Effect of Treatment with Heated Scallop Shell Powder on the Inactivation of Naturally Existing Bacteria and Listeria monocytogenes Inoculated on Chicken Meat

This study investigated the efficacy of heated scallop shell powder (HSSP) treatment in preserving chicken thigh meat. Chicken thigh meat was treated with HSSP slurry (1% and 5%) for 60 min, and the variation in aerobic bacteria and coliform populations was assessed during refrigerated storage (10 °C). There was a substantial increase in aerobic bacteria, reaching nearly 7 log10 colony forming unit (CFU)/g following 7 days of refrigeration, in the untreated chicken meat. Conversely, the aerobic bacterial population of the HSSP-treated chicken was <5 log10 CFU/g. Coliform growth in the untreated chicken reached over 5 log10 CFU/g following 7 days. In contrast, the coliform population of the HSSP-treated chicken did not reach 5 log10 CFU/g at 1% HSSP concentration; it was suppressed to <4 log10 CFU/g at 5% concentration. Listeria monocytogenes, which can grow at low temperatures, was inoculated into the chicken meat (5 log10 CFU/g) treated with alcohol, which was followed by HSSP. In the untreated chicken, L. monocytogenes increased to 9 log10 CFU/g even when refrigerated for 7 days. However, in the chicken treated with 5% HSSP, L. monocytogenes was suppressed to approximately 3 log10 CFU/g. These findings reveal that HSSP treatment is an effective method for disinfecting meat, inhibiting bacterial growth, and enhancing preservation.


Introduction
Currently, although some scallop shells are repurposed for use in food additives and paints, the majority of them are categorized as industrial waste.In areas where scallops are harvested, the heavy metals in internal organs and the odor emanating from discarded shells have become notable pollution issues [1][2][3].Sawai et al. [4,5] demonstrated that heating scallop shells at 800 • C or higher results in the conversion of calcium carbonate (CaCO 3 ), the primary component of the shell, into calcium oxide (CaO), which exhibits antimicrobial properties.Application of these heated shell powders to food products can extend their shelf life.Furthermore, using discarded seashells as a useful resource can mitigate pollution problems.Similarly, other seashells such as oyster shells [6][7][8][9], surf clam shells [10], mussels [11,12], and blood cockle shells [13] have been found to exhibit antimicrobial properties following heat treatment.
There are several reports on the application of heated shell powder in the food sector, including fresh vegetables [32][33][34][35], fruits [36], sausages [37], fish [38], and food packaging materials [39].Specifically, these powders are as effective as or more effective than sodium hypochlorite (NaOCl) treatment in terms of disinfecting and preserving fresh vegetables.However, few reports regarding the effects of these powders on the treatment and preservation of meat [15,40] have been found.Cagri-Mehmetoglu [40] reported that HSSP treatment significantly reduced the growth of Listeria monocytogenes or Salmonella enteritidis inoculated on chicken wings.Ro et al. [15] demonstrated that storing HSSP-supplemented meat patties with beef at 10 • C completely inhibited the growth of three pathogenic Escherichia coli strains.Therefore, in this study, as an application of HSSP on meat, the antimicrobial effect of HSSP treatment and its preservation effect during refrigerated storage on bacteria originally present in chicken thighs and inoculated L. monocytogenes were investigated.

HSSP
Natural Japan Co., Ltd.(Abashiri, Hokkaido, Japan) prepared HSSP (particle size, 4 µm) via heat treatment at 1200 • C.After opening the package containing the powder, it was stored in a desiccator.

