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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 Department  We a c c e p t t h i s  o f Food  thesis  to the r e q u i r e d  STUDIES Science  as conforming standard  THE tiFNfVEflSITY OF /BRITISH COLUMBIA August  ©  <^-V  1985  Pamela K. C r u i c k s h a n k ,  1985  In  presenting  requirements British freely that  this  thesis  f o r an  Columbia, available  scholarly  I  Library  agree  that  reference  and  extensive may  Department  or  understood  that  copying  financial  gain  shall  by  be  his or not  the  I  copying  of  granted  or  her  of  Food  Date: August  1985  of  allowed  Science  The U n i v e r s i t y of B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  by  Columbia  University shall  this  the  this without  for  of  my  It  is  thesis my  of  agree  thesis  Head  the  make i t  further  representatives.  publication be  The  study.  permission.  Department  f u l f i l m e n t of  the  for  purposes  partial  advanced degree at  for  permission  in  for  written  Abstract  Simplex production  of M a i l l a r d  and  mapping  was  aureus.  the  o p t i m i z a t i o n to  number  of  experiments.  the  greatest  The  effect as:  (1)  molar  total  concentration  and  o r g a n i s m was  q u a n t i f i e d as  spiral to  reach  while 28 of  plating  10""  s y s t e m B,  mixture  was  pH  and  total  factors the  r a d i u s of and  minimum  no  of  of  the  test  growth,  by  the  cams of  glucose  the  required +  lysine,  optimized  after  concentration  xylose  to  of  r e a c t i o n mixture the  these  (4)  e x p e r i m e n t s were  was  to  pH,  uniform  inhibitory  + lysine  was  + lysine  (MIC)  5.78  x  reaction  lO'Vg/cfu.  r e g r e s s i o n a n a l y s i s of  in determining  glucose  most  x  a  (3)  Inhibition  + lysine,  minimum  that  factor  of amino a c i d  reactants,  variable  concentration  were  the  significant  significant  the  the  Molar  for this  factors  ii  data  two  inhibitory  + l y s i n e mixture.  t i m e were not The  The  while  8.94  Multiple  the  xylose  optimum g l u c o s e Mg/cfu,  ratio  optimum of model s y s t e m A,  experiments. the  of  s y s t e m . Twenty n i n e  the  model  the  with  production  (5) t i m e of h e a t i n g .  of  the  o p t i m i z a t i o n program  completed  the  sugar,  temperature  the  inhibit  simultaneous  the  on  chosen  use  maximize  reaction conditions likely  were  concurrent  of  be  compounds (2)  to  The  procedures  enabled  have  used  r e a c t i o n compounds w h i c h  Staphylococcus  g r o w t h of shift  optimization  most  significant  compounds p r o d u c e d  ratio,  temperature  combination  for  i n d i c a t e d that  the  of  xylose  by and  reactants. +  lysine  combination total  were pH a n d t e m p e r a t u r e ,  concentration  contributing response,  Separation according to provide of  (P^)  of  each  variable  t h e r e s u l t s of t h e r e g r e s s i o n  of t h e o p t i m i z e d  to molecular  weight,  any i n f o r m a t i o n  the i n h i b i t o r y  r a t i o and  were n o t s i g n i f i c a n t . C a l c u l a t i o n  proportion  supported  whereas m o l a r  using  about  compounds.  Maillard  of the  to  analysis.  reaction  mixtures  ultrafiltration,  the molecular  the  weight  failed range  Table  of C o n t e n t s  Abstract  i i  List  of T a b l e s  List  of F i g u r e s  v v i i  Acknowledgements I. II.  ix  INTRODUCTION  1  LITERATURE REVIEW  8  A. M i c r o b i a l Products  III.  Effects  of  Maillard  Reaction 8  B. O p t i m i z a t i o n  15  MATERIALS AND METHODS  22  A. B a c t e r i a  22  B. Browning R e a c t i o n M i x t u r e s  ...24  C. Measurement o f I n h i b i t i o n D. O p t i m i z a t i o n o f R e a c t i o n  IV.  E.  Separation  of M a i l l a r d  F.  Statistical  Analysis  29 Conditions  Reaction  Products  32 33  RESULTS AND DISCUSSION  34  A. O p t i m i z a t i o n o f I n h i b i t i o n B. S e p a r a t i o n Mixtures C. M u l t i p l e D. G e n e r a l V.  30  of the O p t i m i z e d  Regression  ....34 Maillard  Reaction 54  Analysis  Discussion  54 64  CONCLUSIONS  66  VI . REFERENCES  68  iv  List  of  Tables  Table 1.  2.  3.  Page  Vertices  generated  computer  program  Vertices  program  Original  factor  the  4.  simplex  active  (combination (combination  Second  factor  the  lysine  Final  factor  Limits  optimization 25  simplex  optimization  A) B)  simplex  of  the  compounds model  experiments  production  from  system  a and  glucose  a xylose +  simplex the'  compounds  from  model  model  system  experiments  production a and  Factor  simultaneous 8.  Simultaneous (combination  +  a xylose +  system simplex  system  37 optimization  active and  a  of  the  compounds from xylose  +  a  lysine  system  levels  of  glucose  38 new  starting  simplex  established  after  mapping p r o c e d u r e . 7.  +  36  of  of m i c r o b i o l o g i c a l i y  for  of  system  starting  f o r the  model  27  starting  model  active  limits  + lysine  simplex  super  optimization  (combination  glucose  6.  B)  l i m i t s and  simplex  production  model  A)  (combination  lysine  the  optimization  microbiologicaliy  5.  by  l i m i t s and  lysine  for  super  (xylose + lysine)  microbiologicaliy lysine  the  (glucose + lysine)  generated  computer  for  by  of  factor A)  48  starting  factor  and  the  shift  simplex  established  after  procedure  shift  vertices  xylose + lysine  v  the 49  for glucose (combination  +  lysine B)...50  9.  Multiple  regression  t h r o u g h X5 10. M u l t i p l e  on  on  11. C o n t r i b u t i n g production S.  aureus  inhibition  regression  t h r o u g h X5  of  of  inhibition  the  independent  (glucose the  proportion  + lysine)  independent  (xylose  vi  variables  browning  X1 59  f a c t o r toward  of M a i l l a r d r e a c t i o n compounds  X1 56  + lysine)  ( P j ) of e a c h  i n the o p t i m i z e d  variables  inhibitory  mixtures  the to 62  List  of F i g u r e s  Figure  Page  1.  Carbonylamino  2.  Amadori  3.  Standard for  4.  reaction  rearrangement curve  Schematic (a) F u l l  4  of a b s o r b a n c e  Staphylococcus  (b)  2  (600 nm) v e r s u s  mL  aureus  23  r e p r e s e n t a t i o n of s p i r a l spiral  c f u per  showing  Theoretical  pattern.  no i n h i b i t i o n  zone  of  of S.  aureus....31  of S.  inhibition  aureus  and  method o f measurement 5. '  Maps o f f a c t o r  31  level  versus  response  for glucose  +  lysine. (a)  Molar  ratio  (amino  acid/sugar)  versus  (inhibition)  41  (b) C o n c e n t r a t i o n o f r e a c t a n t s v e r s u s (c)  6.  Initial  pH v e r s u s  versus  (e) R e a c t i o n  time  versus  response  level  versus  factor  41 42  temperature  of  response  response  (d) R e a c t i o n  Maps  response  response  42 43  response  f o r xylose +  lysine. (a)  Molar  ratio  (amino  acid/sugar)  versus  (inhibition)  44  (b) C o n c e n t r a t i o n o f r e a c t a n t s v e r s u s (c)  Initial  response  pH v e r s u s  response  response  45  (d) R e a c t i o n  temperature  versus  (e) R e a c t i o n  time  response  versus  vii  44  response  45 46  7.  Multiple xylose  regression  + lysine  models f o r g l u c o s e  (B) model  systems  vi i i  + lysine  (A) and 58  Acknowledgements My  sincere  gratitude  B.J.  Skura,  this  s t u d y . I would  Dr. W.D.  for his  Powrie,  Vanderstoep, I would Aishima  is  support also  extended  and  like  t o my a d v i s o r , D r .  encouragement  t o thank  my c o m m i t t e e  D r . S. N a k a i , D r . P.M.  Townsley,  for their  valuable suggestions.  also  t o e x p r e s s my  like  throughout  gratitude  members,  and D r . J .  to  Dr.  T.  f o r a l l h i s t i m e and h e l p .  Lastly, unflagging  I give  sense  thanks  to  of humour when  ix  my  husband  i t was needed  John, most.  for his  I.  The  Maillard  Louis-Camilie  INTRODUCTION  reaction,  Maillard  in  browning  i n foods which are  periods  (Eskin  condensation formation brown  et  and  of  1912,  1971).  polymerization  these  the  reaction  conditions  (Bobbio  complete  can  of  known  a  as  the  reducing  interact  glycosidic  supplied  with  by  a number  foods,  however,  Danehy be  and  reserved  on  the  of  in  the  for  the  composition  reactants  is initiated  or  sugar  (Figure  and large  and  the  Imasoto e_t a l . , the  structure  and  the  sugars,  and  Pigman, for  1951)  those  s u c h as  acid  the  term  condensation 1  carbonyl sugars their  freeing  their  required  can  catechols,  the  (Hodge,  i s most  the  if  groups  suggested  that  the  only  thus  group  i t was  and  groups  compounds,  carbonyl  condensation  r e a c t i o n , between  carbonyl  ascorbic  a  1). Non-reducing  cleaved,  of  by  amino a c i d  a-amino  are  polyphenols,  reducing  series  responsible  the  carbonylamino  the  bonds  vicinal  by  prolonged  results  e t a l . , 1981;  m o n o s a c c h a r i d e components. The be  complex  of  determined.  a-amino g r o u p of a p r o t e i n group  cause  for  c h a r a c t e r i z a t i o n of  Maillard reaction  reaction,  This  by  p i g m e n t s have shown a r e l a t i v e l y depending  The  major  stored  into  variability  r e m a i n s t o be  the  pigments  d e g r e e of  1981), a l t h o u g h  or  described  reactions  Investigations  of  is  heated  melanoidins,  colour.  structure  a_l. ,  originally  1953).  likely  In  supplied  (Stadtman,  1948;  "Maillard reaction" reactions  between  2  RNH I  H-C=0  CHOH  I  (CHOH)  I  RNH,  n  (CHOH)  I  r  I  CH OH 2  CH OH 2  Aldose in aldehyde form  Amino group  Addition compound  H 0 2  RN  RNH  II  I  CH  HC  I  I  (CHOH) _ n  1  I  O  (CHOH)  n  I  CH OH  HC  2  CH OH 2  N-substituted glycosylamine  Figure 1. Carbonylamlno reaction.  Schiff base  3 reducing  sugars  The  is  compounds.  condensation  a  corresponding  Schiff's  "Amadori  enolization  N-substituted  of  product  base  N-substituted  undergoes  rapid  amino  initial  reaction  then  and  of  which  to  its  enolization  step  carbonylamino  cyclizes  glycosylamine.  rearrangement"  to  This  which  keto  i s so  form  rapid,  (enol),  (Figure  2).  it is difficult  to  isolate  (N-substituted  1 - a m i n o - 1 - d e o x y - 2 - k e t o s e s ) have been  from  foods  to the  reversible, colourless  and  the  (Hodge and the  compounds t o t h e  t o be  compounds Osman,  formation  operating  formation  which  go or t o  pyruvaldehyde,  leading  the  from  brown  ( E s k i n e t §_1.,  of m e t h y l  diacetyl to  and form  intermediates  combine w i t h amines t o form  the  isolated reactions are a l l  point  the  pigments  are  and  to  Amadori have  pathways  1971). The  first  formation.  dicarbonyl  directly  reductones  combine w i t h amines 3-deoxyhexosone  of  either  the  1976).  i n v o l v e d i n pigment  1 i n v o l v e s the  melanoidins,  to t h i s  an  compounds  derivatives  elucidated, three d i s t i n c t  pathways a r e d i r e c t l y  then  1969). The  produced  reactions  been c o m p l e t e l y  believed  a_l. ,  1-amino-1-deoxy- 2 - k e t o s e  Although  yet  et_  a  then  Because  however t h e Amadori  (Mills  to  which  glycosylamine,  leading  compound  involves  N-substituted  several  the  the 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  1-amino - 1 - d e o x y - 2 - k e t o s e  isomerizes  the  formation  a-dicarbonyls  such  which  melanoidins.  pathway  melanoidins,  or  two  intermediates  the  formed,  are  Pathway  hydroxydiacetyl,  are  not  In  which  of as then 2,  either  alternatively,  4  RNH  RNH  II  I  HC  4-  I  (CHOH)  n  H+  O  CH l HCOH I  I  HC  (HCOH)  n  I  I  CH OH 2  N-substituted aldosylamine  CH OH 2  Cation of Schiff base  -H +  RNH I CH I C=0 I  (HCOH), 'n I  CH OH 2  N-substituted 1-amino1-deoxy-2-ketose (keto)  Figure 2. Amadori rearrangement.  RNH f CH II COH I (HCOH) n I CH OH 2  N-substituted 1-amino^ 1-deoxy-2-ketose (enol)  5 form  5-hydroxymethyl-2-furaldehyde  w i t h a m i n e s t o form t h e brown  does  third  pathway,  known  not  result  directly  in  for  it  produces  pigment  degradation  of a-amino a c i d s  and Osman,  carbon dioxide  a-amino In  acid,  addition  Strecker  not to  degradation  and  De  Karel,  1972;  formation  the  from  and next  radical  essential  provides such  a-dicarbonyl  2,  cause  lower  the c a r b o x y l  the sugar  and  aldehyde  group  of  (Maillard,  important as  the  as a m o l e c u l e  reducing  the  1912).  