d RPS + RPOL (1:1) 6.6a 6.7b 6.7c 6.7d

Values in a single column with same superscripts do not differ significantly (p = 0.05).

ACKNOWLEDGEMENTS

The authors express their gratitude to the managements [9] Mensink RP, Zock PL (1998) Lipoprotein metabolism and of the Kuwait Institute for Scientific Research (KISR) and trans fatty acids. In: Trans Fatty Acids in Human Nutrition. Edited by J.L. Sebedio and W.W. Christie, Scotland: The the Malaysian Palm Oil Board for their financial support Oily Press Ltd., Dundee, pp. 217-234. and encouragement in executing this research. We also thank the Carotino Company, Malaysia, for supplying the [10] Sloan EA (2002) The top 10 functional food trends: The next red palm olein and red palm shortening samples, and the generation. Food Technol. 56(4): 32-57. Kuwait Flour Mills and Bakeries Co. for providing flour and wheat bran samples, free of charge. [11] Theriault A, Chao JT, Gabor, AA (2002) Tocotrienol is the most effective vitamin E for reducing endothelia expression of adhesion molecules and adhesion to monocytes. Athero- sclerosis 160: 21-30.

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[13] Meiselman HL (1978) Scales for measuring food prefer- [2] Chandrasekharan N (1996) Trans fatty acids and coronary ences. In: Encyclopedia of Food Science. Edited by M.S. Pe- heart disease. Palm Oil Technical Bulletin 2(4): 4-5. tersen and A.H. Johnson. Westport, Connecticut: AVI, pp.675-678. [3] Yusoff MSA, Majid R, Ismail R (1995) Production of high carotene palm olein using moderate deodorization tempera- tures. Palm Oil Developments 23: 7-9.

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[5] Bonnie TYP, Choo, YM (2000) Valuable phytonutrients in commercial red palm olein. Palm Oil Developments 32: 20-25. Received for publication: August 29, 2002 Accepted for publication: October 10, 2002 [6] Lietz G, Henry CJK, Mulokozi G, Mugyabuso JK, Ballar A, Ndossi GD, Lorri W, Tomkins A (2001) Comparison of the effects of supplemental red palm oil and sunflower oil on ma- ternal vitamin A status. Am. J. Clin. Nutr. 74: 501-509. CORRESPONDING AUTHOR

[7] Murakoshi M, Nishino H, Satomi Y (1992) Potent preventive Jiwan S. Sidhu action of alpha-carotene against carcinogensis: Spontaneous Biotechnology Dept. liver carcinogenesis and promoting stage of lung and skin Kuwait Institute for Scientific Research carcinogenesis in mice are suppressed more effectively by al- P.O. Box 24885 pha-carotene than by beta-carotene. Cancer Research 52: 6583-6587. 13109 Safat - KUWAIT

[8] Pantazaris TP, Elias BA (1996) Vitamin E: Dramatic benefits e-mail: [email protected] for heart diseases confirmed. Palm Oil Technical Bulletin 2(4): 8. AFS/ Vol 24/ No 4/ 2002 – pages 138 – 143

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THERMAL RESISTANCE OF Bacillus coagulans IN PHOSPHATE BUFFER AND TOMATO JUICE

A. H. Dinçer and A. Ünlütürk

Department of Food Engineering, Faculty of Engineering, Ege University, İzmir, Turkey

SUMMARY

The thermal destruction of Bacillus coagulans cells Clostridium butyricum. Spore-forming facultative anaer- was determined in phosphate buffer (pH 6.8) and tomato obes such as Bacillus coagulans are also causing decay of juice (pH 4.2). D values at 85, 90 and 95°C in phosphate tomato products. Due to a higher thermal resistance com- buffer and tomato juice were 31.7, 13.7, 4.8 min and 32.4, pared to the saccharolytic and butyric spore-forming an- 10.7, 0.5 min, respectively. In phosphate buffer and to- aerobes, B. coagulans is normally used as an indicator in mato juice Z values of 12.1 and 5.5 °C, respectively, were the design of thermal processes for sterilisation of tomato found after linear regression fitting of experimental data. products. The thermal resistance of B. coagulans in to- Regression equations for corresponding thermal death mato products has been reported by Youland and time curves were also obtained. A significant effect of pH Stumbo [3], Knock et al. [4], York et al. [5], Rodrigo et on the heat resistance of B. coagulans was detrmined. The al. [6] and Sandoval et al. [7]. Thermal resistance values heat resistance of the bacterium B. coagulans also de- are essential for the design of the thermal processes creased with decreasing pH values. Lower D and Z values involved in preservation of tomato products, including were obtained for phosphate buffer and tomato juice sterilisation, hot filling and aseptic methods. The objec- comparable to the values reported in the literature for tive of this study was to determine the thermal resistance tomato juice, crushed and pureed tomatoes. of B. coagulans isolated from a double-concentrated tomato paste.

KEYWORDS: Bacillus. coagulans, thermal resistance, phosphate buffer, tomato juice. MATERIALS AND METHODS

Sporulation of the microorganism The B. coagulans DS-18 strain was originally isolated INTRODUCTION in the Department of Food Engineering, Ege University, İzmir-Turkey by A. H. Dinçer from a double-concentrated The traditional way to calculate the efficacy of ther- tomato paste and identified according to Bergey's Manual mal treatment in food preservation is based on the as- of Systematic Bacteriology and The Prokaryotes [8, 9]. sumption that the mortality of microbial cells and the The test microorganism was maintained on nutrient agar inactivation of bacterial spores are processes governed by slants at 4 °C with transfers every 3 months. a first order kinetics. Consequently, the slope of the se- milogarithmic survival curves of the targeted organisms Sporulation of the test strain was carried out in 10 ml or spores at different temperatures, pH levels or heating Nutrient broth containing 100 mg/1 CaC12.2H2O and medium are used to determine the D value, i.e. the time in 500 mg/l MgSO4.7H2O by incubating at 37 °C for 3 days. min at a given temperature required to destroy 1 log cycle The spores were collected when sporulation reached a (90%) of the target microorganism (of course, in an actual level of 90%. Then they were washed five times by cen- process, all others that are less heat-tolerant are destroyed trifugation at 2,600 rpm and stored in M/15 phosphate to a greater extent) [l, 2]. buffer (pH 6.8) at 4 °C for subsequent use, however, within one week of harvesting. Various microorganisms have been reported to be causative in the deterioration of thermally processed to- The spore concentration was determined by duplicate mato products, especially spore-forming saccharolytic and plate counts in Glucose Tryptone Yeast extract agar butyric anaerobes such as Clostridium pasteurianum and (GTYA), pH 5.0, after incubation at 37 °C for 48 h.

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(a ) (a)

8

7 8

s 7 y = -0,0309x + 6,7096 6 2 6 R = 0 , 8 2 7 5 5 4 4 3 3 2 y = -0,0313x + 7,0573 survivors of no log 2 R2 = 0,9761 log no of survivor 1 1

0 0

0 20 40 60 80 100 120 0 50 100 150

heating time(min) heating time(min)

(b) (b)

7 8

7 y = -0,093x + 5,3334 6 R2 = 0,8031 6 5 5 4 4 3 3

2 2 survivors of no log y = -0,073x + 6,0466

2 survivors of no log 1 R = 0,9346 1

0 0

0204060 0 20406080 -1 heating time(min) heating time(min)

(c ) (c )

8 10

9 y = -2,0605x + 8,9687 7 8 R2 = 0,9147 6 7 5 6 4 5 3 4

2 3 log no of survivors of no log y = -0,2083x + 7,3116

survivors of no log 2 1 R = 0,9717 2

0 1

0102030 0

heating time(min) 0246 heating time(min)

FIGURE 1 - Survivor curves for B. coagulans D-18 spores in FIGURE 2 - Survivor curves for B. coagulans D-18 spores phosphate buffer (pH 6.8) at (a) 85 °C, (b) 90 °C and (c) 95 °C. in tomato juice (pH 4.2) at (a) 85 °C, (b) 90 °C and (c) 95 °.

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1,6 2

1,4 y = -0,1824x + 17,158 R2 = 0,93 1,5 1,2

) 1 ) 1 0,8 0,6 D values (log 0,5 D values (log D values 0,4 y = -0,0824x + 8,523

R2 = 0,996 0,2 0 80 85 90 95 100 0

80 85 90 95 100 -0, 5 Temperature (°C) Temperature (°C)

FIGURE 3 - Thermal death time curves of B. coagulans D-18 heated in phosphate buffer at pH 6.8 (▲) and tomato juice at pH 4.2 (■).

Thermal treatment RESULTS AND DISCUSSION The heat resistance of the test strain, B. coagulans DS- 18, in phosphate buffer or tomato juice was determined at D values at 85, 90 and 95 °C in phosphate buffer 85, 90 and 95 °C by using screw-capped vials having an and tomato juice were 31.7, 13.7-, 4.8 min and 32.4, internal diameter of 0.7 mm, external diameter, 1.5 mm, 10.7, 0.5 min, respectively (Figs.1 and 2). and length, 75 mm. The sterilised vials (121 °C/ 20 min) were filled with 0.5 ml phosphate buffer or tomato juice The curves for the thermal death time of B. coagulans inoculated with spore suspension to obtain a final spore heated at 85, 90 and 95 °C in phosphate buffer and tomato concentration of 107-108 spores per ml. Before inoculation juice are shown in Fig. 3. the spore suspension was heated at 80 °C for 20 min. Then the vials containing phosphate buffer or tomato In phosphate buffer and tomato juice Z values of juice were placed in a temperature-controlled water bath 12.1 °C and 5.5 °C, respectively, were found after linear and stabilised at the required temperature. A thermocou- regression fitting of experimental data. Regression equa- ple was used to control temperature. tions for the corresponding thermal death time curves are also given in Fig. 3. As seen in these curves, a significant The intervals depended on temperatures studied, six effect of pH on the heat resistance of B. coagulans can time intervals and two vials for each time interval were be observed. As the pH decreased, the heat resistance of used for each temperature. Immediately after the desig- B. coagulans also decreased. nated time the vials were transferred from the heated water bath to an ice water bath to stop the thermal treatment. Several authors have studied the thermal resistance of B. coagulans in tomato products. Youland and Stumbo [3] Enumeration of survivors reported values of D 104°C = 1.72 min and D110°C = 0.32 min From the heated and cooled spore suspensions appro- for B. coagulans ATCC 8038. Knock et al. [4] determined priate decimal dilutions in 0.1% sterile peptone water D values in South African tomato juice at 93.3 °C, 96.1 °C, were made and 1 ml volumes of the dilutions were plated 98.9 °C and 106 °C of 13.4, 6.3, 3.1 and 0.51 min, resp., in duplicate with GTY agar (pH 5.0). The plates were and a corresponding Z value of 16.1 °C. York et al. [5] incubated at 37 °C and the colonies counted after 48 h. D found values of D100°C = 0.53 min and Z = 15.6 °C for the values were based on the reciprocal of slopes obtained heat resistance of B. coagulans ATCC 8038 in tomato when the log number of colony forming units surviving juice with pH 4.2 employing an inoculum of 104 spores were plotted against time. For example, a D value at 72 °C per ml. Rodrigo et al. [6] determined the thermal heat of 1 min means that for each min of processing at 72 °C the resistance of strain ATCC 8038 in pureed and crushed target bacteria population will be reduced by 90%. The Z tomatoes at two different pH values, determining a heat values represented the reciprocal of slopes when the logs resistance of D90°C = 12.3 min and Z = 13.7 °C for a pH of of D values were plotted against temperature. 4.5 and D90°C = 9.0 min and Z = 13.9 °C for a pH of 4.3.

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Lower D and Z values were obtained in this study for [5] York, G., Heil, J., Marsh, G., Ansa, A., Merson, R., Wolcott, T. phosphate buffer and tomato juice in comparison with the and Leonard, S., J. Food Sci. 40, 764 (1975) above reported values for tomato juice, crushed and pureed [6] Rodrigo, M., Martinez, A., Sanchis, J., Trama, J. and Giner, V., tomatoes. This could be due to the different pH values, J. Food Sci. 55, 1029 (1990) soluble solid content and water activity of tomato juice. Microbial heat resistance is influenced by many environ- [7] Sandoval, A.J., Barreiro, J.A. and Mendoza S., J. Food Sci. mental factors, such as pH, water activity, heating medium 57, 1369 (1992) composition, etc. The influence of these factors can be even [8] Sneath, P., Bergey's Manual of Systematic Bacteriology, vol. higher than that of their genotype. Additionally, the pH 2, ch. 13, William and Wilkins, Baltimore, MD (1986) value of the heating medium is an important factor with a strong effect on heat resistance. In this study lower D and Z [9] Slepecky, R.A. and Hemphill, H.E., in Balows, A., Trüper, H. G., Dworkin, M., Harder, W. and Schleifer, K. H. (eds.), values were obtained in tomato juice (pH 4.2) than in The Prokaryotes, 2nd ed., p. 1663, Springer Verlag, Berlin, phosphate buffer (pH 6.8). Although the influence of pH Heidelberg, New York (1990) has long been known, more information is needed. Some data published are in disagreement and many other as- [10] Montville, T.J. and Sapers, G.M., J. Food Sci., 46, 1710 pects remain unknown. Most authors agree that spores (1981) have their maximum heat resistance at pH values close to [ll] Sala, F.J., Ibarz, P., Palop, A., Raso, J. and Condon, S., J. neutrality [2]. There is still no agreement on the magni- Food Prot. 58, 239 (1995) tude of the influence of pH upon heat resistance and, thus, upon Z values [10, 11]. Some authors have reported no [12] NCA (National Canners Association), Laboratory Manual for Food Canners and Processors, vol. 1, Microbiology and influence of pH on Z values [6, 10], but others have found Processing, The AVI Pub. Co., Inc., Westport, CT (1968) an increase toward acidity [11].

CONCLUSION

Decay caused by B. coagulans is less frequent, but not less important. The minimum heat treatment of certain tomato products is based on reaching 95 °C in the coldest point. But this conventional process may become insufficient for controlling quality impairment caused by B. coagulans, when there is a high initial contamination. B. coagulans is the most heat-resistant of all microorganisms capable of spoiling tomato products with a pH below 4.5 [12]. The hot-fill-hold cool process has been successfully applied to acid products such as fruit purees, tomato juices and tomato concentrates. This system of continuous heat treatment would avoid hold ups and provide a better quality product at lower energy cost. Further, heat treat- ment based on the thermal resistance of B. coagulans would ensure the stability of the product with respect to this microorganism and to butyric anaerobes, because of the lower heat resistance of Clostridium butyricum and C. pasteurianum [12].

