Analysis of Dominant Spoilage Bacteria in Beijing Sausages

Wenhui Liang

†

, Fang Wang

†

, Ting Li

, Jiayun Kang

, Yu Hao

, Siyu Shi

and Jinghua Qi

1,2 g

Beijing Key Laboratory of Agricultural, Product Detection and Control of Spoilage Organisms and Pesticide Residue,

Beijing University of Agriculture, Beijing, China

Beijing Innovation Consortium of Swine Research System, Beijing, China

2816155579@qq.com,

g,*

abc960718@sina.com

Corresponding author

†

Contributed equally to this work

Keywords:

Beijing Sausages, Isolation, Identification, Spoilage Bacteria.

Abstract:

Low temperature-heated meat products, which are popular among consumers because of their unique flavor,

texture, and nutrition, have a relatively short shelf life due to the low sterilization temperature and are therefore

prone to spoilage. The profile of bacteria associated with spoilage of Beijing sausages is here summarized

through isolation, identification and spoilage microflora analysis. The main spoilage bacteria isolated and

identified from Beijing sausages were one genus of Pseudomonas sp., one genus of Staphylococcus sp. and

two genera of Brochothrix sp. By denaturing gradient gel electrophoresis (PCR-DGGE) analysis, we

identified 13 spoilage bacteria of Beijing sausages, of which Pseudomonas sp. was the most abundant.

1 INTRODUCTION

Beijing sausage is a low-temperature meat product

which has the advantages of simple production

technology, low sterilization temperature, slight

protein denaturation, compact meat quality, elasticity,

and delicious taste. However, due to the

characteristics of the processing technology, the

heating temperature of Beijing sausages fails to

effectively sterilize the product, and the nutrition

profile provides good conditions for the growth of

microorganisms. Thus, Beijing sausages are prone to

spoilage during storage, transportation, and sale, so

the shelf life of these products is often short, greatly

limiting the development of such products.

The key to extending the shelf life of meat

products is to study the species and characteristics of

the dominant bacteria involved in spoilage, and then

https://orcid.org/0000-0001-8982-7693

https://orcid.org/0000-0002-0438-6786

https://orcid.org/0000-0002-5644-5768

https://orcid.org/0000-0003-0174-9710

https://orcid.org/0000-0001-9311-3562

https://orcid.org/0000-0002-2461-4643

https://orcid.org/0000-0001-9204-3697

select effective preservation measures. In recent

years, the species and characteristics of spoilage

bacteria in some meat products have been studied in

domestic and foreign literature (Adams et al., 2007;

Danilo et al., 2009; Doulgeraki et al., 2012; Liu et al.,

2010; Peirson et al., 2003), collectively showing that

the types of dominant bacteria involved in spoilage

vary between meat products. Here, the bacterial

distribution after spoilage in Beijing sausages was

analyzed. The dominant bacteria causing the spoilage

were isolated, purified, and identified to provide the

theoretical basis for taking effective anti-spoilage

measures and thus extending the shelf life of Beijing

sausages.

Liang, W., Wang, F., Li, T., Kang, J., Hao, Y., Shi, S. and Qi, J.

Analysis of Dominant Spoilage Bacteria in Beijing Sausages.

DOI: 10.5220/0011182900003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 84-88

ISBN: 978-989-758-595-1

2 MATERIALS AND METHODS

2.1 Media

Nutrient Broth (NB) was made by dissolving 10 g

peptone (Beijing Oboxing Biotechnology Co., Ltd,

China), 3 g beef extract (Beijing Oboxing

Biotechnology Co., Ltd, China), and 5 g sodium

chloride in 1 L distilled water. pH was adjusted to 7.4

with 1 mol/L sodium hydroxide solution, then

sterilized at 121

C for 15 min. Nutrient Agar (NA)

consisted of NB with the addition of 1.5%-1.7% agar.

