D CONTROL (dry soup and gravy mixes)

Summary

Signiﬁcant hazards

r Salmonellae; spp., others such as Cl. perfringens and B. cereus may be signiﬁcant depending on the ﬁnal use of the soups or preparations.

Control measures

Initial level (H 0 ) r Depending on the composition of the products, low levels of pathogens are likely to be present if untreated herbs and spices used as ingredients. Low

levels are ensured by adherence to GHP and the application of HACCP. Reduction (ΣR)

r For dry-mixed products, no reduction during processing. In the case of heat-processed products, killing is achieved, the extent being dependent

on the initial levels.

Increase (ΣI) r In dry products, pasty or liquid products with very low water activity, low pH, or high salt content increase is only caused by contamination from the

processing environment.

Testing

r Testing is only done to verify compliance to pre-established

speciﬁcations, e.g. according to AIIBP (1992).

Spoilage

r Spoilage with molds is possible, in particular for pasty and liquid products

at water activities >0.7.

III Soy sauces

A Deﬁnition Soy sauces are important seasonings in Eastern Asia. The prototype soy sauce originated in China

∼ 2000 years ago, and has not been substantially changed since it was brought into Japan. Soy sauces are salty liquid seasonings ranging from amber to dull brown color, produced from soy beans/defatted soy grits with or without wheat, barley, and/or rice by a two-stage fermentation, an aerobic solid-state fungal fermentation is followed by an anaerobic mixed lactic-yeast, submerged fermentation in a strong salt brine.

B Important properties There are two main types of soy sauces, the Chinese and the Japanese, which differ in organoleptic

qualities and composition, depending on the proportion of raw materials and types of fermentations. Soy sauces produced in various South-East Asian countries most closely resemble the Chinese type and their production varies from one region to another. The salt content of Japanese style soy sauces is 16–18% and the water activity is as low as 0.80 or below. Chinese and various South-East Asian soy sauces vary in salt content between 10% and 26%, except Indonesian “kecaps”, which has 6–7% NaCl. The pH of Japanese soy sauces is 4.7–5.0 (Yokotsuka, 1986b). The pH values of some commercial brands of soy sauce from the Philippines were reported to be in the range of 5.2–6.1 (Soriano and Pardo, 1978). The pH values of various types of soy sauces from Thailand and Malaysia range from 4.0–5.5 (Merican, 1978; Sundhagul et al., 1978).

375 The Chinese style soy sauces are generally prepared from soybeans with little or no wheat, and have

SPICES, DRY SOUPS, AND ORIENTAL FLAVORINGS

very low alcohol content. Good quality Chinese “chiang-yiu” is dark in color and has a high specific gravity, viscosity, and high nitrogen content. The Japanese soy sauces are produced from soybeans and roasted wheat in different proportions, and their alcohol contents are higher (up to ∼3%) than those of Chinese styles. Their viscosity and nitrogen content is lower than those of the Chinese type, but their amino acid content may be higher. There are five main types of Japanese soy sauce (called shoyu): koikuchi (produced in the largest proportion), usukuchi, tamari, shiro, and saishikomi. Koikuchi is reddish-brown and has a strong flavor. Usukuchi is light and has a maximum total nitrogen content of 1.2%. Both are made from approximately equal parts of soy beans and wheat kernels. Koikuchi and usukuchi type shoyus have higher alcohol (1–3%) and higher lactic acid (0.8–1.5%) content. The tamari-type shoyu is essentially a Chinese type soy sauce made primarily of cooked soybeans and small proportions of wheat or barley flour. It contains less alcohol (<0.5%) and somewhat less lactic acid (0.5–1.2%) than the previous types. Shiro shoyu is made mostly from wheat kernels with a very small amount of soybeans and is very light. Shaishikono shoyu is prepared from the product of the solid-state fungal fermentation (koji) and unpasteurized shoyu instead of salt water. It is very dark and high in solid content (Yokotsuka, 1986a,b).

C Methods of processing and preservation Koikuchi-type Japanese soy sauce. The industrial production of the Japanese-type soy sauce (koikuchi

shoyu) can be divided into the following stages (Yokotsuka, 1986a; Hose, 1992): r Treatment of raw materials.

r Koji production. r Moromi preparation and fermentation (aging). r Pressing of aged mash. r Pasteurization and reﬁning.

