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Optimization of conditions for production of Maillard reaction products inhibitory to the growth of Staphylococcus… Cruickshank, Pamela K. 1985

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OPTIMIZATION OF CONDITIONS FOR PRODUCTION OF MAILLARD REACTION PRODUCTS INHIBITORY'TO THE'GROWTH OF STAPHYLOCOCCUS AUREUS by PAMELA K. CRUICKSHANK A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in FACULTY OF GRADUATE STUDIES Department of Food Science We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE tiFNfVEflSITY OF /BRITISH COLUMBIA August 1985 < -^V © Pamela K. Cruickshank, 1985 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the The U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree that p e r m i s s i o n fo r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s for f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Food Science The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: August 1985 A b s t r a c t Simplex o p t i m i z a t i o n was used to maximize the p r o d u c t i o n of M a i l l a r d r e a c t i o n compounds which i n h i b i t the growth of Staphylococcus aureus. The simultaneous f a c t o r s h i f t and mapping procedures of the o p t i m i z a t i o n program enabled the o p t i m i z a t i o n to be completed with a minimum number of experiments. The r e a c t i o n c o n d i t i o n s l i k e l y to have the g r e a t e s t e f f e c t on the p r o d u c t i o n of these compounds were chosen as: ( 1 ) molar r a t i o of amino a c i d to sugar, (2) t o t a l c o n c e n t r a t i o n of r e a c t a n t s , (3) pH, (4) temperature and (5) time of h e a t i n g . I n h i b i t i o n of the t e s t organism was q u a n t i f i e d as the r a d i u s of no growth, by the concurrent use of the v a r i a b l e and uniform cams of the s p i r a l p l a t i n g system. Twenty nine experiments were r e q u i r e d to reach the optimum of model system A, glucose + l y s i n e , while model system B, xylose + l y s i n e , was optimized a f t e r 28 experiments. The minimum i n h i b i t o r y c o n c e n t r a t i o n (MIC) of the optimum glucose + l y s i n e r e a c t i o n mixture was 5.78 x 10"" Mg/cfu, while that of the xylose + l y s i n e r e a c t i o n mixture was 8.94 x lO'Vg/cfu. M u l t i p l e r e g r e s s i o n a n a l y s i s of the data i n d i c a t e d that pH and t o t a l c o n c e n t r a t i o n were the two most s i g n i f i c a n t f a c t o r s i n determining the i n h i b i t o r y compounds produced by the glucose + l y s i n e mixture. Molar r a t i o , temperature and time were not s i g n i f i c a n t f o r t h i s combination of r e a c t a n t s . The most s i g n i f i c a n t f a c t o r s f o r the xylose + l y s i n e i i combination were pH and temperature, whereas molar r a t i o and t o t a l c o n c e n t r a t i o n were not s i g n i f i c a n t . C a l c u l a t i o n of the c o n t r i b u t i n g p r o p o r t i o n (P^) of each v a r i a b l e to the response, supported the r e s u l t s of the r e g r e s s i o n a n a l y s i s . S e p a r a t i o n of the opt i m i z e d M a i l l a r d r e a c t i o n mixtures a c c o r d i n g to molecular weight, using u l t r a f i l t r a t i o n , f a i l e d to p r o v i d e any in f o r m a t i o n about the molecular weight range of the i n h i b i t o r y compounds. Table of Contents A b s t r a c t i i L i s t of Tables v L i s t of F i g u r e s v i i Acknowledgements ix I. INTRODUCTION 1 I I . LITERATURE REVIEW 8 A. M i c r o b i a l E f f e c t s of M a i l l a r d Reaction Products 8 B. O p t i m i z a t i o n 15 I I I . MATERIALS AND METHODS 22 A. B a c t e r i a 22 B. Browning Reaction Mixtures ...24 C. Measurement of I n h i b i t i o n 29 D. O p t i m i z a t i o n of Reaction C o n d i t i o n s 30 E. Sep a r a t i o n of M a i l l a r d Reaction Products 32 F. S t a t i s t i c a l A n a l y s i s 33 IV. RESULTS AND DISCUSSION 34 A. O p t i m i z a t i o n of I n h i b i t i o n ....34 B. Separ a t i o n of the Optimized M a i l l a r d Reaction Mixtures 54 C. M u l t i p l e Regression A n a l y s i s 54 D. General D i s c u s s i o n 64 V. CONCLUSIONS 66 VI . REFERENCES 68 iv L i s t of Tables Table Page 1. V e r t i c e s generated by the super simplex o p t i m i z a t i o n computer program (glucose + l y s i n e ) 25 2. V e r t i c e s generated by the super simplex o p t i m i z a t i o n computer program (xylose + l y s i n e ) 27 3. O r i g i n a l f a c t o r l i m i t s and s t a r t i n g simplex experiments f o r the simplex o p t i m i z a t i o n of the p r o d u c t i o n of m i c r o b i o l o g i c a l i y a c t i v e compounds from a glucose + l y s i n e (combination A) model system and a xylose + l y s i n e (combination B) model system 36 4. Second f a c t o r l i m i t s and s t a r t i n g simplex experiments for the simplex o p t i m i z a t i o n of the' p r o d u c t i o n of m i c r o b i o l o g i c a l i y a c t i v e compounds from a glucose + l y s i n e (combination A) model system and a xylose + l y s i n e (combination B) model system 37 5. F i n a l f a c t o r l i m i t s f o r the simplex o p t i m i z a t i o n of the p r o d u c t i o n of m i c r o b i o l o g i c a l i y a c t i v e compounds from a g l u c o s e + l y s i n e model system and a x y l o s e + l y s i n e model system 38 6. L i m i t s f o r new s t a r t i n g simplex e s t a b l i s h e d a f t e r the mapping procedure. 48 7. F a c t o r l e v e l s of s t a r t i n g simplex e s t a b l i s h e d a f t e r the simultaneous f a c t o r s h i f t procedure 49 8. Simultaneous f a c t o r s h i f t v e r t i c e s f o r glucose + l y s i n e (combination A) and x y l o s e + l y s i n e (combination B)...50 v 9. M u l t i p l e r e g r e s s i o n of the independent v a r i a b l e s X1 through X5 on i n h i b i t i o n (glucose + l y s i n e ) 56 10. M u l t i p l e r e g r e s s i o n of the independent v a r i a b l e s X1 through X5 on i n h i b i t i o n ( x y l o s e + l y s i n e ) 59 11. C o n t r i b u t i n g p r o p o r t i o n (Pj) of each f a c t o r toward the pr o d u c t i o n of M a i l l a r d r e a c t i o n compounds i n h i b i t o r y to S. aureus i n the optimized browning mixtures 62 v i L i s t of F i g u r e s F i g u r e Page 1. Carbonylamino r e a c t i o n 2 2. Amadori rearrangement 4 3. Standard curve of absorbance (600 nm) versus c f u per mL f o r Staphylococcus aureus 23 4. Schematic r e p r e s e n t a t i o n of s p i r a l p a t t e r n . (a) F u l l s p i r a l showing no i n h i b i t i o n of S. aureus....31 (b) T h e o r e t i c a l zone of i n h i b i t i o n of S. aureus and method of measurement 31 5. ' Maps of f a c t o r l e v e l versus response for glucose + l y s i n e . (a) Molar r a t i o (amino acid/sugar) versus response ( i n h i b i t i o n ) 41 (b) C o n c e n t r a t i o n of r e a c t a n t s versus response 41 (c) I n i t i a l pH versus response 42 (d) Reaction temperature versus response 42 (e) Reaction time versus response 43 6. Maps of f a c t o r l e v e l versus response f o r x y l o s e + l y s i n e . (a) Molar r a t i o (amino acid/sugar) versus response ( i n h i b i t i o n ) 44 (b) C o n c e n t r a t i o n of r e a c t a n t s versus response 44 (c) I n i t i a l pH versus response 45 (d) Reaction temperature versus response 45 (e) Reaction time versus response 46 v i i 7. M u l t i p l e r e g r e s s i o n models f o r glucose + l y s i n e (A) and xylose + l y s i n e (B) model systems 58 v i i i Acknowledgements My s i n c e r e g r a t i t u d e i s extended to my a d v i s o r , Dr. B.J. Skura, f o r h i s support and encouragement throughout t h i s study. I would a l s o l i k e to thank my committee members, Dr. W.D. Powrie, Dr. S. Nakai, Dr. P.M. Townsley, and Dr. J . Vanderstoep, f o r t h e i r v a l u a b l e s u g g e s t i o n s . I would a l s o l i k e to express my g r a t i t u d e to Dr. T. Aishima f o r a l l h i s time and h e l p . L a s t l y , I give thanks to my husband John, f o r h i s u n f l a g g i n g sense of humour when i t was needed most. ix I. INTRODUCTION The M a i l l a r d r e a c t i o n , o r i g i n a l l y d e s c r i b e d by L o u i s - C a m i l i e M a i l l a r d i n 1912, i s the major cause of browning i n foods which are heated or s t o r e d f o r prolonged p e r i o d s ( E s k i n et a_l. , 1971). T h i s complex s e r i e s of condensation and p o l y m e r i z a t i o n r e a c t i o n s r e s u l t s i n the formation of melanoidins, the pigments r e s p o n s i b l e f o r the brown c o l o u r . I n v e s t i g a t i o n s i n t o the composition and s t r u c t u r e of these pigments have shown a r e l a t i v e l y l a r g e degree of v a r i a b i l i t y depending on the r e a c t a n t s and the r e a c t i o n c o n d i t i o n s (Bobbio et a l . , 1981; Imasoto e_t a l . , 1981), although complete c h a r a c t e r i z a t i o n of the s t r u c t u r e remains to be determined. The M a i l l a r d r e a c t i o n i s i n i t i a t e d by a condensation r e a c t i o n , known as the carbonylamino r e a c t i o n , between the a-amino group of a p r o t e i n or amino a c i d and the c a r b o n y l group of a reducing sugar (Figure 1). Non-reducing sugars can i n t e r a c t with the a-amino groups only i f t h e i r g l y c o s i d i c bonds are c l e a v e d , thus f r e e i n g t h e i r monosaccharide components. The c a r b o n y l groups r e q u i r e d can be s u p p l i e d by a number of compounds, such as c a t e c h o l s , the v i c i n a l p o l y p h e n o l s , and a s c o r b i c a c i d (Hodge, 1953). In foods, however, the c a r b o n y l group i s most l i k e l y s u p p l i e d by reducing sugars, and i t was suggested (Stadtman, 1948; Danehy and Pigman, 1951) that the term " M a i l l a r d r e a c t i o n " be r e s e r v e d f o r those condensation r e a c t i o n s between 1 2 H-C=0 I (CHOH)n I CH 2OH RNH, RNH I CHOH I (CHOH) r I CH 2OH Aldose in aldehyde form Amino group Addition compound H 2 0 RNH I HC I (CHOH)n_1 I HC CH 2OH N-substituted glycosylamine O RN II CH I (CHOH)n I CH 2OH Schiff base Figure 1. Carbonylamlno reaction. 3 reducing sugars and amino compounds. The i n i t i a l condensation product of the carbonylamino r e a c t i o n i s a S c h i f f ' s base which c y c l i z e s to the corresponding N - s u b s t i t u t e d g l y c o s y l a m i n e . T h i s compound then undergoes "Amadori rearrangement" which i n v o l v e s a r a p i d e n o l i z a t i o n of the N - s u b s t i t u t e d glycosylamine to an N - s u b s t i t u t e d 1-amino -1-deoxy-2-ketose ( e n o l ) , which then isomerizes to i t s keto form ( F i g u r e 2). Because the e n o l i z a t i o n step i s so r a p i d , i t i s d i f f i c u l t to i s o l a t e the N - s u b s t i t u t e d glycosylamine, however the Amadori compounds ( N - s u b s t i t u t e d 1-amino-1-deoxy-2-ketoses) have been i s o l a t e d from s e v e r a l foods ( M i l l s et_ a_l. , 1969). The r e a c t i o n s l e a d i n g to the 1-amino-1-deoxy- 2-ketose d e r i v a t i v e s are a l l r e v e r s i b l e , and the compounds produced to t h i s p o i n t are c o l o u r l e s s (Hodge and Osman, 1976). Although the r e a c t i o n s l e a d i n g from the Amadori compounds to the formation of the brown pigments have not yet been completely e l u c i d a t e d , t h r e e d i s t i n c t pathways are b e l i e v e d to be o p e r a t i n g (Eskin et §_1., 1971). The f i r s t two pathways are d i r e c t l y i n v o l v e d i n pigment formation. Pathway 1 i n v o l v e s the formation of methyl d i c a r b o n y l i n t e r m e d i a t e s which then go e i t h e r d i r e c t l y to the formation of melanoidins, or to reductones and a - d i c a r b o n y l s such as pyruvaldehyde, d i a c e t y l and h y d r o x y d i a c e t y l , which then combine with amines to form m e l a n o i d i n s . In pathway 2, 3-deoxyhexosone i n t e r m e d i a t e s are formed, which e i t h e r combine with amines to form m e l a n o i d i n s , or a l t e r n a t i v e l y , 4 RNH I HC I (CHOH)n O I HC I CH 2OH N-substituted aldosylamine 4- H + RNH II CH l HCOH I (HCOH)n I CH 2OH Cation of Schiff base -H + RNH I CH I C = 0 I (HCOH), I CH 2OH 'n N-substituted 1-amino-1-deoxy-2-ketose (keto) RNH f CH II COH I (HCOH) I CH2OH n N-substituted 1-amino^ 1-deoxy-2-ketose (enol) Figure 2. Amadori rearrangement. 