Free Access
Issue
Dairy Sci. Technol.
Volume 89, Number 6, November-December 2009
Page(s) 541 - 553
DOI https://doi.org/10.1051/dst/2009030
Published online 23 September 2009

© INRA, EDP Sciences, 2009

1. INTRODUCTION

Organic methods of food production have gained increased public interest over the last couple of decades, and this concept of agriculture has grown considerably, mainly, in the western world. Organic products have been identified to contain a higher nutritional quality compared to conventional food and, as a consequence, have attracted consumer acceptability [5]. Organic and conventional dairy productions differ considerably in feeding regimens, in the use of antibiotic and chemotherapeutic treatments and in the handling of animals [12]. However, all the standards of organic farming specify strict rules for milk production and feeding of dairy cattle. The feed must be obtained from organic farming where the use of any artificial input is forbidden [23]. The chemical composition of organic and conventional dairy products has been analyzed and compared by many researchers [32, 36] in order to support and prove the authenticity on the benefits of a natural livestock system. These studies showed that there are little or any differences in the compositional data.

Milk and dairy products are an important source of conjugated linoleic acid (CLA), which consists of a mixture of positional and geometric isomers of linoleic acid (C18:2) with conjugated double bonds. The main isomer is the cis-9, trans-11, but less amounts of trans-9, trans-11 and trans-10, cis-12 are also formed [5, 6, 38]. Milk and dairy products have been shown to contain the highest amounts of CLA [5, 38], and it is produced as an intermediate component in the ruminal biohydrogenation pathway, carried out by the rumen bacteria. The dietary lipids that are hydrolyzed into free fatty acids (FA) are subjected to biohydrogenation in the digestive tract of ruminants and, as a consequence, the biohydrogenation pathway is the major source of CLA in milk fat [15, 30].

CLA formation by bacterial strains has been documented since the early 1960s [27]. For example, propionibacteria (i.e. as adjunct culture and possibly lactic acid bacteria) [28] and some yoghurt starter cultures [7, 37] showed potential as food-grade organisms to be used for CLA enhancement in dairy products. In yoghurt, there is a certain potential to increase the CLA content by adding adjunct cultures under the condition that free linoleic acid or oil and suitable lipase are added; this approach of increasing the CLA level in the fermented milk has its limitation(s) [37]. The substrate level in the growth media may be a key factor for CLA production, since a high level of unsaturated long-chain fatty acids (LCFA) is believed to be inhibitory not only to cell growth but also to the overall biohydrogenation steps, especially for gram-positive bacteria, which lack an outer membrane [21].

The interest in the possible ability of dairy starter cultures to increase the CLA content in dairy products is vast. As a significant proportion of the CLA isomers are formed during biohydrogenation of linoleic acid in the rumen by the bacteria Butyrivibrio fibrisolvens, it was to be expected that bacteria used as dairy starter cultures would also have the ability to form CLA [7]. Many researchers investigated the synthesis of CLA using selected bacterial strains under controlled conditions in laboratory media or model systems [11, 28, 37]. Some of these selected strains include lactobacilli, bifidobacteria and propionibacteria, which were found to convert efficiently the added linoleic acid to CLA.

CLA in fermented milk and dairy products should provide “functional” aspects as inhibition of initiation of carcinogenesis process, effects on anti-atherogenic, anti-adipogenic, anti-diabetogenic and anti-inflammatory activities, beneficial regulatory effects on immune function, and alters the low-density lipoprotein/high-density lipoprotein cholesterol ratio [8, 15].

During fermentation, many bioactive components are formed [34], as organic acids (lactic, pyruvic, formic, acetic and propionic), peptides derived from milk proteins due to proteolysis [16], and acetaldehyde, acetone, acetoin and biacetyl through carbohydrates degradation [20]. The CLA amounts could be enhanced through biohydrogenation of linoleic acid by bifidobacteria and other bacteria [24]. To date, few studies have explored the effect of bifidobacteria and yoghurt starter cultures on growth and acidification kinetics as well as the identification of bioactive components in organic fermented milk.

