Characterization of spoilage bacterial communities in chilled duck meat treated by kojic acid

Wenfu Hou,Qiqi Yue,Wen Liu,Ji Wu,Yng Yi,Hongxun Wng,∗College of Food Science nd Engineering,Wuhn Polytechnic University,Wuhn,Huei,430023,Chin

bSchool of Biological and Pharmaceutical Engineering,Wuhan Polytechnic University,Wuhan,Hubei,430023,China

Keywords:

Kojic acid

Duck meat

Spoilage

High-throughput sequencing

ABSTRACT

Chilled duck meat is a popularly consumed meat in China but easily perishes at room/low temperature.Kojic acid is usually used as an antimicrobial.This study investigated the sensory scores,total bacteria number,and total volatile base nitrogen(TVB-N)value of chilled duck meat under kojic acid treatment during storage.The results presented that kojic acid suppressed the growth of spoilage bacteria and prolonged the shelf-life of chilled duck meat by at least two days.In further investigation,the bacterial community composition was explored through high-throughput sequencing.Pseudomonas spp.were the predominant spoilage bacteria after 6-day storage and were significantly suppressed by kojic acid.Besides,Brochothrix showed drug resistance and became the dominant bacteria in the kojic acid treated sample after 6-day storage.In conclusion,kojic acid maintained the sensorial,chemical,and microbial quality of duck meat stored at low temperatures. Meanwhile, kojic acid enriched the bacteria composition and showed strong antimicrobial activity against Pseudomonas spp.

1.Introduction

Fresh meat is considered as an ideal culture medium for various spoilage and pathogenic microorganisms because of its rich nutrients and sufficient water. China is the largest producer of duck meat,with 3 million tons annually[1,2].Raw duck meat is well accepted by consumers in China and Southeast Asia due to their deliciousness.However,the raw duck meat is easily contaminated by microorganisms owing to its characteristics,which resulted in spoilage.Different methods have been employed to preserve the fresh meats,and low temperature is the most important measure.However,spoilage is commonly observed in chilled duck meat[3].Thus,multi-preservative solution is necessary.The increasing anxiety about the safety of synthetic preservatives promoted the food industry to look for natural alternatives.Besides low temperature,the husbandry industry needs other valid measures with few side effects.Introducing natural antimicrobials is the hot spot in meats preservation,as it may suppress the growth of spoilage bacteria and prolong the shelf-life[4].

Kojic acid is a natural organic acid secreted by fungi such asAspergillus oryzeaandAspergillus flavus[5,6].Kojic acid and its derivatives have been widely used as food additive presenting interesting bioactivities for preventing enzymatic browning reaction,antiviral and antimicrobial activities among mushrooms,spinach and shrimps[24,25].Previous reports revealed that kojic acid maintained a strong inhibition against food-borne bacteria such asEscherichia coliandStaphylococcus aureusand couldn’t be utilized by bacteria[6,7].On the other hand,studies have shown that the acetic acid and kojic acid presented a good performance in inhibiting the growth of spoilage bacteria in chilled duck meat and maintaining its good quality among 50 different natural antimicrobials in 6 and 8 days,respectively[8].Meanwhile,chemical safety brings kojic acid wide employment in fresh products preservation.

The dominant species of spoilage in chilled meat includeEnterobacteria,Lactic acidbacteria(LAB),Pseudomonas,Brochothrix,andListeria,which easily reach a density of 8 log CFU/g[9].Although the role of microorganisms causing meat spoilage is well-studied, most of these studies focus on a few types of bacteria,lacking the com-prehensive map of bacteria numbers and community composition.Natural antimicrobials mainly influence the bacterial community in meat.Therefore,the dynamic bacterial community monitoring helps to reveal the antibacterial activities.

The purpose of this research is to investigate the antibacterial activity of kojic acid against meat spoilage bacteria and monitor the bacterial community variation during the spoilage with highthroughput sequencing method.

2.Materials and methods

2.1.Sample preparation

Samples of chilled duck meat were purchased from a supermarket in Wuhan,China.The samples were transported to the laboratory in polyethylene bags and strictly kept under 4◦C during transport.Chilled duck meat was washed with running sterile water and dried with the purpose of eliminating the contamination during package or transportation,followed by dividing into 4 groups.The 4 groups of chilled duck meat were immersed in 0.1%,0.4%,0.8% kojic acid(Aladdin,China)solution(experimental group)and sterile water(control group)for 5min under15◦C.The samples were then sealed and packed in 4◦C.All samples were performed in three replicates.

