3 OF ETHNIC CURED MEATS - nmis.gov.phnmis.gov.ph/attachments/article/10/Code of RCPPHEC.pdf · 8...

Draft RCP as of August 17, 2010

1

1 RECOMMENDED CODE OF PRACTICE FOR THE PROCESSING AND HANDLING 2

OF ETHNIC CURED MEATS 3

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5

1. SCOPE 6

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This Code of Practice is concerned with the receipt of raw materials and ingredients, 8

preparation and processing of ethnic cured meats as defined in this Code, in order to 9

conform with the required standards stated in PNS/NMIS No. ___. Standards for 10

Ethnic Cured Meats. The product shall be prepared from animal meat, which is cured 11

through the addition of curing agents. This Code is intended to provide guidelines to 12

achieve compliance with the standards for ethnic cured meats packed in any suitable 13

container. 14

15

2. DEFINITION OF TERMS 16

17

For the purpose of this Code, the following definitions apply: 18

19

Casing – a tubular membrane, natural or manufactured, shirred or in loose form, 20

stretchable or unstretchable, permeable or impermeable to gas or liquid, may be 21

edible or non-edible. 22

23

Comminution – a general term for the process of particle size reduction. This may 24

include cutting, chopping, grinding, milling and other physical treatment. 25

26

Container – any form of packaging material, which completely or partially encloses 27

the food (including wrappers). A container may enclose the food as a single item or 28

several units or types of prepackaged food when such is presented for sale to the 29

consumer. 30

31

Cure accelerator – it is an ingredient used primarily to speed up the curing process 32

and stabilize the cured color of the finished product. 33

34


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Curing – it is a preservation technique that involves the treatment of muscle meat 35

with common salt, and nitrite/nitrate, with or without the addition of sugar, spices and 36

other ingredients, to prolong the keeping quality of the product. 37

38

Current Good Manufacturing Practices (cGMP) – a quality assurance system 39

aimed at ensuring that products are consistently manufactured, packed or repacked or 40

held to a quality appropriate for the intended use. It is thus concerned with both 41

manufacturing and quality control procedures. 42

43

Dry sausage – a sausage that has undergone a controlled air drying process, and 44

may or may not be fermented. 45

46

Edible casing – it is a casing or tubing prepared from collagen, cellulose, or food-47

grade synthetic material or from natural sources (e.g., hog or sheep intestines) that 48

contain the sausage mix (CODEX STAN 192-1995 (Rev. 5-2004)). Edible casings are 49

casings that do not have to be removed before consumption and are fit to be 50

consumed together with the sausage mix. 51

52

Extenders – refer to any non-meat ingredient added to reduce formulation cost, 53

improve emulsion stability and other technological purposes. 54

55

Flavor and flavoring substances – substances which are added to impart flavor 56

which are either natural, nature identical or artificial flavoring substances. 57

(a) natural flavor – these flavoring substances derived through appropriate physical 58

processes from spices, herbs, fruit or fruit juices, vegetable or vegetable juices, edible 59

yeast, bark, bud, root, leaf or plant materials, meat, fish, poultry, eggs, dairy products 60

or fermentation products thereof. 61

(b) nature-identical flavoring substances – these are substances chemically derived 62

from aromatic materials or obtained synthetically, which are chemically identical to 63

substances present in' natural products intended for human consumption. 64

(c) artificial flavoring substances – these are substances that impart flavor but which 65

have not been identified in natural products or natural sources of flavorings. 66

67

Food – any substance, whether processed, semi-processed or raw, which is intended 68

for human consumption, and includes drink, chewing gum and any substance which 69


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has been used in the manufacture, preparation or treatment of “food” but does not 70

include cosmetics or tobacco or substances used only as drugs. 71

72

Food Additives – any substance the intended use of which results or may reasonably 73

be expected to result, directly or indirectly, in its becoming a component or otherwise 74

affecting the characteristics of any food (including any substance intended for use in 75

producing, manufacturing, packing, processing, preparing, treating, packaging, 76

transporting, or holding food; and including any source of radiation intended for any 77

such use), if such substance is not generally recognized, among experts qualified by 78

scientific training and experience to evaluate its safety, as having been adequately 79

shown through scientific procedures to be safe under the conditions of the intended 80

use (R.A. 3720. Food, Drugs and Cosmetic Act). 81

82

Food and Drug Administration or FDA – formerly known as Bureau of Food and 83

Drug (BFAD) of the Department of Health (DOH); which was renamed in accordance 84

to RA 9711 (Food and Drug Administration (FDA) Act of 2009). 85

86

Food Standard – a regulatory guideline that defines the identity of a given food 87

product (i.e. its name and the ingredients used for its preparation) and specifies the 88

minimum quality factors and, when necessary, the required fill of the container. It may 89

also include specific labeling requirements other than or in addition to the labeling 90

requirements generally applicable to all prepackaged foods. 91

92

Ingredient - any substance including food additive, used as a component in the 93

manufacture or preparation of a food and present in the final product in its original or 94

modified form. 95

96

Label – includes any tag, brand, mark, pictorial, or other descriptive script, written, 97

printed, marked, embossed or impressed on, or attached to the container. 98

99

Labeling – any written, printed or graphic matter (1) upon any article or any of its 100

container or wrappers and/or (2) accompanying the packaged food. 101

102

Lot – food produced during a period of time and under more or less the same 103

manufacturing condition indicated by a specific code. 104

105


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Meat – it is fresh, chilled, or frozen edible carcass including offal derived from food 106

animals (Joint DA-NMIS and DOH-FDA A.O. 01 s.2009). Skeletal & non-skeletal 107

muscle tissues may include beef (cattle), carabeef (carabao or water buffaloes), pork, 108

poultry (chicken, duck, turkey), lamb/mutton (sheep), and chevon (goat). 109

110

Packaging – the process of packing that is part of the production cycle applied to a 111

bulk product to obtain the finished product. Any material, including painted material, 112

employed in the packaging of a product including any outer packaging used for 113

transportation of shipment. Packaging materials are referred to as primary or 114

secondary according to whether or not they are intended to be in direct contact with 115

the product. 116

117

Processing aids – these are additives that are used in the processing of food to 118

achieve a specific technological purpose and which may or may not result in the 119

presence of residues or derivatives in the final product (BFAD A.O. No. 88-A s. 1984) 120

121

Potable water – it is the water fit for human consumption and potability determined by 122

health authorities cited in Philippine National Standards for drinking water 123

(Department of Health-Administrative Order No. 2007-0012: Philippine National 124

Standards for Drinking Water 2007) 125

126

Sausage – it is fresh or preserved meat, chopped or comminuted fine, to which has 127

been added salt and spices and may contain sugar, seasoning, saltpeter (potassium 128

or sodium nitrate) potassium or sodium nitrite, with or without binder (BFAD A.O. 154 129

s. 1971). Sausage is comminuted seasoned meat that has been stuffed into casings, 130

and may have undergone smoking, curing, fermentation and heating (FAO, 1985). 131

132

Spices – refer to any aromatic vegetable substance in whole, broken, ground or in 133

any other form, except those other substances which have been traditionally regarded 134

as food. 135

136

Water Activity – it is the ratio of vapor pressure of water in the food substrate to the 137

vapor pressure of pure water at the same temperature (Jay et. al., 2005). It is also a 138

measure of water available for chemical reactions and microbial growth (Fennema, 139

1996). 140

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3. RAW MATERIALS, INGREDIENTS AND PACKAGING MATERIAL 142

REQUIREMENTS 143

144

3.1 Raw materials and ingredients. Raw materials for processing shall not contain 145

parasites, microorganisms, toxins, and decomposed or extraneous substances. 146

147

3.1.1 Basic Ingredients 148

149

Meat. Meat to be used shall be sound, clean, and fit for human consumption. Meat 150

should have a meat inspection mark or certificate of inspection by the NMIS and/or 151

authority for the meat source, to confirm their suitability for processing. 152

153

Curing agents. Salt to be used shall be fine or coarse sodium chloride (NaCl) 154

available from natural sources or manufactured as food grade, and meets the purity 155

requirements as specified in Section 4.1 of the Implementing Rules and Regulations 156

of the ASIN Law, Republic Act (RA) 8172, an Act Promoting Salt Iodization 157

Nationwide. 158

159

Nitrites and/or nitrates to be used shall be as food-grade salt of sodium or potassium. 160

Curing agents used must comply with the regulations set for food additives (BFAD 161

Bureau Circular No. 016 s.2006. Updated List of Food Additive) and other applicable 162

food standards 163

164 165 3.1.2 Optional Ingredients 166

167

Spices, Condiments and Flavorings. All spices, condiments and flavor/flavoring 168

substances used shall be certified as food grade by the Food and Drugs 169

Administration (FDA). 170

171

Water. Only clean, potable water (Annex A) shall be used for the preparation and for 172

all the pretreatment and processing steps of ethnic cured meat production. 173

174

Non-potable water may be used only for operations not in direct contact with the food 175

materials provided that this does not pose a hazard to health as determined and 176

approved by the official agency having the jurisdiction over it. 177

178


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Binders and extenders. These may include soy proteins, cereal flours and other 179

suitable food-grade materials complying with applicable food standards 180

181

Fat. Fat to be used should come from any edible and food-grade vegetable and 182

animal fat source, and must comply with applicable food standards. 183

184

Sugar. Sugar to be used must be food grade and comply with applicable food 185

standards. 186

187

Vinegar. Vinegar to be used must be food grade and comply with applicable food 188

standards 189

190 Other food ingredients. These ingredients must be of food-grade quality and comply 191

with applicable food standards required by FDA and/or authority. 192

193

3.1.3. Casings 194

Casings used may be edible or non-edible, must be of food grade-quality, and should 195

conform to food standards required by FDA, NMIS, and/or authority. 196

197

3.2 Packaging materials. The packaging materials should be appropriate for the product 198

to be packed and for the expected conditions of handling during distribution and 199

storage. These should provide the products adequate protection from contamination 200

and should be sufficiently durable to withstand mechanical, chemical and thermal 201

stresses encountered during processing and normal distribution. All packaging 202

materials must be clean and free from defects that may affect the product or package 203

integrity. These shall be stored in a clean and sanitary manner. 204

205

4. HYGIENE 206

207

It is recommended that the products covered by the provisions of this code of practice 208

should be processed and handled according to the appropriate sections of 209

Recommended Code of Practice – General principles of Food Hygiene (CAC/RCP 1-210

1969 (Rev. 4, 2003)), the Code of Hygienic Practice for Meat (CAC/RCP 58-2005), 211

and/or BFAD A.O. No. 153 s. 2004 - Revised Guidelines on Current Good 212

Manufacturing Practice In Manufacturing, Packing, Repacking, or Holding Food, 213

covering the plant facilities and operations requirement including the construction and 214


