Microbiological and biochemical aspects of the fermentation of milk with saccharomycopsis lipolytica

MICROBIOLOGICAL AND BIOCHEMICAL ASPECTS OF THE FERMENTATION OF MILK WITH SACCHAROMYCOPSIS LIPOLYTICA

•

C T.VERRIPS

MICROBIOLOGICAL AND BIOCHEMICAL ASPECTS OF THE FERMENTATION OF MILK WITH SACCHAROMYCOPSIS LIPOLYTICA

n "o +- O I-* Oc ^ o o o BIBLIOTHEEK TU Delft P 1804 5107 543444

MICROBIOLOGICAL AND BIOCHEMICAL ASPECTS OF THE FERMENTATION OF MILK WITH SACCHAROMYCOPSIS LIPOLYTICA

PROEFSCHRIFT

TER VERKRIIGING VAN DE GRAAD VAN DOCTOR IN DE TECHNISCHE WETFNSCHAPPEN AAN DE TECHNISCHE HOGESCHOOL DELET, OP GEZAG VAN DE RECTOR MAGNIFICUS IR H B BOEREMA, HOOGLERAAR

IN DE AEDELING DER ELEKTROTECHNIEK, VOOR EEN COMMISSIE AANGEWEZEN DOOR HET COLLEGE

VAN DEKANEN, TE VERDEDIGLN OP DINSDAG 27 MEI 1975 TE 14.00 UUR

DOOR

CORNELIS THEODORUS VERRIPS SCHEIKUNDIG INGENIEUR

Aan mijn ouders,

CONTENTS

1 GENERAL INTRODUCTION 9

2 CHEESE MAKING 10 . 1 Introduction 10 .2 The microbial s t a r t e r s used in cheese making 10

.3 Some chemical and biochemical aspects of cheese ripening 12

.4 Calf rennin and its r e p l a c e r s 15 .5 Some important aspects of the p r o p e r t i e s of milk 16

.1 Milk fat 16 .2 Milk p r o t e i n s 19 3 SACCHAROMYCOPSIS LIPOLYTICA, A MICRO-ORGANISM WHICH

MAY BE USED FOR RAPID CHEESE MAKING 26

.1 Screening, isolation and taxonomy 26 .2 Some important biological and biochemical properties of the s e

-lected yeast s t r a i n 29 .3 Isolation and characterization of the exolipaseproduced by S a c c h a

-r o m y c o p s i s l i p o l y t i c a 36 .4 The specificity of S a c c h a r o m y c o p s i s l i p o l y t i c a lipase 46

.5 Purification andpropertiesof theexoproteaseof S a c c h a r o m y

-c o p s i s l i p o l y t i -c a 61 ,6 Analysis of s o m e p a r t i c u l a r aspects of growthandproduction of lipase

and protease in S a c c h a r o m y c o p s i s l i p o l y t i c a 72 4 GROWTH OF SACCHAROMYCOPSIS LIPOLYTICA IN BATCH

CUL-TURES ON MILK 77 .1 Introduction 77 .2 Growth p a r a m e t e r s 78 5 COMPARISON BETWEEN BATCH AND CONTINUOUS

FERMENTA-TION OF MILK BY SACCHAROMYCOPSIS LIPOLYTICA 87

. 1 Introduction 87

CHAPTER 1 - GENERAL INTRODUCTION

For many years a great number of - mainly industrial - laboratories have investigated the very complicated process of cheese ripening as well as the substrate for cheese making, i.e. milk.

The amount of cheese produced every year is enormous: in the original six E.E.C. countries over 2,000,000 tons (1969) in an innumerable variety. The industrial production of cheese is quite common nowadays, but from an economic point of view it has one serious drawback, namely the time-con-suming ripening process.

This thesis describes a technique to accelerate this process.

T a b l e 2 . 1 - S u r v e y of s o m e i m p o r t a n t c h e e s e s a n d t h e i r p r o c e s s i n g

Milk

£

Milk ripened and treated with rennet

Curd scalded

31

P r e s s e d

T

Cheddar Emmental P a r m e s a n Port du salut

Unripened milk, treated with rennet

r

i

Curd scalded 1 Pressed 1 Not pressed Brick Edam Gouda

Curd not scalded

Caciocavallo Pecorino

T

Not pressed

£

Surface ripened Brie Camembert Coulommiers Limburg Neufchatel Internal mould-ripened Stilton Roquefort Blue Gorgonzola

T a b l e 2 . 2 - C o r r e l a t i o n b e t w e e n t h e s t a r t e r a n d t h e c h e e s e f l a v o u r

CHEESE STARTER *)

FLAVOUR CONTRIBUTION BY PRODUCTION OF

Cheddar and similar hard pressed cheeses

Herrgard

Bfinza

Emmental and similar varieties

S t r e p t o c o c c u s l a c t i s Mainly acid S t r e p t o c o c c u s c r e m o r i s Mainly acid S t r e p t o c o c c u s l a c t i s (var.faecalis) Flavotir and acid S t r e p t o c u c c u s d u r a n s S t r e p t o c o c c u s d i a c e t y l a c t i s S t r e p t o c o c c u s p a r a c i t r o v o r u s S t r e p t o c o c c u s c i t r o v o r u s Various lactobacilli S t r e p t o c o c c u s t h e r m o p h i l u s L a c t o b a c i l l u s b u l g a r i c u s S t r e p t o c o c c u s t h e r m o p h i l u s L a c t o b a c i l l u s h e l v e t i c u s Propionic acid bacteria

Flavour and acid

Flavour and acid (-i-a lot of diacety 1) Flavour and acid

Flavour and acid Flavour and acid Acid and flavour

Acid and flavour ^ peptides and amino acids **) Acid and flavour

Acid and flavour Gas and flavour * ) Nomenclature of the micro-organisms according Davis (1965).

** ) Some other bacteria listed in this Table also produce peptides and amino acids but compared with L a c t o b a c i l -l u s b u -l g a r i c u s on-ly in sma-l-l amounts.

acid bacteria.

Franke (1962) showed that A s p e r g i l l u s n i g e r bymeauisof a NAD-dependent alcohol dehydrogenase converts ketones into alcohols. The de-composition of lactose is very important for all cheese varieties. The con-version of lactose by homofermentative Lactobacillaceae follows the Emb-den-Meyerhof-Parnas- or E.M.P.-scheme (glycolysis); by heterofermen-tative species also the hexose-monophosphate- or H.M.P.-pathway can be used. Lactose is first split into glucose and galactose. Pulay (1958) found that in Emmental cheese the glucose is converted more rapidly than galac-tose. In Camembert lactose disappeared completely in the first stage (14 days) of ripening. Besides lactic acid also pyruvic acid, valeric acid, a-ke-toglutaric acid and others are formed. In Emmental cheese in the first stage of ripening lactic acid bacteria produce lactic acid, but in the subsequent stage of ripening thepropionic acid bacteria replace the lactic acid bacteria and form propionic acid, acetic acid and carbon dioxide from lactic acid. The pyruvic acid can be converted into diacetyl via a-acetolactic acid on the one hand (Suomalainen, 1968) and via various pathways into amino acids on the other.

2.4. Calf r e n n i n and its r e p l a c e r s

For the manufacture of cheese a proteolytic enzyme is necessary. Nor-mally the enzyme of the fourth stomach (abomasum) of a milk-feeding calf, i.e. rennin or chymosin, is used. This enzyme splits K-casein, a protein which stabilizes the casein micelles, into a glycomacropeptide (64 amino acid residues) and para-K-casein, by hydrolysing the Phe-Meth bond. The hydrolyses of K-casein causes destabilization of the casein micelles in milk and an elastic curd begins to form. The essential property of calf rennin is its great preference for the hydrolysis of the Phe-Meth linkage of K-casein and its weak action on other peptide bonds.

