SLAUGHTERING TECHNIQUES , POST-MORTEM INSPECTIONS &  MEAT HYGIENE /SANITATION IN MEAT AND POULTRY  

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SLAUGHTERING TECHNIQUES , POST-MORTEM INSPECTIONS &  MEAT HYGIENE /SANITATION IN MEAT AND POULTRY

 

Post no-1454 Dt-29/02/2020
Compiled & shared by-DR RAJESH KUMAR SINGH,
JAMSHEDPUR, JHARKHAND,INDIA
9431309542,rajeshsinghvet@gmail.com

               

 

PRE SLAUGHTER HANDLING AND INSPECTION OF ANIMALS

 

Pre-slaughter handling of meat animals includes procedures adopted during transportation, pre-slaughter rest and ante mortem inspection.

 

Transportation of Meat Animals

Animals are taken to the slaughter house either on hoof or through road/rail/sea transport. Whatever the means, what is of paramount importance is that the animals must not be subjected to cruelty. There are legislations against cruelty to animals and procedures recommended need to be strictly adhered to. If the animals are carried by road or rail care needs to be taken to avoid overcrowding which may lead to suffocation and physical injuries in the form of bruises, fractures etc. Tying or chaining of the animals is to be avoided. There are specially designed trucks and wagons available to take care of such issues and should be used as far as possible.

 

Pre-Slaughter Rest

�       Pre-slaughter rest is the period before slaughter when animals are rested in order to improve the meat quality and reduce the chances of contamination with gastrointestinal bacteria.

�       During pre-slaughter rest fatigue could have negative influence on protective functions of the animals (low immunity) allowing rapid penetration of the microorganisms especially E Coli through mucous membrane of the intestine to the blood stream and ultimately to the organs.

�       Normal feeding and resting for 48 hours brings gradual freeing of muscles and other organs from microorganisms, improve bleeding and keeping quality of meat.

�       Pre-slaughter rest also helps in preserving glycogen level in the muscles and during the later phase this glycogen undergoes anaerobic respiration and results in production of lactic acids which has mild preservation effect on meat quality

�       Under stressed conditions muscles do not receive enough oxygen which leads to higher lactic acid production often associated with comprised quality in terms of colour, texture and water holding capacity.

 

Ante-Mortem Inspection

It refers to inspection of food animals conducted prior to (12 to 24 hr) slaughter to ascertain fitness or otherwise of the animals for slaughter.

 Objectives of ante-mortem inspection

1)      To detect the animals suffering from infectious or scheduled diseases (communicable to animals or human beings) such as FMD, Rabies, Anthrax, etc.

2)      To detect diseases causing toxic or infectious conditions and which may escape detection in post mortem examination.

3)      To prevent outbreaks of food poisoning resulting from the consumption of meat from animals which were ill at slaughter

4)      To make post-mortem inspection more efficient and less laborious.

5)      To document information on animal diseases prevalent in the region.

To prevent the use of meat from animals suffering from febrile condition.

 

Facilities for conducting ante-mortem inspection

  1. For successful ante-mortem examination, procedure for adequate identification of the animals to be slaughtered need to be followed.
  2. The lairage should be properly designed, well lighted and ventilated
  3. The lairage should have provision for isolation pens
  4. There should be competent assistant staff for handling of livestock and above all a competent veterinary officer must be available.
  5. A well designed code on veterinary ante-mortem inspection procedures, judgement principles and documentation of findings need to be maintained.

 

Procedure for ante-mortem inspection

Immediately after the animals arrive at the slaughterhouse, they are examined by qualified veterinary personnel before being sent to the lairage where ante-mortem inspection is carried out 12 to 24 hours before slaughter by the qualified veterinarians.

Ante-mortem inspection can be carried out in two stages:

 

Stage-I: General examination

The animals are to be examined collectively to assess the overall health status so as to segregate them into three groups:

1)      Healthy

2)      Unhealthy (diseased)

3)      Apparently healthy (doubtful cases)

�      The animals are examined while they are at rest and also in motion

�      They are observed for the gait, posture, fatigueness and for their response to external stimuli.

�      They are examined for abnormal behavior like walking in circles, state of alertness, symptoms of tiredness and agitation

�      Animals are examined for abnormal discharge from natural orifices like eyes, nose, mouth, anus, vagina etc. and/or swellings on any part of the body.

�      It is also important to observe for any evidence of cruelty to animals such as any sign of bruises, torn skin, and fractured bone indicating carelessness during transportation.

At the end of the first stage of examination, healthy animals are cleared as fit for slaughter whereas the other two groups are taken to the next stage of examination.

 

Stage-II. Detailed clinical examination

The animals classified as unhealthy (diseased) are subjected to further examination to correctly diagnose the illness while the animals placed in group three (Apparently healthy or doubtful cases) are examined to ascertain whether these animals are really sick and if so what could be the nature of illness.

Animals are subjected to the following investigations:

(i)        Temperature, pulse and respiration rate should be recorded and animals suffering from fever must be retained for treatment preferably outside the meat plant.

(ii)      The lymph nodes are palpated and examined for any swelling and abnormalities and on this basis the animals are either rejected or passed with clear instructions for careful post-mortem examination

(iii)    Pregnant animals or animals having delivered a calf within the last 48 hours are not permitted for slaughter. Animals undergoing treatment or with a recent history of treatment as also experimental animals are not to be slaugthered unless a �no objection certificate� has been issued by veterinarians.

(iv)    Blood, urine and faeces samples to be drawn for laboratory tests

(v)      Pathoganomonic clinical symptoms of the diseases for suspected animal to be carefully studied.

(vi)    The species, class, age, condition, colour and markings re recorded in case of diseased or sick animals and in case of the animals in poor condition.

 

Diseases and abnormalities encountered in ante-mortem inspection

General conditions Specific infections
Cattle Calves Sheep Swine
Moribund and exhausted status Actinomycosis

 

Immaturity

 

 

Pneumonia

 

 

Arthritis,

 

 

Emaciation Actinobacillosis

 

Calf diphtheria Caseous lymphadenitis Atrophic
Anasarca Tuberculosis Arthritis Enterotoxaemia Rhinitis
Poorness Blackleg Ringworm FMD Swine Fever
External injuries FMD White Scour   Swine
Localized swellings

a)         tumours

b)         abscesses

c)         oedema

d)        haematoma

e)         hernia

Anthrax

 

Salmonellosis   Erysipelas

 

Inflammatory conditions of skin

a)   urticaria

b)   eczema

Rinderpest

 

    Swine

 

Pregnancy Mastitis     Tuberculosis
Fractures Ringworm

Pneumonia

    FMD
Dysnoea Retained  placenta      
Discharges from natural orifices        

 

 

Judgment decisions at ante-mortem inspection

Post anti-mortem inspections, the animals can be subjected to any of the following three decisions.