These are illustrated in Figure 7.1. Treatment of raw materials. The soybeans/defatted grits are soaked in water and cooked with steam

under pressure. This process greatly inﬂuences the digestibility of soy protein and, therefore, affects both the koji and moromi stages (see below) of fermentation. The wheat kernels are roasted brieﬂy at 160–180 ◦

C, and then coarsely crushed. Koji fermentation. The cooked soybean-roasted wheat mixture is inoculated with a starter culture

of Asp. oryzae and/or Asp. soyae, and spread in 30–40-cm thick layer on large perforated stainless steel plates in the koji room. This mass is aerated by moisture-controlled air for 2–3 days at ∼30 ◦

C, allowing the molds to grow throughout the mass to develop their enzymes necessary to hydrolyze the proteins and starch of the raw materials (Tochikura and Nakadai, 1988). This mold-cultured material is called koji.

Moromi stage. Koji is mixed with cold salt water with 22–23% salt content to a ﬁnal volume of 120–130% of that of the raw materials. The mash, called moromi, is kept in fermentation tanks for 4–

8 months, depending on the temperature, and occasionally agitated by bubbling compressed air through the moromi, to promote microbial growth. In moromi undergoing normal fermentation, lactic acid bacteria grow ﬁrst and produce lactic acid and acetic acid to lower the pH, enabling the main fermentation yeast to grow. This is followed by a “maturation” fermentation by a second group of yeasts. The order and extent of growth of these three groups of organisms and their balance are crucial as they affect the quality of the soy sauce (Noda et al., 1980).

MICROORGANISMS IN FOODS 6

Figure 7.1 Flow sheet for manufacture of Koikuchi-type soy sauce.

During this fermentation period, koji mold enzymes hydrolyze most of the proteins to amino acids and oligopeptides, the starch remaining after koji production is also hydrolyzed. A considerable proportion of sugar is fermented to lactic acid, acetic acid, and alcohol by lactic acid bacteria and yeasts. Current practice in the Japanese industry uses pure cultures of Pediococcus halophilus and Zygosaccharomyces rouxii, which are added to the mash. Initially, the pediococci decrease the pH from 6.5–7.0 to 4.7–4.9. The lactic acid fermentation is gradually superceded by yeast fermentation.

Soy sauce yeasts grow vigorously at pH 4.0–5.0 in the presence of high brine concentrations (Ohnishi, 1957). However, in practice, to ensure sufﬁcient yeast growth and good fermentation, the yeasts (at concentrations of ∼10 6 cfu/g of moromi) are added when the pH of the moromi is between pH 5.0 and

5.2 (Matsumoto and Imai, 1981). This is followed by vigorous agitation of the moromi using compressed air blown through it at appropriate timing to enhance yeast growth and, hence, fermentation. Soy sauce yeasts include two osmophilic groups, the main fermentation yeasts such as Zygosaccha- romyces rouxii and maturation yeasts such as Candida versatilis and Can. etchellsii. Z. rouxii grows at water activity between 0.78 and 0.81 and in media containing 24–26% salt (Yoshii, 1979). The water activity of moromi with 17–18% salt is >0.80, making it ideal for Z. rouxii to grow and effect fermentation. The maturation yeasts, Can. versatilis and Can. etchellsii, also have high salt tolerance and are capable of growing in moromi with water activity of 0.78 or salt content of 26% (w/v) (Yoshii, 1979).

Pressing. The fermented aged moromi mash is filtered under high pressure through nylon cloth filters to separate into filtrate and cake. The filtrate is stored in tanks to allow sedimentation and separation of the oil layer. Both the sediments and oil layers are removed to obtain raw soy sauce.

377 Pasteurization and reﬁning. The raw soy sauce is adjusted to obtain a standard concentration of

SPICES, DRY SOUPS, AND ORIENTAL FLAVORINGS

brine and total nitrogen, then pasteurized at 70–80 ◦

C for 20–30 min. Alternatively, the raw sauce can

C for a few seconds to affect killing of bacterial spores that may be present in the raw sauce. After pasteurization, the sauce is stored (without stirring) at 50–60 ◦

be subjected to a high temperature short time treatment of 120 ◦

C for 3–4 days to obtain better color and aroma. Further, sedimentation will occur and the clariﬁed sauce is then packed in appropriate containers or further treated depending on the types.

Packaging and shelf life. The industrially produced clarified sauce is bottled or dehydrated by various drying processes, usually after pasteurization. Refined and pasteurized, preservative-free “shoyu” packaged in fiber drums has a shelf life of 1 year at room temperature when stored unopened. If stored opened at room temperature, it has a shelf life of about 2 weeks. The Japanese Soy Sauce Association’s official method requires a shelf life of 3 years in glass bottles and 1.5 years in plastic containers. During storage the color of soy sauce will gradually darken due to the amino-carbonyl reaction between amino acids and sugars. The flavor will also deteriorate.

