Nissim Silanikove Department 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,

Log SCCCMTLog SCCCMT 5.51 b 0.91 b 5.33 b 0.65 b Uninfected 6.12 a 1.59 a 6.38 a 2.23 a Infected P [F] GoatsSheepBacteriological Status The LS Means of CMT and log SCC in uninfected and infected udders and their different significance level (P [F]).

GoatsSheepBacteriological Status The LS Means of fat, protein and lactose in uninfected and infected udders and their different significance level (P [F]). LactoseProteinFatLactoseProteinFat 45.9 a a 49.4 a 49.0 b 55.9 Uninfected 44.0 b b 42.9 b 50.3 a 56.9 Infected NS NS P [F]

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 (%) = [C × (100-C) × IUY]/100 Total curd yield loss (%) = [C × (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 , ,300, ,100,00075 Goat , , ,300,00075 HerdHalf-udder model HerdHalf-udder model Total curd loss (%)Milk loss (%)Projected SCC Infection rate

+ - 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: NumberBacteria 33 NBF 23 Streptococci 11 CNS 8 E. Coli 9 S. aureus Cork 2005

Lactose, g/L Lactose concentration: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep %, P < Goat %, 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

The ratio in the reduction in milk yield between goats 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

fat, g/L Fat concentration: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep - 5%, NS Goat %, NS

protein, g/L Protein concentration: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep - 9%, P < Goat + 2.3%, P <0.07

casein, g/L Casein concentration: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep - 12%, P < Goat %, NS

Whey, g/L Whey concentration: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep + 7.5%, P < 0.07 Goat +11.5%, P <

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

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 < Goat +151%, P <

Ca, mmol Ca activity: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep %, P < Goat -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

PA activity, units/mL Plasminogen activator activity: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep + 239%, P < Goat +128%, P < 0.05

PL activity, units/mL Plasminogen activity: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep %, P < Goat 0%, NS

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

PL activity, units/mL Plasmin activity: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep %, P < Goat + 195%, P <

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.

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

CD14+ (%) CD8+ (%) PMN (%) SCC (x1000) NumberBacteria 5.0± ±0.8 b 32±3.3 c 116±20 c 33NBF 9.4±1.68.9±1.6 ab 58±5.2 ab 3379±350 a 23Strep. 11.3± ±3.6 a 54±8.5 ab 543±129 b 11CNS 10.3±7.29.3±7.5 ab 63±20.1 a 4333±443 a 9E. coli 5.8±0.61.3±0.1 c 82±1.7 a 1148±163 b 9S. aureus 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 Curd firmness (V) Clotting time (sec) Bacteria 6.58± ±63 NBF 1.02± ±340 Strep. 3.80± ±468 CNS E. coli 3.28± ±193 S. aureus Cork 2005

Effects of mammary gland infection on plasminogen (PLG), plasminogen activator (PA) and plasmin (PL) PA (unit/mL) PLG (unit/mL) Bacteria NBF Strep CNS E. coli S. aureus 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) a a a a b

Correlation matrix Curd firmness (V) Clotting time (sec) ; P = ; P = 0.020log SCC 0.61; P = ; P = 0.002Lactose ; P = ; P = 0.001Whey ; P = ; P = 0.005pp ; P = ; P = 0.001Plasmin 0.08; NS0.01; NSCasein Cork 2005

Protein, r - NS Whey, r = 0.83 Casein, r - NS Cork 2005

SCC, r = 0.46 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% 50% 100%

FPLC ה'ד'ג'ב' א'

OPTYGRAPH EFFECT OF ADDING VARIOUS P-P FRACTIONS (O.5 MG/ML) TO BACTERIAL FREE MILK 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 caeinolysis Cork 2005

Thank you: I hope that this lecture will contribute to our ability to raise healthier cows and produce better dairy products BOLFA 2006