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Nuts carry with them from the fields a wide array of microorganisms, many of which have their origin in soil. Some are contaminated with excreta from animals, birds, and insects. Various treatments are given nuts and nut meats in separating the nut meats from the shells. Most of these treatments lower microbial numbers in the nut meats. For example, sorting of lightweight pieces from the heavier nuts removes much of the dust that carries microorganisms. Flotation in water is used with pistachios to remove immature fruits and with pecans to remove fragments of shells. Blanching in hot water loosens pellicles from almonds and peanuts, and some nuts are salted in a brine solution. Treatments with water can remove microorganisms, but they can also elevate aw, and reuse of water results in increasing populations of microorganisms that can be spread to other nuts. Therefore, frequent changes of water are needed.

Low aw is the major limiting factor in preservation of nut meats; therefore, drying is required to prevent mold growth if harvested nuts are not sufficiently dry. Moisture content of tree nuts normally ranges from 3.8 to 6.7%. Thus, the aw is usually less than 0.70 and microbial growth does not occur (Beuchat, 1978). Microorganisms usually die during storage. High temperatures and aw just below the level sufficient for growth are factors that increase death rates of microorganisms (King and Shade, 1986).

Because of the wide variety of nuts, the different environments they come from, and the several treatments given them, types and numbers of microorganisms present on nuts vary widely. Counts range upward to several thousand per gram, and insect-damaged nuts carry more microorganisms than undamaged ones (King et al., 1970). Nuts harvested from orchards where farm animals have been kept have an increased likelihood of contamination with E. coli. However, neither tree nuts nor ground nuts are considered to be likely vehicles of pathogenic microorganisms. Treatments with propylene oxide, permitted on tree nuts but not on peanuts, destroys most of the residual microflora (Beuchat, 1973; USHEW, 1978). Roasting, a treatment given peanuts and some other nuts, destroys vegetative cells of microbes.

Mycotoxins, especially aflatoxins, are of concern because of the chance of mold growth on nuts that contain high amounts of moisture. Nut meats removed from refrigeration can have condensate form on them, especially when placed in areas of high humidity. If these nuts are not used soon and are stored at favorable temperatures, molds are likely to grow on them.

G. Confections and Bakery Products

Confections and baked goods are low in bacterial numbers and seldom carry pathogenic bacteria. Methods of preparation and very low aw greatly limit survival and growth of microorganisms. However, these ingredients are usually added after freezing so that any contaminants they carry are given no positively lethal treatment.

H. Eggs and Egg Products

The ice cream industry uses egg yolks primarily for their flavor in the manufacture of French vanilla ice cream (also known as frozen custard; 1.4% egg yolk solids required) and in parfaits. Egg yolk is used also as a source of emulsifying and stabilizing agents, because egg yolk contains a high amount of lecithin. Sorbets usually contain 2.5-3% egg white. Pasteurized egg yolk is commercially available in three forms, liquid, frozen, and dried, that are useful in manufacture of frozen desserts. Egg white is available in dry and frozen forms. It is also possible to break and separate yolks from albumen of fresh shell eggs; however, this is usually feasible and economical only in production of small batches of ice cream.

Addition of 10% sucrose to egg yolks is effective in preventing gelation that occurs during storage of frozen yolks. Gelation of frozen plain yolk occurs most rapidly at approximately -18°C. Sugar is usually added to both the liquid and frozen forms. Salt also prevents gelation of egg yolk and is effective at approximately 2% concentration, but the salty flavor is undesirable in frozen desserts, making the sugar form the product of choice if frozen yolks are used.

The interiors of shell eggs (eggs in the shell) are usually sterile (with the possible exception of harboring certain salmonellae) at the time of laying (Brooks and Taylor, 1955; Morris, 1989). However, the shells of eggs become contaminated with several thousand to millions of bacteria during laying, collection, and processing.