Traditional soy sauces produced in China and South-East Asian Countries. The fermentation of soy sauce in China and Asian countries other than Japan traditionally employs “natural” fermentations using fungi in the environment from previous batches. In South-East Asia, the initial process involves the breakdown of protein and starch as a solid-state fermentation of boiled soybeans mixed with wheat ﬂour and spread on bamboo trays. The traditional Korean fermentation involves kneading steamed soybeans into brick-like blocks and leaving it at ambient temperature to allow a variety of fungi to grow. The fermentation for these types of soy sauce depends on chance contamination of the necessary molds. Usually Aspergillus spp. and other molds are present on the bamboo tray used for a prior fermentation. The microorganisms commonly present include Asp. oryzae and other aspergilli; Rhizopus oligosporus, R. oryzae, and other Rhizopus spp.; Mucor spp.; and occasionally Penicillium spp. The use of traditional fermentation techniques is increasingly being replaced by use of pure cultures of Asp. oryzae to inoculate the bean–ﬂour mixture in modern sterile koji rooms, or in stainless steel trays.

The brine phase (moromi) stage of the Chinese-type soy sauce fermentation depends on halotolerant bacteria and yeasts that are naturally present. Species reported to be present in the submerged brine fermentation are B. subtilis, B. pumilus, B. citreus, B. licheniformis, Sarcina maxima, and Z. rouxii (Korea); Pediococcus halophilus, Ped. soyae, Pichia spp., Candida spp., and B. licheniformis (Malaysia); Hansenula anomala, H. subpelliculosa, and Lb. delbrueckii (Philippines); Saccharomyces spp. and Lac- tobacillus spp. (Singapore); and Ped. halophilus, Staphylococcus spp., and Bacillus spp., (Thailand). Unlike the Japanese method, these microorganisms are not generally added intentionally; the fermentation is dependent on the selection of desirable organisms from the environment by speciﬁc physical and chemical parameters present in the various phases of the fermentation process. The moromi stage for the South-East Asian and Korean sauces is left under the sun for ≥2 months to mature. South-East Asian soy sauces have a shelf life of at least 1 year. Generally, their shelf life depends mainly on their salt content and added preservatives. When a preservative is used, it is most often sodium benzoate (400–1000 mg/kg).

D Types of ﬁnal products Soy sauces or their dehydrated versions are used as natural seasoning agents to enhance the ﬂavor of

foods such as soups, meat and poultry, vegetables, and seafoods. They provide a meaty, hearty ﬂavor when added to various sauces, gravies, salad dressings, and other condiments.

A range of products can be produced using soy sauce as the main ingredient, and in many South- East Asian countries these fall under the generic name of soy sauce. Thick sauces in this area are

MICROORGANISMS IN FOODS 6

concentrated soy sauces prepared traditionally by evaporation in the sun, but nowadays the product is often thickened with caramel, molasses, and/or starch. Light or “white” sauces are prepared by diluting soy sauce with water to the required consistency, whereas sweet soy sauce such as Indonesian “kecap manis” is prepared from black soybeans (without wheat) in the same manner as the other South-East Asian soy sauces, with the addition of palm sugar and herb extract to the mature sauce, before ﬁltering, concentrating by heating, and packed.

Blended soy sauce, i.e. soy sauce blended with hydrolyzed vegetable protein, is produced in many South-East Asian countries. These products, as well as products using hydrolyzed vegetable protein as an ingredient such as hydrolyzed vegetable protein sauce, soups, gravy mixes, and bouillon cubes, were reported to contain 3-monochloropropane-1,2-diol (3-MCPD) in levels higher than that allowed in many countries (Hamlet, 1999; MAFF, 1999). 3-MCPD should not be present in naturally fermented soy sauce.

E Initial microﬂora The initial microﬂora of the raw ingredients of soy sauce, soybeans, wheat, barley, and rice, is described

in detail in Chapter 8 (Cereals and Cereal Products). Mesophilic vegetative microorganisms including coliforms are present, commonly in low populations, along with low numbers of spores of Bacillus spp. and Clostridium spp. Psychrotrophic bacteria and Actinomycetes are almost always present (ICMSF, 1980b, Vol. 2, p. 672). Soy grits also can contain thermophilic bacteria, including spores and fecal streptococci, coliforms and E. coli (Hose, 1992, Table 7.8).

In traditional technologies, the main source of contamination during koji fermentation is the bamboo fermentation tray. The trays are never washed and are depended upon to inoculate new batches. Another source of contamination is the crude salt that is used in the second stage of fermentation. It can be a source of halophilic/halotolerant microorganisms that are required for fermentation and aroma development.