2.2.Sensory analysis

For the sensory assessment,chilled duck meat was assessed according to four scales: luster,5=very bright,4=bright,3=slightly bright,2=dull,1=very dull;odor,5=fresh,4=slightly fresh,3=slightly smelly,2=smelly,1=off-odor;morphology,5=very tight,4=tight,3=slightly tight,2=slightly loose,1=loose;and elasticity,5=very resilient,4=resilient,3=slightly resilient,2=soft,1=very soft.The elasticity was evaluated by pressing the surface of the meat with the panelists’ hands. Scores≥16 were determined as first-degree fresh;total scores around 12–16 were determined as second-degree fresh; scores under 12 were determined as spoilage.Panelists were trained,healthy individuals aging 18–40 years old.

2.3.Total volatile base nitrogen(TVB-N)value

TVB-N determination procedure was in accordance with the semi-microtitration method in Method for Analysis of Hygienic Standard of Meat and Meat Products(GB/T 5009.228-2016).Ten grams of samples were homogenized with 100mL sterile water.The TVB-N value of filtrate was evaluated[10].All samples were estimated in three replicates.

2.4.Bacterial enumeration

Twenty-five grams of samples were diluted with 225mL sterile saline solution and homogenized with a stomacher in 7 times/s for 2min.Plate count agar(PCA)was used and incubated at 37◦C,48h.All samples were estimated at three replicates.

2.5.Characterization of bacterial community

Samples were divided into five groups.Un-treated chilled duck meat at Day 0 was considered as negative control.Samples were treated with kojic acid at the concentration of 0,0.1%,0.4% and 0.8%.For each sample,three replicates were analyzed.

DNA of samples was extracted and confirmed using the Bacterial DNA Extraction Kit(Omega Bio-tek,Norcross,GA,U.S.)and 2% agarose gel electrophoresis.Barcoded primers targeting hypervariable V3 and V4 regions were employed to 16S rDNA PCR amplification,detailed information of the primers is presented inTable 1.PCR amplification was performed in a 20-μL reaction system with annealing temperature of 55◦C for 25 cycles.Amplicons were detected with 2% agarose gel electrophoresis,then depurated using an AxyPrepDNA PCR purification kit(AxygenBiosciences,USA).Sequencing libraries were produced using a TruSeq®DNA PCR-Free Sample Preparation Kit(Illumina,USA),and quantified using QuantiFluor-ST system(Thermo Scientific)and the Agilent Bioanalyzer 2100 system.

Table 1Primers used in this study.

Lastly amplicon was sequenced by Majorbio Company(Shanghai,China)on an Illumina HiSeq2500 platform that generated 250 bp paired-end reads.Raw sequences obtained from HiSeq2500 were processed on a free online platform of Majorbio I-Sanger Cloud Platform(www.i-sanger.com).

3.Results and discussion

3.1.The quality of chilled duck meat under kojic acid treatment

3.1.1.Sensory analysis

Sensory quality of chilled duck meat was showed at Fig.1.At Day 0,chilled duck meat had the best quality and the kojic acid had little effect as the sensory scores of the control group and treated group were very close.The sensory quality declined with storage period.At Day 8,the sensory scores of all samples(except for 0.8% kojic acid-treatment samples)were dropped to around 5 and the samples turned to very dull,off-odor,loosely and very soft,which was determined to be an unacceptable quality.However,kojic acid were capable to postpone the deterioration depending on concentration.It was noted that at Day 2,the sensory quality of the 0.8% kojic acid-treatment samples were almost equal to Day 0,the sensorial shelf-life of the 0.8% kojic acid-treatment sample was two days longer than the control.The results were in accordance with previously reported[11].

Fig.1.Sensory quality of chilled duck meat under kojic acid treatment during storage at 4◦C.

Fig.2.Total bacteria count of chilled duck meat samples under kojic acid treatment during storage at 4◦C.

3.1.2.Bacterial enumeration

Initial total bacterial counts of the 0.4% and 0.8% kojic acidtreatment chilled duck meat were lower than the 0.1% kojic acid-treatment samples and the negative control. Dramatic increasing trend was observed on negative control during the whole storage.However,kojic acid showed concentration-dependent inhibition to the bacteria.The total bacterial counts 7.0 log CFU/g is the upper limit of fresh meat according to the instruction of International Commission on Microbiological Specification for Foods[12].The negative control group reached to 7.0 log CFU/g at Day 5,while the 0.1% and 0.4% kojic acid-treatment samples reached to the limit at days 5 and 6,respectively.At Day 8,the number of bacterial counts of the 0.8% kojic acid-treatment sample increased to 6.5 log CFU/g.Nevertheless,the others reached to around 8 log CFU/g.It seemed that 0.8% kojic acid showed the best antimicrobial activity and delay the deterioration nearly 3–4 days(Fig.2).