Received for publication: September 24, 2002 REFERENCES Accepted for publication: January 27, 2003

[l] Stumbo, C.R., Thermobacteriology in Food Processing, 2nd ed. Academic Press, New York (1973) CORRESPONDING AUTHOR [2] Jay, J.M., Modern Food Microbiology, 4th ed., Van Nostrand Reinhold, New York (1992) A. Handan Dinçer 1682 Sok. No: 18/4 [3] Youland, G. and Stumbo, C., Food Technol. 7, 286 (1953) 35600 Karşiyaka İzmir - TURKEY

[4] Knock, G., Lambrechts, M., Hunter, R. and Riley F.R., in Hersom, A.C. and Hulland, E.D. (eds.), Canned Foods, an In- e-mail: [email protected] troduction to their Microbiology, 5th ed. Chem. Pub. Co. Inc., New York (1959) AFS/ Vol 24/ No 4/ 2002 – pages 144 - 147

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A COMPARATIVE STUDY OF THE MICROBIOLOGICAL QUALITY AND CONSUMER ACCEPTABILITY OF SOYCHEESE AND MILK-BASED CHEESE

O. O. Aboaba and M. S. Adeleye

Department of Botany and Microbiology, University of Lagos, Lagos, Nigeria

SUMMARY

Cheese samples were prepared from soy and raw dairy most unusual and interesting of the many different prepara- milk. Coagulation was facilitated using vinegar, corn liquor tions. Soy milk has been made in China for generations and or a plant leaf extract (Calotropis procera). The samples its consumption is fast gaining grounds in Nigeria [1]. It has were examined for their microbiological quality during been recommended by physicians for years to patients who storage, acid level and consumer acceptability. are lactose-intolerant and, more recently, to those who are prone to degenerative heart disease and, thus, need milk In the cheese samples four bacterial genera, one fungus with unsaturated fat as a replacement to cow’s milk. and yeasts were isolated and identified. Coliforms could not be detected. The total bacterial and lactic acid microbe Soymilk that is devoid of the characteristic objection- counts were more evident in the cheese types during stor- able beany flavor, is very tasty and of high nutritional value. age, especially at ambient temperature (28 ± 2 ûC). Yeasts When it is prepared with the same water content as cow’s and molds were not isolated in the first 24 hours at both milk, it contains 52% more of protein, but 12% fewer calo- refrigerated (4 ûC) and ambient temperature (28 ± 2 ûC), ries, 24% less fat, 48% less saturated fat, 16% less carbo- but their populations increased thereafter during storage. hydrates and no cholesterol. When compared with breast milk, it contains 21.4% more protein, 16% less calories, The level of acidity increased in all samples with the 19% less fat and 47% less carbohydrates [2]. storage period. It can be used, therefore, as a practical substitute for The samples prepared from soy milk with vinegar and cow’s milk and could solve malnutrition problems in raw milk with leaf extract were able to retain their colour, developing countries like Nigeria. The need for Nigerians appearance, taste and texture throughout the storage pe- to consume more proteins in their diet was highlighted in riod of 5 days, while the other two samples i.e. soy milk the FAO report of 1966, and 1990 by Aderiye [3]. The with corn liquor and naturally fermented cow’s milk dete- development of Soy-Ogi, a breakfast cereal made from riorated after 2 days at ambient temperature. soybeans and corn by the Federal Institute of Industrial Statistical analysis showed that the cheese samples Research in the eighties was very good especially for made from raw dairy milk were preferred by the panelists. infants. It was nutritionally adequate, free from toxic residues and was affordable. The preparation of fermented soy milk to make cheese was studied and found to be feasible. It had since KEYWORDS: been commercialized in local Nigerian markets. The idea Consumer acceptability, coagulant, soy milk. of substitution of fermented cow’s milk with soycheese was supported by reports on the consumer acceptability and microbiological quality of a cheese made from 20% soy milk with 80% cow’s milk compared with cheese made INTRODUCTION from fresh cow’s milk [4]. The Kwara Agricultural Development Project in Soy milk is an aqueous extract from soybeans (Gly- Ilorin, Nigeria developed cheese made from 100% soy cine max), which are legumes of Manchurian origin. It is milk using vinegar and fermented corn liquor as coagu- a creamy emulsion, which resembles cow’s milk in both lants. This type of product is a welcome change in a de- appearance and consistency. Furthermore, it is one of the veloping country to improve the protein intake of many

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that cannot afford animal proteins like poultry, fish, beef Microbiological quality of the cheese types etc., therefore, the aim of this project was to determine the All the cheese types were assessed for microbial popu- microbiological quality and consumer acceptance of soy lation at day zero and during storage, every 24 h for 5 days, cheese when compared with the traditionally prepared using the pour plate method. One gram of each sample was fermented raw cow’s milk cheese called “warankasi”, macerated and homogenized in 9 ml of bacteriological which is very popular amongst the people. peptone and serially diluted up to 10–5 dilution level. An aliquot of 0.1 ml of an appropriate dilution was used for the enumeration of the microbial types. Duplicate plates were MATERIALS AND METHODS used and incubation was done at 37 ûC for 18 – 24 h for the bacterial isolates and 25 ûC for 48 h for yeast and molds. Collection of Materials The microbial counts obtained were converted to log Soybeans,vinegar and fermented corn liquor were col- numbers and plotted against time and statistically ana- lected from the Biotechnology Department of the Institute lysed (variance of the total viable counts of cheese sam- of Industrial Research, Oshodi, Lagos. Raw cow’s milk ples at 28 °C and 5-6 °C. De Mann Rogosa and Sharpe was collected from a cattle ranch at Abeokuta, Ogun State medium was used for isolation of lactic acid bacteria, the in a sterile conical flask. The Sodom apple leaves plate count agar for total plate count and malt extract agar (Calotropis procera) were obtained from the plant on the for yeasts and molds. Salmonella/ Shigella agar and bril- premises of the Institute. liant green bile agar was used for total coliforms. Pure cultures of the isolates were prepared and identified using Preparation of soy milk and soy cheese morphological and biochemical characteristics.

The beans were sorted, rinsed in tap water and 100 g were placed in 500 ml of boiling water containing a few Biochemical quality of the cheese types drops of 0.5% NaHCO3 solution. Boiling was continued for pH: The hydrogen ion concentrations of the cheese 15 min. The blanched seeds were manually dehulled and samples were monitored during storage every 24 h for 5 milled in Stephan milling machine (Gallenkamp model) days using a calibrated digital pH meter (Model 121-E). before being homogenised using a Moulinex electric blender with 500 ml of water. The slurry was filtered Total acidity: One g of the cheese samples was through a six-fold finely wooven cheese-cloth to obtain the crushed in 10 ml of distilled water and titrated against milk as a filtrate. To a 100 ml portion of milk 10ml of co- 0.1N NaOH using phenolphthalein as indicator. The agulant, i.e. vinegar or fermented corn liquor, was added. amount of NaOH needed to neutralise the acid in the The corn liquor was obtained from fermented corn gruel. sample was recorded. Readings were taken in duplicate One hundred g of corn grains was steeped in 1 L of sterile and averaged. The amount of lactic acid expressed as total distilled water for 48 h at 28 ± 2 °C. It was wet-milled and titratable acidity (%) was calculated from the equation. allowed to ferment naturally for 3 days. The clear liquid supernatant of the gruel was the corn liquor. The mixture was heated to 70 °C for 15 min. The scum on top of the % Acid = ml of NaOH x 9.0008 mixture was removed manually using a spoon and the curds 100 formed were left to boil for another 5-10 min. Then the curds were separated from the whey by filtering with a clean Sensory evaluation: The cheese samples were assessed muslin cloth, washed with tap water and manually moulded for organoleptic qualities, such as colour, taste, texture into compact forms. The sample prepared with vineger was and appearance by a fifteen-member panel based on ex- labelled A , while the one with corn liquor was labelled A . 1 2 perience. The test was carried out in a closed air- The cheese samples were placed in sterile beakers cov- conditioned room (10 0C) that was partitioned into cubi- ered with aluminium foil and stored at 28 ± 2 ûC or refrig- cles. The light source for each cubicle was white light erated at 5-6 ûC. from fluorescent tubes. The members of the panel were instructed not to eat any cheese or its products 48 h before Preparation of fermented cow’s milk cheese samples testing. During the test, each member was asked to rinse Two different samples were prepared. To a 100 ml por- mouth twice with sterile saline water before each tasting tion of raw milk, 10ml of sodom apple leaf extract was session. A Questionnaire was prepared and distributed to added. This extract was obtained by blending 50 g of each member for scoring. The mean percentage of panel- Calotropis procera leaves with a Phillips HP–2815 model ists was calculated for each attribute as follows: in 250 ml of sterile distilled water and centrifugation. The cheese was prepared and stored using the procedure de- 90% = 4; 80 – 90% = 3; 70 – 80% = 2; < 70% = 1 scribed above and labelled B1. The other sample was pre- pared by natural fermentation of a similar volume of raw Statistical analyses involving t-test, F value and least milk at 28 ± 2 ûC for 18 h. This sample was labelled B2 and significant difference were used to evaluate the responses of stored also as above. the panelists with regard to their preferences of cheese types.

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RESULTS 6 Microbiological quality of the cheese types: Four bacte- 5 rial types were isolated from the cheese samples, but no 4 coliforms, and also four fungal isolates. The bacterial 3 2 isolates were identified as Bacillus sp., Streptococcus sp., 1 Lactobacillus sp. and Micrococcus sp. The total bacterial 0 count increased at the two temperatures used for storage. of sample CFU\ml LOG 050100 150 Samples A1 and B1 had a drop in population (total count) Storage Periods in Hours during the first 48 h and 72 h, respectively, at room and refrigerated temperatures. Later on, there was observed an increase during storage (Figs. 3 and 4). Total lactic acid FIGURE 4 - Total bacteria count of count in these samples (A1 and B1) steadily increased and milk curds stored at refrigeration temperature (symbols see Fig. 1). began to decrease after 96 h (Figs. 1 and 2). Bacterial populations were higher in the soy cheese, especially the one prepared with corn liquor. 6 5 4 6 3 5 2 4 1 3 0 2 of sample CFU\ml LOG 1 050100 150 0 Storage Periods in Hours of sample CFU\ml LOG 050100 150

Storage Periods in Hours FIGURE 5 - Yeast and mold counts of milk curds stored at room temerature (symbols see Fig. 1). FIGURE 1 - Lactic acid bacteria counts of milk curds stored at room temperature

(A1 = -♦-♦-; A2 = -■-■-; B1 = -▲-▲-; B2 = -X-X-).

6 4 6 2 4 0 2 050100 150

0 of sample CFU\ml LOG Storage Period in Hours 050100 150 LOG CFU\ml of sample CFU\ml LOG Storage Periods in Hours

FIGURE 6 - Yeast and mold counts of FIGURE 2 - Lactic acid bacteria counts of milk curds stored at refrigeration temperature (symbols see Fig. 1). milk curds stored at refrigeration temperature (symbols see Fig. 1).

6 8 5 6 4 3 4 2 2 1 Changes pH 0 0 LOG CFU\ml of sample of CFU\ml LOG 050100 150 050100 150 Storage Periods in Hours Storage Periods in Hours

FIGURE 3 - Total bacteria counts of FIGURE 7 milk curds stored at room temperature (symbols see Fig. 1). pH changes in milk curds during storage (symbols see Fig. 1).

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Yeasts and molds were not isolated in samples A1 and ganoleptic qualities were analysed statistically consider- B1 in the first 0-24 h of storage at room and refrigerated ing the observed LSD value. temperatures (Figs. 5 and 6). Those isolated later during storage were identified as Candida, Rhodotorula, Sac- Chemical quality of the cheese types charomyces and Aspergillus. Coliforms were not isolated throughout storage period. A statistical analysis of vari- Total acidity: There was an increase observed in acid ance of the total viable counts of the samples showed that content in all the cheese types at both temperatures during there were significant differences between the two storage storage, more pronounced in samples stored at room tem- temperatures. perature (Fig. 7).

The treatment i.e. use of different coagulants given pH changes: There was a gradual decrease in the pH did not produce any significant effect when the of all samples irrespective of storage temperature. organoleptic qualities were analysed statistically consider-

TABLE 1 Organoleptic studies of milk curd cheese samples stored at refrigerated temperature (5-6 °C).

Samples Colours Appearance Taste Texture Storage Periods (Hrs.)

A1 3 3 1 3 120

A2 2 2 1 1 72

B1 4 4 2 3 120

B2 3 2 1 3 72

KEY: 4 = Very Good; 3 = Good; 2 = Acceptable; 1 = Not acceptable

TABLE 2 Organoleptic studies of milk curd cheese samples stored at room temperature (28 ± 2 °C).

Samples Colors Appearance Taste Texture Storage Periods (Hrs.)

