1FINAL DRAFT 
RECOMMENDED CODE OF PRACTICE FOR THE PROCESSING AND  
HANDLING OF ETHNIC FLOUR-BASED CONFECTIONERIES  
(POLVORON, PIAYA, AND BARQUILLOS ) 
 
1. SCOPE  
 
This Code of Practice is concerned with the receipt of raw  materials and ingredients, 
preparation and processing of ethnic flour-based confectio neries ( polvoron, piaya, 
and barquillos ) as defined in this Code, in order to conform with th e required 
standards stated in PNS/FDA No. ___. Standards for Ethn ic Flour-based 
Confectioneries ( Polvoron, Piaya, and Barquillos ). The product shall be prepared 
from a basic mixture of flour and sugar. This Code is i ntended to provide guidelines 
to achieve compliance with the standards for ethnic flo ur-based confectioneries 
(polvoron, piaya, and barquillos ) packed in any suitable packaging material and/or 
container. 
 
2.       DEFINITION OF TERMS 
 
For the purpose of this Code, the following definitio ns apply: 
 
Confectionery - a group of food items primarily made of sugar and ot her 
sweeteners, and includes candies, caramels, toffees, and choc olate bars.  It is a 
generic term for sweetened food products.  Sugar confecti onery refers to products 
such as sweets, candy and chocolates.  These products are sh elf-stable and usually 
have water activity below 0.85. (Dictionary of Food S cience and Technology , 
International Food Information Service, Blackwell Pub lishing, UK, 2005.)  
 
Container  – any form of packaging material, which completely or partially encloses 
the food (including wrappers). A container may enclose t he food as a single item or 
several units or types of prepackaged food when such is p resented for sale to the 
consumer. 
  2Current Good Manufacturing Practices (cGMP)  – a quality assurance system 
aimed at ensuring that products are consistently manufac tured, packed or repacked 
or held to a quality appropriate for the intended u se. It is thus concerned with both 
manufacturing and quality control procedures.  
 
Flavor and flavoring substances – substances which are added to impart flavor 
which are either natural, nature identical or artifici al flavoring substances (A.O. No. 
88-B s. 1984; Rules and Regulations governing the Labe ling of Prepackaged Food 
Products distributed in the Philippines): 
(a) natural flavor  
flavoring substances derived through appropriate physical  processes from spices, 
herbs, fruit or fruit juices, vegetable or vegetable juices, edible yeast, bark, bud, root, 
leaf or plant materials, meat, fish, poultry, eggs, da iry products or fermentation 
products thereof. 
(b) nature-identical flavoring substances  
substances chemically derived from aromatic materials or ob tained synthetically, 
which are chemically identical to substances present in' na tural products intended for 
human consumption. 
(c) artificial flavoring substances  
substances that impart flavor but which have not been i dentified in natural products 
or natural sources of flavorings. 
 
Flour – a powdery substance that is the product of grinding or milling of cereal 
grains (such as but not limited to wheat, rice, barley, and maize), root crops and 
legumes.  
 
Food - any processed substance which is intended for human consum ption and 
includes drink for man, beverages, chewing gum and any su bstances which have 
been used as an ingredient in the manufacture, prepar ation or treatment of food. 
(R.A. 9711. Food and Drug Administration (FDA) Act of  2009). 
 
Food Additives  – any substance the intended use of which results or may  
reasonably be expected to result, directly or indirectl y, in its becoming a component 
or otherwise affecting the characteristics of any food (i ncluding any substance  3intended for use in producing, manufacturing, packing, processing, preparing, 
treating, packaging, transporting, or holding food; a nd including any source of 
radiation intended for any such use), if such substance is not generally recognized, 
among experts qualified by scientific training and expe rience to evaluate its safety, 
as having been adequately shown through scientific proce dures to be safe under the 
conditions of the intended use (R.A. 3720. Food, Drug and Cosmetic Act).   
 
Food and Drug Administration or FDA  - formerly known as Bureau of Food and 
Drug (BFAD) of the Department of Health (DOH); which  was renamed in accordance 
to RA 9711 (Food and Drug Administration (FDA) Act of  2009). 
 
Food Standard  – a regulatory guideline that defines the identity of a given food 
product (i.e. its name and the ingredients used for it s preparation) and specifies the 
minimum quality factors and, when necessary, the require d fill of the container. It 
may also include specific labeling requirements other tha n or in addition to the 
labeling requirements generally applicable to all pre packaged foods. 
 
Ingredient  - any substance including food additive, used as a compon ent in the 
manufacture or preparation of a food and present in the final product in its original or 
modified form. 
 
Label  – includes any tag, brand, mark, pictorial, or other d escriptive script, written, 
printed, marked, embossed or impressed on, or attached to the container. 
 
Labeling  – any written, printed or graphic matter (1) upon a ny article or any of its 
container or wrappers and/or (2) accompanying the package d food. 
 
Lot – food produced during a period of time and under mo re or less the same 
manufacturing condition indicated by a specific code. 
 
Milk – the normal mammary secretion of milking animals obtai ned from one or more 
milkings without either addition to it or extraction f rom it, intended for consumption as 
liquid milk or for further processing (CODEX STAN 206-1 999). 
  4Milk product – a product obtained by any processing of milk, which may  contain 
food additives, and other ingredients functionally nece ssary for the processing 
(CODEX STAN 206-1999). 
 
Muscovado sugar –  a non-centrifugal sugar which is primarily composed of su crose 
and molasses. It includes three forms, namely: amorphous; lump or molded locally 
known as “ panocha”  or “ sangkaka ”; and thick, concentrated syrup locally known as 
“inuyat”. (BAFPS Draft Standards for Muscovado Sugar) 
 
Packaging  – the process of packing that is part of the production cycle applied to a 
bulk product to obtain the finished product. It also ref ers to any material, including 
painted material, employed in the packaging of a prod uct including any outer 
packaging used for transportation of shipment. Packaging m aterials are referred to 
as primary or secondary according to whether or not they are intended to be in direct 
contact with the product. 
 
Rancidity  – formation of off-flavors in food due to lipid oxi dation (oxidative rancidity) 
and/or release of free fatty acids by lipolysis (hydrol ytic rancidity). 
 
Water Activity – the ratio of vapor pressure of water in the food sub strate to the 
vapor pressure of pure water at the same temperature ( Jay et. al. , 2005). It is also a 
measure of water available for chemical reactions and m icrobial growth (Fennema, 
1996). 
      
3.      RAW MATERIALS, INGREDIENTS AND PACKAGING MA TERIAL 
REQUIREMENTS 
 
3.1 Raw materials and ingredients .  
Raw materials for processing shall not contain parasites, m icroorganisms, toxins, 
and decomposed or extraneous substances. 
 
