Rhizopus Chinensis

Sufu (Chinese soy cheese) and stinky tofu (chaotofu) are block-type fermented soy foods. In the manufacture of these fermented foods, sufu utilizes mainly mold(s), whereas stinky tofu utilizes mainly bacteria. Molds multiply under aerobic condition and produce amylase, protease, and other hydrolases. These enzymes then hydrolyze their substrates. However, molds do not grow under anaerobic conditions but can use the enzymes they produced to hydrolyze their substrates. Therefore, under aerobic conditions, molds metabolize through respiration, whereas under anaerobic condition, they conduct anaerobic respiration and metabolize through fermentation. Using molds to conduct fermentation is a combined utilization of these enzymatic reactions to process food.

In the manufacture of traditionally fermented foods by the so-called traditional fermentation processes, molds and bacteria come from the spores present naturally in the raw materials, in the air in the manufacturing environment, or on the utensils. These traditionally produced products from this household-type industry have difficulty in expanding their production to meet market demand. In order to stabilize product quality, it is therefore necessary to apply specific molds and bacteria through pure culturing techniques. The fermentation technique is then transformed from natural fermentation to pure culture fermentation.

A bacterium is much smaller than a mold, and bacteria are not as complicated as yeasts or molds. Even though some of bacteria have similar shapes, their functions can be significantly different. Morphologically, bacteria may have the same shape, but they can be either beneficial or harmful. It is therefore necessary to understand their physiological characteristics.

The physiological characteristics of bacteria are quite complicated. Bacteria may be classified as aerobic, anaerobic, psychrophilic, thermophilic, and nutrient-selective. Their products are also very complicated.

Some bacteria produce spores, and others do not. In general, bacteria that produce endogenous spores are more harmful. The bacterial cells themselves protect these spores. They are heat-resistant during thermal sterilization. A temperature of 110-120 °C for 3-5 min under pressurized steam is needed to kill these spores. Under normal pressure and at 100 °C, non-sporeforming bacteria cells can be killed easily, but these spores can withstand 30 min under the same temperature condition without much killing effect. Therefore, it is necessary to wait for the spores to germinate and then apply an intermittent sterilization process to inactivate them.

When bacteria are utilized during fermentation, their growth is not easily observable compared to molds. Usually, we have to observe physical changes in the substrate such as hardness and mucilage production, or sensory changes in color, odor, and taste, in order to understand the bacterial growth condition.

In the fermentation industry, we utilize microbes obtained from pure culture isolation techniques. In principle, the whole fermentation process should be maintained under pure culturing condition. However, in the manufacture of fermented foods, the pure culturing condition is not as critically controlled as in the microbiology laboratory. This is especially true for mold-ripened foods. Mold is used to start the process and is commonly followed by yeast and bacteria. These bacteria and yeasts have a close relationship to the characteristics of the final product. Even though the presence of some bacteria is considered beneficial, their effects on product quality are not well understood. They are considered nonfunctional microorganisms. However, in the case of multi-microbial conditions, these microbes must provide some functions and warrant future investigation.

II. SUFU A. Introduction

Sufu is one of the traditional soy foods made by fermentation in China and Taiwan. Soybean, the major raw material, is processed into soymilk and then to soybean curd (tofu). Tofu cubes are inoculated with fungi and fermented until they are covered with fungous mycelia. Subsequently, they are soaked in brine (or salt is added to partially dried tofu) and aged in the mashes from wine, miso, or soy sauce. Sufu has a smooth and sticky texture as well as salty taste and is also known as Chinese soybean cheese or vegetable cheese. Sufu is also called stinky tofu and has other names in different regions.

According to the historical records, tofu was first made by Liu An around 179 b.c-122 b.c. by adding gypsum to soymilk. Around 1500 years ago, a piece of salted and dried tofu was soaked in the soy sauce mash to make the earliest sufu in history. In 1590, sufu was called ream cake in the Japanese language of the Chinese Herbal Encyclopedia. Homemade sufu is very common in Chinese households located along central and southern coast provinces of mainland China and Taiwan. Nowadays, it is sold in jars manufactured by the industry.

