, &

2466

: ,

, 2011

UNIVERSITY OF PATRAS
DEPARTMENT OF CHEMISTRY
DIVISION OF APPLIED, ANALYTICAL
& ENVIRONMENTAL CHEMISTRY

DISSERTATION:
PRODUCTION OF KEFIR SINGLE CELL PROTEIN USING
MIXED WASTES OF THE FOOD INDUSTRY

DIAMANTOPOULOU DIONYSIA
2466

Supervisor: Dr. Argyro Bekatorou, Lecturer

PATRAS, 2011





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ABSTRACT
Food industry wastes are rich in carbohydrates and have high organic
loads so they cannot be processed through biological treatment, and are
usually discarded causing environmental pollution. On the other hand,
their abundance and rich composition renders them important substrates
for biotechnological conversion to added-value products. The present
study describes the treatment of mixed food industry wastes (cheese
whey, molasses, orange pulp and potato pulp) with the natural mixed
culture kefir for single cell protein production. The treated materials can
be used as protein enriched animal feeds or as food additives and the
developed processes will lead to reduced environmental pollution cause
by the disposal of the above substrates. The choice of the kefir culture
was based on the fact that its microflora includes microbes that are able
to utilize the different carbohydrates contained in the proposed
substrates, as well as microbes with probiotic properties.

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Key words: food industry wastes, kefir culture, single cell protein,
cheese whey, molasses, orange pulp, potato pulp, animal feed

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........................................................................................3
1 - ..........................................................................9
1.1 ...................................................................9
1.1.1 ................9
1.1.2 .............................................................................10
1.1.3 .................................................................................11
1.1.4 (SCP).............................................................11
1.2 .............................................................12
1.2.1 .................................................................................12
1.2.2 ................................................................................. 14
1.2.3 ................................................................................ 14
1.3 .....................................................................14
1.3.1 ...............................................................................15
1.3.2 ............................................................................ 15
1.3.3 ...................................................................................................... 17
1.3.4 ...............................................................................17
1.4 SCP...........................................................................................17
1.4.1 SCP..............................................................................18
1.4.2 ......................................................................................... 18
1.4.3 ...........................................19
1.5 ...........................................20
1.5.1 ...................................................................................................... 20
1.5.1.1 .....................................................................21
1.5.1.1.1 ................................................................................22
() Saccharomyces cerevisiae...................................................................22
() Kluyveromyces..........................................................................22
1.5.1.1.2 ..................................................................23
() Streptococcus.......................................................................23
() Lactococcus..........................................................................24

() Lactobacillus.........................................................................24
1.5.1.2 ..................................................................25
1.5.2 ....................................................................25
1.5.2.1 ...............................................................26
1.5.2.2 ........................................................................26
1.5.2.3 ................................................................26
1.6 .........................................27
1.6.1 ...........................................................................27
1.6.1.1 .........................................................................27
1.6.1.2 .....................27
1.6.1.3 ............................28
1.6.2 ................................................................................................ 29
1.6.3. .................................................................................................. 30
1.6.4 ................................................................................ 30
1.6.5 .......................................................................31
1.6.5.1 .................................................................31
1.6.6 .....................................32
1.6.6.1 ................................................33
1.6.6.2 ......................................................................33
1.6.6.3 ............................34
1.7 ..............................................35
2 - ..................................................................36
2.1 ..................................................36
2.1.1 ....................................................................................................... 36
2.1.1.1 ..........................................................................36
2.1.1.2 ........................................................36
2.1.1.3 .....................................................................36
2.1.1.4 .........................................................................36
2.1.1.5 .................................................37

2.1.1.5.1 .................................................................................... 37
2.1.1.5.2 ....................................................................................... 37
2.1.1.5.3 .................................................................38
2.1.1.5.4 ........................................................................38
2.1.2 ............................................................................................... 38
2.1.2.1 HPLC (High Pressure Liquid
Chromatography)...................................................................................... 38
2.1.2.2 Kjeldahl...................................38
2.1.2.3 .........................................................40
2.1.2.4 ..................................................................40
2.1.2.5 .................................................................40
2.1.2.6
/ pH.....41
2.2 SCP
................................................................44
2.2.1 .......................................44
2.2.1.1 ................44
2.2.1.2 ....................44
2.2.1.3 ...............................45
2.2.2 ................................................................45
2.2.2.1 4Be.............................................................45
2.2.2.2 .......................................................46
2.2.2.3 .................................................46
2.2.3
......................................................................................................... 46
2.2.3.1 .....................................46
2.2.3.2
.......46
2.2.3.3 /
pH....................................................................................... 48
3 - ...................................49
3.1 ...............................................................49

