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Evaluation of aroma production and survival of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus in fermented milks
International Dairy Journal 9 (1999) 125}134
Evaluation of aroma production and survival of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus in fermented milks Fausto Gardini *, Rosalba Lanciotti, Maria Elisabetta Guerzoni , Sandra Torriani Dipartimento di Protezione e Valorizzazione Agroalimentare, Universita% degli Studi di Bologna, Via San Giacomo 7-40126 Bologna, Italy Istituto di Produzioni e Preparazioni Alimentari, Universita% degli Studi di Bari, Sede di Foggia, Via Napoli 25-71100 Foggia, Italy Istituto di Industrie Agrarie, Universita% degli Studi di Catania, Via Santa Soxa, Catania, Italy Received 28 August 1998; accepted 24 February 1999
Abstract The fat, the non-fat milk solid and the inoculum size of Lactobacillus acidophilus IPVR 224 were modulated according to a Central Composite Design. The aim was to evaluate the e!ects of these variables and their interactions on the decrease in pH during fermentation, the qualitative and quantitative composition of the aroma pro"le, as well as the loss in viability of Streptococcus thermophilus IPVR 161, Lactobacillus delbrueckii subsp. bulgaricus IPVR 132 and L. acidophilus IPVR 224 strains during "ve weeks of storage. In addition, the overall acceptability of the fermented milks was assessed by means of a sensory panel. The polynomial quadratic equations obtained allowed to individuate the variables that signi"cantly a!ected the aroma pro"le, organoleptic properties and microbiological counts of the various fermented milks. The survival of the L. acidophilus strain during storage was higher at low concentration of non-fat milk solids and its presence did not a!ect the acetaldehyde content of fermented milks. From the response surfaces analysis it was possible to select optimum conditions for enhanced positive organoleptic traits and for improved survival of the probiotic culture. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Fermented milks; Lactobacillus acidophilus; Modelling; Survival; Aroma compounds
1. Introduction The microorganisms used for the production of fermented milks are claimed to impart nutritional and health bene"ts to consumers. The probiotic activity is related to lactic acid bacteria, that are able to proliferate or even survive for a long period in the human intestinal tract (Mital & Garg, 1992). Among the lactic acid bacteria, Lactobacillus acidophilus is characterized by its capacity to colonise in the intestine even under low surface tension caused by the presence of bile salts (Gilliland & Speck, 1977). Consumption of acidophilus milk products is considered to promote several bene"cial effects such as control of enteric infections, normalization of gut micro#ora, lowering of serum cholesterol level, prevention of colon cancer, immunomodulation and enhanced availability of nutrients (Mital & Garg, 1992; Klaver & van der Meer, 1993; Lee & Salminen, 1995;
* Corresponding author.
Mitsuoka, 1992; Gonc & Akalin, 1995). Moreover, milk inoculated with L. acidophilus has been found to improve lactose tolerance among lactose malabsorbers (Shah, 1994; Montes, Bayless, Saavedra & Perman, 1995) and inhibit enteric and foodborne microbial pathogens (Kilic, Pavlova, Ma & Tao, 1996). In a study of Dettmann (1995), the nutritional bene"ts of using mixed cultures of L. acidophilus and Bixdobacterium bixdum are stressed and among others their predominant production of L(#) lactic acid. However, several L. acidophilus strains do not grow in milk and survive poorly in fermented products. Shah, Lankaputhra, Britz and Kyle (1995) suggested that the number of viable L. acidophilus cells should be greater than 5 log cfu g\ to have therapeutic bene"ts. Therefore, the use of probiotic cultures that provide high viable counts during the storage of the product is essential. The sensory characteristics of fermented milks play an important role in product acceptance by consumers. Several #avour compounds have been isolated from yoghurt-type products; however only acetaldehyde, ethanol,
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F. Gardini et al. / International Dairy Journal 9 (1999) 125}134
acetone, diacetyl and 2-butanone are found in substantial amount (Kneifel, Ulberth, Erhard & Jaros, 1992). Acetaldehyde is considered as the most prominent compound for the typical yoghurt aroma (Bottazzi, Battistotti & Montescani, 1973). The #avour quality of yoghurt also depends on the relationship between other volatile components; for example, a ratio between acetaldehyde and acetone of 2.8 is considered optimum (Accolas, Hemme, Desmazeaud, Vassal, Bouillanne & Veaux, 1980). Generally, acidophilus milk products are characterized by lack of #avour due to the fact that L. acidophilus possesses an alcohol dehydrogenase which converts acetaldehyde to ethanol (Marshall & Cole, 1983). It was shown that the addition of whey proteins and threonine to milk can prevent this problem; otherwise L. acidophilus can convert excess of threonine to acetaldehyde via glycine with the enzyme threonine aldolase (Hickey, Hillier & Jago, 1986). Because of the increasing interest in adding L. acidophilus to yoghurt in order to improve the probiotic activity of the intestinal micro#ora, a proper selection of strains is required. The principal aim of the present study was to evaluate the e!ects of fat, non-fat milk solids and inoculum size of L. acidophilus, modulated according to a Central Composite Design. In particular, the decrease in pH after fermentation, the loss in viability of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and L. acidophilus during a 5 weeks storage at 43C, the qualiquantitative aroma pro"le during storage at 43C and the overall acceptability of the fermented milks were analysed. 2. Materials and methods 2.1. Bacterial strains and culture media Lactobacillus delbrueckii subsp. bulgaricus IPVR 132, L. acidophilus IPVR 224 and S. thermophilus IPVR 161 were kindly provided by F. Dellaglio, Dipartimento Scienti"co e Tecnologico, University of Verona (Italy). The yoghurt culture (L. delbrueckii subsp. bulgaricus IPVR 132 and S. thermophilus IPVR 161) was chosen with regard to their well-established protosymbiosis, whereas L. acidophilus was selected on the basis of its ability to grow in milk, its high resistance to low pH and potential health promoting properties. The yoghurt cultures and L. acidophilus were maintained as frozen stock cultures at !803C in reconstituted (10% w/w) skim milk (Oxoid, Basingstoke, UK) and MRS broth (Oxoid) containing 20% glycerol, respectively. Cultures were transferred using 1% inocula and propagated twice in reconstituted skim milk at 373C before use. 2.2. Experimental design Di!erent fermented milks were prepared by modulating inoculum size of L. acidophilus [LA], percentage of
Table 1 Central Composite Design (three factor, "ve levels) used for the evaluation of the e!ects of milk solids, milk fat and L. acidophilus inoculum on the characteristics of the fermented milk Run
Milk solids (%) [S]
Milk fat (%) [F]
Lactobacillus acidophilus inoculum (%) [LA]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
11.5 14.5 11.5 14.5 11.5 14.5 11.5 14.5 13.0 13.0 10.0 16.0 16.0 13.0 13.0 13.0 13.0
2 2 6 6 2 2 6 6 4 4 4 4 0 8 4 4 4
1.0 1.0 1.0 1.0 2.0 2.0 2.0 2.0 1.5 1.5 1.5 1.5 1.5 1.5 0.5 2.5 1.5
milk fat [F] and non-fat milk solids [S] added to the basal medium according to a three-factor, "ve level Central Composite Design (Box, Hunter & Hunter, 1978). The 17 combinations obtained are shown in Table 1. The Central Composite Design permits to reduce the number of possible combinations to a manageable size. In fact, this design is characterized by using only a fraction of the total number of factor combination for experimentation. In statistical literature, the technique of reducing the number of factor combinations in a factorial experiment is known as confounding (Gacula, 1988). Five replicates of each combination were used. Skim milk powder (Sacco srl, Cadorago, Italy) was reconstituted to 10% (w/w) and was used as the basal medium. To obtain the levels required by the di!erent runs of Central Composite Design (Table 1), suitable amounts of commercial UHT cream (Parmalat, Parma, Italy) and skim milk powder (Sacco srl) were added to the basal medium. The mixes were heat-treated at 903C for 5 min, cooled to 373C in a water bath and inoculated with the yoghurt culture to attain a level of about 7 log cfu g\ for each strain. Immediately after this step, the milks were divided into 17 aliquots (250 g), and inoculated with di!erent percentages of the L. acidophilus culture, according to the runs of the Central Composite Design. With the aim to determine the #avour compounds, aliquots (5 g) of the inoculated milks for each combination were also dispensed into sterile glass vials (5 replicates) and sealed with aluminium caps. Then, the inoculated milks were fermented at 373C for 5 h. The "nal pH, depending on the combination, ranged from 5.3 to 4.2. The fermented milks were then transferred to a cold store at 43C.