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layout of processing plant, hygienic facilities, equipment, utensils and working 215

surfaces. 216

217

5. PREPARATION AND PROCESSING 218

219

The preparation of ethnic cured meat is described from the receipt of raw materials 220

until the packing operations. The production process should be supervised by 221

personnel with adequate technical training and experience. 222

223

5.1 Preparation of Raw Materials and Ingredients 224

225

5.1.1 Meat 226

227

Receipt 228

Meat from any food animal species shall only be accepted if it is sound and suitable 229

for processing, according to the requirements stipulated in sub-subsection 3.1.1. 230

Fresh meat used for the processing of ethnic cured meats should have been chilled 231

or frozen immediately after slaughter. The internal temperature of chilled meat should 232

be near 0°C, while internal temperature of frozen meat should be -18°C or below. In 233

addition, chilled and frozen meat should be maintained and transported by the 234

supplier in chilled (near 0°C), and frozen conditions (-18°C or below), respectively. 235

Those found with contamination should be rejected. Special precautions must be 236

taken to reject meat showing signs of deterioration and spoilage. 237

238

Inspection and sorting 239

The meat shall be inspected and sorted according to meat source (species), meat 240

parts or cuts, and intended use of the meat pieces. 241

242

If prepackaged meat pieces or mechanically deboned meat is to be used as raw 243

material, choose only those contained in clean, non-toxic, and properly labeled 244

packaging materials. 245

246

Storage/holding 247

Fresh or chilled meat must be kept chilled at temperatures of 0° to 4°C, while frozen 248

meat must be kept at -18°C or below. 249

250


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Meat held for processing should be stored in a suitable type of container and must be 251

protected from domestic animals, parasites, chemical or microbiological 252

contaminants, debris, and dust. Meat may be placed in corrosion resistant trays. 253

Cartons may also be used as long as appropriate inner lining is used or if the meat is 254

individually wrapped. 255

256

The containers should be arranged to allow adequate air circulation and to prevent 257

drip from one meat piece from falling onto another piece. Conditions in the storage 258

and holding areas such as temperature and humidity should be regulated to prevent 259

spoilage and contamination. 260

261

Regular inspection of the storage facility should be done to avoid infestation. 262

Temperature and humidity levels conducive to spoilage and contamination should be 263

avoided. 264

265

Washing and/or sanitizing 266

Meat may be washed to remove dirt, dust, and filth that might contaminate the 267

meat, or water may be used to facilitate thawing of frozen meat. Water used for 268

washing, thawing, and rinsing should be of potable quality. 269

270

5.1.2 Optional Ingredients 271

272

Receipt 273

Optional ingredients and casings to be used in the preparation of ethnic cured meat 274

shall conform to the requirements stipulated in sub-subsections 3.1.2, and 3.1.3. 275

Whenever applicable, certificates of analyses (COA) from ingredient suppliers shall 276

be secured to confirm their suitability for processing. Ingredients shall be rejected if 277

they do not conform to the requirements and are found to have signs of deterioration, 278

decomposition, or contamination to an extent which renders them unfit for human 279

consumption. 280

281

Storage/Holding 282

Optional ingredients shall be stored in closed containers as protection against 283

infestation by domestic animals, parasites, filth, and chemical and microbiological 284

contaminants. Storage requirements such as temperature and humidity may vary 285


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depending on the ingredient, and these should be provided accordingly by the 286

storage facilities to be used. 287

288

Stored stocks of ingredients should be used on a “first in-first out” (FIFO) or a “first to 289

expire-first to use” (FEFU) basis. 290

291

5.2 Processing Operations 292

293

5.2.1. Preparation of meat 294

If frozen meat is used, the thawing process should take place in conditions that 295

minimize contamination and microbial growth. Frozen meat may be thawed in air or 296

by immersing in running water. Time and temperature conditions must be controlled 297

to minimize microbial growth and allow the product to thoroughly thaw out. Chilled 298

meat and thawed meat must be kept at 0° to 4°C while waiting for further processing. 299

300

5.2.2. Slicing, trimming, and comminution 301

For tocino, tapa, and other similar products, meat shall be sliced and trimmed 302

according to the desired size and thickness. 303

304

For longganisa or similar sausage-type products, the meat shall be comminuted to 305

smaller, uniformly-sized particles through the use of a meat grinder, a food 306

processor, or a silent cutter. Traditionally, meat used for longganisa is more coarsely 307

ground as compared to emulsified sausages like hotdogs and bologna. 308

309

The slicing, trimming, and comminution of meat must be performed in a way that 310

minimizes contamination and growth of microorganisms. 311

312

5.2.3 Mixing and Curing 313

Sliced or comminuted meat is mixed or blended with curing agents and other non-314

meat ingredients including sugar, vinegar, and spices and flavorings. The meat 315

mixture is allowed to cure for 1 to 2 days at 4° to 10°C. In cases when water is used 316

to dissolve the dry ingredients, it is added in the form of ice water to help control the 317

temperature of the curing brine/mixture. The curing brine should be maintained at not 318

more than 5°C. Mixing and curing conditions should be controlled to prevent 319

contamination and growth of microorganisms. 320

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5.2.4 Shaping (for longganisa and other sausage-type products) 322

Shaping may be accomplished by either stuffing and linking, or moulding: 323

324

Stuffing and Linking. The meat mixture is stuffed into casings by hand, through a 325

screw feed or by using an automatic stuffing machine. Care must be observed not to 326

use too much pressure that will damage the casings, and at the same time, the 327

sausage mix must completely and compactly fill the casings. 328

329

Sausage-type products that use casings with small diameters and long strands are 330

further divided into segments that are uniform in size and length. This may be 331

accomplished by twisting the casing or tying with a string to create sausage links. If 332

casings with large diameters are used, the meat mixture is filled into individual 333

casings that have the ends sealed with strings or metal clips. 334

335

Moulding. The meat mixture is shaped into cylinders using plastic or paper wraps, 336

which will give the product its shape in lieu of a casing. 337

338

5.2.5 Tumbling and massaging (for tocino, tapa and other similar products) 339

To increase meat tenderness and enhance the distribution of curing ingredients 340

inside the meat, the meat slices may also be subjected to tumbling and massaging. 341

The process involves equipment with rotating chambers or drums with baffles or 342

massaging arms which subject the meat pieces to a gentle beating process as the 343

chamber rotates. Tumbling can take place for several hours, but the temperature 344

should be kept at less than 4°C. Tumbling and massaging time and temperature 345

must be controlled to minimize microbial growth. 346

347

5.2.6 Other Treatments 348

349

Drying. An optional treatment for ethnic cured meat is drying. For longganisa and 350

other sausage-type products in casings, drying is done particularly if the product 351

would be subjected to smoking. Longganisa and similar products are allowed to 352

stand at room temperature for 1 to 2 hours in order to allow the casings to dry before 353

the product is subjected to smoking. Drying may also be accomplished by placing the 354

products inside a warm smokehouse kept at 50°C without smoke. Drying the casing 355

will depend on the size/diameter of the sausage and may range from 15 minutes to 356

one hour. 357


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358

Drying of tapa and similar products may be done by sun drying or through the use of 359

artificial dryers. The drying process should be done in a manner that prevents 360

contamination and microbial growth. 361

362

Smoking. Ethnic cured meats may or may not be smoked. Using raw sawdust, 363

ethnic cured meats are smoked at temperatures ranging from 50° to 70°C until the 364

desired product flavor and color is obtained. The relative humidity of the smokehouse 365

should be maintained at around 80%, since very low humidity can dry out the product 366

while excessively high humidity dilutes the effect of smoke. 367

368

Liquid smoke, a water soluble compound with smoke flavor, is also available, and 369

can be sprayed or applied to the meat or sausage surface. 370

371

5.3 Packing 372

Packing can be done either mechanically or manually. It is important to standardize 373

filling for economic reasons. Gas-packing or vacuum-packing may be done. The 374

room temperature of the packing area should be maintained at 10°C or below. 375

376

5.4 Closing or Sealing of Containers 377

Seams and other closures shall be sealed air-tight to meet the requirements of the 378

processors. 379

380

The seal area of flexible containers must be free of food material and wrinkles. 381

Sealing temperature and pressure shall conform to the sealing equipment to be used. 382

383

5.5 Coding of Sealed Containers 384

Coding of sealed container shall be indelible with details of production date and time, 385

batch code, product code, the product line in which product is packed, the 386

manufacturing plant and other information necessary for product traceability. Where 387

the container does not permit the code to be embossed or inked, the label shall be 388

legibly perforated or otherwise marked, and securely affixed to the product container. 389

390

5.6 Post-Process Container Handling 391

Ethnic cured meats must be kept at chilled (0° to 4°C) or frozen conditions (below 392

-18°C). Fluctuations in storage temperature that will expose the product to freeze- 393


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thaw cycles should be avoided. The repeated freezing and thawing process can 394

cause deterioration of the product quality. 395

396

Mechanical shocks leading to breakage of semi-rigid containers due to container 397

abuse must be avoided. These occur by knocking against each other during 398

conveying, packaging and labeling operations, among others. Flexible 399

containers/pouches shall be handled singly rather than in bunches, and care must be 400

exercised so as to prevent damage by roughened contact surfaces. 401

402

403

6. FOOD ADDITIVES 404

405

6.1 Food additives when used shall be in accordance with the regulations established by 406

the Food and Drugs Administration (B.C. No.2006-016 Updated List of Food 407

Additives), the Codex Alimentarius Commission (CAC/STAN 192-1995, Rev. 5 408

(2004)), and/or authority for these products. The following food additives listed in, but 409

not limited to, Table 1, may be used for the manufacture of ethnic cured meats. 410

411

6.2 All others that have not been included in the above list (Table 1) shall be allowed as 412

carry over provided they are approved by the FDA regulation (B.C. No. 016 s. 2006; 413

Updated List of Food Additives) and shall be in accordance to Section 5.2 of the 414

“Principle Relating to the Carry-Over of Food Additives into Foods (CAC/Volume 1, 415

1991). These additives include those that are used for raw materials and other 416

ingredients such as edible casings. Table 2 shows the list of additives that may be 417

used for edible casings. 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435


