Free Access
Issue
Dairy Sci. Technol.
Volume 89, Number 6, November-December 2009
Page(s) 613 - 625
DOI https://doi.org/10.1051/dst/2009042
Published online 10 November 2009

© INRA, EDP Sciences, 2009

1. INTRODUCTION

Small-scale milk production in developing countries where ambient temperatures are well above 30 °C suffers high losses, particularly when the market chain lacks adequate infrastructure to preserve milk. For this reason, most small-scale agro-pastoralists process their leftover milk into artisanal fermented dairy products for home consumption, some of which enter the informal market for economic benefit. However, lack of process control results in inconsistent quality of traditional fermented dairy products. Recent implications of dairy and other acidic foods in Escherichia coli O157:H7 outbreaks [2, 15] have challenged the safety of goat milk products that were processed under uncontrolled conditions. Once acid adapted, E. coli O157:H7 can survive in high acid foods for extended periods of time and can survive lethal pH of the stomach to cause disease in the intestine [18, 26]. Since E. coli O157:H7 has common occurrence in raw milk, it can also contaminate milk post-pasteurization following poor milk handling. Therefore, proper handling of raw and pasteurized milk and the application of appropriate preservation methodologies are important to inhibit E. coli O157:H7 because low pH alone is no longer sufficient to eliminate its occurrence in fermented dairy products.

The lactoperoxidase (LP) system can be activated in raw milk and post-pasteurization of milk as an additional bacteriological control in dairy processing. LP is a naturally occurring enzyme in milk that catalyses the oxidation of thiocyanate into hypothiocyanite in the presence of hydrogen peroxide [19]. Hypothiocyanite has a bacteriostatic effect on E. coli in milk [25]. In spite of heat sensitivity at temperatures above 70 °C [13], the LP enzyme is reported to maintain the activity at pasteurization temperatures of 63 °C for 30 min with ~ 30% loss of activity at 72 °C for 15 s [1].

Studies have shown that the LP system is not only antagonistic against undesirable microbes, but it also affects the growth and lactic acid production of some lactic acid bacteria (LAB) at both ambient and refrigeration temperatures [16, 24]. Acid production is critical in dairy fermentation because it is used to assess the quality of starter cultures [7]. Therefore, there are concerns that LP activation in milk will not only affect milk quality, but inhibition of acid production will also prolong the formation of casein gels and enable outgrowth of acid-adapted enteropathogens that occur in milk [9].

To our knowledge, there have been no studies on the effect of LP activation on indigenous lactic starter cultures that were used in artisanal fermented dairy products. Although there have been limited studies on the effect of LP activation on single strain lactic starter cultures [24], further studies are needed to enable the selection of LP-resistant lactic cultures that can be developed for the fermentation of specialized dairy products. In order to respond to the concerns of the application of LP system in milk intended for processing into fermented dairy products, this study was designed to first of all investigate the sensitivity of single Lactococcus sp. and Bifidobacterium longum to LP activation in pasteurized goat milk. Subsequently, selected sensitive and resistant LAB were used to ferment LP-activated goat milk that had been inoculated with E. coli O157:H7 as a model system to determine whether inhibition of acid production would affect the growth of E. coli O157:H7 in fermented milk. Finally, the application of LP system was tested in the fermentation of a traditional dairy product called Madila using indigenous lactic culture. Goat milk with inoculated E. coli O157:H7 was used to determine whether the effect of LP system on LAB and E. coli O157:H7 in the traditional product would differ from that of the commercial product.

2. MATERIALS AND METHODS

2.1. Milk source

Fresh Saanen goat milk was sourced from the University of Pretoria, experimental farm. The Saanen goats were milked following standard procedures with a milking machine. Milk from individual goats was pooled and delivered within 1 h of milking. One hundred millilitre portions of fresh goat milk were transferred into sterile 150 mL blue-capped Schott bottles and pasteurized at 63 °C for 30 min in a thermostatically controlled water bath before inoculation and activation of the LP system. Pasteurized milk was used for the processing of commercial fermented milk, and raw milk was used for traditional Madila fermentation.