It is noteworthy that the Phe-Meth bond is not hydrolysed by rennin in syntheticpeptides very rapidly, but the hydrolysis rate increases enormously on a synthesized decapeptide which contains the same sequence of amino acids as K-casein (Schatterkerk, 1973). This particular sequence in the K-casein molecule makes the Phe-Meth bond also highly susceptible to other proteases, e.g. pepsin and chymotrypsin, but under conditions so far examined these proteases hydrolysed the other peptide bonds in K-casein

of t h e m e m b r a n e i s rough, but after s t i r r i n g the milk the m e m b r a n e s u r f a c e b e c o m e s smooth. T h e m e m b r a n e c o n s i s t s e s s e n t i a l l y of two s e p a r a t e l a y e r s . T h e inner layer which is firmly bound to the globule is s t r u c t u r a l l y s i m i l a r to the biological c e l l m e m b r a n e and has its own specific s e r o l o g i c a l r e a c t -i o n s . Th-is layer -i s bas-ically a closely kn-it complex of globular p r o t e -i n s and a phospholipid double l a y e r . The outer l a y e r has been investigated l e s s thoroughly, but i s p a r t i c u l a r l y important for the stability of the fat e m u l s i -on. T h e r e is evidence that the principal m o l e c u l a r a x e s m this l a y e r a r e o r i e n t e d p a r a l l e l to the s u r f a c e instead of p e r p e n d i c u l a r to it a s in the inner l a y e r . Keenan (1970, 1971) m his c o m p a r a t i v e study of the milk fat globule m e m b r a n e and p l a s m a m e m b r a n e s gives the data listed m T a b l e 2.3 for the c h e m i c a l composition of the globule m e m b r a n e :

T a b l e 2 . 3 C h e m i c a l c o m p o s i t i o n of t h e m i l k f a t g l o -b u l e m e m -b r a n e a c c o r d i n g t o K e e n a n ( 1 9 7 0 , 1 9 7 1 ) Protein: lipid ratio 1.77 Phospholipid as % of the total lipid 20.3

Density (g/ml) 1.08-1.10 Fatty acid composition of the membrane lipids <%)

CIO 0 2.6 C16:l 1.5 C12 0 3.2 C17:0 0.9 C14:0 10.8 C18:0 21.0 C14-1 0.3 018:1 22.5 C15 0 1.3 C18:2 1.5 C16-0 34.4

T h e s t r u c t u r e of the m e m b r a n e is far from c l e a r , but a c c o r d i n g to W a l -lach and Gordon (1968) the m e m b r a n e p r o t e i n s exist p a r t l y in r a n d o m coil and p a r t l y i n o - h e l i x configuration, some of the peptide chains in the r a n d o m configuration lying in o r n e a r the s u r f a c e and the r e m a i n d e r m the a - h e l i x form lying m o r e o r l e s s p e r p e n d i c u l a r to it. The coils t h e m s e l v e s each have a hydrophobic face and the h y d r o c a r b o n t a i l s of the lipids a r e bound t o t h e s e c o i l s . The lipids have a specific composition and o r g a n i s a t i o n , the l a t t e r d e t e r m i n e d by t h e s u r r o u n d i n g p r o t e i n , so that the lipid t a i l and p r o t e i n

pancreatic lipase and phospholipase A. Pitas (1967) using this technique determined the distribution of the individual fatty acids in milk fat trigly-cerides. This distribution is given in Table 2.5.

T a b l e 2.5 - The d i s t r i b u t i o n (%) of the f a t t y a c i d s in the milk f a t t r i g l y c e r i d e s a c c o r d i n g to P i t a s (1967) Fatty a c i d C4:0 0 6 : 0 0 8 : 0 C10:0 012:0 C14:0 C16:0 C16:l C18:0 018:1 C18:2

P osition of fatty acid in the 1 9.8 16.3 17.2 20.7 24.6 27.6 45.5 48.5 56.2 41.8 26.7 2 5.6 25.8 4 2 . 1 49.7 47.5 53.8 41.7 35.6 25.7 27.9 77.9 glycerol molecule 3 84.6 58.0 40.7 29.6 28.0 18.6 12.8 15.9 18.0 30.3 - 1 4 . 6

Unfortunately all these investigations did not give sufficient data to elucidate the con^osition of the individual triglycerides in milk fat. For the t r i -glycerides containing medium or short chain fatty acids, Breckenridge

(1968, 1969) gives some details about the composition as regards fatty acids. One of the most remarkable results of this analysis is the presence of a large amount of triglycerides containing 018:1 a s w e l l a s C4:0 (7.66%). The lack of detailed data about the conposition of the triglycerides makes a correct interpretation of the hydrolysis of milk fat with S . l i p o l y t i c a lipase very difficult.

2.5.2. Milk proteins

Milk proteins can be divided into two groups, the caseins and the milk serum proteins. A survey of the milk proteins is given in Table 2.6.

T a b l e 2.7 A m i n o a c i d c o m p o s i t i o n ( R e s i d u e s / m o n o -m e r ) of t h e -m a i n -m i l k p r o t e i n s a c c o r d i n g t o M c K e n z i e

( 1 9 7 1 )

AMINO ACID

Milk serum proteins Caseins ft-Lacto- a-Lact- *a .-Casein /i-Casein K-Casein ^ s,l globulin albumin 9% 3% 4 2 % 249f 12% Alanine Arginine Aspartic acid Asparagine Cysteine or i (Cys-Cys) Glycine Glutamic acid Glytamine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine 14 3 16 3 3 25 2 10 22 15 4 4 8 7 8 2 4 10 3 1 21 13 3 8 13 12 1 4 2 7 7 4 4 6 10 6 16 0 10 45 6 13 17 17 5 7 20 17 6 3 11 12 0 5 39 5 10 22 11 6 9 35 15 9 1 4 19 IJ 5 11 2 3 25 3 12 9 9 2 4 18 11 12 2 9 10

*a means the calcium sensitive fraction of a-casein (Waugh, 1958), s

T h e amino acid s e q u e n c e of all the c a s e i n s h a s been elucidated by F r e n c h i n v e s t i g a t o r s .

M e r c i e r (1971) g i v e s t h e sequence for a c a s e i n of v a r i o u s g e n e t i c v a r i -a n t s showingth-at b e t w e e n the -amino -a c i d s 45 -and 69, -a - c -a s e i n c o n t -a i n s s i x p h o s p h o r y l a t e d s e r i n e m o l e c u l e s (the whole m o n o m e r contains 8 of t h e s e g r o u p s ) .

1 3

T h e sequence of /^-casein, genetic v a r i a n t s A , A , B and C, h a s been e l u c i d a t e d by B r i g n o n (1971). T h i s p r o t e i n c o n t a i n s six p h o s p h o r y l a t e d s e

T h e size d i s t r i b u t i o n of c a s e i n m i c e l l e s h a s been a subject of intensive s t u d i e s , using both e l e c t r o n m i c r o s c o p y and other p h y s i c a l m e t h o d s . The model of the c a s e i n m i c e l l e s given by Gar n i e r (1970) might be the best m o -del available at the moment (Fig. 2.1). His d e s c r i p t i o n of these m i c e l l e s i n c l u d e s the following s t a t e m e n t s :

1. The open s p o n g e - l i k e s t r u c t u r e of the micelle should account for its high w a t e r content (2-2.5 g w a t e r / g protein) and for the easy penetration of r e n n i n , /5-lactoglobulin and carboxypeptidase A. 2. The functional unit of K-casem s e e m s to be a t r i m e r (mol. weight

55,000 - 60,000). T h i s t r i m e r should play a key r o l e in the building of m i c e l l e s and in the clotting of milk.

T h e proposed model might explain the clotting action of r e n n i n . It is known that p a r a - K - c a s e i n still binds a - and /J-casein. It can be a s s u m e d that in an e a r l y stage of clotting all the Kcasein nodes have been t r a n s f o r m e d by rennin into p a r a K c a s e i n but the m i c e l l e s t r u c t u r e is m a i n tained. Through c o l l i s i o n of m i c e l l e s (due to mechanical t r e a t m e n t o r n o r -mal Browman movement) two t r i m e r s of p a r a - K - c a s e i n may be linked on t h e i r exterior s u r f a c e , c a u s i n g the m i c e l l e s to c o a l e s c e . The d i s t r i b u t i o n of t h e various c a s e i n over the m i c e l l e s is not homogeneous. K i r c h m e i e r (1970) using 2 , 4 - d i n i t r o - f l u o r o b e n z e n e (DNFB), which p e n e t r a t e s into m i c e l l e s , s t a t e s t h a t a m i c e l l e contains a coat, consisting of two l a y e r s , and a c o r e .

U s i n g the s a m e p e n e t r a t i o n technique, but s t a r c h gel e l e c t r o p h o r e s i s for t h e separation of the c a s e i n s , we found the d i s t r i b u t i o n shown in T a b l e 2.9 ( y - c a s e i n not c o n s i d e r e d ) . T a b l e 2.9 - D i s t r i b u t i o n (%) o f f l t - , / J - , a n d K - c a s e i n o v e r a c a s e i n m i c e l l e LOCATION TOTAL (%) %a Outer layer 12 41 Inner layer 35 52 Core 53 57 23 %/i 6 30 37 %K 53 18 6

Due to the higher content of K-caseins the outer layer contains more carbohydrates than the core. So the hypothesis of a hydrophobic core with a hydrophilic outer layer of the micelle has been confirmed by the results of our experiments,

Although our data differ considerably from those of Kirchmeier (1970), it is obvious that the distribution of the caseins over the micelles is not homogeneous, so the accessibility to the various caseins for proteolytic enzymes is not equal.