compounds, odour  pyrrole,  and  imidazole,  Figueiredo Lee  the degree Toledo  novel  1979;  temperature  of b r o w n i n g  that  c o n d i t i o n s have been  et a l . ,  increased  greater  reactions  undergo to  form  nitrogenous polymers.  reaction on  intermediates  polymerization  of b r o w n i n g  and  Bobbio,  Namiki results  and  free  radicals,  E i c h n e r and 1975).  In  increase  i n the  Hayashi  (1975)  are produced  reaction,  extent at temperatures  (Bobbio et a l . ,  Hayashi,  and  which  shown t o have a  1981;  i n an  p r o d u c t s . Namiki  e a r l y .stages of the. c a r b o n y l a m i n o much  1  pigments,  are necessary  temperatures  s t a g e s of b r o w n i n g ,  effect  1981;  of  pyrazine.  Various  general,  formation  c a r b o n atom l o s t  compounds  flourescent,  significant  to  The  providing  condensation  coloured,  found  from  reacts  "Strecker degradation",  pathways  originates  In t h e f i n a l aldol  from  1976).  flavour-producing pyridine  the  f o r m a t i o n . At e l e v a t e d provided  of  as  r e d u c i n g compounds w h i c h  compounds,  (Hodge  then a l s o  pigments.  The  however  which  i n the  developed  above 8 0 ° C . O t h e r  to  a  work  6 (Lee  e_t  browning 70°C,  a_l. ,  products  with  decreasing may  be  the  reached rate  formation exceeding  also  plays  It  with  was  found  increasing  Hayashi, effect  produced.  melanoidins produced precipitated produced  The are  by  the carbonylamino  reaction  this  above  was  lowering  pH  8.0  (Namiki  and  mixture  may a l s o  have  properties  a _ l . (1981)  of  found  the that  t h e pH t o 2.0, w h i l e  melanoidins  and c o u l d n o t be d i s s o l v e d  t h e pH t o 11.0.  important  to  monosaccharide  initiation  of  carbohydrates generally  substantiated  production increased  chemical e_t  in  a t pH 6.0 were s o l u b l e a n d c o u l d n o t be  sugars or non-reducing  Namiki  Bobbio  necessarily  Maillard  t y p e o f r e a c t a n t s and t h e m o l a r  also  their  and  there  of temperatures f o r  role  radical  a t pH 3.0 were i n s o l u b l e  raising  o f c a r b o n y l compounds  The pH of t h e r e a c t i o n  on t h e p h y s i c a l  melanoidins  free  and  the  pH, and  that  60°  of  temperature.  known t h a t  pH, e s p e c i a l l y  1975).  between  70°C. T h e r e f o r e ,  important  f a v o u r e d by an a l k a l i n e i t  formation  p r o d u c t s which doesn't  an  i s well  the  o r range  t o an e v e r - i n c r e a s i n g  reaction.  by  of  f o r m a t i o n o f browning  when  showed  a maximum l e v e l  an optimum t e m p e r a t u r e  pH  an  the  however,  a t temperatures  correspond  is  1979),  being  and H a y a s h i  degree  sugars which components  the  also  the  a  of  of browning. have  are  carbonylamino  have  ratio  higher  been  reactants Reducing  cleaved  essential  to  f o r the  reaction.  Certain  reactivity,  the order  aldopentoses>aldohexoses>disaccharides. (1975) n o t i c e d  that  the formation of f r e e  7  radicals an  in  a browning  amino a c i d The  t o sugar  objective  solution  of  this  of  Maillard  inhibitory  to  Staphylococcus  according containing  research  reaction  procedure  to molecular  the a n t i m i c r o b i a l  ,  using  which  to  systems identify  components.  were the  are  the Simplex  e t a l . (1984). F o l l o w i n g  browned weight  was t o o p t i m i z e t h e  products  aureus  t e c h n i q u e of Nakai  optimization  p r e f e r e n t i a l l y at  r a t i o o f 2:1.  production  Optimization  occurred  the  fractionated fraction(s)  II.  LITERATURE REVIEW  A. MICROBIAL EFFECTS OF  The  Maillard  polymerization extremely these  MAILLARD REACTION PRODUCTS  reaction  reactions  wide v a r i e t y  which  have been  (Omura e t a l . , 1983), as properties.  reaction  has  association it  has  been  products  with  well  this  realized  Overheated  on  agar  interest  Maillard  2  a  3  enteri  t i di s  found  that  reducing  sterilization sugars  the growth failure  to  assimilate nitrogenous carbonyl  and  of  certain  the  was  sugars  compounds  1928).  t h o s e media bacteria. due  but by  compounds, of t h e  not  instead  a  [lactose, Salmonella  to  (1930)  unable  a  lack  to  binding  also  to  support  that  of a b i l i t y of  products,  sugars.  times  media c o n t a i n i n g  to  reducing  8  medium  concluded  degradation  reaction  at  Lewis  t o be He  their  microorganisms.  inhibitory  of m i c r o b i o l o g i c a l  caused  grow  and  synthetic  Bundt,  Although  Maillard  inhibitory,  fl  (Fisher  i n the  r e c e n t phenomenon,  that  ( N H ) S O , N a P O ] were shown t o be 4  of  (Molund,  rejuvinated.  or  some  an  anti—mutagenic  as  t h e g r o w t h of c e r t a i n  milk  f o r m a t i o n of  mutagenic  f o r some t i m e an  and  shown t o p o s s e s s  is a relatively  have  effect  t o the  In r e c e n t y e a r s  reason,  greatly cancer  (MRP)  stimulatory  For  been  lead  of p r o d u c t s .  diverse products  1985)  i n c l u d e s complex c o n d e n s a t i o n  the to  essential probably  9  Fulmer  et  al.  (1931)  [NH„C1,  K PO„,  glucose]  better  growth  o f Aerobacter  2  medium s t e r i l i z e d direct the  noted  that  sterilized  by  by f i l t r a t i o n . They  of  commercially glucose d i d sterilized  stimulation.  prepared not  heating,  peclinovorum  They  caramel  stimulate  sterilized heat in  col i  as  well  the  same  media  (Baumgartner, had  only  solution,  a  in  solutions  containing  1938). very  Removal  small  suggesting  that  was  less  from  as  heat  the  resistant coli  on  the  caramel  to  suspended  sterilized  of the caramel  the  prepared  media c o n t a i n i n g h e a t  filter  effect  a  growth s t i m u l a n t .  (54°C f o r 8 min) than were E.  treatment  that  of t h e c a r a m e l i z e d medium  suspended  r e d u c i n g sugar  t h e r e was a  found  w i t h c h a r c o a l d i d not remove t h e b a c t e r i a l Escherichia  produced  d i d t h e same  that  caramel  growth  medium  of c a r a m e l i z a t i o n and  also  and  medium. D e c o l o u r i z a t i o n  than  found  r e l a t i o n s h i p between t h e d e g r e e  degree  synthetic  sugars  with charcoal  toxicity  of  the  was n o t t h e a c t i v e  agent. Hill  and  Streptococcus of  Patton  faecal  is  (1947)  found  that  better  growth o f  was o b t a i n e d when t h e s u g a r  component  t h e t r y p t o p h a n a s s a y medium was a u t o c l a v e d s e p a r a t e l y and  then combined cooling. faecal  is  aseptically  Later,  they  with the rest  suggested  i n tryptophan assay  of  nutrients  of  t h e browning  that  of t h e medium  decreased  medium was due t o  growth o f S. inactivation  and n o t t h e f o r m a t i o n o f i n h i b i t o r s reaction  ( P a t t o n and H i l l ,  after  1948).  as a  result  1 0  N-D-glucosylglycine, reaction of  between g l u c o s e  Lactobacillus  mixtures either  L.  glucose  stimulated  Phytophthora of  its  was  found  contained  instead, separately, fragariae A  with  the  when  nitrogenous Lankford,  certain  to the  culturing  glycine  media  of  and  would  In a r e l a t e d effect  assimilation  of of  growth  If  the  1956). I t sugars  were  were  the  cause  non-reducing  glycerol,  support  fungus  reducing  dextrose  used  autoclaved  growth  heat  Vibrio  to  containing  of  P.  cholerae  was  phosphate  and  (Finkelstein  and  glucose,  sterilized  A nonvolatile  attributed  p e p t o n e or y e a s t  or  1956).  medium  factor  glycine  the  (McKeen,  organism.  exception  compounds was  inhibitory  than  Heated  isoleucine)  containing  bacteriotoxic  1957).  other  r e s i d u e of an  t h e m a t e r i a l c o n t a i n e d c a r b o n y l compounds,  authors  the  toxic  media  substance  and  media  the  if  (McKeen,  produced  of  t o grow on heated  or  acids  growth  1953).  l e d to s t u d i e s to determine  a substance  carbohydrates,  Maillard  a l a n i n e , s e r i n e ) the  isolating  fragariae  that  al.,  valine,  cystine,  the  ejt a l . , 1953).  in  failure  in  s t i m u l a t e d the  et  amino  (/3-alanine,  (Rogers  Difficulty  glycine,  (Rogers  and  (methionine,  gayoni  intermediate  and  gayoni  of  depressed of  an  the was  bacteriotoxic  counteracted  by  ether e x t r a c t to  which  activity. reducing  the The  agents,  extract. study, Maillard  Lankford  e t a l . (1957)  reaction  cyst(e)ine.  They  products  on  concluded  investigated the  bacterial that  two  11  mechanisms may have been or  rendering  arabinosus  type  responsible for destroying  i tn u t r i t i o n a l l y  and Leuconosloc  reaction  of  cyst(e)ine  o f g l u c o s e t o form  V a r i o u s Lactobacillus phase  in  the  presence  heated  The  of t h e r e a c t i o n  complex .and  reducing  although not  author  i t s UV The  the rate  by t h e r e s i d u a l the  1969). also  (MRP)  The s o l u b l e at  Earlier  possess fraction  of x y l o s e  buffer  final  and t h e glucose  pH, a n d i t s  reaction  work  a  1969).  col i  was  mixture, p h a s e was  by t h e same  that  Maillard  activity for  glucose-glycine  pyruvate  (0.3M)  activity  and  amino  compounds  col i , and  Ps eudomonas  Candida  in  a t 120° C f o r 20 min  a g a i n s t Staphylococcus  Escherichia  cerevisiae  decarboxylase  wentii.  (1M, pH 10.6) h e a t e d  showed a n t i m i c r o b i a l subtilis,  of  (0.4M)  (Jemmali,  amount o f  lag  90°C f o r 48h s t i m u l a t e d t h e a c t i v i t y o f  from Aspergillus  Saccharomyces  decreased  stimulatory  d e c a r b o x y l a s e and o x i d a t i v e  Bacillus  a  demonstrated  pyruvate  carbonate  cyst(e)ine  compounds.  of t h e browning  heated  A solution  of  Escherichia  of  mixture  extracted  degradation  was n o t c o n t r o l l e d ,  l a g phase  1967)  Maillard  (0.4M) - g l y c i n e  absorption,  (Jemmali,  products  some o r g a n i s m s .  a  of growth d u r i n g t h e l o g a r i t h m i c  (Jemmali,  reaction  inhibitory  mixture  i n the presence  affected  aldehyde  f o r v a r i o u s l e n g t h s of time  power.  increased  namely,  destruction  of glucose  was c h a r a c t e r i z e d  glycine,  des;  s p e c i e s showed  mixtures pH  eroi  with  p r o d u c t s of g l u c o s e , o r thermal independent  Lactobacillus  unavailable to  mesent  cystine  utiIis  aureus, aeruginosa,^ (Kato  and  1 2 Shibasaki, increase The  1974). A n t i b a c t e r i a l in  x y l o s e c o n c e n t r a t i o n and  antimicrobial  (acetone) phenolic  than  less  bacteria  effect  amount of  interest  most  the  c h a n g e s and Maillard  negative caramels these  (Kato  from  work  the  has  reaction 1976)  with  was  a  with  Melanoidins  produced  their  affected  e t h a n o l p r o d u c t i o n of while activities  melanoidin, products  not  of  were  depressed.  fermentation,  study  such  i t was  (Shvets  and  cells  and  as h i g h e r  f o r 16  and h,  Saccharomyces Catalase  medium  formation  somehow  glucose  of  others.  and  functioning.  water b a t h  in  have a  presumed t h a t  of  some s t r a i n s  The  of  mechanisms  cells  mixture  grown  Although  melanoidins  metabolic  boiling  fair  presence  the  yeast  a  physiological  the  while  affecting  of y e a s t s  the  in  yeast  reaction  industry. on  Since  heating a a  substances  attracted  studying  normal  over  has  one  to  (4:1,  invertase  w:w)  by  as c a t e c h o l ,  Maillard  potential,  glycine  cerevisiae,  yeast  at  compounds c o u l d a d s o r b  interfere  other  potential,  positive  Four  methylhydroquinone.  focused  effects.  electrokinetic  and  fermentation  aimed  solvent  chromatography.  yeast  products,  these  have  of  of  increased. by  identified  an  Shibasaki,1974).  