D CONTROL (dough)

D CONTROL (dough)

Summary

Significant hazards

r Salmonella.

Control measures

Initial level (H 0 )

r Use pasteurized ingredients such as eggs and milk. r Specify supplier control programs for other sensitive ingredients.

Increase (ΣI) r Control water usage, maintaining dry processing conditions. Reduction (ΣR)

r Subsequent baking to set dough structure destroys Salmonella species.

Testing

r Environmental monitoring for Salmonella. r Lactic acid bacteria for refrigerated dough. r Routine testing of other dough is not recommended. r Coliforms as indicators is of little value.

Spoilage

r Strict cleaning and disinfection of equipment and maintenance of dry conditions is essential to control potential spoilage organisms in refrigerated

dough.

Control measures. The microbial quality of doughs can be maintained by using ingredients (particu- larly flour) of good quality, and by maintaining good bakery sanitation. Moist environmental niches can readily support the establishment of harborage sites for salmonellae. The subsequent baking process achieves temperatures of ca. 90 ◦

C to set dough structure, which greatly exceeds lethality needed to destroy Salmonella species. Refrigerated doughs should have a specified shelf-life so that they can be removed from retail display before they spoil. The quality of sourdough breads can be maintained by careful adherence to traditional procedures or by the use of pure cultures.

Testing. Manufacturing environmental monitoring for salmonellae is useful to prevent establishment of harborage sites, focusing on wet or moist locations, such as condensation, and accumulated static material in equipment. Surveillance for lactic acid bacteria is useful in refrigerated dough products that support lactic growth. Routine testing of other dough is not recommended. Use of coliforms is of little value as an indicator because of the natural variation in the incoming materials and lack of processing steps to reduce levels.

VI Breads

A Effects of processing on microorganisms Standard bread dough.

A typical bread contains wheat flour, water or milk, salt, fat, sugar and yeast (usually Saccharomyces cerevisiae). Ingredients are mixed; the dough may then be permitted to rise (ferment) for several hours at 24–29 ◦

C, before being divided, shaped and finally proofed (fermented) before baking. During this period, which may last as long as 20 h, yeast enzymes ferment sugars and produce carbon dioxide in the dough.

MICROORGANISMS IN FOODS 6

For economic reasons, fermentation time has been reduced to the point where primary fermentation is reduced or eliminated in some products. In conventional bread production, the properties of bakers’ yeast are fully utilized, but with high-speed production, some of the rheological functions of the yeast are replaced by high-intensity mixers. As insufficient time elapses for metabolic reactions necessary for flavor development, breads produced by these processes lack the flavor that only full microbial action can contribute (Sugihara, 1977). At the end of fermentation, bread doughs contain very high numbers of yeasts, and a wide variety of other microorganisms acquired from ingredients. Yeasts are the principal contributors to flavor. Bacteria (usually Lactobacillus species) contribute flavor in breads with fermentation periods >8 h (Kent-Jones and Amos, 1957). Lactic acid bacteria produce short-chain fatty acids, which probably contribute to good flavor in small amounts, but would give undesirable flavors and aromas in large amounts (Robinson et al., 1958).

During baking, the internal temperature of the loaf closely approaches l00 ◦

C. When the crumb reaches

C, the optimal baking time has been reached (Stear, 1990). All vegetative microbial cells have been destroyed during this process. Subsequent fungal spoilage problems arise from aerial contamination after baking, from the slicing machine and from cooling and wrapping equipment. The level of fungal

spores in the air in bread manufacturing plants has been reported as 100–2500 spores/m 3 (Knight and Menlove, 1961). However, modern facilities have much greater control over spore numbers. Only spore-forming bacteria survive baking. If the ingredients of the dough are heavily contaminated with spores of the “rope” species, B. subtilis, some survival is to be expected during baking. The sources of the spores are multiple, but the highest inoculum may be from the equipment that has been contaminated by previous batches of bread.