5 form 5-hydroxymethyl-2-furaldehyde which then a l s o r e a c t s with amines to form the brown pigments. The t h i r d pathway, known as "S t r e c k e r degradation", does not r e s u l t d i r e c t l y i n the formation of pigments, however i t produces reducing compounds which are necessary for pigment formation. At e l e v a t e d temperatures a - d i c a r b o n y l compounds, provided from pathways 1 and 2, cause the degradation of a-amino a c i d s to the next lower aldehyde (Hodge and Osman, 1976). The carbon atom l o s t as a molecule of carbon d i o x i d e o r i g i n a t e s from the c a r b o x y l group of the a-amino a c i d , not from the sugar r a d i c a l ( M a i l l a r d , 1912). In a d d i t i o n to p r o v i d i n g e s s e n t i a l reducing compounds, St r e c k e r degradation p r o v i d e s important odour and f l a v o u r - p r o d u c i n g compounds such as p y r r o l e , imidazole, p y r i d i n e and p y r a z i n e . In the f i n a l stages of browning, i n t e r m e d i a t e s undergo a l d o l condensation and p o l y m e r i z a t i o n r e a c t i o n s to form c o l o u r e d , f l o u r e s c e n t , nitrogenous polymers. V a r i o u s r e a c t i o n c o n d i t i o n s have been shown to have a s i g n i f i c a n t e f f e c t on the degree of browning (Bobbio et a l . , 1981; De F i g u e i r e d o Toledo and Bobbio, 1981; Eichner and K a r e l , 1972; Lee et a l . , 1979; Namiki and Hayashi, 1975). In g e n e r a l , increased temperature r e s u l t s i n an i n c r e a s e i n the formation of browning p r o d u c t s . Namiki and Hayashi (1975) found that novel f r e e r a d i c a l s , which are produced i n the e a r l y .stages of the. carbonylamino r e a c t i o n , developed to a much grea t e r extent at temperatures above 80°C. Other work 6 (Lee e_t a_l. , 1979), however, showed the formation of browning products reached a maximum l e v e l between 60° and 70°C, with the r a t e of formation of c a r b o n y l compounds dec r e a s i n g at temperatures exceeding 70°C. T h e r e f o r e , there may be an optimum temperature or range of temperatures f o r the formation of browning products which doesn't n e c e s s a r i l y correspond to an e v e r - i n c r e a s i n g temperature. pH a l s o p l a y s an important r o l e i n the M a i l l a r d r e a c t i o n . I t i s w e l l known that the carbonylamino r e a c t i o n i s favoured by an a l k a l i n e pH, and t h i s was s u b s t a n t i a t e d when i t was found that f r e e r a d i c a l p r o d u c t i o n i n c r e a s e d with i n c r e a s i n g pH, e s p e c i a l l y above pH 8.0 (Namiki and Hayashi, 1975). The pH of the r e a c t i o n mixture may a l s o have an e f f e c t on the p h y s i c a l and chemical p r o p e r t i e s of the melanoidins produced. Bobbio e_t a_l. (1981) found that melanoidins produced at pH 6.0 were s o l u b l e and co u l d not be p r e c i p i t a t e d by lowering the pH to 2.0, while melanoidins produced at pH 3.0 were i n s o l u b l e and c o u l d not be d i s s o l v e d by r a i s i n g the pH to 11.0. The type of r e a c t a n t s and the molar r a t i o of r e a c t a n t s are a l s o important to the degree of browning. Reducing sugars or non-reducing sugars which have been c l e a v e d to t h e i r monosaccharide components are e s s e n t i a l f o r the i n i t i a t i o n of the carbonylamino r e a c t i o n . C e r t a i n carbohydrates a l s o have a higher r e a c t i v i t y , the order g e n e r a l l y being aldopentoses>aldohexoses>disaccharides. Namiki and Hayashi (1975) n o t i c e d that the formation of f r e e 7 r a d i c a l s in a browning s o l u t i o n o c c u r r e d p r e f e r e n t i a l l y at an amino a c i d to sugar r a t i o of 2:1. The o b j e c t i v e of t h i s r e s e a r c h was to opt i m i z e the pro d u c t i o n of M a i l l a r d r e a c t i o n products which are i n h i b i t o r y t o Staphylococcus aureus , using the Simplex O p t i m i z a t i o n technique of Nakai et a l . (1984). F o l l o w i n g the o p t i m i z a t i o n procedure browned systems were f r a c t i o n a t e d a c c o r d i n g to molecular weight to i d e n t i f y the f r a c t i o n ( s ) c o n t a i n i n g the a n t i m i c r o b i a l components. I I . LITERATURE REVIEW A. MICROBIAL EFFECTS OF MAILLARD REACTION PRODUCTS The M a i l l a r d r e a c t i o n i n c l u d e s complex condensation and p o l y m e r i z a t i o n r e a c t i o n s which l e a d to the formation of an extremely wide v a r i e t y of products. In recent years some of these d i v e r s e products have been shown to possess mutagenic (Omura et a l . , 1983), as w e l l as anti—mutagenic (Molund, 1985) p r o p e r t i e s . For t h i s reason, i n t e r e s t i n the M a i l l a r d r e a c t i o n has been g r e a t l y r e j u v i n a t e d . Although t h e i r a s s o c i a t i o n with cancer i s a r e l a t i v e l y recent phenomenon, i t has been r e a l i z e d f o r some time that M a i l l a r d r e a c t i o n products (MRP) have an i n h i b i t o r y , and at times a s t i m u l a t o r y e f f e c t on the growth of c e r t a i n microorganisms. Overheated milk agar or s y n t h e t i c medium [ l a c t o s e , (NH 4) 2SO a, Na 3PO f l] were shown to be i n h i b i t o r y to Salmonella enteri t i di s ( F i s h e r and Bundt, 1928). Lewis (1930) a l s o found that s t e r i l i z a t i o n of m i c r o b i o l o g i c a l media c o n t a i n i n g reducing sugars caused those media to be unable to support the growth of c e r t a i n b a c t e r i a . He concluded that the f a i l u r e to grow was due not to a lac k of a b i l i t y to a s s i m i l a t e the sugars but i n s t e a d to b i n d i n g of e s s e n t i a l nitrogenous compounds by degradation products, probably c a r b o n y l compounds, of the reducing sugars. 8 9 Fulmer et a l . (1931) noted that s y n t h e t i c medium [NH„C1, K 2 P O „ , glucose] s t e r i l i z e d by h e a t i n g , produced b e t t e r growth of Aerobacter peclinovorum than d i d the same medium s t e r i l i z e d by f i l t r a t i o n . They found that there was a d i r e c t r e l a t i o n s h i p between the degree of c a r a m e l i z a t i o n and the degree of s t i m u l a t i o n . They a l s o found that a commercially prepared caramel and caramel prepared from glucose d i d not s t i m u l a t e growth as w e l l as the heat s t e r i l i z e d medium. D e c o l o u r i z a t i o n of the c a r a m e l i z e d medium with c h a r c o a l d i d not remove the b a c t e r i a l growth s t i m u l a n t . Escherichia col i suspended in media c o n t a i n i n g heat s t e r i l i z e d reducing sugar s o l u t i o n s was l e s s r e s i s t a n t to heat treatment (54°C f o r 8 min) than were E. coli suspended in the same media c o n t a i n i n g f i l t e r s t e r i l i z e d sugars (Baumgartner, 1938). Removal of the caramel with c h a r c o a l had only a very small e f f e c t on the t o x i c i t y of the s o l u t i o n , suggesting that the caramel was not the a c t i v e agent. H i l l and Patton (1947) found that b e t t e r growth of Streptococcus faecal is was obtained when the sugar component of the tryptophan assay medium was autoclaved s e p a r a t e l y and then combined a s e p t i c a l l y with the r e s t of the medium a f t e r c o o l i n g . L a t e r , they suggested that decreased growth of S. faecal is i n tryptophan assay medium was due to i n a c t i v a t i o n of n u t r i e n t s and not the formation of i n h i b i t o r s as a r e s u l t of the browning r e a c t i o n (Patton and H i l l , 1948). 1 0 N - D - g l u c o s y l g l y c i n e , an intermediate i n the M a i l l a r d r e a c t i o n between glucose and g l y c i n e , s t i m u l a t e d the growth of Lactobacillus gayoni (Rogers et a l . , 1953). Heated mixtures of glucose and amino a c i d s other than g l y c i n e e i t h e r s t i m u l a t e d (/3-alanine, v a l i n e , i s o l e u c i n e ) or depressed (methionine, c y s t i n e , a l a n i n e , s e r i n e ) the growth of L. gayoni (Rogers ejt a l . , 1953). D i f f i c u l t y i n i s o l a t i n g and c u l t u r i n g the fungus Phytophthora fragariae l e d to s t u d i e s to determine the cause of i t s f a i l u r e to grow on c e r t a i n media (McKeen, 1956). I t was found that heated media c o n t a i n i n g reducing sugars contained a substance t o x i c to the organism. I f non-reducing carbohydrates, with the exception of g l y c e r o l , were used i n s t e a d , or i f g l y c i n e and dextrose were a u t o c l a v e d s e p a r a t e l y , the media would support the growth of P. fragariae (McKeen, 1956). A substance b a c t e r i o t o x i c to Vibrio cholerae was produced when medium c o n t a i n i n g glucose, phosphate and nitrogenous compounds was heat s t e r i l i z e d ( F i n k e l s t e i n and Lankford, 1957). A n o n v o l a t i l e r e sidue of an ether e x t r a c t of the m a t e r i a l contained carbonyl compounds, to which the authors a t t r i b u t e d the b a c t e r i o t o x i c a c t i v i t y . The i n h i b i t o r y f a c t o r was counteracted by reducing agents, peptone or yeast e x t r a c t . In a r e l a t e d study, Lankford et a l . (1957) i n v e s t i g a t e d the e f f e c t of M a i l l a r d r e a c t i o n products on the b a c t e r i a l a s s i m i l a t i o n of c y s t ( e ) i n e . They concluded that two 11 mechanisms may have been r e s p o n s i b l e for d e s t r o y i n g c y s t i n e or r e n d e r i n g i t n u t r i t i o n a l l y u n a v a i l a b l e to Lactobacillus arabinosus and Leuconosloc mesent eroi des; namely, a M a i l l a r d type r e a c t i o n of c y s t ( e ) i n e with aldehyde degradation products of glucose, or thermal d e s t r u c t i o n of c y s t ( e ) i n e independent of glucose to form i n h i b i t o r y compounds. Va r i o u s Lactobacillus s p e c i e s showed a decreased l a g phase i n the presence of glucose (0.4M) - g l y c i n e (0.4M) mixtures heated for v a r i o u s lengths of time (Jemmali, 1969). The pH of the r e a c t i o n mixture was not c o n t r o l l e d , and the complex was c h a r a c t e r i z e d by the r e s i d u a l amount of glucose .and g l y c i n e , i t s UV a b s o r p t i o n , the f i n a l pH, and i t s reducing power. The l a g phase of Escherichia col i was inc r e a s e d in the presence of the browning r e a c t i o n mixture, although the rate of growth d u r i n g the l o g a r i t h m i c phase was not a f f e c t e d (Jemmali, 1969). E a r l i e r work by the same author (Jemmali, 1967) a l s o demonstrated that M a i l l a r d r e a c t i o n products (MRP) possess s t i m u l a t o r y a c t i v i t y f o r some organisms. The s o l u b l e f r a c t i o n of a g l u c o s e - g l y c i n e mixture heated at 90°C f o r 48h s t i m u l a t e d the a c t i v i t y of pyruvate decarboxylase and o x i d a t i v e pyruvate decarboxylase e x t r a c t e d from Aspergillus wentii. A s o l u t i o n of x y l o s e (0.3M) and amino compounds i n carbonate b u f f e r (1M, pH 10.6) heated at 120° C f o r 20 min showed a n t i m i c r o b i a l a c t i v i t y a g a i n s t Staphylococcus aureus, Bacillus s u b t i l i s , Escherichia col i , Ps eudomonas aeruginosa,^ Saccharomyces cerevisiae and Candida utiIis (Kato and 1 2 S h i b a s a k i , 1974). A n t i b a c t e r i a l a c t i v i t y i n c r e a s e d with an i n c r e a s e i n xylose c o n c e n t r a t i o n and a l s o as pH i n c r e a s e d . The a n t i m i c r o b i a l substances were separated by s o l v e n t (acetone) e x t r a c t i o n and l i q u i d chromatography. Four p h e n o l i c compounds were i s o l a t e d and i d e n t i f i e d as c a t e c h o l , 3-methylcatechol, 4-methylcatechol, and methylhydroquinone. Fungi were l e s s s e n s i t i v e to the a n t i m i c r o b i a l substances than b a c t e r i a or yeasts (Kato and Shibasaki,1974). The e f f e c t of melanoidins and other M a i l l a r d r e a c t i o n products on the v i a b i l i t y of yeast has a t t r a c t e d a f a i r amount of i n t e r e s t from the fermentation i n d u s t r y . Although most of the work has been focused on the p h y s i o l o g i c a l changes and m u l t i p l i c a t i o n of yeast i n the presence of M a i l l a r d r e a c t i o n products, one study (Shvets and Slyusarenko, 1976) was aimed at studying the mechanisms i n v o l v e d with these e f f e c t s . Since yeast c e l l s have a negative e l e c t r o k i n e t i c p o t e n t i a l , while melanoidins and caramels have a p o s i t i v e p o t e n t i a l , i t was presumed that these compounds c o u l d adsorb to yeast c e l l s and somehow i n t e r f e r e with t h e i r normal metabolic f u n c t i o n i n g . M e l a n o i d i n s produced by h e a t i n g a mixture of glucose and g l y c i n e (4:1, w:w) over a b o i l i n g water bath f o r 16 h, a f f e c t e d e thanol p r o d u c t i o n of some s t r a i n s of Saccharomyces cerevisiae, while not a f f e c t i n g o t h e r s . C a t a l a s e and i n v e r t a s e a c t i v i t i e s of y easts grown i n medium c o n t a i n i n g m e l a n o i d i n , were depressed. The formation of secondary products of fermentation, such as higher a l c o h o l s , organic 1 3 a c i d s , aldehydes and e s t e r s , was a l s o a f f e c t e d . Since the c h r o m a t i c i t y (measured as the c o e f f i c i e n t of l i g h t r e f l e c t i o n from the surface of the yeast) of the yeast c e l l s decreased a f t e r fermentation in the presence of melanoidins, the authors concluded that melanoidins adsorb to yeast c e l l s through an e l e c t r i c a l i n t e r a c t i o n . Mirna and C o r e t t i (1976) found that the growth of Micrococcus sp. and Staphylococcus aureus was i n h i b i t e d by 5-hydroxymethylfurfural, d e r i v e d from the r e a c t i o n between amino sugars and n i t r i t e i n cured meat products. H y d r o x y m e t h y l f u r f u r a l i s an intermediate of the M a i l l a r d r e a c t i o n and thus may c o n t r i b u t e to the o v e r a l l i n h i b i t o r y e f f e c t of nonenzymatic browning products. M e l a n o i d i n s are known to be e f f e c t i v e c h e l a t i n g agents (Gomyo and H o r i k o s h i , 1976; Kajimoto et a l . , 1975), i n d i c a t i n g t h i s may pl a y a r o l e i n t h e i r i n h i b i t o r y c a p a c i t y . To t e s t t h i s p o s s i b i l i t y , L e i t e e_t a l . , (1979) prepared melanoidins by he a t i n g a s o l u t i o n of glucose (1.25M) and g l y c i n e (0.66M) in c i t r a t e b u f f e r (0.05M), i n a water bath at 90°C f o r 38 h, u n t i l an absorbance reading of 0.200 was obtained with a thousand f o l d d i l u t i o n of the r e a c t i o n mixture. When added to the growth medium at a c o n c e n t r a t i o n of 1.5%, the conc e n t r a t e d melanoidin decreased the s p e c i f i c growth rate of Staphylococcus aureus by 46%. When the growth medium was d i a l y s e d a g a i n s t a s i m i l a r medium c o n t a i n i n g 3.0% melanoidin to remove the i r o n , there was a 10% decrease i n the s p e c i f i c growth r a t e , i n d i c a t i n g that 14 some mechanism other than c h e l a t i o n of i r o n was p r i m a r i l y r e s p o n s i b l e f o r the i n h i b i t o r y a c t i v i t y ( L e i t e et a l . , 1979). The _in v i v o e f f e c t s of M a i l l a r d r e a c t i o n products on the i n t e s t i n a l m i c r o f l o r a of r a t s were s t u d i e d by H o r i k o s h i et a l • (1981). Rats were administered browned s o l u t i o n , obtained by h e a t i n g g l u c o s e (1M), g l y c i n e (1M) and NaHC0 3 (0.1M) at 90°C f o r 48 h. There was no s i g n i f i c a n t d i f f e r e n c e in the numbers of e n t e r o c o c c i , s t a p h y l o c o c c i , c o l i f o r m s and C l o s t r i d i a between r a t s i n the t e s t group and those i n the c o n t r o l group. The l a c t o b a c i l l i however, showed a s i g n i f i c a n t i n c r e a s e i n growth in the t e s t group. The browning r e a c t i o n mixtures were then f r a c t i o n a t e d i n t o low (<500) and high (>500) molecular weight compounds by u l t r a f i l t r a t i o n and added to the c u l t u r e medium of l a c t o b a c i l l i i s o l a t e d from the i n t e s t i n e s of the r a t s , as w e l l as to stock c u l t u r e of Lactobacillus lactis. The low (<500) and high (>500) molecular weight f r a c t i o n s showed trends s i m i l a r , but not as marked, to that of the u n f r a c t i o n a t e d mixture, suggesting a s y n e r g i s t i c e f f e c t between the two f r a c t i o n s . Browning r e a c t i o n mixtures prepared with a r g i n i n e and xyl o s e or h i s t i d i n e and gl u c o s e were t e s t e d u n f r a c t i o n a t e d and p a r t i a l l y p u r i f i e d by d i a l y s i s , a g a i n s t pathogenic and s p o i l a g e b a c t e r i a f r e q u e n t l y found in food (Einarsson e_t a l . , (1983). I t was shown t h a t the i n h i b i t o r y e f f e c t of the M a i l l a r d r e a c t i o n products depended on the type of b a c t e r i a 1 5 as w e l l as the type of MRP. The high (>1000) molecular weight f r a c t i o n was more i n h i b i t o r y than the low (<1000) molecular weight compounds when t e s t e d with Bacillus subtil i s , Escherichia coli, and Staphylococcus aureus. S e v e r a l patents have been granted f o r a n t i m i c r o b i a l mixtures produced by the M a i l l a r d r e a c t i o n (Yazima e_t a l . , 1974; Yazima e_t a_l. , 1975a; b; c ) , however there i s no i n d i c a t i o n in the l i t e r a t u r e that these products are being produced commercially. B. OPTIMIZATION O p t i m i z a t i o n of any process i s the deter m i n a t i o n of the most e f f i c i e n t route of o b t a i n i n g a d e s i r e d outcome. The outcome to be op t i m i z e d may be an HPLC solvent system or the for m u l a t i o n of a new food product. O p t i m i z a t i o n of the process a l l o w s the f i n a l r e s u l t to be reached with the minimum of time and e f f o r t expended (Long, 1969; Yarbro and Deming, 1974). U n t i l the e a r l y 1920's experiments i n v e s t i g a t i n g the e f f e c t s of s e v e r a l f a c t o r s on a response were designed using the " o n e - f a c t o r - a t - a - t i m e " approach. T h i s e n t a i l e d v a r y i n g the l e v e l of one f a c t o r while h o l d i n g a l l the other f a c t o r s s t a t i o n a r y , and n o t i n g the e f f e c t on the response. T h i s approach i s s u i t a b l e only when the f a c t o r s do not i n t e r a c t with one another. In general however, f a c t o r s do i n t e r a c t , i n which case the "o n e - f a c t o r - a t - a - t i m e " approach w i l l not 16 always r e s u l t i n the optimum set of r e a c t i o n c o n d i t i o n s (Deming and Morgan, 1973). Box and Wilson (1951) were the f i r s t to d e s c r i b e the process whereby s e v e r a l v a r i a b l e s c o u l d be v a r i e d at the same time to a r r i v e at the optimum. The i r " e v o l u t i o n a r y o p e r a t i o n " (EVOP) methods employ a combination of f a c t o r i a l designs and r e g r e s s i o n techniques to estimate the d i r e c t i o n of steepest ascent and to l o c a t e the optimum region of a response s u r f a c e . Because the EVOP techniques were o r i g i n a l l y designed to analyze the r e s u l t s of small v a r i a t i o n s in the o p e r a t i n g c o n d i t i o n s of i n d u s t r i a l processes, a l a r g e number of measurements are r e q u i r e d to reach a s t a t i s t i c a l l y v a l i d d e c i s i o n as to the best d i r e c t i o n to move. Spendley et §_1. (1962) designed a more e f f i c i e n t procedure, the s e q u e n t i a l simplex method. T h i s technique does not r e q u i r e t r a d i t i o n a l t e s t i n g of s i g n i f i c a n c e through the r e p l i c a t i o n of o b s e r v a t i o n s , and t h e r e f o r e i s f a s t e r and simpler than methods p r e v i o u s l y put forward. A simplex i s a geometric f i g u r e made up of n+1 p o i n t s , where n i s the number of f a c t o r s . T h e r e f o r e a simplex d e f i n e d by two f a c t o r s takes the shape of a t r i a n g l e ; a t h r e e - f a c t o r simplex i s a t e t r a h e d r o n , and so on. F a c t o r i a l a n a l y s i s can be used to determine which f a c t o r s s i g n i f i c a n t l y a f f e c t the response, which in turn e s t a b l i s h e s n. I t has been shown (Morgan and Deming, 1974; Parker et a l . , 1975), however, that t h i s i s not always necessary because the i n c l u s i o n of p o t e n t i a l l y unimportant f a c t o r s 17 does not adversely a f f e c t the simplex. T h i s may be d e t r i m e n t a l , however, i f there i s a lar g e number of f a c t o r s because the number of experiments to be performed during the o p t i m i z a t i o n i n c r e a s e s e x p o n e n t i a l l y with the number of f a c t o r s . Once the number of f a c t o r s has been decided upon, upper and lower l i m i t s , which c o n f i n e the search f o r the optimum w i t h i n those boundaries, are assigned to each f a c t o r . The (n+1) v e r t i c e s , (experiments) are c a r r i e d out and the response of each i s e v a l u a t e d . Since the o b j e c t i v e of the s e q u e n t i a l simplex method i s to f o r c e the simplex to move to the r e g i o n of optimum response, v a r i o u s r u l e s govern the d e c i s i o n to be made a f t e r each vertex i s a p p r a i s e d . The o r i g i n a l (n+1) v e r t i c e s c o n s t i t u t e the " s t a r t i n g simplex", and based on the r e s u l t s of each of these v e r t i c e s , a d e c i s i o n as to which d i r e c t i o n to move can be made. The worst response of the s t a r t i n g simplex i s d i s c a r d e d and r e p l a c e d with i t s m i r r o r image across the face of the remaining p o i n t s . The response at t h i s new v e r t e x i s then e v a l u a t e d i n comparison to the responses of the remaining v e r t i c e s of the simplex. If the response of the new vertex i s the worst response, the simplex would be r e f l e c t e d back to the o r i g i n a l p o s i t i o n . The simplex would then o s c i l l a t e back and f o r t h between these two p o s i t i o n s f o r e v e r . To avoid t h i s s i t u a t i o n , the second worst response of the new simplex i s d i s c a r d e d i n s t e a d of the worst response. T h i s w i l l allow the simplex to continue toward the optimum. 