| Applications of biotechnology to traditional fermented foods |
|I. Research priorities|
|Research Priorities in Traditional Fermented Foods|
|1 Upgrading Traditional Biotechnological Processes|
|2 Genetic Improvement of Microbial Starter Cultures|
|3 Sudan's Fermented Food Heritage|
|4 Lesser-Known Fermented Plant Foods|
|5 Lactic Acid Fermentations|
|6 Mixed-Culture Fermentations|
|III. Milk derivatives|
|7 Fermented Milks Past, Present, and Future|
|8 Lactobacillus GG Fermented Whey and Human Health|
|9 The Microbiology Ethiopian Ayib|
|10 Moroccan Traditional Fermented Dairy Products|
|11 Fermented Milk Products in Zimbabwe|
|IV. Plant derivatives|
|12 Cassava Processing in Africa|
|13 Improving the Nutritional Quality of Ogi and Gari|
|14 Solid-State Fermentation of Manioc to Increase Protein Content|
|15 Leaf and Seed Fermentations of Western Sudan|
|16 Continuous Production of Soy Sauce in a Bioreactor|
|V. Animal derivatives|
|17 Using Mixed Starter Cultures for Thai Nham|
|18 Starter Cultures in Traditional Fermented Meats|
|19 Fermented Fish Products in the Philippines|
|20 Fish-Meat Sausage|
|21 An Accelerated Process for Fish Sauce (Patis) Production|
|VI. Human health, safety, and nutrition|
|22 Nutrition and Safety Considerations|
|23 Mycotoxic Flora of Some Indigenous Fermented Foods|
|24 Commercialization of Fermented Foods in Sub-Saharan Africa|
|25 Biotechnology for Production of Fruits, Wines, and Alcohol|
|26 Future Directions|
|Board on Science and Technology for International Development|
Nham is traditionally made from fresh lean pork that is trimmed; minced; mixed thoroughly with salt, potassium nitrate, cooked rice and seasonings; and packed in either banana leaves (1) or cylindrical plastic bags (2). Nham production in Thailand depends on chance contamination with wild organisms - lactic acid bacteria and nitrate reducing bacteria. It is a long process; generally the fermentation lasts 3 to 5 days depending on the season. When nham is packed into cylindrical plastic bags, which exclude air, and is held in the bags during fermentation, a microenvironment is selected for microorganisms that are not only salt tolerant but can also grow in the absence of air. In these gram-positive fermentative types of microorganisms, lactic acid bacteria are predominant (3,4). The fermentable carbohydrates are used by those organisms to produce organic acids, mainly lactic acid, that contribute to a variety of flavors and textures. The nham finally develops approximately 1.0 percent total acidity as lactic acid and the pH is 4.3 (5).
Problems in marketing traditional nham include its short shelf life and high price and the intensive labor required for its production. It has high energy costs if kept under refrigeration in the marketplace. Additionally, the manufacturers have a heavy exposure to risk of losing a large stock through a process failure. Pork meat is quite expensive, and the raw material cost is increasing more quickly than the selling price. In addition, large-scale production of nham has the problem of its short storage life. A longer shelf life is required so that the nham can be distributed to the marketplace. Therefore, the nham market needs the product to have consistent quality, safety, and longer shelf life. The nham should stay fresh and not turn rancid or develop an off flavor or change in color when it is in the marketplace.
On the other hand, nham production depends on natural fermentation; the product quality therefore varies from batch to batch. The shelf life of nham is quite short - approximately a week at Thai ambient temperatures. Chilled conditions can extend the shelf life, but normally the product is stored at ambient temperatures. The sanitation conditions of the processing are also poor because of a lack of knowledge and technology. The initial native lactic acid bacteria may be insufficient to bring about the normal ripening process. This may allow pathogenic bacteria to grow before lactic acid bacteria occur, resulting in the possibility of food poisoning. Since most nham is consumed without further cooking, proper fermentation is of paramount importance in ensuring the product's safety.
