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
3
.
1
. Chemical composition of plant juice The total annual amount of brown and green
juice produced by Dangrønt Products Ltd, Den- mark is about 100.000 m
2
. Table 1 shows the composition of green juice
from different crops as well as one sample of brown juice derived from pressing of mixed crops.
Italian rye-grass shows the highest amount of water soluble carbohydrates. The juices also con-
tain some amounts of organic acids, especially malic acid and citric acid. The content of lactic
acid in the brown juice probably derives from spontaneous lactic aid fermentation in the grass
before pressing.
The content of mono, di- and tri-saccharides and fructans have been analysed in juices deriving
from 3 – 4 cuts of alfalfa, clover grass and Italian rye-grass, as well as the dry content of the juices
has been examined. Table 2 shows the variations in the examined samples.
The highest amount of fructans and total sug- ars are found in Italian rye-grass about Julian day
250, first week of September, where we found a total amount of carbohydrates of 40 of the dry
matter, where 50 of this is fructans. Alfalfa contains no fructans.
Fig. 4. Growth curves for C. glutamicum V5, ATCC 700239 in traditional growth medium with hydrolysed soy protein as
amino acid source and in the same substrate where the soy protein hydrolysate is substituted with lactic acid fermented
green juice from alfalfa.
Samples of brown juice were analysed for con- tent of dry matter, free and fructan bound sugars
and organic acids. From Fig. 1, it can be seen, that the dry matter
in the brown juice varies between 3 and 7, with an average on 4.8. The amount of free sugars
varies between 100 and 360 g kg
− 1
dry matter, the fructans between 70 and 170 g kg
− 1
. The highest total amount of carbohydrates is found to
be 47 in dry matter in June. A remarkable drop in the content of free sugars is seen at day number
211 and day number 280. As alfalfa is harvested only in short periods around these days, it is likely
to assume that the drop in the content of fructan and free sugars in the brown juice is caused by a
considerable increase of juice derived from alfalfa in the mixed brown juice.
The composition of the brown juice is not only influenced by the variation in the amounts of the
different species of crops. The weather conditions, temperature, the fall of rain and the time after
harvest where the crops are left on the field, before processing influence the final composition
of the brown juice. It is thus reasonable to believe that low content of free sugars at the 211 Julian
day is partly caused by lactic acid fermentation, before pressing, as the amount of organic acid
and particularly lactic acid is shown to increase at the same time as the amount of free sugars
drops.
3
.
2
. Lactic acid fermentation of plant juice Two strains giving the highest yield of lactic
acid have been selected and compared concerning productivity and utilisation of carbohydrates and
organic acids. The highest growth rate and lactic acid produc-
tivity is found with Lactobacillus sali6arius in sterile brown juice. Growth in sterile MRS broth,
normally used for cultivation of lactic acid bacte- ria and sterile brown juice with the same content
of WSC are compared in Fig. 2a and b, where it can be shown, that the growth rate is higher in
brown juice than in MRS broth.
The chemical composition of brown juice be- fore and after sterilisation and lactic acid fermen-
tation shown in Table 3 documents that the two strains have very different abilities to utilise sug-
ars and organic acids in the juice. Lactobacillus paracasei ssp. paracasei P4155 is found to utilise
all available free carbohydrates as well as fruc- tans, citric acid and malic acid, whereas Lacto-
bacillus sali6arius BC 1001 is not able to utilise fructans and citric acid.
Lactobacillus paracasei ssp. paracasei P4155 produces more formic acid and more acetic acid
than Lactobacillus sali6arius BC 1001. When unsterile brown juice is fermented by the
two different lactobacilli as shown in Fig. 3a and b, the same tendency is seen. The only difference
is that we reach a higher yield with both strains, presumably because other micro-organisms and
enzymes in the brown juice take part in the degra- dation of the different carbon sources.
The fermented juice has been stored for up to half a year under approximately anaerobic condi-
tions in a 8 m
3
circular tank with just enough air space above the liquid to allow an UV lamp to be
placed. The juice has also been concentrated by
evaporation.
3
.
3
. Growth experiments with lactic acid fermented juice
Both evaporated and stored juice have shown to be excellent growth media for par example
Corynebacterium glutamicum, used for amino acid production. If peptone, yeast extract or hy-
drolysed soy protein in a general growth medium is substituted with fermented brown or green juice
with the same content of protein a very high growth rate can be obtained as shown in Fig. 4,
where Corynebacterium glutamicum V5, ATCC 700239 is grown in lactic acid fermented green
juice from alfalfa.
4. Discussion and conclusion