2.2 Samples and Sensory

Observation

Beijing sausages produced on the same day were

obtained from the Pengcheng food branch of

BeijingShunxin Agricultural Limited Company, with

the specification of weighing 350-400 g/piece. Ten

Beijing sausages were put into an open homogeneous

sack and stored at 4

C. The sensory indexes of the

samples were observed and recorded in time during

storage. According to the national standard SB/T

10481-2008 (Ministry of Commerce of the People's

Republic of China, 2009), a sensory record table was

set up. The results are shown in Table 1. After sensory

evaluation, they were taken out for microbiological

analysis.

Table 1: Sensory changes of Beijing sausages in storage.

Storage days/d

1-3 4 5 6 7 8 9 10 11 12 13 14 15

Color ++++ ++++ +++ +++ ++ ++ ++ ++ + + +

—

Texture ++++ +++ +++ ++ ++ ++ ++ + + + +

—

Smell ++++ ++++ +++ +++ +++ ++ ++ ++ + + + + —

(Notes: ++++: very bright color, tight and resilient tissue, no mucus and mildew, no odor and rancidity, with meat flavor;

+++: bright color, tight tissue, no mucus, and mildew, with meat flavor, no odor and rancidity; ++: dark color, whitened

epidermis, soft tissue, no meat flavor; +: dark color, whitened epidermis, soft and inelastic tissue, mild rancidity odor; —:

with white odor, juicy phenomenon, and rancid flavor.)

2.3 Analysis of the Dominant

Spoilage Bacteria in Beijing

Sausages

2.3.1 Sample Pretreatment

As shown in Table 1, when stored at 4

C, the samples

had reached complete spoilage after 15 days. The

spoiled sausages were evenly divided into two

groups. Under sterile conditions, each sausage’s

epidermis and central part were evenly sampled with

sterile scissors. The samples from each group were

cut and mixed thoroughly.

2.3.2 Microbiological Culture

From each of the two mixed samples, 25 g was moved

into the aseptic homogenization bag with filter

membrane, 225 mL 0.85% sterile physiological saline

solution added, and samples homogenized for 150 s

by the beat nomogenizer. Next, 1 mL of filtrate was

extracted with a pipettor, slowly injected into a test

tube containing 9 mL sterile physiological saline

solution along the tube wall, and shaken evenly on the

oscillator to make 10

-2

samples of homogenization

solution. Finally, the required concentration was

diluted 10 times in a turn, according to the estimation

of sample contamination, 2-3 samples with the

appropriate dilution were selected, and 100 μL of

sample homogenate was injected into the surface of

the medium by pipettor. Samples were allowed to set

for 10 min then incubated at 36±1

C for 48 h.

2.3.3 Isolation, Purification, and

Morphological Analysis of Dominant

Spoilage Bacteria

Typical single colonies cultured on NA medium were

selected and repeatedly streaked on the medium for

separation and purification to obtain pure colonies.

For the four isolated and purified individual colonies,

colony morphology was observed and recorded,

including shape, size, color, luster, transparency, edge

shape, surface smoothness, wettability, and uplift

degree. Single colonies were stained with safranin,

and the morphology of the cells was observed by

ZEISS Axioplan 2 optical microscope (10 times

eyepiece × 100 times objective lens).

Analysis of Dominant Spoilage Bacteria in Beijing Sausages

2.3.4 Identification of Dominant Spoilage

Bacteria by 16S rDNA Sequence

Analysis

The four strains purified as described above

(numbered R1, R2, R3, R4) were inoculated in 5 mL

corresponding medium and cultured at the

appropriate temperature.

(1) Genomic DNA extraction: Briefly, bacterial

solution (1.0 mL) was centrifuged at 13000 rpm for 2

min in a 1.5 mL centrifuge tube. The supernatant was

discarded, the pellet transferred to a fresh tube, and

1.0 mL 0.85% NaCl was added. The tubes were then

centrifuged at 13000 rpm for 2 min. The precipitate

was suspended in 550 μL 1×TE. Then, 17 μL

lysozyme (35 mg/mL), 3 μL proteinase K (20

mg/mL), and 30 μL 10% SDS were added, with 30

min incubation at 37

C after each. 100 μL NaCl (5

mol/L) and 80 μL CTAB/NaCl solution were added

and mixed well, and samples incubated at 65

C in a

water bath for 10 min. An equal volume of

chloroform: isoamyl alcohol (24:1) was added and

samples mixed. After mixing, the tubes were

centrifuged at 13000 rpm for 10 min at room

temperature. The aqueous layer was transferred to a

fresh tube, and 2 volumes of cold ethanol were added.

