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Effect of gamma irradiation on microbial decontamination, and chemical and sensory characteristic of lycium fruit
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Radiation Physics and Chemistry 75 (2006) 596–603 www.elsevier.com/locate/radphyschem
Effect of gamma irradiation on microbial decontamination, and chemical and sensory characteristic of lycium fruit Hsiao-Wei Wena, Hsiao-Ping Chungb, Fong-In Choub,, I-hsin Linc, Po-Chow Hsiehc a Department of Food Science and Technology, Cornell University, Geneva, NY 14456, USA Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan, ROC c Committee on Chinese Medicine and Pharmacy, Department of Health, Executive Yuan, Taiwan, ROC
b
Received 17 June 2005; accepted 4 December 2005
Abstract Lycium fruit, popular traditional Chinese medicine and food supplement generally is ingested uncooked, was exposed to several doses of gamma irradiation (0–14 kGy) to evaluate decontamination efficiency, changes in chemical composition, and changes in sensory characteristic. In this study, lycium fruit specimens contained microbial counts of 3.1 103–1.7 105 CFU/g and 14 kGy was sufficient for microbial decontamination. Before irradiation, the main microbe isolated from lycium fruit was identified as a strain of yeast, Cryptococcus laurentii. After 10 kGy of irradiation, a Gram-positive spore-forming bacterium, Bacillus cereus, was the only survivor. The first 90% reduction (LD90) of C. laurentii and B. cereus was approximately 0.6 and 6.5 kGy, respectively, the D10 doses of C. laurentii and B. cereus was approximately 0.6 and 1.7 kGy, respectively. After 14 kGy irradiation, except the vitamin C content, other chemical composition (e.g., crude protein, b-carotene, riboflavin, fructose, etc.) and the sensory characteristic of lycium fruit specimens did not have significant changes. In conclusion, 14 kGy is the optimal decontamination dose for lycium fruit for retention of its sensory quality and extension of shelf life. r 2006 Elsevier Ltd. All rights reserved. Keywords: Microbial decontamination; Lycium fruit; Gamma irradiation; Chemical composition; Sensory characteristic; Food supplement; Traditional Chinese medicine
1. Introduction The popularity of Chinese medicine recently has been increasing since the good manufacturing practice system has begun to be applied in Chinese medicine, and the knowledge regarding the actions of Chinese medicine is increasing. However, microbiological contamination of herbal raw material is still a serious problem due to the Corresponding author. Tel.: +886 3 5721962; fax: +886 3 5725974. E-mail address: fi[email protected] (F.-I. Chou).
quality reduction of raw materials. This quality reduction can result in a decreasing production of therapeutical preparations. The goal of this research is to determine the optimal conditions for microbial decontamination of lycium fruit using gamma irradiation. Lycium fruit, the dried fruit of Lycium chinense Mill and Lycium barbarum L., is commonly employed in traditional Chinese medicinal treatment for improving vision, and used as a food supplement (Hsu and Peacher, 1994; Cheng et al., 2005). Lycium fruit could be a potential antioxidant for use in retarding the aging process and preventing diseases caused by reactive
0969-806X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2005.12.031
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oxygen species and free radicals (Ha et al., 2005; Luo et al., 2004). Crude extracts of lycium fruit were identified to be rich in antioxidants (e.g., carotenoids, riboflavin, etc.) (Luo et al., 2004). There are several ways to consume lycium fruit. People can ingest lycium fruit directly by chewing it without further processing. Moreover, the cold or hot water extract of lycium fruit can be used as drinks or can produce various types of health-beneficial foods. Lycium fruit contains abundant amounts of proteins, glucose and fructose thus making itself favorable to microbe propagation and affecting hygiene, particularly as the whole-grain fruit was eaten without cooking or in the form of tea. The recently increasing demand for lycium fruit as a health-beneficial food has motivated interest in the improvement in its quality. The quality of the lycium fruit is dependent on three major factors: the source, harvest time, and storage conditions. Microorganisms in the place of harvest, or the contamination and decay caused by microorganisms during transportation or storage commonly reduce the quality of lycium fruit. The high relative humidity and high temperature are the favorable conditions for microorganisms to grow, under which, lycium fruit easily becomes contaminated and decomposed, raising concerns for the safety of the consumers. Therefore, an effective method to eliminate the contamination of microorganisms from lycium fruit is a crucial requirement. Irradiation has become an effective means of processing and preserving food products (WHO, 1988; Molins, 2001; Fan et al., 2003). For example, gamma irradiation can extend shelf life of treated foods without inducing the formation of any radionuclide in food products. Food can be treated with irradiation either before or after packaging without heating, the addition of preservatives, or other processing. A Joint FAO/ IAEA/WHO Expert Committee on the Wholesomeness of Irradiation of Food has ruled that foods subjected to low dosages (10 kGy) of irradiation are safe and do not require toxicological testing (WHO, 1981). In 1997, a Joint FAO/IAEA/WHO Study Group was convened to assess the safety and nutritional adequacy of food irradiated to doses above 10 kGy. They concluded that food irradiated to any dose appropriate to achieve the intended technological objective is both safe to consume and nutritionally adequate (WHO, 1999). Numerous countries already apply irradiation to a wide range of food products and medicinal materials (Molins, 2001). Our previous studies have addressed the decontamination using radiation is a highly effective means of microbial decontamination for pollen (Chou, 1998). High-quality decontamination treatment should be effective against microorganisms, and adaptable to large amounts of material with high efficiency without reducing the quality of the treated commodities. This study investigated the decontamination of lycium fruit
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by radiation. In addition to determining the suitable irradiation dose for lycium fruit, we investigated how radiation affects the nourishing components and sensory characteristics. The nourishing components including b-carotene and riboflavin before and after irradiation were examined. Moreover, the predominant microbe strain was isolated and identified. The results in this study are valuable references for the food industry on irradiation processes, legislators and regulators.
