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This study aimed to evaluate the potential of Weissella cibaria D30 as an adjunct culture in cottage cheese, including an assessment of antioxidant, antilisterial, and compositional parameters. Cottage cheese samples were manufactured using a commercial starter culture and probiotic strains Lactobacillus rhamnosus GG (GG) or W. cibaria D30 (W) and without probiotic (control). Samples were stored at 4 ± 1°C for 28 d. Bacterial cell counts (log cfu/g) of control, GG, and W samples were counted at 0, 7, 14, 21, and 28 d. Counts of W. cibaria D30 in the W samples remained at 6.85 log cfu/g after 28 d. Total solids, fat, protein, ash, and pH were measured and no significant differences were observed in compositional parameters or pH after 28 d of storage in all cheeses except those inoculated to Listeria monocytogenes. To measure the antilisterial effect, Listeria monocytogenes was inoculated into the cottage cheese samples and bacterial cell counts were obtained at 0, 6, 12, 24, 48, 72, 96, 120, and 144 h. Listeria monocytogenes counts were less than the analytical limit of detection (<10 cfu/g) in the inoculated GG and W samples, whereas the counts of L. monocytogenes in the inoculated control sample remained at 3.0 log cfu/g after 144 h. We used the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging activity assays to assess antioxidant activity: GG and W samples exhibited significant increases in antioxidant activity compared with the control sample. These results indicate that W. cibaria D30 has potential as an adjunct culture in the dairy industry.
Report on joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria.
Food and Agriculture Organization of the United Nations,
Rome, Italy2001
). Although probiotics must be able to survive adequately in food carriers, their viability after passing through the gastrointestinal tract is also crucial. Various dairy products have been investigated for their effect on probiotic viability after exposure to the gastrointestinal tract, and cheese has been identified as a good carrier of probiotics (
). In this regard, cheese has certain beneficial characteristics, including physical and chemical properties. High fat content, high water activity, and the pH of cheese may offer added protection to probiotics and help them maintain higher viability throughout gastric transit (
). Furthermore, cheese has a higher buffering capacity, which controls the high acidic environment of the gastrointestinal tract and helps to create a favorable environment for probiotic viability (
Fresh cheeses are particularly vulnerable to contamination by foodborne pathogens such as Listeria monocytogenes, which is considered a postprocessing contaminant. This bacterium is usually associated with refrigerated, ready-to-eat (RTE) foods and can grow within wide temperature (0 to 42°C) and pH (4 to 9) ranges and under high salt concentrations (10%;
). Listeria monocytogenes is frequently associated with listeriosis outbreaks; thus, the European Union has stipulated microbial criteria for RTE foods (EC regulation 2073), stating that in those foods “able to support the growth of L. monocytogenes,” the pathogen must be absent in 25 g of product during their shelf life (
). Hence, control of L. monocytogenes growth and multiplication in fresh cheese has been demanded. Some studies have focused on the addition of adjunct cultures isolated from herbs, fruits, and vegetables to prevent the growth of L. monocytogenes in cheese (
Characterization of Lactococcus lactis isolates from herbs, fruits and vegetables for use as bio preservatives against Listeria monocytogenes in cheese.
Korean kimchi is emblematic of Korean culture and is a famous side dish popular in Korea; it is made of salted cabbage with a variety of seasonings. Many studies have generated plausible evidence of the antimicrobial, anti-inflammatory, and antiadhesive activity against foodborne pathogens and the antioxidant and anticancer effects of probiotics isolated from Korean kimchi (
). Weissella species are reported to be prominent during kimchi fermentation and have antioxidative, antifungal, antibacterial, anticancer, anti-inflammatory, and immune-boosting potential (
). Lactic acid bacteria (LAB) isolated from fermented foods are generally recognized as safe; thus, extensive investigations have been carried out to use these novel strains as adjunct cultures in the cheese industry (
Characterization of Lactococcus lactis isolates from herbs, fruits and vegetables for use as bio preservatives against Listeria monocytogenes in cheese.