�      Passed/Accepted/ Fit for slaughter: The animals free from any disease and normal can be directly sent for slaughter.

�      Rejected/ Condemned/ Unfit for slaughter: Animals suffering from fever (106oF or more), emaciated or dead animals, immature or pregnant animals are considered unfit and not passed for slaughter. Animals with established symptoms of diseases are not sent for slaughter.

�      Suspect: Animals falling under this category are those for which decision regarding fitness for slaughter cannot be made at the ante-mortem inspection stage. The following possibilities exist under these circumstances.

(a)   Slaughter under special precautions: Animals under suspect category if having symptoms of diseases.

(b)   Delayed/Detained slaughter: If the animals require treatment, or have history of recent treatment/vaccination, animals in febrile condition, fatigued and exited state may require treatment before they are slaughtered.

(c)   Segregated slaughter: Such decisions are made under special conditions such as dirty stock or animal suspected for some contagious diseases etc. Such animals are slaughtered at the end of the day�s kill or separately slaughtered and a thorough post- mortem examination is performed.

(d)   Casualty and emergency slaughter: Casualty slaughter is required when an animal is not in acute pain or immediate danger of death but affected with a more chronic condition like benign superficial tumors, obturator paralysis and post- partum paraplegia etc. When an animal is in acute pain or suffering from condition like, fractures, severe injuries, uterine prolapsed etc., where a delay in slaughter would be contrary to the animal welfare, then animals requires emergency slaughter. Prolonged recumbency in cows and sows after parturition, abscess formation in pigs due to Corynaebacterium pyogenes, pregnancy toxaemia and enterotoxaemia in sheep and injuries and affections of udder and uterus in cattle are the several other causes of emergency slaughter.

  1. Fitfor slaughter.
  2. Unfitfor slaughter and the animal is condemned at ante-mortem.

III. Suspect Animals which fall under 2 categories

(a)       slaughter under strict supervision

(b)      retain for a specified period enabling +he symptoms to develop or subside and thereupon take a final decision on slaughter (fit/unfit/slaughter under strict supervision).           

 

 

 

 

SLAUGHTERING TECHNIQUES AND POST-MORTEM INSPECTIONS

 

 

Slaughtering Procedures

After the animals have been rested for sufficient time, they are quietly taken to the stunning area. Animals may be facilitated through mechanical means like canvass straps or rolled plastic or prodded to move forward. However tail twisting or beating is forbidden. Slaughter animals are properly restrained before stunning or bleeding. Different types of restraints are used for different species.

 

Stunning

It is a process to inactivate animals so that it is not able to move. It is an obligatory process with large animals. Stunning ensures that the animal is unconscious before it is slaughtered in order to eliminate pain, discomfort and stress from the procedure. Many countries have legislation that requires pre-slaughter stunning. Care should be taken not to affect the heart and it should function normally to ensure complete bleeding which ensures better meat quality. Stunning is done in special stunning pan (box with movable side wall).

 

Stunning methods

  1. Most common method employed is strikingon the head with a wooden hammer or captive bolt. However blow should not damage the frontal bones as it may cause brain hemorrhage.
  2. Electrical stunning: An electric current of 75-120 volt is passed for 15-70 seconds through the hind part of the animal head in the regions of parietal boxes by puncturing the skin. This causes unconsciousness of the animals which may last for 5 min and it is enough to transfer the animal from stunning pan to bleeding runway.
  3. Anesthetization:Anaesthetization may be carried out on swine using a mixture of CO2 and air in equal volume with 0.18% chloroform and the inhalation period may last for one min.

 

Slaughter

The most common methods of slaughter practiced worldwide are the Halal (Islamic), the Kosher (Jewish) and the Jhakta (Sikh) methods.

Halal: Halal is one of the most popular method of slaughter. This method prescribes slaughtering of animals with a sharp knife to make a swift, deep incision that cuts the front of the throat, the carotid artery, wind pipe and jugular veins but leaves the spinal cord intact. The animal is then hung upside-down and left to exsanguinate i.e drainage of blood. The Halal slaughter requires that the name of Allah (or God) should be mentioned at the initiation of the operation. This method of slaughter ensures that the blood flows out completely from the animal.

Jhatka: It is an instant decapitation process limited mostly to sheep and goats and practiced in countries like India by few religious sects. The animals are killed by a single strike of a sword or axe by severing the head Jewish Slaughter (Kosher): �Kosher� is the term applied to the procedures and techniques of slaughter practiced under the Jewish faith. In Hebrew language, Kosher means fit to be used as food. Under this method of slaughter, the animals in fully conscious state are killed and bled thoroughly by one clean stroke of the knife. Animals are however hoisted and shackled first. A 16-inch (40.6 cm) razor-sharp steel knife called the chalaf is stuck into the throat by a trained slaughterer, the shohet, in an operation in which the animal is killed and bled at the same time. Skinning is made from the chest down to the level of the belly, and the chest is cut open first for inspection and later evisceration.

 

Bleeding

Bleeding is a procedure in the slaughter process which is performed by cutting jugular vein in the neck and carotid artery in order to allow blood to drain from the carcass, resulting in the death of the animal from cerebral anoxia. The bleeding knife should be continuously sharpened as a blunt knife may prolong the incision and damage the cut ends of the blood vessels. This may result in premature clotting and blockage of the vessels thus delaying the bleeding process. A prolonged delay in bleeding could result in the animal regaining consciousness. The delayed bleeding may also result in an increase in blood pressure causing the blood vessels to rupture and haemorrhage of muscle. The extra blood in the tissues may lead to meat getting decomposed quickly. Incisions should be therefore swift and precise. In poultry, sheep, goats and ostriches, the throat is cut behind the jaw.