3.2 SSF . . .52

3.2.1 .........................52
3.2.2
................................................................................................... 57
..................................................................60
4 - .................................................................63
........................................................................................... 63
.................................................................................... 64

 1 -
1.1
1.1.1

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: Saccharomyces (S. cerevisiae, S. carlsbergensis, S. uvarum
..), Kluyveromyces (K. marxianus, K. fragilis, K. lactis, K. bulgaricus),
Candida (C. kefyr, C. pseudotropicalis, C. Utilis, C. lipolytica) ..

. S. cerevisiae, K. marxianus C.
utilis
. S. cerevisiae
,
SCP
. K.
marxianus ,
,
,

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. K. marxianus,

45C,
.
C. utilis ,
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,
C. utilis.

Yarrowia Lipolytica, C. tropicalis, C. rugosa C. guilliermondii,
.

 Pishia sp., Hansenula

polymorpha, H. capsulata C. boidinii.
,
. ,
/ . , ,
.
SCP ,
Acinetobacter calcoaceticus Alcaligenes sp.
.
SCP ,
,
, Kiel.
, Lactobacillus delbruckii subsp. bulgaricus,
, .
, C. krusei
.
,
, .

, L.
delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. casei
subsp. casei, Leuconostoc sp. .
Propionibacteria, P. freudenreichii subsp.
shermanii P. freudenreichii subsp. freudenreichii,
12,
SCP.
,
SCP, .

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,
(Anderson.,
2009; Garca-Garibay et al., 2003).
1.1.2


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(Stepaniak et al., 2002).
1.1.3
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, 10. 000 .


,
(M,
2001).
.
, Leningrad,
1008 .
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. 1500 . .
15% .
(2100 . .)

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. 1864 . .
Luis Pasteur,



(M, 2001).

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. Hammurabi 1770 . .

. 1000
,
1919 o 1933 (M, 2001).
1.1.4 (SCP)
SCP
.
, S. cerevisiae

60% .


C.utilis ( Torula)
. ,

Torula.

,
SCP.
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. 60 70,
SCP , ,
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SCP ,
, 50 60.
1970,
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. SCP
1980.
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, SCP,
86 , .
,
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SCP, .
,
SCP

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SCP
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1.2
1.2.1

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Acetobacter aceti,
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(GC)
(HPLC) (, 2001).

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CO2 H2O ,
,
(M, 2001).
1.2.2
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, 95% ,
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16 ,
12 . ,
, Gram,
.

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et al., 2003).

1.2.3
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(Alur, 1999).

1.3

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(
., 2009). ,
(),

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(, 2009)
1.3.2

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1 (lag phase)

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pH .
4 (Death phase)



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S. cerevisiae,
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(Narvhus et al.,
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SCP ,
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SCP
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SCP

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S. cerevisiae, K. marxianus C. utilis

. S. cerevisiae

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(Garca-Garibay et al., 2003).
1.4.1 SCP
SCP
, .
50%-85%
45%-70%. , ,
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,

,
. , SCP

(Garca-Garibay et al., 2003).
1.4.2
SCP, ,
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C. utilis,
Torula,
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C. utilis.

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, 50%
( , ),
( , ). ,

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, , (Koutinas
et al., 2003; Garca-Garibay et al., 2003).

1.4.3
SCP ,
.

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SCP ,
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L. delbruecki, bulgaricus, kefir K. marxianus
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(,
, pH, ),
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L. delbrueckii subsp. bulgaricus Streptococcus
thermophilus,
(Garca-Garibay et
al.,2003).

1.5
1.5.1

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12 .

"" .
.
kefiran.
,
.
(Stepaniac et al.,
2002).

1.5.1.1
,
.