2. Materials and methods 2.1. Sampling and irradiation of lycium fruit The dried fruit of L. chinense Mill was used for experiments. Dr. Y.S. Chung, Institute of Chinese Pharmaceutical Sciences, China Medical University, Taiwan, confirmed their authenticity. The fresh lycium fruit was ordered from various sources, Ning Xia, Mongolia and Xinjiang Autonomous Region, People Republic of China. The harvested lycium fruit was dried with sunshine for three consecutive days, transported to the harbor by car (for 7–9 days) under the ambient temperature (35 1C710 1C), and shipped to Taiwan (for 5 days) under 15 1C73 1C. Lycium fruit was sent from the importers to the retail shops of Chinese medicine within 15 days under the ambient temperature (25 1C710 1C). Ten samples of lycium fruit purchased from the importers or the local retail shops of Chinese medicine in Taiwan were stored under 15 1C and analyzed within 5 days. Gamma irradiation was performed in a 30,000 Ci cobalt-60 hot cell at the Nuclear Science and Technology Development Center, National Tsing Hua University. Specimens, each weighed 500 g, were used. Three replicates of 30 g sample were taken from each of the 500 g specimens and aerobically packed in a PVC (poly-vinyl chloride) container. Samples were placed in the cobalt-60 hot cell on irradiation stands and were irradiated at room temperature (25–30 1C). The irradiated stands were rotated ten times per min to ensure that the irradiated sample received a well-distributed radiation dose. The samples were treated with a constant dose rate of 5 kGy/ h for various time intervals in order to obtain 0, 2, 4, 6, 8, 10, 12, and 14 kGy of gamma rays. The absorbed dose was measured using silver dichromate dosimeter (ISO/ ASTM, 2003). The microbial content of the samples was measured immediately after irradiation. 2.2. Enumeration of microbes The solid culture media used in this study included plate count agar (PCA), tryptic soy agar (TSA), and potato dextrose agar (PDA; Difico, Detroit, USA) with a final pH value of 5.6; while, the liquid culture medium
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was plate count broth (PCB, Difico, Detroit, USA). The 0.067 M phosphate buffer saline (PBS, pH 7.0) contained 4.73 g of Na2HPO4 and 4.54 g of KH2PO4 per liter of distilled water. The two methods were applied to count the microorganisms in both the irradiated and non-irradiated lycium fruit: spread plate method and direct observation method. In spread plate method, each 30 g sample was immersed in 100 ml PBS at 10 1C for 10 min and homogenized with a Lab-blender stomacher MIX-1 (AES Laboratoire, France) for 3 min. Following adequate dilution of the resulting homogenate with PBS, the suspensions were spread and plated onto the PCA culture plates for total aerobic microbial enumeration. Molds and yeasts were enumerated by the spread plate method using a PDA with chloramphenicol (Beuchat, 1993). The plates were incubated at 25 1C, and not disturbed until colonies were counted. Each sample was tested on violet red bile glucose agar (Difico, Detroit, USA) to determine the total number of Enterobacteriaceae with incubation at 35 1C for 24–48 h. Counts were recorded in colony forming units per gram (CFU g1). The presented data were the average counts in three petri dishes for each test. The second method, direct observation method, was applied to determine the survivor of the microorganisms in the specimens. The lycium fruit specimens were placed directly on the surface of a plate culture medium and incubated at 30 1C for 1–7 days. The longer incubation time was needed because irradiation-induced cell injury delayed visible colony formation. The growth of microorganisms on and around the specimens was determined by visual observation and using a microscope. Five replicates were made for each specimen. 2.3. Identification of microbial strains and determination of radio resistance A strain, named as LY1 was isolated as the predominant strain from lycium fruit before irradiation. A strain, named as LB1, was the only survivor after 10 kGy of irradiation. The LB1 strain was cultured onto the TSA plate. In addition to identification of microbial strain with Gram staining and microscopy, we conducted the identification PC program at the Culture Collection and Research Center of the Food Industry Research and Development Institute, Taiwan. The characteristics of strain LB1 was shown in Table 1 and was identified as a Gram-positive rod, Bacillus cereus (Logan and Berkeley, 1984; Sneath, 1986; VITEK, 1995). Strain LY1 was identified according to its morphological and physiological characteristics and through the yeast identification PC program (Barnett et al., 1996). First, LY1 was cultured in glucose peptone yeast (GPY) broth (2% glucose, 1% peptone and 0.5% yeast extract) to determine the size, morphology and propagation of the vegetative cells, and then it was
cultured in GPY agar to determine color, brightness and surface characteristics of the colony (Vishniac, 2002). In addition, it was cultured in corn meal agar (Difico, Detroit, USA) to determine the growth of mycelium, and in malt extract agar (Difico, Detroit, USA) and corn meal agar to determine the spore formation. The physiological characteristics of the yeast, including its semi-anaerobic fermentation, aerobic utilization and growth in various nitrogen and carbon sources at temperatures from 20 to 42 1C, were tested. Finally, strain LY1 was identified as a strain of yeast, Cryptococcus laurentii (Barnett et al., 1990, 1996; Kurtzman and Fell, 1998). The radio resistance of isolated C. laurentii LY1 and B. cereus LB1 strains was evaluated in this study. C. laurentii LY1 was cultured in potato dextrose broth (PDB, Difico, Detroit, USA) at 25 1C and sampled in the exponential phase. After centrifugation for removing the media, C. laurentii LY1 was suspended in a PBS. B. cereus LB1 was cultured in PCB medium at 30 1C for 4–7 days. Sporulation was confirmed by microscopic examination. Spores were harvested by dipping the culture broth in a water bath at 80 1C for 10 min. After centrifugation, spores were suspended in PBS and stored at 4 1C before use. The microbe suspension was placed in test tubes and taken to the hot cell for irradiation, as described above. After exposure to various doses of radiation, the specimens were immediately examined to determine the survival rate by spreading 0.1 ml of the diluted microbe suspension onto a solid medium on a
Table 1 Characteristic of the isolate LB1 isolated from lycium fruit Test item
Characteristic
Cell morphology Motility Gram stain Oxidase test Spore Catalase test MacConkey growth Anaerobic grown O/F test Biology (4 h) 1st test Biology (24 h) 1st test Biology (4 h) 2nd test Biology (24 h) 2nd test API 50CHB Vitek ID BACIL Fatty acid analysis 1st test Fatty acid analysis 2nd test Identification
Rod + + + + + + Fermentation Bacillus 0.393 Bacillus cereus 99.9% Bacillus cereus 81% Bacillus thuringiensis 0.646 Bacillus cereus (or Bacillus thuringiensis)
+, positive reaction; , negative reaction.
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culture plate. The culture plates of C. laurentii LY1 and B. cereus LB1 strains were incubated at 25 and 30 1C, respectively. At the 48th, 72nd and 96th hours, the plates were removed to observe the growth condition and to count the colonies. The countable colony number is 30–300 per culture plate. The survival rate was estimated and the survival curve was plotted (Chou and Tan, 1990; Moseley and Copland, 1975). 2.4. Sample analysis The pH value, titratable acidity, moisture, crude proteins, b-carotene, riboflavin, and vitamin C in the lycium fruit samples were determined by official analysis methods of the AOAC 981.12, 942.15, 925.10, 976.06, 941.15, 981.15 and 984.26, respectively (AOAC, 1990). In addition, sugars and organic acid were analyzed by high-performance liquid chromatography (LC-10AT, Shimadzu, Japan) (Medlicott and Thompson, 1985). Moreover, color parameters of the surface of irradiated and non-irradiated lycium fruit samples, including Hunter’s lightness (L), redness (a) and yellowness (b), were measured with a color and color difference meter (model TC-8600A, Tokyo Denshoku Co.). 2.5. Sensory evaluation The triangle test was conducted to determine whether the panelists could distinguish between 14 kGy gamma irradiated and non-irradiated lycium fruit (Meilgaard et al., 1999). Twenty staff members from various divisions of the Nuclear Science and Technology Development Center were employed as the panelists for this work. Criteria for participation included willingness to sign an informed consent form and to evaluate the irradiated samples, and availability during the scheduled testing. Testing was undertaken in a place free from extraneous odors and sound. Panelists were requested not to talk during the procedure. The panel test was carried out by using natural light, since the panelists cannot detect such color difference visually. Five grams of the dried lycium fruit samples were placed in numerically coded weight glass beakers. Three lycium fruit samples, coded with three-digit random numbers, were presented to each panelist, along with water. Two of the samples were identical. Panelists individually evaluated the samples and marked score sheets. The panelists were instructed to evaluate the samples by chewing the dried whole-grain lycium fruit in the order and should clear their palates between samples using water and determine which one is the odd sample. The panelists were allowed to re-test the samples. Panelists who could not detect differences were allowed to guess. No panelist or datum was eliminated. The mean value obtained from three independent tests was recorded.