Salt-rising bread. This product is unique in that the leavening agent is Clostridium perfringens rather than yeast (Robinson, 1967). Although this species can cause foodborne gastroenteritis, it is harmless when used as a leavening agent because it dies during baking. Food poisoning has never been attributed to salt-rising bread (Dack, 1961). B. cereus can also be used for the same purpose (Goepfert et al., 1972).

Soda crackers. The formulation for soda crackers is similar to that of bread except that baking soda is added. Yeast (Sacc. cerevisiae) is a principal ingredient, but since the industry began in 1840, man- ufacturers have relied on chance contamination by Lactobacillus species for acid formation in a 24 h fermentation period. Later investigations revealed the predominance of Lactobacillus plantarum and the secondary role of Lb. delbruckii and Lb. leishmannii in this function. Pure culture starters with these species reduce the fermentation period to about 6 h, maintain better quality and permit controlled variations in flavor (Sugihara, 1977, 1978a,b).

For crackers and similar biscuits, a continuous fermentation process has been developed using a mixed yeast and Lactobacillus culture. The bacteria used are Lb. plantarum, Lb. fermentans, and Lb. casei (Fox et al., 1989).

Sour dough bread. The traditional method for sourdough bread uses a “starter” built up from the previous batch every 8 h. It requires a high proportion of active starter sponge for each batch of new starter (about 40%) followed by a fermentation period of 7–8 h at 27 ◦

C. The initial pH is 4.4–4.5, and the final pH 3.8–3.9. The dough requires a starter that makes up about 11% of the final dough mix. This is followed by a similar 7–8 h period of fermentation with a similar pH drop. The very high proportion of starter sponge contributes a massive inoculum and ensures a highly acid environment that contains a substantial amount of acetic acid. The low pH and the acetic acid prevent growth of spoilage microorganisms and loss of the starter (Kline et al., 1970).

417 In San Francisco sourdough bread the sourdough yeast is Sacc. exiguus (Torulopsis holmii), and

CEREALS AND CEREAL PRODUCTS

the heterofermentative bacterium is Lb. sanfrancisco (Sugihara, 1977). Sacc. exiguus does not ferment maltose, and Lb. sanfrancisco ferments only maltose. Thus, the two microorganisms do not compete for the same carbohydrate, a fact that probably contributes to the survival of both species in the starter. The yeast is unusually resistant to acetic acid (Sugihara et al., 1970). Excessive acidification produced by lactobacilli had a deleterious effect on dough rheology and the presence of yeast in the starter improved bread quality (Collar et al., 1994).

Sour rye bread. This was first mentioned in 800 BC, and since the fifth century BC dried starters made up of cakes of fermented whole grain have been available to bakers. The souring microorganisms are Lb. plantarum, Lb. brevis, and Lb. fermentans. Lb. sanfrancisco, Lb. pontis, Lb. amylovorus, Lb. reuteri, Lb. johnsonii, and Lb. acidophilus may also be found in some cultures (Vogel, 1997). Lactic and acetic acids are the principal compounds formed. The technological importance of the starter varies with

the pH, acidity, and lactic acid:acetic acid ratio. Only Lb. brevis var. lindneri was found to give ad- equate acidity over a wide range of processing conditions (Spicher, 1982). The addition of yeast speeds the fermentation process. Numbers of lactic acid bacteria increase and yeasts decrease

with increased fermentation times, with populations in the range of 10 8 and 10 7 cfu/g, respec- tively (Rosenquist and Hansen, 2000). This bread is generally made with self-perpetuating starter sponges, but pure cultures or the previously mentioned cake, are available commercially (Sugihara, 1977).

Italian panettone. In Italy, panettone dough is the basis for Christmas fruitcake, columba (Easter cake), breakfast rolls and snack cakes. Its production is similarly based on a yeast (Sacc. exiguus) and one or more species of bacteria (Lb. brevis, Enterobacter and Citrobacter species). The madre or mother sponge has been perpetuated for centuries in very clean surroundings by a special staff (Sugihara, 1977). The preparation procedure is strikingly like that of sourdough French bread.