 

Skinning

After successful bleeding, first the head is skinned, separated from the body, marked with the same number as the body and then hung on hook for post mortem examination. �Skinning� is a term mostly used for small ruminants and the method of skinning is known as �case-on�. The skinned materials are called as �skins�. Skin is the most valuable byproduct economically. In sheep and goats, skin is first cut around the leg to expose and loosen the tendon of the hock and used for hanging the carcass. This process is called legging. The second step that follows is called skinning which involves removal of the entire skin and preparation of the animal body for evisceration. Skinning can be done either in the horizontal or hanging position depending on the convenience and available facilities. If animal body is in hanging position, legging is generally started at the back of the free leg by removing the skin around the hock and continued towards the toes. This exposes the tendon on the back leg and the foot is cut off at the joint above the toe. The body skin is removed by making an opening in the front legs, cutting towards the jaw and continuing over the brisket to the naval. Once the brisket has skinned, knife is seldom used to protect the �fell� (a fine membrane between the skin and the carcass). This helps in improving carcass appearance and reducing surface shrinkage. This is largely accomplished by using fist/hand. After the skin has been removed, the carcass is washed and placed on a hook. In horizontal skinning the animal is placed on its back on a flat raised surface and similar process repeated.

This operation is absent in pigs, because skin is a part of the carcass. In the case of large ruminants (cattle and buffaloes), cuts are made on the skin along the mid-ventral line and also on the medial side of the limbs connecting to the respective points (sternum and pelvis) in the mid-ventral line. Skinning of large ruminants is known as �flaying� and the incisions made on the skins are known as �ripping lines�. The deskinned materials are called as �hides.

 

Evisceration

It should be carried out without damaging the internal organs or disturbing the internal surface of the carcass. Damage to the gastro-instestinal tract (GIT) may contaminate the carcass with the microorganisms. The first step in evisceration is to cut around the tied bung or rectum and free it completely from all attachments. The breastbone is then cut along the midline up to its tip. Another cut is made from the cod or udder down the midline into the breast cut. Then the ureter connections to the kidneys are severed and the intestines loosened. The stomach and intestinal mass are removed. The lever could be detached from its connecting tissues and then pulled out along with the contents of the abdominal cavity. The gall-bladder is carefully removed from the liver so that its content does not spill out and contaminate the carcass. The pluck consisting of heart, lungs, trachea and esophagus can be pulled out as a unit. The carcass is then washed and carried manually or mechanically to the inspection area.

 

Carcass splitting and sizing

In the slaughterhouses, carcasses of small ruminants are not split into sides or quarters; carcasses of large ruminants are split into four quarters; and carcasses of pigs are split into two sides. Therefore, at the retail meat stalls selling buffalo meat, pork and mutton, we find quarters, sides and whole carcasses of respective animals. Carcasses are sawed by electric or pneumatic saws starting from the hind part to the central vertebrae. This facilitates transport, storage and efficient refrigeration.

 

Post Mortem Examination

Postmortem examination/inspection refers to inspection of carcass and organs by qualified veterinarians to ensure that carcass and organs are fit for human consumption. During inspection, care should be taken not to contaminate the carcass and organs from diseased animals. The knives and other instruments used for cutting and examining organs, glands and tissues should be properly sterilized before and after use. The particular sequence should be followed during postmortem examination so that each carcass and thereof organs are checked thoroughly.

 

Objectives of postmortem examination

Carcasses should not be sent to the chilling section without inspection after dressing. Some of the diseases are not apparent during ante mortem examination can be detected easily in postmortem examination. Thus, post mortem inspection ensures safe meat to the  consumers and also controls diseases right at the farm level itself. It also directs to adopt a proper disposal procedure for condemned meat and offal. Since postmortem inspection is performed for carcasses as well as their viscera, it ensures a systematic way of evisceration and handling of offals.

 

Facilities required for postmortem examination

�       The area where the examination is being conducted should have sufficient and well distributed light. The natural light is considered better than artificial light. The intensity of light must be 540 lux

�       The person carrying inspection need clean, sharp, stainless knives

�       There must be provision for hot and cold water

�       There should be a sterilizer to sterilize the knives, saws and cleavers. The postmortem examination should be carried out under hygienic conditions. The knives should be sterilized by dipping them in� boiling water for 30 minutes or by autoclaving them for 10-15 minutes. (The sterilization of anthrax contaminated knives requires special consideration).

�       To put a mark on carcass and its viscera, marking dyes should also be provided, which should be cheap, non-toxic and non-corrosive in nature. Marking indicates that the carcass has been inspected and guarantees the consumer about is wholesomeness. Marking of meat is done by (i) using a stamp (ii) branding or (iii) labeling. Common method � Metal stamp dipped in a stamping ink.

�       There should be provision of detained room side by the inspection site.

 

General consideration

Following points should be considered during postmortem examination/ inspection:

�         The examination must be done as soon as possible. Carcasses of beef and pork set rapidly and if the inspection is delayed especially in cold weather the examination of lymph nodes becomes difficult.

�         Carcass and organs are to be examined methodically following a definite sequence. Healthy carcass should be examined before inspecting the diseased or suspected ones.

�         Great care must be taken at the time of inspection particularly in cases suspected for zoonotic diseases.

�         The identity of carcass and its viscera should be maintained.

�         Inspector should avoid unnecessary cuts considering the value of high quality food. One should incise the carcass in such a way that the surface of the carcass appears clean and undistorted.

 

Postmortem  principles

 

Visual perception

First the carcass and visceral organs should be examined visually for any visible abnormalities. Examination is done for any change of colour, atrophy, hypertrophy, neoplastic condition etc.

 

Palpation

The organs are palpated for any change in consistency, sliminess or gelation, cyst, etc.

 

Incisions

The organs are incised, if needed. This is done to examine any parasite inside organ, structural deformity etc.

 

Laboratory tests

These are done for confirmation and support the observation made by macroscopic examination. While examining the organs of carcass, lymph node of adjoining area must be examined.

 

Postmortem Examination of Carcasses

 

Large animals

In case of large animals like cattle, sequence of postmortem examination is as follows:

Head

�         Verify the number, age and sex of the animal

�         Inspect gums, lips and tongue for FMD, necrotic and other forms of stomatitis, actinomycosis and actinobacillosis (Palpate the tongue for the latter).

�         Incise the internal and external masticatory muscles and tongue for Cysticercus bovis.

�         Incise retropharyngeal, submaxillary and parotid lymph nodes for tuberculosis (TB) lesions.

  • For sheep & goat, the lips, gums and nasal cavities should be examined for contagious ecthyma.

Lungs

�         Examine visually and then palpate for the detection of pleurisy, pneumonia, tuberculosis, fascioliasis and hydatid cysts.