and  been  with  antimicrobial  and  viability  multiplication  Slyusarenko,  and  the  of m e l a n o i d i n s  the  a l s o as pH  liquid  to  increased  separated  4-methylcatechol,  or y e a s t s  on  involved  and  sensitive  products  of  were  compounds were i s o l a t e d  were  The  substances  extraction  3-methylcatechol, Fungi  activity  and  containing  of  alcohols,  secondary organic  1 3 acids,  aldehydes  chromaticity reflection decreased  (measured  from  fermentation  concluded  an e l e c t r i c a l  Mirna  (1976)  sugars  and  Hydroxymethylfurfural  effect  and  thus  Melanoidins and  derived  nitrite i s an  of the yeast  cells  may  capacity.  To t e s t  this  prepared  melanoidins  aureus  reaction  mixture.  concentration the  specific  1976; play  cured  inhibited  meat of  a  by  a  When  Kajimoto role  heating  thousand added  products.  the  Maillard inhibitory  Leite a  to  c h e l a t i n g agents  et  in  a l . ,  their e_t  solution buffer  1975),  inhibitory al. , of  (1979) glucose  (0.05M),  fold the  dilution  10%  decrease  i n the s p e c i f i c  of the  decreased  aureus  t o remove t h e i r o n , growth  a  g r o w t h medium a t a  melanoidin  r a t e o f Staphylococcus  melanoidin  in  an a b s o r b a n c e r e a d i n g o f  o f 1.5%, t h e c o n c e n t r a t e d  3.0%  by  between  by 46%.  When t h e g r o w t h 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 containing  of  products.  possibility,  with  growth  cells  growth  reaction  intermediate  a t 90°C f o r 38 h, u n t i l  0.200 was o b t a i n e d  the was  from t h e  (1.25M) and g l y c i n e (0.66M) i n c i t r a t e bath  t o yeast  that  a r e known t o be e f f e c t i v e  this  water  of m e l a n o i d i n s ,  adsorb  in  browning  Horikoshi,  indicating  light  may c o n t r i b u t e t o t h e o v e r a l l  of nonenzymatic  (Gomyo  of  found  and Staphylococcus  sp.  reaction  coefficient  i n the presence  the  interaction.  5-hydroxymethylfurfural, amino  the  Since  that melanoidins  and C o r e t t i  Micrococcus  as  affected.  t h e s u r f a c e of t h e y e a s t )  after  the a u t h o r s through  and e s t e r s , was a l s o  rate,  medium  t h e r e was a  indicating  that  14  some mechanism o t h e r responsible  for  than  the  chelation  inhibitory  of  iron  was  activity  primarily  (Leite  et a l . ,  1979). The  _in  vivo  the  intestinal  et  al •  microflora  (1981).  obtained  Rats  by h e a t i n g  (0.1M) a t 90°C in  e f f e c t s of M a i l l a r d  o f r a t s were s t u d i e d  were  glucose  administered  control  group.  The  browning  reaction  (<500)  and  (1M)  in  (<500)  and  and  NaHC0  3  trends  similar,  to  g r o u p . The  test  fractionated weight  the  into  compounds  culture  medium  a  low by of  t h e i n t e s t i n e s o f t h e r a t s , as lactis.  molecular  mixture,  i n the  the  o f Lactobacillus  not  and  showed  molecular  from  (>500) but  coliforms  however,  in  were t h e n  added  culture  high  growth  (>500)  and  as t o stock  staphylococci,  lactobacilli  isolated  unfractionated  Horikoshi  i n t h e t e s t g r o u p and t h o s e  mixtures  high  ultrafiltration  well  rats  increase  lactobacilli  by  f o r 48 h . T h e r e was no s i g n i f i c a n t d i f f e r e n c e  between  significant  p r o d u c t s on  browned s o l u t i o n ,  (1M), g l y c i n e  t h e numbers o f e n t e r o c o c c i ,  Clostridia  reaction  as  weight  marked,  suggesting  a  The  low  f r a c t i o n s showed  to  that  synergistic  of  the effect  between t h e two f r a c t i o n s . Browning xylose and  mixtures prepared with arginine  o r h i s t i d i n e and g l u c o s e  partially  spoilage al.,  reaction  p u r i f i e d by d i a l y s i s ,  bacteria  frequently  ( 1 9 8 3 ) . I t was  Maillard  were  reaction  shown  products  found  that  and  tested  unfractionated  against  p a t h o g e n i c and  in  food  (Einarsson  e_t  the i n h i b i t o r y e f f e c t of the  d e p e n d e d on t h e t y p e o f  bacteria  1 5 as w e l l a s t h e t y p e weight  fraction  molecular subtil  is,  1974;  coli,  patents  Yazima  (>1000) than  have  been  tested  granted  by t h e M a i l l a r d b;  molecular  t h e low (<1000) Bacillus  with  and Staphylococcus  the l i t e r a t u r e  aureus.  for antimicrobial  r e a c t i o n (Yazima e_t a l . ,  c ) , however  that these  there  products  i s no  are being  commercially.  OPTIMIZATION  Optimization most  efficient  o f any p r o c e s s route  outcome t o be o p t i m i z e d formulation process  Deming,  the  a  new  of  food  the  early  expended  Optimization  be  reached  (Long,  1920's e x p e r i m e n t s  of s e v e r a l f a c t o r s  o f one f a c t o r  stationary,  with  to  system or t h e of the  with  the  1969; Y a r b r o a n d  1974).  level  approach  product.  result  and e f f o r t  of the  o b t a i n i n g a d e s i r e d outcome. The  on a r e s p o n s e  "one-factor-at-a-time"  the  i s the determination  may be an HPLC s o l v e n t  the f i n a l  o f time  Until effects  of  allows  minimum  in  high  when  e_t a_l. , 1975a; in  The  inhibitory  Escherichia  produced  produced  more  compounds  indication  B.  was  MRP.  weight  Several mixtures  of  and  noting  i s suitable  one  another.  which case  investigating the  were d e s i g n e d  using  approach. This e n t a i l e d  varying  h o l d i n g a l l the other  factors  while the  effect  on t h e r e s p o n s e .  o n l y when t h e f a c t o r s do In g e n e r a l  however,  the "one-factor-at-a-time"  not  This  interact  f a c t o r s do i n t e r a c t , approach  will  not  16 always r e s u l t  i n the  (Deming  Morgan,  first  and  to d e s c r i b e be  varied  Their  "evolutionary of  to estimate the  techniques of  t o move.  Spendley  et  the  n  is by  two  three-factor analysis  al. ,  can  been  1975),  because the  factors simplex  significantly I t has  number  testing  to  affect  the  response,  however, inclusion  that of  this  locate  the  results  conditions  are  method.  This  technique  of  significance  to  of  efficient  the  through  therefore is faster  and  forward.  of  which  potentially  a  n+1  points, simplex  triangle;  a  so on.  Factorial  which  factors  in turn  Deming, is  of  Therefore a  determine  (Morgan and  to  more  i s a t e t r a h e d r o n , and used  shown  and  a  shape  be  a  as  factors. the  employ  decision  f i g u r e made up  of  optimum.  measurements  designed  simplex  takes  of  valid  of o b s e r v a t i o n s , and  the  the  regression techniques  to analyze  number  i s a geometric  variables  methods  operating  methods p r e v i o u s l y put  A simplex  n.  the  (1962)  at  the  s u r f a c e . B e c a u s e t h e EVOP  designed  require traditional  than  defined  §_1.  (1951) were  steepest ascent  in  sequential  replication  where  and  conditions  several  (EVOP)  reach a s t a t i s t i c a l l y  direction  simpler  of  Wilson  to a r r i v e  designs  processes, a large to  does not the  factorial  reaction  whereby  operation"  variations  procedure,  and  same t i m e  were o r i g i n a l l y  industrial  best  at the  of  r e g i o n of a r e s p o n s e  small  required  process  the d i r e c t i o n  optimum  set  1973). Box  the  could  combination  optimum  establishes  1974;  Parker  not a l w a y s unimportant  et  necessary factors  17  does  not  adversely  detrimental, because  affect  however,  the  i f there  simplex.  i s a large  t h e number of e x p e r i m e n t s  optimization  increases  This  may  number of  t o be p e r f o r m e d  exponentially  with  be  factors  d u r i n g the  the  number of  factors. Once t h e number of and  lower  within  limits,  those  (n+1)  of  sequential the  decision  each  of  and  on  the  as  to  based  decision worst  method  response  which  of t h e  remaining  p o i n t s . The  vertices  of  is  worst  the  back and this  forth  situation,  the  optimum The  and  the  out  constitute  its  of  response,  I f the  position.  between t h e s e second  is  discarded instead  the  simplex  response  simplex two  worst  the  be made.  discarded  of  the  is  The  the then  remaining  new  vertex  reflected  back  then  oscillate  forever.  To a v o i d  of t h e new  simplex  response. T h i s w i l l optimum.  a  and  f a c e of  of t h e  would be  response  simplex",  vertex  would  The  vertices,  the  positions  of the worst  t o c o n t i n u e toward  is  responses  simplex  The  these  new  the  appraised.  of  at t h i s  the  govern  "starting  across  of  t o move t o  the  simplex  to the  the  is  t o move c a n  image  response  comparison  the  each  starting mirror  the s i m p l e x  vertex  upper  factor.  various rules  direction  the s i m p l e x .  to the o r i g i n a l  each  results  with  in  carried  response,  vertices  replaced  evaluated  are  i s to f o r c e  t o be made a f t e r (n+1)  search for  i s e v a l u a t e d . S i n c e the o b j e c t i v e  optimum  original  been d e c i d e d upon,  are a s s i g n e d to each  (experiments)  simplex  region  has  which c o n f i n e the  boundaries,  vertices,  response  factors  allow  18 When t h e optimum simplex  optimization  position.  At t h i s  size  of  the  There  i s a lower  If  has  effects,  force  time  the simplex  in  limit  and t h e  determined  order  to  of t h e  indeterminate errors will  rules  of that  decrease  the  t o home i n on t h e optimum.  t o the s i z e  simplex  the  t o c i r c l e about  i t may be n e c e s s a r y  simplex  i t i s too small,  been  fail  simplex  however.  w i l l mask t h e t r u e  to  move  toward  the  optimum. When t h e s i m p l e x impossible optimum  if  from  domains  original  simplex  provides  rules  and  optimum.  been  accelerate  regions  Morgan,  1973).  difficulty reached,  prodedure.  The  rapidly  a t the  toward  reflection  of the o r i g i n a l  is  drawback o f the  lack  of  (1965) overcame when  modification  expansion, rule  be  variables'  knowing  modified  The e x p a n s i o n  the  Another  not  with their  for reflection,  of  and Mead  of  may  when t h e s e a r c h i s  method  Nelder  i t is  Confirmation  i s reached  optimization  contraction.  the response  point  differing  other  has  and  obtained i s the g l o b a l  the  for acceleration.  and t h e  response  if  local  simplex  optimum  to  two d i m e n s i o n s  t h e same optimum  (Deming  provision  massive  a  widely  Spendley's  this  tell  or merely  obtained begun  to  i s i n more t h a n  simplex  the  of the method  c o n t r a c t i o n , and  enables  the  search  t h e optimum. F o r i n s t a n c e i f is  simplex,  better  than  an e x p a n s i o n  the  best  i s indicated  t h e new v e r t e x E i s g e n e r a t e d :  E  = P + 2  (  P-W)  (1)  19  where P and W a r e d e f i n e d  where W = w o r s t best  vertex;  of W;  C  vertex;  N = next-to-the-worst  P = centroid  = contraction  R  as:  of N-B  of  R;  vertices; C  R  vertex; w  B  = contraction  reflection;  and  E  =  expansion. If  the response  the  new  simplex  than  the response  and  BNR  becomes  at E i s better  i s BNE.  than  I f the response  a t B, t h e e x p a n s i o n the  new  simplex.  the response  at  B,  a t E i s not b e t t e r  i s s a i d t o have  failed  I f the response  a t the  reflection  (R) i s worse t h a n  the response  direction  chosen  t o be i n c o r r e c t and t h e s i m p l e x  i s judged  at  N,  then  the  20  is  c o n t r a c t e d . W i t h t h e s e added f e a t u r e s t h e a c c u r a c y  modified search  simplex is  stopped  predetermined or of  procedure when  value  Routh procedure  et  vertex Nakai  the s t e p s i z e  i n response  al.  the  (1977)  frequently Morgan  vertex  a  To overcome factorial  this,  The  than a  variable) the v a l u e  curve  simplex  fitting  the  he  i n the p r e c e d i n g  that  the search f o r the  simplex. optimum  when t h e p r o c e d u r e s  i n t r o d u c e d a s u b p r o g r a m of analysis" that  optimum  into  this  without  of  used.  "quadratic,  Routh's super  modified  quickly  to  reflection  (1974) o r R o u t h et. a_l. (1977) were  and f o u n d  the  the  vertex  found  regression  identified  modified  following  at the b o u n d a r i e s  Deming  optimization,  less  of e a c h  quadratic  immediately  however,  stalled  and  becomes  do n o t e x c e e d  further  t o r e p l a c e t h e worst (1982),  controllable.  deviation.  