3.1.1 Basic Ingredients 
  5Flour . May come from cereal grains, root crops, or legumes, an d must conform to 
RA 8976 (Philippine Food Fortification Act of 2000) ( for wheat flour), and other 
applicable food standards. 
 
Sugar and other sweeteners . May include table sugar, muscovado sugar, corn 
syrup, and other similar food items, and must conform to  RA 8976 (Philippine Food 
Fortification Act of 2000) (for refined sugar), and o ther applicable food standards. In 
some cases, sweetened condensed milk and similar milk produ cts also act as the 
source of sugar and/or sweetener.  
 
Milk and milk products . Applicable as basic ingredient only for polvoron. Must 
conform to requirements prescribed by PNS/BAFPS 36:2008  (Philippine National 
Standard for Fresh Milk), FDA A.O. No. 132 s. 1970 (Re gulation Prescribing the 
Standard of Identity and Quality of Milk and Milk Pro ducts, B-4.12-01), and other 
applicable food standards.  
 
Milk and milk products used in the production of ethnic fl our-based confectioneries 
(polvoron, piaya, and barquillos ) should come from healthy animals. Milk and milk 
products should have been produced under hygienic condit ions as required by the 
regulatory agency and/or authority for this type of f ood items. 
 
Butter, margarine, lard, oil, and other similar foo d items . Applicable as basic 
ingredient for polvoron and piaya. Must comply with RA 8976 (Philippine Food 
Fortification Act of 2000) (for cooking oil), FDA A.O. No. 230 s. 1974 (Regulation: B-
4 Definition and Standards for Food; B-4.15 Fats and Oils, B-4.15-01 Shortening), 
FDA A.O. No. 243 s. 1975 (Regulation: B-4 Definition  and Standards of Food; B-4.18 
Margarine), and other applicable food standards. 
 
3.1.2 Optional Ingredients 
  
Eggs . Must be fresh eggs, and must comply with the requiremen ts prescribed by 
PNS/BAFPS 35: 2005 (Philippine National Standards fo r Table Egg), and other 
applicable food standards. 
  6Fruit, vegetables, nuts, and root crops. May include fresh items or preserves and 
must conform to all applicable food standards 
 
Flavor and Flavoring Substances . - All flavor/flavoring substances as defined in 
subsection 2.4 shall be food grade, and must comply with  FDA’s B.C. No. 016 s.2006 
(Updated List of Food Additives). 
 
Water . Must be water fit for human consumption and potabil ity determined by health 
authorities as cited in Philippine National Standards f or drinking water (Department 
of Health A.O. No. 2007-0012. Philippine National S tandards for Drinking Water 
2007). 
 
Only clean, potable water (Annex A) shall be used for the preparation and for all the 
pretreatment and processing steps of confectionery produ ction.  
 
Non-potable water may be used only for operations not  in direct contact with the food 
materials provided that this does not pose a hazard to  health as determined and 
approved by the official agency having the jurisdiction  over it. 
 
Sesame Seeds . Must conform to applicable food standards. 
 
3.2     Packaging materials . The packaging materials used shall be made of 
suitable, food-grade materials that would not adversel y affect product quality and 
safety. The packaging materials should be appropriate fo r the product to be packed 
and for the expected conditions of handling during dist ribution and storage. These 
should provide the products adequate protection from co ntamination and should be 
sufficiently durable to withstand mechanical, chemical an d thermal stresses 
encountered during processing and normal distribution. A ll packaging materials must 
be clean and free from defects that may affect the pro duct or package integrity. 
These shall be stored in a clean and sanitary manner.  
 
4. HYGIENE 
It is recommended that the product covered by the prov isions of this code of practice 
be prepared and handled in accordance with the appropr iate sections of the  7Recommended International Code of Practice – General P rinciples of Food Hygiene 
(CAC/RCP 1–1969, Rev 4 (2003)), Code of Hygienic Pra ctice for Milk and Milk 
Products (CAC/RCP 57-2004),  Recommended International Code of Hygienic 
Practice for Egg Products CAC/RCP 15-1976 (amended 1978 , 1985),  and/or the 
FDA A.O. No.  153 s. 2004 - Guidelines, Current Good Manufacturing P ractices in 
Manufacturing, Packing, Repacking or Holding Food, cover ing the plant facilities and 
operations requirement including the construction and l ayout of processing plant, 
hygienic facilities, equipment, utensils and working surf aces. 
 
5. PREPARATION AND PROCESSING  
 
The preparation of ethnic flour-based confectioneries ( polvoron, piaya, and 
barquillos ) is described from the receipt of raw materials until the packing operations. 
The production process should be supervised by personnel  with adequate technical 
training and experience.  
 
5.1  Preparation of Raw Materials and Ingredients 
 
5.1.1 Flour  
 
Receipt  
Flour shall only be accepted if it is sound and suitabl e for processing, according to 
the requirements stipulated in sub-subsection 3.1.1. Tho se found with contamination 
should be rejected. Special precautions must be taken to  reject flour showing signs 
of deterioration and contamination. Choose only those contained in clean, non-toxic, 
and properly labeled packaging materials. 
 
Storage/holding 
Flour held for processing should be stored in any suitabl e type of closed container to 
protect from domestic animals, parasites, chemical or micr obiological contaminants, 
debris, and dust. Flour should be stored at a cool, dr y place. 
 
5.1.2 Sugar and other sweeteners 
  8Receipt and Inspection  
Sugar and sweeteners used for the production of ethnic flour-based confectioneries 
(polvoron, piaya, and barquillos ) shall only be accepted if it is sound and suitable for  
processing, according to the requirements stipulated in sub-subsection 3.1.1. Those 
found with contamination which could not be removed to  acceptable levels by normal 
plant sorting or preparation procedures should be rej ected.  
 
Sugars and other sweeteners  must conform to applicable , existing standard 
specifications for white sugar (PNS/BAFPS 82:2010), raw cane sugar (PNS/BAFPS 
81:2010), coconut sap sugar (PNS/BAFPS 76:2010), and o ther types of sweeteners. 
Choose only those contained in clean, non-toxic, and pro perly labeled packaging 
materials. 
 
Storage/holding 
Sugars and sweeteners held for processing should be stored in any suitable type of 
closed container to protect from domestic animals, parasite s, chemical or 
microbiological contaminants, debris, and dust. These ing redients should be stored 
at temperature and relative humidity conditions that will minimize deterioration of 
quality. The storage area should also be free from stro ng odors or aroma. Regular 
inspection of the storage facility should be done to avo id infestation. 
 
