| Applications of biotechnology to traditional fermented foods |
|III. Milk derivatives|
Fermentation is the oldest means of preserving milk (1). Originally, unpasteurized milk was left to ferment naturally, and fermentation involved microorganisms present in the raw milk and surrounding air. With the development of modern technologies, specific lactic-acid-producing microorganisms are now introduced to carry out fermentations under controlled conditions. In this way fermented products of superior nutritional, physical, chemical, and sanitary qualities are produced.
In Zimbabwe one finds the modern fermented products such as yogurt and different types of cheese. The rural population, however, still ferment their milk traditionally. Fresh unpasteurized cow's milk is allowed to stand, at ambient temperature, in an earthenware pot loosely covered by a plate. This allows microorganisms inherent in the milk, from the pot, and from the surrounding air to ferment the milk. Fermentation takes 1 to 2 days depending on the ambient temperature (20 to 25°C). The fermented milk is not refrigerated and has an estimated shelf life of 3 days at ambient temperature.
In response to the urban population's desire for fermented milk, the Zimbabwe Dairy Marketing Board produces a fermented milk called Lacto on an industrial scale. Milk is standardized, pasteurized at 92°C for 20 minutes, cooled to 22°C, and inoculated with 1.2 percent of an imported mesophilic starter culture, similar to that used to produce "filmjolk," a Scandinavian fermented milk. The milk is immediately packaged into sachets, left to ferment at ambient temperature for 18 hours, and stored at 5°C ready for the market. The shelf life of refrigerated Lacto is 7 days.
Our studies have compared traditionally fermented milk with Lacto. We included traditionally fermented pasteurized milk, since substitution of unpasteurized with pasteurized milk might be an alternative for upgrading hygienic standards. The initial study was concerned with the effects of pasteurization and of the container used during fermentation on the total microbial cell counts, the counts of lactic acid bacteria, the amount of lactic acid produced, and the acceptability of the fermented milk by a panel (2).
We have characterized 10 predominant lactic acid bacterial isolates from traditionally fermented milk and four isolates from Lacto (3). We have also carried out studies to determine the fate of pathogenic and nonpathogenic Escherichia cold during fermentation of Lacto and traditionally fermented pasteurized and unpasteurized milk. The survival of E. cold was also tracked during storage of the fermented products at ambient (20°C) and refrigeration temperatures (5°C) for 4 days (4), since it is possible that pathogenic bacteria may gain access to these products before, during, and after fermentation. In the case of traditionally fermented milk, coliform contamination from cattle dung or from the milker's hands is possible. Contamination with coliforms during Lacto production can occur through bulk starter cultures and from inadequately sanitized equipment.
TRADITIONALLY FERMENTED MILK AND LACTO
In an earlier study (2) unpasteurized milk and pasteurized milk were fermented in clean nonsterile earthenware pots and sterile glass containers. At the same time, Lacto was fermented in plastic sachets and sterile glass containers. Bacterial counts and lactic acid levels were determined. The acceptability of the fermented milks was ranked by 11 panelists. Comparisons of all parameters were made after 24 and 48 hours of fermentation, when Lacto and traditionally fermented milk are likely to be consumed.
The numbers of lactic acid bacteria, lactic acid production, and acceptability were always higher for unpasteurized than pasteurized traditionally fermented milk irrespective of the container used.
Earthenware pots are better containers for traditional fermentation of milk. This is because earthenware pots have micropores in their walls, which, if not sterilized, may harbor lactic acid bacteria from the previous fermentation, which then act as inocula for the next fermentation. Our results suggest that earthenware pots are good containers to ferment milk in and may still have a place in milk fermentation in the home.
Unpasteurized milk fermented traditionally in either container was significantly more acceptable to the panel than Lacto, although the products were similar in all the other parameters assessed. It was therefore impossible to explain the differences in the acceptability of traditionally fermented milk and Lacto on the basis of this work. We suggested that the differences were probably due to the types of microorganisms involved in the fermentation of the two milk products rather than pasteurization or the container used for fermentation. Thus, we set out to isolate and characterize the lactic acid bacteria in traditionally fermented milk and Lacto.
ISOLATION AND IDENTIFICATION OF LACTIC ACID BACTERIA
From the previous study (3), 10 predominant morphologically different lactic acid bacteria colony types from plates inoculated with traditionally fermented milk and four morphologically different types of colonies from Lacto plates were selected and isolated into pure culture. The isolates were identified using numerical taxonomic techniques and reference strains. The isolates and reference strains were examined for 32 characteristics. Data were analyzed using the simple matching coefficient, and clustering was by unweighted pair group average linkage (5).
All the isolates from traditionally fermented milk belonged to the genus Lactobacillus. Seven of the isolates could be identified as belonging to L. helveticus, L. plantarum, L. delbrueckii subspecies lactis (two isolates), L. cased subsp. cased (two isolates) and L. cased subsp. pseudoplantarum. Three of the isolates could only be identified as either betabacteria or streptobacteria. The four isolates from Lacto were identified as Lactococcus lactis. They could not, however, be identified to subspecies level.
From this study we concluded that the differences in acceptability of traditionally fermented milk and Lacto are probably due to differences in the biochemical pathways and resulting types and levels of end products produced by the different bacteria responsible for fermentation of the two products. We suggested that more work should be done to determine the particular flavors and aroma present in traditionally fermented milk that are absent in Lacto and to determine whether any of our isolates are responsible for producing these desired properties.
