Lacto-dynamographic study of lactic acid bacteria as a tool for their selection

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Lacto-dynamographic study of lactic acid bacteria as a tool for their selection

Pietro Micari, Andrea Caridi

“Mediterranea” University of Reggio Calabria, Department of Agro-Forestry and Environmental Technology and Science, Italy,

acaridi@unirc.it

1. Introduction

The utilization of the lacto-dynamographic technique in the dairy industry is a routine way to give a fast preventive assessment of milk coagulation characteristics and curd rheological properties. Indeed the lacto-dynamograph provides, through a diapason tracing, a graphic image of the milk coagulation mechanisms by means of three parameters that are correlated to several curd characteristics:

rennet coagulation time (r in min), curd firming rate (k₂₀ in min) and curd firmness (a₃₀ in mm). This allows a simple and quick

identification of animals that produce over-reactive or under-reactive milk, regarding rennet.

The analysis of milk using a lacto-dynamograph shows different types of tracings; this makes possible to categorize milks destined to cheesemaking in typologies regarding their different behaviour. Several milks show optimal lacto-dynamographic performances; others are under-reactive regarding rennet and, consequently, extremely slow in all the coagulation phases and low in curd firmness; someone else show very fast coagulation time with an elevated curd firmness (Chiofalo & Micari, 1987; Chiofalo & Micari, 1990/91).

If samples of the same typology, inoculated with different strains of lactic acid bacteria (LAB), are examined by lacto-dynamograph at the same time and in the same operational condition regarding the control sample (same milk, not inoculated with LAB), it is possible to evaluate and study the different behaviour of the LAB in the coagulation process.

The LAB are involved in the production of various types of fermented foods. The selection of LAB suitable as starter for cheesemaking is performed on the basis of numerous characteristics, referred particularly to their role during cheese ripening. Topics of interest include: acidification activity (Neviani, Divizia, Abbiati & Gatti, 1995), production of bacteriocin-like substances (Simonetta, Frison & Moragues de Velasco, 1998), rate of cell autolysis (Cibik & Chapot Chartier, 2000), amino acids catabolism (Curtin, De Angelis, Cipriani, Corbo, McSweeney & Gobbetti, 2001) and subsequent flavour formation (Amarita, Requena, Taborda, Amigo & Pelaez, 2001), proteolytic activity (Boutrou, Sepulchre, Gripon & Monnet, 1998), exopolysaccharides production (Ruas Madiedo, Hugenholtz & Zoon, 2002) and texture promoting capacity (Martensson, Oste & Holst, 2002). Screening tests were proposed to detect in LAB enzymic patterns (Bianchi Salvadori, Camaschella & Cislaghi, 1995), lytic behaviour and proteolytic activity (Boutrou, Sepulchre, Gripon & Monnet, 1998).

Moreover, considering that the choice of a starter culture could affect heavy coagulum formation, it was suggested that coagulation tubes could be used to measure the amount of heavy coagulum formed and select LAB involved in coagulation of skimmed milk to produce lactic casein (Gosling & Munro, 1995).

However, along with our bibliographic researches, no work takes into consideration the lacto-dynamographic analysis of milk inoculated with LAB.

So we decided to study the correlations existing between coagulation properties of the milk and LAB employed as starter; this to try the definition of a method useful to evaluate the influence of LAB on the milk coagulation characteristics and, consequently, to quickly and simply exclude the strains less suitable for cheesemaking, speeding up the selection times.

In the present work several autochthonous LAB, previously isolated from Calabrian raw milk and raw milk cheeses, were studied using a lacto-dynamograph for coagulation parameters induced in pasteurised goats’ and ewes’ milk.

2. Materials and Methods

2.1 Microorganisms

Thirty-one strains of autochthonous LAB, previously isolated from Calabrian goats’ and ewes’ milks and cheeses not added with selected LAB, were employed.

The strains were stored as frozen stocks at –20°C in MRS broth (rods) or M17 broth (cocci) containing 25% (v/v) glycerol. Working cultures were grown in appropriate (MRS or M17) broth media.

2.2 Methodology

The goats’ and ewes’ milks used were distributed in test tubes in quantities of 10 ml, thermized at 80°C for 10’ and suddenly cooled until 4°C. Within 24 h the milks were inoculated in duplicate with each strain of LAB, using 0.2 ml of preculture in MRS broth (rods) or M17 broth (cocci), and suddenly incubated at 37°C for 30’.

After incubation the pH of the inoculated milk was measured using a pH-meter at 3 decimals (model 355, Mettler Toledo, Greifensee, Switzerland).