5.1.3 Milk and Milk Products 
 
Receipt  
Milk and milk products shall only be accepted if it is sou nd and suitable for 
processing, according to the requirements stipulated in sub-subsection 3.1.1. Those 
found with contamination should be rejected. Special pr ecautions must be taken to 
reject milk and milk products showing signs of spoilage, an d deterioration. 
 
If prepackaged processed milk products are to be used as ra w material, choose only 
those contained in clean, non-toxic, and properly label ed packaging materials. 
 
Storage/holding  9Milk and milk products held for processing should be store d in any suitable type of 
closed container to protect from domestic animals, parasite s, chemical or 
microbiological contaminants, debris, and dust.  
 
The storage temperature for milk and milk products shoul d be sufficient to maintain 
product safety and suitability. Fresh milk and similar f luid milk products must be 
stored at temperatures less than 7°C. Canned and dried  milk products may be stored 
at a cool, dry place. 
 
5.1.4. Butter, margarine, lard, oil, and other simi lar food items 
 
Receipt  
Butter, margarine, lard, oil, and other similar food  items shall only be accepted if it is 
sound and suitable for processing, according to the requir ements stipulated in sub-
subsection 3.1.1. Those found with contamination should b e rejected. Special 
precautions must be taken to reject items showing signs of rancidity, spoilage, and 
deterioration. Choose only those contained in clean, no n-toxic, and properly labeled 
packaging materials. 
 
Storage/holding 
Butter, margarine, lard, oil, and other similar food  items held for processing should 
be stored in any suitable type of closed container to pr otect from domestic animals, 
parasites, chemical or microbiological contaminants, debris,  and dust.  
 
These ingredients should be stored at temperature and relative humidity conditions 
that will minimize deterioration of quality. The sto rage area should also be free from 
strong odors or aroma. Regular inspection of the storage  facility should be done to 
avoid infestation. 
 
5.1.5 Optional Ingredients  
 
5.1.5.1 Eggs 
 
Receipt   10 Egg used for the production of  ethnic flour-based confe ctioneries ( polvoron, piaya, 
and barquillos ) shall only be accepted if it is fresh, and suitable for  processing, 
according to the requirements stipulated in sub-subsection  3.1.2. Those found with 
contamination which could not be removed to acceptable l evels by normal plant 
sorting or preparation procedures should be rejected. Sp ecial precautions must be 
taken to reject eggs with broken shells, or those with sign s of degradation, abnormal 
color or odor. 
 
Inspection and sorting  
Eggs shall be inspected and sorted according to quality and size specifications 
before processing (PNS/BAFPS 35:2005. Philippine Natio nal Standard: Table Egg). 
Choose only those contained in clean, non-toxic, and pro perly labeled packaging 
materials. 
 
Storage/holding 
Eggs held for processing should be stored in any suitable type of closed container to 
protect from domestic animals, parasites, chemical or micr obiological contaminants, 
debris, and dust.  
 
Eggs should be stored at temperature and relative humi dity conditions that will 
minimize deterioration of egg quality. Eggs are kept  at refrigerated temperatures 
during storage particularly when these are not immedi ately used for processing. The 
storage area should also be free from strong odors or a roma. Regular inspection of 
the storage facility should be done to avoid infestatio n.    
 
Washing and/or sanitizing 
Eggs are washed to remove dirt, dust, soil, insect, and f ilth that might contaminate or 
affect the color, aroma or flavor of the eggs. Water u sed for washing and rinsing 
should be of potable quality. Only approved sanitizin g agents may be used in the 
wash or rinse water for eggs. 
 
5.1.5.2 Other optional Ingredients 
 
Receipt  11 Optional ingredients to be used in the preparation o f ethnic flour-based 
confectioneries ( polvoron, piaya, and barquillos ) shall conform to the requirements 
stipulated in sub-subsection 3.1.2. Whenever applicable,  certificates of analyses 
(COA) from ingredient suppliers shall be secured to confi rm their suitability for 
processing. Ingredients shall be rejected if they do not conform to the requirements 
and are found to have signs of deterioration, decomposi tion, or contamination to an 
extent which renders them unfit for human consumption.   
 
Storage/Holding 
Optional ingredients shall be in closed containers as p rotection against infestation by 
domestic animals, parasites, filth, and chemical and microb iological contaminants. 
Storage requirements such as temperature and humidity  may vary depending on the 
ingredient, and these should be provided accordingly b y the storage facilities to be 
used. 
 
Stored stocks of ingredients should be used on a “first in -first out” (FIFO) or a “first to 
expire-first to use” (FEFU) basis. 
 
5.2  Processing Operations 
 
5.2.1 Preparation 
 
If raw milk is used as basic ingredient, pasteurization m ust be done to ensure the 
safety of the product. Sufficient time and temperature  combinations for 
pasteurization must be used to ensure that the milk used  does not pose a health 
hazard. Pasteurization may be done by heating the mi lk at 63–66°C for at least 30–
32 minutes. Under FDA A.O. No. 132 s. 1970 (Regulation Prescribin g the Standard 
of Identity and Quality of Milk and Milk Products, B-4. 12-01), pasteurized milk should 
have been subjected to a temperature not lower than 63°C and held continuously at 
that temperature for not less than 30 minutes. The mil k should then be immediately 
cooled to 10°C or lower. Pasteurization could also be done by keeping the milk 
temperature at not lower than 72°C for at least 15 se conds, and immediately cooling 
the milk to 10°C or lower. 
  12 Breaking eggs, and/or separating the egg yolk from the egg white must be performed 
in a hygienic manner to prevent contamination. 
 
 
5.2.2 Mixing, Cooking, and Forming 
 
5.2.2.1 Polvoron 
Using a pan or skillet, the flour is toasted over moderat e heat until it is light brown. 
The toasted flour is transferred to a bowl, and allow ed to cool. Sugar, and milk and 
milk products are then added and mixed thoroughly with  the toasted flour. Butter 
and/or margarine is lightly heated to melt it, and t hen added to the flour-sugar-milk 
mixture. After thorough mixing, the polvoron  mixture can then be formed manually 
into oval shapes, or polvoron  press or molders could be used. The formed polvoron  
may be chilled to produce a firmer texture that will allow the product to hold its 
shape. 
 
5.2.2.2. Piaya 
To form the base dough, flour and shortening (in th e form of vegetable lard, oil, 
butter or margarine) are mixed together. Water may also be added to the dough 
mixture. The dough is then divided into small, round  portions. The filling is placed 
inside the small round portion and then flattened.  T raditionally, the filling used in 
piaya is muscovado sugar. Other filling formulations may also  consist of a 
combination of water, starch, and other forms of sugar su ch as white sugar.  
 