T a b l e 3.1 - Y e a s t s f r e s h l y i s o l a t e d f r o m F r e n c h c h e e s e s ( N o m e n c l a t u r e a c c o r d i n g t o L o d d e r , 1 9 7 0 )

YEASTS CHEESE SOURCE C a n d i d a i n t e r m e d i a B, C C a n d i d a v i n i C C a n d i d a z e y l a n o i ' d e s B D e b a r y o m y c e s c a n t a r e l l i C D e b a r y o m y c e s h a n s e n i i B, C, R K l u y v e r o m y c e s l a c t i s B, C P i c h i a o h m e r I C S a c c h a r o m y c e s c e r e v i s l a e B S a c c h a r o m y c e s r o s e l B S a c c h a r o m y c e s u n i s p o r u s C S a c c h a r o m y c o p s i s l i p o l y t i c a ( C a n d i d a l i p o l y t i c a ) BB T o r u l o p s i s C a n d i d a B B = Brie C = Camembert R = Itoquefort BB = Bresse Bleu The d i a m e t e r of t h e s e h a l o e s is m o r e o r l e s s a m e a s u r e of the e n z y m a t i c activity of the y e a s t s c o n c e r n e d . If a l l e x o e n z y m e s h a v e the s a m e m o l e c u lar w e i g h t and the s a m e shape and the s u b s t r a t e i s homogeneously d i s t r i b u ted o v e r the a g a r , then t h e zone d i a m e t e r would be a p r a c t i c a l l y c o r -r e c t m e a s u -r e of t h e e n z y m a t i c activity. B e c a u s e n o -r m a l l y t h e s e a s s u m p t i o n s a r e not realized, we subsequently investigated e v e r y p o s i t i v e s t r a i n m o r e t h o r o u g h l y .

M o r e qualitative m e t h o d s for l i p a s e a r e t h a t a c c o r d i n g to Y a m a d a (1962) and f o r p r o t e a s e t h a t of Kunitz (1947). T h e a b s e n c e of l a c t o s e f e r m e n t a t i o n or a s s i m i l a t i o n w a s t e s t e d in tube c u l t u r e s u s i n g l a c t o s e a s the only c a r b o n s o u r c e .

After the s c r e e n i n g s mentioned only a few y e a s t s r e m a i n e d which had t h e d e s i r e d p r o p e r t i e s and t h e s e y e a s t s w e r e f u r t h e r t e s t e d in whey, milk o r c r e a m f o r the d e t e r m i n a t i o n of organoleptic quality of the f e r m e n t e d m i l k . Finally a yeast w a s c h o s e n belonging to t h e genus C a n d i d a , namely a

of the isolated yeasts 75100% were identified as strains of S. l i p o -l y t i c a . Twenty other brands were investigated of which except Bresse Bleu only Bleu de Bresse also contained S. l i p o l y t i c a . However, only 40% of the yeast strains isolated from the latter brand belonged to the species S . l i p o l y t i c a .

3.2. S o m e i m p o r t a n t b i o l o g i c a l a n d b i o c h e m i c a l p r o p e r -t i e s of -t h e s e l e c -t e d y e a s -t s -t r a i n

After selection o f S . l i p o l y t i c a as a micro-organism which might be used for rapid manufacture of a cheese flavour and taste concentrate, we started to study the growth of S . l i p o l y t i c a in whey, in synthetic media and m milk, using Biotec FL 110 fermentors.

We determined some growthparameters such as 0„-consumption,pH and viable count of S . l i p o l y t i c a as well as the exo-enzyme production m the media mentioned.

F i r s t we investigated thepioduction of lipase as a function of the amount and type of fat in various growth media. (Compositions of the media are given in Ch^ter 7.) We used tributyrin and olive oil (mainly triolein) as fats. Because preliminary experiments had shown that the optimal tempe-rature of the lipase activity of S. l i p o l y t i c a on tributyrin was lower than the optimal temperature for the hydrolysis of olive oil, two different kinds of lipases might be synthesized by S. l i p o l y t i c a . If the two en-zymes are inducible, the cells growing on tributyrin should produce a lipase with a high preference for tributyrin, whereas those growing on olive oil should produce a lipase with preference for olive oil as substrate. The r e -sults of the experiments are given in Table 3.3. From these experiments it may be concluded that olive oil is a more favourable substrate for lipase production than tributyrin (cf. however below).

Secondly the conclusion may be drawn, that there is only one lipase pro-duced by S. l i p o l y t i c a , because, independent of the growth medium, the ratio between the lipase activities with tributyrin and olive oil, respectively, as substrates is almost constant. The conclusion that olive oil is superior to tributyrin for the lipase synthesis, is not completely correct, because the yeast grows better in the media containing olive oil, so the number of cells per ml in these media is higher than in the tributyrin media. Considering the number of yeast cells, the amount of lipase produced by a single cell

T a b l e 3.4 - G r o w t h , l i p a s e a n d p r o t e a s e a c t i v i t i e s a n d o x y g e n c o n s u m p t i o n of S. l i p o l y t i c a in a f u l l f a t s o y

-b e a n m e a l m e d i u m

Incubation time N / n i l pH Lipase activity Protease a c t . 0„-consuniption

5 ^ h xlO e.u. e.u. fimoXO^(\. ^^

0 4 8 12 16 20 24 28 32 44 4 7 11 15 31 55 120 230 350 400 7.50 7.43 7.34 7.20 7.14 7.09 7.04 6.99 6.93 6.87 -1.13 5.48 6.79 7.95 8.73 8.51 8.13 7.42 6.17 0 0 3 39 73 109 132 150 157 169 -0.028 0,090 0,243 0.596 0.748 0.977 0,785 0.528 0.342 T a b l e 3,5 - G r o w t h , l i p a s e a n d p r o t e a s e a c t i v i t i e s a n d o x y g e n c o n s u m p t i o n of S. l i p o l y t i c a In w h e y

Incubation time N / m l pH Lipase activity P r o t e a s e act. O^-consumption xlO^ e.u. e.u. /imol O g / ( 1 . s)

0 8 12 16 20 24 28 32 48 72 5 3S 85 140 170 195 290 370 510 710 6.50 6.43 6.31 6.20 6.12 6.18 6.31 6.39 6.52 7.12 -1.81 3.57 4.34 4.36 3.32 2.79 1.46 -12 21 32 51 72 104 147 -0.010 0.298 0.637 0.945 0,813 0,523 0.370 0.235 0.162 3 1

T a b l e 3.8 - G r o w t h , l i p a s e a n d p r o t e a s e a n d o x y g e n c o n s u m p t i o n of S . l i p o l y t i c a on a d e f a t t e d s o y b e a n m e a l m e d i u m ( P - m e d i u m ) Incubation time h 0 4 8 12 16 20 24 28 32 44 N / m l X 10^ 1 3 9 18 36 160 280 400 400 400 pH 6.92 6.88 6,80 6,53 6,30 6,98 7.64 7.98 8.09 8.33 Lipase activity e.u. -6.37 11.45 12.13 8.89 2.54 0.10 -P r o t e a s e activity e.u. -5 10 99 177 221 227 230 233 236 O^-consumption ^ m o l Og/Cl. s) -0.042 0.960 1,947 1.568 0.975 0,570 0,432 0.418 T a b l e 3.9 - G r o w t h , 1 ip a s e a n d p r o t e i n a c t i v i t i e s a n d o x y g e n c o n s u m p t i o n of S . l i p o l y t i c a on a d e f a t t e d s o y b e a n m e a l m e d i u m ( P - m e d i u m ) + 0 . 2 % g l u c o s e Incubation time h 0 4 8 12 16 20 24 28 32 44 N / m l X 10^ 2 5 11 23 71 153 317 420 510 520 pH 7.00 6.95 6.83 6.71 6.70 6.99 7.53 7.74 7,86 7.88 Lipase activity e.u. -5.11 7,85 9.31 6.22 4.01 n.d.* -P r o t e a s e activity e.u. -3 27 120 191 257 284 295 302 305 On-consumption (umol 02/(1. s) -0.035 0.743 0.997 0.495 0.220 0.197 0.123 -*n.d. - not determined 33

Number of viable yeasl cells

.o

25 30 Incubation tim* (h)

Fig. 3.2 Relation between the exollpase activity and the oxygen consun^-tion of S. 1 i p o 1 y 11 c a . OLlpase activity; • Oxygen consumpconsun^-tion.

so V 72 Incubation time (h)

Fig. 3.1 Growth of S. l i p o l y t i c a under optimum conditions In Biotec FL 110 fermentors on various media, o Whey; • O-medlum; A P-medlum.

oxygen consumption pmol 02^(1 s) carbon dioxide production pmoiOyds) 0 2 - consu fnpt ion C02-production " ^ X . 25 30 35 Incubation time (h) Number of viable yeast cells 1 0 8 ^ protease activity (e u )

Fig. 3.3 Correlation between the oxygen consumption and the carbondio-xyde production by cells of S . l i p o l y t i c a growing on the O-medlum. O Og-consumption • COg-productlon

40 50 Incubation time(h)

Fig. 3.4 Relation between the exoprotease activity and the number of yeast cells. ONumber of yeast cells/ml; • P r o t e a s e activity.

Fig. 3.6 Determination of the purity of the exollpase after chromatography on Sephadex G200 with ultracentrifugal analysis.