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Table 1. Food Additives for Ethnic Cured Meat. (BFAD B.C. No.016 s. 2006. Updated List of 436 Food Additives) 437

Function Additive Maximum allowable level Antioxidant BHA 200 mg/kg (Fat or oil basis)*

BHT 100 mg/kg (Fat or oil basis)* Gallate, Propyl 200 mg/kg * Isopropyl Citrates 200 mg/kg † Tertiary Butylhydroquinone (TBHQ) 100 mg/kg (Fat or oil basis)* Tocopherols 3000 mg/kg*

Color Allura Red AC 500 mg/kg (For use in glaze, coatings, or decorations for fruit, vegetables, meat, or fish) **

Annatto extracts 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) †

Canthaxanthin 1000 mg/kg *** Carmines 1000 mg/kg ***

100 mg/kg † Carotenes, vegetable 1000 mg/kg (For use in tocino (fresh, cured sausage) only) ***

20 mg/kg † Carotenoids 1000 mg/kg ***

20 mg/kg † Curcumin 1000 mg/kg (For use in tocino (fresh, cured sausage) only) ***

20 mg/kg † Erythrosine 30 mg/kg * Grape Skin Extract 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) †

Iron Oxides 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) †

Ponceau 4R 250 mg/kg *** 200 mg/kg †

Sunset Yellow FCF 500 mg/kg (For use in glaze, coatings, or decorations for fruit, vegetables, meat, or fish) *** 135 mg/kg †

Tannic Acid (Tannins, food grade) 10 mg/kg* Emulsifier/ Stabilizer

Diacetyltartaric and fatty acid esters of glycerol

GMP **

Polysorbates 10000 mg/kg * Propylene Glycol Alginate 5000 mg/kg *

Humectant Phosphates 1100 mg/kg (As phosphorus)* Preservative Benzoates 750 mg/kg (As benzoic acid) †

1000 mg/kg (As benzoic acid) † Hydroxybenzoates, p- GMP (Surface treatment; As p-hydroxybenzoic acid) † Nitrates 1254 mg/kg (As residual NO3 ion) ***

365 mg/kg (As residual NO3 ion) † Nitrites 134 mg/kg (As residual NO2 ion) * Sodium Diacetate 1000 mg/kg * Sorbates 2000 mg/kg (As sorbic acid) * Sulphites 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

438

439 440 441 442


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Table 2. Food Additives for Edible Casings* (BFAD B.C. No.016 s. 2006. Updated List of Food 443 Additives) 444

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

Ponceau 4R 500 mg/kg (For use in glaze, coatings, or decorations for fruit, vegetables, meat, or fish)

Sunset Yellow CF 500 mg/kg (For use in glaze, coatings, or decorations for fruit, vegetables, meat, or fish)

Emulsifier/ Stabilizer

Diacetyltartaric and fatty acid esters of glycerol

GMP

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, p- 36 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 7. LABELING 447

448

7.1 Each container shall be labeled and marked with the following information in accordance 449

with BFAD A.O. 88-B s. 1984 (Rules and regulations Governing the Labeling of 450

Prepackaged Food Products distributed in the Philippines): 451

452

(a) The product shall be known as “cured meat” or “ethnic cured meat”. It shall be 453

labeled by common names that include “tocino”, “longganisa”, or “tapa”, provided that 454

the product conforms to the product definitions in subsection 3.1 of the Standards for 455

Ethnic Cured Meats. Other local or regional names referring to products similar to 456


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those defined in subsection 3.1 (Standards for Ethnic Cured Meats) may also be 457

included, provided that these names are acceptable in the area of distribution. The 458

label shall also indicate the species or type of meat source used, such as “pork”, 459

“chicken”, “turkey”, or “beef”. 460

(b) The complete list of ingredients and food additives used in the preparation of the 461

product in descending order of proportion. 462

(c) The net content by weight in the metric system. Other systems of measurement 463

required by importing countries shall appear in parenthesis after the metric system 464

unit. 465

(d) The name and address of the manufacturer, packer and/or distributor of the food. 466

(e) Open date marking 467

The words “Best/Consume Before/Expiry Date” indicating end of period at which the 468

product shall retain its optimum quality attributes at defined storage conditions. 469

(f) Lot or code number identifying product lot. 470

(g) The words “Product of the Philippines”, or the country of origin if imported. 471

(h) Directions for use; 472

Directions for use should be indicated on the label. For products that utilize casings 473

or wrappers, the label should indicate that non-edible casings or wrappers should be 474

removed from the product before consumption. 475

(i) Storage instructions; and 476

(j) Additional requirements 477

A pictorial representation of the product and/or raw materials used placed on the 478

label should not mislead the consumer with respect to the product and/or raw 479

material so illustrated. 480

481

7.2 Nutrition Labeling 482

Nutrition labeling shall conform to the established regulations of FDA and/or authority for 483

this commodity. 484

485

8. QUALITY ASSURANCE 486

487

8.1 Inspection of Finished Products 488

All processed products shall be inspected before labeling and casing and defective 489

products shall be withdrawn or rejected. The company must have an approved policy and 490

procedures based on the BFAD A.O. No. 153 s. 2004 - Guidelines, Current Good 491

Manufacturing Practices in Manufacturing, Packing, Repacking or Holding Food. 492


16

493

8.2 Record Keeping 494

Permanent and legible dated records of time, temperature code mark and other pertinent 495

details shall be kept concerning each load. Such records are essential as a check on 496

processing operations. 497

498

Written records of all container closure examinations shall specify the code lot, the date 499

and time of container closure inspections, the measurements obtained and all the 500

corrective actions taken. 501

502

Records shall be maintained identifying initial distribution of the finished product to 503

facilitate, if necessary, the segregation of specific food lots that may have been 504

contaminated or otherwise unfit for intended use. 505

506

All process deviations involving failure to satisfy the minimum requirements of the process 507

shall be recorded detailing those deviations and the actions taken. 508

509

8.3 Good Manufacturing Practices (GMP) 510

Processing establishments shall have developed, documented and implemented 511

prerequisite programs based on Food and Drugs Administration’s Current Good 512

Manufacturing Practices (cGMP) and Hygiene Control. An effective GMP and Hygiene 513

Control program will decrease the number of critical control points that a manufacturer 514

must face during the hazard analysis of the product/process. 515

516

9. STORAGE AND TRANSPORT OF FINISHED PRODUCT 517

518

Storage and transport conditions of the finished product shall be such that the integrity of 519

the product container, and the safety and quality of the product are not adversely affected. 520

521

Cases and cartons must be of proper size so that the containers fit snugly and are not 522

subject to damage from movement within the case. They must be strong enough to 523

withstand normal transport. 524

525

Chilled products must be kept at 0° to 4°C while frozen products must be kept at -18°C or 526

below. Extreme fluctuations in temperature and humidity during storage and transport of 527

the product must be avoided to prevent product deterioration. 528


17

529

10. LABORATORY CONTROL PROCEDURES 530

531

Each food processing establishment shall have access to laboratory control of both the 532

processes used and the finished products. All food ingredients and food products declared 533

unfit for human consumption by the laboratory shall be rejected. 534

535

Representative samples for each lot or batch shall be taken to assess the safety and 536

quality of the product. 537

538

Microbiological laboratory shall be separated from the processing area. No pathogens 539

shall be handled within the premises of manufacturing plant. 540

541

Laboratory procedures for quality control of the processes and the product must follow 542

recognized or standard methods for easy interpretation of results. 543

544

11. END PRODUCT SPECIFICATIONS 545

546

Appropriate methods shall be used for sampling analysis and determinations to meet the 547

following specifications: 548

1. To the extent possible in good manufacturing practices, the products shall be free 549

from any objectionable characteristics. 550

2. The product shall not contain any toxic substances originating from microorganisms 551

and chemicals. 552

3. The product shall be free from chemical pollutants in amounts which may pose 553

hazard to health. 554

4. The product shall comply with the requirements set forth by the Food and Drugs 555

Administration, and the Codex Alimentarius Commission on Pesticide Residues and 556

Food Additives. 557

558

12. REFERENCES 559

560

Abiva, C. C. 2001. A Quick Guide to Filipino Food and Cooking. Anvil Publishing, Inc. 561

Pasig City, Philippines. 562

563


18

A.O. No. 88-A s. 1984. Regulatory Guidelines Concerning Food Additives. Bureau of Food 564

and Drugs. Department of Health. Alabang, Muntinlupa City, Philippines. 565

566

A.O. No. 88-B s. 1984. Rules and Regulations governing the Labeling of Prepackaged 567

Food Products distributed in the Philippines. Bureau of Food and Drugs. Department of 568

Health. Alabang, Muntinlupa City, Philippines. 569

570

A.O. No. 153 s. 2004. Guidelines, Current Good Manufacturing Practice in Manufacturing, 571

Packing, Repacking or Holding Food. Bureau of Food and Drugs. Department of Health. 572

Alabang, Muntinlupa City, Philippines. 573

574

A.O. No. 154 s. 1971. Regulation B-4 Definition and Standards of Identity for Food 4.14 575

Meat and Meat Products, 4.14.01 Sausages. Bureau of Food and Drugs. Department of 576


578

Archer, G.P. and C.J. Kennedy. 1998. Maximising Quality and Stability of Frozen Foods: 579

A Producers Guide to the State of the Art. Report no 2. EU Concerted action CT96-1180. 580

Accessed: 9 June 2010. Available at: http://www.nutrifreeze.co.uk/Documents/Maximising 581

%20Quality.pdf> 582

583

Association of Analytical Chemists. Official Methods of Analysis Manual. 16th ed., 1995. 584

AOAC International. 481 North Frederick Ave., Suite 500, Gaithersburg, MD 20877-2417. 585

U.S.A. 586

587

B.C. No. 01-A s. 2004. Guidelines for the Assessment of Microbiological Quality of 588

Processed Foods. Bureau of Food and Drugs. Department of Health. Alabang, Muntinlupa 589

City, Philippines. 590

591

B.C. No. 016 s. 2006. Updated List of Food Additives. Bureau of Food and Drugs. 592

Department of Health. Alabang, Muntinlupa City, Philippines. 593

594

DTI. 2007. Starting A Business: Tocino. Bureau of Small and Medium Enterprise 595

Development. Department of Trade and Industry. Sen. Gil Puyat Ave., Makati City, 596

Philippines. Available at: <http://www.dti.gov.ph/dti/uploads/file/Starting%20a%20Business 597