Somathiti (6) found that the initial coliform count was high in nham - approximately 107 cells per gram - and decreased to 102 cells per gram on the fifth day. An investigation of Salmonella in nham in the Bangkok market showed that it was present in 56 (or 12 percent) of 450 samples. In nham produced in Chiang Mai, Chiang Rai, and Ubonratchathani, Salmonella was found in 25 percent, 42 percent, and 11 percent, respectively, of the total samples. However, Shigella sp. was not found in nham bought from any of these markets.
Thus, the nham process needs to be studied to improve product quality, to give a more uniform standard quality, and to develop the technology for applying of the process on an industrial scale before launching extensively in the Thai and export markets.
In developing of an improved nham process, not only is there a need for the knowledge of modern scientific discoveries and technological developments but also the knowledge of consumers' needs and wishes. The final product must be acceptable to consumers. A unified system is required that combines scientific and consumer information for systematic development of the nham product.
Effect of Starter Cultures
In our research mixed starter cultures and the carbon sources used in nham formulation were important factors in determining product quality (7). The starter cultures had a potential to make a good nham quality. Cooked rice, a carbon source for lactic acid production by starter cultures, was an important factor in nham fermentation.
The addition of L. plantarum to the nham mass accelerated very distinctly the decrease in the pH of nham. Consequently, the firmness and color developed, influenced directly by acid production. Those findings were in agreement with the work of many researchers (8-12). P. cerevisiae increased the firmness later during the last period of fermentation. The optimum growth of P. cerevisiae is at pH 5.0 (13), the conditions during this period allow good growth and acid production causing the increase in firmness. L. plantarum inoculation had a very distinct effect in terms of firmness development when it was used together with P. cerevisiae.
M. varians in the nham system significantly reduced nitrate to nitrite during the initial fermentation and increased the tristimulus values at the beginning of fermentation. L. plantarum then continued to intensify the color. This finding agreed with the work of Deibel et al. (14); they reported that nitrate-reducing activity generally occurred during the first 2 to 16 hours, while acid production was initiated after 8 to 16 hours. It was clear that it was important to ensure the nitrate reducing activity of the M. varians that took place prior to its inhibition by the growth of lactic acid bacteria. The nitrite formed was decomposed spontaneously in acid surroundings into nitric oxide, which subsequently reacted with myoglobin to form a pink compound - nitrosomyoglobin. So the residual nitrate in the nham system reduced quickly when acid was produced. The rate of nitrosomyoglobin formation increased with falling pH, and this reaction takes place best in the pH range of 5.0 to 5.5 (15) and was therefore accelerated by L. plantarum. The L. plantarum inoculation had a very distinct effect in terms of color development when it was used together with M. varians.
On the other hand, L. brevis seemed to be a poor lactic acid producer and decreased the color of the product and also produced gas, which decreased the firmness of the nham.
The starter cultures L. plantarum and P. cerevisiae increased during the initial fermentation and were highest on the third day of fermentation with 106 to 109 cfu/g-' (colony forming units) and then decreased slowly during the later period of fermentation. In the nham sample, on the other hand, the M. varians decreased during the fermentation approximately 2 log cycles by the third day. The total bacterial count was related to the starter cultures counts, but there was a little higher count of approximately 1 log cycle. No yeasts or molds were detected in the finished nham.
The pathogenic bacteria, including Enterobacteriaceae and Staphylococcus aureus, decreased during fermentation. In the nham fermented for 3 days the Enterobacteriaceae and 5. aureus counts were 102 and 103 cfu/g-', respectively.
In Thailand large amounts of cooked rice are added to the raw nham mixture. It is degraded only slowly and may result in growth of undesirable organisms during fermentation, particularly at high ripening temperatures. Glucose is therefore added to the cooked rice. This ensures a sufficiently rapid initial growth and nitrate reduction by M. varians and rapid later pH drop, without inhibiting the chemical reactions necessary for the development of firmness and desired color.
