Biltong:• A popular ready to eat salted and dried meat product from South Africa.

• Uses lean strips with excess fat trimmed that may cause rancidity and extend the time for the meat to absorb the salt.

• Dried at ambient humidity and temperature

• Recently, increase in interest to manufacture biltong in US

• Requires USDA-FSIS validation

Microbial Validation of Biltong Processing to Achieve 5-Log Reduction of L. monocytogenes and E. coli O157:H7

Kavya Gavai2 and Peter Muriana 1,2

1Robert M. Kerr Food & Agricultural Products Center and the 2Dept. of Animal and Food Sciences, Oklahoma State University, Stillwater, OK

Production of Biltong Salting Spicing Marination Drying

Popular choice for consumersFavorable go to snack food

Healthy

USDA-FSIS Biltong Requirements

• Biltong is produced without a heat lethality step but still must be determined to be safe for consumer consumption with a sufficient (5-log) reduction of pathogenic bacteria as required by the UDSA-FSIS.

In the US, beef jerky is manufactured under USDA-FSIS compliance guidelines to achieve >5-log reduction for pathogen of concern(Salmonella, L.mono, E. coli O157:H7 , Staphylococcus aureus)

• Relative humidity : >90% throughout cooking or thermal heating process• Cooked at >175F using sealed oven or steam injector

Salting Decreases water

activityIncrease the shelf life

of the product

Influence:Flavor ,appearance and

texture

Vinegar Inhibits microbial growth

Influence:Flavor

Water binding capacity

USDA-FSIS Process of Biltong

Process demonstrate a >2-log reduction of pathogen and perform testing of every lot of edible ingredient to ensure no pathogen

Process must demonstrate a >5-log reduction of pathogen

Preliminary confirmation test for Listeria monocytogenes using MOX agar

On modified MOX agar Listeria monocytogenes strains are :• Round but the surface is concave and depressed into

the media• Approximately 1mm in diameter• Black in color and surrounded by a black zone

Growth conditions: TSB broth ; 30 ℃ for 24 hours Incubation: 30 ℃ for 24 hours

Preliminary confirmation test for E. coli O157:H7 using CHROMagar O157

On CHROMagar 0157 E. coli O157:H7 strains are:• Mauve colored due to the utilization of chromogenic

substrates in the medium.• Non-E. coli O157:H7 bacteria may utilize other

chromogenic substrates resulting in blue to blue-green colored colonies .

Growth conditions: TSB broth ; 37 ℃ for 24 hours Incubation: 37 ℃ for 24 hours

383-2 39-2 V7-2 SCA-2

ATCC 35150 ATCC 43889ATCC 45756ATCC 43894

Microbial Validation of Biltong Processing to Achieve 5-Log Reduction of L. monocytogenes and E. coli O157:H7

1. Bacterial strains and growth conditions• Strains are acid adapted by growing overnight at suitable temperature in TSB and re-

inoculated into pre-warmed TSB(1% glucose)• Centrifugation (8000 RPM, 5 ℃,20 min)• Preparation of culture cocktail

2. Inoculation of culture cocktail on beef pieces.• Trimming of beef pieces and cutting into similar size of (~1.9cm thick X ~5.1cm wide X

~7.6 cm long)• Inoculation of beef pieces with 150ul of culture cocktail on each side• Refrigeration at 5 ℃ for 30 minutes

3. Processing Of Biltong• Water dip for 30 seconds• Rotated manually and excess liquid is drained

4. Marination and Drying• Short time marination-30 minutes• Vacuum tumbling (15 inches Hg ) of beef pieces with either mixture of spice, salt and

Vinegar or with individual components of marinade• Spice: (Coriander 1.1 % and black pepper 0.8% ), Salt (2.2 % ) and Vinegar -100 grain ( 4% )

of total formulation• Stomaching Dilutions Plating• Drying for 10 days: Temperature- controlled humidity oven (75°F/23.9°C; 55% RH)

Antibiotic resistance:-Listeria monocytogenes: 100ug/ml streptomycin and 10 ug/ml Rifamycin.-E. coli O157:H7 : 5ug/ml of Novobiocin and 2.5ug/ml Rifamycin.