In the early 1920s, a sufu market survey in Shan-Hai (or Shanghai) showed several different processing techniques used by Chinese people. By the existence of fungi, type of fungi, and mash, sufu can be classified as follows:

1. No fungi added in soaking and aging a. Jen-Nin tofu: predried tofu aged in salt and miso or soy sauce mash b. Sufu: predried tofu aged in the salt and koji

2. Fungous mycelia allowed to grow on tofu, followed by soaking in brine and aging in the mash a. Jiang sufu: Soaked in brine and aged in miso or soy sauce mash b. Red mash sufu: Soaked in brine and aged in a mixture of soy sauce mash and red koji from Monascus c. Wine mash sufu: Soaked in brine and aged in rice wine mash d. Red sufu: Soaked in brine and aged in red rice wine mash e. Flavored sufu: Soaked in brine and aged in the mash that contains olive leaf or aromatic mushroom f. Sake sufu: Soaked in brine and aged in the mash that contains rice sake g. Kwantung sufu: Similar to red mash sufu with addition of hot pepper, anise, and xanthoxylon seeds h. Rose sufu: Similar to red mash sufu with addition of rose mash

Sometimes meat, crab, shrimp or sesame seed are added to produce moldy tofu. Mash formulated with alcohol, salt, and spice can also be used for sufu soak-in-brine and aging processes. Sesame oil or food-grade paraffin can also be used to cover the top of a jar to prevent air contact for better preservation.

B. Making of Sufu

A flowchart for sufu manufacturing is presented in Fig. 1.

Medium-size soybean with yellow or white hilum, thin seed coat that are hard in texture, high in soluble protein and low in fat content are the most suitable for manufacturing sufu.

Processing steps of sufu manufacturing (Fig. 1) can be divided into four stages. The first stage is to process soybeans to tofu cubes (1 day). The second stage is to inoculate the tofu cubes with fungi and allow mycelia to cover the tofu cube surface (3 days). The third

Whole soybean

Addition of coagulant(s) Fungal inoculation

Figure 1 Flowchart for sufu manufacturing. (From Ref. 1.)

step is to allow moldy tofu cubes to dry with spreading of salt onto it (or brining) followed by thermal dehydration to harden the moldy tofu cubes. The last step is to soak and age the moldy tofu cubes in the mash.

1. Preparation of Soymilk and Making of Tofu a. Preparation of Soymilk Soybeans are processed into soymilk and then into tofu. Fully hydrate the soybean in water until their weight reaches 2.2 times the original soybean weight. Soaking time depends on the temperature of water used: 10 °C for 18 hr, 20°C for 10 hr, or 30°C for 6 hr. Soaked soybeans and water in a 1:2 or 1:4 ratio is then disintegrated with a grinder. In order to extract hot-water soluble proteins, more water is added, followed by heating. Protein extraction rate depends on the amount of water added. Optimal amount of water is about 10 times soybean weight. Insufficient amount of water will lower the extraction rate; for example, 80% of total protein can be extracted from soybeans and water in a 1:10 ratio, and 35% of total protein extracted from soybeans and water in a 1:5 ratio. A soybean slurry of 11 kg should be recovered from 1 kg of soybeans.

The silicone antifoaming agent can be added while heating to 100 °C for 5-10 min. Direct or indirect steam can be applied for the thermal treatment. Heating temperatures lower than 90 °C will produce soymilk with beany flavor, poor coagulation, and low ex-tractable solids. Temperatures exceeding 100°C will not increase the extraction rate. Properly heated soybean slurry should be filtered through cheesecloths to obtain the soymilk.

b. Making of Tofu When the temperature of soymilk drops to 70-75°C, warm suspension of calcium sulfate (2.5 to 2.7% by the weight of raw total soybean) is added with vigorous stirring with a wooden spatula. After stirring, the mixture is let set for 10 min to coagulate the soy protein. In Japan, 2% of coagulant is used with less stirring to form bigger coagulated curds for the making of tofu. The making of sufu requires vigorous stirring to form small soy protein curds, which are easier to drain during pressing. Lowering the amount of moisture in the tofu will produce a harder-texture product. Either acidification of the soymilk to pH 5.0 by the addition of acetic acid along with coagulant or addition of 50 ppm antibacterial agent to the soymilk can prevent the unwanted microorganisms in curd molding.

2. Preparation of Moldy Tofu

This procedure is unique in sufu processing. The press-dehydrated tofu is cut into 2 cm x 2 cm x 2 cm cubes. If necessary, tofu cubes can be sun-dried or heat-dried to further remove surface moisture. The dried cubes are laid out in a rice straw-layered container, 2 to 3 cm apart from each other. Several covered containers can be piled up and placed in a warm environment. Fungi from the rice straw will be naturally transferred to the tofu cubes and grow. In 3 to 7 days, mold growth is complete, with white Mucor mycelia covering the surface of tofu cubes (Fig. 2). Homemade sufu uses natural fermentation to produce the moldy tofu. In industrial practice, pure culture is applied onto the surface of sterilized filter papers or bamboo sheets. They are layered with the tofu cubes for inoculation and fermentation in the incubator.