18 When the optimum has been determined the r u l e s of simplex o p t i m i z a t i o n f o r c e the simplex to c i r c l e about that p o s i t i o n . At t h i s time i t may be necessary to decrease the s i z e of the simplex i n order to home i n on the optimum. There i s a lower l i m i t to the s i z e of the simplex however. If i t i s too small, indeterminate e r r o r s w i l l mask the true e f f e c t s , and the simplex w i l l f a i l to move toward the optimum. When the simplex i s i n more than two dimensions i t i s impossible to t e l l i f the p o i n t o btained i s the g l o b a l optimum or merely a l o c a l optimum. C o n f i r m a t i o n may be obtained i f the same optimum i s reached when the search i s begun from widely d i f f e r i n g regions of the v a r i a b l e s ' domains (Deming and Morgan, 1973). Another drawback of Spendley's simplex o p t i m i z a t i o n method i s the lack of p r o v i s i o n f o r a c c e l e r a t i o n . Nelder and Mead (1965) overcame t h i s and the other d i f f i c u l t y of not knowing when the optimum has been reached, with t h e i r m o d i f i c a t i o n of the o r i g i n a l simplex prodedure. The m o d i f i e d simplex method provi d e s r u l e s for r e f l e c t i o n , expansion, c o n t r a c t i o n , and massive c o n t r a c t i o n . The expansion r u l e enables the search to a c c e l e r a t e r a p i d l y toward the optimum. For ins t a n c e i f the response at the r e f l e c t i o n i s b e t t e r than the best response of the o r i g i n a l simplex, an expansion i s i n d i c a t e d and the new vertex E i s generated: E = P + 2 ( P - W ) (1) 19 where P and W are d e f i n e d as: where W = worst vertex; N = next-to-the-worst v e r t e x ; B best v e r t e x ; P = c e n t r o i d of N-B v e r t i c e s ; C w = c o n t r a c t i o n of W; C R = c o n t r a c t i o n of R; R r e f l e c t i o n ; and E = expansion. If the response at E i s b e t t e r than the response at B, the new simplex i s BNE. If the response at E i s not b e t t e r than the response at B, the expansion i s s a i d to have f a i l e d and BNR becomes the new simplex. I f the response at the r e f l e c t i o n (R) i s worse than the response at N, then the d i r e c t i o n chosen i s judged to be i n c o r r e c t and the simplex 20 i s c o n t r a c t e d . With these added f e a t u r e s the accuracy of the m o d i f i e d simplex procedure i s much more c o n t r o l l a b l e . The search i s stopped when the step s i z e becomes l e s s than a predetermined value (eg. 1% of the domain of each v a r i a b l e ) or when the d i f f e r e n c e s in response do not exceed the value of the standard d e v i a t i o n . Routh et a l . (1977) f u r t h e r m o dified the simplex procedure by i n c o r p o r a t i n g a q u a d r a t i c curve f i t t i n g to determine the vertex immediately f o l l o w i n g the r e f l e c t i o n v e r t e x to r e p l a c e the worst vertex i n the preceding simplex. Nakai (1982), however, found that the search f o r the optimum f r e q u e n t l y s t a l l e d at the boundaries when the procedures of Morgan and Deming (1974) or Routh et. a_l. (1977) were used. To overcome t h i s , he introduced a subprogram of " q u a d r a t i c , f a c t o r i a l r e g r e s s i o n a n a l y s i s " i n t o Routh's super simplex o p t i m i z a t i o n , and found that t h i s m o d i f i e d super simplex i d e n t i f i e d the optimum q u i c k l y without s t a l l i n g at the boundaries. Nakai et a l . (1984) a l s o observed that the search for the optimum moves r a p i d l y in the i n i t i a l stages , but then slows c o n s i d e r a b l y as the optimum i s approached. V i s u a l i z a t i o n of the response s u r f a c e can be very h e l p f u l in determining the general l o c a t i o n of the optimum and thus speeding up the search. However, graphic i l l u s t r a t i o n of data i n more than three dimensions i s very d i f f i c u l t . To exp e d i t e the search i n t h i s r e g i o n they developed a mapping procedure whereby the responses are p l o t t e d a g a i n s t each 21 f a c t o r , once a c e r t a i n number of experiments have been performed. F a c t o r l e v e l i s separated i n t o l a r g e and small l i m i t s which include the m a j o r i t y of data p o i n t s and the m a j o r i t y of good data p o i n t s , r e s p e c t i v e l y . These l i m i t values are then used to generate a s e r i e s of matched data which enable the i n v e s t i g a t o r to draw most probable l i n e s which i n d i c a t e where the optimum i s most l i k e l y to be l o c a t e d . A l s o d e s c r i b e d i n t h i s paper (Nakai et. a_l. , 1984) i s the simultaneous f a c t o r s h i f t program which s h i f t s the f a c t o r l e v e l 1/5 the d i s t a n c e between the present best (B) value and the t a r g e t (T) value, s t a r t i n g from the B v a l u e . A l l f a c t o r s are s h i f t e d s imultaneously and the program generates three experiments beyond the T value to c o r r e c t f o r cases where T values are i n c o r r e c t l y set too c l o s e to the B v a l u e s . The simultaneous f a c t o r s h i f t program, used i n c o n j u n c t i o n with the mapping procedure, s i g n i f i c a n t l y (p<0.0l) improved o p t i m i z a t i o n e f f i c i e n c y when compared with the Morgan and Deming (1974) simplex o p t i m i z a t i o n . I I I . MATERIALS AND METHODS A. BACTERIA Staphylococcus aureus ATCC 25923 c u l t u r e s were obtained by r e h y d r a t i n g b a c t r o l d i s k s (Difco) i n t r y p t i c soy broth (TSB) ( D i f c o , D e t r o i t , MI) and growing the c e l l s i n a 250 mL erlenmeyer f l a s k i n a shaker incubator at 37°C and 120 rpm (New Brunswick S c i e n t i f i c Co., Inc., E d i s o n , NJ). Between experiments c u l t u r e s were maintained on t r y p t i c soy agar (TSA) (BBL, R o c k v i l l e , MD) s l a n t s at 4°C, with t r a n s f e r s to f r e s h s l a n t s at two month i n t e r v a l s . A standard curve of absorbance (600nm) (Bausch and Lomb Sp e c t r o n i c 20) versus c f u per mL was prepared at the o u t s e t , to be used f o r e s t i m a t i n g c u l t u r e d e n s i t y ( F i g u r e 3). For i n d i v i d u a l experiments, c u l t u r e s were grown overnight i n a c o n t r o l l e d environment incubator shaker (37°C, 120 rpm) (New Brunswick S c i e n t i f i c Co., Inc., Edison, NJ). The c e l l s were harvested by c e n t r i f u g a t i o n (10,000 x g; 10 min; room temperature) and the broth decanted. The p e l l e t was washed with 0.1% peptone broth, c e n t r i f u g e d , and resuspended i n s t e r i l e peptone. C u l t u r e d e n s i t y was estimated from the standard curve, and a d j u s t e d to 10 5 c f u per mL with a p p r o p r i a t e d i l u t i o n s . 22 23 10°l 1 1 1 i 1 i i 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Absorbance (600 nm) F i g u r e 3. Standard curve of absorbance (600 nm) versus c f u per mL f o r Staphylococcus aureus. 24 B. BROWNING REACTION MIXTURES Model systems of a-D(+)-glucose + L - l y s i n e monohydrochloride (combination A) and D(+)-xylose + L - l y s i n e monohydrochloride (combination B) were prepared a c c o r d i n g to the c o n d i t i o n s generated by the simplex o p t i m i z a t i o n computer program (Tables 1, 2). Glucose, xylose and l y s i n e were purchased from Sigma Chemical Company (St. L o u i s , MO). Predetermined amounts of the r e a c t a n t s were d i s s o l v e d in 50 mL d i s t i l l e d water, a d j u s t e d to the a p p r o p r i a t e pH with concentrated NaOH, and r e f l u x e d i n 100 mL round bottom f l a s k s in a constant temperature water bath (Blue M E l e c t r i c Company, Blue I s l a n d , IL) f o r a f i x e d length of time. Three s t r a i g h t L i e b i g condensers (Corning Glass Works, Corning, NY) were p l a c e d i n s e r i e s to allow maximum u t i l i z a t i o n of the water bath. The round bottom f l a s k s were secured to the condensers then lowered i n t o the water. Styrofoam packing ch i p s were spread on the water surface to minimize evaporation, however, p e r i o d i c lowering of the f l a s k s was re q u i r e d to maintain the water l e v e l around the f l a s k s . The browning r e a c t i o n mixtures, a f t e r the h e a t i n g p e r i o d , were adjusted back to the s t a r t i n g pH, and s t o r e d i n foil-wrapped, screw cap g l a s s b o t t l e s at 4°C under an atmosphere of n i t r o g e n u n t i l used. 25 Table 1. V e r t i c e s generated by the super simplex o p t i m i z a t i o n computer program (glucose + l y s i n e ) , ( v e r t i c e s 1-6 = " s t a r t i n g simplex") Factor Vertex XI X2 X3 X4 X5 1 1 .00 30.00 9.00. 75.0 25.00 2 2.37 34. 10 9.20 77. 1 26.02 3 1.31 48.24 9.20 77. 1 26.02 4 1.31 34. 1 0 9.91 77. 1 26.02 5 1.31 34. 1 0 9.20 84. 1 26.02 6 • 1.31 34. 1 0 9.20 77. 1 29.56 7 2.04 43.86 9.69 81 .9 28.46 8 2.50 50.00 10.00 85.0 30.00 9 1 .00 46. 1 2 9.81 83. 1 29.03 10 1 .00 50.00 10.00 85.0 30.00 1 1 1 .66 50.00 10.00 76.3 30.00 1 2 1 .84 50.00 10.00 75 . 0 30.00 13 1 . 80 50.00 9.45 83. 1 30.00 14 1 .65 50 . 0 0 9.59 81 .3 30.00 15 1 .94 50 . 0 0 10.00 84 . 8 28.85 16 2.26 50.00 10.00 85 . 0 28.49 17 2.19 50 . 0 0 10 .00 85 . 0 30 . 0 0 Table 1. cont'd. F a c t o r Vertex XI X2 X3 X4 • X5 18 2.50 50.00 1 0.00 85.0 30.00 1 9 2.19 50.00 10.00 85.0 29.54 20 2.46 50.00 10.00 85.0 29.31 21 1 .22 50.00 10.00 81.5 29.36 22 1 .00 50.00 10.00 79.7 29.03 23 1 .00 50.00 1 0.00 79.7 29.03 24 1 .89 50.00 10.00 80.9 29.26 25 1 .22 50.00 1 0.00 84.7 29.26 26 1 .22 50.00 10.00 80.9 29.94 27 1 .42 50.00 10.00 82.4 29.55 28 1 .42 50.00 10.00 83.7 29.55 29 1 .49 50.00 10.00 82.8 29.62 X1 = molar r a t i o of amino a c i d to sugar X2 = t o t a l c o n c e n t r a t i o n of r e a c t a n t s (% w/v) X3 = i n i t i a l pH X4 = r e a c t i o n temperature (°C) X5 = r e a c t i o n time (hours) Table 2. Vert i c e s generated by the super simplex o p t i m i z a t i o n computer program ( xylose + l y s i n e ) ( v e r t i c e s 1-6 = " s t a r t i n g simplex") Factor Vertex XI X2 X3 X4 X5 1 1 .00 30.00 9.00 75.0 25.00 2 2.37 34. 1 0 9.20 77. 1 26.02 3 1.31 48.24 9.20 77. 1 26.02 4 1.31 34. 10 9.91 77. 1 26.02 5 1.31 34.10 9.20 84. 1 26.02 6 1.31 34. 10 9.20 77. 1 29.56 7 1 .00 38. 12 9.41 79. 1 27.03 8 1 .00 40. 1 3 9.51 80. 1 27.53 9 1 .48 46.27 9.81 83. 1 29.07 10 1 .74 50.00 10.00 85.0 30.00 1 1 1 .36 48.53 9.93 84.3 25.00 12 1 .38 50.00 10.00 85.0 25.00 13 1 .39 50.00 10.00 77.5 27.81 14 1 .43 50.00 10.00 75.0 28.70 15 1 .43 50.00 9.57 83 .8 28.88 16 1 .40 50.00 9.66 82. 1 28. 16 17 1 .47 47.81 10.00 85.0 29.73 Table 2. cont'd. Factor Vertex • XI X2 X3 X4 X5 18 1 .55 47.59 10.00 85.0 30.00 19 1 .99 50.00 1 0.00 84.8 29.21 20 1.81 50.00 10.00 83.9 28.90 21 1.73 49.04 9.86 85.0 28. 1 3 22 1 .88 48.55 9.79 85.0 27.84 23 1 .47 47.81 10.00 85.0 27.84 24 1 .85 46. 1 3 9.95 85.0 28.31 25 1 .56 40.70 9.95 * 85.0 28.31 26 1 .56 46. 1 3 9.81 85.0 28.31 27 1 .56 46. 