F. Gardini et al. / International Dairy Journal 9 (1999) 125}134
2.3. Microbiological analysis For each run of the experimental design, samples in duplicate were analysed immediately after fermentation and after "ve weeks of storage at 43C. Fermented milk samples (1 g) were added to 99 ml of sterile peptone diluent (0.1% w/v) and homogenized with a Stomacher Lab-Blender 400 (Seward Medical, London, UK) for 2 min. Appropriate dilutions were made in 1/4 strength sterile Ringer solution, and subsequentely plated in duplicate onto selective media. Lactobacillus delbrueckii subsp. bulgaricus was enumerated on MRS (Oxoid) at pH 5.2 after incubation at 453C for 72 h in an anaerobic jar containing H and CO gases (generated by an Oxoid BR38 kit). Streptococcus thermophilus was counted on M17 (Oxoid) plates after aerobic incubation at 373C for 48 h. Lactobacillus acidophilus was enumerated on MRSsorbitol plates (Dave & Shah, 1996) after incubation at 373C for 72 h. The selectivity of the growth conditions was con"rmed by the microscopic appearance of the cells from single colonies.
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with an autosampler HS250 (Carlo Erba Instruments) in which the samples were maintained at a controlled temperature (803C) for conditioning and sampling. For each analysis 2.5 ml of head space were automatically sampled with a gastight syringe 1750 (Hamilton, Bonaduz, Switzerland) at a controlled temperature of 803C. The volatile compounds were identi"ed using pure standard and quanti"ed on the basis of calibration curves previously prepared. 2.7. Statistical analysis Modelling was aimed at describing the decrease in pH after fermentation, the loss in viability of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and L. acidophilus during a 5 weeks storage at 43C, the quali-quantitative aroma pro"le during storage at 43C and the overall acceptability of the fermented milks as function of the independent variables of the Central Composite Design. A software package (Statistica for Windows, Statsoft, Tulsa, USA) was used to "t the second-order model to the dependent variables using the following equation:
2.4. pH measurement
y" B x # B x# B x x , G G GG G GH G H
Measurement of pH was carried out using a pH meter Model 2001 (Crison, Barcelona, Spain).
where y is the dependent variable to be modelled, B , B G GG and B are regression coe$cients of the model, and GH x and x are the independent variables in coded values. G H The variables with a signi"cance lower 95% (P'0.05) were not included in the "nal models. The three-dimensional surface plots were drawn to illustrate the main and interactive e!ects of the independent variables on the dependent ones. These graphs were drawn imposing a constant value (i.e. the central points of the interval taken into consideration) to one independent variable.
2.5. Sensory evaluation The sensory evaluation to examine the di!erences among the fermented milks of the various runs and the overall acceptability of each product was carried out by a panel of 20 judges. The sensory evaluation was carried out, after overnight storage, on samples (250 g) served at about 83C. Samples were scored on a hedonic scale of 1}7; 7 was considered excellent, 4 acceptable and 1 extremely poor.
3. Results 2.6. Gas chromatographic analysis of the aroma compounds The aroma constituents of the head space of sealed vials containing 5 g of product (empty/full ratio 1 : 1) were determined using a gas chromatograph Vega series 2000 (Carlo Erba Instruments, Milan, Italy) equipped with a Flame Ionization Detector (FID). Before the analysis, all the samples were preconditioned for 2 h at 803C. A glass column 2 m;2 mm i.d. packed with 80/120 Carbopack B AW/6.6% and PEG 20 M (Supelco, Bellefonte, USA) was used to separate the aroma compounds. The analytical conditions were the following: column temperature: 80}2003C at 43C min\; detector and injector temperature: 2003C; carrier gas (nitrogen) #ow rate: 20 ml min\. The gas chromatograph was connected
The 17 fermented milks were prepared according to the runs of the Central Composite Design (Table 1). Immediately after fermentation at 373C, the pH decrease was evaluated. The samples were stored at 43C and analysed at di!erent time intervals for aroma compounds and loss of viability of the yoghurt bacteria (IPVR 132 and IPVR 161 strains) and L. acidophilus IPVR 161. This strain, isolated from the human intestinal tract, was chosen for its potential health promoting properties, as evidenced by its in vitro ability to adhere to epithelial cells and assimilate cholesterol (F. Dellaglio, personal communication). The data obtained were modelled according to polynomial quadratic equations in order to identify the variables that signi"cantly a!ected the aroma, sensory and microbiological characteristics of the
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various combinations. In the "nal models, only the coe$cients characterized by P(0.05 were used. This model, already applied by Torriani, Gardini, Guerzoni and Dellaglio (1996) for the study of milk cultures, allows us to evaluate the e!ects of linear, quadratic and interactive terms of the independent variables on the chosen dependent variables. 3.1. pH decrease The decrease in pH after the fermentation with respect to the initial value was in#uenced by all the variables considered in the Central Composite Design (Table 2, Eq. (1)). The [LA] variable was signi"cant in its linear term and in its interaction with [F]. The [S] variable was signi"cant in its linear and quadratic terms and in its interaction with [F]. The positive coe$cient of the individual factor and the negative coe$cient of the quadratic term indicated that it is possible to identify an optimum level of non-fat milk solids to limit the decrease in pH. This optimal value (about 13%) is highlighted by the response surfaces, derived from the relative polynomial equation, for the [S];[LA] and [S];[F] interactions (Fig. 1a and b). These graphs were obtained from Eq. (1) of Table 2 by imposing, respectively, the central values of 4% for [F] and 1.5% for [LA]. The e!ect of the [LA];[F] interaction on decrease in pH during the fermentation process is shown in Fig. 1c. The highest pH decrease was observed when both the factors were at their lowest levels. However, the inoculum size of L. acidophilus seems to weakly enhance the pH decrease when the amount of fat is high. 3.2. Variations in latic acid bacteria cell numbers The loss of viability of lactic acid bacteria was evaluated as the di!erence in log cfu g\ between the counts after the fermentation at 373C and the counts after "ve weeks of storage at 43C. In all the runs of the Central Composite Design, at the end of storage, the three strains used as starters were characterized by a cell concentra-
tion higher than 10 cfu g\. In particular, in all the samples after the fermentation, the L. acidophilus IPVR 224 concentration was always higher than 8.30 log cfu g\ and, at the end of storage, the mean loss of viability was lower than 0.76 log cycles. The loss of viability of this strain was less pronounced with respect to the yoghurt cultures and ranged between 0.5 and 1.1 log cfu g\. The best-"t equations relative to the loss of viability of the inoculated cultures are shown in Table 2. Under the conditions used in this study, the decrease of L. acidophilus count, as shown by the Eq. (2) of Table 2, was a!ected only by the linear term of the [S] variable; its increase determined a loss of viability, even though the concentration of this microorganism was always higher than the initial inoculum. The amount of non-fat milk solids had the same positive e!ect on the viability of L. delbrueckii subsp. bulgaricus IPVR 132 strain (Table 2, Eq. (3)), though only its interaction with [LA] was signi"cant. The latter variable was present also in its interaction with [F] and played a signi"cant role, as shown in Fig. 1d, relative to the [LA];[F] interaction. These two factors had compensatory e!ects on L. delbrueckii subsp. bulgaricus IPVR 132 cell number decrease. In fact, the loss of viability reached the maximum when one of the two factors was at the highest level and the other at the lowest level. On the contrary the combination of these two variables at their lowest and highest levels was associated with the minimum loss of viability. The model describing the decrease in the streptococci number during storage (Table 2, Eq. (4)) permitted the individuation of a threshold value of [F] which determines the maximum decrease of S. thermophilus IPVR 161 was maximum, as indicated by the positive sign of the linear term and the negative sign of its quadratic term. The value was about 4% as shown by Fig. 1e and f, for [LA];[F] and [F];[S] interactions, respectively. Streptococcus thermophilus IPVR 161 took fair advantage from a high inoculum level of L. acidophilus IPVR 224, which determined a lower loss of viability. Moreover, it is
Table 2 Best-"t equations relative to the e!ects of the di!erent variables on the pH decrease after fermentation and the loss of viability of lactic acid bacteria strains during storage of fermented milks. Only terms with P(0.05 were included
pH Decrease after fermentation Eq. L. acidophilus IPVR 224 Eq. L. delbrueckii subsp. bulgaricus IPVR 132 Eq. S. thermophilus IPVR 161 Eq.