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3. Results and discussion 3.1. Microorganisms in lycium fruit The spread plate method and direct observation were used to evaluate the viability of microorganisms in the specimens of lycium fruit following irradiation. From the direct observation of the microorganism growth in the specimen of non-irradiated lycium fruit on a PCA plate, the species and amounts of microorganisms clearly varied around the lycium fruit specimens. Some of specimens were covered with yellow yeast, which produced a large volume of gas in the culture plate. The yellow yeast was identified as C. laurentii. In addition, some of the specimens were polluted by mold. The lycium fruit specimens contained total mesophilic microbial counts of 3.1 103–1.7 105 CFU/g and the microbial profiles of specimens sampled from different sources varied markedly. The viability of microorganisms in lycium fruit, following irradiation with different gamma-ray radiation doses of 0, 4, 6, 8, 10, 12 and 14 kGy, was evaluated. Although most of the specimens contained no living microbes after irradiation at a dose of 8 kGy, some specimens required a 14-kGy irradiation for complete decontamination. However, for Enterobacteriaceae inactivation, 4 kGy was a sufficient dose. As the radiation dose increased, the microbial profiles in the lycium fruit significantly changed. Yeast and molds initially grew on all non-irradiated lycium fruit. Following irradiations at the doses of 4 and 6 kGy, the concentration of yeast was obviously reduced; therefore, fungi and bacteria gradually became the predominant. Following the irradiation at 8 kGy, some of the specimens still contained a few viable fungi, and the main microbial species had become a bacterial strain with a cream-colored colony. Following the irradiation at 10 kGy, the lycium fruit specimens contained only a viable bacterium of the aforementioned cream-colored bacterial strain. This surviving strain was isolated and identified as a Gram-positive spore-forming bacterium, B. cereus, and it could be completely decontaminated after the irradiation at a dose of 14 kGy. This research employed two approaches to evaluate the viability of microorganisms in the irradiated samples, because a single method cannot yield complete results. The first method is the spread-plate method, in which the samples were immersed with shaking. After a proper dilution, the immersed suspensions were evenly spread on the plate. After the microbial colony had formed, the colony morphology was observed and the colony number was counted. The strength of this approach is that the microbial colony exhibits clear growth and allows easy counting. However, the weakness is that the growth of some strains of the microbes depends on particular component of the lycium fruit,
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so the microbial colonies cannot form on the general medium for counting, potentially leading to underestimation. The second method is to put the sample of lycium fruits directly onto the medium and to observe the growth of microbes. The microbes are counted by eye, and an optical microscope is used to observe the microbial growth on the lycium fruit and the proliferation from the lycium fruit to the medium. The strength of this approach is that the strains of microbes that depend on particular components in lycium fruit can grow selectively on both the fruit and the adjacent medium. Restated, it provides opportunities for growth. Also, a microscope can be used to observe clearly the situation with regard to growth of the microbe on the lycium fruit and the erosion of the lycium fruits. However, the weakness is that when the microbe grows on lycium fruit with a less plain surface or a less consistent color, the microbial colonies are not clearly seen and so can be easily missed. Additionally, when the amount of microbe is high or many microbes are present, the microbial colonies on the lycium fruit frequently overlap as they grow, so differentiating and counting accurately the microbial colonies become difficult. Therefore, the two methods mentioned above should be simultaneously applied to determine the number of microbe contained in lycium fruit during the determination of the relationship between radiation dose and decontamination of the microbes. Furthermore, the growth of the microbes in the irradiated lycium fruit is slower than that in the non-irradiated lycium fruit. Therefore, more time is required to observe the microbial colonies and a 2-day delay is required before counting to ensure that the number of microbial colonies is not underestimated. Although the amount of microbes in the lycium fruit is the main factor that determines the required radiation dose, the presence of spore-forming microbial strains is also an important reason for the required increase in radiation dosage, confirming that the irradiation of lycium fruit at a dose of 14 kGy can effectively eliminate microbes. In this study, gamma irradiation effectively inhibited the process of fermentation of lycium fruit by killing yeasts, greatly improving the quality of the lycium fruit and its stability during transport and storage.
0 Log Inactivation (CFU/ml)
600
-1 -2 -3 -4 -5 -6
C. laurentii B. cereus
-7 0
10 12 4 6 8 2 Dose of Co-60 Gamma Radiation (kGy)
14
Fig. 1. Surviving fraction of the strain of yeast, Cryptococcus laurentii, and endospores of Bacillus cereus at various doses of gamma irradiation.
(0.6 kGy) for the first 90% reduction (LD90) and D10. However, the survival curve of the spores of B. cereus falls slowly and has a shoulder. Therefore, it has different values for the LD90 (6.5 kGy) and D10 (1.7 kGy). The D10 value is normally calculated over the linear part of the death curve, but the presence of a shoulder may result in underestimation of the actual dose required. Therefore, these two parameters (LD90 and D10) are required to precisely describe the radiation resistance of microorganisms. The spore-forming bacteria are the more resistant ones, because bacteria in spore form tend to be much more resistant to radiation than those in vegetative form. Therefore, the radiation dose required to decontaminate lycium fruits should be determined from the dose required to eliminate the endospore of B. cereus strain. However, comparing B. cereus with the highly radio-resistant bacterial strain, Deinococcus radiodurans, which is also appearing in food (Moseley, 1983), reveals that the radioresistance of B. cereus is clearly lower than that of D. radiodurans. The LD90 for D. radiodurans is about 13 kGy (Chou and Tan, 1990; Tan and Maxcy, 1982). The presence of B. cereus bacteria in lycium fruit specimens should not raise problems for radiation decontamination.
3.2. Radio resistance of the predominant yeast and the spore-forming bacterial strain in the lycium fruit specimen
3.3. Composition changes of lycium fruit after gammaray irradiation
The radiation resistance of C. laurentii, the predominant microbial strain in the pre-irradiated lycium fruit specimens, and B. cereus, the bacterial strain isolated from the 10 kGy irradiated lycium fruit specimens, was measured and shown in Fig. 1. The radio resistance of these two strains differed greatly. The survival curve of the yeast, C. laurentii, has no shoulder and its slope declines sharply. This curve shows the same value
Specimens of lycium fruit that had been gamma irradiated at 4, 8 and 14 kGy, and those of non-irradiated lycium fruit, were examined to determine their pH value, titratable acidity, moisture, crude protein, b-carotene, riboflavin, vitamin C, fructose, glucose, citric acid, malic acid, succinic acid, fumaric acid and oxalic acid contents. The pH value of the specimens of non-irradiated lycium fruit was 5.08, and the titratable acidity that expressed as
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Table 2 Effects of gamma-irradiation on the composition in lycium fruit Items
Moisture (g/100 g) Crude protein (g/100 g) b-carotene (mg/100 g) Riboflavin (mg/100 g) Vitamin C (mg/100 g) Fructose (g/100 g) Glucose (g/100 g) Citric acid (g/100 g) Malic acid (g/100 g) Succinic acid (g/100 g) Fumaric acid (g/100 g) Oxalic acid (g/100 g)
Irradiation dose (kGy) 0
4
8
14
16.51 13.44 1.13 0.63 8.35 25.03 22.84 4.26 2.54 1.18 0.02 0.14
16.61 13.24 1.13 0.64 4.3 24.91 22.76 4.24 2.47 1.21 0.02 0.15
16.58 13.31 1.12 0.63 ND 25.01 22.83 4.31 2.49 1.18 0.02 0.14
16.60 13.19 1.12 0.60 ND 24.99 22.78 4.35 2.55 1.22 0.02 0.15
Each value was the average of triplicate of experiments. ND, indicates no vitamin C detected.