2.2. Cultures

E. coli O157:H7 strains UP10 and 1062 were obtained from the Onderstepoort Veterinary Institute, Agricultural Research Council (Republic of South Africa (RSA)). Cultures were maintained on MacConkey agar (Oxoid, Hampshire, England) plates that were stored at 2 °C. Working cultures were prepared by transferring a single colony of each E. coli O157:H7 strain from MacConkey agar into sterile Tryptone Soy Broth (TSB; Biolab, Wadeville, RSA) and incubated for 24 h at 37 °C. The activating inoculum was prepared after two successive 24 h transfers of 0.5 mL of each of the E. coli O157:H7 strains UP10 and 1062 into 100 mL sterile TSB at 37 °C. This culture was used as inoculum for challenge tests.

The following lactic starter cultures were used in this study: single strain Lactococcus lactis subsp. lactis 345, Lactococcus lactis subsp. cremoris 326, Lc. lactis subsp. cremoris 328, Lactococcus lactis subsp. diacetylactis 339 and Lc. lactis subsp. diacetylactis 340 in vacuum-sealed ampoules were obtained from the Department of Food Bioscience, University of Free State, RSA; Lc. lactis subsp. lactis AM1 isolated from traditional Amasi; and B. longum BB536 obtained from Morigana (South Korea). Activated cultures were prepared by growing cultures in 100 mg·L−1 sterile skim milk at 22 °C for 16 h.

2.3. Inoculation and fermentation

All the 100 mL volumes of pasteurized goat milk were inoculated with 1 mL LAB culture. Each LAB culture was inoculated into two separate bottles containing 100 mL pasteurized milk; the LP system was activated in one of the two bottles, and the second bottle served as the LP-untreated control. Before activation of the LP system, the thiocyanate content of goat milk was determined according to the International Dairy Federation [12]. The LP activity was determined by spectroscopic measurement using one-step ABTS (2,2′-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid, Sigma, St. Louis, USA) solution as substrate [25]. The LP system was activated by adding sodium thiocyanate (Saarchem, Krugersdorp, RSA) to a final concentration of 14 mg·L−1. After thorough mixing, 30 mg·L−1 sodium percarbonate (Aldrich Chemical Company Inc., Milwaukee, USA) was added as a source of hydrogen peroxide [12]. The inoculated goat milk was then incubated at 30 °C for 6 h in a thermostatically controlled water bath.

To determine the effect of activated LP system on LAB in commercial fermented milk and its impact on the survival of E. coli O157:H7, 100 mL pasteurized goat milk samples inoculated with 1 mL selected single strain lactic cultures were also inoculated with 1 mL E. coli O157:H7 cocktail containing strains UP10 and 1062 prior to the activation of LP system. The initial concentration of LAB and E. coli O157:H7 was determined before incubation at 30 °C in a thermostatically controlled water bath for 24 h.

To prepare traditional Madila, fresh unpasteurized goat milk was transferred into two plastic buckets in 400 mL volumes. LP system was activated in one bucket containing 400 mL goat milk, and the second milk sample was used as the LP-untreated control. The LP-activated and control goat milk samples were each inoculated with 10% (v/v) traditional skim milk culture and 1% (v/v) E. coli O157:H7 strain UP10. Goat milk samples were allowed to ferment at 30 °C for 5 days. After 24 h and on each subsequent day for a total of 5 days, 1-day-old soured milk was added to fermenting Madila in a 4:1 (fermenting Madila:sour milk) ratio [17]. The 1-day-old soured milk was prepared by inoculating unpasteurized goat milk with 1% (w/v) freeze-dried traditional fermented milk and incubating at 25 °C for 24 h. On d 5, the whey from the fermented Madila was strained through a sterile jute bag. Madila was then mixed with cold unpasteurized goat milk in a ratio of 4 parts Madila:1 part goat milk.

2.4. Acid challenge

The surviving E. coli O157:H7 from LP-activated and control fermenting Madila samples were tested for acid adaptation after 24 h. Acid challenge test was conducted by transferring 1 mL of milk samples into 10 mL TSB acidified with 6 mol·L−1 lactic acid (Saarchem, Wadeville, RSA) to pH 4.0 for 4 h at 37 °C. Survival of E. coli O157:H7 from LP-activated and control Madila was compared to the survival of non-adapted E. coli O157:H7 challenged in acidified TSB (pH 4.0) for 4 h at 37 °C.

