Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1229-1153(Print)
ISSN : 2465-9223(Online)
Journal of Food Hygiene and Safety Vol.35 No.3 pp.279-283

Antimicrobial Effects of EcoCal® and GF Bactostop® Formulated in Emulsified Sausages against Lactic Acid Bacteria

Yewon Lee1, Sunghee Cheong2*, Yohan Yoon1,3*
1Department of Food and Nutrition, Sookmyung Women’s University, Seoul, Korea
2Geo Food Tec Institute, Seongnam, Korea
3Yoon Biotech, Seoul, Korea

Authors Sunghee Cheong and Yohan Yoon corresponded equally to this work.

*Co-correspondence to: Sunghee Cheong, Geo Food Tec Institute, Seongnam 13631, Korea Tel: +82-31-714-0205, Fax: +82-31-714-1305 E-mail:
*Co-correspondence Author to: Yohan Yoon, Sookmyung Women’s University, Seoul 04310, Korea Tel: +82-2-2077-7585, Fax: +82-2-710-9479 E-mail:
April 1, 2020 April 27, 2020 May 12, 2020


In this study we evaluated the antimicrobial effects of EcoCal® (calcium oxide) and GF Bactostop® (organic acids mix) in sausages during storage at 10°C. The sausages were formulated with 0.1% EcoCal® (0.1ECO), 0.1% EcoCal®+0.5% GF Bactostop® (0.1ECO+0.5GF), 0.2% EcoCal® (0.2ECO), and 0.2% EcoCal®+0.5% GF Bactostop® (0.2ECO+0.5GF). Total aerobic and lactic acid bacteria in the sausages were enumerated on tryptic soy agar and Lactobacilli MRS agar, respectively, during storage at 10°C for 10 weeks. The 0.1ECO+0.5GF showed the most effective antimicrobial effects on the sausages, and 0.1ECO showed the second most effective antimicrobial effect. Total aerobic bacterial cell counts gradually increased in the control, 0.2ECO, and 0.2ECO+0.5GF groups, but cell growth was generally inhibited in 0.1ECO by approximately day 42 (P<0.05) and 0.1ECO+0.5GF by approximately day 49 (P<0.05). Lactic acid bacterial cell counts gradually increased in the control, 0.2ECO, and 0.2ECO+0.5GF groups, but the lactic acid bacteria growth was inhibited in 0.1ECO by approximately day 49 (P<0.05) and in 0.1ECO+0.5GF by approximately day 64. These results suggest that using 0.1% EcoCal®+0.5% GF Bactostop® in sausage formulation is useful for inhibiting lactic acid bacteria growth, thereby extending the shelf-life of the sausage product.


    Geo Food Tec Institute

    Sausages are manufactured from fresh ground meat, such as beef, chicken, and pork1). Sausages are produced through a series of processes; raw meat and additives are chopped, mixed, and emulsified, followed by the mixture being stuffed into the casing for cooking2,3). During processing, sausages are at risk of being exposed to and contaminated by various bacteria, especially during the vacuum-packaging process4,5). During the storage of vacuum-packaged sausages, lactic acid bacteria can cause spoilage6). Holzapfel7) reported that 68% and 16.9% of the total lactic acid bacteria examined in their study were Lactobacillus sake and Lactobacillus curvatus, respectively, for vacuum-packaged processed meats. Lactic acid bacteria in meat products contribute to organoleptic downgrading of the meat products, and produce souring and gas8). Therefore, lactic acid bacteria in sausages must be controlled to maintain high quality.

    EcoCal® is a commercial antimicrobial composed primarily of calcium oxide. Calcium oxide, a primary component of shell powder, is a food preservative with excellent antimicrobial activity9). This component also makes it useful as an acid regulator and dough conditioner10). GF Bactostop® is an organic acid mix composed of salt, ascorbic acid, sodium acetate, calcium lactate, tri-sodium citrate, and citric acid, and it can be used for food formulation as an antimicrobial. Mani- López et al.11) reported that acetic acid, diacetates, acetate, and dehydroacetic acid showed obvious antimicrobial activities against bacteria and yeast in meat products. Additionally, Gálvez et al.12) showed that the antimicrobial activities of bacteriocins increased when combined with organic acids.

    Therefore, the objective of this study was to evaluate the antimicrobial effects of EcoCal® and GF Bactostop® on lactic acid bacteria in sausages.

    Materials and Methods

    Sausage preparation

    Lean pork and pork fat were purchased from Dodram Food Co., Ltd. in Seongnam, Korea. They were ground separately through a grinder with 5 mm hole plate, and common ingredients (ground meat and ice, some additives and spices) and two types of commercial antimicrobial preservatives [Calcium oxide (EcoCal®, Micro-Tech Foods Ingredients, Inc., New Taipei city, Taiwan) and/or organic acids mix (GF Bactostop®, Pacovis Co., Stetten, Switzerland)] were measured and finely chopped to the ratios presented in Table 1, and the manufacturing process is shown in Fig. 1. The pre-ground pork, fat, and ice with 1.2% salt, 0.2% nitrite pickle salt (5% nitrite and 95% salt), 0.25% sodium pyrophosphate, and other ingredients were finely chopped using a bowl chopper (Type TK 20L, 2000S, Kilia Fleischwarenfabrik, Kiel, Germany) according to the formulations (Table 1) and manufacturing process of the sausage (Fig. 1). The finely emulsified sausage batter was stuffed into collagen casing #26 (Devro, INC, Sandy Run, SC, USA). The stuffed sausages were placed in a smoke chamber (Type IMAGO F3000, Metatek Co., Ltd., Nonsan, Korea) at 55ºC for 20 min, dried at 55°C for 10 min, and smoked at 60°C for 15 min. The sausages were then primary-heated at 72°C for 20 min, secondary-heated at 78°C for 10 min, then cooled by washing with cold water. The sausages were then vacuum-packaged, post-pasteurized at 90°C for 10 min, then cooled in an ice/water bath. Manufactured sausages were stored at 10°C for 10 weeks for microbiological analysis.

    Microbiological analysis

    Overall, 25 g of the sausage samples were removed aseptically from the vacuum-packaged samples, placed in a filter bag (3M, St. Paul, MN, USA) containing 50 mL of 0.1% buffered peptone water (BPW; Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and pummeled in a pummeler (Interscience, St. Nom, France) for 1 min13). The homogenates were serially diluted with 0.1% BPW, and the diluents were plated on tryptic soy agar (TSA; Becton, Dickinson and Company) and Lactobacilli MRS agar (Becton, Dickinson and Company) for the isolation of total aerobic bacteria and lactic acid bacteria, respectively. All plates were incubated at 37°C for 24 h. The number of typical colonies on the plates were manually counted.

    Statistical analysis

    Bacteria cell count data were analyzed using a general linear model procedure with SAS® version 9.3 (SAS Institute, Cary, NC, USA). Least square (LS) means were used for mean comparisons among the treatment groups by pairwise t-test at α=0.05.

    Results and Discussion

    Although there was some sample variation in total bacterial cell counts, the cell counts of total aerobic bacteria in the sausages gradually increased with increasing storage time (Table 2). These variations in cell counts were likely due to sample variations14). In general, 0.1ECO and 0.1ECO+0.5GF groups had lower (P<0.05) cell counts for total aerobic bacteria compared to the other groups. Total bacterial cell counts gradually increased in control, 0.2ECO, and 0.2ECO+0.5GF groups, but bacterial growth was generally inhibited in 0.1ECO by approximately day 42 (P<0.05) and 0.ECO+0.5GF by approximately day 49 (P<0.05). The lactic acid bacterial cell counts in the sausages increased as storage time increased (Table 3). In addition, 0.1ECO and 0.1ECO+0.5GF groups had lower (P<0.05) cell counts in lactic acid bacteria than the other groups. In these treatments, the lactic acid bacterial cell counts were below the detection limit (0.5 Log CFU/g) by day 49. Therefore, 0.1ECO and 0.1ECO+0.5GF groups were analyzed for an additional three weeks. At the end of the storage period, there were significant differences in the cell counts of lactic acid bacteria between the two groups. The 0.1ECO+0.5GF group had cell counts of lactic acid bacteria below the detection limit by day 64, but the cell counts of lactic acid bacteria in the 0.1ECO group were below the detection limit by day 49, then increased to 4 Log CFU/g. These results showed that the addition 0.1% of EcoCal® and 0.5% of GF Bactostop® may be effective in inhibiting lactic acid bacteria growth in sausages. A study by El-Aziz et al.15) showed that CaO, a major component of EcoCal®, had antimicrobial effects on minced meat, and this result is similar to our result. The antimicrobial effects of CaO are caused by active oxygen and alkaline pH, which in turn cause the destruction of the bacterial cell membranes16). However, the antimicrobial effect of CaO were not concentration dependent. According to Bodur et al.17) the antimicrobial effect of 0.05% CaO on E. coli was higher than that of 0.1% CaO, but antimicrobial effects on Listeria monocytogenes and Salmonella were showen at 0.1% CaO. This means that the antimicrobial effect of Ecocal may not be concentration dependent for total aerobic bacteria. In addition, Schirmer and Langsrud18) reported that the organic acids mix, which is similar to GF Bactostop®, showed greater antimicrobial activity when combined with 50% CO2 modified atmosphere-packed (MAP) during storage. Therefore, the use of GF Bactostop® in vacuum-packaged sausages may increase the antimicrobial activity of vacuum-packaging, resulting in the extension of shelf-life. Insoluble CaO had been reported in several studies to increase the absorptivity as well as the solubility in acid through reactions with organic acid19-21). Therefore, a mixture of CaO and organic acids mix may increase the solubility and antimicrobial activity of lactic acid and total aerobic bacteria.