F Primary processing Effects of processing on microorganisms. The heat treatments of raw materials of koji fermentation

eliminate most of their initial microﬂora which is then replaced by the natural microﬂora in the manufacturing environment or by the purposeful addition of starter cultures. In the industrial production of soy sauce, “tane koji”, the fungal starter for the soybean/wheat mixture, is produced by culturing selected strains of Asp. oryzae or Asp. sojae on either steamed, polished rice or a mixture of wheat bran and soybean meal (Yong and Wood, 1976; Beuchat, 1984). Microbiological changes during production of koji are illustrated in Figure 7.2 (Hose, 1992).

Microﬂora changes in moromi fermentation of shoyu mash are shown by Figure 7.3 (Tamagawa et al., 1975). The microorganisms which grow in the brine mash are limited to halotolerant lactic acid bacteria and yeasts such as Ped. halophilus (previously Ped. soyae) and yeasts including Z. rouxii, Torulopsis halophilus, Tor. nodaensis and Tor. halonitratophila (Ho et al., 1984), and Candida spp. such as Can.

Table 7.8 Ranges of bacterial counts in nine batches of soy grits a

Type of microbial count

log 10 cfu/g

Total mesophilic bacteria

Total thermophilic bacteria

Mesophilic spores

Thermophilic spores

Fecal streptococci

a Hose (1992).

SPICES, DRY SOUPS, AND ORIENTAL FLAVORINGS

Figure 7.2 Change in mold, total bacterial and mesophilic spore counts during production of tane koji. (From Hose, 1992.)

Figure 7.3 Change in microorganisms in Moromi-mash. Curve 1: Wild yeasts; 2: Staphylococcus spp.; 3: Bacillus spp.;

4: Pediococcus halophilus; 5: Zygosaccharomyces rouxii; 6: Candida versatilis.

versatilis and Can. etchellsii (Fukushima, 1989), which are tolerant to the high salt concentrations. Bacillus licheniformis and B. subtilis were also found growing in Malaysian soy sauce fermentation (Ho et al., 1984). Molds may appear on the surface of the mash, but they are believed to have no relation to proper fermentation at this stage or during aging (Yokotsuka, 1960).

The use of pure culture inocula at all stages of modern production of soy sauces has reduced the risk of carrying unwanted contaminants from one batch to the next, and shortened the fermentation time. Selection parameters of starter microorganisms for use in the fermentation of soy sauce include, among others, non-mycotoxigenicity of the Asp. oryzae/Asoyae strains selected. Pediococcus halophilus must produce lactic acid and other organic acids, the yeast strains should produce alcohol and/or desired ﬂavor substances in a high salt concentration brine (Sugiyama, 1984).

Contaminating microorganisms reported to be isolated from koji include Micrococcus, Streptococcus, Lactobacillus, and Bacillus (Fukushima, 1989). Most of these contaminants are not salt tolerant and are

MICROORGANISMS IN FOODS 6

thus not able to survive the brine fermentation. The initial acetic acid concentrations present during the shoyu-koji-making process aid to suppress the growth of non-acid tolerant contaminating bacteria of the koji substrate (Hayashi et al., 1979). Probably only the spores of contaminating bacilli can survive the brine.

Spoilage. In traditional products, non-pasteurized products or products with a low salt content, aspergilli, ﬁlm and pellicle-forming yeasts may cause spoilage (Roling et al., 1994). In low-salt (<15%) sauces, some types of spoilage bacteria may also grow unless the pH is low or preservatives (e.g. sodium benzoate) are present.

The principal contaminants of koji are coagulase-negative Staphylococcus spp. and B. subtilis (Chiba, 1977). The contaminating Staphylococcus spp. grow symbiotically with the koji mold, and the combi- nation becomes a problem when koji fermentation takes place at low temperatures (at or below 25 ◦ C), whereas B. subtilis grows in competition with the koji molds, especially at higher temperatures.

Wild salt-tolerant lactic acid bacteria may grow in soy sauce and produce biogenic amines such as tyramine and histamine (Uchida, 1982; Stratton et al., 1991). Some wild salt-tolerant lactic acid bacteria may produce ornithine by decomposing arginine in an abnormal fermentation, resulting in the accumulation of citrulline as an intermediate product. When such raw soy sauce is pasteurized, ethyl carbamate can be produced by the reaction between citrullin and alcohol (Matsudo et al., 1993).