(1) (2) (3) (4)
Equation
R
F
SE
!0.16[LA]#0.38[S]!0.015[S]!0.007[S][F]#0.033[LA][F] 0.058[S] 0.36[F]#0.05[S][LA]!0.21[LA][F] !3.24[LA]#1.74[F]!0.07[F]!0.09[S][F]#0.24[S][LA]
0.999 0.901 0.973 0.965
5441.3 69.39 82.08 32.50
0.049 0.38 0.32 0.37
[S], non-fat milk solids (%); [F], fat added (%); [LA], L. acidophilus inoculum size (%). Regression coe$cient. F-value. Standard error.
F. Gardini et al. / International Dairy Journal 9 (1999) 125}134
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Fig. 1. Three-dimensional contour plots concerning pH decrease after fermentation and loss of viability of starter cultures after a 5 week storage at 43C. The "gures are relative to the e!ects of the interaction [S][LA] (a), [S][F] (b) and [LA][F] (c) on pH decrease and [LA][F] (d) on L. delbrueckii subsp. bulgaricus loss of viability, [LA][F] (e) and [S][F] (f) on S. thermophilus loss of viability.
possible to state that the conditions favouring S. thermophilus viability determined a greater cell decrease of L. delbrueckii subsp. bulgaricus. 3.3. Aroma proxle after a week of storage Gas chromatographic analysis showed the presence of acetaldehyde, acetic acid, diacetyl, acetone and 2-butanone in all the runs of the Central Composite Design. The best-"t equations relative to the amounts of these compounds detected in the head space of fermented milks, as a function of the independent variables of the Central Composite Design, after one week of storage are reported in Table 3. Eq. (1) of Table 3 shows the individual and interactive e!ects of the variables of the Central Composite Design on acetaldehyde concentration. It is interesting to observe that the [LA] variable was not signi"cant and the acetaldehyde production was in#uenced only by [F], both in its linear and quadratic term, by [S], in its quadratic term, and by their interaction. The negative
sign of the coe$cient of the quadratic term of [F] de"nes a maximum in acetaldehyde presence, as shown by Fig. 2a, relative to the [S];[F] interaction. This maximum corresponds, approximately, to 5% of [F], at the lowest levels of [S], and is shifted towards lower fat levels (about 3%) when the non-fat milk solids content reaches its maximum. Moreover, the milk solids content enhanced acetaldehyde production, at least for a fat concentration up to 6%; for higher fat values this trend was the opposite. The other aroma compounds showed di!erent pattern. As shown by Eq. (2) of Table 3, acetic acid was a!ected by the [S] and [F] variables in their quadratic and interactive terms; also for this compound the [LA] variable showed no in#uence. The e!ects of the [S];[F] interaction on acetic acid presence in the head space are reported in Fig. 2b. In contrast to acetaldehyde production, it was possible to obtain minimum values of acetic acid concentration for values of [F] ranging between 4 and 6%, depending on [S]. However, analogously with acetaldehyde, the non-fat milk solids [S] showed a
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Table 3 Best-"t equations relative to the e!ects of the di!erent variables on the aroma compounds of fermented milks after one week of storage. Only terms with P(0.05 were included Aroma compound Acetaldehyde Acetic acid Diacetyl Acetone 2-Butanone
Eq. Eq. Eq. Eq. Eq.
(1) (2) (3) (4) (5)
Equation
R
F
SE
0.55[F]#0.0071[S]!0.036[F]!0.024[S][F] 0.000082[S]#0.00035[F]!0.00028[S][F] 0.048[LA]!0.0049[S]#0.0006[S]#0.00075[F]!0.00076[S][F]!0.0037[S][LA] 0.102[LA]#0.0012[S]#0.003[F]!0.003[S][F]!0.0078[S][LA] !0.0083[S]#0.0238[F]#0.005[LA]#0.001[S]!0.0022[S][F]
0.990 0.993 0.990 0.936 0.997
156.59 176.17 86.78 32.85 361.25
0.25 0.016 0.0034 0.0024 0.0042
Expressed as mg of aroma compound in equilibrium in the head space for kg of fermented milk. [S], non-fat milk solids (%); [F], fat added (%); [LA], L. acidophilus inoculum size (%). Regression coe$cient. F-value. Standard error.
Fig. 2. Three-dimensional contour plots relative to the amount of aroma compounds in the head space of the fermented milks after one week of storage at 43C; the results are expressed as mg of compound in the head space per kg of fermented milk. The "gure is relative to the e!ects of the interaction [S][F] on acetaldehyde (a), acetic acid (b), diacetyl (c) and acetone (d), the interaction [LA][S] on diacetyl (e) and [S][F] on 2-butanone (f).
positive e!ect on the presence of acetic acid at the lowest levels of [F]. The amounts of diacetyl and acetone in the head space were in#uenced by [LA] variable in its linear term and by
its interaction with [S] and [F] (Table 3, Eqs. (3) and (4)). However, the presence of the [S] and [F] quadratic terms with positive signs, allowed to recognise the combinations of these two variables able to minimize the
F. Gardini et al. / International Dairy Journal 9 (1999) 125}134
presence of diacetyl and acetone in the head space, as evidenced by the surface responses reported in Fig. 2c and d. The in#uence of the [LA] variable on the production of diacetyl is shown in Fig. 2e, relative to the [LA];[S] interaction. The maximum concentrations of diacetyl were reached when [S] was at its maximum and [LA] at its minimum level, and vice versa. Within this interval the lowest levels of diacetyl in the head space were obtained in correspondance with the lowest values of milk solids. The same behaviour was obtained for acetone (Fig. 2d). The production of 2-butanone is weakly in#uenced (Table 3, Eq. (5)) by the [LA] variable, while it was positively stimulated by [S] and negatively a!ected by [F] (Fig. 2f). 3.4. Aroma proxle after xve weeks of storage The best-"t equations relative to the principal aroma compounds detected after "ve weeks of storage at 43C in the head space of the fermented milks are reported in Table 4. All the considered variables a!ected the presence of these compounds, with the exception of acetaldehyde which was not in#uenced by [LA]. After "ve weeks of storage, acetaldehyde was still quantitatively the principal constituent of the aroma. Eq. (1) of Table 4 describes the individual and interactive e!ects of the considered variables on this compound. The acetaldehyde presence was in#uenced by [S], both in the linear and quadratic terms, by [F], in its linear term, and by their interaction. Fig. 3a, relative to the [S];[F] interaction, shows a maximum in acetaldehyde presence in correspondence to 13}14% of milk solids, while fat had a negative e!ect on the acetaldehyde concentration, as indicated by the negative sign of Eq. (1) in Table 4. As shown by Eq. (2) of Table 4, acetic acid production was a!ected, at the end of storage, by the same variables that were signi"cant after one week, as well as by [LA]
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and [S];[LA]. This can account for the contribution of L. acidophilus to the production of this compound during refrigerated storage. For diacetyl production (Table 4, Eq. (3)), the most important variable is fat content, which is present in its quadratic term. The presence of a quadratic term determines a change in the sign of the "rst derivative of the curve and, consequently, the presence of a maximum or a minimum, depending on its sign. In fact, the presence of the positive sign for its quadratic term in the "nal model determines a minimum value (as shown in Fig. 3b, relative to the [S];[F] interaction) in correspondence to a concentration of about 4% of fat. Acetone and 2-butanone in the head space showed the same behaviour, as indicated by the Eqs. (4) and (5) of Table 4 in which the same terms (with the same signs) were signi"cant. In particular, for the [LA];[S] interaction (Fig. 3c and d) the increase of milk solids determined an increase in the production of these two compounds, whereas an increase of L. acidophilus inoculum had an opposite e!ect. 3.5. Sensory properties The organoleptic properties of the products prepared according to the di!erent combinations of the Central Composite Design were evaluated by 20 untrained panelists, who quanti"ed the overall quality of fermented milks with a score ranging from 1 to 7. The polynomial model, which describes the e!ects of the selected variables on the panellist evaluation is reported in Table 5. The overall quality was not a!ected by the L. acidophilus inoculum level, but only by the [S] and [F] variables in their linear and quadratic terms and by the [S];[F] interaction. As shown in Fig. 4, relative to the [S];[F] interaction, the obtaining of products with desired traits is positively a!ected by the increase of both variables. In fact, combinations with the best organoleptic and textural characteristics were obtained
Table 4 Best-"t equations relative to the e!ects of the di!erent variables on the aroma compounds of fermented milks after "ve week of storage. Only terms with P(0.05 were included Aroma compound Acetaldehyde Acetic acid Diacetyl Acetone 2-Butanone
Eq. Eq. Eq. Eq. Eq.