2.5. Chemical analyses

In order to determine the concentration of thiocyanate to add to milk, the thiocyanate concentration of milk was determined according to the IDF method [12]. About 8 mL of raw milk were thoroughly mixed with 4 mL of 20% (w/v) trichloroacetic acid (Saarchem, Gauteng, RSA) and allowed to stand for 30 min. The mixture was then filtered through a Whatman No. 40 filter paper and 1.5 mL of the clear filtrate mixed with 1.5 mL of ferric nitrate reagent (16 g of Fe(NO3)3·9H2O (Saarchem)) in 50 mL distilled water. The absorbance was measured at 460 nm wavelength with a Lamda EZ150 UV spectrophotometer (Perkin Elmer, USA), and the thiocyanate concentration was determined from a standard curve.

The titratable acidity (TA), used to measure lactic acid production, was determined by titrating 9 mL of milk with 0.1 mol·L−1 NaOH (Promark Chemicals, Robertsham, RSA). TA was expressed as percent lactic acid [3].

The pH readings were taken at the time of sampling of thoroughly mixed samples by inserting the pH electrode (Hanna Instruments, Italy) directly into the fermenting milk samples.

2.6. Microbiological analyses

Fermenting milk was sampled for viable E. coli O157:H7 and LAB counts after 0, 2, 4, 6 and 24 h for commercial Amasi/Maas-type fermented milk and 0, 1, 2, 3, 4 and 5 days for traditional Madila. Serial dilutions were prepared with 0.1% buffered peptone water (Oxoid, Hampshire, UK) and spread plated on M 17 agar (Oxoid) for Lactococci sp. counts, MRS agar (Oxoid) for Lactobacillus sp. and Leuconostoc sp. counts and Sorbitol MacConkey agar (SMAC, Oxoid) for E. coli O157:H7 counts. M 17 plates were incubated at 30 °C for 24–48 h, MRS plates were incubated at 37 °C for 48 h and SMAC plates were incubated at 37 °C for 24 h preceding enumeration of sorbitol negative E. coli O157:H7. Detection limit for microbial counts was 10 cfu·mL−1.

2.7. Statistical analyses

Analysis of variance (ANOVA) was used to determine whether activated LP had a significant effect on lactic acid production, and viability of lactic starters and E. coli O157:H7 cultures throughout the processing of commercial fermented milk (24 h) and the Madila processing period (5 days). Each sample was analysed in duplicate, and the experiment was conducted three times. The significance level was set at P ≤ 0.05. ANOVA was performed using Statistica (Tulsa, USA, 2008).

3. RESULTS

3.1. Quality of raw and pasteurized Saanen goat milk

The LP activity, TA and pH of raw and pasteurized Saanen goat milk are presented in Table I. The TA and pH of raw and pasteurized goat milk were within standard values. The standard deviation (SD) for pH of pasteurized goat milk (0.20) could be due to inconsistencies in time taken to reach pasteurization temperature, leading to an increase in pH. The average counts for E. coli, Lactococcus sp. and Lactobacillus sp. in fresh goat milk are presented in Table I. Zero counts were found in goat milk pasteurized at 63 °C for 30 min.

Table I.

Chemical and microbiological quality of raw and pasteurized Saanen goat milk (N = 6).

3.2. The effect of LP activation on single strain LAB in goat milk

All LAB cultures tested grew in pasteurized and LP-activated goat milk reaching populations of 9.1–9.4 log cfu·mL−1, with the exception of Lc. cremoris 326 that reached a final concentration of 8.5 log cfu·mL−1 after 6 h. Although there was a significant strain (P ≤ 0.05) effect on growth and acid production of the seven individual LAB strains tested (Tab. II), they did not show significant sensitivity to LP system (P > 0.05). The highest acid production was observed in Lc. lactis AM1 that was isolated from traditional Amasi, while the lowest acid reduction was observed in Lc. cremoris 326 (Tab. II). The acid production correlated positively with the decrease in pH.

Table II.

Changes in the mean values (†SD) of pH, acid production and LAB counts in pasteurized and lactoperoxidase-activated Saanen goat milk fermented at 30 °C by single strains of LAB.