�         Incise the bronchial and mediastinal lymph nodes and expose the lung by giving deep incision from the base to apex (for checking TB lesions).

�         Check the tumors, abscesses etc. by palpation.

Heart

�         Examine the pericardium for traumatic or tubercular pericarditis.

�         Incise the ventricles of the heart and pay attention to look for petechial hemorrhages on the epicardium and endocardium and cuts in the myocardium. Flavy condition of the myocardium is indicative of septic conditions.

Liver

�         A visual examination should be made for fatty changes, abscesses, hydatid cysts, actinobacillosis etc.

�         For examination of fascioliasis, incise thin portion of left lobe of liver and examine the contents.

�         For sheep and goat, lungs, heart and liver:

(i) Palpate lungs, heart and liver and accompanying lymph glands for abscesses.

(ii) Cut the bile duct for examining possible fluke infestation

Stomach and intestines

�         Check the serous surface of the intestine for TB lesions and actinobacillosis.

�         Palpate the mesenteric lymph node and if necessary incise and examine the same.

Spleen

�         Examine the surface and substance for TB lesion, anthrax, hematoma and presence of infarcts.

Uterus

�         Check for septic conditions by viewing, palpating and incising if necessary.

Udder

�         Check the supramammary lymph nodes by incising for the evidence of TB lesions.

�         Check for abscesses if any.

General inspection of carcass

�         Look for the injuries and bruises. Bruises are dark colour after 24 hours and there is watery condition after 24 to 38 hours. After 3 days, the area becomes rusty orange colour and soapy to touch.

�         Look for inflammation, abscesses and TB lesions in the thoracic and abdominal cavities.

�         Examine the kidneys.

�         Incise and examine renal lymph nodes.

 

Postmortem  judgement

Similar to ante-mortem examination, a competent veterinarian has to submit its judgment report:

�         Fit for human consumption

�         Unfit for human consumption or total condemnation

�         The affected organs must be condemned while rest can be passed for human consumption (partially condemned).

  • The condemned carcass/ organs should be disposed off following scientific procedure.

 

Diseases and conditions for which carcass is totally or partially condemned

The carcass is totally condemned for rabies, anthrax, glanders, rinderpest, foot and mouth disease, acute enzootic meningoencephalitis, acute pleurisy, contagious bovine pleuropneumonia, ovine foot rot, sheep pox, swine fever, swine erysipelas, salmonellosis, fibrinous rhinitis, black leg, bovine viral diarrhea, haemorrhagic septicaemia, listeriosis, pasteurellosis, coccidiosis, calf diphtheria, calf diarrhoea, malignant oedema, tetanus etc.

The carcass in case of actinomycosis, actinobacillosis and Johne�s disease, is totally condemned if accompanied with emaciation and in generalized form, otherwise the carcass is passed for human consumption after local condemnation. In case of corynebacterium infection, carcass is passed after removal of affected organs.

In case of heavy infestation (more than 10 cysts) with Cysticercus bovis and Cysticercus cellulose, carcass is totally condemned, otherwise the carcass is passed after removal of head, heart, diaphragm and esophagus. Trichinella spiralis infected carcass is totally condemned. If carcass show the sign of fascioliasis with emaciation then it is totally condemned, otherwise it is passed after trimming of liver.

Carcass is passed for consumption after removal of lung in case of emphysema and broncheolitis. If there is no fever, then carcass with sign of gastroenteritis is unconditionally passed.

The judgement of carcass suffered from tuberculosis depends on method of spread, extent of disease, character and age of lesion and general condition of the animal. Carcass is totally condemned when tuberculosis spreads through portal or pulmonary or systemic circulation. In case of localized tuberculosis, the carcass is passed after removal of the affected organs and associated lymph nodes.

 

Metabolic and nutritional disorders and intoxication

The carcasses are totally condemned if they show any of the following sign:

Anaemia with emaciation, (ii) grass tetany, (iii) haemoglobinuria, (iv) jaundice, (v) ketosis with chronic indigestion, (v) poisoning, (vii) bloat or (viii) impaction etc.

Abnormal conditions: Abnormal conditions like, abnormal colour, taste and smell, buck smell, bore smell etc. results in total condemnation of the carcass.

 

RIGOR MORTIS: BIOCHEMICAL AND HISTOLOGICAL CHANGES

 

 

Meat is basically defined as the flesh of animals used as food. The term meat generally differs from the muscle in the sense its structural and physicochemical nature as it (muscle) has undergone certain chemical and biochemical changes following death of an animal which is a postmortem aspect. Thus, during the time elapsed between death of an animal and its processing, a series of biochemical and physico-chemical changes takes place which lead to conversion of muscle into meat.

 

Muscle: Structure, Composition & Functioning

Muscle is made of number of fiber bundles (1.0 mm thick), comprised of a group of fibers, (0.1 mm thick) held together by a structure of connective tissues or perimysium (figure 22.1). Connective tissues which provide edible texture, structure and flexibility to the muscles, comprised of fibrous protein collagen, reticulin, and elastin. Muscle fiber, a unit of muscle contraction, is a multinucleate, cylindrical cell bounded by an outer membrane or sarcolemma and is consist of myfribils of 1-2 micron size. Myofibrils are separated by sarcoplasmic reticulum, a fine network of tubules. Each fiber is filled with sarcoplasm containing mitochondria, enzymes, glycogen, ATP, creatine, and myoglobin. The myofribrils are cross striated to give rise to understanding of physical structure of muscles (dark or A and light or I/Z bands). The unit of fibril is sacromere which lies between adjacent two Z- bands. Fibrils are consist of two set of filaments i.e. myosin and F-actin. Contraction and relaxation of striated muscles takes place due to interaction between actin, myosin and ATP. In the presence of magnesium and calcium ions, myosin liberates ATP which results in muscle contraction.

The composition of muscle is highly variable depending upon specie, type of muscle, animal�s maturity and the treatments given to the animal before its slaughtering. Variation in the composition ultimately affects the nutritional and functional profile muscle tissues.

The lipid components of muscle tissue vary more widely than do the amino acid in fish muscle, the differences have been made in the concept of lean or white fish and fatty fish. In lean fish, storage fat is carried in the liver. Muscle of lean fish contains <1% lipid, mostly phospholipids, located in the membrane. In fatty fish, depot fat apparently occurs as extracellular droplets in the muscle tissue.

 

In mammals and birds, both the amount and type of collagen have important influence on textural properties of the muscle. In fish however, collagen is readily softened by normal cooking procedures.