by i n c o r p o r a t i n g  determine  more  ( e g . 1% of t h e domain  when t h e d i f f e r e n c e s the standard  i s much  o f the  super  simplex simplex  stalling  a t the  boundaries. Nakai the  et  optimum  slows  moves r a p i d l y  considerably  Visualization determining speeding data  a l . (1984) a l s o  in  expedite procedure  in the i n i t i a l  as  the  of t h e r e s p o n s e the  general  up t h e s e a r c h . more  than  the s e a r c h f o r  stages  ,  is  but  then  approached.  s u r f a c e can be v e r y h e l p f u l i n  However,  o f t h e optimum graphic  three dimensions  the  that  optimum  location  the search i n t h i s whereby  observed  thus  illustration  of  i s very d i f f i c u l t .  r e g i o n they developed  responses  and  are plotted  a  To  mapping  against  each  21  factor,  once  performed. limits  are  level  the  data  then the  number  points,  used  where  the  simultaneous  factor  level  value  and  All  1/5 the  factors  generates  the  a series  t o draw optimum  factor  shift  most is  paper  t a r g e t (T) v a l u e ,  are  for  cases  the  B v a l u e s . The  shifted  and  These  experiments  with  (p<0.0l) i m p r o v e d  beyond  simultaneous the  mapping  factor  et.  Deming  to  1984)  best  (B)  the  too  be  the  and  procedure,  lines  shifts  the  set  data  a_l. ,  from  shift  optimization efficiency  limit  likely  the T v a l u e  incorrectly  the  of m a t c h e d  program w h i c h  starting  small  probable  (Nakai  been  p o i n t s and  most  simultaneously  where T v a l u e s a r e  t h e Morgan and  large  have  t h e d i s t a n c e between t h e p r e s e n t  three  conjunction  into  respectively.  to generate  located. Also described in this is  experiments  m a j o r i t y of d a t a  investigator  indicate  of  i s separated  include  of good  which enable which  certain  Factor  which  majority values  a  B  value. program  to  correct  close  program,  to  used  in  significantly  when c o m p a r e d  (1974) s i m p l e x o p t i m i z a t i o n .  with  III.  A.  AND  METHODS  BACTERIA  Staphylococcus by  MATERIALS  rehydrating  (TSB)  aureus  ATCC  bactrol disks  25923 c u l t u r e s were  (Difco) in  ( D i f c o , D e t r o i t , MI) and g r o w i n g  erlenmeyer (New  flask  Brunswick  incubator  Scientific  Co., I n c . , E d i s o n ,  (BBL, R o c k v i l l e , MD)  fresh  s l a n t s a t two month A standard  curve  20) v e r s u s  t o be u s e d  for estimating  For  individual  overnight (37°C, NJ).  in  a  120 rpm)  The c e l l s  soy  washed  c f u p e r mL  (600nm) ( B a u s c h and Lomb  was p r e p a r e d  culture density  experiments,  Scientific  a t the o u t s e t ,  (Figure 3).  cultures  environment  Brunswick  were h a r v e s t e d  resuspended estimated per  mL w i t h  with in  from  0.1%  sterile  were  grown  incubator  shaker  Co., I n c . , E d i s o n ,  by c e n t r i f u g a t i o n (10,000 x g;  peptone  broth,  peptone.  the standard  appropriate  agar  transfers to  10 min; room t e m p e r a t u r e ) and t h e b r o t h d e c a n t e d . The was  rpm  intervals.  controlled (New  120  mL  N J ) . Between  tryptic  s l a n t s a t 4°C, w i t h  broth  i n a 250  a t 37°C and  on  of absorbance  Spectronic  soy  the c e l l s  i n a shaker  e x p e r i m e n t s c u l t u r e s were m a i n t a i n e d (TSA)  tryptic  obtained  curve,  dilutions.  22  pellet  centrifuged,  Culture  and  density  and a d j u s t e d  t o 10  was 5  cfu  23  10°l 0  1  1  1  i  1  i  i  0.2  0.4  0.6  0.8  1.0  1.2  1.4  Absorbance (600 nm)  Figure  3. S t a n d a r d cfu  curve  p e r mL  of absorbance  f o r Staphylococcus  (600 nm) v e r s u s aureus.  24  B. BROWNING REACTION MIXTURES  Model  +  L-lysine  monohydrochloride  ( c o m b i n a t i o n A) and D ( + ) - x y l o s e  + L-lysine  monohydrochloride  ( c o m b i n a t i o n B) were p r e p a r e d a c c o r d i n g t o  the  systems  conditions  computer  of  a-D(+)-glucose  generated  program  (Tables  by  the  simplex  optimization  1, 2 ) . G l u c o s e , x y l o s e and  were p u r c h a s e d  from Sigma C h e m i c a l  (St. Louis,  MO).  Predetermined  amounts of t h e r e a c t a n t s were d i s s o l v e d  i n 50  mL d i s t i l l e d  water,  concentrated  NaOH,  flasks  Blue  straight  Liebig  were  were  evaporation,  in  water  (Corning  in series  lowered  spread  on  however,  bottom into the  water  periodic  level  adjusted  back  the  atmosphere  of nitrogen  starting glass  until  Glass  with bottom  Electric  of t i m e .  Works,  Three  Corning,  were s e c u r e d t o  used.  Styrofoam packing  surface  around  of the  to  minimize  l o w e r i n g of t h e f l a s k s  mixtures, after  cap  round  (Blue M  length  the water.  reaction  screw  mL  bath  flasks  browning  foil-wrapped,  100  pH  t o a l l o w maximum u t i l i z a t i o n  t o m a i n t a i n t h e water  to  appropriate  IL) f o r a f i x e d  condensers  then  the  refluxed  b a t h . The r o u n d  condensers  required  Island,  placed  t h e water  chips  and  to  i n a constant temperature  Company,  NY)  adjusted  Company  lysine  was  the f l a s k s .  The  the h e a t i n g p e r i o d ,  were  pH, bottles  and at  stored 4°C  under  in an  25  Table  1. V e r t i c e s  generated  optimization (vertices  by t h e s u p e r  computer  program  1-6 = " s t a r t i n g  simplex  (glucose + l y s i n e ) ,  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  50.00  9.59  81 .3  30.00  15  1 .94  50.00  10.00  84.8  28.85  16  2.26  50.00  10.00  85.0  28.49  17  2.19  50.00  10.00  85.0  30.00  Table  1. c o n t ' d . Factor X2  X4 •  XI  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 = m o l a r  ratio  X2 = t o t a l  concentration  X3 = i n i t i a l  X3  X5  Vertex  o f amino a c i d  of r e a c t a n t s  pH  X4 = r e a c t i o n  temperature  X5 = r e a c t i o n  time  t o sugar  (hours)  (°C)  (%  w/v)  Table  2. V e r t i c e s g e n e r a t e d optimization (vertices  by t h e  super simplex  computer p r o g r a m ( x y l o s e  1-6 = " s t a r t i n g  + lysine)  simplex")  Factor  XI  X2  X3  X4  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  Vertex  X5  Table  2. c o n t ' d . Factor  Vertex  • XI  X2  X3  X4  X5  8  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  1  X1 = m o l a r  ratio  X2 = t o t a l  concentration  X3 = i n i t i a l  o f amino a c i d  X4 = r e a c t i o n  temperature  X5 = r e a c t i o n  time  t o sugar  of reactants  pH  (hours)  (°C)  *  (%  w/v)  29  C. MEASUREMENT OF INHIBITION  The  procedure  the browning the  t o determine  reaction  mixture  u n i f o r m and v a r i a b l e  (Spiral poured  Systems,  Inc., C i n c i n n a t i ,  s u r f a c e . Uneven a g a r deposition plastic  an  experiment,  to  s t a n d a t room t e m p e r a t u r e  a  plates  the  may r e s u l t  spiral  culture prior  browning  deposition  reaction  variable exactly  plater.  cam, t h e same  Following the browning 18  h  at  Inhibition the  aureus  centre  stored  night  before  t h e bags and a l l o w e d to  ensure  adequate  not  sufficiently  dry.  5  using  from  into  was on  the uniform  then  cam 1  f o r about  reaction  of  aureus  t h e d e p o s i t e d 5.  the agar  of the M a i l l a r d  deposition  10  min  p r o d u c t s . The  deposited,  t o p of the b a c t e r i a ,  of  the b a c t e r i a l  mixture, the p l a t e s  (GCA/Precision  o f growth of  were  in  with  the  following  spiral.  reaction  37°C  from  agar  ( 1 0 c f u / m L ) was d e p o s i t e d o n t o  The l i q u i d  directly  were  variations  Plates  u s e d . The  is  soy a g a r  mixture  plates  o f t h e sample on t h e s u r f a c e of  i f the agar  of t r y p t i c  in  plater.  overnight  was a l l o w e d t o soak to  Agar  Plater  t o p t o ensure a l e v e l  were removed  of the agar. P o o l i n g  10 cm p l a t e  OH).  bags a t 4°C u n t i l  s u s p e n s i o n o f S.  The  bench  by t h e s p i r a l  sealed  the p l a t e  t h e c o n c u r r e n t use of  s u r f a c e s can r e s u l t  in  drying  involved  c a p a c i t y of  cams o f t h e model DU S p i r a l  on a c o m p l e t e l y f l a t  sample  the i n h i b i t o r y  the  s u s p e n s i o n and  were i n c u b a t e d f o r  Scientific,  Chicago, I L ) .  was r e c o r d e d a s t h e d i s t a n c e petri  plate  (mm)  t o t h e r a d i u s where  from growth  30 began.  The  final  v a l u e was  the average  taken at 0°, 90°,  180°, and  270  of  the p l a t e  D.  OPTIMIZATION OF  a)  ° around  four the  measurements circumference  4).  REACTION CONDITIONS  Optimization The  simplex o p t i m i z a t i o n  PC-1500  pocket  generated  the  computer  the c o n d i t i o n s  performed. on  (Figure  of  Factors  inhibitory  were j u d g e d  to  most  (Sharp C o r p o r a t i o n ,  f o r the Osaka,  Sharp  Japan),  f o r each of the e x p e r i m e n t s  to  likely  effect  capacity  t o have a s i g n i f i c a n t  be  of t h e M a i l l a r d r e a c t i o n p r o d u c t s  be:  (1) m o l a r  ratio  (2) t o t a l  concentration  (3)  program, w r i t t e n  of amino a c i d of  to sugar;  reactants;  pH;  (4) t e m p e r a t u r e (5) l e n g t h  of  reaction;  of r e a c t i o n time at the  specified  temperature. Upper and and  the  (Tables xylose  1,  where n i s t h e  2 ) . The  were p l a c e d  program  experiments two  first  on  constituted  of the  optimizations  the responses  s i x experiments,  on  generated  number  + l y s i n e ) were p e r f o r m e d  Based the  limits  computer  (vertices), initial  lower  these (n+1)  parameters experiments  factors.  These  "starting (glucose  six  simplex" +  lysine;  concurrently.  (inhibition the program  of  growth,  g e n e r a t e d the  mm)  of  seventh  F i g u r e 4.  S c h e m a t i c r e p r e s e n t a t i o n o f (a) f u l l s p i r a l p a t t e r n s h o w i n g no i n h i b i t i o n , a n d (b) t h e o r e t i c a l z o n e o f i n h i b i t i o n a n d method o f m e a s u r e m e n t .  32  vertex and of  by  discarding  replacing the  i t with  starting  additional  the v e r t e x g i v i n g i t s mirror  simplex  response  was  had  program t o g e n e r a t e  until  the  optimum  been  point  was  five  experiments  t o r e a c h t h e optimum  b)  factors  This process  generally  7  allowed  was  repeated  optimization  requires  (Nakai  one  t o move. In  at vertex  An  vertices  only  program  reached.  involving  response  the  evaluated  response  v e r t e x 8.  worst  image. A f t e r  r e q u i r e d f o r the  o t h e r words, e v a l u a t i o n of t h e the  the  e_t a l . ,  22  to  24  1984).  Mapping After  the c o m p l e t i o n  procedure toward  was  employed  t h e optimum  factor,  showing  response,  was  §_1.(1984)  to  the  the  a  22nd  visualize  ( F i g u r e s 5,  vertex  a  mapping  movement of  6). A separate  relationship  prepared  and  of  between  graph  factor  the  data  for  each  level  and  u s i n g t h e mapping p r o g r a m of N a k a i  Sharp  CE-150 p r i n t e r  interfaced  e_t  with  the  S h a r p PC-1500 c o m p u t e r . The 1984)  was  simultaneous  factor  then  u s i n g the  established  by  employed  t h e mapping  E. SEPARATION OF  best  and  (Nakai  et a l . ,  target  values  procedure.  was  mixtures  used  browning  reaction  lysine,  according to molecular was  program  MAILLARD REACTION PRODUCTS  Ultrafiltration  (25 mL)  shift  diluted  to  1000  to f r a c t i o n a t e  of g l u c o s e  mL  + lysine  weight. with  The  the  optimized  and  xylose  reaction  distilled  +  mixture  water,  then  33  passed MA)  through  to  remove  contaminated passed  a 0.45M f i l t e r  t h e m a j o r i t y o f any  the  mixture.  through  (Amicon  a  ultrafiltration The  p e r m e a t e was The  cut-off)  and  of  500  mL)  YC05  25  through  (500  a  was m.w.  was  using 4°C,  to  freeze-dried  then filter  50  Amicon psi using  the  (5,000  repeated  then  under  have  was  the  distilled  YM5  cut-off)  compounds and  mixture  adjusted  sterile  w h i c h may  original  water and m.w.  w i t h YM2  filters.  cut-off) (1,000  The  m.w.  permeate  to c o n c e n t r a t e the  a d j u s t e d back  the  to  a  less  volume  ml.  spiral range  was  at  Bedford,  c u t - o f f ) Amicon  MA),  52),  with  procedure  m.w.  