The aims of this study were to investigate the performance of different strains of Bifidobacterium animalis subsp. lactis on acidification and FA profile including CLA in organic and conventional fermented milk.

2. MATERIALS AND METHODS

2.1. Milk

Commercial organic and conventional pasteurized whole milks were purchased from a local supermarket and used for the manufacture of yoghurt and probiotic fermented milks.

2.2. Chemical composition of milk (organic and conventional)

Fat, protein, total solids and density contents were determined using an ultrasonic milk analyzer Ekomilk (Eon Trading, Bulgaria), an equipment that analyzes the milk composition by ultrasound; the analysis was done according to the recommendations of Venturoso et al. [35]. Lactose and lactic acid contents were determined according to the guidelines of Instituto Adolfo Lutz [17], and a digital potentiometer (Quimis, Diadema, São Paulo) was used to measure the pH values.

Calcium (Ca), magnesium (Mg), copper (Cu), iron (Fe) and zinc (Zn) contents in the fresh milk were determined using an atomic absorption spectrophotometer (Polarized Zeeman AAS, Hitachi Z-5000; Hitachi, Tokyo, Japan). A hollow cathode lamp was employed at 422.7, 202.6, 324.8, 283.4 and 213.9 nm, and slits of 0.7, 1.3, 1.3, 0.2 and 1.3 nm for measuring the Ca, Mg, Cu, Fe and Zn contents, respectively, after wet digestion (HNO3:H2O2 – 5:1; mL·100 mL−1) and addition of 0.1% (g·100 g−1) lanthanum as La2O3 (for Ca and Mg analyses) as recommended by the AOAC [3]. The working standard solutions (100 mg·mL−1) were prepared with CaCl2, MgCl2, CuCl2, FeCl3 and ZnCl2, and were obtained from Titrisol Merck, Darmstadt, Germany. All the chemical analyses were performed in duplicate.

2.3. Sources of microbial cultures and enumeration

Concentrated and freeze-dried single strains of probiotic and yoghurt microflora were employed: S. thermophilus (St-) strain TA040 (Danisco, Sassenage, France), Lactobacillus delbrueckii subsp. bulgaricus strain LB340 (Danisco, Sassenage, France), B. animalis subsp. lactis strain BB12 (Chr. Hansen A/S, Hoersholm, Denmark), strain BL04 (Danisco, Madison, USA), strain B94 (DSM Food Specialities, New South Wales, Australia) and strain HN019 (Danisco, Madison, USA). Each lyophilized strain was weighed and dissolved in 50-mL sterilized skimmed milk (121 °C for 15 min) that had been tempered to 42 °C for 15 min before use. One milliliter of each rehydrated culture was inoculated in 500 mL of organic and conventional milk (treated at 85 °C for 15 min). This procedure allowed us to obtain initial counts of ~ 8.0 log10 colony forming units (CFU)·mL−1.

Enumeration was made, in duplicate, one day after fermentation of the stored products at 4 °C, as no changes in bacteria counts between the end of fermentation and 24 h after the process were observed. Each sample (1.0 mL) was added to 9.0 mL of 0.1% (g·100 mL−1) sterile peptonized water and then appropriate dilutions were made. Subsequently, S. thermophilus and L. delbrueckii subsp. bulgaricus were plated into M17 agar and MRS agar (Oxoid Ltd., Basingstoke, UK); the latter agar was previously acidified to pH 5.4 with acetic acid, and afterward the plates were incubated at 37 °C for 48 h [10]. B. animalis subsp. lactis strains were enumerated in RCA agar adjusted to pH 7.1 added with 0.3 g·100 g−1 of aniline plus 1 μL·mL−1 of dicloxacillin and incubated under anaerobic conditions at 37 °C for 72 h [29].

