Chemistry
AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-1998.jsonl.gz.gz
Agricultural And Food Sciences
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Growth Inhibition of Film-Forming Yeasts During Production of Ume-Zuke (Pickled Japanese Apricots) by an Antimicrobial Fraction Extracted from Paprika Seeds.

Paprika extract (50 to 100 μg/ml) obtained by suspending ground paprika seeds in water, adding ethanol (50%, v/v), and then filtering the suspension, inhibited the growth of film-forming yeasts (Kloeckera, Pichia, Debaryomyces, and Candida species) isolated during the production of ume-zuke, a processed food product made by salting Japanese apricots (Prunus mume). The antimicrobial activity of the extract towards Pichia anomala was influenced by the initial number of yeast cells, as well as the temperature, pH, and sodium chloride concentration of the culture medium (ume vinegar). The inhibitory effect was cumulatively enhanced, although not synergistically, when the paprika seed extract was used in combination with either SO2, sorbic acid, thiamine dilauryl sulfate, or acetic acid. The antimicrobial activity of the extract was not influenced by the vinegar components. The extract proved to be very effective as a preservative to prevent the contamination and spoilage of ume-zuke by film-forming yeasts.

Untll recently, the possibility of microbial contamination in the production of ume-zuke, a processed ume (Japanese apricot) product, has not been considered because it has traditionally been produced and preserved using a high concentration of salt and In the presence of organic acids which give the product a low pH. However, due to health considerations, an increasing number of people prefer umezuke with a lower salt concentration, which increases the risk of microbial contamination and spoilage mainly due to the presence of film-forming yeasts. During the production of ume-zuke, acid-resistant and/or salt-resistant film-forming yeasts occur during the salt preservation stage (i.e., the primary processing stage) (Onda et a/., 1997a), and develop as co]onies on the fruit surface at or after desalinization and seasoning (the secondary processing stage). Therefore, a natural preservative with hlgh antimicroblal activity against film-forming yeasts is essential. Yajima et a/ ( 1996Yajima et a/ ( , 1997 reported that the precipitate fractionated from an aqueous water-extract of paprika seeds by salting out with ammonium sulfate demonstrated antimicrobial actlvity against several yeasts. This preclpitate was partially purified by performing successive column chromatography steps. The present study describes the utilization of a paprika seed extract to inhibit the growth of film-forming yeasts isolated from ume-zuke during the primary processing stage in order to prevent its contamination and spoilage.

Materials and Methods
Preparation of antimicrobia/ fraction from 50% ethanol aqueous paprika seed extract Paprika seeds ( I kg) were ground to a powder in a mechanical grinder (Type D3V-B; Miyako Bussan Co., Ltd., Osaka). The powder was mixed with 2/ of water and stirred for 4 h at 40'C. To this suspension, 5/ of 70% ethanol was added and the solution was stirred for an additional 2 h. After suction filtering, the filtrate was concentrated to one-third its original volume by rotary evaporation at 40'C, and then sterilized by autoclaving.
Yeasts Assays for minimum inhibitory concentration Five film-forming yeast strains (K. apiculata YITC 203, P. anomala YITC 201 and YITC 256, C. guilliermondii YITC 222, and D. hansenii YITC 214) were pre-cultivated in YM medium containing 5% sodium chloride (pH 6.0) at 25'C for 2 days. The ume vinegar medium (5 ml) was placed in a test tube (17.5x 130 mm) and the 50% ethanol extract of paprika seeds was added to yield a concentration of 6.25, 12.5, 25, 50, IOO, 200, or 300 mg dry weight/1. The mixture was sterilized by heatlng at 65'C for 15 min, and 50 /ll of the pre-cultured yeast ( 105 cells/ml as determined using a Thoma counting chamber) was inoculated into the mixture. The mixture was statically incubated at 25'C for 10 days after sealing the test tube with a silicone foam plug, and the minimum inhibitory concentration of the 50% ethanol extract of paprika seeds was determined by observing the existence and degree of film on the surface of the culture medium. The yeast cell growth was monitored by following the optical density at 660 nm.