(1) (2) (3) (4) (5)
Equation
R
F
SE
!0.54[F]#0.44[S]!0.023[S]#0.032[S][F] 0.0086[LA]#0.000054[S]#0.00017[F]!0.00018[S][F]!0.00062[S][LA] 0.026[LA]#0.0032[S]!0.0116[F]#0.0009[F]!0.002[S][LA] !0.076[LA]#0.017[S]!0.0024[S][F]#0.014[F][LA] 0.0165[S]!0.053[LA]#0.0106[LA][F]!0.0021[S][F]
0.989 0.973 0.991 0.972 0.991
140.31 57.24 136.31 40.74 187.27
0.22 0.02 0.003 0.001 0.014
Expressed as mg of aroma compound in equilibrium in the head space for kg of fermented milk. [S], non-fat milk solids (%); [F], fat added (%); [LA], L. acidophilus inoculum size (%). Regression coe$cient. F-value. Standard error.
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Fig. 3. Three-dimensional contour plots relative to the amount of aroma compounds in the head space of the fermented milks after "ve week of storage at 43C; the results are expressed as mg of compound in the head space per kg of fermented milk. The "gures are relative to the e!ects of the interaction [S][F] on acetaldehyde (a), diacetyl (b) and [LA][S] on acetone (c) and 2-butanone (d).
Table 5 Best-"t equations relative to the e!ects of the di!erent variables on the overall acceptability of fermented milks. Only terms with P(0.05 were included
Overall acceptability
Eq. (1)
Equation
R
F
SE
0.38[S]!0.013[S]#0.019[S][F]
0.997
608.44
0.36
[S], non-fat milk solids added (%); [F], fat added (%). Regression coe$cient. F-value. Standard error.
with the highest levels of both non-fat milk solids and fat content.
4. Discussion The health bene"ts deriving from intake of fermented dairy products containing L. acidophilus are well documented and depend on a high viability of the microorganism. Furthermore, the presence of an high
concentration of viable bacterial cells at the time of consumption is necessary for complying with the FILIDF standard (IDF, 1992). The choice of proper strains selected on the basis of the desired features is therefore essential to meet these requirements. The strains used in this study remained viable during storage and the relevant survival of L. acidophilus IPVR 244 cells in the fermented milk is of great interest even if this result appears to be in contrast with some literature data. In fact, Kneifel, Jaros and Erhard (1993) compared the
F. Gardini et al. / International Dairy Journal 9 (1999) 125}134
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concentration of diacetyl could be responsible for aroma defects linked to a too pronounced &butter #avour'. However, such #avour defects of fermented milks can be controlled, as observed by Hickey et al. (1986), through compositional modi"cation of raw milk. In fact, as evidenced by the results obtained in this work, the presence of compounds such as diacetyl and acetone can vary in a wide range of values in response to modulation of chemical, physical and biological variables.
5. Conclusion
Fig. 4. Three-dimensional contour plots relative to the [F][S] interaction on overall acceptability of fermented milks.
traditional yoghurts with fermented milks containing L. acidophilus and B. bixdum and demonstrated a lower surviving ability of the latter species. Gilliland and Lara (1988) also reported that both the L. acidophilus viability and its enzymatic activities in milk diminished remarkably during storage at 43C. On the other hand, Hull, Roberts and Mayes (1984) showed that the survival of L. acidophilus was higher when it was incorporated in the starter culture during manufacture (as in this study), because of the higher acquired tolerance to environmental conditions when grown in mixture with the other starter organisms. Another advantage of the use of L. acidophilus as a starter culture is the reduction or overacidi"cation to which classical yoghurts can frequently undergo. This phenomenon is, in turn, related to other defects such as syneresis, reduction of viable counts and accumulation of D(!) lactic acid in the product. The consequent undesirable drop in pH is mainly due to uncontrollable growth of strains of L. delbrueckii subsp. bulgaricus at high acidity levels during refrigerated storage (Kurmann, Rasic & Kroger, 1992). &Mildly acidifying' fermented milks obtained by means of cultures containing L. acidophilus and bi"dobacteria o!er the advantage of lower &over-acidi"cation' (Kneifel et al., 1993). Furthermore, these cultures give reduced contents of D(!) lactate in the products. However, the milk fermented by L. acidophilus or bi"dobacteria is often characterized by lack of acetaldehyde, which is quantitatively the principal and the most important constituent of yoghurt aroma. The absence of alcohol dehydrogenase in lactic acid bacteria involved in yoghurt making is a desirable feature for starter cultures. However, some L. acidophilus strains possess an alcohol dehydrogenase which converts the acetaldehyde to ethanol resulting in lack of #avour in acidophilus milk (Marshall & Cole, 1983). Also a high
The use of a Central Composite Design allowed to point out the e!ect of the compositional variables on the overall microbial viability as well as #avour characteristics of fermented milks during the storage at 43C. In particular, the polynomial models obtained and the relative response surfaces permitted to individuate the levels of the three independent variables able to minimize the loss of viability of the di!erent bacterial species used as starters. Moreover, it was possible to de"ne the e!ects of the same variables on the aroma pro"le. The results of the sensory evaluation on the di!erent fermented milks examined con"rmed that the organoleptic characteristics of the products were not a!ected by the L. acidophilus inoculum level, but only by the fat and milk solids.
Acknowledgements The authors would like to thank Prof. F. Dellaglio (Dipartimento Scienti"co e Tecnologico, University of Verona, Verona, Italy) for providing the strains used in the present study.
References Accolas, J. P., Hemme, D., Desmazeaud, M. J., Vassal, L., Bouillanne, C., & Veaux, M. (1980). Thermophilic lactic starters: properties and behaviour in dairy technology: a review. Lait, 60, 487}524. Bottazzi, V., Battistotti, B., & Montescani, G. (1973). In#uence of single and associated strains of L. bulgaricus and S. thermophilus as well as milk treatments on the production of acetaldehyde in yoghurt. Lait, 53, 295}308. Box, G. E. P., Hunter, W. G., & Hunter, J. S. (1978). Statistics for experimenters. An Introduction to design data analysis and model building. New York: Wiley. Dave, R. I., & Shah, N. P. (1996). Evaluation of media for selective enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and Bi"dobacteria. Journal of Dairy Science, 79, 1529}1536. Dettmann, J. (1995). Rechtsdrehende milchsaure. Molkerei Zeitung Welt der Milch, 49, 161}162. Gacula, M. C., Jr. (1988). Experimental design and analysis. In H. Moskowitz, Applied sensory analysis of foods, vol. II (pp. 83}140). Boca Raton: CRC Press Inc.
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Gilliland, S. E., & Lara, R. C. (1988). In#uence of storage at freezing and subsequent refrigeration temperatures on beta-galactosidase activity of Lactobacillus acidophilus. Applied and Environmental Microbiology, 54, 898}902. Gilliland, S. E., & Speck, M. L. (1977). Antagonistic action of Lactobacillus acidophilus toward intestinal and foodborne pathogen in associative cultures. Journal of Food Protection, 40, 820}823. Gonc, S., & Akalin, A. S. (1995). The e!ects of Lactobacillus acidophilus and Lactobacillus bixdus found survival in yoghurt on the organism and health. Gida, 20, 75}80. Hickey, M. W., Hillier, A. J., & Jago, G. R. (1986). Transport and metabolism of lactose, glucose and galactose in homofermentative lactobacilli. Applied and Environmental Microbiology, 51, 825}831. Hull, R. R., Roberts, A. W., & Mayes, J. J. (1984). Survival of Lactobacillus acidophilus in yoghurt. Australian Journal of Dairy Technology, 39, 164}166. IDF (1992). General standard of identixcation for fermented milks. Standard 163. Brussels: International Dairy Federation. Kilic, A. O., Pavlova, S. I., Ma, W. G., & Tao, L. (1996). Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of a phage isolated from yoghurt. Applied and Environmental Microbiology, 62, 2111}2116. Klaver, F. A. M., & van der Meer, R. (1993). The assumed assimilation of cholesterol by lactobacilli and Bixdobacterium bixdum is due to their salt-deconjugating activity. Applied and Environmental Microbiology, 59, 1120}1124. Kneifel, W., Jaros, D., & Erhard, F. (1993). Micro#ora and acidi"cation properties of yogurt and yogurt-related products fermented with commercially available starter cultures. International Journal of Food Microbiology, 18, 179}189.