Anaerobic conditions were ensured by the use of AnaeroGen (Oxoid Ltd., Basingstoke, UK). The enumeration of microbial counts was performed on plates containing 30–300 colonies. Microscopic examination of the cells in the colonies of every strain of bifidobacteria was performed using a light microscope.

2.4. Fermented milk manufacture

Organic and conventional pasteurized whole milks were heat treated to 85 °C for 15 min in a water bath (Lauda®-Königshofen, Germany) under constant stirring, cooled to 10 °C and stored overnight at 4 °C. On the following day, each type of milk was tempered to 42 °C, divided into five batches and inoculated with different combinations of starter cultures. Each batch of milk was incubated at 42 °C in a thermostatically controlled water bath until the pH reached 4.7. The rate of acidification of each of the microbial blends was monitored using the Cinac system (Ysebaert, Frépillon, France). After reaching a pH of 4.7, each fermented milk was agitated manually using a stainless steel plunger (i.e. consisting of a rod and perforated disk) that was moved upward and downward for 60 s, dispensed into 50-mL polypropylene cups (heat sealed using Selopar equipment – BrasHolanda, Pinhais, Brazil), quickly cooled in an ice bath and stored at 4 °C until required for analysis. The samples were prepared in duplicate, and the experiment was replicated twice on different days.

Five kinetic parameters were considered: (a) Vmax [maximum acidification rate, measured in pH units per min (upH·min−1)], (b) (time to reach the maximum acidification rate), (c) pH corresponding to Vmax and (d) TpH5.0 and TpH4.7 (time in hours to reach pH 5.0 and 4.7, respectively).

2.5. FA and CLA analysis

Lipids were extracted from fresh milk, yoghurts and probiotic fermented milk products (organic and conventional) according to the ISO method 14156 ISO [18], and the fatty acid methyl esters (FAMEs) were prepared by esterification according to the ISO method 15884 ISO [19].

Analyses of FAMEs were carried out in a gas chromatograph, Model 3400CX (Varian, São Paulo, Brazil) equipped with a split-injection port, a flame-ionization detector and a software package for system control and data acquisition (model Star Chromatography Workstation version 5.5, Varian Inc., Palo Alto, CA). Injections were performed in a 30 m long fused silica capillary column with 0.25-mm internal diameter, coated with 0.25 μm of Chrompack CP-Wax 52CB (Chrom Tech, Apple Valley, MN, USA), using helium as carrier gas at a flow rate of 1.5 mL·min−1 and a split ratio of 1:50. The injector temperature was set at 250 °C and that of the detector at 280 °C. The oven temperature was initially set at 75 °C for 3 min, then programed to increase to 150 °C at a rate of 37.5 °C·min−1 and then to 215 °C at a rate of 3 °C·min−1. Samples (1 μL) were injected manually after a dwell time of ca. 2 s.

Qualitative FA composition of the samples was determined by comparing the retention times of the peaks produced after injecting the methylated samples with those of the respective standards of FA (SIGMA 05632 and SIGMA 189-19). CLA (cis-9, trans-11) was detected at 23.10 min of retention time. The quantitative composition of each FA was calculated from the area of each peak and expressed as a percentage according to the Official Method Ce 1-62 [4]. The FA composition was classified as reported by Ackman [1] who described short-chain fatty acids (SCFA) as C2 to C4, medium-chain fatty acids (MCFA) as C6 to C12 and LCFA as C14 to C24. All samples were analyzed in quadruplicate, and the results, expressed as g·100 g−1 of total FA, were reported as mean value.

2.6. Statistical analysis

Analysis of variance for multiple comparisons using Statistica 6.0, Statsoft (Tulsa, USA), was performed in order to confirm the statistical significance of differences among samples (P < 0.05). Mean values were compared using the Tukey test at P ≤ 0.05.