T a b l e 3 . 1 1 - A m i n o a c i d c o m p o s i t i o n of t h e t w o p e p t i d e c h a i n s of t h e l i p a s e AMINC ) ACID A s p (Asn) T h r S e r * Glu (Gin) P r o Gly Ala V a l * Meth lieu * Leu* T y r P h e * L y s His A r g C y s T r p I 2 1 10 12 25 12 15 15 14 2 13 22 5 11 10 4 9 2 3 11 21 10 13 25 12 15 15 16 2 12

n

5 10 10 4 9 2 3

Total number of amino

acids per peptide chain 205 204

*Amino acids which were not equally shared on the two peptide chains. e m u l s i f i e r s (e.g. bile salts) the enzymatic activity was not improved. In t h i s respect our l i p a s e is different from p a n c r e a s lipase (Julien, 1972). We a l s o tried to e s t a b l i s h with the u l t r a f i l t r a t i o n technique whether the lipase f r o m S. l i p o l y t i c a contained subunits. T h e s e e x p e r i m e n t s showed that t h e lipase d o e s not contain a p r o t e i n - l i k e cofactor. However, d u r i n g t h i s f i l t r a t i o n the e n z y m e slowly l o s e s its activity. When the concentrated Amicon P M I O filtrate (mol weight < 10,000) was added to the high-molecular enzyme f r a c t i o n , which did not p a s s the filter, the activity is improved to a s m a l l e x t e n t (about 10%).

Because the i n t e r a c t i o n of a lipid-like s t r u c t u r e of the l i p a s e and the lipid substrate might explain the i n t e r a c t i o n between l i p a s e s and lipids we c a r r i e d out s o m e e x p e r i m e n t s to d e t e c t components with such a s t r u c t u r e ,

Rel activity

.^o-, — O - r

/

. /

^s

Fig. 3.7 Influence of the pH on the activity of the exollpase of S . l l p o -1 -1 -1 l e a , oTrlbutyrin; T = 30 C. • T r i o l e i n ; T 43°C.

Relative activity

Temperature C O

Fig. 3.8 Influence of the temperature on the activity of the exollpase of S, l i p o l y t i c a . •Tributyrin; pH 6.0, OTrlolein;pH 7.8.

T a b l e 3 . 1 3 I n f l u e n c e of b i v a l e n t m e t a l i o n s on t h e a c -t i v i -t y of -t h e l i p a s e f r o m S . l i p o l y -t i c a

Metal ion

Substrate: triolein Substrate; tributyrin

2+, 2+1 _(Me^"")

iMe""^! = [Me'"^! = Pre-incuba- Pre-incubated (Me""^) = (Me""^) = Pre-incuba- Pre-incubated 10"^mol/l lO'^mol/l tion lO"^mol/1 enzyme 24 h 10"'^mol/l 10"^mol/l tion lO""^mol/l enzyme 24 h

Me enzyme dialysed Me enzyme dialysed NaVK* Ba^- Ca^- Cd2- Co^- Cu^- Fe^-Hg^^ M g 2 ^ Mn2+ Ni2-Sn^^ Zn^-100 101 110 58 71 30 87 0 109 102 36 97 0 100 102 107 82 90 56 100 16 1*02 100 71 95 62 100 66 99 60 74 35 65 0 91 78 105 74 25 100 89 102 85 100 79 70 19 115 121 106 93 76 100 107 106 81 91 51 98 9 84 106 108 108 56 100 110 98 100 100 84 99 17 106 117 111 118 98 100 86 94 8a 65 68 67 4 91 92 94 53 72 100 116 100 96 90 92 76 5 96 100 96 89 105

Subsequently a conversion of the monoglycerides into glycerol and fatty acids takes place. The choice of the position of hydrolysis is now determined by the preference of the lipase for the 2 or 3 position and by the fatty acids in these positions. It is possible that the liberation of the first and the last two fatty acids proceeds more or less simultaneously.

b) Primary attack on the 2-position

R 2 - C - 0 II 0 0-C-R1 II 0 0-C-R3 II 0 • 0-C-R1 II 0 •0-C-R3 II 0 • RjCOOH

Of course the choice of theplaceof the primary attack on the 2-position is a property of the enzyme, although the hydrolysis rate can be influenced by the fatty acids in the 1 or 3 position of the glycerol molecule. After the first attack on the 2-position the next hydrolysis step" is fully determined by the fatty acids themselves, because the OH-group in the 2-position will have the same influence on the 1 and 3-position.

0 r O - C - R , O-C-R3 II O • RlCOOH 0-C-R3 0 p 0 - C - R , HO- |{ .R3COOH L OH

Also these monoglycerides can be further hydrolysed to glycerol and fatty acids: -OH lipase HO-O-C-R3 I 0 .R3COOH 47

Number of miceiles/ml l » 1 0 " r 1«108 1x10' I x l o S -Number of micelles/ml ixio^r I I IS 1«109 1x10^ 1»105 I I I I L-J sterilized and homogenized milk U 16 ^ m Fig. 3.9a Size distribution of micelles of tributyrin and olive oil.

C o u l t e r - c o u n t e r e x p e r i m e n t a r e given m F i g s . 3.9a and 3.9b. The data p r e s e n t e d m t h e s e f i g u r e s can be used for calculating the amount of s u b -s t r a t e which can be attacked by l i p a -s e -s . In thi-s calculation we con-sider the t r i g l y c e r i d e s in the monomolecular l a y e r on the surface of the (globular) m i c e l l e as the only available t r i g l y c e r i d e s for the l i p o l y s i s .

Although it IS c l e a r that this i s a v e r y s i m p l e p i c t u r e of the r e a l s i t u a t -ion, the data of the r e a c t i o n r a t e s obtained by using this model a r e much m o r e in a g r e e m e n t with the data values for the lipolysis of milk fat than t h o s e obtained without the c o r r e c t i o n mentioned. Table 3.14 shows the dif-f e r e n c e between the K and V v a l u e s (i.e. the Michaelis-Menten

con-m con-max ^

s l a n t and the m a x i m u m r a t e of reaction, r e s p e c t i v e l y ) , calculated with the L m e w e a v e r - B u r k - p l o t , with and without c o r r e c t i o n for the p h y s i c a l s t a t e of the s u b s t r a t e s .

T h e specificity of the purified S . l i p o l y t i c a lipase w a s studied by m e a n s of the m e t h o d s d e s c r i b e d in Chapter 7. F i r s t we examined the r a t e of hydrolysis a s a function of the chain length of the fatty a c i d s using highly pui ified t r i g l y c e r i d e s containing only one kind of fatty a c i d s . The r e s u l t s of t h e s e e x p e r i m e n t s a r e given in Table 3.15.

T a b l e 3 . 1 5 T h e r e l a t i v e r a t e s of h y d r o l y s i s of t r i g l y -c e r i d e s , -c o n t a i n i n g o n l y o n e k i n d of f a t t y a -c i d s , by

S. l i p o l y t i c a l i p a s e

Triglyceride with Relative reaction rate C12:0 39 C14:0 18 C16:0 12 018:0 2 018:1 100

F r o m these d a t a the conclusion may be d r a w n that the h y d r o l y s i s r a t e d e c r e a s e s with i n c r e a s i n g chainlength of the fatty acid, w h e r e a s the double bond in the acid a c c e l e r a t e s the r a t e of h y d r o l y s i s significantly. S u b s e -quently the lipolysis of t r i o l e i n was studied in g r e a t e r detail (Table 3.16). T he r e s u l t s suggest that the lipase has a pronounced p r e f e r e n c e for the 1 or 3 position, but is not absolutely specific for these p o s i t i o n s .

Remarkable i s the r e l a t i v e l y high amount of /3-MG and to a s m a l l e r e x t e n t aMG f o r m e d . T h i s can only be explained by a m o r e o r l e s s s i m u l

T a b l e 3 , 1 8 T h e l i p o l y s i s of s y n t h e t i c C 1 8: x g l y c e r i -d e s , I , T h e i n f l u e n c e of a -d o u b l e b o n -d i n t h e f a t t y a c i -d . I I . T h e i n f l u e n c e of t h e p o s i t i o n of a f a t t y a c i d . G l y c e r i d e OOO SOO OSO SSO SOS SSS SES OEO 0 ( 0 H ) 0 S(OH)0 S(OH)S 0(OH)(OH) R e l a t i v e r e a c t i o n r a t e 100 67 89 21 26 2 4 63 119 51 14 -D i g l y c e r 1.2 DG 4.50 3.01 1.29 6.87 J 1.05 SO(OH) (OO(OH) (OH) SO 1 0.34 SS (OH) 1.70 0.16 0.31 4.38 -Ides (%) 1.3 DG 0.50 0.20 0.18 0.19 0.15 0.01 0.01 0*09 -Monoglycerides a - M G ^ 0.30 0.06 S(OH)(OH) 0.01 0(OH)(OH) 0.12 J 0.03(OH)(OH)O 1 0.01 S(OH)(OH) 0.02 -4.06 2.10 0.56 -(%) - M G 3.00 0.53 1.25 0.12 0.28 -0.02 0.57 0.04 0.02 0.01 -L i b e r a t e d fatty acid % 0 1 8 : 0 -32 5 76 82 100 97 -29 100 -% C 1 8 : l 100 68 95 24 18 3 (E) 98(0) 2(E) 100 71 -CO

tn Oi

Fig. 3.10 Gas chromatogram of the fatty acids liberated by the exollpase of S . l i p o l y t i c a (80 min at 25°C). C7:0andC13:0 are used as in-ternal standards.

internal standards). The decrease of the pH of the milk during the enzy-matic lipolysis is shown in Figs. 3.11, while Table 3.19 gives the concen-trations of the individual fatty acids in milk after various times of incu-bation with the purified enzyme. The amount of fatty acids liberated from the triglycerides during the first twenty minutes of incubation divided by the amountoffatty acids present inmilkfat gives ratios which are a measure of the r a t e of liberation of the individual fatty acids (Table 3.20).

pH 67 \ \ « . \ \ s \ \ ' \ \ 60 80 Incubation time (mm)

Fig. 3.11 Influence of the lipolysis of mllkf at by S, l i p o l y t i c a exollpase on the pH of milk. Incubation temperature: O 25°C; • 43°C, Figs. 3.12a and 3.12b show that there is a linear logarithmic relation-ship between the r a t e of hydrolysis and the chain length for saturated fatty acids from C8 to C14 at 25 Caswell as at 43 C. However, for unsaturated fatty acids there i s no such relation.