-MeatProcessing3.pdf> 598

599


19

DTI. 2009. Starting A Business: Native Longganisa. Bureau of Small and Medium 600

Enterprise Development. Department of Trade and Industry. Sen. Gil Puyat Ave., Makati 601

City, Philippines. Available at: <http://www.dti.gov.ph/uploads/DownloadableFiles/SAB_ 602

Native_ Longanisa_09.pdf> 603

604

Essien, E. 2003. Sausage Manufacture: Principles and Practice. Woodhead Publishing 605

Ltd. Cambridge, England. 606

607

FAO. 1990. Manual on simple methods of meat preservation.. FAO Animal Production and 608

Health Paper 79. Food and Agriculture Organization of the United Nations. Rome, Italy. 609

Available at: <http://www.fao.org/docrep/003/x6932e/x6932e00.htm>. 610

611

FAO. 1991. Guidelines for slaughtering, meat cutting and further processing. FAO Animal 612

Production and Health Paper 91. Food and Agriculture Organization of the United 613

Nations. Rome, Italy. Available at: <http://www.fao.org/docrep/004/t0279e/T0279E00.htm> 614

615

FAO/WHO Codex Alimentarius Commission Manual. 1995. Codex Alimentarius 616

Commission. Food and Agriculture Organization. Viale delle Terme di Caracalla, 00100 617

Rome, Italy. 618

619

Food, definition. ALINORM 04/27/41, para. 88 and Appendix VI. 2005. Codex Alimentarius 620

Commission. Food and Agriculture Organization. Viale delle Terme di Caracalla, 00100 621

Rome, Italy. 622

623

FSIS. 1999. General HACCP Model for Beef Slaughter. Accessed: 16 August 2010. 624

<http://www.fsis.usda.gov/OPPDE/nis/outreach/models/HACCP-13.pdf>. 625

626

FSIS. 2008. The Regulated Industries: Characteristics and Manufacturing Processes. 627

Accessed: 8 February 2010. Available at : <http://www.fsis.usda.gov/pdf/phvt-regulated_ 628

industries.pdf> 629

630

FSIS. 2009. Hot Dogs and Food Safety. Accessed 19 March 2010. Available at: 631

<http://www.fsis.usda.gov/pdf/hot_dogs_and_food_safety.pdf> 632

633


20

Heinz, G. and P. Hautzinger. 2007. Meat Processing Technology: for Small- and Medium- 634

Scale Producers. Food and Agriculture Organization of the United Nations: Regional office 635

for Asia and the Pacific. Bangkok, Thailand. 636

637

Joint DA-NMIS and DOH-FDA A.O. 01 s.2009. Delineation of Functions and Shared 638

Responsibilities in the Regulation of Meat Products. National Meat Inspection Service. 639

Department of Agriculture. Visayas Ave., Diliman, Quezon City. 640

641

M.C. no. 09 s. 2008. Guidelines on the Assessment of Microbiological Quality of Fresh, 642

Chilled, and Frozen Meat. National Meat Inspection Service. Department of Agriculture. 643

Visayas Ave., Diliman, Quezon City. 644

645

New Zealand Food Safety Authority. Draft Code of Practice: Production of Processed 646

Meats. Accessed: 9 June 2010. Available at: <http://www.nzfsa.govt.nz/consultation/ 647

processed-meat-cop-part1-4/part-3-process-control/index.htm> 648

649

Philippine National Standards No. 991:1993. Agricultural and Other Food Products – 650

Bottled Drinking Water Specifications. Bureau of Product Standards. Department of Trade 651

and Industry. Makati City, Philippines. 652

653

R.A. 3720. Food, Drugs and Cosmetic Act. Bureau of Food and Drugs. Department of 654


656

Ranken, M.D. 2000. Handbook of Meat Product Technology. Blackwell Science Ltd. 657

Oxford, England. 658

659

Sanchez, P.C. 2008. Philippine fermented foods: Principles and technology. The 660

University of the Philippines Press. Diliman, Quezon City, Philippines. 661

662

S.A.O. No. 412 s. 1980. Standardization of Tocino. Philippine Bureau of Standards. 663

Ministry of Trade. Quezon City, Philippines. 664

665

Savic, I.V. 1985. Small-scale Sausage Production. Food and Agriculture Organization of 666

the United Nations. Rome, Italy. 667

668


21

USDA/FSIS/AFDO. 1999. Safe Practices for Sausage Production: Distance Learning 669

Course Manual. Accessed 22 March 2010. Available at: 670

<http://www.midwesternresearch.com/PDF/SausageFSIS.pdf> 671

672

USFDA. 2001. Staphylococcus aureus. Chapter 12. In Bacteriological Analytical Manual. 673

Accessed 24 June 2010. Available at: http://www.fda.gov/Food/ScienceResearch/ 674

LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm> 675

676

USFDA. 2003. Detection and Enumeration of Listeria monocytogenes in Foods. Chapter 677

10. Bacteriological Analytical Manual. Accessed 23 August 2010. Available at: 678

<http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalytical679

ManualBAM/default.htm> 680

681

USFDA. 2007. Salmonella. Chapter 5. Bacteriological Analytical Manual. Accessed 682

23 August 2010. Available at: http://www.fda.gov/Food/ScienceResearch/ 683

LaboratoryMethods/ BacteriologicalAnalyticalManualBAM/default.htm> 684

685

686

687

688

689

690

691

692

693

694

695

696

697

698

699

700

701

702

703

704


22

705

706

ANNEXA707 Standard Parameters and Values for Drinking Water 708

Philippine National Standards for Drinking Water 2007 (DOH AO 2007-0012) 709 710

Table 1. Standard values for bacteriological quality 711 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 Reservoir Water treatment works Consumers’ taps nearest meter Refilling stations Water vending machines

712 Table 2. Standard values for Physical and Chemical Quality for Acceptability Aspects for 713

Drinking Water 714

715 716 Table 3. Standard Values for Organic and Inorganic Chemical Constituents of Health 717 Significance in Drinking Water 718

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 CaCO3 Zinc 5.0

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


23

719 720 721

722 723 724 725 726 727 728 729 730

Cyanide (Total) 0.07 Selenium 0.01 Organic Chemicals

Constituents Maximum Level (mg/L)

Constituents Maximum Level (mg/L)

Benzene 0.01 Ethylbenzene 0.30 Carbon tetrachloride 0.004 Nitrilotriacetic acid (NTA) 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 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

2.0 Registered

Pendimethalin 20.0 Registered Pentachlorophenol (PCP) 9.0 Banned


24

731 732

733

ANNEXB734 Determination of Crude Protein Content 735

(AOAC 981.10; Block Digestion Method) 736

737 A. Reagents 738 (a) Catalyst tablets.-containing 3.5 g K2SO4 and 0.175 g HgO. 739 (b) Boric acid solution.-4%. Dissolve 4 g H3BO3 in H2O containing 0.7 mL 0.1% alcoholic solution 740 of methyl red and 1.0 mL 0.1% alcoholic solution of bromocresol green, and dilute to 100 mL with 741 H2O. 742 (c) Sodium hydroxide-sodium thiosulfate solution.-Dissolve 2000 g NaOH and 125 g Na2S2O3 in 743 H2O and dilute to 5 L (ca 5o mL is used per analysis). 744 (d) Hydrochloric acid standard solution.- o.2N ( 936.15 [see A.1.o6]). 745 (e) Hydrogen peroxide. – 3o-35%. 746 (f) Sulfuric acid.-concentrated. 747 748 B. Equipment 749 (a) Digestion block and associated glassware.-Tecator Ds-6 or Ds-2o (Tecator), or equivalent. 750 (b) Distillation unit and associated glassware.-Kjeltec 1oo3 (Tecator), or equivalent. 751 752 C. Determination 753 Accurately weigh ca 2 g well-ground and mixed sample on 7 cm N-free filter paper (e.g., whatman 754 541), fold, and transfer to 250 mL digestion tube. Place tubes in fume hood and add 2 or 3 boiling 755 stones, 2 catalyst tablets, 15 mL H2SO4, and slowly 3 mL 3o-35% 756 H2O2. Let reaction subside and place tubes in block digestor preheated at 410°. (Digestor must be 757 placed in perchloric acid fume hood or be equipped with exhaust system. Boiling concentrated 758 acid is very corrosive and also emits corrosive fumes. Rapid addition of 3o-35% H2O2 may cause 759 the reaction to become violent.) Digest at 410° until mixture is clear, ca 45 min. Remove tubes and 760 let cool ca 1o min. Do not let precipitate form; if precipitate forms, reheat. Carefully add 5o-75 mL 761 H2O. 762 763 Place NaOH-Na2S2O3 solution in alkali tank of steam distillation unit. Make sure that 5o-75 mL is 764 dispensed from unit before conducting distillation. Attach digestion tube containing diluted digest to 765 distillation unit. Place 25o mL receiving flask containing 25 mL H3BO3 solution with mixed indicator 766 on receiving platform, with tube from condenser extending below surface of absorbing solution. 767 Steam distil until 100-125 mL collects (absorbing solution turns green from liberated NH3). Remove 768 digestion tube and receiving flask from unit. Titrate absorbing solution with 0.2 N Hcl to neutral 769 gray end point and record volume acid required to 0.01 mL. Titrate reagent blank similarly. 770 771 D. Computation: 772 % N = ( vA – vB ) x 1.4oo7 x N /g sample 773 % Protein = ( vA – vB ) x 1.4oo7 x N x 6.25/g sample 774 where vA and vB = volume standard acid required for sample and blank, respectively; 1.4007 = 775 milliequivalent weight N x 1oo(%); N = normality of standard acid; and 6.25 = protein factor for 776 meat products (16% N). 777 778

779

780 781


25

782 783 784 785 786

ANNEXC787 788

Determination of Moisture Content 789 (AOAC 950.46b; Air Drying) 790

791 792 --First Action 793 --Final Action 1991 794 795 1. With lids removed, dry sample containing ca 2 g dry material 16 –18 h at 100 – 102° in air 796

oven (mechanical convection preferred). Use covered Al dish ≥50 mm diameter and ≤4o mm 797 deep. Cool in desiccator and weigh. Report loss in weight as moisture. 798 799

2. With lids removed, dry sample containing ca 2 g dry material to constant weight (2 – 4 h 800 depending on product) in mechanical convection oven or in gravity oven with single shelf at ca 801 125°. Use covered Al dish ≥50 mm diameter and ≤4o mm deep. Avoid excessive drying. 802 Cover, cool in desiccator, and weigh. Report loss in weight as moisture. (Dried sample is not 803 satisfactory for subsequent fat determination.) 804