3.3. The effect of LP activation on single strain LAB in goat milk that has E. coli O157:H7 present

There was no significant difference between LP-activated and control populations of all LAB strains tested, although cell numbers differed significantly (P ≤ 0.05) for individual cultures (Tab. III). Also, there was a significant (P ≤ 0.05) overall LAB strain effect on acid production by single lactic cultures (Tab. III). All LAB tested in the presence of E. coli O157:H7 had a significantly higher (P ≤ 0.05) acid production after 6 h compared to cultures that had no E. coli O157:H7. In the presence of E. coli O157:H7, acid production by Lc. lactis subsp. cremoris 326 culture was similar to those produced by Lc. lactis subsp. diacetylactis 340 and Lc. lactis subsp. lactis 345 after 6 h of fermentation. Also, all cultures tested with the exception of Lc. lactis AM1 showed a greater increase in acid production in the LP-activated milk after 6 h of fermentation compared to the LP-untreated controls. Lc. cremoris 326 showed the greatest difference in acid production.

Table III.

Changes in the mean values (†SD) of acid production and E. coli O157:H7 counts in pasteurized and LP-activated Saanen goat milk fermented by single strain LAB at 30 °C.

Like the single strain LAB cultured in the absence of E. coli O157:H7, LP system did not have a significant effect on acid production in the presence of E. coli O157:H7 throughout the fermentation period. Nonetheless, a marginal reduction in acid production was observed in the LP-activated 24 h culture of B. longum BB536 and Lc. lactis AM1. Lc. diacetylactis 340 on the other hand showed resistance to activated LP system with 4.18% increase in acid production compared to control cells after 24 h (Tab. III).

The E. coli O157:H7 numbers generally increased in goat milk during the first 6 h of fermentation. Inhibition of E. coli O157:H7 numbers was subsequently observed after 24 h in all single strain LAB cultured goat milk (Tab. III). Inhibition of E. coli O157:H7 was however not uniform for all the LAB strains tested. Here, LP system had a significant effect (P ≤ 0.05) on E. coli O157:H7 over time. Although LP inhibition of E. coli O157:H7 was not apparent in the Lc. diacetylactis 340 culture (19% in LP-activated culture compared to 18% in the control), significant reductions were observed in Lc. lactis 345 (18% in LP-activated culture compared to 13% in the control); B. longum BB536 (26% in LP-activated culture compared to 24% in the control culture) and Lc. lactis AM1 (24% in LP-activated culture compared to 19% in the control). Overall, Lc. cremoris 326 showed the greatest difference in E. coli O157:H7 inhibition between the LP-activated culture (23%) and the control culture (10%).

3.4. The effect of LP system on the processing of a traditional fermented product that has E. coli O157:H7 present

The LP system did not significantly affect the growth and acid production of indigenous LAB in Madila fermentation. The numbers of LAB increased reaching an optimum of 9.28 log cfu·mL−1 after 24 h of fermentation (Tab. IV). The LAB concentration subsequently declined marginally maintaining a level of ~ 8 log cfu·mL−1 throughout the fermentation period until d 5 when LAB numbers declined further. Similarly, pH of fermenting Madila was unaffected by activated LP throughout the fermentation period (P > 0.05) (Tab. IV). The pH of the LP-activated Madila declined to 4.22 and 4.19 in LP-untreated Madila after 24 h. The pH did not change significantly during the subsequent fermentation period. The TA of both LP-activated and control Madila increased after 24 h followed by constant acid production until d 3 (Tab. IV). On d 4 and d 5, both LP-activated and control Madila showed a progressive increase in TA; however, the LP-activated Madila had a higher TA compared to the control. This LP effect on TA was significant (P ≤ 0.05) over the 5-day period.

Table IV.

Changes in the mean values (†SD) of pH, titratable acidity and counts of E. coli O157:H7 and indigenous LAB during processing of traditional Madila at 30 °C.

The E. coli O157:H7 numbers in both LP-activated and control Madila increased marginally after 24 h of fermentation. Subsequently, E. coli O157:H7 counts in LP-activated Madila declined progressively until they reached < 1.0 log cfu·mL−1 at the end of the fermentation period (Tab. IV). The E. coli O157:H7 numbers in the control Madila also declined until d 3 after which the cell numbers levelled reaching 4.25 log cfu·mL−1 at the end of the fermentation period. The LP effect on E. coli O157:H7 survival during fermentation of Madila was statistically significant (P ≤ 0.05).