An additional experiment was carried out to ascertain the influence of the lipolysis on pH and on the amount of free fatty acids on prolonged incu-bation (Fig. 3.13). Table 3.21 gives the amount of the individual free fatty acids after 24 h of lipolysis. From this Table it can be seen that the main part of the short chain fatty acids had been liberated from the milk fat.

22 23 Incubation time ih)

Fig, 3.13 Influence of the prolonged lipolysis of milkfatby S, l i p o l y t i-ca exollpase on the pH of milk.

Protease activity (eu)

Elution volume

Fig. 3.14 Sephadex GlOO chromatogram of S . l i p o l y t i c a protease, showing the absorption at 278 nm ( ) and the protease acti-vity ( o o ).

The results obtained from the lipolysis of milk fat by purified lipase are in agreement with the data obtained in the experiments with the artificial triglycerides. In both cases the rate of hydrolysis for the long-chain fatty acids is:

C18:l > C18:2 > C14:0 > C16:0 > C18:0

These data £ire very valuablefor explaining the role of the fatty acids in the metabolism o f S . l i p o l y t i c a growing in milk.

3 . 5 P u r i f i c a t i o n a n d p r o p e r t i e s of t h e e x o p r o t e a s e of S a c c h a r o m y c o p s i s l i p o l y t i c a

The exoprotease produced by S. l i p o l y t i c a under the cultivation conditions described before was purified and isolated according to the me-thods given in Chapter 7. The second peak of the Sephadex G 100 chroma-togram contains the exoprotease (Fig. 3.14). Thispeakwas not pure as was demonstrated by isoelectric focussing. We have therefore further purified the G 100 protease fraction on a DEAE-cellulose ion-exchange column. We succeeded in the crystallization of the pure enzyme. The purity of the p r o -tease fraction was checked by isoelectric focussing (see Fig. 3.15) and ultracentrtfuge analysis. With the latter method the molecular weight was determined. It was found to be 25,600. This was checked on a Sephadex G 100 column according to the method of Andrew (1964). This method gives a molecular weight of 24,900 for the protease (see Fig. 3.16). The amino acid ajialysis of the protease was carried out according to the method of Stein and Moore (1954) using a Beckman amino acid analyser. The results are presented in Table 3.22.

We could not detect any prosthetic group or cof actor, so we assumed that the protease contains only amino acids, which is quite normal for proteases. The recovery d u r i r ^ the purification procedure is given in Table 3.23. The protease has a pl of 5.95 (see Fig. 3.15) and a pH-optimum of 7.05. On the acid side of the optimum it has a considerable activity, on the basic side the activity decreases rabidly (see Fig. 3.17). The pH stability of the en-zyme is quite good for pH < 7 but for pH >8 the protease is unstable. The relation between the activity and the temperature is given in Fig. 3.18. The

T a b l e 3 . 2 3 R e c o v e r y d u r i n g t h e p u r i f i c a t i o n of S , l i -p o l y t i c a e x o -p r o t e a s e

P u r i f i c a t i o n s t e p s V o l u m e P r o t e a s e activity Vol, x Act, P r o t e i n content R e c o v e r y of P u r i f i c a t i o n 3

m l e.u. X 10 mg ** the activity factor

C u l t u r e s u p e r n a t a n t Acetone f r a c t i o n F i l t r a t e D E A E - c e l l batch t r e a t m e n t Sephadex GlOO f r a c t i o n s D E A E - c e l l column f r a c t i o n s F r e e z e d r y i n g 16000 2060 1400 3500 270 -310 2050 2820 1060 12200 -4960 4223 3948 3710 3294 2 9 8 5 * 14320 8750 3730 1380 640 5 8 0 * * * 100 85,3 79,6 74,8 66,4 60,2 1.0 1.4 3.1 7.8 16.4 16.4

* Calculated (1 m g p u r e p r o t e a s e / m l h a s an activity of 5146 e.u.). ** D e t e r m i n e d with the F o l m ihethod.

*** R e a l d r y weight.

05

optimal temperature is 43.7 C. The protease is thermo-unstable.

Another subject of investigation concerned the sensitivity of the protease to metal ions. Table 3.24 shows that on the whole all the bivalent metal ions tested have inhibitory effects compared with the metal ions of the controls (Na and K ). The strongest inhibition was found in the case of Fe . The role of metalions is not limited to the kinetics of the proteolysis, but the metal ions are also important in governing the type of hydrolysis of the substrates. Compared with the ions of the control (Na / K phosphate), Ca , Fe , Mg , Mn and Zn influence the action of the protease in such a way that the substrates are hydrolysed into small peptides (or even amino acids), so the ratio (4% - 12%): 4% TCA soluble compounds de-c r e a s e s de-considerably as a funde-ction of time, whereas Co , Cr and Cu show the opposite effect. Of the tested ions only Ca , Fe and Mg can be used in practice.

The specificity of the protease was determined with a -casein and a-lactalbumin as substrates. For the determination of the free NH„-groups we used the method of Sanger, whereas the amino acids with a free COOH-group were measured by means of carboxypeptidase and thin-layer chro-matography (see Chapter 7). Only if the incubation time was relatively short and a large excess of substrate was used, a clear preference was found for the peptide bonds before the amino acids glycine, alanine, leucine and tryp-tophan in the amino acid sequence of these proteins. On prolonged incu-bation also other peptide bonds were hydrolysed and the peptides were often degraded into amino acids. The tendency of protease to split peptide bonds p r i o r to apolar amino acids was alsofoundby Turkova (1969) for A s p e r -g i l l u s protease and for a S e r r a t i a protease by Miyata (1970).

From inhibition tests with e.g. diisopropylphosphofluoridate (DIPF), which combines with the serine hydroxyl group, it can be concluded that S . l i p o l y t i c a protease is a so-called s e r i n e p r o t e a s e , which means that a serine and a histidine residue are involved in the active centre of the enzyme. For this kind of protease a detailed reaction mechanism was described by Polgar (1971).

The hydrolysis of the milk serum protein a-lactalbumin was studied in greater detail. The reaction products formed after 15, 30 and 45 minutes of hydrolysis were isolated with Amicon ultrafiltration. Sephadex G 25 and Biorad ion-exchange chromatography (see Fig. 3.19). In further separation

Relative protease activity

10

45 50 Temperature (*C)

Fig, 3,18 Influence of the temperature on the activity of the exoprotease of S , l i p o l y t i c a (pH = 7,05).

Absorption 278 nm

10 number of peak

Elution volume

Fig. 3.19 Separation on Sephadex G25 of the peptides, with a molecular weight ^5000, formed during the hydrolysis of a—lactalbumin with S . l i p o l y t i c a protease,

T a b l e 3 . 2 5 T h e m a i n p e p t i d e s f o r m e d d u r i n g p r o t e o -l y s i s of a - -l a c t a -l b u m i n Elution volume (ml) • Tryp P e p t i d e s P I + p n P III P IV P V p VI, v n P VIII P K P X

P XI, XII, XIII P XIV 15 m m 28.5% 7,6 10.5 26.2 5.2 11.1 8.7 -2.3 -Incubation t i m e 30 m i n 21.2% 7.9 7.9 19.8 8.3 1 4 . 3 6.0 9.9 3.6 2.0 45 m i n 12.3% 8.3 10.7 17.4 13.0 16.2 6.8 8.0 4.0 3.3

The total dry weight of all peptides = mol. weight < 5.000. Incubation time (min) Dry weight (mg)

15 m 2.86 30 m 7.35 45 m 9.98

Oi

CO

Fig. 3.20 Determination of the molecular weights of the peptides formed during the hydrolysisofo—lactalbumin with S . l i p o l y t i c a p r o -tease (cf Fig. 3.20).