). Therefore, the aim of the present study was to investigate the potential of W. cibaria D30, isolated from Korean kimchi, as an adjunct culture in cottage cheese and to examine its characteristics with respect to physicochemical properties, antioxidant activity, and antilisterial activity in cheese.
MATERIALS AND METHODS
Microorganisms and Culture Conditions
Lactobacillus rhamnosus GG (KCTC 12202 BP) and W. cibaria D30 were used as probiotic strains in the present study. The W. cibaria D30 strain was isolated from Korean kimchi and has demonstrated probiotic characteristics (
). The cheese starter culture was supplied by Culture Systems Inc. (Mishawaka, IN) in freeze-dried form and comprised Lactobacillus acidophilus, Bifidobacterium longum, and Streptococcus thermophilus. The probiotic strains were streaked onto de Man, Rogosa, and Sharpe (MRS; BD BBL, Franklin Lakes, NJ) agar plates directly from the −80°C stock and incubated at 37°C for 24 h. One colony from each strain was inoculated into 10 mL of MRS broth and incubated under the same conditions as above. Subsequently, a 5-mL aliquot was inoculated into 500 mL of MRS broth and incubated in a shaking incubator at 37°C and 150 rpm for 10 to 12 h, followed by centrifugation at 5,000 × g for 10 min at 4°C. The supernatant was decanted and the pellet was resuspended in low-fat milk. The colony count of the inoculum was determined by spread plating on MRS agar at required dilutions, followed by incubating at 37°C for 72 h.
A 3-strain mixture of L. monocytogenes (ATCC 15313, Scott A NADC 2045, and H7969 serotype 4b) was used to contaminate cheese samples. The L. monocytogenes strains were grown in 10 mL of tryptic soy broth (BD BBL) at 37°C for 24 h. The cells were obtained by centrifugation at 5,000 × g for 10 min at 4°C. The supernatant was discarded and the pellet was resuspended in 0.1% peptone water. Cell suspensions of each strain were mixed in the same volume and serially diluted to achieve a count of 5 to 5.5 log cfu/g in cottage cheese. The colony count of the inoculum was determined by spread plating on listeria selective agar (Oxoid Ltd., Basingstoke, UK) at required dilutions and then incubating at 37°C for 48 h.
Preparation of Cottage Cheese
Commercially available pasteurized low-fat milk (4% fat, Seoul, South Korea) was used to produce cottage cheese. Cheese was manufactured according to
with some modifications. The milk was preheated to 37°C, starter culture and liquid animal rennet (Maysa, Istanbul, Turkey) were added at 0.2% (about 109 cfu/g) and 100 μL/L, respectively. Cheese milk was incubated at 37°C until the pH reached approximately 4.7 to 4.5. When the curd was produced, it was cut manually with aid of a cheese wire knife into 1-cm3 pieces. Subsequently, the whey was removed by 2 to 3 repetitive washings with cold water (4°C).
Probiotic was added at 109 cfu/g after the addition of the starter culture. The control cheese sample was prepared following the same procedure without adding probiotics. For antilisterial activity, cheese samples were cut into small slices (3 × 6 cm), and 100 µL of inoculum was spread over cheese slices by using a bent glass rod. Inoculated individual pieces were vacuum-packed and stored at 4°C.
Six types of cheese were prepared and labeled as follows: cheese inoculated with starter culture and L. rhamnosus GG as adjunct (GG); W = cheese inoculated with starter culture and W. cibaria D30 as adjunct (W); cheese inoculated with starter culture only (control, C); GG cheese inoculated with L. monocytogenes (GGL); W cheese inoculated with L. monocytogenes (WL); and C cheese inoculated with L. monocytogenes (CL).