The lacto-dynamographic trials, both for the inoculated milks and for the control milks, were carried out using a lacto-dynamograph (Maspres, Firenze, Italy) in 10 ml of milk at 35°C with the addition of 200 µL of standard calf rennet (CHR Hansen, Corsico, Italy) with an

original strength of 160±8 IMCU ml-1_{, diluted to 0.8% (for the ewes’ milk) or 1.6% (for the goats’ milk).}

3. Results and discussion

The values of pH measured on the milks at the end of the incubation showed minimum differences (± 0.05 unit of pH) compared with the corresponding controls.

In Tabs. 1-2 the lacto-dynamographic values are shown, whereas in Figs. 1a ÷ 3b the deviation of the same values compared with the corresponding controls, set at zero, are reported.

The behaviour of r, rennet coagulation time, in the goats’ and ewes’ milks inoculated with lactobacilli (Fig.1a) demonstrates that the

majority of the strains induce a remarkable reduction of the coagulation time. Less evident is the reduction of this parameter when the enterococci are inoculated (Fig. 1b). Observing altogether Figs. 1 and 2 it is possible to note a peculiar behaviour of several LAB. These strains, e.g. L252 and C192, act in opposed way if inoculated in goats’ or ewes’ milk. Other strains, e.g. L355 and C417, show the same behaviour in both milks.

The curd firmness (a₃₀) increases notably compared to the control milks (Figs. 2a-2b), above all for the goats’ milk, after the addition of

the LAB. Indeed, compared to a control value of 27.78 mm (Tab. 2), there is an increase both for lactobacilli, until 36.88 mm for strain L260, and for enterococci, until 35.12 for strain C498. These two strains are also able to notably reduce the coagulation time, enhancing

consequently the inverse relationship between r and a30.

A technological point of interest is the high range of strain variability for the parameter a_30.This gives the possibility to suppose a specific

pre-selection of the LAB for the cheesemaking. Really the goats’ milk produces a softer curd and a faster draining of the whey and the use of LAB able to increase the curd firmness could improve the cheese quality.

The curd firmness in the ewes’ milk, in itself high (49.78), is further increased above all by lactobacilli (Tab. 1). Indeed the parameter a₃₀

oscillates from 32.26 to 59.86 after addition of lactobacilli and from 42.08 to 53.78 after addition of enterococci (Figs. 2a-2b). Also for the ewes’ milk the noticed strain variability gives the possibility to suppose a specific strain pre-selection.

The LAB activity induces a remarkable reduction of the parameter k₂₀, as reported in Figs. 3a-3b, in both milks, with an interesting

variability among the strains, above all for the goats’ milk (Tab. 2).

The behaviour manifested by the tested strains to increase the curd firming rate could be attributed, in our opinion, to interaction between the external constituents of the bacterial cells and the milk constituents responsible for the coagulation mechanism.

4. Conclusions

The trials carried out with the present work could constitute a first approach to the proposed method, that, after further investigations and optimizations, could represent a simple and quick method to easily perform a pre-selection of a high number of strains, discriminating lactic acid bacteria regarding their coagulation properties.

References

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Ewes' milk inoculated with lactobacilli

-400 -300 -200 -100 0 100 200 300 400 500 L134 L252 L260 L342 L343 L355 L356 L410 L559 d e lt a r (s e c )

Fig. 1a - Deviation of the r (coagulation time) values of the

goats’ and ewes’ milk inoculated with the lactobacilli compared with the corresponding controls, set at zero.

Goats' milk inoculated with lattobacilli

-400 -300 -200 -100 0 100 200 300 400 500 L134 L252 L260 L342 L343 L355 L356 L410 L559 d e lt a r (s e c )

Ewes' milk inoculated with enterococci

-400 -300 -200 -100 0 100 200 300 400 500 C 8 2 C 8 5 C 1 1 6 C 1 2 3 C 1 4 2 C 1 4 3 C 1 6 2 C 1 9 2 C 2 6 8 C 3 1 0 C 3 2 1 C 3 8 4 C 4 0 0 C 4 1 7 C 4 2 5 C 4 4 3 C 4 7 2 C 4 9 8 C 5 7 1 d e lta r ( se c)

Goats' milk inoculated with enterococci

-400 -300 -200 -100 0 100 200 300 400 500 C82 C85 C116 C123 C142 C143 C162 C187 C192 C268 C310 C321 C384 C400 C417 C425 C443 C472 C498 C571 d e lt a r (s e c )

Fig. 1b - Deviation of the r (coagulation time) values of the

goats’ and ewes’ milk inoculated with the enterococci compared with the corresponding controls, set at zero.