titration ml of 0.1 N NaOH was 4.2. No significant changes in pH and acidity were observed following 4, 8 and 14 kGy gamma irradiation. The approximate compositions in 100 g lycium fruit samples were 16.51 g moisture, 13.44 g crude protein, 1.13 mg b-carotene, 0.63 mg riboflavin, 8.35 mg vitamin C, 25.03 g fructose, 22.84 g glucose, 4.26 g citric acid, 2.54 g malic acid, 1.18 g succinic acid, 0.02 g fumaric acid and 0.14 g oxalic acid. Table 2 presents the results. Lycium fruit is the essential source of b-carotene and riboflavin (Luo et al., 2004). b-carotene is an anti-oxidant and a precursor of vitamin A, and is believed to improve vision (Hammond et al., 2001; Keijer et al., 2005). Notably, gamma irradiation had no effect on b-carotene content. Riboflavin, an anti-oxidant and a water-soluble vitamin, is essential for normal cellular functions (Foraker et al., 2003). Riboflavin was 0.63 mg per 100 g before irradiation and slightly decreased to 0.60 mg per 100 g after the 14 kGy irradiation. Other composition was not significantly changed by gamma irradiation except the vitamin C content. We observed that the vitamin C content was decreased following the increasing doses of gamma irradiation; this result was similar as the effect of gamma irradiation on dried potato slices (Wang and Du, 2005). The reduce of the vitamin C content should not be a significant problem, because lycium fruit that commonly employed in traditional Chinese medicinal treatment for improving vision is the essential source of b-carotene, not vitamin C (Hsu and Peacher, 1994).
(Table 3). Gamma irradiation slightly changed the color specifications of the surface of the dried lycium fruit samples: the lightness, redness, and yellowness of the samples slightly declined at high dosage (14 kGy). From a three-dimensional scatter plot, all the L, a, b values varied by two variables (storage time and irradiation doses) were not significantly different from one another at 95% confidence level. However, the b value seemed to slightly alter only when gamma dose was changed.
3.4. Hunter’s color value
4. Conclusion
Hunter’s color parameters of the irradiated and nonirradiated lycium fruit, such as lightness (L), redness (a), yellowness (b) and color difference (DE), were determined
In this research, the optimal doses for inactivating microorganisms in lycium fruit have been studied. The sufficient dose for inactivating Enterobacteriaceae was
3.5. Sensory evaluation Sensory analysis was performed immediately following irradiation. Triangle test panelists could not detect any significant difference between the 14-kGy gamma irradiated and non-irradiated lycium fruit. For N ¼ 20, a total of 11 correct responses were required to conclude a significant difference at pp0.05 (Meilgaard et al., 1999). In our study, of the 20 panelists, only 7 correctly selected the odd sample. This number was smaller than that required to confirm significance at a 5% level. Moreover, some panelists, who presented the correct results, could not specify the difference between these two samples. This uncertainty indicated that panelists could not distinguish non-irradiated lycium fruits from lycium fruit irradiated at 14 kGy, as expected just by chance. The evaluation showed that lycium fruit retained their sensory quality after irradiation.
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Table 3 Changes in Hunter’s color value of non-irradiated and gamma irradiated lycium fruit Color parametera
L
Storage period (month)
Irradiation dose (kGy) 0
4
25.22b 25.06 18.29 19.73 9.79 9.89 0.00 1.452
24.85 23.75 18.28 18.49 9.85 9.76 0.375 1.484
8
14
24.98 23.83 23.08 23.17 a 18.02 17.95 17.99 17.82 b 9.82 9.45 9.60 9.35 DE 0.362 1.471 1.464 2.149 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi a 2 2 L, degree of lightness; a, degree of redness; b, degree of yellowness; DE, the overall color difference ( ðDLÞ þ ðDaÞ þ ðDbÞ2 ). b Each value was the average of triplicate determinations. 0 3 0 3 0 3 0 3
4 kGy and 14 kGy was required for eliminating the total mesophilic microbes. In addition, 8 kGy was a sufficient dose for the inactivation of yeasts, resulting in eliminating the unwanted fermentation process of lycium fruit. To investigate the effect of gamma irradiation on chemical composition of lycium fruit, the crude protein, b-carotene, riboflavin, vitamin C, fructose, glucose, citric acid, malic acid, succinic acid, fumaric acid, and oxalic acid contents of lycium fruit were analyzed before and after irradiation. Results showed that vitamin C content reduced after irradiation at 14 kGy. However, amount of other composition including b-carotene and riboflavin did not change significantly under the same treatment. Lycium fruit has been used as the major source of b-carotene and riboflavin, and people normally ingest lycium fruit to improve their vision. In addition, the sensory characteristic of lycium fruit also did not significantly alter after the 14 kGy irradiation. Therefore, gamma irradiation at 14 kGy can be a potential method as the cold decontamination for lycium fruit to prolong the shelf life, to improve the hygienic quality, and to reduce the risk of food borne disease.
Acknowledgements We would like to thank the Committee on Chinese Medicine and Pharmacy of the Department of Health, Taiwan, for their financial support (CCMP-RD-045), Professor Y.S. Chung for confirmation of the authenticity of lycium fruit, and our colleagues in the Nuclear Science and Technology Development Center for their assistance in performing gamma irradiation and sensory evaluation.