,
Kluveromyces, Candida, Torulopsis Saccharomyces,
Lactobacillus, (L. brevis, L. acidophilus, L. casei, L.
helveticus, L. delbruecki), Streptococcus S. salivarius,
Lc. Lactis ssp. thermophilus, Leuconostoc
mesenteroides, L. cremoris (
et al., 2007; Stepaniak et al., 2002).
1. K (Stepaniak et al.,
2002)
Lactobacilli
(1085 109 g1)

Lb. acidophilus
Lb. delbrueckii subsp.
bulgaricus
Lb. helveticus
Lb. kefiranofaciens
Lb. kefirgranum

Lb. kefir
Lb. parakefir
Lb. brevis
Lb. plantarum
Lb. casei and
subspecies

1.5.1.1.1

Lactococcus &
Leuconostoc
(108109 g1)

Lc. lactis subsp. Lactis

Lc. lactis subsp.
cremoris
Sc. thermophilus

Lc. lactis subsp. lactis

biovar. diacetilactis
Ln. lactis
Ln. mesenteroides
subsp. mesenteroides
Ln. mesenteroides
subsp. cremoris
Ln. mesenteroides
subsp. dextranicum

Z
(106108 g1)

K. marxianus
(anamorph: Candida
kefyr, synonyms: K.
lactis, K. bulgaricus) &
subspecies
Brettanomyces
anomalus (synonym:
Dekkera anomala)
Debaryomyces
hansenii (anamorph:
Can. famata)

S. unisporus
Sac. turicensis
Sac. cerevisiae
Sac. exiguus
(synonyms: Can.
holmii, Torula holmii)
Torulaspora
delbrueckii
(anamorph: Can.
colliculosa)
Pichia fermentans
(anamorph: Can.
lambica)

() Saccharomyces cerevisiae
S. cerevisiae
, , . ,
Meyen
Reess 1870
. S.
cerevisiae .

16 Saccharomyces
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Saccharomyces ,
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Saccharomyces

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, D ,
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S. cerevisiae
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12 14 (Koutinas
et al.,2009; Yuji Oda et al., 1999).
() Kluyveromyces
Kluyveromyces Ascomycetes.
, Ascomycetes
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Kluyveromyces

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Kluyveromyces
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.
17 Kluyveromyces.

. Kluyveromyces , K.
polysporus, K. africanus
.
Kluyveromyces

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S. cerevisiae ,
Kluyveromyces
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.
K. lactis K. marxianus
Kluyveromyces.
.
,

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-.

K. marxianus K. lactis, .
,
Kluyveromyces , ( Batt,
2004).
1.5.1.1.2
() Streptococcus
Streptococcus,
. 1980,
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Enterococcus Lactococcus. ,
,

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, S. thermophilus.
,
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delbrueckii ssp. bulgaricus,
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Streptococcus
(Narvhus et al.,
2003).

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Lactococcus,
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. L. cremoris L. lactis
. L. cremoris L.
lactis .
, L. lactis
L. cremris. L. cremoris
(Narvhus et al., 2003).
() Lactobacillus
Lactobacillus,
60 .
(Weissela Carnobacterium),
.
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1919, Orla-Jensen, Lactobacillus :
Thermobacteria, Steptobacteria Betabacteria. ,
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bulgaricus,
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Leuconostoc /
,
-, (Narvhus et al., 2003).
1.5.1.2
, ,
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,
, .
, 70%

.


,
.
, ,
,

,

Kluyveromyces, Candida, Torulopsis Saccharomyces,
L. brevis, L. acidophilus, L. casei, L. helveticus, L.
delbruecki, S. salivarius,
Lc. Lactis ssp. thermophilus, Leuconostoc mesenteroides and L. cremoris
(Koutinas et al., 2005).
1.5.2
, , ,

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1235 ,
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(Akuzawa et al., 2002).
1.5.2.1
, ,
Candida spp., K. lactis Torula spp.
L. delbrueckii subsp. bulgaricus,
.

.
Lb. acidophilus Lactococcus lactis (Akuzawa et al., 2002).
1.5.2.2
,

.
30 C 15
37 C 7 .
,

28 C 4 ,
1,4 %. 30%
28 C
.
,
3 20C.
6 C .

2,5% 10%
. 26-28 C
0,8%
. , 17 C

0,9%. .


