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AgriQuora . 28th Jul, 2021

Bacterial Spoilage in Milk: Proteolysis, Gas Production And Ropiness

Any form of bacterial spoilage in milk is very significant because dairy products have a relatively limited keeping time and very high perishability. If you fail to apply sufficient preservation methods, these products can go bad really quick and result in massive losses.

The losses are not only in terms of quality but also monetary, as you may have to condemn the whole batch. There is no way you will sell substandard products to your customers for ethical reasons.

Again, defective products may cause cause food poisoning and/or adverse health effects on unsuspecting consumers. Souring of fresh milk is a form of spoilage, especially if it curdles. However, if you intend to make fermented milk products, lactic acid fermentation is a very necessary technology for the process.

Milk is rich in nutrients such as lactose, citrate, butterfat (with fatty acids), among other nitrogenous compounds. Given the fluidity of fresh milk, it exhibits very high water activity (aw) which provides a suitable environment for bacterial proliferation.

Which Bacteria are Responsible for Spoilage?

Other than the lactic acid bacteria (LABs), many other bacteria will produce acid to ferment milk if the conditions are not favorable for the LABs. Therefore, eliminating LABs (if that were possible) will not assure you of safety from acid producing bacteria. For instance,

  • Coliforms will produce a mixture of acids, gases, and alcohols
  • Some species of micrococci, micro-bacterium, and bacilli can produce acid in milk as well
  • Clostridium spp. inhibits the growth of LABs and may produce butyric acid

Please note that insufficient heat treatment of milk will still lead to milk spoilage. If the heat destroys all the vegetative forms of bacteria BUT fails to destroy the Clostridium botulinum spores, the spores will still vegetate and cause butyric acid fermentation.

The defects/bacterial spoilage in milk:

1. Gas Production

Gas production is usually accompanied by acid formation, which is undesirable in milk and milk products.

Some of the most notorious gas formers include coliforms, yeasts, clostridium species, and gas forming bacillus that produce a mixture of carbon (IV) oxide and hydrogen gases.

To identify milk that has gas, check for foam at the top layer of the milk, ripping of the curd, and rapid frothy/stormy fermentation of the milk.

2. Proteolysis

This is the hydrolysis of milk proteins by microorganisms to produce peptides that gives the milk a bitter taste/flavor.

Storage of milk at low temperatures and destruction of lactic acid producers in the milk through heat treatment favor this process. Moulds and yeasts may also destroy any lactic acid formed in the milk and accelerate the process.

Proteolytic bacteria produce the following forms of bacterial spoilage in milk:

  1. Acid proteolysis, which involves production of acid and proteolysis
  2. Proteolysis that produces both acidity and alkalinity
  3. Sweet curdling, which results from the activity of the rennin-like enzymes that these bacteria produce at the early stages of proteolysis
  4. Slow proteolysis, which result from the activity of the bacterial endo-enzymes after autolysis

Acid proteolysis leads to production of a lot of whey and formation of shrunken curd. The bacteria further digests the curd, which changes the color of the curd from opaque to a little translucent. Some bacteria may completely dissolve the curd.

There are three major causes of acid proteolysis, namely:

  • Micrococcus spp.: - these are very notorious and may even cause the proteolysis of freshly drawn milk as some of them inhabit the cow’s udder
  • Streptococcus faecalis and Streptococcus liquifasciens: - very active proteolytic bacteria that may cause proteolysis of pasteurized milk
  • Spores of some strains of Bacillus spp.: - especially the lactose fermenting and proteolytic strains such as Bacillus cereus, which can survive high temperature pasteurization and cause acid proteolysis.

Some bacteria that may not be able to ferment lactic acid may still cause proteolysis and this varies with different species of bacteria. Some strains may act on the casein directly and produce little proteolysis.

The following three scenarios are very likely to occur following the action of these bacteria:

  • They may produce very little or non-existent acidity making the milk alkaline
  • Most of these bacteria cause sweet curdling of the milk before they digest the casein
  • Others hydrolyze the protein extremely fast that no curdling is observed. This results into a clear liquid that lacks curd.

The most active proteolytic bacteria are found among the following species of bacteria.

Non-spore forming bacteria include:

  • Micrococcus
  • Alcaligenes
  • Achromobacter
  • Pseudomonas
  • Proteus
  • Flavobacterium

Spore forming bacteria include:

  • Bacillus spp.
  • Clostridium spp.