Idli. This is an Indian fermented bread, which together with similar products in other Asian and Middle Eastern countries, is prepared from rice and black gram mungo (Phaseolus mungo), a legume. The ingredients are soaked in water, combined and permitted to ferment overnight, then steamed and served hot. Leavening and acidification are primarily accomplished by Leuc. mesenteroides, with Streptococcus faecalis, and Pediococcus cerevisiae playing secondary acidifying roles (Mukherjee et al., 1965). Idli is unique in that the leavening action is solely from the activity of a lactic acid bacterium (Leuc. mesenteroides).

B Spoilage Bread, has a short shelf-life owing to staling or mold growth. The interior or crumb has an a w about

0.94–0.95, whereas the crust has an a w <

0.70. Fungal growth is relatively slow, so that in dry climates, the surface of a slice of bread may dry before fungal growth is evident. In humid conditions or on wrapped bread, however, fungal growth occurs in a few days.

In a 2-year study of German packaged sliced breads made from rye, wheat or blends, 437 samples were stored at 25 ◦

C and 70% relative humidity for 7 days, and fungal growth was analyzed (Spicher, 1984a). More than 65% of the samples showed visible growth. Predominant was Pen. roqueforti (85% of isolates); other Penicillium species and Aspergillus species made up the remainder. Breads which had been heat treated showed no spoilage, and only Pen. roqueforti was able to grow on breads preserved with propionic or sorbic acid.

MICROORGANISMS IN FOODS 6

C for 10 weeks yielded similar results (Hartog and Kuik, 1984). Initial fungal counts were low, with >90% of samples having counts <10 3 /g. Shelf-lives of samples from smaller bakeries were shorter (42% spoilage within 6 weeks versus 5%), attributed to higher pH and lower levels of preservatives used. Pen. roqueforti was again the dominant spoilage species. Its resistance to weak acid preservatives is well documented (Engel and Teuber, 1973; Pitt and Hocking, 1997).

A study of 204 samples of Dutch rye bread stored in plastic bags at 24 ◦

“Chalky mold” is also an important cause of bread spoilage in Europe (Spicher, 1984b). This spoilage is due to Endomyces fibuliger (32% of isolates), Zygosaccharomyces bailii (24%), Hyphopichia burtonii (20%); and Sacc. cerevisiae (10%). The lowest growth temperature for these species was 5 ◦

C. Calcium propionate (0.3%) delayed spoilage, whereas sorbic acid (0.05%) prevented it. Similar studies have been carried out on various bakery products (Spicher and Isfort, 1987). Of 150 samples of partially baked bread rolls or baguettes stored for 7 days at 25 ◦

C and 70% relative humidity, ∼60% showed mold growth, including 92% of those packaged in CO 2 , 62% of those without preservatives and 50% of those preserved with propionic acid, and also packaged under CO 2 . The predominant spoilage fungi were Pen. roqueforti (38%), Paecilomyces variotii (14%), Asp. niger (8%), Eur. amstelodami (6%), and Moniliella suaveolans (6%). Packaging under CO 2 may fail because of leaks in packaging material. The addition of vinegar or dilute acetic acid (5–8%, at the rate of 0.9–1.8 ml/100 cm 2 ) to bakery goods prevented growth of most fungi during storage (Spicher and Isfort, 1988). However, growth of Pen. roqueforti and Paec. variotii was prevented only by the addition of 10% vinegar at the higher application rate. Addition of ethanol (1.5% w/w) to the surface of baguettes or packing material also increased mold-free shelf-life (Doulia et al., 2000).

Rope is a bacterial spoilage problem of bread caused by B. subtilis (B. subtilis var. mesentericus) or

B. licheniformis. The most common source of contamination with these bacteria is flour and equipment that has been in contact with contaminated dough (Stear, 1990). Whole grain breads may also have higher spore counts than other products. The spores survive baking, and germinate and grow within 36–48 h inside the loaf to form a characteristic soft, stringy, brown mass with an odor of ripe cantaloupe. The bacteria are heavily encapsulated, which contributes the mucoid nature of the material. They also produce amylases and proteases that cause the breakdown of the bread structure. Conditions favoring the appearance of rope are: (i) a slow cooling period or storage >25 ◦ C; (ii) pH > 5; (iii) high spore level and (iv) moist loaf. The water activity inside a loaf is marginal for B. subtilis so that rope may appear in localized areas where the moisture content is high. Calcium propionate, good sanitation and good bakery practice keep it under control (Graves et al., 1967; Pyler, 1973).