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ARTICLE IN PRESS H.-W. Wen et al. / Radiation Physics and Chemistry 75 (2006) 596–603 Kurtzman, C.P., Fell, J.W. (Eds.), 1998. The Yeasts, a Taxonomic Study, fourth ed. Elsevier Science, Amsterdam. Logan, N.A., Berkeley, R.C.W., 1984. Identification of Bacillus strains using the API system. J. Gen. Microbiol. 130, 1871–1882. Luo, Q., Cai, Y., Yan, J., Sun, M., Corke, H., 2004. Hypoglycemic and hypolipidemic effects and antioxidant activity of fruit extracts from Lycium barbarum. Life Sci. 76, 137–149. Medlicott, A.P., Thompson, A.K., 1985. Analysis of sugars and organic acids in ripening mango fruits (Mangifera indica L. var Keitt) by high performance liquid chromatography. J. Sci. Food Agric. 36, 561–566. Meilgaard, M., Civille, G.V., Carr, B.T., 1999. Statistical tables T8. Critical number of correct responses in a triangle test. In: Lourdes, F. (Ed.), Sensory Evaluation Techniques, third ed. CRC Press, Boca Raton, Fl. 369pp. Molins, R.A. (Eds.), 2001. Food Irradiation: Principles and Applications. Wiley-Interscience, New York. Moseley, B.E.B., 1983. Photobiology and radiobiology of Micrococcus (Deinococcus) radiodurans. Photochem. Photobiol. Rev. 7, 223–274. Moseley, B.E.B., Copland, H.J.R., 1975. Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans. J. Bacteriol. 121, 422–428. Sneath, P.H.A., 1986. Endospore-forming gram-positive rods and cocci. In: Sheath, P.H.A., Mair, N.S., Sharpe,
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M.E., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 2. Williams & Wilkins, Baltimore, pp. 1105–1139. Tan, S.T., Maxcy, R.B., 1982. Inactivation and injury of a hemolytic radiation-resistant Micrococcus isolated from chicken meat. J. Food. Sci. 47, 1345–1349 1353. Vishniac, H.S., 2002. Cryptococcus tephrensis, sp.nov., and Cryptococcus heimaeyensis, sp.nov.; new anamorphic basidiomycetous yeast species from Iceland. Can. J. Microbiol. 48, 463–467. VITEK Procedures Manual, 1995. BioMe´rieux Vitek Inc., USA. Wang, J., Du, Y., 2005. The effect of gamma-ray irradiation on the drying characteristics and final quality of dried potato slices. Int. J. Food Sci. Technol. 40, 75–82. WHO, 1981. Wholesomeness of irradiated food: report of a Joint FAO/IAEA/WHO Expert Committee. In: World Health Organization Technical Report Series 659. WHO, Geneva, Switzerland, 36pp. WHO, 1988. Food Irradiation: A Technique for Preserving and Improving the Safety of Food. WHO, Geneva, Switzerland. WHO, 1999. High-Dose Irradiation: Wholesomeness of food irradiated with dose above 10 kGy: report of a Joint FAO/ IAEA/WHO Study Group. In: World Health Organization Technical Report Series 890. WHO, Geneva, Switzerland, 7pp.
Radiation Physics and Chemistry 75 (2006) 596–603 www.elsevier.com/locate/radphyschem
Effect of gamma irradiation on microbial decontamination, and chemical and sensory characteristic of lycium fruit Hsiao-Wei Wena, Hsiao-Ping Chungb, Fong-In Choub,, I-hsin Linc, Po-Chow Hsiehc a Department of Food Science and Technology, Cornell University, Geneva, NY 14456, USA Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan, ROC c Committee on Chinese Medicine and Pharmacy, Department of Health, Executive Yuan, Taiwan, ROC
b
Received 17 June 2005; accepted 4 December 2005
Abstract Lycium fruit, popular traditional Chinese medicine and food supplement generally is ingested uncooked, was exposed to several doses of gamma irradiation (0–14 kGy) to evaluate decontamination efficiency, changes in chemical composition, and changes in sensory characteristic. In this study, lycium fruit specimens contained microbial counts of 3.1 103–1.7 105 CFU/g and 14 kGy was sufficient for microbial decontamination. Before irradiation, the main microbe isolated from lycium fruit was identified as a strain of yeast, Cryptococcus laurentii. After 10 kGy of irradiation, a Gram-positive spore-forming bacterium, Bacillus cereus, was the only survivor. The first 90% reduction (LD90) of C. laurentii and B. cereus was approximately 0.6 and 6.5 kGy, respectively, the D10 doses of C. laurentii and B. cereus was approximately 0.6 and 1.7 kGy, respectively. After 14 kGy irradiation, except the vitamin C content, other chemical composition (e.g., crude protein, b-carotene, riboflavin, fructose, etc.) and the sensory characteristic of lycium fruit specimens did not have significant changes. In conclusion, 14 kGy is the optimal decontamination dose for lycium fruit for retention of its sensory quality and extension of shelf life. r 2006 Elsevier Ltd. All rights reserved. Keywords: Microbial decontamination; Lycium fruit; Gamma irradiation; Chemical composition; Sensory characteristic; Food supplement; Traditional Chinese medicine
1. Introduction The popularity of Chinese medicine recently has been increasing since the good manufacturing practice system has begun to be applied in Chinese medicine, and the knowledge regarding the actions of Chinese medicine is increasing. However, microbiological contamination of herbal raw material is still a serious problem due to the Corresponding author. Tel.: +886 3 5721962; fax: +886 3 5725974. E-mail address: fi[email protected] (F.-I. Chou).
quality reduction of raw materials. This quality reduction can result in a decreasing production of therapeutical preparations. The goal of this research is to determine the optimal conditions for microbial decontamination of lycium fruit using gamma irradiation. Lycium fruit, the dried fruit of Lycium chinense Mill and Lycium barbarum L., is commonly employed in traditional Chinese medicinal treatment for improving vision, and used as a food supplement (Hsu and Peacher, 1994; Cheng et al., 2005). Lycium fruit could be a potential antioxidant for use in retarding the aging process and preventing diseases caused by reactive
0969-806X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2005.12.031
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oxygen species and free radicals (Ha et al., 2005; Luo et al., 2004). Crude extracts of lycium fruit were identified to be rich in antioxidants (e.g., carotenoids, riboflavin, etc.) (Luo et al., 2004). There are several ways to consume lycium fruit. People can ingest lycium fruit directly by chewing it without further processing. Moreover, the cold or hot water extract of lycium fruit can be used as drinks or can produce various types of health-beneficial foods. Lycium fruit contains abundant amounts of proteins, glucose and fructose thus making itself favorable to microbe propagation and affecting hygiene, particularly as the whole-grain fruit was eaten without cooking or in the form of tea. The recently increasing demand for lycium fruit as a health-beneficial food has motivated interest in the improvement in its quality. The quality of the lycium fruit is dependent on three major factors: the source, harvest time, and storage conditions. Microorganisms in the place of harvest, or the contamination and decay caused by microorganisms during transportation or storage commonly reduce the quality of lycium fruit. The high relative humidity and high temperature are the favorable conditions for microorganisms to grow, under which, lycium fruit easily becomes contaminated and decomposed, raising concerns for the safety of the consumers. Therefore, an effective method to eliminate the contamination of microorganisms from lycium fruit is a crucial requirement. Irradiation has become an effective means of processing and preserving food products (WHO, 1988; Molins, 2001; Fan et al., 2003). For example, gamma irradiation can extend shelf life of treated foods without inducing the formation of any radionuclide in food products. Food can be treated with irradiation either before or after packaging without heating, the addition of preservatives, or other processing. A Joint FAO/ IAEA/WHO Expert Committee on the Wholesomeness of Irradiation of Food has ruled that foods subjected to low dosages (10 kGy) of irradiation are safe and do not require toxicological testing (WHO, 1981). In 1997, a Joint FAO/IAEA/WHO Study Group was convened to assess the safety and nutritional adequacy of food irradiated to doses above 10 kGy. They concluded that food irradiated to any dose appropriate to achieve the intended technological objective is both safe to consume and nutritionally adequate (WHO, 1999). Numerous countries already apply irradiation to a wide range of food products and medicinal materials (Molins, 2001). Our previous studies have addressed the decontamination using radiation is a highly effective means of microbial decontamination for pollen (Chou, 1998). High-quality decontamination treatment should be effective against microorganisms, and adaptable to large amounts of material with high efficiency without reducing the quality of the treated commodities. This study investigated the decontamination of lycium fruit
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by radiation. In addition to determining the suitable irradiation dose for lycium fruit, we investigated how radiation affects the nourishing components and sensory characteristics. The nourishing components including b-carotene and riboflavin before and after irradiation were examined. Moreover, the predominant microbe strain was isolated and identified. The results in this study are valuable references for the food industry on irradiation processes, legislators and regulators.