Most of these bacteria can grow and cause proteolysis and bitterness of milk that is held at chilling temperature. However, most of them are thermoduric (except some species of micrococci) and should not be present in pasteurized milk.

Proteolysis by the endo-enzymes after bacterial autolysis is slow and insignificant in milk. However, when long time is allowed for the process, it can be significant, especially in the curing of cheese.

3. Ropiness

Ropiness is a form of bacterial spoilage in milk that makes the milk highly viscous or sticky. Ropy milk has characteristic silk-like threads that may vary in length from a few inches to several feet.

You would test for this by dipping a pointed device (like a needle) on the surface of the milk (after incubating for 12-48 hours) and raising the needle to see if there is presence of a “rope.”

To test for #ropiness in milk: dip a pointed device on the surface & raise to check for a “rope."

This defect affects milk, cream and whey. The effect is very significant in milk and cream meant for the market.

Ropiness can be classified as either bacterial or non-bacterial in nature

Bacterial Ropiness

Caused by a slimy capsular material produced by the bacterial cells (which is usually either gums or mucus).

You can further classify bacterial ropiness under these two categories:

  • Surface Ropiness: - observed at the top of the milk and is caused by Alcalegenes viscolactis, which is majorly found in the soil and water.
  • Ropiness observed throughout the milk: - caused by:
  1. Some coliforms, (e.g. Enterobacter aerogenes, Enterobacter cloacae, and in rare curcumstances Escherichia coli.)
  2. Some species of LABs, (e.g. Streptococcus lactis, Lactobacillus bulgaricus, Lactobacillus cereus, Streptococcus cremoris, and Lactobacillus plantarum). Most of these microorganisms grow in chains, which leads to the formation of the ropes in milk.
  3. Other microbes like Micrococci and Bacilli.

Non-bacterial Ropiness

This form of bacterial spoilage in milk may occur as a result of:

  1. Presence of mastitis in the milk, especially fibrin and leucocytes from the cow’s blood present in the milk.
  2. Thick cream at the top of the milk
  3. Casein film or lactalbumin that occur in milk during cooling

Common Defects in Fermented Milks

Fermented milks have a better keeping quality than fresh milk due to the low pH that do not favor growth of a wide range of bacteria. However, there still exist a variety of defects in fermented milks that can affect appearance, texture, or flavor.

To get a clear understanding of these defects, it is best that we classify the defects as follows:

  1. Defects resulting in changes of appearance
  2. Flavor and aroma defects
  3. Defects of consistency, body and texture, and viscosity

Some of the very common defects under these categories, their causes, and prevention methods include:

Appearance defects in fermented milks

Wheying-off/whey separation- Disturbance of the coagulum during incubation
- High inoculation rate leading to excessive acidification
- Incorporation of excessive air
- Too low total solids
- Avoid mechanical shaking during coagulation
- Proper incubation period
- Correct storage temperature
- Correct standardization
Stale/aged- A film is formed on the surface due to freezing of the product
- The package is smeared over/damaged
- Avoid freezing of the product
- Ensure careful handling of the product during packaging and storage
Cream line- Insufficient stirring of milk
- Insufficient or absence of homogenization
- Homogenize effectively
- Thoroughly and evenly stir the product to obtain a sufficiently homogenous product
Mould/yeast colonies formed on the surface- Poor hygiene leading to mould and yeast growth- Maintain hygiene during production and product handling
Inhomogenous appearance- Insufficient stirring of the product after fermentation- Thoroughly and evenly stir the product after fermentation
Gas formation- Contamination by gas producing microorganisms- Maintain hygiene during production and product handling
Discoloration- Heating/low pH- Use stable coloring agent