6 C (Akuzawa et
al., 2002).
1.5.2.3


.
.
1990
Lc. lactis subsp. lactis, Lb. delbrueckii subsp.
bulgaricus .
20-25
2-3 . ,
.
.

.
.
0,6-0,8% 0. 7-1%.
0,8-1% 1,1-1,8%
1,0-1,2%
1,8-2,5% (Akuzawa et al., 2002).
1.6
,
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.
,
,

(Hunt, 2003).
1.6.1
1.6.1.1

, , 672
.


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(Hunt, 2003).
1.6.1.2
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(Hunt, 2003).
1.6.1.3 .
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(Hunt, 2003).
1.6.2

.

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(
70 % ) , ,
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, SCP
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,

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,

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,
,

. ,
, SCP, , ,
. ,
,
.
, ,
,
luyveromyces .. K. marxianus, K.
fragilis K. lactis, S. cerevisiae
- (Koutinas et
al., 2008).

1.6.3.
,
.

,
.
SCP,
. ,
,

,
.
80%
, 50% 30%
20% . ,
, , .

, ,
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, , , ,
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. ,

.
, 40%
,

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(Koutinas et al., 2004).
1.6.4
, ,
.
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, .
.
, ,
,
,
.
, , ,
, (
), - , -
, ,
, ,
(Burlingame et al., 2009).

 , , ,

,
(Thompson et al., 2008; Rama, 2003).

,
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1.6.5

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1.6.5.1
, ,
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C, 75%
9,14 100 .
, , , , , ,
.
pH 2,5. 88%

10%.
, 1:1:2.
, ,
.
,
,
. pH 3,5

.
,
.

C. ,

 120% C .
C ,

. B
.
,
. , ,

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, ,
(Pao
et al., 2003).

,
.

.

70
(USDA/FAS 2003).
, 70%.

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,
,
.
1.6.6
,
.

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,
.

,
,
.
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,
,
(Hunt et al., 2003).

, ,

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,
SCP .
1.6.6.1

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, .
, , ,
, ,
. ,
SCP,
, .
,
,
.
SCP,
(C. utilis),
, (Saccharomyces, Kluyveromyces),
(), ,
, (, ,
)
. ,

SCP
. ,
,

,
.

,
.
1.6.6.2
,
,
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1.6.6.3
,
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.

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,
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,
,
,

.

70 (USDA/FAS 2003).
,
70%. ,

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1.7

SCP
(, , ).


,
.

 2 -
2.1
2.1.1
2.1.1.1

.

,
,
.
2.1.1.2
.

,
,
. 48 h
30C
2-4C. .
2.1.1.3

2% v/v .

5 mL . 48 h
30 C, 50 mL .
,
500 mL , ,
2 L,
. 2 L, 20
g .
2.1.1.4

:

(oBaume)

oBe, 100 mL

(vortex): IKA-VIBRO-FIX

: EXACTA, Alessandrini Strumentazione

Cobo

: METTLER 200. Orma model BC

-:

WBT BINDER, B 115-89080, 10-70 oC

TERMAKS, 10-70 oC.

: TECHNIC U PROFESSIONAL MICROSCOPE

: EPI-BION SENTRON pH-System 1001

: LABCONCO WATER PRO PS (

, )

: SHIMADZU LIQUID
CHROMATOGRAPH LC-9A

: Shim-pack SCR-101N, 142/B

:
(
)

: Refractive Index Detector RID-6A

: Column oven SHIMADZU CTO-10A

: DGU-2A

: Schleicher & Schuell SPARTAN 13/B, Disposable Filter

Holders (0,45 m)

Kjeldahl : InKjel M

: SIGMA 3K12, BIOBLOCK SCIENTIFIC

Shaker : SELECTA ROTABIT MOTOR (150-200 U/min)

Raypa Incuterm Digit

Stomacher : Bag Mixer interscience

Gosselin

2.1.1.5
2.1.1.5.1

.
2.1.1.5.2



4 ,
. .
2.1.1.5.3

.
2.1.1.5.4

.
2.1.2
2.1.2.1

Chromatography)

HPLC

(High

Pressure

Liquid

(HPLC) SHIMADZU LC9A. : (i) Shim-pack, SCR-101 N, (ii)
: , (iii)
: 0. 8 mL/min (iv) : 60C, (v)
: .
1% v/v
0,45 m.
-1 1 % v/v.
2.1.2.2 Kjeldahl
Kjeldahl, 1883,

. ,
,
.
,
6,83

.
, Kjeldahl,

.
,
,
,
.
() .