Each  F.  (25  t h e YC05 f i l t e r  than  (10,000 m.w.  (model  Corp.,  bacteria  diluted  Danvers,  cell  passed  filter.  The  retentate  concentration  of  PM10  Corporation,  nitrogen.  (Millipore,  fraction  plating of  the  tested  with  to determine  S.  aureus,  using  the m o l e c u l a r  the  weight  compounds.  ANALYSIS  backward e l i m i n a t i o n  stepwise  multiple  were u s e d  to analyze  Columbia  Amdahl  proportion  of e a c h  according  to  (1964).  then  technique,  inhibitory  STATISTICAL  The  was  the  and  stepwise  regression analysis the data 470  V/8  variable method  with  technique  response  The was  of B a r y l k o - P i k i e l n a  of  (Ray,  the U n i v e r s i t y  computer.  to the  methods  of  the 1982)  British  contributing calculated and M e t e l s k i  IV.  A.  OPTIMIZATION OF  To the  factor  should of  use  be  chosen  factor  perceived boundaries  where  the  them  with  relatively  however, the  of  the to  that  fact  broad  "home  determine  S.  simplex each  that  in"  however the  overcome by  the  failed  next. This  the  starting  optimum  one  cases  the  some i d e a the  search of  the  boundaries this  t o the  study, due  to  small  (or  vertex  of  the  of  the  r e s u l t e d in responses simplex  is  i s unknown.  unsuccessful  from  factor  those  In  the  v e r t i c e s and  of the  program.  preliminary appropriate  true  starting  respond  choice  i s wrongly  Setting  in  of  limits,  locate  q u a n t i t i e s or p o t e n c i e s  produced  the  to  optimum.  to  i n the  optimum  then narrowing  on  i n the  to the  of  the  boundaries to o b t a i n  aureus  of  stalling  the  optimum.  levels  those  optimum w h i c h  p a r t i c u l a r a p p r o a c h was  that  Several  failure  l o c a t i o n of  optimum, and  compounds  eventual  in  a local  u s u a l l y be  inhibitory  for  Misjudgement  is difficult,  changes  zero  care.  assurance  nonexistent)  starting  range between  (overall)  approximate  p r o b l e m can  order  of  s u c c e s s f u l l y , the  the  result  global  within  as  great  can  the  This  in  with  with  the  location  well  in detection  as  contained  optimization  as  limits  optimum, or  DISCUSSION  INHIBITION  simplex limits  RESULTS AND  experiments boundary  34  limits  were  required  w h i c h would  to  allow  35  the  program t o p r o c e e d .  six Of  vertices  The o r i g i n a l  of t h e s t a r t i n g  a l l s i x p r e l i m i n a r y experiments  mixture, the  none of t h e M a i l l a r d  g r o w t h of S.  aureus.  gave a m e a s u r a b l e the  relative  to proceed.  t i m e ) were  narrowed  to  the  judged  the  the f a c t o r to t h i s ,  required.  antimicrobial  limits  factor  established that  a  1.00,  ratio  1983; F o s t e r ,  upper  limit  one  too  indicate  hope  a  of amino a c i d  factor  would  production  t o sugar,  1:1  also  which  t h a t t h e lower  for factor  1980) p r o v i d e d  t i m e . In  were  5. The r e s u l t s  of amino a c i d  limit  the  were  however,  a third  sought  and 5  results  be m a x i m i z e d . The  indicated  a t 2.50. S i n c e  4. S i m i l a r simplex,  that  i n Table  therefore,  (total  and  considerations were  unable  2  broad,  t o be narrowed  of a n t i o x i d a t i v e M a i l l a r d  al.,  to  temperature),  starting  would a l s o  ratio  a s t h e lower  higher  production  the  experiments  approximately  lysine  inhibited  ratio),  i n Table  conditions  compounds  1, t h e m o l a r  +  3.  only vertex 3  responses  be  other  c h o s e n a r e shown  preliminary  mixtures  (molar  to  second  maximize browning, w i t h  ultimately  1  shown  several  Reaction  Table  of e a c h v e r t e x , t h e p r o g r a m was  values  were o b t a i n e d w i t h  addition  W i t h no  with the  the g l u c o s e  of r e a c t a n t s ) , 4 ( r e a c t i o n  (reaction  requiring  with  in  With x y l o s e + l y s i n e ,  response.  success  limits  a r e shown  reaction  Limits for factors  concentration  of  simplex  factor  molar  of  limits of  the  limit for should ratio  be was  1. The o b s e r v a t i o n  t o sugar  o p t i m i z e d the  compounds  (Waller  et  the b a s i s f o r s e t t i n g the 1 only d i c t a t e d  r e a c t a n t t o the o t h e r , a second  factor  the  ratio  was r e q u i r e d  Table  3.  Original  factor  experiments  limits  and s t a r t i n g  simplex  f o r t h e s i m p l e x 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 compounds system  from a g l u c o s e + l y s i n e  and a x y l o s e + l y s i n e  active  model  model  system.  Factor  XI  X2  X3  X4  X5  Lower  Limit  0.02  3.00  7.00  60.0  5.00  Upper  Limit  50.00  90.00  10.00  95.0  48.00  3.00  7.00  60.0  5.00  Vertex  1  X1  0 = 02  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  = m o l a r r a t i o o f amino ia c i d t o s u g a r  X2 = t o t a l  concentration  X3 = i n i t i a l  of r e a c t a n t s  pH  X4 = r e a c t i o n  temperature  X5 = r e a c t i o n  time  (hours)  (°C)  (%  w/v)  Table  4. S e c o n d  factor  experiments of  limits  and s t a r t i n g  f o r the simplex  simplex  optimization  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  active  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.  Factor  X2  X3  X4  X5  Lower  Limit  0.10  20.00  7.00  75.0  20. 00  Upper  Limit  10.00  90.00  1 0.00  95.0  50. 00  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  Vertex  X1  = m o l a r r a t i o o f amino a c i d t o s u g a r  X2 = t o t a l  concentration  X3 = i n i t i a l  of r e a c t a n t s  pH  X4 = r e a c t i o n  temperature  X5 = r e a c t i o n  time  (hours)  (°C)  (%  w/v)  Table  5. F i n a l of  factor  l i m i t s f o r the simplex  the production  compounds and  of m i c r o b i o l g i c a l l y  from a g l u c o s e + l y s i n e  a xylose  + lysine  model  system.  Lower L i m i t  XI  1.00  2.50  X2  30.00  50.00  X3  9.00  10.00  Upper  Limit  X4  75.0  85.0  X5  25.00  30.00  = molar  r a t i o of amino a c i d  X2 = t o t a l c o n c e n t r a t i o n X3 = i n i t i a l  pH  X4 = r e a c t i o n  temperature  X5 = r e a c t i o n  time(hours)  t o sugar  of r e a c t a n t s  (°C)  active  model  Factor  X1  optimization  (%  w/v)  system  39  to  control  limit  of  30%  compounds  total  was  chosen  still  solubility 50%  the  of  the  The  favoured have  by  by  an  the  an  the  inhibitory  on.  inhibition  by  inhibitory  these  aureus.  The  mixture  was  of  94%  presumed  that  greater  would  This, of  approximately filtration  plating S.  was  the  not  to maintain  insolubilization  to  mixture  Therefore,  the  of  the  pH  would the  ultimately  the  pH  and  the  of  most tested  neutral  pH  same m i x t u r e a t  pH  main study  cause i t was  of  the  generally  temperature,  the  i n h i b i t o r y compounds. a  compounds  temperature were  clogging  failed  to  the  range  formed,  the  inhibit fear  removed by  a temperature  is  of  avoid  being  reaction  s o l e cause  one  c a s e . At  of  was  g r o w t h of  to n e u t r a l  of  pH,  a  the  with  of  physically  was  not  The  limit  3,  the  reaction  because  were  was  this  insoluble  necessary  on  10.00  the  production  93°C  was  decided  the  t o be  compounds  of  upper  high  w i t h the  not  higher  inhibitory  the  obtained  apparatus. This  aureus.  too  inhibition  outset  however, p r o v e d  making  of  be  adjusted  pH  an  factor  mixtures,  amount of that  pH  lower  inhibitory  Maillard  to  that  was  the the  that  9.00  reaction  i n d i c a t i n g that At  for the  A  concentration.  e f f e c t in i t s e l f ,  mixtures  inhibition.  that  confirm  w i t h S.  10,  fact  but  of  concentration  limits  of  reactants.  such that  of  range  To  this  maximum  a l k a l i n e pH,  microorganism. A decided  was  of  production  at  reactants  choice  constrained  because  occurred  approached  possible.  concentration  the  spiral growth  that  filtration, that  Maillard reaction  the it  prevented  products.  The  40  limits on  controlling  t h e l e n g t h of t i m e  t h e b a s i s of r e s u l t s Once  the  experiments reaction  from  limits  preliminary  were  inhibitory  were 29 f o r g l u c o s e  + lysine  chosen  experiments.  established  required to optimize  products  of h e a t i n g were  the  the p r o d u c t i o n  number of  Maillard  t h e g r o w t h of S.  to  and 28  for  xylose  of  aureus,  +  lysine,  respect i v e l y . Following two  execution  sugar/amino  acid  employed  in  movement  of t h e d a t a  plots  small while  entered  medium data  values  grouped implied  four  from  t h a t they  direction  were u s e d  grouping  as  used  groups.  If  data  plot  to  one f a c t o r  to  separate  data  The  points,  p o i n t s . The  the  average  to d i v i d e  of  The s m a l l , the p l o t t e d  points the  remained  next,  this  had been o b t a i n e d under a p p r o x i m a t e l y  be  could  to  linked  those  factors.  together  to  the  Therefore  estimate the  s u r f a c e w h i c h would h o p e f u l l y i n d i c a t e  containing  of  ( F i g u r e s 5, 6 ) . The  were t h e n  these  The  level  the  contained a l l the data  regard  response  was  t h e m a j o r i t y of good  same c o n d i t i o n s w i t h points  a mapping p r o c e d u r e  of t h e s m a l l and l a r g e l i m i t s .  into  together  the  t h e optimum  t h e medium  and l a r g e l i m i t s points  of  s m a l l , medium, and l a r g e g r o u p i n g s .  contained  calculated  f o r each  determine  factor  the l a r g e grouping  computer  to  toward  versus  points into  grouping  combinations,  attempt  of r e s p o n s e  the d a t a  the  an  of 22 e x p e r i m e n t s  the  region  t h e optimum.  plotted  data  the g e n e r a l v i c i n i t y  of t h e f i r s t  22 e x p e r i m e n t s  o f t h e optimum. T h e r e f o r e  indicated  the l i m i t s  on  41  (a)  1.0  (b)  2.6  M O L A R RATIO (amino acid/sugar)  36  E £ LU  V)  z o  CL  <n us cc  20 43.0  60.0  CONCENTRATION (%)  F i g u r e 5 . Maps o f f a c t o r l e v e l v e r s u s r e s p o n s e f o r g l u c o s e + l y s i n e , ( a ) M o l a r r a t i o o f amino a c i d t o s u g a r v e r s u s r e s p o n s e ( 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  10.0  9.5  PH  20  70.7  86.0 TEMPERATURE  Figure'5  cont'd,  (  C)  ( c ) I n i t i a l pH v e r s u s r e s p o n s e . (d) R e a c t i o n t e m p e r a t u r e v e r s u s  response.  43  20  I 28.6  30.0  TIME (H)  Figure  5 cont'd,  (e) R e a c t i o n  time  versus  response.  44  18  36  (b)  27.<-  I  6  121~i28 2 3  i—«\-21  E E LU CO  z  o  0. CO LU CC  • 13,16  •3 20 40.7  60.0  CONCENTRATION (%)  Figure  6.  Maps of f a c t o r l e v e l v e r s u s r e s p o n s e f o r x y l o s e + l y s i n e , (a) M o l a r r a t i o of amino a c i d t o s u g a r v e r s u s r e s p o n s e ( 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 .  45  Figure  6 cont'd,  (c) I n i t i a l pH v e r s u s r e s p o n s e . (d) R e a c t i o n t e m p e r a t u r e v e r s u s  response.  46  Figure  6 cont'd,  (e) R e a c t i o n  time  versus  response.  47  the  factors  starting was  were  simplex  hoped  optimum,  substantial of  this  the  this  responses  levels  shift  from  t h e optimum,  were t h e n  of  factor  level  t h e new v e r t i c e s  + lysine,  two d i f f e r e n t  response  of  i t  vertices  showed  no  indicating  the f a i l u r e  carried  out,  that  simultaneous  shift  response The  only  result  the  exceeded  5,  shift  7  present shift  levels  t o be  the next  that 8  of  A1,  of  the  4,  which  response,  was l e s s  than  optimization  was  confirmed  result  produced was  factor  that  that starts  a  lower  required.  shift  experiment  the present best stopped  Since  dictates  the  to  of g l u c o s e  the  and  program  of the simultaneous  (Figures  6,  out u n t i l  vertex  mapping p r o c e d u r e reached  given  used  2 were t o be p e r f o r m e d .  