Nature of muscle-

  1. I)  Striated or voluntary muscle �lean meat.
  2. II)  Unstriated or involuntary muscle- stomach wall.

III) Cardiac muscle- heart wall.

In white muscle, fat is apparently diffusely located among the muscle cells. Basically the lipid composition of meat of mammalian and avian muscles can be categorized into lipids from muscle tissue and lipids from adipose tissues lipid in the lean portion contains greater portions of phospholipids than lipids than lipids in adipose tissue lean muscle contains about 0.5-1.0% phospholipids and the fatty acids of phospholipids are more unsaturated than those of triglycerols. Consequently, lipid in the lean portion of meat has a higher degree of unsaturation than those in adipose tissue. The degree of unsaturation of fatty acids in cold blooded fish is much greater than that of fatty acids in avian and mammalian muscles. The much greater percentage of polyenoic fatty acids is found in avian and mammalian muscles. The much greater percentage of polyenoic fatty fish reflects differences in phospholipid � triacylglycerol ratios. Poultry fat is more unsaturated than pork fat, beef and mutton.

 

Composition (%) of muscle tissue

SPECIES WATER (%) PROTEIN (%) LIPID (%) ASH (%)
Beef 70-73 20-22 4-8 1.0
Pork 68-70 19-20 9-11 1.4
Chicken 73.7 20-23 1.0
Lamb 73.0 20.0 5-6 1.6
Cod 81.2 17.6 0.3 1.2
Salmon 64.0 20-22 13-15 1.3

 

General Consequences Following Death of an Animal (Post Mortem Changes)

Following the death of animal, circulation of the blood ceases resulting in the complex series of changes within the muscle (Fig.22.2). As much as possible blood is removed from the animal carcass to increase the edibility and keeping qualities of the meat, since blood is an ideal medium for the growth of spoilage microorganisms. Failure of blood circulation and its removal from the muscle tissue results in depletion of oxygen supply to the tissue leading to depletion of ATP and creatine phosphate levels (due to stoppage of electron transport chain and oxidative phosphorylation) and most importantly to onset of anaerobic metabolisms of glycogen. Anaerobic metabolism of glucose and breakdown of ATPs by the continuing action of sarcoplasmic ATPase leads to depletion of ATP and creatine phosphate results in onset of rigor mortis on the other hand breakdown of glycogen in the absence of oxygen leads to production of lactic acid thus decrease in pH. Other postmortem physical changes in muscle are:

 

Change in pH

Decrease in pH due to lactic acid formation is accompanied by various exothermic reactions such as anaerobic glycolysis. pH changes from physiological pH i.e 7.2-7.4 to ultimately post-mortem pH i.e 5.3-5.5 in 24 hrs. This has profound effect on muscle portion of meat. Usually glycolysis ceases even before the glycogen is depleted.

 

Change in temperature

Temperature of animal increases from 37.6-39.0�C. This is the reason why animal cools slowly during refrigeration as a result of continuous production of heat. This phenomenon is known as �animal heat�. Removal of �animal heat� by chilling or refrigeration is essential to ensure longer shelf-life of meat.

 

Change in proteins

Due to the change in pH and high temperature, colour of meat changes and water holding capacity (WHC) also decreases. Sarcoplasmic proteins get denatured and attached to the surface of myofilament, which produces change in meat colour which becomes light. Water holding capacity of myofibril proteins decreases resulting in exudation of fluid.

Sarcoplasmic proteins are more lable with respect to physiological conditions prevailing in the post- mortem muscles. These proteins are highly susceptible to disruption as compared with myofribrilar proteins. Sarcoplasmic proteins during rigor mortis denatured below pH 6.0 and at 37�c.

 

Change in water holding capacity

Water holding capacity is the function of respective proteins which binds with water. In pre-rigor stage meat possesses a high water holding capacity but later it decreases during first hour following death of animals. Lowest water holding capacity is found at its iso-electric pH i.e. 5.3-5.5. After post-rigor aging water holding capacity. is found to be increased because of increase in osmotic.

During post mortem movement of Na. K, Mg, and Ca in muscles takes place. But during aging there is continuous release of of Na & Ca. and uptake of K ions continued up to 6-8 days. The movement of cations produces an increased electrical charge on muscle protein which facilitated the formation of hydrated ions. This is believed to be the reason of increased water holding capacity during aging of meat.

 

Post mortem glycolysis

After the death of animal, blood circulation stops, thus oxygen supply to muscles tissues decreases hence anaerobic conditions prevails in the muscle. The glycogen present in tissues is no longer converted into CO2 and water instead, converted in to lactic acid through anaerobic glycolysis. The conversion of glycogen takes place through two different pathways.

(a)    Amyloytic pathway i.e. hydrolytic

(b)   Phosphorolytic pathway

Post mortem glycolysis

Due to this glycolysis pH changes from physiological pH i.e. 7.2-7.4 to ultimately post- mortem pH i.e. 5.3-5.5. This pH is attained within 24 hours and being related to ATP production, which falls in this pathway. The net fall in ATP is responsible for onset of rigor mortis. The pH 5.3-5.5 is ideal pH which can be obtained by well rested and well fed animal before slaughter (fig. 22.3).

 

Conversion of Muscle into Meat (Rigor Mortis)

The most important change that occurs in postmortem muscle is the development of rigor mortis, means stiffness of the muscle. The primary cause of onset of rigor is post mortem decline in the level of ATP. The process takes from 7-24 hrs depending on the species; however it is linked with the rate of depletion of ATP in muscle. The entire process of conversion of muscle into meat is broadly divided into three stages:

 

Pre-rigor stage

During early stages of postmortem or pre-rigor stage, the concentration of ATP more or less remains constant as the muscle tries to maintain ATP levels by an active creatine kinase. However it will lead to liberation of creatine from muscle. Thus, in this state creatine phosphate levels fall more rapidly than that of ATPs. ATPs are providing cushioning effect for the filaments of two proteins i.e. actin and myosin. This results in a meat which is soft and pliable. In pre-rigor stage, myosin dissociates from actin and can be extracted in solution of high ionic strength.  Water holding capacity of the muscle proteins remains high during this stage.

