Z Lebensm Unters Forsch (1992) 195:569-571 Zeitschrift for

�9 Springer-Verlag 1992

Original paper

Caseinolytic activity of Lactococcus lactis subsp, lactis: study of different culture media Rosario G6mez, Rosina L6pez *, and Carmen Pelfiez

Instituto del Frio (CSIC), Ciudad Universitaria s/n, E-28040 Madrid, Spain

Received May 18, 1992

Caseinolytische Aktivit,~it von Lactococcus lactis subsp, lactis: Einflufl des Nachweismediums

Zusammenfassung. Die verschiedene caseinolytische Ak- tivitfit ist von dem Nachweismedium abh/ingig. D ie / / - Casein-Aktivit/it der Kul tur war in jedem Fall gr6ger als die Aktivit/it der anderen Casein-Fraktionen. Die h6ch- ste Aktivit/it wurde beim Wachstum in einem Casein- N/ihrboden mit 0,1% Hefeextrakt, 20 mmol CaC12 und 1% Lactose gefunden. Der EinfluB des N/ihrbodens auf die proteolytische Aktivit/it der untersuchten Keime wur- de ebenfalls ermittelt.

Summary. Different caseinolytic activity levels of L a c - tococcus lactis subsp, lactis I F P L 367 were obtained de- pending upon the culture medium tested. Activity on the //-casein fraction was in all cases higher than activity on other casein fractions. The highest activity level was re- corded for bacterial cells cultured in a casein-based me- dium that also contained 0.1% yeast extract, 20 mmol / CaC12, and 1% lactose. The effect of the components of the growth media on the proteolytic activity of the bac- terial strain employed in the experiment is also discussed.

Introduction

The ability of lactic acid bacteria to grow in milk is de- pendent on the proteolytic system of these bacteria. In milk, non-protein nitrogen is responsible for about 20- 25% of the total cell mass. Consequently, when this has been consumed, lactic acid bacteria hydrolyse casein with proteinase and peptidases to obtain essential amino acids

* Present address: Instituto de Fermentaciones Industriales, Juan de la Cierva 3, E-28006 Madrid, Spain Correspondence to: R. G6mez

[1]. Such enzymes play an important role in cheese ripen- ing, in that they give rise to precursors of cheese flavour and a roma components [2]. Therefore, the presence of casein in the medium is clearly important in nitrogen nu- trition since its removal necessitates supplementation with hydrolysed protein to obtain adequate growth of starters [3]. Most strains grow poorly, if at all, with casein as the sole amino acid source. The so-called "casein medium", which is cited in many reports concern- ing starter proteinases, contains yeast extract consisting largely of free amino acids and peptides [4]. On the other hand, these nitrogen compounds can produce inhibition of cell wall proteinase synthesis when supplied in excess [5]. Earlier investigations into the proteolytic system of lactic acid bacteria carried out in our laboratory [6] have reported variations in the caseinolytic activity of different strains according to the growth medium used to culture the bacteria (unpublished results). Therefore, the object of the present study was to consider the caseinolytic ac- tivity of L a c t o c o c c u s lactis subsp, lactis IFPL 367 em- ploying different culture media.

Materials and methods

Microorganisms. The strain used was L. lactis subsp, lactis IFPL 367 from the collection at the Instituto del Frio, originally isolated from goats' milk cheese made from raw milk.

Culture media tested. Six different culture media were tested. Me- dium no. 1 consisted of Elliker broth (dilution 1/10), supplemented with 2% Hammarsten casein (Merck) dissolved in 1 mol NaOH, pH 7.0 [7] and 0.04% added KH2PO 4. The remaining media had the same composition as medium no. 1 but also contained the following additives: 1% lactose (medium no.2); 20 mmol CaC12 (medium no. 3); 1% lactose and 20 mmol CaC12 (medium no. 4); 0.1% yeast extract and 20 mmol/CaC12 (medium no. 5); 0.1% yeast extract, 20 mmol CaC12, and 1% lactose (Medium no. 6). - Lactocoecus lae- tis subsp, laetis IFPL 367 was cultured in Elliker broth (Oxoid) at 30 ~ C for 24 h. The cells were harvested by centrifugation at 13 000 g (0~ ~ C for 15 min). The resulting pellet was resuspended in the original volume with 0.1 mol sodium phosphate buffer, pH 7.2. The above-mentioned media were then inoculated (10%) with this sus-

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pension and incubated at 30 ~ C for a further 24 h. A second subcul- ture was then grown under these same conditions but the incubation was halted after 17 h. Microbial growth was measured by spectro- photometric readings (OD) at 480 rim. The cells were harvested by centrifugation as described above and the resulting pellets were washed twice and resuspended to a density of about l09 cells/m1 in phosphate buffer. These cell suspensions were used for analysis of the caseinolytic activity. Care was taken to keep the temperature during preparation of cell suspensions between 0 and 4 ~ C to pre- vent the release of proteinase activity [8].