2. Materials and methods 2.1. Sampling and irradiation of lycium fruit The dried fruit of L. chinense Mill was used for experiments. Dr. Y.S. Chung, Institute of Chinese Pharmaceutical Sciences, China Medical University, Taiwan, confirmed their authenticity. The fresh lycium fruit was ordered from various sources, Ning Xia, Mongolia and Xinjiang Autonomous Region, People Republic of China. The harvested lycium fruit was dried with sunshine for three consecutive days, transported to the harbor by car (for 7–9 days) under the ambient temperature (35 1C710 1C), and shipped to Taiwan (for 5 days) under 15 1C73 1C. Lycium fruit was sent from the importers to the retail shops of Chinese medicine within 15 days under the ambient temperature (25 1C710 1C). Ten samples of lycium fruit purchased from the importers or the local retail shops of Chinese medicine in Taiwan were stored under 15 1C and analyzed within 5 days. Gamma irradiation was performed in a 30,000 Ci cobalt-60 hot cell at the Nuclear Science and Technology Development Center, National Tsing Hua University. Specimens, each weighed 500 g, were used. Three replicates of 30 g sample were taken from each of the 500 g specimens and aerobically packed in a PVC (poly-vinyl chloride) container. Samples were placed in the cobalt-60 hot cell on irradiation stands and were irradiated at room temperature (25–30 1C). The irradiated stands were rotated ten times per min to ensure that the irradiated sample received a well-distributed radiation dose. The samples were treated with a constant dose rate of 5 kGy/ h for various time intervals in order to obtain 0, 2, 4, 6, 8, 10, 12, and 14 kGy of gamma rays. The absorbed dose was measured using silver dichromate dosimeter (ISO/ ASTM, 2003). The microbial content of the samples was measured immediately after irradiation. 2.2. Enumeration of microbes The solid culture media used in this study included plate count agar (PCA), tryptic soy agar (TSA), and potato dextrose agar (PDA; Difico, Detroit, USA) with a final pH value of 5.6; while, the liquid culture medium
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was plate count broth (PCB, Difico, Detroit, USA). The 0.067 M phosphate buffer saline (PBS, pH 7.0) contained 4.73 g of Na2HPO4 and 4.54 g of KH2PO4 per liter of distilled water. The two methods were applied to count the microorganisms in both the irradiated and non-irradiated lycium fruit: spread plate method and direct observation method. In spread plate method, each 30 g sample was immersed in 100 ml PBS at 10 1C for 10 min and homogenized with a Lab-blender stomacher MIX-1 (AES Laboratoire, France) for 3 min. Following adequate dilution of the resulting homogenate with PBS, the suspensions were spread and plated onto the PCA culture plates for total aerobic microbial enumeration. Molds and yeasts were enumerated by the spread plate method using a PDA with chloramphenicol (Beuchat, 1993). The plates were incubated at 25 1C, and not disturbed until colonies were counted. Each sample was tested on violet red bile glucose agar (Difico, Detroit, USA) to determine the total number of Enterobacteriaceae with incubation at 35 1C for 24–48 h. Counts were recorded in colony forming units per gram (CFU g1). The presented data were the average counts in three petri dishes for each test. The second method, direct observation method, was applied to determine the survivor of the microorganisms in the specimens. The lycium fruit specimens were placed directly on the surface of a plate culture medium and incubated at 30 1C for 1–7 days. The longer incubation time was needed because irradiation-induced cell injury delayed visible colony formation. The growth of microorganisms on and around the specimens was determined by visual observation and using a microscope. Five replicates were made for each specimen. 2.3. Identification of microbial strains and determination of radio resistance A strain, named as LY1 was isolated as the predominant strain from lycium fruit before irradiation. A strain, named as LB1, was the only survivor after 10 kGy of irradiation. The LB1 strain was cultured onto the TSA plate. In addition to identification of microbial strain with Gram staining and microscopy, we conducted the identification PC program at the Culture Collection and Research Center of the Food Industry Research and Development Institute, Taiwan. The characteristics of strain LB1 was shown in Table 1 and was identified as a Gram-positive rod, Bacillus cereus (Logan and Berkeley, 1984; Sneath, 1986; VITEK, 1995). Strain LY1 was identified according to its morphological and physiological characteristics and through the yeast identification PC program (Barnett et al., 1996). First, LY1 was cultured in glucose peptone yeast (GPY) broth (2% glucose, 1% peptone and 0.5% yeast extract) to determine the size, morphology and propagation of the vegetative cells, and then it was
cultured in GPY agar to determine color, brightness and surface characteristics of the colony (Vishniac, 2002). In addition, it was cultured in corn meal agar (Difico, Detroit, USA) to determine the growth of mycelium, and in malt extract agar (Difico, Detroit, USA) and corn meal agar to determine the spore formation. The physiological characteristics of the yeast, including its semi-anaerobic fermentation, aerobic utilization and growth in various nitrogen and carbon sources at temperatures from 20 to 42 1C, were tested. Finally, strain LY1 was identified as a strain of yeast, Cryptococcus laurentii (Barnett et al., 1990, 1996; Kurtzman and Fell, 1998). The radio resistance of isolated C. laurentii LY1 and B. cereus LB1 strains was evaluated in this study. C. laurentii LY1 was cultured in potato dextrose broth (PDB, Difico, Detroit, USA) at 25 1C and sampled in the exponential phase. After centrifugation for removing the media, C. laurentii LY1 was suspended in a PBS. B. cereus LB1 was cultured in PCB medium at 30 1C for 4–7 days. Sporulation was confirmed by microscopic examination. Spores were harvested by dipping the culture broth in a water bath at 80 1C for 10 min. After centrifugation, spores were suspended in PBS and stored at 4 1C before use. The microbe suspension was placed in test tubes and taken to the hot cell for irradiation, as described above. After exposure to various doses of radiation, the specimens were immediately examined to determine the survival rate by spreading 0.1 ml of the diluted microbe suspension onto a solid medium on a
Table 1 Characteristic of the isolate LB1 isolated from lycium fruit Test item
Characteristic
Cell morphology Motility Gram stain Oxidase test Spore Catalase test MacConkey growth Anaerobic grown O/F test Biology (4 h) 1st test Biology (24 h) 1st test Biology (4 h) 2nd test Biology (24 h) 2nd test API 50CHB Vitek ID BACIL Fatty acid analysis 1st test Fatty acid analysis 2nd test Identification
Rod + + + + + + Fermentation Bacillus 0.393 Bacillus cereus 99.9% Bacillus cereus 81% Bacillus thuringiensis 0.646 Bacillus cereus (or Bacillus thuringiensis)
+, positive reaction; , negative reaction.