Kneifel, W. M., Ulberth, F., Erhard, F., & Jaros, D. (1992). Aroma pro"les and sensory properties of yogurt and yogurt-related products. I. Screening of commercially available starter cultures. Milchwissenschaft, 47, 362}365. Kurmann, J. A., Rasic, J. L., & Kroger, M. (1992). Encyclopedia of fermented milk products. New York: Van Nostrand Reinhold. Lee, Y. K., & Salminen, S. (1995). The coming age of probiotics. Trends in Food Science and Technology, 6, 241}244. Marshall, V. M., & Cole, W. M. (1983). Threonine aldolase and alcohol dehydrogenase activities in Lactobacillus bulgaricus and Lactobacillus acidophilus and their contribution to #avours production in fermented milk. Journal of Dairy Research, 50, 375}379. Mital, B. K., & Garg, S. K. (1992). Acidophilus milk products: manufacture and therapeutics. Food Revue International, 8, 347}389. Mitsuoka, T. (1992). The human gastrointestinal tract. In B. J. B. Wood, The lactic acid bacteria in health and disease (pp. 69}113). Amsterdam: Elsevier. Montes, R. G., Bayless, T. M., Saavedra, J. M., & Perman, J. A. (1995). E!ects of milk inoculated with Lactobacillus acidophilus or yogurt starter culture in lactose maldigesting children. Journal of Dairy Science, 78, 1657}1664. Shah, N. (1994). Lactobacillus acidophilus and lactose intolerance: a review. Asean Food Journal, 9, 47}54. Shah, N. P., Lankaputhra, W. E. V., Britz, M. L., & Kyle, W. S. A. (1995). Survival of Lactobacillus acidophilus and Bixdobacterium bixdum in commercial yoghurt during refrigerated storage. International Dairy Journal, 5, 515}521. Torriani, S., Gardini, F., Guerzoni, M. E., & Dellaglio, F. (1996). Use of a response surface methodology to evaluate some variables a!ecting the growth and acidi"cation characteristics of yoghurt cultures. International Dairy Journal, 6, 625}636.
Evaluation of aroma production and survival of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus in fermented milks Fausto Gardini *, Rosalba Lanciotti, Maria Elisabetta Guerzoni , Sandra Torriani Dipartimento di Protezione e Valorizzazione Agroalimentare, Universita% degli Studi di Bologna, Via San Giacomo 7-40126 Bologna, Italy Istituto di Produzioni e Preparazioni Alimentari, Universita% degli Studi di Bari, Sede di Foggia, Via Napoli 25-71100 Foggia, Italy Istituto di Industrie Agrarie, Universita% degli Studi di Catania, Via Santa Soxa, Catania, Italy Received 28 August 1998; accepted 24 February 1999
Abstract The fat, the non-fat milk solid and the inoculum size of Lactobacillus acidophilus IPVR 224 were modulated according to a Central Composite Design. The aim was to evaluate the e!ects of these variables and their interactions on the decrease in pH during fermentation, the qualitative and quantitative composition of the aroma pro"le, as well as the loss in viability of Streptococcus thermophilus IPVR 161, Lactobacillus delbrueckii subsp. bulgaricus IPVR 132 and L. acidophilus IPVR 224 strains during "ve weeks of storage. In addition, the overall acceptability of the fermented milks was assessed by means of a sensory panel. The polynomial quadratic equations obtained allowed to individuate the variables that signi"cantly a!ected the aroma pro"le, organoleptic properties and microbiological counts of the various fermented milks. The survival of the L. acidophilus strain during storage was higher at low concentration of non-fat milk solids and its presence did not a!ect the acetaldehyde content of fermented milks. From the response surfaces analysis it was possible to select optimum conditions for enhanced positive organoleptic traits and for improved survival of the probiotic culture. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Fermented milks; Lactobacillus acidophilus; Modelling; Survival; Aroma compounds
1. Introduction The microorganisms used for the production of fermented milks are claimed to impart nutritional and health bene"ts to consumers. The probiotic activity is related to lactic acid bacteria, that are able to proliferate or even survive for a long period in the human intestinal tract (Mital & Garg, 1992). Among the lactic acid bacteria, Lactobacillus acidophilus is characterized by its capacity to colonise in the intestine even under low surface tension caused by the presence of bile salts (Gilliland & Speck, 1977). Consumption of acidophilus milk products is considered to promote several bene"cial effects such as control of enteric infections, normalization of gut micro#ora, lowering of serum cholesterol level, prevention of colon cancer, immunomodulation and enhanced availability of nutrients (Mital & Garg, 1992; Klaver & van der Meer, 1993; Lee & Salminen, 1995;
* Corresponding author.
Mitsuoka, 1992; Gonc & Akalin, 1995). Moreover, milk inoculated with L. acidophilus has been found to improve lactose tolerance among lactose malabsorbers (Shah, 1994; Montes, Bayless, Saavedra & Perman, 1995) and inhibit enteric and foodborne microbial pathogens (Kilic, Pavlova, Ma & Tao, 1996). In a study of Dettmann (1995), the nutritional bene"ts of using mixed cultures of L. acidophilus and Bixdobacterium bixdum are stressed and among others their predominant production of L(#) lactic acid. However, several L. acidophilus strains do not grow in milk and survive poorly in fermented products. Shah, Lankaputhra, Britz and Kyle (1995) suggested that the number of viable L. acidophilus cells should be greater than 5 log cfu g\ to have therapeutic bene"ts. Therefore, the use of probiotic cultures that provide high viable counts during the storage of the product is essential. The sensory characteristics of fermented milks play an important role in product acceptance by consumers. Several #avour compounds have been isolated from yoghurt-type products; however only acetaldehyde, ethanol,
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 3 3 - 3
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acetone, diacetyl and 2-butanone are found in substantial amount (Kneifel, Ulberth, Erhard & Jaros, 1992). Acetaldehyde is considered as the most prominent compound for the typical yoghurt aroma (Bottazzi, Battistotti & Montescani, 1973). The #avour quality of yoghurt also depends on the relationship between other volatile components; for example, a ratio between acetaldehyde and acetone of 2.8 is considered optimum (Accolas, Hemme, Desmazeaud, Vassal, Bouillanne & Veaux, 1980). Generally, acidophilus milk products are characterized by lack of #avour due to the fact that L. acidophilus possesses an alcohol dehydrogenase which converts acetaldehyde to ethanol (Marshall & Cole, 1983). It was shown that the addition of whey proteins and threonine to milk can prevent this problem; otherwise L. acidophilus can convert excess of threonine to acetaldehyde via glycine with the enzyme threonine aldolase (Hickey, Hillier & Jago, 1986). Because of the increasing interest in adding L. acidophilus to yoghurt in order to improve the probiotic activity of the intestinal micro#ora, a proper selection of strains is required. The principal aim of the present study was to evaluate the e!ects of fat, non-fat milk solids and inoculum size of L. acidophilus, modulated according to a Central Composite Design. In particular, the decrease in pH after fermentation, the loss in viability of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and L. acidophilus during a 5 weeks storage at 43C, the qualiquantitative aroma pro"le during storage at 43C and the overall acceptability of the fermented milks were analysed. 2. Materials and methods 2.1. Bacterial strains and culture media Lactobacillus delbrueckii subsp. bulgaricus IPVR 132, L. acidophilus IPVR 224 and S. thermophilus IPVR 161 were kindly provided by F. Dellaglio, Dipartimento Scienti"co e Tecnologico, University of Verona (Italy). The yoghurt culture (L. delbrueckii subsp. bulgaricus IPVR 132 and S. thermophilus IPVR 161) was chosen with regard to their well-established protosymbiosis, whereas L. acidophilus was selected on the basis of its ability to grow in milk, its high resistance to low pH and potential health promoting properties. The yoghurt cultures and L. acidophilus were maintained as frozen stock cultures at !803C in reconstituted (10% w/w) skim milk (Oxoid, Basingstoke, UK) and MRS broth (Oxoid) containing 20% glycerol, respectively. Cultures were transferred using 1% inocula and propagated twice in reconstituted skim milk at 373C before use. 2.2. Experimental design Di!erent fermented milks were prepared by modulating inoculum size of L. acidophilus [LA], percentage of