Flavour defects in fermented milks

No coagulation/no sourness/slow coagulation- Inhibitors in milk
- High inoculation temperature or low inoculation rate
- Starter culture strain imbalance cocci:rods
- Fresh, inhibitor-free milk
- Young and active starter culture
- Correct inoculation temperatures at 23-25°C
- 2-3% of starter culture
Bitter taste- Too long storage
- Contamination with highly proteolytic bacteria or enzymes
- Normal storage (two weeks under cold storage)
- Avoid contaminants by ensuring high production hygiene
Yeast/fruity flavour- Contamination by yeasts- Ensure high standards of production hygiene as yeasts can withstand very high acid conditions
Rancid flavour- Fat breakdown by lipolytic microrganisms
- Insufficient heat treatment of milk
- Maintain high production hygiene
- Ensure proper high temperature pasteurization of milk (85°C/30minutes or 90°C/5minutes or 121°C/5seconds)
High acid/very sour- Too high acidification during and after incubation
- High inoculation rates and high storage temperatures
- Correct incubation period
- Cool down immediately after incubation

Consistency and viscosity defects in feremented milks

Slimy/ropy/stringy- Contamination with slime (exopolysaccharides) producing bacteria e.g. Leuconostocs and lactobacilli
- Too low incubation temperature
- Maintain proper starter culture ratio
- Use active starter culture
- Maintain high production and product handling hygiene
Sandy- Contraction of the gel particles
- Early stirring of the coagulum
- Stirring coagulum at high temperatures above 38°C and holding at this temperature
- Avoid mechanical disturbance before coagulation
- Stir after cooling of the product
Gummy- Unsuitable stabilizers
- Excessive addition of stabilizers
- Faulty incorporation of stabilizers
- Ensure correct amount of stabilizer and application procedure
Thin/liquid- Too strong stirring of the gel
- Low total solids due to adulteration of the milk
- Too short cold storage period
- Gentle and thorough stirring of the gel
- Use unadulterated milk with normal total solids
Wheying-off- High acidification before cooling
- Insufficient cooling
- High inoculation rate
- Low temperature pasteurization
- Proper incubation period
- Proper pasteurization temperatures (85°C/30minutes or 90°C/5minutes or 121°C/5seconds)
- Cool after the set incubation time
Soft/soupy- Low protein content in milk as a result of adulteration
- Low inoculation rate
- Too short incubation period
- Mechanical shaking before coagulation is complete
- Standardize to proper total solids
- Use normal milk disturbance

How to Troubleshoot Major Defects of Fermented Milk Products

Fermented dairy products require careful handling during processing and storage. Failure to observe some of the basic practices as required would lead to serious defects of fermented milk products.

To better understand how to troubleshoot and handle these defects, we are going to divide them into three categories;

  1. Flavor defects
  2. Appearance defects
  3. Texture defects

Each of these categories contain many defects. We will enumerate the defects and illustrate their causes and possible prevention measures.

It is important to understand each defect, its causes, and prevention measures so that you do not end up with poor quality product.