A salt-tolerant wild yeast Z. rouxii var. halomembranis can grow in moromi that has progressed to the maturation process. This wild yeast is harmless to health, but has a high salt tolerance, and grows to form a membranous ﬁlm on the surface of moromi or soy sauce resulting in deterioration of the soy sauce aroma and ﬂavor.

Pathogens. There have been no reports of illnesses due to enteropathogenic microorganisms associated with soy sauces. Carry over of mycotoxins from contaminated raw materials does not appear to be a problem. Badly molded soybeans and grains are not used because of their impact on product quality. The strains of Asp. oryzae and Asp. soyae used by the Japanese industry do not produce aﬂatoxin, and testing them for production of other kinds of mycotoxins indicate no serious problems (Steinkraus et al., 1983). It is important to select strains that do not produce mycotoxins such as cyclopiazonic acid, kojic acid, β-nitropropionic acid or aspergillic acid. However, traditional fermentation does not use pure mold cultures, and a variety of fungi, including undesirable types, can be present. Hence, the possibility of low levels of mycotoxin contamination of traditional soy sauce cannot be ruled out.

Fermented shoyu was determined to have bactericidal activity for some enteropathogenic bacteria, including, S. Typhi-Shikata, Shigella ﬂexnerii, and Vibrio cholerae-inaba. Clostridium botulinum type

C (Steinkraus et al., 1983).

A and B spores inoculated into shoyu survived, but did not grow within 3 months at 30 ◦

G Control Under normal circumstances, pH, water activity (salt content), and the presence of competing harmless or

desirable microorganisms may prevent growth of undesirable microbes during and after the fermentation may of soy sauces and thereby ensure preservation during the primary storage in closed containers. The main control factor is high salt content. Pasteurization is becoming more widely accepted as a means of prolonging microbiological stability (Husin and Yeoh, 1989).

Important control points in the industrial production of soy sauce are as follows. Raw materials and their heat treatment. Soy beans/grits contain substantial populations of mesophilic

aerobic bacteria and mesophilic and thermophilic aerobic spores. Undesirable growth of these bacteria

SPICES, DRY SOUPS, AND ORIENTAL FLAVORINGS

be changed at least every 2–3 h, otherwise undesirable spore-forming Bacillus spp. can grow to high levels (Beuchat, 1984). Autoclaving soybeans after soaking should be monitored by time–temperature measurement, e.g. 1 h at 1 kg/cm 2 steam pressure (Steinkaraus et al., 1983). The water content of steamed soybeans should not be allowed to exceed 62% to prevent bacterial spoilage. The function of the wheat ﬂour is to coat the surface of the soybeans, thereby suppressing bacterial growth. Therefore, if crushed wheat is used, its particle size should be small enough to perform this function. The time–temperature of roasting of wheat (170 ◦ C±5 ◦

C for 40–50 s.) before crushing and particle size (30% less than 30 mesh size) can be considered as control points.

Cooling. Recontamination of raw materials by equipment following heat treatment can occur. If cooling is inadequate, growth of contaminating microorganisms is possible before controlled fermentation can be initiated. Insufﬁcient cooling could also have an adverse effect on germination of the mold starter culture (tane koji). Time of cooling to 30 ◦

C and ﬂow-rate of cooling air should be monitored. Koji fermentation. Although substrates for the koji fermentation undergo a heat treatment before

fermentation, contamination from equipment plus contamination by bacteria present in the mold starter culture can occur. Conditions in the koji rooms (machines) must therefore favor the koji molds. This requires control of temperature, humidity, and air ﬂow in the koji room, and testing the germination rate of “tane koji”. Good growth of koji molds is achieved by incubating koji at 30–32 ◦

C at the beginning of the fermentation when enzyme production starts. After the initial 24 h, the incubation temperature of the koji room is lowered to between 28 ◦

C. If a conveyor belt is used to transport raw materials and equipment to the koji room, it should be washed thoroughly and disinfected.

C and 30 ◦

Brine fermentation (moromi phase). In the event of incorrect addition of salt (e.g. <22% salt content in brine), undesirable growth of microorganisms other than the salt-tolerant bacteria and yeasts of the moromi starter is possible. Correct addition of salt and water, and adjusting the dry matter content is a control point. The sodium chloride content of the mash should be >17% to prevent growth of undesirable putrefactive bacteria during fermentation. A concentration of sodium chloride in excess of 23%, however, can retard the growth of desirable halophilic bacteria and yeasts (Beuchat, 1984). Controlling the moromi fermentation requires monitoring temperature (30 ◦ C±2 ◦ C), aeration timing, and ﬂow rate. It is also important to ensure that the transportation route of moromi, the fermentation tanks, and all contact surfaces are kept clean and sanitized as a routine maintenance procedure.