The effects of initial number of yeast cells, temperature, pH and NaCl concentration on the minimum inhioitory concentration of 50% ethanol paprika seed extract and other preservatives The effects ofthe culture conditions such as initial number of yeast cells, temperature, pH and NaCl concentration on the minimum inhibitory concentration of 50% ethanol paprika seed extract and other preservatives toward P. anomala YITC 256 were investigated. In the experiments, P. anomala YITC 256 cells were added to the solution to yield the final cell number of 102 to 107/ml, the pH of the ume vinegar medium was adjusted from I .5 to 4.0, the NaCl concentration was increased from 4.4% for the original concentration in the ume vinegar medium to 12.4 to 22.4% by adding table salt, and the culture medium temperature was varied between lO'C and 35'C. The culture medium was incubated for 10 days, and the minimum inhibitory concentration of each preservative was determined by observing the existence and extent of film on the surface of the culture medium.
Antimicrobia/ activity of 50% ethanol paprika seed extract and another antimicrobia/ substance The antimicrobial activity of the 50% ethanol extract of paprika seeds toward the film-forming yeasts was assayed in combination with potassium metabisulfite, potassium sorbate, acetic acid, or thiamine dilauryl sulfate. The 50% ethanol extract of paprika seeds was added to yield a final concentration of 6.25, 12.5, 25, 50, 100, or 200 mg dry weight/1. The four other preservatives were also added to give the various concentrations. The initial number of each of the five film-forming yeasts was I 05 cells/ml. The mixture was statically incubated at 25'C for 10 days, and its minimum inhibitory concentration was determined.

Results and Discussion
Minimum inhibitory concentration of 50% ethano/ aqueous paprika seed extract and other antimicrobial substances toward isolated flm-forming yeasts In a previous study (Yajima et al, 1996), the ammonium sulfate preclpitate from an aqueous paprika seed extract was investigated. In the present study, however, the antimicrobial fraction was not prepared by ammonium sulfate precipitation because this method would be impractical in commercial use due to its high production cost and low yield. Paprika seed extract obtained using only water can not be easily filtered through filter paper, which causes a considerable amount of antirnicrobial activity to be lost during filtration. Therefore, ethanol was added to the aqueous extract to yield a final ethanol concentration of approximately 50% (v/v) prior to filtration and subsequent concentratlon. The concentrate was used as the antimicrobial fraction. The dry weight of the concentrate after heating at 100'C for 24 h was approximately 97 g/kg'paprika seeds. The recovery of activity was much higher in the concentrate of the filtrate from the 50% ethanol aqueous extract than in that from an aqueous extract alone.
The antimicrobial activity toward film-forming yeasts isolated from ume-zuke containing sulfur dioxide, sorbic acid, acetic acid, and thiamine dilauryl sulfate as well as the 50% ethanol aqueous paprika seed extract (paprika seed extract) was investigated. This is because acetic acid is a common commercial food preservative, and thiamine dilauryl sulfate is often used for preserving processed ume products. Furthermore, sulfur dioxide and sorbic acid are also common commercial food preservatives, including ume products.
Thirteen strains from 4 species were isolated from ume fruits just after the addition of salt for preservation and from ume vinegar obtained between I and 6 weeks after the salt addition (Onda et al, 1997b). Film formation began 3 weeks after the salt addition. The film attained a width of about 0.5 cm in 6 weeks. Table I shows the MIC (minimum inhibitory concentration) of the paprika seed extract and the four antimicrobial substances toward the film-forming yeasts. The paprika seed extract demonstrated an inhibitory effect toward 5 strains from 4 species of the film-forming yeasts isolated at different times during salt preservation, and inhibited the growth of Kloeckera apiculata 203 at 50 pglml, and the other yeasts at 100pg/ml. Sulfur dioxide, sorbic acid, thiamine dilauryl sulfate, and acetic acid inhibited the growth of the five film-forming yeasts at a concentration between 3.1 and 12.5 Jug/ml, 25 and 100 pglml, 10 and 50pg/ml, and 3000 and 7000 pglml, respectively. The MIC of sulfur dioxide and thiamine dilauryl sulfate was lower than that of the paprika seed extract, whereas the inhibitory effect of sorbic acid was identical to that of the paprika seed extract on 2 of the 5 yeast strains tested and higher in the other 2 strains. In contrast to these substances, the growth of the yeasts was not inhibited by the 7 organic acids-adipic acid, fumaric acid, succinic acid, lactic acid, gluconic acid, malic acid, and citric acid-or ethanol and glycine, at higher concentrations. Little if any antimicrobial activity against the film-forming yeasts was expected in the case of only the organic acids because the tested yeasts were isolated from ume vinegar with a high concentration of salt (20%) and low pH (about 2). Furthermore, P. anomala and C. guilliermondii can assimilate citric acid and malic acid, which are contained in ume vinegar. The MIC of thiamine dilauryl sulfate was between that of sulfur dioxide and sorbic acid. The MIC of thiamine dilauryl sulfate has not been reported. Effect of initia/ number of yeast cells, temperature, pH, and sodium chloride concentration on antimicrobial activity of paprika seed extract Figure I shows the effect of the initial number of P. anomala YITC 256 yeast cells ( Fig. 1-a), temperature (b), pH (c), and sodium chloride concentration (d) on the antimicrobial activity of the paprika seed extract.