The dough stuffed with filling is pressed into flat, ro und shapes, and sprinkled with 
sesame seeds. The piaya  is then cooked either by grilling or by baking in an o ven, 
until the product is light brown in color. 
 
5.2.2.3. Barquillos  
Flour and sugar are mixed with other ingredients such as milk, butter and/or eggs, 
until a batter with smooth consistency is formed. The ing redients may be mixed by 
hand or by using an electric mixer. 
  13 Cooking may be done through the use of a wafer iron ( barquillera ) or by baking in an 
oven. When using a wafer iron ( barquillera ), a small portion of the batter is dropped 
into the center of the hot iron, and then the hot pl ates are pressed together and 
heated until the product is light brown in color. Whil e the product is still warm and 
pliable, the cooked product is immediately rolled int o cylinders using stainless steel 
rods or sticks. The barquillos is then allowed to cool.  
 
Another method of cooking barquillos is by baking in an oven. Small portions of the 
batter are dropped into baking sheets and thinly spread  into circular shapes using a 
spatula or spoon. The batter is then baked in a preheat ed oven until the edges of the 
wafer are brown in color. The wafers are removed from  the baking sheets, formed 
into cylinders while the product is still warm, and the n allowed to cool.   
 
Barquiron  is made by stuffing or filling the hollow portion of  the barquillos with 
polvoron . 
 
5.2.3 Wrapping 
Polvoron and barquiron  shall be individually wrapped in suitable, food-gra de 
packaging materials. 
 
5.3 Packing 
Packing can be done either mechanically or manually. It is important to standardize 
the fill-in weight for economic reasons.  
 
5.4      Closing or Sealing of Containers 
Seams and other closures of containers shall be sealed air- tight to meet the 
requirements of the processors. 
 
The seal area of flexible containers must be free of fo od material and wrinkles. 
Sealing temperature and pressure shall conform to the sealing equipment to be 
used. 
 
5.5   Coding of Sealed Containers  14 Coding of sealed container shall be indelible with det ails of production date and time, 
batch code, product code, the product line in which produ ct is packed, the 
manufacturing plant and other information necessary fo r product traceability. Where 
the container does not permit the code to be embossed or  inked, the label shall be 
legibly perforated or otherwise marked, and securely a ffixed to the product container. 
 
5.6  Post-Process Container Handling 
Mechanical shocks leading to breakage of semi-rigid contain ers due to container 
abuse must be avoided. These occur by knocking against each other during 
conveying, packaging and labeling operations, among oth ers. 
 
Flexible containers/pouches shall be handled singly rather  than in bunches, and care 
must be exercised so as to prevent damage by roughened co ntact surfaces. 
 
6.   FOOD ADDITIVES 
 
6.1 Food additives when used shall be in accordance with  the regulations 
established by the Food and Drugs Administration (FDA)  (Bureau Circular No. 016 
s.2006. Updated List of Food Additives) and/or the Cod ex Alimentarius Commission.  
 
The following food additives listed in, but not limit ed to, Table 1, may be used for the 
manufacture of ethnic flour-based confectioneries ( polvoron, piaya  and barquillos ). 
 
6.2 All others that have not been included in the abo ve list shall be allowed as carry-
over provided they are approved by FDA regulation (B .C. No. 016 s. 2006; Updated 
List of Food Additives) and shall be in accordance to th e Section 4 of the Preamble 
of the General Standard for Food Additives (GFSA) (C odex Stan 192-1995, Rev. 5 
(2004)).  These additives include those that are used fo r the raw materials and other 
ingredients.  
Table 1. Food Additives for Ethnic flour-based confectioneri es ( polvoron, piaya, and 
barquillos ) (B.C. No.016 s. 2006. Updated List of Food Additives)   
 
Function  Food Additive  Max. level of usage  
Anti-caking 
agent Polydimethylsiloxane 10 mg/kg b 
Antioxidant Ascorbyl Esters 200 mg/kg (As ascorbyl stearat e) d  15 BHA 100 mg/kg (Fat or oil basis) b 
200 mg/kg (Fat or oil basis)  b 
50 mg/kg c 
BHT 200 mg/kg (Fat or oil basis)  b 
50 mg/kg c 
25 mg/kg d 
Gallate, propyl 200 mg/kg (Fat or oil basis)  b 
Tertiary Butylhyroquinone 200 mg/kg (Fat or oil bas is)  b 
200 mg/kg c 
Tocopherols 500 mg/kg (Fat or oil basis) b 
Color Allura Red AC 348 mg/kg b 
Amaranth 100 mg/kg  b 
Annatto Extracts 25 mg/kg (As total bixin or norbixin) b 
Brilliant Blue FCF 300 mg/kg b 
Caramel Colour, Class III GMP  b 
GMP  c 
GMP  d 
Caramel Colour, Class IV GMP  b 
GMP  c 
Carotenoids GMP  c 
200 mg/kg  d 
Fast Green FCF 100 mg/kg  b 
100 mg/kg  d 
Sunset Yellow FCF 400 mg/kg  b 
Preservative Benzoates 1500 mg/kg (As benzoic acid)  b 
Hydroxybenzoates, p- 2000 mg/kg (As p-hydroxybenzoic acid) 
b 
300 mg/kg (As p-hydroxybenzoic acid)  
d 
Stabilizer Polysorbates 10000 mg/kg  b 
5000 mg/kg (Flour basis)  c 
5000 mg/kg  d 
Sorbates 2000 mg/kg (As sorbic acid) b 
Sorbitan Esters of Fatty 
Acids 20000 mg/kg a 
5000 mg/kg (Flour basis)  c 
10000 mg/kg d 
Sweetener Alitame 300 mg/kg a 
Acesulfame Potassium 3500 mg/kg b 
Aspartame 10000 mg/kg b 
5000 mg/kg d 
Saccharin 3000 mg/kg  b 
2000 mg/kg d 
Sucralose 1500 mg/kg  b 
a Based on the Food Category System: 5.0 Confectioner y;  
b Based on the Food Category System:5.2 Sugar-based confectionery including hard and soft candy, 
nougats, etc. other than food categories  05.1, 05.3, and 05.4;  
c Based on the Food Category System: 7.1.2. Crackers,  excluding sweet crackers  
d Based on the Food Category system: 7.2 Fine bakery wares 
  16 7. LABELING 
 