 

Rigor mortis

This period is very important as meat becomes rigid and stiff. Onset of rigor mortis may be 8-10 hours postmortem and it may last in 15-20 hours in meat. Onset of rigor is demonstrated by fall in ATP, loss of extensibility of muscles and contraction of tissues. Time difference between death of an animal and onset of rigor state is termed as �delay phase�. This period depend upon number of factors such as age, health, size of carcass, the amount of fat cover, nutritional status of animal, pH and glycogen level, temperature as well. ATP plays very important role in this stage. As ATP level falls two muscle proteins gradually forms an associated actomyosin complex which is inextensible and is responsible for contraction. This is the necessary criterion for development of rigor mortis.  The extent of contraction of the muscles is determined by estimation of length of the sacromere within the myofibril. Meat which is cooked in this state is very tough in texture. The water holding capacity of the muscle protein remains minimum during this stage due to  drop in pH as it comes closer to their iso-electric point i.e. pH 5.3-5.5. If the ultimate pH (5.60) falls too quickly, carcass would still be warm adversely affecting water holding capacity and prevailing partial denaturation of protein resulting into pale, soft and exudative (PSE) muscle ultimately leading to lower yield of the meat. This is often encountered in pigs having sufficient reserves of glycogen. On the other hand if inadequately feed or fasting animal having minimum reserve of glycogen is subjected for slaughtering; dark, firm and dry (DFD) meat conditions. DFD meat is having pH not below 6.0 and is darker in colour and susceptible for microbial growth.

 

Post rigor  (conditioning/ageing)

During post rigor stage meat become tenderizes and organoleptically acceptable when it is kept cold for sometime after rigor mortis. The muscle again becomes soft and pliable with improved flavour and juiciness. The post rigor meat provides lesser problems in toughness, when cooked compared to with that cooked in rigor. Meat gradually reaches to an optimum tenderness period after an ageing period of 10-18 days stored at 0�- 5�C following the dissolution of rigor. However, prolonged storage of meat in some species may results in some problems viz. microbial spoilage, desiccation of proteins, and development of off flavours. Thus it is recommended to consume meat before it gets spoiled. The ageing which also called as conditioning or ripening of meat is sometime accelerated by raising storage temperature for e.g. holding meat at 15� for 3 days period in UV to control the microbial growth at surface. While in the case of pork, ageing is not recommended rather to eat fresh as it develops rapid onset of fat rancidity even at low temperature. On the other hand beef is generally aged and lamb & mutton are occasionally aged.

Ageing is considered as very important aspect of meat processing as it imparts desirable flavour, textural and other sensory attributes to the finished product. The responsible factors for this desirable changes are still been researchable issue, however it is now a fact that in post- rigor state actomyosin complex does not dissociate but other subtle changes occur like, increase in the water holding capacity due to increase osmotic pressure in the muscle fibre due to net inside movement of cations and breakdown of proteins by liberated proteolytic enzymes, the cathepsins may lead to tenderness.  While cooking of meat tenderizing agents such as enzyme calpain etc are added which breaks down the stiff muscle protein to yield a soft and orgnoleptically acceptable meat.

 

MEAT HYGIENE AND SANITATION IN MEAT AND POULTRY INDUSTRY

 

Meat Hygiene

Meat Hygiene refers to a set of activities that require the implementation of specific standards, codes of practices and regulatory action by the competent authority to ensure �safety and suitability� of the meat the consumers eat.  Hygiene requirements are to be met at different stages of production, processing and transportation and must include hygiene of personnel, slaughter & meat processing equipments and environment. To ensure this, proper cleaning and sanitization practices are to be followed by plant personnel and should include disinfection of meat plant premises, equipments and storage area. Failure in maintaining meat hygiene may pose serious public health hazards and therefore evaluation of meat for meat borne pathogens which can cause diseases of public health importance is very important. Food Safety and Standards (Food Products Standards and Food Additives) Regulations, 2011 also warrant that every product being sold in the Indian market must meet/conform to legal standards of quality.

 

Principles of meat hygiene

There are three principles of meat hygiene, which are crucial for meat processing operations.

�         Prevention of microbial contamination during meat product manufacture by adopting proper cleaning and sanitation practices.

�         Minimization of microbial growth in meat products by storing them at a low temperature.

�         Reduction or elimination of the risk of microbial contamination by applying suitable heat treatment and packaging systems at the final processing stage.  

 

Possible source of contamination

Failures in maintaining slaughter hygiene, meat cutting and meat handling/transportation and in the hygiene of by-products and additives contribute to quality losses and deterioration of the final processed meat products

 

Microbiological contamination in the meat processing chain

Carcass contamination

during slaughtering

Unavoidable � keep as low as possible.

 

Meat cutting

 

No reduction of contamination possible, but further contamination should be prevented.
Further processing

 

No reduction of contamination possible, but prevent further Contamination and create challenges / hurdles for microbial growth and survival (aw, preservatives).
Heat treatment of final product Pasteurization (approx. 80�C): Substantial reduction of contamination, but products need refrigeration. Sterilization (above 100�C): Total elimination of contamination, products can be stored without refrigeration (in sealed food containers).

 

 

Control measures

Two useful schemes are usually adapted at various levels of meat production:

�         Good Hygienic Practices (GHP) and

�         Hazard Analysis and Critical Control Point (HACCP) Scheme.

 

Good hygienic practices in meat processing

Microbial meat spoilage or food poisoning through meat can be prevented if the microbial load/bacterial contamination, which occurs during slaughtering and meat handling, is kept as low as possible.  The key for achieving this is strict meat hygiene including an uninterrupted cold chain throughout the entire meat production and handling chain through the following interventions.

Personnel hygiene

�       Wear clean protective clothes

�       Washing hands before starting work and repeatedly washing hands during work

�       No finger rings, watches, bracelets

�       Access to production areas with working clothes only

�       Cleaning/disinfection of hands/tools/clothes if there was contact with highly contaminated subjects or abnormal animal parts likely to contain pathogens.

�       Fresh wounds through knife cuts etc. must be covered by a water tight bandage.  Workers with purulent wounds are not allowed to work with meat. (Risk of spread of Staph. aureus bacteria).

�       Strict toilet hygiene must be observed (removal of apron, hand washing and hand disinfection).  Toilets must be kept clean and must not have direct access to production areas. (Risk of spread of Salmonella).

�       Periodic medical examinations of staff

Hygiene during meat processing 

�       Ideally meat cutting/deboning should be carried out in climatized rooms (approx. + 10�C) with low air humidity.

�       If visual contamination of meat has occurred during manufacturing, do not try to wash it off but remove it with knives by cutting off superficial meat parts in the case of minor contamination. Discard the meat in case of heavy contamination.