805 806

807

808

809

810

811

812

813

814

815

816

817

818

819

820

821

822

823

824

825


26

826

827 828 829

ANNEXD830 831

Determination of Crude Fat 832 (AOAC 960.39) 833

834 835 A. Sample Drying 836 837 1. Weigh 3 – 4 g sample by difference into thimble containing small amount of sand. Mix 838

with glass rod, place thimble and rod in 50 mL beaker, and dry in oven 6 h at 100 – 102° or 1.5 839 h at 125°. Proceed as in (B); or 840

2. Weigh 3 – 4 g sample by difference into small disposable Al dish, add sand, and mix, 841 spreading mixture on bottom of dish with glass or Al paddle. Dry with paddle as in (1). Roll 842 edges of dish and insert with paddle into thimble. Proceed as in B. 843 844

B. Determination: 845 (Large amounts H2O-soluble components such as carbohydrates, urea, lactic acid, glycerol, and 846 others may interfere with extraction of fat; if present, extract 2 g sample on small paper in funnel 847 with five 20 mL portions H2O prior to drying for ether extraction. Caution: see Appendix B, safety 848 notes on monitoring equipment, distillation, and diethyl ether.) 849 850 1. Extract ca 2 g sample, dried as in (A), with anhydrous ether. Use thimble with porosity 851

permitting rapid passage of ether. Extraction period may vary from 4 h at condensation rate of 852 5 – 6 drop/s to 16 h at 2 – 3 drop/s. Petroleum ether, 945.16A (see 27.4.04), may be used 853 instead of anhydrous ether, if desired. 854 855

2. Dry extract to constant weight at 100°, cool, and weigh. 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878


27

ANNEXE879 Determination of Water Activity (AOAC 978.18) 880

881 A. Principle 882 Water activity, aw, is ratio of vapor pressure of H2O in product to vapor pressure of pure H2O at 883 same temperature. It is numerically equal to 1/100 of relative humidity (RH) generated by product 884 in closed system. RH can be calculated from direct measurement of 885 partial vapor pressure or dew point or measured indirectly by sensors whose physical or electric 886 characteristics are altered by RH to which they are exposed. Instruments are checked or 887 calibrated on basis of RH generated by standard salt slushes. 888 889 B. Instruments and Systems 890 (Select 1 of following instruments or systems to perform test. Each has different application 891 limitations because of interferences from other volatile components of products being measured. 892 check with instrument manufacturer for more specific limitations.) 893 894 (a) Change in electrical conductivity of immobilized salt solution. – Instrument available from 895

Beckman Industrial, Rosemount Analytical Div., 89 Commerce Rd, Cedar Grove, NJ 07009; 896 Nova Sina AG, Andreastrasse 7-11, CH 8050, Zurich,Switzerland; Rotronic Instrument Corp., 897 160 E. Main St, Huntington, NY 11743. Immobilized salt sensors are affected by polyols such 898 as glycerol and glycol and by volatile amines 899

(b) Change in electrical capacitance of polymer thin films. – Instrument available from General 900 Eastern Instruments, 50 Hunt St, Watertown, MA 02172. Polymer thin film sensors are affected 901 by CH3COOH. 902

(c) Dew point by chilled mirror technique. – Instrument available from EG&G, Environmental 903 Equipment Division, 217 Middlesex Turnpike, Burlington, MA 01803 or General Eastern 904 Instruments. Dew point measurements can be affected by condensables with lower critical 905 temperature than H2O. 906

(d) Longitudinal change in dimensions of water-sorbing fiber. – Instrument available from G Lufft 907 Metallbarometerfabrik, D-7, Postfach 692, Neue Weinsteige 22, Stuttgart, Germany. 908

(e) Partial water vapor pressure by manometric system. – Partial H2O vapor pressure 909 measurements can be made useless by living products that respire, such as grains or nuts; by 910 active fermentation; or by products that expand excessively when subjected to high vacuum. 911

(f) Relative weight of moisture sorbed by anhydrous hydrophilic solid, e.g., microcrystalline 912 cellulose.-see J. Agr. Food chem. 22, 326(1974). 913

914 C. Apparatus and Reagents 915 (As needed for instrument or system selected.) 916 917 (a) Dew point instrument. – Equipped to measure temperature to ±0.1°. See 978.18B(c). 918 (b) Forced-draft cabinet. – Constant temperature, set to maintain 25 ± 1°; capacity ≥0.06 m3 (2 cu ft); 919

with access port to accommodate instrument sensor leads. Use in conjunction 920 with (c). 921

(c) Insulated box with cover. – Large enough to hold test container, (e), and small enough to fit in 922 forced-draft cabinet, (b); with access port to accommodate instrument sensor leads. Protect 923 test container from short-term temperature fluctuations. 924

(d) Manometric system. – Sensitive to pressure differential of ± 0.01 mm Hg (1.33 Pa). See 925 978.18B(e). 926

(e) Test containers. – 120 or 240 mL (4 or 8 oz) wide-mouth or Mason glass jars with Al- or Teflon-927 lined screw caps and gaskets. Check integrity of cap seals and sensor leads by any means 928 available, e.g., ability of system to hold vacuum, using Tesla coil. 929

(f) Water bath. – Capable of maintaining temperature constant within 0.1° at 25±1°; capacity 930 sufficient to hold measuring chamber of selected apparatus. 931

(g) Hydrophilic solid. – Microcrystalline cellulose, Type PH-101 (FMC Corp.,Pharmaceutical and 932 Bioscience Division, 1735 Market St, Philadelphia, PA 19103, or equivalent). 933

(h) Reference salts. – ACS reagent grade, fine crystal. see Table 978.18. 934


28

935 D. Preparation of Reference Salt Slushes 936 937 Place selected reference salt in test container to depth of ca 4 cm for more soluble salts (lower aw), 938 to depth of ca 1.5 cm for less soluble salts (higher aw), and to intermediate depth for intermediate 939 salts. Add H2O in ca 2 mL increments, stirring well with spatula after each addition, until salt can 940 absorb no more H2O as evidenced by free liquid. Keep free liquid to minimum needed to establish 941 saturation of salt with H2O. Slushes are ready for use upon completion of mixing, and are usable 942 indefinitely (except for some high aw salts susceptible to bacterial attack), if contained in manner to 943 prevent substantial evaporation losses. Some slushes, e.g., NaBr, may solidify gradually by crystal 944 coalescence, with no effect on aw. 945 946 E. Calibration 947 948 Select ≥ 5 salts to cover aw range of interest or range of sensor being used. Measure humidity 949 generated by each salt slush in terms of instrument readout, as in 978.18F. Plot readout against aw 950 values given in Table 978.18 for selected salts, using cross-section paper scaled for reading to 951 0.001 aw unit. Draw best average smooth line through plotted points. Use this calibration line to 952 translate sensor instrument readout of samples to aw or to check vapor pressure or dew point 953 instruments for proper functioning. 954 955 F. Determination 956 957 Place calibration slush or sample in forced-draft cabinet, (b), or H2O bath, (f), until temperature is 958 stabilized at 25±1°. Transfer salt slush or sample to test container, (e), seal container with sensing 959 device attached, and place in temperature control device. Use volume of sample or slush >1/20 960 total volume sample container plus any associated void volume of sensing system, but not so 961 much as to interfere with operation of system. Record instrument response at 15, 30, 60, and 120 962 min after test container is placed in temperature control device, or record response on strip chart. 963 Two consecutive readings, at indicated intervals, which vary by <0.01 aw unit are evidence of 964 adequately close approach to equilibrium. Continue readings at 60-min intervals, if necessary. 965 Convert last reading to aw by calculation from physical measurements or by reference to calibration 966 line. Make all measurements within range of calibration points; do not extrapolate calibration line. 967 Make all measurements in same direction of change, and, if required by properties of sensor, 968 expose sensor to controlled RH below ambient before starting each measurement. 969 970 971 972 973

974 975 976 977


29

ANNEXF978 Isolation of Salmonella (USFDA, 2007) 979

A. Sample Preparation (For meats, meat substitutes, meat by-products, animal substances, 980 glandular products, and meals (fish, meat, bone)). 981

Aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile lactose broth and 982 blend 2 min. Aseptically transfer homogenized mixture to sterile wide-mouth, screw-cap jar (500 983 ml) or other appropriate container and let stand 60 ± 5 min at room temperature with jar securely 984 capped. If mixture is powder or is ground or comminuted, blending may be omitted. For samples 985 that do not require blending, add lactose broth and mix thoroughly; let stand for 60 ± 5 min at room 986 temperature with jar securely capped. 987

Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Add up 988 to 2.25 ml steamed (15 min) Tergitol Anionic 7 and mix well. Alternatively, use steamed (15 min) 989 Triton X-100. Limit use of these surfactants to minimum quantity needed to initiate foaming. Actual 990 quantity will depend on composition of test material. Surfactants will not be needed in analysis of 991 powdered glandular products. Loosen jar caps 1/4 turn and incubate sample mixtures 24 ± 2 h at 992 35°C. Continue as in B., below. 993

B. Isolation of Salmonella 994

1. Tighten lid and gently shake incubated sample. 995

Guar gum and foods suspected to be contaminated with S. Typhi. Transfer 1 ml mixture to 996 10 ml selenite cystine (SC) broth and another 1 ml mixture to 10 ml TT broth . Vortex. 997

All other foods. Transfer 0.1 ml mixture to 10 ml Rappaport-Vassiliadis (RV) medium and 998 another 1 ml mixture to 10 ml tetrathionate (TT) broth. Vortex. 999

2. Incubate selective enrichment media as follows: 1000

Foods with a high microbial load. Incubate RV medium 24 ± 2 h at 42 ± 0.2°C (circulating, 1001 thermostatically-controlled, water bath). Incubate TT broth 24 ± 2 h at 43 ± 0.2°C (circulating, 1002 thermostatically-controlled, water bath). 1003

Foods with a low microbial load (except guar gum and foods suspected to be 1004 contaminated with S. Typhi). Incubate RV medium 24 ± 2 h at 42 ± 0.2°C (circulating, 1005 thermostatically controlled, water bath). Incubate TT broth 24 ± 2 h at 35 ± 2.0°C. 1006

Guar gum and foods suspected to be contaminated with S. Typhi. Incubate SC and TT 1007 broths 24 ± 2 h at 35°C. 1008