Coefficient (r) was calculated in order to estimate the correlation between linoleic acid (C18:2), trans-vaccenic acid (TVA) (C18:1t), CLA (C18:2 c9, t11) and fermentation time (TpH4.7) in organic and conventional milk, considering P ≤ 0.01.

3. RESULTS AND DISCUSSION

3.1. Chemical composition of milk

The chemical composition (g·100 g−1) of conventional and organic milk was closely related, did not present any significant differences and is shown in Table I. The protein content, however, was significantly higher in organic milk. Ca, Zn, Mg and Cu contents did not significantly differ in both types of milks, but the Fe content was higher in organic milk. Such data are similar to what has been reported by the researchers in different countries [9, 33].

Table I.

Gross chemical composition* and certain minerals of organic and conventional fresh milk.

3.2. Acidification kinetics

The acidification rates in milk (organic and conventional) of yoghurt starter organisms and different strains of B. animalis subsp. lactis (B94, BB12, BL04 and HN019) in co-culture with S. thermophilus are shown in Table II. Comparison of the acidification kinetics of the four strains of B. animalis subsp. lactis grown in conventional and organic whole milk revealed different results. Co-cultures St-HN019 and St-BB12 showed a higher acidification rate (Vmax) in both milks. The time to reach Vmax () for St-B94, St-BL04 and St-LB340 was significantly higher (P ≤ 0.05) in conventional milk than in organic milk. The pH at which Vmax was reached, , did not significantly differ between co-cultures and between milks.

Table II.

Comparison of the acidification kinetic parameters in organic and conventional milk by the yoghurt cultures (St-LB340 – control), and S. thermophilus in co-culture with the four strains of B. animalis subsp. lactis (St-BB12, St-B94, St-BL04 and St-HN019).

The time to reach pH 5.0, TpH5.0, was significantly higher for St-B94 and St-BL04 in conventional milk when compared with organic milk. Fermentation of the conventional milk with co-culture St-B94 required 6.00 h to reach pH 4.7, but the same co-culture required 4.17 h to reach the same value of pH in organic milk, i.e. a decrease of 24.5% in the fermentation time (Fig. 1). Similarly, yoghurt starter cultures and co-culture St-BL04 showed a significantly shorter time of fermentation in organic milk than in conventional milk. In spite of the absence of the published data, a lower fermentation time of organic milk was unexpected. This result may be related to the presence of some organic acids, as formic acid, in organic milk due to handling of the animals (feeding regimen, no use of antibiotic and chemotherapeutic treatments).

thumbnail Figure 1.

Fermentation time (TpH4.7) of yoghurt cultures (St-LB340 – control), and S. thermophilus in co-culture with the four strains of B. animalis subsp. lactis (St-BB12, St-B94, St-BL04 and St-HN019) in organic and conventional milk. Mean values of duplicate experiments with different letters are significantly different; P ≤ 0.05.

To our knowledge, there are no data available on the kinetic profile of B. animalis subsp. lactis in organic milk. On the other hand, the acidification times results obtained from the co-cultures St-LB340 and St-BL04 in conventional milk were much lower than those reported by Almeida et al. [2] and by Oliveira and Damin [25].

3.3. Probiotic and yoghurt starter culture counts

Table III details the microbial counts of the yoghurt starter cultures and the bifidobacteria in the inoculated milk (D0) and (D1), i.e. 24 h after the fermentation period in the stored products at 4 °C. The average increase in the counts of all the microorganisms in organic and conventional milk products was 1.09 log10 and 1.22 log10 cycles, respectively. Some differences were observed for B. animalis subsp. lactis counts concerning all products. Strains of B. animalis subsp. lactis reached on average 8.60 log10 CFU·mL−1 in organic and 8.75 log10 CFU·mL−1 in conventional fermented milk with no significant differences. S. thermophilus counts did not present significant differences between the different milks, i.e. varying between 9.00 and 10.14 log10 CFU·mL−1 and being at least one order of magnitude higher than the other bacteria (data not shown).