Processing time required for the preparation of moldy tofu depends on the fungi strains and fermentation temperature. Rhizopus chinensis var. chungyuen requires 7 days at 12°C, whereas Mucor hiemalis and Mucor silvaticus take only 3 days at 20°C, Mucor praini takes 2 days at 25°C, and Actinomucor repens needs 2 days at 27°C.

Fermented Sufu
Figure 2 Molded tofu.

3. Brining and Maturation

Moldy tofu cubes can be sun-dried to remove the surface moisture followed by brine curing and mash aging. There are two methods for brine curing. One is to soak the moldy tofu cubes in saturated brine that contains 25-30% of salt to help the sodium chloride penetrate into the tofu interior. The other one is to spread salt on top of the moldy tofu cubes directly, followed by applying pressure to remove the moisture and effectively dehydrate the cubes. If moldy tofu is cured in high-alcohol-content mash or high-salt content mash, the brine-curing procedure can be eliminated (Fig. 3). There are some products called moldy tofu or salty sufu made only by brine curing and aging for more than 1 month.

After brine curing and partial dehydration, the tofu cubes are soaked in the mash for the aging process. Soaking in different mash results in different flavors and textures. Various mash formulations developed in different regions show their unique taste and flavor. Three categories of mash have been developed: (a) wine mash produced from grains; (b) soy sauce mash or miso, which is made from wheat and soybean; and (c) formulated mash containing alcohol and seasonings. There are many varieties of sufu manufactured with wine mashes. Sufu made with Shao-Shin liquor mash mixed with black bean koji, seasoning, and salt is highly rated. The salt-cured, moldy tofu cubes and olive leaves are layered in big ceramic jars. The jars are then filled with the mash, sealed, and aged for months or 1-2 years. Some manufacturers inoculate Monascus purpureus or Monascus anka on steamed rice to have red koji mash or Japanese-style cloudy wine mash. The product obtained from red koji mash is called red sufu. The mash in (b) that uses soybean koji or wheat koji blended with high concentration of salt and water for preservation is very salty. The formulated mash in (c) is a modified method for mass production. There are many formulations for the mash; for example, 10% alcohol blend with 5% salt or with 4% salt and 1% acetic acid. Either one of the formulations is good for preserving the sufu

Rhizopus Chinensis
Figure 3 Bottling of seasoning tofu.

at 20-35°C for over 6 months. After packaging, addition of a layer of sesame oil or paraffin (food grade, MP 60°C) at the top of the jars can prolong shelf life.

C. Changes in Sufu During Fermentation

Sufu is an easily digestible soy food made from hot water-extractable soy proteins in the form of tofu that has passed through the fungal degradation, brine-curing, and mashaging processes. The compositional changes are the result of salted migrating into the tofu cubes. When moldy tofu is converted to sufu, salt and the mash components migrate into the tofu cubes, whereas the soluble materials in the tofu cubes are transferred to the soaking liquid. Therefore, the characteristics of the aging mash will affect the composition of sufu during aging. Protein losses are 5% from tofu to moldy tofu, and 20% from moldy tofu to sufu, respectively (on dried weight basis).

Protein degradation occurs during sufu fermentation. In moldy tofu, protein nitrogen decreases significantly, and the amino nitrogen and ammonia nitrogen increase accordingly. Fungi alone cannot produce of ammonia nitrogen more than 7%, and thus bacteria may have contributed to the increase in ammonia nitrogen. In the final product sufu, protein nitrogen increase results from the soluble solids moved from the tofu cubes to the liquid and changes the overall percentages.

During sufu fermentation, a significant amount of cystine is lost due to degradation by microorganisms. None or trace amount of cystine can be found in sufu after a long period of aging.

D. Product Quality and Compositions

Sufu contains high levels of salt and spices. The nutritional composition of sufu variants sold in Taiwan is listed in Table 1. Sufu contains 60-70% moisture and 12-17% protein,

Table 1 Compositions of Sufu Variants Marketed in Taiwan

Composition

Rose sufu

Tsao sufu

Red sufu

Kwantung sufua

Moisture

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