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culture plate. The culture plates of C. laurentii LY1 and B. cereus LB1 strains were incubated at 25 and 30 1C, respectively. At the 48th, 72nd and 96th hours, the plates were removed to observe the growth condition and to count the colonies. The countable colony number is 30–300 per culture plate. The survival rate was estimated and the survival curve was plotted (Chou and Tan, 1990; Moseley and Copland, 1975). 2.4. Sample analysis The pH value, titratable acidity, moisture, crude proteins, b-carotene, riboflavin, and vitamin C in the lycium fruit samples were determined by official analysis methods of the AOAC 981.12, 942.15, 925.10, 976.06, 941.15, 981.15 and 984.26, respectively (AOAC, 1990). In addition, sugars and organic acid were analyzed by high-performance liquid chromatography (LC-10AT, Shimadzu, Japan) (Medlicott and Thompson, 1985). Moreover, color parameters of the surface of irradiated and non-irradiated lycium fruit samples, including Hunter’s lightness (L), redness (a) and yellowness (b), were measured with a color and color difference meter (model TC-8600A, Tokyo Denshoku Co.). 2.5. Sensory evaluation The triangle test was conducted to determine whether the panelists could distinguish between 14 kGy gamma irradiated and non-irradiated lycium fruit (Meilgaard et al., 1999). Twenty staff members from various divisions of the Nuclear Science and Technology Development Center were employed as the panelists for this work. Criteria for participation included willingness to sign an informed consent form and to evaluate the irradiated samples, and availability during the scheduled testing. Testing was undertaken in a place free from extraneous odors and sound. Panelists were requested not to talk during the procedure. The panel test was carried out by using natural light, since the panelists cannot detect such color difference visually. Five grams of the dried lycium fruit samples were placed in numerically coded weight glass beakers. Three lycium fruit samples, coded with three-digit random numbers, were presented to each panelist, along with water. Two of the samples were identical. Panelists individually evaluated the samples and marked score sheets. The panelists were instructed to evaluate the samples by chewing the dried whole-grain lycium fruit in the order and should clear their palates between samples using water and determine which one is the odd sample. The panelists were allowed to re-test the samples. Panelists who could not detect differences were allowed to guess. No panelist or datum was eliminated. The mean value obtained from three independent tests was recorded.
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3. Results and discussion 3.1. Microorganisms in lycium fruit The spread plate method and direct observation were used to evaluate the viability of microorganisms in the specimens of lycium fruit following irradiation. From the direct observation of the microorganism growth in the specimen of non-irradiated lycium fruit on a PCA plate, the species and amounts of microorganisms clearly varied around the lycium fruit specimens. Some of specimens were covered with yellow yeast, which produced a large volume of gas in the culture plate. The yellow yeast was identified as C. laurentii. In addition, some of the specimens were polluted by mold. The lycium fruit specimens contained total mesophilic microbial counts of 3.1 103–1.7 105 CFU/g and the microbial profiles of specimens sampled from different sources varied markedly. The viability of microorganisms in lycium fruit, following irradiation with different gamma-ray radiation doses of 0, 4, 6, 8, 10, 12 and 14 kGy, was evaluated. Although most of the specimens contained no living microbes after irradiation at a dose of 8 kGy, some specimens required a 14-kGy irradiation for complete decontamination. However, for Enterobacteriaceae inactivation, 4 kGy was a sufficient dose. As the radiation dose increased, the microbial profiles in the lycium fruit significantly changed. Yeast and molds initially grew on all non-irradiated lycium fruit. Following irradiations at the doses of 4 and 6 kGy, the concentration of yeast was obviously reduced; therefore, fungi and bacteria gradually became the predominant. Following the irradiation at 8 kGy, some of the specimens still contained a few viable fungi, and the main microbial species had become a bacterial strain with a cream-colored colony. Following the irradiation at 10 kGy, the lycium fruit specimens contained only a viable bacterium of the aforementioned cream-colored bacterial strain. This surviving strain was isolated and identified as a Gram-positive spore-forming bacterium, B. cereus, and it could be completely decontaminated after the irradiation at a dose of 14 kGy. This research employed two approaches to evaluate the viability of microorganisms in the irradiated samples, because a single method cannot yield complete results. The first method is the spread-plate method, in which the samples were immersed with shaking. After a proper dilution, the immersed suspensions were evenly spread on the plate. After the microbial colony had formed, the colony morphology was observed and the colony number was counted. The strength of this approach is that the microbial colony exhibits clear growth and allows easy counting. However, the weakness is that the growth of some strains of the microbes depends on particular component of the lycium fruit,
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so the microbial colonies cannot form on the general medium for counting, potentially leading to underestimation. The second method is to put the sample of lycium fruits directly onto the medium and to observe the growth of microbes. The microbes are counted by eye, and an optical microscope is used to observe the microbial growth on the lycium fruit and the proliferation from the lycium fruit to the medium. The strength of this approach is that the strains of microbes that depend on particular components in lycium fruit can grow selectively on both the fruit and the adjacent medium. Restated, it provides opportunities for growth. Also, a microscope can be used to observe clearly the situation with regard to growth of the microbe on the lycium fruit and the erosion of the lycium fruits. However, the weakness is that when the microbe grows on lycium fruit with a less plain surface or a less consistent color, the microbial colonies are not clearly seen and so can be easily missed. Additionally, when the amount of microbe is high or many microbes are present, the microbial colonies on the lycium fruit frequently overlap as they grow, so differentiating and counting accurately the microbial colonies become difficult. Therefore, the two methods mentioned above should be simultaneously applied to determine the number of microbe contained in lycium fruit during the determination of the relationship between radiation dose and decontamination of the microbes. Furthermore, the growth of the microbes in the irradiated lycium fruit is slower than that in the non-irradiated lycium fruit. Therefore, more time is required to observe the microbial colonies and a 2-day delay is required before counting to ensure that the number of microbial colonies is not underestimated. Although the amount of microbes in the lycium fruit is the main factor that determines the required radiation dose, the presence of spore-forming microbial strains is also an important reason for the required increase in radiation dosage, confirming that the irradiation of lycium fruit at a dose of 14 kGy can effectively eliminate microbes. In this study, gamma irradiation effectively inhibited the process of fermentation of lycium fruit by killing yeasts, greatly improving the quality of the lycium fruit and its stability during transport and storage.
0 Log Inactivation (CFU/ml)
600
-1 -2 -3 -4 -5 -6
C. laurentii B. cereus
-7 0
10 12 4 6 8 2 Dose of Co-60 Gamma Radiation (kGy)
14
Fig. 1. Surviving fraction of the strain of yeast, Cryptococcus laurentii, and endospores of Bacillus cereus at various doses of gamma irradiation.
(0.6 kGy) for the first 90% reduction (LD90) and D10. However, the survival curve of the spores of B. cereus falls slowly and has a shoulder. Therefore, it has different values for the LD90 (6.5 kGy) and D10 (1.7 kGy). The D10 value is normally calculated over the linear part of the death curve, but the presence of a shoulder may result in underestimation of the actual dose required. Therefore, these two parameters (LD90 and D10) are required to precisely describe the radiation resistance of microorganisms. The spore-forming bacteria are the more resistant ones, because bacteria in spore form tend to be much more resistant to radiation than those in vegetative form. Therefore, the radiation dose required to decontaminate lycium fruits should be determined from the dose required to eliminate the endospore of B. cereus strain. However, comparing B. cereus with the highly radio-resistant bacterial strain, Deinococcus radiodurans, which is also appearing in food (Moseley, 1983), reveals that the radioresistance of B. cereus is clearly lower than that of D. radiodurans. The LD90 for D. radiodurans is about 13 kGy (Chou and Tan, 1990; Tan and Maxcy, 1982). The presence of B. cereus bacteria in lycium fruit specimens should not raise problems for radiation decontamination.
