Nissim SilanikoveDepartment of Animal Physiology,Agricultural Research Organization,

The Volcani Center, Israel

Interactions between bacteria type,

caseinolysis and physico-chemical

properties in caprine, ovine and bovine milk

Cork 2005

Milk quantity:Yield of milk, fat, total proteins,

casein and curd.

Milk quality: Internal bacterial contamination,

somatic cell count, secreted enzymes.

Cork 2005

Bacterial infection may affect

caseinolysis and micelle properties

by three main routes:

1. directly, by secreting extracellular enzymes different bacteria will cause different "type"

of physico-chemical damage to the milk

Cork 2005

3. a combination of 1 and 2

2. activate the host innate immune system

milk from different type of bacteria with similar

SCC will result in similar damage to the milk

Cork 2005

Hypothesis:

Healthy gland ~ 20,000 Cows

~ 800,000 goats and sheep

Infected gland ~ 3,000,000

CASEINOLYSIS INDEX

Cell depended

Bacteria and Cells depended

Cork 2005

Somatic cell count and gross composition as bases for grading milk quality in sheep and goats

Aim: to calculate the losses of milk and cheese loss as related to the level of subclinical udder infection in a herd.

Elucidated the major factors that influence milk yield and, consequently, curd yield in Assaf sheep and Saanen and Shami × Anglo-Nubian goats,

CMTLog SCCCMTLog SCC

Uninfected0.65 b5.33 b0.91 b5.51 b

Infected2.23 a6.38 a1.59 a6.12 a

P [F]0.00010.00010.00010.0001

Bacteriological

Status

SheepGoats

The LS Means of CMT and log SCC in uninfected and infected udders and their different significance level (P [F]).

Bacteriological

Status

SheepGoats

The LS Means of fat, protein and lactose in uninfected and infected udders and their different significance level (P [F]).

FatProteinLactoseFatProteinLactose

Uninfected55.949.0 b49.4 a37.4 a38.145.9 a

Infected56.950.3 a42.9 b35.1b38.344.0 b

P [F]NS0.00010.00010.04NS0.0001

Quantifying the damage caused by IMI with CNS

From data collected in the present study and those published recently two equations could be developed to calculate milk yield loss and total curd yield loss. These equations combine milk loss and reduction in curd yield per litre of milk in sheep or goats with sub clinical IMI:

Milk yield loss (%) = 100 - [C × 100 + (100-C) × IUY]/100 Total curd yield loss (%) = 100 - [C × 100 + (100-C) × (IUY-ICY × D)]/100

where: C = % uninfected udders; IUY = percentage to which milk production is reduced by sub clinical udder infection; ICY = percentage of curd lost because of sub clinical udder infection; D = litres of milk needed to produce 1 kg of cheese (30 %moister)

Calculated percent milk and curd loss in sheep and goats herd due to rate of infection with CNS according to the equations

Sheep

25 760,0001281712

501,300,00025153424

752,100,00038235136

Goat

25 640,000832116

50 920,0001564132

751,300,0002386248

Half-udder model

Herd Half-udder model

Herd

Infection rate

Projected

SCC

Milk loss (%)Total curd loss (%)

+ -

The model: Each goat, sheep or cow tested had at least one uninfected quarter (NBF) and one of the other quarters infected with one of the following bacteria:

BacteriaNumber

NBF33

Streptococci23

CNS11

E. Coli8

S. aureus9

Cork 2005

05

1015202530

35404550

Uninfected Infected

Sheep

GoatLac

tose

, g/L

Lactose concentration: sheep or goat with onegland infected with CNS specie and the

contra-lateral being free Sheep - 25.1%, P < 0.0001Goat - 11.3%, P < 0.004

Milk yield (half) of sheep or goat infected with CNS specie in one gland and the contra-lateral being free.Open bars – S; Hatched bars – G

0.0

0.5

1.0

1.5

2.0

Uninfected Infected

Milk

yie

ld (

kg/d

ay)

00.10.20.30.40.50.6

Ratio ofreduction inMY between

goats andsheep

Ratio ofreduction in

lactosebetween

goats andsheep

The ratio in the reduction in milk yield betweengoats and sheep in comparison to the ratio of

reduction in lactose concentration

Conclusion

The greater reduction in lactose concentration in infected glands of sheep than in goats, explains the higher loss of milk yield in sheep

