INNOVATIVE ANALYTICAL SOLUTIONS FOR THE DAIRY INDUSTRY Pierre Broutin, Managing Director/Western Europe Senior Scientist IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Bentley CombiFTS Rapid and Accurate Determination of Milk Chemical Composition and Somatic Cells content Milchprüfring Baden-Württemberg, Germany 5xCombiFTS 500, 1 IBC150 IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Bentley Bartec CombiFTS 600 Up to 600 samples/hour IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Bentley Bartec CombiFTS 500 IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Automation Combination with Combination with Raudszus Conveyor IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Bentley ILAS CombiFTS 600 A Unique Solution – up to 600 samples/hour

Bentley CombiFTS Ilas 600 The optimum solution Complete and fully automated standardisation of your sample preparation and QC: Samples warmed up in a water bath, shaken and identified automatically (RFID, Barcode) Samples warmed up in a water bath, shaken and identified automatically (RFID, Barcode) Samples uncapped automatically Samples uncapped automatically Samples replaced in the tray after analysis Samples replaced in the tray after analysis Pilot samples automatically inserted in the process Pilot samples automatically inserted in the process Highest throughput Highest throughput IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Bentley CombiFTS Somacount FCM (Flow Cytometry) Bentley FTS (FTIR) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Bentley FTS Milk Components Absorption wavebands Protein (NH) Fat A (C=0) Fatty Acids Fat B (CH) Lactose (OH) Frequency of infrared radiation absorbed depending upon type of molecular bonds /vibration mode IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Homogenizer pressure curve IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Somatic Cells Height Distribution (DNA content) Somatic Cells Width Distribution (cells size) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Full Sample & environment traceability For each samples, all the following information is recorded: For each samples, all the following information is recorded: Infrared Spectrum Infrared Spectrum All environmental variables that could affect the end results: All environmental variables that could affect the end results: - Sample, water bath, ambient, homogenizer, deck temperatures - Optical deck Moisture level and pressure - Centerburst energy level (automatic alignment) - Homogenizer pressure - Vibrations…. Somatic cells peak distributions Somatic cells peak distributions IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

FTS Infrared Applications 1) Traditional Components → Fat, Protein, Lactose, Solids, SNF, MUN, FPD 2) New Components available: o Citric Acid o Casein o pH o FFA o Fatty Acids profile ( Saturated, Unsaturated, Mono & Poly Unsaturated,C16:0, C18:0, C18:1, short, medium, long chain FA ….) o Milk Ketone bodies (Acetone/BHB) 3) New developments - Unique Solutions o Blood BHB (patent pending) o Lactoferrin o Iodine value ( o Iodine value (often used to determine the amount of unsaturation in fatty acids (C=C)) o 50 more Fatty Acids calibrations under development) … IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Blood BHB Determination by infrared spectroscopy for the monitoring of the cows metabolic activity and detection of ketosis (patent pending) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin Why predicting Blood BHB instead of Milk BHB? Milk BHB is unstable and levels very low, close to the infrared technology detection level Milk BHB content will decrease rapidly if the milk is not stored at 4°C right after milking Thus, The determination of milk BHB is not very reliable and only targets the Milk BHB content The development of a calibration based on the Blood BHB allows not only to take into account the variation of the ketone bodies but also all the variations in the milk composition that are directly or indirectly linked to the metabolic disorder, and particularly the milk fatty acids profile

Blood BHB Determination by infrared spectroscopy for the monitoring of the cows metabolic activity and detection of ketosis (patent pending) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin Blood BHB Infrared calibration on 696 cow samples

Blood BHB Determination by infrared spectroscopy for the monitoring of cows metabolic activity and detection of ketosis (patent pending) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin Correlation spectrum between the Blood BHB content and the milk spectra The spectral zone with the highest correlation are related to the milk fatty acids profile

Lactoferrin for the early detection of mastitis and monitoring of the sanitary status of the mammary gland Lactoferrin (LF) is an iron binding antimicrobial glycoprotein present in milk Lactoferrin (LF) is an iron binding antimicrobial glycoprotein present in milk Plays a role in pre-immune host defense Plays a role in pre-immune host defense Strong association between LF concentration and occurrence of mastitis Strong association between LF concentration and occurrence of mastitis Could be used to check sanitary status of the mammary glands in the herd Could be used to check sanitary status of the mammary glands in the herd Previously reported normal milk LF concentrations in lactation period were between µg/ml. Previously reported normal milk LF concentrations in lactation period were between µg/ml. 150 DHIA samples selected: 150 DHIA samples selected: - 50 from healthy cow (<200 kSCC) - 50 from healthy cow (<200 kSCC) - 50 with accute mastitis () - 50 with accute mastitis () - 50 with chronical mastitis (> 800 kSCC/3-4 controls) - 50 with chronical mastitis (> 800 kSCC/3-4 controls) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Lactoferrin (LF) for the detection of mastitis and monitoring of the sanitary status of the mammary gland LFir (µg/mL) LFref (µg/mL) Log LFir (µg/mL) Log LFref (µg/mL) IBC/µLLog IBC/µLSCC/µL Log SCC/µL Healthy144,04120,851,861,993142,34951,88 Accute210,32234,462,092, , ,19 Chronic226,98250,662,152, , ,31 Min41151,131,17631,80111,04 Max ,452, , ,00 Mean ,042, , ,79 Sd66,0299,030,240,286213,440,742242,390,72 LFir (µg/mL) LFRef (µg/mL) logLFir (µg/mL) LogLFRef (µg/mL) IBC/µLLog IBC/µLSCC/µL Log SCC/µL Sy,x/LFref77,13 0,21 0,260,220,250,21 r/Log LFRef 0,651,000,420,640,430,68 o Correlation between Log LFir and Log SCC with Log LFref significantly better than normal scale o Correlation between Log (Lfir) and Log(LFref) equivalent to correlation between Log SCC and Log LFref o LF seems to be a good indicator of Cow Mastitis Status and could especially be used to differentiate healthy from accute or chronical mastitis o It has been suggested that bovine milk LF play an important role in host defense against Escherichia Coli mastitis, On the other end, LF thought to have little antibacterial effect on Staphylococcus aureus or Streptococcus agalactia IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Lactoferrin for the detection of mastitis and monitoring of mammary gland sanitary status IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Quantitative vs Qualitative Analysis Limits of Quantitative Analysis : Requires to know the target components to be measured Requires to collect representative calibration samples and classical reference values Advantages of the Qualitative Analysis (spectral database): No classical reference values required No need initially to know the target components/contaminants but requires developing a spectral database of “normal/target” samples Qualitative Analysis can be used : → to detect abnormal samples → to detect adulterated samples (melamine…) → to improve calibration robustness (on-going calibration) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