Composition and pH
Cheese samples were analyzed for protein, fat, total solids contents, and ash. Total protein and fat were analyzed by the Kjeldahl method (method 991.20;
) and Soxhlet extraction method, respectively. Total solids content was analyzed by oven drying a known weight of samples at 102°C for 3 to 4 h until at a constant weight. Ash content was obtained by heating appropriate weights of samples in a muffle furnace at 550°C overnight. The pH of all cottage cheese samples was measured using a digital pH meter (WTW-720, WTW, Weilheim, Germany) equipped with a glass electrode, which was immersed in cheese samples homogenized with distilled water. All analyses were conducted in triplicate.
Viability and Verification of LAB
Lactic acid bacteria counts of cheese samples were obtained at 0, 7, 14, 21, and 28 d of storage according to the method of
Influence of probiotic strains added to cottage cheese on generation of potentially antioxidant peptides, anti-listerial activity, and survival of probiotic microorganisms in simulated gastrointestinal conditions.
. Briefly, cheese samples (10 g) were homogenized in 90 mL of 0.1% peptone water and subsequently macerated in a stomacher for 1 min. The LAB were enumerated by spread plating on MRS agar at required dilutions followed by incubating at 37°C for 72 h. The count of W. cibaria D30 in the W sample was taken at the end of shelf life (28 d) and verification was done based on morphology of the strain.
Antioxidant Activity
Water-Soluble Extracts
Water-soluble extracts (WSE) were prepared using the method of
with some modifications. Briefly, 10 g of cheese sample was suspended in 30 mL of distilled water and kept at 40°C under gentle stirring for approximately 1 h. The homogenates were centrifuged at 5,000 × g at 4°C for 30 min. Subsequently, the uppermost fat layer was removed, the supernatant was filtered through Whatman No. 2 filter paper, and the WSE were further used to analyze antioxidant activity.
Antioxidant Activity Assays
Total antioxidant activity was determined according to the method of
. Two hundred microliters of WSE was added to 1 mL of freshly prepared 100 µM 2,2-diphenyl-2-picrylhydrazyl radical (DPPH) and allowed to stand in the dark for 15 to 20 min. Absorbance was measured by spectrophotometer at 517 nm. Three replicates were carried out for each sample. The absorbance of the blank was measured by using distilled water and following the same procedure as above. The results were expressed as percentage of scavenging activity, which was calculated as follows:
with some modifications. The ABTS radical solution was prepared by reacting 14 mM ABTS and 5 mM potassium persulfate in the dark at room temperature for 12 to 16 h. Before the assay, the ABTS•+ radical cation solution was diluted with 0.1 M ethanol at a ratio of 1:10 to obtain absorbance of 0.7 ± 0.02 at 734 nm. Then, 20 µL of sample (WSE) was suspended in 980 µL of the prepared ABTS•+ radical cation solution and incubated at 37°C for 5 min. The absorbance was then measured at 734 nm. This experiment was conducted in 3 replicates. The formula used to calculate the scavenging activity was as follows:
where Acontrol and Asample represent the absorbance of control (distilled water) and WSE, respectively.
Viability of L. monocytogenes
The viability of L. monocytogenes in cheese samples was evaluated at 0, 6, 12, 24, 48, 72, 96, 120, and 144 h. Enumeration was carried out according to the method of
. Briefly, 10 g of cheese sample was homogenized with 90 mL of 0.1% sterile peptone water, followed by maceration in a stomacher for 1 min. Decimal dilutions were spread-plated on listeria selective agar (Oxoid Ltd.) and incubated at 37°C for 48 h.
Statistical Analysis
The results were obtained for each treatment in triplicate and are presented as means ± standard deviations. Statistical analyses were conducted using IBM SPSS statistics 20 (SPSS/IBM Corp., Chicago, IL). The data were assessed using one-way ANOVA. A difference was considered significant at P ≤ 0.05 using Duncan's multiple range test.
