Antibacterial Effects against Various Foodborne Pathogens and Sensory Properties of Yogurt Supplemented with Panax ginseng Marc Extract

Yogurt culture (ABT-B: a mixture of Lactobacillus acidophilus, Bifidobacterium longum, and Streptococcus thermophilus) was purchased from Culture Systems, Inc. (Mishawaka, USA). Bacillus cereus KCCM11341, Enterobacter sakazakii ATCC51329, Staphylococcus aureus 1573, Salmonella Typhimurium (National Veterinary Research and Quarantine Service, Korea), Listeria monocytogenes H7962 (Centers for Disease Control and Prevention, USA), and Escherichia coli O157:H4 FRIK125 (Wisconsin Food Research Institute, USA) were used for evaluation of the antibacterial effect. All pathogenic strains were activated in trypticase soy broth (Difco Labs, USA).

Ginseng marc was obtained from Il-Hwa Research Institute (Korea). Ginseng marc was extracted three times with 4 volumes of 75% ethanol at 60°C for 12 h, and then lyophilized using a freeze-drying method. To prepare the yogurt, 11% skim milk (Seoul Milk Co., Korea), 0.1% pectin (Buy Chem, Korea), and 10% sucrose (Cheil Jedang, Korea) were added to 0.2 L of milk (Seoul Milk Co., Korea), with or without ginseng marc extract (GME) at various concentrations (0.5% and 1.0%), and heated at 85°C for 10 min (Karnopp et al., 2017; Shah et al., 2000). After cooling, the 0.02% yogurt culture was inoculated into each yogurt sample and then incubated at 40°C in a water bath (Lab Companion, Korea) until the pH decreased to 4.5. After fermentation was complete, the yogurt samples were stored at 4°C for 21 d.

The moisture and ash contents were measured by the AOAC method (2006). Crude protein and crude fat contents were determined by the Kjeldahl method and Soxhlet extraction, respectively (Jung et al., 2010). All tests were performed in five times and the results are expressed as means ± standard deviation (SD).

Yogurt samples with and without GME supplementation were collected every 2 h during fermentation and then every 7 d during the storage period for analysis of the antibacterial activity. Cell numbers of each sample were determined by the standard plate-count method. The media of agar plates used in this study were bromoceresol purple agar for LAB (Choi et al., 2014), Salmonella Shigella agar for S. Typhimurium, mannitol salt agar for S. aureus 1573, MacConkey sorbitol agar for E. coli O157:H4 FRIK125, tryptic soy agar for B. cereus KCCM11341 and E. sakazakii ATCC51329, and Oxford agar for L. monocytogenes H7962 (Chung et al., 2015; Sah et al., 2016). A 100-μL diluted yogurt sample was spread on agar plates. After incubation at 37°C for 2 d, the cell colonies were counted.

The sensory evaluation for consumer acceptability was conducted by 49 untrained panelists (28 female, 21 male, 21 to 60 years old) and approved by the Institutional Biosafety Committee (approval number: 2017-003) (Korea). Completely fermented yogurt samples were ripened for 1 d at 4°C. Two concentrations of GME were used for this evaluation: 0.5% and 1.0%. The color, flavor, texture, overall taste and acceptability were evaluated on a 7-point scale as follows: 1 = extremely dislike, 2 = very dislike, 3 = slightly dislike, 4 = neither like nor dislike, 5 = slightly like, 6 = very like, 7 = extremely like.

All results are expressed as means±SD. For statistical comparisons, the proximate composition was analyzed using one-way analysis of variance with Duncan’s posthoc test (p<0.05), and sensory evaluation was analyzed using the non-parametric Kruskal-Wallis/Mann-Whitney test (p<0.05). All statistical tests were performed using the SPSS 21.0 program (SPSS Inc., USA).

The compositions of the GME, yogurt, and yogurt supplemented with GME at 1% are presented in Table 1. According to Lee et al. (2013b), carbohydrates have the largest contribution in Panax ginseng at 67.3±0.0%, followed by crude protein (12.5±0.3%), moisture (5.9±0.6%), ash (3.7±0.2%), and crude fat (1.3±0.1%). Water is traditionally used as the main extracting solvent for ginseng concentrate. However, the general components of ginseng marc were found to differ when using alcohol as the extract solvent, according to the alcohol concentration (Kang et al., 2004; Kim, 2007). In this study, the crude fat and protein compositions of the GME were increased by 31.8% and 13.6%, respectively.