Normally, 10-20 days pass between the time an egg is contaminated and the time when there is a significant increase in bacterial numbers inside the egg. One reason is that little iron is available at the shell membranes and in the albumen, and most bacteria require iron for growth. Glycoproteins of the membrane fibers bind iron tightly. Ovotransferrin, a protein of the albumen (white), also chelates iron. Certain species of Pseudomonas produce an iron chelate, pyover-dine, that has been claimed to scavenge iron from ovotransferrin (Board and Tranter, 1995). Thus, they are able to overcome one of the major barriers to growth in egg albumen. Chemotaxis played a role in movement of Pseudomonas putida and Salmonella Enteritidis toward yolk surfaces (Lock et al., 1992). The chemical attractant was not identified.

Additional hurdles that microbes face in the albumen of the shell egg involve binding of biotin by avidin (Chignell et al., 1975) and of riboflavin by ovoflavoprotein (Clagett, 1971). Bacteria that require either or both of these vitamins would, therefore, be inhibited in albumen of the egg. Furthermore, the highly alkaline (pH 9.5) albumen contains lysozyme, an enzyme that can lyse the cell membrane of certain gram-positive bacteria. Once a bacterium has reached the yolk of the egg, inhibitors are of no effect and nutrients abound, so growth can proceed rapidly.

Fresh eggs are seldom used in ice cream except in small operations. Because of the relatively high risk of the presence of salmonellae on and in fresh eggs, it is important that egg breaking be done in a room separate from the freezing and filling rooms. Furthermore, all eggs must be pasteurized if they are added to a frozen dessert after the mix is pasteurized. The FDA reported three recalls of liquid whole eggs for contamination with salmonella bacteria in the 19971999 enforcement reports (FDA Enforcement Reports, 1997, 1998, 1999).

Micrococci are nearly always present on freshly laid eggs, but spoilage of shell eggs is nearly always caused by gram-negative rods, especially species of Pseudomonas and Proteus (Board and Tranter, 1995).

Samples of unpasteurized liquid egg from commercial egg breakers have been reported to range in aerobic plate count from 103 to 106/g (Froning et al., 1992). Although the number of salmonellae in unpasteurized liquid eggs is usually less than one per gram, the risk that these organisms may be present is significant. Recently, the incidence of contamination of eggs with S. enteritidis through transovarian infection has caused considerable concern. S. enterica sero-var Enteritidis, commonly known as S. Enteritidis, has adapted to survive in the hen's internal organs from which it is occasionally deposited into the contents of an egg. A conservative estimate of the average incidence of infection across the United States is 1:20,000 eggs (American Egg Board, 1999). Foodborne illnesses from S. Enteritidis have been on the decline in the United States since 1995.

Most manufacturers use pasteurized egg products, including liquid whole egg, frozen sugar egg yolk, or dried egg yolk. Approved pasteurization standards for egg products produce 6-8 logj0 reductions in numbers of Salmonella (Speck and Tarver, 1967; Shafi et al., 1970). All pasteurized egg products should meet the following microbiological limits: aerobic plate count, less than 10,000/g; coli-form count, less than 10/g; yeast and mold count, less than 10/g; and salmonellae, negative in 25 g.

Freezing reduces numbers of viable microorganisms in egg products (Winter and Wilkin, 1947). Although most species of bacteria survive freezing in some numbers, the major survivors of both pasteurization and freezing are Bacillus, Micrococcus, and Enterococcus (Wrinkle et al., 1950; Froning et al., 1992). Salmonella Oranienburg survived storage in frozen yolk (Cotterill and Glauret, 1972).

I. Coloring Materials

Coloring materials are often added to frozen dessert mixes after pasteurization;

therefore, it is important that colorants be free of pathogens and low in total numbers of microorganisms. The following are typical microbiological specifications for food, drug, and cosmetic (FD&C) dry powders, blends, granulars, and FD&C lakes and lake blends: aerobic plate count less than 1000/g; coliforms, less than 10/g; yeasts and molds, less than 100/g; E. coli or Salmonella, negative in 25 g. Most firms do not test each batch for microbial counts but are willing to arrange for batch certification by an independent laboratory.

Colors and lakes provide very limited nutrients for growth of microorganisms, and, when sold in the liquid form, they contain low concentrations of benzo-

ates as preservatives. When purchased in the powder or granular form, the water and containers used in hydrating them should be practically sterile. The water should be free of sources of nitrogen and energy that might enable microorganisms to grow. When rehydrated colorants are to be kept for several weeks, it is advisable to store them refrigerated.