A1 2 3 2 3 72

A2 2 1 2 1 48

B1 4 3 2 3 72

B2 3 2 1 1 48

KEY: 4 = Very Good; 3 = Good; 2 = Acceptable; 1 = Not acceptable

TABLE 3 Statistical analyses of values obtained for organoleptic quality assessment by the panelists.

t-test F-value Source of information LSDvalue tcal T0.05(3) Fcal F0.05(1,6)

A1 and B1 0.295 2.353 1.02 5.99 1.984

A2 and B1 3.129 2.353 9.8 5.99 1.316

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Sensory evaluation: Samples A1 and B1 were found to can be suppressed or displaced in dairy products during be acceptable in terms of colour, appearance, taste and storage by lactic acid bacteria, which are components of texture for 3 days when stored at room temperature, but dairy starter cultures and supposedly possessed antimicro- for 5 days when refrigerated. Sample B1 was preferred by bial activities. the panelists as shown by the F-value, but unfortunately LSD values contradict this (refer to Table 3). Samples A1 Bayoumi [7] confirmed that nisin, an antibacterial sub- and B2 were favoured only for 2 days at room temperature stance can lower the total bacterial counts and prevent and 3 days when refrigerated. There was a rapid decrease clostridial gas formation in cheese. in deterioration, appearance, taste and texture (Tables 1 and 2) of these samples An increase in acid content in the cheese samples was observed as the lactic acid bacteria population increased, which finally resulted in a gradual decrease of pH values. DISCUSSION The pH changes are related to the increase in acidity as a result of production of lactic acid from carbohydrates dur- The cheese types studied were found to contain differ- ing fermentation. The production of lactic acid plays a ent microbial types which increased in population with major role in the acid content of soft cheeses [8]. storage period. The presence of microorganisms was not surprising as they are actively involved in the fermentation The presence of a highly acidic coagulant facilitated process and will grow in any medium with abundance of the rate of curd formation. Samples prepared with vine- fermentable sugars and other nutrients. The ability of Lac- gar (pH 3.68) or extracts of Calotropis procera leaves tobacillus sp. to ferment sugars with production of lactic (pH 4.97) were formed within 18 h and the curds were acid makes it possible to use them in production of fer- bigger and firmer (plate 1), while those prepared with corn mented plant and dairy products. Also their thermoduric liquor (pH 5.86) were formed after 24 h with smaller curds. nature allows their survival during the heat processes used Jacob [9] reported that vinegar served as an ingredient that in cheese production. The viridan group of Streptococcus is facilitated coagulation. important in cheese and production of yoghurt, as they can survive heat processes at about 60 ûC for 30 min and also the thermophilic species of Bacillus can produce a consid- Sensory evaluation studies showed that samples A1 erable amount of lactic acid from sugars. and B1 were most acceptable to the panelists; the rate of deterioration was slower, especially when stored at refrig- erated temperature. The high acid content and the low The yeasts ferment sugars and, thus, are implicated in temperature collectively suppressed spoilage flora for a the fermentation processes and spoilage of highly acidic considerable period. Sample B , however, remained the foods [5]. Aspergillus spp. are widespread in nature and 1 favorite in terms of colour, taste and texture. This was not their spores are abundant in the environment. They, there- surprising as it, additionally, had been a local treat for fore, readily colonize and cause spoilage. many years and the people were more familiar with it. A new product requires some period of time in the market The growth pattern was more stable at room tempera- for consumer acceptability. However, more work needs to ture, and the sharp drop in total bacterial count in samples be done in this laboratory to improve the organoleptic A1 and B1 could have been due to the harsh effect of the properties as the LSD values did not indicate any signifi- increased acidity on those microbes that were not acid- cant difference between the samples. tolerant. Their population was, therefore, reduced and those that survived were able to cope and hence the increase thereafter. Ambient temperatures favour the proliferation of microorganisms, while lower temperatures retard their REFERENCES growth and metabolic activities [6]. [1] Iwe, M. O. Nig. Food Journal, 9, 92-104. (1990) The absence of yeasts and molds at the initial stage of storage is likely due to the inability of their vegetative cells [2] Shurtleff, W. and Aoyagi, A. J. Amer. Oil Chem. Soc., 61, to survive the heat used in processing. The surviving spores 1798 – 1800. (1984) could, however, germinate and grow in the medium. The rate of increase of the lactic acid bacteria count was more [3] Aderiye, M. A. M.Sc. Thesis, University of Lagos, Lagos. 32. (1990) pronounced than that of the total viable count. It is wellknown that lactic acid bacteria are essential in large [4] Aworh, O. C. and Akinniyi, O.O. Nig. Food Journal, 9, 50- numbers to ferment the milk sugar and this, coupled with 57. (1989) the presence of other metabolic end products, represses spoilage and food poisoning organisms. It has also been [5] Frazier, W. C. and Westhoff, D.C. Food Microbiology. TMH established that some microbes or other aerobic bacteria (Ed.) John Murray Publication, London. pp.383-385. (1978)

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[6] Pelzar, M. J. (Jr.); Reid, R. D. and Chan, E.C.S. Microbiol- ogy. 5th Edition, McGraw Hill Book Company, New York, pp. 129-134. (1986):

[7] Bayoumi, S. Chem. Mikrobiol. Technol. Lebensm., 19, 65- 69. (1991)

[8] Metwalli, N. H.; Shalabi, S.I; Zahran, A.S., and Demerdash, E.I. J. of Food Technol. 17, 297-305. (1982).

[9] Jacob, M. B. Chemistry and Technology of Foods and Food Products 2nd Edition, Interscience Publication, New York, pp. 870-891. (1951).

Received for publication: October 30, 2002 Accepted for publication: January 31, 2003

CORRESPONDING AUTHOR

O. O. Aboaba Department of Botany and Microbiology University of Lagos Lagos - NIGERIA

e-mail: [email protected]

AFS/ Vol 24/ No 4/ 2002 – pages 148 - 153

153 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

MÖGLICHKEITEN UND GRENZEN DES EINSATZES VON GASSENSOR-ARRAYS ZUR QUALITÄTSBEURTEILUNG VON LEBENSMITTELN

S. Nitz1 und D. Hanrieder2

1 Lehrstuhl für Chemisch-Technische Analyse und Chem. Lebensmitteltechnologie, Technische Universität München, Weihenstephaner Steig 23, 85350 Freising-Weihenstephan, Germany 2Fachbereich Landwirtschaft/ Ökotrophologie/ Landespflege, Hochschule Anhalt (FH), Strenzfelder Allee 28, 06406 Bernburg, Germany

1. EINLEITUNG

In der modernen Analytik sind Sensoren unverzichtba- Reis oder Getreide soll ebenfalls möglich sein [11,12]. In re Instrumente zur Messung verschiedenster Stoffe und den meisten dieser Arbeiten ist jedoch ungeklärt, ob von Stoffgruppen, zur Bestimmung physikalischer Größen und den eingesetzten Sensorsystemen tatsächlich produktcha- zur Charakterisierung stofflicher Zustände. Dabei sind in rakteristische bzw. qualitätsrelevante Verbindungen er- Hinblick auf deren Aufbau, Funktionsweise und Anwen- fasst wurden oder ob die gefundenen Diskriminierungen dungsgebiet eine Vielzahl von Sensortypen im Einsatz. nur auf der Wechselwirkung der Sensoren mit nicht quali- tätsrelevanten Verbindungen beruhen, die herkunfts-, Während bei Einzelsensor-Systemen die Messgröße sorten-, bzw. verarbeitungsspezifisch sein können, es je- direkt oder indirekt mit der Probeneigenschaft korreliert, doch nicht sein müssen. Im Folgenden sollen die kom- besteht bei Systemen auf der Basis von Multi-Sensor- merziell erhältlichen Systeme vorgestellt und die prinzi- Arrays kein derartiger Zusammenhang. Bei letzteren piellen Möglichkeiten und Grenzen dieser Sensorsysteme werden die von den einzelnen Sensoren eines Arrays in bezug auf die Gasanalytik, insbesondere die Geruchs- gelieferten Messdaten („Signalmuster“) verarbeitet und analytik anhand ausgewählter Anwendungen aus dem anschließend mittels Verfahren der multivariaten Daten- Bereich der Ernährungsindustrie aufgezeigt werden. analyse ausgewertet [1-7]. Die Entwicklung und Kom- merzialisierung von Multi-Sensor-Arrays wurde maßgeb- lich von modernen Bestrebungen zur Entwicklung einfa- KEYWORDS: Elektronische Nasen, Gassensor-Arrays, QMB- cher und zeitsparender Messtechniken und Analysenme- Sensoren, MOS, MS-Sensor, Differenzierung von Hopfen- und thoden vorangetrieben. Besonders vielversprechend er- Erdbeersorten, Kork-Taint. scheint deren Einsatz in Bereichen mit hohem Probenauf- kommen, denn diese schnelle und verhältnismäßig güns- tige Messtechnik bringt eine erhebliche Zeitersparnis und somit Kostenreduzierung gegenüber klassischen Analy- 2. SENSORSYSTEME senmethoden mit sich. Die verschiedenen Sensorsysteme werden häufig – in Anlehnung daran, dass sie hauptsäch- lich flüchtige und damit auch geruchsaktive Substanzen Die verschiedenen Sensorsysteme, die derzeit kom- detektieren können – unter dem Sammelbegriff „elektro- merziell erhältlich sind, können in vier Hauptgruppen nische Nasen“ oder Chemosensoren vertrieben. In der eingeteilt werden. Ihr Messprinzip sowie einige wichtige einschlägigen Literatur wird davon berichtet, dass künst- Eigenschaften sind in Tabelle 1 zusammengefasst. liche Nasen in der Lage sind, z.B. Kaffee unterschiedli- Metall-Oxid-Sensoren (MOS) verändern ihren elekt- cher Herkunft und Röstung voneinander zu unterscheiden, rischen Widerstand durch Redox-Reaktion mit im Gas- zwischen Orangensäften unterschiedlicher Herstellungs- raum enthaltenen organischen Molekülen und chemosor- verfahren zu differenzieren sowie unterschiedliche Hop- biertem Sauerstoff. MOS-Sensoren sind sehr empfindlich fensorten und Biere zu erkennen [8-10]. Die Authentizi- gegenüber oxidierbaren Substanzen, müssen daher mit tätsbestimmung von Weinen, die Einteilung von Mu- Luft und definiertem Feuchtigkeitsgehalt betrieben wer- scheln in Qualitätsklassen sowie die Erkennung von „off den. Als nachteilig ist dabei der nicht über den gesamten flavours“ bei unter ungünstigen Bedingungen gelagertem Konzentrationsbereich lineare Zusammenhang zwischen

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Signal und Substanzkonzentration anzusehen. Dadurch Allen Sensorsystemen gemeinsam sind die Probenauf- lässt sich die Konzentrationsabhängigkeit der Sensorsig- gabe und die Auswertung. Die Probenaufgabe für flüssige nale nur näherungsweise eliminieren. und feste Proben erfolgt mittels Headspace-Technik. Diese Sensoren auf der Basis leitfähiger Polymere verän- Probenahmetechnik, obwohl sehr reproduzierbar, schränkt die Anwendungen auf solche Bereiche ein, in denen die dern ihre Leitfähigkeit durch Adsorption leichtflüchtiger Gasphase produktcharakteristische bzw. qualitätsrelevante Moleküle. Aufgrund ihrer hohen Polarität sind sie relativ Verbindungen in messbaren Mengen enthält. unempfindlich gegenüber unpolaren Substanzen, jedoch extrem empfindlich gegenüber Luftfeuchtigkeit. Das Sensorsignal weist ebenfalls keine Linearität mit der Sub- 2.1. Einfluss der chemischen Struktur auf die Ansprechemp- findlichkeit von Multisensor-Arrays stanzkonzentration auf. Das Sensorverhalten von QMB´s gegenüber einzel- Quartz-Crystal-Microbalance-Sensoren (QMB) sind nen flüchtigen Substanzen kann anhand der Polarität der mit unterschiedlichen gas-sensitiven Materialien (GC- verwendeten GC-Phasen näherungsweise prognostiziert Phasen) beschichtet und verändern ihre Grundfrequenz werden [13]. durch die Wechselwirkung mit flüchtigen Substanzen. Die Adsorption an den gas-sensitiven Schichten der Schwing- Erwartungsgemäß reagieren die mit unpolaren GC- quarze führt zu einer Massenerhöhung und dadurch zu Phasen belegten Sensoren (OV1, SE54) sowie die mittel- einer niedrigeren Schwingfrequenz. Die Frequenzänderung polare Phase (OV17) auf unpolare Substanzen wie z.B. ist somit linear bezüglich der Konzentration. Hexan (Abb.1). Bei Vorliegen einer endständigen Alde- hydgruppe sprechen auch die mit polareren Phasen beleg- Die jüngste Klasse von Sensorsystemen basiert auf ten Sensoren (CW1500 und CW20M) an. Bei gleicher der Massenspektrometrie. Gemessen wird hierbei die Dampfraumkonzentration reagieren die Sensoren auf unaufgetrennte Probe über eine offene oder geschlossene Aldehyde, insbesondere auf Hexenal, deutlich empfindli- Kopplung zum Massenspektrometer. Durch Selektion der cher als auf den homologen Kohlenwasserstoff ohne für die zu diskriminierende Probeneigenschaft charak- funktionelle Gruppe. teristischen Fragmentionen kann ein „Sensor-Array“ definiert werden, welches dann für die Musteranalyse Monoterpenkohlenwasserstoffe liefern ein ähnliches verwendet wird. Diese Methode ist nicht nur standardi- Signalmuster wie Hexan (Abb. 2). Unterschiede ergeben sierbar, sie ist auch für die unterschiedlichsten Anwen- sich jedoch in der Intensität der Signale, die die beim dungen durch Selektion/ Deselektion der charakteristi- Hexenal beobachteten Werte z.T. deutlich überschreiten. schen Fragmentionen, die für eine bestimmte Eigenschaft Das Monoterpenoxid 1,8-Cineol unterscheidet sich von der zu diskriminierenden Proben charakteristisch sind, in entsprechenden sauerstofffreien Monoterpenen durch ein bezug auf Empfindlichkeit (Full-scan oder Single Ion deutlich höheres Signal am mittelpolaren (OV17) Sensor. Monitoring-SIM) und Selektivität adaptierbar.

TABELLE 1 - Kommerziell erhältliche Multi-Sensor –Arrays.

Metall Oxide Conducting Polymer Quartz Crystal Micro- Sensortyp MS-Sensor® (MOS) (CP) balance (QMB) Substrat Keramik Silizium Quarz - SnO , WO , ZnO, Polymere aus: 2 3 Silicone, (poly)Glycole Gassensitive Schicht dotiert mit Spuren Pyrrol, Indol, Thiophen, keine (GC-Phasen) von Metall (Pt, Pd) Anilin elektrischer Fragmentionen/ Messgröße elektrischer Widerstand Frequenz Widerstand Intensität Arbeitstemperatur des Raumtemperatur 175 - 450°C Raumtemperatur 150-250 °C Sensors bis 100°C Lebensdauer 24 Monate 6-9 Monate 9-12 Monate 15 Jahre mit polaren in begrenztem Maße Temperatur Dekontamination durch Ausheizen Lösungsmitteln durch Ausheizen Vakuum Identität ausgetauschter nicht gegeben nicht gegeben Sensoren laut 98% entfällt (Einzelproduktion) (Einzel-produktion) Herstellerangabe Selektivität + - + - + - ++++ Empfindlichkeit ++ ++ bis -- +- +- (allgemein: ppm) SIM +++

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100 100 75 75 % 50 % 50 25 25 0 0 CW1500 CW20M OV1 SE54 OV17 CW1500 CW20M OV1 SE54 OV17 2E-Hexenal: 100% = 240 Hz Hexan: 100% = 14 Hz

ABBILDUNG 1 - Ansprechempfindlichkeit von QMB-Sensoren auf Hexan und 2-E Hexenal.