RESULTS AND DISCUSSION
Viability of LAB in Cottage Cheese at Refrigerated Storage (4°C)
The changes in microflora during 28 d of storage at 4°C are illustrated in Figure 1. On d 0, total LAB counts were 8.5 ± 0.01, 9.02 ± 0.05, and 8.99 ± 0.06 log cfu/g in C, GG, and W samples, respectively. At 7 d of storage, LAB count increased in all cheese samples and reached 8.76 ± 0.04, 9.34 ± 0.05, and 9.03 ± 0.05 log cfu/g in C, GG, and W samples, respectively. The LAB count continued to increase until 14 d of storage in the GG and W samples, whereas a significant reduction (1.2 log cfu/g) was observed in the C sample (P < 0.05). The declining LAB in the C sample after 7 d can be explained by the high vulnerability of starter LAB to the harsh cheese environment such as high pH (pH 4.4–4.5), no residual lactose, and low temperature (4°C;
). In contrast, the steady increase in LAB until 14 d in the GG and W samples can be attributed partially to high tolerance of nonstarter LAB, such as L. rhamnosus GG and W. cibaria D 30 (
Figure 1Bacterial cell counts of lactic acid bacteria in cottage cheese during storage at 4°C. Results are expressed as mean ± SD (n = 3). C = cheese inoculated with starter culture (control); GG = cheese inoculated with starter culture and Lactobacillus rhamnosus GG as adjunct; W = cheese inoculated with starter culture and Weissella cibaria D30 as adjunct.
Nonetheless, the LAB count decreased in all samples after 14 d of storage (P < 0.05). At 21 d of storage, LAB counts were 6.93 ± 0.07, 8.38 ± 0.17, and 8.03 ± 0.05 log cfu/g in the C, GG, and W samples, respectively. At 28 d of storage, LAB counts reached 6.84 ± 0.05, 7.51 ± 0.053, and 7.24 ± 0.015 log cfu/g in the C, GG, and W samples, respectively.
showed a decline in viable count of Lactobacillus casei 431 in yogurt beyond 14 d of storage. This behavior could be due to the inhibitory activities of LAB, such as production of organic acids (lactic acid), hydrogen peroxide, nutrient competition, bacteriocin, diacetyl, and alcoholic compounds (
). However, the rate of decline of viable cell count was lower in the cottage cheese samples with probiotics (GG and W) than in the C sample. In addition, we detected no difference in LAB viability in the GG and W samples (P > 0.05).
The general shelf life of fresh cheese stored in a refrigerator is 10 to 12 d. Nevertheless, at the end of the study period (after 28 d), the viable count of W. cibaria D30 in W cheese was 6.85 log cfu/g.
Compositional Analysis and pH
The compositional data of all samples after 28 d of storage are shown in Table 1. Values of experimental cheese samples were compared with those of the control cheese. The addition of probiotics did not have a significant effect on protein, TS, or ash content. The addition of probiotics reduced the fat content of the experimental samples but not significantly (P > 0.05).
Influence of probiotic strains added to cottage cheese on generation of potentially antioxidant peptides, anti-listerial activity, and survival of probiotic microorganisms in simulated gastrointestinal conditions.
reported the influence of Lactobacillus casei ATCC 373 and L. rhamnosus GG ATCC 53103 added to cottage cheese. We did not detect a significant difference (P > 0.05) between experimental samples in pH levels at 28 d of storage. The pH values were 4.58 ± 0.01, 4.57 ± 0.01, 4.56 ± 0.01, 4.52 ± 0.02, and 4.54 ± 0.02 in C, GG, W, GGL, and WL samples, respectively.
Table 1Composition and pH of cottage cheese samples after 28 d of storage at 4°C
C = cheese inoculated with starter culture (control); GG = cheese inoculated with Lactobacillus rhamnosus GG as adjunct; W = cheese inoculated with Weissella cibaria D30 as adjunct; GGL and WL = GG and W cheeses inoculated with Listeria monocytogenes.
Means within a row with different superscripts differ (P < 0.05).
a Means within a row with different superscripts differ (P < 0.05).