Table 1 Central Composite Design (three factor, "ve levels) used for the evaluation of the e!ects of milk solids, milk fat and L. acidophilus inoculum on the characteristics of the fermented milk Run
Milk solids (%) [S]
Milk fat (%) [F]
Lactobacillus acidophilus inoculum (%) [LA]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
11.5 14.5 11.5 14.5 11.5 14.5 11.5 14.5 13.0 13.0 10.0 16.0 16.0 13.0 13.0 13.0 13.0
2 2 6 6 2 2 6 6 4 4 4 4 0 8 4 4 4
1.0 1.0 1.0 1.0 2.0 2.0 2.0 2.0 1.5 1.5 1.5 1.5 1.5 1.5 0.5 2.5 1.5
milk fat [F] and non-fat milk solids [S] added to the basal medium according to a three-factor, "ve level Central Composite Design (Box, Hunter & Hunter, 1978). The 17 combinations obtained are shown in Table 1. The Central Composite Design permits to reduce the number of possible combinations to a manageable size. In fact, this design is characterized by using only a fraction of the total number of factor combination for experimentation. In statistical literature, the technique of reducing the number of factor combinations in a factorial experiment is known as confounding (Gacula, 1988). Five replicates of each combination were used. Skim milk powder (Sacco srl, Cadorago, Italy) was reconstituted to 10% (w/w) and was used as the basal medium. To obtain the levels required by the di!erent runs of Central Composite Design (Table 1), suitable amounts of commercial UHT cream (Parmalat, Parma, Italy) and skim milk powder (Sacco srl) were added to the basal medium. The mixes were heat-treated at 903C for 5 min, cooled to 373C in a water bath and inoculated with the yoghurt culture to attain a level of about 7 log cfu g\ for each strain. Immediately after this step, the milks were divided into 17 aliquots (250 g), and inoculated with di!erent percentages of the L. acidophilus culture, according to the runs of the Central Composite Design. With the aim to determine the #avour compounds, aliquots (5 g) of the inoculated milks for each combination were also dispensed into sterile glass vials (5 replicates) and sealed with aluminium caps. Then, the inoculated milks were fermented at 373C for 5 h. The "nal pH, depending on the combination, ranged from 5.3 to 4.2. The fermented milks were then transferred to a cold store at 43C.
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2.3. Microbiological analysis For each run of the experimental design, samples in duplicate were analysed immediately after fermentation and after "ve weeks of storage at 43C. Fermented milk samples (1 g) were added to 99 ml of sterile peptone diluent (0.1% w/v) and homogenized with a Stomacher Lab-Blender 400 (Seward Medical, London, UK) for 2 min. Appropriate dilutions were made in 1/4 strength sterile Ringer solution, and subsequentely plated in duplicate onto selective media. Lactobacillus delbrueckii subsp. bulgaricus was enumerated on MRS (Oxoid) at pH 5.2 after incubation at 453C for 72 h in an anaerobic jar containing H and CO gases (generated by an Oxoid BR38 kit). Streptococcus thermophilus was counted on M17 (Oxoid) plates after aerobic incubation at 373C for 48 h. Lactobacillus acidophilus was enumerated on MRSsorbitol plates (Dave & Shah, 1996) after incubation at 373C for 72 h. The selectivity of the growth conditions was con"rmed by the microscopic appearance of the cells from single colonies.
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with an autosampler HS250 (Carlo Erba Instruments) in which the samples were maintained at a controlled temperature (803C) for conditioning and sampling. For each analysis 2.5 ml of head space were automatically sampled with a gastight syringe 1750 (Hamilton, Bonaduz, Switzerland) at a controlled temperature of 803C. The volatile compounds were identi"ed using pure standard and quanti"ed on the basis of calibration curves previously prepared. 2.7. Statistical analysis Modelling was aimed at describing the decrease in pH after fermentation, the loss in viability of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and L. acidophilus during a 5 weeks storage at 43C, the quali-quantitative aroma pro"le during storage at 43C and the overall acceptability of the fermented milks as function of the independent variables of the Central Composite Design. A software package (Statistica for Windows, Statsoft, Tulsa, USA) was used to "t the second-order model to the dependent variables using the following equation:
2.4. pH measurement
y" B x # B x# B x x , G G GG G GH G H
Measurement of pH was carried out using a pH meter Model 2001 (Crison, Barcelona, Spain).
where y is the dependent variable to be modelled, B , B G GG and B are regression coe$cients of the model, and GH x and x are the independent variables in coded values. G H The variables with a signi"cance lower 95% (P'0.05) were not included in the "nal models. The three-dimensional surface plots were drawn to illustrate the main and interactive e!ects of the independent variables on the dependent ones. These graphs were drawn imposing a constant value (i.e. the central points of the interval taken into consideration) to one independent variable.
2.5. Sensory evaluation The sensory evaluation to examine the di!erences among the fermented milks of the various runs and the overall acceptability of each product was carried out by a panel of 20 judges. The sensory evaluation was carried out, after overnight storage, on samples (250 g) served at about 83C. Samples were scored on a hedonic scale of 1}7; 7 was considered excellent, 4 acceptable and 1 extremely poor.
3. Results 2.6. Gas chromatographic analysis of the aroma compounds The aroma constituents of the head space of sealed vials containing 5 g of product (empty/full ratio 1 : 1) were determined using a gas chromatograph Vega series 2000 (Carlo Erba Instruments, Milan, Italy) equipped with a Flame Ionization Detector (FID). Before the analysis, all the samples were preconditioned for 2 h at 803C. A glass column 2 m;2 mm i.d. packed with 80/120 Carbopack B AW/6.6% and PEG 20 M (Supelco, Bellefonte, USA) was used to separate the aroma compounds. The analytical conditions were the following: column temperature: 80}2003C at 43C min\; detector and injector temperature: 2003C; carrier gas (nitrogen) #ow rate: 20 ml min\. The gas chromatograph was connected
The 17 fermented milks were prepared according to the runs of the Central Composite Design (Table 1). Immediately after fermentation at 373C, the pH decrease was evaluated. The samples were stored at 43C and analysed at di!erent time intervals for aroma compounds and loss of viability of the yoghurt bacteria (IPVR 132 and IPVR 161 strains) and L. acidophilus IPVR 161. This strain, isolated from the human intestinal tract, was chosen for its potential health promoting properties, as evidenced by its in vitro ability to adhere to epithelial cells and assimilate cholesterol (F. Dellaglio, personal communication). The data obtained were modelled according to polynomial quadratic equations in order to identify the variables that signi"cantly a!ected the aroma, sensory and microbiological characteristics of the