CausesPrevention Measures
1. Unclean/Feed flavor
Old Raw Milk- Do not process old milk (max 48h)
- Change to another batch of old milk
- Only use milk with low bacterial and yeast counts
Contamination of equipment/packaging material- Control cleaning and disinfection
Inadequate de-aeration of milk- Apply/optimize vacuum de-aeration
Too low acidification from starter culture- Increase propagation temperatures increase inoculation rate
Contaminated fruit preparation or sugar- Micro-count, yeast and mould count of all ingredients
2. Bitter/Cheesy flavor
Poor raw milk quality- Do not process old milk (max, 48h) Only use milk with low bacteria counts
- Examine raw milk for protein decomposing germs(Pseudomonas flourescens)
- Exclude milk with bitter agents from feed (potatoes, legumes etc.)
Poor ingredient quality- Make sure all ingredients are fresh; with low bacteria count
Too high whey protein content/ inadequate WPC- Reduce content of whey protein powder
- Change whey protein content (WPC) source to a less proteolytic product
Poor sanitation/ contamination with proteolytic micro-organisms- Make sure that all equipment is sterile and in good repair/condition
Water contaminated with Pseudomonas flourescens- Control water quality
Application of old/contaminated bulk starter- Propagate new bulk starter
Starter cultures with high proteolytic activity- Change starter culture
Too warm or too long storage of finished product- Ensure constant cooling chain, keep best before date
3. Rancid flavor
Poor ingredients- Assure high quality ingredients Do sensory test for milk powder before use
Pasteurised milk contaminated with raw milk- Control regeneration area in pasteurizer for leaks
Fat degradation by contaminants- Increase bacteriological control of production
Insufficient heat treatment- Pasteurization 90-95 0C/5-10 minutes
Excessive mechanical treatment of raw milk- Look for and eliminate all points where excessive shear occurs in the raw milk stream
4. Oxidized/Tallow/Metallic flavor
Poor Ingredients- Do not process old milk (max. 48h after milking)
- Control raw milk for off-flavors
- Use high quality milk powder and other ingredients
Transfer of metal ions (iron, copper etc.) to the milk or yoghurt- Remove all product contact surfaces that are not stainless
Incorporation of air by defect pumps/pipes or leaky valves/fittings- Control pumps/pipes/valves/fittings
Too low homogenization temperature- Homogenization temperature>60o C
Exposure to sunlight- Use light-proof retail packaging material
- Avoid application of vats, pumps and pipes that are not protected against light
Residuals of cleaning agents- Ensure sufficient rinsing before processing milk
- Use adequate cleaning agents/disinfectants
5. Soapy flavor
Residues of cleaning agents- Adequate rinsing of equipment
- Application of suitable cleaning agents
Old or degenerated cultures from bulk starter- Save bulk starter≤2 days
6. Cooked/ Burned/ Scorched flavor
Poor quality off ingredients- Do not add scorched milk powder/ condensed milk
- Use high quality ingredients
Excessive heat treatment of milk- Pasteurization 90-95 o C/5-10
Burned on pasteurizer- Clean pasteurizer
7. Malty flavor
Contamination with Lc. Lactis var. maltigenes malt flavor forming germs- Processing of adequate, fresh milk (max. 48h)
8. Mouldy / Yeasty flavor
Too high concentration of whey protein/ whey protein concentration in formula- Reduce whey protein content
9. Too sweet/ Lack of sweetness
Improper formulation- Increase or decrease sweetness with  sugar: adjust final acidity ( higher acid-less sweet)
Improper pH of final product- Make sure of proper pH at break (pH≤4.5) Use more acidic/less acidic starter culture
10. High acid/ sour flavor
Excessive incubation/ Post acidification- Ensure quick reduction in temperature at the end of fermentation
- Avoid high storage temperatures/keep cooling chain
- Change to a less acidic starter culture with lower post-acidification
11. Low acid/ Flat/Stale (No aroma)
Low solids content of the process milk- Increase milk solids content in formula
Improper pH of final product- Make sure of proper pH at break (pH≤4.5)
Inhibitory substances (penicillin) present in milk- Check raw milk (inhibitor-test)
Starter culture does not form enough acid and/ or yoghurt flavor- Apply more acidic / aromatic starter culture
- Increase fermentation temperature starter or direct vat inoculation (DVI)
- Change bulk starter media
- Only use fresh (< 2days) bulk starter
Phage attack on starter culture- Use phage alternative culture Take action to improve sanitation
Too low inoculation rate of starter culture- Increase inoculation of bulk starter or DVS innoculation
Excessive heat treatment of the fruit base and flavoring concentrate- Reduce heating temperature/time
Blunting the fruit acid by neutralisation- Do not neutralize the acid
12. Artificial flavor
Application of unsuitable flavor- Change flavor compound
Too high concentration of fruit base and flavorings- Reduce dosage

Having understood the causes and prevention measures for the flavor defects, let us now look at the appearance defects of fermented milk products.

These are the defects that you see on the product.