100

75 Limonen: 100% = 670 Hz % 50

25 Geruch: “ citrusartig”

0 CW1500 CW20M OV1 SE54 OV17

100

75

% 50 1,8-Cineol: 100% = 725 Hz 25 Geruch: „Eucalyptusartig“ 0 O CW1500 CW20M OV1 SE54 OV17

ABBILDUNG 2 - Ansprechempfindlichkeit von QMB-Sensoren auf Monoterpene.

3. AUSWERTUNG

Eine systematische Zuordnung der Ansprechempfind- Signale von Chemosensoren werden fast immer mittels lichkeit von MOS- oder Polymersensoren zu bestimmten Transformation vorverarbeitet. Ziel der eigentlichen Signal- Substanzklassen ist dagegen nicht möglich, wie beispiel- verarbeitung ist die Unterscheidung von Proben bzw. die haft der Vergleich der zu unterschiedlichen Zeitpunkten Zuordung unbekannter Proben zu bekannten Klassen. Je und nach Probenaufgabe mit zwei unterschiedlichen Pro- nach Anwendung eignen sich für diese Analyse verschiede- bengebern gemessenen Sensorantworten eines MOS- ne Strategien, die aus der Mustererkennung bzw. multiva- Arrays (Fox 4000) auf Methyljasmonat mit den Herstel- riaten Datenverarbeitung abgeleitet werden können. In der lerangaben zeigt (siehe Abb. 3). Praxis haben sich zum einen lineare Verfahren wie die Hauptkomponentenanalyse (Principal Component Analy- sis, PCA) als Methode des unüberwachten Lernens oder

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0,8

0,7

0,6

0 0,5

0,4 R/R ∆ ∆ ∆ ∆ 0,3 0,2

0,1

0

SY/LG

polare Verb.

polare Verb. 1 1 TA2 k. Angabe TA2 k.

unpolare Verb. F-und Chloride F- und Chloride 1 1 PA2 polare Verb. P30/2 k. Angabe k. P30/2 1 T70/2 Foodflavour T70/2 0/ 0/2 unpolare Verb. 0/2 T30/ P30/ 1 1 SY/G Amine/S-Verb. SY/G SYAA unpolare Verb. SYAA unpolare SYGCT polare Verb. polare Verb. SYGCT P P P40/ T40/

ABBILDUNG 3 - Sensorantworten für Methyljasmonat auf Array Fox 4000.

die Diskriminanzanalyse (DA) als Methode des überwach- scheinlichkeit (P) für die willkürliche Trennung eines ten Lernens und zum anderen nichtlineare Verfahren, dazu Datensatzes in zwei unterschiedliche Klassen in Abhän- zählen künstliche neuronale Netze (Artificial Neuronal gigkeit von der Musteranzahl (n). Networks, ANN), durchgesetzt.

PCA und DA sind Datenreduktionstechniken, die es p erlauben, den Informationsgehalt eines mehrdimensiona- n,r len Datensatzes in wenigen „künstlichen“ Dimensionen zu komprimieren. Diese Dimensionen (Hauptkomponen- 1 ten, Diskriminanzfunktionen) stellen dabei optimale Line- arkombinationen der Ausgangsdaten dar, wobei der In- formationsgehalt von der ersten in Richtung der höheren r=30 Dimensionen abnimmt. Sie ermöglichen damit in der 0,5 Regel die grafische Darstellung eines multivariaten Klas- sifizierungsproblems in einer zweidimensionalen Fläche, maximal im dreidimensionalen Raum. Während die PCA die natürliche Ordnung der Daten widerspiegelt, werden 0,1 bei der DA die neuen Dimensionen so berechnet, dass sie n die ursprünglichen Datenmuster nach einer vorgegebenen 0 306090120150 Klassenzuordnung maximal separieren.

Um bei Sensorarray-Messungen mittels PCA bzw. ABBILDUNG 4 - Wahrscheinlichkeit p für eine willkürliche DA eine aussagekräftige Klassentrennung (in Sorten, Trennung in 2 Klassen in Abhängigkeit von der Musteranzahl n. Qualitäten, Stoffgruppen o. ä.) zu erreichen, muss ent- sprechend der Anzahl der Sensoren (r) eine ausreichende Anzahl (n) an Kalibrationsdatenmustern (jeweils r- Hieraus geht gleichzeitig deutlich hervor, dass man dimensional) vorliegen. Abb. 4 zeigt beispielhaft für ein von einer signifikanten Trennung (Irrtumswahrschein- aus 30 Sensoren bestehendes Sensorarray die Wahr- lichkeit < 10 %) erst dann sprechen kann, wenn die Pro-

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ben-, d. h. Musteranzahl mindestens das 3-fache der Anzahl Nonan versetzten Pfefferprobe dargestellt. Wie nicht der Sensoren beträgt. In praxi bedeutet dies, dass für einen anders zu erwarten, verursacht das zugesetzte geruchsak- zu klein gewählten Kalibrationssatz die Hauptachsentrans- tive Vanillin einen stark von der typischen Pfeffernote ab- formation stets einen Merkmalsraum liefert, in dem die als weichenden Geruchseindruck, obwohl es mittels GC/FID unterschiedlich definierten Klassen getrennt vorliegen, im Headspace nicht nachweisbar ist. unabhängig davon, ob sich deren Signalmuster tatsächlich signifikant unterscheiden [14]. Viele der in der Literatur Dagegen weicht die mit Nonan dotierte Pfefferprobe beschriebenen Diskriminierungen beruhen auf einem derar- sensorisch kaum von der reinen Pfefferprobe ab, obwohl der tigen Missverhältnis zwischen Sensorenzahl und ge- prozentuale Anteil im Dampfraum nahezu 50 % beträgt. messener Musteranzahl. Auf die Verwendung hochdimen- Vom Gassensor-Array detektiert werden - in Abhängigkeit sionaler Sensorarrays sollte in der industriellen Praxis ver- von der Empfindlichkeit der Sensoren - generell Unter- zichtet werden, da die Erstellung großer Kalibrationsdaten- schiede in der Zusammensetzung der Gasphase, sodass im sätze zeitaufwendig und kostspielig ist. Um den Proben- vorliegenden Fall wie die Messdatenauswertung mittels aufwand zu reduzieren, sollten soweit möglich orthogonale PCA zeigt (siehe Abb. 5) empfindlichkeitsbedingt eine (unterschiedlich selektive) Sensoren eingesetzt werden. Diskriminierung nach den Hauptkomponenten im Dampf- raum und nicht nach dem sensorischen Eindruck erfolgt. Ähnliche Ergebnisse erhält man bei der Musteranaly- 4. ANWENDUNGEN se der Messdaten von Pfeffer unterschiedlicher Herkunft, die hier nicht im einzelnen wiedergegeben werden sollen. Unsere Untersuchungen an Modellsystemen haben Auch in diesem Fall erfolgt die Diskriminierung aufgrund ergeben, dass eine universell anwendbare „elektronische einer unterschiedlichen prozentualen Verteilung vornehm- Nase“, die darüber hinaus frei von äußeren Störungen zur lich nicht geruchsrelevanter Monoterpene im Dampfraum. Bearbeitung von geruchsrelevanten Fragestellungen in Die Gruppe der geruchsrelevanten sauerstoffhaltigen vielen Bereichen der Lebensmittelindustrie herangezogen Sesquiterpene und Phenolderivate (z.B. Kresol, Guajacol, werden kann, bis dato noch nicht existiert. Unglückli- Eugenol, Vanillin) gelangt unter den gewählten Messbe- cherweise hat die Bezeichnung „elektronische“ oder dingungen nicht in messbaren Mengen in die Gasphase. „künstliche Nase“ vielfach zu Verwirrungen in der Beur- teilung der Leistungsfähigkeit von Multi-Sensor-Arrays An diesem Beispiel wird deutlich, dass Gassensor- geführt. Sie erweckte bei vielen potentiellen Anwendern Systeme, wie die auf der Basis von QMB’s, MOS oder irrtümlich den Anschein, die Diskriminierung von Proben leitenden Polymeren, die nicht durch Selektion in bezug erfolge aufgrund ihrer unterschiedlichen Geruchseigen- auf Selektivität und Empfindlichkeit für eine bestimmte schaften. Das folgende Beispiel belegt, dass der psycho- Anwendung optimiert wurden, sehr reproduzierbar nur für physische Vorgang der Erkennung und Charakterisierung die Erkennung von Ähnlichkeiten oder Unterschieden der von Geruchsnoten derzeit noch der menschlichen Nase Gasphasenzusammensetzung geeignet sind. Das erzielte vorbehalten bleibt. In Tabelle 2 sind die prozentuale Zu- Ergebnis gibt aufgrund der Unselektivität/Semiselektivität sammensetzung des Dampfraumes sowie die humansenso- der Sensoren aber keine Auskunft darüber, welche im rische Bewertung von 3 unterschiedlichen Pfefferproben Headspace vorhandenen Substanzen zu einer Diskriminie- zusammengefasst. Neben der Bewertung einer reinen Pfef- rung geführt haben und ob diese überhaupt mit der zu ferprobe sind die einer mit Vanillin und einer mit Nonan untersuchenden Produkteigenschaft korrelieren.

TABELLE 2 Mit GC/FID ermittelte Headspace-Zusammensetzung (%) und sensorische Bewertung von unterschiedlichen Pfefferproben.

Verbindung Pfeffer Pfeffer + Vanillin Pfeffer + Nonan Nonan n.n. n.n. 45 α-Pinen 21 19 11 ß-Pinen 21 21 12 3-Caren 33 34 18 Limonen 15 16 9 Vanillin n.n. n.n. n.n. Fehlgeruch * Note: 1,5 Note: 3,6 Note: 1,9 *Note: Mittelwert aus Bewertung (1= kein Fehlgeruch, 5= starker Fehlgeruch), Anzahl der Probanden: 10

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Pfeffer schwarz Pfeffer + Nonan

Pfeffer weiss Pfeffer + Vanillin

ABBILDUNG 5 - PCA der QMB-Messdaten unterschiedlicher Pfefferproben.

2 3 1 6 5

2 4

1 3

Pro Sorte 12 reale Messungen Pro Sorte 50 reale Messungen

ABBILDUNG 6 - Sortenunterscheidung von Hopfen. 1= H. Nugget, 2= Select, 3= H.Magnum, 4= H.Perle, 5= H. Tradition, 6= N. Brewer

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8.9 x 8.0 xx 3 MOS-Sensor 4 2 6.0 x x x

4.0 5 1 x 6 xx x 5 2.0 x x x x x 4 x x x x 0.0 x -2.0 x x x xxx x x 2 x x -4.0 x xx x x x x 1 3 -6.0 1 6 -7.3 -18.2 -16.0 -14.0-12.0-10.0 -8.0 -6.0 -4.0 -2.0 0.0 2.0 4.0 6.7

ABBILDUNG 7 - Diskriminierung von 6 Hopfensorten mittels QMB- und MOS-Sensoren.

4.1 Analyse von Hopfensorten In Abb. 6 sind die Ergebnisse der PCA von QMB- Bemühungen, eine analytische Methode zur Überprü- Messungen an Hopfendolden, einmal mit 12 Messungen, fung der Sortenreinheit von Hopfen und Hopfenprodukten ein anderes Mal mit 50 Messungen je Probe, gegenüber- zu entwickeln, beruhten bisher auf der zeitaufgelösten gestellt. Hier ist deutlich der im vorigen Abschnitt erläu- GC- bzw. HPLC-Trennung der Hopfeninhaltsstoffe. Die terte Einfluss des Verhältnisses Sensorelemente/ Proben- entwickelten Methoden sind bis dato jedoch nicht voll- anzahl zu erkennen. Bei 50 realen Messungen je Probe ständig ausgereift. Die in einer Anwendungsbroschüre werden die Sorten Nugget, Select und Magnum zwar eines Herstellers „elektronischer Nasen“ veröffentlichte noch immer unterschieden, der Abstand zwischen den Diskriminierung unterschiedlicher Hopfensorten [15] gab Klassen ist jedoch deutlich kleiner als im Falle von nur 12 den Anlass, diese Anwendung einerseits auf ihre Zuver- Messungen. lässigkeit zu überprüfen und andererseits die mit unter- Die Problematik, die sich bei der Diskriminierung schiedlichen Sensorsystemen erhaltenen Ergebnisse auf gleicher Proben mit unterschiedlichen Sensortypen ergibt, ihre Vergleichbarkeit zu untersuchen. wird aus dem Vergleich der Sortendiskriminierung mit QMB’s und MOS-Sensoren deutlich (siehe Abb. 7).

100,0%

80,0% ni 2Me-3-Buten-2-ol 60,0% 6-Me-5-Hepen-2-on

40,0% Myrcen 3Me--Butyl-Isobutanoat 20,0% E-ß-Ocimen

0,0% Perle Nugget S.Select Magnum Tradition N-Brewer

ABBILDUNG 8 - Headspace-Zusammensetzung von 6 Hopfensorten bei 60°C.

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Sowohl mit dem QMB- als auch dem MOS-Array werden bestimmte, aber nicht identische, Diskriminierun- gen zwischen den Sorten vorgenommen. Auffällig ist insbesondere, dass mit dem MOS-Array die Sorte 5 als sehr verschieden von den übrigen Sorten detektiert wird, während die QMB-Messung keinen solch gravierenden Unterschied ausweist. Anhand dieser Ergebnisse wird die Bedeutung der für die Messung verwendeten Sensoren im Array recht deutlich, insbesondere dann, wenn Analysen unterschiedlicher Anwender verglichen werden sollen. Da jeder „Sensor-Typ“ aufgrund seiner Unse- lektivität/Semiselektivität sowie unterschiedlichen An- sprechcharakteristika zwangsläufig zu unterschiedlichen Gut Überdarrt Diskriminierungen führt, ist eine Standardisierung dieser Methode ebenso wie die Vergleichbarkeit von Cluster- darstellungen, die auf der Datenauswertung von Messun- gen mit unterschiedlichen Sensortypen beruhen, nicht gewährleistet.