Cooked rice and glucose had no effect on pH reduction during nham fermentation. As the fermentation time increased, the pH decreased. The pH dropped rapidly after 18 hours of fermentation at 30°C, 43 percent relative humidity with pH 5.1. The beginning of cooked rice reduction coincided with the increase in reducing sugars after 12 hours of fermentation. The reducing sugars declined after another 12 hours of fermentation, and this coincided with the decrease in pH. This indicated that if both cooked rice and glucose were used at high levels ( 10 percent and I percent, respectively) at the beginning of the fermentation, the pH dropped more slowly than if lower levels were used (8 percent cooked rice and 0.5 percent glucose). Increasing the amount of cooked rice, on the other hand, reduced the firmness of the nham. There was an increase in weight loss at the high level of glucose. There were 1.0 to 1.3 percent reducing sugars and 2 to 3 percent cooked rice in the finished nham, and this residual carbohydrate could be used by the undesirable organisms during storage. Therefore, the carbon source levels in nham should be reduced.
When the glucose level was maintained at 0.5 percent but the level of cooked rice increased, a longer period was required to attain adequate fermentation end products (16).
It was also found that 6 percent cooked rice with 0.5 percent glucose in the nham formulation, when fermented with starter cultures at 30°C and 97 percent relative humidity, caused rapid pH reduction. Acid production was good, firmness and color development were satisfactory, and the product was microbiologically safe.
The rate of fermentation and the ultimate pH of nham are directly influenced not only by the specific formulation but also by the processing conditions. Since the safety and quality of nham depend on the rate and extent of acid production, a thorough understanding of these environmental parameters is essential for total control of the product. In our research, higher temperatures increased the rate of fermentation, reduced pH, and improved firmness and color development. The initial temperature of nham was very important in determining the final product. The achievement of lowering pH was affected by the initial product temperature and the time at that temperature. For experimentation with frozen meat, the temperature of nham mixtures was 15°C; with fresh meat the temperature was 26°C. The pH dropped more quickly in nham made with fresh meat than with frozen meat.
Nham made using frozen meat was fermented at 30°C and 97 percent relative humidity. It took 3 days to reduce the pH to 4.3 to 4.4, while the nham using fresh meat fermented under the same conditions needed only 2 days to reduce the pH to 4.1.
Nham is usually held at a high temperature during processing to ensure rapid fermentation, but this can also accentuate the growth of pathogens. In addition, nham is usually eaten without further cooking by the consumer. These conditions make strict control of the product essential. Although proper sanitation, employee hygiene, and the control of raw materials definitely reduce contamination, ultimate control of product safety must be inherent in the formulation and process. The addition of starter cultures can provide sufficient microbial numbers to ensure numerical dominance over the natural flora, including pathogens, and in combination with the proper processing controls can guarantee the safety and quality of the final nham.
Nham is usually sold in Thai markets at ambient temperatures (20° to 30°C). It was found that nham prepared using the improved conditions described here when stored at these temperatures had a shelf life of 9 to 11 days while commercial nham usually has a shelf life of only 3 days. In supermarkets nham is stored at chilled temperatures (5°C), and it can be exported at low temperatures (1°C). Additionally, consumers usually store the product in a household refrigerator (10°C). It was found that shelf life was extended to 63 to 103 days at storage temperatures of 1° to 10°C. The higher the storage temperature, the greater the change in nham quality.
Nham fermented with 103 cfu/g M. varians, 103 cfu/g L. plantarum, and 106 cfu/g P. cerevisiae with 6 percent cooked rice and 0.5 percent glucose at 30°C, 97 percent relative humidity for 3 days, was accepted by the trained panel, with an overall acceptability mean ideal ratio score of 0.95+0.01. For quality degradation during storage, the overall acceptability of the product depended on sourness and off-flavor detected in the sample.
Nham using fresh meat fermented at a low temperature was given a higher than ideal score for sourness. However, the newly developed formulation for nham was superior to that of the commercial nham.