0

10

20

30

40

50

60

70

Uninfected Infected

Speep

Goat

fat,

g/L

Fat concentration: sheep or goat with one gland infected with CNS specie and the

contra-lateral being free Sheep - 5%, NSGoat - 0.03%, NS

0

10

20

30

40

50

60

Uninfected Infected

Sheep

Goat

pro t

ein,

g/L

Protein concentration: sheep or goat with one gland infected with CNS specie

and the contra-lateral being free Sheep - 9%, P < 0.0009Goat + 2.3%, P <0.07

05

1015202530

35404550

Uninfected Infected

Sheep

Goatcase

in ,

g/L

Casein concentration: sheep or goat with one gland infected with CNS specie

and the contra-lateral being free Sheep - 12%, P < 0.0002Goat + 0.003%, NS

0

2

4

6

8

10

12

14

Uninfected Infected

Sheep

Goat

Whe

y, g

/L

Whey concentration: sheep or goat with one gland infected with CNS specie

and the contra-lateral being free Sheep + 7.5%, P < 0.07Goat +11.5%, P < 0.0001

CONCLUSIONS

In goats the increase in total protein concentration relates to increase in total whey concentration

In sheep the reduction in total protein concentration relates to a decrease in casein concentration, which overweighs the increase in whey concentration

0

0.5

1

1.5

2

2.5

Uninfected Infected

Sheep

Goat

P-p

, g/L

Proteose-peptone concentration: sheep or goat with one gland infected with CNS specie

and the contra-lateral being free Sheep + 247%, P < 0.0001Goat +151%, P < 0.0001

00.20.40.60.8

11.2

1.41.61.8

2

Uninfected Infected

Speep

GoatCa,

mm

o l

Ca activity: sheep or goat with one gland infected with CNS specie and the

contra-lateral being free Sheep - 30.1%, P < 0.002Goat -14.2%, P < 0.002

Conclusions

In both goats and sheep, infection is associated with increased casein degradation

The increase in casein degradation is greater in sheep then in goats

Measurement of Ca activity is potentially a convenient and cheap method to track casein degradation

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Uninfected Infected

Sheep

Goat

PA

act

ivit

y, u

n its

/mL

Plasminogen activator activity: sheep or goat with one gland infected with CNS specie

and the contra-lateral being free Sheep + 239%, P < 0.002Goat +128%, P < 0.05

0102030405060

708090

100

Uninfected Infected

Sheep

Goat

PL

ac t

ivit

y, u

n its

/mL

Plasminogen activity: sheep or goat with one gland infected with CNS specie

and the contra-lateral being free Sheep - 32.2%, P < 0.001Goat 0%, NS

Conclusions

Plasminogen activator activity in goats is unusually high and consequently all the plasminogen in the gland is converted to plasmin

0

10

20

30

40

50

60

Uninfected Infected

Sheep

Goat

PL

ac t

ivit

y, u

n its

/mL

Plasmin activity: sheep or goat with one gland infected with CNS specie and the

contra-lateral being free Sheep + 73.7%, P < 0.0007Goat + 195%, P < 0.0003

Conclusions The basal level of PL activity is higher in

sheep than in goats, which explains the higher basal level of proteose-peptones

PL activity in infected glands is higher in sheep than in goats, which explain the higher increase in proteose-peptones

In sheep, the source of increased PL activity in the infected gland is accelerated conversion of plasminogen to plasmin, whereas in goats the source is external

Question: How comes that in goats accelerated degradation of casein is not reflected in casein concentration, whereas in sheep it does?

Answer: In goats the reduction in casein output (30%) is essentially similar to the reduction in milk yield, whereas in sheep the reduction in casein output (60%) is higher than in milk volume (53%).

Thus, both in goats and sheep part of the increased loss in casein yield is related to increased degradation of casein

Effect of subclinical mastitis on curd yield Yc, and clotting time Tc,

in milk of infected vs. uninfected udder halves of sheep.

0

10

20

30

300

400

500

600

700

800

900

1000

1100

1200

P < 0.0001

P < 0.0001

Gra

ms

Sec

onds

Curd yield Clotting time

Infected Uninfected

Effect of subclinical mastitis on curd yield, Yc (a) and clotting time, Tc (b), in milk of infected vs. uninfected udder halves of goats.Open bars – uninfected; Hatched bars – infected

150

175

200

225

250

aP < 0.0001

Cur

d yi

eld

(g/L

)

0

50

100

150

200

250

300

350

b P < 0.02

Clo

tting

tim

e (s

)

BacteriaNumberSCC (x1000)

PMN

(%)

CD8+

(%)

CD14+

(%)