,
. , ,
3 0,3
. ,


. , 7 ml 10 ml ,
, .
Kjeldahl.
() Kjeldahl
, 6 .
,

.

40 oC
60, 80 100 oC .
100 oC,
30 . , ,
, . ,

. ,
,
. ,

.
() .
,
. ,
20 ml Cl 0,1 .
NaOH,
, 60 mL.
NaOH, .
, ,

HCl. , ,
HCl
.
() HCl
,
HCl NaOH 0,1
. NaOH
,
. ,
, 2 ,

 . ( .)
2.1.2.3

, 0 72 ,
. , 1
ml . ,
, 1/10
, 1/100.
, Vortex,
.
.



.

.
5 , 5
1000,
(0,100*0,0025)
10 100 .
2.1.2.4
,
.
,
, 1
, .
x1 =
x2 = -
x3=
, :
=x3- x1
(x2-x1) , (x3-x1)
, 100 (x3-x1)/ (x2-x1).
100/100 - 100 (x3-x1)/ (x2-x1).
,
,
.
2.1.2.5


.
(AOAC 942. 05, 1995)
.

, 15 .
550C, 5 ,
.
100
:
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100(3. 4)

0 = (g)

1 = (g)

2 = (g)

2.1.2.6
/ pH.

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2 ,

72 .
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Duran.
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PDA Potato Dextrose Agar

PDA ,
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,

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PDA 39,0 g /L pH 4,5 3,5.
4 g
, 20 g 15 g .
,

0,1 ml .

 PDA, 35 30 oC.

M17
17,
, . ,
,
. 17 57,2 g / L pH
7,1.
2,50 g , 2,50 g peptic digest of meat, 5,0 g
papaic digest of soybean meat, 2,50 g , 5,0 g
, 5 g , 19 g ,
0,25 g , 0,50 g 15 g .
37 oC 48-72 ,
, 30 oC
48-72 , .
17 .
, 1 mL
.

MRS agar
MRS agar,

. 70,3 g/L.
10 g , 10 g , 5 g
, 20 g , 1,08 g tween 80, 2 g
, 5 g , 2 g , 0,20 g
, 0,05 g 15 g .
37 oC 48-72 ,
.

()

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2.2.1
2.2.1.1
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500 ml
.
1:

/ ml

200 ml

2 % w/v

2 % w/v

0,1 % w/v

0,1 % w/v

1,02 % w/v

Yeast extract

0,4 % w/v

2 % w/v

,

40 oC.
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/mL

400 ml

2 % w/v

2 % w/v

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1,02 %w/v

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2 %w/v

. ,
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100 ml
10 ml
10 ml
10 ml
100 ml
30 ml
10 ml
10 ml
100 ml
10 ml
10 ml
30 ml
100 ml
30 ml
100 ml
30 ml
100 ml
--30 ml
100 ml
30 ml
10 ml
-100 ml
30 ml
-10 ml
100 ml
10 ml
--30 ml

.
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HPLC.


Kjeldahl.
2.2.3.3 /
pH

pH 4, 5,5 7.
.
pH
pH NaOH
.
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.

 3 -
3.1
1:
.

(%w/w)

93. 50. 4

22. 81. 0

82. 91. 4

82. 8

(%w/w)

6. 50. 4

77. 21. 0

17. 11. 4

17. 2

pH

4. 2

4. 7

4. 1

5. 5

1. 90. 5

3. 81. 7 c

14. 22. 5

5. 180. 48

7. 50. 5
c
d

0. 36

1. 04 d

0. 0 e

0. 8 c

0. 58 c

7. 32. 3 e

2. 85

2. 47 d

42. 56. 1 e

8. 31

0. 88

b
aq

1012aq

COD (mg/L)

525585165

>80000

1034

aq (mg/g
DM)

na

7. 721. 50

15. 782. 16

6. 021. 03

org (mg/g
DM)

na

1. 760. 08

4. 981. 04

2. 740. 65

4. 350. 40

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2. 030. 78

3. 790. 90

org (IC50
mg/ml)

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(Sawalha et al., 2009).
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(Sawalha
et al., 2009).
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.