7.1 Each container shall be labeled and marked with the  following information in 
accordance with FDA’s Labeling Regulation (A.O. 88-B s. 1984; Rules and 
Regulations governing the Labeling of Prepackaged Food  Products distributed in the 
Philippines): 
(a) The name of the product shall be known as Polvoron, Piaya, or Barquillos . 
Additional descriptors pertaining to the ingredients use d or the product form may 
also be included (e.g. Barquiron, Purple yam piaya; Chocolate  polvoron, Pandan-
flavored barquillos ). Other local or regional names referring to similar products may 
also be included, provided that these names are acceptabl e in the area of 
distribution and in accordance with the customs or practi ces of the area or country in 
which the product is distributed. 
(b) The complete list of ingredients and food additiv es used in the preparation of the 
product in descending order of proportion. 
(c)  The net quantity of content by weight in the met ric system. Other systems of 
measurement required by importing countries shall appe ar in parenthesis after the 
metric system unit. 
(d)  The name and address of the manufacturer, packer a nd/or distributor of the 
food. 
(e) Open date marking  
The words “Best/Consume Before/Expiry Date” indicating  end of period at which the 
product shall retain its optimum quality attributes at defined storage conditions. 
(f) Lot or code number identifying product lot. 
(g) The words “Product of the Philippines”, or the coun try of origin if imported. 
(h)  Additional requirements 
A pictorial representation of raw material or end-pro duct on the label should not 
mislead the consumer with respect to the raw material or  end-product so illustrated. 
(i) Optional information 
Any special conditions for the storage of the food may be indicated in the label. Where 
practicable, storage instructions shall be in close proximi ty to the open date marking. 
 
7.2  Nutrition Labeling  17 Nutrition labeling shall conform to the established re gulations of FDA and/or 
authority.  
 
8.   QUALITY ASSURANCE  
 
8.1    Inspection of Finished Products 
All processed products shall be inspected before labeling and casing and defective  
products shall be withdrawn or rejected. The company mu st have an approved policy 
and procedures based on the FDA A.O. No. 153 s. 2004 – Guidelines on Current 
Good Manufacturing Practices in Manufacturing, Packing, Re packing or Holding 
Food. 
 
 8.2 Record Keeping 
Permanent and legible dated records of time, tempera ture code mark and other 
pertinent details shall be kept concerning each load. Su ch records are essential as a 
check on processing operations. 
 
Written records of all container closure examinations shal l specify the code lot, the 
date and time of container closure inspections, the measu rements obtained and all 
the corrective actions taken. 
 
Records shall be maintained identifying initial distrib ution of the finished product to 
facilitate, if necessary, the segregation of specific foo d lots that may have been 
contaminated or otherwise unfit for intended use. 
 
All process deviations involving failure to satisfy the minimum requirements of the 
process shall be recorded detailing those deviations and t he actions taken. 
 
 8.3  Good Manufacturing Practices (GMP) 
Processing establishments shall have developed, documented and implemented 
prerequisite programs based on Food and Drug Administr ation’s Current Good 
Manufacturing Practices (cGMP) and Hygiene Control. An  effective GMP and 
Hygiene Control program will decrease the number of cri tical control points that a 
manufacturer must face during the hazard analysis of t he product/process.  18  
9. STORAGE AND TRANSPORT OF FINISHED PRODUCT 
 
Storage and transport conditions of the finished produc t shall be such that the 
integrity of the product container, and the safety an d quality of the product are not 
adversely affected. 
 
Cases and cartons must be thoroughly dry. They must be of  proper size so that the 
containers fit snugly and are not subject to damage from movement within the case. 
They must be strong enough to withstand normal transpo rt. 
 
Extreme fluctuations in temperature and humidity duri ng storage and transport of the 
product must be avoided to prevent product deteriorati on. 
 
10.       LABORATORY CONTROL PROCEDURES 
 
Each food processing establishment shall have access to labor atory control of both 
the processes used and the finished products. All food i ngredients and food products 
declared unfit for human consumption by the laborator y shall be rejected. 
 
Representative samples for each lot or batch shall be ta ken to assess the safety and 
quality of the product. 
 
Microbiological laboratory shall be separated from the processing area. No 
pathogens shall be handled within the premises of manu facturing plant. 
 
Laboratory procedures for quality control of the process es and the product must 
follow recognized or standard methods for easy interpre tation of results. 
 
11. END PRODUCT SPECIFICATIONS 
 
Appropriate methods shall be used for sampling analysis and determinations to meet 
the following specifications:  19 1. To the extent possible in good manufacturing practice s, the products shall be free 
from any objectionable characteristics. 
2. The product shall not contain any toxic substances or iginating from 
microorganisms and chemicals. 
3. The product shall be free from chemical pollutants in  amounts which may pose 
hazard to health. 
4. The product shall comply with the requirements set fo rth by the Food and Drugs 
Administration, the Fertilizer and Pesticide Authority  of the Department of 
Agriculture, and the Codex Alimentarius Commission on P esticide Residues and 
Food Additives. 
 
12.      REFERENCES 
 
Abiva, C. C. 2001. A Quick Guide to Filipino Food and Cooking . Anvil Publishing, 
Inc. Pasig City, Philippines. 
 
A.O. No. 88-A s. 1984. Regulatory Guidelines Concerning Food Additives . 
Department of Health. San Lazaro Compound, Sta. Cru z, Manila, Philippines. 
 
A.O. No. 88-B s. 1984. Rules and Regulations governing the Labeling of 
Prepackaged Food Products distributed in the Philip pines . Bureau of Food and 
Drugs. Department of Health. Alabang, Muntinlupa Cit y, Philippines. 
 
A.O. No. 132 s. 1970. Regulation Prescribing the Standard of Identity and   
Quality of Milk and Milk Products (B-4.12-01). Department of Health. San Lazaro 
Compound, Sta. Cruz, Manila, Philippines. 
 
A.O. No. 230 s. 1974. B-4 Definition and Standards of Food; B-4.15 Fats a nd 
Oils; B-4.15-01 Shortening. Department of Health. San Lazaro Compound, Sta. 
Cruz, Manila, Philippines. 
 
A.O. No. 243 s. 1975. B-4 Definition and Standards of Food; B-4.18 Margar ine. 
Department of Health. San Lazaro Compound, Sta. Cru z, Manila, Philippines. 
 
A.O. No. 153 s. 2004. Guidelines, Current Good Manufacturing Practice in 
Manufacturing, Packing, Repacking or Holding Food. Department of Health. San 
Lazaro Compound, Sta. Cruz, Manila, Philippines. 
 
A. O. No. 2007-0012. Philippine National Standards for Drinking Water 20 07. 
Department of Health, Manila, Philippines 
 
Association of Analytical Chemists. Official Methods of Analysis Manual.  16 th  ed., 
1995. AOAC International. 481 North Frederick Ave., S uite 500, Gaithersburg, MD 
20877-2417. U.S.A.  20  
BAFPS. Draft Standards for Muscovado Sugar . Bureau of Agriculture and 
Fisheries Product Standards. Department of Agriculture. Visayas Ave., Quezon City. 
 