J. Spices

Spices can carry widely varying numbers and types of microorganisms. Spore formers are especially prone to be present and to survive over long periods. Spices, like nuts, can be treated with ethylene oxide to reduce the microbial load. Furthermore, spices can be irradiated to kill microorganisms.

Cinnamon contains cinnaminic acid, a microbial inhibitor. However, dilution of cinnamon with ice cream mix greatly reduces this antimicrobial effect.

V. FROZEN YOGURT A. Composition and Properties

Consumers often choose to eat frozen yogurt because they expect that it will contain less lactose than ice cream containing a similar amount of fat, and because they expect some benefit from the viable bacteria contained in the yogurt. Therefore, it is important to consider how much lactose is fermented to lactic acid during preparation of the mix, how many viable cells reside in the product, and how much galactosidic (lactase) activity those cells retain.

Frozen yogurt has a composition similar to low-fat ice cream. However, there is no Standard of Identity for frozen yogurt. The labeling regulations based on content of milkfat are the same as for ice cream. The unique characteristic of frozen yogurt is that it contains cultures of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The shorter name, L. bulgaricus is usually used for the latter bacterium.

These two bacteria are typically grown together in skim milk fortified with 1-4% added nonfat milk solids. Therefore, the nonfat milk contains about 6.5% lactose, and about 1.2% of it is converted to lactic acid during fermentation. This fortified skim milk is heated to approximately 85°C for 5 min and cooled before inoculation. Temperature of incubation is high, approximately 42°C, so generation time and, consequently, incubation time are short. From 10 to 20% of the finished and cooled yogurt is added to the processed and aged base mix at the time flavoring and coloring agents are added. Freezing follows.

It is also possible to add the yogurt culture to the base mix, which is then incubated until the titratable acidity, expressed as lactic acid, reaches approximately 0.30%. However, this process involves cooling the mix after pasteurization to the incubation temperature, then completing the cooling of the full batch, and holding it to permit aging. Therefore, time of production is longer and the capacity of the fermentation tank must be larger than with the previously described method.

The product is frozen in the same way as ice cream, and the overrun is typically in the range of 70-100%. Freezing kills many of the streptococci and lactobacilli of the yogurt culture. Sheu and Marshall (1993) observed that numbers of viable L. bulgaricus of two strains decreased approximately 45 and 90% during the continuous freezing of a simulated frozen yogurt mix. Viable cell numbers decreased approximately 5% more during storage at -20°C for 2 weeks after freezing. However, when the same two cultures were entrapped in beads (average diameter <18 |m) of calcium alginate gel, viable counts were approximately 45% higher than those of the nonentrapped cultures (Fig. 2). Cells of the strain of L. bulgaricus that were most susceptible to freeze damage were much larger than those of the smaller strain, suggesting that stresses of freezing are more damaging to large than to small cells.

Researchers have shown that exopolysaccharide (capsules) on bacteria renders cells comparatively resistant to thermal and physical shock (Robinson, 1981). Hong (1995) isolated three nonencapsulated mutants of S. thermophilus

Figure 2 Numbers of survivors among Lactobacillus delbrueckii ssp. bulgaricus enrobed in calcium alginate. (From Sheu and Marshall, 1993.)

and compared them with the encapsulated parental strain for abilities to withstand freezing under a variety of conditions. The parent and mutant strains did not respond differently when frozen without agitation. However, freezing to -7°C with agitation in a batch freezer and hardening to -29°C resulted in survival of 28% of the encapsulated and only 17% of the nonencapsulated strains (Fig. 3). Early log phase cells were more sensitive to freezing than late log phase or stationary phase cells. Cell viability after batch freezing was unaffected by (a) culture growth temperatures between 40 and 45°C, (b) fat content between 5 and 14%, or (c) neutralization of the acid produced by the cells during growth in skim milk. S. thermophilus survived significantly better in reduced-fat ice cream frozen in a continuous freezer to 50% overrun than in the same mix frozen to 100% overrun. The added agitation and scraping of the freezer barrel walls needed to attain higher overrun may have been responsible for the lowered rate of survival. Additional oxygen whipped into the mix might have increased cellular exposure to free radicals and thus increased the death rate. However, no significant difference was found between numbers of survivors when the gas

Figure 3 Numbers of survivors among encapsulated and nonencapsulated strains of Streptococcus thermophilus subjected to freezing in nonfat ice cream mix by a batch freezer. (From Hong, 1995.)

whipped into the ice cream was nitrogen or air. Storage of the frozen ice cream at —23 or -29°C resulted in significantly more survivors than storage at — 17°C.