Als nächstes stellt sich die Frage, ob die beobachteten ABBILDUNG 9 - PCA von QMB-Messungen Sortendifferenzierungen tatsächlich auf sortenspezifi- an gut gedarrtem und überdarrtem Hopfen. schen Unterschieden, die von den Sensorelementen er- fasst wurden, beruhen. Die GC-Analyse der untersuchten Wie die Musteranalyse von 300 QMB-Messungen je Hopfensorten zeigt, dass neben einigen wenigen Kompo- Probe (Abb. 9) zeigt, kann hier eine gute Unterscheidung nenten im leichtflüchtigen Bereich der Hauptanteil des mit Multi-Sensor-Arrays erzielt werden. Dampfraumes aus Myrcen besteht (siehe Abb. 8). Die Sensorsignale werden folglich bei allen gemessenen Sor- Um eine echte Unterscheidung der Sorten zu errei- chen, muss ein selektives Sensorsystem, welches nicht auf ten hauptsächlich von Myrcen und nicht von anderen Myrcen sondern auf sortenspezifische Komponenten in Verbindungen, die Sortenrelevanz besitzen könnten, wie noch messbarer Konzentration im Dampfraum anspricht, z.B. 3-Methyl-Butyl-Isobutanoat, hervorgerufen. Es muss eingesetzt werden. Die Hopfeninhaltsstoffe, die als also davon ausgegangen werden, dass die beobachteten Diskriminierungen nicht auf unterschiedliche Sortenei- Grundlage für die Sortendiskriminierung herangezogen genschaften, sondern auf unterschiedliche Gehalte von werden könnten, sind Nebenkomponenten mit Headspace- Anteilen < 5 %. In Abb. 10 sind beispielhaft für die Sor- vornehmlich Myrcen zurückzuführen sind. Unterschiedli- ten Tettnanger und Nugget diejenigen Komponenten ge- che Gehalte an der Hauptkomponente Myrcen sind ein kennzeichnet, die als Indikatoren für eine Sortenunter- Unterscheidungsmerkmal gut gedarrter von überdarrten scheidung dienen könnten. Es handelt sich dabei vorwie- Hopfenproben. gend um Methyl- und Isobutylester von Carbonsäuren mit

320 10

Tettnanger 280 5 240 0 200 Tettnanger Magnum mV 160 Nugget -5

120 Hersbrucker Perle Nugget -10 80 6 7 8 9 10 11 12 14 15 16 17 18 19 20 20 10 0 -10

ABBILDUNG 10 ABBILDUNG 11 GC-Vergleich der Sorten Tettnanger und Perle. PCA der Messungen von Hopfensorten mit dem MS-Sensor.

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Kettenlängen im Bereich C3-C10. Daneben könnten wei- Sensorsignalen ist bereits ein deutlicher Unterschied zwi- terhin die bicyclischen Monoterpene Sabinen, α-Pinen schen den Sensorantworten des Fox 4000 auf eine NaCl- und β-Pinen sowie die acyclischen Monoterpene Lösung und Erdbeerhomogenat ersichtlich (Abb. 12). β-Ocimen und Linalool miteinbezogen werden. Ebenso deutet sich eine Differenzierung zwischen den von Erdbeerproben der Sorte „Polka“ verursachten Sen- Es wurde versucht, diese Sortenunterscheidung mit sorsignalen einerseits und von Proben der Sorten „Kent“ dem MS-Sensor zu erreichen. Das „Array“ zur Sortendif- und „Elsanta“ andererseits an. ferenzierung im SIM-Modus setzte sich aus den zwölf Fragmentionen m/z 100, 101, 106, 110, 111, 112, 114, Zur Überprüfung der Zuverlässigkeit der aufgefunde- 115, 127, 137, 139 und 142 zusammen. Die Sortenindika- nen Diskriminanzfunktionen (Abb. 13) wurde nacheinan- toren zeichneten für 48-95 % der Peakflächensummen der für jede Probe die Zugehörigkeit des Sensorsignal- einzelner Fragmentionen verantwortlich. Nicht sortenty- Musters dieser Probe (Testprobe) zu den Erdbeersorten pische Verbindungen besaßen nur in 5 von 36 Messungen für „unbekannt“ erklärt, die Diskriminanzanalyse ohne Peakflächenanteile von mehr als 40 %, mehrheitlich be- Einbeziehung dieses Musters erneut durchgeführt und die trugen ihre Anteile 17-35 % an der Gesamtpeakfläche. Zugehörigkeit der Probe dann mit Hilfe dieser Diskrimi- Die in Abbildung 11 dargestellten Ergebnisse der PCA nanzanalyse ermittelt (leave-one-out-Methode). Die sich zeigen, dass eine gute Trennung der Sorten mit dem aus- dabei ergebende Fehlerrate (Verhältnis der Anzahl fehl- gewählten „Array“ erzielt werden kann [16,17]. klassifizierter Proben zur Gesamtzahl an Testproben) ist jedoch nur eine einzelne Stichprobe der „tatsächlichen“ 4.2. Differenzierung von Erdbeersorten Fehlerrate. Mit ihrer Hilfe und unter Annahme einer Bi- Anhand mehrjähriger Versuche, die an der Hochschu- nomialverteilung lässt sich eine statistisch begründete le Anhalt gemeinsam mit dem Institut für Pflanzenanaly- Fehlerrate (Konfidenzintervall der Fehlerrate) berechnen. tik der Bundesanstalt für Züchtungsforschung an Kultur- Die Größe dieses - übrigens unsymmetrischen - Kon- pflanzen (BAZ) in Quedlinburg durchgeführt wurden, fidenzintervalls hängt dabei hauptsächlich von der Ge- konnte nachgewiesen werden, dass aus den Signalen eines samtzahl n der gemessenen und nach der leave-one-out- Metalloxidsensor-Arrays die Sorte von Erdbeeren mit Methode abgefragten Proben ab [20]. Die bei der Zuord- einer hohen Wiederfindungsrate, ausgehend sowohl von nung der gemessenen Sensorsignal-Muster zu den Erd- zuvor gefrorenen als auch von frischen Proben, vorherge- beersorten aufgetretenen Fehlerraten (siehe Tab. 2) bestä- sagt werden kann [18, 19]. Untersucht wurde der Dampf- tigen erneut, dass aus den Metalloxid-Sensorsignalen des raum über Erdbeerhomogenaten in gesättigter NaCl- Fox 4000 die Erdbeersorte mit hoher statistischer Sicher- Lösung bei 40 °C. Aus den statistisch unverarbeiteten heit vorhergesagt werden kann.

0 0 R/R R/R ∆ ∆ NaCl-Lösung ‘Kent’

t in s t in s 0 0

R/R ‘Elsanta’ R/R ‘Polka’ ∆ ∆

t in s t in s

ABBILDUNG 12 Fox 4000 Sensorsignale unterschiedlicher Erdbeersorten im Vergleich zum Blindwert (NaCl-Lösung).

162 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

Kent Polka Diskriminanzfunktion 2 Diskriminanzfunktion (26,8 % der Gesamtvarianz)

Elsanta

Diskriminanzfunktion 1 (73,2 % der Gesamtvarianz)

Abb. 13: DiskriminanzanalyseABBILDUNG 13 - Diskriminanzanalyse der Fox-4000-Signale der Fox-4000-Signale vonvon Erdbeerproben. Erdbeerproben.

TABELLE 2 Vorhersage von Sorte und Erntetag von Erdbeerproben ausgehend von Fox 4000 Sensorsignalen.

Gruppe Fehlerrate fehlklassifiziert als 'Elsanta', Proben des 1. Erntetages 0/18 - 'Elsanta', 4. Erntetag; 'Elsanta', Proben des 2. Erntetages 2/14 'Polka', 2. Erntetag 'Elsanta', Proben des 3. Erntetages 0/10 - 2 x 'Elsanta', 2. Erntetag 'Elsanta', Proben des 4. Erntetages 3/14 1 x 'Elsanta', 1. Erntetag 'Kent', Proben des 1. Erntetages 0/14 - 'Kent', Proben des 2. Erntetages 0/11 - 'Kent', Proben des 3. Erntetages 0/10 - 'Kent', Proben des 4. Erntetages 0/14 - 'Polka', Proben des 1. Erntetages 0/11 - 'Kent', 3. Erntetag 'Polka', Proben des 2. Erntetages 2/11 'Elsanta', 4. Erntetag 'Polka', Proben des 3. Erntetages 1/10 'Polka', 4. Erntetag 'Polka', Proben des 4. Erntetages 0/14 - Gesamtfehlerrate 8/151 (95 % Konfidenzintervall) (3 % < 6 % < 11 %)

163 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

Area 1,20E+07 1,00E+07 8,00E+06 6,00E+06 4,00E+06 2,00E+06 0,00E+00 Buttersäure Ethylbutyrat 2-Methylbutters. Methylcapronat Benzaldehyd Capronsäure MeO-Furaneol 2-Nonanon Linalool Terpineol Myrtenal Hexyl(iso)butyrat 2-Undecanon Methylanthranilat Hexylcapronat Octylisobutyrat g-Decalacton

Substanz

ABBILDUNG 14 - SPME-GC-FID von 2 Erdbeersorten (Elsanta , Fragaria vesca L.).

Dagegen ist mit dem QMB-Sensorsystem eine Sor- und wird derzeit bearbeitet. Diese vorläufigen Ergebnisse tendiskriminierung nicht möglich. Auch mit frischem deuten daraufhin, dass MOS-Sensoren möglicherweise für Probenmaterial gelingt weder eine Unterscheidung der bestimmte Substanzen eine im Vergleich zur FID- oder Sensorsignale unterschiedlicher Erdbeerhomogenate noch MS-Detektion höhere Empfindlichkeit aufweisen. eine Unterscheidung der Sensorsignale von Erdbeeren und der Referenz (NaCl-Lösung). Aufgrund der niedrigen Die sichere Erkennung auch des jeweiligen Ernteta- Werte der QMB-Sensorsignale kann davon ausgegangen ges durch die MOS-Sensoren ist vermutlich - obwohl in werden, dass die Konzentration der im Dampfraum be- allen Fällen optisch reif aussehende Früchte geerntet findlichen Verbindungen sehr gering war. Dieses konnte wurden – ebenfalls auf Unterschiede in der Headspace- sowohl mittels dynamischer Headspace-GC-MS als auch Zusammensetzung zurückzuführen. durch Anreicherung und Thermodesorption-GC-MS bestätigt werden. Neben geringen Mengen an nicht 4.3 Unterscheidung von Essig unterschiedlicher Qualität sortentypischen Verbindungen sind keine Substanzen im Ziel dieser Untersuchung war es zu prüfen, ob Mes- Headspace identifizierbar, die eine Erklärung für die sungen mit einem Multi-Sensor-Array geeignet sind, eine eindeutige Klassifizierung mit den MOS-Sensoren liefern humansensorische Prüfung von Essig, welcher gelegent- würden. Aus diesem Grund konnte auch mit dem MS- lich ein strenges lösungsmittelartiges (nach Klebstoff) Sensor im Scan-Modus keine sortenabhängige Unter- Aroma besitzen oder nach Lagerung entwickeln kann, zu scheidung erzielt werden. Erst Untersuchungen mittels ersetzen. Dieser Fehlgeruch entsteht, wenn nach der mit einer Festphasen-Mikroextraktion (SPME) lieferten Hilfe von Essigsäure-Bakterien durchgeführten Vergä- eindeutige Unterschiede in der Dampfraumzusammen- rung alkoholhaltiger Lösungen zu Essigsäure der Restge- setzung unterschiedlicher Erdbeersorten (Abb. 14). Ob die halt von Ethanol im Handelsprodukt deutlich mehr als mittels SPME-GC-MS identifizierten Substanzen für eine 0,5 % beträgt. Tab. 3 zeigt die sensorische Beurteilung Sortendifferenzierung herangezogen werden können und und die deutlich verschiedene Headspace-Zusammenset- welche zur reproduzierbaren Sortenklassifizierung mit zung (Hauptkomponenten) von zwei extrem unterschied- den MOS-Sensoren geführt haben, ist noch nicht geklärt lichen Essigqualitäten aus dem Handel.

TABELLE 3 - Essigsäure (Handelsproben) unterschiedlicher Qualität.

sensorische Ethanol Essigsäure Ethylacetat Probenbezeichnung Beurteilung % % ppm Probe 1 o.k. 0,05 10 228 Probe 2 Fehlgeschmack stark 1,3 10 1303

164 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

Probe 2 A Essig B

Essig

Probe 1 Probe 2

Probe 3 Wasser Probe 1

ABBILDUNG 15 - Diskriminierung von Essigproben mittels QMB6 (A, links) und MS-Sensor (B, rechts).