1 All values are means of 3 replicates (± SD).
2 C = cheese inoculated with starter culture (control); GG = cheese inoculated with Lactobacillus rhamnosus GG as adjunct; W = cheese inoculated with Weissella cibaria D30 as adjunct; GGL and WL = GG and W cheeses inoculated with Listeria monocytogenes.
The antioxidant activity of cottage cheese samples during storage at 4°C was analyzed at 0, 7, 14, 21, and 28 d by using the DPPH and ABTS radical scavenging assays. Figure 2, Figure 3 show the antioxidant activities for the DPPH and ABTS assays, respectively.
Figure 2Antioxidant activity of water-soluble extracts (WSE) of cottage cheese samples measured by the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay. Antioxidant activity is expressed as percent of scavenging activity. Results are expressed as mean ± SD (n = 3). C = cheese inoculated with starter culture (control); GG = cheese inoculated with starter culture and Lactobacillus rhamnosus GG as adjunct; W = cheese inoculated with starter culture and Weissella cibaria D30 as adjunct.
Figure 3Antioxidant activity of water-soluble extracts (WSE) of cottage cheese obtained by the ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] assay. Antioxidant activity is expressed as percent of scavenging activity. Results are expressed as mean ± SD (n = 3). C = cheese inoculated with starter culture (control); GG = cheese inoculated with starter culture and Lactobacillus rhamnosus GG as adjunct; W = cheese inoculated with starter culture and Weissella cibaria D30 as adjunct.
Radical scavenging activities increased significantly (P < 0.05) in both assays with increased storage time. Nonetheless, the cottage cheese samples with added probiotic strains had higher scavenging activities than the control sample (P < 0.05). At the end of the storage period (28 d), DPPH and ABTS radical scavenging activities in C, GG, and W samples were, respectively, 26.04 ± 0.98%, 41.71 ± 1.02%, and 44.6 ± 0.93% in the DPPH assay, and 42.43 ± 1.07%, 62.05 ± 1.65%, and 65.10 ± 1.03% in the ABTS assay. There was no difference in antioxidant activity among cheese samples with different strains of probiotics (P > 0.05). These findings are in agreement with those of
Himalayan cheese (Kalari/Kardi)—Effect of different probiotic strains on oxidative stability, microbiological, sensory and nutraceutical properties during storage.
, who studied the antioxidant activity of Lactobacillus casei 279, Lactobacillus brevis 021, and Lactobacillus plantarum 01 strains added to Kalari cheese. This can be explained by the fact that cheese contains a high amount of protein and these proteins (especially casein) are subjected to proteolysis by enzymes present in milk, such as plasmin. In addition, the residuals of rennet added during the cheese-making process and microbial proteolytic enzymes result in the production of bioactive peptides. These bioactive peptides are responsible for antioxidant activity by inhibiting the formation of free radicals such as oxygen and nitrogen, for example, via different mechanisms (
). However, samples with probiotics showed an increase (P < 0.05) in antioxidant activities, which may be explained by the fact that some LAB have specific abilities to produce antioxidant enzymes (i.e., superoxide dismutase, catalase, glutathionine reductases, and thioredoxin) to scavenge free radicals, thereby resulting in oxidative defense (
). Overall, results showed that the antioxidant activity of fresh cheese was enhanced by the addition of probiotic strains.
Viability and Behavior of Inoculated L. monocytogenes in Cottage Cheese During Storage
When pasteurized milk is used for cheese manufacturing, a major cause of cheese spoilage is cross-contamination due to improper handling, which mainly affects the surface of cheese (
). In this study, the surface of the cottage cheese was inoculated with a multi-strain cocktail of L. monocytogenes to simulate this postprocessing contamination. Figure 4 shows the behavior of L. monocytogenes throughout storage. The initial counts of L. monocytogenes were 5 to 5.5 log cfu/g in all 3 samples (CL, GGL, and WL). We detected no significant difference in L. monocytogenes count in the control sample (CL) at 6 h (P > 0.05), whereas the counts of GGL and WL samples were significantly reduced by 0.87 and 0.77 log cfu/g, respectively (P < 0.05). Beyond 6 h, L. monocytogenes counts declined steadily in all 3 samples, and at 120 h the count reached 2.0 log cfu/g in the GGL and WL samples and 3.4 log cfu/g in the CL sample. At 144 h, the L. monocytogenes counts in GGL and WL samples were below the limit of detection (<10 cfu/g), whereas that of the CL sample was 3.0 log cfu/g.