1 3 9.95 85.0 29.84 28 1 .61 48.44 10.00 85.0 30.00 X1 = molar r a t i o of amino a c i d to sugar X2 = t o t a l c o n c e n t r a t i o n of r e a c t a n t s (% w/v) X3 = i n i t i a l pH X4 = r e a c t i o n temperature (°C) X5 = r e a c t i o n time (hours) 29 C. MEASUREMENT OF INHIBITION The procedure to determine the i n h i b i t o r y c a p a c i t y of the browning r e a c t i o n mixture i n v o l v e d the concurrent use of the uniform and v a r i a b l e cams of the model DU S p i r a l P l a t e r ( S p i r a l Systems, Inc., C i n c i n n a t i , OH). Agar p l a t e s were poured on a completely f l a t bench top to ensure a l e v e l agar s u r f a c e . Uneven agar s u r f a c e s can r e s u l t i n v a r i a t i o n s in sample d e p o s i t i o n by the s p i r a l p l a t e r . P l a t e s were s t o r e d in s ealed p l a s t i c bags at 4°C u n t i l used. The night before an experiment, p l a t e s were removed from the bags and allowed to stand at room temperature o v e r n i g h t to ensure adequate d r y i n g of the agar. P o o l i n g of the sample on the s u r f a c e of the p l a t e may r e s u l t i f the agar i s not s u f f i c i e n t l y dry. The suspension of S. aureus (10 5 cfu/mL) was d e p o s i t e d onto a 10 cm p l a t e of t r y p t i c soy agar u s i n g the uniform cam of the s p i r a l p l a t e r . The l i q u i d from the d e p o s i t e d 51. aureus c u l t u r e was allowed to soak i n t o the agar f o r about 10 min p r i o r to d e p o s i t i o n of the M a i l l a r d r e a c t i o n p r o d u c t s . The browning r e a c t i o n mixture was then d e p o s i t e d , with the v a r i a b l e cam, d i r e c t l y on top of the b a c t e r i a , f o l l o w i n g e x a c t l y the same s p i r a l . F o l l o w i n g d e p o s i t i o n of the b a c t e r i a l suspension and the browning r e a c t i o n mixture, the p l a t e s were incubated f o r 18 h at 37°C (GCA/Precision S c i e n t i f i c , Chicago, I L ) . I n h i b i t i o n of growth was recorded as the d i s t a n c e (mm) from the c e n t r e of the p e t r i p l a t e to the ra d i u s where growth 30 began. The f i n a l value was the average of four measurements taken at 0°, 90°, 180°, and 270 ° around the circumference of the p l a t e (Figure 4 ). D. OPTIMIZATION OF REACTION CONDITIONS a) O p t i m i z a t i o n The simplex o p t i m i z a t i o n program, w r i t t e n f o r the Sharp PC-1500 pocket computer (Sharp C o r p o r a t i o n , Osaka, Japan), generated the c o n d i t i o n s f o r each of the experiments to be performed. F a c t o r s most l i k e l y to have a s i g n i f i c a n t e f f e c t on the i n h i b i t o r y c a p a c i t y of the M a i l l a r d r e a c t i o n products were judged to be: (1) molar r a t i o of amino a c i d to sugar; (2) t o t a l c o n c e n t r a t i o n of r e a c t a n t s ; (3) pH; (4) temperature of r e a c t i o n ; (5) l e n g t h of r e a c t i o n time at the s p e c i f i e d temperature. Upper and lower l i m i t s were p l a c e d on these parameters and the computer program generated (n+1) experiments ( v e r t i c e s ) , where n i s the number of f a c t o r s . These s i x i n i t i a l experiments c o n s t i t u t e d the " s t a r t i n g simplex" (Tables 1, 2). The two o p t i m i z a t i o n s (glucose + l y s i n e ; xylose + l y s i n e ) were performed c o n c u r r e n t l y . Based on the responses ( i n h i b i t i o n of growth, mm) of the f i r s t s i x experiments, the program generated the seventh F i g u r e 4 . Schematic r e p r e s e n t a t i o n of (a) f u l l s p i r a l p a t t e r n showing no i n h i b i t i o n , and (b) t h e o r e t i c a l zone of i n h i b i t i o n and method of measurement. 32 vertex by d i s c a r d i n g the vertex g i v i n g the worst response and r e p l a c i n g i t with i t s m i r r o r image. A f t e r the v e r t i c e s of the s t a r t i n g simplex had been e v a l u a t e d only one a d d i t i o n a l response was r e q u i r e d f o r the program to move. In other words, e v a l u a t i o n of the response at vertex 7 allowed the program to generate vertex 8. T h i s process was repeated u n t i l the optimum p o i n t was reached. An o p t i m i z a t i o n i n v o l v i n g f i v e f a c t o r s g e n e r a l l y r e q u i r e s 22 to 24 experiments to reach the optimum (Nakai e_t a l . , 1984). b) Mapping A f t e r the completion of the 22nd v e r t e x a mapping procedure was employed to v i s u a l i z e movement of the data toward the optimum ( F i g u r e s 5, 6). A separate graph for each f a c t o r , showing the r e l a t i o n s h i p between f a c t o r l e v e l and response, was prepared using the mapping program of Nakai e_t §_1.(1984) and a Sharp CE-150 p r i n t e r i n t e r f a c e d with the Sharp PC-1500 computer. The simultaneous f a c t o r s h i f t program (Nakai et a l . , 1984) was then employed using the best and t a r g e t v a l u e s e s t a b l i s h e d by the mapping procedure. E. SEPARATION OF MAILLARD REACTION PRODUCTS U l t r a f i l t r a t i o n was used to f r a c t i o n a t e the o p t i m i z e d browning r e a c t i o n mixtures of glucose + l y s i n e and xylose + l y s i n e , a c c o r d i n g to molecular weight. The r e a c t i o n mixture (25 mL) was d i l u t e d to 1000 mL with d i s t i l l e d water, then 33 passed through a 0.45M f i l t e r ( M i l l i p o r e , Corp., Bedford, MA) to remove the m a j o r i t y of any b a c t e r i a which may have contaminated the mixture. The d i l u t e d mixture was then passed through a PM10 (10,000 m.w. c u t - o f f ) Amicon f i l t e r (Amicon C o r p o r a t i o n , Danvers, MA), using the Amicon u l t r a f i l t r a t i o n c e l l (model 52), at 4°C, under 50 p s i using n i t r o g e n . The r e t e n t a t e was a d j u s t e d to the o r i g i n a l c o n c e n t r a t i o n (25 mL) with s t e r i l e d i s t i l l e d water and the permeate was passed through a YM5 (5,000 m.w. c u t - o f f ) f i l t e r . The procedure was repeated with YM2 (1,000 m.w. c u t - o f f ) and YC05 (500 m.w. c u t - o f f ) f i l t e r s . The permeate of the YC05 f i l t e r was f r e e z e - d r i e d to c o n c e n t r a t e the l e s s than 500 m.w. compounds and then a d j u s t e d back to a volume of 25 ml. Each f r a c t i o n was then t e s t e d with S. aureus, using the s p i r a l p l a t i n g technique, to determine the molecular weight range of the i n h i b i t o r y compounds. F. STATISTICAL ANALYSIS The backward e l i m i n a t i o n and stepwise methods of the stepwise m u l t i p l e r e g r e s s i o n a n a l y s i s technique (Ray, 1982) were used to analyze the data with the U n i v e r s i t y of B r i t i s h Columbia Amdahl 470 V/8 computer. The c o n t r i b u t i n g p r o p o r t i o n of each v a r i a b l e to the response was c a l c u l a t e d a c c o r d i n g to the method of B a r y l k o - P i k i e l n a and M e t e l s k i (1964). IV. RESULTS AND DISCUSSION A. OPTIMIZATION OF INHIBITION To use simplex o p t i m i z a t i o n s u c c e s s f u l l y , the l e v e l s of the f a c t o r l i m i t s as w e l l as the range between those l i m i t s , should be chosen with great c a r e . Misjudgement in the choice of f a c t o r l i m i t s can r e s u l t i n f a i l u r e to l o c a t e the optimum, or i n d e t e c t i o n of a l o c a l optimum which i s wrongly p e r c e i v e d as the g l o b a l ( o v e r a l l ) optimum. S e t t i n g f a c t o r boundaries with the assurance that the true optimum i s c o n t a i n e d w i t h i n them i s d i f f i c u l t , however in those cases where the approximate l o c a t i o n of the optimum i s unknown. T h i s problem can u s u a l l y be overcome by s t a r t i n g the search with r e l a t i v e l y broad boundaries to o b t a i n some idea of the l o c a t i o n of the optimum, and then narrowing the boundaries i n order to "home i n " on the optimum. In t h i s study, however, that p a r t i c u l a r approach was u n s u c c e s s f u l due to the f a c t that S. aureus f a i l e d to respond to the small (or n o n e x i s t e n t ) changes in the q u a n t i t i e s or p o t e n c i e s of the i n h i b i t o r y compounds produced from one vertex of the s t a r t i n g simplex to the next. T h i s r e s u l t e d i n responses of zero f o r each of the s t a r t i n g simplex v e r t i c e s and the e v e n t u a l s t a l l i n g of the program. S e v e r a l p r e l i m i n a r y experiments were r e q u i r e d to determine the a p p r o p r i a t e boundary l i m i t s which would allow 34 35 the program to proceed. The o r i g i n a l f a c t o r l i m i t s with the s i x v e r t i c e s of the s t a r t i n g simplex are shown i n Table 3. Of a l l s i x p r e l i m i n a r y experiments with the glucose + l y s i n e mixture, none of the M a i l l a r d r e a c t i o n mixtures i n h i b i t e d the growth of S. aureus. With xylose + l y s i n e , only vertex 3 gave a measurable response. With no responses to i n d i c a t e the r e l a t i v e success of each vertex, the program was unable to proceed. L i m i t s f o r f a c t o r s 1 (molar r a t i o ) , 2 ( t o t a l c o n c e n t r a t i o n of r e a c t a n t s ) , 4 ( r e a c t i o n temperature), and 5 ( r e a c t i o n time) were judged to be too broad, and were narrowed to the values shown i n Table 4. S i m i l a r r e s u l t s were obtained with the second s t a r t i n g simplex, however, r e q u i r i n g the f a c t o r l i m i t s to be narrowed a t h i r d time. In a d d i t i o n to t h i s , s e v e r a l other c o n s i d e r a t i o n s were a l s o r e q u i r e d . Reaction c o n d i t i o n s were sought which would maximize browning, with the hope that the pr o d u c t i o n of a n t i m i c r o b i a l compounds would a l s o be maximized. The l i m i t s u l t i m a t e l y chosen are shown i n Table 5. The r e s u l t s of the p r e l i m i n a r y experiments i n d i c a t e d that the lower l i m i t f o r f a c t o r 1, the molar r a t i o of amino a c i d to sugar, should be approximately 1.00, t h e r e f o r e , a 1:1 molar r a t i o was e s t a b l i s h e d as the lower l i m i t f o r f a c t o r 1. The o b s e r v a t i o n that a higher r a t i o of amino a c i d to sugar optimized the pr o d u c t i o n of a n t i o x i d a t i v e M a i l l a r d compounds (Waller et a l . , 1983; F o s t e r , 1980) provided the b a s i s f o r s e t t i n g the upper l i m i t at 2.50. Since f a c t o r 1 only d i c t a t e d the r a t i o of one rea c t a n t to the other, a second f a c t o r was r e q u i r e d Table 3. O r i g i n a l f a c t o r l i m i t s and s t a r t i n g simplex experiments f o r the simplex o p t i m i z a t i o n of the p r o d u c t i o n of m i c r o b i o l o g i c a l l y a c t i v e compounds from a glucose + l y s i n e model system and a xylose + l y s i n e model system. F a c t o r XI X2 X3 X4 X5 Lower L i m i t 0.02 3.00 7.00 60.0 5.00 Upper L i m i t 50.00 90.00 10.00 95.0 48.00 Vertex 1 0 = 02 3.00 7.00 60.0 5.00 2 45.61 20.83 7.61 67.2 13.81 3 10.27 82.35 7.61 67.2 13.81 4 10.27 20.83 9.74 67.2 13.81 5 10.27 20.83 7.61 91 .9 13.81 6 10.27 20.83 7.61 67.2 44.22 X1 = molar r a t i o of amino i a c i d to sugar X2 = t o t a l c o n c e n t r a t i o n of r e a c t a n t s (% w/v) X3 = i n i t i a l pH X4 = r e a c t i o n temperature (°C) X5 = r e a c t i o n time (hours) Table 4. Second f a c t o r l i m i t s and s t a r t i n g simplex experiments f o r the simplex o p t i m i z a t i o n of the p r o d u c t i o n of m i c r o b i o l o g i c a l i y a c t i v e compounds from a glucose + l y s i n e model system and a xylose + l y s i n e model system. Factor X2 X3 X4 X5 Lower L i m i t 0.10 20.00 7.00 75.0 20. 