3.1.3.TVB-N

Fig.3.TVB-N values of chilled duck meat samples under kojic acid treatment duringstorage at 4◦C.

TVB-N primarily includes trimethylamine(TMA),ammonia and dimethylamine(DMA)and was regarded as an important quality parameter of meat freshness[13].TVB-N re flects later stages of spoilage rather than freshness.Fig.3 showed TVB-N values of chilled duck meat samples under kojic acid treatment.The TVBN values of all samples remained at 3–5mg/100g in the first two days.Then,the average TVB-N of the untreated and 0.1% kojic acid groups sharply increased in the subsequent period and reached around 10mg/100g at Day 6.In contrast,TVB-N value in higher concentration groups maintained stable trends until Day 6.At Day 8,all groups recorded the highest TVB-N values.However,the 0.8% kojic acid group showed the lowest values among all samples(8.75mg/100g),and TVB-N value of it decreased 34.55% with significantly difference(P＜0.05)compared with the 0.4% kojic acid group(even its TVB-N value was still within the standard range of first-class freshness in 8 days),illustrating that 0.8% kojic acid presented better performance in maintaining the freshness of chilled duck meat.TVB-N are the products produced from protein decomposed by enzymes and bacteria.A high concentration of kojic acid showed inhibition to the activity to bacteria.Consequently,TVB-N values tended to reduce during the preservation period.

Consumption of food contaminated with spoilage bacteria or their toxins results in foodborne illness,which is a global public health issue.Thirty percent of foodborne illnesses were caused by bacteria[26].Therefore,it is necessary to establish novel and safe methods to suppress spoilage and pathogen bacteria.It was reported that kojic acid exhibited strong antimicrobial activity againstListeria monocytogenes,Bacillus subtilis,Staphylococcus aureus,Escherichia coliandSalmonella typhimuriumand even possessed anti-biofilm formation ability[14,15].According to the above chemical and biological analysis,the higher kojic acid concentration brought the stronger antimicrobial ability.The shelf-life of chilled duck meat under kojic acid treatment was prolonged for at least two days.In this study,it is recommended that 0.8% kojic acid be used as a preservative on chilled duck meat.

Fig.4.Ace index of OUT level(A)and Shannon index of OUT level(B).CK0d:Negative control group;CK6d,CL6d_L,CL6d M,CL6d_H were the samples treated with 0,0.1%,0.4%,0.8% kojic acid at days 6,respectively.*P＜0.05,**.P＜0.01,***.P＜0.001.

Fig.5.Relative abundance of microbiota at the genus level.Rows in the heat-map represent different genus-level phylotypes,while columns represent different samples.The color intensity is proportional to the abundance of OTUs in each row.(CK0d:Negative control group;CK6d,CL6d_L,CL6d_M,CL6d_H were the samples treated with 0,0.1%,0.4%,0.8% kojic acid at days 6,respectively).

3.2.Characterization of bacterial community

3.2.1.Sequencing analysis

The V3-V4 region of 16S rRNA was amplified and sequenced from 5 groups’samples(Negative control group,Day 6-0%-treated group,Day 6-0.1%-treated group,Day 6-0.4%-treated group,Day 6-0.8%-treated group).An average of 35455,39297,32768,35487,40021 high quality sequences were obtained respectively,with a sequence mean length of 445,449,449,449 and 449 bp.

Fig.6.Relative abundances of the OTUs in phylum level (A) and genus level(B).(The category “others”represented by a percent＜ 1%).CK0d:Negative control group;CK6d,CL6d_L,CL6d_M,CL6d_H were the samples treated with 0,0.1%,0.4%,0.8% kojic acid at days 6,respectively.

The ACE and Shannon index are used to re flect the community richness and diversity,respectively[16,17].The ACE index of negative control group was significantly higher(P＜0.01)than the Day 6 groups,indicating that after 6-day storage,community richness decreased significantly(Fig.4A).As for the Shannon index,the negative control group was significantly higher than the Day 6 groups.Day 6-0.4%-treated and Day 6-0.8%-treated groups were higher than the low concentration groups(Fig.4B),which had higher bacterial counts,indicating that the concentration of kojic acid influenced the bacterial community biodiversity.In the rarefaction analysis,individual rarefaction curves presented similarly before reaching a plateau.The results indicated that this level of sequencing was reasonable to use to re flect the most of bacterial phylotype[18].