B. Probiotic Nature

Although it was 1908 when Eli Metchnikoff suggested that certain bacteria in the human intestine could prolong the life of persons who consumed them in their foods, only recently have food microbiologists coined the term probiotic and have selected specific bacteria to add to foods as dietary adjuncts. The inference of the word probiotic is that a microorganism confers a positive effect on a biological entity, most importantly on human life. Most bacteria thought to have a probiotic effect are part of the natural microflora of the human intestine. Many of them are also useful as starter bacteria in food fermentations. Prebiotic is a term coined to describe substances needed to support the growth of probiotic microorganisms.

Benefits to consumers of fermented dairy foods and of those to which dietary adjunct bacteria are added include the following: (a) improved nutritional qualities (synthesized vitamins and enzymes as well as hydrolyzed proteins), (b) competitive exclusion of infective bacteria, (c) production of antibacterial substances (Shahani et al., 1977), (d) enhanced antibody production, (e) moderated response to endotoxin, and (f) anticarcinogenic activity.

Humans influence the nature of the intestinal microflora in several ways. Salivary, gastric, and intestinal secretions, bile, and mucus provide selective environmental factors. The stomach is strongly acidic, but pH increases as food moves to the distal end of the large intestine. Intestinal motility moves both food and microorganisms along the gastrointestinal tract, expelling billions of bacteria daily. Oxidation reduction potential is also a selective force. In general, the greater the distance intestinal contents travel from the stomach, the higher their microbial numbers.

Fermented dairy foods usually contain viable cells of the bacteria used as starter. Commonly used starter cultures contain lactococci, streptococci, lactoba-cilli, or leuconostocs. Some species of these genera have been shown to affect consumers favorably.

Frozen yogurt is the most popular dessert made from fermented milk. Most manufacturers produce frozen yogurt by adding 10-20% of plain yogurt to a pasteurized low-fat ice cream mix. Flavoring is then added just before the mix is frozen. Assuming 5 X 108/g of viable S. thermophilus and L. bulgaricus in the plain yogurt and addition of 20% yogurt to the mix, the number of yogurt bacteria in the mix before freezing would be 108/g. If freezing were to kill 50% of the yogurt bacteria, the viable number remaining would be 5 X 107/g. This large number of viable cells may provide benefit to consumers. Lopez et al.

(1997) stored three batches of commercial frozen yogurt at -23°C for over 1 year. The numbers of lactic acid bacteria, which exceeded 107/g initially, decreased only slightly during the storage period. A strong correlation (r2 = 0.62) existed in 11 brands of frozen yogurt between P-galactosidase activity and numbers of lactic acid bacteria (Schmidt et al., 1997).

Additionally, frozen desserts can be used as carriers of dietary adjuncts. Modler et al. (1990) used ice cream as a carrier for three species of bifidobacteria. At the end of 70 days of storage at -17°C, viable counts of these bacteria had decreased only 10%. Bifidobacteria have been receiving major attention as potential dietary adjuncts. These anaerobic, nonmotile, nonsporing, gram-positive, bifurcated (y-form) or curved rods produce acetic acid and L(+) -lactic acid as they ferment sugars. They comprise nearly 100% of the microflora in the stools of healthy breast-fed infants but only 30% to 40% of stool flora of formula-fed infants (Jao et al., 1978). As humans age, the percentage of bifidobacteria in stools decreases to low values. Their growth can be stimulated by oligosaccharides (Gyorgy et al., 1974), including P-linked N-acetylglucosaminides (Zilliken et al., 1955), glycoproteins (Bezkorovainy et al., 1979), and cysteine-containing peptides of kappa-casein (Poch and Bezkorovainy, 1991). Therefore, some foods are being supplemented with such ''prebiotic'' substances with the intention of enhancing growth of bifidobacteria in the human intestine.

