BIOTECHNOLOGY LETTERS

Volume 15 No.3 (March 1993) pp.239-244

received lOth February

INFLUENCE OF MEDIA C O M P O S I T I O N ON I~ACTIC ACID

P R O D U C T I O N RATE FROM WHEY BY Lactobaciilus helveticus

A. AMRANE and Y. PRIGENT ~'

Universit6 de Rennes I
Laboratoire des proc~d6s de separation
I.U.T., D6partement Chimie
B.P. t 144. 35014 Rennes cedex. France

SUMMARY
The effect of preculture and culture media formulation on Lactohacillus helveticmv
lactic acid production rate was investigated in batch fermentations. Maximum lactic acid
productivity of 5.5 g/l.h, was obtained from hydrolyzed whey. Clarified whey
ultrafiltrate gave 4.4 g/l.h, at less expense.

INTRODUCTION
Lactic acid bacteria are known as very fastidious concerning their nutritional
requirements (Desmazeaud, 1983), and especially their nitrogen supplementation :
according to Vahvaselkh and Linko (1987), Aeschlimann and von Stockar 11990). a
culture medium giving rise to fast growth and lactic acid production rates has to be
supplemented with yeast extract ; as shown by Francou t1986), Baralle and Borzani
(1988), lactic yeast autolyzate may be used instead of veast extract : Leh t 1987) also
reported that whey proteins enzymatic hydrolyzates may be regarded as a fair nitrogen
source.

In contrast to this, little attention has been paid to inoculum preparation ( Reddy et al.,
1976 ; Roy et al., 1986 ; Vahvaselk~i and Linko, 1987) : usually, lactic acid bacteria are
precultivated on skim m i l l
The main purpose of this paper is to investigate how media composition /nitrogen
sources, total phosphorus concentration) influences lactic acid production rate.

MATERIAI,S AND METItOI)S

The Lactobacilht~ helveticus strain milano used throughout this work was kindly
supplied by EVEN (Ploudaniel, France).
Seed culture and culture media were prepared from the tollowing carbon sources :
fresh sweet cheese whey (FW), reconstituted sweet cheese whey powder ~WPL or their
hydrolyzates (suffix H) by a protease of Bacillus subtilis (B500. GIST-BROCADES,
S6clin. France) according to Leh (1987). For some experiments, whey was clarified

239
(prefix K) by heat/calcium process and cross-flow microfiltration (Fauquant et al.. 1985),
and then hydrolyzed as before, or ultrafiltered (suffix U),
Either of the following nitrogen supplementations were used for the seed culture
media: YEP contained (g/l) : yeast extract : 20, trypsic casein peptone : 5. pancreatic
casein peptone : 5 and Tween 80 : I, so its total and a-amino nitrogen contents were
respectively 4.54 and 1.67 gll. Kluyveromyces fragilis (CBS 397) were grown on a
WPU medium supplemented according to Wasserman et al. (1958), and autolyzed as
described by Orberg et al. (1984). Then the medium was supplemented with 1 g/1 Tween
80 and an amount of yeast autolyzate such that its total and a-amino nitrogen contents
were close to those of YEP : this supplementation was referred to YA.
The various media are summarized in Table 1 as regards their composition.

TABLE 1
Batches Preculture medium Culture medium
Carbon source Nitrogen Carbon Nitrogen
supplementation source supplementation
FIG, la B025 FWH YEP FW H
B032 FWH FWH
FIG. lb B088 WPH YEP W P H
B177 WPH YA W P H
B025 FWH YEP FW H
FIG.2 B297 FWH YEP FWU CSL* (4 ~/i)
B086 KFWH YA KFWU CSL* (4 g/l)
* CSL = corn steep liquor.

250 ml sterile preculture medium were inoculated with 2 ml skim milk culture and
incubated overnight (16h) at 42C. Then 200 ml (11%, v/v) preculture were aseptically
transferred into 1600 ml culture medium in a 21 fermentor (SET 2M SGI, Toulouse,
France). Lactic acid fermentation was then camed out at 42C. agitation speed 300 rpm :
pH was maintained at 5.9 by automatic addition of 10M NaOH. In addition, the
fermentor was equipped with an aseptic recirculation loop involving a laboratory-made
turbidimeter : as turbidity was continuously recorded, total biomass was calculated on-
line after dry cellular weight calibration.
After fermentation had been processed, lactose and lactic acid concentrations were
determined spectrophotomeWically on the supernatant by the phenol-sulfuric acid method
(Herbert et al., 1971) and the Fe3+-lactate complex method (Ayroulet-Martin and
Fournaud, 1979) respectively.
Hydrolyzed whey samples and yeast autolyzate were analyzed for nitrogen contents,
total nitrogen NTK by the Kjeldahl method (Rodier, t975), and primary (x-amino groups
NaNH2 by the TNBS method (Satake et al., 1960). after enzyme and remaining proteins
had been precipitated with zinc hexacyanoferrate (Desmaison et al.. 1970). Peptide's
average molecular weight was calculated according to Leh (1987) :