B.C. No. 6-A s. 1998. Permissible Net Content Variation in Prepackaged Fo od . 
Bureau of Food and Drugs. Department of Health. Alab ang, Muntinlupa City, 
Philippines. 
 
B.C. No. 01-A s. 2004. Guidelines for the Assessment of Microbiological Qu ality 
of Processed Foods . Bureau of Food and Drugs. Department of Health. Al abang, 
Muntinlupa City, Philippines. 
 
B.C. No.016 s. 2006. Updated  List of Food Additives. Bureau of Food and Drugs, 
Department of Health. Alabang, Muntinlupa City, Phi lippines. 
 
Belitz, H.-D., W. Grosch, and P. Schieberle. 2009. Food Chemistry . 4 th  revised and 
extended ed. Springer-Verlag, Heidelberg, Germany.   
 
Codex Alimentarius Commission. 1995. FAO/WHO Codex Alimentarius 
Commission Manual . Food and Agriculture Organization. Viale delle Ter me di 
Caracalla, 00100 Rome, Italy. 
 
Dictionary of Food Science and Technology , International Food Information 
Service, Blackwell Publishing, UK, 2005  
 
DOH. 2000. Implementing Rules and Regulations for the  Food Fortification Act of 
2000 (RA 8976 s. 2006). Department of Health. San La zaro Compound, Sta. Cruz, 
Manila, Philippines. 
 
FNRI. 1997. The Philippine Food Composition Tables . Food and Nutrition 
Research Institute, Department of Science and Technology,  Bicutan, Taguig, 
Philippines. 
 
Food, definition . ALINORM 04/27/41, para. 88 and Appendix VI. 2005.  Codex 
Alimentarius Commission. Food and Agriculture Organizat ion. Viale delle Terme di 
Caracalla, 00100 Rome, Italy. 
 
Konkel, P. J. 2001. Confectionery Products . Chapter 56. In Compendium of 
Methods for the Microbiological Examination of Food s . 4 th  ed. edited by F.P. 
Downes and K. Ito. American Public Health Association, Wa shington, DC, USA. 
 
PNS/BAFPS 35:2005. Philippine National Standard: Table Egg.  Bureau of 
Product Standards, Department of Trade and Industry, Makati City, Philippines. 
 
PNS/BAFPS 36:2008. Philippine National Standard: Fresh Milk.  Bureau of 
Product Standards, Department of Trade and Industry, Makati City, Philippines 
 
R.A. 3720. Food, Drug and Cosmetic Act. Bureau of Food and Drugs. Department 
of Health. Alabang, Muntinlupa City, Philippines. 
  21 R.A. 9711. Food and Drug Administration (FDA) Act of 2009 . Bureau of Food and 
Drugs. Department of Health. Alabang, Muntinlupa Cit y, Philippines.      
 
Vaclavik, V. and E. Christian. 2008. Essentials of Food Science . 3 rd  edition. 
Springer Science+Business Media, LLC., New York, USA. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  22 ANNEX A 
Standard Parameters and Values for Drinking Water 
Philippine National Standards for Drinking Water 2007  (DOH AO 2007-0012) 
 
 Table 1. Standard values for bacteriological quali ty     
Parameter Value/Unit Point of Compliance 
Total Coliform < 1.1 MPN/100 ml Service Reservoir 
Water treatment works 
Consumers’ taps 
Refilling stations 
Water haulers 
Water vending machines 
Fecal Coliform < 1.1 MPN/100 ml Service Reservoir 
Water treatment works 
Consumers’ taps 
Refilling stations 
Water haulers 
Water vending machines 
Point sources - Level 1 
Heterotropic Plate Count < 500 CFU/ml Service Reservoi r 
Water treatment works 
Consumers’ taps nearest meter 
Refilling stations 
Water vending machines 
 
Table 2.  Standard values for Physical and Chemical  Quality for Acceptability Aspects 
for Drinking Water 
 
 
 
 
 
 Constituents  Maximum Level (mg/L)  
or Characteristic Constituents  Maximum Level 
(mg/L)  
or Characteristic 
Taste No objectionable taste Hydrogen Sulfide 0.05 
Odor No objectionable odor Iron 1.0 
Color Apparent = 10 color 
units  
True = 5 color units Manganese 0.4 
Turbidity 3 NTU pH 6.5 – 8.5 
Aluminum 0.2 Sodium 200 
Chloride 250 Sulfate 250 
Copper 1.0 Total Dissolved 
Solids 500 
Hardness 300 as CaCO 3  Zinc 5.0  23 Table 3.  Standard Values for Organic and Inorganic  Chemical Constituents of Health 
Significance in Drinking Water  
Inorganic Chemicals 
Constituents  Maximum Level (mg/L)  Constituents  Maximum Level (mg/L)  
Antimony 0.02 Fluoride 1.0 
Arsenic 0.05 Lead 1.01 
Barium 0.7 Mercury (total) 0.001 
Boron  0.5 Nickel 0.02 
Cadmium 0.003 Nitrate 50 
Chromium (Total) 0.05 Nitrite 3.0 
Cyanide (Total) 0.07 Selenium 0.01 
 
Organic Chemicals 
Constituents  Ma ximum Level 
(mg/L) Constituents  Maximum 
Level (mg/L) 
Benzene 0.01 Ethylbenzene 0.30 
Carbon tetrachloride 0.004 Nitrilotriacetic acid (N TA) 0.20 
1,2-Dichlorobenzene 0.1 Polyaromatic hydrocarbons ( PAHs)  0.20 
1,4-Dichlorobenzene 0.5 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 1.01 Vinyl chloride 0.0003 
Edetic Acid (ADTA) 0.001 Xylene 0.5 
 
Organic Pesticides 
Constituents  Maximum Level 
(ug/L) Status in the 
Philippines 
Aldrin and Dieldrin (combined) 30.0 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 Register ed 
Endrin 30. Banned 
1,2-Dibromomethane (Ethylene dibromide) 0.6 Banned 
Heptachlor and Heptachlor epoxide (combined) 0.03 B anned 
Lindane 2.0 Restricted 
MCPA (4-(2-methyl-4-chloro) phenoxyl acetic acid 2. 0 Registered 
Pendimethalin 20.0 Registered 
Pentachlorophenol (PCP) 9.0 Banned  24 ANNEX B 
Determination of Water Activity (AOAC 978.18) 
  