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various combinations. In the "nal models, only the coe$cients characterized by P(0.05 were used. This model, already applied by Torriani, Gardini, Guerzoni and Dellaglio (1996) for the study of milk cultures, allows us to evaluate the e!ects of linear, quadratic and interactive terms of the independent variables on the chosen dependent variables. 3.1. pH decrease The decrease in pH after the fermentation with respect to the initial value was in#uenced by all the variables considered in the Central Composite Design (Table 2, Eq. (1)). The [LA] variable was signi"cant in its linear term and in its interaction with [F]. The [S] variable was signi"cant in its linear and quadratic terms and in its interaction with [F]. The positive coe$cient of the individual factor and the negative coe$cient of the quadratic term indicated that it is possible to identify an optimum level of non-fat milk solids to limit the decrease in pH. This optimal value (about 13%) is highlighted by the response surfaces, derived from the relative polynomial equation, for the [S];[LA] and [S];[F] interactions (Fig. 1a and b). These graphs were obtained from Eq. (1) of Table 2 by imposing, respectively, the central values of 4% for [F] and 1.5% for [LA]. The e!ect of the [LA];[F] interaction on decrease in pH during the fermentation process is shown in Fig. 1c. The highest pH decrease was observed when both the factors were at their lowest levels. However, the inoculum size of L. acidophilus seems to weakly enhance the pH decrease when the amount of fat is high. 3.2. Variations in latic acid bacteria cell numbers The loss of viability of lactic acid bacteria was evaluated as the di!erence in log cfu g\ between the counts after the fermentation at 373C and the counts after "ve weeks of storage at 43C. In all the runs of the Central Composite Design, at the end of storage, the three strains used as starters were characterized by a cell concentra-
tion higher than 10 cfu g\. In particular, in all the samples after the fermentation, the L. acidophilus IPVR 224 concentration was always higher than 8.30 log cfu g\ and, at the end of storage, the mean loss of viability was lower than 0.76 log cycles. The loss of viability of this strain was less pronounced with respect to the yoghurt cultures and ranged between 0.5 and 1.1 log cfu g\. The best-"t equations relative to the loss of viability of the inoculated cultures are shown in Table 2. Under the conditions used in this study, the decrease of L. acidophilus count, as shown by the Eq. (2) of Table 2, was a!ected only by the linear term of the [S] variable; its increase determined a loss of viability, even though the concentration of this microorganism was always higher than the initial inoculum. The amount of non-fat milk solids had the same positive e!ect on the viability of L. delbrueckii subsp. bulgaricus IPVR 132 strain (Table 2, Eq. (3)), though only its interaction with [LA] was signi"cant. The latter variable was present also in its interaction with [F] and played a signi"cant role, as shown in Fig. 1d, relative to the [LA];[F] interaction. These two factors had compensatory e!ects on L. delbrueckii subsp. bulgaricus IPVR 132 cell number decrease. In fact, the loss of viability reached the maximum when one of the two factors was at the highest level and the other at the lowest level. On the contrary the combination of these two variables at their lowest and highest levels was associated with the minimum loss of viability. The model describing the decrease in the streptococci number during storage (Table 2, Eq. (4)) permitted the individuation of a threshold value of [F] which determines the maximum decrease of S. thermophilus IPVR 161 was maximum, as indicated by the positive sign of the linear term and the negative sign of its quadratic term. The value was about 4% as shown by Fig. 1e and f, for [LA];[F] and [F];[S] interactions, respectively. Streptococcus thermophilus IPVR 161 took fair advantage from a high inoculum level of L. acidophilus IPVR 224, which determined a lower loss of viability. Moreover, it is
Table 2 Best-"t equations relative to the e!ects of the di!erent variables on the pH decrease after fermentation and the loss of viability of lactic acid bacteria strains during storage of fermented milks. Only terms with P(0.05 were included
pH Decrease after fermentation Eq. L. acidophilus IPVR 224 Eq. L. delbrueckii subsp. bulgaricus IPVR 132 Eq. S. thermophilus IPVR 161 Eq.
(1) (2) (3) (4)
Equation
R
F
SE
!0.16[LA]#0.38[S]!0.015[S]!0.007[S][F]#0.033[LA][F] 0.058[S] 0.36[F]#0.05[S][LA]!0.21[LA][F] !3.24[LA]#1.74[F]!0.07[F]!0.09[S][F]#0.24[S][LA]
0.999 0.901 0.973 0.965
5441.3 69.39 82.08 32.50
0.049 0.38 0.32 0.37
[S], non-fat milk solids (%); [F], fat added (%); [LA], L. acidophilus inoculum size (%). Regression coe$cient. F-value. Standard error.
F. Gardini et al. / International Dairy Journal 9 (1999) 125}134
129
Fig. 1. Three-dimensional contour plots concerning pH decrease after fermentation and loss of viability of starter cultures after a 5 week storage at 43C. The "gures are relative to the e!ects of the interaction [S][LA] (a), [S][F] (b) and [LA][F] (c) on pH decrease and [LA][F] (d) on L. delbrueckii subsp. bulgaricus loss of viability, [LA][F] (e) and [S][F] (f) on S. thermophilus loss of viability.
possible to state that the conditions favouring S. thermophilus viability determined a greater cell decrease of L. delbrueckii subsp. bulgaricus. 3.3. Aroma proxle after a week of storage Gas chromatographic analysis showed the presence of acetaldehyde, acetic acid, diacetyl, acetone and 2-butanone in all the runs of the Central Composite Design. The best-"t equations relative to the amounts of these compounds detected in the head space of fermented milks, as a function of the independent variables of the Central Composite Design, after one week of storage are reported in Table 3. Eq. (1) of Table 3 shows the individual and interactive e!ects of the variables of the Central Composite Design on acetaldehyde concentration. It is interesting to observe that the [LA] variable was not signi"cant and the acetaldehyde production was in#uenced only by [F], both in its linear and quadratic term, by [S], in its quadratic term, and by their interaction. The negative
sign of the coe$cient of the quadratic term of [F] de"nes a maximum in acetaldehyde presence, as shown by Fig. 2a, relative to the [S];[F] interaction. This maximum corresponds, approximately, to 5% of [F], at the lowest levels of [S], and is shifted towards lower fat levels (about 3%) when the non-fat milk solids content reaches its maximum. Moreover, the milk solids content enhanced acetaldehyde production, at least for a fat concentration up to 6%; for higher fat values this trend was the opposite. The other aroma compounds showed di!erent pattern. As shown by Eq. (2) of Table 3, acetic acid was a!ected by the [S] and [F] variables in their quadratic and interactive terms; also for this compound the [LA] variable showed no in#uence. The e!ects of the [S];[F] interaction on acetic acid presence in the head space are reported in Fig. 2b. In contrast to acetaldehyde production, it was possible to obtain minimum values of acetic acid concentration for values of [F] ranging between 4 and 6%, depending on [S]. However, analogously with acetaldehyde, the non-fat milk solids [S] showed a
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Table 3 Best-"t equations relative to the e!ects of the di!erent variables on the aroma compounds of fermented milks after one week of storage. Only terms with P(0.05 were included Aroma compound Acetaldehyde Acetic acid Diacetyl Acetone 2-Butanone
Eq. Eq. Eq. Eq. Eq.
(1) (2) (3) (4) (5)
Equation
R
F
SE
0.55[F]#0.0071[S]!0.036[F]!0.024[S][F] 0.000082[S]#0.00035[F]!0.00028[S][F] 0.048[LA]!0.0049[S]#0.0006[S]#0.00075[F]!0.00076[S][F]!0.0037[S][LA] 0.102[LA]#0.0012[S]#0.003[F]!0.003[S][F]!0.0078[S][LA] !0.0083[S]#0.0238[F]#0.005[LA]#0.001[S]!0.0022[S][F]
0.990 0.993 0.990 0.936 0.997
156.59 176.17 86.78 32.85 361.25
0.25 0.016 0.0034 0.0024 0.0042
Expressed as mg of aroma compound in equilibrium in the head space for kg of fermented milk. [S], non-fat milk solids (%); [F], fat added (%); [LA], L. acidophilus inoculum size (%). Regression coe$cient. F-value. Standard error.
Fig. 2. Three-dimensional contour plots relative to the amount of aroma compounds in the head space of the fermented milks after one week of storage at 43C; the results are expressed as mg of compound in the head space per kg of fermented milk. The "gure is relative to the e!ects of the interaction [S][F] on acetaldehyde (a), acetic acid (b), diacetyl (c) and acetone (d), the interaction [LA][S] on diacetyl (e) and [S][F] on 2-butanone (f).