NBF33116±20

c

32±3.3

c

4.4±0.8

b

5.0±1.1

Strep.233379±350

a

58±5.2

ab

8.9±1.6

ab

9.4±1.6

CNS11543±129

b

54±8.5 ab

17.7±3.6 a

11.3±2.9

E. coli94333±443 a

63±20.1 a

9.3±7.5 ab

10.3±7.2

S. aureus91148±163

b

82±1.7

a

1.3±0.1

c

5.8±0.6

SCC and cell differentiation according to bacteria isolates in the milk

Cork 2005

No significant differences were fund among the

different bacteria in fat, protein and casein

Lactose concentrations was significantly

Lower in milk from all infected quarters

compared to milk from uninfected quarters,

regardless of type of bacteria

Cork 2005

Clotting time (in seconds) Curd firmness (volts) 30 min after

coagulating enzyme additions

Two parameters were tested using

the Optigraph© (Alliance Instruments)

according to bacteria isolates in the

quarter milk

Cork 2005

1 2 3

Curd firmness(volts)

Clotting time(sec)

Cork 2005

Clotting time and Curd firmness

BacteriaClotting time (sec)

Curd firmness(V)

NBF 650±63 6.58±0.2Strep. 2490±340 1.02±0.3CNS 1255±468 3.80±0.8E. coli S. aureus 1078±193 3.28±0.7

Cork 2005

Effects of mammary gland infection on plasminogen (PLG), plasminogenactivator (PA) and plasmin (PL)

BacteriaPLG

(unit/mL)

PA

(unit/mL)

NBF146.2 531

Strep.167.8 518

CNS 174.4 530

E. coli327 542

S. aureus156.7 555

1 unit is the amount of PLG, PA or PL that produces a

Change of 0.1 in absorbance at 405 nm in 60 min Cork 2005

Plasmin

)unit/mL(

10.5

23.3

18.8

20.0

17.2

a

a

a

a

b

Correlation matrix

Clotting time (sec)

Curd firmness)V(

log SCC0.48 ;P = 0.020 - 0.46 ;P = 0.001

Lactose -0.64 ;P = 0.002 0.61 ;P = 0.003

Whey0.83 ;P = 0.001 -0.72 ;P = 0.001

pp0.59 ;P = 0.005 -0.60 ;P = 0.004

Plasmin0.65 ;P = 0.001 -0.65 ;P = 0.001

Casein0.01 ;NS0.08 ;NS

Cork 2005

0 1 2 3 4 5 6 7 8 90

5

10

15

Whe

y pr

otei

ns (

g/L)

Curd firmness (A30 Optigraph)

0 1 2 3 4 5 6 7 8 90

10

20

30

40

50

Tot

al p

rote

in (

g/L)

Curd firmness (A30 Optigraph)

Protein, r - NS

0 1 2 3 4 5 6 7 8 90

10

20

30

40

50

Cas

ein

(g/L

)

Curd firmness (A30 Optigraph)

Whey, r = 0.83

Casein, r - NS

Cork 2005

0 1 2 3 4 5 6 7 8 9

10

100

1000

10000lo

g S

omat

ic C

ell C

ount

Curd firmness (A30 Optigraph)

SCC, r = 0.46

Cork 2005

100

1000

0 1 2 3 4 5 6

Strep. dysgalactiae S. aureus

Curd firmness (A30 Optigraph)

log

Som

atic

Cel

l Cou

nt

Cork 2005

CURD FROM HELTHY GLANDS

CURD FROM INFECTED GLAND

AFTER ONE DAY OF MATURATION

HEALTHY INFECTED

HEALTHY INFECTED

AFTER 3 MONTH OF MATURATION

SDS PAGE Tricine

0% 0% 50% 50% 100% 100%

52

35

28

2114

FPLC

-

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

450.00

500.00

550.00

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

ml

mA

U

2395 - 100%

2395 - 0% (/2)

ב'ג'ד'ה'

א'

OPTYGRAPH EFFECT OF ADDING VARIOUS P-P FRACTIONS (O.5 MG/ML) TO BACTERIAL FREE MILK

20 25 30 35 40 45

1

2

3

4

5

Clotting Time (min)

CONTROL

Hypothesis:

Healthy gland ~ 20,000

Infected glands ~ 200,000

Bacteria depended

Cork 2005

The results suggest that different

bacteria affects caseinolysis in a

different manner directly or

indirectly, influencing the quantity

and quality of milk products

Cork 2005

The main conclusions from this

study are: 2. Casein has low value

for predicting milk quality for cheese

production 3. Whey is a convenient

4. This study shows that in order to

understand milk quality it will be

important to study the interactions

among specific bacteria the innate

immune system and caeinolysisCork 2005

Thank you: I hope that this lecture

will contribute to our ability to

raise healthier cows and produce

better dairy products

BOLFA 2006