The tubes were centrifuged at 13000 rpm for 10 min.

The supernatant was discarded and placed at room

temperature for 30 min to remove residual ethanol.

DNA was dissolved in sterile water or TE.

(2) PCR amplification: Bacterial DNA extracted

as described above was used as the template. PCR

was performed with 16S rDNA universal primers

(forward primer 27F: 5'-AGA GTT TGA TCC TGG

CTC AG-3', reverse primer 1541R: 5'-AAG GAG

GTG ATC CAG CC-3').

Reaction conditions for PCR were as follows: 95

oC for 5 min; 30 cycles of 1 min at 95

C, 1 min at 57

C, and 1 min 20 s at 72

C; 72

C for 5 min.

The reaction mixture was 100 μL and consisted of

0.8 μL Taq (5 U/μL), 10 μL 10× PCR Buffer (Mg

＋

Plus), 8 μL dNTP Mixture (2.5 mM/each), 2.5 ng

template DNA, 2 μL each forward and reverse

primers (10 μmol/L), and ddH

O to 100 μL (ARa et

al., 2006; Cheng et al., 2006).

(3) Sequence alignment: The PCR amplification

products were purified and sequenced by Beijing

Haocheng Mingtai Technology Co., Ltd. The 16S

rDNA sequence of the strains with high homology

were downloaded from NCBI and analyzed by

MEGA 7 software.

(4) Phylogenetic analysis: Phylogenetic analysis

was performed by MEGA 7.0.26 software. The

phylogenetic tree was built using the neighbor-

joining method and with 1000 bootstrapping

replicates.

2.4 PCR-DGGE Analysis of Spoilage

Bacteria in Beijing Sausages

Bacteria were identified using PCR denaturing

gradient gel electrophoresis. Twenty-five grams of

the pretreated sample described in Section 2.3.1 was

packed in a sterile homogenization sack, added 225

mL 0.85% sterile physiological saline solution, and

the sample homogenized for 150 s. Approximately 4

mL of sample was transferred to a sterile 5 mL

centrifuge tube. After storage at -80 oC for 24 h,

samples were sent to the Beijing Haocheng Mingtai

Technology Co., Ltd for PCR-DGGE identification.

The DGGE recovered bands were PCR amplified.

3 RESULTS

3.1 Identification Results and Analysis

of 16S rDNA

Morphological characteristics of strains R1-R4 are

shown in Table 2. The microscopic images of the

safranin single dye are shown in Figure 1.

Table 2: Morphology characteristics of different strains.

Colonial morphology R1 R2 R3 R4

Shape circle circle circle circle

Size big small small small

Color light milky white white white white

Luster glossy glossy glossy dim

Transparency translucent opaque opaque opaque

Edge shape regular regular regular regular

Sshirunshiruurface smooth smooth smooth smooth

Wettability moist dry moist moist

Uplift raised flat raised raised

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

a b

c d

Figure 1: Microscope images of strains R1 (a), R2 (b), R3 (c) and R4 (d).

Four strains were isolated and purified for 16S

rDNA sequence analysis. NCBI was used to identify

the bacteria based on the sequences; analysis results

are shown in Table 3.

Table 3: Result of NCBI sequence comparison analysis of four strains.

. Strain label Similarity

Population and login number of the strain

with the highest similarity in GenBank

English name

1 R1 99.78% Pseudomonas lundensis ATCC 49968(T) Pseudomonas sp.

2 R2 100% Staphylococcus vitulinus ATCC51145(T) Staphylococcus sp.

3 R3 100% Brochothrix thermosphacta ATCC 11509(T) Brochothrix sp.

4 R4 100% Brochothrix thermosphacta ATCC 11509(T) Brochothrix sp.

3.2 Identification

Results and

Analysis of PCR-DGGE

The result of DNA extraction is shown in Figure 3.