The consumer panel was also used to determine the effect of reducing the fermentation time from 3 days to 2 days. The results showed that only visual texture was significantly different from the ideal product.
In consumer testing the majority of the consumers (90 percent) accepted the developed nham in terms of sourness, spiciness, and saltiness.
In conclusion, the development of traditional fermented pork sausage, nham, was very successful in that the product was developed by using mixed starter cultures and had a very high quality in terms of consistency, microbiological safety, and longer shelf life. It was also acceptable by the target consumers. The product could be processed in a simple plant and with equipment that was available at the fermented meat factory with only an improvement in the technology of culture preparation and temperature control. In addition, the developed nham had a longer shelf life than commercial nham. The product, therefore, could be shipped from the cottage industry producers in the north to all provinces in Thailand, particularly to Bangkok, and also gave the potential for overseas shipment if refrigeration is used.
1. Adams, M. R. 1986. Progress in Industrial Microbiology. Vol. 23. Microorganisms in the Production of Food. New York: Elsevier Science Publishers.
2. Pakrachpan, L. 1981. Fermented Food Industry. (In Thai). Biotechnology Department, Faculty of Agro-Industry, Kasetsart University, Thailand.
3. Comenuanta, J. 1966. Thai Fermented Pork. I. Microbiology of the Thai Fermented Pork. B.Sc. thesis, Kasetsart University, Thailand.
4. Techapinyawat, S. 1975. Microbial Study During Fermentation of Thai Fermented Pork. M.Sc. thesis, Kasetsart University, Thailand.
5. Wiriyacharee, P. 1990. The Systematic Development of a Controlled Fermentation Process Using Mixed Bacterial Starter Cultures for Nham, a Thai Semi-dry Sausage. Ph.D. thesis, Massey University, New Zealand.
6. Somathiti, S. 1982. A Survey of Some Enteropathogenic Bacteria in Thai Fermented Pork. M.Sc. thesis, Kasetsart University, Thailand.
7. Wiriyacharee, P., M. D. Earle, D. J. Brooks, G. Page, and L. Rujanakraikarn. 1991. Identifying the important factors affecting the characteristics of nham. Food 21(1):48-58.
8. Klemet, J. T., R. G. Cassens, and 0. R. Fennema. 1973. The association of protein solubility with physical properties in a fermented sausage. Journal of Food Science 38:1128-1131.
9. Klemet, J. T., R. G. Cassens, and 0. R. Fennema. 1974. The effect of bacterial fermentation on protein solubility in a sausage model system. Journal of Food Science 39:833-835.
10. Klettner, P. G., and W. Rodel. 1978. Testing and controlling parameters important to dry sausage ripening. Fleischwirtschaft 58:576O, 63-64, 66.
11. Klettner, P. G., and P. A. Baumgartner. 1980. The technology of raw dry sausage manufacture. Food Technology Australia 32:380384.
12. Palumbo, S. A., L. L. Zaika, J. C. Kissinger, and J. L. Smith. 1976. Microbiology and technology of the pepperoni process. Journal of Food Science 41:12-17.
13. Buchanan, R. E., and N. E. Gibson. 1974. Bergey's Manual of Determinative Bacteriology. Baltimore, Md.: Williams and Wilkins Co.
14. Deibel, R. H., C. F. Niven, and D. D. Wilson. 1961. Microbiology of meat curing. III. Some microbiological and related technological aspects in the manufacture of fermented sausages. Applied Microbiology 9:156-165.
15. Niinivaara, F. P. 1955. The influence of pure bacterial cultures on aging and changes of the red color of dry sausage. Thesis, University of Helsinki, Finland, Acta Agralia Finnica No. 84.
16. Pezacki, W. 1974. Technological control of dry sausage ripening. VIII. Effect of pre-drying on the dynamics of carbohydrate changes taking place at the beginning of ripening. Fleischwirtschaft 58:124-126, 129-132, 135.