After 24 h of fermentation, E. coli O157:H7 cells in LP-activated and control Madila were challenged to lethal acid treatment at pH 4.0 for 4 h to determine whether E. coli O157:H7 in the fermenting medium had become acid adapted. Acid challenge caused 1.81 log cfu·mL−1 and 1.65 log cfu·mL−1 reductions in E. coli O157:H7 counts in LP-activated and control Madila, respectively. The non-adapted E. coli O157:H7 cells were reduced beyond detection after 4 h acid challenge at pH 4.0 (data not shown).

4. DISCUSSION

While lactic acid produces fresh flavour to fermented milk products [11], it is also important for the coagulation of milk. Therefore, rapid production of lactic acid is the most important attribute of lactic starter cultures [7]. In this study, all the single strain LAB tested with the exception of Lc. cremoris 326 were fast acid producers that reduced the pH of pasteurized goat milk to an average of pH 4.5 in 6 h. The relatively low acid production of Lc. cremoris 326 correlated positively with the cell concentration in pasteurized goat milk. Since all conditions were the same, the difference in the rate of growth and lactic acid production was characteristic of the strain.

The lack of activated LP inhibition of all the LAB strains tested is supported by other authors. For example, Nakada et al. [16] observed no significant difference in viability for single strain cultures Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus in yoghurt with or without subjection to the LP system, although acid production was inhibited in LP-activated cultures at 41 °C. In this study, acid production by single strain Lactococcus sp. and B. longum BB56 was not significantly suppressed in LP-activated goat milk. This apparent resistance could be due to the low LP activity of Saanen goat milk used in this study. LP activity of milk has been found to be highly variable depending on the type of milk and the period of lactation [6]. However, the level of LP activity in Saanen goat milk recorded in this study falls within the range of 0.04–0.16 U·mL−1 reported by Fonteh et al. [10] for raw goat milk during the lactation period. Regardless of the low LP activity of milk, the marginal reduction of acid production by B. longum BB536, Lc. cremoris 328 and Lc. lactis 345, compared to acid production in control milk, suggests that these LAB could potentially be sensitive to the LP system at a higher LP activity.

The presence of E. coli O157:H7 did not affect the growth of LAB in goat milk. The increased acid production of LAB in E. coli O157:H7 inoculated milk compared to the milk that had no E. coli O157:H7 was due to the additional lactic acid production by E. coli O157:H7 metabolism of lactose. The LP effect on acid production in the presence of E. coli O157:H7 was variable for the individual LAB tested. This difference lies in the strain-to-strain variation of lactic cultures [21], and the interaction between the lactic cultures, E. coli O157:H7 and the stresses encountered in the fermenting medium. Although the nature of this interaction was not investigated, the lactic cultures were clearly influenced by the presence of E. coli O157:H7 because lactic acid production in LP-activated milk differed from the cultures that had no E. coli O157:H7 present. The greater increase of acid production in LP-activated milk was unexpected. Given that E. coli O157:H7 cells were significantly inhibited by the LP system, the difference in acid production could not be attributed to acid production by E. coli O157:H7 alone. It appears that increased acid production was stimulated by lactic starter cultures in the presence of an antagonistic pathogen.

Apart from lactic acid inhibition of E. coli O157:H7 in fermented milk, other factors such as the production of bacteriocins and ethanol could have contributed to E. coli O157:H7 inhibition. Some species of Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris are known to produce nisin and lactococcin, respectively, that have broad antimicrobial spectra [15, 20]. Although studies have indicated that these antimicrobial peptides inhibit Gram positives, when coupled with activated LP, these bacteriocins could have an additional inhibitory effect on E. coli O157:H7 as observed in the LP-activated Lc. lactis subsp. cremoris 326 24 h fermented milk culture compared to the control culture. The increased inhibition of E. coli O157:H7 observed in Lc. lactis AM1 and B. longum BB536 cultures in both LP-activated and control milk could be due to their characteristic antimicrobial properties. Both Lc. lactis subsp. lactis and B. longum, particularly strain B. longum BB536, have been classified as probiotics that are antagonistic against pathogenic microbes [14, 22].