P. anomala YITC 256 is one of the most frequently found strains in ume vinegar with a low pH. This strain was present at the time the film formation began, and is an important part of the inhibition of film-forming by ume-zuke (Onda et al, 1997b). The MIC of the paprika seed extract was 25 to 100 pglml when the initial number of yeast cells was 102 to 106 cells/ml in the ume vinegar medium containing 4.4% NaCl (pH 2.0) arid the culture was incubated at 25'C. However, the growth was not inhibited even at an extract concentration of 300 pglml when the initial number was 107 cells/ml. The ume vinegar medium with 105 yeast cells/ml was a clear solution and that with 106 cells/ml was slightly cloudy (A660=about 0.1), while that with 107 cells/ml was very cloudy. These results suggest that the effective prevention of film formation can be expected if the paprika seed extract is added before the ume vinegar becomes cloudy. When the initial solution of P. anomala YITC 256 was inoculated into the medium at 105 cells/ml and incubated at different temperatures, which varied at 5'C intervals from lO to 35'C, for 10 days, the MIC toward the yeast of the paprika seed extract was markedly lower at the lower incubation temperature ( Fig. l-b).
Similarly, the MIC toward the yeast of the paprika seed extract was lower In an ume vinegar medium with a lower pH (range: 1.5 to 4.0) (Fig. 1-c). This pH effect is similar to that observed in carboxylic acid-based antimicrobial substances. Pitt ( 1974) reported that the maximum concentration of sorbic acid that still permits the growth of yeast (Pichia membranaefaciens) is 100 /lg/ml at a pH of 2.5 to 3.0, 200 pg/ml at pH 3.5, and 300 pglml at pH 4.0. Nomoto et al ( 1955) reported that the MIC toward the yeast (Saccharomyces cerevisiae) of sorbic acid is 20-100 pglml at pH 3.8 and 200-1000pg/ml at pH 5.3. Yamamoto et al (1984) reported that the MIC toward yeasts that are not resistant to the salt of acetic acid is between 3.5 and 4.0% and I .3 to I .4% toward yeasts resistant to salt. In contrast, the MIC of sorbic acid and acetic acid in the present study was lower. This may be due to differences in the composition and pH of the media used. In the present study, the proportion of undissociated acetic acid and sorbic acid in the ume vinegar medium was greater than that in the media reported by Pitt ( 1974) and Yamamoto et al ( 1984) for the same concentration of each organic acid because the pH of the ume vinegar medium was 10wer than those ofthe media used by Pitt and Yamamoto et   at a pH below 3.0 because the pKa of sorbic acid is 4.75 and the proportion of undissociated sorbic acid is approximately 98% of the total (Sofos & Busta,198 l). The MIC of carboxylic acid-based preservatives should be almost the same at a pH that is comparatively lower than their pKa, because at such a pH, almost all the preservative molecules become undissociated. Although the component of the paprika seed extract that demonstrates antimicrobial activity has not yet been identified, the MIC of this extract decreased wlth a decrease in the pH ofthe medium (range: 1.5 to 4.0). Thus, the pH effect on the antimicrobial activity differs from that of the well-known carboxylic acid-based preservatives. The antimicrobial activity of fatty acid esters is not generally influenced by the pH of the medium. However, Kato and Shibasaki ( 1975) reported that the antimicrobial activity of glycerol monolaurate (a fatty acid ester) was much higher than that of lauric acid (fatty acid) and slightly increased with a decrease in pH. The relationship between the antimicrobial activity of the paprika seed extract and the structure of the antimicrobial substance remains unclear and should be examined more closely in the future.
The sodium chloride concentration infiuenced the film formation of Pichia anomala YITC 256 such that the yeast did not grow in the presence of 22.4% sodium chloride in the ume vinegar medium. In general, the higher the concentration of sodium chloride, the lower the MIC of the paprika seed extract ( Fig. 1-d).