Red bread is usually caused by Serratia marcescens, which may grow if the moisture level is high enough. Although this is uncommon today, the red color formation has been described as “bleeding bread” (Legan, 2000).

The chapatti, a disc of soft, pliable unleavened bread, is a common wheat food in India. In tropical areas where the temperature is 40–45 ◦

C, chapattis that are packaged in polyethylene pouches, where the equilibrium relative humidity reaches 90–95%, spoil after ∼7 days owing to the growth of aspergilli (Kameswara Rao et al., 1964, 1966).

The tortilla, a flat maize pancake, is the staple cereal food in all of Central America. Dry maize is cooked with limestone, soaked in water for about 14 h, ground into a wet pasty flour, rolled, and patted into a pancake and then cooked for 4–5 min on a stove top or coals. The uncooked tortilla often has high levels of a general microflora that are largely destroyed during cooking. Tortillas are

a very rich, moist medium for microbial spoilage, which will take place within 24 h under tropi- cal conditions (Capparelli and Mata, 1975) and generally <8 days at room temperature. Addition of preservatives, such as propionic acid or fumaric acid, or refrigeration extend shelf-life (Haney, 1989).

CEREALS AND CEREAL PRODUCTS

C Pathogens and toxins Some of the molds that grow on bread are capable of producing mycotoxins (Robinson, 1967; Bullerman

and Hartung, 1973). The most important of these is Pen. crustosum, which has several times caused toxicosis in dogs owing to penitrem A production in moldy bread or hamburger buns (Arp and Richard, 1979; Richard et al., 198l; Hocking et al., 1988). However, this is not a problem for humans who usually will not eat moldy or stale bread. Samples of bread stored at 25 ◦

C and 70% relative humidity were also examined for mycotoxin formation (Spicher, 1984c). Of 110 moldy samples studied, 11 contained citrinin (<5 µg/kg), four contained ochratoxin (<8 µg/kg), and five contained zearalenone (<5 µg/kg). These levels are not cause for concern. Aflatoxins, patulin, sterigmatocystin, and penicillic acid were not detected.

In canned non-acid bread, previously inoculated with spores of Cl. botulinum, toxin was formed on incubation if the a w of the bread was ≥0.95. This a w is equivalent to a moisture content of about 40%. As long as the water content did not exceed 36%, no toxin was formed (Wagenaar and Dack, 1954; Denny et al., 1969). There have been no reports of botulism from this product.

Acid breads, whose pH is ≤4.8, are safe from botulism, even when the moisture content is 40% (Wagenaar and Dack, 1954; Ingram and Handford, 1957; Weckel et al., 1964).

A substantial trade exists in packaged “part-baked” breads. One concern is with the possible survival of the partial cook by Cl. botulinum and other spores. However, retail shelf-life as long as 90 days without refrigeration are common. Growth of spores is prevented by a combination of reduced a w and reduced pH.

Tortillas are often prepared under very primitive conditions, especially in rural areas of Central America. Fortunately, treating the maize with lime and cooking the tortillas destroys aflatoxins that might be present in the original maize, a fact of major importance to millions of people in Latin America (Ulloa-Sosa and Schroeder, 1969). Cooking also destroys bacterial pathogens; however, tortillas are usually immediately recontaminated, and because of their high moisture content and the prevailing warm temperatures, they can support the growth of E. coli, Staph. aureus, B. cereus, and possibly other bacterial pathogens. If tortillas are not consumed promptly after cooking, they may be responsible for widespread disease outbreaks (Capparelli and Mata, 1975).

Kaur (1986) reported that B. cereus levels in flour were low and only occasionally >10/g. The organism was not isolated from baked 400 g loaves made with dough containing ca.10 4 /g, but did survive in 800 g loaves. It is not likely that the low levels of B. cereus that occur naturally will survive the baking process and grow in baked bread.