3. Mix (vortex, if tube) and streak 3 mm loopful (10 µl) incubated TT broth on bismuth sulfite (BS) 1009 agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar. Prepare BS 1010 plates the day before streaking and store in dark at room temperature until streaked. 1011 4. Repeat with 3 mm loopful (10 µl) of RV medium (for samples of high and low microbial load 1012 foods) and of SC broth (for guar gum). 1013 5. Refer to 994.04 in Official Methods of Analysis (1) for option of refrigerating incubated sample 1014 preenrichments and incubated sample selective enrichments (SC and TT broths only) of low 1015 moisture foods. This option allows sample analyses to be initiated as late as Thursday while still 1016 avoiding weekend work. 1017 6. Incubate plates 24 ± 2 h at 35°C. 1018 7. Examine plates for presence of colonies that may be Salmonella. 1019 8. Lightly touch the very center of the colony to be picked with sterile inoculating needle and 1020

inoculate TSI slant by streaking slant and stabbing butt. Without flaming, inoculate LIA slant by 1021 stabbing butt twice and then streaking slant. Since lysine decarboxylation reaction is strictly 1022


30

anaerobic, the LIA slants must have deep butt (4 cm). Store picked selective agar plates at 5-1023 8°C. 1024

9. Incubate TSI and LIA slants at 35°C for 24 ± 2 h. Cap tubes loosely to maintain aerobic 1025 conditions while incubating slants to prevent excessive H2S production. Salmonella in culture 1026 typically produces alkaline (red) slant and acid (yellow) butt, with or without production of H2S 1027 (blackening of agar) in TSI. In LIA, Salmonella typically produces alkaline (purple) reaction in 1028 butt of tube. Consider only distinct yellow in butt of tube as acidic (negative) reaction. Do not 1029 eliminate cultures that produce discoloration in butt of tube solely on this basis. Most 1030 Salmonella cultures produce H2S in LIA. Some non- Salmonella cultures produce a brick-red 1031 reaction in LIA slants. 1032

10. All cultures that give an alkaline butt in LIA, regardless of TSI reaction, should be retained as 1033 potential Salmonella isolates and submitted for biochemical and serological tests. Cultures that 1034 give an acid butt in LIA and an alkaline slant and acid butt in TSI should also be considered 1035 potential Salmonella isolates and should be submitted for biochemical and serological tests. 1036 Cultures that give an acid butt in LIA and an acid slant and acid butt in TSI may be discarded 1037 as not being Salmonella . Test retained, presumed-positive TSI cultures as directed in D-11, 1038 below, to determine if they are Salmonella species, including S. arizonae. If TSI cultures fail to 1039 give typical reactions for Salmonella (alkaline slant and acid butt) pick additional suspicious 1040 colonies from selective medium plate not giving presumed-positive culture and inoculate TSI 1041 and LIA slants as described in D-8, above. 1042

11. Apply biochemical and serological identification tests to: 1043

a. Three presumptive TSI cultures recovered from set of plates streaked from RV medium (or 1044 SC broth for guar gum), if present, and 3 presumptive TSI agar cultures recovered from plates 1045 streaked from TT broth, if present. 1046

b. If 3 presumptive-positive TSI cultures are not isolated from one set of agar plates, test other 1047 presumptive-positive TSI agar cultures, if isolated, by bioche mical and serological tests. 1048 Examine a minimum of 6 TSI cultures for each 25 g analytical unit or each 375 g composite. 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


31

ANNEXG1078 Detection and Enumeration of Listeria monocytogenes 1079

(Conventional method; USFDA (2003)) 1080

Media and Reagents 1081

1. Acetic Acid, 5 N 1082 2. Acriflavine monohydrochloride 1083 3. Agar (Difco Laboratories, Detroit, MI) 1084 4. N-(1-naphthyl) ethylene diamine (R48) 1085 5. α-Naphthol reagent (R48) 1086 6. Blood agar base No. 2 (Unipath) 1087 7. Cycloheximide 1088 8. Natamycin (pimaricin) 1089 9. Sheep blood, defibrinated 1090 10. Ethanol, absolute 1091 11. Fluorescent antibody (FA) buffer (Difco) 1092 12. Glycine anhydride 1093 13. Gram stain kit 1094 14. Hydrogen peroxide solution, 3% for catalase test (R12) 1095 15. KOH 40% solution (R65) 1096 16. Listeria-typing sera set (Difco) 1097 17. Lithium chloride-phenylethanol-moxalactam (LPM) agar (M81) with added esculin and iron 1098

(M82) 1099 18. Nalidixic acid (sodium salt) 1100 19. Nitrate reduction medium (M108) and nitrate detection reagents (R48) 1101 20. Nutrient broth (M114) 1102 21. Physiological saline solution, 0.85% (R63) 1103 22. Purple carbohydrate fermentation broth base (M130), containing 0.5% solutions of 1104

dextrose, esculin, maltose, rhamnose, mannitol, and xylose 1105 23. SIM medium (Becton-Dickinson Microbiology Systems, M137) or motility test medium 1106

(MTM, Difco) (M103) 1107 24. Sulfanilic acid reagent (R48) 1108 25. Trypticase soy agar with 0.6% yeast extract (TSAye) (M153) 1109 26. Trypticase soy broth with 0.6% yeast extract (TSBye) (M157) 1110 27. Oxford medium (OXA) (M118) 1111 28. Buffered Listeria Enrichment Broth (BLEB) (M52) 1112 29. PALCAM agar (M118a) 1113 30. Carageenan (Sigma type II) 1114 31. BCM agar (M17a) 1115 32. MOX agar (M103a) 1116 33. ALOA agar (M10a) 1117 34. Chromogenic Listeria Agar (M40b) 1118 35. Rapid L'mono (M131a) 1119 36. CHROMagar Listeria (M40a) 1120 37. Tryptose broth and agar (Difco) (M167) 1121

Note: Alternative companies may be used when the products are equivalent. 1122

A. Sample treatment. 1123

Sample refrigeration at 4°C is recommended for handling, storing, and shipping materials to be 1124 analyzed for L. monocytogenes, which will grow, although slowly, at this temperature if other 1125 conditions permit. However, if the sample is already frozen, it should not be thawed until 1126 analysis. 1127

a. Composited samples. 1128


32

Generally, composited samples are prepared as follows. A food lot sample is collected 1129 consisting of 10 sub-samples (liquid, cream or solid food) and 50-g or ml portions of each sub-1130 sample are used to make two composite samples (250 g each). Take care to make sub-1131 samples representative of a food's outer surface as well as its interior. For the first composite 5 1132 × 50-g portions from 5 sub-samples are pooled and blended or stomached in 250 ml buffered 1133 Listeria enrichment broth base containing sodium pyruvate without selective agents (BLEB, 1134 M52). The second composite is made from the remaining 5 sub-samples in the same way. 1135 Both blended composites contain 250-g food portions and 250 ml basal BLEB. 1136

Normally 50 g of each composite blend (equivalent to 25g food plus 25 ml basal BLEB) is 1137 mixed with a 200 ml amount of basal BLEB. Since there are two composites, the end result is 1138 two 25-g analytical portions each contained in 225 ml amounts of basal BLEB. Thus, for each 1139 sub-sample a total of 50 g of composited food is analyzed. An aliquot (50 ml) of the composite 1140 blend should be retained, preferably at 5° C and not below 0° C, for possible pathogen 1141 enumeration. 1142

b. Non-composited samples. 1143

If composite samples are not required, single 25-g analytical portions of food are simply 1144 blended or stomached in 225 ml of basal BLEB and pre-enriched/enriched as described later. 1145 A 25-g sample should be retained for possible pathogen enumeration. Store it at 5° C if it is not 1146 frozen or, if frozen, in a non-defrosting freezer. 1147

B. Pre-enrichment and enrichment. 1148

Incubate for 4 h at 30° C, add the selective agents and continue incubating for a total time of 1149 48 h at 30° C. If cycloheximide is unavailable, the preferred substitute is pimaricin (natamycin) 1150 at 25 mg/L (27). Natamycin is much safer to use than cycloheximide. Another possibility, if the 1151 matrices of interest (e.g. pasteurized milk and cream, yogurt, and precooked frozen seafood) 1152 are low in yeast and mold, is to do without an anti-fungal agent. This is not advisable for mold-1153 ripened cheeses, smoked or dried seafood or fresh produce. 1154

C. Enrichment with enumeration. 1155

Surveillance enumeration of Listeria monocytogenes levels in contaminated food is now 1156 required. Detection may be done first and if contamination is detected, a reserve sample 1157 portion can be enumerated. This is probably the preferable method as, generally, only a few 1158 percent of samples can be expected to be positive and then most often at a level of only about 1159 1 cfu/25g. However, the option of combining regulatory detection and enumeration is provided 1160 in Enumeration. 1161

D. Isolation procedure 1162 1163

At 24 and 48 h, streak BLEB culture onto one of the following esculin-containing selective isolation 1164 agars: either OXA (16, M118) or PALCAM (42, M118a) or MOX (41, M103a) or LPM (31, M81) 1165 fortified with esculin and Fe3+ (M82). These esculin-containing media are listed in order of preferred 1166 use, subject to their availability. Incubate OXA, PALCAM or MOX plates at 35° C for 24-48 h and 1167 fortified LPM plates at 30° C for 24-48 h. It is strongly recommended that one of the L. 1168 monocytogenes-L. ivanovii differential selective agars, such as BCM (33, M117a), ALOA (M10a), 1169 RapidL'mono (M131a), or CHROMagar Listeria (M40a) be streaked at 48 h (optionally at 24 h, too) 1170 in addition to the chosen esculin-containing selective agar. This will reduce the problem of masking 1171 of L. monocytogenes by L. innocua. [Note: BCM has been collaboratively validated by FDA. An ISO 1172 TC34 SC9 comparative validation showed that all the media (and a selective blood agar - LMBA, 1173 Sifin, Germany) inhibited Listeria competitors more or less equally well. ALOA was preferred only 1174 because its formulation is public. Another differential selective medium, Chromogenic Listeria Agar 1175 (M40b) is due to be marketed in the future.] 1176 1177