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,

, (Lisiska, 1989; Nandutu et al., 2007;

Eichhorn et al., 2005; Rodriguez et al., 1994).

(Wang et al., 2008),
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1999), .
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(Negro et al.,
2003).
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2
:
) HPLC

)
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)


. 2, 4, 7 8
30 mL
36,5, 36,6, 35,5
37,2 . ,
, 5 6 28,3 27,9
.

. :

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0,38
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6
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3

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5 , .
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6. , , ,

(Koutinas et al., 2007) .
1:

0,5
9

Cells x 10 /g sugar utilised

Cell biomass /g sugar utilised

0,4

Yield

0,3

0,2

0,1

0,0
1

Substrate



:
( - )

3
(- )

(- )

(- )

 2:

(ml)

(4oe)

(1:2)

mL

mL

mL

mL

100

10

10

10

g/L

30,1
2
3
4
5
6
7
8

100
100
100
100
100
100
100

30
10
30
30
30

10
30
30
10

36,5

35,49

30,2

30,77

36,6

36,60

28,3

27,27

27,9

27,25

10
30
10
35,4

34,23

37,2

37,03

(cells1
09/g
sugar
utilized)

(g
biomass
/g sugar
utilized)

g/L

cells
109

cells
109

0,47

1,63

1,2
3

6,60

6,2
3

0,17

0,17

1,01

1,48

1,1
2

6,50

6,1
8

0,20

0,14

0,43

1,14

1,0
1

7,16

12,
8

0,20

0,38

0,00

1,4

1,1
3

3,60

3,3

0,06

0,06

0,03

2,43

1,4
2

7,28

12,
4

0,18

0,40

0,65

1,47

1,1
0

4,80

6,3
7

0,12

0,19

1,17

2,72

1,1
3

5,16

3,8
4

0,07

0,08

0,17

2,21

1,2
1

4,88

4,7
6

0,07

0,10

29,63

10

100

10

30

1,74
31,5

29,76

2,72

1,6
0

5,12

9,3
1

0,05

0,26

3.2.2

,
pH 4, 5,5 7
, 3
SCP.
,
:
) HPLC

) SCP
Kjeldahl
)
)
)
)
:
pH = 4 SCP 20,28%
0,196
pH = 5,5 SCP 21,3%
0,336
pH = 7 SCP 20,56%
0,206
pH = 5,5
SCP
. , ,
100 mL , 10 mL , 30 mL
10 mL ,

.
, , pH 2-8,
, 4-4,5 (Loureiro et al.,2003).
, Saccharomyces,
pH 4,5-5,0 (Richard et al., 2004). ,

,
5,5 4 7.

 pH 4-7
pH 5,5

2:
, .
40

yield (g biomass / g sugar utilised)x100

protein increase

35
30

Yield

25
20
15
10
5
0
3,5

4,0

4,5

5,0

5,5

6,0

6,5

7,0

pH
3:

4 pH=4
Y4 pH=5. 5
Y4 pH=7

g/L
35,85
35,20
37,28

g/L
0,16
3,31
2,87

g
1,250
1,343
1,327

(g
biomass/g
sugar
utilized)

g
8,232
12,077
8,398

0,196
0,336
0,206

%
12,32
10,30
11,72

%
32,60
31,60
32,28

4:

(log CFU/mL)

PDA (30 C) ME (30 C) MRS (37 C) MRS (pH 5.

2, 37 C)

(17 37 C)

(17 30 C)

6,28

6,38

6,67

6,36

6,59

6,86

9,13

8,97

8,29

8,28

8,46

8,21

 4
.
,
30 C .

, ,
.
,
37 C, pH 5,5-5,8. ,
,
.
,
37 C
30 C.
.

,
,

.
, SCP
.
, ,

. ,
SCP,
,
(Paraskevopoulou et al.,
2003).
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, SCP
, ,
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,

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,
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,
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,

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40% ,
.

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.

 4 -

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