Table III.

Counts of single strains of L. delbrueckii subsp. bulgaricus (LB340) and B. animalis subsp. lactis strains (BB12, B94, BL04 and HN019) fermented in co-culture with S. thermophilus in organic and conventional milk 24 h after the end of fermentation.

3.4. FA profile and CLA contents

Table IV shows the FA contents (g·100 g−1) in organic and conventional fresh milk (M), yoghurts (Y) and fermented milk (FM) products prepared with St-LB340 (control) and St-BB12, St-B94, St-BL04 and St-HN019.

Table IV.

FA contents (g·100 g−1) in organic and conventional fresh milk (M), yoghurts (Y) and fermented milk (FM).

No significant differences were found for SCFA contents in organic fresh milk, yoghurt and fermented milk; whereas significant differences (P ≤ 0.05) were found for SCFA contents in conventional fresh milk, yoghurt and fermented milk. In addition, MCFA and LCFA varied slightly in organic and conventional products (Tab. IV).

The highest amounts of saturated fatty acids (SFA) were found in fermented organic milk made with co-cultures St-BL04 and St-BB12 (P ≤ 0.05). Contents of polyunsaturated fatty acids (PUFA) averaged 3.61 g·100 g−1 with no significant differences (P ≤ 0.05), whereas the lowest content of monounsaturated fatty acids (MUFA) (26.34 g·100 g−1) was found in organic yoghurt with a statistically significant difference (P ≤ 0.05) compared to other samples (Tab. IV). The contents of SFA, MUFA and PUFA in organic milk are similar to the data reported by Fanti et al. [13].

No significant difference was found in the concentration of cis-9, trans-11 CLA isomer between the fresh and fermented milk by different co-cultures in the conventional milk. On the other hand, the highest concentration of cis-9, trans-11 CLA was found in organic milk fermented with co-cultures St-BB12, St-B94, St-BL04, St-LB340 and St-HN019 with a statistically significant difference (P ≤ 0.05) versus organic fresh milk. The highest amounts of CLA (65% higher than in the control) were found in organic milk fermented by St-bifidobacteria and yoghurt cultures (Fig. 2). The ability of probiotic bacteria to produce CLA confirms those previously obtained from a survey carried out on dairy products [7, 24, 26].

thumbnail Figure 2.

CLA (g·100 g−1) in organic and conventional fresh milk (M), yoghurts (St-LB340 – control) and by fermented milk S. thermophilus in co-culture with the four strains of B. animalis subsp. lactis (St-BB12, St-B94, St-BL04 and St-HN019). Mean values (n = 4) with different letters are significantly different; P ≤ 0.05.

To explain the observed variations in CLA levels, it should be taken into account that the different enzyme activities of starter cultures have been identified as factors that may contribute to influence the CLA content of cultured dairy products [31]. In particular, Lin et al. [22] demonstrated that a higher level of linoleic acid isomerase produced by Lactobacillus acidophilus in crude extract increased the CLA content of fermented milk.

These results suggest that different strains of bifidobacteria as well as yoghurt cultures grown were able to produce appreciable quantities of CLA in the organic fermented milk.

3.5. Correlation between linoleic acid (C18:2), TVA (C18:1t), CLA (C18:2 c9, t11) and fermentation time (TpH4.7) in organic and conventional milk

CLA is produced in the rumen mainly through incomplete biohydrogenation of linoleic acid in CLA (C18:2) by ruminal bacteria and by enzymatic action of Δ-9-desaturase upon TVA – C18:1t in mammary glands [15].

In this study, the amount of linoleic acid in fresh organic milk was 1.85 g·100 g−1, and after fermentation a significant decrease (P ≤ 0.05) in this FA was found for all employed strains, including yoghurt strains, resulting in an increase in CLA amounts. In fact, a strong negative significant correlation (r = −0.803, P ≤ 0.01) was observed between linoleic acid and CLA.