00 Upper L i m i t 10.00 90.00 1 0.00 95.0 50. 00 Vertex 1 0.10 20.00 7.00 75.0 20. 00 2 9.13 34.35 7.61 79. 1 26. 1 5 3 2.13 83.85 7.61 79. 1 26. 1 5 4 2.13 34.35 9.74 79. 1 26. 15 5 2.13 34.35 7.61 93.2 26. 15 6 2.13 34.35 7.61 79.1 47. 36 X1 = molar r a t i o of amino a c i d to sugar X2 = t o t a l c o n c e n t r a t i o n of r e a c t a n t s (% w/v) X3 = i n i t i a l pH X4 = r e a c t i o n temperature (°C) X5 = r e a c t i o n time (hours) Table 5. F i n a l f a c t o r l i m i t s f o r the simplex o p t i m i z a t i o n of the production of m i c r o b i o l g i c a l l y a c t i v e compounds from a glucose + l y s i n e model system and a xylose + l y s i n e model system. Fa c t o r Lower L i m i t Upper L i m i t XI 1.00 2.50 X2 30.00 50.00 X3 9.00 10.00 X4 75.0 85.0 X5 25.00 30.00 X1 = molar r a t i o of amino a c i d to sugar X2 = t o t a l c o n c e n t r a t i o n of r e a c t a n t s (% w/v) X3 = i n i t i a l pH X4 = r e a c t i o n temperature (°C) X5 = r e a c t i o n time(hours) 39 to c o n t r o l the t o t a l c o n c e n t r a t i o n of r e a c t a n t s . A lower l i m i t of 30% was chosen because production of i n h i b i t o r y compounds s t i l l o c curred at t h i s c o n c e n t r a t i o n . The s o l u b i l i t y of the r e a c t a n t s was such that an upper l i m i t of 50% approached the maximum c o n c e n t r a t i o n p h y s i c a l l y p o s s i b l e . The choice of l i m i t s f o r f a c t o r 3, pH, was c o n s t r a i n e d by the f a c t that the M a i l l a r d r e a c t i o n i s favoured by an a l k a l i n e pH, but that too high a pH would have an i n h i b i t o r y e f f e c t in i t s e l f , on the growth of the microorganism. A range of 9.00 to 10.00 was u l t i m a t e l y decided on. To c o n f i r m that pH was not the s o l e cause of i n h i b i t i o n by these r e a c t i o n mixtures, one of the most i n h i b i t o r y mixtures was a d j u s t e d to n e u t r a l pH and t e s t e d with S. aureus. The amount of i n h i b i t i o n with the n e u t r a l pH mixture was 94% of that obtained with the same mixture at pH 10, i n d i c a t i n g that pH was not the main cause of the i n h i b i t i o n . At the outset of t h i s study i t was g e n e r a l l y presumed that the higher the r e a c t i o n temperature, the g r e a t e r would be the p r o d u c t i o n of i n h i b i t o r y compounds. T h i s , however, proved to be not the case. At a temperature o f approximately 93°C i n s o l u b l e compounds were formed, making f i l t r a t i o n necessary to a v o i d c l o g g i n g the s p i r a l p l a t i n g apparatus. T h i s mixture f a i l e d to i n h i b i t the growth of S. aureus. T h e r e f o r e , because of the f e a r that the i n h i b i t o r y compounds were being removed by f i l t r a t i o n , i t was decided to maintain a temperature range that prevented the i n s o l u b i l i z a t i o n of the M a i l l a r d r e a c t i o n products. The 40 l i m i t s c o n t r o l l i n g the le n g t h of time of h e a t i n g were chosen on the b a s i s of r e s u l t s from p r e l i m i n a r y experiments. Once the l i m i t s were e s t a b l i s h e d the number of experiments r e q u i r e d to optimize the p r o d u c t i o n of M a i l l a r d r e a c t i o n products i n h i b i t o r y to the growth of S. aureus, were 29 f o r glucose + l y s i n e and 28 f o r x y l o s e + l y s i n e , respect i v e l y . F o l l o w i n g execution of 22 experiments f o r each of the two sugar/amino a c i d combinations, a mapping procedure was employed i n an attempt to determine the d i r e c t i o n of movement of the data toward the optimum ( F i g u r e s 5, 6). The p l o t s of response versus f a c t o r l e v e l were used to separate the data p o i n t s i n t o s m a l l , medium, and l a r g e groupings. The small grouping contained the m a j o r i t y of good data p o i n t s , while the l a r g e grouping c o n t a i n e d a l l the data p o i n t s . The computer c a l c u l a t e d the medium grouping as the average of the entered values of the small and larg e l i m i t s . The s m a l l , medium and l a r g e l i m i t s were then used to d i v i d e the p l o t t e d data p o i n t s i n t o four groups. If data p o i n t s remained grouped together from one f a c t o r p l o t to the next, t h i s i m p l i e d that they had been o b t a i n e d under approximately the same c o n d i t i o n s with regard to those f a c t o r s . T h e r e f o r e these p o i n t s c o u l d be l i n k e d together to estimate the response s u r f a c e which would h o p e f u l l y i n d i c a t e the r e g i o n c o n t a i n i n g the optimum. The p l o t t e d data of the f i r s t 22 experiments i n d i c a t e d the g e n e r a l v i c i n i t y of the optimum. Therefore the l i m i t s on 41 (a) (b) E £ LU V) z o CL <n us cc 1.0 2.6 M O L A R RATIO (amino acid/sugar) 3 6 20 4 3 . 0 60.0 CONCENTRATION (%) F i g u r e 5 . Maps of f a c t o r l e v e l v e r s u s response f o r g l u c o s e + l y s i n e , (a) Molar r a t i o of amino a c i d t o sugar v e r s u s response ( i n h i b i t i o n ) , (b) C o n c e n t r a t i o n of r e a c t a n t s v e r s u s r e s p o n s e . 42 20 9.5 10.0 PH 20 70.7 86 .0 T E M P E R A T U R E ( C ) F i g u r e ' 5 cont'd, (c) I n i t i a l pH versus response. (d) Reaction temperature versus response. 43 20 I 28.6 30.0 TIME (H) F i g u r e 5 cont'd, (e) Reaction time versus response. 44 (b) 36 E E LU CO z o 0. CO LU CC 20 18 27. <- 121~i28 I6 2 3 i —«\-21 • 3 • 13,16 40.7 60.0 CONCENTRATION (%) F i g u r e 6. Maps of f a c t o r l e v e l versus response for x y l o s e + l y s i n e , (a) Molar r a t i o of amino a c i d to sugar v e r s u s response ( i n h i b i t i o n ) , (b) C o n c e n t r a t i o n of r e a c t a n t s v e r s u s re sponse . 45 F i g u r e 6 cont'd, (c) I n i t i a l pH versus response. (d) R e a c t i o n temperature versus response. 46 F i g u r e 6 cont'd, (e) Reaction time versus response. 47 the f a c t o r s were narrowed around that area and a new s t a r t i n g simplex was generated (Tables 6, 7),. Although i t was hoped that t h i s would cause r a p i d movement toward the optimum, the responses from the v e r t i c e s showed no s u b s t a n t i a l move toward the optimum, i n d i c a t i n g the f a i l u r e of t h i s approach. F a c t o r l e v e l s were then changed using the simultaneous f a c t o r s h i f t program. The t a r g e t (T) values, obtained from the maps of f a c t o r l e v e l versus response, and the l e v e l s c orresponding to the present best response, were used to generate the new v e r t i c e s (Table 8 ) . In the case of glucose + l y s i n e , two d i f f e r e n t experiments had given the present best response of r=33mm. Th e r e f o r e the simultaneous s h i f t p rovided two combinations (A1 and A2) of f a c t o r l e v e l s to be c a r r i e d out, the r e s u l t s of which would determine the next move. The r e s u l t of combination A2 exceeded that of A1, i n d i c a t i n g that v e r t i c e s 4, 5, 6, 7 and 8 of the simultaneous s h i f t combination 2 were to be performed. Since the simultaneous f a c t o r s h i f t program d i c t a t e s t hat experiments should be c a r r i e d out u n t i l the r e s u l t s t a r t s d e t e r i o r a t i n g , only vertex 4, which produced a lower response than the pre v i o u s best response, was r e q u i r e d . The r e s u l t of the simultaneous f a c t o r s h i f t experiment for x y l o s e + l y s i n e was l e s s than the present best response, t h e r e f o r e the o p t i m i z a t i o n was stopped at that p o i n t . A f i n a l mapping procedure confirmed that the optimum p o i n t had been reached ( F i g u r e s 5, 6). S e v e r a l v e r t i c e s gave responses Table 6. L i m i t s for new s t a r t i n g simplex e s t a b l i s h e d a f t e r the mapping procedure. Glucose + L y s i n e Xylose + Lysine Lower Upper Lower Upper Factor L i m i t L i m i t L i m i t L i m i t XI 1 .00 1 . 94 1 .47 1 .88 X2 50.00 50.00 40.12 47.80 X3 10.00 10.00 9.79 10.00 X4 79.7 85.0 85.0 85.0 X5 29.00 30.00 27.80 30.00 X1 = molar r a t i o of amino a c i d to sugar X2 = t o t a l c oncentrat ion of r e a c t a n t s (% w/v) X3 = i n t i a l pH X4 = r e a c t i o n temperature (°C) X5 = r e a c t i o n time (hours) Table 7. Factor l e v e l s of s t a r t i n g simplex e s t a b l i s h e d a f t e r the mapping procedure. F a c t o r Vertex # XI X2 X3 X4 X5 1 A 1 .00 50.00 1 0.00 79.7 29.00 B 1 .47 47.81 10.00 85.0 27.80 2 A 1 .89 50.00 1 0.00 80.9 29.30 B 1 .85 46.13 9.95 85.0 28.30 3 A 1 .22 50.00 1 0.00 84.7 29. 30 B 1 .56 40.70 9.95 85.0 28.30 4 A 1 .22 50.00 10.00 80 .9 29.90 B 1 .56 46.13 9.81 85.0 28.30 5 A B 1 .56 46. 1 3 9.95 85.0 29.80 A = glucose + l y s i n e B = x y l o s e + l y s i n e X1 = molar r a t i o of amino a c i d to sugar X2 = t o t a l c o n c e n t r a t i o n of r e a c t a n t s (% w/v) X3 = i n i t i a l pH X4 = r e a c t i o n temperature (°C) X5 = r e a c t i o n time (hours) Table 8. Simultaneous f a c t o r s h i f t v e r t i c e s f o r glucose + l y s i n e (combination A) and xylose + l y s i n e (combi nat i on B) . Fac tor Combi nation A X1 X2 X3 X4 X5 Al 1 .42 50 . 00 10.00 82.4 29.60 Shi f t 4 1 .49 50.00 10.00 82.8 29. 60 S h i f t 5 1 . 55 50.00 1 0. 00 83 . 1 29.70 S h i f t 6 1 . 62 50. 00 1 0. 00 83.4 29.70 S h i f t 7 1 .69 50.00 10.00 83.7 29.80 S h i f t 8 1 .75 50.00 10.00 84 . 1 29.90 A2 1 . 42 50.00 1 0. 00 83.7 29.60 S h i f t 4 1 .49 50.00 1 0. 00 83.4 29.60 S h i f t 5 1 .55 50.00 10.00 83 . 1 29.70 S h i f t 6 1 .62 50.00 10.00 82.8 29.70 S h i f t 7 1 . 69 50.00 1 0. 00 82.4 29.80 S h i f t 8 1 . 75 50.00 10.00 82. 1 29.90 Combinat ion B B1 1.61 48.44 10.00 85.0 30.00 S h i f t 4 1 .62 48.72 1 0. 