A. Principle 
Water activity, a w, is ratio of vapor pressure of H 2O in product to vapor pressure of pure H 2O 
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 indire ctly 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 standa rd 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 sa lt solution. – Instrument available from 
Beckman Industrial, Rosemount Analytical Div., 89 Comm erce Rd, Cedar Grove, NJ 
07009; Nova Sina AG, Andreastrasse 7-11, CH 8050, Zuri ch,Switzerland; Rotronic 
Instrument Corp., 160 E. Main St, Huntington, NY 117 43. Immobilized salt sensors are 
affected by polyols such as glycerol and glycol and by vo latile amines 
(b) Change in electrical capacitance of polymer thin fi lms. – Instrument available from 
General Eastern Instruments, 50 Hunt St, Watertown, M A 02172. Polymer thin film 
sensors are affected by CH 3COOH.  
(c) Dew point by chilled mirror technique. – Instrument  available from EG&G, Environmental 
Equipment Division, 217 Middlesex Turnpike, Burlingto n, MA 01803 or General Eastern 
Instruments. Dew point measurements can be affected by co ndensables with lower 
critical temperature than H 2O.  
(d) Longitudinal change in dimensions of water-sorbing fiber. – Instrument available from G 
Lufft Metallbarometerfabrik, D-7, Postfach 692, Neue W einsteige 22, Stuttgart, Germany.  
(e) Partial water vapor pressure by manometric system. – Partial H 2O vapor pressure 
measurements can be made useless by living products that re spire, such as grains or 
nuts; by active fermentation; or by products that expa nd excessively when subjected to 
high vacuum.  
(f) Relative weight of moisture sorbed by anhydrous hy drophilic 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 temp erature to ±0.1°. See 978.18B(c).  
(b) Forced-draft cabinet. – Constant temperature, set t o maintain 25 ± 1°; capacity ≥0.06 m 3 (2 
cu ft); with access port to accommodate instrument sensor l eads. Use in conjunction with 
(c).  
(c) Insulated box with cover. – Large enough to hold t est container, (e), and small enough to 
fit in forced-draft cabinet, (b); with access port to accom modate instrument sensor leads. 
Protect test container from short-term temperature flu ctuations.  
(d) Manometric system. – Sensitive to pressure differenti al 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 
Teflon-lined screw caps and gaskets. Check integrity of  cap seals and sensor leads by 
any means available, e.g., ability of system to hold v acuum, using Tesla coil.  
(f) Water bath. – Capable of maintaining temperatur e constant within 0.1° at 25±1°; capacity 
sufficient to hold measuring chamber of selected apparatu s.   25 (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.  
 
D. Preparation of Reference Salt Slushes 
 
Place selected reference salt in test container to depth of ca 4 cm for more soluble salts 
(lower a w), to depth of ca 1.5 cm for less soluble salts (higher a w), and to intermediate depth 
for intermediate salts. Add H 2O in ca 2 mL increments, stirring well with spatula afte r each 
addition, until salt can absorb no more H 2O as evidenced by free liquid. Keep free liquid to 
minimum needed to establish saturation of salt with H 2O. Slushes are ready for use upon 
completion of mixing, and are usable indefinitely (ex cept for some high a w salts susceptible 
to bacterial attack), if contained in manner to preve nt substantial evaporation losses. Some 
slushes, e.g., NaBr, may solidify gradually by crystal coa lescence, with no effect on a w.  
 
E. Calibration 
 
Select ≥ 5 salts to cover a w range of interest or range of sensor being used. Measu re 
humidity generated by each salt slush in terms of instru ment readout, as in 978.18F. Plot 
readout against a w values given in Table 978.18 for selected salts, using cross-section paper 
scaled for reading to 0.001 a w unit. Draw best average smooth line through plotted  points. 
Use this calibration line to translate sensor instrument r eadout of samples to a w or to check 
vapor pressure or dew point instruments for proper funct ioning.  
 
F. Determination 
 
Place calibration slush or sample in forced-draft cabinet , (b), or H 2O 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 tem perature control device. Use 
volume of sample or slush >1/20 total volume sample con tainer 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 t est container is placed in temperature 
control device, or record response on strip chart. Two con secutive readings, at indicated 
intervals, which vary by <0.01 a w unit are evidence of adequately close approach to 
equilibrium. Continue readings at 60-min intervals, i f necessary. Convert last reading to a w 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 requir ed by properties of sensor, expose 
sensor to controlled RH below ambient before starting each measurement.  
 
  26 ANNEX C 
Enumeration of Yeast and Mold Count 
Enumeration of Yeasts and Molds in Food--Dilution P lating Technique (USFDA, 2001)  
A. Equipment and materials   
1. Basic equipment (and appropriate techniques) for pr eparation of sample homogenate 
2. Equipment for plating samples 
3. Incubator, 25°C 
4. Arnold steam chest 
5. pH meter 
6. Water bath, 45 ± 1° C 
 
B. Media and reagents 
 
1. Dichloran rose bengal chloramphenicol (DRBC) agar 
2. Dichloran 18% glycerol (DG18) agar 
3. Plate count agar (PCA), standard methods; add 100 mg chloramphenicol/liter when 
this medium is used for yeast and mold enumeration. Th is medium is not efficient 
when "spreader" molds are present. 
4. Malt agar (MA) 
5. Malt extract agar (Yeasts and Molds) (MEAYM) 
6. Potato dextrose agar (PDA), dehydrated; commerciall y available 
 