The lower fermentation time observed in organic milk fermented by St-BL04, St-B94 and yoghurt starter cultures (St-LB340) could indicate that there is a relation between short fermentation time and the occurrence of an incomplete biohydrogenation of linoleic acid into CLA by bifidobacteria and yoghurt strains, similarly, what occurs during fermentation process in the rumen. Indeed, a significant negative correlation was found between fermentation time and CLA (r = −0.450, P ≤ 0.01), and a significant positive correlation (r = 0.673, P ≤ 0.01) between fermentation time and linoleic acid contents.

These results agree with those reported by Oliveira et al. [26], who observed a higher CLA content in probiotic fermented milk supplemented with different prebiotics, which showed a fermentation time lower than control fermented milk (i.e. not supplemented milk). Meanwhile, Florence [14] found that for fermentation carried out with pure cultures of bifidobacteria and L. bulgaricus in which fermentation times were very long (TpH4.7 > 10 h), no significant increase in CLA amounts was found in both milks (conventional and organic) after the process.

Then, probably there is a factor in organic milk which stimulates fast fermentation and allows metabolic biohydrogenation pathway of CLA production.

In parallel, there are higher contents of TVA in fresh and fermented organic milk when compared with conventional products. The higher amounts of TVA are associated with higher contents of CLA with a strong significant positive correlation (r = 0.883, P ≤ 0.01) as previously reported by Molketin and Giesemann [23].

4. CONCLUSION

The chemical composition of conventional and organic milk was closely related. All bacteria showed adequate counts according to regulatory recommendations. Despite the fact that no significant differences were noted with the main FA, organic fermented milk showed higher amounts of TVA and CLA. A significant positive correlation was found between fermentation time (TpH4.7) and linoleic acid, as well as between TVA and CLA. Beside, a significant negative correlation was observed between linoleic acid and CLA, and between TpH4.7 and CLA, whatever the type of milk. The differences noted in FA composition and acidification profile are not enough to predict the effect of each bacteria on CLA increase in organic fermented milk. Thus, new studies are required to elucidate the observed data.

Acknowledgments

The authors thank Danisco Brasil Ltda (Cotia, São Paulo, Brazil), Chr. Hansen and DSM Food Specialities for the donation of cultures, FAPESP and CNPq for financial support, and Professor C. Colli from São Paulo University, Department of Food Science for the analysis of major and trace mineral elements.

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All Tables

Table I.

Gross chemical composition* and certain minerals of organic and conventional fresh milk.

Table II.

Comparison of the acidification kinetic parameters in organic and conventional milk by the yoghurt cultures (St-LB340 – control), and S. thermophilus in co-culture with the four strains of B. animalis subsp. lactis (St-BB12, St-B94, St-BL04 and St-HN019).

Table III.

Counts of single strains of L. delbrueckii subsp. bulgaricus (LB340) and B. animalis subsp. lactis strains (BB12, B94, BL04 and HN019) fermented in co-culture with S. thermophilus in organic and conventional milk 24 h after the end of fermentation.

Table IV.

FA contents (g·100 g−1) in organic and conventional fresh milk (M), yoghurts (Y) and fermented milk (FM).

All Figures

thumbnail Figure 1.

Fermentation time (TpH4.7) of yoghurt cultures (St-LB340 – control), and S. thermophilus in co-culture with the four strains of B. animalis subsp. lactis (St-BB12, St-B94, St-BL04 and St-HN019) in organic and conventional milk. Mean values of duplicate experiments with different letters are significantly different; P ≤ 0.05.

In the text
thumbnail Figure 2.

CLA (g·100 g−1) in organic and conventional fresh milk (M), yoghurts (St-LB340 – control) and by fermented milk S. thermophilus in co-culture with the four strains of B. animalis subsp. lactis (St-BB12, St-B94, St-BL04 and St-HN019). Mean values (n = 4) with different letters are significantly different; P ≤ 0.05.

In the text

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