Cultures

Biltong beef pieces Inoculation Gloved finger technique

Dip tanks and baskets Water dip

Vacuum tumbler marination

Treatments hanging in oven

Temperature and humidity probe

Water Activity (Aw) and ‘Non-Intact Beef’• Non-intact beef: ground beef, blade-tenderized beef, vacuum-tumbled beef

• Safety of ground beef (hamburger) & bladed-tenderized beef (steak) can be obtained by cooking to a certain internal temperature

• Biltong is not ‘cooked’, so safety depends on maintaining low water activity

• USDA-FSIS requires biltong Aw to be < 0.85 (to prevent enterotoxin formation should Staphylococcus aureus be present since S.a. can tolerate low Aw.

Aw 0.805 Aw 0.850 Aw 0.890

0.7000

0.7500

0.8000

0.8500

0.9000

0.9500

1.0000

0 1 2 3 4 5 6 7 8 9 10

Wat

er A

ctiv

ity (A

w)

Time (days)

Water Activity During Processing of Biltong (75oF, 55% RH)

L. monocytogenesE. coli O157:H7

Figure A: Water activity during processing of Biltong for the reduction of Listeria monocytogenes and E. coli O157:H7 (75oF, 55% RH)

(A)

Biltong Processing to Achieve 5-Log Reduction of L. monocytogenes

7.33

5.98

3.63

2.84

2.20

1.831.26

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8 9 10

List

eria

mon

ocyt

ogen

es(L

og C

FU/m

L)

Time (days)

Listeria - Trials 1 & 2…Listeria monocytogenes: Starting level (log 7.33)

Listeria monocytogenes: Target level (log 2.33 = 5-log reduction)

5-log Reduction

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8 9 10

List

eria

mon

ocyt

ogen

es(L

og C

FU/m

L)

Time (days)

Be ef/Lm (CTL)

Be ef/Lm (+Spices)

Be ef/Lm (+Salt)

Be ef/Lm (+Vinegar)

Be ef/Lm (+Spice, Salt,Vinegar)

Listeria monocytogenes: Starting level (log 7.29)

Listeria monocytogenes: Target level (log 2.29 = 5-log reduction)

5-log Reduction

Figure B : Processing of biltong to achieve 5 log reduction of Listeria monocytogenes for the combined components of the marinade

Figure C: Processing of biltong to achieve 5 log reduction of Listeria monocytogenes for the individual components of the marinade

(B) (C)

Biltong Processing to Achieve 5-Log Reduction of E.coli O157:H7

7.18

5.96

3.28

1.89 1.79

1.531.26

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8 9 10

E. c

oli O

157:

H7 (L

og C

FU/m

L)

Time (days)

E.coli O157:H7 - Trials 1 & 2…E. coli O157:H7 : Starting level (log 7.18)

E. coli O157:H7 : Target level (log 2.18 = 5-log reduction)

5-log Reduction

Figure D : Processing of biltong to achieve 5-Log the reduction of E.coli O157:H7 for the combined components of the marinade

(D)

45

50

55

60

65

70

75

80

85

45

50

55

60

65

70

75

80

85

0 1 2 3 4 5 6 7 8 9 10

Rela

tive

Hum

idity

(%)

Tem

pera

ture

(o F)

Time (days)

Chamber Temp Probes 1 & 2

Biltong Beef Temp Probes 1 & 2

Chamber % Relative Humidity

Temperature and Relative Humidity Measurements for Biltong

Figure E: Temperature (°C) and relative humidity (% RH) during biltong processes maintained at 75oF, 55% RH

(E)

Conclusion• The combination of spice, salt and vinegar in the marinade resulted

in a reduction of > 5 log and & >6 log with subsequent dryingperiod of 10 days, for E. coli O157:H7 and Listeria monocytogenesrespectively.

• The greatest reduction of Listeria monocytogenes from theindividual components of the marinade was exhibited from thetreatment with vinegar(>5log) followed by salt-only (4.68 log),spice-only (4.1 log) and conditions of drying, without any addedingredients, rendered a 4.2-log reduction over a period of 10 daysand water activity levels of 0.79.

• No one previously was able to achieve 5-log reduction:

Ø Meat loses 60% of moisture at the end of the process

Ø Method of enumeration

a)CFU/gm : residual counts are increased which goes againstthe log reduction

b) CFU/ml: fixed volume diluent which eliminates the issueregard loss of moisture