the previous best  xylose + lysine  therefore  factor  A2  4,  combination  s h o u l d be c a r r i e d  than  were  o f w h i c h would d e t e r m i n e  vertices  simultaneous  deteriorating,  response,  had  from  and t h e l e v e l s  (A1 a n d A2) of f a c t o r  of combination  indicating  response,  ( T a b l e 8 ) . In t h e c a s e  the r e s u l t s  move. The r e s u l t  experiments  versus  obtained  r=33mm. T h e r e f o r e t h e s i m u l t a n e o u s  two c o m b i n a t i o n s  been  7),. A l t h o u g h  (T) v a l u e s ,  experiments  provided  final  a new  changed u s i n g the simultaneous  to the present best  generate  for  and  r a p i d movement toward t h e  the  p r o g r a m . The t a r g e t  corresponding  the  area  approach.  maps  best  that  ( T a b l e s 6,  would c a u s e  move t o w a r d  Factor factor  around  was g e n e r a t e d  that the  narrowed  at that  response, point. A  t h e optimum p o i n t had  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 after  f o r new  s t a r t i n g simplex  t h e mapping Glucose  procedure.  +  Lower Limit  Factor  established  Lysine  Xylose  +  Lysine  Upper Limit  Lower Limit  Upper Limit  1 .47  1 .88  XI  1 .00  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  1 . 94  X1 =  molar r a t i o  X2 =  t o t a l c o n c e n t r a t i o n of  X3 = i n t i a l  of amino a c i d  reactants  pH  X4 = r e a c t i o n  temperature  X5 = r e a c t i o n  time  (hours)  t o sugar  (°C)  (%  w/v)  Table  7. F a c t o r after  levels  of s t a r t i n g  t h e mapping  simplex  established  procedure. Factor  Vertex  XI  #  X1  X3  X4  X5  1  A B  1 .00 1 .47  50.00 47.81  1 0.00 10.00  79.7 85.0  29.00 27.80  2  A B  1 .89 1 .85  50.00 46.13  1 0.00 9.95  80.9 85.0  29.30 28.30  3  A B  1 .22 1 .56  50.00 40.70  1 0.00 9.95  84.7 85.0  29. 30 28.30  4  A B  1 .22 1 .56  50.00 46.13  10.00 9.81  80.9 85.0  29.90 28.30  5  A B  1 .56  46. 1 3  9.95  85.0  29.80  A = glucose B  X2  = xylose  = molar  + +  lysine lysine  r a t i o o f amino a c i d  X2 = t o t a l c o n c e n t r a t i o n X3 = i n i t i a l  of r e a c t a n t s  pH  X4 = r e a c t i o n  temperature  X5 = r e a c t i o n  time  t o sugar  (hours)  (°C)  (%  w/v)  Table  8. S i m u l t a n e o u s lysine  factor  shift  vertices  for glucose +  ( c o m b i n a t i o n A) and x y l o s e +  lysine  (combi nat i on B) . Fac t o r Combi n a t i o n A  X1  X2  X3  X4  X5  Al Shi f t 4 Shift 5 Shift 6 Shift 7 Shift 8  1  .42 1 .49 1 . 55 1 . 62 1 .69 1 .75  50 . 00 50.00 50.00 50. 00 50.00 50.00  10.00 10.00 1 0. 00 1 0. 00 10.00 10.00  82.4 82.8 83 . 1 83.4 83.7 84 . 1  29.60 29. 60 29.70 29.70 29.80 29.90  A2 Shift Shift Shift Shift Shift  1 . 42 1 .49 1 .55 1 .62 1 . 69 1 . 75  50.00 50.00 50.00 50.00 50.00 50.00  1 0. 00 1 0. 00 10.00 10.00 1 0. 00 10.00  83.7 83.4 83 . 1 82.8 82.4 82. 1  29.60 29.60 29.70 29.70 29.80 29.90  1.61 1 .62 1 .64 1 .66 1 .68 1 .69  48.44 48.72 49.00 9.28 49.56 49.85  10.00 1 0. 00 10.00 10.00 10.00 10.00  85.0 85.0 85.0 85.0 85.0 85.0  30.00 30.00 30.00 30.00 30.00 30.00  4 5 6 7 8  Combinat i o n B B1 Shift Shift Shift Shift Shift X1  4 5 6 7 8  = molar  X2 = t o t a l  ratio  o f amino a c i d  concentration  X3 = i n i t i a l  of reactants  pH  X4 = r e a c t i o n  temperature  X5 = r e a c t i o n  time  t o sugar  (hours)  (°C)  (%  w/v)  51 of  less  than  The  20 mm  the  amino a c i d  most  50%  was  10;  length  of  (xylose  + lysine)  length  reaction vertex was  28,  first  (initial  22  temperature) allowable indicated the to  possible Ashoor  and  point  beyond that  on  Maillard  nm)  of b r o w n i n g  Therefore  may  f a c t o r s X2  i t may  of  total 85°C;  optimum was  s e t of  reached  at  lysine  and  factor  this  X4  maximum  factors  clearly  suggesting  boundary.  that  If allowed  however,  it  begin  deteriorate.  found was  X3  (reaction  to  s t u d y of t h e e f f e c t  solutions  after  at t h e i r  limit,  would  intensity,  logical  out  ( c o n c e n t r a t i o n ) and  limit,  in their  carried  these  t h e upper  response  reaction be  maps  upper  browning  B  conditions  for xylose +  were f i x e d  l i e beyond  (1984),  the  18.  The  the  the  Zent  The  lysine  glucose + l y s i n e ,  toward  and  temperature  of t h e mapping p r o c e d u r e  values.  initial  c o n c e n t r a t i o n of  response  of x y l o s e + l y s i n e  a trend  reaction  h.  +  the  of  h. F o r c o m b i n a t i o n  of  30  t h e optimum  of  optimum proceed  time  when: t h e  83.7°C;  reaction  for glucose  experiments,  pH)  10;  A)  concentration  was  1.55;  t h e maps.  (combination  mixture;  29.55 set  on  mixture  the  reaction  ratio  reaction  a result  Maillard  was  pH  obtained with vertex As  the  time  initial  while  mixture  1.42;  t h e optimum  conditions  not a p p e a r  temperature  t o sugar  of  was  of t h e t o t a l  reaction  47.6%;  reaction  ratio  reaction  amino a c i d  reactants and  the  do  inhibitory  t o sugar  r e a c t a n t s was  was:  therefore  glucose + lysine  produced  pH  and  that  pH  also  of  absorbance  maximized a t  t o assume t h a t  is  pH  pH (420 10.  10 r e p r e s e n t e d  52  the  optimum  the  actual  unknown.  in t h i s response  For  the  knowledge t h a t the  upper  efficient  The  r e q u i r e d . The uniform  cam.  sample,  the  of  of  mixtures  stylus  uniform  the  entire  been  sample  cam  such  applied  at  a  the  the  manpower with  a  cm  agar  and  variable  plate.  The  a  50  M1  aureus spiral,  that  the  i s known  and  area with  the  point  the  edge of  at a constant effecting of agar the  no  to  deposited  such  of m a g n i t u d e  s u r f a c e at  challenged  and  the c e n t r e  the M a i l l a r d  t h a t each u n i t  plating materials  dilution  to  a  inhibition.  location  of  S.  the  provided  particular  S.  uniform  to d e p o s i t  amount, from  spiral  the  of  Archimedes s p i r a l ,  agar  of  c a p a c i t y of  semi-automated  three orders  deposited  was  at  inhibitory  deposits approximately  10  form of an  length  were  extremely  plater  reduces  rotating  from t h e  the  an  f o r measuring  i s equipped  of more t h a n  lifts  however,  factors  Utilization  is a  ever-decreasing  of  remains  r e a c t i o n mixture,  v a r i a b l e cam  beginning  factors  provided  spiral  system  same. C o n t i n o u s  The  the  model  sample  always the  from the  the  greatly  a  i s i n the  amount  of  the M a i l l a r d  The  i n an  edge  sample  DU  obtained,  study,  these  a n a l y z i n g the  plating  which  this  system  reproducible technique  technique  of  for  cams  spiral  these  maps  sufficient.  plating  c u l t u r e and and  of  reaction solution.  variable  rapid  of  From t h e  optimum p o i n t s of  method  each browning  aureus  purposes  the  spiral  as w e l l .  surface  b o u n d a r y was  The  and  study  reaction  i s obtained where the  the  plate.  rate  along  dilution  t o w h i c h MRP  of had  same number of c f u of  53  S.  aureus.  Using  this  browning  reaction  measuring no  browned  varying were  potencies  directly  of t h e  assessed  t h e c e n t r e of the p l a t e ,  by  i n which  a p p e a r e d . The r a d i u s measurements o f t h e optimum  of combinations  t h e minimum  the  mixtures  t h e r a d i u s , from  growth  vertices  technique,  inhibitory  solutions,  A and B were t h e n  concentrations  using  the formula  q = m(r)/r  x  W  V  /D  V  used  (MIC)  to calculate  o f e a c h of t h o s e  (Anon.,  x 1/2TTV  1980):  (2)  A  where,  q = MIC Ug/cf u) , m(r)  = radial  r a t e o f sample d e p o s i t i o n (ml/mm),  r  = radial  position  V  = m a t e r i a l weight  W D V  = culture  V  A  =  The were  c e n t r e o f p l a t e (mm),  per unit  deposited per unit  x  lO'Vg/cfu  Maillard  f o r glucose  of judging  the potency  mg o f t h e g l u c o s e  8.94  mg o f t h e x y l o s e + l y s i n e growth of 10  7  S.  + lysine  mixture.  + lysine Maillard  aureus  reaction  o f these.compounds  5.78  the  a r e a of  2  lO""Mg/ c f u f o r t h e x y l o s e + l y s i n e way  (yg/ml),  (ml/mm ).  MIC's f o r t h e optimum  5.78  volume o f sample  density (cfu/ml),  volume o f c u l t u r e plate  from  An  a n d 8.94 x alternative  i s t o say t h a t  browning mixture or  browning mixture  colony  mixtures  forming  will  units.  inhibit  54  B. SEPARATION  OF THE OPTIMIZED MAILLARD  Separation into  to  inhibitory the  glucose  way,  Maillard  + lysine  the  These some  weight the  S.  inhibited  restore  aureus.  in  identify  optimum M a i l l a r d fractions  molecular  combination  results  suggest  destroyed  reaction the  that  the  mixtures. lower  If  by t h e work o f Jemmali  some l o s s may  step. Also,  molecular  the  low  weight  to i n h i b i t  of t h o s e compounds  fractions,  compounds  the  as  e_t a l .  freeze-drying  exists  between t h e  compounds,  would  S.  procedure,  (1969) and H o r i k o s h i  relationship  lysine  result  l o s s of in  a  in activity.  rapid  s h o u l d be  Multiple determine  time  required  method o f f r a c t i o n a t i n g  for  ultrafiltration,  the M a i l l a r d  reaction  developed.  C. MULTIPLE REGRESSION  to  +  capacity  weight  of the  fractions  inhibitory  compounds and o t h e r  Because of the l o n g  products  failed  an  fractions  a l l five  have o c c u r r e d d u r i n g  weight  molecular  the  in  range  the s e p a r a t i o n  molecular  i f a synergistic  low  more  mixtures  or the x y l o s e  inhibitory  suggested  a  weight  o r i g i n a l mixture a l s o  in  decrease  reaction  was p e r f o r m e d  Pooling  aureus.  existed  (1981),  MIXTURES  compound(s) . However, none o f t h e f i v e  combination to  the  various molecular  attempt  of  of  REACTION  regression  ANALYSIS  analysis  the s i g n i f i c a n c e  of the data  was  of each of t h e f i v e  performed reaction  55 conditions reaction The  with  the m u l t i p l e  one  model on  variable the  selection  of t h e  variables a  each  The  that  stepwise  outside  the  variable  i n t h e model  level.  The  calculating  model  ends  has  statistics  contribution  t o t h e model  until  all  the  F-statistics  significant  predetermined The  the  remaining  a t t h e 0.05  not the  method  deletes  any  F-statistic.  the  variables  F-statistic  and  every  predetermined  procedure  variable  in  stepwise  the  the  forward  included  and  at  i s deleted.  variables  a r e added t o  of  f o r t h e model  The  sequentially  Unlike  none  elimination  variables.  procedure  a significant  is significant  independent  selection  model  when  of  stepwise  i s added, t h e the  method  s t e p w i s e method d o e s  a significant  backward the  in  aureus.  5.  i n t h e model and  not p r o d u c e  procedure  Maillard  The  once t h e y a r e  variable  does  used.  forward  however, t h e  examines a l l the v a r i a b l e s variable  were  F-statistics.  retain variables  After  of  of  time. V a r i a b l e s  of t h e i r  procedure,  necessarily model.  basis  the growth  procedure  at  production  t h e backward e l i m i n a t i o n  is a modification w i t h no  the  inhibit  method and regression  which b e g i n s adds  to  compounds w h i c h  stepwise  method  respect  begins  by  a l l  the  including showing  the  smallest  Variables  are  deleted  in  t h e model  level  (or  produce  some  other  level).  results  of the  the  regression  variables  on  combination  A,  elimination  method, v a r i a b l e s  are  dependent  shown  in  Table  of  variable 9.  the  independent  inhibition,  Using  were e l i m i n a t e d  for  the  backward  from  t h e model  Table  Step  9. M u l t i p l e regression  R*  Variables Removed  of  the  Independent  Source of Var1 a t 1 on  variables  X1 t h r o u g h  D e g r e e s of Freedom  X5 on I n h i b i t i o n  Sum o f Squares  F  (glucose  +  lysine)  B Va 1 ue  None  R e g r e s s 1 on Error Total  5 23 28  937 . 302 177 . 6 6 3 1114 965  24 . 27  I n t . - 8 9 . 689 X1 - 0 . 593 X2 0 . 386 X3 8 . 474' X4 0 . 0O2 X5 0 . 007  0.837 •  X5  R e g r e s s Ion Error Total  4 24 28  933 . 3 3 5 181 . 6 3 0 1114 . 9 6 5  30.83  Int . X1 X2 X3 X4  2  0.822  X4  Regress1on Error Total  3 25 28  916 .717 198 . 248 1114 . 9 6 5  38 . 53  I n t . - 8 6 . 310 X1 - 0 . 434 X2 0 . 488 X3 9 . 367  3  0.808  X1  R e g r e s s 1 on Error Total  2 26 28  9 0 0 . 