�       Do not hose down floor and wall areas or equipment next to meat processing operations or final products with a power hose. (Risk of contamination by aerosol/droplets).

�       Never take meat pieces, which accidentally had contact with the floor or other contaminated surfaces, back onto working tables or into meat processing machines.

�       Containers for meat, fat, or semi�or fully processed meat products must not be placed directly on the floor but on hygienic stands, pallets etc.).

Hygiene of meat processing premises (design and construction) 

Meat processing facilities must meet the basic hygienic standards in order to ensure and maintain clean and hygienic working conditions:

�       Provision of change room for duty staff.

�       Wall windows must be positioned at least 2 m high over floor level in order to allow profound washing and disinfection of floors and walls. Window frames should be of non-corrosive material e.g. aluminium or similar materials and must not be painted.

�       Walls in all rooms, where meat and by-products are handled, must have smooth and easily washable surfaces up to a minimum height of 2 m in processing plants. Walls should preferably be covered with wall tiles or at least with washable paint.

�       Floors in the mentioned sections must be impermeable for water and reasonably smooth for good cleaning, but anti-slip for workers safety.

�       In order to facilitate proper cleaning, the junction between floor and walls must be rounded (not rectangular)

�       Rooms for meat processing should have sufficient ventilation. Air conditioning is only required in meat cutting/deboning rooms (10 – 12�C).

�       Supply systems for electrical wiring and pipes for hot and cold water as well as for compressed air should not hamper cleaning operations and be out of reach of possible dirt contamination. Insulations for hot water pipes must have smooth surfaces and be washable.

�       Openings for ventilation must be bird- and insect-proof.

Equipment hygiene

�       Equipment should have proper sanitary design and construction. Designs must allow easy and profound cleaning and avoid any accumulation of difficult to remove organic matters. They should make use of food grade construction material in designing food contact surfaces and equipments and should allow easy cleaning after processing operations.

�       Stainless steel must be used for all food contact surfaces e.g. working tables, meat hooks (at least their parts contact in meat), blades of knives, saws, cleavers and axes etc.

�       Food grade synthetic materials should be used for meat containers and other utensils

 

Hazard analysis and critical control point scheme (HACCP)

HACCP are factory and product specific strictly sanitary control schemes that shall prevent, detect, control and/or reduce to safe levels of accidentally occurring hazards to consumers� health. Despite GHP in place, accidental hazards cannot be ruled out and may occur at any processing step of the individual meat product. Specifically for meat processing plants, such hazards may be provoked by failures such as:  batches of incoming raw meat materials with abnormal tissues or heavy contamination, breakdowns in refrigeration, failure in cooking/sterilization operations, abnormal pH or aw in raw or finished products, errors in levels of application of curing salts and other additives, technical problems in sealing of vacuum packages or cans with the risk of recontamination. HACCP schemes serve as additional alarm systems in the interest of consumer protection to prevent such problems occurring.  In case potential hazards should occur, they can be detected, contained or eliminated at any stage.

 

Food poisoning through micro-organisms present in the meat

Food poisoning sets alarming situation for consumers. After consumption of meat contaminated with food poisoning bacteria, food poisoning results in severe illness with consumers needing intensive and costly medical treatments. Type of food poisoning observed due to bacteria are of two types, ie.

�         food borne infection or

�         food borne intoxication.

Bacteria that cause food borne infections, cause sickness through microbial metabolic substances i.e. toxic substances released by the living microorganisms inside the human digestive tract. The best known examples of food borne infections are those caused by Salmonella bacteria and entero-pathogenic form, mostly type O157: H7 residing in faecal material, Listeria monocytogenes, Campylobacter jejun and, Yersinia enterolytica. The Norovirus group can be responsible for food infections with similar, mainly gastro-intestinal symptoms, as bacterial food infection agents.

Microorganisms causing food borne intoxications produce and release the poison during their multiplication in the food. Upon ingestion by consumers of such food, which was heavily intoxicated outside the human body, severe gastro-intestinal food poisoning symptoms occur. Food borne intoxications are frequently caused by Staphylococcus aureus, and Cl. botulinum.  Moulds are sometimes found on the surface of meat products after prolonged storage. Mold produce two types of toxins i.e. Aflatoxins (toxin of Aspergillus flavus) and Ochratoxin (toxin of Penicillium vividicatum).

 

Cleaning and Sanitation

Generally cleaning refers to removal of visible, physical/chemical dirt and to some extent bacteria from the equipment surfaces, sometime from products itself and from the processing environment. On the contrary, sanitization term is used with disinfection of the product or product contact surfaces by all killing spoilage and pathogenic microorganisms in order to avoid all possible risks of microbial contamination. Inactivation of microorganisms requires antimicrobial treatments, carried out in food industries through hot water or steam or through the application of disinfectants or sanitizers.

 

Cleaning procedures

The first step in floor and equipment cleaning is to physically remove scrap, i.e. coarse solid particles, with a dry brush or broom and shovel. This is usually referred to as �dry cleaning�. Wet cleaning is followed after removal of physical scrap material. Wet cleaning could be done manually or by using high pressure nozzles. However, this would require water in sufficient quantities.

Cleaning with equipment producing a pressurized steam/water-mix is even more efficient as impact temperatures of approx. 100�C can be achieved. The disadvantage of this method is the intense fog and aerosol formation, which may not only cause unwanted microbial spreading by water droplets (aerosol) but also affect installations and equipment through high humidity and excessive condensation. For these reasons a steam/water-mix is not suitable for meat processing facilities and cold or hot pressurized water cleaning is preferred.

A relatively new cleaning method for the food industry, in particular the larger-scale plants, is foam cleaning.  Water foam containing detergents and other cleaning agents is sprayed on wetted walls, floors and surfaces of equipment. The foam does not immediately run off but clings to the surfaces.  This allows a longer term contact on the surfaces to be cleaned.  After a sufficient impact period (min. 15 minutes) the foam is washed down with water (water hose or low-pressure water spray). As no high pressure water spraying is needed for washing off the foam, the spreading of water droplets (aerosol) in the room to be cleaned is minimized.

 

Cleaning agents

Traditional cleaning substances/detergents for manual use are alkalines, such as sodium carbonates (Na2CO3, washing soda). These substances are efficient in dissolving proteins and fats, but may cause corrosion in tools and equipment, if their pH is 11 and above.

Ideal detergents should have the following desirable properties:

�         Wetting and penetrating power-must wet, penetrate and dispose soil and remove it from walls of equipments.

�         Emulsifying power

�         Saponifying power

�         Deflocculating power

�         Sequestering and chelating power

�         Quick and complete solubility

�         Should be non-corrosive to metal surface

�         Economical

�         Stability during storage

�         Should be mild on hands

�         Should possess germicidal action

In Meat industry, various types of detergents and cleaners are used and each one has one or more limitations.  These are presented in table 25.2:

Table 25.2 Common detergents & sanitizers used in meat industry

Detergent
Functions
Limitations
1. Alkalies:

a) Sodium hydroxide

b) Sodium carbonate

c) Sodium bicarbonate

d) Sodium silicates

e) Sodium phosphates

Digest, disrupt or dissolve soil especially protein, act as emulsifier, bactericidal agents, generally used at 0.2 – 2% (NaOH) i)  Some of these have poor solubility and wetting power

ii) NaOH corrosive towards AI, Tin and Zn especially at higher concentration. (>2.0%)

2. Acids:

a) Nitric Acid

b) Sulphuric Acid

c) Hydrochloric Acid

d) Phosphoric Acid

e) Acetic Acid

Remove hard deposits such as water stones, such deposits do not dissolve in alkalies, generally HNO3 (0.5%) Phosphoric acid (2.0%) used Strong acids are corrosive to metal surface and dangerous also
3. Complex Phosphates:

a) Tetra sod. pyrophosphate

b) Sod. tripolyphosphate

c) Sod. tetraphosphate

d) Sod. hexametaphosphate

Water softening, soil displacement by emulsification, peptization prevention of redeposition of soil

 

Excellent but unstable in hot solution and in presence of strong alkalies.
4. Chelating Agents:

EDTA (Ethylene diamine tetra acetic acid)

Sequestering, water softening, removal of mineral deposition.  

5. Wetting Agents:

a) Anionic (sod. salt of various complex organic materials

b) Non ionic e.g. teepol

— Wetting and penetrating properties in soil

— Stable dispersion

— Emulsion formation

 

 

QAC are expensive.

Alkaline cleaning agents are generally suitable for removing organic dirt, protein residues and fat, while acid cleaning agents are used particularly for removal of encrusted residues of dirt or protein or of inorganic deposits (�scaling�) such as waterstone, milkstone, lime etc. On the other hand, Neutral cleaning agents have much less effect than alkaline or acid cleaning agents, but have mild impact on skin and materials and are useful for manual cleaning of smooth surfaces without encrusted dirt. In practice alkaline and acid cleaning substances should be used alternatively.

 

Disinfection techniques

The elimination of microorganisms is achieved through disinfection, either by using

�   hot water (or better steam) or

�   chemical disinfectants.

Chemical disinfectants are preferred for most applications in the meat industries as they are easy to use and do not involve the risk of accidents or other negative side effects such as damage to equipment by generating high humidity or water condensation, which may occur when using steam. Best results are achieved when chemical disinfection is preceded by intensive dry/wet cleaning.

 

Disinfectants for the meat industry

Disinfectants should be effective and rapidly acting in killing microorganisms. In principle the following groups of substances are generally used as disinfectants:

  1. Chlorine containing compounds e.g. Na/Ca hypochlorite or chlorine gas, has a corroding effect on equipment.
  2. Aldehydes(used in animal production, e.g. Formaldedyde) Phenoles / Kresols (used in medicine, households Alcohols (used in medicine, e.g. skin) Alkalines (pH 10 or higher) (e.g. NaOH, used in animal production) Acids (some organic acids used in food industries). Quaternary ammonium compounds Amphotensids (used in food industries, as not corrosive) Low efficiency on spores. They have effect on cell walls, not corrosive, odourless, additional cleaning properties (surfactant)
  3. Oxygen releasing compounds e.g. Peroxide compounds (H2O2) Per-acetic acid (use in food industries). Penetrate into cells, good effect on all microorganisms including on  spores and virus, odourless, may be corrosive in concentrations >1%

An example of the optimal combination of disinfectant commercially used is

�         organic acids

�         surfactants (= surface active agents)

�         peroxide compounds

The organic acids, apart from their sanitizing effect, decrease the pH as some disinfectants are more efficient at lower pH. The surfactants assist in penetrating organic material. The peroxide compounds have the direct antimicrobial effect by coagulation and denaturation of proteins (virus) and penetration through cell walls causing cell destruction (bacteria).

 

Cleaning and disinfection (sanitation) schemes 

�         Several daily disinfections (by hot water or chemicals) are necessary for hand tools, meat saws and cutting boards.

�         Daily disinfection is useful for dismantled equipment such as parts of grinders, fillers, stuffers, etc.

�         Disinfection once a week is recommended for other equipment and floors and walls of processing and chilling rooms.

 

Cleaning and disinfection plans

Specific cleaning and sanitization plans should be developed for specific processing areas eg. Meat storage, processing etc. An example of such plan is given in table for disinfection of meat processing equipment, in this case for a meat grinder. This type of equipment is an integral part of almost every meat processing line.  Meat grinders require particular careful and frequent cleaning and sanitation, as the output product minced meat is hygienically very sensitive.

Cleaning and disinfection plan

Equipment: Meat grinder

Pre-cleaning  Potable water

Temp.:  40-50�C

Pressure:  20-30 bars

 
Cleaning Daily

Agent: A

Concentr.:  1.0%

Temp.:  40-50�C

Time:  20-30 min

pH: approx. 12

1 x monthly

Agent:  B

Concentr.: 1.5%

Temp.: 40-50�C

Time:  20-30 min

pH: approx. 1.8

Rinsing Potable water

Temp.:  30-50�C

Pressure:  5-10 bars

 
Drying    
Disinfection  2 x weekly

Agent: C

Concentration.:  0.5%

Temp.:  30-40�C

Time:  30 min

pH: approx. 5.7

3 x weekly

Agent:  D

Concentr.: 1.0%

Temp.: 30-40�C

Time:  30 min

pH: approx. 10.2

Rinsing Potable water

Temp.: 30-50�C

Pressure: 5-10 bars

 

Agent A: Alkaline cleaning substance (Source: Heinz and Hautzinger, 2007)

Agent B: Acid cleaning substance

Agent C: Disinfectant

Agent D: Disinfectant chemically different from C and supplementing impact of C

Source-ecoursesonline.iasri.

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