00 85.0 30.00 S h i f t 5 1 .64 49.00 10.00 85.0 30.00 S h i f t 6 1 .66 9.28 10.00 85.0 30.00 S h i f t 7 1 .68 49.56 10.00 85.0 30.00 S h i f t 8 1 .69 49.85 10.00 85.0 30.00 X1 = molar r a t i o of amino a c i d to sugar X2 = t o t a l c o n c e n t r a t i o n of r e a c t a n t s (% w/v) X3 = i n i t i a l pH X4 = r e a c t i o n temperature (°C) X5 = r e a c t i o n time (hours) 51 of l e s s than 20 mm and t h e r e f o r e do not appear on the maps. The glucose + l y s i n e r e a c t i o n mixture (combination A) produced the most i n h i b i t o r y M a i l l a r d mixture when: the amino a c i d to sugar r a t i o was 1.42; the c o n c e n t r a t i o n of r e a c t a n t s was 50% of the t o t a l r e a c t i o n mixture; the i n i t i a l pH was 10; the r e a c t i o n temperature was 83.7°C; and the l e n g t h of r e a c t i o n time was 29.55 h. For combination B (xylose + l y s i n e ) the optimum set of r e a c t i o n c o n d i t i o n s was: amino a c i d to sugar r a t i o 1.55; c o n c e n t r a t i o n of t o t a l r e a c t a n t s 47.6%; i n i t i a l pH 10; r e a c t i o n temperature 85°C; and l e n g t h of r e a c t i o n time 30 h. The optimum set of r e a c t i o n c o n d i t i o n s f o r glucose + l y s i n e was reached at ver t e x 28, while the optimum response f o r x y l o s e + l y s i n e was obtained with vertex 18. As a r e s u l t of the mapping procedure c a r r i e d out a f t e r the f i r s t 22 experiments, f a c t o r s X2 ( c o n c e n t r a t i o n ) and X3 ( i n i t i a l pH) of glucose + l y s i n e , and f a c t o r X4 ( r e a c t i o n temperature) of xylose + l y s i n e were f i x e d at t h e i r maximum a l l o w a b l e v a l u e s . The maps of these f a c t o r s c l e a r l y i n d i c a t e d a t r e n d toward the upper l i m i t , s uggesting that the optimum p o i n t may l i e beyond t h i s boundary. I f allowed to proceed beyond the upper l i m i t , however, i t i s a l s o p o s s i b l e that the response would begin to d e t e r i o r a t e . Ashoor and Zent (1984), i n t h e i r study of the e f f e c t of pH on M a i l l a r d browning i n t e n s i t y , found that absorbance (420 nm) of browning r e a c t i o n s o l u t i o n s was maximized at pH 10. T h e r e f o r e i t may be l o g i c a l to assume that pH 10 represented 52 the optimum in t h i s study as w e l l . From the maps obtained, the a c t u a l response s u r f a c e of these f a c t o r s remains unknown. For the purposes of t h i s study, however, the knowledge that the optimum p o i n t s of these f a c t o r s were at the upper boundary was s u f f i c i e n t . The s p i r a l p l a t i n g system provided an extremely e f f i c i e n t method f o r a n a l y z i n g the i n h i b i t o r y c a p a c i t y of each browning r e a c t i o n s o l u t i o n . U t i l i z a t i o n of the uniform and v a r i a b l e cams of the s p i r a l p l a t e r to d e p o s i t the S. aureus c u l t u r e and the M a i l l a r d r e a c t i o n mixture, provided a r a p i d and r e p r o d u c i b l e technique f o r measuring i n h i b i t i o n . The s p i r a l p l a t i n g system i s a semi-automated p l a t i n g technique which g r e a t l y reduces manpower and m a t e r i a l s r e q u i r e d . The DU model i s equipped with a v a r i a b l e and a uniform cam. The v a r i a b l e cam d e p o s i t s approximately 50 M1 of sample, i n an e v e r - d e c r e a s i n g amount, from the centre to the edge of a r o t a t i n g 10 cm agar p l a t e . The d e p o s i t e d sample i s i n the form of an Archimedes s p i r a l , such that the amount of sample at a p a r t i c u l a r l o c a t i o n i s known and always the same. Continous d i l u t i o n of the M a i l l a r d r e a c t i o n mixtures of more than three orders of magnitude i s obtained from the beginning of the s p i r a l to the p o i n t where the s t y l u s l i f t s from the agar s u r f a c e at the edge of the p l a t e . The uniform cam d e p o s i t e d S. aureus at a constant r a t e along the e n t i r e l e n g t h of the s p i r a l , e f f e c t i n g no d i l u t i o n of the sample such that each u n i t area of agar to which MRP had been a p p l i e d was c h a l l e n g e d with the same number of c f u of 53 S. aureus. Using t h i s technique, the v a r y i n g p o t e n c i e s of the browning r e a c t i o n mixtures were d i r e c t l y assessed by measuring the r a d i u s , from the ce n t r e of the p l a t e , in which no growth appeared. The radius measurements of the optimum v e r t i c e s of combinations A and B were then used to c a l c u l a t e the minimum i n h i b i t o r y c o n c e n t r a t i o n s (MIC) of each of those browned s o l u t i o n s , using the formula (Anon., 1980): q = m(r)/r x W V / D V x 1/2TTV A (2) where, q = MIC Ug/cf u) , m(r) = r a d i a l r a t e of sample d e p o s i t i o n (ml/mm), r = r a d i a l p o s i t i o n from c e n t r e of p l a t e (mm), W V = m a t e r i a l weight per u n i t volume of sample (yg/ml), D V = c u l t u r e d e n s i t y ( c f u / m l ) , V A = volume of c u l t u r e d e p o s i t e d per u n i t area of p l a t e (ml/mm2). The MIC's for the optimum M a i l l a r d r e a c t i o n mixtures were 5.78 x lO'Vg/cfu f o r glucose + l y s i n e and 8.94 x lO""Mg/ c f u f o r the xylose + l y s i n e mixture. An a l t e r n a t i v e way of judging the potency of these.compounds i s to say that 5.78 mg of the glucose + l y s i n e M a i l l a r d browning mixture or 8.94 mg of the xylose + l y s i n e browning mixture w i l l i n h i b i t the growth of 10 7 S. aureus colony forming u n i t s . 54 B. SEPARATION OF THE OPTIMIZED MAILLARD REACTION MIXTURES Sep a r a t i o n of the optimum M a i l l a r d r e a c t i o n mixtures i n t o v a r i o u s molecular weight f r a c t i o n s was performed i n an attempt to i d e n t i f y the molecular weight range of the i n h i b i t o r y compound(s) . However, none of the f i v e f r a c t i o n s of the glucose + l y s i n e combination or the xylose + l y s i n e combination i n h i b i t e d S. aureus. P o o l i n g a l l f i v e f r a c t i o n s to r e s t o r e the o r i g i n a l mixture a l s o f a i l e d to i n h i b i t S. aureus. These r e s u l t s suggest that the s e p a r a t i o n procedure, i n some way, d e s t r o y e d the i n h i b i t o r y c a p a c i t y of those M a i l l a r d r e a c t i o n m i x t ures. I f the i n h i b i t o r y compounds e x i s t e d in the lower molecular weight f r a c t i o n s , as suggested by the work of Jemmali (1969) and H o r i k o s h i e_t a l . (1981), some l o s s may have occu r r e d d u r i n g the f r e e z e - d r y i n g step. A l s o , i f a s y n e r g i s t i c r e l a t i o n s h i p e x i s t s between the low molecular weight compounds and other compounds, l o s s of the low molecular weight compounds would r e s u l t i n a decrease i n a c t i v i t y . Because of the long time r e q u i r e d f o r u l t r a f i l t r a t i o n , a more r a p i d method of f r a c t i o n a t i n g the M a i l l a r d r e a c t i o n products should be developed. C. MULTIPLE REGRESSION ANALYSIS M u l t i p l e r e g r e s s i o n a n a l y s i s of the data was performed to determine the s i g n i f i c a n c e of each of the f i v e r e a c t i o n 55 c o n d i t i o n s with respect to the p r o d u c t i o n of M a i l l a r d r e a c t i o n compounds which i n h i b i t the growth of 5. aureus. The stepwise method and the backward e l i m i n a t i o n method of the m u l t i p l e r e g r e s s i o n procedure were used. The stepwise method i s a m o d i f i c a t i o n of the forward s e l e c t i o n procedure which begins with no v a r i a b l e s in the model and s e q u e n t i a l l y adds one v a r i a b l e at a time. V a r i a b l e s are added to the model on the b a s i s of t h e i r F - s t a t i s t i c s . U n l i k e the forward s e l e c t i o n procedure, however, the stepwise method does not n e c e s s a r i l y r e t a i n v a r i a b l e s once they are i n c l u d e d i n the model. A f t e r each v a r i a b l e i s added, the stepwise method examines a l l the v a r i a b l e s i n the model and d e l e t e s any v a r i a b l e t h a t does not produce a s i g n i f i c a n t F - s t a t i s t i c . The stepwise procedure ends when none of the v a r i a b l e s o u t s i d e the model has a s i g n i f i c a n t F - s t a t i s t i c and every v a r i a b l e i n the model i s s i g n i f i c a n t at the predetermined l e v e l . The backward e l i m i n a t i o n procedure begins by c a l c u l a t i n g the s t a t i s t i c s f o r the model i n c l u d i n g a l l the independent v a r i a b l e s . The v a r i a b l e showing the s m a l l e s t c o n t r i b u t i o n to the model i s d e l e t e d . V a r i a b l e s are d e l e t e d u n t i l a l l the v a r i a b l e s remaining i n the model produce F - s t a t i s t i c s s i g n i f i c a n t at the 0.05 l e v e l (or some other predetermined l e v e l ) . The r e s u l t s of the r e g r e s s i o n of the independent v a r i a b l e s on the dependent v a r i a b l e i n h i b i t i o n , f o r combination A, are shown in Table 9. Using the backward e l i m i n a t i o n method, v a r i a b l e s were e l i m i n a t e d from the model T a b l e 9 . M u l t i p l e r e g r e s s i o n of t h e Independent v a r i a b l e s X1 t h r o u g h X5 on I n h i b i t i o n ( g l u c o s e + l y s i n e ) V a r i a b l e s S o u r c e of Degrees of Sum o f S t e p R * Removed Var1 a t 1 on Freedom S q u a r e s F B Va 1 ue 0 0 . 8 4 1 None R e g r e s s 1 on 5 937 . 302 24 . 27 In t . - 8 9 . 689 E r r o r 23 177 .663 X 1 - 0 . 593 T o t a l 28 1114 965 X2 0 . 386 X3 8 . 474' X4 0 . 0O2 X5 0 . 007 1 0 . 8 3 7 • X5 R e g r e s s Ion 4 933 .335 3 0 . 8 3 In t . - 9 0 . 180 E r r o r 24 181 . 6 3 0 X1 - 0 . 583 T o t a l 28 1114 .965 X2 0 . 443 X3 8 . 851 X4 0 . 002 2 0 . 8 2 2 X4 R e g r e s s 1 o n 3 916 .717 38 . 53 In t . - 8 6 . 310 E r r o r 25 198 . 248 X1 - 0 . 434 T o t a l 28 1114 .965 X2 0 . 488 X3 9 . 367 3 0 . 8 0 8 X1 R e g r e s s 1 on 2 900 . 968 54 . 73 In t . - 8 5 . 1 13 E r r o r 26 213 .997 X2 0 . 474 T o t a l 28 1114. . 965 X3 9 . 182 A l l v a r i a b l e s 1n t h e model a r e s i g n i f i c a n t a t the 0 . 0 5 l e v e l I n t . = I n t e r c e p t 57 i n the f o l l o w i n g order: r e a c t i o n time (X5), r e a c t i o n temperature (X4), molar r a t i o (X1). Only v a r i a b l e s X2 ( t o t a l c o n c e n t r a t i o n ) and X3 ( i n i t i a l pH) were s i g n i f i c a n t at the 0.05 l e v e l . The square of the m u l t i p l e c o r r e l a t i o n c o e f f i c i e n t , R 2, was 0.84 f o r the model c o n t a i n i n g a l l f i v e independent v a r i a b l e s , and 0.81 f o r the model c o n t a i n i n g the two s i g n i f i c a n t independent v a r i a b l e s . The r e g r e s s i o n model fo r glucose + l y s i n e i s shown i n Fig u r e 7. The comparatively l a r g e r e g r e s s i o n c o e f f i c i e n t f o r v a r i a b l e X3, i n i t i a l pH, i n d i c a t e s the major e f f e c t t h i s r e a c t i o n c o n d i t i o n had on the p roduction of i n h i b i t o r y M a i l l a r d r e a c t i o n products. The la c k of s i g n i f i c a n c e of v a r i a b l e s XI, X4, and X5 was somewhat s u r p r i s i n g s i n c e i t i s known that these f a c t o r s g r e a t l y a f f e c t the degree of browning during the M a i l l a r d r e a c t i o n . I t appears, however, that these f a c t o r s have much l e s s importance in the p r o d u c t i o n of compounds a n t i m i c r o b i a l to S. aur e us . Xylose + l y s i n e was a l s o examined using the backward e l i m i n a t i o n procedure (Table 10). S t a t i s t i c s c a l c u l a t e d f o r the model i n c l u d i n g a l l the independent v a r i a b l e s i n d i c a t e d t h a t the r e g r e s s i o n c o e f f i c i e n t f o r v a r i a b l e X1, molar r a t i o , was so small as to be undetectable by the computer. T h e r e f o r e , a c c o r d i n g to the r u l e s for e l i m i n a t i o n from the model , t h i s v a r i a b l e was removed f i r s t . V a r i a b l e X2, t o t a l c o n c e n t r a t i o n , was the only other v a r i a b l e removed, l e a v i n g v a r i a b l e s X3, X4 and X5 w i t h i n the model. To v e r i f y . t h e r e g r e s s i o n c o e f f i c i e n t f o r v a r i a b l e X1, the stepwise 58 A: Y = 0.474X 2 + 9.182X3 - 85.113 (3) R 2 = 0.81 B: Y = 7.259X 3 2 + 0 .-631X, + 0.01 5X 5 2 - 1 07 . 61 2 (4) R 2 = 0.79 F i g u r e 7. M u l t i p l e r e g r e s s i o n models f o r glucose + l y s i n e (A) and xylose + l y s i n e (B) model systems. T a b l e 1 0 . M u l t i p l e r e g r e s s i o n of Independent v a r i a b l e s X1 t h r o u g h X5 on i n h i b i t i o n ( x y l o s e + l y s i n e ) V a r i a b l e s S o u r c e of Degrees of Sum of S t e p R' Removed V a M a t Ion Freedom S q u a r e s F B V a l u e 0 0 . 8 0 0 None R e g r e s s 1 on 4 66 1 .432 22 . 98 Int . - 9 6 . 0 8 1 E r r o r 23 1 6 5 . 5 3 2 X1 0 . 0 0 0 T o t a l 27 8 2 6 . 9 6 4 X2 0 . 124 X3 5 . 8 5 5 X4 0 . 5 9 7 X5 0 . 0 1 4 1 0 . 8 0 0 X 1 R e g r e s s i o n 4 66 1 .432 22 . 98 In t . - 9 6 . 0 8 1 E r r o r 23 1 6 5 . 5 3 2 X2 0 . 