T a b l e 3 . 2 6 a - P r o t e o l y s i s of m i l k and a 3% c a s e m a t e s o l u t i o n w i t h S. l i p o l y t i c a e x o p r o t e a s e Incubation tune m i n 0 (corrected value) 10 20 30 45 60 3% casemate solution N-compounds soluble in 4% TCA mg/kg 0 153.4 216.7 258.6 294.5 327.3 N-compounds soluble in 12% TCA mg/kg 0 86.4 109.1 133.4 157.0 179.9

Milk (about 3% caseins) N-compounds N-compounds soluble in 4% TCA mg/kg 0 69,0 110.2 141,4 177,6 203.1 soluble in 12% TCA mg/kg 0 46.8 85.4 100.8 123.6 145,5 T a b l e 3 . 2 6 b - I n f l u e n c e of t h e h e a t t r e a t m e n t of m i l k o n t h e r a t e of p r o t e o l y s i s by S, l i p o l y t i c a p r o t e a s e Incubation t i m e mm 0 10 25 20 30 45 60 90 120 240 N-compound F r e s h milk 0 t 93.0) 137.8 188.1 235.6 345.0 435.7 s soluble in 4% Diluted (1:10) f r e s h milk * 0 (- 108) 135.0 196.2 239.7 479.3 629.1 TCA m g / k g Sterilized milk 0 (= 68.8) 69.0 110.2 141.4 177.6 203,1 246.7 282.1 381.2

* After the hydrolysis reactions had been stopped, the diluted milk was concentrated with the Amicon ultrafiltration cell (UM 05 filter) to the original concentration and after that treated with 4% TCA.

because it also splits other peptide bonds at a considerable rate, it cannot be used as a rennin replacer.

3. The protease hydrolyses fresh milk faster than it does sterile milk, producing different hydrolysisproducts as well in the two cases. 4. The protease hydrolyses the milk proteins m the following order

(pH 6.5) Kcasein = /3 casein > a casein » /3lactoglobulin> a l a c t -albumin.

5. Because the caseins m milk are present in a micellar state, the rate of hydrolysis of the caseins is significantly lower in milk than m the c a s e of isolated caseins. T a b l e 3.27 - Amount of l i b e r a t e d N H g - g r o u p s d u r i n g p r o t e o l y s i s of the main m i l k p r o t e i n s by S. l i p o l y t i c a p r o t e a s e Incubation t i m e m m 0 10 20 30 45 60 90 180 300 Amount of f r e e NHg-group o r - L a c t a l b u m i n 45.2 42,8 4 7 , 3 50,8 52 7 54.8 56,8 63,4 67.6 li-- L a c t o g l o b u m m 51,6 47,9 50.0 56.3 59.5 61.9 64.3 74.6 84.1 s (Ninhydrin a - c a s e i n 37.3 42.1 45.2 50.6 51.6 55.9 57.9 73.8 74.6 method) /i-- c a s e i n 36.5 42.1 49.2 50.0 52.5 55.7 61,0 74.6 74.7 K - c a s e m 37.7 38,3 50.0 54,0 57.9 61.1 61.9 71.4 73.0 3.6 A n a l y s i s of s o m e p a r t i c u l a r a s p e c t s of g r o w t h a n d p r o d u c t i o n of l i p a s e a n d p r o t e a s e by S a c c h a r o m y c o p s i s l i p o l y t i c a

As IS shown m Table 3.7 and 3.12 in 7200 ml Omedium S. l i p o l y t i -c a produ-ces 660 m g p u r e lipase, whi-ch -corresponds to (92x10 )/(mol. weight) mol lipase p e r litre medium. Unfortunately, the exact molecular weight of the lipase could not be determined, because the enzyme floated during the ultracentrifugal analysis. However, from the ammo acid analysis

Rate of lipase synthesis molecules lipase

yeast cell s 2 5 r - x l 0 3

Rate cA protease synthesis molecules protease

yeast cHI s - , 2 5 x l 0 3

Incubation time(h)

Fig. 3.23 The rate of synthesis of the exoenzymes of S. l i p o l y t i c a . OLlpase production per cell per second in the O-medium; • Protease production per cell per second in the P-medium.

CHAPTER 4 - GROWTH OF SACCHAROMYCOPSIS LIPOLYTICA IN BATCH CULTURES ON MILK

4 . 1 I n t r o d u c t i o n

T h e results of the e n z y m e s t u d i e s and the fermentation e x p e r i m e n t s m s o y b e a n meal and whey w e r e used a s far a s p o s s i b l e a s a b a s i s for the o p -t i m i z a -t i o n of -the f e r m e n -t a -t i o n p r o c e s s of S . l i p o l y -t i c a in m i l k . T h e r e IS a g r e a t difference between the f e r m e n t a t i o n brought about in milk by S . l i p o l y t i c a and the o r d i n a r y f e r m e n t a t i o n p r o c e s s e s . In m o s t f e r m e n t a t -ions c a r b o h y d r a t e s a r e used a s the main c a r b o n - and e n e r g y - s o u r c e . The m e t a b o l i s m of c a r b o h y d r a t e s under a n a e r o b i c conditions follows the E m b d e n -M e y e r h o f - P a r n a s (E.-M.P.) pathway for about 90% (Simon, 1968), w h e r e a s under aerobic conditions the EmbdenMeyerhof P a r n a s pathway is r e s p o n s i b l e for 3050% of the breakdown (Blumenthal, 1954). However, S . l i -p o l y t i c a cannot u s e l a c t o s e for f e r m e n t a t i o n and l a c t o s e is a l m o s t the only s u g a r component of milk. C i t r a t e , p r e s e n t m milk (about 0.20%), can be a s s i m i l a t e d , but a s the main c a r b o n and energy s o u r c e for S . l i p o l y -t i c a in milk s e r v e -the -t r i g l y c e r i d e s .

F r o m our detailed s t u d i e s (see C h t ^ t e r 3) we know that S . l i p o l y t i c a can s p l i t the milk fat into fatty a c i d s and d i - and m o n o g l y c e r i d e s by m e a n s of an exollpase. T h e fatty a c i d s enter the i n t e r i o r of the y e a s t cell a s un-d i s s o c i a t e un-d aciun-ds. B e c a u s e the main b a r r i e r un-during this p e n e t r a t i o n is a lipid lipoprotein m e m b r a n e , the uptake of the m o r e lipophilic fatty a c i d s is e a s i e r than that of the s h o r t - c h a i n fatty a c i d s . However, by i n c r e a s i n g the c h a i n length of the fatty a c i d s , s t e r i c hindrance effects may d e c r e a s e t h e i r uptake r a t e , s o m o s t probably t h e r e will be an optimal chain length for t h i s t r a n s p o r t p r o c e s s .

I n s i d e the cell the fatty acids undergo the /3-oxidation, after which the active acetate i s c o n v e r t e d into e.g. COg v i a the Krebs cycle, which gives the y e a s t a great a m o u n t of e n e r g y . During these p r o c e s s e s many i m p o r t a n t

T a b l e 4 . 1 B a t c h g r o w t h a n d l i p a s e a n d p r o t e a s e a c t i v i t i e s of S. l i p o l y t i c a in m i l k (3 . 2% ) . B i o t e c F L l l O f e r

-m e n t o r - A i r a t -m o s p h e r e

Incubation N/ml pH Lipase 0„-consumption Amount of free Protease Nitrogenous 6

time 10 activity /imol/(l. s) fatty acids activity compounds h e.u. /i mol/1 e.u. soluble

corrected for t=0 3% TCA (mg/kg)

0 4 8 12 16 20 24 28 32 36 40 44 48 0.6 1.7 8.2 43.0 110.0 130.0 105.0 80.0 70.0 56.0 40.0 40.0 38.0 6.65 6.60 6.25 6.10 6.00 5.96 5.93 5.90 5.87 5.85 5.82 5.79 5.77 0.00 0.25 0.85 1.75 2,00 2,00 1.60 0,75 0.50 0.40 0.40 0,45 0.45 0.14 0.97 2.22 1.61 1.36 1.16 1.08 0.94 1.00 1.16 1.27 1.27 (967 = 0) n,d, * 2746,6 n,d. 5945.7 7042.7 n.d. n.d. 9052,7 n.d. 7800.8 n.d. 9085.2 0 2 19 40 195 240 250 250 240 240 240 240 240 45 30 25 50 230 250 290 305 310 320 360 420 440 * n.d. = not determined to

The results of an analysis of the growth and the lipase and protease ac-tivities of S. l i p o l y t i c a in milk under the aeration conditions chosen (for details see Chapter 7) are shown inTable 4.1. When these data are compared with the results of the fermentation in the O-medium by S . l i p o l y t i c a (Table 3.6) some remarkable differences can be observed, although the fat and the protein contents in both media are nearly the same.

About six to seven times more lipase is produced in the O-medium than in milk. Taking thenumber of yeasts cells into account, the quantity of lipa-se produced per cell in the O-medium is as a maximum about hundred times larger than in milk (Table 4.2). More or less parallel to the lipase production, the oxygen consumption varies per yeast cell. The difference

in the protease production is less pronounced. After the same time of incubation in both media approximately the same quantity of protease is p r e -sent. However, per yeast cell considerably more protease is produced in the P-medium than in milk.

The changes in the amount of some free fatty acids in the fermented milk as a function of the time are interesting (Table 4.3). There is a drastic reduction in the amount of C8:0 (caprylic acid) and C10:0 (capric acid) after 32 hours of incubation. The phenomenon that the rate of uptake of these fatty acids by S . l i p o l y t i c a surpasses their rate of production in the milk, will be discussed in greater detail in Chapter 5.