Figure 4Bacterial cell counts of Listeria monocytogenes during storage at 4°C. Results are expressed as mean ± SD (n = 3). CL, GGL, and WL = C, GG, and W cheeses inoculated with L. monocytogenes, where C = cheese inoculated with starter culture (control); GG = cheese inoculated with starter culture and Lactobacillus rhamnosus GG as adjunct; W = cheese inoculated with starter culture and Weissella cibaria D30 as adjunct.
These results demonstrated that L. monocytogenes was able to survive for a longer period in CL (15 d) than in the GGL and WL samples. These findings are in agreement with results from other studies in Galotyri cheese, a soft acid curd cheese (
Fate of Listeria monocytogenes on fully ripened Greek Graviera cheese stored at 4, 12, or 25°C in air or vacuum packages: In situ PCR detection of a cocktail of bacteriocins potentially contributing to pathogen inhibition.
Fate of Listeria monocytogenes on fully ripened Greek Graviera cheese stored at 4, 12, or 25°C in air or vacuum packages: In situ PCR detection of a cocktail of bacteriocins potentially contributing to pathogen inhibition.
explained that the combined hurdle effect of lactic acid, pH, and water activity inhibited the growth of the pathogen but that it survived for a longer period with a low death rate under refrigerated storage. Thus, faster inhibition (144 h) of L. monocytogenes in the GGL and WL samples demonstrated that L. rhamnosus GG and W. cibaria D30 act as protective adjunct cultures through the potential formation of antimicrobial compounds such as acetic acid, lactic acid, and bacteriocin.
CONCLUSIONS
The present study revealed that the W. cibaria D 30 strain isolated from Korean kimchi has potential for use as an adjunct culture in cheese manufacture. In particular, W. cibaria D 30 could provide additional hurdle effects to prevent the growth of L. monocytogenes and ensure the microbial safety of RTE soft cheeses. Ascertaining the long-term survival of L. monocytogenes in RTE soft cheese is crucial because of the potential risk of transmittance of the pathogen from cheese slicers to other RTE foods that might support its growth. In addition, the use of W. cibaria D 30 enhanced antioxidant activity and did not affect the compositional parameters or pH, which ensures its potential use as an adjunct culture in cottage cheese production to improve quality. The viability of W. cibaria D 30 above the minimum threshold level of 106 cfu/g until the end of the shelf life shows its potential to deliver probiotic health benefits to the consumer at the time of consumption.
ACKNOWLEDGMENTS
This paper was supported by Konkuk University (Seoul, Korea) in 2016.
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Influence of probiotic strains added to cottage cheese on generation of potentially antioxidant peptides, anti-listerial activity, and survival of probiotic microorganisms in simulated gastrointestinal conditions.
Report on joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria.
Food and Agriculture Organization of the United Nations,
Rome, Italy2001 (World Health Organization, Geneva, Switzerland)
Fate of Listeria monocytogenes on fully ripened Greek Graviera cheese stored at 4, 12, or 25°C in air or vacuum packages: In situ PCR detection of a cocktail of bacteriocins potentially contributing to pathogen inhibition.
Characterization of Lactococcus lactis isolates from herbs, fruits and vegetables for use as bio preservatives against Listeria monocytogenes in cheese.
Himalayan cheese (Kalari/Kardi)—Effect of different probiotic strains on oxidative stability, microbiological, sensory and nutraceutical properties during storage.
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