C. Procedures  
Sample preparation  
Analyze 25-50 g from each subsample; generally, larger sample sizes increase 
reproducibility and lower variance compared with small samples. Test individual 
subsamples or composite according to respective Compliance Pr ogram for the food 
under analysis. Add appropriate amount of 0.1% pepton e water to the weighed sample 
to achieve 10 -1 dilution, then homogenize in a stomacher for 2 min. Alternatively, 
blending for 30-60 sec can be used but is less effective.  Make appropriate 1:10 (1+9) 
dilutions in 0.1% peptone water. Dilutions of 10 -6 should suffice. 
Plating and incubation of sample  
Spread-plate method . Aseptically pipet 0.1 ml of each dilution on pre- po ured, solidified 
DRBC agar plates and spread inoculum with a sterile, bent glass rod. DG18 is preferred 
when the water activity of the analyzed sample is less than 0.95. Plate each dilution in 
triplicate. 
Pour-plate method . Use sterile cotton-plugged pipet to place 1.0 ml por tions of sample 
dilution into prelabeled 15 x 100 mm Petri plates ( plastic or glass), and immediately add 
20-25 ml tempered DG18 agar. Mix contents by gently sw irling plates clockwise, then 
counterclockwise, taking care to avoid spillage on dish l id. After adding sample dilution, 
add agar within 1-2 min; otherwise, dilution may beg in to adhere to dish bottom 
(especially if sample is high in starch content and dishes a re plastic) and may not mix 
uniformly. Plate each dilution in triplicate. 
From preparation of first sample dilution to pouring  or surface-plating of final plate, no 
more than 20 min (preferably 10 min) should elapse. Note: Spread plating of diluted 
sample is considered better than the pour plate method . When the pour plate technique 
is used, fungal colonies on the surface grow faster and o ften obscure those underneath 
the surface, resulting in less accurate enumeration. Surfa ce plating gives a more uniform  27 growth and makes colony isolation easier. DRBC agar shou ld be used for spread plates 
only. 
Incubate plates in the dark at 25°C. Do not stack pl ates higher than 3 and do not 
invert. Note:  Let plates remain undisturbed until counting. 
Counting of plates  
Count plates after 5 days of incubation. If there is no growth at 5 days, re-incubate for 
another 48 h. Do not count colonies before the end of the incubation period because 
handling of plates could result in secondary growth fro m dislodged spores, making final 
counts invalid. Count plates containing 10-150 colonies. If mainly yeasts are present, 
plates with 150 colonies are usually countable. However,  if substantial amounts of mold 
are present, depending on the type of mold, the uppe r countable limit may have to be 
lowered at the discretion of the analyst. Report result s in colony forming units (CFU)/g or 
CFU/ml based on average count of triplicate set. Round off counts to two significant 
figures. If third digit is 6 or above, round off to d igit above (e.g., 456 = 460); if 4 or below, 
round off to digit below (e.g., 454 = 450). If thir d digit is 5, round off to digit below if first 2 
digits are an even number (e.g., 445 = 440); round off to digit above if first 2 digits are an 
odd number (e.g., 455 = 460). When plates from all dilutions have no colonies, report 
mold and yeast counts (MYC) as less than 1 times the lowe st dilution used. 
Isolate individual colonies on PDA or MA, if further a nalysis and species identification is 
necessary. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  28 ANNEX D 
Enumeration of Coliform and E.coli  Count 
(Enumeration of Escherichia coli  and the Coliform Bacteria (USFDA, 2001)) 
Conventional Method for coliforms, fecal coliforms and E. coli  
A. Equipment and materials 
1. Covered water bath, with circulating system to maint ain temperature of 45.5 ± 0.2°C. 
Water level should be above the medium in immersed tu bes. 
2. Immersion-type thermometer, 1-55°C, about 55 cm l ong, with 0.1°C subdivisions, 
certified by National Institute of Standards and Techn ology (NIST), or equivalent 
3. Incubator, 35 ± 1.0°C 
4. Balance with capacity of > 2 kg and sensitivity of 0.1 g 
5. Blender and blender jar 
6. Sterile graduated pipets, 1.0 and 10.0 mL 
7. Sterile utensils for sample handling 
8. Dilution bottles made of borosilicate glass, with pol yethylene screw caps equipped with 
Teflon liners. Commercially prepared dilution bottles containing sterile Butterfield's 
phosphate buffer can also be used. 
9. Quebec colony counter, or equivalent, with magnifyi ng lens 
10. Longwave UV light [~365 nm], not to exceed 6 W. 
11. pH meter 
B. Media and Reagents  
Brilliant green lactose bile (BGLB) broth, 2% (M25 ) 
Lauryl tryptose (LST) broth (M76 ) 
EC broth (M49 ) 
Levine's eosin-methylene blue (L-EMB) agar (M80 ) 
Butterfield's phosphate-buffered water (R11 ) or equivalent diluent (except for shellfish) 
Lauryl tryptose MUG (LST-MUG) broth (M77 ) 
Peptone Diluent, 0.1% (R56 ) 
MPN - Presumptive test for coliforms, fecal colifor ms and E. coli  
Weigh 50 g food into sterile high-speed blender jar. (see Chapter 1 and current FDA 
compliance programs for instructions on sample size and com positing) Frozen samples can 
be softened by storing it for < 18 h at 2-5°C, but do not thaw. Add 450 mL of Butte rfield's 
phosphate-buffered water and blend for 2 min. If <50  g of sample are available, weigh 
portion that is equivalent to half of the sample and add sufficient volume of sterile diluent to 
make a 1:10 dilution. The total volume in the blend er jar should completely cover the blades. 
Prepare decimal dilutions with sterile Butterfield's p hosphate diluent. Number of dilutions to 
be prepared depends on anticipated coliform density. S hake all suspensions 25 times in 30 
cm arc or vortex mix for 7 s. Do not use pipets to del iver <10% of their total volume. Transfer 
1 mL portions to 3 LST tubes for each dilution for at least 3 consecutive dilutions. Hold pipet 
at angle so that its lower edge rests against the tube.  Let pipet drain 2-3 s. Not more than 15 
min should elapse from time the sample is blended unti l all dilutions are inoculated in 
appropriate media. 
NOTE:  Use 5-tube MPN for analysis of shellfish and shellfish ha rvest waters.  29 Incubate LST tubes at 35°C. Examine tubes and record r eactions at 24 ± 2 h for gas, i.e., 
displacement of medium in fermentation vial or efferv escence when tubes are gently 
agitated. Re-incubate gas-negative tubes for an addi tional 24 h and examine and record 
reactions again at 48 ± 2 h. Perform confirmed test on  all presumptive positive (gas) tubes. 
MPN - Confirmed test for coliforms  
From each gassing LST tube, transfer a loopful of suspen sion to a tube of BGLB broth, 
avoiding pellicle if present. Incubate BGLB tubes at 3 5°C and examine for gas production at 
48 ± 2 h. Calculate most probable number (MPN) of coli forms based on proportion of 
confirmed  gassing LST tubes for 3 consecutive dilutions. 
MPN - Confirmed test for fecal coliforms and E. coli  
From each gassing LST tube from the Presumptive test, t ransfer a loopful of each 
suspension to a tube of EC broth (a sterile wooden appl icator stick may also be used for 
these transfers). Incubate EC tubes 24 ± 2 h at 45.5 ° C and examine for gas production. If 
negative, reincubate and examine again at 48 ± 2 h. Use results of this test to calculate fecal 
coliform MPN. To continue with E. coli  analysis, proceed to Section F under Enumeration of 
Escheria coli and the Coliform Bacteria of the USFDA Ba cteriological Analytical Manual 
(2001). The EC broth MPN method may be used for seawa ter and shellfish since it conforms 
to recommended procedures ( 1). (Caution: see Note below). 
NOTE:  Fecal coliform analyses are done at 45.5± 0.2°C for al l foods, except for water 
testing and in shellfish and shellfish harvest water anal ysis, which uses an incubation 
temperature of 44.5± 0.2g°C.