Goats' milk inoculated with lactobacilli

-20 -16 -12 -8 -4 0 4 8 12 L134 L252 L260 L342 L343 L355 L356 L410 L559 d e lt a a30 ( m m )

Fig. 2a - Deviation of the a₃₀ (curd firmness) values of the

Ewes' milk inoculated with enterococci

-20 -16 -12 -8 -4 0 4 8 12 C 8 2 C 8 5 C 1 1 6 C 1 2 3 C 1 4 2 C 1 4 3 C 1 6 2 C 1 9 2 C 2 6 8 C 3 1 0 C 3 2 1 C 3 8 4 C 4 0 0 C 4 1 7 C 4 2 5 C 4 4 3 C 4 7 2 C 4 9 8 C 5 7 1 d e lta a30 ( m m )

Goats' milk inoculated with enterococci

-20 -16 -12 -8 -4 0 4 8 12 C 8 2 C 8 5 C 1 1 6 C 1 2 3 C 1 4 2 C 1 4 3 C 1 6 2 C 1 8 7 C 1 9 2 C 2 6 8 C 3 1 0 C 3 2 1 C 3 8 4 C 4 0 0 C 4 1 7 C 4 2 5 C 4 4 3 C 4 7 2 C 4 9 8 C 5 7 1 d e lt a a30 (m m )

Fig. 2b - Deviation of the a₃₀ (curd firmness) values of the goats’

and ewes’ milk inoculated with the enterococci compared with the corresponding controls, set at zero.

Ewes' milk inoculated with lactobacilli

-300 -240 -180 -120 -60 0 60 L134 L252 L260 L342 L343 L355 L356 L410 L559 d e lt a k20 (s e c )

Fig. 3a - Deviation of the k₂₀ (curd firming rate) values of the

Fig. 3b - Deviation of the k₂₀ (curd firming rate) values of the

goats’ and ewes’ milk inoculated with the enterococci compared with the corresponding controls, set at zero.

Tab. 1 - Lacto-dynamographic values of the ewes’ milk inoculated with the lactic acid bacteria.

Tab. 2 - Lacto-dynamographic values of the goats’ milk inoculated with the lactic acid bacteria. r a30 k20 test 15.15 49.78 3.15 L134 14.00 54.08 1.45 L252 22.30 32.26 2.30 L260 13.45 53.82 2.00 L342 10.00 50.00 1.45 L343 10.45 54.00 1.45 L355 12.45 59.86 1.45 L356 11.45 56.58 1.45 L410 15.30 54.36 2.00 L559 14.45 58.86 1.45 C82 19.15 42.94 2.15 C85 18.15 45.62 2.00 C116 19.45 44.00 2.00 C123 19.45 42.08 2.00 C142 19.45 45.34 2.00 C143 17.15 50.00 2.00 C162 15.00 47.44 2.30 C192 14.30 53.78 2.15 C268 16.00 50.00 2.00 C310 15.00 50.60 2.00 C321 14.15 50.84 2.45 C384 14.15 50.34 2.45 C400 14.15 51.62 2.45 C417 14.15 51.62 2.45 C425 14.15 51.38 2.45 C443 14.30 48.08 2.00 C472 15.00 50.60 2.15 C498 15.00 52.60 2.00 C571 16.00 46.08 2.00 r a30 k20 test 14.00 27.78 7.45 L134 16.15 23.94 8.45 L252 9.30 32.00 3.00 L260 11.30 36.88 3.00 L342 15.00 32.88 4.30 L343 15.15 27.94 6.30 L355 12.00 29.98 5.30 L356 14.15 32.86 4.15 L410 14.30 31.94 4.00 L559 13.00 32.86 4.15 C82 13.15 31.94 6.15 C85 13.00 29.92 6.30 C116 13.15 30.36 6.30 C123 13.15 27.92 7.00 C142 12.45 32.36 5.30 C143 13.00 30.36 6.15 C162 13.45 32.44 4.15 C187 15.00 31.62 5.45 C192 13.15 31.96 4.30 C268 15.15 27.92 6.15 C310 12.45 33.70 7.00 C321 12.45 30.36 5.15 C384 13.45 31.92 5.15 C400 14.45 26.36 7.45 C417 13.45 33.52 5.15 C425 14.00 31.62 6.00 C443 12.45 29.94 5.45 C472 13.45 29.94 5.30 C498 12.30 35.12 5.30 C571 15.00 27.66 6.30