In the traditional Madila product, the indigenous LAB were resistant to LP system. The resistance of indigenous LAB from traditional fermented milk to activated LP system has not been reported. Previous studies examining the sensitivity of mixed and single strain lactic starter cultures to activated LP system found that acid production and survival of lactic starter cultures in activated LP milk vary from one investigation to another. Seifu et al. [24] reported that activated LP inhibits acid production of commercially mixed lactic starter cultures. In this study, the indigenous LAB were not only insensitive to activated LP, but lactic acid production was not inhibited in LP-activated milk. The lack of LP inhibition of lactic cultures could be due to the reversal of antimicrobial hypothiocyanite by the enzyme NADH-OSCN oxidoreductase into thiocyanate [5]. This reversal factor exhibited by NADH oxidoreductase together with NADH oxidase and peroxidase enzymes is stimulated during oxidative stress [23]. Investigation of the molecular basis for resistance of these indigenously mixed lactic starters could shed more light on the mechanism of resistance against LP activation. These indigenous LAB cultures could be developed for upscale Madila processing from LP-activated milk.

Although inhibition of acid production was not observed in LP-activated fermented milk in this study, acid challenge of the 24 h culture during Madila fermentation indicated that the inoculated E. coli O157:H7 had become acid adapted. This finding is consistent with those of other authors who have reported acid resistance of E. coli during fermentation of dairy products [29]. Though acid adapted, the E. coli O157:H7 cells were inhibited in LP-activated Madila. Previous studies have indicated a limited period of LP efficacy in milk [8]. It was stated in the guideline for raw milk preservation [4] that the activated LP system can extend the keeping quality of raw milk stored at 30 °C for 7–8 h. In this study, activated LP inhibition of acid-adapted E. coli O157:H7 was evident after d 4 of Madila fermentation. This observation suggests that when activated LP system was coupled with low pH, the combined inhibitory effect was extended for at least 5 days at 30 °C. The delayed LP inhibition of E. coli O157:H7 in LP-activated Madila suggests that low pH sensitized acid-adapted E. coli O157:H7 to activated LP. The increased enzymatic production of HOSCN/OSCN and the easy passage of uncharged hypothiocyanite into the cell at low pH [27, 28] could have contributed to the inhibition of acid-adapted E. coli O157:H7 in LP-activated Madila. Since the combination of LP activation and low pH caused > 5.0 log cfu·mL−1 reduction in E. coli O157:H7, it can be applied in traditional milk processing and storage at ambient temperature to improve the microbiological safety of fermented milk with respect to E. coli O157:H7.

5. CONCLUSION

This study has shown evidence that the application of activated LP did not inhibit lactic acid production by single strain and indigenous LAB during the first 6 h of fermentation, which is a crucial period for growth and acid production of LAB in the processing of fermented dairy. These cultures can therefore be developed for the processing of specialized dairy products from activated LP milk. Though E. coli O157:H7 cells were inhibited in LP-activated milk, the high numbers in fermented milk after 24 h indicate that the application of LP system in the industrial processing of milk may not be sufficient to reduce E. coli O157:H7 that occur in milk. However, in the traditional processing of milk products, like Madila, where milk is slowly fermented at ambient temperatures over long periods, LP system can be applied to improve the safety of the product.

Acknowledgments

This research was supported by the National Research Fund and the Third World Organization for Women in Science.

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All Tables

Table I.

Chemical and microbiological quality of raw and pasteurized Saanen goat milk (N = 6).

Table II.

Changes in the mean values (†SD) of pH, acid production and LAB counts in pasteurized and lactoperoxidase-activated Saanen goat milk fermented at 30 °C by single strains of LAB.

Table III.

Changes in the mean values (†SD) of acid production and E. coli O157:H7 counts in pasteurized and LP-activated Saanen goat milk fermented by single strain LAB at 30 °C.

Table IV.

Changes in the mean values (†SD) of pH, titratable acidity and counts of E. coli O157:H7 and indigenous LAB during processing of traditional Madila at 30 °C.

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