CausesPrevention Measures
1. Syneresis/whey separation
Contamination of the milk with gas-forming micro-organisms- Check delivered milk and culture for gas formers, aerobic spore formers (B. cereus)
- Only use fresh milk (<48h)
Aqueous yoghurt milk- Control freezing point
Insufficient fat content in formula- Increase fat content
Low milk solids non-fat content in formula- Increase milk solids non-fat (MSNF)
Insufficient stabilization level and/or unsuitable stabilizer- Add/ increase addition/ change stabilizer system
Insufficient heat treatment- Pasteurize at 90-95o C/5-10 minutes
Insufficient homogenization pressure- Check homogenization pressure (> 200 bar)
Homogenization temperature too high- Homogenization temperature 60-68o C
Starter culture too fast- Change to a slower fermenting starter culture
- Lower fermentation temperature
Coagulum disturbed at pH >4.6- Avoid any movement of the coagulum while it is being formed
- Don’t agitate the coagulum before pH 4.5 is reached
The coagulum has not been agitated sufficiently after fermentation- Increase agitation before/ during cooling
Excessive mechanical treatment- Avoid agitating for too long or too vigorously when: - Breaking pH (stirred yoghurt), - Cooling, - Adding fruit preparation/ flavors, - Filling Apply gentle pumps and agitation devices
Unsuitable fruit preparation- Use suitable fruit preparation:
- Optimized hydrocolloids,
- 35-50%  fruit content
- 10-60% soluble solids (typically 35-40%SS)
- pH 3.8-4.0 (for twin pot yoghurt pH 3.2-3.5)
Post-acidification due to delayed or lengthy cooling (stirred yoghurt)- Efficient cooling immediately after breaking the coagulum
- Use starter culture with lower post-acidification
Air incorporation during processing and filling- Limit air intake during processing
pH too low on filling(set yoghurt)- Avoid filling at pH <6.0
Temperature too low on filling (stirred yoghurt)- Increase the filling temperature to22-25o C
Excessive vibration of the finished product- Allow the yoghurt to redevelop viscosity by keeping it undisturbed in cold storage for min, 24 hours
- Instruct handlers to avoid vibrations due to their negative influence on yoghurt quality
Inconstant temperatures (especially too high) during storage of the finished product- Ensure constant cooling chain
2. Cream separation
Homogenization pressure too low- Homogenization pressure should be >150 bar  (150-250 bar)
3. Light and frothy
Incorporation of air due to excessive agitation of the milk after the addition of culture- Avoid strong agitation as well as pumping after adding the culture
- Avoid the incorporation of air in any way
Contamination by gas-forming bacteria like coliforms or lactic yeasts- Microbiological control of milk and equipment
- Increase disinfection and cleaning of equipment
- Increase pasteurization temperature/time
Frothy surface due to unsuitable filling device- Replace pump seals (regular control) Repair/change filling machinery
4. Graininess
Improper formulation- Decrease MSNF in formulation
- Change formulation to a more suitable starch type
- Optimize stabilizer system/ usage rate
Starter culture not uniformly distributed- Agitate sufficiently when adding starter culture
- Make sure the culture is completely thawed before stopping agitation
Fermentation too fast- Lower the fermentation temperature
- Decrease inoculation rate of starter culture Change to a slower starter culture with higher polysaccharide (EPS) formation
Breaking of coagulum at excessively high pH- Break only at pH ≤4.5
Insufficient mechanical treatment after breaking up coagulum (stirred yoghurt)- Use a smoothing valve in line
5. Color leaching
Difference in osmotic pressure too high in the fruit preparation and the white mass- Choose a fruit preparation that is designed not to leach color (proper sweeteners)
- Substitute some or all the sucrose in the white mass with fructose
Fruit preparation insufficiently stabilized- Use suitable fruit preparation (optimized hydrocolloids)
6. Yeasty/mouldy
Process milk or yoghurt contaminated with yeast/moulds: - contaminated ambient air/production rooms, - residues of rinse water in pumps and pipes, - filling devices or packaging material, - fruit preparation- Ventilate production rooms sufficiently (laminar flow or filtrated air)
- Ensure that packaging material is sealed
- Correct storage of packaging material
- Use adequate cleaning agents/ disinfection of equipment and production room
- Control all incoming ingredients/ fruit preparations for yeast and mould counts
- Use protective starter cultures

Finally, let us now look at the common texture defects of fermented dairy products and how to prevent them. They are some of the serious defects of fermented milk products you will ever encounter as a processor.