124 T o t a l 27 8 2 6 . 9 6 4 X3 5 . 8 5 5 X4 0 . 5 8 7 X5 0 . 0 1 4 2 0 . 7 9 0 X2 R e g r e s s i o n 3 6 5 3 . 6 7 5 3 0 . 18 Int . - 1 0 7 . 6 1 2 E r r o r 24 173 . 289 X3 7 . 259 T o t a l 27 8 2 6 . 9 6 4 X4 0 . 6 3 1 X5 0 . 0 1 5 A l l v a r i a b l e s 1n the model a r e s i g n i f i c a n t a t the 0 . 0 5 l e v e l I n t . = I n t e r c e p t . 60 r e g r e s s i o n procedure was performed on the same data. As with the backward e l i m i n a t i o n procedure, X3, X4 and X5 were the only v a r i a b l e s r e t a i n e d in the model, and the R 2 value f o r t h i s t h r e e - v a r i a b l e model was i d e n t i c a l to that computed f o r the backward e l i m i n a t i o n procedure. The r e g r e s s i o n model f o r xylose + l y s i n e i s shown i n F i g u r e 7. As with glucose + l y s i n e , the l a r g e r e g r e s s i o n c o e f f i c i e n t f o r v a r i a b l e X3 i n d i c a t e d that i t played an important r o l e i n the production of i n h i b i t o r y compounds. However, when the other v a r i a b l e s were compared, l a r g e d i f f e r e n c e s e x i s t e d between the two combinations of sugar and amino a c i d . F a c t o r X2 ( t o t a l c o n c e n t r a t i o n ) , the only f a c t o r other than pH to remain i n the model f o r the glucose + l y s i n e mixture, was not s i g n i f i c a n t enough to remain i n the model f o r combination B (xylose + l y s i n e ) . On the other hand, f a c t o r s X4 ( r e a c t i o n temperature) and X5 ( r e a c t i o n time) were r e t a i n e d by the model f o r combination B, whereas they were not s t a t i s t i c a l l y s i g n i f i c a n t i n combination A (glucose + l y s i n e ) . Although the r e g r e s s i o n c o e f f i c i e n t i s u s u a l l y regarded as a measure of the e f f e c t a p a r t i c u l a r v a r i a b l e had on the response, Y, B a r y l k o - P i k i e l n a and M e t e l s k i (1964) suggested using the c o n t r i b u t i n g p r o p o r t i o n of each independent v a r i a b l e , P j , as a measure of the v a r i a b l e s ' c o n t r i b u t i o n s to the response. P^ can be c a l c u l a t e d a c c o r d i n g to equation (5) where i s the r e g r e s s i o n c o e f f i c i e n t , s^ the standard d e v i a t i o n of each independent v a r i a b l e , and r^y the p a r t i a l c o r r e l a t i o n c o e f f i c i e n t between X^ and Y. The sum of P^ 61 equals 100 x R 2. Pi = \ai s j r i y l / I l a ^ i r i y | x 100R 2 (5) The P^ values for each of the f i v e f a c t o r s f o r glucose + l y s i n e and xylose + l y s i n e , c a l c u l a t e d f o r the model i n c l u d i n g a l l f i v e independent v a r i a b l e s , are shown i n Table 11. In the case of combination A, i n i t a l pH and t o t a l c o n c e n t r a t i o n of r e a c t a n t s c o n t r i b u t e d , by f a r , the l a r g e s t p r o p o r t i o n s to the o v e r a l l response (44.44% and 39.73%, r e s p e c t i v e l y ) . Molar r a t i o made a s m a l l negative c o n t r i b u t i o n to the response, i n d i c a t i n g t h a t an inverse r e l a t i o n s h i p e x i s t e d between t h i s f a c t o r and p r o d u c t i o n of compounds i n h i b i t o r y to S. aureus. Since the values of the molar r a t i o between amino a c i d and sugar ranged from 1.00 to 2.50, the r e s u l t s of the r e g r e s s i o n a n a l y s i s i n d i c a t e that a gr e a t e r p r o p o r t i o n of sugar, r a t h e r than amino a c i d , would have had a p o s i t i v e e f f e c t on i n c r e a s i n g i n h i b i t i o n . Time and temperature had very small e f f e c t s on the o v e r a l l response, i n d i c a t i n g that the upper and lower l i m i t s of these two f a c t o r s were such that maximum p r o d u c t i o n of i n h i b i t o r y compounds was p o s s i b l e anywhere w i t h i n those ranges. As with glucose + l y s i n e , i n i t i a l pH was one of the most i n f l u e n t i a l f a c t o r s i n determining the p r o d u c t i o n of i n h i b i t o r y compounds by x y l o s e + l y s i n e . These r e s u l t s are Table 11. C o n t r i b u t i n g p r o p o r t i o n (P^) of each f a c t o r toward the p r o d u c t i o n of M a i l l a r d r e a c t i o n compounds i n h i b i t o r y to 5. aureus i n the optimized browning mixtures. P i ( % ) F a c t o r Glucose + Lys i n e Xylose + Lys i n e X1 0.32 0.00 X2 39.73 11.79 X3 44.44 31.90 X4 0.06 35.98 X5 0.15 0.31 X1 = molar r a t i o of amino a c i d to sugar X2 = t o t a l c o n c e n t r a t i o n of r e a c t a n t s (% w/v) X3 = i n i t i a l pH X4 = r e a c t i o n temperature (°C) X5 = r e a c t i o n time (hours) 63 c o n s i s t e n t with the f i n d i n g s of Katchalsky and Sharon (1953) durin g t h e i r study of the k i n e t i c s of aldose-amino a c i d i n t e r a c t i o n s . Previous work (Katchalsky, 1941) had shown that t h i s r e a c t i o n occurs between u n d i s s o c i a t e d amino groups and a l d o s e s . If the z w i t t e r i o n i c form of the amino a c i d or p e p t i d e i s represented by HR (eg. +NH 3CH 2COO~), i t i s only the negative ion R' (eg. NH 2CH 2COCr) which can combine with the aldose in aqueous s o l u t i o n . T h e r e f o r e , s i n c e the c o n c e n t r a t i o n of the negative ion i s markedly i n f l u e n c e d by the hydrogen ion c o n c e n t r a t i o n , the importance of pH i s r e a d i l y apparent. In marked c o n t r a s t to glucose + l y s i n e , f a c t o r X2 ( t o t a l c o n c e n t r a t i o n ) of xylose + l y s i n e c o n t r i b u t e d much l e s s , to the response (11.79%) and f a c t o r X 4 ' ( r e a c t i o n temperature) c o n t r i b u t e d the l a r g e s t p r o p o r t i o n of a l l f i v e f a c t o r s (35.98%). A l s o , f a c t o r X5 ( r e a c t i o n time) was approximately twice as important i n xylose + l y s i n e than i n glucose + l y s i n e (0.31% versus 0.15%). The l a r g e v a r i a n c e between the two combinations i n the importance of t o t a l c o n c e n t r a t i o n of r e a c t a n t s , may be e x p l a i n e d by the d i f f e r e n c e i n r e a c t i v i t y of the two sugar-types. I t has been recognized f o r some time that aldopentoses have a higher degree of r e a c t i v i t y i n the M a i l l a r d r e a c t i o n than do aldohexoses, and t h e r e f o r e the t o t a l amount of xylose present i n the r e a c t i o n mixture may be of l e s s importance in determining the o v e r a l l r e a c t i o n r a t e than the amount of glucose present i n a s i m i l a r mixture. 64 The l a r g e d i f f e r e n c e i n the importance of r e a c t i o n temperature (0.06% f o r glucose + l y s i n e versus 35.98% for xylose + l y s i n e ) i s l e s s e a s i l y e x p l a i n e d . The r e s u l t s suggest t h a t , w i t h i n the temperature range of t h i s experiment, combination B, c o n t a i n i n g x y l o s e , was much more responsive to a temperature change than was combination A. Other work ( K n i p f e l e_t al. , 1983) a l s o suggests that pentoses are more temperature s e n s i t i v e than hexoses. K i n e t i c s t u d i e s , however, are r e q u i r e d to f u l l y e x p l a i n t h i s phenomenon. D. GENERAL DISCUSSION The complexity of the M a i l l a r d r e a c t i o n makes i t very d i f f i c u l t , indeed impossible at t h i s stage, to d e s c r i b e the mode of a c t i o n of these compounds on the i n h i b i t i o n of b a c t e r i a l growth. S e v e r a l authors have expounded t h e i r own t h e o r i e s . Lewis (1930) concluded that i n h i b i t i o n was due to the c o n v e r s i o n of n i t r o g e n - c o n t a i n i n g compounds i n t o forms not r e a d i l y a s s i m i l a t e d by c e r t a i n b a c t e r i a , while Baumgartner (1938) a t t r i b u t e d i t to the d i r e c t a c t i o n of a t o x i c compound produced d u r i n g s t e r i l i z a t i o n of the medium. Lankford e_t a l . (1957) expanded on Lewis' work and concluded that b i n d i n g or d e s t r u c t i o n of c y s t i n e by products of glucose degradation was the l i k e l y cause of i n h i b i t i o n . However, F i n k e l s t e i n and Lankford (1957) went on to conclude that b a c t e r i o t o x i c substances were r e s p o n s i b l e . 65 Other suggestions e x p l a i n i n g p o s s i b l e mechanisms of i n h i b i t i o n by M a i l l a r d r e a c t i o n products i n c l u d e : p h y s i c a l a d s o r p t i o n of melanoidins to b a c t e r i a l s u r f a c e s v i a opposing e l e c t r o k i n e t i c p o t e n t i a l s (Shvets and Slyusarenko, 1976) and, c h e l a t i o n of m e t a l l i c ions necessary f o r b a c t e r i a l growth (Gomyo and H o r i k o s h i , 1976; L e i t e e_t a l . , 1979). From these v a r i e d suggestions and o p i n i o n s i t i s apparent that the question of how M a i l l a r d r e a c t i o n products i n h i b i t b a c t e r i a , w i l l not be e a s i l y answered. E x t e n s i v e work toward t y i n g these many t h e o r i e s together i s r e q u i r e d . V. CONCLUSIONS Simplex o p t i m i z a t i o n proved to be an e f f i c i e n t technique f o r the o p t i m i z a t i o n of the p r o d u c t i o n of M a i l l a r d r e a c t i o n products which i n h i b i t the growth of S. aureus. However, a m i c r o b i a l system such as the one used i n t h i s study may not be as s e n s i t i v e a measure of change as the d i r e c t q u a n t i f i c a t i o n of a s p e c i f i c compound. For t h i s reason, e s t a b l i s h i n g the a p p r o p r i a t e f a c t o r l i m i t s may r e q u i r e p r e l i m i n a r y experimentation. The s p i r a l p l a t i n g system p r o v i d e d a simple, r e p r o d u c i b l e procedure f o r measuring the potency of each M a i l l a r d r e a c t i o n mixture. Quadratic and l i n e a r m u l t i p l e r e g r e s s i o n a n a l y s i s and c a l c u l a t i o n of the c o n t r i b u t i n g p r o p o r t i o n of each of the f i v e f a c t o r s were very u s e f u l i n determining the r e l a t i v e importance of each f a c t o r with respect to the p r o d u c t i o n of M a i l l a r d r e a c t i o n products which i n h i b i t the growth of Staphylococcus aureus . The two most important f a c t o r s f o r the p r o d u c t i o n of i n h i b i t o r y compounds by the glucose + l y s i n e model system were t o t a l c o n c e n t r a t i o n and i n i t i a l pH, whereas f o r the xylose + l y s i n e model system, they were i n i t i a l pH and r e a c t i o n temperature. Attempts to i d e n t i f y the molecular weight range of the i n h i b i t o r y compounds were u n s u c c e s s f u l , p o s s i b l y due to l o s s of these compounds d u r i n g the f r a c t i o n a t i o n procedure. Development of a s e p a r a t i o n procedure which allows the 66 67 i n h i b i t o r y compounds t o remain i n t a c t would be d e s i r a b l e . The M a i l l a r d r e a c t i o n model s y s t e m s s t u d i e d i n t h i s e x p e r i m e n t p r o d u c e d h i g h l y p o t e n t a n t i m i c r o b i a l compounds. F u r t h e r s t u d y may u l t i m a t e l y l e a d t o t h e use of t h e s e compounds a s n a t u r a l l y - o c c u r r i n g p r e s e r v a t i v e s i n s e l e c t e d f o o d s y s t e m s . VI. REFERENCES Anonymous. 1980. S p i r a l p l a t e computations i n the e v a l u a t i o n of m a t e r i a l i n t e r a c t i o n s with t e s t microorganisms. S p i r a l Systems Marketing, Bethesda, Maryland. 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