Spoilage of vacuum packaged meat

Alfas Ahmed SiddiqueSupervisor- Dr. Shane Powell

Meat in Australian economy• Australia is the 3rd largest meat exporter• >1 million metric ton exported by 2015• Exporting to Japan, China, Indonesia, USA, South Korea, New Zealand,

Saudi Arabia, Hong Kong, United Arab Emirates ….• 61% Australian land for farming and grazing • Beef is vacuum packaged commercially to distribute and sell.

Vacuum packed meat• Vacuum packaging - Meat in a plastic film or bag - Remove air and oxygen - SealingAim is to make meat safer to consume and protect from any contamination.

Aims• Overall Aims - Determining off odor producing microbes in vacuum packaged beef (numerous and culturable only) - Identification of microbes cause off odor under refrigeration

Sample

Discoloration

Purge

Fig – Spoiled vacuum packaged sample

Previous studies• The condition is anaerobic• Psychotropic Lactic Acid Bacteria (LAB) mostly reported as dominant

and off odor producer Brochothrix, Lactobacillus, Leuconostoc, Carnobacteria, Weisellia (a large group of bacteria produce lactic acid)

• Clostridia spp. involvement in blown pack.• Enterobacteria in spoilage Serratia spp The LAB become dominant and off odor producer.

Method

Sampling

Plate count

Isolation and pure culture Characterization

Cooked meat growth

Potential spoilage microbe

Inoculation on irradiated sterile

meat

Off odor detection

Gene sequencing for identification

Sampling, plate count and pure culture

Two spoiled sampled were examined. Up to dilution 4 were spread plated on

PCA – Non selective VRBG – Selective for EnterobacteriaceaceBHI – Non selective CFC – Selective for PseudomonasMRS – Selective for LAB STAA – Selective for Brochotrix -Incubation at 4ᵒC, 10ᵒC and 25ᵒC.

(PCA-plate count agar, BHI- Brain heart infusion agar, MRS-Man Rogosa Sharpe agar, VRBG-Violet Red Bile Glucose agar, CFC- Centrimide Fucidine Cephalosporin, STAA- Sterptomycin thallous acetate actidione)

Characterization • Colony morphology• Gram stain• KOH test• Hetero and homo fermentation of LAB X - APT broth (hot loop test). Bubbles – hetero; No bubbles - Homo

X - HHD (Blue colony- Hetero; White colony – Homo) - M5 media (Green color change – Hetero; No color change – Homo) media color changes from blue to green by hetero fermentation

Isolates with similar colony morphology and gram stain assumed as same microbes.

M5 media

Homo fermentation

Hetero fermentation

Fig – Homo and hetero fermentation differentiation using M5

Testing spoilage potential

Cooked meat medium - Inoculation and incubation at 4ᵒC, 10ᵒC, 20ᵒC and 25ᵒC. - Off odor detection

Sterile irradiated meat inoculation - Potential spoilage isolates were inoculated on irradiated meat - Incubation at 25ᵒC.

Gene sequencing -Sequencing of potential isolates.

Cooked meat Irradiated meat Inoculation

Results

• Psychrotolerant LAB at 4ᵒC

• Diverse flora in BHI

PCA BHI MRS0

1

2

3

4

5

6

7

8

4C10C25C

Media and temperatures

Log

CFU/

g of

mea

t

Log CFU/G of meat of 1st spoiled sample

• Psychrotolerant at 10ᵒC

• LAB is higher

• Higher Log CFU/g

PCA BHI MRS0

1

2

3

4

5

6

7

8

4C10C25C

Media and temperatures

Log

CFU/

g of

mea

t

Log CFU/G of meat of 2nd spoiled odor sample

Characterizations results

• 90 isolates

• Mesophilic and psychotropic

• Psychrotolerant

• Gram +ve/-ve cocci/rods in pairs/chains

• Cooked meat medium

• M5 media (LAB only)

Isolate no. Media Colony morphology Gram stain Cooked meat broth

P10 PCA Dull white, round, smooth, flat, glistering, small in size

-ve Short rods chains

Sulfurous

P15 PCA Transparent, round, smooth, flat, smallest tiny dot like

-ve/+ve Coccoid rod/short rod Chains

Unpleasant smell

P19 PCA Transparent, round, smooth, flat, smallest tiny dot like

-ve Rods Sulfurous

P20 PCA Watery milky, glistering, round, flat -ve Cocci Chains Unpleasant smell

B10 BHIB Whitish, round, transparent, glistering, small -ve cocci Spoil smell

B25 BHIB Transparent, round, glossy, flat, glistering, medium size

+ cocci Unpleasant smell

B29 BHIB Dull white, round, flat, opaque, no glistering, smooth

+ve cocci Unpleasant smell

Short Isolation chart

Homo- Hetero differentiation

M6 MRS Completely transparent, round, smooth, flat, dot like

+ve Cocci Spoil smell APT – HomoM5 – HeteroHHD – Hetero

M12 MRS Transparent, round, smooth, flat, smaller size

+ve Small Rods

No smell APT – No inoculationM5 – HomoHHD – Homo

Sign of spoilage on radiated meat and compare with sample

Discoloration and purge

Irradiated meat inoculation resultsIsolates Amount of inoculum

cells/cm²Off odor Color change Purge production

P10 3x10^6 Yes, Sickly sweet, Greyish + + +P19 3.38x10^6 No or as control - P20 5.8x10^6 Yes, Chunky cheesy, sour and vomit

typeGreyish + +

P27 4.36x10^8 No or as control - -M1 4.02x10^8 No or as control - -M6 3x10^6 No or as control - -M12 5.76x10^8 Yes, Chunky cheesy, sour and vomit

typeGreyish +

B10 6.83x10^8 Yes, Fruity sweet Slightly greyish + +

B1 6.63x10^8 No or as control - -B29 4.95x10^8 Yes, Yeasty alcoholic Slightly greyish +

B25 5.25x10^8 Yes, Yeasty alcoholic Slightly greyish +

** The red colored ones were analyzed by gene sequencing to identify the microbes

Gene sequencing results• P10 Carnobacterium spp.

• B25 Carnobacterium spp.

ConclusionOne of the off odor producing microbes was Carnobacterium spp.

It grows between -1ᵒC to 25ᵒC.

Aerobically, Anaerobically and Micro aerobically.

Cause spoilage in vacuum packed meat under refrigeration.

Thank you