Pasteurization of raw sauce. After moromi fermentation/ageing and pressing, the sauce should undergo a pasteurization process (e.g. heating to 70–80 ◦

C for 20–30 min). At this low temperature treatment

B. subtilis spores will not be killed but be reduced by filtration using celite as filter aid (Haga, 1971). Pasteurization of raw sauce should be monitored by time–temperature measurement. Pasteurization or sterilization of soy sauce and canned food flavored with it should take into account the properties of thermophilic and facultative anaerobic bacilli, which are reported to be present in the brine fermentation of soy sauce in Malaysia (Ho et al., 1984).

Post-pasteurization steps. Recontamination of soy sauce following pasteurization during sedimentation, ﬁltration, and bottling or spray-drying is also possible by contact with contaminated equipment or with air-borne ﬂora. Preservatives such as sodium benzoate (0.4–1.0 g/L) can be added to prevent the growth of yeasts during storage, depending on the pH of the product. During bottling, it is desirable to sterilize the nozzle of the bottling equipment using steam or alcohol, which is practised in Japanese

MICROORGANISMS IN FOODS 6

IV Fish and shrimp sauces and pastes

A Deﬁnitions Fish sauces and pastes are traditional products obtained by maceration and/or autolysis of muscles, with

or without microbial action, in the presence of high salt concentrations. These products usually have a strong characteristic aroma and are used as flavorings in many oriental foods. Under this category are shrimp and fish pastes, fish sauces, and pickled shrimp or fish sauces.

B Important properties Fish sauces and pastes are produced by hydrolysis in the presence of high salt concentrations. Under

such conditions, the proteolytic activities of the ﬁsh enzymes may be low, and the fermentation lengthy. For example, fermentation of ﬁsh sauce can take 6–12 months to complete.

Hydrolysis mainly occurs by proteolytic enzymes in the ﬂesh and entrails of raw ﬁsh (Amano, 1962). The substrates have to be properly handled and thoroughly mixed with salt in the initial stages of

processing. Usually factors such as pH, acidity, and temperature are not monitored during processing. Salt concentration is a critical factor. Traditional processes rely heavily on the high salt concentration,

e.g. 13–15% in Malaysian shrimp paste (Merican et al., 1984) and 20–25% in Philippines’s “bagoong” (Soriano et al., 1986), and low a w , e.g. 0.67 for Malaysian shrimp paste (Adnan and Owen, 1984), to prevent the growth of pathogenic and spoilage microorganisms. Methods of preparation have been

perfected through generations, and include safety features in manufacturing that take into consideration climate, environment, and handling problems. The products generally have good keeping quality. In addition, they may be pasteurized prior to bottling. Blending with other foods may be practised.

C Methods of processing and preservation Fish/shrimp sauces and pastes are principally fermented by the action of enzymes in the ﬂesh and

entrails, in the presence of high salt concentrations. Microorganisms are assumed to contribute to the characteristic flavors because these flavors are lacking in products prepared under sterile conditions (Amano, 1962). In some products such as shrimp sauce, fermentation is influenced by the combined effects of shrimp enzymes and microbial enzymes.

Fish/shrimp paste. Fish/shrimp paste is made from ﬁsh or shrimp with 6–10% salt by a process of salt-controlled anaerobic fermentation. The manufacture of shrimp paste is seasonal, depending on the availability of Acetes shrimp.

The fish or shrimp is first washed in sea-water at coastal landing sites. This step can introduce a high level of contamination if the coastal waters are heavily polluted. Sorting to remove foreign materials is then carried out. This is typically done by hand which can also introduce additional microorganisms. The fish/shrimp are drained, mixed thoroughly with salt and spread out on mats to dry in the sun for 5–8 h, until the moisture content is about 50%. The fish/shrimp are then minced, packed tightly in tubs, and allowed to ferment under anaerobic conditions for about 7 days. The paste is pressed tightly to ensure the total exclusion of air and to prevent oxidation, which would result in a putrid product. The paste is taken out in lumps and dried further in the sun for 5–8 h. This is followed by a second mincing, pressing into tubs, and fermenting for another 7 days. The process of drying, mincing, pressing, and fermenting is repeated 6–7 times, depending on the aroma and texture required. The longer the fermentation, the stronger is the aroma. If stronger aroma is desired, the shrimp are mixed with salt and held overnight at ambient temperature before the initial drying and mincing steps (Adnan, 1984).