M

ABBILDUNG 16 - Unterscheidung von Verpackungsfolien mit unter- schiedlichem Kaltsiegelkleber mittels QMB- (links) bzw. MOS-Sensorsystem (rechts)

Durch den relativ hohen Ethanolgehalt in Probe 2 rem Material beschichteten Sensoren vornehmlich durch entsteht eine vergleichsweise große Menge an Ethylace- Essigsäure und Wasser hervorgerufen werden, und die tat, welches zum lösungsmittelartigen Fehlaroma führt. unpolaren Sensoren keine Empfindlichkeit gegenüber Da bei dieser Anwendung die im Dampfraum von Ethanol bzw. Ethylacetat aufweisen. 10 %iger Essigsäure vorkommenden Hauptkomponenten die Qualität der Probe bestimmen, sollte eine Diskriminie- Eine sehr gute Diskriminierung kann dagegen mit rung mittels Multi-Sensor-Arrays prinzipiell möglich dem MS-Sensor erreicht werden (Abb. 15B), wenn die sein. Die Ergebnisse der PCA der QMB-Messdaten (Abb. Fragmentionen m/z 46, 60 und 70 als „Array“ für die 15A) zeigen jedoch eine starke Streuung und Überlap- selektive Detektion von Ethanol, Essigsäure und Ethyl- pung. Eine Unterscheidung nach der Qualität der Probe acetat verwendet werden. Auch Proben, die noch keinen gelingt in diesem Falle nicht, sogar die Unterscheidung großen Fehlgeruch entwickelt haben, können sehr gut von Essig und Wasser ist mit dem Array nicht möglich. diskriminiert werden (Probe 3 in Abb. 15B). Es ist anzunehmen, dass die Sensorsignale der mit pola-

165 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

4.4 Analyse von Verpackungsmaterialien 4.5 Untersuchung von Korkproben mit Kork-Fehlgeruch Im Gegensatz zu Lebensmitteln besitzen Verpa- Korken, welcher mit Guajakol, 1-Octen-3-ol/1-Octen- ckungsmaterialien eine sehr definierte Matrix. In der 3-one, 2-Methylisoborneol, Geosmin oder 2,4,6-Trichlor- Regel sind Rohstoffe und Verarbeitungshilfsstoffe hinrei- anisol kontaminiert ist, besitzt einen Fehlgeruch, der sich chend bekannt, wodurch eine Grundvoraussetzung für als typischer „Kork-Fehler“ bei Weinen und anderen eine erfolgreiche Anwendung von Sensorsystemen gege- Getränken bemerkbar macht [21, 22]. Dieser Qualitäts- ben ist. Aufgrund ihrer Temperaturstabilität können bei mangel führt bei Weinen zu Verlusten von 1 Milliarde der Probenaufgabe auch höhere Temperaturen als bei Dollar im Jahr. Hauptverantwortlich für den Kork-Fehler Lebensmitteln angewandt werden, sodass durch die folg- ist 2,4,6-Trichloroanisol (2,4,6-TCA), welches von Mik- lich höhere Dampfraumkonzentration eine höhere Emp- roorganismen, die auf dem Kork wachsen, gebildet wird. findlichkeit erreicht wird. Bei den qualitätsbeinflussenden Es wird jedoch angenommen, dass 2,4,6-TCA und andere Komponenten handelt es sich im allgemeinen nicht um chlorierte Verbindungen auch während der Verarbeitung geruchsaktive Spurenkomponenten, sondern um flüchtige des Korkmaterials, beim Bleichen, aus Ligninbestandtei- Substanzen wie beispielweise Lösungsmittel aus Druck- len entstehen können [23]. Eine schnelle Methode zur farben oder Restmonomere. Solche Störkomponenten Erkennung des Aromafehlers im Rohmaterial wäre in können mit Sensorsystemen leichter erfasst werden. Als jedem Falle von großer wirtschaftlicher Bedeutung. Beispiel sei hier die Unterscheidung von zwei Verpa- ckungsfolien wiedergegeben, die aufgrund unterschiedli- Obwohl die Headspace-Technik in puncto Schnellig- cher Verbundklebstoffe (Kaltsiegelkleber) zu einer guten keit gegenüber Extraktionsverfahren anerkanntermaßen bzw. einer schlechteren Siegelnahtfestigkeit geführt hat- deutliche Vorteile hat, besteht bei dieser Anwendung das ten. Wie aus Abb. 16 deutlich zu erkennen ist, gelang die Problem, dass 2,4,6-TCA erst ab 135 °C in den Dampf- Diskriminierung der beiden Folien sowohl mit dem raum freigesetzt wird. Bei dieser Temperatur beobachtet QMB- als auch mit dem MOS-Sensorsystem. man im Headspace eine große Anzahl an Verbindungen, einige wie Furfural und 5-Methyl-Furfural sind zum Teil Die vergleichsweise beste Unterscheidung lieferte der auf den Abbau von Pentosen und Hexosen zurückzufüh- MS-Sensor (siehe Abb. 17). Da die Kaltsiegelkleber der ren (siehe Abb. 18). Außerdem werden bei 135 °C kork- Folien sich in Bezug auf das Vorkommen von größeren eigene Sesquiterpene wie β-Caryophyllen, β-und δ-Ele- Restmengen an Lösungsmitteln wie Ethanol, CS2 und men und α-Copaen aus der Matrix ausgetrieben. In bezug Cyclohexan unterschieden, wurde die beste Diskriminie- auf die Hauptkomponenten im Headspace unterscheiden rung mit den Fragmentionen m/z 56,76,78 und 84 als sich unbelastetes und mit 2,4,6-TCA belastetes Korkmate- „Sensor-Array“ erreicht. rial nicht. Daher kann eine Diskriminierung derartiger Proben mittels Headspace-Analyse nur mit einem Sensor-

ABBILDUNG 17 Unterscheidung von Verpackungsfolien mit unterschiedlichem Kaltsiegelkleber mit dem MS-Sensor.

166 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

Aceton Essigsäure Methyl-furfural

10,0 Headspace GC-FID von unbelastetem Kork

Furfural Temperatur: 135°C

Säule: DB1, 30 m, i.d 0.32 mm, 3µm, Carrier He

7,5 Temp. Programm: 60°C (5)-15°C/min-200°C Hexanal

RetentionszeitRetentionszeit von von 2,4,6-TCA TCA

5,0

2,5

Minuten 5 10 15 20 25

ABBILDUNG 18 - Headspace-GC von unbelastetem Korkmaterial.

normal

Off-flavour

ABBILDUNG 19 - Unterscheidung von Korkmaterial mit und ohne „off-flavour”.

system erzielt werden, welches mit der notwendigen mensionale Fragmentionen-„Array“ setzte sich aus m/z Empfindlichkeit auf 2,4,6-TCA anspricht. Je spezifischer 195, 197 und 210 zusammen (Abb. 19). Es bestand damit die Sensoren dabei auf 2,4,6-TCA reagieren, desto weni- ausschließlich aus Fragmentionen des Spektrums von ger stören die im Headspace vorhandenen Hauptkompo- 2,4,6-Trichloranisol, deren Intensität in den Massenspekt- nenten die Diskriminierung. ren der im Headspace über nicht kontaminierten Korken nachweisbaren flüchtigen Verbindungen gleich Null war. Andere Hauptkomponenten (siehe Abb. 18), die während Eingesetzt wurde für diese Anwendung ein MS- der Dampfraumgenerierung bei 135 °C freigesetzt werden Sensor im SIM–Modus. Das zum Nachweis von 2,4,6- und die bei konventionellen Sensorsystemen die Signal- Trichloranisol in Korkmaterial verwendete dreidi- antwort hauptsächlich bestimmen würden, stören auf-

167 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

grund der hohen Selektivität der Methode die Messung Im Unterschied zur menschlichen Nase reagieren den und folglich auch die Unterscheidung anhand der PCA Proben-Headspace messende „Multi-Sensor-Arrays“ nicht der Sensorantworten nicht. Somit ist die Unterscheidung ausschließlich mit geruchsaktiven Substanzen, es werden von Korkproben mit bzw. ohne Fehlgeruch auf der Basis vielmehr alle flüchtigen Komponenten der Gasphase erfasst, der Bestimmung von 2,4,6-Trichloranisol mit dem MS- die mit dem Sensorsystem in Wechselwirkung treten und Sensor durch Headspace-Direktinjektion/ Mustererken- für die die Sensoren empfindlich genug sind. Eine Aus- nung grundsätzlich möglich. nahme bilden Sensoren bzw. Arrays, die für eine spezifische Anwendung konstruiert, selektiert und optimiert wurden.

5. SCHLUßFOLGERUNGEN Mit den sensorischen Befunden in bezug auf den Ge- ruch von Proben korreliert eine Diskriminierung mit Voraussetzung für den Einsatz von „Multi-Sensor- Chemosensoren nur dann, wenn die Unterschiede in den Arrays“ zur Unterscheidung von Proben bzw. zur Zu- Sensorsignal-Mustern überwiegend auf qualitative und/ ordnung unbekannter Proben zu einer Klasse ist, dass oder quantitative Unterschiede bezüglich geruchsrelevan- das zu untersuchende Probenmaterial flüchtige Verbin- ter Hauptkomponenten zurückgeführt werden können dungen enthält, die in ihrer qualitativen und quantitati- oder mit dem Sensorarray erfassbare Hauptkomponenten ven Zusammensetzung charakteristisch für die Probe (Indikatorkomponenten) eine Korrelation mit den ge- sind. Der Erfolg einer Diskriminierung hängt in starkem ruchsprägenden Minorkomponenten aufweisen. Maße von der ausgewählten Sensorkombination ab. Eine Diskriminierung ist immer dann möglich, wenn die Gas- Beim derzeitigen Entwicklungsstand stellen „elektro- phasenzusammensetzung der Proben unterschiedlich ist nische Nasen“ keine Alternative zu humansensorischen und Sensoren selektiert wurden, die auf diese Unter- Methoden der Geruchserfassung und –bewertung dar. Sie schiede ansprechen. können jedoch überall dort eingesetzt werden, wo die Gasphasenzusammensetzung in enger Beziehung zu den Bei den meisten der bisher zu kommerziell erhältli- für die Probendiskriminierung relevanten Probenmerk- chen Arrays kombinierten Sensortypen (QMB, MOS, malen steht. Deshalb sind genaue Kenntnisse über die halbleitende Polymere) werden die Sensorsignale auf- Headspace-Zusammensetzung der Proben bei allen Sys- grund der geringen Spezifizität der Sensoren in der Regel temen der Schlüssel zu einer erfolgreichen Anwendung. durch die Hauptkomponenten der Headspace-Zusammen- setzung verursacht. Daher können in größerer Konzen- Aufgrund der Komplexität und Inhomogenität der tration vorliegende Störkomponenten zu Pseudodiskrimi- Zusammensetzung von Naturprodukten ist mit den bis- nierungen der Proben führen, d. h. zu Klassenunterschei- lang verfügbaren Sensorsystemen nicht mit einer breiten dungen, die nicht auf die eigentlich relevante Produkt- Anwendung in der Qualitätskontrolle von Lebensmitteln eigenschaft zurückzuführen sind. Die in der qualitativen zu rechnen. Anwendungen in bezug auf einfachere Sys- und quantitativen Headspace-Zusammensetzung zu su- teme, wie z. B. im Bereich der Verpackung oder beim chenden chemischen Ursachen für eine erfolgreiche Pro- Recycling von Kunststoffen, bieten größere Chancen für bendiskriminierung mit derartigen in der Regel unspezifi- deren sinnvollen Einsatz. schen bis breitenselektiven Chemosensoren sind meist unbekannt und können nur mittels klassischer Analysen- Derzeit sind vornehmlich statische Headspace- verfahren ermittelt werden. Probengeber im Zusammenhang mit Sensorsystemen im Einsatz. Erste Kopplungen mit Anreicherungssystemen MS-Sensoren bieten im Gegensatz dazu den Vorteil, wie SPME oder Thermodesorption sind bereits beschrie- dass im Scan-Modus Rückschlüsse auf die Identität der ben worden und versprechen eine zukünftige Steigerung Substanzen, die durch ihre Anwesenheit im Dampfraum der Empfindlichkeit der Systeme. Andere Schnellmetho- der Proben die Diskriminierung beinflusst haben, gezogen den wie Fast-GC mit anschließender Mustererkennung werden können. werden ebenfalls neue Wege in der Qualitätssicherung eröffnen. Qualitätsbestimmende Spurenkomponenten oder ge- ruchsbeeinflussende Minorkomponenten neben Haupt- komponenten können prinzipiell nur mit ausreichend empfindlich und spezifisch auf diese Substanzen anspre- DANKSAGUNG chenden Sensorsystemen erfasst werden (z.B. MS- Sensoren im SIM-Modus). Anderenfalls werden die Die hier vorgestellten Untersuchungen sind Teil der betreffenden Verbindungen entweder überhaupt nicht Untersuchungen, die aus Mitteln der industriellen Ge- detektiert oder ihr relativer Einfluss auf die gemessenen meinschaftsforschung (BMWi/AiF) über den For- Sensorsignale ist so gering, dass er im Rauschen unter- schungskreis der Ernährungsindustrie e. V. (FEI) geför- geht. Eine Probenklassifizierung nach Qualität bzw. dert wurden (AiF-FV 11589 BI/II). Dafür sei an dieser Geruch ist in diesen Fällen nicht möglich. Stelle herzlichst gedankt.