Hekmut and McMahon (1992) fermented a representative ice cream mix with L. acidophilus and Bifidobacterium bifidum and then froze it in a batch freezer. Counts of L. acidophilus dropped from 1.5 X 108/g immediately after freezing to 4 X 106/g after 17 weeks, whereas those of B. bifidum dropped from 2.5 X 108/g to 1 X 107/g during the same period. Coincidentally, P-galactosidase activity dropped by about 25%. Freezing caused a loss in viable cell numbers of 0.7-0.8 logi0 in ice cream inoculated with four strains of probiotic bacteria (L. reuteri, L. acidophilus, L. rhamnosus, and B. bifidum). However, during 1 year of frozen storage, counts did not drop significantly and all remained above 106/g (Hagen and Narvhus, 1999). Incorporation of glycerol in the ice cream did not improve survival. In another study (Ravula and Shah, 1998), 10 strains of S. thermophilus and 7 of L. bulgaricus along with probiotic bacteria (13 strains of L. acidophilus and 11 strains of bifidobacteria) were screened for abilities to survive freezing at - 18°C when the pH was 4.5 or 4.0 and sucrose levels were 8 and 16%. Counts of the yogurt bacteria decreased about 1 logj0 during the first 3-5 weeks and then remained fairly constant. However, probiotic strains varied widely in response, with some losing up to 6 log10 cycles in numbers of recoverable cells.

Another popular dietary adjunct that may be added to frozen desserts is L. acidophilus. Certain strains of this bacterium were reported to assimilate cholesterol in a laboratory medium (Gilliland et al., 1984) as well as to lower serum cholesterol in rats (Grunewald, 1982). It is important that bacteria added to foods for probiotic effects be able to survive the effects of low pH and bile and to attach to and grow in a niche of the intestinal tract.

Each of the lactose-fermenting bacteria is a potential carrier of P-galactosi-dase. If these bacteria survive through the stomach and resist lysis by bile acids and enzymes, they may be permeated by lactose molecules. Intercellular P-galac-tosidase can then hydrolyze lactose to glucose and galactose so it can be absorbed through the human intestinal cell wall. Thus, symptoms of lactose malabsorption, a common malady among persons of Asian and African descent, can be reduced or eliminated.

VI. SHERBETS, SORBETS, AND ICES

Whereas sherbets contain 2-5% total milk solids, neither sorbets nor ices contain milk solids. All three product groups are high in sweetener; contain fruits, fruit juices, or fruit flavoring; and are generally acidic. Sherbet mix of typical composition can be made by adding one part of ice cream mix to four parts of water ice mix. Because sherbet contains milk solids, it must be pasteurized. A product called yogurt sherbet is defined in the California Food and Agricultural Code as having an acidity of 0.6% calculated as lactic acid and a yogurt content of not less than 40%.

Water ices typically contain 20-30% sugar, 0.35-0.5% citric acid, fruit flavoring, gum stabilizer, and water. Sorbet is a frozen fruit product that can be considered to be an ''upscale'' version of Italian ice (water ice). White tablecloth restaurants often serve it as an intermezzo between the appetizer and the main course. There is no federal standard for the product. It usually contains 30-50% fruit or fruit juice, 30% sugar, 2.6% egg white solids and pectin, modified cellulose, and/or gum stabilizer. At least one company has produced a chocolate sorbet. Overrun is 20% or less. Because it is expected to contain no milk ingredient, persons who suffer allergies to components of milk consider it to be safe to eat. However, since it is usually made in equipment used also to make ice cream, there is a risk that traces of milk proteins may enter a sorbet. This happened in Rochester, MN, when a 3-year old boy consumed 4-6 oz of lemon sorbet (Lao-prasert et al., 1998). The quantity of protein ingested was only 120-180 |g, but symptoms of itching throat, facial angioderma, and vomiting were experienced within 20 min of consumption.

Water ices and sorbets may not be required to be pasteurized. Their very low pH restricts growth of microorganisms to yeasts and molds. Furthermore, mixes are commonly prepared immediately before freezing, thus limiting the potential for microbial growth. They remain susceptible to contamination from ingredients, equipment, personnel, and the environment. Acid-tolerant bacteria, especially spores, can survive in them but will have little opportunity to grow.

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