W3 L3: The microbiology of milk and honey 

What is milk?

• Emulsion of fat & water containing dissolved carbohydrates, proteins, vits & minerals - that are produced in/ transported to mammary gland to provide complete nutrition & immunological protection to newborns

Components of milk:

• Water ~ 87%
• Protein ~ 3.5 %
• Fat ~ 4%
• Lactose ~ 4.7%
• Minerals ~ 0.8%

Water activity (aw) → 0.99

pH → 6.4-6.6

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How is milk produced? lactation

• Following digestion necessary nutrients are absorbed from intestines into blood stream

• Nutrients are delivered to udder (high supply of blood) → allows large vol. of milk to be produced

• Nutrients used to produce accumulated milk, then secreted

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• Udder is highly developed & modified sweat gland - in cattle is composed of 4 individual glands → quarters

• Interior of each quarter comprises → teat cistern, gland cistern, milk ducts & glandular tissue

• Glandular tissue→ contains millions of microscopic sacs - alveoli

  • Each alveolus is lined w/ milk-producing epithelial cells & surrounded by muscle cells that contract to squeeze milk into milk ducts when stimulated during milking (calf sucking)

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Milk formation

• Milk secreted from epithelial cells into lumen of alveoli
• Substances e.g. water, minerals, vits & immunoglobulins can pass cell membrane from blood stream
• Substance inc. proteins, lactose & fat are produced in secretory cells - then transported into lumen
• Amt. of milk regulated by lactose by influencing osmotic pressure b/w blood & alveoli

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Bacteria in mammary gland

• Cross-talk b/w milk microbiota, epithelial cells & immune cells maintain a balanced, healthy environment
• Microbial imbalance that leads to infection→ commensal bacteria barely inhibit the pathogen (1); immune & epithelial cells only respond to the pathogen (2-3)
  • results in massive production of pro-inflammatory mediators (cytokines, chemokines, AMPs)- causes the attraction of additional activated immune cells (4)→ leads to mastitis

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Origin of human milk bacteria

• Dendritic cells go across gut epithelium to directly take up bacteria from gut lumen

• Once associated w/ dendritic cells, live bacteria spread to other locations through bloodstream

• Dendritic cells migrate using enteromammary pathway via mesenteric lymph node, so bacteria arrive at mammary gland

  • mechanism explains presence & abundance of maternal gut bacteria in colostrum & breast milk

• Milk microbiota, breast milk microbiota & infant oral microbiota all continue travelling until infant gut is reached

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Enteromammary pathway

• When pathogen enters maternal gut, antigens are presented to immune cells that travel via blood
• IgA production induced at mammary cells & secreted as component of milk to protect infant

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Maternal gut microbiome regulates neonatal gut microbiome via IgGs

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• Maternal antibodies transferred placentally before birth to fetus & via breast milk to neonate after birth
• After birth, maternal milk provides 1st source of antibody-mediated protection in intestinal tract of infants against infection
• Gut microbiome can induce antigen-specific immunoglobulin G (IgG) → cross-reacts w/ pathogen antigens to promote systemic pathogen eradication in humans & animals
• Gut microbiome-induced IgG antibodies exhibit bias against Gram -ve Enterobacteriaceae e.g. E.coli - common causative bacterium in neonatal infections
• Maternal IgG antibodies cooperate w/ IgAs in neonatal gut
• Recent studies → gut microbiome-induced IgG antibodies transferred from serum to maternal milk in process facilitated by neonatal Fc receptor (FcRn)→ ↑ levels of IgG & IgA in neonatal intestine than in adult intestine & robust IgG & IgA coating of gut commensal bacteria
• FcRn expressed at high levels in epithelial cells in human mammary glands → facilitates transfer of serum IgG to maternal milk & in neonatal intestinal enterocytes to facilitate uptake of maternal milk & transcytosis to circulation

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• IgGs in maternal milk recognise proteins of Gram -ve bacteria
• FcRn mediates transfer of gut microbiome-reactive maternal IgG to neonatal intestine- directly targets gut bacteria
• Induction of faecal bacteria-specific serum IgG after transplantation of IgG+ or IgG- luminal bacteria from WT to germ-free neonates
• Imaging of luminescence-expressing C rodentium on mucosa of cecum & colon of WT & Fcgrt -/- neonatal mice after infection

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Milk natural antimicrobial systems

• Antibodies→ IgA, IgG

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• Lactoperoxidase→ generates short lived [O] intermediates e.g. hypothiocyanite - effective in killing aerobic & anaerobic bacteria

• Xanthine oxidase→ produces antimicrobial radicals such as superoxide, nitric oxide and peroxynitrite

• Lysozyme→ degrades bacterial cell wall of Gram-positives

• Lactoferrin→ binds iron and withholds

• Phagocytes

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Milk Distribution

==Historically== Now

• ==No temperature control== → Industrialisation
• ==Short distribution chains== → Long distribution chains, from farms to urban centres
• ==Preservation not that important==→ preservation is essential

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Milk distribution: factors affecting milk quality

MILK PRODUCTION→ MILK COLLECTION→ MILK CHILLING & STORAGE→ MILK PACKAGING→heat treatment→ TRANSPORT→ CONSUMER PRACTICES→ Prevent Mastitis