383 Fish sauce. Fish sauce is traditionally prepared by autolysis and fermentation of small ﬁsh that are

SPICES, DRY SOUPS, AND ORIENTAL FLAVORINGS

salted and stored under anaerobic conditions for 6–12 months. In Malaysia, fish sauce is prepared from small anchovies, i.e. Anchoviella commersonii and Anch. indicus. When larger fish are used such as in Thailand, the fish are comminuted before being mixed with salt, and may be supplemented with hepatopancreatic tissues (Raksakulthai and Haard, 1992).

Shrimp sauce. Acetes shrimp is prepared for fermentation in much the same way as shrimp paste. After salting (10–20%), the shrimp are allowed to drain overnight before adding cooked rice (6–15%). The mixture is fermented in an enclosed container for 7 days. The ferment is then stirred to ensure homogeneity and the fermentation is continued for another 2 weeks. The mature product (known as cinkaluk in Malaysia) may be blended with tomato ketchup and cooked before bottling.

D Types of ﬁnal products Under this category of ﬁsh and shrimp, sauces and pastes are products of South-East Asian countries

known by different names in different localities, with some variations in the methods of processing and utilization of the ﬁnal products. Examples of these products are as follows.

r “Bagoong” of the Philippines, which is the partially or completely fermented product of small ﬁsh or small shrimp and salt, with or without added condiments, ﬂavoring materials or coloring matter

(Soriano et al., 1986). Bagoong is either eaten raw or cooked, and is generally used as a ﬂavoring or condiment in many traditional recipes. It is also used as an appetizer, served in a mixture with onions, garlic, tomatoes, or green mangoes.

r Fish or shrimp paste of Indonesia (trassi), Malaysia (belacan), and Thailand (kapi) is the macerated mince of small ﬁsh or shrimp and salt that has undergone an anaerobic fermentation. This product is

used as an ingredient in a spicy “dipping sauce”, or in traditional dishes. r The ﬁsh sauces, “budu” (Malaysia), “nampla” (Thailand, Laos), “nuoc-mam” (Cambodia, Vietnam),

and “patis” (Philippines), are the amber to brown liquid supernatant of a salted ﬁsh fermentation. The products vary in consistency from country to country and are used in much the same way as soy sauce, i.e. as a table condiment, as a dipping sauce or as ingredient in cooking traditional dishes.

r The fermented (pickled) fish or shrimp sauces, “cincaluk” (shrimp)(Malaysia) and “burong isda” or “burong dalag” (fish) (Philippines) are a fermented shrimp/fish-salt mixture with cooked rice. The

product undergoes a lactic acid fermentation (Soriano et al., 1986). These sauces are also used as condiments, as dipping sauces or as ingredients in cooking. The Philippine product (burong isda) is eaten as is or with rice. The ﬁsh may still be whole and intact. Some sauce comes from partial hydrolysis of the ﬁsh and rice but the product is largely solid.

E Initial microflora The microflora, including both identity, numbers, and distribution, of the final products are influenced

by the initial microﬂora, human contact such as the hands of ﬁshermen and handlers, and the equipment used in these operations.

The initial microflora is that present on the raw materials, which reflects that of the marine environment and salt. When fish and shrimp are caught in coastal waters, polluting microorganisms such as Streptococcus faecalis have been reported to be present during the early stages of fermentation (Orillo and Pederson, 1968; Ohhira et al., 1990). For an account of the microorganisms of fish and shrimp, please refer to Chapter 3. See earlier comments in this chapter regarding the microflora of salt preparations.

MICROORGANISMS IN FOODS 6

Poor handling of raw materials and an unsanitary environment during processing and storage are the most critical problems associated with these products, e.g. drying of salted shrimp or ﬁsh in the sun exposes the products to ﬂies and other pests.

F Primary processing Effects of processing on microorganisms. The high salt content and microaerophilic or anaerobic

conditions during fermentation encourage the proliferation of lactic acid bacteria, e.g. Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum in Malaysian shrimp pastes, Leuc. mesenteroides subsp. mesenteroides in Indonesian shrimp paste; Lb. plantarum in Malaysian ﬁsh sauce; Leuc. mesenteroides subsp. mesenteroides in pickled shrimp; and Strep. faecium and Leuc. mesenteroides subsp. mesenteroides in pickled ﬁsh (Ohhira et al., 1990). Burong dalag undergoes a lactic fermentation in which Leuc. mesenteroides, Ped. cerevisiae, and Lb. plantarum play major roles (Soriano et al., 1986).