HSSP Treatment of Chicken Thighs 2.2.1. Preparation of Samples and Inoculation with Pathogens
Chicken thigh meat cut into approximately 20 g pieces was purchased from a city supermarket.The cut chicken meat was used without any pretreatment (chicken meat samples) to investigate the naturally existing total aerobic bacteria and coliform counts.The chicken meat sample without HSSP treatment, shown in Section 2.2.2, was used as a control when examining chicken thighs for naturally existing bacteria.
The bacteria were inoculated using the following procedure: L. monocytogenes ATCC (American Type Culture Collection) 19114, the inoculum organism was stored in a 10% glycerol solution at −80 • C.Then, the bacterial cells were thawed and preincubated in a nutrient broth (Eiken Chemicals Co., Ltd., Tokyo, Japan) at 37 • C for 20 h, washed (3000 rpm, 10 min), and resuspended in sterile 0.85% saline at a concentration of 10 9 colony forming unit (CFU)/mL.The cut chicken meat (approximately 500 g) was soaked in 500 mL of 70% ethanol for 15 min, transferred to a colander, and allowed to stand on a clean bench for 1 h.The colander was soaked in 500 mL of sterile water to remove the alcohol remaining in the chicken meat (15 min).Next, the colander containing chicken meat was soaked in sterile water (500 mL) and inoculated with 1 mL of the bacterial suspension of L. monocytogenes ATCC 1911 (approximately 10 9 CFU/mL) for 15 min.The colander containing the chicken meat was drained for 20 min, and L. monocytogenes was allowed to settle.The L. monocytogenes-inoculated chicken meat was used for sampling in this study.The abovementioned processes were performed on a clean bench at room temperature (25 • C ± 2 • C).The L. monocytogenes-inoculated chicken meat without HSSP treatment, shown in Section 2.2.2, was used as a control when inoculated with L. monocytogenes.

HSSP Treatment
The HSSP treatment was performed according to the protocol described by Yamanaka et al. [7].The HSSP was added to a sterilized stainless-steel vessel containing sterilized water (2.8 L) at a concentration of 1 wt/v% or 5 wt/v% and agitated using a magnetic stirrer at 500 rpm.A disinfected colander containing chicken meat samples or L. monocytogenes-inoculated chicken meat samples (approximately 100 g) was immersed in the HSSP slurry for 60 min.Subsequently, the HSSP-treated chicken meat samples were drained for 1 h.The abovementioned procedure was performed at 25 Approximately 20 g of the HSSP-treated or untreated chicken meat was sampled and homogenized with 100 mL of sterile physiological saline for 1 min using a stomacher (Pro Media, SH-IIM; Elmex Ltd., Tokyo, Japan).Then, a 1 mL aliquot of the solution in a stomacher filter bag (Elmex) was serially diluted with sterile 0.85% saline and incubated with Standard Methods Agar (Eiken Chemicals), X-GAL Agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and PALCAM Listeria-Selective Agar (Merck KGaA, Darmstadt, Germany) to count the total aerobic bacteria, coliforms, and Listeria, respectively.After incubation at 37 • C for 48 h, the bacterial colonies were counted.This was set as day 0.
The drained chicken meat was stored in a polyethylene bag (Ziploc ® , Asahi Kasei Home Products Co., Ltd., Tokyo, Japan) at 10 • C to investigate the storage quality of the chicken meat after treatment.Then, the populations of aerobes, coliforms, and Listeria present in chicken meat after 3, 5, and 7 days of storage were estimated using the procedure described above.

Color Measurement
The color change was measured on days 0, 3, 5, and 7 of storage at 10 • C for the untreated and HSSP-treated chicken meat samples without alcohol treatment.The Hunter color values (L*, a*, and b*) of the chicken meat surface were measured using a colorimeter (CR-400, Konica Minolta, Inc., Tokyo, Japan) at three different regions on the chicken meat's surface.

Statistical Analysis
All experiments were performed in triplicate (n = 3).Data are presented as mean ± standard error.Furthermore, data were subjected to a two-way analysis of variance with Tukey's test using BellCurve for Excel ® version 2.0.3 (Social Survey Research Information Co., Ltd., Tokyo, Japan); p < 0.05 was considered statistically significant.