ν (cm-1) HOW TO DEVELOP A SPECTRAL DATABASE? STEP1: SPECTRA COLLECTION OF « NORMAL /TARGET » SAMPLES IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

DETECTION OF ABNORMAL/ADULTERATED SAMPLES STEP3: PROJECTION OF NEW/UNKNOWN SAMPLES IN SPECTRAL DATABASE IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin ν (cm-1)

DETECTION OF ABNORMAL/ADULTERATED SAMPLES Spectral Approach: IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin -Highly sensitive and exhaustive approach across the entire FTIR spectrum = fingerprinting Spectral approach can be applied: - At the Individual Cow Level(monitoring between controls) - At the Herd Level (checking cow vs herd) - Between herds (checking herd vs herd) - On bulk tank milk samples - To check dairy plant milk process

Bentley FTS Fully Optimir Compatible ( The project aims to develop innovative farm management web applications that will use the spectral analysis of the Milk Recording samples to enable a sustainable and profitable management of the milk production The project aims to develop innovative farm management web applications that will use the spectral analysis of the Milk Recording samples to enable a sustainable and profitable management of the milk production The Mid Infrared (MIR) spectrum is used as a mirror of the cows' state giving indicators on their: The Mid Infrared (MIR) spectrum is used as a mirror of the cows' state giving indicators on their: Fertility Fertility Feeding Feeding Health Health Rejection of pollutants (methane) Rejection of pollutants (methane) Milk Quality Milk Quality FTS spectra are readily available on the instrument as well as all the environmental variables that could affect the quality of the spectra. All data can be exported in real time together with the results IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Bentley FTS/DairySpec FT On-going & Future Developments 1) New calibrations/applications - Quantitative (Fatty acids ( C14:0, C4:0, C6:0, C8:0, C10:0, C12:0, C18:3, C18:3n-3), FFA, Ketones bodies….) - Qualitative (fingerprinting) - Blood BHB, TEM, Lactoferrin, Iodine value…. 2) Rapid determination of milk adulteration - by contaminants - by milk from different animal species 3) Detection of abnormal samples: - Outside the calibration range - Outside laboratory «normal » spectral database 4) Use of 3 rd party calibration/models (Phenofinlait/Optimir…) 5) Somatic cells differentiation IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Somatic Cells Differentiation Mastitis in dairy cattle is the a persistent inflammatory reaction of the udder tissue. This mammary gland infection is the most common disease in dairy cattle. It is also the most costly to the dairy industry (loss of milk production, culling, treatments, inhibitors…) Milk from cows with mastitis has a higher somatic cells content. The total Somatic Cells Count not always sufficient to identify the origin of the infection and reduce the number the incidence of mastitis in the herd. New tools have been developed to improve the mastitis diagnostic or to anticipate the onset of mastitis: Use of the Pathoproof PCR test to identify and quantify the 16 most predominant mastitis bacteria on individual cow or on bulk milk Use of the Lactoferrin which plays a role in the pre-immune host defense system to detect on the onset of mastitis (prevention) Use of BactoCount to screen DHIA Milk Samples Somatic Cells differentiation (PMN, Macrophages (M), Lymphocytes…) Chronic Mastitis: High SCC, High Macrophages, Low PMN Acute Mastitis: Higher SCC, High PMN (up to 90% of SCC) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Somatic Cells Differentiation IBC vs SCC on Individual Cow Milk Samples

Somatic Cells Differentiation IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

CombiFTS Main Features & Benefits Full FT-MIR High Resolution Spectrum (8 cm-1) Full FT-MIR High Resolution Spectrum (8 cm-1) Up to 600 samples/hour Up to 600 samples/hour Fat/Protein/Lactose/Solids/MUN/FPD/Fatty Acids profile+++ Fat/Protein/Lactose/Solids/MUN/FPD/Fatty Acids profile+++ Fully compliant with ICAR/IDF/ISO requirements Fully compliant with ICAR/IDF/ISO requirements Automatic performance checks and adjustment Automatic performance checks and adjustment Automatic rinse/auto-zero function (programmable) Automatic rinse/auto-zero function (programmable) Completely open system+full potential of an FTIR deck Completely open system+full potential of an FTIR deck (spectra access/calibration development) (spectra access/calibration development) Permanent database storage of all raw data (spectra) and diagnostics (complete traceability) Permanent database storage of all raw data (spectra) and diagnostics (complete traceability) Spectral Database retroactive valorisation with new calibration models Spectral Database retroactive valorisation with new calibration models Extensive network remote control and data access Extensive network remote control and data access Full potential of an FTIR deck – no limitations IDF PRIVILEGED SUPPORTING PARTNER IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

Thank you for your attention ! IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin