Draft RCP as of August 17, 2010
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RECOMMENDED CODE OF PRACTICE FOR THE PROCESSING AND HANDLING
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OF ETHNIC CURED MEATS
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1. SCOPE
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This Code of Practice is concerned with the receipt of raw materials and ingredients,
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preparation and processing of ethnic cured meats as defined in this Code, in order to
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conform with the required standards stated in PNS/NMIS No. ___. Standards for
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Ethnic Cured Meats. The product shall be prepared from animal meat, which is cured
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through the addition of curing agents. This Code is intended to provide guidelines to
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achieve compliance with the standards for ethnic cured meats packed in any suitable
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container.
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2. DEFINITION OF TERMS
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For the purpose of this Code, the following definitions apply:
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Casing – a tubular membrane, natural or manufactured, shirred or in loose form,
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stretchable or unstretchable, permeable or impermeable to gas or liquid, may be
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edible or non-edible.
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Comminution – a general term for the process of particle size reduction. This may
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include cutting, chopping, grinding, milling and other physical treatment.
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Container – any form of packaging material, which completely or partially encloses
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the food (including wrappers). A container may enclose the food as a single item or
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several units or types of prepackaged food when such is presented for sale to the
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consumer.
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Cure accelerator – it is an ingredient used primarily to speed up the curing process
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and stabilize the cured color of the finished product.
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Draft RCP as of August 17, 2010
Curing – it is a preservation technique that involves the treatment of muscle meat
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with common salt, and nitrite/nitrate, with or without the addition of sugar, spices and
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other ingredients, to prolong the keeping quality of the product.
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Current Good Manufacturing Practices (cGMP) – a quality assurance system
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aimed at ensuring that products are consistently manufactured, packed or repacked or
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held to a quality appropriate for the intended use. It is thus concerned with both
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manufacturing and quality control procedures.
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Dry sausage – a sausage that has undergone a controlled air drying process, and
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may or may not be fermented.
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Edible casing – it is a casing or tubing prepared from collagen, cellulose, or food-
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grade synthetic material or from natural sources (e.g., hog or sheep intestines) that
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contain the sausage mix (CODEX STAN 192-1995 (Rev. 5-2004)). Edible casings are
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casings that do not have to be removed before consumption and are fit to be
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consumed together with the sausage mix.
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Extenders – refer to any non-meat ingredient added to reduce formulation cost,
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improve emulsion stability and other technological purposes.
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Flavor and flavoring substances – substances which are added to impart flavor
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which are either natural, nature identical or artificial flavoring substances.
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(a) natural flavor – these flavoring substances derived through appropriate physical
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processes from spices, herbs, fruit or fruit juices, vegetable or vegetable juices, edible
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yeast, bark, bud, root, leaf or plant materials, meat, fish, poultry, eggs, dairy products
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or fermentation products thereof.
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(b) nature-identical flavoring substances – these are substances chemically derived
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from aromatic materials or obtained synthetically, which are chemically identical to
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substances present in' natural products intended for human consumption.
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(c) artificial flavoring substances – these are substances that impart flavor but which
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have not been identified in natural products or natural sources of flavorings.
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Food – any substance, whether processed, semi-processed or raw, which is intended
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for human consumption, and includes drink, chewing gum and any substance which
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Draft RCP as of August 17, 2010
has been used in the manufacture, preparation or treatment of “food” but does not
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include cosmetics or tobacco or substances used only as drugs.
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Food Additives – any substance the intended use of which results or may reasonably
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be expected to result, directly or indirectly, in its becoming a component or otherwise
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affecting the characteristics of any food (including any substance intended for use in
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producing, manufacturing, packing, processing, preparing, treating, packaging,
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transporting, or holding food; and including any source of radiation intended for any
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such use), if such substance is not generally recognized, among experts qualified by
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scientific training and experience to evaluate its safety, as having been adequately
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shown through scientific procedures to be safe under the conditions of the intended
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use (R.A. 3720. Food, Drugs and Cosmetic Act).
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Food and Drug Administration or FDA – formerly known as Bureau of Food and
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Drug (BFAD) of the Department of Health (DOH); which was renamed in accordance
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to RA 9711 (Food and Drug Administration (FDA) Act of 2009).
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Food Standard – a regulatory guideline that defines the identity of a given food
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product (i.e. its name and the ingredients used for its preparation) and specifies the
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minimum quality factors and, when necessary, the required fill of the container. It may
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also include specific labeling requirements other than or in addition to the labeling
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requirements generally applicable to all prepackaged foods.
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Ingredient - any substance including food additive, used as a component in the
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manufacture or preparation of a food and present in the final product in its original or
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modified form.
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Label – includes any tag, brand, mark, pictorial, or other descriptive script, written,
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printed, marked, embossed or impressed on, or attached to the container.
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Labeling – any written, printed or graphic matter (1) upon any article or any of its
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container or wrappers and/or (2) accompanying the packaged food.
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Lot – food produced during a period of time and under more or less the same
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manufacturing condition indicated by a specific code.
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Draft RCP as of August 17, 2010
Meat – it is fresh, chilled, or frozen edible carcass including offal derived from food
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animals (Joint DA-NMIS and DOH-FDA A.O. 01 s.2009). Skeletal & non-skeletal
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muscle tissues may include beef (cattle), carabeef (carabao or water buffaloes), pork,
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poultry (chicken, duck, turkey), lamb/mutton (sheep), and chevon (goat).
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Packaging – the process of packing that is part of the production cycle applied to a
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bulk product to obtain the finished product. Any material, including painted material,
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employed in the packaging of a product including any outer packaging used for
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transportation of shipment. Packaging materials are referred to as primary or
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secondary according to whether or not they are intended to be in direct contact with
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the product.
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Processing aids – these are additives that are used in the processing of food to
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achieve a specific technological purpose and which may or may not result in the
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presence of residues or derivatives in the final product (BFAD A.O. No. 88-A s. 1984)
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Potable water – it is the water fit for human consumption and potability determined by
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health authorities cited in Philippine National Standards for drinking water
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(Department of Health-Administrative Order No. 2007-0012: Philippine National
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Standards for Drinking Water 2007)
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Sausage – it is fresh or preserved meat, chopped or comminuted fine, to which has
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been added salt and spices and may contain sugar, seasoning, saltpeter (potassium
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or sodium nitrate) potassium or sodium nitrite, with or without binder (BFAD A.O. 154
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s. 1971). Sausage is comminuted seasoned meat that has been stuffed into casings,
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and may have undergone smoking, curing, fermentation and heating (FAO, 1985).
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Spices – refer to any aromatic vegetable substance in whole, broken, ground or in
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any other form, except those other substances which have been traditionally regarded
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as food.
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Water Activity – it is the ratio of vapor pressure of water in the food substrate to the
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vapor pressure of pure water at the same temperature (Jay et. al., 2005). It is also a
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measure of water available for chemical reactions and microbial growth (Fennema,
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1996).
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3.
RAW
MATERIALS,
INGREDIENTS
AND
Draft RCP as of August 17, 2010
PACKAGING
MATERIAL
REQUIREMENTS
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3.1 Raw materials and ingredients. Raw materials for processing shall not contain
parasites, microorganisms, toxins, and decomposed or extraneous substances.
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3.1.1 Basic Ingredients
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Meat. Meat to be used shall be sound, clean, and fit for human consumption. Meat
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should have a meat inspection mark or certificate of inspection by the NMIS and/or
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authority for the meat source, to confirm their suitability for processing.
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Curing agents. Salt to be used shall be fine or coarse sodium chloride (NaCl)
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available from natural sources or manufactured as food grade, and meets the purity
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requirements as specified in Section 4.1 of the Implementing Rules and Regulations
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of the ASIN Law, Republic Act (RA) 8172, an Act Promoting Salt Iodization
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Nationwide.
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Nitrites and/or nitrates to be used shall be as food-grade salt of sodium or potassium.
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Curing agents used must comply with the regulations set for food additives (BFAD
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Bureau Circular No. 016 s.2006. Updated List of Food Additive) and other applicable
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food standards
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3.1.2 Optional Ingredients
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Spices, Condiments and Flavorings. All spices, condiments and flavor/flavoring
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substances used shall be certified as food grade by the Food and Drugs
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Administration (FDA).
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Water. Only clean, potable water (Annex A) shall be used for the preparation and for
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all the pretreatment and processing steps of ethnic cured meat production.
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Non-potable water may be used only for operations not in direct contact with the food
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materials provided that this does not pose a hazard to health as determined and
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approved by the official agency having the jurisdiction over it.
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Draft RCP as of August 17, 2010
Binders and extenders. These may include soy proteins, cereal flours and other
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suitable food-grade materials complying with applicable food standards
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Fat. Fat to be used should come from any edible and food-grade vegetable and
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animal fat source, and must comply with applicable food standards.
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Sugar. Sugar to be used must be food grade and comply with applicable food
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standards.
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Vinegar. Vinegar to be used must be food grade and comply with applicable food
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standards
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Other food ingredients. These ingredients must be of food-grade quality and comply
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with applicable food standards required by FDA and/or authority.
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3.1.3. Casings
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Casings used may be edible or non-edible, must be of food grade-quality, and should
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conform to food standards required by FDA, NMIS, and/or authority.
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3.2 Packaging materials. The packaging materials should be appropriate for the product
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to be packed and for the expected conditions of handling during distribution and
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storage. These should provide the products adequate protection from contamination
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and should be sufficiently durable to withstand mechanical, chemical and thermal
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stresses encountered during processing and normal distribution. All packaging
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materials must be clean and free from defects that may affect the product or package
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integrity. These shall be stored in a clean and sanitary manner.
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4.
HYGIENE
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It is recommended that the products covered by the provisions of this code of practice
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should be processed and handled according to the appropriate sections of
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Recommended Code of Practice – General principles of Food Hygiene (CAC/RCP 1-
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1969 (Rev. 4, 2003)), the Code of Hygienic Practice for Meat (CAC/RCP 58-2005),
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and/or BFAD A.O. No. 153 s. 2004 - Revised Guidelines on Current Good
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Manufacturing Practice In Manufacturing, Packing, Repacking, or Holding Food,
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covering the plant facilities and operations requirement including the construction and
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Draft RCP as of August 17, 2010
layout of processing plant, hygienic facilities, equipment, utensils and working
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surfaces.
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5.
PREPARATION AND PROCESSING
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The preparation of ethnic cured meat is described from the receipt of raw materials
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until the packing operations. The production process should be supervised by
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personnel with adequate technical training and experience.
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5.1 Preparation of Raw Materials and Ingredients
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5.1.1 Meat
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Receipt
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Meat from any food animal species shall only be accepted if it is sound and suitable
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for processing, according to the requirements stipulated in sub-subsection 3.1.1.
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Fresh meat used for the processing of ethnic cured meats should have been chilled
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or frozen immediately after slaughter. The internal temperature of chilled meat should
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be near 0°C, while internal temperature of frozen meat should be -18°C or below. In
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addition, chilled and frozen meat should be maintained and transported by the
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supplier in chilled (near 0°C), and frozen conditions (-18°C or below), respectively.
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Those found with contamination should be rejected. Special precautions must be
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taken to reject meat showing signs of deterioration and spoilage.
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Inspection and sorting
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The meat shall be inspected and sorted according to meat source (species), meat
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parts or cuts, and intended use of the meat pieces.
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If prepackaged meat pieces or mechanically deboned meat is to be used as raw
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material, choose only those contained in clean, non-toxic, and properly labeled
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packaging materials.
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Storage/holding
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Fresh or chilled meat must be kept chilled at temperatures of 0° to 4°C, while frozen
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meat must be kept at -18°C or below.
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Draft RCP as of August 17, 2010
Meat held for processing should be stored in a suitable type of container and must be
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protected
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contaminants, debris, and dust. Meat may be placed in corrosion resistant trays.
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Cartons may also be used as long as appropriate inner lining is used or if the meat is
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individually wrapped.
from
domestic
animals,
parasites,
chemical
or
microbiological
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The containers should be arranged to allow adequate air circulation and to prevent
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drip from one meat piece from falling onto another piece. Conditions in the storage
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and holding areas such as temperature and humidity should be regulated to prevent
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spoilage and contamination.
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Regular inspection of the storage facility should be done to avoid infestation.
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Temperature and humidity levels conducive to spoilage and contamination should be
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avoided.
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Washing and/or sanitizing
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Meat may be washed to remove dirt, dust, and filth that might contaminate the
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meat, or water may be used to facilitate thawing of frozen meat. Water used for
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washing, thawing, and rinsing should be of potable quality.
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5.1.2 Optional Ingredients
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Receipt
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Optional ingredients and casings to be used in the preparation of ethnic cured meat
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shall conform to the requirements stipulated in sub-subsections 3.1.2, and 3.1.3.
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Whenever applicable, certificates of analyses (COA) from ingredient suppliers shall
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be secured to confirm their suitability for processing. Ingredients shall be rejected if
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they do not conform to the requirements and are found to have signs of deterioration,
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decomposition, or contamination to an extent which renders them unfit for human
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consumption.
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Storage/Holding
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Optional ingredients shall be stored in closed containers as protection against
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infestation by domestic animals, parasites, filth, and chemical and microbiological
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contaminants. Storage requirements such as temperature and humidity may vary
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Draft RCP as of August 17, 2010
depending on the ingredient, and these should be provided accordingly by the
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storage facilities to be used.
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Stored stocks of ingredients should be used on a “first in-first out” (FIFO) or a “first to
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expire-first to use” (FEFU) basis.
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5.2 Processing Operations
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5.2.1. Preparation of meat
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If frozen meat is used, the thawing process should take place in conditions that
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minimize contamination and microbial growth. Frozen meat may be thawed in air or
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by immersing in running water. Time and temperature conditions must be controlled
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to minimize microbial growth and allow the product to thoroughly thaw out. Chilled
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meat and thawed meat must be kept at 0° to 4°C while waiting for further processing.
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5.2.2. Slicing, trimming, and comminution
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For tocino, tapa, and other similar products, meat shall be sliced and trimmed
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according to the desired size and thickness.
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For longganisa or similar sausage-type products, the meat shall be comminuted to
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smaller, uniformly-sized particles through the use of a meat grinder, a food
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processor, or a silent cutter. Traditionally, meat used for longganisa is more coarsely
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ground as compared to emulsified sausages like hotdogs and bologna.
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The slicing, trimming, and comminution of meat must be performed in a way that
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minimizes contamination and growth of microorganisms.
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5.2.3 Mixing and Curing
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Sliced or comminuted meat is mixed or blended with curing agents and other non-
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meat ingredients including sugar, vinegar, and spices and flavorings. The meat
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mixture is allowed to cure for 1 to 2 days at 4° to 10°C. In cases when water is used
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to dissolve the dry ingredients, it is added in the form of ice water to help control the
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temperature of the curing brine/mixture. The curing brine should be maintained at not
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more than 5°C. Mixing and curing conditions should be controlled to prevent
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contamination and growth of microorganisms.
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Draft RCP as of August 17, 2010
5.2.4 Shaping (for longganisa and other sausage-type products)
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Shaping may be accomplished by either stuffing and linking, or moulding:
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Stuffing and Linking. The meat mixture is stuffed into casings by hand, through a
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screw feed or by using an automatic stuffing machine. Care must be observed not to
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use too much pressure that will damage the casings, and at the same time, the
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sausage mix must completely and compactly fill the casings.
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Sausage-type products that use casings with small diameters and long strands are
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further divided into segments that are uniform in size and length. This may be
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accomplished by twisting the casing or tying with a string to create sausage links. If
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casings with large diameters are used, the meat mixture is filled into individual
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casings that have the ends sealed with strings or metal clips.
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Moulding. The meat mixture is shaped into cylinders using plastic or paper wraps,
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which will give the product its shape in lieu of a casing.
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5.2.5 Tumbling and massaging (for tocino, tapa and other similar products)
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To increase meat tenderness and enhance the distribution of curing ingredients
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inside the meat, the meat slices may also be subjected to tumbling and massaging.
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The process involves equipment with rotating chambers or drums with baffles or
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massaging arms which subject the meat pieces to a gentle beating process as the
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chamber rotates. Tumbling can take place for several hours, but the temperature
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should be kept at less than 4°C. Tumbling and massaging time and temperature
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must be controlled to minimize microbial growth.
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5.2.6 Other Treatments
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Drying. An optional treatment for ethnic cured meat is drying. For longganisa and
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other sausage-type products in casings, drying is done particularly if the product
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would be subjected to smoking. Longganisa and similar products are allowed to
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stand at room temperature for 1 to 2 hours in order to allow the casings to dry before
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the product is subjected to smoking. Drying may also be accomplished by placing the
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products inside a warm smokehouse kept at 50°C without smoke. Drying the casing
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will depend on the size/diameter of the sausage and may range from 15 minutes to
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one hour.
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Draft RCP as of August 17, 2010
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Drying of tapa and similar products may be done by sun drying or through the use of
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artificial dryers. The drying process should be done in a manner that prevents
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contamination and microbial growth.
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Smoking. Ethnic cured meats may or may not be smoked. Using raw sawdust,
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ethnic cured meats are smoked at temperatures ranging from 50° to 70°C until the
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desired product flavor and color is obtained. The relative humidity of the smokehouse
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should be maintained at around 80%, since very low humidity can dry out the product
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while excessively high humidity dilutes the effect of smoke.
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Liquid smoke, a water soluble compound with smoke flavor, is also available, and
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can be sprayed or applied to the meat or sausage surface.
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5.3 Packing
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Packing can be done either mechanically or manually. It is important to standardize
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filling for economic reasons. Gas-packing or vacuum-packing may be done. The
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room temperature of the packing area should be maintained at 10°C or below.
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5.4 Closing or Sealing of Containers
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Seams and other closures shall be sealed air-tight to meet the requirements of the
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processors.
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The seal area of flexible containers must be free of food material and wrinkles.
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Sealing temperature and pressure shall conform to the sealing equipment to be used.
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5.5 Coding of Sealed Containers
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Coding of sealed container shall be indelible with details of production date and time,
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batch code, product code, the product line in which product is packed, the
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manufacturing plant and other information necessary for product traceability. Where
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the container does not permit the code to be embossed or inked, the label shall be
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legibly perforated or otherwise marked, and securely affixed to the product container.
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5.6 Post-Process Container Handling
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Ethnic cured meats must be kept at chilled (0° to 4°C) or frozen conditions (below
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-18°C). Fluctuations in storage temperature that will expose the product to freeze11
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Draft RCP as of August 17, 2010
thaw cycles should be avoided. The repeated freezing and thawing process can
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cause deterioration of the product quality.
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Mechanical shocks leading to breakage of semi-rigid containers due to container
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abuse must be avoided. These occur by knocking against each other during
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conveying,
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containers/pouches shall be handled singly rather than in bunches, and care must be
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exercised so as to prevent damage by roughened contact surfaces.
packaging
and
labeling
operations,
among
others.
Flexible
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6.
FOOD ADDITIVES
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6.1 Food additives when used shall be in accordance with the regulations established by
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the Food and Drugs Administration (B.C. No.2006-016 Updated List of Food
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Additives), the Codex Alimentarius Commission (CAC/STAN 192-1995, Rev. 5
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(2004)), and/or authority for these products. The following food additives listed in, but
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not limited to, Table 1, may be used for the manufacture of ethnic cured meats.
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6.2 All others that have not been included in the above list (Table 1) shall be allowed as
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carry over provided they are approved by the FDA regulation (B.C. No. 016 s. 2006;
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Updated List of Food Additives) and shall be in accordance to Section 5.2 of the
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“Principle Relating to the Carry-Over of Food Additives into Foods (CAC/Volume 1,
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1991).
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ingredients such as edible casings. Table 2 shows the list of additives that may be
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used for edible casings.
These additives include those that are used for raw materials and other
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Draft RCP as of August 17, 2010
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Table 1. Food Additives for Ethnic Cured Meat. (BFAD B.C. No.016 s. 2006. Updated List of
Food Additives)
Function
Antioxidant
Color
Additive
BHA
BHT
Gallate, Propyl
Isopropyl Citrates
Tertiary Butylhydroquinone (TBHQ)
Tocopherols
Allura Red AC
Annatto extracts
Canthaxanthin
Carmines
Carotenes, vegetable
Carotenoids
Curcumin
Erythrosine
Grape Skin Extract
Iron Oxides
Ponceau 4R
Sunset Yellow FCF
Tannic Acid (Tannins, food grade)
Diacetyltartaric and fatty acid
esters of glycerol
Polysorbates
Propylene Glycol Alginate
Humectant
Phosphates
Preservative Benzoates
Emulsifier/
Stabilizer
Hydroxybenzoates, pNitrates
Nitrites
Sodium Diacetate
Sorbates
Sulphites
Maximum allowable level
200 mg/kg (Fat or oil basis)*
100 mg/kg (Fat or oil basis)*
200 mg/kg *
200 mg/kg †
100 mg/kg (Fat or oil basis)*
3000 mg/kg*
500 mg/kg (For use in glaze, coatings, or decorations for fruit,
vegetables, meat, or fish) **
1000 mg/kg (As total bixin or norbixin; For use in tocino (fresh,
cured sausage) only) ***
20 mg/kg (For use in glaze, coatings, or decorations for fruit,
vegetables, meat, or fish) †
1000 mg/kg ***
1000 mg/kg ***
100 mg/kg †
1000 mg/kg (For use in tocino (fresh, cured sausage) only) ***
20 mg/kg †
1000 mg/kg ***
20 mg/kg †
1000 mg/kg (For use in tocino (fresh, cured sausage) only) ***
20 mg/kg †
30 mg/kg *
1000 mg/kg (For use in tocino (fresh, cured sausage) only) ***
GMP (For use in glaze, coatings, or decorations for fruit,
vegetables, meat, or fish) †
1000 mg/kg (For use in tocino (fresh, cured sausage) only) ***
GMP (For use in glaze, coatings, or decorations for fruit,
vegetables, meat, or fish) †
250 mg/kg ***
200 mg/kg †
500 mg/kg (For use in glaze, coatings, or decorations for fruit,
vegetables, meat, or fish) ***
135 mg/kg †
10 mg/kg*
GMP **
10000 mg/kg *
5000 mg/kg *
1100 mg/kg (As phosphorus)*
750 mg/kg (As benzoic acid) †
1000 mg/kg (As benzoic acid) †
GMP (Surface treatment; As p-hydroxybenzoic acid) †
1254 mg/kg (As residual NO3 ion) ***
365 mg/kg (As residual NO3 ion) †
134 mg/kg (As residual NO2 ion) *
1000 mg/kg *
2000 mg/kg (As sorbic acid) *
500 mg/kg (As residual SO2) *
* For food category system: 8.3. Processed comminuted meat, poultry and game products
**For food subcategory: 8.3.1. Non-heat treated processed comminuted meat, poultry, and game products
*** For food subcategory: 8.3.1.1. Cured (including salted) non-heat treated processed comminuted meat, poultry, and game
products
† For food subcategory: 8.3.1.2. Cured (including salted) and dried non-heat treated processed comminuted meat, poultry,
and game products
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439
440
441
442
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Draft RCP as of August 17, 2010
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Table 2. Food Additives for Edible Casings* (BFAD B.C. No.016 s. 2006. Updated List of Food
Additives)
Function
Additive
Maximum allowable level
Antioxidant
Ascorbyl Esters
5000 mg/kg (As ascorbyl stearate)
Tocopherols
5000 mg/kg
Color
Allura Red AC
500 mg/kg (For use in glaze, coatings or
decorations for fruit, vegetables, meat or fish)
Annatto extracts
60 mg/kg (As total bixin or norbixin)
Canthaxanthin
GMP
Carmines
500 mg/kg (For use in glaze, coatings, or
decorations for fruit, vegetables, meat, or fish)
Carotenes, vegetable
GMP
Carotenoids
500 mg/kg (For use in glaze, coatings, or
decorations for fruit, vegetables, meat, or fish)
Curcumin
500 mg/kg (For use in glaze, coatings, or
decorations for fruit, vegetables, meat, or fish)
Erythrosine
GMP
Fast Green FCF
GMP (Surface treatment; For decoration,
stamping, marking or branding the product)
Grape Skin Extract
GMP
Iron Oxides
1000 mg/kg (Ready-to-eat basis)
Orange B
150 ppm
500 mg/kg (For use in glaze, coatings, or
Ponceau 4R
decorations for fruit, vegetables, meat, or fish)
500 mg/kg (For use in glaze, coatings, or
Sunset Yellow CF
decorations for fruit, vegetables, meat, or fish)
Emulsifier/
Diacetyltartaric and fatty acid
GMP
Stabilizer
esters of glycerol
Dioctyl Sodium Sulfosuccinate
200 mg/kg
Polysorbates
1500 mg/kg
Propylene Glycol Alginate
20000 mg/kg
Sorbitan Esters of Fatty Acids
3500 mg/kg
Sucrose Esters of Fatty Acids
5000 mg/kg (Fat or oil basis)
Humectant
Phosphates
1100 mg/kg (As phosphorus)
Preservative
Hydroxybenzoates, p36 mg/kg (As p-hydroxybenzoic acid)
Nitrates
146 mg/kg (As residual NO3 ion)
Nitrites
134 mg/kg (As residual NO2 ion)
Sorbates
GMP (As sorbic acid)
Sulphites
500 mg/kg (As residual SO2)
*For food category system: 8.4. Edible casings (e.g. sausage casings)
445
446
447
7.
LABELING
448
449
7.1 Each container shall be labeled and marked with the following information in accordance
450
with BFAD A.O. 88-B s. 1984 (Rules and regulations Governing the Labeling of
451
Prepackaged Food Products distributed in the Philippines):
452
453
(a)
The product shall be known as “cured meat” or “ethnic cured meat”. It shall be
454
labeled by common names that include “tocino”, “longganisa”, or “tapa”, provided that
455
the product conforms to the product definitions in subsection 3.1 of the Standards for
456
Ethnic Cured Meats. Other local or regional names referring to products similar to
14
457
Draft RCP as of August 17, 2010
those defined in subsection 3.1 (Standards for Ethnic Cured Meats) may also be
458
included, provided that these names are acceptable in the area of distribution. The
459
label shall also indicate the species or type of meat source used, such as “pork”,
460
“chicken”, “turkey”, or “beef”.
461
(b)
product in descending order of proportion.
462
463
The complete list of ingredients and food additives used in the preparation of the
(c)
The net content by weight in the metric system. Other systems of measurement
464
required by importing countries shall appear in parenthesis after the metric system
465
unit.
466
(d)
The name and address of the manufacturer, packer and/or distributor of the food.
467
(e)
Open date marking
468
The words “Best/Consume Before/Expiry Date” indicating end of period at which the
469
product shall retain its optimum quality attributes at defined storage conditions.
470
(f)
Lot or code number identifying product lot.
471
(g)
The words “Product of the Philippines”, or the country of origin if imported.
472
(h)
Directions for use;
473
Directions for use should be indicated on the label. For products that utilize casings
474
or wrappers, the label should indicate that non-edible casings or wrappers should be
475
removed from the product before consumption.
476
(i)
Storage instructions; and
477
(j)
Additional requirements
478
A pictorial representation of the product and/or raw materials used placed on the
479
label should not mislead the consumer with respect to the product and/or raw
480
material so illustrated.
481
482
7.2 Nutrition Labeling
483
Nutrition labeling shall conform to the established regulations of FDA and/or authority for
484
this commodity.
485
486
8.
QUALITY ASSURANCE
487
488
8.1 Inspection of Finished Products
489
All processed products shall be inspected before labeling and casing and defective
490
products shall be withdrawn or rejected. The company must have an approved policy and
491
procedures based on the BFAD A.O. No. 153 s. 2004 - Guidelines, Current Good
492
Manufacturing Practices in Manufacturing, Packing, Repacking or Holding Food.
15
Draft RCP as of August 17, 2010
493
494
8.2 Record Keeping
495
Permanent and legible dated records of time, temperature code mark and other pertinent
496
details shall be kept concerning each load. Such records are essential as a check on
497
processing operations.
498
499
Written records of all container closure examinations shall specify the code lot, the date
500
and time of container closure inspections, the measurements obtained and all the
501
corrective actions taken.
502
503
Records shall be maintained identifying initial distribution of the finished product to
504
facilitate, if necessary, the segregation of specific food lots that may have been
505
contaminated or otherwise unfit for intended use.
506
507
All process deviations involving failure to satisfy the minimum requirements of the process
508
shall be recorded detailing those deviations and the actions taken.
509
510
8.3 Good Manufacturing Practices (GMP)
511
Processing establishments shall have developed, documented and implemented
512
prerequisite programs based on Food and Drugs Administration’s Current Good
513
Manufacturing Practices (cGMP) and Hygiene Control. An effective GMP and Hygiene
514
Control program will decrease the number of critical control points that a manufacturer
515
must face during the hazard analysis of the product/process.
516
517
9.
STORAGE AND TRANSPORT OF FINISHED PRODUCT
518
519
Storage and transport conditions of the finished product shall be such that the integrity of
520
the product container, and the safety and quality of the product are not adversely affected.
521
522
Cases and cartons must be of proper size so that the containers fit snugly and are not
523
subject to damage from movement within the case. They must be strong enough to
524
withstand normal transport.
525
526
Chilled products must be kept at 0° to 4°C while frozen products must be kept at -18°C or
527
below. Extreme fluctuations in temperature and humidity during storage and transport of
528
the product must be avoided to prevent product deterioration.
16
Draft RCP as of August 17, 2010
529
530
10. LABORATORY CONTROL PROCEDURES
531
532
Each food processing establishment shall have access to laboratory control of both the
533
processes used and the finished products. All food ingredients and food products declared
534
unfit for human consumption by the laboratory shall be rejected.
535
536
Representative samples for each lot or batch shall be taken to assess the safety and
537
quality of the product.
538
539
Microbiological laboratory shall be separated from the processing area. No pathogens
540
shall be handled within the premises of manufacturing plant.
541
542
Laboratory procedures for quality control of the processes and the product must follow
543
recognized or standard methods for easy interpretation of results.
544
545
11. END PRODUCT SPECIFICATIONS
546
547
Appropriate methods shall be used for sampling analysis and determinations to meet the
548
following specifications:
549
1.
from any objectionable characteristics.
550
551
2.
3.
The product shall be free from chemical pollutants in amounts which may pose
hazard to health.
554
555
The product shall not contain any toxic substances originating from microorganisms
and chemicals.
552
553
To the extent possible in good manufacturing practices, the products shall be free
4.
The product shall comply with the requirements set forth by the Food and Drugs
556
Administration, and the Codex Alimentarius Commission on Pesticide Residues and
557
Food Additives.
558
559
12.
REFERENCES
560
561
Abiva, C. C. 2001. A Quick Guide to Filipino Food and Cooking. Anvil Publishing, Inc.
562
Pasig City, Philippines.
563
17
564
Draft RCP as of August 17, 2010
A.O. No. 88-A s. 1984. Regulatory Guidelines Concerning Food Additives. Bureau of Food
565
and Drugs. Department of Health. Alabang, Muntinlupa City, Philippines.
566
567
A.O. No. 88-B s. 1984. Rules and Regulations governing the Labeling of Prepackaged
568
Food Products distributed in the Philippines. Bureau of Food and Drugs. Department of
569
Health. Alabang, Muntinlupa City, Philippines.
570
571
A.O. No. 153 s. 2004. Guidelines, Current Good Manufacturing Practice in Manufacturing,
572
Packing, Repacking or Holding Food. Bureau of Food and Drugs. Department of Health.
573
Alabang, Muntinlupa City, Philippines.
574
575
A.O. No. 154 s. 1971. Regulation B-4 Definition and Standards of Identity for Food 4.14
576
Meat and Meat Products, 4.14.01 Sausages. Bureau of Food and Drugs. Department of
577
Health. Alabang, Muntinlupa City, Philippines.
578
579
Archer, G.P. and C.J. Kennedy. 1998. Maximising Quality and Stability of Frozen Foods:
580
A Producers Guide to the State of the Art. Report no 2. EU Concerted action CT96-1180.
581
Accessed: 9 June 2010. Available at: http://www.nutrifreeze.co.uk/Documents/Maximising
582
%20Quality.pdf>
583
584
Association of Analytical Chemists. Official Methods of Analysis Manual. 16th ed., 1995.
585
AOAC International. 481 North Frederick Ave., Suite 500, Gaithersburg, MD 20877-2417.
586
U.S.A.
587
588
B.C. No. 01-A s. 2004. Guidelines for the Assessment of Microbiological Quality of
589
Processed Foods. Bureau of Food and Drugs. Department of Health. Alabang, Muntinlupa
590
City, Philippines.
591
592
B.C. No. 016 s. 2006. Updated List of Food Additives. Bureau of Food and Drugs.
593
Department of Health. Alabang, Muntinlupa City, Philippines.
594
595
DTI. 2007. Starting A Business: Tocino. Bureau of Small and Medium Enterprise
596
Development. Department of Trade and Industry. Sen. Gil Puyat Ave., Makati City,
597
Philippines. Available at: <http://www.dti.gov.ph/dti/uploads/file/Starting%20a%20Business
598
-MeatProcessing3.pdf>
599
18
600
Draft RCP as of August 17, 2010
DTI. 2009. Starting A Business: Native Longganisa. Bureau of Small and Medium
601
Enterprise Development. Department of Trade and Industry. Sen. Gil Puyat Ave., Makati
602
City, Philippines. Available at: <http://www.dti.gov.ph/uploads/DownloadableFiles/SAB_
603
Native_ Longanisa_09.pdf>
604
605
Essien, E. 2003. Sausage Manufacture: Principles and Practice. Woodhead Publishing
606
Ltd. Cambridge, England.
607
608
FAO. 1990. Manual on simple methods of meat preservation.. FAO Animal Production and
609
Health Paper 79. Food and Agriculture Organization of the United Nations. Rome, Italy.
610
Available at: <http://www.fao.org/docrep/003/x6932e/x6932e00.htm>.
611
612
FAO. 1991. Guidelines for slaughtering, meat cutting and further processing. FAO Animal
613
Production and Health Paper 91. Food and Agriculture Organization of the United
614
Nations. Rome, Italy. Available at: <http://www.fao.org/docrep/004/t0279e/T0279E00.htm>
615
616
FAO/WHO Codex Alimentarius Commission Manual. 1995. Codex Alimentarius
617
Commission. Food and Agriculture Organization. Viale delle Terme di Caracalla, 00100
618
Rome, Italy.
619
620
Food, definition. ALINORM 04/27/41, para. 88 and Appendix VI. 2005. Codex Alimentarius
621
Commission. Food and Agriculture Organization. Viale delle Terme di Caracalla, 00100
622
Rome, Italy.
623
624
FSIS. 1999. General HACCP Model for Beef Slaughter. Accessed: 16 August 2010.
625
<http://www.fsis.usda.gov/OPPDE/nis/outreach/models/HACCP-13.pdf>.
626
627
FSIS. 2008. The Regulated Industries: Characteristics and Manufacturing Processes.
628
Accessed: 8 February 2010. Available at : <http://www.fsis.usda.gov/pdf/phvt-regulated_
629
industries.pdf>
630
631
FSIS. 2009. Hot Dogs and Food Safety. Accessed 19 March 2010. Available at:
632
<http://www.fsis.usda.gov/pdf/hot_dogs_and_food_safety.pdf>
633
19
634
Draft RCP as of August 17, 2010
Heinz, G. and P. Hautzinger. 2007. Meat Processing Technology: for Small- and Medium-
635
Scale Producers. Food and Agriculture Organization of the United Nations: Regional office
636
for Asia and the Pacific. Bangkok, Thailand.
637
638
Joint DA-NMIS and DOH-FDA A.O. 01 s.2009. Delineation of Functions and Shared
639
Responsibilities in the Regulation of Meat Products. National Meat Inspection Service.
640
Department of Agriculture. Visayas Ave., Diliman, Quezon City.
641
642
M.C. no. 09 s. 2008. Guidelines on the Assessment of Microbiological Quality of Fresh,
643
Chilled, and Frozen Meat. National Meat Inspection Service. Department of Agriculture.
644
Visayas Ave., Diliman, Quezon City.
645
646
New Zealand Food Safety Authority. Draft Code of Practice: Production of Processed
647
Meats. Accessed: 9 June 2010. Available at: <http://www.nzfsa.govt.nz/consultation/
648
processed-meat-cop-part1-4/part-3-process-control/index.htm>
649
650
Philippine National Standards No. 991:1993. Agricultural and Other Food Products –
651
Bottled Drinking Water Specifications. Bureau of Product Standards. Department of Trade
652
and Industry. Makati City, Philippines.
653
654
R.A. 3720. Food, Drugs and Cosmetic Act. Bureau of Food and Drugs. Department of
655
Health. Alabang, Muntinlupa City, Philippines.
656
657
Ranken, M.D. 2000. Handbook of Meat Product Technology. Blackwell Science Ltd.
658
Oxford, England.
659
660
Sanchez, P.C. 2008. Philippine fermented foods: Principles and technology. The
661
University of the Philippines Press. Diliman, Quezon City, Philippines.
662
663
S.A.O. No. 412 s. 1980. Standardization of Tocino. Philippine Bureau of Standards.
664
Ministry of Trade. Quezon City, Philippines.
665
666
Savic, I.V. 1985. Small-scale Sausage Production. Food and Agriculture Organization of
667
the United Nations. Rome, Italy.
668
20
669
Draft RCP as of August 17, 2010
USDA/FSIS/AFDO. 1999. Safe Practices for Sausage Production: Distance Learning
670
Course Manual. Accessed 22 March 2010. Available at:
671
<http://www.midwesternresearch.com/PDF/SausageFSIS.pdf>
672
673
USFDA. 2001. Staphylococcus aureus. Chapter 12. In Bacteriological Analytical Manual.
674
Accessed 24 June 2010. Available at: http://www.fda.gov/Food/ScienceResearch/
675
LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm>
676
677
USFDA. 2003. Detection and Enumeration of Listeria monocytogenes in Foods. Chapter
678
10. Bacteriological Analytical Manual. Accessed 23 August 2010. Available at:
679
<http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalytical
680
ManualBAM/default.htm>
681
682
USFDA. 2007. Salmonella. Chapter 5. Bacteriological Analytical Manual. Accessed
683
23 August 2010. Available at: http://www.fda.gov/Food/ScienceResearch/
684
LaboratoryMethods/ BacteriologicalAnalyticalManualBAM/default.htm>
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
21
Draft RCP as of August 17, 2010
705
706
707
ANNEXA
708
Standard Parameters and Values for Drinking Water
709
Philippine National Standards for Drinking Water 2007 (DOH AO 2007-0012)
710
711 Table 1. Standard values for bacteriological quality
Parameter
Value/Unit
Point of Compliance
Total Coliform
< 1.1 MPN/100 ml
Fecal Coliform
< 1.1 MPN/100 ml
Heterotropic Plate Count
< 500 CFU/ml
Service Reservoir
Water treatment works
Consumers’ taps
Refilling stations
Water haulers
Water vending machines
Service Reservoir
Water treatment works
Consumers’ taps
Refilling stations
Water haulers
Water vending machines
Point sources - Level 1
Service Reservoir
Water treatment works
Consumers’ taps nearest meter
Refilling stations
Water vending machines
712
713
714
715
716
717
718
Table 2. Standard values for Physical and Chemical Quality for
Drinking Water
Constituents
Maximum Level (mg/L)
Constituents
or Characteristic
Taste
No objectionable taste
Hydrogen Sulfide
Odor
No objectionable odor
Iron
Color
Apparent = 10 color units
Manganese
True = 5 color units
Turbidity
3 NTU
pH
Aluminum
0.2
Sodium
Chloride
250
Sulfate
Copper
1.0
Total
Dissolved
Solids
Hardness
300 as CaCO3
Zinc
Acceptability Aspects for
Maximum Level (mg/L)
or Characteristic
0.05
1.0
0.4
6.5 – 8.5
200
250
500
5.0
Table 3. Standard Values for Organic and Inorganic Chemical Constituents of Health
Significance in Drinking Water
Inorganic Chemicals
Constituents
Antimony
Arsenic
Barium
Boron
Cadmium
Chromium (Total)
Maximum Level (mg/L)
0.02
0.05
0.7
0.5
0.003
0.05
Constituents
Fluoride
Lead
Mercury (total)
Nickel
Nitrate
Nitrite
Maximum Level (mg/L)
1.0
1.01
0.001
0.02
50
3.0
22
Draft RCP as of August 17, 2010
Cyanide (Total)
0.07
Selenium
0.01
Organic Chemicals
Constituents
Benzene
Maximum
Level (mg/L)
0.01
Constituents
Maximum Level (mg/L)
Ethylbenzene
0.30
Carbon tetrachloride
0.004
Nitrilotriacetic acid (NTA)
0.20
1,2-Dichlorobenzene
0.1
0.20
1,4-Dichlorobenzene
0.5
Polyaromatic
hydrocarbons
(PAHs)
Polynuclear aromatic
0.0007
1,2-Dichloroethane
0.003
Tetrachloroethene
0.02
1,1-Dichloroethene
0.05
Styrene
0.04
1,2-Dichloroethene
0.07
Tetrachloroethene
0.70
Dichloromethane
1.0
Trichloroethene
0.07
Di(2-ethyhexyl)
phthalate
Edetic Acid (ADTA)
1.01
Vinyl chloride
0.0003
0.001
Xylene
0.5
Organic Pesticides
Constituents
Aldrin and Dieldrin (combined)
Maximum Level
(ug/L)
30.0
Status in the Philippines
Banned
Atrazine
0.03
Registered
Carbofuran
2.0
Registered
Chlordane
7.0
Banned
DDT **
0.2
Banned
1,2-Dibromo-3-chloropropane (DBCP)
1.0
Banned
2,4-Dichlorophenoxyacetic acid (2,4-D)
1.0
Registered
Endrin
30.
Banned
1,2-Dibromomethane (Ethylene dibromide)
0.6
Banned
Heptachlor and Heptachlor epoxide (combined)
0.03
Banned
Lindane
2.0
Restricted
MCPA (4-(2-methyl-4-chloro) phenoxyl acetic
acid
Pendimethalin
2.0
Registered
20.0
Registered
Pentachlorophenol (PCP)
9.0
Banned
719
720
721
722
723
724
725
726
727
728
729
730
23
Draft RCP as of August 17, 2010
731
732
733
734
ANNEXB
735
Determination of Crude Protein Content
736
(AOAC 981.10; Block Digestion Method)
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
A. Reagents
(a) Catalyst tablets.-containing 3.5 g K2SO4 and 0.175 g HgO.
(b) Boric acid solution.-4%. Dissolve 4 g H3BO3 in H2O containing 0.7 mL 0.1% alcoholic solution
of methyl red and 1.0 mL 0.1% alcoholic solution of bromocresol green, and dilute to 100 mL with
H2O.
(c) Sodium hydroxide-sodium thiosulfate solution.-Dissolve 2000 g NaOH and 125 g Na2S2O3 in
H2O and dilute to 5 L (ca 5o mL is used per analysis).
(d) Hydrochloric acid standard solution.- o.2N ( 936.15 [see A.1.o6]).
(e) Hydrogen peroxide. – 3o-35%.
(f) Sulfuric acid.-concentrated.
B. Equipment
(a) Digestion block and associated glassware.-Tecator Ds-6 or Ds-2o (Tecator), or equivalent.
(b) Distillation unit and associated glassware.-Kjeltec 1oo3 (Tecator), or equivalent.
C. Determination
Accurately weigh ca 2 g well-ground and mixed sample on 7 cm N-free filter paper (e.g., whatman
541), fold, and transfer to 250 mL digestion tube. Place tubes in fume hood and add 2 or 3 boiling
stones, 2 catalyst tablets, 15 mL H2SO4, and slowly 3 mL 3o-35%
H2O2. Let reaction subside and place tubes in block digestor preheated at 410°. (Digestor must be
placed in perchloric acid fume hood or be equipped with exhaust system. Boiling concentrated
acid is very corrosive and also emits corrosive fumes. Rapid addition of 3o-35% H2O2 may cause
the reaction to become violent.) Digest at 410° until mixture is clear, ca 45 min. Remove tubes and
let cool ca 1o min. Do not let precipitate form; if precipitate forms, reheat. Carefully add 5o-75 mL
H2O.
Place NaOH-Na2S2O3 solution in alkali tank of steam distillation unit. Make sure that 5o-75 mL is
dispensed from unit before conducting distillation. Attach digestion tube containing diluted digest to
distillation unit. Place 25o mL receiving flask containing 25 mL H3BO3 solution with mixed indicator
on receiving platform, with tube from condenser extending below surface of absorbing solution.
Steam distil until 100-125 mL collects (absorbing solution turns green from liberated NH3). Remove
digestion tube and receiving flask from unit. Titrate absorbing solution with 0.2 N Hcl to neutral
gray end point and record volume acid required to 0.01 mL. Titrate reagent blank similarly.
D. Computation:
% N = ( vA – vB ) x 1.4oo7 x N /g sample
% Protein = ( vA – vB ) x 1.4oo7 x N x 6.25/g sample
where vA and vB = volume standard acid required for sample and blank, respectively; 1.4007 =
milliequivalent weight N x 1oo(%); N = normality of standard acid; and 6.25 = protein factor for
meat products (16% N).
779
780
781
24
Draft RCP as of August 17, 2010
782
783
784
785
786
ANNEXC
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
Determination of Moisture Content
(AOAC 950.46b; Air Drying)
--First Action
--Final Action 1991
1. With lids removed, dry sample containing ca 2 g dry material 16 –18 h at 100 – 102° in air
oven (mechanical convection preferred). Use covered Al dish ≥50 mm diameter and ≤4o mm
deep. Cool in desiccator and weigh. Report loss in weight as moisture.
2. With lids removed, dry sample containing ca 2 g dry material to constant weight (2 – 4 h
depending on product) in mechanical convection oven or in gravity oven with single shelf at ca
125°. Use covered Al dish ≥50 mm diameter and ≤4o mm deep. Avoid excessive drying.
Cover, cool in desiccator, and weigh. Report loss in weight as moisture. (Dried sample is not
satisfactory for subsequent fat determination.)
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
25
Draft RCP as of August 17, 2010
826
827
828
829
ANNEXD
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
Determination of Crude Fat
(AOAC 960.39)
A. Sample Drying
1. Weigh 3 – 4 g sample by difference into thimble containing small amount of sand. Mix
with glass rod, place thimble and rod in 50 mL beaker, and dry in oven 6 h at 100 – 102° or 1.5
h at 125°. Proceed as in (B); or
2. Weigh 3 – 4 g sample by difference into small disposable Al dish, add sand, and mix,
spreading mixture on bottom of dish with glass or Al paddle. Dry with paddle as in (1). Roll
edges of dish and insert with paddle into thimble. Proceed as in B.
B. Determination:
(Large amounts H2O-soluble components such as carbohydrates, urea, lactic acid, glycerol, and
others may interfere with extraction of fat; if present, extract 2 g sample on small paper in funnel
with five 20 mL portions H2O prior to drying for ether extraction. Caution: see Appendix B, safety
notes on monitoring equipment, distillation, and diethyl ether.)
1. Extract ca 2 g sample, dried as in (A), with anhydrous ether. Use thimble with porosity
permitting rapid passage of ether. Extraction period may vary from 4 h at condensation rate of
5 – 6 drop/s to 16 h at 2 – 3 drop/s. Petroleum ether, 945.16A (see 27.4.04), may be used
instead of anhydrous ether, if desired.
2. Dry extract to constant weight at 100°, cool, and weigh.
26
Draft RCP as of August 17, 2010
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
ANNEXE
Determination of Water Activity (AOAC 978.18)
A. Principle
Water activity, aw, is ratio of vapor pressure of H2O in product to vapor pressure of pure H2O at
same temperature. It is numerically equal to 1/100 of relative humidity (RH) generated by product
in closed system. RH can be calculated from direct measurement of
partial vapor pressure or dew point or measured indirectly by sensors whose physical or electric
characteristics are altered by RH to which they are exposed. Instruments are checked or
calibrated on basis of RH generated by standard salt slushes.
B. Instruments and Systems
(Select 1 of following instruments or systems to perform test. Each has different application
limitations because of interferences from other volatile components of products being measured.
check with instrument manufacturer for more specific limitations.)
(a) Change in electrical conductivity of immobilized salt solution. – Instrument available from
Beckman Industrial, Rosemount Analytical Div., 89 Commerce Rd, Cedar Grove, NJ 07009;
Nova Sina AG, Andreastrasse 7-11, CH 8050, Zurich,Switzerland; Rotronic Instrument Corp.,
160 E. Main St, Huntington, NY 11743. Immobilized salt sensors are affected by polyols such
as glycerol and glycol and by volatile amines
(b) Change in electrical capacitance of polymer thin films. – Instrument available from General
Eastern Instruments, 50 Hunt St, Watertown, MA 02172. Polymer thin film sensors are affected
by CH3COOH.
(c) Dew point by chilled mirror technique. – Instrument available from EG&G, Environmental
Equipment Division, 217 Middlesex Turnpike, Burlington, MA 01803 or General Eastern
Instruments. Dew point measurements can be affected by condensables with lower critical
temperature than H2O.
(d) Longitudinal change in dimensions of water-sorbing fiber. – Instrument available from G Lufft
Metallbarometerfabrik, D-7, Postfach 692, Neue Weinsteige 22, Stuttgart, Germany.
(e) Partial water vapor pressure by manometric system. – Partial H2O vapor pressure
measurements can be made useless by living products that respire, such as grains or nuts; by
active fermentation; or by products that expand excessively when subjected to high vacuum.
(f) Relative weight of moisture sorbed by anhydrous hydrophilic solid, e.g., microcrystalline
cellulose.-see J. Agr. Food chem. 22, 326(1974).
C. Apparatus and Reagents
(As needed for instrument or system selected.)
(a) Dew point instrument. – Equipped to measure temperature to ±0.1°. See 978.18B(c).
(b) Forced-draft cabinet. – Constant temperature, set to maintain 25 ± 1°; capacity ≥0.06 m3 (2 cu ft);
with access port to accommodate instrument sensor leads. Use in conjunction
with (c).
(c) Insulated box with cover. – Large enough to hold test container, (e), and small enough to fit in
forced-draft cabinet, (b); with access port to accommodate instrument sensor leads. Protect
test container from short-term temperature fluctuations.
(d) Manometric system. – Sensitive to pressure differential of ± 0.01 mm Hg (1.33 Pa). See
978.18B(e).
(e) Test containers. – 120 or 240 mL (4 or 8 oz) wide-mouth or Mason glass jars with Al- or Teflonlined screw caps and gaskets. Check integrity of cap seals and sensor leads by any means
available, e.g., ability of system to hold vacuum, using Tesla coil.
(f) Water bath. – Capable of maintaining temperature constant within 0.1° at 25±1°; capacity
sufficient to hold measuring chamber of selected apparatus.
(g) Hydrophilic solid. – Microcrystalline cellulose, Type PH-101 (FMC Corp.,Pharmaceutical and
Bioscience Division, 1735 Market St, Philadelphia, PA 19103, or equivalent).
(h) Reference salts. – ACS reagent grade, fine crystal. see Table 978.18.
27
Draft RCP as of August 17, 2010
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
D. Preparation of Reference Salt Slushes
Place selected reference salt in test container to depth of ca 4 cm for more soluble salts (lower aw),
to depth of ca 1.5 cm for less soluble salts (higher aw), and to intermediate depth for intermediate
salts. Add H2O in ca 2 mL increments, stirring well with spatula after each addition, until salt can
absorb no more H2O as evidenced by free liquid. Keep free liquid to minimum needed to establish
saturation of salt with H2O. Slushes are ready for use upon completion of mixing, and are usable
indefinitely (except for some high aw salts susceptible to bacterial attack), if contained in manner to
prevent substantial evaporation losses. Some slushes, e.g., NaBr, may solidify gradually by crystal
coalescence, with no effect on aw.
E. Calibration
Select ≥ 5 salts to cover aw range of interest or range of sensor being used. Measure humidity
generated by each salt slush in terms of instrument readout, as in 978.18F. Plot readout against aw
values given in Table 978.18 for selected salts, using cross-section paper scaled for reading to
0.001 aw unit. Draw best average smooth line through plotted points. Use this calibration line to
translate sensor instrument readout of samples to aw or to check vapor pressure or dew point
instruments for proper functioning.
F. Determination
Place calibration slush or sample in forced-draft cabinet, (b), or H2O bath, (f), until temperature is
stabilized at 25±1°. Transfer salt slush or sample to test container, (e), seal container with sensing
device attached, and place in temperature control device. Use volume of sample or slush >1/20
total volume sample container plus any associated void volume of sensing system, but not so
much as to interfere with operation of system. Record instrument response at 15, 30, 60, and 120
min after test container is placed in temperature control device, or record response on strip chart.
Two consecutive readings, at indicated intervals, which vary by <0.01 aw unit are evidence of
adequately close approach to equilibrium. Continue readings at 60-min intervals, if necessary.
Convert last reading to aw by calculation from physical measurements or by reference to calibration
line. Make all measurements within range of calibration points; do not extrapolate calibration line.
Make all measurements in same direction of change, and, if required by properties of sensor,
expose sensor to controlled RH below ambient before starting each measurement.
975
976
977
28
Draft RCP as of August 17, 2010
978
ANNEXF
979
Isolation of Salmonella (USFDA, 2007)
980
981
A. Sample Preparation (For meats, meat substitutes, meat by-products, animal substances,
glandular products, and meals (fish, meat, bone)).
982
983
984
985
986
987
Aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile lactose broth and
blend 2 min. Aseptically transfer homogenized mixture to sterile wide-mouth, screw-cap jar (500
ml) or other appropriate container and let stand 60 ± 5 min at room temperature with jar securely
capped. If mixture is powder or is ground or comminuted, blending may be omitted. For samples
that do not require blending, add lactose broth and mix thoroughly; let stand for 60 ± 5 min at room
temperature with jar securely capped.
988
989
990
991
992
993
Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Add up
to 2.25 ml steamed (15 min) Tergitol Anionic 7 and mix well. Alternatively, use steamed (15 min)
Triton X-100. Limit use of these surfactants to minimum quantity needed to initiate foaming. Actual
quantity will depend on composition of test material. Surfactants will not be needed in analysis of
powdered glandular products. Loosen jar caps 1/4 turn and incubate sample mixtures 24 ± 2 h at
35°C. Continue as in B., below.
994
B. Isolation of Salmonella
995
1. Tighten lid and gently shake incubated sample.
996
997
Guar gum and foods suspected to be contaminated with S. Typhi. Transfer 1 ml mixture to
10 ml selenite cystine (SC) broth and another 1 ml mixture to 10 ml TT broth . Vortex.
998
999
All other foods. Transfer 0.1 ml mixture to 10 ml Rappaport-Vassiliadis (RV) medium and
another 1 ml mixture to 10 ml tetrathionate (TT) broth. Vortex.
1000
2. Incubate selective enrichment media as follows:
1001
1002
1003
Foods with a high microbial load. Incubate RV medium 24 ± 2 h at 42 ± 0.2°C (circulating,
thermostatically-controlled, water bath). Incubate TT broth 24 ± 2 h at 43 ± 0.2°C (circulating,
thermostatically-controlled, water bath).
1004
1005
1006
Foods with a low microbial load (except guar gum and foods suspected to be
contaminated with S. Typhi). Incubate RV medium 24 ± 2 h at 42 ± 0.2°C (circulating,
thermostatically controlled, water bath). Incubate TT broth 24 ± 2 h at 35 ± 2.0°C.
1007
1008
Guar gum and foods suspected to be contaminated with S. Typhi. Incubate SC and TT
broths 24 ± 2 h at 35°C.
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
1020
1021
1022
3. Mix (vortex, if tube) and streak 3 mm loopful (10 µl) incubated TT broth on bismuth sulfite (BS)
agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar. Prepare BS
plates the day before streaking and store in dark at room temperature until streaked.
4. Repeat with 3 mm loopful (10 µl) of RV medium (for samples of high and low microbial load
foods) and of SC broth (for guar gum).
5. Refer to 994.04 in Official Methods of Analysis (1) for option of refrigerating incubated sample
preenrichments and incubated sample selective enrichments (SC and TT broths only) of low
moisture foods. This option allows sample analyses to be initiated as late as Thursday while still
avoiding weekend work.
6. Incubate plates 24 ± 2 h at 35°C.
7. Examine plates for presence of colonies that may be Salmonella.
8. Lightly touch the very center of the colony to be picked with sterile inoculating needle and
inoculate TSI slant by streaking slant and stabbing butt. Without flaming, inoculate LIA slant by
stabbing butt twice and then streaking slant. Since lysine decarboxylation reaction is strictly
29
Draft RCP as of August 17, 2010
1023
1024
1025
1026
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
anaerobic, the LIA slants must have deep butt (4 cm). Store picked selective agar plates at 58°C.
9. Incubate TSI and LIA slants at 35°C for 24 ± 2 h. Cap tubes loosely to maintain aerobic
conditions while incubating slants to prevent excessive H2S production. Salmonella in culture
typically produces alkaline (red) slant and acid (yellow) butt, with or without production of H2S
(blackening of agar) in TSI. In LIA, Salmonella typically produces alkaline (purple) reaction in
butt of tube. Consider only distinct yellow in butt of tube as acidic (negative) reaction. Do not
eliminate cultures that produce discoloration in butt of tube solely on this basis. Most
Salmonella cultures produce H2S in LIA. Some non- Salmonella cultures produce a brick-red
reaction in LIA slants.
10. All cultures that give an alkaline butt in LIA, regardless of TSI reaction, should be retained as
potential Salmonella isolates and submitted for biochemical and serological tests. Cultures that
give an acid butt in LIA and an alkaline slant and acid butt in TSI should also be considered
potential Salmonella isolates and should be submitted for biochemical and serological tests.
Cultures that give an acid butt in LIA and an acid slant and acid butt in TSI may be discarded
as not being Salmonella . Test retained, presumed-positive TSI cultures as directed in D-11,
below, to determine if they are Salmonella species, including S. arizonae. If TSI cultures fail to
give typical reactions for Salmonella (alkaline slant and acid butt) pick additional suspicious
colonies from selective medium plate not giving presumed-positive culture and inoculate TSI
and LIA slants as described in D-8, above.
11. Apply biochemical and serological identification tests to:
1044
1045
1046
1047
1048
1049
1050
1051
1052
1053
1054
1055
1056
1057
1058
1059
1060
1061
1062
1063
1064
1065
1066
1067
1068
1069
1070
1071
1072
1073
1074
1075
1076
1077
a. Three presumptive TSI cultures recovered from set of plates streaked from RV medium (or
SC broth for guar gum), if present, and 3 presumptive TSI agar cultures recovered from plates
streaked from TT broth, if present.
b. If 3 presumptive-positive TSI cultures are not isolated from one set of agar plates, test other
presumptive-positive TSI agar cultures, if isolated, by bioche mical and serological tests.
Examine a minimum of 6 TSI cultures for each 25 g analytical unit or each 375 g composite.
30
Draft RCP as of August 17, 2010
1078
1079
1080
1081
1082
1083
1084
1085
1086
1087
1088
1089
1090
1091
1092
1093
1094
1095
1096
1097
1098
1099
1100
1101
1102
1103
1104
1105
1106
1107
1108
1109
1110
1111
1112
1113
1114
1115
1116
1117
1118
1119
1120
1121
1122
ANNEXG
Detection and Enumeration of Listeria monocytogenes
(Conventional method; USFDA (2003))
Media and Reagents
1. Acetic Acid, 5 N
2. Acriflavine monohydrochloride
3. Agar (Difco Laboratories, Detroit, MI)
4. N-(1-naphthyl) ethylene diamine (R48)
5. α-Naphthol reagent (R48)
6. Blood agar base No. 2 (Unipath)
7. Cycloheximide
8. Natamycin (pimaricin)
9. Sheep blood, defibrinated
10. Ethanol, absolute
11. Fluorescent antibody (FA) buffer (Difco)
12. Glycine anhydride
13. Gram stain kit
14. Hydrogen peroxide solution, 3% for catalase test (R12)
15. KOH 40% solution (R65)
16. Listeria-typing sera set (Difco)
17. Lithium chloride-phenylethanol-moxalactam (LPM) agar (M81) with added esculin and iron
(M82)
18. Nalidixic acid (sodium salt)
19. Nitrate reduction medium (M108) and nitrate detection reagents (R48)
20. Nutrient broth (M114)
21. Physiological saline solution, 0.85% (R63)
22. Purple carbohydrate fermentation broth base (M130), containing 0.5% solutions of
dextrose, esculin, maltose, rhamnose, mannitol, and xylose
23. SIM medium (Becton-Dickinson Microbiology Systems, M137) or motility test medium
(MTM, Difco) (M103)
24. Sulfanilic acid reagent (R48)
25. Trypticase soy agar with 0.6% yeast extract (TSAye) (M153)
26. Trypticase soy broth with 0.6% yeast extract (TSBye) (M157)
27. Oxford medium (OXA) (M118)
28. Buffered Listeria Enrichment Broth (BLEB) (M52)
29. PALCAM agar (M118a)
30. Carageenan (Sigma type II)
31. BCM agar (M17a)
32. MOX agar (M103a)
33. ALOA agar (M10a)
34. Chromogenic Listeria Agar (M40b)
35. Rapid L'mono (M131a)
36. CHROMagar Listeria (M40a)
37. Tryptose broth and agar (Difco) (M167)
Note: Alternative companies may be used when the products are equivalent.
1123
A. Sample treatment.
1124
1125
1126
1127
Sample refrigeration at 4°C is recommended for handling, storing, and shipping materials to be
analyzed for L. monocytogenes, which will grow, although slowly, at this temperature if other
conditions permit. However, if the sample is already frozen, it should not be thawed until
analysis.
1128
a. Composited samples.
31
Draft RCP as of August 17, 2010
1129
1130
1131
1132
1133
1134
1135
1136
Generally, composited samples are prepared as follows. A food lot sample is collected
consisting of 10 sub-samples (liquid, cream or solid food) and 50-g or ml portions of each subsample are used to make two composite samples (250 g each). Take care to make subsamples representative of a food's outer surface as well as its interior. For the first composite 5
× 50-g portions from 5 sub-samples are pooled and blended or stomached in 250 ml buffered
Listeria enrichment broth base containing sodium pyruvate without selective agents (BLEB,
M52). The second composite is made from the remaining 5 sub-samples in the same way.
Both blended composites contain 250-g food portions and 250 ml basal BLEB.
1137
1138
1139
1140
1141
1142
Normally 50 g of each composite blend (equivalent to 25g food plus 25 ml basal BLEB) is
mixed with a 200 ml amount of basal BLEB. Since there are two composites, the end result is
two 25-g analytical portions each contained in 225 ml amounts of basal BLEB. Thus, for each
sub-sample a total of 50 g of composited food is analyzed. An aliquot (50 ml) of the composite
blend should be retained, preferably at 5° C and not below 0° C, for possible pathogen
enumeration.
1143
b. Non-composited samples.
1144
1145
1146
1147
If composite samples are not required, single 25-g analytical portions of food are simply
blended or stomached in 225 ml of basal BLEB and pre-enriched/enriched as described later.
A 25-g sample should be retained for possible pathogen enumeration. Store it at 5° C if it is not
frozen or, if frozen, in a non-defrosting freezer.
1148
B. Pre-enrichment and enrichment.
1149
1150
1151
1152
1153
1154
Incubate for 4 h at 30° C, add the selective agents and continue incubating for a total time of
48 h at 30° C. If cycloheximide is unavailable, the preferred substitute is pimaricin (natamycin)
at 25 mg/L (27). Natamycin is much safer to use than cycloheximide. Another possibility, if the
matrices of interest (e.g. pasteurized milk and cream, yogurt, and precooked frozen seafood)
are low in yeast and mold, is to do without an anti-fungal agent. This is not advisable for moldripened cheeses, smoked or dried seafood or fresh produce.
1155
C. Enrichment with enumeration.
1156
1157
1158
1159
1160
1161
Surveillance enumeration of Listeria monocytogenes levels in contaminated food is now
required. Detection may be done first and if contamination is detected, a reserve sample
portion can be enumerated. This is probably the preferable method as, generally, only a few
percent of samples can be expected to be positive and then most often at a level of only about
1 cfu/25g. However, the option of combining regulatory detection and enumeration is provided
in Enumeration.
1162
1163
1164
1165
1166
1167
1168
1169
1170
1171
1172
1173
1174
1175
1176
1177
D. Isolation procedure
At 24 and 48 h, streak BLEB culture onto one of the following esculin-containing selective isolation
agars: either OXA (16, M118) or PALCAM (42, M118a) or MOX (41, M103a) or LPM (31, M81)
fortified with esculin and Fe3+ (M82). These esculin-containing media are listed in order of preferred
use, subject to their availability. Incubate OXA, PALCAM or MOX plates at 35° C for 24-48 h and
fortified LPM plates at 30° C for 24-48 h. It is strongly recommended that one of the L.
monocytogenes-L. ivanovii differential selective agars, such as BCM (33, M117a), ALOA (M10a),
RapidL'mono (M131a), or CHROMagar Listeria (M40a) be streaked at 48 h (optionally at 24 h, too)
in addition to the chosen esculin-containing selective agar. This will reduce the problem of masking
of L. monocytogenes by L. innocua. [Note: BCM has been collaboratively validated by FDA. An ISO
TC34 SC9 comparative validation showed that all the media (and a selective blood agar - LMBA,
Sifin, Germany) inhibited Listeria competitors more or less equally well. ALOA was preferred only
because its formulation is public. Another differential selective medium, Chromogenic Listeria Agar
(M40b) is due to be marketed in the future.]
32
Draft RCP as of August 17, 2010
1178
1179
1180
1181
1182
1183
1184
1185
1186
1187
1188
1189
1190
1191
1192
1193
1194
1195
1196
1197
1198
1199
1200
Listeria colonies are black with a black halo on esculin-containing media. Certain other bacteria can
form weakly brownish black colonies, but color development takes longer than 2 days. Transfer 5
or more typical colonies from OXA and PALCAM or modified LPM or MOX to Trypticase soy agar
with yeast extract (TSAye), streaking for purity and typical isolated colonies. If BCM plates are
streaked as recommended above and blue colonies are observed, they are presumptive L.
monocytogenes colonies since L. ivanovii is not often reported in foods. L. monocytogenes and L.
ivanovii colonies on ALOA are blue and have a zone of lipolysis around them. Purification on
TSAye is a mandatory step in the conventional analysis because isolated colonies on selective
agar media may still be in contact with an invisible weak background of partially inhibited
competitors. At least 5 isolates are necessary because more than one species of Listeria may be
isolated from the same sample. Use of BCM and ALOA plates will help to reduce the number of
colonies that need to be picked. L. monocytogenes and L. ivanovii can be distinguished using a
commercial Confirmatory Medium (Biosynth International, Inc.) or by conventional rhamnose/xylose
fermentation broths or agars. Incubate TSAye plates at 30° C for 24-48 h. The plates may be
incubated at 35° C if colonies will not be used for wet-mount motility observations. For the
approved rapid methods (Table 2), use the selective isolation agar recommended by the
manufacturer but, as noted above, auxiliary use of the new L. monocytogenes-L.ivanovii differential
agars is also recommended.
1201
1202
1203
1204
1205
1206
1207
1208
1209
1210
1211
1212
1213
1214
1215
1216
1217
1218
1219
1220
1221
1222
1223
1224
1225
1226
1227
1228
1229
1230
1231
1232
1233
1234
1235
1. Examine TSAye plates for typical colonies. Observation with Henry oblique transmitted
illumination can be helpful at this stage but is not mandatory.
2. Pick typical colony from culture plate incubated at 30°C or less and examine by wet mount,
using 0.85% saline for suspending medium and oil immersion objective of phase-contrast
microscope. Choose a colony with enough growth to make a fairly heavy suspension;
emulsify thoroughly. If too little growth is used, the few cells present will stick to the glass
slide and appear non-motile. Listeria spp. are slim, short rods with slight rotating or
tumbling motility. Always compare with known culture. Cocci, large rods, or rods with rapid,
swimming motility are not Listeria spp. Alternatively, use the 7-day motility test medium.
3. Test typical colony for catalase. Listeria species are catalase-positive.
4. Gram stain 16- to 24-h cultures. All Listeria spp. are short, Gram-positive rods; however,
with older cultures the Gram stain reaction can be variable and also cells may appear
coccoidal. The cells have a tendency to palisade in thick-stained smears. This can lead to
false rejection as a diphtheroid.
5. Pick typical colony to a tube of TSBye for inoculating carbohydrate fermentation and other
test media. Incubate at 35° C for 24 h. This culture may be kept at 4°C several days and
used repeatedly as inoculum. Commercial kits are available for isolate identification.
6. Inoculate heavily (from TSAye colony) 5% sheep blood agar by stabbing plates that have
been poured thick and dried well (check for moisture before using). Draw grid of 20-25
spaces on plate bottom. Stab one culture per grid space. Always stab positive controls (L.
ivanovii and L. monocytogenes) and negative control (L. innocua). Incubate for 24-48 h at
35° C. Attempt to stab as near to bottom of agar layer as possible, without actually
touching bottom of agar layer and possibly fracturing the agar.
7. Examine blood agar plates containing culture stabs with bright light. L.monocytogenes and
L. seeligeri produce a slightly cleared zone around the stab. L. innocua shows no zone of
hemolysis, whereas L. ivanovii produces a well-defined clear zone around the stab. Do not
try to differentiate species at this point, but note nature of hemolytic reaction. Resolve
questionable reactions by the CAMP test. (Note: Hemolysis is more easily determined
when the depth of the blood agar is thinner than the usual 5mm. Optionally, this may be
achieved by use of a blood agar overlay (1-2 mm) technique).
8. Nitrate reduction test. This test is optional. Only L. grayi ssp. murrayi reduces nitrates. The
test distinguishes L. grayi ssp. murrayi from L. grayi ssp. grayi. Use a TSBye culture to
inoculate nitrate broth (M108). Incubate at 35° C for 5 days. Add 0.2 ml reagent A, followed
by 0.2 ml reagent B (R48). A red-violet color indicates presence of nitrite, i.e. nitrate has
been reduced. If no color develops, add powdered zinc and hold for 1 h. A developing red-
E. Identification procedure
Identify purified isolates by the following classical tests. Rapid kits are available to facilitate
biochemical testing to genus or species level.
33
Draft RCP as of August 17, 2010
1236
1237
1238
1239
1240
1241
1242
1243
1244
1245
1246
1247
1248
1249
1250
1251
1252
1253
1254
violet color indicates that nitrate is still present and has not been reduced. As an alternative
procedure (R48), add 0.2 ml reagent A followed by 0.2 ml reagent C. An orange color
indicates reduction of nitrate. If no color develops, add powdered zinc as above.
Development of an orange color indicates unreduced nitrate.
9. Inoculate SIM or MTM from TSBye. Incubate for 7 days at room temperature. Observe
daily. Listeria spp. are motile, giving a typical umbrella-like growth pattern. MTM provides
the best defined umbrellas. Alternatively, observe the 30° C TSBye cultures, by phase
contrast microscopy (×1000) for tumbling motility.
10. From TSBye culture, inoculate the following carbohydrates as 0.5% solutions in purple
carbohydrate broth (the use of Durham tubes is optional): dextrose, esculin, maltose,
rhamnose, mannitol, and xylose. Incubate 7 days at 35° C. Positively reacting Listeria spp.
produce acid with no gas. Consult Table 1 for xylose-rhamnose reactions of Listeria spp.
All species should be positive for dextrose, esculin, and maltose. All Listeria spp. except L.
grayi should be mannitol-negative. If pigmentation of the isolate on OXA, PALCAM, MOX
or LPM plus esculin/Fe3+ is unequivocal, the esculin test may be omitted.
11. Purified isolates identified as Listeria monocytogenes by alternative rapid methods should
be retained for regulatory reference.
F. Interpretation of test data
1255
1256
1257
1258
1259
1260
1261
1262
The importance of completely characterizing each isolate cannot be overemphasized. Partial
characterization, even if accurate, may be misleading. Since all Listeria species test negative for
indole, oxidase, urease, and H2S production from organic sulfur compounds (H2S is produced
from thiosulfate in the MICRO-ID test kit) and test positive for methyl red and Voges-Proskauer,
these tests are discretionary. Brochothrix, which is closely related phylogenetically to Listeria, is
distinguishable from Listeria by its inability to grow at 35° C and by its lack of motility.
Distinguishing features of the Gram-positive non-sporeforming rods, Erysipelothrix and Kurthia,
which occur rarely in Listeria analysis, can be found elsewhere.
1263
1264
1265
1266
1267
1268
1269
1270
1271
1272
1273
1274
1275
1276
1277
1278
1279
1280
1281
1282
1283
1284
1285
1286
1287
All Listeria spp. are small, catalase-positive, Gram-positive rods that are motile in wet mounts
and in SIM. They utilize dextrose, esculin, and maltose, and some species utilize mannitol,
rhamnose, and xylose with production of acid. An isolate utilizing mannitol with acid production is
L. grayi. L. monocytogenes, L. ivanovii, and L. seeligeri produce hemolysis in sheep blood stabs
and consequently are CAMP test-positive. Of the three, only L. monocytogenes fails to utilize
xylose and is positive for rhamnose utilization. The difficulty in differentiating L. ivanovii from L.
seeligeri can be resolved by the CAMP test. L. seeligeri shows enhanced hemolysis at the S.
aureus streak. L. ivanovii shows enhanced hemolysis at the R. equi streak. Of the non-hemolytic
species, L. innocua may provide the same rhamnose-xylose reactions as L. monocytogenes but
it is negative in the CAMP test. L. innocua sometimes gives negative results for utilization of
rhamnose . The significance of the undocumented reference to hemolytic L. innocua isolates is
unclear since it is commonly accepted that L. innocua is non-hemolytic and L. monocytogenes is
hemolytic. A L. welshimeri isolate that is rhamnose-negative may be confused with a weakly
hemolytic L. seeligeri isolate unless resolved by the CAMP test. Sometimes aberrant listeria
strains are isolated which are extremely difficult to speciate. (See Guideline for BAM Users on
Identification of Atypical Hemolytic Listeria Isolates.) [Note: The clinical significance of a strain of
L. monocytogenes that is phenotypically hemolytic-negative is debatable. If it is due to a defect of
the hemolysin gene, especially a deletion rather than a point mutation, it is likely clinically less
significant than a normal strain would be, judging from laboratory studies of constructed
hemolysin mutants in mice. However, if it is due to a regulatory defect that affects the expression
of the hemolysin gene in vitro, the possibility of conditional expression in vivo is raised. Until
convenient methods are devised to distinguish these structural and regulatory alternatives, the
isolate need only be carefully confirmed as being a strain of L. monocytogenes phenotypically
hemolysin-negative in vitro so that a soundly based regulatory decision can then be made, based
upon all the relevant circumstances.]
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Only after all other results are available does serotyping and other kinds of typing of Listeria
isolates become meaningful. All data collection must be completed before species identities are
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Draft RCP as of August 17, 2010
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determined. FDA no longer conducts routine bacteriophage-susceptibility typing of L.
monocytogenes isolates.
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G. Enumeration (required)
1. If a sample tests positive for L. monocytogenes, use a reserve portion of sample for
enumeration. Current methods of enumeration are only presumptive for Listeria
monocytogenes and some degree of further testing of isolated Listeria colonies is
necessary. Conventional enumeration is described and alternative rapid methods are
indicated. The proportion of presumptive isolates that are actually L. monocytogenes may
be determined by conventional or rapid tests. Flexibility in choice of methods and
adaptations of them is permitted but the observed count must be reported with 95%
confidence limits, the method used named and any modifications indicated. The correction
factor for converting the observed count to L. monocytogenes numbers must be reported
as the whole number ratio of number of isolates identified as L. monocytogenes to the total
number of Listeria isolates tested.
2. All enumeration methods, including microscopic, colony and Most Probable Number (MPN)
counts are fundamentally governed by the Poisson distribution law of infrequent events.
This describes the distribution of Listeria among the arrays of compartments (tubes, wells,
counting chamber squares, filter grid squares, and virtual squares on culture agar
surfaces). Compartmentalization separates or delineates colony-forming units in the
various methods. In general, the confidence limits (CLs) of these estimates are considered
proportional to the square root of the observed count. [The tabulated CLs for MPN results
are asymmetric about the mean because they are usually obtained with low numbers of
tubes (3 or 5) near the dilution endpoint.] As the count increases its confidence limits,
expressed as a percentage of the count, decrease. Thus, choosing among methods largely
reduces to a consideration of material and labor expenses and to how inoculation
manipulations for an optimal number of compartments can be reduced by techniques such
as filtration, semi-automation and robotics.
3. Surveillance Enumeration. This is required for accumulating data on cell numbers of L.
monocytogenes in regulatory samples that test positive for the pathogen. To estimate the
degree of sample contamination by presumptive L. monocytogenes, quantify the initial
enrichment broth, before starting incubation, by direct spread plate count on ALOA, BCM
or equivalent differential agar. Also, use a 3 or more-tube/well MPN culture procedure on 1,
0.1, 0.01and 0.001-g samples in BLEB (30° C, 48 h, with or without pyruvate and without
delayed addition of selective agents) followed by streaking on the chosen selective agar. If
all the MPN tubes are Listeria positive, use reserve sample to repeat the MPN
determination using an appropriate range of more dilute analytical portions, e.g. 10-4, 10-5,
10-6, 10-7, and 10-8 g.
4. If selective agar plates are in short supply, an economic alternative to spreading dilution
aliquots on individual selective agar plates is the drop plating method. Using a multichannel pipette is well suited to this method. Decimally dilute 10 µl amounts of the contents
of the enrichment containers in 90 µl amounts of TSBye in micro-titer plates with roundbottomed wells. Mix with a gentle circular motion of the micropipette tip before changing
the tip for the next dilution. Carefully plate 10 µl of the dilutions as drops on plates of ALOA,
BCM or equivalent agar. Let the droplets be absorbed before inverting the plates for
incubation. Square plates are most convenient and efficient for this technique.
Table 1. Alternative rapid enumeration methods.
Method
Reference
Validation
Specificity Matrix
MPN filter
Entis & Lerner (20)
AOAC INTL.
Filter/colony-lift
Carroll et al. (13)
Peer review
DNA probe colony
hybridization
BAM Chapter 24
(17,18)
FDA
All Listeria, FDA foods
L. monocytogenes
Meat
L. monocytogenes,
FDA foods
35
Draft RCP as of August 17, 2010
5. Alternatively, the methods shown in Table 1 may be used. Identify isolates by conventional
or rapid methods. When all Listeria are enumerated estimate the proportion that is L.
monocytogenes by determining the species of 10 typical Listeria colonies. M.L. Grant
(FDA/DFS/ORA Laboratory Information Bulletin 17: (3) LIB No. 4240, 9pp) has developed
a filter enumeration method, based on the BAM Listeria enrichment and isolation method,
which enumerates Listeria at cell numbers of >100 cfu/g.
6. Tolerance enumeration. Enumeration to determine if a regulated level of tolerance is being
met is not needed with the current "zero-tolerance" policy of no detectable L.
monocytogenes in 2 x 25-g analytical portions of food or beverage. It would require
narrower confidence limits than does surveillance enumeration. Narrower confidence limits
for tolerance and surveillance enumeration can be accomplished by counting more colony
forming units, which can be accomplished by increasing the number of replicate tubes or
other containers. For the current FDA method, the wells of one or more 96-well micro-titer
plates, with round-bottomed wells, can be inoculated, by multi-channel pipette or
robotically, with 0.1 ml of homogenate of complete BLEB and sample. After incubation at
30° C for 48 h, use the same kinds of transfer methods described in Surveillance
Enumeration to inoculate enriched samples to ALOA, BCM or equivalent differential agar to
determine which wells are positive. Using the proportion of L. monocytogenes-positive
wells, the mean concentration can be calculated using the Poisson equation.
7. Alternatively, the 1600 filter grid compartments MPN method for Listeria may be used for
presumptive enumeration of L. monocytogenes.
8. Identify isolates by conventional methods, including the use of ALOA, BCM or equivalent
agar, or by rapid methods. When necessary estimate the proportion of L. monocytogenes
among 10 Listeria isolates.
9. Simultaneous detection and enumeration. Most samples are likely to be negative and thus
it is efficient to delay enumeration of reserve samples until the Listeria detection stage is
completed. Even then, most positive samples will only contain a few cfu/25g. Nevertheless
it may sometimes be more convenient to do simultaneous detection and enumeration. To
accomplish this, prepare the enrichment homogenate as described above and immediately
spread 0.1 ml on ALOA, BCM or an equivalent L. monocytogenes selective agar. Incubate
plates at 35° C for 24-48 h. The combined minimal method will allow the cell number of
presumptive L. monocytogenes to be categorized as <0.04 cfu/g, 0.04 - 100 cfu/g, 10025,000 cfu/g, or > 25,000 cfu /g. More replica plates and more decimal dilutions in TSBye
are optional to obtain a more precise enumeration. Test 5 representative colonies for ability
to ferment L-rhamnose by the conventional fermentation method, by the BCM rhamnose
confirmatory agar or by a rapid L. monocytogenes identification kit to definitively rule out
the uncommon occurrence of L. ivanovii in foods.
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36
Draft RCP as of August 17, 2010
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ANNEXH
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Determination of Staphylococcus aureus
(Direct Plate Count Method (USFDA, 2001))
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A. Equipment and materials
1. Same basic equipment as for conventional plate count (Chapter 3).
2. Drying cabinet or incubator for drying surface of agar plates
3. Sterile bent glass streaking rods, hockey stick or hoe-shaped, with fire-polished ends, 3-4 mm
diameter, 15-20 cm long, with an angled spreading surface 45-55 mm long.
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B. Media and reagents
1. Baird-Parker medium (M17)
2. Trypticase (tryptic) soy agar (TSA) (M152)
3. Brain heart infusion (BHI) broth (M24)
4. Coagulase plasma (rabbit) with EDTA
5. Toluidine blue-DNA agar (M148)
6. Lysostaphin (Schwartz-Mann, Mountain View Ave., Orangeburg, NY 10962)
7. Tryptone yeast extract agar (M165)
8. Paraffin oil, sterile
9. 0.02 M phosphate-saline buffer (R61), containing 1% NaCl
10. Catalase test (R12)
A. Preparation of sample
(see Chapter 1 of USFDA/CFSAN Baceriological Analytical Manual, 2001).
B. Isolation and enumeration of S. aureus
1. For each dilution to be plated, aseptically transfer 1 ml sample suspension to 3 plates of
Baird-Parker agar, distributing 1 ml of inoculum equitably to 3 plates (e.g., 0.4 ml, 0.3 ml, and 0.3
ml). Spread inoculum over surface of agar plate, using sterile bent glass streaking rod. Retain
plates in upright position until inoculum is absorbed by agar (about 10 min on properly dried
plates). If inoculum is not readily adsorbed, place plates upright in incubator for about 1 h. Invert
plates and incubate 45-48 h at 35°C. Select plates containing 20-200 colonies, unless only plates
at lower dilutions (>200 colonies) have colonies with typical appearance of S. aureus. Colonies of
S. aureus are circular, smooth, convex, moist, 2-3 mm in diameter on uncrowded plates, gray to
jet-black, frequently with light-colored (off-white) margin, surrounded by opaque zone and
frequently with an outer clear zone; colonies have buttery to gummy consistency when touched
with inoculating needle. Occasionally from various foods and dairy products, nonlipolytic strains of
similar appearance may be encountered, except that surrounding opaque and clear zones are
absent. Strains isolated from frozen or desiccated foods that have been stored for extended
periods frequently develop less black coloration than typical colonies and may have rough
appearance and dry texture.
2. Count and record colonies. If several types of colonies are observed which appear to be S.
aureus on selected plates, count number of colonies of each type and record counts separately.
When plates of the lowest dilution contain <20 colonies, these may be used. If plates containing
>200 colonies have colonies with the typical appearance of S. aureus and typical colonies do not
appear at higher dilutions, use these plates for the enumeration of S. aureus, but do not count
nontypical colonies. Select > 1 colony of each type counted and test for coagulase production. Add
number of colonies on triplicate plates represented by colonies giving positive coagulase test and
multiply by the sample dilution factor. Report this number as number of S. aureus/g of food tested.
37
Draft RCP as of August 17, 2010
1445
E. Coagulase test
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Transfer suspect S. aureus colonies into small tubes containing 0.2-0.3 ml BHI broth and emulsify
thoroughly. Inoculate agar slant of suitable maintenance medium, e.g., TSA, with loopful of BHI
suspension. Incubate BHI culture suspension and slants 18-24 h at 35°C. Retain slant cultures at
room temperature for ancillary or repeat tests in case coagulase test results are questionable. Add
0.5 ml reconstituted coagulase plasma with EDTA (B-4, above) to the BHI culture and mix
thoroughly. Incubate at 35°C and examine periodically over 6 h period for clot formation. Only firm
and complete clot that stays in place when tube is tilted or inverted is considered positive for S.
aureus. Partial clotting, formerly 2+ and 3+ coagulase reactions, must be tested further (4). Test
known positive and negative cultures simultaneously with suspect cultures of unknown coagulase
activity. Stain all suspect cultures with Gram reagent and observe microscopically. A latex
agglutination test (AUREUS TESTTM, Trisum Corp., Taipei, Taiwan) may be substituted for the
coagulase test if a more rapid procedure is desired.
38