E. COLI STRAINS IN LACTO AND TRADITIONALLY FERMENTED MILK
In another study (4) the growth and survival of pathogenic and nonpathogenic strains of E. cold were determined in traditionally fermented pasteurized and unpasteurized milk and Lacto. Unpasteurized and pasteurized milk and freshly inoculated Lacto, together with sterile control milk, were each inoculated with two strains of pathogenic and one strain of nonpathogenic E. cold to give approximately 103 cells/ milliliter. All the milk treatments were left to ferment at ambient temperature (20°C) for 24 hours. One set of the fermented products was stored at ambient temperature, and the other set was refrigerated (5°C) for another 96 hours. Samples were taken at 24-hour intervals and tested for numbers of E. coli, pH, and percentage of lactic acid.
Lacto inhibited all three E. cold strains. Two strains (one pathogenic and one nonpathogenic) could not be recovered, and the third (pathogenic) survived only in very low numbers after 24 hours of storage of Lacto at both 20° and 5°C.
All three E. cold strains survived and multiplied to maximum cell numbers in the range 107 to 109/milliliter during traditional fermentation of unpasteurized milk. Cell numbers decreased to 103 to 10e6 and 10e2 to 10e5 during storage of the fermented product at 20° and 5°C, respectively. These results indicated that traditional methods of fermenting milk in Zimbabwe pose a potential health hazard because, if milk is contaminated during milking or fermentation, E. coli, and possibly other enteric pathogens, are able to multiply to infective doses and retain relatively high numbers during storage of the product at both refrigeration and ambient temperatures. The results also indicated that more than acid production alone is involved in the fate of E. cold during fermentation and storage of Lacto and traditionally fermented unpasteurized milk since more E. cold survived in unpasteurized fermented milk despite similar final lactic acid and pH levels of both milk products. We suggested that, since in our earlier studies we found that different lactic acid bacteria were responsible for fermentation of the two milk products, it is likely that these organisms produce different types and quantities of other inhibitory products (antibiotics, volatile acids, hydrogen peroxide) during fermentation.
Higher maximum numbers, 109 to 10'° of the three strains of E. coli, were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10e5 to 10e3 and 10e4 to 10e7 during storage of the fermented product at 20° and 5°C, respectively. Under our experimental conditions there appeared to be more danger in traditionally fermenting pasteurized milk than unpasteurized milk; since less acid was produced, more E. cold multiplied and survived during fermentation and during storage of the pasteurized fermented milk. The practical relevance of this result should be interpreted with caution, since pasteurization also removes milk-borne organisms such as E. cold and Salmonella spp. and since it is unlikely that airborne recontamination of the milk by E. cold would result in initial numbers as high as 103 cells/milliliter. Thus, use of pasteurized milk in practice may not be as inappropriate as it might appear in theory.
Generally, fewer E. cold survived when the fermented milk products were stored at refrigeration than at ambient temperature. However, most people in rural areas of Zimbabwe do not have access to refrigerators.
We are currently determining the amounts of some B vitamins and of aroma and flavor compounds in traditionally fermented unpasteurized milk and Lacto. Preliminary results indicate that traditionally fermented milk contains more thiamine, riboflavin, pyridoxine, and folic acid than Lacto. Again, traditionally fermented unpasteurized mill: is performing better than Lacto.
From the work we have done so far there are two options to follow in our future studies. We know that traditionally fermented milk has similar amounts of lactic acid and a pH level similar to that of Lacto and that it might also have higher amounts of some B vitamins; however, it is not hygienically acceptable. We know some of the lactic acid strains involved in the fermentation, but we also know that in a situation where raw milk is used and fermentation is carried out under conditions where asepsis is not observed, other microorganisms, in addition to lactic acid bacteria, contribute to the production of aroma and flavor compounds. Supposing we were to develop a starter culture based mainly on members of the genus Lactobacilllus, it is debatable whether we would have the same organoleptic properties in a traditionally fermented pasteurized milk as found in traditionally fermented unpasteurized milk. If we developed and sold this starter culture for home use in fermentation of boiled milk, it is also unlikely that poor rural people would adopt such a fermentation since it has an added cost when compared with traditional fermentation.
Alternatively, we could incorporate some isolates from traditionally fermented milk into the Lacto starter culture and see whether the organoleptic properties of Lacto can be improved. Such a product would have to taste much better than traditionally fermented unpasteurized milk so as to entice rural populations to abandon traditional fermentation and adopt Lacto. Educational programs would have to be instituted for the public to appreciate the wisdom of spending money on buying Lacto, a hygienically safer product. At present, it is unlikely that Lacto will replace traditionally fermented milk in the foreseeable future.
1. Robinson, R. K., and A. Y. Tamime. 1981. Microbiology of fermented milks. Pp. 245-278 in: Dairy Microbiology, Vol. 2. R. K. Robinson, (Ed.). London: Applied Science Publishers.
2. Feresu, S., and M. I. Muzondo. 1989. Factors affecting the development of two fermented milk products in Zimbabwe. MIRCEN Journal of Applied Microbiology and Biotechnology 5:349-355.
3. Feresu, S., and M. I. Muzondo. 1990. Identification of some lactic acid bacteria from two Zimbabwean fermented milk products. World Journal of Microbiology and Biotechnology 6: 178-186.
4. Feresu, S., and H. Nyati. 1990. Fate of pathogenic and nonpathogenic Escherichia cold strains in two fermented milk products. Journal of Applied Bacteriology 69:814-821.
5. Sneath, P. H. A., and R. R. Sokal.1973. Numerical Taxonomy: The Principles and Practice of Numerical Classifications. San Francisco: W. H. Freeman, pp. 228-234.