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LITERATUR

[1] Shurmer H.V., The electronic nose, Analytical Proceedings [17] Dittmann B., Horner G., Nitz S., Development of a New 31 (1), S. 39-40, 1994 Chemical Sensor on a Mass Spectrometric Basis, Advances in Food Science, 20, S. 115, 1998. [2] Hodgins D., The development of an electronic nose for industrial and environmental applications, Sensors and [18] Hirschfelder M., Ulrich D., Hoberg E., Hanrieder D., Rapid Actuators B, 26-27, S. 255-258, 1995 Discrimination of Strawberry Varieties Using a Gas Sensor Array, Gartenbauwissenschaft 63, 185-190, 1998 [3] Bartlett P.N., Blair N., Gardner J.W., Electronic noses - prin- ciples, applications and outlook, Association Scientifique In- [19] Hirschfelder M., Ulrich D., Hanrieder D., Discrimination of ternationale du Café, S. 616-625, 1993 Strawberry Varieties by a Gas Sensor Array in Correlation to Human Sensory Evaluation, Seminars in Food Analysis 3, S. 27-36, 1998 [4] Shurmer H.V., Gardner J.W., Odour discrimination with an electronic nose, Sensors and Actuators B, 8, S. 1-11, 1992 [20] Ahrens H., Läuter J., in Mehrdimensionale Varianzanalyse, Akademie-Verlag Berlin, 1981 [5] Gardner J.W., Bartlett P.N., A brief history of electronic noses, Sensors and Actuators B, 18-19 (1994), S. 211-220 [21] Tanner H., Zanier C., Buser H.R., Schweiz. Z. Obst- u. Weinbau 117, S. 97, 1981 [6] Horner G., Vonach B., Ein intelligentes Sensorsystem er- kennt Gerüche, Labor-Praxis, S. 28-30, April 1995 [22] Kugler D., Diss. TH Karlsruhe, 1993 [7] Tan T., Lucas Q., Moy L., Gardner J.W., Bartlett P., The electronic nose - a new instrument for sensing vapours, LC- [23] Heimann W., Rapp A., Völter I., Knipser W., Dt. Lebensm. GC-Int., Vol.8 No.4, S. 218-225, April 1995 Rundschau 79, S. 103, 1983

[8] Gardner J.W., Shurmer H.V., Tan T., Application of an elec- tronic nose to the discrimation of coffees, Sensors and Actua- tors B, 6, S. 71-75, 1992

[9] Egashira, Makoto; Shimizu, Yasuhiro; Takao, Yuji, Fish freshness detection by semiconductor gas sensors, Olfaction Taste XI, Proc. Int. Symp. 11th (1994), Meeting Date 1993, S. 711-14, Editor(s): Kurihara, Kenzo; Suzuki, Noriyo; Oga- was, Hisashi, Publisher: Springer, Tokyo

[10] Aishima T., Dicrimination of food aromas by applying chemometric pattern recognition to gas sensor array, Olfac- tion Taste XI, Proc. Int. Symp. 11th (1994), Meeting Date 1993, 711-14, Editor(s): Kurihara, Kenzo; Suzuki, Noriyo; Ogawas, Hisashi, Publisher: Springer, Tokyo

[11] Shiers V. P., Electronic Nose Technology-Evaluations and De- velopments for the Food Industry, Proceedings of Food Ingredi- ents Europe Conference, Miller Freeman, S. 198-200, 1995

[12] Tulett C., Olfaktroskopie, Labor Praxis, S. 34-39, März 1995 Received for publication: January 16, 2003 Accepted for publication: January 28, 2003 [13] S. Nitz, H. Kollmannsberger, C. Lachermeier, G.Horner, Odour Assessment with Piezoelectric Quartz Crystal Sensor Arrays, a Suitable Tool for Quality Control in Food Technol- ogy?, Adv. Food. Sci. (CMTL) 21, S. 136-150, 1999 CORRESPONDING AUTHOR

[14] Horner G., Qualitative and Quantitative Evaluation Methods S. Nitz for Sensorarrays in Proceedings of the 6th International Lehrstuhl für Chemisch-Technische Analyse Symposium „Olfaction and electronic nose“, Tübingen, 20- 22 September, Germany und Chemische Lebensmitteltechnologie Technische Universität München [15] Alpha M.o.S. Application Note Nr 8, August 1995 Weihenstephaner Steig 23 85350 Freising, Weihenstephan - GERMANY [16] Zeidler C., Dittmann B., Nitz S., Characterization of Hop Va- rieties Using a Mass Spectrometry Based Sensor in Proceed- e-mail: [email protected] ings of the 6th International Symposium „Olfaction and elec- tronic nose“, Tübingen, 20-22 September, Germany AFS/ Vol 24/ No 4/ 2002 – pages 154 - 169

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CONSTITUENTS OF ESSENTIAL OIL OF Echinophora tenuifolia L. subsp. sibthorpiana (Guss.) Tutin.

H. Ç. Özen and Z. Toker

Dicle University, Faculty of Science and Art, Department of Biology, 21280, Diyarbakõr-Turkey

SUMMARY

In this study the essential oil of air-dried aerial parts Short description of the aerial parts of E. tenuifolia of Echinophora tenuifolia L. subsp. sibthorpiana (Guss.) subsp. Sibthorpiana (Guss.) Tutin. used for steam distilla- Tutin was obtained by steam distillation and analyzed by tion to obtain the essential oil: bushy, often rounded, softly GC/FID and GC/MS. Among the fifty compounds iden- pubescent, perennial or biennial, 20-45 cm; stem: terete, tified, the major components were 3-carene (23.5%), smooth or grooved; basal leaves: oblong-ovate or triangular- methyleugenol (18.4%), α-phellandrene (11.4%) and p- ovate, 12-40 x 3.5-15 cm, 2-pinnate; segments: 0.5-1 cm cymene (10.1%). long, ovate, cuneat, irregularly toothed to deeply incised or pinnatified; uppermost leaves: simple, toothed in the upper half; 2-3(-5) rays, equalling bracts; 1-5 bracts, ovate- lanceolate, 3-5 mm; 6 bracteoles 6, ovate, 3 mm thick and KEYWORDS: Echinophora tenuifolia, essential oils, 3-carene, fusing with top of the rays in fruits and reflexing; 12 pedi- methyleugenol, α-phellandrene, p-cymene. cels shorter than the bracteoles; stylopodium of outer flow- ers flat and conspicuous at flowering stage; fruits pyramidal, 5-10 x 4-8mm; 8-9 flowers; growing in dry hills, chalky ravines, steppe, and fallow fields; widespread in Turkey except N.E. Anatolia [1]. INTRODUCTION

The genus Echinophora is represented in the flora of Methods Turkey by six species. These are Echinophora tenuifolia The essential oil was obtained from the dried aerial L. subsp. sibthorpiana (Guss.) Tutin., Echinophora parts by steam distillation according to the method of tournefortii Jaub. et Spach., Echinophora orientalis Cockins and Middleton [3]. Analyses and identifications Hedge. et Lamond and the three endemic, Echinophora were done using a GC/MS combination and rentention chrysantha Freyn. et Sint., Echinophora trichophylla J.E. indices were compared with in-house and commerical Smith and Echinophora carvifolia Boiss. et Bal [1]. spectra and retention data of mass spectrum libraries for SE54 and OV1 columns. The GC/MS system was a In this study the essential oil of E. tenuifolia L. subsp. Varian 3400 GC fitted with an OPTICâ injector and sibthorpiana (Guss.) Tutin., known as “Çörtük, Çördük, coupled with a Finnigian ion trap mass spectrometer. Tarhana otu,Turşu otu or Peynirotu“ in Turkey, has been The column was an HP Ultra 2 (5% diphenyl- and 95% investigated. 5% infusions of this plant is used against dimethyl-polysiloxane, 50 m x 0.2 mm x 0.33 µm; helium gastric ulcers [2]. Fresh and dried leaves, and also flavoring as carrier gas, 1.6 ml/min; temperature programme 50- branches, of this plant are added to meaty meals, pickles, 270 °C at 2°/min). Quantification and retention index (RI) soup flavourings and a special kind of cheese. determinations were carried out using an HP 5890 GC fitted with OPTICâ injector, FID and an HP-5 fused silica capillary (5% diphenyl– and 95% dimethyl- MATERIALS AND METHODS polysiloxane groups, 25 m x 0.2 mm x 0.33 µm; nitro- gen as carrier gas, 0.3 ml/min (constant flow mode); Plant material temperature programme 30–280 °C at 3°/min). The data E. tenuifolia subsp. sibthorpiana (guss.) Tutin. was quoted are percentages of the relative peak areas (% collected in the Diyarbakõr region during June 1999. RPR). By using this GC-FID system we were able to Voucher specimens are kept at the Dicle University Her- confirm the identity of substances, when spectrometric barium of the Faculty of Sciences until analysis. characterization was inconclusive, by comparsion of

170 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

precise retention indices with in-house libraries com- p-cymene, terpinolene, octyl acetate, (Z)-2-nonenal, cam- piled using identical analytical conditions (column di- phor, β-caryophyllene, terpinene-4-ol, benzene, acetalde- mension, phase, etc). hyde, carvotanacetone, cuminaldehyde, cis-sabinol, p- cymen-8-ol, α-phellandrene epoxide, methyl eugenol, eugenol, thymol, carvacrol. Newly isolated and identified in RESULTS the volatile oil are: α-thujene, benzaldehyde, verbenene, 1,8-cineol, phenylacetaldehyde, artemisia ketone, p-2,4(8)- The steam distillation described elsewhere [3] of the ae- menthadiene, p-iso-propenyltoluene, linalool, nonanal, cis- rial parts (stems, leaves, fruits, flowers) of E. tenuifolia p-menth-2-enol, limonene epoxide, trans-p-menth-2-enol, subsp. sibthorpiana (Guss.) Tutin. gave yellowish oils with α-phellandrene-8-0l, ß-phellandrene-8-ol, borneol, m- a yield of 0.06%. Fifty compounds were identified in the cymene-8-ol, α-terpineol, trans-piperitol, verbenone, thu- essential oil and the composition is given in Table 1. The jenoner, myristicin, caryophyllene oxide and viridiflorol. P- following components have been previously isolated: 3- cymene, methyleugenol and 3-carene have been detected as carene, α-pinene, camphene, hexanal, β-pinene, sabinene, major components [4-7]. These above-described results are myrcene, α-phellandrene, α-terpinene, heptanal, limonene, similar to our findings as shown in Table 1. β-phellandrene, (Z)-β-ocimene, γ-terpinene, (E)-β-ocimene,

TABLE 1 Chemical composition of the essential oil of E. tenuifolia subsp. sibthorpiana (Guss.) Tutin.

RI Compound Percentage RI Compound Percentage

931 α-thujene 0.20 1124 cis-p-menth-2-enol* 0.30 938 α-pinene 0.15 1142 limonene epoxide* 0.26 954 camphene 0.08 1148 trans-p-menth-2-enol* 0.20 964 benzaldehyde* tr 1155 camphor 0.81 968 verbenene* 0.26 1162 α-phellandrene-8-ol* 5.70 977 sabinene 0.29 1165 β-phellandrene-8-ol* 2.26 982 β-pinene tr 1176 borneol* tr 992 myrcene 0.89 1184 terpinene-4-ol 0.30 1009 α-phellandrene 11.39 1186 m-cymene-8-ol* 0.74 1015 3-carene 23.48 1190 p-cymene-8-ol 0.88 1021 α-terpinene tr 1197 a-terpineol* 0.30 1029 p-cymene 10.11 1204 cis-piperitol* tr 1033 limonene 1.00 1205 phellandrene epoxide** 3.55 1034 β-phellandrene 2.00 1208 trans-piperitol* 0.38 1037 1,8-cineol* 0.24 1218 verbenone* 0.56 1039 cis-ocimene 0.06 1222 thujenone* 0.84 1048 phenylacetaldehyde* tr 1235 phellandrene epoxide** 3.55 1049 trans-ocimeme 0.10 1249 cuminaldehyde tr 1061 artemisia ketone* tr 1262 carvotanacetone 0.18 1063 γ-terpinene 0.32 1295 thymol 0.13 1089 p-2,4(8)-menthadiene* 0.22 1303 carvacrol 0.49 1091 terpinolene 0.51 1409 methyleugenol 18.42 1094 p-iso-propenyltoluene* 0.10 1532 myristicin* 0.05 1101 linalool* 0.38 1604 caryophyllene oxide* 0.73 1104 nonanal* tr 1610 viridiflorol* 0.37

Total identified 89.59

RI = retention index; tr = traces; * = newly isolated components; ** = isomers not characterized

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DISCUSSION

In the oil of E. tenuifolia subsp. sibthorpiana (Guss.) [9] Kivanc, M. (1988) Antimicrobial activity of “Cortuk” (Echi- Tutin. fifty components were characterized representing nophora sibthorpiana (Guss.) pice, its essential oil and me- thyleugenol, Die Nahrung, 32(6), 635-637. 89.6% of the oil with 23.5% of 3-cene, 18.45 of me- α thyleugenol, 11.4% of -phellandrene, and 10.1% of p- [10] Friedman, M., Henika, P.R. and Mandrell, R.E. (2002) Bac- cymene as major constituents. Methyleugenol (4-allyl- tericidal activities of plant essential oils and some of their 1,2-dimethoxybenzene) was previously reported as one of isolated constituents against Campylobacter jejun, Es- the primary oil components [4]. In volatile oils of this cherichia coli, Listeria monocytogenes and Salmonella en- terocolitica, J. Food Protect. 65(10), 1663-1666. species collected from different regions in Turkey also the occurrence of methyleugenol (17.5%) and, additionally, [11] DeVincenzi, M., Silano, M., Stacchini, P. and Scazzochino, B. α-phellandrene (15.5%) and p-cymene (14.7%) has been (2000) Constituents of aromatic plants: I. Methyleugenol, Fi- reported [5]. In another study of essential oils from E. toterapia, 71(2), 216-221. tenuifolia samples collected in Turkey α-phellandrene (51.0%) and methyleugenol (24.7%) were found as main components [6].

Moreover, compared with all the previous reports the essential oils of E. tenuifolia subsp. sibthorpiana (Guss.) Tutin. were generally rich in monoterpenes, particularly 3-carene. Some of the newly isolated constituents (li- nalool, nonanal, limonene, artemisia ketone, α-terpineol, verbenone, myristicin and viridiflorol) are practically used as antimicrobial agents [7-10]. Methyleugenol, one of the main compounds, dominates the pleasant odour of the plants and also oils and is. therefore, used in food- stuffs as flavouring agent [11].

REFERENCES

[1] Hedge, I. C. and Lamond, J. M.(1972) Echinophora, Flora of Turkey and East Aegean Islands. vol. 4, (Edition of P.H. Davis) Edinburgh University Press, Edinburgh , 309-311.

[2] Baytop, T. (1984) Türkiye’de Bitkiler ile Tedavi. İstanbul Üniversitesi Yay. 3255, İstanbul, 210.

[3] Cocking and Middleton (1935) Steam Distillation of Plant Material, Quart. J. Pharm. Pharmacol. 8, 435-442.

[4] Tanker, N., Tanker, M., Şener, B., Svendsen, A.B. (1976) Echinophora tenuifolia L. subsp. sibthorpiana (Guss.) Tutin.‘in Uçucu Yagõnõn Gaz Kromatografisi ile Araştõrõl- masõ. Ankara Eczcõlõk Fak Dergisi 6(2), 161-180. Received for publication: October 03, 2002 Accepted for publication: January 17, 2003 [5] Başer, K. H. C., Erdemgil, F. Z. and Özek, T., (1994) Echi- nophora tenuifolia L. subsp. sibthorpiana (Guss.) Tutin. J. Essent. Oil Res., vol. 6, pp. 399-400. CORRESPONDING AUTHOR [6] Başer, K. H. C., Kürkçüoglu, M., Malyer, H. And Bicakci A. (1998) Essential oils of six Echinophora species. J. Essent. Hasan Çetin Özen Oil Res., vol.10, 345-391. Dicle University Science and Art Faculty [7] Janson, A.M., Scheffer, J.J. and Baerheim, S.A. (1987) An- Department of Biology timicrobial activity of essential oils, Planta Medica, 53(5), 21280 Diyarbakõr - TURKEY 395-398.

