Fig. 2. a Growth curve and base-titration curve for a batch fermentation of MRS broth by L. sali6arius BC 1001. b
Growth curve and base-titration curve for a batch fermenta- tion of brown juice by L. sali6arius BC 1001. The brown juice
was added glucose to reach the same sugar concentration as in the MRS broth.
Lier, 1994; Mu¨ller and Steller, 1995. The aim of this study is to find a way of simultaneous preser-
vation and utilisation of plant juice for fermenta- tion purposes.
2. Materials and methods
2
.
1
. Sources of juice Italian rye-grass Lolium multiflorum cultivar
Bofur, clover grass consisting of 50 of each of perennial
ryegrass Lolium
perenne cultivar
Chantal and white clover Trifolium repens cultivar Milkanova, and alfalfa Medicago sati6a
cultivar Daisy
were grown,
harvested and
cut by Borris Experimental Station, Danish Insti- tute of Plant and Soil Sciences. The fresh
crops were pressed in a screw press, Kent Ma- chines type 164 Innopro AB. Sweden, and sepa-
rated in juice and press cake. The juice was used fresh or kept in a freezer at − 20°C for up to one
year.
Brown juice was received at regularly intervals from an industrial green pellet plant, Dangrønt
Products, Ringkøbing, Denmark. The samples were placed in a freezer at the laboratory, at
− 20°C, immediately after receiving them. The
brown juice is produced when the green biomass is heated to 80°C by steam, followed by pressing
in a screw press.
2
.
2
. Dry matter The dry matter content of the juice was deter-
mined after drying at 105°C for 20 h, in an Heraeus Type 6060 laboratory oven.
2
.
3
. Sugar analyses Content of mono-, di-, and trisaccharides were
determined after centrifugation of the sample and the content of water soluble carbohydrates WSC
was determined after mild acid hydrolysis. The increase in the total amount of sugars after hy-
drolysis was ascribed to the content of fructans. products, other possibilities are now studied in-
tensively. It has been shown, that acidifying the juice by means of lactic acid fermentation can
make the juice storable Andersen and Kiel, 1997. The chemical composition of green crops
as well as juice from green crops has been analysed thoroughly Cheeseman, 1976. The vari-
ation in the content of water soluble carbohy- drates
in rye-grass
under different
cutting conditions McGrath, 1988, as well as the distri-
bution and metabolism of the reserve carbohy- drate, fructan in grasses have also been studied
Pollock and Cairns, 1991. Silaging of grasses has been studied intensively as well as the utilisation
of fructans by lactic acid bacteria Mu¨ller and
M .
Andersen ,
P .
Kiel Industrial
Crops and
Products
11 2000
129 –
137
Table 2 Variations in contents of free sugars: mono-, di- and trisaccharides and fructan in cuts of alfalfa, clover grass and Italian rye-grass
a
Alfalfa g kg
− 1
dry matter Clover grass
Italian rye-grass Julian day cut
Free sugars Fructan
DM Julian day cut Free sugars
Fructan DM
Julian day cut Free sugars
Fructan DM
117 5.94
183 264
161 5.38
161 196
6.02 147
230 45
4.57 210
249 105
175 4.62
6.15 182
211 153
178 177
78 5.50
252 198
218 5.07
9.58 218
280 259
133 60
3.71 299
232 164
6.59
a
Amounts in g kg
− 1
dry matter.
2
.
3
.
1
. Sample pre-treatments Hydrolysis of the grass juice samples were car-
ried out by adding 2.5 ml of a 1 M H
2
SO
4
to 20 ml grass juice. The solution was diluted to 50 ml
and placed in a boiling water bath for 15 min. After cooling, the solution was neutralised with
diluted ammonia. The hydrolysed samples as well as the untreated samples were centrifuged at
6000 × g in a Heraeus Sepatech Contifuge 17 RS. Before the sugar analyses, the ions in the samples
were eliminated by an ion exchange treatment in a mixed bed resin consisting of Amberlite CG 120
and Dowex 1 × 4 both from Fluka Chemie AG, Switzerland, and passed through a 0.45 microme-
ter Sartorius filter membrane.
2
.
3
.
2
. High performance liquid chromatography For sugar analyses a high performance liquid
chromatography HPLC system consisting of a Perkin – Elmer isocratic LC pump 250, a Perkin –
Elmer Series 200 RI detector and a Perkin – Elmer Model 1020 integrator were used. The pre treated
samples were injected through a Rheodyne model 7125 injector into a Bio-Rad Aminex HPX-87C
column. The column was provided with a Bio- Rad IG Carbo C micro guard column. The
column was kept at 85°C and the sugars were eluted with demineralised water with a flow rate
of 0.6 mlmin. Identification of the sugars were accomplished by comparing the retention times
with those for known standards. For control of the method test samples were simultaneously
analysed by means of an enzyme test method Boehringer Mannheim no. 716260.
2
.
4
. Analyses for organic acids An HPLC system consisting of a 410 series LC
pump Perkin – Elmer, a RI detector Series 200 Perkin – Elmer and a PC integrator model 1022
Perkin – Elmer were used. The system was equipped with two Aminex HPX-87 H cation-ex-
change columns Bio-Rad, connected in series. To protect the column a Bio-Rad 1G cation H
micro guard column was inserted. The first and the second column were operated at 35 and 85°C,
respectively as described by Blake et al. 1987. After centrifugation and filtration as described
earlier, 1 ml of the sample was injected through a Rheodyne model 7125 Injector with a 20 ml loop.