After fermentation, LABs number of yogurt (control) and yogurt supplemented with 1% GME were 9.2±0.3 and 9.1±0.2 Log CFU/mL, respectively. pH of yogurt (control) and yogurt supplemented with 1% GME were 4.5±0.0. Six strains of foodborne pathogens (3 gram-positive and 3 gram-negative strains) were inoculated into yogurt (control) and yogurt supplemented with 1% GME to determine the antibacterial effects. During the yogurt fermentation and storage periods, yogurt supplemented with 1% GME showed a lower cell number of pathogens than observed for the control yogurt sample for S. aureus 1573, B. cereus KCCM11341, L. monocytogenes H7962, E. coli O157:H4 FRIK125, and E. sakazakii ATCC51329, but not for S. Typhimurium 15 (Fig. 1). B. cereus KCCM11341 and E. sakazakii ATCC51329 were found to be the most sensitive pathogens tested, as their viable cell numbers decreased most rapidly when the yogurt sample supplemented with 1% GME was stored in a refrigerator for 1 d. Although almost pathogens are affected by the acidic pH, low temperature, and inhibitory peptides derived from milk (Beales, 2004; Hashemi et al., 2016), these antibacterial effects might be enhanced by GME supplementation. GME contains acidic polysaccharides, oligosaccharides, and polyacetylene (Kang et al., 2004; Seo et al., 2015). The addition of plant polyphenol such as ginseng, olive, and peanut sprouts in yogurt production have been reported to enhance antioxidant and antimicrobial effect (Cimo et al., 2013; O’Sullivan et al., 2014). In addition, polyacetylene, a lipid-soluble compound of ginseng marc, was previously reported to exert antimicrobial effects against S. aureus, Bacillus subtilis, Cryptococcus neoformans, and Aspergillus fumigatus (Fukuyama et al., 2012). Kim et al. (2014) reported that supplementation of red ginseng marc enhanced antibacterial activity to improve the meat quality of broiler chicken. Chung et al. (2015) also reported that the growth of Salmonella enterica and E. coli was inhibited in poultry litters treated with red ginseng marc.

Fig. 1. Anti-bacterial effects of yogurt supplemented with 1% Panax ginseng marc extract during fermentation and storage.

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A sensory evaluation was conducted to compare the consumer acceptability between control yogurt and yogurt supplemented with GME. As shown in Table 2, there was no significant difference in any of the sensory parameters (p<0.05) between yogurt and yogurt supplemented with 0.5% GME. The control yogurt showed the highest scores with respect to flavor (5.2), overall taste (5.6), and overall acceptance (5.5). The yogurt supplemented with 0.5% GME had the highest scores with respect to color (5.5), which was due to the color of the ginseng marc itself (yellow-brown). The yogurt supplemented with 1% GME showed low scores in the sensory evaluation, which might be related to the presence of triterpenoid peptides or propylene glycol in GME, which are known to cause the bitter taste of ginseng extract (Szente and Szejtli, 2004; Tamamoto et al., 2010). Various masking agents have been investigated to minimize this bitter taste. Many carbohydrate products such as α-, β-, and γ-cyclodextrin, glucose, lactose, honey, corn starch, oligosaccharide, sucrose, and xylose have been used to improve the taste and flavor of red ginseng extract (Chung et al., 2011; Lee et al., 2009). In this study, when 0.54% β-cyclodextrin and 4.7% corn syrup were added to reduce or mask the bitter taste of the GME, there was no significant difference (p<0.05) between samples supplemented with 0.5% GME and 1% GME (data not shown).

In conclusion, these results indicate that ginseng marc is a suitable yogurt supplement based on evaluations of consumer acceptance and the fact that yogurt supplemented with ginseng marc could effectively inhibit common foodborne pathogens such as S. aureus, B. cereus, L. monocytogenes, E. coli, S. Typhimurium, and E. sakazakii. The present study demonstrates that ginseng marc fermented with lactic acid bacteria can be a beneficial material for dairy products and can be used widely in the food industry as a supplement and natural antibacterial extract.