33

Listeria colonies are black with a black halo on esculin-containing media. Certain other bacteria can 1178 form weakly brownish black colonies, but color development takes longer than 2 days. Transfer 5 1179 or more typical colonies from OXA and PALCAM or modified LPM or MOX to Trypticase soy agar 1180 with yeast extract (TSAye), streaking for purity and typical isolated colonies. If BCM plates are 1181 streaked as recommended above and blue colonies are observed, they are presumptive L. 1182 monocytogenes colonies since L. ivanovii is not often reported in foods. L. monocytogenes and L. 1183 ivanovii colonies on ALOA are blue and have a zone of lipolysis around them. Purification on 1184 TSAye is a mandatory step in the conventional analysis because isolated colonies on selective 1185 agar media may still be in contact with an invisible weak background of partially inhibited 1186 competitors. At least 5 isolates are necessary because more than one species of Listeria may be 1187 isolated from the same sample. Use of BCM and ALOA plates will help to reduce the number of 1188 colonies that need to be picked. L. monocytogenes and L. ivanovii can be distinguished using a 1189 commercial Confirmatory Medium (Biosynth International, Inc.) or by conventional rhamnose/xylose 1190 fermentation broths or agars. Incubate TSAye plates at 30° C for 24-48 h. The plates may be 1191 incubated at 35° C if colonies will not be used for wet-mount motility observations. For the 1192 approved rapid methods (Table 2), use the selective isolation agar recommended by the 1193 manufacturer but, as noted above, auxiliary use of the new L. monocytogenes-L.ivanovii differential 1194 agars is also recommended. 1195 1196 E. Identification procedure 1197

1198 Identify purified isolates by the following classical tests. Rapid kits are available to facilitate 1199 biochemical testing to genus or species level. 1200

1. Examine TSAye plates for typical colonies. Observation with Henry oblique transmitted 1201 illumination can be helpful at this stage but is not mandatory. 1202

2. Pick typical colony from culture plate incubated at 30°C or less and examine by wet mount, 1203 using 0.85% saline for suspending medium and oil immersion objective of phase-contrast 1204 microscope. Choose a colony with enough growth to make a fairly heavy suspension; 1205 emulsify thoroughly. If too little growth is used, the few cells present will stick to the glass 1206 slide and appear non-motile. Listeria spp. are slim, short rods with slight rotating or 1207 tumbling motility. Always compare with known culture. Cocci, large rods, or rods with rapid, 1208 swimming motility are not Listeria spp. Alternatively, use the 7-day motility test medium. 1209

3. Test typical colony for catalase. Listeria species are catalase-positive. 1210 4. Gram stain 16- to 24-h cultures. All Listeria spp. are short, Gram-positive rods; however, 1211

with older cultures the Gram stain reaction can be variable and also cells may appear 1212 coccoidal. The cells have a tendency to palisade in thick-stained smears. This can lead to 1213 false rejection as a diphtheroid. 1214

5. Pick typical colony to a tube of TSBye for inoculating carbohydrate fermentation and other 1215 test media. Incubate at 35° C for 24 h. This culture may be kept at 4°C several days and 1216 used repeatedly as inoculum. Commercial kits are available for isolate identification. 1217

6. Inoculate heavily (from TSAye colony) 5% sheep blood agar by stabbing plates that have 1218 been poured thick and dried well (check for moisture before using). Draw grid of 20-25 1219 spaces on plate bottom. Stab one culture per grid space. Always stab positive controls (L. 1220 ivanovii and L. monocytogenes) and negative control (L. innocua). Incubate for 24-48 h at 1221 35° C. Attempt to stab as near to bottom of agar layer as possible, without actually 1222 touching bottom of agar layer and possibly fracturing the agar. 1223

7. Examine blood agar plates containing culture stabs with bright light. L.monocytogenes and 1224 L. seeligeri produce a slightly cleared zone around the stab. L. innocua shows no zone of 1225 hemolysis, whereas L. ivanovii produces a well-defined clear zone around the stab. Do not 1226 try to differentiate species at this point, but note nature of hemolytic reaction. Resolve 1227 questionable reactions by the CAMP test. (Note: Hemolysis is more easily determined 1228 when the depth of the blood agar is thinner than the usual 5mm. Optionally, this may be 1229 achieved by use of a blood agar overlay (1-2 mm) technique). 1230

8. Nitrate reduction test. This test is optional. Only L. grayi ssp. murrayi reduces nitrates. The 1231 test distinguishes L. grayi ssp. murrayi from L. grayi ssp. grayi. Use a TSBye culture to 1232 inoculate nitrate broth (M108). Incubate at 35° C for 5 days. Add 0.2 ml reagent A, followed 1233 by 0.2 ml reagent B (R48). A red-violet color indicates presence of nitrite, i.e. nitrate has 1234 been reduced. If no color develops, add powdered zinc and hold for 1 h. A developing red-1235


34

violet color indicates that nitrate is still present and has not been reduced. As an alternative 1236 procedure (R48), add 0.2 ml reagent A followed by 0.2 ml reagent C. An orange color 1237 indicates reduction of nitrate. If no color develops, add powdered zinc as above. 1238 Development of an orange color indicates unreduced nitrate. 1239

9. Inoculate SIM or MTM from TSBye. Incubate for 7 days at room temperature. Observe 1240 daily. Listeria spp. are motile, giving a typical umbrella-like growth pattern. MTM provides 1241 the best defined umbrellas. Alternatively, observe the 30° C TSBye cultures, by phase 1242 contrast microscopy (×1000) for tumbling motility. 1243

10. From TSBye culture, inoculate the following carbohydrates as 0.5% solutions in purple 1244 carbohydrate broth (the use of Durham tubes is optional): dextrose, esculin, maltose, 1245 rhamnose, mannitol, and xylose. Incubate 7 days at 35° C. Positively reacting Listeria spp. 1246 produce acid with no gas. Consult Table 1 for xylose-rhamnose reactions of Listeria spp. 1247 All species should be positive for dextrose, esculin, and maltose. All Listeria spp. except L. 1248 grayi should be mannitol-negative. If pigmentation of the isolate on OXA, PALCAM, MOX 1249 or LPM plus esculin/Fe3+ is unequivocal, the esculin test may be omitted. 1250

11. Purified isolates identified as Listeria monocytogenes by alternative rapid methods should 1251 be retained for regulatory reference. 1252

1253 F. Interpretation of test data 1254

The importance of completely characterizing each isolate cannot be overemphasized. Partial 1255 characterization, even if accurate, may be misleading. Since all Listeria species test negative for 1256 indole, oxidase, urease, and H2S production from organic sulfur compounds (H2S is produced 1257 from thiosulfate in the MICRO-ID test kit) and test positive for methyl red and Voges-Proskauer, 1258 these tests are discretionary. Brochothrix, which is closely related phylogenetically to Listeria, is 1259 distinguishable from Listeria by its inability to grow at 35° C and by its lack of motility. 1260 Distinguishing features of the Gram-positive non-sporeforming rods, Erysipelothrix and Kurthia, 1261 which occur rarely in Listeria analysis, can be found elsewhere. 1262

All Listeria spp. are small, catalase-positive, Gram-positive rods that are motile in wet mounts 1263 and in SIM. They utilize dextrose, esculin, and maltose, and some species utilize mannitol, 1264 rhamnose, and xylose with production of acid. An isolate utilizing mannitol with acid production is 1265 L. grayi. L. monocytogenes, L. ivanovii, and L. seeligeri produce hemolysis in sheep blood stabs 1266 and consequently are CAMP test-positive. Of the three, only L. monocytogenes fails to utilize 1267 xylose and is positive for rhamnose utilization. The difficulty in differentiating L. ivanovii from L. 1268 seeligeri can be resolved by the CAMP test. L. seeligeri shows enhanced hemolysis at the S. 1269 aureus streak. L. ivanovii shows enhanced hemolysis at the R. equi streak. Of the non-hemolytic 1270 species, L. innocua may provide the same rhamnose-xylose reactions as L. monocytogenes but 1271 it is negative in the CAMP test. L. innocua sometimes gives negative results for utilization of 1272 rhamnose . The significance of the undocumented reference to hemolytic L. innocua isolates is 1273 unclear since it is commonly accepted that L. innocua is non-hemolytic and L. monocytogenes is 1274 hemolytic. A L. welshimeri isolate that is rhamnose-negative may be confused with a weakly 1275 hemolytic L. seeligeri isolate unless resolved by the CAMP test. Sometimes aberrant listeria 1276 strains are isolated which are extremely difficult to speciate. (See Guideline for BAM Users on 1277 Identification of Atypical Hemolytic Listeria Isolates.) [Note: The clinical significance of a strain of 1278 L. monocytogenes that is phenotypically hemolytic-negative is debatable. If it is due to a defect of 1279 the hemolysin gene, especially a deletion rather than a point mutation, it is likely clinically less 1280 significant than a normal strain would be, judging from laboratory studies of constructed 1281 hemolysin mutants in mice. However, if it is due to a regulatory defect that affects the expression 1282 of the hemolysin gene in vitro, the possibility of conditional expression in vivo is raised. Until 1283 convenient methods are devised to distinguish these structural and regulatory alternatives, the 1284 isolate need only be carefully confirmed as being a strain of L. monocytogenes phenotypically 1285 hemolysin-negative in vitro so that a soundly based regulatory decision can then be made, based 1286 upon all the relevant circumstances.] 1287

Only after all other results are available does serotyping and other kinds of typing of Listeria 1288 isolates become meaningful. All data collection must be completed before species identities are 1289


35

determined. FDA no longer conducts routine bacteriophage-susceptibility typing of L. 1290 monocytogenes isolates. 1291

G. Enumeration (required) 1292 1. If a sample tests positive for L. monocytogenes, use a reserve portion of sample for 1293

enumeration. Current methods of enumeration are only presumptive for Listeria 1294 monocytogenes and some degree of further testing of isolated Listeria colonies is 1295 necessary. Conventional enumeration is described and alternative rapid methods are 1296 indicated. The proportion of presumptive isolates that are actually L. monocytogenes may 1297 be determined by conventional or rapid tests. Flexibility in choice of methods and 1298 adaptations of them is permitted but the observed count must be reported with 95% 1299 confidence limits, the method used named and any modifications indicated. The correction 1300 factor for converting the observed count to L. monocytogenes numbers must be reported 1301 as the whole number ratio of number of isolates identified as L. monocytogenes to the total 1302 number of Listeria isolates tested. 1303