F r o m table 4.1 it appears furthermore that the amount of small nitro-genous compounds decreases during the first eight hours of incubation, which means that the yeast cells take up amino acids or short peptides which are already present in the milk before inoculation with yeasts.

For a better understanding of the proteolysis in milk during the fermentation we carried out growth experiments using S. l i p o l y t i c a in an a r tificial milk (as regards preparation see Chapter 7), in which the milk p r o teins a r e replaced by amino acids (the same composition as in milk p r o -teins). During these ejqjeriments we followed the commongrowth parameters and, in addition, the uptake of the amino acids by the yeast cells. The results are given in Tables 4.4, 4.5 and 4.6.

It is remarkable that in spite of the abundance of all necessary growth factors such as vitamins, citrate, phosphate and amino acids, the yeast starts with the synthesis of the lipase to ensure its energy supply. From Table 4.4 it is obvious that, as expected, the yeast cells did not synthesize

T a b l e 4 . 4 - G r o w t h a n d l i p a s e a n d p r o t e a s e a c t i v i t i e s of S. l i p o l y t i c a in a n a r t i f i c i a l m i l k

Incubation N/ml 0„-consumption CO„-production Lipase Free fatty acids Protease pH 6

time 10 /imol/(l. s) ^mol/(l. s) activity ^mol/l activity

h e.u. e.u. 0 2 4 6 8 10 12 14 16 18 20 22 24 30 36 2.1 2.5 3.8 7.1 12.5 24.0 44.0 71.0 120.0 250.0 320.0 300.0 310.0 280.0 240.0 0 0.30 0.90 2.00 3.10 4,25 6.75 9.00 10.25 12.00 14.00 13.50 12.25 8.00 5,80 0 0 0.71 1.79 2.90 4.01 6.13 7,92 9.'51 11,15 12.82 12.63 11,44 7.25 5.38 n.d. n.d. n.d. 0.20 0.50 0.80 0.90 1,10 1,30 1,30 1.10 0,95 0,50 0.30 0.25 n.d. n,d. 6756 n.d. 19903 25551 30636 38341 43290 n.d. n.d. 56615 n.d. n.d. 19233 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6.40 6.39 6,21 6.00 5.75 5.48 5.20 5.02 4.93 4.86 4.81 4.80 4.80 4.35 3.50 n.d. = not determined 00 CO

Alanine Glulamine (mg/kg) (mg/kg)

40 r

30 35 Incubation time (h)

Fig. 4.1 Uptake of amino acids by cells of S . l i p o l y t i c a growing on an artificial milk. Uptake of: alle amino acids (o); glutamic acid (•); alanine (A). The shaded area represents the period In which the metabolism changes from amino acids to fatty acids.

CHAPTER 5 - COMPARISON BETWEEN BATCH AND CONTINUOUS FERMENTATION OF MILK BY SACCHAROMYCOPSIS LIPOLYTICA 5.1 I n t r o d u c t i o n

We started the fermentation of milk as a batch process because from a technical point of view a batch fermentation proceeds more easily than a continuous fermentation. However, continuous fermentation has some ad-vantages over batch fermentation. Briefly summarized these adad-vantages a r e :

1. The fermentor in continuous fermentation is smaller than in batch fermentation on the same production scale.

2. The time necessary for cleaning the apparatus in continuous fermen-tation is only a fraction of the cleaning time in batch fermenfermen-tation. 3. A continuous fermentor may give a product with a constant quality over a long period of time, whereas the fermented product originating from different batches may show slight deviations, even if r e -producibility as regards cultural conditions is considered

thourough-ly.

4. It is possible that the quality of a product made by continuous fer-mentation is better than the quality of a product obtained by batch fermentation, because undesirable metabolic substances formed in the first stage of the latter fermentation may remain in the end product, whereas they are absent or at least diluted, in a product obtained with continuous fermentation.

All these points are of economic in^iortance. Besides the advantages mentioned, there are, however, some technical problems involved in the continuous fermentation of milk with S. l i p o l y t i c a . The main problems are:

1. The sterilization of the whole equipment and the maintenance of sterile conditions. Because the doubling time of the yeast (ca. 4 h)

T a b l e 5 , 1 B a t c h g r o w t h a n d l i p a s e a n d p r o t e a s e a c t i v i t i e s of S. l i p o l y t i c a i n UHT m i l k . D u l v e n s e m l p l o t p l a n t s c a l e f e r m e n t o r ( A v e r a g e v a l u e s of t w o e x p e r i -m e n t s ) 0 4 6 8 10 12 14 16 18 20 22 24 30 34 4.5 6.0 8.5 14.0 21.0 30.0 36.0 51.0 75,0 80.0 155.0 230,0 510.0 380.0 6.52 6,40 6,24 6,02 5,89 5.78 5,70 5.53 5.37 5.24 5.14 5,00 4.67 4.42 0.00 0.75 1.25 2.00 2.50 3.10 3,50 7,70 10.80 14.20 14.80 14,80 14.80 14.40 0,00 0.60 1.10 1,60 2,30 2.50 3.10 6,00 9.00 13.60 14.20 13.60 12,50 11.80 0.00 1.00 2.25 3.50 3.90 4.60 4.75 4,35 4.00 3.25 2,75 2.75 2.50 2.50 104.4 769.1 1297.5 1674.1 2125,3 2368,5 2589.5 2905.7 3008,0 3002.0 2989.0 3026.0 2797.6 2624.3 n.d. n.d/ n.d. n.d. n.d. n.d. n.d. 30 150 180 205 195 190 200 270 270 265 260 255 260 290 320 425 685 900 1135 1575 1740 260 260 255 250 240 240 270 290 340 530 730 870 1150 1300 n.d. n.d. n,d. n,d. n.d. n.d. n.d. 22,2 44,9 113,1 222.9 296.3 532,7 659.9 00 CO o £ a. 6 s a. © • a ° ^ o _o -gs 7-. Oi <y> C 5 S< o -Q ^ •« Z to i-H C

T a b l e 5 , 2 a - F a t t y a c i d s ( / x m o l / 1 ) l i b e r a t e d f r o m m i l k f a t d u r i n g b a t c h g r o w t h of S . l i p o l y t i c a i n U H T m i l k i n t h e D u i v e n s e m i - p i l o t - p l a n t s c a l e f e r m e n t o r a e r a t e d w i t h p u r e o x y g e n ( A v e r a g e v a l u e s of t w o e x p e r i m e n t s ) Incubation time (h) Fatty acid C6:0 C8:0 CIO.O C10:l C12:0 C14:0 C14:l C15:0 C16:0 C16:l C18:0 C18:l C18:2 0 16 7 9 2 34 46 9 6 279 39 116 408 73 4 171 290 355 55 336 645 100 90 1554 173 760 2724 438 6 626 670 714 123 596 1208 162 157 3065 235 1300 3655 464 8 769 739 895 138 617 1551 246 237 3462 395 1791 5368 533 10 1177 703 916 126 1168 2236 367 319 4952 635 1861 6207 586 12 1293 672 896 123 1267 2488 328 316 5854 646 2534 6557 711 14 1389 463 767 130 1486 2706 451 324 6191 681 2720 7618 969 16 1521 364 692 128 1429 3139 450 413 7095 747 2747 9258 1074 18 1382 141 334 129 1172 3504 412 474 8422 1020 2719 9431 940 20 1240 98 225 143 768 3471 388 470 8593 933 2826 9923 942 22 1157 90 198 315 714 3498 339 498 8532 978 2731 9859 981 24 976 84 169 450 573 3445 302 488 8504 916 2682 10594 1077 30 323 75 123 1635 258 1676 240 287 7577 1059 2478 11166 1082 34 12 34 44 3152 84 414 112 212 6503 804 2276 11512 1084 Total fatty acids 1044 7691 12975 16741 2125J 23685 25895 29057 30080 30020 29890 30260 27979 26243 li mol/1

during the growth of S. l i p o l y t i c a inmilk. Fig. 5.2. shows the amounts of some of the free fatty acids present after 22 hours of fermentation of the milk by growing yeast cells and after 24 hours incubation of the milk with the purified lipase.

The odd fatty acid C15:0, which is metabolized by the yeasts during the first period of fermentation at a comparatively low rate, serves as refe-rence. (After about 24 hours C15:0 is also metabolized at a considerable rate.) The amount of C15:0 is almost equal in both experiments, and the amount of lipase in the experiment with purified exolipase is 7.5x10 mg/ ml, whereas during a fermentation with yeast cells the maximum amount

—3

of lipase IS 28.9x10 mg/ml.

The average amount of lipase over the whole incubation period is in both experiments almost the same, so the differences in the amounts of the even fatty acids observed between the two experiments is a measure of the

con-sumption of fatty acids by the yeast cells. A more specific picture of this consumption as a function of time is given m Fig. 5.3, which shows the relative amounts of saturated fatty acids of medium chain length compared with the amount of the odd fatty acid C15:0. From Pig. 5.3 it is, further-more, obvious that there are breakpoints betweenproduction and consumpt-ion of fatty acids. For C8:0 and C10:0 this point comes after six to eight hours of fermentation, whereas for C12:0 it comes aftei fourteen hours. The breakpoint for C14:0 lies between 24 and 30 hours (see Table 5.2a). F i g s . 5.4a and 5.4b show more precisely what happens with the fatty acids. In these figures the liberation of fatty acids in the milk, fermented by yeast cells, is directly compared with that m milk with the purified S. l i p o l y t i c a lipase.