CausesPrevention Measures
1. Low Viscosity
Improper formulationIncrease milk solids non-fat (MSNF) by adding suitable milk proteins Add/ optimize stabilizer system
Low fat contentIncrease fat content
Aqueous yoghurt milkControl freezing point, change batch or compensate with addition of MSNF
Milk containing inhibitorsExamine raw milk for inhibitors
Insufficient heat treatmentPasteurization 90-95 o C/5-10 minutes
Insufficient homogenization pressureControl homogenization pressure (>200 bar) Is the homogenizer working properly?
Improper starter cultureChange to a starter culture with higher polysaccharide (EPS) formation
Insufficient inoculation of starter cultureIncrease culture dosage
Bacteriophage attack on starter cultureChange culture
Incorrect fermentation temperatureDepending on the starter culture, increase or reduce fermentation temperature
Disturbing the coagulum at pH>4.6- Avoid any disturbance of the coagulum while its being formed
- Don’t agitate coagulum before pH 4.5 is reached
If the coagulum has been cooled before agitation, it becomes much more sensitive to mechanical treatment, and will quickly lose its viscosityStir the coagulum before cooling
Excessive mechanical treatment- Avoid too long and too strong agitation at Breaking pH (stirred yoghurt)
- Cooling
- Addition of fruit preparation/flavors
- Filling - apply gentle pumps and agitation devices
Insufficiently concentrated flavorings/fruit base- Use concentrated flavorings and fruit preparations
- Use stabilized fruit preparation with texturing effect in the yoghurt mass (stirred yoghurt)
Air incorporation during processing and fillingLimit air intake during processing
Too low temperature at filling (stirred yoghurt)Increase the filling temperature to 22-25 o C
Too fast cooling from filling temperature to final storage temperatureIncrease cooling time of secondary cooling ( from primary cooling 25o C to final cooling 5o C)
Excessive vibrations of the finished productAllow the yoghurt to redevelop viscosity by keeping it undisturbed in cold storage for 24 hours
Unsuitable stabilizer blend (high gelatin content) when stored at high temperatures (> 15-20 o C)If  the storage temperature are high, change to a more suitable stabilizer blend
2. Too firm/ hard curd
Improper formulationOptimize stabilizer usage Reduce milk solids non fat
3. Soft/ smeary product
Contamination by residues of cleaning agentsControl cleaning of equipment (CIP device) Careful rinsing of the whole equipment with suitable water
Contamination by alkali-forming or protein-decomposing micro-organismsControl fermentation equipment for possible raw milk contamination
Improper formulationIncrease milk solids non-fat (MSNF) by adding suitable milk proteins Add/ optimize stabilizer system
Too low incubation (set yoghurt) or inconstant temperature in incubation chamberControl filling and incubation temperatures (37-43o C depending on culture)
Cooling at too high pH (set yoghurt)Control pH and cool in time. Optimal cooling pH 4.65-4.80 depending on the culture
4. Stringy/Slimy/Ropy
Too long pre-storage of the raw milk at deep temperaturesUse adequate fresh milk
Contamination of the milk by slime-forming bacteria (influence of feeding)Control raw milk. Acid cleaning and disinfection of equipment
Too high sugar content in formulationReduce sugar content or change to a starter culture that is less sensitive to high sugar /solids content
Degeneration in the bulk starterPermanent control of bulk starter production and correct (and not too long) storage
Too low incubation temperatureIncrease incubation temperature (37-43o C depending on culture)
Improper cultureChange to a culture that produces less exo-polysaccharides
5. Lumpy
Too high gelatin content on formulaUse less gelatin in the stabilizer or reduce dosage of stabilizer
Insufficient agitation when breaking up coagulum (stirred yoghurt)Increase mechanical treatment Use a smoothing valve in line.
Filling temperature too high (stirred yoghurt)Fill at a lower temperature (22-24o C)
Filling into cups at improper pH value (set yoghurt)Filling of set yoghurt at pH > 5.7
6. Sandy/ gritty
Inadequate protein combination in formulaChange formula to a apply more adequate milk protein source
Excessive pasteurizationPasteurization 90-95 o C/5-10 minutes
Vibrations during incubation(set yoghurt)Avoid application of any mechanical force during incubation
Irregular temperature during incubationControl/monitor temperature during incubation
Agitating the gel above 38o C followed by holding the temperature above 38o CCool the yoghurt immediately after breaking the coagulum
Uneven agitation of the coagulum (stirred yoghurt)Agitate the coagulum more evenly
7. Dry
Improper formulation (inadequate or too much starch or protein)Optimize stabilizer blend Use adequate milk protein source
Improper hydration of proteinEnsure proper hydration (temperature/time) for dry ingredients used in formula
Insufficient homogenisationHomogenization> 150 bar (150-250 bar)/ 60-68o C
8. Jelly/ gel-like
Too high  stabilizationAdd less stabilizer (especially if it contains gelatin)
Fruit preparation with gelling properties (set yoghurt, fruit on bottom)Use a fruit preparation with no interfacial (to the white mass) interaction

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