positive e!ect on the presence of acetic acid at the lowest levels of [F]. The amounts of diacetyl and acetone in the head space were in#uenced by [LA] variable in its linear term and by
its interaction with [S] and [F] (Table 3, Eqs. (3) and (4)). However, the presence of the [S] and [F] quadratic terms with positive signs, allowed to recognise the combinations of these two variables able to minimize the
F. Gardini et al. / International Dairy Journal 9 (1999) 125}134
presence of diacetyl and acetone in the head space, as evidenced by the surface responses reported in Fig. 2c and d. The in#uence of the [LA] variable on the production of diacetyl is shown in Fig. 2e, relative to the [LA];[S] interaction. The maximum concentrations of diacetyl were reached when [S] was at its maximum and [LA] at its minimum level, and vice versa. Within this interval the lowest levels of diacetyl in the head space were obtained in correspondance with the lowest values of milk solids. The same behaviour was obtained for acetone (Fig. 2d). The production of 2-butanone is weakly in#uenced (Table 3, Eq. (5)) by the [LA] variable, while it was positively stimulated by [S] and negatively a!ected by [F] (Fig. 2f). 3.4. Aroma proxle after xve weeks of storage The best-"t equations relative to the principal aroma compounds detected after "ve weeks of storage at 43C in the head space of the fermented milks are reported in Table 4. All the considered variables a!ected the presence of these compounds, with the exception of acetaldehyde which was not in#uenced by [LA]. After "ve weeks of storage, acetaldehyde was still quantitatively the principal constituent of the aroma. Eq. (1) of Table 4 describes the individual and interactive e!ects of the considered variables on this compound. The acetaldehyde presence was in#uenced by [S], both in the linear and quadratic terms, by [F], in its linear term, and by their interaction. Fig. 3a, relative to the [S];[F] interaction, shows a maximum in acetaldehyde presence in correspondence to 13}14% of milk solids, while fat had a negative e!ect on the acetaldehyde concentration, as indicated by the negative sign of Eq. (1) in Table 4. As shown by Eq. (2) of Table 4, acetic acid production was a!ected, at the end of storage, by the same variables that were signi"cant after one week, as well as by [LA]
131
and [S];[LA]. This can account for the contribution of L. acidophilus to the production of this compound during refrigerated storage. For diacetyl production (Table 4, Eq. (3)), the most important variable is fat content, which is present in its quadratic term. The presence of a quadratic term determines a change in the sign of the "rst derivative of the curve and, consequently, the presence of a maximum or a minimum, depending on its sign. In fact, the presence of the positive sign for its quadratic term in the "nal model determines a minimum value (as shown in Fig. 3b, relative to the [S];[F] interaction) in correspondence to a concentration of about 4% of fat. Acetone and 2-butanone in the head space showed the same behaviour, as indicated by the Eqs. (4) and (5) of Table 4 in which the same terms (with the same signs) were signi"cant. In particular, for the [LA];[S] interaction (Fig. 3c and d) the increase of milk solids determined an increase in the production of these two compounds, whereas an increase of L. acidophilus inoculum had an opposite e!ect. 3.5. Sensory properties The organoleptic properties of the products prepared according to the di!erent combinations of the Central Composite Design were evaluated by 20 untrained panelists, who quanti"ed the overall quality of fermented milks with a score ranging from 1 to 7. The polynomial model, which describes the e!ects of the selected variables on the panellist evaluation is reported in Table 5. The overall quality was not a!ected by the L. acidophilus inoculum level, but only by the [S] and [F] variables in their linear and quadratic terms and by the [S];[F] interaction. As shown in Fig. 4, relative to the [S];[F] interaction, the obtaining of products with desired traits is positively a!ected by the increase of both variables. In fact, combinations with the best organoleptic and textural characteristics were obtained
Table 4 Best-"t equations relative to the e!ects of the di!erent variables on the aroma compounds of fermented milks after "ve week of storage. Only terms with P(0.05 were included Aroma compound Acetaldehyde Acetic acid Diacetyl Acetone 2-Butanone
Eq. Eq. Eq. Eq. Eq.
(1) (2) (3) (4) (5)
Equation
R
F
SE
!0.54[F]#0.44[S]!0.023[S]#0.032[S][F] 0.0086[LA]#0.000054[S]#0.00017[F]!0.00018[S][F]!0.00062[S][LA] 0.026[LA]#0.0032[S]!0.0116[F]#0.0009[F]!0.002[S][LA] !0.076[LA]#0.017[S]!0.0024[S][F]#0.014[F][LA] 0.0165[S]!0.053[LA]#0.0106[LA][F]!0.0021[S][F]
0.989 0.973 0.991 0.972 0.991
140.31 57.24 136.31 40.74 187.27
0.22 0.02 0.003 0.001 0.014
Expressed as mg of aroma compound in equilibrium in the head space for kg of fermented milk. [S], non-fat milk solids (%); [F], fat added (%); [LA], L. acidophilus inoculum size (%). Regression coe$cient. F-value. Standard error.
132
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Fig. 3. Three-dimensional contour plots relative to the amount of aroma compounds in the head space of the fermented milks after "ve week of storage at 43C; the results are expressed as mg of compound in the head space per kg of fermented milk. The "gures are relative to the e!ects of the interaction [S][F] on acetaldehyde (a), diacetyl (b) and [LA][S] on acetone (c) and 2-butanone (d).
Table 5 Best-"t equations relative to the e!ects of the di!erent variables on the overall acceptability of fermented milks. Only terms with P(0.05 were included
Overall acceptability
Eq. (1)
Equation
R
F
SE
0.38[S]!0.013[S]#0.019[S][F]
0.997
608.44
0.36
[S], non-fat milk solids added (%); [F], fat added (%). Regression coe$cient. F-value. Standard error.
with the highest levels of both non-fat milk solids and fat content.
4. Discussion The health bene"ts deriving from intake of fermented dairy products containing L. acidophilus are well documented and depend on a high viability of the microorganism. Furthermore, the presence of an high
concentration of viable bacterial cells at the time of consumption is necessary for complying with the FILIDF standard (IDF, 1992). The choice of proper strains selected on the basis of the desired features is therefore essential to meet these requirements. The strains used in this study remained viable during storage and the relevant survival of L. acidophilus IPVR 244 cells in the fermented milk is of great interest even if this result appears to be in contrast with some literature data. In fact, Kneifel, Jaros and Erhard (1993) compared the
F. Gardini et al. / International Dairy Journal 9 (1999) 125}134
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concentration of diacetyl could be responsible for aroma defects linked to a too pronounced &butter #avour'. However, such #avour defects of fermented milks can be controlled, as observed by Hickey et al. (1986), through compositional modi"cation of raw milk. In fact, as evidenced by the results obtained in this work, the presence of compounds such as diacetyl and acetone can vary in a wide range of values in response to modulation of chemical, physical and biological variables.
5. Conclusion
Fig. 4. Three-dimensional contour plots relative to the [F][S] interaction on overall acceptability of fermented milks.
traditional yoghurts with fermented milks containing L. acidophilus and B. bixdum and demonstrated a lower surviving ability of the latter species. Gilliland and Lara (1988) also reported that both the L. acidophilus viability and its enzymatic activities in milk diminished remarkably during storage at 43C. On the other hand, Hull, Roberts and Mayes (1984) showed that the survival of L. acidophilus was higher when it was incorporated in the starter culture during manufacture (as in this study), because of the higher acquired tolerance to environmental conditions when grown in mixture with the other starter organisms. Another advantage of the use of L. acidophilus as a starter culture is the reduction or overacidi"cation to which classical yoghurts can frequently undergo. This phenomenon is, in turn, related to other defects such as syneresis, reduction of viable counts and accumulation of D(!) lactic acid in the product. The consequent undesirable drop in pH is mainly due to uncontrollable growth of strains of L. delbrueckii subsp. bulgaricus at high acidity levels during refrigerated storage (Kurmann, Rasic & Kroger, 1992). &Mildly acidifying' fermented milks obtained by means of cultures containing L. acidophilus and bi"dobacteria o!er the advantage of lower &over-acidi"cation' (Kneifel et al., 1993). Furthermore, these cultures give reduced contents of D(!) lactate in the products. However, the milk fermented by L. acidophilus or bi"dobacteria is often characterized by lack of acetaldehyde, which is quantitatively the principal and the most important constituent of yoghurt aroma. The absence of alcohol dehydrogenase in lactic acid bacteria involved in yoghurt making is a desirable feature for starter cultures. However, some L. acidophilus strains possess an alcohol dehydrogenase which converts the acetaldehyde to ethanol resulting in lack of #avour in acidophilus milk (Marshall & Cole, 1983). Also a high
The use of a Central Composite Design allowed to point out the e!ect of the compositional variables on the overall microbial viability as well as #avour characteristics of fermented milks during the storage at 43C. In particular, the polynomial models obtained and the relative response surfaces permitted to individuate the levels of the three independent variables able to minimize the loss of viability of the di!erent bacterial species used as starters. Moreover, it was possible to de"ne the e!ects of the same variables on the aroma pro"le. The results of the sensory evaluation on the di!erent fermented milks examined con"rmed that the organoleptic characteristics of the products were not a!ected by the L. acidophilus inoculum level, but only by the fat and milk solids.
Acknowledgements The authors would like to thank Prof. F. Dellaglio (Dipartimento Scienti"co e Tecnologico, University of Verona, Verona, Italy) for providing the strains used in the present study.
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