[8] Meeker, H.G. and Linke, H.A. (1988) The antibacterial ac- e-mail: [email protected] tion of eugenol, thyme oil and related essential oils used in dentistry, Compendium, 9(1): 32, 34-35. AFS/ Vol 24/ No 4/ 2002 – pages 170 – 172

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FATTY ACID COMPOSITIONS OF Hypericum triquetrifolium TURRA

Hasan Çetin Özen and Mehmet Başhan

Dicle University Science and Art Faculty, Department of Biology, 21280, Diyarbakõr, Turkey

SUMMARY MATERIALS AND METHODS

In this study, the fatty acid composition of Hypericum H. triquetrifolium Tura was collected in the Diyar- triquetrifolium Tura. was determined by means of GC/MS bakõr region (Turkey) in July 2002. Voucher specimens analysis. As major components α-linolenic (45.5%), are kept at the herbarium of the Science Faculty, Dicle palmitic (28.1%) and linolenic acid (12.5%) were found. University – Turkey until analysis.

Isolation and transmethylation of fatty acids: Fatty acids were isolated and transmethylated using a method de- scribed by Graces and Mancha [9]. Flowering tops of KEYWORDS: fresh plant parts (10g) were heated with a reagent contain- Hypericum tricuetrifolium, GC/MS analysis, fatty acid composition. ing methanol/heptane/2,2-dimethoxypropane/H2SO4 = 37/36/20/5/2 vol%.). At 80 °C the digestion and lipid transmethylation simultaneously took place in a single phase. After cooling to room temperature, the upper phase was collected for GC-MS analysis.

INTRODUCTION GC-MS analysis: GC-MS analysis was performed with a Fisons MD 800 mass (quadrupole)-GC 8000 series Hypericum (Guttiferae) is a genus extensively dis- instrument equipped with a flame ionization detector and tributed as herbs or shrubs, which occur widely in tem- a fused silica column (30m x 0.25mm I.D.) coated with perate regions of the world [1]. Hypericum is usually OV1. The initial temperature was 40 °C for 4 min after placed in the large family Guttiferae, which is alterna- injection, then increased to 280 °C (8°C/min) with a final tively called Clusiaceae. hold at 280 °C for 20 min. The injector and detector tem- ° ° In Turkey there are found 77 species [2] and many of peratures were maintained at 270 C and 250 C, respec- them have been used in Turkish herbal medicines, mainly tively. Helium was used as carrier gas with a flow-rate of used in the treatment of depression, hepatitis and skin 0.8 ml/min. disease, for example "Binbirdelik otu" [3].

The recent increase of interest in the chemistry of this RESULTS AND DISCUSSION genus seems to be recent and has led to the isolation of more than 100 components with different biological ac- The fatty acid compositions of H. triquetrifolium tivities. Nowadays, the purified extract of its aerial parts Tura samples are shown in Table 1. The major compo- is primarily used because of its antidepressant activity [4]. nents were α−linolenic, palmitic and linoleic acids. Low Furthermore, the anticancer [5] and antimicrobial activity concentrations of oleic, stearic, palmitoleic, myristic, and [6] of it are currently under investigation. lauric acid were also detected. As major components of Hypericum species found in previous studies were linoleic In spite of the several works on the chemical con- and palmitic acid in H. elatum, linoleic and linolenic acid stituents, very little is known about the fatty acid compo- in H. androsemum [7], palmitic, linoleic and stearic acid sition of this genus [7, 8]. in H. perforatum [8]. The qualitative fatty acid profiles of Hypericum species are very similar, but their quantitative Therefore, this work was aimed on the determination compositions are quite different. High concentrations of of the fatty acid profile of H. triquetrifolium Tura. α−linolenic acid (45.5%), an omega-3 fatty acid, have

173 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

been detected in H. triquetrifolium Tura samples com- [9] Garces, R., Mancha, M. (1993) One-Step Lipid Extraction pared to the other Hypericum sp. (7.9-29.0%). Generally, and Fatty Acid Methyl Esters Preparation from Fresh Plant Tissues, Anal. Biochem., 211,139-143. omega-3 fatty acids are effective in the treatment of de- pressions [10]. Hypericine, present in Hypericum, is a wellknown potent antidepressive agent [4]. Recent studies [10] Edwards, R., Peet, M., Shay, J., Horrobin, D. (1998) Omega – 3 Polyunsaturated Fatty Acid Levels in the Diet and in Red indicated that antidepressants plus omega-3 fatty acids as Blood Cell Membranes of Depressed Pattients, J. Affect Dis- additives enhance the effects of depression treatment [11]. ord., 48, 149-155.

TABLE 1 - Fatty acids as percentages of total fatty [11] Nemets, B., Stahl, Z., Belmaker, R.H. (2002) Addition of acid content of H.triquetrifolium Tura. (n=5). Omega – 3 Fatty Acid to Maintenance Medication Treatment for Recurrent Unipolar Depressive Disorder, Am. J. Psycia- Fatty acids % composition* try, 159, 477-479.

12:0 0.47 (0.09) 14:0 1.25 (0.05) 16:0 28.10 (0.10) 16:1 2.81 (0.06)

18:0 4.08 (0.23) 18:1 5.31 (0.27) 18:2 12.48 (0.48) 18:3n-3 45.46 (0.73)

* Mean values; ( ) standard deviations

REFERENCES

[1] Campbell, M.H., Delfosse, E.S. (1984) The Biology of Aus- tralian Weeds 13. Hypericum perforatum L., J. Aust .Inst. Agric. Sci., 50:63.

[2] Robson, N.K.B. (1967) Flora of Turkey and the East Eagean Islands. Vol. 2 (Edited by P.H. Davis) Edinburgh University Press, Edinburgh, 355-401.

[3] Baytop, T.(1984) Therapy with medicinal plants in Turkey, Istanbul University Press., Istanbul. 185-186.

[4] Stevenson, C., Edzard, E. (1999) Safety of Hypericum in Pa- tients with Depression: A comparison with Conventional An- tidepressants. Drugs, 11,125-132.

Received for publication: December 20, 2002 [5] Kamuhabwa, A.R., Agostinis, P., D’Hallewin, M., Karsan, A., Accepted for publication: January 07, 2003 De Witte, P. (2000) Photodynamic Activity of Hypericin in Human Urinary Bladder Carcinoma Cell. Anticancer Res., 20, 2579-2584. CORRESPONDING AUTHOR [6] Sokmen, A., Jones, B.M., Erturk, M. (1999) Antimicrobial Activity of Extracts from the Cell Cultures of Some Turkish Hasan Çetin Özen Medicinal Plants. Phytoter. Res., 13,355-357. Dicle University Science and Art Faculty [7] Gunstone, F.D., Taylor, G.M. (1967) Lipid Constituents of Department of Biology Hypericum androsaemum. Phytochem., 6,1297-1298. 21280 Diyarbakõr - TURKEY

[8] Girzu, M., Carnat, A.P., Chabard, J.L. (1995) Composition e-mail: [email protected] en Acides Gras de la Sommite Fleurie du Millepertuis (Hype- ricum perforatum L., Hyperaceae). OCL., 2, 317-318. AFS/ Vol 24/ No 4/ 2002 – pages 173 - 174

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SUBJECT INDEX AUTHOR INDEX

A A α-phellandrene 170 Aboaba, O.O. 148 B Adeleye, M.S. 148 Al-Amiri, H.A. 138 Bacillus. coagulans 144 Al-Hooti, S.N. 138 β-carotenes 138 Al-Othman, A. 138 C Al-Saqer, J.M. 138 3-carene 170 coagulant 148 B consumer acceptability 148 Başhan, M. 173 cookies 138 D D Dinçer, A.H. 144 Differenzierung von Hopfen- und Erdbeersorten 154 H E Hanrieder, D. 154 Echinophora tenuifolia 170 J Elektronische Nasen 154 essential oils 170 Johari, M. 138 F M fatty acid composition 173 Mansour, I.B. 138 functional foods 138 N G Nitz, S. 154 Gassensor-Arrays 154 GC/MS analysis 173 O H Özen, H.Ç. 170 Hypericum tricuetrifolium 173 Özen, H.Ç. 173 K S Kork-Taint 154 Sidhu, J.S. 138 M T methyleugenol 170 Toker, Z. 170 MOS 154 MS-Sensor 154 U N Ünlütürk, A. 144 nutritive value 138 P author-index

pan bread 138 p-cymene 170 phosphate buffer 144

Q QMB-Sensoren 154 R

red palm olein 138 S sensory quality 138 soy milk 148 T thermal resistance 144 tomato juice 144 V vitamin E 138 subject-index

176 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

SUBJECT INDEX for Advances in Food Sciences 2002

A F Adavena 04 food processing waste 79 aglycones 106 foods of animal origin 66 agricultural waste 79 Fresh egg pasta 37 albumins 20 fresh pasta 37 alcoholic fermentation 116 functional foods 138 α-phellandrene 170 fungi 121 antioxidants 24 fungicides 94 aroma 55 G Aspergillus niger 30 Aspergillus niger 121 Gassensor-Arrays 154 Aspergillus terreus 121 GC/MS analysis 173 globulins 20 B glutelins 20 Bacillus. coagulans 144 glycosides 106 β-carotenes 138 glycosidically bound volatiles 106 ß-Galactosidase 12 H B. macerans 12 herbs 55 biosorption 121 histology 73 Book reviews 41 honey 125 Book reviews 85 hop flavour 106 Book reviews 128 Humulus lupulus L. 106 C hydroperoxides 24 Hypericum tricuetrifolium 173 3-carene 170 cassava peels 94 I coagulant 148 insecticides 94 cobalt 121 colour 55 K consumer acceptability 148 KDGal aldolase 30 cookies 138 Kork-Taint 154 D L degradation 30 lactase 12 Differenzierung von Hopfen- 154 lactic acid bacteria (LAB) 04 und Erdbeersorten lead 121 DMACA reagent 73 linoleic acid 24 drying 55 M E malon-dialdehyde 24 Echinophora tenuifolia 170 manganese 121 Elektronische Nasen 154 marula 116 enzymatic hydrolysis 106 metal ions 121 Escherichia coli 66 methyleugenol 170 essential oils 170 microbial community 62 exopolysaccharides (EPSs) 04 MOS 154 MS-Sensor 154 F mukumbi 116 fatty acid composition 173 mushrooms 79 ferrous sulfate method 50 flavanols 73 N flavonoids 24 nickel 121 flavonols 73 non-dairy 04

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N W nuclei 73 wine 116 nutritional value 79 Y nutritive value 138 yeasts 116 O oats 04 subject-index off-flavour 55 oil quality assurance tests 50 oil quality improvement 50 olive fruits 99 olive leaves 99 P pan bread 138 parsley 55 p-cymene 170 Pediococcus damnosus 2.6 04 peroxidised oils 50 pH 69 Phanerochaete chrysosporium 121 phosphate buffer 144 physico-chemical analysis 125 Pleurotus sajor-caju 94 polyphenols 99 purification 30 Q QMB-Sensoren 154 quality assurance tests 99 R rancidity 99 red palm olein 138 S sausage 62 sensory quality 138 Shigella flexneri 69 solid state fermentation 79 soybean 20 soy milk 148 Staphylococcus aureus 37 sunflower oil 99 survival 69 T temperature 69 thermal resistance 144 Tokat 125 tomato juice 144 Trichoderma reesei 121 Turkey 125 V vacuum – microwave 55 valuable food protein 20 Verocytotoxigenic Escherichia coli group 66 vitamin E 138

178 © by PSP Volume 24 – No 4. 2002 Advances in Food Sciences

AUTHOR INDEX for Advances in Food Sciences 2002

A J Abdel-Fatah, O.M. 30 Johari, M. 138 Abd-Elmoien, N. M. 99 Abdou, H. M. 79 K Aboaba, O.O. 148 Khalesi, Z. 121 Adeleye, M.S. 148 Kollmannsberger, H. 106 Adenipekun, C. O. 94 Kunz, B. 55 Al-Amiri, H.A. 138 Kvesitadze, G. 20 Al-Hooti, S.N. 138 Kvesitadze, G. 24 Al-Othman, A. 138 Al-Saqer, J.M. 138 L Altieri, C. 37 La Salandra, G. 62 Altieri, C. 66 Leupold, G. 24 Leupold, G. 41 B Leupold, G. 85 Bade, M. 55 Leupold, G. 128 Başhan, M. 173 Losito, S. 69 Bassiuny, A. M. M. 50 Bauer, J. 73 M Beneduce, L. 37 Mabrouk, S.S. 12 Beneduce, L. 69 Mahmoud, E. A. 99 Betsiashvili, M. 20 Mansour, I. B. 138 Böhm, M. E. 55 Mårtensson, O. 04 Massa, S. 37 D Massa, S. 62 Dinçer, A.H. 144 Massa, S. 69 Dueñas-Chasco, M. 04 Mpofu, A. 116 Dupuy, A. 69 N E Natola, K. 62 Elshafei, A.M. 30 Nitz, S. 106 El-Shayeb, N.M.A. 12 Nitz, S. 154 Emtiazi, G. 121 Novelli, A. 37 Nutsubidze, N. 20 F Farag, R. S. 50 O Farag, R. S. 99 Öste, R. 04 Fasidi, I. O 94 Özen, H.Ç. 170 Feucht, W. 73 Özen, H.Ç. 173 G P Gigolashvili, G. 20 Parlar, H. 24 Goffredo, E. 62 Polster, J. 73 H S Habibi, M. H. 121 Sadunishvili, T. 20 Haghighipour, M. A. 121 Shalashvili, A. 24 Hanrieder, D. 154 Sheble, A.S. 12 Holst, O. 04 Sidhu, J.S. 138 Simonishvili, S. 24 I Sobieh, O. 12 Irastorza, A. 04 Spano, G. 37

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S Spano, G. 62 Spano, G. 69 Staaf, J. 04 T Tarantino, D. 69 Toker, Z. 170 Tüzen, M. 125 U Ugrekhelidze, D. 24 Ünlütürk, A. 144 Z Zambakhidze, N. 24 Zvauya, R. 116 author-index

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