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Important Risk Factors

• Health status, housing & herd size, silage, water source & waste management

• Milk practices, mastitis control measures, Equipment cleaning and maintenance

• Efficiency of chilling practices, equipment, personnel hygiene & sanitation

• Maintenance of chill temperatures equipment, personnel hygiene & sanitation

• Efficiency of pasteurisation

• Maintenance of chill temp- adherence to use-by-dates

  →PREVENT MICROBIAL HAZARDS

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Quality in distribution chain

• Mastitis prevention
• temp control
• heating

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Mastitis

• Inflammation of mammary glands due to ↑ level of bacteria & somatic cells, w/ the subsequent ↓ in milk quality
• Causes major losses in milk production → clinical (25 cases per 100 cows/year) or subclinical (15-20% cows)
• Caused by 137 different organisms but 5 cause over 80% of infections:
  • Staphylococcus aureus
  • Streptococcus agalactiae
  • Streptococcus dysgalactiae
  • Streptococcus uberis
  • E. coli

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Methods to prevent mastitis

• Provision of clean litter
• Rapid removal of slurry
• Prevention of muddy areas
• Shave udders
• trim tails Wash teats with disinfectant
• Dry teats
• Keep parlour floor clean
• Clean teat cups
• Discard foremilk

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Detection

• Main indicators of milk quality

  • Somatic cell count (SCC) → Plate count (PC)

• Somatic cells are a mixture of milk-producing cells (1-2%) & immune cells (98-99%)

  • SCC < 100,000 cells/mL = no infection
  • 200,000 cells/mL = mastitis

• EU regulations:

  • PC < 100,000 mesophiles per mL
  • SCC < 400,000 cells per mL

• Milk buyers pay a premium of 3-5% of milk price below threshold of 200,000 and apply reductions of 5-10% if above

  

The influence of farm hygiene practice

• Bacterial counts (CFU/ml)

  

Temp control

• Farm bulk tank→ refrigerated (< 4ºC for < 48h) = 103 CFU/mL
• Road tanker→ insulated (< 6ºC for 1-8h) = 103 -104 CFU/mL
  • Rejected if > 7ºC
• Silo at dairy→ insulated (6-8ºC) or refrigerated (2-4ºC)
  • < 104 CFU/mL in the silo
  • < 105 CFU/mL before pasteurisation

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Heating: pasteurisation/UHT

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Milk spoilage → Psychrotrophic bacteria

• Refrigerated raw milk may contain psychrotrophic bacteria that produce thermoresistant exo-proteases and lipases → compromise the quality of dairy products during storage
• Carbohydrates
  • 𝘓𝘢𝘤𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘭𝘢𝘤𝘵𝘪𝘴 converts: lactose→ Lactic acid (produces sour taste/smell?)
• Proteins
  • 𝘓𝘢𝘤𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴, 𝘌𝘯𝘵𝘦𝘳𝘰𝘣𝘢𝘤𝘵𝘦𝘳, 𝘚𝘦𝘳𝘳𝘢𝘵𝘪𝘢, 𝘈𝘦𝘳𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘢𝘯𝘥 𝘉𝘢𝘤𝘪𝘭𝘭𝘶𝘴 converts: Caseins & whey proteins → Short peptides, amino acids, amines (produces bitter, putrid smell/ taste?)
• Lipids
  • 𝘓𝘢𝘤𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴, 𝘈𝘦𝘳𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘢𝘯𝘥 𝘈𝘤𝘪𝘯𝘦𝘵𝘰𝘣𝘢𝘤𝘵𝘦𝘳 converts: Short-chain fatty acids (produces rancid flavour)

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Microbial hazards in milk today

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Milk legislations & testing

• EU Council Directive 92/46/ECC lays down the health rules for the production and distribution of milk and dairy product on the market

• Milk can only come from herds that are officially TB-free (and Brucellosis-free)

• Pasteurised milk must pass the phosphatase test to assure the effectiveness of the process

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Honey

Sweet, viscous substance made from floral nectar by bees & some related insects

Produced after ingestion, enzymatic activity, regurgitation & H2O evaporation

• Water~18%
• Fructose~40%
• Glucose~ 30%
• Other sugars~ 10%
• Minerals~ 2%
• water activity (aw) → 0.60
• pH → 3.4 - 5

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History

• Earliest evidence of humans collecting honey is a cave-painting in Valencia, on Spain's eastern coast, thought to date from around 8000 BC
• Since about 4000 BC, the ancient Hindi medical theory of Ayurveda outlined honey's medicinal qualities in treating burns, allergies & infections
• Western cultures have eventually caught up by devising honey-based wound dressings & oral medicines.
• Composition of honey varies greatly - depends on the local flora in the bees' immediate environment
• Bees visit various flowers making honey w/ diff. healing properties - scope for finding new uses for honey is vast.

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How is it produced?

1. Bees collect nectar using their tongue
2. It goes to their honey stomach (40 mg of nectar)
3. Enzymes break down sucrose into glucose & fructose
4. Digested nectar is regurgitated, placed in honeycomb cells & left unsealed
5. Fermentation → LAB & yeasts (acidity)
6. Bees flutter their wings to circulate air & evaporate H2O (sugar conc. ↑ & then sealed with wax)
7. Food supply (E) or removed by beekeepers

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