    In conclusion, a mixture of 0.1% of EcoCal® with 0.5% of GF Bactostop® in sausages showed the highest antimicrobial effects on lactic acid bacteria, which are usually spoilage bacteria in sausages, and 0.1% of EcoCal® showed the second highest antimicrobial effect. Therefore, the use of 0.1% EcoCal® and a mixture of 0.1% of EcoCal® and 0.5% GF Bactostop® may be useful in inhibiting lactic acid bacteria growth in sausages, which can extend the shelf-life of the sausage.


    본 연구는 EcoCal® (산화 칼슘) 및 GF Bactostop® (유 기산염 믹스)을 소시지에 사용하였을 때 항균 효과를 확 인하였다. 소시지 제조 시, 대조군과 0.1% EcoCal® (0.1ECO), 0.1% EcoCal®+0.5% GF Bactostop® (0.1ECO + 0.5GF), 0.2% EcoCal® (0.2ECO) 및 0.2% EcoCal®+0.5% GF GF Bactostop® (0.2ECO+0.5GF) 등 총 5개 군을 첨가 하여 소시지를 제조하였다. 제조가 완료된 소시지를 진공 포장하고 10주 동안 10°C에서 저장하며 7-10일 마다 시료 를 균질화 하고 tryptic soy agar 및 Lactobacilli MRS agar 에 도포하여 호기성 일반세균 및 젖산균을 각각 확인하였 다. 그 결과, 0.1ECO+0.5GF 첨가한 소시지가 가장 효과 적인 항균 효과를 나타냈으며, 0.1ECO 첨가 소시지가 두 번째로 효과적인 항균 효과를 나타냈다(P<0.05). 일반세균 수는 대조군, 0.2ECO 및 0.2ECO+0.5GF 첨가 소시지에서 는 저장 후 42일까지 점차 증가했지만(P<0.05), 0.1ECO 및 0.1ECO+0.5GF 첨가 소시지에서는 저장 후 49일까지 일반세균의 생장이 억제되었다(P<0.05). 젖산균 수는 대조 군, 0.2ECO 및 0.2ECO+0.5GF 첨가 소시지에서 약 49 일 까지 증가하였으나(P<0.05), 0.1ECO 및 0.1ECO+0.5GF 첨 가 소시지에서 젖산균 생장이 억제되었다. 본 결과는 소 시지 제형에 0.1% EcoCal®+0.5% GF Bactostop®을 첨가 하면 젖산균의 생장을 억제하여 소시지의 보존 기한을 연 장시키는데 유용할 것으로 판단된다.


    This research was financially supported by Geo Food Tec Institute.



    Manufacturing process of emulsified sausages with different levels of Ecocal® and GF Bactostop®.


    Formula of experimental vacuum-packaged sausages (Unit: %)

    Total aerobic bacterial cell counts (mean±SD; Log CFU/g) in vacuum-packaged sausages during storage at 10°C for 71 days

    Lactic acid bacterial cell counts (mean±SD; Log CFU/g) in vacuum-packaged sausages during storage at 10°C for 71 days