On the basis of the data on the lyi^olysis of milk fat by means of purified lipase as a function of time (Table 3.19) and those on the amount of lipase produced by the yeast cells during their growth in milk (Table 5.1), it is possible toconstructacurvewhichrepresents the real amount of fatty

acids liberated from milk fat during this fermentation (see Figs. 5.4a and 5.4b). Now the amounts of fiee fatty acids actually present in the milk are known (Table 5.2a). The difference between the two curves shows the amount of fatty acids used as energy source for the growth of the yeast cells (Fig. 5.5a and 5.5b).

Some remarks should be made by the data about C10:l and C18:l. It is

15 20 Incubation time (h)

Fig. 5.3 Production and consumption of even medium-chain saturated fatty acids, compared with the odd fatty acid C15:0, during batch fer-mentation of UHT milk by S. l i p o l y t i c a ,

possible that the drastic increase in the amount of C10:l after 20 hours is the result of a four times repeated/5-oxidation of C18:l followed by excret-ion of the superfluous residue C10:l into the medium. This assumptexcret-ion is supported by the fact that in spite of the increase in oleic acid C18:l during the entire fermentation as a matter of fact a considerable amount of this fatty acid is consumed.

The proteolysis of the milk during the fermentation is shown in Fig. 5.6. During the first hours of fermentation there is a slight disappearance of small peptides probably due to uptake by the yeast cells. In this period hardly any proteolysis takes place. After fourteen to sixteen hours a strong proteolysis starts. In the period between 16 and 24 hours the increase in the amount of amino acids and of small and large peptides is more or less equal. However, after 24 hours the already formed peptides are hydrolysed with formation of amino acids. It is not possible to say whether this is the result of the activity of the exoprotease alone, oi of the combined effect of this protease, the uptake of peptides by the yeast cells, the intracellular proteolysis of these compounds and the excietion of superfluous amino acids back into the medium.

From the results of an analysis of the amino acids libeiated during the fermentation of the milk (Table 5.2b) and oui expei iments with an artificial milk (Table 4.6) the following conclusions may be

drawn-1. In the first stage of fermentation all ammo acids except threonine, alanine and perhaps lysine were taken up rapidly by the yeast cells. 2. Amino acids such as aspartic acid, isoleucme and leucine, which are abundantly present in milkproteins, were absent m the fermented milk during the fii st 18 hours of incubation. These amino acids seem to have special relevance to the growing yeast.

3. Milkproteins, especially the caseins, contain a large amount of pro-line, From our model experiments we know that the pure protease preferentially hydrolyses the caseins. Yet we did not find a trace of proline m the fermented milk. Using the same technique, experi-ments with L a c t o b a c i l l u s b u l g a r i c u s for the fermentation of milk showed that a large amount of proline had been liberated du-ring the proteolysis of milk (Veriips, 1975). As already concluded in Chapter 4, the yeast S. l i p o l y t i c a seems to lack enzymes ne-cessary for the biosynthesis of proline and, theiefore, has to utilize

5. The extremely high levels of lysine and histidine indicate that these amino acids play a minor role in the biosynthetic processes of the yeast investigated.

On the basis of the results obtained in the batch experiments we could s t a r t the continuous fermentation of UHT-milk with S. l i p o l y t i c a . In the culture medium the pH was kept at about 5,20. This was not effected through direct regulation of the pumps by thepH, but the regulation mecha-nism of the continuous fermentation was an accurate level sensor. Direct pH-regulation was notpossible in this system because after a certain period of fermentation the electrodes were poisoned by the fermented milk. The level sensor and membrane pumps were sufficiently accurate to ensure a very constant fermentation. Because the e d a c i t y of the fermentor was 10 l i t e r s and the doublingtime of the yeast cells nearly four hours, this system produced about 60 liters/day. The amount of milk in the fermentor could be increased without any difficulty to 15 liters and the production to 90 liters/day. However, for our experiments this was not necessary.

Fig. 5.7 shows a flow sheet of the equipment with a post-incubation ves-sel. This vessel was included because the fermented milk (pH 5.20) did not have all the desired taste properties. Inthispost-incubation vessel, having a temperature of 42 C, the normal fermentation was stopped, but the exo-enzymes, especially the protease were very active under these conditions, resulting in a high production of all kinds of taste compounds (mainly pep-tides). With this equipment five experiments were carried out. Two long runs of six weeks with spray-drying of the end product and taste evaluation experiments. For the analysis of the fermentor process we carried out two rims, one of two weeks and one of four weeks and special experiments were carried out in order to get information for calculation of all kinds of mass transfer coefficients. As the latter experiments were carried out under supervision of Ir. J. Lowik, the results obtained are not included in this thesis.

Table 5.3 shows the results of the analysis of the two continuous fermen-tation runs. From these data the conclusion may be drawn that all process parameters are nearly constant. The small differences between the values observed for these parameters lie within the experimental e r r o r s . The constancy of the quality of the fermented product was also confirmed by the results of the taste panel experiments. When batch and continuous

T a b l e 5 . 3 - C o n t i n u o u s f e r m e n t a t i o n of U H T - m i l k w i t h g r o w i n g c e l l s of S, 1 ip o l y t i c a , D u i v e n s e m i - p i lo t - p l a n t s c a l e f e r m e n t o r , T w o s e p a r a t e f e r m e n t o r r u n s m 3 <4-, ° to ^ d o Q o 1 2 3 5 7 14 1 2 3 7 14 24 29 c .2 S a <u E I H <D 2 S 125 130 120 135 140 125 140 130 130 150 120 140 160 X, a 5,21 5,21 5,19 5,21 5,22 5.20 5.20 5.22 5.20 5.23 5.20 5.21 5.23 a o to • c ^ o \

' 1

14.60 14.70 14.70 14.80 14.70 14.70 14.55 14.70 14.80 14.60 14.70 14.50 14.70 o (N 0 O S o a. 13.10 13.20 12.90 13.10 13,10 13.00 12.90 13,00 13.10 12.90 13.00 12.80 12.90 Lipas e activit y e.u . 3.00 3.10 3,00 2.95 3.00 3.05 3.00 3.00 3.10 3.05 3,10 3,00 3.10 Amoun t o f fre e fatt y acid s ^mol/1 29164 -32923 -33166 33121 28017 -32658 32628 32518 32647 P roteas e activit y e.u . 190 190 195 190 195 190 190 190 190 185 190 190 185 to

g

t

o o 1 2 solubl e i n 3% TC A mg/k g 815 845 810 850 845 860 8.45 8.40 8.60 8.30 8.45 8.40 8.40 to •H 3 O

t

o o Z solubl e i n 12 % TC A mg/k g 620 640 620 640 650 650 650 650 660 640 640 630 640 Amoun t o f fre e amin o acid s mg/k g 185,7 -179.6 -181.9 174.7 178.3 -184.8 190.4 182.1 185.3

T a b l e 5 , 5 - A m o u n t o f f r e e f a t t y a c i d s ( ; u m o l / l ) a t v a r i o u s c o n t i n u o u s f e r m e n t a t i o n s of U H T - m i l k w i t h g r o w i n g c e l l s of S, l i p o l y t i c a Fatty acid 06:0 C8;0 C10:0 C10:l C12:0 C14:0 C14:l C15:0 C16.0 C16:l C18:0 C18:l C18:2 Total fatty acids ^mol/1 Exp.I 1 1458 314 851 153 914 3137 418 375 6482 838 2679 10449 1096 29164 Exp,l 3 1399 371 853 150 1194 3575 784 503 6937 1034 2931 11994 1198 32923 E x p . I 7 1417 368 858 152 1200 3632 793 508 6978 1031 2942 12056 1211 33166 Exp.l 14 1405 374 842 143 1208 3651 799 512 7003 1052 2930 11998 1204 33121

Continuous fermentation time in E x p , II 1 1446 306 832 148 899 2962 308 344 6357 829 2523 9989 1074 28017 E x p , II 7 1383 364 850 141 1182 3514 778 495 6854 1017 2899 11974 1207 32658 E x p . II 14 1388 368 84 b 140 1194 3488 791 491 6838 1003 2934 11955 1198 32636 E x p . II 24 1390 356 845 122 1195 3407 756 491 6844 1005 29X8 11937 1252 32518 days E x p . n 29 1396 360 852 131 1178 3485 771 490 6856 999 2951 11965 1213 32647 E x p , II 650 rev,/mm 1392 335 859 125 1211 3505 780 492 6880 1009 2933 11971 1228 32720 Exp. II Exp. II 550 475 r8V,/mm rev,/mm 1352 351 919 131 1185 3434 580 480 6670 1021 2827 11850 1142 31942 1036 397 938 128 1120 2887 461 450 6240 867 2774 11582 1071 29951 o