The result of PCR amplification is shown in Figure 4.

DGGE patterns of bacterial DNA are shown in Figure

5. The primer was 16S rDNA corresponding to the

universal primer 338F-534R, and the PCR product in

the map was 250bp, which was judged as the target

fragment. The PCR amplification results are shown in

Figure 4.

Figure. 3: Result of DNA extraction. Figure. 4: Result of PCR amplification. Figure. 5: DGGE patterns of bacterial

DNA.

(Notes: The Arabic numerals in the figure indicate the number of the rubber recovery, with only one band cut at the same

level. Due to the low brightness of band 6 in DGGE, the dominant bacteria were not considered, and the gray value analysis

was not carried out.)

Analysis of Dominant Spoilage Bacteria in Beijing Sausages

4 CONCLUSIONS

Two methods, microbial cultivation and polymerase

chain reaction-denaturing gradient gel

electrophoresis (PCR-DGGE), were used to analyze

the dominant spoilage bacteria in Beijing sausages.

Using 16S rDNA sequence analysis, the main

spoilage bacteria isolated and identified were one

genus of Pseudomonas sp., one genus of

Staphylococcus sp., and two genera of Brochothrix

sp. PCR-DGGE analysis identified 13 species of

bacteria, including Pseudomonas sp., Brochothrix sp.,

and Staphylococcus sp., with Pseudomonas sp. being

the most dominant spoilage bacteria, followed by

Brochothrix sp. and Staphylococcus. The results of

the two methods are consistent with one another and

with the results of other studies in the past two years

identifying spoilage bacteria in low-temperature meat

products (Samelis et al., 2000; Jenni et al., 2015).

ACKNOWLEDGMENTS

This work was supported by Beijing Key Laboratory

of Agricultural, Product Detection and Control of

Spoilage Organisms and Pesticide. The authors

express their thanks to Beijing Innovation

Consortium of Swine Research System and

Pengcheng food branch of Beijing Shunxin

Agricultural Limited Company for providing the

primary sample.

REFERENCES

Adams M R, Moss M. Food Microbiology[M]. RSC, 2007.

ARa C, SeungKu Y, EuiJoong K. Cloning, sequencing and

expression in Escherichia coli of a thermophilic lipase

from Bacillus thermoleovorans ID‐1[J]. FEMS

Microbiology Letters, 2006,186(2).

Cheng H, Jiang N. Extremely Rapid Extraction of DNA

from Bacteria and Yeasts[J]. Biotechnology Letters,

2006,28(1).

Danilo E, Federica R, Antonella N, et al. Mesophilic and

psychrotrophic bacteria from meat and their spoilage

potential in vitro and in beef.[J]. Applied and

environmental microbiology, 2009,75(7).

Doulgeraki A I, Ercolini D, Villani F, et al. Spoilage

microbiota associated to the storage of raw meat in

different conditions[J]. International Journal of Food

Microbiology, 2012,157(2).

Jenni H, Riitta R, Javeria A, et al. Meat Processing Plant

Microbiome and Contamination Patterns of Cold-

Tolerant Bacteria Causing Food Safety and Spoilage

Risks in the Manufacture of Vacuum-Packaged Cooked

Sausages[J]. Applied and Environmental

Microbiology, 2015,81(20).

Liu F, Wang D, Du L, et al. Diversity of the Predominant

Spoilage Bacteria in Water‐Boiled Salted Duck during

Storage[J]. Journal of Food Science, 2010,75(5).

Ministry of Commerce of the People's Republic of China.

SB/T 10581-2008, Quality safety requirement of

pasteurized meat products[S]. Beijing : Standards Press

of China, 2009.

Peirson M D, Guan T Y, Holley R A. Aerococci and

carnobacteria cause discoloration in cooked cured

bologna [J]. Food Microbiology, 2003,20(2).

Samelis J, Kakouri A, Rementzis J. Selective effect of the

product type and the packaging conditions on the

species of lactic acid bacteria dominating the spoilage

microbial association of cooked meats at 4°C[J]. Food

Microbiology, 2000,17(3).

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