2. All enumeration methods, including microscopic, colony and Most Probable Number (MPN) 1304 counts are fundamentally governed by the Poisson distribution law of infrequent events. 1305 This describes the distribution of Listeria among the arrays of compartments (tubes, wells, 1306 counting chamber squares, filter grid squares, and virtual squares on culture agar 1307 surfaces). Compartmentalization separates or delineates colony-forming units in the 1308 various methods. In general, the confidence limits (CLs) of these estimates are considered 1309 proportional to the square root of the observed count. [The tabulated CLs for MPN results 1310 are asymmetric about the mean because they are usually obtained with low numbers of 1311 tubes (3 or 5) near the dilution endpoint.] As the count increases its confidence limits, 1312 expressed as a percentage of the count, decrease. Thus, choosing among methods largely 1313 reduces to a consideration of material and labor expenses and to how inoculation 1314 manipulations for an optimal number of compartments can be reduced by techniques such 1315 as filtration, semi-automation and robotics. 1316

3. Surveillance Enumeration. This is required for accumulating data on cell numbers of L. 1317 monocytogenes in regulatory samples that test positive for the pathogen. To estimate the 1318 degree of sample contamination by presumptive L. monocytogenes, quantify the initial 1319 enrichment broth, before starting incubation, by direct spread plate count on ALOA, BCM 1320 or equivalent differential agar. Also, use a 3 or more-tube/well MPN culture procedure on 1, 1321 0.1, 0.01and 0.001-g samples in BLEB (30° C, 48 h, with or without pyruvate and without 1322 delayed addition of selective agents) followed by streaking on the chosen selective agar. If 1323 all the MPN tubes are Listeria positive, use reserve sample to repeat the MPN 1324 determination using an appropriate range of more dilute analytical portions, e.g. 10-4, 10-5, 1325 10-6, 10-7, and 10-8 g. 1326

4. If selective agar plates are in short supply, an economic alternative to spreading dilution 1327 aliquots on individual selective agar plates is the drop plating method. Using a multi-1328 channel pipette is well suited to this method. Decimally dilute 10 µl amounts of the contents 1329 of the enrichment containers in 90 µl amounts of TSBye in micro-titer plates with round-1330 bottomed wells. Mix with a gentle circular motion of the micropipette tip before changing 1331 the tip for the next dilution. Carefully plate 10 µl of the dilutions as drops on plates of ALOA, 1332 BCM or equivalent agar. Let the droplets be absorbed before inverting the plates for 1333 incubation. Square plates are most convenient and efficient for this technique. 1334

Table 1. Alternative rapid enumeration methods.

Method Reference Validation Specificity Matrix

MPN filter Entis & Lerner (20) AOAC INTL. All Listeria, FDA foods

Filter/colony-lift Carroll et al. (13) Peer review L. monocytogenes Meat

DNA probe colony hybridization

BAM Chapter 24 (17,18) FDA L. monocytogenes,

FDA foods


36

5. Alternatively, the methods shown in Table 1 may be used. Identify isolates by conventional 1335 or rapid methods. When all Listeria are enumerated estimate the proportion that is L. 1336 monocytogenes by determining the species of 10 typical Listeria colonies. M.L. Grant 1337 (FDA/DFS/ORA Laboratory Information Bulletin 17: (3) LIB No. 4240, 9pp) has developed 1338 a filter enumeration method, based on the BAM Listeria enrichment and isolation method, 1339 which enumerates Listeria at cell numbers of >100 cfu/g. 1340

6. Tolerance enumeration. Enumeration to determine if a regulated level of tolerance is being 1341 met is not needed with the current "zero-tolerance" policy of no detectable L. 1342 monocytogenes in 2 x 25-g analytical portions of food or beverage. It would require 1343 narrower confidence limits than does surveillance enumeration. Narrower confidence limits 1344 for tolerance and surveillance enumeration can be accomplished by counting more colony 1345 forming units, which can be accomplished by increasing the number of replicate tubes or 1346 other containers. For the current FDA method, the wells of one or more 96-well micro-titer 1347 plates, with round-bottomed wells, can be inoculated, by multi-channel pipette or 1348 robotically, with 0.1 ml of homogenate of complete BLEB and sample. After incubation at 1349 30° C for 48 h, use the same kinds of transfer methods described in Surveillance 1350 Enumeration to inoculate enriched samples to ALOA, BCM or equivalent differential agar to 1351 determine which wells are positive. Using the proportion of L. monocytogenes-positive 1352 wells, the mean concentration can be calculated using the Poisson equation. 1353

7. Alternatively, the 1600 filter grid compartments MPN method for Listeria may be used for 1354 presumptive enumeration of L. monocytogenes. 1355

8. Identify isolates by conventional methods, including the use of ALOA, BCM or equivalent 1356 agar, or by rapid methods. When necessary estimate the proportion of L. monocytogenes 1357 among 10 Listeria isolates. 1358

9. Simultaneous detection and enumeration. Most samples are likely to be negative and thus 1359 it is efficient to delay enumeration of reserve samples until the Listeria detection stage is 1360 completed. Even then, most positive samples will only contain a few cfu/25g. Nevertheless 1361 it may sometimes be more convenient to do simultaneous detection and enumeration. To 1362 accomplish this, prepare the enrichment homogenate as described above and immediately 1363 spread 0.1 ml on ALOA, BCM or an equivalent L. monocytogenes selective agar. Incubate 1364 plates at 35° C for 24-48 h. The combined minimal method will allow the cell number of 1365 presumptive L. monocytogenes to be categorized as <0.04 cfu/g, 0.04 - 100 cfu/g, 100-1366 25,000 cfu/g, or > 25,000 cfu /g. More replica plates and more decimal dilutions in TSBye 1367 are optional to obtain a more precise enumeration. Test 5 representative colonies for ability 1368 to ferment L-rhamnose by the conventional fermentation method, by the BCM rhamnose 1369 confirmatory agar or by a rapid L. monocytogenes identification kit to definitively rule out 1370 the uncommon occurrence of L. ivanovii in foods. 1371

1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390


37

1391 1392

ANNEXH1393 Determination of Staphylococcus aureus 1394 (Direct Plate Count Method (USFDA, 2001)) 1395

1396

A. Equipment and materials 1397 1. Same basic equipment as for conventional plate count (Chapter 3). 1398 2. Drying cabinet or incubator for drying surface of agar plates 1399 3. Sterile bent glass streaking rods, hockey stick or hoe-shaped, with fire-polished ends, 3-4 mm 1400

diameter, 15-20 cm long, with an angled spreading surface 45-55 mm long. 1401

B. Media and reagents 1402 1. Baird-Parker medium (M17) 1403 2. Trypticase (tryptic) soy agar (TSA) (M152) 1404 3. Brain heart infusion (BHI) broth (M24) 1405 4. Coagulase plasma (rabbit) with EDTA 1406 5. Toluidine blue-DNA agar (M148) 1407 6. Lysostaphin (Schwartz-Mann, Mountain View Ave., Orangeburg, NY 10962) 1408 7. Tryptone yeast extract agar (M165) 1409 8. Paraffin oil, sterile 1410 9. 0.02 M phosphate-saline buffer (R61), containing 1% NaCl 1411 10. Catalase test (R12) 1412 1413 A. Preparation of sample 1414 (see Chapter 1 of USFDA/CFSAN Baceriological Analytical Manual, 2001). 1415 1416 B. Isolation and enumeration of S. aureus 1417 1418 1. For each dilution to be plated, aseptically transfer 1 ml sample suspension to 3 plates of 1419 Baird-Parker agar, distributing 1 ml of inoculum equitably to 3 plates (e.g., 0.4 ml, 0.3 ml, and 0.3 1420 ml). Spread inoculum over surface of agar plate, using sterile bent glass streaking rod. Retain 1421 plates in upright position until inoculum is absorbed by agar (about 10 min on properly dried 1422 plates). If inoculum is not readily adsorbed, place plates upright in incubator for about 1 h. Invert 1423 plates and incubate 45-48 h at 35°C. Select plates containing 20-200 colonies, unless only plates 1424 at lower dilutions (>200 colonies) have colonies with typical appearance of S. aureus. Colonies of 1425 S. aureus are circular, smooth, convex, moist, 2-3 mm in diameter on uncrowded plates, gray to 1426 jet-black, frequently with light-colored (off-white) margin, surrounded by opaque zone and 1427 frequently with an outer clear zone; colonies have buttery to gummy consistency when touched 1428 with inoculating needle. Occasionally from various foods and dairy products, nonlipolytic strains of 1429 similar appearance may be encountered, except that surrounding opaque and clear zones are 1430 absent. Strains isolated from frozen or desiccated foods that have been stored for extended 1431 periods frequently develop less black coloration than typical colonies and may have rough 1432 appearance and dry texture. 1433 1434 2. Count and record colonies. If several types of colonies are observed which appear to be S. 1435 aureus on selected plates, count number of colonies of each type and record counts separately. 1436 When plates of the lowest dilution contain <20 colonies, these may be used. If plates containing 1437 >200 colonies have colonies with the typical appearance of S. aureus and typical colonies do not 1438 appear at higher dilutions, use these plates for the enumeration of S. aureus, but do not count 1439 nontypical colonies. Select > 1 colony of each type counted and test for coagulase production. Add 1440 number of colonies on triplicate plates represented by colonies giving positive coagulase test and 1441 multiply by the sample dilution factor. Report this number as number of S. aureus/g of food tested. 1442 1443 1444


38

E. Coagulase test 1445

Transfer suspect S. aureus colonies into small tubes containing 0.2-0.3 ml BHI broth and emulsify 1446 thoroughly. Inoculate agar slant of suitable maintenance medium, e.g., TSA, with loopful of BHI 1447 suspension. Incubate BHI culture suspension and slants 18-24 h at 35°C. Retain slant cultures at 1448 room temperature for ancillary or repeat tests in case coagulase test results are questionable. Add 1449 0.5 ml reconstituted coagulase plasma with EDTA (B-4, above) to the BHI culture and mix 1450 thoroughly. Incubate at 35°C and examine periodically over 6 h period for clot formation. Only firm 1451 and complete clot that stays in place when tube is tilted or inverted is considered positive for S. 1452 aureus. Partial clotting, formerly 2+ and 3+ coagulase reactions, must be tested further (4). Test 1453 known positive and negative cultures simultaneously with suspect cultures of unknown coagulase 1454 activity. Stain all suspect cultures with Gram reagent and observe microscopically. A latex 1455 agglutination test (AUREUS TESTTM, Trisum Corp., Taipei, Taiwan) may be substituted for the 1456 coagulase test if a more rapid procedure is desired. 1457

3 OF ETHNIC CURED MEATS - nmis.gov.phnmis.gov.ph/attachments/article/10/Code of RCPPHEC.pdf · 8...

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