3.2. Radio resistance of the predominant yeast and the spore-forming bacterial strain in the lycium fruit specimen
3.3. Composition changes of lycium fruit after gammaray irradiation
The radiation resistance of C. laurentii, the predominant microbial strain in the pre-irradiated lycium fruit specimens, and B. cereus, the bacterial strain isolated from the 10 kGy irradiated lycium fruit specimens, was measured and shown in Fig. 1. The radio resistance of these two strains differed greatly. The survival curve of the yeast, C. laurentii, has no shoulder and its slope declines sharply. This curve shows the same value
Specimens of lycium fruit that had been gamma irradiated at 4, 8 and 14 kGy, and those of non-irradiated lycium fruit, were examined to determine their pH value, titratable acidity, moisture, crude protein, b-carotene, riboflavin, vitamin C, fructose, glucose, citric acid, malic acid, succinic acid, fumaric acid and oxalic acid contents. The pH value of the specimens of non-irradiated lycium fruit was 5.08, and the titratable acidity that expressed as
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Table 2 Effects of gamma-irradiation on the composition in lycium fruit Items
Moisture (g/100 g) Crude protein (g/100 g) b-carotene (mg/100 g) Riboflavin (mg/100 g) Vitamin C (mg/100 g) Fructose (g/100 g) Glucose (g/100 g) Citric acid (g/100 g) Malic acid (g/100 g) Succinic acid (g/100 g) Fumaric acid (g/100 g) Oxalic acid (g/100 g)
Irradiation dose (kGy) 0
4
8
14
16.51 13.44 1.13 0.63 8.35 25.03 22.84 4.26 2.54 1.18 0.02 0.14
16.61 13.24 1.13 0.64 4.3 24.91 22.76 4.24 2.47 1.21 0.02 0.15
16.58 13.31 1.12 0.63 ND 25.01 22.83 4.31 2.49 1.18 0.02 0.14
16.60 13.19 1.12 0.60 ND 24.99 22.78 4.35 2.55 1.22 0.02 0.15
Each value was the average of triplicate of experiments. ND, indicates no vitamin C detected.
titration ml of 0.1 N NaOH was 4.2. No significant changes in pH and acidity were observed following 4, 8 and 14 kGy gamma irradiation. The approximate compositions in 100 g lycium fruit samples were 16.51 g moisture, 13.44 g crude protein, 1.13 mg b-carotene, 0.63 mg riboflavin, 8.35 mg vitamin C, 25.03 g fructose, 22.84 g glucose, 4.26 g citric acid, 2.54 g malic acid, 1.18 g succinic acid, 0.02 g fumaric acid and 0.14 g oxalic acid. Table 2 presents the results. Lycium fruit is the essential source of b-carotene and riboflavin (Luo et al., 2004). b-carotene is an anti-oxidant and a precursor of vitamin A, and is believed to improve vision (Hammond et al., 2001; Keijer et al., 2005). Notably, gamma irradiation had no effect on b-carotene content. Riboflavin, an anti-oxidant and a water-soluble vitamin, is essential for normal cellular functions (Foraker et al., 2003). Riboflavin was 0.63 mg per 100 g before irradiation and slightly decreased to 0.60 mg per 100 g after the 14 kGy irradiation. Other composition was not significantly changed by gamma irradiation except the vitamin C content. We observed that the vitamin C content was decreased following the increasing doses of gamma irradiation; this result was similar as the effect of gamma irradiation on dried potato slices (Wang and Du, 2005). The reduce of the vitamin C content should not be a significant problem, because lycium fruit that commonly employed in traditional Chinese medicinal treatment for improving vision is the essential source of b-carotene, not vitamin C (Hsu and Peacher, 1994).
(Table 3). Gamma irradiation slightly changed the color specifications of the surface of the dried lycium fruit samples: the lightness, redness, and yellowness of the samples slightly declined at high dosage (14 kGy). From a three-dimensional scatter plot, all the L, a, b values varied by two variables (storage time and irradiation doses) were not significantly different from one another at 95% confidence level. However, the b value seemed to slightly alter only when gamma dose was changed.
3.4. Hunter’s color value
4. Conclusion
Hunter’s color parameters of the irradiated and nonirradiated lycium fruit, such as lightness (L), redness (a), yellowness (b) and color difference (DE), were determined
In this research, the optimal doses for inactivating microorganisms in lycium fruit have been studied. The sufficient dose for inactivating Enterobacteriaceae was
3.5. Sensory evaluation Sensory analysis was performed immediately following irradiation. Triangle test panelists could not detect any significant difference between the 14-kGy gamma irradiated and non-irradiated lycium fruit. For N ¼ 20, a total of 11 correct responses were required to conclude a significant difference at pp0.05 (Meilgaard et al., 1999). In our study, of the 20 panelists, only 7 correctly selected the odd sample. This number was smaller than that required to confirm significance at a 5% level. Moreover, some panelists, who presented the correct results, could not specify the difference between these two samples. This uncertainty indicated that panelists could not distinguish non-irradiated lycium fruits from lycium fruit irradiated at 14 kGy, as expected just by chance. The evaluation showed that lycium fruit retained their sensory quality after irradiation.
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Table 3 Changes in Hunter’s color value of non-irradiated and gamma irradiated lycium fruit Color parametera
L
Storage period (month)
Irradiation dose (kGy) 0
4
25.22b 25.06 18.29 19.73 9.79 9.89 0.00 1.452
24.85 23.75 18.28 18.49 9.85 9.76 0.375 1.484
8
14
24.98 23.83 23.08 23.17 a 18.02 17.95 17.99 17.82 b 9.82 9.45 9.60 9.35 DE 0.362 1.471 1.464 2.149 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi a 2 2 L, degree of lightness; a, degree of redness; b, degree of yellowness; DE, the overall color difference ( ðDLÞ þ ðDaÞ þ ðDbÞ2 ). b Each value was the average of triplicate determinations. 0 3 0 3 0 3 0 3
4 kGy and 14 kGy was required for eliminating the total mesophilic microbes. In addition, 8 kGy was a sufficient dose for the inactivation of yeasts, resulting in eliminating the unwanted fermentation process of lycium fruit. To investigate the effect of gamma irradiation on chemical composition of lycium fruit, the crude protein, b-carotene, riboflavin, vitamin C, fructose, glucose, citric acid, malic acid, succinic acid, fumaric acid, and oxalic acid contents of lycium fruit were analyzed before and after irradiation. Results showed that vitamin C content reduced after irradiation at 14 kGy. However, amount of other composition including b-carotene and riboflavin did not change significantly under the same treatment. Lycium fruit has been used as the major source of b-carotene and riboflavin, and people normally ingest lycium fruit to improve their vision. In addition, the sensory characteristic of lycium fruit also did not significantly alter after the 14 kGy irradiation. Therefore, gamma irradiation at 14 kGy can be a potential method as the cold decontamination for lycium fruit to prolong the shelf life, to improve the hygienic quality, and to reduce the risk of food borne disease.
Acknowledgements We would like to thank the Committee on Chinese Medicine and Pharmacy of the Department of Health, Taiwan, for their financial support (CCMP-RD-045), Professor Y.S. Chung for confirmation of the authenticity of lycium fruit, and our colleagues in the Nuclear Science and Technology Development Center for their assistance in performing gamma irradiation and sensory evaluation.
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