MV = NTK * 120
Nc~NH2 * [.31

240
RESULTS

I) Com.mri~on ~ t w e e n nitrogen supplementations for seed culture media"

The batch fermentations displayed in Fig.la were obtained on the same culture
medium (FWH) - the seed culture media, both based on FWH. differ only as regards
their nitrogen supplementations (B025 YEP ; B032 none). As expected, bacteria
precultivated on an unsupplemented medium give rise to slow growth and lactic acid
production kinetics, leading to the following conclusion : the mean volumetric
productivity obtained from a minimal culture medium is mainly determined by the
nitrogen supplementation of the seed culture broth.
As shown in Table 1, the batch fermentations B088 and B177 displayed in Fig.lb
were both obtained from a W P H culture medium ; these fermentations differ mainly as
regards the nitrogen supplementation of their seed culture media total and a-amino
nitrogen contents were respectively 4.9 and 1.9 g/l for YEP. and 4.54 and 1.67 g/l for
YA. So the average molecular weights MW were nearly the same (YEP" 236" YA 249L
Fig.lb clearly shows that bacteria precultivated on a W P H + Y E P medium lead to a
faster growth on a W P H culture medium, and also to an earlier autolysis than the ones
prepared on a WPH+YA seed culture medium.
As a consequence of this, lactic acid production starts earlier for B088 " but both
fermentations cease at the same time, and at the same final lactic acid concentrations
(mean volumetric productivities- VP = 4.3 g/l.h.).

Fig. 1 " Precutture medium FWH supplemented with

a
b
50 lO.x (g/l) 50 10.x (g/l) . o o oo o
40 P (g/l),~/~
40 p (gjl)o
3O / S
0 0 0 0 0 0 0 0

2O

10 I0
time (h) t ~~ Z ime (h)
0 J ' i i O I i I I

0 5 I0 15 20 0 5 I0 15 20
B032 none ( * growth. ~ production) B088 YEP (o growth. + productionl
B025 YEP ( [] growth. + production} B177" YA (0 growth. * production)

241
 From this another conclusion may be drawn : both lactic yeast autolyzate (YA) and
yeast extract (Saccharomyces cerevisiae) + peptones (YEP) are convenient nitrogen
supplementations for precultivating lactic bacteria, probably because the corresponding
peptides have nearly mean molecular weights ; however it should be noticed that YA is
less expensive than YEP, and may contribute to a further valorization for lactose from
whey.
According to Leh (1987), the mean volumetric productivity observed for a culture
medium is in close connection with MW of their peptide content, for a constant total
nitrogen content. Fig.la and b provide an additional information : for a given
fermentation medium, volumetric productivity is greatly influenced by the MW of the
peptidcs used in the supplementation of the seed culture medium.

2) Effect of the total t)host)horus content of the culture medium on lactic acid
production rate :
Owing to the results of Fig.lb. the preculture media corresponding to the
fermentations shown in Fig.2 should have equivalent performances.
So the main difference between batches B086 and B297 may be found in the total
phosphorus content of the culture media (Table 1), resulting from heat/calcium
clarification process for KFWU : this medium contains 23 mg/1 total phosphorus, versus
530 in unclarified whey permeate FWU.
As shown in Fig.2, a FWU + CSL medium gives rise to a slow lactic acid production
rate (VP = 2.7 g/1.h.) ; this is clearly due to both qualitative and quantitative deficiencies
in the nitrogen supplementation of the FWU + CSL medium. A drastic decrease in the
total phosphorus content of the medium (KFWU) leads to a noteworthy increase in the
volumetric productivity (4.4 versus 2.7 g/1.h., i.e. +39%), despite the fact that nitrogen
supplementation remains inadequate. A similar conclusion has been previously described
by White e al. (1976) from phosphorus-limited continuous culture of Streptococcus
mutan&
Fig.2 also shows that KFWU results in a mean volumetric productivity only 20%
smaller than FWH (4.4 versus 5.5 gll.h.), at less expense, since KFWU does not need
enzymatic hydrolysis at all and since valuable seroproteins may be recovered during
whey ultraf'fltration.

242
 50 "l p ~g/l)
!

40 o ~ o a
Ill

30

20

10

e (h)
O~ i I

0 5 10 15
Fig.2 : Production histories for bacteria cultivated on :
= B025 : hydrolyzed whey
[] B086 : clarified whey permeate + CSL
* B297 : whey permeate + CSL

From all the results reported before it may be concluded that the best lactic acid
production rate at the lowest cost is obtained from the following media : lactic acid
bacteria have to be best precultivated on hydrolyzed whey supplemented with lactic yeast
autolyzate, and transferred into clarified whey permeate with corn steep liquor as a
nitrogen source.

Acknowledgements : A grant from the Algerian Ministry of Education and financial

support from EVEN (France/have been greatly appreciated.

NOMENCI.ATURE

MW" peptides average molar weight.

N'rK, NaNH2 : total and primary, a-amino nitrogen concentrations (g/l).
p : lactic acid concentration (g/l).
VP : lactic acid mean volumetric productivity (g/1.h.).
x : total cell mass concentration (g/l).

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243
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