    1. Sachindra, N.M., Sakhare, P.Z., Yashoda, K.P., Rao, D.N., Microbial profile of buffalo sausage during processing and storage. Food Control, 16, 31-35 (2005).
    2. Hugo, C.J., Hugo, A., Current trends in natural preservatives for fresh sausage products. Trends. Food Sci. Technol., 45, 12-23 (2015).
    3. Jeong, Y., Han, Y., Effect on the emulsification stability and quality of emulsified sausages added with Wanggasi-Chunnyuncho (Opuntia humifusa f. jeollaensis) fruit powders. Food Sci. Anim. Resour., 39, 953-965 (2019).
    4. Park, H.J., Go, E.K., Wee, S.H., Yoon, H.C., Heo, E.J., Kim, Y.J., Lee, H.S., Moon, J.S., Analysis of foodborne pathogenic contamination of cooked hams and sausages in Korean processing facilities. Food Sci. Anim. Resour., 32, 103-111 (2012).
    5. Kim, S., Lee, H. J., Kim, M., Yoon, J.W., Shin, D.J., Jo, C., Storage stability of vacuum-packaged dry-aged beef during refrigeration at 4oC. Food Sci. Anim. Resour., 39, 266 (2019).
    6. Wang, T., Guo, H., Zhang, H., Ren, F., Zhang, M., Ge, S., Luo, H., Zhao, L., Dynamics of bacterial communities of lamb meat packaged in air and vacuum pouch during chilled storage. Food Sci. Anim. Resour., 39, 209 (2019).
    7. Holzapfel, W.H., 1998. The gram-positive bacteria associated with meat and meat products, In. A. Davies and R. Board (Eds.), The microbiology of meat and poultry. Blackie Academic & Professional, London, UK, pp. 35-84.
    8. Huis in’t Veld, J., Microbial and biochemical spoilage of foods: an overview. Int. J. Food Microbiol., 33, 1-18 (1996).
    9. Sawai, J., Shiga, H., Kojima, H., Kinetic analysis of the bactericidal action of heated scallop-shell powder. Int. J Food Microbiol., 71, 211-218 (2001).
    10. Ministry of Food and Drug Safety, (2019, December 19). Korean Food Code. Retrieved from
    11. Mani-López, E., García, H.S., López-Malo, A., Organic acids as antimicrobials to control Salmonella in meat and poultry products. Food Res. Int., 45, 713-721 (2012).
    12. Gálvez, A., Abriouel, H., Lucas, R., José, M., Burgos, G., 2011. Bacteriocins for bioprotection of foods. In M. Rai, & M. Chikindas (Eds.), Natural antimicrobials in food safety and quality. Oxfordshire, UK: CAB International. pp. 39-61.
    13. Lee, H.J., Jung, N.Y., Park, S.H., Song, S.M., Kang, S.I., Kim, J.S. Heu, M.S., Characteristics and preparation of calcium acetate from butter clam (Saxidomus purpuratus) shell powder by response surface methodology. J. Kor. Soc. Food Sci. Nutr., 44, 888-895 (2015).
    14. Heo, E.J., Ko, E.K., Seo, K.H., Kim, Y.J., Park, H.J., Wee, S.H., Moon, J.S., Validation of PCR and ELISA test kits for identification of domestic animal species in raw meat and meat products in Korea. J. Food Hyg. Saf., 29, 158-163 (2014).
    15. El-Aziz, D.M.A., Yousef, N.M., Antimicrobial effects of calcium oxide nanoparticles and some spices in minced meat. ARC J. Animals. Veteri. Sci., 3, 38-45 (2017).
    16. Dizaj, S.M., Lotfipour, F., Barzegar-Jalali, M., Zarrintan, M. H., Adibkia, K., Antimicrobial activity of the metals and metal oxide nanoparticles. Mater. Sci. Eng., 44, 278-284 (2014).
    17. Bodur, T., Yaldirak, G., Kola, O., Cagri-Mehmetoglu, A., Inhibition of Listeria monocytogenes and Escherichia coli O157:H7 on frankfurters usıng scallop-shell powder. J. Food Safety., 30, 740-742 (2010).
    18. Schirmer, B.C., Langsrud. S., A dissolving CO2 headspace combined with organic acids prolonged the shelf-life of fresh pork. Meat Sci., 85, 280-284 (2010).
    19. Guthrie, H.A., 1971. Introductory Nutrition, (2nd Eds). Mosby, S. Louis, MO, USA, pp. 109-111.
    20. Lee, J., Lee, H., Lee, S., Kim, S., Ha, J., Choi, Y., Oh, H., Kim, Y., Lee, Y., Seo, K., Yoon, Y., Quantitative microbial risk assessment for Campylobacter jejuni in ground meat products in Korea. Food Sci. Anim. Resour., 39, 565 (2019).
    21. Park, S.H., Jang, S.J., Lee, H.J., Lee, G.W., Lee, J.K., Kim, Y.J., Kim, J.S., Heu, M.S., Optimization of calcium acetate preparation from littleneck clam (Ruditapes philippinarum) shell powder and its properties. Kor. J. Food Sci. Technol., 47, 321-327 (2015).