Naturally Existing Bacteria
Variations in the aerobic and coliform populations of the chicken meat following HSSP treatment are shown in Tables 1 and 2, respectively.The population of aerobic bacteria in the untreated chicken meat increased by one order of magnitude to over 5 log 10 CFU/g on day 3, even during refrigeration, and increased by approximately two orders of magnitude to reach 6.7 log 10 CFU/g on day 7. Conversely, the HSSP treatment (1% and 5%) maintained levels 1-3 orders of magnitude lower than those of the untreated group, even after 7 days.No treatment (Control) 4.6 ± 0.3 a,A 5.6 ±1.4 a,AB 6.5 ± 1.3 a,B 6.7 ± 1.0 a,B HSSP 1% 3.9 ± 0.2 a,A 3.5 ± 0.4 b,A 6.1 ± 0.1 a,B 5.6 ± 0.1 b,C HSSP 5% 3.8 ± 0.4 a,A 4.2 ± 0.1 b,AB 5.1 ±1.7 b,B 3.8 ± 1.9 c,AC Abbreviation: HSSP, heated scallop shell powder.Means in the same column followed by different letters ( a-c ) are significantly different (p < 0.05).Means in the same row followed by different letters ( A-C ) are significantly different (p < 0.05).No treatment (Control) 3.9 ± 0.8 a,A 5.0 ± 1.7 a,B 5.4 ± 1.6 a,B 5.4 ± 1.1 a,B HSSP 1% 3.5 ± 0.4 ab,A 3.5 ± 0.2 b,A 4.3 ± 0.1 b,B 4.8 ± 0.1 a,B HSSP 5% 2.9 ± 0.3 b,A 3.2 ± 1.0 b,AB 3.9 ± 0.4 b,B 3.6 ± 1.1 b,B Abbreviation: HSSP, heated scallop shell powder.Means in the same column followed by different letters ( a,b ) are significantly different (p < 0.05).Means in the same row followed by different letters ( A,B ) are significantly different (p < 0.05).
The population of coliforms in the untreated chicken exceeded 5 log 10 CFU/g by day 7. On the contrary, in the HSSP treatment, the coliforms increased over time but remained < 5 log 10 CFU/g at 1% and <4 log 10 CFU/g at 5%, even after 7 days (Table 2).
Based on previous reports, the antimicrobial effects of CaO are caused by its alkalinity (pH ≥ 12) as a result of hydration.In addition to alkalinity, reactive oxygen species (ROS) released from CaO are considered another antimicrobial mechanism [41], and their formation has been detected in HSSP, including CaO as the main component [14,42].ROS are highly oxidizing free radicals with significant reactivity to numerous biomolecules [43].These ions can be lethal to bacterial cells, which is probably because of the damage they cause to bacterial membranes, DNA, and proteins.

HSSP 1% HSSP 5%
Cagri-Mehmetoglu [40] reported that chicken wings inoculated with L. monocytogenes and S. enteritidis at 8 log 10 CFU/g and treated with HSSP showed a reduction of three to five orders of magnitude, respectively.Yamanaka et al. [7] prepared fried chicken using chicken thighs treated with heated oyster shell powder; sensory evaluation revealed that the fried chicken prepared using chicken thighs treated with heated oyster shell powder was softer and tastier than that prepared using untreated chicken thighs.Furthermore, Mine et al. [47] reported that adding heated oyster shell powder to minced meat strengthened the binding power and suppressed weight loss after heating.As mentioned previously, the sensory evaluation was satisfactory, and no serious problems with the HSSP-treated meat were anticipated at this stage.

Conclusions
In this study, HSSP treatment effectively inhibited naturally existing bacteria and the inoculated L. monocytogenes in chicken thigh meat during cold storage (~7 days), indicating that HSSP treatment is a valuable meat disinfection method.However, there is growing concern about the accumulation of used antimicrobials and antiseptics in rivers and other sources, the development of drug resistance in environmental microorganisms, and the spread of drug-resistant genes [48][49][50][51].The heated shell powder, whose main component is CaO, exhibits antimicrobial activity, and it is used to control microorganisms in food and the environment.When released into the environment, the heated shell powder absorbs CO 2 and returns to its original shell component, CaCO 3 , which has no antimicrobial activity.Then, it returns to the sea through rivers.It may also be used as a component of shellfish and may be caught and landed again.Shells can be regarded as a circulating antimicrobial agent, which is a material associated with the SDGs.Apart from calcium fortification, heated shell powder can help food producers and consumers produce and consume wholesome food with a good taste.

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Abbreviation: HSSP, heated scallop shell powder.Table 4. Changes in the Hunter color values of chicken meat after HSSP treatment and storage at 10 °C.Hunter Color Values Treatment Storage Time Day 0 Day 3 Day 5 Day 7

Table 1 .
Variation in naturally existing total aerobic bacterial population (log 10 CFU/g) of chicken meat after HSSP treatment and storage at 10 • C.

Table 2 .
Variation in naturally existing total coliform population (log 10 CFU/g) of chicken meat after HSSP treatment and storage at 10 • C.

Table 4 .
Changes in the Hunter color values of chicken meat after H °C.

Table 4 .
Changes in the Hunter color values of chicken meat after HSSP treatment and storage at 10 °C.

Table 3 .
Changes in color values of chicken meat after HSSP treatment and storage at 10 °C.