The eluent was a 5 mM sulphuric acid and the flow rate was 0.5 ml min
− 1
. The organic acids
were identified
by comparison
with known standards. Control of the results was done
by comparison with results from enzymatic meth- ods.
Table 3 Chemical composition of two different brown juice samples 1 and 2 in g kg
− 1
of dry matter, and the chemical composition of the same juices after sterilisation at 120°C for 20 min and fermentation by 1: L. sali6arius BC1001 and 2: L. paracasei ssp. paracasei P
4155 Composition of dry
Brown juice 2 fermented by Brown juice 1
Brown juice 1. fermented by Brown juice 2.
L. sali6arius matter g kg
− 1
L. paracasei. 76.3 9 3.0
322.8 9 2.8
a
322.6 9 4.8 Water soluble carbohy-
drates 244.6 9 2.4
Free carbohydrates 248.4 9 3.2
‘Fructan’ 74.1 9 1
76.3 9 3.0 78.2 9 5.2
30.0 9 0.2 11.4 9 0.1
11.6 9 0.3 11.4 9 0.1
Succinic acid 8.2 9 0.1
6.4 9 1.0 Malonic acid
9.3 9 0.1 10.2 9 0.3
20.2 9 0.3 Citric acid
19.0 9 0.1 19.2 9 0.3
Malic acid 27.6 9 0
28.8 9 0.7 35.1 9 5.2
1.8 9 1.6 2.7 9 1.3
Formic acid 1.0 9 1.6
233.6 9 5.9 Lactic acid
15.0 9 0.2 15.2 9 0.4
265.8 9 0.3 40.8 9 0.9
34.6 9 2.4 66.8 9 2.5
Acetic acid 34.6 9 0.7
388.4 9 8.2 119.6 9 2.9
331.0 9 8.3 117.6 9 4.2
Total organic acids
a
The numbers are averages of two independent fermentation with triple determinations.
Fig. 3. a Contents of free sugars, fructans and lactic acid in brown juice, before sterilisation light grey bars, after fermentation with L. sali6arius BC 1001 of sterilised brown juice, dark grey bars and after fermentation with the same strain in not sterilised
brown juice open bars. b Contents of free sugars, fructans and lactic acid in brown juice, before sterilisation light grey bars, after fermentation with L. paracasei ssp. Paracasei P 4155 of sterilised brown juice, dark grey bars and after fermentation with the
same strain in not sterilised brown juice open bars.
2
.
5
. Micro-organisms and culti6ation media Lactobacillus sali6arius BC 1001 was isolated
from grass juice at our laboratory Lactobacillus paracasei ssp. paracasei P4155 was kindly sup-
plied by Dr Marina Mu¨ller, Centre for Agricul- tural Landscape and Land Use Research, Institute
for Microbial Ecology, Paulinenaue, Germany.
The lactic acid bacteria were grown in MRS broth for 6 h at their optimum temperature,
which is 40°C for L. sali6arius and 35°C for L. paracasei ssp paracasei before inoculation of the
plant juice. Corynebacterium glutamicum V5, ATCC 700235
originally derived from Professor Martin Becker, Institute of Microbiology and Biotechnology,
University of Latvia, Riga was pre-cultured in shaking flasks at 30°C and 200 rpm. The composi-
tion of the growth medium in g l
− 1
was as follows: Glucose 60; hydrolysed soy protein 20,
NH
4 2
SO
4
10; MgSO
4
, 7H
2
O 0.6; KH
2
PO
4
1; biotin 0.00015; thiamine 0.0002; FeSO
4
, 7H
2
O 0.010 and MnSO
4
, H
2
O 0.007.
2
.
6
. Lactic acid fermentations Where nothing else is stated, the fermentation
experiments were carried out with fresh, unsterile plant juice. The juice was inoculated with a fresh
grown culture of L. sali6arius BC 1001 or L. paracasei ssp paracasei P4155. The amount of
inoculum used was 5 vv in the plant juice. The fermentations were carried out in double in
2-l fermentors with a working volume of 1 l. The fermentors were equipped with controllers for pH,
temperature and agitation. The pH was main- tained at 6.25 by automatic addition of 4 M
NaOH. The agitation speed was 92 rpm and the temperature was maintained at optimum.
The medium and the product were analysed for sugars and organic acids. All analyses were made
in triple.
2
.
7
. Growth experiments with Corynebacterium glutamicum V
5
The lactic acid fermented juice of alfalfa was used in growth experiments with the strain ATCC
700235 of Corynebacterium glutamicum V5. The growth in alfalfa juice was compared with
growth in the traditional soy hydrolysate medium described in Section 2.5. In the new medium 2
soy hydrolysate was replaced by 46 lactic acid fermented alfalfa juice with a dry matter content
of 12.3. The growth was carried out in a 100-l fermentor with a 40-l working volume. The
medium was sterilised at 121°C for 20 min. After cooling to 30°C and adjustment of pH to 7.0, the
inoculum, 1 was added. The agitation was in- creased with the increase in growth and varied
from 400 to 1000 rpm. PH was kept constant with 25 ammonia and dissolved oxygen was kept at
20 of saturation.
Every hour, a sample was taken to determine the optical density at 650 nm of the medium. Both
fermentations were repeated.
3. Results