968 213 .997 1114.. 965  54 . 73  Int . X2 X3  0  0.841  1  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 Int. = Intercept  at  the 0 . 0 5  level  - 9 0 . 180 - 0 . 583 0 . 443 8 . 851 0 . 002  - 8 5 . 1 13 0 . 474 9 . 182  57  in  the  following  temperature  order:  ( X 4 ) , molar  reaction  ratio  c o n c e n t r a t i o n ) and X3  (initial  0.05  square  level.  coefficient, independent  The R ,  was  2  0.84  variables,  for  glucose  regression  indicates the  the  production  lack  of  major  greatly  affect  to  S.  of  since  (total at  the  correlation all  five  f o r t h e model c o n t a i n i n g The  in Figure  effect  the  X2  significant multiple  variables.  regression  7. The  i t is  condition  XI,  known  X4,  that  i n the p r o d u c t i o n  these  pH,  had on  products. and  X5  these  d e g r e e of b r o w n i n g d u r i n g that  model  initial  reaction  variables  the  comparatively  X3,  this reaction  I t a p p e a r s , however,  The was  factors  the M a i l l a r d  factors  have  much  of compounds  antimicrobial  examined u s i n g  t h e backward  aur e us .  Xylose elimination the  model  that  the  ratio,  +  lysine  procedure including  was  variables regression  X3,  was X4  10). S t a t i s t i c s  coefficient  for  to the r u l e s was  the o n l y and  variable  coefficient  first.  other  variable  for  within  X1,  from  Variable  X1,  molar  by t h e c o m p u t e r .  X2,  removed,  t h e m o d e l . To  variable  for  indicated  for elimination  removed  X5  calculated  variables  as t o be u n d e t e c t a b l e  , this variable  concentration,  also  a l l the independent  so s m a l l according  was (Table  regression  Therefore, model  the  of i n h i b i t o r y M a i l l a r d  surprising  importance  were  reaction  variables  coefficient for variable  somewhat  less  of  i s shown  significance  reaction.  pH)  and 0.81  s i g n i f i c a n t independent  large  (X1). Only  (X5),  f o r t h e model c o n t a i n i n g  two  + lysine  time  the  the total  leaving  verify.the stepwise  58  A:  Y = 0.474X R  B:  2  R  Figure  3  - 85.113  (3)  = 0.81  Y = 7.259X 2  + 9.182X  2  2 3  + 0 .-631X, + 0.01 5 X  2 5  - 1 07 . 61 2  (4)  = 0.79  7. M u l t i p l e r e g r e s s i o n m o d e l s f o r g l u c o s e + l y s i n e (A) and x y l o s e + l y s i n e (B) model s y s t e m s .  Table  10. M u l t i p l e r e g r e s s i o n  Variables Step  R'  0  X1  0.800  2  Int.  None  0.800  1  All  Removed  X2  0.790  variables =  of  Independent  Source  of  Degrees  V a M a t Ion  R e g r e s s 1 on  X1 t h r o u g h  of  X5 on i n h i b i t i o n  Sum  (xylose +  lysine)  of  Freedom  Squares  F  4  66 1 . 4 3 2  22 . 98  B Value  Int .  Error  23  165.532  X1  Total  27  826.964  X2  -96.08 1 0.000 0 . 124  X3  5.855  X4  0.597  X5  0.014  4  66 1 . 4 3 2  Error  23  165.532  X2  Total  27  826.964  X3  5.855  X4  0.587  X5  0.014  Regression  Regression  3  653.675  22 . 98  3 0 . 18  Int .  -96.081 0 . 124  Int . - 1 0 7 . 6 1 2  Error  24  173 . 289  X3  7 . 259  Total  27  826.964  X4  0.631  X5  0.015  1n t h e model a r e s i g n i f i c a n t  Intercept.  variables  at  the 0 . 0 5  level  60  regression  p r o c e d u r e was  performed  on the same d a t a .  the  backward  elimination procedure,  only  variables  retained  this  three-variable  the  +  lysine,  the large  indicated of  lysine  that  is  shown  to that  in Figure  regression  i t played  an  important  compared,  combinations  large  computed f o r  of sugar  and  amino  concentration),  the  only  the  the  glucose  model  for  significant (xylose  enough t o remain  + lysine).  temperature) model  and  On X5  f o r combination  significant  acid.  A  the r e g r e s s i o n  production variables  Factor  X2  (total  t h a n pH t o remain i n mixture,  X4  not B  (reaction  retained  were n o t +  was  f o r combination  t i m e ) were  (glucose  X3  between the two  hand, f a c t o r s  B, whereas t h e y  i n combination  Although  existed  lysine  (reaction  variable  other  i n t h e model  the other  model f o r  r o l e i n the  f a c t o r other +  for  when t h e  differences  for  7. As w i t h g l u c o s e +  coefficient  i n h i b i t o r y compounds. However,  were  as  identical  value  2  backward e l i m i n a t i o n p r o c e d u r e . The r e g r e s s i o n  xylose  with  X3, X4 and X5 were t h e  i n t h e m o d e l , and t h e R  model was  As  by t h e  statistically  lysine).  coefficient  i s usually  regarded  a measure of t h e 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 t h e  r e s p o n s e , Y, B a r y l k o - P i k i e l n a using  the  variable,  contributing  and M e t e l s k i  proportion  P j , as a measure of t h e  of  (1964) each  variables'  to  t h e r e s p o n s e . P^ c a n be c a l c u l a t e d a c c o r d i n g  (5)  where  deviation correlation  i s the r e g r e s s i o n of e a c h  independent  coefficient  coefficient,  suggested independent  contributions to equation  s^ t h e  standard  v a r i a b l e , and r ^ y t h e p a r t i a l  between X^ and  Y.  The  sum  of  P^  61 equals  100 x  R. 2  Pi  The P^ v a l u e s lysine  and  including 11.  In  =  \a  xylose  the  +  concentration  of  the  respectively).  overall ratio  the  response,  existed  t o S.  the r e s u l t s  and  these  of s u g a r ,  positive  temperature  response,  had  factors  inhibitory  for  the  A,  made  very  factor  were  compounds  such was  an  inverse  production  ranged  of  from  1.00 t o  indicate acid,  that  a  would  i n h i b i t i o n . Time  effects  on  the  and l o w e r  maximum  possible  39.73%, negative  that  than amino  that  largest  t h e v a l u e s of t h e  analysis  upper  pH and t o t a l  small  increasing  the  i n Table  ( 4 4 . 4 4 % and  and  +  model  by f a r , t h e  Since  small  that  inital  a  and s u g a r  on  a r e shown  indicating  rather  effect  indicating  two  calculated  of t h e r e g r e s s i o n  proportion a  glucose  aureus.  2.50,  (5)  2  for  between t h i s  inhibitory  had  | x 100R  response  r a t i o between amino a c i d  have  y  factors  combination  molar  greater  i  variables,  Molar to  relationship  r  of r e a c t a n t s c o n t r i b u t e d , to  contribution  lysine,  independent  case  proportions  Ila^i  f o r e a c h of t h e f i v e  a l l five  compounds  sjriyl/  i  overall l i m i t s of  production  anywhere  within  of  those  ranges. As most  with  glucose  +  i n f l u e n t i a l factors  inhibitory  compounds  lysine,  initial  in determining  by x y l o s e  pH was  the  one of t h e  production  + l y s i n e . These  of  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 toward t h e p r o d u c t i o n compounds  inhibitory  optimized  browning  (P^) of e a c h  of M a i l l a r d t o 5. aureus  factor  reaction i n the  mixtures.  Pi(%) Factor  X1  Glucose  + Lysine  Xylose  + Lysine  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  = molar  X2 = t o t a l  ratio  of amino a c i d  concentration  X3 = i n i t i a l  of r e a c t a n t s  pH  X4 = r e a c t i o n t e m p e r a t u r e X5 = r e a c t i o n time  t o sugar  (hours)  (°C)  (%  w/v)  63 consistent during  with  their  interactions that and  this  the  findings  study  . Previous  reaction  is  negative  the  aldose  in  hydrogen  less,  (Katchalsky,  between u n d i s s o c i a t e d amino  groups  by HR  form  marked  to  of t h e amino  (eg. NH CH COO~), +  3  2  2  2  solution.  negative  the  acid  or  i t i s only  combine  Therefore,  ion i s markedly  concentration,  contrast  to  the  response  with  since  the  influenced  importance  glucose  factors  (35.98%).  approximately glucose  +  twice  lysine  between t h e  two  for  by  of pH  is  some  and  time  factor  X2  in  in  the  factor  X5  the  be  the may  be  mixture.  of  than  of  explained  total the  I t has  been  have a  amount  r a t e than  in  by  reaction  less  was  large variance  sugar-types.  total  five  time)  importance  may  reaction  in a similar  X4'(reaction  (reaction  0 . 1 5 % ) . The  two  much  p r o p o r t i o n of a l l  Maillard  mixture  overall  contributed  that aldopentoses  therefore  reaction  the  present  versus  of t h e  lysine,  in xylose + lysine  reactants,  reactivity  i n the  determining  (0.31%  of  aldohexoses,  factor  important  in r e a c t i v i t y  recognized d e g r e e of  as  and  largest  combinations  concentration difference  Also,  +  lysine  (11.79%)  t e m p e r a t u r e ) c o n t r i b u t e d the  glucose  1941)  acid shown  c o n c e n t r a t i o n ) of x y l o s e +  present  (1953)  apparent.  In (total  Sharon  of a l d o s e - a m i n o  ( e g . N H C H C O C r ) w h i c h can  the  ion  and  had  aqueous  of  kinetics  zwitterionic  i o n R'  concentration  readily  work  represented  the  the  the  occurs  a l d o s e s . I f the  peptide  of  of K a t c h a l s k y  higher  than of  do  xylose  importance  in  t h e amount  of  64 The  large  temperature xylose  difference  (0.06%  + lysine)  suggest  for  is  that,  within  combination  responsive  to  work  pentoses Kinetic  the  easily the  B,  are  more  studies,  e_t  of  reaction  versus  explained.  temperature  35.98% f o r  The  range  c o n t a i n i n g x y l o s e , was  a temperature  (Knipfel  importance  glucose + lysine  less  experiment,  Other  in  change t h a n  al. ,  1983)  temperature  however, a r e  was  of  this  much  more  combination  also  suggests  sensitive  required  results  than  to f u l l y  A.  that  hexoses.  explain  this  phenomenon.  D.  GENERAL DISCUSSION  The  complexity  difficult, mode  of  bacterial  indeed  of the M a i l l a r d  impossible at t h i s  action  of  these  reaction  makes i t v e r y  stage, to d e s c r i b e  compounds  on  the  inhibition  g r o w t h . S e v e r a l a u t h o r s have expounded  theories.  Lewis  (1930) c o n c l u d e d  that  readily  Baumgartner toxic  (1938) a t t r i b u t e d  compound p r o d u c e d  Lankford that  assimilated  by  certain  or  of  g l u c o s e d e g r a d a t i o n was  the  However, F i n k e l s t e i n  Lankford  that  bacteriotoxic  and  substances  likely  were  own  due  to  forms  bacteria,  while  during s t e r i l i z a t i o n  destruction  was  of  into  i t to the d i r e c t  e_t a l . (1957) e x p a n d e d on L e w i s '  binding  their  inhibition  t h e 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 not  the  action  of  a  o f t h e medium.  work and  cystine  by  cause  of  (1957) went on responsible.  concluded  products  of  inhibition. to  conclude  65  Other inhibition adsorption  suggestions by  Maillard  chelation  growth  apparent inhibit  of  these that  work t o w a r d  ions  H o r i k o s h i , 1976;  tying  will  not  be  mechanisms  physical  surfaces via  opposing  Slyusarenko,  necessary Leite  for  1976)  bacterial  e_t a l . , 1979).  and  Maillard  easily  of  include:  and  suggestions  t h e q u e s t i o n of how  bacteria,  products  (Shvets  metallic  varied  possible  to b a c t e r i a l  potentials  (Gomyo and  From  reaction  of m e l a n o i d i n s  electrokinetic and,  explaining  opinions reaction  answered.  t h e s e many t h e o r i e s t o g e t h e r  it  is  products Extensive  is required.  V.  Simplex  CONCLUSIONS  optimization  proved  technique  f o r the o p t i m i z a t i o n of  reaction  products  which system  study  may  as  direct  quantification  reason, require  not  be  the  efficient  t h e g r o w t h of S.  aureus.  the  one  used  in  this  a measure of change as  specific  compound.  appropriate  factor  For  the this  limits  may  preliminary experimentation.  The  spiral  reproducible Maillard  plating  procedure  reaction  Quadratic  and  five  were v e r y  importance Maillard  the  of e a c h  reaction  Staphylococcus  useful  factor  . The  potency  reaction to i d e n t i f y  inhibit  of  the  relative  most  growth  of  important  factors  for  compounds  by  the  c o n c e n t r a t i o n and  lysine  model  system,  glucose initial they  + pH,  were  temperature. the m o l e c u l a r  weight  range  compounds a  and  the  of  of  each  r e s p e c t t o t h e p r o d u c t i o n of  compounds were u n s u c c e s s f u l , p o s s i b l y  Development  of  the  inhibitory these  simple,  analysis  determining  two  were t o t a l  whereas f o r the x y l o s e +  Attempts  a  p r o p o r t i o n of e a c h  which  inhibitory  system  and  in  with  products  t h e p r o d u c t i o n of  pH  the  multiple regression  contributing  aureus  l y s i n e model  provided  f o r measuring  linear  of  factors  system  mixture.  