3.2.2.Bacterial composition

The heat map clusters analysis(Fig.5)presented the relative abundance of genera through different depths of color.The heat map plot groups genera according to the similar abundance[19].Obviously,four Day 6 groups were clustered together,showing the similar bacteria composition.Compared to the Day 6-0.4%-treated group and the Day 6-0.8%-treated group,the relative abundance of theLactobacillus,Kurthia,Vagococcus,Enterococcus,Weissella,Arthrobacter,andPseudoclavibacteriumin the Day 6-0%-treated group and the Day 6-0.1%-treated group were lower.In contrast,Janthinobacteriumproliferated in the Day 6-0%-treated group and the Day6-0.1%-treated group(Fig.5).The heat map analysis visually showed the different bacterial communities among samples under different treatments.The bacteria of the negative control were mainly from the environment and slaughter processing.However,after storage,the antimicrobial compound or bacterial interaction may reduce the composition.

In order to further estimate the changes of the composition of bacterial community,the relative abundance of phylum(Fig.6A)and geneus(Fig.6B)were presented in bar charts.Compared to the negative control group,irrespective of with phylum or genus level,bacterial communities and compositions of Day 6 groups became more similar during storage.In the phylum level,three predominant phylums in negative control group wereProteobacteria(56.8%),Bacteroidetes(25.8%)andFirmicutes(10.3%).Six-day storage and the kojic acid treatment influenced the bacterial community compositions.The relative abundance ofProteobacteriadecreased with the increase of kojic acid concentration(from96.6% to 21.5%).However,Firmicuteswere proliferating with the increase of kojic acid concentration(from 1.20% to 77.5%).

At the genus level, three predominant genera in negative control group wereAcinetobacter(15.26%),Chryseobacterium(12.50%)andPseudomonas(11.21%).The bacteria contaminated the meat surface during the slaughter and transportation procedure.As mentioned earlier,at Day 6,the chilled duck meat turned into organoleptic spoilage.According to sensory analysis,bacterial enumeration,and the TVB-N value,0.1% kojic acid showed little effect on preservation,which is in accordance with the bacteria compositions results.The Day 6-0%-treated group and the Day 6-0.1%-treated group presented the similar bacterial compositions,in whichPseudomonasshowed the overwhelming predominance(over 90%),followed bySerratia(1.96%),andBrochothrix(2.61%),respectively.Notably,the relative abundance ofBrochothrix,Lactococcus,andSerratiaincreased with the increasing kojic acid concentration.It’s noted thatPseudomonasdecreased to 15.3% in the Day 6-0.8%-treated group compared to 61.0% in the Day 6-0.4%-treated group.Pseudomonas,Brochothrix,Lactococcus,andSerratiaare common spoilage bacteria in meat products and were universally detected by culture-dependent methods.In this study,the highlight finding was the gaping differences among the Day 6 groups.After 6-day storage,on the one hand,the chilled duck meat became spoiled,but kojic acid still retarded some bacterial growth.

Interaction between species might influence the composition[20].Pseudomonasspecies were the most common bacteria found in spoiled meat.The relative abundance ofPseudomonassharply reduced to 15.3% at Day 6.The reason may be thatPseudomonasspecies might be more sensitive to kojic acid.Some reports showed that kojic acid revealed better efficiency against Gram-negative bacteria than Gram-positive bacteria[14,21].

It’s proved that kojic acid damage the cell membrane integrity of Gram-negative bacteria more easily than that of Gram-positive bacteria[23].Kojic acid increased cell surdrophobicity by 2–3 folds,reducing the selective permeability of the cell membrane and destroying the structure of the cell membrane.Moreover,Russo reported that the increase ofBrochothrixspecies might suppress the growth ofPseudomonas[22].

4.Conclusion

In this study,bacterial community compositions of chilled duck meat during spoilage were observed and evaluated with meat samples stored at low temperatures and different kojic acid concentrations.The high-throughput sequencing data combined with chemical analysis and bacteria counts drew a comprehensive map of the microbiological and biochemical changes during spoilage.Kojic acid is capable of maintaining the quality of the shelf life(8 days)of the chilled duck meat,re flecting in the sensory quality,bacteria numbers and TVB-N values.The chilled duck meat under kojic acid treatment was prolonged by at least two days(nearly a 30% increase),which is conducive to maintain better quality and reduce the loss of producers to a certain degree.Additionally,kojic acid influenced bacteria community compositions of chilled duck meat and showed unignorable antimicrobial activity against some spoilage bacteria.Pseudomonasspecies were significantly suppressed by kojic acid.In the same time,Brochothrixshowed higher drug resistance and became the dominant bacteria in kojic acid treated samples after 6-day storage.Therefore,further study needs to be done to understand the active mechanism of kojic acid onPseudomonasspp.and the resistance mechanism ofBrochothrixto kojic acid.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgement

This study was supported by The Major Technological Innovation Projects of Hubei Province(2017ABA136).