Electrophoretic evaluation of the caseinolytic activity of intact cells. Caseinolytic activity was estimated by means of sodium dodecyl sul- phate polyacrylamide gel electrophoresis as described by Hill and Gasson [9]. Electrophoresis gels were developed at 30 mA for 4 h 35 min in a model SE-61195M slab gel electrophoresis unit (from Hoefer Scientific Instruments, San Francisco, Calif., USA). Quanti- tative analysis of the bands present in the gels was carried out using a Chromato Scanner CS-930 double wavelength densitometer and a Data Recorder DR-2 integrator with a graphic printing system (both from Shimadzu Corporation, Kyoto, Japan).

Results and discussion

L. lactis subsp, lactis IFPL 367 displayed differing casei- nolytic activity levels, depending upon the growth me- dium used to culture the cells. Bands of differing intensity and mobility appeared for the degradation products (Fig. 1). These bands were located in the intermediate re- gion between the/?- and the x-casein and there were also a further three bands corresponding to peptides with a relative moleculare mass (Mr) of 25010, 23000, and 21 920. The last two bands were in the Mr range of 23 000-21400 recorded by Hill and Gasson [9] for the degradation products of/?-casein. In addition, there were other bands with lower Mrs, including a strong band with an Mr of 18570 and other, fainter bands with lower Mrs.

Fig. 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of Hammarsten casein digested by intact bacterial cells of Lae- toeoeeus taetis subsp, laetis IFPL 367 cultured in different growth media: lanes 1, 2, 4, 5, 6 and 7, cells grown in media nos. 1, 2, 3, 4, 5, 6, respectively, incubated with casein at 30 ~ C for 8 h; lane 3, cells with casein that did not undergo incubation. The numbers represent relative molecular mass

C a s e i n b r e a k d o w n (%) 8 0

70

60

50

40

30

20

I0-

0

/ /

I 2 3

2

,I 4

,I t - . N I I

A[ 5 6

C e l l s g r o w n i n c u l t u r e m e d i a

Fig. 2. Percentage breakdown of casein with respect to total casein in sample B by (cells and casein with at 0 incubation time) by bac- terial cells grown in different culture media. (-.) as-; (1~) fl-; ( ~ ) K-; (@) total casein

The cells grown in culture media nos. 3 and 4, had high levels of casein degradation, 54.5 and 55.0% of the total casein present in sample B, respectively (Fig. 2). Hydroly- sis of the/?-casein was responsible for the major share of these percentage degradation values, although hydrolysis of the ~s and K-casein also accounted for contributions. These results were consistent with those reported for a se- lection of Streptococcus lactis [10] and S.cremoris [11] strains. These same studies indicated that/?-casein was the preferred substrate for the cell wall protease of these microorganisms, although other workers have shown that there may sometimes be a second type of proteolytic activity that acts as a catalyst of hydrolysis of c~- and ~c- casein fractions [12].

In comparison, breakdown of a~-casein by the bac- terial cells cultured in growth media nos. 1 and 2, which do not containd added Ca 2 +, was slight and breakdown of the K-casein was negligible in two cases (Fig. 2). These results are in agreement with those reported by Monnet et al. [10] and Laan et al. [13], who found that the protein- ase activities of L. lactis subsp, lactis NCDO 763 and L. lactis subsp, cremoris HP and AC1 are stimulated by C a 2 +.

The presence of yeast extract in culture medium no. 5 brought about a slight reduction (6.3%) in the overall proteolytic activity of L. lactis subsp, lactis IFPL 367 (Fig. 2). This decrease mainly affected the K-casein, only 4.5% of which was hydrolysed by these cells, as opposed to 8.2% by the cells cultured in medium no. 3. This lower proteolytic activity with the addition of yeast extract has been attributed to the inhibitory action of the low-Mr ni- trogen compounds present in that substrate [4]. These re-

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sults are similar to those reported by other investigators such as Mills and Thomas [14], who found that the addi- tion of amino acids and peptides to milk usually resulted in lower levels of cellular proteinase with lactococci.

When the culture media contained lactose and yeast extract (medium no. 6), the total casein hydrolysis was 69.2%, the highest percentage attained for all the growth media tested (Fig. 2). This increase substantially affected the c~-casein, with hydrolysis of 26.7% as opposed to 11.5% by the cells cultured in medium no. 3. I t may be as- sumed that this proteolytic activity is higher in the pres- ence of these two substrates due to the different physio- logical cell age reached at the different growth rates in each medium (OD = 3.0 in medium no. 6, as compared to 1.2 in medium no. 3). Also, more rapid depletion of lac- tose and yeast extract sources of carbon and nitrogen more readily assimilable than casein, may in the end have brought about a higher level of syntesised proteinases. Our observations are in keeping with the results of Hu- genholtz et al. [15], who found that the highest level o f proteinase product ion were measured in cells that had reached the stat ionary phase of growth.

I t has been established in the present study that growth in medium no. 6 promotes a level of proteolytic activity in this strain higher than those recorded for the other media tested. Furthermore, this wild-type Lac- tocoecus lactis subsp, lactis strain isolated f rom sponta- neously fermented goats ' milk shows similar caseinolytic properties as commerical starter cultures for cows' milk.

Acknowledgement. The authors would like to acknowledge the financial support for this study provided by the Comisi6n Inter- ministerial de Cieneia y Tecnologia under Project AL188-0158.

References

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