Studies on Philippines’ bagoong revealed that bacteria actually decreased during the course of fermentation at ambient temperature from an initial count of 3.5 × 10 6 –5.8 × 10 4 cfu/g after 1 week, to

6.2 × 10 3 cfu/g at 2 weeks, to 100 cfu/g after 8 weeks (Amano, 1962). In contrast, “shiokara”, a Japanese product increased in bacterial populations from an initial count of 9.0 × 10 4 to 3.2 × 10 7 cfu/g in

41 days (Amano, 1962). During the course of shiokara fermentation, a succession of organisms appeared, viz. Bacillus, Micrococcus, and Lactobacillus at the beginning, giving rise to salt tolerant Micrococcus at later stages of fermentation. Vibrio, Achromobacter/Moraxella, and Flavobacterium were also isolated

from commercial shiokara, with total bacterial counts ranging from 1.5 × 10 3 to 2.4 × 10 7 cfu/g. Vibrio, Flavobacterium, Achromobacter/Moraxella, and Micrococcus were all able to grow in 10–20% NaCl concentrations, whereas Bacillus spp. only grew well at 5% NaCl (Amano, 1962). All these isolates were capable of producing ammonia.

Growth of anaerobes in the presence of high salt appears to contribute to the typical flavor of fermented fish products. Products such as pickled shrimp and fish to which a carbohydrate source has been added undergo a lactic acid fermentation. This favors the growth of Lb. plantarum, Lb. pentoaceticus, Strep. faecium, and yeasts (Amano, 1962).

Fermented fish products such as pickled shrimp and shrimp paste may contain histamine >500 mg/kg (Azudin and Saari, 1990), with a 10-year-old fish sauce having as much as 700 mg/kg (Amano, 1962). Although it has been reported that salted bonito had been implicated in bacterially formed histamine poisoning, properly prepared fish pastes and sauces have not been linked to a serious problem with histamine toxicity. However, the practice of using semi-spoiled fish as raw materials is a cause of concern.

Spoilage. Salt plays a significant role in these products and acts as preservative to inhibit the growth of spoilage and pathogenic microorganisms. Although the high salt content prevents the growth of most spoilage microorganisms, moderately halophilic bacteria such as Bacillus and Staphylococcus spp. have been isolated in spoiled fish sauce (Mabesa et al., 1986), and extremely halophilic strains of Halobacterium salinarum have been reported as spoilage agents in salted fish (Sanderson et al., 1988). These bacteria are associated with poor hygienic practices. Halophilic microorganisms surviving in fermented fish tend to produce ammonia and thus contribute to off-flavors (Amano, 1962).

Pathogens. The high salt content inhibits the growth of pathogens. Although reports of food poisoning from fermented ﬁsh products are rare, the trend of reducing the salt content of these products may pose

a serious problem. A home-made product, made with reduced salt, was reported to cause Cl. botulinum type E poisoning in Japan (Amano, 1962). Shrimp paste does not support the growth of molds and attempts to detect Asp. ﬂavus and aﬂatoxin have yielded negative results (Sim et al., 1985).

SPICES, DRY SOUPS, AND ORIENTAL FLAVORINGS

G Control Under normal circumstances, the high salt concentration of ﬁsh sauces and pastes is sufﬁcient to inhibit

the growth of spoilage and pathogenic microorganisms. However, quality control and standardization of the process are necessary to ensure a hygienically consistent product. A code of practice should be developed to guide processors on good manufacturing practices. Pasteurization or cooking is usually practised as a means of prolonging shelf life. In products such as ﬁsh/shrimp pastes and blended ﬁsh sauces, benzoic acid/sodium benzoate is added to extend shelf life.

Control points that should be considered or used include the following.

1. Avoiding harvesting ﬁsh and shrimp from polluted waters. Use only clean water, salt, and utensils. Monitoring by visual inspection the catch, equipment, and raw ingredients. Use only ﬁsh of good quality for fermentation.

2. Enforcing good hygienic practices and proper sanitation and hygiene of food handlers involved in the preparation and processing of products. Monitor by inspection the cleanliness of food handlers, operations, and premises.

3. Control a w through sodium chloride addition and/or drying, (a w <

0.85 for ﬁsh/shrimp sauces and

0.75 for ﬁsh/shrimp pastes). Assure that sufﬁcient levels of salt are added to decrease aw. Monitor a w to ensure maximum values are not exceeded.

4. Proper control the anaerobic fermentation, including the use of correctly designed equipment. Tradi- tional methods and equipment currently in use are often not properly designed for maintaining anaerobic conditions. Monitor by visual inspection areas of discoloration indicative of aerobic spoilage. Ideally, plants should invest in proper equipment.

D CONTROL (dry soup and gravy mixes)