calculation  initial  a  an  Maillard  as  sensitive of  establishing  such  be  t h e p r o d u c t i o n of  inhibit  However, a m i c r o b i a l  to  during  the  separation  66  fractionation  procedure  which  due  of  the  to  loss  procedure. allows  the  67  inhibitory The  Maillard  experiment Further compounds food  compounds  produced  study  may  t o remain reaction highly  intact model  potent  ultimately  as n a t u r a l l y - o c c u r r i n g  systems.  would  be  systems  desirable.  studied  antimicrobial lead  to  the  preservatives  in  this  compounds. use of in  these  selected  VI.  Anonymous. of  Spiral plate  material  Spiral Ashoor,  1980.  interactions  and  Zent,  common amino a c i d s Barylko-Pikielna,  N.  contribution quality.  P.A.,  36:  attainment B.  J.P.  Research", York,  Maryland.  Maillard  K.  1964.  sterilized  thermal  and  de  of  1206.  Determination  scoring  of  of  over-all  reducing sugars  resistance  and  of b a c t e r i a .  Andrade L e i t e ,  P r e p a r a t i o n and  g l u c o s e and  Wilson,  fructose  J.  S.R.  1981.  characterization with g l y c i n e .  of An.  83.  K.B.  1951.  of optimum c o n d i t i o n s .  and  browning  109.  C i e n c . 53:  13:  microorganisms.  s u g a r s . J . Food S c i . 49:-  Heat  the  evaluation  369.  from  G.E.P. and  Danehy,  1938.  R e a c t i o n V:  Acad. B r a s i l .  Series  1984.  Metelski,  Imasato, H.  melanoidins  Box,  and  and  e f f e c t s on  Maillard  J.B.  i n the  test  c o e f f i c i e n t s in sensory  J.G.  Bacterid.  with  Bethesda,  J . - F o o d - S c i . 29:  Baumgartner,  Bobbio,  computations  Systems M a r k e t i n g ,  S.H.  their  REFERENCES  On  the  J . Royal  experimental Statist.  Soc.  1. Pigman,  v o l . 3,  pp.  W.W.  1951.  241-290.  N.Y.  68  "Advances  Academic  i n Food  Press,  New  69  De F i g u e i r e d o reaction  T o l e d o , M.C. and B o b b i o , P.A.  1981.  V I I : Browning  lysine,  and g l u t a m i c Deming,  with  a c i d . An. A c a d . B r a s i l .  Cienc.  glycine  53: 707.  S.N. and Morgan, S.L. 1973. S i m p l e x o p t i m i z a t i o n o f  variables Eichner,  in analytical  K.  content  and  Agric.  Karel,  and w a t e r  reaction  in  chemistry. M.  1972.  activity  model  Anal.  Chem. 45: 278A.  The i n f l u e n c e o f water  on t h e  sugar-amino  systems under v a r i o u s  browning  conditions. J .  Food Chem. 20: 218.  Einarsson,  H.,  Inhibition products. Eskin,  of glucose  Maillard  Snygg,  of  B.G.,  bacterial  and  growth  Eriksson,  by  C.  Maillard  1983.  reaction  J . A g r i c . Food Chem. 31: 1043.  N.A.M., H e n d e r s o n ,  "Biochemistry  H.M.,  and  Townsend,  R.J.  o f F o o d s " , pp. 83-108, Academic  1971.  Press,  New  York. Finkelstein,  R.A.  substance  and L a n k f o r d ,  in autoclaved  and p h o s p h a t e . A p p l . Fisher,  M.N.  and Bundt,  Natur  der  Bac.  enterit.  C.E. 1957. A b a c t e r i o t o x i c  c u l t u r e media  containing  glucose  M i c r o b i o l . 5: 74. A.J.  Microben  1928.  des  die  biochemische  P a r a t y p h u s B. s c h o t m u l l e r  Breslau,  Differenzierungsmedium  Uber  fur  sowie  uber  dieselben.  ein  und neues  Biochem.  Zeit.  isolation  and  198: 428. Foster,  R.C.  1980.  Preliminary  70 characterization Maillard Thesis, Fulmer,  of  reaction  an  between  Oklahoma S t a t e  E.I., Williams,  effect  of  promoting  antioxidative arginine  University,  A . L . and  sterilization  properties  and  of the  xylose,  M.S.  S t i l l w a t e r , Oklahoma.  Werkman,  of  toward  component  media  C.H. upon  bacteria.  1931. their  The  growth  J. Bacterid.  21:  299. Gomyo, T. a n d H o r i k o s h i , melanoidin  with  M.  1976.  metallic  On  ions.  the Agric.  interaction Biol.  of  Chem. 40:  33. Hill,  E.G.  and P a t t o n , A.R.  microbiological Hodge,  J.E.  Hodge,  J.E.  and  S.  87.  J . Agric.  E.M.  (ed.),pp.  of  browning  F o o d Chem. 1: 928.  1976.  Carbohydrates.  1.  Food  in  Chemistry",  41-137, M a r c e l D e k k e r , New  1981. E f f e c t s o f b r o w n i n g  H.,  reaction from  foods. Chemistry  York.  M. , Ohmura, M., Gomyo, T., Kuwabara, Y. a n d Ueda,  microflora Imasato,  Osman,  reaction in  105: 481.  o f Food S c i e n c e . P a r t  Fennema  Horikoshi,  Dehydrated  i n model s y s t e m s .  "Principles O.R.  assay. Science  1953.  reactions  1947. The M a i l l a r d  of the r a t . Prog. Leite,  Food N u t r .  S.R.A. a n d B o b b i o ,  VI. Structural  fructose  products  S c i . 5: 223.  P.A. 1981. M a i l l a r d  determinations  and g l y c i n e .  on t h e i n t e s t i n a l  in  An. A c a d . B r a s i l .  melanoidins C i e n c . 53:  71  J e m m a l i , M.  1967. I n f l u e n c e d e s p r o d u i t s de l a  Maillard  sur  decarboxylase 264: Jemmali,  pyruvate  decarboxylase  M.  1969. on  Influence  some  of  the  G., Y o s h i d a ,  V.  browning  reaction  products  from  Katchalsky, acids  metals.  A. or  Katchalsky,  Reaction  1941.  Interaction 3.  of aldoses  Determination  of  with  a-amino  equilibrium  B i o c h e m . J . 35: 1024.  interaction.  Biochim.  Biophys.  substances  by  heat  Ferment. T e c h n o l . J.E.,  Nutritional  to  Reaction Feather  of  10: 290. of a n t i m i c r o b i a l  xylose  solution. J.  52: 177.  McLeod,  value  Relationship  treatment  of a l d o s e - a m i n o  Acta  K a t o , N. and S h i b a s a k i , I . 1974. P r o d u c t i o n  M.S.  the  g l y c i n e and r e d u c i n g  A. and S h a r o n , N. 1953. K i n e t i c s  Maillard  between  J . J a p a n O i l Chem. S o c . 24: 15.  peptides.  constants.  flora. J.  H. and T a k a m o r i , Y. 1975. S t u d i e s on complex.  sugars.and  reaction  32: 151.  protein  and  S e r . D.  Maillard  b a c t e r i a of the i n t e s t i n a l  the metal  Knipfel,  et l a pyruvate  o x y d a t i v e . C.R. A c a d . S c i . , P a r i s ,  Bacteriol.  Kajimoto,  acid  de  2672.  products Appl.  la  reaction  J.G.  of foods  and  and f e e d s  composition  and  McCaig, of  1983.  plant  origin:  processing.  i n "The  i n Foods and N u t r i t i o n " , (eds.),pp.  T.N.  361-377,  G.R. ACS  Waller  Symposium  72  Series Lankford,  215, Amer. Chem. S o c . W a s h i n g t o n , C.E., R a v e l ,  effect  of  glucose  assimilation Lee,  CM.,  J.A.  and  Ramsey,  and h e a t  H.H.  sterilization  of c y s t e i n e . A p p l . M i c r o b i o l .  L e e , T.C. and C h i c h e s t e r , C O .  the p r o d u c t i o n products  D.C.  of b i o l o g i c a l l y  in apricot  1957. on  The  bacterial  5: 65.  1979. K i n e t i c s o f  active  Maillard  and g l u c o s e - L - t r y p t o p h a n .  browned J . Agric.  Food Chem. 27: 478. Leite,  S.R.A., I m a s a t o , H. and B o b b i o ,  of Staphylococcus obtained Rev. Lewis,  Microbiol.  I.M.  culture Long,  from  (S. Paulo)  1969. S i m p l e x  between  1979. I n h i b i t i o n by  a  glucose  melanoidin and g l y c i n e .  10: 100. o f Phytomonas  malvaceara  J . Bacteriol.  in  19: 423.  o p t i m i z a t i o n of t h e r e s p o n s e  s y s t e m s . A n a l . Chim. A c t a  L.C  formation  McKeen,  reaction  growth  media c o n t a i n i n g s u g a r s .  chemical  Rend.  a  S6  1930. The i n h i b i t i o n  D.E.  Maillard,  aureus  P.A.  from  46: 193.  1912. A c t i o n d e s a c i d s a m i n e s s u r l e s s u c r e s : des m e l a n o i d i n e s  par v o i e  methodique.  Compt.  154: 66. W.E.  dextrose  1956. toxic  Interaction t o Phytophthora  product  of  fragaraie.  glycine  and  Science  123:  509. Mills,  F.D.,  Baker,  B.G.  and  Hodge,  J . E . 1969. Amadori  73  compounds a s n o n v o l a t i l e foods. J . A g r i c . Mirna,  products  additives, Prod., Molund,  Food Chem.  A. a n d C o r e t t i ,  reaction  V.P.  1976. I n h i b i t o r y  and of d e g r a d a t i o n  and  a  of  with  food  2nd I n t . Symp. N i t r i t e  Meat  model Food  a  products  of  careinogen-induced  coffee,  melanoidin.  Science,  University  Nakai,  of  British  optimization  of  m e t h o d s . A n a l . Chem. 46: 1170.  S. 1982. C o m p a r i s o n  application  Ph.D. T h e s i s ,  Columbia.  Morgan, S.L. and Deming, S.N. 1974. S i m p l e x chemical  water-insoluble  system  Vancouver, B r i t i s h  analytical  of n i t r i t e of  Inhibition  responses  Department  effect  Pudoc, W a g e n i n g e n . 1985.  biological  Columbia,  K.  precursors i n processed  17: 723.  pp. 39-45, P r o c .  Zeist,  fraction  flavour  of  optimization  to food product  techniques  for  and p r o c e s s development. J .  Food S c i . 47: 144. Nakai,  S.,  Koide,  super-simplex  K.  a n d E u g s t e r , K. 1984. A new mapping  o p t i m i z a t i o n f o r food product  and  process  d e v e l o p m e n t . J . F o o d S c i . 49: 1143. Namiki,  M. a n d H a y a s h i ,  radicals  during  the  w i t h amino a c i d s . Nelder,  J.A.  function  and  T. 1975. Development amino-carbonyl  J . Agric.  Mead,  R.  of  novel  reaction  free  of sugars  F o o d Chem. 23: 487. 1965.  m i n i m i z a t i o n . Computer  A  simplex  J . 7: 308.  method f o r  74  Omura, H,  Jahan,  Formation Maillard and  Patton, by  Reaction  215.  K.  and  (eds),  Hill,  E.G.  537-563,  L.R.,  Morgan,  optimization absorption A.A.  of  S.L.  1982.  "SAS  Statistical  and  106:  User's  A n a l y s i s System  of  Waller  nutrients  68. 1975.  Simplex  in  atomic  factors  Statistics",  (SAS)  "The  Symposium  A p p l . S p e c t r o s c . 29: Guide:  in  D.C.  Deming, S.N.  experimental  spectrometry.  ACS  Inactivation  h e a t i n g with g l u c o s e . Science  1983.  G.R.  Washington,  1948.  H.  reaction,  Nutrition",  pp.  Amer. Chem. Soc.,  and  Murakami,  the M a i l l a r d  i n Foods and  Feather  A.R.  Parker,  Ray,  Shinohara,  of mutagens by  M.S.  Series  N.,  429.  pp.  Institute  101-110,  Inc.,  Cary,  NC. Rogers,  D.,  King,  stimulation  T.S.  and  C h e l d e l i n , V.H.  Lactobacillus  of  N-D-glucosylglycine.  Proc.  1953.  Growth  gayoni  Soc.  Exptl.  Biol.  by  Med.  82:  1 40. R o u t h , M.W., of  the  Shvets,  Swartz, super  V.N.  melanoidin cerevisiae. Kiev.  P.A.  and  D e n t o n , M.B.  modified simplex. and  Slyusarenko,  f o r m a t i o n and  Mikrobiologiya,  12:  from 73.  Performance  A n a l . Chem. 49: T.P.  1976.  Inst,  of  Saccharomyces  of t h e Food  Prikladnaya  1422.  Effects  c a r a m e l i z a t i o n on  Technological Translated  1977.  Industry,  Biokhimiya  i  75  Spendley,  W.,.  Hext,  Sequential  application  optimization '  G.R.  and  and of  Himsworth, simplex  F.R.  1962.  designs  evolutionary operation.  in  Technometrics  4: 441.  Stadtman, E.R. 1948. "Advances 325-372, Academic Waller,  G.R.,  for  arginine-xylose Maillard and  Yarbro,  Chim. A c t a M.,  reaction  Yazima,  Adeleye, of  B.O.  products.  i n Foods and N u t r i t i o n " , pp.  and  Deming,  125-140,  of f a c t o r s  S.N.  in  G.R.  "The  Waller  ACS Symposium D.C.  1974.  f o r simplex  1983.  antioxidative  215, Amer. Chem. S o c . , W a s h i n g t o n , L.A.  Abstr.  and  reaction  (eds),  v o l . 1 , pp.  York.  synthesis  Maillard  Feather  preprocessing  Yazima,  R.W.  the  Reaction  M.S.  Series  P r e s s , New  Beckel,  Conditions  i n Food R e s e a r c h " ,  Selection  and  optimization. Anal.  73: 391. Ohta,  R.  and  Akatsuka,  s o l u t i o n s . Japan.  Kokai  S.  Patent  1974.  Maillard  74 61,313. Chem.  81: 176153p.  M.,  Ohta,  from M a i l l a r d  R.  and A k a t s u k a ,  reaction  S. 1975a.  mixtures. Japan.  Kokai  Antibiotics Patent  74  71,121. Chem. A b s t r . 82: 21807b. Y a z i m a , M., O h t a , R. substances Patent  from  and  Akatsuka,  Maillard  reaction  S.  1975b.  Antibiotic  m i x t u r e s . Japan.  74 75,501. Chem. A b s t r . 82: 64540b.  Kokai  76  Y a z i m a , M., Maillard  Ohta,  R.  and  reaction  66,601. Chem. A b s t r .  Akatsuka, mixtures. 83:  S.  1975c. F u n g i c i d e s  Japan.  176642p.  Kokai  Patent  from 74  

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